Translating Pre-Clinical Research to Clinical Patient Care™
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Plenary Presentations
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Commercialization and Protection of Intellectual Assets in Applications of Mass Spectrometry
Bringing improvements in assessing conditions using Mass Spectrometry (MS) requires substantial investment in research, development of devices and methods for diagnostic and therapeutic interventions. Investments are unlikely to be made unless returns on those investments can be realized. Returns are made possible through licensing of patented inventions, trade secrets, and other intellectual assets (IP). With proper strategies combining business, technology, and the law, commercializing important innovations will result in improved patient care, recognition of innovators' contributions, and business success, all of which can lead to further innovation and improvements in health care.
Direct Mass Spectrometric Characterization of Fluids, Cells and Tissues - the Benefits and the Price of Real-Time Analysis
Development of ambient mass spectrometric techniques opened a new way of looking at clinical samples regarding the simplicity (and time demand) of analysis and the nature of data produced by these techniques. Application of these approaches in cancer surgery, histopathology, clinical microbiology and clinical chemistry has been successfully demonstrated. While these techniques mean valid alternatives to currently used technologies - with clear benefit on both cost and reliability sides - their widespread application can potentially change the current landscape of medical diagnostics. Future role of these methods in stratified clinical patient journeys will be discussed with particular emphasis on the currently unresolved problems and their future solutions regarding both regulatory and technology aspects.
When Clinical Lab Tests Fail: New Paradigms of Metabolic Regulation
Traditionally, mass spectrometry-based metabolomics has been applied to identify metabolites whose levels are altered as a function of disease. While this platform has provided great insight into many pathologies, it fails to identify important changes in metabolites whose levels are homeostatically controlled by multiple converging pathways. Here we describe an isotope-based platform that enables tracking of pathway dynamics at the systems level. To accomplish this approach, we have developed an extension of the widely used XCMS metabolomic software that processes data from isotopically enriched samples in an automated fashion. We show that monitoring isotope incorporation at the systems level provides new insights into cancer pathogenesis that would otherwise be missed by standard metabolomic approaches.
Genetic Defects in Bile Acid Synthesis Causing Liver Disease - Diagnosis and Treatment - Translational Medicine from Mass Spectrometry Discovery to the Bedside
The combined application of FAB-MS with metabolic profiling using GC-MS, and electrospray HPLC-MS with accurate mass measurement led to the discovery of six genetic defects in the complex pathway for bile acid synthesis from cholesterol. The biochemical basis of each defect was characterized, mutations in genes encoding the respective enzymes confirmed, and a successful therapy developed. These genetic defects accounted for 2% of >12,000 cases screened for unexplained liver disease. Early diagnosis is important because patients with these fatal forms of metabolic liver disease respond successfully to oral primary bile acid therapy, thereby avoiding the need for liver transplantation - the only other therapeutic option. Based on the successful application of this therapeutic strategy, oral cholic acid therapy was granted approval in November 2013 by the European Medicines Agency (EMA) for the treatment of these bile acid synthetic defects and is currently under review by the US FDA.
This presentation of more than two decades of work is an example of translational medicine - taking a clinical problem at the bedside, to the utilization of mass spectrometry to define a new class of metabolic liver disease, to the development of a successful and safe therapy, and finally taking this back to the bedside by obtaining regulatory approval. Accelerating Clinical Mass Spectrometry; Workflows and Whitespace
Over the last decade, LC-MS/MS technologies have evolved from their innate promise of reference level measurement to become a circumscribed technology in patient management. The implementation of LC-MS/MS reflects the abstruse and inchoate status of the platform; we are challenged with ensuring future advancements are not ersatz as we balance sanguine hype and egregious hubris.
This presentation will highlight our efforts to enable LC-MS/MS and peripheral technologies in clinical diagnosis, subsequently converting the apostates. Specific examples involving contiguous automation from patient to result, atavistic enhancements in hormone analysis, targeted protein analysis despite opprobrium and functional immune-response determination will be shown as we adduce the locution "mass spectrometry est sui generis".
Moving Mold Identification into the 21st Century with MALDI-TOF MS
MALDI-TOF MS has revolutionized clinical microbiology laboratories, enabling accurate bacterial and yeast identification within minutes at minimal costs and labor. However, mold identification by mass spectrometry has lagged, forcing many laboratories to rely on slow phenotypic methods that are prone to misidentifications. This presentation will describe the development of the NIH Mold Database - a clinically comprehensive, freely available database for the identification of molds from solid media. Its clinical performance will be highlighted through a series of multicenter studies and case presentations, with the NIH Mold Database outperforming a commercially available database. The NIH Mold Database has pushed the boundaries of clinical mycology, providing faster therapeutic guidance, positively impacting patient care, and opening new doors and challenges for clinical research.
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Podium Presentations
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Automated Mass Spectrometry Analysis of Full-length Hemoglobin for Clinical Applications
We developed a method for the identification of hemoglobin variants by electron-transfer dissociation (ETD) of the full length protein (Coelho-Graça et al, JASMS, 2012) and a method for the quantification of Hemoglobin A2 using precursor-ion isolation and detection (Acosta-Martin et al., Anal. Chem., 2013). These two workflows were now integrated into a fully automated pipeline, which includes data acquisition for HbA, HbS, HbC identification and HbA2 relative quantification. This workflow will now be tested in a clinical environment for hemoglobin variant identification as well as diagnosis of thalassemia.
Quantitation of Monoclonal Antibody Therapeutic Infliximab by LC-MS/MS
We have verified that unique proteotypic peptides from variable regions of Infliximab (chimeric IgG1 kappa targeting TNF-α) may be used to quantify the drug in patient serum using standard SRM analysis on an ABSciex API 5000. The assay was developed, validated and compared to an electrochemiluminescent method. Samples from patients receiving Infliximab therapy were tested at various time-points following treatment. The ability to quantify Infliximab in patients using proteotypic peptides and LC-MS/MS was demonstrated. This analytical approach has the potential to be quickly adaptable to other monoclonal antibody drugs and to significantly improve patient care.
Identification and Quantitation of Insulin Analogues by Immunocapture Coupled with LC-MS/MS
Insulin is frequently measured for the investigation of spontaneous hypoglycaemia in adults and children. Several synthetic insulin analogues are in routine clinical use for the management of Type I and Type II diabetes mellitus. In clinical medicine, hypoglycemia remains one of the most frequently encountered sequelae of insulin therapy. Commercial insulin immunoassays exhibit variable cross-reactivities to analogue insulins. Given these limitations, we have developed an immuocapture-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of five insulin analogues. We will present our work in developing this assay, and its application to clinical and forensic cases in our laboratory.
High-Throughput Sensitive LC-MS/MS Estradiol Analysis
We have developed a LC-MS/MS assay for the measurement of estradiol and estrone. The assay has a novel sample preparation utilising on-line solid phase extraction combined with a short chromatography column that results in a rapid run time making it suitable for routine clinical use. Despite the rapid run time, estradiol and estrone are adequately separated to remove a potential isobaric interference. The simultaneous measurement of estradiol and estrone gives additional clinical information towards calculating total estrogen status.
Three Years Experience in Screening and Diagnosis of Primary Aldosteronism by LC-MS/MS
Primary aldosteronism (PA) is the most common form of secondary (ie potentially treatable) hypertension and is generally caused by adrenal hyperplasia or aldosterone producing adenoma. Biochemical testing for this disease has traditionally relied on immunoassays for aldosterone and plasma renin activity or renin mass. However these have been fraught with problems of intermethod variation and analytical cross-reactivity. LC-MS/MS offers an excellent solution. We describe three years experience in screening, diagnosis and tumor localization for primary aldosteronism by LC-MS/MS. We delineate the technical challenges associated with the methods, data processing, and autointerpretation. Advantages and potential pitfalls will be described along with the the many fortuitous diagnoses provided by LC-MS/MS which would have otherwise been delayed or missed.
Aldosterone by LC-MS/MS: Cost Saving Not at the Cost of Patient Care
Measurement of low abundance endogenous analytes in high throughput laboratories present challenges associated with specificity, efficiency of analysis and cost of testing. We evaluated Liquid Liquid Extraction (LLE) vs Supported Liquid Extraction (SLE) in measuring serum aldosterone using an AB SCIEX Triple Quad 5500 Mass Spectrometer with and without the use of differential ion mobility spectrometry (DMS). Under the evaluated conditions, over 30% of samples analyzed by various methods had ratio of the mass transitions outside of the expected limits which lead us to evaluate several HPLC column chemistries and mobile phases. LLE and SLE extractions produced comparable results. LLE was 30% more laborious although less expensive. The use of LLE and DMS could reduce expenses but at the cost of high starting serum volume. Our final choice for the assay using low sample volume and low cost was in favor of LLE in conjunction with 2 dimensional chromatography and use of an AB Sciex 5500 MS without the use of DMS.
Development of an Assay and Viral Database for the Identification of Biothreat Viruses Using MALDI Mass Spectrometry of Intact Virions
Since many laboratories employ the MALDI Biotyper system for bacterial identification, we sought to develop a viral typing assay which would be compatible with this platform. We employed a strategy of producing spectra from highly purified viral stocks for spectral database compilation, which could then be used to type unknowns using a more rapid method of harvest. A custom made set of reference spectra was successfully generated from 7 highly purified viral isolates using a modified BioTyper method. Spectral patterns were unique to each virus and peaks corresponding to the molecular weight of capsid proteins were detected and confirmed by LC/MS-MS. Successful identification of West Nile virus, and Rift Valley Fever virus was achieved from 1ml of infected media using a sucrose cushion and standard micro-centrifugation with BioTyper scores of 1.8 and above.
Salvage Microbiology: Direct Detection of Pathogens from Sterile Clinical Specimens Submitted for Culture Following Initiation of Antimicrobial Treatment
We prospectively identified 56 cases of suspected bacterial infection from which 80 sterile specimens were submitted for culture after the initiation of antimicrobial treatment (multiple specimens were submitted for culture in 21 cases). Compared to culture, PCR/ESI-MS was more likely to detect potential pathogens: 74% (59/80) vs. 32.5% (26/80). Pathogenic organisms were detected by PCR/ESI-MS that were not identified in culture in 45% (25/56) of the patients included in our study: 36% (20/56) of the PCR/ESI-MS positive patients had completely negative cultures; and 9% (5/56) had polymicrobic infections. Cohen's Kappa statistic confirms that there is discordance between culture and PCR/ESI-MS results (K = 0.348). Our prospective series demonstrates that PCR/ESI-MS can detect pathogenic bacteria that are not grown in conventional culture directly from clinical specimens obtained following initiation of antibiotic treatment.
Computational Considerations for Intensity-Based Microbial Identification Using MALDI-TOF MS
MALDI-TOF driven microbial identification is rapidly changing the workflow of clinical microbiology laboratories. The majority of users will understand that the problem the analyzer is attempting to solve is rooted in pattern recognition. The complexities of how this is achieved computationally may be more problematic to understand intuitively. This presentation will approach this computational problem using the historically well performing dot product approach of spectral library search algorithms. The caveats explored will be evaluating how performance characteristics change due to m/z window size, baseline subtraction algorithms, and comparison of median-based and eigenvector based reference spectrum generation. These permutations were evaluated based on the bootstrapped comparison of spectra of E. avium and E. Faecium generated by a Bruker Biotyper.
So You Want a Mass Spectrometer?
Use of liquid chromatography-mass spectrometry for analysis of small molecules in the clinical laboratory has increased in the past few years. Many labs have considered implementation of this technology, but some do not have enough experience to decide in which type of mass spectrometry system they should invest. This presentation will describe the basics of liquid chromatography, the types of mass spectrometer available and what type of analysis they are most suited to (quantitative versus qualitative), and the basics of return on investment calculations that may be required to justify a mass spectrometer purchase to laboratory administration.
Instrument Selection, Due Diligence, and Setting Up a Clinical Lab for Successful Implementation of MS
After determining that your laboratory is ready to expand into clinical applications of mass spectrometry and determining what type mass spectrometer will be optimal for the work you propose, the next tasks are to select a vendor, make necessary modifications to your lab, perform instrument qualification, and begin validating clinical assays. This presentation will focus on our laboratories approach to performing due diligence and to setting up a laboratory to successfully implement clinical applications of mass spectrometry.
The MS Test Life Cycle Process: How to Keep Your Lab Moving Forward
As more laboratories develop mass spectrometry-based tests, they need to have a standardized program to define, document, validate, and monitor laboratory-developed tests going forward to prepare for and meet the increased regulatory oversight. This presentation will describe the seven phases of a mass spectrometry test life cycle process at the Mayo Clinic which takes a laboratory-developed test from design, development, verification, validation, launch, and maintenance to retirement. It will also focus on the importance of post analytical monitoring and how proficiency testing programs can be used in combination with other assessments to monitor assay performance over time.
Comparing LC/MS and Immunoassays for Clinical Biomarkers
In clinical studies, biomarker assays are routinely measured using immunoassays. These assays generally meet the criteria necessary for clinical biomarkers, but in some cases are not sufficient. Mass spectrometry based assays are inherently more selective and while these assays generally lack sensitivity compared to immunoassays, the selectivity of the platform has advantages in the ability to multiplex, and independently measure PTMs and isotope labeled tracers as part of kinetics studies. Recent technical advances such as immuno-affinity enrichment and low-flow chromatography allow mass spectrometry to compete with immunoassays in terms of sensitivity.
Next Generation Blood Plasma Collection and Sample Preparation in Mass Spectrometry Based Diagnostics
A new technology is described that allows blood plasma extraction from a single 25-50 uL drop of blood obtained from a finger-stick. Blood fractionation was achieved by capillary driven flow through multiple membrane layers that sequester blood cells via filtration as plasma migrates to a collection disc at the bottom of the membrane stack. Fractionation is completed in roughly 3 min when a 6.4 mm diameter collection disc is saturated with either a 2.4 or 4.8 uL sample of plasma, depending on the disc size. Analyte concentration and sampling volumes are hematocrit independent. Stripping (delaminating) the upper, cell bearing membrane layers allowed evaporation from the plasma loaded collection disc to dryness in 15 min. Internal standards and antibodies were either preloaded in the collection disc or added after plasma sampling; allowing affinity selection and analysis of analytes with a Perfinity Workstation (PWS) coupled to a Shimadzu 8050 triple quadrapole instrument.
Measurement and Normalization of Low-abundance Protein Analyte Panels in Dried Blood Spots (DBS) Using an Automated SISCAPA Workflow
An improved high-throughput automated SISCAPA workflow was devised and applied to dried blood spots samples (DBS). This protocol provides significant sensitivity and throughput improvements for protein measurement in DBS, and overcomes sample hematocrit and volume variability issues that can limit the accuracy of protein-based DBS technology in clinical settings. We measured two 11-protein panels using 3-minute cycles, including targets in the low ng/ml range, and normalized results by separately measuring a set of high-abundance proteins comprising 80% of the total plasma protein, as well as the dominant red cell proteins, to yield surrogate hematocrit and total plasma load.
Moving High Resolution Mass Spectrometry into Routine Clinical Practice: Optimizing Targeted Data Analysis
Although there has been much interest in high resolution mass spectrometry (HRMS), a major challenge in implementing this technology in the clinical laboratory is the data analysis process. Even for targeted analysis, the process is complex because so much data is generated from a single sample and many parameters are involved in identifying compounds. Using a training set and a test set of urine samples, we try to determine the optimal parameters for targeted data analysis. The goal of this study is to streamline the data analysis process and make HRMS more accessible to the clinical lab.
Determination of Metabolites of the Bath Salt Pentylone, Using LC/MSAll
Bath salts have emerged recently as recreational drugs, which are known to cause hallucinogenic effects. The pathways metabolizing bath salts have not been well characterized, which complicates our ability to screen for bath salt use. We sought to understand the metabolism of the bath salt pentylone by using a mouse model and a novel untargeted metabolomic platform based on LC/MSAll. MSAll alternates between high and low energy scans during acquisition, resulting in MS1 data and fragmentation data for each precursor ion. We used MS1 data to quantify metabolite alterations in pentylone-treated mice and MSAll data to obtain semi-putative structural assignments.
Mass Spectrometry vs Designer Synthetic Cannabinoids
Identification and quantification of new designer drugs is an important public health and safety issue; however, the metabolism of synthetic cannabinoids and appropriate urinary target analytes are frequently unknown. With more than 40 entirely new designer drugs identified by the US Drug Enforcement Agency in the first five months of 2013, designer drugs are the emerging face of drug abuse. We utilized LC-MS/MS to confirm and quantify metabolites, optimize cutoffs for synthetic cannabinoid immunoassays and, when metabolic profiles were unknown, human hepatocyte incubation and high resolution mass spectrometry to decipher urinary targets.
Metabolomics and Metabolite Imaging Correlates Biofilm Polyamine Synthesis with Colon Cancer
Mass spectrometry-based metabolomics was used to investigate the effects of colonic biofilms on metabolites in colon cancer. Untargeted analysis revealed upregulation of polyamines in tumors compared to normal tissues, which was enhanced in biofilm positive tumors. Antibiotic-treated tumors were metabolically similar to biofilm negative than positive tumors, indicating that polyamines were produced by both host and biofilm. Targeted analysis obtained concentrations of these metabolites, and nanostructure initiator mass spectrometry (NIMS) imaging provided spatial specificity of the metabolites in the tissues. The results show that biofilms can alter the colon metabolome to produce known regulators of cell proliferation and tumor growth.
Colorectal Cancer Detection Using Targeted Metabolic Profiling
Colorectal cancer (CRC) is one of the most prevalent and deadly types of cancer worldwide. In this study, we proposed a targeted LC-MS/MS metabolic profiling approach for sensitive and selective CRC diagnosis. 234 serum samples from three groups (66 CRCs, 76 polyps, and 92 controls) were analyzed. 42, 48 and 8 out of 158 targeted metabolites showed statistical significance between pairwise group comparisons. PLS-DA models were established for the separation of CRC patients from other groups, and validated using Monte Carlo cross validation. ROC curves were generated, with excellent AUROCs (>0.93), high sensitivities (>0.89) and high specificities (>0.80).
Targeted Metabolomics Identifies Glutathione Inhibition as a Mode of Sensitizing Drug-resistant Ovarian Cancer Cells to Nutrient Deprivation by L-asparaginase
L-asparaginase (L-ASP) is used for treatment of acute lymphoblastic leukemia. Our recent metabolomic investigations showed that cell lines, such as OVCAR-8 which exhibited sensitivity towards L-ASP are glutamine addicted while drug resistant cells, such as OVCAR-4 are glutamine independent. To investigate the biological pathways associated with glutamine independence, we profiled the metabolites of TCA cycle, pentose phosphate pathway, nucleotides, and GSH/GSSG using a label-free, HILIC-based LC-MS/MS assay. Results revealed interesting differences in GSH metabolism between OVCAR-4 and OVCAR-8 cells. Treatment of OVCAR-4 cells with L-ASP combined with BSO, an inhibitor of GSH synthesis, induced cytotoxicity in these cells.
CDC Efforts Towards the Development of a Reference Method for Measurement of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in Serum
Vitamin D status is routinely assessed by measuring serum concentrations of the most stable and abundant vitamin D metabolites. To assure the accuracy of measurements and to harmonize laboratory results regardless of measurement procedure or location, worldwide standardization of 25-hydroxyvitamin D measurements is underway. Several JCTLM-recognized reference measurement systems have been established consisting of reference materials and methods. We are presenting key details from our new isotope dilution LC-MS/MS candidate reference method for measurement of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum. The method will be used to support standardization activities, including the CDC Vitamin D Standardization Certification Program.
An Update on Standardization Efforts for Steroid Hormones
Correct diagnosis and treatment of patients rely on accurate testing. Furthermore, the effective use of research findings in patient care and public health requires testing in research and patient care to be accurate and comparable. The CDC, together with the Partnership for the Accurate Testing of Hormones (PATH), is working to improve the accuracy and reliability of steroid hormone testing. In addition to its testosterone standardization program, CDC successfully conducts programs to standardize estradiol and vitamin D, and it works with PATH to develop new programs, promote accurate testing, and establish reference ranges.
Standardization of Immunosuppressant Drug Measurements
The movement of LC–MS/MS into clinical laboratories has been accelerated by the need to monitor immunosuppressant drugs. The analytical sensitivity and specificity of LC-MS/MS allows laboratories to quantify cyclosporine, tacrolimus, sirolimus, everolimus and mycophenolic acid, often with multiple drugs quantified within a single run. Yet even for small drug molecules the path to standardization has not been easy, as shown by calibrator traceability studies, proficiency testing schemes and inter-laboratory comparisons. This presentation will cover the state of standardization of immunosuppressant quantification from the perspective of both immunoassay and LC-MS/MS, each of which has had challenges to success.
Using the Accurate Molecular Mass of Monoclonal Light Chains to Detect Residual Disease in Patients with Multiple Myeloma
Multiple myeloma is a plasma cell proliferative disorder where the M-protein (monoclonal immunoglobulin) serves as a surrogate marker for the plasma clone responsible for its production. In multiple myeloma along with clinical symptoms, the diagnosis, monitoring and treatment decisions are dependent on the detection of an M-protein. The absence of an M-protein is indicative of remission, whereas an increase of M-protein can signal progression. Here, we report a mass spectrometry based method to determine the accurate molecular mass of the M-protein in patients diagnosed with multiple myeloma. We are able detect these patient-specific M-proteins in sera months after successful therapy when the patient was deemed in remission using stringent criteria. This highlights the enhance sensitivity mass spectrometry can offer in detecting monoclonal immunoglobulins in patients sera.
Meeting the Needs of In-house Thyroid Cancer Patients: Identifying Appropriate Thyroglobulin and Thyroglobulin Antibody Testing Algorithms
Thyroglobulin (Tg) immunoassay methods are susceptible to Tg antibody (TgAb) interference necessitating referral of high TgAb specimens. The most common referral tests are radioimmunoassay (RIA) and LC/MS/MS. This study will identify the limitations of UPMC in-house Tg to develop appropriate testing algorithms.
High TgAb specimens (n=20) from adults post total thyroidectomy were analyzed for Tg using immunoassay (DxI800; Beckman Coulter), RIA (USC Endocrine Laboratories), and LC/MS/MS (Quest Diagnostics). At low Tg concentrations there was poor correlation between the DxI800 and other methods (RIA, R2 = 0.64; LC/MS/MS, R2 = 0.76). Comparison of RIA and LC/MS/MS showed some discrepancy (R2 = 0.82) but had high correlation for low Tg concentrations (<13 ng/mL; R2 = 0.96).
Reference testing using LC/MS/MS for TgAb positive specimens that exceed 13ng/mL Tg is clinically necessary.
Shotgun Proteomics-based Clinical Testing for Diagnosis and Classification of Amyloidosis
Shotgun proteomics technology has matured in research laboratories and is poised to enter clinical laboratories. However, the road to this transition is sprinkled with major technical unknowns like long-term stability of the platform, reproducibility and clinical utility over traditional IHC platform. As a result, diagnostic laboratories have avoided using shotgun proteomics for routine diagnostics. We developed two shotgun proteomics assays (FFPE tissue-based and subcutaneous fat aspirate-based) for amyloid subtyping by standardizing the platform for better quality control and earning clinical acceptance. These assays have >91% success rate of subtyping compared to 48% of traditional IHC technique. These assays were utilized to detect 19 different amyloid subtypes in 5600 patients. Retrospective data mining of amyloid proteomes revealed ALect2 as a new third major type in the kidney.
Reducing the Need for Calibration Standards
Mass spectrometers are extremely reliable tools for clinical testing, especially when isotopically labeled internal standards are used. This lead us to consider if a traditional number of calibration standards are always necessary. With linear data, a single calibrator should be sufficient to establish detector response for quantitation. Here we compare efforts utilizing a weighted historical response factor to a conventional approach. For nonlinear data we utilize a unique solution accounting for isotope contributions to internal standard. In this case, use of one or two experimentally determined constants and a single adjustable parameter still allows use of a single calibration standard.
Finding the Fakes of Drug Testing; New Strategies to Identify Substituted Samples
Urine- based drug testing is a key component of pre-employment screening, probationary monitoring, and pain management compliance programs. There exists an enormous incentive for deception if a sample donor believes they are at risk of failing. Substituted synthetic urine samples are the most common deception attempted today. Synthetic urine products are increasingly complex, capable of passing routine sample validity tests. We have characterized synthetic urine and mined natural urine identifying novel targets to distinguish real from artificial samples. Exogenous markers and esoteric endogenous markers, functioning as validity indicators, have been incorporated into LC-MS/MS assays at high risk for substitution.
Drug Screening Using Multiple Methods (immunoassay, GC-MS, LC-MS/MS): Is there an Easier Way?
In the clinical laboratory, drug screening is done by a combination of methodologies. Recently, drug screening methods that utilize high resolution mass spectrometry(HRMS) have been published, however, few studies have compared HRMS screening to traditional drug screening methodologies. The objective of this study was to develop LC-HRMS and LC-MS/MS drug screening methods that are identical except for the mass spectrometer used and to compare them to immunoassay screening for their ability to identify drugs/metabolites in routine specimens. The MS methods were more sensitive and specific compared to immunoassay and had a better positive and negative predictive value. They were also capable of detecting additional compounds with a high degree of confidence in positive identifications.
Multicenter Evaluation of MALDI-TOF MS for the Identification of Bacteria and Yeast
Inter-laboratory performance of MALDI-TOF MS for organism identification is unstudied. Herein, we assess Bruker Biotyper MALDI-TOF MS validation data from > 5,500 isolates from 9 medical centers. Of 1,820 Gram positives, 94.8% were correctly identified, 1.9% incorrectly identified while 3.2% could not be identified. Of 2,635 Gram negatives, 95.9% were correctly identified, 2.3% incorrectly identified while 1.8% could not be identified. Of 382 yeast, 95.8% were correctly identified, 2.4% incorrectly identified. Of 863 anaerobes 92.78% were correctly identified, 0.1% incorrectly identified while 8.1% could not be identified. In conclusion, MALDI-TOF MS is reliable and reproducible for organism identification across laboratories.
Comparison of Preparation Methods and Instrumentation Platforms for MALDI-TOF MS Identification of Medically Relevant Yeast Species
Our objective was to evaluate multiple “direct smear” techniques for yeast identification using the VITEK MS and Bruker Biotyper MALDI-TOF MS platforms.
Yeast isolates (117) were analyzed after 24 and 48h incubation. Isolates were analyzed by the direct smear method +/- a 25% formic acid on-plate extraction on both platforms and with 100% formic acid on Biotyper.
VITEK MS correctly identified 113 isolates at 24h with one misidentification. With a revised threshold of >1.7, Biotyper correctly identified 103 isolates, with 3 misidentifications. Formic acid significantly increased the identification rate. MALDI-TOF MS analysis for yeast identification drastically improves time to identification.
Identification of Filamentous Fungi Using MALDI-TOF Mass Spectrometry
Identifying filamentous fungi using MALDI-TOF MS has been difficult and spectral libraries have been inadequate for generating consistent matches. This study aims to verify this application of MALDI-TOF using a liquid broth-based growth protocol. Additionally, we hypothesize that advances in a current mold spectrum library will yield more frequent matches for identification. MALDI-TOF analysis of 158 mold isolates matched microscopic identification 72% of the time and DNA sequencing proved MALDI-TOF to be accurate in 82% of total cases. MALDI-TOF following growth in liquid broth is a viable method for identifying molds in the clinical laboratory.
Vitamin D by LC-MS/MS: Practical Tips and Stories from the Front-Line
Are you thinking of measuring a small molecule such as Vit D by mass spectrometry? Maybe you have an instrument and are wondering how to bridge the [large!] gap between installation and launching a validated method? Want some advice on how to deal with problems encountered along the way?
This session is intended to provide practical advice, examples and first-hand accounts of experiences encountered during development, validation and on-going performance assessment of Vitamin D testing by LC-MS/MS. Method selection/development tips, validation design examples and troubleshooting case studies will be presented.
How to Make a Clinical LC-MS Testosterone Assay that Actually Works for Real Patients: Lessons Learned from the Front Lines
This presentation will discuss the whole process of developing, validating and operationalising a Dx MS assay using an example testosterone assay currently being offered in our lab. Discussion of the approaches, instrumentation and methodologies for analysis of small molecules will be examined. Examples will be given of the sample preparation strategies, analysis modes and software options as well as how they behaved in our laboratory.
Development and Validation of Broad Spectrum Drug Screening Method on a Time-Of-Flight Mass Spectrometer
Recent developments in the high resolution mass spectrometry have lead to instruments with improved sensitivity and specificity as compared with contemporary low resolution LC-MS/MS instruments. HRMS instruments have potential to be universal detectors for broad spectrum drug screening in clinical and toxicology laboratories. We investigated a panel of more than sixty compounds in urine with a dilute, hydrolyze, and shoot methodology using a TOF instrument. Qualitative results obtained by our method had very good correlation with those obtained by LC-MS/MS methods. We observed that the addition of a fragment ion was essential in eliminating false positive results. This report will outline the challenges faced during the method development for a broad spectrum drug screen using high resolution TOF MS.
Unraveling Trypsin Digestion of Apolipoproteins A-I and B to Evaluate the Impact on Imprecision of MS-based Quantification
Trypsin digestion of apolipoproteins A-I and B was evaluated in normo- and hypertriglyceridemic serum to characterize the cleavage efficiency of multiple signature peptides. Addition of stable-isotope labeled peptides prior to digestion enhanced reproducibility, and only minor differences were observed across the various specimens. The total imprecision of apolipoprotein A-I and B measurement by LC-MS/MS, after quadruplicate sample preparations on five separate days, was <10% for all measured peptides. In general, 70 to 90% could be attributed to variations before LC-MS/MS analysis.
The results provide valuable information about the impact of proteolysis on apolipoprotein A-I and B measurement imprecision, of particular importance for application in clinical chemistry.
Development of a High-Throughput Mass Spectrometry Method to Screen for Familial Hypercholesterolemia and Detect a Common Genetic Variant of Apolipoprotein B
Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder largely underdiagnosed (prevalence 1:500), resulting from mutations in LDLR, APOB or PCSK9 genes and characterized phenotypically with elevated low-density lipoprotein cholesterol and apolipoprotein B particles. We developed an LC-MS/MS method in patient specimens utilizing peptides specific to apolipoprotein B (apoB100) and the most common APOB mutation (R3500Q). Method optimization was achieved by reduction in trypsin digestion times without compromising signal intensity and accuracy. Identification of an R3500Q mutation on LC-MS/MS was confirmed with genetic testing. Our data supports LC-MS/MS as a promising FH screening method, particularly in pediatric populations.
What Contributes to Inter-laboratory Variability of Targeted Protein
LC-MS/MS Assays: A Case Study Using IGF-1
The use of targeted LC-MS/MS assays for proteins will simplify the harmonization and standardization of clinical measurements. Careful investigation into the variability of the bottom-up proteomics process and its effect on inter-laboratory agreement is lacking. We developed a detailed standard operating procedure to quantify serum IGF-1, which is a well-characterized biomarker of growth hormone abuse, and deployed it in 5 laboratories in 3 countries. We identified the variability due to instrumentation and the influence of the approach to calibration on inter-laboratory agreement. The primary source of variability was not derived from the sample preparation but from the method of calibration.
Accelerator Mass Spectrometry to Detect Platinum-induced DNA Adducts and Identify Chemoresistance: from Bench Research to a Phase II Clinical Trial
Accelerator mass spectrometry (AMS) can measure carbon-14 at the sub-attomole level in mg-size specimens, and is commonly used for radiocarbon dating. We use AMS to measure DNA adducts induced by platinum chemotherapeutic agents. As alkylating agents, platinum agents kill cancer cells through induction of DNA adducts. We hypothesize that cancer cells with low platinum-induced DNA adducts will survive chemotherapy and are chemoresistant. Pre-clinical studies in cell lines and animal models support this hypothesis. After completion of pre-clinical studies and a Phase I clinical trial, a Phase II trial is going on and will be presented at the meeting.
Reduced Sample Volume and Simplified Sample Preparation in the Simultaneous Measurement of Zinc, Antimony, Bismuth, and Manganese in Whole Blood Using ICP-MS
To achieve required sensitivity from whole blood samples with Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), historic extraction methods often required a time-consuming chemical and/or microwave digestion. The reported method describes a simplified dilution for the simultaneous analysis of Zn, Sb, Bi, and Mn in whole blood. A 100 uL sample was extracted with 4.9 mL of basic diluent containing an Indium internal standard. Goat blood was added to all calibrators to achieve a matrix matched method. Inter and Intra-assay imprecision was less than 5% for all four elements.
Use of Isocratic Pump Direct Injection and Inductively Coupled Plasma-Mass Spectrometry for Analysis of Lead in 20 µL of Whole Blood
Lead in whole blood is currently analyzed to monitor exposures in children and in the workplace. Routine analysis by inductively coupled plasma-mass spectrometry (ICP-MS) utilizes a peristaltic pump for sample introduction into the ICP-MS, requiring 1-2 mL of prepared sample for each injection, despite the use of discrete injection valve systems. We investigated the use of an isocratic pump for discreet 100 µL injections into the ICP-MS. This method modification resulted in an 80% reduction in required sample and reagent volumes while maintaining acceptable analytical sensitivity, imprecision, and accuracy with respect to the original assay.
Targeted Profiling of Inflammation Related Oxylipins in Human Biofluids: Application to Surgery Patients
Current clinical risk scores are inadequate for surgical outcomes prediction, and robust biomarkers for the early detection of complications are lacking. To help understand these conditions, a sensitive UPLC-MS profiling method for the quantification of 48 oxylipins involved in inflammation signalling has been developed. Blood samples of patients undergoing surgery were analysed and significant lipid mediator evolutions were related to different stages of the inflammatory response and invasiveness of the intervention. Serial oxylipin measurements are of clinical merit in surgery and may serve as a novel biomarker for prediction of poor surgery outcomes, by accounting for patient-specific pre-operative inflammatory states.
Validation and Application of a LC-HRMS Platform for Simultaneous Absolute Quantification and Biomarker Discovery in the Clinical Laboratory
We have recently developed and validated a Q-TOF mass spec platform that simultaneously collects high resolution/high mass accuracy data for discovery work while simultaneously performing absolute quantification of over 50 relevant analytes commonly measured for variety of inherited metabolic disorders. These include amino acids, organic acids, purines/pyrimidines, and acylcarnitines. Previously, multiple forms of instrumentation were required to measure all of these different chemical classes, but advances in both chromatography and the linear dynamic range of high resolution mass specs make this attainable with a singe system, We show this method in its application to a drug study involving Alkaptonuria patients.
Lipidomics of Omega-3 Transgenic Mice Reveals a Panel of Anti-inflammatory and Pro-resolving Mediators
A large number of studies have demonstrated reduced disease risk and health benefits of long term omega-3 fatty acid supplementation. In this study, we used high-throughput MS assays, including a prototype microfluidic MS platform, to characterize the molecular phenotype of plasma samples from an animal model of long term omega-3 supplementation. Our results highlight a novel panel of anti-inflammatory and pro-resolving mediators, which may be involved in the health benefits associated with alterations of the omega-6/omega-3 fatty acids ratio.
Translating Between IT and MS Subcultures - An Overview of IT Technologies and Terminology Designed for Clinical Laboratory Personnel
In the quest to automate mass spectrometry instrument interfaces in the clinical laboratory, IT technologies play a critical role in the way disparate systems integrate. At play are a wide range of networking technologies, a dizzying array of communications protocols, laboratory information systems, middleware systems, and challenging instrument software systems. In most cases, laboratorians are the ones needing to orchestrate how all this technology must play together and to figure out what kind of assistance they need from their IT departments, vendors, and technical staff for a successful implementation. This presentation will provide laboratorians with the basic skills and understanding of the underlying IT technologies to effectively communicate with their technology providers.
A Road Map to Interfacing Your Mass Spectrometer
A barrier to implementing mass spectrometry (MS) in the clinical laboratory is the challenge of interfacing MS and automated sample preparation instruments to laboratory information systems (LIS) for electronic order and result transmission. This presentation will review basics of interface terminology, protocols, and workflows and will explain common differences encountered between interfacing mass spectrometers versus automated chemistry analyzers. Interfacing options now available from MS vendors will be discussed. Case histories will be used to illustrate recommendations to laboratorians for choosing between interface protocol options (.txt export, XML export, HL7), designing MS data storage architectures, selecting MS fields to export, interface testing and version control, and using middleware/LIS rules to automate data review.
Keeping Techs from Early Retirement: Approaches to Data Review in Multiplexed Mass Spectrometry Assays
Development of multiplexed mass spectrometry assays seems like every lab’s dream – however, increasing the number of analytes directly increases the complexity of the analysis. In an assay that measures 20 opioids and metabolites, there are nine metrics monitored for each of four sample preparation methods per analyte, totaling 720 data points per patient specimen. Without automated software tools, result preparation involved several manual processes and decision-making steps requiring extensive technologist time. Implementation of either custom or commercial software to perform quality control calculations reduces the amount of time required to manually review data, and ultimately, reduces the risk for errors.
Molecular Analysis in 3D Using Imaging Mass Spectrometry and Randomised Compression Methods
Development of 3D imaging mass spectrometry has provided a technique with great potential to determine molecular localisations throughout a tissue volume. By simultaneously mapping multiple molecules a new perspective of tissue function and disease alterations is obtained which can reveal the volumetric interactions of molecules. However, the increased data volume and complexity requires suitable algorithms for extracting meaningful information.
In this work tissue volumes are automatically identified based on their individual spectral differences using automated spatial segmentation. This analysis provides a molecular profile of each tissue type as well as visualisation of the 3D structure. This analysis is greatly accelerated for 3D images by a data compression scheme based on randomised data sampling.
High Performance MALDI MS, MS/MS, and Multiplexed MS/MS Tissue Imaging
Imaging mass spectrometry (IMS) has demonstrated the unique ability to provide untargeted and spatially resolved molecularly specific information. Recent advances in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry instrumentation have provided for systems capable of high throughput, high sensitivity, and high specificity imaging. Specifically, the SimulTOF 300 Tandem Mass Spectrometer (SimulTOF Systems, Sudbury, MA) allows for multiple high resolution precursor ion selection tandem mass spectrometry (MS/MS) experiments to be performed in a single laser shot. The goal of this technology is to allow for targeted analyte MS/MS imaging as well as untargeted MS/MS discovery in tissues.
Simultaneous Profiling of Multiple N-linked Glycans on FFPE Tissue Slides and Tumor Tissue Microarrays Using MALDI Mass Spectrometry Imaging
A new MALDI imaging mass spectrometry workflow for simultaneous analysis of multiple N-linked glycans in formalin-fixed tissue microarrays (TMA) and standard FFPE tissue slides will be presented. Following antigen retrieval processing and on-tissue digestion with peptide N-glycanase, 30-50 glycan species can be detected. The approach is amenable to any FFPE tissue, and represents an additional molecular correlate assay for use with the TMA format. Additionally, depending on the construction of the TMA and targeted tumor type, the approach has the potential to create novel glycan biomarker panels for cancer detection and prognosis.
DEEM - A New Approach to Automated Method Development for LC-MS/MS Sample Preparation
The new automated method development process Direct Extraction Elution Measurement (DEEM) will be presented. Method development using the AC Extraction Plate is simplified by evaluating the three 'pipette and shake' steps simultaneously. Using a Tecan EVO, an array of spiked solutions were prepared with different pH values (acid, neutral and basic) and organic solvent percentages ranging from 5% to 100%. These are extracted and directly analyzed to determine the extraction efficiency. Percentage extraction is plotted against organic solvent content and pH values to determine suitable extraction, rinse and elution conditions. Examples of developed methods will be shown.
An improved method to quantify total cotinine and trans-3'-hydroxycotinine in urine using UHPLC/MS/MS, automated sample preparation and automated data analysis
We developed and validated an automated high-throughput, low-cost, sensitive and selective method to analyze the nicotine metabolites total cotinine and hydroxycotinine in urine. Sample preparation was performed on a PerkinElmer Staccato System Robotics station. We used UHPLC coupled to an AB Sciex API 6500 triple quadupole mass spectrometer for peak separation and detection, and we used Indigo ASCENT software for automated peak integration and quality assurance screening. The limit of detection was 0.030 ng/mL for both analytes on a sample size of 0.2 mL. Our method will be used to measure secondhand smoke exposure in large population studies.
Deproteination of Serum Samples for LC-MS/MS Analyses Applying Magnetic Micro-particles
A novel, micro-particle based approach for sample preparation in LC-MS/MS was evaluated. In a commercially available kit, a proprietary protein denaturation reagent is combined with ferromagnetic particles which capture and remove denatured proteins from the sample. The principle was tested for amiodarone as an exemplary analyte. On-line solid phase extraction was applied as a second clean-up step. This set-up was found highly efficient and convenient. The principle competes with protein precipitation and seems highly attractive for automation, since no centrifugation or application of positive or negative pressure is required.
An Assessment of MALDI-TOF MS for the Identification of Infrequently Encountered Gram-negative Bacteria Unidentifiable by Conventional Methods
Gram-negative bacteria that cannot be identified by biochemical methods in our laboratory have traditionally been identified by 16S rRNA sequencing. Despite it's accuracy, this method is laborious, time consuming and costly. We compared the performance of 16S rRNA sequencing to MALDI-TOF MS for 65 Gram-negative isolates that could not be identified by conventional means. MALDI-TOF MS correctly identified 100% and 90.9% of isolates to the genus and species level, respectively. Additionally, MALDI-TOF MS provided species level identification for 7 of 10 isolates for which 16S rRNA sequencing could only provide a genus level or bacterial complex group identification. These results highlight the utility of MALDI-TOF MS in providing rapid and accurate identification to the species level for infrequently isolated Gram-negative organisms that are difficult to identify by traditional methods.
High Intra- and Inter-Laboratory Reproducibility of Mass Spectrometric-Based Identification of Bacteria and Yeasts
Data is scarce describing the intra-/inter-laboratory reproducibility of MALDI-TOF MS for identification of microorganisms. Our objective was to assess the performance of the VITEK MS® v2.0 system at three distinct sites for identification of bacteria and yeast. The study included 10 isolates analyzed by two operators at three sites on five different days, analysis of 300 'challenge' isolates and analysis of 1,089 “rare†isolates. All experiments demonstrated excellent reproducibility and analytical performance characteristics for identification of all organism types. MALDI-TOF MS-based microbial identification exhibits high intra-/inter-laboratory reproducibility and can tolerate multiple operators using multiple reagent lots on multiple instruments.
VITEK Mass Spectrometry for the Identification of Bacteria and Yeast Directly from Positive Blood Culture Broths
We evaluated VITEK MS (IVD Knowledgebase v2.0 and Saramis RUO database v4.10) for detection of bacteria (28 genus, 61 species) and yeast (4 genus, 7 species) directly from 632 blood culture broths representing 407 bacteremic episodes. MS-IVD identified more organisms (84.7%) than MS-RUO (80.1%), and combined use of MS-IVD and MS-RUO increased identification to 86.4%. Correlation between IVD/RUO-MS (bloods) and conventional testing (isolates) was 99.4% at genus-level and/or species-level. MS-IVD and MS-RUO results (isolates) correlated 95.8% to genus-level and/or species-level. MS-IVD/RUO results (isolates) correlated 97.9% with conventional testing and MS-IVD/RUO correctly identified 4 isolates, misidentified to genus by conventional testing.
Experimental Designs for Metabolomics: Untargeted Does Not Mean Unplanned
The purpose of this educational course is to discuss the basic principles that investigators new to metabolomics should consider when designing untargeted studies. Factors to be discussed will include sample choice for comparison, size of cohorts analyzed, instrument(s) applied, and public resources available for data processing. Special attention will be devoted to optimizing experimental design to answer specific types of scientific questions. A list of minimum criteria will be recommended that should be in place prior to analyzing clinical samples. Strategies to deal with the challenges of studying clinical laboratory samples will be discussed, such as sample normalization, sample handling, extraction procedures, biological variation, and sample heterogeneity.
Data Processing and Analysis for Metabolomics
The purpose of this section of the metabolomic educational course is to discuss and demonstrate the basic principles behind metabolomic data processing and analysis. This presentation will cover how informatic technologies allow for metabolomic data processing, including automated feature detection, retention time correction, alignment of chromatograms, peak area calculations and, ultimately, statistical analysis and identification of significantly altered features. This presentation will be geared to the non-expert, providing an overview of the most straightforward approaches to performing mass spectrometry-based metabolomic experiments.
Applications of Metabolomics in Biomedical Research
In this course the utility of metabolomics will be discussed and its application to different areas of biomedical research. One of the major uses of metabolomics is biomarker discovery, which can help determine mechanisms of diseases; here their ultimate value in biomedical research will be discussed. Another topic introduced will be drug metabolism studies, which have heavily benefited from metabolomic analysis. Numerous novel metabolites and toxic modes of action have been discovered, and the emerging field of pharmacometabolomics can aid in predicting responders and non-responders. The course will end with a discussion on the integration of other –omic technologies with metabolomics, such as the rising field of metagenomics for correlating bacterial-conferred metabolic dysregulation with disease. This course is meant to introduce the non-expert to potential applications of the field.
The Development and Utilization of Liquid Chromatograph-Tandem Mass Spectrometry Methods for Value Assignment of Vitamers in Clinical Standard Reference Materials®
The National Institute of Standards and Technology (NIST) has utilized isotope-dilution liquid chromatography tandem mass spectrometry to value assign vitamin D, folate, and vitamin B6 metabolites in a variety of clinical Standard Reference Materials (SRMs). NIST currently offers and is developing SRMs which include single level materials, as well as materials specifically focused on classes of related vitamers spanning multiple levels and encompassing value ranges relevant to the clinical measurement community. The availability of new standard materials, as well as advances in separations and mass spectrometry, allow NIST to continually expand the number of value assigned vitamers in clinical SRMs.
Critical Considerations for the Use of Isotopically Labeled Protein Standards for SI-traceable Quantification in Serum: Human Growth Hormone as a Model System
To manage and inform diagnostic or therapeutic decisions, measurement results which are accurate, specific and comparable between laboratories are required. Two challenges associated with this are the definition of the measurand and the commutability of the reference standard used. Once the measurand is defined, the next step in improving standardization is developing traceable quantification methods for proteins in biological fluids. A reference method for the quantification of human growth hormone (rhGH) in serum has been developed using multi-step sample clean-up, tryptic digestion, and isotope dilution mass spectrometry. Critical considerations for using isotopically labeled rhGH as the internal standard are described.
Development of a Reference Measurement System for Urinary Albumin
Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urinary albumin is vital to clinical diagnosis. Therefore to address urinary albumin measurement precision, we have developed a reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to quantify urinary albumin and a primary reference material to be used as a calibrator for higher-order urinary albumin methods.
The MS-based quantitation of urinary albumin provides both accurate and repeatable measurements at both micro- and normoalbuminuria levels, which can facilitate early diagnosis of kidney dysfunction.
Direct Analysis of Dried Blood and Urine Samples by PaperSpray-MS: Applications in Pain Management, Compliance, and Therapeutic Drug Monitoring
PaperSpray is a rapid method for the direct analysis of dried matrix spots by MS. Analysis is performed by depositing the sample on a porous substrate (e.g. paper) that has been cut to a sharp point followed by simultaneous extraction and ionization directly into the MS. Two applications will be discussed. 1) Screening of illicit drugs and pain medications from dried urine spots on an orbitrap MS. 2) Quantitative analysis of tacrolimus and other immunosuppressive drugs from dried blood spots with LLOQs as low as 1.5 ng/mL on a triple quad. Cross-validation data with HPLC-MS/MS assays will also be presented.
Overcoming All Issues Associated with Dried Blood Sampling? the Case of CYP1A2 Phenotyping
The standard approach for CYP1A2 phenotyping is the determination of the paraxanthine/caffeine ratio in plasma, at a fixed time point after intake of the CYP1A2 substrate caffeine. Here we set up an LC-MS/MS method for DBS-based CYP1A2 phenotyping, with special attention for methodological issues associated with DBS sampling.
Apart from classical validation parameters, we evaluated the (combined) impact of hematocrit, volume spotted and punch location and we compared capillary DBS concentrations (non-volumetric application, direct from the fingertip) with those in venous blood, DBS and plasma in >70 volunteers. While several of the evaluated parameters had a significant impact on the concentration of the individual compounds, the paraxanthine/caffeine CYP1A2 phenotyping ratio essentially remained unaffected. These findings suggest that CYP1A2 phenotyping is an ideal DBS application.
Recent Developments in Automated Dried Plasma Spot Analysis Using a Novel Membrane Size Exclusion Card
Recent advancements in LC/MS technologies have revived interest in using dried blood spots (DBS) in routine bioanalysis. This approach is useful for patients who are challenged with conventional larger volume blood collections. To circumvent possible issues such as hematocrit and compound instability a novel two-layered membrane-cellulose substrate device is described that enables the collection of dried plasma spots (DPS).
The new device is suitable for applications of micro samples of whole blood (5 to 25 µL). In order to construct an effective device, materials from several major polymer membrane suppliers as well as high-quality scientific paper suppliers were acquired and tested. The preferred membrane and paper materials were then constructed into standard dimension ‘cards’ which are amenable to modern automated bioanalyses using on-line LC/MS/MS.
Protein Biomarkers Validation Platform
Described in this presentation is a Protein Biomarkers Validation Platform for performing MS-based assays at a scale, throughput, and cost that justifies their use in biomarker validation and clinical laboratories. The platform is comprised of four key modules: 1) Sample preparation, 2) Immunoaffinity isolation and protein elution, 3) Mass Spectrometry analysis, and 4) Data processing. The platform can tackle the high-demanding task of analyzing thousands of samples per day, with analytical performances and cost basis matching those of existing enzymatic immunoassays. At the same time, the platform can detect and quantify protein variants, which could have yet-undiscovered pathophysiological implications and potential clinical utility for specific diseases. The platform's performance is demonstrated through analysis of insulin-like growth factor 1 (IGF1) from >1,000 human plasma samples.
Towards the Development of a µHuman: Dynamic Metabolic Response Analysis of 3-Dimensional Hepatocyte Bioreactor to Acetaminophen Exposure Using UPLC-IM-MS
An integral aspect of the integration of multiple organ-mimics for streamlined drug testing is the determination of methods for bioreactor viability, baseline, and condition interrogation in a non-destructive manner. Herein, we describe the analysis of effluent from a three-dimensional liver bioreactor using ultraperformance liquid chromatography-ion mobility-mass spectrometry and multivariate statistical analysis and self-organizing map-based approaches to data distillation. Bioreactor responses to acetaminophen are described, and metabolic perturbations are correlated to physiological reasoning and proposed metabolic pathway disruption. These analyses demonstrate an untargeted approach for benchmarking organ-mimic metabolic profiles and uncovering biological ramification of xenobiotic stimuli.
Inertial Imaging of Individual Biomolecules with Nanomechanical Systems
The spatial distribution of mass within an individual analyte can be imaged - in real time and with molecular-scale resolution - when it adsorbs onto a nanomechanical resonator. Each single-molecule adsorption event induces discrete, time-correlated perturbations to the modal frequencies of the device. We show that by continuous monitoring of multiple vibrational modes, the spatial moments of mass distribution can be deduced for individual analytes, one-by-one, as they adsorb. We validate this new method for inertial imaging using both experimental multimode frequency-shift data and finite-element simulations - to analyze the inertial mass, position-of-adsorption, and the shape of individual analytes. The spatial resolution is limited only by frequency noise and can achieve atomic scale with advanced NEMS devices.
De novo Biomarker Development: Learning the Ropes: Part 1
There is a need to develop new biomarkers to provide quantitative and actionable information essential for good clinical decisions. This information can aid in screening, prognosis, diagnosis and for therapeutic monitoring or improving the cost effectiveness of providing healthcare. The technologies involved in developing new markers are generic but unique characteristics of the targeted clinical area impacts the development strategy. Overall, the biomarker development process consists of multiple steps, each increasing the probability of producing clinically useful assays. This session provides insight into the strategies for discovery to increase the probability of ultimate success and outline traditional and emerging approaches for validation to hone the panel. Finally, an expert panel will discuss the challenges and obstacles in the transition of new markers to clinical practice.
Biomarker Development: Learning the Ropes: Validation: Part 2
Validation of candidate biomarkers requires moving to a quantitative assay able to assess increasingly large number of samples. Traditionally, this has involved ELISA assays, which uses antibodies to different epitopes but which can be influenced (low sensitivity/specificity) by a number of factors including cross reactivity and interferences from the matrix or analyte. Recent developments in protein-based mass spectrometry methods with respect to sample preparation and instrumentation allows for absolute quantification of an analyte based on accurate measurement of representative and unique peptides. These targeted MS methods, called multiplex or selective reaction monitoring (MRM or SRM, respectively) can produce equivalent data to an ELISA. MRM assays have additional advantages with respect to multiplexing and quantification of disease-induced post-translational modifications.
Expert Panel Discussion: Learning the Ropes: Part 3
There is a need to develop new biomarkers to provide quantitative and actionable information. The technical ability and strategic approach on how to move from discovery through validation has been developing over recent years. Mass spectrometry has been central to this. The next stage is to move these new assays into clinical realm. This session will be a panel discussion with Shijun (Simon) Sheng (Senior application scientist, Thermo), Cory Bystrom (Director, Research and Development, Cleveland Heart Lab) and Ian Wright (ACBI ). Their experience with IVD and MS manufacturers as well as their interaction with academic and clinical labs will provide insight into the various aspects involved. They will highlight the challenges and successes of using MS- based methods for accurate quantification. A vision for the diagnostic industry and MRM assays will be discussed.
Taking Protein-targeted Multiplexed Multiple Reaction Monitoring Mass Spectrometry to the Clinic
There is tremendous potential for MRM MS analysis of proteins in clinical diagnostics. Progress has been limited by inability to translate protein biomarkers from discovery through validation required for clinical implementation. We present successful translation and validation of a multiplexed protein MRM assay available through a CLIA-certified laboratory. The assay identifies benign pulmonary nodules with high negative predictive value to avoid unnecessary invasive procedures. Importantly, the assay provides molecular information independent of clinical data currently used to assess risk of malignancy. The assay proteins are associated with cancer-related pathways, including cell growth and proliferation, lung inflammation, and oxidative stress response.
High-Resolution Mass Spectrometric Quantification of Peptides in Clinical Samples
New hybrid mass spectrometers with high resolution and accurate mass capabilities have opened new avenues in quantitative proteomics. Targeted clinical analyses, routinely performed on triple quadrupole instruments in selected reaction monitoring mode, were replicated on a high-resolution quadrupole-orbitrap instrument operated in parallel reaction monitoring (PRM) mode. The trapping capability is well indicated to isolate and analyze peptides in tiny amounts and thus dramatically increased the dynamic range while providing highly selective measurements. The PRM technique was applied to the evaluation of large panels of putative makers in clinical plasma samples and showed a gain in selectivity and an increase in the confidence of measurements in complex matrices. The PRM analysis of lung cancer candidate markers resulted in a clear discrimination of the disease stages and subtypes.
Analysis of Serum Glycopeptides from Aggressive and Non-aggressive Prostate Cancer Patients Using Automated Glycopeptide Extraction and Parallel Reaction Monitor
Current prostate cancer screening techniques cannot distinguish between aggressive and non-aggressive forms of tumors. In this study, we used an automated solid phase extraction (SPEG) of glycopeptides from serum samples of men with varying degrees of prostate pathology and analyzed with parallel reaction monitoring (PRM) in an attempt to discover prostate cancer biomarkers. Seventy-five serum samples were obtained representing patients with non-cancerous, aggressive, or non-aggressive prostate cancer. Glycopeptides were isolated from 40 µl of serum from each patient via automated glycopeptide capture using liquid handling robotic system (Versette, Thermo Fisher Scientific). Samples were then analyzed using a Q Exactive mass spectrometer by PRM assay (Thermo Scientific). We identified six potential serum biomarkers that that may distinguish aggressive from non-aggressive forms of prostate cancer. In addition, we identified three potential serum biomarkers that may be used to distinguish between cancer and non-cancer.
Detection of Allenic Norleucine, a Nephrotoxic Amino Acid from Amanita Smithiana Mushrooms
Amanita smithiana is a poisonous mushroom, growing along the Pacific coast of North America, which is sometimes confused with the edible and highly valuable Pine mushroom. It causes acute renal failure, usually requiring dialysis. We present one such case of misidentification from British Columbia. Up until now, laboratory confirmation of A. smithiana in poisoning cases has relied on thin-layer chromatography. The chemical identity of the characteristic spot seen on TLC has been uncertain. We used LC-MS/MS to confirm that the toxin spot consists of a modified amino acid, allenic norleucine. Chlorocrotylglycine, an additional amino acid unique to this species, was also detected by LC-MS/MS from an adjacent TLC spot. Mass spectrometric detection of these two compounds should now be able to replace the more labour intensive and subjective thin-layer chromatography assay.
High Resolution Mass Spectrometry: The Comprehensive Toxicology Screen of the Future?
High resolution mass spectrometry (HRMS), which allows exact determination of molecular weight, has been proposed as an alternate to immunoassay screening of biological samples. One advantage of HRMS is the ability to screen for unknown compounds. The molecular formula of the target ion is predicted from the measured mass, then the user must determine which formula and structural isomer best fit the data. In our laboratory the search process has been empirical, often requiring multiple iterations. We present here a strategy for optimizing the parameters required for untargeted searches as well as final parameters and method performance on blinded samples.
All Ions MS/MS in TOF/MS: A Reliable and Cost-effective Alternative to Collecting Accurate Mass Data on Molecular and Fragment Ions from QTOF/MS
All Ions MS/MS in TOF/MS is a technique that uses multiple fragmentor voltages to induce in-source fragmentation allowing simultaneous generation of molecular and fragment ions in full scan MS mode. Cycling of fragmentor voltages during each time segment in a TOF/MS run from low to high energy allows formation of molecular and fragment ions, respectively. We successfully demonstrated the ability of All Ions MS/MS in an Agilent LC 1200- TOF/MS 6230 to generate and identify fragment ions of 75 designer drugs in a pyschostimulants drug panel. This allowed for significantly improving the accuracy of drug identification by LC-TOF/MS including discrimination between structural isomers.
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Untargeted Metabolomics Identifies Numerous Solutes that Accumulate when the Kidneys Fail
Solutes normally cleared by the kidneys accumulate and cause illness when the kidneys fail. Untargeted metabolomics is an ideal way to expand our knowledge of these "uremic" solutes. The current study employed the Orbitrap based mass spectrometer (Q Exactive MS) to profile solutes that accumulate in hemodialysis samples. The discovery phase revealed 400 uremic components in negative ESI mode and 175 uremic components in positive ESI mode. Chemical identification using MS/MS scans and accurate mass information has so far revealed novel uremic solutes including N-glutamyl heptenoic and N-glutamyl octenoic acid.
Characterization of Metabolomic Profiles of Nipple Aspirate Fluid Using UPLC-QToF; Potential Insights for Novel Breast Cancer Risk Biomarkers
Nipple aspirate fluid (NAF) components are constantly secreted, metabolized and re-absorbed by the epithelial lining of the breast duct making NAF a viable biologic to mine for novel early detection and cancer surrogate biomarkers. We characterized metabolomic profiles of NAF and plasma from healthy pre (N=10) and postmenopausal (N=19) women using UPLC-QToF. NAF was composed of lipids, drug metabolites and food additives as well as multiple bioactive food components. Plasma primarily contained endogenous metabolites. This work suggests NAF contains novel biomarkers distinct from plasma; further profile characterization, metabolite identification, and determination of potential breast cancer relationship are ongoing.
The Cancer Isotopolome in Whole Cells by Metabolomics
NMR and mass spectrometry (MS) are powerful analytical technologies that provide complementary information about the cancer metabolome and biomass profile. Here we describe software for integrating MS and NMR data that filters MS-based metabolomic features which are not identified by NMR. By applying this approach to cancer cells grown on specific isotope labels, we are able to reduce the thousands of peaks detected in metabolic extracts by MS to a small number that are consistent with the analytes observed in whole-cell NMR. The analysis of whole cells by NMR also allows for quantitative assessment of the amount of label that ends up in protein, DNA, and lipid membrane biomass. Comparing quiescent cells to cancer cells, our approach has revealed that the metabolic reprogramming observed in cancer results in major alterations in the precursors used for biomass production.
MALDI-TOF 101 for Microorganism Identification
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging diagnostic tool in the clinical microbiology laboratory. MALDI-TOF MS provides identification of microorganisms within minutes, decreasing turnaround time compared to conventional phenotypic methods, and has been shown to be reliable and cost effective. The method generates a spectrum, or “fingerprint,” of ribosomal proteins, which is compared against a reference database to assign an identification and confidence value to the organism. This session will focus on the basics of MALDI-TOF MS in the clinical microbiology laboratory and discuss the two platforms that currently exist, the Bruker Biotyper and bioMérieux’s VITEK MS.
MALDI-ToF-MS for Mycobacteria & Mold
The use of MALDI-ToF-MS for the identification of routine bacterial isolates in the clinical microbiology laboratory is becoming increasingly commonplace. Similarly, there is a large body of published literature describing the performance of this method for these organisms. However, the application of MALDI-ToF-MS to mycobacteria and mold species poses unique challenges such as the need for initial extraction, safety considerations and database composition. In this session, we will review published extraction methods as well as the performance of both commercial MALDI-ToF-MS systems for these organisms. The practicalities of implementing MALDI-ToF-MS for mycobacteria and mold identification will also be discussed.
Advanced Applications for MALDI-TOF MS in Clinical Microbiology
This educational session will focus on advanced applications of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as they pertain to the diagnosis of infectious diseases. Three primary topics will be discussed: 1) the use of MALDI-TOF MS for the detection of beta-lactamases and other resistance determinants, 2) phylogenetic typing of bacterial organisms, 3) direct identification of organisms from positive blood cultures and patient specimen. The educational session will focus on describing the methods used in these techniques as well as reviewing the literature that addresses their performance.
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Poster Presentations
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Free and Total Sialic Acid Study in Human Plasma with LC-MS/MS
Sialic acids (SAs) often terminate the glycan structures of cell surface and secreted glycoconjugates. Plasma SA has been considered as a biomarker for cancer and cardiovascular diseases. Here we analyzed both free and total SA in plasma samples from 30 cardiovascular diseases patients and 6 normal people with LC-MS/MS. Free SA was ranged from 437 to 1350 ng/ml, and total SA was from 128 to 501 μg/ml in patients, compared with 323 ng/ml and 258 μg/ml respectively in normal people. Measurement of a large number of samples is going be carried out to make statistical assumption.
Identification of Noninvasive Biomarkers of Coordinate Metabolic Reprogramming in Colorectal Cancer
Colorectal cancer is a leading cause of cancer mortality worldwide. Although the disease shows reasonably good response at early stages, currently no reliable method for noninvasive high-throughput screening and diagnosis is available. In this study we used a mass spectrometry-based global urinary metabolic profiling to identify noninvasive signatures associated with colorectal carcinogenesis in mice. Using a combination of transcriptomics, tissue metabolomics and genetic engineering, we showed that these signatures including those representing aberrant methylation reflected coordinate reprogramming of metabolic network during tumorigenesis. The combined biomarker panel could identify mice undergoing tumorigenesis well before adenoma formation. Analysis of human colorectal tumors also showed similar metabolic reprogramming including derangement of ten biomarkers identified in mice.
GC/MS-based Serum Metabolomics for Discovery of Pancreatic Cancer Biomarker
To discover effective biomarker for pancreatic cancer, we performed gas chromatography mass spectrometry (GC/MS)-based human serum metabolomics, and constructed a diagnostic model with the multivariate analysis using the results of it. In the validation set study based on an independent sample set from model composition, this diagnostic model possessed highest sensitivity (71.4%) for pancreatic cancer patients. Furthermore, it had higher sensitivity in stage 0-2 PC (77.8%) and lower false positive rate in chronic pancreatitis patients (17.4%) than conventional tumor markers. GC/MS-based serum metabolomics is a promising screening method for pancreatic cancer.
Laser Microdissection and Cervix Carcinoma Proteomics: Heat Shock Proteins as Biomarker Candidates
Cellular proteins of cervical cancer tissue and healthy epithelium/or stroma of the cervix from patients were identified after laser microdissection of ~2500 cells. Statistical analysis resulted in a number of peptides that were more abundant in tumor cells versus normal epithelial cells. As an example, heat shock protein 90 was validated by immunoblotting, immunohistochemistry, ELISA and 2D-LC-MS/MS in a large set of sera from cervical cancer patients at various stages of disease and healthy subjects. The overexpression of HsP90 discovered in tissue was successfully translated into serum samples of patients with cervical cancer versus healthy subjects.
Using PRISM-SRM Analysis for Potential Novel Ovarian Cancer Biomarkers
Over 75% of ovarian cancer patients are diagnosed at advanced stage due to lack of effective biomarkers for early detection. In this study, we combined immunoaffinity depletion and PRISM-SRM analysis to monitor 16 potential biomarker candidates (including CA125 and HE4) derived from genomic data in blinded serum samples from 20 serous ovarian cancer patients and 20 benign serous control patients. We established the first feasible SRM assay for the classical ovarian cancer biomarkers HE4 and CA125, and additionally demonstrated that the multiplexing capability and high sensitivity of PRISM-SRM analysis is suitable for use in biomarker validation studies.
Discovery and Validation of Serum Protein Biomarkers Panel for Cervical Cancer Diagnostics
We applied a workflow consisting of (i) protein biomarker discovery in depleted pooled serum samples using stable-isotope labeling; (ii) verification of biomarker candidates by label-free analysis of non-depleted serum samples; (iii) validation of selected protein biomarkers with targeted LC-MS/MS analysis to discover and validate a serum protein biomarker panel for cervical cancer diagnostics. The application of biomarker panel allowed to differentiate between patients with cervical intraepithelial neoplasia (pre-malignant stage), patients with early- and late-stage cervical cancer versus healthy controls with sensitivities of 0.88, 0.77, 0.91 and specificities of 0.82, 0.55, 0.87, respectively.
Quantitation of Tryptophan and Kynurenine in Blood and Urine by Tandem Mass Spectrometry
Indoleamine-2,3-dioxygenase (IDO-1) catalyzes conversion of tryptophan (TRP) to kynurenine (KYN) and its expression is stimulated by infection. As a surrogate for IDO activity, we developed a rapid MS/MS technique to determine KYN and TRP in serum and urine. KYN (m/z 209/94) and TRP (m/z 205/146) were quantitated using D6-KYN (m/z 215/98) and D5-TRP (m/z 210/150). Quantitation was accurate and precise in serum from 0.1-25 µM and 0.5-250 µM for KYN and TRP, respectively. In afebrile children serum KYN and TRP ranged respectively from 0.5-3.8 and 11-105 µM, and urinary excretion ranged from 0.2-2.7 and 3.2-25 µmol/mmol creatinine, respectively.
Diagnosis of Male Reproductive System Disorders with Protein Biomarkers Quantified by SRM Assays in Seminal Plasma
Non-invasive methods for differential diagnosis of male reproductive system disorders present the unmet needs in the urology clinics. In our presentation, we will discuss these needs, describe our biomarker discovery pipeline, present in detail our work on male infertility biomarkers, and introduce our approaches to discovery of prostate cancer and prostatitis biomarkers. As an example, we recently discovered and validated by mass spectrometry-based SRM assays 18 proteins in 119 seminal plasma samples, identified two protein biomarkers, ECM1 and TEX101, and proposed an algorithm for differential diagnosis of azoospermia forms and subtypes. Clinical assays for ECM1 and TEX101 will replace the majority of diagnostic testicular biopsies, improve the prediction of sperm retrieval and increase the reliability of assisted reproduction techniques.
Prevalence of Vitamin D2 in a Patient Population
Accurate measurement of 25-hydorxyvitamin D2 and 25-hydroxyvitamin D3 is critical to the assessment of vitamin D nutritional status. We sought to evaluate the current prevalence of vitamin D2 usage and its impact on the accuracy of vitamin D measurement. A total of 266,269 patient results analyzed between June 2009 and December 2012 using in house LC-MS/MS methods were included for final analysis. 25OH-D2 was still present in 17% of patients tested in December 2012. To achieve accurate vitamin D measurement, clinical laboratories should determine their local prevalence of vitamin D2 and assess the accuracy of their assays for both isomers.
Developing GPCR Phosphorylation Biomarkers for Cardiovascular Diseases
Novel biomarkers for cardiovascular disease such as heart failure offer great promise to the earlier diagnosis and treatment of these diseases. We use mass spectrometry-based approaches, such as protein phosphorylation analysis, in combination with different animal models and diseased human heart tissue, to search for biomarkers for cardiovascular diseases. We are currently focused on determination of the phosphorylation sites on GPCRs. We subsequently design stable isotope labeled phosphor-peptides and develop MRM assays for sensitive and multiplexed quantitation of these receptors and their phosphorylated forms. We then apply these novel assays on a large, well-characterized, bio-bank of normal and diseased human heart tissue. The data provided by these studies should help to develop GPCR phosphorylation biomarkers for diagnosis and treatment of cardiovascular diseases.
High-Fat Diet Induces Metabolic Alterations in Urine of Young and Old Mice
To understand the metabolic changes associated with obesity development, we carried out metabolomics changes of 50 organic acids induced by high-fat diet (HFD) in urine samples of mice using GC/MS. We had analyzed urine from four groups of mice: young control, young high fat diet (HFD), old control and old HFD. We found that HFD feeding has changed a lot of metabolic pathways, and we also identified metabolites that characterize a healthy aging by comparing old control group and young control group. We are in the process correlating the metabolomics data with clinical data.
Functional Network Analysis of Paediatric Idiopathic Nephrotic Syndrome Urine by Multi-Omic LC-MS
INS is a prevalent glomerular disease in children. The lack of understanding pathogenic mechanisms can lead to incorrect diagnoses, poor therapeutic response and adverse side-effects. A quantitative multi-omics approach is presented to reveal molecular factors involved. Urine samples were collected from healthy and INS diagnosed children, which were affinity depleted, proteins recovered and tryptic digested. LC-MS data were obtained with ion mobility data independent acquisitions to determine protein abundances. Similarly, neat urines were analysed using a small molecule profiling approach. A large number of proteins were over-expressed in INS urine, including a high fraction of glycosylated proteins. Metabolites showing significant changes include homocysteine, glutamate and uridine. Pathway analysis suggests correlation with neuronal system disorders, specifically acute fatigue.
Verification of a Parkinson's Disease Protein Signature by Multiple Reaction Monitoring LC-MS
Diagnosis of Parkinson's disease is based on the appearance of motor symptoms. A 2-DE analysis panel of proteins in the T-lymphocyte proteome was recently proposed as a disease signature. Here, an LC-MS based method was used to quantitatively evaluate this signature by MRM in T-lymphocytes and peripheral blood mononuclear cells. A discriminant function was applied to MRM data from T-lymphocytes protein extracts, assigning seven controls out of nine as true negatives and nine patients out of nine as true positives. Good discriminant power was obtained by selecting a subset of peptides from the protein signature (GELS, MOES, SEPT6, TWF2, LSP1, VIME, TALDO), with an ROC AUC of 0.877. The signature was not able to classify subjects by analyzing whole mononuclear cells. The results suggest portability of the method to large cohort validation using alternative technologies such as LC-MS.
High-Throughput LC-MS MRM Analysis of Calcyclin in Pre-Eclampsia Patient Sera
PE is a pregnancy-specific disease that complicates 2-8% of all pregnancies and is associated with perinatal and maternal morbidity and mortality. We previously showed increased calcyclin expression in placental tissue. A microfludic LC-MS based assay was developed to evaluate this observation by MRM in tryptic digested SCX fractionated serum. Measurements were applied for the quantitative analysis of calcyclin using 2 isotopically labeled peptides. The linear correlation coefficients were ≥ 0.99 and LODs equal to 0.36 and 0.70 ng/ml in serum. The correlation between both peptides was 0.997 and endogenous calcyclin was quantified in all samples. The peptide concentrations were significantly different between pregnant and non-pregnant sera but preterm pregnant control and PE samples could not be distinguished. Compared with nanoscale LC, a throughput increase of about 4 fold was achieved.
Usefulness of a LC-MS/ MS Thyroglobulin Assay for Evaluation of Suspected Heterophile Interference
Thyroglobulin (Tg) measurement by immunoassay can be affected by heterophilic antibodies (HAB). LC-MS/MS measurement of Tg should overcome HAB interferences. Specimens suspected of HAB interference were assayed by immunoassay, HAB workup (dilutions and blocking reagents) and LC-MS/MS. Results of immunoassay and LC-MS/MS measurements matched for patients with negative HAB workup but were discordant on suspected HAB cases. Suspected HAB+ samples had Tg levels below LOQ in the LC-MS/MS assay, while measuring up to 455ng/mL by immunoassay. In all cases the LC-MS/MS results matched the clinical presentation. The LC-MS/MS Tg method allows for accurate quantitation in the presence of HAB interferences.
Absolute Quantification of Abeta38, Abeta40 and Abeta42 in Cerebrospinal Fluid of AD Patients and Healthy Controls Using Selected Reaction Monitoring
Cerebrospinal fluid (CSF) amyloid beta (Abeta) peptides are well-established biomarkers for Alzheimer disease (AD). Several immunoassays exist, but their broad-scale use is hampered by interference from matrix effects. Here we report on a method for absolute quantification of Abeta1-42, Abeta1-40 and Abeta1-38 in human CSF using selected reaction monitoring mass spectrometry. The performance of the method was evaluated on two independent clinical materials. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Abeta42, Abeta40 and Abeta38 in CSF, while it retains the ability to distinguish AD patients from controls.
CSF Biomarker Discovery Using Label-Free Proteomics for Lewey Body Dementia and Parkinson’s Disease and Validation by a Targeted Peptide MRM Assay
Novel markers are needed to distinguish Lewey Body dementias (LBD) and progression. Biomarker screening using label free quantitation was conducted. Validation was performed using a targeted peptide Multiple Reaction Monitoring LC-MS/MS assay using 50μl of CSF and included existing markers. 16 age-matched controls (61-78yrs) and 7 Alzheimer’s disease samples were included. 4 proteins were able to distinguish early LBD (0-2yr) including ApoE. One protein decreased in later LBD to normal levels the other three increased in later LBD. Fifteen proteins were able to distinguish late LBD (3-5yrs) including previously described markers tau, YKL40 osteopontin and cystatin C.
Profiling of Urinary Biogenic Amines and Metabolites for Neuroblastoma Screening by Polarity-Switching LC-MSMS
Because of variability in production and metabolism, a combination of urinary catecholamines and their metabolites is measured for biochemical diagnosis of neuroblastoma. A rapid biogenic amine profiling method was developed to measure catecholamines, metanephrines and their acidic metabolites by polarity-switching LC-MSMS. Dual SPE clean-up of urine, equilibrated with labelled internal standards, was used to recover amines and acids before combining extracts for simultaneous LC-MSMS analysis with a core-shell HPLC column. The utility of the method was illustrated by analysing 48 spot paediatric urine specimens collected from children with and without neuroblastoma or paraganglioma.
Phospholipids Profiling in the Clinical Laboratory Using a MALDI-QIT-TOF Platform
Phospholipids and their metabolites are increasingly recognized as key diagnostic molecules in a number of pathologies including acute inflammation, CVD and atherosclerosis. Our investigation aims at evaluating the potential of a MALDI-MS based platform for screening of Phospholipids biomarkers in the clinical laboratory. Plasma from carefully phenotyped children and adolescents with familial hyperlipidemias were evaluated. PL-profiles were characterized and correlations with specific LDL levels were observed. OxPC levels were positively correlating with IMT and HDL-C but negatively with LDL-C. The results show the potential of OxPLs as markers for early vascular disarrangements in patients with familial hyperlipidemias.
Immunoaffinity-Based Multiplexed Targeted Mass Spectrometric Assay for Ovarian Cancer Biomarker Verification
Ovarian cancer is a deadly disease as it is frequently asymptomatic until it has advanced to an untreatable stage and spread well beyond the ovaries. One of the few FDA-approved ovarian cancer biomarkers, CA-125 does not provide the accuracy required for early cancer diagnosis or differentiate it from malignant non-ovarian disease. We used an immunoaffinity-based targeted mass spectrometric approach to verify the biomarkers CA-125 and Mesothelin in a clinically well-characterized cohort of serum samples and quantitatively characterized their behavior. We also compared the MRM-based proteomics assay with the corresponding ELISA assay to determine the robustness and complementarity of this approach.
Measurement of 25-hydroxyvitamin D2 and D3 in Perimenopausal and Menopausal Women in Korea Using PerkinElmer MSMS Vitamin D Kit
We investigated the status of 25OHD2 and 25OHD3 in perimenopausal and menopausal women. We compared measurements of LC-MS/MS and popular assays (RIA and ECLIA). RIA and ECLIA which are employed in clinical laboratories for total 25OHD concentration measure have shown an acceptable correlation with LC-MS/MS. The 95% confidence intervals of 25OHD2,25OHD3, and total 25OHD were 0.6 to 0.8 ng/mL, 22.2 to 23.5 ng/mL, and 22.8 to 24.1 ng/mL. Vitamin D deficiency and insufficiency (<20 ng/mL) was observed in 41.8% of women.
Serum N-glycan Profiling for the Screening of Ovarian Cancer by MALDI-TOF Mass Spectrometry
Aberrant N-glycan profiles are associated with many types of cancer. N-glycan profiles were investigated by MALDI-TOF mass spectrometry (MS) and compared between disease-free controls and ovarian cancer patients. An optimal combination of multiple biomarkers was derived from the training sets. With these combined multiple biomarkers, a validation set for the ovarian cancer showed ~88% sensitivity and ~88% specificity. In particular, the combined multiple biomarkers for ovarian cancer revealed ~95% accuracy in the prediction of post-operative prognosis, showing a great potential for clinical applications.
LC-MS/MS Measurement of Urine Free Collagen Crosslinks Pyridinoline and Deoxypyridinoline: Urine Markers of Bone Resorption
The pyridinium cross-links Pyridinoline (PYD) and Deoxypyridinoline (DPD) are established markers of bone resorption, useful in assessing and monitoring response to osteoclast inhibitory treatment (e.g. bisphosphonates). A LC-MS/MS method have been developed to simultaneously quantify concentrations of PYD and DPD is urine. The method have been thoroughly validated to show excellent assay recovery, low intra/inter assay variability, and demonstrated good correlation with commercial immunoassay for total PYD and DPD. Ratio of PYD:DPD is a valuable diagnostic tool for Ehlers-Danlos Syndrome (EDS), where a reverse ratio is specific and diagnostic for the kyphoscoliotic type of the condition.
LC/MS/MS Method for the Quantification of Endogenous Steroids in Oral Fluids Using the QTRAP® 5500 LC/MS/MS System with SelexION™ Ion Mobility Technology
Steroids are present in oral fluids at very low concentrations, therefore a sensitive, selective and robust method was developed to enable the quantification of 7 steroids in oral fluid by LC/MS/MS. 1 mL of oral fluid was extracted by SPE, and the dried extract was derivatized with dansyl chloride. 2 µL of the derivatized sample was analyzed using LC/MS/MS in positive ESI mode using an AB SCIEX QTRAP® 5500 mass spectrometer coupled with an Eksigent ekspert™ microLC 200 system. SelexION™ ion mobility technology was used to enhance the selectivity of the method and to thereby improve the data quality. The lower limits of quantification (LLOQ) were established at 1 pg/mL for Testosterone, 5 pg/mL for 17-Hydroxyprogesterone, Cortisol, and Progesterone, and 0.5 pg/mL for derivatized Estradiol, Estrone, and Estriol.
Analysis of the Heterogeneity of Immunoglobulins in Human Serum
In normal healthy people, immunoglobulins (Igs) are the second most abundant protein in serum. There are multiple classes of Ig, and each Ig molecule contains two separate hypervariable regions that are normally very heterogeneous. In some diseases, like multiple myeloma, a single Ig becomes dominant. This monoclonality is currently detected using electrophoresis in blood. We present here alternative techniques for measuring Ig heterogeneity based on linear mode MALDI mass spectrometry for the analysis of whole serum or enriched Ig preparations and reflector mode MALDI mass spectrometry for unseparated trypsin-digested samples. In the latter, Ig sequences can be confirmed by MS-MS.
A Sensitive LC/MS/MS Method for the Quantification of Free T3 and Free T4 in Serum, Using a Simple Ultrafiltration Sample Preparation Procedure
Here we present a sensitive method for the measurement of free thyroxine (FT4) and free 3,3´,5-triiodothyronine (FT3) in serum, using the AB SCIEX QTRAP® 6500 system. The method employs a simple and rapid ultrafiltration sample preparation to isolate the free T3 and free T4 prior to analysis by LC/MS/MS. Liquid chromatography separation was accomplished using a Phenomenex Kinetex C18 column (2.6um, 3.0 x 50mm) at a flow rate of 500uL/minute. T3 and T4 were monitored in positive MRM mode, using electrospray ionization. Unlike earlier attempts to analyze FT3 and FT4 by LC/MS/MS, this sensitive method requires a relatively small injection volume of only 50uL of the final sample. A comparison study was performed versus an established immunoassay, and an excellent correlation has been observed. The method LOQ was <0.5 pg/mL for both FT3 and FT4.
Differentiation of Virulence of Helicobacter Pylori by Matrix-assisted Laser Desorption/ionization Mass Spectrometry and Multivariate Analyses
The pathogenesis of Helicobacter pylori can be attributed to the upregulation of genes for the cag pathogenicity island and the vac virulence factor. Since different expressed alleles for the aforementioned genes will determine the degrees of virulence for H. pylori strains, it is therefore important to determine the differences in virulence between H. pylori strains in order to provide appropriate medical treatment. Significant differences in protein profiles between reference, clinical, and inoculated H. pylori strains were successfully determined using MALDI-TOF/MS coupled to multivariate statistical analysis.
The Quantification of a Panel of Urinary Organic Acids by LC-MS/MS
Organic acids include short to medium chain mono- and dicarboxylic acids and hydroxylated analogs with a carbon chain length up to 12. Their measurement in urine has the potential to identify a large number of metabolic disorders. The most common method of analysis of organic acids is by GC/MS of the trimethylsilyl esters of the ethyl acetate extract of acidified urine, however LC-MS/MS is appealing because of the significantly shorter run-times, and the simple sample preparation which does not require derivatization of the samples prior to analysis. We have developed a rapid LC-MS/MS method for the measurement of a panel of organic acids in urine, including: lactic acid, 2-hydroxyglutaric acid, uracil, orotic acid, ethylmalonic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, pyroglutamic acid, 3-hydroxypropionic acid, 3-hydroxyglutaric acid, and N-isovalerylglycine.
Exponentially Modified Gaussian Peak Fitting in the Production LC/MS/MS Setting
We present a peak fitting model (the exponentially modified Gaussian or EMG) for the reporting of high throughput production chromatography. By monitoring the four EMG parameters, it is possible to determine reproducibility, accuracy and precision over time. These trends establish and confirm schedules for column replacement, preventive and routine maintenance. Inclusion of a goodness of fit estimator is sufficient to track the total system stability. This model supports ongoing efforts in the production analytical chemistry community to reduce the number of calibrators needed for production assays, thus increasing instrument capacity.
Validation of ICP-MS for Measuring Calcium Concentration in Human Breast Milk
We studied human breast milk calcium determination by ICP-MS and compared the results with that of the Beckman Coulter AU5800 automated analyzer. Although using dynamic reaction cell (DRC) system was able to reduce the interference of 40Ca with 40Ar, the measurement of 42Ca and 43Ca afforded the best linearity. Accurate and precise calcium measurements in breast milk can be achieved in our system. ICP-MS outperformed AU5800 automated analyzer in terms of precision and accuracy. This analytical difference may be due to the heterogeneous nature of breast milk and highlights the importance of using ICP-MS to measure calcium concentrations in breast milk.
Determination of Trace Elements in Human Blood Plasma by Inductively Coupled Plasma Mass Spectrometry with Collision Cell Using Matrix-matched Calibration
A method was developed for determination of Mn, As, Cd, W, Hg, Pb, and U in blood plasma using Agilent 7700 ICP-MS with collision cell. Plasma specimens were diluted 1:10 with alkaline solution that consists of NH4OH, H4EDTA, n-butanol, Triton X-100, and internal standards. A similar strategy as previously reported for determination of trace elements in blood was used to matrix-match plasma specimens by adding NaCl to calibration standards. MDL’s for Mn, As, Cd, W, Hg, Pb, and U were 0.0155, 0.0042, 0.0024, 0.0044, 0.0068, 0.0033, and 0.0021 µg L-1, respectively. Analytical accuracy was evaluated with certified reference materials.
MALDI Imaging Mass Spectrometry: Technical Advances and Clinical Problem Solving
MALDI Imaging Mass Spectrometry (IMS) uniquely combines the spatial advantages of microscopy with chemical specificity that is leveraged to investigate biological systems (e.g., tissues). Advances in MS instrumentation, sample preparation, and image analysis are converging to enable novel, clinically important applications of IMS. Presently, numerous reports of IMS providing biological and clinical insight are in the published literature; however, IMS has not yet been applied in clinical practice. This presentation presents the state-of-the-art of MALDI analysis of tissues, the diagnostic potential of MALDI IMS, and remaining challenges in the transfer of the technology to the clinical laboratory.
MALDI Imaging Mass Spectrometry for the Detection of Metabolites in Kidney Tissues
We applied matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to localize metabolites in kidney tissues between high fat diet (HFD)-fed mice and standard diet (STD)-fed mice. We found differences of distribution in 8 m/z values between HFD-fed mice and STD-fed mice, and the major differences were observed in the renal cortex. In addition, we found that 6 m/z values were distributed specifically in the glomeruli of both STD-fed mice and healthy human, and furthermore, the signal intensity was increased in HFD-fed mice compared with STD-fed mice.
High Quality Sections and Molecular Distribution Images of Neuropeptides from Heat Stabilized Tissue
MALDI imaging (MSI) is gaining interest in pharmaceutical research, as it is a valuable tool when mapping molecules in situ on tissue sections.
Residual enzymatic activity in tissue samples cause changes. Hence sample preservation prior to MSI is crucial in order to make accurate measurements. Rapid heat stabilization prevents changes and reveals a molecular composition closer to in vivo.
To establish a protocol for heat stabilized tissue in the IMS workflow parameters of high impact on sample morphology were identified as; fresh state stabilization with embedding in CMC, freezing in dry ice cooled liquid bath, section transfer and thaw mounting technique. A tape transfer system CryoJane (Leica) was also proven compatible with sections of superior quality. It is concluded that good quality sections can be produced from heat stabilized tissue and MSI images could be obtained.
Development of Multi-compartmental Model for the Diagnosis of Fatty Acid Beta-oxidation Disorders Using U13C-Palmitic Acid in Vitro and UPLC-ESI -MS/MS
We developed an eight-compartmental model to describe the FAO enzyme activity. A multi-compartment model is a mathematical model used for describing the way materials transfer among the compartments of a system. Acylcarnitines, produced by fibroblasts of healthy subjects and with confirmed FAO defects incubated with [U-13C] palmitate, were quantified by UPLC-MS/MS and analysed by the software SAAM II. Fractional rate constants for each step were calculated allowing us to describe subclasses of enzyme activities and differentiate kinetic within the same enzyme deficiency.
A Novel Duplex Tandem Mass Spectrometry Assay for the Clinical Diagnosis of Neuronal Ceroid Lipofuscinoses (NCL)
NCL encompasses a group of the most common inherited neurodegenerative lysosomal storage disorders of childhood. Infantile NCL (INCL) and classical Late Infantile NCL (c-LINCL) are two most severe forms of NCL and have marked deficiency of PPTI enzyme/TPPI enzyme. Current fluorescence-based screening protocols have limitations primarily because of variable background fluorescence and for INCL require the addition of a coupling enzyme to yield a fluorescent compound. In order to circumvent these limitations, a duplex tandem mass spectrometry assay was developed for clinical diagnosis of INCL and c-LINCL in Dried Blood Spots (DBS) from newborns.
Simultaneous LC-MS/MS Determination of Urine Sialic Acid, Pipecolic Acid and Creatinine and Serum Pipecolic Acid, for Diagnosis of Inherited Metabolic Disorders
This novel analytical method is designed to facilitate and streamline diagnostic laboratory testing for several diverse inherited disorders of metabolism. These conditions include a lysosomal disorder, Sialic Acid Storage Disease; a group of peroxisomal biogenesis disorders collectively known as Zellweger spectrum disorders; and a specific disorder of lysine metabolism, pyridoxine-dependent epilepsy. Quantitation of sialic acid, pipecolic acid and creatinine is performed by LC-MS/MS in positive electrospray ionisation mode with multiple reaction monitoring, using isotopic dilution with appropriate internal standards. Inclusion of appropriate controls provides capability to analyze both urine and plasma samples within a single method.
Open-Tube Flow Injection Liquid Chromatography-Tandem Mass Spectrometry for In-Born Error Metabolism Disorder Research Using a Meta Calculation Software
The research reported here is to demonstrate a new approach in analyzing donor samples for the quantitation of aminoacids and acylcarnitines using flow injection LC-MS/MS with a meta calculation software. This beta version software is developed for automatic calculation of mass ion ratio and user defined formula using data file generated from Tandem MS (Thermo Scientific); its performance is compared with manual multiple-steps calculation using Excel. The preliminary research result shows the agreement between these two approaches is within 10% of bias. This software reduces manual calculation time and applies to all analytes that can be detected by Tandem MS.
Neonatal Screening Tests for Inherited Metabolic Disorders by Tandem Mass Spectrometry in Korea
We report the prevalence of inherited metabolic disorders in Korea. We used Agilent 1260 Infinity HPLC System (Agilent Technologies, CA, USA) for liquid chromatography, and API 2000 (AB Sciex, On, Canada) for mass spectrometry for screening 55 diseases, which are 25 items for amino acid, 16 items for organic acid, and 14 items for fatty acid disorders. Total number of samples tested were 82,807 from January, 2012 to November, 2013. Among the 82,807 cases, 4,144 cases were repeated. From the repeated 4,144 cases, 510 cases were presumptive positive for screening tests, from which 426 blood spot specimens could be reobtained. 53 cases were identified as abnormal again and were recommended the confirmative tests, which were 0.06% of total cases and 12.4 % of reobtained cases. Tandem Mass Spectrometry is useful for screening the inherited metabolic disorders in newborns in Korea.
Measurement of Succinylacetone Levels in Dried Blood Spots with Transient Neonatal Tyrosinemia Using UPLC-MS/MS
We revised the method to measure the succinylacetone in dried blood spots (DBS) using UPLC-MS/MS and analysed succinylacetone levels with transient neonatal tyrosinemia (TNT, n=128) and with normal controls (NC, n=92) to improve the newborn screening of tyrosinemia. This method showed good precision, acceptable linearity, and no ion suppression. The levels of succinylacetone with TNT (122.0±48.8 ng/mL) were significantly higher than those with NC (53.2±35.0 ng/mL). The adoption of different cutoff values of succinylacetone according to the tyrosine levels (116.8 ng/mL for NC and 252.8 ng/mL for TNT) could be useful to decrease the false positive rate.
Quantitative Analysis of Methyl-malonic Acid in Serum and Evaluation and Comparison of Sample Preparation Techniques Using LCMSMS QQQ in 1D and 2D Configuration
Liquid chromatography triple quadrupole (QQQ) mass spectrometry (LC/MS/MS) is suited for rapid analysis of multiple analytes. A highly sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of Methyl-malonic acid by QQQ. Various sample preparation techniques and chromatographic configurations are evaluated and compared based on their ease of use, analyte recovery and post-extraction cleanliness. The described method achieves the required sensitivity and is capable of quantitating the analyte over a relevant dynamic range. Excellent reproducibility was observed for all compounds (CV < 10%). All calibration curves displayed linearity with an R2 > 0.995.
A Metabolic Approach Associated with Systems Genetics Analysis to Identify Biomarkers and Candidate Genes in Cardiometabolic Diseases
Cardiometabolic diseases (CMD) can be present long before becoming clinically apparent. Accurate predictors of disease are important to prevent morbidity. In order to identify novel genetic determinants of metabolism or new clinical biomarkers underpinning CMD, we studied a panel of recombinant inbred rat lines using metabolomic (UPLC-MS) and genomic approaches. We have profiled the metabolome of 10 organs and biofluids by UPLC-MS. Further data analysis using mQTL-mapping of significant metabotypes revealed 1500 mQTLs co-localized with physiological QTLs, providing new insights on potential pathophysiological mechanisms related to diastolic blood pressure and insulin.
Targeted and Untargeted Metabolomics of Cystic Fibrosis Sputum Guided by Molecular Networking
Cystic Fibrosis sputum contains a complex community of pathogenic microbes and host cells which interact in a dynamic molecular environment. Identifying microbial molecules and human inflammatory signatures with clinical relevance is challenging due to the complexity of the metabolome and patient variability. Molecular networking, a way of organizing MS2 fragmentation patterns into chemical relationships, can guide mass spectrometry (MS) analysis of these complex systems. Here we use tandem MS and molecular networking along with ecological statistics to compare and analyze CF and non-CF sputums. We identify microbial virulence factors and their chemical relatives from well-studied and unknown CF pathogens and also identify major human metabolites that distinguish CF from non-CF sputum. Molecular networking allowed for better annotation and streamlined analysis of this complex metabolome.
Untargeted and Targeted Lipidomic/metabolomic Study of lkb-/- Mouse Sciatic Nerve
Mitochondrial metabolic dysfunctions are a common cause of numerous neurodegenerative disorders and are important contributors to peripheral neuropathy. Liver Kinase B1 (LKB1), a tumor suppressor is a key regulator in energy metabolism. By applying untargeted metabolomics platform to analyze wild-type and lkb1 knockout mouse sciatic nerves, we identified several key lipids/metabolites that showed a consistent and significant decrease in the lkb-/- animals. These lipids/metabolites were further quantified with specific lipid assays using a targeted metabolomic platform enabling accurate quantification. Lipidomic data combining with transcriptional and immunohistochemsitry data establish that lkb1 plays an important role in Schwann cell differentiation and myelination and that mitochondrial dysfunction leads to deficits in myelin lipid synthesis in lkb mutant sciatic nerves.
The Human Induced Sputum Lipidome - Providing a Baseline for Lipid Biomarker Studies of Respiratory Disease and Infection
Sputum induction is a non-invasive procedure to sample the airways, and is routinely used to evaluate airway inflammation in diseases such as asthma or COPD, and to diagnose microbial infections of the respiratory system. Despite the potential of induced sputum to yield a wealth of metabolic markers for disease, its lipid composition has not yet been fully characterized. We therefore analyzed the induced sputum lipidome of healthy volunteers, using high-resolution shotgun MS followed by directed UHPLC-MS/MS, to gain insight into induced sputum sources, its sampling reproducibility and intra-cohort variability, and to provide baseline data for future respiratory disease studies.
Prostarix: A Clinical Mass Spectrometric Assay of Urine Sarcosine and Amino Acids for Prostate Cancer Risk Assessment
Tests are needed for patients with modestly elevated PSA to provide guidance regarding whether to biopsy the prostate. We previously demonstrated that urine metabolites aided in risk stratification for prostate cancer biopsy results in this patient group. Here we report the analytical and clinical validation of the Prostarix test and its implementation as a laboratory developed test in the Metabolon clinical laboratory. Assay ruggedness and performance characteristics were established during analytical validation. Associations between analyte measurements, the algorithmically-derived Prostarix Risk Score, and biopsy outcomes were clinically validated and continue to be monitored using patient results from a community urology clinic.
Bioavailability and Metabolomic Targets of Sulforaphane in Humans
Sulforaphane (SFN), an isothiocyanate derived from crucifers, has many health benefits. Yet, knowledge is limited regarding its bioavailability and molecular targets in humans. We sought to determine SFN absorption and its molecular targets in humans consuming broccoli sprout extract (BSE) supplements pre-treated with myrosinase, the enzyme that forms SFN, or fresh broccoli sprouts. SFN absorption from BSEs was ~3 times lower than from sprouts. Metabolomic profiling revealed novel pathways altered with SFN consumption but distinct plasma metabolomic profiles following BSE or sprout consumption. This research provides important information for conducting SFN supplementation studies to understand SFN's role in disease prevention.
Metabolic Profiling Reveals Latent Metabolites and Interactions in Atherogenesis
Atherosclerosis remains the number one cause of mortality and morbidity in the western world. In this study, using UPLC-MS-based metabolomics, established as well as novel metabolites were identified being involved in atherogenesis. Cholesterol, oxidized cholesterol esters, purines, pyrimidines, and sphingolipids were just a few of the metabolites detected dysregulated, known for their involvement in atherosclerosis and validating our experimental design. Beta-oxidation intermediates (acylcarnitines) manifested a pattern indicating truncation of the process. Lastly, a previously unassociated lipid moiety, namely phosphatidylethanolamine-ceramide, was detected with statistical significance (t-test;p=9.8x10-12). This metabolite demonstrated the highest Spearman pairwise correlation to cholesterol, and will be the main focus in validation studies.
Investigation of Serum Metabolome Changes in Postmenopausal Women After Administration of Phytoestrogenic Dietary Supplements
Post analysis of samples from a clinical trial assessing the safety and efficacy of black cohosh and red clover for reduction of vasomotor symptoms in postmenopausal women were analyzed for metabolomic profiling of these botanical supplements. LC-HRMS data was obtained for serum samples at varying timepoints from the year-long study. The metabolomic profile was characterized to identify characteristic compounds from the botanical supplements compared to placebo and hormone therapy groups. Compounds of interest were identified for possible metabolic pathways of interest in order to determine a possible mechanism of action and biomarkers for vasomotor symptoms.
Capillary Ion Chromatography Coupling with High Resolution Mass Spectrometry for Anionic Polar Metabolites Profiling
Human oral squamous cell carcinoma (HOSCC) accounts for 94% of all oral malignancies. A highly sensitive platform coupling capillary ion chromatography (Cap IC) with Q Exactive mass spectrometer has been developed for metabolomic profiling of metabolic biomarkers for OSCC metastasis, 3 paired OSCC cell lines (UM1, UM5, CSC) with wild-type controls were examined. The Cap IC has demonstrated outstanding separation and peak shape for anionic polar metabolites and the sensitivities increased by more than 100-fold compared to RPLC and HILIC methods. Differential analysis finds significant changes in energy metabolism pathways, e.g., glycolysis cycle and tricarboxylic acid cycle.
Development of a Novel Metabolomics Profiling Work-flow Using Ultrafast Online SPE/MS and LC/MS/MS for Human Urine
The ability of an ultrafast SPE/MS system to profile the human urinary metabolome within 15 seconds was evaluated in the present study. MS and SPE methods for metabolomic profiling were optimized on an Agilent High Throughput RapidFire Mass Spectrometry System. This novel work-flow allowed for profiling of 1293 small molecule features using C18 cartridge and 1105 features using HILIC cartridge. A representative set of 10 metabolites were being investigated for confirmation by subsequent LC/MS/MS analysis. This SPE/MS system may be utilized for ultrafast and efficient analysis of urinary metabolites in large sample sets for many patho-physiological conditions.
Metabolite Analysis in Biological Fluids with Ultra-fast Tandem Mass Spectrometry
Some amino acids and acylcarnitines can be biomarkers in amino acid metabolism and lipid metabolism. We used Dried Blood Spots (DBS) samples and measured them by FIA (Flow Injection Analysis). As the result, we could obtain the specific profiles about each sample.
Moreover, there are some methods with column to examine in detail.
For example, to measure enzyme activity of methylmalonyl-CoA mutase and propionyl-CoA carboxylase, methylmalonic acid and 3-OH propionic acid can be targeted. Here we show the method that measures their target compounds with MRM (Multiple Reaction Monitoring). We analyzed test samples by using the method optimized for each target compound. As the result, we could obtain the specific profiles about each sample.
Arterial and Venous Plasma Metabolomics: Reflecting Intra-Tissue Metabolic Activity
Arterial and venous blood mediate metabolic exchange and homeostatic maintenance of all tissue interstitial fluids, yet their biochemical differences have been largely unexplored since the 1970’s. Here we use mass spectrometry based untargeted and targeted metabolomics to examine the arterio-venous metabolic balance across human peripheral tissue in vivo. Paired arterial and venous plasma profiling revealed subtle, yet highly significant differences, specifically in the water soluble plasma metabolome. Predominant differences were observed in amino and non-amino organic acid composition, likely reflecting skeletal muscle metabolism. This study shows that the untargeted arterio-venous metabolic profiling provides a comprehensive view into metabolic exchange, including discovery of unanticipated roles of specific metabolites, and offers new insights into organ homeostasis.
A New and Easy Strategy for the Rapid Identification of Bacteria Causing Bloodstream Infections Using MALDI-TOF MS
A shortened culture of at least 3h for direct identification of bacteria from positive blood cultures by MALDI-TOF MS (MALDI) was compared to routine subculture and identification. 375 mono-microbial positive blood cultures included 91 Gram-negative bacilli, 140 CNS, 54 S. aureus, 31 Enterococci, 53 Streptococci and 6 other bacteria. The direct identification by MALDI was correct in 65-100% (Gram-negative bacilli 100%, S.aureus 96%, S.pneumonia 96%, CNS and Enterococci 65%). Direct identification of bacteria from blood cultures by MALDI is reliable with the advantage of reporting the causative pathogen the same day the sample was positive.
Salvage Microbiology: Detecting Fusobacterium
Nucleatum in CNS Culture Negative
Infection by PCR Coupled with Electrospray Ionization
Mass Spectrometry
A previously healthy man presented with a one-month history of fevers,
cough and headaches. Chest X-ray revealed a right middle lobe cavitary
lesion and magnetic resonant imaging (MRI) of the brain revealed multiple
abscesses. Blood, bronchoalveolar lavage (BAL) and cerebrospinal (CSF)
fluid were both negative by traditional culture techniques. He was placed
on broad spectrum antimicrobials. CSF specimens were submitted for
PCR followed by Electrospray Ionization-Mass Spectrometry (PCR/ESIMS).
In addition, tissues from brain biopsy and brain abscess excision
were submitted for eubacterial 16S rDNA sequencing and
immunohistochemistry staining. Both techniques detected Fusobacterium
nucleatum, a fastidious, anaerobic gram-negative bacillus.
Tuberculosis: Application of SRM Assays Towards Biomarker Discovery
Selected reaction monitoring (SRM) is increasingly utilized in targeted proteomics to consistently and reproducibly measure a predetermined set of proteins. We have developed publicly available bioinformatics resources, PeptideAtlas, SRMAtlas, SRMQuantAtlas and AutoCalib to facilitate prompt and efficient fully quantitative SRM assay deployment. We demonstrate this workflow for fully quantitative SRM assays for proteins relevant to M. tuberculosis infection, mapping these with over 1200 specific peptides to screen clinical samples. This process eliminates the first stage of biomarker discovery (identification) by directly proceeding to assay deployment of potential markers. Presented results will demonstrate novel, reliable tools for disease diagnosis and stratification.
Rapid Identification of Mould Species from Agar Cultures by MALDI-TOF Mass Spectrometry
VITEK® MS is a MALDI-TOF mass spectrometer for microbial identification. Sample preparation for moulds is more challenging than for bacteria and yeasts. A specific protocol was set up on 140 well-characterized strains that were grown on solid media and tested at two different incubation times. These strains represented 22 clinically relevant species belonging to 17 genera. Preliminary cross-validation performances showed 97.8% correct identification. VITEK® MS with Knowledge Base v3.0.0 was able to discriminate different species within the same genus and to distinguish close species, like Aspergillus fumigatus versus Aspergillus lentulus or Scedosporium apiospermum versus Pseudallescheria boydii.
Diagnosis of Breast Cancer Based on Lipid Profiles Obtained by MALDI-TOF Mass Spectrometry
Breast cancer is a type of cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules, and is mainly diagnosed in women. Conventional diagnostic methods used to detect breast cancer include ultrasound, mammography, computed tomography (CT), magnetic resonance imaging (MRI) and core needle biopsy (CNB). However, drawbacks such as the lack of experienced doctors, adverse effects from radiation following examination, and the time-consuming and invasive natures of some examinations often increase the risks of medical treatment. In this study, two mass spectrometric techniques-MALDI-TOF and statistic methods-principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used to detect and classify lipids in biological specimens for breast cancer diagnosis.
Targeted Quantitation of Insulin and Its Therapeutic Analogs
Detection and quantification of insulin and its analogs has become paramount for medical and athletic doping. Traditional assays lack the ability to differentiate insulin and insulin analogs due to the lack of selectivity. Therefore, a Mass Spectrometric Immnoassay was developed. Capitalizing on conserved sequences, a single antibody is used to simultaneously enrich for insulin and insulin therapeutics, while LC/MS enables differentiation of the sequence variances. A robust clinical research assay able to concurrently measure multiple insulin analogs, providing a 15 pM lower limit-of-quantification and a dynamic range of 15-960 pM, is demonstrated.
PapilloTyper System for Prediction of Cervical Cancer and Genotyping of Wide Spectrum of Human Papillomaviruses
We report a MALDI-TOF MS-based human papillomavirus (HPV) diagnostic system, termed PapilloTyper system™ consisting of PapilloTyper™ kit for high-resolution HPV genotyping, PapilloPredict™ kit for detecting oncogenic E6/E7 mRNA expression and sophisticated automatic result reporting software. PapilloTyper was able to identify 74 different genotypes of HPV including 42 anogenital types. PapilloPredict was found to analyze E6/E7 mRNA from HPV types 16, 18, 31, 52, 53, 56, 58 and 66. The software for automation of the PapilloTyper system resulted in a peak analysis time reduction of 52 min (from 60 to 8 min; 87%) and improved reproducibility (less than %CV of 5). The capacity to identify wide range of HPV genotypes and oncogene expression along with high throughput precision analysis make the PapilloTyper system suitable for routine screening and early intervention of cervical cancer.
Abscription-based Detection of DNA Methylation: Rapid Multiplex MS Readout
A 3-gene bisulfite-free test for DNA methylation was developed to detect a CpG Island Methylator Phenotype that is associated with survival in low grade gliomas. Methylated and unmethylated FFPE DNAs were separated using magnetic beads and the relative amounts of each DNA were determined by CAP; coupled Abscription-PCR. Defined trinucleotide RNA signals generated by Abscription were quantified by LC-MS using single ion monitoring for multiple m/z to maximize sensitivity. Multiplexed LC-MS readout increased throughput 3-fold compared to serial analysis of each target. Results of the multiplexed were in agreement with single target detection.
Low Cost, Low Sample Volume, Rapid Vitamin D Assay
Traditional low cost immunoassay-based analysis of total 25-OH Vitamin D is known for high inter-lab assay variability, according to NIST studies. While LC/MS analysis of vitamin D provides more accuracy and clinical information, the higher cost of LC/MS assays has been a barrier to its wider use. In our setting, we developed a fast, 3 min method of 25-OH Vitamin D analysis with an internal cost comparable to that for highly discounted immunoassays (Diasorin) . Typically, cost lowering is achieved by simplification of sample preparation. Paradoxically, we found that plasma protein precipitation in the long run is not cost efficient, since this approach reduces column lifetime and requires much more frequent instrument maintenance. We were able to lower sample volume to 40 ul and implement supported liquid-liquid extraction (Biotage) as a very simple sample preparation step.
Ultra-sensitive Direct, Simultaneous LC-MS/MS Quantification of Intact Human Insulin, Glargine, Lispro, Aspart, Detemir and Glulisine in Human Plasma
This work provides a clinical research method for simultaneous, direct quantification of intact human insulin and 5 analogs in human plasma. LLOQs of 50-200 pg/mL (1.4 5.6 µIU/mL) were achieved from 250 µL human plasma, with average accuracy and precision for standard curve and QC samples being 94-100 % and 5.3-7.5, respectively. Results of patient samples analyzed in a blind study concurred with their diabetes multi-dosing regimes. We also demonstrated that high levels of human and bovine insulin do not interfere with quantification of analogs. We propose this method for pharmacokinetic monitoring of diabetic patients, sport anti-doping and forensic toxicology analysis.
Evaluation of Atmospheric Pressure Photo Ionization (APPI) for the Direct Analysis for Drugs of Abuse in Urine
Methodologies for the measurement of drugs of abuse have shifted to LC-MS/MS, minimizing the sample preparation needed for a GC-MS approach. Direct analysis of urine by LC-MS/MS can offer a significant advantage in time savings but can lead to potential matrix effects, as observed with electrospray, reducing the sensitivity and accuracy of the method. Performing the analysis using Atmospheric Pressure Photo Ionization (APPI) has greatly reduced the matrix effects relative to electrospray permitting direct analysis of urine using rapid LC separations and with no separations (flow injection analysis).
LC-MS Analysis of Intact Amino Acids on a Novel Mixed-mode HPLC Column
There are several established methods for analyzing amino acids, but each of these methods has disadvantages. The pre-labeled method has problems with derivitization efficiency and cost, while the post-labeled method is usually not compatible with LC-MS due to non-volatile mobile phases.
We have developed a novel amino acid separation column for LC-MS(/MS) which can analyze the complete array of 55 amino acids: 1) high throughput separation with Leu/Ile separation in 5min, and 2) simple gradient separation in 1 min or 10min. In addition, no prederivitization is required, and a standard LC-MS(/MS) system is sufficient for the analysis.
In this presentation, we will show the sensitivity and application for amino acids in serum.
Development of a Turboflow™ LC-MS/MS Method for Determination of 17-hydroxyprogesterone in Human Serum
Despite being characterized by a high rate of positive results, the most widespread methods for 17-hydroxyprogesterone quantification are the immunometric ones. We have developed a TurboFlow™ LC-MS/MS method using a Vantage triple quadrupole mass spectrometer (Thermo Scientific) equipped with an atmospheric pressure chemical ionization source. The method was fully validated and results compared with radioimmunoassay (RIA) currently used in our laboratory.
The method was linear from 0.02ng/mL to 50ng/mL. Total imprecision was lower than 5%. The Bland-Altman plot indicates an overestimation of RIA method with respect to TurboFlow™ LC-MS/MS method.
The method is rapid, sensitive and suitable for routine purpose.
Application of a GC-MSMS Method for the Simultaneous Determination of Persistent Organic Pollutants in California First Time Mothers and their Cord Blood
In our study of “Survey of Body Burdens in California Communities”, we measured levels of 18 PCBs, 7 OCPs and 5 PBDEs in 2 mL volume of maternal and cord blood from healthy, first-time mothers using a new GC-MSMS method. We confirmed that California PBDE blood levels are decreasing and that children may have higher exposures than their mothers to environmental contaminants-POPs. The method of simultaneously measure 30 POPs can be applied to large clinical and biomonitoring studies.
Sensitive Measurement of Plasma Catecholamines by 2D-LC-MS/MS
We developed and validated a simple and sensitive method to accurately quantify all three individual catecholamines in heparin plasma by 2D-LC-MS/MS, with a LLOQ of 5 pg/mL and a chromatographic run time of 5 min. This method is the most sensitive and the highest throughput to our knowledge, suitable for routine clinical laboratory use.
Novel Simplified and High-throughput 25-OH-Vitamin D2/D3 Analysis Including C-3 Epimer Separation Using Staggered Injections in a Daily Clinical Laboratory
Implementation of Vitamin D analysis for LC-MS including 3-C epimer separation is still widely discussed due to time of analysis (5-10 min per sample) and cost-intensive investments. One drawback of current immuno assays is the (miss-)detection of the epimer may leading to inaccurate results. We evaluated a novel simplified CE-IVD LC-MS Vitamin D2/D3 assay on pediatric samples including epimer separation and developed a LC protocol using a dual-channel UHPLC system with staggered injections to further decrease time per sample (2:20min including epimer separation), and to enable high-throughput analysis, running MS instruments on higher capacities.
An Evaluation of Biphenyl Chemistry to Aid in High-Throughput Bioanalytical LC-MS/MS Analyses
LC-MS/MS has become commonplace in the bioanalytical laboratory, as it can produce high throughput, high data quality analyses. The use of fully porous UHPLC and superficially porous HPLC columns are often used to increase the efficiency and peak capacity, and to decrease the analysis times. While these column advancements do impact efficiency, they do not directly impact selectivity or retention, which are the prominent parameters to analyte resolution.
We investigated a biphenyl based column chemistry to determine the effect on sample throughput and data quality in bioanalytical separations. From these applications we hope to demonstrate the advantages of the biphenyl-based stationary phase on throughput, data quality, sensitivity, and compound resolution.
Simultaneous Screening of Trace Levels of Multiple Green Plant-Based Allergens and Gluten in Food Using LC-MS
Mass spectrometry has become an important analytical technique in allergen research, aiding in the identification and characterization of allergenic proteins and peptides, as well as providing a sensitive alternative to ELISA for detecting and quantifying trace levels of these analytes in food and consumer products. This presentation will describe a proteomic approach used to to detect and quantify trace levels of marker peptides representing wheat, soy, peanut, ten different kinds of tree nuts, as well as wheat, barley and rye gluten in a single assay, using liquid chromatography-MS. Proteins were extracted from food samples and enzymatically digested with pepsin, trypsin and chymotrypsin. LC-QTOF accurate mass and MS/MS analysis permitted the identification of 2 or more marker peptides representing each analyte.
Analysis of Immunosuppressants in Whole Blood by Microflow LC-MS/MS
Here we present a microflow LC-MS/MS method that has been developed for the analysis of the immunosuppressant drugs Tacrolimus, Sirolimus, Cyclosporin A and Everolimus, with an injection-to-injection time of 1 minute. The sensitivity for all compounds has been shown to be <1ng/ml in whole blood, which represents <1pg on column for these compounds. 50µL whole blood was precipitated with 200µL methanolic zinc sulphate (80:20), and centrifuged, diluted with water, and then injected onto the Eksigent ekspert™ microLC 200 system, which was interfaced to the AB SCIEX QTRAP® 4500 LC/MS/MS system. The micro-flow LC method employed a flow rate of only 20uL/minute, yielding a significant solvent savings compared to traditional HPLC methods. Long-term method performance was evaluated, and compared favourably to traditional high-flow LC methods.
Fast Quantitation of PETH, an Alcohol Biomarker, in Dried Human Blood Spots by Microflow LC-MS/MS
Phosphatidylethanol (PEth) is a glycerophospholipid homologue where ethanol has been bound at the position that normally contains an amino-alcohol. As the formation of PEth is specifically dependent on ethanol, the diagnostic specificity of PEth as an alcohol biomarker is theoretically 100%. The quantitation of PEth in dry blood spots is of significant interest for forensic purposes. The published analytical LC-MS/MS methods for PEth are slow, therefore we have recently developed a rapid and simple approach to quantitation of PEth using an Eksigent ekspert™ microLC 200 system, interfaced to an AB SCIEX Triple Quad™ 5500 mass spectrometer. The run-time for the microflow LC-MS/MS method was 2.0 min, at a flow rate of only 50 uL/minute. The method displayed excellent correlation with an existing HPLC-MS/MS method, with the advantage of reduced solvent consumption and reduced run-time.
Quick & Simple Quantitation of Steroids, Using Dried Blood Spots and MicroLC-MS/MS
Quantitation of steroids in dried blood spots (DBS) is of significant interest in many disciplines, e.g. a panel of steroids is monitored in dried blood spots for Congenital Adrenal Hyperplasia (CAH) screening. The published analytical LC-MS/MS methods for the analysis of steroids typically have long run-times. Furthermore, most analytical methods employ high-end MS/MS systems to achieve the necessary level of sensitivity. The goal of this work was to develop a rapid method, enabling the quantification of steroids in dried blood spots using a low-end MS/MS system. We have chosen to explore the use of MicroLC, using the Eksigent ekspert™ microLC 200 system, because this technique typically provides shorter LC run-times, and a significant enhancement in sensitivity.
Alkyl Protocatechuates as Urinary Biomarkers of Exposure
to Parabens
Human exposure to parabens is a concern, owing to adverse health effects of these compounds. Parabens are metabolized and eliminated from the human bodies within a few hours of exposure. In this study, for the first time, methyl- and ethyl- protocatechuates (OH-MeP and OH-EtP) and their parent compounds, methyl- (MeP) and ethyl- parabens (EtP), were determined in urine samples collected from the U.S. children and adults. The concentrations of urinary OH-MeP and OH-EtP were higher than the corresponding concentrations of MeP and EtP in adults. Significant correlation between OH-MeP/OH-EtP and MeP/EtP was observed. The occurrence of a significant proportion of alkyl protocatechuates and 3,4-DHB among various paraben derivatives analyzed in this study suggests the need for consideration of these derivatives in the estimation of human exposure to parabens.
Urinary Concentrations of Bisphenol a and their Association with Indicators of in Vitro Fertilization
We evaluated the association of urinary BPA concentrations with outcomes of in vitro fertilization. BPA concentrations of urine samples from 40 couples undergoing IVF was measured using LC/MS/MS. Along with implantation result, D3 FSH level, peak E2, No. of retrieved oocytes, sperm concentration and motility, percentage of fertilization and percentage of good quality embryo were also assessed as indicators of IVF. Thirty-four couples excluding 6 couples with unusual BPA exposure were analyzed. The average concentration of female urinary BPA was 4.8 ng/mL (range, N.D.-20.8). That of male urinary BPA was 8.2 ng/mL (range, N.D.-24.6). There was no significant difference in indicator parameters of IVF depending on urinary BPA concentration. It suggests that unintentional low-dose BPA exposure may not affect outcomes of IVF.
Quantitative Analysis of Bisphenol a and Bisphenol A-glucuronide in Serum Using UHPLC-MS-MS
Used in plastics and thermal paper, bisphenol A (BPA) has received much attention due to its endocrine disruption potential and widespread human exposure. The measurement of BPA in biological samples is essential for risk assessment, but such analyses are challenging due to the trace levels of BPA in these samples and the possibility of background contamination. To overcome these limitations and to enable fast analyses, we developed and validated a highly sensitive and selective method using ultrahigh pressure liquid chromatography (UHPLC) interfaced with a new generation triple quadruple mass spectrometer (Shimadzu LCMS-8050) for the quantitative analysis of BPA and its major metabolite BPA-glucuronide. Each UHPLC-MS-MS analysis was completed within 2 min (3.5 min total cycle time), and only 25 uL of serum was required per analysis.
Automatic On-Line Solid Phase Extraction (SPE) Coupled with LCMSMS Method for the Quantitative Determination of Urinary Phthalate Metabolites
To assist California Biomonitoring program, we established an automated on-line solid phase extraction procedure coupled with LC-MS/MS for the quantitative determination of urinary phthalate metabolites with minor modifications from the published automated on-line SPE method to improve laboratory throughput. The automated on-line SPE method can minimize the time consuming sample preparation step using off-line SPE and potentially reduce operator error during the process. Ten target urinary phthalate metabolites are quantitatively measured with corresponding stable isotope labeled internal standards. Method LOD, precision, and other parameters will be reported.
Improved Dilute-and-Shoot Method for the Analysis of Carboxy-THC Using Rapid Fire Mass Spectrometry
RapidFire Mass Spectrometry (RF/MS/MS) offers throughput many times faster than conventional MS technologies. Analysis of THC metabolite (carboxy-THC) in urine by RF/MS/MS indeed found to be rapid, but actually proved costly because SPE was required. An improved and cost effective dilute and shoot sample preparation method was developed that showed good agreement between RF/MS/MS and the conventional LC/MS/MS method of analysis.
Rational Utilization of Mass Spectrometry for Compliance Monitoring in Chronic Pain Patients
Compliance monitoring has become a common means to try to curb prescription opioid abuse. While mass spectrometry offers ideal analytical performance for this purpose, the cost of such assays is substantial and immunoassay methods still dominate in the clinical laboratory. This work discusses the substantial non-compliance rate in chronic pain patients, the rate of discordance between mass spectrometry and immunoassays and a risk-based testing strategy, which applies mass spectrometry only on high-risk patients and low risk patients who screen non-compliant. This risk-based strategy allows for the utilization of mass spectrometry to improve patient care without incurring unnecessary health care costs.
RapidFire™: Experience with Urine Drug Testing for Gabapentin and Pregabalin
RapidFire™ (RF), a highly automated solid phase extraction device is capable of dramatically increased throughput with lower overall per sample costs. A method for Gabapentin (G) and Pregabalin (P) was developed which has a net run time of 25 sec and has demonstrated analysis of full 96 well plates in less than 45 minutes. Analysis of the same full 96 well plate using UPLC/MSMS would require at least 3 hours. Validation aspects of the method will be discussed. In addition, patient data acquired over 6 months will be discussed and compared with data acquired with the previous UPLC/MSMS method.
Method Validation of 13 Benzodiazepines by LDTD-MS/MS Analysis in 6 Seconds Per Sample with Correlative Data from LC-MS/MS for 200 Real Patient Urine Samples
The Food and Drug Administration (FDA) guidance for industry in developing bioanalytical method validation in human clinical pharmacology is applied to the LDTD-MS/MS analysis method of 13 benzodiazepines. Sample preparation consists of dispersive SPE fast processing followed by a basic LLE. Upper layer is directly applied to dedicated plate and dried prior to analysis. All FDA validation criteria were evaluated successfully and included verification of any potential drug interference, carry over and matrix effect. Cross-validation with gold standard analysis method by LC-MS/MS shows passing-Bablok regression with no significant deviation from linearity (Cusum test, P=0.11). All compounds are analyzed simultaneously in 6 seconds per samples.
Urine versus Oral Fluid Testing for Pain Management
Urine and oral fluid specimens were collected from 412 pain patients. Illicit drugs in urine and oral fluid were screened by immunoassay and positive specimens were confirmed by LC-MS/MS. Specimens were analyzed by LC-MS/MS for opiates, opioids, and benzodiazepines by LC-MS/MS. Prescription information was provided for each specimens. Oral fluid correlated 100% with urine for opiates, opioids, and benzodiazepines. Least correlation was for THC and urine was tested for carboxy-THC and oral fluid was tested for parent THC. This study gave the appropriate cutoffs for medication monitoring in oral fluid. Based on this study oral fluid is a viable alternate matrix for pain management testing.
Quantitation of Pentobarbital in Serum Using Liquid Chromatography-Tandem Mass Spectrometry (LC/MS-MS)
Our aim was to validate a user-friendly method for quantitation of pentobarbital by LC/MS-MS. Pentobarbital was extracted, diluted and analyzed. The CVs were <5%. The LOQ was <0.5 ug/mL. Recoveries were within 96-106%. Correlation with GC/FID was excellent. The quantitative pentobarbital method by LC/MS-MS is easy and user-friendly with an excellent analytical sensitivity. It requires a one-step extraction of pentobarbital from serum, followed by an injection into the LC/MS-MS. We were able to meet our institution’s turn-around time goal.
Improved Extraction of Pain Management and Illicit Drugs from Saliva Using Popular Oral Fluid Collection Devices
Oral fluid is emerging as one of the more popular and less intrusive sample matrices for measurement and screening of various controlled substances. However, there is not a consensus and universally adopted device for collection of saliva. Presence of stabilizing buffer and/or lack thereof may improve or hinder the extraction of different compounds from these devices. Several of the most popular oral fluid collection devices were examined. In this study, a group of compounds with wide ranges of hydrophobicity and acid/base reactivity were utilized to determine overall recovery and extraction cleanliness.
Mental Health Medication Monitoring: Aripiprazole in Urine by UPLC/MS/MS
A quantitative “dilute and shoot” urinalysis method was developed to monitor adherence to common antipsychotic medications, including Aripiprazole. Under 1% of an oral Aripiprazole dose is excreted unchanged in urine; requiring low detection limits for analysis. OPC 3373, the major urinary Aripiprazole metabolite at 19% dose recovery, was synthesized and assessed as a biomarker for the first time. We present an application of ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC/MSMS) facilitating fast, sensitive testing for Aripiprazole. The method demonstrated good reliability with authentic specimens. OPC 3373 is a superior urine biomarker for evaluation of patient adherence with Aripiprazole therapy.
Oral Fluid Pain Management Panel by “Dilute and Shoot” LC-MS/MS
A QuantisalTM sampling device was used to collect patient oral fluid samples. This device uses a sponge to collect 1mL of sample, which is then diluted into 3mL of buffer to stabilize the sample. Samples were diluted an additional 10X with internal standard and water/methanol and analyzed by LC-MS/MS on an AB Sciex 4500 platform using an Agilent 1290 chromatographic system. A set of 30 analytes and 15 internal standards were monitored for quantitative and qualitative ions without any sample clean-up/extraction.
This poster will review the validation of this oral fluid-pain management panel, including linearity, precision and accuracy, matrix effects, and studies on the potential for interference caused by other pain-related medications. Finally, using patient samples, the utility of LC/MSMS for analysis of oral fluid is demonstrated reducing the time and labor per sample.
Rapid Analysis of Selected Benzodiazepines by Automated SPE/MS/MS
The application of a highly automated solid phase extraction mass spectrometric technique for rapid benzodiazepine analysis was explored. Urine samples were enzyme-hydrolyzed and diluted prior to analysis on an Agilent RapidFire™ 300 coupled to an Agilent 6460 triple quadrupole. The method was validated and applied to positive authentic urine samples to evaluate concordance with HPLC/MS/MS results. Cycle time per injection was decreased from 96s (HPLC/MS/MS) to 13s (SPE/MS/MS). Over 5,000 injections were completed per SPE cartridge, contributing to a cost per sample substantially less than with traditional analysis. The SPE/MS/MS method is a viable alternative for rapid, more cost-efficient benzodiazepine analysis.
An Ultrafast Online SPE-QTOF Urine Drug Method for 35 Analytes in a Common Medication Indicated (CMI) Panel
A CMI panel of 35 analytes has been developed using the RapidFire™, a highly automated, front end solid phase extraction accessory that interfaces with MS technology. This new method dramatically increases throughput, with a net run time of 12 seconds per sample and full analysis of a 96 well plate in less than 20 minutes. Analysis of the same plate by UPLC-TOF would require 16 hours using the current production method. Data using both LC and RF methods will be discussed and a comparison of limits and patient samples will be presented.
Analysis of Pain Panel Medications in Urine on Raptor™ Biphenyl by LC-MS/MS
For nearly a decade, the Restek™ Biphenyl has been the column of choice for clinical diagnostic and Pain Management drug screening testing because of its ability to provide highly retentive, selective, and rugged reversed-phase separations of drugs and metabolites. By bringing the speed of Superficially Porous Particles to the Biphenyl family, Restek’s Raptor™ Biphenyl provides clinical labs with an even faster option for a wide variety of clinical assays. Drug Screening applications can be difficult to optimize and reproduce due to the limited selectivity of C18 and phenyl-hexyl phases. Using Raptor™ Biphenyl columns pain management analysis can be performed with a 5-minute cycle time and complete isobaric resolution. Popular competitor methods have tailing peaks, longer run times, and co-elutions; only the Raptor™ Biphenyl exhibits the selectivity and performance needed for these tests.
Easy to Automate Extraction of THC Metabolites for LC-MS/MS Analysis
An application is described for the sample preparation of Tetrahydrocannabinolic acid (THCA) from urine for subsequent LC-MS/MS analysis. THCA concentrations were in the forensic relevant range of 5-500 ng/mL. Extraction was done with the Tecan Immobilized Coating Extraction (TICETM) Technology as it is easy to use, reproducible and suitable for cost efficient automation due to simple pipette and shake steps. Finally, a method was set up for simultaneous quantitative extraction of both analytes, THCA and THCA-glucuronide, followed by LC-MS/MS analysis by adjusting organic solvent content and pH conditions in the extraction, wash and elution step.
Direct Analysis of Opioids and Metabolites in Oral Fluid by Mixed-mode µElution SPE Combined with UPLC/MS/MS
26 opioid drugs and metabolites were analyzed in oral fluid using mixed-mode solid phase extraction (SPE) followed by revered-phase UPLC/MS/MS analysis. Oral fluid samples (100 ìL), diluted in a stabilization buffer were extracted using mixed-mode, strong cation exchange µElution plates. LC separation was achieved on a C18 hybrid particle column using a linear solvent gradient. All analytes eluted in less than 5.5 minutes, and baseline separation was achieved for all isobaric compounds. Calibration curves were linear from 5-500 ng/mL with R2 values of 0.99 or greater. Quality control results at were accurate and precise. Nearly all results were within 10% of target values and 95% of %CVs were under 10%.
A Quantitative Determination of Methadone and Its Metabolite (EDDP)
Opioid withdrawal is a major problem in the pediatric intensive care unit (PICU). Withdrawal symptoms are not only unpleasant but can be life-threatening. Methadone is commonly substituted for narcotic infusions during the weaning process to prevent withdrawal symptoms. A sensitive LC-MS/MS method suitable for the simultaneous determination of methadone and EDDP concentrations in pediatric patients using DBS is developed. 0.1 ng/ml of LOQ was achieved for both Methadone and EDDP compounds on IONICS 3Q 220 LC-MS/MS with acceptable precision and accuracy.
Solid Phase Extraction of N-linked Glycopeptides Using Hydrazide Tip
Glycoproteome contains valuable information where biomarkers may be discovered for disease diagnosis and monitoring. To facilitate high throughput N-linked glycopeptide isolation, in this study, a novel hydrazide tip was devised and an integrated workflow of N-linked glycopeptide isolation using hydrazide tips was presented. We demonstrated that, with the hydrazide tips, the processing time was significantly decreased from 3-4 days to less than 8 h with excellent reproducibility. The hydrazide pipette tips have great potential in achieving automation of N-linked glycopeptide isolation for high throughput sample preparation when used in combination with liquid handling robotic systems.
Comparative Proteomic Analysis of the Anti-inflammatory Action of Lovastatin in Macrophages
Lovastatin is widely used in clinical practice to treat patients with hypercholesterolemia and its clinical efficacy in regulating atherosclerosis, an inflammatory disorder has been observed. Since macrophages play a central role in atherosclerosis, we performed a systematic bottom-up proteomic analysis of the anti-inflammatory effect of lovastatin in macrophages using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A novel 290 kDa cytoskeletal protein, a group of well-defined G-proteins and members belong to G-protein associated cascades were identified. This finding provides a direct evidence that lovastatin exerts anti-inflammatory activity through a mechanism involving the expression of these specific target molecules and their network.
Targeted Quantification of TMPRSS2-ERG Fusion Protein Products in Prostate Cancer Using an Antibody-independent PRISM-SRM Approach
An antibody-independent PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy was used for quantifying TMPRSS2-ERG gene fusion protein products in prostate cancer cell lines and tumors. The PRISM-SRM assays enabled identification of three 'signature' peptides for highly sensitive and specific detection of ERG protein, revealing that 1) ERG protein expression is highly correlated with TMPRSS2-ERG gene rearrangements, and 2) at least two groups of ERG protein isoforms are simultaneously expressed at variable levels in TMPRSS2-ERG gene fusion positive samples. The results indicate that ERG protein may be a promising marker for stratifying prostate cancer risk.
The Transformative Role of Data Repositories in Enabling Clinical Proteomics Research
Fundamental limitations in the computational analysis of tandem mass spectrometry data typically result in up to 90% of all spectra being discarded as unidentified. As such, there are many terabytes of unidentified spectra from a large variety of diseases, cell lines and clinical samples that cannot be translated into biomedical knowledge. Similarly to the community-wide annotation and analysis of the Human genome, we advance that overcoming this stalemate in mass spectrometry requires a shift in both computational and social approaches towards a paradigm where public data repositories become the underpinning platform for the collaborative annotation of all mass spectrometry data.
MudCHiP: Online 2D Chip-LC Peptide Trapping as a Robust Approach for Proteomic Biomarker Applications
Online cation exchange chromatography (MudPIT) is a proven tool for large-scale biomarker discovery applications. Hence, we integrated two-dimensional separations used in MudPIT-MS/MS into a targeted proteomics workflow using SRM. Here, peptides from many human plasma proteins revealed significant mean peak area increases of 90% compared to conventional RP-SRM-MS experiments. Implementation of chip-based MudPIT columns further improved this workflow and enabled reliable and accurate inter-chip reproducibility essential for biomarker monitoring. Further, MudPIT-chip columns have been assessed in discovery experiments and enabled 1600 proteins to be identified from a cancer cell lysate in under 6h analysis time in a completely online system.
Identification of A. Fumigatus Proteins in Patient Bronchoalveolar Lavage Fluid
Aspergillus fumigatus causes the potentially fatal disease invasive pulmonary aspergillosis in immunocompromised patients. Fungal proteins found in lung or plasma may be useful diagnostic biomarkers for this disease. In this study, bronchoalveolar lavage fluid (BALF) from patients suspected of having invasive aspergillosis are being analyzed via bottom-up proteomic techniques using 2DLC- MS/MS. We identified Aspergillus proteins in patient BALF, and correlated the finding to clinical and microbiology culture results. To our knowledge, this report represents the first identification of Aspergillus proteins from human BALF.
Spectral Libraries Retention Time Prediction Based on Endogenous Peptide Standards
Spectrum libraries are an invaluable starting point for developing targeted assays (e.g. SRM, PRM) by providing information about fragmentation patterns and retention times. When library data are collected under a variety of LC conditions, the use of peptide standards can greatly improve the ability to accurately predict retention time in new experiments. Unfortunately, any samples not including the peptide standards cannot be used in the predictions. We present a method for selecting peptides endogenous to a sample to act as standards and demonstrate their use for predicting retention times of other peptides including those with post-translational modifications.
Multiplexed Protein Quantitation Assay Using Selected Reaction Monitoring (SRM) Mass Spectrometry Technology
SRM platform, a targeted protein quantitation technology, has shown great potential for biomarker development. In a triple-quadruple (QQQ) mass spectrometer, SRM is a tandem selection process where selecting peptides by precursor m/z is followed by selecting fragmentations of the targeted peptide. When it is coupled with liquid chromatography and the addition of heavy isotope labeled internal standards, SRM becomes a highly selective, sensitive, and high throughput technology for protein quantitation. SRM arises as an alternative to antibody-based assays for develpment of clinically relevant biomarkers. Jadebio has developed sets of SRM panels such as Neuro-CSF-SRM-150 panel for neurodegenerative diseases.
Identification of Plasma Biomarkers of Chronic Drug Exposure in a Rat Model
The goal was to test the feasibility of detecting protein changes in rat plasma from 0 to 30 days following chronic drug exposure.
Only 1 microliter crude plasma was sufficient for proteomic quantitation using LC-MS/MS. Protein changes discovered above were verified on independent cohorts using MRM.
In total, ~500 proteins were identified. There were 64, 61, and 8 differentially expressed proteins after termination of cocaine, nicotine, or morphine administration. 114 proteins were detected by MRM verification. Several proteins showed consistent results with discovery studies. This methodology is able to determine biosignatures that reliably report on drug exposure for up to 30 days following last drug use.
Measurement of Thyroglobulin in Fine-Needle Aspiration Samples: Comparing LC-MS/MS to an Immunoassay
Measurement of thyroglobulin (Tg) concentration in ultrasound-guided fine-needle aspiration (FNA) samples is commonly used in the evaluation of suspicious lymph nodes in papillary thyroid carcinoma. We adapted an LC-MS/MS method for measurement of plasma Tg to the analysis of Tg in FNA samples and compared results of quantitative measurement of Tg by LC-MS/MS with those from the Beckman Access Tg immunoassay. Imprecision of triplicate analysis of FNA samples by LC-MS/MS was below 11.2%. Reasonable agreement between the methods was observed in samples containing greater than 50 ng/mL of Tg; some disagreement was observed at concentrations of Tg below 50 ng/mL.
A Comparison of Peptides to Whole-Protein Stable Isotope Labeled Internal Standards in Quantitative Protein MS Workflows
LC-MS/MS methods employing the sensitivity of multiple reaction monitoring (MRM) for quantification of proteins has typically utilized stable isotope labeled peptides (SIL-Pep), commonly called AQUA peptides. A more ideal strategy involves the use of a stable labeled full length protein internal standard that can be introduced early in the analytical workflow, overcoming processing variability. In this study, we compared the performance of SIL peptides to whole-molecule SIL protein as the internal standard in a quantitative assay for Erythropoietin (EPO) in plasma. The results demonstrate that the performance of whole-protein SIL-EPO is better than that of SIL peptides in this application.
Highly Multiplexed and Sensitive Plasma Protein Quantification by 2D LC-MRM/MS
Toward improved depth and breadth of plasma protein quantitation, a new 2D LC system was evaluated within a bottom-up proteomic approach. Our method is antibody-free, utilizes a complex mixture of isotopically coded peptide standards, two dimensions of standard-flow RPLC (operated under high and low pH conditions), and targeted MRM technology. After preliminary optimizations, 261 plasma proteins (inferred from 641 interference-free peptides) were reproducibly quantified across 13 fractions. These proteins cover an 8 order of magnitude concentration range (from 290 pg/mL to 15 mg/mL), which is in excess of 10-fold lower than that attainable without pre-treatment where 172 proteins were quantified.
Multiplexed Mass Spectrometry Immunoassay for Quantitative Determination of Apolipoprotein C-I, C-II and C-III and their Isoforms
Multiplexed quantitative assays provide extensive information about several proteins in a single-assay. In this work, a fully quantitative multiplex mass spectrometric immunoassay (MSIA) method for analysis of apolipoprotein C-I (apoC-I), apolipoprotein C-II (apo-CII) and apolipoprotein C-III (apoC-III) from human plasma was developed. The assay was utilized to accurately and reproducibly quantify apoC-I, apoC-II and apoC-III and their posttranslational modifications in a high throughput manner in several hundred human plasma samples. The results provided a unique insight into the quantitative distribution of these apolipoproteins and their isoforms across a large population.
Proteomics Profiling of Cancer Cell Lines Using High Flow Chromatography and Electrospray Ionization Technique
Nanospray LC-MS/MS techniques are widely used for proteomics analysis to cover the wide dynamic range and for a maximum number of protein identifications in complex biological samples. Although nanospray LC-MS/MS technique is sensitive, it requires nanoflow and long chromatographic gradients. In proteomics profiling and integrated systems biology studies, nanospray is extremely challenging due to large number of sample sets. Here we introduce a combination of high flow chromatography and electrospray ionization (AJS-ESI) coupled to a high resolution iFunnel Q-TOF for high throughput LC/MS analysis of 4 different human glioblastoma (GBM) cell lines. At the 1% FDR level, 4,264 unique human proteins and 24,916 unique peptides were identified in the preliminary analysis of 4 different human glioblastoma cell lines.
GlycoProtein Analysis (GPA): Automated MS Platform for Identification of Intact N-Glycopeptides and Its Application to Human Plasma for Biomarker Discovery
We developed the high throughput method to globally identify N-glycopeptides in human plasma by high resolution tandem mass spectrometry with GlycoProtein Analysis (GPA) algorithm. Three scoring systems were applied to automatically search the site-specific N-glycopeptides agianst human plasma N-glycopeptide database combined with retrosynthetic glycan library. We identified the highest number of 141 distinct glycopeptides with five N-glycosites at <1% false discovery rate in alpha-1-acid glycoprotein. Also we found the site specific glycoforms in total 393 glycopeptides of 67 N-glycoproteins from Immunoglobulins (~1 mg/ml) to AFP(~10 ng/ml) present in plasma. The comparisons of normal and HCC plasma samples represented different expression of site-specific N-glycopeptides with the highly branched and fucosylated glycan structure in proteins to be associated with liver cancer progression
Targeted Multiplexed Quantitation of Glycoproteins and Corresponding Glycoforms Using MSIA LC-HR/AM MS Analysis
Qualitative and quantitative analysis of glycoproteins and corresponding glycoforms presents many challenges for LC-MS due to its complexity and relative abundance to matrix proteins. Previous studies utilized global glycopeptide enrichment from biological matrices followed by LC-MS quantitation. While this strategy provides increased detection and identification capabilities, there are still issues with detecting all possible glycoforms per targeted protein and lacks normalization strategies. Our approach is to utilize MSIA extraction tips for serial glycoprotein extraction followed by multiplexed HR/AM MS analysis on the Q Exactive mass spectrometer. The study will show comparative detection, normalization, and quantification of four different plasma glycoproteins.
Characterization of Stable Isotope Labeled APO-A1 for Use as an Internal Standard in a Quantitative MS Workflow
The ideal internal standard for quantitative LC/MS assays would be a stable-isotope-labeled full-length protein that is equivalent to the native target protein which can be introduced at the beginning of the workflow. We have extensively characterized heavy APO-A1 expressed in HEK293 cells to show that it is sufficiently similar to native APO-A1 using peptide mapping, intact mass, and protein-antibody binding affinity assays on a Biacore. We have also studied the digestion time course to demonstrate that SIL-APO-A1 peptides are liberated at the same rate as those from the endogenous protein. The optimized LC/MS/MS conditions for APO-A1 will be presented.
Improving Throughput for Targeted Quantification Methods by Intelligent Acquisition
Introduction
Targeted quantification has become a very popular technique to verify putative biomarker candidates in a large cohort of samples. These candidates are usually generated from a discovery experiment that identifies proteins empirically observed to be differentially expressed, or they can originate from a biological hypothesis (e.g. a pathway or an interactome that provides a list of associated proteins). These lists of candidates are usually large, sometimes containing 100-500 proteins. Traditionally, targeted quantification is done using SRM assays or, more recently, PRM experiments. Both of these experiments have limited throughput, usually around 10-50 proteins. Thus the number of proteins that can be verified by such techniques remains limited, leading to a subjective filtering of proteins that proceed to this stage. We present a new method that relies on real time decisio
Quantification of Large Peptides in Human Serum Using High Resolution IFunnel LC-QTOF
High resolution LC-MS techniques can potentially provide more accurate understanding of the metabolism of therapeutic proteins due to their selectivity in complex background. In this study a glycopeptide MUC5AC-13 was spiked into human plasma to mimic DMPK of therapeutic proteins. For the MUC5AC-13 LOD of <25pg on a 2.0 mm column and <20fg on a HPLC-Chip was achieved in MS mode. The average RSD was 3.67% with 4 orders of magnitude linearity on 2.0 mm column. The average RSD was 6.52% with >5 orders of magnitude linearity on HPLC-Chip. At the same time MS/MS provided qualitative information for the validation of the peptide sequence in a combined qual-quant experiment. All Ions MS/MS also yielded excellent sensitivity with LODs of 5pg (UHPLC) and 100fg (Chip). These results have shown the excellent potential of the iFunnel Q-TOF as a Qual/Quant technique for large therapeutic peptide.
Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Biomarker Quantification for Breast Cancer Patient Assessment
Comprehensive molecular evaluation of cancer patients is rapidly evolving area of biomarker research; the greatest impact can be made by examining critical test points in the proteome of these patients to enable selection of therapy. For that purpose, we have undertaken the measurement of a panel of protein biomarkers in breast cancer that are expressed over a wide dynamic range. Biomarkers for tissue quality control (actin, GAPDH), epithelial-to-mesenchymal transition (vimentin), proliferation (Ki67), and known biomarkers for classification (Her2, ER, and PR expression and phosphorylation) are quantified in cell lines and frozen tumor specimens by liquid chromatography-multiple reaction monitoring mass spectrometry.
Development of Peptide Biosensors for Multiplexed Monitoring of Kinase Activities
Kinase inhibition has been a fruitful area of research in the fight against cancer. Selective inhibition of kinases can reverse the effects of oncogenic mutations. Inhibitors are rarely perfect, and associated problems include non-specific inhibition and vulnerability to mutations in the kinase gene. To address these problems, peptide biosensors are being developed to act as selective substrates to quantify the activities of target kinases. Phosphorylation of biosensors is easily measured by observing the mass shift on a mass spectrometer. As of now, biosensors for ABL1, JAK2, and SYK kinase activities have been successful, while additional biosensors are being developed.
Elucidation of Plasma Biomarkers for Acute Graft-Versus-Host Disease Using Nano-Ultra Performance Liquid Chromatograpy and Tandem Mass Spectrometry
There are no obvious plasma biomarkers specific to acute graft-versus-host disease. Therefore we tried to identify plasma biomarkers for acute GVHD using nano-ultra performance liquid chromatography and tandem mass spectrometry. Plasmas were pooled from five patients without acute GVHD before and after HSCT and likewise from five patients with acute GVHD that were matched for age, the conditioning regimen, and time of sample acquisition. Plasma proteins were identified by one-dimensional gel electrophoresis combined with reverse-phase nano-ultra performance liquid chromatography and tandem mass spectrometry followed by peptide fragmentation. Dozens of candidate plasma proteins were identified which can be used for the prediction, diagnosis, or monitoring of GVHD. Further validation studies are required for finding clinically useful protein biomarkers in plasma.
LRN-C Materials Program Stability Studies: Urinary Nerve Agent Metabolites
The Laboratory Response Network-Chemical (LRN-C), a Centers for Disease Control and Prevention (CDC) sponsored program, is a national network of public health laboratories established to respond to human exposure to toxicants. The mission of the LRN-C materials program is to ensure the reliability of the calibration and quality control materials used by this national network of laboratories. Determining the shelf life of calibration and quality control materials is an essential part of the quality assurance process. Based on stability testing guidelines from the Clinical Laboratory Standards Institute, the stability of a panel of nerve agent metabolites in urine was evaluated.
Use of Titrimetric Primary Standards for ESI-MS
One common problem with clinical mass spectrometry is the dearth of primary or other certified standards for calibration or stability testing purposes. Many new analysis methods are at the mercy of the purity assessment of the provider of custom synthesized molecules and ongoing analyses are at risk from the degradation of materials during storage frozen in matrix, solution or native state. Since electrospray ionization often relies upon ionization of molecules in solution this investigation assesses the utility of primary titrimetric standards as standards for evaluation of the purity or stability of electrospray active molecules.
Burden of Proof: Providing Clinical Confidence in the Face of Analytical Fallacies
Although much confidence is placed in the capacity of LC-MS/MS to provide unambiguous quantitation, this faculty of LC-MS/MS is largely dependent upon a priori knowledge of the analytes, specimen/matrices, and extraction/chromatographic methods in question. In the absence of this knowledge (which is often), LC-MS/MS quantitation is fallible and, thus, is only as valid as one demonstrates it to be. Presented herein is a holistic approach to analytic validation of a multiplexed assay quantifying endogenous metabolites, with a focus on esoteric validation studies.
Evaluation of a Spike Recovery Approach to Quality Control in a Multiplexed LC/MS/MS Opioid Assay
Monitoring compliance with chronic pain medications necessitates assays that confidently assess the presence or absence of analytes. Conventionally, matrix effects such as ion suppression are accounted for using peak area requirements for deuterated internal standards, but this is difficult to monitor without commercially available internal standards. In this work we describe an analysis of over 2100 opioid LC/MS/MS samples for which the recovery of spiked compounds was used in parallel with proxy internal standards as a quality control parameter. We find that this requirement detects an additional 1.1% of sample failures relative to using a proxy internal standard.
Dried Blood Spot Analysis Using Temperature-enhanced Flow-through Desorption Coupled Online to SPE-MS/MS – Tackling the Hematocrit Issues
The effect of hematocrit (Ht) variation on precision and recovery in dried blood spot (DBS) analysis is a serious obstacle in the way to wider acceptance of DBS for quantitative bioanalysis. Using online flow-through desorption (FTD) for DBS analysis, we have investigated high temperature desorption as a means to obtain high recoveries, which are less dependent on Ht. Also, a new blood application device was evaluated for full-spot DBS analysis. Results with online FTD-SPE-MS/MS for immunosuppressants show significant reduction of the Ht influence effect . Strategies to reduce the Ht effect on partial-spot DBS analysis will also be addressed.
Small Scale LC/MS Sample Preparation Workstation Based on Hamilton Microlab NIMBUS
LC/MS Sample Prep Workstation processes upto 192 sample in one run and supports protein precipitation extraction (PPE), liquid-liquid extraction (LLE) and solid phase extraction (SPE). System can be enclosed in a hood or connected to in-house exhaust system. Easy to use interface facilitates quick set up and supports wide range of labware. Hamilton's unique technologies such as CO-RE, ADC, MAD, pLLD and cLLD enable efficient pipetting of volatile, organic and inorganic solvents, and phase separation of liquids. NIMBUS is a compact, affordable and personal bench top pipettor that suits most lab needs for throughput, size and budget.
Automated Sample Preparation of Biological Fluids for LC-MS/MS Analysis
A novel technology for extraction of small molecules from biological fluids has been adapted to a 96 wellplate format. The individual wells contain an innovative coating into which analytes absorb. The simple equilibrium process and protein removal will be described. The approach reduces the number, as well as complexity, of the process steps to simple pipetting and shaking operations. This simple work flow has been adapted to robotic processing for sample preparation using a Tecan EVO. A suitable work table layout and work flow will be explained. The samples produced are clean and ideally suited to use in clinical LC-MS/MS analysis.
How Protein Sample Preparation Is Going to Become as Fast, Easy and Reproducible as SPE
The digestion of proteins is both a time consuming bottleneck and source of irreproducibility in the sample preparation process, thus creating a significant barrier for the translation of proteomics into clinical assays. Fundamentally, applications of enzymatic catalyses in biotechnology are often limited by the activity of the enzyme under desired operating conditions. Notably, Taq polymerase has become one of the most important enzymes in molecular biology due to its ability to withstand the protein-denaturing conditions required during polymerase chain reaction (PCR). Historical attempts to apply thermally stable enzymes to proteomics have resulted in restricted success. However, in this presentation we will describe the application of a highly stable trypsin reactor and its use in the analysis/quantification of biological samples.
Enhanced Resolution and Matrix Interference Reduction for the Analysis of Vitamin D Metabolites
Analysis of Vitamin D metabolites has continued to be a topic of interest in recent publications, primarily as biomarkers for possible disease states and vitamin sufficiency. In this study, an LC/MS method for the analysis of Vitamin D metabolites is expanded to include dihydroxy metabolites along with the epi-homologs. Chromatographic resolution is utilized for the quantitation of hydroxy and dihydroxy Vitamin D2 and D3 metabolites including the isobaric epimers. In addition, sample preparation techniques are evaluated for the impact of biological matrix ionization effects.
The unique combination of the selectivity of the F5 stationarly phase along with the novel sample preparation technique allow of a robust and accurate LC/MS method for quantitation of all the associated Vitamin D metabolites
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation
Improving sample quality, throughput and reproducibility is of highest concern for clinical research; however, in order for progress to occur, it will be necessary to investigate new analytical strategies. This presentation will focus on an ultra-fast-immuno-MS platform that combines next-generation plasma separator cards with fully automated immuno-affinity and rapid digestion with reversed phase liquid chromatography coupled directly for mass spectrometric analysis. Two common IgG peptides, VVSVLTVLHQDWLNGK and TTPPVLDSDGSFFLYSK, were monitored using MRM transitions. Calibration levels were linear with r2 values of 0.994 and 0.996 for each peptide and LOD for each peptide was approximately 10 fmol of material on column.
Sample Preparation for 25-OH Vitamin D Mass Spectrometry Analysis: The Phospholipid Conundrum
Recent developments in sample cleanup for 25-OH vitamin D mass spectrometry methods has generated interest in selectively removing interfering compounds to improve complications from ion suppression and increase the lifetimes of LC-columns. More specifically, the ability to selectively separate vitamin D analytes from potentially interfering phospholipids is an attractive alternative to standard protein-crash methods. SPEware Inc. has recently developed a novel phospholipid removal sorbent (Maestro ®) available in a flangeless 1 mL cartridge. Our goal was to evaluate the efficacy of the Maestro® cartridge in removing phospholipids, optimize parameters for extraction of 25-OH Vitamin D, and compare the analytical performance of the method to a simple protein-crash method for 25-OH Vitamin D.
Generic SLE Extraction Method for Ultra-fast Drug Screening in Saliva Using LDTD-MS/MS Analysis
The Laser Diode Thermal Desorption (LDTD) ion source combined with Mass Spectrometry (MS) is an effective screening tool for drug analysis in Oral Fluids. Oral fluid analysis is a non-invasive method that has facilitated laboratory testing for many drugs of abuse. A new extraction approach using Supported Liquid Extraction (SLE) is evaluated as a generic, rapid, and streamlined extraction procedure prior to fast analysis on LDTD. Full method validation using 13 common drugs of abuse spiked in oral buffer and extracted using SLE 96-array into 96 LazWell plate holder. Data for Linearity, Recovery, Stability, Carryover, Matrix Effect, Precision and drug interference is presented. The evaluation of this new workflow to provide a fast and generic screening method for analysis of drugs in oral fluid generated excellent data and can be useful for labs with a sample analysis bottleneck.
Development and Validation of an Automated Immuno-MALDI Assay for the Clinical Measurement of Plasma Renin Activity
We have developed an automated immuno-MALDI (iMALDI) assay for the determination of plasma renin activity (PRA) using an Agilent Bravo liquid handling platform and a Bruker Microflex MALDI-TOF instrument. The iMALDI approach circumvents the disadvantages of radioimmunoassays (RIAs), which are commonly used for PRA determination. Initial results indicate strong correlation between iMALDI and RIA (R2 = 0.92, n = 30) at a limit of detection of 83 amol/µL (108 pg/mL). The assay will be validated on 200 patient samples by comparing iMALDI and RIA results.
Improved Sample Preparation Methods for Athlete Doping Analysis of Common Compounds in Urine by LCMS
: Anti-doping testing by urine analysis requires fast and robust screening methods with repeatable sample preparation. Since, every sample has to be screened, methods are designed to be sufficiently sensitive and specific to identify all suspect samples. One must be careful to minimize false suspects. Ensuring samples are spiked with internal standards accordingly will help verify that samples are being extracted and tested correctly and with accurate uniformity.
The Australian Sports Drug Testing Laboratory, our collaborators, have invested time in determining a limited number of comprehensive screening methods. These methods, using Thomson’s eXtreme Filter Vials (patented), comply with the World Anti-Doping Agency’s (WADA) Prohibited List.
In exploring new methods labs have looked at both detection and sample prep as routes to quicker and more accurate analysis. Liquid chromatography
Quantitative Analysis of 25-Hydroxy-Vitamin D in Serum and Evaluation and Comparison of Sample Preparation Techniques Using LCMSMS QQQ and QTOF in 1D and 2D Configuration
Liquid chromatography triple quadrupole (QQQ) and time-of-flight (QTOF) mass spectrometry (LC/MS/MS) are suited for rapid analysis of multiple analytes. A highly sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of 25-Hydroxy-Vitamin D2 and D3 by QQQ and QTOF. Various sample preparation techniques and chromatographic configuration are evaluated and compared based on their ease of use, analyte recovery and post-extraction cleanliness. The described method achieves the required sensitivity and is capable of quantitating analytes over a relevant dynamic range. Excellent reproducibility was observed for all compounds (CV < 5%). All calibration curves displayed linearity with an R2 > 0.995.
Heat Fixation Inactivates Viral and Bacterial Pathogens and Is Compatible with Downstream MALDI Mass Spectrometry Tissue Imaging
Rapid stabilization of molecules of interest is the key to high quality tissue proteomic studies. Proteomic analysis of tissue for infectious disease research must overcome the additional challenge of pathogen inactivation before downstream analysis can take place. We therefore examined the potential of heat fixation for pathogen inactivation. BalbC mice were infected with an alpha virus (VEE) or the bacterial pathogen (B. thailandensis). Tissue samples were heat fixed at 95°C for 30s using the Denator system, and infected control samples were untreated. The presence of each pathogen in control tissue was demonstrated by positive plaque or colony formation, whereas heat fixation completely inactivated the organisms. MALDI-MSI results were comparable to fresh frozen and produced spectra with fewer degradation products. Heat fixation will enable the use of infected tissue from studies performed at bio-safety levels 3 and 4 to be safely used for downstream analysis.
A Versatile Automated Platform for Reproducible, High-throughput Protein Digestion, Peptide Fractionation, and Peptide Cleanup for LC/MS Analysis
We present an automated sample preparation platform for protein analysis by LC/MS facilitating in-solution protein digestion, peptide cleanup, and fractionation of peptides. This platform permits parallel processing of up to 96 samples in microtiter plate formate enabled by a precision liquid handler adapted to use disposable microchromatography cartridges operated through a simple user interface. We report key analytical figures-of-merit for this platform using model systems for protein and peptide quantification and characterization by LC/MS. Signals from target peptides typically yield % CVs < 5% for digestion/cleanup workflow and < 10% for digestion/cleanup/fractionation/cleanup workflow without the use of internal standards.
Extraction of Antiepileptic Drugs from Human Urine, Serum and Oral Fluid Using Supported Liquid Extraction (ISOLUTE® SLE+) in 96-Well Plate Prior to LC-MS-MS
Dosing and therapeutic drug monitoring (TDM) for doctors prescribing antiepileptic drugs is of importance to effective patient treatment. Effective sample preparation strategies are needed to extract the various types of antiepileptic drugs from complex matrices prior to analysis using techniques like liquid chromatography mass spectrometry. Supported liquid extraction is a fast alternative to solid phase extraction sample preparation methods that allows for the extraction of both zwitterionic and neutral forms of antiepileptic drugs simultaneously from various complex matrices. This poster details the fast methodology of supported liquid extraction for antiepileptic drug sample preparation in various complex matrices prior to LC-MS-MS
Extraction of Synthetic Cannabinoids (SPICE), Opiates, and Benzodiazapine Drugs from Oral Fluid Using Supported Liquid Extraction (ISOLUTE® SLE+)
Oral fluid testing for the presence of drugs is steadily becoming a common requested analysis by law enforcement. Currently there are oral fluid collection kits that use a buffer to preserve the oral fluid samples. A fast and reliable sample preparation method using supported liquid extraction (SLE) was developed to extract a broad suite of basic drugs and synthetic cannabinoids from spiked neat oral fluid and oral fluid samples collected with commercial collection kits. Recovery data and ion suppression data generated from the novel extraction method is detailed in this poster.
Extraction of Mycophenolic Acid and Mycophenolic Acid Metabolite from Serum Using Supported Liquid Extraction (ISOLUTE® SLE+) Prior to LC-MS-MS Analysis
Immunosuppressant drug therapy is instrumental in lowering the risk of tissue or organ transplant rejection for patients having undergone transplant surgery. Therapeutic drug monitoring (TDM) is critical for doctors to assess the dosage amounts. A series of effective immunosuppressant drugs can be monitored in the patient serum as the free drug and the glucoronidated metabolite. An effective sample preparation strategy for extracting mycophenolic acid and the glucoronidated metabolite from serum is proposed here using ISOLUTE Supported Liquid Extraction prior qualitative analysis by liquid chromatographic mass spectrometry.
Effectiveness of Solid Phase Extraction in Analysis of Urinary Cortisone and Hydrocortisone
Solid Phase Extraction (SPE) has long been viewed as a simple removal and/or pre-concentration technique. The plethora of different SPE products presents both opportunities and challenges in selecting the right SPE material and cleanup method. The cleanliness and recovery of cortisone and hydrocortisone extract from urine by different SPE phases are examined. The tested phases include silica based and surface-modified polymeric resin beads. Each phase represents one or more modes of retention mechanism such as hydrophobic, cationic or anionic, or all combined.
Method Development Strategies for Drugs of Abuse Extraction from Urine Using Supported Liquid Extraction Prior to UPLC-MS/MS Analysis
Drugs of abuse screening can be complicated due to the wide variation of functional groups associated with different analyte classes. This poster demonstrates method development strategies for drugs of abuse screening using supported liquid extraction prior to UPLC-MS/MS analysis. Urine extracts were analysed using a Waters ACQUITY UPLC system coupled to a Quattro PREMIER XE triple quadrupole mass spectrometer. Optimal method performance resulted in recoveries greater than 70% for all analytes and corresponding RSDs below 10%. Calibration curves demonstrated excellent linearity and coefficients of determination from 1-500 ng/mL for hydrolysed and non-hydrolysed urine.
Nicotine and Metabolites: Evaluation of Supported Liquid Extraction Approaches Prior to UPLC-MS/MS Analysis
Nicotine is of importance as the addictive chemical in tobacco and can be administered to aid smoking cessation. It is also a potential medication for several conditions including ulcerative colitis, Alzheimer’s and Parkinson’s diseases. This poster will demonstrate a simplified protocol for the simultaneous extraction of nicotine and its major metabolites. LC-MS/MS analysis was performed using a Waters ACQUITY UPLC coupled to a Quattro Premier XE triple quadrupole MS using electrospray ionization, operated in the MRM mode. Extraction recoveries greater than 70% were obtained for plasma, serum, whole blood and urine. Calibration curves demonstrated good linearity across the respective ranges.
Rapid and Complete Serum Digestion by a Microwave-Assisted Enzyme Reaction
Microwave radiation was applied to trypsin digestion of non-depleted human serum to achieve fast and complete digestion. Microwave power, duration and temperature were controlled by Rapid enzyme digestion system (REDS). The extent of the digestion was estimated by 1D-gel electrophoresis. More than 90% of proteins were digested within 3 hr by REDS with 300W at 37degrees Celsius which was similar with the result from the conventional overnight method. Mechanical shaking motion of several Hz was added to the microwave irradiation to improve the efficiency of the digestion. More than 95% of proteins were digested within 3 hr by shaking REDS digestion.
Investigation of Sample Preparation for High-Throughput N-Glycan Profiling
N-glycan profiling can be a useful clinical method for multi-cancer diagnosis. We applied 96-sample handling devices for semi-automatic sample preparation of N-glycan profiling. Commercial serum was loaded to a 96-well plate and processed in a 96/batch mode from serum dilution to N-glycan profiling, except for solid phase extraction. The N-glycans were profiled by MALDI-TOF MS. Each N-glycan sample was loaded at four MALDI spots and a total of 384 MALDI spots were used to calculate reproducibility of 96 samples. Serum treatment using the semi-automated sample preparation resulted in 5.4% and 8.5% CV for spot-to-spot (n=4) and sample-to-sample (n=96), respectively.
Evaluation of Supported Liquid Extraction for the Simultaneous Extraction of Vitamin D Metabolites: 25-hydroxy and 1α,25-dihydroxyvitamin D2/D3, PTAD Derivatization and Analysis Using UPLC-MS/MS
Vitamin D analysis has important clinical relevance with levels needing to be measured for a wide variety of reasons. This poster demonstrates a simple supported liquid extraction protocol, derivatization with PTAD and subsequent detection of both the traditional hydroxy metabolites and the biologically active dihydroxy metabolites in serum. Extracts were analysed using a Waters ACQUITY UPLC system coupled to a Quattro PREMIER XE triple quadrupole mass spectrometer. Positive ions were acquired using ESI operated in the positive MRM mode. Method performance demonstrated high analyte recoveries and low ion suppression, allowing a quantifiable range matching levels therapeutically expected for each metabolite.
Determination of Thyroid Hormones in Biological Fluids by LC/MS with Online Solid Phase Extraction
An online SPE-LC/MS method was developed for the determination of thyroid hormones in biological fluids. The method exploited and compared the RP-Amide and C8 as the trapping column with phenyl phase as the separation column. The preliminary data demonstrated that both RP-Amide and C8 effectively trapped the thyroid hormones extracted from spiked rabbit plasma samples prepared by standard protein precipitation, and gave sharp peak shapes. However, the RP-Amide trapping gave much higher signals and thus recoveries. In addition, the RP-Amide trapping was compatible with 20% methanol washing and with 100% aqueous mobile phases, thus more versatile in trapping method development.
Reduced Workflow Strategies for the Sample Preparation of Serotonin, Histamine and Related Catecholamine Metabolites in Plasma and Urine
Modern bioanalytical laboratories equipped with ultra performance liquid chromatography – tandem mass spectrometers (UPLC-MS/MS) allow for the high throughput screening of candidate drug molecules at each checkpoint of the discovery pipeline. The bottleneck of this analytical process is typically the time invested in sample preparation (both development/routine analysis) to harmonize biological matrices with the injection requirements of commercially available systems. In a collaborative effort with MD Anderson Cancer Center, we detail here the application of new column technology to purify histamine and serotonin from mouse plasma (previously reported as 2 separate assays) in one extraction method with the elimination of the sorbent conditioning and equilibration steps familiar to traditional solid phase extraction method protocols prior to measurements by UPLC-MS/MS. The salient metabolites homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid (VMA), are endogenous metabolites that are currently measured for disease detection (also reported here).
A High Throughput Solid Phase Extraction Method for Vitamin B3 and Related Metabolites from Pooled Human Serum Prior to Mixed Mode LC-MS/MS
Sample preparation development strategies that target vitamin B compounds and related metabolites are a challenge since selected parent metabolite pairs have both acid and basic pKa values. Of particular interest to this study is the determination of vitamin B3, a precursor to the synthesis of hormones that cascade through a number of metabolic pathways in vivo. Workflow strategies to facilitate population screening of the parent compound nicotinic acid (pka(s) = 2.2, 4.8) as well as 2 relevant metabolites niacinamide (pka = 3.54) and nicotinuric acid (pka(s) = 3.1, 3.5) in a single analysis will be presented. The analytes of interest were fortified into pooled mixed gender serum and extracted using a 96 well solid phase extraction plate. The reconstituted extracts were measured using a gradient mixed-mode LC-ESI-MS/MS method. Relative recovery and method repeatability were determined.
Optimization of an HFIP/MTBE Fractionation Method for Sequential Extraction of Proteins, Lipids and Nucleic Acids for Systems Biology Applications
Systems biology studies combine proteomics with genomics, transcriptomics, and the newer fields of lipidomics and metabolomics. Unfortunately, current sample preparation techniques rely upon mutually incompatible reagents to isolate proteins, lipids and nucleic acids. This is especially true for lipidomic studies, where the most commonly accepted methods (Folch and Bligh/Dyer) are incompatible with recovery of proteins in a state usable for proteomic applications. We have developed a novel sample preparation technique which allows sequential isolation of proteins, nucleic acids and lipids from solid and liquid samples. This method was previously shown to extract lipids and proteins from neonatal fecal material.
Fully Automated Analysis of 25-hydroxyvitamin D2-D3 and Immunosuppressant Drugs with ZinMass-200 Clinical LC-MS/MS Analyzer
Fully automated, reliable, rapid and sensitive LC-MS/MS methods were developed for analysis of Immunosuppressant drugs and 25-hydroxy metabolites of Vitamin D. All sample preparation, data acqusition and calculation steps were made by a ZinMass-200 Series Clinical LC-MS/MS Analyzer without any human control for reducing user errors in sample preparation.
Analysis run time was 3 minutes for 25-hydroxyvitamin D2 and D3. Recovery for 25-hydroxyvitamin D2 and D3 analytes were between 97%-105%. Linearity from 2,5 to 400 µg/L for both analyte. Correlation coefficients were 0,9989 and 0,9995 respectively. Limit of detection for 25-hydroxyvitamin D2 was determined as 1 µg/L while limit of detections for 25-hydroxyvitamin D3 was 0.8 µg/L.
A fully automated analysis method for Immunosuppressant drugs also succesfully validated. Analysis run time was 6 minutes.
Automation of Sample Clean-Up for a Comprehensive Forensic Toxicology Urine Screening LC-MS/MS Method
For research use only, not for use in diagnostic procedures. This presentation describes rapid cleanup of uniquely hydrolyzed urine samples using disposable pipette extraction (DPX) automated tips for comprehensive screening of illicit drugs. Attempting to eliminate the susceptibility of the results to human error, the sample preparation was automated using the Tecan Freedom EVO® Liquid Handling Workstation allowing for high throughput analysis. The automated workflow monitors 130 analytes; detecting and quantifying these compounds in a single Liquid Chromatography-Tandem Mass Spectrometry run. Recoveries for over 60% of the analytes were greater than 80%; typical accuracies and precisions were within 15%.
Automated Extraction and Quantitation of 1,25-Dihyroxyvitamin D3 in Serum Samples
While the 25-OH form of vitamin D is commonly used to assess the vitamin D status of patients, in some cases it may be desirable or even necessary to determine the serum levels of the active metabolite 1,25-dihyroxyvitamin D. For example, the levels of 1,25-dihyroxyvitmin D may be increased or decreased depending on conditions related to hyperparathyroidism or chronic renal failure . Here we present a method to assess the levels of the active form of vitamin D, which automates the addition of an internal standard and extraction followed by a five minute LC-MS/MS detection step.
3 Strategies to Stop a Pervasive High-Throughput Sample Preparation Quality Problem You Likely Didn’t Know You Had
Clinical testing using liquid chromatography-tandem mass spectrometry is generally perceived to be more specific than antibody-based detection methods. During our transition from individual vials to 96-well plate-based methods for various quantitative assays that use forced evaporation with commercially available equipment, we have observed a carryover phenomenon we refer to as “hotspot” carryover. Hotspot carryover causes an increased likelihood of falsely elevated results for samples in close proximity to wells with significantly elevated analyte concentrations at an estimated rate of approximately 4%. We present three independent methods investigated to detect, reduce and eliminate this potentially disastrous quality concern with clinical mass spectrometry.
Membrane for Rapid Immunoaffinity Purifications and Processing
Described here-in is the construction of a membrane that uses covalently linked Protein A to capture antibodies, in either a specific and non-specific manner, for use in immunoaffinity purification schemes as well as in the study of the heterogeneity of immunoglobulin preparations. Membrane construction, characterization and experimental schemes for immunoglobulin processing are presented. The objective of this work is to enable an inexpensive and rapid technology for immunoassay type experiments as well as for immunoglobulin heterogeneity analysis to be performed with MALDI-MS detection.
A Novel Online Cleanup Valve Solution for Quantitative Analysis of Testosterone in Serum Utilizing LC-MS/MS
Determination of testosterone levels in serum is an important measurement in clinical research. An improved level of detection is possible, with minimal sample preparation, using a previously unreported configuration that significantly reduces the sample complexity using single binary pump and online cleanup component. The online cleanup system presented here has a built-in, single piston pump with solvent selection capability and user-selectable valves integrated in a single module. The analytical method contains quantifier and qualifier transitions to ensure accurate quantitation of testosterone. The UHPLC system was equipped with binary pump, the novel integrated module with two switching valves, enrichment and analytical columns.
A Validated Bioanalytical Method for the Measurement of Bisphenol-A-glucuronide in Human Urine Using UHPLC-MS-MS
Used as a plasticizer and monomer in some polymers, bisphenol-A (BPA) is under investigation for possible adverse effects on reproduction, diabetes, cardiovascular disease, and obesity. To assess human exposure, a rapid method was developed to measure BPA in urine as its monoglucuronide, which is the primary excreted form. Urine samples were prepared using solid phase anion exchange extraction. BPA-glucuronide was measured in the extracts using UHPLC with selected reaction monitoring MS-MS on a Shimadzu Nexera LCMS-8040 triple quadrupole mass spectrometer system. [13C]-BPA-glucuronide was used as a surrogate standard for quantitative analysis. Calibration curves were linear with R2 values of 0.9999.
A Rapid and Sensitive SLE Method to Eliminate the Matrix Effects of Liquid Chromatograpy-tandem MS for the Determination of Levonorgestrel in Plasma
The experiment is aimed at developing a rapid and sensitive sample preparation method for the determination of levonorgestrel in plasmaby solid supported liquid extractioncolumns packed with diatomaceous earth.Proteins and phospholipidscan be removed effectively by SLE resulting in the elimination of the matrix effects of mass spectrometry detection. An average recoveryof 94.2%of levonorgestrelis reached.
Determination of β-blockers from Human Plasma with SPE 96-well Plate Format coupled with LC-MS/MS
With the growth of high blood pressure and heart disease population, related drugs such as β-blockers become the focus of attention. So a simple and efficient sample preparation technique is necessary. This study was carried out by using solid phase extraction cartridges with a format of 96-well plate and LC-MS/MS detection for the analysis of metoprolol and propranolol in human plasma. The plasma was spiked with the two β-blockers at a concentration range from 1 to 5 ng/mL. The method is rapid and efficient with the recovery range from 84.2% to 92.2%.
The results indicate that with 5 mg packing material of Cleanert PEP and a format of 96-well plate β-blockers in human plasma can be extracted effectively and eluted with a small volume of the eluant.
Liquid Chromatography-High Resolution Mass Spectrometry and Microsampling; a Perfect Couple for Small Molecule Analysis in Clinical Research?
Clinical research depends on the availability of human biofluids. Collection of biofluids can be invasive and therefore collection methods which minimally interfere with the individual are desired. Micro-sampling of dried blood spots, capillary plasma and saliva have seen a significant increase in popularity in clinical, bioanalytical and medical applications, lately. Liquid chromatography (LC)-high resolution mass spectrometry (LC-HR/MS) offers the analytical advantage of high mass accuracy measurements based on the exact mass of analytes rather than on their fragmentation patterns (MS/MS). Present work demonstrates some applications of LC-HR/MS in combination with micro-sampling of biofluids in clinical research areas.
Mass Spectrometric Quantification of Acetaminophen and Structurally Related Substances in Human Serum and Plasma
The method described allows the quantification of Acetaminophen and nine structurally related compounds. After dilution and reduction of the samples, chromatographic separation of the analytes was achieved by gradient elution on a pentafluorphenyl phase column with subsequent detection by electrospray triple quadrupole mass spectrometry. Quantification was performed by means of deuterated analogues of the analytes as internal standards. The method was fully validated and allowed precise quantification of the analytes with good limits of quantification The method was used to analyze over 70 samples (spiked and patient samples) and the results were consistent with the expected values.
HPLC Methods to Separate Several Steroid Isomers
Accurate quantification of steroid isomers is very important in the clinical diagnostic labs. Compare to immunoassay, HPLC-MS/MS is more specific, selective, and sensitive. However, there are still false results due to incapable to separate isomers by mass spectrometry, which requires chromatography separation, also, preferable without ion pairing reagents. We have screened several different types of regular reversed phase HPLC columns, and found out Matrix Polychrome column is capable to separate most steroids isomers (testosterone and epitestosterone; hydrocortisone and prednisolone; betamethasone and dexamethasone; 3-epi-25-OH Vitamin D3 and 25-OH vitamin D3)with simple isocratic mobile phase.
Comparison of Alternative Calibration Strategies for Quantitative Analysis of Testosterone and 25-Hydroxyvitamin D3 in Serum
Recently, an increasing number of publications have discussed the potential of using alternative calibration in LC-MS/MS assays in clinical laboratory. The aim of this investigation is to evaluate three different alternative calibration strategies for measurement of testosterone and 25-hydroxyvitamin D3 in serum. We observed excellent agreement between conventional multiple point calibration and both single point and contemporaneous response factor alternative calibration methods in two analytes commonly measured in the clinical laboratory. Internal calibration is promising but involves technical challenges that must be carefully evaluated for the most robust implementation.
A Liquid Chromatography-Tandem Mass Spectrometry Method for the Measurement of Dimethandrolone and Its Undecanoate Ester: Application to a Phase 1 Study in Men
Background: Dimethandrolone (DMA) and its ester Dimethandrolone Undecanoate (DMAU) are being developed by the NICHD for hormonal male contraception because of its dual androgenic and progestational actions. Objective: We developed and validated LC-MS/MS methods for the measurement of DMAU and DMA in serum for phase 1 pharmacokinetics study in men. See Long Abstract for Method/Results/Conclusion.
Rapid Measurement of Aldosterone in Human Serum by Two Dimensional Liquid Chromatography-Tandem Mass Spectrometry
Determination of aldosterone concentration is a critical part in evaluation of primary aldosteronism, which accounts for at least 2% of hypertensive patients1. Due to its low plasma concentration level, aldosterone has traditionally been measured by radioimmunoassay or other formats of Immunoassays (IAs) with fair sensitivity. However, IAs lack specificity. Several liquid chromatography tandem mass spectrometry (LC-MS/MS) based assays were reported to quantify aldosterone with the employment of solid phase extraction (SPE) or liquid-liquid extraction (LLE)2,3,4. The SPE based cleanup is complex, time consuming, and not cost effective; while LLE based cleanup usually needs longer runtime to resolve interference from analyte during LC separation. We developed a rapid and simple two dimensinal LC-MS/MS assay to measure aldosterone in human serum.
An Alternative USP Method for the Analysis of Impurities in Riboflavin (Vitamin B2) Using LC-MS-MS
Riboflavin (vitamin B2) is a water-soluble vitamin. It is synthesized by all plants and many microorganisms, but it is not produced by higher animals. It is a precursor of coenzymes that are required for the enzymatic oxidation of carbohydrates, so it is essential to basic metabolism. The current USP method for impurity analysis is non-quantitative while the European Pharmacopeia (EP) calls for the use of ion-pair chromatography to separate the riboflavin from four major impurity peaks. An alternative to USP and EP methods using LC/MS/MS to identify and quantify the riboflavin and impurities is presented. In addition to improving the specificity of the method with MS/MS detection, two additional goals were to decrease analysis time and eliminate the need for ion pairing reagents that are not compatible with MS. This new proposed method is also compatible with UV-Vis detection.
Detection and Quantitation of a Minor Metabolite of Vitamin D2, 3-Epi-25-hydroxyvitamin D2, in Human Serum Using LC-MS/MS
Accurate measurement of vitamin D is important in the diagnosis and treatment of diseases. Vitamin D status is generally assessed by measuring its major circulating metabolites, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2, in serum or plasma. The presence of the minor structural analog of 25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin D2, can pose problems if complete separation is not achieved. The presence of 3-epi-25-hydroxyvitamin D2 in sera was confirmed. LC baseline separation of 3-epi-25-hydroxyvitamin D2 from its isomers was achieved. The 3-epi-25-hydroxyvitamin D2 concentration was determined to be 2 % to 5 % of the 25-hydroxyvitamin D2 levels in sera with high 25-hydroxyvitamin D2.
Determination of Serum and Plasma Methotrexate Levels Using LC-MS/MS
Our LC-MS/MS method for the determination of methotrexate concentration levels in human serum and plasma samples includes the addition of isotopically labeled methotrexate as an internal standard and a solid-phase extraction (SPE) step prior to injection to the LC-MS/MS system. Methotrexate analytical standards were prepared in serum and plasma matrices demonstrating linearity across the standard curve range of 0.01 - 10 umol/L, with r > 0.999. Between-run analysis of three levels of serum and plasma controls prepared across the standard curve range and four additional levels of commercial controls demonstrated results within 3.2% of target concentrations with CVs within 3.4%.
Improved Extraction of Cannabinoids from Oral Fluid
Oral fluid analysis is increasingly incorporated into drug testing programs because of its simple, noninvasive sample collection process and its indication of recent drug use over other forms of biological fluids like blood and urine. Delta-9-tetrahydrocannabinol (THC), the most commonly used illicit drug, is difficult to detect in oral fluid due to low concentration, small sample volume, and sensitivity issues. In this study, we developed a simple yet effective solid phase extraction (SPE) protocol for THC and its metabolites from spit.
Quantitative Analysis of Water Soluble Vitamins in Human Blood Using Liquid Chromatography Tripe Quadrupole Mass Spectrometry
Liquid chromatography triple quadrupole (QQQ) mass spectrometry (LC/MS/MS) are suited for rapid analysis of multiple analytes. A highly sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of the relevant Water Soluble Vitamins. This method uses a simple offline sample preparation in blood. The described method achieves the required sensitivity and is capable of quantitating of the Vitamins over their relevant dynamic range. Excellent reproducibility was observed for all compounds (CV < 10%). All calibration curves displayed linearity with an R2 > 0.995.
Replacing an In-house Equilibrium Dialysis Method of Free Testosterone Using Estimation Based on Total Testosterone and Sex Hormone Binding Globulin
We compared free testosterone (fT) measured by equilibrium dialysis (ED), to fT calculated from total testosterone (TT) and sex hormone binding globulin (SHBG) in 40 male and 40 female serum samples. TT in both methods was measured on Waters tandem mass spectrometry coupled with liquid chromatography and SHBG was measured on Abbott Architect Plus i1000 SR. Calculated fT correlates well with ED measurement, with R2 of 0.876 in males and 0.996 in females. The close correlation between the two methods warrants the replacement of in-house ED measurements of fT using calculation based on TT and SHBG.
The Robust and Sensitive LC-MS/MS Quantitation of Thyroid Hormones (T3 and T4) in Serum
Thyroid hormones play an important role in many biological processes. LC-MS/MS of thyroid hormones (T3 and T4) at low concentration levels, has shown to be superior to immunoassay and is becoming the method of choice for measurement of free T3 and T4 due to its high selectivity and sensitivity. In this paper, a simple LC-MS/MS method is reported for sensitive T3 and T4 quantitation in serum. Results show sub-pg/mL detection limits of T3 and T4 with a concentration linearity up to 700 pg/mL. Comprehensive LC-MS/MS results (LOD, LOQ, linear dynamic range, accuracy and matrix effect) will be discussed.
Determination of Urinary Ethyl Glucuronide and Ethyl Sulfate by LC/MS/MS for Clinical Research
Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is ideally suited for the rapid analysis of multiple analytes. A highly sensitive and specific LC/MS/MS method has been developed for the quantitation of ethyl glucuronide and ethyl sulfate. A dilution procedure and a solid phase extraction (SPE) procedure are evaluated and compared based on ease of use, analyte recovery and post-extraction cleanliness. The described method achieves the required sensitivity and is capable of quantitating analytes over a wide dynamic range. Excellent reproducibility was observed for all compounds (CV < 5%). All calibration curves displayed linearity with an R2 > 0.995.
A Fast, Specific, Sensitive and Robust LC-MSMS Method for Quantification of Urinary Free Catecholamines: Full Validation and Application in Diurnal Study
A fast, specific, sensitive and robust LC-MSMS method was developed and fully validated for the quantitative determination of urinary free catecholamines. Linearity, specificity, matrix effect, carryover, precision, recovery, stability and method comparison were evaluated. Excellent sensitivity and precision guaranteed the reliability to conduct a urinary catecholamine diurnal study. 557 urine samples were collected every 2-4 hours in 3 consecutive days from 25 volunteers. Consistent and significant diurnal patterns for epinephrine and norepinephrine excretion were observed in the 3-day period, with highest peak at around noon while nadir in early morning. No statistical significant diurnal rhythm was observed for dopamine.
Analysis of Underivatized Methyl Malonic Acid (MMA) in Human Serum Under Reversed-Phase LC and Tandem Mass Spectrometry
Methylmalonic acid (MMA) is deemed as one of the markers to evaluate the vitamin B-12 (cobalamin) deficiency. The analysis is traditionally carried out after derivatization of MMA with butanol to improve the chromatographic retention or analyzed under HILIC condition. Here, MMA in an underivatized form is analyzed under reversed-phase LC condition and tandem MS in negative ionization mode. The sample preparation method utilizes a liquid-liquid extraction procedure. Based on a limited number of patient sample comparison, this method correlated well against an established LC/MS method which used a derivatization procedure.
A Routine Combined LC-MS/MS Assay for Male Androgens
To determine the androgen status in men the measurement of other androgens such as DHT and DHEA with testosterone is beneficial. We report a combined LC-MS/MS assay for the measurement of testosterone, androstenendione, DHT and DHEA.
Samples were extracted using on-line solid phase extraction on a C18 cartridge (Waters OSM) and analytes were measured using a Waters TQS mass spectrometer.
Chromatographic separation was achieved between all four androgens and adequate LLOQs and precision was achieved.
The assay requires a small volume of sample and all analytes are measured simultaneously without derivitisation achieved by using a high sensitivity mass spectrometer.
A Novel Method for the Measurement of Plasma Metanephrines Using Online Solid Phase Extraction- Liquid Chromatography Tandem Mass Spectrometry
Measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine is very useful in the diagnosis of phaeochromocytomas. Our aim was to develop a method for plasma metanephrines using a small sample volume with minimal hands-on preparation. Samples were deproteinised prior to online SPE using a Waters Online SPE Manager coupled to a Xevo TQ-S mass spectrometer. We achieved a lower limit of quantification of 37.5 pmol/L for metanephrine and 75 pmol/L for normetanephrine and 3-methoxytyramine using only 150 µL of sample. The assay was linear up to 30000 pmol/L for all analytes and results compared well to a previously published LC-MS/MS method.
A Rapid and Sensitive LC-MS/MS Method for the Simultaneous Analysis of Testosterone, Androstenedione, 17 OHP and DHEAS
Measurement of serum testosterone (T), DHEAS, androstenedione(A4) and 17-hydroxyprogesterone (17OHP) are important in the investigation of hyperandrogensim in females. We describe a simple method for their simultaneous measurement. After protein precipitation samples were extracted using on-line solid phase extraction by a Waters Acquity/on line sample manager coupled to a Waters Xevo TQ tandem mass spectrometer. Separation of all four steroids was achieved within a run time of 5.5 minutes and adequate sensitivity and precision were achieved. We have developed a rapid assay for the LC-MS/MS measurement of T, A4, 17OHP and DHEAS suitable for the investigation of hyperandrogensim in females.
LC-MS/MS Method for Quantitative Analysis of Thiopurine Drug Metabolites in Whole Blood
Thiopurine drugs, azathioprine and 6-mercaptopurine (6-MP), are clinically important for immunosuppression following solid organ transplant and treatment of childhood acute lymphatic leukemia and autoimmune diseases such as inflammatory bowel disease. Metabolism of thiopurine drugs varies significantly amongst individuals, with elevated levels of some metabolites linked to toxicity, therefore monitoring of metabolite levels is an important part of therapy. Current production methods utilize HPLC-UV to quantify thiopurine metabolite levels in packed erythrocytes. In order to facilitate ease of quantification, here we present a LC-MS/MS assay for the quantitation of azathioprine/6-MP and the metabolites 6-thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine nucleotides (6-MMPN) in whole blood. The method is linear from 25.5-912 ng/mL (6-MP), 33.6-1200 pmol/200µL (6TG), 840-30,000 pmol/200µL (6-MMP), with LOQs of <17.2ng/mL (6-MP), <18.6 pmol/200µL (6-TG), <440 pmol/200µL (6-MMP). Intra and inter-assay imprecision using patient samples was found to be <7.5%. Method comparison to a HPLC-UV method analyzed by Deming Regression with 95% confidence intervals gave 6-TGN: y = 1.38(x) + 5.94 (R 0.966), n = 42 and 6MMPN: y = 0.69(x) – 62.0 (R 0.980), n = 27.
Simultaneous Analysis of Urinary Metanephrines, Catecholamines and Serotonin by ESI-LC-MS/MS with Solid Phase Extraction Sample Preparation
A unique, reliable, rapid and sensitive LC-MS/MS method was developed for simultaneous analysis of urinary dopamine, norepinephrine, epinephrine, metanephrine, normetanephrine, 3-methoxytyramine and serotonin.Solid phase extraction and ESI-LC-MS/MS was used for the simultaneous analysis of urinary Metanephrines, Catecholamines and Serotonin in urine samples. Confirmation and quantification ions for dopamine, metanephrine, normetanephrine, 3-methoxytyramine and serotonin determined in positive electrospray ionization mode while norepinephrine and epinephrine ions were determined in negative electrospray ionization mode. Recoveries for all analytes were between 70%-95%. The predetermined calibration range for the analytes in 24-hour urine was 50-10000 ng/ml. The calibration curves were linear in the range tested and the correlation coefficients were minimum 0.994 for all analytes.
Quantitative Screening Method for Analysis of Drugs of Abuse in Urine Using LDTD with Benchtop Quadrupole Orbitrap Mass Spectrometer
LDTD coupled with HRAM is used for direct analysis of over 50 drugs of abuse in urine. Sample processing is enzymatic hydrolysis followed by LLE. Samples are plated onto LazWell 96-well plate pre-coated with EDTA and introduced to the mass spectrometer using thermal desorbtion from the plate using a Phytronix™ LDTD source.. Screening cutoffs were met for the compounds identified showing this method to be useful for rapid screening of urine in a forensic toxicology setting.
Maximizing Triple Quadrupole Mass Spectrometry Productivity Through the Automated Use of an Expanded Dual-Channel HPLC System with Online Sample Cleanup
This work explores increasing mass spectrometer productivity through the automated use of an expanded dual channel high performance liquid chromatography system. The complete, integrated LC/MS/MS system is comprised of a triple quadrupole mass spectrometer coupled to a configurable HPLC system, all controlled by a single software application. A previously developed method for the analysis of 25-OH vitamin D has been used for testing the capabilities of this new instrument. By staggering injections on parallel streams and switching between the two streams at the appropriate time, throughput of the integrated expanded system can double the throughput of the standard method.
Verification of a LC/MS/MS Method for 19 Opioids, Opiates, and their Metabolites in Human Urine without Hydrolysis
The use of LC-MS/MS in bioanalysis is often hindered by the need for complex sample preparation and extraction methods which can introduce errors in sample handling that lead to large sample to sample variability. The development of a rugged system for the sample preparation and chromatographic analysis prior to MS detection that requires minimal sample handling is essential in a clinical environment. In this work, we present the verification of a panel for 14 opioids, opiates, and their metabolites in urine. This method alleviates the need for the labor intensive, somewhat variable, and time consuming hydrolysis step that is the typically performed prior to analysis.
Verification of a LC/MS/MS Method for 14 Antidepressants Utilizing Dried Blood Spots
Important factors in the analysis of therapeutic drugs in whole blood are accurate measurements, storage capabilities, small sample volume, and easy extraction. Dried blood spots are becoming an adopted technique for the analysis of drugs in biological matrices. Due to the complexity of the solution resulting from dissolving the blood spot, the sample must undergo further clean up by chromatographic separation before introduction into the mass spectrometer. An application will be demonstrated using the Thermo Scientific Prelude SPLC™ system and the Thermo Scientific NG Endura™ MS to quantitatively analysis fourteen clinically relevant antidepressant drugs collected from dried blood spots.
Universal Calibration: Populations Don’t Lie, People Do
Assay calibration has proven to vary based on any one component in the manufacturing of materials. Standardization programs have been implemented in an effort to ensure accuracy of methods across laboratories. As the acceptance criteria for these programs can be stringent (< 10% bias) all areas for potential error need to be explored. These methods of calibration rely on gravimetry, and consideration of the quality of material and are subject to compounding errors. This paper we propose the expanded use of “clinical calibration” where multiple reference intervals are expected, together with the use of normal population mean for assay calibration.
Of Knockout Mice and Men: Comparison of Vitamin D Metabolomes in CYP24A1 Mice and Patients with Loss-of-function Mutations of CYP24A1 (IIH) by LC-MS/MS
LC-MS/MS has emerged as a sensitive, specific tool for studying vitamin D metabolism in animal models and humans. We have applied this method to sera from CYP-knockout mice and patients with rare genetic defects in vitamin D metabolism. We report data for 25-OH-D3, 24,25-(OH)2D3 and 3-epi-25-OH-D3 in samples from mice and men with ablation or loss-of-function mutations of CYP24A1, the enzyme which degrades both 25-OH-D3 and 1,25-(OH)2D3. The animals/patients show low levels of 24,25-(OH)2D3 indicating that they cannot clear vitamin D at the normal rate. We conclude that LC-MS/MS of vitamin D offers valuable diagnostic information to the clinician.
Metanephrine and Normetanephrine Quantification in Human Plasma by SPE-LC-MS/MS
Metanephrine (ME) and Normetanephrine (NME), are routinely screened in both urine and plasma serving as the best biochemical markers for Pheochromocytomas. However, measurement of metanephrines and normetanephrine in plasma is challenging due to low physiological concentrations, the hydrophilic nature of the compound1, and time consuming sample preparation requirements. This paper outlines a high sensitivity SPE LC-MS/MS method for the quantitation of Metanephrine and Normetanephrine in plasma using IONICS 3Q 220 triple quadrupole mass spec. Results achieved by this method include an R2 value of > 0.996 over the 0.005 to 1ng/mL concentration range. The LOQ’s achieved by this method for Metanephrine and Normetanephrine were 0.005 and 0.01pg/μL respectively.
Free Thyroid Hormones by Ultrafiltration and 2D-LC-MS/MS, Reinvented
We redeveloped and validated a simple, fast and sensitive method to accurately quantify both FT4 and FT3 in serum by UF and 2D-LC-MS/MS, with a LLOQ of 1 pg/mL and a chromatographic run time of <3 min. This method utilizes the UF procedure that produces comparable results to the ED procedure, and provides better turnaround time that should better serve routine clinical laboratory use
Method for Multiplex Analysis of Testosterone and 5 Chemical Castration Drugs Using UPLC-MS/MS
We developed a method for simultaneous measurement of plasma testosterone and 5 chemical castration drugs such as cyproterone acetate, medroxyprogesterone acetate, goserelin, leuroplide and triptorelin by using UPLC-MS/MS. Sample preparation using methanol extraction yielded good recovery and ionization efficiency, with chromatographic separation achieved within 12 min/sample. Within-run imprecisions were 2.8–8.2%. LLOD and LLOQ were 0.0312 and 0.125 ng/mL for testosterone, and 0.25 and 1.0 ng/mL for other drugs. In a limited pilot study, human samples showed variable serum concentrations for each drug. The performance of our devised method was generally acceptable which enables effective drug monitoring during treatment.
High Throughput Reliable Quantitation of 25-hydroxyvitamin D in Serum by Offline Sample Preparation and a LC-MS/MS Instrument
Increased clinical awareness of the prevalence of vitamin D deficiency and insufficiency has led to a need for large volume 25-hydroxyvitamin D (25(OH)VD) test. LC-MS/MS method has emerged as a reliable method of choice for high throughput clinical laboratory due to its high specificity and high accuracy at low ng/mL level. Present study proposes a simple and fast off-line sample preparation and direct injection of filtered serum sample directly into IONICS 3Q 200 series triple quadrupole MS system for fast analysis.
Reasonably good recovery rate of ~60% was found for 25(OH)VD using Phospholipid Depletion kit. The LOQs obtained are 0.25 and 0.5ng/mL for 25(OH)VD3 and 25(OH)VD2, respectively. Good linearity was also found over a board range of concentration.
Quantitation of Glutathione and Related Thiols in Acid-Preserved Samples by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry
Glutathione (GSH), the most abundant non-protein thiol in the body, is important for protection from oxidative damage. Determination of GSH, and its oxidized form (GSSG), provides an important indicator of redox status of cells, tissues and organisms. Samples collected for analysis by an established LC-fluorescence method were preserved with perchloric acid. Ammonium bicarbonate neutralized the acid, yielding a pH compatible with NEM derivatization of free thiols and producing MS-friendly samples. Good results were achieved for GSH and cysteine, but responses for the oxidized forms, GSSG and cystine, were non-linear. Each required a different solution in sample preparation, chromatography, and/or MS conditions to yield linear calibration curves.
High Sensitivity Assay for Bis-Ketosteroids Using Derivatization and LC-ESI/MS/MS
Herein presented a highly sensitive LC-ESI/MS/MS method for the simultaneous analysis of 8 bis-ketosteroids which are used as biomarkers for adrenal gland disorders. The method involves extraction of 200 µL serum by Solid Liquid Extraction with diisopropylether and derivatization with quaternary aminooxy reagent. LC-ESI/MS/MS analysis is performed using a reverse phase column, H2O/Acetonitrile/Formic acid gradient and QTRAP®5500 LC/MS/MS (AB SCIEX, Framingham, MA) operating in Multiple Reaction Monitoring (MRM) mode. Unique fragments are selected for the MRM transitions of each bis-ketosteroid. Quantitation of the derivatized bis-ketosteroids is enabled by using an isotopically enriched Internal Standard. Limit of Detection values were <1 pg/mL.
Sensitive Quantitation of a Steroid Panel in Urine by LC-MS/MS Using the AB SCIEX Triple QuadTM 6500 System
The measurement of steroids in urine can allow researchers to identify potential endocrine dysfunction in humans. Presented here are two LC-MS/MS methods optimized for the detection of low levels (pg/mL) of steroids in human urine. Analysis of progesterone, testosterone, cortisol, DHEA, pregnenolone and androsterone was performed using positive electrospray ionization, while the remaining steroids (estriol, estradiol, estrone, aldosterone) were evaluated using negative electrospray ionization. The steroids considered were found to have a limit of quantitation ranging from 2 – 100 pg/mL. The sensitivity of the two methods allows the analysis of steroids in urine over clinically relevant concentration ranges.
Analysis of Bile Acid Profiles in Patients with Necrotizing Enterocolitis (NEC) by Liquid Chromatography-Tandem Mass Spectrometry
We have implemented a liquid-chromatography tandem mass spectrometry method (LC-MS/MS) for the separation and detection of bile acids in plasma/serum and stool samples. A C18 analytical column is used for bile acid separation. Detection of bile acids is performed in negative ion mode by multiple reaction monitoring (MRM) on a 4000 QTRAP (ABSCIEX) mass spectrometer. Seventeen bile acids were monitored and detected in a bile acid standard mixture within a twelve minute analysis time. The same LC-MS/MS method was applied for the analysis of bile acids extracted from plasma, serum and stool samples of patients with necrotizing enterocolitis (NEC).
Mixed Mode Solid Phase Extraction UPLC/MS/MS Method for the Analysis of Serum Testosterone for Clinical Research
Here we evaluate a UPLC/MS/MS method to measure serum testosterone at concentrations within the adult male and female range for clinical research purposes. An analytically sensitive method with minimal matrix effects was developed using a mixed-mode Solid Phase Extraction (SPE) sorbent in 96-well plate format, allowing researchers to prepare samples for analysis through either a manual or automated process. Thus providing flexibility in sample preparation depending on the laboratory environment. The method demonstrates good linearity, precision (< 6% CV) and accuracy (< 10% bias) with minimal ion suppression.
For Research Use Only. Not for use in diagnostic procedures.
Comparison of Liquid Chromatography High-Resolution Mass Spectrometry and LC-MS/MS for Quantifying Serum Testosterone
LC-MS/MS methods on triple quadrupole based mass spectrometers have become a mainstay for quantification of steroids in reference clinical laboratories. We compared the performance characteristics of serum testosterone assay using LC-MS/MS method (Applied Biosystems API 5000) with a LC-HR-MS method (Q-Exactive quadrupole-orbitrap mass spectrometer).
Testosterone concentration in serum samples (N =72 males, ages 18-90) was measured using LC-MS/MS and LC-HR-MS methods.
Method comparison between LC-HR-MS and LC-MS/MS gave an r2 of 0.93 (TTSTLC-MS = 1.10 *TTSTLC-MS/MS + 20.87). The inter-assay imprecision on LC-HR-MS method was 2.97 % at 1093 ng/dL, 6.12 % at 360 ng/dL.An inter-assay imprecision of 6.7 % at 800 ng/dL and 5.9 % 100 ng/dL on the LC-MS/MS method was noted. The limit of quantitation (LOQ) for the LC-HR-MS method was 50 ng/dL and 5 ng/dL using the LC-MS/MS method.
Use of Online Multidimensional Separation Coupled to Mass Spectrometry in Protein Profiling of Formalin Fixed Paraffin Embedded Lung Cancer Tissues
Formalin fixed paraffin embedded (FFPE) tissues were used in this study to analyze the tissue proteome of lung tumor samples. Multidimensional liquid chromatography (LC) coupled to tandem mass spectrometry (MS) was used in this study to explore changes at the protein level in lung tumor tissues. The enhanced separation by liquid chromatography was used to improve characterization of the complex tissue proteome to discover protein level changes across a wide dynamic range.
Quantitative Analysis of Five Tobacco-specific N-nitrosamines in Urine by Liquid Chromatography–atmospheric Pressure Ionization Tandem Mass Spectrometry
A liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of five tobacco-specific N-nitrosamines (TSNA), including free and conjugated forms, in urine. The limit of detection for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N’-nitrosoanatabine (NAT) and N’-nitrosoanabasine (NAB) were 0.6, 0.6, 10.0, 0.4 and 0.4 pg/mL respectively, with a linear calibration range of up to 20,000 pg/mL. Intra- and inter-day precision for TSNA measurements ranged from 0.82% to 3.67% and 2.04% to 7.73%.The validated method was then applied to urine samples collected from 50 smokers and 30 nonsmokers. Our results suggest that this sensitive and relatively simple analytical method is suitable for measuring TSNA exposure resulting from exposure smokers.
Simultaneous Detection of 19 Drugs of Abuse on Dried Urine Spot by Liquid Chromatography-Tandem Mass Spectrometry
Lack of specificity in drugs of abuse testing by EMIT and CEDIA immunoassays, and concerns over its cost in conjunction with reflex confirmatory tests, prompted us to investigate the combinatorial use of dried urine spot and LC-MS/MS. The method development and validation were performed in accordance with the guidelines published by FDA and CLSI. While our method of high sensitivity and specificity provides an alternative diagnostic utility to immunoassays, it also offers superior solutions in specimen transportation, preservation, and storage. The benefits of our method are apparent in reducing turnaround time and test costs that result in better patient care.
Rapid On-site Screening of Illicit Drugs by Portable Mass Spectrometer
Abuse of illicit drugs is a global problem. To develop a reliable screening tests of illicit drugs, we developed a portable mass spectrometer about 10 kg in weight. The instrument consists of a low pressure dielectric barrier discharge ionization source and a compact linear ion trap mass analyzer. We also employed a discontinuous sample gas introduction technique that enhanced the ion transfer efficiency. Both liquid and solid samples can be measured with our mass spectrometer. In a test, our spectrometer detected 0.1 ppm methamphetamine in urine and 1 ng of methamphetamine powder.
Direct Analysis of Melphalan in Human Whole Blood by Paper Spray Ionization Using Mass Spectrometry
High dose melphalan is the common active agent used in the treatment of multiple myeloma, however, the optimal dose that produces optimal response with acceptable safety and devoid of toxicity is still unknown. We recently developed a direct, rapid, robust and simple method based on paper spray ionization mass spectrometry to determine melphalan levels in human whole blood in order to permit pharmacokinetically-guided dose individualization. Our results have shown that paper spray mass spectrometry shortens the workflow and decreases the complexity of MS quantification and has potential to significantly increase the availability of fast clinical test.
Evaluation of Three Quadrupole Time-Of-Flight Mass Spectrometry (QTOF) Platforms for Targeted Screening of Drugs and Metabolites
Liquid Chromatography Quadrupole Time-of-flight Mass Spectrometry (LC-QTOF) was investigated for targeted screening of drugs and metabolites. 19 spiked plasma and urine samples with a combination of over 80 compounds were analyzed by an AB Sciex 5600 Triple TOF, Agilent 6530 QTOF, and Waters G2-S QTOF. Instrument evaluation and data review were conducted at each vendor site. Despite differences in reconstitution of the samples, injection volume, chromatography, acceptance criteria and the library used for compound matching, all three platforms performed well, with 90% of expected compounds detected in most plasma samples, and 80% of compounds detected in urine.
Rapid and Simple Confirmation and Quantification of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic Acid (THC-COOH) in Urine by LC-MS/MS
Cannabinoids are among the most commonly detected compounds in toxicology screening programs and workplace drug testing. Urine is tested for 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) including its ester-glucuronide. Traditionally this has involved hydrolysis, SPE, evaporation, derivatisation and detection by GC-MS. We sought to replace this with a rapid 'dilute and shoot' LC-MS/MS method. To avoid sample transfer, alkaline hydrolysis was performed in the injection vial; the sample was then neutralised and injected into the LC-MS with a total runtime of 3 min. The assay was validated in the range 5-1000 ng/ml and matrix effects were minimal. The assay has been in use for >6 months analysing ~80 samples daily for medical screening and workplace confirmations to AS/NZS 4308:2008 standards. It has proven robust and reliable with intraday CV's of <10%.
LC-MS/MS Analysis of Ethyl Glucuronide in Urine: A Case Study in Ion Suppression
Ethyl glucuronide and Ethyl sulfate are metabolic conjugates of ethanol with either glucuronic or sulfonic acid. This validated 'dilute & shoot' method employs LC-MS/MS with negative ion electrospray. This presentation demonstrates the importance of matrix matched controls in routine analysis. An ion suppression event consistently occurred in submitted human specimens at the retention time for ethyl glucuronide. This event does not occur in synthetic urine, processed urine (i.e., filtered, frozen, diluted) or water. It is important that calibrators and QCs be prepared in appropriate, validated matrix to ensure an equivalent instrument response between control and patient samples.
Quantitation of 25-Hydroxyvitamin D2 and D3 in Serum and Plasma by Liquid Chromatography-Tandem Mass Spectrometry
Vitamin D deficiency is a common health problem worldwide. A liquid-liquid extraction and LC-MS/MS method was developed and validated for quantifying 25-hydroxyvitamin D2 (25OHD2) and 25OHD3 in serum and plasma over a linear dynamic range of 5-160 ng/mL. The validated assay demonstrated acceptable linearity (r2 values >0.99), intra- and inter-day imprecision (3.6-15.1%RSD), and bias (88.0-126.0%). Extraction efficiency was 76.5-110.5% and matrix effect ranged from -46.7 to -32.0%. Analytes were stable in authentic serum and plasma for 7 days at room temperature and 4°C. No significant difference in 25OHD3 concentrations was observed between serum and plasma. This method provides reliable and accurate measurement of 25OHD2 and 25OHD3 in serum or plasma for use in clinical practice.
Development of a high-throughput screening method for multiple 'bath salts' in urine by LC-MS/MS
'Bath salts' are designer drugs that are structurally and pharmacologically related to amphetamines. Recently we have seen more illicit use of bath salts and some new analogs are being introduced and replacing the older drugs. Therefore, a fast, sensitive, and comprehensive high-throughput screening method for multiple bath salts is needed. In this study, we present a quantitative LC/MS/MS method for simultaneous monitoring of 20 bath salts using AB SCIEX 5500 QTRAP® LC-MS/MS system. The method is fast, sensitive and allows for the analytical panel to be further expanded to accommodate newer bath salts.
Analysis of Synthetic Cannabinoids in Urine and Whole Blood for Forensic Toxicology
Synthetic cannabinoids, constitute a growing challenge for law enforcement agencies and forensic laboratories. Two methods are presented for the analysis of a variety of synthetic cannabinoids and metabolites in urine and whole blood using solid phase extraction and phospholipid removal plates, respectively. Analytical separation was achieved using a newly developed solid-core particle UHPLC column in under 7.5 minutes. The methods were shown to be linear, accurate, and precise across their calibration ranges. The universal nature of the extraction methods enabled the quantification of neutral, basic, and acidic compounds and their metabolites.
Successful Implementation of Serum/Plasma Voriconazole Levels Using Liquid Chromatography Mass Spectrometry (LC/MS/MS) at Children's Hospital Los Angeles (CHLA)
A very simple, fast, sensitive, in-house MRM method by LC/MS/MS was successfully implemented at CHLA for quantitation of serum/plasma voriconazole, which resulted in better optimization of therapeutic drug levels, decreased sample volumes, and significant cost savings. Average TAT was reduced from five to seven days to ten hours with annual volumes increasing from 400 to 1,200 and an annual cost savings of approximately $180,000. Measurement sensitivity has also increased with the LOD of in-house voriconazole now being 0.008 mcg/ml (previously reported as <0.5 mcg/ml). And finally, the minimum sample requirement was reduced from 1 ml of whole blood to 0.5 ml.
Evaluation of an Advanced Software Processing Tool for MS/MS Library Searching to Identify Compounds in Urine Toxicology Samples
LC/MS/MS analysis combined with automated library searching of MS/MS spectra for compound identification has emerged as a technique of choice for both screening and confirmation in forensic analyses. For laboratories performing analyses of large panels of compounds, a complete and simple software solution is required, offering an automated workflow to streamline data collection, processing, and reporting. However, it is also desirable to provide the user with the flexibility to interact with the data, and to investigate alternative library matches. In this study we evaluate the use of a new software tool that (i) provides automated library searching for the routine reporting of results, and (ii) allows the user the flexibility to interact with the MS/MS library searching results, to browse through the library entries, and to select additional spectral matches for reporting.
Quantitative Analysis of THC-COOH in Urine by GC-Ion Trap MS/MS: Selecting the Most Suitable Precursor Ion
In order to maximize sensitivity in MS/MS analysis, precursor ions are usually chosen solely on the basis of relative abundance. Starting from different, though predominant, precursor ions we compared two GC - MS/MS (ion trap) assays of THC-COOH in urine. The dynamic range and the precision were clearly better in the method based on the lesser abundant precursor ion. Our study shows that, aside from its abundance, the fragmentation pattern of the precursor ion determines the characteristics of the assay.
High-Throughput Quantitative Analysis of Tricyclic Antidepressants and Selective Serotonin Reuptake Inhibitors in Human Serum Using Ultra-Fast SPE-MS/MS
Steady increases in the need for greater analytical capacity and throughput have placed demands on common analytical technologies like HPLC or LC/MS/MS. An efficient, fast, accurate, and sensitive SPE/MS/MS method with a wide calibration range was developed for the simultaneous quantitation of analytes from tricyclic antidepressant and serotonin re-uptake inhibitor (SSRI) classes in human serum. This analytical method employs protein precipitation followed by analysis using a SPE/MS/MS system, enabling analysis in 13 seconds providing a throughput of greater than 240 samples per hour. Comparable accuracy, precision, and linearity results were achieved at rates 20-30 fold faster than traditional LC/MS/MS methods.
A Comprehensive Solution for Quantitation of Ketamine and Ketamine Metabolites in Urine Using LC-MS/MS and GC-MS/MS
The demand for fast ketamine screening continues to grow for forensic and clinical purposes. In the current work, a complete solution was developed for quantification of ketamine and two ketamine metabolites in urine matrix using Bruker EVOQ LC-MS/MS and Scion GC-MS/MS. The LC-MS/MS strategy features the easy “dilute-and-shoot” sample prep and fast analysis time (< 5min). Method robustness was evaluated with %RSD < 3% of all 3 compounds for 60 injections in different days. Alternately, the GC-MS/MS method works as an independent drug confirmation, and is an orthogonal tool to LC-MS/MS for comprehensive drug quantitation. This work demonstrates the LC-MS/MS and GC-MS/MS platforms are both feasible technologies for fast quantitation of ketamine in urine matrix. It provides the solution for comprehensive ketamine quantitation with cross confirmation required by forensic purposes.
Metabolomic Analysis of the Toxicity of Acute and Chronic Pesticides Exposure in B50 Cultured Neuroblastoma Cells
Cultured rat neuroblastoma B50 cells were exposed to the neurotoxic insecticides permethrin and malathion for varying doses and times, in order to determine both acute and chronic effects. Cell damage was assessed by morphological and biochemical analysis and resultant metabolite analysis was performed using gas chromatography-mass spectrometry. Metabolites that most contributed to the variance between treated and control cells were identified using principal component analysis. This analysis indicated that cells damaged by pesticide could be grouped according to their metabolite profile. The metabolites detected were further investigated for changes in trend following exposure, and a proposed pathway of neurotoxicity determined.
Ketamine Metabolites, Tramadol and Z-drugs in Urine by LC/MS
Several unrelated drugs of abuse not detected by routine immunoassay screens are merged into a single LC/MS assay based on commonalities of metabolism and sample preparation. We present a simple method for zopiclone, zolpidem phenylcarboxylic acid, zaleplon, tramadol and ketamine (and its norketamine and dehydronorketamine metabolites) in human urine, where sample is precipitated with 9:1 acetonitrile/methanol and analyzed by reverse-phase LC/MS. Calibration curves were linear to at least 50x the lower limits of quantitation, which were 1-5 ng/mL for most compounds. No significant carryover was observed at 2500 ng/mL for any compound. Within-run and between-run precision was <10% and 12%, respectively, using samples prepared from canine (ketamine group) or human (all others except zaleplon) post-ingestion urine. The detection window for zopiclone, zolpidem and tramadol was 2 days.
The Separation and Analysis of Opiates and their Glucuronide Metabolites in Urine Matrix Negating the Need for Hydrolysis Sample Preparation Using Tandem LCMS
Opiate glucuronides pose a great separation challenge in reverse phase chromatography due to their highly polar nature and are, therefore, routinely hydrolyzed during sample preparation, a process that removes any sugar group returning the metabolite to its original parent form.
Acid and Enzyme hydrolysis stages of sample preparation can be relatively lengthy processes so this research project set out to investigate the scientific feasibility of separating and measuring common opiates, metabolites and glucuronide metabolites individually and in one quick 4 minute analytical method.
Results were obtained for five batches of spiked urine samples over the concentration range of 0.5-1000ng/ml
Advantages of Ultra High Resolution Mass Spectrometer QExactive in Analysis of Unlimited Number of Compounds in Urine Quantitative Screening Application.
Implementation of ultra high resolution mass spectrometers for quantitative toxicology screening applications allows for unlimited number of analytes, short acquisition times and simple sample preparation. At the same time, ultra high resolution mass spectrometry provides high confidence in reported hits.
We developed a quantitative screening method for analysis of unlimited number of compounds in urine samples. The method was validated for 45 representative compounds including opioids, benzodiazepines and amphetamines.
Obtained LOQ’s and method precision met or exceeded application requirements. The dynamic range was 3-4 fold for all tested analytes. Limited matrix effects in donor samples were observed.
A Novel Separation for Ethyl Glucuronide and Ethyl Sulfate Using a Multi-Mode Reversed-Phase Column
Ethyl glucuronide and ethyl sulfate were separated using a multi-mode C18 column. The analytes were eluted of the column using an ammonium formate/acetonitrile gradient mobile phase. The separation was primarily affected by the buffer pH and salt concentration. Increasing the pH caused a decrease in retention time for both analytes. In addition, changing the composition of the organic modifier affected the retention of ethyl glucuronide and ethyl sulfate. The greater retention of ethyl glucuronide and ethyl sulfate was achieved using this system compared to traditional reversed-phase methods.
The Analysis of Cannabinoids and their Metabolites in Human Urine by LC-MS/MS
The determination of cannabinoids and their metabolites, from a natural or synthetic source, has become routine in many forensic toxicology laboratories. The optimization of analysis time, resolution between metabolites, and method robustness is of ultimate importance when developing an efficient method for validation. The Raptor™ Biphenyl column combines the speed of superficially porous particles (SPP) with the resolution of highly selective USLC® technology to produce simple dilute and shoot methods with analysis times of less than 7 minutes for cannabinoids and their metabolites in urine.
“Dilute-digest-and-filter” Urine Analysis for Rapid Screening and Quantitation of 18 Benzodiazepines Using LC-MS/MS System
Screening and quantitation of benzodiazepines is growing in demand for both clinical and forensic applications. A “dilute-digest-and-filter” LC-MS/MS strategy was developed for benzodiazepine analysis in urine in this work. A mild glucuronide hydrolysis was used to avoid destruction of benzodiazepine. Urine samples were directly injected after a quick filtering cleanup. A 4-min LC condition was developed to realize fast turn-around time. The calibration range was from 25 to 2500 ng/mL which covered the expected range for routine clinical and forensic needs. The linear response of benzodiazepines is all above 0.996 and good accuracy (<15%) was achieved. The validated LC-MS/MS platform maintains excellent sensitivity and reproducibility running thousands of samples without diverting or source cleaning, which satisfies the high-throughput demands of a general clinical/forensic laboratory.
A Method for the Determination of Desomorphine, Heroin, Methadone, Buprenorphine and Metabolites in Urine Using LC/MS QQQ
Analytical measurement of opioid panels commonly include a number of compounds such as heroin, methadone and buprenorphine. Research methods with sufficient flexibility are of value to respond to the need to measure new compounds resulting from clandestine production. Desomorphine, also known as Krokodil, is an emerging synthetic opioid, sometimes used as a heroin substitute.
This poster describes the development of an LC/MS QQQ research method that rapidly quantifies desomorphine together with heroin, buprenorphine, methadone and their metabolites 6-acetylmorphine, norbuprenorphine and EDDP over a concentration range of 1-5000ng/ml. Precision data, LLOQ and calibration linearity will be reported for each analyte.
Quantitation of (-)-Δ9-Tetrahydrocannabinol (THC) in Dried Bloodspots Using LC-MS/MS
This paper outlines a simple, three step extraction method requiring no SPE or evaporation step for the analysis of THC in dried bloodspots(DBS) using IONICS 3Q 210 triple quadrupole mass spectrometer. 4 uL of sample were extracted from the dried bloodspots and used to create 6 calibration curves. The R2 average obtained over these 6 runs was >0.999, while central variance (CV) was less than 5% at 1 ng/mL. The average signal to noise obtained over the runs was 17. Overall, this method provides the sensitivity, accuracy and rapid sample preparation required to perform the routine analysis of THC extracted from dried bloodspots in a clinical research laboratory.
Simultaneous Analysis of Seven Amphetamine Class Drugs in Urine for Forensic Toxicology
In many drug testing laboratories, amphetamines and their derivatives are screened for, and identified by, testing a urine sample from a suspected user. The analysis of these compounds can be done by GC or LCMS. In the case of LC/MS analysis, simple dilution of the urine sample is typically the only sample preparation method employed. However, as urine is a sample matrix containing many potential interfering compounds, this type of relatively crude sample preparation can lead to issues in analysis. Potential matrix interferences from urine can compromise method sensitivity, and allow potential cross contamination contribute to an increased instrument maintenance burden. These interfering compounds can also compromise the effectiveness of an analytical LC column very quickly.
In this effort, a forensic toxicology method incorporating an online SPE system (ACQUITY Online SPE Manager-OSM) with LC/MS for analysis of a group of amphetamine class compounds in urine was developed. Results from the method utilizing SPE for sample preparation were compared with those obtained from results obtained using urine samples that were simply diluted before LC/MS analysis. Results were compared in terms of background signal, matrix interferences and analytical sensitivity for amphetamine drugs.
A Workflow-driven High-throughput Screening Method for Synthetic Cannabinoids and Metabolites in Urine with Q-TOF Mass Spectrometer and Multiplexed LCs
Synthetic cannabinoids are a major health problem and a rapid screening test is needed. Here we present an easy workflow for LC-MS/MS-based high-throughput screening method for multiple synthetic cannabinoids/metabolites in urine on a Q-Tof mass spectrometer and multiplexed LCs. Analyte quantification was performed with molecular ions and screening results were confirmed in the same run with mass accuracy, retention time, isotopic ratio and MS/MS matching using MasterView software. The final injection-to-injection cycletime was ~2 min. This workflow offers flexibility for adding new compounds, with higher sensitivity and specificity than immunoassays and could be easily utilized for other designer drugs.
LC-MS/MS Method for Quantitative Analysis of Mycophenolic Acid and Two of Its Major Metabolites in Serum
Mycophenolic acid (MPA) is the active metabolite of the immunosuppressant drug mycophenolate mofetil, which is commonly prescribed after organ transplantation. MPA therapy is monitored to balance therapeutic efficacy, to prevent organ rejection, while minimizing the adverse effects associated with high concentrations. We developed a 2.5 minute LC-MS/MS method used for the quantification of MPA and its clinically relevant metabolites mycophenolic acid glucuronide (MPAG) and mycophenolic acid acyl-glucuronide (AcMPAG), in serum. Chromatographic separation and validation data including internal and external method comparison are presented.
Increased Throughput for the Analysis of Delta-9-THC in Oral Fluids Using Triple Quadrupole Mass Spectrometry Coupled with an Automated Dual-Channel HPLC System
In this work we demonstrate increased mass spectrometer productivity through the automated use of a dual channel high performance liquid chromatography system. The integrated LC-MS/MS system is comprised of a triple quadrupole mass spectrometer coupled to a configurable dual HPLC system, all controlled by a single software. The analysis of delta-9-THC from oral fluids was used to demonstrate capabilities of this system. The new HPLC system mirrors certain components of this single stream system to provide a second stream, operating in parallel to the first stream. By staggering injections on parallel streams, throughput can double as compared to standard method.
A Highly Sensitive Quadrupole GC-MS for Quantitative and Qualitative Toxicology
Extraordinarily high sensitivity was observed with the new Thermo Scientific ISQ quadrupole GC-MS. A study was performed to evaluate the sensitivity of the ISQ, and determine its effectiveness for unknown toxicology screening via comparison with the Thermo Scientific ITQ ion trap platform. The signal/noise ratios of drug standards were compared, and then commercial controls and proficiency test (PT) samples were analyzed. The ISQ quadrupole analyzer exhibited higher sensitivity than the ion trap instrument. Identical drugs were detected by both instruments from extracted samples. The sensitive ISQ quadrupole analyzer allows the use of a single instrument for quantitative and qualitative analyses.
Tandem GC-MS/MS Comprehensive Drugs Screen of Biological Fluids by Full Scan (untargeted) and MRM (targeted)
This is a highly sensitive, comprehensive drug screen on the Bruker Scion GC-MS/MS instrument in biological fluids. A full scan method was developed for screening and a retention time (RT) matched confirmation MRM method for quantitation. 150+ compounds were included in various drug classes. Chromatographic conditions permitted anlysis of both basic and acid/neutral drugs in one method. Two MRM transitions were developed for each compound. Ion ratios and MRMs were combined into a single method with the same conditions to allow RT matching with the full scan method. Metycaine and Cyclopal were Internal Standards and solid phase extractions. Optimized parameters (transitions, RT and collision energy) for 150+ compounds are presented. Method validation (LODs, recovery and matrix effects). Performance was evaluated using 100 positive patient urine samples and compared to LC-MS/MS.
Simultaneous Targeted and Non-Targeted Drug Screening in Urine Samples Using a Q-TOF Mass Spectrometer and Software Tools for Confident Compound Identification
Laboratories performing drug screening analyses often wish to identify as many compounds as possible from a single experiment. The complexity of the analysis and the data interpretation increases with the number of target compounds, and this is compounded further when the lab also wishes to identify “unknown” compounds. A complete solution for simultaneous targeted and non-targeted screening has been elusive, requiring either a duplicate injection or at least multiple passes of data processing. In this study we show a comprehensive targeted and non-targeted sample analysis for a set or urine toxicology samples, using the AB SCIEX TripleTOF® 5600+ high resolution Q-TOF mass spectrometer. Spectra generated in these experiments are searched against a library of over 1200 compounds and linked to an automated ChemSpider session to help identify non-targeted compounds in the samples.
Measuring Hepcidin-25 in Serum Patients: Is LC-MRM Ready for Clinical (absolute) Quantitation?
Since its discovery, Hepcidin has generated many hopes in terms of diagnostic. However, its accurate quantification for clinical use remains so far cautioned by the limited sensitivity, specificity or reproducibility of the techniques described.
In the present work, we measured hepcidin in the serum of 30 patients suffering from various iron-related diseases by two methods: a competitive C-ELISA and a highly specific and quantitative mass spectrometry method (MRM). The results obtained allowed us to validate our MRM method and to propose it as a suitable alternative method for accurate determination of hepcidin in blood sample at the clinical laboratory.
Our Journey of Building an LC-MS Program for Clinical Testing: The Cases of Iothalamate Quantification and Amyloid Identification
Our group started building a clinical LC-MS program in 2006 and has established an infrastructure for method development, validation, and clinical service. To illustrate the valuable experience, we will present our journey to the successful initiation and implementation of LC-MS in the clinical lab. To demonstrate the unique advantages that the LC-MS technology may bring to patient care, two novel assays will be particularly discussed with one measuring non-radioactive iothalamate for glomerular filtration rate determination and the other subtyping amyloid proteins.
An Ultra-sensitive Assay for the Detection of Serum Oestrogens
We are developing an ultra-sensitive LC-MS/MS based assay for the detection and quantification of very low concentrations (below picomole/L) of oestrogens in biological samples with an aim of routine clinical application for selection and evaluation of appropriate breast cancer treatments for post- and peri-menopausal women. A number of derivatisation methods have been evaluated, including dansyl chloride, Girard’s reagent P, and picolinic acid. Preliminary data obtained using these approaches will be reported and optimisation strategies in terms of sample preparation, derivatisation and process automation will be discussed.
Application of Untargeted and Targeted Metabolomics to the Study of Liver Disease Caused by a Deficiency in HSD3B7-diagnosis and Response to Therapy
9 genetic defects in bile acid biosynthesis pathway which cause various degree of liver injury in patients were identified and described. HSD3B7 deficiency is the most commonly occurring defect and can lead to morbidity or mortality in patients if not diagnosed and treated. By applying untargeted metabolomics platform to analyze normal and patient urine samples, we were able to confirm these biomarkers, namely Δ5-cholen-24-oic acid derivatives in patient urine. These signature compounds were then obtained with sophisticated multistep synthesis and used to set up a sensitive, robust analytical assay enabling accurate quantification. Data from two systems were highly correlated and the combined approach proved to be very effective in facilitating diagnosis of the deficiency, monitor and assessment of therapeutic response.
Introducing Liquid Chromatography - Mass Spectrometry into the Clinical Laboratory: Using Vitamin D Testing as a Practical Example
Five years ago our vitamin D testing-associated reference laboratory cost increased exponentially, therefore we developed an LCMSMS-based method to decrease our expenditures. Our in-house assay is standardized to NIST reference material and its performance exceeds that of the commercial vitamin D immunoassays. Start up cost and learning curve were steep but return on investment was less than 12 months. Our largest obstacles were tech training and maintenance scheduling while keeping production constant. We now perform testing 5 days/week with two technologists, producing 20,000 patient results annually. We also expanding our test menu and installing a new LCMSMS.
Plasma Citric Acid Cycle Intermediates Levels by GC-MS and LC-MS/MS - A Prospective Application in Diagnosis of Mitochiondrial Disorders
We developed a GC-MS and LC-MS/MS methods for quantification of plasma citric acid cycle (CAC).Our assay was applied to measure plasma levels of seven citric acid cycle intermediates in human plasma of control patients and patients with known and unknown causes of mitochondrial dysfunction. Our test is a simple way to detect mitochondrial dysfunction and also provides otherwise unappreciated, important information about the effect of specific mitochondrial defects on the function of the citric acid cycle, which in turn offers insights into pathophysiology and possible therapeutic measures.
Application of Mass Spectrometry to the 24/7 Pharmacology and Toxicology Laboratory: Far Better Than Immunoanalysis!
Since 2005, our laboratory has evaluated the feasibility and then accomplished the replacement of automated immunoanalysis by liquid chromatography routinely coupled with mass spectrometry in all the activities of the Pharmacology and Toxicology laboratory. As a technique of reference, it permits the development of routine, recourse specialized activities and of clinical and fundamental research programs.This presentation describes the project steps: preliminary studies and technical, scientific, financial and human costs and benefits of the operation over the past seven years of 24/7 operation. The LC MS/MS coupling allows us to respond to the large majority of routine and specialized analyses performed in the laboratory.
Tear as a Diagnostic Fluid
Biochemical approaches to disease diagnosis and management based on mass spectrometry are increasingly common, but most activity is centered on LC-MS (and LC-MS/MS). We have developed a practical, reproducible and high throughput MALDI-based approach to tear fluid analysis and we are exploring its potential in clinical chemistry. Our efforts are focused on identifying and quantifying individual peptides and proteins the correlate with disease or outcomes.
Relative Quantitation of Diverse Classes of Lipids Using a Single Step Extraction Protocol and Targeted LC-MS/MS: Ready for Clinical Application
Lipids comprising sphingomyelins, intact and glycated ceramides and phosphatidyl choline play key role in processes that lead to myocardial infarction and other cardiac diseases. Unique signatures of these lipids in plasma, erythrocytes and HDL could aid in predicting the course of cardiovascular events such as myocardial infarction and inform proper course of therapy and reduce cost. The technique employed must co-analyze a large number of lipid species, for a large number of patients, and be thoroughly characterized for clinical application. Most established assays require extensive sample preparation and the use of multiple chromatographic phases precluding its routine clinical use. We have developed and characterized a single step extraction and separation assay for co-analysis of over 200 lipid species by mass spectrometry from plasma, RBC and HDL. A pilot study of retrospective samples demonstrated that specific erythrocyte membrane lipid species were higher in patients with sudden cardiac arrest than in healthy study subjects.
The Importance of Collaborations Between Private and Public Health Laboratories in Response to Designer Drugs of Abuse
The increasing emergence of synthetic drugs is a difficult challenge for public health officials, clinicians, and law enforcement. The collaboration between Cayman Chemical Co. and researchers at the Arkansas Public Health Laboratory has characterized biomarkers for the development of validated human specimen testing. This collaboration has produced peer-reviewed publications, educational presentations, and development of metabolic reference standards for clinical testing. Importantly, the Center for Drug Detection and Response has been established to continue this critical partnership to continue to support clinical testing and impact legislative policy.
Monitoring Cocaine Abuse Using LC-Tandem MS in an Addiction Medicine/Psychiatry Setting: How Does Use of Oral Fluid Compare with Urine Immunoassay Testing?
We compared cocaine-related test results on 401 simultaneously collected urine and Oral Fluid (OF) sample pairs from 257 patients in addiction medicine/psychiatry clinics. The Orasure Intercept device was used for sample collection. OF was tested for cocaine (COC) and benzoylecgonine (BZE) with a 2.0 ng/ml LOD using LC/MS/MS. The OF was judged positive if either COC or BZE was found. Urine was tested using a Roche KIMS immunoassay with an LOD of 150 ng/ml BZE. Where needed, urine results were confirmed using GC-MS. OF and urine results agreed 377 times (345 negatives and 32 positives). There were 24 discrepant pairs: in 22 cases OF tested positive and urine negative, and in 2 cases OF was negative and urine positive. In 11 of the 22 OF positive-urine negative cases, only COC was present. Overall we found OF detected more cocaine abuse than traditional urine testing.
The Development of a Multiplexed Mass Spectrometry-based Test to Identify and Monitor Kidney Disease in Paediatric Fabry Disease Patients and Those Most at Risk
Nephropathy remains a major Type 1 Diabetes Mellitus (T1DM) and Fabry disease (FD) related complication. Whilst current diagnosis is based on albuminuria and creatinine levels, novel biomarkers are necessary for earlier detection when treatment may be more beneficial. A targeted, multiplexed, UPLC-MS/MS assay was developed to validate 25 protein biomarkers in urine samples from control, T1DM and FD patients (n=10/group). Ganglioside GM2 activator protein, cubilin and megalin were found significantly altered (<0.05) in paediatric FD and T1DM groups compared to controls. This triple quadrupole-based assay could be translated into a 10min clinical test to identify and monitor pre-symptomatic kidney disease.
Non-Invasive, In-Situ Screen for Celiac Disease and Disease Management
The diagnosis of celiac disease (CD) is dependent on observing structural changes and abnormalities in the intestine epithelium. We report on MS analysis to develop a non-invasive screening method for individuals suffering from gluten. The cholesterol-lowering drug Simvastatin (SV) is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in the small intestinal epithelium, which is highly expressed near the tips of the villi. This provides an effective means to monitor the health of CD patients by monitoring SV and its metabolites following oral administration. Dr. Khosla and coworkers published a seminal clinical study demonstrating the effectiveness of this approach. In this work we present the latest results employing liquid chromatography/mass spectrometry (LC/MS) as the key enabling diagnostic tool for CD screening and disease management.
A Novel Method for Monitoring Therapy and Guiding Treatment Decisions in a New Era of Anticoagulants
A number of orally administered anticoagulants have recently been introduced to the market and hold the promise of overcoming some of the disadvantages associated with traditionally used drugs. While these new anticoagulants do not require routine monitoring due to predictable pharmacological profiles, there are specific situations where detecting and quantifying them may be required for clinical decision making. We have developed a simple and sensitive method to quantify four oral anticoagulants simultaneously by LC-MS/MS from plasma samples. This method provides clinicians with a novel way to monitor anticoagulant therapy and guide treatment decisions in the new era of anticoagulants.
Critical Aspects of Bioanalytical LC-MS Method Validation - Analyte Stability and Method Robustness Studies
Over the past 15 years liquid chromatography mass spectrometry (LC-MS) has evolved from a scientific curiosity into a routinely applied technique finding increasingly more use in the routine analysis laboratories. Almost all fields of chemical analysis (biochemical, medical, environmental, food, drug discovery etc) have experienced big changes. Method validation is a key activity in chemical analysis, indispensable for obtaining reliable results. The higher the complexity of the method the more important is the validation procedure.
The regulations in the field of bioanalytical method validation have been revised recently in Europe and USA (the new EMA and FDA guidelines have been published). Although the validation guidelines have been updated for the detection method (LC-MS), several aspects on validation procedure have been left aside.
Determination of Urine Creatinine via Tandem Mass Spectrometry to Normalize Results in a Production Biomonitoring Setting
Urine creatinine correction is an important tool used to normalize human exposures to environmental contaminants and is becoming more widely used in public health biomonitoring. The Washington State Department of Health Public Health Laboratories developed a tandem mass spectrometry method using positive electrospray and an isotopically labeled creatinine internal standard to quickly and accurately measure creatinine without the hazards and interferences of working with picric acid. Many of the environmental contaminants prioritized by the Centers for Disease Control and Prevention for biomonitoring are also analyzed by mass spectrometry. This method eliminates a separate creatinine instrument, simplifying implementation of clinical projects.
LC-MS/MS Quantification of Sirolimus: A Comparison of Internal Standards
LC-MS/MS assays have been adopted in order to increase specificity in the measurement of immunosuppressant drugs such as sirolimus. However, the variability associated with these methods remains a cause for concern. Since accurate quantification is highly dependent on the internal standard (IS) used, the chosen IS should be as structurally and chemically close to the target analyte as possible. This study evaluated the performance of a deuterated rapamycin IS (rapamycin-d3) against two alternative internal standards: desmethoxyrapamycin (DMR) and tacrolimus-d2. The comparison study showed that all three IS options are viable candidates for quantification of sirolimus by LC-MS/MS.
Multi-Center Evaluation of the Prelude™ SPLC-MS/MS System for Immunosuppressant Drug Analysis
LC-MS/MS methods for immunosuppressant drug (ISD) analysis have increasingly adopted on-line extraction utilizing 2-dimensional chromatography for more effective sample 'clean-up'. The Prelude™ SPLC system is a 2-channel on-line extraction platform that utilizes turbulent flow technology coupled with MS/MS. We describe the multi-center evaluation of the Prelude™ system for measurement of ISDs at 3 beta test sites. The performance characteristics of the instruments were comparable between sites. Method comparison data showed acceptable correlation for each compound between instruments and with existing Legacy methods. In summary, this method is ideal for robust, high-throughput analysis of ISDs in a clinical laboratory setting.
LC-MS/MS: A Tool to Mitigate Interferences in Complex Matrices
Complex matrices and sample variability make biological samples analytically challenging. NIST investigated an unknown compound that interfered with the quantitative determination of 3-epi-25-hydroxyvitamin-D3 in DEQAS human serum samples, when analyzed by the NIST Reference Measurement Procedure. The compound was detected in differing amounts and not detected in all samples. A high mass accuracy mass spectrometer was used to determine the exact mass of the interfering compound and relevant fragments. The presence of the unknown was determined to be associated with the type of blood collection bag, which were extracted and further analyzed to identify the interferent.
LC-MS/MS Detection of Increased Androstenedione Levels in Patients Receiving Danazol Therapy
We report eight cases of significantly elevated androstendione (AND) levels following LC-MS/MS measurement in patients receiving danazaol for aplastic anemia. Samples were prepared and analysed as per previously published method. The use of a quantifier MRM transition and qualifier MRM for AND as well as the ratio of response of the two was used to identify AND. The ratio of AND to internal standard retention time for each specimen run was 1.00, confirming the identity of the peak. Values ranged between 464-1176 nmol/l (reference interval: female 0.59- 6.11; male :0.87-4.36) at 6 or 12 month follow- up post Danazol initiation.
Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Assay for the Detection of 9 Oral Antidiabetic Drugs in Plasma and Urine
Diabetes type II patients are frequently prescribed oral antidiabetics for the management of hyperglycemia. However, in overdose situations, life threatening hypoglycemia can occur. Detection of the presence of these medications allows for the correct identification of the underlying cause of the hypoglycemia from antidiabetic drugs versus other causes, such as insulin-induced or insulinoma. Here, we report the development and validation of a sensitive and specific LC-MS/MS assay for the qualitative detection of 9 oral antidiabetics of both sulfonylurea and meglitinide (glinide) drug classes. This assay can therefore be useful in investigating clinical cases of hypoglycemia of unknown origin, using plasma or urine.
Determination of Insulin-Like Growth Factor-1 in Plasma by HRAM LC/MS for Clinical Research
High Resolution Accurate Mass (HRAM) Liquid Chromatography Mass Spectrometry (LC/MS) is ideally suited for the rapid analysis of biomolecules. A highly sensitive and specific HRAM LC/MS method has been developed for the quantitation of Insulin-Like Growth Factor-1 (IGF-1). This method uses a simple sample preparation combined with an online sample cleanup procedure coupled to a high resolution accurate mass quadrupole time-of-flight mass spectrometer. The described method achieves the required functional sensitivity and is capable of quantitating IGF-1 over a sufficiently wide dynamic range at R2 > 0.999. Intra- and inter- day CVs are below 5 and 10% respectively.
Inter-Laboratory Comparison of Two Different Extraction Methods for Methylmalonic Acid Quantitation Using Liquid Chromatography Tandem Mass Spectrometry
We utilized LC-MS/MS in 2 different laboratories to evaluate and compare methods for methylmalonic acid (MMA) quantitation. In Method 1, we used a traditional liquid-liquid extraction where methyl-tert-butyl ether and phosphoric acid were used to extract MMA. Method 2 differed in that it utilized an anion exchange solid phase extraction automated in a 96-well format on a liquid handler. The imprecision studies were acceptable at both sites, with an imprecision <10%, as determined by CV and a 22 split sample correlation showed the values were not significantly different. These methods are comparable and offer diverse options for different size laboratories.
Simultaneous Analysis of Persistent Organic Pollutants in California Biomonitoring Study Using GC-MS/MS
Although many sample extraction methods coupled with gas chromatography (GC) technique for the analysis of POPs in human serum were developed in labs throughout the world, they were typically used to analyze only 1 or 2 classes of POPs using separate instrumental analyses between organochlorine and PBDE groups. Therefore, we successfully developed a simultaneous analytical method using automated solid phase extraction coupled with an Agilent GC-triple quadrupole mass spectrometer that can be applied to a large cohort study for human exposure and its negative health effects.
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