Translating Pre-Clinical Research to Clinical Patient Care™
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Addition of Solid Phase Extraction to Opiate Sample Preparation for UPLC-MS/MS
In this study, we are assessing the inclusion of solid phase extraction (SPE) in the preparation of patient’s urine samples for opiate testing using UPLC/MS-MS method. We found comparable recoveries of most opiates using Dilute and shoot (D/S) only versus SPE. The extraction efficiency for all compounds ranged from 83.5-110%. Minimal matrix effect was observed using SPE except for buprenorphine at the high creatinine concentration. Method comparison analysis using D/S versus SPE demonstrated good agreement with all correlation coefficients (R2) > 0.95. Given the current analysis, we do not find sufficient benefit in adding SPE to our opiate testing.
Column and Solvent Considerations that Contribute to the Success of LC-MS Analyses
Particle packed reversed phase columns are commonly used in LC-MS. Two significant developments in HPLC columns that offer advantages in clinical LC-MS applications will be presented: monolithic columns and hydrophilic interaction chromatography (HILIC). Monolithic columns offer faster analysis without requiring a dedicated UHPLC instrument, and have less matrix sensitivity with biological samples. HILIC columns efficiently separate polar hydrophilic compounds, which are not retained in reversed phase columns. Examples are nucleotides, peptides, metabolites and sugars.
The role of LC-MS solvents in the success of LC-MS analyses will also be discussed. It will be shown that contamination and impurities compromise data quality.
A Comprehensive Study for Validation of a LC-MS/MS Method for the Simultaneous Determination of Four Immunosuppressive Drugs in Whole Blood
In this study, we aimed to develop a rapid, sensitive, selective, and cost-effective LC-MS/MS method, to determine four immunosuppressive drugs in whole blood samples simultaneously.
Non-isotopic internal standards ascomycin (tacrolimus, sirolimus, everolimus) and cyclosporine D (cyclosporine A) were obtained from ClinMass® while whole blood controls and calibrators were obtained from ClinChek® (lyophilised) and ClinCal® (lyophilised), respectively.
Intra-day precisions were between 3.0-5.3%, 1.1-4.4%, 3.7-4.0, 3.1-6.3% and inter-day precisions were between 4.1-4.8%, 1.1-3.2%, 3.7-4.0%, 6.0-7.8 while the accuracy was varied from -6% to +10%, -7% to +4%, -6% to +5%, -4% to +11%, for Cyclosporine A, Tacrolimus, Sirolimus and Everolimus, respectively.
Using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry to Study the Distribution of Human Neutrophil Peptides in Tears
Natural antibiotics are produced as part of the innate immune response in plants and animals. Defensins are important biological antibiotics that are regulators of inflammation and play important roles in the immune response. Additionally, defensins contribute to the antimicrobial action of granulocytes and mucosal host defense in the skin against foreign bodies. Defensins are divided into many types according to their structures. One type known as human neutrophil peptides (HNPs) has a broad spectrum of antimicrobial activity and a high abundance in mammalian epithelia and granulocytes, especially in inflamed or infected tissues. This study explores the distribution and physiological mechanisms of HNPs in human tears.
Cortisol Measurement in Urine: LC-MS/MS Method Validation and Preliminary Clinical Application
In this study, we aimed to develop a rapid, sensitive and selective LC-MS/MS method for the determination of free cortisol levels in urine samples.
Cortisol, methanol and ammonium formate were purchased from Sigma, and d4-cortisol was purchased from Cambridge Isotope Laboratories. Mobile phases were 5 mmol/L ammonium formate and methanol. An Agilent 6420 tandem mass spectrometer was used in the positive-ion mode with an APCI interface. Quantitative analysis was performed in the MRM mode.
The intra-day and inter-day precisions were 1.9–4.5% and 2-5.4%, respectively while the accuracy was 103.4%. The method was linear from 2.0 to 500.0 ng/mL (r2) > 0.999). The limits of detection and quantification were 1 µg/L and 2 µg/L.
Differential Breast Cancer Glycosylation Detected by Gas Chromatography Nodal Glycan Analysis
Aberrant Glycosylation is a hallmark of tumor cells that has been profoundly observed in breast cancer. Breast cancer as a heterogeneous disease has been classified based on the glycoprotein reseptors ER,PR and Her2. We propose differential glycosylation is associated with different breast cancer subtype. To implement this concept we measure glycan content of the enriched plasma membrane of the breast cancer cell lines from each subtype. Glycan permethylation and then semi purification and enrichment is followed by GC-MS to detect glycan “nodes” of N-, O-glycans pooled together.
A LC-MS/MS Method for the Measurement of Testosterone Undecanoate and Dihydrotestosterone Undecanoate
We developed and validated LC-MS/MS method for measurement of TU and DHTU in serum for a Phase 2 pharmacokinetics study of orally administered TU in men. d21-TU and d21-DHTU were used as internal standards and serum sample processed by liquid/liquid extraction. The LC-MS/MS system used a Shimadzu high-performance LC 20 series system with an Applied Biosystems API 5500 with ESI source, operated in positive ion detection mode. Serum TU and DHTU levels were lower if the TU was administered without food. When administered with a meal, serum TU levels peaked at 4.4 hour which was followed by serum DHTU levels.
Selection of Internal Standards for LC-MS/MS Applications
Internal standards are utilized across a wide range of clinical mass spectrometry applications including therapeutic drug monitoring, newborn screening, endocrinology, and pain management testing. The ability of an internal standard to improve the accuracy of analyte quantitation depends upon proper internal standard selection and critical assessment and mitigation of the challenges that arise due to internal standard use. Examples will be presented to illustrate the importance of label placement, isotopic distribution and purity, impact of natural isotope abundance, cross talk, and scrambling.
Global Mapping of Nutrient Utilization by Untargeted Metabolomics
Metabolomics has revealed that the complexity of cellular metabolism exceeds that expected from conventional textbooks. By integrating stable isotopes with untargeted metabolomic technologies, we can now identify novel pathway connections and nutrient fates. A limitation of isotope-based metabolomics applied to the clinic has been that each experiment required both a labeled cohort as well as an unlabeled control cohort. Here, we introduce a database called isoMETLIN that enables the distinction of labeled metabolites so that patients can be analyzed without unlabeled controls. This resource reduces the number of patients needed per experimental analysis and allows multiplexing with numerous isotopic labels.
An Interactive Digital Pathway Map: A Resource for Interpreting Metabolomic Data
Interpretation of mass spectrometry-based metabolomic data in the context of biochemistry requires a high level of familiarity with metabolic pathways. Metabolomic investigators need to know specific details about the particular pathway of interest and understand how it is integrated within the bigger picture of comprehensive metabolism. Our objective is to provide an all-in-one resource for biochemical interpretation of metabolomic data. Contrary to physical pathway maps, we have created a digital version of comprehensive metabolism that can be viewed at any level. Metabolism can be viewed at the aerial perspective or, with multiple mouse clicks, at the “zoomed-in” molecular level of specific metabolic reactions. This resource can also be used effectively for teaching metabolism in the classroom.
Brain Region Mapping Using Global Metabolomics
Historically, studies of brain metabolism have been
based on targeted analyses of a limited number of
metabolites. Here we present an untargeted mass
spectrometry-based metabolomic strategy that has
successfully uncovered differences in a broad array
of metabolites across anatomical regions of the
mouse brain. The NSG immunodeficient mouse
model was chosen because of its ability to undergo
humanization leading to numerous applications in
oncology and infectious disease research. Metabolic
phenotyping by hydrophilic interaction liquid
chromatography and nanostructure imaging mass
spectrometry revealed both water-soluble and lipid
metabolite patterns across brain regions. This study helps define regional homeostasis for the normal mouse brain to give context to the reaction to pathological events.
Complete Annotation of the Untargeted, LC-MS Based Metabolomic Analysis of Escherichia coli
The large number of signals commonly detected in LC/MS based metabolomics has led to widely varying estimates of the number of metabolites being assayed in these experiments. Understanding the composition of these datasets is critical to making informed decisions during experimental design and data interpretation.
In this work we thoroughly annotate the peaks detected in an LC/MS based untargeted metabolomic analysis of E. coli metabolic extract. MS/MS analysis of peaks is used to empirically annotate fragment peaks. Further, an estimate of known and unknown compounds is made by matching fragmentation spectra to metabolite databases. This information is critical to the planning and interpretation of metabolomic experiments.
Rapid Detection of Microbial Resistance to Lactam Antibiotics by LC-MS/MS
Rapid Detection of Microbial Resistance to Lactam Antibiotics by LC-MS/MS
Michael Jarvis, SCIEX, 71 Four Valley Dr., Vaughan Ontario L4K 4V8
Current methods of assessing antibiotic resistance using turbidometric or disk diffusion methods to assess bacterial growth require prolonged incubation periods of up to 24 hours or more. A rapid detection strategy using multiplexed antibiotic mixtures incubated with bacteria for periods of 1 hour followed by antibiotic extraction and detection by electrospray LC-MS/MS yields comparable information in time periods as short as 90 minutes. The assay strategy includes quantitation of parent antibiotic levels and detection of metabolites produced by beta lactamases. Antibiotics screened in this manner include penicillin, ampicillin, amoxicillin, cloxacillin, piperacillin and cefotaxime in the presence and absence of tazobactam.
Protein Disulfide Bond Mapping Using Online LC–Electrochemistry–MS Applied to the Characterization of notch3 Protein Fragments
Disulfide bonds are crucial for the structure and biological function of proteins. For example in CADASIL (Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), genetic mutations in the NOTCH3 gene result in disruption of the triply-paired cysteine structure of the corresponding notch3 protein which leads to aggregation. Protein analysis with a bottom-up approach inherently lacks information on the presence and connectivity of disulfide bonds. We have developed an LC-Electrochemistry-MS system that enables online reduction and characterization of disulfide bonds. The approach was optimized using standard proteins and has been applied to notch3 protein fragments.
Proteomics and Metabolomics Analysis of Patients Sera Revealed Activation of Anti - Oxidative Pathways in Vivax Malaria
Oxidative damage of platelets plays a crucial role in the pathogenesis of thrombocytopenia found in Plasmodium vivax malaria. In this study serum samples from patients diagnosed with vivax malaria and healthy controls from different endemic regions of India were investigated using different gel-based (2DE and 2D-DIGE) and MS-based quantitative proteomics (iTRAQ and label-free LC-MS/MS) and metabolomics approaches. In our study, hemopexin, ceruloplasmin, and superoxide dismutase-1 were found to be up-regulated in malaria patients. In metabolomics analysis we have identified oxidative stress markers like nitrotyrosine, tyrosine etc. in malaria patients. Altered with severity differential expression of multiple serum proteins, antioxidative enzymes and oxidation protein products cumulatively represent the oxidative stress status of the malaria patients and reflect severity of infection.
Development of a Sensitive, Accurate and Robust LC-MS-based Method for Profiling of Angiotensin Peptides in Plasma of Atherosclerotic ApoE/LDLR (--/--) Mice
The aim of this study was to develop an analytical methodology for accurate quantification of angiotensins: Ang I, Ang II, Ang 1-7, Ang III and Ang IV in plasma of atherosclerotic mice. A triple quadrupole mass detector equipped with chip-based nanospray source connected to nanoHPLC was used. Plasma angiotensin profile was substantially modified in ApoE/LDLR double knock-out mice with increase in concentration of Ang II from 37.6±21.3 pg mL-1 in WT to 200.2±47.6 pg mL-1. Concentrations of Ang I, III and IV were also increased 3-10 fold in ApoE/LDLR (--/--) mice while that of Ang 1-7 was unchanged.
Quantitative Analysis of Precursor Prostate Specific Antigen Isoforms to Improve Prostate Cancer Detection by Immunoprecipitation and Mass Spectrometry
Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa). More recently, promising data is emerging regarding one precursor form of free PSA, proPSA is associated with PCa. ProPSA is comprised of native proPSA as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, which have been shown to be more cancer-associated than native proPSA. In this study, we developed and optimized a method for an immunoprecipitation-based platform and MRM-MS assay capable of sensitive and accurate quantification of proPSA in serum.
Development of a Fast Analytical Method for Separation of Urinary Arsenic Metabolites by Using HPLC-ICP-MS
Identification of arsenic species in human urine has been used as a measure of exposure to inorganic arsenic. The purpose was to develop and validate a fast analytical method to quantify arsenic species. Urine samples were filtered, diluted with water and injected to HPLC-ICP-MS. The run-time was 5 minutes per injection with baseline resolved chromatographic separation. The analytical measurement range was 3.4 to 80 µg/L with r2 values >0.999. Intra-assay imprecision (%CV) was less than 4.6% and inter-assay imprecision was less than 7.1%. The method demonstrated fast and acceptable performance for arsenic speciation in human urine.
Effect of Sodium and Carbon on Analytical Determination of Ultratrace Metals in Bio Specimens by ICP-MS
Disparity between composition of external calibration standards and bio specimens, like blood or human plasma, is a main impediment in accurate determination of metals at ultratrace level by ICP-MS. We have overcome the problem by addition of 1% of NaCl to the intermediate calibration standards and use of at least 3% n-butanol in the diluent as a source of carbon. The addition of carbon is necessary to maximize elemental signal enhancement due to charge transfer from C+ ions to the analytes and internal standards, while Na+ provides a matrix-match effect with the inorganic easy ionized elements present in the bio specimens.
Development and Validation of a Robust Method to Measure Chromium and Cobalt in Whole Blood of Patients with Metal Implants Using Dual Reaction Mode ICP-MS
While all prosthetic implants bear risks, metal-on-metal (MoM) implants have unique hazards associated with their use. In MoM implants, friction between component parts results in wear causing the release of metal particles from the device into the space surrounding the implant. Metal ions (i.e. chromium and cobalt) from the device or from the metal particles enter the bloodstream. In accordance with FDA recommendations, we developed a method using a NexION 350 inductively coupled plasma-mass spectrometer (ICP-MS) to quantitate chromium and cobalt in EDTA anti-coagulated whole blood from patients with MoM implants. Chromium and cobalt ions were separated from polyatomic interferences using the dynamic reaction cell (DRC) and the kinetic energy discrimination (KED) modes, respectively. The preliminary data highlight the exceptional specificity and sensitivity of this method.
The Role of ICP-MS in Clinical Applications
Trace elemental analysis of urine, blood or serum in clinical research by ICP-MS is demonstrated to be a powerful tool for answering questions on the implication of exposure to metals via food, drugs or the environment at large.
Development and Implementation of Amino Acid Quantitation by LC-MS/MS at BC Children’s Hospital
Quantitative analysis of amino acids in biological fluids is utilized in the diagnosis of amino acid disorders, a class of inborn errors of metabolism. The most widely used method for amino acid analysis is ion-exchange chromatography coupled with post-column ninhydrin derivitization. This technique was utilized by our laboratory, but the combination of low throughput and increasing sample numbers led to a backlog and unacceptable turn-around-times. Thus began our endeavour to develop a high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for amino acid quantitation. The method was validated and, as of Spring 2014, implemented LC-MS/MS for plasma amino acid analysis. In this presentation, we will compare our current platform with our previous methodology, with respect to method performance, testing capacity, turn-around-times, and laboratory workflows.
Comparison of Non-derivatization and Derivatization Tandem Mass Spectrometry Methods for Analysis of Amino Acids and Acylcarnitines in Dried Blood Spot
Flow injection tandem mass spectrometry has been frequently used to analyze amino acids (AA), acylcarnitines (AC), and succinylacetone (SUAC) in dried blood spots. While butyl esterification method is routine, it is possible to detect AAs, ACs, and SUAC as their native free acids. Using quality control DBS samples, we conducted a comprehensive study to evaluate and compare non-derivatization and derivatization methods on a triple quadrupole mass spectrometer. 12 AAs, 18 ACs, and SUAC were detected and quantified. Massive data analysis involving 26,000 calculations were performed using a streamlined meta-calculation software. Both methods had excellent analytical precision (within-run: <10%; run-to-run, <15%). Minor differences (<15%) between quantitative values resulting from derivatization and non-derivatization methods were observed for the majority of the analytes.
Cut-off Validation of Newborn Screening by Tandem Mass Spectrometry with Application of Worldwide Collaborative Project
Cut-off validation of newborn screening test (NST) of metabolic disorders is important to reduce false negative and false positive cases. We reviewed NST data and adjusted cut-off values and interpretation criteria. Presumptive cut-off values of 10 amino acids and 30 acylcarnitines were estimated by calculation of mean, standard deviations (SD), cumulative percentiles, and positive rate of 148,534 neonates born in 2013-2014. New cut-off values were then decided in consideration of reduction for false negative rate. We believe that our new cut-off is enough to detect inborn error metabolism and reduce false negative rate. Our experience for cut-off validation will be helpful for other laboratories doing newborn screening test.
Innovations in Newborn Screening for Inborn Metabolic Disorders in Dried Blood Spots
Newborn screening of lysosomal storage disorders (LSDs) is being broadly implemented as new therapies became available and timely initiation of treatment improves outcome. A series of enzyme activity assays based on tandem mass spectrometry has been developed in our lab, greatly outperforming other available tests in terms of performance, simplicity and cost efficiency. We will demonstrate the potential of multiplexed enzyme activity assays coupled with analysis of metabolic markers to reliably screen for Pompe and Fabry diseases, sphingolipid disorders and mucopolysaccharidoses (I; II; IIIA; IVA; VI) in newborn dried blood spots. Several large scale pilot studies based on these assays have been completed or are underway.
Measurement of Lymphocyte Argininosuccinate Synthetase Activity by Tandem Mass Spectrometry
We established a liquid chromatography-tandem mass spectrometry method for the measurement of argininosuccinate synthetase (ASS) activity in phytohemagglutinin-activated peripheral lymphocytes and examined the effect of fasting on ASS activity. The assay was based on the measurement of argininosuccinate (ASA) after 1-h incubation with substrates. The mean value of ASS specific activities in activated lymphocytes from healthy subjects (N=10) was 88.3 mU/g (SD 23.8 mU/g). The mean value of ASS activities in patients with citrullinemia (N=3) was 16.6 mU/g (SD 6.7 mU/g). No significant difference in ASS activities was found in subjects with fasting and non-fasting status (66.2 vs. 76.2 mU/g).
Ceramide Trihexosides and Sulfatides Quantitation in Urine by LC-MS/MS
We have developed an analytical method to quantify ceramide trihexosides and sulfatides in lipid extracts prepared from human urine. These analytes are useful for the diagnosis of Fabry disease, Metachromatic Leukodystrophy, multiple sulfatase deficiency, sphingolipid activator deficiency, and some cases of mucolipidosis II. This method has greater specificity and sensitivity than our current thin layer chromatography method. Other advantages are markedly decreased sample volume and easy sample preparation. Samples are extracted using chloroform-methanol with appropriate internal standards. Mass spectrometry analysis is performed using liquid chromatography tandem mass spectrometry in positive and negative multiple reaction monitoring mode.
Triplex Tandem Mass Spectrometry Assays for Screening of 3 Lysosomal Storage Disorders in a Korean Population
We evaluated the performance of triplex tandem mass spectrometry assays for screening of Pompe, Fabry, and Gaucher diseases with Acquity UPLC CSH C18 column (Waters, USA) and TQD triple quadrupole mass spectrometer (Waters, USA) in the multiple-reaction-monitoring mode. We evaluated the precisions, linearity, limit of detection, recovery, carryover, and ion suppression with 3 enzyme activities in 376 anonymous newborn dried blood spots. Intra- and inter-assay precisions were between 0% and 18.9%. The linearity of each enzyme activity was good (R2=0.9952, 0.9982, 0.9974, respectively). The lower limit of detection was 0.22 umol/h/L, 0.39 umol/h/L, 0.79 umol/h/L, respectively. The recovery and the carryover was good. There was no ion suppression. The performance for screening of 3 lysosomal storage disorders was acceptable in a Korean population.
Tandem Mass Spectrometry Analysis of Urinary Glycosaminoglycans as Biomarkers for Mucopolysaccharidose Patients
Mucopolysaccharide disorders are the result of primary defects in lysosomal enzymes. The primary objectives of this research project were to evaluate the GAG urinary excretion and the relationship with disease severity, response to treatment and the impact of co-morbidities in mucopolysaccharidoses. We thus devised a quantitative methodology for the analysis of urine samples using a tandem mass spectrometry multiplex approach. Dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin sulfate disaccharides were specifically targeted. Our results show an efficient differentiation between MPS patients and controls. High sensitivity and good resolution allow the detection of small quantities of urinary GAGs.
UDP-Galactose-4’-epimerase Activity Determination in Red Blood Cells by LC-MS/MS
UDP-galactose-4’-epimerase (GALE) activity can be measured by the use of a rapid LC-MSMS separation of UDP-glucose and UDP-galactose that allows direct quantification of enzymatic products and is an improvement upon recently published enzyme assays using LC-MS/MS. Six abnormal results were identified in red blood cells (RBC) during a blinded sample exchange with Emory University; 4 true positive and 2 suspected carriers. Intra and inter assay precision was assessed at four activity levels and CVs were <6% and <10% respectively (N=20). A reference range evaluation has yielded a mean of 16.7 nmol/h/mg Hb (min-max = 7.1-36.8, stdev = 4.7, N=181). Demonstration of reduced GALE activity in RBC is a useful diagnostic test following an abnormal newborn screening result for galactosemia.
Urinary Glucose Tetrasaccharide Assay Using Rapid Ultraperformance LC-MS/MS for Pompe Disease
We evaluated the urinary glucose tetrassaccharide (Glc4) assay using UPLC-MS/MS (AB SCIEX TQ 6500). Calibration curve were linear over a range from 5 to 500 ¥ìmol/L. Within- and between-day precision CVs were 6.52-14.6% and 11.5-13.2%, respectively. The mean concentration of urinary Glc4 of 27 normal controls and 3 pseudodeficiency patients were 1.5 and 12.1 mmol/mol creatinine, respectively. Urinary Glc4 concentration in a patient with Pompe disease was 171.3 which decreased to 130.9 after enzyme replacement therapy. The urinary Glc4 assay measured by UPLC MS/MS can be a reliable biomarker for diagnosis and therapeutic monitoring of Pompe disease.
Development of a Direct Assay of Iduronate-2-sulfatase for Mucopolysaccharidosis Type II (Hunter Syndrome) Using UPLC-MS/MS
Mucopolysaccharidosis type II (Hunter syndrome) is caused by a deficiency in iduronate-2-sulfatase. We developed and evaluated the performance of UPLC-MS/MS with commercially available substrate (4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate) for the detection of Hunter syndrome. All reagents were fully separated within 3 min. No ion suppression was observed. The intra- and inter-assay precisions were 7.9%–10.5% and 4.8–10.2%, respectively. The enzyme activities measured in the DBSs were consistently lower in patients with Hunter syndrome than in normal newborns (P=0.001). Additionally, we performed multiplex tests for five lysosomal storage disease including Hunter, Hurler, Fabry, Pompe, and Gaucher disease.
Tandem Mass Spectrometric Determination of Atypical 3-beta-Hydroxy-delta-5-bile Acids in Patients with 3-beta-HSD Deficiency
3beta--Hydroxy-delta-5-C27-steroid oxidoreductase (HSD3B7) deficiency, a progressive cholestatic liver disease, is the most common genetic defect in bile acid synthesis. Early diagnosis is important because patients respond to oral primary bile acid therapy, which targets the negative feedback regulation for bile acid synthesis to reduce the production of hepatotoxic 3-beta-hydroxy-delta-5-bile acids.
A tandem mass spectrometry method is described for the measurement of 3-beta-hydroxy-delta-5-bile acid sulfates in urine applicable to the diagnosis and accurate monitoring of responses to primary bile acid therapy in HSD3B7 patients.
Metabolic Phenotyping Reveals a Lipid Mediator Response to Ionizing Radiation
Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here we screened mouse serum for metabolic alterations following an acute exposure to gamma radiation using a multi-platform, mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and eicosanoids, which could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.
Targeted Serum Metabolite Profiling for Colorectal Cancer Progression Monitoring
Colorectal cancer (CRC) is one of the most prevalent cancers worldwide, and a major cause of human morbidity and mortality. In addition to early detection, close monitoring of disease progression in CRC can be critical for patient prognosis and treatment decisions. In this study we applied a targeted LC-MS/MS metabolic profiling approach using serial serum samples to monitor CRC patient disease progression. A PLS-DA model using a panel of 5 metabolites was established, and excellent model performance (sensitivity=0.83, specificity = 0.94, AUROC=0.91) was obtained, superior to traditional biomarker CEA. Our results suggest the potential usefulness of metabolic profiling for CRC disease progression monitoring.
A Data Independent LC-MS Based Method for a Multi-omic Approach to Investigate Obesity Treatment Within a Mouse Model
Obesity is one of the risk-factors associated with metabolic syndrome. Pharmacological manipulation of glycosphingolipid metabolism has been shown to improve the symptoms of metabolic syndrome. A global view of the alterations in the proteome, metabolome and lipidome during development is still to be fully explored. The work presented here is to provide a multi-omic analysis of protein and lipid liver extracts from control and obese mouse models undergoing treatment to prevent or revert obesity. Integrated pathway analysis was used to review the complimentary datasets and provide an understanding of the underlying biology of differentially expressed proteins and lipids.
A Metabolomic Approach to Identify Potential Therapeutic Biomarkers for Diabetic Foot Ulcers
Diabetic foot ulcer (DFU) is a common complication of diabetes and may lead to amputation of the lower extremity. To prevent the progression of DFU is imperative because of the great impact in an individual¡¦s life and a significant burden to the healthcare system. We used a combined direct injection and UPLC-MS/MS assay to quantify 186 metabolites in DFU patients (n=55) and diabetic patients (DM) without complications (n=57). PCA and PLS-DA analyses enable a clear differentiation of DMF and DM patients based on changes of their amino acids and acylcarnitines. These metabolites hold the potential to be therapeutic markers.
Targeted Oxylipin Profiling for Clinical Diagnostic: A Novel Insight in Ventilator Associated Pneumonia
Ventilator associated pneumonia (VAP) is the most common infection in intensive care units (ICU), however robust diagnostic techniques for early detection are lacking. Blood samples of 58 brain injured patients were analysed through their ICU stay using a sensitive UPLC-MS profiling assay for the quantification of 48 inflammatory signalling molecules. Lipid mediators were able to differentiate patients with brain injuries or pneumonia and predictive modelling of VAP onset was performed. Serial oxylipins are of clinical merit in ICU, generating insight for potential disease biomarkers, stratifying critical patients based on their risk of adverse outcome and personalizing their therapeutic management.
UPLC-MS-based Characterization of the Maternal Plasma Lipidome During the Early 2nd Trimester of Pregnancy Window
Pregnancy involves metabolic changes which are reflected in the maternal plasma metabolome. Herein, plasma trajectories from women who experienced a normal term delivery were mapped. A large cohort of women (n=359) of mixed ethnic background were recruited with plasma and accompanied clinical metadata collected at 12, 16 and 20 gestational weeks. Utilizing a UPLC-MS lipidomics approach it was demonstrated that the maternal plasma lipidome undergoes characteristic alterations during the early 2nd trimester to support the increased maternal/fetal energy requirements and placental development. Observed ethnic-specific differences could pave the way of personalized medicine applications.
Metabolomics Approaches for Biomarker Discovery for Toxicant Exposure of Di-isononyl Phthalates Using Liquid Chromatography-high Resolution Mass Spectrometry
Di-isononyl phthalate (DINPs) are widely used plasticizers and have effects on reproductive system. Three metabolomics approaches were used to explore DINP exposure markers in an LTQ/Orbitrap HRMS dataset obtained from liver enzyme incubation. The data processing techniques included signal mining algorithm with isotope tracing (SMAIT), mass defect filtering (MDF), and web-based XCMS. Fourteen metabolites were validated as DINP exposure markers using a rat model. Among the 14 exposure-related DINP metabolite signals, 8 have not been reported in the literature. The metabolomics platform can efficiently and systematically filter probable metabolite signals from a complex LC-HRMS dataset for toxic exposure marker discovery.
Development of Chemical Isotope Labeling LC-MS for Human Serum Metabolome Profiling from Dried Serum Spots
Dried blood spots have been used as a means of sample collection and storage for diagnosis of inborn errors of metabolism and "omics" research with advantages such as convenience of storing and transporting samples. In this work, we describe a method for profiling the amine/phenol submetabolome of human serum from dried serum spots (DSS). Similar numbers of putative metabolites could be detected from DSS and serum matrix. Detailed workflow for DSS metabolomics will be presented. This method is shown to be useful to differentiate different groups of metabolomic samples with almost the same performance as using serum directly.
Skyline for Small Molecules: A Flexible Tool for Cross-platform LC-MS/MS Method Creation and Data Analysis for Metabolomics
This presentation will demonstrate the initial implementation of the Skyline software package for the creation of custom LC-MS/MS methods for small molecule analysis using several classes of metabolites. The software includes the ability to define precursor and product ions based on empirical formula or m/z, define collision energy specifically by molecule or based on a linear equation, and define expected retention time. As with peptide data, Skyline reads in the raw data from all major instrument vendors and allows native instrument method export. Thus the package eases the translation and validation of targeted metabolomics methods between instruments and laboratories.
X13CMS and IsoMETLIN: Platforms for Isotope-Based Metabolomics
Mass spectrometry-based untargeted metabolomic technologies have been applied widely to compare the levels of hundreds to thousands of small molecules between sample groups unbiasedly, which has thereby enabled systems-level analyses. Recently, however, isotope-based metabolomics has been introduced to the standard metabolomic workflow, which a stable isotope is introduced into a biological system and metabolomic technologies are used to track its fate unbiasedly. Here we introduce X13CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS, and isoMETLIN, an analog of the widely used METLIN database, that is designed to facilitate the identification of both isotopologues and isotopomers.
Application of Liquid Chromatography/mass Spectrometry for Evaluation of Plasma ATP Concentration and Turnover Rate
Nucleotides and its derivatives in the extracellular fluid, play a prominent role in many physiological processes. Nucleotides released from cells to extracellular space can trigger thrombosis, inflammation and ectopic calcification. Quantitative analysis represents a challenge due to relatively low concentration of these compounds. Reduction of flow in chromatographic separations linked to mass detectors offers massive gain in sensitivity but at the expense of complexity of work flows. Nanoflow LC/MS is extensively used in proteomic while its application in small molecule separation is not common. This study aimed to test several options and to develop procedure to quantitate ATP concentration in plasma using LC/MS capable to resolve ATP isotopomers to facilitate turnover rate studies.
A Pipeline for Untargeted UPLC-MS Profiling to Expand Tissue Metabolome Coverage – Optimization of Chromatographic Separation and Tissue Extraction
In order to address the challenges of tissue sample analysis and metabolome coverage, a novel pipeline consisting of two consecutive extraction steps (aqueous followed by organic) was developed. Analysis was achieved using two UPLC-MS methodologies: HILIC chromatography for analyzing the aqueous extracts and RP lipid profiling for the organic. Both extraction and separation steps were extensively optimized. This pipeline was successfully applied on tissues including vascular, liver and adipose. The two chromatographic modes proved highly complementary, with >5000 features robustly detected and >250 metabolites structurally assigned and mapped to metabolic pathways covering a wide range of biological and disease processes.
Metformin Treated Wistar Rats Demonstrate Remarkable Alterations in Lipid and Bile Acid Plasma Levels
Type 2 diabetes is one of the greatest threads of public health. The beneficial effects of metformin towards mitigating disease manifestation have been connected to gut microbiota and inflammation onset. In total, 12 animals were treated with Metformin for 5 weeks with 12 used as controls. Using UPLC-TOF-MS methodologies, highly significant alterations were detected for the levels of pro- and anti-inflammatory lipid precursors in plasma samples, and of sulfated and glucosylated bile acids in plasma and liver tissue. This study may provide insights of the pathways able to function beneficially for diabetic patients, as well as of blood markers reflecting modifications in gut microbiota.
Comprehensive Human Fecal Metabolome Analysis Using Chemical Isotope Labeling LC-MS
Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other components. We report a sensitive chemical isotope labeling LC-MS method for comprehensive and quantitative analysis of the amine-, phenol, and carboxylic acid-containing metabolites in fecal samples. A sequential water-acetonitrile extraction method is found to be optimal for extracting metabolites from fecal samples. Pre-analytical issues including sample storage conditions are investigated to study their effects on fecal metabolome profiling. The effect of diet on human fecal metabolome is studied and its implication on applying fecal metabolomics for disease biomarker discovery is discussed.
A Novel High Resolution Metabolite MS/MS Spectral Library for Accurate Metabolite Identification in Human Biofluids
Identification of metabolites in human biofluids remains a major analytical challenge that we here address in a library-based approach. We describe a workflow for rapid and accurate metabolite profiling based on the use of a high resolution spectral MS/MS library of about 800 metabolites selected from the Human Metabolome Database (http:/www.hmdb.ca/).
A High Throughput LC-MS/MS Method for the Analysis of Multiple Vitamin D Analytes in Serum and Placenta Using Supportive Liquid-liquid Extraction
Serum analysis of 25OHD3 is routinely used to define patient vitamin D status. Quantification of multiple vitamin D metabolites may provide more accurate definitions of vitamin D status for specific patients and disease scenarios. The aim of this study was to develop an LC-MS/MS method for multiple Vit-D metabolites in serum and placenta. This method ensures accurate 25OHD3 measurements, separating inactive C-3 epimers and isobar interferences. LC-MS/MS analysis was carried out using a Waters AQUITY UPLC coupled to a XEVO TQ-S detector. Separation of multiple vitamin D analytes was achieved in a 9 minute run time and optimised sample preparation.
Rapid, Simultaneous Analysis of Urinary Catecholamines and Metanephrines by Mixed-mode SPE and HILIC LC-MS/MS
A single extraction and analysis method has been developed for urinary catecholamines and metanephrines. Analytes were extracted using weak cation exchange mixed-mode SPE and analyzed using HILIC LC/MS/MS. Recoveries were good and matrix effects were sgnificanly reduced compared to reverse-phase analysis. Linearity and QC results were excellent for all compounds down to 0.5 ng/mL, with excellent accuracy and precision. This method enables rapid, simultaneous and accurate LC/MS/MS analysis of these challenging compounds without the challenges associated with traditional reversed-phase separation or ion-pairing techniques.
A Chromatography/tandem Mass Spectrometry Method for the Simultaneous Quantitative Determination of Ten Endogenous Steroids Using Low Serum Volume
We report a validated gas chromatography selected reaction monitoring – tandem mass spectrometry assay (GC-MS/MS) for the simultaneous quantification of progesterone, dehydroepiandrosterone, androstenediol, androstenedione, testosterone, dihydrotestosterone, androsterone, 5alpha-androstan-3beta-17beta-diol (3β-diol), estrone and estradiol. After addition of stable isotope internal standards to 250 µl of serum, the method involved a liquid-liquid extraction, derivatization and solid-phase extraction. Individual steroids were measured with high sensitivity, accuracy and reproducibility while its applicability was successful on serum from men, pre- and post-menopausal women. This method will allow measurement of ten steroids in clinical epidemiology and laboratory research while providing low quantification limits with a limited volume of sample.
Metabolomics of Exosomes from Uterine Aspirates and Plasma Samples of Endometrial Cancer Patients
Endometrial cancer (EC) is the most frequent gynecological cancer diagnosed in the USA. The goal of this study is to characterize exosomal metabolomes derived from human uterine fluids and plasma, and identify diagnostic bio-signatures of EC patients. Exosomes from both biofluids were analyzed by liquid chromatography mass spectrometry and were found to have unique features. Metabolomic profiles differentiating EC from benign patients was obtained; validation of these findings is ongoing. These findings could have broad application for clinical studies focused on biomarker development.
LC-MS/MS Method After Derivation for the Determination of L- and D- Isomers of 2-hydroxyglutarate in Biological Fluids: Application as Biomarker of IDH Mutation
Activating mutations of isocitrate deshydrogenase leading to the production of 2-hydroxyglutarate have been described in hematologic malignancies and solid tumors. Therefore, we developed a liquid chromatography tandem mass spectrometry method allowing a rapid, accurate and precise simultaneous quantification of both L and D enantiomers of 2-HGA in human serum for clinical applications as diagnostic and predictive biomarker. A derivatization step with (+)-o,o’-diacetyl-L-tartaric anhydride allowed to separate the two enantiomers without chiral stationary phase, on a C18 column (Agilent ZorbaxSB®, 4.6 x 150 mm, 5 µM) combined to a XevoTQ mass spectrometer with an electrospray ionization (ESI) source. This method was validated according ICH Q2(R1)is linear over the range 0.338-135.04 µmol/L, accurate (bias <5.6% RSD) and precise (repeatability <6.2% and intermediate precision <5.6%).
Multiple Reaction Monitoring Assay for Identification of Borrelia burgdorferi
Lyme disease is caused by Borrelia burgdorferi. Currently, Lyme infection is identified by the presence of skin lesion called erythema migrans, and is later confirmed by immunological assays. However, such an approach has a low therapeutic value because delayed diagnosis may lead to serious complications. Herein we developed a mass spectrometric method to detect the presence of Borrelia burgdorferi using multiple reaction monitoring with a quantification concatamer as an internal standard. Two quantification concatamers containing signature peptides from the six most abundant Borrelia burgdorferi proteins have been constructed and will be used to improve the detection limit of Lyme infection.
Identification of Staphylococcus Aureus by Shotgun Spectral Matching
Staphylococcus aureus is a Gram-positive bacterium that can be pathogenic when expressing specific toxins. Production of altered penicillin binding proteins causes resistance to most active antibiotics. Rapid characterization of virulence and resistance factors may aid in providing clues for treatment of individual patients. A standard bottom-up, shotgun proteomics method was applied on 12 different isolates of S. aureus to create a reference database of spectral libraries with one library from each isolate. Additional isolates, treated as unknowns, were then searched against all reference libraries for sample identification based on relative numbers of spectra finding a match the different spectral libraries.
High Throughput Pulse-chase Analysis of Metabolite Turnover in Microorganisms Followed by LAESI Mass Spectrometry
Metabolic fluxes and the pools of the associated chemical species are closely linked to their turnover in a living organism. Pulse-chase analysis, using stable isotope labeling, has been applied to monitor dynamic changes in metabolite buildup and decay. The lipid metabolism of Chlamydomonas reinhardtii has been extensively studied using conventional extraction and separation techniques. Here, we introduce a high-throughput method for pulse-chase analysis based on laser ablation electrospray ionization (LAESI) mass spectrometry (MS) with ion mobility separation (IMS). Simultaneous detection, identification, and quantitation of some molecular constituents, e.g., metabolites, lipids, and peptides, in a microorganism can be achieved within minutes. Pulse-chase analysis by LAESI-IMS-MS can be applied to follow changes in metabolic fluxes during disease.
Highly Sensitive Screening for Antibiotic Resistance Using Parylene-matrix Chip
¥â-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to ¥â-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Parylene-matrix chip and MALDI-TOF mass spectrometry are used to quantitate ¥â-lactamase mediated hydrolysis of penicillin (m/z: [PEN+H]+=335.1 and [PEN+Na]+=357.8) into penicilloic acid (m/z : [PA+H]+=353.1 and [PA+Na]+=375.4) with minimal interference of low molecular weight noise peaks. The ¥â-lactamase assay was carried out with an antibiotic-resistant E.coli strain and an antibiotic-susceptible E.coli strain, revealing that the minimum number of E.coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/parylene-matrix chip assay.
An Illustrative Example of the Need for Ongoing Clinical Microbiology Competency in the Era of MALDI-TOF MS Microorganism Identification: Neisseria spp.
We report the case of a penile lesion culture containing Neisseria cinerea misidentified as Neisseria meningitidis by MALDI-TOF MS. To further investigate this phenomenon, four characterized isolates, including the patient isolate of N. cinerea, a laboratory isolate of Neisseria mucosa, and ATCC strains of Neisseria lactamica, and Neisseria sicca were analyzed at various days growth by the Bruker MALDI Biotyper (Bruker, Billerica, MA) and the VITEK MS (bioMérieux, Marcy l’Etoiile, France). N. meningitidis was the first or second match for three isolates during at least one day tested by the Bruker Biotyper. These isolates were correctly identified by VITEK MS. N. sicca failed identification on both platforms, and on Day 5 was misidentified as N. mucosa by VITEK MS. These findings suggest MALDI-TOF MS may not be an accurate stand-alone identification method for Neisseria species.
MALDI-TOF-MS and MS-ASTRA Assay Development for the Generation of Biotyper Reference Spectra and Antibiotic Sensitivity Determination for Category B Bacteria
To aid in the reliability of bacterial identification using the MALDI Biotyper platform, we are generating spectral database entries for over 200 isolates of the Gram-negative facultative intracellular pathogens Burkholderia pseudomallei and Burkholderia mallei. These bacterial strains are inherently resistant to many antibiotics and cause opportunistic infections in immunocompromised individuals. Therefore, in conjunction with our database effort we are testing the applicability of Mass Spectrometric-Antibiotic Susceptibility Rapid Assay (MS-ASTRA) for antibiotic sensitivity testing in Burkholderia isolates. The overall goal is to develop assay conditions which simultaneously identify a bacterial species and screen for sensitivity to antimicrobial drugs.
Personalized Chemotherapy Through the Combination of Microdosing and Accelerator Mass Spectrometry
Platinum-based chemotherapy is the primary therapeutic intervention for over 300,000 cancer patients per year in the US. Although some patients are cured by this type of chemotherapy, 50-70% of patients show no benefit and only suffer the side effects. Currently, there is no diagnostic test that predicts therapy response towards these therapeutics before the treatment is started. These compounds are DNA alkylating agents that kill cancer cells through formation of DNA-adducts. We are developing a predictive test for cancer patient’s response to platinum-based chemotherapy drugs. The technology utilizes the ultra-sensitive accelerator mass spectrometry method to measure drug-DNA adduct frequency.
Pharmacodynamic Strategy for Monitoring the Extent of Immunosuppression: Requirement of a Kinase Inhibitor for Measuring Calcineurin Phosphatase Activity
An approach for measuring the immunosuppressants effects on their cellular targets, such as calcineurin for calcineurin inhibitors, has been developed in order to improve the monitoring of immunosuppression after transplantation. The determination of calcineurin activity consists in the direct measurement of the dephosphorylation of a phosphorylated substrate of calcineurin, the RII substrate. None of the methods described so far have discussed the possibility of rephosphorylation of the substrate. Here, we demonstrate the importance of such an influence on the measurements of calcineurin activity for monitoring the extent of immunosuppression after transplantation. Accordingly, we report the development and the validation of a LC-MS/MS assay for measuring calcineurin activity in biological samples wherein a kinase inhibitor is present in the assay reaction mix.
Swab Touch Spray - Mass Spectrometry for Direct Analysis of Bacteria and Drugs in Oral Fluid
Touch spray mass spectrometry (TS-MS) is an ambient ionization technique in which a sample is transferred to a probe where ionization occurs via electrospray-like mechanisms. The probe can be metallic or a medical swab, the latter having potentials for direct and in vivo oral fluid analysis. Here we present oral fluid testing by TS-MS to detect Streptococcus pyogenes, the leading cause of strep throat, and drugs of abuse, for clinical-toxicological applications. The approach outlined is intended for point-of-care testing using oral fluid in clinical-toxicological applications. Proof-of-concept results presented will require extension and refinement in analytical procedures to meet clinical/legal requirements.
Utility of the Microflex LT Platform in the Development of Serum Proteomic Companion Diagnostic (CDx) Tests in NSCLC
Clinical oncology assays using mass spectrometry have exclusively been run on platforms designed for use in research laboratories. Here, we employ the microflexTM LT (LT), a MALDI-TOF MS which is a component of Bruker’s FDA cleared MALDI Biotyper CA system, for development of a CDx test. In this work, we evaluate the LT through comparative studies with similar RUO platforms, utilizing samples from NSCLC patients. In summary, the LT appears to be suitable for measurement of NSCLC serum proteomic profiles and inclusion of the LT in CDx test development will expand utility of MALDI-based testing in the clinic.
Direct Mass Spectrometry Analysis of Wet Biofluid Samples Using Slug-Flow Microextraction
We have previously developed paper spray and extraction spray to analyze dried biofluid for point-of-care analysis. Recently, we introduced a slug flow microextraction method in conjunction with nanoelectropray for a single-step analysis of wet biofluid samples of low volumes (~5µL). Direct quantitative analysis of therapeutic drugs and illicit drugs in blood and urine sample has been demonstrated with the limits of detection better than 1 ng/mL and RSD better than 15% achieved. Real-time chemical derivatization was incorporated for the detection of anabolic steroids in urine at sub-ppb levels. Furthermore, online monitoring of enzyme function in wet blood has also been enabled using this method.
Using MALDI-TOF MS to Screen for Monoclonal Proteins in Serum
This study aims to evaluate MALDI-TOF MS as a clinical screening method for serum monoclonal proteins (M-proteins). A set of 556 serum samples previously tested by gel electrophoresis (PEL) and immunofixation (IFE) were analyzed by MALDI-TOF MS. The mass distributions of immunoglobulin light chains from each sample were compared to normal serum. Deviations from the normal distribution were considered positive. Upon analysis, 100% of PEL positive, 91% of IFE positive and 19% of PEL/IFE negative samples screened positive by MALDI-TOF MS. The results suggest that MALDI-TOF MS has near equivalent sensitivity compared to current methods and would be an effective screen for M-proteins.
Chemical Biopsy Based on SPME Approach: A New Medical Tool
Patient monitoring, metabolomics and biomarkers discovery are an integral part of medical diagnosis and biomedical research. However tissue analysis remains as the bottleneck of such studies due to the invasiveness of sample collection, as well as the laborious and time-consuming sample preparation protocols. In the proposed technology we have integrated in vivo sampling, sample preparation and global extraction of metabolites. This approach based on small coated fibre format of solid phase microextraction was presented and evaluated during liver and lung transplantation in pig model. Sampling approaches, including selection of the probe, transportation, storage conditions and analyte coverage are going to be discussed. The applicability of the method for spatially resolved metabolomics studies was demonstrated during lung transplantation experiments.
Strategies for the Direct Coupling of Solid Phase Microextraction (SPME) to Mass Spectrometry: Applications in the Clinical Lab
In this work we introduce two novel strategies for the direct coupling of SPME devices to MS for trace analysis of target compounds: coated blade spray (CBS) and solid phase microextraction-transmission mode (SPME-TM). Contrary to the general believes that sample prep is slow, analyte-enrichment and sample-clean-up is performed in 1 minute, and the total analysis time does not exceed 3 minutes in both approaches. Detection limits at the low ppt levels are feasible in complex matrices of clinical relevance. Given the structural configuration of the apparatus, sample prep can be used to perform extractions independently of the sample complexity or its dimensions.
Standardized and Quantitative Metabolic Phenotyping of Bile Acids in Blood - An International Inter-laboratory Ring Trial Test
Bile acids are relevant endogenous markers for liver cell functions, inflammation, apoptosis, gut microbiome but also are signalling molecules regulating triglycerides, cholesterol and glucose metabolism. We have developed the worldwide first kit including analytical reagents to harmonize the analysis of individual bile acids from only 10 µL of either human plasma or human serum (16 bile acids) or mouse plasma samples (19 bile acids) for UHPLC-MS/MS analysis. A ring trial test with 14 participants has been successfully performed. Bile acid profile of mice is quite different from that of human; relevant aspects for translational medicine.
Preliminary Experience with the Waters Unispray™ Source
We have tested the Waters UnisprayTM source in a routine clinical laboratory for a number of applications including steroids, therapeutic drugs and plasma metanephrines. In the UnisprayTM source the gas flow and droplets from a grounded nebuliser strike a rod target held at approximately 1kV upstream from the sample cone. The source has proved to be robust and we have seen a three fold increase in sensitivity for plasma metanephrines and sirolimus (pos). Gains in sensitivity are not seen for all compounds and we have seen no real improvement over the electrospray technique for testosterone, androstenedione or aldosterone.
Usability Study of a New HPLC, a New Tandem MS and a New Data Processing Software
Stability, robustness and time efficiency are three major challenges in laboratories where a large number of samples are analyzed routinely. Here we report results from a usability study of a new two channels HPLC, a new tandem mass spectrometer and a new data processing software to address these challenges. A total of 2000 crashed synthetic serum samples spiked with alprazolam and isotopically-labeled internal standard were analyzed continuously over 100 hours, with additional 44 QC samples inserted. On-line turboflow column helps removing sample matrix. Cross-channel RSD% of retention time and concentration was observed at 0.85%, 1.49%, respectively. Instrument-to-instrument precision studies (3 units) are reported using two example compounds (testosterone, estradiol) with APCI probe in both positive and negative ionization modes. For in vitro diagnostic use.
Exploring the Potential of the Last Generation UHR-Q-TOF for Rapid Generation of Accurate Information on Proteoforms Distribution and Relative Abundancy
All along their life cycle, proteins undergo various transformations that can alter their functions while keeping a good part of their primary sequence intact. This multiplication of PTM patterns, alternative splicing forms or products of proteolitic processing cannot be simply resolved with a bottom-up approach, as very few peptides are specific from the given proteoform. In this work we illustrate last-generation UHRQ-TOF’s capability to generate accurate proteoform distribution information within minutes from an highly complex sample, therefore enabling to complement the information obtained with the more traditional bottom-up approaches.
PhoTorrent Atmospheric Pressure Photo Ionization (APPI) Source to Achieve High Efficiency Photo Ionization on Testosterone and 25-OH Vitamin D3
An IONICS PhoTorrent modular APPI source was used to explore APPI detection capabilities of 2 clinical compounds, Testosterone and 25-OH Vitamin D3. Sensitivity as low as 100 ag/µL for Testosterone was achieved, while maintaining excellent linearity to 10 ng/mL. The 25-OH Vitamin D3 gave sensitivity as low as 100 fg/µL, with excellent linearity to 100 ng/mL. The PhoTorrentTM APPI source is well suited to flow rates of 100-500 L/min. The modularity of the PhoTorrent APPI Source facilitates switching from APPI to ESI and APCI, increasing the number and range of compounds that can be analyzed effectively with minimal hardware changes.
Quantification of Multiple Therapeutic mAbs in Serum
Using microLC-ESI-Q-TOF Mass Spectrometry
Therapeutic monoclonal immunoglobulins (mAbs) are an important class of drugs used to treat diseases ranging from autoimmune disorders to B cell lymphomas. Here we demonstrate the simultaneous quantification of four therapeutic mAbs in serum using the abundance of the kappa light chain from each IgG kappa mAb. We also report the concentration of each therapeutic mAb in pharmaceutical preparations using IgG specific proteotypic peptides. Our results show that microLC-ESI-Q-TOF mass spectrometry is a robust platform for monitoring multiple therapeutic mAbs in a single assay.
Mass Spectrometry and Stable Isotope Labeling for Quantitative Analysis of Ribosomal RNA Modifications
Quantitative Mass Spectrometry (qMS) platform has been developed to perform global profiling of modifications in ribosomal RNA. Using qMS, we have carried out mechanistic studies aimed to characterize temporal and functional relationships between individual RNA modification steps and the ribosome assembly process in bacteria. Now, we are advancing the technology to quantitatively monitor RNA modifications in yeast and human ribosomes. This will be instrumental in defining the key roles ribosomal modifications play in fundamental biological processes of ribosome biogenesis and translation, and to understand what defects in RNA modification machinery are implicated in hereditary diseases and cancer.
Development of a Sensitive Analytical Method for Serum Bisphenol A Using LC-MS/MS
We developed a sensitive assay for serum BPA. BPA in serum were extracted with toluene and derivatized with dansyl chloride. After derivatization, the samples were analyzed by liquid chromatography-tandem mass spectrometry. The assay was linear in a range from 0.05 ng/mL to 5.0 ng/mL. Within-run and between-run imprecision were within acceptable level. The median BPA concentration was 0.044 ng/mL in male (range: 0.003 to 0.551 ng/mL) and 0.029 ng/mL (range: under detection limit to 0.055 ng/mL) in female. Our method provides a valuable tool for public health studies about BPA exposure.
Development and Validation of an Opioid LC-MS/MS Assay: Evaluation of Different β-glucuronidase Enzymes and Protein Precipitation Plates
We report the development and validation of an opioid confirmatory assay using enzyme hydrolysis and LC-MS/MS for the qualitative identification of thirteen opioids. We evaluated three different β-glucuronidase enzymes and three protein precipitation plates that are currently on the market. The combination of IMCSzme β-glucuronidase and Supleco protein precipitation plate provided an efficient and cost-effective hydrolysis and sample clean-up. LOD, Linearity, precision, matrix effect, recovery and carry-over of the final method were validated. This LC-MS/MS assay detected compounds missed by GC-MS due to interference or co-eluting peaks. This procedure is significantly faster and less expensive compared to the GC-MS method.
The Analysis of Fentanyl and Its Analogues in Human Urine by LC-MS/MS
Synthetic opioid drugs, such as fentanyl and sufentanil, have very high analgesic potency. Abuse of these prescription opioids and their illicit analogue, acetyl fentanyl, is a growing public health problem. In this study, a simple dilute and shoot method was developed with an analysis time of less than 3.5 minutes for fentanyl, norfentanyl, acetyl fentanyl, and sufentanil in human urine by LC-MS/MS using the RaptorTM Biphenyl column.
Improved Method for the Analysis of a Pain Management Supplemental Panel in Urine Using the Thomson EXtreme Filter Vials® by LC-MS/MS
This improved sample preparation method allows for the quantitative measurement of 17 drugs in a supplemental pain panel in urine. The urine samples were prepared using the eXtreme|FV®, followed by LC/MS/MS analysis. The most critical aspects of reliable urine analysis are the reduction of interferences from the sample matrix and analyte recovery. Traditionally, SPE, SLE and centrifugation have been used to reduce matrix interference prior to analysis. However, these techniques are time consuming, adversely impact recovery, use large amounts of solvent and are expensive. The improved sample preparation method using the Thomson eXtreme|FV® allows for the analysis of 17 drugs.
High Throughput Screening and Confirmation of 41 Pain Panel Drugs in Oral Fluid by an Integrated On-Line Extraction UHPLC-MS/MS System
A rapid and sensitive procedure for the quantification of Pain Panel Drugs (PPDs) in Synthetic Saliva was developed using Thomson filter vial for sample preparation and using an integrated On-Line Extraction (OLE)-UHPLC-MS/MS System. The lower limit of quantitation (LLOQ) was 0.2 ng/mL and upper level of quantitation (ULOQ) was 100 ng/mL. The linearity regression coefficient R2 was >0.99. The sub ng/mL level PPDs detection with about three orders of dynamic detection range will cover the clinical research needs. The method run cycle time was 8.5 minutes.
Development of a Rapid Mass Spectrometry Based Screen of Tricyclic Antidepressants as an Alternative to Immunoassay Screening
While immunoassay is a prevalent screening technique it is also prone to issues such as high false positive rates due to lack of analyte specificity. Mass spectrometry was therefore investigated as an alternative screening technique for the ability to improve analyte specificity on a comparable time scale. In this study, a Rapid Online Sample Preparation and Injection method was developed using a commercially available guard cartridge on a conventional LC-MS/MS system. Using a two-point calibration curve to provide semi-quantitation, a robust method was developed and validated that improved upon the high false positive rate observed in immunoassay screening.
Paper Spray Ionization - Tandem Mass Spectrometry for Quantification of Prescription Drugs in Oral Fluid
Paper Spray (PS) ionization is an ambient technique which creates ions in a mechanism similar to electrospray ionization. When coupled with a triple quadrupole mass spectrometer, quantification of analytes in a complex matrix can be achieved with no time consuming sample preparation or chromatography. This automated 3 minute multiple reaction monitoring method was used to quantitatively measure opioids, benzodiazepines, and tranquillizers at ng/mL levels in <10uL saliva. This PS method may be adapted for other biological matrices and the speed and simplicity of the technique is ideal for point of care diagnostics, roadside drug testing and many more clinical applications.
Validation of an Automated Method to Remove β-Glucuronidase from Hydrolyzed Pain Management Urine Samples
The escalating abuse of pain medicines has mandated the health care providers and practitioners to routinely perform urine drug testing. Most laboratories opt for a dilute-and-shoot approach, along with enzymatic hydrolysis that requires no sample clean up. The addition of extra protein to the urine can result in fouling of the LC column and loss of productivity. In this work, we present an automated method that uses an Impact Protein Precipitation plate to remove majority of the proteins from hydrolyzed urine.
A Fast, Sensitive, and High-throughput LC-MS/MS Assay for Benzodiazepines/Z-Drugs/Barbiturates
Benzodiazepines, Z-drugs, and barbiturates belong to a group of psychotropic drugs for the treatment of anxiety, depressant, and insomnia. However, these drugs have the potential for over dosage or abuse, which requires the development of fast and accurate methods for the screening and confirmation analysis. LC-MS/MS offers superior sensitivity, selectivity, and robustness for simultaneously detecting benzodiazepines and non-benzodiazepines in complex biological matrices. This work here presents a fast, reliable, and accurate LC-MS/MS method on an IONICS 3Q 120 triple quadrupole mass spectrometer with Restek Biphenyl column for the analysis of a total of 43 compounds, using fast polarity switching.
A Fast Polarity Switching LC-MS/MS Analysis of Benzodiazepines and Barbiturates
ESI-LC-MS/MS has been widely used to monitor pain management drugs on a routine basis in many labs worldwide. Because some of these drugs ionized better in negative mode than that in positive electrospray ionization mode, the panel of interest usually is split into positive mode and negative mode panels. However, recent advancement of fast and robust polarity switching technology allows high throughput reliable implementation of combining all drugs into one panel disregard of positive and negative ionization modes. Current study demonstrates a fast, robust polarity switching ESI-LC-MS/MS method on an IONICS 3Q 120 triple quadrupole mass spectrometer.
High-throughput Targeted Screening and Definitive Method for Barbiturate Drugs in Urine Using LDTD-MS/MS with Ultra-fast Analysis at 9 Seconds Sample to Sample
An ultra-fast quantitative targeted screening and a definitive confirmation method for the detection of Barbiturates drugs in urine have been developed for the LDTD-MS/MS system. Extraction is a liquid-liquid procedure at acidic pH. Organic upper layer is applied to the LazWell plate and dried prior to analysis. Mass spectrometer with ion mobility system is used for adequate isobaric drug resolution (amobarbital and pentobarbital) as confirmation method. Standard method validation was performed. All analyses were cross-validated with a LC-MS/MS method. Cross-validation results show low percentage differences between the two methods. All samples on LDTD-MS/MS were analyzed at 9 seconds per sample.
Development of a Whole Blood Microsampling Bioanalytical Method for the Analysis of Opiates with a Goal of Point-of-Care Therapeutic Drug Monitoring
Knowledge of steady state concentration in chronic opiate therapy is crucial to maintaining drug efficacy, minimizing breakthrough and ensuring proper compliance. To support steady-state measurements, conventinal blood sampling by venipuncture is needed and the patient is required to remain at the facility until sampling is complete. DBS sampling can potentially simplify this procedure, allowing at home sampling. We've evaluated a new microsampling device that eliminates some of the inherent issues with DBS. The device allows precise collection of 10uL volumes of blood. Commonly prescribed and/or abused opiates were quantified from whole blood sampled onto the microsampling device. Accuracy and precision were evaluated and the data support the potential in the clinical setting.
The Positive Inconsistent in Urine Drug Testing at a Community Specialty Pain Clinic
Urine Drug Testing (UDT) data assists clinicians develop a treatment plan, tailor specific drug panels, determine UDT testing frequency and validate medication therapy compliance or diversion in initiating and ongoing opioid therapy. A recent retrospective review of Soloniuk Pain Center UDT results showed that 28.8% of UDT in Redding, CA, and 36.0% of UDT in Red Bluff, CA were noncompliant. For this study, 480 consecutive samples were analyzed using the Waters liquid chromatography tandem mass spectrometer (LC-MS/MS). Out of the 50 drugs/metabolites, 28 were accounted for all of the inconsistent results. Out of the 14 drugs found in both clinics, the 6 common inconsistent positives with the highest incident percentages were THC (20.8%), morphine (13.4%), codeine (10.1%), lorazepam (9.4%), tramadol (6.7%), and oxycodone (5.4%).
Sensitive Measurement of HU-210 from Oral Fluids via LC-MS/MS: A Fully Automated SPE Sample Preparation and MS Sensitizing Derivatization Process
HU-210 is reported as 100-800 times more potent than THC. It is a challenging task to detect and properly measure HU210 from a human specimen at low levels of the drug exposure. We would like to report that by using a new MS sensitizing reagent (MB338), we developed an integrated method to quantify HU210 in oral fluid. The process included SPE, derivatization, and LCMSMS quantification via an AB SCIEX API5000. The range of the quantification linearity is between low pg/mL to ng/mL. This process is also compatible to quantify THC and THCA and may be fully automated.
Value Assignment of Candidate Standard Reference Material® 3949 Folate Vitamers in Frozen Human Serum by Isotope-Dilution LC-MS/MS
The National Institute of Standards and Technology (NIST) is developing a candidate Standard Reference Material (SRM) 3949 Folate Vitamers in Frozen Human Serum to replace SRM 1955 Homocysteine and Folate in Human Serum. Isotope dilution-liquid chromatography tandem mass spectrometry analysis at the Centers for Disease Control and Prevention and NIST will be utilized to assign values for folic acid and 5-methyltetrahydrofolate (5-mTHF), as well as several minor folates. Endogenous levels of folic acid and 5-mTHF in SRM 3949, enhanced folate stability via ascorbic acid addition, and additional minor folate values are improvements over SRM 1955 that should better serve customers.
A Novel 6x5 Peptide Mixture for Instrument Performance Monitoring
Performance monitoring and standardization of LC-MS/MS instrumentation continues to be challenging across all MS laboratories. Towards this end, we have prepared a peptide mixture and produced a novel software tool (PReMiS ™) that reports on LC column performance and MS instrument parameters (including sensitivity and dynamic range). In this current study we will use the reagent and software to optimize LC and MS methods, compare instruments across multiple laboratories and, compare different instrument types. The absolute sensitivity of the instruments (neat and spiked into complex mixtures) as well as a comparison of the peptide separation on multiple reversed phase columns will be presented. The reagent/software combination has also been critical for monitoring trends in instrument parameters.
Accuracy Evaluation of High- and Low-density Lipoprotein Cholesterol Assays in Clinical Laboratories by Comparison with Isotope Dilution Mass Spectrometry
We evaluated the performance of high-density (HDL) and low-density lipoprotein (LDL) cholesterol assays commonly used in Korea. Five levels of commutable frozen serum pools were measured with five routine assays (Toshiba-Kyowa, Hitachi-Sekisui, Siemens, Roche, and Beckman Coulter). Target values were measured with CDC reference method by gas chromatography-isotope dilution mass spectrometry. The mean bias of HDL cholesterol was 1.5~3.5 mg/dL (3.2~8.0%). The LDL cholesterol values of one material with high triglyceride of 330 mg/dL were significantly overestimated (20.4 mg/dL) in all assays, at both refrigerated and frozen state, but the degree was quite different depending on the assays.
Value Assignment of Vitamin D Metabolites in Vitamin D Standardization Program (VDSP) Serum Samples
Assay variability has been cited as an obstacle to establishing optimal vitamin D exposure. As part of the Vitamin D Standardization Program (VDSP) effort to standardize the measurement of total 25(OH)D, value assignment of total 25(OH)D in 50 single donor serum samples was performed using two isotope-dilution LC-MS/MS methods. Both methods are recognized as reference measurement procedures (RMPs). These samples and their assigned values serve as the foundation for several aspects of the VDSP. To our knowledge, this is the first time that two RMPs have been used to assign values to such a large number of serum samples.
Characterization of Stable Isotope Labeled Insulin-Like Growth Factor-1 for Use as an Internal Standard in Quantitative High-Resolution MS Workflows
High-resolution mass spectrometry (HRMS) methods for quantitative analysis of intact proteins in clinical samples are becoming more widely adopted. The accurate quantitation of a plasma protein in clinical applications is enabled by early introduction of an internal standard that behaves identically to the native target protein throughout the analytical workflow. Surrogate proteins are typically used as internal standards in HRMS assays, but stable-isotope labeled (SIL) proteins provide a more ideal alternative. We have characterized SIL-N15-IGF1 expressed in E. coli and will demonstrate the use of SIL-IGF1 as an internal standard in a quantitative LC-HRMS method for intact IGF1 in human serum.
Proteomic and Metabolic Changes in Malnutrition
Evidence suggests that the risk of death from infections in children with severe acute malnutrition may be sustained after apparent medical stabilization and nutritional rehabilitation. Biomarkers may allow identification of children at highest risk as well as identify physiological processes involved, thereby guiding development of new intervention strategies to reduce mortality in malnourished children. Using mass spectrometry we assessed the relationship between plasma protein and metabolite levels and subsequent survival of severely malnourished Kenyan children enrolled in a clinical trial of daily antimicrobial prophylaxis in severe malnutrition after apparent medical stabilization. Preliminary results will be presented.
Two-dimensional Liquid Chromatography Coupled to High-resolution Mass Spectrometry for Mapping LKB1 Dependent Signaling Networks in Non-small Cell Lung Cancer
Liver kinase B1 (LKB1) is a tumor suppressor gene that encodes for a key regulator kinase in cells. Somatic mutations of this gene lead to loss of its expression, which is commonly found in 30-40% of non-small cell lung cancer. However, the understanding of the role of LKB1 loss in the development of lung cancer is unclear. Online 2D LC-MS/MS is used to study LKB1-directed phosphorylation events at protein level that may reveal these interactions. It is the goal here to provide a comprehensive understanding of the underlying molecular biological changes specific to LKB1 loss, and to provide a clearer understanding of the process by which LKB1 loss leads to lung cancer.
Application of Nanoflow LC-MS for Targeted Proteomic Quantification of Cd39 and Cd73 in Human Calcified and Non-calcified Aortic Valves
CD39 and CD73 converts sequentially ATP to adenosine. The expression of enzymes could be crucial for pathology of aortic valves. Our aim was to develop method for quantification of ecto-enzymes in aortic valves and to compare it with immunohistochemistry. Nanoflow LC/MS analysis of tryptic peptides provided specific and sensitive method for quantitation of CD39 and CD73 protein. Values determined in calcified and non-calcified valves respectively, were: 0.72±0.19 and 1.03±0.44 pmol/mg (CD39) and 0.98±0.24 and 1.29±0.16 pmol/mg (CD73) that was consistent with immunohistochemistry. Nanoflow LC/MS proved effective for proteomic quantification of CD39 and CD73 and confirmed its depletion in valve pathology.
Mass Spectrometry-based Proteomic/Metabolic Studies of Hearts from ApoE/LDLR (--/--) Mice Reveal Potential Mechanism for Its Increased Susceptibility to Hypoxia
The aim of this study was to characterize global cardiac proteome changes in atherosclerotic ApoE/LDLR (--/--) mice and correlate these findings with changes in metabolic substrate preference and response to reduced oxygen tension. Hypoxia in ApoE/LDLR (--/--) mice caused maximum decrease of ST segment of ECG by 0.14 ± 0.02 mV while there was no change in ST segment (<0.01 mV) in control mice. Differential analysis of cardiac proteome revealed coordinated increased expression of mitochondrial proteins including those responsible for fatty acid oxidation. Metabolic experiments with [1-13C]-glucose were consistent with increased use of fatty acids.
Mass Spectrometric Quantification of Enriched Microglia Using a Metabolically-labeled Immortalized Cell Mix
Isobaric labeling requires a high speed, high resolution MS/MS scan for reporter ion quantification; this is not an option on all instruments. In order to quantify ethanol induced changes to microglial activation, we created a simulated proteome of enriched microglia using heavy metabolically-labeled immortalized cells to approximate protein expression in enriched microglia. Microglia from chronically ethanol exposed mice and control mice were mixed with equal amounts of the immortalized cell mix and analyzed on an Orbitrap XL. Over 2,500 proteins and 9,000 peptides were identified from the microglia, and of these 81% of proteins and 62% of peptides were successfully quantified in the control and ethanol treated groups. The immortalized cells proved to be a successful tool to quantify in vivo protein levels with earlier generation high resolution instrumentation.
Targeted Quantitation of 1-84 Parathyroid Hormone (PTH) by SID-MRM Mass Spectrometry
The measurement of PTH is used to assess hypocalcemia, hypercalcemia, metabolic bone disease, parathyroid gland tumors and intraoperative parathyroid hormone during parathyroidectomies. The bioactive amino acid sequence of PTH is 1-84. There are immunoassays specific for measuring PTH 1-84, however, these immunoassays can be susceptible to interference by cross-reacting PTH fragments. In this study, we developed a targeted quantitation method for PTH 1-84 using LC/MS/MS. The C-terminal peptide SLGEADKADVNVLTK was used as the surrogate peptide. PTH in normal patient samples was enriched by immunocapture, digested, and quantified by LC-MRM with a stable isotopically labeled IS.
Comparison of SDVB-Monolithic & Bead-based Columns Used in Nanoflow LC-MS for Proteomic Study
NanoLC-ESI-MS/MS is a powerful tool in proteomic analysis. The preparation conditions including the choice of monomer, inside diameter of capillary, and porogen is optimized. The performance of polymeric column was also compared and evaluated with micro particle-filled capillary columns, including a totally porous silica C18 column and a HALO® fused core C18 column. With all optimized conditions, the monolithic capillary column was prepared by in-situ polymerization of styrene and divinylbenzene (SDVB) inside a 4 meter-long, 50 µm i.d. fused silica capillary. This continuous unitary porous structure has more robust and high separation efficiency when comparing with the bead-based columns. The characterization of this novel monolithic media will be a promising addition to the stationary phase used in capillary column for proteome research.
Proteomic Platform for Comprehensive and Quantitative Urinary Proteomes
Urine is a desirable body fluid for clinical research as it can be obtained non-invasively in large quantities from every patient.
Here we build on recent developments in our group in the sample-preparation workflow to enable reproducible, parallelized and sensitive processing of urine proteins.
We identified a total of 3284 proteins in urine and calculated the LFQ values of 2200 proteins in average per sample, with MS-signals spanning 6 orders of magnitude.Remarkably, we identified 1354 proteins, which have not been reported in urine before.
Median coefficient of variation (CV) was 27% for technical replicates, which is excellent for label-free shotgun proteomics.
Our developments contribute to a robust and sensitive high-throughput urine proteomics platform which we hope will open urine proteomics to routine, quantitative analysis of patient samples in clinical settings.
Rapid and Robust Plasma Proteomics Platform for Clinical Settings
Mass spectrometry (MS)-based proteomics should in principle be an ideal technology to discover disease indicators in the blood plasma proteome. Recent developments in our group such as the ‘in StageTip’ (iST) sample-preparation workflow and optimized high-throughput LC-MS measurement technologies enable fast and multiplexed sample processing with very high reproducibility (R2 ≥ 0.99) and low coefficients of variation (CV < 20 %) for the majority (81 %) of the top 200 quantified proteins. We anticipate that these developments will facilitate MS-based blood plasma proteomics for routine, quantitative analysis of patient samples in clinical settings.
Identifying the Proteome of Different Mycobacterial Species Using Orbitrap™ Mass Spectrometry
Advances in next generation sequencing, of whole bacterial genomes, have led to a plethora of useful genetic data. However, these data often lack correlation with expressed protein profiles. It has been well demonstrated that even very closely related genomes express drastically different phenotypes which often have major roles in pathogenicity. Therefore, it is just as important to have a method for examining the proteome of a bacterium. We setout to develop a method for the identification and characterization of the mycobacterial proteome using reversed-phase liquid chromatography and a Q Exactive™ Plus Orbitrap™ MS with both Top-down and Bottom-up approaches.
Phosphoproteomic Analysis by HAMMOC Enrichment and LC-MS/MS
The combination of HAMMOC enrichment and LC-MS/MS analysis were employed for the phosphorproteome analysis in this study. A total of 2953 phosphopeptides and 1231 phosphoproteins were found in milligram proteins of RAW264.7 cell lysate by combining two desalting methods coupled with HAMMOC-sIEF approach. The use of centrifugal filter and HAMMOC coupled with three various fractionation strategies, including sIEF¡BSCX and HILIC, had shown good complementarity (86.8% more phosphopeptide identifications than SCX only). Finally, 114 phosphoproteins were found differentially expressed between control and lipopolysaccharide-treated RAW264.7 from 200 microgram proteins by using label-free comparative approach after HAMMOC and LC-MS/MS. Results indicate that HAMMOC followed by LC-MS/MS analysis is suited for the in-depth analysis of qualitative and quantitative phosphoproteome profiling.
Differential Proteomic Analysis of PLC/PRF/5 Cell Lines Treated with Various Anti-cancer Drugs by iTRAQ Labeling and Mass Spectrometry
Sorafenib, sunitinib and tivozanib are oral small molecular VEGF receptor tyrosine kinase (RTK)-targeted drugs, and two of them are approved to treat anti-angiogenesis. The iTRAQ technology was used to investigate the protein profiles in PLC/PRF/5 cell lines treated with these three anti-cancer drugs. A total of 11233 unique peptides were identified which were associated with 2010 proteins in two biological replicate experiments. Among them, 187, 112 and 128 differential expressed proteins were selected for Metacore analysis. They are found to be associated with oxidative phosphorylation, cytoskeleton remodeling and transcription role of AP-1 in regulation of cellular metabolism.
Evaluation of Two LC-MS/MS Thyroglobulin Assays Performance in the Presence of Anti-thyroglobulin Autoantibodies
Thyroglobulin (Tg) measurement by MS is attractive due to its insensitivity to Tg autoantibodies (TgAB) interference, a problem observed with immunoassays (IA) and radioimmunoassays (RIA). Standardization of Tg-MS assays is paramount for clinical adaptation. Two Tg-MS assays were compared across Tg and TgAB concentration ranges. Mixtures controlling Tg and TgAB levels were run to assess effects in IA/RIA vs. Tg-MS. Tg-MS methods correlated well (Slope = 1.1; R2 = 0.97), with minimal variation in Tg concentration (-0.2 to +0.2-fold) while IA showed significant deviation from expected Tg concentrations (-0.3 to -7-fold, and +0.3 to +10.7-fold in IA and RIA, respectively).
Analysis of COL6A Proteins as a Potential Therapeutic Marker for Ullrich Muscular Dystrophy and Bethlem Myopathies
Abnormalities in the extracellular matrix are common in human muscular dystrophies (MD). Mutations in COL6A are prevalent causes of congenital MD such as Ullrich MD, Bethlem myopathies and yet poorly defined intermediate form. Most of the COL6A mutations lead to altered extracellular deposition of COL6A: thus COL6A proteins have enormous potential in monitoring and evaluating efficacy of new therapeutic trials for COL6A-related MD. Approaches to accurately access the level of COL6A in cell-based assays are critically needed. Here, we propose that a LC-SRM-MS assay can detect the cellular level and extracellular release of COL6A in human fibroblasts and cultured media.
A Hybrid Approach to Proteomic Sequencing of Immunoglobulins
Primary sequence determination of an antibody is a precursor to accurate characterization. When the source cell is unavailable, direct proteomic sequencing is the only option. Unlike other proteomic applications, no comprehensive database of antibody sequences exists. We utilize a hybrid approach that combines the strengths of database-mediated peptide identification and de novo protein sequencing in order to sequence an antibody and determine sites of common post-translational modifications. In this study we demonstrate how new acquisition modes that combine CID, ETD, and HCD fragmentation can improve sequencing accuracy. In addition, we expand on our algorithm to sequence a mixture of a heavy and light chain as well as a mixture of three monoclonal antibodies.
Free Urinary Light Chain Analysis Assisted via Proteolytic Cleavage and LC-MS/MS
In 1847, Dr. Bence Jones described excess protein in the urine of a patient with cancer. Noting that the protein, later discovered to be immunoglobulin light chains, precipitated when warming urine from 40 to 60oC, he thus developed the first method for detecting a cancer marker in bodily fluids. Today, light chains are characterized or measured by clinical labs using techniques such as gel electrophoresis and immunoassay. Recent literature has questioned the validity of quantitative results measured by immunoassay. Therefore we developed a liquid chromatography tandem mass spectrometry method to measure κ and λ free light chains, which employed proteolysis.
Profiling of the Mucosal Metabolome by the DESI-MS of Medical Swabs – New POC Diagnostic Approach for Infections, Dysbiosis and Immunological Diseases
Medical swabs are standard sampling devices in the diagnostics of various diseases, but also used for DNA collection. Processing protocols for swabs are narrowly targeted (e.g. PCR testing for Chlamydia) and also time consuming with reporting times of 3-4 days. Alternatively, medical swabs can also be analysed by DESI-MS. The mass spectrometric data – acquired in the timeframe of 20-30 s after sampling – features mostly metabolite- and lipid-type constituents including bacterial secondary metabolites and human inflammatory mediators. The spectral information was successfully used for the identification of infectious agents and stratifying allergy patients in case of pharyngeal and nasal mucosa, respectively.
The Measurement of Food-intake and Nutrient Absorption in C. elegans by Quantitative Mass Spectrometry
Directly measuring food intake in C. elegans remains challenging, despite it being a preferred genetic model system for studying food-related behaviors. Our “pulse-feeding” method measures food intake by feeding heavy isotope labeled E. coli “food” and then quantifying nutrient absorption by mass spectrometry. The pulse-feeding assay allows absorption of food into the proteome and changes in protein synthesis to be monitored. We show that serotonin induced feeding leads to an increase in protein synthesis in a serotonin receptor dependent manner. The pulse-feeding assay can be an important tool for comparisons of the effects of genetic and pharmaceutical perturbations on eating.
Mapping c-Src Phosphorylation Sites as Potential Disease Biomarkers
Protein post-translational modifications (PTMs) are essential in regulation of many physiological processes and dysregulation of these modifications is usually indicative of disease states. Therefore, mapping PTMs in signaling proteins has become a powerful tool to discover biomarkers for disease diagnosis and progression. Here, we mapped the phosphorylation sites on c-Src using LC/MS/MS and will further use the identified sites to develop MRM-based approaches to monitor the phosphorylation status of c-Src in different cancer biopsies for biomarker discovery. These MRM methods are readily transferrable to other tissues and across instrument platforms and are scalable to search for novel biomarkers.
The Fastest(msec.), Most Efficient (100%) ESI/MS Sample Introduction Tool that Also Produces Excellent MALDI Crystals
ESI and MALDI have various imperfections.
For ESI, electric field lines from the surface of cone jets are inherently dispersive directing most of the sample away from the MS orifice. Also,coulombic interactions of like charged droplets, repel each other limiting LC/ESI sample introduction efficiency.
For MALDI, uL volume samples take up to a minute to dry producing large crystals that phase separate, often yielding poor quality, large crystals, in coffee ring "structures" producing noise and lower than optimal sensitivity. Primitive devices used to "manually" deposit volatile liquids, present an irreproducible sample preparation approach.
In 14 peer review papers users have shown that one IBF approach solves both dilemmas increasing sample introduction efficiency to 100% for ESI or producing excellent MALDI crystals, with major (10-100x)increase in sensitivity for MALDI and SIMS,LITERALLY.
HPLC-UHPLC Hybrid 2D Platform for LC-MS Analysis of Biological Samples. Back to the Future
We developed a cost-efficient hybrid LC platform that’s ideal for biological sample analysis by LC/MS. This platform is based on Agilent 1100 and Agilent 1290 series LC devices. The first dimension of the analytical system used a standard pressure binary HPLC pump, where the sample was injected using a standard analytical autosampler into a pre-analytical column.
The fraction containing analytes of interest was transferred through a UHPLC valve to a fused core column. We successfully used this platform for LC/MS analysis. Instead of retiring an entire functioning Agilent 1100 LC system, we just added one UHPLC pump to achieve much greater overall performance, functionality and lower cost, compared to a single pump UHPLC system purchase.
Optimization and Validation of Online SPE-UHPLC-MS/MS for Trace Level Quantitation of Bisphenol A Analogues in Human Urine
California enacted legislation banning the use of BPA in baby bottles. Manufacturers are considering BPA analogs or derivatives as replacements for BPA in various applications. In the present study, mass spectrometry parameters were optimized to achieve the lowest possible instrument limit of detection. In addition, on-line solid phase extraction protocol was optimized for maximal absolute recovery rates and high throughput. Initial demonstration of capability and control charts were established.
Rapid Quantification of Free and Glucuronidated THC-COOH in Human Urine Using Coated Well Plates and Column-switching LC-MS/MS
Cannabis is the most commonly encountered illicit substance in workplace urine drug testing. Generally, cannabis consumption is detected by measuring the total concentration of the urinary THC-COOH metabolite obtained by hydrolyzing THC-COOH-glucuronide. To meet the workplace drug testing demands regarding rapid throughput, automation and low costs, we have developed a novel high-throughput method for the direct quantification of THC-COOH and THC-COOH-glucuronide in urine. Our method is based on minimal sample preparation employing Tecan AC Extraction Plates and subsequent quantification by two-dimensional chromatography and tandem mass spectrometry. The presented method was successfully validated and applied to several hundred authentic urine samples.
Rapid Quantitative Analysis of 25-OH Vitamin D2 and D3 in Patient Serum Using a Novel Weak Anion Exchange Disposable Pipette Extraction (DPX-WAX) and LC-MS/MS
The increased clinical interest in vitamin D analysis has meant an increased demand on hospital laboratories, requiring quicker and more efficient analysis methods. To this end, we present a novel and rapid method for the extraction of vitamin D2 and D3 using disposable pipette extraction with weak anion exchange (DPX-WAX) and LC-MS/MS detection, evaluated against conventional liquid-liquid extraction in routine patient samples. The limits of detection were determined to be >10 and >5 µM for Vit D2 and D3, respectively. The average recovery was 103% with a mean CV of 13.6 and 7.6% for vitamin D2 and D3, respectively. Coefficients of determination (R2) were greater than 94% between the extraction techniques, with a proportional bias of 1.09-1.12%. These results indicate that DPX-WAX is effective for vitamin D analysis in routine laboratories.
High Throughput Determination of Multiple Drugs in Plasma and in Blood Using Solid Phase Microextraction
Solid phase microextraction in high throughput thin film format (TF-SPME) has demonstrated its potential and usefulness as sample preparation tool in multiple areas of application, including the bioanalytical field. In this study, an analytical protocol for the determination of multiple prohibited drugs in plasma samples using TF-SPME and liquid chromatography-tandem mass spectrometry (LC-MS/MS) is presented. Additionally, SPME devices prepared on an alternative material as a support are introduced and evaluated by extracting selected drugs from whole blood. Overall, results herein presented demonstrate the great convenience of SPME as sample preparation tool in the clinical lab.
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use
Two forensic LC-MS/MS methods to detect buprenorphine and/or ethanol uses were run on a multichannel UHPLC system utilizing positive-displacement pumps. A maximum throughput of 34 urine samples per hour was achieved. Desired quantitation ranges, accuracy and repeatability criteria were met for each application. Internal standard (IS) peak areas showed less than 25% coefficient of variation (CV) among calibrators, QCs and specimens (n = 20) on any of the four channels. Retention time variations throughout these batches were less than 3% CV. Results were within +/- 15% of those determined on a conventional multichannel system.
Enhancing Ion Abundances and Spatial Homogeneity of Glycans by Regulating the Substrate Temperature in MALDI MS: A Physical Chemistry Perspective
Identifying glycans provides critical insights into the pathogenesis of a cancer. Mass spectrometric analyses, especially matrix-assisted laser desorption/ ionization mass spectrometry (MALDI MS), are the major tools for the identification of glycans. However, owing to the thermally labile nature, ionization efficiency, and heterogeneity of ion population, the identification of glycans in MALDI MS is still less effective. A laboratory-built sample preparation compartment was built to facilitate a rapid regulation of the substrate temperature. This preparation method successfully enhanced the abundances and eliminated the spatial heterogeneity of ions. A theoretical simulation was conducted to discuss the homogeneity of ion populations.
Quantitative Analysis of IGF-1 Using Online Digestion Coupled to the Triple Quad LCMS-8050
Automated digestion was used to accelerate sample analysis for a quantitative protein workflow analyzing the clinically important protein IGF-1. Digestion using a Perfinity Workstation was carried out in 4 minutes. The total run time (Workstation to Shimadzu LCMS-8050) was 20 minutes. Four peptides (10 transitions) were monitored. Digestion and LC-MS parameters were optimized using commercial standards, and the assay was evaluated using acetonitrile serum extracts spiked with IGF-1. IGF-1 was detected from 100 – 1000 ng/mL with r2 values >0.99. Accuracy was verified using a blinded “unknown” sample. This work demonstrates the feasibility of online digestion automation for IGF-1 analysis.
Sample Preparation: The Achilles Heel of Rapid Mass Spectral Analysis
This presentation will describe a microfluidic membrane system in which human plasma is extracted from a drop of blood by capillary action and a 2.5 uL sample aliquot prepared for MS analysis through internal standard addition, immunosorbent binding, and chemical modifications, all within a single membrane card without an energy source. This allows self sampling at remote sites via a finger-stick, a substantial degree of sample preparation within a single membrane bearing card, and dry transport of prepared samples to an analytical laboratory on a collection disc smaller than a postage stamp.
Effect of Enzyme Source, Form and Hydrolysis Conditions on the Conversion of Glucuronide Drug Metabolites in Urine to Parent Drugs by β-Glucuronidase
β-Glucuronidase (β-D-glucuronide glucuronosohydrolase) enzymes play an important role in the analysis of biological fluids for the presence of drug metabolites for drug screening and drug metabolism studies. Although there are numerous β-glucuronidase enzymes available, each enzyme has optimum conditions to which hydrolysis of glucuronide metabolites can be effectively conducted. Variables such as β-glucuronidase enzyme concentration, digestion pH, incubation time and temperature all play an important role for effective hydrolysis of glucuronide metabolites. In this study, critical variables are evaluated for determining an appropriate β-glucuronidase enzyme and incubation conditions.
Simple Sample Preparation for Measuring Methylmalonic Acid in Blood Serum by LC-MS
Many researchers need to measure methylmalonic acid (MMA) in blood serum to assess vitamin B12 sufficiency. Most perform sample preparations that involve laborious and expensive solid-phase or liquid extraction followed by derivatization to form the butyl ester. Butyl-MMA can then be measured by LC-MS or GC-MS. We developed a simpler approach involving protein precipitation with acetonitrile followed by evaporation of the supernatant. After the residue was butylated and the remaining butylation reagent was evaporated, the residue was reconstituted with a water/methanol diluent and subjected to a five-minute LC-APCI-MS/MS method reliably achieving a quantitative range of 50 to 1000 nM.
Improved Method for the Analysis of 31 Drugs of Abuse/Pain Management Panel in Oral Fluid Samples Using the Thomson EXtreme® Filter Vials by LC-MS/MS
The goal of this study was to improve the sample preparation for the analysis of drugs of abuse in oral fluids. Oral fluid samples were collected with Intercept® i2he™ Oral Fluid Collection Devices. The samples were prepared using eXtreme|FV®, followed by LC/MS/MS analysis. The most critical aspects of reliable analysis are the reduction of interferences from the sample matrix and analyte recovery. Traditionally, SPE, SLE and centrifugation have been used to reduce matrix interference. These techniques are time consuming, adversely impact recovery, use large amounts of solvent and are expensive. The improved method allows for the analysis of 31 drugs.
Quantitation of 17β-Estradiol in Serum by LC-MS/MS: Achieving 2 pg/mL Sensitivity Using an Aggressive Sample Preparation Procedure
A method for the quantitation of 17β-Estradiol in human serum, suitable for application on a LC-MS/MS clinical system, is examined. The sample preparation procedure includes a liquid-liquid extraction and derivatization in dansyl chloride, followed by a solid phase extraction. This procedure ensures maximally efficient extraction of the analyte from the serum while significantly reducing matrix interferences in order to achieve the clinically necessary 2 pg/mL sensitivity. The linear range of the assay is demonstrated as 2-5000 pg/mL with a typical patient CV of <7% and excellent calibration agreement (<2% bias) with the Center for Disease Control’s Hormone Standardization (HoSt) program.
Comparison of Different Liquid-liquid Sample Preparations for LC-MS/MS Assays of Total Serum Testosterone Measurements
The removal of protein binding and phospholipids is usually carried out in two steps in the reference method of total testosterone measurements. In the present study, a simpler approach of one-step liquid-liquid extraction is examined against the reference procedure. QCs and unknown samples were compared in terms of interference, accuracy, recovery and lipid removal. The difference of accuracies of QCs and values of unknown samples between the simple procedure and the reference one is less than 5%. This simple procedure could replace the reference procedure for the accurate measurement of testosterone in human serum.
iST Sample Preparation for High Throughput Clinical Proteomics
Rapid and robust workflows are crucial for day-to-day clinical applications. Especially sample preparation procedures are time consuming and limit the overall technical reproducibility of MS-based proteomics. Here we present the in-StageTip (iST) method for streamlined sample processing of complete proteomes. This simplified 3-step procedure is performed in a single, enclosed volume and allows peptide pre-fractionation in a high-throughput fashion. Applying the procedure to the cancer cell line HeLa allowed us to estimate copy-numbers of 9,667 proteins with excellent reproducibility (R2 = 0.97) in quadruplicates measurements. The in-StageTip method allows high-throughput applications with near complete proteomic coverage of highly complex samples.
Fast, Simple Method for the Analysis of Benzodiazepines in Meconium and an Interlaboratory Method Comparison
A novel method for the quantitation of 10 commonly prescribed benzodiazepines and/or their metabolites, 7-aminoclonazepam, clonazepam, α-hydroxyalprazolam, alprazolam, nordiazepam, diazepam, midazolam, oxazepam, lorazepam, and temazepam, in meconium was developed using enzymatic hydrolysis, WAX-S dispersive pipette extraction (DPX) tips, and LC-MS-MS analysis. The method was linear over a range of 5 ng/g to 1000 ng/g with all correlation coefficients above 0.99. Within-run, between-run, and total imprecision were evaluated; all fell below 13% CV. A blind study with a corresponding laboratory of 35 positive patient samples resulted in approximately 92% correlation, with 100% accuracy in qualitative results.
A Fast and Effective Method for Quantitation of Vitamin A & E from Human Serum Using Novum SLE
Dietary intake of vitamin A and E may be reduced in individuals who have undergone a bariatric procedure. Monitoring the serum concentration of the two micronutrients is an effective way to help maintain a proper healthy condition in affected individuals. We present a simple and effective procedure to extract vitamin A and E from human serum by a SLE (solid liquid extraction) procedure. The quantitation is carried out under reversed-phase chromatography and APCI-LC/MS/MS.
A Rapid Sample Preparation Method for Quantitative Analysis of Cortisol in Saliva and Urine by LC-MS/MS
Late night saliva and 24-hour urine cortisol are among the initial screening tests for cortisol excess [i.e. Cushing’s syndrome or disease]. Early morning saliva cortisol may be used to assess and rule out adrenal insufficiency [i.e. Addison’s syndrome]. Since conventional LC-MS/MS approach requires lengthy manual sample preparation by liquid-liquid extraction [LLE] or solid phase extraction [SPE], we investigated commercially available dispersive pipette extraction tips [DPX tips] to shorten this preparation time. Results were comparable with a LC-MS/MS method in clinical use and may reduce sample preparation time up to 80%, making it much more suitable for a clinical diagnostic lab.
Sample Preparation of Three Steroids for Quantitative Determination by LC-MS/MS – Comparison of Two Extraction Procedures
Two sample preparation procedures for the steroids testosterone, androstendione and 17á -hydroxyprogesterone using commercially available extraction plates were automated on a Liquid Handling System. The performance between a SLE-plate for supported liquid extraction and the AC Extraction Plate™, a deep well plate having wells coated with an absorptive material acting as the extraction phase, was compared. Plasma calibrators and controls as well as human plasma samples were used to determine linearity, accuracy, precision and LOD. The overall handling of both procedures on a Tecan Freedom EVO® liquid handling system was evaluated.
Analysis of Aldosterone in Plasma for Clinical Research Using Automated Extraction
Here we evaluate a newly developed UPLC-MS/MS method for the measurement of plasma aldosterone for clinical research purposes. An analytically sensitive method was developed using a mixed-mode Solid Phase Extraction (SPE) sorbent in 96-well plate format. Automated extraction was employed, enabling high throughput of samples. Analysis was performed using an ACQUITY UPLC® I-Class system, with samples injected onto a 2.1 x 100mm Waters CORTECS UPLC C18 column, separated using a water/methanol gradient and quantified with a Waters Xevo® TQ-S mass spectrometer to obtain quantitative measurement of aldosterone at high sensitivity.
For Research Use Only, Not for use in diagnostic procedures.
An Automated Sensitive Measurement of Estrone and 17β-Estradiol from Human Plasma on LC-MS Using Solid-Phase Extraction and MassBoost Derivatization
Measurements of estrogen levels in plasma are difficult due to their low abundance and lack of ionizable groups. A sensitive method was developed to quantify estrone (E1) and 17β-estradiol (E2) in 100 uL plasma. The process includes solid phase extraction, derivatization via a new reagent, and quantification via LC-tandem mass spectrometry on an API 4000 QTrap MS coupled with an Agilent 1200 HPLC. A linear response to both compounds was observed from 5-500 pg/mL, with an LOQ of 5 pg/mL for estradiol and 10 pg/mL for estrone. Throughput for this method was further improved by coupling it to automated sample preparation.
Optimization and Validation of Cannabinoid Metabolite Confirmation in Urine Using LC-MS/MS and Biotage EVOLUTE EXPRESS AX SPE Cartridges
Solid Phase Extraction (SPE) has shown to be an effective process for sample clean-up and concentration of THC, which is a common illicit found in clinical urine samples. We present an initial validation of the Biotage EVOLUTE EXPRESS AX cartridge for THC-COOH, which omits a pre-conditioning step and allows for direct elution into auto-sampler vials. We also validated previously published strategies to reduce non-specific adsorption of this analyte. Results were comparable to a previously validated SPE product and techniques adopted in this method provided for a robust and precise extraction procedure for analysis by LC-MS/MS.
Comparison of 25-hydroxy Vitamin D Extraction Using Supported Liquid Extraction and Phospholipid Depletion Plate Technology Prior to LC-MS/MS Analysis
Vitamin D analysis has extremely important clinical relevance. Many sample preparation approaches to the extraction of 25-hydroxy vitamin D have been employed prior to LC-MS/MS analysis. This poster compares the use of supported liquid extraction and a novel protein and phospholipid depletion plate, for the extraction of 25-hydroxy-vitamin D from serum. Optimum protocols were compared for recoveries, extract cleanliness, calibration line performance using PBS/BSA, calibrated serum samples and DEQAS external quality control samples. The extraction protocol was ultimately transferred to an SPE automation platform and method performance versus manual processing compared.
Determination of Testosterone in Serum by Automated Sample Preparation and Ultra-fast LDTD-MS/MS in a Cross Validation Study with Real Patient Samples
A fast and quantitative method for the Testosterone analysis in serum samples is applied to the LDTD-MS/MS with differential ion mobility system. Testosterone is extracted using the new AC extraction plate in an automated 3 step process on a TECAN robot system. Mass spectrometer with ion mobility system (SelexION™) is used for isobaric drug resolution. Validation criteria such as carry over, wet stability and quality controls were successfully evaluated. All analyses were cross-validated and compared with a LC-MS/MS method. The cross-validation results show low percentage differences between the two methods. All samples were analyzed in 9 seconds per samples.
Evaluation of Methylisothiazolinone (MI) Extraction from Sunscreen Using Supported Liquid Extraction Prior to GC/MS Analysis
Methylisothiazolinone (MI) has received widespread attention over previous months due to a number of allergic reactions reported from the use of various personal care products such as sunscreens and skin lotions. This poster demonstrates the development of a simple sample preparation protocol using supported liquid extraction columns prior to GC/MS analysis. The final method demonstrates good extraction efficiency, extract cleanliness and acceptable linearity across the required analytical range. Calibration curves were constructed spiking MI into sunscreen from 50-750 ng/mL. Although no internal standard was used, good coefficients of determination (r2) greater than 0.99 were demonstrated.
Catecholamine Analysis: Evaluation of Method Optimization to Improve Sensitivity and Reduce Limits of Quantitation Using LC-MS/MS
Catecholamines are classic biomarkers for the detection of diseases like hypertension, pheochromocytoma and neuroblastoma. The main target analytes are epinephrine, norepinephrine and dopamine and are traditionally analyzed using liquid chromatography with electrochemical detection. This poster investigates various parts of the method development process to evaluate the sensitivity of LC-MS/MS analysis. A highly sensitive LC/MS system, a Shimadzu Nexera UHPLC coupled to an AB SCIEX 5500 triple quadrupole MS was used for analysis. Method sensitivity in terms of pre-cursor ion selection and MRM transitions, chromatography and solid phase extraction protocols were all evaluated for increased sensitivity.
Extraction of Buprenorphine and Norbuprenorphine from Urine Samples Using New Nbe™ (Narrow Bore Extraction) Columns: Fully Automated Sample Preparation
Sample preparation in clinical and toxicology laboratories has historically been very labor intensive. Individual portions of the analytical process, from sample accessioning to the instrumental analysis itself, have been streamlined and accelerated, but a comprehensive unattended process from sample aliquots to ready-to-analyze extracts has not been shown. Here, we present a process for robotic pipetting of urine samples, on-deck hydrolysis, automated extraction of the hydrolyzed samples, and subsequent analysis of the extracts for the presence of buprenorphine and norbuprenorphine. Analyses of the extracts were performed on an AB Sciex 5000 LCMS system with focus on linearity and LLOQ measurements.
Versatile Platform for Fully Automated Sample Preparation of Forensic Whole Blood for LC-MS Analysis
A robotic setup for efficient and fully automated sample preparation of forensic whole blood for LC-MS analysis was developed based on a Tecan Freedom EVO liquid handler. The platform was extended with devices such as a balance, centrifuge and evaporator and is capable of processing whole blood from 96 primary tubes into injection ready extracts in a couple of hours. Automated steps include weighing of aliquots of whole blood for each sample, addition of internal standard, pretreatment by centrifugation, solid phase extraction, evaporation of eluate and finally reconstitution. Results from validated methods in routine production for toxicological screening and quantification of drugs of abuse in whole blood will be presented.
Converting a Liquid-liquid Extraction Method for Vitamin D to a 96-well Plate Supported-liquid Extraction Format: A Case Study with Real Patient Plasma Samples
The objective of this study was to transfer a validated liquid-liquid extraction method to a supported liquid extraction method to leverage advantages in reduced workflow and support a throughput of ~550 samples/month. During this evaluation, it was determined that sample processing time was reduced by ~50% for a de-identified patient set (n=30). To optimize the method to maximize analytes sensitivity, pH adjustment in the sample pre-treatment, elution volumes for extraction, and MRM transitions for analytes detection were evaluated. Once optimized and validated, method was applied to real patient sample sets. The samples were split and processed by each method for verification.
FFPE Protein Recovery and Optimization for Proteomics Analysis
Mayo Clinic has amassed a large archive of Formalin Fixed Paraffin Embedded (FFPE) tissue blocks from a number of different diseases. Protein profiling of such FFPE tissues offers a valuable opportunity to obtain new information regarding the molecular mechanisms of disease. However, the analysis of proteins from FFPE samples is both complex and challenging. With this in mind, we have designed a study to systematically investigate multiple different sample preparation methods to optimize a workflow for the proteomic analysis of FFPE tissues.
Multiplexed Analysis of apo A1, apo B and apo E in Normo- and Hyper-triglyceridemic Specimens Using the Automated SISCAPA-MRM Workflow
Measuring LDL and HDL cholesterol has been the gold standard method for risk estimation in cardiovascular disease (CVD). However, multiple reports indicate that measuring the ratio of apolipoprotein-B100 to apolipoprotein-A1 provides a more accurate risk assessment, especially in patients suffering from hyperlipidemia. Apolipoprotein-E is emerging biomarker for CVD. Here, we present a multiplexed, automated SISCAPA assay for precise measurement of apoA1, apoB100 and apoE both in liquid specimens (e.g. serum) and in dried-blood-spots. We aim to explore the value of longitudinal sample collection for monitoring lipid profile and ultimately cardiovascular health on an individual basis.
Simultaneous Quantification of 17-β-oestradiol and Oestrone in Human Plasma by LC-MS/MS
A simple, rapid and sensitive procedure for the simultaneous quantification of 17-β-oestradiol and oestrone in serum and human plasma using a combination of liquid-liquid extraction and liquid chromatography-tandem mass spectrometry (LLE-LC/MS/MS) has been developed and validated. Employing standards and quality control samples prepared in a matrix of human plasma and charcoal stripped serum, the analytes were isolated and sub-sequently chromatographically separated on a 150 x 2.1 mm column under reversed-phase conditions. The LLE-LC/APCI/MS/MS procedure possesses good accuracy, specificity, sensitivity and precision, and may be adopted as a convincing alternative to immunological approaches for the measurement of oestrogens in routine investigations.
A Novel Analytical Method to Analyze Phosphatidylcholine in Human Breath Using UHPLC-MS/MS
Phosphatidylcholine (PC) is an important phospholipid of a lung surfactant. Dipalmitoylphosphatidylcholine (DPPC) is the most abundant form of PC, and is believed to be the most important pulmonary surfactant. Exhaled breath contains non-volatile substances including PC. Current analytical method do not account for analyzing the DPPC in human breath. This is the first time, we validates a LC-MS/MS method capable of analyzing DPPC (PC 32:0) and PC 16:0/18:1 in exhaled breath. The particles in exhaled breath were collected in a polymer filter and subsequently, 6.5 and 16 pg/filter of MLQs can be achieved for PC 32:0 and PC 16:0/18:1, respectively.
Profiling Sialylation Status of Macrophage Upon Cell Activation
Sialic acids (SAs) are widely expressed on immune cells and their levels and linkages named as sialylation status may vary upon cell activation related to either physiological or pathological processes. In this study, we globally profiled sialylation status of macrophages upon activation. LC-MS/MS results showed cellular SA increased from 369 to 1.08×103 ng/mL after cell activation. This result was supported by the increase of α-2,6 linked SAs on the cell surface measured by flow cytometry and confocal microscopy. Results of this work will contribute to a better understanding of the physiological and pathological roles of SAs in the immune system.
Small Molecule Analysis Using MALDI-TOF MS with Solid Nanostructure Matrices
TiO2 nanowire arrays and functional nanoweb matrices were synthesized and applied to MALDI-TOF mass spectrometry for the analysis of small molecules from human serum and milk samples qualitatively. TiO2 nanowires were synthesized by top-down hydrothermal process, and functional nanoweb matrices were synthesized by simultaneous process of electrospinning of nanoweb and electrospraying of TiO2 nanoparticles on the metal target plate. The feasibility of applying solid matrices to MALDI-TOF MS was demonstrated by the analysis of short peptides (leu-enkephalin) and amino acids. Amino acid in human sera and antibiotic drugs in milk were analyzed qualitatively and quantitatively using synthesized solid matrices.
Altered Adrenal and Gonadal Steroids Biosynthesis in Patients with Burn Injury
Previous studies have reported gender differences in outcome following burn injury, suggesting that steroids may play a role. For this single-center, retrospective descriptive study, we used high-sensitivity liquid chromatography tandem mass spectrometry (LC-MS/MS) based steroid quantification to determine endogenous steroid concentrations in plasma samples of male patients with burn injury. Our data indicate that burn injury alters endogenous steroids biosynthesis, with decreased testosterone concentrations and elevated estrone concentrations during the first 21 days after the injury. Several of the measured steroids were found to correlate positively with the area of the burn injury. Further studies are needed to delineate the underlying mechanisms behind alterations in steroid biosynthesis after burn injury.
Drug Metabolism and Pharmacokinetics (DMPK) Utility of Mass Spectrometry in Discovery of Small Molecule Drugs
Mass spectrometry (MS) is crucial in determination of drug-like properties of small molecules in drug discovery. Medicinal chemists have tried to improve the odds of discovering viable development candidates by producing higher number of compounds. DMPK groups have to mirror that increased productivity to prevent bottlenecks. MS is used as a primary tool to study several in vitro and in vivo attributes of newly synthesized compounds. Creative utility of MS has enabled generation of high quality data in an efficient, timely, and cost effective manner. Qualitative and quantitative MS approaches and their specific utility in discovery DMPK will be discussed in this presentation.
A Practical and Sensitive Plasma Catecholamines Measurement via LC-MS/MS After an Integrated Solid Phase Extraction (SPE) and Derivatization Sample Preparation
Clinical measurement of plasma catecholamines are a challenge due to the low abundance and chemical instability of the endogenous analytes. We have developed a robust and sensitive plasma catecholamines measurement process which provides linear responses at 6-200pg/mL for dopamine, epinephrine and norepinephrine. This process integrated a solid phase extraction (SPE) sample preparation with an elution, derivatization, and direct shoot (EDDS) procedure. Finally all three analytes were separated and quantified via LC-MSMS. The assay took 200uL of plasma sample volume, the sample preparation was done on an automated liquid handler, and the quantification was carried out on an AB Sciex API 5000 Tandem Mass Spectroscopy coupled with Shimadzu LC-20AD LC pumps.
High Throughput Measurement of Five Tobacco-specific Nitrosamines in Urine by Automation and Liquid Chromatography–mass Spectrometry
A high throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method with robotic sample preparation and automatic data processing was developed and validated for the determination of five total tobacco-specific N-nitrosamines (TSNA), including both free and conjugated forms in urine. The sample size was decreased from previously reported 5 ml to 1.7 mL. The limit of detection for NNAL), NNN),NNK,NAT and NAB were 0.6, 1.8, 12.0, 2.0and 1.6 pg/mL respectively, with a linear calibration range of up to 20,000 pg/mL. The new robotic sample preparation method decreased the matrix effects and increased the throughput. The automatic data process by using Indigo Ascent™ combined peak integration and quality assurance,resulting in improved precision and reproducibility.
Medicinal Cannabis and the Need for Enhanced Cannabinoid Profiling
Medicinal cannabis refers to the use of cannabis as a therapy to treat diseases and/or alleviate symptoms. The cannabis industry is projected to be an $8B industry by 2018. With this growth has come an explosion in cannabis testing labs. This presentation will provide an overview of medicinal cannabis, including grow operations, dispensaries and a focus on testing laboratories. Cannabinoid profiling was performed using LCMS and GCMS. A case study involving a 10 year-old epileptic child with leaky gut syndrome and summary of his experiences with CBx oils will be presented. Future opportunities for MS-based testing will be discussed.
Signal Enhancement in HPLC-ESI-MS/MS Analysis of Spironolactone Metabolites Using HFIP and NH4F as Eluent Additives
We developed an HPLC-ESI-MS/MS method for simultaneous determination of spironolactone and its two metabolites, 7α-thiomethylspironolactone and canrenone from human blood plasma samples. Created as a part of a study evaluating the pharmacokinetic profile of spironolactone in children up to two years of age, the required sample volume was designed to be very small. That brought the question of ionization efficiency into our attentions and we investigated two signal boosting mobile phase additives, hexafluoroisopropanol and ammonium fluoride. Both were found to be beneficial and with the latter we observed more than 5 times increase in MS signal.
Successful Implementation of Immunosuppressant Drugs (ISDs) Monitoring Using Liquid Chromatography Mass Spectrometry (LC-MS/MS)
We hereby report our experience at Children’s Hospital Los Angeles (CHLA) in implementing therapeutic monitoring of ISDs including Tacrolimus,CyclosporineA, Sirolimus and Everolimus using LC-MS/MS. A quantitative multiple reaction monitoring (MRM) analytical method was validated on a Thermo-PRELUDE-TSQ-QUANTIVA LC-MS/MS including accuracy, precision, analytical sensitivity, reportable range, and recovery. In summary, a very simple, fast, sensitive LC-MS/MS method was successfully implemented in three months, which, when compared to the previously used immunoassay method, resulted in better optimization of therapeutic drug levels, decreased sample volumes, and significant cost savings.
Salivary Cortisol Measurement Using Mass Spectrometry: Clinical Utilities and Implementation Challenges
Salivary cortisol has emerged as a front line test for detecting cortisol overproduction (eg. Cushing’s) or underproduction (eg. adrenal insufficiency). Mass spectrometry is the analytical method of choice due to its sensitivity and specificity. The implementation of salivary cortisol measurement by mass spectrometry is replete with challenges encompassing sample collection, preparation, and analysis, as well as reference intervals. We report data of test validation and improvement, summary of clinical utilization, and preliminary data of reference interval study.
Rapid Measurement of Tacrolimus in Whole Blood by Paper Spray-Tandem Mass Spectrometry (PS-MS/MS)
LC-MS/MS requires an LC system and sample preparation for tacrolimus monitoring. Paper spray (PS) ionization generates gas phase analyte ions from dried blood spots without sample preparation and LC. We evaluated a PS-MS/MS for tacrolimus TDM in a clinical laboratory, and found PS-MS/MS to be accurate for tacrolimus monitoring with rapid turnaround time amenable to random access testing protocols.
A Simplified, Rapid LC-MS/MS Assay for Serum and Salivary Creatinine
In routine clinical laboratories serum creatinine is usually measured by colourimetric or enzymatic assays using automated analysers, however these methods are prone to interferences. We have developed a straightforward and rapid LC-MS/MS assay for serum creatinine using methanol extraction, and separation with a strong cation exchange column. With a short run time of 1.1 minutes and imprecision of 1.1-1.4% at the concentrations tested, this assay provides an alternative for patient samples where interference is likely in routine creatinine methods. In addition salivary creatinine can be measured and we have found that concentrations are on average 15% of serum levels.
Separation and Quantification of Serum L- and D-2-hydroxyglutarate Enantiomers by LC-MS/MS Following Derivatisation
The metabolite 2-hydroxyglutarate (2-HG) is normally present at low levels within cells and body fluids. It exists as two stereoisomers (L-2-HG and D-2-HG) due to an asymmetric carbon atom in the carbon backbone. Elevated 2-HG is found in inherited metabolic disorders and in malignancies such as acute myeloid leukaemia due to enzyme mutation, and it is important to differentiate between the stereoisomers in these cases. We have used the chiral derivatisation reagent DATAN (diacetyl-L-tartaric anhydride) to form diasteroisomers of L-2-HG and D-2-HG which we were then able to separate and quantify using an LC-MS/MS method.
Development of High Sensitivity Micro-LC-MS/MS Method for Estradiol in Human Serum without Derivatization
Reliable measurement of low concentration of estradiol (E2) in human blood samples has always been a challenge in clinical laboratories. However, there are considerable demands to accurately measure serum E2 at very low concentrations (< 5 pg/mL) in postmenopausal women, men, and pediatric patients. Mass spectrometry-based methods have been increasingly used as the method of choice in clinical laboratories for measuring E2. However, most of them require large sample volume and involve complex sample preparation. Our study aims to develop a high sensitivity method using Micro-LC-MS/MS to reliably measure E2 concentrations below 3 pg/mL using low sample volume and without derivatization.
Analysis of Sex Steroids in Urine by LC-MS/MS
Measurements of steroid panels in urine can provide information about steroid status and metabolism. We developed an automated isotope dilution LC-MS/MS method using solid phase extraction on a polymeric, reverse-phase sorbent to quantitate 12 steroids which includes progestagens, estrogens and androgens as well as some of their conjugates in human urine without derivatization. Steroids are separated on a C18 column and analyzed separately by electrospray ionization in the positive and negative ion mode for maximum sensitivity. The measurement range covers four orders of magnitude. The method is used to measure steroids in urine from men, women and children.
A Fast Analysis of Low Level Estrogens in Serum by Bruker EVOQ Elite LCMS
A rapid and sensitive method for the quantification of Estrogens in human serum was developed using Bruker Advance UHPLC coupled to the EVOQ Elite triple quadrupole mass spectrometer system. Excellent sensitivity, linearity and dynamic detection range were obtained. The limited of quantitation (LOQ) was 1pg/mL, 2.5 pg/mL and 5.0 pg/mL for Estrone (E1), 17-Estradiol (E2) and Estriol (E3), respectively. The low pg/mL level estrogens detection with over four orders of dynamic detection range will cover the clinical research needs. The method run cycle time was 4 minutes.
The Analysis of Vitamin D and Metabolites in Plasma by LC-MS/MS
Vitamin D deficiency has been linked to an increased risk for many chronic diseases including diabetes, heart disease, and some cancers. Vitamin D exists in two forms, vitamin D2 and vitamin D3. Each undergoes metabolism to form 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. For accurate determination of vitamin D levels in the blood, it is important to distinguish between these metabolites and separate them from matrix interferences. The Raptor™ ARC-18 column combines the speed of superficially porous particles with the resolution of highly selective USLC® technology to produce a simple, rugged method for the determination of vitamin D metabolites in plasma.
Determination of Plasma Catecholamines 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 catecholamines (dopamine, epinephrine and norepinephrine). An efficient sample preparation procedure by filtration on Captiva NDLipids and by solid phase extraction (SPE) allows simultaneous extraction of all analytes in plasma. The described method achieves the required functional sensitivity and is capable of quantitating analytes over a wide dynamic range with a single injection. Excellent reproducibility was observed (CV < 5%). All calibration curves displayed linearity with an R2 > 0.9997.
Determination of Plasma Renin Activity by LC-MS/MS for Clinical Research
A highly sensitive and specific LC/MS/MS method has been developed for the determination of plasma renin activity for clinical research. Plasma samples are incubated for 3 hours-37oC. A sample preparation procedure by solid phase extraction (SPE) allows efficient extraction of Angiotensin I in plasma. Plasma renin activity is calculated by subtracting Angiotensin I concentration in a blank plate. The described method achieves the required functional sensitivity and is capable of quantitating Angiotensin I over a wide dynamic range with a single injection. Excellent reproducibility was observed (CV < 6%). All calibration curves displayed linearity with an R2 > 0.9996.
A Rapid and Accurate LC-MS/MS Method for the Analysis of Nicotine, Nicotine Metabolites, and Minor Tobacco Alkaloid in Urine
The analysis of nicotine metabolites has several aspects including monitoring public tobacco exposure, evaluation of nicotine replacement therapy, and drug therapy assessment. Most of the modern methods adapt the usage of high pH chromatography with relatively high concentration of additive reagents, which may not be applicable to all LC-MS instrumentation. An LC-MS/MS method was developed for urine test of nicotine, cotinine, trans-3’-hydroxycotinine, nornicotine, norcotinine, and anabasine. It was demonstrated that a fast and highly efficient analysis of these basic compounds can be achieved with the Raptor™ Biphenyl column using regular LC-MS solutions suitable for all LC-MS instrumentation.
A Rapid and Sensitive LC-MS/MS Method for the Analysis of Free Thyroid Hormones
The unbound or “free” thyroxin (T4) and tri-iodothyronine (T3) are the active forms of thyroid hormones which only exist at low levels (pg/mL) in the circulation. The measurement of these free hormones is necessary to assess thyroid function for both veterinary and human clinicians. A fast, accurate, and sensitive method was developed for the analysis of thyroid hormones at the free form levels using the highly efficient and selective Raptor™ Biphenyl column. The clinical applicability of the method was demonstrated by analyzing the fortified thyroid hormone in phosphate buffer saline containing 4% human albumin.
Bioanalytical UPLC-MS/MS Method Development and Validation for Measuring Penicillins in Human Blood Plasma– Analyte Stability Issues
Penicillins are the important grop of antimicrobials widely used in children and adults for over 50 years. Penicillins are beta-lactam antimicrobials and therefore especially intolerant to the stress conditions since the degradation of penicillins occur in different ways in different conditions.
UPLC-MS/MS method was developed and validated for determination of 5 in human blood plasma. The aim of developing and validating the bioanalytical method for measuring penicillins in blood plasma was to use it for the measurement of intensive care unit patients’ plasma samples, in order to use the data for the population pharmacokinetic modelling and dose optimization.
Investigation of Arachidonic Acid and Its Metabolites as Biomarkers for Potential Efficacy Endpoints for Monoacylglycerol Lipase Inhibition
Brain inflammation can occur with infection, traumatic brain injury or disease and arachidonic acid (AA) and metabolites are increased. Monoacylglycerol lipase (MAGL) is an enzyme that controls brain levels of AA and pro-inflammatory metabolites. To further develop MAGL inhibition as a target for diseases with neuroinflammation, efficacy biomarkers are needed in investigative studies. A quantitative assay was developed using liquid chromatography coupled with mass spectrometry to measure AA and metabolites in human cerebral spinal fluid. AA and metabolites were measured from control patients and patients with brain inflammation and the biggest change was observed in patients with intra-cerebral hemorrhage.
Effective Monitoring for Enantiomeric Forms of Methamphetamine and Related Compounds by LC-MS
Methamphetamine is a powerful CNS stimulant widely abused due to the increased mental alertness and suppression of fatigue it produces. The L-isomer is used legally in several over the counter medicines. L-methamphetamine is also a metabolite of therapeutic drugs such as selegiline. Immunoassay does not differentiate between the legal and illicit versions and a positive result is seen if either form is detected. A chiral LC-MS method is presented on urine samples for methamphetamine. Sample recoveries and detection limits are presented. The method will be used to determine other related enantiomeric compounds, similar in structure, to methamphetamine.
A Five-Minute Analysis that Separates 25-hydroxyvitamin D from Its C3 Epimer
Measurement of 25-hydroxyvitamin D (25OHD) is useful for the nutritional assessment of a patient’s vitamin D status. LC-MS/MS methods that measure 25OHD are considered the “gold standard”, but are susceptible to interference from the C3-epimer of 25OHD. Reported methods that separate the C3-epimer from 25OHD tend to involve tedious sample preparation and/or lengthy chromatography. In this study, we achieve our goal of developing a LC-MS/MS method that separates 25OHD from its C3 epimer in under 5 minutes and involves simple sample preparation for routine clinical use.
Online Analysis of Immunosuppressants in Whole Blood with the Evoq Triple Quad
A robust and reliable research method to quantitate the immunosuppressants cyclosporine A, tacrolimus, sirolimus and everolimus in whole blood samples using the Bruker Advance UHPLC with OLE coupled to the EVOQ Elite triple quad is demonstrated. The integrated online extraction option of the UHPLC together with the ClinMass® LC-MS/MS complete kit (MS11000) provides fast and easy sample cleanup. Interlacing the online extraction and chromatographic separation reduces the overall run time to 3 minutes per sample. The calibrations showed excellent linearity (r²≥0.997) and the assay had a very good precision with RSD <9% as well as high accuracy with bias <±6.5%.
LC-MS/MS Method for the Measurement of Free 25-OH Vitamin D3
The measurement of total 25-OH Vitamin D3 is suboptimal with serum concentrations correlating poorly with PTH. For this reason we developed a method to quantify the free fraction employing ultrafiltration at 37°C and LC-MS/MS. The range of results from 34 healthy volunteers was 1.5 to 17.9 pg/mL. This cohort was supplemented with 8 patients with elevated parathyroid hormone (PTH). The free 25-OH Vitamin D3 concentration correlates excellently with the concentration of PTH and poorly with the total 25-OH Vitamin D3 concentration. A poor correlation was observed between total 25-OH Vitamin D3 and PTH. We can now evaluate the role of free 25-OH Vitamin D3 in patients with bone and/or a variety of malignant diseases.
Validation of a Reliable LC-MS/MS Method for Analysis of Five Steroids Simultaneously in Clinical Laboratory
Our aim was to developed a LC-MS/MS method for 17-hydroxyprogesterone, androstenedione, 11-desoxycortisol, 21-desoxycortisol and cortisol quantification in serum using protein precipitation, isotopic internal standards and two dimensional liquid chromatography consisting of trapping column and reverse-phase C18 analytical column following atmospheric pressure chemical ionization and mass spectrometry detection. Functional sensitivity was less than 0.5 ng/mL, precision was less than 15%, recovery ranged from 93% to 120% linearity ranged from 89% to 111%, accuracy was considered adequate for all compounds tested. In conclusion, we developed a suitable method for routine measurement of 17-hydroxyprogesterone, androstenedione, 11-desoxycortisol, 21-desoxycortisol and cortisol in serum.
HDL and Total Cholesterol Analysis by LDTD-MS/MS Analysis in 7 Seconds Per Sample with Cross-validation Data from a Clinical Laboratory Using Real Patient Serum Samples
A fast and quantitative method for the measurement of Total and HDL cholesterol in human serum sample using the LDTD-MS/MS system. A liquid-liquid extraction is performed for total cholesterol and HDL isolated fraction. Organic upper layer is directly applied to the LazWell plate and dried prior to analysis. Standard method validation criteria are evaluated. All analyses were cross-validated and compared with an enzymatic coloration technique from an external clinical laboratory. The cross-validation results show low percentage differences between the two methods. Samples were analyzed in 7 seconds per sample using LDTD-MS/MS.
Use of 96-well Pipetting Workstation for Liquid-liquid Extraction of Adrenal Steroids
The compounds 17-hydroxypregnenolone and pregnenolone are extracted with a 96-well pipetting workstation using methyl t-butyl ether. After derivatization the compounds, and isotopically labeled internal standards, are derivatized and quantified by HPLC tandem mass spectrometry. The method is rugged and reliable with precision and accuracy within acceptable limits. Studies to evaluate four collection tube types and special matrix types (icterus, hemolysis, and lipemia) are highlighted. Pooled human serum/plasma is used for preparation of control samples while 1%BSA solution is used for calibrator preparation.
Development of a High-throughput UPLC-MS/MS Method for Medroxyprogesterone Acetate (MP 17 Acetate) Quantification in Human Plasma
Medroxyprogesterone acetate (MPA), a synthetic analog of the hormone progesterone, has been commonly employed as a contraceptive agent. MPA quantification can be useful in determining potential drug-drug interactions, and as a result, robust analytical methods are required for drug quantification. While current LC-MS/MS methods are laborious and require high sample volumes, we have developed a rugged method for MPA determination with a sample volume of 0.6 mL of plasma and a lower limit of quantification of 0.2 ng/mL. The method was validated according to the recommendations of the FDA, Guidance for Industry: Bioanalytical Method Validation document.
LC-MS Quantitative Analysis of 25-Hydroxy-Vitamin D, 1, 25-Dihydroxy-Vitamin D, and their Isobars in Serum
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 25-hydroxy-vitamin D and 1, 25-dihydroxy-vitamin D and their respective isobars. This method uses a simple offline sample preparation in serum. The described method achieves the required sensitivity and is capable of quantitating of these compounds over their relevant dynamic range. Excellent reproducibility was observed for all compounds (CV < 15%). All calibration curves displayed linearity with an R2 > 0.995.
A UPLC-MS/MS Method for the Analysis of Plasma Mycophenolic Acid for Clinical Research
Here we present a UPLC-MS/MS method to measure mycophenolic acid in human plasma for clinical research purposes. An analytically sensitive method across the range 0.1 – 20 µg/mL was developed using protein precipitation extraction (PPE), providing a fast and cost effective technique. The method demonstrates good linearity, precision (< 10% CV) and accuracy (≤ 5.1% bias).
For Research Use Only, not for use in diagnostic procedures.
Measurement of Urinary Serotonin for Clinical Research, Using Mixed Mode SPE and a High-strength Silica PFP Column
A single extraction and analysis method has been developed for urinary serotonin for clinical research. Serotonin was extracted using weak cation exchange mixed-mode SPE and analyzed by LC/MS/MS. Recoveries were good and matrix effects were minimal for most compounds. Linearity and QC results were excellent, with % CV and bias values less than 3% at all QC points. This method enables rapid, simultaneous and accurate LC/MS/MS analysis of these challenging compounds without the challenges associated with traditional reversed-phase separation or ion-pairing techniques.
For Research Use Only, not for use in diagnostic procedures.
Rapid, Simultaneous Analysis of Plasma Catecholamines and Metanephrines by Mixed-mode SPE and HILIC LC-MS/MS
A single extraction and analysis method has been developed for plasma catecholamines and metanephrines for clinical research. Analytes were extracted using weak cation exchange mixed-mode SPE and analyzed using HILIC LC/MS/MS. Recoveries were good and matrix effects were minimal for most compounds. Linearity and QC results were excellent for all compounds down to 10 pg/mL, with % CV and bias values less than 10% at all QC points. This method enables rapid, simultaneous and accurate LC/MS/MS analysis of these challenging compounds without the challenges associated with traditional reversed-phase separation or ion-pairing techniques.
For Research Use Only, not for use in diagnostic procedures.
Comparison of Different Whole Blood Sample Pretreatment Methods for Targeted Analysis of Basic Drugs
Whole blood presents a complete specimen for analysis of many drugs, therapeutic or illicit. This specimen type also poses significant challenges in pretreatment prior to extraction and eventual analysis by any method(s). We examined several common sample pretreatment procedures that hemolyze the blood and precipitate the plasma proteins. The resulting clarified samples were then treated by a SPE method designed for basic drugs (e.g. amphetamines, natural and synthetic opiates, benzodiazepines, and PCP). The pretreatment techniques were then evaluated based on the overall recovery, response and reproducibility for each analyte at two concentration levels.
Quantitative Analysis of 25-Hydroxy-Vitamin D in Serum Using LC/QQQ and LC/Q-TOF
Liquid chromatography triple quadrupole (QQQ) and quadrupole time-of-flight (Q-TOF) 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 by Q-TOF. Using simple sample preparation techniques and chromatographic configuration achieves the required sensitivity and is capable of quantitating the compounds over their relevant dynamic range. Excellent reproducibility was observed for all compounds (CV < 15%). All calibration curves displayed linearity with an R2 > 0.995.
LC-MS Quantitative Analysis of Fat Soluble Vitamins in Blood
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 fat soluble vitamins. This analytical 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 < 15%). All calibration curves displayed linearity with an R2 > 0.995.
LC-MS Quantitative Analysis of Water Soluble Vitamins in Blood
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 analytical method uses a simple offline sample preparation in blood. The described analytical 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 < 15%). All calibration curves displayed linearity with an R2 > 0.995.
Determination of 25-OH Vitamin D3 and D2 in Dried Blood Spots: A Simple Method Based on Stable Isotope and UPLC-MS/MS
DBS (dried blood spot) collection is simple, relatively painless, lower blood volume than venipuncture. The sensitive assay of 25-OH vitamin D for DBS are routinely used for screening test of vitamin D deficiency. We developed a new assay for the quantification of 25-OH vitamin D3 and D2 in 3.2 mm DBS. This assay has a more simple extraction method and is highly sensitive.
Accuracy Evaluation of Three Routine 25-hydroxyvitamin D Assays by Comparing with LC-MS/MS
Since the US Endocrine Society guideline defines vitamin D deficiency as 25(OH)D less than 20 ng/ml (50nmol/l), the accurate measurement of 25(OH)D in clinical laboratory is essential. We evaluated the accuracy and the effect of vitamin D binding protein (DBP) levels in three routine 25(OH)D immunoassays (Centaur, Elecsys, Architect) compared with LC-MS/MS using the samples of 48 healthy, 50 pregnant, and 50 ICU patients. All of three immunoassays showed significantly lower levels of 25(OH)D results than those of LC-MS/MS. Mean absolute biases [(routine assay) - (LC-MS/MS)] were -2.56 ng/mL (Centaur), -5.66 (Elecsys), and -6.21 (Architect). Although the mean value of DBP was significantly higher in pregnant women and lower in ICU patients than in healthy controls, the degree of biases in three immunoassays was not affected by the DBP levels.
1,25-di-hydroxy Vitamin D Analysis by LC-MS: Optimization for Sample Prep Automation and Medium Throughput Lab
A manual RIA assay for 1,25di-OH Vitamin D (1,25DiOHVitD) from serum shall be replaced. Goal is to establish a LC-MS based workflow that significantly reduces hands on time (manual work), shortens the time to results and enables to analyze the samples on the existing LC-MS system. Extraction of 1,25DiOHVitD with the easy to automate AC Extraction Plate followed by derivatization with DAPTAD and analysis by LC-MS showed extraction efficiency of 84% at 100pg/mL and appeared to be very reproducible. The calibration curve for standard materials was linear for the concentration range of 5 to 160 pg/mL.
Simultaneous Determination of Methylmalonic Acid and Homocysteine in Plasma by LC-MS/MS
Methylmalonic acid (MMA) and homocysteine (HCY) belong to sensitive indicators of cobalamine deficiency. Cobalamine is an essential nutrient which plays an important role in the hematopoiesis, in the development of central nervous system (CNS), in the synthesis of DNA and regulation of fatty and amino acids metabolism. The aim of this work was to develop the method for simultaneous determination of MMA and HCY as biomarkers of functional cobalamine deficiency by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The full validation of this method was carried out.
LC-MS/MS Measurement of Urinary 2,3-dinor-11β-Prostaglandin F2α in Patients with Systemic Mastocytosis: Improved Diagnostic Accuracy Compared to ELISA
Systemic mastocytosis (SM) results in the accumulation of mast cells in tissues. Currently, urine concentrations of 11-β-prostaglandin F2α (BPG) are measured by ELISA and used for screening and monitoring of SM. Comparison with a novel LC-MS/MS method found no measurable BPG in urine specimens despite measureable immunoassay values. However, ELISA BPG concentrations did correlate with the LC-MS/MS measured 2,3-dinor isoform of BPG. In a clinical study of 203 patients LC-MS/MS analysis of 2,3-BPG improved clinical sensitivity and specificity for SM detection over the immunoassay both alone, and when combined with other urinary markers of SM.
Validation of a LC-MS/MS Method to Determine Whole Blood Oxidized and Reduced Forms of Glutathione for an Assessment of Degree of Oxidative Stress
Because of large differences in reduced glutathione (GSH) and oxidized glutathione (GSSG) between different studies, accurate revision of the methods used and analytical validation of newly modified method is needed. We modified recently reported measurement methods for GSH and GSSG and validated for precision, linearity, limit of quantification, limit of detection, and ion-suppression. In addition we evaluated clinical utility of GSH/GSSG ratio by testing the increase of GSH/GSSG ratio according to the amount of oxidative stress induced by iron in mouse model. Our results showed decrease of GSH/GSSG ratios according to the amount of oxidative stress induced.
An Improved Platform for the Recovery and Analysis of Cannabinoids from Dried Blood Samples
In order to quantitate three cannabinoids; delta-9 tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) from dried blood spots (DBS), we examined the recoveries from four platforms routinely used in dried blood spot collections; 903™ Card, DMS™ Card, HemaSpot-HF™ and HemaSpot-SE™. While recovery was poor (40-60%) from cotton fiber based absorbents, 80% could be recovered from the glass fiber membrane-based HemaSpot-SE™, which separates blood cells from plasma. Cannabinoid levels from wet plasma were equal to levels found from the dried plasma portion of the HemaSpot-SE™ (99% correlation).
Mass-Directed Isolation and Profiling of Small Molecule Analytes with SFC
Electrospray ionization mass spectrometry (ESI-MS) coupled with supercritical fluid chromatography (SFC) is routinely being used in our laboratory for identification and isolation of small molecule analytes in supporting drug discovery efforts in PK analyses as well as other bioanalytical and clinical applications. This study demonstrates the utility of the preparative SFC-MS system to rapidly purify small molecule analytes for toxicology measurements and metabolite identification. The purification of one deuterated chiral isomer from its closely eluting impurity at the gram scale was accomplished within a day using 10 min mass-directed SFC methods. The limit of quantitation was reached below 1 ng.
Improved Detection of 17ß-Estradiol and Estrone in Serum Through Derivatization with Dansyl Chloride Utilizing LC-MS/MS Technology
Sub-picogram levels for determination of 17ß-estradiol and estrone presents several challenges for traditional analysis of the molecule. Molecular similarities leave some methods susceptible to cross-reactivity with other steroids, leading to poor analytical accuracy. A lack of highly ionisable functional groups also poses a challenge for analysis by mass spectrometry. Through the use of high-end triple detection and derivatization, detection limit was improved to the point that 17ß-estradiol and estrone can be quickly and accurately quantified down to 0.5 pg/mL using an LC/MS/MS approach.
Detection and Quantitation of Exemestane, Letrozole and Anastrozole in Human Serum by LC-MS/MS and Atmospheric Pressure Chemical Ionization
Carcinomas expressing estrogen receptors are sensitive to the proliferation of estrogens. An effective therapeutic option for the treatment of these estrogen-responsive cancers in postmenopausal women involves the use of aromatase inhibitors including exemestane, letrozole and anastrozole. These selective drugs interfere in the synthesis of estrogen in peripheral tissues by inactivating aromatase. The resulting decrease in circulating estrogen levels can slow, and even stop, the growth of breast cancer cells. Monitoring the serum concentrations of exemestane, letrozole and anastrozole will help determine the patient’s adherence to the drugs, as well as provide useful information regarding concentration-dependent side effects and therapeutic efficacy.
Sensitive Analysis of Serum 5alpha-Dihydrotestosterone by 2D-LC-MS/MS
In humans, circulating androgen 5alpha-dihydrotestosterone (DHT) exerts major biological effects on skin and prostate. DHT is a more potent androgen than testosterone (T) and is the primary androgen in the prostate. The 2D-LC-MS/MS setup allows extensive clean-up and transfers only a small part of elution profile of the 1st dimension containing targeted analyte to the 2nd dimension for high efficiency separation. A simple and sensitive method to accurately quantify DHT in serum by 2D-LC-MS/MS was developed and validated, with a LLOQ of 5 pg/mL and suitable for routine clinical laboratory use.
Evaluation of a Method for Forensic Quantitative Screening of Over 120 Drugs of Abuse on a Triple Quadrupole Mass Spectrometer
A liquid-liquid extraction scheme (LLE) was developed for analyzing a wide range of compounds in human urine in a forensic toxicology setting. Samples preparation was enzymatic hydrolysis followed by LLE using commercially available extraction tubes. All samples were analyzed on a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer. The method was tested with over 100 compounds from a wide chemical space including polar and non-polar compounds as well as positively and negatively ionizing compounds. Limits of quantitation (LOQ), LLE recoveries, precision, and matrix effects were determined. LOQs met forensic toxicology requirements for 95% of the compounds tested.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research for Rapid Quantitative Analysis of Vitamin D in Human Plasma
Clinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma or serum. We evaluated Q Exactive Orbitrap mass spectrometer in fast, cost-efficient method collecting high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis. The linearity range was 1-100 ng/mL; precision and accuracy was with 15%; matrix effects and interferences were not observed.
Quantitation of Ganciclovir in Human Serum by UPLC-MS/MS
Ganciclovir is an antiviral drug administered for the prevention and treatment of cytomegalovirus disease in immunocompromised patients. Ganciclovir concentration levels measured by this assay are used in pharmacokinetic calculations to obtain dosing information using area-under-the-curve analysis of serum concentrations following ganciclovir administration. We developed and validated an ultra-performance liquid chromatography- mass spectrometry method to measure the concentration of ganciclovir in human serum. Sample preparation, UPLC-MS/MS conditions, linearity, precision, patient samples correlation, recovery, stability, matrix effect and interference were demonstrated. This assay can be used in the clinical setting for therapeutic drug monitoring and pharmacokinetic studies of ganciclovir.
Quantitative Analysis of a Glyburide Analogue, a Potential NLRP3 Inhibitor, Using Micro-sampling, Hybrid Solid Phase Extraction and LC-MS/MS
A LC-MS/MS method was developed to quantify a novel inhibitor of the NLRP3 inflammasome that limits myocardial injury after ischemic and non-ischemic injury in mice. Initially, glyburide analogue was extracted from 20-µL of mouse plasma using HybridSPE 96 well plate. Separation and quantification was achieved by RP-LC followed by positive electrospray ionization and selected reaction monitoring (SRM) of the glyburide analogue (369→169) and structural analogue internal standard, glipizide (445→166). The method validation was linear, 1-1000 ng/ml. Precision and Accuracy of the method was ±15%. A cross-validation is underway to employ dried blood spots to enhance the toxicokinetic studies in mice.
Development of a Consolidated LC-MS/MS Assay for Quantification of Voriconazole, Posaconazole and Teriflunomide
Clinical laboratories are increasingly challenged with decreasing test reimbursements and the concomitant need to generate high quality patient results at lower cost. The consolidation of multiple tests onto a single platform or workflow can increase productivity and lead to reduced expense. The aim of this work is to develop a single LC-MS/MS method to quantify voriconazole and posaconazole, anti-fungal agents commonly used in immunocompromised patients, as well as teriflunomide, a drug with both immunosuppressive and anti-viral properties used in renal transplant patients. We present the data for voriconazole and are working toward adding posaconazole and teriflunomide.
Determination of Concentration and Distribution of Doxorubicin in Lungs by in vivo and in Situ Solid Phase Microextraction
Currently available sample preparation methods for tissue analysis are mainly based on the biopsy therefore are too invasive for repetitive analysis of living systems and too time consuming and laborious to deliver results in real time. Solid phase microextraction (SPME) addresses the abovementioned issues. In this work we used in vivo and in situ SPME for determination of concentration of the chemotherapeutic agent in lung of living pigs and in human lungs obtained from the deceased donors, during In Vivo Lung Perfusion. This results showed feasibility of the method to provide fully quantitative data with good spatial and temporal resolution.
Multimodal Imaging Mass Spectrometry for Probing Aβ-Plaque Pathology
Alzheimer’s disease is the most common neurodegenerative disorder affecting 12% over 65. The exact mechanisms underlying AD remain unknown but cognitive decline has been linked to formation of β-amyloid (Aβ) deposits as senile plaques. Changes in peptide truncation and plaque associated neuronal lipid species have been implicated with proteopathic mechanisms in AD. The aim of this study was therefore to employ SIMS and MALDI based imaging mass spectrometry (IMS) to probe Aβ plaque pathology in tgARCSWE mice with particular focus on associated neuronal lipid species and Aβ truncations. SIMS revealed plaque associated lipid species including sulfatides, triglycerides and cholesterol. MALDI IMS in turn revealed AD implicated Aβ peptide truncation (e.g.pyro-Glu).In conclusion, multimodal IMS is a powerful approach to interrogate chemical plaque pathology in AD.
Imaging Analysis of Metals, Lipids, and Proteins in Biological Tissues via Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry
The relationships between metals, lipids, and proteins in biological tissues are complex and are linked to disease. A technique that can explore the correlations between the different analytes and their normal and abnormal distributions has exciting potential for clinical diagnosis. In this study, analyses of metal, lipid, and protein distributions in biological tissues were studied using MALDI-TOF imaging mass spectrometry. Current studies have focused on the metal, lipid, and protein profiles for oyster tissues, in which future studies will involve the application of the technique to other biological and clinical tissues.
Biomarker Candidates Discovery of Myocarditis Using MALDI Imaging Mass
Spectrometry
Spatial distribution of inflammation relative metabolites from myocarditis heart tissues was visualized by imaging mass spectrometer. MALDI-TOF/TOF, UPLC-Q-TOF M), and high resolution magic angle spinning (HR-MAS) nuclear magnetic resonance were employed to identify metabolites.
Decrease level of creatine and NADH in myocarditis heart tissues were analyzed by imaging analysis. In addition, increase of lipids involved choline, especially sphingomyelins and lysophosphatidylcholines that has associated in inflammatory processes were detected. Increase level of choline and glycerol-3-phosphocholine were proved that involved in phosphatidylcholines and SMs synthesis and carnitine and acyl-carnitine that transported acyl-CoA was derived lipids levels are reduced in myocarditis heart tissue.
Multi-mode Desorption Electrospray Ionization (DESI) Mass Spectrometry Imaging at Different Pixel Resolutions for Human Tissue Imaging
DESI a surface analysis technique using an electrospray probe, can be used as a spatially resolved imaging technique. The droplets impact upon the surface, with ionization occuring due to the charge imparted onto the droplets and requires no modifications to the sample by matrix addition. The profile of the spray on the surface affects the spatial resolution and modification can be achieved using different gas & solvent flow rates, affecting the spatial resolution. DESI does not destruct the tissue surface and the tissue section can be re-analyzed under different conditions or techniques. Here we describe the workflow that allows one tissue image profile to be rapidly acquired at low spatial resolution, followed by the analysis of a region of interest at a higher spatial resolution and finally the same tissue section being H&E stained. Example of human tissue sections will be presented.
Desorption Electrospray Ionization (DESI) for Tissue Imaging on a Time-Of-Flight (TOF) Mass Spectrometer
DESI mass spectrometry imaging, when used with a TOF mass spectrometer, is a very effective technique that determines the spatial localization and molecular distribution of molecules within a variety of samples. It can be applied to animal and human thin tissue sections and be utilized to differentiate tissue types or study the distribution of molecules within tissues. In this study, good sensitivity for a wide range of lipid and small molecule metabolites from tissues was achieved in positive and negative mode, allowing a more comprehensive understanding of the sample by imaging the sample using a TOF mass spectrometer equipped with DESI.
Dual Polarity Mass Spectrometric Imaging from Single Tissue Sections with Desorption Electrospray Ionization (DESI) Mass Spectrometry
Over the years Desorption Electrospray Ionization (DESI), an ambient ionization technique, has been applied to Mass Spectrometry Imaging (MSI) to allow for the direct analysis of surfaces at atmospheric pressure. Here we demonstrate that using standard DESI analysis conditions combined with a SYNAPT G2-Si, it is possible to analyze a tissue section in one ionization mode followed by a second experiment acquired in the opposite ionization mode and access a wealth of molecular information from a single tissue section without the need to alter the imaging analysis conditions.
Stable Isotope Nanostructure Imaging Mass Spectrometry to Monitor Tumor Metabolism
Metabolic variation in tumor cells is tightly coupled to cancer pathology and progression. While it is reasonable to consider a tumor as a homogeneous mass of similar cells for metabolomic analysis these structures are in reality a diverse population of cells in different metabolic states. We have developed a method to monitor anaerobic glycolysis within tumors with single cell spatial resolution to address the unavoidable averaging that occurs in traditional extraction based methods. This analysis utilizes nanostructure imaging mass spectrometry (NIMS), with ESI LC-MS/MS, to map the conversion of stable isotope-labeled glucose to lactate (13C3).
Simultaneous Determination of P-Phenylenediamine, N-Acetyl-p-phenylenediamine and N,N-Diacetyl-p-phenylenediamine in Human Urine by LC-MS/MS
A sensitive LC-MS/MS method for determination of PPD and its metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in human urine has been developed and validated. PPD, MAPPD and DAPPD were extracted for urine by methylene chloride at alkaline pH. Detection was performed by LC-MS/MS using electrospray positive ionization under multiple reaction-monitoring mode. The transition ions m/z 109 ¡ú 92, m/z 151 ¡ú 92, m/z 193 ¡ú 92, and m/z 136 ¡ú 77 were selected for the quantification of PPD, MAPPD, DAPPD, and IS, respectively. Linearity was within 10¨C2000 ng/mL for all analytes. Intra- and inter-assay imprecisions were within 1.58¨C9.52% and 5.43¨C9.45% respectively for PPD, MAPPD and DAPPD. The method was successfully applied to analysis of PPD, MAPPD and DAPPD in urine samples collected from suicidal and accidental cases.
First Metabolic Profile of PV8, a Novel Synthetic Cathinone, by High-Resolution Mass Spectrometry
Novel psychoactive substances (NPS) are constantly emerging onto the illicit drug market making it difficult to identify parent and metabolites new drugs. Recently, PV8 (α-PHpP or 1-Phenyl-2-(1-pyrrolidinyl)-1-heptanone) was identified for the first time in illegal products. In order to identify unique markers of intake, tie adverse effects to specific drug intake, and educate the public of the dangers of this NPS, we evaluated in silico metabolism predictions, determined metabolic stability with human liver microsomes, and determined metabolic schemes with human hepatocyte incubation and high-resolution mass spectrometry for the identification and structural elucidation of PV8 metabolites.
Is It Noroxymorphone or Nornaloxone, and Why Should You Care?
Noroxymorphone/nornaloxone positive results were evaluated to propose concentrations to determine the source (oxycodone, oxymorphone or naloxone) when detected alone.
16,273 positive results yielded 14,587 positive for oxycodone, oxymorphone, and/or noroxycodone. Presumed identity was normoxymorphone. Median concentration was 183ng/mL. 75% of results were >75ng/mL.
170 specimens with presumed identity of nornaloxone had compounds related to buprenorphine. 56 (33%) were positive for naloxone (median concentration 23ng/mL). Median concentration of nornaloxone was 35ng/mL.
Our data suggest concentrations of noroxymorphone/nornaloxone <75ng/mL are from metabolism of naloxone. Ideally, urine testing should include precursor drugs, with results compared to patient prescriptions for accurate interpretation.
Forensic Drugs Screening Analysis by HPLC Coupled to QTOF Mass Spectrometry: Comparison to a Routine EMIT, HPLC, GC/NPD and GC/MS Workflow
Recent advances in QTOF hardware and software have increased the feasibility of applying this technology to routine forensic drugs screening. To prove this, ten blinded whole blood samples previously analyzed by EMIT, HPLC-UV, GC/NPD and GC/MS workflows were analyzed by HPLC-QTOF-MS/MS with both TOF-IDA-MS/MS and TOF-MS/MS-All with SWATH™. With an estimated 50% reduction in analysis time, simpler sample preparation, and higher confidence especially with high resolution accurate mass MS/MS library matching, HPLC-QTOF-MS/MS detected nearly all of the 106 compounds detected by EMIT, HPLC, GC/NPD and GC/MS, and also detected five compounds missed in the original screening analysis.
Comparison of Accurate Mass MS/MS Acquisition and Processing Techniques on Forensic Toxicological Screening
(For research use only, not for use in diagnostic procedures).
High resolution mass spectrometry (TOF or ion trap) yields generic methods that can identify some compounds. However, many compounds cannot be identified with MS1 evidence alone. Obtaining MS/MS for every possible compound in a sample can be challenging. Data dependent techniques often miss candidates. Targeted techniques are limited in the number compounds that can be monitored. Data-independent techniques, such as SWATH™ acquisition, are capable of capturing MS1 and MS/MS evidence for all possible candidates. We compared several acquisition techniques, and several data processing techniques, in a toxicological screening scenario.
In Oral Fluid 7-Aminoclonazepam Is Superior to Clonazepam for Detection of Clonazepam Use
The benzodiazepine clonazepam (CLON) requires careful monitoring to minimize abuse and diversion. We screened for both the parent drug CLON and its primary metabolite 7-aminoclonazepam (7AC) using the Orasure Intercept oral fluid (OF) collection device, followed by LC-Tandem MS in more than 800 OF samples from outpatient addiction medicine clinics. 102 samples were found positive for 7AC and/or CLON. 93 (91.2 %) were confirmed by ion ratio to contain 7AC (median, range: 4.15, 0.5-316.7 ng/ml), while only 49 of the 102 (48.0 %) samples were confirmed by ion ratio to contain CLON (median, range: 3.44, 0.1-217.2 ng/ml). No samples were confirmed positive for CLON and negative for 7AC. In the 49 confirmed pairs the median 7AC/CLON concentration ratio was 2.4. 7AC should be the OF analyte measured because of its higher concentration and superior ion ratio confirmation characteristics.
In Vitro Human Metabolism of Designer Cathinones: LC-MS/(MS) Metabolites Identification and Characterization for Doping Control Purposes
Designer cathinones are synthetic molecules obtained through the modification of the chemical structure of cathinone, a potent stimulant with pharmacological properties closely resembling those of amphetamine. These stimulants, which are becoming increasingly available mostly on non official marketing channels (i.e. websites), were specifically developed to circumvent the current regulatory restriction but, at the same time, they may remain “invisible” at the analytical controls. In this study we have in vitro investigated the human phase I and phase II metabolism of designer cathinones with the aim to evaluate the most appropriate analytical markers for their administration. On the basis of our results, the hydroxylated and demethylated metabolites represent the best analytical markers to detect the intake of designer cathinones.
Development of a Rapid LC-MS/MS Method for Hair Cortisol Determination to Assess the HPA Axis
With the growing popularity of hair cortisol concentration as a biomarker to assess the HPA axis, we are developing a rapid method for the determination of hair cortisol using UHPLC-MS/MS. Our method uses a 1.7 µm C18 column, with the MS in MRM mode to detect cortisol in a 2-minute run. The calibration curve is linear with a correlation coefficient ≥ 0.998. The LOD is 0.12 ng/g for samples pre-treated with SPE, or 5.4 ng/g for filtered samples. The intra-day coefficient of variation was 2.5%. Further, cortisone and prednisone do not contribute to the cortisol peak with this method.
Cost Advantage and Improved Accuracy of Medication Compliance by a Qualitative Time-Of-Flight Mass Spectrometry and Immunoassay-based Screen in Pain Management
Qualitative screens for pain management testing are an effort to provide a less-expensive, faster, and comprehensive evaluation of medication compliance and drug abuse. This study of a definitive TOF-MS and immunoassay-based workflow challenges the wide standing belief that quantitation is essential for urine drug compliance testing. Our data demonstrates that the hybrid screen is superior in confirming compliance per prescription (226/302 vs. 205/302), identifying substance abuse (97 vs. 71), and has a substantial cost advantage (41% cost savings), when compared to a conventional immunoassay-based screen reflexed to quantitation by LC-MS/MS.
A Simplified, Mixed-mode Sample Preparation Strategy for Urinary Forensic Toxicology Screening by LC-MS/MS
A simplified mixed-mode sample preparation method has been developed for the extraction and LC/MS/MS analysis of a forensic toxicology panel. The entire SPE procedure is reduced from 6 steps to three by eliminating conditioning and equilibration steps and combining both wash procedures into a single step. This enables a panel of opioid drugs, amine stimulants, benzodiazepines and other drugs of abuse to be extracted in a single method with recoveries that exceed 90% for 95% of the compounds, and limits of detection that ranged from 1-10 ng/mL.
Optimization of Automated Online SPE-LC-MS/MS Used in Pain Management Drug Monitoring
A systematic approach was taken to optimize the combined reverse phase (RP) solid phase extraction (SPE), RP liquid chromatography (LC), and mass spectrometry / mass spectrometry (MS/MS) conditions to achieve the following goals: maximize automation / minimize labor and cycle time, maximize applicability to the widest range of drugs encountered in pain management (PM), and scale the combination of sample size and SPE / LC separations to pre-concentrate the sample sufficiently to make all of the drugs (even the challenging ones) relatively easy to measure on a routine basis. Total automation of SPE-LC-MS/MS (urine in vial/tube/plate to results) with a 5 minute cycle time was achieved, and all drugs were readily measured across the full relevant concentration range. The process of achieving balance in all the parameters and scaling / optimizing the separations will be described.
Automated Targeted Screening of Benzodiazepines in Urine Using LDTD-MS/MS at 400 Samples Per Hour Rate
Toxicology laboratories use screening methods to obtain fast semi-quantitative screen value for drugs. Lack of specificity of those method demands confirmation of many false positive samples raising cost and time. Uses of LDTD-MS/MS enhance specificity at same throughput. Cross validation with LC-MS/MS validate this method. LC run were adapted to crude sample preparation reducing ionic suppression. Purified beta-glucuronidase enzymes are used for 15 minutes incubation instead of 1 hour. Conventional glucuronidase enzyme incubation is also performed. Complete workflow use Tecan robotic system with 8 channels liquid handler. Two 96 wells plates are process in parallel to feed LDTD-MS/MS system.
A User’s Perspective on UPLC-Q-ExactiveTM High Resolution Mass Spectrometry: Application to Comprehensive Drug Screening in Clinical Laboratory
The availability of comprehensive urine drug screening is crucial for patient care in many clinical settings. This presentation introduces the audience to a complex, powerful, yet user-friendly and cost-effective high-resolution mass spectrometry methodology for comprehensive drug screening in matrices of interest at the Centre for Addiction and Mental Health in Toronto (urine, swabs and/or herbal products). Using an UPLC system coupled to the Q-ExactiveTM mass spectrometer (Quadrupole-Orbitrap) we established an in-house full scan/ddMS2 protocol based on monoisotopic mass, retention time and MS2 databases/libraries comprising of multiple drug classes and metabolites (cannabinoids, cocaine, opiates/opioids, benzodiazepines, antipsychotics, antidepressants, synthetic cannabinoids, bath salts, other designer drugs). Case discussions are included.
The Analysis of Synthetic 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.
The Analysis of Common Drugs of Abuse in Human Urine by LC-MS/MS
The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) in forensic toxicology labs has increased significantly over the years. LC-MS provides sensitivity, speed, and the ability to simplify sample preparation. The Raptor™ Biphenyl column was developed to complement high-throughput LC-MS/MS analyses by combining the increased efficiency of superficially porous particles with the resolution of Ultra Selective Liquid Chromatography™ technology. A simple dilute and shoot method was developed for 10 common drugs of abuse and their metabolites using a Raptor™ Biphenyl 5µm column. The low back pressure of the 5µm particle column allows even conventional 400 bar LC systems to take advantage of this high speed separation with a total analysis time of 5 minutes.
Measurement of Human Urinary Organophosphate Pesticide Metabolites in a Clinical Setting
Organophosphorus (OP) pesticides are among the most widely used and toxic pesticides used in the United States. Human exposure to these compounds can come from consumption of fruits and vegetables, applying the pesticides to crops, exposure to insecticides used inside homes, and even from broadcast spraying for mosquitos. There are many compounds used in this class, and not everyone is exposed to all of them, but an analysis of urine for the six downstream OP metabolites is an effective way to assess the overall exposure to this class. In the case of agricultural workers, an analysis of their urine can assess the effectiveness of chemical hygiene procedures used during crop protection applications of these toxic compounds. A number of published approaches were examined and we also explored recent novel techniques to enhance the response of anions in the positive mode in LC/MS/MS.
New Acquisition and Processing Tools for Targeted and Unknown Screening Approaches in Clinical Research and Forensic Toxicology
Orbitrap instruments have gained in popularity in Forensic and Toxicology Research, offering the possibility to identify new substances in complex matrixes. A new scan mode termed vDIA (variable data-independent acquisition) will be described. It provides selectivity and sensitivity comparable to data-dependent MS2 measurements. Processing tools will also be discussed. TraceFinder identifies and confirms substances using the exact mass of the analyte, the isotopic distribution, the fragment ions and the retention times. Identification of unknown compounds can be performed using mzCloud a unique HRAM MSn spectral database. Structures can be confirmed using Mass Frontier that is capable to automatically generate possible fragments at an expert level.
Comparison of Tacrolimus Quantification Using the Waters MassTrak LC-MS/MS Assay with the Abbott Architect Immunoassay
Immunoassays are the most common methodology utilized in the clinical laboratory for drug measurements. Although most immunoassays offer excellent sensitivity, cross reactivity of metabolites can result in decreased specificity as compared with mass spectrometry based assays. The Waters MassTrak assay is currently the only FDA cleared LC-MS/MS kit for quantification of tacrolimus in whole blood. Many clinical laboratories use the FDA cleared Abbott Architect Tacrolimus immunoassay because it is available on a reliable automated platform. This is the first comprehensive report comparing the Waters MassTrak LC-MS/MS assay with the Abbott immunoassay. Our results demonstrate that the immunoassay values range from 10 to 30% higher than values determined by isotope dilution (p < 0.0001; via two-tailed paired t-test test).
Quantification of Drugs for Drug-Facilitated Crimes in Human Urine by LC-MS/MS
An analytical method based on liquid chromatography tandem mass spectrometry for the quantification of drugs for drug-facilitated crime in human urine is described. The method includes ketamine, its metabolites norketamine and dehydronorketamine, phencyclidine and gamma-butyrolactone (GBL); the method is also suitable to detect gamma-hydroxybutyric acid (GHB) at physiological levels. Sample preparation was based on extraction using methanol containing 0.1% formic acid. Mass spectrometric detection was performed by single reaction monitoring (SRM) on a Thermo Scientific TSQ Quantum Access MAX triple quadrupole using heated electrospray ionization. Linearity of the method was evaluated on duplicate curves for each analyte.
RapidFire-Based Screening of THCA: Comparison with Established Methods
A rapid mass spectrometry-based screening method for THCA has been recently developed and implemented in our pain management laboratory. Following implementation, it was observed that the false positive rate in patient samples increased when compared to the previously used EIA-based method. Data from both EIA and RF methods will be summarized and a discussion of false positive rates observed in authentic patient samples via both screening methods will be presented.
Enzyme Hydrolysis Using a Novel Recombinant β-Glucuronidase for Pain Management Urine Drug Testing
A novel recombinant β-glucuronidase (IMCSzyme), known to have higher activity than traditional abalone β-glucuronidase, was investigated. The IMCSzyme successfully hydrolyzed the glucuronide drug metabolites in patient samples, in an assay that is faster and uses less enzyme. Codeine, morphine, cyclobenzaprine, amitriptyline and naloxone showed higher quantitation following hydrolysis with IMCSzyme, indicating more complete conversion than was previously obtained. Our study demonstrates that the recombinant β-glucuronidase (IMCSzyme) is a viable and cost-effective alternative to current hydrolysis procedures using abalone β-glucuronidase.
A Novel and Fast Workflow for Forensic Toxicological Screening and Quantitation Using QTOF LC-MS/MS System
Forensic toxicological screening is challenging partly because of the extensive and evolving compound list. Current detection tools such as immunoassay, LC-UV, and GC-MS either lack the promptness and flexibility to adapt to the new analytes, or require extensive sample preparation methods, or suffer from insufficient sensitivity and specificity. In this study, we aim to (1) develop a sensitive and selective screening workflow in a forensic toxicological setting by utilizing QTOF LC-MS/MS system, IDA-MS/MS acquisition and an MSMSAll approach with the novel SWATHTM acquisition and to (2) to compare two LC methods (6.5-min and 2-min) for throughput/performance evaluation.
Detection of Ethyl Glucuronide and Ethyl Sulfate in Urine by Hydrophilic Interaction Liquid Chromatography (HILIC)-MS/MS
Ethyl glucuronide (EtG) and ethylsulfate (EtS) are biomarkers of alcohol abuse. LC-MS/MS methods that detect EtG and EtS usually employ reversed phase columns. But EtG and EtS are very polar metabolites, making retention in reversed phase challenging. Polar hydrophilic compounds that are difficult to separate in reversed phase columns are separated on HILIC columns. This paper demonstrates that a zwitterionic HILIC column can be used for EtG and EtS detection in urine (ESI-ion trap detection). The method developed produced sharp peaks and achieved baseline separation in less than 7 min. A lower limit of quantitation 1 ìg/L was achieved.
Rapid Mass Spectrometry Based Urine Drug Screening of 27 Antipsychotic and Antidepressant Medications
This poster will demonstrate figures of merit for screening 27 compounds using a two-minute dilute-and-shoot method on a SCIEX 4600 Triple TOF at a 10x dilution. Each compound’s positive identification relies upon it having significant abundance, acceptable retention time, exact parent mass, agreeable isotope ratio, and sometimes the appropriate fragmentation pattern. Figures of merit including limit of detection/limit of quantitation (LOD/LOQ), upper limit of linearity (ULOL), and possible inter-assay interferences will be discussed. Using patient samples, type I and type II errors will be assessed when compared with a traditional LC confirmation method.
LC-MS/MS Method for Quantitative Analysis of Gabapentin and Pregabalin in Serum or Plasma
Gabapentin and pregabalin are analogs of the inhibitory neurotransmitter gamma-amino butyric acid. Both analogs can be prescribed as full or partial anticonvulsants for some types of seizures as well as for postherpetic neuralgia and Restless Legs Syndrome (gabapentin) or for pain from diabetes, shingles, fibromyalgia, or spinal cord injury (pregabalin). Gabapentin and pregabalin are commonly tested to monitor therapeutic efficacy and patient compliance. We developed a 3.5 minute LC-MS/MS method for quantification of gabapentin and pregabalin in serum or plasma. Chromatographic separation and validation data including internal and external method comparison are presented.
A Novel Separation for the Bath Salts Using a Multi-Mode Reversed-Phase Column
Analysis and separation of bath salts mephedrone, methylone and MDPV was achieved using a multi-mode reverse phase column. The separation was obtained using the aqueous normal phase (ANP) mechanism. Bath salts were separated in four minutes using an isocratic mobile phase. The separation was effected by the concentration of acetonitrile and the pH of the aqueous buffer. Increasing the organic modifier concentration caused an increase in retention time. Increasing the pH caused a decrease in retention time. Using these parameters, a rapid and simple method was developed to quantitate the bath salts.
Ethanol Metabolites by Paper Spray Ionization: Method Development in Negative Ion Mode
Paper spray is a direct ionization technique that simplifies the mass spectrometric analysis of compounds from biological fluids without time consuming sample preparation and chromatography. Paper spray technology is therefore attractive for compound screening and quantitation in forensic toxicology. The sample collection and storage in a simple paper cassette is attractive for the shipment of samples to the forensic toxicology laboratory.
In this work, we develop protocols in negative ion mode for the screening of ethanol metabolites (ethyl sulfate and ethyl glucuronide) in urine by coupling paper spray technology to a new generation triple stage quadrupole (TSQ) mass spectrometer (MS).
Hepatic Metabolism of Licochalcone A, a Chalcone from Licorice (Glycyrrhiza Inflata)
Licochalcone A is a chalcone natural product that has been isolated from roots of the licorice species, Glycyrrhiza inflata. It shows several bioactivities in vitro. Since little information is available concerning the human metabolism of licochalcone A, we carried out preclinical in vitro hepatic metabolism studies. Several Phase I metabolites of licochalcone A were observed and characterized using high resolution LC-MS/MS with accurate mass measurement. While testing for the formation of possible reactive metabolites using human liver microsomes, the cofactor NADPH and the biological nucleophile GSH, several GSH conjugates were detected and characterized using UHPLC-MS/MS. These results indicate that licochalcone A not only can form GSH conjugates due to the reactivity of its alpha,beta-unsaturated ketone structure but also through the formation of electrophilic metabolite.
Comprehensive and Extended LC-MS Analysis of 166 Various Drugs and their Metabolites in Urine, Oral Fluid and Blood
Liquid chromatography triple quadrupole (QQQ) mass spectrometry (LC/MS/MS) is suited for rapid analysis of multiple analytes. A sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of 166 drugs of the following drug classes: stimulants, benzodiazepines, antidepressants, opioids, muscle relaxants, hallucinogens, etc by QQQ. Using simple sample preparation techniques in urine and blood and chromatographic configurations achieves the required sensitivity and separation and is capable of quantitating the drugs over their relevant dynamic range. Excellent reproducibility was observed for all drugs (CV < 15%). All calibration curves displayed linearity with an R2 > 0.995.
The Development of an LC-MS/MS Screening Method for 104 Targeted Compounds in Whole Blood, Using Library Searching on a QTRAP Mass Spectrometer
In order to detect a large variety of drugs in whole blood, many forensic laboratories incorporate multiple screening assays to cover different drug classes. Our objective was to develop a single LC-MS/MS assay capable of accurately identifying >100 target compounds in less than 10 minutes. We have employed a QTRAP mass spectrometer, which enabled ‘on-the-fly’ acquisition of full-scan MS/MS spectrum for every detected compound, which was searched against a spectral reference library. A cross-method comparison with an outside laboratory demonstrated that our method (i) provided more specific information about compound identity, (ii) provided superior sensitivity, and (iii) detected more compounds.
LC-MS Analysis of Barbiturates in Urine, Oral Fluid and Blood
Liquid chromatography triple quadrupole (QQQ) mass spectrometry (LC/MS/MS) is suited for rapid analysis of multiple analytes. A sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of barbiturates by QQQ. Using simple sample preparation techniques in urine, oral fluid, and blood, and chromatographic configurations achieves the required sensitivity and separation and is capable of quantitating the drugs over their relevant dynamic range. Excellent reproducibility was observed for all drugs (CV < 15%). All calibration curves displayed linearity with an R2 > 0.995.
LC-MS Analysis of Phytocannabinoids and their Metabolites in Urine, Oral Fluid and Blood
Liquid chromatography triple quadrupole (QQQ) mass spectrometry (LC/MS/MS) is suited for rapid analysis of multiple analyte’s. A sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of Phytocannabinoids and their metabolites by QQQ. Using simple sample preparation techniques in urine, oral fluid and blood and chromatographic configurations achieves the required sensitivity and separation and is capable of quantitating the drugs over their relevant dynamic range. Excellent reproducibility was observed for all drugs (CV < 15%). All calibration curves displayed linearity with an R2 > 0.995.
LC-MS Analysis of Nicotine and Its Metabolites in Urine, Oral Fluid and Blood
Liquid chromatography triple quadrupole (QQQ) mass spectrometry (LC/MS/MS) is suited for rapid analysis of multiple analytes. A sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of nicotine and its metabolites by QQQ. Using simple sample preparation techniques in urine, oral fluid and blood, and chromatographic configurations achieves the required sensitivity and separation and is capable of quantitating the drugs over their relevant dynamic range. Excellent reproducibility was observed for all drugs (CV < 15%). All calibration curves displayed linearity with an R2 > 0.995.
Fast and Confident Identification of Drugs and their Metabolites Using Ion Trap LC-MSn Analysis and a Library of >4,500 Compounds
Comprehensive screening of urine samples in forensic toxicology and clinical research is focused on the unambiguous identification of parent drugs and their corresponding metabolites. Here, we present the evaluation of a comprehensive and robust forensic toxicology ion trap based LC-MSn spectral library screening to detect and confirm both parent drugs and metabolites in urine as alternative or complementary technology to GC-MS. Acquired data (full scan MS, MS2 and MS3) were searched against the Toxtyper library (900 compounds) and the recently published Maurer/Wissenbach/Weber (MWW, Wiley-VCH, Weinheim, Germany, 2014) LC-MSn library which contains > 4500 compound entries including 3000 metabolites.
Experience and Lessons Learned Developing a Comprehensive Toxicology Panel for Pain Management and Beyond
We aim to set up a comprehensive toxicology testing program to meet the diverse clinical needs for urine drug testing. An LC-MS/MS method that quantitates 78 drugs and metabolites in urine with a simple dilute-and-shoot sample preparation was established. Method comparison using patient and proficiency testing samples demonstrated that this assay was sensitive and accurate. Urine drug screens are triaged to either immunoassays for emergent toxicity assessment or to mass spectrometry screening and quantitation for risk stratification and regulatory compliance. A comprehensive report is generated after testing detailing the quantity of each drug and metabolite, and provided with an interpretation.
Using LC-MS/MS Urine Drug Testing to Identify Licit and Illicit Drug-Use in a Community-based Patient Population
Liquid chromatography tandem mass spectrometry-based (LC-MS/MS) urine drug testing (UDT) results from 165209 unique specimens were retrospectively reviewed to determine the number of positive results reported for each drug, metabolite and drug product tested. The relative frequencies of the detected drugs were: methadone > cannabinoids > opiates > oxycodone > cocaine > clonazepam > buprenorphine > diazepam > fentanyl > amphetamine > methylphenidate > heroin > alprazolam > nitrazepam > ecstasy > meperidine > flurazepam > flunitrazepam. The influences and considerations of drug metabolism on: observed positivity rates; monitoring patient compliance; and LC-MS/MS UDT method design will be presented.
LC-MS/MS Analysis of Pain Management Drugs and their Polar Metabolites Utilizing Supported Liquid Extraction for Sample Pretreatment
Presented is a method utilizing SLE followed by UPLC-MS/MS for the detection of tapentadol, tramadol, meperidine and their polar metabolites. The extraction was optimized to ensure recovery of parent drugs and metabolites by incorporating a pretreatment with a basified brine and methylene chloride/isopropanol solution followed by an acidified methylene chloride/isopropanol elution. This ensured all analytes eluted in the same extract. The method has a LOD at least 50 ng/mL and a ULOL of 5,000 ng/mL for tapentadol, tramadol and their metabolites. The LOD for meperidine and normeperidine is 2 and 5 ng/mL, respectively with a ULOL of 1000 ng/mL. The method offers excellent S:N compared to other sample preparation methods.
Drug Excretion into Breast Milk: Are All Drugs Contraindicated for Breastfeeding?
Although not all drugs may be considered contraindicated while breastfeeding, there remains little data on this topic. The objective of this study is to investigate the risk of methotrexate exposure in nursing infants. We developed a simplified extraction method and an LC-MS/MS method to measure Methotrexate (MTX) and 7-hydroxymethotrexate in breast milk. Patients receiving MTX were recruited. We found that MTX is excreted into breast milk, with the highest concentrations at 1-12 hours post-dose; detectable levels were observed at 48 - 96 hrs. This data provides the foundation to establish a TDM system for measuring drug concentrations in breast milk.
LC-MS/MS Study of 25-OH Vitamin D2 and D3 with Perkin Elmer Vitamin D Kit Using Both Derivatized and Non-derivatized Methods
Quantification of 25-OH Vitamin D2 and D3 is widely used as a means of assessing vitamin D deficiency status because of their clinical significance in a variety of disorders. LC-MS/MS technology has demonstrated superior sensitivity, selectivity, and robustness for simultaneously detecting these Vitamin D metabolites in complex biological matrices. This work presents a rapid, reliable, and accurate LC-MS/MS research method on an IONICS 3Q 120 triple quadrupole mass spectrometer for studying 25-OH Vitamin D2 and D3 with Perkin Elmer Vitamin D kit using both derivatized and non-derivatized methods. Good linearity (coefficients R2>0.993), accuracies (97-102%), and CVs (< 10%) were obtained.
Clinical and Forensic Monitoring of Zopiclone Through the Use of a Degradation Product
The clinical and forensic monitoring of zopiclone in biological samples is severely limited by poor ex vivo stability of both the parent drug and the primary metabolites zopiclone-N-oxide and N-desmethylzopiclone. All three rapidly degrade to 2-amino-5-chloropyridine (ACP) under ambient and refrigerated conditions, with limited degradation also occurring in frozen samples. An acidic stabilization process is effective, but impractical for remote and poorly controlled sample collections. Alternatively, we are implementing a process in which all zopiclone and primary metabolites are chemically converted into ACP prior to analysis. Measured ACP levels are then utilized as a surrogate analyte reflecting zopiclone concentrations.
From Mice to Clinics © Verification of Hypothesis-driven Biomarker-panel by LC-MS MRM
Alport is a X©\linked disease, displaying mutations in COL4A3, COL4A4 or COL4A5©\genes, which in turn results in damage of multiple basement membranes followed by organ pathology. In order to better monitor this slow progressing disease we assessed a hypothesis driven biomarker©\panel by LC©\MS MRM in mice, followed verification in patients urine. Development and validation of a protein 10©\plex in mouse ¨Cand human urine will be described, followed by presentation of the verification of some marker in both species.
Full Validation of a High-throughput Immuno-MS PD-assay
Many compounds for the treatment of muscle-hypotrophy are currently being developed. Biomarker/PD-readouts are needed for monitoring target-engagement, efficacy, ectr. Myostatin as ActRIIb-ligand is one amongst. To be able to monitor slightest changes we developed and validated a high-throughput Immuno-MS method on a 96-well format. After Immunoprecipitation, myostatin is eluted from the antibody-complex by tryptic digest, and the signature-peptide is quantified against a calibration curve. First results from clinical samples are shown.
LCMS Method for the Simultaneous Determination of Metformin and Miglitol in Plasma: Application to Pharmacokinetic Studies
The objective of the current study was to develop a rapid and sensitive LC-MS method for the simultaneous estimation of metformin and miglitol in plasma using voglibose as internal standard. Chromatographic separation of active ingredients was achieved using a Zorbax eclipse C18 (150mm×4.6mmX5μm). The mobile phase constitute 95% ammonium acetate (0.02 mM, pH 6.8) and 5% methanol was pumped at an isocratic flow rate of 0.5 mL/min. The data acquisition was carried out in positive ion mode by Single Ion Monitoring at 130.1 m/z for metformin, 208.1 m/z for miglitol and 268.4 m/z for IS. Simple protein precipitation using higher concentration of acetonitrile was utilized to remove the endogenous materials. The validated method was applied for pharmacokinetic studies. Surprisingly, the pharmacokinetic profiles and parameters observed for both the drugs were comparable.
A System Genomic and Metabolomic Approach to Identify the Gene Networks Determining Metabolomic and Lipidomic Traits in Cardiometabolic Diseases Using MS-based
Cardiometabolic diseases (CMD) are one of the most widespread diseases worldwide. Recent high throughput genetic and metabolomic studies revealed associations between disrupted metabolic profiles and disease phenotypes. In order to identify novel genetic determinants of metabolism or new clinical biomarkers underpinning CMD, we performed untargeted metabolic profiling, by UPLC-MS, of 5 tissues of recombinant inbred rat lines. Further data analysis using mQTL-mapping of significant metabotypes revealed a large number of candidate genes identified and putative biomarkers, demonstrates strong potential to study the genomic basis of cardiometabolic diseases for novel biomarker and disease mechanism discovery.
Optimization of LC-MS/MS Method for Thyroglobulin Quantitation in Human Serum
The incidence of thyroid cancer worldwide is estimated at 213,000 persons per year. Measurement of thyroglobulin (Tg) is used for monitoring of the thyroid cancer recurrence. After thyroidectomy, Tg concentrations drop to a very low or undetectable level, and elevated serum Tg concentration indicates cancer recurrence. However, the presence of endogenous thyroglobulin autoantibodies (Tg-AAbs) can mask Tg epitopes and can lead to false-negative immunoassay results. We have established a LC-MS/MS method for Tg quantitation in human serum using a Tg-specific peptide FSPDDSAGASALLR as the surrogate peptide. Accurate concentration of thyroglobulin in serum can be measured without interference of Tg-AAbs.
A Novel Translational Analysis Pipeline Applied to the Discovery and Verification of Potential Biomarkers for Type 2 Diabetes
The translational proteomics pipeline requires a workflow that can provide unbiased discovery through targeted verification of biomarkers. This process can be long and painful since it typically requires integration of multiple pieces of data and, to date, has required a patchwork of different analysis tools that must be manually integrated since a complete solution has not been available. We have created a novel analysis package to address these challenges and applied it to a large set of plasma samples with increased insulin resistance and type 2 diabetes. Our analysis pipeline integrated visualization tools for the determination of system suitability, optimal performance and high-confidence targeted quantification.
Rapid Discovery of Differentially Expressed Proteins in T2D Plasma Samples Using Improved UHPLC Chromatography and PSMART Data Acquisition
Translational clinical proteomics links global protein discovery to targeted quantification with the ultimate goal of identifying and verifying clinically relevant disease biomarkers. Studies incorporating LC-MS can identify thousands of proteins and peptides per study requiring automated data processing to increase data interrogation. To better facilitate commercially available software, higher flow rates utilizing UHPLC pumps significantly increases the LC reproducibility. Incorporation of pSMART data acquisition results in significant increase in detection/sequencing reproducibility and better relative quantitation. We applied these new workflows to investigate protein differences associated with type 2 diabetes (T2D).
Clinical Shotgun Proteomic Subtyping of Pituitary Adenomas
Accurate subtyping of pituitary adenomas is essential for prescribing therapy. A novel shotgun proteomics assay was developed for subtyping these tumors from formalin-fixed paraffin-embedded specimens. A cohort of 35 cases of five different subtypes was analyzed. Hormone profile of each case was correlated with patient’s clinical record, serum hormonal levels and electron microscopy. 100% of cases were in agreement with their clinical picture. Correlation of hormone profile with IHC battery revealed 89% agreement rate. Exceptions were gonadotrophs that had neither clinical symptoms nor serum elevations of FSH/LH. This assay is being validated for routine use in a CAP/CLIA clinical laboratory.
Quantitative Measurement of Full-Length and C-Terminal Proteolyzed RBP4 from Serum Using HR/AM Detection to Support Translational Research
Serum retinol-binding protein 4 (RBP4) levels have been associated with diabetes and insulin resistance. Two major proteolyzed forms of RBP4 in human serum (RBP4-L and RBP4-LL) account for more than 50% of total RBP4 levels in some insulin resistant individuals. RBP4-L correlates highly with insulin resistance, but RBP4-LL does not. The presence and level of expression of these 3 forms of RBP4 might become a highly specific and sensitive biomarker for diabetes, heart diseases and other conditions. Currently, western analysis and ELISA do not provide high throughput or specific workflows to quantify these forms of the same protein to support translational research. Hence, we have applied a novel mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HRAM) to address this shortcoming.
Discrimination of Human Brain Tumors by Desorption Electrospray Ionization - Mass Spectrometry Imaging
We aim to acquire diagnostic information directly from human brain tissue using desorption electrospray ionization – mass spectrometry (DESI-MS) for intraoperative diagnosis and tumor margin assessment. We have expanded the types of human brain tissue (i.e. normal tissue and pituitary tumors). Preliminary results suggest that normal, meningioma, low-grade glioma, high-grade glioma, and pituitary tumors have characteristic lipid profiles that allow for discrimination. Further, small metabolites (m/z <200) detected in the negative mode were found to differ between the classes – an analogous metabolite profile. One promising metabolite, N-acetyl-aspartic acid (NAA), was detected and appears to decrease in neoplastic areas.
Quantitative Analysis of Human Tear Fluid by MALDI-TOF MS
Identifying biochemical markers for disease is a difficult endeavor. It requires methods that can quantify multiple components simultaneous, are high throughput, cost effective and deliver high precision and accuracy. Ideally, the same methods should also be applicable in a routine clinical setting. We and others are exploring the potential of matrix-assisted laser desorption/ionization (MALDI) for these applications. We demonstrate that precise results (CVs 1-2%) are achievable under ideal conditions, but that in a practical setting, factors such as the selection of internal standard, sample handling and ion suppression mean that CV’s are more typically 10% or greater. Some of the most important factors in determining reproducible and accurate results are illustrated and discussed by way examples.
UPLC-MS/MS Multiplex Methodology for Creatine Synthesis and Transport Disorders, Triple H Syndrome and OTC Deficiency
We aimed to increase the number of treatable disorders screened by the Mass Urinary Screening Program in the Province of Quebec. Creatine synthesis and transport disorders, Triple H syndrome and Ornithine transcarbamylase deficiency (OTC) were targeted by selecting specific urinary biomarkers: creatine, guanidineacetate, uracil, orotic acid and creatinine. A rapid multiplex methodology was developed and validated to analyze these biomarkers from urine samples dried on filter paper. This efficient methodology demonstrates the feasibility of mass or high-risk screening urine samples for early, pre-symptomatic detection and treatment of these inborn errors of metabolism.
Individualized Monitoring of Patients with Monoclonal Gammopathy: Optimizing Sample Preparation for Mass Spectrometry
Plasma cell proliferative disorders are described by the expansion of a single clone of plasma cells. These cells commonly produce large amounts of monoclonal immunoglobulin. These monoclonal antibodies, clinically referred to as M-protein, are currently detected by agarose gel serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). Recently, we have developed a top down mass spectrometry based method and coupled it to an optimized sample preparation procedure to yield a sensitive and high throughput platform capable of screening for monoclonal gammopathies and monitoring residual disease in myeloma patients.
Using LC-ESI-Q-TOF of Immunoglobulin Light Chains to Resolve Ambiguous Serum Protein Electrophoresis Cases
Monoclonal gammopathies are diagnosed by detecting a monoclonal immunoglobulin (M-protein) at levels exceeding the polyclonal background. In clinical practice, M-proteinsi are routinely detected by protein gel electrophoresis (PEL) and immunofixation electrophoresis (IFE). Occasionally, disease modifying factors and artifacts result in PEL and IFEs which appear to have M-proteins but clinical history and other laboratory testing suggest these could be artifacts. Recently, we have described a LC-ESI-Q-TOF method which can profile the mass distribution of serum immunoglobulin light chains with greater resolution of immunoglobulins which greatly aids in the resolution of gel ambiguities.
Uses of Intellectual Assets in Commercializing 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.
Combine HDX-MS and NMR for Polycystin-2 C-terminal Tail Structural Characterization
Polycystin-2 (PC2) is a calcium-regulated calcium-channel. The C-terminal tail of human PC2 is crucial for channel assembly and function. We have combined biophysical and structural approaches to study the calcium-dependent molecular mechanism within the C-terminal tail. The goal of our study is to define the oligomeric states of PC2 C-terminal tail in solution, to characterize its calcium-binding property, and to map its calcium-responding elements. This study provides a structural basis for regulation of the PC2 channel by its cytosolic C-terminal domain, with an improved understanding of the functional role of PC2 in regulating intracellular calcium signaling.
Development of a Rapid LCMS Method for Steroids in Plasma
Steroids are important to the human body for normal biological activity. Steroid measurement is essential when evaluating disorders such as congenital adrenal hyperplasia, Cushing’s disease and polycystic ovarian disease. Traditional methods to measure steroids have been immunoassays, however, these assays lack specificity and could take up to a week to be completed. Liquid chromatography mass spectrometry (LC-MS/MS) has become the industry standard in evaluating steroids due to its specificity, precision and sensitivity. This presentation will focus on a rapid six minute method to analyze 19 steroids and internal standards in plasma at pg/mL levels using UHPLC-MS/MS detection.
UPLC-MS/MS Analysis of Arginine Kinetics and Metabolism in Children with Severe Falciparum Malaria
We studied 10 healthy Tanzanian children (HC) and 10 with severe falciparum malaria (SM), using (13C6,15N4) labeled arginine tracer with measurement of isotopic enrichment in arginine and citrulline. These procedures allowed us to simultaneously determine the conversion of arginine to citrulline via the arginase-dependent (urea cycle) and nitric oxide synthase (NOS) pathways. Preliminary results show that arginine flux is higher in those with SM compared to HC children [median 108 (84-144) vs 85 (68-95) µmol/Kg/hr] and that citrulline production via the NOS pathway is reduced in malaria. These are the first direct measurements of arginine flux and metabolism in SM.
Quantitative Omics Strategies for Investigating the Oral Microbiome in Dental and Systemic Diseases
Microbes play an integral role in many human biochemical and metabolic processes. Perturbations of the normal microbial community have been linked to deleterious health outcomes. The oral microbiome is of particular interest, as the mouth is a primary point of entry into the body. The switch from a positive/symbiotic host-oral microbiome relationship to a negative/pathogenic disease state is likely mediated by proteins and/or small molecules. Yet large-scale surveys of the proteome and metabolome of oral microbiota, and the connection of these to disease states, have been lacking. Recent advances in quantitative, mass spectrometry-based technologies have made such investigations feasible. We present here a beginning analysis of the oral microbiome proteome and metabolome isolated from dental plaque samples collected in collaboration with the Marshfield Clinic Research Foundation.
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Corporate Workshops
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Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Waters Towards Excellence in Steroid Hormone Measurements Monday from 7:00 - 8:00 AM in Seabreeze This workshop will provide valuable details about the use of Waters’ Oasis µElution plate technology and column chemistries for the analysis of serum testosterone, androstenedione, and estradiol. These research methods have been evaluated for analytical sensitivity, reproducibility, and accuracy through participation in the Center for Disease Control and Prevention’s (CDC) Hormone Standardization (HoSt) Program. “Quantitation of 17â-Estradiol in Serum Using an Aggressive Sample Prep Method” “Quantitation of 17â-Estradiol in Serum Using an Aggressive Sample Prep Method” Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Restek Pain Assays Do Not Have to Be a Big Pain! Analyze 230 therapeutic and drugs of abuse compounds by LC-MS/MS with the Restek Raptor Biphenyl Monday from 7:00 - 8:00 AM in Marina 6 The use of pain management drugs is steadily increasing. As a result, hospital and contract labs are seeing an increase in patient samples that must be screened for a wide variety of drugs to prevent drug abuse and to ensure patient safety and adherence to their medication regimen. The Raptor™ Biphenyl column was developed to complement high-throughput LC-MS/MS analyses. In this workshop, we will present the methodology for a 230 compound multi-class drug and metabolite screen and discuss the challenges one must consider when developing a large screening assay. Topics of the discussion will include mobile phase considerations, isobar resolution, drug interference, and Instrumentation. Optimized chromatography will also be presented for nine separate drug panels for use during confirmation and quantitative analyses. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Sigma-Aldrich Sample Preparation Considerations for Multiplexed MRM LC-MS Protein Assays in the Clinical Laboratory Monday from 7:00 - 8:00 AM in Spinnaker Multiplexed protein assays have tremendous potential in clinical diagnostics, in particular measurement of proteins in plasma or serum derived from circulating blood. The dynamic range of protein concentration in these samples, and the dominance of very high abundance proteins such as albumin and immunoglobulins, make measurement of low concentration proteins impossible without some type of enrichment approach. This workshop will discuss the benefits of using Seppro® protein depletion columns in sample preparation for multiplexed LC-MS protein clinical assays. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Phenomenex Solutions for Challenging Biological Matrices in Analytical Method Development Monday from 12:00 - 1:00 PM in Harbor Ballroom 3 (1) When SPE is Not Enough: Considerations in developing a drug analysis in whole blood. We evaluate 7 common pretreatment procedures that hemolyze erythrocytes and precipitate plasma proteins from whole blood. The effectiveness of a procedure was based on overall recovery, response and reproducibility for analytes representative of different drug classes. (2) Optimizing Sample Prep for Quantitation of Buprenorphine and Norbuprenorphine in Meconium. Meconium, the first stool produced by a newborn, is a complex matrix of materials ingested by a fetus, making it a good specimen to detect in utero drug exposure. Traditionally, meconium is homogenized in a laborious methodology. A simplified method where meconium is homogenized directly in enzyme, hydrolyzed and cleaned up by SPE to extract buprenorphine and norbuprenorphine is explored. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Thomson Instrument Co Improved Sample Preparation of Biological Samples using the Thomson eXtreme Filter Vials® and analysis by LC-MS/MS Monday from 12:00 - 1:00 PM in Harbor Ballroom 1 Sample preparation continues to be a critical factor in the quantitative measurement of biological samples. The goal of this seminar is to discuss how to streamline the sample preparation process of oral fluids and urine utilizing the Thomson eXtreme Filter VialsTM to reduce interferences from the sample matrix and increase analyte recovery. The Thomson eXtreme Filter VialsTM saves time, reduces solvent usage, alleviates the need for expensive consumables and lab equipment. Samples preparation for matrices such as urine and oral fluids will be discussed. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Thermo Scientific Time Matters. Simplify Workflow Complexity. Monday from 12:00 - 1:00 PM in Harbor Ballroom 2 Discover three newly listed Class I medical devices for general clinical use: Thermo Scientific™ Prelude MD™ HPLC, Thermo Scientific™ Endura MD™ mass spectrometer, and Thermo Scientific™ ClinQuan MD™ software. Clinical laboratories can use these devices to build their own lab developed tests (LDT). Combined, tools provide laboratories the ability to obtain the quantitative accuracy of LC-MS, easily and confidently. Examples of various compounds and workflows will demonstrate the robustness, stability, and time efficiency of these new class I medical devices.
Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Bruker (1) Robustness of the EVOQ Elite in Clinical Research Analysis Monday from 12:00 - 1:00 PM in Marina 6 (1) Due to the high sensitivity and selectivity provided by LC-MS/MS, this technology is being adopted in more clinical research applications. This talk will describe the unique design and performance characteristics of the EVOQ LC triple quadrupole system for the analysis of various research assays including Vitamin D, alcohol biomarkers, fructose and steroid profiling. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: ZefSci Special Considerations for Clinical LC-MS/MS Method Validation Tuesday from 7:00 - 8:00 AM in Seabreeze CLIA regulations require method validation, but do not specify which tests are required. We will talk about why LC-MS/MS methods may need extra validation steps compared to other high complexity tests. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Tecan Automation Solutions for Mass Spec Sample Preparation in the Clinical Laboratory Tuesday from 7:00 - 8:00 AM in Spinnaker Although there have been monumental advances in mass spectrometry (MS) instrumentation in recent years, its unglamorous counterpart, sample preparation, has not enjoyed the same rate of development. This workshop will illustrate how the status quo is changing and will highlight a number of recent advances by Tecan utilizing its world class Freedom EVO liquid handling workstation to develop a variety of protocols designed to handle a broad range of sample types, sample preparation protocols and analytes to meet the needs of the modern analytical clinical laboratory. This includes both vacuum and positive pressure based SPE protocols, tip based cleanup for rapid processing of a small number of samples, sample tracking and LIMS integration. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: IONICS MS Identification of Potential Biomarkers for Efficacy and Toxicity in Clinical Trials of a Cancer Vaccine using Targeted Metabolomic Profiling Tuesday from 7:00 - 8:00 AM in Spinnaker Dr. Devanand Pinto will discuss what his Biomarker Quantification Team at the National Research Council of Canada has accomplished in their pursuit of a faster, more sensitive and more robust method of measuring immune response in cancer vaccine trials. Immune response is typically measured by cytokine profiling and flow cytometry; however, these techniques require extensive sample preparation and, due to the use of antibodies, have limited multiplexing capabilities. The Biomarker Quantification Team investigated the use of targeted metabolomics profiling to study the metabolic profile of 38 patients enrolled in a Phase I/Ib clinical trial for a novel cancer vaccine.This targeted metabolomic profiling approach utilizes an IONICS 3Q 320 which provides the leading sensitivity and advanced multiplexing capabilities necessary for success. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Agilent Technologies (1) Development of a Multiplexed MRM LCMS LDT Tuesday from 12:00 - 1:00 PM in Harbor Ballroom 3 (1) The potential impact of multiplexed MRM LCMS analysis for clinical diagnostics is enormous and has garnered much attention in recent years. This workshop will discuss the approach for the development of multiplexed MRM LCMS LDT(s) and highlight some of the analytical challenges associated with the design and development of such an LDT. (2)The Pediatric Trial Network (PTN) was established to create an infrastructure for investigators to conduct trials that improve child health. Advancement of low volume sampling technologies is being achieved through these trials by demonstrating the concordance of drug concentrations in plasma to Dried Blood Spots (DBS). In this presentation, Dr. Lewis will give a brief description of challenges and remedies encountered in the quantitative analysis of dried spot specimens for small molecule targets. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Waters Clinical Research and Biomarker Discoveries, Challenges and Opportunities Tuesday from 12:00 - 1:00 PM in Harbor Ballroom 1 In this workshop, the directors of the large MS core facilities at Duke and Georgetown University Medical Centers will discuss the real-life challenges of, and the opportunities for conducting clinical research and biomarker discovery using state-of-the-art MS-based approaches, including proteomics, lipidomics and metabolomics. Application examples, useful tips and tricks on how to manage large MS experiments, and some practical MS troubleshooting advice will be provided. Presentation #1: Supporting the Systems Medicine Paradigm: Metabolomics and Lipidomics Core Technologies for Clinical and Translational Research Presentation #2: The Utilization of Quality Measures in Experimental Design and Interpretation to Aid Quantitative Clinical Proteomics and Metabolomics Studies” Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Shimadzu Secrets to Successful LC-Tandem MS Implementation - Tips and tricks for Triple Quadrupole Mass Spec Techniques Tuesday from 12:00 - 1:00 PM in Harbor Ballroom 2 From screening to confirmation/quantitative testing, there are many pitfalls that can prevent maximum laboratory efficiency. This workshop will discuss several specific scenarios and offer solutions to everyday challenges in medication monitoring and urine drug testing, including: - Better sample preparation, - Validity Testing (to ensure the sample is human and that it has not been diluted nor adulterated), - Detection of early signs of medication misuse or illicit drug use, - State of the art LC/MS technology, - Delivering quality data on an ultra-fast timescale, and - Simplifying reporting and billing. Please join us for this practical workshop and learn how to operate your laboratory at maximum efficiency. Attendees will receive a lunch, t-shirt and GiantMicrobe! Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Biotage Advances in Sample Prep for Pain Management: Developing a Simple, Fast Method for Nonpolar Compounds and Polar Metabolites in Urine prior to LC/MS Tuesday from 12:00 - 1:00 PM in Marina 6 Described here is a method utilizing supported liquid extraction for the detection of tapentadol, its glucuronidated, sulfated, and N-desmethyl metabolites; tramadol, its N- and O- desmethyl metabolites; and meperidine and its N-desmethyl metabolite. The method utilizes ISOLUTE SLE+ (Biotage; Charlotte, NC) for sample clean-up followed by analysis by LC-MS/MS. The extraction was optimized to ensure good recovery of both nonpolar parent drug polar metabolites by utilizing a sample pretreatment with a basified brine solution and elution with acidified methylene chloride/isopropanol. These steps ensured that both polar and nonpolar analytes eluted in the same extract. The method has a limit of detection of at least 50 ng/mL and an upper limit of linearity of at least 5,000 ng/mL. The method is simple, fast and offers excellent S:N. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Agilent Technologies The Agilent StreamSelect LC/MS System – Up To Four Times the Throughput with Outstanding Reliability Tuesday from 7:00 - 8:00 PM in Harbor Ballroom 3 The StreamSelect LC/MS System delivers up to four parallel chromatographic separations to the same triple quadrupole mass spectrometer, with superior robustness and data quality. Intuitive automation software coordinates the completely integrated system, maximizing MS utilization and greatly enhancing throughput and cost-effectiveness. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Thermo Scientific Break the Bottleneck - Accelerate Genomics to Proteomics to Bedside with New Cloud-computing and Informatics Solutions Tuesday from 7:00 - 8:00 PM in Harbor Ballroom 2 An overview of the proteo-genomic data analysis workflows and automation of informatics solutions for clinical service providers as well as researchers to run routine data analysis workflows in a seamless manner. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: IONICS MS Recent Developments in Endocrinology – Diurnal Steroids Fluctuations, Free 25-OH Vitamin D3 & Thyroid Hormone Management Tuesday from 7:00 - 8:00 PM in Marina 6 In this workshop Dr. Steven Soldin from the National Institutes of Health (NIH) will address several recent developments in endocrinology. Topics discussed will include: diurnal fluctuations of steroids, changing trends in management of thyroid patients, the role in measurement of free 25-OH Vitamin D3 and a variety of clinical applications. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Spark Holland (1) Routine Clinical LC-MS Assays Using Online Sample Preparation: Daily User Experiences and Illustrations Wednesday from 7:00 - 8:00 AM in Seabreeze (1) For reproducible highly sensitive LC-MS/MS assays, sample preparation is pivotal. Several sample prep approaches are available, each with its own (dis)advantages. In our clinical chemistry laboratory different sample prep strategies are used with online sample prep as chosen method. In this presentation we provide an overview of our clinical LC-MS/MS assays using online sample prep and discuss different strategies by focusing on relevant examples. (2) Punching of DBS cards for LC/MS analysis is tedious and too manual for a high sample volume lab. This presentation describes how commercial DBS cards as well as a prototype dried plasma spot (DPS) card can be analyzed on-line in a fully automated fashion using either SRM LC/MS or high res. mass spectrometry techniques for the bioanalytical determination of drugs in micro blood samples. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Sigma-Aldrich Developing the Best Sample Preparation Methods to Ensure Robust LC/MS Analyses in Biological Fluids Wednesday from 7:00 - 8:00 AM in Spinnaker Sample preparation continues to be one of the critical factors for effective method development when analyzing biological samples. Too often this portion of the assay is not allocated sufficient attention to ensure a robust analytical method. The goal of this seminar is to discuss several approaches for sample preparation for biological fluids, and to demonstrate the benefit that proper sample clean up can have on an LC/MS based methods. Sample preparation methods for matrices such as plasma and urine will be covered, along with techniques such as solid phase extraction, phospholipid depletion, and enzymatic digestion. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: SCIEX 1) Lipid Clinical Biomarkers: From Discovery to Quantitation. 2) Implementing LC-TOF-MS/MS in the Clinical Research Arena Developing A Broad Spectrum Test Wednesday from 12:00 - 1:00 PM in Harbor Ballroom 3 1) Lipid Clinical Biomarkers: From Discovery to Quantitation - This presentation will focus on the general methods used for lipid biomarker discovery will be presented with special focus on the novel use of DMS as a tool orthogonal to chromatography to achieve better qualitative lipid identification. 2) Implementing LC-TOF-MS/MS in the clinical research arena developing a broad spectrum test - This presentation will focus on the pros and cons of assorted screening approaches for maximum compound coverage, minimizing false positive finding and improving confidence in results. As well as, tips for managing challenging cases that include complex samples and matrices.
Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Shimadzu Prepare Your Laboratory for the Future - Laboratory-on-a-Card Technology and Ultra-Fast Mass Spectrometry Wednesday from 12:00 - 1:00 PM in Harbor Ballroom 1 Please join us for this interactive workshop where you will discover a new technology (Noviplex Duo) that enables multiple plasma extractions and sample preparation steps to be performed from a single blood drop in just minutes. This “lab-on-a-card” technology simplifies and accelerates sample preparation by: - Integrating and automating multiple sample prep steps, - Introducing in-transit sample preparation, - Allowing collection of multiple fractions, - Reducing sample prep time, - Simplifying sample transport, - Reducing chain of custody issues, and - Enabling simultaneous analysis of proteins and metabolites. Combined with ultra-fast mass spectrometry, these technologies can save your laboratory time, money and resources. Attendees will receive a lunch, t-shirt and GiantMicrobe! Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Thermo Scientific Employing Translational Research Workflow on LC-HRAM Platform for Detection of Pathogen Induced Cancer in a Human T-Cell Leukemia Virus Type 1 Disease Model Wednesday from 12:00 - 1:00 PM in Harbor Ballroom 2 Dr. Sucharita Dutta, from Eastern Virginia Medical School will discuss the Human T-Cell Leukemia Virus Type 1 (HTLV-1) as the causative factor for the development of an aggressive lymphoma, Adult T-cell Leukemia (ATL). The translational workflow involves standard data-dependent acquisition (DDA) experiments followed by more in-depth pSMART data acquisition methodologies or the most comprehensive global profiling of proteins from exosome samples to exhaustively mine for proteins that show functional significance via pathway analysis. |
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