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MSACL 2013
MSACL.MBDxA 2013 Conference Registrants have complimentary access to protected videos (
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ABSOLUTE QUANTIFICATION TO STUDY NEURODEGENERATIVE DISEASES BY QCONCAT STRATEGY
Defining the total amount of proteins in cell by mass spectrometry requires known amounts of internal standards. However, absolute quantification was hampered by the availability of standard labeled signature peptides in accurately known amounts. In this project, we designed peptide standards containing concatenated sequences of specific proteotypic peptides that are derived from various neuron disease related proteins. The concatenated peptides were generated in in vitro translation system. Isotopically labeled Lys and Arg were introduced to produce stable isotope-labeled peptides. A known amount of isotope-labeled peptides standards were co-digested with tissue samples containing target proteins. Absolute quantification of these neuron disease related proteins was performed by multiple reaction monitoring (MRM) with a triple quadrupole mass spectrometry.
Evaluation of Oxidative Stress Using Isotopic Dilution Liquid Chromatography Mass Spectrometry for the Determination of Isoprostanes in Human Serum Samples
F2-Isoprostanes (F2-IsoPs) have been widely used in recent years as the most sensitive biomarkers of oxidative damage/stress. We present a novel LC-MS/MS method using isotopic dilution to simultaneously determine six F2-IsoPs (8-iso-PGF2a, 8-iso-15(R)-PGF2a, 15(R)-PGF2a, PGF2a, 11b-PGF2a, 5-iPF2a-VI) in a single human serum sample (500uL). This method can be applied to both clinical and research settings to evaluate health status after surgical procedures, chemical treatment/exposures, chronic health conditions and other risk factors that may cause oxidative stress. In addition, it can aide to elucidate mechanistic pathways of chemical toxicities and effects to human health due to oxidative stress.
Discovering Glycan Biomarkers of Ovarian Cancer by LC-MS/MS: Comparing N-Glycans Released From Proteins in Cancer Cell Lines and Tissues
Epithelial ovarian cancer has the highest mortality rate within gynaecological cancers, owing to the lack of early detection diagnostic biomarkers. The membrane protein glycosylation profiles from serous ovarian cancer cell lines and primary tumor samples were characterized and compared using graphitized carbon LC-ESI-MS/MS. We show that cell surface glycans play a critical role in ovarian cancer as evidenced by the changes in glycan structures and related glycosyltransferase enzyme expression in cancer compared to normal cells. The data demonstrates the potential utility of mass spectrometry for monitoring cancer-specific glycan alterations as diagnostic ovarian cancer biomarkers and therapeutic anti-glycan antibody targets.
Multiplexed protein-assay establishment and validation for clinical trials.
With improved sensitivity and higher scan-rates of the mass-spectrometers, absolute quantification of multiplexed proteins using isotope-dilution LC-MS MRM, has become a more and more used solution for protein-biomarker. This is especially needed for applications, where ligand-binding assays cannot provide reliable data – some of the reasons for this will be highlighted. However, for application in clinical trials fully validated methods need to be applied. For protein-biomarker assays this bears some challenges, which include selection of the calibration-matrix, sensitivity, choice of internal standard, different working ranges and recovery. IGF-1-binding proteins (IGFBP’s) are a family of regulatory proteins, which control the various biological functions of IGF-1. We established /validated a LC-MS assay for the IGFBP’s 1, 2, 3, and 5 in human serum to be applied for clinical trials.
Global quantification of histones, histone PTM’s and histone modification enzymes in mesenchymal stem cells using a multiplexed SRM assay
Histone post translational modifications (PTM’s) are a central theme in the regulation of gene expression. A rapidly growing list of modifications confirms that they play fundamental roles in chromatin remodeling processes. These processes are also thought to play a role in stem cell development and senescence (1). To date, most studies in this area have been carried out by genomic analysis, immunostaining, or top-down LC-MS/MS analysis, and as such are not fully quantitative. We developed a multiplexed SRM assay for ten histone modification proteins and histone gamma H2Ax sequences. The assay was used to interrogate mesenchymal stem cells derived from human adipose tissue. Lysates were prepared from cell cultures at different ages (Self Renewing (SR) and Senescing (S)).
Automated quantitative mass spectrometry-based approaches for the diagnosis of beta thalassemia in a clinical trial setup
Hemoglobinopathies are disorders of hemoglobin, may be categorised into two disease groups, structural variants and thalassemias. These disorders are one of the most common type of inherited disorders, and pose a significant healthcare problem. Mass spectrometry has been previously used in the diagnosis of hemoglobinopathies, and it is currently employed as a method to characterise structural variants. Here we show results using multiple mass spectrometry based approaches for quantitation of the delta globin chain, which may be used as a biomarker for beta-thalassemia. Our results derived from a clinical trial show excellent correlation with existing HPLC measurements.
Understanding Age-related Effects on the Severity of Sepsis in the Plasma Proteome of Elderly CAP Patients
A semi-quantitative plasma proteomics workflow was applied to understand the age-related risk of severe sepsis, a systematic inflammatory disorder, among community-acquired pneumonia (CAP) patients. Seven hundred and seventy-two proteins were identified and 86 proteins had statistically significant differences in expression between groups. Differentially expressed proteins can be categorized according to age and severity of disease and are involved in numerous pathways, such as acute-phase response, coagulation signaling, and production of nitric oxide and reactive oxygen species amongst others. This study has provided valuable insight to factors which may explain age-related outcomes in CAP patients.
Quantitative Nanospray MS/MS of 8-iso-PGF2á in Human Urine: Sensitive Detection of a Clinical Marker of Oxidative Stress
We present a method for quantification of 8-iso-PGF2á in human urine by nanoflow HPLC with chromatographic separation of 8-iso-PGF2á from isomeric isoprostanes / prostaglandins and sensitive nanospray detection (1 fmol injected). Our work expands on a recent publication by using a more thorough sample isolation scheme. The Agilent Technologies HPLC-Chip Cube MS Interface and microfluidic chips proved to be robust enough for routine quantification. Calibration curves using standard 8-iso-PGF2á with internal standard 8-iso-PGF2á-d4 were generated. The content of 8-iso-PGF2á in human urine was determined.
Searching for Cardiovascular Biomarkers Using “Omics” Approaches
Cardiovascular disease remains the leading cause of death in the U.S., affecting nearly 60 million people and claiming nearly one million lives each year. Novel biomarkers for cardiovascular disease such as heart failure and hypertension offer great promise to the diagnosis and treatment of these diseases. We use multiple “Omics” approaches, such as those for phosphoproteome and interactome, in combination with different animal models and cellular systems, to search for biomarkers for cardiovascular diseases. We also investigate the effects of several key modulators for heart diseases. Novel biomarkers are revealed from “Omics” data using systems biological tools.
Quantitative Targeted Proteomics of Mycobacterium Tuberculosis Disease Markers: A Workflow for Large-Scale Clinical SRM Assay Development and Validation
Targeted quantitative proteomics utilizes techniques of tandem mass spectrometry, mostly selected reaction monitoring (SRM), to measure predetermined sets of proteins in a consistent, reproducible and quantitative manner. In order to verify potential markers of Mycobacterium Tuberculosis infection we have established a protocol for quantitative SRM assay development and validation to known Mtb protein surrogates according to generally accepted guidelines. 1228 peptide surrogates were selected with the support of modern proteomics resources including PeptideAtlas and SRMAtlas, a unique compendium of verified SRM measurements that allows the extraction of optimal SRM transitions sorted by abundance to provide full quantitative assay support.
Determining Circadian Patterns of Cardiovascular Biomarkers by a Novel Dried Blood Spot Collection Device
Concentrations of circulating biomarkers for cardiovascular disease (CVD) vary in a circadian rhythm throughout a day and disease state can be predicted from time-specific patterns. To show feasibility for circadian measurements of CVD biomarkers, blood samples were collected at 8 time points within a 24 h period with a novel dried blood spot (DBS) collection device, HemaSpot, that enables self-collection at home. Biomarkers including cortisol, aldosterone, epinephrine, norepinephrine and homocysteine were measured by multiplex LC-MS/MS. These methods will allow unprecedented and efficient determination of CVD risk based on circadian biomarkers.
Discovery of predictive COPD biomarkers by using a mass spectrometry based proteomics
The goal of our study is to identify biomarkers for Chronic Obstructive Pulmonary Disease (COPD), which can predict the inception of the disease, and to understand the molecular mechanism of the disease. We analyzed four different sets of human serum samples; sample from COPD patients, healthy heavy smokers, and baseline samples from the cased and control. The samples were analyzed by a bottom-up label-free proteomics approach, using an LTQ-Orbitrap Velos. Twenty proteins that were overexpressed in serum before the patients acquired COPD, and eleven overexpressing proteins in healthy heavy smokers were identified. Experiments to confirm those proteins in each individual samples are underway.
Immuno vs. SRM Assays: Lessons From Quantitation of Fibulin-1 in Human Plasma Samples
Fibulin-1 is an extracellular matrix (ECM) glycoprotein, recently found as a novel biomarker in cardiovascular disease. Alternative splicing in the fibulin-1 gene produces four splice variants (A, B, C and D) of which the composition in blood is presently unknown. We have developed a two-site immunoassay for absolute quantitation of total fibulin-1 in blood and compared the performance of this assay to that of an isoform-specific fibulin-1 assay developed on a nanoflow LC-SRM-MS platform. Fibulin-1 plasma concentrations were determined using both assays and compared across different patient cohorts. The value of each type of assay is discussed.
A High Sensitivity Analytical Method for the Determination of Derivatized 1,25-Dihydroxyvitamin D3 and 1,25-Dihydroxyvitamin D2 by LC/MS/MS
We report here the results of a recently developed workflow for the analysis of 1,25-dihydroxy vitamin D, using a novel derivatizing reagent to significantly improve the sensitivity of the LC-MS/MS based assay. Serum samples (200 µL) were diluted with deionized water and cleaned up using a combination of Celite and silica gel SPE type cartridges. Samples were then immediately derivatized using Ampliflex™ Diene reagent, and after 1 hour the derivatized samples were diluted with deionized water and analyzed by LC/MS/MS on an AB SCIEX QTRAP® 6500 system.
Using double charcoal stripped serum as the matrix, we were able to routinely obtain LODs as low as 5 pg/mL. Linear calibration curves were generated from the same matrix from 5 to 250 pg/mL. Precision values, based on multilevel calibrators and controls, were typically less than 10%, with accuracies ranging between 94-106% .
Rapid inflammation screening through analysis of oxygenated fatty acids (oxylipins) in human plasma
Oxylipins are signaling lipids that play prominent roles in health and disease. Because of the low concentration, reactivity and the presence of many isomeric species, their analysis requires a rapid, sensitive and specific methodology.
Here, we present a high-throughput solution to simultaneously screen and quantify bioactive oxylipins and free fatty acids in plasma. Using a combination of solid-phase extraction and UPLC-ESI-triple quadrupole mass spectrometry, we were able to measure the levels of 123 oxylipins (e.g., including prostaglandins, prostacyclines, thromboxanes, dihydroprostaglandins and isoprostanes) and 13 relevant free fatty acids (including e.g., DHA, EPA and arachidonic acid) in human plasma samples in less than 10 minutes. Such high-throughput SPE-UPLC-based assay could find routine applications in drug discovery and development for diagnostic or prognostic screening.
Laser microdissection of cervix carcinoma tissues: translation of heat shock protein 90alpha as potential biomarker candidate from tissue to serum
In this study we compared cancer tissue and healthy epithelium/or stroma of the cervix from patients with cervical cancer by laser microdissection of ~2500 cells. Statistical analysis resulted in a number of peptides more abundant in tumor cells versus normal epithelial cells. As an example, heat shock protein 90alpha was validated by immunoblotting, immunohistochemistry, ELISA and by tandem massspectrometry in the multiple reaction monitoring in large number of sera from cervical cancer patients at various stages of disease and healthy subjects.
Verification of Biomarkers for Pancreatic Ductal Adenocarcinoma Using Multiple Reaction Monitoring
Most patients with Pancreatic Ductal Adenocarcinoma (PDAC) have a 5-year survival rate of less than 4%. There is an urgent need for better biomarkers for PDAC. We applied iTRAQ, using 6 PDAC tissues and the corresponding, adjacent normal tissue, and obtained a preliminary list of 1,445 PDAC-specific proteins. Thirty-three candidates of the preliminary list were verified by multiple reaction monitoring (MRM) using 30 PDAC and 20 normal healthy serum samples. Seventeen MRM-verified proteins had the AUC values > 0.7. The 6-MRM verified proteins were combined into a multi-marker panel, which improved the discriminatory power versus the best individual marker.
A Sensitive and Selective LC-Ion Mobility-Mass Spectrometric Analysis of Allopregnanolone and its Isomers in Human Plasma and Serum
The challenges for the analysis of neurosteroids, including allopregnanolone, by LC-MS/MS are poor ionization efficiency, and the presence of numerous isobaric interferences in biological samples. To overcome these challenges, ion mobility separation has been combined with conventional LC-MS/MS detection using a highly sensitive triple quadrupole mass spectrometer equipped with the SelexION™ ion mobility device.
The method employed liquid-liquid extraction of 100µL serum or plasma. After extraction, the sample was derivatized using a commercially available quaternary aminooxy reagent. The calibration range was from 5 pg/mL to 100 ng/mL in serum or plasma, with CV < 10% and accuracy between 90-110%. The recovery was 98%, and the limit of detection is 5 pg/mL for allopregnanolone and pregnanolone. Plasma samples from normal, pregnant, and postpartum women were analysed using this method.
Ultra-Sensitive Quantitation of Aldosterone in Serum by LC-MS/MS using the AB SCIEX Triple Quad™ 6500 System
The analysis of aldosterone in serum was accomplished using UHPLC coupled to the AB SCIEX Triple Quad™ 6500 system. Pre-analytical sample preparation consisted of liquid-liquid extraction in MTBE, followed by evaporation under N2 gas, and reconstitution of the sample in the LC mobile phase. Chromatographic separation was accomplished using a Phenomenex Gemini-NX C18 analytical column. The total run-time for the method including re-equilibration was 10 minutes, to ensure adequate separation of the aldosterone analyte from endogenous interferences.
Human serum samples were analyzed containing concentrations of aldosterone ranging from 14 pg/mL to 300 pg/mL. The limit of quantitation for aldosterone in serum was observed to be less than 1 pg/mL. The method displayed excellent linearity (r=0.99971), accuracy (89-118%), and CV% (0.5-0.1%) over the concentration range from 1-1000 pg/mL.
Detection and Evaluation of Parathyroid Hormone-Like Protein using a novel Mass Spectrometric Immunoassay
Cancer metastasis is a primary concern when it comes to patient diagnosis and prognosis. New biomarkers, and higher content assaying technologies, are of high value if able to identify at risk patients. The ability to segregate such individuals for modified treatment regimens will not only save lives, but drastically reduce the overall treatment costs. Parathyroid Hormone-Related Protein (PTHrP) is a high profile biomarker that is showing promise in this area. Due to the heterogeneous nature of this protein, mass spectrometry (MS) is an ideal method for analytical detection. Described is a method for the immuno-affinity extraction and MS analysis of PTHrP from human biological matrix.
Cell Membrane Glycan Profiling Can Differentiate Cancer Origins And Subtypes
In clinical settings, biopsies are routinely used to determine cancer type and grade. Identification is commonly based on tumor cell morphology, as determined via histochemical or immunohistochemical staining. Unfortunately, in a significant number of cases, biopsy results are inconclusive. Moreover, even when primary cancer types can be identified, phenotypic subtypes (such as drug-resistant variants) are rarely differentiated. Glycomic profiling of the cell membrane offers an alternate route towards cancer diagnosis. In this study, significant differences were observed in the membrane glycosylation patterns of various cancer cell types. Based on characteristic N-glycan profiles, cancer origins and molecular subtypes were rapidly differentiated.
Development and Validation of an LC-MS/MS Assay for Quantitation of Urinary Leukotriene E4.
Alan J. Lueke, Joseph H. Butterfield, Amy K. Saenger
Assessment of leukotriene E4 (LTE4) in urine is a non-invasive means to evaluate systemic production and excretion of cysteinyl leukotrienes, which are derived endogenously from arachidonic acid through the 5-lipoxygenase pathway. LTE4 is the primary stable metabolite of total cysteinyl leukotrienes and their synthesis is initiated by a variety of stimuli including antigens, microbes, cytokines and immune complexes. Elevated concentrations of LTE4 are associated diagnostically with inflammatory conditions which have accelerated activity of mast cells, inclusive of mast-cell proliferation disorders, allergic reactions and mastocytosis.
We have developed a sensitive, precise LC-MS/MS assay for quantitation of leukotriene E4 in urine. This assay has significant potential utility as a marker of mastocytosis, particularly in concert wi
Urinary Exosomes; A Source of Biomarkers and Novel Diagnostics for Polycystic Kidney disease
Polycystic kidney disease (PDK) is estimated to affect 1/400 to 1/1000 individuals to varying degrees. Genetic testing (~$5,000) and MRI’s ($5,000) are expensive options. We have identified several proteins in urinary exosomes of PKD patients that are differentially expressed compared to controls. The ratio of two such proteins (polycystin-1/TMEM2) segregates PKD patients from control. This data was obtained by analyzing exosomes isolated in a D2O/sucrose gradient which is not amenable to translation to the clinical laboratory. Thus, further development of a quick and inexpensive method to isolate urinary exosomes and a rapid LC-MS/MS analytical work flow will yield the basis of a clinically amenable test that will reliably estimate PKD and its severity. Our efforts to accomplish this goal will be demonstrated.
A Novel Method for the Extraction of 1α,25-Hydroxy-vitamin D2 and D3 and Analysis using UHPLC-MS/MS
Vitamin D analysis has extremely important clinical relevance with levels needing to be measured for a wide variety of reasons. This poster presents a novel high throughput method for the extraction of the biologically active components: 1,25-dihydroxy-vitamin D2 and 1,25 dihydroxy-vitamin D3 using an ISOLUTE SLE+ 400 96 well plate. The method was developed from charcoal stripped serum samples spiked at various therapeutic levels and subjected to a partial validation. Overall method performance demonstrated high analyte recoveries and low ion suppression, allowing a quantifiable range matching that therapeutically expected.
Aberrantly Glycosylated TIMP1 as a Serological Biomarker Candidate for Colorectal Cancer by Targeted MRM Analysis Coupled with Lectin-Capturing and Peptide Affinity Enrichment
An aberrant glycosylation state can play central roles in pathological processes involved in cancer or disease. Blood containing glycoproteins aberrantly glycosylated can be used as an attractive sample for development of a cancer biomarker. To fractionate aberrant protein glycoforms related to colorectal cancer progress from serum samples, lectin capturing showing specific affinity to the aberrant glycan structure was used. Through the comparative MRM-MS analysis of lectin-fractionated serum and tryptic peptide affinity enrichment technique with anti-peptide antibody, the target protein, aberrantly glycosylated tissue inhibitor of metalloproteinase 1 (TIMP1), can be a potent colorectal cancer biomarker candidate.
GABA Levels in Cerebrospinal fluid (CSF) of a Pediatric Population Measured by LC/MS
Studies indicate that low levels of brain GABA (gamma-aminobutyric acid) are associated with poor seizure control. However, precise measurement of GABA levels in the developing brain remains especially challenging due to their extremely low levels in cerebrospinal fluid (CSF). Using a new robust ESI-MS/MS method we demonstrate that differences in CSF-GABA levels between patients with seizures ages 4 to 24 months (n=30) and age-matched controls without seizures (n=42) are highly significant (P=0.003). This method has potential for prescreening patients in the preclinical stage and for monitoring patients’ responses in evaluating strategies for treating seizures.
Use of Ultra Performance Convergence Chromatography-Tandem Mass Spectrometry for the Separation & Assay of Serum Vitamin D Metabolites
Mass spectrometry is being increasingly used for the analysis of vitamin D metabolites in clinical samples. In this study, we explored the feasibility of using a CO2-based chromatography system coupled with tandem mass spectrometry to successfully separate and quantify the most abundant clinically-relevant vitamin D metabolites, 25-OH-D and 24,25-(OH)2D3 in serum extracts. We conclude that this is a rapid, convenient and accurate method for vitamin D analysis that avoids the common technical complication often encountered with such analyses, namely comigration with the 3-epimer of 25-OH-D3.
Development of a quantitative gastroesophageal cancer SRM assay for use in FFPE tumor tissues
Aberrant over-expression of receptor tyrosine kinases (EGFR, etc) along with other critical downstream oncogenic mediators (KRAS, etc) are known drivers of gastroesophageal cancer (GEC). We tested a‘GEC-multiplex’ assay for quantification of RON, Met, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC on 100 primary GEC tumors, with paired metastases. Multiplex quantification of tissues identified patients with amplified HER2, cMET, IGF1R and showed inter-patient and intra-patient (from primary to metastasis) heterogeneity of samples, allowing potential therapeutic approaches.
Diagnostic value of testosterone (T), free testosterone (fT), and free
androgen index (FAI) in polycystic ovary syndrome
The objective of the study was to determine reliable
reference intervals for T, fT, and FAI and to test the discriminative value of these parameters in a PCOS-population using LCMSMS and 2nd Gen IA. Serum was obtained daily during a normal menstrual cycle from 25 healthy women (743 data-points). A single serum was obtained from 43 PCOS-patients. T was measured by LC-MS/MS and Architect® 2nd Generation Testosterone IA. Sex hormonebinding globulin was measured to calculate fT (Vermeulen) and FAI ([T]/[SHBG]*100). The results for each parameter are summarized below: PCOS T range(nmol/L) by LC-MS/MS 0.6–3.2, compared to normals 0.3–1.6 (AUC 0.84); by IA 0.7–2.9 compared to normals 0.5–2.0 (AUC 0.83). PCOS fT range(pmol/L) by LC-MS/MS 9–60 compared to normals 5.2–26 (AUC 0.91); by IA 12–62 compared to normals 7.2–33 (AUC 0.90). FAI AUC results were similar to fT.
Development of an Inductively Coupled Plasma Mass Spectrometry Method for Measurement of Iodine in Urine
Iodine deficiency may lead to reduced thyroid hormone production and ultimately hypothyroidism. Iodine status can be assessed by urinary iodine excretion, for which inductively coupled plasma-mass spectrometry (ICP-MS) is the gold standard method. An ICP-MS method for determination of urine iodine was developed, optimised and validated. Measurement of urine iodine in 203 individuals revealed a median concentration 93 µg/L, consistent with iodine deficiency according to World Health Organization classification and in keeping with recent reports of iodine deficiency in the UK. No correlation was found between urine iodine concentration and thyroid biochemistry in these individuals.
Triple Quad ICP-MSMS: Illuminating the Challenges in Clinical Analyses
While ICP-MS is an immensely powerful multi-element analytical technique, it does suffer from some well-documented spectral interferences. Today many of the challenges in ICP-MS analyses have been conquered yet, achieving low detection limits via single quad ICP-MS in clinical applications still presents many obstacles to existing CRC instrumentation. Triple quad ICP-MS with MSMS capability restricts what enters the collision/reaction cell by allowing only the target mass of interest and its associated polyatomic interference to enter the cell. This level of control is critical to performing targeted chemical reactions. Here we present a comparative study between single quad and triple quad analysis of both urine and whole blood samples. The data clearly indicates the superior interference removal of the triple quad ICP-MSMS.
Utilizing UCT-ICP-MS to attenuate polyatomic interferences in the analysis of nickel and cobalt in serum and urine
Polyatomic interferences represent significant obstacles in the analysis of biological matrices by ICP-MS. The primary polyatomic interference for Nickel (60Ni) is calcium oxide (44Ca16O). The major polyatomic interferences for Cobalt (59Co) are calcium oxide (43Ca16O) and magnesium chloride (24Mg35Cl). Analysis for nickel and cobalt by PerkinElmer (PE) Elan DRC-ICP-MS requires empirically measured and calculated correction factors to correct for these interferences. Utilizing the PE NexION UCT-ICP-MS in kinetic energy discrimination (KED) mode with helium gas causes these polyatomic molecules to lose energy, allowing them to be filtered by the kinetic energy barrier therefore attenuating these interferences. Utilization of this technology improves analysis stability and decreases analysis time.
Utilization of a Chromatographic Internal Standard in the Rapid Quantification of Arsenic Species in Urine by HPLC-ICP-MS
Arsenic is a toxic element with wide natural distribution; its toxicity is largely dependent on molecular species. Six species are of primary importance for clinical evaluation of patients. They are, in order of decreasing toxicity: As3+, As5+, monomethylarsine, dimethylarsine, and the non-toxic species arsenocholine, and arsenobetaine. Current methods for quantifying these species have two significant deficiencies: long elution times (> eight minutes) and lack of a chromatographic internal standard. We report a method which corrects these deficiencies by including phenylarsonic acid as a seventh reference peak, and refining the separation to elute all seven peaks in < two minutes.
Direct Analysis of Acylcarnitines and Amino Acids in Dried Blood Spots without Punching using a Novel Autosampler Equipped with Tandem Mass Spectrometry
Expanded newborn screening by analysis of acylcarnitines and amino acids using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is widely practiced. The sampling of analytes directly from dried blood or plasma spots (DBS or DPS) without punching has advantages because of decreased sample handling, leading to lower cost and fewer laboratory errors. We report here successful applications of a DBS autosampler (LEAP Technologies, Inc) that utilizes flow-through solvent extraction to facilitate direct analysis of analytes by ESI-MS/MS. Several analytical options with this autosampler, including acylcarnitines and amino acid from DBS and methylmalonic acid from DPS, using UPLC-MS/MS, will be described.
Newborn screening and high-resolution accurate-mass spectrometry: A novel concept to improve the detection of inborn errors of metabolism
We will present for the first time a novel concept to use both dried blood spots and high-resolution accurate-mass spectrometry in newborn screening. Current mass-spectrometry based protocols are limited to detect amino acids and acylcarnitines. We will show the advantage of simultaneous detected organic acids. These metabolites are essential to identify newborns-at-risk, and may help to reduce false-positive results. A first clinical evaluation in both affected/non-affected newborns will be shown covering 10+ common conditions. Organic acids are usually analyzed by GC-MS in a subsequent urine sample leading to time-delayed diagnosis. Some newborns experience an acute metabolic crisis within the first days of life, thus rapid and accurate diagnostics is needed. It is now possible to identify organic acids within first-line screening to identify newborns-at-risk in a more timely matter.
6 plex assay to measure lysosomal enzymatic activity relevant to Fabry, Gaucher, Pompe, Krabbe, Niemann-Pick, and MPS1 by tandem mass spectrometry
We measured lysosomal enzymatic activity by LC-MS/MS to determine if the patients are affected by six lysosomal storage diseases: Fabry, Gaucher, MPS1, Pompe, Niemann-Pick and Krabble. The run takes about 5.5 minutes per sample. Chromatography revealed good recovery of product and internal standard. The preliminary data obtained from trial experiments showed that our method was specific and accurate. We are currently collecting more serum from normal and affected patients to further validate the method.
Preliminary Evaluation of a Novel Acid Hydrolysis Method for Free and Total Carnitine Determination by Direct-Injection Tandem Mass Spectrometry
An abnormal ratio of free and total carnitine has a major impact on the diagnosis and treatment of carnitine cycle defects. The proposed method of acid hydrolysis for free and total carnitine quantification eliminates the contaminating effects observed from an alkaline hydrolysis. Greater than 99.9% hydrolysis was observed in the monitored acylcarnitine species (C2, C8, and C16) after a 2 hour incubation with 3N butanolic HCl. Linearity of total carnitine was achieved between 12uM to 90uM with an R2 of >0.99. Accuracy of total carnitine ranged from 100-120% and total imprecision was <5% for all concentrations measured.
Stability of Decadienoylcarnitine Enriched Dried Blood Spots Stored at Various Temperatures and Humidity
The Newborn Screening Quality Assurance Program (NSQAP) added decadienoyl carnitine (C10:2) into its proficiency testing dried blood spot (DBS) materials in 2012, as elevated C10:2 is a primary indicator for dienoyl Co-A reductase deficiency (DE-RED) in fatty acid disorders. We examined the short- and long-term stabilities of C10:2-enriched dried blood spots stored at 37C, ambient temperature, 4C, and -20C by way of three different storage period lengths. We conclude that C10:2 enriched DBS are stable for up to 6 months at -20C and 4C storage and retain C10:2 concentration for 1 month working conditions.
Development of LC-MS/MS for Newborn Screening of Mucopolysaccharidosis 1 (Hurler syndrome)
A bioanalytical method based on LC-MS/MS with electrospray ionization in the positive ion mode has been developed to quantify the enzymatic activity of ¥á-L-iduronidase in dried blood spots (DBS). After the assay procedure involving extraction of the enzyme from dried blood spot punch, incubation with substrate and internal standard, and liquid-liquid extraction by ethyl acetate, reversed-phase chromatography coupled to tandem mass spectrometry with multi-reaction monitoring of product and IS was achieved. The accuracy profile results of enzymatic activity in a preliminary study with control samples implied reliable application of the method to clinical routine analysis.
Ultra-Fast Amino Acid Analysis by MicroLC-MS/MS with aTRAQ™ Kit Technology on the AB SCIEX Triple Quad™ 4500 System
In this work we present a MicroLC-MS/MS method for the analysis of amino acids in physiological fluids. The method employed derivatization using amine-reactive isotope-coded tags (aTRAQ™ reagents) to provide improved sensitivity and wider dynamic range, and to generate labeled internal standards for every amino acid analyte. Chromatographic separation was achieved in a run-time of 8 minutes, using a Halo C18 column (0.5x100mm) at a flow rate of 20uL/minute. This represents a significant time savings compared to the equivalent high-flow LC-MS/MS analysis, which typically requires a run-time of 18 minutes to achieve adequate separation of all isobaric (same mass) analytes. Furthermore, the Micro-LC method reduced solvent consumption by 40x compared to the conventional LC flow rate. Mass spectrometric detection was accomplished using the AB SCIEX Triple Quad™ 4500 system.
Development of a method for the diagnosis of adrenoleukodystrophy using Liquid chromatography-mass spectrometry
Adrenoleukodystrophy (ALD) is the most common peroxisomal disorder, and is caused by mutations in ABCD1, a peroxisomal membrane transporter protein. It is also associated with an accumulation of very long-chain fatty acids, particularly cerotic acid (26:0) and its metabolite lysophosphatidylcholine (Lyso PC26:0). We developed mass screening methods for the adrenoleukodystrophy by the quantification of Lyso PC or saturated fatty acid with different length of acyl chain using liquid chromatography-mass spectrometry. Those lipids could be extracted from dried blood spots using a 96-well plate within 5 minutes per sample, making it an ideal method for ALD mass screening.
An enzyme assay mass screening system for adenosine deaminase deficiency from dried blood spots using a DART ion source combined with triple quadrupole mass spectrometry
We have developed an enzyme assay mass screening system for adenosine deaminase (ADA) deficiencyusing a DART ion source combined with triple quadrupole mass spectrometry. Incubation of the substrate adenosine and extract from dried blood spots of healthy donors resulted in the proportional increase of the product inosine and hypoxanthine seen with selective reaction monitoring analysis. In addition, the substrate and products of the enzyme could be analyzed by the system without interference although the difference between their molecular weights is less than one. This system could swiftly distinguish between a patient and healthy donor sample, and thus is practical for ADA mass screening.
Development of Rapid Novel Newborn Screening for Mucopolysaccharidoses
MPS are caused by excessive GAG accumulation from deficiencies of degradative enzyme activities. We recently developed a new high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using RapidFire with tandem mass spectrometry (RF/MS/MS) for MPS. Preliminary research findings show that the combined measurements of these three GAGs are sensitive and specific for detecting subtypes of MPS with significant increases of specific GAG(s). Since the RapidFire system requires no up-front sample preparation and draws samples directly from quenched assay plates without the need for solid-phase extraction or any other sample desalting/pre-processing, each sample is processed in just six to eight seconds. Our innovative method provides the opportunity to develop cost-effective and novel approaches for newborn screening for inherited metabolic diseases.
Quantification of Decadienoyl Carnitine (C10:2) in dried blood spots by tandem mass spectrometry varies by method
In fatty acid oxidation disorders, elevated Decadienoyl Carnitine (C10:2) is a primary indicator for dienoyl Co-A reductase deficiency (DE-RED). We are reporting the quantified C10:2 values and cutoffs by various methods used in participating laboratories.
One hundred five domestic and international laboratories reported specimen 31262 as presumptive positive for C10:2. Quantification differences of C10:2 in DBS by methods allow one to accurately access lab performances across laboratories. NSQAP routinely provides DBS materials to assess laboratory and method performance, guidance and surveillance to ensure continuous improvements to sustain the quality of newborn screening results worldwide.
Rapid analysis of orotic acid in dried blood spots using liquid chromatography - tandem mass spectrometry (MS/MS)
Orotic acid (ORA) is an important biochemical marker for urea cycle diseases such as ornithine transcarbamylase (OTC) deficiency which are new targets of neonatal mass screening using MS/MS. Furthermore, an elevation of ORA is also observed in hereditary orotic aciduria or some amino acid transporter defects.
We have developed and validated a specific, high-throughput and sensitive stable-isotope-dilution LC-MS/MS method for the determination of ORA in Dried Blood Spots. The method can be a promising tool of the MS/MS screening, and in diagnosis of the above diseases.
Multiplex Newborn Screening of Lysosomal Storage Diseases using Flow Injection Tandem Mass Spectrometry
For over a decade our group has been developing enzyme assays based on tandem mass spectrometry for the detection of lysosomal storage disorders using dried blood spots (DBS). We report on current efforts that are focused on developing a multiplex assay to diagnose 6 disorders (Krabbe, Gaucher, Niemann-Pick, Fabry, Pompe and Mucopolysaccharidosis I) using flow injection analysis. DBS are incubated overnight in a common buffer solution containing the substrates after which the assays are extracted with ethyl acetate; solvent evaporated and reconstituted sample is injected onto a tandem mass spectrometer. Mass-orthogonal enzymatic products and internal standards are used for selected reaction monitoring and quantitation of enzyme activities.
LC-MS/MS Analysis for Galactosemia Monitoring
Galactitol and galactose-1-phosphate are key analytes for the monitoring of individuals with galactosemia. We present an LC-MS/MS method for the detection and quantitation of these metabolites in blood and urine samples using normal phase chromatography with gradient elution on a XBridge Amide Column (2.1x50mm; 3.5 µm) with an AB SciEx API 5000 instrument in MRM mode. Minimal sample volume and preparation is required (dilution for urine, dilution and precipitation for blood), and the chromatographic separation is complete in under five minutes. Quantitation is accomplished using labeled internal standards for each compound.
Sulfatides determination in dried blood spots of the patients with metachromatic leukodystrophy by using UPLC-tandem mass spectrometry
Sulfatides in dried blood spots of the patients with metachromatic leukodystrophy was extracted with chloroform-methanol and separated using an HSS T3 column. Quantification of sulfatides species according to the N-linked acyl-moieties was achieved by MRM in negative ion mode. The performance such as precisions and linearity were acceptable. Molecular species of sulfatides in DBSs from controls were barely detected. In contrast, MLD patients showed marked increased of several molecular species of sulfatides including C16:0, OH-C16:0, C17:0, C24:1, and C24:0 acyl-moiety forms. In conclusion, this method allows a fast and effective tool for the diagnosis and treatment of MLD patients.
GC/MS-based serum metabolomic profiling for early detection of colorectal cancer
We performed GC/MS-based serum metabolomic analysis to find effective biomarkers for early diagnosis of colorectal cancer. The metabolites whose levels displayed significant changes between colorectal cancer patients and healthy volunteers were subjected to multiple logistic regression analysis with the stepwise variable selection method. The prediction model was composed of the 4 metabolites selected, and its sensitivity, specificity and accuracy were higher than those of conventional tumor markers. In addition, the model displayed high sensitivity for detecting colorectal cancer at the stage from 0 to 2.
Development of a chiral LC-MS/MS approach for measuring metabolites of the synthetic cannabinoids JWH-018 and AM2201
The (ω)-hydroxyl, (ω)-carboxyl, and (ω-1)-hydroxyl metabolites of the synthetic cannabinoids JWH-018 and AM2201 are common biomarkers used to test human clinical and forensic specimens. However, current analytical procedures do not account for specific enantiomeric metabolites. This study validates a chiral LC-MS/MS approach capable of simultaneously resolving all the primary metabolites and parent compounds, including baseline resolution of each enantiomer. Chromatographic separations were achieved within 8 min using a LUX 3µ cellulose-3 (150 x 2 mm) column and a simple acetonitrile/ammonium bicarbonate (20 mM) gradient. Standard responses are linear and achieve accuracy and precision parameters established by previously validated methods.
An Untargeted Metabolomic Workflow to Improve Structural Characterization of Metabolites
Mass spectrometry-based metabolomics relies on MS2 data for structural characterization of metabolites. Unfortunately, many ions detected in metabolomics experiments are present at low abundances and are not amenable to identification by MS2 fragmentation. Here we introduce a novel two-part approach to increase the number of ions that could be interrogated by MS2 in untargeted metabolomic experiments. We first collect MS2 scans with improved sensitivity, but decreased specificity. Then, by evaluating MS2 fragment intensities as a function of retention time and precursor mass targeted for MS2 analysis, we obtain deconvolved MS2 spectra of previously untargetable metabolites. The value of our approach is highlighted with metabolic extracts from brain, liver, and astrocytes, and performance is evaluated using metabolite standards and simulations.
Untargeted metabolomics of colorectal cancer
Recent technological advancements in metabolomics have improved metabolite identification and opened the doors to finding novel mechanisms of cancer. Here, the relationship between colorectal cancer and biofilm formation was examined. Biopsies of tumored colon with biofilm and non-tumored tissues without biofilm were taken. Global mass spectrometry-based metabolomics analysis was carried out. Metabolites highly correlated to tumored tissues were observed, and were seen to be from the same metabolic pathway. Tissues were stained to locate the biofilm, and analyzed by nanostructure-initiator mass spectrometry to reveal the localization of the metabolites. Metagenomic studies will determine the relationship between bacterial species and metabolites.
Application of serum metabolic profiling in biomarker discovery and pathway analysis of cardiovascular calcification.
Coronary and valvural calcification consist of calcified lesions in coronary artery and arterial valve walls. The aim of this study is to evaluate untargeted metabonomic strategies in two groups of patients, diagnosed with: 1) calcific coronary artery disease and 2) calcific aortic valve disease. Serum organic extracts were run using a lipid profiling UPLC-MS method. Supervised multivariate data analyses revealed distinct profiles between controls and diseased groups. Biomarkers showed good prediction of disease as profiles, and as isolated or panel of markers. Additionally, network construction and pathway analysis revealed interesting observations and assisted on establishing hypotheses to be validate further in larger sample-sets and bottom-up studies.
Broad coverage Biogenic amine profiling by Accq-Tag UPLC-MS/MS for Biomarker discovery
Metabolomics comprises of the targeted and untargeted analysis of the metabolome in relation to e.g. disease diagnosis, -progression and/or –treatment. Within the Netherlands Metabolimcs Centre a variety of platforms is available for detailed analysis of the partial- or the complete metabolome for a variety of phenotypes. Here we report on the results obtained with our biogenic amine platform, which is based on Accq-Tag derivatisation and UPLC-MS/MS analysis of only ul’s of plasma, serum or CSF. Applications towards biomarker discovery in an EAE model for MS, in early detection of pre-clampsia or in responder/non-responder studies in drug interventions will be shown.
A Comprehensive Untargeted Metabolomics Approach to Monitor the Central Metabolism of Bacteria
Unlike targeted metabolomics approach which is optimized to explore a defined set of metabolites, the untargeted approach tends to be holistic and designed to simultaneously measure as many metabolites as possible without bias. However, the diverse chemical nature and wide concentration range of metabolites in complex biological matrices make it a great challenge. Here we present a LC-ESI-TOF/MS comprehensive analytical strategy, combining reverse phase (RP) and hydrophilic interaction chromatography (HILIC), in order to expand the metabolome coverage for untargeted metabolomics analysis. The developed design was applied to different bacteria model species to explore the diversity of their key metabolic pathways.
The use of Temporal Metabolite Profiling in understand failed IVF treatment
In vitro fertilization (IVF) has become a common assisted reproductive technique. However, it is not always a successful technique for many unknown reasons. Understanding of the differences between processes that occur with women whom become pregnant and those who do not can help in the understanding why IVF is not always successful. Mass spectrometry metabolite profiling is becoming a popular way to analyze hundreds of metabolites in a single experiment and gain knowledge on the distributions on each of these potential biomarkers. Temporal metabolite profiling can help describe complex dynamic processes such as pregnancy. Profiling temporal urine samples is a non-invasive technique, which can help the understanding of this process. Eleven metabolites have been identified as statistically significant.
LC-MS/MS-Based Metabolomic Urinalysis For Prostate Cancer Patient Management
Prostate cancer (PRCA) detection methods yield a high rate of negative prostate biopsies. Metabolomic studies have identified sarcosine and amino acids as potential PRCA biomarkers. A protocol was developed to measure alanine, cysteine, glutamate, glycine and sarcosine from urine sediments by LC-MS/MS. The metabolite panel was tested in a prospective study of 74 men prior to prostate biopsy. Individuals in the top tertile in the logistic regression algorithm had an odds ratio of having PRCA of 4.8 (95% CI: 1.4 – 17) relative to the bottom tertile, suggesting that urine metabolites can aid clinical management of patients with suspicion of PRCA.
Neuronal Cell Culture Metabolomics of Illicit Drug Use
A Metabolomics approach has been used to determine the response of neuronal cells to the illicit drug methamphetamine and the cocktail with caffeine.
Neuronal cells have been cultured and then exposed to chronic levels of methamphetamine and the commonly used cocktail, methamphetamine with caffeine (YABA). The cells were harvested after the exposure period and the cells and media analysed using GC-MS based Metabolomics. Changes in metabolite regulation in the exposed cells and their media were determined by comparison of the metabolite levels in control cells and media.
Up-regulated and down-regulated metabolites were identified and mapped to biochemical pathways to investigate the pathway modification caused by the drug and dug cocktail. The pathway redefinition has been compared with literature models of methamphetamine neuronal damage.
A MRM-based targeted metabolomics method for bisphenol A derivatives in Human Urine using UHPLC-MS/MS
In the present study, bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE), and other commercially available BPA derivatives are used to set up a MRM-based targeted metabolomics method. We use an Agilent 1290 UHPLC coupled with an Agilent 6460 triple quadruple mass spectrometer. We optimize liquid chromatography (LC) separation, electrospray ionization (ESI) source parameters, and compound parameters to maximize sensitivity on MS. We use automated liquid-liquid extraction (LLE) for sample extraction. We also use offline solid phase extraction (SPE) to clean up sample matrix. Initial Demonstration of Capability (IDC) is conducted including the linearity, linear range, and coefficient of variation of the standard curve. This method will provide useful preliminary information for future untargeted screening of BPA derivatives in human biofluids.
MALDI MS - a rapid tool for probing the fungicidal property of CdS quantum dots
We report the successful application of MALDI-MS for identifying the antifungal interactions of CdS quantum dots with Saccharomyces cerevisiae and Candida utilis. CdS quantum dots synthesized in-house with particle sizes between 1-7 nm exhibited excellent fungicidal activity. The growth curves and the growth rates of both these fungi were highly inhibited even at concentrations of 10 mg/L. MALDI-MS was applied as a tool to evaluate the antifungal activity of the CdS QDs in order to substantiate the observations obtained by the direct cell count method. It was observed that the trend observed in the case of Saccharomyces and Candida was well-represented in the MALDI-MS spectra. This provides evidence that mass can be used as an effective tool in rapid assessing the antifungal activity.
Mass Spectrometry based Analytical Methods for Adeno-associated Virus (AAV) Gene Therapy Vectors
There are 12 known Adeno-associated virus (AAV) serotypes and approximately 100 genetic variants. Diagnostic tests to validate the serotype or protein composition of AAV capsids are needed. Protein identification by mass spectrometry is a preferred analytical technique for AAV capsid serotype identification, particularly since antibody-based methods are unlikely to be able to distinguish highly homologous sequences or capsids that have been modified. These methods are also useful for identifying the presence of additional proteins that were not previously distinguishable using other methods. These additional proteins may represent novel virus host protein interactions or simply by products from vector manufacturing.
Creation and validation of a database to identify and differentiate yeast and mycelial growth phases of clinically important dimorphic fungi
There are few fungi able to cause infection in immunocompetent individuals and thermal dimorphism is thought to be the primary virulence factor. The identification of thermally dimorphic fungi presents a difficult challenge to laboratories not located in endemic regions of infection. This study aims to create and validate a MALDI-TOF database from stored clinical isolates of Histoplasma capsulatum, H. capsulatum var duboisii, Blastomyces dermatitidis, Coccidioides immitis, C. posadasii, Paracoccidioides brasiliensis, Penicillium marneffei, Emmonsia parva, E. crescens and Sporothrix schenckii; which has the ability to distinguish both yeast and mycelial phases of growth, and identify all organisms to species level.
MALDI-TOF Identification of Bacteria Using 70% Formic Acid On-plate Direct Extraction
Rapid, accurate identification of bacteria in cultures can aid therapeutic decisions. In this study, we compared the accuracy of 2 MALDI-TOF methods using the Bruker Biotyper® for identifying bacterial isolates: 1) the direct spot method, and 2) a direct on-plate 70% formic acid extraction method. Overall, 139 (73.2%) out of 190 total isolate identifications were unaffected by the type of method used. Forty isolates (21%) had improved identification scores and 11 isolates (5.8%) had lower identification scores with the on-plate extraction method. Direct on-plate extraction with 70% formic acid improves MALDI identifications and may reduce the need for tube extraction.
Accuracy of Bruker MALDI-TOF for Identification of Bacteria with Lower Biotyper® Scores
Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) has been shown to be a rapid and accurate method for bacterial identification. We evaluated the accuracy of genus and species identification for isolates tested in duplicate using Bruker Biotyper scores in the 1.700-1.999 range, which is used for genus-only identification. Correct identification was achieved for 202 (95.3%) of the 212 isolates that had at least 1 Biotyper score within this range. The Bruker MALDI-TOF method thus yields accurate genus and species results even when Biotyper scores are in the genus-only range.
Evaluation of culture processing methods and the Bruker MALDI BioTyper™ 3 database for the identification of yeasts.
The Bruker MALDI BioTyper™ 3 database was validated for the identification of yeasts; comparing identifications against those obtained using gold standard culture methods and rRNA ITS1&2 genotyping.
Preliminary data for 191 yeasts has shown that using conventional 70% formic acid extraction and reducing the species acceptable threshold in the database search to >1.9 log score the identification success rate of MALDI-TOF MS was 96% (184/191). A full data set of 500 isolates will be presented.
MALDI-TOF provides an accurate and rapid tool for the identification of yeasts reducing the turn-around time for yeast identification by up to 72 hours.
Identification of Mycobacteria species by VITEK® MS
VITEK® MS consists of a MALDI-TOF mass spectrometer for microbial identification with a database recognizing unique fingerprints. Mycobacterial species require inactivation and extraction due to their pathogenicity and lipid rich cell wall. The study describes a sample preparation method using mechanical disruption and chemical treatment from multiple media for database development. Most of the 37 mycobacterial species tested with 5-20 strains each were clearly differentiated except M. tuberculosis complex. The preliminary results indicated 100% species match for 20 mycobacterial species and 89-98% for the remaining species. The VITEK® MS platform offers rapid, reliable, and robust microbial identification with high throughput.
Assessing the impact of delayed processing on the identification of microorganisms from positive blood cultures by MALDI-TOF
The application of MALDI-TOF to the direct identification of bacteria and fungi present in positive blood cultures is poised to reduce the turnaround time for accurately diagnosing the etiology of infection in patients with bacteremia, fungemia, and sepsis. The majority of published studies addressing this functionality have been performed on blood cultures processed within 8 hours of being registered as positive for growth. However, current methods for processing blood culture media for MALDI-TOF require a substantial amount of hands-on time such that processing may be impractical to run across all shifts. This study evaluated the impact of a variety of extended incubation conditions on organism identification by MALDI-TOF after positive blood cultures were removed from the culture instrumentation.
Comparison of MALDI-TOF MS to Nucleic Acid Sequencing for the Identification of Clinical Isolates of Mycobacterium spp.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS is a rapid and reliable method for identification of microorganisms. However, some Mycobacterium species are very resistant to standard preparation methods used to construct the original reference database. This shortcoming was overcome using an optimized preparation technique coupled with a new reference database. These improved methods were tested with clinical isolates of acid fast bacilli (AFB).MALDI-TOF MS resulted in 79.5% identification results with log(score) values ¡Ý 2.0 which indicates a species identification. 12.8% of mass spectra resulted in a log(score) value of > 1.7 and < 2.0. Reference 16S rRNA gene and rpoB sequencing confirmed the MALDI-TOF identification results for 92.3% isolates with log(score) values >1.7. There were no false positive identification results.
Use of an automated software tool for the evaluation of the MBT-MSBL assay
ß-lactamase activity can be monitored by the mass spectrometric analysis of the hydrolysis reaction of the antibiotics (MBT-MSBL assay). So far, spectra evaluation has been done by visual comparison of the peak intensities of the hydrolyzed and the non-hydrolyzed forms. A new software tool allows for the automated, user-independent evaluation of MBT-MSBL derived spectra supporting the optimization of the assay conditions, the evaluation of inhibition setups, and the analysis of kinetic studies. The calculation of a defined value permits the comparison of MBT-MSBL results to the results of established routine methods.
Identification of Nontuberculous Mycobacteria (NTM) by MALDI-TOF MS Using a New Mycobacteria Library
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and an optimized preparation technique was used for identification of mycobacteria. Mass spectra were acquired from all 18 tested strains which represented 17 different species. MALDI Biotyper 3.1 and Mycobacteria Library 1.0 resulted in 16 (88.9%) identification results with log(score) values ≥ 2.0 which indicates a species identification. Two (11.1%) mass spectra resulted in a log(score) value of > 1.7 and < 2.0. All identification results were correct at species level, indicating the high potential of MALDI-TOF MS for fast and reliable identification of nontuberculous mycobacteria.
Biocomputing for Detection of Identifying Peaks in Microorganism
MALDI-TOF Spectra
While pronouncedly dissimilar groups of microorganisms can reliably be
identified by their MALDI-TOF spectrum using distance measures, this
is not possible where the difference between spectra of closely
related groups is realized as a difference in a few peaks only.
In such cases one falls back to using 'biomarkers', single peaks or a
group of only a few peaks, to identify groups.
Therefore, we suggest the use of probability tables and decision tree
constructing algorithms to find putative biomarkers. This approach
uncovers even complex biomarkes and generates a data basis for
automatic classification using the corresponding machine learning
algorithms.
Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for rapid antibiotic resistance detection
Hydrolytic enzymes play a major role in antibiotic resistance of Gram negative bacterial species. These enzymes hydrolyze certain antibiotics, leading to their inactivation. Our work shows that Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS) is a rapid and well-adapted tool for the analysis of antibiotic degradation. Herein, we describe a fast assay to monitor faropenem hydrolysis by resistant bacteria. For the first time, the bioMérieux VITEK MS instrument was used for such an approach and this instrument was shown to be suitable for qualitative measurements of enzyme-mediated drug degradation.
Mass Spec Based Quantitative and Sensitive Bisulfite-free DNA Methylation Profiling from FFPE Tissues: A New 8-Gene Clinical Test for Brain Cancer Prognostics
Although the importance of epigenetic biomarkers such as DNA methylation has been recognized, the few clinical tests available are reported to have high failure rates with patient samples, even when examining a single gene. We have developed a sensitive, bisulfite-free method for quantitative analysis of DNA Methylation biomarker panels, even in compromised FFPE tissues. The use of Mass Spectrometry generates a percentage methylation for each gene and these data yield a CpG Island Methylator Phenotype (CIMP) status for the tumor, which can be a diagnostic, prognostic or companion diagnostic indicator. Methylation levels of 25 genes can be determined with as little as 10 ng of patient DNA. This technology allows DNA methylation biomarkers discovered with bisulfite-based methods to be translated to the clinical laboratory. An 8 gene brain cancer prognostic test from FFPE is presented.
Proteolytic Peptides in Red Blood Cells - Biomarkers for Hemoglobinopathies?
We have discovered proteolytic peptides in peripheral blood of clinical specimens carrying different types of hemoglobin mutations. Proteolytic peptides carrying these mutations were found for both homozygous patients and heterozygous carriers. Once validated, these proteolytic fragments with specific mutations can serve as ultimate biomarkers for the respective disease genotypes, and can be screened with high throughput using mass spectrometry, similar to neonatal metabolic screening.
Clinical Evaluation of Restriction Fragment Mass Polymerphism Assay for Detection and Genotyping of a Wide Spectrum of Human Papillomaviruses
We describe a novel MALDI-TOF MS-based genotyping strategy, termed Restriction Fragment Mass Polymorphism (RFMP) that is suitable for genotyping variations in a simple, accurate, and high-throughput manner. The RFMP assay proved to be an accurate and reliable tool for diagnosing human papillomavirus (HPV) that was able to detect 74 different genotypes of HPV including 42 anogenital types. The clinical performances of the RFMP assay was highly comparable with Linear Array HPV genotyping test. The software for automation of the RFMP assay resulted in a peak analysis time reduction of 52 min (from 60 to 8 min; 87%) and improved reproducibility. The sensitivity, accuracy, wide range of genotype identification and high throughput capacity make the RFMP assay suitable for mass screening and monitoring of HPV-associated cervical cancer.
Therapeutic Drug Monitoring of 8 new anticancer agents by High-Performance Liquid Chromatography-Tandem Mass Spectrometry
A method was developed for the quantification of 8 tyrosine kinase inhibitors (TKIs) used as anticancer agents for targeted therapy. The method consisted of a plasma protein precipitation followed by dilution and analysis by LC-MS/MS. The chromatographic separation was achieved in gradient mode on a C18 column, followed by electrospray ionization-triple quadrupole mass spectrometry. The resulting method was accurate, precise and sensitive. This method is the first broad range LC-MS/MS assay covering the major currently in-use TKIs. It has been validated according to international regulations and can be used to evaluate patient adherence to therapy and pharmacokinetic studies.
Development of a quantitative colorectal cancer SRM assay for use in FFPE tumor tissues
Aberrant over-expression of receptor tyrosine kinases (EGFR, etc) along with other critical downstream oncogenic mediators (KRAS, etc) are known drivers of colorectal cancer (CRC), subdividing the disease into distinct molecular subsets. We tested a‘CRC-multiplex’ MS assay for quantification of Met, RON, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC on 42 primary human CRC cancer tissues, with paired metastases. Multiplex quantification of tissues highlights inter-patient and intra-patient (from primary to metastasis) heterogeneity of samples, and allows insights into potential therapeutic approaches.
Measurement of teicoplanin by liquid chromatography-tandem mass spectrometry: Development of a novel method
Teicoplanin is an antibiotic used for treatment of endocarditis, osteomyelitis, septic arthritis, and methicillin resistant Staphylococcus aureus (MRSA). Teicoplanin is emerging as a suitable alternative to vancomycin, where trough serum levels are monitored by immunoassay routinely. This report details the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring teicoplanin in patient serum. Ion suppression, linearity, stability, matrix effects, recovery, imprecision, lower limits of quantification and detection, interference, and method comparison against immunoassay were all assessed. This alternative method for the measurement of teicoplanin by LC-MS/MS suggests a negative bias for tandem mass spectrometry compared to immunoassay.
Determination of Extensive Glycosylation on Glycoproteins and Glycolipids in High-density Lipoprotein using Mass Spectrometry
HDL has long been considered to be atheroprotective due to its role in reverse cholesterol transport, with many other recently discovered functions including toxin binding, antioxidant, anti-inflammatory, and immunomodulatory functions.
Many of the functional proteins and lipids in HDL are glycosylated. Yet, very little is known about these glycoconjugates of HDL. HDL binds microbial toxins via its constituent glycoconjugates: glycoproteins and glycolipids.
No adequate methods to determine HDL glycan diversity as a whole in a clinically relevant manner exist. Here we proposed an analytical strategy via mass spectrometry that is detailed and quantitative, examining the exact glycan structures on the glycoproteins and glycolipids of HDL.
GC-MS Based Glycan Analysis in Clinical Samples
Altered glycotransferase (GT) activity is the immediate upstream cause of the ubiquitous expression of aberrant glycans by cancer cells. GTs build glycans in an opportunistic, first-come-first-build manner, with each unique GT adding a specific sugar residue to a specific linkage position on a growing glycan “tree”. We have invented a GC-MS based approach to the analysis of glycans in 10 microliters of unfractionated, undigested whole biofluids in which glycans are characterized and quantified on the basis of monosaccharide-specific branch points and linkages rather than intact structure. These branch points and linkages serve as direct, undiluted surrogates of GT activity. Pilot studies in well-characterized lung cancer patient samples and controls show ROC c-statistics in the 0.8-0.9 range. Applications of this new technology in lung cancer and beyond will be discussed.
Using High Throughput Mass Spectrometry-based Method to Examine Efficacy of Epigenetic Drug on Histone H3
Post-translational modification of histones is a major mechanism regulating the accessibility of chromatin for the purposes of transcription, replication, and DNA repair. Aberrant regulation of histone acetylation correlates to major human diseases, and epigenetic drugs can aid in restoring normal acetylation states. In this study, we monitor altered acetylation status between healthy and cancer cell lines using our label-free high throughput quantitative mass spectrometry-based method. Additionally, we found H3K23 acetylation increase ~6 fold (highest) after a 4-day incubation with trichostatin A in Jak3 EBV cells. This method provides a promisingly way to examine the efficiency of epigenetic drugs.
On-line Separation of Polypeptides by Isoelectric Point Focusing OMJ-CIEF coupled to High Resolution Mass Spectrometry
We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI.
The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.
Data will be presented with a higher number of proteins being identified in a shorter timeframe (from one cancer cell-line) than other techniques currently in use.
Unsupervised Statistical Identification of Aberrant Chromatographic Peaks Using Information from Internal Standards
Deuterated internal standards are ubiquitous in clinical applications of high throughput LC-MS and LC-MS/MS methods, yet their full information content is chronically underutilized in the analysis of chromatographic data. The retention time/intensity profiles of analytes and their corresponding internal standards have, by design, a high degree of mutual information content. This presentation will focus on a novel statistical method for the unsupervised identification of aberrant peaks through the use of this information. Fully harnessing the information generated by the inclusion of internal standards will allow for increasingly automated analysis of patient samples and drive down operating costs.
Caught in the act: Direct identification of Ligand-Receptor interactions
Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties—one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. The LRC technology enables the identification of receptors for many types of ligands under under near-physiological conditions and without the need for genetic manipulations.
Gone in 60 Seconds: Use of an Immobilized Enzyme Reactor to Drive Proteolysis of Cell Lysates and Blood Samples to Completion in Record Time
The rate limiting step in mass spectrometry based analysis of proteins is proteolysis. Whereas analysis by MS can be achieved in milliseconds, the requisite trypsin digestion used in preparing samples for MS analysis typically requires 12-24 hours and is labor intensive. A high efficiency immobilized enzyme reactor (IMER) column provides fast on-line digestion in just 1-4 minutes with continuous sample analysis on reusable trypsin column. This presentation will review the advantages and disadvantages of various proteomic-MS strategies and describe the development of a fully automated workstation capable of performing a proteomic workflow prior to MS detection.
Low sample consumption Vitamin D analysis of babies
Traditional immunoassay-based analysis of 25-OH Vitamin D requires a large sample volume, typically 1 -1.5 ml of serum. Such volume is always and easy available for analysis of adults. In contrast, for infants and babies, sample volume is always was a bottleneck. Clinical analysis was always performed for critical target analytes. Important, but non-essential analytes was always were overlooked due to very limited sample availability.
We are successfully overcame this constrain by combination of highly sensitive triple quad mass spectrometry (Agilent Technologies) for detection, use of a fused-core chromatographic column (Supelco) for separation and supported liquid-liquid extraction (Biotage) as sample preparation step. That combination allowed to use only 40 microliter of serum per analysis and maintain LLOQ of 2 ng/ml.
Quantification of CD4 Receptor on Human CD4+ T Cells Using Conventional Flow Cytometry and Multiple Reaction Monitoring Mass Spectrometry
We report the development of a highly reproducible nanoLC MRM MS-based method to quantify CD4 density in the unit of copy number per cell on human CD4+ T cells. The method utilizes stable isotope labeled full-length standard CD4 as an internal standard to measure endogenous CD4 directly, without the use of antibodies. The development of a new approach for CD4 protein quantification is important as a complementary strategy to validate the analysis from the cytometry-based conventional method. It also indicates the potential of using MRM-MS for measurement of cell protein markers in clinical laboratories.
An introduction of a novel atmospheric pressure
Micro/Nano-ESI ion source for increased sensitivity with reduced chemical background noise and higher S/N ratio
The advent of micro/nano-ESI has considerably extended the applications of ESI in analytical mass spectrometry. However, traditional micro/nano-ESI systems can suffer from unstable Taylor cone effects, clogging of emitters, and incomplete droplet desolvation. In this study, a novel source was developed to accommodate nano to micro LC flow ranges. This new ion source transmits ions to MS interface using a novel gas-flow entraining technique through a specially designed source chamber. Compared to traditional micro/nano-ESI ion source, this novel ion source offers ease of use, 2 fold improvement in signal stability, and higher S/N ratio.
Cross validation method of LDTD-MS/MS with a run time of 9 second per sample versus LC-MS/MS for the Analysis of 25-hydroxyvitamin D2 and D3
The Laser Diode Thermal Desorption™ (LDTD) ionization source has been coupled to a mass spectrometer equipped with the SelexION™ differential ion mobility cell, enabling a high throughput capacity for the analysis of 25-OH-vitamin D2 and D3 in biological matrix, with sample-to-sample analysis time of 9 seconds. Improvement of the analysis specificity is achieved by the action of the DMS. Preparation of sample consisted of a protein precipitation by addition of methanol followed by a liquid-liquid extraction with hexane. 5 µl of the upper layer is deposited in proprietary 96-wells plate and dried prior analysis. Multiple tests are conducted for validation: matrix effect, specificity, reproducibility, stability and concomitant drugs. All those tests meet requirement of the guidelines from regulatory environment. Correlation between LC-MS/MS and LDTD-MS/MS values is expressed by r2=0.952.
Quantification of glycopeptides in human serum using multiple reaction monitoring
Glycosylation is one of the most common post-translational modifications for proteins. It plays an important role in cell-cell and cell-matrix interactions; many diseases have been found to be associated with abnormal glycosylation. Profiling of glycans has further led to disease marker discovery. However, the quantification of glycosylation is less developed limited primarily to relative quantification of glycans released from proteins. This research developed an analytical method to quantify glycopeptides on the site-specific level. The absolute amount of proteins was determined from the peptide calibration curve, while the degree of glycosylation was normalized to the total protein content.
Cloud-Based Informatics Solutions to Accelerate Clinical Research
Mass spectrometry sensitivity, speed, and robustness have increased dramatically in the last decade. However, the second half of the equation -- the informatics solutions -- have traditionally not kept pace, particularly with respect to the following:
(1) securely storing the massive amounts of MS data generated
(2) running the informatics workflows quickly
(3) empowering researchers -- biologists, clinicians, MDs -- to easily query (to ask ad-hoc questions against) the vast amounts of data
Over the last four years, our lab has helped develop a solution to address the above three concerns using a whole new technology paradigm: cloud technology.
Analysis of Estrogens in Plasma Samples with a Simplified Sample Preparation using SelexION™ Ion Mobility Technology
Typically the analysis of estrogens in serum or plasma by LC-MS/MS requires a time consuming and costly sample preparation. We have evaluated the use of differential mobility spectrometry to improve the selectivity of detection in plasma by LC-MS/MS, thus enabling improved limits of detection even when using a highly simplified sample preparation protocol.
Proteins were precipitated from plasma samples using acetonitrile. After centrifugation, the supernatant was directly injected on a 2D chromatographic separation system, and detection was accomplished using an AB SCIEX QTRAP® 5500 system, equipped with the SelexION™ differential ion mobility device.
For 50 pg/mL estrone (E1) and estradiol (E2) spiked into matrix, the S/N increased from 27 to 162, and from 77 to 462, respectively, when the SelexION™ device was employed. The estimated LOQ is <5 pg/mL for E2, and <1 pg/mL for E1.
How to Benefit from Different Column Selectivities in the Clinical Laboratory
Liquid chromatography, in combination with mass spectrometric detection, has emerged as an important tool. The major roadblock, however, is often not the capital investment cost, but rather the lack of appropriate chromatographic methods and the specially trained personnel needed to run them. Dedicated methods and protocols are part of the package when a new LC-MS is installed, but sometime these “complete methods” fail to meet the expected improvement gains, which often due to column chemistries specified are not suitable. This poster will discuss the role of chromatography from a molecular perspective and a number of applications with relevance to routine clinical analyses will be presented.
Ultra fast Immunosuppressants quantitation in whole blood by Laser Diode Thermal Desorption – Tandem Mass Spectrometry: 9 seconds sample to sample
The Laser Diode Thermal Desorption (LDTD) remove the chromatographic step and significantly increasing the analytical throughput for the quantitation of Tacrolimus, Sirolimus, Everolimus and Cyclosporin A. 50 µl of whole blood samples were treated with 100 µl of a precipitation reagent including deuterated internal standard. After vortex and centrifugation, 100 µl of 1-chlorobutane is added to the vial for liquid-liquid extraction. 6 µL of the supernatant is spotted on plate and allowed to dry. All validation parameters in term of Acuracy, precision, reproducibility, specificity and stability were measured and fulfilled. 120 real blood samples of Cyclosporine A were analyzed in cross validation with LC MS/MS method. Both methods agreed, with concordance correlation coefficient of 0.99 (95% confidence interval 0.982 – 0.991). Sample throughput achieved was 9 seconds per sample.
Rapid Analysis of Endogenous Steroids using UPC2 MS/MS
Steroid regulation plays a central role in the health and development of adults and children. Often disease states will differ based on subtle variations in a complex series of interactions among the many different steroids. Differential diagnosis, therefore, depends on the simultaneous quantitative analysis of multiple steroid levels. This study focuses on the application of Convergence Chromatography, utilizing supercritical CO2 as the primary mobile phase, for the rapid chromatographic analysis of endogenous steroids. We will present data collected with the recently commercialized ACQUITY UltraPerformance Convergence Chromatography (UPC2) system coupled to a tandem quadrupole mass spectrometer, demonstrating a significant reduction in analysis time (less than 2 minutes per analysis) and cost of analysis per sample.
A Novel On-Line Sample Cleanup and Liquid Chromatography Platform for LC-MS Analysis in the Clinical Research Laboratory
Clinical research and toxicology laboratories have a need for reproducible, short HPLC methods and an easy to use and maintain HPLC separation platform. A new system, encompasses a novel HPLC pump design and fluidics configuration, enabling the user to perform on-line sample cleanup using TurboFlow technology and high efficiency chromatographic separation on two channels utilizing multiplexing, prior to MS analysis. Reproducibility, linearity, and other performance data are discussed. Several routine clinical research assays (ISD, Vitamin D, steroids, pain management) have been satisfactorily tested. They display significantly reduced solvent consumption and shortened run times.
Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology
Paper spray is a direct ionization technique that simplifies the mass spectrometric analysis of dried blood spots (DBS). Paper-spray technology is therefore attractive for forensic toxicology screening for drugs of abuse. The sample collection and storage of DBS in a simple paper cassette make shipment of samples to the forensic toxicology lab safe and convenient. Both qualitative and quantitative analysis of small molecules from complex matrices such as blood or other biofluids is possible without time consuming sample preparation and chromatography.
In this work, we evaluate the analytical capability of paper spray technology coupled to a very fast and sensitive benchtop Orbitrap™ mass spectrometer for forensic toxicology screening of drugs of abuse in whole blood.
Advances in Steroid Panel Analysis with High Sensitivity LC/MS/MS
Quantification of multiple steroids from a single biological fluid sample is useful for both understanding diseases including areas of sedation, seizure prevention, oncology and reproduction and for clinical treatment. Achieving adequate clinically relevant sensitivity across the whole panel is quite difficult because optimum ionization conditions vary depending on the steroid. In this method Shimadzu 10ADvp HPLC pumps and IONICS 3Q Series 200 Molecular Analyzer were used. Pg/mL levels of sensitivity can be achieved for androgens, glucocorticoids, and progestagens together in one analytical run; thereby enabling the analysis of a panel of steroids by one method. We have successfully used the 3Q to analyze H295R cell culture media for this panel of steroids over the physiological relevant ranges.
The utility of time-of-flight mass spectrometry in discovering new environmental biomonitoring targets through non-targeted analysis
Only about 300 of the 3000 environmental chemicals in the CDC's list of high production volume chemicals are currently being biomonitored. The rest are not biomonitored for lack of available analytical methods that will allow their measurement in biological matrices. Targeted approaches in developing methods for new chemicals to biomonitor do not always yield targets that are detectable in human serum and urine wasting resources and effort spent on method development. We assessed the utility of non-targeted analysis by using time-of-flight mass spectrometry to uncover new chemical targets for biomonitoring. Of the 729 environmental organic acids that are potential endocrine disruptors in our database, we found formula hits to 145 chemicals with detection frequencies > 25% in the serum of 20 pregnant women. About 63 of the 145 chemicals are not currently biomonitored by the CDC.
A high-throughput LC/MS/MS method for the simultaneous determination of environmental phenols in human urine
Because of the ubiquity of environmental phenols and potential risk to human health, environmental phenols are listed as high priority chemicals to be monitored by the California Environmental Contaminants Bio-monitoring Program (CECBP). Previously we have developed a sensitive method for simultaneous determination of a set of phenols including several parabens in human urine by high pressure liquid chromatography tandem mass spectrometry (HPLC/MS/MS). Samples were pre-processed using enzymatic de-conjugation of the glucuronides followed by an off-line solid phase extraction (SPE) on a vacuum manifold. In the current method, samples are prepared using automatic SPE procedure on Zephyr Liquid Handling Workstation in a 96-well plate format. Not only does method automation reduces human error during the sample process, thus increasing reproducibility, it also improves productivity dramatically. The current method provides a high-throughput (HTP) and low cost approach to evaluate human exposure to environmental phenols and is adequate for analyzing a large number of samples for epidemiological studies.
Quantitation of 23 PCB and PBDE congeners in human serum matrix using a GC-MS/MS based MRM method
Polychlorinated biphenyls (PCBs) and Polybrominated diphenyl ethers (PBDEs) are two series of persistent organic environmental pollutants (POPs) origin from industry products. Determination of the quantitation levels of PCBs and PBDEs in human has become part of the comprehensive study for Autism disorder. A GC-MS/MS based multiple reaction monitoring (MRM) method for simultaneous quantitation of 23 PCBs and PBDE congeners in human serum matrix has been developed. Experiment was performed on a Bruker Scion triple quadrupole GC-MS system. This method has a calibration range from 0.04 ppb to 100 ppb. LOQ is between 0.02-0.04 ppb with matrix. Scion TQ shows great sensitivity, robustness, linearity for analysis of PCB/PBDEs. Finalized MRM method has now been applied to a clinical study of hundreds serum samples collected from fertile females with children later showing Autism spectrum disord
Analysis of Hydroxylated Polybromated diphenyl ethers (OH-PBDEs) on the Bruker EVOQ LC/MS
Fire safety standards, including the application of flame-retardant chemicals, have been established in modern societies to reduce deaths and injuries as well as economic impact of fires. Bromine, like other halogens, quenches free radicals generated in a fire with high efficiency, thereby preventing the propagation of a flame.
To investigate the OH-PBDEs a LC-MRM method was developed on the Bruker EVOQ LCMS/MS system. A method was developed to analyze twenty-four dansyl chloride derivatives of OH-PBDEs and OH-PCBs, including an internal standard. The separation of the 5�L injection of OH-PBDEs was on a 100 X 2.1 mm id. 2�m particles C18 with a 0.1% formic in water and 0.1% formic Acetonitrile gradient from 80% organic to 100% organic solvent using a Bruker Advance UHPLC system with a CTC THC Pal autosampler. The analytes were detected with a Bruker EVOQ Triple quadrupole system.
Removal of Beta-Glucuronidase Enzyme from Urine Post Hydrolysis to Improve Assay Performance and Column Lifetime
Acid hydrolysis provides the most efficient conversion of drug metabolites, but the resulting samples increase the frequency and cost of maintenance and destroy important metabolites such as 6-monoacetyl morphine (6-MAM). Enzymatic processes have their own associated problems. If the enzymes are not removed from the sample prior to analysis, they will continue to react with drugs like codeine-6-glucuronide indefinitely resulting in different measured values depending on the time point of analysis. The solublized enzyme can also crash out of solution onto the HPLC column frit causing premature column death. In this work we discuss a simple technique to remove the enzyme post hydrolysis that improves column lifetime and assay performance.
Eliminating Hydrolysis for Pain Management Monitoring of Benzodiazepines by LC/MS/MS
A urine drug confirmation method for common pain management drugs, such as opiates and benzodiazepines, reports values for total drug by hydrolyzing the glucuronide conjugates. The hydrolysis step to convert drug conjugates back to parent compounds is the most time consuming step in the process. Developing an LC/MS/MS assay that is able to detect both parent and glucuronide(s)drug conjugates in urine eliminating the need for hydrolysis would be beneficial. In this presentation we will discuss the challenges of developing and validating LC/MS/MS methodology for the analysis of benzodiazepines without hydrolysis.
Quantitative Extraction of the most Commonly Prescribed Pain Medications in Urine Using Solid Phase Extraction (SPE) and Analysis by LC-MS-MS
The use of pain medication for chronic pain and its gradual dependence and addiction has become a growing concern. Therefore an utmost importance has been put forth by pain medicine practitioners to frequently perform urine drug tests to monitor compliance to prescribed medications and to detect substances that should not be present. To meet the demands of clinical market, we have developed a fast and effective LC-MS-MS method in conjunction with a solid phase extraction protocol. It covers the most commonly used pain panel drugs using a reliable approach enhancing the effectiveness of monitoring programs for pain patients.
Automated, High Throughput Analysis of Benzodiazepines by LC/MS/MS from Urine and Plasma using a Single SPE Procedure
Eight benzodiazepines were extracted from both urine and plasma using a single automated solid phase extraction (SPE) procedure. The automated procedure proved to be reproducible and produced excellent recoveries for the target analytes from both sample matrices. When coupled with a rapid (<3 minute) LC/MS/MS method, this workflow provides the opportunity to substantially increase sample throughput and simultaneously decrease costs per each analysis.
A Simple LC/MS/MS Screening and Quantify Method for the Analysis of 41 Common Pain Drugs in an Oral Fluid Matrix
Currently there is much interest associated with the analysis of various pain medications in oral fluid matrices. Typical collection of such a matrix involves a collection swab that retains the oral fluid matrix. The swab is then subjected to extraction buffer before sending to laboratories for analysis.
Therefore, we have developed a simple method to simultaneously screen and quantify such compounds in one injection using a new QTRAP® platform. The compounds analyzed in this method consists of 41 drugs.
High-Throughput Method for the Quantitation of Pain Panel Drugs in Urine Using Micro Flow LC/MS/MS Analysis
Utilization of novel micro-flow LC technology is employed to improve throughput and reduce laboratory costs associated with research of drugs of abuse and pain panel drugs. Less solvent consumption and shorter run times can be cost and time saving measures in a high-throughput drug screening laboratory. Less solvent consumption can also lower waste disposal costs.
Analysis of barbiturates and 11-nor-9-carboxy-Δ9-THC in urine using automated Disposable Pipette Extraction (DPX) and LC/MS/MS
This report demonstrates the use of disposable pipette extraction (DPX) as a fast and automated sample preparation technique for the analysis of barbiturates and 11-nor-9-carboxy-Δ9-THC (COOH-THC) in urine. Using a GERSTEL MPS 2 equipped with a DPX option and coupled to an Agilent 6460 LC/MS/MS instrument, 8 barbiturates and COOH-THC were extracted and analyzed averaging a cycle time of 7 min/sample providing rapid, just-in-time sample preparation for high throughput screenings. The limits of quantitation (LOQ) for the barbiturates and COOH-THC were 100ng/mL and 10ng/mL, respectively. Extraction efficiencies were greater than 70% with RSDs less than 10%.
Development of a high-throughput LC/MS/MS assay for Pain Management panel from urine
An LC-MS/MS method for the analysis of pain management panel comprising 12 analytes has been developed on Ionics 3Q Series 120 Triple quadrupole system. Combined with SLE for urine sample cleanup, this method provides a faster, more accurate and reproducible solution for the analysis of pain management drugs. Preliminary results show that the LLOQs for 12 drugs in neat solution are in the range of 20 to 500 pg/mL with CVs less than 15% over the entire concentration range. The accuracy is 90 to105%. This method also displayed good linearity for all analytes with R2 > 0.99.
Quantitative Analysis of Opiates and Benzodiazepines in Urine with Prelude SPLC dual channel LC System and Quantum Ultra Mass Spectrometer
Prelude SPLC, a novel Thermo Fisher Scientific dual channel LC system coupled with Quantum Ultra was used to analyze opiates and benzodiazepines in urine. The two panels were analyzed using separate methods executed either on one channel (multiplexing both methods) or on two channels (multiplexing one of the methods). A simple gradient liquid chromatography elution was carried out to separate all the drugs and their internal standards with a Thermo AccucoreTM PFP column, 2.1 x 50 mm, 2.6 µm in less than 6.5 minutes. The method was further decreased to less than 3.5 minutes by two-channel multiplexing.
Extraction of 22 Pain Management Drugs from Human Urine Using Supported Liquid Extraction (ISOLUTE® SLE+) in 96-Well Plate Prior to LC-MS-MS Analysis
The screening of various prescribed pain medications in patient urine samples has grown significantly in the US. As a result, clinical testing laboratories have moved to develop fast, reliable quantitative and qualitative LC-MS/MS methods. Clinical labs prefer to use a single extraction technique for speed and cost effectiveness. A fast and simple sample preparation method was developed using supported liquid extraction, specifically ISOLUTE SLE+ 96 well plates to extract 22 pain management drugs. This poster demonstrates a rapid and reliable Supported Liquid Extraction assay for the simultaneous extraction of pain management drugs from urine prior to LC-MS/MS analysis.
Harmonization of an LC-MS/MS Assay for Therapeutic Drug Monitoring of the Immunosuppressant Drug Tacrolimus
Monitoring blood concentrations of the immunosuppressant drug tacrolimus in solid organ transplant recipients is considered the standard of care. While LC-MS/MS is often considered the “gold-standard” for these measurements, the technique is not yet harmonized across laboratories because of the wide variety of calibrators, sample pre-treatment protocols and instrumentation employed. This presentation will demonstrate the harmonization of tacrolimus measurements by LC-MS/MS using a commercially available test kit through a proficiency testing survey of laboratories using this assay. Also presented is a comparison of the results obtained using this test system to a reference measurement procedure for tacrolimus.
Measuring serum 25-hydroxyvitamin D using a liquid chromatography-tandem mass spectrometry method compared with total 25-hyroxyvitamin D using an RIA method
Serum 25-hydroxyvitamin D concentration is a reliable biomarker for vitamin D status. We compared the performance of RIA and LC-MS/MS methods. Serum 25-hydroxyvitamin D was measured using an MSMS vitamin D kit (PerkinElmer, USA) and an RIA kit (DiaSorin, USA). The LC-MS/MS method was linear from 4.7 to 150 ng/mL with intra- and inter-day CVs of <6% for both 25-hyroxyvitamin D3 and 25-hydroxyvitamin D2, which had lower limits of quantification of 0.38 and 0.38 ng/mL, respectively. The RIA and LC-MS/MS methods agreed well (slope, 0.9931; average bias, 0.3 ng/mL).
Development and evaluation of a high-throughput reference measurement procedure for the determination of estradiol-17¥â in human serum using ID-UPLC-MS/MS
In The Joint Committee for Traceability in Laboratory Medicine, LC-MS/MS method for serum estradiol (E2) has complicated sample purification and long analytical time. We developed a high-throughput reference measurement procedure for serum E2. The E2 was extracted from serum matrix using methyl tert-butyl ether and drivatized with dansyl chloride prior to reversed-phase (C18) LC-MS/MS. The accuracy of our method for E2 agreed with the certified values within the uncertainty of the measurements for the certified reference materials. Reproducibility was obtained with within-set coefficient of variations (CVs) ranging from 0.4 to 0.9% and between set CVs ranging from 2.9 to 3.3%.
Discovery and Validation of Serum Biomarkers for Cervical Cancer Detection using iTRAQ labeling and label-free quantitation
We performed comparative analyses of depleted serum samples from patient sets related to the early (stage Ia-IIb) and late stages (stage IIIa-IV) of cervical cancer using stable-isotope labeling for the discovery of protein biomarkers. We found that 12 proteins were up- and 9 proteins were down-regulated in serum of early-stage patients. 14 proteins were up- and 23 proteins down-regulated in serum of late-stage patients. 32 proteins were associated with disease progression. The up-regulation of biomarker candidates was validated by label-free LC-MS/MS of individual serum samples.
Identification and validation of biomarkers for atherosclerosis by quantitative clinical proteomics
We used 4-plex iTRAQ to quantify proteins in four groups: 1) Healthy, 2) Cardiovascular disease asymptomatic with high coronary calcium score, 3) Stable atherosclerotic disease, and 4) Acute myocardial infarction. ELISA and SRM-MS were used for single patient validation of candidate proteins. Proteins involved in cardiovascular diseases i.e. serum amyloid A protein, C-reactive protein, and apolipoprotein(a) display an increased expression profile from group 1 to 4. Vinculin, insulin-like growth factor-binding protein 1, and integrin alpha-9 display similar profiles. ELISA assays confirmed the expression profile of apolipoprotein(a) and C-reactive protein. Selected proteins will be quantified on single patient level by SRM-MS.
Targeted proteomics analysis of protein kinases
To systematically evaluate optimal proteotypic peptides for expressed protein kinases, we purified kinases with ActivX nucleotide binding probes and identified isolated proteins using discovery-based proteomics. All tryptic peptides were subsequently evaluated on a triple quadrupole mass, leading to identification of proteotypic peptides for 207 protein kinases. Further evaluation using total cell lysates, enabled the analysis of 152 protein kinases. To evaluate the assay in a drug discovery context, cells were treated with clinically relevant inhibitors and changes in the abundance of protein kinases were determined. Together this platform represents a sensitive way to systematically identify alterations in protein kinase levels.
Pharmacodynamic Biosensor Assay for Kinase Activity in Leukemia
Phosphorylation in chronic and acute leukemia involves multiple active kinases linked to disease onset, progression and drug resistance. We designed deliverable peptide substrates for Abl and Syk kinases and applied these in a multiplexed assay to monitor kinase activity and determine sensitivity of cells to kinase inhibitors. This was achieved using multiple reaction monitoring mass spectrometry (MRM-MS) quantifying the degree of phosphorylation of all peptide substrates in leukemia cell lines treated with different kinase inhibitors. MRM-MS enabled us to simultaneously monitor endogenous peptides important to these signaling pathways as validation of the technique.
Small, Standardized Protein Database Provides Rapid and Statistically Significant Identification and Characterization of Targeted Proteins
Mass spectrometry based targeted protein identification using complete database search requires large amount of time and computational power. We built a small standardized human database that can be appended to any targeted protein sequence that provides sufficient number of PSMs to peptide validation algorithm to train its model. The performances of small database were evaluated using different human samples run on LTQ-Orbitrap mass analyser; search with SEQUEST and validated with Percolator. Targeted peptide identification through small the database, is statistically as robust as complete database search moreover it is faster and identify more number of high confident peptides per protein.
Improved Spectral Libraries as a Tool for Targeted Assays
Spectrum libraries have long provided a means of collecting data from a variety of sources and providing a reference to be used, for example, to inform targeted assay method building. We build on the spectrum library model in two ways: 1. Introducing the Peptide Retention Time Kit as a means of standardizing quality across samples. 2. Computing and storing important spectrum data beyond the fragmentation pattern, including statistics on fragment peak variability and retention time relative to the Peptide Kit components. We demonstrate the use of these libraries to streamline the process of building sensitive targeted assays.
How good is public spectrum library for building SRM/MRM assay?
Single Reaction Monitoring (SRM) is an emerging mass spectrometry based technique for high throughput relative/absolute quantification of targeted proteins in biological samples with high sensitivity and specificity. Recent advancement in mass spectrometry makes SRM technology suitable for various clinical application including disease diagnosis and prognosis. The most critical step in building SRM assay is selection of transitions for targeted proteins. We in this study have shown the most efficient way for selecting such transitions for optimal SRM assay based on publicly available peptide spectral library and tools available for building SRM assay.
Protein profiling and discovery of surrogate markers of vascular remodeling in Murine models for Hypertension by Mass Spectrometry
The goal of this study is to report changes in protein expression and to discover surrogate markers for vascular remodeling under hypertension. Mice were used as model organisms to imitate similar conditions to human hypertension and its underlying molecular biological changes. Aortic tissues sampled from three treatment groups, Angiotensin II, L-NAME and DOCA were compared with a normal control by a bottom-up proteomics approach. A liquid chromatography based LTQ Oritrap Velos instrument was used for this analysis. In this study, we identified treatment dependent and independent protein expressions that can be used to study progression of hypertension in similar biological systems.
RECENT DEVELOPMENTS IN HR/AM QUANTIFICATION – IMPLICATION FOR CLINICAL PROTEOMICS
Targeted proteomics has emerged as the priviledge method for quantitative analysis of peptides in clinical samples. The recent development of high-resolution/accurate mass measurements, with various acquisition methods, has opened new avenues for quantitative proteomics studies. An overview of the capabilities of high resolution hybrid instrument (quadrupole/orbitrap) for quantification on precursor ions (SIM mode) or fragment ions (PRM mode) are presented and discussed; more specifically the exquisite sensitivity (sub-amol range) resulting from trapping experiment, the multiplexing capabilities to increase throughput, and the selectivity resulting from high resolution. The specificities and technical aspects are illustrated on different experiments including precise quantification of a panel of markers in urine or blood samples, and large scale screening of several hundred candidates.
Quantitative Mass Spectrometric Immunoassay of Clusterin
Clusterin, also known as apolipoprotein J, is a heterodimeric-secreted glycoprotein. Clusterin circulates in serum as part of the HDL complex, which, when elevated is correlated with a lower risk of cardiovascular disease (CVD).
In this study, we demonstrated the process of identifying and quantifying clusterin in the plasma of subjects with CVD by MALDI-based mass spectrometric immunoassay (MSIA). Areas of investigation included: (1) determining a method for the direct extraction of the clusterin, (2) constructing usable working curves, (3) testing the approach on a small sample population and (4) applying the assay to determine the concentration of several clusterin variants in human samples.
Application Of Mass Spectrometry Of Intact Protein And Typtic Peptides In The Analysis Of Des-Leu Albumin In Plasma - A Novel Marker For Chronic Pancreatitis
We have previously identified a truncated form of albumin lacking the C-terminal leucine known as des-Leu albumin, which is present at high concentrations in patients with pancreatitis. With the recent application of Mass Spectrometry in the routine laboratory we now have the technology to develop simple methods for this novel marker and evaluate its clinical utility for the diagnosis and monitoring of pancreatitis.
We have measured des-Leu albumin in plasma as its intact protein and from its tryptic peptide digest and found levels of 20-90% compared to levels <10% in the normal population.
Quantitation of IgG Subclasses by LC-MS/MS
This fully validated assay gives accurate, robust identification and quantitation for each IgG Subclass in serum. Measurement of subclass proteins is useful in evaluating patients with clinical signs and symptoms of humoral immunodeficiency or combined immunodeficiency. Nephelometry, utilizing an antigen/antibody reaction, has been the method of choice, but also has limitations. Immunoturbidimetric assays can give falsely low concentrations of IgG subclasses due to antigen excess. Here, we describe a superior, fully validated, LC/MSMS method that does not rely on antigen/antibody reactions and thus is not hampered by antigen excess.
CSF Leakage Detection Using Multiple Protein Markers by LC-MS/MS
Identification of central nervous system fistulas is dependent on detecting CSF in body fluids. Currently, Beta 2-transferrin (βTf) and Prostaglandin-H2 D-Isomerase (Beta-Trace protein) are markers of CSF. βTf is identified by immunofixation electrophoresis while Beta-Trace protein is quantitated by nephelometry. Both of these CSF markers are limited in a background contaminated by serum. We have developed a selective reaction monitoring (SRM) LC-MS/MS method that utilizes both a CSF marker (Beta-Trace protein) and serum markers (Alpha 2 macroglobulin and Complement C3). The SRM LC-MS/MS technique had 98.5 percent concordance with our current βTf when compared with 65 consecutive clinical samples.
Characterization of Stable Isotope Labeled Human Protein Standards for Quantitative Mass Spectrometry Based Assays
Stable-isotope labeled (SIL) synthetic peptides are typically used as internal standards for the quantitation of proteins by mass spectrometry. The accuracy of absolute quantitation with such peptide-based approaches is subject to error associated with protein fractionation, enrichment, and proteolysis steps. A more ideal alternative strategy involves the use of SIL proteins that can be introduced early in the analytical workflow as true internal standards. In this study, we describe the characterization of stable-isotope labeled full-length proteins generated by baculovirus-directed expression in human HEK293 cells and demonstrate their use as internal standards for MS-based protein quantitation.
Standardizing, Comparing, and Predicting Retention Times Across LC-MS Proteomic Platforms Using a Characterized Standard
MS based proteomic platforms require regular suitability checks and standardization in order to relate experimental data between analytical runs and efficiently transfer assays. These requirements demand a well characterized standard which; (1) contains detectable, unique peptides that span the full elution profile, (2) provides a quick ‘eye test’ for system suitability, and, (3) displays retention time and chemical stability. We have developed a mixture of 14 SIL peptides to fit these criteria and describe specific procedures for developing a reliable LC-MS platform suitability test and a method to translate the retention times of peptide targets across instrument platforms.
Development of Insulin-Like Growth Factor 1 Mass Spectrometric Immunoassay
Mass Spectrometric Immunoassays (MSIA) are methods that utilize mass spectrometric detection in conjunction with immuno-purification to quantify proteins. This process is achieved through the utilization of affinity pipette devices (MSIA-Tips) that enable high-throughput assaying of hundreds of samples per day. Described here is the development of a SRM-MSIA for insulin-like growth factor 1 (IGF1). The analyte was extracted from samples using anti-IGF1 antibody derivatized MSIA tips. Prior to extraction, Long R3 IGF1 was spiked into samples to serve as internal reference standard for signal normalization and IGF1 quantification. Following capture and elution, tryptic peptides were analyzed via LC/MS. The standard curves spanned the range of 1 to 1,500 ng/mL. The assay exhibited intra- and inter-assay precisions of <10%, and linearity and spiking-recovery in the 90 - 110% range.
Absolute quantification of serum Apolipoprotein AI and B using SID-MRM-MS
Serum apolipoproteins AI and B are alternative risk markers of cardiovascular disease. From a standardization viewpoint, absolute quantification of well defined serum apolipoproteins should be preferred above that of heterogeneous serum lipids and macromolecular lipoprotein complexes. Nowadays, mass spectrometry offers the possibility for sensitive, selective measurement of proteotypic peptides obtained after complete tryptic digestion of the proteins under investigation.
For serum apo AI and B quantification, an SID-MRM-MS method was developed on an Agilent 6490 platform. Accurate and simultaneous measurement of apolipoproteins AI and B in 0.5 µl serum in an 18 min chromatographic run was achieved. Encouraging is the fact that clinically equivalent test results were obtained with LCMS as compared to established immunoturbidimetry in a normotriglyceridemic serumpool.
GlycoProtein Analysis (GPA): High-throughput MS platform for automated identification and label-free quantification of N-linked glycopeptides in complex samples
Here we describe the development of a fully automated MS platform for the identification and quantification of a variety of N-glycopeptides. The GlycoProtein Analysis (GPA) platform, can identify the glycan compositions, glycosylated sites and glycopeptide sequences of N-glycopeptides tryptically digested from glycoproteins by using combined HCD and CID/ETD fragmentation patterns. Label-free quantification of the identified N-glycopeptides was performed by summation of peak area of LC/MS chromatogram. The GPA platform has been successfully evaluated for the different glycoproteins including human plasma samples.
GlycoProtein Analysis (GPA) of Human Plasma for the Discovery of Cancer Biomarker
It is challenging to perform the identification and the quantification of N-glycopeptides from mass spectrometry analysis because the instrumental sensitivity and concentration of N-glycopeptides are a lot lower than those of the general non-glycopeptides due to the intrinsic property and microheterogeniety of glycopeptides.In this study, the GPA platform and algorithm were applied to identify the different glycoforms of a N-glycopeptide tryptically digested from glycoproteins. We also describe a new label-free quantitative method that can be applied to target glycoproteins in order to discover a cancer biomarker in human plasma. The change and quantification of site-specific N-glycosylation in cancer progression as well as quantification of nonglycosylated tryptic peptides of glycoproteins are discussed.
Improving the Sensitivity and Specificity of Peptide Quantification
Quantification of proteins is becoming increasingly important in multiple application areas. The increase in the number of ‘potential’ protein biomarkers discovered is driving a need for better quantification strategies to confirm or refute their ultimate utility. Increased throughput is required as larger sample numbers must be analyzed, which means reduced sample preparation and / or accelerated chromatography, increasing the chance of interferences. Higher sensitivity strategies are also critical to get the coverage of both high and low abundant precursors. In this work, differential mobility separation mass spectrometry and microflow chromatography will be explored for improving specificity, sensitivity and throughput.
The complete Human SRMAtlas
The SRMAtlas is a publicly accessible web-based resource for the selection of SRM assays to conduct targeted proteomics. SRMAtlas supports the selection of both optimized peptides and their transitions for each targeted protein. We have recently developed the complete Human SRMAtlas, an assay library of over 170,000 SRM assays for all human proteins to account for the entire mass spectrometry accessible 20,333 predicted human protein-coding ORF’s (99.9%). For the development of the SRM assays we used a high-throughput approach based on the synthesis of equimolar synthetic computationally selected proteotypic peptides for each human protein coding ORF and over 11,000 isoforms and SNP’s. The synthetic peptides were employed as reference compounds to generate the corresponding fragment ion spectra and to extract the SRM assay coordinates.
Comparison of Automated Sample Preparations for the Detection of Fentanyl Analogs and Metabolites in Human Urine Utilizing LC-MS/MS
Automated sample preparation minimizes variability, increases throughput, improves lab efficiency, and reduces human contact with hazardous chemicals. Nine fentanyl analogs and three metabolites were measured employing a 96-well plate solid phase extraction (SPE) and online SPE. A 96-well plate SPE increases efficiency and throughput through the simultaneous extraction of many samples, but requires separate sample concentration and transfer steps, which achieves lower detection limits. Online SPE extracts and directly injects the sample onto a liquid chromatography column. Although costly, online SPE requires less sample, increases analyst productivity, and reduces analyst exposure to hazardous samples, including fentanyls, which are powerful narcotic.
Mechanistic Investigations of Hydrostatic Pressure Effects on Tryptic Digestion.
Proteolytic digestion is a fundamental bottleneck of proteomic sample preparation. We present the results of a systematic study to deconvolute pressure effects on protease activity from pressure effects on substrate proteins. Model proteins were digested with and without high pressure and analyzed by high resolution MS/MS on the LTQ-Orbitrap XL. Comprehensive data suggest that pressure effects on digestion are substrate specific, resulting in greater improvements for proteins that are typically resistant to trypsin. Pressure-based digestion appears to be particularly useful for analysis of tough proteins, providing opportunities for time savings and increasing the reproducibility of quantitative analysis.
MRM Assay development using ultra-fast digestions
Using the Perfinty Workstation and a Shimadzu triple quadrapole mass spectrometer, we have developed an MRM-based assay to quantify myofilament protein PTMs (phosphorylations and O-GlcNAcylations) implicated in diabetic cardiomyopathy. The Perfinity Workstation automates all proteomic sample preparation in-line and in real time with the mass spectrometric MRM-based assay. By determining the concentration of specific O-GlcNAc and O-phosphate modifications of myosin heavy chain, actin, myosin light chain proteins 1 and 2, and cardiac troponin I as a function of diabetes, we hope to elucidate the mechanism of disease progression as it correlates to cardiac dysfunction. Replacing bench-top preparative steps with column-mediated digestion, desalting, and Reverse Phase HPLC separation improves the reproducibility and throughput of our developed MRM-based assay significantly.
Minimizing hematocrit effects in Dried Blood Spot analysis using online DBS-SPE-MS/MS with maximum recovery
Full-spot desorption and quantitative recovery was investigated as possible solution to the hematocrit (Ht) issue in quantitative Dried Blood Spot (DBS) analysis. Flow-through desorption of Dried Blood Spots (DBS) coupled online to SPE and (LC)MS/MS was optimized for full-spot analysis of 5 µl DBS samples. Desorption conditions including temperature were optimized to achieve maximum dissolution of dried blood. Near 100% recovery was obtained for all test compounds in blood independent of Ht values ranging from 0.3 - 0.7. Using these “harsh” extraction conditions, the precision of the online DBS-SPE-MS/MS assay stays within acceptance criteria for blood samples with Ht ranging from 0.3 – 0.7.
Robust and Sensitive Measurement of the Growth Promoting Hormone IGF2 via a Mass Spectrometric Immunoassay
Mass spectrometry-based methods are becoming more prominent in the targeted measurement of protein biomarkers in biofluids, nonetheless due to lack of sensitivity, robustness, speed of analysis, and throughput, its adoption in the clinical field has been slow. However, recent technological advances in instrumentation and sample preparation and enrichment have begun to enable MS-based targeted protein assays to become a more attractive alternative to classical ELISAs and other automated immunoassays. The use of these technological advancements in the robust, sensitive, and high-throughput measurement of the growth promoting hormone, insulin-like growth factor 2 (IGF2) is presented.
Integration of an Automated Sample Preparation Workstation for the Analysis of Immunosuppressant Drugs by LC-MS/MS
The objective of this work was the automation of an LC-MS/MS method for the analysis of the immunosuppressant drugs Tacrolimus, Cyclosporine A, Sirolimus, and Everolimus, to eliminate human error, increase reproducibility, eliminate subjectivity during data processing, and save time. All steps of sample processing could be automated using a Beckman Coulter BioMek NXP platform. The sample preparation consisted of a simple protein precipitation using ZnSO4 solution. After centrifugation, the clear supernatant was injected directly onto the LC-MS/MS system. Samples were loaded in test tube format and the final samples were prepared in a 96-well plate format. The LC-MS/MS data acquisition, processing, and reporting were automatically performed using the Cliquid® software.
Automated Sample Prep, Data Acquisition and Processing for the Analysis of 25-OH Vitamin D2 and D3 by LC-MS/MS using the Beckman Coulter Biomek NXP Workstation
An LC-MS/MS method for the analysis of 25-OH Vitamin D2 and D3 was developed, making use of commercially available serum calibrators and controls. The sample preparation consisted of a Salt-Assisted Liquid-Liquid Extraction (SALLE), using acetonitrile and a saturated aqueous solution of ZnSO4. All of the liquid handling steps were automated using the Biomek NXP laboratory automation system. Centrifugation was performed offline, however the Biomek system also supports online centrifugation. Samples were loaded in barcoded test tube format and the final samples were prepared in a 96-well plate format, and then transferred to the LC-MS/MS system for analysis of 25-OH Vitamin D2 and D3. Chromatographic separation was accomplished using a Phenomenex Luna C18, 50x2.1mm, 3μm analytical column, and mass spectrometric detection was performed on the AB SCIEX API 3200™ LC/MS/MS system.
A Method for Automated Extraction of Pain Medication from Urine for Analysis by LC MS/MS
Sample preparation methodology that extracts and concentrates an analyte prior to analytical analysis is typically necessary to facilitate accurate quantitative and qualitative results. The ability to fully automate a sample preparation process that extracts a suite of basic drugs from urine affords a high throughput sample analysis scenario. Here we describe an automation process where 192 urine samples are extracted for a suite of pain management drugs using a Tecan Freedom EVO workstation in combination with the Biotage SLE+ plate. The process fully automates sample barcode identification, the enzymatic hydrolysis step, the addition of internal standard, and the solid liquid extraction (SLE) protocol. Quantitative data collected post automated extraction is comparable to data generated from manual protocol in a fraction of the time. Sample tracking and chain of custody requirements are met fully, all samples are treated identically and sample preparation time is significantly reduced when compared to the manual process.
Development of an SPE-LC/MS/MS Method for Quantitation of Four Synthetic Insulins in Human Plasma: Challenges of Working with Large Peptides
Intact insulins are difficult to analyze by LC-MS/MS due to poor sensitivity and poor fragmentation. Insulin and its analogs also suffer from non-specific binding and poor solubility, making LC and sample preparation method development difficult. The SPE LC-MS/MS method described here was successfully used to quantify insulin glargine, detemir, aspart , and glulisine in human plasma. Standard curves prepared in solvent were linear from 50 pg/mL to 500 ng/mL. The detection limit for extracted human plasma samples was 0.25 ng/mL for each of the four insulin analogs.
Automated Online Solid Phase Extraction UPLC/MS/MS for the Analysis of Mycophenolic Acid in Human Plasma
The Waters ACQUITY UPLC Online SPE Manager (OSM) is a new online solid phase extraction (SPE) system coupled with Ultra Performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) for the preparation and analysis of biological matrices. Here we successfully demonstrate the potential of the OSM for the automated sample preparation and analysis of mycophenolic acid for clinical research purposes. This method displays good linearity, precision and accuracy with minimal ion suppression.
For Clinical Research purposes, not for use in Diagnostic Procedures.
Automated Quantitative Flow-Through LC/MS of Drugs from Dried Blood Spot Cards
A quantitative MS study of drugs in dried bloods spots was performed using a new automated flow-through DBS device. Whole blood was spiked with anti-depressant drugs at therapeutic levels and spotted to cards. DBS cards were automatically imaged to determine the co-ordinates of blood spot centers. Spots were automatically extracted using a 2mm clamp to seal in a metered liquid flow of aqueous solvent and internal standard. The extracts were concentrated and desalted using automated SPE, followed by fast LC separation and accurate mass LC/TOF analysis. Drugs were quantitated in multiple spots using multilevel DBS calibration curves generated using peak areas of accurate mass extracted ion current traces. Results were used to determine the quantitative range, precision and carry-over for the system.
LC/MS Bioanalysis of Steroid Hormones; Investigation on the Impact of Matrix Effects
Immunoassay approaches for steroid determination are typically hindered due to the lack of specificity of antibodies for the measurement of the particular steroids. With the wide acceptance of LC-MS/MS in the clinical setting, there is a growing trend toward converting traditional IA techniques toward more specific and robust LC-MS/MS approaches. Though LC-MS/MS improves assay specificity and allows of multiplexed analyte assays to be conducted simultaneously, it is not without limitations specifically toward interferences form endogenous sample matrix. Often LC-MS/MS assays can be hindered due to ionization effects due to endogenous matrix from biological samples; this can result in random and arbitrary discrimination in analyte response. In this study, the impact of biological sample matrix interference is investigated with regards to the assay of steroid hormones progesterone, aldosterone, corticosterone and deoxycorticosterone. Traditional techniques for preparing plasma /serum samples are compared with novel matrix depletion technique in the HybridSPE-Phospholipid with regards to analyte response, precision and accuracy. The study demonstrates the impact of phospholipid matrix interference, and how selective depletion of said matrix during the sample preparation step ultimately improves robustness of the LC-MS/MS assay.
“Dilute-and-Shoot: Hit or Miss?” Minimization of matrix effects using a rapid automated cleanup technique for the analysis of buprenorphine and norbuprenorphine in urine
Buprenorphine (Bup) and norbuprenorphine (Nbup) are opioids commonly monitored for patient treatment on opiate addiction. This study describes the automated extraction and high throughput analysis of Bup and Nbup from small sample volumes of urine (< 1 mL) using a GERSTEL MPS 2 autosampler with a Disposable Pipette Extraction (DPX) option coupled to an Agilent 6460 LC/MS/MS instrument. The automated DPX-LC/MS/MS method removes potential matrix interferences and ion suppression providing higher method sensitivity. Recoveries for both analytes were greater than 85% with %RSDs less than 10%, respectively. Addition of automated valve switching capability further increases the throughput potential.
Matching Old Samples to New Assays: Two Case Studies
Can samples already collected for an old method be adapted to a new LC-MS assay for better quantitation? In one case, protein precipitation worked well for tissue homogenates in a HILIC-MS/MS assay of dimethylarginines, and transfer of the separation to a PFP column greatly improved sensitivity. However, plasma results were poor. The cause was determined to be phospholipid contamination causing ion suppression. A single step SPE cleanup of the extracts was developed to solve the problem. In a second case, plasma for glutathione analysis by an established LC-fluorescence method was preserved with 7.5% perchloric acid. Addition of excess ammonium bicarbonate neutralized the acid in an MS-compatible manner yielded a pH compatible with NEM derivatization of the free thiol, allowing the samples to be analyzed by a new LC-MS method.
Urinary Cortisol Extraction using Supported Liquid Extraction prior to LC-MS/MS Analysis.
Free urinary cortisol levels can be an indicator of a variety of disorders, such as Cushing Syndrome. This poster presents a simple method for the extraction of cortisol from urine demonstrating good recoveries and low ion suppression. LC-MS/MS analysis was performed using a Waters 2795 Liquid Handling Unit coupled to a Quattro Ultima Pt triple quadrupole MS using electrospray ionization, operated in the MRM mode. Supported liquid extraction method development resulted in recoveries greater than 90% for cortisol spiked urine. Calibration curves constructed using this method from 25-2000 ng/mL, demonstrated excellent linearity with coefficients of determination greater than 0.99.
Immunosuppressants: Strategies for Simplified Sample Preparation using Supported Liquid Extraction prior to UPLC-MS/MS Analysis.
Immunosuppressant drugs are extremely important for therapeutic drug monitoring (TDM) regimes, requiring robust and reliable methods for their analysis. This poster aims to demonstrate strategies for the extraction of various immunosuppressant drugs from whole blood providing clean samples prior to UPLC-MS/MS analysis. Spiked whole blood was extracted using a variety of protocols to investigate optimal combination of drug recovery and extract cleanliness. UPLC-MS/MS was conducted using a Waters ACQUITY UPLC coupled to a Quattro Premier XE triple quadrupole mass spectrometer. Acceptable extraction recoveries were obtained showing excellent extract cleanliness, demonstrating a reliable method for mass spectrometric approaches for these analytes.
A Novel Method for the Extraction of 1á, 25-Hydroxy-vitamin D2/D3 and Analysis using UHPLC-MS/MS.
Vitamin D analysis has extremely important clinical relevance with levels needing to be measured for a wide variety of reasons. This poster presents a novel high throughput method for the extraction of the biologically active components: 1,25-dihydroxy-vitamin D2 and 1,25 dihydroxy-vitamin D3 using an ISOLUTE SLE+ 400 96 well plate. The method was developed from charcoal stripped serum samples spiked at various therapeutic levels and subjected to a partial validation. Overall method performance demonstrated high analyte recoveries and low ion suppression, allowing a quantifiable range matching that therapeutically expected.
Extraction of Catecholamine Metabolites Urine Using Ion Exchange Solid Phase Extraction in 96-Well Plate Format Prior to LC-MS-MS Analysis
Serotonin metabolites 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), vanillylmandelic acid (VMA), methanephrine (ME), and normetanephrine (NME), are used to screen for markers of progressive and degenerative pathogenesis related to the growth of tumors, carcinoid syndrome, Whipple disease, and celiac disease. Clinical diagnostics can be complicated due to the need to extract these compounds from complex matrices like urine prior to analytical analysis. A dual solid phase extraction strategy in a 96 well plate format was developed using cation and anion exchange sorbents to extract HVA, VMA, 5-HIAA, ME, and NME prior to simultaneously analyzing for all of the extracted analytes.
Total proteins analysis of Saliva with Microwave enzymatic digestion
Saliva has been noticed as a substitute for serum or urine in the analysis of human specimen because the Information from saliva reflects the altered health state and can be used in therapeutic and toxicological monitoring. Microwave-assisted enzymatic digestion can reduce enzyme reaction time for protein digestion from several hours to tens of minutes. In this study, enzymatic digestion of total proteins in saliva was analyzed with microwave for effective proteomic analysis. The digested salivary total proteins were profiled and identified by MLADI/TOF and LC/MS. Microwave-assisted enzymatic digestion finish the reactions within a few minutes without overdigestion and less oxidation.
Robust Targeted Protein Quantification by LC-MS using a New Automated Sample Preparation Platform for Protein Denaturation, Digestion and Cleanup
Mass spectrometry (MS) coupled with liquid chromatography has emerged in the recent decade as the most versatile analytical strategy for targeted protein analysis. From monitoring large-scale production processes to the validation and clinical assaying of protein biomarkers, the use of targeted MS for protein analysis is beginning to displace ELISA-based assays.
Here we present a high throughput, automated sample preparation platform for targeted protein quantification by LC-MS. Using AssayMAP technology, protein denaturation, reduction, and alkylation have been automated and are compatible with commonly used reagents and chemistries. Automation of the sample preparation workflow permits reproducible parallel processing of up to 96 samples from protein denaturation through peptide cleanup.
Rapid LC-MS/MS analysis of non-hydrolyzed urine samples for opiates and opiate-glucuronide conjugates using novel HummingbirdRA tips on a Tecan robotic system
Analysis of opiates in urine is commonly performed using enzymatic hydrolysis followed by solid-phase extraction (SPE). An ideal way to save time and reduce costs would be to perform SPE on non-hydrolyzed opiate samples using a high-throughput robotic platform. Since high throughput robotic systems utilize pipette tips for handling liquid samples, it would be optimal to perform the SPE directly in pipette tips to ensure maximum throughput. In this study, the high throughput capabilities of dispersive pipette extraction (DPX) is demonstrated by using HummingbirdRA tips to extract 6 common opiates and their primary metabolites and glucuronide conjugates with a Tecan robotic system. The resulting clean sample extracts were rapidly analyzed using a 2 minute LC-MS/MS run.
Rapid LC-MS/MS analysis of urine samples for benzodiazepines using HummingbirdRA tips on a Tecan robotic system
Analysis of benzodiazepines in urine is commonly performed using enzymatic hydrolysis followed by solid-phase extraction (SPE). SPE methods are often incorporated after hydrolysis to remove the matrix and its effects that result in ion suppression. An ideal way to save time and reduce costs would be to perform SPE on benzodiazepine samples using a high-throughput robotic platform. Since high throughput robotic systems utilize pipette tips for handling liquid samples, it would be ideal to perform the SPE directly in pipette tips to ensure maximum throughput. In this study, the high throughput capabilities of dispersive pipette extraction (DPX) are demonstrated by using HummingbirdRA tips to extract 5 common benzodiazepines with a Tecan robotic system. The resulting clean extracts allowed for a rapid LC-MS/MS run.
Automatisation of human Cerebrospinal Fluid (CSF) pre-treatment using Agilent AssayMAP technology: trypsin digestion and reverse phase
Development of clinical proteomics methods based on targeted mass spectrometry (Multiple Reaction Monitoring) hold promise for the discovery of novel biomarkers that might form the foundation for new clinical tests. As an important part of the various origins of errors and biases has been well identified in the pre-analytical step, the use of an automation device is of great interest. Ou results show that the automatisation of CSF sample trypsic digestion and clean up is robust and easy of use for the pre-treatment of large cohort of sample needed for clinical validation of new proteomic methods.
Heat Inactivation Enables Reliable Measurement of Tissue Proteome
In this work, a heat stabilization system, utilizing conductive heat, at controlled pressure, has been used to generate rapid, homogenous thermal denaturation of enzymes and thereby stop degradation in different kinds of tissue. The heat-stabilized samples were compared to snap-frozen samples and, in time study manner, compared with different post-sampling intervals. The protein and peptide content, including their PTMs, were examined using mass spectrometry, western blot and RPPA.
Testing Androgenic Anabolic Steroids in Oral Fluid using Turbulent Flow Orbitrap MS
Anabolic androgenic steroids (AASs) are tested often in the clinical practices of endocrinology and sports doping. These fields rely heavily on blood and urine levels of both endogenous and exogenous compounds. As the diagnosis of disease states and knowledge of athletes challenge the current testing parameters, new methodology needs to be developed. However, access to blood or even urine is not always possible. Therefore, methods have to allow for the testing of alternative matrices including oral fluid. Moreover, AAS identification has become increasingly difficult as new compounds and masking is employed in some clinical situations. In this talk, we will explore new options for detecting AASs in oral fluid and advantages and future directions of this work.
Automated mass spectrometric quantification of cortisol and melatonin in saliva
Disruption of circadian rhythm can have serious health implications. Cortisol and melatonin show circadian rhythm and are of interest in several research areas such as depression and the metabolic syndrome. We developed an online LC-MS/MS method for the combined analysis of cortisol and melatonin in a routine set-up. Online sample extraction and chromatographic separation was achieved within 7.5min. Intra- and inter-day precision were <10% at three different levels for both analytes. Linearity was excellent for cortisol and melatonin (r2 > 0.99). LLOQ for cortisol and melatonin was determined at 0.15nM and 5pM which is adequate for quantifying these hormones in saliva.
Determination of urinary catecholamines and metanephrines in a single run 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), metanephrine, normetanephrine and 3-methoxytyramine. A single, efficient sample preparation procedure allows simultaneous extraction of all analytes in urine. The described method achieves the required functional sensitivity and is capable of quantitating analytes over a sufficiently wide dynamic range with a single injection. Excellent reproducibility was observed for all compounds (CV < 5%). All calibration curves displayed excellent linearity with an R2 > 0.9997.
The novel application of HPLC-MS/MS towards the detection of corticotropin-releasing hormone in cerebrospinal fluid.
Corticotropin-releasing hormone (CRH) is a 41-amino acid peptide. Classically associated with homeostasis through its interaction with the hypothalamic-pituitary-adrenal axis, it is also linked to a range of stress related disorders including depression and anxiety. Moreover, accumulating evidence suggests that placental expression of CRH regulates the timing of parturition. Current approaches to CRH quantification are unsuitable for routine use and there remains no reference method. Here, principles of proteomics have been combined with HPLC-MS/MS to generate a qualitative working assay. This has scope for future development into a quantitative method that would permit routine CRH measurement.
Development of a High Resolution Accurate Mass Method for the Multiplexed Monitoring of Antiretroviral Agents in Human Serum
There are currently several antiretroviral (ARV) drug classes used for the management of HIV infection, including compounds used for protease and reverse transcriptase inhibition. Development of a method including chromatographic separation for identification of the various classes of ARVs is challenging due to their variable chemical structures. The goal of this work is to qualitatively screen and confirm the presence of a panel of ARV drugs using a multiplexed approach. Validation data indicate the ability to screen and subsequently confirm the presence of 15 ARV agents in a single specimen in a high-throughput, multiplexed format.
Metabolic phenotyping by automated chip-based nanoelectrospray ionization high resolution mass spectrometry
A high-throughput quantitative metabolomic approach has been developed and its versatility has been shown through analysis of different types of clinical samples. The combination of single-stage high resolution mass spectrometry, nanoelectrospray and linear statistical pattern recognition methods enables selective, sensitive and quantitative detection of small metabolites and characterization of the general similarities and differences of the spectral pattern. Dried blood spot and urine samples were analyzed and the method found to be a promising alternative to standard newborn screening methods. Extracts of bacteria cell lysates were examined and typical features of small molecule and phospholipid patterns were detectable.
Designer stimulants – evolving abuse patterns.
This presentation summarizes analytical data on the positivity rate for 25 designer stimulants in over 25,000 routinely tested urine samples in 2011 and 2012. Tracking monthly positivity revealed changing patterns of supply and abuse of a tremendous number of new recreational drugs appearing on the market annually. The data shows how drugs succeed each other in popularity especially after scheduling. Metabolic patterns of 16 synthetic cathinones including their long-term metabolites, markers of the parent drugs, and analytical issues will be discussed.
Accurate Reference Standards for Accurate Quantitation of Thyroid Hormones: Impact on Clinical Reference Ranges
Development of LCMSMS methods that differentiate and accurately quantitate thyroid hormones are a high priority for clinical diagnostic laboratories. Reverse-T3 was synthesized, characterized and developed into a certified solution standard. The certified solution standard was ~30-50% higher concentration than in-house standards prepared from commercially-available reverse-T3 powder. An inter-laboratory study traced the problem to impurity profile, inorganic content and in-homogeneity of the research-grade reverse-T3, and material handling. Switching from powder to certified solution standard caused a shift in reference intervals. Additional data was collected to establish an EP transformation equation, followed by verification of the transformed reference intervals in healthy subjects.
An LC-MS method for simultaneous determination of ofloxacin and cefixime in human plasma: Development and validation
Simple, rapid, and sensitive HPLC-MS assay method has been developed and validated for simultaneous determination of ofloxacin and cefixime in human plasma using moxifloxacin as an IS. Chromatographic separation of analytes and IS was achieved on C18 column using an isocratic solvent mixture [acetonitrile, methanol and 0.5%formic acid (23:10:67%)]. Simple protein precipitation using acetonitrile was utilized for extraction of analytes. The calibration curves were linear (>0.99) in the concentration range of 4-500 ng/mL for ofloxacin and 40–6000 ng/mL for cefixime. The results of the validation parameters met the requirements of biological analysis, hence can be used for pharmacokinetic and clinical studies
Minimizing Collection Tube Interfering Substances in the Analysis of Testosterone using Atmospheric Pressure Chemical Ionization (APCI)
The monitoring of testosterone serves as an aid in diagnosing and treating disease states related to hormone imbalance. Development of a clinically diagnostic method requires the ability to perform measurements of hormone levels across a broad dynamic range. As a result of our status as a national reference laboratory and our complex testing menu we do not always have control of the specimen types that we receive in our laboratory. The need arose to develop a method that would be free from interfering substances and to be able to assay multiple specimen types regardless of the collection tube submitted.
Effects of preanalytical factors on serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC-MS/MS for the clinical laboratory testing
Vitamin D testing is increasing worldwide. Development of a simple and high-throughput MS-based method is needed for the measurement of serum 25-hydroxyvitamin D (25OH-D) as an indicator of vitamin D status. Here, we present high-throughput MS-based quantitative analysis of serum and plasma 25OH-D3 and D2 levels with satisfactory analytical performances. No significant effects of the common preanalytical factors were found for these assays. This LC-MS/MS based 25OH-D assay has the potential to be used as a routine clinical laboratory assay for assessing vitamin D status.
Estimation of vitamin D Status in Chinese population
Vitamin D status, estimated on 6706 human serum specimens collected during January to July 2012 from areas of 10 provinces of China using LC-MS/MS for measurement of 25hydroxy-vitamin-D, were: healthy male (n = 904), 7.41% (30-80 ng/mL), 45.4% (20-30ng/mL), and 47.2% (<20 ng/mL); healthy female (n = 560), 4.8% (30-80 ng/mL), 37.1% (20-30ng/mL), and 58.0% (<20 ng/mL); pregnancy women (n = 4853), 21.72% (30-80 ng/mL), 36.0% (20-30ng/mL), and 42.2% (<20 ng/mL); Osteopathy patients (n = 125), 8.0% (30-80 ng/mL), 41.6% (20-30ng/mL), and 50.4% (<20 ng/mL); kidney disease (n = 267), 9.7% (30-80 ng/mL), 22.1% (20-30ng/mL), and 68.2% (<20 ng/mL).
Quantitation of total testosterone and estradiol simultaneously in human serum by ID LC-MS/MS
Reliable and accurate measurements of total estradiol and testosterone are essential for the diagnosis, treatment, and prevention of hormone related diseases and disorders. Herein a highly specific and high-throughput procedure for simultaneously measuring estradiol and testosterone in human serum was developed. Estradiol and testosterone are released from serum under acidic buffer condition, isolated with automatic liquid-liquid extractions, and quantified by carbon-13 isotope dilution liquid chromatography tandem mass spectrometry.
A Rapid and Selective Method for the Measurement of Testosterone in Human Serum using Laser Diode Thermal Desorption-Differential Ion Mobility-Tandem MS
In this work we present a rapid method for the measurement of testosterone in human serum using a combination of Laser Diode Thermal Desorption (LDTD) ionization, differential ion mobility spectrometry, and tandem mass spectrometry.
To confirm the validity of the method, a comparison study was performed by analysing a set of 24 anonymized serum samples (i) by LC-MS/MS, and (ii) by LDTD-DMS-MS/MS. The measured concentrations varied by less than 10% (accuracies ranged from 90-110%) for the two methods across the entire sample set. The method exhibited a linear response over the concentration range from 0.1 ng/mL to 100 ng/mL of testosterone, with %CV<14% at the Lower Limit of Quantitation and %CV<6% across the remainder of the concentration range.
Cost Effective, High-throughput Analysis of Vitamin D using Micro-flow LC/MS/MS
Here we present the details of the analysis of 25-monohydroxy Vitamin D2 and 25-monohydroxy Vitamin D3 using the Eksigent ekspert™ microLC 200 system in combination with the AB SCIEX QTRAP® 4500 LC/MS/MS system.
The Limit of Quantitation (LOQ) for the analysis was determined using ‘blank’ stripped serum and spiked serum and found to be below 4 ng/mL, and 5 ng/mL for 25-monohydroxy Vitamin D3 and 25-monohydroxy Vitamin D2, respectively. Accuracies were within 92-109% across the calibration range (1 – 100 ng/mL), with CVs below 15% based on n=5 for each calibration concentration. Serum samples from donors were also analyzed using the method described above, and the results indicate that method performance is comparable to that of a conventional HPLC method.
LC-MS/MS quantitation of 1a,25-Dihydroxyvitamin D3 and 1a,25-Dihydroxyvitamin D2 in human serum by immunoextraction and PTAD derivatization.
Measurement of 1a,25-Dihydroxyvitamin D (1a,25(OH)2D) is essential to the clinical investigation of PTH-independent hypercalcemia. In addition to the well-known endocrine functions, there is an increasing body of literature demonstrating paracrine and autocrine actions of 1a,25(OH)2D and interest in quantifying this hormone is growing accordingly. Low circulating concentration, interferences from more abundant vitamin D metabolites, and poor ionization efficiency hamper the mass spectrometric analysis of 1a,25(OH)2D. We present a method for the ABSCIEX API5000 to quantify 1a,25(OH)2D3 and 1a,25(OH)2D2 employing delipidation, immunoextraction, and PTAD derivatization and having a LOD of <2.5 and <5.0 pg/mL for the respective forms.
Analytical Measurement of Serum 25-OH-vitamin D3, 25-OH-vitamin D2 and their C3-epimers by LC-MS/MS in Infant and Pediatric Specimens
A simple and sensitive LC-MS/MS procedure was developed for quantitation of serum 25(OH)D3/2 and their C3-epimers. Serum 25(OH)D metabolites were extracted with MTBE. The ion-transitions 401.2→365.2, 407.2→371.3, 413.2→331.2, and 419.2→337.1 were monitored for 25(OH)D3 and C3-epi-25(OH)D3, 25(OH)D2, C3-epi-25(OH)D2, and d6-25(OH)D2, respectively. The assay provided an LLOQ of ≤2.8 nmol/L, linearity to 400 nmol/L, and a CV<15%. As a proof-of-principle, 25(OH)D3 and C3-epi-25(OH)D3 were quantified in 200 pediatric subjects. A higher concentration of C3-epi-25(OH)D3 was observed in infants <1 year, with concentrations dropping by 50% at 12 months. Liquid vitamin D3 supplements were not a source of the epimer.
An LC-MS/MS Method for Quantifying Azole Antifungal Medications in Plasma
We developed an LC-MS/MS method for therapeutic drug monitoring of the antifungals voriconazole, fluconazole, itraconazole, and posaconazole in 500 uL alkalinized plasma. Liquid/liquid extraction: 3 mL ethyl acetate/hexane 30:70. LC-MS conditions: 35-100% methanol (0.1% formic acid) over 6 min at 0.2 mL/min, C18 column (2.1 x 150 mm), Varian 1200L triple quadropole mass spectrometer, positive electrospray ionization, multiple reaction monitoring (voriconazole 349.9>126.9, voriconazole-d3 352.9>283.8, fluconazole 306.9>219.9, itraconazole 704.8>392.0, posaconazole 700.8>682.7). Post-column infusion studies showed the absence of ion suppression. Preliminary lower limits of quantitation are < 0.1 ug/mL.
Measurement of Thyroxine (T4), Triiodothyronine (T3) and Reverse Triiodothyronine (rT3) by Liquid Chromatography with Online Sample Cleanup-Tandem Mass Spectrometry in Negative Mode
Accurate and precise measurement of thyroid hormones in serum is important in clinical research. Liquid chromatography with online sample cleanup was employed to minimize manual sample preparation. This was coupled to a triple quadrupole mass spectrometer utilizing jet stream and dual ion funnel technologies to increase ion sampling and sensitivity. LODs of 1 pg/mL for T3 and 2 pg/mL for T4 and rT3 were obtained. LLOQs of 2 pg/mL for T3 and <5 pg/mL for T4 and rT3 were obtained. The linear correlation coefficients in the range from 2 pg/mL to 100 pg/mL were >0.99 for the three analytes.
Oxidative loss of 5-methyltetrahydrofolate in serum due to suboptimal specimen handling and storage
The main circulatory folate form 5-methyltetrahydrofolate is susceptible to oxidative degradation to the pyrazino-s-triazine derivative of 4¥á-hydroxy-5-methyltetrahydrofolate, also known as MeFox. Using an LC-MS/MS method that measures five folate vitamers and MeFox we assessed the effects of suboptimal preanalytical conditions on 5-methyltetrahydrofolate and MeFox in three serum QC pools: (1) long-term frozen (-20¨¬C) or refrigerated (5¨¬C) storage for up to 1 year; (2) short-term exposure to room temperature and 37¨¬C for up to 1 day; (3) multiple freeze/thawing cycles. Depending on the sample handing condition 5-methyltetrahydrofolate undergoes oxidative degradation and MeFox only recovers minor 5-methyltetrahydrofolate losses.
Simultaneous determination of antidepressants in human serum and urine by monolithic silica spin column extraction and LC-MS/MS
In clinical and forensic laboratories, the identification and quantification of drugs and medicines are necessary to determine the death or decide a treatment protocol. In addition, an immediate analysis result is required to decide the subsequent course of action. Therefore, simple and fast method coupling spin column extraction with LC-MS/MS was developed for the simultaneous extraction fifteen antidepressants (TCA, SSRI, SNRI, NaSSA) as model drugs from serum and urine. In this study, monolithic silica spin column (C18) was used. All the tested drugs were simultaneously determined. The proposed method will be able to apply for clinical and forensic cases.
Measurement of 3-Epi-25-OH-Vitamin D3 and 25-OH-Vitamin D3 by LC-MS/MS on the AB SCIEX Triple Quad™ 4500 System
The method presented here was developed using the AB SCIEX Triple Quad™ 4500 system and the Eksigent ekspert™ ultraLC 100 system, and permitted the independent measurement of both 25-OH Vitamin D3 and 3-epi-25-OH Vitamin D3 in a single LC-MS/MS analysis.
‘Blank’ stripped serum and spiked serum samples have been analyzed to determine the Limit of Quantitation (LOQ) for the method. The estimated LOQ was approximately 2 ng/mL for each analyte. The accuracy of the measurements for each analyte ranged from 83-119% over the entire concentration range, including at the LLOQ. The method displayed excellent linearity over the concentration range from 1-100 ng/mL, with r>0.998 for each analyte.
25-Hydroxyvitamin D2/D3 LC-MS/MS Method Optimization Using the Analysis of Standard Reference Materials
Our LC-MS/MS method includes the use of isotopically labeled hexadeuterated 25-hydroxyvitamin D2 and D3 internal standards, liquid-liquid extraction of serum and plasma samples, followed by solvent reconstitution of the dried sample extracts, and injection to the LC-MS/MS system. Analytical standards were prepared in serum and plasma matrices demonstrating linearity across the range of 2 - 180 ng/mL, with r>0.999. LC-MS/MS method optimization is achieved with the ongoing between-run analysis of external quality assessment control samples and 25-hydroxyvitamin D metabolite Standard Reference Materials including the testing of the NIST SRM 972, SRM 1950, and SRM 968e human serum and plasma samples.
Low pg/ml Detection of 17ß-Estradiol in Serum through Increased Ion Sampling Efficiency Using Dual Ion Funnel LC/MS/MS Technology
Low-level determination of 17ß-estradiol 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 dual ion funnel technology, ion sampling efficiency has been improved to the point that 17ß-estradiol can be quickly and accurately quantified down to 0.25 pg/ml using an LC/MS/MS approach.
The Sensitivity Improvement of 17beta-estradiol LC-MS/MS quantitation in human serum with 4-(Dimethylamino) benzoyl chloride (DMABC) derivatization
17beta-estradiol (E2) quantitation is still a challenge in clinical field because of its low concentration in human serum (children, male adult, etc), requirement of good accuracy and high sensitivity, etc. A sensitive LC-MS/MS method for E2 with 4-(dimethylamino)benzoyl chloride (DMABC) derivatization was introduced to improve sensitivity. And test was performed on 3Q mass spectrometer coupled with a Shimadzu UFLCxr system. Preliminary results showed the direct measurement of E2 (MRM at m/z 271-145) gave a LLOQ at 3.1 pg/mL in neat solution with CV at 13.7% and accuracy at 97.1 %. With DMABC derivatization(MRMs at m/z 420.1-166.0) the LLOQ can be reached at 0.24pg/mL with CV at 12.9 % and accuracy at 104.5 %. The human serum sample will be used to do further method evaluation.
A Novel Drug of Abuse Procedure in Whole Blood Using a Short Column and Rapid Gradients
A methodology was developed for the cost-effective extraction and rapid determination of a wide range of drugs of abuse from a single extraction from post-mortem whole blood. The types of compounds include barbiturates, benzodiazepines, amphetamines, cocaine based compounds, opiates, and THC based compounds. Some work was also done on extending the capabilities for ‘bath salts’ (cathinones) and synthetic THC compounds (JWH analogs). The methods use a short column (Sunfire HPLC cartridge column, 4.6x20 mm, 3µ) and very rapid gradients. An AB SCIEX 4000 Q-Trap LC/MS/MS was used for detection using scheduled multiple reaction monitoring (sMRM).
Comparison of different measurement procedures used in NHANES to assess methylmalonic acid blood levels
Methylmalonic acid (MMA), a functional indicator of vitamin B12 insufficiency, has been measured in the US population as part of the National Health and Nutrition Examination Survey (NHANES). From 1999–2004, plasma was analyzed by a GC/MS method; during 2011-2012 serum was analyzed by LC-MS/MS. To provide continuity in interpreting NHANES data, we compared the two methods using 326 residual plasma samples previously analyzed by GC-MS. The two procedures correlated well (r=0.99) and showed no significant bias (Bland Altman bias (95% CI)=0.9 (-1.4 to 3.3) nmol/L), indicating that no adjustment will be needed to compare previous to current NHANES data.
Method for Determination of Leflunomide Metabolite Over a 40,000-Fold Dynamic Range
A method to quantify the leflunomide metabolite (teriflunomide) was developed and validated across the concentration range of 0.005 to 200 ug/mL. The method provides chromatographic separation of the parent drug from the metabolite. To cover the broad quantitative range of this assay with a single extraction procedure, two calibration curves are used with partial overlap in concentration range. The assay was validated using several collection tube types and interference studies with special matrix conditions of lipemia, icterus, and hemolysis were evaluated. The final assay has a cycle time of four minutes and imprecision of less than 6% across all controls.
Ultrafast Simultaneous Analysis of 6 Antiepileptic Drugs in Human Serum Using Online SPE/MS/MS
Clinical research laboratories traditionally rely on HPLC and immunoassay, more recently LC/MS for quantitative analysis of antiepileptic drugs. An efficient, fast, accurate, and sensitive SPE/MS/MS method with a wide calibration range was developed for the simultaneous quantitation of 6 antiepileptic drugs (AED) in human serum. This method employs protein precipitation followed by dilute and shoot on the SPE/MS/MS system, enabling analysis of 6 AEDs in 14 seconds per sample producing significant savings in analysis time and solvent consumption compared to traditional LC/MS/MS methods. This method is capable of throughputs greater than 240 samples per hour.
Development of a Semi-Quantitative, High Throughput Method for Simultaneous Detection of 34 Commonly Prescribed Cardiovascular Drugs in Hospitalized Patients
We developed a rapid Semi-quantitative screening process for the most commonly prescribed cardiovascular drugs in patient's serum. To evaluate the reliability of the assay in a clinical setting a blind trial was performed with 294 samples from hospitalized patients, with each patient receiving some or none of the drugs being monitored. The results were compared to the Medication Administration Record as a gold standard. With a PPV of at least 90% for 31 drugs and a NPV of over 93% for all drugs monitored, we have developed a reliable semi-quantitative tool for clinical research.
A Selective Attomole LC-MSMS Quantitation of Peptide Drugs in Human Plasma
Because traditional radioimmunoassay for peptides drugs may lacks of specificity because of cross reactivity, and capability for high throughput. The LC-MSMS technique becomes favored due to its high selectivity and sensitivity. With multiply charge states and extensive fragments, it may become difficult to develop a fast and reliable quantitative method for peptide drugs. Here a LC-MSMS method recent advancement of tandem quadrupole mass spectrometer and unique ion source is introduced and evaluated for several therapeutic peptides in human fluid. Preliminary results showed that the LLOQ of angiotensin-I in human plasma can be reached at 7.5 attomol/uL with accuracy at 106.5% and CVat 11.6 %, and the LLOQ of angiotensin-I can be reached at 7.5 attomol/uL with accuracy at 106.5% and CV at 11.6 %. More detail will be provided for evaluated peptides.
The Utilization of Novel Platform in a LC-MS/MS Workflow for the Analysis of Vitamin D, Testosterone, Immunosuppressants, Chemotherapeutics and Cortisol
The use of LC-MS/MS in bioanalysis is often hindered by the need for complex sample preparation and extraction methods which can introduce errors in sample handling that lead to large sample to sample variability. The development of a rugged system for the sample preparation and chromatographic analysis that requires minimal sample handling is essential in a clinical environment. In this work, we present the application of new platform in the development of faster, reproducible, and lower solvent consuming methods for the analysis of 25-OH vitamin D2 (25OH-D2), 25-OH vitamin D3 (25OH-D3), testosterone, immunosuppressants, chemotherapeutics, and cortisol.
Ultrafast Analysis of an Immunosuppressant Drug Panel in Whole Blood Using a High-Throughput SPE/MS/MS System
The ability to simultaneously analyze tacrolimus, everolimus, sirolimus, and cyclosporin A in whole blood using an ultrafast SPE/MS/MS system was evaluated. Excellent linearity, precision, accuracy, and signal-to-noise ratios were determined for all four analytes in just under 13 seconds per sample. Identical human samples analyzed using this method correlated well with a traditional LS/MS/MS method, however the analysis time was approximately 10 times faster. This methodology is capable of throughputs of greater than 270 samples per hour.
Analysis of Lewisite Metabolites in Urine by Solid Phase Extraction–Liquid Chromatography-Positive Electrospray Ionization–Tandem Mass Spectrometry
Lewisite (L) is a toxicologically significant vesicant and blistering organoarsenic chemical warfare agent. Urinary Lewisite metabolites are hydrolysis products of the native agent, existing in arsenic (III) and (V) oxidation states. The method presented detects two of the As(V) Lewisite metabolites by positive electrospray ionization tandem mass spectrometry (MS/MS). The metabolites, namely, 2-chlorovinylarsonic acid (CVAOA) and bis-2-chlorovinylarsinic acid (BCVAOA) were resolved by dual column reverse-phase C18 liquid chromatography (LC) with a run-time of 3.9 minutes. Urine specimens were oxidized with hydrogen peroxide to form the As(V) metabolites and then extracted using silica solid phase extraction (SPE) prior to LC-MS/MS analysis.
Quantitation of Underivatized 1,25-dihydroxyvitamin D by a High Sensitivity LC-MS/MS Instrument
1,25-dihydroxyvitamin D (1,25OHD), a vitamin D metabolite created in the kidneys. It presents a quantitative challenge due to its low concentration (pg/mL) in blood. This study utilizes an ammonium adduct in combination with a high sensitivity LC-MS/MS instrument, creating an alternative to traditional derivatization methods, increasing sensitivity and reducing sample preparation requirements. This method achieves a linearity range of 5-50,000 pg/mL for 1,25OHD3 with accuracies ranging from 86.7-111.9%. Inter and intra-day variation coefficients were achieved at less than 12%. Providing highly sensitive, reliable results this method enables clinicians to accurately determine the vitamin D status of patients.
Development of LC-MS/MS method for the quantitation of Androstadienone in human serum
Androstadienone has been shown to modulate psychological, physiological, and hormonal outcomes, but there is currently no reliable way to measure its production. The purpose of this research study is to develop a method using liquid chromatography tandem mass spectrometry (LC-MS/MS) for reliably detecting minute amounts of androstadienone in human blood, and utilize such a method to measure individual variation in rates of production of androstadienone associated with various emotional stimuli. The developed LC-MS/MS method demonstrated good linearity from 0.1-10ng/ml with R2 > 0.995.
Use of Differential Mobility Spectrometry for the Rapid Analysis of Histamine and its Metabolites in Biological Matrices
An LCMS method for analyzing histamine and its metabolites is highly desirable due to the advantages of LCMS over other techniques such as HPLC-fluorescence which requires derivatization with ortho-phthaldialdehyde. The method monitors histamine, the major metabolite 1-methylhistamine as well as N-á-methylhistamine and 3-methylhistamine. The use of ion mobility spectrometry allows for rapid analysis without the need to chromatographically separate the isobaric N-methylated metabolites which also have some common fragments leading to interference otherwise.
Quantitative Analysis of Prostaglandins in Plasma by LC/MS/MS Utilizing Dual Ion Funnel Technology
In this study, a robust, sensitive, reliable and fast method is presented for the quantitation of prostaglandins in plasma using a LC-MS/MS triple quadrupole mass spectrometer with negative electrospray ionization. Major target prostaglandins were PGD2, PGE2, PGF2a and LTB4. A reverse-phase column with sub 2 micron particles is used to achieve the necessary chromatographic separation from interferences and isobaric compounds. The total chromatography time is 3 minutes. The plasma samples were analyzed. All prostaglandins are baseline separated. The LODs and LOQs were evaluated. All calibration curves showed good linearity with over four orders of dynamic range.
Study of matrix effects on perchlorate assay in human urine using IC coupled with MS/MS with ESI and APCI
Perchlorate interferes with iodine uptake into the thyroid gland.With large amounts,it can cause thyroid disorders, and may raise other health concerns.Perchlorate is eliminated from the body almost exclusively via urine, except for lactating women.Therefore,urine perchlorate concentration is a useful biomarker of perchlorate exposure in the environment.Our previous method, IC/ESI/MS/provided reasonable accuracy and precision.However,retention time shift along with a narrower calibration curve as well as a much higher baseline noise in synthetic urine compared with a pure solvent may indicate that matrix effects exist.The purpose of our study was to compare the severity of matrix effects using ESI and APCI sources for the same urine sample matrices, and then to find a better ionization technique with proper source parameters to subdue matrix effects in urine perchlorate assay.
Measurement of 1,25-Dihydroxyvitamin D2 and D3 in Blood by LC-MS/MS Utilizing Ion Funnel Technology
Liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) has become an essential tool for small molecule quantitation due to its high sensitivity and specificity, excellent reproducibility and the ability to perform simultaneous analysis of multiple analytes. 1,25-dihydroxyvitamin D – a metabolite of vitamin D – has proven to be a challenging compound to analyze due to the low pg/ml range relevant to clinical research. A sensitive method has been developed for the quantitation of 1,25-dihydroxyvitamin D2 and D3 in blood using ultra high performance liquid chromatography (UHPLC) and triple quadruple (QQQ) mass spectrometry enhanced with dual ion funnel technology coupled with an immuno extraction LC-MS/MS Kit for the preparation of plasma samples.
Multiplexed Immunosuppressant Assay in Dried Blood Spots by LC-MS/MS - Clinical Validation and Implementation
To simplify monitoring of immunosuppressive drugs levels and improve compliance, we are implementing an easy at home dried blood spot sample collection with no need for hospital visits. We developed and validated a liquid chromatography-mass spectrometry assay for simultaneous measurement of tacrolimus, sirolimus, and cyclosporine A from dried blood spots. Sample preparation, LC-MS/MS conditions, linearity, precision, patient samples correlation, stability, effect of hematocrit, spotted blood volume, punch location, and matrix effect were evaluated. We hope that dried blood spot analysis will contribute to successful outcome of organ transplant and overall patient satisfaction.
Analysis and characterization of 25-Hydroxy-Vitamin D3 and D2 and 3-Epi-25-Hydroxy-Vitamin D3 and D2 by LCMSMS
An LC-MS/MS method was developed for the quanitation of 25-Hydroxy-Vitamin D and C-3-25-Hydroxy-Vitamin D epimer’s for both D3 and D2 in serum. The C-3 epimer of 25-Hydroxy-Vitamin D3 and D2 are mainly present in young children and some adults and the analysis of the C-3 epimer’s may have some clinical relevance to the overall metabolism of Vitamin D itself. An analytical method was developed using an Agilent 1260 HPLC system with an Agilent 6460 Tandem Mass Spectrometer with Jet Stream technology in positive electrospray ionization (ESI) mode. Various Agilent columns were evaluated with a water: methanol containing 0.1% Formic Acid gradient to achieve baseline separation of the four metabolites. Accuracy of the method was verified using NIST reference materials and the sample preparation consisted of liquid-liquid extraction from 150 ul of serum. Analytical sensitivity was achieved at less than 1 ng/ml for all the metabolites and the calibration dynamic range was from 1 to 250 ng/ml.
Quantitative Analysis and Comparison of Free and Total Thyroid Hormones in Serum using positive and negative ESI LC-MS/MS
Sensitive, reproducible and selective methods were developed for quantification of Total and Free thyroid hormones in serum that simultaneously measured the multiple analytes using different electrospray ionization modes. Separate sample preparation techniques were used for the determination of the thyroid hormones- Liquid-Liquid extraction and protein precipitation for the total concentration ultra-centrifugation for the free concentration. A labeled internal standard was included for each of the analyte’s to ensure accurate quantification. An Agilent 1290 HPLC system and a 6490 Tandem mass spectrometer with Ion Funnel and Jet Stream technology and a Poroshell 120 EC-C18 100 x 3.0 mm, 2.7 um with a water:acetonitrile containing 0.1% Acetic acid gradient to achieve baseline separation of the three analytes was employed. The method was calibrated over the range of 1 to 1000 pg/ml for free T4, T3 and rT3 and 1 to 1000 pg/ml for total T3 and rT3 and 1 pg/ml to 1000 ng/ml for total T4 in positive and negative mode. The calibration curves showed excellent linearity and reproducibility with analytical sensitivity being achieved at 1 pg/ml for all the analyte’s in positive mode and 5 pg/ml for the analyte’s in negative mode. Accuracy of the methodology was verified using NIST reference materials.
Quantitative Analysis and Comparison of Free and Total Thyroid Hormones in Serum using positive and negative ESI LC-MS/MS
Sensitive, reproducible and selective methods were developed for quantification of Total and Free thyroid hormones in serum that simultaneously measured the multiple analytes using different electrospray ionization modes. Separate sample preparation techniques were used for the determination of the thyroid hormones- Liquid-Liquid extraction and protein precipitation for the total concentration ultra-centrifugation for the free concentration. A labeled internal standard was included for each of the analyte’s to ensure accurate quantification. An Agilent 1290 HPLC system and a 6490 Tandem mass spectrometer with Ion Funnel and Jet Stream technology and a Poroshell 120 EC-C18 100 x 3.0 mm, 2.7 um with a water:acetonitrile containing 0.1% Acetic acid gradient to achieve baseline separation of the three analytes was employed. The method was calibrated over the range of 1 to 1000 pg/ml for free T4, T3 and rT3 and 1 to 1000 pg/ml for total T3 and rT3 and 1 pg/ml to 1000 ng/ml for total T4 in positive and negative mode. The calibration curves showed excellent linearity and reproducibility with analytical sensitivity being achieved at 1 pg/ml for all the analyte’s in positive mode and 5 pg/ml for the analyte’s in negative mode. Accuracy of the methodology was verified using NIST reference materials.
Method Development for a Sensitive Assay of Benzoylated Polyamine Derivatives by LC-MS/MS-ESI
Polyamine levels provide abundant information relating to stages in cell growth and developmental processes, and a more sensitive analysis would be ideal especially for samples in limited quantities. Current analytical methods rely upon dansylated products, which do not provide consistent MS results. The objective of this study was to develop a sensitive method with a short chromatographic run time detecting 6 polyamines (putrescine, cadaverine, spermidine, N8-acetylspermidine, spermine, and N1-acetylspermine) after derivatization as benzoylated derivatives. With an improved clean-up method polyamines in drosophila can be detected in the low nM range for the least sensitive amines and in the pM range for the others.
Nano rutile self assembled monolayer (SAM) cancer chip for MALDI-MS based direct sensitive cancer detection
Mass spectrometry based detection of cancer cells is a challenging task since the cells grown in the growth medium do not yield effective signals, owing to interference from the nutrients in the culture medium. Thus, prior to mass spectrometry these cells need to be washed, centrifuged and pretreated inorder to obtain good signals. We have devised a titanium based cancer chip by nano-patterning a rutile self assembled monolayer (SAM) on its surface making the chip hydrophilic, capable of capture and detection of concentrations as low as 101 cells/mL from the external environment. The captured cells on the chip can be directly loaded onto a homemade target plate and overlaid with matrix and analyzed by MALDI-MS. The current approach evades any form of pretreatment and sample preparation processes, hence is time saving and does not require the conventional MALDI target plate.
Novel mass spectrometry method for rapid analysis of native biopsy samples
A novel, mass spectrometry-based method is presented for the analysis of core biopsy samples. The method is based on the Rapid Evaporative Ionization Mass Spectrometry and Laser Desorption Mass Spectrometry analysis of core biopsy samples, subsequently after sampling. The biopsy samples are analysed within a few seconds and the collected tissue specific data is processed by the combination of principal component analysis and linear discriminant analysis. Tissue identification is based on comparison of processed data with authentic, tissue-specific datasets, previously collected in our database. An automated ion source setup was also constructed thus enabling the method for further clinical use.
DESI-MS with fine needle aspirations of renal neoplasms: Tumor lipid composition discriminates histological classification
A variety of benign and malignant neoplasms arise in the kidney. While most neoplasms are managed clinically with resection, renal compromised patients or patients who are poor surgical candidates often have a biopsy in an effort to determine if they can be managed more conservatively. Definitively diagnosing these tumors based on morphology alone is challenging, and the results immunohistochemical panels are often difficult to interpret. We propose a simpler approach to discriminate renal neoplasms on the basis of their lipid content using routine specimen handling procedures and DESI-MS. This is the first use of DES-MS on cytological specimens and the first to correlate the histological type of renal tumors with their intracytoplasmic lipids.
Phosphoproteome Analysis of Immune Cell Signaling Targets in Thymus Development and Function
RNase L is one of the key enzymes involved in the function of interferons against viruses and other microbial pathogens. Deficiency of RNase L contributes to the abnormality of the thymus and alternation of immune responses. In this study, we performed a systematic phosphoproteomic analysis of tyrosine phosphorylation events in thymic cells. By employing IMAC to enrich phosphoprotein, SDS-PAGE separation, trypsin digestion, Fe-NTA phosphopeptide-enrichment, and tandem LC-MS/MS, we identified a number of novel proteins as the target molecules of RNase L. This finding provides direct evidence that RNase L impacts thymus development and function through regulating phosphorylation of specific proteins.
Development and Validation of a Rapid LC-MS/MS Assay for Simultaneous Quantitation of Nicotine and its Metabolites for Heart-Lung Transplant Evaluation
Concentrations of nicotine (Nic) and its metabolites in blood reflect patients’ current smoking status and tobacco exposure. Demonstration of abstinence is one of the requirements for qualification of heart-lung transplantation surgery and can increase the survival rate from this surgical treatment. For the purpose of pre- and post-transplant evaluation, we developed and validated a rapid LC-MS/MS assay that only requires 100 microliters of sample volume to accurately identify and simultaneously quantitate Nic and its major metabolites, Cotinine (Cot) and 3-OH-cotinine (3OHCot), in serum.
Analysis of Nicotine Wipe Samples by Paper Spray Electrospray Ionization-MS
There is a demand for a more rapid and less expensive testing methods to monitor environmental tobacco smoke exposure. Paper spray ESI tandem MS has been applied to the analysis of nicotine extracted from cotton wipe field samples. The calibration curve from 2.5 to 8,000 ng was linear (R2 = 0.9993). For field samples, run to run accuracy varied by an average of ± 32% from results obtained by a validated LC-tandem MS method. The averaged values from replicate analyses improved the accuracy to <10% at mid- to higher concentrations. The run to run precision was determined to be the limiting cause.
Drug Testing in an Addiction Medicine/Psychiatric Setting: Comparison of Urine Immunoassay Results with Oral Fluid Ones Tested Using Liquid Chromatography-MS/MS
We simultaneously collected urine and oral fluid from 119 outpatients (155 paired specimens). Urines were tested qualitatively for eight drug classes by immunoassays on a cobas 501 analyzer. The paired oral fluid specimen was tested for twenty-four drug-related analytes using a Thermo TLX2 chromatograph, and a Quantum Ultra tandem MS in the multiple reaction monitoring mode, with Q1 resolution set at 0.2 FWHM. There were only 51 discrepant pairs of results out of 1226 urine-oral fluid test pairs. Most discrepant results involved benzodiazepines (8) and buprenorphine (17). We conclude oral fluid can be used instead of urine in this clinical setting.
Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for busulfan
Background: Our institution currently sends adult and pediatric plasma samples for busulfan testing to different reference laboratories. Objective: Develop a LC-MS/MS assay for busulfan. Results: Six calibrators were used (0-2000 ng/mL) and the reportable range was 5-5000 ng/mL. The LOD =1 ng/mL and LOQ=5 ng/mL. Extraction recovery was ~90% and ion suppression was ~5%. Run time was 4 minutes. Precision studies yielded CVs <20% at LOQ and <10% through the linear range. Conclusions: An accurate, sensitive and rapid LC-MS/MS assay for busulfan was developed. Method comparison, cost-analysis and electronic data upload to the laboratory information system studies are ongoing.
Development of a qualitative Gas Chromatography-Mass Spectrometry (GC-MS) method for the detection of “legal highs” in urine
“Legal highs” are designer drugs intended as alternatives to ecstasy or cannabis. Use is becoming increasingly prevalent with growing reports of intoxication and fatality. A liquid:liquid extraction procedure and qualitative GC-MS screening method for the detection of ten legal highs (designed as ecstasy alternatives) in urine in a single run of less than 30 minutes is reported. The method validation showed that the method was both specific and sensitive, with few potential interferences and good detection limits. A number of ante-mortem and post-mortem urine samples have been screened with mephedrone, BZP, TFMPP, mCPP and MDPV detected.
Determination of the “legal high” 4-methylethcathinone (4-MEC) by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and report of 4-MEC related fatality
4-Methylethcathinone (4-MEC) is a synthetic cathinone which can be found in so-called “legal high” preparations. 4-MEC is structurally related to mephedrone which has been linked to a number of fatalities; however little information is available as to methods of detection or toxicity of 4-MEC. The development of a method for the quantitation of 4-MEC in blood by LC-MS/MS is reported. The inter-batch coefficient of variance was 12% and the limit of quantitation was 4 ng/mL. In addition a case report of a fatality attributed to 4-MEC toxicity is presented in which the blood 4-MEC level of 167 ng/mL was determined.
Investigation of an Apparent Anti-Hypertensive Overdose Using LC-MS/MS
Objective: Develop a quantitative LC-MS/MS assay for the anti-hypertensive compounds, labetalol and nifedipine, to investigate a potential iatrogenic overdose.
Methods: A quantitative LC-MS/MS assay was developed on an ABSciex TripleTOF® 5600 high resolution mass spectrometer. Both analytes were linear over the curve range of 10 to 100 nM. Metoprolol was used as internal standard.
Results: Labetalol and nifedipine levels were quantitated in a series of serum samples taken over the course of several days. Nifedipine levels were either not detectable or were within the therapeutic range. Labetalol levels were in the high therapeutic/low toxic range, potentially explaining the clinical presentation.
Method development for multiplex determination of second-line anti-tuberculosis drugs by UPLC-MS/MS
Monitoring anti-tuberculosis drug concentrations is helpful for multidrug-resistant cases. We developed a rapid method that simultaneously measures 9 major second-line anti-tuberculosis drugs using LC-MS/MS. We revised the conditions and procedures for multiplex determination of first-line anti-tuberculosis drugs. Drug concentrations were determined by MRM in positive ion mode, and assay performance was evaluated. Within-run and between-run imprecisions were 2.8-11.1% and 3.7-11.6%, respectively at the concentrations of low and high levels for each nine drug. The lower limits of detection and quantitation were 0.05-0.5 ¥ìg/mL and 0.25-5.0 ¥ìg/mL, respectively. Linearity was acceptable at 5 level concentrations for each drug (R2 = 0.98-0.99). The devised method allows for rapid and sensitive quantification and should be helpful for drug monitoring in tuberculosis treatment.
Comprehensive Toxicological Screening using Generic MS/MS^ALL with SWATH™ Acquisition on the TripleTOF® 5600+ LC/MS/MS System
A novel experimental technique is presented, employing MS/MS^ALL with SWATH™ acquisition for comprehensive toxicological screening, using the TripleTOF® 5600+ system. This technique enables the acquisition of MS/MS data throughout the entire LC run for all compounds in a given sample, whether known or unknown, by employing Sequential Windowed Acquisition of all Theoretical masses. Using this approach, high-resolution extracted ion chromatograms can be monitored for the characteristic MS/MS fragment ions of all compounds of interest. Furthermore, retrospective data analysis may be performed to query the data for the presence of any previously unknown compounds, since the acquisition is non-targeted in nature.
Linker Fate in Bioconjugates by Mass Spectrometry Immunoassay
Bioconjugates are formed by conjugation of a pharmacophore or toxin via a linker and tether to an antibody scaffold or targeting antibody. In drug metabolism and pharmacokinetic (DMPK) analysis of the bioconjugates, the concentrations of the antibody scaffold and pharmacophore and/or toxin are measured separately to determine if there is difference in half-life or degradation/metabolism taking place to inactivate the phamacophore or release the toxin. We are also interested in the fate of bioconjugate linker and tether after catabolism of the antibody in-vivo. We have developed a mass spectrometry immunoassay (MSIA) method to determine the conjugation sites of bioconjugates by capturing the pharmacophore portion of the bioconjugate digest and sequence (MS/MS) the Ab peptide to determine location. We have also developed a MSIA method to capture the linker/tether from PK samples.
Quantification of serum allopurinol and oxypurinol concentration using liquid chromatography-tandem mass spectrometry
Allopurinol is a hypouricemic agent used for the treatment of gout. The aim of this study was to develop an accurate method for quantification of allopurinol and oxypurinol in serum using LC-MS/MS. The analytes were detected using LC-MS/MS in negative ionization mode. Serum concentrations of allopurinol and oxypurinol were quantified with MRM method. Linearity, precision, accuracy, LOD, carryover, and stability were evaluated for method validation. Interference by xanthine and hypohxanthine was also examined. The assay performance was acceptable enough to cover the therapeutic range of allopurinol and oxypurinol. The analytes were stable at room temperature for a day and a cycle of freeze and thawing. The method was suitable for quantification of serum allopurinol and oxypurinol using LC-MS/MS, and it may be used for therapeutic monitoring of patients with gout and renal impairment.
Rapid analysis of Synthetic Cannibinoids in Urine using Solid Phase Extraction and LC-MS/MS.
Synthetic cannabinoids are psychoactive compounds that are designed to mimic the affects of marijuana. These compounds are typically sprayed onto natural herbs for the purpose of smoking. Because of their large potential for abuse, these compounds have been listed as schedule 1 narcotics by the Drug Enforcement Agency. Therefore, a fast and effective testing method for these compounds is required. In conjunction with a Solid Phase Extraction method, an analytical testing method for synthetic cannabinoids and their metabolites utilizing LC-MS/MS is presented. The testing method results in a clean extract that displays excellent peak shape and a fast analysis time.
Comparison of a Targeted Drug Screen on Two High Resolution Mass Spectrometer Platforms
This study compared performance of targeted compound drug screens on the ABSciex TripleTOF® 5600 and the Thermo Exactive Orbitrap. Liquid chromatography conditions were held constant and drug standards were analyzed to determine accurate masses, retention times, and spectra/confirmation ions which were used to build libraries for each platform. Reproducibility was assessed on each system and clinical urine samples were run to determine false positives/negatives. The instruments performed similarly as broad-spectrum drug screens. The Exactive Orbitrap was more sensitive than the TripleTOF®, giving lower LLOD but also higher susceptibility to false hits. Spectral matching and confirmation ions added confidence in results.
Rapid Analysis of 11-Nor-Tetrahydrocannibinol-9-Carboxylic Acid, 11-Nor-Tetrahydrocannibinol-9-Carboxylic Acid Glucuronide and 11-Hydroxy-Cannibinol in Urine by LC/MS.
A rapid LC/MS method was developed that could detect and quantitate THCCOOH, THCCOOH glucuronide and THC-OH in urine. All three compounds could be separated in under four minutes using a C18A stationary phase along with an acetonitrile/methanol/water/0.1% formic acid mobile phase. The samples were prepared for analysis using a two step solid phase extraction procedure, which allowed for the THC metabolites to be analyzed in conjunction with other drugs of abuse. In addition, this method gave accurate values for THCCOOH glucuronide without performing any hydrolysis of the glucuronide conjugate. The method was validated for linearity, precision, accuracy, specificity and recovery.
Determination of Opioids in Dried Blood Spots using Novel Flow-Through Technology coupled to LC/MS/MS
Dried blood spot technology (DBS) coupled to highly sensitive LC/MS/MS systems promises significant advantages in bioanalytics both in the toxicology and pharmaceutical industry. Two major disadvantages that limit the acceptance of DBS technology are: (1) the laborious, time-consuming, error prone sample pre-treatment required, and (2) poor assay sensitivity due to reduced sample volume. An automated card extraction system virtually eliminates off-line sample preparation with direct online sample extraction of DBS cards without the need for hole punching while triple quadrupole mass spectrometry technology provides unparalleled sensitivity and superior quantitation for the low sample volumes typically encountered with DBS analyses. This presentation will describe our application of this integrated analysis system to the quantification of opiates in blood in the ng/mL concentration range.
Application of ultra high performance liquid chromatography tandem mass spectrometry to the analysis of tamoxifen and metabolites in serum and plasma, to support a genotyping study.
Tamoxifen (TAM) is administered as therapy for and prevention of breast cancer. A 4 minute procedure for monitoring TAM and metabolites: endoxifen, 4-hydroxytamoxifen (4-OH-TAM) and N-desmethyl tamoxifen (N-DM-TAM) was developed using a Waters Acquity TQD. To a 100-µL aliquot of serum, 500 µL of precipitating reagent containing internal standards was added, vortexed and centrifuged, and 10 µL of supernatant was injected into the UPLC-MS/MS. The analytical measurement range was 2-60 mg/mL for endoxifen and 4-OH-TAM and 20-600 ng/mL for TAM and N-DM-TAM. Intra and inter-assay imprecision (CV) was < 15% at four different concentrations.
The Use of Automated Direct Sample Analysis (DSA/TOF) for the Rapid Screening of Illicit Street Drugs
Screening methods for drugs of abuse currently require long chromatographic methods and sample preparation. These methods can be time-consuming and laborious. The ability to analyze samples rapidly is desirable, as drug seizures may result in thousands of samples. A method was developed for screening various classes of street seized illicit designer drugs, drugs of abuse and abused human growth hormones in solid, liquids as well as in urine of possible illicit drug offenders using the DSA-ToF. We present a fast screening and confirmation method by AxION® direct sample analysis with time-of-flight mass spectrometry (DSA™ TOF-MS). Fast screening of seized samples in seconds, without sample preparation, is easily performed using DSA TOF-MS. A real life case study for the rapid analysis of 369 unknown drugs from seized pills, vials, powders and urine samples.
Quantification of 7 Designer Cathinones in Urine Using a Q Exactive Mass Spectrometer
We developed a method for analysis of MDPV, methylone, mephedrone, methedrone, ethylone, butylone and naphyrone in urine using liquid/liquid extraction and a Q Exactive benchtop Orbitrap mass spectrometer. All compounds were detected at 0.5 ng/mL. MDPV, methylone, mephedrone, methedrone, ethylone and butylone were linear to 1000 ng/mL and showed good precision and recoveries. Lack of a suitable internal standard for naphyrone limited the results for this compound to a qualitative rather than quantitative method.
A Robust and Simple Accurate Mass MS/MS Drug Screening Method Using the TripleTOF® 5600 System.
For Research Use Only. Not For Use In Diagnostic Procedures. Laboratories performing drug screening analyses on samples often wish to identify as many compounds as possible from a single experiment. Laboratories also desire the capability and flexibility to perform retrospective data analysis. Some have proposed accurate-mass measurements provide sufficient selectivity to resolve interferences in an LC-MS method, however we have observed numerous cases where accurate mass measurements alone cannot resolve interferences. Here we present a "dilute and shoot" robust high-throughput method for analyzing 41 drugs in urine using the AB SCIEX TripleTOF® 5600. This method employs a TOF-MS scan along with several scheduled product ion scans, during each cycle. All compounds examined in this effort were correctly identified from the increased selectivity provided by the MS/MS full scan data.
High sensitivity analysis of THC-COOH and THC in oral fluid
Oral fluid is becoming increasingly popular as a sample for forensic drug testing. While the levels of THC in oral fluid pose no great challenge for LC-MS-MS analysis, the analysis of metabolites is often preferred to rule out passive exposure. Unfortunately, THC-COOH is present at very low levels (<100 pg/mL), and oral fluid collection devices limit the amount collected and dilute the sample in a stabilization solution containing surfactants.
In this work, we demonstrate a method that utilizes a sensitive and fast hybrid triple quadrupole linear ion trap mass spectrometer to determine THC-COOH and THC in oral fluid. The method gives a LLOQ of 10 pg/mL for THC-COOH and 200 pg/mL for THC in oral fluid.
A comprehensive routine LC-MSn screening solution for clinical and forensic toxicology
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with library searching is an emerging screening solution for toxicology. The Toxtyper workflow is based on an amaZon speed ion trap MS and holds a spectral library of currently 840 compounds. Data evaluation and reporting is carried out by an automated spectral library searchalgorithm. Blinded samples were automatically processed and analysed by 7 independent Toxtyper systems at 5 different locations. The combination of MS²/MS³ spectra and retention time information allows a robust identification of drugs and metabolites. The high degree of automation is ideally suited for the transfer of this solution to routine laboratories.
Quantitative Analysis of THC and Main Metabolites in Whole Blood Using Tandem Mass Spectrometry and Automated Online Sample Preparation
An on-line extraction procedure with Thermo Scientific TurboFlow technology was developed for the sensitive quantification of THC and its two main metabolites, 11-OH-THC and THC-COOH, from whole blood. Blood samples were treated by protein precipitation followed by an online extraction and analysis by Reverse Phase Liquid Chromatography (RP-LC) coupled to mass spectrometry. Linear calibration curves were obtained within a concentration range of 0.25 ng/mL to 100 ng/mL. The limits of quantification for the three molecules were lower than the usual recommended cut-off values in whole blood. The final method had a good precision and accuracy.
Identification and Quantification of Dimethylamylamine (1, 3-DMAA) in Blood/Serum/Urine by Liquid Chromatography Tandem Mass Spectrometry.
DMAA has been marketed as nutritional and anabolic supplement which has stimulant like effects. In 2011, DMAA was associated with two deaths in military population. Since DMAA exists as enantiomers which makes its analysis very challenging in biological samples. A sensitive and reliable method of liquid chromatography–electrospray ionization/tandem mass spectrometry (HPLC-ESI/MS/MS) was developed and validated for determining 1,3-dimethylamylamine (1,3-DMAA) in biological samples (Blood/Serum/Urine). The samples were extracted with buffer and purified by liquid-liquid partition with ethyl acetate. The parameters for reverse-phase (C18) HPLC and positive ESI/MS/MS were optimized. The matrix effect, specificity, linearity, precision, accuracy and reproducibility of the method were determined and evaluated. The method was linear over a range of 50.0–5000.00 ng/mL with R2 of 0.99.
Ultrafast Analysis of Panels of Synthetic Drugs of Abuse in Urine Using Online SPE/MS/MS
The ability of an ultrafast SPE/MS/MS system to analyze panels of synthetic drugs of abuse in urine was evaluated in the present study. MS and SPE methods were optimized on an Agilent High Throughput RapidFire Mass Spectrometry System. Excellent linearity, precision, and accuracy were determined for all analytes in both synthetic cathinone (bath salt) and synthetic cannabinoid (spice) panels. At injection cycle times of less than 15 seconds, this method is capable of throughputs greater than 240 samples per hour providing a fast and efficient screen for these analytes
Quantitative Determination of Serum/Plasma Voriconazole Levels Using Liquid Chromatography Mass Spectrometry (LC/MS/MS)
A quantitative MRM method by LC/MS/MS was developed for quantitation of voriconazole for efficacy and safety of treatment for fungal diseases. 100 µL of plasma / serum is needed. An isocratic chromatographic program through Luna C8 column was carried out for 2 minutes. The assay yields linear results up to 75 mcg/ml. Recovery was 103%, 105% and 102%, respectively. Intra-day imprecision was 3.4%, 2.7%, and 3.9%, and inter-day imprecision was 4.3%, 4.7%, and 3.8%, respectively. Method comparison was carried out by comparing 20 patients results with HPLC method. The agreement was excellent with a correlation coefficient R=0.9956.
LC/MS/MS Analysis of THCA and THCA Glucuronide in Urine
A fast, quantitative method eliminating the hydrolysis, extraction and derivatization performed in traditional gas chromatography mass spectrometry (GC/MS) analysis of 11-nor-9-carboxy-Ä9-tetrahydrocannabinol (THCA) was developed. Diluted urine samples were analyzed by ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC/MSMS) on an Agilent 1290/6490. THCA and THCA Glucuronide were monitored in positive multiple reaction monitoring mode. The linear range was 4-1000 ng/mL for both THCA and THCA Glucuronide with correlation coefficients exceeding 0.99. Inter/intra-assay precision did not exceed 9 % coefficient of variation and accuracy was within 16% of the target concentrations for the two compounds. The LC/MS/MS method and original GC/MS method correlated well; the former resolving some GC/MS peak interferences.
A high throughput automated sample preparation and analysis workflow for comprehensive toxicology urine screenings with LC/MS/MS
This presentation demonstrates the use of Disposable Pipette Extraction (DPX) for rapid, automated sample preparation of biological matrices for comprehensive LC-MS/MS screening. Combining Scheduled MRM™ algorithm and MS/MS spectral acquisition allows for compound identification with highest confidence based on mass spectral library matching.The automated workflow monitors extremely large panels of analytes;detecting and quantifying these compounds in a single run.
Advanced Automated MS/MS Library Searching for Compound Identification in Forensic Toxicology Samples
In this study we have evaluated the advantages of a new automated library searching software to improve the level of confidence for forensic toxicology screening by LC-MS/MS. The software enables the user to dynamically review the acquired data, search multiple libraries, create subsets of libraries, and adjust and refine search parameters.
Samples were analyzed using a hybrid linear ion trap-triple quadrupole system, and a hybrid quadrupole-time-of-flight instrument. MS/MS spectra were searched against an AB SCIEX Forensic Drug Spectral Library comprised of over 1250 compounds. The data processing was performed with the LibraryView™ software equipped with two library search algorithms. Both targeted and unknown drugs and metabolites were identified in the samples with high level of confidence based on MS/MS library searching results.
High confidence time of flight (TOF) screening data: just add fragment(s)
Recent development in TOF technology has provided with an ability to incorporate product ion exact mass (EM) in screening. MSE functionality enables the acquisition of both low energy (precursor ion) and high energy (product ion) data in single rapid screen to help with unambiguous identification of detected compounds. We compared TOF screening with results obtained using traditional selected reaction monitoring (SRM) experiments. Samples were screened against a targeted compound database for precursor ion EM and retention times to obtain positive hits which were further classified using product ion EM. Generally precursor ion EM TOF experiments showed good agreement with SRM, but for oxycodone a confirmatory product ion EM was necessary to prevent false positive results.
Transferability of a mass spectral library between a nominal mass and high resolution instrument
In this study, we investigate whether a nominal mass library can be used effectively with a high resolution instrument. We generated a high resolution mass spectral library using an AB Sciex TripleTOF®5600 System and compared its performance to that of our pre-existing, nominal mass library generated with an AB Sciex 3200 QTrap. The ability of each library to identify drugs in a set of 100 patient samples was investigated. Overall, the nominal mass library performed very similarly to the high resolution library in identifying compounds.
Development and validation of a LC/QToF Mass Spectrometry method to quantify buprenorphine and its metabolite in urine
Quantitation of buprenorphine (BUP) and its metabolite, norbuprenorphine (NorBUP), in urine by Waters Acquity UPLC/Xevo G2 QToF mass spectrometry was developed and validated by this lab. All analytical parameters: intra- and inter-assay precision, LOD/LOQ, linearity, accuracy/recovery, stability, dilution, interference, and ion-suppression were investigated and met the established acceptance criteria. The method was compared to the reference lab’s LCMSMS method. The correlations for BUP/NorBUP were y=0.9272x+0.9239, R2=0.9872 and y = 1.003x -4.9, R2 =0.9889. We demonstrated that this LC accurate mass time-of-flight mass spectrometry method is accurate, stable, and robust; suitable for routine urine drug of abuse confirmation testing.
Design of Experiments: A Powerful Technique for Developing LC/MS Methods
Traditional method development usually involves changing one factor at a time while holding everything else constant. This can be a laborious, time consuming process that often relies on luck or tenacity to discover the optimal conditions. A more efficient approach is to use Design of Experiments (DoE), a statistical technique for planning, conducting and analysing experimental data. DoE allows the important parameters in an assay to be quickly and efficiently optimized, producing higher quality methods in less time. This presentation will provide an introduction to the use of DoE for optimizing clinical LC/MS assays.
A turbulent flow liquid chromatography-tandem mass spectrometry method for the quantitation of hydroxychloroquine in serum and whole blood
Hydroxychloroquine (HQ) is used in the treatment of a variety of immune-mediated diseases. We developed a quantitative HQ method based on turbulent flow liquid chromatography-tandem mass spectrometry. Our method is linear from 15.7 to 2000 ng/ml, has total precision of 10%, and shows recovery within ±15%. Carryover for serum specimens is minimal; it is also acceptable for whole blood specimens within the range of 15.7 to 1000 ng/ml. Comparison of 85 paired whole blood and plasma specimens yielded a good correlation (r = 0.80), suggesting that plasma may potentially be used in patient compliance monitoring.
Detection of peptides in plasma with differential ion mobility spectrometry coupled to mass spectrometry
Biological samples require separations to simplify mass spectra for optimized analyte detection. Chromatographic and electrophoretic techniques are commonly utilized, but these separations can add minutes to hours to MS analysis times. Non-MS techniques such as immunoassays can be fast, but method development for a given analyte can be time-consuming and specificity remains questionable for some assays. The use of differential ion mobility spectrometry (DIMS) as a filter prior to MS analyses adds only milliseconds to MS analysis times. Here, we show that DIMS-MS provides a significant improvement in detection limits for peptides in plasma as compared to MS alone.
Internal Calibration: Toward “Random Access” LC/MS Analysis.
Quantitative LC/MS usually requires the analysis of multiple calibrators before the concentration of analyte in a sample can be determined. This consumes valuable instrument time & resources, can delay reporting results and limits LC/MS to a batch mode of operation. We will describe a new method of calibration (“internal calibration”) that can overcome these limitations by using calibrators that are added directly to a sample. A precise and accurate quantitative result can be obtained in a single analysis, thereby allowing the possibility of random access operation.
LC-MS quantitative screening for analysis of 18 anabolic steroids in oral fluid using MS2 spectra data collected with QExactive Orbitrap Mass Spectrometer
Processed by LLE, oral fluid samples were analyzed with 15 min LC method. The QExactiveTM Orbitrap equipped with APCI probe collected MS2 spectra using resolution of 35K. Two fragments from each analyte’s MS2 spectrum were selected for quantitation and confirmation.
The 16 of 18 compounds was quantified with LOQ of 1 ng/mL. Inter assay precision was within 20% and inter assay precision was within 25%. Percent recovery in spiked oral fluid ranged 80-127%.
Prediction of the hematocrit of dried blood spots via potassium measurement on a routine clinical chemistry analyzer
A major challenge in the use of dried blood spots (DBS) for quantitative bioanalysis is the impact of the hematocrit, as strongly deviating hematocrit may significantly impact DBS-based quantitation. We set up a rapid and simple protocol to extract potassium from 3-mm DBS punches, followed by quantitation using a routine clinical chemistry analyzer. The extracts’ potassium concentrations were used to calculate the approximate Hct of the blood used to generate DBS. The procedure was successfully validated (linear calibration range 0.19-0.63) and applied to DBS of patient samples, followed by Bland-Altman analysis, demonstrating a good correlation between ‘predicted’ and ‘actual’ hematocrit.
Analysis of polyfluoroalkyl substances and bisphenol A in dried blood spots by liquid chromatography tandem mass spectrometry
Single dried blood spots (DBS), from the New York State newborn screening program were used to measure PFOS, PFOA and BPA levels in baby’s blood shortly after birth. The typical volume of blood in a 16-mm diameter DBS is approximately 50 uL. A liquid-liquid extraction of the DBS and HPLC-MS/MS) negative ion ESI method was used to detect and quantitate the two polyfluoroalkyl compounds and BPA using 13C-labeled internal standardization. PFOS and PFOA were detected in 100% of the 200 DBS specimens analyzed. BPA was found in 86% of the specimens at concentrations ranging from 0.2 to 36 ng/mL..
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Copy Number Alterations in DNA Repair/Repair-Related Genes Identify Disease-Specific Biomarkers with Potential Clinical Relevance to Hematologic Malignancies
Microarray copy number alterations (CNAs) were analyzed for 155 DNA repair-related genes in 425 cases representing diverse hematologic malignancies. Expected disease associations in RB1, TP53 and ATM were seen. Newly defined relationships included deletion of ERCC5 seen in B-ALL, with co-deletion of ERCC5 and LIG4 confined to NHL. CNAs specific to NHL (GTF2H4, POLQ, REV1L, FANCL, BRCA2, and NUDT1), AML [UBE2V2, APTX, APTX, MGMT and RPA2, with the latter found only in cases with translocations (PML/RARA or MLL)] myeloma (ALKBH3) and MDS (ALKBH5, TOP3A) were seen. Other CNAs occurred in multiple disorders (NEIL1, PMS2, ENDOV, CHEK2, and HELQ). The results reveal disease specificity for CNAs in DNA repair related genes. Further study is warranted to validate these alterations as disease specific diagnostic/prognostic biomarkers.
Detection of “oncometabolite” 2-hydroxyglutarate by Magnetic Resonance Analysis as a Biomarker of IDH1/2 Mutations in Glioma
Somatic mutations in isocitrate dehydrogenase (IDH)1 and 2 have been detected in a subset of gliomas, rendering these tumors with elevated levels of “oncometabolite”, D-2-Hydroxyglutarate (2HG). We demonstrate the detection of 2HG as a biomarker of this subset of gliomas ex vivo using a magnetic resonance (MR) approach. This study thus highlights the feasibility of using MR detection of 2HG for the diagnosis and classification of IDH1/2 mutation-positive brain tumors and opens up the possibility of performing analogous non-invasive MR-based imaging and spectroscopy studies directly in humans in the neuro-oncology clinic.
Mass Spectrometry Used To Define Plasma Protein-Based Classifiers That Discriminate Patients With Colon Polyps or Adenomas As Compared to Colonoscopy
Plasma samples were collected from patients undergoing colonoscopy and their plasma protein profiles were quantified via LCMS. 100 matched samples were used in this analysis. Approximately 150,000 molecular features were observed in at least 50% of the samples in at least one group. Ten rounds of 10-fold cross validation using Elastic Net feature selection, top 100 feature ranking, and SVM classifier assembly were performed to determine potential classifier performance. The average cross-validated AUC was 0.92 ± 0.12, indicating a high degree of predictive performance. These results demonstrate the feasibility of blood-based protein tests to help manage colonoscopy screen compliance.
ApoE typing from small amounts of blood samples
In clinical testing, ApoE levels in blood are determined by immunoassay. Combining ApoE immunoassay reagents with mass spectrometric analysis, we attempted to resequence ApoE protein from small amounts of sera for typing. Most ApoE mutant isoforms, such as ApoE2 and ApoE4, involve substitutions of lysine and arginine residues, and on trypsin digestion will result in fragments with a larger mass difference than a single amino acid substitution of other amino acids. ApoE types from sera, including heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly with the APOE genotyping results in each of the subjects. ApoE types from sera may be a novel clinical test that is performed using blood samples remaining from those collected for routine clinical tests and may enable retrospective studies using preserved body fluids.
Immuno-MALDI Assay for Plasma Renin Activity: Proof of Principle for a Translatable Proteomic Assay without Chromatography
The renin-angiotensin-aldosterone system (RAAS) plays an essential role in maintaining arterial blood pressure. Determination of plasma renin activity (PRA) is essential for screening and diagnosis of primary aldosteronism and other derangements of the RAAS pathway. Clinical laboratories have traditionally used radioimmunoassays (RIAs) for angiotensin-I in the determination of PRA. Recently however, there has been pressure to discontinue radioisotope-dependent assays in favour of stable isotope techniques. We have developed a MS approach for the determination of plasma renin activity utilizing immuno-MALDI (iMALDI) MS and have compared with both RIA and LC-MS/MS. The iMALDI method has the benefit of requiring no chromatography.
Differential solublization method to extract low-molecular-weight proteins/peptides for successful serum SRM analyses
Selected reaction monitoring mass spectrometry (SRM-MS) has been used to measure low abundance proteins/peptides in serum/plasma, owing to its high sensitivity and selectivity. However, the presence of highly abundant proteins in serum/plasma and the huge dynamic range of serum proteins/peptides make SRM analysis challenging. We previously developed differential solubilization (DS) method to extract low-molecular-weight proteins/peptides in serum with good reproducibility. Here we present data indicating that the DS method is useful as pretreatment for serum SRM analyses for biomarker validation.
Validation of Arsenic Speciation Method for Human Urine Using HPLC-ICP-MS
Arsenic is carcinogenic depending on its chemical nature and oxidation states. Quantitative determination of inorganic and organic Arsenic species in clinical samples could be used as potential biomarker for its toxic exposure. Arsenic can be ingested in form of one or more species. Arsenic species can be metabolized into several different inorganic and organic forms, including As-III and As-V, and the methylated species such as Monomethyl arsonic acid (MMA), and Dimethyl arsinic acid (DMA) or Arsenobetaine (AsB) and Arsenocholine (AsC) which most likely stay unaffected in the body. We have developed an analytical method for quantitative determination of six Arsenic species in clinical samples using isocratic LC coupled with ICP-MS. The results showed that each of the six Arsenic species was well separated and LOD of each of the species was approximately 1.0 ppb.
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