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Use of Micro-Liquid Chromatography/Tandem Mass Spectrometry Method to Assess Diurnal Effects on DHEA
Our findings showed a significant diurnal fluctuation for DHEA similar to that observed by Stolze et al. The morning values were higher than evening. The mean at 08h00 was 566ng/mL and at midnight 230ng/dL, the mean decrease was 60%, P= <0.001 and n=19. We demonstrated large DHEA diurnal concentration fluctuations requiring strict adherence to time of the blood draw and use of separate time dependent reference intervals.
Hidden Aspects of Electrospray Ionization and Quantitation of 25 Hydroxy Vitamin D: Lessons Learned
In LC/MS analysis of 25 OH Vitamin D, dehydration is the major side reaction. Comparing acetonitrile to methanol, acetonitrile does not support hydrogen bond formation; therefore, proton-induced water elimination in-source becomes a major side-reaction. MeOH, in contrast, supports hydrogen bond formation with the 25(OH)D3 hydroxyl groups. Since droplets evaporation process can vary with mass spectrometer hardware design, ratio between intact [M+H]+ and dehydrated precursor can be unpredictable. We also noticed that degree of dehydration is concentration-dependent. Chromatographic separation between analyte and its deuterated internal standard might cause different levels of dehydration and resulted in quantitative error.
Analysis of Urinary Free Catecholamines and Metanephrines by Tandem Mass Spectrometry: Validation and Implementation in a Clinical Laboratory
Analysis of both urinary free catecholamines and metanephrines are used in the diagnosis of phaeochromocytoma. This poster describes the development, validation and implementation of a new SPE-LC-MS/MS (solid phase extraction – liquid chromatography - tandem mass spectrometry) method for urinary free catecholamines and advantages of this method compared to the HPLC-ECD method previously used. The poster also demonstrates the capability of this new method to incorporate both catecholamines and metanephrines in a single analysis.
Determination of Etonogestrel in Blood Plasma by High Performance Liquid Chromatography - Mass Spectrometry
Purpose. Development and validation of methods for determining plasma concentrations of etonogestrel (3-keto desogestrel).
The method used - HPLC/MS. Analytical equipment - liquid chromatograph and a mass spectrometer with a triple quadrupole. Chromatographic conditions: analytical column C18 (75*2,1 mm, 3.5 µm), flow rate 0.4 ml/min, eluting with a gradient system (acetonitrile :water). Detection mode is MRM. Sample preparation is a liquid-liquid extraction with diethyl ether was concentrated and redissolved in 50% acetonitrile.
Results. The recovery rate is 77%. Calibration curve is linear in the range 40-4000 pg/ml. The LLOQ is 40 pg/ml and LOD is 20 pg/ml.
Conclusions. Etonogestrel - active substance in the composition of many last-generation oral contraceptives. The method developed by HPLC/MS is sensitive enough to determine the picogram of etonogestrel in human plasma.
Sex Steroid Hormone Stability: Gel versus Non-gel Tubes
We examined the stability of four sex steroids in serum stored at 4°C for up to 5 days. Serum collected into gel and non-gel containing tubes was analysed by LC-MS/MS for testosterone, androstenedione, 17-hydroxyprogesterone (n=20); and estradiol (n=27). Differences in measurand concentration at day 0 and following storage (day 1 and day 5) were evaluated for statistical / clinical significance. Androstenedione concentrations in gel-containing tubes were reduced by an average of 14% by day 5 (p<0.001), determined to be clinically significant. In addition, estradiol and testosterone concentrations were significantly increased in plain serum tubes by day 1 (p<0.01).
Fat Soluble Vitamin Detection in Human Serum and Plasma by LC-MS/MS Using Biotage ISOLUTE SLE+ 96-well Plate Extraction
Traditionally HPLC methods have been used to analyse serum vitamins A, E and K, which involves extensive sample clean-up and long chromatographic run times. The aim of this study was to develop a single LC-MS/MS method for the determination of these fat soluble vitamins. Relatively low serum concentrations, the lack of availability of some authentic standards and occurrence of interfering lipids provided significant hurdles to method development. Here we present a simple and rapid method for the extraction and analysis of fat soluble vitamins in human serum and plasma, and the investigation of possibilities offered by MRM3 transitions for improved selectivity and sensitivity either as a front-line test or as a confirmation method.
Interferences of Blood Collection Tubes in the Measurement of Androgen Concentrations After Administration of a Novel Androgen Ester
Our aim was to determine the most appropriate blood collection tube for measurement of DMAU and DMA in blood after oral administration of Dimethandrolone Undecanoate (DMAU), a new androgen being developed as a potential male hormonal contraceptive. In vitro experiment showed that when venous blood was transferred into six types of blood collection tubes and known amounts of DMAU were added to each tube, NaF+Oxalate, NaF+EDTA and P800 tubes showed the least interference in measuring DMA when kept for 30 min at 4C. In vivo experiment in 6 subjects showed that when serial blood samples were collected over 24h after oral administration of DMAU and kept for 30 min at 4C, DMA level differences between the different blood collection tubes were within the imprecision of the assays. The pronounced interference seen in vitro was not reflected in samples collected after oral administration of DMAU.
Method Validation for Quantitation of Testosterone Calibrators: A Modification of Reference Measurement Procedures
Development of accuracy based calibrators in biological matrices for clinical diagnostic applications requires reference measurement procedures with high accuracy and sensitivity. Testosterone presents a unique challenge with regards to the wide range of endogenous levels across female, male and age based patient populations.
We wish to present our efforts towards validating a method for quantitation of Testosterone in calibrators from 0.020 ng/mL to 20 ng/mL in serum by modifying a valid reference measurement procedure. The 1000 fold range of these calibrators in serum presents unique challenges towards designing a method that will accurately measure Testosterone.
Data presented will address various elements of the method validation such as precision and accuracy, linearity, limit of quantitation, recovery, matrix effects, extract stability, tracebility.
Profiling Thyroid Hormones by LC/MS/MS Analysis in Various Preclinical Species and Humans
Compounds in drug development may enhance the metabolism or clearance of thyroid hormones in animal models, triggering a sequence of toxicity events, and accurate measurements of thyroid hormones are important to aid in understanding the response. In this project, we utilized a validated LC/MS/MS method to profile the five thyroid hormones (T4, T3, rT3, 3,3’-T2, and 3,5-T2) in serum samples from various animal species and humans. These results enabled us to establish the distribution in normal animals and evaluate the influence of different factors, such as gender, on the profile.
High Serum Lipids Cause Erroneously Low Total 25-OH Vitamin D Levels by a Chemiluminescent Immunoassay.
The DiaSorin Liaison® chemiluminescent immunoassay (CIA) for total 25-OH vitamin D (25-OH vit D) has begun to replace mass spectrometry as the method of choice in many clinical laboratories. This work examined the performance of this assay in routine clinical practice. A total of 153 paired samples were analyzed by the CIA and an in-house tandem mass spectrometry assay. While the DiaSorin showed acceptable performance overall, substantial negative bias was observed in samples with elevated lipids (total cholesterol, trigycerides and high density lipoprotein (HDL)); mixing studies confirmed the CIA underestimates 25-OH vit D levels in samples with elevated cholesterol. Clinical laboratorians should be aware that the CIA can yield erroneously low 25-OH vit D levels in the presence of elevated lipids.
Sensitive Measurement of Plasma 1,25-Dihydroxyvitamin D2&3 (125DHVD) via LC-MS/MS: A Simple SPE Sample Preparation and MS Sensitizing Derivatization Process
The physiologically active forms of Vitamin D, 1,25-dihydroxyvitamin D2/3 (DHVD) are important biomarkers for a number of disease states. With recent advancements in LC-MS/MS technology, reliable measurements of DHVD are still challenging due to low abundance and poor ionization capacity, even with the aid of PTAD, a Cookson type derivatization reagent. There were reports of quantification of DHVD with a combination of immuno-affinitive capture and PTAD derivatization. In this presentation, we will report quantification of plasma DHVD with a simple SPE and quick derivatization with an in house developed novel crown-ether based PTAD reagent.
A Total and Free Testosterone Method that Utilizes Automation and a Novel Microdialysis Plate to Achieve Efficient Workflow in a Clinical Laboratory
Isotope dilution equilibrium dialysis (IDED) has long been considered the gold-standard method for free testosterone measurement. However, IDED requires a high sample volume and is labor intensive, making it a challenge for clinical laboratories processing hundreds of samples daily. We combined two necessary components of comprehensive testosterone testing, the measurement of the total testosterone concentration by LC-MS/MS and free testosterone by isotope dilution equilibrium dialysis, into a single automated method with the use of a microdialysis plate and robotic liquid handler.
Evaluation of Measurement for Serum 3-epi-25-hydroxyvitamin D3, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 Using UPLC-MS/MS in a Korean Reference Laboratory
We evaluated the performance of Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) method to measure serum 3-epi-25-hydroxyvitamin D3 (epi-25(OH)D3), 25-hydroxyvitamin D3 (25(OH)D3) and 25-hydroxyvitamin D2 (25(OH)D2). Using Kinetex XB-C18 column (Phenomenex, USA) and isocratic methanol/water (77.5/22.5, v/v) flowing at 0.25 mL/min, run time was 13 minutes/sample. TQD triple quadrupole mass spectrometer (Waters, USA) with MRM transitions, MSMS Vitamin D kit (PerkinElmer, Finland) and PTAD derivatization method were used. Intra- and inter-run precisions were less than 15 %. The carryover was 0.10%, -0.27% and 0.10%, respectively. There was no ion suppression. The linearity was good with R2>0.9999. The peak of epi-25(OH)D3 was clearly separated from that of 25(OH)D3 in the XIC. The performance of UPLC-MS/MS for epi-25(OH)D3, 25(OH)D3, and 25(OH)D2 was acceptable.
High-sensitivity, High-Throughput Quantitation of Catecholamines and Metabolites in Urine by LC/MS/MS for Clinical Research
In this study, we demonstrated the analytical performance of two LC/MS/MS methods that quantitated urinary catecholamines and their various metabolites for efficient clinical research. One method involved acid hydrolysis of urine samples followed by solid phase extraction to quantitate deconjugated norepinephrine (NE), epinephrine (E), dopamine (DA), metanephrine (MN) and normetanephrine (NMN). The other method was a dilute-and-shoot method to quantitate free MN, NMN, vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA). Both methods had quantitative range to cover biologically relevant concentrations and analysis time of less than 5 minutes.
High-sensitivity, High-Throughput Quantitation of Catecholamines and Metabolites in Plasma by Automated WCX-SPE Coupled to LC/MS/MS for Clinical Research
Plasma levels of catecholamines and their metabolites, namely norepinephrine, epinephrine, dopamine, metanephrine and normetanephrine, are of significant relevance in clinical research in the field of endocrinology and disease screening. Fast, high-sensitivity analytical system is warranted. To this end, we developed an optimized method for simultaneous determination of these compounds using Shimadzu LCMS-8060, achieving complete chromatographic separation and low pg/mL LLOQ in plasma within 5 minute analysis time. Sample preparation was automated by Biotage Extrahera using WCX-SPE in 96-well format. Pre-validation study was performed, including demonstration of quantitative consistency with HPLC-based predicate device.
Development and Validation of LC-MS/MS Method for Determination of Testosterone in Serum
We developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for serum total testosterone. The accuracy, imprecision, limit of quantification (LOQ), and linearity of LC-MS/MS method were validated and compared with enzyme chemiluminescence immunoassay(ECLIA) and radioimmunoassay(RIA) methods. The accuracy, precision, LOQ, and linearity were excellent. Especially the accuracy for LC-MS/MS method was better than ECLIA and RIA methods. The LC-MS/MS method showed excellent performance compared with ECLIA and RIA.
Generic Sample Preparation Methodology for the Analysis of Steroid Hormones by LC-MS/MS for Clinical Research
Care must be taken when developing steroid hormone analytical methods for clinical research, to limit interferences which affect accuracy and precision. MS/MS provides a way to selectively differentiate between these hormones, while chromatography can provide separation of isobaric species. However, sample preparation is the key in providing LC-MS/MS analytical sensitivity by reducing matrix effects. We have developed an LC-MS/MS approach for the analysis of a wide range of steroid hormones. Extraction analytical sensitivity and phospholipid removal capability have been explored, which has shown that for a generic approach to steroid analysis, Oasis® PRiME HLB SPE is the most favourable option in providing optimal LC-MS/MS sensitivity.
For Research Use Only, Not for Use in Diagnostic Procedures.
The AA2-Ratio: Improved Screening for Primary Aldosteronism in Hypertension
The Aldosterone-to-Angiotensin-II-Ratio (AA2-Ratio) is a novel LC-MS/MS based high-throughput test for primary aldosteronism (PA), a severe but curable form of hypertension. The test can be applied to standard collected serum samples and does not interfere with anti-hypertensive therapies including ACE inhibitors. These are major advantages over currently employed diagnostic procedures, building the basis for the implementation of the AA2-Ratio into clinical practice of therapeutic management of hypertension.
Metanephrines in Urine by Liquid Chromatography Tandem Mass Spectrometry
Neuroendocrine tumors, such as pheochromocytomas or paragangliomas, represent a clinically and etiologically diverse group of disorders. These tumors exhibits increased production of catecholamines and their metabolites and therefore quantification of biogenic amines is used for their diferentional diagnosis. The aim of this work was to develop the LC-MS/MS method based on hydrophilic interaction liquid chromatography which provides possibility for quantification of wider group of analytes related with neuroendocrine tumors. Method for quantification of metanephrines in urine fulfilled validation requirements.
Extraction of Urinary Hormone Metabolites from Urine Using Supported Liquid Extraction Prior to HPLC-MS/MS Analysis
Estrogen, androgen, and glucocorticoid metabolism can be used to assess overall hormonal balance during hormone therapy. Urine is the recommended testing matrix for quantification of primary estrogen levels as well as secondary estrogen metabolites when monitoring overall hormone balance, therapy, and detoxification; non-invasive collection allows for sampling over a 24-hour period, providing insight to circadian rhythm. Since many samples are generated for a single patient in one day, a fast and robust testing protocol is needed during clinical testing. Here we demonstrate a rapid and reliable sample preparation method using SLE+ to extract a suite of 30 hormone analytes from a hydrolyzed urine matrix. LC-MS/MS analysis in a single injection shows that matrix effects are eliminated by the SLE+ protocol and that analyte recovery and sensitivity are adequate for clinical interpretation.
Accuracy Evaluation of Routine Vitamin D Immunoassays Compared with LC-MS/MS in Pregnant Women and Intensive Care Unit Patients
Serum samples of 160 healthy controls, 50 pregnant women, and 50 intensive care unit (ICU) patients were collected. Total 25(OH)D levels of each sample were measured by LC-MS/MS and three automated assays, including ADVIA Centaur (Siemens, Germany), Elecsys (Roche, Switzerland), and Architect i200SR (Abbott, US).The vitamin D results of all three routine immunoassays showed poor concordance compared with those of LC-MS/MS in ICU patients than those in healthy controls. In pregnant women, the vitamin D results of ADVIA Centaur showed the high positive bias compared with vitamin D values measured by LC-MS/MS. In conclusion, standardization of each immunoassays is essential for clinical use to estimate vitamin D status. Moreover, for the exact vitamin D status evaluation in ICU patients and pregnant women, using LC-MS/MS measurement would be recommended.
Quantitation of Serum 17-hydroxyprogesterone, Testosterone, Dehydroepiandrostenedione and Androstenedione Using UPLC-MS/MS
Quantitation of steroid metabolites including 17-hydroxyprogesterone (17OHP), testosterone (TTST), androstenedione (ANDRO) and dehydroepiandrostenedione (DHEA) is useful in the diagnosis and clinical management of adrenal disorders like congenital adrenal hyperplasia. We have developed a UPLC-MS/MS method that can be used for simultaneous measurement of 17OHP, TTST and ANDRO. DHEA was also quantified using the same sample preparation and chromatographical method but with the requirement of relatively higher sample volume. Since our method does not need a derivatization step, complete automation of the sample extraction process using a liquid handling system is feasible in the future.
Development and Validation of a LC-ESI-MS/MS Quantification Method of 25-hydroxyvitamin D2&D3 and of their C3-epimer
Development and validation of a LC-ESI-MS/MS quantification method of 25-hydroxyvitamin D2&D3 and of their C3-epimer (25-OH-D) has been performed to overcome the issue with the overestimation of vitamin D levels cause by the occurrence of C3-epimers. Briefly, samples were deproteinized using ZnSO4 and MeOH, extracted using heptane, dried down, and finally resolubilized in the mobile phase (MeOH 68 %/ formic acid 0.1 %). Liquid chromatography was performed using isocratic gradient with a pentafluorophenyl column. CV and Bias were respectively inferior to 20 % and to 15 % for 3 nM of 25-OH-D. Method comparison was performed with another lab using an LC-ESI-MS/MS technique and we have obtained a slope and intercept of respectively 0.998 and 0.05 nM for total 25-OH-D. This LC-ESI-MS/MS method allows quantification of 25-OH-D2&D3 and of both C3-25-OH-D2&D3 which a few labs offer.
Tackling the Interference Problem for Estradiol Analysis by LC-MS/MS, Using Differential Ion Mobility Spectrometry
The measurement of low pg/mL concentrations of estradiol in serum and plasma, by LC-MS/MS, requires a highly sensitive method. To be sure, the sensitivity challenge is significant. Nevertheless this requirement can be readily addressed with the use of high performance tandem mass spectrometry instrumentation, larger sample volumes, and extensive sample preparation. However, those familiar with this analysis will be acutely aware of the more urgent selectivity challenge posed by the presence of ubiquitous, low-level, non-specific chromatographic interferences that frequently mask the presence of the target estradiol peak. In the work presented here, differential ion mobility spectrometry (DMS) has been employed to enhance the selectivity of the analysis, while enabling the measurement of <1 pg/mL estradiol in serum, using a simple one-step liquid liquid extraction sample preparation.
Steroid Hormones in Serum, a Simply, Accurate, Sensitive and Not Extractable Kit Ready to Use by LC-MS/MS
Alifax - Eureka Steroid Hormones in serum kit ready to use permits the simultaneous analisis of the most important steroids in a un single cromatography sample run (17-OH-Progesterone, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone sulfate (DHEAS), Androstenedione, Cortisol, 11-Deoxycortisol, Corticosterone, Aldosterone, Testosterone, Dihydrotestosterone, Androsterone, Estrone, Estradiol, Pregnenolone, 17-OH-Pregnenolone, Progesterone, 11-Deoxycorticosterone)after a simple matrix deproteinisation. To analyze correctly full panel in routine it's necessary to have a medium/high level mass QQQ detector coupled with an UHPLC. Seven levels of serum calibrators and two levels of serum controls are available, covering clinical range concentration values.
Mass Spectrometric N-Terminal Sequencing of Peptides Using a Bacterial Aminopeptidase
Aminopeptidases catalyze the hydrolysis of peptide bonds joining the N-terminal amino acid of any peptide to the next amino acid in the sequence. Processive aminopeptidase can be used to create a population of peptides of different lengths differing by one amino acid mass, so that the entire population can be studied in a single mass spectrum, to determine the peptide’s amino acid sequence. Sometimes, structure present in the peptide can also interfere. In this study, we describe the use of a non-specific, processive, deblocking Bacillus subtillis-derived aminopeptidase (BsuAP) in a reaction.
TEst
Test.
Accurate and Precise Sample Introduction at the Micro-Level: A New Approach to Routine, High Throughput Analysis for Trace Element Quantification with ICP-MS
Particularly in clinical analysis, there is a growing demand for routine trace element quantification from samples of a very low volume. By pairing an advanced automation platform with an appropriate nebulizer/spray chamber it is now simple to reproducibly introduce liquid samples from the low microlitre level (< 5 µL) into the milliliter level to an inductively coupled plasma mass spectrometer (ICP-MS). Such sample introduction technology allows the analyst to work with greater confidence with precious samples and limited volumes with reduced or no compromise as to the analyte suite of interest and the number of replicate measurements. We report on the application of this technology to sample handling/introduction and measurement of trace element analytes, notably platinum, in human blood serum – a routine test in toxicology studies associated with platinum based chemotherapies.
Challenges of ICP-MS Method Development for Routine Clinical Analysis
We developed a quantitative method for analysis of As, Cd, Hg and Pb in clinical whole blood samples using ICP-MS. We evaluated previously published strategies for ICP-MS internal standard selection, acid wash concentration and matrix-matching calibrators. Internal standardization can help correct for non-spectral interferences. Rhodium and Iridium performed optimally for all four analytes. Wash cycle duration proved more effective than acid rinse concentration for reducing Mercury memory effects and preventing carryover. We showed that four commonly used whole blood anticoagulants can be suitable for matrix-matching calibrators. Development of analytical methods for ICP-MS can be challenging and several factors must be considered in the validation phase.
Liquid Chromatography Mass Spectrometry Applications in a Newborn Screening for Mucopolysaccharidoses
Mucopolysaccharidoses (MPSs) are progressive disorders where early diagnosis allows early treatment that provides better outcomes and prognosis. We have developed a pilot study using dried blood spots (DBS) from 2640 random newborn samples and 13 newborn MPS samples. All DBS were evaluated by liquid chromatography tandem mass spectrometry to determine levels of heparan sulfate (0S, NS) and keratan sulfate (mono-KS, di-KS and ratio di-KS/total KS). Cutoffs were established for HS to diagnose MPS I, II and III. HS-0S showed 98% specificity and 77% sensitivity and it is a potential biomarker for newborn screening of MPSs.
An Improved Tandem Mass Spectrometry Method for GALC Enzyme Assay and Psychosine Analysis in Dried Blood Spots for Identification of Krabbe Disease
Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the GALC gene. Deficiency in GALC result in an abnormal accumulation of a highly cytotoxic metabolite, called psychosine. Our lab has developed mass spectrometry methods that are being used in newborn screening (NBS) of lysosomal storage diseases. Now we focus on high accuracy GALC enzyme assays using purified white blood cells from patients suspected to have Krabbe disease. Psychosine measurement in dried blood spots and cerebrospinal fluid were also developed and could serve as a second tier assay. The methods should reduce the false positive rate of NBS and help determine disease severity and monitor Krabbe patients.
Preliminary Results from the Slovenian Expanded Newborn Screening Pilot Study
Newborn screening programme in Slovenia currently includes only two diseases, phenylketonuria and congenital hypothyroidism. Last year a pilot study of expanded newborn screening for inborn errors of metabolism using tandem mass spectrometry (MS/MS) started. 10000 dried blood spots from newborns were analysed retrospectively. Newborns with highest elevations of measured analytes were immediately investigated and three newborns with inborn errors of metabolism were found (not counting hyperphenylalaninemia/phenylketonuria); one case of VLCAD, a case of 3-MCC deficiency and a case of GA1. Based on these results the cumulative incidence of inborn errors of metabolism (detected by MS/MS) is high in Slovenia. Follow-up tests on selected newborns to set the cut-off values for chosen disorders are ongoing.
Newborn Screening Tests for Metabolic Disorders Using Tandem Mass Spectrometry in Korea-Report from One Laboratory
We summarized the data of newborn screening tests in EONE Laboratories from January 2013 to september 2015, and compared the results with the previous reports in Korea. A total of 155,331 dried blood specimens (DBS) were analyzed for 11 amino acids and 14 acylcarnitines using MS/MS (API 2000, AB Sciex, Canada).
0.6% of all screened samples were positive in the initial screening test. We recommend the second screening test to confirm the positive marker for these samples. Of these, 976 repeat samples were available for retest. After the second screening test, one hundred eighty five samples were positive again; 92 positive samples for amino acids, with phenylalanine being the most common; and 93 positives for acylcarnitines, C5-acylcarnitnine as the most common. Regarding positivity rate and a spectrum of abnormal analytes, the results were similar to those reported previously.
Evaluation of Automated Data Processing of Acylcarnitines and Amino Acids in Flow Injection Tandem Mass Spectrometry Analysis Using a Meta-calculation Software
The research by Flow Injection Tandem Mass Spectrometry provides large quantities of information. The main advantage include no-chromatographic separation, rapid analysis time, low solvent consumption, and identification and quantitation of a large number of target analytes using their unique selected reaction monitoring transitions from precursor ions to product ions. We evaluated a meta-calculation software for automatic data processing. A total of 18 acycarnitines and 12 amino acids were evaluated for each sample. Among 280 samples evaluated, a total of 8400 calculations of concentrations were processed. The manual transcription errors were eliminated, and processing time was improved from hours to minutes.
For research use only. Not for use in diagnostic procedures.
The Role of Specimen Handling Time on the Interpretation of Plasma Acylcarnitine Profiles for the Diagnosis of Inborn Errors of Fatty Acid Metabolism
Analysis of plasma acylcarnitine profiles plays an important role in the diagnosis and management of patients with inborn errors of fatty acid metabolism. A pattern of elevated long-chain acylcarnitines (C16 to C18:2-carnitine) is associated with impaired carnitine cycle function as well as defective long-chain fatty acid metabolism. Blood collected from 3 subjects was immediately processed (spun, plasma separated, frozen) or left at room temperature for 2, 4, 6, 8, 24, or 48 hours and subsequently processed and analyzed by FI-ESI-MS/MS. Specimens left unprocessed for greater than 8 hours were associated with elevated C16 to C18:2-carnitine in all three subjects. No elevation of long-chain acylcarnitines was observed in plasma left at room temperature. Careful control of pre-analytical handling time is imperative for the correct interpretation of plasma acylcarnitine profiles.
Developed Method for Acylcarnitine Analysis in Serum Using LC-MS/MS as a Clinical Exam
Method for determination of acylcarnitine by LC-MS/MS was developed to improve existing techniques that are not suitable for the clinical diagnosis tests due to quantitative differences between analytical instruments and the lack of internal calibrator.
The validation of AC determination assay in serum was conducted and the intra- and inter-day repeatability, accuracy, linearity, recovery and precision were confirmed. The previously developed method was simplified and precision was significantly improved by addition of the calibrator.
We believe that this method has the potential to become standard method for clinical exam of positively screened infants or follow-up patients.
Detection and Direct Quantitation of Guanidinoacetate, Creatine and Creatinine in Human Urine by LC-MS/MS and Electrospray Ionization
The cerebral creatine deficiency syndromes (CCDS), inborn errors of creatine metabolism, include the two creatine biosynthesis disorders GAMT deficiency and AGAT deficiency), and the X-linked creatine transporter [SLC6A8] deficiency.
Specimens were prepared by diluting urine with ultrapure water. A minimum sample volume of 100 µL was used. The diluted sample mix was injected onto an Agilent 1200 Series HPLC system using a reverse-phase column. Analysis was performed by positive electrospray ionization using an Agilent 6410 triple quadrupole mass spectrometer.
Measurements of these three analytes in urine allow for the biochemical diagnosis of CCDS. The ability to measure all three analytes directly in the urine, with a very simple method of sample preparation, offers advantages over previous methods involving derivatization and indirect calculations.
Quantification of Glycosaminoglycans (CS, DS and HS) in Dried Urine Spots by UPLC-MS/MS
Dried urine spots (DUS) on filter paper are readily shippable in the mail to a distant laboratory for analysis. We have adapted a previously developed UPLC-MSMS method of analyzing the glycosaminoglycans (GAGs) CS, DS and HS, in liquid urine to extracts of DUS for this purpose. A fixed amount of d3- creatinine is added to a 2 cm diameter disk excised from a DUS, and creatinine and GAGs are co-extracted. An aliquot of the extract is analyzed for creatinine and the remainder is dried, then methanolysed to produce dimeric subunits derived from the principal GAGs. A mixture of isotope-labeled dimers, prepared from standard GAGs by deuteriomethanolysis, enables quantification by pseudo-isotope-dilution. This assay has sufficient sensitivity, precision and accuracy to reliably measure elevated urinary GAGs in DUS from patients with known or suspected mucopolysaccharidoses.
Quantification of Testosterone in Serum by Liquid Chromatography-tandem Mass Spectrometry
Androgens (such as testosterone) are natural hormones. They are important in sexual development in both men and women. In women, androgens are produced in small amounts by the ovaries and the adrenal glands. Similar to higher blood estrogen levels, higher amounts of androgens in the blood may be linked to an increased risk of breast cancer in women. An ultra sensitive quantitative analytical method is required for total testosterone in human serum to be able to measure the low levels found in women. Therefore, a method was developed on an Agilent 6495 Ion Funnel Mass Spectrometer to quantify and characterize testosterone underivatized in serum and to ascertain which method may be the most appropriate for laboratory use.
Study of Drug Resistance Changes in Ovarian Cancer Patients
Ovarian cancer is a serious and one of the top cancer diseases among women which cause more deaths compared to all other types cancers in women. It does not show any symptoms in the early stage and therefore it is very well known as the ‘silent killer’ and not many people will survive from this cancer. The current diagnostic strategies have several limitations and disadvantages. But the good news is that a drug called ‘Olaparib’ has been approved by FDA to treat ovarian cancer patients. This drug stops the activity of a protein called PARP in human body that helps cancer to grow very rapidly.
Integration of Genomics, Proteomics and Lipidomics for Biomarker Discovery of Esophageal Squamous Cell Carcinoma
It is gradually recognized that integration of the data from genomics, transcriptomics, proteomics and metabolomics may expand our knowledge to understand of disease. Recently, genomics sequencing and proteomics analysis to esophageal squamous cell carcinoma (ESCC) revealed several significant evidence related with tumor gene mutations as well as expression, such as APO clearly dis-regulated in ESCC. Herein, we proposed an approach that integrates genomics, proteomics and lipidomics to find how these lipid associated genes and proteins impact the lipids" abundance in serum during the development of ESCC. We selected the sera of health, mild/moderate/severe dysplasia and ESCC for lipidomics analysis by MSE, then combined proteomics and genomics data in publications for further analysis of lipid candidates during the ESCC development.
A Novel Liquid Chromatography Mass Spectrometry Method for the Analysis of Succinate: Fumarate Ratios in the Detection of SDHx-associated Tumours
Paragangliomas and pheochromocytomas are endocrine tumours associated with mutations of the Succinate dehydrogenase (SDH) gene. SDH catalyses the conversion of succinate to fumarate in the Krebs cycle. Therefore a mutation in the SDHx gene will result in an accumulation of succinate and decreased production of fumarate. We have developed a novel method for the analysis of these metabolites by liquid chromatography mass spectrometry. Increased succinate:fumarate ratios correlate with patients that have been confirmed to have SDHx mutations with immunochemistry staining. This method will aid in the early detection of patients at risk of developing SDHx associated tumours.
Ion Mobility Mass Spectrometry: Alternative Drift Gas Selection for Improved Separation of Isomers in Clinical Analysis
Ion mobility spectrometry (IMS) has been coupled with mass spectrometry to improve isomer separation capabilities without sacrificing time. To improve this potential, several strategies have been employed, including varying the drift gas environment. Although helium and nitrogen have been the traditional options, other drift gases have shown promise in improving resolution between isomers. This study will compare the most common drift gases, helium and nitrogen, to gases such as carbon dioxide, sulfur hexafluoride, and argon for their individual merits in improving separation of targeted clinical compounds, especially for Vitamin D metabolite epimers and steroid diastereomers such as testosterone and dehydroepiandrosterone.
Validating the Role of FraB in the Metabolism of F-Asn by Salmonella
Fructose-asparagine (F-Asn), an amadori product from food, has been found to play an essential role for Salmonella"s growth in the inflamed intestine. Determining the role of enzymes involved in F-Asn metabolism in Salmonella will facilitate the discovery of therapeutic targets. And quantification of F-Asn from mouse feces is necessary for the study of effects on Salmonella"s F-Asn metabolism in mice model. Here a Salmonella deglycatase, FraB is verified to catalyze the deglycation of F-6-phosphate-Asp producing glucose 6-phosphate and aspartate. Also the method to extract F-Asn from mouse feces is developed and isotope-labelled F-Asn is used for its quantification.
Uromics: Metabolomics in Urine for Seroquel®, Latuda®, and Haldol®
Metabolomic studies of drugs in the body customarily focus on blood samples to identify these compounds. Metabolomic studies in urine (e.g, uromics) are less common. Using an exact mass LC-QTOF instrument and enzymatic hydrolysis, as needed for confirmation, work in this lab has identified metabolites of a variety of antipsychotic drugs in urine that were either not previously identified in the literature or were known but thought to be minor metabolic pathways. This work will specifically report on advances in Seroquel®, Haldol®, and Latuda® monitoring demonstrating both new metabolites and increased importance of known metabolites.
Non-Targeted, Targeted or Semi-Targeted Screening in Clinical Metabolomics – Where to Start
Clinical Metabolomics usually has to deal with highly diverse sample sets in its search for distinguishing features with a potential as biomarker. Additional to a specific disease or treatment of interest a myriad of lifestyle and genetic factors influence the metabolic profile of each individual sample. Preanalytical and analytical variation in larger sample cohorts adds further variability. A semi-targeted screening of samples based on mass transitions covering known metabolic pathways can stratify (group) sample sets and identify outliers. The global/non-targeted screening could afterwards evaluate known or so far unknown metabolites with potential as biomarkers using smaller sample sets.
Separating Vitamin D2 D3, their 25-OH Metabolites and C-3 Epimers
The accuracy of current vitamin D measurements by immunoassays and LCMS has been questioned due to the overlapping LC peaks with identical m/z values epimers and isobars. To solve this problem, we have developed a new HPLC method to achieve baseline separation of vitamin D2/D3, their 25-OH metabolites and C3-epimers in one single run.
A Novel [15N] Glutamine Flux Using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases
A novel liquid chromatography mass spectrometry (LC-MS) method is developed to quantify glutamine-derived [15N] nitrogen flux into nucleosides and nucleobases. This method will be a valuable tool to identify the nitrogen flux derived from glutamine and it can be further adaptable for high throughput analysis of large set of DNA in a clinical setting.
Serum Metabolomics for Early Identification of Esophageal Squamous Cell Carcinoma Using High-Throughput UHPLC/Q-TOFMS Technique
A high-throughput untargeted metabolomics workflow is developed using an UHPLC/Q-TOFMS technique. The untargeted metabolic profiling of one sample can be completed within 12 minutes, which enables an analytical throughput of >100 samples per day. This workflow is used for the metabolic profiling of serum samples collected from esophageal squamous cell carcinoma (ESCC) subjects with the aim to discover potential metabolite biomarkers for early identification. This metabolomics study includes a total of 97 ESCC cases and 105 healthy controls (HCs). 16 differential metabolites (potential biomarkers) were identified and found to be disturbed in several metabolic pathways among ESCC donors, which may possess great potential for early identification of ESCC.
Analysis of Selected Carboxylic Acids and Amino Acids in Clinical Samples of Patients at Risk for Cardiovascular Disease
To identify novel biomarker for early identification of individuals at risk for future cardiovascular events, we performed non-targeted Gas Chromatography/Mass spectrometry-based (GC-MS) analysis on 1000 subjects at risk for CVD in a semi-quantitative fashion to screen for plasma small-molecule metabolites that predict risk for CVD. Among these metabolites, several mono-, di-, and tri-carboxylic acids presented a significant prediction for CVD risk. We are further developing a targeted Stable-isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method (LC-ESI-MS/MS) for the quantitation of carboxylic acids and amino acids, in order to demonstrate their clinical utility by analyzing them in a cohort study of 2000 subjects.
Metabolomic Profile Change in Type 2 Diabetes Revealed by Commercial Metabolomics Kit with Mass Spectrometry
We measured 188 metabolites including acylcarnitines, amino acids, biogenic amines, glycerophospho- and sphingolipids, and hexose with the AbsoluteIDQ p180 kit from Biocrates Life Science using Agilent HPLC 1260 and AB Sciex 5500 QTrap in five patients with T2DM and five healthy controls. Each 188 metabolites were compared by Mann-Whitney test. We found eighteen statistically different metabolites, six metabolites increased and twelve metabolites decreased in patients with T2DM. In addition, the patients with T2DM and healthy controls were separated with little over-lap using principal component analysis. Especially branched chain amino acid, leucine, isoleucine and valine were increased in patients with T2DM.
The Application of Ion Mobility Mass Spectrometry to Lipidomics – a Demonstration of Instrumental Capabilities for a Diabetic Mouse Model
The use of ion mobility coupled with high resolution LC-MS/MS has been applied to investigate the lipidomic changes in blood plasma for a mouse model of type 2 diabetes. The ability to separate ions across four dimensions (retention time, mass to charge ratio, fragmentation, ion mobility) has allowed us to improve detection of lipid species obtained from a complex matrix. In addition, multiplexing and all ion fragmentation were applied to increase sensitivity and selectivity of the analysis. Phospholipid distribution of plasma samples from diabetic mice model was compared with controls and significant differences were identified. Identification of biomarkers and differentiation of structural isomers was facilitated by collisional cross section data. Results clearly show the potential of the technique in the field of lipidomics.
The Application of Untargeted Data Acquisition for Identifying Metabolites of ‘Designer Drug’ 25I-NBOMe in Clinical Samples
Untargeted data acquisition is used to identify the metabolites of novel psychoactive substance 25I-NBOMe in clinically relevant samples. Lack of phase I and II metabolite identification studies of emerging psychoactive substances limits effective monitoring in clinical and forensic testing. Data independent SWATH MS/MS acquisition has been used to identify phase I and II metabolites of 25I-N-BOMe. The present study demonstrates that 25I-NBOMe is extensively metabolised, mainly by O-demethylation, O,O-bis-demethylation, debenzylation, acetylation, glutathione adduction, hydroxylation with or without demethylation, as well as by glucuronidation and sulphation of the main phase I metabolites.
Change in Redox Balance Couples with Redistribution of Metabolic Flux to Protect Glutathione-deficient Gclm-knockout Mice from Alcoholic Liver Disease
Alcoholic liver disease (ALD) is known to progress via steatosis. Although factors contributing to the outcome are not fully understood, oxidative stress is known to be involved. Our recent study showed that glutathione-deficient (Gclm-null) mice, surprisingly, protected from alcoholic steatosis compared to wild-type mice. In order to investigate the mechanism, the reorganization of the polar metabolome was examined using HILIC-ESI-MS. The analysis revealed that shift in redox balance due to glutathione depletion takes place in tandem with redistribution of acetyl CoA flux into alternative metabolic pathways. This siphons alcohol-derived acetyl CoA away from de novo lipid biosynthesis leading to the observed protection of Gclm-null mice from alcoholic steatosis.
Discovering Diabetic Lipid Biomarker Using HRAM LC-MS-MS Approach on a High Field Hybrid Quadrupole-Orbitrap Mass Spectrometer
Lipids play a key role in cell, tissue and organ physiology with diseases such as diabetes which involve disruption of their metabolic enzymes and pathways. Identification of unique lipid biomarkers to distinguish healthy humans compared to those with a disease can have an impact on the early detection of diseases and personalized medicine. Here we demonstrate that HRAM LC MSn approach on a high filed hybrid quadrupole Orbitrap mass spectrometer enables rapid putative biomarker discovery through lipidomics profiling experiments. Two phenotypes of the rat (ZDF vs. lean wild type) plasma samples were used as a model case.
A Workflow for Drug Discovery from Environmental Samples Using Molecular Networks
The problem of identifying non-reference encoded biomolecules using tandem mass spectrometry can be avoided by comparing spectral similarities and building a network based on these similarities. Nodes in such a network represent spectra, while edges connect similar spectra with small mass differences.
The spectral network approach allows the identification of new molecules by creating a network containing labeled spectra. These labeled nodes allow the determination of similar molecules occurring as proximal nodes in the graph.
We demonstrate this molecular network approach using an environmental sample, utilizing spectral libraries from known natural product compounds to find new, similar, candidates from the environment.
Discrimination of Diabetic Lipid and Metabolite Profiles in Plasma in the Zucker Rat Model Using PaperSpray-High Resolution Mass Spectrometry
The relative distribution of metabolites and lipids in serum has been linked to a broad range of disease states, among them diabetes. The analysis of the samples typically requires various levels of sample preparation followed by time-consuming and costly analysis using HPLC-MS. A simple and informative screen could provide rapid information to make differential health determinations is discussed here. Serum samples from diabetic, fatty and control rats is analyzed using paper spray interfaced to high resolution mass spectrometry. The analysis is simple and uses positive and negative ion modes. For a small sample set (n=3 each for control, fatty and diabetic Zucker rats) significant differences in phosphatidyl and lysophosphatidyl cholines, ethanolamines, serines and other lipids, and in small molecule metabolites including fatty acids. The 2 minute analysis uses 15 mcl of sample.
Analyte or Amalgamation? Exploring Relationships and Redundancy in Metabolomic Datasets
Features in a metabolomic dataset are highly redundant. Annotation of these relationships and redundancies is key to data reduction, lower statistical significance thresholds, and a better understanding of metabolomic results.
This poster presents an overview of: the types of relationships to be annotated in these datasets; poor assumptions of current annotation approaches and their corresponding failures; the computational challenge of this search problem; and a tool to explore these relationships.
Of interest are how background ions contribute detected features, how peaks which have poor EIC correlation can still be related, and additional relationships which should be considered within these datasets.
Use of a Novel C18-Based Stationary Phase for Human Urine Metabolite Profiling by UHPLC-High Resolution Accurate Mass Spectrometry (HRAM)
Profiling urine can provide useful information to aid clinical diagnosis. The use of UHPLC and High Resolution Accurate Mass (HRAM)instrumentation in clinical laboratories has grown considerably in recent times. Here, a novel C18 based polar embedded column (ACE Excel 1.7um C18-Amide) is used. This unique stationary phase has been designed to retain and separate a range of polar and non-polar species. Using an 8 minute broad gradient with HRAM it was possible to demonstrate the profiling capability of the method for a wide range of naturally occurring small polar molecules in urine.
Measurement of Kynurenine-to-tryptophan Ratio as a Biomarker for Urinary Tract Infection
Tryptophan (Trp) catabolism yields kynurenine metabolites through the activity of indoleamine-2,3-dioxygenase (IDO). The ratio of the product kynurenine (Kyn) to the substrate Trp is used as a surrogate for biological IDO enzyme activity. IDO expression is locally induced during urinary tract infection (UTI) to suppress immunity and facilitate bacterial colonization. We developed a tandem mass spectrometry assay to measure Trp and Kyn in biological samples and compared Kyn/Trp ratios in urine samples of healthy controls to symptomatic patients with and without UTI. Kyn/Trp ratios were non-specifically increased in symptomatic patients versus healthy controls. No difference was seen between symptomatic patients with and without UTI, indicating that Kyn/Trp ratios may serve as a general indicator of a systemic inflammatory process.
Rapid Evaporative Ionisation Mass Spectrometry (REIMS) as a Novel Approach to Microbial Community Profiling
Mass spectrometric methods are already integrated into diagnostic workflows of clinical microbiology laboratories; hastening and simplifying the turnaround for sample specimens and therefore leading to better patient outcomes. However, current commercial systems rely on isolating and culturing target microorganisms, followed by further sample preparation for MALDI-ToF, leaving room for further improvement. Rapid evaporative ionisation mass spectrometry (REIMS) has been shown to offer robust species identification for a range of clinically important microorganisms. Taxonomic markers have been identified in mixed cultures, demonstrating potential for taxonomic classification in non-pure samples. Work is currently underway to develop and optimise the application of the REIMS technology directly from samples, removing the need to first isolate and culture the microorganism of interest.
Identification of Nontuberculous Mycobacteria Species by Tinkerbell LT
NTM species identification is important for the appropriate treatment of corresponding type and its clinical significance. In this study, 63 species of mycobacteria reference strains and 105 clinical isolates, identified by multi-locus sequencing, were analyzed by MALDI-TOF MS (Tinkerbell LT, ASTA, Suwon, Korea). We acquired specific signals of each individual mycobacterial species cultured on 3% ogawa and MGIT on the basis of unique mass fingerprints. In addition, several mass peaks were selected as diagnostic markers for species confirmation. In conclusion, Tinkerbell LT-MS could be applied for rapid and reliable identification of mycobacteria in clinical laboratory.
Categorizing and Differentiating of Clinically Isolated Mycobacteria by Differential Pattern Analysis of MALDI-TOF MS Data
The use of MALDI-TOF MS has become a powerful and popular method to identify various pathogenic bacteria in many clinical laboratories. However, mycobacterium species are still challenging to correctly identify by MALDI-TOF MS. Fifty mycobacterial isolates obtained from patients were selected to include 20 species, identified by PCR analysis, and analyzed by a MALDI-TOF mass spectrometer to investigate the effect of culture condition, sample preparation, and MALDI peak selection. Phylogenetic trees based on MALDI spectrum were used to show the differentiation and the categorization of the isolates.
Analysis of Broad Spectrum / High Potency Antibiotics – Vancomycin and Gentamicin in Blood and Plasma Using PaperSpray Interfaced to Mass Spectrometry
The growth of infections resistant to traditional antibiotics (i.e. MRSA) has created the need to monitor therapeutic concentrations of the drugs and assure their effectiveness. Two of the most common drugs used are Vancomycin and Gentamicin. The active therapeutic windows for these require prompt monitoring of both peak levels due to toxicity and troughs which are ca. 10 mcg/mL. These compounds are currently monitored using immunoassays. Here, a method is developed and characterized using paperspray interfaced to mass spectrometry to monitor both drugs in plasma and whole blood. Limits of detection below 5 mcg/mL of blood or plasma are achieved. This study demonstrates: 1) the first analysis of aminoglycoside antibiotics using paperspray; 2) proof of principle for multiplexing and monitoring more than one of these drugs; 3) proof of principle for analysis of new drugs without assays.
Identification of Anaerobic Bacteria from BACTEC and BacT/Alert Anaerobic Blood Culture Media Using the Bruker MALDI Sepsityper Kit and MALDI- TOF MS
In this study, the accuracy of identification of anaerobic microorganisms from two different anaerobic blood culture (BC) media using the Bruker MALDI Sepsityper kit followed by MALDI-TOF MS was evaluated. Comparison of the BACTEC and BacT/ALERT systems for detection of anaerobic bacteria showed that the BACTEC instrument was more rapidly able to detect anaerobic organisms spiked into the BACTEC BC bottles (median 43.13h) as compared to the BacT/ALERT BC bottles (median 65.12h). Conversely, anaerobic organisms present in the BacT/ALERT BC bottles were more accurately identified by MALDI-TOF MS after Sepsityper processing (15/26; 58%) then the BACTEC BC bottles (6/26; 23%). The MALDI Sepsityper method must be further refined for identification of anaerobes from positive blood culture bottles to enable more accurate identification of these organisms.
Improving the Detection of Thyroglobulin in Human Plasma for Clinical Research by Combining SISCAPA Enrichment and Microflow LC/MS
Use of an optimized SISCAPA enrichment that is highly specific for a signature peptide of thyroglobulin combined with microflow LC/MS using a vetted dual-pump trapping configuration provides a sub 1 ng/mL quantification limit of Tg protein with a cycle time of 6.75 min. This quantification limit is comparable with the best in literature for standard flow LC/MS. Microflow also offers other tangible benefits including the use of five times less starting plasma and half the injection volume of the standard flow method to reach this detection limit while being more precise. Accordingly, microflow is a viable and attractive solution for clinical research.
Paper Spray-tandem Mass Spectrometry for Therapeutic Drug Monitoring of Tacrolimus, Cyclosporin and Sirolimus in Whole Blood
Paper spray (PS) is open atmosphere ionization for direct and rapid mass spectrometry analysis. It is performed by simultaneously extracting and ionizing dried sample deposited on paper matrix. A PS-MS/MS method for TDM of cyclosporin, sirolimus, and tacrolimus was developed and evaluated. The method uses PS disposable cartridges and automated sample extraction and ionization interface (Prosolia Inc.) and TSQ Vantage tandem mass spectrometry (Thermo Scientific). Assay sensitivity, specificity, linearity, imprecision, and accuracy met set criteria. PS-MS/MS is a viable alternative to immunoassays or LC-MS/MS methods for immunosuppressive drug TDM.
A New PI Fractionator for Analysis of Blood Samples from AD-patients
Online desalinator device coupled with a capillary isoelectric focusing fractionator enables
direct pI-based separation of proteins in body fluids such as blood plasma.
Proteomics analysis retained its high reproducibility, (median CV=3.5 %), and high
correlation between the replicates (median R = 0.961).
Increased depth of analysis fast, reproducible and low cost.
Additional advantage is another domain for biomarker discovery, which is the difference
between the positions of the same protein in patient and healthy samples representing the pI
shift of the protein.
Parallel Reaction Monitoring and Selected Reaction Monitoring Exhibit Comparable Quantitative Performance in Clinical Research and Forensic Applications
Selected reaction monitoring (SRM) has emerged as the MS “gold-standard” for targeted quantification. An alternative quantitative method is parallel reaction monitoring (PRM). In PRM, the third quadruple of a triple quadrupole is substituted with a high resolution mass analyzer to permit parallel detection of all product ions in one high resolution mass analysis.
Here, we evaluate the analytical performance of the PRM method and draw a comparison to SRM. We demonstrated comparable quantitative performance between PRM and SRM in terms of run-to-run reproducibility, matrix effects, and measurement accuracy using epi-vitamin D quantitation in plasma and barbiturate measurement in urine.
Quantification via Signature Peptides: The Advantage of Using Differential Ion Mobility Spectrometry
Some bioactive peptides and proteins exhibit their activity at very low concentrations. Selective and sensitive assays are also required for biomarkers.
Although signature peptides are often used to increase the sensitivity in LC-MS/MS determination of large molecules, there is a risk that isobaric interferences might be accounted for the target compound.
The use of Differential Ion Mobility Spectrometry (DMS) as an additional separation dimension helps to increase selectivity and specificity.
The SelexION® option with 6500 triple quadrupole mass spectrometer from Sciex permitted to remove endogenous interferences and achieve an LLOQ of 5 pg/mL in LC-MS/MS method for Exenatide, a GLP-1 receptor agonist.
Meconium Targeted Drug Screening in 9 Seconds Per Sample Using Laser Diode Thermal Desorption Mass Spectrometry (LDTD-MS/MS)
Drug abuse during pregnancy is a major medical issue associated with significant maternal and infant complications. Meconium is a common specimen used to identify drug-exposed infants. The proposed mechanism for drug presence in the meconium is that the fetus excretes the drug into bile and amniotic fluid. Current analysis method uses several immunoassays to cover many drugs. To reduce the number of screening reagents, required quantity of meconium and analysis time, a Laser Diode Thermal Desorption Mass Spectrometry (LDTD-MS/MS) method was developed. A fast extraction method is used with calibration range of: 20 to 200 ng/g for amphetamines/cocaine/opiate/oxycodone/PCP/methadone and 50 to 500 ng/g for Barbiturates/Benzodiazepines classes. The lower limit of the calibration curves represents the cutoff for reporting results. Sample analysis time is 9 seconds per sample.
Comparison of Dried Blood Spot Collection Devices by Paper Spray Ionization Mass Spectrometry
Paper spray ionization (PS) is an emerging technique that combines sample collection, preparation and ionization on a paper substrate for rapid analysis of small analytes by mass spectrometry. Currently, there are many commercially available DBS devices that may be integrated with PS. Coupling PS and DBS devices eliminates the need of offline extraction of the analytes from the DBS. Additionally, with this new approach, the PS measurement may utilize specialized DBS devices for improved sample volume precision and accuracy or filtration of the biological matrix. Analytical figures of merit of DBS collection devices coupled with PS are investigated.
Results from a Seven Month Trial of Single Point Calibration
We have evaluated the potential for use of a single point calibration over a period of seven months and a total of 340 analytical runs using production laboratory data. The single point calibration data is incorporated into a weighted response factor that provides a more stable estimate of instrument response and therefore better analytical precision. This is shown for three androgen analytes along with several observations that were made over the seven month period. The weighted response factor is shown to behave in a predictable fashion. Several points to consider in adoption of this type of strategy are discussed.
A New Instrument that Combines Fast Microfluidic Separations with High Pressure Mass Spectrometry for Clinical Diagnostic Applications
We have developed a prototype instrument that combines microfluidic capillary electrophoresis (CE) with high pressure mass spectrometry (HPMS) into a single benchtop instrument. This instrument is roughly the size of a conventional HPLC pump, and it contains all of the components necessary for running microchip CE-ESI-HPMS, including a small, custom-built scroll pump, and an on-board computer. We have used this platform to generate some preliminary data for applications relevant to clinical diagnostics including simulations of newborn screening via dried blood spot analysis and monitoring of pain management drugs in urine. This presentation will provide an overview of the instrumentation and examples of the data generated by this system.
Reduction in Pipette Tip Consumable Cost and Waste Through Innovation
As the number of samples processed in labs using high-throughput processes has increased, there has been a corresponding rise in the level of waste, such that in 2010, four million pounds of plastic pipette tips were disposed of after just one use. The TipNovus high-throughput automated pipette tip cleaning system from Grenova enables laboratories to safely reuse sanitized tips and thereby reduce cost and waste output.
Validation of LC-MS/MS Assay Method for the Determination of Triclocarban in Human Urine
Previously, we have developed an LC/MS/MS method to analyze 13 environmental phenols in human urine. In this study, we expanded our method analyze triclocarban (TCC) along with previous analytes in human urine. Briefly, samples were processed using enzymatic de-conjugation of glucuronides, followed by solid phase extraction (SPE) on a C18 cartridge. Analytes in the concentrated eluate were separated by reversed-phase HPLC, detected by atmospheric pressure chemical ionization (APCI) MS/MS (QTRAP5500) in negative ion mode, and quantified by isotope dilution method. The method proved to be accurate, precise and without obvious matrix effects. The limits of detection are in the low ng/mL range.
Exposure Biomarker Discovery for Toxic Phthalate Plasticizers Using Liquid Chromatography-High Resolution Mass Spectrometry and Metabolomics Approaches
Phthalates, including DINP, DPHP, and DINCH, are used as plasticizers and could cause undesired health impacts, such as endocrine disrupting effects. Workers and general population are exposed to them, so assessment of their exposure is a public health issue. Therefore, exposure biomarker discovery for toxic phthalates is of great interest in occupational and environmental health. We used HRMS with metabolomics approaches, signal mining algorithm with isotope tracing (SMAIT), MDF, and XCMS, to filter out meaningful metabolite signals from complex LC-HRMS data. They were further verified as markers of toxic phthalate exposure using animal models and tested in general population.
Incorporating Analysis of Mitragynine into LC-ESI-MS/MS Methodology Routinely Used for Quantification of Pain Medication and Illicit Drugs in Urine
We describe development and validation of a 6.5 minute LC-ESI-MS/MS method for quantification of mitragynine in human urine for monitoring of kratom use. Methadone-d3 is used as internal standard. A between-laboratory comparison of concentrations was performed with mitragynine concentrations across the analytical measurement demonstrating acceptable correlation. Our laboratory has routinely measured mitragynine in urine samples for > 1 year with an overall CV for QC samples of 9.3% and accuracy of 102.3%. The methodology we describe allows for measurement of mitragynine within routinely used sample analysis techniques for pain medication and illicit drugs, enabling easy implementation of mitragynine testing.
Differential Recovery of Gabapentin and Pregabalin Utilizing SPE Extraction Demonstrated in a 42 Analyte Urine Confirmatory LC-MS/MS Panel
Methods that quantify a wide variety of drug chemistries from a single analysis are increasingly being implemented. An impediment to implementation of these methods is inclusion of gabapentin and pregabalin to these comprehensive methods, as specimens routinely have concentrations in the hundreds of microgram per milliliter range. The concentration and types of organic solvents used in the elution step can be modified to predictably and reproducibly reduce the recovery of gabapentin and pregabalin, while not affecting the complete recovery of analytes requiring high levels of sensitivity.
LC-MS/MS Method Development Challenges for the Separation of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most common medications worldwide for the treatment of pain, fever, and inflammation. Although NSAIDs are generally considered safe, long-term use can result in renal failure or serious gastrointestinal and cardiovascular side effects. Monitoring their use can help prevent drug interactions and to minimize side effects. For this analysis, the combination of the Raptor™ Biphenyl column, use of scheduled polarity switching, and selection of mobile phases that maximized sensitivity in both positive and negative ion modes allowed the simultaneous detection of 27 NSAIDs (plus acetaminophen) in one fast 8.5 minute analysis.
The Analysis of Common Antiepileptic Drugs in Human Urine by LC-MS/MS
The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) in therapeutic drug monitoring and toxicology labs has increased significantly over the years. LC-MS provides sensitivity, speed, and the ability to simplify sample preparation. The Raptor™ Biphenyl column was developed to complement high-throughput LC-MS/MS analyses by combining the increased efficiency of superficially porous particles (SPP) with the resolution of Ultra Selective Liquid Chromatography™ (USLC™) technology. In this example, a simple dilute and shoot method was developed for 14 common antiepileptic drugs in urine using a Raptor™ Biphenyl column.
A Multi-Class Drug and Metabolite Screen of 231 Analytes by LC-MS/MS
Therapeutic drug monitoring can be challenging due to the low cut-off levels, potential matrix interferences and isobaric drug compounds. To address these challenges, many drug testing facilities are turning to liquid chromatography coupled with mass spectrometry (LC-MS/MS) for its increased speed, sensitivity, and specificity. The Raptor™ Biphenyl column was developed to complement high-throughput LC-MS/MS analyses by combining the increased efficiency of superficially porous particles with the resolution of Ultra Selective Liquid Chromatography™ column technology. In this example, a method was developed for a 231 compound multi-class drug and metabolite screen.
Pain Panel Drug Screening by Nanopost Array Laser Desorption Ionization Mass Spectrometry (NAPA LDI-MS) on REDIchip
REDIchips are comprised of nanopost arrays (NAPA), fabricated on silicon substrates to form patterned targets for high throughput LDI-MS of small molecules in MALDI instruments. The REDIchip’s NAPA targets exhibit excellent performance for quantitation and screening of pain panel drugs such as opiates/opioids, anesthetics, and cocaine metabolites in biofluids. The pain drugs are quantitated over at least four orders of magnitude dynamic range to determine appropriate cut-off values for pass/fail parameters for drug screening from a variety of human biofluids. REDIchip’s NAPA LDI-MS workflow allows for efficient and sensitive detection and quantitation of several classes of pain drugs.
Validation of an Algorithm for Determining Urinary Medication Cutoffs Using Quantitative LC-MS/MS
The advent of quantitative methods of determining medications and their metabolites in urine using LS-MS/MS analysis presents a quandary of establishing an appropriate cutoff to assess medication compliance. We suggest a simple frequency distribution plot after log transformation will make the data approximately Gaussian. Extrapolation of the lower values allows estimate of a 2.5% cutoff. Quantitative urinary excretion of hydrocodone (734 patients) and hydromorphone (732) were evaluated. Values less than 20ng/mL were excluded. The frequency distribution plot of hydrocodone roughly followed a Gaussian distribution with an estimated 2.5% cutoff of 20ng/mL. However, the hydromorphone was not Gaussian.
Is Enantiomeric Testing of Methamphetamine Necessary?
When scheduled substances are prescribed, Urine Drug Test (UDT) results are necessary for clinical decisions. While UDT can detect recent methamphetamine use, it could not distinguish between illegal S-methamphetamine and legal R-methamphetamine use. At Soloniuk Pain Center (SPC) and Redding Opioid Recovery Medical Clinic (RORMC) in Northern California, review of enantiomeric confirmation was conducted to determine if chiral testing is necessary in these two unrelated patient populations. Of the 100 methamphetamine positive samples, S-methamphetamine was found in 70% of the SPC samples and 100% of the RORMC samples. With a 30% R-methamphetamine confirmation rate, chiral analysis for chronic pain patients is essential for informed clinical decision-making.
Improved Sample Preparation for the Analysis of 12 Opiates in Urine Using the Thomson eXtreme Filter Vials® by LC-MS/MS
This improved sample preparation method allows for the quantitative measurement of opiates in urine. The urine samples were prepared using the eXtreme|FV®, followed by LC/MS/MS analysis. The most critical aspects of reliable urine analysis are the reduction of interferences from the sample matrix and analyte recovery. eXtreme|FV®, were compared to an existing SPE sample preparation method to reduce the sample matrix causing interference prior to analysis. SPE is time consuming, adversely impacts recovery, uses large amounts of solvent and are expensive. The improved sample preparation method using the Thomson eXtreme|FV® allows for the analysis of 12 Opiates.
Direct Injection of Serum and Online Solid Phase Extraction for the Quantification of 35 Benzodiazepines and Metabolites by Liquid Chromatography MS/MS
An analytical method for the quantification of 35 benzodiazepines and metabolites that allows for direct injection of human serum is reported. Conventional HPLC methods require manual offline sample preparation; in this case, internal standards are automatically added to each sample by the autosampler prior to injection of the intact serum onto an online SPE liquid chromatographer using a Thermo Scientific™ Transcend™ II system. Analytes are detected by mass spectrometry in single reaction monitoring acquisition mode on a Thermo Scientific™ TSQ Endura™ triple quadrupole with a heated electrospray ionization source. The method was analytically validated in terms of limits of quantification, linearity ranges, accuracy and precision for each analytes using the MS9050 ClinMass® TDM Platform from RECIPE with the MS9550 Add-On Set for Benzodiazepines.
Measurement Uncertainty and Mass Spectrometry; Key Concepts Applicable to Routine Laboratories and Knowledge Gained from Participation in CCQM Intercomparisons
Measurement Uncertainty (MU) is useful for many reasons, whether it is determining if data from separate time points or different laboratories are genuinely different or statistically the same, or to determine which sources of method bias or imprecision may require attention. Laboratories working to the ISO 15189 standard are required to estimate MU for each assay.
In this presentation the basic MU concepts will be discussed along with examples of top down and bottom up approaches to MU calculation using mass spectrometry generated data. Sources of imprecision and bias relevant to mass spectrometry assays will also be discussed.
Harmonization in Individual Bile Acids Analysis in Mouse and Man – an Inter-Laboratory Ring Trial, Method Comparison and Clinical Relevance of Bile Acids
Interest in bile acids has been growing since the discovery of their significance, hormone-like regulatory factors acting via nuclear-farnesoid-X receptor (FXR), and G-protein-coupled plasma-membrane bound receptors (TGR5). Therefore, simultaneous quantitation of individual bile acids is of highly interest in many diseases, life-style, and clinical-related questions e.g. in nutrition, use of antibiotics, gut microbiome, metabolic syndrome, type 2 diabetes, Alzheimer´s disease, NAFLD or other liver related diseases. Standardization is mandatory to develop bile acid related biomarkers and to bring it into clinical routine application. Here, we describe the world-wide first bile acid assay in standardized format and will present the inter-laboratory ring trial data, method comparison and discuss their clinical relevance.
Thyroid Hormone Analysis in NIST Standard Reference Materials
The aim of this work was to develop higher order analytical methods and apply these to value assign target iodine status biomarkers, namely thyroid hormones, in applicable reference materials for the delivery of traceability and calibration tools. Method development for the quantification of thyroid hormones specifically T4 and T3 has been accomplished by way of selective elemental detection utilizing liquid chromatography (LC) coupled to an inductively coupled plasma mass spectrometer (ICP-MS). This higher order analytical method was applied to value assign target T3 and T4 hormones in currently available SRMs.
Peace of Mind in the Era of LDT Regulation - A Home-brew Barcoding Solution for Tracking Everything LC-MS/MS
With regulation of LDTs on the horizon, the need for laboratories to develop a feasible method of logging LC-MS/MS reagents and solvents is inevitable. Such a system must be able to document and track all lot changes and lend itself to retrospective troubleshooting when the components of a prepared solvent or reagent cause subpar performance. We have developed a workflow that involves the use of individual barcodes - to document lot and expiration of reagents, track where and when they were used, and document that validation was performed - all in a MS Access database. This system can be customized to track anything in the LC-MS/MS lab - from chemicals to consumables.
Tip-based Fractionation for Comprehensive Phosphoproteome Analysis
We developed a new workflow for phosphoproteomics studies which is able to find a considerable number of phosphosites with reduced time, cost and effort. The workflow is a combination of Titanium dioxide (TiO2) phosphopeptide enrichment followed by pipette tip-based Strong Cation-Exchange (SCX) fractionation. Using this method more than 9000 phosphosites and 7500 phosphopetides were detected out of 3 mg of HeLa lysate. The new workflow was successfully applied for the first time for the analysis of the effects of inhibition of cholesterol egress from lysosomes using U18666A inhibitor on the phosphoproteome of Mouse Embryonic Fibroblasts cells (Mef cells). 12881 phosphosites were found in this experiment which 751 were significantly regulated (349 phosphosites were up-regulated and 402 down-regulated).
Quantitative Analysis of Protein Expression in Zebrafish Embryos Neuronally Expressing the Human EWSR1-ERG Oncogene
Ewing sarcoma, a pediatric bone sarcoma, is characterized by a reciprocal translocation event between EWSR1 and a gene of the ETS family. A binary transgenic zebrafish model for Ewing’s sarcoma has been recently developed, which expresses EWSR1-ERG neuronally and GFP for monitoring. A bottom-up proteomics approach was performed on embryos expressing EWSR1-ERG and on wild type embryos. Spectral counting was used to calculate the changes between the mutated and wild type fish. A variety of the up and downregulated proteins were involved in pathways, such as oncogenesis, transcription and translation and cellular respiration, which were repeatedly associated with Ewing’s sarcoma.
Multiplexed Quantification of a Serum Protein Panel to Monitor Treatment of Duchenne Muscular Dystrophy Patients
Duchenne muscular dystrophy (DMD) is a genetic disorder that is characterized by continuous muscle damage. Recently, a novel drug therapy aimed at muscle restoration and delay of disease progression has been conditionally approved for treatment of patients that suffer from this severe and fatal disease. Besides the established functional outcome measures, protein biomarkers offer an attractive alternative for monitoring the effectiveness of treatment. In this study, an established combination of immunocapture and mass spectrometry (MS) will be employed for serum protein quantification in a multiplexed assay for evaluation of various DMD biomarker candidates.
Development of a Sensitive, Accurate and Robust LC/MS-based Method for Profiling of Angiotensin Peptides in Plasma of Atherosclerotic ApoE-/-/LDLR-/- Mice
The aim of this study was to develop an analytical methodology for accurate quantification of angiotensins: Ang I, Ang II, Ang-(1-7), Ang III and Ang IV in plasma of atherosclerotic mice. A triple quadrupole mass detector equipped with chip-based nanospray source connected to nanoHPLC was used. Plasma angiotensin profile was substantially modified in ApoE/LDLR double knock-out mice with increase in concentration of Ang II from 37.6 ± 21.3 pg mL-1 in WT to 200.2 ± 47.6 pg mL-1. Concentrations of Ang I, III and IV were also elevated 3-10 fold in ApoE-/-/LDLR-/- mice while that of Ang-(1-7) was unchanged.
LC-MS/MS Measurements of Parathyroid Hormone-Related Protein (PTHrP): Negative Correlation Between Age and PTHrP Concentrations in CSF
Parathyroid hormone related protein (PTHrP) is involved in intracellular calcium regulation. Calcium is necessary for the nerve signaling and brain function, but limited information is available related to PTHrP expression in brain. Recently we developed LC-MS/MS method for the assessment of PTHrP in plasma. Using this method we analyzed PTHrP in a set of paired serum and CSF samples, and evaluated distribution of PTHrP concentrations, and the ratio of concentrations (PTHrPCSF/PTHrPserum) in adults. A trend to higher PTHrP concentrations with increasing age was observed in serum, while a trend to lower concentrations was observed for PTHrPCSF and the PTHrPCSF/PTHrPserum ratio.
Mass Spectrometry Mediated Study of Secreted Vesicles of Salmonella Typhimurium
Salmonella Typhimurium is Gram-negative pathogen which infects up to 1.3 billion people worldwide. Salmonella have ability to invade, survive and replicate within host. Effector proteins encoded by Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) have role in invasion and survival within host. Only 30-40 effector proteins, secreted by T3SS have been identified till date and out of which few has fully characterized. Therefore, there is an urgent need to explore more and more effector proteins. Mass spectrometry can help both in discoveries of novel effectors proteins from the secreted vesicles of the Salmonella Typhimurium and in their characterization.
Calmodulin-Like Protein 5, a New Marker of Keratinocyte Differentiation, Disturbed in Atopic Eczema
Atopic eczema is a skin disease that affects approximately 25% of children and between 1 and 3% of the adult population in the UK. In atopic eczema the skin barrier is disturbed to the extent that it no longer functions to retain as much water as usual, resulting in an itchy, red skin rash. Label free proteomics was used to identify protein markers that may potentially be able to elucidate the mechanism behind this change. Fourteen proteins we identified as being significantly changed between control and atopic eczema elbow scrapings were selected for validation by immunohistochemical staining. Alpha-1 acid glycoprotein, bleomycin hydrolase, calmodulin-like protein 3, cathepsin D and dermcidin all showed changes, however calmodulin–like protein 5 was the most significant.
Diagnostic Protein Quantitation of 26 Actionable Targets in Patient Biopsies Using Clinical Mass Spectrometry
Many available oncology therapies target specific proteins. Additionally, protein biomarkers affecting chemotherapy efficacy have been identified. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. In this study we present a multiplexed Liquid Tissue-Selected Reaction Monitoring assay to simultaneously quantify 26 clinically-actionable proteins from a single microgram of FFPE patient tissue. The assay’s performance and tumor expression levels of these biomarkers in over 300 clinical biopsies will be presented. By providing absolute quantitation of multiple actionable proteins from patient biopsies, clinical mass spectrometry is delivering personalized cancer care.
Detection of UCN3 as a Biomarker for Obstructive Sleep Apnea in Children Using Multiple Reaction Monitoring
Obstructive sleep apnea (OSA) is a disorder where a person is deprived of oxygen that may cause additional health problems if left untreated. Children with OSA are thought to be underdiagnosed due to the lack of sleep centers around the country and noncompliance with take-home testing. Urocortin-3, (UCN3) has been previously identified as a urine biomarker for OSA in children. Here, we utilized multiple reaction monitoring to identify a peptide fragment that gave 3 transitions with high signal intensity. We find that the linearity and sensitivity are sufficient to make a differentiation between children with and without OSA based on previous findings. We also developed two methods in parallel that deplete abundant proteins and concentrate UCN3 in both urine and plasma spiked samples. Both methods yield highly purified UCN3 that are scalable for high throughput assays.
Improved Efficiency in Translation of Biomarker Development by Triple Component Human Serum Proteome Profiling for Diagnosis and Prognosis of Chronic Leukemia
Successful translation during biomarker development with complex human serum proteome requires ultra-high resolution protein identification technologies. Here we devised a triple component serum proteome profiling method that integrated serum abundant protein depletion, MudPIT, and segment survey scan mass screening, by which its effectiveness to increasing peptide and protein coverages as well as run-to-run reproducibility at very low false discovery rate was studied, comparing to one-dimension shotgun proteomic approach. We further applied this method to biomarker development for diagnosis and prognosis of chronic myelogenous leukemia and chronic lymphocytic leukemia, due to the facts that clinical symptoms of chronic leukemia are often non-specific where the American Cancer Society estimates that at least one-fifth of the people with leukemia have been underdiagnosed.
The Future Is Now - Monitoring Insulin Levels for Diabetes Clinical Care with High Precision Using Mass Spectrometry
In the past, the clinical utility of monitoring insulin and proinsulin levels in blood has been low due to the variability between available assays (typically ELISA and Western Blot) and lack of standardization (1-3). The establishment of an accurate and standardized approach would have clinical value in the assessment of endogenous and synthetic analogs of insulin as well as proinsulin and C-peptide. To address this shortcoming, we have developed and validated a multiplexed assay for intact proinsulin, insulin, C-Peptide and synthetic insulin analogs using semi-automated immunoenrichment coupled to high-resolution mass spectrometric detection. The multiplexed assay is rapid (ca 10 mins), precise and sensitive across the complete clinical range for all analytes.
Development of Clinical Assays Based on Parallel Reaction Monitoring
Targeted proteomics analyses of biomarkers are routinely performed on triple quadrupole mass spectrometers, which present limited selectivity. The analyses of clinical samples performed on a quadrupole-orbitrap instrument using parallel reaction monitoring (PRM) showed a significant gain in sensitivity and selectivity. A new acquisition method leveraging the presence of internal standards showed a dramatic improvement of the reliability and the precision of the analyses. The method has a broad applicability to the quantitative analysis of clinical samples, such as lung cancer plasma samples to discriminate the disease stages and subtypes, and of tissue samples to map driver mutations (EGFR and KRAS).
Quality Controls for the Quantification of Apolipoprotein L1 and Its Genetic Variants Based on Peptide Mass Spectrometry Measurements in Kidney Disease
In the translation of building a clinical LC-MS/MS assay on MRM peptides in compliance with high throughput for patients, the initial small-scale experiments have to demonstrate the traceability along the workflow. Apolipoprotien L1 (ApoL1), an HDL lipoprotein with three genetic variants, is used as a model protein to assess the strategies of standards, matrix effects, and the quality controls for the enzymatic digestion. A simple, high-throughput, quantitative LC-MS/MS ApoL1 assay has been successfully developed, validated and then applied to 235 participants to examine the associations between chronic kidney disease with ApoL1 plasma levels and its genetic variants.
New Approach for Intact Protein Separation, Detection, and Quantitation Based on Multiple Reaction Monitoring Triple Quadrupole Mass Spectrometry
There is an increasing demand for protein quantitation in biological fluid for disease detection, protein therapeutics monitoring, and response control during clinical trials. Current triple quadrupole mass spectrometer (QQQ-MS) protein quantitation methods require protein digestion step that is often incomplete thus introduce error. Our method bypasses protein digestion to directly quantify intact proteins on QQQ-MS with multiple reaction monitoring. A series of model proteins are shown to be sensitively and specifically detected, even in the presence of biological matrices. A chromatographic method was developed to address the complex biological matrix for this system. The success of this project can aid clinical diagnostic and treatment advancements.
Development of a Quantitative MS-based Assay for Intact BNP and Its Proteolytic Variants: Early Impressions with LC and CE from “High Flow” to “No-flow”
B-type Natriuretic Peptide is a biologically active circulating factor whose concentration is routinely used in the diagnosis of heat failure. Although this analysis is routinely performed using ELISA platforms, these assays are incapable of differentiating between proteolytic forms of BNP. High-resolution mass spectrometry can easily resolve proteolytic variants with accurate quantitation, provided it is supplied with sufficiently resolved peaks. We are working on several different separation techniques that 1. Are compatible with mass spectrometry; 2. Can resolve intact BNP variants derived from a plasma matrix; 3. Can accommodate the large dynamic range with sufficient sensitivity.
Applying a Proteoform Profiling Method for Neurological Disorder Biomarker Discovery
In this study we have used a quantitative proteoform profiling experiment to detect and identify several potential biomarkers for Alzheimer’s disease based on cerebrospinal fluid (CSF). We were able to comfortably and reproducibly detect more than 1500 proteoforms in each sample leading to the detection and identification of 77 differentially regulated proteoforms (potential biomarker candidates) which are currently being identified and validated through a scheduled top-down MS/MS approach. Many of those correspond to proteoforms of proteins carrying a specific modification, a mutation or having undergone a proteolytic modification which would have made their characterization much more challenging, if not impossible, with a bottom-up workflow.
Quantitative LC-MS of Apolipoprotein L1 in Human Serum with High Throughput Automated Sample Preparation.
Chronic kidney disease (CKD) is a prevalent disorder that often remains undiagnosed until its most severe renal and cardiovascular complications arise. Although biomarkers for CKD are few, Apolipoprotein-L1 variants have been directly linked to an increased risk for this disease. In order to facilitate its routine detection, we built and evaluated a quantitative LC-MS/MS assay for Apo-L1 that is compatible with automated high-throughput upstream sample preparation capable of processing 96 samples in under 6 hours.
Preserving Specimen Integrity in Plasma Renin Activity Measurements
Plasma Renin Activity (PRA) measures the capacity of circulating Renin and Angiotensinogen to generate Angiotensin 2 (Ang2) using the quantity of its precursor, Angiotensin 1 (Ang1), which is linearly correlated with Ang2 abundance and, thus, predicts hypertension (1,2). Historically, EDTA plasma is the required specimen type for measurement of renin activity due to the chelation of divalent cations from the plasma, which reduces degradative loss of Ang1 to Ang2 during the “generation” procedure by conversion from the metalloprotease, Angiotensin Converting Enzyme (3). As a reference laboratory that receives a many incorrect specimen types, we tested the stabilizing effect of supplemental EDTA on Ang1 in EDTA and non-EDTA samples types. Our preliminary results indicate that supplemental EDTA is required to prevent degradation of Ang1 regardless of sample type.
Quantitative Mass Spectrometry-Based Proteomics Approaches for Personalized Medicine
LC-MS/MS is a powerful tool for the discovery and quantification of biomarkers. Two case studies will be presented. In the first study, an unbiased approach was used to discover plasma proteins from patients undergoing treatment for metastatic colorectal cancer (mCRC). 1723 unique proteins were identified, and 235 were differentially-expressed between Intrinsic Resistance and Responder groups at baseline. In the second study, an MRM assay for the absolute quantitation of 9 cancer-related proteins (55 unique peptides) in cell lysates was developed. The LLOQ was ≤ 10 ng/mL and good precision (< 30%) and accuracy (70-130%) was achieved. Both LC-MS/MS methods have an important role in the advancement of personalized medicine.
Quantifying Translational Differences Between Single Blastomeres in the 16-cell Xenopus Embryo by Mass Spectrometry
Characterization of the proteomic machinery underlying cell differentiation promises to elevate our understanding of the normal and impaired development of the vertebrate embryo. However, this requires specialized, highly sensitive tools, particularly based on mass spectrometry, to measure single cells. Here, we utilize a custom-built capillary electrophoresis microelectrospray mass spectrometry technique to study proteomic differences between selected blastomeres that were extracted from the 16-cell normal frog embryo. This technology enabled the reproducible quantification of ~150 nonredundant proteins, which allowed us to uncover translational differences between multiple blastomere types. These results set the stage for translational studies where the hindrance of developmental pathways is suspected to lead to inborn defects in the embryo.
Continuing Development of BDX003 a Serum-based MALDI-TOF Test to Detect Hepatocellular Carcinoma in High-risk Patients
BDX003 is a serum protein test combining MALDI-TOF with an AFP ELISA which classifies patients into two groups (HCC or NoHCC). The test was initially developed with two sample sets totaling 158 from HCC patients at various stages, and 135 patients with liver disease but no HCC. This development effort resulted in a test which detects HCC with 80% sensitivity and 79% specificity in the validation cohort. Here we present ongoing work to further characterize the BDX003 assay. HCC detection sensitivity will be tested in at least one independent sample cohort with a focus on early stage (I/II) cancer. We will also present results from pilot studies to evaluate reproducibility, stability, and cross platform portability of the test.
Determination of Hemagglutinin Content in Influenza Vaccines Using Size Exclusion Chromatography and Quantitative Mass Spectrometry
Size exclusion chromatography (SEC) has been employed to separate HA species under the same conditions employed in the SRID assay. In order to determine the amount of the active form of HA in the vaccine, we have used a protocol where vaccine samples are first separated by SEC followed by LC-MS/MS quantitation of the protein components. The same SEC fractions may also be subject to SRID analysis to determine the fractions with SRID activity. A measure of potency can be calculated by summing the concentrations of all fractions that are SRID active. We have also demonstrated that the SRID active fractions can reproducibly be isolated via size exclusion chromatography and that the assay is also stability indicating, based on changes observed in the SEC profile of vaccines subjected to pH based stress.
Method Development of LC-MS Based Peptide Quantitation Assay to Differentiate Kininogen and Kallikrein Cleaved Kininogen
The purpose of this study was to develop a robust LC-MS based peptide quantitation assay to differentiate kininogen (HK) and kallikrein cleaved kininogen in order to determine a 2HK cut-point in plasma of healthy volunteers from patients with disease. MRM on an ABSciex 5500 QTrap mass spectrometer was done on various types of plasma samples. The target candidate peptides (SSRIGE, SSRIGEIKE mis 2, KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE, KKIYPTVNCQPLGMISLMK, and SYYFDLTDGLS) and 3-5 product ions for each peptide were validated empirically for plasma kininogen. Ratio of AUC for each peptide between different sample types was calculated. . The ratio of SSRIGE peptide against HMWK vs LMWK unique peptide SYYFDLTDGLS has a potential to be used in an assay to differentiate plasma of healthy volunteers from patients with disease.
A Multiplexed LC-MS/MS Method for Quantitative Analysis of Apolipoproteins in High-Density Lipoprotein
High-density lipoprotein (HDL) consists of many apolipoproteins which play important roles in regulation of cholesterol transportation. Elevated cholesterol is a well-established risk factor for cardiovascular diseases. Individuals with cardiovascular risk factors have an increased risk of developing Alzheimer’s disease (AD); however cholesterol-lowering therapies do not appear to reduce AD risk. An analytical method to allow quantitative analysis of HDL will allow future investigation of HDL apolipoproteins as therapeutic targets for AD. We isolated HDL fractions in plasma specimens using ultracentrifugation. Following trypsin digestion, the fractions were assayed using LC-MS/MS, and quantified using peptide standard curves. Four HDL proteins (Apo-A1, Apo-A2, Apo-C2, and Apo-C3) were compared to immunoassay results with three indicating correlation.
Proteomic Profiling Reveals Potential Novel Biomarkers of Aortic Stenosis in Affected Heart Tissue
Aortic stenosis (AS), the most common form of valve disease in the Western world, occurs due to progressive narrowing of the aortic valve (AV) orifice, resulting in slowly increasing load onto the left ventricle (LV). The LV responds with a myriad of adaptive changes, which eventually become maladaptive. Current management is focused on assessing the severity of valve stenosis, but neglects the myocardial response, that is pivotal for timing of surgery and prognosis. The aim of this pilot study is to find new biomarkers of myocardial remodeling that could refine our understanding of maladaptive changes in aortic stenosis and ultimately allow to optimize the timing of surgery.
Comparison of Extraction Methods for the Quantification of Eculizumab from Serum Using Microflow LC-ESI-Q-TOF Mass Spectrometry
As monoclonal antibody therapeutics become humanized, tryptic peptide approaches for quantitation of biologics in serum become more challenging. An alternative is to monitor the unique molecular mass of the intact light chain. The ability to selectively extract only the IgG4 antibodies from serum should allow for an advantage in detection. The IgG4 extraction compared to the extraction of all IgG immunoglobulins allowed for a ~10-fold lower limit of quantitation for eculizumab. This approach could be beneficial for the therapeutic monitoring for IgG4 biologics that need a lower limit of detection and the specificity that other approaches are unable to provide.
Characterizing Secreted Factors Contributing to Drug Resistance in Pancreatic Cancer Tumor Micro-Environment
Poor prognosis in pancreatic cancer has largely been linked to therapeutic resistance to first-line treatments. Tumor heterogeneity in pancreatic adenocarcinoma microenvironment (consisting of fibroblasts, pancreatic stellate cells (PSC), and extracellular matrix proteins (EMPs)) may confer this resistance. Studies have implied that secreted factors targeting tumor growth and metastasis pathways may play an important role in conferring drug resistance as well as the secretion of EMPs that contribute to fibrosis and act by physically limiting drug entry. We carried out a bottom-up mass spectrometry discovery analysis on the secretome to identify secreted factors from PSCs and MiaPaCa—a pancreatic carcinoma cell line—in the presence of the chemotherapeutic drug triptolide. Highly targeted pathways were identified that may provide insight to additional therapeutic strategies or targets.
Quantification of Vitamin D-Binding Protein Isoforms by MRM
Vitamin D-binding protein (VDBP) is a plasma protein that transports vitamin D metabolites to target tissues. Recent work has shown that quantification of VDBP using immunoassays gives variable results depending on the antibody specificity; therefore, standardization of the methods used for the measurement of this protein is needed. Because there are three common isoforms, GC-1s, GC-1f, and GC-2, with up to two sites of glycosylation on the peptide unique to each isoform, quantification of the protein is challenging. In this work, a method for quantification of the three main VDBP isoforms was investigated using multiple reaction monitoring (MRM).
A Novel 6x5 Peptide Mixture for Full Instrument Characterization and Performance Monitoring
Clinical mass spectrometry requires robust instrument performance. Currently, as both low and high-resolution instruments are being utilized to carry out clinical tests, a standardized reagent is needed for system suitability monitoring. In this study we present a full workflow for the analysis of HPLC, MS1 and MS2 instrument stages using a peptide mixture of isotopologues that span 4 orders of dynamic range. The HPLC and MS1 data were analyzed using PReMiS™ software which reports directly on critical LC and MS1 parameters. For the MS2 evaluation, both SRM (Triple-stage-Quadrapole) and PRM (high-resolution orbitrap) data were analyzed with Skyline to analyze MS/MS related metrics associated with both types of instruments.
A Reference Measurement System for Urine Albumin
Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urine albumin is vital to clinical diagnosis. To address urine albumin measurement precision, we have developed the following components of the urine albumin reference measurement system: a multiplexed candidate reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to quantify urine albumin; a primary reference material to be used as a calibrator for higher-order urine albumin methods; and a secondary reference material to be used as a matrix-based quality control for commercially-available urine albumin assays.
Patients with Celiac Disease Display a Varying Oligoclonal Antibody Response to Tissue Transglutaminase: Characterization Utilizing a Proteomic Approach
Celiac Disease (CD) is characterized by an intolerance to dietary gluten. Patients with CD often have high levels of IgA autoantibody against tissue transglutaminase (tTG). To characterize tTG reactive IgA, the autoantibodies were isolated and then analyzed by TOF-LC/MS and LC-MS/MS, using top-down and bottom-up proteomic approaches. This provided information from the variable regions of both IgA light and heavy chains to characterize tTG reactive antibodies. Comparison of positive and negative sample groups demonstrated significant light chain profile differences, indicating that celiac patients exhibit an oligoclonal response to tTG.
Development of a Digested Yeast Protein Extract as a Mass Spectrometry Reference Material
The emergence of mass spectrometry (MS) based proteomic platforms utilized in biochemical and biomedical research has increased the need for high quality MS measurements. In an attempt to standardize MS metrics for the proteomics community, Saccharomyces cerevisiae yeast protein extract has been used in several successful inter-laboratory studies that demonstrated the yeast proteome as a suitable proteomic reference material (RM). To further improve on MS performance materials, we are developing a tryptic digest of yeast protein extract (RM 8313: Digested Yeast Protein Extract) to function as a quality control material to benchmark the performance of MS-based instrumentation. RM 8313 will facilitate the intra- and inter-laboratory assessment of MS-based instrument performance and measurement quality by eliminating the variability in laboratory protein digestion protocols.
A Validated Amino Acid Analysis Assay for Accurate Quantification of Stable Isotope Labeled Thyroglobulin and Other Protein Reference Materials
With the emergence of mass spectrometry for the clinical measurement of proteins in biological matrices, the development of biological reference materials will continue to grow in importance. Certified Reference Materials with values assigned by metrologically valid procedures will be critical to minimize and control experimental variations in all steps of the workflow including protein extraction, fractionation, enrichment, proteolysis and analysis. To this end, we have verified sequence fidelity and isotopic incorporation of an SIL full length Thyroglobulin for use as an internal standard in quantitative MS workflows. We have also developed an amino acid analysis (AAA) method traceable to NIST SRM 2389 and validated the method against NIST SRM 927. This AAA method may be used for accurate quantification of proteins to enable development of accuracy-based protein reference materials.
Targeted LC-MS/MS Screening and Quantification of Proteins from Complex Biological Matrices Using a Retention Time Library in Place of Protein Standards
LC-MS/MS is a robust platform for quantifying targeted proteins in complex matrices. Typically, quantitative protein assays are developed using comparable synthetic or expressed protein standards to establish method conditions appropriate for measurement of clinical samples. Here a mixture of common proteins was used to develop a retention time library for a set UHPLC method, which Skyline utilized to estimate peptide retention times as well as MRM transitions and instrument parameters from known sequences effected in chicken plasma by Salmonella endotoxins. Utilizing key instrument and software capabilities, a targeted method was quickly developed without the use of comparable standards.
Development and Validation of PreTRM™ - A Multi-Protein Predictor of Spontaneous Preterm Birth
Spontaneous preterm delivery, the leading cause of mortality and morbidity in neonates, lacks an adequately performing diagnostic test. Targeted quantitative proteomics approaches allow for interrogating multiple biological pathways in a single assay. 5,501 pregnant women were enrolled in the Proteomic Assessment of Preterm Risk (PAPR) clinical trial. Utilizing subsets of PAPR serum samples and several QC pools, the performance of 147 candidate proteins was determined in development and verification studies. Using two proteins with acceptable performance, a PreTRM™ score for spontaneous preterm delivery risk was validated.
Antibody-Independent SRM Strategies for Ultrasensitive and Multiplexed Quantification of Cancer Biomarker Candidates
Selected reaction monitoring (SRM)-based targeted quantification provides good selectivity, reproducibility, sensitivity, and multiplexing capability rendering it highly suitable for robust, high-throughput and cost-effective verification of cancer biomarker candidates. The ability of SRM-MS to detect and quantify low abundance proteins present in blood plasma/serum at low ng/mL or even lower levels, however, relies on the front-end enrichment through specific affinity reagents (e.g., antibodies). To address this limitation, we have recently developed two antibody-independent, targeted quantification capabilities (i.e., PRISM-SRM and LG-SRM) that enable protein quantification at low or sub ng/mL levels in plasma/serum. Relevant applications for protein biomarker quantification will be presented.
LC/MS Analysis of Monoclonal Antibody Structure Utilizing HALO® BioClass Fused-Core™ Particles; Multilevel Analysis for Proteins and Glycovariants
The structural characterization of monoclonal antibodies (mAbs) is a challenging and complex task. Efforts have been directed at optimizing high performance HALO® BioClass Fused-Core™ silica materials in order to improve the separations component of LC-MS analysis of proteins, mAbs, glycopeptides and released glycans. Recently improved superficially porous silica packing materials have been applied to HPLC / UHPLC characterization of biomolecules including glycosylation variants from mAbs. HALO BioClass Fused-Core™ materials show considerable utility for analysis of these highly complex molecules, using conditions that permit excellent separations and analysis via LC-MS. Alternatives to standard trifluoroacetic acid (TFA) and formic acid (FA) mobile phase conditions are shown for intact molecule separations, subunit analyses, and for analysis of tryptic digests.
Cleanert HFMF® A New Sample Clean-up Technique for Plasma Sample Prior to LC-MS/MS in Bio-analysis
Plasma represents a major sample form in bio-analysis. The major challenge in the preparation of plasma samples prior to LC-MS/MS is to remove both proteins and phospholipids. This article introduces a new sample pretreatment technique by utilizing Cleanert HFMF£¨hollow fiber membrane filtration£©, a hollow fiber membrane grafted with cyclic acid to filtrate proteins and phospholipids by centrifugation. A comparison study between Cleanert HFMF, PPT, LLE and SPE was carried out in terms of the efficiency to eliminate the matrix effects of proteins and phospholipids. The results showed HFMF enjoys the best capacity for removing the major impurities in plasma.
Matrix Effects Reduction with 2-D Online SPE-UHPLC-ESI-MS/MS for Trace Level Quantitation of Bisphenol A Analogues in Human Urine
In the current study, a 2-D online SPE method has been developed to reduce matrix effects, and therefore, to improve limit of detection (LOD). We found out that the second dimension SPE helped to reduce the matrix effects significantly.
Multichannel Optimization of a New Four Channel HPLC with a Single Mass Spectrometer to Simplify Workflow Complexity and to Improve Throughput of LC-MS
There are four things that matter the most to routine clinical lab operation: the quality of test results, the efficiency of generating those results, the turn-around time to report the results and how much it costs to do all this. Currently, clinical laboratories are facing challenges in handling a large number of samples with crowded lab space and shortage of experienced staff. We address clinical labs’ concerns for speed, efficiency, flexibility and laboratory space restrictions with a compact four-channel HPLC, tandem mass spectrometry and software that streamlines LC-MS workflow.
For in vitro diagnostic use. Not available in all countries.
Advances in Solid Phase Extraction – Removal of Residual Phospholipids Using a Novel Reverse-phase Sorbent
A novel reversed-phase SPE sorbent has been developed that is designed to additionally remove phospholipids from biological samples. Pretreated samples were loaded directly onto the SPE sorbent without conditioning and equilibration. The three-step protocol (load-wash-elute) eliminated >95% of phospholipids compared to protein precipitation. This reduction was shown to have direct impacts on ion suppression of co-eluting analytes.
Fully Automated Broad Spectrum Extraction of Drugs and their Metabolites from Oral Fluid Samples Using Narrow Bore OFX Solid Phase Extraction Columns
Increasingly, clinical and toxicology laboratories are asked to analyze oral fluid samples. The ease and non-invasive nature of oral fluid collection makes it an attractive alternative to more traditional collection of urine or blood. Here, we present a process for robotic pipetting of oral fluid samples, automated extraction of the samples, online evaporation and reconstitution of the extracts (“selective elute and shoot”) and subsequent analysis of the extracts for the presence of a representative list of drugs and drug metabolites. Analyses of the extracts were performed on an AB Sciex 5000 LCMS system with focus on linearity and LLOQ measurements.
All Roads Lead to Robots: Automation of Customized, Effective Trypsin Digestion
Precise protein quantification is essential for both discovery and targeted protein workflows. It requires reproducible protein proteolysis that consistently generates the same peptide in each sample type. Here, we have developed a robotic MS samples preparation workflow by using a Biomek NXP workstation. The workflow is completely hands free with flexible denaturation and proteolysis time for sample specific conditions (e.g. plasma, urine, IPSC, and tissue). It improved reproducibility and throughput by approximately 4 and 7 fold compared to manual processing. We will present expanded automation workflows, to assist in quality control (QC), digestion optimization, and digestion reproducibility testing for large scale LC-MS based analysis.
High-Throughput Preparation of Cellular FAMEs and Sterols for GC/MS Analysis
In recent years, metabolomic and isotopic enrichment analysis has led to breakthroughs in a variety of fields of research including cancer biology, immunology, and aging/regenerative medicine. This research has necessitated the preparation and analysis of ever increasing numbers of samples. There are considerable challenges to preparing metabolomics samples, especially in the preparation of cellular lipid samples. Here we describe a high-throughput procedure for the preparation of cellular fatty acids and sterols that reduces costs, labor and preparation time while increasing sample consistency. The resulting FAMEs and Sterols can then be analyzed by GC/MS for quantification and isotopic enrichment analysis.
A Fast and Effective Quantitation Method for Vitamin A & E from Human Serum Using Novum SLE in Conjunction with a Kinetex Evo C18 Column
Nutritional supplements like vitamins and minerals are critical measure of a patients overall health condition that underwent a gastric bypass and other types of bariatric surgery. In order to quantify the nutritional intake of a patient, we have developed a simple and reliable method to extract vitamin A & E from human serum, using Novum SLE. A Kinetex 5u, Evo C18, 100x2.1 mm HPLC column was utilized to obtain the best selectivity of the two vitamer of vitamin E, alpha and gamma tocopherol along with vitamin A, while a polarity switching technique in mass spectrometric ionization was employed.
Generation of a Novel Digestion Protocol for Enhanced Proteome Coverage
Identification and quantification of biomarkers is often hindered by poor sample solubility and incomplete protein digestion. Clean-up and enrichment strategies can improve analytical sensitivity, however these approaches cannot enhance the rate of trypsin digestion or its reproducibility.
A novel digestion technique has been developed to overcome these barriers by increasing the efficiency and reproducibility of digestion, whilst significantly improving the workflow efficiency.
Improved Speed & Reproducibility of Protein Digestion Using Novel Sample Preparation Technology
Currently overnight in-solution trypsin digestion of proteins is used during peptide mapping; however this protocol requires a number of steps, which can differ between laboratories, making method transfer and data analysis between user groups problematic. Additionally, due to the number of steps required, in-solution digestion can increase the potential for user error. As a result, this methodology often leads to variations in the chromatographic profile and complicates the adoption of robust, generic workflows. Here we describe a workflow including novel, rapid and precise digestion of Cytochrome C, followed by micro-elution solid phase extraction (SPE) clean-up and analysis with next generation UHPLC and high resolution mass spectrometry detection (UHPLC-HRMS).
Rapid Determination of Drug Protein Binding Affinity Using Solid Phase Micro Extraction
In this study, a novel BioSPME micro extraction device is evaluated as a rapid means of determining drug protein binding affinities from plasma. Here, a model set of drugs with varied range of protein binding affinities were utilized to evaluate the utility of the BioSPME sampling technique. Initial studies demonstrate that drug binding affinities can be determined in less than 30 minutes using the micro extraction technique.
Comparison of Several Approaches for Vitamin D Metabolite Analysis
LC-MS/MS method has emerged as a reliable method of choice for vitamin D metabolite analysis. However, it ‘s easily affected by phospholipids content in the serum samples. We present two different vitamin D metabolites extraction methods. One is using Vitamin D extraction plate. The process is simple and high throughput. For 96-well plate , the process time is less than 10 minutes. Most matrix interferences are removed, while the recoveries of mono- and di- hydroxyl vitamin D are good. Another procedure is using polymeric SPE extraction product. The final recoveries of vitamin D metabolites are all above 86%.
Simple Extraction of Antidepressants from Whole Blood for LC-MS/MS Analysis Using Coated Well Plates
Antidepressants are widely prescribed medications for the treatment of depression and other mental illnesses. Although in general having good safety profiles, they still carry some risk of side effects and fatal intoxication due to suicidal/accidental overdose. Therefore, the analysis of antidepressant drugs in blood samples is important in clinical and forensic toxicology. This poster describes the determination of different classes of antidepressants in human whole blood employing a straightforward sample preparation procedure based on the AC Extraction Plate and subsequent LC-MS/MS analysis.
A Novel Drug Screening Protocol for Acidic, Basic, and Neutral in Hydrolyzed Urine Using Supported Liquid Extraction Prior to LC-MS/MS Analysis
Introduction:
Screening urine samples quickly for drugs is typically performed by immunoassay analyzers. Once sample test positive for drug presence then an additional step is needed to qualitatively and quantitatively confirm the identity and amount of drug identified from the initial screening. Ideally, the workflow of a laboratory could be optimized by conducting the screening and confirmation analysis via tandem LC-MS simultaneously. To accomplish this, a reliable sample preparation method is needed to extract acidic, basic and neutral drugs from urine. This poster will outline the details for a novel sample preparation method can be used to extract acidic, basic and neutral drugs from hydrolysed urine samples. Over 32 different drugs were extracted from urine samples quickly and analysed qualitatively and quantitatively.
Effective Extraction Strategies for Buprenorphine and Norbuprenorphine in Urine, Oral Fluid and Whole Blood Using Cation Exchange Solid Phase Extraction and Sup
Introduction: Buprenorphine and Norbuprenorphine are typically problematic for analysis due to analyte stability issues during sample preparation. A fast and robust testing protocol is needed to address extracting the targets out of complex matrices typically encountered during toxicological testing. The sample preparation method would also be expected to yield good analyte recovery and minimum matrix effects. Here, we demonstrate new rapid and reliable sample preparation methods that were used to extract the target analytes from small amounts of complex biological matrix. Qualitative and quantitative data demonstrates the utility of these methods prior to LC-MS/MS analysis.
Solid Phase Extraction Optimization and Separation of Vitamin D Metabolites by LC-MS/MS, for Clinical Research
Many challenges are posed by matrix interferences when analyzing vitamin D metabolites by LC-MS/MS for clinical research. In particular, lysophosphatidylcholines (LysoPCs) cause ion suppression effects in mass spectrometry. Elution profile testing was carried out using Waters® Oasis® HLB and Oasis PRiME HLB µElution plates for the analysis of vitamin D metabolites by LC-MS/MS to optimize and compare SPE conditions to protein precipitation. Oasis PRiME HLB was shown to increase analytical sensitivity of vitamin D metabolites through the removal (>99%) of the targeted LysoPCs when compared to protein precipitation and Oasis HLB. Furthermore, the workflow created was minimal, simply load the protein precipitate, wash and elute.
For Research Use Only, Not for Use in Diagnostic Procedures.
In-line, Automated Method for the Sample Preparation and LC-MS/MS Analysis of Dried Matrix Blood Microsamples for Immunosuppressant Drug Monitoring
LC-MS/MS is replacing HPLC and immunoassays for immunosuppressant drug monitoring to achieve greater sensitivity and specificity. This study evaluated an in-line LC-MS/MS method where 10 µL blood samples collected by a microsampling device (Mitra) were prepared for analysis using a process (MagSi-TDMPREP) to eliminate interferences. Automation of this method resulted in a processing time of 30-45 minutes for 96 samples, robot dependent (4 or 8 span), while resulting in ≥ 80% recovery (RSD <15%) for four immunosuppressants. This method also included the tools required to handle, track, and prepare samples for direct LC-MS/MS injection or batching in vials/microtiter plates.
Comparison of Sample Preparation Strategies for the Extraction of Methylmalonic Acid from Serum Prior to LC-MS/MS Analysis
This poster summarises various sample preparation strategies for the extraction of methylmalonic acid from serum prior to LC-MS/MS analysis. A range of extraction techniques were evaluated: protein precipitation, phospholipid depletion, supported liquid extraction and solid phase extraction using both silica and polymer-based mixed-mode anion exchange chemistries. Analysis was performed using an ACQUITY IClass UPLC interfaced to a Xevo TQS triple quadrupole mass spectrometer via electrospray ionization operating in multiple reaction monitoring mode. Method performance was evaluated for evaporative effects, assay recovery and ion suppression effects using post column infusion experiments. Phospholipid removal was also determined. Full results will be presented.
Catecholamine Analysis: Method Optimization to Improve Sensitivity and Reduce Limits of Quantitation Using LC-MS/MS
Catecholamines are classic biomarkers for the detection of diseases like hypertension, pheochromocytoma and neuroblastoma. The main target analytes - epinephrine, norepinephrine and dopamine, are traditionally analyzed using liquid chromatography with electrochemical detection. This poster discusses the impact of optimization of various parts of the method development process to maximise the sensitivity of LC-MS/MS analysis. A highly sensitive LC/MS system, a Shimadzu Nexera UHPLC coupled to an AB SCIEX 5500 triple quadrupole MS was used for analysis. Method parameters; pre-cursor ion selection, MRM transitions, chromatography and solid phase extraction protocols were optimised for increased sensitivity, allowing quantitation down to 20 pg/mL.
Exploring the Sources of Cross Contamination in 96-Well Sample Preparation Prior to LC-MS/MS Analysis
In all areas of analytical testing it is vital to ensure proper measures are in place to reduce or eliminate cross contamination between samples, which could result in false positive and/or false negative results. Sample carryover in the LC/MS system is usually monitored early in the method development process. However, one area often overlooked is sample preparation. This involves multiple aspects: pipetting, sample transfer, extraction, evaporation and mixing steps. This poster will discuss various stages of the sample preparation process to determine the potential for cross contamination and present approaches to minimize and or eliminate the effect.
Determination of PBDEs in Human Milk by Automated Solid Phase Extraction and Gas Chromatography/High Resolution Mass Spectrometry
Although they have been banned since 2004, PBDEs are still ubiquitous in humans and in wildlife. Due to their possible adverse health effects, biomonitoring programs and research studies require that PBDEs be continuously measured in biological matrices including breast milk, serum, and tissue. We have developed a new method to determine PBDE levels in human milk that improves throughput, precision, and that reduces background contamination. This method has been validated using breast milk samples from an epidemiological study with high, medium, and low PBDE levels measured previously using the traditional method. PBDE levels from the two methods are in agreement.
Fast Determination of 17-Hydroxyprogesterone by LC-MS/MS for Diagnosis of Congenital Adrenal Hyperplasia
STAT 17-hydroxyprogesterone [17-OHP] is commonly ordered for confirmation of congenital adrenal hyperplasia [CAH] mostly in neonates and pediatric patients. Rapid test turnaround time [TAT] is critical, especially in life-threatening salt-wasting cases. Since conventional LC-MS/MS requires a time consuming manual sample preparation step, we investigated commercially available dispersive pipette extraction tips [DPX tips] to shorten the sample preparation time with the aim of building a STAT test to meet a clinical diagnostic need. Results indicate that DPX tips are effective for 17-OHP analysis, and may reduce sample preparation time by 75%. This rapid method is able to meet the STAT test requirement for CAH confirmation in children.
Moving Towards the Standardization of Protein Quantification Workflows and Improving their Analytical Reproducibility
High variability in protein quantification analytical data and a general lack of expertise strongly support the requirement for a standardized, kit-based approach. In this work, commercially available kits were used to quantify monoclonal antibody-based drugs in human and rat plasma. Single digit accuracy, precision, and repeatability data was achieved in experiments performed by different analysts, on different days, and with 5 unique lots of kits. Quantification limits from 10 to 500 ng/mL were achieved for all antibodies tested. Linearity of calibrators was always greater than 0.99 while accuracy and precision of quality control samples was better than 5%.
High Throughput Screening of Urine Marker Codes by Laser Ablation Electrospray Ionization Mass Spectrometry
Laser ablation electrospray ionization mass spectrometry (LAESI-MS) is a technique for high throughput screening from complex liquid matrices. LAESI-MS was used in high throughput screening of urine containing polyethylene glycol markers, which are used in drug screening to assure urine identity. Detectability of polyethylene glycol markers in urine indicate utility for expected concentrations within patient samples. For high throughput screening, data acquisition time was 2.3 seconds per well and 13 minutes for 96-well plate, considerably increasing efficiency.
Automated Comprehensive Urine Sample Preparation Using DPX Extraction on the Hamilton NIMBUS96 with LC-MS/MS Analysis
A high throughput sample preparation method for drugs of abuse (38 compounds) in urine was developed using DPX tip technology coupled with the Hamilton NIMBUS96 robotics system. The sample preparation of two well plates of 96 hydrolyzed samples takes approximately 15 minutes to complete for LC-MS/MS analysis. The method was evaluated for linearity, precision, extraction efficiency, and limits of detection and quantitation. The method established herein is highly reproducible and provides the necessary sensitivity for forensic and clinical purposes.
Automated, High Throughput Quantitative Analysis of 39 Drugs of Abuse in Oral Fluid Using DPX Extraction and LC-MS/MS
A high throughput sample preparation method for drugs of abuse (39 compounds) in oral fluid was developed using DPX tip technology coupled with the Hamilton NIMBUS96 robotics system. A well plate of 96 samples takes 10 minutes to complete sample preparation followed by solvent evaporation, reconstitution and injection into the LC-MS/MS system. The method was evaluated for linearity, precision, extraction efficiency, and limits of detection and quantitation. The method established herein is highly reproducible and provides the necessary sensitivity for forensic and clinical purposes.
Automated Desorption, SPE Extraction, and LC/MS/MS Analysis of Dried Blood Spots
In this report, the complete automation of dried blood spot (DBS) analysis is demonstrated. DBS cards are inserted automatically into a flow through cell in which individual blood spots are rapidly and effectively desorbed. The DBS elution is integrated into a complete cleanup and analysis system using online SPE with replaceable cartridges combined with automated injection to an LC/MS/MS system. Automated DBS extraction methods were optimized for a variety of analytes from rat and bovine blood. The resulting precision and accuracy data are provided.
Enhanced Recovery of Trypsin Digested Proteins Using Dispersive Pipette Extraction for Downstream Proteomic Analysis
For clinical proteomics, targeted peptides/proteins extraction and desalting efficiency are essential to obtaining high quality mass spectra data. In this study, we report a novel peptide purification method using IMCStipsTM, a disposable pipette extraction tip with sorbent loosely contained inside a pipette tip. Using human serum albumin as a model protein, the trypsin digested samples were readily purified using IMCStips™ packaged with reverse phase resin. The purification improved MS peak intensities, S/N ratios and sequence coverage significantly in comparison to other commercially available tips. Similar results have been obtained with other proteins, like human prealbumin, ¦Á-acid-glycoprotein and transferrin. Since this method can be used in an automated online format, we envision it to be applied in the clinical proteomics studies, especially for low abundance peptides.
Quick and Easy Sample Preparation of Urine for the Analysis of Psychoactive Drugs Using the Thomson eXtreme Filter Vials® by LC-MS/MS
This improved sample preparation method allows for the quantitative measurement of psychoactive drugs, Benzodiazepines in urine. The urine samples were prepared using the eXtreme|FV®, followed by LC/MS/MS analysis. The most critical aspects of reliable urine analysis are the reduction of interferences from the sample matrix and analyte recovery. eXtreme|FV®, were compared to SPE for sample preparation to reduce the sample matrix causing interference prior to analysis. SPE is time consuming, adversely impacts recovery, uses large amounts of solvent and are expensive. The improved sample preparation method using the Thomson eXtreme|FV® allows for the analysis of 9 Benzodiazepines.
Hydrolyze Your Way to Compliance – a Call for Pain Management Certified Reference Materials
There are many published studies on the performance of commercially available beta-glucuronidases obtained from different sources used for the hydrolysis of glucuronide-conjugated opioids. We have applied several studies testing three enzymes (beta-glucuronidase from Helix pomatia from Sigma-Aldrich, beta-glucuronidase from Haliotis rufescens from Kura Biotec, and a recombinant beta-glucuronidase from IMCS) for hydrolysis efficiency using spiked standards of parent drug and their corresponding glucuronides. We also performed a selected patient comparison focused on the glucuronide-species of interest, concentrating on parameters that could influence interpretation of results in a clinical setting. We saw up to 12-fold differences in recovery for total codeine, hydromorphone, morphine, and oxymorphone.
Evaluation of Novel Solid Phase Micro Extraction Sample Preparation Method for LC-MS Analysis of Drugs of Abuse in Urine and Plasma Samples for Forensics.
The novel sample preparation method using solid phase micro extraction (SPME) blades was evaluated for analysis of 20 chemically diverse drugs of abuse in urine and plasma samples. Analytes were detected on a hybrid quadrupole-Orbitrap-based mass spectrometer. Urine and plasma samples were spiked with internal standards then diluted with buffer and water, respectively. The sample preparation method consisted of 4 steps which included: SPME blades conditioning, analytes extraction, wash step and elution step. Method was evaluated for limits of quantitation, precision and matrix effects.
Limits of quantitation were in range of 0.1-5 ng/mL and method precision was better than 15% in both urine and plasma. Matrix effects in urine samples were compounds dependent and were in the range of 27.1-100 % recovery. Negligible matrix effects in plasma were observed: 79.0-119 % recovery.
A Novel Automated Sample Prep Process for an Improved LC/MS/MS 25-hydroxy Vitamin D Method
Our aim was to develop and validate an automated front-end extraction process for vitamin D including an improved LC/MS/MS method. Complete automation was achieved with the Tecan AC extraction plate on a Tecan EVO 100 system, whereas 25-OH Vit D3 and 3-epi-25-OH Vit D3 were chromatographically separated with a Phenomenex, Kinetex, 2.6 µ pentfluorophenyl (F5) column on a Waters Acquity UPLC Xevo TQ-S LC/MS/MS system. We demonstrated improved assay efficiency, accuracy, and turn-around-time by reducing the sample prep time from a 2.5 hour manual extraction to a 45 min hands-free process and analytical time from 8 to 4 minutes per sample.
Heat Stabilization Preserves the Molecular Integrity of the Sample
Active enzymes rapidly change the composition of biomolecules after sampling. Subsequent analytical results reflect a mix of the in vivo status and degradation products with increased inter-sample variation. The proteome, metabolites, and existing or potential biomarkers are affected. We describe efficient enzyme inactivation and standardization of sample handling using a heat-stabilization technology to eliminate degradation. Comparisons were made to standard snap-freezing and inhibitors. The results show that heat inactivated samples reflect the in vivo status as closely as possible which enables analyses to differentiate true biomarkers from those degradation products found in any situation where cells are under stress.
Characterizing Matrix Depletion Using a Novel 96-well Format Extraction Media - Tecan® AC Extraction Plate (AC Plate).
We interrogated the AC Plate to characterize serum phospholipid (PL) depletion. Extraction recovery, matrix effect, and chromatography were evaluated for testosterone, 13C3-testosterone, and eight PL MRMs. We compared the effects of varying pH, organic content (% methanol or acetonitrile) and two protein-binding releasing agents (ZnSO4 or LiCl/ammonium formate) in the AC Plate extraction reagent versus simple acetonitrile or ZnSO4/methanol protein precipitation with and without filtration through a Sigma HybridSPE plate. The AC Plate reduces background PL by 4 orders of magnitude compared with simple acetonitrile precipitation while providing sufficient sensitivity to measure 1 ng/dL of testosterone in 100 uL of serum.
The Novel LC-MS/MS System Integrated with Automated Sample Preparation for Drugs Analyses
The limits of traditional analytical methods, such as LC-MS/MS, are progressively being pushed further with regard to robustness, ease of use, sensitivity, throughput, cost effectiveness and accuracy. Here we propose a novel approach to automated sample preparation by combining an on-line automated sample preparation instrument (CLAM-2000, Shimadzu) for LC-MS/MS analysis (LCMS-8040, Shimadzu). In this study, we investigated ability to analyze for antiepileptic drugs and antiarrhythmic drugs using the CLAM-2000 for automated sample preparation for LC-MS/MS. We validated feasibility of this system by measuring precision and accuracy of the drugs which have various chemical properties.
Determination of Urinary Opioids by Solid-Phase Extraction LC-MS/MS for Clinical Research: Comparison of Automated and Manual Sample Preparation
Automated sample preparation improves laboratory operations by a) reducing errors in sample tracking and preparation, b) producing more consistent results free of analyst-to-analyst variation, c) allowing analysts to work more efficiently, and d) minimizing laboratory hazards in regard to solvent exposure and repetitive motions associated with manual pipetting. For labs considering automated sample preparation, the aim of this study was to compare the performance and benefits of a Tecan Freedom EVO® 100 liquid handler to manual sample preparation using a routine clinical research application.
High Throughput Analysis for Novel Oral Anticoagulants Using LC-MS/MS System Integrated with Automated Sample Preparation
LC-MS/MS has become essential tool for monitoring the concentration of drugs in biological samples due to its high level of sensitivity and specificity; however, manual sample preparation often involves several complicated manual steps which can introduce error into the results. In this study, we investigated the ability to analyze for Novel Oral Anticoagulants by LC-MS/MS using automated sample preparation to process large sample sets. We validated the automated method by comparing the data collected on the automated system to the data collected using a manual sample preparation protocol.
The Novel LC-MS/MS System Integrated with Automated Sample Preparation for Drugs Analyses
The limits of traditional analytical methods, such as LC-MS/MS, are progressively being pushed further with regard to robustness, ease of use, sensitivity, throughput, cost effectiveness and accuracy. Here we propose a novel approach to automated sample preparation by combining an on-line automated sample preparation instrument (CLAM-2000, Shimadzu) for LC-MS/MS analysis (LCMS-8040, Shimadzu). In this study, we investigated ability to analyze for antiepileptic drugs and antiarrhythmic drugs using the CLAM-2000 for automated sample preparation for LC-MS/MS. We validated feasibility of this system by measuring precision and accuracy of the drugs which have various chemical properties.
Integration of Steroids Analysis in Serum Using LC-MS/MS with Full-automated Sample Preparation
Currently sample preparation for the detection of steroids in serum by LC-MS/MS involves complex offline extraction methods such as solid phase extraction or liquid/liquid extraction, all of which require additional sample concentration and reconstitution in an appropriate solvent. These sample preparation methods are time-consuming, often taking 1 hour or more per sample, and are more vulnerable to variability due to errors in manual preparation. Our approach to offering a high sensitivity steroid detection method and timely, automated analysis of multiple samples is to use the automated sample preparation system coupled to the detection capabilities of a high-sensitivity LC-MS/MS.
Development and Validation of a Quantitative Method for Doxorubicin/Doxorubicinol in Serum and Saliva: Special Considerations for Working with Unstable Analytes
We describe the development and validation of a method for quantification of doxorubicin and doxorubicinol in human serum and oral fluid. During development of the method, we performed stability studies for this compound in various primary solutions and matrices, based upon previous studies that indicate degradation under varying conditions.
Development and Validation of LC-MS Based Autotaxin Functional Assay and Autotaxin Inhibitors Screening by PUF-LCMS
After identification of the biomarker and treatment target of asthma by LC-MS based lipidomics and we develop and validate LC-MS based Autotaxin functional assay and screen the inhibitors from mixture (botanical extracts) by PUF-LCMS.
Simultaneous Therapeutic Drug Monitoring for Voriconazole, Posaconazole, Fluconazole, Itraconazole, and Metabolites in Human Serum by HPLC-MS/MS
A simple and fast sample extraction and HPLC-MS/MS method was developed for simultaneous quantitation of voriconazole, voriconazole-N-oxide, posaconazole, fluconazole, itraconazole, and hydroxyitraconazole in human serum for therapeutic drug monitoring of antifungal treatments. Sample preparation involves only protein precipitation and dilution; LC-MS/MS method takes only three minutes. The inter-day CVs ranged from 4% to 6%, and the intra-day CVs ranged from 2% to 4%. The recoveries were between 95% and 103% for three concentrations tested. This method is accurate and robust, and offers a cost-effective tool for dosing adjustment for single-drug antifungal regimens and combination salvage therapies on a daily basis.
LCMS Based CSF Neurotransmitter Quantification for Following Psycho-pharmacotherapy in Psychiatric Disorders
Psychiatric disorders such as depression and bipolarity are leading causes of disability and have huge socioeconomic consequences. The molecular mechanisms underlying psychiatric diseases still remain elusive. Pharmacotherapy of these conditions aim at modulating the neurotransmitter equilibrium in the synaptic cleft. However, appropriate molecular markers to monitor psycho-pharmacotherapy effects are not available. We developed a SPE-LCMS method for quantification of neurotransmitters in CSF. Neurotransmitter quantification was employed to establish comprehensive CSF profiles in suicidal patients that underwent psychopharmaca treatment. The data aid to improve diagnosis, prognosis of psychiatric conditions as well as monitor treatment effects and provide insight in the underlying molecular pathology.
Quantitation of Serum Indoxyl Sulfate and P-Cresyl Sulfate in Chronic Kidney Disease by UPLC-MS/MS Method
Chronic kidney disease causes tremendous impact because it increased risk of cardiovascular disease and mortality. Loss of kidney function induces accumulation of potentially toxic compounds, resulting in uremic retention. Indoxyl sulfate and p-cresyl sulfate are important protein-bound uremic solutes which can stimulate the progression of chronic kidney disease. A sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for quantitation of indoxyl sulfate and p-cresyl sulfate in serum has been developed and can be used to follow-up the progression of disease in chronic kidney disease patients.
A Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for the Simultaneous Determination of Serum Estradiol, Estrone, and Estriol
We developed and validated a highly sensitive LC-MS/MS method for the simultaneous measurement of serum E1, E2, and E3 for clinical and research purposes in both women and men. The three estrogens were separated on a selected column within 3 minutes and detected on API5000 with ESI source at negative mode. The assay sensitivity reached 2 pg/mL without using sample derivatization and other sensitivity enhancing techniques. The assay precision (%CV) were 2.6 to 5.6 for E1, 4.3 to 5.2 for E2, and 4.1 to 8.7 for E3. The accuracy (%) were from 95.6 to 99.0 for E1, from 96.8 to 98.4 for E2, and from 94.7 to 97.3 for E3 respectively at spanning different estrogen concentrations.
Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Determination of Tryptophan and Its Kynurenine Metabolites
Maternal tryptophan and kynurenine pathway metabolites have been found to be associated with the risk of pre-eclampsia. In order to explore the role of these metabolites in predicting pre-eclampsia, we aimed to establish a simple and rapid ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to measure tryptophan, kynurenine, and kynurenic acid in both plasma and urine samples. Samples/deuterated internal standards were deproteinized with methanol and injected without derivatization (Acquity Xevo TQ MS, Waters Corporation). Validation of the method was based on Clinical and Laboratory Standards Institute protocol C62-A (Liquid Chromatography-Mass Spectrometry Methods).
Optimization of Derivatization Reaction Used in Sample Preparation Method in Analysis of Methylmalonic Acid in Plasma for Clinical Research
Methylmalonic acid (MMA) is polar molecule that poses challenges for the development of quantitative LC-MS methods. Two analytical approaches have been implemented: analysis of MMA in negative ionization mode and analysis of derivatized MMA in positive ionization mode. The product of the derivatization reaction with n-butanol shows improved reverse phase chromatographic retention and higher ionization efficiency than underivatized MMA, making this method a preferred quantitative solution. The derivatization reaction parameters described in the literature resulted in variable, up to 100 fold, reaction efficiency. Several reaction parameters were investigated and optimized to ensure reproducible and efficient butylation reaction of MMA.
Development of Reference Measurement Procedure for 24R,25-Dihydroxyvitamin D3 and Value Assignment on SRMs of Vitamin D Metabolites in Human Serum
Accurate and precise quantitative evaluations of 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) are important for reliable diagnosis and appropriate treatment of diseases. 24R,25(OH)2D3 is an important vitamin D metabolite used as a catabolism marker and indicator of kidney disease. NIST has developed a candidate RMP for the determination of 24R,25(OH)2D3 in human serum using ID-LC/MS/MS. This method of high precision and high accuracy was used to value assign the concentrations of 24R,25(OH)2D3 in 2 SRMs of Vitamin D Metabolites in Human Serum (SRM 972a and candidate SRM 2973), which can serve as an accuracy base for the routine methods used in clinical laboratories.
Determination of Monosialogangliosides in Human Plasma by a Novel UPLC/MS/MS Assay in Combination with Chemical Derivatization
In this study, a novel reverse phase UPLC/MS/MS method for determination of three monosialoganglioside species, GM1, GM2, and GM3, in human plasma has been developed and validated. This assay employed DMTMM & PAEA chemical derivatization for signal enhancement and D3-labeled monosialogangliosides as internal standards (IS). The analytes and ISs were extracted from plasma using protein precipitation procedure, cleaned up with liquid-liquid extraction, and derivatized with DMTMM & PAEA. Thereafter, the samples were injected into a Shimadzu Nexera UHPLC system interfaced to an AB Scix Qtrap 5500 mass spectrometer that operating in ESI positive and Multiple Reaction Monitoring (MRM) mode to achieve highly sensitive and specific detection.
Fast Analysis of Low pg/mL Level Testosterone in Serum by Bruker TQ LC/MS
Testosterone concentrations in adult females and children (male and female) are an order of magnitude lower than adult male testosterone concentrations and require a sensitive and specific analytical method to accurately determine the testosterone level. A sensitive method for the quantification of total testosterone in serum was developed using Bruker TQ LC/MS system. Excellent sensitivity, linearity and dynamic range were obtained with a LOD of 1 pg/mL, R2 > 0.99, and greater than four orders of dynamic range. The low pg/mL level detection with wide dynamic detection range will cover the clinical research needs.
Determination of Asymmetric Dimethylarginine (ADMA) and Symmetric Dimethylarginine (SDMA) in Human Serum by Ion-Pair Chromatography-Mass Spectrometry
Here we report a reliable LC-MS/MS method for the determination of ADMA and SDMA in human serum by ion-pair chromatography-mass spectrometry. With heptafluorobutyric acid as an ion-pair additive to mobile phases, polar molecules ADMA and SDMA can be well retained on a reversed-phase column. This eliminates the need for pre-column derivatization which is time consuming. Baseline separation of ADMA from SDMA can be easily achieved compared to normal-phase chromatography. The method was validated on an Agilent multi-stream LC-MS/MS System. The method provides not only good linearity, accuracy, and precision, but also increased productivity with the use of multi-stream LC system.
Sensitive and Specific LC-MS/MS Analysis of Plasma Free Metanephrines Using Either On-line or Off-line Sample Cleanup
RECIPE’s CE IVD certified ClinMass® LC-MS/MS Complete Kits – Free Metanephrines in Plasma allow quantitation of metanephrine, normetanephrine and 3-methoxytyramine in human plasma after on-line or off-line sample preparation. Both kits include simultaneous extraction and concentration, while the separation and mass selective detection is achieved using LC-MS/MS. The method shows outstanding validation results for all analytes. Close agreement of analyte concentrations with results reported for RCPAQAP proficiency testing scheme could be obtained. With RECIPE’s MS11000/MS11100 a fast, easy and reliable analysis of plasma free metanephrines has been realized which is very well suited for clinical routine analysis.
Sensitive and Specific LC-MS/MS Analysis of Methylmalonic Acid in Serum, Plasma and Urine
RECIPE’s CE IVD certified ClinMass® LC-MS/MS Complete Kit, advanced – Methylmalonic Acid in Serum/Plasma/Urine (MS5100) allows the sensitive and highly selective quantitation of methylmalonic acid (MMA) in serum, plasma and urine. In samples of healthy patients the concentration of MMA - compared to succinic acid (SA) - is present in low concentrations and often requires off-line extraction or derivatisation before analysis. With our approach, the structural isomer SA is chromatographically baseline seperated and simple protein precipitation is sufficient for sample preparation. Therefore, RECIPE’s MS5100 Complete Kit allows the fast and reliable analysis of MMA and can be easily implemented in clinical routine analysis for methylmalonic acidemias.
Effects of Nutrient Stress on Arginine and Dimethylarginine in Primary Hepatocytes
Neonatal intrahepatic cholestasis caused by citrin deficiency, is characterized by galactosemia, hyperlipidemia, hyperammonemia, and multiple aminoacidemia. Galactose-free formulas are effective for resolving clinical symptoms. This study aimed to investigate the impacts of substitution of 18 mM glucose in culture medium by 9 mM glucose/galactose, 13.5 mM glucose or 9 mM glucose on cellular arginine, dimethylarginine and oxidative stress in primary hepatocyte. The measurement of arginine and metabolites was performed using API 5000 tandem mass spectrometer with positive ionization mode. Oxidative stress increased in hepatocytes cultured in glucose-insufficient medium, compared to 18 mM glucose medium. Intracellular concentrations of arginine and lysine did not change, but citrulline, ADMA, and SDMA increased in AML12 cultured with glucose insufficient medium.
A Dilute-And-Shoot Method for Simultaneous Analysis of Vanillylmandelic Acid, Homovanillic Acid and 5-Hydroxyindoleacetic Acid in Human Urine
Vanillylmandelic acid (VMA), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) are final metabolites of epinephrine, dopamine, and serotonin, respectively. Measurement of these metabolites in urine is used for the diagnosis of carcinoid and neuroendocrine tumors including neuroblastoma, pheochromocytoma, and paraganglioma. The development of a simple dilute-and-shoot and yet accurate, specific, and fast LC-MS/MS analysis would greatly reduce the time and cost for this type of clinical test. Using a Raptor™ Biphenyl column, it was demonstrated that VMA, HVA, and 5-HIAA can be simultaneously analyzed in human urine without extensive sample clean up and resulted in accurate and reproducible quantitation.
High Throughput Quantitation for Therapeutic Drug Monitoring with Open Access LC/MS/MS System
In this paper, we introduce four research use only LC/MS/MS methods for therapeutic drug monitoring (TDM), mycophenolic acid, sunitinib and axitinib, voriconazole, itraconazole, using a mobile phase switching system. With an open access user interface software (Open Solution QuantAnalytics, Shimadzu), analysts can start LC/MS/MS measurement quickly only by selecting a method and placing vials in the specified autosampler plate positions and review the data in office as soon as it becomes available on the designated data server. High-throughput LC/MS/MS methods for TDM with a new data acquisition and processing software was developed.
Development and Validation of a LC-MS/MS Method for the Quantification of the Checkpoint Kinase 1 Inhibitor (CHK1) CCT245737 in Human Plasma
A LC-MS/MS method was developed and validated to quantify CCT245737, a small molecule orally active CHK1 inhibitor, in human plasma. The method requires 20µL of plasma and involve acetonitrile deproteinisation after addition of the labelled CCT245737 (IS). Detection was obtained by SRM, following the transitions m/z 379.8→360.2 for CCT245737 and m/z 384.0→324.2 for the IS. It is sensitive, precise and accurate with overall precision ≤8.5%, accuracy in the range 96%–102% and high recovery ≤93.4%. The LLOQ is 20ng/ml. The assay was validated in the range 20-20000 ng/ml. This is the first method validated to measure CCT245737 in human plasma.
Bioanalytical UPLC-MS/MS Method Development and Validation for Measuring Commonly Used Antimicrobials in Human Blood Plasma
Bioanalytical UPLC-MS/MS method was developed and validated for the most commonly used antimicrobials in England’s intensive care units. The list of the antimicrobials consisted: co-amoxiclav (amoxicillin with clavulanic acid), piperacillin with tazobactam, flucloxacillin, meropenem, ertapenem, ceftriaxone, cefotaxime, benzylpenicillin, clarithromycin and fluconazole. The separation between the compounds was obtained with reversed phase chromatography using the weak ion-pairing agent hexafluoroisopropanol and basic pH (9.5). The method was fully validated - stability, matrix effects, accuracy, precision, linearity, limits of quantification and method’s uncertainty estimation was evaluated.
Simultaneous Analysis of Nineteen Amino Acids in HuangQi Injection Using AQC Pre-column Derivatization and UPLC/Q-tof-MS
It is difficult to separate, qualify and quantitate amino acids in complex matrix. A reliable method using UPLC/Q-tof-MS coupled with AQC pre-column derivatization was established to determine amino acids in Huangqi injection ( a traditional Chinese injection) and its intermediates. AQC derivatization¡¦s product could be analyzed by MS in ESI mode which is superior to UV detection used in other methods. Furthermore, the accurate mass analysis of Q-tof facilitates the identification of unknown amino acids in Huangqi injection. The method provides good separation, reliable qualification and quantification of 19 amino acids in complex matrices.
Ultra-Fast Quantification of Antidepressants in Urine at 9 Seconds Per Sample Using LDTD-MS/MS
According to a 2011 National Center for Health Statistics (NCHS) report, the rate of Antidepressants use in USA increased by almost 400% between 2005 and 2008 for people older than 12 years old. The federal government’s health statisticians figure that about one in every 10 Americans takes at least one antidepressant. By their calculation, antidepressants were the third most common prescribed medication taken by Americans. These numbers are only likely to increase over the years. Using mass spectrometry combined with high-throughput LDTD ion source enhances specificity at equivalent or better speed for the quantification of 7 Antidepressants in human urine. Using only a basic liquid-liquid extraction for the sample preparation, we are able to achieve a robust method with precision and accuracy at a speed of 9 seconds per sample.
Quantitation of 22 Long Chain Fatty Acids by GC-NCI-MS in Serum and Plasma
Long chain fatty acids (LCFAs) play diverse and critical biological roles. Here we present a GC-NCI-MS method for quantitation of 22 LCFAs (C12-C22) in serum/plasma, including the essential and conditionally essential polyunsaturated LCFAs. Hydrolysis was used to release bound LCFAs, which were extracted in hexane and derivatized to form pentafluorobenzyl esters. Our GC strategy gave baseline separation for most analytes, prior to being ionized by NCI and detected in SIM mode. This method offers several advantages upon published studies by using a single injection, additional internal standards, and backflush technology. Validation data and updated reference ranges are presented.
A New Strategy for the Detection of the Family of Benzophenone-3 Structurally Related Compounds and their Metabolites in Human Urine Samples
A sensitive method for testing urinary concentrations of benzophenone-3 (BP-3) developed by our laboratory is routinely used in biomonitoring studies. In this presentation, we describe a new strategy to expand the testing panel to the family of structurally-related compounds using BP-3 as a model system. BP-3 and its analogs are used in personal care products, such as sunscreens, as well as food packaging, etc., to protect the skin and products from UV light. As some of the these chemicals have carcinogenic, endocrine disrupting, or developmental or reproductive toxicity, wide exposure to vulnerable populations, particularly women and infants, present potential health concerns.
Analysis of Flakka and Related Compounds by HILIC, Reversed-Phase and Chiral Chromatographic Modes
In this study liquid chromagtography coupled to mass spectrometry (LC/MS) was applied to develop a method or set of methods that could be utilized for the analysis of Flakka and related compounds in biological fluids. HILIC, RPLC and chiral liquid chromatography were explored. Suitable conditions were obtained in each of the modes using a variety of stationary phase and mobile phase combinations. In addition to the general analysis, chiral conditions were also developed to separate the enantiomers of each of the pyrrolidino variants explored.
Performance Evaluation of the Multiplex Assays for First-line Anti-tuberculosis Drugs in Dried Blood Spots Using UPLC–MS/MS
We developed an UPLC-MS/MS method for simultaneously measuring blood concentrations of ethambutol, pyrazinamide, rifampicin, isoniazid in Dried Blood Spots (DBSs). Each DBS sample was analyzed on the UPLC system and the assay performance was evaluated. All drugs were clearly separated within 3 min. Within-run and Between-run precisions were 6.1%–11.3% and 12.2%-28.8%. Linearity was acceptable for each drug. Inter-assay calibration variability data on five consecutive days showed a linear and reproducible curve in the observed analytical ranges. Favorable correlations between drug concentrations in DBSs and sera were observed except for rifampicin and isoniazid.
A Novel Integrated LC-MS/MS Strategy for the Ultra-sensitive Determination of Catecholamines in Human Peripheral Blood Mononuclear Cells (PBMC)
We develop and validate the first LC-MS/MS method for determination of catecholamines in human peripheral blood mononuclear cells (PBMC). The analytical novelty includes the first solid phase extraction on a 96-well hydrophilic-lipophilic-balanced microplate upon complexation with phenylboronic acid, optimal LC condition and MRM summation, allowing simultaneous quantitation of catecholamines with ultra-sensitivity at 1– 5 pg/mL without requiring derivatization and evaporation. Satisfactory validation results and successful assessment of reference intervals (n = 40) suggested good reproducibility and reliability of the assay. The novel approach, being simple, rapid, sensitive and reliable, will facilitate better understanding of the new arena of neural-immune network.
Improved Sensitivity for Immunosuppressant Monitoring in Two Dried Matrices: A Proof of Concept
We aim to improve the clinical utility of remote collection of dried blood spots (DBS) for immunosuppressant monitoring with an enhanced method requiring less blood in both DBS and Mitra blood collection device. Immunosuppressants were quantified using deuterated internal standards on SCIEX QTRAP 6500. Limits of detection and quantitation were determined, with similar results using 3-mm DBS (3 µL) and Mitra device (10 µL). The method correlated well with previously published method using 8-mm DBS extractions. We believe this new method will eliminate the majority of rejected samples due to QNS and allow more families to participate in the remote monitoring program.
Detecting THC Metabolites and Other Cannabinoids
In the first part, Δ9-THC, 11-hydroxy-Δ9-THC and 11-nor-9-carboxy-Δ9-THC are detected using a simple gradient. In the second part, four cannabinoids, CBD, CBN, Δ9-THC, and Δ8-THC were baseline separated making quantification easy.
Analyte Stability from Dried Blood Collected on HemaSpot-HF™ Devices
Representative analytes including small molecules, proteins and nucleic acids, were examined for their long-term storage stability on HemaSpot-HF devices stored at various temperatures and humidities. Small molecules cortisol, nifedipine, and tolbutamide enzyme protein β-D-Galactosidase and nucleic acids micro RNA; miR155, messenger RNA; β -Actin and ribosomal RNA; 18S were followed over the course of 12 months while stored at temperatures from -20 oC to 45 oC. Shorter term stability trials at >95% humidity were also followed over 4 weeks from room temperature to 45 oC. Analysis of the small molecules was performed by LC-MS/MS as was analysis of the enzymatic activity of β-D-Gal, by monitoring the formation of 4-methylumbelliferone from 4-MU- β-D-galactose. Analysis of the nucleic acids was performed by qPCR.
Use of the HemaSpot HF™ Blood Collection Device to Monitor Cortisol Chronobiology
Cardiovascular diseases (CVD) are the number one cause of death globally, representing 30% of all deaths and the majority of events occur in the early morning. To examine circadian effects on CVD biomarkers, blood samples were collected at 8 time points within a 24 h period with the HemaSpot HF collection device that enables self-collection at home. Cortisol displayed a diurnal expression pattern in all five donors that was consistent over three separate days. Collection and analysis of blood samples at relevant time points over a 24 h time period will provide important data for circadian levels of key analytes for chronic disease.
Ionization Response of Stable Isotope Labeled Small Molecules and the Potential Impact on LC/MS/MS Assays
LC/MS/MS assays of stable isotope labeled compounds and their native counterparts were compared to investigate relative ionization responses. Native, deuterated, and 13C analogs of Testosterone, Pregabalin, and L-Thyroxine were chosen as representative small molecules. Ionization responses were not equivalent between native and labeled compounds analyzed under identical conditions and concentrations. The magnitude of ionization response differences is affected by experimental parameters such as collision energy. Assays performed at multiple collision energies demonstrated the relative ionization responses can be varied by greater than 20%. These ionization response differences must be evaluated, understood, and optimized when considering the use of stable labeled standards for quantitation.
Simple and Cost-effective Generation of LC-MS/MS Interference Testing Materials
Interference testing is a critical component of the method validation process for a routine clinical laboratory test. One source of potential interferences in LC-MS/MS are the phase II metabolites of the analyte. While it is possible to purchase some of the more common glucuronide metabolites for testing, they are expensive and not all are stable. By using human liver microsomes fortified with the appropriate co-factors, the glucuronide conjugates can be produced in sufficient quantities to perform interference testing at a relatively low cost. Results will be shown for steroid hormones Androstenedione, Desoxycortisol, Cortisol, DHEA, Deoxycorticosterone, 17-Hydroxypregnenolone, Progesterone, 17-Hydroxyprogesterone, and Testosterone.
Evaluation of a New Four-Channel LC and a Mass Spectrometer for Open-Access Analysis of Multiple Drug Classes in Clinical Research
To evaluate performance of a new four-channel LC systems we developed short, efficient methods optimized for different classes of compounds including antipsychotics, antibiotics, anticonvulsants, immunosuppressants, antiarrhythmics, and antidepressants in plasma, serum or blood. The methods were implemented on a new 4-channel LC interfaced to a triple quadrupole mass spectrometer. Each LC channel ran a method optimized for a specific group of compounds. This enabled fast, open access style analysis of any group without having to change or re-equilibrate LC columns or mobile phases. The system performance evaluation was assessed by obtaining overall system productivity and also LOQ, precision and matrix effects for individual methods. Stability of the calibration curve over time was also investigated to evaluate open access functionality.
A Liquid Chromatography-tandem Mass Spectrometry Method for the Detection of Beta-carotene in Serum
Beta carotene is a fat-soluble compound routinely measured in serum because of its physiological importance as a vitamin A precursor. Most analytical techniques measure beta-carotene by liquid chromatography coupled to a diode array detector. These analytical techniques, however, suffer from poor analyte specificity and accuracy, and an inadequate variety of commercial kits that are expensive. To address this, we have developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method that measures beta-carotene in serum; a matrix that contains other carotenoids including alpha-carotene and lycopene (isobars of beta-carotene) and a variety of lipophilic molecules.
Quantitation of Antidepressant Drugs and their Metabolites by High-Resolution Mass Spectrometry Utilizing PRM and Full-MS Scan Modes for Forensic Research
Quantitative analysis of antidepressant drugs is performed in forensic laboratories to support criminal cases. We have performed quantitative analysis of antidepressants drugs and their metabolites in human plasma on a Q ExactiveTM Orbitrap mass spectrometer utilizing high mass-accuracy high-resolution mass spectrometry. Quantitation was performed with both full MS scan followed by MS/MS for confirmation and PRM scan modes. The LOQs obtained with both full-MS and PRM scan modes were well below the values required by forensic labs for all compounds examined. Method specificity, linearity and precision were comparable between the two scan modes.
Direct Injection of Serum and Online Solid Phase Extraction for the Quantification of Antidepressants by Liquid Chromatography Tandem Mass Spectrometry
An analytical method for the quantification of 14 tricyclic antidepressants based on direct injection of human serum is reported. The method involves automated addition of the internal standards followed by injection of the serum sample onto an online SPE liquid chromatographer using a Thermo Scientific™ Transcend™ II system. Mass spectrometric detection is performed by single reaction monitoring on a Thermo Scientific™ TSQ Endura™ triple quadrupole using heated electrospray ionization. The method was analytically validated using the MS9050 ClinMass® TDM Platform from RECIPE with the MS9150 Add-On Set for Tricyclic Antidepressants and limit of quantification, linearity range, accuracy and precision were evaluated.
A UHPLC-MS/MS Method for Asymmetric Dimethyl Arginine (ADMA) a Prognostic Biomarker Among Patients with End-Stage Renal Disease
ADMA has been previously implicated in all-cause mortality in patients with ESRD and is useful as a biomarker to predict adverse outcomes in a high risk population. Current methods for measuring ADMA concentrations are not optimized for clinical workflows as they often involve cumbersome sample preparation or lengthy liquid chromatography to separate the symmetric dimethyl arginine (SDMA) isomer. We developed a facile, rapid and reproducible UPLC-MS/MS method for ADMA that is ideal for implementation in a clinical lab. Detection of ADMA and the C13-ADMA internal standard was achieved in ESI mode using an API 3200 (AB Sciex) coupled with a Dionex Ultimate 3000 RS UHPLC system (ThermoScientific). Total analysis time was 7.17 minutes per sample with only 2 minutes sent to the mass spectrometer allowing for multiplexing of the assay to further reduce analysis time.
Structure Specific Immunolabeling and Mass Spectrometric Probing of Amyloid Beta Plaque Pathology in Alzheimer’s Disease
Alzheimer's disease (AD) is a chronic, neurodegenerative disease, of which the underlying pathological mechanism is still not understood. The disease is characterized by accumulation of amyloid-β (Aβ) peptides into different extracellular plaques. Plaques have also been found in non-demented pathological ageing patients. Therefore, discrimination between structural and molecular plaque architecture are of essential interest to resolve Aβ plaque pathology in AD. Here, hyperspectral imaging paradigm employing the Aβ aggregate binding luminescent conjugated oligothiophenes (LCO) in combination with an in-house software was used to differentiate between different types of plaques. The approach was further shown to be applicable for laser microdissection and offline mass spectrometric analysis, which validated the presence of various C-terminal Aβ in the plaques.
Direct Correlation of Cytological Specimens and Lipids with Morphologically Compatible Ambient Pressure Ionization Mass Spectrometry
Cytological specimens are among the most abundant in anatomic pathology services worldwide because they are obtained through non- and minimally invasive methods. However, cytology challenges pathologists because limited sampling strategies that are so appealing to clinicians and patients are small. Thus, the cytologic diagnostic process is amenable to ancillary testing, especially when it does not interfere with classical morphology. Herein we present an ambient pressure ionization technique validated for both morphology and mass spectrometric analysis and compatible with routine cytological processing. We also demonstrate the need to choose a preservative that is amenable both to morphological and mass spectrometric analyses.
Mass Spectrometry Imaging of Biological Tissue Sections and Small Cell Clusters on Nanophotonic Laser Desorption Ionization Substrates
Mass spectrometry imaging (MSI)-based techniques provide versatile spatially-resolved chemical information directly from tissues. Matrix-assisted laser desorption ionization is the dominant technique in MSI, but due the narrow dynamic range of quantitation and the spectral interferences, it is poorly suited for the MSI of small molecules. Here we present the first evidence for a new matrix-free MSI technique using nanophotonic laser desorption ionization (LDI) from a silicon nanopost array (NAPA). In mouse brain and kidney, over ions were mapped and correlated to anatomical features. Additionally, human and microalgae cells were cultured and analyzed on these silicon substrates, allowing correlation of cell number within a given pixel to the corresponding ion intensities. These results demonstrate the utility of LDI-MSI from NAPA for tissue sections and small cell population analysis.
Multi-Modal Mass Spectrometry Imaging for Clinical and Biomedical Research Applications – a Comparison of MALDI and DESI Techniques for Tissue Imaging
Mass spectrometry imaging (MSI) is now increasingly used for clinical research applications due to significant technological improvements that have made the technique more accessible. MALDI, initially introduced by Caprioli et al1, is the dominant MSI technique used today, due to the ability of MALDI to analyse proteins and also its widespread commercial availability. In the last few years, several alternative ambient ionization techniques have been developed that can ionize clinically important molecules such as lipids directly from tissue. One of these techniques, desorption electrospray ionization (DESI), requires minimum sample preparation and is therefore more compatible within a clinical research environment Here we compare and contrast these two imaging techniques which can be operated on an oa-QTOF mass spectrometer.
Polarity Switching Mass Spectrometry Imaging of Healthy and Cancerous Hen Ovarian Tissue Sections by IR-MALDESI
Mass spectrometry imaging (MSI) is a rapidly evolving field for monitoring the spatial distribution and abundance of analytes in biological tissue sections. It allows for direct and simultaneous analysis of hundreds of different compounds in a label-free manner. In order to obtain a comprehensive metabolite and lipid data, a polarity switching MSI method using infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) was developed and optimized where the electrospray polarity was alternated from one voxel to the next. Healthy and cancerous ovarian hen tissue sections were analyzed using this method. Distribution and relative abundance of different metabolites and lipids within each tissue section were discerned, and differences between the two were revealed.
Quantitative Mass Spectrometry Imaging of Chemotherapeutics in Tissue Sections Using IR-MALDESI
Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) is a powerful analytical platform for the visualization of analyte distributions within tissue sections. Recent method development and optimization extended the analytical capabilities of IR-MALDESI MSI to provide quantitative images of xenobiotics in tissue sections. In this work, we present quantitative MSI for novel poly-2-oxazoline (POx) polymeric micelle formulations of paclitaxel (PTX) in a mouse model. The concentration and distribution of PTX in tumor, spleen and liver was determined using MSI with concentrations validated with LC-MS/MS analysis of serial sections.
Phospholipids as Potential Biomarkers for Ovarian Cancer: Spatial Localization and Use in Diagnosis
Ovarian cancer is the fifth most common cancer among women, mainly due to the poor and vague prognosis and diagnosis. DESI-MS is an excellent technique to characterize different cancer types. It provides detailed spatial information within the sample, providing the opportunity to investigate tumour biology from an entirely new perspective with accurate biochemical information about each tissue type. Using DESI coupled to a TQ for diagnosis makes this technique quicker and less expensive. Furthermore, it opens the possibility to use DESI for targeted imaging and quantification.
Rapid Evaporative Ionisation Mass Spectrometry (REIMS) in Endoscopy: Preparing for Clinical Translation
Rapid Evaporative Ionisation Mass Spectrometry (REIMS) in endoscopy utilises the aerosol released during electrosurgical tissue dissection through integration with an endoscopic polypectomy snare. A significant barrier to its clinical usefulness is the potential delayed signal transfer and loss of signal intensity resulting from aspiration of aerosol from a closed cavity with a long sampling line. We show experiments comparing a novel direct method for introduction of aerosol to the mass spectrometer. The novel method produced a robust signal comparable to the original Venturi setup, while reducing cut-to-signal time by >50%. It has shown good results in 10 procedures on patients.
Ambient Mass Spectrometry Imaging for Assessing P53 Mutation Status in Breast Cancer
TP53 gene mutations are the most frequent genetic alterations in cancer, occurring in 20%-30% human breast cancer. Cancer bearing TP53 mutations are prone to be aggressive and are associated with poor overall and disease-free survival in breast cancer. In this study, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was utilized to chemically map breast cancer tissues, including p53 mutations positive tumor tissues, p53 mutations negative tumor tissues, and normal breast tissues. The methodology allowed prediction of the occurrence of TP53 gene mutations directly from breast cancer tissue based on the detection of specific lipid patterns, and may become a valuable method for assessing p53 status in clinical specimens.
DESI-imaging: Histology Applications
Desorption Electrospray Ionization (DESI) has been utilized across several subjects ever since its development in 2004. One major potential application of this break-through technique is as a diagnostic tool for cancer. By looking at biochemical composition of tissues, DESI-MSI is able to differentiate between types of tissue, cancers and cancer stages hence providing a simpler, faster, user independently diagnosis having major implications in cancer management and costs.
Targeted Protein Quantification from FFPE Tumor Tissue Using Mesodissection and Liquid Tissue SRM Assay: Comparison to a Laser Microdissection Platform
Many oncology therapies target specific proteins; therefore, technologies to quantify tumor-specific protein targets are required. Our current platform uses laser-assisted microdissection to isolate and collect tumor cells for analysis from FFPE tissues mounted on DIRECTOR® slides. However, sections cut onto glass microscope slides are often the only materials available, limiting the utility of high resolution laser microdissection. In this study, we present the evaluation of a tissue mesodissection platform to effectively dissect tumor cells standard glass slides, followed by Liquid Tissue® SRM assays to simultaneously quantify 26 protein biomarkers. Quantitative results from mesodissection and laser microdissection are compared.
Automated Tumor Typing of Tissue Sections Based on MALDI Mass Spectrometry Imaging Data and Machine Learning Using Characteristic Spectral Patterns
We present an automated classification method for MALDI mass spectrometry imaging data with applications to tumor typing of FFPE tissue sections. The proposed method consists of a) data pre-processing, b) identification of characteristic spectral patterns using non-negative matrix factorization (NMF), and c) applying linear discriminant analysis (LDA) for classification. We apply this method to the discrimination of breast, lung, colon and pancreas cancer. MALDI data has been acquired from eight tissue micro arrays (TMAs), two for each tumor type, with a total of 943 cores from 285 patients. Four TMAs have been used for training, the remaining four TMAs for validation. A sensitivity on core level of 100.0% (lung), 99.5% (pancreas), 100.0% (colon), and 100.0% (breast) was achieved. Only limited effects of different preprocessing variants (normalization, filtering) were observed.
Translational Bioinformatics for Mass Spectrometry Imaging in a Clinical Research Setting
Mass Spectrometry Imaging (MSI) is an emerging technology in pathology research that generates hundreds of gigabytes of detailed molecular data of potential importance. Effective translation of MSI data into biologically or clinically useful information requires advanced computational solutions. Here, an integrated bioinformatics research platform is presented that allows intuitive histology-directed interrogation of MSI datasets for tissue-specific biomarker recovery, automated tissue classification and entropy driven tumour heterogeneity assessment.
Quantitative Imaging of Platinum Based on Laser Ablation-inductively Coupled Plasma-mass Spectrometry to Investigate Toxic Side Effects of Cisplatin
This work presents a quantitative bioimaging method for platinum based on laser ablation-inductively coupled plasma-mass spectrometry and its application for a biomedical study concerning toxic side
effects of cisplatin. To trace the histopathology back to cisplatin, platinum was localized and quantified in major functional units of testicle, cochlea, kidney, nerve and brain sections from cisplatin treated mice. The direct consideration of the histology enables precise interpretation of the Pt images and the novel quantitative evaluation approach allows significantly more precise investigations than the pure image.
Improving Biochemical Content - Discriminating Lipid and Metabolite Distribution Using DESI and High Resolution Mass Spectrometry in Healthy and Diseased Tissue
The determination of the distribution of lipids and metabolites in tissue samples is of seminal importance to understanding disease and biochemistry. DESI imaging is important for its ability to provide objective molecular information in histological context without perturbing the integrity of the tissue. The majority of the experiments done using DESI have provided nominal mass information. Both lipids and small molecule metabolites have many nominal mass isobars including phosphatidylserines, cholines, and sulfatides which can differ by less than 50 mDa. Here the need for accurate mass (< 3 ppm), high mass resolution (> 40,000) information are demonstrated. Examples showing differential tissue distribution of isobaric metabolites and lipids differing by < 50 mDa are provided in a variety of tissues and the benefits to understanding disease and fundamental biochemistry are discussed.
Unified Drug Testing by Online SPE-LC/MS/MS for High Productivity & Ease of Use: One Totally Automated Method Measures ALL Drugs in Urine & Oral Fluids
Continued growth of LC/MS/MS for drugs of abuse in urine and OF seems certain. However, there are technical challenges that need to be met. These include easily measuring low dose drugs at or near 1 ng/g concentration (medical purposes, Pesce, 2012 AACC conf,& zero tolerance testing), simplicity performing measurements with lab technicians, and ability to achieve high productivity for all work while minimizing the labor and number of workflows required.
To meet these needs, we have developed an automated on-line SPE-LC/MS/MS method. It uses SPE to clean / pre-concentrate samples so that low dose drugs at or near 1 ng/g concentration are easily measured at S/N ≥20. At the same time, the method’s design is balanced to address all of the drugs (acidic/basic drugs & polar / non-polar drugs), as well as urine and/or OF samples, all in one method, all in one workflow.
Rapid and Sensitive Analysis of a 93-Compound Forensic Panel in Urine
In this poster, we describe a rapid and sensitive analysis of a comprehensive forensic compound panel in human urine using ExionLCTM AC and QTRAP®/Triple QuadTM 4500 LC/MS/MS system. This forensic panel contains 93 compounds with both ionization polarities therefore the method involves fast polarity switching. There are a total of 212 MRM transitions in the method; monitoring 2 transitions per analyte and 1 transition for each internal standard used and the total LC runtime is 6.5 minutes (can be shortened for smaller panel). Sample preparation is based on enzymatic hydrolysis and a simple “dilute and shoot” methodology.
A Coupled Analysis of Low and High Abundance Isotopes Extending the Dynamic Range of an Assay for Methamphetamine in Meconium via LCMSMS
A one-hundred fold increase in the dynamic range of an assay measuring methamphetamine in meconium has been achieved by relying on the natural isotopic distribution of carbon-13. Transitions corresponding to the predominant methamphetamine isotope (12C10-methamphetamine) are utilized for the lower region of the assay (5-1000 ng/g). Transitions corresponding to 13C2, 12C8-methamphetamine are additionally monitored, effectively reducing the analyte signal based upon a 0.54% probability that any given molecule of methamphetamine will possess two 13C isotopes. By monitoring the uncommon isotope, quantitation is extended from a ULOQ of 1000 to 100,000 ng/g with acceptable peak shape, and consistent qualifier ratios throughout.
Point-of-care Identification of Ingested Intoxicants by Thermal Desorption Electrospray Ionization/Mass Spectrometry in the Emergency Room
To expedite rescue of intoxicated patients in the emergency department, we developed an analytical method for rapid identification of ingested intoxicants by thermal desorption-electrospray ionization mass spectrometry. Since no pretreatment of the specimen is required, the whole analytical process could be completed within 15 seconds. This technique, together with informational support provided by online mass spectral database, allows for early non-invasive point-of-care identification of ingested intoxicants in the oral fluid or gastric lavage content of intoxicated patients, and is promising in providing important toxicological information for decision-making during critical resuscitation in a timely manner, and hence ensuring the appropriateness of the succeeding management.
Immunosuppressant (Tacrolimus/Cyclosporin A) Monitoring by LC-MS/MS Using Mitra Microsampling Devices
Tacrolimus and Cyclosporin A are immunosurpressants commonly prescribed for solid organ (cardiac, liver, renal) transplants. Therefore, therapeutic drug monitoring of these medications is important based on their narrow therapeutic range where suboptimal concentrations can lead to organ rejection and elevated concentrations can lead to toxicity. Since transplant patients need to take these medications for life, this study looked at the feasibility of measuring Tacrolimus and Cyclosporin A using a high-performance liquid chromatography tandem mass spectrometry assay using driedblood from a microsampling device (20 mcL) compared to a validated method using 200 mcL venous collected EDTA whole blood.
Enzyme Hydrolysis of Haloperidol Glucuronide; a Major Urine Metabolite of Haldol®
Haloperidol (Haldol®) is a typical antipsychotic prescribed for the treatment of acute symptoms of schizophrenia. adherence to haloperidol therapy is monitored by evaluating levels of haloperidol and one or more metabolites reported to be present in urine at approximately 2 and 4% of the total dose. Unchanged Haldol® has been reported to be present in urine at less than 1% of the administered dose with no evidence of glucuronidation of the parent drug (Baselt, 10th ed). This work demonstrates that the parent drug is excreted in urine as the glucuronide and that hydrolysis can yield much higher levels of parent drug upon LC/MSMS analysis than observed upon direct injection.
Analysis of a Toxicology Panel Using High-efficiency Cortecs Phenyl Columns
High-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become a powerful tool for quantitative analysis of drugs of abuse in the field of forensic toxicology. While many reversed-phase columns have been used for these applications, columns that contain a phenyl functionality have shown to have unique selectivity for many drugs of abuse due to pi-pi bond interactions between the aromatic rings contained within the target analytes and the stationary phase. This poster highlights a novel, high efficiency 1.6 µm solid-core column for the separation of opiods, benzodiazepines, amines and PCP.
Evaluation of Lab Developed Test for Simultaneous Determination of Sirolimus and Everolimus by Liquid Chromatography Tandem Mass Spectrometry
We developed and validated lap developed test for simultaneous determination of sirolimus and everolimus using liquid chromatography tandem mass spectrometry (LC-MS/MS). The accuracy, limit of quantification, precision, linearity, and carry-over were evaluated. The accuracy, precision, and linearity were excellent. The limit of quantification and carry-over were acceptable. But, result of proficiency test was unacceptable. After calibrator change, the concentrations of PT materials were within acceptable range. Although the LC-MS/MS assay shows excellent analytical performance, attending PT program is helpful for harmonization with peer group.
The Application of DPX WAX Tips in Clinical Toxicology for Protein Precipitation
A novel method for the protein precipitation of serum was investigated using WAX dispersive pipette extraction (DPX) tips and analyzed by LC-MS-MS. Significantly, protein precipitation was performed within the DPX WAX tip to remove matrix interferences in less than 2 minutes. Viability was determined by testing the method on a comprehensive panel and comparing the results to standard protein precipitation preparations. Comparison between the two methods revealed that highly lipophilic compounds (LogP > 4) can be predicted to achieve a superior sample cleanup using the DPX WAX tips.
the Application of Untargeted Data Acquisition for Identifying Emerging Psychoactive Substances in Clinical Samples
Untargeted high resolution accurate mass spectral data acquisition, combined with untargeted or targeted data analysis, is used for the systematic investigation of Novel Psychoactive Substances – NPS- in clinical samples. We will demonstrate the utility of this analytical method by detecting emerging psychoactive substances such as novel synthetic cannabinoids and phenethylamines, in anonymised urine and plasma specimens. This work will enable the collation of information about NPS implicated in episodes of acute toxicity in the UK, assisting patient management.
Rapid, Simplified and Highly Efficient Analysis of Urinary THC and Metabolites Using a Novel Reversed-phase Extraction Sorbent
A novel reversed-phase SPE sorbent has been used to rapidly analyze urinary THC and its major metabolites. Extraction of pretreated samples was rapid and UPLC/MS/MS analysis was complete in less than 3 minutes. Extraction recoveries were efficient and consistent, and matrix effects were minimal. All quantitative parameters such as linearity, accuracy, and precision were well within established limits for bioanalytical and forensic methods. This method represents a simplified and highly efficient approach for the analysis of these important compounds.
An Ion Mobility Screening Approach for the Detection of Toxicologically Relevant Substances
The utility and potential benefit of collision cross-section (CCS) data, as generated through use of ion mobility separation (IMS) techniques, was evaluated for >50 toxicologically-relevant substances. Solvent standards were analysed using an established UPLC-method in combination with an IMS-QTof (Waters). Reference CCS values were measured and then incorporated into an existing toxicology library comprising retention time and exact mass data. Collision cross-section measurements were shown to be highly reproducible (within 2% of reference), both in the presence or absence of biological matrix. Use of IMS led to cleaner, low and elevated energy spectra due to the alignment of ions in drift time. CCS values provide an additional dimension of specificity which proves valuable in systematic toxicological analysis.
Method Validation for Nicotine and Its Metabolites by LC-MSMS Reveals a Low Clinical Utility for the Tobacco Alkaloid Anabasine
The need to provide accurate and quantifiable data in the detection of patient adherence to smoking cessation programs necessitates the utilization of high-resolution instrumentation. Reliance on immunoassay-based approaches can lack the sensitivity and specificity to provide a complete clinical picture in the context of various nicotine replacements and smoke cessation therapies. In the healthcare setting, the ability to provide clinicians with this information can have a significant impact on organ allocation and transplantability in addition to informing surgical eligibility. Anabasine, an alkaloid present in tobacco plants, is often utilized as a primary marker of active smoking. Surprisingly, during our validation of a new LC-MSMS assay, we determined that this utilization resulted in a very high false negative rate when compared to patients’ self-reported smoking status.
Utility of Suspect Screening by High Resolution Mass Spectrometry: Adulterated Xanax
A series of patients in the San Francisco Bay Area were treated for complications after ingesting counterfeit Xanax tablets purchased illegally off the street. Serum, urine, and two counterfeit tablets were analyzed using a previously validated method on an ABSciex TripleTOF®5600 system. Targeted analysis, i.e. analysis by known accurate mass, retention time, isotope and fragmentation pattern, revealed the presence of fentanyl in these tablets and biological samples. Suspect analysis, i.e. analysis by exact mass and theoretical isotope pattern, preliminary identified etizolam, a non-FDA approved benzodiazepine analog. Presence of etizolam was later confirmed using a reference standard. This case exemplifies the utility of suspect analysis in clinical toxicology by preliminary identification of a compound that was not identified by targeted analysis.
Toxicology Testing in Complex Patient Populations Requires Definitive Testing
Our objective was to assess the differences in detection rates in a diverse population for the screen and confirm paradigm versus direct to definitive testing for urine toxicology testing.
4794 urine specimens were qualitatively screened for the presence 17 different drug or drug classes on the Olympus AU680 analyzer (Beckman Coulter, Inc., Brea, CA). All samples were then quantitatively tested on an LC-MS/MS platform (Sciex, Framingham, MA or Agilent Technologies, Santa Clara, CA) for the free and conjugated forms of 74 analytes. Data were reviewed to determine what positive specimens would have been missed if only screened positive results were reflexed for LC-MS/MS testing.
With the current complexity of pharmaceuticals immunoassay testing missed a compound in 44% of the specimens. Mass spectrometry methods are essential to obtain an accurate results for treatment decisions.
Qualitative Analysis for Multiple Drugs in Urine by Liquid Chromatography Time-Of-Flight Mass Spectrometry (LC-TOF/MS)
Liquid Chromatography Time of Flight Mass Spectrometry (LC-TOF/MS) allows for the high-throughput detection of multiple drugs and non-targeted analysis making it a good choice as a primary screening tool. LC-TOF/MS was utilized here as a broad-spectrum screen to determine the drug involvement in cases of accidental or intentional overdoses. This comprehensive test includes a simple dilute and shoot method identifying the presence of 111 specific pharmaceutical compounds, illicit drugs and their metabolites in a patient’s urine. The investigation of the utility of reference mass infusion to monitor individual sample ion suppression will also be discussed.
LC-MS/MS Analysis of 25 Opioids from Dried Urine Spots
Dried urine spots are not commonly used in the clinical toxicology laboratory despite being a convenient and economical alternative to traditional liquid specimens. Here we describe the development of an LC-MS/MS assay for detection of 25 opioids (including pain management drugs, drugs of abuse and designer drugs) from DUS.
Efficient Forensic Toxicological Screening and Quantitation Workflow Using QTOF LC-MS/MS System
Quadrupole Time-of-Flight (QTOF) mass spectrometry is becoming the desired technology for sensitive and selective screening workflows in a forensic toxicological setting. It affords accurate mass and mass resolution capabilities combined with sensitivity as well as the capability to perform simultaneous highly specific targeted quantification and non-targeted screening and the ability to perform retrospective data mining. Here we describe a new QTOF system with intuitive new software for easy adoption of accurate mass technology to forensic testing. In this paper we demonstrate that the new hardware and software combined allow the highest level of confidence for compound identification and quantification from blood and urine samples.
Cost Effective Dilute-and-shoot Approach for Determination of Illicit Drugs in Oral Fluids Using LC-MS/MS
Due to a recent increase in the demand of Oral Fluid analysis, many challenges have been set forth in developing robust cost effective assays for determination of illicit drugs. As an alternate matrix to traditional urine for monitoring drugs, oral fluids poses far more challenges due to stability, detection limits and sample clean up. A method was developed for cost effective, dilute and shoot assay with linear ranges of 1-300ng/mL for selective drugs with R2 > 0.99 was validated.
LDTD-MS/MS Method for Quantitative Analysis of Four Immunosuppressant Drugs in Whole Blood and Cost Analysis Comparison to LC-MS/MS
Accuracy, turnaround time, and analytical cost are important factors to consider when developing a therapeutic drug monitoring assay for immunosuppressive drug therapy. Sirolimus, Cyclosporin A, Tacrolimus, and Everolimus therapies are monitored to balance therapeutic efficacy and prevent organ rejection, while minimizing the adverse effects associated with high concentrations in whole blood. Laser Diode Thermal Desorption Tandem Mass Spectrometry (LDTD-MS/MS) technology can provide rapid results and reduced analytical costs associated with mobile phases and liquid chromatography columns. An eight second LDTD-MS/MS method was developed for the quantification of four immunosuppressive drugs, in whole blood. Method validation data and cost analysis are presented.
An Investigation into Removing the Excipients from Select Oral Fluids Collection Devices by SPE and LC/MS Detection
Oral fluids present a convenient sample to collect and generally a non-invasive method of sample collection. Furthermore, OF is becoming increasingly popular in clinical and forensic laboratories. To address the demand, there are also increasing number of sample collection devices and some are offered with a preservative solution. These solutions may contain varying degree of excipients to maintain constant pH, stabilize the analytes and/or prevent bacterial growth in the samples. The nature and concentration of these excipients may pose difficulties with LC/MS detection of the analytes. We investigated multi-mode SPE media to substantially reduce or eliminate the preservative solution excipients.
Amphetamine- and Methamphetamine-like Compounds Identified in Urine from an Over-The-Counter Dietary Supplement
Two compounds were identified in urine specimens from a pain management clinic that resembled both Amphetamine and Methamphetamine. Upon speaking with the physician, it was noted that the patient denied illicit Methamphetamine use, but admitted to taking an over-the-counter dietary supplement known as “Meltdown”. Two of the compounds listed in the ingredients were R-Beta-Methylphenylethylamine and Synephrine HCl. This work shows the similarities between the ingredients in the dietary supplement with Amphetamine and Methamphetamine that were discovered through a SPE LC-MS/MS procedure, and how accurate identification is achieved.
Quantitative Analysis of Human Tear Fluid by MALDI-TOF Mass Spectrometry
Identifying biochemical markers for disease is a difficult endeavor. It requires methods that can quantify multiple components simultaneously, are high throughput, cost effective and deliver high precision and accuracy. We and others are exploring the potential of matrix-assisted laser desorption/ionization (MALDI) for these applications. The approach developed in this study is a non-targeted MALDI based method to analyze human tear fluid. Our results demonstrate that precise measurements are achievable (CV values 10% or less) despite the challenges of internal standard selection, sample handling, and ion suppression. This method gives us the ability to detect, identify, and quantify biomarkers that relate to eye injury and disease. The results of this study will further reinforce the effectiveness of tear as a diagnostic tool and the potential of MALDI-TOF MS for clinical applications.
The Role of UHPLC-MS/MS in Preclinical and Clinical Studies of Drug Interactions with Botanical Dietary Supplements
Inhibition or induction of cytochrome P450 (CYP) enzymes and transporters are responsible for most drug-botanical interactions, and MS-based assays facilitate the pre-clinical and clinical assessment of these interactions. For inhibition of CYP enzymes by botanical dietary supplements, we utilize UHPLC-MS/MS with CYP substrate cocktail inhibition assays to measure activity of multiple CYP isoforms. Preclinical models of induction of CYP enzymes and transporters are carried out using human hepatocytes supported by UHPLC-MS/MS based functional CYP assays. To confirm interactions predicted in vitro, clinical trials of drug-botanical interactions are carried out based on pharmacokinetic studies of known CYP substrates supported by UHPLC-MS/MS.
Molecular Basis for Polycystin-2 Channel Regulation and Assembly via Its C-terminal Tail
The C-terminal tail of polycystin-2 (PC2 Cterm) is crucial for channel regulation. Mutations in PC2 Cterm can cause autosomal dominant polycystic kidney disease. PC2 forms a calcium-permeable channel in the membrane and its function is regulated by cytosolic calcium-levels. To gain insight into how calcium-binding regulates the channel via its C-terminal tail, we characterized the conformational and dynamic responses of calcium within the PC2 Cterm, using hydrogen-deuterium exchange mass spectrometry. Our study, for the first time, provides a complete map of dynamic responses to calcium-binding within the full length C-terminal tail. Our results suggest mechanisms for functional regulation of the PC2 channel and the roles of PC2 in pathophysiology of polycystic kidney disease.
A Split Hair Comparison of Human Hair Cortisol Levels Using an Immunoassay versus Liquid Chromatography-Mass Spectrometry
Measuring hair cortisol levels in humans to determine long term systemic exposure is increasingly being used as a biomarker of chronic stress. Since human hair has a fairly uniform growth rate (~1 cm/month), one can investigate periods of time in which stress is absent, present, or most pertinent. The objective of this study was to develop and validate an LC-MS/MS method for the measurement of cortisol in hair and compare the method to the Siemens Centaur cortisol competitive immunoassay.
Translational Research Workflows on LC-HRAM Platform for Detection of Pathogen Induced Cancer in Human T-Cell Leukemia Virus Type 1 Disease
Exosomes are micro vesicles secreted by the cell membrane and their contents could be used as an effective means to detect many different cancers. By profiling the Exosome cargo from blood samples of patients from different stages of Leukemia we are exploring for Leukemia specific biomarkers that are secreted into the blood. An integrated profiling of the microRNA, messengerRNA and protein content of the exosome would lead to unique signatures that make exosomes ideal for cancer detection. Here, we review the unique proteomic contents of exosomes originating from Leukemia cancer cells as well as their functional effects to promote tumor progression.
Investigating the Lipidomic Profile Derived Through Rapid Evaporative Ionisation Mass Spectrometry of Breast Tissue Samples
Rapid Evaporative Ionisation Mass Spectrometry (REIMS) is a technique used to analyse ions produced in electrosurgical smoke. This technique has recently been developed for near real time identification of normal and cancerous tissue in the form of the iKnife technology. The technology uses multivariate statistics for computational analysis of the whole lipid profile detected in the spectra derived from tissue. Here we describe how REIMS lipid profiles obtained from ex vivo breast specimens can be matched to other datasets to provide information on differences in lipid composition of breast tissue when compared with data obtained through DESI-imaging of the same clinical samples with confirmed histology.
Rapid Evaporative Ionization Mass Spectrometry During Brain Surgery: Our Experience of Real-Time Intraoperative Tumour Characterisation
Rapid intraoperative identification of brain tumor tissue has the potential to improve the extent of resection of brain tumors and reduce damage to normal tissue thus improving survival and making surgery safer.
A single centre study was designed utilizing Rapid Evaporative Ionisation Mass Spectrometry with 3D Ultrasound Neuronavigation to help accurately characterize brain tissue.
Precise intraoperative readings from different tumor zones were taken and compared to matched core biopsy samples verified by routine histopathology.
This has revealed unique in-vivo spectra for different intrinsic brain tumors and normal brain tissue. Intra-tumoral variations may shed important light into intrinsic brain tumor biology.
Successful Implementation of Immunosuppressant Drugs (ISDs) Monitoring Using Liquid Chromatography Mass Spectrometry (LC-MS/MS)
We hereby report our experience at Children’s Hospital Los Angeles (CHLA) in implementing therapeutic monitoring of ISDs including Tacrolimus, CyclosporineA, Sirolimus and Everolimus using LC-MS/MS. A quantitative multiple reaction monitoring (MRM) analytical method was validated on a Thermo-PRELUDE-TSQ-QUANTIVA LC-MS/MS including accuracy, precision, analytical sensitivity, reportable range, and recovery. In summary, a very simple, fast, sensitive LC-MS/MS method was successfully implemented in three months, which, when compared to the previously used immunoassay method, resulted in better optimization of therapeutic drug levels, decreased sample volumes, and significant cost savings.
Characterization of the Host Proteome Within Virion Particles of Sindbis Virus
As virus particles bud from an infected cell, they contain not only virus-specific protein and nucleic acid, but they also incorporate host proteins. While this phenomenon has been observed for a handful of viruses, it remains unclear if host proteins are specifically packaged and if they are critical to the viral life cycle. In this study, the protein content of a prototypical alphavirus, Sindbis virus, was analyzed by liquid chromatography mass spectrometry to determine if host proteins are specifically packaged by these viruses and if these host proteins are critical for the viral life cycle.
Application of Microfluidic Tandem Quadrupole LC-MRM-MS Based Translational Research Analysis of Putative Heart Failure Peptide Biomarkers in Human Plasma
The application of tandem quadrupole mass spectrometry with integrated microfuidic chromatography (IonKey) for the analysis of proteolytic peptides in human plasma is described. A tandem quadrupole platform was used for its performance in terms of sensitivity, precision, and linearity and IonKey was selected due to its balance of sensitivity and throughput. This LC-MS configuration was utilized to demonstrate that proteolytically digested, non-depleted plasma samples from heart failure patients could be classified with good discriminative power using a subset of proteins previously suggested as candidate biomarkers for cardiovascular disease.
Targeted SPE-UPLC-MS/MS Analysis of Oxylipins: from Profiling to Quantification for Translational Research Studies
Oxylipins are signaling molecules that play a role in the regulation of many key biological processes, most notably inflammation. Here, we describe targeted, quantitative SPE-UPLC-MS-MS assays for the analysis of various oxylipins subsets and classes. These subsets represent down-stream products or oxylipins from particular metabolic pathways. Matrix samples were prepared using mixed mode OASIS MAX µElution SPE and analysed using an ACQUITY UPLC I-Class system interfaced to a Xevo TQS Micro Tandem Quadrupole Mass Spectrometer. We demonstrate these methods to be sensitive, selective, linear and precise and therefore suitable for use in translational research studies.
Development of a Routine Hemoglobin Profiling Workflow
Routine hemoglobin profiling is used to determine putative disease states based on glycation and/or sequence variation. Thus, analytical workflows must be sensitive to detect low-level variants and selective to characterize compounds as truncated chains, modified, altered sequence, or any combination. To achieve this, we have developed a workflow incorporating sample preparation routines using cation exchange resins to rapidly and robustly concentrate, remove matrix interference, and facilitate molecular weights cutoff extraction centered at hemoglobin chains. To test this workflow, we have evaluated neat whole blood (WB) as well as WB spiked with bovine hemoglobin at various levels.
Development of a Robust, High-Throughput Sample Preparation Method for Peptidome Biomarker Analysis
Peptidomic profiling offers an alternative approach to identifying native peptide biomarkers in various fluids. The peptidome contains analytical targets preserving the endogenous formation such as signaling peptides and can be used to identify the specific proteolytic cleavage sites or unique PTMs. We present a comprehensive sample preparation, data acquisition, and analysis for studying the urine and plasma peptidome. Experiments were performed using bottom-up sample preparation and analysis to build the sub-proteome used to process the top-down data. Evaluation of the depth and reproducibility of the method is presented as compared to standard data processing approaches.
Detection of Lyme Disease Infection Through Quantification of Borrelia burgdorferi Membrane Proteins
Lyme disease is a tick-borne illness that is caused by the bacteria Borrelia burgdorferi. Diagnosis of Lyme disease in its early stages is problematic, and it generally takes several weeks before Borrelia-specific antibodies can be detected. Untreated individuals with Lyme disease can develop serious complications. We investigated the detection and quantification of B. burgdorferi membrane proteins in human serum by MRM mass spectrometry as an alternative approach to detection of bacterial infection. The bacterial protein ospA was detected at concentrations of approximately 4 fmol ospA/mg serum protein in Lyme disease patient samples; B. burgdorferi proteins were not detected in the control patient samples.
Tau Isoform Differentiation in CSF and Plasma
The microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative disorders. Tau represents not only an attractive therapeutic target for AD, but also a candidate biomarker for AD and other neurodegenerative disorders. Tau is a very heterogenous protein with multiple splice forms and numerous post-translational modifications. Specific tau isoforms have been hypothesized to play a role in the onset and progression of AD. An ideal tau biomarker assay would be capable of measuring distinct tau isoforms. We have set out to identify what soluble isoforms are measureable in AD and control patients using an immunoprecipitation mass spectrometry (IPMS) based assay.
Utilizing Western Blot and Mass Spectrometry to Improve Immunohistochemical Detection of Predictive Biomarkers in a Clinical Setting
Antibody specificity is a potential source of variability in immunohistochemical (IHC) clinical assays. To verify specificity for downstream IHC testing of TLE3 protein, anti-TLE3 antibodies from three separate vendors were analyzed by western blot and subsequent mass spectrometry of gel-excised bands to confirm target specificity. All three anti-TLE antibodies identified a common ~83 kD band by western blot, in addition to several bands of differential molecular weights. High-resolution mass spectrometry was used to verify specific target bands and identify which antibodies resulted in detection of non-specific targets. The results demonstrate the utility of western blot combined with mass spectrometry as a supplemental quality control for IHC testing.
Judging a Book by Its Data: Planning Experiments to Fully Evaluate Prospective Instrument Vendors
To aid in the selection of a new mass spectrometer, we developed an experimental model which allowed the concurrent evaluation of both small molecule and peptide performance across top-tier quadrupole mass spectrometers from four LC-MS/MS vendors. Each Vendor was supplied a seven-sample test set, composed of linear admixtures of two trypsin-digested serum samples. One sample contained the human vitamin D binding globulin variant GC2 (tryptic peptide LPDATPK) and the other sample, deficient for this variant, was spiked with amphetamine to a final concentration of 150pg/mL. Vendors were asked to analyze each test mix in quadruplicate, concurrently monitoring multiple transitions for amphetamine, GC2 and GC2 internal standard. Key parameters that were compared included the y-intercept of the linear regressions, %CV at each of the concentrations, and %CV of transition ion ratios.
Use of an Animal-free Synthetic Surrogate Serum Matrix for Assay Calibrators, Controls, and Patient Sample Diluent in ELISA and LC-MS Based Clinical Assays
The objective is to determine the utility of a well-defined, stable, animal-free matrix for use as a serum (or stripped serum) substitute in clinical assays. The synthetic surrogate matrix was prepared with 2% rHSA expressed from rice. It was tested in commercially available IVD ELISA kits with two different analytes (β-2 microglobulin and thyroglobulin), and in direct comparison to human serum in LC-MS based clinical assays for methotrexate, testosterone and estradiol in blood. For all comparative analyses the synthetic surrogate serum matched the performance of the matrix provided by the kit manufacturers, pooled human serum and stripped human serum with no interferences observed and with correlations of R2 > 0.998. We have formulated a simple, animal-free, analyte-free matrix which has been shown to be suitable as a calibrator/blank matrix as well as a patient sample diluent.
Identification of Two Frequent Hemoglobin Variants in Korea by Liquid Chromatography – Tandem Mass Spectrometry
We developed an UPLC-MS/MS method to identify hemoglobin (Hb) G-Coushatta and Hb Yamagata, common Hb variants in Korea, among suspected specimens detected by conventional HbA1c analyzers. All Hb variant specimens and normal controls were tested in UPLC-MS/MS system after the treatment with endoproteinase Glu-C. Two peaks, 689.8 m/z [M+2H+] and 832.4 m/z [M+3H+] at 6.36 min, and one peak, 832.4 m/z [M+3H+] at 10.78 min, were found to distinguish Hb G-Coushatta and Hb Yamagata from other Hb variants and normal, respectively. And the zygosity for these variants was determined by the intensity of two peaks: 657.8 m/z and 460.2 m/z.
Development of a Rapid LC-MS/MS Method for Human Serum Lipid Mediator Profiling
Lipid mediators are bioactive lipids which play a role in many biological functions, including immune dysfunction after severe inflammation. Recent development of a high sensitivity ultra-fast mass spectrometer enables lower detection limits for lipid mediator species. A comprehensive and highly sensitive application for the analysis of lipid mediators and their metabolites using a triple quadrupole mass spectrometer is presented here. Method conditions were developed to simultaneously analyze 158 lipid mediator-related compounds. A single chromatographic analysis is capable of separating a wide range of positive and negative species such as hydrophilic metabolites, tetranor-PGs and hydrophobic arachidonic acid.
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