HPLC-Electrospray Tandem Mass Spectrometry for Rapid Determination of Dihydropyrimidine Dehydrogenase Activity

Abstract Background: Patients with a partial dihydropyrimidine dehydrogenase (DPD) deficiency have an increased risk of developing severe 5-fluorouracil–associated toxicity. We developed a rapid and specific method to measure the DPD activity in peripheral blood mononuclear cells using HPLC tandem-mass spectrometry (HPLC-MS/MS). Methods: The activity of DPD was measured with thymine as the substrate, followed by reversed-phase HPLC combined with electrospray ionization MS/MS and detection of the product dihydrothymine with multiple-reaction monitoring. Stable-isotope labeled dihydrothymine was used as the internal standard. Results: Dihydrothymine was measured within an analytical run of 10 min, with a lower limit of quantification of 54 μg/L (0.4 μmol/L). The intraassay and interassay variations of the DPD activity assay were both <7%. A linear correlation (R2 = 0.980; P <0.001) was observed between the HPLC-MS/MS data and those obtained with a reference method using radiolabeled thymine. There were no systematic differences between the 2 methods, and both methods yielded similar results. Conclusion: The analysis of the DPD activity with HPLC-MS/MS is rapid, accurate, and sufficiently sensitive to be used as a screening method for patients with a DPD deficiency.

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A Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry Quantitative Method for Determination of Baricitinib in Plasma, and Its Application in a Pharmacokinetic Study in Rats

Molecules ◽

10.3390/molecules25071600 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1600 ◽

Cited By ~ 1

Author(s):

Essam Ezzeldin ◽

Muzaffar Iqbal ◽

Yousif A. Asiri ◽

Azza A Ali ◽

Prawez Alam ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Janus Kinase ◽

Pharmacokinetic Studies ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

Baricitinib, is a selective and reversible Janus kinase inhibitor, is commonly used to treat adult patients with moderately to severely active rheumatoid arthritis (RA). A fast, reproducible and sensitive method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of baricitinib in rat plasma has been developed. Irbersartan was used as the internal standard (IS). Baracitinib and IS were extracted from plasma by liquid–liquid extraction using a mixture of n-hexane and dichloromethane (1:1) as extracting agent. Chromatographic separation was performed using Acquity UPLC HILIC BEH 1.7 µm 2.1 × 50 mm column with the mobile phase consisting of 0.1% formic acid in acetonitrile and 20 mM ammonium acetate (pH 3) (97:3). The electrospray ionization in the positive-mode was used for sample ionization in the multiple reaction monitoring mode. Baricitinib and the IS were quantified using precursor-to-production transitions of m/z 372.15 > 251.24 and 429.69 > 207.35 for baricitinib and IS, respectively. The method was validated according to the recent FDA and EMA guidelines for bioanalytical method validation. The lower limit of quantification was 0.2 ng/mL, whereas the intra-day and inter-day accuracies of quality control (QCs) samples were ranged between 85.31% to 89.97% and 87.50% to 88.33%, respectively. Linearity, recovery, precision, and stability parameters were found to be within the acceptable range. The method was applied successfully applied in pilot pharmacokinetic studies.

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Method for the Determination of Total Homocysteine in Plasma and Urine by Stable Isotope Dilution and Electrospray Tandem Mass Spectrometry

Clinical Chemistry ◽

10.1093/clinchem/45.9.1517 ◽

1999 ◽

Vol 45 (9) ◽

pp. 1517-1522 ◽

Cited By ~ 111

Author(s):

Mark J Magera ◽

Jean M Lacey ◽

Bruno Casetta ◽

Piero Rinaldo

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Signal To Noise Ratio ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

High Volume ◽

Hplc Method ◽

Tandem Mass ◽

Electrospray Tandem Mass Spectrometry ◽

Total Homocysteine

Abstract Background: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround. Methods: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 μL of either plasma or urine with homocystine-d8 (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d4 were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d4 was 1.5 min in a 2.5-min analysis. Results: Daily calibrations between 2.5 and 60 μmol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 μmol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x − 1.377 (r = 0.975; Sy|x =1.595 μmol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; Sy|x =1.146 μmol/L; n = 367). Inter- and intraassay CVs were 2.9–5.9% and 3.6–5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 μmol/L. Mean recovery of tHcy was 94.2% (20 μmol/L) and 97.8% (50 μmol/L). Conclusions: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.

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Rapid Screening of High-Risk Patients for Disorders of Purine and Pyrimidine Metabolism Using HPLC-Electrospray Tandem Mass Spectrometry of Liquid Urine or Urine-soaked Filter Paper Strips

Clinical Chemistry ◽

10.1093/clinchem/46.4.445 ◽

2000 ◽

Vol 46 (4) ◽

pp. 445-452 ◽

Cited By ~ 88

Author(s):

Tetsuya Ito ◽

André B P van Kuilenburg ◽

Albert H Bootsma ◽

Anja J Haasnoot ◽

Arno van Cruchten ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Filter Paper ◽

Screening Method ◽

Urine Samples ◽

Rapid Screening ◽

Specific Method ◽

Tandem Mass ◽

Pyrimidine Metabolism ◽

Inherited Disorders

Abstract Background: A rapid and specific screening method for patients at risk of inherited disorders of purine and pyrimidine metabolism is desirable because symptoms are varied and nonspecific. The aim of this study was to develop a rapid and specific method for screening with use of liquid urine samples or urine-soaked filter paper strips. Methods: Reverse-phase HPLC was combined with electrospray ionization (ESI), tandem mass spectrometry (MS/MS), and detection performed by multiple reaction monitoring. Transitions and instrument settings were established for 17 purines or pyrimidines. Stable-isotope-labeled reference compounds were used as internal standards when available. Results: Total analysis time of this method was 15 min, approximately one-third that of conventional HPLC with ultraviolet detection. Recoveries were 96–107% in urine with added analyte, with two exceptions (hypoxanthine, 64%; xanthine, 79%), and 89–110% in urine-soaked filter paper strips, with three exceptions (hypoxanthine, 65%; xanthine, 77%; 5-hydroxymethyluracil, 80%). The expected abnormalities were easily found in samples from patients with purine nucleoside phosphorylase deficiency, ornithine transcarbamylase deficiency, molybdenum cofactor deficiency, adenylosuccinase deficiency, or dihydropyrimidine dehydrogenase deficiency. Conclusions: HPLC-ESI MS/MS of urine allows rapid screening for disorders of purine and pyrimidine metabolism. The filter paper strips offer the advantage of easy collection, transport, and storage of the urine samples.

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Comprehensive Detection of Disorders of Purine and Pyrimidine Metabolism by HPLC with Electrospray Ionization Tandem Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2005.058842 ◽

2006 ◽

Vol 52 (6) ◽

pp. 1127-1137 ◽

Cited By ~ 52

Author(s):

Susen Hartmann ◽

Jürgen G Okun ◽

Christiane Schmidt ◽

Claus-Dieter Langhans ◽

Sven F Garbade ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Electrospray Ionization ◽

Multiple Reaction Monitoring ◽

Dihydropyrimidine Dehydrogenase ◽

Urine Samples ◽

Tandem Mass ◽

Pyrimidine Metabolism ◽

Phosphoribosyl Transferase ◽

Ionization Tandem Mass Spectrometry

Abstract Background: Clinical presentation and disease severity in disorders of purine and pyrimidine metabolism vary considerably. We present a method that allows comprehensive, sensitive, and specific diagnosis of the entire spectrum of abnormalities in purine and pyrimidine metabolism in 1 analytical run. Methods: We used reversed-phase HPLC electrospray ionization tandem mass spectrometry to investigate 24 metabolites of purine and pyrimidine metabolism in urine samples from healthy persons and from patients with confirmed diagnoses of inherited metabolic disorders. Urine samples were filtered and diluted to a creatinine concentration of 0.5 mmol/L. Stable-isotope–labeled internal standards were used for quantification. The metabolites were analyzed by multiple-reaction monitoring in positive and negative ionization modes. Results: Total time of analysis was 20 min. Recovery (n = 8) of a compound after addition of a known concentration was 85%–133%. The mean intraday variation (n = 10) was 12%. The interday variation (n = 7) was ≤17%. Age-related reference intervals were established for each compound. Analysis of patient urine samples revealed major differences in tandem mass spectrometry profiles compared with those of control samples. Twelve deficiencies were reliably detected: hypoxanthine guanine phosphoribosyl transferase, xanthine dehydrogenase, purine nucleoside phosphorylase, adenylosuccinate lyase, uridine monophosphate synthase, adenosine deaminase, adenine phosphoribosyl transferase, molybdenum cofactor, thymidine phosphorylase, dihydropyrimidine dehydrogenase, dihydropyrimidinase, and β-ureidopropionase. Conclusion: This method enables reliable detection of 13 defects in purine and pyrimidine metabolism in a single analytical run.

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A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Nimbolide in Mouse Serum: Application to a Preclinical Pharmacokinetics Study

Pharmaceutics ◽

10.3390/pharmaceutics10030123 ◽

2018 ◽

Vol 10 (3) ◽

pp. 123 ◽

Cited By ~ 4

Author(s):

Lingzhi Wang ◽

Do-Dang Phan ◽

Nicholas Syn ◽

Xiaoqiang Xiang ◽

Hongyan Song ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Mouse Serum ◽

Tandem Mass ◽

Liquid Chromatography Tandem Mass ◽

Tandem Mass Spectrometric

A sensitive and robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of nimbolide in mouse serum. Exemestane was used as the internal standard (IS). Here, we employed acetonitrile-based protein precipitation (PPT) for serum sample preparation, and performed chromatographic separation using an ODS Hypersil C18 column (100 mm × 2.1 mm, 5 µm) with gradient elution (0.1% formic acid in water vs 100% acetonitrile). The run time was 6 min. Instrumental analysis was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring (MRM) under positive mode. A good linear calibration was achieved in the 5–1000 ng/mL range. The intra- and inter-day precisions for nimbolide were ≤12.6% and ≤13.9% respectively. Intra-day accuracy ranged from 96.9–109.3%, while inter-day accuracy ranged from 94.3–110.2%. The matrix effect of nimbolide, detected but consistent at low and high concentrations, do not affect linearity of standard curve. In conclusion, we have developed and validated a sensitive analytical method for determination of a novel natural compound nimbolide in mouse serum, and it has been successfully applied to our preclinical study in investigating the pharmacokinetic properties of nimbolide, which could greatly facilitate the preclinical development of the promising lead compound for anticancer therapy.

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HPLC–Tandem Mass Spectrometric Method to Characterize Resveratrol Metabolism in Humans

Clinical Chemistry ◽

10.1373/clinchem.2006.071936 ◽

2007 ◽

Vol 53 (2) ◽

pp. 292-299 ◽

Cited By ~ 68

Author(s):

Mireia Urpi-Sarda ◽

Raul Zamora-Ros ◽

Rosa Lamuela-Raventos ◽

Antonio Cherubini ◽

Olga Jauregui ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Solid Phase ◽

Biological Effects ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Assessment Tools ◽

Tandem Mass ◽

Mass Spectrometric Method ◽

Tandem Mass Spectrometric

Abstract Background: Nutritional biomarkers are alternatives to traditional dietary assessment tools. We sought to develop a method for nutritional analysis of resveratrol, a phenolic compound with purported health-promoting properties, and to determine all resveratrol metabolites. Methods: We obtained LDL and urine samples from 11 healthy male volunteers who had consumed 250 mL of Merlot red wine. We measured resveratrol and its metabolites with 96-well solid-phase extraction plates coupled with HPLC-tandem mass spectrometry. Hexestrol was used as the internal standard. Gradient chromatography in multiple reaction monitoring mode was performed on a Luna C18 column, maintained at 40 °C; m/z transitions were as follows: resveratrol, 227/185; resveratrol glucosides, 389/227; resveratrol glucuronides, 403/227; resveratrol sulfates, 307/227; taxifolin, 303/285; and hexestrol, 269/134. Results: Standard calibration curves were linear at 4.4–3289.5 nmol/L. Residual analyses were 100% (3.2) for trans-resveratrol and 100% (11.1) for trans-piceid. In both matrices, imprecision (CV) was <10.8% at all concentrations. Detection limits for resveratrol were 0.2 nmol/L (LDL), 0.3 nmol/L (synthetic urine), and 4.0 nmol/L (blank urine). Resveratrol and metabolites were checked for stability, and no degradation was observed. Conclusions: The HPLC–tandem mass spectrometry method enabled us to identify resveratrol sulfates in human LDL and to characterize the complete profile of resveratrol metabolism in human LDL and urine. This method provides an accurate index of exposure to resveratrol and its metabolites, which can be used as nutritional biomarkers for evaluating the biological effects of moderate wine intake on human health.

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Testosterone measurement by liquid chromatography tandem mass spectrometry: the importance of internal standard choice

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1258/acb.2012.012037 ◽

2012 ◽

Vol 49 (6) ◽

pp. 600-602 ◽

Cited By ~ 24

Author(s):

L J Owen ◽

B G Keevil

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Method Development ◽

Internal Standard ◽

Reference Method ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

Acquity Uplc

Background Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of testosterone. Variation is seen between assays and is likely to be due to method differences. One area of inconsistency among assays is the choice of internal standard. We investigated the effects of three internal standards. Methods Testosterone with two deuterium (D2), five deuterium (D5) and three carbon 13 enrichment (C13) were separately assessed. Samples were extracted using ether following the addition of 10 μL of internal standard. All aliquots were prepared in triplicate, one for each type of internal standard. After mixing, the ether was transferred to a 96-deep well block, and then evaporated to dryness. Extracts were reconstituted with 50% mobile phases and analysed using a Waters Acquity UPLC and Quattro Premier tandem mass spectrometer. This method had previously been shown to have excellent agreement with a reference method using the D2 internal standard and this was considered the target. Results Lower results were obtained when using D5 testosterone when compared with D2 testosterone. The C13 internal standard also gave lower results, but was closer to the D2 target than the D5 internal standard. Conclusions The choice of internal standard alone can have a significant affect on the results obtained by LC-MS/MS assays for testosterone using this chromatography. The effects of the combination of chromatography and internal standard choice should be investigated during method development.

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Quantification of Globotriaosylsphingosine in Plasma and Urine of Fabry Patients by Stable Isotope Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2012.192138 ◽

2013 ◽

Vol 59 (3) ◽

pp. 547-556 ◽

Cited By ~ 63

Author(s):

Henrik Gold ◽

Mina Mirzaian ◽

Nick Dekker ◽

Maria Joao Ferraz ◽

Johan Lugtenburg ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Fabry Disease ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Response To Treatment ◽

Tandem Mass ◽

Healthy Controls ◽

Enzyme Replacement

BACKGROUND Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker. METHODS We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] 13C5-lysoGb3 (internal standard). After addition of the internal standard to 25 μL plasma or 400 μL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography–electrospray ionization–tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H]+ for lysoGb3, m/z 791.4 > 287.3 [M+H]+ for [5,6,7,8,9] 13C5-lysoGb3, and 784.4 > 280.3 [M+H]+ for lyso-ene-Gb3. RESULTS The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7–136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1–23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3–0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%–25% of total lysoGb3. The urine concentration of lysoGb3 was 40–480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations. CONCLUSIONS This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.

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Evaluation of a rapid micro-scale assay for tacrolimus by liquid chromatography-tandem mass spectrometry

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1258/000456302320314502 ◽

2002 ◽

Vol 39 (5) ◽

pp. 487-492 ◽

Cited By ~ 45

Author(s):

BG Keevil ◽

SJ McCann ◽

DP Cooper ◽

MR Morris

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Immunosuppressive Drug ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Limit Of Quantitation ◽

Liquid Chromatography Tandem Mass

Background: The immunosuppressive drug tacrolimus has complex and unpredictable pharmacokinetics, therefore regular monitoring is required in patients receiving tacrolimus therapy. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring tacrolimus concentrations in whole blood and have compared it with a microparticle enzyme immunoassay. Methods: For the LC-MS/MS assay, samples were prepared in a 96-deep well microtitre plate by adding 10 µL of blood to 40 µL of 0·1 mol/L zinc sulphate solution. Proteins were precipitated by adding 100 µL acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 20 µL of the supernatant was injected into the LC-MS/MS system. A C18 cartridge (3 mm × 4 mm) was eluted with a step gradient of 50% to 100% methanol containing 2 mmol/L ammonium acetate and 0·1% (v/v) formic acid, at 0·6 mL/min. The column was maintained at 55°C. Results: The retention times were 0·98 min for ascomycin and 0·98 min for tacrolimus. Cycle time was 2·5 min, injection to injection. The analytes were monitored using a Quattro micro tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: m/z821 > 768 (tacrolimus) and m/z809 > 756 (ascomycin). The limit of quantitation was 0·5 µg/L and the assay was linear up to 30 µg/L. Precision of the method, over the concentration range 2·5-15·0 µg/L, was < 7% within-batch and < 6% between-batch. Total time to analyse 24 samples including result generation was 90 min. Conclusion: We conclude that the LC-MS/MS method is quick, precise and robust and will provide a fast turn around of results for the transplant physician.

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Confirmatory Determination of Six Penicillins in Honey by Liquid Chromatography/Electrospray Ionization–Tandem Mass Spectrometry

Journal of AOAC International ◽

10.1093/jaoac/87.1.45 ◽

2004 ◽

Vol 87 (1) ◽

pp. 45-55 ◽

Cited By ~ 9

Author(s):

Jian Wang

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Electrospray Ionization ◽

Solid Phase ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Penicillin G ◽

Tandem Mass ◽

Ionization Tandem Mass Spectrometry

Abstract A confirmatory method for 6 penicillin antibiotics (amoxicillin, ampicillin, penicillin G, oxacillin, cloxacillin, and dicloxacillin) in honey is presented that allows determination and confirmation of identity of the antibiotics at trace levels. The method includes the use of a stable isotope-labeled internal standard benzyl (d7-phenyl) penicillate and removal of sugar and other substances by solvent and solid-phase extraction. The honey extracts are then analyzed for penicillin residues by liquid chromatography/electrospray ionization–tandem mass spectrometry. Mass spectral acquisition was achieved in an electrospray positive ion mode by applying multiple reaction monitoring of 2 or 3 fragment ion transitions to provide a high degree of sensitivity and specificity. Typical recoveries of 6 penicillins at fortification levels of 6, 16, 40, and 80 μg/kg ranged from 51.4 to 132.9%. The recoveries varied with the individual penicillins and were affected by different honey matrixes. The ion ratios were consistent and could be used for confirmation of identity of the penicillins. The method limits of detection (μg/kg) were 0.25 for amoxicillin, 0.19 for ampicillin, 0.068 for penicillin G, 0.028 for oxacillin, 0.052 for cloxacillin, and 0.085 for dicloxacillin. The method limits of confirmation (μg/kg) were 0.44 for amoxicillin, 0.52 for ampicillin, 0.23 for penicillin G, 0.14 for oxacillin, 0.14 for cloxacillin, and 0.15 for dicloxacillin when a sample size of 5 g honey was used.

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