Dataset Information

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples.

ABSTRACT: Over the past decade, the number of known eicosanoids has expanded immensely and we have now developed an ultra-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometric (UPLC-QTRAP/MS/MS) method to monitor and quantify numerous eicosanoids. The UPLC-QTRAP/MS/MS approach utilizes scheduled multiple reaction monitoring (MRM) to optimize sensitivity, number of metabolites that can be analyzed and the time requirement of the analysis. A total of 184 eicosanoids including 26 deuterated internal standards can be separated and monitored in a single 5min UPLC run. To demonstrate a practical application, human plasma samples were analyzed following solid-phase extraction (SPE) and the recovery rate and matrix effects were determined for the 26 deuterated internal standards added to the plasma. The method was validated and shown to be sensitive with the limit of quantitation at pg levels for most compounds, accurate with recovery rates of 70-120%, and precise with a CV<30 for all compounds. Also, the method showed a linear response over a range spanning several orders of magnitude. In a QC human plasma sample, we identified and rigorously quantified over 120 eicosanoids.

SUBMITTER: Wang Y

PROVIDER: S-EPMC4592635 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples.

Wang Yan Y Armando Aaron M AM Quehenberger Oswald O Yan Chao C Dennis Edward A EA

Journal of chromatography. A 20140712

Over the past decade, the number of known eicosanoids has expanded immensely and we have now developed an ultra-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometric (UPLC-QTRAP/MS/MS) method to monitor and quantify numerous eicosanoids. The UPLC-QTRAP/MS/MS approach utilizes scheduled multiple reaction monitoring (MRM) to optimize sensitivity, number of metabolites that can be analyzed and the time requirement of the analysis. A total of 184 eicosanoids ...[more]

PMID: 25074422

Similar Datasets

Project description:BACKGROUND:The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma. METHODS:K2EDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1?×?50?mm, 3.5??m column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1?×?50?mm, 2.6??m column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines. RESULTS:Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25?ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs???14.4% and %DEVs ??±?7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also applied to quantify in vivo concentrations of prodrugs and TFV in a preclinical study post-rectal administration. CONCLUSIONS:Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the multiplexed quantification TAF and TFV, as well as an independent assay for CMX157 quantification, in plasma. The described methods meet sufficient throughput criteria to support large research trials.

Project description:Acidosis is an important cause of mortality in severe falciparum malaria. Lactic acid is a major contributor to metabolic acidosis, but accounts for only one-quarter of the strong anion gap. Other unidentified organic acids have an independent strong prognostic significance for a fatal outcome. In this study, a simultaneous bio-analytical method for qualitative and quantitative assessment in plasma and urine of eight small organic acids potentially contributing to acidosis in severe malaria was developed and validated. High-throughput strong anion exchange solid-phase extraction in a 96-well plate format was used for sample preparation. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative mass spectroscopy was utilized for separation and detection. Eight possible small organic acids; l-lactic acid (LA), ?-hydroxybutyric acid (aHBA), ?-hydroxybutyric acid (bHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and ?-ketoglutaric acid (aKGA) were analyzed simultaneously using a ZIC-HILIC column with an isocratic elution containing acetonitrile and ammonium acetate buffer. This method was validated according to U.S. Food and Drug Administration guidelines with additional validation procedures for endogenous substances. Accuracy for all eight acids ranged from 93.1% to 104.0%, and the within-day and between-day precisions (i.e. relative standard deviations) were lower than 5.5% at all tested concentrations. The calibration ranges were: 2.5-2500?g/mL for LA, 0.125-125?g/mL for aHBA, 7.5-375?g/mL for bHBA, 0.1-100?g/mL for pHPLA, 1-1000?g/mL for MA, 0.25-250?g/mL for MMA, 0.25-100?g/mL for EMA, and 30-1500?g/mL for aKGA. Clinical applicability was demonstrated by analyzing plasma and urine samples from five patients with severe falciparum malaria; five acids had increased concentrations in plasma (range LA=177-1169?g/mL, aHBA=4.70-38.4?g/mL, bHBA=7.70-38.0?g/mL, pHPLA=0.900-4.30?g/mL and aKGA=30.2-32.0) and seven in urine samples (range LA=11.2-513?g/mL, aHBA=1.50-69.5?g/mL, bHBA=8.10-111?g/mL, pHPLA=4.30-27.7?g/mL, MMA=0.300-13.3?g/mL, EMA=0.300-48.1?g/mL and aKGA=30.4-107?g/mL). In conclusion, a novel bioanalytical method was developed and validated which allows for simultaneous quantification of eight small organic acids in plasma and urine. This new method may be a useful tool for the assessment of acidosis in patients with severe malaria, and other conditions complicated by acidosis.

Project description:BackgroundThe non-nucleoside reverse transcriptase inhibitor dapivirine has been evaluated as a topical microbicidal agent to prevent HIV-1 acquisition. Several clinical trials have evaluated the pharmacokinetics of dapivirine when administered as a 25-mg intravaginal ring. Recent studies have focused on the distribution of dapivirine into breast milk. Drug distribution during lactation and breastfeeding can have implications in terms of infant drug exposure. Thus, sensitive bioanalytical tools are required to characterize the pharmacokinetics of dapivirine in breast milk.MethodsWhole breast milk was spiked with dapivirine and internal standard. Lipid content was disrupted via pre-treatment with n-hexane, and supernatants were subjected to solid phase extraction. Extracted materials were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Separation occurred using a Waters BEH C8, 50 × 2.1 mm UPLC column with a 1.7 µm particle size and dapivirine was detected on an API 5000 mass analyzer. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations.ResultsThe analytical method was optimized for dapivirine extraction from breast milk. The analytical measuring range of the assay was 10-1000 pg/mL. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.6% and %DEVs ≤ ±12.7%. Stability and matrix effects studies were also conducted and deemed acceptable. The method was applied to a previously reported phase 1 clinical trial and demonstrated appropriate performance in the quantitation of dapivirine in breast milk samples from lactating women.ConclusionsAn ultrasensitive LC-MS/MS assay has been developed and validated for the quantitation of dapivirine in breast milk. The described method meets validation acceptance criteria and has been applied to a phase 1 clinical trial.

Dataset Information

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples.

Publications

Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure