Enantiomeric separation and quantitation of warfarin and its metabolites in human plasma by LC-MS/MS
Open Access
- 30 September 2016
- journal article
- Published by European Journal of Chemistry in European Journal of Chemistry
- Vol. 7 (3), 357-362
- https://doi.org/10.5155/eurjchem.7.3.357-362.1470
Abstract
The enantiomeric separation of warfarin (WAR) enantiomers and its hydroxy metabolites positional isomers is described in this work. The utilization of chiral chromatography coupled to Tandem mass spectrometry helps to achieve that. The developed method was able to separate R,S-warfarin and enantiomers of 4,7,10-hydroxy warfarin in human plasma. Plasma samples were processed with simple protein precipitation with acetonitrile. The chromatographic separation was done on chiral Astec Chirobiotic V column with gradient flow of 0.1% aqueous formic acid and 0.1% formic in acetonitrile:water (95:5, v:v). The mass spectroscopic detection was done via negative mode multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer coupled with positive electrospray ionization (ESI). Linearity of the method was valid over a concentration range of 125.00-0.13 µg/mL for racemic WAR, 47.000-0.088 µg/mL for racemic 4’-OH-WAR, 56.00-0.18 µg/mL for racemic 7-OH-WAR and 56.00-0.02 µg/mL for racemic 10-OH-WAR. The LLOQs were found to be 0.13 µg/mL for racemic WAR, 0.088 µg/mL for racemic 4’-OH-WAR, 0.18 µg/mL for racemic 7-OH-WAR and 0.02 µg/mL for racemic 10-OH-WAR. The method was validated for matrix effect, intra- and inter-day precision, freeze/thaw and storage stability. Accuracy and precision were within acceptable range (R- and S-WAR identified peak 2 as S-WAR while metabolite peaks could not be definitively identified. Peak 2 for 7-OH-WAR gave higher blood levels, while the opposite applied to 10-OH-WAR.Keywords
This publication has 16 references indexed in Scilit:
- Warfarin Toxicity and Individual Variability—Clinical CaseToxins, 2010
- Assessing Cytochrome P450 and UDP-Glucuronosyltransferase Contributions to Warfarin Metabolism in HumansChemical Research in Toxicology, 2009
- Variant CYP2C9 Alleles and Warfarin Concentrations in Patients Receiving Low-Dose Versus Average-Dose Warfarin TherapyClinical and Applied Thrombosis/hemostasis, 2008
- Novel Antithrombotic Agents: Indirect Synthetic Inhibitors of Factor Xa and Direct Thrombin Inhibitors. Evidences from Clinical StudiesCurrent Medicinal Chemistry-Cardiovascular & Hematological Agents, 2004
- Long-Term, Low-Intensity Warfarin Therapy for the Prevention of Recurrent Venous ThromboembolismThe New England Journal of Medicine, 2003
- Influence of CYP2C9 and CYP2C19 genetic polymorphisms on warfarin maintenance dose and metabolic clearanceClinical Pharmacology & Therapeutics, 2002
- Interindividual variability in sensitivity to warfarin-Nature or nurture?Clinical Pharmacology & Therapeutics, 2001
- Human P450 metabolism of warfarinPharmacology & Therapeutics, 1997
- Hydroxylation of warfarin by human cDNA-expressed cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactionsChemical Research in Toxicology, 1992
- Very Low Doses of Warfarin Can Prevent Thrombosis in Central Venous CathetersAnnals of Internal Medicine, 1990