TY - JOUR
T1 - Development of a chiral micellar electrokinetic chromatography-tandem mass spectrometry assay for simultaneous analysis of warfarin and hydroxywarfarin metabolites
T2 - Application to the analysis of patients serum samples
AU - Wang, Xiaochun
AU - Hou, Jingguo
AU - Jann, Michael
AU - Hon, Yuen Yi
AU - Shamsi, Shahab A.
N1 - Funding Information:
This work was supported by a grant from financial support by NIH (grant # 2R01-GM-062314 ) and Petroleum Research Foundation (PRF-Grant # 47774-AC7 ). The authors would like to thank Dr. Michael Jann (Mercer University) and Andrea Redman for providing us the blank plasma and patient plasma samples.
PY - 2013/1/4
Y1 - 2013/1/4
N2 - The enantioseparation of warfarin (WAR) along with the five positional and optical isomers is challenging because of the difficulty to simultaneously separate and quantitate these chiral compounds. Currently, no effective chiral CE-MS methods exist for the simultaneous enantioseparation of WAR and all its hydroxylated metabolites in a single run. Polymeric surfactants (aka. molecular micelles) are particularly compatible with micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) because they have a wider elution window for enantioseparation and do not interfere with the MS detection of chiral drugs. Using polysodium N-undecenoyl-l,. l-leucylvalinate (poly-l,. l-SULV) as a chiral pseudophase in MEKC-MS baseline separation of WAR, its five metabolites along with the internal standard was obtained in 45. min. This is in comparison to 100. min required for separation of the same mixture with packed column CEC-MS using a vancomycin chiral stationary phase. Serum samples were extracted with mixed-mode anion-exchange (MAX) cartridge with recoveries of greater than 85.2% for all WAR and hydroxywarfarin (OH-WAR) metabolites. Utilizing the tandem MS and multiple reaction monitoring mode, the MEKC-MS/MS method was used to simultaneously generate calibration curves over a concentration range from 2 to 5000. ng/mL for R- and S-warfarin, 5 to 1000. ng/mL for R- and S-6-, 7-, 8- and 10-OH-WAR and 10 to 1000. ng/mL for R and S-4'-OH-WAR. For the first time, the limits of detection and quantitation for most WAR metabolites by MEKC-MS/MS were found to be at levels of 2 and 5. ng/mL, respectively. The method was successfully applied for the first time to analyze WAR and its metabolites in plasma samples of 55 patients undergoing WAR therapy, demonstrating the potential of chiral MEKC-MS/MS method to accurately quantitate with high sensitivity.
AB - The enantioseparation of warfarin (WAR) along with the five positional and optical isomers is challenging because of the difficulty to simultaneously separate and quantitate these chiral compounds. Currently, no effective chiral CE-MS methods exist for the simultaneous enantioseparation of WAR and all its hydroxylated metabolites in a single run. Polymeric surfactants (aka. molecular micelles) are particularly compatible with micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) because they have a wider elution window for enantioseparation and do not interfere with the MS detection of chiral drugs. Using polysodium N-undecenoyl-l,. l-leucylvalinate (poly-l,. l-SULV) as a chiral pseudophase in MEKC-MS baseline separation of WAR, its five metabolites along with the internal standard was obtained in 45. min. This is in comparison to 100. min required for separation of the same mixture with packed column CEC-MS using a vancomycin chiral stationary phase. Serum samples were extracted with mixed-mode anion-exchange (MAX) cartridge with recoveries of greater than 85.2% for all WAR and hydroxywarfarin (OH-WAR) metabolites. Utilizing the tandem MS and multiple reaction monitoring mode, the MEKC-MS/MS method was used to simultaneously generate calibration curves over a concentration range from 2 to 5000. ng/mL for R- and S-warfarin, 5 to 1000. ng/mL for R- and S-6-, 7-, 8- and 10-OH-WAR and 10 to 1000. ng/mL for R and S-4'-OH-WAR. For the first time, the limits of detection and quantitation for most WAR metabolites by MEKC-MS/MS were found to be at levels of 2 and 5. ng/mL, respectively. The method was successfully applied for the first time to analyze WAR and its metabolites in plasma samples of 55 patients undergoing WAR therapy, demonstrating the potential of chiral MEKC-MS/MS method to accurately quantitate with high sensitivity.
KW - CYP mediated warfarin metabolism
KW - MEKC-MS versus CEC-MS
KW - Optical/positional isomers
KW - Polymeric surfactant
KW - SIM versus MRM
KW - Warfarin/hydroxylated-warfarin metabolites
UR - http://www.scopus.com/inward/record.url?scp=84871371645&partnerID=8YFLogxK
U2 - 10.1016/j.chroma.2012.11.046
DO - 10.1016/j.chroma.2012.11.046
M3 - Article
C2 - 23246089
AN - SCOPUS:84871371645
SN - 0021-9673
VL - 1271
SP - 207
EP - 216
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 1
ER -