Simultaneous quantitation of folic acid and 5-methyltetrahydrofolic acid in human plasma by HPLC-MS/MS and its application to a pharmacokinetic study

摘要

A sensitive method based on high-performance liquid chromatography–tandem mass spectrometry(LC–MS/MS) has been developed for the simultaneous determination of folic acid(FA) and its active metabolite, 5-methyltetrahydrofolic acid(5-M-THF), in human plasma. The analytes were extracted from plasma with methanol solution containing 10 mg/m L of 2-mercaptoethanol and 0.025%(v/v) ammonium hydroxide. FA and 5-M-THF were more stable after the addition of 2-mercaptoethanol and ammonium hydroxide in the sample preparation procedures of this study than they were in the previously published methods. Chromatographic separation was performed on a Hedera ODS-2 column using a gradient elution system of acetonitrile and 1 m M ammonium acetate buffer solution containing 0.6% formic acid as mobile phase. LC–MS/MS was carried out with an ESI ion-source and operated in the multiple reaction monitoring(MRM) mode. The assay was linear over the concentration ranges of 0.249–19.9 ng/m L for FA,and 5.05–50.5 ng/m L for 5-M-THF. The developed LC–MS/MS method offers increased sensitivity for quanti fi cation of FA and 5-M-THF in human plasma and was applicable to a pharmacokinetic study of FA and 5-M-THF.