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. 2015 Aug;5(4):269-275.
doi: 10.1016/j.jpha.2015.05.004. Epub 2015 May 23.

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

Xiao-Hong Zheng  1 , Li-Yuan Jiang  2 , Lan-Ting Zhao  1 , Quan-Ying Zhang  3 , Li Ding  1
Affiliations

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

Xiao-Hong Zheng et al. J Pharm Anal. 2015 Aug.

Abstract

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/mL 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 mM 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/mL for FA, and 5.05-50.5 ng/mL for 5-M-THF. The developed LC-MS/MS method offers increased sensitivity for quantification of FA and 5-M-THF in human plasma and was applicable to a pharmacokinetic study of FA and 5-M-THF.

Keywords: 2-Mercaptoethanol; 5-Methyltetrahydrofolic acid; Folic acid; LC–MS/MS; Pharmacokinetics.

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Figures

Fig. 1
Fig. 1
Chemical structures of (A) FA, (B) 5-M-THF and (C) IS and their proposed fragmentation patterns.
Fig. 2
Fig. 2
The degradation of (A) FA (9.95 ng/mL) and (B) 5-M-THF (20.2 ng/mL) in plasma at different concentration levels of 2-mercaptoethanol at room temperature.
Fig. 3
Fig. 3
Typical chromatograms of (A) blank plasma; (B) LLOQ for FA (0.249 ng/mL), 5-M-THF (5.05 ng/mL) and IS in plasma; and (C) plasma obtained from a volunteer at 1.5 h after an oral administration of 0.8 mg folic acid.
Fig. 4
Fig. 4
Mean plasma concentration-time profiles (mean±SD, baseline values corrected) of FA and 5-M-THF in human plasma after an oral administration of 0.8 mg ofb folic acid (n=20).
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