Development and Validation of Bioanalytical Method for Simultaneous Estimation of Leucovorin and its Primary Metabolite 5-Methyl Tetrahydrofolic Acid from Human Plasma by Liquid Chromatography Tandem Mass Spectrometry with Solid Phase Extraction

Abstract

A simple, highly sensitive, precise and accurate liquid chromatographic mass spectroscopic (LC-MS/MS) method has been developed and validated for simultaneous quantification of Leucovorin and its metabolite 5-Methyl tetrahydrofolic acid in human plasma using Leucovorin-d4 and 5-Methyl-d3 tetrahydrofolic acid as internal standard (IS), respectively. The analytes were extracted from human plasma by solid phase extraction (SPE) technique using ion exchange cartridges. Chromatographic separation was achieved with reversed phase column chromatography. API 5500 LC-MS/MS in multiple reactions monitoring (MRM) was used for detection. Obtained results revealed that the method had a short sample preparation time as well as a chromatographic run time of just 3.0 min. In summary our results suggest that the validated method fulfilled all preset criteria for sensitivity, specificity, linearity (5.0-700.0 ng/mL), inter-and intra-accuracy and precision for both molecules.

Keywords:

• Liquid chromatographic Mass spectroscopic (LC-MS/MS)
• Multiple Reaction Monitoring (MRM)
• Internal Standard (IS)
• Solid Phase Extraction (SPE)