Application of 4-(4-Nitrophenyl)-1,2,4-triazoline-3,5-dione to Analysis of 25-Hydroxyvitamin D₃ in Human Plasma by Liquid Chromatography/Electron Capture Atmospheric Pressure Chemical Ionization-Mass Spectrometry

Abstract

The utility of 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD) as a derivatization reagent in the analysis of 25-hydroxyvitamin D₃ [25(OH)D₃] using liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometry (LC/ECAPCI-MS) was examined. The derivatives of 25(OH)D₃ with NPTAD underwent electron capture in the APCI source in the negative-ion mode and provided 30-fold higher sensitivity compared to an intact compound. This derivatization-LC/ECAPCI-MS method was applied to a plasma 25-hydroxyvitamin D₃ assay, and gave satisfactory results in sensitivity, specificity, measurable range and throughput of the analysis.