An electron-capture dienophile derivatization agent for increasing sensitivity: Determination of a vitamin D analog (Ro 24-2090) in plasma samples with liquid chromatography mass spectrometry

A practical analytical method has been developed for the determination of an analog of vitamin D-3 (Ro 24-2090) in plasma samples. The method employs liquid-liquid extraction, precolumn derivatization, HPLC separation using an automated column-switching system, and particle-beam negative ionization MS determination. An electron-capture derivatization reagent, 4-pentafluorobenzyl-1,2,4-triazoline-3,5-dione is utilized to attain high sensitivity through a unique ionization mechanism. The method achieved a lower Limit of quantitation of 25 pg (63 fmol) of Ro 24-2090 in plasma aliquots ranging from 0.125 to 1.0 ml. Method validation data were obtained for the quantitation of Ro 24-2090 in plasma from humans and seven animal species. Intraassay precision [mean percent relative standard deviation (%RSD)] ranged hom 1.49 to 7.12% among the various species. Interassay precision (mean %RSD) ranged hom 4.28 to 17.5%. Mean overall recovery ranged from 71 to 89%. The method has been successfully used to analyze plasma samples from several pharmacokinetic studies. The potential use of this method for determination of vitamin D-2, vitamin D-3, 1 alpha,25-dihydroxyvitamin D-3, and other drugs containing a diene structural moiety is discussed. (C) 1996 Academic Press, Inc.