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

• Published in: Analytical biochemistry 1996-12, Vol.243 (1), p.28-40
• Main Authors: Wang, Ka, Davis, Preston P., Crews, Theodore, Gabriel, Lana, Edom, Richard W.
• Format: Article
• Language: English
• Subjects: Animals; Cholecalciferol - analogs & derivatives; Cholecalciferol - blood; Chromatography, High Pressure Liquid - methods; Cricetinae; Dogs; Fluorobenzenes; Haplorhini; Humans; Mass Spectrometry - methods; Mice; Rabbits; Rats; Swine; Triazoles
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1996.0478

A practical analytical method has been developed for the determination of an analog of vitamin D3(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 from 1.49 to 7.12% among the various species. Interassay precision (mean %RSD) ranged from 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 D2, vitamin D3, 1α,25-dihydroxyvitamin D3, and other drugs containing a diene structural moiety is discussed.