Determination of ergocalciferol in human plasma after Diels-Alder derivatization by LC–MS/MS and its application to a bioequivalence study

摘要

An accurate, sensitive and selective method is developed for determination of ergocalciferol (vitamin D 2 ) in human plasma using LC–MS/MS. After liquid-liquid extraction with n- hexane, ergocalciferol was derivatized by reacting with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a strong dienophile based on Diels-Alder reaction. Ergocalciferol and its deuterated internal standard, ergocalciferol-d6, were analyzed on X Select CSH C 18 (100mm×4.6mm, 2.5μm) column using acetonitrile and 0.1% (v/v) formic acid in water containing 0.14% methylamine within 6.0min under gradient elution mode. Tandem mass spectrometry in positive ionization mode was used to quantify ergocalciferol by multiple reaction monitoring (MRM). Entire data processing was done using Watson LIMS? software which provided excellent data integrity and high throughput with improved operational efficiency. The major advantage of this method includes higher sensitivity (0.10ng/mL), superior extraction efficiency (≥83%) and small sample volume (100μL) for processing. The method was linear in the concentration range of 0.10–100ng/mL for ergocalciferol. The intra-batch and inter-batch accuracy and precision (% CV) values varied from 97.3% to 109.0% and 1.01% to 5.16%, respectively. The method was successfully applied to support a bioequivalence study of 1.25mg ergocalciferol capsules in 12 healthy subjects.