The determination of 2β-(3-hydroxypropoxy)-1α,25-dihydroxy vitamin D 3 (ED-71) in human serum by high-performance liquid chromatography–electrospray tandem mass spectrometry

Abstract

A liquid chromatography–electrospray ionization tandem mass spectrometric (LC/ESI–MS/MS) method for the determination of 2β-(3-hydroxypropoxy)-1α,25-dihydroxy vitamin D 3 (ED-71) in human serum has been developed. ED-71 in human serum was extracted using two solid-phase extraction steps on Bond Elut C18 and NH2 cartridge. The separation of ED-71 and preED-71 isomer was attained by LC using 2 mmol/L ammonium acetate–methanol (15:85, v/v) as a mobile phase on a Symmetry C18 column (5 μm, 150 mm × 2.1 mm i.d.). ESI–MS/MS analysis was operated using selected reaction monitoring (SRM) in positive ion mode. The method achieved a lower limit of quantitation of 25 pg/mL. The calibration curve (25–3200 pg/mL) gave acceptable linearity ( r > 0.9964). Intra-assay precision ranged from 2.3 to 9.7%. Inter-assay precision ranged from 1.0 to 3.4%. The accuracy was within 90.8–107.0%. This highly sensitive and reproducible method is able to determine only biologically active ED-71 by separating it from preED-71, which is considered to be applicable for the determination of serum samples from pharmacokinetic studies in human.