SIMULTANEOUS DETERMINATION OF EIGHT VITAMIN D₂ ISOMERS BY HIGH-SPEED LIQUID CHROMATOGRAPHY

Abstract

The simultaneous determination of vitamin D₂ and its isomers by high-speed liquid chromatography (HSLC) is described. Preferred operating conditions for the resolution and quantitation of vitamin D₂, 5, 6-traps-vitamin D₂, ergosterol, lumisterol₂, isovitamin D₂, isotachysterol₂, tachysterol₂, and previtamin D₂ are as follows:
column, “Zorbax” SIL (25cm×2.1mm i.d.); pressure, 100-120kg/cm²; temperature, ambient; detector, UV 254nm; mobile phase, 0.15% methanol+2% ether in pentane (for the simultaneous determination), 10% ether in hexane (for quantitation of the practical sets of isomers), or 55% CHCl₃ (distilled) in pentane (for separation of isovitamin D₂ and 5, 6-traps-vitamin D₂); sample size, 1 μl (>1-5 ng); internal standard, p-cresol or α-naphthol.
High-speed, good resolution, precise and accurate trace analysis, greater analysis flexibility, no necessity for preparing derivatives, and gentle operating conditions are main potential advantages of our method which has proved to be very efficient and surpasses all analytical procedures hitherto proposed.