A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 using Sep-Pak C18 cartridges and a single high-performance liquid chromatographic step.
A simple one step high-performance liquid chromatographic (HPLC) procedure for the analysis of plasma concentrations of 25-hydroxyvitamin D3 (25-OHD3) and 25-hydroxyvitamin D2 (25-OHD2) is described. Plasma (2-4 ml) was extracted with methyl cyanide which was passed through a Sep-Pak C18 cartridge, washed with methanol:water (70:30, v/v) and the 25-OHD fraction eluted with methyl cyanide. After isomerisation to their isotachysterol derivatives, the secosteroids were estimated using a straight-phase HPLC system, monitoring the eluent at 301 nm. Radioactive 25-OHD3, added to plasma at the start of the procedure, was used to correct for losses. Recovery of added 25-OHD3 was quantitative and values obtained using this method were similar to those obtained on the same plasma samples using a mass fragmentographic technique. Normal ranges were similar to those described by other workers and within- (5.8% for 25-OHD3) and between-(8.0% for 25-OHD3) batch reproducibilities were satisfactory.
D015652:25-Hydroxyvitamin D 2; D000097:Acetonitriles; D002112:Calcifediol; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004872:Ergocalciferols / Q000031:analogs & derivatives* / Q000097:blood; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006887:Hydroxycholecalciferols / Q000097:blood*; D007536:Isomerism; D011933:Reagent Kits, Diagnostic
D000097:Acetonitriles; D004872:Ergocalciferols; D006887:Hydroxycholecalciferols; D011933:Reagent Kits, Diagnostic; D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol