Elsevier

Clinica Chimica Acta

Volume 120, Issue 1, 26 March 1982, Pages 65-76
Clinica Chimica Acta

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 Chromatographie step

https://doi.org/10.1016/0009-8981(82)90078-XGet rights and content

Abstract

A simple one step high-performance liquid Chromatographie (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.

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