A semi-automated high-performance liquid chromatographic system for the determination of 25-hydroxyvitamin D in human plasma: elimination of interference by barbiturates and use of photodiode array detection.

A semi-automated high-performance liquid chromatographic (HPLC) method for the measurement of 25-hydroxyvitamins D(2) and D(3) is described. Plasma was extracted using acetonitrile and a Bond-Elut C(18) cartridge system, eluted with methanol and fractionated on Sep-Pak SIL. After formation of isotachysterol isomers straight-phase HPLC was carried out monitoring the mobile phase with a photodiode array detection system. Two internal standards have been used, namely [(3)H]25-hydroxyvitamin D(3) and 25-hydroxydihydrotachysterol(3) both of which are shown to give satisfactory results. The use of the extraction system described eliminates interference from barbiturates which had previously been reported to interfere. Photodiode array detection allows confirmation of the identity of the analytes.