A METHOD FOR THE SIMULTANEOUS DETERMINATION OF VITAMIN-D2, VITAMIN-D3 AND THEIR METABOLITES IN PLASMA AND ITS APPLICATION TO PLASMA SAMPLES OBTAINED FROM NORMAL SUBJECTS AND PATIENTS

A method has been established for the simultaneous determination of vitamins D2,D3 and their metabolites in plasma. Vitamin D2/D3, 25-hydroxyvitamin D2/D3 (25-OH-D2/D3), 24R,25-dihydroxyvitamin D2/D3 [24,25(OH)2D2/D3] and 1-alpha,25-dihydroxyvitamin D2/D3 [1,25(OH)2D2/D3] in 1.5 ml of a plasma sample were simultaneously assayed. The method includes extraction of lipid, followed by three steps of high-performance liquid chromatography (HPLC) for clean-up and separation. Vitamin D2/D3 and 25-OH-D2/D3 were quantified by a UV detector, while 24,25(OH)2D2/D3 and 1,25(OH)2D2/D3 were assayed by competitive protein binding assay (CPBA) and radioreceptor assay (RRA) methods, respectively. Existing interfering substances in a sample could be effectively eliminated by HPLC and reliable results with small coefficients of variation were obtained. The method was applied to plasma samples obtained from normal subjects and patients with epilepsy, liver cirrhosis, leukaemia, diabetes, sensorineural deafness and hypophosphataemic osteomalacia; satisfactory results were obtained.