GAS-CHROMATOGRAPHIC MASS FRAGMENTOGRAPHIC DETERMINATION OF SERUM 1-ALPHA,25 DIHYDROXYVITAMIN D3

We extracted 1alpha,25-dihydroxyvitamin D3[1alpha,25(OH)2D3] from 10 m L serum using Sep-Pak C18 and Sep-Pak Silica minicolumns and normal-ph ase high performance liquid chromatography (HPLC) separation for analy sis by gas chromatography-mass fragmentography (GC-MS). A GC-MS method was optimised using manual tuning for ion mass calibration and select ive ion monitoring (SIM) for quantitation. Serum 1alpha,25(OH)2D3 was identified by superimposition of the m/z 452 and 501 ion peaks and by overlapping the m/z 452 ion peak with that of its authentic standard. It was quantitated from the relative peak areas of its m/z 452 ion and the m/z 363 ion of vitamin D2, the internal standard. Twenty picogram s of 1alpha,25(OH)2D3 gave a peak with a signal-to-noise ratio of 26:1 . Between-batch coefficient of variation (CV) for 1alpha,25(OH)2D3 sta ndard was <13%. However, serum analysis was less precise, within-batch CV being 20%. The analytical recovery was about 70% and detection lim it 0.5 pg/mL. When compared with a commercial radioreceptor assay we s till found our method to be sensitive, specific, and adequate for conf irmative and semiquantitative analysis of serum 1alpha,25(OH)2D3.