Gas chromatographic-mass fragmentographic determination of serum 1 alpha,25 dihydroxyvitamin D3

We extracted 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] from 10 mL serum using Sep-Pak C18 and Sep-Pak Silica mini-columns and normal-phase high performance liquid chromatography (HPLC) separation for analysis by gas chromatography-mass fragmentography (GC-MS). A GC-MS method was optimised using manual tuning for ion mass calibration and selective ion monitoring (SIM) for quantitation. Serum 1 alpha,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 picograms of 1 alpha,25(OH)2D3 gave a peak with a signal-to-noise ratio of 26:1. Between-batch coefficient of variation (CV) for 1 alpha,25(OH)2D3 standard was < 13%. However, serum analysis was less precise, within-batch CV being 20%. The analytical recovery was about 70% and detection limit 0.5 pg/mL. When compared with a commercial radioreceptor assay we still found our method to be sensitive, specific, and adequate for confirmative and semiquantitative analysis of serum 1 alpha,25(OH)2D3.