CHARACTERIZATION OF VITAMIN-D(3) METABOLITES USING CONTINUOUS-FLOW FAST-ATOM-BOMBARDMENT TANDEM MASS-SPECTROMETRY AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A mass spectrometric method for the detection of vitamin D, metabolite s is described. This method involves the derivatization of the metabol ites by cycloaddition with 4-phenyl-1,2,4-triazoline-3,5-dione, follow ed by their characterization by continuous-flow fast atom bombardment (CF-FAB) tandem mass spectrometry (MS-MS) and high-performance liquid chromatography (HPLC). Using HPLC, this derivatization has been shown to increase the UV detectability of 25-hydroxyvitamin D3 by about 5-fo ld. The FAB spectra of the adducts are dominated by peaks correspondin g to a protonated molecule and a fragment ion derived in part from the loss of the side chain. Multiple-reaction monitoring (MRM) of this tr ansition by MS-MS may be utilized for trace level analysis of vitamin D metabolites. Sample introduction by flow injection yields detection limits in the low nanogram to high picogram range, whereas the use of on-line capillary LC has been found to decrease the detection limits t o the low picogram level.