Identification of conjugation positions of urinary glucuronidated vitamin D metabolites by LC/ESI-MS/MS after conversion to MS/MS-fragmentable derivatives.

A liquid chromatography/electrospray ionization-tandem mass spectrometry-based method was developed for the identification of the conjugation positions of the monoglucuronides of 25-hydroxyvitamin D [25(OH)D ] and 24,25-dihydroxyvitamin D [24,25(OH) D ] in human urine. The method employed derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis.