Usefulness of Derivatization in High-Performance Liquid Chromatography/Tandem Mass Spectrometry of Conjugated Vitamin D Metabolites

larly the oxidation of its side chain, has been investigated by many groups, the conjugates of D metabolites still remain poorly understood. Axelson1 and we2 have reported that 25-hydroxyvitamin D3 [25(OH)D3] 3-sulfate (S) is a major circulating metabolite in humans. Furthermore, we identified the monoglucuronides (G) of D compounds {vitamin D2, vitamin D3 (D3), 25hydroxyvitamin D2, 25(OH)D3, and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3]} as biliary metabolites from rats dosed with each compound in comparison with standard samples based on their chromatographic behavior during high-performance liquid chromatography (HPLC) and/or liquid chromatography (LC)/mass spectrometry (MS).3,5 On the other hand, the structures of the other unknown conjugated D metabolites existing in small amounts in biological fluids should also be clarified. In this respect, it is necessary to establish a characterization method of metabolites whose standard samples are not available. Although LC/MS, especially LC/electrospray ionization (ESI)-MS, is the most promising analytical method for polar metabolites, such as these conjugates, this method gives fewer fragment ions; therefore, it is unsuitable for structure elucidation. Recently, much interest has been focused on LC/ion trap MS for the analysis of biological substances, because of its easy performance for multi-stage tandem MS (MSn), which is highly specific and provides more structural information. Furthermore, it is thought that derivatization is also effective to obtain more abundant and reliable information on LC/MS. In addition, the derivatization is expected to improve the detection limit in LC/MS. In the present paper, the advantages of derivatization for LC/ion trap MS analysis of the conjugates of D metabolites shown in Fig. 1 were examined. First, the effect of the derivatization for detection responses was examined using 24,25(OH)2D3-3G and -24G as model compounds. Second, the advantages of derivatization for the characterization of the structure were studied using some conjugated D metabolites.