Measurement of the concentration of 25(OH)D in serum/ plasma is widely used as a means of assessing vitamin D status in man. Although the values of D 2 and its metabolites in biological fluids are usually less than one-tenth of those of D 3 and its metabolites, their significant rise is observed in the subjects who had taken D 2 enriched foods and/or had received D 2 for nutritional and/or clinical purposes. Therefore, separate determination of 25(OH)D 2 and 25(OH)D 3 in serum/plasma is important in the field of nutritional and clinical studies.The serum/plasma 25(OH)D levels have conventionally been measured by HPLC with UV detection, but the method requires large sample volumes (0.5-2 ml) and time-consuming chromatographic purifications prior to quantification. [2][3][4] RIA for 25(OH)D utilizing a radioiodinated tracer has also been developed, which requires only 50 ml of plasma or serum and eliminates the need for chromatographic purifications before assay. 5) However, this method is not able to individually quantify 25(OH)D 2 and 25(OH)D 3 because the antibody used in the RIA reacts equally with these two metabolites, 5,6) and its assay validity in the samples with low concentration has been questioned.
7)LC-MS is considered to be a rapid and specific method for the determination of vitamin D compounds in biological fluids. However, the ionization efficiencies of vitamin D compounds are low in various ionization methods, such as electrospray ionization and atmospheric pressure chemical ionization (APCI). Because of the relatively high level at which it circulates in serum (10-40 ng/ml), the levels of 25(OH)D 3 can be determined by the usual LC-MS. On the other hand, those of 25(OH)D 2 cannot be determined without large sample volumes, so that several chromatographic purifications are necessary to remove interfering substances derived from the specimens.We have developed the LC-MS method for 24,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] in human plasma using the derivatization with a Cookson-type reagent (4-substituted 1,2,4-triazoline-3,5-dione).8) In the study, 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione (MBOTA-D), 9) which is rich in proton-affinitive atoms (oxygen and nitrogen atoms), was very effective in increasing the ionization efficiency of the resulting derivative in APCI operating in the positive-ion mode. These data indicate that the derivatization with a Cookson-type reagent having a different substituent at the 4-position, for example, one having more proton-affinitive atoms is capable of providing higher sensitivity in the positive APCI-MS. In the present paper, we describe an LC-APCI-MS-MS method for the simultaneous determination of 25(OH)D 2 and 25(OH)D 3 in human plasma employing derivatization with a Cookson-type reagent which does not require chromatographic purifications for sample pretreatment.
MATERIALS AND METHODS
Materials and Reagents
