2020 年 4 巻 1 号 p. 58-64
A highly-sensitive analytical method using a liquid chromatography (LC)/electrochemistry (EC)/electrospray ionization (ESI)-mass spectrometry (MS) is developed to analyze vitamin D3 (VD3) and its active metabolites, 25-hydroxyvitamin D3 (25(OH) D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in blood. LC/ESI-MS, which is used for the general sensitive analysis of biological compounds, has a low ionization efficiency for low polarity molecules such as VD3 metabolites. The LC/EC/ESI-MS method, which connects the electrochemical detector (ECD) between the LC and ESI-MS, enables simultaneous and sensitive detection of VD3 and its metabolites. The sample separated by LC is oxidized by an electrochemical cell, then the oxidant is ionized by ESI and detected by a mass spectrometer. Based on the results of analyzing VD3 metabolites, ion peaks of the oxidized products generated by EC are detected, and their peak intensities are 10 times or greater than the unoxidized metabolites. This proposed method for the VD3, 25(OH) D3, and 1,25(OH)2D3 analysis achieves the low detection limit of 1 nmol/L, 1 nmol/L, and 20 nmol/L, respectively, a good linearity (r2>0.9903), and a good repeatability within<10% (n=5). Analysis of serum samples spiked with VD3 metabolites detect their oxidants peaks with high sensitivity. This result indicates that this LC/EC/ESI-MS method is also useful for biological sample analysis and allows for quantification of trace amounts of endogenous fat-soluble metabolites other than vitamin D3 metabolites.
