Determination of vitamin D3 and 25-hydroxyvitamin D3 in sera by column-switching high performance liquid chromatography with fluorescence detection

Tetsuharu IWATA, Masatoshi YAMAGUCHI, Hiroshi HANAZONO, Yuki IMAZATO, Masaru NAKAMURA, Yosuke OHKURA
1990 Analytical Sciences  
A column-switching high performance liquid chromatographic method with fluorescence detection for the determination of vitamin D3 and 25-hydroxyvitamin D3 in human and rat sera is described. The vitamins in a lipid extract from serum, obtained by solid-phase extraction technique using a Bond-Slut C18 cartridge, are converted with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl azide into the corresponding fluorescent derivatives. The derivatives are separated from endogenous
more » ... rom endogenous interfering substances by column-switching chromatography. The chromatographic system consisted of a precolumn chromatography for clean-up of the derivatives and an analytical column chromatography for the complete separation of the derivatives. The derivatives are detected fluorometrically at excitation and emission wavelengths of 360 and 440 nm, respectively. The detection limits (S/ N=3) for vitamin D3 and 25-hydroxyvitamin D3 are 15 and 8 fmol, respectively, in a 10 µl injection volume. The sensitivity permits simultaneous determination of the vitamins in 1 ml of normal human and rat sera. Keywords Vitamin D3, 25-hydroxyvitamin D3, column-switching high performance liquid chromatography, fluorescence detection, 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl azide, human serum, rat serum
doi:10.2116/analsci.6.361 fatcat:kwkxbmixb5azpcexcqegqjwmta