Determination of Vitamin D3 and 25-Hydroxyvitamin D3 in Sera by Column-Switching High Performance Liquid Chromatography with Fluorescence Detection

Autor: Tetsuharu Iwata, Masatoshi Yamaguchi, Yosuke Ohkura, Yuki Imazato, Hiroshi Hanazono, Masaru Nakamura
Rok vydání: 1990
Předmět:
Zdroj: Analytical Sciences. 6:361-366
ISSN: 1348-2246
0910-6340
DOI: 10.2116/analsci.6.361
Popis: 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 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 440nm, respectively. The detection limits (S/N=3) for vitamin D3 and 25-hydroxyvitamin D3 are 15 and 8fmol, respectively, in a 10μl injection volume. The sensitivity permits simultaneous determination of the vitamins in 1ml of normal human and rat sera.
Databáze: OpenAIRE
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