Fluorometric assay of 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in plasma.
The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery.
Shimizu
M
M
Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, Japan.
Gao
Y
Y
Aso
T
T
Nakatsu
K
K
Yamada
S
S
D015650:24,25-Dihydroxyvitamin D 3 / Q000097:blood*; D002112:Calcifediol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D006801:Humans; D011810:Quinoxalines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014230:Triazoles / Q000737:chemistry
D005456:Fluorescent Dyes; D011810:Quinoxalines; D014230:Triazoles; C070204:4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl)-1,2,4-triazoline-3,5-dione; D015650:24,25-Dihydroxyvitamin D 3; D002112:Calcifediol