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

Abstract

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 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. KeywordsVitamin 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 Vitamin D3 is hydroxylated in the liver to 25-hydroxyvitamin D3 (25-OHD3), which is further hydroxylated in the kidney either to 1 a,25-dihydroxyvitamin D3 or to 24,25-dihydroxyvitamin D3. The determination of these compounds in human plasma/ serum is very important for the assessment of "vitamin D3 status" in healthy and diseased persons. Among these compounds, vitamin D3 and 25-OHD3 occur in serum at relatively high concentrations. Thus, their determination is still widely used for the assessment of vitamin D3 status and provides significant results in many clinical situations.Various methods for the determination of vitamin D3 and/ or 25-OHD3 in human plasma/ serum have been reported.l-22 The methods include lipid extraction, purification by chromatography and quantification by physicochemical methods such as UV-absorption'-14 and mass fragmentography15, competitive protein binding assaysg-14"7-22 or radioimmunoassay.14 Among these methods, the UV-high performance liquid chromatographic (HPLC) methods have been widely used for the simultaneous determination of vitamin D3 and 25-OHD3 in plasma/ serum. Such methods, however, include an extremely complicated pretreatment of plasma! serum, and require a very long time for the determination.We have developed 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl azide (DMEQ-CONS) as a highly sensitive and reactive fluorescence derivatization reagent for alcohols.23 The reagent has then been applied to the determination of cholesterol and cholestanol in human serum24,25, and 7-dehydrocholesterol (previtamin D3) in rat26 and human skin surfaces.27 Recently, we found that vitamin D3 and 25-OHD3 react with DMEQ-CONS to give the corresponding highly fluorescent carbamic acid...

Simultaneous Estimation of Geniposide and Genipin in Mouse Plasma Using High-Performance Liquid Chromatography

“…[20][21][22][23][24][25] Analyses of geniposide and genipin together with other iridoids have already been reported. 17 However, there have been no reports published regarding the detection and separation of these two compounds from each other in plasma from an animal.…”

Aminopropyl-silica as an advantageous alternative to nonpolar sorbents for continuous cleanup/preconcentration of vitamin D3 metabolites

“…Shimada et al have reviewed the methods presently available for the measurement of vitamin D3 and its metabolites in human fluids, [4]. These methods are based on protein saturation [5][6][7], radioimmunoassay [8,9], mass-spectrometry [10] or separation techniques which involve HPLC with several detection systems (photometric [11,12], fluorimetric [13,14], radiochemical [15,16] or mass-spectrometry [17]) as well as GC-MS [18, 19l. The low concentration levels of these metabolites in human fluids and the presence of other species with similar structures and properties make mandatory the use of purification steps prior to their determination in biological fluids. These steps have been based on either solid-phase extraction [5,[19][20][21] or liquid-liquid extraction [10,12,20], which are slow, time-consuming operations.…”