A validated liquid chromatography method for the simultaneous determination of vitamins A and E in human plasma

Abstract

A fast (15 min) and simple HPLC method for determination of all-trans-retinol and alpha-, gamma- and delta-tocopherols in human plasma has been developed. The assay utilized 200 microl of plasma to which 20 microl of internal standard solution (retinol acetate) was added followed by 200 microl of water, 400 microl of ethanol and 800 microl of hexane. The hexane layer was collected, evaporated, the residue dissolved in 200 microl of methanol and analyzed on a Zorbax Eclipse XDB-C18 column using a step gradient with a polar organic mobile phase composed of acetonitrile and methanol and variable wavelength fluorescence detection. The quantification limits for all-trans-retinol and gamma-tocopherol were 20 ng/ml and for alpha- and delta-tocopherols 500 ng/ml and 10 ng/ml, respectively. The procedure was validated and applied to the analysis of plasma samples from the Baltimore Longitudinal Study of Aging.

High Serum Level of Retinol and α-Tocopherol Affords Protection Against Oral Cancer in a Multiethnic Population

“…Matched case-control serum samples were analysed for serum level of retinol, α-tocopherol and β-carotene using high performance liquid chromatography (HPLC) method. The serum samples prepared for analysis was a modified version of an earlier reported procedure (Siluk et al, 2007). Briefly, a mixture of 100µl serum, 50µl internal standards, 100µl distilled water was vortex-mixed and the resulting solution was added to a 200µl aliquot of ethanol.…”

Fluorometric Determination of Vitamin Constituents in Human Plasma Using Ultra Performance Liquid Chromatography

“…All standard solutions were stored at -80°C before use during assay development. Ethanol was observed to be a suitably soluble and stable solvent for the dissolution and storage of all vitamins, as noted during prior investigations [36,49]. All vitamin stock solutions were stored in dark, airtight bottles to inhibit photodegradation [50].…”

“…The extraction procedure used during assay development was adapted and modified from an extraction procedure presented by Siluk and colleagues [36]. Twenty microliters of 25 μg/mL retinol acetate internal standard solutions was pipetted onto a 100 μL aliquot of human plasma.…”

“…Although normal phase methods have been employed for the separation of some compounds [9][10][11][12][13], the vast majority of high performance liquid chromatography based fat soluble vitamin assay methods have employed reversed phase chromatography to facilitate analyte separation . Ultraviolet detection of these compounds has been most common in the literature [42][43][44][45][46][47], but fluorescence detection has also been used in the detection of tocopherol and retinol [32][33][34][35][36][37][38][39][40][41][42]. The use of fluorescence detection has allowed for various compounds to be analyzed at low limits of quantification and detection in plasma-human [32][33][34][35][36][37]40] or otherwise [39] as an alternative to ultraviolet detection methods.…”

“…Several methods have been developed for the simultaneous monitoring of tocopherol and retinol in human plasma [5][6][7][8][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]29,30,[32][33][34][35][36][37]40,[43][44][45][46]. These methods have often featured chromatography run-times and flow rates requiring considerable consumption of time and/or solvents during sample analyses [16-18, 20,21,29,35,47].…”