Application of cloud-point extraction-reversed-phase high-performance liquid chromatography. A preliminary study of the extraction and quantification of vitamins A and E in human serum and whole blood.

S R Sirimanne, D G Patterson, L Ma, J B Justice

Index: J. Chromatogr. B. Biomed. Sci. Appl. 716(1-2) , 129-37, (1998)

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Abstract

Methods available for quantification of vitamins A and E in serum or blood requires preconcentration and clean-up by liquid-liquid extraction, evaporation of the extract, and reconstitution of the extract in a solvent of choice before analysis. This process not only involves the use of toxic organic solvents but also requires a long sample preparation time. The lipids and other non-polar coextractants often require additional steps for sample clean-up and evaporation, which may cause sample losses. The use of cloud-point extraction eliminates most of these sample clean-up problems. We recently demonstrated that cloud-point extraction (CPE) can be used for extraction and quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) from human serum. We now demonstrate how CPE can be used with human serum and blood, at volumes as low as 50 microl, and report a methodology for extracting and quantifying two clinically important vitamins, (A and E) from human serum and blood. Vitamins A and E were extracted from human serum and blood by using Genapol X-80 as the cloud-point extractant under salting out conditions. Serum and blood samples were diluted in organic-free water to get sufficiently large sample volumes for CPE. The surfactant-rich phases were separated by centrifugation, and the samples were analyzed by HPLC-UV after deleterious coextractants were removed by precipitating them with acetonitrile. The recoveries of spiked vitamins A and E were found to be 85.6+/-0.4% and 82.6+/-5.2%, respectively. The average concentration of vitamins A and E in a serum pool after correction for recoveries were found to be 43.4+/-1.8 microg/dl (1.5+/-0.1 micromol/l) and 564.3+/-65.3 microg/dl (13.1+/-1.5 micromol/l), respectively. Vitamin A and E concentrations in whole blood were found to be 26.3+/-0.4 microg/dl (0.92+/-0.01 micromol/l) and 457.5+/-15.6 microg/dl (10.6+/-0.4 micromol/l), respectively. These values are comparable with those obtained by the reference method used at the Centers for Disease Control and Prevention. The success of the preliminary study will lead to a comprehensive validation of this method for vitamins A and E in serum and blood.