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A method is described for the simultaneous determination of cholesterol, phytosterols and tocopherols in dairy and non-dairy foods by high-performance liquid chromatography. A facile saponification and extraction scheme is completed rapidly within a single reaction tube. Clean-up is not required, with analysis completed by one of two alternative reversed-phase approaches. Sterols are monitored by short-wave ultraviolet spectrophotometry, and tocopherols by means of a series fluorescence detector. Cholesterol is resolved from the principal plant phytosterols, while the tocopherol congeners are well separated, thereby offering complementary information regarding source-lipid composition in foods under regulatory quality-control conditions. Squalene, an essential metabolic sterol precursor, can also be determined concurrently at the same viewing wavelength.
Solid food or milk powder (0.5 g), milk (5 g) or oil or fat (0.1 g) was mixed with ethanol (10 ml) and aq. 50% KOH (2 ml) and incubated with periodic agitation, at 70° for 8 min. The mixture was extracted with 20 ml of hexane - isopropyl ether (3:1) for 5 min, H2O (30 ml) was added and the mixture was centrifuged at 180 g for 10 min. The organic phase was evaporated to dryness and the residue was dissolved in hexane or ethanol (1 ml). A portion (20 to 50 µl) of soln. was analysed by HPLC on a Rad-PAK column of ODS (5 µm), with hexane - propan-2-ol (999:1) or methanol as mobile phase (1 ml min–1) and detection at 210 to 214 nm for sterols and fluorimetric detection at 330 nm (excitation at 295 nm) for tocopherols. Cholesterol (I) was separated from the principal phytosterols and the tocopherol congeners were well separated; squalene could be determined concurrently. Response was rectilinear over two orders of magnitude of I and α-tocopherol (II) and the detection limit was 0.04 µg of I. Recoveries were 95.2 to 97.7% of I and 93.8 to 94.2% of II. The sampling rate was ≤20 day–1.
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