Various methods have been developed for cholesterol determination in foods due to the necessity of precise and accurate results. The majority of the methods used in food are gravimetric, colori-metric, enzymatic, and chromatographic. The first method used to determine cholesterol in foods was the gravimetric method, in which the cholesterol is separated by precipitation with digitonin or tomatin.5 This analytical method is time consuming and has low sensitivity. Although colori-metric methods are widely used for measuring the cholesterol extracted from meat,6-13 they have a tendency to overestimate the cholesterol content of foods, due to the presence of other interfering chromogens present in the samples. Enzymatic methods using cholesterol oxidase have been used to determine cholesterol in meat and meat products.14-18 As with colorimetric methods, this reaction is also not specific for cholesterol, since all sterols with a 3 p-OH group react with this enzyme, leading to overestimation of the cholesterol content in foods, especially those containing mixtures of animal and vegetable material. The extent of this overestimation, however, would depend on the method used and the sample analyzed. For example, results of the cholesterol obtained from meats by colorimetric methods were comparable to those obtained by gas chromatography
(GC), as pointed by Bohac et al.,19 or high performance liquid chromatography (HPLC), as demonstrated by Bragagnolo and Rodriguez-Amaya.20 Using an enzymatic method, Saldanha et al.14 found no significant difference in bovine meat and milk cholesterol values compared to the results obtained by HPLC. However, Almeida et al.21 obtained higher cholesterol levels by the enzymatic method than those measured by HPLC in semimembranosus, chicken drumsticks, and chicken thighs, but for biceps femoris no difference was observed between the two methods. However, colorimetric and enzymatic methods need strict control of the analytical conditions to give accurate results.
Currently, chromatographic techniques are preferred for dietary cholesterol analysis, due to their ability to separate and quantify cholesterol specifically. To determine cholesterol in meat and meat products, GC methods have been used19,22-24 as well as HPLC methods.25-30 Over the past few years, HPLC has been used as an alternative to GC in cholesterol analysis since the former is carried out at relatively low temperatures, thus preventing the oxidation of cholesterol; cholesterol concentrations obtained by the two methods did not show significant differences.31 However, in samples containing cholesterol and phytosterols in very small amounts, the GC method is preferred since it is more sensitive.
Normally, the chromatographic cholesterol assay would include the following steps: (1) extraction of total lipids with an organic solvent or solvent mixture, (2) removal of the solvent, (3) alkaline saponification of the total lipids, (4) extraction of the unsaponfiables with an organic solvent, (5) removal of the solvent, (6) derivatization of the unsaponfiable matter if GC is to be used (although Kanada et al.32 showed that derivatization was not essential), and (7) chromatographic estimation of the analyte.
The most common extraction method is direct saponification of the food, followed by extraction of the unsaponfiable matter with an organic solvent and subsequent determination of cholesterol by GC15,21,33-35 or HPLC.21,36 Van Elswyk et al.37 and Bragagnolo and Rodriguez-Amaya36 observed that saponification of a lipid extract resulted in a significantly lower value than direct saponification of the sample. The saponification step is very important in the determination of total cholesterol, since in this step the cholesterol esters are converted to free cholesterol; when this step was not carried out, the results must be expressed as free cholesterol or the results of total cholesterol are substituted.
GC detection of cholesterol has also been carried out by flame ionization detector (FID), and separation can be performed in either medium of low or nonpolar columns, but in the majority of studies the slightly polar HP-5, DB-5 fused silica bonded phase capillary column has been used. Identification of the cholesterol was performed by comparison of the retention times of the samples with those of the cholesterol standard, co-chromatography, and confirmed by MS.15,38 Quantification was done by internal standardization using 5a-cholestane in most studies,15,31-33,38,39 although the choices depend to a great extent on the type of sample to be analyzed.40 Thompson and Merola40 recommended 5a-cholestanol instead of 5a-cholestane because it is an alkane and so does not have the same chemical and physical properties as cholesterol or one of the plant sterols when the samples do not already contain such sterols.
Over the past few years, several HPLC methods for quantitative analysis of cholesterol in meat have been developed using direct saponification14,21,41 or with extraction of the lipid and subsequent saponification.27,28,42,43 A reversed-phase (C18, 150 mm, 4.6 mm, and 5 ^m) column, with a mixture of acetonitrile and 2-propanol (in the following proportions: 80:20, 70:30, and 60:40) as mobile phases, and ultraviolet (UV) detection set at 210 nm are the HPLC conditions widely used for the analysis of cholesterol in meat. Identification of the cholesterol is performed by comparison of the retention times of the samples with those of the cholesterol standard, co-chromatography, and spectra taken at 190—300 nm with the photodiode array detector. To confirm its identity and purity, cholesterol was accumulated from several HPLC runs of the sample and analyzed by GC-MS.36 Quantitative determinations are usually made using external standardization or, when possible, internal standardization using 6-ketocholesterol,14 stigmasterol,30 or pregnolone.31
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