Analytical, Nutritional and Clinical MethodsEvaluation of GC and GC–MS methods for the analysis of cholesterol oxidation products
Introduction
Cholesterol oxidation products (COP) commonly found in foods are cholest-5-en-3β,7α-diol (7α-HC), cholest-5-en-3β, 7β-diol (7β-HC), 5α,6α-epoxy- 5α-cholestan-3β-ol (α-CE), 5β,6β-epoxy- 5β-cholestan-3β-ol (β-CE), 5α-cholestan-3β,5,6β-triol (CT), cholest-5-en-3β,20α-diol (20α-HC), cholest-5-en-3β,25-diol (25-HC), 3β-hydroxycholest-5-en-7-one (7-KC) (Larkeson, Dutta, & Hansson, 2000). In the process of oxidation of cholesterol in foods, presence of unsaturated fatty acids, cholesterol level, heat, oxygen, light, UV light, γ-radiation, water activity, technologically related events, etc., influence the formation of COP in foods (Larkeson et al., 2000, Paniangvait et al., 1995, Savage et al., 2002).
In both in vivo and in vitro studies the COP have been shown to have a variety of potentially atherogenic, cytotoxic, mutagenic and possibly carcinogenic effects (Schroepfer, 2000). Cholesterol-containing foods, when consumed fresh, contain low levels of COP and the levels go up during processing, storage and cooking. The consumption of pre-cooked foods of animal origin is becoming more popular in the world due to the low cooking time involved in preparing them at home. Therefore, the intake of foods containing COP has increased (Savage et al., 2002). Thus for health reasons, it is important that these products in food are identified and quantified accurately (Dutta & Savage, 2002).
Analysis of COP generally passes through four major steps; prior extraction of lipids from foods, saponification of extracted lipids, and subsequent enrichment of COP and quantification by GC or by HPLC. An alternative to saponification of extracted lipids is transesterification of total lipids and subsequent enrichment of COP. In both of these methods, total COP in the lipids are accounted for, i.e., COP originating from both free and esterified cholesterol in the lipids. Various saponification methods are in use, e.g., hot saponification or cold saponification using either ethanolic or methnolic potassium hydroxide (KOH). Details of the methods of saponification can be found in the literature (Park & Addis, 1992).
Literature reports on the levels of COP in same type of food show that a number of methods have been used for each step in the analysis of COP, and thus the reported results are significantly different. This indicates the importance of the in-house method validation of COP analysis until a harmonised method is available (Dutta and Savage, 2002, McClusky and Devery, 1993). The main objective of the present study is to evaluate and validate our in-house method by comparing it with a number of commonly used methods for analysis of COP. In order to achieve the objective, the methods of purification and enrichment of COP by various saponification methods and transesterification of lipids with spiked samples of standard COP in tallow were investigated.
Section snippets
Materials
Crude tallow fat was donated by Baeten (Overmere, Belgium). Standard samples of cholesterol, 7α-HC cholester, 19-hydroxycholesterol (19-HC), 7β-HC, α-CE, β-CE, 20α-HC, CT, 25-HC and 7-KC were obtained from Steraloids (Wilton, NH, USA); 5α-cholestane was from Sigma-Aldrich (Stockholm, Sweden); Tri-Sil reagent was purchased from Pierce (Rockford, IL, USA). Ethanol and methanol were from Kemetyl (Haninge, Sweden). All other chemicals and solvents were of analytical grade and were purchased from
Results and discussion
Baseline separations among the standard samples of 5α-cholestane, 7α-HC, 19-HC, 7β-HC, β-CE, α-CE, 20α-HC, CT, 25-HC and 7-KC were achieved with the capillary column used in this investigation (Fig. 2). The resolution and elution pattern of COP are consistent in this column compared with published results where same column type, apart from higher film thickness, was used (Pie and Seillan, 1992, Regueiro and Maraschiello, 1997). In contrast, DB-1 type of column of same dimension used in this
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