Determination Of Cholesterol Oxides Research Articles

Determination of cholesterol oxides by gas chromatography-flame ionization detection/mass selective detection and their occurence in lanolin-containing cosmetics and ointments.

Cholesterol oxides (COPs) are thought to be of toxicological relevance in cholesterol-containing foods. For cholesterol-containing cosmetics and the like, no information is available up to this date. Therefore, the first of two main aims of this study was to develop and validate a method for determining COPs in lanolin-containing cosmetics such as lipsticks and fatty creams as well as in nipple ointments. The second aim was to study the occurrence of COPs and their concentration levels in the respective product classes. The procedure is based on a published method for food comprising some necessary modifications. Sample preparation consisted of transesterfication, solid-phase extraction and silylation of target compounds. Separation of the derivatized COPs and their quantification were performed with gas chromatography (GC) using a flame ionization detector (FID) or a mass spectrometer (MS). The successful validation and the trouble-free application during the market survey showed that the method was fit for purpose. Total COP levels found were in the low per cent range (up to 3%) and surprisingly high, being many orders of magnitude higher than those published for foods. To our knowledge, we present for the first time a method for the determination of COPs in non-food consumer products. Furthermore, our study demonstrates that lanolin-containing cosmetics may be an additional exogenous source of COPs. We further show evidence, that at least part of the COPs are already formed on the sheep's wool.

Determination of cholesterol oxides in anchovies (Engraulis encrasicolus) treated with a commercial mixture of citric acid, trisodium acid and hydrogen peroxide

Determination of cholesterol oxides in anchovies (Engraulis encrasicolus) treated with a commercial mixture of citric acid, trisodium acid and hydrogen peroxide

Determination of cholesterol oxides in anchovies (Engraulis encrasicolus) treated with a commercial mixture of citric acid, trisodium acid and hydrogen peroxide

A new additive formulation containing hydrogen peroxide, citric acid and trisodium acid is illegally used in fishery products due its whitening and antioxidant features. Aim of this study was to evaluate the possible presence of COPs and their role as markers of illegal treatment in anchovies (Engraulis encrasicolus) stored at different temperatures. Sensory analysis was also performed by the specific QIM test. The quantitative determinations (%) of cholesterol oxides (COPs) showed changing amounts during storage. Not always The COPs measured in the treated samples were significantly higher than control samples. Considering the volatility of hydrogen peroxide and the poor repeatability of COPs analyses, as shown in the present study, it is crucial to intensify the control by the Authorities.

Simultaneous determination of cholesterol oxides, cholesterol and fatty acids in processed turkey meat products

Cholesterol oxides, cholesterol, total lipids and fatty acids were determined in the lipid extracts from eight processed turkey meat products (blanquet, frankfurter, ham, meatball, smoked breast, smoked ham and roule). Cholesterol and cholesterol oxides were determined simultaneously by high performance liquid chromatography using a diode array detector at 210 nm and a refractive index detector. Only two cholesterol oxides were identified, 7-ketocholesterol (not detected up to 184 μg/100 g) and β-epoxycholesterol (not detected up to 450 μg/100 g). With the exception of the meatballs, hamburger and frankfurters, all the turkey meat products showed less than 5% fat, and could thus be considered as low fat foods. The cholesterol content varied from 32 to 43 mg/100 g, the ratio of polyunsaturated:saturated fatty acids varied from 1.1 to 1.8 and the ratio of ω6:ω3 from 18 to 28. Trans fatty acids were present in significant amounts in both hamburger and meatball.

Determination of cholesterol oxides in processed food using high-performance liquid chromatography–mass spectrometry with atmospheric pressure chemical ionisation

The present work describes the development and application of an on-line atmospheric pressure ionisation (APCI) LC–MS interface for the simultaneous determination of seven toxicologically relevant cholesterol oxides (7α-hydroxycholesterol, 7β-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, 5,6α-, 5,6β-epoxycholesterol and cholestan-3β,5α,6β-triol). The HPLC method has been optimised to reach better separation of all tested compounds. The influences of APCI parameters (nebulising temperature, cone voltage, source temperature) on signal intensity and fragmentation pattern were investigated for all tested cholesterol oxides compounds. This is the first report on optimisation and determination of two compounds 7α-hydroxycholesterol and 5,6β-epoxycholesterol in processed food using LC–MS. After extraction with hexane, clean-up was carried out using solid-phase extraction on a silica column. For the chromatographic separation of cholesterol oxides an Aquasil C 18 column was used with acetonitrile–methanol (60:40) as mobile phase. For the first time we report the use of such a C 18 column with a relatively hydrophilic nature for the separation of cholesterol oxides. APCI-MS detection was then applied in selected ion monitoring and positive ion modes by using the molecular ions and the main fragments. The developed method shows good linearity, high repeatability and good recovery for all tested cholesterol oxides. The method was applied for determination of seven selected cholesterol oxidation products in different foodstuffs such as butter, butteroil, lard and egg powder.

Development of gas chromatographic method for determination of cholesterol oxides in meat products

A method of extraction, purification and gas chromatographic analysis of five cholesterol oxidation products (COPs) (7β-hydroxycholesterol, 5α, 6α-epoxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol and cholestane-3β, 5α, 6β-triol) has been developed. The method is aimed at the determination of COPs in meat and meat products, in particular dry sausages, where large differences in the content of cholesterol and its oxidation derivatives may occur. Linearity of response of trimethylsilyl ethers was defined. The minimum concentration tested was 2.5 μg g−1 in the injected solution. The main feature of the present work is the addition of a purification step of unsaponifiables by SPE on silica cartridges. Extraction recoveries of COP standard mixtures, performed with or without the SPE step, are evaluated. The introduction of the clean-up step results in high purity samples without further losses in the entire process.

Determination of Cholesterol Oxides in Dairy Products. Effect of Storage Conditions

A methodology to quantify cholesterol oxidation products (COPS) in dairy products has been established by gas−liquid chromatography (GLC). Lipids were extracted according to the Folch method and saponified at room temperature using sodium hydroxide in methanol/benzene (3:2, v/v). Cholesterol oxides were derivatized to trimethylsilyl ethers and resolved by GLC, confirming the structures of cholesterol oxides detected in selected samples by GLC/MS. This methodology allows quick, simple, and reliable quantification of free and esterified cholesterol oxidized derivatives. This method was applied to milk powders, butter oil, and isolated milk protein powders. Effects of adverse storage conditions were studied in whole and skim milk powders. Storage temperature severely affected levels of COPS even under mild storage conditions. Packaging under nitrogen retarded cholesterol oxidation. Slight differences in COPS profiles were observed between different storage conditions. 7-Ketocholesterol was the major choleste...

Procedure for the determination of eight cholesterol oxides in poultry meat using on-column and solvent venting capillary gas chromatography.

A procedure for the determination of eight relevant cholesterol oxides in poultry meat has been developed. The method consists of the enrichment of cholesterol oxides by means of the combined use of solid-phase fractionation and thin-layer chromatography. Florisil and silica columns of 10 g permitted the handling of the total cholesterol oxides content included in the lipid bulk obtained after the Folch's extraction of 20 g of muscle meat. The determination of cholesterol oxides under their trimethylsilyl derivatives was performed by using capillary gas chromatography. The use of a fused-silica open tubular capillary column 30 m×0.25 mm I.D. coated with 5% phenylmethylsilicone and with a film width of 0.25 μm permitted the separation of all the species. Two modes of injection (on-column and solvent venting) were evaluated and compared for the analysis of cholesterol oxides. On-column capillary gas chromatography (cGC) gave better absolute areas relative standard deviation (R.S.D.) values: 3% to 6% vs. 5% to 7% for solvent venting cGC. Regression analysis for each cholesterol oxide was performed for the two modes of injection. The possibility of large volume injection (10 μl) by using the solvent venting mode was also evaluated in order to increase the sensitivity of the detection of cholesterol oxides. R.S.D. values for absolute areas ranging from 6% to 14% were obtained. The validation of the method was carried out within the range of 0.1–1 ppm. Absolute and relative recovery values ranging from 80% to 100% were obtained. Statistical analysis revealed that the method was reproducible. cGC–mass spectrometry was also used to confirm the peaks detected by cGC: the total ion chromatogram mode was used for the analysis of samples containing concentrations down to 0.1 ppm of cholesterol oxides. The analysis of fresh and cooked chicken meat revealed the presence of cholesterol oxides proceeding from the autoxidation of the cholesterol B-ring. Finally, saponification was found to be not as accurate as the described procedure for cholesterol oxides analysis.

Enzymatic Determination of Cholesterol Oxides

For measurement of the concentration of oxidised cholesterols in foodstuffs, a method was developed by adapting the well-known enzymatic determination of cholesterol. A linear correlation was found between the absorbance measured after enzymatic reaction and the concentrations of 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α, 6α-epoxide in the range 1-12 μg. The usefulness of this method was demonstrated by determination of the four cholesterol oxidation products separated by TLC from the non-saponifiable lipid fraction of 5-month-old whole-egg powder (stored under different conditions) and of biscuit enriched with egg powder. The main advantages of the combination of TLC and the enzymatic method, beyond its rapidity and sensitivity, are that the determination of cholesterol oxidation products can be carried out in parallel with many samples containing small amounts of oxidised cholesterols even in the presence of a large quantity of cholesterol.

Determination of cholesterol oxides in heated lard by liquid chromatography

The presence of cholesterol oxidation products (COPS) in heated lard by liquid chromatography was studied. Lard was heated at 180°C for 0, 2, 4, 8, 10, 20, 50, 70, 100, 150 and 200 h. The various COPS present in heated lard were analysed by thin-layer chromatography and high-performance liquid chromatography. 6-Ketocholesterol was used as an internal standard to quantify all the compounds. Results implied that in most cases the COPS content increased along with the increase of heating time. The only exception was that both 5,6α-epoxycholesterol (5,6β-epoxycholestanol) and 7β-hydroxycholesterol contents increased during the first 100 h and then began to decline. Cholestan-3β-5α-6β-triol could be detected only after 20 h heating, and reached plateau as the heating time increased to 200 h The loss of cholesterol reached 70% after 150 h heating, and the amount of cholesterol lost during heating was not equal to the amount of COPS formed. Surprisingly, the highly toxic 25-hydroxycholesterol was not found.