Analysis of sterol oxidation products in foods

The main aspects related to the analysis of sterol oxidation products (SOP) in foods are comprehensively reviewed. Special emphasis is placed on the critical and controversial points of this analysis because these points affect crucial analytical parameters such as precision, accuracy, selectivity, and sensitivity. The effect of sample preparation and the conditions of quantification by gas chromatography and liquid chromatography on these parameters are also reviewed. The results show that, in order to choose an adequate method to analyze SOP in a certain food, the analyst must consider its SOP concentration and matrix complexity. The term SOP includes both cholesterol oxidation products (COP) and phytosterol oxidation products (POP). The state of the art of COP and POP analysis is quite different; many more studies have dealt with the analysis of COP than of POP. However, most of the results presented here about COP analysis may be extrapolated to POP analysis because both groups of compounds show similar structures and characteristics.     

Further data on a histochemical reaction as applied to the thin-layer chromatography of sterols.

A method for the characterization of sterols on silica gel G layers is proposed. After the chromatograms have been sprayed with a permanganate-sulphuric acid oxidative reagent and the reaction has been terminated with sodium hydrogen sulphite, the plates are sprayed with the colour-developing reagent (an acid solution of alcian blue or toluidine blue). The plates are also viewed under UV radiation (254 and 366 nm). Only 3 of the 28 sterol samples assayed (spot content 8 mug) did not show positive reactions after oxidation, which suggests that this step can be used as a "universal" detection method for sterols. After staining the plate, several sterols are shown to be easily differentiated from one another. The exposure of the plates to UV radiation assists characterization. In general, the reaction exhibits satisfactory sensitivity for the qualitative and differentiating detection of sterols; it is also rapid and easy to carry out.     

An HPLC method for the quantification of sterols in edible seaweeds

This study presents an HPLC method for the quantification of sterols in edible seaweeds. Sterols were identified by HPLC/mass spectrometry (HPLC-MS) in positive APCI mode. The samples were saponified by refluxing with 1 m ethanolic KOH, and the non-saponifiable fraction was extracted with hexane. Sterols were quantified by HPLC with UV detection (HPLC-UV), on a 15 x 0.4 cm Kromasil 100 C(18) 5 micro m column (mobile phase 30:70 v/v methanol:acetonitrile; fl ow rate 1.2 mL/min; column temperature 30 degrees C; detection wavelength 205 nm). Method repeatability for fucosterol was good (coefficient of variation 2.4%). Sterol contents were determined in canned or dried brown seaweeds (Himanthalia elongata, Undaria pinnatifida, Laminaria ochroleuca) and red seaweeds (Palmaria sp., Porphyra sp.). The predominant sterol was fucosterol in brown seaweeds (83-97% of total sterol content; 662-2320 micro g/g dry weight), and desmosterol in red seaweeds (87-93% of total sterol content; 187-337 micro g/g dry weight).     

High resolution GC of unsaponifiable matter and sterol fraction in vegetable oils

Summary The analysis of both the total unsaponifiable matter and the sterol fractions in vegetable oils has been performed with a new polar column (TAP, Chrompack). The use of a polar column, which is characterized by high thermal stability, has led to the identification of a greater number of constituents than the use of a nonpolar column (i.e. SE 52, SE 54). The polar column enhances the separation of the main classes of unsaponifiable matter (sterols, 4-methyl sterols and 4,4-dimethyl sterols). When used for the unsaponifiable and sterol fraction analyses, it offers a powerful tool for characterizing the lipid source.     

A single step solid-phase extraction method for complete separation of sterol oxidation products in food lipids

One of the crucial steps in determination of sterol oxidation products (SOPs) in foods is their enrichment and purifications by various preparative methods for further analysis by GC and GC–MS. Among the preparative methods, SPE of various adsorbents and solvent systems, are being used most widely. At present, no single step SPE method is suitable to completely separate the SOPs. In this study, a SPE (1 g silica) method, suitable for both transesterified and cold saponified oil samples, was developed to separate completely SOPs from other lipid components. This method resulted in high recovery from rapeseed oil of added 5β,6β-epoxycholestan-3β-ol (94-96%), cholest-5-en-3β-ol-7-one(94%), cholestane-3β,5α,6β-triol (88–91%), cholest-5-en-3β,7α-diol and 5α,6α-epoxycholestan-3β-ol (88–90%). The method has a high sample capacity of up to 1 g transesterified or cold-saponified oil sample. The method was tested and applied to different vegetable oils and to monitor the effects of refining processes on POPs in hazelnut oil.     

Influence of salinity and natural organic matter on the solid phase extraction of sterols and stanols: application to the determination of the human sterol fingerprint in aqueous matrices

Faecal sterols have been proposed as direct chemical markers for the determination of faecal contamination in inland and coastal waters. In this study, we assess the impact of (a) the concentration of dissolved organic carbon (DOC), (b) the nature of DOC, (c) the salinity and (d) the concentration of sterols and stanols on their solid phase extraction. When natural organic matter (NOM) is modelled by humic acid, increasing DOC concentration from 2.7 to 15.4 mg/L has no significant impact on the recovery of sterols and stanols. The modelling of NOM by a mixture of humic acid and succinoglycan induces a significant (24%) decrease in the recovery of sterols and stanols. For all concentrations of target compounds, no significant increase in recovery is associated with increasing the salinity. Moreover, an increase in the recovery of target compounds is induced by an increase in their concentration. The nine target compounds and the recovery standard (RS) exhibit the same behaviour during the extraction step. Thus, we propose that (a) the concentration of target compounds can be corrected by the RS to calculate more realistic concentrations without modifying their profile and (b) the sterol fingerprint can be investigated in the colloidal fraction of aqueous samples without altering the information it could provide about the source. The application of this analytical method to waste water treatment plant influent and effluents yields results in agreement with previous studies concerning the use of those compounds to differentiate between sources of faecal contamination. We conclude that this analytical method is fully applicable to the determination of sterol fingerprints in the dissolved phase (<0.7 μm) of natural aqueous samples.     

The impact of sterol structure on the interactions with sphingomyelin in mixed langmuir monolayers

In this work, the Langmuir monolayer technique was applied to study the interactions between sphingomyelin and various sterols differing in the structure of the side chain (cholesterol, beta-sitosterol, stigmasterol). The mean area per molecule and the excess free energy of mixing values were analyzed in the context of sterol-induced condensing effect and interactions between molecules in the mixed monolayers. Moreover, the compression modulus values were calculated and widely discussed from the point of view of the ordering effect of sterols. It was found that all of the sterols investigated form the most stable monolayers with sphingomyelin at 2:1 sphingomyelin:sterol proportion and the strongest interactions exist between molecules in cholesterol-containing films. Moreover, cholesterol provokes the strongest area condensation and reveals the highest ordering properties, while plant sterols were found to differ only slightly with regards to their ordering properties. Additionally, the ordering effect of the sterols on dipalmitoylphosphatidylcholine (DPPC) films was analyzed and compared to that on sphingomyelin films.     

Multimeric high-performance liquid chromatography of plant sterols using UV detection

A method for the multimeric high-performance liquid chromatography of plant sterols is proposed which permits the separation of compounds close in structure with similar chromatographic properties. The first stage includes the chemical modification of the sterols with the aid of a bis(p-nitrophenyl) phosphate group. The phosphotriesters formed as the result of the reaction are separated by normal-phase HPLC. The compounds isolated are treated with ammonia, as a result of which the sterol phosphotriesters are converted into the corresponding phosphodiesters. These phosphodiesters are then subjected to reversed-phase HPLC. The chromatographic separation of UV-absorbing sterol derivatives using several variants of HPLC substantially increases the resolving power of the method.State Scientific-Research Institute on the Standardization and Control of Drugs, USSR Ministry of Health, Moscow. L. V. Lomonosov Moscow State University. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 831–836, November–December, 1988.     

Validation of a quantitative procedure for the extraction of sterols from edible oils using radiolabelled compounds

The work described in this paper is integrated in an analytical programme organised by the Community Bureau of Reference with the aim of developing reference materials certified for sterol content. Preliminary inter-comparison of methods showed that the level of agreement of the results was insufficient for certification purposes. Errors could occur in the different steps before the final determination by gas-liquid chromatography. It was, therefore, decided to validate a quantitative procedure for the isolation of sterols. A well defined saponification-extraction method was tested using labelled sterols ([3H]cholesterol and [3H]cholesteryl oleate) and radiochemical measurements. The study has shown that total cholesterol recovery reached 100.5 +/- 1.4%, that cholesteryl ester was saponified quantitatively and that there were no appreciable amounts of degradation products. The procedure has been used as the basis for the certification of three reference materials and it has been shown that the saponification and extraction procedure leads to the quantitative recovery of sterols regardless of the nature of the fatty material tested.     

High Resolution Separation of Non-Polar Lipid Classes by HPLC-ELSD Using Alumina as Stationary Phase

This study describes the performance and capacity of alumina as stationary phase in an HPLC-ELSD (evaporative light-scattering detection) method optimized for the separation of the non-polar lipid classes hydrocarbons, wax esters, sterol esters, triacylglycerols, and sterols, including quantitative determination of these lipid classes in natural samples. By using gradient elution and constant equilibration times between injections, highly reproducible separations of triacontane, stearyl oleate, and cholesterol oleate were accomplished with a binary mobile phase system. Phase A contained 0.5% tetrahydrofuran in hexane and phase B 20% isopropanol and 20% tetrahydrofuran in hexane. The same system was also used to determine the non-polar lipid classes in a zooplankton sample, the major lipid class being wax esters, followed by triacylglycerols, sterol esters, sterols, and hydrocarbons. Substantial amounts of an unknown compound, possibly acylated glyceryl ethers, were also found. The equilibration time of alumina was relatively slow compared to a polyvinyl alcohol stationary phase used earlier by the authors and calibration curves for different lipid classes were more uniform and linear with alumina.     

Isolation and synthesis of 23-methyl-22-dehydrocholesterol -a marine sterol of biosynthetic significance

Anew sterol, 23-methyl-22-dehydrocholesterol, and several closely relatedsterols, have been isolated from cultures of the dinoflagellat symbiont (zooxanthella) from the marine zoanthid $\text{Zoanthus sociatus}$. The structural features of these sterols suggest that direct bioalkylation of a Δ²² double bond may be a new biosynthetic route to novel sterol side chains.     

Critical factors for detection of biphasic changes in membrane properties at specific sterol mole fractions for maximal superlattice formation

Here we use the excitation generalized polarization (GPex) of 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan) fluorescence in fluid cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine large unilamellar vesicles to explore the experimental conditions that would be required in order to detect a biphasic change in membrane properties at specific sterol mole fractions (Cr) (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol %) for maximal sterol superlattice formation. Laurdan's GPex changes with sterol content in an alternating manner, showing minima (termed as GPex dips) at approximately Cr. GPex dips are detectable if the vesicles are preincubated for a sufficient time period and protected from sterol oxidation. In most cases, vesicles with a higher lipid concentration take a longer time to show a GPex dip at Cr. The depth of the GPex dip increases with increasing incubation time and eventually reaches a plateau, at which the maximum area covered by superlattices is expected to be achieved. However, if the vesicles are not protected against sterol oxidation, the GPex dips are attenuated or obliterated. These effects can be attributed to the increased inter-bilayer lipid exchange and the increased vesicle-vesicle interactions present at high lipid (vesicle) concentrations as well as the decreased interactions between oxysterols and phospholipids. These possible explanations have been incorporated into a kinetic model that is able to calculate the effects of sterol oxidation and lipid concentration on the depth of the GPex dip. The depth of the GPex dip, the required incubation time for the dip formation, and the lipid concentration dependence of the GPex dip vary with Cr, suggesting different physical properties for different sterol superlattices. To detect a biphasic change in membrane properties at Cr, one should also use small sterol mole fraction increments over a wide range, keep all of the vesicles in the same sample set under the same thermal history, and consider lipid concentration, probe type, and Cr value. These results improve our mechanistic understanding of sterol superlattice formation and explain why some studies, especially those requiring high lipid concentrations, did not detect a biphasic change in membrane properties at Cr.     

A ROLE FOR STEROLS IN THE PORPHYRIN MEDIATED PHOTOSENSITIZATION OF YEAST

The yeast Saccharomyces cerevisiae was used as a model system to determine the role of sterols in the porphyrin mediated photosensitization of yeast. A sterol auxotroph, RD5-R, was grown on sterols with different levels of unsaturation and assayed for photosensitivity in the presence of either protoporphyrin IX or hematoporphyrin (both at 100 micrograms/ml). Cells grown on the completely saturated sterol (stanol), cholestanol, were substantially more resistant to the photosensizing effects of the porphyrin. We hypothesize that this resistance arises from the inability of the porphyrin to mediate the oxidation of the membrane sterol. Our results indicate that photodegradation of the native yeast sterol, ergosterol, can account for substantial losses of cell viability.     

A new LC/APCI-MS method for the determination of cholesterol oxidation products in food

Cholesterol oxidation products (COPs) can be formed in the body or in animal foods from cholesterol during food processing. A new method for the extraction and quantification of cholesterol, 7-ketocholesterol, cholestane-3beta-5alpha-6beta-triol, 25-hydroxycholesterol, 5,6alpha-epoxycholesterol, and 7beta-hydroxycholesterol by means of reversed-phase LC/atmospheric pressure chemical ionization mass spectrometry is presented. A baseline separation of all COPs was achieved, allowing a separate quantification also for isobaric compounds. The limits of detection were 15-30 ng/mL, quantification was performed from 100 ng/mL to 10 microg/mL with RSD < 2%. The method was applied successfully to the determination of cholesterol and COPs in processed foods such as pork, beef, chicken, and egg.     

Analysis of free and esterified sterols in vegetable oil methyl esters by capillary GC

The introduction of quality standards for vegetable oil methyl esters is gaining in importance due to their increased use as diesel fuel substitutes and as technical products. Free and esterified sterols, the main constituents of the unsaponifiable matter in vegetable oils, are recovered in vegetable oil methyl esters and may influence the technical properties of vegetable oil methyl ester products. A rapid gas chromatographic method for the qualitative and quantitative determination of free and esterified sterols in vegetable oil methyl esters has therefore been developed. The concentration of the free sterols as well as their qualitative and quantitative composition and the concentration of the sterol esters have been determined in rape seed oil methyl ester samples by GC–FID. Prior to analysis, the free sterols were silylated with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane; betulinol was used as an internal standard. Calibration was performed by analysis of standard solutions containing β-sitosterol, cholesteryl stearate, and betulinol. The reproducibility of the quantitative results has been evaluated by repeated injections of the same test solution and by repeated complete analysis of the same sample.     

An efficient diethyl ether-based soxhlet protocol to quantify faecal sterols from catchment waters

A study was conducted to evaluate the efficiency and reproducibility of a diethyl ether-based soxhlet extraction procedure for faecal sterols occurring from catchment waters. Water samples spiked with a mixture of faecal sterols were filtered and analytes were extracted using the diethyl ether-based soxhlet method and the Bligh and Dyer chloroform extraction process. For diethyl ether-based soxhlet extraction procedure, solvent extracts were saponified with 100 microL of 10% KOH in methanol (100 degrees C/120 min) and then acidified with 60 microL of 6M HCl. Lipid contents were extracted by ethanol (0.5 mL) from the saponification products. The lipid extracts were then reacted with 100 microL of bis(trimethyl)trifluoroacetamide (BSTFA) containing 1% trimethyl chlorosilane (100 degrees C/60 min) to form the trimethylsilyl (TMS) derivatives. The derivatised extracts were then analyzed by gas chromatography-mass spectrometry. For sterol concentrations ranging from 35 to 175 microg mL(-1), the soxhlet-based extraction process yielded the following recovery efficiencies for coprostanol (101%), epicoprostanol (97%), cholesterol (97%), dihydrocholesterol (97%) and 5alpha-cholestane (111%), whereas the Bligh and Dyer process yielded recoveries of 32, 41, 0, 36 and 51%, respectively. The results suggested that the diethyl ether-based soxhlet extraction method was more efficient and reproducible than the Bligh and Dyer chloroform extraction process for the analyses of trace levels of faecal sterols from water samples. Moreover, it was revealed that the diethyl ether-based soxhlet extraction method used less solvent and was logistically easier.     

Sterol composition of a cultured marine dinoflagellate,Heterocapsa circularisquama

The composition of sterols was determined in a cultured marine dinoflagellate Heterocapsa circularisquama. Ten kinds of the sterol in H. circularisquama were identified by capillary gas chromatography-mass spectrometry. The major sterol was a 4-methyl sterol, 4alpha,23,24-trimethyl-5alpha-cholest-22E-en-3beta-ol (dinosterol) which is the common sterol in many dinoflagellates.     

Classification of vegetable oils according to their botanical origin using sterol profiles established by direct infusion mass spectrometry

A simple and quick method to classify vegetable oils according to their botanical origin, based on direct infusion of sterol extracts into a mass spectrometer, was developed. Using mass spectrometry (MS) with either an electrospray ionization or an atmospheric pressure photoionization source, followed by linear discriminant analysis of the mass spectral data, oil samples corresponding to eight different botanical origins were perfectly classified with an excellent resolution among all the categories. An excellent correlation between the sterol profiles obtained by MS and by the official gas chromatography (with flame ionization detection) method was obtained. Thus, the proposed method is a promising alternative for sterol fingerprinting of vegetable oils, with the advantage that prior chromatographic separation is not required.     

Application of comprehensive two-dimensional gas chromatography to the quantification of overlapping faecal sterols

Standard solutions containing a mixture of seven sterols and 5alpha-cholestane as internal standard, and sample mixtures that comprised varying ratios of sterol and stanols from green lip mussel tissue and dried cow faeces were analysed by using comprehensive two-dimensional gas chromatography (GC x GC). Quantitative results were compared with single-column GC analysis. The latter samples included sterols of interest, but which cannot be readily obtained elsewhere. It may also mimic potential environmental samples where dairy production and aquaculture (oyster, mussel cultivation) share the same catchment; environmental sterol signatures may exhibit characteristics of both sample types comprising this mixture. Whereas single-column GC-flame ionisation detection was unable to reliably quantitate target sterols, the GC x GC experiment permitted small amounts of sterols and stanols to be detected and separated. Likewise GC-MS analysis was unable to detect some of the minor sterols which coeluted on a single column. The GC x GC mode allows complete separation of several important sterols and stanols, such as 24-ethylcoprostanol, campesterol and 24-methylenecholesterol, demonstrating the enhanced resolving power of the GC x GC system. Separation of 24-ethyl-epi-coprostanol from several algal-derived interfering components was achieved, leading to higher degree of confidence in the quantitative analysis of faecal sterols. The effects of a number of operating variables--column length, carrier flow-rate and elution temperature--on component resolution and presentation of data in the two-column analysis are described.     

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry.

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS-MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.