Cholesterol oxidation in meat-based baby foods

Cholesterol oxidation in commercial meat-based homogenized and freeze-dried baby foods was examined. The 7 major products of this reaction were determined by gas chromatography coupled with mass spectrometry (GC-MS). As far as single cholesterol oxidation products (COP) are concerned, 7-ketocholesterol was the major product of direct cholesterol oxidation in the 2 groups of analyzed samples, and this study confirmed that it is a useful marker of the whole cholesterol oxidation process. Nevertheless, the amounts of cholesterol-5beta,6beta-epoxide were often similar to and sometimes higher than the amounts of 7-ketocholesterol, thus showing a strong development of both direct and indirect cholesterol oxidation pathways. Total COP content was significantly higher in freeze-dried than in homogenized products. Moreover, in freeze-dried samples, the COP content per serving was quite variable and, in 2 samples, it was close to or even higher than 500 microg. The greater development of cholesterol oxidation in the freeze-dried samples was confirmed by their highest total COP/cholesterol percent ratios. A constant correlation between the fatty acid composition and the development of cholesterol oxidation was not found, although a positive correlation between unsaturated fatty acid content and total COP content occasionally exists in samples of the same brand.     

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.     

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

Summary 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.     

液相色谱-串联质谱法测定肉及肉制品中利谷隆及其代谢产物3,4-二氯苯胺残留

建立了液相色谱-串联质谱分析多种肉及肉制品中利谷隆及其代谢产物3,4-二氯苯胺残留的方法。样品用丙酮-乙腈(5:95, v/v)提取,冷冻去脂,经弗罗里硅土固相萃取柱净化后进行液相色谱-串联质谱检测,采用内标法定量。利谷隆及3,4-二氯苯胺在1～500 μg/L范围内呈良好的线性关系,相关系数(r)均大于0.998,方法的定量限(信噪比(S/N)＞10)为10 μg/kg,检出限(S/N＞3)为5 μg/kg。在各种肉及肉制品基质中添加低、中、高3种不同水平的利谷隆及3,4-二氯苯胺,其回收率范围分别为88.3%～101.2%和91.6%～101.6%,相对标准偏差(RSD)分别为4.8%～13.7%和4.7%～11.8%。结果表明所建立的方法可以满足肉及肉制品中利谷隆和3,4-二氯苯胺残留量的检测需要。     

Determination of 7-Ketocholesterol and 7-Hydroxycholesterol in Meat Samples by TLC with Densitometric Detection

JPC – Journal of Planar Chromatography – Modern TLC - Concentrations of two oxycholesterols: 7-ketocholesterol and 7- hydroxycholesterol in meat samples (raw and cooked) were determined...     

Assay of cholesterol 7 alpha-hydroxylase utilizing a silica cartridge column and 5 alpha-cholestane-3 beta, 7 beta-diol as an internal standard

A simplified and accurate assay of cholesterol 7 alpha-hydroxylase activity using 5 alpha-cholestane-3 beta,7 beta-diol as an internal standard is described. Endogenous microsomal cholesterol was used as the substrate. Following incubation and addition of the internal standard, lipids extracted from the incubation mixture were applied to Bond-Elut silica cartridge columns. 7 alpha-Hydroxycholesterol, 7 beta-hydroxycholesterol, 7-ketocholesterol and the internal standard were quantitatively recovered by eluting the column with 6 ml of benzene-ethyl acetate (2:3, v/v) after removal of cholesterol with 6 ml of benzene-ethyl acetate (9:1, v/v). After trimethylsilylation, the mass of 7 alpha-hydroxycholesterol was determined by capillary gas chromatography with selected-ion monitoring. The method permits a faster, easier and more sensitive determination of the activity of cholesterol 7 alpha-hydroxylase in small samples.     

Comparison of cholesterol oxidation product preparation methods for subsequent gas chromatographic analysis

An evaluation was made of the stability of cholesterol hydroperoxides (CHPs) under the analytical conditions and preparation methods commonly used for determination and quantification of cholesterol oxidation products (COPs). CHPs were prepared by photoxidation and separated by silica thin-layer chromatography. CHPs were individually collected by normal-phase liquid chromatography and then subjected either to reduction or to cold saponification. The corresponding hydroxyl derivatives were generated by reduction, whereas cold saponification gave rise predominantly to 7-ketocholesterol. In another test, silylated and non-silylated CHPs were separately injected into a gas chromatograph at 310 degrees C, collected, and re-injected into a gas chromatography-mass spectrometry system. The silylated CHPs were more stable than the non-silylated ones, giving 7-ketocholesterol, 7alpha- and 7beta-hydroxycholesterol as main degradation products. Two unknown degradation peaks were detected in both silylated and nonsilylated CHPs, having 384 as main m/z fragment. The study of their mass spectra led to the conclusion that peaks A and B correspond to 6alpha- and 6beta-hydroxycholesterol, respectively.     

气相色谱-质谱法测定酱猪蹄和酱猪肘中5种胆固醇氧化物

建立了气相色谱-质谱（GC-MS）测定酱猪蹄和酱猪肘中7 β-羟基胆固醇、5 α，6 α-环氧化胆固醇、胆甾烷-3 β，5 α，6 β-三醇、25-羟基胆固醇和7-酮基胆固醇5种胆固醇氧化物（COPs）含量的方法。样品经甲醇-氯仿（1：2，v/v）混合溶液提取，硅胶固相萃取小柱净化后，加入N，O-双（三甲基硅基）乙酰胺-三甲基氯硅烷-三甲基硅基咪唑（3：2：3，v/v/v）（Sylon BTZ）进行衍生化处理，设置合理的柱温升温程序，采用选择离子扫描（SIM）模式进行检测。在优化条件下，5种COPs在22 min内实现分离，且分离度良好。5种COPs的线性范围满足测定要求，3个加标水平下的平均回收率为61.16%~96.96%，相对标准偏差≤ 7.80%（n=3），检出限（以信噪比为3计）和定量限（以信噪比为10计）分别为0.02~47.07 ng/g和0.06~156.90 ng/g。该法快速简便，线性范围广，灵敏度高，可作为测定实际样品中COPs的有效方法。     

Development of analytical procedures to study changes in the composition of meat phospholipids caused by induced oxidation

Lipid peroxidation affects quality of meat products. The aim of this study was to develop a model system and analytical procedures for evaluating the oxidation level of meat samples, by studying the changes in meat phospholipids (PL) composition and the compounds generated by induced oxidation. Different techniques (liquid-, dry column-, accelerated solvent extraction) were investigated to identify a suitable lipid extraction system for extracting PL from bovine meat and to induce lipid oxidation by using tert-butyl hydroperoxide, 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) or Fe(2+) and Cu(2+) salts. Accelerated solvent extraction (ASE) gave results not significantly different from the other extraction methods, but offered the advantage of being a rapid and solvent-saving procedure. The method using a silica column proved to be valid in eluting and separating the components of the phospholipidic fraction and the PL standard mixture. The analytical techniques used to analyse oxidation products of PL included GC-FID, HPLC with corona charged aerosol detector (CAD), MDA determination and the spectrophotometric measurement of peroxide levels (PxL). By means of CAD, PL were quantified and their concentration in the lipid extract was 0.98%+/-0.17 (w/w+/-SD, n=10). The oxidation method induced by ABAP proved to be fast and did not produce any artifacts. Three oxidation times were monitored (0, 90 and 180 min). The oxidation levels after 180 min correlated with a significant increase in the peroxide levels PxL (+71%), MDA (+29%) and aldehydes (+75%), whereas a decrease or even total disappearance of some unsaturated fatty acids was observed. The results obtained demonstrate that the model used in this work is useful for studying oxidation of meat phospholipids. Also, the use of the innovative detector CAD proved to be a good complementary technique in the investigation of lipids.     

Derivatization-independent cholesterol analysis in crude lipid extracts by liquid chromatography/mass spectrometry: applications to a rabbit model for atherosclerosis

Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2Δ and tgl1Δ cells, which are consistent with the function of the respective enzymes.     

MS–MS Fragmentation Patterns of Cholesterol Oxidation Products

Summary. This study was aimed at analyzing the daughter ion spectra of 7 toxicologically relevant cholesterol oxidation products (COPs) i.e. cholestanetriol (cholestane-3β,5α,6β-triol), 7-ketocholesterol (cholest-5-en-3β-ol-7-one), 7α-hydroxycholesterol (cholest-5-en-3β,7α-diol), 7β-hydroxycholesterol (cholest-5-en-3β,7β-diol), 25-hydroxycholesterol (cholest-5-en-3β,25-diol), α-epoxycholesterol (cholestane-5α,6α-epoxy-3β-ol) and β-epoxycholesterol (cholestane-5β,6β-epoxy-3β-ol). In addition, 19-hydroxycholesterol (cholest-5-en-3β,19-diol) was analyzed as this serves as internal standard in COPs determination by HPLC-MS. Mass spectrometry was performed using a triple quadrupol mass spectrometer that was equipped with an APCI ion source. Our results indicate a common fragmentation pattern for COPs. The main breaking sites identified were in the sterol ring system between the carbon atoms with the position numbers 11–12, 12–13, and 8–14. Typical daughter ions of m/z = 81, 95, and 195 were used for multiple reaction monitoring analysis.     

High performance liquid chromatographic separation of cholesterol oxidation products

Summary A fast, sensitive, high performance liquid chromatographic method for the simultaneous determination of cholesterol hydroperoxides and other major oxysterols, using two different detection systems (ultraviolet at 210 nm and light scattering), is here described. The hydroperoxy derivatives were obtained by cholesterol photoxidation, isolated by thin layer chromatography and joined with a standard mixture of 10 oxysterols (epoxy, hydroxy and keto derivatives). Aliquots were directly injected onto a 5-μm particle size, 25×0.46 cm i.d. Spherisorb S5 CN normal phase column, usingn-hexane/anhydrous ethanol (97:3, v/v) as mobile phase and a flow rate of 0.8 mL min⁻¹. This method allowed, in a single isocratic analysis, the separation and quantification of the primary and secondary cholesterol oxidation products in 30 minutes. The light scattering detector was particularly useful for the determination of nonderivatized 5,6-epoxides and cholestane-3β,5α,6β-triol. The sensitivity of both detectors was very similar for most of the oxysterols, except for the 5,6-epoxides and the 7-ketocholesterol. The method suitability for the determination of cholesterol oxidation products in food matrices was successfully tested on a saponified lipid extract from egg yolk powder.     

Comparison of total folate concentrations in foods determined by microbiological assay at several experienced U.S. commercial laboratories

Analysis of total folate concentration measured by microbiological assay in a variety of foods submitted in a routine manner to experienced laboratories that regularly perform folate analysis on fee-for-service basis was evaluated. Homogenates of fresh strawberries, frozen spinach, orange juice, frozen meat and vegetable pizza, dry macaroni, and dried pinto beans were prepared and stored under conditions previously determined to maintain stability of folate content. An aliquot of each composite and of 3 certified reference materials were sent on each of 4 occasions to 4 laboratories. Results for macaroni and pizza, the only folic acid-fortified foods, had considerably lower between-laboratory variation (CV(B)) with CV(B) of 9-11% versus >45% for other foods. Mean total folate ranged from 14 to 279 microg/100 g for a mixed vegetable reference material, from 5 to 70 microg/100 g for strawberries, and from 28 to 81 microg/100 g for wholemeal flour. Only 1 laboratory reported using a tri-enzyme extraction, and all laboratories used folic acid fortified foods as internal control materials. Users of commercial total folate analysis should understand the uncertainty in values determined by microbiological assay, particularly for foods containing primarily naturally occurring folate, which may not be apparent when replicate samples are not submitted for analysis.     

The Assessment of Changes in the Fatty Acid Profile and Dietary Indicators Depending on the Storage Conditions of Goose Meat

The deterioration of food quality due to lipid oxidation is a serious problem in the food sector. Oxidation reactions adversely affect the physicochemical properties of food, worsening its quality. Lipid oxidation products are formed during the production, processing, and storage of food products. In the human diet, the sources of lipid oxidation products are all fat-containing products, including goose meat with a high content of polyunsaturated fatty acids. This study aims at comparing the fatty acid profile of goose breast muscle lipids depending on the storage conditions: type of atmosphere, temperature, and storage time. Three-way variance analysis was used to evaluate changes in the fatty acids profile occurring in goose meat. The health aspect of fatty acid oxidation of goose meat is also discussed. In general, the fatty acid composition changed significantly during storage in the meat packed in the high-oxygen modified atmosphere at different temperatures (1 °C and 4 °C). Higher temperature led to a higher degree of lipid oxidation and nutrient loss. During the storage of samples in vacuum, no changes in the fatty acid content and dietary indices were found, regardless of the storage temperature, which indicates that the anaerobic atmosphere ensured the oxidative stability of goose meat during 11 days of refrigerated storage.     

Evaluation of different sample treatments for determining pesticide residues in fat vegetable matrices like avocado by low-pressure gas chromatography-tandem mass spectrometry

A multi-residue method has been developed for determining 65 pesticide residues in greasy vegetable matrices such as avocado. Conventional organic solvent extraction assisted by a high-speed homogenizer was compared to pressurized liquid extraction (PLE) as extraction techniques. Following this, the lipophilic extract was purified using gel permeation chromatography (GPC). Alternative clean-up methods were also evaluated, as solid-phase extraction cartridges individually used and downstream coupled, but less effective lipophilic separation was archived. The pesticide residue determination was carried out using low-pressure gas chromatography coupled to tandem mass spectrometry (LP-GC-MS-MS), showing the applicability of this type of GC columns for the analysis of fat vegetable matrices. The proposed methodology was validated in avocado matrix. The recoveries were in the range 70-110%, with RSD values lower than 19%, at 12 and 50 microg/kg spiking levels. The limits of quantitation (LOQs) were in the range 0.04-8.33 microg/kg and the limits of detection (LODs) were between 0.01 and 2.50 microg/kg. All of them were lower than the maximum residue levels (MRLs) set by the European Union (EU) in avocado. The proposed method was evaluated analyzing pesticide residues in real avocado samples.     

Coupled-column liquid chromatography method with photochemically induced derivatization for the direct determination of benzoylureas in vegetables

The coupled-column (LC-LC) system, consisting of a first column packed with internal surface reversed phase (ISRP) (50 x 4.6mm ID) and a Chrompack C18 (100 x 4.6 mm ID) as second column, allowed the simultaneous determination of five benzoylurea insecticides in dichloromethane (CH2Cl2) extracts of vegetable samples without any clean-up step. This system was combined with a photochemically induced fluorescence (PIF) post-column derivatization in order to provide strongly fluorescent photoproducts from the non-fluorescent benzoylureas. Limits of detection ranged from 0.21 to 0.98 microg L(-1) of pesticide (equivalent to 0.14-0.65 microg kg(-1) in vegetable samples) and limits of determination ranged from 4.0 to 10.0 microg L(-1) (equivalent to 2.7-6.7 microg kg(-1)). Linearity of the method was established between 2 and 1800 microg L(-1), depending upon the compound. Validation of the total method was performed by randomly analyzing recoveries of four vegetable samples (aubergine, cucumber, green bean, and tomato) spiked at two levels of concentration (10.0 and 33.3 microg kg(-1)). The combination of the LC-LC system with PIF detection provides a sensitive, selective, and rapid method for the determination of pesticides in vegetable samples at levels lower than the maximum residue levels (MRLs) established for these compounds by Spanish legislation.     

紫外光照射下胆固醇氧化产物分析

考察了紫外光照射下胆固醇的氧化情况.实验证明,经过0～5h的紫外光照射,用毛细管气相色谱法检测到有7 酮基胆固醇、5α,6α 环氧胆固醇和胆甾烷三醇生成,其含量随照射时间增加而增加.     

气相色谱-串联质谱法测定肉制品中10种挥发性N-亚硝胺类化合物

建立了气相色谱-串联质谱（GC-MS/MS）检测肉制品中10种挥发性N-亚硝胺类化合物残留量的方法。肉制品样品经同时蒸馏萃取法（SDE）萃取，采用冷冻去脂净化法，在多反应监测模式下分析，外标法定量。结果表明，采用优化后的条件，10种挥发性N-亚硝胺类化合物在1.00~1000 μg/L范围内线性关系良好，相关系数均在0.99以上。方法的检出限（LOD，S/N=3）和定量限（LOQ，S/N=10）分别为0.01~0.02 μg/kg和0.04~0.07 μg/kg。选取3种不同类型的肉制品（火腿肠、中式香肠和腌渍腊肉），在空白样品添加水平为LOQ水平、1.0、2.0 μg/kg时，10种挥发性N-亚硝胺类化合物的平均回收率为74.8%~94.3%，相对标准偏差小于8.3%。市售3类肉制品中6种挥发性N-亚硝胺类化合物（N-亚硝基二甲胺、N-亚硝基二乙胺、N-亚硝基吡咯烷、N-亚硝基哌啶、N-亚硝基二丁胺、N-亚硝基二丙胺）均有不同程度检出，且腌渍腊肉中每种挥发性N-亚硝胺类化合物的检出值最高。该方法操作简单，萃取充分，灵敏度高，试剂用量少，可满足实验室大量样品的日常检测需求。     

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 (7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, 5,6alpha-, 5,6beta-epoxycholesterol and cholestan-3beta,5alpha,6beta-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 7alpha-hydroxycholesterol and 5,6beta-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 C18 column was used with acetonitrile-methanol (60:40) as mobile phase. For the first time we report the use of such a C18 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.     

Determination of the antibiotic chloramphenicol in meat and seafood products by liquid chromatography-electrospray ionization tandem mass spectrometry

A confirmatory method based on isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry is described for the determination of the antibiotic chloramphenicol (CAP) in foods. The method is quantitative and entails liquid-liquid extraction followed by a clean-up step on a silica gel solid-phase extraction cartridge. Mass spectral acquisition is done in the negative ion mode applying multiple reaction monitoring of two diagnostic transition reactions for CAP (m/z 321 --> 257 and m/z 321--> 152). In addition, the presence of two chlorine atoms in the CAP molecule provides further analyte certainty by assessing the 37Cl/35Cl ratio using the transition reactions m/z 323 --> 257 and m/z 323 --> 152. Validation of the method in chicken meat is conducted according to the latest European Union criteria for the analysis of veterinary drug residues at levels of 0.05, 0.10, and 0.20 microg/kg, employing [2H5]-chloramphenicol as internal standard. The decision limit and the detection capability were calculated at 0.01 microg/kg and 0.02 microg/kg, respectively. At the lowest fortification level (i.e. 0.05 microg/kg), precision values below 14 and 17% were achieved under repeatability and within-laboratory reproducibility conditions, respectively. The accuracy of the method was within 20, 15, and 5% of the target values at the 0.05, 0.10, and 0.20 microg/kg fortification levels, respectively. The applicability of this procedure was demonstrated by the analysis of other meat (turkey, pork, beef) and seafood (fish, shrimps) products. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.