Determination of tetracyclines in pig and other meat samples using liquid chromatography coupled with diode array and tandem mass spectrometric detectors

Two high performance liquid chromatographic methods (HPLC–DAD and LC–MS/MS) were developed to analyze tetracycline (TC) residues in pig meat (pork) samples. The method involved a sample preparation using a solid–liquid extraction (SLE) by McIlvaine buffer, followed by a solid-phase extraction (SPE) clean-up using Strata-XL cartridges. The developed sample clean-up resulted in a selective chromatogram in the HPLC–DAD separation and a reduced matrix effect (ME) in LC–MS/MS analysis. Moreover, HPLC columns packed with core–shell particles were tested for separation, which further enhanced the sensitivity and the selectivity of determinations. The validation of the methods for pig samples was carried out according to European Union 2002/657/EC decision. In addition, validation was also performed for bovine, chicken, and turkey meat samples using HPLC–DAD method. The performance characteristics of determinations were evaluated with both spiked and incurred samples, and were systematically compared. LC–MS/MS technique was found to be more accurate for spiked samples; however, HPLC–DAD method resulted in more reliable concentrations for incurred samples.     

Determination of <Emphasis Type="Italic">Bacillus cereus</Emphasis> Emetic Toxin in Food Products by Means of LC–MS²

Cereulide is the heat-stable toxin produced by certain strains of Bacillus cereus. It is the main virulence factor of emetic B. cereus strains, which causes the emetic food poisoning syndrome, including rare fatal cases of food intoxications. Due to presumably low intoxication doses, a sensitive, specific, and robust technique is needed for its detection. In 2002, a LC–MS method was developed which allowed absolute quantification of cereulide using valinomycin as standard. This study describes the validation, according to the Commission Decision 2002/657/EC, of the LC–MS2 method, a tandem mass spectrometry technique, which guarantees lower detection limit and higher specificity. The LC–MS2 method, calibrated with valinomycin, was validated in rice and tested on various matrices (i.e., red beans, spices, and chili con carne) containing cereulide. The process combines a simple extraction step from the food matrix followed by LC–MS2 analysis and detection by ion trap mass spectrometer. The detection limit for cereulide in rice was 0.5 ng eq/g, which is 20 to 2,500 times lower than currently understood intoxicative doses between 10 and 1.280 ng/g previously reported for cereulide. The validated method was specific, sensitive, repeatable, and reproducible with recoveries ranging from 77% to 101%.     

Determination of mineral oil paraffins in foods by on-line HPLC–GC–FID: lowered detection limit; contamination of sunflower seeds and oils

A method is described to lower the detection limit for mineral oil saturated hydrocarbons (MOSH) in foods as compared to the on-line HPLC–LC–GC–FID method described previously: samples are preseparated (enriched) by conventional liquid chromatography on activated silica gel and activated aluminum oxide. The silica gel retains up to 1 g of fat or oil, the aluminum oxide up to 2 mg n-alkanes of at least 24 carbon atoms, i.e. plant paraffins which may severely hinder the analysis of the mineral paraffins. The efficacy of the method is shown for an apple and sunflower oil. Oils extracted from manually harvested seeds grown in fields or gardens contained between 0.14 and 0.77 mg/kg MOSH. In the oils from seeds sampled in an oil mill, this value was increased to 3.3–9.3 mg/kg, indicating a contamination during harvest, transport and/or storage. Concentrations in commercial refined sunflower oils ranged between 2.7 and 32 mg/kg, averaging 11.2 mg/kg. Since deodorization removes a substantial part of the MOSH, this suggests a further contamination in the oil mill. The contamination affected all samples at a similar level, indicating that it occurs systematically by the presently used technology.     

Validation of Two Variations of the QuEChERS Method for the Determination of Multiclass Pesticide Residues in Cereal-Based Infant Foods by LC–MS/MS

Over the past years to ensure food safety and particular for food that intend to be consumed by infants and young children, the European Union has adopted specific legislation concerning the control of pesticide residue levels in that kind of food. In this paper, a liquid chromatography tandem quadrupole mass spectrometry (LC–MS/MS) multiresidue method for the simultaneous analysis of 23 pesticides and metabolites chosen according to the Commission Directives 2006/141/EC, 2006/125/EC, and 96 multiclass pesticides and metabolites chosen according to their physicochemical properties is presented and validated. The extraction procedure is based on three modifications of the quick, easy, cheap, effective, rugged, and safe method according to the analyte. The analytical performance was demonstrated by the analysis of extracts from cereal-based infant foods, spiked at two concentration levels for each pesticide or metabolite. Good sensitivity and selectivity of the method were obtained with limits of quantification at 10 or 3 μg/kg, depending on the analyte. All pesticides and metabolites, except six cases, gave recoveries in the range of 60.4–125.4%, with relative standard deviations less than 29.7%, for both validation levels.     

Mycotoxins produced by Fusarium genus in maize: determination by screening and confirmatory methods based on liquid chromatography tandem mass spectrometry

A confirmatory method for fusariotoxin analysis in maize meal, based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), was developed, and compared with a previously published screening method, based on the same technique. By eluting selectively from a Carbograph-4 clean-up cartridge trichothecenes, fumonisins and macrocyclic lactones, and optimizing LC–MS/MS conditions for every chemical class, a sensitive and reliable determination was performed. Method quantification limits for confirmatory and screening methods were in the range 0.001–0.019 mg/kg and 0.003–0.125 mg/kg, respectively.     

Development and validation of a new LC–MS/MS method for the simultaneous determination of six major ergot alkaloids and their corresponding epimers. Application to some food and feed commodities

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers in food and feed samples. The method involves extraction under alkaline conditions and subsequent clean-up by applying a simple and rapid liquid–liquid partitioning procedure prior to LC–MS/MS analysis. Evaluation of the method revealed good linearity, accuracy and precision. The limits of quantification varied from 0.1 to 1 μg/kg depending on the analyte and matrix. The average extraction and clean-up recoveries in different matrices were between 45 (only for ergometrine in biscuit) and 90%. The uncertainty associated with the analytical method was not higher than 51% and 30%, at concentration levels of 2.5 and 150 μg/kg respectively. Analyte epimerization proved to be minimal during the analytical procedure. The method has been successfully applied to the determination of ergot alkaloids in some Belgian food and feed commodities. Ergot alkaloids were found in 104 out of 122 samples investigated. Ergosine was the most frequently occurring alkaloid, while the highest levels were observed for ergotamine, ergocristine or ergosine, depending on the product type. The total alkaloid content in positive samples varied from 1 to 1145 μg/kg.     

Determination of Botanical Insecticides Residues in Fish by Liquid Chromatography�CElectrospray Tandem Mass Spectrometry

A method for determining residues of four botanical insecticides oxymatrine, matrine, rotenone, and azadirachtin in fish by liquid chromatography–electrospray tandem mass spectrometry (LC–MS/MS) is described. The extraction was achieved using acetonitrile, with the addition of sodium chloride to induce a salting out effect before using n-hexane to defat. Chromatographic separation is achieved on Phenomenex Luna C18 column in the mobile phase composition of acetonitrile and 5 mmol/L ammonium acetate buffer consisting formic acid to provide protons for LC–MS/MS analysis. Accomplishing with the matrix matched calibration curves to compensate for the matrix effect, the quantitative data showed good linear response within the concentration ranges studied. Detection is carried out using positive-ion electrospray tandem mass spectrometry. Calibrations were linear over a working range of 0.5–50 μg/L for oxymatrine, matrine, rotenone, and 2–200 μg/L for azadirachtin. The average recoveries and the relative standard deviation ranged from 88.6–95.7% and 7.58–10.2%, respectively, in spiked fish samples at concentration levels ranging from 0.5 to 10 μg/kg for oxymatrine, matrine, and rotenone and from 2 to 50 μg/kg for azadirachtin. The limits of detection for oxymatrine, matrine, rotenone, and azadirachtin were 0.29, 0.37, 0.21, and 1.4 μg/kg, respectively. The method is accurate, specific, and sensitive for the analysis of the studied botanical insecticides residues in fish samples.     

Determination of epoxidized soy bean oil (ESBO) in oily foods by GC–FID or GC–MS analysis of the methyl diepoxy linoleate

A simplified method is presented for determining epoxidized soy bean oil (ESBO) in food or food simulants, such as olive oil. ESBO is transesterified with methoxide/methanol directly in the homogenated food, i.e. without prior extraction, and analyzed on a cyanopropyl or phenyl polysiloxane stationary phase without formation of dioxolanes. For most foods, flame ionization detection (FID) was appropriate, but for samples with interfering components, GC–MS was needed. Chemical ionization (CI) with ammonia is more sensitive and more selective than electron impact ionization (EI). In CI, positive and negative ion monitoring (PCI and NCI) are similar in sensitivity, but different in selectivity, i.e. the combination of the two is well suited for confirmation. Results are shown to be in agreement with on-line LC–GC–FID.     

Naturally-occurring folates in foods: Method development and analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

An accurate method for detecting and quantifying both synthetic (folic acid) and naturally-occurring folates in foods is described. A system capable of analysing the five most commonly occurring folates (pteroylglutamic acid, 5-methyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and 5-formyltetrahydrofolate) in 20 min using liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Quantification of folates was performed using ¹³C labelled internal standards. This paper outlines the development of a comparatively fast LC–MS/MS method, method validation using commercially available folate standards and establishment of the method’s suitability for quantification using selected reaction monitoring (SRM) mass spectrometry. The application of the system was verified by analysing several certified reference materials and comparing results with certified values as determined by microbiological assay. LC–MS/MS promises to be an ideal tool for the quantitative analysis of folates in food.     

Migration of ε-caprolactam from nylon cooking utensils: validation of a liquid chromatography-ultraviolet detection method

A large amount of modern cooking utensils are made of nylon. This is the generic name of polyamides that are polymers having characteristic amide linkages. Caprolactam is the monomer used in the synthesis of nylon 6. This work presents the validation of an LC-UV method for the determination of caprolactam in the aqueous food simulant 3% acetic acid (w/v) employing capryllactam as internal standard. LC–MS/MS was the confirmation technique. The obtained detection and quantification limits were satisfactory regarding caprolactam specific migration limit; these were 0.6 and 2 mg kg⁻¹, respectively. The working range was 2–30 mg kg⁻¹ and the correlation coefficients (r) were in all cases at least 0.999. The intra-day repeatability was between 2.5 and 4.8% depending on the validation level. The global mean recovery was 100.6% with a RSD (%) of 5.0%. Different nylon cooking utensils were analyzed for the migration of caprolactam in the food simulant acetic acid 3% p/v. Conditions used were repeated exposure to the simulant for 2 h at 100 °C, and the results obtained after the third exposure were in compliance regarding Directive 2002/72/EC. With the aim of identifying the polymer used in these utensils, IR spectra were also obtained for all samples.     

HPLC analysis and safety assessment of coumarin in foods

Coumarin is a component of natural flavourings including cassia, which is widely used in foods and pastries. The toxicity of coumarin has raised some concerns and food safety authorities have set a maximum limit of 2 mg/kg for foods and beverages in general, and a maximum level of 10 mg/l for alcoholic beverages. An efficient method for routine analysis of coumarin is liquid chromatography with diode array detection. The optimal sample preparation for foods containing cinnamon was investigated and found to be cold extraction of 15 g sample with 50 mL of methanol (80%, v/v) for 30 min using magnetic stirring.     

A novel method for the detection of Roundup Ready™ soya using liquid chromatography–electrospray ionisation mass spectrometry

Current methods for the detection and quantification of genetically modified (GM) ingredients predominantly make use of enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) based techniques. For PCR-based techniques, real-time detection systems represent the state of the art for quantitative measurement of DNA-based products. This study sought to assess the viability of using mass spectrometry (MS) for the detection and potential quantification of GM-derived material by developing an assay for the detection of Roundup Ready™ soya (RRS). This was based upon the indirect detection of event-specific PCR products using a single base pair extension followed by online clean-up and detection using liquid chromatography–electrospray–mass spectrometry (LC–ESI–MS). We were able to demonstrate that LC–ESI–MS detection provides conclusive identification of the primer extension products enabling the effective detection and differentiation of GM- and non-GM-derived material. This was carried out using both reference materials and real food matrices.     

HPLC/MS测定食品中的苯甲酸、山梨酸、甜蜜素、糖精钠

建立了测定食品中苯甲酸、山梨酸、甜蜜素、糖精钠的HPLC/MS分析方法。样品中的添加剂用水提取,经HPLC分离后,在ESI负离子模式下检测。4种添加剂测定的线性范围均为10~5 000 ng/mL,检出限均优于0.1 mg/kg;各组分加标回收率分别为:苯甲酸92.1%~96.7%,山梨酸92.3%~94.4%,糖精钠94.0%~97.3%,甜蜜素95.1%~96.9%。     

Detection and quantification of pentosidine in foods

 Pentosidine, a crosslink amino acid in which one arginine and one lysine residue are linked together by a pentose, has been detected in foods for the first time using ion-exchange chromatography with direct fluorescence detection and subsequent ninhydrin derivatization. The method allows the simultaneous quantification of pentosidine along with all other amino acids of acid hydrolyzates at levels lower than 50 μg kg protein. Levels of pentosidine in all food samples investigated were very low (milk products between not detectable and 2–5 mg/kg protein; roasted coffee and some bakery products up to 35 mg/kg protein), indicating that pentosidine does not play a major part in the polymerization of food proteins. Received: 26 March 1996     

Detection and quantification of pentosidine in foods

 Pentosidine, a crosslink amino acid in which one arginine and one lysine residue are linked together by a pentose, has been detected in foods for the first time using ion-exchange chromatography with direct fluorescence detection and subsequent ninhydrin derivatization. The method allows the simultaneous quantification of pentosidine along with all other amino acids of acid hydrolyzates at levels lower than 50 μg kg protein. Levels of pentosidine in all food samples investigated were very low (milk products between not detectable and 2–5 mg/kg protein; roasted coffee and some bakery products up to 35 mg/kg protein), indicating that pentosidine does not play a major part in the polymerization of food proteins. Received: 26 March 1996     

The analysis of phenolic constituents in glabrous canaryseed groats

Nineteen glabrous canaryseed samples, comprising brown- and yellow-coloured seeds, were investigated to determine the nature of phenolic constituents present. Total phenolic content (TPC) was determined, using the Folin–Ciocalteau assay. Flavonoid and phenolic acid compositions were determined, using high performance liquid chromatographic and mass spectrometric (LC–MS/MS) techniques. TPC ranged from 174 to 209 mg/100 g for canaryseed wholemeal samples. The canaryseed bran contained twice as much TPC as the wholemeal. The brown- and yellow-coloured whole canaryseeds exhibited the same flavonoid profiles. LC–MS/MS analysis showed that the canaryseed acetone extract was rich in flavonoid glycosides, with the bran being mainly composed of O-pentosyl isovitexin and the flour having a compound at m/z 468. No proanthocyanidins were detected in the 19 samples. Ferulic acid was the dominant phenolic acid, followed by caffeic and p-coumaric acids. The wholemeal obtained from the brown-coloured group had significantly higher contents of ferulic (>196 mg/kg) and caffeic (>96 mg/kg) acids in comparison to the yellow-coloured canaryseed group. The latter had ferulic and caffeic acids at levels less than 165 and 78 mg/kg, respectively, with one exception which had relatively higher levels (190 and 94 mg/kg). Whilst canaryseed flour contained significantly very low levels of ferulic acid (22–34 mg/kg), the bran was enriched in ferulic (593–766 mg/kg), caffeic (304–452 mg/kg) and p-coumaric (119–142 mg/kg) acids.     

Preparation of quercetin glucuronides and characterization by HPLC–DAD–ESI/MS

Quercetin is the main flavonol in the human diet, and the most commonly used in studies of biological activity. The major circulating forms of quercetin found in the human plasma after consumption of food containing distinct quercetin glycosides are glucuronides and sulfates. In this work quercetin glucuronides have been obtained from green beans (quercetin 3-glucuronide) and by enzymic synthesis (quercetin 4′-glucuronide) using a modification of the method described by Plumb et al. (Methods in polyphenol analysis, The Royal Society of Chemistry, Cambridge, pp 187, 2003) so as to improve the original low yields of that methodology. The method finally optimised got yields of 19% in the preparation of quercetin 4′-glucuronide, which allows its further isolation for their use in biological assays. In addition, quercetin 3′-glucuronide, 3-glucuronide and a diglucuronide were synthesised with lower yields. The compounds prepared have been employed to perform assays in order to obtain data for their identification by HPLC coupled to photodiode array detection and tandem mass spectrometry. It was observed that the analysis by HPLC–ESI/MS/MS could allow the identification of different quercetin glucuronides based on the presence of some minor key MS² fragments.     

Solvent assisted flavour evaporation – a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices

 A compact and versatile distillation unit was developed for the fast and careful isolation of volatiles from complex food matrices. In connection with a high vacuum pump (5×10^–3 Pa), the new technique, designated solvent assisted flavour evaporation (SAFE), allows the isolation of volatiles from either solvent extracts, aqueous foods, such as milk or beer, aqueous food suspensions, such as fruit pulps, or even matrices with a high oil content. Application of SAFE to model solutions of selected aroma compounds resulted in higher yields from both solvent extracts or fatty matrices (50% fat) compared to previously used techniques, such as high vacuum transfer. Direct distillation of aqueous fruit pulps in combination with a stable isotope dilution analysis enabled the fast quantification (60 min including MS analysis) of compounds such as the very polar and unstable 4-hydroxy-2,5-dimethyl-3(2H)-furanone in strawberries (3.2 mg/kg) and tomatoes (340 μg/kg). Furthermore, the direct distillation of aqueous foods, such as beer or orange juice, gave flavourful aqueous distillates free from non-volatile matrix compounds. Received: 4 December 1998 / Revised version: 22 January 1999     

Baseline levels of melamine in food items sold in Canada. II. Egg,soy, vegetable,fish and shrimp products

A variety of egg-containing, soy-based, fish, shrimp and vegetable products sold in Canada were analysed for melamine (MEL) using a sensitive solid-phase extraction LC–MS/MS analytical method. MEL was detected above the method quantification limit of 0.004 mg/kg in 98 of the 378 samples analysed. Concentrations in the various food product groups ranged 0.00507–0.247 mg/kg (egg-containing items), 0.00408–0.0479 mg/kg (soy-based meat substitutes), 0.00409–1.10 mg/kg (fish and shrimp products), and 0.00464–0.688 mg/kg (vegetable products). MEL was detected less frequently in egg- and soy-containing products. The presence of MEL in most of the Canadian Total Diet Study shrimp composites collected after 2001 suggested the residues in shrimp were caused by a relatively recent exposure to MEL. All concentrations of MEL reported were lower than the 2.5 mg/kg interim standard established for MEL in items containing milk and milk-derived ingredients and the respective maximum residue limits for cyromazine and its metabolite, melamine, in vegetables set by the Canadian Government (2009; http://www.hc-sc.gc.ca/fn-an/securit/chem-chim/melamine/qa-melamine-qr-eng.php#8). The consumption of foods containing these low levels of MEL does not constitute a health risk for consumers.     

Comparison of headspace-SPME-GC–MS and LC–MS for the detection and quantification of coumarin,vanillin, and ethyl vanillin in vanilla extract products

A headspace-solid phase micro-extraction (HS-SPME) GC–MS method has been developed for the determination of coumarin, vanillin and ethyl vanillin in vanilla products. Limits of detection ranged from 1.33 to 13.2 ng mL⁻¹. Accuracy and precision data for the method were measured and compared to those obtained using LC-ESI-MS. A survey of 24 commercially available vanilla products was completed using both techniques. No coumarin was detected in any of the samples. Examination of the GC–MS chromatograms revealed the presence of 18 other flavor related compounds in the samples. The method validation and sample analysis data using HS-SPME-GC–MS were comparable to those obtained using the LC–MS method. Because the two methods are conceptually different from one another, both methods would not be subject to the same interferences. This would allow them to be used as confirmatory methods for each other.