Preparation and characterisation of fried chicken as a laboratory reference material for the analysis of heterocyclic amines.

The preparation of a laboratory reference material (LRM) for the determination of naturally occurring heterocyclic amines (HAs) in processed foods is presented in this work. A LRM was prepared from raw chicken breast meat, which was fried under controlled cooking temperature and time. The cooked meat was ground, lyophilised, sieved, homogenised, bottled, and labelled. The HAs DMIP, PhIP, MeIQx, 4,8-DiMeIQx, Norharman and Harman were analysed in the LRM. Homogeneity and stability studies of the bulk LRM were carried out and no statistical differences were observed in the content of the studied HAs in between-bottle and within-bottle comparisons at different storage temperatures (-18, +4, +25 and +40 degrees C) and times (1, 3, 6 and 9 months) by means of HAs determination and analysis of the results. Consequently, the material can be considered homogeneous and stable and can be used in intercomparison exercises for the determination of HAs as well as for quality control purposes in the routine analysis of HAs in foodstuffs. This is the first LRM for the analysis of HAs where these analytes were naturally formed in the material.

Liquid chromatography coupled to tandem mass spectrometry for the analysis of acrylamide in typical Spanish products.

This paper describes the use of liquid chromatography coupled to tandem mass spectrometry for the determination of acrylamide in several typical foods produced and consumed in Spain. Christmas sweets, olives, traditionally made potato crisps, pastry products, sweet fritters ("churros") and one of Spain's most famous dishes, Spanish omelette, were selected. Using the mass spectra information provided by an ion trap analyzer in combination with the accurate mass measurements from time-of-flight (TOF) spectrometry a co-extractive interference present in some potato products was identified as valine. A porous graphitic carbon column, which enabled the co-extractive and acrylamide to be separated, and ion trap or triple quadrupole analyzers, depending on the acrylamide concentration, were used to determine this genotoxic compound in foodstuffs. The highest values were found in potato products, sweet fritters, Christmas sweets and pastry products, with values ranging between 70 and 2000 microg/g. Spanish omelette presented relatively low levels, similar to those obtained for dried fruits.

New method for the analysis of heterocyclic amines in meat extracts using pressurised liquid extraction and liquid chromatography-tandem mass spectrometry.

Heterocyclic amines (HAs) were analysed in meat extract samples using a new method based on pressurised liquid extraction (PLE) and liquid chromatography-tandem mass spectrometry. This method combines the use of a pressurised fluid with a triple quadrupole MS/MS system, resulting in benefits from both systems: high extraction efficiency and sensitivity. The effects of solvent type and PLE operational parameters, such as temperature and extraction time, were studied to obtain maximum recovery of the analytes with minimum contamination. HA extraction was best achieved using dichloromethane/acetone (50/50, v/v) at 80 degrees C for 10 min. Recoveries ranged from 45% to 79% with good quality parameters: limit of detection values between 0.02 and 1 ng g(-1), linearity (r(2)>0.997), and run-to-run and day-to-day precisions with relative standard deviations lower than 13% achieved at both low (0.20 microg g(-1)) and medium (1.0 microg g(-1)) concentrations. This method reduces sample manipulation and total extraction time by nearly four-fold compared to conventional solid phase extraction. The optimised method was validated using laboratory reference material based on a meat extract, and was successfully applied to HA analysis in several cooked beef samples.

Liquid chromatography-tandem mass spectrometry analysis of 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine in cooked meats.

Several cooked meats such as beef (fried, coated-fried), pork (fried, coated-fried), and chicken (fried, griddled, coated-fried, roasted) were analyzed for the heterocyclic amine 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5- b]pyridine (4'-OH-PhIP) not commonly determined in food and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). The highest content of 4'-OH-PhIP was found in fried and griddled chicken breast, the concentration being 43.7 and 13.4 ng/g, respectively, whereas the corresponding PhIP concentrations were 19.2 and 5.8 ng/g. The estimated concentration of both pyridines in fried pork loin, in fried pork sausages, and in coated-fried chicken was below 2.5 ng/g. In the rest of the samples, 4'-OH-PhIP was not detected. The analyses were performed by solid-phase extraction and LC-MS/MS. The fragmentation of 4'-OH-PhIP in an ion trap mass analyzer was studied in order to provide information for the identification of 4'-OH-PhIP. Additionally, the effect of red wine marinades on the formation of 4'-OH-PhIP in fried chicken was examined, finding a notable reduction (69%) in the amine's occurrence.

Field amplified sample injection-capillary electrophoresis-tandem mass spectrometry for the analysis of acrylamide in foodstuffs.

This paper shows the applicability of capillary electrophoresis (CE) coupled to mass spectrometry (MS) for the analysis of acrylamide (AA) in foodstuffs. In order to obtain an ionisable compound amenable to be analysed by CE, acrylamide was derivatised with 2-mercaptobenzoic acid. Spectra in positive and negative modes were studied in order to select the best ionisation mode and multistep tandem mass spectrometry was used to obtain structural information. Maximum signal was observed when negative mode was used and MS/MS and MS3 were selected for quantitation and confirmation, respectively. For the separation, a fused-silica capillary of 80 cm and 50 microm I.D. and 35 mM ammonium formate/ammonia solution at pH 10 as running electrolyte were used. The applicability of field amplified sample injection (FASI) in reversed polarity was evaluated in order to decrease detection limits. The developed FASI-CE-MS/MS method provided a detection limit of 8 ng g(-1) and good linearity (r=0.999) and precision (day-to-day lower than 15%). The method has been applied to the analysis of different representative food products and the results were compared with those obtained by LC-MS/MS.

Heterocyclic amines in griddled beef steak analysed using a single extract clean-up procedure.

Heterocyclic amines (HAs), which are potent mutagenic and carcinogenic substances, are formed in muscle meats during their cooking under ordinary conditions. In this work, we measured the concentration of 15 HAs in different samples of griddled beef steak, which is one of the most consumed meat items is Spain. Three samples were obtained from different restaurants, and the other sample was cooked under controlled conditions to a well-done degree of doneness. A low-time consuming solid-phase extraction procedure was used to purify the samples, and liquid chromatography-tandem mass spectrometry with an ion trap mass analyzer was used as determination technique. A second well-established purification procedure was used to demonstrate the applicability of the method to the analysis of these kind of samples. 8-MeIQx, 4,8-DiMeIQx, PhIP and the comutagens Harman and Norharman were found in all the samples, at levels ranging from 0.28 to 21.2ngg(-1). AalphaC was found in three samples (0.18-1.41ngg(-1)), whereas Trp-P-1 was detected in two samples (0.35ngg(-1)). MeAalphaC was found in three samples but could only be quantified in one (0.15ngg(-1)). Trp-P-2 and DMIP were also detected in some cases at levels below their limit of quantification. The remaining HAs analyzed were not detected in any of the samples.

Analysis of 5-hydroxymethylfurfural in foods by gas chromatography-mass spectrometry.

A new, simple and selective method for the analysis of 5-hydroxymethylfurfural (HMF) in foods by gas chromatography coupled to mass spectrometry (GC-MS) is proposed. Several derivatising procedures based on the formation of an HMF silylated derivative using different reagents were studied. Among the derivatising reagents examined, N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA) provided the best derivatisation yield. Sample clean-up was also optimised, using either liquid-liquid extraction with dichloromethane or solid-phase extraction (SPE) with several commercially available cartridges, and the best results were obtained using ENV+ cartridges. Quality parameters such as day-to-day and run-to-run precision (RSD<10%), linearity (between 25 and 700 ng g(-1)) and detection limit (6 ng g(-1)) were established. This method was successfully applied to the analysis of HMF content in several Spanish food samples from a local market, such as jam, honey, orange juice and bakery products.

Use of fluorescent probes for determination of yeast cell viability by gravitational field-flow fractionation.

The quality of wine greatly depends on the features of the yeast used in its production, and yeast cell viability is one of the most important quality control issues to consider in this regard. In the first steps of winemaking, the use of a low-cost and simple methodology for monitoring the cell viability of yeast inoculates is of paramount importance. Gravitational field-flow fractionation is a useful technique for the determination of cell viability because it provides gentle experimental conditions, although the proper use of fluorophore probes as biomass indicators is required. In this paper the use of different fluorescent probes such as carboxyfluorescein diacetate (cFDA), calcein-AM, and SYTO-13 were considered as viability biomarkers. Calceina-AM allowed the establishment of a direct GrFFF method to determine cell viability, with a limit of detection of 5.0 x 10(4) viable cell/mL. SYTO-13 could be used as biomass indicator with a limit of detection of 3.5 x 10(4) total cells/mL. The suitability of the procedure was tested with three commercial yeast samples, and the results were compared with those obtained using standard techniques.

Analysis of acrylamide in food samples by capillary zone electrophoresis.

Conditions for the determination of acrylamide (AA) after derivatisation with 2-mercaptobenzoic acid by capillary zone electrophoresis were established. A derivatisation reagent-acrylamide ratio of 35:1 was selected as optimum and the reagent excess was not removed as it did not affect the determination of acrylamide by CZE. The best separation was achieved using a 40 mM phosphate buffer at pH 8.0, working at 25 kV in un-coated fused silica capillaries. Linear calibration curves over the range studied (0.3-100 microg mL(-1)), the limit of detection (0.07 microg mL(-1)), and both run-to-run (RSD values of 5.8 and 2.2% for concentration at low and medium concentration levels, respectively) and day-to-day precisions (up to 11.2 and 6.7% at low and medium concentration levels, respectively) were established. Finally, the applicability of the CZE proposed methodology was demonstrated by analyzing levels of acrylamide present in different foodstuff products such as home made french fries, breakfast cereals and biscuits.

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection.

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.

Formation and stability of heterocyclic amines in a meat flavour model system. Effect of temperature, time and precursors.

A model system based on a commercial meat flavour was used to evaluate the formation of heterocyclic amines, simulating the application of this seasoning in household cooking. The effects of different treatments in both dry and aqueous conditions were studied. The lyophilized meat flavour extract was heated at temperatures ranging between 100 and 200 degrees C for times ranging from 10 min to 2 h. Similarly, an aqueous suspension of the extract was heated at 175 degrees C for 1, 2 and 3 h. Precursors of HAs, such as creatinine, glucose, and the amino acids glycine, alanine and phenylalanine were added to the meat extract and their effect was tested by heating the mixture at 200 degrees C for 30 min, when dry conditions were used, and at 175 degrees C for 2 h in wet systems. All conditions led to the formation of HAs, PhIP being the amine that was detected at the highest level of concentration in most model systems (i.e. 173 ng g(-1) at 200 degrees C, 30 min). Moreover, the addition of creatinine and amino acids to the meat extract flavour produced an important increase in IQ and MeIQx content.

Evaluation of reversed-phase columns for the analysis of heterocyclic aromatic amines by liquid chromatography-electrospray mass spectrometry.

Liquid chromatography coupled to mass spectrometry (LC-MS), especially by the use of electrospray ionisation source (ESI), is currently used for the analysis of heterocyclic aromatic amines (HAs) in complex samples. The present paper describes the study of the performance of different narrow-bore reversed-phase columns to achieve the best chromatographic separation for the determination of 16 HAs by LC-ESI-MS in food samples. Different parameters such as peak symmetry, resolution and number of theoretical plates have been evaluated for each column, using different chromatographic conditions. The column that provided the best results was TSK Gel Semi-Micro ODS-80TS of Tosohaas. Quality parameters have been established, obtaining good short-term precision in all cases (relative standard deviation (R.S.D.) lower than 7.7%) and low limits of detection (<13 pg injected in MS and <16 pg injected in MS/MS). The content of HAs in two beef extracts have been determined.

Preparation of a beef-extract as a laboratory reference material for the determination of heterocyclic amines.

The present paper describes the preparation of a suitable laboratory reference material (LRM) to validate analytical methods for the determination of heterocyclic amines (HAs) in foods. Three different lots of reference material were prepared using a beef extract which was contaminated with a well-known quantity of amines at different levels ranging from 10 to 75 ng/g. These materials were then lyophilised under determined conditions and, after grinding and sieving, homogenised and, finally, bottled and labelled. Homogeneity and stability studies were performed and no statistical differences were observed in the analysis of variances for within- and between-bottle results, thus demonstrating the homogeneity of the material. Stability at different storage temperatures (-18, +4, +25 and +40 degrees C) and times (1, 2, 3 and 6 months) was also tested. Therefore, the material can be considered homogeneous and stable and can be proposed for use in inter-comparison exercises for the determination of HAs.

Analysis of heterocyclic amines in food products: interlaboratory studies.

A feasibility study and two interlaboratory exercises on the determination of selected heterocyclic amines (HAs) in beef extract, organised in the framework of a European project, are presented. The aim of these exercises was to improve the quality of the laboratories and to evaluate the performance of a standardised analytical method and also the methods currently used by each of the participants for the analysis of these compounds. Three lyophilised portions of a commercial beef material previously spiked with HAs at different concentration levels ranging from 10 to 75 ng g(-1) were used as laboratory reference materials (lot A, B and C). Firstly, a feasibility study was carried out using a test standard solution and the beef extract (lot A), which contained only five HAs. Then, two interlaboratory exercises were carried out using the laboratory reference materials lot B and lot C, containing 10 selected HAs at two different concentration levels, 75 and 10 ng/g, respectively. The results obtained by all participant laboratories using the proposed method showed satisfactory agreement and the CV(%) between-laboratories obtained were from 8.3 to 24.1% for lot B and from 8.7 to 44.5% for lot C. The standardised method evaluated in these collaborative studies is therefore proposed for the analysis of HAs in food material. Moreover, LC-MS is recommended as the most suitable technique for the analysis of a large number of HAs in food samples.

Occurrence of heterocyclic amines in several home-cooked meat dishes of the Spanish diet.

Heterocyclic amines (HAs) were determined in several of the most frequently eaten meat dishes in Spain such as fried beef hamburger, fried pork loin, fried chicken breast, fried pork sausages, griddled chicken breast, griddled lamb steak and griddled beef steak. All of the products tested were household cooked. The HAs were analysed in the selected meat dishes using an analytical method based on solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry. DMIP, MeIQx, 4,8-DiMeIQx, Norharman, Harman, PhIP, Trp-P-1, AalphaC and MeAalphaC were the amines most frequently found at concentrations of up to 47 ng g(-1) of cooked meat. Glu-P-2, IQ, MeIQ, Glu-P-1, 7,8-DiMeIQx and Trp-P-2 were only found in a few of the meat dishes and their concentrations were lower than 1 ng g(-1) of cooked meat. The highest amounts of HAs, especially PhIP and DMIP, were formed in fried chicken breast and the lowest were formed in fried beef hamburger and in fried pork sausages. Daily intake of HAs in Spain was estimated at 606 ng of mutagenic HAs per capita and day, DMIP and PhIP being the main contributors.

Evaluation of different liquid chromatography-electrospray mass spectrometry systems for the analysis of heterocyclic amines.

Three liquid chromatography-electrospray ionisation (LC-ESI) MS systems are evaluated for the analysis of heterocyclic amines (HAs). The electrospray sources and analysers (ion trap, single quadrupole and triple quadrupole) have been compared in terms of performance and quality parameters. In all cases, a C8 reversed-phase column and (acetic acid-ammonium acetate 30 mM pH 4.5)-acetonitrile (ACN) as mobile phase were used. Ionisation source parameters, post-column addition and working conditions for each acquisition mode (full scan, product ion scan, selected ion monitoring, and multiple reaction monitoring) were optimised for each instrument. The MS-MS spectra obtained with the ion trap and the triple quadrupole systems were very similar in both fragment ions and relative abundances, except for carbolines that showed adduct formation in the ion trap. Quality parameters were established and good precision (relative standard deviations (R.S.D.) < 12%) and very low limits of detection were obtained, mainly when using the triple quadrupole (< 9 pg injected). The content of HAs in a lyophilised beef extract was determined using the three instruments in order to compare their applicability for routine HAs analysis.

Sonication effect on cellular material in sedimentation and gravitational field flow fractionation.

Sonication procedures are generally used prior to field flow fractionation (FFF) separation in order to produce suspensions without aggregates. Yeast cells manufactured in active dry wine yeast (ADWY) were placed in an ultrasound water bath in order to disrupt possible clumps and to obtain a single-cell suspension to be used in optimal conditions during fermentation processes. In order to determine whether this sample preparation procedure meets absolute needs, different yeast samples before and after sonication were analysed by two field flow fractionation techniques. It is shown that 2 min of sonication in the sample preparation process is sufficient to obtain an optimal dispersion of the yeast cells, that is, without critical percentage of aggregates. To demonstrate this effect, photographs of the yeast cell suspensions were performed with non-sonicated and sonicated yeast sample dispersion. The resulting data are compared with the elution profiles obtained from the two different FFF techniques. It is demonstrated that fractogram profiles prove the effectiveness of sonication methodologies.

Quantitation in multianalyte overlapping peaks from capillary electrophoresis runs using artificial neural networks.

The potentiality of artificial neural networks for multicomponent analysis in unresolved peaks from capillary electrophoresis (CE) is evaluated. The system chosen consists of mixtures of three ebrotidine metabolites, which cannot be successfully separated by CE. Data selected for analysis consist of UV spectra taken at the maximum of the CE peak. The most dissimilar analyte, in terms of spectral differences, is accurately quantitated in any type of mixture with an overall prediction error of 5%. Because of the strong interference of the two most overlapped compounds, a preliminary procedure for spectral data filtering based on principal component analysis is performed to improve their quantitation.

Determination of ebrotidine and its metabolites by micellar electrokinetic capillary chromatography.

Ebrotidine and its potential metabolites were determined by micellar electrokinetic capillary chromatogaphy (MECC) using sodium dodecylsulfate (SDS) as surfactant. The influences of buffer composition, SDS concentration and addition of a neutral surfactant such as Brij 35 were studied. A 40 mM phosphate buffer at pH 7.50 containing 50 mM of SDS was selected as carrier electrolyte, and provided the optimum separation with regard to resolution and migration time. Linear calibration curves over the range studied (5.0-50 microg ml(-1)), limits of detection between 0.25 and 2.0 microg ml(-1) and run-to-run precision lower than 10% were obtained. The MECC method was applied to the determinaton of these compounds in spiked human urine.

Improved performance of gravitational field-flow fractionation for screening wine-making yeast varieties.

Performance of gravitational field-flow fractionation (GFFF) is improved here with respect to the ability to fractionate and distinguish different varieties of wine-making yeast from Saccharomyces cerevisiae. A new GFFF channel with non-polar walls has been employed to enhance fractionation selectivity and reproducibility. Since GFFF retention depends from first principles on particle size, Coulter counter measurements were performed in order to compare size distribution profiles with GFFF profiles. From such a comparison, GFFF was shown to be able to reveal differences in yeast cells other than size. This could make use of GFFF for screening different varieties of wine-making yeast towards future quality assessment procedures based on a possible correlation between yeast cell morphology indexes and quality indexes.