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Analytica Chimica Acta (v.672, #1-2)
Gel-based immunotest for simultaneous detection of 2,4,6-trichlorophenol and ochratoxin A in red wine by N.V. Beloglazova; I.Yu. Goryacheva; T.Yu. Rusanova; N.A. Yurasov; R. Galve; M.-P. Marco; S. De Saeger (pp. 3-8).
A new rapid method which allows simultaneous one step detection of two analytes of different nature (2,4,6,-trichlorophenol (TCP) and ochratoxin A (OTA)) in red wine was developed. It was based on a column test with three separate immunolayers: two test layers and one control layer. Each layer consisted of sepharose gel with immobilized anti-OTA (OTA test layer), anti-TCP (TCP test layer) or anti-HRP (control layer) antibodies. Analytes bind to the antibodies in the corresponding test layer while sample flows through the column. Then a mixture of OTA-HRP and TCP-HRP in appropriate dilutions was used, followed by the application of chromogenic substrate. Colour development of the test layer occurred when the corresponding analyte was absent in the sample. HRP-conjugates bound to anti-HRP antibody in the control layer independently of presence or absence of analytes and a blue colour developed in the control layer. Cut-off values for both analytes were 2μgL−1. The described method was applied to the simultaneous detection of TCP and OTA in wine samples. To screen the analytes in red wine samples, clean-up columns were used for sample pre-treatment in combination with the test column. Results were confirmed by chromatographic methods.
Keywords: Red wine; Immunoassay; Rapid test; 2,4,6,-Trichlorophenol; Ochratoxin A; Food control
Gel-based immunotest for simultaneous detection of 2,4,6-trichlorophenol and ochratoxin A in red wine by N.V. Beloglazova; I.Yu. Goryacheva; T.Yu. Rusanova; N.A. Yurasov; R. Galve; M.-P. Marco; S. De Saeger (pp. 3-8).
A new rapid method which allows simultaneous one step detection of two analytes of different nature (2,4,6,-trichlorophenol (TCP) and ochratoxin A (OTA)) in red wine was developed. It was based on a column test with three separate immunolayers: two test layers and one control layer. Each layer consisted of sepharose gel with immobilized anti-OTA (OTA test layer), anti-TCP (TCP test layer) or anti-HRP (control layer) antibodies. Analytes bind to the antibodies in the corresponding test layer while sample flows through the column. Then a mixture of OTA-HRP and TCP-HRP in appropriate dilutions was used, followed by the application of chromogenic substrate. Colour development of the test layer occurred when the corresponding analyte was absent in the sample. HRP-conjugates bound to anti-HRP antibody in the control layer independently of presence or absence of analytes and a blue colour developed in the control layer. Cut-off values for both analytes were 2μgL−1. The described method was applied to the simultaneous detection of TCP and OTA in wine samples. To screen the analytes in red wine samples, clean-up columns were used for sample pre-treatment in combination with the test column. Results were confirmed by chromatographic methods.
Keywords: Red wine; Immunoassay; Rapid test; 2,4,6,-Trichlorophenol; Ochratoxin A; Food control
Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food by Anastasia Meimaridou; Willem Haasnoot; Linda Noteboom; Dimitrios Mintzas; Jana Pulkrabova; Jana Hajslová; Michel W.F. Nielen (pp. 9-14).
Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC50 of 0.3μgL−1 using a monoclonal antibody (Mab22F12) against BaP, similar to the IC50 of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography–mass spectrometry (GC–MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are under development.
Keywords: Polycyclic aromatic hydrocarbons; Flow cytometry; Persistent organic pollutants
Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food by Anastasia Meimaridou; Willem Haasnoot; Linda Noteboom; Dimitrios Mintzas; Jana Pulkrabova; Jana Hajslová; Michel W.F. Nielen (pp. 9-14).
Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC50 of 0.3μgL−1 using a monoclonal antibody (Mab22F12) against BaP, similar to the IC50 of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography–mass spectrometry (GC–MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are under development.
Keywords: Polycyclic aromatic hydrocarbons; Flow cytometry; Persistent organic pollutants
Molecularly imprinted polymer solid-phase extraction for detection of zearalenone in cereal sample extracts by Paolo Lucci; Delphine Derrien; Florent Alix; Céline Pérollier; Sami Bayoudh (pp. 15-19).
The aim of this work was to develop a method for the clean-up and preconcentration of zearalenone from corn and wheat samples employing molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE). Cereal samples were extracted with acetonitrile/water (75:25, v/v) and the extract was diluted with water and applied to an AFFINIMIP™ ZON MIP-SPE column. The column was then washed to eliminate the interferences and zearalenone was eluted with methanol and quantified using HPLC with fluorescence detection ( λexc=275/ λem=450nm). The precision and accuracy of the method were satisfactory for both cereals at the different fortification levels tested and it gave recoveries between 82 and 87% (RSDr 2.5–6.2%, n=3) and 86 and 90% (RSDr 0.9–6.8%, n=3) for wheat and maize, respectively. MIP-SPE column capacity was determined to be not less than 6.6μg of zearalenone and to be at least four times higher than that of immunoaffinity column (IAC). The application of AFFINIMIP™ ZON molecularly imprinted polymer as a selective sorbent material for detection of zearalenone fulfilled the method performance criteria required by the Commission Regulation (EC) No. 401/2006, demonstrating the suitability of the technique for the control of zearalenone in cereal samples.
Keywords: Zearalenone; Molecularly imprinted polymers; Solid-phase extraction; High-performance liquid chromatography; Fluorimetry
Molecularly imprinted polymer solid-phase extraction for detection of zearalenone in cereal sample extracts by Paolo Lucci; Delphine Derrien; Florent Alix; Céline Pérollier; Sami Bayoudh (pp. 15-19).
The aim of this work was to develop a method for the clean-up and preconcentration of zearalenone from corn and wheat samples employing molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE). Cereal samples were extracted with acetonitrile/water (75:25, v/v) and the extract was diluted with water and applied to an AFFINIMIP™ ZON MIP-SPE column. The column was then washed to eliminate the interferences and zearalenone was eluted with methanol and quantified using HPLC with fluorescence detection ( λexc=275/ λem=450nm). The precision and accuracy of the method were satisfactory for both cereals at the different fortification levels tested and it gave recoveries between 82 and 87% (RSDr 2.5–6.2%, n=3) and 86 and 90% (RSDr 0.9–6.8%, n=3) for wheat and maize, respectively. MIP-SPE column capacity was determined to be not less than 6.6μg of zearalenone and to be at least four times higher than that of immunoaffinity column (IAC). The application of AFFINIMIP™ ZON molecularly imprinted polymer as a selective sorbent material for detection of zearalenone fulfilled the method performance criteria required by the Commission Regulation (EC) No. 401/2006, demonstrating the suitability of the technique for the control of zearalenone in cereal samples.
Keywords: Zearalenone; Molecularly imprinted polymers; Solid-phase extraction; High-performance liquid chromatography; Fluorimetry
Electronic nose as an innovative tool for the diagnosis of grapevine crown gall by S. Blasioli; E. Biondi; I. Braschi; U. Mazzucchi; C. Bazzi; C.E. Gessa (pp. 20-24).
For the first time, a portable electronic nose was used to discriminate between healthy and galled grapevines, experimentally inoculated with two tumourigenic strains of Agrobacterium vitis. The volatile profile of target cutting samples was analysed by headspace solid phase microextraction coupled with gas chromatography–mass spectrometry. Spectra from tumoured samples revealed the presence of styrene which is compatible with decarboxylation of cinnamic acid involved in secondary metabolism of plants. Principal Component Analysis confirmed the difference in volatile profiles of infected vines and their healthy controls. Linear Discriminant Analysis allowed the correct discrimination between healthy and galled grapevines (83.3%, cross-validation). Although a larger number of samples should be analysed to create a more robust model, our results give novel interesting clues to go further with research on the diagnostic potential of this innovative system associated with multi-dimensional chemometric techniques.
Keywords: Agrobacterium vitis; Grapevine tumours; Gas cromathography-mass spectrometry; Solid phase microextraction; Electronic nose; Statistical analysis
Electronic nose as an innovative tool for the diagnosis of grapevine crown gall by S. Blasioli; E. Biondi; I. Braschi; U. Mazzucchi; C. Bazzi; C.E. Gessa (pp. 20-24).
For the first time, a portable electronic nose was used to discriminate between healthy and galled grapevines, experimentally inoculated with two tumourigenic strains of Agrobacterium vitis. The volatile profile of target cutting samples was analysed by headspace solid phase microextraction coupled with gas chromatography–mass spectrometry. Spectra from tumoured samples revealed the presence of styrene which is compatible with decarboxylation of cinnamic acid involved in secondary metabolism of plants. Principal Component Analysis confirmed the difference in volatile profiles of infected vines and their healthy controls. Linear Discriminant Analysis allowed the correct discrimination between healthy and galled grapevines (83.3%, cross-validation). Although a larger number of samples should be analysed to create a more robust model, our results give novel interesting clues to go further with research on the diagnostic potential of this innovative system associated with multi-dimensional chemometric techniques.
Keywords: Agrobacterium vitis; Grapevine tumours; Gas cromathography-mass spectrometry; Solid phase microextraction; Electronic nose; Statistical analysis
Towards development of incurred materials for quality assurance purposes in the analysis of food allergens by Zsuzsanna Bugyi; Judit Nagy; Kitti Török; Lívia Hajas; Sándor Tömösközi (pp. 25-29).
Food allergy and intolerance became very important problems in food safety and healthcare during the last few decades. Beside the pharmaceutical treatment of the symptoms, effective cure of these illnesses is the avoidance of the problematic food proteins. According to this reason, proper legislation is crucial for protecting sensitive people. In the European Union 14 allergenic components must be labelled which requires introduction of properly validated analytical methods for the appropriate quantification of allergenic proteins. The aim of our work is studying such parameters which may affect the analytical results, therefore have to be taken into account during the validation process. For investigating these issues, an incurred sample matrix was produced, namely a wheat flour based cookie, which contains allergenic proteins (milk or egg) in a dedicated amount. Using these samples the effects of food processing steps and the analytical performance of the applied Enzyme-Linked Immunosorbent Assay (ELISA) methods were studied. A major finding of our work is that heat treatment caused a large-scale decrease in the amount of measurable allergen content of the samples. The background of this phenomenon has not been clarified yet. Besides, the gathered data indicates that the performance of the ELISA method is highly related to the state of the sample matrix. These problems altogether must be taken into consideration for making a proper validation protocol and revealing their background also has a great importance in further evaluation of the analytical methods.
Keywords: Food allergy; Enzyme-Linked Immunosorbent Assay; Method validation; Incurred samples
Towards development of incurred materials for quality assurance purposes in the analysis of food allergens by Zsuzsanna Bugyi; Judit Nagy; Kitti Török; Lívia Hajas; Sándor Tömösközi (pp. 25-29).
Food allergy and intolerance became very important problems in food safety and healthcare during the last few decades. Beside the pharmaceutical treatment of the symptoms, effective cure of these illnesses is the avoidance of the problematic food proteins. According to this reason, proper legislation is crucial for protecting sensitive people. In the European Union 14 allergenic components must be labelled which requires introduction of properly validated analytical methods for the appropriate quantification of allergenic proteins. The aim of our work is studying such parameters which may affect the analytical results, therefore have to be taken into account during the validation process. For investigating these issues, an incurred sample matrix was produced, namely a wheat flour based cookie, which contains allergenic proteins (milk or egg) in a dedicated amount. Using these samples the effects of food processing steps and the analytical performance of the applied Enzyme-Linked Immunosorbent Assay (ELISA) methods were studied. A major finding of our work is that heat treatment caused a large-scale decrease in the amount of measurable allergen content of the samples. The background of this phenomenon has not been clarified yet. Besides, the gathered data indicates that the performance of the ELISA method is highly related to the state of the sample matrix. These problems altogether must be taken into consideration for making a proper validation protocol and revealing their background also has a great importance in further evaluation of the analytical methods.
Keywords: Food allergy; Enzyme-Linked Immunosorbent Assay; Method validation; Incurred samples
Validation of a two-plate microbiological method for screening antibiotic residues in shrimp tissue by Pham Kim Dang; Guy Degand; Sophie Danyi; Gilles Pierret; Philippe Delahaut; Vu Dinh Ton; Guy Maghuin-Rogister; Marie-Louise Scippo (pp. 30-39).
Microbiological inhibition screening tests could play an important role to detect residues of antibiotics in the different animal food products, but very few are available for the aquaculture products in general, and for shrimps in particular. A two-plate microbiological method to screen shrimp for residues of the most commonly used antibiotics has been developed and validated according to criteria derived from the European Commission Decision 2002/657/CE. Bacillus subtilis was used as a sensitive strain to target antibiotics. Culture conditions on Petri plates (pH of medium) were selected to enhance the capacity of antibiotic detection. Antibiotic residues were extracted from shrimps using acetonitrile/acetone (70/30, v/v) before application on Petri plates seeded with B. subtilis. The method was validated using spiked blank tissues as well as antibiotic treated shrimps with enrofloxacin and tetracycline, two antibiotics often found to be used in shrimp production. For tetracyclines and (fluoro)quinolones, the detection capability was below the maximum residue limit (MRL), while it was around the MRL for sulfonamides. The specificity of the microbiological screening was 100% in all cases while the sensitivity and accuracy was 100% in almost all cases. The capacity of the method to detect contaminated samples was confirmed on antibiotic treated shrimps, analyzed in parallel with a confirmatory method (Liquid Chromatography coupled to mass spectrometry (LC–MS)).
Keywords: Antibiotic residue; Tetracyclines; (Fluoro)quinolones; Sulfonamides; Shrimp; Microbiological method
Validation of a two-plate microbiological method for screening antibiotic residues in shrimp tissue by Pham Kim Dang; Guy Degand; Sophie Danyi; Gilles Pierret; Philippe Delahaut; Vu Dinh Ton; Guy Maghuin-Rogister; Marie-Louise Scippo (pp. 30-39).
Microbiological inhibition screening tests could play an important role to detect residues of antibiotics in the different animal food products, but very few are available for the aquaculture products in general, and for shrimps in particular. A two-plate microbiological method to screen shrimp for residues of the most commonly used antibiotics has been developed and validated according to criteria derived from the European Commission Decision 2002/657/CE. Bacillus subtilis was used as a sensitive strain to target antibiotics. Culture conditions on Petri plates (pH of medium) were selected to enhance the capacity of antibiotic detection. Antibiotic residues were extracted from shrimps using acetonitrile/acetone (70/30, v/v) before application on Petri plates seeded with B. subtilis. The method was validated using spiked blank tissues as well as antibiotic treated shrimps with enrofloxacin and tetracycline, two antibiotics often found to be used in shrimp production. For tetracyclines and (fluoro)quinolones, the detection capability was below the maximum residue limit (MRL), while it was around the MRL for sulfonamides. The specificity of the microbiological screening was 100% in all cases while the sensitivity and accuracy was 100% in almost all cases. The capacity of the method to detect contaminated samples was confirmed on antibiotic treated shrimps, analyzed in parallel with a confirmatory method (Liquid Chromatography coupled to mass spectrometry (LC–MS)).
Keywords: Antibiotic residue; Tetracyclines; (Fluoro)quinolones; Sulfonamides; Shrimp; Microbiological method
Glazing of frozen fish: Analytical and economic challenges by Lynn Vanhaecke; Wim Verbeke; Hubert F. De Brabander (pp. 40-44).
Adequate glazing (6–10%) of fish fillets prior to frozen storage protects the final product from dehydration, oxidation and quality loss. Excessive glazing (>12%) on the other hand may significantly affect the economic value and end user satisfaction of frozen fish fillets. This paper describes the optimization, validation and application of a gravimetric procedure for the quantification of the ice-glaze content of frozen fish fillets (accredited under ISO 17025). This procedure has been utilized to determine the glazing percentage of multiple batches ( n=50) of 11 different fish species sampled from 2005 until 2009. Average glazing percentages were 8.7±2.0% for the pooled samples ( n=712), and ranged between 6.6±2.2% (salmon/cod) and 10.6±1.6% (plaice). The lower threshold value of 6% glazing for sufficient protection was violated in only one batch, whereas none of the batches exceeded the 12% excessive glazing threshold. The annual market place value of one %-point glazing is estimated at 1 million Euro in a low to moderate fish consumption market like Belgium. The large variability of glazing, combined with this technology's possible implications with respect to end-product-quality and economic value urges for technology improvement, monitoring and more controlled application of the glazing process in the frozen fish industry.
Keywords: Belgium; Determination; Frozen fish; Glaze
Glazing of frozen fish: Analytical and economic challenges by Lynn Vanhaecke; Wim Verbeke; Hubert F. De Brabander (pp. 40-44).
Adequate glazing (6–10%) of fish fillets prior to frozen storage protects the final product from dehydration, oxidation and quality loss. Excessive glazing (>12%) on the other hand may significantly affect the economic value and end user satisfaction of frozen fish fillets. This paper describes the optimization, validation and application of a gravimetric procedure for the quantification of the ice-glaze content of frozen fish fillets (accredited under ISO 17025). This procedure has been utilized to determine the glazing percentage of multiple batches ( n=50) of 11 different fish species sampled from 2005 until 2009. Average glazing percentages were 8.7±2.0% for the pooled samples ( n=712), and ranged between 6.6±2.2% (salmon/cod) and 10.6±1.6% (plaice). The lower threshold value of 6% glazing for sufficient protection was violated in only one batch, whereas none of the batches exceeded the 12% excessive glazing threshold. The annual market place value of one %-point glazing is estimated at 1 million Euro in a low to moderate fish consumption market like Belgium. The large variability of glazing, combined with this technology's possible implications with respect to end-product-quality and economic value urges for technology improvement, monitoring and more controlled application of the glazing process in the frozen fish industry.
Keywords: Belgium; Determination; Frozen fish; Glaze
Detection of recombinant bovine somatotropin in milk and effect of industrial processes on its stability by Marie-Hélène Le Breton; Andrea Beck-Henzelin; Janique Richoz-Payot; Sandrine Rochereau-Roulet; Gaud Pinel; Thierry Delatour; Bruno Le Bizec (pp. 45-49).
A LC–MS/MS method has been developed for the direct detection of recombinant bovine somatotropin (rbST) in milk and dairy products. The sample preparation protocol is based on a solid phase extraction step followed by precipitation with cold methanol and enzymatic digestion. The analysis is focused on the tryptic N-terminal peptide, specific of the recombinant form of the hormone and the detection is performed by LC–ESI(+)-MS/MS. This method has been validated according to the European Union criteria described in the Directive 2002/657/EC. Acceptable performances, with a decision limit (CCα) of 1.24ngmL−1 and detection capability (CCβ) of 1.92ngmL−1 were obtained. Calculation of repeatability and intermediate reproducibility of the signal at 100ngmL−1 lead to relative standard deviations lower than 20%, showing the robustness of the method. Samples subjected to various industrial processes namely, heating, freezing, defatting, pasteurization and spray-drying were then analysed in order to determine the consequences of these treatments on the stability of the hormone. Results showed that temperature related processes, such as pasteurization and spray-drying induce a loss of the hormone up to 95%.
Keywords: Growth hormone; Somatotropin; Mass spectrometry; Milk processing
Detection of recombinant bovine somatotropin in milk and effect of industrial processes on its stability by Marie-Hélène Le Breton; Andrea Beck-Henzelin; Janique Richoz-Payot; Sandrine Rochereau-Roulet; Gaud Pinel; Thierry Delatour; Bruno Le Bizec (pp. 45-49).
A LC–MS/MS method has been developed for the direct detection of recombinant bovine somatotropin (rbST) in milk and dairy products. The sample preparation protocol is based on a solid phase extraction step followed by precipitation with cold methanol and enzymatic digestion. The analysis is focused on the tryptic N-terminal peptide, specific of the recombinant form of the hormone and the detection is performed by LC–ESI(+)-MS/MS. This method has been validated according to the European Union criteria described in the Directive 2002/657/EC. Acceptable performances, with a decision limit (CCα) of 1.24ngmL−1 and detection capability (CCβ) of 1.92ngmL−1 were obtained. Calculation of repeatability and intermediate reproducibility of the signal at 100ngmL−1 lead to relative standard deviations lower than 20%, showing the robustness of the method. Samples subjected to various industrial processes namely, heating, freezing, defatting, pasteurization and spray-drying were then analysed in order to determine the consequences of these treatments on the stability of the hormone. Results showed that temperature related processes, such as pasteurization and spray-drying induce a loss of the hormone up to 95%.
Keywords: Growth hormone; Somatotropin; Mass spectrometry; Milk processing
Depletion study of PCDD/Fs and dioxin-like PCBs concentrations in contaminated home-produced eggs: Preliminary study by S. Menotta; M. D’antonio; G. Diegoli; L. Montella; S. Raccanelli; G. Fedrizzi (pp. 50-54).
Dioxins (PCDD/Fs) and polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. The contamination of food products with dioxins and PCBs is a well studied issue, because food is generally considered the major source of dioxin intake for humans. In Italy, the Regional Monitoring Plan (part of the national residue monitoring plan) used in the field for 2009 has also included the control of environmental pollutants in small egg producers. Following an irregular result, 12 laying hens were transferred into a laboratory controlled environment. Eggs were collected for 60 days and they were weekly analysed for the evaluation of dioxins, dioxin-like PCBs (DL-PCBs), and non-dioxin-like PCBs (NDL-PCBs, six congeners) levels. The dioxins and PCBs contents were determined, according to EPA methods, by gas chromatography ic determination coupled with high resolution mass spectrometry (HRGC-HRMS). The content of PCDD/Fs, DL-PCBs and NDL-PCBs was evaluated weekly by mean from week to week. The concentration of dioxins was lower than DL-PCBs (2.5pgTEQg−1 of fat against 4.5pgTEQg−1 of fat), but we observed the same depletion trend for both pollutants. On the opposite, NDL-PCBs had a different course: we noted there was an increase between weeks 6 and 7, but the mean levels remained very low (about 20ngg−1 of fat). The dioxins, and sum of dioxin and DL-PCBs concentration were below the fixed European limits (i.e. 3pgTEQg−1 of fat for dioxins and 6pgTEQg−1 of fat for sum of dioxins and DL-PCBs), beginning from the 3rd week of trial.
Keywords: Polychlorinated dibenzo-p-dioxin; Polychlorinated dibenzofurans; Polychlorinated biphenyls; Eggs; Depletion
Depletion study of PCDD/Fs and dioxin-like PCBs concentrations in contaminated home-produced eggs: Preliminary study by S. Menotta; M. D’antonio; G. Diegoli; L. Montella; S. Raccanelli; G. Fedrizzi (pp. 50-54).
Dioxins (PCDD/Fs) and polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. The contamination of food products with dioxins and PCBs is a well studied issue, because food is generally considered the major source of dioxin intake for humans. In Italy, the Regional Monitoring Plan (part of the national residue monitoring plan) used in the field for 2009 has also included the control of environmental pollutants in small egg producers. Following an irregular result, 12 laying hens were transferred into a laboratory controlled environment. Eggs were collected for 60 days and they were weekly analysed for the evaluation of dioxins, dioxin-like PCBs (DL-PCBs), and non-dioxin-like PCBs (NDL-PCBs, six congeners) levels. The dioxins and PCBs contents were determined, according to EPA methods, by gas chromatography ic determination coupled with high resolution mass spectrometry (HRGC-HRMS). The content of PCDD/Fs, DL-PCBs and NDL-PCBs was evaluated weekly by mean from week to week. The concentration of dioxins was lower than DL-PCBs (2.5pgTEQg−1 of fat against 4.5pgTEQg−1 of fat), but we observed the same depletion trend for both pollutants. On the opposite, NDL-PCBs had a different course: we noted there was an increase between weeks 6 and 7, but the mean levels remained very low (about 20ngg−1 of fat). The dioxins, and sum of dioxin and DL-PCBs concentration were below the fixed European limits (i.e. 3pgTEQg−1 of fat for dioxins and 6pgTEQg−1 of fat for sum of dioxins and DL-PCBs), beginning from the 3rd week of trial.
Keywords: Polychlorinated dibenzo-p-dioxin; Polychlorinated dibenzofurans; Polychlorinated biphenyls; Eggs; Depletion
Furan levels in fruit and vegetables juices, nutrition drinks and bakery products by Jan-Willem Wegener; Patricia López-Sánchez (pp. 55-60).
Furan, an oxygen containing monocyclic aromatic hydrocarbon, is considered possibly carcinogenic to humans. In the framework of the EU-project “Role of Genetic and Non-Genetic Mechanisms in Furan Risk”, furan levels in food have been collected from the literature. Three food type categories have been selected on the basis of the collected data for sampling and analysis on furan with headspace GC–MS. This paper describes the results for the selected food categories, fruit and vegetables juices, nutrition drinks and bakery products. An attempt has been made to correlate the furan levels with the ingredients of the products.
Keywords: Headspace; Furan; Fruit juices; Vegetables juices; Nutrition drinks; Bakery products
Furan levels in fruit and vegetables juices, nutrition drinks and bakery products by Jan-Willem Wegener; Patricia López-Sánchez (pp. 55-60).
Furan, an oxygen containing monocyclic aromatic hydrocarbon, is considered possibly carcinogenic to humans. In the framework of the EU-project “Role of Genetic and Non-Genetic Mechanisms in Furan Risk”, furan levels in food have been collected from the literature. Three food type categories have been selected on the basis of the collected data for sampling and analysis on furan with headspace GC–MS. This paper describes the results for the selected food categories, fruit and vegetables juices, nutrition drinks and bakery products. An attempt has been made to correlate the furan levels with the ingredients of the products.
Keywords: Headspace; Furan; Fruit juices; Vegetables juices; Nutrition drinks; Bakery products
Development of a new analytical method for the determination of sulfites in fresh meats and shrimps by ion-exchange chromatography with conductivity detection by Marco Iammarino; Aurelia Di Taranto; Marilena Muscarella; Donatella Nardiello; Carmen Palermo; Diego Centonze (pp. 61-65).
An accurate and reliable analytical method, based on ion chromatography and suppressed conductivity detection, has been developed and validated for the quantitative determination of sulfites in fresh meats and shrimps. The chromatographic separation was accomplished by using an anion-exchange column eluted with sodium carbonate and sodium hydroxide. The optimized step-change elution, followed by column re-equilibration at the initial mobile phase composition, guaranteed a good resolution even toward endogenous interfering peaks, and an excellent retention time repeatability (1.1%, n=6). Good results in terms of sample extract stability, recovery efficiency were achieved with an extraction solvent mixture based on sodium hydroxide, fructose and EDTA. The method validation, performed by an in-house model according to Decision 657/2002/EC and Regulation 882/2004/EC, provided excellent results with respect to linearity (correlation coefficient up to 0.9998), limits of detection and quantification (2.7 and 8.2mgkg−1, respectively, expressed as SO2), expanded measurement uncertainty (below 10%), recovery values (ranging from 85% to 92%) and repeatability (down to 8%), demonstrating the conformity of the proposed method with the European directives. Finally, by major changes ruggedness studies, the method applicability to the quantitative analysis of cow hamburger, pork and horse sausage, and shrimps was demonstrated.
Keywords: Sulfites; Ion chromatography; Conductivity; Validation; Fresh meats; Shrimps
Development of a new analytical method for the determination of sulfites in fresh meats and shrimps by ion-exchange chromatography with conductivity detection by Marco Iammarino; Aurelia Di Taranto; Marilena Muscarella; Donatella Nardiello; Carmen Palermo; Diego Centonze (pp. 61-65).
An accurate and reliable analytical method, based on ion chromatography and suppressed conductivity detection, has been developed and validated for the quantitative determination of sulfites in fresh meats and shrimps. The chromatographic separation was accomplished by using an anion-exchange column eluted with sodium carbonate and sodium hydroxide. The optimized step-change elution, followed by column re-equilibration at the initial mobile phase composition, guaranteed a good resolution even toward endogenous interfering peaks, and an excellent retention time repeatability (1.1%, n=6). Good results in terms of sample extract stability, recovery efficiency were achieved with an extraction solvent mixture based on sodium hydroxide, fructose and EDTA. The method validation, performed by an in-house model according to Decision 657/2002/EC and Regulation 882/2004/EC, provided excellent results with respect to linearity (correlation coefficient up to 0.9998), limits of detection and quantification (2.7 and 8.2mgkg−1, respectively, expressed as SO2), expanded measurement uncertainty (below 10%), recovery values (ranging from 85% to 92%) and repeatability (down to 8%), demonstrating the conformity of the proposed method with the European directives. Finally, by major changes ruggedness studies, the method applicability to the quantitative analysis of cow hamburger, pork and horse sausage, and shrimps was demonstrated.
Keywords: Sulfites; Ion chromatography; Conductivity; Validation; Fresh meats; Shrimps
Statistical evaluation of fatty acid profile and cholesterol content in fish (common carp) lipids obtained by different sample preparation procedures by Aurelija Spiric; Dejana Trbovic; Danijela Vranic; Jasna Djinovic; Radivoj Petronijevic; Vesna Matekalo-Sverak (pp. 66-71).
Studies performed on lipid extraction from animal and fish tissues do not provide information on its influence on fatty acid composition of the extracted lipids as well as on cholesterol content. Data presented in this paper indicate the impact of extraction procedures on fatty acid profile of fish lipids extracted by the modified Soxhlet and ASE (accelerated solvent extraction) procedure. Cholesterol was also determined by direct saponification method, too.Student's paired t-test used for comparison of the total fat content in carp fish population obtained by two extraction methods shows that differences between values of the total fat content determined by ASE and modified Soxhlet method are not statistically significant. Values obtained by three different methods (direct saponification, ASE and modified Soxhlet method), used for determination of cholesterol content in carp, were compared by one-way analysis of variance (ANOVA). The obtained results show that modified Soxhlet method gives results which differ significantly from the results obtained by direct saponification and ASE method. However the results obtained by direct saponification and ASE method do not differ significantly from each other. The highest quantities for cholesterol (37.65 to 65.44mg/100g) in the analyzed fish muscle were obtained by applying direct saponification method, as less destructive one, followed by ASE (34.16 to 52.60mg/100g) and modified Soxhlet extraction method (10.73 to 30.83mg/100g).Modified Soxhlet method for extraction of fish lipids gives higher values for n-6 fatty acids than ASE method ( tpaired=3.22 tc=2.36), while there is no statistically significant difference in the n-3 content levels between the methods ( tpaired=1.31). The UNSFA/SFA ratio obtained by using modified Soxhlet method is also higher than the ratio obtained using ASE method ( tpaired=4.88 tc=2.36). Results of Principal Component Analysis (PCA) showed that the highest positive impact to the second principal component (PC2) is recorded by C18:3 n-3, and C20:3 n-6, being present in a higher amount in the samples treated by the modified Soxhlet extraction, while C22:5 n-3, C20:3 n-3, C22:1 and C20:4, C16 and C18 negatively influence the score values of the PC2, showing significantly increased level in the samples treated by ASE method. Hotelling's paired T-square test used on the first three principal components for confirmation of differences in individual fatty acid content obtained by ASE and Soxhlet method in carp muscle showed statistically significant difference between these two data sets ( T2=161.308, p<0.001).
Keywords: Modified Soxhlet extraction; ASE; Total lipids; Cholesterol; Fatty acids; Fish
Statistical evaluation of fatty acid profile and cholesterol content in fish (common carp) lipids obtained by different sample preparation procedures by Aurelija Spiric; Dejana Trbovic; Danijela Vranic; Jasna Djinovic; Radivoj Petronijevic; Vesna Matekalo-Sverak (pp. 66-71).
Studies performed on lipid extraction from animal and fish tissues do not provide information on its influence on fatty acid composition of the extracted lipids as well as on cholesterol content. Data presented in this paper indicate the impact of extraction procedures on fatty acid profile of fish lipids extracted by the modified Soxhlet and ASE (accelerated solvent extraction) procedure. Cholesterol was also determined by direct saponification method, too.Student's paired t-test used for comparison of the total fat content in carp fish population obtained by two extraction methods shows that differences between values of the total fat content determined by ASE and modified Soxhlet method are not statistically significant. Values obtained by three different methods (direct saponification, ASE and modified Soxhlet method), used for determination of cholesterol content in carp, were compared by one-way analysis of variance (ANOVA). The obtained results show that modified Soxhlet method gives results which differ significantly from the results obtained by direct saponification and ASE method. However the results obtained by direct saponification and ASE method do not differ significantly from each other. The highest quantities for cholesterol (37.65 to 65.44mg/100g) in the analyzed fish muscle were obtained by applying direct saponification method, as less destructive one, followed by ASE (34.16 to 52.60mg/100g) and modified Soxhlet extraction method (10.73 to 30.83mg/100g).Modified Soxhlet method for extraction of fish lipids gives higher values for n-6 fatty acids than ASE method ( tpaired=3.22 tc=2.36), while there is no statistically significant difference in the n-3 content levels between the methods ( tpaired=1.31). The UNSFA/SFA ratio obtained by using modified Soxhlet method is also higher than the ratio obtained using ASE method ( tpaired=4.88 tc=2.36). Results of Principal Component Analysis (PCA) showed that the highest positive impact to the second principal component (PC2) is recorded by C18:3 n-3, and C20:3 n-6, being present in a higher amount in the samples treated by the modified Soxhlet extraction, while C22:5 n-3, C20:3 n-3, C22:1 and C20:4, C16 and C18 negatively influence the score values of the PC2, showing significantly increased level in the samples treated by ASE method. Hotelling's paired T-square test used on the first three principal components for confirmation of differences in individual fatty acid content obtained by ASE and Soxhlet method in carp muscle showed statistically significant difference between these two data sets ( T2=161.308, p<0.001).
Keywords: Modified Soxhlet extraction; ASE; Total lipids; Cholesterol; Fatty acids; Fish
Characterization of isoflavone composition in soy-based nutritional supplements via ultra performance liquid chromatography by G. Fiechter; B. Raba; A. Jungmayr; H.K. Mayer (pp. 72-78).
The specific isoflavone composition of nutritional supplements is commonly not-labeled, although the stated amounts are strongly dependent on the present isoflavone conjugates. Hence, 11 soy-based dietary supplements were characterized via a newly established ultra performance liquid chromatography (UPLC™) method, on both their native conjugated isoflavone spectra, as well as on quantitative amounts derived as total aglycones after enzymatic hydrolysis utilizing Helix pomatia juice. Capitalizing on sub-2 μm particles, the established RP-UPLC™ technique facilitated efficient chromatographic separation of all 12 soy intrinsic isoflavone forms within 10min. Derived native isoflavone profiles implied a certain variability, comprising conjugated forms, especially glycosides, as the predominant isoflavonic constituents throughout the majority of supplements, whereas only two samples indicated the more bioavailable free aglycones as prevailing compounds. Moreover, the robust quantification as total aglycones subsequent to enzymatic hydrolysis, unexceptionally yielded negative deviations referring to the labeled specifications, thus implying that stated amounts were typically calculated on basis of the high molecular isoflavone conjugates. Thus, especially in regard to better comparability, regulations concerning an uniform labeling basis are needed.
Keywords: UPLC™; Nutritional supplements; Soy; Isoflavones; Helix pomatia
Characterization of isoflavone composition in soy-based nutritional supplements via ultra performance liquid chromatography by G. Fiechter; B. Raba; A. Jungmayr; H.K. Mayer (pp. 72-78).
The specific isoflavone composition of nutritional supplements is commonly not-labeled, although the stated amounts are strongly dependent on the present isoflavone conjugates. Hence, 11 soy-based dietary supplements were characterized via a newly established ultra performance liquid chromatography (UPLC™) method, on both their native conjugated isoflavone spectra, as well as on quantitative amounts derived as total aglycones after enzymatic hydrolysis utilizing Helix pomatia juice. Capitalizing on sub-2 μm particles, the established RP-UPLC™ technique facilitated efficient chromatographic separation of all 12 soy intrinsic isoflavone forms within 10min. Derived native isoflavone profiles implied a certain variability, comprising conjugated forms, especially glycosides, as the predominant isoflavonic constituents throughout the majority of supplements, whereas only two samples indicated the more bioavailable free aglycones as prevailing compounds. Moreover, the robust quantification as total aglycones subsequent to enzymatic hydrolysis, unexceptionally yielded negative deviations referring to the labeled specifications, thus implying that stated amounts were typically calculated on basis of the high molecular isoflavone conjugates. Thus, especially in regard to better comparability, regulations concerning an uniform labeling basis are needed.
Keywords: UPLC™; Nutritional supplements; Soy; Isoflavones; Helix pomatia
A sensitive method for free amino acids analysis by liquid chromatography with ultraviolet and mass spectrometric detection using precolumn derivatization with diethyl ethoxymethylenemalonate: Application to the honey analysis by Riin Rebane; Koit Herodes (pp. 79-84).
In case of some foods and drinks, their amino acid content is demonstrated to correlate with the botanical and/or geographical origin of the plant.In present work a method for amino acid analysis in honey was developed and validated. The method consists of sample preparation (including solid phase extraction), derivatization with diethyl ethoxymethylenemalonate and liquid chromatographic analysis. Full separation of 23 amino acids was achieved. All steps were extensively studied and optimized for analytical performance. Although ultraviolet (UV) detection provides sufficient sensitivity for honey analysis, all the steps of the method were designed to be compatible with mass spectrometric (MS) detection.The developed method has been applied to analysis of nearly 200 Estonian honey samples.
Keywords: Amino acid analysis; Diethyl ethoxymethylenemalonate; Post-column derivatization; Honey; Solid phase extraction; Method development
A sensitive method for free amino acids analysis by liquid chromatography with ultraviolet and mass spectrometric detection using precolumn derivatization with diethyl ethoxymethylenemalonate: Application to the honey analysis by Riin Rebane; Koit Herodes (pp. 79-84).
In case of some foods and drinks, their amino acid content is demonstrated to correlate with the botanical and/or geographical origin of the plant.In present work a method for amino acid analysis in honey was developed and validated. The method consists of sample preparation (including solid phase extraction), derivatization with diethyl ethoxymethylenemalonate and liquid chromatographic analysis. Full separation of 23 amino acids was achieved. All steps were extensively studied and optimized for analytical performance. Although ultraviolet (UV) detection provides sufficient sensitivity for honey analysis, all the steps of the method were designed to be compatible with mass spectrometric (MS) detection.The developed method has been applied to analysis of nearly 200 Estonian honey samples.
Keywords: Amino acid analysis; Diethyl ethoxymethylenemalonate; Post-column derivatization; Honey; Solid phase extraction; Method development
Determination of non-steroidal anti-inflammatory drugs residues in animal muscles by liquid chromatography–tandem mass spectrometry by Piotr Jedziniak; Teresa Szprengier-Juszkiewicz; Małgorzata Olejnik; Jan Żmudzki (pp. 85-92).
A confirmatory method for the determination of residues of nine non-steroidal anti-inflammatory drugs and one metabolite in animal muscles has been developed. After enzymatic hydrolysis samples were extracted with acetonitrile and cleaned up using alumina and C18 SPE cartridges. Liquid chromatography–tandem mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine muscles, according to the Commission Decision 2002/657/EC criteria. Applicability of the method in the analysis of swine, horse and chicken muscles was checked by precision and recovery experiment. The influence of matrix effect on the quantification of non-steroidal anti-inflammatory drugs residues was investigated. The method was used for the confirmation of phenylbutazone and oxyphenbutazone in horse muscle sample.
Keywords: Non-steroidal anti-inflammatory drugs; Residues; Liquid chromatography–tandem mass spectrometry; Matrix effect
Determination of non-steroidal anti-inflammatory drugs residues in animal muscles by liquid chromatography–tandem mass spectrometry by Piotr Jedziniak; Teresa Szprengier-Juszkiewicz; Małgorzata Olejnik; Jan Żmudzki (pp. 85-92).
A confirmatory method for the determination of residues of nine non-steroidal anti-inflammatory drugs and one metabolite in animal muscles has been developed. After enzymatic hydrolysis samples were extracted with acetonitrile and cleaned up using alumina and C18 SPE cartridges. Liquid chromatography–tandem mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine muscles, according to the Commission Decision 2002/657/EC criteria. Applicability of the method in the analysis of swine, horse and chicken muscles was checked by precision and recovery experiment. The influence of matrix effect on the quantification of non-steroidal anti-inflammatory drugs residues was investigated. The method was used for the confirmation of phenylbutazone and oxyphenbutazone in horse muscle sample.
Keywords: Non-steroidal anti-inflammatory drugs; Residues; Liquid chromatography–tandem mass spectrometry; Matrix effect
Multi-residue determination of seventeen sulfonamides and five tetracyclines in fish tissue using a multi-stage LC–ESI–MS/MS approach based on advanced mass spectrometric techniques by Marilena E. Dasenaki; Nikolaos S. Thomaidis (pp. 93-102).
A strategy was newly developed to rapidly screen seventeen sulfonamides and five tetracyclines in a single run from fish tissues using ultra-high performance liquid chromatography (UHPLC) coupled with comprehensive mass spectrometric approaches, including precursor-ion scan and data dependent scan. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of the analytes. All sulfonamides share the same diagnostic product ion at m/ z 156 in positive MS/MS scan, while for tetracycline antibiotics the diagnostic product ion was proved to be at m/ z 153.8. Further characterization of each compound was performed using a data dependent scan. Separation was performed on a Zorbax Eclipse Plus C18 column with a gradient elution using acetonitrile – 0.1% formic acid mobile phase at a flow rate of 0.1mLmin−1. This approach has proven to be a powerful, highly selective, and sensitive tool for rapid screening and detection of non-targeted components in fish tissue and requires a minimum sample preparation such as one generic extraction step with MeOH:ACN 50:50 (v/v) acidified with 0.05% formic acid. The method has also been applied successfully to porcine and poultry meat. The validation of such a screening method was performed for the first time according to Commission Decision 2002/657/EC and satisfactory method performance characteristics were achieved.
Keywords: Veterinary drugs; Meat; Multi-class determination; Tandem mass spectrometry; Generic extraction
Multi-residue determination of seventeen sulfonamides and five tetracyclines in fish tissue using a multi-stage LC–ESI–MS/MS approach based on advanced mass spectrometric techniques by Marilena E. Dasenaki; Nikolaos S. Thomaidis (pp. 93-102).
A strategy was newly developed to rapidly screen seventeen sulfonamides and five tetracyclines in a single run from fish tissues using ultra-high performance liquid chromatography (UHPLC) coupled with comprehensive mass spectrometric approaches, including precursor-ion scan and data dependent scan. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of the analytes. All sulfonamides share the same diagnostic product ion at m/ z 156 in positive MS/MS scan, while for tetracycline antibiotics the diagnostic product ion was proved to be at m/ z 153.8. Further characterization of each compound was performed using a data dependent scan. Separation was performed on a Zorbax Eclipse Plus C18 column with a gradient elution using acetonitrile – 0.1% formic acid mobile phase at a flow rate of 0.1mLmin−1. This approach has proven to be a powerful, highly selective, and sensitive tool for rapid screening and detection of non-targeted components in fish tissue and requires a minimum sample preparation such as one generic extraction step with MeOH:ACN 50:50 (v/v) acidified with 0.05% formic acid. The method has also been applied successfully to porcine and poultry meat. The validation of such a screening method was performed for the first time according to Commission Decision 2002/657/EC and satisfactory method performance characteristics were achieved.
Keywords: Veterinary drugs; Meat; Multi-class determination; Tandem mass spectrometry; Generic extraction
Confirmatory method for the determination of streptomycin in apples by LC–MS/MS by Detlef A. Bohm; Carolin S. Stachel; P. Gowik (pp. 103-106).
The method was specifically developed for the determination and confirmation of streptomycin in apple samples using the whole mellow apple. The method is simple, rapid, sensitive and was validated for streptomycin in accordance with SANCO/3131/2007. After extraction with phosphate buffer and a pH change, the clean-up was performed by the way of SPE with polymeric phase. The LC–MS/MS analysis was carried out using a HILIC column for the separation of the analytes and a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of streptomycin a matrix calibration curve in the linear range of 1.0–20μgkg−1 and the internal standard dihydrostreptomycin (10μgkg−1) were used. The calculated validation parameters like the recovery (101–105%) for 2, 5, 10 and 20μgkg−1 and the relative standard deviation (RSD, 4.1–11.4%) of the 6 replicates fulfil the requirements of SANCO/3131/2007. The LOQ was determined as 2μgkg−1.
Keywords: Streptomycin; Apples; LC–MS/MS; Validation; SANCO/3131/2007; Limit of quantification
Confirmatory method for the determination of streptomycin in apples by LC–MS/MS by Detlef A. Bohm; Carolin S. Stachel; P. Gowik (pp. 103-106).
The method was specifically developed for the determination and confirmation of streptomycin in apple samples using the whole mellow apple. The method is simple, rapid, sensitive and was validated for streptomycin in accordance with SANCO/3131/2007. After extraction with phosphate buffer and a pH change, the clean-up was performed by the way of SPE with polymeric phase. The LC–MS/MS analysis was carried out using a HILIC column for the separation of the analytes and a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of streptomycin a matrix calibration curve in the linear range of 1.0–20μgkg−1 and the internal standard dihydrostreptomycin (10μgkg−1) were used. The calculated validation parameters like the recovery (101–105%) for 2, 5, 10 and 20μgkg−1 and the relative standard deviation (RSD, 4.1–11.4%) of the 6 replicates fulfil the requirements of SANCO/3131/2007. The LOQ was determined as 2μgkg−1.
Keywords: Streptomycin; Apples; LC–MS/MS; Validation; SANCO/3131/2007; Limit of quantification
Validation of an off line solid phase extraction liquid chromatography–tandem mass spectrometry method for the determination of systemic insecticide residues in honey and pollen samples collected in apiaries from NW Spain by María García-Chao; María Jesús Agruña; Gonzalo Flores Calvete; Vasilis Sakkas; María Llompart; Thierry Dagnac (pp. 107-113).
The use of pesticides to protect crops against plagues and insects is one of the most important ways to assure agricultural quality and productivity. However, bad application practices may cause the contamination of different environmental compartments and animal species, as a consequence of migration or accumulation of those compounds.Fipronil, imidacloprid and thiametoxam are systemic or systemic-like insecticides widely used in maize crops. Their heavy action in the nervous system of target insects also means a high toxicity to non-target pollinator insects such as honey bees which can get in touch with them through pollen and nectar during foraging activities. These insecticides have even been suspected to cause a significant decrease of honeybee colonies that has been observed in many countries since the past decade.Since September 1st 2008, the European Commission set new MRLs in food and feed of plant and animal origin. The pesticides included in this study have MRLs in honey and pollen between 10 and 50ngg−1.In the present work, an analytical method was developed with the aim of determining residues of fipronil and some of its metabolites (fipronil sulfone, fipronil sulfide, fipronil desulfinyl and fipronil carboxamide), thiamethoxam and imidacloprid in honey and pollen samples.The extraction optimization was performed using a Doehlert experimental design by studying two factors, the mixture and the ratio of solvents used. Prior to the extraction procedure, raw hive samples containing honey, pollen and wax were centrifuged at 4000rpm. The upper solid material was removed, and 1g of the lower phase was mixed with 3mL of the optimized mixture of methanol/water (10/90). The extract was passed through a florisil cartridge and the target compounds were eluted with methanol and analysed by LC–MS/MS in selective reaction monitoring (SRM) mode.The method was validated according to the guidelines included in the SANCO/10684/2009 document and the ISO 11843 standard for the following parameters: decision limit (CCα), detection capability (CCβ), recovery, repeatability and reproducibility at 0.5, 1 and 1.5 folds the MRLs. Ion suppression/enhancement effects into the ion source were also assessed.The CCβ values were included between 0.83 and 4.83ngg−1, well below the current MRLs.The validated method was applied to the determination of the target pesticides in 91 samples collected in colonies from 73 apiaries of NW Spain (two sampling campaigns during 2008). None of the target insecticides were detected among all the collected samples.
Keywords: Insecticide residues; Pollen; Honey; Fipronil; Liquid chromatography–tandem mass spectrometry; Method validation
Validation of an off line solid phase extraction liquid chromatography–tandem mass spectrometry method for the determination of systemic insecticide residues in honey and pollen samples collected in apiaries from NW Spain by María García-Chao; María Jesús Agruña; Gonzalo Flores Calvete; Vasilis Sakkas; María Llompart; Thierry Dagnac (pp. 107-113).
The use of pesticides to protect crops against plagues and insects is one of the most important ways to assure agricultural quality and productivity. However, bad application practices may cause the contamination of different environmental compartments and animal species, as a consequence of migration or accumulation of those compounds.Fipronil, imidacloprid and thiametoxam are systemic or systemic-like insecticides widely used in maize crops. Their heavy action in the nervous system of target insects also means a high toxicity to non-target pollinator insects such as honey bees which can get in touch with them through pollen and nectar during foraging activities. These insecticides have even been suspected to cause a significant decrease of honeybee colonies that has been observed in many countries since the past decade.Since September 1st 2008, the European Commission set new MRLs in food and feed of plant and animal origin. The pesticides included in this study have MRLs in honey and pollen between 10 and 50ngg−1.In the present work, an analytical method was developed with the aim of determining residues of fipronil and some of its metabolites (fipronil sulfone, fipronil sulfide, fipronil desulfinyl and fipronil carboxamide), thiamethoxam and imidacloprid in honey and pollen samples.The extraction optimization was performed using a Doehlert experimental design by studying two factors, the mixture and the ratio of solvents used. Prior to the extraction procedure, raw hive samples containing honey, pollen and wax were centrifuged at 4000rpm. The upper solid material was removed, and 1g of the lower phase was mixed with 3mL of the optimized mixture of methanol/water (10/90). The extract was passed through a florisil cartridge and the target compounds were eluted with methanol and analysed by LC–MS/MS in selective reaction monitoring (SRM) mode.The method was validated according to the guidelines included in the SANCO/10684/2009 document and the ISO 11843 standard for the following parameters: decision limit (CCα), detection capability (CCβ), recovery, repeatability and reproducibility at 0.5, 1 and 1.5 folds the MRLs. Ion suppression/enhancement effects into the ion source were also assessed.The CCβ values were included between 0.83 and 4.83ngg−1, well below the current MRLs.The validated method was applied to the determination of the target pesticides in 91 samples collected in colonies from 73 apiaries of NW Spain (two sampling campaigns during 2008). None of the target insecticides were detected among all the collected samples.
Keywords: Insecticide residues; Pollen; Honey; Fipronil; Liquid chromatography–tandem mass spectrometry; Method validation
Two-dimensional gas chromatographic profiling as a tool for a rapid screening of the changes in volatile composition occurring due to microoxygenation of red wines by Hans-Georg Schmarr; Jörg Bernhardt; Ulrich Fischer; Alexander Stephan; Patrick Müller; Dominik Durner (pp. 114-123).
Microoxygenation (MOX) is a widely applied technique to deliver continuously trace amounts of oxygen to red wine during vinification and ageing in order to improve color stability and sensory properties. Proven by sensory means, the added oxygen modifies not only tannin structure and color of the wines, but also their composition of volatiles. In this study different microoxygenation treatments prior and after malolactic fermentation were carried out for Pinot noir, Cabernet Sauvignon and Dornfelder wines of the 2007 vintage. Volatile components of subsequent wines were analyzed using headspace-solid phase microextraction coupled to comprehensive two-dimensional gas chromatography–quadrupole mass spectrometry (GC×GC–qMS). Quantitative data were retrieved from two-dimensional images obtained from GC×GC chromatograms of volatile compounds applying a software package, which is commonly used in the field of proteomics for two-dimensional electrophoresis gels. This approach revealed a discrimination of the applied treatments by multivariate statistics based on volatiles alone, such as the clear distinction among wines treated before or after malolactic fermentation in case of Cabernet Sauvignon and Dornfelder or the effect of different oxygen doses. Besides the differentiation of MOX treatments from the untreated control, specific varietal and technological effects could be distinguished. The image processing of the GC×GC data offered valuable tools which were able to identify those areas in the 2D images that were most responsible for discrimination among different MOX treatments. Based on the loadings of individual aroma compounds a set of markers for the MOX-induced modifications of volatiles could be suggested.
Keywords: Red wine; Microoxygenation; Aroma; Profiling analysis; Comprehensive two-dimensional gas chromatography
Two-dimensional gas chromatographic profiling as a tool for a rapid screening of the changes in volatile composition occurring due to microoxygenation of red wines by Hans-Georg Schmarr; Jörg Bernhardt; Ulrich Fischer; Alexander Stephan; Patrick Müller; Dominik Durner (pp. 114-123).
Microoxygenation (MOX) is a widely applied technique to deliver continuously trace amounts of oxygen to red wine during vinification and ageing in order to improve color stability and sensory properties. Proven by sensory means, the added oxygen modifies not only tannin structure and color of the wines, but also their composition of volatiles. In this study different microoxygenation treatments prior and after malolactic fermentation were carried out for Pinot noir, Cabernet Sauvignon and Dornfelder wines of the 2007 vintage. Volatile components of subsequent wines were analyzed using headspace-solid phase microextraction coupled to comprehensive two-dimensional gas chromatography–quadrupole mass spectrometry (GC×GC–qMS). Quantitative data were retrieved from two-dimensional images obtained from GC×GC chromatograms of volatile compounds applying a software package, which is commonly used in the field of proteomics for two-dimensional electrophoresis gels. This approach revealed a discrimination of the applied treatments by multivariate statistics based on volatiles alone, such as the clear distinction among wines treated before or after malolactic fermentation in case of Cabernet Sauvignon and Dornfelder or the effect of different oxygen doses. Besides the differentiation of MOX treatments from the untreated control, specific varietal and technological effects could be distinguished. The image processing of the GC×GC data offered valuable tools which were able to identify those areas in the 2D images that were most responsible for discrimination among different MOX treatments. Based on the loadings of individual aroma compounds a set of markers for the MOX-induced modifications of volatiles could be suggested.
Keywords: Red wine; Microoxygenation; Aroma; Profiling analysis; Comprehensive two-dimensional gas chromatography
Determination of benzene in different food matrices by distillation and isotope dilution HS-GC/MS by Raquel Medeiros Vinci; Michael Canfyn; Bruno De Meulenaer; Thibault de Schaetzen; Ilse Van Overmeire; Jacques De Beer; Joris Van Loco (pp. 124-129).
Benzene is classified by the IARC as carcinogenic to humans. Several sources may contribute for the occurrence of benzene in foods, such as, environmental contamination and the reaction of benzoate salts with ascorbic acid (naturally present or added as food additives). Matrix effect on benzene recovery (e.g. in fatty foods) and artefactual benzene formation from benzoate during analysis in the presence of ascorbate are some of the challenges presented when determining benzene in a wide range of foodstuffs. Design of experiment (DOE) was used to determine the most important variables in benzene recovery from headspace GC/MS. Based on the results of the DOE, a versatile method for the extraction of benzene from all kind of food commodities was developed. The method which consisted of distillation and isotope dilution HS-GC/MS was in-house validated. Artefactual benzene was prevented by addition of a borate buffer solution (pH 11) under distillation conditions. The method presented in this study allows the use of a matrix-independent calibration with detection limits below the legal limit established by the European Council for benzene in drinking water (1μgL−1).
Keywords: Benzene; Food matrices; Headspace; GC/MS
Determination of benzene in different food matrices by distillation and isotope dilution HS-GC/MS by Raquel Medeiros Vinci; Michael Canfyn; Bruno De Meulenaer; Thibault de Schaetzen; Ilse Van Overmeire; Jacques De Beer; Joris Van Loco (pp. 124-129).
Benzene is classified by the IARC as carcinogenic to humans. Several sources may contribute for the occurrence of benzene in foods, such as, environmental contamination and the reaction of benzoate salts with ascorbic acid (naturally present or added as food additives). Matrix effect on benzene recovery (e.g. in fatty foods) and artefactual benzene formation from benzoate during analysis in the presence of ascorbate are some of the challenges presented when determining benzene in a wide range of foodstuffs. Design of experiment (DOE) was used to determine the most important variables in benzene recovery from headspace GC/MS. Based on the results of the DOE, a versatile method for the extraction of benzene from all kind of food commodities was developed. The method which consisted of distillation and isotope dilution HS-GC/MS was in-house validated. Artefactual benzene was prevented by addition of a borate buffer solution (pH 11) under distillation conditions. The method presented in this study allows the use of a matrix-independent calibration with detection limits below the legal limit established by the European Council for benzene in drinking water (1μgL−1).
Keywords: Benzene; Food matrices; Headspace; GC/MS
Development of a stir bar sorptive extraction method coupled to gas chromatography-mass spectrometry for the analysis of volatile compounds in Sherry brandy by Raúl Delgado; Enrique Durán; Remedios Castro; Ramón Natera; Carmelo G. Barroso (pp. 130-136).
Sherry brandy (Jerez, SW, Spain) is a high quality distilled beverage derived from wine. Its perceived quality depends, inter alia, on hundreds of flavour compounds. A Stir Bar Sorptive Extraction (SBSE) method coupled to gas chromatography-mass spectrometry has been developed for the analysis of volatile compounds in Sherry brandy. The optimization of the extraction procedure has been carried out using a statistical approach, based on a factorial design. The best overall analytical conditions obtained were the following: 35mL of sample, diluted 1:1 with Milli-Q water and extraction at 1100rpm for 100min. The method has been successfully validated in a further stage of this work. Several performance characteristics such as calibration, linearity, precision (inter- and intra-assay), detection and quantification limits and recovery were studied. Finally, the method developed has been applied to different Sherry brandies.The results obtained show SBSE to be a suitable technique for the reliable analysis of volatile compounds in brandies.
Keywords: Stir bar sorptive extraction; Brandy; Gas chromatography-mass spectrometry; Flavour; Optimization
Development of a stir bar sorptive extraction method coupled to gas chromatography-mass spectrometry for the analysis of volatile compounds in Sherry brandy by Raúl Delgado; Enrique Durán; Remedios Castro; Ramón Natera; Carmelo G. Barroso (pp. 130-136).
Sherry brandy (Jerez, SW, Spain) is a high quality distilled beverage derived from wine. Its perceived quality depends, inter alia, on hundreds of flavour compounds. A Stir Bar Sorptive Extraction (SBSE) method coupled to gas chromatography-mass spectrometry has been developed for the analysis of volatile compounds in Sherry brandy. The optimization of the extraction procedure has been carried out using a statistical approach, based on a factorial design. The best overall analytical conditions obtained were the following: 35mL of sample, diluted 1:1 with Milli-Q water and extraction at 1100rpm for 100min. The method has been successfully validated in a further stage of this work. Several performance characteristics such as calibration, linearity, precision (inter- and intra-assay), detection and quantification limits and recovery were studied. Finally, the method developed has been applied to different Sherry brandies.The results obtained show SBSE to be a suitable technique for the reliable analysis of volatile compounds in brandies.
Keywords: Stir bar sorptive extraction; Brandy; Gas chromatography-mass spectrometry; Flavour; Optimization
Single-run determination of polybrominated diphenyl ethers (PBDEs) di- to deca-brominated in fish meal, fish oil and fish feed by isotope dilution: Application of automated sample purification and gas chromatography/ion trap tandem mass spectrometry (GC/ITMS) by Sonia Lucía Blanco; Juan M. Vieites (pp. 137-146).
The present paper describes the application of automated cleanup and fractionation procedures of the Power Prep system (Fluid Management Systems) for the determination of polybrominated diphenyl ethers (PBDEs) in feeding stuffs and fish meal and oil. Gas chromatography (GC) separation followed by ion trap tandem mass spectrometry detection in EI mode (ITMS) allowed the analysis of di- to deca-BDEs in the samples matrices used in fish aquaculture. The method developed enabled the determination of 26 native PBDE congeners and 1113C12-labelled congeners, including deca-BDE 209, in a single-run analysis, using isotope dilution. The automated cleanup, consisting of a succession of multilayer silica and basic alumina columns previously applied by Wyrzykowska et al. (2009) in combustion flue gas, was succesfully applied in our complex matrices. The method allowed an increase in productivity, i.e. lower time was required to process samples, and simultaneous purification of several samples was achieved at a time, reducing analyst dedication and human error input. Average recoveries of 43–96% were obtained. GC/ITMS can overcome the complexity originating from the sample matrix, eliminating matrix effects by tandem MS, to enable the detection of congeners penta- to nona-BDEs where interferent masses were present. The provisional detection limits, estimated in the samples, were 5–30pg for di-, tri-, tetra-, and penta-BDEs, 20–65pg for hexa-, hepta-, octa- and nona-BDEs, and 105pg for deca-BDE. Reduction of deca-BDE 209 blank values is of concern to ongoing research. Good accuracy was obtained by application of the whole procedure, representing an efficient, low-cost and fast alternative for routine analyses.
Keywords: Polybromodiphenyl ethers; Deca-BDE 209; Ion trap tandem mass spectrometry; Fish meal; Fish feed; Fish oil
Single-run determination of polybrominated diphenyl ethers (PBDEs) di- to deca-brominated in fish meal, fish oil and fish feed by isotope dilution: Application of automated sample purification and gas chromatography/ion trap tandem mass spectrometry (GC/ITMS) by Sonia Lucía Blanco; Juan M. Vieites (pp. 137-146).
The present paper describes the application of automated cleanup and fractionation procedures of the Power Prep system (Fluid Management Systems) for the determination of polybrominated diphenyl ethers (PBDEs) in feeding stuffs and fish meal and oil. Gas chromatography (GC) separation followed by ion trap tandem mass spectrometry detection in EI mode (ITMS) allowed the analysis of di- to deca-BDEs in the samples matrices used in fish aquaculture. The method developed enabled the determination of 26 native PBDE congeners and 1113C12-labelled congeners, including deca-BDE 209, in a single-run analysis, using isotope dilution. The automated cleanup, consisting of a succession of multilayer silica and basic alumina columns previously applied by Wyrzykowska et al. (2009) in combustion flue gas, was succesfully applied in our complex matrices. The method allowed an increase in productivity, i.e. lower time was required to process samples, and simultaneous purification of several samples was achieved at a time, reducing analyst dedication and human error input. Average recoveries of 43–96% were obtained. GC/ITMS can overcome the complexity originating from the sample matrix, eliminating matrix effects by tandem MS, to enable the detection of congeners penta- to nona-BDEs where interferent masses were present. The provisional detection limits, estimated in the samples, were 5–30pg for di-, tri-, tetra-, and penta-BDEs, 20–65pg for hexa-, hepta-, octa- and nona-BDEs, and 105pg for deca-BDE. Reduction of deca-BDE 209 blank values is of concern to ongoing research. Good accuracy was obtained by application of the whole procedure, representing an efficient, low-cost and fast alternative for routine analyses.
Keywords: Polybromodiphenyl ethers; Deca-BDE 209; Ion trap tandem mass spectrometry; Fish meal; Fish feed; Fish oil