Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method is described for simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T2 and HT2-toxin in cereals. One-step extraction using solvent mixtures of acetonitrile:water:acetic acid (79:20:1) without any clean-up was employed for extraction of these mycotoxins from cereals. The mean recoveries of mycotoxins in spiked cereals ranged from 76.8% to 108.4%. Limits of detection (LOD) and quantification (LOQ) ranged 0.01–20 and 0.02–40 ng/g, respectively. The developed method has been applied for the determination of mycotoxins in 100 cereal samples collected from Malaysian markets. A total of 77 cereal samples (77%) contaminated with at least one of these mycotoxins. Occurrence of mycotoxins in commercial cereal samples were 70%, 40%, 25%, 36%, 19%, 13%, 16, and 16% for aflatoxins, OTA, ZEA, DON, FB₁, FB₂, T2 and HT2-toxin, respectively. The results demonstrated that the procedure was suitable for the determination of mycotoxins in cereals and could be implemented for the routine analysis.     

Dietary intake of ochratoxin A from conventional and organic bread

Ochratoxin A (OTA) was extracted from 100 bread samples by using accelerated solvent extraction (ASE) and analyzed with liquid chromatography coupled with fluorescence detection. The presence of OTA was confirmed by methyl-ester derivatization. Bread samples were bought from different bakeries and supermarkets, 74 of non-organic and 26 of organic bread. The incidence of OTA varied between 20.3% and 23.0% for non-organic and organic bread, respectively. The highest values were obtained with non-organic versus organic products, five samples exceeded the European maximum permitted limit of OTA (3 ng/g) for this product. Estimated daily intake of OTA in this study was 1.6 ng/kg b.w./day. This value represents 32% and 10% of the tolerable daily intake (TDI) according to the Scientific Committee on Food of the European Commission and the FAO/WHO Committee of Experts on Food Additives, respectively. The daily intake estimated from this study reflects the necessity to take a vigilant attitude to guarantee food safety.     

Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin A and zearalenone in cereals using HPLC-FLD

Method validation for quantitative analysis of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in cereals using HPLC with fluorescence detector (FLD) is described. Mycotoxins were extracted with methanol?:?water (80?:?20) and purified with a multifunctional AOZ immunoaffinity column before HPLC analysis. The validation of the analytical method was performed to establish the following parameters: specificity, selectivity, linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision (within- and between-day variability), stability, robustness, measurement of performance, and measurement of uncertainty. Calibration curves were linear (r?>?0.999) over the concentration range, from the LOQ to 26, 40 and 400?ng/g for AFs, OTA and ZEA, respectively. LOD and LOQ were 0.0125 and 0.05?ng/g for aflatoxin B1 (AFB1) and G1 (AFG1), 0.0037 and 0.015?ng/g for aflatoxin B2 (AFB2) and G2 (AFG2), as well as 0.05 and 0.2?ng/g for OTA and 0.5 and 2?ng/g for ZEA, respectively. The mean recovery values were 77–104% for different concentrations of AFs, OTA and ZEA in spiked cereal samples. Both intra- and inter-day accuracy and precision were within acceptable limits. This method was successfully applied for the simultaneous determination of mycotoxins for 60 cereal samples collected from Malaysian markets. Fifty per cent of the cereal samples were contaminated with at least one of these mycotoxins, at a level greater than the LOD. Only one wheat sample and two rice samples were contaminated with levels greater than the European Union regulatory limits for AFs and OTA (4 and 5?ng/g). The means and ranges of mycotoxins obtained for the cereal samples were 0.4?ng/g and 0.01–5.9?ng/g for total AFs; 0.18?ng/g and 0.03–5.3?ng/g for OTA; and 2.8?ng/g and 2.4–73.1?ng/g for ZEA, respectively. The results indicate that the method is suitable for the simultaneous determination of AFs, OTA and ZEA in cereals and is suitable for routine analysis.     

Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin A and zearalenone in cereals using HPLC-FLD

Method validation for quantitative analysis of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in cereals using HPLC with fluorescence detector (FLD) is described. Mycotoxins were extracted with methanol : water (80 : 20) and purified with a multifunctional AOZ immunoaffinity column before HPLC analysis. The validation of the analytical method was performed to establish the following parameters: specificity, selectivity, linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision (within- and between-day variability), stability, robustness, measurement of performance, and measurement of uncertainty. Calibration curves were linear (r > 0.999) over the concentration range, from the LOQ to 26, 40 and 400 ng/g for AFs, OTA and ZEA, respectively. LOD and LOQ were 0.0125 and 0.05 ng/g for aflatoxin B1 (AFB1) and G1 (AFG1), 0.0037 and 0.015 ng/g for aflatoxin B2 (AFB2) and G2 (AFG2), as well as 0.05 and 0.2 ng/g for OTA and 0.5 and 2 ng/g for ZEA, respectively. The mean recovery values were 77-104% for different concentrations of AFs, OTA and ZEA in spiked cereal samples. Both intra- and inter-day accuracy and precision were within acceptable limits. This method was successfully applied for the simultaneous determination of mycotoxins for 60 cereal samples collected from Malaysian markets. Fifty per cent of the cereal samples were contaminated with at least one of these mycotoxins, at a level greater than the LOD. Only one wheat sample and two rice samples were contaminated with levels greater than the European Union regulatory limits for AFs and OTA (4 and 5 ng/g). The means and ranges of mycotoxins obtained for the cereal samples were 0.4 ng/g and 0.01-5.9 ng/g for total AFs; 0.18 ng/g and 0.03-5.3 ng/g for OTA; and 2.8 ng/g and 2.4-73.1 ng/g for ZEA, respectively. The results indicate that the method is suitable for the simultaneous determination of AFs, OTA and ZEA in cereals and is suitable for routine analysis.     

Levels of ochratoxin A in wheat and maize bread from the central zone of Portugal

Ochratoxigenic fungi are natural contaminants of cereal and the produced toxins are harmful to humans and animals. Ochratoxin A (OTA) is among the most important mycotoxins, and the International Agency for Research on Cancer (IARC) classifies it as possibly carcinogenic to humans (group 2B). A total of 61 samples of bread from the central zone of Portugal were analysed for OTA by liquid chromatography (LC) with fluorescence detection (FD). For confirmation two procedures were applied, methyl ester derivatization with boron trifluoride-methanol and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). As far as we know, this is the first report where on-line LC/electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was used for OTA analysis in bread. Limits of detection (LOD) and quantification (LOQ) were 0.015 and 0.03 ng/g, using LC-FD, and 0.03 and 0.09 ng/g by LC-MS/MS. The incidence of OTA was 12.9% and 70.0% for wheat and maize bread, respectively. The highest OTA levels were obtained for maize bread, having one sample exceeded the European maximum limit established for OTA in cereal products. The estimate daily intake (EDI) was below the tolerable daily intake.     

Determination of ochratoxin A in wheat after clean-up through a DNA aptamer-based solid phase extraction column

A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling gel and used as sorbent for the preparation of solid phase extraction (SPE) columns. The SPE columns packed with 300 μl oligosorbent (24 nmol DNA) showed a linear (r = 0.999) behaviour in the range of 0.4–500 ng OTA. After optimisation of the extraction step, SPE columns were used for clean-up of OTA from wheat prior to liquid chromatographic (HPLC) analysis with fluorescence detection (FLD). Average recoveries from wheat samples spiked at levels of 0.5–50 ng/g ranged from 74% to 88% (relative standard deviation <6%) with limits of detection and of quantification of 23 and 77 pg/g, respectively. The comparative HPLC/FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and immunoaffinity (IMA) columns showed a good correlation (r = 0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance.     

Determination of Ochratoxin A in Bread: Evaluation of Microwave-Assisted Extraction Using an Orthogonal Composite Design Coupled with Response Surface Methodology

An analytical method using microwave-assisted extraction (MAE) and liquid chromatography (LC) with fluorescence detection (FD) for the determination of ochratoxin A (OTA) in bread samples is described. A 2⁴ orthogonal composite design coupled with response surface methodology was used to study the influence of MAE parameters (extraction time, temperature, solvent volume, and stirring speed) in order to maximize OTA recovery. The optimized MAE conditions were the following: 25 mL of acetonitrile, 10 min of extraction, at 80 °C, and maximum stirring speed. Validation of the overall methodology was performed by spiking assays at five levels (0.1–3.00 ng/g). The quantification limit was 0.005 ng/g. The established method was then applied to 64 bread samples (wheat, maize, and wheat/maize bread) collected in Oporto region (Northern Portugal). OTA was detected in 84 % of the samples with a maximum value of 2.87 ng/g below the European maximum limit established for OTA in cereal products of 3 ng/g.     

The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D₃) and ergocalciferol (D₂). The main source of vitamin D₃ is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D₃, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MSⁿ) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D₃ in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MSⁿ and LC–MS/MS, respectively. Recoveries of vitamin D₃ added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D₃ was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).     

Aflatoxins contamination in spices and processed spice products commercialized in Korea

A survey for total aflatoxins (aflatoxins B₁, B₂, G₁, and G₂) was conducted on 88 spices and processed spice products commercialized in Korea. The presence of aflatoxins was determined by high-performance liquid chromatography (HPLC) with fluorescence detector using immunoaffinity column clean-up. Total aflatoxins (AFs) are detected in 12 samples (13.6% of incidence) including seven red pepper powder, two red pepper pastes (Kochujang), two curry and one ginger product. The contamination levels are 0.08–4.45 μg/kg as aflatoxin B₁ and 0.08–4.66 μg/kg as AFs. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis on contaminated samples was conducted for the confirmation of detected aflatoxins. The 12 samples which showed aflatoxins by HPLC/FLD were confirmed as aflatoxins by LC–MS/MS.     

Occurrence and daily intake of ochratoxin A of organic and non-organic rice and rice products

Ochratoxin A (OTA) was extracted from 84 rice samples and rice products by using accelerated solvent extraction (ASE) and analysed with liquid chromatography coupled with fluorescence detection. Samples were collected from rice cultivars, local markets and supermarkets; 64 were of non-organic and 20 of organic production. 7.8% of non-organic samples had OTA levels from 4.3 to 27.3 microg/kg and in 30% of organic samples was detected the presence of this mycotoxin varying from 1.0 to 7.1 microg/kg. OTA presence was confirmed by methyl-ester derivatization. Rice and rice products labelled with denomination of origin (DO) were not detected OTA due to the fact that its production has implemented food safety measures such as good agricultural practices (GAPs), good manufacturing practices (GMPs), and the hazard analysis and critical control point (HACCP) system. Estimated daily intake of OTA was 0.17 ng/kg b.w./day. This value reflects that the analysed samples have a minimal contribution to toxicological risk.     

Study of ochratoxin A content in South Moravian and foreign wines by the UPLC method with fluorescence detection

In 2009–2010, 72 wine samples of Moravian and foreign origin were analysed for ochratoxin A contamination. A fast analytical method based on immunoaffinity column clean-up and followed by the ultra performance liquid chromatography coupled to fluorescence detection was used. LOD and LOQ values were 0.3 and 1.0 ng/L. Ochratoxin A was detected in 11% of Moravian wines and the detected OTA level ranged from 1.2 to 71.2 ng/L. In foreign wines, OTA level ranged from 1.6 to 227.0 ng/L. The values of OTA in all studied samples were significantly below the maximum allowable limit, 2.0 μg/kg, set by the European Union.     

Determination of aflatoxin levels in nuts and their products consumed in South Korea

A total of 85 nuts and their products marketed in South Korea were assessed for aflatoxins using a monitoring scheme consisting of enzyme-linked immunosorbent assay (ELISA) for rapid screening, high performance liquid chromatography (HPLC) for quantification and LC–mass spectrometry (MS) for confirmation. Thirty-one out of 85 samples gave ELISA readings above 0.06 and were screened as possible positive samples. Aflatoxin contents of possible positive samples were determined using HPLC with a detection limit of 0.08–1.25 μg/kg and a quantification limit of 0.15–2.50 μg/kg. Nine samples including 1 raw peanut, 4 roasted peanuts, 2 peanut butters, 1 pistachio and 1 seasoned assorted nut were contaminated with aflatoxins (10.6% of incidence), ranging in various levels up to 28.2 μg/kg. LC–MS analysis on contaminated samples revealed that peaks eluting at 4.4, 5.2, 9.1 and 11.9 min were confirmed as aflatoxin G₁, aflatoxin B₁, aflatoxin G₂ and aflatoxin B₂, respectively.     

Survey: Ochratoxin A in European special wines

The occurrence of Ochratoxin A (OTA) was examined in 121 special wines made using different winemaking techniques and from many European origins. The wine groups with the highest OTA content and occurrence, above 90%, were those were the must was fortified before fermentation (mean: 4.48 μg/l) and those made from grapes dried by means of sun exposure (mean: 2.77 μg/l). Fortified wines with long aging in wooden casks were about 50% contaminated, with OTA levels below 1.00 μg/l. Wines affected by noble rot, late harvest wines and ice wines did not contain OTA. Overall, 19.8% of the wines studied contained OTA levels above the maximum permissible limit for the European Union (2 μg/kg) in wine (excluding liqueur wines).     

Identification and quantitative determination of glucosinolates in seeds and edible parts of Korean Chinese cabbage

Glucosinolates (GSLs) have attracted major interest due to the chemopreventive properties of some of their transformation products. GSLs in the seeds and edible parts of Korean Chinese cabbage (Brassicacampestris L. ssp. pekinensis) were identified and quantified by LC–ESI–MS and LC–UV. As a result, nine GSLs were identified: progoitrin, glucoraphanin, glucoalyssin, gluconapin, 4-hydroxy-3-indolylmethyl, glucobrassicanapin, glucoerucin, glucobrassicin, and 4-methoxyglucobrassicin. The total GSL levels were 268–198 and 23.0–15.8 μmol/g dry weight (DW) for seeds and edible parts, respectively. Gluconapin (197 μmol/g DW) was the highest individual GSL in seeds, whereas 4-methoxyglucobrassicin (6.08–4.94 μmol/g DW) and glucobrassicanapin (8.18–3.09 μmol/g DW) were found in the edible parts. In addition, LC–MS profiles of the nine GSLs identified from Korean Chinese cabbage were subjected to principal components analysis (PCA) to evaluate differences among samples. The metabolome among the four cultivar seed or edible parts was clearly separated by PCA.     

7-Ketocholesterol and 7-hydroxycholesterol in pork meat and its gravy thermally treated without additives and in the presence of onion and garlic

The effect of onion and garlic on the formation of two cholesterol oxidation products (COPs): 7-ketocholesterol and 7-hydroxycholesterol was evaluated by comparing their concentrations in meat and gravy samples obtained from three pork dishes prepared in the presence and absence of these flavourings. The concentration of these compounds in meat samples was between 82.4 and 1331.6 ng/g of cooked meat. Gravies contained lower amounts: from 18.3 to 45.6 ng/g of cooked meat. The addition of onion (30 g/100 g of meat) caused a decrease in 7-ketocholesterol and 7-hydroxycholesterol concentrations in all of the investigated pork dishes by 9.5–79%, whilst the addition of 15 g of garlic to 100 g of meat lowered the concentration by 17 to 88%. The greatest decrease was found in grilled minced chops. The quantitative assessment of 7-ketocholesterol and 7-hydroxycholesterol was carried out by thin-layer chromatography with densitometric detection.     

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.     

Residual pattern of fenhexamid on pepper fruits grown under greenhouse conditions using HPLC and confirmation via tandem mass spectrometry

Fenhexamid (25%, SC) was sprayed on pepper fruits grown under greenhouse conditions at the recommended dose rate of 20 g/20 L water. Fruit samples were collected randomly at 0 (2 h after application), 1, 2, 4, 6, 8, 11, and 14 days post-application. The samples were extracted with acetonitrile, partitioned with water, passed through a cleanup procedure, and analysed via HPLC. Residues were confirmed via LC–tandem mass spectrometry (LC–MS/MS) in positive-ion electrospray ionisation (ESI+) mode. The rate of disappearance of fenhexamid on pepper fruits was described as first-order kinetics (r² = 0.992) with a half-life of 4.7-day. Based on the pattern of decline of the fungicide residues in relation to the estimated maximum residue limits (MRL = 5 mg/kg), a safety pre-harvest interval of 1 day is suggested for peppers at the recommended dosage.     

A survey of ochratoxin A contamination in feeds and sera from organic and standard swine farms in northwest Italy

BACKGROUND: A survey was carried out on conventional (n = 11) and organic (n = 4) swine farms in northwest Italy in order to investigate the occurrence of ochratoxin A (OTA) in feed and serum samples collected from September 2006 to March 2009. Each farm was sampled twice and a total of 30 feed samples and 285 serum samples were collected. OTA levels were determined through extraction, immunoaffinity column purification and high‐performance liquid chromatography analysis coupled with fluorimetric detection. RESULTS: All feed samples resulted to be contaminated with OTA at levels ranging from 0.22 to 38.4 µg kg^?1. The OTA concentrations found in organic feed samples were significantly higher (P < 0.05) than those found in conventional feed samples. All serum samples resulted to be contaminated with OTA at levels ranging from 0.03 to 6.24 ng mL^?1. The OTA concentrations found in organic serum samples were significantly higher (P < 0.001) than those found in conventional serum samples. CONCLUSION: None of the feed samples contained more than the maximum level (50 µg OTA kg^?1, considering a feed moisture content of 120 g kg^?1) recommended by the European Commission for OTA in complementary and complete swine feedstuffs. The OTA contamination of organic feed and serum samples was found to be significantly higher than that of conventional feed and serum samples. Copyright © 2010 Society of Chemical Industry     

Ochratoxin A in raw materials and cooked meat products made from OTA-treated pigs

The aim of this study was to determine ochratoxin A (OTA) concentrations in the raw materials and cooked meat products made from pigs sub-chronically exposed to OTA. The treated animal group (n = 5) was administered with 300 μg OTA/kg of feed for 30 days, whereas the control group (n = 5) was left untreated. OTA concentrations were quantified using immunoassay (ELISA) and high performance liquid chromatography with fluorescence detection (HPLC-FD). OTA concentration was the highest in the kidney, followed by the lungs, liver, blood, spleen, heart, and adipose tissue. As for the final meat products, the highest average OTA concentration was detected in black pudding sausages (14.02 ± 2.75 μg/kg), then in liver sausages (13.77 ± 3.92 μg/kg), while the lowest was found in pâté (9.33 ± 2.66 μg/kg). The results pointed out that a sub-chronic pig exposure leads to the accumulation of OTA in raw materials and consequently in meat products, whose level of contamination is directly dependent on OTA contents in raw materials used for their production.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle,liver and egg

A modified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed using a highly sensitive and specific monoclonal antibody (McAb) to determine doxycycline (DC) residues in chicken tissues and egg. The McAb against DC was produced by hybridoma technique and a modified ic-ELISA was characterised in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed at concentrations ranged from 0.01 to 100 ng/ml. The IC₅₀ value was 1.32 ± 0.18 ng/ml. The limit of detection was 0.14 ± 0.02 ng/g. The recoveries of DC from spiked chicken liver, muscle, and egg at levels of 50–600 ng/g were 84.6–85.5%, 88.2–89.1%, and 84.4–89.3%, respectively. The coefficient variations (CVs) were 5.1–9.3%, 3.7–11.3%, and 4.7–9.8%, respectively. Linear regression analysis showed good correlation, with r² values 0.9909 for chicken liver and 0.9916 for chicken muscle.