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.     

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.     

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.     

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).     

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.     

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.     

Quantitative analysis of acrylamide in tea by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An effective sample preparation procedure was optimized and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantitative analysis of acrylamide in tea. [¹³C₃]-acrylamide was used as internal standard. Acrylamide was extracted at 25 °C for 20 min by 10 ml water followed by 10 ml acetonitrile, and then 4 g of magnesium sulfate and 0.5 g of sodium chloride were added to the above mixture under stirring thoroughly. In order to increase the response of acrylamide, 9 ml acetonitrile layer was taken and concentrated to 0.5 ml. Solid-phase extraction with an Oasis MCX cartridge was carried out for clean-up. The limit of detection (LOD) and limit of quantification (LOQ) were 1 and 5 ng/ml, respectively. The recovery efficiency of the extraction procedure ranged between 74% and 79%. The levels of acrylamide in 30 tea samples were less than 100 ng/g. Black, oolong, white and yellow tea samples had quite low acrylamide contents (<20 ng/g). Higher acrylamide levels occurred in baked, roasted, and one sun-dried green tea samples (46–94 ng/g).     

Occurrence of ochratoxin A in raw ham muscle,salami and dry-cured ham from pigs fed with contaminated diet

Pork meat-derived products can contribute to the overall ochratoxin A intake, either by carry-over effect, or by environmental mould population cross-contamination. In order to assess the role of these different contamination routes, a study was carried out with pigs challenged orally with OTA contaminated feed at subchronical level. After slaughtering, thighs and minced meat from control and treated groups were transformed into dry-cured hams and salami, respectively, which were analysed for OTA determination after ripening. From collected data, the carry-over in muscle was generally low, whereas a significant contribution to the OTA contamination in dry-cured hams was due to toxinogenic mould population growing on their surface during ripening. Finally, a survey of different types of dry-cured ham (n = 110), from the Italian market, was performed, showing the occurrence of OTA on the surface portion in 84 out of 110 samples with a median value of 0.53 μg/kg and in the inner core in 32 out of 110 samples with a median value lower than 0.1 μg/kg.     

Effect of microwave pre-thawing of frozen potato strips on acrylamide level and quality of French fries

The objective of this study was to reduce frying time, and hence acrylamide level of French fries by microwave pre-thawing of frozen potato strips. Effect of this pre-treatment on acrylamide content and quality attributes of French fries was determined. Frozen par-fried potato strips (8.5 × 8.5 × 70 mm) were thawed in a microwave oven prior to final frying in sunflower oil at 170, 180, and 190 °C. Potato strips that were final fried without pre-thawing were considered as the control. Acrylamide analysis was performed by liquid chromatography–mass spectrometry (LC–MS) method. Microwave pre-thawing of frozen strips reduced the acrylamide level of French fries by 10% (from 17.7 to 15.9 ng/g), 89% (from 72.1 to 8.0 ng/g), and 64% (from 50.5 to 18.4 ng/g) for frying at 170, 180, and 190 °C, respectively, in comparison to the control samples. Quality attributes (texture, color, and oil content) of pre-treated strips were found to be comparable to those of the control.     

赭曲霉毒素A单克隆抗体免疫亲和柱的制备

制备了赭曲霉毒素A(OTA)单克隆抗体免疫亲和柱,并用间接竞争ELISA和HPLC法评价了免疫亲和柱的性能,其柱容量(结合OTA的能力)约为200ng,加标回收率为90.38%～100.1%,可反复使用3次。 建立了免疫亲和柱-HPLC联用分析谷物中OTA的方法,最低检出限为0.2μg/kg,线性范围为0.6～400μg/kg,OTA加标量为1～10μg/kg时谷物样品中的回收率为78.7%～87.1%,变异系数小于6.5%。用此法检测了大米、小麦、玉米和玉米饲料等15份市售样品,检出率为46.7%,其中OTA的最高含量为0.785μg/kg。     

Levels of creatine,organic contaminants and heavy metals in creatine dietary supplements

High performance liquid chromatography (HPLC) has been optimised for the analysis of the creatine content and possible organic contaminants in 33 samples of creatine supplements from the market. Creatinine resulted to be the major organic contaminant (44% of the samples over 100 mg/kg). About 15% of the samples had dihydro-1,3,5-triazine concentrations exceeding the detection limit of 4.5 mg/kg (maximum 8.0 mg/kg) and a dicyandiamide concentration over 50 mg/kg, while none of the samples were contaminated with thiourea. The heavy metals (arsenic, cadmium, mercury and lead) content was also assessed by means of inductively coupled plasma mass spectrometry (ICP-MS). Only mercury was present in detectable amounts (at levels lower than 1 mg/kg).     

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%).