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.     

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97?a_w . Mancozeb at 40?mg?l^?1 inhibited fungal growth and provided lag phases >24 days at 10–20?mg?l^?1 and 15°C. OTA was observed only at 25°C and doses <10?mg?l^?1. At 15°C, copper oxychloride proved inhibitory at 800?mg?l^?1, while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5–8?g?l^?1 (at 15°C) while at 25°C growth took place even at 8?g?l^?1, although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb?>?copper oxychloride?>?sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.     

Validation of a high-performance liquid chromatography analytical method for ochratoxin A quantification in cocoa beans

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the quantitative analysis of ochratoxin A (OTA) in cocoa beans is described. OTA was extracted with methanol-3% sodium hydrogen carbonate solution and then purified with immunoaffinity columns before its analysis by HPLC. The validation of the analytical method was based on the following criteria: selectivity, linearity, limit of detection and quantification, precision (within- and between-day variability) and recovery, robustness and uncertainty. Detection and quantification limits were 0.04 and 0.1 μg kg^-1, respectively. Recovery was 88.9% (relative standard deviation = 4.0%). This method was successfully applied to the measurement of 46 cocoa bean samples of different origins. A total of 63% of cocoa bean samples was contaminated with a level greater than the limit of detection. The means and medians obtained for cocoa bean were 1.71 and 1.12 μg kg^-1, respectively. Surveillance controls should be set up in both crops and factories involved in transformation processes to avoid this mycotoxin in final products.     

Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK

Three hundred and twenty samples from the 1999 UK harvest comprising wheat (201 samples), barley (106) and oats (13) were analysed for ochratoxin A and deoxynivalenol. A small number of organic samples was also obtained. Samples were collected from farms, central stores, mills, maltings and ports from across the UK from February to April 2000. Ochratoxin A and deoxynivalenol analysis was by affinity column clean up and high-performance liquid chromatography with fluorescence and ultraviolet light detection, respectively, with limits of detection of 0.2 and 20 μg kg^-1. The survey found ochratoxin A at below 5 μg kg^-1 in 97% of the samples indicating satisfactory storage conditions. The remaining 3% of the samples contained ochratoxin A at levels between 5.2 and 231 μg kg^-1, but none of these samples was intended for human consumption. Deoxynivalenol was detected in 88% of all samples, with 83% below 100 μg kg^-1; the maximum level was 600 μg kg^-1. Twenty samples containing deoxynivalenol at or above 150 μg kg^-1 by high-performance liquid chromatography were all confirmed by gas chromatography/mass spectrometry. Nivalenol was also detected by gas chromatography/mass spectrometry at levels of 50 μg kg^-1 or higher in 18 of 20 samples where deoxynivalenol was confirmed.     

Simultaneous detection of 12 mycotoxins in cereals using RP-HPLC-PDA-FLD with PHRED and a post-column derivatization system

A new method for the simultaneous quantification of 12 mycotoxins was developed and optimized using reverse phase high performance liquid chromatography (RP-HPLC) with a photodiode array (PDA) and fluorescence detector (FLD), a photochemical reactor for enhanced detection (PHRED) and post-column derivatization. The mycotoxins included aflatoxins (AFB₁, AFB₂, AFG₁, and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁, FB₂, and FB₃), T-2 and HT-2 toxins. A double sample extraction with a phosphate-buffered saline solution (PBS) and methanol was used for co-extraction of mycotoxins, and a multifunctional immunoaffinity column was used for cleanup. Optimum conditions for separation of the mycotoxins were obtained to separate 12 mycotoxins in FLD and PDA chromatograms with a high resolution. The method gave recoveries in the range 72–111% when applied to spiked corn samples. The limits of detection (LOD) were 0.025?ng/g for AFB₁ and AFG₁, 0.012?ng/g for AFB₂ and AFG₂, 0.2?ng/g for OTA, 1.5?ng/g for ZEA, 6.2?ng/g for FB₁, FB₃ and HT-2 toxin, 9.4?ng/g for FB₂ and T-2 toxin, and 18.7?ng/g for DON. In addition, the limits of quantification (LOQ) ranged from 0.04?ng/g for AFB₂ and AFG₂ to 62?ng/g for DON. The method was successfully applied to the determination of these mycotoxins in 45 cereal samples obtained from the Malaysian market. The results indicated that the method can be applied for the multi-mycotoxin determination of cereals.     

Aflatoxins and ochratoxin A in tea prepared from naturally contaminated powdered ginger

The migration of several major mycotoxins, aflatoxins B₁ (AFB₁), B₂, G₁, and G₂ (AFT, total of the aflatoxins) and ochratoxin A (OTA), from naturally contaminated powdered ginger to surrounding liquid (tea) was investigated. The toxins are commonly found in cereal grains and are toxic, carcinogenic and thermostable. Ginger root is widely used for digestive problems. Powdered ginger (2 g) found to contain AFT and OTA was placed in an empty heat sealable tea bag. The tea bag was heat-sealed and used to prepare tea under different conditions: temperature (50 and 100°C), time (5 and 10 min) and volume (100 and 200 ml). The tea bag was placed in hot water and stirred every 1 min for 5 s during the first 5 min of steeping. After steeping, the tea bag was removed and the tea and ginger residue (in the tea bag) were analysed separately for AFT and OTA. After extraction and immunoaffinity column (IAC) clean-up, the isolated AFT and OTA were separated by reversed-phase liquid chromatography and quantified using a fluorescence detector. At 100°C, approximately 30–40% of AFB₁ and AFT and 20–30% of OTA in the contaminated ginger were found in the ginger tea; the total amounts of AFT and OTA in tea varied less than 5% under the three conditions of preparation. At 50°C, about 10% of OTA and AFT were found in tea. This is the first study on the migration of AFT from botanicals to tea. It is also the first to study the distribution of AFT and OTA from powdered ginger to tea and ginger residue.     

Enzyme-assisted extraction for the HPLC determination of ochratoxin A in pork and dry-cured ham

The extraction of ochratoxin A from meat products is generally carried out using chlorinated organic solvents, such as chloroform or methyl chloride, acidified with hydrochloric or o-phosphoric acid. In this study, an innovative method was developed to extract ochratoxin A from pork and dry-cured ham samples. The method was based on an enzyme-assisted extraction with pancreatin in phosphate buffer pH 7.5. Pancreatin hydrolyses the proteins, so that ochratoxin A, kept in the ionised form, is easily extracted by the aqueous solution. After purification through an immunoaffinity column, ochratoxin A is determined by HPLC with fluorescence detection. The average recovery values were higher than 90.0% and the relative standard deviations were below 5.5%. The limits of detection and of quantification were 0.06 and 0.12?µg?kg^?1, respectively. A comparison between the new enzyme-assisted extraction and an established chloroform method was carried out on six naturally contaminated samples of pork and on 40 samples of dry-cured ham. Significantly higher (p?<?0.001) values of ochratoxin A were obtained on dry-cured ham samples by the enzyme-assisted method.     

Early detection of Aspergillus carbonarius and A. niger on table grapes: a tool for quality improvement

Aspergillus carbonarius and A. niger aggregate are the main fungal contaminants of table grapes. Besides their ability to cause black rot, they can produce ochratoxin A (OTA), a mycotoxin that has attracted increasing attention worldwide. The objective of this work was to set up a simple and rapid molecular method for the early detection of both fungi in table grapes before fungal development becomes evident. Polymerase chain reaction (PCR)-based assays were developed by designing species-specific primers based on the polyketide synthases (PKS_S) sequences of A. carbonarius and A. niger that have recently been demonstrated to be involved in OTA biosynthesis. Three table grape varieties (Red globe, Crimson seedless, and Italia) were inoculated with A. carbonarius and A. niger aggregate strains producing OTA. The extracted DNA from control (non-inoculated) and inoculated grapes was amplified by PCR using ACPKS2F-ACPKS2R for A. carbonarius and ANPKS5-ANPKS6 for A. niger aggregate. Both primers allowed a clear detection, even in symptomless samples. PCR-based methods are considered to be a good alternative to traditional diagnostic means for the early detection of fungi in complex matrix for their high specificity and sensitivity. The results obtained could be useful for the definition of a ‘quality label’ for tested grapes to improve the safety measures taken to guarantee the production of fresh table grapes.     

Survey of ochratoxin A in rice from Iran using affinity column cleanup and HPLC with fluorescence detection

This paper records the occurrence and levels of ochratoxin A (OTA) in rice using a HPLC technique preceded by an immunoaffinity clean-up step. The method was based on the extraction of finely ground rice sample with an acetonitrile/water (60?:?40, v/v) solution. Recovery was 98.9% while the limit of detection (LOD) was 0.2?ng?g^?1. A total of 182 rice samples were analyzed with a frequency of contamination of 6%. Levels of OTA in positive samples ranged 0.2–4.8?ng?g^–1, with an average contamination of all analyzed samples of 1.6?ng?g^–1.     

两种同时检测茶叶中赭曲霉毒素A与桔霉素方法的比对

目的 对比两种同时检测茶叶中赭曲霉毒素A(ochratoxin A, OTA)与桔霉素(citrinin, CIT)的方法。方法 考察不同提取方法、溶剂、料液比的提取效果,比较适配体结合超滤法与免疫亲和柱纯化效果;对比高效液相色谱-荧光检测器(high performance liquid chromatography-fluorescence detector, HPLC-FLD)与超高效液相色谱-三重四极杆质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)测定OTA与CIT的检出限、精密度、准确度等差异,评价方法的适用性。结果 采用70%甲醇超声与振荡的提取方法,在料液比为1:3的条件下,OTA、CIT的提取率最佳,回收率分别为99.49%的92.77%;经适配体结合超滤纯化后,CIT与OTA的回收率为82.07%～92.95%,与免疫亲和柱纯化方法效果类似; UPLC-MS/MS测定CIT和OTA的基质效应(20.0%、-20.0%)小于HPLC-FLD测定CIT和OT...     

Optimization and validation of a HPLC method for simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone using an experimental design

A reversed-phase HPLC optimization strategy is presented for investigating the separation and retention behavior of aflatoxin B₁, B₂, G₁, G_2, ochratoxin A and zearalenone, simultaneously. A fractional factorial design (FFD) was used to screen the significance effect of seven independent variables on chromatographic responses. The independent variables used were: (X1) column oven temperature (20–40°C), (X2) flow rate (0.8–1.2?ml/min), (X3) acid concentration in aqueous phase (0–2%), (X4) organic solvent percentage at the beginning (40–50%), and (X5) at the end (50–60%) of the gradient mobile phase, as well as (X6) ratio of methanol/acetonitrile at the beginning (1–4) and (X7) at the end (0–1) of gradient mobile phase. Responses of chromatographic analysis were resolution of mycotoxin peaks and HPLC run time. A central composite design (CCD) using response surface methodology (RSM) was then carried out for optimization of the most significant factors by multiple regression models for response variables. The proposed optimal method using 40°C oven temperature, 1?ml/min flow rate, 0.1% acetic acid concentration in aqueous phase, 41% organic phase (beginning), 60% organic phase (end), 1.92 ratio of methanol to acetonitrile (beginning) and 0.2 ratio (end) for X1–X7, respectively, showed good prediction ability between the experimental data and predictive values throughout the studied parameter space. Finally, the optimized method was validated by measuring the linearity, sensitivity, accuracy and precision parameters, and has been applied successfully to the analysis of spiked cereal samples.     

Mycotoxin detection in infant formula milks in Italy

After birth, infant formulas constitute an important or often sole food source for infants during the first months of life. In this study, a survey on the presence of aflatoxin M₁ (AFM1) and ochratoxin A (OTA) in the 14 leading brands of infant formulas marketed in Italy was conducted. Mycotoxins were determined by immunoaffinity column clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection. AFM1 was found in two of 185 samples, but at levels below the European legislation limit of 25 ng l^?1. OTA was detected in 133 (72%) samples (range = 35.1–689.5 ng l^?1). It has been observed that OTA contamination was 80% in the ready-to-use preparations and 63% in the powdered samples. The Scientific Committee for Food (SCF) reviewed the toxicology on OTA and concluded that it would be prudent to reduce exposure to OTA ensuring that exposure is towards the lower end of the range of tolerable daily intakes of 1.2–14 ng kg^?1 body weight day^?1. OTA was also evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and a provisional tolerable weekly intake (PTWI) of 100 ng kg^?1 body weight was established. The OTA levels in pre-term ready-to-use infant formulas were sufficient to cause a higher OTA intake than the suggested TDI. The results point out the need to perform controls for prevention programmes especially when attempting to identify risk markers of the infant feed quality.     

The effect of chemical treatment on reduction of aflatoxins and ochratoxin A in black and white pepper during washing

The effect of 18 different chemicals, which included acidic compounds (sulfuric acid, chloridric acid, phosphoric acid, benzoic acid, citric acid, acetic acid), alkaline compounds (ammonia, sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide), salts (acetate ammonium, sodium bisulfite, sodium hydrosulfite, sodium chloride, sodium sulfate) and oxidising agents (hydrogen peroxide, sodium hypochlorite), on the reduction of aflatoxins B₁, B₂, G₁ and G₂ and ochratoxin A (OTA) was investigated in black and white pepper. OTA and aflatoxins were determined using HPLC after immunoaffinity column clean-up. Almost all of the applied chemicals showed a significant degree of reduction on mycotoxins (p?<?0.05). The lowest and highest reduction of aflatoxin B₁, which is the most dangerous aflatoxin, was 20.5%?±?2.7% using benzoic acid and 54.5%?±?2.7% using sodium hydroxide. There was no significant difference between black and white peppers (p?<?0.05).     

Comparison of manual and automatic sampling for monitoring ochratoxin A in barley grain

Automatic and manual sampling for ochratoxin A (OTA) in barley grain was compared under industrial conditions considering sampling uncertainty as well as practical and technical aspects. Ten tonnes of barley inoculated with Penicillium verrucosum were incubated until the OTA concentration reached approximately 15?µg?kg^?1 and sampled with manual and automatic sampling. A nested experimental design and ANOVA was used to estimate variance components from sampling, sample reduction, sample preparation and analysis. Manual sampling resulted in a high sampling uncertainty and OTA concentrations in aggregate samples ranged from 2 to 80?µg?kg^?1. When aggregate samples were formed by automatic sampling the uncertainty arising from nugget effects and spatial distribution was practically eliminated. Results from this study show that an automatic sampler mounted after a mixer or conveyer can provide representative samples of OTA from a moving stream of barley. Automatic sampling might present a practical and economical alternative to manual sampling for feed mill operators when monitoring low levels of mycotoxins in grain or other commodities. Despite careful precautions, sample preparation and analysis resulted in a relative uncertainty of ±40% (p?=?0.95), which was attributed to the sub-sampling following the two grinding steps. Size fractionation of the coarsely ground barley showed that 40% of the total amount of OTA was present in a small fraction of fine particles with a strong tendency to aggregate or stick to equipment and containers. Thus, in order to take advantage of the automatic sampling, it is crucial to apply an appropriate sub-sampling to prevent segregation of particles which may affect the OTA measurements.     

Influence of roasting and different brewing processes on the ochratoxin A content in coffee determined by high-performance liquid chromatography-fluorescence detection (HPLC-FLD)

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in green and roasted coffee. The method consists of extraction of the sample with methanol–5% aqueous sodium hydrogen carbonate/1% PEG8000 (20:80), followed by immunoaffinity column (IAC) clean-up and, finally, high-performance liquid chromatography (HPLC) determination with fluorimetric detection. Mean recoveries for green and roasted coffee spiked at different levels ranging from 94 and 105% were obtained. The limit of determination (S/N = 3) was 0.032 ng g^?1 and the precision (within-laboratory relative standard deviation) was 6%. The method described has been used to assess the influence of roasting and different brewing processes on OTA content in commercial lots of green and roasted coffee. The results provided evidence that roasting led to a significant drop on OTA levels (65–100%). Also, the way coffee is prepared affects the OTA content: brewing using a Moka Express (Italian coffee) led to a significant reduction of OTA concentration (50–75%) since hot water stays in contact with coffee for a short time. On the contrary, Turkish coffee-making (infusion for about 10 min) cause poor reduction in OTA.     

Formation of fumonisins and other secondary metabolites by Fusarium oxysporum and F. proliferatum: a comparative study

The principal aim of this study was to estimate the formation of fumonisins (FB₁ and FB₂), moniliformin (MON), and ergosterol (ERG) by Fusarium oxysporum and Fusarium proliferatum, while the formation of beauvericin (BEA) was estimated by the latter Fusarium species only. Moreover, the effect of temperature on the biosynthesis of mycotoxins was also evaluated. Fumonisins were formed by F. proliferatum, with the highest yield at 18°C (720.0–1976.6 µg g^?1 for FB₁, 74.2–670.8 µg g^?1 for FB₂) and only by three of four F. oxysporum strains at a very low level (0.02–4.77 µg g^?1 for FB₁, 0.02–2.15 µg g^?1 for FB₂). The amount of MON formed by F. proliferatum was the highest (p < 0.001) at 32°C (3056.87 µg g^?1), while MON biosynthesis by F. oxysporum was lower 227.54 µg g^?1 (p < 0.001). BEA was produced by F. proliferatum with the highest level at 25°C (p < 0.001). ERG–recognized as an indicator of fungal biomass development and as a consequence of mycotoxin formation–was found at the highest concentration at a biosynthesis temperature of 25°C for F. proliferatum and F. oxysporum (p < 0.001).     

Co-occurrence of ochratoxin A and citrinin in unprocessed cereals established during a three-year investigation period

The aim of this study was to investigate ochratoxin A (OTA) and citrinin (CIT) co-occurrence in different unprocessed cereals (n = 189) originating from Croatia during a three-year investigation period (2014–2016) using validated enzyme immunoassay (ELISA) methods. CIT and OTA were determined in 49% and 7% of samples, respectively. Significantly higher (p < 0.05) overall mean concentrations were determined for CIT (66.8 ± 76.0 µg/kg) in comparison to OTA (5.2 ± 1.1 µg/kg). Based on the analysis of all investigated cereals, CIT was found about 15 times more frequently than OTA and in similarly (15-fold) higher concentrations, irrespective of the cultivation year. The results revealed a moderately positive correlation between OTA and CIT concentrations in maize (r_s = 0.44) and wheat (r_s = 0.59), whereas in barley and oat this correlation (p > 0.01) was not significant.     

A survey of ochratoxin A occurence in Greek wines

A total of 150 wines, including 123?dry wines (64 red, 49 white and 10 rosé) and 27 dessert wines (14 red and 13 white), were obtained from various viticulture and oenological practices across Greece during the period 1999–2006 and analyzed for ochratoxin a (OTA) using immunoaffinity clean-up and HPLC with fluorescence detection. There was a high frequency of OTA in commercially available wines (69% positive samples). However, the level of contamination was relatively low, with only one sample marginally reaching the EU permitted maximum level (2.0?µg?l^?1). A total of 91% of the samples had OTA concentrations <1.0?µg?l^?1. The higher concentrations were found in wines from the southern regions, especially in dessert-type wines. There were no significant differences based on wine color or production years. Furthermore, there was no difference between conventional or organic cropping systems in terms of OTA presence.     

Occurrence of ochratoxin A in Greek wines

The presence of ochratoxin A in foods and wines is of current interest owing to its toxic effects. Ochratoxin A is a widely distributed mycotoxin with nephrotoxic activity. It constitutes a new restrictive agent for the export of vinicultural products and is thus important from an economic as well as a scientific point of view. In this study the determination of ochratoxin A in Greek wines was made by means of commercial immunoaffinity columns for purification followed by HPLC with fluorescence detection for quantification of the toxin. The method was applied to 28 dry wines (14 red, 13 white, one rosé) and seven sweet wines (three red, four white) obtained by various viticultural and oenological practices. The levels of ochratoxin A ranged from <0.02 to 3.2 ng ml^?1, with sweet wines containing higher levels than dry wines. Most of the wines contained relatively low concentrations of ochratoxin A (0.02–0.5 ng ml^?1), which do not represent a serious risk to consumer health. © 2003 Society of Chemical Industry