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.     

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 (AFB(1), AFB(2), AFG(1), and AFG(2)), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB(1), FB(2), and FB(3)), 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 AFB(1) and AFG(1), 0.012 ng/g for AFB(2) and AFG(2), 0.2 ng/g for OTA, 1.5 ng/g for ZEA, 6.2 ng/g for FB(1), FB(3) and HT-2 toxin, 9.4 ng/g for FB(2) 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 AFB(2) and AFG(2) 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.     

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.     

Co-occurrence of aflatoxins, ochratoxin A and zearalenone in barley from a northern region of Spain

One-hundred and twenty-three barley samples from a region of Spain (Navarra) were analysed in order to evaluate the possible co-occurrence of aflatoxins (AFB1, AFG1, AFB2 and AFG2), ochratoxin A (OTA) and zearalenone (ZEA). The results indicated that 80% of the samples presented detectable, although very low levels, of two or more mycotoxins. The most frequent combinations were AFB1 and OTA; AFB1, ZEA and OTA; and AFB1 and ZEA. In general, the statistical study did not show significant differences between levels or incidence for the mycotoxins in different years of harvest, variety of barley, farming or origin. The calculated values for daily intake were low and the risk to consumers could be assumed to be very low. However, the co-occurrence of several mycotoxins, and therefore synergic or additive effects, should be taken into account when determining permitted levels or risk assessment.     

Determination of mycotoxins in biscuits,dried fruits and fruit jams: an assessment of human exposure

ABSTRACT

A reliable, fast and simple method using UHPLC-MS/MS was developed for the determination of aflatoxins B₁ (AFB1), G₁ (AFG1), B₂ (AFB2) and G₂ (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), HT-2 toxin and T-2 toxin in crude extracts of biscuits with fruit filling, cookies, dried fruits and fruit jams. The method was successfully demonstrated on 39 samples of biscuits with fruit filling, 34 cookies, 14 dried fruits and 10 fruit jams. The mycotoxins detected in biscuits samples were ZEA, OTA, T-2 and AFB1 with an average concentrations of positive samples of 2.64, 4.10, 8.13 and 1.32 µg kg^?1, respectively; while the mycotoxins detected in jam samples were AFB1, OTA, T-2 and AFB2 with an average concentrations of positive samples of 2.00, 17.7, 4.37 and 1.15 µg kg^?1, respectively. The results showed that the majority of samples were in compliance with relevant regulations. However in eight samples of biscuits and three samples of fig jam the contents of OTA were higher than the existing OTA limits. The combined dietary exposure of selected mycotoxins was estimated for the first time for children, adolescents and adults. The estimated combined dietary exposures were all lower than the proposed value assumed to predict a possible risk scenario.     

高效液相色谱法同时检测粮食中常见8 种真菌毒素的含量

建立免疫亲和柱净化-高效液相色谱法同时测定粮食中黄曲霉毒素B1（aflatoxins,AFB1）、黄曲霉毒素B2（AFB2）、黄曲霉毒素G1（AFG1）、黄曲霉毒素G2（AFG2）、赭曲霉毒素A（ochratoxin A,OTA）、玉米赤霉烯酮（zearalenone,ZEN）、呕吐毒素（deoxynivalenol,DON）和T-2毒素的检测方法。样品经乙腈-水提取后,用免疫亲和柱净化,Agilent Elipse Plus C18（100 mm×4 mm,3.5 μm）色谱柱分离,以甲醇-乙腈-水-乙酸为流动相,流速1 mL/min,柱温35 ℃,进样量20 μL,检测系统为可变波长检测器串联光化学衍生器串联荧光检测器。根据信噪比为3的峰响应值,确定各真菌毒素的检出限为：AFB1 0.446 ng/mL、AFB2 0.152 ng/mL、AFG1 0.523 ng/mL、AFG2 0.334 ng/mL、ZEN 7 ng/mL、OTA 0.7 ng/mL、DON 200 ng/mL、T-2毒素100 ng/mL。样品中各真菌毒素的平均加标回收率,玉米为80.0%～104.5%,小麦为83.2%～102.8%,方法精密度为2.6%～10.2%。从样品前处理到分析整个过程耗时约2 h。本方法简便、快速、灵敏度高,适用于粮食中多种真菌毒素的快速测定。     

Validation of a UHPLC-FLD method for the simultaneous quantification of aflatoxins,ochratoxin A and zearalenone in barley

A fast and simple UHPLC-FLD method has been developed for the simultaneous determination in barley of aflatoxins (B1, G1, B2 and G2), ochratoxin A (OTA) and zearalenone (ZEA), some of the most important mycotoxins due to their toxicity and occurrence. The procedure is based on the extraction of the six mycotoxins with a mixture of acetonitrile and water, and the purification of the extract with immunoaffinity columns before analysis. Detection of AFB1 and AFG1 is improved using a photochemical reaction. The method has been validated with satisfactory results. Limits of detection were 340 ng kg⁻¹ for ZEA, 13 ng kg⁻¹ for OTA and varied from 0.5 to 15 ng kg⁻¹ for aflatoxins. Recovery percentages were between 78.2% and 109.2%. After being validated, the method has been successfully applied to 20 barley samples cultivated in a region of northern Spain (Navarra).     

高效液相色谱法同时检测粮谷中的黄曲霉毒素和赭曲霉毒素

建立粮谷类食品中黄曲霉毒素(B1、B2、G1 和G2)和赭曲霉毒素A 的同时检测方法。样品经过甲醇- 水(80:20,V/V)提取,液液萃取净化和富集后,三氟乙酸衍生,采用Agilent ZORBAX SB-C18 色谱柱(4.6mm ×250mm),以乙腈和体积分数2% 冰醋酸溶液为流动相梯度洗脱,在线变换波长荧光检测。根据3 倍信噪比的峰 响应值,确定黄曲霉毒素(B1、B2、G1 和G2)和赭曲霉毒素A 检出限分别为0.06、0.03、0.18、0.05μg/kg 和0.51μg/kg,上述5 种毒素分别在质量浓度0.05～100、0.125～25.00、0.05～100、0.125～25.00μg/L 和0.05～50.00μg/L 范围内呈线性相关,相关系数r 分别为0.9998、0.9998、0.9998、0.9996 和0.9998;在玉米、大米、小麦3 类样品中加标回收率平均为71.73%～115.37%,相对标准偏差为3.00%～9.88%,方法学验证结果表明,黄曲霉毒素和赭曲霉毒素A 5 种毒素同时检测结果与现行国标的单独检测方法检测结果无显著性差异(P ＞ 0.05)。     

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.     

重庆地区辣椒、花椒、八角中真菌毒素污染状况分析

目的 了解重庆地区辣椒、花椒和八角中真菌毒素污染状况。方法 采用随机抽样方法,在重庆地区盘溪、菜园坝和江津三个市场,抽取180批次香辛料样品,采用HPLC-MS/MS检测黄曲霉毒素（AFB1、AFB2、AFG1和AFG2）、赭曲霉毒素A（OTA）和伏马毒素（FB1和FB2）。结果 重庆地区辣椒、花椒和八角中的真菌毒素总检出率分别为60.0%、100%、96.7%。AFs和OTA是三种香辛料最主要的污染毒素,其中AFG2是最主要的黄曲霉代谢产物。盘溪市场的AFs污染总体情况稍好,菜园坝市场AFs污染最严重;江津市场的OTA污染最为严重。结论 香辛料中存在真菌毒素污染,有必要建立香辛料中真菌毒素的限量标准。     

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.     

基于核酸适配体的侧流层析技术同步检测赭曲霉毒素A和黄曲霉毒素B1

由于简单、低成本和快速的特点,侧流层析分析技术被广泛应用于食品、饲料和农产品中真菌毒素的检测。本实验基于适配体与靶标物特异性结合的原理,建立了基于适配体互补链同时检测赭曲霉毒素A（Ochratoxin A, OTA）和黄曲霉毒素B1（Aflatoxin B1, AFB1）两种真菌毒素的荧光多残留试纸条,通过优化两种适配体的浓度以及缓冲体系的PH值,提高了共同检测OTA和AFB1的灵敏度和准确性。OTA和AFB1的T线（TO和TA）和C线荧光强度比值与对应真菌毒素的浓度对数具有良好的线性关系,线性范围为0.5~50 ng/mL,相关系数r2分别为0.9887和0.9910,检出限低至0.51和0.38 ng/mL。通过对花生和葡萄干进行加标回收和实际样品检测,使用多残留试纸条检测的OTA和AFB1的回收率分别为82.06%~109.69%和83.34%~110.06%,相对标准偏差（Relative Standard Deviation, RSD）为1.89%~8.17%,检测结果与高效液相色谱-串联质谱法（HPLC-MS/MS）一致。该生物传感器可以在20 min内同时检测OTA和AFB1,并且具有成本低、检测速度快和易于操作等优点,可以满足花生和葡萄干等实际样品中OTA和AFB1残留量的现场快速检测的要求,为真菌毒素多残留检测提供理论依据和技术支撑。     

Co-occurrence of aflatoxins,ochratoxin A and zearalenone in Capsicum powder samples available on the Spanish market

The aim of this study was to determine the co-occurrence of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in paprika and chilli samples purchased in Spain, using HPLC with fluorescence detection. The occurrence of mycotoxin in 64 paprika samples was 59% for AFs, 98% for OTA and 39% for ZEA, whereas in the 35 chilli samples, the contamination was 40% for AFs, 100% for OTA and 46% for ZEA. None of the samples had AFs levels higher than the legally allowable limits. Regarding the co-occurrence of mycotoxins, 75% of paprika samples and 65% of chilli samples contained more than one mycotoxin. Chilli samples generally had lower concentrations of AFB1, AFB2, total AFs and OTA than had paprika samples. The high incidence of OTA contamination suggests that additional legislation may be required to for these kinds of spices.     

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.     

Aflatoxins and ochratoxin A and their Aspergillus causal species in Tunisian cereals

ABSTRACT

Occurrence of aflatoxins (AFs) AFB1, AFB2, AFG1, AFG2 and ochra toxin A (OTA) was studied in 65 samples of stored and freshly harvested wheat, barley and maize collected in Tunisia. The mycotoxins were simultaneously extracted and quantified by high performance liquid chromatography. Determination of AF-producing (section Flavi) and OTA-producing Aspergillus species (sections Nigri and Circumdati) was conducted in these samples by species-specific polymerase chain reaction (PCR). Results showed that most of maize samples were contaminated with AFs, data after storage showing lower values than those collected at harvest. All contaminated maize samples contained AFG1 and AFG2, among which 27.78% also had AFB1 and AFB2. This AFs pattern was consistent with the A. parasiticus toxin profile. A. flavus however showed the highest frequency in maize but was also found in barley and wheat where no AFs were detected. In contrast, OTA was neither found in maize nor in barley and only one wheat sample contained OTA. A. niger was the only OTA-producing species detected.     

食用植物油中黄曲霉毒素和赭曲霉毒素的污染状况及特征分析北大核心CSCD

针对市场在售的调和油、玉米油、大豆油、花生油和菜籽油等食用植物油,随机购买20种共290份食用植物油样品。采用液相色谱法测定AFB_(1)、AFB_(2)、AFG_(1)、AFG_(2)以及OTA、OTB真菌毒素的含量,对食用植物油中黄曲霉毒素和赭曲霉毒素的污染水平和分布特征进行分析。结果表明:20种290份食用植物油样品中,有16种共67份样品存在不同程度的真菌毒素污染,总污染率达到23.1%,不同种类食用植物油污染呈现“多种类、共分布”的特点,其中AFG_(1)污染率(14.8%)最高,其次为OTA(13.4%)。绝大多数阳性样本受1~4种真菌毒素污染,仅有少数阳性样本受真菌毒素污染数量达到5种。总体上食用植物油样品受到多种真菌毒素的混合污染情况比较严重,应引起一定的重视。     

Binding of zearalenone, aflatoxin B1, and ochratoxin A by yeast-based products: a method for quantification of adsorption performance

A methodology was developed to quantify the efficiency of yeast-based products for adsorption of three mycotoxins: zearalenone (ZEA), aflatoxin B(1) (AFB(1)), and ochratoxin A (OTA). Eight products were tested (yeast cell wall or inactivated yeast). The described experimental protocol based on in vitro tests provided reliable isotherms for each mycotoxin. The most suitable models were the Hill model for ZEA, the Langmuir model for AFB(1), and the Freundlich model for OTA. From these models, original mathematical affinity criteria were defined to quantify the product adsorption performances for each mycotoxin. The best yeast product, a yeast cell wall from baker's yeast, can adsorb up to 68% of ZEA, 29% of AFB(1), and 62% of OTA, depending on the mycotoxin concentrations. The adsorption capacity largely depended both on yeast composition and mycotoxin, but no direct correlation between yeast composition and adsorption capacity was found, confirming that adsorption of mycotoxin on yeast-based products involves complex phenomena. The results of this study are useful for comparing the adsorption efficiency of various yeast products and understanding the mechanisms involved in adsorption.     

超高效液相色谱-串联质谱法测定牛奶中24 种真菌毒素

运用基质分散固相萃取净化,建立牛奶中24 种真菌毒素（黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1、赭曲霉毒素A、玉米赤霉烯酮、玉米赤霉酮、α-玉米赤霉烯醇、β-玉米赤霉烯醇、α-玉米赤霉醇、β-玉米赤霉醇、T-2毒素、HT-2霉素、伏马毒素B1、伏马毒素B2、伏马毒素B3、脱氧雪腐镰刀菌烯醇、3-乙酰脱氧雪腐镰刀菌烯醇、15-乙酰脱氧雪腐镰刀菌烯醇、交链孢霉单甲基醚、交链孢酚、腾毒素、细交链孢菌酮酸）多残留检测的超高效液相色谱-串联质谱检测方法。样品经80%乙腈溶液（体积分数）提取,通过基质分散固相萃取净化,氮吹至近干,1 mL 50%乙腈溶液（体积分数）复溶,采用超高效液相色谱-串联质谱进行测定。经ACQUITY UPLC HSS T3反相柱（2.1 mm×100 mm,1.8 μm）分离,梯度洗脱,采用电喷雾离子源-多反应监测模式采集。24 种目标物的相关系数（R2）均大于0.985,加标回收率为71.0%～123.0%,相对标准偏差均小于10%。该方法具有操作简单、重复性好、灵敏度高、杂质干扰小等特点,可以用于牛奶中24 种真菌毒素的检测。     

Occurrence of Aflatoxins and Ochratoxin A in Baby Foods in Portugal

Infants have a more restricted diet and they generally consume more food on a body weight basis than adults. Therefore, the significance and potential health risk of any contaminant in foods consumed by infants is increased and diligent attention must be paid to this particular area. The present study aims to determine the occurrence of aflatoxin M₁ (AFM₁), aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in processed cereal-based foods (flours) and infant formulae (milk powder) available in the Portuguese market, both sold as conventional and organic origin. Mycotoxin determination was carried out using a method previously applied to duplicate diet samples. This method employed chloroform extraction, liquid–liquid extraction, immunoaffinity column (IAC) cleanup and HPLC analysis with fluorescence detection after post-column derivatisation. Quantification limits were 0.014, 0.004 and 0.028 μg kg⁻¹ for AFM₁, AFB₁ and OTA, respectively. These toxins could only be quantified in 12 of 27 analysed samples (15 positive results): two samples with AFM₁, two samples with AFM₁ and OTA, one sample with AFB₁ and OTA and seven samples with OTA. Positive results concerned four for AFM₁ (26%), one for AFB₁ (7%) and ten for OTA (67%). For these samples, contents ranged between 0.017–0.041 μg AFM₁ kg⁻¹, 0.034–0.212 μg OTA kg⁻¹, and one sample had a value of 0.009 μg AFB₁ kg⁻¹. Considering the presented results, we could provisionally conclude that the presence of these mycotoxins in baby foods does not constitute a public health problem. These are the first results concerning the occurrence of mycotoxins in marketed baby foods in Portugal and this is the first study using the HPLC method, proposed for duplicate diets, in baby food sample analysis.     

Simultaneous Determination of Aflatoxins and Ochratoxin A in Bee Pollen by Low-Temperature Fat Precipitation and Immunoaffinity Column Cleanup Coupled with LC-MS/MS

A simple and sensitive method has been developed for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, and AFG2) and ochratoxin A (OTA) in bee pollen. The analytes in the sample were extracted with a mixture of acetonitrile/water (60:40, v/v), using low temperature for fat precipitation, followed by immunoaffinity column cleanup of extracts. The mycotoxins were quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with an electrospray ionization (ESI) and a triple quadrupole (QqQ) analyzer. Matrix effect, accuracy, and precision were evaluated and achieved good results calculated by matrix-matched calibration standards which reduced the influence of matrix effect. Recoveries at three levels were in the range of 74.3–96.5 % with RSD less than 10.0 %. The correlation coefficients (r ²) of the five mycotoxins were higher than 0.997. The method showed high sensitivity with LOD below 0.05 μg/kg.