基质固相分散液相色谱法检测辣椒产品中的黄曲霉毒素

建立了中性氧化铝-石墨化碳黑的基质固相分散共柱提取净化前处理和以溴为衍生试剂的液相色谱-柱后在线衍生荧光检测法,并将该方法用于辣椒产品中黄曲霉毒素B1,B2,G1,G2的分析。对固相分散剂及共柱净化剂进行了选择和优化。该方法对B1,B2,G1,G2的平均回收率分别为95.4%,87.3%,91.5%和92.6%;方法对B1,G1的检出限为0.25 ng/g,对B2,G2的检出限为0.10 ng/g;对B1,B2,G1,G2进行测定的相对标准偏差(RSD)分别为3.3%,5.8%,4.7%和6.1%。对基质固相分散法和免疫亲和柱法的净化效果进行了比较,结果表明基质固相分散提取净化可以作为一种有效的方法用于辣椒产品中黄曲霉毒素的测定。     

Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by liquid chromatography-tandem mass spectrometry

An easy method for the determination of aflatoxins B1, G1, B2 and G2 in Rhammus purshiana by LC coupled to mass spectrometry has been developed. Aflatoxins were extracted with a mixture of methanol and water and then it was purified by solid-phase clean-up using a polymeric sorbent, not described previously, for the determination of these toxins. The eluted extract was injected into the chromatographic system using a reversed-phase C18 short column with an isocratic mobile phase composed of methanol-water (30:70). A single-quadruple mass spectrometry using an electrospray ionization source operating in the positive ion mode was used to detect aflatoxins due to derivatization presenting several disadvantages. Recoveries of the full analytical procedure were 110% for aflatoxin B1, 89% for aflatoxin B2, 81% for aflatoxin G1 and 77% for aflatoxin G2. Detection limit (S/N = 3) was 10 ng and quantification limit (S/N = 10) was 25 ng, calculated as amount in medicinal herb.     

High-performance liquid chromatography of aflatoxins in human urine

A method was developed for the extraction and determination of unconjugated aflatoxins in human urine by high-performance liquid chromatography. The analysis is based on the elimination of lipid-soluble constituents other than unconjugated aflatoxins in urine by light petroleum extraction. The unconjugated aflatoxins were subsequently extracted from the aqueous phase with chloroform-acetone. Chromatography was performed isocratically with a silica column at 40 degrees C. The resolved aflatoxins were detected and identified by ultraviolet and fluorometric detectors. The recoveries of aflatoxins B1 and G1 added prior to the extraction were 72% and 83%, respectively. This procedure is simple, sensitive and practically useful for epidemiological survey of unconjugated aflatoxins in human urine from areas with a high risk of aflatoxin consumption.     

Simultaneous determination of aflatoxins B2 and G2 in peanuts using spectrofluorescence coupled with parallel factor analysis

In the present study a method for the simultaneous determination of aflatoxins B₂ and G₂ in peanuts has been developed. The method uses second order standard addition method and excitation–emission fluorescence data together with parallel factor analysis (PARAFAC). The aflatoxin analysis was based on extraction with methanol–water and carried out using immunoaffinity clean-up. The results of PARAFAC on a set of spiked and naturally contaminated peanuts indicated that the two aflatoxins could be successfully determined. The method was validated and analytical figures of merit were obtained for both analytes. The limits of detection (LOD) were 0.05 and 0.04 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. The limits of quantification (LOQ) were 0.16 and 0.12 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. Coupling of spectrofluorimetry with PARAFAC can be considered as an alternative method for quantification of aflatoxins in the presence of unknown interferences obtained through analysis of highly complex matrix of peanuts samples at a reduced cost per analysis.     

高效液相色谱-串联质谱法测定山茶油中黄曲霉毒素B1

建立了山茶油中黄曲霉毒素B1含量的高效液相色谱-串联质谱分析方法。通过对前处理方法的优化,选择了甲醇和水作为山茶油中黄曲霉毒素B1的提取溶剂,经免疫亲和柱富集浓缩后,采用高效液相色谱-串联质谱进行分析,经C18色谱柱分离,在电喷雾离子化正离子模式(ESI+)及多反应监测模式(MRM)下进行测定,基质匹配标准溶液外标法定量。在优化条件下,该方法线性范围为0.4～6.4μg/L,相关系数r2>0.998,最低检出限为0.026μg/kg,在添加水平为0.008,0.016和0.032μg时,方法回收率在85.9%～93.8%之间;相对标准偏差为1.8%～5.0%。方法可满足山茶油中黄曲霉毒素B1的检测要求。     

Potential of near infrared spectroscopy for the analysis of mycotoxins applied to naturally contaminated red paprika found in the Spanish market

The potential of the near infrared spectroscopy (NIRS) technique for the analysis of red paprika for aflatoxin B(1), ochratoxin A and total aflatoxins is explored. As a reference, the results from a chromatographic method with fluorescence detection (HPLC-FD) following an immunoaffinity cleanup (IAC) were employed. For the NIRS measurement, a remote reflectance fibre-optic probe was applied directly onto the samples of paprika. There was no need for pre-treatment or manipulation of the sample. The modified partial least squares (MPLS) algorithm was employed as a regression method. The multiple correlation coefficients (RSQ) and the prediction corrected standard errors (SEP(C)) were respectively 0.955 and 0.2 microg kg(-1), 0.853 and 2.3 microg kg(-1), 0.938 and 0.3 microg kg(-1) for aflatoxin B(1), ochratoxin A and total aflatoxins, respectively. The capacity for prediction of the developed model measured as ratio performance deviation (RPD) for aflatoxin B(1) (5.2), ochratoxin A (2.8) and total aflatoxins (4.4) indicate that NIRS technique using a fibre-optic probe offers an alternative for the determination of these three parameters in paprika, with an advantageously lower cost and higher speed as compared with the chemical method. Content of aflatoxin B(1) and total aflatoxins are the parameters currently employed by the food regulations to limit the levels of the four aflatoxins in many foodstuffs. In addition, aflatoxin B(1) itself is an excellent indicator for aflatoxins' contamination since it is always the most abundant and toxic.     

The Use of HPLC/Thermospray Ms for the Confirmation of Aflatoxins in Peanuts

Abstract

An HPLC/Thermospray MS method is described for the determination of aflatoxins B₁, B₂, G₁ and G₂ in peanut extracts. Samples are extracted and prepared for analysis using an SPE method. The final determination utilizes reversed phase HPLC with a thermospray MS detector. The use of the MS allowed for unequivocal identification of aflatoxins in the extracts.     

液相色谱-串联质谱法测定动物肝脏中黄曲霉毒素

建立了动物肝脏中黄曲霉毒素G2、G1、B2、B1的高效液相色谱-串联质谱检测方法。样品经体积比为84∶16的乙腈-水溶液提取,离心后通过真菌毒素多功能净化柱,净化液氮气吹干,用流动相定容,采用C18柱分离,10mmol/L的甲酸铵溶液和甲醇作为流动相,以50∶50比例等度洗脱,在多重反应监测(MRM)正离子模式下进行分析。各组分在9min内完全分离,方法线性关系良好,黄曲霉毒素G2、G1、B2、B1的检出限分别为0.030、0.026、0.016、0.027μg/kg,三个加标水平下平均回收率在81%～98%之间,相对标准偏差小于2%。该方法简便快速,准确可靠,可用于动物肝脏中黄曲霉毒素的测定。     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in hazelnut paste: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 and total aflatoxins in hazelnut paste at European regulatory limits. The test portion was extracted with methanol-water (6 + 4). The extract was filtered, diluted with phosphate-buffered saline (PBS) solution to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxins. The aflatoxins were removed from the immunoaffinity column with methanol, and then quantified by reversed-phase LC with post-column derivatization (PCD) involving bromination. The PCD was achieved with electrochemically generated bromine (Kobra Cell) followed by fluorescence detection (except for one participant who used pyridinum hydrobromide perbromide for bromination). Hazelnut paste, both naturally contaminated with aflatoxins and blank (<0.1 ng/g) for spiking by participants with aflatoxins, was sent to 14 collaborators in Belgium, The Netherlands, Spain, Turkey, the United Kingdom, and the United States. Test portions were spiked at levels of 4.0 and 10.0 ng/g for total aflatoxins by participants using supplied total aflatoxins standards. Recoveries for total aflatoxins and aflatoxin B1 averaged from 86 to 89%. Based on results for naturally contaminated samples (blind duplicates at 3 levels ranging from 4.0 to 11.8 ng/g total aflatoxins), the relative standard deviation for repeatability (RSDr) ranged from 2.3 to 3.4% for total aflatoxins and from 2.2 to 3.2% for aflatoxin B1. The relative standard deviation for reproducibility (RSD(R)) ranged from 6.1 to 7.0% for total aflatoxins and from 7.3 to 7.8% for aflatoxin B1. The method showed exceptionally good within-laboratory and between-laboratory precision for hazelnut paste, as evidenced by HORRAT values, which in all cases were significantly below target levels, the low levels of determination for both aflatoxin B1 and total aflatoxins.     

Fast and efficient immunoaffinity column cleanup and liquid chromatography–tandem mass spectrometry method for the quantitative analysis of aflatoxins in baby food and feeds

An ultra high performance liquid chromatography‐tandem mass spectrometric method has been developed for the highly sensitive and selective determination of regulated aflatoxins. The extraction of aflatoxins from baby food matrices were performed using liquid–liquid extraction procedure followed by immunoaffinity column cleanup. The higher sensitivity for the determination of target aflatoxins was fulfilled by applying a preconcentration step with immunoaffinity columns after acetonitrile–water extraction. The enhanced selectivity was attained with the triple quadrupole mass analyzer operated in electrospray positive ionization mode. Method validation was tested in five different baby food matrices by recovery experiments. Satisfactory recoveries, between 92 and 103%, with relative standard deviations lower than 8% were achieved in all the tested matrices. The proposed method was found to be specific as no interference peaks were observed for blank samples. The limit of detection of the method was found to be in the range of 0.003–0.008 ng/mL. The validated method was fruitfully applied to the screening of aflatoxins in baby foods and feeds sample retailed in local markets of Riyadh, Saudi Arabia. The obtained levels of all analyzed aflatoxins were below the regulation limits set by European Agency.     

Combined biocompatible medium with molecularly imprinted polymers for determination of aflatoxins B1 in real sample

A kind of molecularly imprinted polymers modified with biocompatible medium was prepared by suspension polymerization. The obtained hybrid materials were used as the adsorbents for the solid‐phase extraction of aflatoxins B1 in real samples. A structural analog of the target, 6‐methyl‐4‐phenylchroman‐2‐one was used as the pseudo‐template, owing to their lower toxicity and cheaper price compared with aflatoxins B1; and methacrylic acid and glycidyl methacrylate were used as the co‐monomers. Scanning electron microscopy and size distribution analysis were used to characterize the obtained polymers. The extraction parameters were optimized to achieve the desired extraction performance. The polymer solid‐phase extraction coupled with high‐performance liquid chromatography was successfully applied to determine aflatoxins B1 from soy sauce without the process of protein removal. Under the optimum extraction conditions, the detection results of aflatoxins B1 in lab‐made column in soy sauce samples was carried out, with a recovery rate of 96%. The established method presented a linear range from 10 to 1000 ng/mL with the coefficient of determination of 0.9994 and the limit of detection of 0.05 ng/mL. Likewise, the inherent selectivity of lab‐made column towards aflatoxins B1, Ochratoxin A, and Zearalenone was demonstrated.     

Matrix-free analysis of aflatoxins in pistachio nuts using parallel factor modeling of liquid chromatography diode-array detection data

In the present study a second-order calibration strategy for high performance liquid chromatography with diode-array detection (HPLC-DAD) has been developed using parallel factor analysis (PARAFAC) and has been applied for simultaneous determination of aflatoxins B₁, B₂, G₁ and G₂ in pistachio nuts in the presence of matrix interferences. Sample preparation was based on solvent extraction (SE) followed by solid phase extraction (SPE) on Bond Elut C18 cartridges. Since the sample preparation procedure was not selective to the analytes of interest, exploiting second-order advantage to obtain concentrations of individual analytes in the presence of uncalibrated interfering compounds seemed necessary. Appropriate pre-processing steps have been applied to correct background signals and the effect of retention time shifts. Transferred calibration data set obtained from standardization of solvent based calibration data has been used in prediction step. The results of PARAFAC on a set of spiked and naturally contaminated pistachio nuts indicated that the four aflatoxins could be successfully determined. The method was validated and multivariate analytical figures of merit were calculated. The advantages of the proposed method are using a low-cost SPE step relative to standard method of aflatoxin analysis (immune affinity column assay), a unique and simple isocratic elution program for all samples and a calibration transfer for saving both chemicals and time of analysis. This study show that coupling of SPE-HPLC-DAD with PARAFAC as a powerful second-order calibration method can be considered as an alternative method for resolution and quantification of aflatoxins in the presence of unknown interferences obtained through analysis of highly complex matrix of pistachio samples and cost per analysis can be reduced significantly.     

Determination of aflatoxins in food by overpressured-layer chromatography

Legislative measures for monitoring and control of aflatoxin levels in foods and foodstuffs have been introduced in many countries. The aim of the present work was to make developments in the field of aflatoxin analysis, focusing upon the use of overpressured-layer chromatography (OPLC) for quantitative determination. OPLC methods have been developed for the determination of aflatoxins B1, B2, G1, G2 in different foods. These methods are suitable for sample clean-up and separation as well. Using OPLC we could analyze 10 samples simultaneously. The methods were investigated with fish, corn and wheat samples spiked with 2-10 ng/g aflatoxins. Quantitative evaluation of aflatoxins was accomplished by densitometry. Average recoveries from each food were greater than 73%. The OPLC technique seems to be a rapid, reproducible and cost-effective analysis for quantitative determination of aflatoxins in foods.     

Fast screening of aflatoxins in dairy cattle feeds with CE‐LIF method combined with preconcentration technique of vortex assisted low density solvent–microextraction

Aflatoxin contamination in agricultural products poses a great threat to humans and livestock. The aim of this study was to establish a simple, rapid, highly sensitive, and inexpensive method for the simultaneous detection of aflatoxin B₁, B₂, G₁, and G₂ in agricultural products. We used a vortex assisted low density solvent–microextraction (VALDS‐ME) technique for sample preconcentration and sample detection was achieved with a CE‐LIF method. Aflatoxins were separated in an uncoated fused‐silica capillary with the MEKC mode and were excited by a 355 nm UV laser to produce native fluorescence for detection. The obtained LOD and LOQ for the four aflatoxins were in the range of 0.002–0.075 and 0.007–0.300 μg/L, respectively, and the analysis time was within 6.5 min. Using the established method, aflatoxins were screened in naturally contaminated dairy cattle feed samples including alfalfa, bran, and corn kernel. The result shows that the alfalfa and bran samples were contaminated with aflatoxins to varying degrees. Compared with other analytical techniques for aflatoxin screening in agricultural products, this CE‐LIF method combined with VALDS‐ME preconcentration technique is simple, rapid, highly efficient, and inexpensive.     

Rapid determination of aflatoxins in corn and peanuts

A rapid and simple method using ultra-high-pressure liquid chromatography with UV detection for the determination of aflatoxins B1, B2, G1 and G2 in corn and peanuts has been developed. In this method, aflatoxins were extracted with a mixture of acetonitrile and water (86:14) and then purified by solid-phase clean-up with a MycoSep#226 AflaZon(+) column. The toxins were determined by UPLC-UV without derivatizing aflatoxins in real samples, which has not been used in other studies. The mean recoveries of aflatoxins from non-infected peanut and corn samples spiked with aflatoxins B1, B2, G1 and G2 at concentrations from 0.22 to 5 microg/kg were between 83.4% and 94.7%. The detection limits (S/N=3) for B1, B2, G1 and G2 were 0.32, 0.19, 0.32 and 0.19 microg/kg, and the corresponding quantification limits (S/N=10) were 1.07, 0.63, 1.07 and 0.63 microg/kg, respectively. We also applied this method on real samples. Among 16 peanut samples, 2 (12.5% incidence) were contaminated with aflatoxin; among 18 corn samples, 4 (22% incidence) were contaminated. The proposed method is rapid, simple and accurate for monitoring aflatoxins in corn and peanuts.     

Application of gold-labeled antibody biosensor in simultaneous determination of total aflatoxins using artificial neural network

Preparation of antibody-coated gold nanoparticles (GNPs) specific to aflatoxins B1, B2, G1 and G2 and its use in developing aflatoxins diagnostic method were presented in this paper. The formation of gold-labeled antibodies was accomplished at optimal condition. Due to severe overlapping between the emission profiles for the aflatoxins, they cannot be determined by direct inspection of data. The strategy used in this study, constituted by artificial neural network (ANN), was easy to implement and to originate reliable results. ANN can be successfully applied to spectrofluorimetric spectra matrices to simultaneous determination of total aflatoxins. Quantitative results obtained using ANN method for aflatoxins in pistachio nuts samples were compared to those obtained using the HPLC method. Obtained results using these two methods did not show significant differences.     

Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis

HPLC using fluorescence detection has already become the most accepted method for the determination of aflatoxins due to its several advantages over other analytical methods. Both normal- and reversed-phase HPLC can be used. However the reversed-phase HPLC methods are more popular. Liquid chromatographic determination of aflatoxins using fluorescence detection and its application in food analysis is reviewed in this article.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 and total aflatoxins at European regulatory limits. The test portion is extracted with methanol-water (8 + 2) for dried figs and paprika, and with methanol-water (8 + 2) plus hexane (or cyclohexane) for peanut butter and pistachios. The sample extract is filtered, diluted with phosphate buffer saline, and applied to an immunoaffinity column. The column is washed with water and the aflatoxins are eluted with methanol. Aflatoxins are quantitated by reversed-phase LC with post-column derivatization (PCD) involving bromination. PCD is achieved with either an electrochemical cell (Kobra cell) and addition of bromide to the mobile phase or pyridinium hydrobromide perbromide. Determination is by fluorescence. Peanut butter, pistachio paste, dried fig paste, and paprika powder samples, both naturally contaminated with aflatoxins and containing added aflatoxins, were sent to 16 collaborators in 16 European countries. Test portions of samples were spiked at levels of 2.4 and 9.6 ng/g for total aflatoxins which included 1.0 and 4.0 ng/g aflatoxin B1, respectively. Recoveries for total aflatoxins ranged from 71 to 92% with corresponding recoveries for aflatoxin B1 of 82 to 109%. Based on results for spiked samples (blind duplicates at 2 levels) as well as naturally contaminated samples (blind duplicates at 4 levels, including blank), the relative standard deviation for repeatability ranged from 4.6 to 23.3% for total aflatoxins and from 3.1 to 20.0% for aflatoxin B1. The relative standard deviation for reproducibility ranged from 14.1 to 34.2% for total aflatoxins, and from 9.1 to 32.2% for aflatoxin B1. The method showed acceptable within-laboratory and between-laboratory precision for all 4 matrixes, as evidenced by HORRAT values <1, at the low levels of determination for both total aflatoxins and aflatoxin B1.     

Optimization of Matrix Solid-Phase Dispersion method for simultaneous extraction of aflatoxins and OTA in cereals and its application to commercial samples

A method based on Matrix Solid-Phase Dispersion (MSPD) has been developed for the determination of 5 mycotoxins (ochratoxin A and aflatoxins B and G) in different cereals. Several dispersants, eluents and ratios were tested during the optimization of the process in order to obtain the best results. Finally, samples were blended with C₁₈ and the mycotoxins were extracted with acetonitrile. Regarding to matrix effects, the results clearly demonstrated the necessity to use a matrix-matched calibration to validate the method. Analyses were performed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-QqQ-MS/MS). The recoveries of the extraction process ranged from 64% to 91% with relative standard deviation lower than 19% in all cases, when samples were fortified at two different concentrations levels. Limits of detection ranged from 0.3 ng g⁻¹ for aflatoxins to 0.8 ng g⁻¹ for OTA and the limits of quantification ranged from 1 ng g⁻¹ for aflatoxins to 2 ng g⁻¹ for OTA, which were below the limits of mycotoxins set by European Union in the matrices evaluated. Application of the method to the analysis of several samples purchased in local supermarkets revealed aflatoxins and OTA levels.     

Determination of aflatoxins in animal feeds by liquid chromatography/tandem mass spectrometry with isotope dilution

The objective of the present study is to develop a simple, fast method for detection of aflatoxins in animal feeds. Simultaneous quantitation of four aflatoxins (AFB(1), AFB(2), AFG(1) and AFG(2)) in animal feeds was achieved in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) run. The solid-phase extraction cleanup step is eliminated with the stable isotope dilution method. Matrix effects were observed and overcome by isotope dilution. The method was tested in a variety of animal feed matrices and proved to be accurate and reliable. Method ruggedness tests resulted in recoveries of 78% to 122% with an intra-day assay precision of 2% to 15% and an inter-day assay precision of 3% to 17%. These results indicate that this method is suitable for quantitation of aflatoxins in animal feeds.