Simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography-tandem mass spectrometry

A reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products was developed. The sample was extracted by 84% of acetonitrile aqueous solution and the extract was purified by a reliable solid phase extraction-based clean-up method. Then, the analytes were separated on Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm particle size), and eluted with a mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile/methanol (50/50, v/v). The separated compounds were detected with a Waters Micromass Quattro Ultima Pt tandem quadrupole mass spectrometer operating in positive electro-spray ionization using multiple reaction monitoring mode. The established method was extensively validated by determining the linearity (R² ≥ 0.9990), average recovery (74.7-86.8%) and precision (relative standard deviation ≤ 10.9%). It was shown to be a suitable method for simultaneous determination of the six aflatoxins in peanuts and their derivative products. Finally, a total of 73 samples randomly collected from different areas in Zhejiang province were screened for aflatoxins with the proposed method. The results showed that 31 samples of peanut butter, 14 samples of fresh peanut and 5 samples of musty peanut were contaminated with aflatoxins. Meanwhile, this was the first report on aflatoxins M1 and M2, which were found in unprocessed peanuts and their derivative products.     

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.     

Rapid extraction of aflatoxin from creamy and crunchy peanut butter

A rapid extraction technique was developed for the isolation and subsequent liquid chromatographic determination of aflatoxins B1, B2, G1, and G2 in creamy and crunchy peanut butter. Peanut buftter samples were extracted with a methanol 15% sodium chloride (7 + 3) solution followed by a second extraction with methanol. The extract was subjected to a cleanup using a Vicam Aflatest immunoaffinity column. Control samples for both smooth and crunchy peanut butter were fortified at 4 different levels for aflatoxin B1, B2, G1, and G2. The average aflatoxin B1, B2, G1, and G2 recoveries from smooth peanut buffer were 95.2, 89.9, 94.1, and 62.4%, respectively, and 92.4, 84.3, 85.5, and 53.7%, respectively, from crunchy peanut butter. This extraction method and the official AOAC Method 991.31 produced comparable results for peanut butter samples. This method provides a rapid, specific, and easily controlled assay for the analysis of aflatoxins in peanut butter with minimal solvent usage. Organic solvent consumption was decreased by 85% and hazardous waste production was decreased by 80% in comparison with the AOAC method. Along with the decreased solvent consumption, significant savings in time were observed.     

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。     

Determination of aflatoxins in peanuts by matrix solid-phase dispersion and liquid chromatography

A new method based on matrix solid-phase dispersion (MSPD) extraction was studied to determine aflatoxin B1, B2, G1 and G2 from peanuts. Optimization of different parameters, such as type of solid supports for matrix dispersion and elution solvents were carried out. The method used 2 g of peanut sample, 2 g of C18 bonded silica as MSPD sorbent and acetonitrile as eluting solvent. Recoveries of each aflatoxin spiked to peanut samples at 2.5 ng/g (5 ng/g for aflatoxin G2) level were between 78 and 86% with relative standard deviations ranging from 4 to 7%. The limits of quantification ranged from 0.125 to 2.5 ng/g for the four studied aflatoxins using liquid chromatography (LC) with fluorescence detection. In addition, LC coupled to mass spectrometry with an electrospray interface was used for confirmation of aflatoxins present in real samples. Eleven peanut samples from different countries were analyzed by the proposed method and by using an enzyme-linked immunosorbent assay (ELISA). ELISA test is a good screening method for investigation of these mycotoxins in peanut samples.     

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.     

Immunoaffinity column cleanup with liquid chromatography using postcolumn bromination for the determination of aflatoxins in black and white sesame seed: single-laboratory validation

A single-laboratory validation was conducted to establish the effectiveness of an immunoaffinity column cleanup procedure followed by LC with fluorescence detection for the determination of aflatoxins B1, B2, G1, and G2 in sesame seeds. The sample is homogenized with 50% water (w/w) to form a slurry, then the test portion is extracted with methanol-water (60 + 40, v/v) using a high-speed blender. The sample extract is filtered, diluted with 15% Tween 20 in phosphate-buffered saline solution, and applied to an immunoaffinity column. Aflatoxins are removed with neat methanol, then directly determined by RP-LC with fluorescence detection using postcolumn bromination (Kobra cell). Test portions of blank white sesame seed slurry were spiked with a mixture of aflatoxins to give total levels of 4 and 10 microg/kg. Recoveries for individual and total aflatoxins ranged from 92.7 to 110.3% for spiked samples. Based on results for spiked sesame paste (triplicates at two levels), the RSD for repeatability (RSD(r)) averaged 1.1% for total aflatoxins and 1.4% for aflatoxin B1. The method was demonstrated to be applicable to naturally contaminated samples of black and white sesame seeds obtained from local markets in China.     

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。     

免疫亲和柱净化、在线电化学衍生化高效液相色谱法检测花生中的黄曲霉毒素

 采用免疫亲和柱净化、在线电化学衍生化高效液相色谱法测定了花生中黄曲霉毒素（AFT）B 1,B2,G1和G2。以体积分数为80%的甲醇提取样品中的AFT,经免疫亲和柱净化洗脱 后,以Kobra Cell装置在线衍生,反相HPLC分离定量。4种毒素的分离在13 min内完成,检 出限均达到0.1 μg/kg。5次测定花生样品的RSD值为9.2%～15%;样品添加标样0.5 ～9.0 μg/kg,回收率为74.8%～97.3%。     

花生及其制品中多种霉菌毒素残留同时测定方法

利用液相色谱-质谱联用（LC-MS／MS）建立了花生及其制品中多种霉菌毒素包括黄曲霉毒素（B1,B2,G1,G2）、赭曲霉毒素A、伏马毒素B1、脱氧雪腐镰刀菌烯醇、T-2毒素、HT-2毒素及玉米赤霉烯酮的同时测定方法。样品经PBS溶液和甲醇-水溶液提取,提取液经稀释、过滤后,用免疫亲和柱净化,通过淋洗去除免疫亲和柱上的杂质,随后用洗脱液过柱,将目标物分离下来,氮吹干后定容。以液相色谱-质谱／质谱测定,外标法定量。方法的检出限黄曲霉毒素B1为0．0005mg／kg,黄曲霉毒素B2,G1,G2为0．001mg／kg,赭曲霉毒素A为0．002mg／kg,伏马毒素B1为0．020mg／kg,脱氧雪腐镰刀菌烯醇为0．050mg／kg,T-2毒素为0．010mg／kg,HT-2毒素为0．010mg／kg,玉米赤霉烯酮为0．002mg／kg。在样品中添加检出限水平的毒素混标溶液,加标回收率为72．35％-97．82％,测定结果的相对标准偏差为8．95％～18．41％（n=10）.     

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.     

Factors influencing performance in the rapid separation of aflatoxins by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) is applied to the high-speed analysis of aflatoxins. Baseline separation of the four common aflatoxins G(1), G(2), B(1) and B(2), is accomplished in less than 30 sec. Small (25 mum) internal diameter capillaries are found to be critical in maintaining high efficiency under rapid MECC separation conditions. Van Deemter-like plots are generated in order to study the effects of capillary diameter and organic solvent on efficiency under high electric field conditions. Other experimental parameters affecting efficiency are investigated, including buffer concentration, surfactant concentration, and detector time constant. Simple on-column laser-based fluorescence detection, employing helium-cadmium laser radiation at 325 nm for excitation, allows for limits of detection in the range of 0.05-0.9 femtomoles injected for underivatized aflatoxins. Considerations important in the analysis of aflatoxins in real matrices are presented.     

电化学衍生-高效液相色谱和荧光光度法检测花生酱中的黄曲霉毒素

建立了免疫亲和柱净化-柱后电化学衍生-高效液相色谱结合荧光光度法检测花生酱中4种黄曲霉毒素(B1、B2、G1和G2)的方法。样品经过体积分数为60%的甲醇提取,通过免疫亲和柱净化后,以KobraCell装置柱后衍生,高效液相色谱法分离定量。黄曲霉毒素B1、B2、G1和G2能达到完全的基线分离,检测限分别为0.5、0.15、0.5和0.15μg/kg,线性相关系数0.999,回收率可达74.2%～96.5%,相对标准偏差低于11%。该方法能够满足花生酱中黄曲霉毒素检测的需要。     

免疫亲和萃取-超高效液相色谱-串联质谱法分离测定中成药及中药材中的5种黄曲霉毒素

利用免疫亲和萃取结合超高效液相色谱-串联四极杆质谱技术(UHPLC-ESI/QqQ-MS/MS)建立了中成药及中药材中5种黄曲霉毒素(B1、B2、G1、G2和M1)的提取、分离、确证与定量方法。样品经80%(体积分数)的甲醇水溶液提取和免疫亲和固相萃取后,采用UHPLC-ESI/QqQ-MS/MS的多反应监测模式实现分离、鉴定和外标法定量。5种目标毒素标准溶液的检出限(LOD)为0.05～0.3 μg/L。在0.5～100 μg/L的基质添加浓度范围内具有良好的线性关系(r2＞0.99);以甘草为例,当添加水平为1.0 μg/kg和5.0 μg/kg时,得到62.3%～82.4%的回收率(相对标准偏差(RSD)＜10%, n=6)。该方法灵敏度高、选择性和重复性好、回收率较高、检测速度快,适用于中成药及中药材等复杂基体中多种黄曲霉毒素的快速分析与筛查。     

Overpressured layer chromatographic determination of aflatoxin B1, B2, G1 and G2 in red paprika

Determination of aflatoxin B1 and total aflatoxin (B1 + B2 + G1 + G2) in red paprika powder is described using column chromatographic sample clean-up, overpressured layer chromatography (OPLC) separation and fluorescence densitometric evaluation. Two OPLC methods were developed for separation of the four aflatoxins. The detection limit and quantification limit of aflatoxins in red paprika were 0.5 and 1 μg/kg in both methods, respectively. Recovery experiment was carried out with sample containing 1.74 μg/kg aflatoxin B1 and 3.56 μg/kg total aflatoxins measured by European standard HPLC method. Mean recovery amounted to 78.5% (SD 16.1%, n = 5) for aflatoxin B1 and 81.8% (SD 17.1%, n = 5) for total aflatoxins in the case of method 1. It was 105.3% (SD 10.7%, n = 5) for aflatoxin B1 and 97.4% (SD 18.6%, n = 5) for total aflatoxins using the method 2. Despite of that the Hungarian climate is not proper for the toxin production of moulds high aflatoxin B1 contaminated red paprika purchased from the market was found, which may originate from mixing of imported paprika containing very high level toxin with Hungarian one.     

A sensitive confirmatory method for aflatoxins in maize based on liquid chromatography/electrospray ionization tandem mass spectrometry

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for measurement of aflatoxins B1, B2, G1, and G2 in maize is described. Aflatoxins (AFs) were extracted from 1 g samples by using tri-portions of acetonitrile/water (80:20, v/v) (10 + 7 + 7 mL), and 2/5 of the extract diluted to 500 mL by water was cleaned up with a 100 mg Carbograph-4 cartridge. After the addition of the internal standard AFM1, the final extract was analyzed by LC/ESI-MS/MS in positive ion mode using multiple reaction monitoring with a triple-quadrupole instrument. A C(18) column thermostatted at 45 degrees C with a mobile phase gradient of acetonitrile/water with 2 mmol/L ammonium formate was used. Although the matrix suppression effect was negligible, quantitation was achieved by an external calibration procedure using matrix-matched standard solutions to improve accuracy. Sample recoveries at four spiking levels ranged from 81 to 101% (relative standard deviation (RSD) 相似文献   

Development and in-house validation of a robust and sensitive solid-phase extraction liquid chromatography/tandem mass spectrometry method for the quantitative determination of aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods

A sensitive and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods. Samples were extracted with a mixture of acetonitrile/water (84:16, v/v) and cleaned up through a polymeric solid-phase extraction column. Detection and quantification of the nine mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS), using fully (13)C-isotope-labelled mycotoxins as internal standards. The method was validated in-house for five different cereal processed products, namely barley, oat and durum wheat flours, rye- and wheat-based crisp bread. Recoveries and repeatability of the whole analytical procedure were evaluated at contamination levels encompassing the EU maximum permitted levels for each tested mycotoxin. Recoveries ranged from 89 to 108% for deoxynivalenol, from 73 to 114% for aflatoxins, from 85 to 114% for T-2 and HT-2 toxins, from 64 to 97% for zearalenone, from 74 to 102% for ochratoxin A. Relative standard deviations were less than 16% for all tested mycotoxins and matrices. Limits of detection (signal-to-noise ratio 3:1) ranged from 0.1 to 59.2 μg/kg. The trueness of the results obtained by the proposed method was demonstrated by analysis of reference materials for aflatoxins, deoxynivalenol, zearalenone. The use of inexpensive clean-up cartridges and the increasing availability of less expensive LC/MS/MS instrumentation strengthen the potential of the proposed method for its effective application for reliable routine analysis to assess compliance of tested cereal products with current regulation.     

Cleanup procedure for determination of aflatoxins in major agricultural commodities by liquid chromatography

A simple, fast, reliable, and inexpensive chemical cleanup procedure was developed for quantitation of aflatoxins in major important agricultural commodities by liquid chromatography (LC). Aflatoxins were extracted from a ground sample with methanol-water (80 + 20, v/v), and after a single cleanup step on a minicolumn packed with basic aluminum oxide, they were quantitated by LC equipped with a C18 column, photochemical reactor, and fluorescence detector. Water-methanol-1-butanol (1,400 + 720 + 25, v/v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5.0, 2.5, 7.5, and 2.5 microg/kg were 87.2 +/- 2.3, 82.0 +/- 0.8, 80.0 +/- 1.8, and 80.4 +/- 2.8%, respectively. Similar recoveries, precision, and accuracy were achieved for corn, cottonseed, almonds, Brazil nuts, pistachios, and walnuts. The quantitation limit for aflatoxin B1 was 1 microg/kg. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.     

Determination of 16 Mycotoxins in Maize by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

An ultrahigh-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of sterigmatocystin, verruculogen, enniatin A, fusarenon-X, fumonisins B1, B2, B3, aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, and zearalenone. The mycotoxins were extracted and cleaned up using a multitoxin column, separated on a C18 column, and then detected on a triple-quadrupole mass spectrometer. The limits of detection and quantification ranged within 0.2–2?µg/kg and 1–10?µg/kg, respectively. The recoveries ranged from 70.8 to 118.4%, with relative standard deviations below 15%. The method was used to analyze 80 samples obtained from Shandong Province in China. Fifty-eight samples were contaminated with 10 mycotoxins at concentrations ranging from 1.4 to 6566.1?µg/kg. Some samples exceeded the maximum limits in China and in European regulations for mycotoxins in unprocessed maize.     

Ultra-performance liquid chromatography/tandem mass spectrometry for the simultaneous analysis of aflatoxins B1, G1, B2, G2 and ochratoxin A in beer

The simultaneous analysis of aflatoxins B1, G1, B2, G2 and ochratoxin A in beer was carried out by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). Mycotoxins were extracted, purified and concentrated from the beer sample in one step using a solid-phase extraction (SPE) cartridge that contained a polymeric sorbent. Optimization of different parameters, such as type of SPE sorbent, type and amount of wash solvent and pH of the sample, was carried out. The mobile phase consisted of a gradient of methanol + water (0.1% HCOOH) and a reversed-phase C18 column was used for the separation. The mass spectrometer used an electrospray ionization source operated in the positive mode to detect aflatoxins and in the negative mode to detect ochratoxin. UPLC/MS/MS is a rapid and sensitive technique that allows the separation of the five toxins in only 3.2 min. The limit of detection is 1 pg.