Simultaneous determination of aflatoxins, ochratoxin A, and zearalenone in grains by new immunoaffinity column/liquid chromatography

The simultaneous determination of mycotoxins was performed in 3 steps: extraction, cleanup, and detection. For extraction, a mixture of acetonitrile-water (60 + 40, v/v) was proved appropriate. For cleanup, a new Afla-Ochra-Zea immunoaffinity column was used. After derivatization with trifluoroacetic acid, the mycotoxins aflatoxins, ochratoxin A (OTA), and zearalenone (ZEA) were determined simultaneously by liquid chromatography with fluorescence detection. The detection limits in different matrixes after cleanup with the new immunoaffinity column were very low: aflatoxins, 0.002-0.7 microg/kg; OTA, 0.07-0.25 microg/kg; ZEA, 1-3 microg/kg. The limits of determination were: aflatoxins, 0.25 microg/kg; OTA, 0.5 microg/kg; ZEA, 5 microg/kg. The recovery rates for aflatoxins, OTA, and ZEA for rye and rice were between 86 and 93% when a 0.5 g sample matter per immunoaffinity column was used.     

Determination of zearalenone in botanical dietary supplements, soybeans, grains, and grain products by immunoaffinity column cleanup and liquid chromatography: single-laboratory validation

The accuracy, repeatability, and reproducibility characteristics of a method for measuring levels of zearalenone (ZON) in botanical root products, soybeans, grains, and grain products were determined by an AOAC single-laboratory validation procedure. Replicates of 10 test portions of each powdered root product (black cohosh, ginger, ginseng), brown rice flour, brown rice grain, oat flour, rice bran, soybeans, and wheat flour at each spiking level (ZON at 0, 50, 100, and 200 microg/kg) were analyzed on 3 separate days. Test samples were extracted with methanol-water (75 + 25, v/v). The extracts were centrifuged or filtered, diluted with phosphate-buffered saline (PBS) containing 0.5% Tween 20, and filtered; the filtrates were applied to an immunoaffinity column containing antibodies specific for ZON. After the column was washed with methanol-PBS (15 + 85, v/v) containing 0.5% Tween 20 and then with water, the toxin was eluted from the column with methanol, and the eluate was diluted with water. The eluate containing the toxin was then subjected to RPLC with fluorescence detection. All commodities that were found to contain ZON at < 10 microg/kg were used for the recovery study. The average within-day and between-days recoveries of ZON added at levels of 50-200 microg/kg ranged from 82 to 88% and from 81 to 84%, respectively, for all test commodities. The total average of within- and between-day SD and RSDr values for all test commodities ranged from 2.5 to 7.3 microg/kg and from 4.6 to 6.2%, respectively. HorRat values were <1.3 for all matrixes examined. The tested method was found to be acceptable for the matrixes examined.     

Determination of ochratoxin A in wine and beer by immunoaffinity column cleanup and liquid chromatographic analysis with fluorometric detection: collaborative study.

The accuracy, repeatability, and reproducibility characteristics of a liquid chromatographic method for the determination of ochratoxin A (OTA) in white wine, red wine, and beer were established in a collaborative study involving 18 laboratories in 10 countries. Blind duplicates of blank, spiked, and naturally contaminated materials at levels ranging from < or =0.01 to 3.00 ng/mL were analyzed. Wine and beer samples were diluted with a solution containing polyethylene glycol and sodium hydrogen carbonate, and the diluted samples were filtered and cleaned up on an immunoaffinity column. OTA was eluted with methanol and quantified by reversed-phase liquid chromatography with fluorometric detection. Average recoveries from white wine, red wine, and beer ranged from 88.2 to 105.4% (at spiking levels ranging from 0.1 to 2.0 ng/mL), from 84.3 to 93.1% (at spiking levels ranging from 0.2 to 3.0 ng/mL), and from 87.0 to 95.0% (at spiking levels ranging from 0.2 to 1.5 ng/mL), respectively. Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 6.6 to 10.8% for white wine, from 6.5 to 10.8% for red wine, and from 4.7 to 16.5% for beer. Relative standard deviations for between-laboratories reproducibility (RSDR) ranged from 13.1 to 15.9% for white wine, from 11.9 to 13.6% for red wine, and from 15.2 to 26.1% for beer. HORRAT values were < or =0.4 for the 3 matrixes.     

Use of multitoxin immunoaffinity columns for determination of aflatoxins and ochratoxin A in ginseng and ginger

Conditions were optimized for the simultaneous, alkaline, aqueous methanol extraction of aflatoxins (AFL), i.e., B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2), and ochratoxin A (OTA) with subsequent purification, isolation, and determination of the toxins in ginseng and ginger. Powdered roots were extracted with methanol-0.5% NaHCO3 solution (7 + 3). After shaking and centrifugation, the supernatant was diluted with 100 mM phosphate buffer containing 1% Tween 20 and filtered through glass microfiber filter paper. The filtrate was then passed through an immunoaffinity column, and the toxins were eluted with methanol. The AFL were separated and determined by reversed-phase liquid chromatography (RPLC) with fluorescence detection after postcolumn UV photochemical derivatization. OTA was separated and determined by RPLC with fluorescence detection. Recoveries of AFL added at 2-16 ng/g and OTA added at 1-8 ng/g to ginseng were 72-80 and 86-95%, respectively. Recoveries of AFL and OTA added to ginger were similar to those for ginseng. A total of 39 commercially available ginger products from 6 manufacturers were analyzed. Twenty-six samples were found to be contaminated with AFL at 1-31 ng/g and 29 samples, with OTA at 1-10 ng/g. Ten samples contained no AFL or OTA. Ten ginseng finished products were also analyzed; 3 contained AFL at 0.1 ng/g and 4 contained OTA at levels ranging from 0.4 to 1.8 ng/g. LC/tandem mass spectrometry with multiple-reaction monitoring of 3 collisionally induced product ions from the protonated molecular ions of OTA, AFB1, and AFG1 was used to confirm the identities of the toxins in extracts of the finished products.     

免疫亲和柱净化-柱后光化学衍生-高效液相色谱法同时检测粮谷中的黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

建立了同时检测粮谷中黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A的免疫亲和柱净化-柱后光化学衍生-高效液相色谱方法。样品经过甲醇-水(体积比为80∶20)提取,通过免疫亲和柱富集和净化,采用Waters Nova-Pak色谱柱(3.9 mm i.d.×150 mm,4 μm),以甲醇、乙腈和1%的磷酸溶液为流动相,梯度洗脱,柱后光化学衍生、改变波长荧光检测。黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A检出限分别为0.24,4.0和0.5 μg/kg,标准曲线的线性范围分别为0.24~6.0,4.0~100.0和0.5~40.0 μg/L;在小麦、玉米、黑麦样品中,平均加标回收率为70.8% ~94.0%,相对标准偏差为2.79% ~9.38%。     

Simultaneous determination of four aflatoxins and ochratoxin A in ginger and related products by HPLC with fluorescence detection after immunoaffinity column clean‐up and postcolumn photochemical derivatization

Ginger, a widely used spice and traditional Chinese medicine, is prone to be contaminated by mycotoxins. A simple, sensitive, and reproducible method based on immunoaffinity column clean‐up coupled with HPLC and on‐line postcolumn photochemical derivatization with fluorescence detection was developed for the simultaneous determination of aflatoxins (AFs) B₁, B₂, G₁, G₂, and ochratoxin A (OTA) in 25 batches of gingers and related products marketed in China for the first time. The samples were first extracted by ultrasonication with methanol/water (80:20, v/v) and then cleaned up with immunoaffinity columns for analysis. Under the optimized conditions, the LODs and LOQs for the five mycotoxins were 0.03–0.3 and 0.1–0.9 μg/kg, respectively. The average recoveries ranged from 81.3–100.8% for AFs and from 88.6–99.5% for OTA at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all >0.9995. All moldy gingers were contaminated with at least one kind of the five investigated mycotoxins, while none of them were found in normal gingers. Ginger powder samples were contaminated slightly with the contamination levels below the LOQs, while ginger tea bags were mainly contaminated by OTA at 1.05–1.19 μg/kg and ginger black tea bags were mainly contaminated by AFs at 3.37–5.76 μg/kg. All the contamination levels were below the legally allowable limits.     

Determination of fumonisin B1 in botanical roots by liquid chromatography with fluorescence detection: single-laboratory validation

The accuracy, repeatability, and reproducibility characteristics of a published method for measuring levels of fumonisin B1 (FB1) in botanical root products were determined by an AOAC single-laboratory validation procedure. Replicates of 10 test portions of each powdered root product (black cohosh, echinacea, ginger, ginseng, valerian, dong quai, and turmeric) at each spiking level (FB1 at 0, 50, 100, and 200 ng/g) were analyzed on 3 separate days. Test samples were extracted with methanol-acetonitrile-water (25 + 25 + 100, v/v/v). The extracts were centrifuged, the supernatants diluted with phosphate-buffered saline (PBS) containing 1% Tween 20 and filtered, and the filtrates applied to an immunoaffinity column containing antibodies specific for fumonisins. After the column was washed sequentially with PBS and water, the toxin was eluted from the column with 80% methanol, and the eluate dried by lyophilization. The residue was reconstituted with 50% acetonitrile. FB1 was derivatized with a mixture of o-phthaldialdehyde and mercaptoethanol by using an LC autoinjector. Separations were performed with an RP-LC column, and the FB1 derivative was quantified by fluorescence detection. All root products were found to contain FB1 at <10 ng/g. Average within- and between-day recoveries of FB1 from the botanical roots ranged from 67 to 95% and from 68 to 100%, respectively. Total RSD values for within- and between-day repeatability ranged from 5.5 to 26.4%. HorRat values were <1.3 for all of the matrixes examined. The method meets the AOAC method performance criteria at levels of >50 ng/g for the seven botanical roots tested.     

Sampling and analytical variability associated with the determination of aflatoxins and ochratoxin A in bulk lots of powdered ginger marketed in 1-lb bags

Ginger has been used as a food, dietary supplement, and condiment for centuries. Mycotoxins such as the aflatoxins (AF) and ochratoxin A (OTA) have been reported in ginger roots in several studies. It is important to design effective sampling methods that will accurately and precisely predict the true mycotoxin level in a bulk lot. The objective of this study was to measure the sampling and analytical variability associated with the test procedure used to measure AF and OTA in a bulk lot of powdered ginger using a 5-g laboratory sample and HPLC analytical methods. Twelve 5-g laboratory samples were taken from each of two lots. Duplicate aliquots were removed from each 5-g laboratory sample/solvent blend, and each aliquot was simultaneously analyzed for AF and OTA by HPLC analytical methods. Using a balanced nested design, the total variance associated with the above AF and OTA test procedures was partitioned into sampling and analytical variance components for each lot. Averaged across both lots, the sampling and analytical variances accounted for 87% and 13% of the total variance, respectively, for AF and 97% and 3%, respectively, for OTA. The sampling and analytical coefficients of variation were 9.5% and 3.6%, respectively, for AF, and 16.6% and 2.9%, respectively, for OTA when using a single 5-g laboratory sample and HPLC analytical methods. Equations are derived to show the effect of increasing laboratory sample size and/or number of aliquots on reducing the variability of the test procedures used to estimate OTA and AF in powdered ginger.     

Determination of aflatoxins and ochratoxin A in ginseng and other botanical roots by immunoaffinity column cleanup and liquid chromatography with fluorescence detection

Mycotoxins are toxic secondary metabolites produced by certain molds and are common contaminants of many important food crops, such as grains, nuts, and spices. Some mycotoxins are found in fruits, vegetables, and botanical roots. These contaminants have a broad range of toxic effects, including carcinogenicity, immunotoxicity, neurotoxicity, and reproductive and developmental toxicity. The public health concerns related to both acute and chronic effects of mycotoxins in animals have prompted more than 100 countries to establish regulatory limits for some of the well-known mycotoxins, such as the aflatoxins (AFL). Our research focused on method development for 2 of these toxins, AFL and ochratoxin A (OTA), in ginseng and other selected botanical roots. Methods using an immunoaffinity column (IAC) cleanup, liquid chromatographic separation, and fluorescence detection were modified and evaluated. Two types of IAC cleanup were evaluated: IAC for AFL, and IAC for both AFL and OTA. Three derivatization techniques to enhance the fluorescence of the AFL were compared: precolumn trifluoroacetic acid, postcolumn bromination, and postcolumn ultraviolet irradiation. No derivatization was needed for OTA. Results for AFL using the single analyte IAC cleanup and the 3 derivatization techniques were all comparable for ginseng and for other roots such as ginger, licorice, and kava-kava. Recoveries of added AFL for ginseng at levels from 2 to 16 ng/g were about 80%. Using IAC cleanup for both AFL and OTA recoveries of added AFL for ginseng at 4-16 ng/g were about 70%, and for ginger, licorice, and kava-kava were about 60%. Recoveries of added OTA for ginseng, ginger, and echinacea at 4 ng/g were about 55%.     

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.     

Determination of fumonisins B1 and B2 in corn and corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative study.

A liquid chromatographic (LC) method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn and corn flakes was collaboratively studied by 23 laboratories, which analyzed 5 blind duplicate pairs of each matrix to establish the accuracy, repeatability, and reproducibility characteristics of the method. Fumonisin levels in the corn ranged from <0.05 (blank) to 1.41 microg/g for FB1 and from <0.05 to 0.56 microg/g for FB2, whereas in the corn flakes they ranged from <0.05 to 1.05 microg/g for FB1 and from <0.05 to 0.46 microg/g for FB2. The method involved double extraction with acetonitrile-methanol-water (25 + 25 + 50), cleanup through an immunoaffinity column, and LC determination of the fumonisins after derivatization with o-phthaldialdehyde. Relative standard deviations for the within-laboratory repeatability (RSDr) of the corn analyses ranged from 19 to 24% for FB1 and from 19 to 27% for FB2; for the corn flakes analyses, RSDr ranged from 9 to 21 % for FB1 and from 8 to 22% for FB2. Relative standard deviations for the between-laboratories reproducibility (RSDR) of the corn analyses ranged from 22 to 28% for FB1 and from 22 to 30% for the FB2; for corn flakes analyses, RSDR ranged from 27 to 32% for FB1 and from 26 to 35% for FB2. Mean recoveries of FB1 and FB2 from corn spiked with FB1 at 0.80 microg/g and with FB2 at 0.40 microg/g were 76 and 72%, respectively; for corn flakes spiked at the same levels recoveries were 110 and 97% for FB1 and FB2, respectively. HORRAT ratios for the analyses of corn ranged from 1.44 to 1.53 for FB1 and from 0.96 to 1.48 for FB2, whereas for corn flakes they ranged from 1.60 to 1.82 for FB1 and from 1.39 to 1.68 for FB2.     

Determination of fumonisins B1 and B2 in corn-based foods for infants and young children by LC with immunoaffinity column cleanup: interlaboratory validation study

A liquid chromatographic method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn-based foods for infants and young children was subjected to an interlaboratory validation study involving 11 laboratories. Five blind duplicate sample pairs of each matrix were analyzed to establish the accuracy, repeatability, and reproducibility of the method. Mass fractions in the baby food samples ranged from 89.1 to 384.4 microg/kg FB1 and from 22.5 to 73.6 microg/kg FB2. The method involved a warm extraction with citrate phosphate buffer-methanol-acetonitrile (50 + 25 + 25, v/v/v), a cleanup through an immunoaffinity column, and an end-determination of fumonisins by LC after automated precolumn derivatization with o-phthaldialdehyde reagent. RSDs for within-laboratory repeatability (RSDr) ranged from 6.8 to 23.5% for FB1 and 7.6 to 22.9% for FB2. RSDs for between-laboratory reproducibility (RSDR) ranged from 15.4 to 26.2% for FB1 and 21.6 to 36.3% for FB2. Mean FB1 recoveries from baby foods spiked at 100.0 and 250.0 microg/kg were 89 and 96%, respectively; for FB2 spiked foods at 25.0 and 62.5 microg/kg recoveries were 90 and 85%, respectively. HorRat values ranged from 0.8 to 1.2 for FB1, whereas for FB2 they ranged from 0.9 to 1.4 when calculated according to Horwitz, and from 1.0 to 1.7 when calculated according to Thompson, indicating an acceptable among-laboratory precision for all matrixes (HorRat values <2).     

Aptamer‐affinity column clean‐up coupled with ultra high performance liquid chromatography and fluorescence detection for the rapid determination of ochratoxin A in ginger powder

Aptamers are single‐stranded oligonucleotides with high affinity and specificity and are widely used in targets separation and enrichment. Here, an aptamer‐affinity column (AAC) was firstly prepared in‐house through a covalent immobilization strategy. Then, ochratoxin A (OTA) in ginger powder was absorbed and enriched using the new aptamer‐based clean‐up technology for the first time, and was further analyzed by ultra high performance liquid chromatography with fluorescence detection. After optimization, the average recoveries for blank samples spiked with OTA at 5, 15, and 45 μg/kg ranged from 85.36 to 96.83%. Furthermore, the AAC exhibited a similar accuracy as an immunoaffinity column to clean up OTA in ginger powder. Above all, it exhibited better reusability, twice that of the immunoaffinity column, had lower toxicity and cost, and took less time. Of 25 contaminated ginger powder samples, OTA contamination levels ranged from 1.51 to 4.31 μg/kg, which were lower than the European Union (EU) regulatory limits. All the positive samples were further confirmed by ultra‐fast LC with MS/MS. In conclusion, the method of clean‐up based on the AAC coupled to ultra‐HPLC with fluorescence detection was rapid, specific, and sensitive for the quantitative analysis of OTA in a complex matrix.     

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.     

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.     

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.     

Simultaneous determination of aflatoxins B1, B2, G1, G2 in Fructus Bruceae by high‐performance liquid chromatography with online postcolumn photochemical derivatization

A simple, reliable, and highly sensitive method for the simultaneous determination of aflatoxin B₁, B₂, G₁, G₂ in Fructus Bruceae was developed using high‐performance liquid chromatography coupled to online postcolumn photochemical derivatization and fluorescence detection. Aflatoxins were first extracted by a methanol/water mixture and then cleaned up with an AflaTest? immunoaffinity column. Different clean‐up and derivatization methods were compared and optimized. The established method was extensively validated to show satisfactory performance of linearity (R² ≥ 0.9997), recovery (74.3–100.8%), and precision (RSDs ≤ 2.8%) for the investigated aflatoxins. This proposed method was also applied to 11 F. Bruceae samples and the results showed that 10 out of 11 were contaminated with aflatoxins ranging from 0.26 to 27.52 μg/kg and the occurrence of aflatoxin B₁, the most toxic one, was as high as 91% in all the samples, highlighting the severe contamination and the necessity to set legal limits for aflatoxins in F. Bruceae.     

A survey of ochratoxin A and aflatoxins in domestic and imported beers in Japan by immunoaffinity and liquid chromatography.

Analyses of ochratoxin A (OTA) and aflatoxins (AFs) in 94 imported beer samples from 31 producing countries and in 22 Japanese beer samples were performed by immunoaffinity column and reversed-phase liquid chromatography (LC) with fluorescence detection. Recoveries of OTA from beer samples spiked at 25 and 250 pg/mL were 86.1 and 88.2%, respectively. Recoveries of AFs were 98.4 and 98.9%, 95.4 and 95.5%, 101.2 and 97.8%, and 98.9 and 96.0%, respectively, from beer samples spiked at 4.1 and 41 pg AF B1, 4.45 and 44.5 pg AF B2, 4.7 and 47 pg AF G1, and 4.65 and 46.5 pg AF G2/mL. Detection limits were 1.0 pg/mL for OTA, 0.5 pg/mL for AFs B1 and B2, and 1.0 pg/mL for AFs G1 and G2. OTA was detected in 86 (91.5%) of 94 imported beer samples at a mean level of 10.1 pg/mL and in 21 (95.5%) of 22 Japanese beer samples at a mean level of 12.5 pg/mL. AF B1 was detected in 11 of 94 imported beer samples at a level of 0.5-83.1 pg/mL and in 2 of 22 Japanese beer samples at 0.5 and 0.8 pg/mL. Except for one beer sample from Peru, the samples contaminated with AFs were also contaminated with OTA. Although OTA was detected in most samples from various countries, AFs were detected in the beer samples from only a limited number of countries where AF contamination might be expected to occur because of their warm climate.     

Application of dispersive liquid-liquid microextraction for the determination of aflatoxins B1, B2, G1 and G2 in cereal products

The application of dispersive liquid-liquid microextraction (DLLME) technique for the rapid analysis of aflatoxins B(1), B(2), G(1) and G(2) in maize, rice and wheat products has been evaluated. After extraction of aflatoxins from cereal matrices with a mixture of methanol/water 8:2 (v/v), the analytes were rapidly transferred from the extract to another small volume of organic solvent, chloroform, by DLLME. Aflatoxins were determined using high performance liquid chromatography with florescence detection and photochemical post-column derivatization. Parameters affecting both extraction and DLLME procedures, such as extraction solvent, type and volume of DLLME extractant, volume of water and salt effect, were systematically investigated and optimized to achieve the best extraction efficiency. Under the optimal experimental conditions, the whole analytical method provides enrichment factors around 2.5 times and detection limits (0.01-0.17 μg kg(-1)) below the maximum levels imposed by current regulation for aflatoxins in cereals and cereal products intended for direct human consumption. Recoveries (67-92%) and repeatability (RSD<10, n=3), tested in three different cereal matrices, meet the performance criteria required by EC Regulation No. 401/2006 for the determination of the levels of mycotoxins in foodstuffs. The proposed method was successfully applied to the analysis of retail cereal products with quantitative results comparable to the immunoaffinity chromatography (IAC). The main advantages of developed method are the simplicity of operation, the rapidity to achieve a very high sample throughput and low cost.     

Determination of ochratoxin A at parts-per-trillion levels in cereal products by immunoaffinity column cleanup and high-performance liquid chromatography/mass spectrometry

Ochratoxin A (OTA) is a toxic and potentially carcinogenic fungal toxin found in a variety of food commodities. A new sensitive method has been developed to quantify OTA in cereal products by reversed-phase liquid chromatography (LC) with mass spectrometric (MS) detection. Ochratoxin B was used as the internal standard. OTA was extracted from cereal products with acetonitrile-water, and the extract was diluted with a buffer; the diluted extract was cleaned up on an immunoaffinity column before LC/MS analysis. Two multiple-reaction monitoring transitions were used, one for quantification of OTA and one for confirmation of identity. The method was shown to be highly sensitive, with a low decision limit (CCalpha) of 0.012 microg/kg and a detection capability (CCbeta) of 0.021 microg/kg. Within-laboratory repeatability coefficient of variation values were 7.1, 3.7, and 3.1%, and the corresponding recoveries were 104, 106, and 103% for rice samples fortified with OTA at 0.05, 0.10, and 0.15 microg/kg, respectively. Method validation was performed according to the criteria of European Commission Decision 2002/657/EC. All criteria as presented in the Commission Decision were fulfilled. This method is the first fully validated method using immunoaffinity chromatography for cleanup and MS for detection in the analysis of cereals for OTA. The method was also successfully applied to cereal-derived products. The analytical results for determination of the OTA content of cereal products commercially available in Hong Kong are also reported.