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Spices are very important for cuisines around the world as well as for health enhancement. The Egyptians, Chinese, and Indians have used spices in medicinal remedies and procedures starting in around 2000 BC. Through the centuries, spices have found use as food ingredients to modify the aroma and taste of the final products; however, some spices are suitable substrates for mold growth and mycotoxin development, which could be detrimental to human and animal health. This report covers regulatory control of mycotoxins in food and spices by means of monitoring and regulatory limits, sampling and analysis, management, and prevention of mycotoxins from plant growth (preharvest) through harvest and postharvest as well as decontamination for mycotoxins when necessary. There is no one single-best strategy that can solve mycotoxin contamination problems, but a well-designed and integrated plan of all these strategies could result in a substantial reduction of mycotoxins in spices to regulation safety levels.

Background: The Reveal Q+ MAX for Aflatoxin is a lateral flow immunochromatographic test intended for quantitative analysis within 6 min after aqueous extraction. Objective: Work was conducted to validate the performance of the Reveal Q+ MAX for Aflatoxin method in selected corn and nut matrixes. Methods: This method was validated under the requirements of the AOAC Research Institute Performance Tested MethodSM program. Fivematrixes, including corn naturally contaminated with aflatoxin at 0, 5.2, 21.0, 51.6, 103.6, and 282 ppb as well as peanuts, pistachios, walnuts, and almonds spiked at 0, 5, 20, 50, and 300 ppb were analyzed. Results: Average percentage recoveries of the added aflatoxin from the matrixes ranged from 80.8 to 116.9%. Average LOD for all matrixes is 2 ppb and LOQ is 7 ppb. With the exceptionof sample size for almonds, robustness trials demonstrated that deliberate changes to the assay parameters minimally affected the Reveal Q+ MAX assay performance. Finally, stability results from three independently manufactured lots support Reveal Q+ MAX for Aflatoxin performance consistency and shelf-life of 18 months when stored at room temperature. Conclusions: This study appropriatelyvalidates the Performance Tested MethodSM claim forcorn and selected nut matrixes on Reveal Q+ MAX forAflatoxin, an aqueous lateral flow test kit. Highlights: Aqueous lateral flow test kit detects total aflatoxin between 80 to 120% yieldwith an LOD of 2 ppb.

Bioo Scientific Corp. has developed a rapid enzymatic quantitative assay for the determination of histamine in seafood. Fresh/frozen tuna, canned tuna, pouched tuna, and frozen mahi mahi samples were used for the validation study under the specific guidelines of the AOAC Research Institute Performance Tested MethodsSM program. Recoveries ranged from 82 to 107% at concentrations ranging from 6 to 72 ppm, with RSDr values between 0.8 and 6.5% (6–72 ppm). The linearity of the assay ranged from 0 to 108 ppm, with R2 values exceeding 0.99. The LOD was 0.9 ppm and the LOQ was 2.6 ppm for frozen tuna, which gave the lowest background level of contaminant. Cross-reactivity of the assay was tested against 14 other biogenic amines and was found to be minimal for all (

The aim of this study was to investigate the feasibility of using multitime-regenerated immunoaffinity column (IAC) for aflatoxins B1, B2, G1 and G2 in peanut confection. After each use, the IAC was washed immediately with phosphate-buffered saline and stored for >12h prior to reuse. The evaluation procedure consisted of using extracts of naturallycontaminated peanut confection (4 replicates), aflatoxin-free peanut confection (duplicates), and aflatoxin-free peanut confection sample spiked with the 4 aflatoxins (AFT) at 3 levels in 4 replicates. Each day, 18 test extracts were analyzed using 18 designated IACs. After each use, the IACs were regenerated and reused for corresponding test extracts on the following day. This procedure was repeated daily over the course of 9days. Analytical steps included passing the test extracts through the IACs, washing the columns with water, and eluting AFT with methanol. The eluates were diluted with water and were subjected to reversed phase LC separation, post-column photochemical derivatization and fluorescence detection. After eluting AFT, IACs were immediately regenerated by washing with phosphate buffer solution and storing overnight at 8°C for re-use the following day. Results were analyzed using ANOVA and Tukey tests. The numbers of reuse varied for each AF: For AFB1 AFB2, AFG1and AFG2 could be reused for 9, 6, 6 and 0 times, respectively. According to AOAC method performance criteria, recoveries ranging from 70% to 125% are considered acceptable at the spiking levels used in this study.

Portions of corn, a commodity in which aflatoxin is frequently found, were held at 16, 26 and 32°C after the moisture contents were adjusted to achieve water activities (aw) ranging from too low to ample for support of mold growth. Suspensions of mold spores from toxigenic cultures of Aspergillus flavus , A. ochraceus , Penicillium citrinum , P. cyclopium and P. urticae were added to the test portions, either as A. flavus alone, as A. flavus with one of the other molds or as a mixture of all 5 species. Additional water was used to obtain the proper moisture levels. A temperature of 16°C was generally too low for aflatoxin production by either the added or native strains of A. flavus , although the mold was able to grow at 16°C at aw values as low as 0.80, 0.77 and 0.85 on corn, soybeans and pinto beans, respectively. Aflatoxin production was essentially the same at 26 and 32° C with limiting aw values in the range of 0.85-0.89. Limiting aw values for mold growth at 26 and 32°C were 0.73, 0.69 and 0.75 for corn, soybeans and pinto beans, respectively. This study provided no evidence that substrate suitability at limiting temperatures and aw levels is a factor in the observed difference in the risk of aflatoxin contamination for these commodities. The study did indicate that the associated mold flora, when the seed is exposed to mold invasion, is a risk determinant.

A total of 118 imported cheeses from 13 countries were analyzed for aflatoxin M

Aflatoxin B1 was detected, and its identity confirmed, in hypoallergenic milk substitutes composed, among other things, of the following ingredients susceptible to possible aflatoxin contamination: soya protein isolate, soy and coconut oils, cornstarch, and corn syrup. Except for one determination all findings were under 1 ng of aflatoxin/ml of formula at drinking concentration. Manufacturers' reserve samples of soya protein isolate, the ingredient thought to be the most likely source of the aflatoxin, were found to be uncontaminated. Reserve samples of other ingredients were not examined. Because hypoallergenic formula may be the major nutrient of an individual with a metabolic defect at infancy, a vulnerable stage of life, available control measures should be used to avoid aflatoxin-contaminated ingredients.

The study's objectives were to evaluate a method for the determination of aflatoxins (AFs) in the Brazilian peanut confection “Paçoca” and to apply the method in investigating AF concentrations in Paçoca marketed in São Paulo State throughout 2013. Results of another survey conducted between 1994 and 2002 with another method were also reported. The current method consists of immunoaffinity column cleanup, LC with postcolumn derivatization for AF fluorescence enhancement, and fluorescence determination for the toxins. The mean recovery and mean RSDr values were 88.6 and 7.9%, respectively. The LODs for aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 were 0.04, 0.01, 0.02, and 0.01 ng/g; and the LOQs were 0.15, 0.04, 0.07, and 0.04 ng/g, respectively. Results of the two survey studies indicate that the contamination of AFs in Paçoca remains a public health problem. In the 2013 survey, 71 of 100 samples (71%) had AFs contamination ranging from 0.3 to 41.8 ng/g, with 12 samples (12%) containing >20 ng/g of the toxins, whereas in the 1994–2002 survey, 73 of 150 samples (51%) had AFs contamination ranging from 9 to 1439 ng/g with 65 samples (45%) containing levels >20 ng/g.

Ultrahigh-performance liquid chromatography using positive electrospray ionization and quadrupole orbital ion trap high-resolution mass spectrometry was evaluated for analyzing mycotoxins in finished cereal and nut products. Optimizing the orbital ion trap mass analyzer in full-scan mode using mycotoxin-fortified matrix extracts gave mass accuracies, δM, of± 2.0 ppm at 70,000 full width at half maximum (FWHM) mass resolution (RFWHM). The limits of quantitation were matrix- and mycotoxin-dependent, ranging from 0.02 to 11.6 μg/kg. Mean recoveries and standard deviations for mycotoxins from acetonitrile/water extraction at their relevant fortification levels were 91 ± 10, 94 ± 10, 98 ± 12, 91 ± 13, 99 ± 15, and 93 ± 17% for corn, rice, wheat, almond, peanut, and pistachio, respectively. Nineteen mycotoxins with concentrations ranging from 0.3 (aflatoxin B1 in peanut and almond) to 1175 μg/kg (fumonisin B1 in corn flour) were found in 35 of the 70 commercial grain and nut samples surveyed. Mycotoxins could be identified at δM± 5 ppm by identifying the precursor and product ions in full-scan MS and data-dependent MS/MS modes. This method demonstrates a new analytical approach for monitoring mycotoxins in finished grain and nut products.

We developed a stable isotope dilution assay with liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine multiple mycotoxins in baby foods and animal feeds. Samples were fortified with [(13)C]-uniformly labeled mycotoxins as internal standards ([(13)C]-IS) and prepared by solvent extraction (50% acetonitrile in water) and filtration, followed by LC-MS/MS analysis. Mycotoxins in each sample were quantitated with the corresponding [(13)C]-IS. In general, recoveries of aflatoxins (2-100 ng/g), deoxynivalenol, fumonisins (50-2000 ng/g), ochratoxin A (20-1000 ng/kg), T-2 toxin, and zearalenone (40-2000 ng/g) in tested matrices (grain/rice/oatmeal-based formula, animal feed, dry cat/dog food) ranged from 70 to 120% with relative standard deviations (RSDs)20%. The method provides sufficient selectivity, sensitivity, accuracy, and reproducibility to screen for aflatoxins at ng/g concentrations and deoxynivalenol and fumonisins at low μg/g concentrations in baby foods and animal feeds, without using conventional standard addition or matrix-matched calibration standards to correct for matrix effects.

The objective of this study was to determine the extent of aflatoxin M1 (AFM1) and ochratoxin A (OTA) contamination in human breast milk in the city of Ribeirao Preto, Sao Paulo State, Brazil. During 2012,100 samples of human milk were collected at the local Human Milk Bank. The method comprised, immunoaffinity column purification and isolation, liquid chromatography separation and fluorescence detection. The average percentage recoveries of AFM1 and OTA spiked at 20 and 50 ng/L in control human milk were 78.1 � 11.7% and 73.7 � 9.6%, respectively. The average relative standard deviations of AFM1 and OTA spiked at the same levels were 11.7 and 9.6% respectively. The limits of detection was 0.3 ng/L for AFM1 and OTA. The limit of determination was 0.8 ng/L for both mycotoxins. This method was used to analyze 100 human milk samples, of which, two samples were found to contain AFM1 at level greater than 0.3 ng/L. OTA was detected in 66 samples (66%), wherein 32 were above the limit of detection and 34 were in the range of from 0.8 to 21 ng/L. Results of our study indicate that breast-fed Brazilian infants had only an insignificant exposure to AFM1 and OTA.

Stable isotope dilution with LC/MS/MS was used to determine the following 11 mycotoxins in food grade gums: aflatoxins B1, B2, G1, and G2; deoxynivalenol; fumonisins B1, B2, and B3; ochratoxin A; T-2 toxin; and zearalenone. Samples were fortified with 11 [13C]-uniformly labeled internal standard ([13C]-IS) mycotoxins that corresponded to the 11 target mycotoxins and extracted by acetonitrile–water (4 + 1, v/v), followed by LC/MS/MS analysis. Mycotoxins were quantitated with the fortified [13C]-IS in each sample. The average recoveries of aflatoxins B1, B2, G1, and G2 (1, 5, and 25 μg/kg); deoxynivalenol and fumonisins B1, B2, and B3 (25, 100, and 500 μg/kg); and ochratoxin A, T-2 toxin, and zearalenone (10, 50, and 250 μg/kg) ranged from 84 to 117% with RSDs less than 20%. Method-dependent LOQs were from 0.1 (aflatoxin B1) to 25 μg/kg (fumonisin B3). Among 20 market samples, aflatoxin B1 (< LOQ) was detected in a Guar gum and a Tragacanth gum, and zearalenone (6 ± 0.6 μg/kg) was detected in a Xanthan gum. The detected mycotoxins were further confirmed by comparing their enhanced product ion spectra to those of reference standards. The single laboratory validated stable isotope dilution and LC/MS/MS method provides sufficient selectivity, sensitivity, accuracy, and reproducibility with a simple sample preparation to screen the 11 mycotoxins in gums.

A stable isotope dilution assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of 12 mycotoxins, aflatoxins B₁, B₂, G₁, G₂, and M₁, deoxynivalenol, fumonisins B₁, B₂, and B₃, ochratoxin A, T-2 toxin, and zearalenone, in milk-based infant formula and foods. Samples were fortified with 12 ¹³C uniformly labeled mycotoxins ([¹³C]-mycotoxins) that correspond to the 12 target mycotoxins and prepared by dilution and filtration, followed by LC-MS/MS analysis. Quantitation was achieved using the relative response factors of [¹³C]-mycotoxins and target mycotoxins. The average recoveries in fortified milk, milk-based infant formula, milk powder, and baby yogurt of aflatoxins B₁, B₂, G₁, and G₂ (2, 10, and 50 μg/kg), aflatoxin M₁ (0.5, 2.5, and 12.5 μg/kg), deoxynivalenol, fumonisins B₁, B₂, and B₃ (40, 200, and 1000 μg/kg), ochratoxin A, T-2 toxin, and zearalenone (20, 100, and 500 μg/kg), range from 89 to 126% with RSDs of20%. The individual recoveries in the four fortified matrices range from 72% (fumonisin B₃, 20 μg/kg, milk-based infant formula) to 136% (T-2 toxin, 20 μg/kg, milk powder), with RSDs ranging from 2 to 25%. The limits of quantitation (LOQs) were from 0.01 μg/kg (aflatoxin M₁) to 2 (fumonisin B₁) μg/kg. Aflatoxin M₁ was detected in two European Reference materials at 0.127 ± 0.013 μg/kg (certified value = 0.111 ± 0.018 μg/kg) and 0.46 ± 0.04 μg/kg (certified value = 0.44 ± 0.06 μg/kg), respectively. In 60 local market samples, aflatoxins B₁ (1.14 ± 0.10 μg/kg) and B₂ (0.20 ± 0.03 μg/kg) were detected in one milk powder sample. Aflatoxin M₁ was detected in three imported samples (condensed milk, milk-based infant formula, and table cream), ranging from 0.10 to 0.40 μg/kg. The validated method provides sufficient selectivity, sensitivity, accuracy, and reproducibility to screen for aflatoxin M₁ at nanograms per kilogram concentrations and other mycotoxins, without using standard addition or matrix-matched calibration to compensate for matrix effects.

Mycotoxins in foods have long been recognized as potential health hazards due to their toxic and carcinogenic properties. A simple and rapid method was developed to detect 26 mycotoxins (aflatoxins, ochratoxins, fumonisins, trichothecenes, and ergot alkaloids) in corn, rice, wheat, almond, peanut, and pistachio products using high-performance liquid chromatography-triple-quadrupole mass spectrometry. Test portions of homogenized grain or nut products were extracted with acetonitrile/water (85:15, v/v), followed by high-speed centrifugation and dilution with water. Mean recoveries (± standard deviations) were 84 ± 6, 89 ± 6, 97 ± 9, 87 ± 12, 104 ± 16, and 92 ± 18% from corn, rice, wheat, almond, peanut, and pistachio products, respectively, and the matrix-dependent instrument quantitation limits ranged from 0.2 to 12.8 μg/kg, depending on the mycotoxin. Matrix effects, as measured by the slope ratios of matrix-matched and solvent-only calibration curves, revealed primarily suppression and were more pronounced in nuts than in grains. The measured mycotoxin concentrations in 11 corn and wheat reference materials were not different from the certified concentrations. Nineteen mycotoxins were identified and measured in 35 of 70 commercial grain and nut products, ranging from 0.3 ± 0.1 μg/kg (aflatoxin B1 in peanuts) to 1143 ± 87 μg/kg (fumonisin B1 in corn flour). This rapid and efficient method was shown to be rugged and effective for the multiresidue analysis of mycotoxins in finished grain and nut products.

The objective of this study was to investigate the incidence and occurrence of aflatoxin M 1 (AFM 1 ) in Brazilian milk and infant formula. The distribution and stability of AFM 1 in cheese and yoghurt were also determined. Milk samples and infant formula samples were purchased in Ribeirao Preto-SP, Brazil and were analyzed for AFM 1 using immunoaffinity column purification, liquid chromatography separation and fluorescence detection. AFM 1 was detected in 83% of the milk samples (>3 ng/kg) with levels ranging from 8 to 437 ng/kg for fluid milk, and 20–760 ng/kg for powdered milk. No AFM 1 was found in infant formula. Processing and storage was shown to have little effect on AFM 1 content in milk and milk products. Total AFM 1 mass in milk was reduced by 3.2% in cheese and by 6% in yoghurt (pH 4.4). The mean concentration of AFM 1 in curds was 1.9-fold higher and whey was 0.6-fold lower than in unprocessed milk.

The aim of this study was to investigate the feasibility of using multitime-regenerated immunoaffinity column (IAC) for aflatoxins B

The presence of mycotoxins (such as aflatoxins, deoxynivalenol, fumonisins, and patulin) is routinely monitored by the U.S. Food and Drug Administration (FDA) to ensure that their concentrations in food are below the levels requiring regulatory action or advisories. To improve the efficiency of mycotoxin analysis, the researchers at the FDA's Center for Food Safety and Applied Nutrition have been evaluating modern LC-MS technologies. Consequently, a variety of LC–tandem MS and LC–high-resolution MS methods have been developed, which simultaneously identify and quantitate multiple mycotoxins in foods and feeds. Although matrix effects (matrix-induced ion suppression or enhancement) associated with LC-MS-based mycotoxin analysis remain, this review discusses methods for managing these effects and proposes practical solutions for the future implementation of LC-MS-based multimycotoxin analysis.

The objective of this study was to investigate the incidence and occurrence of aflatoxin M1 (AFM1) in dairy products produced in Brazil. A total of 123 samples of three different groups of dairy products (cheese, yoghurt, and dairy drinks) consumed by Brazilians were collected during 2010. All samples including 58 cheese samples, 53 samples of yoghurt and 12 dairy drinks were purchased from grocery stores in the Ribeirao Preto-SP area. Cheese samples were classified into three categories depending on their moisture and fat contents: Minas Frescal cheese, Minas Frescal light cheese and Minas Padrao cheese. Samples were analyzed for AFM1 by a published method. The method comprised aqueous methanol extraction, immunoaffinity column purification and isolation, reversed phase liquid chromatography separation and fluorescence detection. AFM1 was detected in 84% of the analyzed cheese samples (>3 ng/kg) with levels ranging from 10 to 304 ng/kg in 67% of the samples. AFM1 was detected in 95% of the yoghurt and dairy drink samples with levels ranging from 10 to 529 ng/kg in 72% of the samples. Despite the lack of a Brazilian regulatory limit for AFM1 in yoghurt and dairy drinks the survey data of this study may offer information useful in the determination of whether the occurrence of AFM1 in Brazilian dairy products may be considered as a possible risk for consumer health and whether Brazilian regulatory guidelines for AFM1 in dairy products are needed.

Deoxynivalenol (DON), also known as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium spp. DON, 12, 13-epoxy-3,7 trihydroxytrichothec-9-en-8-one, is one of the most frequently detected mycotoxins in agricultural commodities worldwide. A method consisting of extraction, filtration, column cleanup, and RP-HPLC-UV separation and quantitation was validated for the determination of DON in grains (rice and barley), grain products (whole wheat flour, white flour, wheat germ, and wheat bran), and processed foods (bread, breakfast cereals, and pretzels). A 25 g test portion was extracted with 100 mL acetonitrile-water (84 + 16, v/v). After blending for 3 min, the supernatant was applied to a multifunctional column (MycoSep 225). The purified filtrate (2 mL) was evaporated to dryness and redissolved in the mobile phase. The toxins were then subjected to RP-HPLC-UV analysis. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON added at levels ranging from 0.5 to 1.5 microg/g for all test matrixes were from 75 to 98%. SD and RSD(r) ranged from 0.7 to 11.6% and 0.9 to 12.7%, respectively. Within-laboratory HorRat values were from 0.1 to 0.7 for all matrixes analyzed. The method was found to meet AOAC method performance criteria for grains, grain products, and processed foods. The identity of DON in naturally contaminated test sample extracts was confirmed by HPLC/MS/MS analysis.

The objective of this study was to determine the distribution of an economically important class of mycotoxins, the aflatoxins, in rice milling fractions. Rice plants grown under field production conditions are frequently infected with types of pathogenic fungi that produce toxic metabolites (mycotoxins). Paddy (seeds) rice from healthy plants in the field was collected and stored on a farm under humid, poorly ventilated conditions. Samples were milled into four fractions (hulls, brown rice, bran and white rice) and analysed for aflatoxins (B(1), B(2), G(1) and G(2)) using a validated method. Rice fractions from healthy plants, which contained low levels of aflatoxins (less than 1 µg kg(-1)), were used to determine the efficiency of the extraction method. Seeds stored under poor conditions were found to be contaminated with aflatoxins B(1) and B(2) as were the fractions. The sums of AFB(1) and AFB(2) in stored paddy rice, hulls, brown rice, bran and white rice were 141, 39, 158, 367 and 56 µg kg(-1), respectively. The ratio of aflatoxin B(1) and B(2) was about 10 : 1. AFG(1) and AFG(2) were less than 1 µg kg(-1). Thus, brown rice contained 92.9% of the aflatoxins in paddy rice, whereas white rice contained only 27.9%.

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

Deoxynivalenol (DON), commonly referred to as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium fungi. The presence of DON in foods is a human health concern. The frequency of occurrence of DON in wheat is high, although cleaning prior to milling can reduce DON concentration in final products, and food processing can partially degrade the toxin. This paper describes a method for the determination of DON in some major wheat food products, including bread, breakfast cereals, pasta, pretzels, and crackers. Test samples containing 5 polyethylene glycol were extracted with water. After blending and centrifuging, the supernatant was diluted with water and filtered through glass microfiber filter paper. The filtrate was then passed through an immunoaffinity column and the toxins eluted with methanol. The toxins were then subjected to RPLC separation and UV detection. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON spiked at levels from 0.5 to 1.5 g/g in the five processed foods were >70. SD and RSD values ranged from 2.0 to 23.5 and from 2.0 to 23.2, respectively. HorRat values were

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 methanolacetonitrilewater (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 50 ng/g for the seven botanical roots tested.

Edible oils are consumed directly, and used as ingredients in food, soaps, and skin products. However, oils such as olive oil, peanut oil, and sesame oil could be contaminated with aflatoxins, which are detrimental to human and animal health. A method using immunoaffinity column cleanup with RPLC separation and fluorescence detection (FLD) for determination of aflatoxins (AF) B1, B2, G1, and G2 in olive oil, peanut oil, and sesame oil was developed and validated. Test samples were extracted with methanolwater (55 + 45, v/v). After shaking and centrifuging, the lower layer was filtered, diluted with water, and filtered through glass microfiber filter paper. The filtrate was then passed through an immunoaffinity column, and the toxins were eluted with methanol. The toxins were then subjected to RPLC/FLD analysis after postcolumn UV photochemical derivatization. The accuracy and repeatability characteristics of the method were determined. Recoveries of AFB1 spiked at levels from 1.0 to 10.0 g/kg in olive oil, peanut oil, and sesame oil ranged from 82.9 to 98.6. RSDs ranged from 0.6 to 8.9. HorRat values were

AOAC Official MethodSM 991.31 for the determination of aflatoxins (AFs; sum of aflatoxins B1, B2, G1, and G2) in corn, raw peanuts, and peanut butter by using immunoaffinity column cleanup with LC has been modified and applied to the determination of AFs in botanical roots. The modifications were necessary to improve the performance of the method for matrixes beyond corn and peanuts. The extraction solvent was changed from a mixture of methanol and water to acetonitrile and water. The accuracy, repeatability, and reproducibility characteristics of this method were determined. Replicates of 10 test portions of each powdered root (black cohosh, echinacea, ginger, ginseng, kava kava, and valerian) at each spiking level (AFs at 0, 2, 4, 8, and 16 ng/g) were analyzed on 3 separate days. Test portions were extracted with acetonitrilewater (84 + 16, v/v), and the extracts were centrifuged, diluted with phosphate-buffered saline, filtered, and applied to an immunoaffinity column containing antibodies specific for AFs. After the column was washed with water, the toxins were eluted from the column with methanol and quantified by HPLC with fluorescence detection. All test materials except kava kava were found to contain AF at

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2008 and mid-2009. It covers the major mycotoxins: aflatoxins, alternaria toxins, cyclopiazonic acid, fumonisins, ochratoxin, patulin, trichothecenes and zearalenone. Developments in mycotoxin analysis continue, with emphasis on novel immunological methods and further description of LC-MS and LC-MS/MS, particularly as multimycotoxin applications for different ranges of mycotoxins. Although falling outside the main emphasis of the review, some aspects of natural occurrence have been mentioned, especially if linked to novel method developments.

Dried pink beans naturally contaminated with ochratoxin A (OTA) and dried carioca beans artificially contaminated with OTA by inoculation with Aspergillus ochraceus (ATCC 22947) were tested for ochratoxin A levels as follows: dried beans were washed with water for 2, 60 or 120 min, soaked in water for 60, 120 min or 10 h, and cooked for 60 or 120 min. At each step, test water and beans were separated. Test water, raw beans and cooked beans were analyzed for OTA. The amount of OTA partitioned into water and in residual beans was determined by methanol-sodium bicarbonate extraction, buffer dilution, immunoaffinity column cleanup, liquid chromatographic separation and fluorescence detection. The results demonstrated that the distribution of OTA in processing water and beans depends on the method of preparation. All treatments (washing, soaking and cooking) when applied individually reduced the amounts of OTA retained in bean flour and whole beans. Higher amounts of OTA remained in whole beans than in bean flour after removing the processing water. The combination of the three treatments eliminated about 50% of the toxin from whole beans. This study provides evidence that discarding the washing, soaking and cooking water leads to a significant reduction in OTA contamination in dried beans.

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2007 and mid-2008. It covers the major mycotoxins: aflatoxins, Alternaria toxins, cyclopiazonic acid, fumonisins, ochratoxin, patulin, trichothecenes, and zearalenone. Some aspects of natural occurrence, particularly if linked to novel aspects of analytical methods, are also included. The review demonstrates the rise of LC-MS methods, the continuing interest in developing alternative and rapid methods and the modification of well-established mycotoxin analytical methods by individual laboratories to meet their own requirements.

Botanical products are in great demand in health food markets. Botanicals are used for treatment of diseases as well as to promote health (1). Raw materials for medicinal use and herbal supplements frequently are contaminated with toxigenic fungi originating in the soil; the plants themselves are susceptible to fungal growth both pre- and post-havest under the right environmental conditions (2). Contamination with mycotoxins produced by these fungi could pose human health problems. In recent years, many incidences of natural occurrence of mycotoxins in botanicals have been reported (3). Aflatoxins (AF), the naturally occurring aflatoxins B1, B2, G1, and G2 (AFB1, AFB2, AFG1, AFG2), and ochratoxin A (OTA) have been found in ginseng, ginger, licorice, turmeric, and kava-kava in the United States (4, 5). Fumonisins have been found in medicinal wild plants in South Africa (6) and medicinal plants in Turkey (7) and Portugal (8). Zearalenone was identified in ginseng root (9). The presence of AF and OTA in botanicals may pose risks to human health. AF and OTA were found to co-occur in ginger (5). A multitoxin method for the determination of AF and OTA in botanical roots is needed. A method that is capable of determining AF and OTA simultaneously in powdered ginger and ginseng had been developed (5). The method uses a single immunoaffinity column for cleanup, high-performance liquid chromatography (LC) with post-column derivatization for AF fluorescence enhancement, and fluorescence determination for the toxins. The purpose of this collaborative study was to establish the accuracy, repeatability, and reproducibility parameters of the method by the analysis of spiked powdered ginseng and ginger and naturally contaminated powdered ginger.

Botanicals are used in many countries for medicinal and general health-promoting purposes. Numerous natural occurrences of mycotoxins in botanicals and dried fruits have been reported. Aflatoxins or ochratoxin A (OTA) have been found in botanicals such as ginseng, ginger, liquorice, turmeric, and kava-kava in the USA, Spain, Argentina, India, and some other countries, while fumonisins have been found in medicinal wild plants in South Africa and in herbal tea and medicinal plants in Turkey. Zearalenone was identified in ginseng root. Dried fruits can be contaminated with aflatoxins, OTA, kojic acid, and, occasionally, with patulin or zearalenone. One main area of concern is aflatoxins in dried figs; bright greenish yellow fluorescence under ultraviolet light is associated with aflatoxin contamination. OTA in dried vine fruits (raisins, sultanas, and currants) is another concern. There are also reports of aflatoxins in raisins and OTA in dried figs, apricots, dried plums (prunes), dates, and quince. Maximum permitted levels in the European Union include 4 microg kg(-1) for total aflatoxins in dried fruit intended for direct consumption and 10 microg kg(-1) for OTA in dried vine fruit. This review discusses the occurrence of mycotoxins in botanicals and dried fruits and analytical issues such as sampling, sample preparation, and methods for analysis. Fungal contamination of these products, the influence of sorting, storage, and processing, and prevention are also considered.

The market for botanical dietary supplements in the US has grown rapidly during the last 15 years. Use of newly introduced botanical ingredients has often outpaced an adequate scientific understanding of the ingredients themselves. This may lead to problems, including misidentification, mislabeling, adulteration, and toxicity related to the intended ingredient or one substituted for it. This article reviews recent work with several botanical ingredients (Ephedra, Citrus species, Hoodia gordonii, Teucrium, isoflavones) that illustrates the complexity of the current situation and approaches that contribute to ensuring the quality of botanical ingredients. Recent work with contamination of botanical products by mycotoxins is also reviewed. The need for tools for botanical authentication and methods for reproducible extraction of bioactive constituents is critical. Such tools, and improved analytical techniques for identifying potentially bioactive constituents in fresh plant material and in concentrated extracts and for detection of hazardous contaminants, are expected to improve the overall quality and safety of botanical dietary supplement ingredients.

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 416 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%.

Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as “Bio-Kit” for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 μg whole peanut per g peanut protein per g food; the spiked levels were 0.0, 5, 10, and 20 μg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.

AOAC Official Method 999.14 is applicable for the determination of choline in milk and infant formulas. To date, its use has not been extended beyond these matrixes. We modified Official Method 999.14 and applied it to the determination of choline in a range of choline-containing dietary supplements. Dietary supplement tablets, capsules, wafers, softgels, liquid products, and drink powders were included. We found that the standard curve could be extended to cover a wider range of choline concentrations and defined a procedure for the use of Norit for samples in which the vitamin C content was high enough to interfere with the analysis. Recoveries of choline added to infant formula powders and to representative dietary supplement tablets, capsules, powdered drink mix, and wafer products were 85-114%. The use of Norit during the procedure did not affect the recovery of choline added to infant formula powders or to dietary supplements. An alkaline digestion was included for use with a product containing lecithin as the sole source of choline. Ten of 11 dietary supplement products analyzed by the modified method contained amounts of choline at or above declarations found on the product labels. The remaining product contained about 40% of the label-declared amount of choline.

StarLink is a genetically modified corn that produces an insecticidal protein, Cry9C. Studies were conducted to determine the variability and Cry9C distribution among sample test results when Cry9C protein was estimated in a bulk lot of corn flour and meal. Emphasis was placed on measuring sampling and analytical variances associated with each step of the test procedure used to measure Cry9C in corn flour and meal. Two commercially available enzyme-linked immunosorbent assay kits were used: one for the determination of Cry9C protein concentration and the other for % StarLink seed. The sampling and analytical variances associated with each step of the Cry9C test procedures were determined for flour and meal. Variances were found to be functions of Cry9C concentration, and regression equations were developed to describe the relationships. Because of the larger particle size, sampling variability associated with cornmeal was about double that for corn flour. For cornmeal, the sampling variance accounted for 92.6% of the total testing variability. The observed sampling and analytical distributions were compared with the Normal distribution. In almost all comparisons, the null hypothesis that the Cry9C protein values were sampled from a Normal distribution could not be rejected at 95% confidence limits. The Normal distribution and the variance estimates were used to evaluate the performance of several Cry9C protein sampling plans for corn flour and meal. Operating characteristic curves were developed and used to demonstrate the effect of increasing sample size on reducing false positives (seller's risk) and false negatives (buyer's risk).

Peanuts contain proteins that can cause severe allergic reactions in some sensitized individuals. Studies were conducted to determine the percentage of recovery by an enzyme-linked immunosorbent assay (ELISA) method in the analysis for peanuts in energy bars and milk chocolate and to determine the sampling, subsampling, and analytical variances associated with testing energy bars and milk chocolate for peanuts. Food products containing chocolate were selected because their composition makes sample preparation for subsampling difficult. Peanut-contaminated energy bars, noncontaminated energy bars, incurred milk chocolate containing known levels of peanuts, and peanut-free milk chocolate were used. A commercially available ELISA kit was used for analysis. The sampling, sample preparation, and analytical variances associated with each step of the test procedure to measure peanut protein were determined for energy bars. The sample preparation and analytical variances were determined for milk chocolate. Variances were found to be functions of peanut concentration. Sampling and subsampling variability associated with energy bars accounted for 96.6% of the total testing variability. Subsampling variability associated with powdered milk chocolate accounted for >60% of the total testing variability. The variability among peanut test results can be reduced by increasing sample size, subsample size, and number of analyses. For energy bars the effect of increasing sample size from 1 to 4 bars, subsample size from 5 to 20 g, and number of aliquots quantified from 1 to 2 on reducing the sampling, sample preparation, and analytical variance was demonstrated. For powdered milk chocolate, the effects of increasing subsample size from 5 to 20 g and number of aliquots quantified from 1 to 2 on reducing sample preparation and analytical variances were demonstrated. This study serves as a template for application to other foods, and for extrapolation to different sizes of samples and subsamples as well as numbers of analyses.

Peanuts are one of the 8 most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut-contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts, thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance, which requires reference materials for within- and between-laboratory validations. In this study, National Institute of Standards and Technology Standard Reference Material 2387 peanut butter was used. A polytron homogenizer was used to prepare a homogenous aqueous Peanut Butter suspension for the evaluation of method performance of some commercially available immunoassay kits such as Veratox for Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 20 mg carboxymethylcellulose sodium salt, 643 μg peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was homogenous, stable, reproducible, and applicable for adding to ice cream, cookies, breakfast cereals, and chocolate for recovery studies at spike levels ranging from 12 to 90 μg/g.

The occurrence of aflatoxins and fumonisins in Incaparina was investigated. Incaparina is a mixture of corn and cottonseed flour with added vitamins, minerals and a preservative. It has been marketed as a high-protein food supplement, particularly for children on protein-deficient diets. According to estimates, 80% of Guatemalan children in their first year are given Incaparina to provide an adequate diet. Eight samples of Incaparina manufactured in Guatemala were collected. Five were from three different geographical locations in the USA and three were from Guatemala. Seven were examined for fungal contamination and analysed for aflatoxins and fumonisins. Aspergillus flavus was the predominant fungus in all samples purchased in the USA and in one sample purchased from Guatemala, whereas Fusarium verticillioides was present in only two samples (one from the USA and one from Guatemala). All samples contained aflatoxins, ranging from 3 to 214 ng g(-1) and2 to 32ng g(-1) for aflatoxin B(1) and aflatoxin B(2), respectively; and one sample contained aflatoxin G(1) (7 ng g(-1)). Total aflatoxins present ranged from 3 to 244 ng g(1). All samples contained fumonisins, ranging from 0.2 to 1.7 microg g(-1),0.1 to 0.6 microg g(-1), and0.1 to 0.2 microg g(-1) for fumonisins B(1), fumonisin B(2), and fumonisin B(2), respectively. Total fumonisins present ranged from 0.2 to 2.2 microg g(-1). The identity of aflatoxin B(2) was confirmed using both the chemical derivatization method and liquid chromatographic (LC)/mass spectrometric (MS) analysis. Appropriate regulatory action was recommended for the import of Incaparina and has been in effect since 22 December 1998.

A dopant-assisted atmospheric pressure photoionization (APPI) with liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine patulin in apple juice and apple-based food. Different dopants, dopant flow rates, and LC separation conditions were evaluated. Using toluene as the dopant, the LC-APPI-MS/MS method achieved a linear calibration from 12.5 to 2000 μg/L (r(2)0.99). Matrix-dependent limits of quantitation (LOQs) were from 8 μg/L (solvent) to 12 μg/L (apple juice). [(13)C]-Patulin-fortified apple juice samples were directly analyzed by the LC-APPI-MS/MS method. Other apple-based food was fortified with [(13)C]-patulin, diluted using water (1% formic acid), centrifuged, and filtered, followed by LC-APPI-MS/MS analysis. In clear apple juice, unfiltered apple cider, applesauce, and apple-based baby food, average recoveries were 101 ± 6% (50 μg/kg), 103 ± 5% (250 μg/kg), and 102 ± 5% (1000 μg/kg) (av ± SD, n = 16). Using the suggested method, patulin was detected in 3 of 30 collected market samples with concentrations ranging fromLOQ to 18 μg/L. The use of [(13)C]-patulin allowed quantitation using solvent calibration standards with satisfactory precision and accuracy.

The statistical distribution known as the compound gamma function was studied for suitability in describing the distribution of sample test results associated with testing lots of shelled corn for fumonisin. Thirty-two 1.1 kg test samples were taken from each of 16 contaminated lots of shelled corn. An observed distribution consisted of 32 sample fumonisin test results for each lot. The mean fumonisin concentration, c, and the variance, s2, among the 32 sample fumonisin test results along with the parameters for the compound gamma function were determined for each of the 16 observed distributions. The 16 observed distributions of sample fumonisin test results were compared with the compound gamma function using the Power Divergence test. The null hypothesis that the observed distribution could have resulted from sampling a family of compound gamma distributions was not rejected at the 5% significance level for 15 of the 16 lots studied. Parameters of the compound gamma distribution were calculated from the 32-fumonisin sample test results using the method of moments. Using regression analysis, equations were developed that related the parameters of the compound gamma distribution to fumonisin concentration and the variance associated with a fumonisin test procedure. An operating characteristic curve was developed for a fumonisin sampling plan to demonstrate the use of the compound gamma function.

Gas chromatography/mass spectrometry (GC/MS) with negative ion chemical ionization permits detection of underivatized patulin in apple juice extracts while minimizing co-extractive responses. The technique has been used with a variety of capillary columns in quadrupole, ion trap, and magnetic sector GC/MS instruments to confirm presumptive findings of patulin in apple juice at concentrations ranging from 68 to 3700 μg/L. The demonstrated ability to use any of these 3 mass spectrometers and several capillary columns to confirm the identity of patulin are significant strengths of the technique.