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.     

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.     

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 aflatoxin B1 in medical herbs: interlaboratory study

A method was developed for the determination of aflatoxin B1 in medical herbs (senna pods, botanical name Cassia angustifolia; devil's claw, botanical name Harpagophytum procumbens; and ginger roots, botanical name Zingiber officinale). The method, which was tested in a mini-collaborative study by 4 laboratories, is based on an immunoaffinity cleanup followed by reversed-phase high-performance liquid chromatography separation and fluorescence detection after post-column derivatization. It allows the quantitation of aflatoxin B1 at levels lower than 2 ng/g. A second extractant (acetone-water) was tested and compared to the proposed methanol-water extractant. Several post-column derivatization options (electrochemically generated bromine, photochemical reaction, and chemical bromination) as well as different integration modes (height versus area) were also investigated. No differences were found depending on the choice of derivatization system or the signal integration mode used. The method was tested for 3 different matrixes: senna pods, ginger root, and devil's claw. Performance characteristics were established from the results of the study and resulted in HorRat values ranging from 0.12 to 0.75 with mean recoveries from 78 to 91% for the extraction with methanol-water and HorRat values ranging from 0.10-1.03 with mean recoveries from 98 to 103% for the extraction with acetone-water. As a result, the method, with all tested variations, was found to be fit-for-purpose for the determination of aflatoxin B1 in medical herbs at levels of 1 microg/kg and above.     

Determination of aflatoxins B1, B2, G1, and G2 and ochratoxin A in ginseng and ginger by multitoxin immunoaffinity column cleanup and liquid chromatographic quantitation: collaborative study

The accuracy, repeatability, and reproducibility characteristics of a method using multitoxin immunoaffinity column cleanup with liquid chromatography (LC) for determination of aflatoxins (AF; sum of aflatoxins B1, B2, G1, and G2) and ochratoxin A (OTA) in powdered ginseng and ginger have been established in a collaborative study involving 13 laboratories from 7 countries. Blind duplicate samples of blank, spiked (AF and OTA added) at levels ranging from 0.25 to 16.0 microg/kg for AF and 0.25 to 8.0 microg/kg for OTA were analyzed. A naturally contaminated powdered ginger sample was also included. Test samples were extracted with methanol and 0.5% aqueous sodium hydrogen carbonate solution (700 + 300, v/v). The extract was centrifuged, diluted with phosphate buffer (PB), filtered, and applied to an immunoaffinity column containing antibodies specific for AF and OTA. After washing the column with water, the toxins were eluted from the column with methanol, and quantified by high-performance LC with fluorescence detection. Average recoveries of AF from ginseng and ginger ranged from 70 to 87% (at spiking levels ranging from 2 to 16 microg/kg), and of OTA, from 86 to 113% (at spiking levels ranging from 1 to 8 microg/kg). Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 2.6 to 8.3% for AF, and from 2.5 to 10.7% for OTA. Relative standard deviations for between-laboratory reproducibility (RSDR) ranged from 5.7 to 28.6% for AF, and from 5.5 to 10.7% for OTA. HorRat values were < or = 2 for the multi-analytes in the 2 matrixes.     

Restricted access supramolecular solvents for sample treatment in enzyme-linked immuno-sorbent assay of mycotoxins in food

A restricted access supramolecular solvent (SUPRAS-RAM) made up of tetradecanoic acid reverse micelles is proposed as a wide-scope and low-cost strategy for the treatment of agrifood samples prior to enzyme-linked immunosorbent assays (ELISA). The approach was assessed for the determination of ochratoxin A (OTA) in wines and spices and aflatoxin B1 (AFB1) in cereals, two ubiquitous mycotoxins that were selected as representative contaminants for this study. The samples were selected to cover a variety of matrices in terms diverse composition and high complexity. Macromolecules such as proteins and carbohydrates were not-co-extracted due to the restricted access properties of the SUPRAS that are provided by chemical and physical mechanisms. In this sense, analyte extraction and clean-up were carried out in a single step. Parameters determining the extraction efficiency were studied and optimized. Certified reference materials were used for method validation. Recoveries of OTA ranged between 83% and 96% in wines (with relative standard deviation, RSD, of about 10%) and between 81% and 93% in spices (RSD 7%). Recoveries for AFB1 in wheat ranged from 75% to 85% (RSD 8%). The detection limits were all below the maximum levels established for OTA and for AFB1 by EU directives. This method offers a green and low-cost alternative to the organic solvent-based extraction and/or immunoaffinity columns-based cleanup of complex samples prior to ELISA.     

Concentration of ochratoxin A in wines from supermarkets and stores of Valencian Community (Spain)

Ochratoxin A (OTA) is a mycotoxin produced by fungi species belonging to the genera Aspergillus and Penicillium being isolated in alcoholic beverages. The aim of this work is developed and applied a procedure for the analysis of OTA in wines. An analytical method based on immunoaffinity column (IAC) for clean-up, liquid chromatography with fluorescence detection (LC-FD), and LC-FD after of OTA methylation was used to determine the occurrence of OTA in wines. Recoveries of this mycotoxin spiked to red wines at 0.5 ng/ml level were >90% with an average of relative standards deviations of 4%. Furthermore, 116 wine samples from designation of origin (DO) and three samples from food stores of Valencian Community (Spain) were examined for the occurrence of OTA being the levels of this mycotoxin ranged from <0.01 to 0.76 ng/ml. Finally, the estimated daily intake of OTA in this study was 0.15 ng/kg bw per day.     

Determination of ochratoxin A in licorice root using inverse ion mobility spectrometry

Despite the recent, successful efforts to detect mycotoxins, new methods are still required to achieve higher sensitivity, more simplicity, higher speed, and higher accuracy at lower costs. This paper describes the determination of ochratoxin A (OTA) using corona discharge ion mobility spectrometry (IMS) in the licorice root. A quick screening and measuring method is proposed to be employed after cleaning up the extracted OTA by immunoaffinity columns. The ion mobility spectrometer is used in the inverse mode to better differentiate the OTA peak from the neighboring ones. After optimization of the experimental conditions such as corona voltage, injection port temperature, and IMS cell temperature, a limit of detection (LOD) of 0.010 ng is obtained. Furthermore, the calibration curve is found to be in the range of 0.01-1 ng with a correlation coefficient (R²) of 0.988. Licorice roots were analyzed for their OTA content to demonstrate the capability of the proposed method in the quantitative detection of OTA in real samples.     

Determination of aflatoxin B1 in baby food (infant formula) by immunoaffinity column cleanup liquid chromatography with postcolumn bromination: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B, in a milk powder based infant formula at a possible future European regulatory limit (0.1 ng/g). The test portion was extracted with methanol-water (8 + 2 [v + v]), filtered, diluted with water, and applied to an immunoaffinity column. The column was washed with water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. The separation and determination of the aflatoxin B1 was performed by reversed-phase LC and detected by fluorescence after postcolumn derivatization (PCD) involving bromination. PCD was achieved with either pyridinum hydrobromide perbromide (PBPB) or an electrochemical (Kobra) cell by addition of bromide to the mobile phase. The baby food (infant formula) test samples, both spiked and naturally contaminated with aflatoxin B1, were sent to 14 laboratories in 13 different European countries. Test portions were spiked at levels of 0.1 and 0.2 ng/g for aflatoxin B1. Recoveries ranged from 101 to 92%. Based on results for spiked test samples (blind pairs at 2 levels) and naturally contaminated test samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 3.5 to 14%. The relative standard deviation for reproducibility (RSDR) ranged from 9 to 23%. Nine participants used PBPB derivatization, and     

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%。     

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.     

Determination of ochratoxin A at part-per-trillion level in Italian salami by immunoaffinity clean-up and high-performance liquid chromatography with fluorescence detection

A fast high-performance liquid chromatography method has been devised for the determination of ochratoxin A (OTA) in Italian salami in the low part-per-trillion (pg/g) level. The samples were extracted with ethyl acetate and purified by immunoaffinity column (IAC). The IAC eluate could be directly injected or previously concentrated 10-fold. Recovery at 0.5 and 1 ng/g was 77 +/- 4%. The between-day coefficient of variation measured over 5 days on samples spiked at 1 ng/g was 8%. The developed method required a relatively small volume of non-halogenated organic solvent and the whole procedure was simpler and faster compared to other existing procedures. The limit of detection was 0.06 ng/g that could be even lowered using a preconcentration step. A total of 30 salami samples were analysed using this procedure; the most contaminated sample was found to have OTA concentration at 0.4 ng/g level.     

Multiresidue determination of zeranol and related compounds in bovine muscle by gas chromatography/mass spectrometry with immunoaffinity cleanup

A gas chromatography/mass spectrometry (GC/MS) method with immunoaffinity cleanup was developed for the determination of zeranol and related compounds, taleranol, zearalanone, and alpha-zearalenol in bovine muscle. Muscle samples were extracted with methanol and cleaned up with immunoaffinity chromatography (IAC) columns containing monoclonal antibodies raised against zeranol coupled to CNBr-activated Sepharose 4B. After derivatization, the compounds were analyzed by GC/MS. The dynamic column capacities for zeranol, taleranol, zearalanone, and alpha-zearalenol were 2639.7, 2840.3, 2731.5, and 2736.3 ng/mL Sepharose gel, respectively. The limits of detection and quantification were 0.5 and 1.0 ng/g, respectively, for all 4 compounds. Mean recoveries were 79.6-110.7% with coefficients of variation of 3.2-11.4% at spiked levels of 1.0-5.0 ng/g. This IAC-GC/MS method may be used for the determination of zeranol, taleranol, zearalanone, and alpha-zearalenol residues in bovine muscle, and possibly other tissues.     

Immunomagnetic microbeads for screening with flow cytometry and identification with nano-liquid chromatography mass spectrometry of ochratoxins in wheat and cereal

Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants.     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin B1 in corn samples: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 levels in corn samples, enforced by European Union legislation. A test portion was extracted with methanol-water (80 + 20); the extract was filtered, diluted with phosphate-buffered saline solution, filtered on a microfiber glass filter, and applied to an immunoaffinity column. The column was washed with deionized water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase LC with fluorescence detection after either pre- or postcolumn derivatization. Precolumn derivatization was achieved by generating the trifluoroacetic acid derivative, used by 8 laboratories. The postcolumn derivatization was achieved either with pyridinium hydrobromide perbromide, used by 16 laboratories, or with an electrochemical cell by the addition of bromide to the mobile phase, used by 5 laboratories. The derivatization techniques used were not significantly different when compared by the Student's t-test; the method was statistically evaluated for all the laboratories. Five corn sample materials, both spiked and naturally contaminated, were sent to 29 laboratories (22 Italian and 7 European). Test portions were spiked with aflatoxin B1 at levels of 2.00 and 5.00 ng/g. The mean values for recovery were 82% for the low level and 84% for the high contamination level. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the values for relative standard deviation for repeatability (RSDr) ranged from 9.9 to 28.7%. The values for relative standard deviation for reproducibility (RSDR) ranged from 18.6 to 36.8%. The method demonstrated acceptable within- and between-laboratory precision for this matrix, as evidenced by the HorRat values.     

Determination of the cross-reactivities for alpha-zearalenol, beta-zearalenol, zearalanone, alpha-zearalanol, and beta-zearalanol on three commercial immunoaffinity columns targeting zearalenone

Immunoaffinity extraction has become increasingly important as a sample preparation and cleanup method in mycotoxin analysis. In this study, the antibody specificities of 3 commercial immunoaffinity columns (IACs) targeting zearalenone (ZON) were compared for alpha-zearalenol, beta-zearalenol, zearalanone, alpha-zearalanol, and beta-zearalanol. The recoveries of ZON and its 5 analogs were determined in triplicate when extracted from 10 mL circumneutral river water samples spiked with 20 ng analyte individually or in a mixture. The analytes were analyzed by means of electrospray ionization liquid chromatography/tandem mass spectrometry using deuterated internal standards for quantitation. Recoveries ranged from 69 to 115% for all analytes with relative standard deviations of 1-39%. Cross-reactivities for the analogs were > 80% when applied both individually and in a mixture. No significant competition effects were observed when the compounds were applied as a multianalyte mixture well below the stated IAC capacities. The results obtained here demonstrate that all IACs tested are highly cross-reactive towards the 5 ZON derivatives and may be applied for their simultaneous extraction or cleanup.     

Occurrence of ochratoxin A in bread consumed in Morocco

One hundred samples of commercial bread purchased from January to October (2006) from retail bakeshops in five different cities (Rabat, Témara, Salé, Casablanca and Meknès) in Morocco were surveyed for the presence of ochratoxin A (OTA) using liquid chromatography coupled to fluorescence detection. The identification of OTA in positive bread samples was confirmed by methyl ester derivatization. Analytical results showed that forty eight (48%) samples were positive with OTA greater than the limit of quantification (LOQ = 0.051 ng/g). Levels of OTA in positive samples ranged between 0.14 and 149 ng/g. The average contamination of bread samples with OTA was 13 ± 1.5 ng/g. The highest frequency of positive samples (61.5%) and the most contaminated sample (149 ng/g) were found in bread commercialized in the Casablanca area. Twenty six of the positive samples exceeded the maximum level of 3 ng/g set by European regulations for OTA in cereals and derivatives. Based in the results presented in this study, the estimated daily intake of OTA from bread was 126 ng/kg bw/day. The present paper is the first ever drafted on the natural occurrence of OTA in bread consumed in Morocco. Data on the daily intake of OTA by the Moroccan population are also estimated for the first time.     

Preparation and evaluation of a mixed-bed immunoaffinity column for selective purification of sixteen sulfonamides in pork muscle

This paper describes the preparation of a novel mixed-bed immunoaffinity chromatography (IAC) column by coupling four monoclonal antibodies against different sulfonamides (SAs) to Sepharose 4B. The IAC column can be used to simultaneously extract and purify 16 SAs in pork muscle. The dynamic column capacities for all SAs in mixed standard solution were between 312 and 479 ng/mL gel. After simple extraction and IAC cleanup, the sample solution can be directly injected for liquid chromatography-ultraviolet analysis. The recoveries of SAs from spiked samples at levels of 25, 50 and 100 μg/kg ranged from 83.3 to 103.1% with variation coefficient less than 8.6%. The comparison of IAC with liquid-liquid extraction and solid phase extraction indicated that IAC has better purification effect and needs less organic solution than conventional methods, thus it would be an ideal method for selective purification of SAs in pork muscle.     

Rapid gas chromatography-mass spectrometry screening method for human pharmaceuticals, hormones, antioxidants and plasticizers in water

A rapid gas chromatography-mass spectrometry (GC-MS) method was developed and validated allowing quantification at the ng/l level of 19 analytes in water including human pharmaceuticals, hormones, antioxidants and a plasticizer. On-line continuous liquid-liquid extraction with dichloromethane of 10-401 unfiltered water samples was used to achieve a 10000-40000-fold concentration factor. No sample cleanup or derivatization was required. Recoveries ranged from 57 to 120%. Application of the method to water recycling plant effluent demonstrated the presence of nearly all targeted compounds at ng/l to microgram/l levels. Screening for nontarget compounds in the treated effluent samples indicated the method could be readily extended to include additional analytes.     

Occurrence of ochratoxin A in bread consumed in Morocco

One hundred samples of commercial bread purchased from January to October (2006) from retail bakeshops in five different cities (Rabat, Témara, Salé, Casablanca and Meknès) in Morocco were surveyed for the presence of ochratoxin A (OTA) using liquid chromatography coupled to fluorescence detection. The identification of OTA in positive bread samples was confirmed by methyl ester derivatization. Analytical results showed that forty eight (48%) samples were positive with OTA greater than the limit of quantification (LOQ = 0.051 ng/g). Levels of OTA in positive samples ranged between 0.14 and 149 ng/g. The average contamination of bread samples with OTA was 13 ± 1.5 ng/g. The highest frequency of positive samples (61.5%) and the most contaminated sample (149 ng/g) were found in bread commercialized in the Casablanca area. Twenty six of the positive samples exceeded the maximum level of 3 ng/g set by European regulations for OTA in cereals and derivatives. Based in the results presented in this study, the estimated daily intake of OTA from bread was 126 ng/kg bw/day. The present paper is the first ever drafted on the natural occurrence of OTA in bread consumed in Morocco. Data on the daily intake of OTA by the Moroccan population are also estimated for the first time.