Immunoaffinity Column Clean-up Research Articles

Development and validation of extraction and clean-up procedures for UPLC-MS/MS analysis of aflatoxins in spices.

UPLC-MS/MS analytical conditions for the analysis of aflatoxins in spices were optimized and validated in this study. Liquid-liquid partition-based protocols for the cleaning up of extracts using common organic solvents such as acetonitrile, hexane, and ethyl acetate were developed and validated. The developed liquid-liquid partition methods were compared with immuno-affinity column and QuEChERS clean-up methods for the UPLC-MS/MS analysis of aflatoxins in 8 spices. The reduction of lipophilic components using the partition with hexane is particularly useful in spices like red pepper that have higher levels of fatty acids, carotenoids, sterols, triterpenoids, etc. The subsequent partitioning with ethyl acetate considerably reduced the matrix interference from the polar components and increased the sensitivity. The cleaning up of spice extracts using liquid-liquid partition techniques resulted in limits of quantification (LOQ) of 2-5 µgL-1 in UPLC-MS/MS analysis. Trueness, repeatability, and reproducibility of the methods were in acceptable ranges. The accuracy of the developed methods was further verified by analyzing aflatoxins in naturally incurred samples of spices and comparing the results with those obtained from the immuno-affinity column cleanup-HPLC-FD method.

Determination of the occurrence of and exposure to bisphenol A and its analogues in carbonated beverages and canned tuna using liquid chromatography − tandem mass spectrometry

Bisphenol A (BPA), a known endocrine disruptor, is commonly used in food containers and packaging. Recently, alternatives such as bisphenol AF (BPAF), bisphenol B (BPB), and bisphenol E (BPE) have been introduced to replace BPA. However, these substitutes have been reported to exhibit toxicity levels similar to BPA. In this study, we developed and validated a method for the analysis of trace bisphenols (BPA, BPAF, BPB, and BPE) in food using immunoaffinity column (IAC) clean-up. The method demonstrated satisfactory accuracy and precision. We applied this validated method to analyze 56 carbonated beverage samples and 30 canned tuna samples. In the carbonated beverages, average concentrations of BPA and BPAF were 0.4 and 0.2 μg kg−1, respectively. In canned tuna, BPA and BPAF were found at average concentrations of 22.2 and 0.7 μg kg−1, respectively, while BPB and BPE were not detected in any samples. Estimated exposure levels ranged from 0.13 to 0.18 ng kg bw−1 day−1 in the general population and from 205.2 to 232.0 ng kg bw−1 day−1 among consumers. The commercial IAC-based analytical method used in this study can contribute to the safety management of BPA, BPAF, BPB, and BPE.

High-Performance Liquid Chromatographic Quantification of the Plant Hormone Abscisic Acid at ppb Levels in Plant Samples after a Single Immunoaffinity Column Cleanup.

High-performance liquid chromatography with ultraviolet detection (HPLC-UV) is a common analysis technique due to its high versatility and simple operation. In the present study, HPLC-UV detection was integrated with immunoaffinity cleanup (IAC) of the sample extracts. The matrix effect was greatly reduced, and the limit of detection was as low as 1 ng/g of free abscisic acid (ABA) in fresh plant tissues. A monoclonal antibody 3F1 (mAb 3F1) was developed to specifically recognize free ABA but not ABA analogues. The mAb 3F1-immobilized immunoaffinity column exhibited a capacity of 850 ng/mL and an elution efficiency of 88.8-105% for standards. The extraction recoveries of the column for ABA ranged from 80.4 to 108.9%. ABA content was detected in various plant samples with IAC-HPLC-UV. The results were verified with ultraperformance liquid chromatography-electrospray tandem mass spectrometry. IAC-HPLC-UV can be a sensitive and cost-efficient method for plant hormone analysis.

Evaluation of aflatoxin content in “Cheka” (traditional beverage in South-Western Ethiopia) and its major ingredient (maize)

BackgroundAflatoxin is a toxic cancerous fungal secondary metabolite produced by Aspergillus species. Consumption of aflatoxin-contaminated cereals and their products can cause several health-related effects including acute and chronic aflatoxicosis. This study aims to evaluate the total aflatoxin content in “Cheka” and its major ingredients using the Immuno-Affinity Column Clean-up technique by sensitive HPLC (High-Performance Liquid Chromatography) with fluorescence detection.MethodsThis study followed a completely randomized experimental design to evaluate the level of aflatoxin in traditionally brewed “Cheka” and its major ingredients (maize) by HPLC from November 2019 to June 2020. Traditionally brewed 6 “Cheka” beverages and 6 maize seed samples were collected randomly from Konso and transported to Bless Agri Food Laboratory in Addis Ababa for laboratory evaluation of aflatoxin in samples. The data were analyzed by SPSS version 25.ResultsIn the present study aflatoxin contamination was detected in all maize and “Cheka” samples. A mean total aflatoxin level of 6.78 ± 8.28 μg/kg and 10.76 ± 4.50 μg/kg were observed in maize and “Cheka” samples respectively. The maize taken from above-ground storage (“Gotera”) and underground storage (pitfall) showed a mean total aflatoxin level of 10.23 ± 10.12 μg/kg and 3.35 ± 5.79 μg/kg respectively. The samples taken from “Cheka” prepared from maize of above-ground storage (“Gotera”) and underground storage (pitfall) showed a mean total aflatoxin level of 11.34 ± 3.06 μg/kg and 10.19 ± 6.35 μg/kg respectively. Aflatoxin G family was not detected in both maize and “Cheka” samples while aflatoxin B1 and B2 contributed to the total aflatoxin level. AFB1 predominantly contributed to the contamination of all samples. Although there was no significant difference while comparing between mean total aflatoxin of any other sample types, a significant difference was observed for the AFB1 mean level of pitfall maize and their respective “Cheka” samples (P = 0.033).ConclusionThe mean level of AFB1 in pitfall maize differs from a respective “Cheka” sample. As most of the samples evaluated for aflatoxin contamination showed levels above healthy cut-off points of aflatoxin set by different authorities like the European Union, CODEX, and Ethiopian Standards Agency, there must be a protracted interventional approach to reduce aflatoxin contamination in maize and its products in the region.

Determination of ochratoxin A in licorice extract based on modified immunoaffinity column clean-up and HPLC analysis

Contamination of ochratoxin A (OTA) is a common concern for the quality and safety of licorice and its derivatives, while their complex sample matrices always restrict the monitoring and regulation of OTA. Taking the much more concentrated and complicated licorice extract as the representative, a modified analysis method was established for OTA by HPLC. Parameters were comprehensively investigated based on liquid-liquid extraction and immunoaffinity column clean-up. In comparison to other methods, the developed method achieved effective clean-up efficiency and selectivity without tedious procedures and specialized instrumentation. Good linearity (R2 ≥0.9995), low LOD/LOQ (0.10 μg/kg/0.33 μg/kg), and satisfactory recovery (90.0%-96.4%, RSDs <7.0%) indicated the satisfactory sensitivity and reliability of the method. In addition, the applicability and robustness of the method was demonstrated by the analysis of large numbers of licorice extract samples. It is noteworthy that 66.5% of 176 samples were contaminated with OTA, while the concentrations of 9.1% of samples exceeded the maximum limit (ML, 80 μg/kg) defined by the EU. On account of the high contamination frequency and broad concentration range of OTA, the daily intake limit of licorice extract was preliminarily determined to be 123.18–123.93 g/day (chronic exposure) and 24.24 g/day (acute exposure), indicating a potential of acute risk through daily exposure. This calls for improved supervision and regulation for OTA contamination in licorice samples. This study suggests a prospective option for the efficient determination and routine monitoring of OTA in licorice and its derivatives, simultaneously providing a valuable data base for its health risk assessment.

Verification of a standard method based on immunoaffinity column cleanup and HPLC-FLD analysis for determination of aflatoxins in peanut kernels

Determination of aflatoxins as a group of potent contaminations in many food products is a common analysis in food quality control laboratories. For quantification of the four most toxic aflatoxins (AFs) (i,e. AF B1, B2, G1, and G2) in food and feed, a standard method involving a liquid extraction step followed by immunoaffinity purification and high-performance liquid chromatography (HPLC) is often used. In this work, this method is verified based on the requirements of ISO/IEC 17025 for the determination of aflatoxins in peanut kernel samples. All the method figures of merit including calibration, evaluation of limit of determinations (LODs) and limit of quantifications (LOQs), accuracy, precision, and uncertainty of the method are explained in detail. The accuracy of the applied method was verified by analysis of a CRM and calculation of the recovery. The relative standard deviations of the method (n = 6) were in the range of 4.99–7.85%. The LODs and LOQs of the method were found to be 0.15 and 0.50 μg. kg−1 for AFB1 and G1 and 0.06 and 0.2 μg kg−1 for AFB2 and G2, respectively. Finally, the uncertainties in the determination of aflatoxins concentrations, at the maximum permissible levels were calculated using the GUM procedure as 0.42, 0.094, 0.42, and 0.083 μg. kg−1 for AFB1, B2, G1, and G2, respectively.

Investigation of ochratoxin A (OTA) in bakers' urine samples in Istanbul by immunoaffinity column clean-up and high-performance liquid chromatography

Aim: Ochratoxin A (OTA), is the most toxic mycotoxin in the ochratoxins produced by Penicillium and Aspergillus species. It is known that this mycotoxin has carcinogenic, nephrotoxic, teratogenic, and immunotoxic effects. The aim of our study is the inhalation exposure of bakery workers to ochratoxin A, which can be found in flour. Materials and Methods: In this study, OTA occurrence and level in urine samples collected from bakers in Istanbul were analyzed by NaHCO₃ dilution, IAC (Immunoaffinity Column) clean-up, and high-performance liquid chromatography with fluorescent detection. Creatinine and malondialdehyde levels were measured spectrophotometrically in collected urine samples. Also, 8-hydroxy 2-deoxyguanosine which is a biomarker of DNA damage was tested by ELISA (Enzyme-Linked Immunosorbent Assay). Results: As a result of our study, the limit of detection (LOD) and the limit of quantification (LOQ) of the method were measured as 0.06 ngmLˉ¹ and 0.2 ngmLˉ,¹ respectively. Of the total collected urine samples 185, 4.9% were contaminated with OTA. Also, the statistically significant difference between OTA levels has not been determined in urine samples of bakers working at the production department and the bakers working at the other departments of the bakery (p&gt;0.05). In order to define the exposure profile of OTA in bakers a questionnaire was conducted among the volunteers as well. But after the evaluation of the answers related to gender, age, dietary habits, coffee consumption, smoking, and alcohol consumption, no correlation was found with the OTA exposure (p&gt;0.05). Conclusion: In the present study although OTA presence was not statistically significant, OTA detection in the urine of bakers’ indicates the necessity of regular follow-up for these workers.

Determination of Zearalenone and Its Derivatives in Feed by Gas Chromatography-Mass Spectrometry with Immunoaffinity Column Cleanup and Isotope Dilution.

In this study, a gas chromatography-mass spectrometry (GC-MS) method was established for the determination of zearalenone and its five derivatives in feed, including zearalanone, α-zearalanol, β-zearalanol, α-zearalenol, and β-zearalenol. An effective immunoaffinity column was prepared for sample purification, which was followed by the silane derivatization of the eluate after an immunoaffinity chromatography analysis for target compounds by GC-MS. Matrix effects were corrected by an isotope internal standard of zearalenone in this method. The six analytes had a good linear relationship in the range of 2-500 ng/mL, and the correlation coefficients were all greater than 0.99. The limits of detection (LODs) and limits of quantification (LOQs) were less than 1.5 μg/kg and 5.0 μg/kg, respectively. The average spike recoveries for the six feed matrices ranged from 89.6% to 112.3% with relative standard deviations (RSDs) less than 12.6%. Twenty actual feed samples were analyzed using the established method, and at least one target was detected. The established GC-MS method was proven to be reliable and suitable for the determination of zearalenone and its derivatives in feed.

Simultaneous Detection of Ochratoxin A and Aflatoxins in Industrial and Traditional Red and isot Pepper Flakes along with Dietary Exposure Risk Assessment.

This study focused on the co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in traditionally and industrially dried red pepper flakes (DRPFs) and isot pepper flakes (IPFs). Following the multitoxin immunoaffinity column (IAC) clean-up, high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) was used to quantify AFs and OTA. The limit of detection (LOD) and limit of quantification (LOQ) values were 0.11 and 0.18 μg kg–1 (AFB1), 0.04 and 0.08 μg kg–1 (AFB2), 0.13 and 0.18 μg kg–1 (AFG1), 0.04 and 0.11 μg kg–1 (AFG2), and 0.10 and 0.21 μg kg–1 (OTA), respectively. AFB1, AFB2, AFG1, and OTA were found to be positive in 93, 74, 17, and 94% of all samples, respectively. The contamination levels in positive samples ranged from 0.23 to 38.69, 0.04 to 2.14, 0.13 to 0.88, and 0.18 to 52.19 μg kg–1 for AFB1, AFB2, AFG1, and OTA, respectively, while no AFG2 was found above the detection limit (0.04 μg kg–1). None of the industrial isot peppers exceeded the European Union limits, while the levels of AFB1 (5 μg kg–1), total AFs (10 μg kg–1), and OTA (20 μg kg–1) of the traditional peppers were above the limit by 30% (16/54), 26% (14/54), and 4% (2/54), respectively. Co-occurrence of AFB1-AFB2-OTA was the most frequent, accounting for 54% (29/54) of all samples. At the upper bound (UB), estimated average exposure to AFB1, total AFs, and OTA was determined to be 0.175, 0.189, and 0.124 ng kg–1 bw day–1 in all samples, respectively. The margin of exposure (MoE) value of AFB1 and total AFs was found to be 977 and 909, indicating high health concerns compared to OTA (MoE >10,000). AFB1 and total AFs may result in 0.0058 and 0.0062 liver cancer cases/100,000 person/year at UB, respectively, and weekly OTA exposure was 0.868 ng kg–1 bw, well below the provisional tolerable weekly intake, hence not of health concern. AFs exposure could endanger health, whereas OTA posed no toxicological concerns through dried red pepper consumption

Aflatoxin M1 Determination in Ice Cream Based on Immunoaffinity Column Clean-up Followed by HPLC-FLD

Background: Aflatoxin M1 (AFM1) in industrial ice cream was analyzed by immunoaffinity column (IAC) clean-up followed by high-performance liquid chromatography (HPLC) with a fluorescence detector (FLD) as a highly-sensitive method to confirm and quantify AFM1 in the ice cream samples. Methods: A total of 150 industrial ice cream samples were randomly collected from supermarkets in seven Iranian cities (Tehran, Tabriz, Karaj, Urmia, Sanandaj, Qazvin, and Khoramabad). Results: AFM1 was detected in more than 80.0% of samples. The average AFM1 concentration in ice cream was 29.79 ng/L. According to the results, in 12.62% of samples, AFM1 levels exceeded the maximum European Union (EU) limits for adults (50 ng/mL). This study recommends regular monitoring of AFM1 concentration in ice cream in Iran. Conclusion: Results of this study indicate the need to develop a database to assist law enforcement agencies, traders, and policymakers in local governments to minimize or prevent health-related risks caused by AFM1.

Simultaneous determination of aflatoxins B1, B2, G1 and G2 in commercial rices using immunoaffinity column clean-up and HPLC-MS/MS

Rice is frequently contaminated with aflatoxins, that are highly toxic fungal substances and strongly involved on hepatic cancer. In this work, different extraction and clean-up methods were evaluated for the simultaneous extraction and clean-up of aflatoxins B1, B2, G1 and G2 from rice. Favourable results were obtained by using methanol – water (80:20, v/v) extraction followed by immunoaffinity columns for clean-up, with recoveries of 86–92%, standard deviations between 5 and 11%, LOD ranged between 0.09 and 0.32 µg/kg, and LOQ between 0.31 and 1.06 µg/kg. Method validation and sample analysis were performed by using HPLC-MS/MS. Nine rice samples from different origin, varieties and specific characteristics, acquired in Spanish supermarkets were analysed. In two basmati samples from the same batch aflatoxin B1 was detected at (1.62 ± 0.08) µg/kg and (0.77 ± 0.03) µg/kg, both lower than the levels established by European Regulation for aflatoxin B1 in cereals.

Highly sensitive determination of three kinds of amanitins in urine and plasma by ultra performance liquid chromatography-triple quadrupole mass spectrometry coupled with immunoaffinity column clean-up

建立了超高效液相色谱-三重四极杆质谱高灵敏测定尿液和血浆中α-鹅膏毒肽、β-鹅膏毒肽和γ-鹅膏毒肽的方法。经过免疫亲和柱净化,尿液样品浓缩20倍、血浆样品浓缩10倍,以Kinetex Biphenyl色谱柱(100 mm×2.1 mm, 1.7 μm)作为分析柱,甲醇-0.005%(v/v)甲酸水溶液作为流动相进行梯度洗脱分离,电喷雾电离、负离子、多反应监测模式下检测,外标法定量。3种鹅膏毒肽的线性范围为0.1~200 ng/mL,相关系数(r)>0.999。尿液和血浆中3种鹅膏毒肽的基质效应和提取回收率分别为92%~108%和90%~103%,变异系数均小于13%。尿液中3种鹅膏毒肽的准确度为-9.4%~8.0%,重复性和中间精度分别为3.0%~14%和3.5%~18%,当取样量为2.00 mL时,方法的检出限均为0.002 ng/mL;血浆中3种鹅膏毒肽的准确度为-13%~8.0%,重复性和中间精度分别为3.9%~9.7%和5.5%~12%,当取样量为1.00 mL时,方法的检出限均为0.004 ng/mL。该法操作简单、灵敏、准确,已在中毒患者摄入野生蘑菇后138 h的尿液中检出0.0067 ng/mL α-鹅膏毒肽和0.0059 ng/mL β-鹅膏毒肽。该法已成功解决中毒患者尿液和血浆中超痕量鹅膏毒肽的检测难题,对于疑似中毒病人的早诊断、早治疗、降低死亡率都具有非常重要意义,也为今后开展此类毒素毒理作用及机体代谢规律的研究提供了可靠的技术支撑。

Assessment of Citrinin in Spices and Infant Cereals Using Immunoaffinity Column Clean-Up with HPLC-Fluorescence Detection.

Historically, the analysis of citrinin has mainly been performed on cereals such as red yeast rice; however, in recent years, more complex and abnormal commodities such as spices and infant foods are becoming more widely assessed. The aim of this study was to develop and validate clean-up methods for spices and cereal-based infant foods using a citrinin immunoaffinity column before HPLC analysis with fluorescence detection. Each method developed was validated with a representative matrix, spiked at various citrinin concentrations, based around European Union (EU) regulations set for ochratoxin A (OTA), with recoveries >80% and % RSD < 9% in all cases. The limit of detection (LOD) and the limit of quantification (LOQ) were established at 1 and 3 µg/kg for spices and 0.1 and 0.25 µg/kg for infant cereals, respectively. These methods were then tested across a variety of spices and infant food products to establish efficacy with high recoveries >75% and % RSD < 5% across all matrices assessed. Therefore, these methods proved suitable for providing effective clean-up of spices and infant cereals, enabling reliable quantification of citrinin detected. Samples such as nutmeg and infant multigrain porridge had higher levels of citrinin contamination than anticipated, indicating that citrinin could be a concern for public health. This highlighted the need for close monitoring of citrinin contamination in these commodities, which may become regulated in the future.

Determination of Aflatoxins and Ochratoxin a in Animal Liver using HPLC-FD Method with Immunoaffinity Column Clean-Up

Abstract Analytical methods based on immunoaffinity column clean-up and quantitative determination with liquid chromatography-fluorescence detection were used to determine aflatoxins and ochratoxin A in liver samples. The validation of the procedures was performed. The linearity of the methods was checked, and a good coefficient of correlation was found for all aflatoxins and OTA as well. The LOD and LOQ were acceptable: 0.003 µg/kg and 0.009 µg/kg for AFB1; 0.001 µg/kg and 0.005 µg/kg for AFB2; 0.006 µg/kg and 0.020 µg/kg for AFG1; 0.007 µg/kg and 0.022 µg/kg for AFG2; 0.08 µg/kg and 0.27 µg/kg for OTA. The results for the repeatability estimated by the relative standard deviation (RSDr) were satisfactory and the obtained values were in the acceptable range (1.97–14.41% for all aflatoxins and 3.76-8.31% for OTA) at three proposed concentration levels. RSDR values showed acceptable correlation between two analysts for all four aflatoxins and OTA. The RSDR values were as followed: 2.37% and 5.60% for AFB1, 6.71% and 8.78% for AFB2, 4.40% and 7.00% for AFG1 and 10.30% and 13.91% for AFG2 (for the first and second analyst, respectively). The RSDR values for OTA were 4.91% and 3.15% (1 µg/kg); 3.76% and 4.12% (5 µg/kg) and 8.31% and 8.21% (10 µg/kg). The mean recovery for total aflatoxins and OTA were 78.10% and 93.34%, respectively. All validation parameters were in accordance to European legislation. They indicate that the proposed analytical procedures are suitable and they could be methods of choice for the determination of aflatoxins and OTA in liver samples.

English

The frequency of occurrence and four principal kinds of aflatoxin concentration in maize seeds grown in Burkina Faso was investigated. Ten (10) samples collected, were analyzed by high performance liquid chromatography (HPLC) with post-column derivatisation after immunoaffinity column cleanup. Eight strains of Aspergillus section Flavi were previously isolated from these samples and cultivated on &ldquo;Aspergillus flavus and parasiticus agar (AFPA)&rdquo; to ascertain if they belong to A. flavus or A. parasiticus species. The qualitative ability of aflatoxin production was also previously performed by fluorescence emission under ultra violet light at 365 nm after four (4) days of incubation at 30 &deg;C on Coconut Agar Medium (CAM). Results showed that 70% of samples were contaminated by aflatoxins. The levels ranged from 0.93 to 58.94 &micro;g/kg. Samples M1 and M10 had high concentrations, 58.94 &micro;g/kg and 70.73 &micro;g/kg; whereas M4 and M5 had low concentrations from 1.68 to 0.93 &micro;g/kg, respectively. In these samples, four were contaminated with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1), two with AFB1 and aflatoxin B2 (AFB2) and one (01) with AFB1 only. We notice that AFB1 was the most prevalent member of aflatoxins, and AFG2 was absent in all samples. &nbsp; Key words: Maize, Aspergillus, aflatoxins, HPLC, Burkina Faso.

Single-Laboratory Validation of an Immunoaffinity Column Cleanup LC Method for the Analysis of Aflatoxins and Ochratoxin A in Cannabis Plant Material, Resins, Vapes, Isolates, and Edible Products.

Potential fungal infection of cannabis plants during drying has raised concerns of resulting mycotoxin contamination in leaves and flowers and subsequent contamination of derived products including cannabis-containing edible products. Validated routine methods are essential to monitor cannabis and cannabis products to ensure consumer safety consistent with long-standing controls for mycotoxins such as aflatoxins and ochratoxin A in foodstuffs. To provide single-laboratory validation data to demonstrate the suitability of a method for determining aflatoxins and ochratoxin A in cannabis plant material, resins, vapes, isolates, and edible products such as chocolate. Extraction of solid and liquid matrixes with acetonitrile:water, centrifugation, and then dilution of an aliquot of supernatant with phosphate-buffered saline solution containing Tween 20 surfactant. Cleanup by passing through an immunoaffinity column containing antibodies to both aflatoxins and ochratoxin A and analyzing in a single LC chromatographic run with fluorescence detection. For within-day analysis, recoveries were in the range 77 to 99% with RSDs from 0.7 to 9.6% for aflatoxin B1. Similarly, ochratoxin A recoveries were from 64 to 94% and RSDs from 0.9 to 9.5% for mycotoxin mixtures spiked into cannabis flowers, resins, vapes, isolates, chocolate, gummies, edible oils, and beverages. A method for the determination of aflatoxins and ochratoxin A was successfully developed and single-laboratory validation data has been presented for cannabis plant material, resins, vapes, isolates, and edible products. A multi-mycotoxin immunoaffinity column cleanup with LC-fluorescence has been validated and shown to be suitable for routine control of aflatoxins and ochratoxin A in cannabis flowers and a diverse range of edible cannabis products.

Determination of Aflatoxin B1 Contamination in Wheat and Rice Flour Collected from Iranian Market Using Simple and Reliable HPLC Method

Aflatoxin B1 (AFB1), as the most toxic mycotoxin, has a large variety of toxic effects to human and animals. To evaluate incidence of AFB1 in wheat flour and rice flour sold in Iranian markets, 40 samples including 16 wheat flour samples and 24 rice flour samples were collected from commercial brands commonly marketed in Iran and analyzed for their AFB1 content using post-column photochemical derivatization and HPLC. The mobile phase consisting of water, methanol and acetonitrile mixture (53:28:19, v/v/v). The sample preparation was done with simple extraction with acetonitrile and water (80/20, v/v) accompany with immunoaffinity column cleanup. Our results indicated a good recovery (minimal 90% in wheat flour and 97 % in rice flour) in spike range 0.2-8 µg.kg-1 with RSDs lower than 5%. The results showed both high incidence and high levels of AFB1 in rice flour samples compared to wheat flour. AFB1 was detected in 18.5% with maximum level of 0.26 µg.kg-1. AFB1 was found in 100% of rice samples with a mean and maximum level of 4.09±2.565 µg.kg-1 and 10.16 µg.kg-1, respectively. The results indicate that although the level of AFB1 in wheat flour is not comparatively critical point regarding quality of wheat flour, high incidence and high contamination level of AFB1 in rice flour samples could make serious health problems for Iranian consumers.

Aflatoxin M1 in pasteurized and UHT milk marked in Morocco

Milk plays an essential role in a balanced diet for human health. Mycotoxins are among the most toxic compounds found in milk of lactating cows. The purpose of this work was to assess aflatoxin M1 (AFM1) levels in 67 liquid milk samples (46 pasteurized and 21 UHT) randomly collected during 2019 from supermarkets and dairy shops in four Moroccan cities (Casablanca (n = 27), El Jadida (n = 10), Fez (n = 18) and Meknès (n = 12)). AFM1 was determined in milk by liquid chromatography (LC) after immunoaffinity column (IAC) cleanup. Analytical results indicated that out of the 67 analyzed samples, 9 (13.4%) were considered as positively contaminated, while 58 samples (86.6%) had AFM1 under the detection limit. For positive samples, AFM1 levels ranged between 10 and 77 ng/L, with an overall average value of 4.46 ± 14.09 ng/L. It has to be underlined that only one pasteurized milk sample contained AFM1 at concentration (77 ng/L) that exceeded the Moroccan maximum regulatory limit of 50 ng/L, while 100% of milk samples were below the limit of 500 ng/kg set by Codex Alimentarius standards. Based on the contamination levels found in this study and milk consumption data in the country, the estimated exposure level of Moroccan adults to AFM1 from milk was estimated at 0.88 ng/person/day (0.0125 ng/kg b.w./day). This exposure level is considered higher than the dietary intakes estimated by JECFA for the Middle Eastern and the African diets.

Comparison of aflatoxins contamination levels in betel nuts (Areca catechu L.) imported from Asian countries

BackgroundAspergillus and their linked metabolites such as aflatoxins (AFs) are one of the extremely significant contaminants affecting food production around the world. The contamination of AFs has been identified in various food commodities, which have been recognized as carcinogenic, mutagenic, teratogenic and immunosuppressive. The present study was undertaken to assess the AFs contamination in betel nuts (Areca catechu L.) being imported to Pakistan from South Asian countries during 2018–2019.MethodsA total of 143 betel nuts consignments (India = 80, Indonesia = 39 and Sri Lanka = 24) were obtained and analyzed for the AF contamination using immunoaffinity column (IAC) clean-up procedure subsequent by liquid chromatography with fluorescence detection. Results: In Indian-origin betel nuts, about 96.3% samples were contaminated with AFs, ranging from 1.18‒331.57 µg/kg with mean contamination of 76.11 ± 1.12 µg/kg; whereas, in Indonesian and Sri Lankan shipments, 100% samples of betel nuts were found infected with AFs, ranging between 1.88‒378.94 and 4.74‒106.58 µg/kg with an average level of 123.76 ± 1.25 and 47.95 ± 0.98 µg/kg, respectively.ConclusionsIn conclusion, the AFs levels present an acute toxicity to human health and also hazard factors for the economy since contaminated foodstuffs do not fulfill the requirements of export/import. Therefore, instant actions should be engaged and re-evaluate agricultural procedures and regular monitoring of AFs level in food stuffs to minimize the chances of various diseases such as oral pre-cancerous oral wounds, submucous fibrosis and squamous cell carcinoma (cancer).

Determination of deoxynivalenol and its major conjugates in cereals using an organic solvent-free extraction and IAC clean-up coupled in-line with HPLC-PCD-FLD

ABSTRACT A method for the determination of deoxynivalenol (DON) and its major conjugates in cereals was developed including an immunoaffinity column (IAC) clean-up coupled in-line with high-performance liquid chromatography, post-column derivatisation and fluorescence detection. An IAC for DON with cross-reactivity to 15-AcDON, 3-AcDON and DON-3-G enabled this approach. The isolated analytes were introduced into the chromatographic system without aliquotation employing the hot water elution technique, resulting in the desired low LOQ values for monitoring these analytes in cereals. The absence of any organic solvent during sample preparation in combination with an in-line IAC clean-up renders the method simple, fast, and environmentally friendly. Special attention was paid to inherent IACs properties such as cross-reactivity, analytes’ competition and capacity. The method was applied to determine DON and its major conjugates in barley, wheat and maize in the range of 10–1000 µg kg−1 of DON, 10–300 µg kg−1 of DON-3-G and 15-AcDON and 10–100 µg kg−1 of 3-AcDON. The apparent recoveries varied from 87% to 110% (average of 98%) and the intermediate precision was below 13.5% RSD (except for DON-3-G in wheat). Fifteen maize, wheat and barley samples were analysed revealing levels of DON conjugates that accounted from 9% to 60% of the “total DON” content (m/m). In general, the frequency and the measured mass fractions decreased in the following order: DON>DON-3-G>15-AcDON>3-AcDON.