Immunoaffinity Column Research Articles

Immunological Separation of Bioactive Natural Compounds from Crude Drug Extract and Its Application for Cell-Based Studies.

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.

Natural Occurrence of Ochratoxin A in Spices Marketed in the Czech Republic during 2019-2020.

Spices are a popular ingredient in cuisine worldwide but can pose a health risk as they are prone to fungal infestation and mycotoxin contamination. The purpose of this study was to evaluate ochratoxin A (OTA) in 54 single-kind traditional and less traditional spices, each of which was purchased in six samples of different batches (324 samples in total) at the Czech market during 2019–2020. The HPLC-FLD method with pre-treatment by immunoaffinity columns was employed to determine OTA. The limits of detection and quantification were 0.03 ng g−1 and 0.10 ng g−1, respectively. A total of 101 (31%) samples of 19 spice kinds were positive at concentrations ranging from 0.11–38.46 ng g−1. Only turmeric was contaminated with an OTA level exceeding the European Union limits. However, most spices have no regulation, thus further extensive monitoring of various mycotoxins in various kinds of spices is necessary. Chilli and black pepper are the most studied spices for OTA contamination, however, many other kinds of spice can also be highly contaminated, but studies on them are less common, rare, or have not yet been performed. The uniqueness of this study lies in the wide range of spice types studied for the presence of OTA on the Czech market.

A new magnetic Fe3O4@SiO2@TiO2-APTMS-CPA adsorbent for simple, fast and effective extraction of aflatoxins from some nuts

A novel modified magnetic adsorbent was synthesized by binding 3-aminopropyltrimethoxysilane and 1,8-bis (3-chloropropoxy) anthracene-9,10-dione to magnetic nanoparticles. The adsorbent has been characterized by transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, fourier transform infrared spectroscopy. The extraction parameters of nanoparticle amount, pH, vortex time, and desorption conditions (solvent, volume, time) were optimized by combining aflatoxin (AFB1, AFB2, AFG1, AFG2) analyzes in samples, high-performance liquid chromatography-fluorescence detection method, and post-column derivatization. The proposed method was compared with the conventional immunoaffinity column method. Under optimum conditions, the detection limits (LODs) of the aflatoxins range from 0.05 μg kg−1 to 0.15 μg kg−1. The recovery of the four aflatoxins for the added nut samples ranged from 87.7 % to 97.5 %. In addition, the intra-day and inter-day relative standard deviations were <7.1 %. The satisfactory results show that the synthesized adsorbent has advantages such as good sensitivity and accuracy, environmental friendliness, simplicity, easy of application, reliability, and speed in the extraction of aflatoxins in some nuts.

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.

Preparation and properties of aflatoxins imprinted polymer grafted onto the surface of mesoporous silica SBA-15 functionalized with double bonds.

Mesoporous silica Santa Barbara-15 was functionalized by methacryloxypropyl trimethoxysilane. Taking this as the carrier material, a new mesoporous silica surface imprinted polymer was synthesized by using the C=C bond, functional monomer α-methacrylic acid, and crosslinker ethylene glycol dimethacrylate, which was used to extract aflatoxin from grain efficiently. It is different from the preparation of surface imprinted polymers which is physically wrapping carrier materials with polymer layers. The chemical grafting method makes the coating of the polymer layer more controllable. A new method for selective separation, enrichment, and determination of trace aflatoxin in grain was established by using the polymers as the filter of the solid-phase extraction column and high-performance liquid chromatography. The linear range of the method was 0.5-100 μg/kg, R2 =0.9990-0.9993. The recovery of aflatoxin G2, G1, B2, and B1 was 98.9-119.7% and the relative standard deviation was 3.07-5.76%. By comparing the self-made column with the immunoaffinity column, it was found that the self-made column had better extraction performance for aflatoxins than the immunoaffinity column. It can be used for the analysis and detection of aflatoxins in cereal.

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.

Evaluation of fungal contamination and ochratoxin A detection in different types of coffee by HPLC-based method.

BackgroundMycotoxins are secondary fungal metabolites that are produced by some toxigenic fungi on foodstuffs which are poisoning and potentiate for human's health hazards. In coffee samples, ochratoxin A and fungal contamination were examined.MethodsImmunoaffinity columns were used for treating of all 50 samples from four types of coffee, after that high‐performance liquid chromatography was used for determining the amount of ochratoxin. For the identification of fungi, all coffee samples were cultured in appropriated media.ResultsThe results showed that all samples were contaminated by ochratoxin A but only up to 50% of them had toxins higher than acceptable level as detected in black beans (47%), green beans (33.3%), torch (33.3%), and espresso (25%). Black coffee had a higher mean concentration of ochratoxin A than green coffee.ConclusionPredominant fungi isolated from coffee samples were Aspergillus species. Finally, careful monitoring of mycotoxins in coffee samples is essential to improve the quality of this favorable beverage in future.

Analysis of Selected Mycotoxins in Maize from North-West South Africa Using High Performance Liquid Chromatography (HPLC) and Other Analytical Techniques

Contamination of foods by mycotoxins is linked to various health and economic implications. This study evaluated the incidence of mycotoxins in commercial and small-scale maize and evaluated potential health risks for consumers based on South African and international regulations. The sensitivity/specificity of HPLC over other analytical methods used was also ascertained. In total, 100 maize samples were analyzed using immuno-affinity column for extraction and clean-up, thin layer chromatography (TLC), HPLC, and enzyme linked immunosorbent assay (ELISA) for quantification. Results revealed that fumonisinB1 was the most contaminant mycotoxin in both small-scale and commercial samples with incidence rates of 100% and 98.6%, respectively. Aflatoxins contamination occurred at incidences of 26.7% in small-scale and 25.0% in commercial samples. Furthermore, ochratoxin A had high incidence rates of 97.8% and 93.0% and ranged from 3.60–19.44 µg/kg and 1.60–9.89 µg/kg, respectively, in small-scale and commercial samples, while ZEA occurred in 50% and 55% of small-scale and commercial samples, respectively. Results demonstrate that maize, especially from small-scale farmers, may contribute to dietary exposure to mycotoxins. Farmers and consumers should be alerted to the dangers of mycotoxins contamination in maize with resultant health risks. Additionally, HPLC method was also found to be more specific for mycotoxin detection than ELISA.

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.

Multitoxin immunoaffinity analysis of aflatoxins and ochratoxin A in spices

AbstractA multitoxin method for aflatoxins (AFs) and ochratoxin A (OTA) was modified and validated to analyze black pepper, colorífico, nutmeg, paprika, and turmeric. The method consisted of acetonitrile, NaHCO3 and methanol extraction solution, multimycotoxin immunoaffinity column purification and isolation, reversed‐phase liquid chromatography separation, and fluorescence detection. The limit of detection was &lt;0.1 ng/g for AFs and OTA. Mean recoveries of added AFs ranged from 68 to 88% with relative standard deviation of repeatability (RSDr) values &lt;15%; aflatoxin B1 (AFB1) from 69 to 92% with RSDr &lt;17%; and OTA from 75 to 110% with RSDr &lt;10% at spiked levels ranged from 8 to 40 ng/g for AFs, 4 to 20 ng/g for AFB1, and 4 to 20 ng/g for OTA, respectively. Recoveries of added aflatoxin G2 were approximately 50% at spiked levels ranged from 1 to 5 ng/g. Recoveries of aflatoxin B2 and aflatoxin G1 were &gt;70%. The linearity of the method for AFs ranged from 0.4 to 100 ng/g, for OTA from 0.2 to 40 ng/g with R2 values exceeding 0.99. Results of a single laboratory validation study validated the performance of this method meeting the method acceptability criteria. No differences in toxin detection were determined in robustness testing in which minor changes were introduced to the method.

Ochratoxin A levels in serum of Polish dialysis patients with chronic renal failure

Ochratoxin A (OTA) is a mycotoxin produced by the fungi Aspergillus and Penicillium. It occurs naturally in many products of plant origin and in animals because of the carry-over from feed to meat or milk. Ochratoxin A has nephrotoxic, carcinogenic, hepatotoxic, neurotoxic, and genotoxic properties. Data on ochratoxin concentrations in blood or serum from patients with different kidney disorders are available for several European countries, as well as for Africa and Asia. In this study, we determined OTA concentrations in serum samples from chronic renal failure patients receiving dialysis and from healthy controls, collected in central Poland. Ochratoxin A was analyzed after extraction and purification using immunoaffinity columns by liquid chromatography with fluorescence detection (limit of quantification: 0.1ng/mL) in 88 patients and 16 healthy volunteers. The dialysis group consisted of 40 women and 48 men aged between 23 and 85 years. The mean OTA concentrations were 0.75ng/mL (maximum 2.78ng/mL) in dialysis patients and 0.70ng/mL (maximum 1.44ng/mL) in healthy controls. The mean concentrations in patients treated by dialysis were 0.76 and 0.74ng/ml for women and men, respectively (maximum 2.53ng/ml for women and 2.78ng/ml for men). Statistical analysis using Student's t-test showed no statistically significant differences between the control group (non-dialysis patients) and all dialysis patients.

High-Throughput Analysis of Aflatoxins in Cereals, Nuts, and Processed Products Involving Automated Immunoaffinity Cleanup and Inline HPLC-Fluorescence Detection.

The testing of aflatoxins (AFs) in fresh and processed foods is highly in demand to comply with trade regulations. Consequently, commercial laboratories face huge AF sample loads in food consignments. Worldwide, there is a rising interest in implementing automation to increase sample throughput in AF analysis. This study sought to evaluate the performance of an automated cleanup and HPLC analysis system for determination of regulated AFs (B1, B2, G1, G2) in rice, flattened rice, sorghum, raw and processed peanut, almond, peanut butter, and wheat-based cookies. The samples were extracted with methanol-water (80:20), diluted with Triton X-100, and subjected to automated analysis, where the cleanup step through immunoaffinity column (IAC) and HPLC-fluorescence analyses (involving postcolumn bromination-derivatization) were performed in 10 and 11 min, respectively. The method was validated in all test matrices at the LOQ and higher levels. The method performance was also evaluated against a conventional workflow where cleanup and HPLC analysis were manually performed. The LOQ for peanut, sorghum, rice, and flattened rice was 0.125 ng/g, while it was 0.5 ng/g for peanut butter, almond, and wheat-based cookies. In all matrices, the recoveries at LOQ and higher levels were satisfactory. The double-cartridge exchange system completed the analysis of ∼96 injections in 18 h. Each IAC could be reused 15 times without incurring any recovery loss. The automated system provided a better precision (RSD < 9%) than the conventional workflow (RSD = 12-15%). Because of its high-throughput nature, this method is recommended for routine analysis of AFs. A high-throughput method is reported where cleanup and HPLC analysis of AFs were automatically performed. Each IAC could be used 15 times without any loss of recovery. The method performance was better than the conventional approach and complied with the analytical quality control guidelines.

Monitoring of Aflatoxin B1 in Dry Aged Meats using a modified HPLC-FLD method

The presence of aflatoxins residues in meat and meat products occurs through ingestion of feeds contaminated with Aflatoxin B1 (AFB1) or during the processing of the meat products. Little research has been conducted concerning the detection of AFB1 on meat. The aim of this study was to modify the method for AFB1 determination by high performance liquid chromatography with fluorescence detection in dry aged meats. The aged meat samples used for determination of AFB1 were obtained from a commercial dry aged meat plants and produced for 28 days in a meat plant chiller. All meat samples were analyzed according to the modified method. The samples were cleaned to remove impurities by using a immunoaffinity column. Photochemical reactor was used for the recording and evaluation of the chromatograms. The analytical performance parameters showed that the method was specific for AFB1 detection in the meat samples. Recovery rate, reproducibility (RSDr%) and repeatability (RSDR%) were found to be 85.889%, 3.822% and 6.601%, respectively. Limit of detection and limit of quantification were 0.15 ng/mL and 0.501 ng/mL, respectively. AFB1 was not detected in the meat samples. Our results indicated that the method modified for monitoring AFB1 level in the meats was simple, time-saving and reliable.

Occurrence of Zearalenone and Its Metabolites in the Blood of High-Yielding Dairy Cows at Selected Collection Sites in Various Disease States.

Zearalenone (ZEN) and its metabolites, alpha-zearalenol (α-ZEL) and beta-zearalenol (β-ZEL), are ubiquitous in plant materials used as feed components in dairy cattle diets. The aim of this study was to confirm the occurrence of ZEN and its selected metabolites in blood samples collected from different sites in the hepatic portal system (posthepatic–external jugular vein EJV; prehepatic–abdominal subcutaneous vein ASV and median caudal vein MCV) of dairy cows diagnosed with mastitis, ovarian cysts and pyometra. The presence of mycotoxins in the blood plasma was determined with the use of combined separation methods involving immunoaffinity columns, a liquid chromatography system and a mass spectrometry system. The parent compound was detected in all samples collected from diseased cows, whereas α-ZEL and β-ZEL were not identified in any samples, or their concentrations were below the limit of detection (LOD). Zearalenone levels were highest in cows with pyometra, where the percentage share of average ZEN concentrations reached 44%. Blood sampling sites were arranged in the following ascending order based on ZEN concentrations: EJV (10.53 pg/mL, 44.07% of the samples collected from this site), ASV (14.20 pg/mL, 49.59% of the samples) and MCV (26.67 pg/mL, 67.35% of the samples). The results of the study indicate that blood samples for toxicological analyses should be collected from the MCV (prehepatic vessel) of clinically healthy cows and/or cows with subclinical ZEN mycotoxicosis. This sampling site increases the probability of correct diagnosis of subclinical ZEN mycotoxicosis.

Validation of an analytical method based on QuEChERS and LC-MS/MS to quantify nine mycotoxins in plant-based milk

Plant-based beverages (popularly known as vegetable milk) have become increasingly important in recent years. However, the nonexistence of information on mycotoxin contamination is noticeable. We herein describe the development and validation of an analytical methodology that employs QuEChERS and LC-MS/MS for the simultaneous determination of nine mycotoxins (aflatoxins B1, B2, G1, and G2, fumonisins B1 and B2, ochratoxin A, zearalenone, and citreoviridin) in seven types of vegetable milk (peanut, oat, rice, cashew, maize, soybean, and coconut). The method provided the following quantification limits, recoveries at the lowest validated concentration and relative standard deviations under repeatability conditions at the lowest validated concentration, respectively: aflatoxin B1 (0.023 μg/l, 84.98 and 9.23%); aflatoxin B2 (0.024 μg/, 93.00 and 4.85%); aflatoxin G1 (0.057 μg/l, 98.85 and 5.53%); aflatoxin G2 (0.031 μg/l, 96.64 and 4.08%); fumonisin B1 (2.166 μg/l, 75.55 and 16.78%); fumonisin B2 (1.105 μg/l, 70.47 and 11.89%); ochratoxin A (0.104 μg/l, 72.05 and 5.12%); zearalenone (8.093 μg/l, 107.10 and 6.37%); citreoviridin (1.305 μg/l, 97.25 and 7.28%). The method uses small amounts of samples, solvents, and other inexpensive reagents with no need for laborious clean-up and pre-concentration steps. Its attractive characteristics (simplicity, low cost compared to procedures that use immunoaffinity columns, and full compatibility with routine analyses) make it potentially valuable. As a proof-of-principle, the validated methodology was applied to seven commercial samples of different compositions showing that some were contaminated with aflatoxins and ochratoxin A.

Development a multi-immunoaffinity column LC-MS-MS method for comprehensive investigation of mycotoxins contamination and co-occurrence in traditional Chinese medicinal materials

Mycotoxins are poisonous secondary fungal toxic metabolites and harmful to human health. Traditional Chinese medicinal materials (TCMs), including more than two hundred functional foods, are vulnerably bred fungi, causing spoilage and multi-mycotoxins contamination. This study established a simultaneous analytical method by using multi-mycotoxins immunoaffinity column (multi-IAC) and HPLC-MS/MS to evaluate mycotoxins' contamination levels and natural incidence in TCMs. Aflatoxins (AFs, including AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), fumonisins (FB1 and FB2), zearalenone (ZEN), deoxynivalenol (DON) and T-2 toxins in three TCMs or functional foods of Polygalae Radix (PR), Coicis Semen (CS) and Eupolyphaga Steleophaga (ES) were detected. The systematically investigated results of 30 batch AFB1 positive samples revealed co-occurrence and correlation of multi-mycotoxins are significant differences in various matrices. All the samples in this study contain more than 5 mycotoxins. AFB1-AFs, AFB1-FBs, AFB1-DON, and AFB1-T-2 are the most observed co-occurrence, AFB1-OTA is also of concern due to its synergistic toxicity. This study's results can be used to establish guidelines for screening mycotoxin contaminants and limitations on acceptable levels in TCMs. Simultaneously, mycotoxin's correlation results in different matrices can also provide a reference for the standardization of TCM production and processing.

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 aflatoxins, deoxynivalenol, ochratoxin A and zearalenone in organic wheat flour under different storage conditions

SummarySamples of flour derived from an organic wheat seed cultivated in the region Abruzzo (Central Italy) were examined for the detection of aflatoxins, deoxynivalenol, ochratoxin A and zearalenone after storage in the presence of oxygen or under vacuum, for one year. They were analysed by high‐performance liquid chromatography with fluorescence detection for aflatoxins, ochratoxin A and zearalenone and with diode array detector for deoxynivalenol. An immunoaffinity column clean‐up was applied for all the compounds, except for aflatoxin determination, which required a solid‐phase extraction. Recoveries (range: 76–107%) and within‐laboratory reproducibility (RSD% &lt; 10.3) met the performance criteria of Commission Regulation (EU) No. 519/2014 amending Commission Regulation (EC) No. 401/2006 for the detection of mycotoxins in foodstuffs. Analytes were below the detection limit of the method, and this result was probably due to the quality of organic seed and good agricultural practices performed for the prevention of mycotoxin formation.

Survey of aflatoxins and ochratoxin A contamination in spices by HPLC-based method in Shiraz, Southern of Iran.

Among food and agricultural products, spices play important roles in the diets of millions of people worldwide. These products may be colonized by fungi genus and subsequently mycotoxin production. Due to the large demand and supply of spice for cooking, preservative effects, or medicine purpose, it is essential that further investigation is designed to examine mycotoxins in spice. In the present study, the possible contamination of spices by aflatoxins (AFTs) and ochratoxin A (OTA) were analyzed. A total of 80 spice samples (curry, sumac, ginger, and saffron) were purchased and cultured on appropriate medium. Simultaneously mycotoxins from spices were extracted with immunoaffinity columns (IAC), and the occurrence of AFTs (B1 + B2 + G1 + G2) and OTA was then determined using high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). The results depicted that 62 (77.5%) and 58 (72.5%) spice samples were contaminated with AFTs and OTA, respectively. Out of the 80 analyzed spices samples, the mean concentration of AFTs and OTA was higher in the curry samples. Among spices that contaminated with mycotoxins, 5 (6.25%) and 2 (10%) of the samples were above the acceptable limit of AFTs (≥ 10 μg/kg) and OTA (≥ 15 μg/kg), respectively. Aspergillus species were the predominant species isolated, followed by Penicillium, and finally Mucor species.Among the examined samples, only few curry samples were contaminated with mycotoxins above acceptable limit. Despite this low level of contamination, this spice is used daily in the cuisine of this region of the world, and consequently, even the small amount of these heat stable toxins for a long time may cause many adverse effects. Hence, it is recommended to monitor the toxicogenous fungi contamination and level of mycotoxins in the spices.

Development of a multi-mycotoxin LC-MS/MS method for the determination of biomarkers in pig urine

An LC-MS/MS method has been developed for the sensitive and selective determination of 35 mycotoxins (biomarkers of exposure) in pig urine samples. Sample preparation includes creatinine adjustment (with the developed LC-UV method) with enzymatic hydrolysis of pig urine samples followed by liquid-liquid (LLE) extraction. The LLE protocol, as well as enzymatic hydrolysis for indirect mycotoxin glucuronides determination, was optimized in this study. Additionally, two other sample preparation protocols were compared with the developed LLE method: immunoaffinity columns and solid-phase extraction cartridges (Oasis HLB). The detection and quantification of the biomarkers were performed using triple quadrupole mass spectrometry.The method was validated with regard to the guidelines specified by the EMEA (European Medicines Agency). The extraction recoveries were higher than 60% for 77% of the analytes studied, with the intra- and inter-day relative standard deviation being lower than 20% for most of the compounds at four different concentration levels. The limits of quantification ranged from 0.1 ng/mL for zearalenone and sterigmatocystin to 8 ng/mL for nivalenol. To the best knowledge of the authors, the matrix effect was evaluated for the first time in this study for six different urine samples, and the coefficient of variation was found to be lower than 15% for most analytes studied. Finally, the developed method was applied to analyse 56 pig urine samples. Deoxynivalenol (1–20 ng/mL), zearalenone (0.1–1.5 ng/mL) and ochratoxin A (1.5–15 ng/mL) were the main analytes detected in these samples. Moreover, the co-occurrence of alternariol monomethyl ether and alternariol in pig urine is reported herein for the first time.