Immunoaffinity Column Research Articles

Mycotoxins Contamination in Rice: Analytical Methods, Occurrence and Detoxification Strategies.

The prevalence of mycotoxins in the environment is associated with potential crop contamination, which results in an unavoidable increase in human exposure. Rice, being the second most consumed cereal worldwide, constitutes an important source of potential contamination by mycotoxins. Due to the increasing number of notifications reported, and the occurrence of mycotoxins at levels above the legislated limits, this work intends to compile the most relevant studies and review the main methods used in the detection and quantification of these compounds in rice. The aflatoxins and ochratoxin A are the predominant mycotoxins detected in rice grain and these data reveal the importance of adopting safety storage practices that prevent the growth of producing fungi from the Aspergillus genus along all the rice chain. Immunoaffinity columns (IAC) and QuECHERS are the preferred methods for extraction and purification and HPLC-MS/MS is preferred for quantification purposes. Further investigation is still required to establish the real exposition of these contaminants, as well as the consequences and possible synergistic effects due to the co-occurrence of mycotoxins and also for emergent and masked mycotoxins.

Investigation of ochratoxin A in commercial cheeses and pork meat products by liquid chromatography-tandem mass spectrometry.

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium and commonly detected in a wide range of foodstuffs. The purpose of this work was to monitor the presence of OTA in cheeses and pork meat products. A simple and accurate "dilute and shoot" method with no need of immunoaffinity column and isotopic labeled internal standard, by liquid chromatography-tandem mass spectrometry, was validated in accordance with the criteria set out in Commission Regulation (EC) No. 401/2006. The method showed good linearity in solvent and in matrix (R2 ≥0.995), limit of detection was 0.2µg/kg for cheese and 0.3µg/kg for pork meat products, limit of quantification was fixed at 1µg/kg, and recovery was estimated at two different concentration levels (1 and 5µg/kg) and ranged from 75% to 101%. The interday and intraday laboratory precisions were lower than 7%. The matrix effect, the recovery of the extraction process, and the overall process efficiency were evaluated. No significant ME was observed in the two matrices considered. This method was applied to the analysis of 75 samples, coming from official controls implemented by the Lazio Region (Central Italy). In one sample of dry-cured ham, the concentration found (69.3µg/kg) was well above the guidance value recommended by the Italian Ministry of Health (1µg/kg). These data together with the detection of OTA in three grated cheeses suggest the importance of monitoring these products. Considering the high dietary intake of these matrices, especially among vulnerable populations, further research should be devoted to estimate exposure and risk assessment for OTA.

First step in development and evaluation simultaneous determination of mycotoxins in cereals by liquid chromatography - mass spectrometry

Currently, solid phase extraction (SPE) with immunoaffinity columns is applied in most standardized methods for mycotoxin determination to purify extracts and analysis by HPLC-FLD, HPLC-UV/VIS or LC-MS/MS. Therefore, sample preparation and analysis by instruments are time-consuming and high operating costs. The novel method allow simultaneously identify nine mycotoxin compounds with selective, stable and accurate results. The new method has been evaluated through three stages including validation as requirements of CEN/TR 16059:2010 (phase 1), comparison with current standard methods (phase 2), evaluate the method using an interlaboratory comparison program (phase 3). Cereal samples were extracted by QuEChERS and analyzed by LC-MS/MS. The limit of quantitation (LOQ) was 0,5 μg/kg for each aflatoxin compound and 40 μg/kg, 25 μg/kg, 1 μg/kg, 75 μg/kg for deoxynivalenol, zearalenone, ochratoxin A, each toxin fumonisin (B1&B2), respectively. The recovery is in the range of 70-120%, relative standard deviation RSD < 20%. The novel method also gives the same results compared to the individual standardized methods, using the immunoaffinity column in the extraction stage. At the present, the method is being evaluated through an interlaboratory comparison program with two rounds: round 1 (for survey) and round 2 (official round), which is expected to be implemented in 2022.  

Immunoaffinity Cleanup and Isotope Dilution-Based Liquid Chromatography Tandem Mass Spectrometry for the Determination of Six Major Mycotoxins in Feed and Feedstuff.

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of deoxynivalenol, aflatoxin B1, zearalenone, ochratoxin A, T-2 toxin and fumonisin B1 in feed and feedstuff was established. The sample was extracted with an acetonitrile–water mixture (60:40, v/v), purified by an immunoaffinity column, eluted with a methanol–acetic acid mixture (98:2, v/v), and reconstituted with a methanol–water mixture (50:50, v/v) after drying with nitrogen. Finally, the reconstituted solution was detected by LC-MS/MS and quantified by isotope internal standard method. The six mycotoxins had a good linear relationship in a certain concentration range, the correlation coefficients were all greater than 0.99, the limits of detection were between 0.075 and 1.5 µg·kg−1, and the limits of quantification were between 0.5 and 5 µg·kg−1. The average spike recoveries in the four feed matrices ranged from 84.2% to 117.1% with relative standard deviations less than 11.6%. Thirty-six actual feed samples were analyzed for mycotoxins, and at least one mycotoxin was detected in each sample. The proposed method is reliable and suitable for detecting common mycotoxins in feed samples.

Quantification of six types of cytokinins: Integration of an ultra-performance liquid chromatographic-electrospray tandem mass spectrometric method with antibody based immunoaffinity columns equally recognizing cytokinins in free base and nucleoside forms

Cytokinins (CTKs) exist in various types in plants. The accurate quantification of free base and nucleoside types of cytokinins are helpful for better understanding their physiological role. In the present study, antibodies against trans-zeatin riboside (tZR) and N6-isopentenyladenine riboside (iPR) antibodies with equal recognition to free base and nucleoside cytokinins were developed. The cross-reactivity of tZR mAb 3G101G7 with tZR, trans-zeatin (tZ), dihydrozeatin riboside (DHZR), dihydrozeatin (DHZ), iPR, and N6-isopentenyladenine (iP) was 100.0%, 95.7%, 19.1%, 18.0%, 1.1%, and 0.7%, and that of iPR mAb 5C82F1 with above-mentioned 6 types of cytokinins was 1.5%, 1.4%, 5.7%, 3.1%, 100.0% and 92.6%, respectively. The obtained antibodies were used to prepare two immunoaffinity columns (IAC). The elution efficiencies of tZR 3G101G7-IAC for tZ and tZR, DHZ and DHZR and of iPR 5C82F1-IAC for iP and iPR were almost no difference with the same loading amount on their corresponding IACs. Subsequently, six types of cytokinins in mepiquat chloride (MC)-treated cotton (Gossypium hirsutum L.) roots were determined by IACs combined with ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS). The contents of tZR, iPR and DHZR were increased by 9.3∼38.5%, 6.6∼23.5%, and 30.1∼110.0%, respectively, whereas those of tZ and iP were reduced by 5.3∼20.0% and 27.7∼32.1%, respectively. The decreased tZ and iP levels led to the ratio of auxin-to-active cytokinins increase to promote lateral root initiation in MC-treated cotton seeding. Integration of the IACs equally recognizing cytokinins in their free base and nucleoside forms with UPLC-ESI-MS/MS can accurately quantify different cytokinins in plant tissues.

Determination of Mycotoxin Contamination Levels in Rice and Dietary Exposure Assessment.

The contamination by aflatoxins, ochratoxin A, and zearalenone of samples of paddy and polished rice stored in silos located in Chiriquí, Panama, was evaluated. A total of 23 samples were extracted using immunoaffinity columns and analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector (FLD) and post-column photochemical derivatization. For the method used, the detection limits were lower than 0.25 μg/Kg for aflatoxins (AFB1, AFB2, AFG1, AFG2) and ochratoxin A and 9.35 μg/Kg for zearalenone; the limits of quantification were between 0.25 and 18.75 μg/Kg, respectively. Of the samples analyzed, all of the paddy rice samples were positive for at least one of the mycotoxins studied, zearalenone being the one found with the highest incidence (90.91%); for the polished rice samples, the mycotoxin with the highest incidence was zearalenone (50%), although in concentrations lower than those established in European legislation (100 μg/Kg). The estimate of the daily zearalenone intake according to the concentrations found was always less than 0.07 μg/Kg/bw. This is the first report on the determination of 6 mycotoxins in rice grains from Panama by the HPLC-FLD methodology. Considering the high incidence of mycotoxins in the analyzed rice samples, regular control in the production process is recommended to improve quality and ascertain its safety.

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

The presence and transfer characteristics of aflatoxins in medicinal herbs: From raw materials to edible dispensing granules.

In this study, a sensitive and accurate immunoaffinity columns coupled with high-performance liquid chromatography method was established to monitor the presence of aflatoxins-aflatoxin B1 , aflatoxin B2 , aflatoxin G1 , and aflatoxin G2 -in different medicinal herbs. The proposed method was found to be suitable for the detection of aflatoxins in eight kinds of herbs and their corresponding granules. Two batches of Arecae semen were positive for aflatoxins, with high residue levels of different aflatoxins. To better understand the presence and transfer of aflatoxins during the formulation of dispensing granules from the herbs, the aflatoxins-free herbs were artificially inoculated with Aspergillus flavus to explore aflatoxins production. Both aflatoxin B1 and aflatoxin B2 were detected in all inoculated samples, while aflatoxin G2 was only detected in Astragali radix samples. Additionally, the presence of aflatoxin B1 was extremely high compared to other aflatoxins. More specifically, the transfer rate of the aflatoxin B1 and the total aflatoxins from original herbs to granules were both approximately 40%. These findings indicated that the preparation of herbs into dispensing granules reduced the content of aflatoxins. The high-level presence of aflatoxins in inoculated herbs indicated that attention is needed to the safety of A. flavus-contaminated herbs.

Conjugated type A trichothecenes in oat-based products: Occurrence data and estimation of the related risk

Currently, increasing attention is being paid to conjugated mycotoxins in plant-based foods because of their assumed bioavailability and health risks. However, analytical standards for most mycotoxin glycosides are not available and risk assessment processes have not yet been completed because of the lack of occurrence data. Our study presents a method for efficient pre-concentration of type A trichothecene glycosides using immunoaffinity columns, followed by indirect determination after enzymatic hydrolysis. This method was applied to 52 various oat-based products. Analytical determinations of both free and glycosylated mycotoxins were performed by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry. Free T-2 toxin and T-2-monoglucosides were present in 92% and 69% of samples, respectively. Free HT-2 toxin and HT-2-monoglucosides were present in 92% of samples and HT-2-diglucosides were detected in 38% of samples. The percentage of HT-2 present in the form of glycosides was up to 56% of the total HT-2 content.

Measurement of Fumonisins in Maize Using a Portable Mass Spectrometer.

Fumonisins are a group of mycotoxins that routinely contaminate maize. Their presence is monitored at multiple stages from harvest to final product. Immunoassays are routinely used to screen commodities in the field while laboratory-based methods, such as mass spectrometry (MS), are used for confirmation. The use of a portable mass spectrometer unlocks the potential to conduct confirmatory analyses outside of traditional laboratories. Herein, a portable mass spectrometer was used to measure fumonisins in maize. Samples were extracted with aqueous methanol, cleaned up on an immunoaffinity column, and tested with the portable MS. The limits of detection were 0.15, 0.19, and 0.28 mg/kg maize for fumonisins B1 (FB1), FB2/FB3, and total fumonisins, respectively. The corresponding limits of quantitation in maize were 0.33, 0.59, and 0.74 mg/kg. Recoveries ranged from 93.6% to 108.6%. However, RSDs ranged from 12.0 to 29.8%. The method was applied to the detection of fumonisins in 64 samples of maize collected as part of the Illinois Department of Agriculture’s monitoring program. Good correlations were observed between the portable MS and a laboratory-based LC-MS method (r2 from 0.9132 to 0.9481). Results suggest the portable MS can be applied to the measurement of fumonisins in maize at levels relevant to international regulations.

Identification and removal of aflatoxin coprecipitates derived from plant samples on immunoaffinity chromatographic purification

The non-polar compounds that coprecipitate with aflatoxins and interfere aflatoxin analysis using an immunoaffinity column (IAC) were identified and an effective pretreatment method was developed in combination with IAC. The proanthocyanidins fractionated from cinnamon coprecipitated with four major aflatoxins (B1, G1, B2 and G2) and were effectively removed using zirconia-coated silica gel. A pretreatment method which combined zirconia-coated silica gel and an IAC was developed for LC-MS/MS analysis of aflatoxins and the combined method substantially improved the recovery of the analytes. The method validation for the quantification of aflatoxins in four types of spiked samples (bark, dried fruits, seeds and rhizomes) and a certified reference material showed favorable accuracy. Furthermore, the developed method was applied to the real samples which encouraged mold growth, and aflatoxins B1 and G1 were successfully detected in some of the samples on which mold grew. This is the first study revealing the causative agent of aflatoxin coprecipitation and developing a new technique to remove the matrix from plant samples. Thus, the method has the potential to become a standard analytical method for aflatoxins in food and medicinal plant samples.

Analysis of aflatoxins and ochratoxin a in chilli powder using ultrahigh performance liquid chromatography with fluorescence detection and tandem mass spectrometry.

Chilli powder, a popular spice, is predominantly contaminated with aflatoxins (AFs) and ochratoxin A (OTA), posing a menace to public health. As no validated method exists for the simultaneous and direct analysis of AFs and OTA in chilli powder, it was imperative to develop one to ensure their effective monitoring and promote trade. In this research, we developed and validated a multi-mycotoxin analysis method that allows the simultaneous determination of AFs (AFB1, AFB2, AFG1 and AFG2) and OTA in chilli powder with high sensitivity, accuracy and precision. The optimised sample preparation workflow started with the extraction of chilli powder (25g) with methanol-water (100mL, 80:20). An aliquot (3mL) was cleaned on a multi-mycotoxin, immunoaffinity column (AFLAOCHRA PREP®) and analysed using ultrahigh performance liquid chromatography with fluorescence (UHPLC-FLD) and tandem mass spectrometric (LC-MS/MS) detection in a single chromatographic run. The method performance was evaluated through intra- and inter-laboratory validation (ILV) studies, and also by analysing a certified reference material. A direct analysis using UHPLC-FLD (without derivatisation) provided the limits of quantification (LOQ) of 0.25 and 1ng/g for AFs and OTA, respectively, while the LOQ for all these mycotoxins in LC-MS/MS was 0.5ng/g. These LOQs are much lower than the maximum levels (MLs) specified by the European Commission. The recoveries of these analytes at LOQ and higher levels were above 75% (RSDr < 12%). The ILV study demonstrated satisfactory method-reproducibility (RSDR < 25%). The analysis of the certified reference material provided accuracies of AFs and OTA in the range of 83-101%. The analysis by UHPLC-FLD and LC-MS/MS provided very similar results. The incurred levels of B1 in market samples were estimated with a precision-RSD of < 6%. Considering its efficiency and alignment with the regulatory requirements, this method can be implemented for the routine analysis of AFs and OTA in chilli powder.

Multi-mycotoxin analysis method using liquid chromatography with tandem mass spectrometry and fluorescence detection in Indian medicinal herbs: Development and validation

While medicinal plants are in high demand worldwide for their therapeutic properties, they can constitute a health concern to consumers when contaminated with mycotoxins. The unavailability of standardised methods for multiclass mycotoxin analysis to assess health risks has thus been realised. This study reports a simple, robust and precise method to estimate nine regulated mycotoxins in a range of Indian medicinal plant matrices including giloy (Tinospora cordifolia), ashwagandha (Withania somnifera), safed musli (Chlorophytum borivilianum), satavari (Asparagus racemosus) and tulsi (Ocimum sanctum). The sample preparation method involved extraction of homogenised matrices (12.5 g) using methanol:water (8:2, 100 mL) followed by cleanup through a multi-mycotoxin immunoaffinity column (IAC), which significantly reduced matrix interferences. The method was initially developed and validated using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous analysis of aflatoxins (B1, B2, G1, G2), ochratoxin A, zearalenone, deoxynivalenol, T-2 and HT-2 toxin. Later, it was validated using LC-fluorescence (LC-FLD) for aflatoxins, ochratoxin A and zearalenone. The optimised sample preparation protocol and analytical method provided acceptable results. Compared to LC-FLD, it was possible to attain a lower limit of quantification (LOQ) with LC-MS/MS for all the tested analytes except aflatoxins. However, LOQs of both instruments were lower than the maximum limits (MLs), with recoveries ranging between 71 and 110% and precision (RSD) of ≤10% across matrices. Despite matrix-induced signal suppressions in LC-MS/MS analysis, the matrix-matched calibrations corrected all recoveries. Considering its accuracy, reliability, robustness and time-effectiveness, this method is recommended for regulatory testing purposes.

Preparation and characterization of an immunoaffinity column for the selective extraction of azaspiracids

The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC–MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26–C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC–MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90–92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.

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.

Aflatoxins and Ochratoxin A in Red Chili (Capsicum) Powder from Tunisia: Co-Occurrence and Fungal Associated Microbiota

.&#x0D; Background: Mycotoxins are produced in foods as a result of mold infection of crops before and after harvest. The aim of this report was to assess, for the first time in Tunisia, the contamination of red chili powder with Aflatoxins (AFs) and Ochratoxin A (OTA) and to identify the associated microbiota.&#x0D; Methods: Fifty-five samples of red Capsicum powder (Capsicum annuum) were screened for AFs and OTA and toxigenic fungal species. Mycotoxins were extracted using immunoaffinity columns and quantified by High Performance Liquid Chromatography (HPLC). Dilution method was realized for fungal isolation and confirmed Polymerase Chain Reaction (PCR) analysis. Data were statistically analysed using statistical software (version 5.0).&#x0D; Results: Mycotoxins levels in 5 out of 55 samples were above the European Union (EU) limit. The highest co-occurrence of mycotoxins was found between AFB1 and OTA (39/55 samples). Aspergillus flavus and Aspergillus niger were the most toxigenic species identified. The highest level of molds contamination found in Capsicum powder reached 7.91×106 Colony Forming Unit (CFU)/g.&#x0D; Conclusion: The co-occurrence of two important mycotoxins (OTA and AFB1) observed in this study, alarm us about the hygienic risk raised by Tunisian consumers and raise the need to improve the production process for red Capsicum powder in Tunisia.

Boron-doped activated carbon nanocomposite as a selective adsorbent for rapid extraction of aflatoxins in nut samples

Activated carbon-boron (AC-B) nanocomposite has been developed as a new solid-phase extraction (SPE) sorbent for the extraction and specification of aflatoxins (AFs) in nuts. Porous activated carbon was produced by the chemical activation method from potato peels, which is important biomass waste, and boron was incorporated into the structure. Fourier transform infrared spectroscopy, nitrogen adsorption-desorption, transmission electron microscopy, X-ray diffraction, and scanning electron microscopy methods were used to characterize the adsorbent. The BET specific surface area of AC-B was found to be 1014.7 m2 g−1and the pore volume was 0.42 cm3 g−1. After the extraction parameters (adsorbent amount (1–20 mg), vortex time (1–10 min), pH (3–8), desorption solvent, desorption volume (1–10 ml), and desorption time (1–10 min)) were optimized, recoveries in the range of 89.5–96.5 % of AFG1, AFG2, AFB1, and AFB2 were obtained in real pistachio, cashew nut, and walnut samples. The highest recoveries of AFs were obtained under conditions with AC-B adsorbent amount of 5 mg, vortex time of 3 min, pH 5, desorption solvent acetonitrile, volume 3 ml, and time 3 min. AFs have a low detection limit range (0.04–0.16 μg kg−1). In addition, the method had good repeatability (the intra-day and inter-day RSDs were 0.3–7.0 %, n = 8) and was validated with CRM. The results were evaluated statistically. The proposed AC-B/SPE method has been presented as an alternative to routine analysis due to its advantages such as being environmentally friendly, simple, faster, sensitive, reliable, selective, reproducible, and ease of application in the extraction of aflatoxins compared to the traditional immunoaffinity column method.

Development of a highly sensitive and specific monoclonal antibody-based ELISA coupled with immuno-affinity extraction for the detection of anticancer drug 5-fluorouracil in blood samples

5-Fluorouracil (5-FU) is an effective anticancer drug widely used in cancer treatment. In this study, two 5-FU derivatives containing a spacer arm with the carboxylic group at the end were synthesized, which were linked to the carrier proteins to form 5-FU-protein conjugates used as the immunogens for the production of monoclonal antibody (mAb). Based on the produced mAb, the highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for 5-FU detection was established. The IC50 and LOD values of the assay were found to be 19.5 ng mL−1 and 0.5 ng mL−1, respectively. There was no cross-reactivity (CR) of the ELISA with cytosine, thymine and uracil, which avoided the interference from inherent pyrimidines. The CR values of the assay with three substitutes of 5-FU (tegafur, 5-fluoro-2′-deoxyuridine, carmofur) were within 9.7%–17.6%. The produced mAb was also applied in sample extraction. The immuno-affinity column capable specific capturing 5-FU was prepared by immobilizing the mAb on Sepharose-4B gel and filling into a SPE column. The recoveries of 5-FU in spiked samples measured by ELISA were 72.4%–90.7% with RSD of 3.6%–8.3%. Five blood samples collected from patients were extracted by immuno-affinity column, then measured by ELISA and confirmed by HPLC-MS/MS. There was a good correlation between HPLC-MS/MS and ELISA. It is demonstrated that the developed ELISA combined with immuno-affinity extraction can be a powerful alternative method for the detection of 5-FU in blood samples.

Investigation of ochratoxin a in blood sausages in the Czech Republic: Comparison with data over Europe

Blood sausages consisting of groats, pork, porcine offal, fat, blood, and spices are very popular in the Czech Republic. All these ingredients are potential sources of dietary exposure to ochratoxin A (OTA). OTA has a strong affinity to serum proteins in porcine blood. Thus, the contamination of blood sausages with OTA can be expected. This study aims to evaluate OTA in 200 samples of porcine blood sausages purchased at the Czech market during 2020–2021. The analytical method high-performance liquid chromatography coupled with fluorescence detection with pre-treatment using immunoaffinity columns was employed to determine OTA. The limit of detection was 0.03 ng/g and the limit of quantification 0.10 ng/g. Recovery was 71.6 %. All samples were positive at contents ranging from 0.15 to 5.68 ng/g with a mean of 1.47 ng/g, and a median of 1.26 ng/g. A total of 66% of these samples contained OTA content exceeding the maximum limit of 1 ng/g set in Italy. This study demonstrates that the Czech population is exposed to OTA from blood sausages. The proposed preliminary action limit for OTA in blood sausages should be set at 1 ng/g. No regulatory limits for OTA in blood sausages have been established yet in the European Union legislation. To protect human health, further monitoring of OTA in these products is necessary.