Development and validation of a new qualitative ELISA screening for multiresidue detection of sulfonamides in food and feed

An indirect competitive enzyme-linked immunosorbent assay (ELISA) to screen sulfonamide residues in food (muscle, eggs, milk and honey) and feed has been developed and validated according to Commission Decision 2002/657/EC. The immunoreagents were appropriately produced to detect a wide range of sulfonamide antibiotic congeners, obtaining half-maximum inhibition concentration (IC₅₀) values below 10 μg L⁻¹ for 11 sulfonamides widely used in veterinary practices. Taking into account the complexity of involved matrices, specific sample preparation protocols have been optimised combining high method throughput with low detectable concentrations. Accordingly, depending on the congener, the obtained detection capabilities (CCβs) were lower or equal to 20 μg kg⁻¹ (muscle, eggs and milk), 10 μg kg⁻¹ (honey) and 2 mg kg⁻¹ (feed). Finally the developed qualitative test was applied to real samples collected within the official monitoring programmes: results exceeding the established screening cut-off were re-analysed with a suitable confirmatory method. The presence of one or more sulfonamides was found in all the suspect screening samples thus demonstrating that the proposed ELISA can be successfully applied in class-specific detection of sulfonamides in food and feed.     

Multiclass determination of 27 antibiotics in honey

A multiclass approach for the screening and confirmation of antimicrobial substances in honey has been developed and validated according to Commission Decision 2002/657/EC. A total of 27 basic drugs belonging to sulfonamide, nitroimidazole and quinolone families were determined. Sample preparation consisted in an acidic hydrolysis of honey followed by a double purification step (defatting and strong cation exchange solid-phase extraction). Instrumental determination was performed by liquid chromatography tandem mass spectrometry (LC–MS/MS) operating in positive electrospray ionization mode. Chromatographic separation was performed on a Poroshell 120 EC-C18 column (100 × 3.0 mm, 2.7 μm) using a gradient with acetonitrile and water both containing 0.1% of formic acid. The method was validated in the range 0.1–10 μg kg⁻¹ evaluating selectivity, linearity, precision, trueness, matrix effect, decision limits and detection capabilities. Satisfactory performances were obtained for all the analytes, although important differences were observed in function of the honey type.The procedure was applied to the analysis of 74 honey samples of different botanical origins and geographical provenience collected from the Italian market. In nine honeys (12%) trace levels of sulfonamides were confirmed. The found levels (lower than 2 μg kg⁻¹) do not raise concerns with respect to the public health.     

Establishment of a method to detect sulfonamide residues in chicken meat and eggs by high-performance liquid chromatography

Sulfonamides are a group of antimicrobials used for treatment and prevention of infectious diseases in humans and animals. In veterinary practice, sulfonamides are extensively used due to its broad spectrum of activity and low cost. A multi-residue analysis of seven sulfonamides (sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamethizole, and sulfamethoxypyridazine) in chicken and eggs using HPLC- DAD detection method has been proposed. Chicken and egg samples were homogenized and extracted with distilled water:ethyl acetate (1:3, v/v) liquid-liquid extraction. The extracts were defatted with n-heptane and dried under nitrogen flow at 55 °C. The dry residue was dissolved in 500 μL methanol:acetic acid:water mixture with a ratio of 10:4:36 (v/v/v) and 10 μL of the sample was subjected to HPLC determination under the following conditions: column, Luna 5 μ C18; particle size, 5 μm; mobile phase, 17 mM acetic acid: methanol: acetonitrile (83:10:7, v/v/v); flow rate, 1.0 mL/min; and detection, 270 nm. The specificity was evaluated by analyzing 30 different blank samples of chicken and eggs in order to verify the absence of potential interfering compounds. No interfering peaks were found around the retention time of analytes in the matrices under investigation. The linear correlation coefficients (r²) for 50–250 ppb range were above 0.99 for all sulfonamides tested. The mean recoveries for chicken and eggs spiked at 50, 100, and 150 ppb were in the range of 86–108% for all analytes. Repeatability and within laboratory reproducibility of the developed method was determined at 100 ppb and quantified as the relative standard deviation was lower than 15%. The decision limits were between 108 and 116 ppb for all analytes whilst the detection capability of all analytes ranged from 129 to 140 ppb. An inexpensive and simple liquid-liquid extraction with isocratic elution mode for rapid analysis of residues of seven sulfonamides in chicken and egg samples using HPLC-DAD detection was established in this study.     

A sensitive and validated method for determination of melamine residue in liquid milk by reversed phase high-performance liquid chromatography with solid-phase extraction

A sensitive and validated method for the determination of melamine residue in liquid milk is developed using reversed phase high-performance liquid chromatography-diode array detection (RP-HPLC-DAD) with solid-phase extraction (SPE). The conditions of the extraction, SPE and HPLC were investigated and optimized. The linearity is satisfactory in the range of 0.1–50 μg/mL with a correlation coefficient of 0.9998. Under the optimal conditions, the method limit of detection (LOD) and method limit of quantification (LOQ) were 18 μg/kg and 60 μg/kg, respectively. The recovery of melamine for milk samples spiked with 0.10–3 mg/kg was in the range of 85.5–99.3% with the RSDs (n = 3) of 2.3–3.7%. The intra-day assay precision (RSD) was 5.6% for five replicates of quality control milk sample at 2 mg/kg level. Confirmation of the identities of melamine was achieved by monitoring the two transitions in multiple-reaction monitoring (MRM) mode, and has been applied successfully for the determination of melamine residue in liquid milk samples. The confirmatory method can permit the detection of melamine residues at levels as low as 60 μg/kg in different liquid milks.     

Optimization of the liquid–liquid extraction method and low temperature purification (LLE–LTP) for pesticide residue analysis in honey samples by gas chromatography

This work optimized a simple and practical method for identification and quantification of the pesticides chlorpyrifos, λ-cyhalothrin, cypermethrin and deltamethrin in honey samples. The method was based on liquid–liquid extraction and low temperature purification using acetonitrile: ethyl acetate (6.5 mL:1.5 mL) as the solvent for extraction. A final clean up step with 2 g florisil was performed before analysis by gas chromatography using electron-capture-detector. The technique was proven satisfactory with efficiency exceeding 85% and linear chromatographic response for the tested pesticides, ranging from 0.033 to 1.7 μg g⁻¹ with correlation coefficients above 0.99. Detection and quantification limits were lower than 0.016 and 0.032 μg g⁻¹, respectively. The proposed method was applied to 11 honey samples. Chlorpyrifos and λ-cyhalothrin residues were found in two samples at concentrations below maximum residue limit (MRL) established for food products. The presence of these compounds was confirmed by mass spectrometry in SIM mode (GC–MS-SIM).     

Sodium-benzoate and citrus extract increase the effect of homogenization towards spores of Fusarium oxysporum in pineapple juice

This paper reports on a combined approach (homogenization-HPH, sodium-benzoate and citrus extract) to inactivate spores of Fusarium oxysporum in pineapple juice. In 1st step HPH and antimicrobials were applied as single hurdles. Homogenization alone reduced spores at the undetectable level only through a 3-step treatment at 120/150 MPa; treatments at 1 or 2 steps reduced F. oxysporum spores by 1 and 2 log cfu/mL, respectively. Concerning the effectiveness of the antimicrobials, NIC (not-inhibitory concentration) and MIC values (minimal inhibitory concentration) of Na-benzoate and citrus extract were 181/289 mg/L and 1450/3700 mg/L, respectively.In the last step of the research benzoate (0–100 mg/L) and citrus extract (0–2000 mg/L) were combined through a two variables-five levels CCD (central composite design) and used in combination with a single step HPH treatment at 120 or 150 MPa. The use of Na-benzoate and citrus extract strengthened the effect of homogenization in pineapple juice and reduced spores of F. oxysporum below the detection limit immediately after homogenization.     

Rapid determination of melamine in soil and strawberry by liquid chromatography–tandem mass spectrometry

A fast and sensitive method to determine melamine in soil, strawberry fruit and plant materials using dispersive solid phase extraction (DSPE), and liquid chromatography–electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed. Validation parameters, including method precision, accuracy, and matrix effect were evaluated. The extraction solvent composition, clean-up adsorbents, and LC-ESI-MS/MS conditions were optimized such that this methodology can be adopted to monitor melamine residue concentration in both environmental, crop and food samples. This method exhibits good linear range from 5 to 500 μg/L (γ > 0.999) with an overall precision between 4 and 7%, 7–10% and 4–8% for soil, strawberry and plant materials, respectively. The mean method recovery varied from 92 to 96% for soil, 87–89% for strawberry, and 76–104% for strawberry plant materials. The limit of detection (LOD) and the limit of quantification (LOQ) for these matrices ranged from 0.2 to 1.3 μg/kg and 0.8–4.4 μg/kg, respectively. The applicability of this melamine residue analytical method was successfully demonstrated using field samples collected from an outdoor field trial.     

Antibiotic residues in meat,milk and aquatic products in Shanghai and human exposure assessment

In this study, we screened 20 common antibiotic (three tetracyclines, four fluoroquinolones, three macrolides, three β-lactams, four sulfonamides, and three phenicols) residues in 125 samples from common types of livestock and poultry meat, milk and aquatic products in Shanghai by ultra-performance liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry in 2016 and assessed their role in human exposure by Monte Carlo Simulation. Overall, 15 out of screened antibiotics were found in these samples and the overall detection frequency was 39.2%. Antibiotics were found in 28.6% of livestock and poultry meat (35.3% for pork and 22.2% for chicken), 10.6% of milk, and 52.1% of aquatic products. Of aquatic products, the overall detection frequency of antibiotics was 91.7% for snakeheads, 81.8% for loaches, 76.9% for carps, 40.0% for yellow-head catfishes, and 16.7% for shrimps, but none was detected in swamp eels. Four human antibiotics were detected: azithromycin was detected in 50.0% of snakeheads and 5.1% of loaches, roxithromycin in 5.9% of pork, and chloramphenicol and cefradine respectively in 5.3% of milk. Enrofloxacin and trimethoprim exceeded the maximum residue limits in 7.7% of carps and 8.3% of snakeheads, respectively. The estimated daily exposure dose by Monte Carlo Simulation was less than 1 μg/kg/day. Antibiotic residues in aquatic products and their consumption accounted for 74.71% and 70.35% of overall variance of estimated antibiotic exposure for men and women, respectively. These findings indicated a high level of antibiotic residues in meat, milk and aquatic products and aquatic products were an important source for exposure of human to antibiotics.     

Determination of Fusarium mycotoxins enniatins,beauvericin and fusaproliferin in cereals and derived products from Tunisia

In this study, 51 samples of cereals (wheat, Barley, maize and Sorghum) and by-products (mainly pasta and couscous) purchased from Tunisian supermarkets were examined for contamination with the emerging Fusarium mycotoxins: Enniatins ENs (EN A, EN A₁, EN B and EN B₁), beauvericin (BEA) and fusaproliferin (FUS).The extraction of the samples was performed with methanol using an Ultra-turrax homogenizer. Mycotoxins were analyzed with a liquid chromatography (LC) coupled to a diode array detector (DAD).The frequencies of contamination of total samples with ENs were 96%. EN A₁ was the most common EN found with the highest prevalence of 92.1%, levels ranged between 11.1 and 480 mg/kg. EN B was evidenced in 35 samples and levels ranged from 1.5 to 295 mg/kg. EN B₁ was detected in 20 samples (39.2%) and levels varied from 4.8 to 120.1 mg/kg and EN A was detected in 14 samples with contamination levels ranging between 19.6 and 121.3 mg/kg. The maximum concentration of total ENs in a single sample was 683.9 mg/kg (sorghum). The analytical results also showed that all the analyzed samples were free of BEA and FUS.The present work is the first one ever drafted on the presence of the emerging Fusarium mycotoxins in Tunisian cereals and derived products.     

Development of an enzyme-linked immunosorbent assay for the detection of nitrofurantoin metabolite, 1-amino-hydantoin, in animal tissues

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of 1-amino-hydantoin (AHD) is described. AHD, the marker residue for nitrofurantoin, is a tissue bound toxic metabolite. To monitor the illegal use of nitrofurantoin, a monoclonal antibody-based ELISA method was developed to detect AHD residue in animal tissues. The highly specific antibody against AHD was prepared by monoclonal antibody technology. The antibody exhibited negligible cross reactivity with other nitrofurans, their metabolites and derivatives, and 50% inhibitory concentration was 0.68 μg/L. The limit of detection in four kinds of animal tissues were all below 0.2 μg/kg and recoveries ranged from 75% to 116.7% for fortified samples at levels of 0.2-5 μg/kg with coefficient of variation values below 15%. Analysis of natural contaminated samples by the ELISA method gave similar results to those of the liquid chromatography-tandem mass spectrometry method. These results indicate the ELISA method is suitable for the detection of AHD residue in animal tissues.     

Veterinary drug residues determination in raw milk in Croatia

A total of 1259 raw milk samples were examined over a three-year period for various antibiotics chloramphenicol, penicillins, cephalosporines, tetracyclines, sulfonamides, beta-lactams, quinolones, aminoglycosides and macrolides. Microbiological and immunoassay methods used for determination of these antibiotics were validated according to the guidelines laid down by European Commission Decision 2002/657/EC. Microbiological screening detected 36 positive samples and in only one sample presence of residues was confirmed. Also, immunoassay determination found one positive sample on tetracycline residues. In total, 37 positive samples were determined, which was equal to a frequency of 0.69% of the total number of targeted analyses. The HPLC-DAD method confirmed the presence of residues above the maximum residue limits (MRLs) in two samples with concentrations of 12 μg kg^?1 for penicillin G and 19 μg kg^?1 for amoxicillin, and 1671 μg kg^?1 for tetracycline. Calculated estimated daily intakes (EDIs) for antibiotics presented showed lower exposure levels than the fixed values of acceptable daily intakes (ADIs). These suggested that raw milk in Croatia contain very low levels of veterinary drugs so that toxicological risk with regard to consuming of milk could not be considered as a public health problem.     

Multiresidue determination of antibiotics in feed and fish samples for food safety evaluation. Comparison of immunoassay vs LC-MS-MS

Antibiotic residues (sulfonamides and tetracyclines) were determined in Gilthead sea bream (Sparus aurata) and feed samples by means of immunoassays and LC-MS-MS (liquid chromatography-mass spectrometry²). A preliminary study to know the withdrawal time of oxytetracycline in Gilthead sea bream samples was also conducted. It was carried out using immunoassays based on photometric detection of horseradish peroxidase (HRP) activity and time-resolved fluorometric detection of coproporphyrin of Platinum (II) (ELISA and TR-FIA, respectively). The results were compared to those obtained using an LC-MS-MS methodology. They showed that approximately 37 days would be the withdrawal time in order not to exceed the MRL and fish could be commercialized with safety.Regarding feed samples analysis, an LC-MS-MS method was optimized including sample treatment. Average recoveries (n = 6) ranging from 78 to 108% were obtained and precision of the method was between 0.2 and 3%. The same sample treatment was applied to the feed immunoanalysis obtaining satisfactory results.Finally, 20 fish and 4 feed samples were analysed in order to confirm the feasibility of the immunoassays for detecting antibiotic residues. Sulfonamide residues were not detected in any fish sample. Tetracycline residues were detected in some fish samples from marine farms, with total concentrations between 2.1 and 152 ng g^?1. In all cases, the obtained results correlated well with those achieved by LC-MS-MS. Therefore, the new methodology allows for food safety of the medicated fish.     

Further data on the occurrence of Fusarium emerging mycotoxins enniatins (A,A1, B,B1), fusaproliferin and beauvericin in raw cereals commercialized in Morocco

In this study, 64 samples of raw cereals (wheat, maize and barley) purchased from local markets in Rabat–Salé area from Morocco were analyzed for the occurrence of six emerging mycotoxins: four enniatins ENs (ENA, ENA1, ENB and ENB1), beauvericin (BEA) and fusaproliferin (FUS). Samples were extracted with a mixture of water/acetonitrile (85/15, v/v) by using an Ultra-turrax homogenizer. Mycotoxins were then identified and quantified with a liquid chromatography (LC) with diode array detector (DAD). Positive samples were confirmed with an LC–MS/MS. Analytical results showed that the frequencies of contamination of total samples with ENs, BEA and FUS were 50, 26.5 and 7.8%, respectively. ENA1 was the most common EN found with a percentage of contamination of 39%, levels ranged between 14 and 445 mg/kg. ENB contaminated 14 samples (21.8%) and levels ranged from 5 to 100 mg/kg. ENB1 was present in four samples (6.2%) and levels varied from 8 to 32 mg/kg. ENA was detected in only one sample with 34 mg/kg. BEA levels ranged from 1 to 59 mg/kg and FUS levels varied from 0.6 to 2 mg/kg. The present report is the first one ever drafted on the presence of emerging Fusarium mycotoxins in raw cereals available in Morocco.     

Natural co-occurrence of aflatoxins and ochratoxin A in spices

The present study aims to determine the co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in spices marketed in Turkey. During the period from November 2010 to August 2011, 105 samples of spices were checked for targeted mycotoxins. The mycotoxins were determined by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) after immunoaffinity column (IAC) clean-up. The method was validated for selectivity, sensitivity, accuracy and repeatability as relative standard deviation (RSD). Good recoveries (80.7–95.7%) were obtained for mycotoxins in each food matrix, with RSD values lower than 15%.Aflatoxins were detected in 79.2% of red chilli flake, 63.6% of red chilli powder, 30.4% of black pepper powder and 21.1% of cumin samples, while none of the cinnamon powder samples contained AFs at detectable levels. Four red chilli flake and three red chilli powder samples were above the EU regulatory limit of 5 μg kg⁻¹ for AFB₁. OTA was found in 75% of red chilli flake, 54.5% of red chilli powder, 17.4% of black pepper powder, with four red chilli flake and three red chilli powder samples exceeding EU limit of 30 μg kg⁻¹. No OTA was found in cinnamon powder samples, while detectable levels of OTA were found in only one cumin sample. The co-occurrence of AFB₁ and OTA was detected in 62.5% of red chilli flake, 40.9% of red chilli powder and 4.3% of black pepper powder samples. This study reports for the first time the natural co-occurrence of AFs and OTA in spices from Turkey.     

A survey of aflatoxin M1 of raw cow milk in China during the four seasons from 2013 to 2015

In the present study, a total of 1550 raw cow milk samples were collected from Southern, Northern, Northeast, and Western regions of China during the four seasons from 2013 to 2015. Samples were analyzed for aflatoxin M1 (AFM1) using high performance liquid chromatography (HPLC). In 2013, AFM1 was detected in 21% of 366 raw cow milk samples with levels ranging between 0.01 and 0.24 μg/L. In 11.7% of samples, AFM1 levels were >0.05 μg/L, which is the legal limit in the European Union. The mean and median of positive samples were 0.069 ± 0.052 μg/L and 0.056 μg/L, respectively. In 2014, AFM1 was detected in 28.5% of 624 raw cow milk samples, with levels ranging from 0.01 to 0.25 μg/L. Of these samples, 7.7% had AFM1 levels exceeding 0.05 μg/L, with a mean of 0.042 ± 0.039 μg/L and median of 0.028 μg/L. AFMI was detected in 14.1% of 560 raw cow milk samples in 2015, with levels ranging from 0.01 to 0.144 μg/L. In 1.8% of these samples, AFM1 levels were above 0.05 μg/L, with a mean of 0.026 ± 0.024 μg/L and median of 0.017 μg/L. Our results demonstrate that AFM1 levels of the samples did not exceed the legal limit of 0.5 μg/L in China, the United States, and Codex Alimentarius Commission. Geographically, AFM1 contamination was more predominant in raw cow milk samples from Southern China than in those from other regions, with a higher number of samples containing AFM1 levels above 0.05 μg/L in 2013, 2014, and 2015. AFM1 levels were higher in autumn than in the other seasons during the entire study period. According to our survey, AFM1 contamination has been well-controlled in China during recent years; however, some samples still exceeded the European Union (EU) legal limit. Better prevention and management of aflatoxins in both feed and milk should be considered especially in Southern regions of China and in autumn.     

A HPLC with fluorescence detection method for the determination of tetracyclines residues and evaluation of their stability in honey

A high-performance liquid chromatography with fluorescence detection method for the simultaneous determination of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) residues in honey was developed and validated. Sample preparation was done in a single solid phase extraction step. The chromatographic separation was performed on a C₈ column. Matrix matched calibration curves showed linearity higher than 0.99. The accuracy values lay between 86% and 111% and the precision was lower than 11%. Limits of detection and quantitation were 8 μg kg⁻¹ and 25 μg kg⁻¹, respectively. The method was employed to evaluate the stability of the tetracyclines in honey during 60 days of storage. The residue levels of OTC, TC and CTC were reduced 97%, 76% and 71%, respectively. Honey samples (n = 22) collected from the retail market were analyzed. No tetracyclines residues were detected.     

Rapid screening and quantification of multi-class multi-residue veterinary drugs in royal jelly by ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

A simple and rapid multi-class multi-residue analytical method was developed for the screening and quantification of veterinary drugs in royal jelly by ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). A total of 90 veterinary drugs investigated belonged to more than 14 families such as lincomycins, macrolides, sulfonamides, quinolones, tetracyclines, β-agonists, β-lactams, sedatives, β-receptor antagonists, sex hormones, glucocorticoids, nitroimidazoles, benzimidazoles, nitrofurans, and the others. A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure was used for the sample preparation without solid-phase extraction step. The linearity, sensitivity, accuracy, repeatability, and reproducibility of the method were fully validated. The response of the detector was linear for each target compounds in wide concentration range (at least, two orders of magnitude) with correlation coefficient (R²) of 0.9921–0.9999. The range of the limit of quantification for these compounds in the royal jelly ranged from 0.21 to 20 μg/kg. The repeatability and reproducibility were in the range of 3.01–11.6% and 5.97–14.9%, respectively. The average recoveries ranged from 70.21 to 120.1% with relative standard deviation of 1.77–9.90% at three concentration levels. For the screening method, the data of the precursor and product ions of the target analytes were simultaneously acquired under the All Ions MS/MS mode in a single run. A homemade database including the elemental composition, accurate masses, retention time, isotopic pattern data of the target ions the characteristic in-source fragment ions was utilized for the confirmation and identification of the target compounds. The applicability of the screening method was verified by applying to real royal jelly samples, and certain veterinary drugs were detected in some cases.     

Dual-dummy-template molecularly imprinted polymer combining ultra performance liquid chromatography for determination of fluoroquinolones and sulfonamides in pork and chicken muscle

In this study, a dual-dummy-template molecularly imprinted polymer capable of simultaneous recognizing 8 fluoroquinolones and 8 sulfonamides was synthesized. Its recognition performance was investigated by comparing the 3D conformations of four dummy templates and the two classes of drugs based on computational simulation. Then a solid phase extraction column was prepared and optimized that was combined with ultra performance liquid chromatography for determination of the 16 drugs in pork and chicken. The column could be reused for at least eighty times, and it showed high absorbency capacities (34.9–74.2 μg) and high recoveries (92%–99%) to these drugs. The limits of detection of this method for the two classes of drugs in meat were in the range of 1.0–3.4 ng/g, and the recoveries from the standards fortified blank samples were in the range of 86.1%–109.4%. Therefore, this method could be used as a specific, sensitive and accurate method for determination of fluoroquinolones and sulfonamides in meat.     

Presence of aflatoxin M1 in milk and infant milk products in Tehran,Iran

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic compounds. The purpose of this survey was to determine natural occurrence and level of AFM₁ in pasteurized liquid milk, infant formula and milk-based cereal weaning food consumed in Tehran, Iran.A total of 328 branded milk products and liquid milk samples were collected and investigated by Enzyme Linked Immuno Sorbent Assay (ELISA).The samples of pasteurized liquid milk (n = 128), infant formula (n = 120) and milk-based cereal weaning food (n = 80) showed that the incidence of contamination with AFM₁ is 96.3%, the presence of AFM₁ in each group was 72.2 ± 23.5, 7.3 ± 3.9 and 16.8 ± 12.5 ng/kg, ranging between 31–113, 1–14 and 3–35 ng/kg, respectively.In general, the amount of AFM₁ in 100 (78%) of liquid milk samples and 24 (33%) of milk-based weaning food was higher than the maximum tolerance limit accepted by European Union, but in all of the infant formula samples was lower (European Communities and Codex Alimentarius has prescribed a limit of 50 ng/kg for AFM₁ in milk and 25 ng/kg in infant milk products).     

A potential methodology for differentiation of ciguatgoxin-carrying species of moray eel

A food poisoning incident due to ingestion of an unknown moray eel occurred in Taiwan. To identify the species of causative moray eel implicated in food poisoning, a 376-bp fragment sequence of cytochrome b gene was successfully obtained from four species of moray eel by using a pair of primers (L newest-1/H 15149). This fragment could be amplified using fish meat treated with different heating processes (100 °C for 30 min, 100 °C for 60 min, 121 °C for 15 min, and 121 °C for 30 min). After sequencing, it was found that no variation in sequence was detected among individuals within each species. The species of causative moray eel implicated in food poisoning was judged as Gymnothorax javanicus based on sequence analysis. In addition, using restriction enzyme analysis, including Taq I and Sau96 I, could distinguish these four species of moray eel and identify the fish species of poisoning sample as G. javanicus. The toxicity of viscera in 24 specimens of four moray eel species was not detected, but the food poisoning sample was found to be toxic. Overall, this study proved that DNA sequence and restriction enzyme analyses are useful in identifying that the causative moray eel species was G. javanicus.