A Study of the Occurrence of Aflatoxin M1 in Milk Supply Chain over a Seven-Year Period (2014-2020): Human Exposure Assessment and Risk Characterization in the Population of Central Italy.

Aflatoxin food contamination represents a rising global issue that will continue to increase due to climate change. Aflatoxin M1 (AFM1) is of high concern for the whole dairy industry. In light of AFM1's harmful potential, a human health exposure assessment and risk characterization were performed for all age populations of central Italy with regard to milk and cheese consumption by means of the margin of exposure (MOE). In total, 16,934 cow and ewe's milk samples were collected from 2014 to 2020 and analyzed by an enzyme-linked immunosorbent assay (ELISA) screening method, confirmed by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The average concentration of AFM1 in cow's milk ranged from 0.009 to 0.015 µg/kg, while in ewe's milk, the average concentration ranged from 0.009 to 0.013 µg/kg. The average amount of AFM1 exposure ranged from 0.00005 to 0.00195 g/kg bw/day, with the main contributor represented by drinking milk, followed by the consumption of soft cheeses. A high level of public health concern related to the youngest consumers has arisen from risk characterizations highlighting the need for constant monitoring of AFM1's occurrence in milk by inspection authorities, alongside regular updates with regard to exposure assessments.

Incidence of ionophore and non-ionophore anticoccidials residues in poultry meat and eggs and their risk characterization.

A multi-residue method was applied to investigate the incidence and the concentration of ionophores and non-ionophore anticoccidials residues in poultry meat and hen eggs for the three-year period 2017-2019 in Italy. The risk related to the ingestion of such molecules was also characterized for the entire population. The average incidences of positive samples ranged from 1.35 to 9.45% while the maximum average concentration was of 4.28 µg/kg for nonionophore molecules. No uncompliant sample was recorded. The overall risk characterization related to the intake of anticoccidials trought chicken meat and eggs reveal a minor concern for consumers of all age. However, the monitoring of coccidiostates residues through official control activity in poultry meat and egg is crucial and it should be continuously conducted to ensure safety of such products and safeguard consumers̛ health.

Occurrence and Residue Concentration of Coccidiostats in Feed and Food of Animal Origin; Human Exposure Assessment.

Occurring central Italy, 262 unmedicated feed samples and 353 samples of animal tissues and eggs are tested for coccidiostats between 2012 and 2017. A validated multi-residue HPLC-MS/MS method is applied for the simultaneous determination of the 11 coccidiostats licensed in the EU. The dietary exposure to coccidiostats through poultry meat and eggs is calculated for high consumers, and the contribution to acceptable daily intake of coccidiostats is evaluated. The occurrence of positive feed samples ranges from 17.2% in 2012 to 28.3% in 2017, with an average percentage of positive samples of 25%, while 3.8% of feed samples are non-compliant with a concentration ranging from 0.015 mg/kg for diclazuril to 56 mg/kg for narasin. Positive samples of animal tissues, on average, are 34.7%, fully compliant, while 16% of eggs are positive and violative residues are found in 2%. These noncompliant samples show a concentration varying from 2.4 µg/kg to 1002 µg/kg. The contribution of poultry meat and egg consumption to the acceptable daily intake of each coccidiostat is below 1%, highlighting a low direct risk to public health.

Development and validation of a multiresidue liquid chromatography/tandem mass spectrometry method for 11 coccidiostats in feed.

A confirmatory method for the determination of 11 regulated coccidiostats including the ionophore antibiotics lasalocid, maduramicin, monensin, narasin, salinomycin, and semduramicin and the chemical coccidiostats decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine in animal feed was developed and validated. The procedure was intended for the identification and quantification of the coccidiostats at concentrations relating both to the unintentional carryover as stated in Regulation 574/2011 and to the authorized levels in target feed. The analytes were determined by LC/MS/MS in the positive or negative electrospray ionization mode. The method performance characteristics were estimated in the relevant application field from 0.003 to 200 mg/kg. Validation criteria of linearity, specificity, trueness, precision, LOD, and LOQ along with measurement uncertainty were estimated for all analytes. Absolute and relative matrix effects were also studied. The results proved that the method performance was satisfactory, and it was successfully applied to carryover control by analyzing 165 feed samples collected within regulatory monitoring plans. Finally, since the carryover phenomenon in feed may result in the presence of residues in food products of animal origin, a survey has been carried out on the occurrence of coccidiostats in 167 eggs and animal muscles.

Development, validation and data quality assurance of screening methods: a case study.

Despite the growing importance of qualitative screening tests in routine laboratories involved in the EU official control, their validation is not as deeply explained in Commission Decision 2002/657/EC as the validation of quantitative confirmatory methods. At the same time, the issue of quality assurance of screening assays defining internal quality control (IQC) procedures as required by accreditation bodies is undoubtedly less developed in this analytical field. As an example the present study describes the development, the validation and the IQC implemented for a commercial enzyme linked immunosorbent assay (ELISA) able to detect 17-α-19-nortestosterone (α-NT) and 17-ß-19-nortestosterone (ß-NT) isomers in bullock urine. In order to select a suitable sample treatment, two SPE purification protocols were preliminary compared. The chosen method was therefore fully validated determining the mandatory parameters required by Commission Decision 2002/657/EC: specificity, detection capability and robustness. An in-depth discussion was carried out illustrating the possible validation approaches and their implications especially in the assessment of the key performance characteristic: detection capability. Finally, the control charts implemented for continuous method verification during analyses of real samples were reported.

Development and validation of a multi-residue liquid chromatography-tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs.

A confirmatory method for the determination of residues of eleven coccidiostats including ionophore antibiotics: lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin and chemical coccidiostats: decoquinate, diclazuril, halofuginone, nicarbazin and robenidine in poultry eggs was developed and validated. The sample was extracted with acetonitrile, defatted with hexane and cleaned-up on a silica SPE cartridge. The analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method performance characteristics required by Commission Decision 2002/657/EC were estimated adopting a more flexible and simple validation design. In this alternative approach the experimental study focuses on a larger dynamic range with progressively increasing validation levels. Each of them presents equal concentrations of all the analytes. On the contrary the classical Decision plan investigates a restricted concentration range necessarily different for each analyte being determined by the individual permitted limit (0.5-1.5 times the permitted limit). As a consequence each validation level involves the simultaneous fortification with complex mixtures containing different concentrations of each analyte. Adopting this alternative strategy the validation of several substances with significantly different permitted limits is considerably simplified and a deeper knowledge of the method is achieved. The results proved that the method enables the confirmation of regulated coccidiostats in eggs at the levels required in the official control of residues (CCα in the range of 2.2-174 µg kg(-1), depending on the coccidiostat). The repeatability (CV(r) in the range of 1.1-19%) and within-laboratory reproducibility (CV(Rw) in the range of 1.8-30%) are also acceptable. The procedure was successfully verified in the proficiency test and implemented in the national residue control plan.

Simultaneous determination of eleven quinolones in animal feed by liquid chromatography with fluorescence and ultraviolet absorbance detection.

A rapid and simple multi-residue procedure is described for assaying eleven quinolones (cinoxacin, ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid and sarafloxacin) in feeds at sub-additive levels (1-5 mg kg(-1)). Five grams of sample were extracted by a metaphosphoric acid/acetonitrile mixture (70/30, v/v) and purified onto OASIS HLB cartridges. The determination was achieved by liquid chromatography (LC) using a GEMINI C18 analytical column both with fluorescence detection (FD) and photodiode-array (DAD). Limits of detection for each drug were in the range 0.04-0.8 mg kg(-1). Above the limit of quantification (LOQ), in poultry feed the recoveries were from 69 to 98% with relative standard deviations less than or equal 10%. Finally the measurement uncertainty was estimated using the bottom-up approach.

Development and validation of a confirmatory method for the determination of sulphonamides in milk by liquid chromatography with diode array detection.

A simple and rapid multiresidue method for the determination of seven sulphonamides residues (sulfadiazine, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfadimethoxine and sulfaquinoxaline) in milk samples was developed and validated. The drugs were extracted with a mixture chloroform/acetone and simply cleaned up on a cation exchange solid phase extraction column. The analytes determination was carried out using liquid chromatography with diode array detection (DAD). The procedure has validated as a quantitative confirmatory method according to the European Union (EU) Decision 2002/657/EC. The developed method shows good linearity, specificity, precision (repeatability and intra-laboratory reproducibility), ruggedness and is able to confirm each sulphonamide residue above 30mugkg(-1). Decision limits (CCalpha) around 110mugkg(-1) and recovery above 56% were obtained for all the analytes. The results of the validation process demonstrate that the method is suitable for application, as confirmatory method, in European Union statutory veterinary drug residue surveillance programmes. In addition, a hypothetical situation of sample judgement (compliance or not) in the case in which, at the same time, two different sulphonamides are found, is discussed.

A confirmatory method for the determination of tetracyclines in muscle using high-performance liquid chromatography with diode-array detection.

Using high-performance liquid chromatography with diode-array detection (HPLC-DAD) technique, a confirmatory method for the determination of trace levels of tetracyclines (oxytetracycline, tetracycline, chlortetracycline and doxycycline) and their 4-epimers (4-epioxytetracycline, 4-epitetracycline and 4-epichlortetracycline) in animal tissues (muscle) was developed. The samples are extracted with a mixture of succinic acid 0.1M (pH 4) and methanol after the addition of metacycline as internal standard. The clean-up is carried out by metal chelate affinity chromatography with a following concentration step on an OASIS HLB polymeric reversed phase column. The chromatographic separation of the seven analytes is achieved in 10 min on a short monolithic column (50 mm x 4.6 mm i.d.) using a gradient elution. The method was validated in bovine muscle following the Commission Decision 2002/657/EC criteria: samples spiked at four concentration levels (0.25, 0.5, 1 and 1.5 times the maximum residue limit) were analysed. Method trueness and precision (repeatability and intra-laboratory reproducibility) as well as decision limits (CCalpha) and detection capabilities (CCbeta) are reported.

Validation of a confirmatory method for the determination of sulphonamides in muscle according to the European Union regulation 2002/657/EC.

A simple multiresidue method is described for assaying 10 sulphonamides (SAs) (sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfachlorpyridazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in muscle samples. Samples were prepared by homogenizing the tissue, extracting with ethyl acetate and cleaning up with a cation-exchange solid-phase extraction (SPE) column. The detection of analytes was achieved by HPLC-diode array detection (DAD) at 270 nm. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit, detection capability, trueness and precision. The results of validation process demonstrate that the method is suitable for application in European Union statutory veterinary drug residue surveillance programmes.