Bruno Neri

Validation study on avermectine residues in foodstuffs

Avermectines are antiparasitic agents widely used as veterinary drugs for food producing animals. The European Community, due to their side effects, limited the use of these molecules establishing maximum residue limits (MRLs) in some foods. A validated qualitative and quantitative high performance liquid chromatography method with fluorescence detection (HPLC-FL) is presented for the simultaneous determination of ivermectin (IVM), abemectin (ABA), moxidectin (MOX), eprinomectin (EPR), doramectin (DOR) and emamectin (EMA) in foodstuffs (muscle, eggs and milk). Samples were extracted with acetonitrile, purified with liquid-liquid extraction (LLE), and analysed by HLPC-FL previous derivatization with trifluoroacetic anhydride (TFAA) in presence of 1-methyl-imidazole (MI) and acetic acid. To date, the presented method is the first validated for the matrix eggs, and in accordance with the requirements set by Commission Decision 2002/657/EC. Recoveries of the methods, calculated spiking the samples in the range 5.0-100.0 μg kg(-1), were 64-83% for muscle, 65-89% for milk and 63-84% for eggs. The precision (CV) ranged between 9.2 and 17.1% for muscle, 9.9 and 16.6% for milk and from 9.4 to 17.4% for eggs. Linearity for the six analytes was calculated from 5.0 to 200.0 μg kg(-1). The main advantages of the presented method are its rapidity, the specificity, the good precision and recovery that make it very suitable to the detection and determination of avermectines.

Determination of nandrolone metabolites in human urine: comparison between liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry

Nandrolone (19-nortestosterone) is an androgenic anabolic steroid illegally used as a growth-promoting agent in animal breeding and as a performance enhancer in athletics. Therefore, its use was officially banned in 1974 by the Medical Commission of the International Olympic Committee (IOC). Following nandrolone administration, the main metabolites in humans are 19-norandrosterone, 19-norethiocolanolone and 19-norepiandrosterone, and their presence in urine is the basis of detecting its abuse. The present work was undertaken to determine, in human urine, nandrolone metabolites (phase I and phase II) by developing and comparing multiresidue liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) methods. A double extraction by solid-phase extraction (SPE) was necessary for the complete elimination of the interfering compounds. The proposed methods were also tested on a real positive sample, and they allow us to determine the conjugated/free fractions ratio reducing the risk of false positive or misleading results and they should allow laboratories involved in doping control analysis to monitor the illegal use of steroids. The advantages of LC/MS/MS over GC/MS (which is the technique mainly used) include the elimination of the hydrolysis and derivatization steps: it is known that during enzymatic hydrolysis several steroids can be converted into related compounds and deconjugation is not always 100% effective. The validation parameters for the two methods were similar (limit of quantification (LOQ) <1 ng/mL and percentage coefficient of variance (CV%) <16.4), and both were able to confirm unambiguously all the analytes, thus confirming the validity of both techniques.

Extraction, clean-up and gas chromatography-mass spectrometry characterization of zilpaterol as feed additive in fattening cattle.

Zilpaterol is an adrenergic drug currently licensed in Mexico and South Africa as a feed additive for cattle close to consignment. In this study an analytical method to detect zilpaterol in commercial feeds was set up. The influence of extraction solvent and matrix was evaluated. The drug as a trimethylsilyl derivative was characterized by GC-MS, on a quadrupole detector, in the electron impact mode. Acidic extraction, solid-phase extraction C(18) non-endcapped clean-up and mass characterization on ions m/z 308, 291, 405, 390 provided zilpaterol recoveries >75.3% and repeatability <3.3% in feeds spiked in the range 30.0-120.0 ng/g. The limits of detection and quantification were 7.5 and 25.0 ng/g, respectively. Such limits are well below the dose of 5.0-20.0 microgram/g proposed as effective.

Analytical method for the determination of mycotoxins in indoor/outdoor airborne particulate matter by HPLC-MS-MS

An effective analytical method for the screening of mycotoxins, in indoor/outdoor airborne particulate matter, was developed and method performance data are presented. Mycotoxins are natural compounds produced, in particular conditions, as secondary metabolites by filamentous fungi and moulds, and, after their production, they can be transported far from their source. To simulate real samples, an urban dust (reference material 1649a) free from mycotoxins was used as matrix and spiked by the most common mycotoxins, chosen on the basis of studies carried out previously in other real matrices: deoxynivalenol, aflatoxin B1, ochratoxin A, T-2 toxin, zearalenone and sterigmatocystin. The analytical method was optimised and structured in four successive steps: (1) accelerated solvent extraction of the (spiked) analytes from matrix, (2) solid-phase purification (SPE) of the previous extract, (3) pre-concentration of the eluates from SPE and (4) analysis of the concentrated eluates by high performance liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode. After a proper sampling campaign, the method was applied to real indoor and outdoor particulate matter samples, where the clean-up step showed to be very effective and fundamental to avoid misleading analytical results.

Analysis of beta-agonist residues in bovine hair: Development of a UPLC–MS/MS method and stability study

Beta-sympathomimetic compounds are widely used in therapy because of the bronchodilator, smooth muscle-relaxant and tocolytic properties. However, their growth promoting and performance enhancing effects are often subject to illegal use. The present work describes the development of a fast and reliable analytical multiresidue method for the confirmation 20 β-agonist compounds in animal hair. The procedure is based on alkaline digestion, LLE with organic solvents, SPE clean up and liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, and is suitable for the public Official control of β-agonist residues in hair sample. Validation was performed according to Commission Decision 2002/657/EC requirements. Independent samples spiked with the investigated compounds in the range 0.2-10.0μgkg-1 are showing intra-day and inter-day precision (RSD) lower than 17.8% and 19.7%, respectively. Linearity, measured in the range of 0.1-10.0μgkg-1, resulted with a Pearsons r>0.996. The decision limits (CCα) for the all investigated beta agonists resulted in the range 0.2-1.0μgkg-1. Furthermore, the method was tested on real hair samples obtained from cattle, known as positive to clenbuterol, in order to check its effectiveness and the β-agonists stability.

Intake estimates of dioxins and dioxin-like polychlorobiphenyls in the Italian general population from the 2013-2016 results of official monitoring plans in food

The implementation of the European Union strategy for polychlorodibenzo-dioxins and -furans (PCDD/Fs), and dioxin-like polychlorobiphenyls (DL-PCBs) is determining a general reduction of their presence in the environment and in the food chain. The most important route for human exposure to these substances is food consumption and, as a consequence, a progressive decrease of their dietary intake has been observed in the last decades. In this context, it seemed worth updating the PCDD/F and DL-PCB intake estimation for the Italian population. A total of 2659 samples of food of animal and vegetable origin analyzed for PCDD/Fs and DL-PCBs in the period 2013-2016 by accredited official laboratories and the national food consumption database were considered for the dietary intake assessment in different age groups of the Italian general population The median cumulative intake estimates expressed as pg WHO-TEQ/kg body weight per day and computed with a deterministic and a probabilistic approach were 1.40-1.52 for children, 0.82-0.85 for adolescents, and 0.64-0.61 for adults, respectively. Such results confirm the decreasing trend of PCDD/F and DL-PCB dietary intake even though the Tolerable Daily Intake (TDI) value of 2 WHO-TEQ/kg body weight per day is exceeded at the 95th percentile for all age groups, with children as sensitive group. Most contributing food categories to the intake resulted fish, food of vegetable origin, and cheese. A sensitivity analysis was also performed to calculate the target contamination levels able to keep the dietary exposure below the TDI. Computed target levels fall between P50 and P97 of the occurrence distribution of the main food groups, meaning that most of the Italian food production can be considered safe.