Teresa Gazzotti

INVESTIGATION OF 4‐METHYL STEROLS FROM CULTURED DINOFLAGELLATE ALGAL STRAINS

The marine dinoflagellates Prorocentrum micans, Gonyaulax polyedra, Gymnodinium sp., and Alexandrium tamarense, collected from the Adriatic Sea during red‐tide blooms, were cultured to investigate the 4‐methyl sterol constituents. To ascertain a possible influence of cell age on the 4‐methyl sterol content, for one strain (Gymnodinium sp.)we investigated the composition of these constituents at exponential and stationary growing phases. The lipid material extracted with acetone from the lyophilized algal samples was fractionated by thin‐layer chromatography. The 4‐methyl sterols recovered from the layer were converted into the corresponding OTMS derivatives. Nine of 11 constituents were identified by gas chromatography and gas chromatography‐mass spectrometry; only two minor constituents were characterized by their gas chromatographic parameters. All free methyl sterols identified in the algal samples had been detected previously in various dinoflagellates. The 4‐methyl sterol fractions generally contained very few constituents. Except for the Gymnodinium sp. sample, collected at the exponential growing phase (GyD2 exp), which contains 4,24‐dimethylcholestan‐3‐ol as a unique constituent, dinosterol was the major component. Moreover, 4,24‐ethylcholestan‐3‐ol was also an important constituent of both Prorocentrum and Gonyaulax strains, whereas considerable amounts of dinostanol characterized all the Gymnodinium sp. strains. In addition, the latter contained several minor constituents such as 4‐methylcholestan‐3‐ol, 4,24‐dimethylcholesta‐22‐en‐3‐ol, and 4‐methyl‐24‐ethylcholestan‐3‐ol. 4‐Methyl‐24‐methylene‐cholestan‐3‐ol was a constituent of the Gymnodinium sp. sample, collected at the stationary growing phase (GyD2 stat)only, whereas 4‐methylgorgostanol was identified only in the Alexandrium tamarense Gt4 strain. Except for 4‐methyl‐24‐ethylcholesta‐8(14)‐en‐3‐ol, all the methyl sterol constituents from our algae show a saturated polynuclear system. The pathways by which side‐chain modifications occur in dinoflagellate 4‐methyl sterols are considered, and a map of the fragmentation pattern of the trimethylsilyl‐4‐methyl sterols under electronic impact is also reported.

Organophosphorus pesticides residues in Italian raw milk

Organophosphorus pesticides (OPPs), widely used in agriculture, can cause toxic effects to humans and animals. The main purpose of the present work was to determine the contamination in raw milk by the main organophosphorus pesticides used in Italy and to evaluate the opportunity to start specific procedures of risk management along the milk production chain. The samples, collected in 4 Italian dairy plants directly from the tank trucks during the delivering, were representative of 920 tonnes of raw milk. The isolation of the OPPs (acephate, chlorpyriphos, chlorpyriphos-methyl, diazinon, methamidophos, methidathion, phorate, pirimiphos-methyl) was performed by liquid partition followed by clean-up with solid phase extraction. The analyses were carried out by dual column gas chromatography using two nitrogen-phosphorus detectors. Among the 135 samples analysed, 37 were positive in traces and 10 showed an OPP contamination ranging from 5 to 18 microg/kg. The higher results were recorded in the samples collected during the autumn-winter period. The main pollutants detected were acephate and chlorpyriphos. In every positive sample found, the OPP contamination was lower than the maximum residue level (MRL) fixed by the European Commission.

Perfluoroalkyl substances in human milk: a first survey in Italy.

Due to their widespread diffusion, perfluoroalkyl substances (PFASs) have been frequently found in the environment and in several animal species. It has been demonstrated that they can easily reach also humans, mainly through diet. Being lactation a major route of elimination of these contaminants, their occurrence in human milk is of particular interest, especially considering that it generally represents the unique food source for newborns. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), the two most important compounds of this family, have been frequently found in human milk at variable concentrations, but still limited data are available. The present study, the first conducted in Italy capable to detect these pollutants at ultra-trace levels by UPLC-MS/MS, confirmed the role of lactation as a relevant source of exposure for breastfed children. The measured concentrations ranged between 15 and 288 ng/L for PFOS and between 24 and 241 ng/L for PFOA. Moreover, mean concentrations and frequencies of both analytes resulted higher in milk samples provided by primiparous women, suggesting that the risk of intake might be higher for first-borns. Finally, comparing these results with previous data, PFOS gradual decrease over time since year 2000 was confirmed.

Plasma concentrations and therapeutic effects of budesonide in dogs with inflammatory bowel disease.

OBJECTIVE To evaluate the pharmacokinetics and clinical efficacy of budesonide in dogs with inflammatory bowel disease (IBD). ANIMALS 11 dogs (mean ± SD age, 5.7 ± 3.9 years; various breeds and body weights) with moderate or severe IBD. PROCEDURES Each dog received a controlled-release formulation of budesonide (3 mg/m(2), PO, q 24 h) for 30 days (first day of administration was day 1). The concentration of budesonide and its metabolite (16-α-hydroxyprednisolone) was measured via liquid chromatography-tandem mass spectrometry in plasma and urine samples obtained on days 1 and 8 of treatment. On those days, plasma samples were obtained before the daily budesonide administration and 0.5, 1, 2, 4, and 7 hours after drug administration, whereas urine samples were obtained after collection of the last blood sample. A clinical evaluation was performed on the dogs before onset of drug administration and on days 20 and 30 after start of drug administration. RESULTS The highest plasma concentration of budesonide and 16-α-hydroxyprednisolone on day 1 was detected at 1 hour and at 2 hours after drug administration, respectively. After standardization on the basis of specific gravity, the ratio between urinary concentrations of budesonide and 16-α-hydroxyprednisolone was 0.006 and 0.012 on days 1 and 8, respectively. The clinical response was adequate in 8 of 11 dogs. CONCLUSIONS AND CLINICAL RELEVANCE Budesonide was rapidly absorbed and metabolized in dogs with IBD. The drug gradually accumulated, and there was an adequate therapeutic response and no adverse effects.

Development of a rapid LC–MS/MS method for ribavirin determination in rat brain

A rapid and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of ribavirin (RBV) in rat brain was developed. Sample preparation required only two centrifuge steps before LC-MS/MS analysis and the chromatographic separation was achieved in isocratic conditions using an Atlantis T3 column with a nearly totally aqueous (95%) mobile phase. The method showed a good linearity over a concentration range of 5-1000ppb and satisfactory results in terms of accuracy.

Development and validation of a liquid chromatography/tandem mass spectrometry method for quantitative determination of amoxicillin in bovine muscle

A simple, quick and economical liquid chromatographic/tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of amoxicillin in bovine muscle was developed and validated. The sample preparation procedure involved a liquid extraction with water, followed by a protein precipitation step with acetonitrile. The extract was purified by a liquid-liquid partition with dichloromethane and the upper aqueous layer was directly injected into the LC-MS/MS system. Chromatographic separation was achieved on a reversed phase column, using a mixture of acetonitrile, water and 0.005% formic acid in water as mobile phase. Gradient elution was performed at a flow rate of 0.2 mL min⁻¹. Amoxicillin was detected using positive electrospray ionization in selected reaction monitoring (SRM) mode and was quantified using terbutaline as internal standard. The responses for standards prepared in solvent and in matrix were equivalent and additionally the absence of signal suppression was confirmed by the post column infusion technique. Amoxicillin stability in standard solution and in matrix was investigated at different times and storage conditions. Amoxicillin standards prepared in water were stable on storage up to 20 days at -20°C. Amoxicillin stability in matrix (spiked bovine muscle samples) was assessed up to 15 days at -20°C. The method was validated according to the parameters requested by European Commission Decision 2002/657/EC in terms of specificity, linearity, trueness, precision, decision limit (CCα) and detection capability (CCβ). All the trueness values fell within a range between 14.5% and 6.3%. Precision values for all levels of concentration tested were lower than the relative limit calculated by the Horwitz equation. The amoxicillin MRL is set at 50 μg kg⁻¹ and the CCα and CCβ of the method were 61.2 μg kg⁻¹ and 72.4 μg kg⁻¹, respectively.

Technical note: development and validation of a method using ultra performance liquid chromatography coupled with tandem mass spectrometry for determination of vitamin B12 concentrations in milk and dairy products.

A method using ultra performance liquid chromatography coupled with tandem mass spectrometry was developed to measure cobalamins in naturally enriched raw milk and to evaluate their fate during thermal treatments and along the process of cheese making. After addition of methotrexate as internal standard, samples were submitted to heat treatment in the presence of cyanide, which converts all the less-stable cobalamins into cyanocobalamin; then, purification was performed by a solid-phase extraction step. Reverse-phase ultra performance liquid chromatography separation coupled with tandem mass spectrometry provided a fast and reliable determination. Mass spectrometric analysis was carried out in multiple reaction monitoring mode. The monitored transitions were m/z 678.36 → 147.10 and 678.36 → 359.30 for vitamin B12 and m/z 455.22 → 175.13 and 455.22 → 308.22 for methotrexate (internal standard). The limit of quantification was 2 ng/g. The method showed good linearity from 2 to 20 ng/g (R(2) ≥ 0.98) and intra- and interday precisions were always less than 19%.

Short communication: Monitoring the presence of perfluoroalkyl substances in Italian cow milk

Perfluoroalkyl substances (PFAS) are fully fluorinated compounds widely used during the last 60 yr in the production of multiple industrial and consumer applications, such as food packaging, nonstick cookware, cleaning agents, and many more. These emerging contaminants have recently become of concern for human health because of their potential negative effects. The risk of exposure to PFAS for humans is mainly related to diet, and the increasing interest in food safety has led the European Commission to call Member States to monitor these contaminants in food matrices. The purpose of the present work was to perform the first monitoring on the presence of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), the 2 main and most widely investigated molecules of this family, in cow milk commercially available in Italy. We used an analytical protocol consisting of liquid-liquid extraction followed by 2 purification steps through solid-phase extraction cartridges and injection on an ultra-performance liquid chromatography-tandem mass spectroscopy system. The analysis of 67 samples of different types of cow milk from Italy demonstrated that contamination by PFOS was often present, although at relatively low concentrations (up to 97 ng/L), whereas PFOA was rarely found. On the basis of these results and data reported in the literature on this matrix, milk does not seem to be a major source of PFAS compared with other food categories such as fish and seafood. However, variability among different types of milk must be taken into account, and surveys of milk-derived products would be helpful to better define the risk for consumers.

Proposal of an analytical method for determination of residues of organophosphorus pesticides in milk by GLC-NPD.

G. Pagliuca*, T. Gazzotti, E. Zironi, N. Pavoncelli and R. Rosmini Department of Veterinary Public Health and Animal Pathology – Alma Mater Studiorum – University of Bologna *Correspondence: Dipartimento di Sanita Pubblica Veterinaria e Patologia Animale – Sezione di Igiene e T ecnologia Alimentare – Alma Mater Studiorum – Universita di Bologna, via T olara di Sopra, 50 – 40064 Ozzano dell’Emilia, Bologna, Italy E-mail: pagliuca@vet.unibo.it

Bisphenol A in edible part of seafood

Bisphenol A (BPA) is a man-made compound, mainly used as a monomer to produce polycarbonate (PC), epoxy resins, non-polymer additives to other plastics, which have many food related applications, such as food storage containers, tableware and internal coating of cans, as well as non-food applications such as electronic equipment, construction materials and medical devices. BPA exposure can occur when the residual monomer migrates into packaged food and beverages. Moreover, due to the ubiquitous presence of this compound, the general population can be exposed to environmental sources such as water, air and soil. Many studies have investigated the potential health hazards associated with BPA, which can elicit toxic and cancerogenic effects on humans. According to the European Food Safety Authority opinion, diet is considered to be the main source of exposure, especially canned food; moreover, among non-canned food, meat and fish products have the highest levels of BPA contamination. This review focuses on BPA contamination in seafood, analysing worldwide literature (from January 2010 to October 2015) on BPA contamination of edible parts. The authors try to identify differences between canned and non-canned seafood in literature, and gaps in the state of art. The data evaluated underline that all concentrations for both canned and non-canned seafood were below the specific migration limit set by the European Community Directive for BPA in food. Moreover, the canned seafood is more contaminated than the non-canned one.