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Analisi di vitamina B12 in prodotti lattiero-caseari mediante cromatografia liquida ad ultra prestazione accoppiata alla spettrometria di massa tandem Elisa Zironi, Teresa Gazzotti, Andrea Barbarossa, Federica Farabegoli, Andrea Serraino, Giampiero Pagliuca* Dipartimento di Scienze Mediche Veterinarie, Alma Mater StudiorumUniversità di Bologna, Ozzano Emilia (BO), Italy *Corresponding author: giampiero.pagliuca@unibo.it La vitamina B12, o cobalamina, è una molecola idrosolubile composta da un complesso tetrapirrolico con un atomo di cobalto al centro. È un nutriente essenziale assai importante per l’uomo che, a differenza delle altre vitamine del gruppo B, viene sintetizzato unicamente dai batteri e per questo motivo è presente solo negli alimenti fermentati battericamente o in quelli derivati da animali che l’hanno assunto tramite la dieta o attraverso la microflora gastrointestinale. Gli alimenti di origine animale sono quindi l’unica fonte di questa vitamina per l’uomo, il cui fabbisogno giornaliero è circa di 1-2 mg. Poiché il latte ed i prodotti lattiero-caseari forniscono un apporto dietetico significativo di questa vitamina, sono stati analizzati diversi campioni raccolti in vari stadi durante quattro processi di caseificazione, al fine di valutare la ripartizione di questa molecola nei differenti prodotti; in particolare sono stati esaminati: latte crudo, caglio, siero, cagliata, ricotta, mozzarella e caciotta, tutti prelevati in doppio. La procedura di estrazione è stata condotta su 5 g di ciascuna matrice, aggiungendo un tampone sodio acetato e cianuro di potassio; i campioni sono poi stati sottoposti a trattamento termico che, in presenza di cianuro, converte tutte le cobalamine in cianocobalamina. Dopo purificazione su cartucce per estrazione in fase solida i campioni sono stati iniettati nel sistema di cromatografia liquida ad alta prestazione accoppiata alla spettrometria di massa tandem (UPLC-MS/MS) ed analizzati in elettrospray positivo con modalità multiple reaction monitoring (MRM). Dai risultati ottenuti, il dato più interessante è quello del livello di B12 rilevato nel siero e nella ricotta, in cui si ha una concentrazione circa 10 volte maggiore rispetto a quella del latte di partenza. Parallelamente si assiste ad un calo in cagliata, caciotta e mozzarella. Essendo nota l’affinità della vitamina B12 per alcune proteine, questi dati confermerebbero la tendenza della cobalamina a concentrarsi nella frazione proteica del siero

Milk and dairy products (cheese, yoghurt, etc.) can be naturally modified and enriched with nutritional factors useful for consumer health; these food (called “functional”) together with a healthy life style may enhance the consumer health and welfare. The main objectives of the study were: i) define the possible feeding strategies to naturally enrich milk of cows with conjugated linoleic acid (CLA), omega-3 and vitamin B12; ii) set up a quantitative analytical method to detect B12 vitamin in milk and dairy products; iii) evaluate the content of CLA, omega3 and vitamin B12 in milk after thermal treatment and in cheese . Different feeding strategies were tested to obtain milk enriched; the evolution of the microflora during cheese making and its influence on CLA, omega3, fatty acids and vitamin B12 content of dairy products was also evaluated. Results showed that it is possible to modify the cow diet with different fat supplements enhancing the content of CLA and omega-3 fatty acids without significant adverse effects on the total fat content which may be incompatible with the production of high quality milk. In particular it is possible to produce milk with increased content of CLA, omega 3 and stearic acid; at the same time palmitic and myristic acids can be reduced; results showed also that this feeding strategy is more expensive compared to the usual rations utilized for the production of the standard milk. Thermal treatment and cheese making don’t influence the CLA content in dairy products and their content in CLA depends from the content of milk used for production. The analytical method set up for the quantitative analysis of Vitamin B12 in milk and dairy products proved to be satisfactory (sensitive and rapid); cobalamin supplementation of feed for cows caused an increase of Vitamin B12 in milk after four weeks. The most interesting data are relative to the concentration, during cheese making, of Vitamin B12 in whey and in whey cheese (ricotta cheese) where Vitamin B12 is tenfold concentrated respect to the milk used for processing.

L'esposizione umana ai PFC avviene principalmente attraverso la dieta ed in particolar modo il pesce è risultato tra i prodotti alimentari più inquinati. Il perfluorottano sulfonato (PFOS) e l’acido perfluorottanoico (PFOA) sono i composti più importanti e studiati. Lo scopo del presente lavoro è stato quello di effettuare un monitoraggio preliminare sulla presenza di queste due molecole in 50 campioni di muscolo di branzini, di cui 30 allevati e 20 pescati, prelevati in diverse località italiane del mar Mediterraneo. Il metodo utilizzato prevede un’estrazione con solvente seguita da due passaggi di purificazione: uno con sali ed uno con fase solida dispersiva. L’estratto è stato poi analizzato mediante un sistema UPLC-MS/MS. I dati mostrano ampia contaminazione di questa specie ed evidenziano una netta prevalenza di PFC nei branzini pescati (PFOS da 112,4 ng/L a >2000 ng/L e PFOA da 3,3 ng/L a 487,0 ng/L) rispetto a quelli allevati (PFOS da 11,1 ng/L a 104,5 ng/L e PFOA da

The term perfluorinated (PFCs) defines synthetic fully-fluorinated compounds; since the ‘50s they have been largely employed in a wide range of industrial and consumer applications, due to their chemical and physical properties. Only recently researches on their potential adverse effects on both the environment and human health have been conducted, highlighting carcinogenic activity, toxic effects on reproduction, liver and kidney. Human exposure to PFCs is mainly through diet, and in particular fishery products are one of the most contaminated food. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the two most important and investigated compounds of this group. The aim of this work was to carry out a preliminary monitoring on the presence of these two molecules in 50 bass muscle samples, of which 30 reared and 20 fished, collected from different Italian areas of the Mediterranean Sea. The method used was based on an extraction with organic solvent followed by two purification steps: one with salts and one with dispersive solid phase. The extract was finally analysed by means of a UPLC-MS/MS system. Data showed a relevant contamination of this species and evidenced a significant difference of concentrations between fished (PFOS from 112.4 ng/L to >2000 ng/L and PFOA from 3.3 ng/L to 487.0 ng/L) and reared basses (PFOS from 11.1 ng/L to 104.5 ng/L and PFOA from

I composti perfluorurati (PFC) sono sostanze completamente fluorurate di origine antropica e presentano svariate applicazioni industriali e domestiche, quali imballaggi per alimenti, rivestimenti antiaderenti per tegami, schiume antiincendio, detergenti, cosmetici etc. Anche se i loro primi impieghi risalgono agli anni ’50, scarsa attenzione è stata data ai loro potenziali effetti nocivi sia sull’ambiente che sull’uomo fino a pochi anni fa, quando un crescente interesse verso queste sostanze ha sollevato serie preoccupazioni circa la loro attività cancerogena, gli effetti sulla riproduzione e la tossicità epatica e renale. L'esposizione umana ai PFC avviene principalmente attraverso la dieta, per questo motivo la Commissione Europea ha pubblicato la raccomandazione 161/2010 sul controllo di tali contaminanti nei prodotti alimentari da parte degli Stati Membri. Il perfluorottano sulfonato (PFOS) e l’acido perfluorottanoico (PFOA) sono i composti più importanti e studiati, anche per il fatto che il primo è stato classificato come POP (inquinante organico persistente) durante la Convenzione di Stoccolma del 2009. Lo scopo del presente lavoro è stato quello di effettuare un monitoraggio preliminare sulla presenza di queste due molecole in latte bovino disponibile commercialmente in Italia. Per la loro determinazione è stato usato un metodo sensibile ed efficiente, basato su un’estrazione liquido-liquido, seguita da due passaggi di purificazione su cartucce SPE ed analisi in un sistema UPLC-MS/MS. I dati, ottenuti dall’analisi di 22 campioni di latte bovino, mostrano la frequente presenza di tali sostanze anche se a concentrazioni relativamente basse (concentrazioni di PFOS e PFOA da 15 ng/L a 67 ng/L e da 24 a 32 ng/L rispettivamente). Questi risultati, in linea con i pochi dati disponibili in letteratura, evidenziano una contaminazione abbastanza ridotta del latte vaccino, sottolineando però la necessità di approfondire diversi aspetti per effettuare una corretta valutazione del rischio legato ai PFC, sia nel latte che nei prodotti lattiero caseari.

Perfluorinated compounds (PFCs) are fully-fluorinated man-made substances largely employed in a wide range of industrial and consumer applications, such as food packaging, fire fighting foams, detergents and non-stick cookware. Even if the introduction of these chemicals dates back to the 1950s, little attention has been given to their potential effects on both environment and human health, but the increasing interest on these substances has recently raised serious concerns about their carcinogenic activity, effects on reproduction and liver and kidney toxicity. Human exposure to PFCs is mainly through diet, for this reason the European Union issued the Commission Recommendation 2010/161/EU on the monitoring of these contaminants in food in the Member States. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the two most important and investigated compounds of this group, being added to the POPs (Persistent Organic Pollutants) listing on the Stockholm Convention. Aim of this work was to carry out a preliminary monitoring on the presence of these two molecules in cow milks commercially available in Italy and France. The cooperation between the two laboratories has led to the development of an efficient method for the detection of various PFCs in milk, based on a liquid- liquid extraction with organic solvent followed by two purification steps through SPE cartridges and injection in LCMS/ MS system. The application of this method to several milk samples from Italy and France has shown similar results in the two countries, with PFOS and PFOA detected in most cases and at similar concentrations. However, these levels of contamination were sensibly lower than the ones observed in various studies on breast milk, and even more if compared to the data available on fish and seafood monitoring. In consideration of the above, cow milk seems not to be a major source of PFCs exposure in human diet

Mycotoxins are chemical compounds characterized by a low molecular weight and synthesized by the secondary metabolism of different molds. Fumonisins are water-soluble mycotoxins produced by Fusarium species spoiling corn and derived products. These mycotoxins can enter the food chain also through the consumption of meat of exposed animals. Fumonisins and their metabolites have been associated with several animal and human diseases. They are suspected risk factors for esophageal and liver cancers, neural tube defects and cardiovascular problems. Improved methods are needed to accurately assess fumonisin concentrations in food from vegetable and animal origin to prevent acute and chronic human exposure. The aim of the present work was to develop a sensitive and selective method for quantification and unambiguous identification of fumonisin B1 (FB1), fumonisin B2 (FB2) and their complete hydrolyzed products (HFB1 and HFB2), in order to determine their presence in pig liver. Furthermore, the developed method was applied, in order to test its efficacy, on liver samples of weaned piglets fed a diet in compliance with the FB1 and FB2 contamination limits set by EU. All the samples showed the presence of at least one of the analytes. In particular FB1 was found in 5 out of 7 samples and the average level of contamination found was 28 ppb.

The aim of the present work was to develop a sensitive and selective method for identification and quantification of fumonisin B1 (FB1) in milk. FB1 was isolated from milk, by a single step immunoaffinity column and was detected using liquid chromatography coupled with tandem mass spectrometry in positive electrospray ionization (ESI+). The analysis were carried out in multiple reaction monitoring (MRM) mode using the two main product ions. The good performances of the proposed method can assure a correct fumonisin detection in milk even at relatively low concentrations.

Perfluorinated chemicals (PFCs) are fully fluorinated man-made chemicals with properties that make them useful in different industrial and household applications. They have been manufactured for over 50 years and used as surfactants and surface protectors in carpets, textiles, leather, paper product, fire-fighting, foams, cosmetics, insecticides and food containers. Given their chemical-physical properties, it is expected that PFCs will be resistant to hydrolysis, photolysis, biodegradation and metabolism. Recent studies have shown the global presence of PFCs. Human exposure to PFCs is not well known. Potential routes of exposure may be through air, water, household dust and food. In recent years growing interest arose in the study of PFCs; there are currently some information about their distribution in the environment as pollutant but there is a lack of knowledge about their presence in food. Also analytical methods’ literature for the PFCs determination in food of animal origin is very poor. Clearly this substantial lack of information on the hazardous potential, environmental distribution and the fate from farm to fork of PFCs is of concern to policy-makers, producers and consumers.

Aflatoxin contamination of milk was an emerging problem in Italy in Autumn 2003 and has represented an important risk factor for consumers. The European Union (EU) has established regulatory limits of the presence of AfM1 in milk and has suggested the methods of control: immunoassay tests (ELISA) for screening and high performance liquid chromatography (HPLC) for confirmation. Hence the importance to constantly control the performances of analytical methods. The aim of this research was to evaluate the performances of two ELISA kits, produced by different suppliers, on naturally contaminated raw milk in a range from 10 to 85 ng/L. The samples were previously analysed by HPLC to determine the level of AfM1. Results showed good responses in both kits in terms of precision. In addition to that some trials have been conduced to investigate the possible influence of time on the response of kits ELISA.

A study was conducted in cow raw milk to measure residues of 15 organophosphorus pesticides, used as dairy cattle ectoparasiticides or in crops used for animal feed. The determination was carried out improving a previous method by dual column capillary gas chromatography with nitrogen-phosphorus detection (NPD). The samples, collected in 9 Italian dairy plants directly from the tank trucks during the delivering, were representative of 3,974 tonnes of raw milk. Approximately 4.4 % of the 298 samples analyzed contained residues, always in traces. The main pollutant was chlorpyriphos. These data were compared with a previous similar investigation

A previous method was improved in order to determine 15 organophosphorus pesticides (OPPs) in raw milk by dual column capillary gas chromatography using Nitrogen-Phosphorus Detection (NPD). With a single injection it was possible to measure the following OPPs: acephate, azinphos ethyl, chlorpyriphos ethyl, chlorpyriphos methyl, demeton-S-methyl sulfoxide, diazinon, disulfoton, methacrifos, methamidophos, methidation, parathion ethyl, phorate, pirimiphos methyl, pyrazophos, triazophos. All of these substances have a maximum residue levels (MRL) fixed by the European Commission. The isolation was performed by liquid partition followed by a single cleanup with a C18 monofunctional solid phase cartridge The GLC analysis was carried out with two capillary columns of different polarity, connected in parallel by a glass dual column adapter. The chromatographic temperature program was from 80°C to 100 °C at 2°C/min., followed by a second rate of 5°C/min. up to 300°C. The quantification was obtained using parathion methyl as an internal standard. The analyte identification was confirmed by comparing the retention times in the two columns. The recovery and precision for each compound were determined using 18 aliquots of a blank milk. The aliquots were divided in 3 groups of six and fortified using 0.5, 1 and 1.5 times the permitted limit each, according to the European Commission Decision 2002/657/EC. Although there was an increase in the number of analytes, the GLC conditions were further simplified without any changes in the analytic performances. The method was developed in a UNI EN ISO 9001:2000 certified laboratory.

Aflatoxins are products of secondary metabolism of various species of Aspergillus, a fungus that grows in a wide range of food and animal feedstuffs. Aflatoxin B1 ingested by cows is epatically transformed in aflatoxin M1 (AfM1) and then secreted in milk. The European Union (EU) has established regulatory limits of the presence of AfM1 in milk and has suggested the methods of control: ELISA for screening and HPLC for confirmation. Hence the importance to constantly test the validity of analytical methods, not only on spiked samples but also on naturally contaminated matrices. In this work several trials have been conducted to evaluate the performances of two different commercial ELISA kits on naturally contaminated milk in the range from 10 ppt to 85 ppt; the samples were previously analysed by HPLC to determine the level of AfM1. Results showed good responses in both kits in terms of accuracy and precision.

La contaminazione da aflatossine nel mais e nel latte è stato un problema emergente nell’autunno del 2003 in Italia. Per la valutazione delle contaminazioni da AFM1 nel latte possono essere utilizzate metodiche di screening (ELISA) e di conferma (HPLC). Nel lavoro sono state messe a confronto le performances di due differenti kit ELISA in campioni artificialmente contaminati a concentrazioni di AFM1 da 10 ppt a 80 ppt. I risultati evidenziano una notevole discrepanza tra i due test utilizzati, sia in termini di linearità della risposta che in termini di accuratezza e precisione.

Most of the enzymes involved in such biotransformation, including cytochrome P450 monooxygenase or mixed function oxygenase (MFO) systems have been identified in marine vertebrates and invertebrates. Several studies suggest that during phase I reactions cytochrome P450 and the flavoprotein reductase components of the MFO system are involved in the formation of oxygen free radicals or metabolite radicals. Since the oxyradical generation causes biological damage, the integrity of the cell is protected by antioxidant defences. Antioxidant enzymes as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), play a crucial role in maintaining cell homeostasis. The aim of this work is to study the induction of cytochrome P450 after B[a]P exposure in Scapharca inaequivalvis and the relationship between the level of the antioxidant defences and P450 content

The aim of this study was to determine to what extent rainbow trout fillets fortifyied with selected n-3 polyunsaturated fatty acids (PUFA) and cooked by moist- or dry-heat cooking methods could meet the adequate intake of adults for alpha-linolenic acid (LNA, C18:3 n-3), eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3). n-3 PUFA enriched rainbow trout (Oncorhynchus mykiss Walbaum) were produced as described by Gatta et al. (2004, same proceedings). At the 75th day of the trial, 6 batches of fish (3 fish each) were randomly sampled for each diet. Fish were immersed in ice-cold water for slaughter, gutted, filleted and boned. Within batch, 3 skin-on fillets were destined for baking-in-foil in a preheated forced air convection oven, to exemplify “convection + moist-heating” (OB), while their counterparts were pan-fried in a Teflon-coated pan to exemplify “conduction + dry-heating” (PF). Cooking was discontinued upon reaching 65-70°C. All fillets from each subsample of fish (i.e. cooking method within batch) were skinned, ground together and thoroughly mixed to provide a homogeneous composite paste, which was analysed for lipid and fatty acid contents (g/100 g edible portion). Lipid content did not differ significantly among diets (mean  standard error: OB=7.390.30; PF=7.190.32). On the whole, PUFA contents of cooked fillets were not affected by cooking method. Linoleic acid (LA, C18:2n-6) and arachidonic acid (AA, C20:4 n-6) did not differ among diets (LA range=0.89-1.06; AA range=0.12-0.16). LNA content was significantly higher in D1 cooked fillets (D1=0.44 x; D2=0.23 y; D3=0.20 y). EPA content predominated in D2 cooked fillets (D1=0.19 z; D2=0.43 x; D3=0.27 y). DHA was significantly more represented in D3 cooked fillets (D1=0.79 y; D2=1.10 x; D3=1.13 x). n-6/n-3 did not differ among diets, ranging between 0.62 and 0.66. With reference to the adequate intake of adults recently issued for LNA, and DHA+EPA (2.22 and 0.65 grams/day, respectively, for a 2000 kcal diet), a 100-g serving of the cooked fillets were able to give the following average contribution: D1=20 and 151%; D2=10 and 235%; D3=9 and 215%, respectively. In conclusion, selective n-3 PUFA fortification generated trout fillets which, regardless of the cooking method used, could provide considerable amounts of the suggested adequate intake.