A rapid HPLC-FLD method for Ochratoxin A detection in pig muscle, kidney, liver by using enzymatic digestion with MISPE extraction

Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by various Aspergillus and Penicillium species with nephrotoxic, carcinogenic, immunotoxic and teratogenic effects. OTA has been found in several food commodities, including cereals, beer, wine and spices. OTA can also be present in animal products (especially in pig derived products) as a result of carryover from contaminated feed. Permitted level of 1 μg/kg OTA in pig meat or pig-derived products was set in Italy, as in other countries. Key parameters which affected MISPE, should be described such as extraction efficiency and were optimized, analyzed by an isocratic HPLC-FLD method. Under the optimized conditions, for all analyzed matrices mean recovery was > 89%. Method can be applied as alternative routine procedure to detect OTA presence in pig products. Points: ∗Aim of the study was to develop and validate a quantitative HPLC-FLD method based on MISPE with complex solid matrices (edible tissues) followed by chromatographic analysis. ∗The new method was developed and validated in pig complex matrix and is very sensitive LOD and LOQ respectively 0.001 and 0.003 μg/kg. ∗This method is relatively simple to use and with good performances. Was possible to reuse MISPE column with a “regeneration” solution, until to 7 times

Design and synthesis of indolylglyoxylamide derivatives as probes for the Translocator protein (TSPO)

ABSTRACT The 18kDa Translocator Protein (TSPO) is mainly located at the contact sites between the outer and the inner mitochondrial membranes, and is strictly associated with a number of proteins to form the mitochondrial permeability transition pore (MPTP). It is involved in a variety of biological processes including cholesterol transport, steroidogenesis, cell growth and differentiation, apoptosis induction, and regulation of immune functions. The expression of TSPO is ubiquitary in peripheral tissues (steroid producing tissues, liver, heart, kidney, lung, immune system) and in the central nervous system is mainly located in glial cells and in neurons. TSPO is up-regulated in a number of neuropathologies, Huntington’s and Alzheimer’s diseases, tumors etc.. Consequently, TSPO has been suggested as a promising diagnostic marker to image and measure the TSPO expression and distribution levels, and thus for evaluation of disease progression by means of specific fluorescent or radiolabeled ligands. The aim of this work is to synthesize and biological characterize a series of 2-phenylindol-3-ylglyoxylamides, and to test their validity as new TSPO probes. Compound 1 is characterized by the presence of a chemoreactive isothiocyanate group and by the NBD-fluorescent moiety. Compounds 2 and 3 are characterized by the presence of (1,4,7-triazonane-1,4,7-triyl)triacetic acid group (NOTA) and were synthesize to prepare and evaluate Ga-68 complexes as potential PET agents for measurement of TSPO

Personalized medicine of monoclonal antibodies in inflammatory bowel disease: pharmacogenetics, therapeutic drug monitoring and beyond

The pharmacotherapy of inflammatory bowel diseases (Crohn’s disease and ulcerative colitis) has experienced significant progress with the advent of monoclonal antibodies (mABs). As therapeutic proteins, mABs display peculiar pharmacokinetic characteristics that differentiate them from chemical drugs, such as aminosalicylates, antimetabolites (i.e., azathioprine, 6-mercaptopurine, and methotrexate), and immunosuppressants (corticosteroids and cyclosporine). However, clinical trials have demonstrated that biologic agents may suffer from a pharmacokinetic variability that could influence the desired clinical outcome, beyond primary resistance phenomena. Therefore, therapeutic drug monitoring (TDM) protocols have been elaborated and applied to adaptation drug doses according to the desired plasma concentrations of mABs. This activity is aimed at maximizing the beneficial effects of mABs while sparing patients from toxicities. However, some aspects of TDM are still under discussion, including time-changing therapeutic ranges, proactive and reactive approaches, the performance and availability of instrumental platforms, the widely varying individual characteristics of patients, the severity of the disease, and the coadministration of immunomodulatory drugs. Facing these issues, personalized medicine in IBD may benefit from a combined approach, made by TDM protocols and pharmacogenetic analyses in a timeline that necessarily considers the frailty of patients, the chronic administration of drugs, and the possible worsening of the disease. Therefore, the present review presents and discusses the activities of TDM protocols using mABs in light of the most recent results, with special attention on the integration of other actions aimed at exploiting the most effective and safe therapeutic effects of drugs prescribed in IBD patients

DEVELOPMENT OF AN ELECTROCHEMICAL SENSOR BASED ON SCREEN-PRINTED ELECTRODES FOR OCHRATOXIN A IN PORK MEAT SAMPLES

Ochratoxin A (OTA) is a nephrotoxic, immunosuppressive and teratogenic mycotoxin produced by As- pergillus and Penicillium spp. fungi during food storage. OTA can be detected in cereal products, coffee, wine, beer, cheese and in poultry and pork meat. Many detection techniques, such as liquid chromatography coupled with immunoaffinity column or solid phase extraction cleanup, have been used for OTA determina- tion in different samples (1). In recent years electrochemical techniques have been used for the rapid and accurate detection of OTA (2). The aim of the present study was to develop a new analytical method for OTA quantitative detection in pork meat based on electrochemical sensing, using graphite-based screen-printed electrodes and differential pulse voltammetry (DPV) as detection technique. Experiments were performed with an electrochemical transducer Palmsens, monitored with a personal computer using PSTrace software (Palm Instrument BV, Houten, The Netherlands) for data acquisition and subsequent analysis. The electrochemical assays were performed with miniaturized disposable graphite based screen-printed electrodes (EcoBioServices & Researches s.r.l., Florence, Italy). The effect of pH (range 2-7) and of ionic strength (KCl concentration range 10-200 mM) of the supporting electrolyte solution (acetate buffer) on the DPV peak current and potentials was investigated to optimize the DPV method. The effect of the DPV parameters on OTA oxidation peak was studied. Potential pulse amplitude (Epulse) was evaluated in the range of 10-100 mV. Step height was evaluated in the range of 2-10 mV. The influence of the scan rate was examined in the range of 0.005-0.1V/s. Standard addition method was applied for quantitative analysis. The method was applied for OTA determination in spiked pork meat samples. Results were compared with those provided by a reference HPLC method. The OTA peak current increased with increasing acetate buffer pH (from 2.0 to 7), thus pH of 7.0 for the supporting electrolyte solution was chosen. Concentrations of 75 mM KCl in the supporting electrolyte was selected. The optimization of DPV parameters indicated that best results for voltammograms were obtained from 0 to 1.1 V by using 5 mV potential step, 50 mV potential pulse, 0.01 V/sec scan rate and 0.07 sec time pulse; each scan was performed after an equilibrium time of 30 sec. Calibration graphs of peak height against concentration for OTA by DPV were plotted over the range 25-1000 μg/l in the supporting electrolyte with a LOQ of 25 μg/l. The findings obtained with voltammetric-based sensing were in good agreement with results obtained by HPLC analysis but matrix effects have been detected at lower OTA concentrations indicating the need of more selective extraction procedure. The proposed method is more rapid and inexpensive in comparison with the classical methods for OTA analysis, and can be considered a promising alternative for the evaluation of OTA in meat. 1) Turner et al, Anal Chim Acta, 2009, 632 ,168-180. 2) Prieto-Simón et al, TrAC, 2007, 26, 689-702

DETERMINATION OF OCHRATOXIN A IN FARMED FISH BY ENZYMATIC DIGESTION (ED) COUPLED TO HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH A FLUORESCENCE DETECTOR (HPLC-FLD)

Several studies have demonstrated that fish feeds contain significant concentrations of chemical contaminants, many of which can bioaccumulate and bioconcentrate in fish tissues (1).  The serious concern regarding the use of fish meal and fish oil in the the aquaculture industry has led to extensive search of alternative raw materials for aquafeeds. The most obvious alternatives are oils and proteins of plants origin. The use of these alternative feed ingredients can introduce contaminants that were previously not associated with fish farming such as mycotoxins (2). Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by various Aspergillus and Penicillium species with nephrotoxic, carcinogenic, immunotoxic and teratogenic potential (3). OTA has been found in several food commodities, including cereals and can also be present in food of animal origin as a result of carryover from contaminated feed (3). The aim of the present study was to determine OTA concentrations in muscle, kidney and liver of 10 seabream and 10 seabass of farmed origin collected on the market. Analysis will be performed by using an enzymatic digestion (ED) method coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). Fish tissues were digested for 1 hour at 37°C with a 1% pancreatin solution in a phosphate buffer and then cleaned up with ethylacetate. After being evaporated to dryness and re-dissolved, the sample was processed using HPLC-FLD. The method was validated for: specificity, recovery, trueness, selectivity, linearity, limit of detection (LOD) and limit of quntification (LOQ), repeatability and reproducibility. Recoveries of analytical method were higher than 85 % for all the matrices. Intra and inter-day repeatability expressed as relative standard deviation were less than 9%. The LOD and LOQ for liver and muscles samples were 0.001 and 0.002 μg/kg, respectively. The LOD and LOQ for kidney samples were 0.01 and 0.02 μg/kg, respectively. The highest concentrations of OTA were found in kidney of the 20 fishes analyzed (rang

Determination of ochratoxin A in pig tissues using enzymatic digestion coupled with high-performance liquid chromatography with a fluorescence detector

We present a new method for the rapid analysis of ochratoxin A (OTA) in pig tissues (muscle, liver and kidney) using enzymatic digestion (ED) coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). OTA was digested with a 1% pancreatin solution in a phosphate buffer and then cleaned with ethylacetate. After being evaporated to dryness and re-dissolved, the sample was determined using HPLC-FLD. The method was validated taking into account the currently permitted limit of 1 μg/kg OTA in pork meat and derived products in Italy. The recovery was higher than 90%. Intra- and inter-day repeatability expressed as RSD were less than 7%. The LOD and LOQ were 0.001 and 0.002 μg/kg, respectively. Our method is more efficient, easier, and cheaper than conventional clean-up procedures (liquid–liquid extraction)

Serum levels of Ochratoxin A in dogs with chronic kidney disease (CKD): a retrospective study

Ochratoxin A (OTA) is a mycotoxin produced by secondary metabolism of several fungi belonging to the genera Aspergillus and Penicillium. OTA is potentially nephrotoxic, neurotoxic, immunotoxic and carcinogenic in several animal species and in humans. This toxin has been detected in several human food and animal feed. The aim of this study was to determine OTA in blood samples of healthy and affected by chronic kidney disease (CKD) dogs. CKD group showed higher incidence of OTA-positivity than healthy dogs (96% vs. 56%) and a significantly higher median value of OTA plasma concentration (0.008 ng/ml vs. 0.144 ng/ml). No significant correlation was observed between OTA levels and creatinine values in CKD dogs. This is first study regarding OTA detection in plasma samples of healthy and CKD dogs; the presence of this toxin is higher in nephropatic patients but is not yet clear, if it is correlated with progression of the disease

OCHRATOXIN A RESIDUES IN HUNTED WILD BOAR (SUS SCROFA) FROM TUSCANY

Ochratoxin A (OTA) is a secondary toxic metabolite synthesized by Aspergillus or Penicillium species, which can contaminate various crops. The International Agency for Research on Cancer classified OTA as a group 2B possible human carcinogen. OTA is nephrotoxic, mutagenic, teratogenic and immunosuppressive. OTA can also be present in meat of animals where its presence comes as a result of animal feeding with contaminated grain and feed mixtures. The Italian Ministry of Health Circular No 10, dated 9 June 1999, establishes, as a guideline, a maximum value of 1 μg/kg OTA for swine meat and meat products. The significant increase in the wild boar population has resulted in an increased prevalence of wild boar meat, offal and ready-made products in the food industry. The aim of the present study was to determine OTA concentrations in muscle, kidney and liver of wild boar hunted in Tuscany region. A total of twenty wild boars (male n=11 female n=9) were collected in the Province of Pisa from November 2014 to April 2015, animals have been slaughtered and the carcass weight were determined (from a min. of 14.9 kg and a max. of 72.0 kg). Samples of kidney, liver and muscles from each wild boar were collected and analyzed with an enzymatic digestion clean-up and high-pressure liquid chromatography with fluorescence detection method (1). The highest levels of OTA were found in the kidneys of the twenty wild boar analyzed (0.07- 2.01 μg/kg, mean 0.58±0.63 μg/kg). The levels found in the liver ranged between 0.08- 1.93 μg/kg, (mean 0.53±0.60). The lowest concentrations were found in muscle (0.04- 0.77 μg/kg, mean 0.24±0.24). In eight samples of the tissue samples examined in this study (4 kidney and corresponding 4 liver), the levels of OTA were higher than the guideline level (1 μg/kg) established by the Italian Ministry of Health. The present results are in agreement with a previous study conducted in Calabria in wild boars (2). Swine are particularly sensitive to OTA, kidneys showed the highest accumulation of the latter 101 Società Italiana delle Scienze Veterinarie toxin, followed by liver and muscle tissue, finally the lowest accumulation is represented in adipose tissue. The present results showed the same type of accumulation in wild boar. Traditionally in Tuscany, as in other regions, wild boar meats are used to produce niche products, especially coppa and salami. In agreement with the research of Monaci et al. (3), dried wild boar meat may contribute to overall OTA intake by carry-over effects into processed meats. Monitoring the quality of meat destined for transformation is a priority in order to decrease the possibility of toxin carry-over to humans. The present study confirms that contamination of meat products by OTA represents a potential emerging source of OTA for distinct segments of the Italian population, who are significant consumers of locally-produced wild boar specialties. 1. Luci G., Vanni M., Ferruzzi G., Mani D., Intorre L., Meucci V. 2016. MethodsX 3: 171-177. 2. Bozzo G., Ceci E., Bonerba E., Di Pinto A., Tantillo G., De Giglio E. 2012. Toxins (Basel) 4: 1440-1450. 3. Monaci L., Tantillo G., Palmisano F. 2004. Analytical and Bioanalytical Chemistry 378: 1777- 1782

A new HPLC-UV method compared with HPLC-MS for daptomycin levels in human plasma samples

Application of a new HPLC-UV method on 122 plasma samples of patients from various AOUP wards, after analytical method settings, validation and comparison with a reference LC-MS/MS method. All research activities were performed according to the “Daptolin” protocol (approved by the Pisa University Hospital Ethics Committee, prot. num. 55945

Precision medicine in lymphoma by innovative instrumental platforms

Since the last years, many efforts have been addressed to the growing field of precision medicine in order to offer individual treatments to every patient on the basis of his/her genetic background. Formerly adopted to achieve new disease classifications as it is still done, innovative platforms, such as microarrays, genome-wide association studies (GWAS) and next generation sequencing (NGS), have made the progress in pharmacogenetics faster and cheaper than previously expected. Several studies in lymphoma patients have demonstrated that these platforms can be used to identify biomarkers predictive of drug efficacy and tolerability, discovering new possible druggable proteins. Indeed, GWAS and NGS allow the investigation of the human genome, finding interesting associations with putative or unexpected targets, which in turns may represent new therapeutic possibilities. Importantly, some objective difficulties have initially hampered the translation of findings in clinical routines, such as the poor quantity/quality of genetic material or the paucity of targets that could be investigated at the same time. At present, some of these technical issues have been partially solved. Furthermore, these analyses are growing in parallel with the development of bioinformatics and its capabilities to manage and analyze big data. Because of pharmacogenetic markers may become important during drug development, regulatory authorities (i.e., EMA, FDA) are preparing ad hoc guidelines and recommendations to include the evaluation of genetic markers in clinical trials. Concerns and difficulties for the adoption of genetic testing in routine are still present, as well as affordability, reliability and the poor confidence of some patients for these tests. However, genetic testing based on predictive markers may offers many advantages to caregivers and patients and their introduction in clinical routine is justified