Development and Validation of an Ion Chromatography Method for the Simultaneous Determination of Seven Food Additives in Cheeses

Cheeses are characterized by several chemical-physical properties that make it difficult for the microorganisms growth, consequently. The actual European legislation allows the addition of few food additives in this type of food products. In this work, the entire procedure of extraction, purification, chromatographic separations and quali/quantitative determination of seven food additives (sorbic acid, benzoic acid, lactic acid, acetic acid, nitrites, nitrates and phosphates) was developed and applied for the analysis of different types of cheese (mozzarella, cheese spread, semi-hard and hard cheeses). Through validation procedure it was possible to evaluate the most important validation parameters. Extended calibration curves (r > 0.990) were obtained for all the analyzed compounds. Recovery values ranged from 72.8% to 98.4% and a good repeatability was obtained, with precision levels in the range of 0.03% - 0.11% (n = 6). The potential and feasibility of the method were tested by analysing real samples, such as mozzarella, cheese spread, semi-hard and hard cheeses, confirming that the method is well suited to satisfy the demands for accurate confirmation analyses of seven food additives in cheeses, which is especially valuable in official check analyses and in monitoring schemes.

Recent Epidemic-Like Anthrax Outbreaks in Italy: What Are the Probable Causes?

In Italy, in an area of about 50 km2 between Basilicata and Campania Southern regions, 28 outbreaks of anthrax occurred from August 28th to September 27th 2011. Different species were affected: laboratory tests confirmed anthrax in cattle, horses and sheep. The genetic analysis of strains isolated from infected animals indicated that outbreaks occurring in the two regions were not correlated. Intriguingly, the incidence was highly significant in horses compared to that of cattle and sheep or goats, which were prevalent species in the animal population. In addition, allinfected horses and many cattle developed a fatal, sub-acute form of anthrax, characterized by the presence of massive edema, usually absent in hyperacute forms. The characteristics of these outbreaks suggested a possible role of tabanids as vectors in the transmission of B. anthracis like-epidemic infection.

Inter-Laboratory Ring Trial to Evaluate Reverse Transcription Polymerase Chain Reaction Methods Used for <i>Dolphin Morbillivirus</i>Detection in Italy

Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mammals (C.Re.Di.Ma) performed the first inter-laboratory ring trial with the aim to standardize a diagnostic biomolecular approach for DMV in Italy. Viral isolation is usually considered the “gold standard” for the definitive diagnosis of most pathogens, but it is not often feasible in DMV diagnosis, due to the poor preservation of virus-targeted tissues in stranded cetacean carcasses, as well as to the lack of appropriate sensitivity of cell lines towards DMV variability. Therefore direct viral detection on tissues by means of reverse transcription-PCR (RT-PCR) represents a valuable option for DMV infection’s diagnosis. For detecting DMV in cetacean die-offs occurred in the Mediterranean basin since 2013, C.Re.Di.Ma developed an RT-PCR based method targeting to a 287 bp fragment of DMV nucleoprotein (N) gene. With the purpose to evaluate its performances in terms of accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility), it was submitted to a ring trial. So, 12 Public Laboratories belonging to the Italian dead stranded marine mammals diagnostic network were asked to analyze a panel of 40 samples (positive and negative for DMV, using different dilutions of a viral suspension obtained from a cell culture supernatant of a DMV strain) with the aforementioned technique. Furthermore, we also aimed at comparing the accuracy of other 7 molecular methods routinely applied for DMV detection in Italy. For this purpose, the second panel of identical 40 DMV +ve and -ve samples was provided to Laboratories that routinely used DMV detection methods other than those developed by C.Re.Di.Ma, in order to be analyzed simultaneously with the method they usually applied. The C.Re.Di.Ma technique showed high accuracy [mean Se = 97.8% (95% CI 84.2% - 99.3%), mean Sp = 98.1% (95% CI 72.5% - 99.9%)] and very good precision [k combined equal to 0.91 (95% CI 0.87 - 0.95)]. In conclusion, this study highlighted a satisfactory reliability of most of the molecular methods used in Italy for DMV detection.

Box Behnken design-based optimized extraction of non-dioxin-like PCBs for GC-ECD and GC-MS analyses in milk samples

A multivariate optimization process of the sample extraction procedure by Box-Behnken design through a global desirability function is described for the determination of six non-dioxin-like polychlorinated biphenyls(NDL-PCBs#28,52,101,153,138 and 180)in milk by GC-ECD and mass spectrometry.Three factors were involved in refining the extraction conditions:the acetone percentage in the extraction mixture,the sample/solvent ratio,and the extraction time.The three-factor design required 26 experiments that were carried out in duplicate and in a randomized order to minimize the bias effects of uncontrolled variables.The optimized factors(acetone percentage:30%;sample-to-solvent ratio:0.11 g mL^-1;extraction time:45 min)ensured a low solvent consumption and a reduced extraction time,allowing a rapid and simultaneous preparation of multiple sample extracts.The method was validated according to the European directives(Decision 657/2002/EC,SANTE 2017/11813/EC)through the evaluation of linearity,selectivity,LOD,LOQ,recovery,precision,and ruggedness.