Daniela Gastaldi

Leptin is a 16 kDa lipophilic protein hormone secreted by adipocytes and its most significant function is to inform the brain with negative feedback that regulates food intake. Recently the protein found in human breast milk was related to breast feeding and onset of obesity, and the evidence of a low probability to develop pediatric obesity in children fed with breast milk was also confirmed. Since leptin could have a critical role, its quantitation both in human breast, bovine milk and in infant formula products is interesting. For this reason, we developed an analytical method based on immunoaffinity purification followed by an analysis with nano-High Pressure Liquid Chromatography coupled with High Resolution Mass Spectrometry analyzer (nano-HPLC-HRMS) to identify and quantify leptin in milk samples and performed a pilot study using samples of human breast milk, bovine milk and infant formulas. With an obtained lower limit of quantitation (LLOQ) of 100 ng mL⁻¹ we quantified leptin in human breast milk finding an average of 6.70 ng mL⁻¹. Our results show that leptin was under LLOQ both in bovine milk and in infant formula products. In conclusion, the developed analytical method here described was suitable to quantify leptin in milk samples with a good sensitivity and selectivity, and without the use of radioactive reagents.

There is a growing body of evidence to suggest that cannabinoids are beneficial for a range of clinical conditions, including pain, inflammation, epilepsy, sleep disorders, the symptoms of multiple sclerosis, anorexia, schizophrenia and other conditions. The transformation of cannabinoids from herbal preparations into highly regulated prescription drugs is therefore progressing rapidly. The development of such drugs requires well-controlled clinical trials to be carried out in order to objectively establish therapeutic efficacy, dose ranges and safety. The low oral bioavailability of cannabinoids has led to feasible methods of administration, such as the transdermal route, intranasal administration and transmucosal adsorption, being proposed. The highly lipophilic nature of cannabinoids means that they are seen as suitable candidates for advanced nanosized drug delivery systems, which can be applied via a range of routes. Nanotechnology-based drug delivery strategies have flourished in several therapeutic fields in recent years and numerous drugs have reached the market. This review explores the most recent developments, from preclinical to advanced clinical trials, in the cannabinoid delivery field, and focuses particularly on pain and inflammation treatment. Likely future directions are also considered and reported.

Interesterification is an industrial transformation process aiming to change the physico-chemical properties of vegetable oils by redistributing fatty acid position within the original constituent of the triglycerides. In the confectionery industry, controlling formation degree of positional isomers is important in order to obtain fats with the desired properties. Silver ion HPLC (High Performance Liquid Chromatography) is the analytical technique usually adopted to separate triglycerides (TAGs) having different unsaturation degrees. However, separation of TAG positional isomers is a challenge when the number of double bonds is the same and the only difference is in their position within the triglyceride molecule. The TAG positional isomers involved in the present work have a structural specificity that require a separation method tailored to the needs of confectionery industry. The aim of this work was to obtain a chromatographic resolution that might allow reliable qualitative and quantitative evaluation of TAG positional isomers within reasonably rapid retention times and robust in respect of repeatability and reproducibility. The resulting analytical procedure was applied both to confectionery raw materials and final products.

Determination of caseins in food matrices is usually performed by using the competitiveenzyme- linked immunosorbent assay (ELISA) technique. However such a technique suffers from a number of limitations. Among these, the applicability to a narrow concentration range, a non linear (logarithmic) response, a non-negligible cross-reactivity and a high cost per kit. At the time of the completion of this study, in case of ELISA positive feedback, there was poor availability in the literature of finding reliable instrumental methods able to determine both qualitatively and quantitatively this class of substances. In the present study, a liquid chromatography-high resolution tandem mass spectrometry (HPLC-HRMS/MS) instrumental method was developed with a high resolution mass spectrometer (Orbitrap). Real samples of sausages in which caseins were detected by ELISA technique were analysed. A casein-free sample of ham was used as a blank. The analytical characteristics of the instrumental method were compared with those of a commercial ELISA test, declared specific for α- and β-casein.

Triacylglycerols (TAG) from milk‐fat from different sources (cow, goat and human milks) were characterised using non‐aqueous reversed phase high‐performance liquid chromatography–atmospheric‐pressure chemical ionisation, coupled to MS/MS (RP HPLC‐APCI MS/MS). The fatty‐acid content of all samples was also established by methyl transesterification and GC‐MS analysis. Optimisation of the HPLC gradient, combined with APCI tandem MS, enables TAGs complex mixtures to be analysed without prior separation. More than 160 different glycerides were identified; between 50 and 70 compounds were identified in the chromatograms of each sample. This method also enabled the principal TAG regioisomers to be recognized. The study focused on the investigation of the structure of TAGs containing very‐long‐chain PUFA, namely all cis‐ 4,7,10,13,16,19‐ DHA (DHA, C_{22:6, n‐3}) and all cis‐5,8,11,14,17‐eicosapentaenoic acid (EPA, C_{20:5, n‐3}), both in human and in n‐3‐enriched cow's milks. Ten TAGs containing DHA were identified in human milk and only one in milk from cows fed an n‐3 enriched diet.