Application of chromatographic and spectroscopic techniques in the evaluation of the lipid fraction of animal products

Lipolysis and oxidation of lipids in foods are the major biochemical and chemical processes that cause food quality deterioration, leading to the characteristic, unpalatable odour and flavour called rancidity. In addition to unpalatability, rancidity may give rise to toxic levels of certain compounds like aldehydes, hydroperoxides, epoxides and cholesterol oxidation products. In this PhD study chromatographic and spectroscopic techniques were employed to determine the degree of rancidity in different animal products and its relationship with technological parameters like feeding fat sources, packaging, processing and storage conditions. To achieve this goal capillary gas chromatography (CGC) was employed not only to determine the fatty acids profile but also, after solid phase extraction, the amount of free fatty acids (FFA), diglycerides (DG), sterols (cholesterol and phytosterols) and cholesterol oxidation products (COPs). To determine hydroperoxides, primary products of oxidation and quantify secondary products UV/VIS absorbance spectroscopy was applied. Most of the foods analysed in this study were meat products. In actual fact, lipid oxidation is a major deterioration reaction in meat and meat products and results in adverse changes in the colour, flavour and texture of meat. The development of rancidity has long recognized as a serious problem during meat handling, storage and processing. On a dairy product, a vegetal cream, a study of lipid fraction and development of rancidity during storage was carried out to evaluate its shelf-life and some nutritional features life saturated/unsaturated fatty acids ratio and phytosterols content. Then, according to the interest that has been growing around functional food in the last years, a new electrophoretic method was optimized and compared with HPLC to check the quality of a beehive product like royal jelly. This manuscript reports the main results obtained in the five activities briefly summarized as follows: 1) comparison between HPLC and a new electrophoretic method in the evaluation of authenticity of royal jelly; 2) study of the lipid fraction of a vegetal cream under different storage conditions; 3) study of lipid oxidation in minced beef during storage under a modified atmosphere packaging, before and after cooking; 4) evaluation of the influence of dietary fat and processing on the lipid fraction of chicken patties; 5) study of the lipid fraction of typical Italian and Spanish pork dry sausages and cured hams.

Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

Sviluppo di tecniche di analisi ifenate per la quantificazione di biomarcatori di esposizione a tossici ambientali: gli acidi mercapturici

Human biomonitoring (HBM) is an ideal tool for evaluating toxicant exposure in health risk assessment. Chemical substances or their metabolites related to environmental pollutants can be detected as biomarkers of exposure using a wide variety of biological fluids. Individual exposure to aromatic hydrocarbon compounds (benzene, toluene, and o-xylene –“BTX”) were analysed with a liquid chromatography coupled to electrospray ionisation-mass spectrometry (μHPLC-ESI-MS/MS) method for the simultaneous quantitative detection of the BTX exposure biomarker SPMA, SBMA and o-MBMA in human urine. Urinary S-phenylmercapturic acid (SPMA) is a biomarker proposed by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene (Biological Exposure Index of 25 microg/g creatinine). Urinary S-benzylmercapturic (SBMA) and o-methyl S-benzyl mercapturic acid (o-MBMA) are specific toluene and o-xylene metabolites of glutathione detoxicant pathways, proposed as reliable biomarkers of exposure. To this aim a pre-treatment of the urine with solid phase extraction (SPE) and an evaporation step were necessary to concentrate the mercapturic acids before instrumental analysis. A liquid chromatography separation was carried out with a reversed phase capillary column (Synergi 4u Max-RP) using a binary gradient composed of an acquous solution of formic acid 0.07% v/v and methanol. The mercapturic acids were determinated by negative-ion-mass spectrometry and the data were corrected using isotope-labelled analogs as internal standards. The analytical method follows U.S. Food and Drug Administration guidance and was applied to assess exposure to BTX in a group of 396 traffic wardens. The association between biomarker results and individual factors, such as age, sex and tobacco smoke were also investigated. The present work also included improvements in the methods used by modifying various chromatographic parameters and experimental procedures. A partial validation was conducted to evaluate LOD, precision, accuracy, recovery as well as matrix effects. Higher sensitivity will be possible in future biological monitoring programmes, allowing evaluation of very low level of BTX human exposure. Keywords: Human biomonitoring, aromatic hydrocarbons, biomarker of exposure, HPLC-MS/MS.

Food lipids: their effects on quality and oxidative stability of animal tissues and emulsions

Lipolysis and oxidation of lipids in foods are the major biochemical and chemical processes that cause food quality deterioration, leading to the characteristic, unpalatable odour and flavour called rancidity. In addition to unpalatability, rancidity may give rise to toxic levels of certain compounds like aldehydes, hydroperoxides, epoxides and cholesterol oxidation products. In this PhD study chromatographic and spectroscopic techniques were employed to determine the degree of lipid oxidation in different animal products and its relationship with technological parameters like feeding fat sources, packaging, processing and storage conditions. To achieve this goal capillary gas chromatography (CGC) was employed not only to determine the fatty acids profile but also, after solid phase extraction, the amount of sterols (cholesterol and phytosterols) and cholesterol oxidation products (COPs). To determine hydroperoxides, primary products of oxidation and quantify secondary products UV/VIS absorbance spectroscopy was applied. Beef and pork meat in this study were analysed. In actual fact, lipid oxidation is a major deterioration reaction in meat, meat products and results in adverse changes in the colour, flavour, texture of meat and develops different compounds which should be a risk to human health as oxysterols. On beef and pork meat, a study of lipid fraction during storage was carried out to evaluate its shelf-life and some nutritional features life saturated/unsaturated fatty acids ratio and sterols content, in according to the interest that has been growing around functional food in the last years. The last part of this research was focused on the study of lipid oxidation in emulsions. In oil-in-water emulsions antioxidant activity of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was evaluated. The rates of lipid oxidation of 1.0% stripped soybean oil-in-water emulsions with DOPC were followed by monitoring lipid hydroperoxide and hexanal as indicators of primary and secondary oxidation products and the droplet surface charge or zeta potential (ζ) of the emulsions with varying concentrations of DOPC were tested. This manuscript reports the main results obtained in the three activities briefly summarized as follows: 1. study on effects of feeding composition on the photoxidative stability of lipids from beef meat, evaluated during storage under commercial retail conditions; 2. evaluation of effects of diets and storage conditions on the oxidative stability of pork meat lipids; 3. study on oxidative behavior of DOPC in stripped soybean oil-in-water emulsions stabilized by nonionic surfactant.

Development of Original Analytical Methods for the Determination of Drugs of Abuse in Biological Samples

Drug abuse is a major global problem which has a strong impact not only on the single individual but also on the entire society. Among the different strategies that can be used to address this issue an important role is played by identification of abusers and proper medical treatment. This kind of therapy should be carefully monitored in order to discourage improper use of the medication and to tailor the dose according to the specific needs of the patient. Hence, reliable analytical methods are needed to reveal drug intake and to support physicians in the pharmacological management of drug dependence. In the present Ph.D. thesis original analytical methods for the determination of drugs with a potential for abuse and of substances used in the pharmacological treatment of drug addiction are presented. In particular, the work has been focused on the analysis of ketamine, naloxone and long-acting opioids (buprenorphine and methadone), oxycodone, disulfiram and bupropion in human plasma and in dried blood spots. The developed methods are based on the use of high performance liquid chromatography (HPLC) coupled to various kinds of detectors (mass spectrometer, coulometric detector, diode array detector). For biological sample pre-treatment different techniques have been exploited, namely solid phase extraction and microextraction by packed sorbent. All the presented methods have been validated according to official guidelines with good results and some of these have been successfully applied to the therapeutic drug monitoring of patients under treatment for drug abuse.

Original analytical methods for the determination of psychoactive compounds in complex matrices

This thesis work aims to develop original analytical methods for the determination of drugs with a potential for abuse, for the analysis of substances used in the pharmacological treatment of drug addiction in biological samples and for the monitoring of potentially toxic compounds added to street drugs. In fact reliable analytical techniques can play an important role in this setting. They can be employed to reveal drug intake, allowing the identification of drug users and to assess drug blood levels, assisting physicians in the management of the treatment. Pharmacological therapy needs to be carefully monitored indeed in order to optimize the dose scheduling according to the specific needs of the patient and to discourage improper use of the medication. In particular, different methods have been developed for the detection of gamma-hydroxybutiric acid (GHB), prescribed for the treatment of alcohol addiction, of glucocorticoids, one of the most abused pharmaceutical class to enhance sport performance and of adulterants, pharmacologically active compounds added to illicit drugs for recreational purposes. All the presented methods are based on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) coupled to various detectors (diode array detector, mass spectrometer). Biological samples pre-treatment was carried out using different extraction techniques, liquid-liquid extraction (LLE) and solid phase extraction (SPE). Different matrices have been considered: human plasma, dried blood spots, human urine, simulated street drugs. These developed analytical methods are individually described and discussed in this thesis work.

Valutazione della presenza di contaminanti perfluoroalchilici in alimenti destinati al consumo umano

Le sostanze perfluoralchiliche (PFAS), composti fluorurati ampiamente utilizzati negli ultimi anni in diverse applicazioni industriali e commerciali, sono ritrovati diffusamente nell’ambiente e in diverse specie animali. Recentemente i PFAS hanno destato preoccupazione anche per la salute umana. Il rischio di esposizione è principalmente legato alla dieta (i prodotti ittici sembrano essere gli alimenti più contaminati). Lo scopo di questo lavoro è stato quello di valutare la presenza del perfluorottanosulfonato (PFOS) e dell’acido perfluorottanoico (PFOA), in diverse matrici alimentari: latte vaccino commercialmente disponibile in Italia, latte materno italiano, diverse specie di pesce commercialmente disponibili in Italia e 140 branzini di diverse aree (principalmente Mediterraneo). I campioni di latte sono stati trattati con estrazione liquido-liquido seguita da due fasi di purificazione mediante cartucce SPE prima dell’iniezione nell’UPLC-MS/MS. L’analisi del latte vaccino ha evidenziato una contaminazione diffusa di PFOS, ma a basse concentrazioni (fino a 97 ng/L), mentre il PFOA è stato ritrovato raramente. In questo studio, in grado di individuare anche i livelli delle ultra-tracce, sono state osservate nel latte materno concentrazioni di 15-288 ng/L per il PFOS e di 24-241 ng/LPFOA. Le concentrazioni e le frequenze più alte, per entrambi i PFAS, sono stati ritrovate in campioni di latte forniti da donne primipare, suggerendo un rischio di esposizione per i primogeniti. Il metodo utilizzato per i campioni di pesce era basato su un’estrazione con solvente organico seguita da due fasi di purificazione: una con i sali e una con fase solida dispersiva. L’estratto, analizzato in UPLC-MS/MS, ha confermato la contaminazione di questa matrice a livelli significativi, ma anche l’alta variabilità delle concentrazioni misurate. Il monitoraggio monospecie ha mostrato una contaminazione rilevante (PFOS 11,1- > 10000 ng/L; PFOA < 9-487 ng/L), soprattutto nei branzini pescati, rispetto a quelli allevati.