Development of methods for the determination of veterinary drugs in food matrices by liquid chromatography – mass spectrometry

The possible presence of veterinary drug residues and other contaminants in edible tissues and even food products is one of the key issues for food safety which arouses great public concern. The main objective of this thesis was the development of sensitive, selective, robust and effective analytical methods for the determination of veterinary drugs in food of animal origin using liquid chromatography hyphenated with mass spectrometric techniques. Η πιθανή παρουσία υπολειμμάτων κτηνιατρικών φαρμάκων και άλλων ρυπαντών σε βρώσιμους ιστούς και στα προϊόντα διατροφής είναι ένα από τα βασικά θέματα για την ασφάλεια των τροφίμων που προκαλεί μεγάλη ανησυχία στην κοινή γνώμη. Ο κύριος στόχος της παρούσας διπλωματικής εργασίας ήταν η ανάπτυξη ευαίσθητων, εκλεκτικών, ανθεκτικών και αποτελεσματικών αναλυτικών μεθόδων για τον προσδιορισμό κτηνιατρικών φαρμάκων σε τρόφιμα ζωικής προέλευσης με τη χρήση υγροχρωματογραφίας συζευγμένης με τεχνικές φασματομετρίας μάζας

Simultaneous determination of 148 pharmaceuticals and illicit drugs in sewage sludge based on ultrasound-assisted extraction and liquid chromatography–tandem mass spectrometry

This paper describes the development and validation of a new method for the simultaneous determination of 148 substances in sewage sludge. The selected compounds belong to different classes of pharmaceuticals, including antibiotics, analgesic and/or anti-inflammatory drugs, antiepileptics, benzodiazepines, antipsychotics, and antidepressants, among others, and illicit drugs, including opiates, opioids, cocaine, amphetamines, cannabinoids, and their metabolites. As far as the authors are aware, this is the first method in the peer-reviewed literature covering such a large number of target drugs for determination in a complex matrix like sewage sludge. The method presented herein combines ultrasoundassisted extraction (USE) and liquid chromatography coupled to tandem mass spectrometry. Good analytical performance was achieved, with limit-of-detection values below 10 ng g−1 d.w. for 91 % of the analytes and absolute recovery in the range 50–110 % for more than 77 % of the studied compounds. A combination of methanol and acidified water, also containing EDTA, proved to be the optimum solvent mixture to perform the extractions. An extra solid-phaseextraction clean-up step was not required, substantially reducing sample-preparation time and solvent consumption. Finally, the developed method was applied to the analysis of different sewage-sludge samples from five wastewater treatment plants of Santorini Island (Greece). Out of the 148 target compounds, 36 were detected. Several compounds, including acetylsalicylic acid, citalopram, and ciprofloxacin among others, had maximum concentrations above 100 ng g−1 d.w

Thorough investigation of the phenolic profile of reputable Greek honey varieties:varietal discrimination and floral markers identification using liquid chromatography–high-resolution mass spectrometry

Honey is a highly consumed commodity due to its potential health benefits upon certain consumption, resulting in a high market price. This fact indicates the need to protect honey from fraudulent acts by delivering comprehensive analytical methodologies. In this study, targeted, suspect and non-targeted metabolomic workflows were applied to identify botanical origin markers of Greek honey. Blossom honey samples (n = 62) and the unifloral fir (n = 10), oak (n = 24), pine (n = 39) and thyme (n = 34) honeys were analyzed using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) system. Several potential authenticity markers were revealed from the application of different metabolomic workflows. In detail, based on quantitative targeted analysis, three blossom honey markers were found, namely, galangin, pinocembrin and chrysin, while gallic acid concentration was found to be significantly higher in oak honey. Using suspect screening workflow, 12 additional bioactive compounds were identified and semi-quantified, achieving comprehensive metabolomic honey characterization. Lastly, by combining non-targeted screening with advanced chemometrics, it was possible to discriminate thyme from blossom honey and develop binary discriminatory models with high predictive power. In conclusion, a holistic approach to assessing the botanical origin of Greek honey is presented, highlighting the complementarity of the three applied metabolomic approaches

Multi-residue determination of 115 veterinary drugs and pharmaceutical residues in milk powder, butter, fish tissue and eggs using liquid chromatography-tandem mass spectrometry

A simple and sensitive multi-residue method for the determination of 115 veterinary drugs and pharmaceuticals, belonging in more than 20 different classes, in butter, milk powder, egg and fish tissue has been developed. The method involves a simple generic solid-liquid extraction step (solvent extraction, SE) with 0.1% formic acid in aqueous solution of EDTA 0.1% (w/v)-acetonitrile (ACN)-methanol (MeOH) (1:1:1, v/v) with additional ultrasonic-assisted extraction. Precipitation of lipids and proteins was promoted by subjecting the extracts at very low temperature (-23°C) for 12h. Further cleanup with hexane ensures fat removal from the matrix. Analysis was performed by liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Two separate runs were performed for positive and negative ionization in multiple reaction monitoring mode (MRM). Particular attention was devoted to extraction optimization: different sample-to-extracting volume ratios, different concentrations of formic acid in the extraction solvent and different ultrasonic extraction temperatures were tested in butter, egg and milk powder samples. The method was also applied in fish tissue samples. It was validated, on the basis of international guidelines, for all four matrices. Quantitative analysis was performed by means of standard addition calibration. For over 80% of the analytes, the recoveries were between 50% and 120% in all matrices studied, with RSD values in the range of 1-18%. Limits of detection (LODs) and quantification (LOQs) ranged from 0.008μgkg-1 (oxfendazole in butter) to 3.15μgkg-1 (hydrochlorthiazide in egg). The evaluated method provides reliable screening, quantification, and identification of 115 veterinary drug and pharmaceutical residues in foods of animal origin and has been successfully applied in real samples. © 2015 Elsevier B.V.

Απομόνωση και ταυτοποίηση δευτερογενών μεταβολιτών από τη Salvia fruticosa Mill.

Στα πλαίσια της παρούσας εργασίας μελετήθηκε η χημική σύσταση του οργανικού εκχυλίσματος του είδους Salvia fruticosa Mill. Τo φυτικό δείγμα συλλέχθηκε στη περιοχή Ριζόσκαρο Χανίων στην Κρήτη τον Μάρτιο του 2015. Έπειτα από εκχύλιση του αποξηραμένου φυτικού υλικού με μείγμα CH2Cl2 και MeOH, το οργανικό υπόλειμμα που παραλήφθηκε υποβλήθηκε σε σειρά χρωματογραφικών διαχωρισμών που οδήγησαν στην απομόνωση δέκα μεταβολιτών, των οποίων οι χημικές δομές ταυτοποιήθηκαν μέσω φασματοσκοπικών μεθόδων NMR και MS. Συγκεκριμένα, απομονώθηκαν και ταυτοποιήθηκαν ένα μονοτερπένιο (α-τερπινεόλη), δύο σεσκιτερπένια (σπαθουλενόλη, βιριντιφλορόλη), πέντε διτερπένια (μανοόλη, καρνοσόλη, ροσμανόλη, 7α-Ο-μεθυλοροσμανόλη, μεθίδιο Α της σατζεκινόνης), ένα nor-διτερπένιο (σατζεόνη) και ένα τριτερπένιο (ολεανολικό οξύ).In the framework of the present thesis the chemical composition of the organic extract obtained from the plant Salvia fruticosa Mill was investigated. The plant material was collected in the area of Risoscaro, in Chania, Crete in March of 2015. Specimens of the dried aerial parts of the plant were exhaustively extracted with mixture of CH2Cl2 and MeOH and the crude organic extract was submitted to a series of chromatographic separations that led to the isolation and structure elucidation of ten metabolites. The structures and relative configurations of the isolated natural products were established by means of spectroscopic analyses, including 1D and 2D NMR experiments, as well as MS data. The isolated secondary metabolites include one monoterpene (α-terpineol), two sesquiterpenes (spathulenol, viridiflorol), five diterpenes (manool, carnosol, rosmanol, 7α-O-methylrosmanol, sagequinone methide A), one nor-diterpene (sageone) and one triterpene (oleanolic acid)

Multi-residue determination of seventeen sulfonamides and five tetracyclines in fish tissue using a multi-stage LC-ESI-MS/MS approach based on advanced mass spectrometric techniques

A strategy was newly developed to rapidly screen seventeen sulfonamides and five tetracyclines in a single run from fish tissues using ultra-high performance liquid chromatography (UHPLC) coupled with comprehensive mass spectrometric approaches, including precursor-ion scan and data dependent scan. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of the analytes. All sulfonamides share the same diagnostic product ion at m/z 156 in positive MS/MS scan, while for tetracycline antibiotics the diagnostic product ion was proved to be at m/z 153.8. Further characterization of each compound was performed using a data dependent scan. Separation was performed on a Zorbax Eclipse Plus C18 column with a gradient elution using acetonitrile - 0.1% formic acid mobile phase at a flow rate of 0.1mLmin-1. This approach has proven to be a powerful, highly selective, and sensitive tool for rapid screening and detection of non-targeted components in fish tissue and requires a minimum sample preparation such as one generic extraction step with MeOH:ACN 50:50 (v/v) acidified with 0.05% formic acid. The method has also been applied successfully to porcine and poultry meat. The validation of such a screening method was performed for the first time according to Commission Decision 2002/657/EC and satisfactory method performance characteristics were achieved. © 2010 Elsevier B.V

Αξιολόγηση της μικροκυκλοφορίας με εγγύς του υπερύθρου φασματοσκοπία σε συνδυασμό με την τεχνική αγγειακού αποκλεισμού

Εισαγωγή: Η μέθοδος εγγύς του υπερύθρου φασματοσκοπία σε συνδυασμό με την τεχνική του αγγειακού αποκλεισμού αποτελεί μια σχετικά νέα πρακτική στον κλινικό τομέα για την αξιολόγηση της αγγειακής ενδοθηλιακής δυσλειτουργίας . Σκοπός: Σκοπός της διπλωματικής εργασίας ήταν η αξιολόγηση της μικροκυκλοφορίας με την μέθοδο εγγύς του υπερύθρου φασματοσκοπία (Near Infrared Spectroscopy-NIRS) σε συνδυασμό με την τεχνική του αγγειακού αποκλεισμού (Vascular Occlusion Technique-VOT) . Υλικό και μέθοδος: Πραγματοποιήθηκε συστηματική ανασκόπηση των μελετών που δημοσιεύτηκαν από τον Ιανουάριο του 2000 μέχρι και τον Δεκέμβριο του 2018, στην αγγλική γλώσσα, στις βάσεις δεδομένων PubMed, Scopus, Google Scholar με τις λέξεις-κλειδιά: «vascular occlusion», «Near Infrared Spectroscopy», «vascular occlusion» και «NIRS», «Microcirculation» . Τα κριτήρια ένταξης των δημοσιευμένων μελετών στη συστηματική ανασκόπηση ήταν: μελέτες που αφορούσαν την αξιολόγηση μικροκυκλοφορίας με την μέθοδο NIRS-VOΤ , μελέτες που αφορούσαν την αξιολόγηση της μικροκυκλοφορίας ασθενών/ατόμων ανεξαρτήτου ηλικίας και φύλου με την μέθοδο NIRS –VOT και η αγγλική αρθρογραφία. Αποτελέσματα: Από τις δημοσιευμένες μελέτες, 7 πληρούσαν τα κριτήρια ένταξης. Από τις 7 μελέτες, οι 5 αποτελούσαν αξιολόγηση της μικροκυκλοφορίας σε ενήλικα άτομα και οι 2 σε παιδιά. Τα αποτελέσματα των ερευνών έδειξαν ότι η αξιολόγηση της μικροκυκλοφορίας με την μέθοδο εγγύς του υπερύθρου φασματοσκοπία σε συνδυασμό με την τεχνική του αγγειακού αποκλεισμού είναι κατάλληλη ως κλινική πρακτική παρακολούθησης και μπορεί να εφαρμοστεί σε ένα μεγάλο φάσμα ασθενών και ηλικιών. Συμπεράσματα: Η μέθοδος εγγύς του υπερύθρου φασματοσκοπία (NIRS) συνδυαζόμενη με την τεχνική του αγγειακού αποκλεισμού (VOT) ενδείκνυται ως εργαλείο παρακολούθησης της μικροκυκλοφορίας και ενδεχομένως ως δείκτης προγνωστικότητας, καθώς αποτελεί μία μη επεμβατική μέθοδο η οποία είναι εύκολη στη χρήση και μπορεί να εφαρμοστεί σε ένα ευρύ φάσμα ασθενών και υγιών ατόμων. Επιπλέον, οι τιμές του περιφερειακού κορεσμού οξυγόνου ποικίλουν και εξαρτώνται κυρίως από τη θέση που πραγματοποιείται η μέτρηση, αλλά και από μία σειρά άλλων παραμέτρων.Introduction: The method of near-infrared spectroscopy combined with the technique of vascular exclusion is a relatively new practice in the clinical field for the evaluation of the vascular endothelial dysfunction. Aim: The purpose of the thesis was to evaluate microcirculation using Near Infrared Spectroscopy (NIRS) combined with Vascular Occlusion Technique (VOT) . Material and Methods: Studies published from January 2000 to December 2018 were systematically reviewed in English, PubMed, Scopus, Google Scholar databases with the keywords: "vascular occlusion", "Near Infrared Spectroscopy", " vascular occlusion "and" NIRS "," Microcirculation ". The criteria for inclusion of the published studies in the systematic review were: studies on microcirculation evaluation using the NIRS-VOT method, studies on the evaluation of microcirculation of patients / subjects of independent age and sex using the NIRS-VOT method and English articles. Results: Of the published studies, 7 met the inclusion criteria. Of the 7 studies, 5 consisted of microcirculation evaluation in adults and 2 in children. The results of the studies have shown that microcirculation evaluation by near-infrared spectroscopy combined with vascular exclusion technique is suitable as clinical monitoring practice and can be applied to a wide range of patients and ages. Conclusions: Τhe near-infrared spectroscopy (NIRS) method combined with the vascular occlusion technique (VOT) is indicated as a microcirculation monitoring tool and possibly as an indicator of predictability as it is a non-invasive and easy-to-use method can be applied to a wide range of patients and healthy people. In addition, the values of peripheral oxygen saturation vary and depend mainly on the location of the measurement, but also on a number of other parameters