Development of High Resolution Mass Spectrometric Methods for the investigation of food authenticity

Η αυθεντικότητα των τροφίμων αποτελεί ένα ιδαίτερα σημαντικό γεγονός τα τελευταία χρόνια, λόγω των πολλών περιστατικών νοθείας τροφίμων και διακίνησης προϊόντων κατώτερης ποιότητας με παραπλανητικές ετικέτες. Η εξασφάλιση της αυθεντικότητας του ελαιολάδου δημιουργεί μεγάλη ανησυχία λόγω της οργανοληπτικής, θρεπτικής και οικονομικής του σημασίας. Ο κύριος στόχος αυτής της διατριβής είναι η ανάπτυξη αναλυτικών μεθόδων υγροχρωματογραφίας φασματομετρίας μάζας υψηλής διακριτικής ικανότητας (LC-HRMS) που θα συμπεριλαμβάνουν στρατηγικές στοχευμένης, «ύποπτης» και «μη στοχευμένης σάρωσης», σε συνδυασμό με προηγμένα χημειομετρικά εργαλεία, για την εύρεση του αποτυπώματος του ελαιολάδου. Aρχικά, αναφέρονται οι βιολογικές δράσεις των ελάσσονων συστατικών του ελαιολάδου, των φαινολικών ενώσεων, και οι επιδράσεις διαφόρων παραγόντων στο φαινολικό προφίλ. Παρουσιάζονται όλες οι σύγχρονες και αναλυτικές μέθοδοι που συνδυάζουν την επιστήμη των τροφίμων με τεχνικές μεταβολομικής, με έμφαση στις μεθόδους HR-MS. Το πειραματικό μέρος της διατριβής αποτελείται από τρία τμήματα: (1) Μελέτες αυθεντικότητας ελαιολάδου με LC-QTOF-MS σε συνδυασμό με στοχευμένη και μη στοχευμένη σάρωση και χημειομετρία για την αναγνώριση δεικτών που κατατάσσουν το ελαιόλαδο ως ελαττωματικό ή έξτρα παρθένο. (Κεφάλαιο 3), (2) Διερεύνηση του βιολογικού και συμβατικού τύπου παραγωγής των έξτρα παρθένων ελαιολάδων με LC-QTOF-MS σε συνδυασμό με στοχευμένη και «ύποπτη» σάρωση, προτείνοντας μια νέα μέθοδο ημι-ποσοτικοποίησης (Κεφάλαιο 4), και (3) Ταξινόμηση έξι ελληνικών ποικιλιών ελαιoλάδου με LC-QTOF-MS μη στοχευμένη στοχευμένη σάρωση και χημειομετρία, για την αναγνώριση δεικτών με προκαθορισμένα όρια συγκέντρωσης, που εγγυώνται την ποικιλιακή και γεωγραφική προέλευση (Κεφάλαιο 5). Πιστεύουμε ότι οι παραπάνω μελέτες έχουν σημειώσει μεγάλη πρόοδο στον τομέα της γνησιότητας των τροφίμων με την ανάπτυξη αναλυτικών μεθόδων HR-MS και μέσω της εισαγωγής μιας νέας ολοκληρωμένης ροής εργασιών που περιλαμβάνει στρατηγικές στοχευμένης, «ύποπτης» και μη στοχευμένης σάρωσης HRMS, σε συνδυασμό με, ολοκληρωμένα εργαλεία εξόρυξης, επεξεργασίας δεδομένων και προγνωστικά μοντέλα.Food authenticity has become increasingly important in recent years due to food fraud incidents and the handling of low quality products with misleading labels. The guarantee of the authenticity of olive oil arises great public concern because of its sensory, nutritional and economic importance. The main objective of this thesis is to develop integrated LC-HRMS workflows, including target, suspect and non-target screening strategies, coupled with advanced chemometric tools, for olive oil fingerprinting. First, the biological activities of some minor constituents in olive oil, phenolic compounds, are reported and their occurrence and wide-scope properties in olive matrices, as well as effects of various factors on olive oil phenolic profile, are discussed. The present state of the art for their determination in foodomics science is presented, focusing on target and non-target screening HR-MS workflows coupled to chemometrics. The experimental section of the thesis consists of three sections: (1) Olive oil authenticity studies by target and non-target LC-QTOF-MS combined with advanced chemometrics, for identifying markers that classify olive oil to defective and EVOOs (Chapter 3), (2) Investigating the organic and conventional production type of EVOOs with target and suspect screening by LC-QTOF-MS, a novel semi-quantification method using chemical similarity and advanced chemometris; in order to identify a marker with a concentration threshold, by ACO/RF, that can be used to discriminate organic and conventional EVOOs (Chapter 4), and (3) Classification of Greek olive oil varieties with non-target UHPLC-QTOF-MS and advanced chemometrics; for the investigation of the fingerprints of six greek olive oil varieties and the identification of markers, with post-defined concentration thresholds, that guarantee the varietal and geographical origin (Chapter 5). We believe that these studies have made great progress in the food authenticity field via the introduction of novel integrated HRMS screening workflows which are followed by advanced data processing, comprehensive data mining and predictive modelling tools

Ανάπτυξη μεθόδων φασματομετρίας μάζας υψηλής διακριτικής ικανότητας για τη μελέτη της αυθεντικότητας τροφίμων

Food authenticity has become increasingly important in recent years due to food fraud incidents and the handling of low quality products with misleading labels. The guarantee of the authenticity of olive oil arises great public concern because of its sensory, nutritional and economic importance. The main objective of this thesis is to develop integrated LC-HRMS workflows, including target, suspect and non-target screening strategies, coupled with advanced chemometric tools, for olive oil fingerprinting.First, the biological activities of some minor constituents in olive oil, phenolic compounds, are reported and their occurrence and wide-scope properties in olive matrices, as well as effects of various factors on olive oil phenolic profile, are discussed. The present state of the art for their determination in foodomics science is presented, focusing on target and non-target screening HR-MS workflows coupled to chemometrics. The experimental section of the thesis consists of three sections: (1) Olive oil authenticity studies by target and non-target LC-QTOF-MS combined with advanced chemometrics, for identifying markers that classify olive oil to defective and EVOOs (Chapter 3), (2) Investigating the organic and conventional production type of EVOOs with target and suspect screening by LC-QTOF-MS, a novel semi-quantification method using chemical similarity and advanced chemometris; in order to identify a marker with a concentration threshold, by ACO/RF, that can be used to discriminate organic and conventional EVOOs (Chapter 4), and (3) Classification of Greek olive oil varieties with non-target UHPLC-QTOF-MS and advanced chemometrics; for the investigation of the fingerprints of six greek olive oil varieties and the identification of markers, with post-defined concentration thresholds, that guarantee the varietal and geographical origin (Chapter 5).We believe that these studies have made great progress in the food authenticity field via the introduction of novel integrated HRMS screening workflows which are followed by advanced data processing, comprehensive data mining and predictive modelling tools.Η αυθεντικότητα των τροφίμων αποτελεί ένα ιδαίτερα σημαντικό γεγονός τα τελευταία χρόνια, λόγω των πολλών περιστατικών νοθείας τροφίμων και διακίνησης προϊόντων κατώτερης ποιότητας με παραπλανητικές ετικέτες. Η εξασφάλιση της αυθεντικότητας του ελαιολάδου δημιουργεί μεγάλη ανησυχία λόγω της οργανοληπτικής, θρεπτικής και οικονομικής του σημασίας. Ο κύριος στόχος αυτής της διατριβής είναι η ανάπτυξη αναλυτικών μεθόδων υγροχρωματογραφίας φασματομετρίας μάζας υψηλής διακριτικής ικανότητας (LC-HRMS) που θα συμπεριλαμβάνουν στρατηγικές στοχευμένης, «ύποπτης» και «μη στοχευμένης σάρωσης», σε συνδυασμό με προηγμένα χημειομετρικά εργαλεία, για την εύρεση του αποτυπώματος του ελαιολάδου.Aρχικά, αναφέρονται οι βιολογικές δράσεις των ελάσσονων συστατικών του ελαιολάδου, των φαινολικών ενώσεων, και οι επιδράσεις διαφόρων παραγόντων στο φαινολικό προφίλ. Παρουσιάζονται όλες οι σύγχρονες και αναλυτικές μέθοδοι που συνδυάζουν την επιστήμη των τροφίμων με τεχνικές μεταβολομικής, με έμφαση στις μεθόδους HR-MS. Το πειραματικό μέρος της διατριβής αποτελείται από τρία τμήματα: (1) Μελέτες αυθεντικότητας ελαιολάδου με LC-QTOF-MS σε συνδυασμό με στοχευμένη και μη στοχευμένη σάρωση και χημειομετρία για την αναγνώριση δεικτών που κατατάσσουν το ελαιόλαδο ως ελαττωματικό ή έξτρα παρθένο. (Κεφάλαιο 3), (2) Διερεύνηση του βιολογικού και συμβατικού τύπου παραγωγής των έξτρα παρθένων ελαιολάδων με LC-QTOF-MS σε συνδυασμό με στοχευμένη και «ύποπτη» σάρωση, προτείνοντας μια νέα μέθοδο ημι-ποσοτικοποίησης (Κεφάλαιο 4), και (3) Ταξινόμηση έξι ελληνικών ποικιλιών ελαιoλάδου με LC-QTOF-MS μη στοχευμένη στοχευμένη σάρωση και χημειομετρία, για την αναγνώριση δεικτών με προκαθορισμένα όρια συγκέντρωσης, που εγγυώνται την ποικιλιακή και γεωγραφική προέλευση (Κεφάλαιο 5).Πιστεύουμε ότι οι παραπάνω μελέτες έχουν σημειώσει μεγάλη πρόοδο στον τομέα της γνησιότητας των τροφίμων με την ανάπτυξη αναλυτικών μεθόδων HR-MS και μέσω της εισαγωγής μιας νέας ολοκληρωμένης ροής εργασιών που περιλαμβάνει στρατηγικές στοχευμένης, «ύποπτης» και μη στοχευμένης σάρωσης HRMS, σε συνδυασμό με, ολοκληρωμένα εργαλεία εξόρυξης, επεξεργασίας δεδομένων και προγνωστικά μοντέλα

Application of an advanced and wide scope non-target screening workflow with LC-ESI-QTOF-MS and chemometrics for the classification of the Greek olive oil varieties

An optimized and validated LC-ESI-QTOF-MS method with an integrated non-target screening workflow was applied in the investigation of the metabolomic profile of 51 Greek monovarietal extra virgin olive oils (EVOOs) from the varieties: Manaki, Ladoelia, Koroneiki, Amfissis, Chalkidikis and Kolovi. Data processing was carried out with the R language and XCMS package. A local database consisting of 1608 compounds naturally occurring in different organs of Olea Europa L. was compiled in order to accelerate the identification workflow. The preliminary examination of the distribution of EVOOs toward their cultivars was achieved by Principal Component Analysis (PCA). Ant Colony Optimization-Random Forest (ACO-RF) was developed to prioritize over 250 features and to establish a classification tree. Apigenin, vanillic acid, luteolin 7-methyl ether and oleocanthal were suggested as the markers responsible for the classification of Greek EVOOs’ cultivars. © 2018 Elsevier Lt

Investigating the organic and conventional production type of olive oil with target and suspect screening by LC-QTOF-MS, a novel semi-quantification method using chemical similarity and advanced chemometrics

The discrimination of organic and conventional production has been a critical topic of public discussion and constitutes a scientific issue. It remains a challenge to establish a correlation between the agronomical practices and their effects on the composition of olive oils, especially the phenolic composition, since it defines their organoleptic and nutritional value. Thus, a liquid chromatography-electrospray ionization-quadrupole time of flight tandem mass spectrometric method was developed, using target and suspect screening workflows, coupled with advanced chemometrics for the identification of phenolic compounds and the discrimination between organic and conventional extra virgin olive oils. The method was optimized by one-factor design and response surface methodology to derive the optimal conditions of extraction (methanol/water (80:20, v/v), pure methanol, or acetonitrile) and to select the most appropriate internal standard (caffeic acid or syringaldehyde). The results revealed that extraction with methanol/water (80:20, v/v) was the optimum solvent system and syringaldehyde 1.30 mg L−1 was the appropriate internal standard. The proposed method demonstrated low limits of detection in the range of 0.002 (luteolin) to 0.028 (tyrosol) mg kg−1. Then, it was successfully applied in 52 olive oils of Kolovi variety. In total, 13 target and 24 suspect phenolic compounds were identified. Target compounds were quantified with commercially available standards. A novel semi-quantitation strategy, based on chemical similarity, was introduced for the semi-quantification of the identified suspects. Finally, ant colony optimization-random forest model selected luteolin as the only marker responsible for the discrimination, during a 2-year study. [Figure not available: see fulltext.]. © 2017, Springer-Verlag Berlin Heidelberg

A Validated Ultrasound-Assisted Extraction Coupled with SPE-HPLC-DAD for the Determination of Flavonoids in By-Products of Plant Origin: An Application Study for the Valorization of the Walnut Septum Membrane

Walnut byproducts have been shown to exert functional properties, but the literature on their bioactive content is still scarce. Among walnut byproducts, walnut septum is a dry ligneous diaphragm tissue that divides the two halves of the kernel, exhibiting nutritional and medicinal properties. These functional properties are owing to its flavonoid content, and in order to explore the flavonoid fraction, an ultrasound-assisted (UAE) protocol was combined with solid phase extraction (SPE) and coupled to high-performance liquid chromatography with diode array detection (HPLC-DAD) for the determination of flavonoids in Greek walnut septa membranes belonging to Chandler, Vina, and Franquette varieties. The proposed UAE-SPE-HPLC-DAD method was validated and the relative standard deviations (RSD%) of the within-day and between-day assays were lower than 6.2 and 8.5, respectively, showing good precision, and high accuracy ranging from 90.8 (apigenin) to 97.5% (catechin) for within-day assay, and from 88.5 (myricetin) to 96.2% (catechin) for between-day assay. Overall, seven flavonoids were determined (catechin, rutin, myricetin, luteolin, quercetin, apigenin, and kaempferol) suggesting that the walnut septum is a rich source of bioactive constituents. The quantification results were further processed using ANOVA analysis to examine if there are statistically significant differences between the concentration of each flavonoid and the variety of the walnut septum

Target and Suspect HRMS Metabolomics for the Determination of Functional Ingredients in 13 Varieties of Olive Leaves and Drupes from Greece

The huge interest in the health-related properties of foods to improve health has brought about the development of sensitive analytical methods for the characterization of natural products with functional ingredients. Greek olive leaves and drupes constitute a valuable source of biophenols with functional properties. A novel ultra-high-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS) analytical method was developed to identify biophenols through target and suspect screening in Greek olive leaves and drupes of the varieties: Koroneiki, Throumbolia, Konservolia, Koutsourelia, Kalamon, Petrolia, Amigdalolia, Megaritiki, Mastoeidis, Agouromanakolia, Agrilia, Adramitiani and Kolovi. The method's performance was evaluated using the target compounds: oleuropein, tyrosol and hydroxytyrosol. The analytes demonstrated satisfactory recovery efficiency for both leaves (85.9-90.5%) and drupes (89.7-92.5%). Limits of detection (LODs) were relatively low over the range 0.038 (oleuropein)-0.046 (hydroxytyrosol) and 0.037 (oleuropein)-0.048 (hydroxytyrosol) for leaves and drupes, respectively For leaves, the precision limit ranged between 4.7 and 5.8% for intra-day and between 5.8 and 6.5% for inter-day experiments, and for drupes, it ranged between 3.8 and 5.2% for intra-day and between 5.1 and 6.2% for inter-day experiments, establishing the good precision of the method. The regression coefficient (r2) was above 0.99 in all cases. Furthermore, the preparation of herbal tea from olive leaves is suggested after investigating the optimum infusion time of dried leaves in boiling water. Overall, 10 target and 36 suspect compounds were determined in leaves, while seven targets and thirty-three suspects were identified in drupes, respectively

Application of High Resolution Mass Spectrometric methods coupled with chemometric techniques in olive oil authenticity studies - A review

Extra Virgin Olive Oil (EVOO), the emblematic food of the Mediterranean diet, is recognized for its nutritional value and beneficial health effects. The main authenticity issues associated with EVOO's quality involve the organoleptic properties (EVOO or defective), mislabeling of production type (organic or conventional), variety and geographical origin, and adulteration. Currently, there is an emerging need to characterize EVOOs and evaluate their genuineness. This can be achieved through the development of analytical methodologies applying advanced “omics” technologies and the investigation of EVOOs chemical fingerprints. The objective of this review is to demonstrate the analytical performance of High Resolution Mass Spectrometry (HRMS) in the field of food authenticity assessment, allowing the determination of a wide range of food constituents with exceptional identification capabilities. HRMS-based workflows used for the investigation of critical olive oil authenticity issues are presented and discussed, combined with advanced data processing, comprehensive data mining and chemometric tools. The use of unsupervised classification tools, such as Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA), as well as supervised classification techniques, including Linear Discriminant Analysis (LDA), Support Vector Machine (SVM), Partial Least Square Discriminant Analysis (PLS-DA), Orthogonal Projection to Latent Structure-Discriminant Analysis (OPLS-DA), Counter Propagation Artificial Neural Networks (CP-ANNs), Self-Organizing Maps (SOMs) and Random Forest (RF) is summarized. The combination of HRMS methodologies with chemometrics improves the quality and reliability of the conclusions from experimental data (profile or fingerprints), provides valuable information suggesting potential authenticity markers and is widely applied in food authenticity studies. © 2020 Elsevier B.V

Authentication of Greek PDO kalamata table olives: A novel non-target high resolution mass spectrometric approach

Food science continually requires the development of novel analytical methods to prevent fraudulent actions and guarantee food authenticity. Greek table olives, one of the most emblematic and valuable Greek national products, are often subjected to economically motivated fraud. In this work, a novel ultra-high-performance liquid chromatography–quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS) analytical method was developed to detect the mislabeling of Greek PDO Kalamata table olives, and thereby establish their authenticity. A non-targeted screening workflow was applied, coupled to advanced chemometric techniques such as Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA) in order to fingerprint and accurately discriminate PDO Greek Kalamata olives from Kalamata (or Kalamon) type olives from Egypt and Chile. The method performance was evaluated using a target set of phenolic compounds and several validation parameters were calculated. Overall, 65 table olive samples from Greece, Egypt, and Chile were analyzed and processed for the model development and its accuracy was validated. The robustness of the chemometric model was tested using 11 Greek Kalamon olive samples that were produced during the following crop year, 2018, and they were successfully classified as Greek Kalamon olives from Kalamata. Twenty-six characteristic authenticity markers were indicated to be responsible for the discrimination of Kalamon olives of different geographical origins. © 2020 by the authors

Determination of Metals in Walnut Oils by Means of an Optimized and Validated ICP-AES Method in Conventional and Organic Farming Type Samples

Agricultural products are indispensable for equilibrated diets since they discharge minerals and several bioactive constituents. Considering the increasing demand for organic products, research has been conducted over recent years to investigate whether organically grown food products are chemically different compared to those produced with conventional farming. In this work, a novel inductively coupled plasma atomic emission spectrometric method was developed and validated for the determination of nutrient and toxic elements in walnut oils produced with conventional and organic farming. The method presented good linearity (r2 > 0.9990) for each element at the selected emission line. The limits of detection and limits of quantification ranged between 0.09 μg g−1 to 2.43 μg g−1 and 0.28 μg g−1 to 8.1 μg g−1, respectively. Method accuracy and was assessed by analyzing the certified reference materials BCR 278-R and spiked walnut oil samples. The determined metals were quantified, and the results were analyzed by Student’s t-test to investigate the differences in the elemental profile of the walnut oils according to type of farming (conventional or organic)

Determination of the Toxic and Nutrient Element Content of Almonds, Walnuts, Hazelnuts and Pistachios by ICP-AES

The trace element content of thirty-two nuts including almonds, walnuts, hazelnuts and pistachios available in a Greek market was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Wet acid digestion using nitric acid (65%) took place in Teflon autoclaves. The limits of detection (LODs) and limits of quantification (LOQs) ranged between 0.01 (Mg)–2.52 (Cu) μg g−1 and 0.02 (Mg)–8.40 (Cu) μg g−1, respectively. Good method linearity (r2 > 0.9990) was observed for each element at the selected emission lines. The metals were quantified and one-way analysis of variance (ANOVA) was used to examine whether or not there were any statistically significant differences among the metal concentrations inside the different nut species