Detecció i Quantificació d’Adulteracions en la Prevenció de Fraus en Cafè mitjançant empremtes HPLC-UV i HPLC-FLD

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2021, Tutors: Oscar Núñez Burcio, Javier Saurina PurroyGlobalization has produced a total change of scenario in food industry producing a tough competence to occupy the market share, instigating the reduction of costs by usage of fraudulent practices derived from food adulteration. These practices are performed by substitution of most valuable components for other with less commercial value and/or lower health beneficial properties supposing an economic fraud and a potential health problem. Coffees are sometimes the target of this kind of fraudulent practices due to the high demand of the product where manufacturers adulterate coffee with wheat, corn, and other grains, seeds and plants. In this work, simultaneous non-targeted HPLC-UV and HPLC-FLD fingerprinting methods were developed to achieve the classification and authentication of different instant coffee, and chicory samples using multivariate chemometric methodologies such as principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and partial least squares (PLS). Both HPLC-UV and HPLC-FLD fingerprints, proved to be excellent chemical descriptors for the discrimination of chicory samples against instant coffee and decaffeinate coffee by PLS-DA. However, better results were obtained with HPLC-UV fingerprints when coffee was discriminated from decaffeinated coffee (94.4% classification rate respect to 83.3% for HPLC-FLD fingerprints). Besides, both methodologies were able to detect and quantify adulterant levels in coffee and decaffeinated samples adulterated with chicory exhibiting good regression linearity (R2≥0.996), and low calibration (0.7-2.1%) and prediction (2.4-3.5%) errors. Overall, both non-targeted HPLC-UV and HPLC-FLD showed to be effective, simple, and trustable to accomplish the characterization, classification and authentication of instant coffee and chicory samples being potential methodologies to prevent food fraud

Metodologies d’Empremtes No-dirigides per a l’Autenticació de Té. Aplicació a la Detecció i Quantificació de Fraus en Mostres de Té Adulterades amb Xicoira

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2022, Tutors: Oscar Núñez Burcio, Javier Saurina PurroyIn recent years, in an increasingly globalised world the food industry like so many others has undergone a paradigm shift. This increasing globalisation has generated high competition, and some industries have turned to fraudulent practices in order to reduce production costs. Food frauds are very diverse, ranging from the substitution and/or addition of components to fraud in product labelling. This malpractice for economic purposes becomes a serious problem for consumers by putting their health at risk. To avoid this kind of negligence, it is very important to develop new fraud detection methodologies, especially in a world where these practices are becoming increasingly sophisticated. This project focuses on tea adulteration with chicory. For this purpose, two methodologies have been developed simultaneously for the authentication of different tea varieties (black, red, green, white and oolong). These are two non-targeted fingerprinting methods that use the chromatograms obtained by high performance liquid chromatography with ultraviolet (HPLC-UV) and fluorescence (HPLC-FLD) detection. The obtained data have been subjected to multivariate chemometric treatments, such as Principal Component Analysis (PCA) and Partial Least Squares regression (PLS). The good results obtained have shown that both methodologies are effective for the detection and quantification of tea adulteration with chicory, exhibiting good linear regressions (R2 > 0.998) and calibration, cross-validation, and external validation errors below 1.4%, 6.4% and 3.7%, respectively. Moreover, very acceptable prediction errors have been obtained (less than 21.7%), except for white tea extracts which show higher errors due to the similarity of their fingerprints to those of chicory. In addition to these two methods, two new studies have been started to further advance the authentication of teas. The first one is a non-targeted FIA-MS fast-screening method and the second one is a non-targeted method using the fingerprints obtained by liquid chromatography coupled to a mass spectrometer (LC-MS) as chemical descriptors. Data from both methodologies have also been chemometrically treated with PCA and Partial Least Squares-Discriminant Analysis (PLS-DA) and the results are very promising since both techniques are able to distinguish between chicory and tea sample extracts (although further work is needed to optimise them). The most interesting approach is the FIA-MS method, which represents a great advantage over previous methods, as it entails significant time saving in tea authentication analysi

Differential localization of flavonoid glucosides in an aquatic plant implicates different functions under abiotic stress

Abstract Flavonoids may mediate UV protection in plants either by screening of harmful radiation or by minimizing the resulting oxidative stress. To help distinguish between these alternatives, more precise knowledge of flavonoid distribution is needed. We used confocal laser scanning microscopy (cLSM) with the “emission fingerprinting” feature to study the cellular and subcellular distribution of flavonoid glucosides in the giant duckweed ( Spirodela polyrhiza ), and investigated the fitness effects of these compounds under natural UV radiation and copper sulphate addition (oxidative stress) using common garden experiments indoors and outdoors. cLSM “emission fingerprinting” allowed us to individually visualize the major dihydroxylated B‐ring‐substituted flavonoids, luteolin 7‐O‐glucoside and luteolin 8‐C‐glucoside, in cross‐sections of the photosynthetic organs. While luteolin 8‐C‐glucoside accumulated mostly in the vacuoles and chloroplasts of mesophyll cells, luteolin 7‐O‐glucoside was predominantly found in the vacuoles of epidermal cells. In congruence with its cellular distribution, the mesophyll‐associated luteolin 8‐C‐glucoside increased plant fitness under copper sulphate addition but not under natural UV light treatment, whereas the epidermis‐associated luteolin 7‐O‐glucoside tended to increase fitness under both stresses across chemically diverse genotypes. Taken together, we demonstrate that individual flavonoid glucosides have distinct cellular and subcellular locations and promote duckweed fitness under different abiotic stresses

Characterization and Classification of Spanish Honey by Non-targeted LC-HRMS (Orbitrap) Fingerprinting and Multivariate Chemometric Methods

A non-targeted LC-HRMS fingerprinting methodology using an Orbitrap mass analyzer, and based on C18 reversed-phase mode under universal gradient elution, was developed to characterize and classify Spanish honey samples. A simple sample treatment consisting of honey dis-solution with water and a 1:1 dilution with methanol was proposed. 136 honey samples belonging to different blossom- and honeydew-honeys from different botanical varieties and produced in different Spanish geographical regions were analyzed. The obtained LC-HRMS fingerprints were employed as sample chemical descriptors for honey pattern recognition by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The results demonstrated a superior honey classification and discrimination capability with respect to previous non-targeted HPLC-UV fingerprinting approaches, being able to discriminate and authenticate the honey samples according to their botanical origins. Overall, noteworthy cross-validation multiclass predictions were accomplished, with sensitivity and specificity values higher than 96.2%, except for orange/lemon blossom (BL) and rosemary (RO) blossom-honeys. The proposed methodology was also able to classify and authenticate the climatic geographical production region of the analyzed honey samples, with cross-validation sensitivity and specificity values higher than 87.1%, and classification errors below 10.5%

Honey fraud detection based on sugar syrup adulterations by HPLC-UV fingerprinting and chemometrics

In recent years, honey-producing sector has faced the increasing presence of adulterated honeys, implying greateconomic losses and questioning the quality of this highly appreciated product by the society. Due to the highsugar content of honey, sugar syrups are among its most common adulterants, being also the most difficult todetect even with isotope ratio techniques depending on the origin of the sugar syrup plant source. In this work, ahoney authentication method based on HPLC-UV fingerprinting was developed, exhibiting a 100% classificationrate of honey samples against a great variety of sugar syrups (agave, corn, fiber, maple, rice, sugar cane andglucose) by partial least squares-discriminant analysis (PLS-DA). In addition, the detection and level quantitationof adulteration using syrups as adulterants (down to 15%) was accomplished by partial least squares (PLS)regression with low prediction errors by both internal and external validation (values below 12.8% and 19.7%,respectively

Liquid chromatographic fingerprints for the characterization of flavanol-rich nutraceuticals based on 4-dimethylaminocinnamaldehyde precolumn derivatization

Flavanols consist of a great family of bioactive molecules displaying a wide range of health-promoting attributes for humans, including antioxidant, antimicrobial or an-ti-inflammatory effects. As a result, botanical species rich in this type of compounds are often used to develop nutraceutical products or dietary supplements with recognized healthy attrib-utes. This paper aims at characterizing nutraceutical products using liquid chromatographic fin-gerprints related to flavanol composition. Catechins and their oligomers were exploited to characterize and authenticate of various commercial products prepared with extracts of red ber-ries and medicinal plants. These compounds resulted in interesting descriptors of some fruits and vegetables, thus providing an additional perspective for the study of nutraceuticals. For such a purpose, a new method based on liquid chromatography with UV/Vis detection (HPLC-UV/Vis) with precolumn derivatization with 4-dimethylaminocinnamaldehyde was de-veloped. Results indicated that the separation of flavanols was very complex due to the degrada-tion of procyanidin derivatives. The resulting data sets were analyzed using chemometric methods such as principal component analysis and partial least square-discriminant analysis. Despite the complexity of chromatographic fingerprints, nutraceutical samples could be dis-criminated according to their main ingredients. In general, catechin and epicatechin were the most abundant compounds in the different samples and procyanidin A2 was highly specific of cranberry

Non-targeted ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) fingerprints for the chemometric characterization and classification of turmeric and curry samples

In this work, non-targeted UHPLC-HRMS fingerprints obtained by C18 reversed-phase chromatography were proposed as sample chemical descriptors for the characterization and classification of turmeric and curry samples. 21 turmeric and 9 curry commercially available samples were analyzed in triplicate after extraction with DMSO. The results demonstrated the feasibility of non-targeted HPLC-HRMS fingerprints for sample classification, showing very good classification capabilities by partial least squares regression-discriminant analysis (PLS-DA). 100% classification rates were obtained by PLS-DA when randomly selected samples were processed as 'unknown' ones. Besides, turmeric curcuma species (curcuma longa vs. curcuma zedoaria) and turmeric curcuma longa varieties (Madras, Erodes, and Alleppey) discrimination was also observed by PLS-DA when using the proposed fingerprints as chemical descriptors. As a conclusion, non-targeted UHPLC-HRMS fingerprinting is a suitable methodology for the characterization, classification and authentication of turmeric and curry samples, without the requirement of using commercially available standards for quantification nor the necessity of metabolite identification

Characterization, Classification and Authentication of Spanish Blossom and Honeydew Honeys by non-targeted HPLC-UV and off-line SPE HPLC-UV Polyphenolic Fingerprinting Strategies.

Honey is a highly consumed natural product produced by bees which is susceptible to fraudulent practices, some of them regarding their botanical origin. Two HPLC-UV non-targeted finger-printing approaches were evaluated in this work to address honey characterization, classification, and authentication based on honey botanical variety. The first method used no sample treatment and a universal reversed-phase chromatographic separation. On the contrary, the second method was based on an off-line SPE preconcentration method, optimized for the isolation and extraction of polyphenolic compounds, and a reversed-phase chromatographic separation opti-mized for polyphenols as well.. For the off-line SPE method, the use of HLB (3 mL, 60 mg) car-tridges, and 6 mL of methanol as eluent, allowed to achieve acceptable recoveries for the selected polyphenols. The obtained HPLC-UV fingerprints were subjected to exploratory principal com-ponent analysis (PCA) and classificatory partial least squares-discriminant analysis (PLS-DA) to evaluate their viability as sample chemical descriptors for authentication purposes. Both HPLC-UV fingerprints resulted to be appropriate to discriminate between blossom-honeys and honeydew-honeys. However, superior performance was accomplished with off-line SPE HPLC-UV polyphenolic fingerprints, being able to differentiate among the different blos-som-honey samples under study (orange/lemon blossom, rosemary, thyme, eucalyptus, and heather). In general, this work demonstrated the feasibility of HPLC-UV fingerprints, especially those obtained after off-line SPE polyphenolic isolation and extraction, to be employed as honey chemical descriptors to address the characterization and classification of honey samples according to their botanical origin

Reliable allele detection using SNP-based PCR primers containing Locked Nucleic Acid: application in genetic mapping

BACKGROUND: The diploid, Solanum caripense, a wild relative of potato and tomato, possesses valuable resistance to potato late blight and we are interested in the genetic base of this resistance. Due to extremely low levels of genetic variation within the S. caripense genome it proved impossible to generate a dense genetic map and to assign individual Solanum chromosomes through the use of conventional chromosome-specific SSR, RFLP, AFLP, as well as gene- or locus-specific markers. The ease of detection of DNA polymorphisms depends on both frequency and form of sequence variation. The narrow genetic background of close relatives and inbreds complicates the detection of persisting, reduced polymorphism and is a challenge to the development of reliable molecular markers. Nonetheless, monomorphic DNA fragments representing not directly usable conventional markers can contain considerable variation at the level of single nucleotide polymorphisms (SNPs). This can be used for the design of allele-specific molecular markers. The reproducible detection of allele-specific markers based on SNPs has been a technical challenge. RESULTS: We present a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD) markers. These markers proved highly efficient for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA) base. We demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII) markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles of 16 chromosome-specific COSII markers and to assign eight of the twelve linkage groups to consensus Solanum chromosomes. CONCLUSION: The method based on individual allelic variants allows for a level-of-magnitude higher resolution of genetic variation than conventional marker techniques. We show that the majority of monomorphic molecular marker fragments from organisms with reduced heterozygosity levels still contain SNPs that are sufficient to trace individual alleles