Assessment of experimental factors affecting the sensitivity and selectivity of the spectrophotometric estimation of proanthocyanidins in foods and nutraceuticals

The assessment of a reliable method for the determination of active proanthocyanidins (PACs) in functional foods, nutraceuticals, and foodstuffs is still a pending analytical challenge. In general, pharmaceutical and food companies accept the assay based on 4-dimethylaminocinnamaldehyde (DMAC) as an overall index of PACs, although these values provided confuse information of the actual bioactivity of the product. Besides, obtaining accurate and precise results is difficult because of the complexity of the sample matrices and the differential selectivity of analytes. Here, we have investigated in detail those experimental factors contributing to the PAC-DMAC derivatives, and we have explored the possibilities for increasing the selectivity of the reaction to detect the fraction of PAC molecules with antibacterial properties. In this regard, the use of harsh conditions, especially working at high temperature, contributes to the formation of the desired derivatives and the degradation of the undesired ones. Unfortunately, the selective detection of A-type species based on DMAC derivatization was not realistic. Another goal of the study was the development of a spectrophotometric assay for the determination of the total content of flavanols. In this case, experimental conditions were chosen to reach high responses for all the compounds. Working under mild conditions of temperature and reaction time (20 °C and 20 min), the method was suitable to estimate the overall flavanol content of food supplements, nutraceuticals, and commercial fruit juices

Characterization of fruit products by capillary zone electrophoresis and liquid chromatography using the compositional profiles of polyphenols. Application to authentication of natural extracts

Capillary zone electrophoresis (CZE) and high performance liquid chromatography (HPLC) were applied to the authentication of fruit products based on the compositional profiles of polyphenols. Various sample treatments were used to maximize the overall recovery of polyphenols or specific fractions, such as phenolic acids or anthocyanins. The resulting CZE and HPLC data were treated with Principal Component Analysis (PCA) showing that samples were mainly clustered according to the fruit of origin, with cranberry- and grape-based products clearly separated in groups. A possible adulterated cranberry extract was analyzed more deeply by high resolution mass spectrometry (HRMS) in order to identify the presence of A-type proanthocyanidins, which are characteristic and more abundant in cranberry-based products. In accordance with PCA interpretation, HRMS results indicated that the suspicious sample was not a cranberry-based product, allowing us to validate and demonstrate the suitability of both CZE- and HPLC-proposed methods for the characterization of fruit-based products

Determination of polyphenols in white wines by liquid chromatography: application to the characterization of Alella (Catalonia, Spain) wines using chemometric methods

The determination of polyphenols in wines is of great interest in the field of food analysis due to health and organoleptic implications. In addition, the applicability of polyphenols as food descriptors to be used for characterization, classification, and authentication purposes is gaining popularity. In this work, a simple and reliable method based on HPLC separation in reversed-phase mode with UV-Vis detection was developed and applied to determine polyphenolic compounds in white wines. The chromatographic separation was performed using a C18 column under a methanol elution gradient and assessed by an experimental design approach. Analytical parameters were established under the optimal experimental conditions. LOD values were between 3 and 220 µg/L, and repeatability values were better than 1% for most of the analyzed polyphenols. Compositional data were further exploited to characterize white wines based on principal component analysis to discriminate among mono- and polyvarietal compositions

Determination of polyphenols in spanish wines by capillary zone electrophoresis. Application to wine characterization by using chemometrics

A capillary zone electrophoresis method for the simultaneous determination of twenty polyphenols in wine was developed. The separation was performed using fused-silica capillaries of 75 µm I.D. and a 30 mM sodium tretraborate buffer solution at pH 9.2 with 5% isopropanol as a background electrolyte. A capillary voltage of +25 kV with pressure-assisted (3.5 kPa) separation from min 18 was applied, thus, achieving a total analysis time lower than 20 min. Instrumental quality parameters such as limits of detection (LOD values between 0.3 and 2.6 mg/L), linearity (r2>0.990), and run-to-run and day-to-day precisions (RSD values lower than 6.5% and 15.7%, respectively) were established. Three different calibration procedures were evaluated for polyphenol quantitation in wines: external calibration using standards prepared in Milli-Q water, standard addition, and pseudo-matrix matched calibration using wine as a matrix. For a 95% confidence level, no statistical differences were observed, in general, between the three calibration methods (p-values between 0.11 and 0.84), while for some specific polyphenols, such as cinnamic acid, syringic acid and gallic acid, results were not comparablewhen external calibration used. CZE method using pseudo-matrix matched calibration was then proposed and applied to the analysis of polyphenols in 49 Spanish wines, showing satisfactory results and a wide compositional variation between wines. Electrophoretic profiles and other compositional data (e.g., peak areas of selected peaks) were considered as fingerprints of wines to be used for characterization and classification purposes. The corresponding data were analyzed by PCA in order to extract information on the most significant features contributing to wine discrimination according to their origins. Results showed that a reasonable distribution of wines depending on the elaboration areas was found, being tirosol, gallic, protocatechuic, p-coumaric and caffeic acids some representative discriminant compounds

Determination of polyphenolic profiles by liquid chromatography-electrospray-tandem mass spectrometry for the authentication of fruit extracts

Liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) was applied to the analysis and authentication of fruit-based products and fruit-based pharmaceutical preparations. A Kinetex C18 reversed-phase column under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases was used for the simultaneous determination of 26 polyphenols, allowing an acceptable separation in less than 22 min. Instrumental quality parameters such as limits of detection (LOD, values between 12-14 µg/L for 19 of the 26 analyzed polyphenols), linearity (r2 > 0.991), run-to-run and day-to-day precisions (RSD values lower than 9.9 and 13.5 %, respectively), and accuracy (relative errors lower than 8 %) were established. A simple extraction method, consisting of a sample sonication with acetone:water:hydrochloric acid (70:29.9:0.1 v/v/v) and centrifugation, was proposed. Two calibration procedures, external calibration using standards prepared in water and standard addition, were evaluated for polyphenol quantification in several grape and cranberry fruits and processed fruit products. For a 95 % confidence level, no statistical differences were observed between the two calibration methods (p values between 0.06 and 0.95), denoting that external calibration was suitable enough for the quantitative analysis of polyphenols in fruit-based products. The proposed LC-ESI-MS/MS method was then applied to the analysis of polyphenols in 23 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic concentration data was then analyzed by principal component analysis (PCA) to extract information of the most significant profile data contributing to authentication of natural extracts according to their fruit of origin

Told through the wine: a liquid chromatography-mass spectrometry interplatform comparison reveals the influence of the global approach on the final annotated metabolites in non-targeted metabolomics

This work focuses on the influence of the selected LC-HRMS platform on the final annotated compounds in non-targeted metabolomics. Two platforms that differed in columns, mobile phases, gradients, chromatographs, mass spectrometers (Orbitrap [Platform#1] and Q-TOF [Platform#2]), data processing and marker selection protocols were compared. A total of 42 wines samples from three different protected denomination of origin (PDO) were analyzed. At the feature level, good (O)PLS-DA models were obtained for both platforms (Q2[Platform#1]=0.89, 0.83 and 0.72; Q2[Platform#2]=0.86, 0.86 and 0.77 for Penedes, Ribera del Duero and Rioja wines respectively) with 100% correctly classified samples in all cases. At the annotated metabolite level, platforms proposed 9 and 8 annotated metabolites respectively which were identified by matching standards or the MS/MS spectra of the compound. At this stage, none of the suggested metabolites was coincident between platforms. When screened on the raw data, 6 and 5 of these compounds were detected on the other platform with a similar trend. Some of the detected metabolites showed complimentary information when integrated on biological pathways. Through the use of some examples at the annotated metabolite level, possible explanations of this initial divergence on the results are presented. This work shows the complications that may arise on the comparison of non-targeted metabolomics platforms even when metabolite focused approaches are used in the identificatio

Characterization, classification and authentication of fruit-based extracts by means of HPLC-UV chromatographic fingerprints, polyphenolic profiles and chemometric methods

HPLC-UV was applied to the analysis and characterization of fruit-based and fruit-processed products. A Kinetex C18 reversed-phase column was proposed under gradient elution for the determination of 17 polyphenols. Acceptable sensitivity (LODs below 0.16 mg/L), and good linearity (r2 higher then 0.995), precision (RSD below 6.8%), and method trueness (relative errors below 11%) were obtained. Data corresponding to polyphenolic peak areas and HPLC-UV chromatographic fingerprints were then analyzed by exploratory principal component analysis (PCA) to extract information of the most significant variables contributing to characterization and classification of analyzed samples regarding the fruit of origin. HPLC-UV chromatographic data was further treated by partial least square (PLS) regression to determine the percentages of adulteration in cranberry-fruit extracts. It was found that even mixture samples containing low percentages of adulterants could be distinguished from genuine cranberry extracts. Highly satisfactory results were obtained, with overall errors in the quantification of adulterations below 4.3%

Millora de l’aprenentatge a les assignatures pràctiques de Química Analítica mitjançant una planificació integral basada en l’avaluació continuada i nous instruments d’avaluació

Projecte: 2016PID-UB/014En aquest projecte s’ha treballat en la millora de l’aprenentatge de les classes pràctiques de laboratori de l’assignatura Química Analítica del Grau de Farmàcia. Aquesta assignatura és de primer curs, on el nombre anual d’estudiants implicats és molt gran, de l’ordre de 500, i les pràctiques de laboratori s’han de programar en grups reduïts, simultàniament al desenvolupament de les classes de teoria i seminaris. D’acord a la nostra experiència, el gran nombre de grups de classes pràctiques de laboratori, de professorat implicat i la varietat de coneixements teòrics previs dels estudiants comporta sovint poca homogeneïtat en l’aprenentatge i en l’assoliment de les diferents competències, així com en la seva posterior avaluació. A més, les pràctiques de laboratori es porten a terme en parelles i el programa inclou més de 30 experiències que es simultaniegen i s’avaluen de forma continuada amb l’objectiu de fomentar el desenvolupament de les competències transversals i específiques dels estudiants. Aquest projecte ha permès desenvolupar i implementar durant els cursos 2016-2017 i 2017-2018 una planificació integral innovadora de les pràctiques de laboratori basada en la coordinació de l’equip docent, en l’establiment d’itineraris de pràctiques tancats, en l’avaluació continuada i en la introducció de nous materials d’aprenentatge i instruments d’avaluació. Totes les innovacions desenvolupades es continuen aplicant a l’actualitat

Una planificación innovadora y nuevos instrumentos de evaluación para la mejora del aprendizaje en un laboratorio de prácticas de Química Analítica básica

Este trabajo presenta las acciones realizadas para la mejora del aprendizaje en el laboratorio de prácticas de una asignatura de Química Analítica Básica, como es la Química Analítica del Grado de Farmacia de la Universidad de Barcelona. Lo que comenzó en el marco de un proyecto de innovación docente, se ha consolidado para vertebrar el funcionamiento de estas prácticas de laboratorio hasta la actualidad, involucrando satisfactoriamente del orden de 350 estudiantes y 20 profesores por curso