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.

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 0.16mg/L), and good linearity (r2 higher than 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%.

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 (Q(2)[Platform#1]=0.89, 0.83 and 0.72; Q(2)[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 compounds. At this stage, there was no coincidence among platforms regarding the suggested metabolites. 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 identification.

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 and 14 µg/L for 19 of the 26 analyzed polyphenols), linearity (r (2) > 0.991), run-to-run and day-to-day precisions (relative standard deviation (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.

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.

Classification and characterisation of Spanish red wines according to their appellation of origin based on chromatographic profiles and chemometric data analysis.

Chromatographic profiles of wines have been used as a fingerprint for the discrimination of Spanish wines based on oenological practices. In order to extract information of different families of phenolic compounds, profiles of different UV-vis absorption wavelengths (280, 310, 370 and 520nm) and fluorescence (ex=260nm; em=360nm) were analysed. A total of thirteen phenolic compounds which allowed the discrimination of wines of three different Spanish appellations (Penedes, Rioja and Ribera del Duero) were selected by means of principal component analysis (PCA). Afterwards, these compounds were used to build partial least squares discriminant analysis (PLS1-DA and PLS2-DA) models which allowed the discrimination of wines according to their appellation with classification rates for independent test sets higher than 96% and 93% for PLS1-DA and PLS2-DA models respectively. Finally, characteristic compounds of each appellation were tentatively identified by means of liquid chromatography-mass spectrometry (LC-MS) analysis. Thus, ten out of thirteen compounds (i.e., gallic acid for Penedes, trans-coumaroyltartaric and trans-caffeoyltartaric acids for Rioja and myricetin for Ribera del Duero wines) have been proposed.

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

A capillary zone electrophoresis (CZE) method for the simultaneous determination of 20 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 minute 18 was applied, thus achieving a total analysis time of <25 min. Instrumental quality parameters such as limits of detection (LOD, values between 0.3 and 2.6 mg/L), linearity (r(2) > 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 pseudomatrix-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), whereas for some specific polyphenols, such as cinnamic acid, syringic acid, and gallic acid, results were not comparable when external calibration was used. The CZE method using pseudomatrix-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 principal component analysis (PCA) 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, tyrosol and gallic, protocatechuic, p-coumaric, and caffeic acids being some representative discriminant compounds.

Determination of polyphenols in wines by liquid chromatography with UV spectrophotometric detection.

This paper describes a new chromatographic method for the determination of polyphenolic compounds in wines. The method is based on the separation of analytes by reversed-phase mode in a C18 column (2.6 µm particle size) and UV absorption spectroscopy. The elution gradient is generated from 0.1% formic acid aqueous solution and acetonitrile as an organic modifier. Experimental conditions including pH, percentage of organic modifier and elution gradient profile have been thoroughly optimized using experimental design. A multi-objective function has been defined as a criterion for obtaining a satisfactory compromise among number of compounds separated, resolution and analysis time. Multi-detection at 280, 310 and 370 nm has been utilized in order to work under the most appropriate wavelengths for each compound. Figures of merit including linearity ranges, precisions, detection limits and recoveries have been established under selected experimental conditions using synthetic standards and commercial red wines. The method has been applied to analyze red wines from various Spanish regions.

Derivatization strategies for the determination of biogenic amines in wines by chromatographic and electrophoretic techniques.

This paper revises the derivatization approaches for the determination of biogenic amines in wines. Since most of these amines display poor spectroscopic features to be detected by UV absorption or emission (fluorescence) spectroscopy, derivatization is necessary to attain the desired sensitivity. Reagents such as o-phthaldialdehyde, fluorenylmethylchloroformate, dansyl-Cl and dabsyl-Cl have widely been used for analytical labeling through amino group. A comparison of features of off- and on-line pre- and post chromatographic/electrophoretic labeling is given using 1,2-naphthoquinone-4-sulfonate (NQS) as an example. The evaluation of the influence of the wine sample composition on the derivatization process indicates that pre-column labeling may undergo more severe matrix effects.

Determination of biogenic amines in wines by pre-column derivatization and high-performance liquid chromatography coupled to mass spectrometry.

A new HPLC method for determining biogenic amines in wines is developed. This method is based on pre-column amine derivatization, further separation of derivatives and on-line hyphenation of HPLC to atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Biogenic amines have been derivatized with 1,2-naphthoquinone-4-sulfonate at 65 degrees C and pH 9.2 for 5 min. The separation of derivatives has been accomplished in a C(18) analytical column using an elution gradient based on increasing the percentage of methanol. Derivatives have been ionized in positive mode and detected by selected ion monitoring. The operating conditions of the APCI-MS system (voltages, temperatures and gases) have been thoroughly optimized to obtain the maximum sensitivity for all analytes. In the selected conditions, APCI-MS spectra display little fragmentation and good signal-to-noise ratio. Depending on the amine characteristics, the main spectral peaks are due to mono- and di-derivative products. Figures of merit of the method have been established under the selected conditions using red wine samples. Recoveries ranging from 94% to 106% have been obtained which prove excellent accuracy of the method in the determination of histamine, putrescine, cadaverine, tryptamine, phenylethylamine, tyramine and serotonin in red wines. The proposed method has been applied to the analysis of commercial wines from different Spanish regions.

Determination of HIV drugs in biological matrices: a review.

The present paper reviews the recent advances on the determination of antiretroviral drugs against the human immunodeficiency virus (HIV). Methods have been reviewed with special emphasis on the principal analytical strategies for dealing with clinical samples as well as the determination of the newest drugs. The most critical steps of the analytical procedures including the sample treatment, separation and validation have been discussed. Finally, a brief description of representative applications is given.

Reversed-phase liquid chromatographic method with spectrophotometric detection for the determination of antiretroviral drugs.

In the present paper, a new chromatographic method for the determination of acquired immune deficiency syndrome (AIDS) drugs in plasma samples is proposed. The method consists of solid-phase extraction for sample pretreatment and further chromatographic analysis. Drugs have been separated on a C(18) column using an elution gradient based on an increase in the acetonitrile percentage. Analytes have been detected spectrophotometrically at 240, 250, 260 and 280nm. Chromatographic conditions have been thoroughly optimized using experimental design and multicriteria functions. Analytical parameters of the method have been established for both synthetic and plasma samples. Limits of detection are around 5ngmL(-1) for reverse transcriptase inhibitors (nucleoside and non-nucleoside) and 20ngmL(-1) for protease inhibitors. The method has been validated through a spiking/recovery procedure at three concentration levels. Results obtained are highly satisfactory, with recovery values around 100% for all drugs. The method has been applied to the determination of various drug mixtures of medical interest in plasma samples.

Characterization of wines through the biogenic amine contents using chromatographic techniques and chemometric data analysis.

This paper describes a new method for wine characterization based on the analysis of the biogenic amine composition and the chromatographic profiles using chemometric methods such as principal component analysis and partial least-squares regression. Amine contents have been determined by liquid chromatography with a precolumn derivatization with 1,2-naphthoquinone-4-sulfonate. The corresponding chromatographic data have been advantageously exploited for extracting relevant information regarding some wine features such as elaboration procedure, vintage, or origin region. Results indicate that amines might be used as descriptors of the certain enological practices. Besides, younger wines can be reasonably distinguished from aged ones on the basis of the amine contents. The wine characterization through the analysis of raw chromatographic profiles is proven to be also effective, and patterns dealing with aging processes have also been encountered.

Flow-injection determination of zidovudine in plasma samples using multivariate curve resolution.

This paper describes a spectrophotometric flow-injection method for the determination of an antiretroviral drug (zidovudine) in plasma samples. The main goal of this study is the development of feasible analytical method to monitor the plasmatic drug levels in a rapid and simple way as an alternative to chromatographic procedures. The flow-injection system proposed consists of a two-channel manifold with the injection of the sample into an acid carrier and on-line generation of a pH-gradient. The corresponding data are monitored over time using a diode array spectrophotometer. The discrimination of zidovudine species from plasma components is accomplished through chemometric data analysis based on the zidovudine features on both spectral and time domains. A pretreatment procedure consisting of liquid-liquid extraction is used for sample clean-up. However, despite the pretreatment, noticeable amounts of unknown substances acting as interferences are still present in the extracts. As relevant analytical parameters, the analyte recovery in the extraction process is 101% at 5.3 microg mL(-1) and the detection limit is 0.013 microg mL(-1). Multivariate curve resolution with alternating least squares is used to recover the analyte profiles for its further determination. As a result, the quantification of zidovudine in plasma can be accomplished even in the presence of plasma components with overall prediction error below 3%.

Determination of biogenic amines in wines by ion-pair liquid chromatography and post-column derivatization with 1,2-naphthoquinone-4-sulphonate.

A liquid chromatographic method with post-column derivatization for the determination of biogenic amines in wines is proposed. The method is based on the separation of amines by ion-pair chromatography using sodium heptanesulfonate (SHS) and on-line labeling of analytes with 1,2-naphthoquinone-4-sulfonate. The principal factors influencing the separation (acetonitrile and SHS concentration) have been considered for the optimization of the elution gradient through factorial design and multicriteria decision-making. Figures of merit have been established using red wine samples. Detection limits range from 0.2 to 3 mg L(-1), the peak area run-to-run repeatability from 1.6 to 4.6% and the retention time repeatability lower than 1.2%. Recoveries ranging from 92 and 108% prove the accuracy of the method for determining ethanolamine, ethylamine, histamine and tyramine in commercial red wines. The proposed method has been applied to the analysis of wines from different Spanish regions.

Capillary electrophoresis determination of biogenic amines by field-amplified sample stacking and in-capillary derivatization.

A sensitive CE method for determining biogenic amines in wines based on in-capillary derivatization with 1,2-naphthoquinone-4-sulfonate is presented. In this method, reagent and buffer solutions are introduced hydrodynamically into the capillary whereas the sample is injected electrokinetically, thus, allowing a selective preconcentration of the analytes by field-amplified sample stacking. Amines are labeled inside the capillary using a zone-passing derivatization approach in mixed tandem mode. The most relevant variables influencing on the derivatization and separation as well as significant interactions have been evaluated using experimental design. Multi-criteria decision making is utilized for the simultaneous optimization of interacting variables through overall desirability response surfaces. The validation of the method has proven an excellent separation performance and accuracy for the determination of biogenic amines such as histamine, tryptamine, phenylethylamine, tyramine, agmatine, ethanolamine, serotonin, cadaverine, and putrescine in red wines. Detection limits range from 0.02 mg/L for ethanolamine to 0.91 mg/L for serotonin. The RSDs for migration time and peak area are around 1.2 and 6.2%, respectively. Red wines from different Spanish regions have been analyzed using the proposed method.

Flow-injection spectrophotometric determination of reverse transcriptase inhibitors used for acquired immuno deficiency syndrome (AIDS) treatment: focus on strategies for dealing with the background components.

This paper describes a rapid method for the quantification of reverse transcriptase inhibitors in mixtures of therapeutic interest containing zidovudine, zalcitabine and nevirapine. The method is based on a pH-gradient flow-injection system with diode array detection and further data analysis using multivariate curve resolution. Data matrices of unknown samples mixtures have been analyzed simultaneously with those of standards and background via the construction of augmented data matrices. In this case, the spectral domain has been chosen for performing column-wise (i.e., wavelength-wise) matrix augmentation. The quantification is accomplished from the comparison of the analyte peaks resolved for unknown mixtures and standards, being peak areas the quantitative information to be correlated with the concentration in both calibration and prediction steps. The influence of background signal on the response is tackled through two main strategies involving modelling or subtraction. The potentiality of this method for the analysis of drugs in the presence of unknown interferences is demonstrated. Results obtained in the analysis of drug samples have proven the excellent performance of this method. Overall prediction errors in the quantification of two- and three-component mixtures are about 5-10%.

High-performance liquid chromatographic determination of biogenic amines in wines with an experimental design optimization procedure.

A novel and sensitive HPLC method for determining biogenic amines in wine samples is described. It involves pre-column labeling of the analytes with 1,2-naphthoquinone-4-sulfonate (NQS) and liquid-liquid extraction of derivatives with chloroform for analyte preconcentration and sample clean-up. A linear gradient elution consisting of a mixture of 2% of acetic acid aqueous solution and methanol is used to separate the amine derivatives in a C18 column. The eluted compounds are detected spectrophotometrically at 270 nm. The optimization of both derivatization and separation conditions is accomplished by means of factorial and central composite designs and multicriteria decision functions. The analytical parameters of the method are established using red wine samples. Detection limits range from 0.006 to 0.315 mg L(-1). The run-to-run repeatabilities of retention times and peak areas are around 0.6 and 5.6%, respectively. Recoveries ranging from 91.9 to 105% prove the accuracy of the method for determining histamine, putrescine, cadaverine, tryptamine, phenylethylamine and serotonin in red wines. The proposed method has been applied to the analysis of commercial wines from different Spanish regions.

Determination of histamine in wines with an on-line pre-column flow derivatization system coupled to high performance liquid chromatography.

A new rapid and sensitive high performance liquid chromatography (HPLC) method for determining histamine in red wine samples, based on continuous flow derivatization with 1,2-naphthoquinone-4-sulfonate (NQS), is proposed. In this system, samples are derivatized on-line in a three-channel flow manifold for reagent, buffer and sample. The reaction takes place in a PTFE coil heated at 80 degrees C and with a residence time of 2.9 min. The reaction mixture is injected directly into the chromatographic system, where the histamine derivative is separated from other aminated compounds present in the wine matrix in less than ten minutes. The HPLC procedure involves a C18 column, a binary gradient of 2% acetic acid-methanol as a mobile phase, and UV detection at 305 nm. Analytical parameters of the method are evaluated using red wine samples. The linear range is up to 66.7 mg L(-1) (r = 0.9999), the precision (RSD) is 3%, the detection limit is 0.22 mg L(-1), and the average histamine recovery is 101.5% +/- 6.7%. Commercial red wines from different Spanish regions are analyzed with the proposed method.

Flow-injection determination of amine contaminants in cyclamate samples based on temperature for controlling selectivity.

This paper describes a flow-injection (FI) method for the simultaneous determination of aniline and cyclohexylamine impurities in cyclamate products. The method consists of the derivatization of amines with 1,2-naphthoquinone-4-sulfonate under selective and non-selective conditions. Here, the selectivity is achieved by working at 20 degree C, at which only aniline reacts, whilst higher temperatures (80 degree C) lead to a non-selective reaction of the two analytes. The FI manifold is composed of two flow cells for the spectrophotometric detection of derivatives at 480 nm. Experimental conditions have been optimized by factorial design and multicriteria making approach. Quantification is accomplished by differential analysis of the analyte contributions in the double peaks generated when the sample reaches cell 1 and cell 2. Results obtained with the proposed method are in satisfactory agreement with those provided by the standard method for the analysis of cyclamate samples.