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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.

An ultra-high performance liquid chromatography–high resolution mass spectrometry (UHPLC-HRMS) method was applied to the analysis of beers. Chromatographic profiles were recorded in both positive and negative electrospray modes and the resulting data was evaluated according to an untargeted metabolomic approach applied to the discrimination and characterization of beers. Principal components analysis and partial least squares–discriminant analysis were used for exploratory characterization and classification of beers according to the brewing procedure. Results showed that the chemometric models were able to distinguish among beer types. The study of MS features indicated that various detected markers were relevant for beer discrimination. Among them, for example, p-coumaric and ferulic acids, and 5-methylthioadenosine were tentatively identified as characteristic descriptors of Pilsner and Belgian ale beers, respectively. These findings were further confirmed by comparison of the UHPLC-HRMS data with those of standards and/or by MS/MS experiments. Conclusions extracted were validated on an independent set of samples. This work shows the applicability of untargeted metabolomic strategies to the characterization of beers at the molecular level. Copyright © 2016 The Institute of Brewing & Distilling

This paper describes the development and application of a simple, rapid and inexpensive chromatographic method to determine polyphenols in beers. The separation was optimized by experimental design considering both resolution and analysis time as the objectives. Figures of merit were established under the selected experimental conditions. In general, repeatabilities of peak areas were better than 2%, detection limits were in the order of magnitude of 0.01 mg L−1 and quantitative recovery percentages were about 100 ± 5. Differences in the polyphenolic composition among beer types were encountered to be relevant. This finding was exploited to perform the characterization of commercial beers using chemometric methods such as principal component analysis. The results indicated that coumaric and ferulic acids were more abundant in lager while syringic and gentisic acids were typical of some ale varieties. Concentrations of polyphenols in the samples were used to build classification models to discriminate among lager and ale classes. Models proved to be highly efficient in terms of sensitivity and specificity. It was found that all the samples were correctly assigned to their actual classes.

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.

A new analytical method for the determination of polyphenolic compounds in pear pulp was developed here. The procedure consisted of solvent extraction for the recovery of analytes and further quantification by reversed-phase high-performance liquid chromatography with multidetection by UV-Vis molecular absorption spectroscopy. Preliminary studies were focused on establishing a straightforward extraction procedure for soluble compounds using organic and hydro-organic media. Dimethylsulfoxide was selected as the most efficient extraction solvent for the diverse polyphenol families. The chromatographic separation relied on a methanol gradient which was optimized by experimental design. Figures of merit were established under the selected experimental conditions using synthetic standards and pear extracts. In general, repeatabilities of peak areas were better than 3%, detection limits were in the order of magnitude of 0.1 mg L−1 and quantitative recoveries were about 100%. The method was applied to analyze commercial pears of various origins with chlorogenic, neochlorogenic and gallic acids, arbutin and catechin being some of the most abundant compounds. Differences in the polyphenol composition among pear varieties were found to be relevant. As a result, such compounds may result in potential descriptors of varietal characteristics.

A flow injection method for the rapid determination of histamine in wines is proposed. This method is based on the reaction of the analyte with 1,2-naphthoquinone-4-sulfonate to form a derivative detected by UV-Vis absorption spectroscopy. The amine derivatization is carried out at pH 9.2 in a PTFE coil heated at 60°C. Experimental conditions are not able to provide selectivity for histamine with respect to other amino compounds so that its response is interfered with by other amines and amino acids present in the wine matrix. Here, multivariate calibration methods have been used to overcome the lack of selectivity. As a result, wines can be directly analyzed without removing interferences. For this purpose, spectra taken at the peak maximum have been used as multivariate data to be further analyzed with partial least square regression. The number of significant latent variables to be used in the construction of the calibration models as well as a preliminary evaluation of the prediction performance have ...

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 aC18 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.

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 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%.

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 of25 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.

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.

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.

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 °C and pH 9.2 for 5 min. The separation of derivatives has been accomplished in a C18 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.

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.

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.

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 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. This work was supported by the Spanish Ministerio de Ciencia y Tecnología (project CTQ2008-04776/BQU), Fundació Bancaixa (project P1-1B2010-50) and the Generalitat Valenciana (Research Group of Excellence, Prometeo/2009/054; PrometeoII/2014/023).

Ultrahigh pressure liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry (UHPLC-APPI-MS/MS) was applied to the analysis and authentication of fruit-based products and pharmaceutical preparations. Two sub-2 μm C18 reversed-phase columns, Syncronis (100 × 2.1 mm, 1.7 μm) and Hypersil Gold (50 × 2.1 mm, 1.9 μm), were proposed under gradient elution with 0.1% formic acid aqueous solution and methanol mobile phases for the determination of 29 polyphenols, allowing us to obtain polyphenolic profiles in less than 13.5 and 23.5 min, respectively. Several atmospheric pressure ionization (API) sources (H-ESI, APCI, and APPI) were compared. For dopant-assisted APPI, four organic solvents, toluene, acetone, chlorobenzene and anisole, were evaluated as dopants. Both H-ESI and acetone-assisted APPI were selected as the best ionization sources for the analysis of targeted polyphenols. Acceptable sensitivity (LOD values down to 0.5 μg kg−1 in the best of cases), linearity (r2 higher than 0.995) and good precision (RSD values lower than 15.1%) and trueness (relative errors lower than 10.2%) were obtained using both UHPLC-API-MS/MS methods. A simple extraction procedure, consisting of sample sonication with acetone/water/hydrochloric acid (70 : 29.9 : 0.1 v/v/v) and centrifugation, was used. The proposed UHPLC-ESI-MS/MS and UHPLC-APPI-MS/MS methods with both C18 reversed-phase columns were then applied to the analysis of 32 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic profile data were then analyzed by principal component analysis (PCA) to extract information on the most significant data contributing to the classification of natural extracts according to the type of fruit.

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%.

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.

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.

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%.

This paper aims at the characterization of acid–base properties of nucleoside analogs using a pH-gradient flow injection -diode array detector (FI-DAD) system and further data analysis with multivariate curve resolution. Drugs selected comprise nucleoside reverse transcriptase inhibitors (NRTIs) extensively used in AIDS treatment, including zidovudine (AZT), didanosine (ddl), stavudine (d4T) and zalcitabine (ddC). The FI system consists of a two-channel manifold in which the sample injection into an acid carrier is responsible of the generation of a pH gradient. A first step is focused on an accurate calibration of the pH-gradient shape inside the FI peak using nucleoside analogs of known p K a values as standard compounds. The pH gradient in the FI peak is estimated from the concentration profiles of standard compounds. In a second step, the pH-gradient profile is exploited for a rapid estimation of unknown p K a values of test drugs. Various approaches for calculating p K a values are evaluated using the peak front or peak tail gradient ranges. As a result, overall errors in p K a estimations are lower than 2.5%.

A flow-injection system with differential spectrophotometric detection is proposed for the simultaneous determination of aniline and cyclohexylamine based on their reaction with 1,2-naphthoquinone-4-sulfonate (NQS). The pH is chosen to achieve selectivity since only aniline reacts at acidic pH whereas the two amines are derivatized in basic medium. The flow manifold comprises two reactors and two detection cells for developing and monitoring the reaction under selective and general (non-selective) conditions. A double beam spectrophotometer is used for differential detection, with two flow cells placed in the sample and reference holders. Figures of merit such as sensitivity, linear range, detection limit and precision are established. The evaluation of accuracy using a series of synthetic mixtures indicates overall prediction errors of 3% and 5% for aniline and cyclohexylamine, respectively. The method is applied to the determination of amine impurities in commercial sweeteners. Good concordance between the proposed and the standard chromatographic methods is found.

A voltammetric method is proposed for the simultaneous determination of tryptophan, cysteine, and tyrosine using multivariate calibration techniques. Various electrodes and voltammetric techniques were explored to ascertain the optimum measurement strategy. Among them, differential pulse voltammetry (DPV) with a Pt electrode was selected as analytical technique since it provided a suitable compromise between sensitivity and reproducibility while allowing the oxidation peaks of the three compounds to be reasonably discriminated. The sensitivity of DPV with Pt electrode for Trp standards was 8.4×10−2 A l mol−1, the repeatability 3.7% and the detection limit below 10−7 M. The lack of full selectivity of the voltammetric data was overcome using multivariate calibration methods on the basis of the differences in the voltammetric waves of each compound. The accuracy of predictions was evaluated preliminarily from the analysis of three-component synthetic mixtures. Subsequently, this method was applied to the analysis of oxidizable amino acids in feed samples. Results obtained were in good concordance with those given by the standard method using an amino acid analyzer.

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.

The potentiality of artificial neural networks for multicomponent analysis in unresolved peaks from capillary electrophoresis (CE) is evaluated. The system chosen consists of mixtures of three ebrotidine metabolites, which cannot be successfully separated by CE. Data selected for analysis consist of UV spectra taken at the maximum of the CE peak. The most dissimilar analyte, in terms of spectral differences, is accurately quantitated in any type of mixture with an overall prediction error of 5%. Because of the strong interference of the two most overlapped compounds, a preliminary procedure for spectral data filtering based on principal component analysis is performed to improve their quantitation.

This paper describes a method for the determination of lysine based on a flow injection (FI) differential potentiometry system. The flow injection manifold is composed of a support electrolyte solution channel and a water channel acting as a carrier into which the sample solution is injected. The lysine biosensor was consisted of lysine oxidase chemically immobilized on a nylon membrane and attached to an all-solid-state ammonium electrode. A circular ammonium electrode was used as a reference. Hence, the possible interference of endogenous ammonium can be partly corrected by differential potentiometry. In order to increase the sensitivity of the response, the reaction was kinetically developed following a stopped-flow method. As a result, the sensitivity increased from 20 to 40 mV per decade when comparing the FI and the stopped-flow values. Furthermore, the peak-to-peak stopped-flow signals generated can be used as a more selective analytical response for lysine. The quantification of lysine in mixture samples containing small amounts of ammonium can be achieved with an acceptable accuracy, with prediction errors lower than 4%. However, when the ammonium concentration exceeded the lysine concentration, multivariate calibration with non-linear partial least squares (PLS) regression was needed to improve the lysine quantification, with an overall prediction error around 10%.

The determination of calcium and total hardness in natural waters is carried out with a potentiometric sensor array which consists of a series of ion-selective electrodes (ISEs) for Ca 2+ , Mg 2+ , NH 4 + , K + , Na + , Li + , and H + . The selectivity of the calcium and magnesium ISEs is not fully achieved as other cation species may interfere with the analysis. The proposed sensor array device can overcome this drawback since it can take advantage of the cross-selectivities of cation species towards each ISE. In this approach, the multivariate data generated by the sensor array results in a richer source of analytical information which allows the quantification of calcium and total hardness in the water samples by means of chemometric methods. Results obtained are in reasonable concordance with those given by the standard method based on complexometry.

A liquid chromatographic method with potentiometric detection using a lysine biosensor is proposed for the determination of lysine in pharmaceutical samples. The method is based on the separation of lysine from other species by micellar liquid chromatography and on-line neutralization of the eluent with a lithium hydroxide solution. This neutralization process is required to render the pH of the chromatographic effluent compatible with the enzymatic detection of lysine. The biosensor is composed of a lysine oxidase membrane attached to an ammonium electrode, in such a way that the ammonium generated in the enzymatic degradation of the substrate is monitored potentiometrically. The effect of experimental variables such as the acidity and composition of the mobile phase and the pH on the biosensor response is studied to optimize the detection conditions. Results obtained in the analysis of lysine in pharmaceutical preparations are compared with those obtained by the standard method. A good concorda...

Various types of glycosaminoglycans (GAGs) including heparins, chondroitin sulfates, dermatan sulfate and hyaluronic acid were studied from their proton nuclear magnetic resonance (1H NMR) spectra using chemometric techniques. Despite the complexity of the 1H NMR signals, data analysis using principal component analysis enabled the different GAG classes to be distinguished and permitted their classification according to their chemical structure. The analysis of the composition of the major disaccharide unit and other relevant chemical structures in the heparin samples was performed using partial least squares regression.

A potentiometric sensor array has been developed for the determination of lysine in feed samples. The sensor array consists of a lysine biosensor and seven ion-selective electrodes for NH4+, K+, Na+, Ca2+, Mg2+, Li+, and H+, all based on all-solid-state technology. The potentiometric lysine biosensor comprises a lysine oxidase membrane assembled on an NH4+ electrode. Because the selectivity of the lysine biosensor towards other cation species is not sufficient, there is severe interference with the potentiometric response. This poor selectivity can be circumvented mathematically by analysis of the richer information contained in the multi-sensor data. The sensor array takes advantage of the cross-selectivity of lysine for each electrode, which differs from the other species and quantification of lysine in complex feed sample extracts is accomplished with multivariate calibration methods, such as partial least-squares regression. The results obtained are in a reasonable agreement with those given by the standard method for amino acid analysis.

This paper examines the potentiality of in-capillary derivatization for improving the sensitivity of the spectrophotometric detection of amino acids in capillary zone electrophoresis. 1,2-Naphthoquinone-4-sulfonate was selected as the labeling agent of amino acids. The underivatized sample and the reagent solution segments are injected by pressure into the capillary prior to applying the running voltage. The corresponding derivatization reaction occurs inside the capillary once the potential is applied, as it induces mixing of the sample with the reagent. Several introduction modes consisting of tandem or sandwich configuration have been evaluated. These techniques result in a straightforward and automated way of carrying out a derivatization. Furthermore, in-capillary procedures may become much more attractive than conventional pre-capillary derivatization in terms of sensitivity and reproducibility. The optimum operation mode found consists of a sandwich system where the sample is injected in between two reagent segments. The method was applied to the determination of amino acids in feed samples. Results show a good concordance with those given by a standard amino acid analyzer.

This paper shows the possibilities of chemometric techniques for enhancing the analytical capabilities of conventional flow injection analysis (FIA). FIA is an excellent methodology for generating responses of different complexity from which relevant analytical information can be extracted chemometrically. The simplest case involves a single scalar datum per sample, as commonly occurs with FI peak heights or areas. More complex multivariate data consist of data vectors or matrices generated by taking a time array over the FI peak at a certain wavelength, or by recording the whole spectrum at a pre-selected time or at regular steps over the peak. The calibration procedures that can be applied to each type of data (i.e. scalars, vectors or matrices) in order to quantify the analyte in unknown samples are generically called zero-, first- and second-order calibration methods, respectively. The characteristics, advantages and shortcomings of each type of calibration are illustrated from various examples of quantitative determinations of amines and amino acids using 1,2-naphthoquinone-4-sulfonate as a derivatizing reagent. In addition, other FI methods using multivariate calibration procedures are also reviewed.

Artificial neural networks (ANN) have been applied to the resolution of overlapping capillary electrophoresis peaks of amino acid derivatives labelled with 1,2-naphthoquinone-4-sulfonate (NQS). The separation was performed with a fused-silica capillary and the corresponding 3D-electropherograms were recorded in a range from 225 to 550 nm with a diode array detector (DAD). Since complete resolution of all the analytes was not accomplished, a chemometric approach was used to improve the quantification mathematically. In the present case, a three-layer back propagation (BP) ANN with a sigmoid transfer function was built in order to perform the amino acid determination. The inputs of the ANN were the spectra or the electropherograms of each sample and the outputs were the concentrations of the amino acid derivatives in the overlapping peaks to be predicted. The results were compared with those from partial least squares regression (PLS) and, in general, ANN provided better predictions than PLS.

This paper shows the use of three-way data analysis methods to improve resolution and quantification in poorly separated peaks from capillary zone electrophoresis (CZE). Second-order data (data matrices) obtained with a diode array detector (DAD) are analyzed. There is a large overlap in the electrophoretic time domain and the data do not have a strict trilinear structure. These shortcomings might hinder the mathematical resolution. However, three-way methods allow the pure electrophoretic profiles of the analytes to be recovered in an unknown mixture, even in the presence of uncalibrated interferences. Furthermore, the analyte can be quantified in unknown mixtures with reasonable accuracy using pure standards. As an example, ebrotidine metabolites were chosen to test the method.

Capillary zone electrophoresis with diode-array detection was applied to the separation of ebrotidine and its metabolites. However, three of these, which are neutral in the conditions studied, co-migrated with the electroosmotic flow signal. Therefore, strongly overlapping peaks were observed. The main aim of this study was to show the potentiality of capillary electrophoresis in combination with chemometrics. Multivariate calibration methods were applied to quantify these analytes in synthetic mixtures. The results obtained using partial least squares (PLS) were in agreement with actual values, with an overall prediction error of 9.7%.

This paper illustrates the possibilities of chemometric methods in the resolution and quantification of various compounds in overlapping peaks from capillary electrophoresis. Ebrotidine and most of its metabolites were efficiently separated by capillary zone electrophoresis (CZE) in a fused-silica capillary. However, the procedure was not suitable for the physical separation of the three less ionizable metabolites, which comigrated and overlapped with the electroosmotic flow signal. Multivariate curve resolution based on an alternating least squares procedure was used for their mathematical resolution. For such a purpose, data obtained in the CZE system with a diode array detector, which consisted of UV spectra registered over time, were analyzed. The ebrotidine metabolites were successfully resolved and quantified in synthetic mixtures and urine samples.

This paper describes a contribution to Chemometrics and Intelligent Laboratory Systems Elsevier's data base of files. Mixtures of nucleosides and a pharmaceutical preparation composed of cytosine and inosine-5′-monophosphate are analyzed using three different pH-gradient spectrophotometric flow-injection and continuous flow systems. Results for one of the flow-injection systems and for the continuous-flow system are reported for the first time in this paper. Resolution and quantitation of the constituents in the mixtures are given using a multivariate curve resolution method based on a constrained alternating least squares optimization.

A voltammetric method for the simultaneous determination of oxidizable amino acids (cysteine, tyrosine and tryptophan) is proposed. The method is based on the analysis of multivariate data, which consists of cyclic voltammograms registered in the range from 400 to 1100 mV with a graphite–methacrylate composite electrode, by using multivariate calibration methods. Cyclic voltammograms of pure standards of these oxidizable amino acids show characteristic shapes. Thus, the resolution of mixtures of them by multivariate calibration lies in the differences in their voltammograms. The main difficulties encountered in these analyses are the high degree of overlapping and the lack of bilinearity of voltammetric data. The prediction abilities of principal component regression (PCR) and partial least squares regression (PLS) were assayed to the analysis of oxidizable amino acids in a series of synthetic samples. The quantification of cysteine and tryptophan was especially satisfactory since they are the most characteristic amino acids.

A 1H NMR method for the quantification of dermatan sulfate impurities in heparin industrial samples is proposed. The method is based on the analysis of 1H NMR spectral data by multivariate calibration. The 1H NMR spectra of heparin and dermatan sulfate standards showed characteristic profiles. Thus, differences in the methyl peaks of acetamido groups of heparin and dermatan sulfate were greatly advantageous for the analysis. Other hydrogens of the sugar ring were also relevant in this study. Thus, the determination of dermatan sulfate by multivariate calibration depended on all these differences. Partial least squares regression (PLS) was chosen as the calibration method. In addition, a data standardization procedure was developed in order that 1H NMR spectra registered with different instruments operating under different measurement conditions were comparable. The quantification of dermatan sulfate in the samples was satisfactory, with an overall prediction error of 6%.

A liquid chromatographic method for the determination of aniline in cyclamate sweeteners based on a pre-column derivatization with 1,2-naphthoquinone-4-sulfonate (NQS) is proposed. Aniline traces were extracted from the cyclamate samples using dichloromethane. After solvent evaporation, the dry residue was derivatized with NQS at pH 9.5 and 85 degrees C for 1 min. The aniline derivative, which was extracted from the reacting mixture, was redissolved in the eluent solution and injected into the chromatographic system. The separation of aniline derivative from other amine impurities was carried out in a C18 column using a 2% acetic acid-methanol (40:60, v/v) mobile phase. Results from the analysis of aniline in the sweetener samples with the proposed method were compared with those from the standard method. A good concordance between the two methods was observed.

The sequential determination of aniline and cyclohexylamine using flow-injection analysis and their simultaneous kinetic determination with a stopped-flow completely continuous flow (stopped-flow CCF) system are proposed. Both methods are based on the reaction of these amines with 1,2-naphthoquinone-4-sulfonate (NQS) for two measurement conditions: (i) under selectivity conditions for one of the analytes (aniline) and (ii) under non-selective reaction conditions in which both amines react simultaneously with NQS. The pH enabled both amines to be distinguished in the flow-injection method, while the difference in reaction rates of the two derivatives was exploited advantageously in the stopped-flow method. Therefore, aniline was directly quantified under selective conditions, whereas the determination of cyclohexylamine involved the calculation of its net analyte response. The figures of merit (sensitivity, linear range, detection limit and precision) of both methods were established. Several mixtures of aniline and cyclohexylamine were quantified using both flow-injection and kinetic methods. The overall prediction errors of the flow-injection method were 1.6% and 4.9% for aniline and cyclohexylamine, respectively. The accuracy of the kinetic procedure in the analysis of synthetic samples was 2.1% and 5.4% for aniline and cyclohexylamine, respectively. The method was applied to the determination of aniline and cyclohexylamine in commercial sweeteners. A good agreement between the proposed and the standard methods was found.

A new method for the determination of amino acids by capillary electrophoresis based on derivatization with 1,2-naphthoquinone4-sulfonate is developed. The separation of amino acid derivatives can be performed in a fused-silica capillary with a 40mM aqueous sodium tetraborate solution‐isopropanol background electrolyte solution (3:1, v/v) under an applied potential of 30 kV. Glutamic and aspartic acids and cysteine can be separated faster in a 20mM sodium tetraborate solution at 15 kV. Amino acid derivatives are spectrophotometrically detected at 480 nm. The precapillary derivatization is developed for 5 min at 65°C and pH 10. The linear range for lysine is up to 1.25  10 —3 M, the repeatability is 3.0%, and the detection limit is 0.67 pmol when injecting 22.5 nL. The method is applied to the determination of amino acids in pharmaceutical samples. Good agreement is found between the method proposed and the standard method.

A procedure for the rapid determination of mixtures of nucleic acid components from the analysis of spectrophotometric multivariate data obtained with continuous-flow and flow injection pH-gradient systems is proposed. Three flow systems have been developed and assayed in which an on-line pH gradient is generated from the mixing and controlled dispersion of acidic and basic titrant solutions. Quantitative determinations of any particular analyte in the unknown samples in the presence of interferences is performed with a single pure standard for this analyte. They are carried out using an alternating least squares multivariate curve resolution procedure. The methods proposed have been validated using synthetic and real sample mixtures. The results obtained are concordant with the labeled values, and the relative prediction errors are around 5%.

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at + 1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6 x 10 -4 M, the sensitivity 11300 μA/M, the repeatability 1.8%, the detection limit 8.2 X 10 -7 M and the response time t 95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.

A method for the determination of cyclamate in sweetener tablets is proposed. The method is based on the conversion of cyclamate to cyclohexylamine and the subsequent reaction with 1,2-naphthoquinone-4-sulfonate, yielding a spectrophotometrically active derivative which is detected at 480 nm. The hydrolysis step is performed batch wise by treatment of cyclamate with hydrogen peroxide and hydrochloric acid, while the cyclohexylamine derivatization is carried out in a flow-injection system. Conditions for both hydrolysis and derivatization have been optimised. The linear range is up to 10 −3 M cyclamate, the overall repeatability 3.1% and the detection limit is 7.7 × 10 −6 M. The accuracy of the method proposed in the analysis of commercial sweetener tablets is about 3.5%.

A voltammetric method for the determination of tryptophan (Trp) in feed samples using multivariate calibration techniques is proposed. The multivariate data consisted of the oxidation wave of cyclic voltammograms registered from 400 to 925 mV using a graphite–methacrylate composite electrode, at which Trp was oxidized around a potential of 900 mV. The interference from other oxidizable amino acids as well as the feed sample matrix was circumvented by using multivariate calibration methods. Principal component regression (PCR) and partial least squares regression (PLS) and non-linear PLS (NL-PLS) were checked for quantifying Trp in these samples. The results obtained using NL-PLS were in agreement with those obtained with the standard method for the determination of amino acids, with an overall prediction error of 7.9%.

A flow injection spectrophotometric method for the simultaneous determination of aniline and cyclohexylamine using multivariate calibration methods is proposed. The method is based on the reaction of these amines with 1,2-naphthoquinone-4-sulfonate, yielding spectrophotometrically active derivatives. The data analysed with multivariate calibration methods consisted of the spectra registered in the range 290–590 nm at the maximum of the flow injection peak. Although the spectrum of each derivative was characteristic, overlapping occurred and no selective wavelengths were found. The predictive abilities of principal component regression and partial least-squares regression (PLS), non-linear PLS, locally weighted regression (LWR) and artificial neural networks were examined for the determination of aniline and cyclohexylamine in sample mixtures. The accuracy for cyclohexylamine and aniline quantifications in unknown mixtures was optimum with LWR, providing overall prediction errors of 3.4 and 5.6%, respectively.

A new potentiometric method is proposed to determine lysine in pharmaceutical samples. This method is based on a lysine biosensor consisting of a chemically immobilized lysine oxidase membrane attached to an all-solid-state ammonium electrode. Lysine is degraded in the sensor to release ammonium, which is detected by means of the ammonium electrode. The presence of endogenous ammonium in the samples interferes with these determinations, since the response measured corresponds to the sum of the ammonium generated enzymatically and that present in the sample. This is a general drawback for all biosensors based on the detection of ammonium. Study of samples containing both lysine and ammonium showed that concentration ranges exist in which a near-logarithmic relationship between potentials measured and lysine concentrations is found. Therefore, within these ranges, lysine can be determined by using the standard addition method, with the subsequent data treatment involving an iterative linearization procedure. Results obtained with the proposed potentiometric method are consistent with those given by the standard method for amino acid analysis.

A new procedure for the quantitative determination of mixtures of nucleic acid components, based on continuous spectrophotometric acid-base titrations and multivariate curve resolution, is proposed. The procedure simultaneously takes into account the spectroscopic and acid-base properties of the compounds, which leads to a higher selectivity. Furthermore, quantitative determination of an analyte in a complex mixture is performed using a synthetic solution as standard containing only the analyte of interest. An intrinsic difficulty in the analysis of spectrometric titration data is the presence of rank deficiency due to closure for the mixtures of two or more compounds. An additional problem can be encountered in some mixtures if species spectra or species concentration profiles are practically identical (rank overlap). However, even in the presence of these rank difficulties, accurate quantitation with prediction errors lower than 5% was obtained. The presence of unknown and uncalibrated interferences in the samples does not affect the quantitative determination of the analyte of interest. The proposed procedure was successfully applied to the analysis of real samples (pharmaceuticals) using synthetic external standards.