Three-dimensional spectrochromatographic determination of chlorogenic acid in Melampyrum stenophyllum Boiss. extracts by parallel factor analysis.

INTRODUCTION: Co-elution is a common challenge in phytochemical chromatography. Full chromatographic separation often requires extensive optimization, long analysis times, and excessive solvent use. A viable alternative could be mathematical elution of analytes using three-dimensional decomposition. OBJECTIVES: This study aimed to develop a method to determine chlorogenic acid in Melampyrum stenophyllum Boiss. extracts without complete chromatographic separation, to validate the method, and to cross-validate assay results against a classical ultra-performance liquid chromatography (UPLC) method. METHODOLOGY: Ultra-performance liquid chromatography-photodiode array (UPLC-PDA) spectrochromatograms were arranged into a three-way data cube with dimensions of time, wavelength, and sample and then decomposed using parallel factor analysis to reveal chromatographic, spectral, and concentration profiles. The chromatographic and spectral profiles were used to identify chlorogenic acid in overlapping signals. The relative concentration profile was used to quantify it in the plant extract. The assay results were statistically compared with those from an in-house classical UPLC method. RESULTS: Chlorogenic acid was co-eluted at 1.45 min and quantified as 16.11 mg per gram dry weight of Melampyrum stenophyllum extracts (SD = 0.28), despite significant interference in a 4-min runtime. The analytical validity was confirmed by recovery calculations from standard solutions and standard addition samples (RSD < 2%), and the t-test resulted in a p-value of 0.09 (α = 0.05), indicating no significant difference between the results obtained from mathematical elution and chromatographic separation. CONCLUSION: Chlorogenic acid was quantified from plant material accurately despite the co-elution. Validation and cross-validation results support the method's applicability.

A novel strategy on the spectrochromatographic analysis of a quaternary mixture by parallel factor analysis model.

Poor chromatographic resolution is one of the main challenges in chromatographic analysis. Partially separated chromatographic peaks frequently occur, due to the nature of analytes and the demand for fast analysis using high flow rates and shorter columns. Modeling of chromatographic three-way data using suitable chemometric tools enables determining co-eluted peaks without using additional experimental efforts. In this paper, parallel factor analysis (PARAFAC) was applied to chromatographic data for the quantitative resolution of a quaternary mixture at the co-elution condition of acetaminophen, aspirin, ascorbic acid, and guaifenesin in a spectrochromatogram. The spectrochromatograms of the calibration set, validation set, and real samples were arranged as a three-way array. In the next step, the PARAFAC model was implemented to decompose the spectrochromatographic array into trilinear components, corresponding to spectral, chromatographic, and relative concentration profiles of the analytes. The chromatographic and spectral modes were used for the qualitative analysis of components, whereas the analytes in commercial tablets were quantified from their individual profiles in their concentration mode. This study indicated that the application of the PARAFAC model provided a novel strategy for determining overlapping peaks in a chromatogram to perform the analysis of multicomponent mixtures with reduced runtime and without additional efforts.

Two-way and three-way approaches to ultra high performance liquid chromatography-photodiode array dataset for the quantitative resolution of a two-component mixture containing ciprofloxacin and ornidazole.

Two-way and three-way calibration models were applied to ultra high performance liquid chromatography with photodiode array data with coeluted peaks in the same wavelength and time regions for the simultaneous quantitation of ciprofloxacin and ornidazole in tablets. The chromatographic data cube (tensor) was obtained by recording chromatographic spectra of the standard and sample solutions containing ciprofloxacin and ornidazole with sulfadiazine as an internal standard as a function of time and wavelength. Parallel factor analysis and trilinear partial least squares were used as three-way calibrations for the decomposition of the tensor, whereas three-way unfolded partial least squares was applied as a two-way calibration to the unfolded dataset obtained from the data array of ultra high performance liquid chromatography with photodiode array detection. The validity and ability of two-way and three-way analysis methods were tested by analyzing validation samples: synthetic mixture, interday and intraday samples, and standard addition samples. Results obtained from two-way and three-way calibrations were compared to those provided by traditional ultra high performance liquid chromatography. The proposed methods, parallel factor analysis, trilinear partial least squares, unfolded partial least squares, and traditional ultra high performance liquid chromatography were successfully applied to the quantitative estimation of the solid dosage form containing ciprofloxacin and ornidazole.

Comparative Application of PLS and PCR Methods to Simultaneous Quantitative Estimation and Simultaneous Dissolution Test of Zidovudine - Lamivudine Tablets.

In the development strategies of new drug products and generic drug products, the simultaneous in-vitro dissolution behavior of oral dosage formulations is the most important indication for the quantitative estimation of efficiency and biopharmaceutical characteristics of drug substances. This is to force the related field's scientists to improve very powerful analytical methods to get more reliable, precise and accurate results in the quantitative analysis and dissolution testing of drug formulations. In this context, two new chemometric tools, partial least squares (PLS) and principal component regression (PCR) were improved for the simultaneous quantitative estimation and dissolution testing of zidovudine (ZID) and lamivudine (LAM) in a tablet dosage form. The results obtained in this study strongly encourage us to use them for the quality control, the routine analysis and the dissolution test of the marketing tablets containing ZID and LAM drugs.

Fast determination of diphenhydramine hydrochloride in reconstitutable syrups by CWT, PLS AND PCR methods.

Diphenhydramine hydrochloride (DPH), a histamine H1-receptor antagonist, is widely used as antiallergic, antiemetic and antitussive drug found in many pharmaceutical preparations. In this study, a new reconstitutable syrup formulation of DPH was prepared because it is more stable in solid form than that in liquid form. The quantitative estimation of the DPH content of a reconstitutable syrup formulation in the presence of pharmaceutical excipients, D-sorbitol, sodium citrate, sodium benzoate and sodium EDTA is not possible by the direct absorbance measurement. Therefore, a signal processing approach based on continuous wavelet transform was used to determine the DPH in the reconstitutable syrup formulations and to eliminate the effect of excipients on the analysis. The absorption spectra of DPH in the range of 5.0-40.0 µg/mL were recorded between 200-300 nm. Various wavelet families were tested and Biorthogonal1.1 continuous wavelet transform (BIOR1.1-CWT) was found to be optimal signal processing family to get fast and desirable determination results and to overcome excipient interference effects. For a comparison of the experimental results obtained by partial least squares (PLS) and principal component regression (PCR) methods were applied to the quantitative prediction of DPH in the mentioned samples. The validity of the proposed BIOR1.1-CWT, PLS and PCR methods were achieved analyzing the prepared samples containing the mentioned excipients and using standard addition technique. It was observed that the proposed graphical and numerical approaches are suitable for the quantitative analysis of DPH in samples including excipients.

A new three-dimensional modeling of fluorescence excitation-emission measurements to explore the interaction of hydroxychloroquine and DNA, and to quantify their binding constant.

A new three-dimensional chemometric approach was introduced to explore the interaction of hydroxychloroquine (HCQ)-calf thymus deoxyribonucleic acid (DNA) and quantify binding constant using fluorescence excitation and emission measurements. The fluorescence excitation-emission spectra were recorded after gradual titration of HCQ with DNA. Then, the excitation and emission curves and relative concentrations of the drug and drug-DNA complex were quantitatively estimated using a three-dimensional model called Parallel Factor Analysis (PARAFAC) to a cubic fluorescence data array. The interaction of HCQ and DNA was predicted by applying newly modified Stern-Volmer equations to the relationship between the actual DNA concentration, and the HCQ concentration in the relative concentration profile of the PARAFAC model. In the PARAFAC application, the binding constants of the HCQ-DNA complex at 288, 298, and 310 K were found as 6.78 × 103, 5.07 × 103, and 3.74 × 103 L mol-1, respectively. From the temperature studies, the thermodynamic parameters (ΔS0= 3.528 J mol-1 K-1, ΔH0= -20.099 kJ mol-1 and ΔG0=-21.11, -21.12, and -21.19 kJ mol-1 at 288, 298, and 310 K, respectively) were calculated. The drug-DNA interaction is spontaneous due to negative ΔG0 values. The positive ΔS0 and negative ΔH0 values revealed the major role of the electrostatic force on the binding of HCQ to DNA. Assay results obtained from the proposed three-way modeling were compared to those provided by the traditional spectrofluorimetric method.

Development of a novel UPLC approach to co-prediction of four active compounds in a multi-component pharmaceutical preparation.

In this work, a new ultra-performance liquid chromatography method based on photodiode array detection (UPLC-PDA) was first developed for the quantitative analysis of the quaternary mixture of ascorbic acid (AA), paracetamol (PAR), caffeine (CAF) and chlorpheniramine maleate (CPA) in a commercial dosage form. The developed UPLC-PDA method offered a new possibility for the co-determination of four active ingredients in a drug combination with short run time and simple sample preparation. The successful chromatographic separation of the four drugs was performed using a Waters Acquity UPLC BEH C18 column (1.7 µm 2.1 × 100 mm) (Mildford, USA) and a mobile phase consisting of water (12 %), acetonitrile (13 %) and 0.1 M H3PO4 (75 %) at a flow rate of 0.25 mL/min. The validation of the proposed UPLC-PDA approach was verified by analyzing synthetic mixtures, inter- and intra-day experiments, and commercial powder samples and provided satisfactory results.

Continuous wavelet transforms for the simultaneous quantitative analysis and dissolution testing of lamivudine-zidovudine tablets.

Dissolution testing has a very vital importance for a quality control test and prediction of the in vivo behavior of the oral dosage formulation. This requires the use of a powerful analytical method to get reliable, accurate and precise results for the dissolution experiments. In this context, new signal processing approaches, continuous wavelet transforms (CWTs) were improved for the simultaneous quantitative estimation and dissolution testing of lamivudine (LAM) and zidovudine (ZID) in a tablet dosage form. The CWT approaches are based on the application of the continuous wavelet functions to the absorption spectra-data vectors of LAM and ZID in the wavelet domain. After applying many wavelet functions, the families consisting of Mexican hat wavelet with the scaling factor a=256, Symlets wavelet with the scaling factor a=512 and the order of 5 and Daubechies wavelet at the scale factor a=450 and the order of 10 were found to be suitable for the quantitative determination of the mentioned drugs. These wavelet applications were named as mexh-CWT, sym5-CWT and db10-CWT methods. Calibration graphs for LAM and ZID in the working range of 2.0-50.0 µg/mL and 2.0-60.0 µg/mL were obtained measuring the mexh-CWT, sym5-CWT and db10-CWT amplitudes at the wavelength points corresponding to zero crossing points. The validity and applicability of the improved mexh-CWT, sym5-CWT and db10-CWT approaches was carried out by the analysis of the synthetic mixtures containing the analyzed drugs. Simultaneous determination of LAM and ZID in tablets was accomplished by the proposed CWT methods and their dissolution profiles were graphically explored.

A multiway spectral analysis model to monitor the kinetic chlorination reaction of caffeine and determine its content in commercial liquid and solid drinks.

A multiway data analysis model, namely parallel factor analysis (PARAFAC) was proposed to decompose a three-way array of second-order kinetic UV measurements, for the chlorination reaction of caffeine with NaOCl, into a set of the spectra, time, and concentration matrices. The multiway resolution provided the simultaneous estimation of spectral, kinetic, and quantitative analysis of caffeine. The ability of the PARAFAC tool was checked by analyzing the validation samples in the presence of interferences. The added recovery and relative standard deviations for caffeine in the spiked samples were calculated as 99.1%-99.5% and 0.52%-1.34% for Iced Coffee Black liquid coffee (ICB), 99.5%-103.0% and 0.42%-1.03% for Jacobs Monarch Gold 100% Instant Coffee (JMG) and 99.5%-101.4% and 0.11%-0.13% for Çaykur Black Filter (Süzen) Bag Tea (BTB). Caffeine in commercial drinks was analyzed using the concentration matrices of the PARAFAC application. The PARAFAC results were statistically compared to those obtained by the developed UPLC method.

Three-dimensional strategies in the quantitative resolution of kinetic UV absorbance measurements for monitoring the oxidation of quercetin by oxidant agents and analyzing dietary supplement product.

Three-dimensional strategies involving the application of parallel factor analysis (PARAFAC) to the kinetic UV absorbance measurements were elaborated to monitor the oxidation of quercetin with oxidant agents (K2Cr2O7 and KIO3) and to quantify analyte in a dietary supplement product. Loadings (spectral, kinetic and concentration profiles) were obtained by the PARAFAC deconvolution. Spectral identification, kinetics and quantification of the relevant analyte in the presence of interferent(s) were performed. The elaborated chemometric strategies were carefully validated to demonstrate the capability of the method. Assay results of the PARAFAC strategies were statistically compared to that of the newly developed UPLC method.

A New Ultra-Performance Liquid Chromatographic Method for the Quantification of Vitamin C in Fresh and Dried Goji Berries (Lycium barbarum L.) Cultivated in Turkey.

BACKGROUND: The potential background of the study is related to comprehensive detection of the content of vitamin C with an actual chromatographic method. OBJECTIVE: Vitamin C is of vital importance in terms of human life and health due to its polyfunctional activity such as antioxidant activity and antiviral effect with other biological functions. In this regard, it may be necessary to update analytical methods or develop up-to-date analytical methods to accurately estimate the amount of vitamin C in natural samples. In this study, a new ultra-performance liquid chromatography with photodiode array detection (UPLC-PDA) method has been developed for the determination of vitamin C content in fresh and dried goji berries (Lycium barbarum L.), which are cultivated in Turkey. METHOD: The chromatographic elution of vitamin C in natural fruit samples was achieved on an ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 100 mm) column using methanol and 0.1 M H3PO4 pH 2.15 (20:80, v/v), which are mobile phase. UPLC determination was done at the 242.8 nm. Flow rate was 0.20 mL/min at a column temperature of 30°C. Linearity range of the calibration graph was found to be at 5-30 µg/mL. The validity of the newly developed UPLC method was tested by analyzing individual test samples and added samples. RESULTS: Applicability of the validated UPLC method was verified by the quantitative analysis of vitamin C content in both fresh and dried goji berries. CONCLUSIONS: We believe that the newly developed and validated UPLC method would be a useful and promising approach for simple quantitative analysis of goji berry samples for vitamin C. HIGHLIGHTS: In previous studies, no UPLC-PDA method was reported for the analysis of vitamin C in goji berries. The method provided a good repeatability for the analysis of real samples.

A new application of continuous wavelet transform to overlapping chromatograms for the quantitative analysis of amiloride hydrochloride and hydrochlorothiazide in tablets by ultra-performance liquid chromatography.

A new application of continuous wavelet transform (CWT) to overlapping peaks in a chromatogram was developed for the quantitative analysis of amiloride hydrochloride (AML) and hydrochlorothiazide (HCT) in tablets. Chromatographic analysis was done by using an ACQUITY ultra-performance LC (UPLC) BEH C18 column (50 x 2.1 mm id, 1.7 pm particle size) and a mobile phase consisting of methanol-0.1 M acetic acid (21 + 79, v/v) at a constant flow rate of 0.3 mL/min with diode array detection at 274 nm. The overlapping chromatographic peaks of the calibration set consisting of AML and HCT mixtures were recorded rapidly by using an ACQUITY UPLC H-Class system. The overlapping UPLC data vectors of AML and HCT drugs and their samples were processed by CWT signal processing methods. The calibration graphs for AML and HCT were computed from the relationship between concentration and areas of chromatographic CWT peaks. The applicability and validity of the improved UPLC-CWT approaches were confirmed by recovery studies and the standard addition technique. The proposed UPLC-CWT methods were applied to the determination of AML and HCT in tablets. The experimental results indicated that the suggested UPLC-CWT signal processing provides accurate and precise results for industrial QC and quantitative evaluation of AML-HCT tablets.

Fractional-continuous wavelet transforms and ultra-performance liquid chromatography for the multicomponent analysis of a ternary mixture containing thiamine, pyridoxine, and lidocaine in ampules.

New chemometric approaches based on the application of partial least squares (PLS) and principal component regression (PCR) algorithms with fractional wavelet transform (FWT) and continuous wavelet transform (CWT) are proposed for the spectrophotometric multicomponent determination of thiamine hydrochloride (B1), pyridoxine hydrochloride (B6), and lidocaine hydrochloride (LID) in ampules without any separation step. In this study PLS and PCR techniques were applied to the raw spectral data, FWT-coefficients, and FWT-CWT-coefficients. These calibration models were labeled as Raw-PLS and Raw-PCR, FWT-PLS and FWT-PCR, and FWT-CWT-PLS and FWT-CWT-PCR, respectively. A new ultra-performance liquid chromatographic (UPLC) method was developed for the comparison of the results obtained by applying the chemometric calibration methods. Chromatographic separation and determination of B1, B6, and LID in ampules were performed on an Acquity UPLC BEH C18 column (50x2.1 mm id, 1.7 pm particle size) using gradient elution with a mobile phase consisting of methanol and 0.01 M HCI at a constant flow rate of 0.6 mL/min. These combined chemometric calibrations and UPLC were validated by analyzing various ternary mixtures, B1, B6, and LID. The proposed chemometric approaches (signal processing-multivariate calibrations) and UPLC method were applied to the quantitative multicomponent analysis of marketed ampules containing the vitamins B1 and B6 with LID.

A Chemometric Strategy in the Development of a RP-UPLC Method for the Quantitative Resolution of a Two-Component Syrup Formulation.

A novel chemometric strategy was implemented in the development of a new ultraperformance liquid chromatography method for the quantitative estimation of guaifenesin and pseudoephedrine hydrochloride in a two-component syrup formulation with minimal experimental effort, time and reagent. A full factorial design with three factors was investigated to find optimal working conditions of chromatographic factors (column temperature, flow rate, and 0.1 M H3PO4% in mobile phase) that affect the chromatographic separation. Then, optimum experimental conditions providing adequate separation of the analyzed drug substances within the short runtime were determined. Under optimal experimental conditions, the retention times for guaifenesin and pseudoephedrine hydrochloride were obtained as 0.817 and 1.430 min, respectively. In the optimized RP-UPLC method, chromatographic response was reported as a linear function of concentration between 5.0 and 80.0 µg/mL for guaifenesin and 10.0-90.0 µg/mL for pseudoephedrine hydrochloride. The proposed method was carefully validated and successfully applied to quality control and analysis of a cough syrup preparation containing guaifenesin and pseudoephedrine hydrochloride. Consequently, the proposed reversed-phase ultraperformance liquid chromatography method provided an opportunity to quantify relevant drugs with small amount of reagents and short runtime.

Multiway resolution of spectrochromatographic measurements for the quantification of echinuline in marine-derived fungi Aspergillus chevalieri using parallel factor analysis.

A multiway resolution of incomplete chromatographic separation was presented for spectrochromatographic quantification of echinuline in marine-derived fungi Aspergillus chevalieri. Two-dimensional spectrochromatographic maps of calibration, validation and real samples were recorded as a function of time and wavelength using UPLC-PDA instrument under non-optimized chromatographic conditions, which gave rise to co-elution of echinuline and the constituents of sample matrix. A three-way array was obtained by concatenating the data matrices of the spectrochromatographic maps. Then, parallel factor analysis was applied to the multiway array to extract the individual contribution of echinuline in three modes (time, wavelength and sample). While time and wavelength profiles were used for the characterization of echinuline, the sample profile was used for its quantitative determination of the analyte in validation set and in real samples. Validity of the analytical method was evaluated by analyzing the validation set, which consist of test samples, standard addition samples, intra-day and inter-day samples. The proposed multiway analysis method was then applied to marine-derived fungi extracts and echinuline content was found to be 31.9 µg/g based on the average of ten assay results. The assay results provided by PARAFAC model were statistically compared with those obtained by a newly developed classical UPLC method, which ensured the complete separation of echinuline in a run time of nine minutes. The assay results were found to be comparable due to the fact that there was no significant difference between the analysis results (F = 1.63, Fcrit = 3.17; t = 0.69, tcrit = 2.11) at the significance level of 95%). Consequently, the PARAFAC method permitted the accurate determination of echinuline in fungal extracts despite the partial chromatographic separation with a run time of only three minutes.

Artificial neural network combined with principal component analysis for resolution of complex pharmaceutical formulations.

A chemometric approach based on the combined use of the principal component analysis (PCA) and artificial neural network (ANN) was developed for the multicomponent determination of caffeine (CAF), mepyramine (MEP), phenylpropanolamine (PPA) and pheniramine (PNA) in their pharmaceutical preparations without any chemical separation. The predictive ability of the ANN method was compared with the classical linear regression method Partial Least Squares 2 (PLS2). The UV spectral data between 220 and 300 nm of a training set of sixteen quaternary mixtures were processed by PCA to reduce the dimensions of input data and eliminate the noise coming from instrumentation. Several spectral ranges and different numbers of principal components (PCs) were tested to find the PCA-ANN and PLS2 models reaching the best determination results. A two layer ANN, using the first four PCs, was used with log-sigmoid transfer function in first hidden layer and linear transfer function in output layer. Standard error of prediction (SEP) was adopted to assess the predictive accuracy of the models when subjected to external validation. PCA-ANN showed better prediction ability in the determination of PPA and PNA in synthetic samples with added excipients and pharmaceutical formulations. Since both components are characterized by low absorptivity, the better performance of PCA-ANN was ascribed to the ability in considering all non-linear information from noise or interfering excipients.

Three-way analysis-based pH-UV-Vis spectroscopy for quantifying allura red in an energy drink and determining colorant's pKa.

Three-way analysis-based pH-UV-Vis spectroscopy was proposed for quantifying allura red in an energy drink product without the need for chromatographic analysis, and determining the colorant's pKa without using any titration technique. In this study, UV-Vis spectroscopic data matrices were obtained from absorbance measurements at five different pH levels from pH 8 to pH 12 and arranged as a three-way array (wavelength × sample × pH). In the three-way analysis procedure, parallel factor analysis (PARAFAC) was implemented to decompose the three-way array into a set of trilinear components. Each set of three components relates to spectral, pH and relative concentration profiles of allura red and sample matrix in the energy drink. First, UV-Vis spectra of the colorant's acid-base pair and sample's matrix were characterized by using the estimated spectral profile. Then, from the pH profile the pKa value was found to be 11.28 for the related colorant. Finally, allura red in energy drink samples was determined using the estimated concentration curve in the relative concentration profile. In the quantitation procedure, the working concentration range was 0.8-19.2 µg/mL. PARAFAC approach was tested in terms of selectivity, precision, and accuracy of the method. Added recovery results obtained by applying the proposed method to spiked samples were between 101.5% and 103.5%. In the application of the method to the analysis of real samples, successful results were reported. For a comparison, an ultra-performance liquid chromatographic method was developed for the quantitation of the colorant. Compared to the chromatographic method, we observed that PARAFAC model was simple and less expensive without requiring separation.

A New Application of PARAFAC Model to UPLC Dataset for the Quantitative Resolution of a Tri-Component Drug Mixture.

In the presented work, a three-way analysis of ultra-performance liquid chromatography-photodiode array (UPLC-PDA) dataset was performed by parallel factor analysis (PARAFAC) for quantitatively resolving a ternary mixture containing paracetamol and methocarbamol with indapamide selected as an internal standard in their co-eluted chromatographic conditions. Paracetamol and methocarbamol were quantified in the working range between 3-24 and 5-50 µg/mL by applying PARAFAC decomposition to UPLC-PDA data array obtained under unresolved chromatographic peak conditions. To compare the experimental results provided by co-eluted UPLC-PARAFAC method, an ordinary UPLC method was developed ensuring proper separation of the peaks. The performance of both PARAFAC and ordinary UPLC methods were assessed by quantifying independent test samples, intra- and inter-day samples and spiked samples of pharmaceutical preparations. Then, both methods were applied for quantitative estimation of the related drugs in a commercial pharmaceutical preparation. In this study, PARAFAC method was proved to be a very powerful alternative for the quality control of pharmaceutical preparations containing paracetamol and methocarbamol even in their co-eluted chromatograms with high precision and accuracy in a short chromatographic runtime of 1.2 min.

PARAFAC and MCR-ALS approaches to the pKa determination of benzoic acid and its derivatives.

In general, the identification of biological activities of a molecule requires the observation of its physicochemical characteristics with its molecular interactions in an organism. The acid-base ionization constant (or pKa) is one of the key parameters that shows the physicochemical behaviors of molecules used in pharmaceuticals, foods, cosmetics etc. Therefore, the development of new methods (or approaches) is necessary to get simple, rapid, inexpensive and reliable determination of the acidity constants of active and inactive ingredients used in commercial products. In this paper, new UV spectroscopic methods were developed for the first time, by applying parallel factor analysis (PARAFAC) and multivariate curve resolution-alternating least squares (MCR-ALS) to the pH-UV spectral data arrays for determining the pKa values of benzoic acid and its five derivatives (4-fluorobenzoic acid, thiosalicylic acid, anthranilic acid, phthalic acid, 4-aminobenzoic acid). The pH profiles obtained by the PARAFAC and MCR-ALS decomposition of the pH-UV data arrays were used for the quantitative estimation of the acid-base ionization constants for the investigated compounds without classical titration procedure. We concluded that the proposed PARAFAC and MCR-ALS provided us an opportunity for simple and rapid pKa determination of relevant compounds, which have functional importance in pharmaceutical and food industries.

New HPLC-chemometric approaches to the analysis of isoflavones in Trifolium lucanicum Gasp.

New HPLC-chemometric approaches were proposed for the simultaneous chromatographic quantification of daidzein, genistein, formononetin, and biochanin A in the samples consisting of the aerial parts of Trifolium lucanicum Gasp. (Leguminosae). Partial least squares and principal component regression algorithms were applied to the multiple chromatographic data set obtained by measuring at 240, 248, 256, and 264 nm to construct HPLC-partial least squares and HPLC-principal component regression calibrations. Chromatographic separation was carried out by using a mobile phase containing methanol, acetate buffer (pH=4.75) and acetonitrile (21:58:21, v/v/v) on the reversed phase column, Supelcosil™ LC-18 (15 cm×4.6 mm id). In addition, conventional HPLC based on the detection at a single wavelength was used for the determination of each compound in the extracts of T. lucanicum. The validity and applicability of the proposed HPLC-chemometric and conventional HPLC methods were performed by analyzing various synthetic plant samples. A good agreement was observed in the application of the proposed HPLC-chemometric tools to the synthetic and extracted samples of T. lucanicum.