Combining (1)H NMR spectroscopy and chemometrics to identify heparin samples that may possess dermatan sulfate (DS) impurities or oversulfated chondroitin sulfate (OSCS) contaminants

Heparin is a naturally produced, heterogeneous compound consisting of variably sulfated and acetylated repeating disaccharide units. The structural complexity of heparin complicates efforts to assess the purity of the compound, especially when differentiating between similar glycosaminoglycans. Recently, heparin sodium contaminated with oversulfated chondroitin sulfate A (OSCS) has been associated with a rapid and acute onset of an anaphylactic reaction. In addition, naturally occurring dermatan sulfate (DS) was found to be present in these and other heparin samples as an impurity due to incomplete purification. The present study was undertaken to determine whether chemometric analysis of these NMR spectral data would be useful for discrimination between USP-grade samples of heparin sodium API and those deemed unacceptable based on their levels of DS, OSCS, or both. Several multivariate chemometric methods for clustering and classification were evaluated; specifically, principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and the k-nearest-neighbor (kNN) method. Data dimension reduction and variable selection techniques, implemented to avoid over-fitting the training set data, markedly improved the performance of the classification models. Under optimal conditions, a perfect classification (100% success rate) was attained on external test sets for the Heparin vs OSCS model. The predictive rates for the Heparin vs DS, Heparin vs [DS+OSCS], and Heparin vs DS vs OSCS models were 89%, 93%, and 90%, respectively. In most cases, misclassifications can be ascribed to the similarity in NMR chemical shifts of heparin and DS. Among the chemometric methods evaluated in this study, we found that the LDA models were superior to the PLS-DA and kNN models for classification. Taken together, the present results demonstrate the utility of chemometric methods when applied in combination with (1)H NMR spectral analysis for evaluating the quality of heparin APIs.     

Determination of diosmin in pharmaceutical formulations using Fourier transform infrared spectrophotometry

A Fourier transform infrared (FT-IR) spectrometric method was developed for the rapid, direct measurement of diosmin in different pharmaceutical drugs. Conventional KBr-spectra were compared for best determination of active substance in commercial preparations. The Beer–Lambert law and two chemometric approaches, partial least squares (PLS) and principal component regression (PCR+) methods, were tried in data processing.     

FT-IR spectrophotometric analysis of dehydroepiandrosterone and its pharmaceutical formulation

A Fourier transform infrared (FT-IR) spectrometric method was developed for the rapid, direct measurement of dehydropeiandrosterone. Conventional KBr spectra and KBr + 2.0 mg microcrystalline cellulose (MCC) spectra were compared for best determination of active substance in drug formulation. Two chemometric approaches, partial least-squares (PLS) and principal component regression (PCR+) methods were used in data processing. The best results were obtained with PCR+ method.     

Dissolution and assaying of multicomponent tablets by chemometric methods using computer-aided spectrophotometer

Dissolution of three component tablets containing paracetamol (APAP), propyphenazone (PP), and caffeine (CAF) was carried out by USP paddle method. Three chemometric methods; inverse least square (ILS), principal component regression (PCR) and partial least squares (PLS) were applied to simultaneous assay of APAP, PP and CAF in tablets. The PCR, PLS and ILS methods were applied to simultaneous dissolution APAP, PP and CAF in tablets using a double beam UV-Vis spectrophotometer without any chemical separation and any graphical treatment of the overlapping spectra of three drugs. Twenty two mixture solutions in different concentrations were prepared in simulated gastric juice (SGJ, USP) for the chemometric calibrations as training set. The absorbance data matrix was obtained by measuring the absorbance at 14 wavelength points (from 222.5 to 292.5 nm) with the intervals of 5 nm (Deltalambda=5 nm) in the spectral region between 200 and 310 nm. Training set and absorbance data were used for the calibrations of chemometric methods. The developed calibrations were tested for the previously prepared solutions of mixture of three drugs for the validation of the assay method. The chemometric calculations were performed by using the 'MAPLE VRSQUO; software. The results of three chemometric methods were statistically compared with each other. These chemometric calibrations were successfully applied to the content uniformity and dissolution of the multicomponent tablets without any separation procedure. The synthetic mixtures of three drugs were used for the validity of the calibrations. Means recoveries (percent) and relative standard deviation of PLS, PCR and ILS methods were found to be 100.1+/-0.6, 101.4+/-1.6 and 100.1+/-0.6 for APAP; 100.9+/-3.2, 102.0+/-3.3 and 100.9+/-3.2 for PP; 99.9+/-3.5, 101.6+/-3.3 and 99.9+/-3.2 for CAF, respectively. Dissolution profiles of three component tablets were performed. More than 95% of drugs were dissolved within 15 min. All of the three-chemometric methods in this study can be satisfactorily used for the quantitative analysis and for dissolutions test of multicomponent dosage form.     

Spectrophotometric quantitative determination of cilazapril and hydrochlorothiazide in tablets by chemometric methods

Four chemometric methods were applied to simultaneous determination of cilazapril and hydrochlorothiazide in tablets. Classical least-square (CLS), inverse least-square (ILS), principal component regression (PCR) and partial least-squares (PLS) methods do not need any priori graphical treatment of the overlapping spectra of two drugs in a mixture. For all chemometric calibrations a concentration set of the random mixture consisting of the two drugs in 0.1 M HCI and methanol (1:1) was prepared. The absorbance data in the UV-Vis spectra were measured for the 15 wavelength points (from 222 to 276 nm) in the spectral region 210-290 nm considering the intervals of deltalambda = 4 nm. The calibration of the investigated methods involves only absorbance and concentration data matrices. The developed calibrations were tested for the synthetic mixtures consisting of two drugs and using the Maple V software the chemometric calculations were performed. The results of the methods were compared each other as well as with HPLC method and a good agreement was found.     

Simultaneous determination of Fe(II) and Fe(III) in pharmaceutical formulations with chromogenic mixed reagent by using principal component artificial neural network and multivariate calibration

The use of chemometric approaches for the simultaneous determination of Fe(II) and Fe(III) ions has been explored by means of a two component reagent. Mixed reagents of 1,10-phenanthroline and thiocyanate were used as a selective chromogenic system for speciation of Fe(II) and Fe(III). Although the complexes of Fe(II) and Fe(III) with mixed reagent show a spectral overlap, they have been simultaneously determined with chemometric approaches, such as principal component artificial neural network (PC-ANN), principal component regression (PCR) and partial least squares (PLS). A set of synthetic mixtures of Fe(II) and Fe(III) was evaluated and the results obtained by the applications of these chemometric approaches were discussed and compared. It was found that the PC-ANN and PLS methods afforded better precision relatively than its of PCR. PC-ANN and PLS methods were also applied satisfactorily in determination of Fe(II) and Fe(III) in pharmaceutical samples.     

Quality Control of Multi-Component,Intact Pharmaceutical Tablets with Three Different Near-Infrared Apparatuses

The purpose of this study was to develop a robust and versatile near infrared (NIR) analysis protocol for the quality control of intact tablets containing two active pharmaceutical ingredients, acetylsalicylic acid (ASA) and caffeine, as well as three excipients. Reference samples were prepared and a calibration model built for each apparatus. All components of the formulation were characterized by transmission measurements with NIR spectroscopy (NIRS). The study was performed with three different Fourier transform NIR apparatuses and chemometric models. Calibration was carried out by the partial least squares regression method and a pre-processing technique to optimize the efficiency of the models. High performance liquid chromatography was the reference method for obtaining active pharmaceutical ingredient concentration values used in model building. It also served as a reference for chemometric model validation. Eighteen samples were analyzed by chemometric modeling to predict each component's concentration. Four out of five ingredients were quantified precisely with the three chemometric models developed. ASA quantification uncertainty ranges were between 1.0 and 1.1%, and the average error was less than 5% for caffeine. More than 99.9% of tablet content were analyzed and quantified. The results show that a versatile in-line or at-line NIRS method, with three different chemometric models built from three different acquisition apparatuses, can be developed without sample preparation for pharmaceutical tablet quality control of existing products.     

Simultaneous spectrophotometric determination of cyproterone acetate and estradiol valerate in pharmaceutical preparations by ratio spectra derivative and chemometric methods

Ratio spectra derivative spectrophotometry and two chemometric methods (classical least squares, CLS and inverse least squares, ILS, were proposed for the simultaneous quantitative analysis of a binary mixture consists of cyproterone acetate (CA) and estradiol valerate (EV) in the commercial pharmaceutical preparations. In the ratio spectra derivative method, linear regression equations for both drugs were obtained by measuring the analytical signals at the wavelenghts corresponding to either maximums and minimums in the first derivative spectra of the ratio spectra. In the chemometric techniques, the concentration matrix was prepared by using the synthetic mixtures containing these drugs. The absorbance matrix corresponding to the concentration matrix was obtained by measuring the absorbances at 14 wavelengths in the range 220-290 nm for the zero-order spectra. Two chemometric calibrations were constructed by using the absorbance matrix and concentration matrix for the prediction of the unknown concentrations of CA and EV in their mixture. The numerical values were calculated by using 'MAPLE V' software. The accuracy and the precision of the methods have been determined and they have been validated by analyzing synthetic mixtures containing these two drugs. The proposed methods were successfully applied to a pharmaceutical formulation, sugar-coated tablet, and the results were compared with each other.     

Quantifying low levels of polymorphic impurity in clopidogrel bisulphate by vibrational spectroscopy and chemometrics

Vibrational spectroscopic methods were developed for quantitative analysis of Form II of clopidogrel bisulphate in Form I and Form II polymorphic mixtures. Results show that both IR and Raman spectroscopy combined with chemometrics are suitable to quantify low levels of Form II in Form I, down to 2 and 3%, respectively, with less than 1% limit of detection. Different preprocessing and multivariate methods were applied for spectral processing and were compared to find the best chemometric model. Common problems of quantitative vibrational spectroscopy in the solid phase are discussed; and procedures appropriate to eliminate them are proposed.     

Evaluation of chemometric techniques to select orthogonal chromatographic systems

Several chemometric techniques were compared for their performance to determine the orthogonality and similarity between chromatographic systems. Pearson's correlation coefficient (r) based color maps earlier were used to indicate selectivity differences between systems. These maps, in which the systems were ranked according to decreasing or increasing dissimilarities observed in the weighted-average-linkage dendrogram, were now applied as reference method. A number of chemometric techniques were evaluated as potential alternative (visualization) methods for the same purpose. They include hierarchical clustering techniques (single, complete, unweighted-average-linkage, centroid and Ward's method), the Kennard and Stone algorithm, auto-associative multivariate regression trees (AAMRT), and the generalized pairwise correlation method (GPCM) with McNemar's statistical test. After all, the reference method remained our preferred technique to select orthogonal and identify similar systems.     

Simultaneous spectrophotometric determination of paracetamol, phenylephrine and chlropheniramine in pharmaceuticals using chemometric approaches

Background and the purpose of the study

The linear multivariate calibration models such as principal components regression (PCR) and partial least squares regressions (PLS1 and PLS2) due to the mathematical simplicity and physical or chemical interpretability are sufficient and generally preferred method for analysis of multicomponent drugs. In this study, simultaneous determination of paracetamol, phenylephrine and chlorpheniramine in pharmaceuticals using chemometric methods and UV spectrophotometry is reported as a simple alternative technique.

Material and methods

Principal components regression (PCR) and partial least squares regressions (PLS1 and PLS2) were used for chemometric analyses of data obtained from the spectra of paracetamol, phenylephrine and chlorpheniramine between wavelengths of 200 to 400 nm at several concentrations within their linear ranges. The analytical performance of these chemometric methods were characterized by relative prediction errors and recoveries (%) and compared with each other.

Results

PCR, PLS1 and PLS2 were successfully applied to a tablet formulation, with no interference from excipients as indicated by the recovery. However, the PLS1 shows better results due to its flexibility and mathematical principals.

Conclusion

The proposed methods are simple and rapid requiring no separation step, and can be easily used as an alternative in the quality control of drugs.     

Investigation of the HPLC response of NSAIDs by fractional experimental design and multivariate regression analysis. Response optimization and new retention parameters

The optimization of the HPLC procedures and the investigation of the predictive models are generally seen as distinct tasks although they could be both approached by very similar chemometric methods.     

Spectrophotometric and chemometric determination of hydrochlorothiazide and spironolactone in binary mixture in the presence of their impurities and degradants

Hydrochlorothiazide (HCT) and spironolactone (SPR) are mostly co‐formulated in antihypertensive formulations. Several methods have been developed and validated for their determination; these methods include spectrophotometric and chemometric‐assisted spectrophotometric methods. The developed spectrophotometric methods were isosbestic point (ISO) and ratio subtraction (RS) methods. The absorbance values at 232.4 (λ_iso1) and 257.6 nm (λ_iso2) were used for determination of the total mixture concentration, while HCT could be directly determined at 317.2 nm (λ_max) and by subtraction SPR concentration could be obtained. Also SPR concentration could be calculated by RS method using the absorbance at 243.8 nm (λ_max). A wavelength selection method based on genetic algorithm (GAs) was developed and compared to the conventional partial least squares method (PLS). In this method, several parameters were adjusted and the optimum parameter settings were determined using experimental design. The developed chemometric methods were successfully applied for the determination of the HCT and SPR, as well as for determination of their impurities and degradation products. The proposed methods were successfully applied for determination of HCT and SPR in commercial tablets and they were statistically compared to each other and to the reported method. No significant difference was found, providing their accuracy and precision. Copyright © 2010 John Wiley & Sons, Ltd.     

Detection of trace crystallinity in an amorphous system using Raman microscopy and chemometric analysis

A novel analytical method to detect and characterize active pharmaceutical ingredient (API) trace crystallinity in an amorphous system using Raman microscopy and chemometric methods, namely band-target entropy minimization (BTEM) and target transformation factor analysis (TTFA) is developed. The method starts with Raman mapping measurements performed on some random areas of the amorphous system. This is followed by chemometric data analysis. In the case of a system without any a priori information, the BTEM algorithm is used to recover a set of pure component Raman spectral estimates followed by component and/or crystal structure identification. In the case of a system with some a priori information, TTFA is used to predict the presence or existence of a suspected component and/or crystal structure in the observed system.Four different amorphous systems were used as models. It is demonstrated that combined Raman microscopy and chemometric methods (BTEM or TTFA) outperformed powder X-ray diffraction (PXRD) in detecting trace crystallinity in amorphous systems. The spatial distributions of drug and polymer can also be directly obtained in order to study the homogeneity of the APIs in the solid dispersions. The present methodology appears very general and applicable to many other types of systems.     

Simultaneous spectrophotometric determination of chlorphenoxamine hydrochloride and caffeine in a pharmaceutical preparation using first derivative of the ratio spectra and chemometric methods

Three new methods are described for the simultaneous determination of chlorphenoxamine hydrochloride (CP) and caffeine (CAF) in their combination. In the first method, ratio spectra derivative spectrophotometry, analytical signals were measured at the wavelengths corresponding to either maxima and minima for both drugs in the first derivative spectra of the ratio spectra obtained by using each other spectra as divisor in their solution in 0.1 M HCl. In the other two methods, chemometric techniques, classical least-squares (CLS) and inverse least-squares (ILS), the concentration data matrix were prepared by using the synthetic mixtures containing these drugs in 0.1 M HCl. The absorbance data matrix corresponding to the concentration data matrix was obtained by the measurements of absorbances in the range 225-285 nm in the intervals with Deltalambda = 5 nm at 13 wavelengths in their zero-order spectra, then, calibration or regression was obtained by using the absorbance data matrix and concentration data matrix for the prediction of the unknown concentrations of CP and CAF in their mixture. The numerical values were calculated by using MAPLE V software in chemometric methods. The procedures do not require any separation step. The accuracy and the precision of the methods have been determined and they have been validated by analyzing synthetic mixtures containing title drugs. These three methods were successfully applied to a pharmaceutical formulation, sugar-coated tablet, and the results were compared with each other.     

Two new spectrophotometric approaches to the multicomponent analysis of the acetaminophen and caffeine in tablets by classical least-squares and principal component regression techniques

Classical least-squares (CLS) and principal component regression (PCR) techniques were proposed for the simultaneous analysis of tablets containing acetaminophen and caffeine without using a chemical separation procedure. The chemometric calibrations were prepared by measuring the absorbances values at the 15 wavelengths in the spectral region 215-285 nm and by using a training set of the mixtures of both drugs in 0.1 M HCI. The obtained chemometric calibrations were used for the estimation of acetaminophen and caffeine in samples. The numerical calculations were performed with the 'MAPLE V' software. By applying two techniques to synthetic mixtures, the mean recoveries and the relative standard deviations in the CLS and PCR techniques were found as 99.5 and 1.29, 99.7 and 1.00% for acetaminophen and 99.9 and 1.92, 100.0 and 1.178% for caffeine, respectively. Our results were compared with those obtained previously by one of us considering HPLC method as a reference method. These two methods were successfully applied to a pharmaceutical tablet formulation of two drugs.     

HPLC and chemometric methods for the simultaneous determination of cyproheptadine hydrochloride, multivitamins, and sorbic acid

Three methods are presented for the simultaneous determination of cyproheptadine hydrochloride (CP), thiamine hydrochloride (B1), riboflavin-5-phosphate sodium dihydrate (B2), nicotinamide (B3), pyridoxine hydrochloride (B6), and sorbic acid (SO). The chromatographic method depends on a high performance liquid chromatographic (HPLC) separation on a reversed-phase, RP 18 column. Elution was carried out with 0.1% methanolic hexane sulphonic acid sodium salt (solvent A) and 0.01 M phosphate buffer containing 0.1% hexane sulphonic acid sodium salt, adjusted to an apparent pH of 2.7 (solvent B). Gradient HPLC was used with the solvent ratio changed from 20:80 to 70:30 (over 9 min), then to 80:20 (over 11 min) for solvent A:B, respectively. Quantitation was achieved with UV detection at 220 and 288 nm based on peak area. The other two chemometric methods applied were principal component regression (PCR) and partial least squares (PLS). These approaches were successfully applied to quantify each drug in the mixture using the information included in the UV absorption spectra of appropriate solutions in the range 250-290 nm with the intervals Deltalambda = 0.4 nm at 100 wavelengths. The chemometric methods do not require any separation step. The three methods were successfully applied to a pharmaceutical formulation and the results were compared with each other.     

Simultaneous spectrophotometric determination of diclofenac potassium and methocarbamol in binary mixture using chemometric techniques and artificial neural networks

In this study, the simultaneous determination of diclofenac potassium (DP) and methocarbamol (MT) by chemometric approaches and artificial neural networks using UV spectrophotometry has been reported as a simple alternative to using separate models for each component. Three chemometric techniques-classical least-squares (CLS), principal component regression (PCR), and partial least-squares (PLS)-along with radial basis function-artificial neural network (RBF-ANN) were prepared by using the synthetic mixtures containing the two drugs in methanol. A set of synthetic mixtures of DP and MT was evaluated and the results obtained by the application of these methods were discussed and compared. In CLS, PCR, and PLS, the absorbance data matrix corresponding to the concentration data matrix was obtained by the measurements of absorbances in the range 260-310 nm in the intervals with Δλ = 0.2 nm in their zero-order spectra. Then, calibration or regression was obtained by using the absorbance data matrix and concentration data matrix for the prediction of the unknown concentrations of DP and MT in their mixtures. In RBF-ANN, the input layer consisting of 251 neurons, 9 neurons in the hidden layer, and 2 output neurons were found appropriate for the simultaneous determination of DP and MT. The accuracy and the precision of the four methods have been determined and they have been validated by analyzing synthetic mixtures containing the two drugs. The proposed methods were successfully applied to a pharmaceutical formulation containing the examined drugs.