Absorbance Data Matrix Research Articles

A novel eco‐friendly methods for simultaneous determination of aspirin, clopidogrel, and atorvastatin or rosuvastatin in their fixed‐dose combination using chemometric techniques and artificial neural networks

AbstractIn this study, the simultaneous determination of aspirin, clopidogrel, and either atorvastatin or rosuvastatin in their fixed‐dose combination (FDC) formulations has been reported. As a straightforward substitute for employing distinct models for each component, UV spectrophotometry was applied with chemometric approaches and artificial neural networks to achieve this. Three chemometric techniques, including principal component regression (PCR), partial least‐squares (PLS), and classical least‐squares (CLS), were applied in addition to the radial basis function‐artificial neural network (RBF‐ANN). The validation of a set of laboratory‐prepared combinations of aspirin, clopidogrel, and atorvastatin in one ternary mixture and aspirin, clopidogrel, and rosuvastatin in a second ternary mixture was assessed, and the results from the use of these approaches were recorded and compared. The absorbance data matrix matching the concentration data matrix in CLS, PCR, and PLS was created using measurements of absorbances in the range of 250–280 nm at intervals of 0.2 nm in their zero‐order spectra. Then, in order to forecast the unknown concentrations, calibration or regression was created utilizing the concentration and absorbance data matrices. Using RBF‐ANN for the simultaneous determination of aspirin, clopidogrel, and atorvastatin or rosuvastatin in their formulations was achieved by providing the input layer with 151 neurons; there are 2 hidden layers and 3 output neurons were obtained. The green profile of the developed methods has been assessed and compared with previously reported spectrophotometric methods. The suggested techniques were effectively applied to FDC dosage forms that contained the cited medications.

Chemometrics Assisted Spectrophotometric Method Development and Validation for Simultaneous Estimation of Abacavir, Lamivudine and Dolutegravir in Dosage Form

Abstract: Aim: Multivariate methods, particularly Classical Least Square, Inverse Least Square (ILS), Partial Least Square and Principal Component Regression are invented for the quantitation of the Abacavir sulphate (ABA), Lamivudine (LAM) and Dolutegravir sodium (DOL) in their combined tablet formulation. Materials and Methods: Chemometrics is the integration of statistical and mathematical approaches to analytical data in order to extract as much information as possible. The calibration and validation sets were built using fractional factorial design. 32 ternary mixtures of calibration sets and 16 mixtures of validation set were prepared. The absorbance data matrix was created by measuring absorbance in the range of 230 to 308 nm (Δλ = 3 nm) at 27 distinct wavelengths. The models were developed with the aid of MATLAB 2018a, Minitab 16.1.1 and MS Excel 2010. Results: The recovery values were close to 100% with low standard deviation (SD) justified the high accuracy of the proposed methods. For chemometrics approaches, the RMSECV, RMSEC, and RMSEP values produced show good accuracy and precision. The values of model diagnostic tools were lowest for ILS method. Conclusion: The ILS was shown to be the most appropriate method among produced chemometric models. The chemometric method is more accurate and precise than conventional methods as the total absorbance of the ternary mixture was measured and can be utilised in simultaneous estimation. With great recoveries and precision, the proposed approach was successfully used to the assay of formulation. Keywords: Abacavir sulphate, Lamivudine, Dolutegravir sodium, Chemometrics, Spectophotometric, Validation.

Coupling of multivariate curve resolution-alternating least squares and mechanistic hard models to investigate antibody purification from human plasma using ion exchange chromatography

A two steps proposal for the purification of immunoglobulin G from human blood plasma is investigated. The first step is precipitation using cold ethanol based on the Cohn method with some modification and the second step is a chromatographic separation by DEAE-Sepharose FF resin as a weak anion exchanger. The presence of interferent in the region3 of chromatographic fractions, which is co-eluted with IgG, restricts the application of the mechanistic chromatography model. Therefore, multivariate cure resolution-alternating least squares (MCR-ALS) as a soft method is employed on measured absorbance data matrix from eluted fractions to recover pure concentration and spectral profiles. Besides, possible solutions for resolved concentration and spectral profiles are investigated. The reaction-dispersive model as a mechanistic hard model for the column is utilized for the evaluation of the ion exchange chromatography. Using a genetic algorithm as a global optimization method, mobile phase modulator (MPM) adsorption model parameters such as β, kdes,0, and Keq,0, were fitted to the concentration profiles from MCR-ALS as 1.96, 2.87×10−4 m3 mol−1s−1, and 1883, respectively. Furthermore, a new resampling incorporated non-parametric statistics is conducted to assess parameters’ uncertainty. Values of 2.00, 1.10×10−3 m3 mol−1s−1, and 549.80 are estimated median, and values of 0.05, 2.5×10−3, and 691.00 are calculated interquartile range (IQR) for β, kdes,0, and Keq,0, respectively. Finally, results show three and two outliers for β and kdes,0, respectively.

Application of PCR and PLS Tools for the Simultaneous Quantification of Praziquantel and Ivermectin Binary Mixtures in Veterinary Tablets

Two chemometric calibration methods, principal component regression (PCR) and partial least squares regression (PLS) were proposed for the simultaneous spectrophotometric determination of praziquantel (PRA) and ivermectin (IVE) in a marketed veterinary formulation without using preliminary separation step. UV spectra of calibration set and samples including PRA and IVE were recorded in the spectral region of 225-315 nm. PCR and PLS algorithms were applied to absorbance data matrix and concentration set including PRA and IVE in the linear concentration range of 20.0-160.0 µg/mL for PRA and 2.0-44.0 µg/mL for IVE. The capability of the PCR and PLS methods were validated by analyzing validation samples. Assay results showed that both PCR and PLS approaches provided an opportunity for quantifying PRA and IVE in veterinary tablet formulation.

Chemometrics-Assisted Spectrophotometric Method Development and Validation for Simultaneous Estimation of Emtricitabine, Tenofovir Alafenamide Fumarate, and Dolutegravir Sodium in Dosage Form

Aim: This study aims on the development of a chemometric-assisted spectroscopic method for the analysis of combined dosage form of emtricitabine (EMT), tenofovir alafenamide fumarate (TEN), and dolutegravir sodium (DOL). The use of a multivariate algorithm to analyse spectrophotometric data is a novel approach to estimating drug concentrations in formulations. Materials and Methods: The quantitative estimation of EMT, TEN, and DOL in tablets was carried out using four chemometric approaches: Classical least square (CLS), inverse least square, partial least square, and principal component regression. Thirty-two ternary mixtures of calibration sets and 16 mixtures of validation sets were prepared. The absorbance data matrix was attained by calculating absorbance at 25 different wavelengths in a range of 240–336 nm (Δλ = 4 nm). The chemometric calculations were performed using Matlab2018a and Minitab software. The developed methods were validated. Results: The great accuracy of the current study was justified by the near-perfect recovery values (100%) and low standard deviation. For chemometrics approaches, the root mean square error of calibration (RMSEC), root mean square error of prediction (RMSEP), and root mean square error of cross-validation (RMSECV) outcomes display decent accuracy and precision. Conclusion: The CLS approach yielded the lowest predicted residual error sum of squares, RMSEC, RMSEP, and RMSECV scores. As a result, CLS might be regarded as the best chemometric approach among all techniques utilized. The label claim determined is in excellent accordance with the mean recoveries for EMT, TEN, and DOL. So, it can be used in quality control laboratories.

SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF PHENYLEPHRINE HYDROCHLORIDE AND NAPHAZOLINE HYDROCHLORIDE IN EYE DROPS BY CHEMOMETRIC TECHNIQUES AND ARTIFICIAL NEURAL NETWORK

Three multivariate calibration-prediction techniques, partial least squares (PLS), principal component regression (PCR) and artifi cial neural networks (ANN), have been applied without separation in the spectrophotometric multi-component analysis of phenylephrine hydrochloride and naphazoline hydrochloride. A set of 25 synthetic mixtures of phenylephrine hydrochloride and naphazoline hydrochloride has been evaluated to determine the predictability of PLS, PCR and ANN. The absorbance data matrix was obtained by measuring zero-order absorbances between 230-300 nm at intervals of 3 nm. The suitability of the models was determined on the basis of root mean square error (RMSE), root mean squared cross validation error (RMSECV) and root mean squared prediction error (RMSEP) values of calibration and validation data. The results showed a very good correlation between true values and the predicted concentration values. Therefore, the methods developed can be used for routine drug analysis without chemical pre-treatment.

Liquid chromatographic and spectrophotometric determination of diflucortolone valerate and chlorquinaldol in creams

A new reversed phase high pressure liquid chromatography and a spectrophotometric method are proposed for the simultaneous determination of diflucortolone valerate (DIF) and chlorquinaldol (CHL) in creams. An isocratic system consisted of an ACE® C18 analytical column and a mobile phase composed of methanol/phosphate buffer (pH 5.5, 0.1 M) (95:5, v/v) at a flow rate 1.0 mL/min was used for the optimal chromatographic separation using UV detection at 220 nm. Ephedrine hydrochloride was used as internal standard. Principal component regression (PCR) was used as the chemometric technique in spectrophotometry. In this technique, the concentration data matrix was prepared by using the synthetic mixtures containing these drugs in methanol/water (3:1). The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbances at 60 wavelengths in the range of 230-348 nm with appropriate interval Δλ of 2 nm for DIF and CHL in the zero-order spectra for the binary combinations. The linear ranges were found 2.40-300 μg/mL for DIF, 0.72-240 μg/mL for CHL for LC method, and 0.88-3.0 μg/mL for DIF and 1.0-11.2 μg/mL for CHL for PCR method. The accuracy, precision and the linear ranges of the methods have been evaluated and they have been validated by analyzing synthetic mixtures containing the title drugs. These two methods were successfully applied to two pharmaceutical cream preparations and the results were compared with each other.

DEVELOPMENT AND VALIDATION OF CHEMOMETRIC-ASSISTED UV-SPECTROPHOTOMETRIC METHOD FOR EPIGALLOCATECHIN GALLATE AND CURCUMIN IN TABLET FORMULATION

A chemometric method, Partial Least Square, was applied for the simultaneous estimation of epigallocatechin gallate and curcumin in tablet formulation. Twenty five mixed sample solutions were prepared for chemometric calibration as training set and sixteen mixed solution for validation set using Full Factorial Design. The absorbance data matrix was obtained by measuring absorbance at 20 different wavelengths in the range of 220 to 410 nm (Δλ = 10 nm). The developed calibration data was used to test tablet formulation containing epigallocatechin gallate and curcumin. The developed methods were validated using RMSECV and RMSEP. The chemometric calculations were performed using Minitab 16.1.1 and Microsoft Excel 2010. The method is also more accurate and precise than conventional UV methods.

DEVELOPMENT AND VALIDATION OF A CHEMOMETRICS ASSISTED SPECTROSCOPIC METHOD FOR THE SIMULTANEOUS ESTIMATION OF GALLIC ACID, ELLAGIC ACID AND CURCUMIN IN POLYHERBAL ANTIDIABETIC FORMULATIONS

Two chemometric methods, Inverse Least Square (ILS) and Classical Least Square (CLS), were applied for the simultaneous estimation of gallic acid, ellagic acid and curcumin in polyherbal antidiabetic formulation. Twenty mixed solutions were prepared for the chemometric calibration as training set and 10 mixed solutions were prepared as validation set. The absorbance data matrix was obtained by measuring the absorbance at 20 different wavelengths, from 241 to 279 nm with the interval of 2 nm (Δλ= 2 nm). The developed calibrations were successfully tested for three antidiabetic polyherbal formulations for their gallic acid, ellagic acid and curcumin contents. Developed methods were validated and root mean square error of precision (RMSEP) was determined. Both chemometric methods in this study can be satisfactorily used for the quantitative analysis in polyherbal dosage forms. The chemometric calculations were performed by using the chemometrics toolbox with MATLAB R2015a software.

SIMULTANEOUS ESTIMATION OF TELMISARTAN, HYDROCHLOROTHIAZIDE AND AMLODIPINE BESYLATE IN TABLET PREPARATION BY CHEMOMETRIC ASSISTED SPECTROPHOTOMETRIC ANALYSIS

A chemometric method was developed by application of Partial Least Square regression to for the simultaneous estimation of telmisartan, hydrochlorothiazide and amlodipine besylate in tablet preparations. Calibration set was prepared considering seven sets; each set with twenty-four mixed solutions and twenty-one ternary mixed solutions, were prepared as a validation set. The absorbance data matrix for training set was obtained by recording absorbance within wavelength range 220-320 nm at 2nm intervals. The developed method was validated according to ICH Q2 (R1) guidelines and results were reported. The developed and validated multivariate method was successfully tested for laboratory mixtures as well as commercial tablet formulation of telmisartan, hydrochlorothiazide and amlodipine besylate.

Development and Validation of Chemometric Spectrophotometric Methods for Simultaneous Determination of Simvastatin and Nicotinic Acid in Binary Combinations.

The development and introduction of combined therapy represent a challenge for analysis due to severe overlapping of their UV spectra in case of spectroscopy or the requirement of a long tedious and high cost separation technique in case of chromatography. Quality control laboratories have to develop and validate suitable analytical procedures in order to assay such multi component preparations. New spectrophotometric methods for the simultaneous determination of simvastatin (SIM) and nicotinic acid (NIA) in binary combinations were developed. These methods are based on chemometric treatment of data, the applied chemometric techniques are multivariate methods including classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS). In these techniques, the concentration data matrix were prepared by using the synthetic mixtures containing SIM and NIA dissolved in ethanol. The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbance at 12 wavelengths in the range 216 - 240 nm at 2 nm intervals in the zero-order. The spectrophotometric procedures do not require any separation step. The accuracy, precision and the linearity ranges of the methods have been determined and validated by analyzing synthetic mixtures containing the studied drugs. Chemometric spectrophotometric methods have been developed in the present study for the simultaneous determination of simvastatin and nicotinic acid in their synthetic binary mixtures and in their mixtures with possible excipients present in tablet dosage form. The validation was performed successfully. The developed methods have been shown to be accurate, linear, precise, and so simple. The developed methods can be used routinely for the determination dosage form.

SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF DICLOFENAC SODIUM, PARACETAMOL, AND CHLORZOXAZONE IN TERNARY MIXTURE USING CHEMOMETRIC AND ARTIFICIAL NEURAL NETWORKS TECHNIQUES

Objective: The aim of this study is to develop and validate simple, accurate, and precise spectrophotometric methods for the simultaneous determination of diclofenac sodium (DIC), paracetamol (PAR), and chlorzoxazone (CHZ) in ternary mixture using chemometric and artificial neural networks (ANN) techniques.Methods: Three chemometric techniques include classical least squares (CLS), principal component regression (PCR), and partial least squares (PLS) in addition to cascade-forward backpropagation ANN (CFBP-ANN) were prepared using the synthetic mixtures containing the three drugs in methanol. In CLS, PCR, and PLS, the absorbances of the synthetic mixtures in the range 267-295 nm with the intervals Δλ=0.2 nm in their zero-order spectra were selected. Then, calibration or regression was obtained using the absorbance data matrix and concentration data matrix for the prediction of the unknown concentrations of DIC, PAR, and CHZ in their mixtures. In CFBP-ANN, two layers, sigmoid layer with 10 neurons and linear layer were found appropriate for the simultaneous determination of the three drugs in their ternary mixture.Results: The four proposed methods were successfully applied to the analysis of the three drugs in laboratory prepared mixtures and tablets with good percentage recoveries in the range of 98-102%. Relative standard deviation for the precision study was found <1%.Conclusion: The four proposed methods showed simplicity, accuracy, precision, and rapidity making them suitable for quality control and routine analysis of the cited drugs in ternary mixtures and pharmaceutical formulation containing them.

Multivariate Chemometric Models and Application of Genetic Algorithm for Simultaneous Determination of Ledipasvir and Sofosbuvir in Pure Form and in Pharmaceutical Preparation; A Comparative Study

Objectives: Four multivariate chemometric methods have been developed for simultaneous determination of sofosbuvir and ledipasvir in their pure and pharmaceutical dosage forms. Methods: Firstly, partial least squares and artificial neural network have been applied for the quantitative analysis of the studied drugs. Results: Experimental design of different synthetic mixtures of sofosbuvir and ledipasvir in different ratios has been done. The zero-order absorption spectra of these prepared mixtures have been recorded over the wavelength range 200-400 nm with 1 nm interval. The obtained absorbance and concentration data matrix have been utilized to obtain calibration or regression analysis data which has been used for the prediction of the unknown concentrations of each drug in their mixtures. Alternatively, the application of genetic algorithm to partial least squares and artificial neural network has been done and greatly increased the precision and predictive ability of the methods. The four methods have been successfully applied to quantify sofosbuvir and ledipasvir in the real market sample. Conclusion: The investigated methods have been found to be accurate, precise and could resolve the overlapped spectra of the mixture without any preliminary separation steps.

Simultaneous spectrophotometric determination of glimepiride and pioglitazone in binary mixture and combined dosage form using chemometric-assisted techniques

In the present work, pioglitazone and glimepiride, 2 widely used antidiabetics, were simultaneously determined by a chemometric-assisted UV-spectrophotometric method which was applied to a binary synthetic mixture and a pharmaceutical preparation containing both drugs. Three chemometric techniques – Concentration residual augmented classical least-squares (CRACLS), principal component regression (PCR), and partial least-squares (PLS) were implemented by using the synthetic mixtures containing the two drugs in acetonitrile. The absorbance data matrix corresponding to the concentration data matrix was obtained by the measurements of absorbencies in the range between 215 and 235nm in the intervals with Δλ=0.4nm 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 pioglitazone and glimepiride in their mixtures. The described techniques have been validated by analyzing synthetic mixtures containing the two drugs showing good mean recovery values lying between 98 and 100%. In addition, accuracy and precision of the three methods have been assured by recovery values lying between 98 and 102% and R.S.D. % ˂0.6 for intra-day precision and ˂1.2 for inter-day precision. The proposed chemometric techniques were successfully applied to a pharmaceutical preparation containing a combination of pioglitazone and glimepiride in the ratio of 30: 4, showing good recovery values. Finally, statistical analysis was carried out to add a value to the verification of the proposed methods. It was carried out by an intrinsic comparison between the 3 chemometric techniques and by comparing values of present methods with those obtained by implementing reference pharmacopeial methods for each of pioglitazone and glimepiride.

Development and Validation of Chemometric-Assisted Spectrophotometric Methods for Simultaneous Determination of Phenylephrine Hydrochloride and Ketorolac Tromethamine in Binary Combinations.

The present work describes new spectrophotometric methods for the simultaneous determination of phenylephrine hydrochloride and ketorolac tromethamine in their synthetic mixtures. The applied chemometric techniques are multivariate methods including classical least squares, principal component regression, and partial least squares. In these techniques, the concentration data matrix was prepared by using the synthetic mixtures containing these drugs dissolved in distilled water. The absorbance data matrix corresponding to the concentration data was obtained by measuring the absorbances at 16 wavelengths in the range 244-274 nm at 2 nm intervals in the zero-order spectra. The spectrophotometric procedures do not require any separation steps. The accuracy, precision, and linearity ranges of the methods have been determined, and analyzing synthetic mixtures containing the studied drugs has validated them. The developed methods were successfully applied to the synthetic mixtures and the results were compared to those obtained by a reported HPLC method.

SIMULTANEOUS ESTIMATION OF EMITRICITABINE, EFAVIRENZ, TENOFOVIR DISPROXIL FUMARATE BY CHEMOMETRY AND REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A chemometric ultra-violet spectrophotometric method of analysis, Partial Least Square (PLS) method was applied to simultaneous assay of emitricitrabine, efavirenz and tenofovir disoproxil fumarate (DF) in their combined dosage tablet formulation. For comparison of this chemometric method a HPLC method for simultaneous determination of emitricitabine, efavirenz and tenofovir DF in combination was developed. Twenty one mixed solutions were prepared for chemometric calibration set and twenty one ternary mixtures were prepared as validation sets. The absorbance data matrix for training set was obtained by recording absorbance within wavelength range 230-290 nm at 2nm intervals. The developed multivariate and HPLC methods were successfully tested for laboratory mixtures as well as commercial tablet formulation of emitricitrabine, efavirenz, tenofovir DF.

Photodegradation study of nystatin by UV-Vis spectrophotometry and chemometrics modeling.

Nystatin, one of the tetraene antifungal antibiotics, is very sensitive to light. Thus, when nystatin is exposed to natural daylight, it is photodegraded to products of lower biological activity. In this work, the photodegradation kinetics of nystatin was monitored by a UV-Vis spectrophotometry method. The absorbance spectra of the nystatin, exposed to a 366 nm UV lamp, were recorded at different periods of time. By application of factor analysis to the absorbance data matrix, three absorbing chemical species, coexisting in the reaction system, were detected. The soft-modeling multivariate curve resolution-alternating least squares analysis of the evolutionary absorbance data revealed that nystatin undergoes photodegradation in a two-step consecutive manner. Hard-modeling data analysis suggested that reaction has first-order kinetics in the first step and zero-order kinetics in the second step. The reaction rate constants of the first and second steps were estimated as 0.0929 (+/-0.0076) and 0.0052 (+/-0.0016)/min, respectively. Finally, the pure spectra of the resolved chemical species were calculated.

Application of rank annihilation factor analysis to the spectrophotometric determination of the formation constant of complex of a new synthesized tripodal ligand with Co2+

The complex formation between a new synthesized tripodal ligand (L22py) and the cation Co2+ in water was studied spectrophotometrically using rank annihilation factor analysis (RAFA). According to molar ratio data the stoichiometry of complexation between the ligand and the cation Co2+ was 1:1. Formation constant of this complex was derived using RAFA on spectrophotometric data. In this process the contribution of ligand is removed from the absorbance data matrix when the complex stability constant acts as an optimizing object and simply combined with the pure spectrum of ligand, the rank of original data matrix can be reduced by one by annihilating the information of the ligand from the original data matrix. The effect of ethanol, dimethylformamide (DMF) and acetonitrile (AN) was investigated on the formation constant of the Co2+ complex. Complex formation constant in water was estimated as log Kf=5.09±0.02. In mixtures of solvents of water and DMF and water and AN, the formation constant of the complex was increased because of lowering donor number of the solvent and in mixture of water and ethanol, the complex formation constant was decreased because of lowering of dielectric constant of the solvent.

Simultaneous spectrophotometric and liquid chromatographic determination of dextromethorphan hydrobromide, phenylephrine hydrochloride, and carbinoxamine maleate in pharmaceutical preparations

Simultaneous determination of dextromethorphan hydrobromide (DEX), phenylephrine hydrochloride (PHEN), and carbinoxamine maleate (CAR) in pharmaceutical preparations was performed by using liquid chromatograpy (LC) and spectrophotometry. In LC, the separation was achieved on a C18 column and the optimal mobile phase for satisfactory separation in a gradient elution program was found to be acetonitrile-sodium perchlorate solution (5: 95, v/v) initially, then a linear gradient up to 60% acetonitrile in 8 min. In spectrophotometric method, a chemometric technique, principal component regression (PCR), was used. In the method, the absorbance data matrix corresponding to the concentration data matrix was obtained by the measurement of absorbances in their zero order spectra by Δλ = 1 nm in the 210–300 nm range. Then, the calibration was obtained by using this data matrix for the prediction of unknown concentrations of DEX, PHEN, and CAR in their ternary mixture. The methods proposed were validated and successfully applied to a pharmaceutical preparation, capsule, and the results were compared.

Liquid Chromatographic and Spectrophotometric Determination of Diflucortolone Valerate and Isoconazole Nitrate in Creams

A new RP-LC method and two new spectrophotometric methods, principal component regression (PCR) and first derivative spectrophotometry, are proposed for simultaneous determination of diflucortolone valerate (DIF) and isoconazole nitrate (ISO) in cream formulations. An isocratic system consisting of an ACE C18 column and a mobile phase composed of methanol-water (95 + 5, v/v) was used for the optimal chromatographic separation. In PCR, the concentration data matrix was prepared by using synthetic mixtures containing these drugs in methanol-water (3 + 1, v/v). The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbances at 29 wavelengths in the range of 242-298 nm for DIF and ISO in the zero-order spectra of their combinations. In first derivative spectrophotometry, dA/dlambda values were measured at 247.8 nm for DIF and at 240.2 nm for ISO in first derivative spectra of the solution of DIF and ISO in methanol-water (3 + 1, v/v). The linear ranges were 4.00-48.0 microg/mL for DIF and 50.0-400 microg/mL for ISO in the LC method, and 2.40-40.0 microg/mL for DIF and 60.0-260 microg/mL for ISO in the PCR and first derivative spectrophotometric methods. These methods were validated by analyzing synthetic mixtures. These three methods were successfully applied to two pharmaceutical cream preparations.