Simultaneous determination of dorzolamide HCL and timolol maleate in eye drops by two different spectroscopic methods

Two-component mixtures of dorzolamide hydrochloride and timolol maleate were assayed by first derivative and ratio derivative spectrophotometric methods. The first method, derivative spectrophotometry, by the zero-crossing measurements, was used due to the drugs closely overlapping absorption spectra. Linear calibration graphs of first derivative values at 250.3 nm for dorzolamide hydrochloride and 315.8 nm for timolol maleate. The second method, is based on ratio first derivative spectrophotometry, the amplitudes in the first derivative of the ratio spectra at 242.9 and at 223.5 nm were selected to determine dorzolamide and timolol maleate in the binary mixture. Calibration graphs were established for 8.0-30.0 microg ml(-1) for dorzolamide hydrochloride and 3.0-24.6 microg ml(-1) for timolol maleate in binary mixture. Good linearity, precision and selectivity were found, and the proposed methods were applied successfully to the pharmaceutical dosage from containing the above-mentioned drug combination without any interference by the excipients. Vierordt's method was also developed for a comparison method.     

Content uniformity and dissolution tests of triplicate mixtures by a double divisor-ratio spectra derivative method

The use of a UV double divisor-ratio spectra derivative calibration for the simultaneous analysis of synthetic samples and commercial tablet preparations without prior separation is proposed. The method was successfully applied to quantify three ternary mixtures, chlorpheniramine maleate and caffeine combined with paracetamol or acetylsalicylic acid and a mixture of acetylsalicylic acid combined with paracetamol and caffeine, using the information in the absorption spectra of appropriate solutions. Beer's law was obeyed in the concentration range of 0.84-4.21 microg/ml for chlorpheniramine maleate, 1.60-15.96 microg/ml for caffeine, 2.0-20.0 microg/ml for acetylsalicylic acid and 1.58-15.93 microg/ml for paracetamol. The whole procedure was applied to synthetic mixtures of pure drugs as well as to commercial preparations (Algon) by using content uniformity and dissolution tests (USP 24) and was found to be precise and reproducible. According to the dissolution profile test more than 84% of paracetamol and caffeine were dissolved within 20 min. Acetylsalicylic acid dissolved more slowly, taking about 45-60 min to dissolve completely. A chemometric method partial least squares (PLS) and a HPLC method were also employed to evaluate the same mixtures. The results of the proposed method were in excellent agreement with those obtained from PLS and HPLC methods and can be satisfactorily used for routine analysis of multicomponent dosage forms.     

Simultaneous determination of paracetamol and methocarbamol in tablets by ratio spectra derivative spectrophotometry and LC

The application of the ratio spectra derivative spectrophotometry and high-performance liquid chromatography (HPLC) to the simultaneous determination of paracetamol (PAR) and methocarbamol (MET) in combined pharmaceutical tablets is presented. The spectrophotometric procedure is based on the use of the first derivative of the ratio spectra obtained by dividing the absorbtion spectrum of the binary mixtures by a standard spectrum of one of the compounds. The first derivative amplitudes were measured at 243.0 and 230.3 nm for the assay of PAR and MET, respectively. Calibration graphs were established for 2-30 microg ml for PAR and 2-36 microg/ml for MET in binary mixture. The detection limits for PAR and MET were found 0.097 and 0.079 microg/ml, respectively; while the quantification limits were 0.573 microg/ml for PAR and 1.717 microg/ml for MET. For the HPLC procedure, a reversed-phase column with a mobile phase of methanol-water (60:40, v/v), was used to separate both compounds with a detection of 274.0 nm. Linearity was obtained in the concentration range of 2 300 and 1.5-375 microg/ml for PAR and MET, respectively. The detection and quantification limits were found to be 0.42 and 1.4 microg/ml for PAR and 0.36 and 1.2 microg/ml for MET, respectively. The relative standard deviations were found to be less than 0.52%, indicating reasonable repeatibility of both methods. The proposed methods were successfully applied to the determination of these drugs in commercial tablets.     

Comparison of zero- and second-order derivative spectrophotometric and HPLC methods for the determination of gemcitabine in human plasma

Zero- and second-order derivative spectrophotometric and high-performance liquid chromatography (HPLC) methods were developed and validated for the determination of gemcitabine in human plasma. Spectrophotometrically, gemcitabine was determined by means of zero-order derivative absorbance values (A) at 288 nm and from values from the second-order derivative absorbance values (2D) at 285 nm. Beer's Law was obeyed in the range 0.50-15.0 microg ml(-1). The proposed other method, normal-phase HPLC method for determination of gemcitabine in human plasma was described. Calibration curve was linear over the concentration range 0.20-15.0 microg ml(-1). Quantitation was achieved by diode array detection at 272 nm using 2'-deoxycytidine as internal standard. Results obtained by spectrophotometric and HPLC methods for determination of gemcitabine in human plasma described in this paper showed adequate accuracy, precision and repeatability. No interference was found in plasma at the selected derivative wavelength and chromatographic conditions. According to the statistical comparison, there is no significant difference between the three methods. This is suggested that the three methods are equally applicable.     

Development of the second-order derivative UV spectrophotometric method for direct determination of paracetamol in urine intended for biopharmaceutical characterisation of drug products

Paracetamol is a widely used nonsalicylate analgesic and antipyretic drug. The existing methods for the determination of paracetamol in biological fluids are mainly HPLC techniques, although there are some reported methods based on spectrophotometric determinations. However, all these methods involve some extraction or derivatisation procedures. In the present study the UV spectra of investigated samples were recorded over the wavelength range 220-400 nm (lambda step 0.21 nm; scan speed 60 nm/min) and second-order derivative spectra were calculated. Second-order derivative spectra of different blank urine samples displayed the presence of a zero-crossing point at 245-247 nm defined as lambdazc. The zero-order absorption spectra of paracetamol in water displays maximum absorbance at 243 nm, while in second derivative spectra, a minimum peak at 246 nm was observed. Therefore, the application of zero-crossing technique to the second-derivative UV absorption spectrum should be useful for the determination of paracetamol using 2Dlambdazc. The proposed method enables determination of total paracetamol in urine directly and simply by reading the 2Dlambdazc of the diluted samples. The obtained results were in good accordance with published data on cumulative urinary excretion after per oral administration of paracetamol obtained applying different spectrophotometric methods of determination. It could be useful for biopharmaceutical characterisation of drug products (monitoring of the levels of paracetamol in urine in bioavailability testing, for the evaluation of in vitro-in vivo correlation and screening of different formulations during drug product development).     

Determination of Lansoprazole in pharmaceutical dosage forms by two different spectroscopic methods

Two different ultraviolet (UV) spectroscopic methods were developed for determination of Lansoprazole in pharmaceutical dosage forms. The solutions of the standard and the sample were prepared in 0.1 M NaOH and phosphate buffer pH 6.6. Both UV spectrophotometric and derivative spectroscopic techniques were applied. Second-order derivative spectra were generated between 200 and 400 nm at N = 9, deltalambda = 31.5. The linear range for the UV spectrophotometric method was 3.0-25.0 microg ml(-1) and that for the derivative spectroscopic method was 0.5-25.0 microg ml(-1). The developed methods were applied to three different pharmaceutical preparations. The percentage recovery was 100.2%.     

Derivative ratio spectra-zero crossing spectrophotometry and LC method applied to the quantitative determination of paracetamol, propyphenazone and caffeine in ternary mixtures

Two methods were used to determine paracetamol, caffeine and propyphenazon in ternary mixtures and tablets. Derivative ratio spectra–zero crossing procedure was based on the simultaneous use of the first derivative of ratio spectra and measurements of derivative ratio analytical signals corresponding to the zero crossing points of wavelengths. By using propyphenazon as a divisor, the amounts of paracetamol and caffeine in the ternary mixture were determined by measuring the first derivative ratio amplitudes at 242.8 nm (zero-crossing point for caffeine) and 251.2 and 273.8 nm (zero-crossing point for paracetamol) respectively. Also by using paracetamol as a divisor, the contents of propyhenazon and caffeine in the same ternary mixture were determined by measuring the first derivative ratio amplitudes at 244.8 and 276.9 nm (zero-crossing point for caffeine) and 250.6 and 274.0 nm (zero-crossing point for propyphenazon), respectively. For the HPLC procedure, a Nucleosil C₁₈ column and a mobile phase consisted of water and methanol (20:80) were used to separate three compounds with cetrimide as an internal standard. The flow rate was 1.0-ml/min with an ultraviolet (UV) detection at 254 nm. Both methods were also applied to the determination of these three compounds in ternary mixtures and tablet formulation. The analytical results were quite good in all cases.     

Stability-indicating methods for the determination of mosapride citrate in the presence of its degradation products according to ICH guidelines

In the present work, different spectrophotometric methods and one spectrofluorimetric method have been developed and validated for the determination of mosapride citrate in the presence of its acid-induced degradation products. The drug was subjected to stress stability study including acid, alkali, oxidative, photolytic, and thermal stress degradation. The developed spectrophotometric methods included the use of first order derivative ((1)D), derivative of ratio spectra ((1)DD), mean centring of ratio spectra (MC) and H-point standard additions (HPSAM) spectrophotometric methods. For (1)D method, the peaks amplitudes at 282.8 and 319.6 nm were measured, while for (1)DD method those at 308 nm and 323 nm were measured. Mean centring of ratio spectra method used the values at 317 nm for calibration, while for HPSAM the absorbance at 273 and 288.6 nm were used. These methods were successfully applied for determination of mosapride in the concentration range of 5-70 μg.ml(-1). The spectrofluorimetric method was based on measuring the native fluorescence of mosapride in 0.1 M NaOH using λ(excitation) 276 nm and λ(emission) 344 nm and 684 nm with linearity ranges of 50-3000 ng.ml(-1) and 50-9000 ng.ml(-1), respectively. All the developed methods were validated according to the International Conference on Harmonization (ICH) guidelines and were applied for bulk powder and dosage form. The results obtained were statistically compared to each other using one-way ANOVA testing.     

Quantitative determination of ambroxol in tablets by derivative UV spectrophotometric method and HPLC

A derivative UV spectrophotometric method for the determination of ambroxol in tablets was developed. Determination of ambroxol in tablets was conducted by using first-order derivative UV spectrophotometric method at 255 nm (n = 5). Standards for the calibration graph ranging from 5.0 to 35.0 microg/ml were prepared from stock solution. The proposed method was accurate with 98.6+/-0.4% recovery value and precise with coefficient of variation (CV) of 1.22. These results were compared with those obtained by reference methods, zero-order UV spectrophotometric method and reversed-phase high-performance liquid chromatography (HPLC) method. A reversed-phase C(18) column with aqueous phosphate (0.01 M)-acetonitrile-glacial acetic acid (59:40:1, v/v/v) (pH 3.12) mobile phase was used and UV detector was set to 252 nm. Calibration solutions used in HPLC were ranging from 5.0 to 20.0 microg/ml. Results obtained by derivative UV spectrophotometric method was comparable to those obtained by reference methods, zero-order UV spectrophotometric method and HPLC, as far as ANOVA test, F(calculated) = 0.762 and F(theoretical) = 3.89, was concerned.     

Chemometric and derivative methods as flexible spectrophotometric approaches for dissolution and assaying tests in multicomponent tablets

Two derivative spectrophotometric (ratio derivative spectra and algorithm bivariate calibration) and a chemometric methods (partial least squares, PLS) are proposed for the simultaneous determination of binary mixtures in tablet analysis and dissolutions tests, without prior separation. These approaches are successfully applied to quantify trimethoprim (TMP) combined with sulfamethoxazole (SMX) or sulfamethazine (SMZ) or sulfafurazole (SFZ) using the information in the absorption spectra of appropriate solutions. Beer's law was obeyed in the concentration range of 0.98-17.5 microg/ml for TMP, 0.95-17.2 microg/ml for SMX, 1.16-17.5 microg/ml for SMZ and 0.97-17.4 microg/ml for SFZ. The first derivative (1D) bivariate algorithm method involves the use of four calibration curves: two for each compound at two different wavelengths, selected by Kaiser's method. Similarly, the first derivative ratio spectrophotometry employs the linear relationship between the ratio spectra of the analytes and the concentration range. The results were compared with those obtained by PLS multivariate calibration. The calibration models from PLS were pre-treated by orthogonal signal correction and evaluated by cross-validation using the 'SIMCA-P 9' software. Synthetic mixtures of TMP and sulfonamides were used in five different sets for the validity of the calibrations. Mean recoveries for derivative ratio, derivative bivariate and PLS methods were found to be between 99.7% and 102.0% for TMP, 99.4% and 100.2% for SMX, 99.3% and 101.0% for SMZ and 98.1% and 102.3% for SFZ. The calibrations of the three methods were successfully applied to the assaying and dissolution of placebo and commercial tablets without any prior separation. More than 85% of TMP, SMX and SMZ were dissolved within 15 min. For SFZ, only 85% of the compound was dissolved after 60 min. In this study, the three spectrophotometric methods can be satisfactorily used for the quantitative analysis and for dissolution tests of multicomponent dosage forms.     

Two derivative spectrophotometric determinations of indapamide in pharmaceutical dosage forms

Simple, fast and reliable derivative spectrophotometric methods were developed for determination of indapamide in bulk and pharmaceutical dosage forms. The solutions of standard and the sample were prepared in methanol. The quantitative determination of the drug was carried out using the first-derivative values measured at 252.8 nm (N=6) and the second-derivative values measured at 260.4 nm (N=9). Calibration graphs constructed at their wavelengths of determination were linear in the concentration range of indapamide using peak to zero 1.00-30.00 microg ml(-1) for first-derivative and 1.00-35.00 microg ml(-1) for second-derivative spectrophotometric method. The developed methods were successfully applied for the assay of pharmaceutical dosage forms which do not require any preliminary separation or treatment of the samples. The details of the statistical treatment of analytical data are also presented. The results obtained from two derivative spectrophotometry were compared with a spectrophotometric method reported in literature and no significant difference was found statistically.     

Application of TLC-densitometry, first-derivative UV-spectrophotometry and ratio derivative spectrophotometry for the determination of dorzolamide hydrochloride and timolol maleate

Three methods are described for the simultaneous determination of dorzolamide hydrochloride (DORZ) and timolol maleate (TIM) in ophthalmic solutions. The first method is based on application of thin layer chromatographic separation of both drugs followed by the densitometric measurements of their spot areas. After separation on silica gel GF(254) plates, using methanol-ammonia 25% (100:1.5 v/v) as the mobile phase, the chromatographic zones corresponding to the spots were scanned at 253 and 297 nm, respectively. The calibration function was established in the ranges of 2-18 microg for DORZ and 0.5-4.5 microg for TIM. The second method depends on first derivative ultraviolet spectrophotometry, with zero-crossing measurement method. The first derivative values D(1) at 250.2 and 312.5 nm were selected for the assay of DORZ and TIM, respectively. Calibration graphs follow Beer's law in the range 10-64 and 2.5-16 microg ml(-1), respectively. The third method is based on ratio first derivative spectrophotometry. The signals in the first derivative of the ratio spectra at 244 and 306.2 nm were selected to determine DORZ and TIM in the mixture and calibration graphs are linear in the range of 5-40 and 5.0-17.5 microg ml(-1), respectively. The proposed methods were successfully applied to the determination of these compounds in synthetic mixtures and in pharmaceutical preparations. The proposed methods are simple, rapid and suitable for quality control application.     

Stability indicating methods for the determination of aceclofenac

Five new selective, precise and accurate methods for the determination of aceclofenac in the presence of its degradation product; diclofenac are described. Method A utilizes third derivative spectrophotometry at 242 nm. Method B is RSD(1) spectrophotometric method based on the simultaneous use of the first derivative of ratio spectra and measurement at 245 nm. Method C is a pH-induced difference (deltaA) spectrophotometry using UV measurement at 273 nm. Method D is a spectrodensitometric one, which depends on the quantitative densitometric evaluation of thin layer chromatogram of aceclofenac at 275 nm. Method E is RP-HPLC that depends on using methanol: water (60:40 v/v) as mobile phase at a flow rate of 1 ml/min and UV detection at 275 nm. Regression analysis of a beer's plot showed good correlation in the concentration ranges 5-40, 10-40, 15-50, 50-200, 1-50 microg/ml for methods A, B, C, D and E, respectively. These methods are suitable as stability indicating methods for the determination of aceclofenac in presence of its main degradation product, diclofenac. The proposed methods were applied for the analysis of the drug in its pharmaceutical formulation and the results obtained were compared with those obtained with the official B.P. method.     

Comparison of derivative spectrophotometric and liquid chromatographic methods for the determination of rofecoxib

Two different UV spectrophotometric methods were developed for the determination of rofecoxib in bulk form and in pharmaceutical formulations. The first method, an UV spectrophotometric procedure, was based on the linear relationship between the rofecoxib concentration and the lambdamax amplitude at 279 nm. The second one, the first derivative spectrophotometry, was based on the linear relationship between the rofecoxib concentration and the first derivative amplitude at 228, 256 and 308 nm. Calibration curves were linear in the concentration range using peak to zero 1.5-35.0 microg x ml(-1). HPLC was carried out at 225 nm with a partisil 5 ODS (3) column and a mobile phase constituted of acetonitrile and water (50 :50 v/v). A linear range was found to be 0.05-35.0 microg x ml(-1). The developed methods were successfully applied for the assay of pharmaceutical dosage form. The statistics of the analytical data is also presented. The results obtained by first derivative spectrophotometry were compared with HPLC and no significant difference was found.     

Simultaneous determination of phenytoin, barbital and caffeine in pharmaceuticals by absorption (zero-order) UV spectra and first-order derivative spectra--multivariate calibration methods

The quantitative predictive abilities of partial least squares (PLS-1) and principle component regression (PCR) on absorption (zero-order) UV spectra are compared with the results obtained by the use of these multivariate calibration methods on first-order derivative spectra. Both approaches were satisfactorily applied to the simultaneous determination of these drugs in synthetic and pharmaceutical mixtures. Significant advantages were found in the simultaneous determination of phenytoin, barbital and caffeine in binary and ternary mixtures, by application of different multivariate calibration methods when the calibration matrix was performed using the first-order derivative spectra. The proposed method was validated by applying it to the analysis of binary and ternary mixtures of phenytoin, barbital and caffeine. Determinations were made over the concentration ranges of 0.24-22.0, 0.01-27.0 and 0.049-27.0 microg ml(-1) for phenytoin, barbital and caffeine, respectively, in the binary and 0.45-22.0, 0.05-26.0 and 0.05-20.0 microg ml(-1) for phenytoin, barbital and caffeine, respectively, in the ternary mixtures. The relative standard errors in the determinations were less than 3% in most cases.     

Simultaneous spectrophotometric determination of pseudoephedrine hydrochloride and ibuprofen in a pharmaceutical preparation using ratio spectra derivative spectrophotometry and multivariate calibration techniques

Spectrophotometric methods are described for the simultaneous determination of pseudoephedrine hydrochloride and ibuprofen in their combination. The obtained data were evaluated by using five different methods. In the first method, ratio spectra derivative spectrophotometry, analytical signals were measured at the wavelengths corresponding to either maximums and minimums 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 four spectrophotometric methods using chemometric techniques, classical least-squares, inverse least-squares, principal component regression and partial least-squares (PLS), the concentration data matrix were prepared by using the synthetic mixtures containing these drugs in methanol:0.1 M HCl (3:1). The absorbance data matrix corresponding to the concentration data matrix was obtained by the measurements of absorbances in the range 240-285 nm in the intervals with deltalambda = 2.5 nm at 18 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 pseudoephedrine hydrochloride and ibuprofen in their mixture. The procedures did not require any separation step. The linear range was found to be 300-1300 microg/ml for ibuprofen and 100-1300 microg/ml for pseudoephedrine hydrochloride in all five methods. The accuracy and the precision of the methods have been determined and they have been validated by analyzing synthetic mixtures. The five methods were successfully applied to tablets and the results were compared with each other.     

Chemometric resolution of a mixture containing hydrochlorothiazide and amiloride by absorption and derivative spectrophotometry

Four chemometric techniques, classical least squares (CLS) and inverse least squares (ILS) and principal component regression (PCR) and partial least squares regression (PLSR) were applied to the absorption and derivative spectrophotometric determinations of amiloride and hydrochlorothiazide in a pharmaceutical preparation. Four chemometric calibrations for both zero-order and first derivative spectra were constructed by measuring the absorbance and their dA/dlambda values at 34 points in the wavelength range 205-395 nm for a training set containing 2-10 microg/ml amiloride and 4-28 microg/ml hydrochlorothiazide corresponding to 25 point mixture design. The building chemometric calibrations were confirmed by using the synthetic mixtures containing two drugs. The results obtained by the proposed techniques based on the use of the measurements at the absorption spectra and at the first derivative spectra were statistically compared with each other.     

A comparison of matrix resolution method, ratio spectra derivative spectrophotometry and HPLC method for the determination of thiamine HCl and pyridoxine HCl in pharmaceutical preparation

A comparison of two spectrophotometric methods and a HPLC method were described in this work for the analysis of pyridoxine hydrochloride and thiamine hydrochloride in a vitamin combination. In the first method, A₁¹ (1%, 1 cm) values of these two compounds were calculated using absorbances measured at 246.8 and 290.5 nm in zero-order spectra. The matrix was written for A₁¹ (1%, l cm) values and the concentration of both compounds were determined using ‘Matlab’ software. In the second method, the measurements in the derivative of the ratio spectra were made at 297.8 and 309.5 nm for pyridoxine hydrochloride and at 245.6 and 257.7 nm for thiamine hydrochloride. The calibration graphs were established in the range 8–40 μg/ml of both vitamins. In the HPLC method, the separation of these compounds was realized on a Nucleosil 100-5 C₁₈ column with 0.1 M (NH₄)₂C0₃–water–methanol (5:15:80 v/v) as the mobile phase. Results of spectrophotometric and HPLC procedures were compared.     

Comparison of spectrophotometric and an LC method for the determination perindopril and indapamide in pharmaceutical formulations.

A new sensitive, simple, rapid and precise reversed-phase high performance liquid chromatographic (HPLC) and two spectrophotometric methods have been developed for resolving binary mixture of perindopril and indapamide in the pharmaceutical dosage forms. The first method is based on HPLC on a reversed-phase column using a mobile phase of phosphate buffer pH 2.4 and acetonitrile (7:3 v/v) was used. Linearity range for perindopril and indapamide was 5.0-70.0 and 8.0-35.0 microg ml(-1). In the second method, the first derivative spectrophotometry with a zero-crossing technique of measurement is used for the simultaneous quantitative determination of perindopril and indapamide in binary mixtures without previous separation step. Linear calibration graphs of first derivative values at 225.7 and 255.4 nm for perindopril and indapamide, respectively. The third method is based on ratio derivative spectrophotometry, the amplitudes in the first derivative of the ratio spectra at 226.5 and at 255.3 nm were selected to determine perindopril and indapamide in the binary mixture. All the proposed methods showed good linearity, precision and reproducibility. The proposed methods were successfully applied to the pharmaceutical dosage forms containing the above-mentioned drug combination without any interference by the excipients.     

Spectrophotometric resolution of metronidazole and miconazole nitrate in ovules using ratio spectra derivative spectrophotometry and RP-LC

Metronidazole and miconazole nitrate in ovules was determined by ratio spectra derivative spectrophotometry and by high-performance liquid chromatography (HPLC). The first method depends on ratio spectra first derivative spectrophotometry, by utilizing the linear relationship between substances concentration and ratio spectra first derivative peak amplitude. The ratio first derivative amplitudes at 242.6 [(1)DD(242.6)], 274.2 [(1)DD(274.2))] 261.8 [(1)DD(261.8))] 273.5 [(1)DD(273.5))]and 281.5 [(1)DD(281.5)] nm were selected for the assay of metronidazole and miconazole nitrate, respectively. The second method is based on high-performance liquid chromatography on a reversed-phase column using a mobile phase of methanol-water-phosphoric acid (30:70:0.20 v/v) (pH 2.8) with programmable detection at 220.0 nm. The minimum concentration detectable by HPLC was 0.9 microg ml(-1) for metronidazole and 0.3 microg ml(-1) for miconazole nitrate and by ratio derivative spectrophotometry 4.0 microg ml(-1) for metronidazole and 0.5 microg ml(-1) for miconazole nitrate. The proposed procedures were successfully applied to the simultaneous determination of metronidazole and miconazole nitrate in ovules with a high percentage of recovery, good accuracy and precision.