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.     

Spectrofluorimetric and spectrophotometric methods for the determination of vigabatrin in tablets

Two sensitive and selective spectrofluorimetric and spectrophotometric methods have been developed for the determination of vigabatrin in tablets. The methods are based on derivatization with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The product showed an absorption maximum at 460 nm and a fluorescence emission peak at 520 nm in ethyl acetate. The color was found to be stable for at least 48 h in this solvent. The optimum conditions of the reaction were investigated and it was found that the reaction proceeds quantitatively at pH 10.0, 70 degrees C in 50 min when the mole ratio of the reagent to drug was 30. The reaction obeys Beer's law over the ranges of 2-10 and 0.05-1.00 microg ml(-1) for the spectrophotometric and spectrofluorimetric measurements, respectively. The detection limits were found to be 0.50 and 0.01 microg ml(-1) for the spectrophotometric and spectrofluorimetric methods, respectively. The proposed methods were applied to the assay of vigabatrin in tablets. The results were compared statistically with those obtained by the modified spectrofluorimetric method reported in the literature.     

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.     

Validated stability-indicating methods for determination of cilostazol in the presence of its degradation products according to the ICH guidelines

Sensitive and selective stability-indicating assay methods (SIAMs) are suggested for the determination of cilostazol (CIL) in the presence of its acid, alkaline and oxidative degradation products. Developing SIAMs is necessary to carry out any stability study. Stress testing of CIL was performed according to the International Conference on Harmonization (ICH) guidelines in order to validate the stability-indicating power of the analytical procedures. Stress testing showed that CIL underwent acid, alkaline and oxidative degradation; on the other hand, it showed stability towards photo- and thermal degradation. Two chromatographic SIAMs were developed, namely HPLC and HPTLC methods. The concentration range and the mean percentage recovery were 1.0-31.0 microg/ml and 99.96+/-0.46 and 0.6-14.0 microg/spot and 99.88+/-1.10 for HPLC and HPTLC methods, respectively. In addition, derivative spectrophotometric methods were developed in order to determine CIL in the presence of its acid degradation product; these were performed by using the third derivative spectra (3D) and the first derivative of the ratio spectra (1DD) methods. The linearity range and the mean percentage recovery were 2.0-34.0 microg/ml and 100.27+/-1.20 for the (3D) method, while they were 2.0-30.0 microg/ml and 99.94+/-1.18 for the (1DD) method. Also, two chemometric-assisted spectrophotometric methods, based on using partial least squares (PLS) and concentration residual augmented classical least squares method (CRACLS), for the determination of CIL were developed. Both methods were applied on zero order spectra of the mixtures of CIL and its acid degradation product, the mean percentage recovery was 100.03+/-1.09 and 99.91+/-1.27 for PLS and CRACLS, respectively. All methods were validated according to the International Conference on Harmonization (ICH) guidelines and applied on bulk powder and pharmaceutical formulations.     

Spectrophotometric and Spectrofluorimetric Determination of Fluorouracil in the Presence of its Degradation Products

Three reliable spectrophotometric and spectrofluorimetric procedures are described for the determination of fluorouracil in bulk powder and ampoules in the presence of its degradation products. One spectrophotometric procedure, based on measurement at 555 nm of the violet-coloured complex formed by fluorouracil with cobalt(II), has a detection limit of 0.03 mg mL^?1. Two sensitive spectrofluorimetric procedures are also proposed. One is based on measurement of the intrinsic fluorescence of the liberated fluorouracil at 375 nm, after precipitation as its cobalt(II) complex, decomposition of the precipitate with sulphuric acid and excitation at 295 nm. The second depends on excitation of the fluorouracil-cobalt(II) complex at 395 nm and measuring its fluorescence at 483 nm. The limits of detection of the two spectrofluorimetric procedures are 0.5 and 2 μg mL^?1, respectively. The three procedures have been used successfully for the determination of fluorouracil ampoules. The validity of the methods has been assessed by applying the standard addition technique.     

Stability indicating methods for the determination of clozapine

Five new selective, precise and accurate methods are described for the determination of clozapine in the presence of its main degradation product. Method A utilizes the second and third derivative spectrophotometry at 315 and 305 nm, respectively. Method B is RSD(1) spectrophotometric method based on the simultaneous use of the first derivative of ratio spectra and measurement at 295 nm. Method C is a pH-induced difference (delta A) spectrophotometry using UV measurement at 325 nm. Method D is a densitometric one, after separation on silica gel plate using methanol: water as mobile phase, and the spot was scanned at 295 nm. Method E is RP-HPLC using acetonitrile: water (40:60 v/v) as mobile phase at a flow rate of 1 ml/min and UV detection was at 295 nm. Regression analysis showed good correlation in the concentration ranges 3-10, 4-10, 10-25 micro g/ml, 200-1000 ng/spot, 5-100 micro g/ml with percentage recoveries of 99.4+/-0.28, 99.8+/-0.20, 100.05+/-0.11, 99.41+/-0.34, 100.11+/-0.07 and 100.07+/-0.05% for methods A, B, C, D and E, respectively. These methods are suitable as stability indicating methods for the determination of clozapine in the presence of its main degradation product either in bulk powder or in pharmaceutical formulations.     

Simultaneous spectrofluorimetric and spectrophotometric determination of melatonin and pyridoxine in pharmaceutical preparations by multivariate calibration methods

Partial least-squares (PLS) calibration and principal component regression (PCR) methods were utilized for the simultaneous spectrofluorimetric and spectrophotometric determination of pyridoxine (PY) and melatonin (MT). Since emission and adsorption spectra of these drugs overlap, PY and MT cannot be directly determined by fluorimetric nor by spectrophotometric methods. Full-spectrum multivariate calibration PLS and PCR methods were developed for both fluorimetry and spectrophotometry. The conditions were optimized for fluorimetric as well as for spectrophotometric determination of both drugs. The simultaneous determination of PY and MT was carried out in mixtures by recording the emission fluorescence spectrum between 324 and 500 nm (lambda(ex) 285 nm) for fluorimetry, and by recording the absorption spectrum between 250 and 350 nm for spectrophotometry (lambda(max(PY)) 310 nm, lambda(max(MT)) 278 nm). The experimental calibration matrixes were designed orthogonally. At the optimum conditions, dynamic ranges were 0.04-1.3 and 0.1-4 microg ml(-1) for fluorimetry and 1-22 and 1-24 microg ml(-1) for spectrophotometry for MT and PY, respectively. The calibration concentrations were prepared in the dynamic ranges. The parameters of the chemometrics procedure for the simultaneous determination of MT and PY were optimized, and the proposed methods were validated with prediction set. Finally the procedures were successfully applied to simultaneous spectrofluorimetric and spectrophotometric determination of PY and MT in synthetic mixtures and in a pharmaceutical formulation.     

Voltammetric, spectrofluorimetric and spectrophotometric methods to determine flufenamic acid

Simple, rapid and sensitive voltammetric, spectrofluorimetric and spectrophotometric methods for determination of flufenamic acid (FF) in bulk powder and capsule dosage form are presented. The methods are based on the cyclisation reaction of FF with concentrated sulphuric acid to produce the corresponding acridone derivative. The voltammetric method is based on the adsorptive stripping differential pulse (DP) technique. The acridone derivative is determined over the concentration range of 8-60 ng ml(-1) using adsorptive preconcentration at the hanging mercury drop electrode (HMDE). The lower detection limit was found to be 1.02 ng ml(-1). The fluorimetric and spectrophotometric methods are based on the measurement of the fluorescence intensity at 450 nm (lambda(ex) = 400 nm)and peak-to-peak measurements of the first- (D1) and second-derivative (D2) curves, respectively. Beer's law is obeyed over the concentration ranges of 2-20 ng ml(-1) and 0.2-8.0 microg ml(-1) for the fluorimetric and spectrophotometric measurements, respectively. The three methods were proved to be accurate and reproducible as indicated by a relative standard deviation of <2%.     

Spectrophotometric and spectrofluorimetric determination of etodolac and aceclofenac

Two simple, sensitive and reproducible spectrophotometric and spectrofluorimetric methods were adopted for the analysis of the anti-inflammatory drugs, etodolac and aceclofenac. The first method is based on the formation of coloured complexes between the drugs and p-dimethylaminobenzaldehyde reagent (PDAB) in the presence of sulfuric acid and ferric chloride. Measurement of the absorbances was carried out at 591.5 and 545.5 nm for etodolac and aceclofenac, respectively. Regression analysis of Beer's plots showed good correlation in the concentration ranges 10-80 and 8-55 microg ml(-1), respectively. The second was the spectrofluorimetric method in which samples of etodolac in ethanol showed native fluorescence at a lambda = 345 nm when excitation was at 235 nm and samples of aceclofenac in the phosphate buffer pH 8 showed native fluorescence at lambda = 355 nm when excitation was at 250 nm. The calibration graph was rectilinear from 96 to 640 ng ml(-1) for etodolac and from 2 to 8 microg ml(-1) for aceclofenac. The proposed methods are applied successfully for the determination of the two drugs in bulk powder with a mean accuracy of 100.48+/-0.85 and 100.03+/-0.38 in the PDAB method and of 100.61+/-0.79 and 99.88+/-0.45 in the spectrofluorimetric method. Applicability of the proposed methods was examined by analysing dosage forms of the investigated drugs. Recoveries were 98.77-101.46 and 98.65-102.10% for the two methods, respectively and RSD values were 0.6-0.7 and 0.35-1.06% respectively.     

Spectrophotometric, septrofluorimetric and LC determination of lisinopril

Three methods are described for the determination of lisinopril in the pharmaceutical tablets. The spectrophotometric method depends on the reaction of the lisinopril with sodium hypochlorite and phenyl hydrazine to form a condensation product measured at 362 nm. The spectrophotometric method was extended to develop a stability indicating method. The spectrofluorimetric method depends on reaction of the lisinopril with o-phthalaldehyde in the presence of 2-mercaptoethanol in borate buffer pH 9.5. The fluorescence of the reaction product was measured upon excitation at a maximum of 340 nm with emission wavelength at 455 nm. The HPLC method depends on using Hypersil silica column with a mobile phase consisting of methanol–water–triethylamine (50:50:0.1 v/v) and the pH was adjusted to 2.6 with 0.1 N perchloric acid. Quantitation was achieved with UV detection at 210 nm based on peak area.     

UV and Three Derivative Spectrophotometric Methods for Determination of Ezetimibe in Tablet Formulation

UV, first, second and third derivative spectrophotometric methods have been developed for the determination of ezetimibe in pharmaceutical formulation. The solutions of standard and sample were prepared in methanol. For the first method, UV spectrophotometry, the quantitative determination of the drug was carried at 233 nm and the linearity range was found to be 6-16 μg/ml. For the first, second and third derivative spectrophotometric methods the drug was determined at 259.5 nm, 269 nm and 248 nm with the linearity ranges 4-14 μg/ml, 4-14 μg/ml and 4-16 μg/ml. The calibration graphs constructed at their wavelength of determination were found to be linear for UV and derivative spectrophotometric methods. All the proposed methods have been extensively validated. The described methods can be readily utilized for the analysis of pharmaceutical formulation. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations.     

Use of mixed anhydrides for the determination of terfenadine in dosage forms and spiked human plasma

Terfenadine reacts with mixed anhydrides (malonic and acetic anhydrides) producing a yellow-coloured product with intense fluorescence. Based on this fact, a spectrophotometric method was developed for the determination of terfenadine in dosage forms. The relation between the absorbance at 395 nm and the concentration is rectilinear over the range 0.5-5 microg ml(-1) (molar absorptivity is 1.405 x 10(5) l mol(-1) cm (-1)). The reaction product was also measured spectrofluorimetrically at 435 nm after excitation at 395 nm. The fluorescence intensity was directly proportional to the concentration over the range 0.5-4 ng ml(-1) with minimum detectability (S/N = 2) of 0.07 microg ml(-1) (approximately 1.5 x 10(-10) M). The different parameters affecting the development and stability of the reaction product were carefully studied and incorporated into the procedure. The proposed spectrophotometric method was successfully applied to the determination of terfenadine in tablets and suspensions; the percentage recoveries were 99.83 +/- 0.75 and 99.65 +/- 0.83, respectively. The proposed spectrofluorimetric method was applied to the determination of terfenadine in spiked human plasma. The percentage recovery was 99.35 +/- 2.19. The method is highly sensitive and specific. No interference was noticed from co-formulated drugs, such as pseudoephedrine and ibuprofen.     

Derivative spectrophotometric, thin layer chromatographic-densitometric and high performance liquid chromatographic determination of trifluoperazine hydrochloride in presence of its hydrogen peroxide induced-degradation product.

Three methods are presented for the determination of trifluoperazine HCl in presence of its hydrogen peroxide induced degradation product. The first method was based on measurement of first (1D) and second (2D) derivative amplitudes of trifluoperazine HCl in 0.1 N hydrochloric acid at the zero crossing point of its sulfoxide derivative, main degradation product, (at 268.4 and 262.5 nm for 1D and 2D, respectively). The second method was based on the separation of trifluoperazine HCl from its sulfoxide derivative followed by densitometric measurement of the intact drug spot at 255 nm. The separation was carried out on Merck aluminum sheet of silica gel 60 F(254), using chloroform-methanol (7:3 v/v) as mobile phase. The third method was based on high performance liquid chromatographic separation of trifluoperazine HCl from its sulfoxide derivative on reversed phase, ODS column, using a mobile phase of acetonitrile-phosphate buffer pH 4.2 (60:40 v/v) at ambient temperature. Quantitation was achieved with UV detection at 255 nm based on peak area. The first derivative spectrophotometric method was utilized to investigate the kinetics of the hydrogen peroxide degradation process at different temperatures. The apparent pseudo first-order rate constant, half life and activation energy were calculated.     

Stability-indicating spectrophotometric and spectrodensitometric methods for the determination of diacerein in the presence of its degradation product

Three sensitive, selective, and precise stability-indicating methods for the determination of the novel osteoarthritis drug, diacerein (DIA) in the presence of its alkaline degradation product (active metabolite, rhein) and in pharmaceutical formulation were developed and validated. The first method is a first derivative (D(1) ) spectrophotometric one, which allows the determination of DIA in the presence of its degradate at 322 nm (corresponding to zero crossing of the degradate) over a concentration range of 4-40 μg/mL with mean percentage recovery 100.21 ± 0.833. The second method is the first derivative of the ratio spectra (DD(1) ) by measuring the peak amplitude at 352 nm over the same concentration range as (D(1) ) spectrophotometric method, with mean percentage recovery 100.09 ± 0.912. The third method is a TLC-densitometric one, where DIA was separated from its degradate on silica gel plates using ethyl acetate:methanol:chloroform (8:1.5:0.5 v:v:v) as a developing system. This method depends on quantitative densitometric evaluation of thin layer chromatogram of DIA at 340 nm over a concentration range of 1-10 μg/spot, with mean percentage recovery 100.24 ± 1.412. The selectivity of the proposed methods was tested using laboratory-prepared mixtures. The proposed methods have been successfully applied to the analysis of DIA in pharmaceutical dosage forms without interference from other dosage form additives and the results were statistically compared with reference method.     

Derivative spectrophotometric and fluorimetric methods for determination of rofecoxib in tablets and in human plasma in presence of its photo-degradation product

Rofecoxib (I) has been determined in the presence of its photo-degradation product (II) using first derivative spectrophotometry ((1)D) and first derivative of the ratio spectra ((1)DD) by measuring the amplitude at 316.3 and 284 nm for (1)D and (1)DD, respectively. (I) can be determined in the presence of up to 70% and 80% of (II) by the (1)D and (1)DD, respectively. The linearity range of both the methods was the same (5.8-26.2 microg ml(-1)) with mean percentage recovery of 100.08 +/- 0.84 and 100.06 +/- 1.06 for (1)D and (1)DD, respectively. (1)D method was used to study kinetics of (I) photo-degradation that was found to follow a first-order reaction. The t(1/2) was 20.2 min while K (reaction rate constant) was 0.0336 mol min(-1). Both methods were applied to the analysis of (I) in bulk powder and in pharmaceutical formulations. Also a spectrofluorimetric method is described to determine (I) at very low concentrations (25-540 ng ml(-1)) where (I) is converted to its photo-degradate (II), which possesses a native fluorescence that could be measured. The proposed method was applied for the analysis of tablets containing rofecoxib as well as to rofecoxib-spiked human plasma.     

Spectrophotometric and spectrofluorimetric determination of atomoxetine in pharmaceutical preparations

Visible spectrophotometric and spectrofluorimetric methods were developed for the determination of atomoxetine in pharmaceutical preparations. The spectrophotometric method was based on a nucleophilic substitution reaction of atomoxetine with 1,2-naphthoquinone-4-sulphonate (NQS) in an alkaline medium to form an orange-colored product. The absorbance-concentration plot is rectilinear over the range 5-40 microg mL(-1). The limits of detection and quantification were calculated to be 0.02 microg mL(-1) and 0.06 microg mL(-1), respectively. The spectrofluorimetric method was based on the derivatization reaction of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) with atomoxetine to produce a fluorescent derivative. The formed highly fluorescent derivative that was measured at 462 nm after excitation at 533 nm. The fluorescence-concentration plot is rectilinear over the range 10-500 ng mL(-1). The limits of detection and quantification were calculated to be 0.19 ng mL(-1) and 0.57 ng mL(-1). The analytical performance of both methods was fully validated, and the results were satisfactory. The methods have been successfully applied for the determination of the studied drug in capsules and the results obtained ware in good agreement with those obtained by the reference method.     

First derivative spectrophotometric and high-performance liquid chromatographic determination of cinchocaine hydrochloride in presence of its acid degradation product

Two methods are presented for the determination of cinchocaine HCl in presence of its acid-induced degradation product using first (¹D) derivative spectrophotometry and high-performance liquid chromatography. Cinchocaine HCl was determined by measurement of its first derivative amplitude at the zero crossing point of 2-hydroxyquinoline-4-carboxylic acid diethylaminoethylamide as its acid degradation product (at 333.5 nm). The HPLC method depends upon using a μBondapak C₁₈ column at ambient temperature with a mobile phase consisting of acetonitrile—0.01 M sodium acetate trihydrate (45:55, v/v) containing 0.06% (w/v) heptane sulphonic acid sodium salt and adjusted to apparent pH 4.5 with acetic acid at a flow rate 2 ml min⁻¹. Quantitation was achieved with UV detection at 254 nm based on peak area. The HPLC method was applied for simultaneous determination of cinchocaine HCl, methylparaben and propylparaben. The two proposed methods were successfully applied to the determination of the cinchocaine HCl in laboratory-prepared mixtures in the presence of its acid degradation product and in cream. Moreover, the proposed methods were utilized to investigate the kinetics of the acid degradation process at different temperatures and the apparent pseudo first-order rate constant, half-life and activation energy calculated.     

Stability indicating methods for the determination of diloxanide furoate

Five new selective, precise and accurate methods are described for the determination of diloxanide furoate (DI) in presence of its degradation products. Method A utilizes the first and second derivative spectrophotometry at 270 and 280 nm, respectively. Method B is a RSD(1) spectrophotometric method based on the simultaneous use of the first derivative of ratio spectra and measurement at 270 nm. Method C is a pH-induced difference spectrophotometry using UV measurement at 295 nm. Method D is a densitometric one, after separation on silica gel plate using chloroform: methanol as mobile phase and the spots were scanned at 258 nm. Method E is reversed phase high performance liquid chromatography using methanol: water (80:20% v/v) as mobile phase at a flow rate of 1 ml/min and UV detection at 258 nm. Regression analysis showed good correlation in the concentration ranges 5-30, 5-25, 10-40 microg/ml, 100-500 ng/spot, 2-50 microg/ml with percentage recoveries of 99.92+/-0.56 and 99.79+/-0.47, 99.23+/-0.38, 99.96+/-0.06, 99.03+/-0.51, 98.81+/-0.68 for methods A, B, C, D and E, respectively. These methods are suitable as stability indicating methods for the determination of DI in presence of its degradation products either in bulk powder or in pharmaceutical formulations.     

First derivative spectrophotometric, TLC-densitometric, and HPLC determination of acebutolol HCL in presence of its acid-induced degradation product

Three methods are presented for the determination of acebutolol HCl in presence of its acid-induced degradation product. The first method was based on measurement of the first derivative amplitude of acebutolol HCl at 266.6 nm. The second method was based on separation of acebutolol HCl from its acid-induced degradation product followed by densitometric measurement of the spots at 230 nm. The separation was carried out on silica gel 60 F254, using ethanol-glacial acetic acid (4:1, v/v) as mobile phase. Second order polynomial equation was used for the regression line. The third method was based on high performance liquid chromatographic (HPLC) separation of acebutolol HCl from its acid-induced degradation product on a reversed phase, ODS column using a mobile phase of methanol-water (55:45, v/v) with UV detection at 240 nm. The first derivative spectrophotometric method was utilized to investigate the kinetics of the acid degradation process at different temperatures.     

Colorimetric and fluorimetric methods for determination of panthenol in cosmetic and pharmaceutical formulation

Two methods are suggested for determination of panthenol. The first is colorimetric method where panthenol is subjected to alkaline hydrolysis; the resulting beta-alanol is allowed to react with vanillin (Duquenois reagent) in presence of McIlvain buffer pH 7.5. The color developed is measured at 406 nm. The linearity range was found to be 50-500 microg/ml while the lower limit of detection was about 10 microg/ml. The second is a sensitive and reliable modified fluorimetric method is also suggested, whereas panthenol, after alkaline hydrolysis is treated with ninhydrin. The fluorescent product was found to have excitation lambda(max) at 385 nm and emission lambda(max) at 465 nm. The method showed high sensitivity with linearity range from 0.01 to 3 microg/ml. The lower limit of detection (LOQ) reached 0.005 microg/ml. Validation of both methods was carried out and the two methods were applied for determination of panthenol in some cosmetic and pharmaceutical formulations.