A comparative study of two analytical techniques for the simultaneous determination of amprolium HCl and ethopabate from combined dosage form and in presence of their alkaline degradation.

Chemometric and separation-based techniques (HPLC) are the most applicable and versatile analytical techniques for the analysis of multicomponent mixtures, in the present contribution, a comparison was highlighted between the two analytical techniques of utmost importance as stability indicating assays: UV-spectrophotometry and HPLC-UV focusing on the greenness of each for the simultaneous determination of amprolium HCl (AMP) and ethopabate (ETHOP) in the presence of their alkaline degradation products. The first method was chemometric methods applied were PLS-1, GA-PLS and GA-ANN. To compare the prediction ability of the models, a 4-factor 5-level experimental design was used to establish a calibration set of 25 mixtures containing different ratios of the drugs and their degradation products. The validity of the proposed methods was assessed using an independent validation set of 5 mixtures. The comparison between the different models showed the superiority of ANN model in solving the highly overlapped spectra of the quaternary mixture, yet using inexpensive and easy to handle instruments like the UV-VIS spectrophotometer. The ANN method was used for the quantitative analysis of the drugs in pharmaceutical dosage form via handling the UV spectral data. The second method was based on liquid chromatographic HPLC determination of AMP and ETHOP using C18 column (250 × 4.6 mm2)-PRONTOSIL 5 µm, a mobile phase consisting of methanol: Hexane sulphonic acid sodium salt at (pH = 3.4 ± 0.2) adjusted by orthophosphoric acid (55: 45 v/v). Quantitation was achieved with UV detection at 270 nm at temperature 24 °C. Linearity, accuracy and precision were found to be acceptable over the concentration range of 10.0-70.0 and 1.0-25.0 µg·mL-1 for AMP and ETHOP, respectively. The proposed methods could be successfully applied for the routine analysis of the studied drugs either in their pure bulk powders or in their pharmaceutical preparations without any preliminary separation step. The results obtained were statistically compared with those obtained by applying the reported method.

Development and Validation of HPLC Fluorescence and UPLC/DAD Stability-Indicating Methods for Determination of Hepatitis C Antiviral Agent Daclatasvir.

Background: Few stability-indicating chromatographic methods were published for determination of daclatasvir. All used UV detection. Objective: This work aimed to develop rapid, specific, and novel stability-indicating methods using HPLC with fluorescence detection and ultra performance liquid chromatography (UPLC) with UV detection for the determination of daclatasvir in bulk powder and in its dosage form. Methods: The drug was subjected to hydrolysis (acidic and alkaline) as per International Conference on Harmonization (ICH) guidelines. The fragmentation pattern of the drug was studied using LC-MS. Separation was carried out first by HPLC using Thermo BDS Hypersil Phenyl (300 mm × 4 mm, 5 µm) column and a mobile phase consisting of ammonium phosphate buffer (0.02 M) pH 3-methanol (40+60, v/v) at flow rate 1 mL/min. Quantitation was achieved by fluorescence detection at 305 and 457 nm for excitation and emission, respectively. The second method used UPLC equipped with diode-array detector. Acquity BEH C18 (50 mm × 2.1 mm, 1.7 µm) column was used with the same mobile phase at flow rate 0.4 mL/min and detection wavelength at 305 nm. ICH guidelines were used for validation. Results: The mean percentage recovery ± SD values for tablet assay were found to be 101.12 ± 0.655 and 99.78 ± 0.632 by HPLC and UPLC methods, respectively. The assay results showed a good agreement with the reported method. Conclusions: The developed HPLC and UPLC methods provide simple, accurate, and reproducible analysis of daclatasvir without interference from degradates. Highlights: This is the first research using fluorescence detection in a stability-indicating chromatographic method for determination of daclatasvir.

Comparative stability-indicating chromatographic methods for determination of 4-hexylresorcinol in pharmaceutical formulation and shrimps.

Three chromatographic stability-indicating methods were developed for determination of 4-hexylresorcinol in pure form and in a pharmaceutical formulation. Method A was based on a gradient elution liquid chromatographic HPLC determination of 4-hexylresorcinol, its related impurities and in presence of its degradation products. UPLC-MS/MS (Method B) was described for determination of the cited drug in presence of its degradation products. Method C was a thin- layer chromatography (TLC)-densitometry method for the separation and determination of the active ingredient, one of its related impurities and in presence of its degradation products. The mechanism of alkali, oxidative and photodegradation of 4-hexylresorcinol was studied according to ICH guidelines. The degradation products were characterized by the LC-MS/MS method. Methods A and B were applicable for determination of 4-hexylresorcinol residues in shrimp meat. The studied drug was easily degraded in alkali medium giving toxic compounds. The results obtained by the proposed methods were statistically analyzed and compared with those obtained by applying a reported method.

Successive ratio subtraction coupled with constant multiplication spectrophotometric method for determination of hydroquinone in complex mixture with its degradation products, tretinoin and methyl paraben.

A sensitive and selective stability-indicating successive ratio subtraction coupled with constant multiplication (SRS-CM) spectrophotometric method was studied and developed for the spectrum resolution of five component mixture without prior separation. The components were hydroquinone in combination with tretinoin, the polymer formed from hydroquinone alkali degradation, 1,4 benzoquinone and the preservative methyl paraben. The proposed method was used for their determination in their pure form and in pharmaceutical formulation. The zero order absorption spectra of hydroquinone, tretinoin, 1,4 benzoquinone and methyl paraben were determined at 293, 357.5, 245 and 255.2 nm, respectively. The calibration curves were linear over the concentration ranges of 4.00-46.00, 1.00-7.00, 0.60-5.20, and 1.00-7.00 µg mL(-1) for hydroquinone, tretinoin, 1,4 benzoquinone and methyl paraben, respectively. The pharmaceutical formulation was subjected to mild alkali condition and measured by this method resulting in the polymerization of hydroquinone and the formation of toxic 1,4 benzoquinone. The proposed method was validated according to ICH guidelines. The results obtained were statistically analyzed and compared with those obtained by applying the reported method.

Chromatographic Determination of Aminoacridine Hydrochloride, Lidocaine Hydrochloride and Lidocaine Toxic Impurity in Oral Gel.

Two sensitive and selective analytical methods were developed for simultaneous determination of aminoacridine hydrochloride and lidocaine hydrochloride in bulk powder and pharmaceutical formulation. Method A was based on HPLC separation of the cited drugs with determination of the toxic lidocaine-related impurity 2,6-dimethylaniline. The separation was achieved using reversed-phase column C18, 250 × 4.6 mm, 5 µm particle size and mobile phase consisting of 0.05 M disodium hydrogen phosphate dihydrate (pH 6.0 ± 0.2 adjusted with phosphoric acid) and acetonitrile (55 : 45, v/v). Quantitation was achieved with UV detection at 240 nm. Linear calibration curve was in the range of 1.00-10.00, 13.20-132.00 and 1.32-13.20 µg mL(-1) for aminoacridine hydrochloride, lidocaine hydrochloride and 2,6-dimethylaniline, respectively. Method B was based on TLC separation of the cited drugs followed by densitometric measurement at 365 nm on the fluorescent mode for aminoacridine hydrochloride and 220 nm on the absorption mode for lidocaine hydrochloride. The separation was carried out using ethyl acetate-methanol-acetic acid (65 : 30 : 5 by volume) as a developing system. The calibration curve was in the range of 25.00-250.00 ng spot(-1) and 0.99-9.90 µg spot(-1) for aminoacridine hydrochloride and lidocaine hydrochloride, respectively. The results obtained were statistically analyzed and compared with those obtained by applying the manufacturer's method.

Spectrophotometric stability-indicating methods for the determination of leflunomide in the presence of its degradates.

Five simple and sensitive methods were developed for the determination of leflunomide (I) in the presence of its degradates 4-trifluoromethyl aniline (II) and 3-methyl-4-carboxy isoxazole (III). Method A was based on differential derivative spectrophotometry by measuring the delta(1)D value at 279.5 nm. Beer's law was obeyed in the concentration range of 2.00-20.00 microg/mL with mean percentage accuracy of 100.07 +/- 1.32. Method B depended on first-derivative spectrophotometry and measuring the amplitude at 253.4 nm. Beer's law was obeyed in the concentration range of 2.00-16.00 microg/mL with mean percentage accuracy of 98.42 +/- 1.61. Method C was based on the reaction of degradate (II) with 2,6-dichloroquinone-4-chloroimide (Gibbs reagent). The colored product was measured at 469 nm. Method D depended on the reaction of degradate (II) with para-dimethyl aminocinnamaldehyde (p-DAC). The absorbance of the colored product was measured at 533.4 nm. Method E utilized 3-methyl-2-benzothiazolinone hydrazone in the presence of cerric ammonium sulfate with degradate (II). The green colored product was measured at 605.5 nm. The linearity range was 40.00-280.00, 2.40-24.00, and 30-250 microg/mL with mean percentage accuracy of 100.75 +/- 1.21, 100.13 +/- 1.45, and 99.74 +/- 1.39 for Methods C-E, respectively. All variables were studied to optimize the reaction conditions. The proposed methods have been successfully applied to the analysis of leflunomide in pharmaceutical dosage forms and the results were statistically compared with that previously reported.

Application of first-derivative, ratio derivative spectrophotometry, TLC-densitometry and spectrofluorimetry for the simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical dosage forms and plasma.

Four sensitive methods are described for the direct determination of telmisartan (TELM) and hydrochlorothiazide (HCT) in combined dosage forms without prior separation. The first method is a first derivative spectophotometry (1D) using a zero- crossing technique of measurement at 241.6 and 227.6 nm for TELM and HCT, respectively. The second method is the first derivative of ratio spectrophotometry (1DD) where the amplitudes were measured at 242.7 nm for TELM and 274.9 nm for HCT. The third method is based on TLC separation of the two drugs followed by the densitometric measurements of their spots at 295 and 225 nm for TELM and HCT, respectively. The separation was carried out on silica gel 60 F254 using butanol: ammonia 25% (8:2 v/v) as mobile phase. The fourth method is spectrofluorimetric determination of TELM, depending on measuring the native fluorescence of the drug in 1 M sodium hydroxide at lambda excitation 230 nm and emission at 365 nm. The proposed methods were applied successfully for the determination of the two drugs in bulk powder and in pharmaceutical formulations. The spectrofluorimetric method was utilized for the analysis of TELM in human plasma.

Determination of trimetazidine dihydrochloride in the presence of its acid-induced degradation products.

Three methods are presented for the determination of trimetazidine dihydrochloride in the presence of its acid-induced degradation products. The first method was based on measurement of first-derivative D1 value of trimetazidine dihydrochloride at 282 nm over a concentration range of 8.00-56.00 microg/mL with mean percentage accuracy of 99.80+/-1.17. The second method was based on first derivative of the ratio spectra DD1 at 282 nm over the same concentration range with the percentage accuracy of 99.14+/-0.68. The third method was based on separation of trimetazidine dihydrochloride from its acid-induced degradation products followed by densitometric measurement of the spots at 215 nm. The separation was performed on silica gel 60 F254 using methanol-ammonia (100+/-1.5, v/v) as mobile phase. This method was applicable for determination of the intact drug in the presence of its degradation products over a concentration range of 2.00-9.00 microg/spot with mean percentage accuracy of 99.86+/-0.92. The proposed methods were successfully applied for the determination of trimetazidine dihydrochloride in bulk powder, laboratory-prepared mixtures containing different percentages of degradation products, and pharmaceutical dosage forms. The validity of results was assessed by applying the standard addition technique. The results obtained agreed statistically with those obtained by the reported method.

Fluorimetric determination of some antibiotics in raw material and dosage forms through ternary complex formation with terbium (Tb(3+)).

A highly sensitive and specific fluorimetric method was developed for the determination of cefazolin sodium I, cefoperazone sodium II, ceftriaxone sodium III, and cefixime IV. The proposed method involves the formation of ternary complex with Tb(3+) in the presence of Tris buffer. The quenching of the terbium fluorescence due to the complex formation was quantitative for the four studied drugs. The effect of pH, concentration of Tris buffer and terbium were studied. The formation of the complex was highly dependent on the pH. The optimum pH was found to be pH 8 for cefazolin sodium I, ceftriaxone sodium III, cefixime IV and pH 10 for cefoperazone sodium II. The optimum concentration for Tb(3+) was found 1 ml of 10(-4) M solution and for Tris buffer 1 ml of the prepared solution. Under the described conditions, the proposed method was applicable over the concentration range 8.79 x 10(-6)-7.91 x 10(-5), 9.7 x 10(-6)-4.49 x 10(-)5, 6.10 x 10(-6)-2.50 x 10(-5), and 4.92 x 10(-6)-2.95 x 10(-5) mol with mean percentage accuracy of 99.79+/-0.24, 98.97+/-1.25, 100.05+/-0.79, and 100.15+/-0.54 for I, II, III, and IV, respectively. The proposed method was applied successfully for the determination of studied drugs in bulk powder and in pharmaceutical formulations. The results obtained by applying the described method were statistically analyzed and compared with those obtained by applying the official method. The proposed method was used as stability indicating method for the determination of the studied drugs in the presence of their degradation products.

Use of pi-acceptors for spectrophotometric determination of dicyclomine hydrochloride.

Simple, rapid, accurate, and sensitive spectrophotometric methods are described for the determination of dicyclomine hydrochloride. The methods are based on the reaction of this drug as an n-electron donor with 2,3-dichloro-5,6-dicyano-p-benzoqunione (DDQ), p-chloranilic acid (p-CA), and chloranil (CL) as pi-acceptors to give highly colored complex species. The colored products are measured spectrophotometrically at 456, 530, and 650 nm for DDQ, p-CA, and CL, respectively. Optimization of the different experimental conditions were studied. Beer's law was obeyed in concentration ranges of 20-100, 50-250, and 80-600 microg/mL for DDQ, pCA, and CL, respectively. Colored complexes are produced in organic solvents and are stable for at least 1 h. The methods were applied to Spasmorest antispasmotic tablets and ampoules with good accuracy and precision.

Stability-indicating methods for the determination of linezolid in the presence of its alkaline-induced degradation products.

Three methods are presented for the determination of linezolid in the presence of its alkaline-induced degradation products. The first method was based on separation of linezolid from its alkaline degradation product by TLC followed by densitometric measurement of the spots of intact drug at 244 nm. The separation was carried out on silica gel 60 F(254) using isobutanol:ammonia (9:1 v/v) as a mobile phase. The second method was based on first derivative (1)D ultraviolet spectrophotometry with zero crossing point and peak to base measurement. The (1)D value at 251.4 nm was selected for the assay of linezolid in the presence of degradation product. The third method was depended on the first derivative of the ratio spectra (1)DD by measurement of the value at 263.6 nm. The proposed methods were successfully applied to the determination of the drug in bulk powder, in laboratory prepared mixtures with its degradation product and in commercial tablets.

Spectrophotometric determination of clobetasol propionate, halobetasol propionate, quinagolide hydrochloride, through charge transfer complexation.

Two spectrophotometric procedures are described for the determination of clobetasol propionate(I), halobetasol propionate(II) (corticosteroids) and quinagolide hydrochloride(III) (prolactin inhibitor). For corticosteroid drugs, the procedures are based on the formation of phenyl hydrazones of the corticosteroids which are subsequently subjected to charge transfer complexation reaction with either 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) as pi-acceptor or with iodine as sigma-acceptor. Prolactin inhibitor was reacted directly with the previous reagents. The molar ratios of the reactants were established and the experimental conditions were studied giving maximum absorption at 588 and 290 nm with DDQ and iodine methods, respectively for the three drugs. The concentration ranges were 20-150,50-300, and 20-80 microg ml(-1) in DDQ method for (I), (II), and (III), respectively and 13-20,15-40, and 8-32 microg ml(-1) in iodine method for (I), (II) and (III), respectively.

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.