Nesrin K. Ramadan

Rapid and Highly Sensitive HPLC and TLC Methods for Quantitation of Amlodipine Besilate and Valsartan in Bulk Powder and in Pharmaceutical Dosage Forms and in Human Plasma

Two simple, sensitive, and specific high-performance liquid chromatography and thin-layer chromatography methods were developed for the simultaneous estimation of Amlodipine besilate (AM) and Valsartan (VL). Separation by HPLC was achieved using a xTerra C18 column and methanol /acetonitrile /water/ 0.05% triethylamine in a ratio 40:20:30:10 by volume as mobile phase, pH was adjusted to 3 ± 0.1 with o-phosphoric acid. The flow rate was 1.2 mL min−1. The linearity range was 0.2 to 2 µg mL−1 for amlodipine besilate and 0.4 to 4 µg mL−1 for Valsartan with a mean percentage recovery of 99.59 ± 0.523% and 100.61 ± 0.400% for amlodipine besilate and valsartan, respectively. The TLC method used silica gel 60 F254 plates; the optimized mobile phase was ethyl acetate/ methanol / ammonium hydroxide (55:45:5 by volume). Quantitatively, the spots were scanned densitometrically at 237 nm. The range was 0.5–4.0 µg spot−1 for amlodipine besilate and 2.0–12.0 µg spot−1 for valsartan. The mean percentages recovery was 99.80 ± 0.451% and 100.61 ± 0.363% for amlodipine besilate and valsartan, respectively. The HPLC method was found to be simple, selective, precise, and reproducible for the estimation of both drugs from spiked human plasma.

Spectrophotometric methods for simultaneous determination of ternary mixture of amlodipine besylate, olmesartan medoxomil and hydrochlorothiazide.

Four, accurate, precise, and sensitive spectrophotometric methods are developed for the simultaneous determination of a ternary mixture containing amlodipine besylate (AM), olmesartan medoxomil (OL) and hydrochlorothiazide (HZ), where AM is determined at its λ(max) 364.6 nm ((0)D), while (OL) and (HZ) are determined by different methods. Method (A) depends on determining OL and HZ by measuring the second derivative of the ratio spectra ((2)DD) at 254.4 and 338.6 nm, respectively. Method (B) is first derivative of the double divisor ratio spectra (D-(1)DD) at 260.4 and 273.0 nm for OL and HZ, respectively. Method (C) based on successive spectrophotometric resolution technique (SSRT). The technique starts with the ratio subtraction method then measuring OL and HZ at their isoabsorptive point at 260.0 nm, while HZ is measured using the amplitude of first derivative at 335.2 nm. Method (D) is mean centering of the ratio spectra (MCR) at 252.0 nm and 220.0 nm for OL and HZ, respectively. The specificity of the developed methods is investigated by analyzing laboratory prepared mixtures containing different ratios of the three drugs and their combined dosage form. The obtained results are statistically compared with those obtained by the official or reported methods, showing no significant difference with respect to accuracy and precision at p=0.05.

Novel potentiometric application for the determination of pantoprazole sodium and itopride hydrochloride in their pure and combined dosage form

Three sensitive and selective polyvinyl chloride (PVC) matrix membrane electrodes were developed and investigated. Sensor I was developed using tetraheptylammonium bromide (THB) as an anion exchanger with 2-nitrophenyl octyl ether (2-NPOE) as a plasticizer for the determination of the anionic drug pantoprazole sodium sesquihydrate (PAN). To determine the cationic drug itopride hydrochloride (ITH), two electrodes (sensors II and III) were developed using potassium tetrakis(4-chlorophenyl) borate (KTCPB) as a cation exchanger with dioctyl phthalate (DOP) as a plasticizer. Selective molecular recognition components, 2-hydroxypropyl-β-cyclodextrin (2-HP βCD) and 4-tert-butylcalix[8]arene (tBC8), were used as ionophores to improve the selectivity of sensors II and III, respectively. The proposed sensors had a linear dynamic range of 1×10(-5) to 1×10(-2) mol L(-1) with Nernstian slopes of -54.83±0.451, 56.90±0.300, and 51.03±1.909 mV/decade for sensors I, II and III, respectively. The Nernstian slopes were also estimated over the pH ranges of 11-13, 3.5-8 and 4-7 for the three sensors, respectively. The proposed sensors displayed useful analytical characteristics for the determination of PAN and ITH in bulk powder, in laboratory prepared mixtures and in combined dosage forms with clear discrimination from several ions, sugars and some common drug excipients. The method was validated according to ICH guidelines. Statistical comparison between the results from the proposed method and the results from the reference methods showed no significant difference regarding accuracy and precision.

Stability-Indicating Methods for the Determination of Ornidazole in The Presence of its Degradate According to ICH Guidelines

Four simple, sensitive, selective and precise methods were developed for the determination of Ornidazole (OZ) in presence of its degradation product. The first method was based on first derivative spectrophotometry D1 and measuring the peak amplitude of D1 spectra at 290.4 and 332 nm. The second method was depended on measuring the peak amplitude of the first derivative of the ratio spectra DD1 at 288.5 and 328 nm. The third method was the mean centering of the ratio spectra one (MCR), which allowed the determination of OZ in presence of its degradate and the concentration of OZ was determined by measuring the amplitude at 312.8 nm. Separation and determination of OZ by HPLC in the forth method was achieved using Lichrosorb RP-18 column and acetonitrile: water, (50:50v/v), 0.2% triethylamine, the pH was adjusted to 4 using o-phosphoric acid. The flow rate was 1 mL min-1. Beer’s law was obeyed in concentration range 5–30 μg/ml for the first three methods. The linearity range in the forth method was 2-20 μg/ml. The proposed methods were used to determine OZ in its pure powdered form with mean percentage recoveries of 99.86 ± 1.249% and 99.98 ± 0.868% for OZ at 290.4 and 332 nm respectively, in D1 method. In DD1 method, the mean percentage recoveries were 100.11 ± 1.020% and 100.15 ± 1.043% at 288.5 and 328 nm respectively. While in MCR and HPLC methods, the mean percentage recoveries were 100.09 ± 0.387% and 100.00 ± 1.302% respectively. The degradation product was obtained in alkaline stress condition, separated, and identified by LC-MS spectral analysis, from which the degradation product was confirmed. The four methods were validated according to International Conference on Harmonization. The four methods were found to be specific for OZ in presence of up to 80% of its degradation product in the first three methods. The four proposed methods were successfully applied for the determination of OZ in Tibezole® tablets. Statistical comparison between the results obtained by these methods and the reported method for the determination of the drug in its pharmaceutical formulation was done, and it was found that there was no significant difference between them.

Simultaneous determination of a binary mixture of pantoprazole sodium and itopride hydrochloride by four spectrophotometric methods

Four simple, sensitive, accurate and precise spectrophotometric methods were developed for the simultaneous determination of a binary mixture containing Pantoprazole Sodium Sesquihydrate (PAN) and Itopride Hydrochloride (ITH). Method (A) is the derivative ratio method ((1)DD), method (B) is the mean centering of ratio spectra method (MCR), method (C) is the ratio difference method (RD) and method (D) is the isoabsorptive point coupled with third derivative method ((3)D). Linear correlation was obtained in range 8-44 μg/mL for PAN by the four proposed methods, 8-40 μg/mL for ITH by methods A, B and C and 10-40 μg/mL for ITH by method D. The suggested methods were validated according to ICH guidelines. The obtained results were statistically compared with those obtained by the official and a reported method for PAN and ITH, respectively, showing no significant difference with respect to accuracy and precision.

Comparative study between recent methods manipulating ratio spectra and classical methods based on two-wavelength selection for the determination of binary mixture of antazoline hydrochloride and tetryzoline hydrochloride.

A comparative study was developed between two classical spectrophotometric methods (dual wavelength method and Vierordts method) and two recent methods manipulating ratio spectra (ratio difference method and first derivative of ratio spectra method) for simultaneous determination of Antazoline hydrochloride (AN) and Tetryzoline hydrochloride (TZ) in their combined pharmaceutical formulation and in the presence of benzalkonium chloride as a preservative without preliminary separation. The dual wavelength method depends on choosing two wavelengths for each drug in a way so that the difference in absorbance at those two wavelengths is zero for the other drug. While Vierordts method, is based upon measuring the absorbance and the absorptivity values of the two drugs at their λ(max) (248.0 and 219.0 nm for AN and TZ, respectively), followed by substitution in the corresponding Vierordts equation. Recent methods manipulating ratio spectra depend on either measuring the difference in amplitudes of ratio spectra between 255.5 and 269.5 nm for AN and 220.0 and 273.0 nm for TZ in case of ratio difference method or computing first derivative of the ratio spectra for each drug then measuring the peak amplitude at 250.0 nm for AN and at 224.0 nm for TZ in case of first derivative of ratio spectrophotometry. The specificity of the developed methods was investigated by analyzing different laboratory prepared mixtures of the two drugs. All methods were applied successfully for the determination of the selected drugs in their combined dosage form proving that the classical spectrophotometric methods can still be used successfully in analysis of binary mixture using minimal data manipulation rather than recent methods which require relatively more steps. Furthermore, validation of the proposed methods was performed according to ICH guidelines; accuracy, precision and repeatability are found to be within the acceptable limits. Statistical studies showed that the methods can be competitively applied in quality control laboratories.

Validated spectrophotometric methods for simultaneous determination of troxerutin and carbazochrome in dosage form

Four simple, accurate, sensitive and precise spectrophotometric methods were developed and validated for simultaneous determination of Troxerutin (TXN) and Carbazochrome (CZM) in their bulk powders, laboratory prepared mixtures and pharmaceutical dosage forms. Method A is first derivative spectrophotometry (D(1)) where TXN and CZM were determined at 294 and 483.5 nm, respectively. Method B is first derivative of ratio spectra (DD(1)) where the peak amplitude at 248 for TXN and 439 nm for CZM were used for their determination. Method C is ratio subtraction (RS); in which TXN was determined at its λmax (352 nm) in the presence of CZM which was determined by D(1) at 483.5 nm. While, method D is mean centering of the ratio spectra (MCR) in which the mean centered values at 300 nm and 340.0 nm were used for the two drugs in a respective order. The two compounds were simultaneously determined in the concentration ranges of 5.00-50.00 μg mL(-1) and 0.5-10.0 μg mL(-1) for TXN and CZM, respectively. The methods were validated according to the ICH guidelines and the results were statistically compared to the manufacturers method.

Simultaneous determination of metronidazole and spiramycin in bulk powder and in tablets using different spectrophotometric techniques

Metronidazole (MZ) is an anti-infective drug used in the treatment of anaerobic bacterial and protozoa infections in humans. It is also used as a veterinary antiparasitic drug. Spiramycin (SP) is a medium-spectrum antibiotic with high effectiveness against Gram-positive bacteria. Three simple, sensitive, selective and precise spectrophotometric methods were developed and validated for the simultaneous determination of MZ and SP in their pure form and in pharmaceutical formulations. In methods A and B, MZ was determined by the application of direct spectrophotometry and by measuring its zero-order (D(0)) absorption spectra at its λ(max) = 311 nm. In method A, SP was determined by the application of first derivative spectrophotometry (D(1)) and by measuring the amplitude at 218.3 nm. In method B, the first derivative of the ratio spectra (DD(1)) was applied, and SP was determined by measuring the peak amplitude at 245.6 nm. Method C entailed mean centering of the ratio spectra (MCR), which allows the determination of both MZ and SP. The methods developed were used for the determination of MZ and SP over a concentration range of 5-25 µg ml(-1). The proposed methods were used to determine both drugs in their pure, powdered forms with mean percentage recoveries of 100.16 ± 0.73 for MZ in methods A and B, 101.10 ± 0.90 in method C, 100.09 ± 0.70, 100.02 ± 0.88 and 100.49 ± 1.26 for SP in methods A, B and C, respectively. The proposed methods were proved using laboratory-prepared mixtures of the two drugs and were successfully applied to the analysis of MZ and SP in tablet formulation without any interference from each other or from the excipients. The results obtained by applying the proposed methods were compared statistically with a reported HPLC method and no significant difference was observed between these methods regarding both accuracy and precision.

Development and Validation of Spectrophotometric and Spectrofluorimetric Methods for the Determination of Cyclobenzaprine HCl

Five simple and sensitive methods were developed for the determination of cyclobenzapirine hcl (CB) in presence of its degradation product anthraquinone (AQ). Method A dual wavelength spectrophotometry (DW); where two wavelengths were selected for the drug 283 and 306 nm in such a way that the difference in absorbance was zero for its degradation. Method B ratio difference spectrophotometry (RD) was depended on measuring the ratio difference between 290 and 305 nm. Method C was depended on measuring the peak amplitude of the first derivative of the ratio spectra (1DD) at 282 and 306 nm. Method D Isoabsorptive Point (ISO) at 280 nm Coupled with Second Derivative (2D). Method E depending on spectrofluorimetric determination of cyclobenzapirine HCl through quenching of uranyl acetate with ʎexi 228 nm and ʎem at 458 nm. Linearties were obtained in concentration range 5 μg/ml – 30 μg/ml in case of methods A, B, C and D, while in case of methods E linearity was obtained in concentration range of 1 μg/ml –10 μg/ml. The five methods were found to be specific for CB in presence of different concentration % of its degradation product. The five proposed methods were successfully applied for the determination of CB in Multirelax tablets. Statistical comparison between the results obtained by the proposed methods and that obtained by the official one for the determination of the drug was done, founding that there were no significant differences between them.

Stability-indicating high-performance liquid chromatography and thin-layer chromatography methods for the determination of cyclobenzaprine hydrochloride and asenapine maleate

Two sensitive, specific, and selective stability-indicating chromatographic methods were developed for the determination of cyclobenzaprine HCl (CZ) and asenapine maleate (AS) in pure forms, in the presence of their degradation products and in their pharmaceutical formulations. The first method was an isocratic reversed-phase high-performance liquid chromatography (RP-HPLC). Analysis was performed on cyano column using a mobile phase consisting of acetonitrile—(0.05 m) potassium dihydrogen phosphate buffer (pH 3 ± 0.1) (70:30, v/v) with a flow rate of 1.5 mL min−1 and ultraviolet (UV) detection at 290 nm for the determination of CZ, and methanol—( 0.05 m) potassium dihydrogen phosphate buffer (pH 6 ± 0.1) (70:30, v/v) with a flow rate of 1.5 mL min−1 and UV detection at 220 nm for the determination of AS. The second method was thin-layer chromatography (TLC), using silica gel 60 F254 plates and toluene—methanol—chloroform-ammonia solution 33% (5:3:6:0.1, by volume) as the mobile phase for the two drugs. The spots were scanned densitometrically at 290 and 220 nm for the determination of CZ and AS, respectively. The methods were validated according to the International Conference on Harmonization (ICH) guidelines, and the acceptance criteria for linearity, accuracy, precision, specificity, and system suitability were met in all cases. The linearity ranges were 2.5–25 μg mL−1 for the RP-HPLC method and 5–50 μg band−1 for the TLC method for both drugs. The limits of detection for the RP-HPLC method were 0.250 and 0.578 for CZ and AS, respectively, while the limits of quantification were 0.758 and 1.572 for CZ and AS, respectively. The limits of detection for the TLC method were 1.355 and 1.284 for CZ and AS, respectively, while the limits of quantification were 4.472 and 3.891 for CZ and AS, respectively. The results were compared statistically at a 95% confidence level with the reported methods. There were no significant differences between the mean percentage recoveries and the precisions of the two methods.