Alkaline-induced Degradation Product Research Articles

Green Simultaneous Chromatographic Separation of Pyridostigmine Bromide and Its Related Substances in Pure Form, Tablets and Spiked Human Plasma.

A green, accurate and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for simultaneous quantitative determination of pyridostigmine bromide (PR), impurity B (IMP B);3-hydroxy-N-methylpyridinium bromide and impurity A (IMP A); pyridin-3-yl-dimethylcarbamate. The two pharmacopeial impurities are also its main inactive metabolites. Furthermore, IMP B is known to be its alkaline-induced degradation product. Achievable separation of the studied components required silica gel HPTLC F254 plates as a stationary phase and acetone: acetic acid (80:20, v/v) as a developing system. Scanning of the separated bands was done at 260nm. According to green solvent selection guidelines, acetone and acetic acid are eco-friendly solvents. Validation of the developed method was insured by its acquiesce to international conference on harmonization (ICH) guidelines. The introduced method was successfully achieved for the quantitative determination of PR, IMP B and IMP A in the range of 0.4-10, 2-11 and 0.4-3.5μg/band, respectively. Successful application of the developed method was done for determination of PR in human plasma in the range of 0.6-10μg/band, so the proposed HPTLC can be applied in the pharmacokinetic studies. The studied drug was also analyzed in Mestinon® tablets using the developed method.

Different chromatographic methods for the simultaneous determination of vitamin E and vinpocetine in their combined dosage form and in the presence of the alkaline-induced degradation product of vinpocetine

Accurate, selective, and sensitive thin-layer chromatography (TLC)—densitometry and reversed-phase high-performance liquid chromatography (RP-HPLC) methods have been developed and validated for the simultaneous determination of vitamin E (VIT E) and vinpocetine (VINP) in the presence of the alkaline-induced degradation product of vinpocetine (DEG). The proposed TLC—densitometric method depends on the separation and quantitation of VIT E, VINP, and VINP alkaline-induced degradation product on TLC silica gel 60 F254 plates, using methanol—chloroform—ethyl acetate—glacial acetic acid—ammonia solution (6:2:2:0.5:0.1, by volume) as the developing system followed by densitometric measurement at 235 nm. The studied components were well resolved from each other with significantly different RF values of 0.81, 0.62, and 0.41 for VIT E, VINP, and DEG, respectively. On the other hand, the developed RP-HPLC method was based on the separation of the studied components using 0.05 M KH2PO4 (adjusted to pH = 3) and methanol in gradient elution mode on C8 column at a flow rate of 1.5 mL min−1 and ultraviolet (UV) detection at 235 nm. The studied components were well resolved from each other with significantly different Rt values of 10.90, 2.89, and 1.90 min for VIT E, VINP, and DEG, respectively. The developed methods were validated according to the International Conference on Harmonization (ICH) guidelines demonstrating good accuracy and precision. The results were statistically compared with those obtained by the reported method, and no significant difference was found. The developed methods are the first developed stability-indicating assay methods (SIAMs) for the analysis of the studied binary mixture.

Stability Indicating Spectrophotometric Methods for Determination of Vitamin E and Vinpocetine in Their Combined Dosage Form

Stability indicating spectrophotometric methods have been developed and validated for determination of Vitamin E (VIT E) and Vinpocetine (VINP) in presence of alkaline induced degradation product of Vinpocetine (DEG) without prior separation. In method (A), two wavelengths were selected for each drug in such a way that the difference in absorbance was zero for the other drugs. Absorbance difference between 217.4 and 234.4 nm were used to determine VIT E while difference between 250.4 and 314.0 nm were selected for VINP determination and the difference between 258.6 and 312.0 nm were selected to determine DEG. In method (B), area under the curve method (AUC) was used for measuring VINP concentration by direct measuring AUC in the range of 330-350 nm, at which no interference from other components. While for determination of VIT E and DEG, the ternary mixture was divided by standard spectrum of 6 μg/mL VINP, in the obtained ratio spectra VIT E and DEG has been directly determined at 210.0 and 230.0 nm, respectively. In method (C), VIT E and DEG were determined by second derivative of ratio spectra (2DD) method by measuring their peak amplitudes at 215.2 and 237.2 nm, respectively, using 6 μg/mL standard spectrum of VINP as a divisor, while the absorbance at isoabsorptive point (λiso=264.4 nm) was used to determine the total concentration of VINP and DEG (where no contribution from VIT E) in the mixture and then by subtraction, VINP concentration could be obtained. The developed methods were validated according to ICH guidelines revealing good accuracy and precision. The results obtained by the proposed methods were statistically compared with those obtained by the reported HPLC methods and no significant difference was obtained. These methods are the first developed SIAM ones for analysis of the studied components.

Stability indicating methods for the analysis of cefprozil in the presence of its alkaline induced degradation product

Three simple, specific, accurate and precise spectrophotometric methods were developed for the determination of cefprozil (CZ) in the presence of its alkaline induced degradation product (DCZ). The first method was the bivariate method, while the two other multivariate methods were partial least squares (PLS) and spectral residual augmented classical least squares (SRACLS). The multivariate methods were applied with and without variable selection procedure (genetic algorithm GA). These methods were tested by analyzing laboratory prepared mixtures of the above drug with its alkaline induced degradation product and they were applied to its commercial pharmaceutical products.

Development and validation of three spectrophotometric methods for determination of pyridostigmine bromide in the presence of its alkaline-induced degradation product

Three simple, accurate and validated spectrophotometric methods were developed for the determination of pyridostigmine bromide (PB) in presence of its alkaline-induced degradation product, 3-hydoxy- N -methylpyridinium bromide (3-OH NMP) in powder form and in pharmaceutical formulations. Method A, is first derivative method ( 1 D), which is based on measuring the peak amplitude of the first derivative spectra ( 1 D) of PB at 260 nm. Method B, is first derivative of ratio spectra ( 1 DD) which allows the determination of PB at 267.4 nm using (50 µg/mL) of 3-OH NMP as a suitable devisor. Finally, method C depends on mean centering of ratio spectra (MCR) of PB with different concentrations, which were recorded over 200-400 nm and divided by the spectrum of 20 µg/mL of 3-OH NMP as a devisor. The obtained ratio spectra were mean centered and the concentrations of PB were then determined from the calibration graphs obtained by measuring the amplitudes at 338 nm. The proposed methods were successfully applied for assay of PB both in pure form and in pharmaceutical formulations. The proposed methods were validated in compliance with International Conference on Harmonization (ICH) guidelines. The results obtained by the developed methods were statistically compared to those obtained by the reported HPLC method using F- and student's t-tests showing no significant difference regarding both accuracy and precision.

Development and Validation of a Stability-Indicating High-Performance Thin-Layer Chromatographic Method for Determination of Pyridostigmine Bromide in the Presence of Its Alkaline-Induced Degradation Product

A validated, sensitive, and highly selective stability-indicating high-performance thin-layer chromatographic (HPTLC) method has been adopted for the quantitative determination of pyridostigmine bromide in the presence of its alkaline-induced degradation product and in pharmaceutical formulations. 3-hydroxy-N-methyl pyridinium bromide (3-OH NMP) is the metabolite, impurity, and alkaline-induced degradation product of pyridostigmine bromide (PB). Pyridostigmine bromide and its alkaline-induced degradation product were separated on silica gel HPTLC F254 plates using methanol–ethyl acetate–triethyl amine–glacial acetic acid (9:1:0.5:0.05 by volume) as the developing system followed by scanning of the separated bands at 270 nm over a concentration range of 2–10 μg band−1 with mean percentage recoveries of 99.84% (SD 1.384). The proposed method was successfully applied to the analysis of pyridostigmine bromide both in bulk powder and in pharmaceutical formulation without interference from other dosage form add...

Different approaches in manipulating ratio spectra applied for the analysis of Cefprozil in presence of its alkaline-induced degradation product: A comparative study

Four simple, accurate and precise stability-indicating spectrophotometric methods manipulating ratio spectra were developed and validated for simultaneous determination of Cefprozil (CZ) and its alkaline-induced degradation product (DCZ) without prior separation namely; ratio difference, mean centering, derivative ratio using Savitsky–Golay filter and continuous wavelet transform. The accuracy, precision and linearity ranges of the proposed methods were determined. The methods were validated and the specificity was assessed by analyzing synthetic mixtures containing the drug and its degradate. The four methods were applied for the determination of the cited drug in tablets and the obtained results were statistically compared with those of a reported method. The comparison showed that there are no significant differences between the proposed methods and the reported method regarding both accuracy and precision.

Spectrophotometric Bivariate Method for Determination of Candesartan Cilexetil in Presence of its Alkaline Induced Degradation Product

Spectrophotometric Bivariate Method for Determination of Candesartan Cilexetil in Presence of its Alkaline Induced Degradation Product

Simultaneous HPTLC and RP‐HPLC methods for determination of bumadizone in the presence of its alkaline‐induced degradation product

Accurate, selective, sensitive and precise HPTLC-densitometric and RP-HPLC methods were developed and validated for determination of bumadizone calcium semi-hydrate in the presence of its alkaline-induced degradation product and in pharmaceutical formulation. Method A uses HPTLC-densitometry, depending on separation and quantitation of bumadizone and its alkaline-induced degradation product on TLC silica gel 60 F(254) plates, using hexane-ethyl acetate-glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP-HPLC separation of bumadizone and its alkaline-induced degradation product using a mobile phase consisting of methanol-water-acetonitrile (20:30:50, v/v/v) on a Phenomenex C(18) column at a flow-rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method.

ACID- ALKALI DEGRADATION STUDY ON OLMESARTAN MEDOXOMIL AND DEVELOPMENT OF VALIDATED STABILITY-INDICATING CHROMATOGRAPHIC METHODS

A reversed phase liquid chromatography (RP-HPLC) and thin layer chromatography (HPTLC) densitometry methods as a stability indicating assays of olmesartan medoxomil in presence of its acid or alkaline induced degradation products were developed. Olmesartan medoxomil and its degradation products were analyzed by HPLC equipped with UV-Variable wave length detector at 257 nm where quantitation was achieved by isocratic elution on Agilent, Exclipse XDB- C 18 column with mobile phase composed of acetonitrile: methanol: water: glacial acetic acid (40:35:25:0.1 v/v/v/v) at flow rate 1ml min.-1. HPTLC was performed on aluminum packed Nano silica gel 60 F 254 TLC plates as stationary phase with significant difference in Rƒ values between olmesartan medoxomil and its degradates using chloroform: methanol: formic acid (8:1.5:0.5 v/v/v) as mobile phase. Densitometric evaluation of intact drug was carried out at 260 nm. The calibration curve of olmesartan medoxomil in bulk form was linear from 0.5- 10 µg ml-1 and 0.05- 1 mg ml-1 with mean percentage accuracy 99.97 ± 1.085 % and 100.35 ± 1.060 % for HPLC and HPTLC methods, respectively. The two proposed methods were successfully applied for the determination of olmesartan medoxomil in drug substance and in drug product. Methods validation was tested for linearity; accuracy; precision; selectivity and robustness, according to USP guidelines.

Stability‐indicating methods for the determination of famciclovir in the presence of its alkaline‐induced degradation product

Five sensitive, selective and precise stability-indicating methods are presented for the determination of famciclovir (FCV) in the presence of its alkaline-induced degradation product. Method A utilizes the first derivative spectrophotometry at 321 nm. Method B depends on using the first derivative of the ratio spectrophotometry (DD(1)) by measurement of the amplitude at 256 nm. Method C is based on the reaction of FCV with hydroxylamine to form hydroxamic acid, causing the hydroxamic acid to react with triferric ion to form ferric hydroxamate that is measured at 503 nm. Method D is based on the separation of FCV from its degradation product followed by densitometric measurement of the bands at 304 nm. The separation was carried out on silica gel 60 F(254), using chloroform: methanol (70:30, v/v) as a mobile phase. Method E is based on a high performance liquid chromatographic (HPLC) separation of FCV from its degradation product using an ODS column with a mobile phase consisting of methanol-50 mM dipotassium hydrogen phosphate (25:75, v/v, pH 3.0)with UV detection at 304 nm. Regression analysis showed good correlation in the concentration ranges 16-72 microg/ml, 40-240 microg/ml, 40-240 microg/ml, 0.75-5.25 microg/band and 20-240 microg/ml with percentage recoveries of 99.65 +/- 0.85, 100.27 +/- 0.91, 99.72 +/- 0.84, 100.65 +/- 1.52 and 99.88 +/- 0.50 for methods A, B, C, D and E, respectively. These methods are suitable as stability-indicating methods for the determination of FCV in the presence of its degradation product either in bulk powder or in pharmaceutical formulation. Statistical analysis of the results has been carried out revealing high accuracy and good precision.

Development and Validation of HPLC, TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

AbstractReversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C18 column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F254 TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.

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 1D ultraviolet spectrophotometry with zero crossing point and peak to base measurement. The 1D 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 1DD 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.

Kinetic study of alkaline induced hydrolysis of the skeletal muscle relaxant chlorzoxazone using ratio spectra first derivative spectrophotometry

2-Amino-4-chlorophenol was found to be the alkaline induced degradation product and the synthetic precursor of chlorzoxazone. The aim of this work is to study different factors affecting the degradation process due to the high toxicity of 2-amino-4-chlorophenol. Chlorzoxazone was found to follow pseudofirst order kinetics. Ratio spectra first derivative spectrophotometry (DR 1) was developed for monitoring the change in chlorzoxazone concentration during the degradation process. Kinetic parameters (rate constant ( K) and half-life ( t 0.5)) were calculated at different temperatures (40–120 °C) and different sodium hydroxide concentrations (3–10 M). Activation energy at 3 and 8 M sodium hydroxide concentration and alkaline induced catalysis constant at 60, 70 and 80 °C were also calculated.

Spectrophotometric and HPLC Determination of Linezolid in Presence of Its Alkaline-Induced Degradation Products and in Pharmaceutical Tablets

In this work three sensitive stability-indicating methods for the determination of linezolid in the presence of its alkaline-induced degradation products are developed. The first method depends on the oxidative coupling reaction of linezolid with 3-methyl benzothiazoline-2-one hydrazone (MBTH) hydrochloride in the presence of ferric chloride in an acid medium. The resulting stable colored products showed absorption λmax at 627 nm. The second method is based on alkaline hydrolysis of linezolid to yield the degradation product which reacts with haematoxylin reagent in the presence of boric acid to give a reddish violet chromogen at λmax 557 nm. The third method is based on (HPLC) resolution efficiency in the determination of linezolid in the presence of its alkaline induced degradation product. The percentage recoveries for the proposed methods were 99.54 ± 0.58, 99.84 ± 0.78, and 100.02 ± 0.49 within the concentration range of 1.6–12 µg mL−1, 10–60 ng mL−1 and 5–40 µg mL−1, respectively. The proposed methods were applied successfully for the determination of intact drug in bulk powder, laboratory prepared mixtures containing different percentages of degradation product and pharmaceutical dosage forms. The results of the analysis were found to agree statistically with those obtained with the reported method. Furthermore, the methods were validated and also assessed by applying the standard addition technique.

STABILITY STUDY OF NILVADIPINE IN BULK DRUG AND PHARMACEUTICAL CAPSULES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple, stability-indicating, liquid chromatographic method has been developed for the assay of nilvadipine in the presence of its alkaline-induced degradation products. A μBondapak-C18 column was used with a mobile phase consisting of acetonitrile/0.01 M sodium acetate (55:45, v/v) adjusted to pH 3.5 with acetic acid at a flow rate of 1.8 mL min−1. With procaine hydrochloride as an internal standard, quantitation was achieved with UV detection at 254 nm based on the peak areas ratios. Determination of nilvadipine was possible over the concentration range 0.096–0.64 μgmL−1 with limit of detection of 0.032 μg/mL (8.3 × 10−8 M). Between-day and within-day relative standard deviations were lower than 2%. The proposed method was successfully applied to the determination of nilvadipine in bulk and capsule form, with a high percentage of recovery. Moreover, the method was utilized to investigate the kinetics of the degradation process at different temperatures. The apparent first-order rate constant, half-life and activation energy were calculated. The proposed method was extended to the in vitro detection of nilvadipine in human urine samples.

SPECTROPHOTOMETRIC DETERMINATION OF CEFPROZIL IN PHARMACEUTICAL DOSAGE FORMS, IN URINE AND IN THE PRESENCE OF ITS ALKALINE INDUCED DEGRADATION PRODUCTS

Four simple, rapid and accurate methods for spectrophoto-metric determination of cepfrozil are presented. Method I and II depend on measuring first derivative peak-trough amplitude of the drug in 0.1 N HCl (D1 a) and in phosphate buffer pH 7(D1 b), respectively. Method III and IV are based on measuring the absorbance difference (ΔA) and first derivative difference (ΔD1) of buffered cefprozil solutions against its acidic solutions as blanks. The proposed methods are applied for the determination of the drug in pure and dosage forms with good accuracy and precision. Among the previously mentioned methods; D1 a, D1 b and ΔD1 are used for the determination of cefprozil in urine. These methods have been successfully applied for the determination of cumulative amounts of cefprozil in urine following an oral dose of 500 mg to a human male volunteer. Moreover, by using D1 a method and applying the zero-crossing technique, both the intact drug and its alkaline induced degradation products could be determined in presence of each other without interference. The proposed methods proved to be accurate and reproducible.

A Stability-Indicating Spectrophotometric Method for the Determination of Fenoterol in Pharmaceutical Preparations.

ABSTRACT A simple and highly sensitive spectrophotometric method was developed for the determination of fenoterol hydrobromide in pharmaceutical formulations. The method is based on measurement of the orange-yellow color peaking at 436 nm, produced when the drug is coupled with diazotized benzocaine in triethylamine medium. The method is applicable over the range of 0.5 – 10 μg/ml, with minimum detectability of 3.6 ng/ml (≈ 9.4×10−9 M). The molar absorptivity is 4.61×104 L. mol−1. cm−1. with relative standard error of 0.196%. The proposed method was successfully applied to the determination of fenoterol in its dosage forms; the mean recoveries of 99.61 ± 1.59 and 98.97 ± 1.52 for tablets and aerosols, respectively, were obtained. The results of the proposed method were favorably compared to those obtained by the official and reference methods. No i8543168 was encountered from the co-formulated drug ipratropium bromide, common excipients and alkaline induced degradation product. A proposal of the reaction pathway is presented.

Derivative spectrophotometric determination of glibenclamide, mebeverine hydrochloride and clopamide in the presence of their alkaline-induced degradation products

First- and second-derivative spectrophotometric methods have been proposed for the determination of glibenclamide, mebeverine hydrochloride and clopamide with their corresponding degradation products. Solutions of these drugs were analysed by measuring the first- and second-derivative spectral response at certain wavelengths where the respective degradation products exhibit no contribution. Also, clopamide can be determined in a mixture with reserpine and dihydroergocristine by utilising the second-derivative method. Kinetic investigation of the alkaline degradation rate of these drugs revealed that the proposed methods were stability indicating. The different pharmaceutical preparations were assayed and were found to give results of the same accuracy and reproducibility as the corresponding official method or “maximum absorbance” methods of assay.