Separation of enantiomers of deprenyl with various CDs in CE and the effect of enantiomer migration order on enantiomeric impurity determination of selegiline in active ingredients and tablets

Opposite affinity pattern of enantiomers of the antiparkinsonian chiral drug deprenyl (DEP) was observed towards various neutral and charged derivatives of -CD. The effect of the enantiomer migration order on the LOD of enantiomeric impurity of R-DEP (selegiline) was studied for the standard substances and in the tablets from three different suppliers. The influence of injection mode on the LOD of a minor enantiomeric impurity was also studied and the CE method was compared with the pharmacopoeial HPLC method using a commercially available chiral column Chiralcel OD-H. The optimized CE method was more suitable for low-level enantiomeric impurity determination in selegiline compared to the pharmacopoeial HPLC method.     

Development and validation of a stability-indicating LC method for the assay of lodenafil carbonate in tablets

A stability-indicating liquid chromatographic method has been developed for the quantitative determination of lodenafil carbonate in tablets. The method employs a Synergi Fusion C18 column (250 × 4.6 mm, i.d., 4 μm particle size), with mobile phase consisting of a mixture of methanol-acetic acid 0.1% pH 4.0 (65:35, v/v) and UV detection at 290 nm, using a photodiode array detector. A linear response (r = 0.9999) was observed in the range of 10-80 μg/mL. The method showed good recoveries (average 100.3%) and also intra and inter-day precision (RSD < 2.0%). Validation parameters as specificity and robustness were also determined. Specificity analysis showed that no impurities or degradation products were co-eluting with the lodenafil carbonate peak. The method was found to be stability-indicating and due to its simplicity and accuracy can be applied for routine quality control analysis of lodenafil carbonate in tablets.     

Development and validation of a stability-indicating LC method for simultaneous determination of related compounds of guaifenesin,terbutaline sulfate and ambroxol HCl in cough syrup formulation

A new liquid chromatographic method has been developed and validated for the determination of terbutaline sulfate (TLS), guaifenesin (GFN) and ambroxol HCl (AML), for its potential impurities in drug substances and drug products. Efficient chromatographic separation was achieved on X-Terra RP-18 column with a simple mobile phase combination containing a gradient mixture of solvents A and B at a flow rate of 1.0 mL min⁻¹ and quantitation was carried out using ultraviolet detection at 222 nm with column temperature of 35 °C. The resolution between TLS, GFN and AML, its associated impurities was found to be greater than 1.5. Regression analysis shows an r value (correlation coefficient) greater than 0.998. This method was capable to detect all the process impurities of TLS, GFN and AML, at a level below 0.015% with respect to a test concentration of 0.125, 5.0 and 1.5 mg mL⁻¹, respectively. The % RSD for the inter-day and intra-day precisions for all the impurities of TLS, GFN and AML were found to be less than 3.0. The method has shown good, consistent recoveries. The drugs were subjected to stress conditions of acid, base, water hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by international conference on harmonization (ICH).     

A stability-indicating high-performance liquid chromatographic assay for the determination of some pharmaceutically important nitrocompounds

Summary A simple, rapid and precise stability-indicating high performance liquid chromatographic assay for the determination of furazolidone (I), nitrofurazone (II), nitrofurantoin (III), niridazole (IV) and nifuroxime (V) in pure form and in pharmaceutical preparations has been devloped.Reversed phase chromatography was conducted using a Lichrosorb R.P. 18 column (250×4 mm), with methanol-water-buffer pH3 (40555) eluent and detection at 365, 375, 367, 368 and 340 nm respectively. Calibration graphs were linear over the concentration ranges 3–18, 1–10, 1–10, 3–18 and 3–18 m·ml^–1. Reoveries from bulk drugs were 99.94–100.00 with a relative standard deviation of 1.02–1.61.Sunlight degradation of most of the studied drugs to 5-nitrofuraldehyde was observed. Accelerated stability studies have been carried out on each drug by exposure to sunlight for different time periods. Graphs of log of the remaining concentration (Log. C) against time indicated that photodecomposition of the nitrocompounds is a first order reaction. The proposed method was applied to some representative dosage forms and the results obtained were in good agreement with those obtained by the official methods.     

Development and validation of a stability-indicating RP-HPLC method for determination of atomoxetine hydrochloride in tablets

This paper describes the development of a stability-indicating RP-HPLC method for the determination of atomoxetine hydrochloride (ATX) in the presence of its degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of acid, base, oxidation, wet heat, dry heat, and photodegradation. In stability tests, the drug was susceptible to acid, base, oxidation, and dry and wet heat degradation. It was found to be stable under the photolytic conditions tested. The drug was successfully separated from the degradation products formed under stress conditions on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) by using acetonitrile-methanol-0.032 M ammonium acetate (55 + 05 + 40, v/v/v) as the mobile phase at 1.0 mL/min and 40 degrees C. Photodiode array detection at 275 nm was used for quantitation after RP-HPLC over the concentration range of 0.5-5 microg/mL with a mean recovery of 100.8 +/- 0.4% for ATX. Statistical analysis demonstrated that the method is repeatable, specific, and accurate for the estimation of ATX. Because the method effectively separates the drug from its degradation products, it can be used as a stability-indicating method.     

Development and validation of a stability-indicating HPLC method for the determination of degradation products in dipyridamole injection

Summary The development and subsequent validation of an isocratic high-performance liquid chromatographic (HPLC) procedure employing ultraviolet (UV) detection for the determination of degradation products in Dipyridamole Injection is reported. The development of this assay involved the evaluation of several factors including buffer type, ionic strength, pH, organic composition, and column type. The described method is simple, reproducible, accurate, and selective. The precision, relative standard deviation (RSD), amongst five sample preparations for total degradation products was not more than (NMT) 10.2 %, while the individual degradation products were NMT 12.1%. Intermediate precision, as determined from fifteen sample preparations, generated by two Analysts on different HPLC systems over three days, exhibited an RSD for total and individual degradation products of 8.2 % and NMT 27.5 %, respectively. The mean absolute recovery of dipyridamole using the described method is 102.1±1. 9%, (mean±SD, n=12) over the concentration range of 0.03 % to 5.0 % of its label claim of 5 mg mL⁻¹. The limit of detection and limit of quantitation were 0.1 and 0.3 μg mL⁻¹, respectively. The linearity of the peak response was verified with respect to dipyridamole concentration over a range of 0.3 and 50 μg mL⁻¹ (0.03 % to 5.0 % label claim). The Standard and Assay Preparations are stable for up to 48 hours at room temperature. The selectivity was evaluated by subjecting the finished product (Dipyridamole Injection) to thermal, acidic, basic, oxidative and fluorescent radiation stress conditions. No interference in the analysis of degradation products was observed, showing the method is stability-indicating.     

Development and Validation of Stability indicating HPLC method for determination of related substances and assay of Monobenzone drug substance

Monobenzone, a mono benzyl ether of hydroquinone, is used as a topical drug for skin depigmentation. It increases the excretion of melanin from melanocytes. A novel stability-indicating high performance liquid chromatographic approach has been established in the current study to identify related compounds and assay of Monobenzone. The Agilent 1260 system was used for the HPLC analysis, and a gradient method using 0.1% orthophosphoric acid in water and acetonitrile as the eluent was used to separate Monobenzone from known contaminants and degradation products on a Zorbax SB-phenyl (250 mm x 4.6 mm) 5 μm column. The flow rate was set at 0.8 mL/min, the column oven temperature was 30 °C, and the detector wavelength was maintained at 215 nm. The relative retention times for Hydroquinone and Monobenzone dimer were around 0.35 and 1.86 respectively, while the Monobenzone retention time was approximately 10.4 min. The total run time was 30 min. For all known analytes spanning the concentration range from LOQ to 200% of the specification level, the calibration plot revealed a linear relationship (minimum r=0.999). All known analytes' LOD and LOQ were found to be within 0.06–0.10 μg/mL and 0.15–0.24 μg/mL, respectively. A recovery study determined the accuracy of the proposed method. For Hydroquinone and Monobenzone dimer, mean recovery was found to be in the range of 95.86%–99.89% and 93.02%–99.84% respectively. The repeatability study showed that the method is precise enough within acceptable limits. Studies on solution stability and robustness produced results that fell within acceptable bounds. The proposed method shows excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ and system suitability findings. Additionally, the forced degradation study showed that the approach was stability indicating in nature.     

Development and validation of a reverse-phase liquid chromatographic method for assay and related substances of abacavir sulfate

A simple isocratic liquid chromatographic method was developed for the determination of abacavir from its related substances and assay for the first time. This method involves the usage of C18 (Intertsil octadecyl silane-3V, 150 mm x 4.6 mm, 5 microm) column. The method was validated over the range of 0.002-0.1 mg/mL for chloro impurity, 0.005-0.1 mg/mL for amino impurity and pyrimidine impurity, and 0.005-0.2 mg/mL for abacavir. The mobile phase consists of a mixture of 10 mM ammonium acetate buffer and ACN in the ratio of 90:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 214 nm. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. The developed method was validated for linearity, range, precision, accuracy, and specificity. This method can be conveniently used in a quality control laboratory for routine analysis of both related substances and assay.     

Development and validation of a novel stability indicating RP-HPLC method for the quantitative determination of marbofloxacin in its tablets

A fast, feasible, isocratic, stability indicating reverse phase-high performance liquid chromatography (RP-HPLC) method has been developed and validated for the quantitative determination of marbofloxacin in marbofloxacin tablets. The method was developed using Zorbax SB C18 (150?mm?×?4.6?mm), 5-µm column thermostated at 30°C, mobile phase A (1?mL of trifluoroacetic acid in 1000?mL of water), mobile phase B (acetonitrile) in the ratio of 83:17?v/v at flow rate of 1.0?mL/min, and an injection volume of 10?µL. The analyte was monitored at a wavelength of 298?nm. The method was validated in accordance with the Food and Drug Administration (FDA) Veterinary International Conference on Harmonization guidelines. To demonstrate stability indicating ability of method, drug product was subjected to the stress condition of acidic, basic, humidity, thermal, oxidative, and photolytic degradation.     

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min⁻¹. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.     

Chiral determination of a novel acaricide cyflumetofen enantiomers in cucumber,tomato, and apple by HPLC

A simple chiral analytical method was developed for the enantiomeric determination of cyflumetofen in cucumber, tomato, and apple by normal‐phase HPLC. The effects of mobile phase composition and column temperature on the enantioseparation were evaluated. Excellent separation was achieved at 25°C on a Chiralpak AD‐H column, with a mixture of n‐hexane and 2‐propanol (95:5, v/v) as mobile phase at a flow rate of 1.0 mL/min detecting at 234 nm. The resolution of cyflumetofen enantiomers was up to 5.5. The elution order of the enantiomers was determined by an online OR‐2090 detector, which was performed under the same chromatographic conditions. The first eluted enantiomer was (–)‐cyflumetofen and the second eluted one was (+)‐cyflumetofen. The method was validated for linearity, repeatability, accuracy, LOD, and LOQ. LOD ranged from 0.1 to 0.15 mg/kg, with the LOD varying from 0.33 to 0.5 mg/kg for each enantiomer, respectively. The average recoveries of the pesticide ranged from 71.4 to 102.0% at all fortification levels. The precision values associated with the analytical method, expressed as RSD values, were below 14.8% in all matrices. The method was then successfully applied to detect cyflumetofen enantiomers in real samples.     

Development of a stability indicating HPTLC method for the estimation of olanzapine in pharmaceutical dosage forms

We report herein an accurate, precise, and economical stability indicating high performance thin layer chromatographic (HPTLC) method developed to assess the safety of olanzapine in pharmaceutical formulations. Olanzapine was subjected to forced degradation studies to assess the effect of environmental conditions on its stability. Stress conditions such as hydrolysis under acidic and alkaline environment, degradation and oxidation by heat, light and air were used to study the stability of olanzapine. Mobile phase comprising of toluene: methanol (5:5 v/v) and aluminum plate pre-coated with silica gel 60 F₂₅₄ as a stationary phase were used for the development of chromatogram by HPTLC technique. Densitometric analysis of olanzapine carried out at 297 ​nm gave sharp symmetrical peak with R_f value of 0.50 and a satisfactory baseline resolution for all components. The drug was found to undergo degradation under acidic, alkaline and oxidative conditions. A single distinct peak in acidic and alkaline media while two peaks obtained as a result of oxidative degradation were well resolved along with the parent drug. The degradation products and parent drug showed significantly different R_f values. The developed HPTLC method gave quick and reproducible results for the olanzapine content in the tablets. The mean recoveries were 100.75% which confirms accuracy of the proposed method. The method was further validated for specificity, ruggedness and robustness. Based on the results, it can be suggested that the developed HPTLC method is quite efficient in separating the olanzapine from its degradation products; hence it can be used by pharmaceutical industries and regulatory bodies for the routine analysis of olanzapine in various pharmaceutical dosage forms.     

A stability-indicating reversed-phase high performance liquid chromatography method for simultaneous assay of two corticosteroids and estimation of their related compounds in a pharmaceutical injectable formulation

Betamethasone Sodium Phosphate and Betamethasone Acetate are the two corticosteroids active pharmaceutical ingredients (APIs) that are present in the injectable formulation, Celestone Chronodose^® Injection. It is extremely challenging to develop a Quality Control friendly HPLC method to separate all the potential impurities and degradation products of the two APIs from each other using a single HPLC method. A novel stability-indicating reversed-phase HPLC (RP-HPLC) method using two oxo-cyclic organic modifiers in the mobile phase was developed and validated. This method can separate a total of 32 potential impurities and degradation products from the two APIs and also from each other. Peak symmetry and separation efficiency were enhanced by using two chaotropic agents (trifluoroacetic acid and potassium hexafluorophosphate) in the mobile phases of this method. The stability-indicating capability of this method has been demonstrated by analyzing aged and stressed degraded stability samples of the drug product. This method uses an ACE 3 C18 (15 cm × 4.6 mm) HPLC column. The method was validated per ICH guidelines and proved to be suitable for routine QC use.     

Development and Validation of a Stability Indicating HPLC Method for Determination of Granisetron

The aim of this study was to study the stress degradation of granisetron and analysis of the drug in the presence of its degradation products. Forced degradation studies were conducted on bulk sample using acidic, alkaline, oxidative, heat and photolytic conditions. Granisetron was relatively unstable under acidic, alkaline and oxidative conditions. Separation of granisetron and degradation products was achieved using a Nova‐Pak C₈ column and acetonitrile‐KH₂PO₄ 25 mM (75:25, v/v) as mobile phase with UV detection at 305 nm. The method was linear over the range of 0.2‐15 μg/mL granisetron (r² > 0.999). The within‐day and between‐day precision values were also in the range of 0.5‐4%. The proposed method was successfully applied for quantitative determination of granisetron in tablets and in vitro dissolution studies.     

Development and validation of a selective and robust LC-MS/MS method for quantifying amlodipine in human plasma

A liquid chromatographic–tandem mass spectrometric method (LC-MS/MS) for quantifying amlodipine in human plasma was developed and validated. Sample preparation was based on liquid–liquid extraction using NaOH and a mixture of ethyl acetate/hexane (80/20; v/v). Chromatography was performed on a C-18 analytical column and the retention times were 1.9 and 3.0 min for amlodipine and nimodipine (internal standard), respectively. The ionization was optimized using ESI(+) and enhanced selectivity was achieved using tandem mass spectrometric analysis via two MRM functions, 409238 and 418343 for amlodipine and nimodipine. The calibration curve ranged from 0.2 to 20.0 ng/mL. The inter-day precision and accuracy and the relative standard deviation (RSD) were <15%. The analyte was shown to be stable over the timescale of the whole procedure. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.     

Validated spectrofluorometric methods for determination of amlodipine besylate in tablets

Two simple and sensitive spectrofluorometric methods have been developed and validated for determination of amlodipine besylate (AML) in tablets. The first method was based on the condensation reaction of AML with ninhydrin and phenylacetaldehyde in buffered medium (pH 7.0) resulting in formation of a green fluorescent product, which exhibits excitation and emission maxima at 375 and 480 nm, respectively. The second method was based on the reaction of AML with 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) in a buffered medium (pH 8.6) resulting in formation of a highly fluorescent product, which was measured fluorometrically at 535 nm (lambda(ex), 480 nm). The factors affecting the reactions were studied and optimized. Under the optimum reaction conditions, linear relationships with good correlation coefficients (0.9949-0.9997) were found between the fluorescence intensity and the concentrations of AML in the concentration range of 0.35-1.8 and 0.55-3.0 microg ml(-1) for ninhydrin and NBD-Cl methods, respectively. The limits of assays detection were 0.09 and 0.16 microg ml(-1) for the first and second method, respectively. The precisions of the methods were satisfactory; the relative standard deviations were ranged from 1.69 to 1.98%. The proposed methods were successfully applied to the analysis of AML in pure and pharmaceutical dosage forms with good accuracy; the recovery percentages ranged from 100.4-100.8+/-1.70-2.32%. The results were compared favorably with those of the reported method.     

Optimization and validation of a new CE method for the determination of pantoprazole enantiomers

A new CE method using sulfobutylether-beta-cyclodextrin (SBE-beta-CD) as chiral additive was developed and validated for the determination of pantoprazole enantiomers. The primary factors affecting its separation efficiency, which include chiral selector, buffer pH, organic additive, and applied voltage, were optimized. The best results were obtained using a buffer consisting of 50 mM borax-150 mM phosphate adjusted to pH 6.5, 20 mg/mL SBE-beta-CD, and a 10 kV applied voltage. The optimized method was validated for linearity, precision, accuracy, and proved to be robust. The LOD and LOQ for R-(+)-pantoprazole were 0.9 and 2.5 μg/mL, respectively. The method is capable of determining a minimum limit of 0.1% (w/w) of R-enantiomer in S-(-)-pantoprazole bulk samples.     

Development of validated stability-indicating chromatographic method for the determination of fexofenadine hydrochloride and its related impurities in pharmaceutical tablets

ABSTRACT: A simple reversed phase high performance liquid chromatographic method with diode array detector (HPLC-DAD) has been developed and subsequently validated for the determination of fexofenadine hydrochloride (FEX) and its related compounds; keto fexofenadine (Impurity A), meta isomer of fexofenadine (Impurity B), methyl ester of fexofenadine (Impurity C) in addition to the methyl ester of ketofexofenadine (Impurity D). The separation was based on the use of a Hypersil BDS C-18 analytical column (250 × 4.6 mm, i.d., 5 μm). The mobile phase consisted of a mixture of phosphate buffer containing 0.1 gm% of 1-octane sulphonic acid sodium salt monohydrate and 1% (v/v) of triethylamine, pH 2.7 and methanol (60:40, v/v). The separation was carried out at ambient temperature with a flow rate of 1.5 ml/min. Quantitation was achieved with UV detection at 215 nm using lisinopril as internal standard, with linear calibration curves at concentration ranges 0.1-50 μg/ml for FEX and its related compounds. The optimized conditions were used to develop a stability-indicating HPLC-DAD method for the quantitative determination of FEX and its related compounds in tablet dosage forms. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. Complete separation was achieved for the parent compounds and all degradation products. The method was validated according to ICH guidelines in terms of accuracy, precision, robustness, limits of detection and quantitation and other aspects of analytical validation.     

Development and validation of a stability-indicating HPLC assay method for simultaneous determination of spironolactone and furosemide in tablet formulation

The objective of the current study was to develop and validate a simple, precise and accurate isocratic stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay method for the determination of spironolactone and furosemide in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on an SGE 150 × 4.6 mm SS Wakosil II 5C8RS 5-μm column using a mobile phase of acetonitrile-ammonium acetate buffer (50:50, v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 254 nm using a photodiode array detector. The drug was subject to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was found to be linear in the drug concentration range of 40-160 μg/mL with correlation coefficients of 0.9977 and 0.9953 for spironolactone and furosemide, respectively. The precision (relative standard deviation; RSD) among a six-sample preparation was 0.87% and 1.1% for spironolactone and furosemide, respectively. Repeatability and intermediate precision (RSD) among a six-sample preparation were 0.46% and 0.20% for spironolactone and furosemide, respectively. The accuracy (recovery) was between 98.05 and 100.17% and 99.07 and 100.58% for spironolactone and furosemide, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of spironolactone and furosemide; therefore, the assay can be considered to be stability-indicating.     

Development and validation of a stability-indicating column high-performance liquid chromatographic assay method for determination of nebivolol in tablet formulation

A simple, precise, and accurate isocratic reversed-phase (RP) stability-indicating column high-performance liquid chromatographic (HPLC) assay method was developed and validated for determination of nebivolol in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on a Phenomenex Luna C8 (2) column (250 mm x 4.6 mm id, 5 microm particle size) using mobile phase composed of acetonitrile-pH 3.5 phosphate buffer (35 + 65, v/v) at a flow rate of 1.0 mL/min, and detection was performed at 280 nm using a photodiode array detector. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The method was linear in the drug concentration range of 40-160 microg/mL with a correlation coefficient of 0.9999. The repeatability relative standard deviation (RSD) for 6 samples was 0.69%, and the intermediate precision (RSD) for 6 samples was 1.39%. The accuracy (recovery) was between 98.57 and 99.55%. Degradation products produced as a result of stress studies did not interfere with detection of nebivolol, and the assay can thus be considered stability-indicating.