Stability-indicating Reversed-phase HPLC Method Research Articles

Separation and Quantification of Octahydro-1h-Indole-2-Carboxilic Acid and Its Three Isomers by HPLC using Refractive Index Detector

Octahydro-1H-indole-2-carboxylic acid is a key starting material for the synthesis of Perindopril and Trandolapril. A rapid, economical, simple, sensitive and reliable stability-indicating reverse phase HPLC method developed and validated for the quantitative determination of all isomers related to Octahydro-1H-indole-2-carboxylic acid and its related substances. The compound is non chromophoric, it has three chiral centers and there is a possibility of four pairs of enantiomers. Refractive index detector was used for the quantification of all its isomers. Optimized mobile phase is 10 mM potassium phosphate buffer with pH-3.0. The C18 column (Inertsil ODS-4, 250 mm×4.6 mm×5 μm) is used as the stationary phase with a mobile phase flow rate of 1.5 mLmin–1 and column temperature maintained at 35°C. The developed method was validated as per ICH guidelines for Accuracy, linearity, range, precision, ruggedness, robustness, solution stability, limit of quantification, and limit of detection. A linear range from LOQ to 150% performed for the analyte and its three isomers. The correlation coefficient obtained for all the isomers was more than 0.999. The recoveries obtained for all isomers were found in between 93.9% and 107.9%. The detection limit for all the isomers were about 0.006 mg/mL and the quantification limits were in between 0.022 mg/mL to 0.024 mg/mL respectively. The proposed method can be successfully applied for the quantification of the three isomers in Octahydro-1H-indole-2-carboxylic acid and provides a simple and cost effective quality control tool for routine analysis.

A Stability Indicating HPLC Method for Dronedarone in Bulk Drugs and Pharmaceutical Dosage Forms

The objective of the current study was to develop a validated, specific and stability-indicating reverse phase HPLC method for the quantitative determination of Dronedarone and its related substances. The determination was done for active pharmaceutical ingredient and its pharmaceutical dosage forms in the presence of degradation products, and its process-related impurities. The drug was subjected to stress conditions of hydrolysis (acid and base), oxidation, photolysis and thermal degradation per International Conference on Harmonization (ICH) prescribed stress conditions to show the stability-indicating power of the method. Significant degradation was observed during acid, oxidative and photo stress studies. In the developed HPLC method, the resolution between Dronedarone and its process-related impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for Dronedarone and it’s all the five impurities. The chromatographic separation was achieved on a C8 stationary phase. The method employed a linear gradient elution and the detection wavelength was set at 288 nm. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.6%. The developed HPLC method was validated with respect to linearity, accuracy, precision and robustness.

Stability-indicating HPLC method for simultaneous determination of terbutaline sulphate, bromhexine hydrochloride and guaifenesin

The aim of the present study was the development and subsequent validation of a simple, precise and stability-indicating reversed phase HPLC method for the simultaneous determination of guaifenesin, terbutaline sulphate and bromhexine hydrochloride in the presence of their potential impurities in a single run. The photolytic as well as hydrolytic impurities were detected as 3,5-dihydroxybenzoic acid, 3,5-dihydroxybenzaldehyde, 1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl) amino]-ethanone from terbutaline, 2-methoxyphenol and an unknown impurity identified as (2RS)-3-(2-hydroxyphenoxy)-propane-1,2-diol from guaifenesin. The chromatographic separation of all the three active components and their impurities was achieved on Wakosil II column, using phosphate buffer (pH 3.0) and acetonitrile as mobile phase which was delivered initially in the ratio of 80:20 (v/v) for 18 min, then changed to 60:40 (v/v) for next 12 min, and finally equilibrated back to 80:20 (v/v) for 10 min. Other HPLC parameters were: Flow rate at 1.0 ml/min, detection wavelengths 248 and 280 nm, injection volume 10 μl. The calibration graphs plotted with five concentrations of each component were linear with a regression coefficient R2 >0.9999. The limit of detection and limit of quantitation were estimated for all the five impurities. The established method was then validated for linearity, precision, accuracy, and specificity and demonstrated to be applicable to the determination of the active ingredients in commercial and model cough syrup. No interference from the formulation excipients was observed. These results suggest that this LC method can be used for the determination of multiple active ingredients and their impurities in a cough and cold syrup.

Development and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Assay of Betamethasone Dipropionate, Chlorocresol, and for the Estimation of Betamethasone Dipropionate Related Compounds in a Pharmaceutical Cream and Ointment

A new stability-indicating reversed-phase HPLC (RP-HPLC) method has been developed and validated for simultaneous assay of betamethasone dipropionate (BD) and chlorocresol and also for the estimation of BD related compounds in a pharmaceutical cream matrix. In addition, this newly developed RP-HPLC method was also demonstrated as suitable for a pharmaceutical ointment product that does not contain chlorocresol. The RP-HPLC method uses a Waters SymmetryShield RP18 analytical column (150 × 4.6 mm). Water (mobile phase A) and acetonitrile (mobile phase B) were used in the gradient elution with a flow rate of 1.5 mL/min and detection wavelength at 240 nm. A Waters XBridge Shield RP18 analytical column (150 × 4.6 mm) was identified as an alternate column. The limit of detection (LOD) and the limit of quantitation (LOQ) are 0.02 μg/mL and 0.05 μg/mL, respectively. The precision of the method for BD is less than 0.3% RSD, and the accuracy of BD ranged from 99.5% to 102.6%. The stability-indicating capability of this method has been demonstrated by analyzing aged samples of the product. This RP-HPLC method was successfully validated per ICH guidelines and proved to be suitable for routine quality control use.

Determination of Individual Homologues and Total Content of Benzalkonium Chloride by Reversed-Phase High-Performance Liquid Chromatography Using a Short Butyl Column

Benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides that usually contains C-10, C-12, C-14, and C-16 homologues), commonly known as BKC, is used as a bacteriostatic agent in many household, food, and drug products. In this paper, we report a simple, rapid, robust, and stability-indicating reversed-phase HPLC method using a short butyl (C4) column for the simultaneous determination of each individual homologue content, as well as the total concentration of individual homologues in commercial bulk raw material batches of BKC samples. The chromatographic separation was performed on a 5 cm ACE C4 column with mobile phase consisting of water, acetonitrile, and potassium chloride. Even though using a short column can potentially cause some challenges to resolving certain critical pairs of peaks, we have successfully separated all of the analyte peaks (including those from stressed, degraded products) on a short column using an optimal mobile phase.

Development and Validation of a Stability-Indicating HPLC Method for Determination of Ciprofloxacin Hydrochloride and its Related Compounds in Film-Coated Tablets

The objective of the current study was the development and subsequent validation of a simple, sensitive, precise and stability-indicating reversed-phase HPLC method for the determination of ciprofloxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities. The chromatographic separation of ciprofloxacin HCl and its related compounds was achieved on an Inertsil ODS3 column using UV detection. The optimized mobile phase consisted of phosphoric acid solution: acetonitril. The proposed method provided linear responses within the concentration range 250–750 μg mL−1 for ciprofloxacin HCl and 0.5–1.5 μg mL−1 for its related compounds. LOD and LOQ values for the active substance were 5.159 and 15.632 μg mL−1, respectively. Correlation coefficients (r) of the regression equations for the impurities were greater than 0.99 in all cases. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed.

Development and validation of a stability-indicating reversed-phase high performance liquid chromatography method for assay of betamethylepoxide and estimation of its related compounds

Betamethylepoxide (16beta-methyl-Delta(1,4)-pregnadiene-9beta-11beta-oxide-17alpha,21-diol-3,20-dione) is a key intermediate for the synthesis of various active pharmaceutical ingredients (APIs) of steroid compounds. A stability-indicating reversed-phase HPLC method for assay of betamethylepoxide and estimation of its related compounds has been developed and validated. This method can accurately quantitate betamethylepoxide in the presence of numerous structurally related compounds (including the alpha-epimer, known as alphamethylepoxide). This method can also adequately separate most of the impurities from each other and estimate their quantities in betamethylepoxide samples. The stability-indicating capability of this method has been demonstrated by adequate separation of the degradation products from betamethylepoxide in stress degraded and aged stability samples. The HPLC column used in the method was a 5 cm YMC Hydrosphere C(18) column (4.6 mm I.D.) and the mobile phase consisted of (A) water and (B) acetonitrile:methanol (8:25, v/v).

Development and validation of a stability-indicating reversed-phase high performance liquid chromatography method for NPC 1161C, a novel 8-aminoquinoline anti-malarial drug

NPC 1161C (±8-[(4-amino-1-methylbutyl)amino-5-(3,4-dichlorophenoxy)-6-methoxy-4-methylquinoline succinate]) is a novel investigational antimalarial drug of interest for its in vivo oral potency, activity against blood and tissue stage parasites, favorable toxicity profile, long duration of action, and utility in both prophylaxis and treatment models. The pharmaceutical development of NPC 1161C warranted the availability of an assay for the detection and quantification of the drug and its separation from the impurities and degradation products. A simple and rapid stability-indicating reversed-phase HPLC method was developed and validated according to ICH guidelines. The method was found to be linear, precise and accurate. It also proved to be selective in the presence of impurities and degradation products during forced degradation studies. The method was found to be robust by factorial experimental design and was well within the recommended parameters of system suitability testing. Degradants of the drug during stress studies were also identified using high resolution mass spectrometry.

Quantitation of 5-Flucytosine in capsules using high-pressure liquid chromatography

AbstractA stability-indicating reversed phase HPLC method for the quantitation of 5-flucytosine in capsules (the only dosage form available) has been developed. The method requires the use of a mobile phase without any counterion and the samples can be assayed at room temperature. The method is simple, reproducible, precise and accurate with percent relative standard deviation of 0.77 based on 6 readings. There was no interference from the excipients present in capsules and from fluorouracil (the major product of decomposition of 5-flucytosine). The recovery of 5-flucytosine from the synthetic mixtures was quantitative. A simple extraction procedure for 5-flucytosine from the capsules has been developed.