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.     

Stability indicating HPTLC determination of clopidogrel bisulphate as bulk drug and in pharmaceutical dosage form

A sensitive, selective, precise and stability indicating high-performance thin layer chromatographic method of analysis of clopidogrel bisulphate both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon tetrachloride-chloroform-acetone (6:4:0.15, v/v/v). This system was found to give compact spots for clopidogrel bisulphate (R_f value of 0.30±0.01). Clopidogrel bisulphate was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also the degraded products were well separated from the pure drug. Densitometric analysis of clopidogrel bisulphate was carried out in the absorbance mode at 230 nm. The linear regression data for the calibration plots showed good linear relationship with r²=0.999±0.001 in the concentration range of 200-1000 ng. The mean value of correlation coefficient, slope and intercept were 0.999±0.001, 0.093±0.011 and 8.83±0.99, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 40 and 120 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of degraded product were resolved from the standard drug with significantly different R_f values. This indicates that the drug is susceptible to acid-base hydrolysis, oxidation and dry heat degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Stability indicating high-performance thin-layer chromatographic determination of nelfinavir mesylate as bulk drug and in pharmaceutical dosage form

A sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method of analysis of nelfinavir mesylate both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-methanol-acetone (7:1.5:1.5, v/v/v). This system was found to give compact spots for nelfinavir mesylate (R_f value of 0.45±0.02). Nelfinavir mesylate was subjected to acid and alkali hydrolysis, oxidation, dry heat treatment and photodegradation. Also the peaks of degraded products were well resolved from the pure drug with significantly different R_f values. Densitometric analysis of nelfinavir mesylate was carried out in the absorbance mode at 250 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r²=0.999±0.002 in the concentration range of 1000-6000 ng per spot. The mean value of correlation coefficient, slope and intercept were 0.999±0.002, 0.014±0.001 and 21.73±1.26, respectively. The method was validated for precision, robustness and recovery. The limits of detection and quantitation were 60 and 140 ng per spot, respectively. Statistical analysis proves that the method is repeatable and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Determination of Exemestane in bulk and pharmaceutical dosage form by HPTLC

An HPTLC method for analysis of Exemestane in bulk and pharmaceutical formulation has been established and validated. The analyte was separated on aluminium plates precoated with silica gel 60 F₂₅₄. The mobile phase was chloroform:methanol 9.2:0.8 (v/v). Quantification was done by densitometric scanning at 247 nm. Response was a linear function of Exemestane concentration in the range of 100–500 μg mL⁻¹. The limit of detection and quantification for Exemestane were 5.8 and 17.58 μg mL⁻¹, respectively. Average recovery of Exemestane was 100.1, which shows that the method was free from interference from excipients present in the formulation. The established method enabled accurate, precise, and rapid analysis of Exemestane in bulk as well as pharmaceutical formulation.     

Stability-indicating HPLC method development and validation for simultaneous estimation of metformin,dapagliflozin, and saxagliptin in bulk drug and pharmaceutical dosage form

A simple, precise, and rapid stability-indicating reversed-phase-HPLC method was developed and validated for the estimation of metformin (MET), dapagliflozin (DAP), and saxagliptin (SAX) combination in bulk and tablet dosage forms. The proposed method uses a Kromasil C18 column (150 × 4.6 mm, 5 μm) with column oven temperature of 30°C and mobile phase containing a mixture of 60% phosphate buffer (pH = 3) and 40% acetonitrile. The flow rate was set at 1.0 mL/min, and the injection volume was 10 μL. The detection was carried out at 230 nm using a photodiode array detector, and the total run time was 4 min. The proposed method was validated according to International Council for Harmonisation (ICH) guidelines for specificity, linearity, precision, accuracy, robustness, and solution stability. The method is linear over the range of 125–750 μg/mL for MET, 1.25–7.5 μg/mL for DAP, and 0.625–3.75 μg/mL for SAX. The observed correlation coefficients (R²) for MET, DAP, and SAX are >0.999. The proposed method is precise, and the percentage relative standard deviation was found to be between 0.4 and 0.8. The observed percentage recoveries were between 98.51 and 100.80 for all three compounds. The product was subjected to stress conditions of acid, base, oxidative, thermal, and photolytic degradation. The product was found to degrade significantly in oxidative, acid, and base hydrolysis degradation conditions, and the degradation products were well determined from the active peaks, thus proving the stability-indicating power of the method. The developed and validated stability-indicating reversed-phase-HPLC method was appropriate for quantitative determination of these drugs in pharmaceutical preparations and also for quality control in bulk manufacturing.     

Stability‐indicating ultra‐performance liquid chromatography method for the estimation of thymoquinone and its application in biopharmaceutical studies

Thymoquinone (THQ) is known for its neuroprotective and anti‐convulsant properties in preclinical studies. We herewith describe a simple, rapid, selective, sensitive and stability‐indicating UPLC method for the estimation of THQ and its application to biopharmaceutical studies such as in vitro release from nanoparticulate system and in vivo pharmacokinetic study. The method employed gradient elution using a Waters Acquity HSS‐T3 C₁₈ (100 × 2.1 mm, 1.8 µm) UPLC column. The mobile phase consisted of water and acetonitrile, pumped at a flow rate of 0.5 mL/min. The injection volume was 5 µL and THQ was monitored at 294 nm wavelength with a total run time of 6 min. In solution as well as in plasma, the method was found to be linear (r ≥ 0.998), precise (CV ≤ 2.45%) and accurate (recovery ≥ 84.8%) in the selected concentration range of 0.1–0.8 µg/mL. Forced degradation studies revealed that THQ undergoes degradation under acidic, basic, oxidation and UV light stress conditions. However, the developed UPLC method could effectively resolve degradation product peaks from THQ. Further, no interference was found at the retention time of THQ from any plasma components, indicating selectivity of the developed method. For solutions, the limits of detection and quantitation of the method were found to be 0.001 and 0.0033 µg/mL, respectively; while in plasma they were 0.006 and 0.02 µg/mL, respectively. The validated method was successfully applied to quantify THQ in dissolution medium as well as oral in vivo pharmacokinetic study of THQ suspension and THQ‐ solid lipid nanoparticle (THQ‐SLN) formulation. A 2‐fold increase in the relative bioavailability was observed with the THQ‐SLN compared with THQ. The results indicate that the SLN significantly increased plasma concentrations and retention within the systemic circulation. Copyright © 2010 John Wiley & Sons, Ltd.     

Stability‐Indicating Methods for the Determination of Rofecoxib

Two stability indicating methods have been developed for determining rofecoxib in the presence of its degradation product. The first suggested method is high performance liquid chromatography (HPLC), in which analysis is carried out using hypersil BDS C18 column (250 × 4.6 mm I.D.) with mobile Phase consisting of 0.05 M phosphate buffer (pH 3.5) and acetonitrile (70:30 v/v). A linear relationship was obtained between the detector response at 225 nm and the corresponding concentration of the studied rofecoxib in the concentration range (1–6 μg / 10 μl) with mean % recovery of 99.80 ± 0.405. The second method depends on the quantitative densitometric evaluation of thin layer chromatograms (HPTLC) with mobil phase consisting of toluene: chloroform: methanol (60: 35: 5 v/v/v) by using fluorescent high performance silica gel 60 plate. A linear relationship was obtained between peak area and the concentration of the cited drug in the range 1–6 μg/spot with a mean % recovery of 99.79 ± 0.185. The suggested methods are precise, accurate, rapid and prove their specificity in the presence of its degradation products. Both procedures are successfully applied to determine the drug in the presence of its degradation product and also in their pharmaceutical formulations.     

Forced-degradation study of valdecoxib as bulk drug and in tablet formulation by HPTLC

A stability-indicating forced-degradation study of valdecoxib was conducted using high performance thin layer chromatography (HPTLC). It was used to analyze valdecoxib as bulk drug and as tablets. Undegraded valdecoxib was eluted with a retardation factor, Rf, of 0.56. Valdecoxib was forcibly degraded by exposure to alkali, acid, oxidation, and light, the greatest degradation occurring under basic conditions. Base-degraded valdecoxib gave an additional peak with an Rf value of 0.76. The calibration curve was linear in the range of 0.2-1 microg/microL with a correlation coefficient of 0.9952. Complete validation was carried out for precision (inter-day, intra-day, repeatability), accuracy, and robustness. All the data were analyzed statistically. This HPTLC procedure shows the reliability needed for use as a stability-indicating method. It can quantify valdecoxib in bulk and in tablets and also resolves the degraded peak of valdecoxib. This method is also useful for studying the degradation pattern and degradation mechanism of valdecoxib.     

Validated HPTLC method for simultaneous estimation of levofloxacin hemihydrate and ornidazole in pharmaceutical dosage form

A simple, rapid, and accurate high-performance thin-layer chromatography (HPTLC) method is described for the simultaneous determination of levofloxacin hemihydrate and ornidazole in tablet dosage form. The method is based on the HPTLC separation of the two drugs followed by densitometric measurements of their spots at 298 nm. The separation is carried out on Merck TLC aluminium sheets of silica gel 60 F254 using n-butanol-methanol-ammonia (5:1:1.5, v/v/v) as mobile phase. The linearity is found to be in the range of 50-250 and 100-500 ng/spot for levofloxacin hemihydrate and ornidazole, respectively. The method is successively applied to pharmaceutical formulation because no chromatographic interferences from the tablet excipients are found. The suitability of this HPTLC method for the quantitative determination of the compounds is proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines.     

HPTLC method for determination of nevirapine in pharmaceutical dosage form

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis of nevirapine both as a bulk drug and in formulations was developed and validated. The solvent system consisted of toluene-carbon tetrachloride-methanol-acetone-ammonia (3.5:3.5:2.0:1.0:0.05, v/v/v/v/v). Densitometric analysis of nevirapine was carried out in the absorbance mode at 289nm. This system was found to give compact spots for nevirapine (R(f) value of 0.44+/-0.02). Nevirapine was subjected to acid and alkali hydrolysis, oxidation, dry heat and wet heat treatment and photodegradation. The drug undergoes degradation under acidic, basic conditions and oxidation. Also the degraded products were well resolved from the pure drug with significantly different R(f) values. Linearity was found to be in the range of 30-1000ng/spot with significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots showed good linear relationship with r(2)=0.998+/-0.002 in the working concentration range of 300ng/spot to 1000ng/spot. The mean value of slope and intercept were 0.073+/-0.005 and 36.78+/-1.50, respectively. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 5 and 10ng/spot, respectively. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of acid degradation process. Arrhenius plot was constructed and activation energy was calculated.     

Comprehensive stability‐indicating high‐performance liquid chromatography coupled with diode array detection method for simultaneous determination of amprolium hydrochloride and ethopabate in powder dosage form for veterinary use

This research deals with the development of a stability‐indicating high‐performance liquid chromatography method for simultaneous determination of amprolium hydrochloride and ethopabate. To the best of our knowledge, no comprehensive stability‐indicating method has been reported for analysis of this mixture. Separation was achieved using Kromasil cyano column with gradient elution of the mobile phase composed of sodium hexane sulfonate solution and methanol. Quantification was based on measuring peak areas at 266 nm. Amprolium and ethopabate peaks eluted at retention times 10.42 and 18.53 min, respectively. The proposed procedure was validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Linearity ranges for amprolium and ethopabate were 1.5–240 and 1–160 μg/mL, respectively. Analytes were subjected to stress conditions of hydrolysis, oxidation and thermal degradation. The proposed method enabled resolution of drugs from their forced‐degradation products and amprolium related substance (2‐picoline). Moreover, specificity was verified by resolution of the analytes from about 22 drugs used in antimicrobial veterinary products. The validated method was successfully applied to assay of the combined veterinary powder dosage form, additionally it was implemented in the accelerated stability study of the dosage form when stored for six months at 40°C and 75% relative humidity.     

Stability‐indicating HPLC assay for lysine–proline–valine (KPV) in aqueous solutions and skin homogenates

A simple, sensitive and stability‐indicating high‐performance liquid chromatographic (HPLC) assay method was developed and validated for a bioactive peptide, lysine–proline–valine (KPV) in aqueous solutions and skin homogenates. Chromatographic separation was achieved on a reversed phase Phenomenex C₁₈ column (4.6 × 250 mm, packed with 5 µm silica particles) with a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). The proposed HPLC method was validated with respect to accuracy, precision, linearity, repeatability, limit of detection (LOD) and limit of quantitation (LOQ). The calibration curve was linear with a correlation coefficient (r) of 0.9999. Relative standard deviation values of accuracy and precision experiments were <2. The LOD and LOQ of KPV were 0.01 and 0.25 µg/mL, respectively. Under stress conditions (acid, alkali and hydrogen peroxide) KPV yielded lys–pro–diketopiperazine as major degradation product, which was identified by flow injection MS analysis. The developed HPLC method was found to be efficient in separating the active peptide from its degradation products generated under various stress conditions. Also, the validated method was able to separate KPV from other peaks arising from endogenous components of the skin homogenate. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of aliskiren and hydrochlorothiazide from their pharmaceutical preparations using a validated stability‐indicating MEKC method

A stability‐indicating MEKC method was developed and validated for the simultaneous determination of aliskiren (ALI) and hydrochlorothiazide (HCTZ) in pharmaceutical formulations using ranitidine as an internal standard (IS). Optimal conditions for the separation of ALI, HCTZ and its major impurity chlorothiazide (CTZ), IS and degradation products were investigated. The method employed 47 mM Tris buffer and 47 mM anionic detergent SDS solution at pH 10.2 as the background electrolyte. MEKC method was performed on a fused‐silica capillary (40 cm) at 28°C. Applied voltage was 26 kV (positive polarity) and photodiode array (PDA) detector was set at 217 nm. The method was validated in accordance with the ICH requirements. The method was linear over the concentration range of 5–100 and 60–1200 μg/mL for HCTZ and ALI, respectively (r²>0.9997). The stability‐indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Precision and accuracy evaluated by RSD were lower than 2%. The method proved to be robust by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of ALI and HCTZ both individually and in a combined dosage tablet formulation to support the quality control.     

A novel validated LC method for quantitation of lopinavir in bulk drug and pharmaceutical formulation in the presence of its potential impurities and degradation products

A simple isocratic liquid chromatographic method was developed for determination of lopinavir from its related impurities and assay for the first time. This method involves the use of a C(8) (Symmetry Shield RP8, 150 x 4.6 mm, 5 microm) column. The method was validated over the range of limit of quantitation (LOQ) to 120% of impurity specification limit and LOQ to 150% of working concentration for assay. The mobile phase consisted of a mixture of 50 mM of potassium phosphate buffer, acetonitrile and methanol in the ratio of 40:50:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 210 nm. The drug 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 was successfully applied for content determination of lopinavir in pharmaceutical formulations. The method can be conveniently used in a quality control laboratory for routine analysis for assay and related substances as well for the evaluation of stability samples of bulk drugs and pharmaceutical formulations.     

Validated HPTLC and HPLC methods for determination of fluorometholone and sodium cromoglycate in presence of their impurities and degradation products; application to kinetic study and on rabbit aqueous humor

Fluorometholone (FLM) and Sodium Cromoglycate (CMG) are co-formulated in ophthalmic preparation and showed marked instability under different conditions. Two specific, sensitive and precise stability-indicating chromatographic methods have been developed and validated for their determination in the presence of their degradation products and FLM impurity. Ten components were efficiently separated by them. The first method was HPTLC-spectrodensitometry, where the separation was achieved using silica gel 60?F₂₅₄ HPTLC plates and developing system of ethyl acetate: methanol (9:1, v/v). The second method was a reversed phase HPLC associated with kinetic study of the degradation process and was successfully applied for determination of the studied compounds in spiked rabbit aqueous humor. The mobile phase was acetonitrile: methanol: 0.05?M potassium dihydrogenphosphate (0.1% trimethylamine); pH 2.5, adjusted with orthophosphoric acid (20: 30: 50, by volume). In both methods, the separated components were detected at 240?nm and system suitability was checked. Good correlation was obtained in the range of 0.10–24.00 and 0.20–48.00?µg band^?1, for FLM and CMG by HPTLC. While for HPLC, the linearity ranges from 0.01–50.00 and 0.05–50.00?µg?mL^?1 for both drugs. The methods were applied in pharmaceutical formulation, where they were compared to the reported method with no significant difference.     

Stability indicating high-performance thin-layer chromatographic determination of gatifloxacin as bulk drug and from polymeric nanoparticles

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method for determination of gatifloxacin both as a bulk drug and from polymeric nanoparticles was developed and validated as per the International Conference on Harmonization (ICH) guidelines. The method employed thin-layer chromatography (TLC) aluminium plates precoated with silica gel 60F-254 as the stationary phase and the mobile phase consisted of n-propanol-methanol-concentrated ammonia solution (25%) (5:1:0.9, v/v/v). This solvent system was found to give compact spots for gatifloxacin (R_f value of 0.60 ± 0.02). Densitometric analysis of gatifloxacin was carried out in the absorbance mode at 292 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9953 with respect to peak area in the concentration range of 400-1200 ng spot⁻¹. The mean value (±S.D.) of slope and intercept were 9.66 ± 0.05 and 956.33 ± 27.67, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 2.73 and 8.27 ng spot⁻¹, respectively. Gatifloxacin was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. The drug undergoes degradation under acidic and basic conditions and upon wet and dry heat treatment. The degraded products were well separated from the pure drug. The statistical analysis proves that the developed method for quantification of gatifloxacin as bulk drug and from polymeric nanoparticles is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating one.     

Stability indicating RP-HPLC method for simultaneous estimation of rupatadine fumarate and montelukast sodium in bulk and tablet dosage form

A simple, selective, accurate and sensitive high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of rupatadine fumarate (RPT) and montelukast sodium (MNT). Chromatographic separation achieved isocratically on a Hypersil BDS C₈ (250 mm × 4.6 mm, 5 μm) column utilizing a mobile phase of methanol: acetonitrile: buffer (40: 30: 30), (pH 3 with H₃PO₄) at a flow rate of 1.0 mL/min and column oven temperature 40°C with UV detection at 270 nm. Statistical analysis proves that the method is reproducible and selective for the simultaneous estimation of RPT and MNT. As the method could effectively separate the drugs from their degradation products, it can be employed as stability indicating method. The developed method was validated as per ICH guidelines in terms of accuracy, precision, linearity and specificity.     

Development and validation of stability indicating RP-HPLC and HPTLC for determination of Niclosamide in bulk and in synthetic mixture

Two simple, specific, sensitive, accurate and precise stability indicating methods were described for quantitative determination of the anthelmintics drug Niclosamide. The first method was high performance liquid chromatographic with the use of a reversed phase hibar^R C-18 column (250 mm × 4.66 mm, 5 μm) and mobile phase of methanol: 1 mM ammonium phosphate buffer (85:15 v/v) at a flow rate of 1.2 mL/min. The retention time of drug was found to be 6.45 ± 0.02 min. Quantification of drug was achieved with diode array detection (DAD) at 332 nm. Linear calibration curve was obtained in concentration range 0.01–100 μg/mL with r² value of 0.999. The limit of detection and limit of quantification were found to be 0.048 μg/mL and 0.01 μg/ml respectively. The second method involved a high performance thin layer liquid chromatographic. Chromatographic separation was carried out with precoated silica gel G60 F254 aluminum sheets using toluene:ethyl acetate (7:3% v/v) as a mobile phase. Linearity of proposed method was found to be 200–700 ng/band at 332 nm with retention factor of 0.59 and r² value of 0.998. The limit of detection and limit of quantification were found to be 36.21 ng/band and 109.7 ng/band respectively. Both the developed methods were successfully validated as per International Conference on Harmonization guideline (ICH). Niclosamide was subjected to different stress conditions. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time. Stress samples were successfully assayed by developed high performance liquid chromatographic and high performance thin layer liquid chromatographic method. Statistically analysis proves that there were no statistical significant differences between two developed methods.     

Development and validation of analytical method for estimation of bilastine in bulk and pharmaceutical (tablet) dosage form

BackgroundThe present work describe a simple, linear, precise, robust, accurate and selective HPLC method for estimation of Bilastine in bulk and tablet dosage form. Bilastine is a second generation antihistamine medication. Generally it is used for treatment of allergic rhinoconjunctivitis and urticaria (hives). Methanol: Acetonitrile: Phosphoric Acid Buffer pH 2.1 in proportion of (21:33:46) was used as mobile phase with flow rate 1.0 ​ml/min. The column used for the method development is 250 ​× ​4.6 mm ​× ​5 ​μm dimension.ResultIn the range of 5–25 ​μg/ml the linearity of Bilastine shows a correlation coefficient of 0.9981.ConclusionThe method was validated as per ICH guidelines for linearity, precision, accuracy, robustness.     

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.