Stability-indicating Reversed-phase High-performance Liquid Chromatography Research Articles

A New Stability-Indicating RP-HPLC Method for Simultaneous Quantification of Diacerein and Aceclofenac in Novel Nanoemulgel Formulation and Commercial Tablet Dosage Form in the Presence of their Major Degradation Products Using DoE Approach.

The present study aimed to develop a simple, robust, sensitive and effective stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous quantification of diacerein (DCN) and aceclofenac (ACE) in novel nanoemulgel formulation and commercial tablets in the presence of their main degradation product: rhein (RH) and diclofenac sodium (DLS), respectively. A fractional factorial design was used to screen the crucial independent factors, whereas a central composite design was used for the optimization of the chromatographic conditions. The separation was carried out on Phenomenex C18 column (5μm, 250 × 4.6mm), using a mobile phase consisting of phosphate buffer pH3 (0.1% v/v orthophosphoric acid) and acetonitrile (40:60v/v) at a flow rate of 1mL/min with detection at 264nm. The analytes were exposed to a variety of stress conditions, including heat, alkali, acid, oxidation, photochemical, humidity and hydrolysis. DCN, ACE, RH and DLS were found to have retention times of 4.32 ± 0.15, 5.77 ± 0.07, 8.28 ± 0.20 and 9.10 ± 0.18min, respectively. The percent recovery for all four analytes was found to be between 98 and 102, and the procedure was discovered to be linear in the range of 0.1-64μg/mL with R2 value > 0.999. The established method was validated as per ICH guidelines and successfully used to assay DCN and ACE in their combined marketed tablet dosage form and developed nanoemulgel formulation.

Determination of Firocoxib and Its Related Substances in Bulk Drug Substance Batches of Firocoxib by a High-Speed Reversed-Phase HPLC Method With a Short Fused-Core Biphenyl Column.

Firocoxib is a nonsteroidal anti-inflammatory drug. It provides control of postoperative pain and inflammation associated with soft tissue and orthopedic surgery in dogs, and control of pain and inflammation associated with osteoarthritis in horses. A high-speed stability-indicating reversed-phase high-performance liquid chromatography method was developed to determine firocoxib and its related substances in bulk batches of firocoxib drug substance. Firocoxib was dissolved in neat acetonitrile (ACN) and analyzed on a short HALO (fused-core) biphenyl column (30 × 4.6mm i.d., 2.7-μm particle size) at flow rate of 2.5mL/min. Column temperature was maintained at 50°C. Mobile phase A is composed of 0.1% of H3PO4 in water and mobile phase B is composed of ACN. Analytes were detected with UV detection at 240nm and quantitated against an external reference standard. Firocoxib and its related compounds were adequately separated within 4min by a gradient elution. The method was validated for specificity, linearity, accuracy, precision and robustness according to method validation guidelines described in The International Conference on Harmonization. The validation data demonstrated that this method is sensitive, accurate, robust, specific and stability-indicating.

Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Determination of Doxycycline and Rifampicin in Polymeric Nanoparticles

Background: The combination of doxycycline (DOXY) and rifampicin (RIF) is recom-mended as a treatment therapy for brucellosis by the World Health Organization. Objective: The aim of the current study was to develop and validate the stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the analysis of a combina-tion of doxycycline & rifampicin. Methods: The RP-HPLC method was developed and validated to estimate the doxycycline and ri-fampicin combination as per ICH guidelines. The drug combination solution was exposed to differ-ent stress conditions: acidic, basic, photo-oxidation, and oxidation. Results: The method was found linear in the range of 2 -10μg/mL for both the drugs with a reten-tion time of 3.5 min for doxycycline and 6.5 min for rifampicin at lambda maximum of 350 nm. The RP-HPLC method was precise and accurate with %RSD < 2%. The intra-day and inter-day precisions were calculated and found within the acceptable range of 5%. Both drugs demonstrated good stability in the mobile phase after 6 hours. The LOD and LOQ of doxycycline and rifampicin were 100 ng/mL & 200ng/mL and 150ng/mL & 500ng/mL, respectively. The forced degradation of the combination of drug solutions was performed. The degraded drug peaks were well-resolved from the peaks of drugs. The percentage encapsulation efficiency of doxycycline and rifampicin in the nanoparticle system was assessed by utilizing the validated RP-HPLC method and found >60% (DOXY) and >70% (RIF). Conclusion: The developed RP-HPLC method of DOXY-RIF combination was rapid, accurate, precise, and stability-indicating. The method can be appropriately applied todetecte drugs in the na-noparticulate system.

Development and Validation of a Stability-Indicating Reversed-Phase HPLC Method for Assay and Estimation of Related Substances of Ivermectin in an Oral Paste.

Ivermectin is a potent semi-synthetic antiparasitic drug used in veterinary medicine. A reversed-phase high performance liquid chromatography (HPLC) method has been developed and validated for the identification and assay of Ivermectin, including the identification and estimation of its related impurities in an oral paste. Analytes were separated using a gradient elution at a flow rate of 1.5mL/min on a Zorbax Extend-C18 column (150mm×4.6mm i.d., 3.5-μm particle size) maintained at 30°C. The mobile phase was composed of water as mobile-phase A and acetonitrile/methanol (85/15, v/v) as mobile-phase B. Ultraviolet detection at 245nm was employed to monitor the analytes. Limit of quantitation (LOQ) and limit of detection (LOD) of the method are 0.6 and 0.2μg/mL, respectively. The validation results demonstrated excellent linearity of the method in the range of 0.1-150% of the analytical concentration (0.6mg/mL) of the method. The stability-indicating capability of the method has been demonstrated by adequately separating the degradation products from the stress degraded samples of the oral paste as per method validation requirements prescribed in the current International Council for Harmonisation guidelines.

Development and Validation of a Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Identification, Assay, and Estimation of Related Substances of Ivermectin Drug Substance.

Ivermectin is a potent semi-synthetic antiparasitic drug used in veterinary medicine. It is widely used for the treatment of parasites. This study aimed to develop a stability-indicating reversed-phase HPLC (RP-HPLC) method for assay and identification of ivermectin including identification and estimation of its related substances in bulk drug substance batches of ivermectin. Ivermectin and its related substances were separated on an Ascentis Express C18 column (100 mm × 4.6 mm id, 2.7 µm particle size) maintained at 45°C (column temperature) on an HPLC system with gradient elution. The mobile phase was composed of water - acetonitrile (ACN; 50 + 50, v/v) as mobile phase A, and isopropanol - ACN (15 + 85, v/v) as mobile phase B. Analytes were detected with a detection wavelength of 252 nm and quantitated against an external reference standard of ivermectin with a quantitation limit of 0.1% of the target (analytical) concentration. The HPLC method was able to separate all analytes of interest by gradient elution within 25 min. The method was validated according to the guidelines described in the International Conference on Harmonization guideline Q2(R1). The HPLC method for assay of ivermectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability indicating. The performance of the HPLC method is significantly faster and possesses a higher degree of selectivity. Implementation of this method for routine analysis in QC laboratories would save significant time, resources and solvents.

Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes.

A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation.

Development and Validation of a Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography Method for Assay of Doramectin and Estimation of its Related Substances in Commercial Batches of Doramectin Drug Substance.

Doramectin is a broad-spectrum antiparasitic drug used in veterinary medicine. It belongs to the family of avermectins, which possess a macrocyclic lactone structure, and is widely used for the treatment of parasites. This study aimed to develop a stability-indicating reversed-phase (RP) HPLC method for the assay and identification of doramectin including identification and estimation of its related substances in commercial batches of doramectin drug substance. Methanol was used to dissolve and prepare doramectin samples. Doramectin and its related substances were separated on a HALO C8 (100 mm × 4.6 mm i.d., 2.7 µm particle size) column maintained at 40 °C using an isocratic HPLC method with a mobile phase composed of acetonitrile-water (70 + 30, v/v). Analytes were detected with UV detection at 245 nm and quantitated against a single point external reference standard of doramectin. The LOQ of the method is 0.1% of the target concentration as described in the method. The HPLC method can separate all analytes of interest by an isocratic elution within 10 min. The method was validated according to the guidelines described in the International Conference on Harmonization Q2(R1). The HPLC method for assay of doramectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability-indicating. This is the first known paper to report an HPLC method for assay of doramectin and estimation of its related substances in commercial batches of doramectin drug substance.

A NEW HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SEPARATION AND SIMULTANEOUS QUANTIFICATION OF EPTIFIBATIDE AND ITS IMPURITIES IN PHARMACEUTICAL INJECTION FORMULATION

Objective: The objective of the present study is to develop a stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for qualitative and quantitative determination of Eptifibatide and its impurities in bulk and pharmaceutical dosage forms.
 Methods: The chromatographic separation was carried on Phenomenex Luna C18 column (250 mm×4.6 mm; 5µ id) as stationary phase, methanol and phosphate buffer at pH 6.4 in the ratio of 65:45 (v/v) as mobile phase at flow rate of 1.0 ml/min, Ultra Violet (UV) detection was carried at the wavelength of 236 nm and the analysis was completed with a run time of 15 min.
 Results: In the developed conditions, the retention time of Eptifibatide and its impurities 1 and 2 were found to be 3.35, 4.93 and 8.18 min, respectively. The method was validated for system suitability, range of analysis, precision, specificity, stability and robustness. Spiked recovery at 50%, 100% and 150% was carried for both standard and impurities and the acceptable % recovery of 98-102 was observed for Eptifibatide and both impurities studied and the % Relative standard deviation (RSD) in each spiked level was found to be less than 2. Stability tests were done through the exposure of the analyte solution to five different stress conditions i. e expose to 1N Hydrochloric acid (HCl), 1 N Sodium hydroxide (NaOH), 3% Hydrogen peroxide (H2O2), 80 °C temperature to UV radiation. In all the degradation conditions, standard drug Eptifibatide was detected along with both the impurities studied and the degradation products were successfully separated. In the formulation analysis, there is no other chromatographic detection of other impurities and formulation excipients.
 Conclusion: The developed method was found to be suitable for the quantification of Eptifibatide and can separate and analyse impurities 1 and 2.

A Validated specific Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography Assay Method for L-Ornithine L-Aspartate and Silymarin, and their related Substances and its Application to Dissolution Studies

The present study was aimed at the development and successive validation of a novel, simple, sensitive, and stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for quantitative calculation of L-ornithine L-aspartate (LOLA) and Silymarin (SL), and also their relevant substances in bulk and pharmaceutical dosage forms. The chromatographic technic was optimized using the impurity-spiked solution. The separation of all the two active components and their impurities was achieved by a chromatographic method with an Agilent Eclipse XDB-C18, 150 × 4.6 mm, 3.5 μ column, using gradient elution with mobile phase A consisting of a mixture of 0.1% orthophosphoric acid and water and acetonitrile as mobile phase B. The instrumental settings included a flow rate of 1 mL/min for both related substances and assay, a detector wavelength of 225 nm, by using a PDA detector. The established method was validated according to the current ICH requirements. The detection limit and the limit of quantification for the two active components and their related impurities were established with respect to test concentration. The calibration graphs plotted were linear with a regression coefficient R2 andgt; 0.999, indicates the linearity of the method was within the limits. Recovery studies were satisfactory and the parameters, such as, specificity, linearity, accuracy, precision, and robustness were determined as part of the method validation. Moreover, using the same method dissolution study was performed on active pharma ingredients to estimate the recovery. The obtained results were within the range of acceptance criteria. These results suggest that the developed method was found to be applicable for routine analysis for testing chromatographic purity of LOLA and SL and it can be utilized for the calculation of both active ingredients and their impurities in tablet dosage forms.

Development and Validation of Stability-Indicating Assay Method by RP-HPLC for Simultaneous Estimation of Rosuvastatin Calcium and Fenofibrate in Pharmaceutical Dosage Form

A Simple, precise, and accurate stability-indicating reversed-phase high-performance liquid chromatography method has been established for the simultaneous estimation of rosuvastatin calcium and fenofibrate in combined bulk and tablet formulation. The chromatographic separation was performed on reverse phase Princeton (C18) (250 mm x 4.6 mm, 5µ) column with mobile phase as a mixture of water (pH adjusted to 3.0 with orthophosphoric acid) and acetonitrile in the ratio (40:60) v/v at the flow rate 1.0 ml/min. Detection was carried out at wavelength 240 nm. The retention time under the optimized condition of Rosuvastatin calcium and Fenofibrate was found to be 2.485 & 3.905 minutes respectively. The calibration curve was linear in the range of 6-16 µg/ml and 87-232 µg/ml for rosuvastatin calcium and fenofibrate with a correlation coefficient of 0.9999 and 0.9994 respectively. Relative standard deviation values for all key parameters were less than 2.0%. The percentage recovery was found to be 99.66-100.37% and 99.13-100.44% for rosuvastatin calcium and fenofibrate respectively. The developed reversed-phase high-performance liquid chromatography method was found to be simple, specific, sensitive, rapid, linear, accurate, precise, and economical, and could be used for regular quality control of rosuvastatin calcium and fenofibrate in bulk and tablet formulations. 
 Keywords: Rosuvastatin calcium, Fenofibrate, RP-HPLC, Method validation, ICH guidelines.

Stability-Indicating Simultaneous Method Development and Validation of Guaifenesin and Dextromethorphan HBr by Reverse-Phase High-Performance Liquid Chromatography

The developed method was validated according to ICH guidelines with respect to specificity, linearity, limits of detection, quantification, accuracy, precision, and robustness. The stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method is precise; it has been developed for the simultaneous estimation of assay of guaifenesin (GN) and dextromethorphan hydrobromic (HBr) (DN) in drug substance and drug product. The chromatographic separation was done in an isocratic mode using the Syncronus C8 (250 × 4.6 mm, 5 μ particle size) column with mobile phase containing a 10 mM ammonium acetate in water (modulated pH 4.30 with orthophosphoric acid) and acetonitrile in the ratio of 60:40 (% v/v) used for efficient chromatographic separation. The flow rate of the mobile phase was 1 mL/min with ambient column temperature and detection of wavelength at 279 nm; injection volume 10 μL was fixed for achieving good elution of eluents. The retention time for GN was found to 3.46 minutes and DN was found to 7.58 minutes. GN and DN were linear in the concentration range from 357 to 1,428 and 19 to 75 μg/mL, respectively. Regression analysis showed that the r value (correlation coefficient) greater than 0.999 for GN r value was found to be 0.999, GN r value was found to be 0.999, DN r value was found to be 0.999. Limit of detection (LoD) and limit of quantification (LoQ) of GN was found to be 0.151 and 0.904 μg/mL, DN was found to be 0.241 and 0.726 μg/mL. The developed method was validated and found to be accurate, specific, and robust. Both the drugs were subjected to the stress conditions like acidic, basic, oxidative, photolytic, and thermal conditions. The degradation results were found to be satisfactory. In peroxide stress condition, GN was found stable over DN, and DN was found to degrade significantly. The degradation products were well resolved from GN, DN, and their impurities. The peak purity test results confirmed that the GN and DN peak were homogenous and pure in all stress conditions, thus, proving the stability-indicating nature of the method. This method could be applied for the simultaneous estimation of GN and DN in drug substance and drug product.

Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Sofosbuvir, Velpatasvir, and Voxilaprevir in Tablet Formulation

Objective: The present study aimed to develop a stability-indicating reverse-phase high performance-liquid chromatography (RP-HPLC) method for the estimation of Sofosbuvir, Velpatasvir, and Voxilaprevir in tablet dosage form and validated in accordance with ICH guidelines. Methods: The optimized conditions for the developed RP-HPLC method are Agilent C18 (250 mm×4.6mm, 5μ) column maintained at 30ºC with a mobile phase consisting of Buffer(0.1%OPA) and Acetonitrile taken in the ratio 55:45%v/v on isocratic mode at flow rate 1.0ml/min. The sample was detected at 220 nm. Results: The retention time of Sofosbuvir, Velpatasvir, and Voxilaprevir was found to be 2.17, 2.731 and 3.55 min respectively. The developed method was validated for accuracy, precision, specificity, ruggedness, robustness and solution stability.

Butyl-based reversed-phase high-performance liquid chromatography and silica normal-phase high-performance thin-layer chromatography methods for the determination of palonosetron in the presence of degradation products and dosage form additives

Stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) and normal-phase high-performance thin-layer chromatography (NP-HPTLC) methods have been developed for the determination of palonosetron which is a potent antiemetic drug used with chemotherapy. Forced degradation studies were performed on palonosetron to study its stability behavior. The drug was subjected to acid, base, neutral hydrolysis, oxidation, thermal and photolytic conditions. Mass analysis has been performed to elucidate the oxidative degradants by Advion compact mass spectrometer. HPLC separation was achieved on PerfectChrom 100 C4 (250 × 4.6 mm, 5 μm particle size) column using buffer (20 mM dipotassium hydrogen orthophosphate, adjusted with phosphoric acid to pH 2.5):acetonitrile:methanol (60:30:10, v/v) as the mobile phase with isocratic mode at a flow rate of 1 mL/min using photodiode array detector (PAD) at 210 nm. The method showed adequate sensitivity concerning linearity, accuracy and precision over the range of 0.1–10 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were determined to be 0.03 μg/mL and 0.09 μg/mL, respectively. HPTLC separation was carried out on aluminum plates pre-coated with silica gel 60 F254 using methanol:ammonia (10:0.5, v/v) as the mobile phase. CAMAG scanner was operated at 254 nm for the densitometric measurement in the absorbance mode. A polynomial relationship was constructed in concentration range of 0.1–2 μg/band, with LOD and LOQ 0.02 μg/band and 0.06 μg/band, respectively. The cited chromatographic methods were successfully applied to the determination of palonosetron in the presence of its degradation products and additives in the commercially available vials. Method validation was performed as per the ICH guidelines confirming methods robustness to be used in quality control laboratories. Statistical comparisons have been performed between the results of the cited chromatographic methods and those of the official one using Student’s t test and F test values at 95% confidence interval level, revealing good accuracy and precision.

An Improved Reversed-Phase High-Performance Liquid Chromatography Method for the Analysis of Related Substances of Prednisolone in Active Ingredient.

Prednisolone, an important active pharmaceutical ingredient, is a synthetic glucocorticoid used for the preparation of various pharmaceutical products with anti-inflammatory and immunosuppressive properties. It is a challenge in high-performance liquid chromatography (HPLC) to separate the prednisolone peak and its structurally related substance (hydrocortisone), which only differs in a double bond at the C-1 position. Successful application of the HPLC method according to the European Pharmacopoeia monograph for related substances of prednisolone is very often limited to the chromatographic system available. This is due to the nonbaseline separation of the prednisolone and hydrocortisone peaks, which is strongly influenced by the instrument parameters and the chosen C18 column. First, an adjusted European Pharmacopoeia method for related substances of prednisolone was developed within the allowable adjustments. Next, an improved stability-indicating reversed-phase HPLC method for related substances of prednisolone was developed and validated for use in quality control laboratories for routine analysis. The optimized separation was performed on a Phenomenex Gemini C18 column (150 mm × 4.6 mm, 3 μm) using a gradient mobile-phase system consisting of acetonitrile/tetrahydrofuran/water (15:10:75 v/v/v), acetonitrile/water (80:20 v/v), and ultraviolet detection at 254 nm. A baseline separation was achieved, and stability indicating capability was demonstrated by a forced degradation study. A full validation procedure was performed in accordance with International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines.

STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF SOFOSBUVIR, VELPATASVIR, AND VOXILAPREVIR IN BULK AND TABLET DOSAGE FORMS

Objective: The present study was aimed to develop a novel, simple, rapid accurate and precise, stability-indicating reversed-phase high-performance liquid chromatography method for the simultaneous estimation of sofosbuvir, velpatasvir, and voxilaprevir in bulk and tablet dosage forms.
 Methods: The chromatographic elution was achieved in isocratic mode using the combination of sofosbuvir, velpatasvir, and voxilaprevir in the ratio of acetonitrile and water (65:35%v/v) using a Phenomenex C18 column which has specification (150 × 4.6 mm, 5 μ particle size) and the flow rate of 1.0 ml/min and wavelength (ultraviolet) detection at 220 nm.
 Results: The retention time obtained for sofosbuvir, velpatasvir, and voxilaprevir was 2.213 min, 2.568 min, and 2.917 min, respectively. Sofosbuvir, velpatasvir, and voxilaprevir and their combination drug product were exposed to acidic, alkali, thermal, photolytic, and oxidative stress conditions. The current method was validated according to the ICH guidelines for accuracy, precision, linearity, specificity, and sensitivity.
 Conclusion: The method developed is more sensitive, accurate, precise, and robust then the methods reported earlier. Retention time and run time were decreased; hence, the method is economical simple and precise. Forced degradation studies indicated for the suitability of the method for stability studies of sofosbuvir, velpatasvir, and voxilaprevir. The proposed method can be used for routine quality control analysis test in pharmaceutical industries.

STABILITY INDICATING RP-HPLC ASSAY OF HYDROCODONE AND IBUPROFEN IN TABLETS

Objective: The combination of hydrocodone and ibuprofen is prescribed to temporarily relieve severe pain. A new selective, precise and accurate stability indicating reverse phase high performance liquid chromatography (RP-HPLC) technique was developed for identification and estimation of hydrocodone and ibuprofen in tablets.
 Methods: The analysis of hydrocodone and ibuprofen was executed on phase Zodiac C18 (250 × 4.6 mm, 5 μm) column with sodium dihydrogen phosphate (0.1M, pH5.0) and methanol (60:40, v: v) as a mobile phase with a flow rate of 1.0 ml/min. The detection and measurement was done at 234 nm. The stability of hydrocodone and ibuprofen was examined under different conditions recommended by ICH, including alkaline, acidic, neutral, oxidative, photolytic and thermal.
 Results: Hydrocodone and ibuprofen are eluted at 4.019 min and 4.999 min, respectively. The assay method was linear (range: 3.75-11.25 µg/ml and 100-300 μg/ml) with R2values of 0.9996 and 0.9999 for hydrocodone and ibuprofen, respectively. Method was accurate (recovery: 99.65% and 99.37% for hydrocodone and ibuprofen, respectively), precise (RSD: 0.262% and 0.261% for hydrocodone and ibuprofen, respectively), selective and robust. In degradation studies, peaks of degradants did not interfere with the peaks of hydrocodone and ibuprofen. This validated method was applied to assay the content of hydrocodone and ibuprofen in tablets.
 Conclusion: This method can be opted in the routine quality control test of fixed dose tablet combination comprising of hydrocodone and ibuprofen.

A reliable HPLC-DAD method for simultaneous determination of related substances in TBI-166 active pharmaceutical ingredient

A sensitive, stability-indicating reversed-phase high-performance liquid chromatography with diode array detection (HPLC–DAD) method has been developed for the determination of TBI-166 and its 10 kinds of related impurities. Chromatographic separation was achieved on a Kromasil ODS column (250 mm × 4.6 mm, 5 μm), with a gradient elution of the mobile phase system consisting of acetonitrile and 1% ammonium formate solution (with 0.2% formic acid). The flow rate was 1.0 mL/min, and the detection wavelength was set at 251 nm. The method was validated according to the International Conference on Harmonization (ICH) guidelines with respect to selectivity, linearity, limits, accuracy, precision, and robustness. The calibration curves were linear from LOQ to 150% of the specification limit of impurity with correlation coefficients not less than 0.999. The limits of quantitation were between 0.123 and 0.257 μg/mL. Accuracy for the related substances was estimated by the recovery ranged from 94.6% to 111.2%. The method was proved to be reliable for the determination of related substances in TBI-166 bulk drug, which is essential and important in the quality control.

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE ESTIMATION OF LEDIPASVIR IN BULK AND TABLET DOSAGE FORM

Objective: The objective of this study was to develop a stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the estimation of ledipasvir (LDP) in bulk and tablet formulation.
 Methods: Stability-indicating RP-HPLC method was developed and validated for the estimation of LDP in bulk and tablet formulation. RP-HPLC was carried out on HiQ SiL C18 columns (250 mm × 4.6 mm, 5 μ particle size) using mobile phase acetonitrile:1 mM ammonium acetate buffer in the ratio of 90:10 v/v at a flow rate of 1 ml/min. The analytes were monitored using MD 2010 PDA detector at 333 nm.
 Results: The retention time was found to be 3.843 min. The proposed method was found to be having linearity in the concentration range of 5–30 μg/ml. The number of theoretical plates obtained was 4236.50 which indicate the efficient performance of the column. The limit of detection was 0.305 μg/ml and limit of quantification was 0.923 μg/ml, which indicate the sensitivity of the method; the high percentage recovery indicates that the proposed method is highly accurate. The developed method has been validated according to the ICH guidelines and found to be simple, specific, precise, and accurate.
 Conclusion: The proposed method is precise, accurate, and stability indicating. Therefore, the proposed method can be used for routine quality control and analysis of LDP during stability studies in bulk samples and tablet dosage forms.

SIMULTANEOUS DETERMINATION OF DIPHENHYDRAMINE, EPHEDRINE, NOSCAPINE, AND GLYCEROL GLYCOLATE USING STABILITY-INDICATING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY: APPLICATION TO NOSCOF TABLETS

Objective: Noscof tablet is a fixed dosage combination formulation having diphenhydramine (DH), ephedrine (ED), noscapine (NP), and glycerol glycolate (GG). A sensitive, selective, accurate, precise, and stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method with photodiode array detection has been developed and validated for simultaneous analysis of DH, ED, NP, and GG in bulk drug and Noscof tablets.
 Methods: Reversed-phase chromatographic separation and analysis of DH, ED, NP, and GG were done on an Altima C18 column with 0.01 M KH2PO4 buffer (pH 3.5) and acetonitrile (50:50%, v/v) as mobile phase at 0.8 ml/min flow rate in isocratic mode. Detection was performed at 260 nm. The method was validated in harmony with International Conference on Harmonization (ICH) guidelines. The tablet sample solution was subjected to diverse stress conditions using ICH strategy such as hydrolytic degradation (neutral - with distilled water, alkaline - with 2 N NaOH, and acidic - with 2 N HCl), oxidation (with 10% H2O2), photodegradation (exposing to UV light), and dry heat degradation (exposing to 105°C).
 Results: Using the above stated chromatographic conditions, sharp peaks were obtained for ED, NP, DH, and GG with retention time of 3.272 min, 4.098 min, 5.467 min, and 6.783 min, respectively. Good regression coefficient values were obtained in the range of 2–12 μg/ml for ED, 3.75–22.5 μg/ml for NP, 3.125–18.75 μg/ml for DH, and 25–150 μg/ml for GG. The quantification limits were 0.181 μg/ml, 0.187 μg/ml, 0.246 μg/ml, and 1.114 μg/ml for ED, NP, DH, and GG, respectively. The values of validation parameters are within the acceptance limits given by ICH. The ED, NP, DH, and GG showed more percent of degradation in acid condition and less percent of degradation in the neutral condition. The peaks of degradants did not interfere with the peaks of analytes. ED, NP, DH, and GG were assessed with a good percentage of the assay (near to 100%) and low percent relative standard deviation (<2%) in Noscof tablets using the proposed method.
 Conclusion: The stability indicating RP-HPLC method developed was suitable for quantifying ED, NP, DH, and GG simultaneously in bulk as well as in tablet formulation.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF NEW STABILITY-INDICATING REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND EMPAGLIFLOZIN IN TABLET DOSAGE FORM

Objective: The objective of this study was to develop and validate a stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of the metformin and empagliflozin in tablet dosage forms.Methods: The chromatographic conditions were optimized and it was run through Std. BDS (250 mm × 4.6 mm, 5 m) column with mobile phase consisting of 0.1% orthophosphoric acid buffer: acetonitrile in the ratio of 50:50. The flow rate was 1 ml/min and optimized wavelength was 210 nm. Temperature was maintained at 30°C.Results: The retention times of metformin and empagliflozin were found to be 2.588 min and 3.679 min and percentage relative standard deviation (RSD) of the metformin and empagliflozin was found to be 0.59 and 1.2, respectively. Percentage recovery was in the range of 100.01–100.65% for metformin and empagliflozin, respectively.Conclusion: A sensitive, rapid, and specific method has been developed for the simultaneous estimation of metformin and empagliflozin using RP-HPLC in tablet dosage form.