Stability-indicating Liquid Chromatography Research Articles

A stability-indicating liquid chromatography - diode array detection method for quantification of febuxostat in pharmaceuticals: a recent application to chemometrics based method development and comparative greenness assessment

With an increased demand for anti-gout drugs, the need for a quality-controlled drug product also arises. In this context, a new liquid chromatography method was developed for quantifying febuxostat that integrates chemometrics principles and assesses the method's eco-friendly nature. Systematized risk assessment identified that organic proportion, flow rate, and pH are the critical method variables and were subsequently optimized using a Box-Behnken design. The validation studies were performed using the optimized chromatographic conditions of 80% organic solvent, 1.0 mL/min flow rate, and a pH value of 6.8 on a C-18 column. The analyte eluted at 2.2 min and was monitored at 262 nm using photodiode array detection. After applying the forced degradation conditions, febuxostat degraded from 1.32 to 11.98% of its initial content. Statistical evaluation found that accuracy, precision, detection and quantitation limits, solution stability, and system suitability were acceptable. The lack of interfering degradation peaks ensured the stability-indicating nature of the method and method specificity. The application of three greenness metrics tools, such as the Green Analytical Procedure Index (GAPI), Analytical Greenness (AGREE) metrics, and analytical eco-scale (AES), revealed excellent method greenness. The current chromatographic method is apt for the routine quality control of febuxostat present in pharmaceutical formulations.

Stability-Indicating Liquid Chromatography Method for Rifaximin and LC-MS Characterization of its Potential Degradants

Rifaximin (RFX) is a structural analog of rifampin that has been shown to be a gastrointestinal-selective antibiotic with broad-spectrum antibacterial properties, a pronounced safety profile, and limited drug interactions. Crohn’s disease, diarrhea, hepatic encephalopathy, irritable bowel syndrome, and traveler’s diarrhea caused by non-invasive diarrheagenic Escherichia coli, can all be treated with rifaximin. FDA has designated RFX as an orphan drug for the treatment of hepatic encephalopathy. There is a substantial need for analytical quality monitoring of Rifaximin to ensure the safety and efficacy of treatment. To generate degradants, forced degradation study of the drug was carried out in acid, alkali, oxidative, thermal, and photolytic stress conditions. The optimum separation of the drug, and its degradants was achieved on an Inertsil ODS-3V C18 column (250 X 4.6 mm X 5 μm) under gradient elution conditions using, 10 mM potassium dihydrogen orthophosphate (pH 5.0 ± 0.05) as mobile phase A, and acetonitrile as mobile phase B, at a flow rate of 1.0 mL/min. The run time was 41 minutes and the detection wavelength was 258 nm. The method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use Guidelines. The drug was stable in neutral hydrolysis, photo and dry heat degradation conditions. The percentage degradation observed during acid, alkali hydrolysis and oxidative stress conditions were 70.46, 15.11 and 24.18, respectively. The degradants were investigated by LC-MS which indicated the presence of four (m/z 784.2, 744.5, 784.3, 753.8), three (m/z 744.5, 784.3, 753.8) and five (m/z 772.4, 838.4, 744.5, 753.8, and 801.9) degradants in acid, alkali and oxidative stress conditions, respectively. The structures of degradants were elucidated using Liquid chromatography–mass spectrometry (LC-MS) and Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses.

Development and Stability of a New Formulation of Pentobarbital Suppositories for Paediatric Procedural Sedation.

Pentobarbital is a drug of choice to limit motion in children during paediatric procedural sedations (PPSs). However, despite the rectal route being preferred for infants and children, no pentobarbital suppositories are marketed, and therefore they must be prepared by compounding pharmacies. In this study, two suppository formulations of 30, 40, 50, and 60 mg of pentobarbital sodium were developed using hard-fat Witepsol® W25 either alone (formulation F1) or with oleic acid (formulation F2). The two formulations were subjected to the following tests described in the European Pharmacopoeia: uniformity of dosage units, softening time, resistance to rupture, and disintegration time. The stability of both formulations was also investigated for 41 weeks of storage at 5 ± 3 °C using a stability-indicating liquid chromatography method to quantify pentobarbital sodium and research breakdown product (BP). Although both formulae were compliant to uniformity of dosage, the results were in favour of a faster disintegration of F2 compared to F1 (-63%). On the other hand, F1 was found to be stable after 41 weeks of storage unlike F2 for which several new peaks were detected during the chromatographic analysis, suggesting a shorter stability of only 28 weeks. Both formulae still need to be clinically investigated to confirm their safety and efficiency for PPS.

QbD-based stability-indicating liquid chromatography (RP-HPLC) method for the determination of flurbiprofen in cataplasm.

A nonsteroidal drug called flurbiprofen (FBN) has analgesic, anti-inflammatory and antipyretic activity. Currently the determination of FBN in cataplasm does not have any pharmacopeial method. However, the drug substance, tablet and ophthalmic solution formulations do have pharmacopeial methods. The development and validation of an accurate, precise and stability-indicating analytical method for the determination of FBN in cataplasm formulations is reported. The gradient method was employed for the quantification of FBN in the presence of internal standards such as biphenyl. A nonpolar separation phase (C18 , 250 × 4.6 mm, 5 μm Inertsil column; GL Sciences) was used. The optimal flow rate, column oven temperature, injection volume and detector wavelengths were 1.0 ml/min, 40°C, 20 μl and 245 nm, respectively. Mobile phase A was a mixture of water and glacial acetic acid (30:1 v/v) pH adjusted to 2.20 with glacial acetic acid or 1m NaOH; mobile phase B was methanol (100%). The gradient elution program was [time (min)/% B]: 5/60, 20/70, 25/70, 30/60 and 40/60. The obtained RSDs for the precision and intermediate precision were 0.7 and 0.5%. The percentage recovery ranged from 99.2 to 100.4%. The linear regression coefficient >0.9996 indicates that all peak responses were linear with the concentration. The sample and standard solutions were stable for up to 24 h on the benchtop and in the refrigerator. The critical peaks were well separated from the generated peaks owing to forced degradation, including diluent and placebo peaks. The method validation data and quality by design-based robustness study results indicate that the developed method is robust and fit for routine use in the quality control laboratory. The proposed method is specific, accurate and precise, and the quality by design utilized the first method for the determination of FBN in cataplasm formulations. Transdermal patches and gels have low extraction capacity and this method is applicable for quantification.

An Innovative Stability-Indicating Liquid Chromatography with Tandem Mass Spectrometry Method Development and Validation for the Determination of Sotorasib in Human Plasma

An Innovative Stability-Indicating Liquid Chromatography with Tandem Mass Spectrometry Method Development and Validation for the Determination of Sotorasib in Human Plasma

Empagliflozin: Validation of Stability-Indicating LC Method and in silico Toxicity Studies.

A new stability-indicating liquid chromatography method was developed for the quantification of empagliflozin and two synthetic impurities. The chromatographic conditions included Spherisorb® RP-18 column (150 × 4.6mm, 5μm) with a PDA detector, using acetonitrile and formic acid (pH4.0) as mobile phase in gradient elution and flow-rate of 1.2mL·min-1. The gradient increasing from 51 to 100% acetonitrile until 11.00min, followed by decreasing the solvent from 100% to the initial ratio from 11.01 to 15.00min. The method was validated according to International Council of Harmonization guidelines. The LOD and LOQ values for impurities A and B were 35 and 15ng·mL-1, respectively, (for LOD) and 115 and 35ng.mL-1, respectively (for LOQ). The method was linear in the range of 80-140, 115-1150 and 35-350ng·mL-1 for EMPA, impurities A and B, respectively, and the correlation coefficient were > 0.999 in all situations, indicating the method good linearity. The developed method showed a good recovery for empagliflozin and added impurities. The method has proven to be precise, demonstrated values less than 2.0% to empagliflozin and 5.0% to synthetic impurities, robust and selective with no interference from other products in the determination of analytes. The in silico toxicity prediction suggested that the impurities do not present any toxicity risk for the parameters evaluated.

Stability-indicating liquid chromatography method development for assay and impurity profiling of amitriptyline hydrochloride in tablet dosage form and forced degradation study.

Amitriptyline hydrochloride is an antidepressant drug with sedative effects used to treat the symptoms of anxiety, agitation with depression and schizophrenia with depression. A reversed-phase high-performance liquid chromatography method was developed to separate and quantitatively determine the assay and four organic impurities of amitriptyline in tablet dosage form and bulk drugs using a C18 column in an isocratic elution mode with mobile phase consisting of a mixture of pH7.5 phosphate buffer and methanol. The pH conditions used in the chromatographic separation are discussed. The stability-indicating characteristics of the proposed method were proved using stress testing [5m HCl at 80°C/1h, 5m NaOH at 80°C/1h, H2 O (v/w) at 80°C/1h, 6% H2 O2 (v/v) at 25°C/1h, dry heat at 105°C/24 h and UV-vis light/4days] and validated for specificity, detection limit, quantitation limit, linearity, precision, accuracy and robustness. For amitriptyline and its four known organic impurities, the quantitation limits, linearity and recoveries were in the ranges 0.25-3.0μg/ml (r2  > 0.999) and 87.9-107.6%, respectively. The mass (m/z) spectral data of amitriptyline hydrochloride and its impurity are discussed. The proposed LC method is also suitable for impurity profiling and assay determination of amitriptyline in bulk drugs and pharmaceutical formulations.

Physicochemical Stability of a Novel Tacrolimus Ophthalmic Formulation for the Treatment of Ophthalmic Inflammatory Diseases.

Tacrolimus is an immunosuppressant used to treat a large variety of inflammatory or immunity-mediated ophthalmic diseases. However, there are currently no commercial industrial forms available that can provide relief to patients. Various ophthalmic formulations have been reported in the literature, but their stability has only been tested over short periods. The objective of this study was to evaluate the physicochemical stability of a preservative-free tacrolimus formulation (0.2 and 1 mg/mL) at three storage temperatures (5 °C, 25 °C and 35 °C) for up to nine months in a multidose eyedropper. Analyses performed were the following: visual inspection and chromaticity, turbidity, viscosity, size of micelles, osmolality and pH measurements, tacrolimus quantification by a stability-indicating liquid chromatography method, breakdown product research, and sterility assay. In an in-use study, tacrolimus quantification was also performed on the drops emitted from the eyedroppers. All tested parameters remained stable during the nine month period when the eyedrops were stored at 5 °C. However, during storage at 25 °C and 35 °C, several signs of chemical instability were detected. Furthermore, a leachable compound originating from a silicone part of the eyedropper was detected during the in-use assay. Overall, the 0.2 mg/mL and 1 mg/mL tacrolimus ophthalmic solutions were physicochemically stable for up to nine months when stored at 5 °C.

Development and Validation of a Stability-Indicating Reversed-Phase Ultra High Pressure Liquid Chromatography Method for Assay of Delmopinol Hydrochloride and Estimation of Its Related Substances in Commercial Bulk Batches.

Delmopinol hydrochloride is widely used as an active ingredient (AI) in many human oral hygiene products. To the best of our knowledge (via literature search), there is no stability-indicating liquid chromatography method available in the public domain for assay and estimation of related substances of Delmopinol hydrochloride. A fast stability-indicating Reversed-Phase Ultra High-Performance Liquid Chromatography (RP-UHPLC) method has been developed and validated for identification, assay and estimation of related substances in commercial bulk batches of this AI. The major peak of the AI and its related compounds are adequately separated in 6min by a gradient elution on a hybrid silica-based C18 column (Waters Acquity UPLC® BEH C18, 50mm × 2.1mm I.D., 1.7μm particle size) maintained at 50°C. Mobile phase-A is composed of aqueous 10mM NH4OH and mobile phase-B is ACN. The AI and its related compounds are detected with UV detection at 220nm and quantitated by an external Delmopinol hydrochloride reference standard. The quantitation limit of the method is 0.1% of the target analytical concentration. This UHPLC method is fast and green and has been demonstrated to be specific, accurate, linear, precise, sensitive and robust as per ICH guidelines.

VALIDATED AND QUANTIFIED STABILITY INDICATING STRESS DEGRADATION STUDY OF ORAL ANTI-DIABETIC AGENT CANAGLIFLOZIN BY RP-HPLC METHOD

Objective: The present investigation is aimed to develop and validate, a simple, consistent and sensitive stability-indicating reverse phase-high performance liquid chromatography (RP-HPLC) method for the determination of oral anti-diabetic drug Canagliflozin in bulk and pharmaceutical dosage form as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH-Q2 (R1)). Methods: The chromatographic separation was achieved by using Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) ZORBAX C18 (250 mm x 4.6 mm, 5μm particle size) with a mobile phase consisting of Acetonitrile: Water in a ratio of 53:47% v/v at a flow rate of 1 ml/min with an injection volume of 20 μl. Results: The Retention time of the drug Canagliflozin was found to be 2.36±0.05 min and detected at 214 nm UV wavelength. The linear regression equation was found to be y = 60702x–2156.2 with a correlation coefficient 0.9999. Stress degradation studies were performed by exposing the Canagliflozin into acidic, alkaline, oxidative, thermal and photolytic stress conditions with active samples withdrawn at different time intervals as per ICH guidelines. Conclusion: The proposed Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was found to be robust, precise and specific for estimation of Canagliflozin in pharmaceutical dosage forms.

A new stability-indicating RP-HPLC method for the determination of dicyclomine hydrochloride and dimethicone combination in tablet dosage forms

BackgroundWe describe a “stability-indicating liquid chromatography” technique for the estimation of dimethicone (DEC) and dicyclomine hydrochloride (DEH) in the established tablet formulations. Individual quantification of DEH and DEC was reported. But simultaneous quantification of DEH and DEC was lacking. DEH and DEC were analysed on an “XTerra C18 column (250 mm × 4.6 mm, 5 μm)” with the mobile phase solvent run isocratically with 0.1M K2HPO4-acetonitrile (55:45, v/v) on a flow speed of 1.0 mL/min.ResultsThe chromatographic run period for the DEC and DEH assay was 6.0 min with retention times of 2.134 and 2.865 min, respectively. The method was validated for accuracy (99.453 to 100.417% and 99.703 to 100.303% recovery values for DEH and DEC, respectively), precision (RSV value 0.135% for DEC and 0.171% for DEH), linearity (5–15 μg/mL for DEH and 20–60 μg/mL for DEC), selectivity (no hinderance from excipients) and specificity (no hinderance from degradants) recovery.ConclusionThe developed stability-indicating liquid chromatography process was well applied to established tablet formulations.

Luliconazole: Stability-indicating LC method, structural elucidation of major degradation product by HRMS and in silico studies

Aim: A new stability-indicating liquid chromatography method was developed and validated for the quantitative determination of luliconazole. Materials and methods: Preliminary forced degradation study demonstrated an additional peak of the degradation product at the same retention time to the drug, due to this, the method was developed optimizing the chromatographic conditions to provide sufficient peak resolution (R ≥ 2). The experimental design was evaluated to assess the robustness and the best chromatographic conditions to be used for the validation. Methodology: Luliconazole solutions were exposed to various stress conditions to evaluate the method indication stability, in which the degradation product (DP-1) formed was isolated, identified, and evaluated in silico to predict degradation pathway and toxicity. The procedure was validated by robustness, selectivity, linearity, precision, and accuracy. Liquid chromatography was performed in a Phenomenex® RP-18 column with a mixture of acetonitrile and 0.3% (v/v) triethylamine solution as a mobile phase in isocratic elution. Results and conclusions: The method demonstrated robustness, good recovery, precision, linear response over a range from 5.0 to 40.0 μg.mL-1, and to be stability indicating. The alkaline stress condition resulted in the formation of DP-1. hrms studies identified this product as an hydroxyacetamide derivative, and in silico studies did not show toxic potential.

Analytical Techniques for the Estimation of Perindopril Erbumine: A Review

This paper includes different analytical methods available for the estimation of the Perindopril Erbumine. Perindopril Erbumine is an Angiotensin-Converting Enzyme (ACE) inhibitor used to treat hypertension and heart failure. The drug is officially listed in the monograph of USP, BP & EP. This drug is available in various marketed formulations. There are so many methods reported for the estimation of Perindopril Erbumine and its related substances. Various methods including UVspectroscopic method, stability-indicating liquid chromatography method, Thermo-Radio, and Photostability study, Stability indicating UHPLC-DAD method, LC-MS/MS electrospray ionization validated method, and LC-MS/MS bioanalytical method are discussed in this paper.

Do Ophthalmic Solutions of Amphotericin B Solubilised in 2-Hydroxypropyl-γ-Cyclodextrins Possess an Extended Physicochemical Stability?

Fungal keratitis is a sight-threatening disease for which amphotericin B eye drops is one of the front-line treatments. Unfortunately, there are currently no commercial forms available, and there is little data concerning the long-term stability of compounded formulations based on intravenous dosages forms. New formulations of amphotericin B ophthalmic solutions solubilised with γ-cyclodextrins have shown promising in-vitro results, but stability data is also lacking. The objective of this study was therefore to investigate the stability of a formulation of ready-to-use amphotericin B solubilised in 2-hydroxypropyl-γ-cyclodextrins (AB-HP-γ-CD), for 350 days. An amphotericin B deoxycholate (ABDC) formulation was used as a comparator. Analyses used were the following: visual inspection, turbidity, osmolality and pH measurements, amphotericin B quantification by a stability-indicating liquid chromatography method, breakdown product research, and sterility assay. AB-HP-γ-CD formulation showed signs of chemical instability (loss of amphotericin B) after 28 and 56 days at 25 °C and 5 °C. Adding an antioxidant (ascorbic acid) to the formulation did not improve stability. ABDC formulation showed signs of physical instability (increased turbidy and amphotericin B precipitation) after 28 days and 168 days at 25 °C and 5 °C. As such, AB-HP-γ-CD formulation does not provide long-term stability for ophthalmic amphotericin B solutions.

Stability of Ophthalmic Atropine Solutions for Child Myopia Control.

Myopia is an ophthalmic condition affecting more than 1/5th of the world population, especially children. Low-dose atropine eyedrops have been shown to limit myopia evolution during treatment. However, there are currently no commercial industrial forms available and there is little data published concerning the stability of medications prepared by compounding pharmacies. The objective of this study was to evaluate the stability of two 0.1 mg/mL atropine formulations (with and without antimicrobiobial preservatives) for 6 months in two different low-density polyethylene (LDPE) multidose eyedroppers. Analyses used were the following: visual inspection, turbidity, chromaticity measurements, osmolality and pH measurements, atropine quantification by a stability-indicating liquid chromatography method, breakdown product research, and sterility assay. In an in-use study, atropine quantification was also performed on the drops emitted from the multidose eyedroppers. All tested parameters remained stable during the 6 months period, with atropine concentrations above 94.7% of initial concentration. A breakdown product (tropic acid) did increase slowly over time but remained well below usually admitted concentrations. Atropine concentrations remained stable during the in-use study. Both formulations of 0.1 mg/mL of atropine (with and without antimicrobial preservative) were proved to be physicochemically stable for 6 months at 25 °C when stored in LDPE bottles, with an identical microbial shelf-life.

Stability of tedizolid phosphate-sodium rifampicin and tedizolid phosphate-meropenem admixtures in intravenous infusion bags stored at different temperatures.

This is a report on the chemical stability and physical compatibility of intravenous tedizolid phosphate 0.8 mg/mL-sodium rifampicin 2.4 mg/mL and tedizolid phosphate 0.8 mg/mL-meropenem 4 mg/mL combinations in polypropylene 0.9% sodium chloride infusion bags stored at different storage conditions. Triplicate solutions of both admixtures were prepared in 0.9% sodium chloride polypropylene infusion bags and stored under light protection at room temperature (25±2 °C), refrigeration (2-8 °C) or freezing (-15 - -25 °C) conditions. The study was performed using a validated and stability-indicating liquid chromatography (LC) method. For both admixtures and for all storage conditions, at least 90% of the initial drug concentration of tedizolid phosphate remained unchanged throughout the entire study period. Stability of sodium rifampicin at 25±2 °C was determined to be seven hours and six days when it was stored at 2-8 °C. Under the same storage conditions, meropenem was stable for 12 h or 6 days, respectively. Under freezing conditions, sodium rifampicin was stable throughout all 28 days, while stability of meropenem was only 8 days. Solutions of 0.8 mg/mL tedizolid phosphate admixtured with 2.4 mg/mL rifampicin or 4 mg/mL meropenem, in polypropylene 0.9% sodium chloride infusion bags, are stable for at least 7 or 12 hours, respectively, when stored at 25±2 °C. When stored at 2-8 °C, stability was increased to 6 days for both admixtures.

Forced degradation studies and stability-indicating liquid chromatography method for determination of tirofiban hydrochloride and synthetic impurities

This study aimed to develop and validate a stability-indicating liquid chromatography method for the determination of tirofiban hydrochloride and two synthetic impurities (impurity A and impurity C). The method utilizes a RP-18 column (250 mm × 4.6 mm; 5 μm) with the PDA detector for quantitation. A mixture of triethylamine 0.1% (acidified to pH 5.5 with phosphoric acid) and acetonitrile was used as the mobile phase at a flow rate of 1 mL min−1 with gradient elution. The method presented satisfactory linearity, precision, accuracy and robustness, as well as low limits of detection and quantification, which demonstrate sensitivity in the determination of tirofiban and impurities A and C. It was selective for the determination of the drug and impurities analysed, without interference of the degradation products generated under forced conditions, demonstrating the stability-indicating capacity of the proposed method. Tirofiban showed to be practically stable to oxidative (30% H2O2 for 24 h) and thermal (75 ºC for 24 h) conditions, but presented degradation to UVA light and acid hydrolysis, obeying the first order kinetics for both. In this way, it can be used as a stability-indicating method in the quality control of the raw material of tirofiban hydrochloride, as well as of the finished product. The obtained results demonstrate the importance of deepening the studies in this area, in order to guarantee the quality of commercialized pharmaceutical products.

Compatibility of treprostinil sodium and dopamine hydrochloride during simulated Y-site administration.

To evaluate the physical and chemical compatibilities of treprostinil sodium and dopamine hydrochloride. Treprostinil sodium (4,000, 76,000, and 500,000 ng/mL) were mixed with dopamine hydrochloride (0.6, 3.2, 6, and 40 mg/mL). Samples were obtained at hours 0, 1, 2, and 4 for physical compatibility and chemical stability testing. Physical compatibility was assessed by visual examination and measurements of turbidity and pH. Drug concentrations were assessed using stability-indicating liquid chromatography mass spectrophotometry (LCMS) for treprostinil sodium and stability-indicating high-performance liquid chromatography (HPLC) for dopamine hydrochloride. Treprostinil sodium 4,000 and 76,000 ng/mL, when mixed with dopamine hydrochloride 0.6, 3.2, 6, and 40 mg/mL, were stable for 4 hours. Treprostinil sodium 500,000 ng/mL was stable when mixed with dopamine hydrochloride 0.6 mg/mL for 4 hours, but when mixed with dopamine hydrochloride 3.2, 6, and 40 mg/mL, significant precipitation was seen. Treprostinil sodium 4,000 and 76,000 ng/mL were stable for 4 hours during simulated Y-site coadministration with dopamine hydrochloride 0.6, 3.2, 6, and 40 mg/mL. Treprostinil sodium 500,000 ng/mL is stable when mixed with dopamine hydrochloride 0.6 mg/mL.

INTERNATIONAL CONFERENCE ON HARMONIZATION RECOMMENDED FORCED DEGRADATION STUDIES AND DEVELOPMENT OF A NEW VALIDATED ISOCRATIC REVERSE-PHASE ULTRA HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF TELMISARTAN AND AMLODIPINE I

Objective: The objective of this research work is to develop a simple, rapid, cost-effective, accurate, precise, and robust stability-indicating reversephase ultra-high performance liquid chromatography method for the quantitative estimation of telmisartan (TEL) and amlodipine (AMD) in combined tablet dosage form.
 Methods: The method uses Poroshell 120EC-C18 column (4.6 mm × 50 mm, 2.7 μm) with an optimized mobile phase containing acetonitrile:50 mM ammonium acetate buffer in the ratio (45:55 v/v), pH adjusted to 4.5 with acetic acid. The flow rate was 0.5 ml/min, column temperature at 25°C and detection was monitored by a PDA detector at a wavelength of 245 nm. International Conference on Harmonization (ICH) recommended stress degradation studies were performed on TEL, AMD standard drugs, and tablet formulations; further stressed samples were analyzed by the proposed method.
 Results: Major degradation of TEL and AMD was observed under acidic, alkali, hydrolytic and oxidation conditions. The described method was validated as per the ICH guideline and validation parameters such as system suitability, linearity, accuracy, precision, specificity, and robustness results were within acceptable limits.
 Conclusion: The method was found to be suitable and accurate for quantitative estimation and stability study of title drugs in pharmaceutical preparations.

QUALITY BY DESIGN APPROACH TO STABILITY-INDICATING REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT, OPTIMIZATION, AND VALIDATION FOR THE ESTIMATION OF SIMEPREVIR IN BULK DRUG

Objective: A simple, robust, precise, and selective stability-indicating liquid chromatography (LC) method (reverse-phase high-performance LC) was developed for the estimation of simeprevir through quality by design paradigm.
 Methods: The chromatographic separation was performed on Water’s 2695(Alliance) equipped with a photodiode array detector at 300 nm. The method was developed on Discovery C18 column (250×4.6, 5 mm) using orthophosphoric acid and acetonitrile (55:45 % v/v) with the flow rate of 1 ml/min at 30°C. The degradation studies of simeprevir were carried out under the stress conditions of hydrolysis (acid, base, and neutral), oxidation, photolytic, and thermal as per the International Conference on Harmonization (ICH) guidelines. The peroxide hydrolysis shows more critical impurities which were well resolved from pure drug with the application of design of experiment and optimized the method.
 Results: Independent variables (critical analytical attributes) selected for the method optimization were mobile phase ratio, flow rate, and temperature of the column based on the risk assessment. The retention time and resolution were selected as the method response. In response surface method, the central composite design and 23 factorial designs were employed for the optimization of the method. The polynomial equation was derived for the estimation of method response.
 Conclusion: The method was optimized for better resolution among the drug, and impurity peaks were then validated as per the ICH parameters.