Mobile phase-B Research Articles

DEVELOPMENT OF NOVEL SINGLE HPLC METHOD FOR SIMULTANEOUS SEPARATION OF MULTIPLE IMPURITIES IN DEXAMETHASONE DRUG PRODUCT

Objective: A simple, reliable, and rapid HPLC method has been established for the detection of Dexamethasone (DEX) and its related impurities. The proposed method has been validated for specificity, linearity, system suitability, accuracy, precision, robustness, LOD, and LOQ as per International Council for Harmonisation (ICH) guidelines. All parameters were found to be within the accepted limits, affirming the method's reliability. Methods: Analysis was conducted using HPLC on X-Bridge C18 column (250 mm×4.6 mm id, 3.5 µm) with a mobile phase-A comprising buffer and acetonitrile (90:10, v/v), mobile phase-B comprising buffer and acetonitrile (25:75, v/v) and a flow rate of 0.8 ml/min by following gradient elution. The detection was performed with a UV detector set at 240 nm. The method has been employed to investigate DEX and DEX-related impurities. These studies were conducted in tablet formulations of DEX. Results: The Retention Time (tR) of DEX was about 41.589 min, and all parameters met acceptable limit values. The response exhibited linearity over a concentration range of 0.162 to 3.052 µg/ml (R2= 0.9999). The percentage of DEX recovered from the pharmaceutical tablet dosage form ranged from 96.3 % to 100.4 %. Sensitivity levels for the developed method were indicated by LOD and LOQ values of 0.081–0.162 µg/ml. The proposed method was validated according to ICH guideline. Conclusion: Hence, a simple, reliable, accurate, and precise HPLC method was developed, proving suitable for the separation of DEX and DEX-related impurities in commercial formulations.

Cleaning Method Validation for Determination of Residues in Anti-Cancer Drug product for Injection by Using RP-HPLC Technique

Cleaning method validation is a part of good practices in pharmaceutical sector and need to seek the contamination present in production area. OBJECTIVE: An RP-HPLC method for the cleaning samples analysis for Anti-Cancer Drug product Melphalan Hydrochloride for Injection was develop and validated. The method was developed and validated for quantitation of Cleaning of residue left in the vessels of the production equipment’s. The analytical method used to determine the complete cleaning for Melphalan Hydrochloride for Injection by HPLC method. METHOD: The Cleaning validation was performed on Inertsil ODS,C18,(150mm×4.6mm,5μm) chromatographic column temperature at 40°C, and the detection wavelength was set at 260nm. Mobile Phase-A:10mL ammonium acetate, 88mL Purified water, 10mL acetic acid, 2mL Triethylamine, and Mobile Phase-B: 1mL Buffer, 10mL acetonitrile,190mL Purified water, and at a flow rate of 1.5 mL/min, and the injection volume was 10μL. RESULTS: The validation results of Melphalan Hydrochloride were linear over the concentration from 0.10 µg/mL to 0.75µg/mL (r = 1.00) and the recoveries from LOQ to 150% and precision ranged from 50% to 76% (RSD < 30%, n =9). CONCLUSION: This method has been validated successfully and all the results met the acceptance criteria. Hence method of Cleaning Validation is suitable for testing of residue sample of Melphalan Hydrochloride for Injection.

Stability-indicating RP-HPLC method development and validation for the quantification of amlodipine besylate and valsartan tablets in solid oral dosage form.

The present study discusses the development of simple, rapid, specific, precision, accuracy, stability indicating the HPLC method for the analysis of amlodipine besylate and valsartan tablet dosage form. The chromatographic separation was achieved using phosphate buffer with 1% triethyl amine (pH3.0) as mobile phase-A and mixed Methanol and buffer in the ratio of (65:35)(v/v) as mobile phase-B. The detection of components was made at 237 nm for amlodipine besylate and valsartan. Analytical techniques should enrich sensitivity and specificity for the estimation of pharmaceutical drug products. Evaluated stress studies under different types of ICH conditions. The optimized HPLC method was validated as per the current ICH guidelines. The validated HPLC method was obtained highly specific with linearity ranging between 25 and 200 μgmL-1 of amlodipine besylate and 40-320 μgmL-1 of valsartan and both components correlation coefficient was > 0.999. The method showed high accuracy more than 97%. In stress studies, amlodipine besylate and valsartan were found to be sensitive to acid stress conditions and oxidation stress conditions. The method was found to be suitable for the quality control of amlodipine besylate and valsartan in the tablet as well as in stability-indicating studies. The method was applied to the analysis of stability samples.

A quality by design assisted RP-HPLC method utilizing central composite design for the assay of eliglustat and its organic impurities in drug

This study aimed to develop a stability-indicating RP-HPLC assay method for eliglustat and its organic impurities using a central composite design approach. The chromatographic separation was achieved with a 10 mM phosphate buffer at pH 4.0 on a Thermo Accucore C18 column (150 x 4.6 mm, 2.6 μm) at a wavelength of 210 nm. Gradient elution was performed using a mobile phase-A consisting of buffer and acetonitrile in a 90:10 v/v ratio and a mobile phase-B composed of water and acetonitrile in a 35:65 v/v ratio, at a flow rate of 1.1 mL/min at 45°C for a total run time of 55 minutes. Based on preliminary experiments, central composite design was utilized to evaluate the effects of independent variables such as flow rate, acetonitrile ratio and column oven temperature on the response factors. Eliglustat and its impurities (EGS-5A, EGS-Diastereomers, and EGS-N-Oxide) had retention time of 3.5 minutes, 21.5 minutes, 23.2 minutes and 25.9 minutes, respectively. The limits of detection and quantitation for eliglustat and its impurities were established in relation to the test concentration, achieving a desirability of 1. The developed method was validated as per regulatory guidelines and successfully applied in bulk drugs and formulation.

New stability indicating RP-HPLC methods for the determination of related substances and assay of trametinib acetic acid: a mass balance approach.

New stability indicating related substances and assay methods for trametinib acetic acid by RP-HPLC have been developed and validated through forced degradation and mass balance studies for the first time. In related substances (RS) method, trametinib acetic acid was successfully separated from its six related substances namely, cyclopropanamide impurity, desiodo trametinib, desacetyl trametinib, trione acetamide intermediate, trione intermediate, and trione PTSA intermediate using YMC-Triart C18 (150 × 4.6mm, 3µm) column. Orthophosphoric acid (0.15%) in water was used as buffer. Gradient elution was programmed using mobile phase-A (buffer and acetonitrile mixture in 80:20 v/v ratio) and mobile phase-B (buffer and acetonitrile mixture in 20:80 v/v ratio). Acetonitrile and methanol mixture (1:1 v/v) was used as diluent. Flow rate, injection volume, column temperature, and wavelengths were kept as 0.8mL/min, 10µL, 55°C, and 240nm, respectively. Desacetyl trametinib and cyclopropanamide impurity were identified as potential degradation impurities in acid and base degradation conditions, respectively. Assay method specific to above six related substances was also developed. Assay of forced degradation samples was also determined, and the mass balance was established by adding total impurities formed in RS method to assay of trametinib acetic acid.

Separation and Validation of Comprehensive Impurities in Erythromycin Tablets by using Rp-HPLC method

In this study a simple, rapid, accurate and precise gradientRP-HPLC method has been developed and validated for the separation of impurities (IMP) in Erythromycin (ERT-A) tablets pharmaceutical dosage form. The chromatographic separation was carried out on WatersX-Terra RP 18 (250 mm x 4.6 mm I.D., 3.5 µm particle size) at 65°C was used for this separation. Mobile Phase-A consists of Buffersolution (35 g of di-potassium hydrogen phosphate in 1000mL of water, adjust the pH7.0with dilute o-phosphoric acid, filtered through 0.45 µm membrane filter) acetonitrile and water in ratio of 5:35:60 v/v/v respectively. Mobile Phase-B consists of a mixture of phosphate buffer pH 7.0, water and acetonitrile in ratio of 5:45:50 v/v/v respectively .The flow rate and injection volume was 1.0 mL/min and 100 μL respectively. The analysis was carried out under the gradient conditions as time (min)/A (v/v): B(v/v); T0/100:00, T45/100:00, T47/0:100, T63/0:100, T65/100:00, and T70/100:00. The wavelength was identified at 215 nm. The ERT-Adegrades under various circumstances. The degradation products were well resolved from the ERT peaks. This method was found to be linear. The cumulative %RSD values are ;identified and found that they are within the range. From the study concluded that the method is accurate, specific, selective, precise, robust, and useful during the process development and quality check in finished dosage form manufacturing.

Stability indicating RP-HPLC-UV method for quantitative estimation of isomer and other organic impurities of atopic dermatitis drug abrocitinib in drug substance and pharmaceutical dosage form using quality by design (QbD) approach

A modest, reliable, robust, stability-indicating RP-HPLC-UV method was developed and validated following ICH Q2(R2) for the separation and quantification of isomer and other organic impurities of Abrocitinib (Janus kinase inhibitor). Quality by design (QbD) approach was used to achieve the desired specifications. The chromatographic separation was achieved for impurities method of Abrocitinib within 35 min of chromatographic run time on Agilent ZORBAX Eclipse plus C18 150*4.6 mm, 3.5 µm column with gradient elution mode: time (min)/B (%): 0.01/0, 2.5/15, 8.0/20, 15.0/40, 20.0/50, 25.0/50, 25.1/0, 35.0/0 with mobile phase-A (MP-A): 10 mmol/L CH3COONH4 and acetonitrile with the ratio of 900:100 (v/v), mobile phase-B (MP-B): 10 mmol/L CH3COONH4 and acetonitrile with the ratio of 100: 900 (v/v). The detection wavelength, mobile phase flow rate, column temperature, and injection volume were used at 287 nm, 1.0 mL/min, 40 °C, and 20 µl respectively. Utilizing forced degradation and mass balance investigations, the new method’s stability-indicating properties were assessed. The developed method was validated as per ICH and found to be specific, precise, rugged, robust, accurate, linear, and with a limit of quantification values,0.0359 µg/mL for the impurity IMP-A, 0.0192 µg/mL for the impurity IMP-B, 0.0322 µg/mL for the impurity IMP-C and 0.0201 µg/mL for abrocitinib.

Development of HPLC method for isomeric impurities of key starting material of novel oral anticoagulant drug; Edoxaban Tosylate Monohydrate

Reverse-phase high-performance liquid chromatography method has been developed for the determination of EDO-S1 stereoisomeric impurities such as isomer 1, isomer 2, isomer 3, isomer 4, isomer 5, isomer 6 and isomer 7 with good resolution using the column, Bakerbond C18 (150 × 4.6 mm; 3 μm). The separation was achieved with mobile phase-A (10 mM dipotassium hydrogen phosphate pH-7.0 with 10% orthophosphoric acid solution in Milli-Q water) and mobile phase-B (n-Propanol: Acetonitrile ratio of 20:30 % V/V), which consisted of mobile phase mixture in the combination of moilephase-A: mobile phase-B (85:15). The total run time was 30 min at 0.8 mL/min flow rate, 20 µL injection volume and 30 ℃ column oven temperature. The column eluate was monitored at 210 nm to quantify the impurities The method showed adequate specificity, sensitivity, linearity, accuracy, precision, and robustness inline to ICH tripartite guidelines. The limit of detection and quantification limits were 0.1 and 0.3 μg mL−1, respectively, for all isomeric impurities and EDO-S1. The developed method was found to be linear over the concentration range of LOQ to 150% of specification range for isomeric impurities with a correlation coefficient >0.999. The method was precise (%RSD < 5.0), robust, and accurate (with 85%–115% recovery).

Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product. Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP). In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil. The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil.

Eco-Friendly Stability-Indicating HPLC Method for Related Compounds in Pemetrexed Ditromethamine (Antineoplastic Agent) for Injection.

An eco-friendly analytical technique was developed with the intention of preserving the environment by using green chemistry principles. Pemetrexed is a folate analogue indicated for the treatment of advanced lung cancer. Development of a green stability-indicating HPLC method for the quantification of pemetrexed ditromethamine (PDT) impurities in Active Pharmaceutical Ingredient (API) and parenteral dosage form. Chromatographic separation was achieved using a Zorbax SB C18 column (150 mm × 4.6 mm i.d., 3.5 µ particle size) with perchlorate buffer (pH 3.0 ± 0.1, 50 mM) as mobile phase A and acetonitrile-perchlorate (90 + 10, v/v) buffer as mobile phase B at a flow rate of 0.8 mL/min with a column temperature of 40°C ± 0.5°C. All analytes were well resolved by gradient elution with a total run time of 75 min. The UV detection wavelength was 230 nm. The RP-HPLC method is capable of resolving all the degradation and process impurities for PDT API and parenteral dosage form. The related compounds method was validated in accordance with International conference on harmonization (ICH) Q2(R1) and United states of Pharmacopoeia (USP) <1225> guidelines, and found to be accurate, specific, precise, linear, robust and stability-indicating. The precision and intermediate results were <5% CV for all the impurities. The accuracy for all the impurities was found to be between 90 and 110%. The linearity of regression co-efficient values for all the impurities were found to be more than 0.999. The proposed related compounds method is found suitable for the determination of process and degradation impurities of commercial formulations, stability samples in QC analysis for PDT API, and drug product. The developed liquid chromatographic method greenness and eco-friendliness were assessed using the green analytical procedure index (GAPI) and the analytical greenness (AGREE) tool, and found to be green. A PDT detoxification procedure was also developed to reduce environmental pollution.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION OF THE DISSOLUTION OF BROMELAIN-A COMPLEX MIXTURE OF PROTEOLYTIC ENZYMES, IN DELAYED-RELEASE TABLETS

Objective: To develop a simple, accurate, precise and linear Reverse Phase High-Performance Liquid Chromatographic (RP-HPLC) method and verify for the quantitative estimation (Dissolution) of Bromelain in delayed-release tablets. Methods: The optimized RP-HPLC method for both acid and buffer stage dissolutions of delayed-release tablets uses Zorbax 300 SB-C8 column (150 mm X 4.6 mm; 3.5μ), a mobile phase-A of 0.1% trifluoroacetic acid in water and a mobile phase-B of 0.1% Trifluoroacetic acid in Acetonitrile in the gradient proportion, flow rate of 1.0 ml/min, injection volume of 25 µl, detection wavelength of 280 nm using a UV/PDA detector, column temperature of 40 °C, sample tray/compartment temperature of 5 °C and a run time of 20 min. Results: The developed method gave Bromelain eluting at about 6 min. Bromelain exhibited linearity in the range 53.4-800.6 μg/ml (r2=0.99992). The precision is exemplified by relative standard deviation of 1.3 and 2.3% for acid and buffer stages, respectively. Percentage recovery of the drug was found to be between 90.0 and 110.0 during accuracy studies. Conclusion: A simple, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (Dissolution) of Bromelain in tablets and hence this method can be explored for the analysis of Bromelain in tablets in various pharmaceutical industries.

Determination of Clorsulon and its related substances in commercial bulk drug substance batches by a rapid ion-pair UPLC method

Clorsulon is a potent anthelmintic agent and is widely used for the treatment and control of parasites including adult liver flukes in cattle and sheep. A rapid ion-paired reversed phase ultraperformance liquid chromatography (IP-UPLC) method has been developed and validated for determination of Clorsulon and its related substances in bulk drug substance batches of Clorsulon with a short octadecyl column. Analytes were eluted by a gradient elution on a Acquity UPLC® BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Column temperature was maintained at 55 °C and all analytes were monitored by UV detection at 268 nm. Mobile phase-A constitutes 3 mM tetrabutylammonium hydroxide in H2O and mobile phase-B constitutes acetonitrile/methanol (50/50, v/v), respectively. All analytes of interest were adequately separated within 5 min. The analytes were quantitated against an external reference standard of Clorsulon. The chemical structures of degradation products of Clorsulon were proposed based on two-dimensional UPLC-MS data. The IP-UPLC method has been successfully validated and demonstrated to be accurate, sensitive, robust, and specific.

Simultaneous determination of praziquantel, fipronil, eprinomectin, (S)-methoprene, their key related substances and butylated hydroxytoluene (BHT) in a topical veterinary drug product by a single stability indicating high performance liquid chromatography method

A simple, robust and QC (quality control) friendly high performance liquid chromatography (HPLC) method was developed and validated for simultaneous determination of four active pharmaceutical ingredient [namely fipronil, (S)-methoprene, eprinomectin, and praziquantel] and their key degradation products in a broad-spectrum topical finished product. Typical sample of the finished product contains a total of 30-plus peaks of interest. Analytes were separated on a HALO C18 column (150 mm × 4.6 mm i.d., 2.7 µm particle size) with a gradient elution at 50 °C column temperature and 0.6 mL/min flow rate. Detection wavelength of 245 nm is used for praziquantel, eprinomectin and their degradation products, 265 nm for (S)-methoprene and its degradation products and 280 nm for fipronil and its degradation products and for the antioxidant, BHT. Mobile phase of the method is composed of 10 mM potassium phosphate buffer and 1,4- Dioxane (98/2, v/v, pH 5.0) as mobile phase-A, and EtOH/MeOH/MeCN/IPA (86/4/6/4, v/v/v/v) as mobile phase-B. All analytes of interest were adequately separated by this single HPLC method. The stability-indicating capability of the method has been demonstrated by successfully separating the degradation products in the stressed degraded samples of the finished product. Limit of quantitation (LOQ) and limit of detection (LOD) of the method is 0.3% and 0.1% of target analytical concentration for each individual API in the finished product. This method has been demonstrated to be sensitive, robust, specific, accurate and stability-indicating for analysis of the topical drug product containing praziquantel, fipronil, eprinomectin and (S)-methoprene.

Development of HPLC Method for Ixabepilone (Oncology Drug) in Bulk and Dosage Form: Quantification of Impurities and Forced Degradation Studies.

The objective of study is to develop a new stability-indicating HPLC method for quantifying ixabepilone degradation products and known process impurities (EPO-2 and Epothilone B) in bulk and injectable dose forms. A gradient stability-indicating RP-HPLC approach was developed to determine the known impurities of ixabepilone in ixabepilone API and ixabepilone for injection. Ixabepilone was subjected to base, acid, oxidation, photolytic and thermal degradations. The gradient approach was used to optimize the mobile phase-A [pH 4.8 acetate buffer (10 mM) and acetonitrile 90:10 v/v] and mobile phase-B [pH 4.8 acetate buffer (10 mM) and acetonitrile 20:80 v/v] of a USP L1 column. A wavelength of 250 nm was chosen based on known impurities and degradation products response, with a 1.0 mL/min flow rate. In compliance with ICH criteria Q2(R1), the developed technique was validated. The stability-indicating-related impurities technique was proven to be appropriate for estimating degrading impurities and known impurities in ixabepilone API and ixabepilone injection.

Liquid chromatography separation and identification of new oxidative degradation impurity in edoxaban tosylate hydrate drug substance by using liquid chromatography with tandem mass spectrometry.

Edoxaban is an anti-coagulant medication and a director factor Xa inhibitor. A novel reverse phase liquid chromatography-mass spectrometry compatible method developed for separation and identification of new oxidative degradation impurities in edoxaban tosylate hydrate drug substance. The separation of three oxidative degradation impurities was achieved by using YMC Triart phenyl (250 × 4.6) mm, 5 µm column with mobile phase containing gradient elution of mobile phase-A as 10 mM ammonium acetate and mobile phase-B as acetonitrile:methanol (1:1)% (v/v). The flow rate of the mobile phase is 0.7 mL/min with a column temperature of 40 °C and detection wavelength of 290 nm. Edoxaban tosylate hydrate shows significant degradation in oxidative stress conditions and forms three oxidative degradation products. The degradation products were identified and characterized by using a high-resolution mass spectrometry quadrupole-time of flight mass detector. The three oxidative degradation impurities of Edoxaban drug substance were well resolved with each other and along with Edoxaban drug substance peak. Among the three oxidative degradation impurities di-N-oxide impurity was the new oxidative degradation impurity identified for the first time and a novel reverse-phase high-performance liquid chromatography method was developed for separation of the three oxidative degradation impurities.

Development, stability-Indicating assessment, and evaluation of influential method conditions using a full factorial design for the determination of Nintedanib Esylate related impurities.

The design of an appropriate analytical method for assessing the quality of pharmaceuticals requires a deep understanding of science, and risk evaluation approaches are appreciated. The current study discusses how a related substance method was developed for Nintedanib esylate. The best possible separation between the critical peak pairs was achieved using an X-Select CSH Phenyl Hexyl (150 × 4.6) mm, 3.5 μm column. A mixture of water, acetonitrile, and methanol in mobile phase-A (70:20:10) and mobile phase-B (20:70:10), with 0.1% trifluoroacetic acid and 0.05% formic acid in both eluents. The set flow rate, wavelength, and injection volumes were 1.0 mL/min, 285 nm, and 5 μL, respectively, with gradient elution. The method conditions were validated as per regulatory requirements and United States Pharmacopeia general chapter <1225>. The correlation coefficient for all impurities from the linearity experiment was found to be >0.999. The % relative standard deviation from the precision experiments ranged from 0.4 to 3.6. The mean % recovery from the accuracy study ranged from 92.5 to 106.5. Demonstrated the power of the stability-indicating method through degradation studies; the active drug component is more vulnerable to oxidation than other conditions. Final method conditions were further evaluated using a full-factorial design. The robust method conditions were identified using the graphical optimization from the design space. This article is protected by copyright. All rights reserved.

High Performance Liquid Chromatography Method Development and Validation for Separation of Liothyronine Sodium Related Substances Using a Quality by Design Approach

The application of the Quality by Design (QbD) principles in developing a novel high performance liquid chromatography (HPLC) method for the analysis of liothyronine sodium related substances for finished product using Fusion QbD® software is explored. The effect of various chromatographic parameters including, column stationary phase, initial hold time and gradient time on separations were systematically investigated. Results show that optimal separations of these compounds in a standard solution can be achieved using a X Bridge C18 column (150 mm × 4.6 μm, 5m), maintained at sample temperature 15°C by using pH 2.0 buffer solution as mobile phase-A and methanol as mobile phase-B with a flow rate of 1.0 ml/min at 225 nm of detection and a gradient time of 38 minutes. The injection volume is 200 μl. Pre-validation studies of the method were performed and all the parameters met the acceptance criteria. The results are demonstrated that optimized method is selective, linear, precise, repeatable and accurate.

Development and Validation of SI/RS-UHPLC-PDA Method for Olmesartan Medoxomil and Metoprolol Succinate-Related Substance.

Olmesartan medoxomil (OLM) and metoprolol succinate (MPS) in fixed-dose combination (FDC) tablet formulation prescribed extensively. Stability indicating (SI) method for impurities and related substance (RS) test quantitates the amount of these analytes in formulation; the manuscript presents SI/RS-ultra-high performance liquid chromatography-photodiode array (UHPLC-PDA) method for OLM and MPS and their impurities. Well-resolved separation of all analytes was achieved with gradient elution on a Shimadzu on Shimpack GIST-C18 (100 mm x 2.1 mm, 2 µm) column maintained at 25°C. Mobile phase-A consist of 0.1% orthophosphoric acid in water and mobile phase-B was acetonitrile at a flow rate of 0.4 mL/min, data integrated at 225 nm and 16 min of short runtime for satisfactory elution of all peaks. The proposed SI/RS-UHPLC-PDA method was developed and validated as per International Conference on Harmonisation (ICH) of Technical Requirements guidelines. The system suitability test complied by all eluted peaks of the interest with acceptable linearity, recovery, and precision. Specificity, robustness, and method sensitivity parameters were determined; all the parameters were found to be within the limits. All the impurities and stress-degraded peaks were well resolved. The proposed method was found to be simple, fast, linear, and accurate. Further, the method is precise, robust, and specific; suitable for routine IPQC during active pharmaceutical ingredient manufacturing, stability and impurity profiling studies of the titled bulk analytes. Furthermore, the method can be extended to assess the levels of impurities formed during life cycle of new FDCs of titled analytes.

Determination of Sodium, Potassium, and Magnesium as Sulfate Salts in Oral Preparations Using Ion Chromatography and Conductivity Detection

An ion chromatography technique with conductivity detection was selected as an analytical tool for the simultaneous indirect determination of sodium sulfate, potassium sulfate, and magnesium sulfate via their respective cations. The method was developed and validated for the quantitative assay of the inorganic salts under study in oral pharmaceutical dosage forms. Chromatographic separation was achieved on a Dionex®IonPac® CS16 column (250 × 5 mm) column using the gradient elution method. A mobile phase-A consisting of methane sulfonic acid (6.7%, v/v) in Milli-Q water, which is used together with Milli-Q water, was used as a mobile Phase-B. The flow rate was 1.2 mL/min. The retention times of sodium, potassium, and magnesium as sulfates were 7.8, 12.8, and 16.2 min, respectively. The method was validated according to ICH guidelines and showed good linearity and accuracy results within concentration ranges of 80.0–240.0, 20.0–60.0, and 4.5–13.5 ppm for sodium, potassium, and magnesium as sulfates, respectively. The relative standard deviation results for intra- and inter-day precision were less than 1.0%. The method was applied successfully for determination of the analytes under study in their mixed pharmaceutical oral solution and found suitable for their routine and stability analysis.

Comprehensive study on degradation profile of firocoxib and structural elucidation of its key degradation products

Firocoxib is widely used in veterinary medicine as a non-steroidal analgesic and anti-inflammatory drug substance. Herein, a comprehensive study on the degradation profile of firocoxib was performed through force degradation studies to understand its degradation profile and characterize its major degradation products (DPs). Firocoxib drug substance was subjected to acidic, alkaline, oxidation (H2O2, KMnO4, and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation, as recommended in the ICH guidelines. Firocoxib and its major DPs were adequately separated by investigational HPLC method, which utilized a HALO C18 (100 × 2.1 mm, 2.0 µm) column. Mobile phase-A for the HPLC method is composed of 0.1% formic acid in water and mobile phase-B acetonitrile. A total of six major DPs were observed for firocoxib drug substance under these stress degradation conditions. Structural elucidation of the DPs performed using liquid chromatography-high resolution mass spectrometry and comparison of their fragmentation profile with that of the parent compound. For further structural confirmation of two major DPs, DP-2 and DP-6 were isolated and purified from the stressed samples using a preparative HPLC and analyzed by comprehensive nuclear magnetic resonance (NMR) spectroscopy studies. Most probable mechanistic pathways for the formation of DPs of firocoxib under various stress degradation conditions were postulated to understand its degradation profile. Based on the results from forced degradation, firocoxib was found to be quite stable under basic and thermal conditions, and somewhat unstable under acidic, oxidative, and photolytic conditions. The results of this study should facilitate quality monitoring and establish a stability profile of firocoxib drug substance and drug products. These results may also assist in the design and development of new formulations made with firocoxib drug substance with desired shelf life.