Development of expeditious RP-HPLC method for simultaneous estimation of bilastine and montelukast in fixed dosage combination

Stability indicating analytical RP-HPLC (Reverse Phase- High performance Liquid Chromatography) method was developed for the analysis of the drug in the presence of its degradation products and subsequent validation of Pemetrexed that was conducted in API and pharmaceutical dosage form. Pemetrexed was subjected to different stress conditions as per International Conference on Harmonization (ICH) guidelines, stress conditions applied were including the effect of acid, base, oxidative, hydrolysis, thermal and photolytic degradation conditions. Chromatographic separation of API (Active Pharmaceutical ingredient) was achieved on Water’s HPLC instrument and ODS C18, 250x4.6 mm, 5µm particle size column with gradient elution of 0.1% ortho phosphoric acid buffer: Acetonitrile 80:20 ratio was taken as the eluent (mobile phase). The buffer pH was maintained at 3, the flow rate was 1.0 ml/min and wavelength were detected at 225 nm. The Retention time (RT) of Pemetrexed was Found at 2.9 minutes, column temperature was kept ambient and the runtime was 6 min. System suitability parameters like theoretical plates, tailing factor, and Asymmetry values were showed at 3391, 1.41, and 1.101 respectively. The percentage drug purity was found to be 99.99%. Validation of the method was proved that linear relationship over the range of 25-150 µg/mL, with linear regression curve (correlation coefficient) r2 value was noticed at 0.999 and LOD (limit of detection), LOQ (limit of quantification) values was established at 0.33µg/mL, 0.99µg/mL respectively. Recovery studies carried out at the concentrations of 50%, 100% and 150% with percentage recovery values of 100.06, 99.8 and 99.87 respectively. The precision of intraday was 0.64% and inter-day was 0.95%. Robustness of flow rate and mobile phase was changed even though the method was Robust. Forced degradation studies were also conducted to check the stability and suitability of this method to resolve the degradation products. Method validation was proved to be accurate, precise, linear, repeatable and robust. Thus, the accepted method can be proceeded for the estimation of Pemetrexed in pharmaceutical dosage form in a daily basis, due to their simplicity, suitability, accuracy, robustness and reproducibility.

In this study, an accurate, simple, economical and precise Reversed-Phase High Pressure Liquid Chromatography (RP-HPLC) method was developed for the simultaneous estimation of Ozenoxacin and Benzoic Acid in a pharmaceutical cream formulation, according to the International Conference on Harmonisation (ICH) guidelines. Chromatographic separation was achieved by gradient elution, on RP-HPLC Instrument, equipped with column C8 (150 mm × 4.6 mm, 5 μm particle size) using Ultra Violet (UV) detector at 235 nm wavelength, by using Mobile Phase A: triethylamine, trifloroacetic acid and water (1:1:1000) and Mobile Phase B: methanol and Diluent: water, acetonitrile and triethylamine (500:500:1), at flow rate 0.8 mL min−1; injection volume of 20 μL; column oven temperature 45 °C and sample temperature: 25 °C; Run time: 15 min. All the validation parameters were within the acceptance criteria, as per ICH requirements, for Ozenoxacin and Benzoic acid. Consequently, this method has found to be validated, simple, rapid and successfully applicable, to the simultaneous estimation of Ozenoxacin and Benzoic acid by RP-HPLC, for routine analytical testing in quality control, with a run time of 15 min and for future research studies. Forced degradation of Ozenoxacin cream 1% w/w formulation was performed and found that validated method has stability indicating potential that needs to be further studied.

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous quantitative estimation of gabapentin and amitriptyline hydrochloride in gabantip-at tablet and validate as per international conference on harmonization (ICH) guidelines and to perform the force degradation studies using the developed method.Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5μm) and mobile phase consisting of 0.05 M potassium dihydrogen orthophosphate pH 2.1 adjusted with orthophosphoric acid and acetonitrile in the ratio (55:45), at flow rate 1 ml/min and column temperature (25 °C). The effluents obtained were monitored at 221 nm with the UV-visible detector.Results: The retention time of gabapentin and amitriptyline hydrochloride was found to be 1.959 min and 4.221 min respectively. The linearity of gabapentin was found in the range of 720-1680 ppm and that for amitriptyline hydrochloride was found to be 24-56 ppm. The correlation coefficient for gabapentin and amitriptyline hydrochloride were 0.999 and 0.9963 respectively. The high recovery values (98%-101%) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveals that the method is precise.Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous estimation of gabapentin and amitriptyline hydrochloride tablet dosage form. Hence the proposed method may find practical applications as a quality-control tool in the simultaneous analysis of the two drugs in combined dosage forms in quality-control laboratories.

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous quantitative estimation of ramipril, atorvastatin and clopidogrel in Atamra-CV tablet and validate as per international conference on harmonization (ICH) guidelines and to perform the force degradation studies using the developed method.Methods: In the present work, the good chromatographic separation was achieved isocratically using a shim-pack HPLC Kromasil 150 mm x 4.6 mm, 5 m. m. And mobile phase consisting of 0.05 M potassium dihydrogen orthophosphate pH 3 adjusted with orthophosphoric acid and acetonitrile in the ratio (52:48), at flow rate 1 ml/min and column temperature (30 °C). The effluents obtained were monitored at 210 nm with the UV-visible detector.Results: The retention time of ramipril, atorvastatin and clopidogrel was found to be 2.893 min, 5.012 min and 6.102 min respectively. The linearity of ramipril, atorvastatin and clopidogrel was found in the range of 25-150 % and the correlation coefficient for ramipril, atorvastatin and clopidogrel were>0.999. The high recovery values (98%-101%) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveals that the method is precise. The three-drug samples were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the analytes from their forced-degradation products.Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous estimation of ramipril, atorvastatin and clopidogrel tablet dosage form. Hence the proposed method may find practical applications as a quality-control tool in the simultaneous analysis of the three drugs in combined dosage forms in quality-control laboratories. The proposed method was made use of photodiode array (PDA) as a tool for peak identification and purity confirmation.

Diabetes mellitus, particularly Type 2 Diabetes Mellitus (T2DM), presents a significant global health challenge, necessitating continuous advancements in therapeutic strategies. Gliclazide, an oral selective inhibitor of sodium glucose co-transporter-2 (SGLT2), has emerged as a promising agent for managing T2DM by facilitating urinary glucose excretion and reducing plasma glucose levels. However, ensuring the quality and efficacy of pharmaceutical formulations remains crucial for regulatory approval and clinical effectiveness. In this study, we present the development of an improved stability-indicating Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for the precise quantification of Gliclazide. The novel analytical method employs 0.1% Potassium Phosphate in water 0.1% Potassium Phosphate in methanol (MeOH) as the mobile phase, demonstrating enhanced sensitivity, cost-effectiveness, and reduced reliance on organic chemicals. Through rigorous validation following International Conference on Harmonisation (ICH) guidelines, the developed method exhibited superior performance in terms of retention times compared to existing methodologies. The proposed analytical approach holds promise for routine analysis of Gliclazide in both bulk and tablet formulations, offering a valuable tool for pharmaceutical quality control and clinical research in the management of T2DM.

The present investigation aimed to develop and comparatively validate a reversed-phase high-performance liquid chromatographic (RP-HPLC) method and a chemometric-assisted UV spectrophotometric method for the simultaneous estimation of Pyridoxine Hydrochloride (PYR) and Nicotinamide (NIC) in pharmaceutical formulations. Chemometric analysis using Partial Least Squares (PLS) regression was employed to overcome spectral overlap limitations encountered in conventional spectrophotometric analysis. RP-HPLC separation was achieved using a C18 column under optimized chromatographic conditions with an isocratic mobile phase system. The developed analytical methods were validated according to International Conference on Harmonisation (ICH) guidelines with respect to linearity, accuracy, precision, robustness, reproducibility, specificity, limit of detection, and limit of quantitation. The chemometric-assisted spectrophotometric method demonstrated excellent predictive capability with satisfactory calibration and validation performance. The RP-HPLC method exhibited sharp peak resolution, acceptable retention times, high theoretical plate count, and minimal tailing. Both analytical methods showed good linearity over the selected concentration ranges with correlation coefficients approaching unity. Recovery studies confirmed the accuracy of the developed methods with percentage recoveries within acceptable limits. Statistical analysis demonstrated no significant difference between the proposed RP-HPLC and chemometric-assisted methods regarding quantitative estimation of both analytes. The comparative evaluation established that the chemometric-assisted spectrophotometric method provides a rapid, economical, and environmentally friendly alternative to chromatographic analysis, whereas RP-HPLC offers superior selectivity and sensitivity for routine quality control applications. The developed methods were successfully applied for simultaneous estimation of Pyridoxine HCl and Nicotinamide in marketed pharmaceutical formulations.

A simple, specific and cost effective RP-HPLC method was developed for simultaneous estimation of Dosulepin and Methylcobalamin in bulk. In addition the developed method was applied to the suitable combined tablet dosage form ie., Prothiaden. As there is no UV or HPLC method for the simultaneous estimation of Dosulepin and Methylcobalamin, a need was felt to develop the method for the analysis of both drugs simultaneously. Chromatography was carried on Zorbax C8 column (150x4.5, 5) with mobile phase comprising of Ortho Phosphoric Acid buffer and Methanol in the ratio of 50:50 v/v. The flow rate was adjusted to 0.8 ml/min with PDA detection at 219.8 nm. The retention times of Dosulepin and Methylcobalamin were found to be 3.8 min, 1.9 min respectively and other replicate standard system suitability parameters are within the limit and uniform. The different analytical parameters such as accuracy, precision, linearity, robustness, limit of detection (LOD), limit of quantification (LOQ) were determined according to the International Conference on Harmonization (ICH) Q2B guidelines. The detector response was linear in the range of 0.9-2.7 mg/ml., 0.018- 0.054 mg/ml for Dosulepin and Methylcobalamin respectively. The proposed method was successfully applied for the reliable quantification of active pharmaceuticals present in the commercial formulations.

Objective: An accurate, precise, rapid and economical reverse phase High Performance Liquid Chromatography (HPLC) method has been developed and validated for the estimation of etravirine in pharmaceutical dosage forms, using PDA detector. Method: Elution was carried out using a mobile phase-A and B consisting of HPLC grade and flow rate was set on 1 ml/minute at 310 nm wave length. The retention time for Etravirine (ETR), impurity-1 and impuriey-2 was found to be 15.813, 12.043 and 17.704 respectively minutes. Result: Analytical method was developed using HPLC Shimadzu (with power stream) gradient chromatographic technique. Data were passed through the spinchrom software. Separation was achieved on Xselect HSS T3 (150 × 4.6 mm, 3.5 μm) column and using mobile phase A (Buffer) was used at pH 4.0 and mobile phase B (Methanol: Acetonitrile: Water (90:5:5 v/v)) by gradient programme. Flow rate was maintained at 1 ml/min with 310 nm PDA detection. The Retention Time (RT) obtained for Etravirine (ETR), impurity 1 and impurity 2 was at 15.813 min, 12.043 min and 17.704 min respectively with injection volume 10 μL and the detection was made at 310 nm. The % recovery of impurity-1 and impurity-2 observed was above 90% from LOQ level to 150%. The correlation coefficient r2 was 0.9993 for impurity-1 and 0.9997 for impurity-2. The method was found unaffected by change in method variance during the robustness study. During the stress study with acid, base, peroxide and temperature, maximum degradation was observed with peroxide indicating the sensitivity of the molecule toward oxidative stress. Conclusion: The developed method is precise, accurate, robust and linear and hence can be routinely used for the related substance analysis of metformin hydrochloride and teneligliptin hydrobromide hydrate tablet in the quality control laboratory at manufacturing site during the commercial manufacturing. Results of all validation parameter were within the limits as per International Conference on Harmonization (ICH) guideline. Keywords Reverse Phase High Performance Liquid Chromatography (RP-HPLC); Etravirine (ETR); Method development and validation

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the identification and quantification of moxifloxacin hydrochloride-related substances in finished dosage forms. Chromatographic separation was achieved on an Agilent C18 column (150 × 4.6 mm, 5 μm) with the mobile phase (0.01 M potassium dihydrogen orthophosphate as buffer and methanol in the ratio of 70:30) eluted in isocratic mode. The HPLC flow rate was 1.0 mL min-1 , and peaks were monitored at 230 nm using a photodiode array (PDA) detector. The column temperature was kept constant at 30°C, and the injection volume was 10 μL. The run time of the method was 16 min. The method was validated as per the International Conference on Harmonization (ICH) guidelines. Linearity was recorded at various concentrations ranging from 0.25 to 1.5 μg mL-1 for all the moxifloxacin impurities. Linearity, regression value, recovery, %relative standard deviation (RSD) of method precision values were found within the acceptance limits. The method for related substances (RS) in moxifloxacin was found to be specific, linear, accurate, precise, rugged, and robust. The validated method was suitable for the quantification of the RSs in moxifloxacin drug products. The method was applied in quality control lab for the analysis of moxifloxacin impurities in stability analysis.

Rapid Development and Validation of Atoltivimab, Maftivimab and Odesivimab in Pharmaceutical Dosage form by using the RP-HPLC Method

The International Conference on Harmonization (ICH) has provided practical guidance on the amount and type of stability data needed to support marketing applications (International Conference on Harmonization Guideline Q1A(R2), 2003; International Conference on Harmonization Guideline Q1C, 1996; International Conference on Harmonization Guideline Q7A, 2001). Additional guidance has been issued by the World Health Organization (WHO, 2009). Recent scientific advances and practices have resulted in improved scientific understanding of the chemical and physical attributes that contribute directly or indirectly to drug substance and drug product stability. Combining this improved understanding with the science- and risk-based approaches detailed in ICH Q8–Q11 allow for alternative and more scientifically driven approaches to meet the scientific and regulatory objectives for drug substance and drug product stability (International Conference on Harmonization Guideline Q8(R2), 2009; International Conference on Harmonization Guideline Q9, 2005; International Conference on Harmonization Guideline Q10, 2008; International Conference on Harmonization Guideline Q11, 2012). This paper follows and expands on the concept and application of the lean stability strategies that were introduced for drug substance (Colgan et al., J Pharm Innov 7:205–213, 2012). Herein, proposals are presented to more fully leverage enhanced product knowledge to design and prosecute improved stability strategies throughout the developmental lifecycle. The chemical and physical attributes that could impact stability and the tools that can be leveraged in pursuit of optimized stability strategies are discussed. Dove-tailing these stability strategies into regulatory filings for products with extensive product knowledge and the reception of these strategies by regulatory authorities in both major and emerging markets are summarized.

Paracetamol and Tapentadol are both analgesic drugs. As there is no UV or HPLC method for the simultaneous estimation of Paracetamol and Tapentadol, a need was felt to develop the method for the analysis of both drugs simultaneously. This work concerns with the development and validation of a simple, specific and cost effective RP-HPLC method for simultaneous estimation of Paracetamol and Tapentadol in bulk and the developed method was applied to the pharmaceutical dosage form ie., Tapcynta. Chromatography was carried on Thermohypersil BDS C18 column with mobile phase comprising of Dihydrogen potassium phosphate buffer and Acetonitrile in the ratio of 50:50 v/v. The flow rate was adjusted to 0.9 ml/min with PDA detection at 218.6 nm. The retention times of Paracetamol and Tapentadol were found to be 4.8 min, 8.0 min respectively and other replicate standard system suitability parameters are within the limit and uniform. The different analytical parameters such as accuracy, precision, linearity, robustness, limit of detection (LOD), limit of quantification (LOQ) were determined according to the International Conference on Harmonization (ICH) Q2B guidelines. The detector response was linear in the range of 88044x + 19214 mg/ml, 49855x + 36868 mg/ml for Paracetamol and Tapentadol respectively. The proposed method was successfully applied for the reliable quantification of active pharmaceuticals present in the commercial formulations.

A simple, specific, accurate, precise stability indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of pyridoxine hydrochloride (PYH) and meclizine hydrochloride (MEH). An isocratic separation of PYH and MEH were achieved on C 18, 250 × 4.6 mm ID, 5 μm particle size columns at column oven temperature 37°C with a flow rate of 0.5 mL min−1 and using a diode array detector to monitor the detection at 254 nm. The mobile phase consisted of buffer : acetonitrile : trifluoroacetic acid at a ratio of 30 : 70 : 0.1 (v/v). The retention times of PYH and MEH was found to be 5.25 and 10.14 min, respectively. Suitability, specificity, linearity, accuracy, precision, stability, and sensitivity of this method for the quantitative determination of the drugs were proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) Q2 (R1) guidelines. The proposed method is reliable and robust and can be used as quality control tool for the estimation of these drugs in combined pharmaceutical solid dosage forms.

This study was designed to develop and validate a new reversed-phase high-performance liquid chromatography (RP-HPLC) method based on Q2 (R1) International Conference on Harmonization (ICH) guideline for determination of curcumin in pharmaceutical samples. The HPLC instrument method was optimized with isocratic elution with acetonitrile: ammonium acetate (45:55, v/v, pH 3.5), C18 column (150 mm×4.6 mm×5 µm particle size) and a flow rate of 1 ml/min in ambient condition and total retention time of 17 min. The volume of injection was set at 20 µl and detection was recorded at 425 nm. The robustness of the method was examined by changing the mobile phase composition, mobile phase pH, and flow rate. The method was validated with respect to precision, accuracy and linearity in a concentration range of 2-100 µg/ml. The limit of detection (LOD) and limit of quantification (LOQ) were 0.25 and 0.5 µg/ml, respectively. The percentage of recovery was 98.9 to 100.5 with relative standard deviation (RSD) < 0.638%. The method was found to be simple, sensitive and rapid for determination of curcumin in pharmaceutical samples and had enough sensitivity to detect degradation product of curcumin produced under photolysis and hydrolysis stress condition.

In the present study, a simple, rapid and cost-effective analytical method was developed for the simultaneous determination of three commonly prescribed cardiovascular drugs: propranolol, rosuvastatin and valsartan. The method employed artificial neural networks (ANN) to model the relation between the UV absorption spectra of the drugs and their concentrations. An experimental design of 25 samples was employed as a calibration set, and a central composite design of 20 samples was used as a validation set. The firefly algorithm (FA) was evaluated as a variable selection procedure to optimize the developed ANN models resulting in simpler models with improved predictive performance as evident by lower relative root mean square error of prediction (RRMSEP) values compared to the full spectrum ANN models. Validation of the developed FA-ANN models demonstrated excellent accuracy, precision and selectivity for the quantification of the target analytes as per international conference on harmonisation (ICH) guidelines. Additionally, the greenness, analytical practicality and sustainability of the developed models were assessed using the analytical greenness (AGREE), blue applicability grade index (BAGI) and the red-green-blue (RGB) tools, confirming their environmentally friendly, practical and sustainable nature. This research shed the light on the potential of ANN coupled with UV fingerprinting for the rapid and simultaneous determination of critical cardiovascular drugs posing a significant impact on pharmaceutical quality control and patient monitoring.