Chromatographic study of sitagliptin and ertugliflozin under quality-by-design paradigm

Abstract The present study entails the systematic development and validation of a stability-indicating RP-HPLC method for the analysis of sitagliptin and ertugliflozin in a fixed-dose combination. Analytical quality by design (AQbD) concepts were used to define critical method variables, employing Pareto risk assessment and a Placket-Burman screening design, preceded by a Box-Behnken design with response surface analysis to optimise critical method parameters such as % acetonitrile (X1), buffer pH (X2) and column oven temperature (X3). Multiple response optimisation (Derringer's desirability) of variables was accomplished by studying critical analytical attributes, such as resolution, retention time and theoretical plates. The title analytes were separated effectively on a PRONTOSIL C18 column at 37 °C using a mobile phase of acetonitrile:acetate buffer, pH 4.4 (36:64 percent v/v), pumped at a flow rate of 1 mL/min, and UV detection at 225 nm. Linearity was observed over a concentration range of 25-150 µg/mL and 3.75-22.5 µg/mL at retention times of 2.82 and 3.92 min for sitagliptin and ertugliflozin, respectively. The method obeyed all validation parameters of the ICH Q2(R1) guidelines. The proposed robust method allows the study of the selected drugs in pharmaceutical dosage forms as well as in drug stability studies under various stress conditions.

Response Surface Methodology in Spectrophotometric Estimation of Saxagliptin, Derivatization with MBTH and Ninhydrin

Objectives: Design of experiments assisted spectrophotometric methods have been established for the quantification of saxagliptin in pharmaceutical formulation via charge transfer complexation and Schiff's base formation. Materials and Methods: Box-Behnken design was exploited in method-1, involved the measurement of absorbance of green/blue-colored complex (at 600 nm), formed by the reaction of saxagliptin with 3-methyl-2-benzothiazolinone hydrazone in the presence of ferric chloride. The central composite design was employed in method-2, involved the determination of absorbance of Ruhemann's purple (at 585 nm), formed by the reaction of the primary amine group of saxagliptin with ninhydrin reagent in presence of sodium hydroxide. Optimization of reaction variables namely, reagent concentration (A), oxidizing agent/alkalinity (B) and reaction/heat time (C) was performed through response surface methodology for the response (Y) i.e. absorbance of colored compound. The reliability of both methods was investigated through validation as per International Council for Harmonisation guidelines. Results: Saxagliptin executed linearity in the concentration range of 0.01-0.25 µg/mL and 1-10 µg/mL by method-1 and 2. A high value of molar absorptivity, low values of Sandell's sensitivity and limit of detection/limit of quantification divulges the good sensitivity methods. The % assay of saxagliptin in the marketed formulation was found to be 100.27 and 99.86 by method-1 and method-2, respectively. Conclusion: The proposed eco-friendly and economical methods can be routinely employed in quality control for the analysis of saxagliptin in the pharmaceutical dosage forms.

Quality-by-Design Approach for Chromatographic Analysis of Metformin, Empagliflozin and Linagliptin.

New analytical quality by design-oriented HPLC method with multiple response optimization (Derringer's desirability function) was demonstrated for simultaneous analysis of three antidiabetic drugs (metformin hydrochloride/empagliflozin/linagliptin) in a fixed-dose combination. Central composite design was employed for systematic optimization of critical method parameters, namely, % organic phase (X1), aqueous phase pH (X2) and flow rate (X3) while resolution, capacity factor and theoretical plate number as critical analytical attributes. Effective chromatographic separation of title analytes was accomplished on Std. Discovery C18 column at 30°C with mobile phase comprising acetonitrile: phosphate buffer pH 5 (38:62% v/v), pumped at a flow rate of 1 mL/min by isocratic elution pattern and UV detection at 222 nm. The model is rectilinear in the range of 1.0-200, 0.2-40 and 0.1-20 µg/mL at retention times of 3.04, 3.93 and 5.99 min for metformin, empagliflozin and linagliptin, respectively. The method obeyed all validation parameters of ICH Q2(R1) guidelines. The proposed HPLC method was highly robust for method transfer, regulatory flexibility within design space and can be used for assay of pharmaceutical dosage forms comprising these analytes. The proposed method was applied for stability studies of drugs under various stress conditions.

Development of a Discriminative and Biorelevant Dissolution Test Method for Atorvastatin/Fenofibrate Combination with Appliance of Derivative Spectrophotometry.

OBJECTIVES: Nowadays, the market is flooded with combinations of drugs in various dosage forms, but there is a lack of official methods to quantify them. A single dissolution test method for the analysis of combined dosage form is preferred for simplification of quality control testing. MATERIALS AND METHODS: If the developed dissolution medium mimics the biorelevant and discriminating dissolution procedure for drug products with limited drug aqueous solubility it is a useful tool for qualitative forecasting of the in vivo behavior of formulations. RESULTS: Dissolution profiles were evaluated for atorvastatin and fenofibrate in capsules, using a paddle-type United States Pharmacopeia dissolution apparatus in 900 mL of medium at 50 rpm and 37±0.5°C. The best medium was 900 mL of 0.5% w/v sodium lauryl sulfate. The cumulative % dissolution was more than 85% within 45 min for marketed tablets. The proposed dissolution test conditions have discriminative power, dissimilarity factor (f1) values are low (12-16%), and similarity (f2) factor values are also low (45-48%). Hence the use of 0.5% w/v sodium lauryl sulfate solution is justified. CONCLUSION: The dissolution method was validated (% relative standard deviation <2). To quantify both drugs simultaneously, a second derivative spectrophotometric method was established (λmax 281 nm and 296 nm, respectively, for atorvastatin and fenofibrate) in acetate buffer, pH 2.8 solution.

Degradation Kinetics, In Vitro Dissolution Studies, and Quantification of Praziquantel, Anchored in Emission Intensity by Spectrofluorimetry.

OBJECTIVES: A simple, rapid, specific, and highly sensitive ecofriendly spectrofluorimetric method has been developed for the quantification of praziquantel. MATERIALS AND METHODS: A linear relationship was found between fluorescence intensity and praziquantel concentration in the range of 1-20 µg/mL in water at emission wavelength of 286 nm after excitation wavelength at 263 nm with a good correlation coefficient (0.999). RESULTS: The proposed method was validated according to International Conference on Harmonization guidelines and statistical analysis of the results revealed high accuracy and good precision with the percentage relative standard deviation values less than 2. The detection and quantification limits were 0.27 and 0.81 µg/mL, respectively. The proposed method was extended to investigate the stability of the drug and its degradation kinetics in the presence of acidic, alkaline, and oxidative conditions. CONCLUSION: The method was utilized for in vitro dissolution studies of praziquantel tablet formulation. The suggested procedures could be used for the assessment of praziquantel in drug substance and drug products as well as in the presence of its degradation products.

Spectrophotometric Quantification of Anti-inflammatory Drugs by Application of Chromogenic Reagents.

OBJECTIVES: Simple, specific, accurate, precise, sensitive, and cost effective spectrophotometric methods were developed and validated for quantification of the drugs lornoxicam (LOR) and mesalamine (MES) in pure form and in pharmaceutical formulations. MATERIALS AND METHODS: A Shimadzu UV-1800 double-beam UV-Visible spectrophotometer having a spectral bandwidth of 0.1 nm with wavelength accuracy ±0.1 nm with a pair of 1 cm path length matched quartz cells was used to measure the absorbance of the resulting solution. Method I was used for the quantification of LOR, based on measurement of the absorbance of bluish green chromogen complex at 760 nm, which is formed by the reaction of LOR with ferric chloride and potassium ferricyanide (redox technique). Method II was used for the quantification of MES, based on measurement of the absorbance of yellow chromogen at 400 nm, which is formed by the condensation reaction of the primary amino group of MES with salicylaldehyde reagent (SA) (Schiff base formation). RESULTS: Both methods obeyed Beer's law in the concentration range of 0.5-4.5 µg/mL and 0.2-1.7 µg/mL with good correlation coefficients of 0.9974 and 0.998 for methods I and II, respectively. CONCLUSION: The developed method is simple, sensitive, and specific, which was validated statistically as per ICH guidelines, and can be used in the routine analysis of LOR and MES in pharmaceutical dosage forms.

Quantification of Tamsulosin Hydrochloride and Solifenacin Succinate by Discriminative Derivative Synchronous Emission Spectroscopy.

OBJECTIVES: The present study was undertaken with the objective to develop and validate a simple spectrofluorimetric method for the simultaneous quantification of tamsulosin hydrochloride and solifenacin succinate. MATERIALS AND METHODS: First-derivative synchronous spectrofluorimetry was attempted for the simultaneous quantification of the analytes. Tamsulosin hydrochloride was quantified at a wavelength of 322 nm (zero-crossing wavelength point of solifenacin succinate) and solifenacin succinate was measured at 570 nm (zero-crossing wavelength point of tamsulosin hydrochloride). RESULTS: Calibration plots were constructed over the concentration range of 2-10 µg/mL for tamsulosin hydrochloride and 30-150 µg/mL for solifenacin succinate. The method gave satisfactory results when it is validated for linearity, specificity, accuracy, precision, LOD and LOQ as per the ICH guidelines. The assay values in the commercial formulation were found to be in the percentage range of 95.0 for tamsulosin hydrochloride and 103.5 for solifenacin succinate by the proposed method. These results were well in agreement with their label claim. CONCLUSION: The proposed synchronous analytical method can be employed for routine quality control analysis of tamsulosin hydrochloride/solifenacin succinate in tablet dose forms.

Quantification and stress degradation studies of cefepime/tazobactam in dry injection form by an RP-HPLC method

A simple, specific, precise, accurate, linear, rapid, economic and validated stability indicating an RP-HPLC method for the simultaneous quantification of cefepime and tazobactam in a dry injection dosage form has been developed. Separation was performed on a 5 µm ACE C18 column with phosphate buffer, pH adjusted to 4.5 with phosphoric acid: methanol (70:30) at a flow rate of 1 mL/min and at a temperature of 25 °C. Regression analysis showed linearity at a detector wavelength of 290 nm in the range of 200-600 μg/mL for cefepime and 25-75 μg/mL for tazobactam. All of the analytes were adequately resolved with acceptable tailing. The percentage content found for cefepime was 99.98% and of tazobactam was 99.49% in the parenteral formulation. The method was validated in terms of linearity, precision, accuracy, specificity, robustness and system suitability according to ICH guidelines. Stress degradation studies were performed on the placebo and drug products, drugs of interest were well resolved from the degradation products. The developed method was effectively applied for the simultaneous quantification of cefepime and tazobactam in a dry injection formulation.

Desenvolveu-se método específico, preciso, exato, linear, rápido e econômico, de validação de estabilidade, indicando o método de CLAE-FR para a quantificação simultânea de cefepima e tazobactam na forma de dosagem injetável seca. A separação foi realizada em coluna C18 de ACE 5 mM com tampão fosfato, pH ajustado para 4,5 com ácido fosfórico:metanol (70:30), em fluxo de 1 mL/min e temperatura de 25 °C. A análise de regressão mostrou linearidade no detector de comprimento de onda de 290 nm, na faixa de 200-600 μg/mL, para cefepima, e 25-75 μg/mL, para tazobactam. Todos os analitos foram, adequadamente, resolvidos com cauda aceitável. O teor percentual encontrado na formulação parenteral foi de 99,98%, para cefepima, e de 99,49%, para o tazobactam. O método foi validado em termos de linearidade, precisão, exatidão, especificidade, robustez e adequação do sistema de acordo com as diretrizes ICH. Estudos de degradação por estresse foram realizados no grupo placebo e nos medicamentos e os fármacos de interesse foram bem resolvidos a partir dos produtos de degradação. O método desenvolvido foi efetivamente aplicado para quantificação simultânea de cefepima e tazobactam na formulação injetável seca.