Degradation kinetic studies of non-pharmacopeial drug products and determination of their forced degradants and Impurities

Abstract

Stability of a drug is assessed to ensure the chemical and physical integrity of the drug product and its capacity to remain protected against exposure to environment, such as air, light, and heat throughout its shelf-life. Development and use of stability-indicating methods are critical parameters in drug regulation to prevent counterfeit medicines. Forced degradation or stress testing according to ICH Q3B (R2) is a part of this process, used to predict the stability of drug substance or drug product with effects on purity, potency, and safety. Three dipeptidyl peptidase IV (DPP-IV) inhibitors, sitagliptin, vildagliptin and linagliptin used to treat type 2 diabetes mellitus (T2DM) were studied, which are not yet included in the official book, i.e. USP, BP. The collected samples from pharmaceutical companies of Bangladesh were evaluated by comparing with innovator products. It is required to establish specificity of a stability indicating method, which also provide a perception into degradation pathways as well as degradation products of the drug molecules and helps in structure elucidation of the degradants by spectral analysis. The aims of the studies were to evaluate the quality of these three DPP-IV inhibitors. The present investigation also deals with method development and optimization by applying quality by design (QbD) approaches and validation of the selective stability-indicating RPUHPLC method according to ICH Q2 (R1) guideline. From degradation kinetics studies halflives

(t1/2) and shelf -lives (t0.9) of these three drug molecules were determined at room temperature by applying Arrhenius equation. Major degradation products of linagliptin were isolated and characterized by IR, 1 H-NMR and 13 C-NMR spectroscopic method and described plausible degradation pathways. All brands which were used in these studies were similar with their innovator products in terms of weight variation, hardness, disintegration and potency. For the comparison of dissolution profile with the reference product, the difference factor (f1) and similarity factor (f2) were calculated in four different dissolution media. Seven brands of sitagliptin, seven brands of vildagliptin and five brands of linagliptin among nine are similar and bioequivalent to innovator brand in respect to drug release pattern where the f1 value less than 15 and f2 value more than 50. The optimized chromatographic condition for separation and quantitation of sitagliptin, vildagliptin and linagliptin was reverse phase ultra high performance liquid chromatography (RP-UHPLC) equipped with X-bridge C18 column (4.6 i.d. × 150 mm, 5 μm) having flow rate 1 ml/min using phosphate buffer (pH 6) and acetonitrile (70:30, v/v) as mobile phase at 246nm, 228nm and 267nm for vildagliptin, linagliptin and sitagliptin, respectively using photodiode diode array plus (PDA+) detector. The column oven temperature was ambient for analysis of all samples. The retention time for vildagliptin, linagliptin and sitagliptin were 2.423±0.04min, 3.203±0.06 min and 4.189±0.12 min respectively. For routine analysis of these three products in pharmaceutical companies single, simple, precise, sensitive, accurate and robust method was developed and optimized by applying Quality by design (QbD) approaches using design of experiments (DoE) where 3 3 full factorial Box -Behnken Design (BBD) model were used. Three factors were utilized for the experimental design of the method as independent variables which comprise percentages of organic modifiers, pH of buffer in mobile phase and flow rate. The co-variates or responses included the retention time, resolution between peak 1 and 2(Rs1) and resolution between peak 2 and 3 (Rs2). This design was statistically analyzed by ANOVA, normal plot of residual, box-cox plot for power transform, perturbation, counter plot and 3D response surfaces plots. The quadratic effect of different variables like percentages of acetonirile in mobile phase(p< 0.0001), flow rate (p< 0.0001 ) and pH of buffer (p< 0.003 ) separately as well as in interaction was most significant on retention time(RT), resolution between peak 1 and 2(Rs1) and resolution between peak 2 and 3 (Rs2). The developed method was validated as per the requirements of ICH-Q2B guidelines for specificity, system suitability, linearity, sensitivity, precision, accuracy, and robustness. The linear regression analysis data for the linearity plot showed correlation coefficient values in case of sitagliptin of 0.999 with LOD value of 0.06 µg/mL and LOQ of 0.225µg/mL, in case of vildagliptin of 0.998 with LOD value of 0.01 µg/mL and LOQ of 0.05µg/mL and in case of linagliptin of 1.0 with LOD value of 0.005 µg/ml and LOQ of 0.015µg/ml. The relative standard deviation (%RSD) for inter-day and intra-day precision was not more than 2.0%. The method was found to be accurate with percentages recovery of 100±2% and the % RSD was less than 2%. The results showed that the proposed method is simple, sensitive and highly robust for routine analysis. Forced degradation or stress testing is performed according to ICH Q1A and ICH Q1B guideline to meet the stability testing of a drug substance or a drug product with effects on purity, potency, and safety. This study was carried out to ensure stability indicating assay method. The stressed condition were hydrolytic (acid and base), oxidative, thermal and photolytic. The degradation kinetics was estimated in acidic, alkaline, oxidative and thermal forced degradation condition. The half-lives (t1/2) and shelf -lives (t0.9) of the drugs were calculated by using an Arhenius plot. The calculated half-life of sitagliptin was maximum (2310h) in thermal and minimum (138.5h) in acid hydrolysis condition, for vildagliptin maximum (990h) in thermal and minimum (115.5h) in acid hydrolysis condition and for linagliptin maximum (1732.5h) in thermal and minimum (385h) in acid hydrolysis conditions. The proposed stability indicating method revealed that these three gliptins were stable in various heat and photolytic condition; however, protection is recommended during storage and handling in strong acidic, alkaline and oxidative condition. Five major degradants of linagliptin in acidic (3) and oxidative (2) forced degradation condition were isolated and characterized by IR, 1 H-NMR and 13 C-NMR spectroscopic methods. After acidic degradation novel compounds are 1-(2-amino-5- (hydroxylmethyl) – 1 - methyl – 1 H – imidazol – 4 – yl ) – 1 – methyl – 3 - ( ( 4 – methyl – 1 , 2 dihydroquinazolin -2-yl) methyl)urea (DA1); 7,7'-((2E,4E)-3 , 4 – dimethylhexa – 2 , 4 - diene-1, 6 – diyl ) bis ( 8 - ( ( R ) – 3 – aminopiperidin – 1 – yl ) – 3 – methyl – 1 - ( ( 4 - methyl quinazolin-2-yl)methyl)-3,7-dihydro-1H-purine-2,6-dione) (DA2) and 1-(3-amino7-methyl-8-oxo-5,8-dihydroimidazo[1,5-a]pyridin-1-yl)-1-methyl-3-((4-methylquinazolin2- yl ) methyl ) urea (DA3). The two novel oxidative degradants are 1 -( but – 2 – yn – 1 –yl

)–4-(1-methyl – 3 - ( ( 4 – methylquinazolin – 2 – yl ) methyl ) ureido ) - 1H – imidazole – 5–

carboxylic acid (DO2) and 5 , 6 – diamino – 1 – methyl – 3 - ( ( 4 – methylquinazolin – 2 –yl)

methyl )pyrimidine-2,4(1H,3H)-dione (DO3).

From this study it can be concluded that the quality of antidiabetic DPP-IV inhibitors manufactured by Bangladeshi pharmaceutical companies fulfill the world class requirement based on the comparison with innovator products which are effectively worked on T2DM to reduce the global burden on diabetes.