DEGRADATION KINETICS OF CARVEDILOL PHARMACEUTICAL DOSAGE FORMS (TABLETS) THROUGH STRESS DEGRADATION STUDY

Objective: The aim of the research work to monitor impurities profiling and degradation kinetics of Carvedilol Pharmaceutical Dosage Form (Tablets) through stress degradation study. 
Methods: To study impurity profiling and degradation kinetics Chromatographic condition used as, Inertsil ODS 3V column (150 mm x 4.6 mm, 5μm) with mobile phase consisting Mobile phase-A (Water, Acetonitrile and Trifluroacetic acid in the ratio of 80:20:0.1 v/v/v respectively and pH adjusted to 2.0 with dilute potassium hydroxide solution) and Mobile phase-B (Water and acetonitrile in the ratio of 100:900 v/v respectively) delivered at flow rate of 1.0 ml min-1 and the detection wavelength 240 nm. The column compartment temperature maintained at 40 °C. 
Results: Stress degradation study conducted using Acid, Alkali, Oxidation, Humidity, Thermal and Photolytic stress degradation conditions. Known, unknown and degradant impurities nature and degradation kinetics in different stressed degradation conditions were monitored through stability indicated method reverse phase HPLC method. Carvedilol molecule found sensitive to Oxidation and Alkali conditions. Impurity-A significantly increased from its not detected level. 
Carvedilol molecule found stable in Acid, Humidity, Thermal and Photolytic stress degradation condition. In all stressed conditions, mass balance was found between 95% to 105% and peak purity of carvedilol peak was found pure. 
Conclusion: Stress degradation study is required to know the degradation pathway of product and to prove the stability indicating nature of the analytical method. Study provide information pertaining to the intrinsic stability of drug product.


INTRODUCTION
Carvedilol chemically it is named, (±)-1-(carbazol-4-yloxy)-3-((2-omethoxyphenoxy) ethyl) amino)-2-propanol. Carvedilol is a racemic mixture where the S(-) enantiomer is a beta-adrenoceptor blocker and the R (+) enantiomer is both a beta and alpha-1 adrenoceptor blocker and is currently used to treat heart failure, left ventricular dysfunction and hypertension [1,2]. Carvedilol shows greater antioxidant activity than other commonly used beta-blockers [3,4]. It has been prescribed as an antihypertensive agent, an antiangina agent [5][6][7][8]. The dual action of carvedilol is advantageous in combination therapies as moderate doses of 2 drugs have a decreased incidence of adverse effects compared to high dose monotherapy in the treatment of moderate hypertension [9]. Stress degradation indicates the chemical behaviour of the molecule, which give the necessary information in development of pharmaceutical dose.
As per guidance given by International Conference on Harmonization (ICH) guidelines (Q1A), Stability studies of pharmaceutical dose needs to be performed to decide to propose shelf life of drug product [10]. Stability study of drug substance and drug product provide vital information pertaining to the intrinsic stability of the molecule. During stability/stress forced degradation study drug substance/drug product will degrade or decompose and will produce other related substance, which is known as impurities/degradants. These impurities then monitored through suitable stability-indicating method in which these impurities are well separated from each other and from the main drug component. Hence stress degradation study is a tool to identify and estimate the degradants impurities that would occur during the stability study of drug product. Stress degradation study of drug substance/drug product provide the following important information. a) Determine the route of degradation of drug substance and drug product. b) Differentiate the degradation product which is generating from drug substance and with excipient interaction with drug substance in drug product. c) Help in the determination of structure of degradation product. d) To show the degradation mechanism such as hydrolysis, oxidation, thermolysis or photolysis of the drug substance and drug product [11]. e) Help in the determination of the stability-indicating nature of the method. f) To understand the chemical nature of the drug molecule. g) To get the possible information degradant in stability study of drug product [12].

Regents and materials
Marketed samples of Carvedilol tablets were used for degradation kinetic of carvedilol. Its associated related substances (impurities) were obtained from OLYMPUS Chemical and Fertilizers from Mumbai, India. Trifluroacetic acid and Acetonitrile were procured from spectrochemLimited, HPLC grade water was obtained from Milli-Q purification system. 0.45 µm PVDF filter used of Merck India make.

Instrumentation
HPLC (Make: Waters) equipped with an integrated autosampler and a quaternary gradient pump was used. The column holder having temperature controlled and an Ultraviolet (UV)/Photodiode array detector (PDA) was used for the development and analytical method validation. Chromatographic data was acquired using empower software.

Chromatographic conditions
Inertsil ODS3V (150 x 4.6) mm, 5 µm column was used. The column holder temperature maintained at 40 °C. The mobile phase consist of a different composition of buffer solution and organic solvents.
Mobile Phase-A is mixture of Water, Acetonitrile and Trifluroacetic acid in the ratio 80:20: 0.1 v/v/v and pH of this mixture adjusted to 2.0 with dilute potassium hydroxide solution.
Mobile Phase-B is mixture water and Acetonitrile in the ratio 10:90 v/v.
HPLC gradient programme run mentioned in table 2.

Standard solution preparations
Solution containing 2µg/ml of Carvedilolstandard prepared in diluent.

Controlled sample solution
Accurately weigh and transferred tablets powder equivalent to 25 mg of Carvedilol into 25 ml volumetric flask, Add about 20 ml of diluent sonicated for 30 min with intermittent shaking and make the volume with diluent. Filtered through 0.45 µm PVDF filter after discarding first five ml of filtrate. (Sample concentration: 1000µg/ml)

Sample preparation for force degradation study
Different degradation samples were prepared as mentioned below (table 3). They were analysed along with a controlled sample (nonstressed sample). %Degradation was calculated in terms of % total impurities (known and unknown impurities) along with Mass Balance. This solution was neutralized with 1N sodium hydroxide (NaOH).

Base degradation
Solution containing 1000 µg/ml of Carvedilolwas treated with 1N sodium hydroxide (NaOH). The degradation condition mentioned in table 3.
This solution was neutralized with 1N hydrochloric acid (HCI).

Oxidative condition
Solution containing 1000 µg/ml of Carvedilolwas treated with 5 % v/v hydrogen peroxide (H2O2). The degradation condition mentioned in table 3.

Thermal degradation study
Transferred tablets powder equivalent to 25 mg of Carvedilol into dry 25 ml volumetric flask and kept in oven, the degradation conditions mentioned in table 3.

Photolytic degradation study
Transferred tablets powder equivalent to 25 mg of Carvedilol into dry 25 ml volumetric flask and exposed light providing anoverall illumination of not less than 1.2 million lux hours and ultraviolet energy of not less than 200 Wh/m2 asperphoto stability testing guideline [29].

RESULTS AND DISCUSSION
Carvedilol molecule can be synthesized in many routes one of them is mentioned below [30].
Other synthetic route to synthesize Carvedilol molecule is mentioned below. Impurities in pharmaceuticals are the unwanted chemical that remain with the active pharmaceutical ingredients (APIs), or develop during formulation or upon aging of the both API and formulated APIs to medicines. The presence of these unwanted chemicals, even in small amounts, many influences the efficacy and safety of the pharmaceutical products. Impurity profiling (i.e. the identity as well as the quantity in the pharmaceuticals) is one of the most important aspect of quality of drug product.
During synthesis of carvedilol molecule by different rout yields many impurities. There are possibility of generation of many impurities, which are process-related and degradants impurities depending on process route, which is also mention in European and US pharmacopeia. The possible generation of impurities are mentioned below.
The chemical name of carvedilol impurities are shown in table 4.  Force degradation samples were analysed with a stability indicating method using PDA detector [31,32]. Peak purity results (purity angle<purity threshold) for anlaytes indicates the homogeneity and purity of Carvedilol peak and its related impurities. There is no interference observed for analyte peak and its known impurities form blank and placebo, proving the specificity of the method.
The fig. 8, fig. 9 and fig. 10 shows representative chromatograms of Blank, Placebo and spiked sample (with all known impurities). The Force degradation study (stress study) revealed that all the peaks i.e. Carvedilol peak, known impurities, unknown impurities and degradants, are well resolved from each other which is visiblyconfirmedin fig. 11 and fig. 12. Carvedilol can easily oxidised and form biproduct and this biproduct is Carvedilol impurity-A which increases in presence of oxygen. In fig. 11 it was observed that in oxidation treated sample known Impurity-A, unknown impurity at 1.78 RRT and in fig. 12 Alkali treatment sample known Impurity-D, unknown impurity at 1.78 RRT was formed as a major degradation impurity. In Acid, Humidity, Thermal and Photolytic conditions drug product found stable.
The % degradation with acceptable mass balance of Force degradation study (stress study) results reported in table 4.

CONCLUSION
Stress degradation studies provide vital information in developing new drug molecule or drug product. It give the necessary information of degradation pathway of the molecule which will play very important role in stability of drug product. In Drug product development by different synthetic route of API (Active Pharmaceutical Ingredient) was used; hence it is important to perform the stress degradation study to know different impurities profiling of known and unknown impurities. The degradants which is formed during stress degradation studies can be identified their structure which will give the information pertaining to toxic nature of degradants and can be controlled during the drug development stage. Stress degradation study also helps in deciding the manufacturing process, optimization of process parameter in different stages of drug product production, packaging condition, packaging material of drug product.

ACKNOWLEDGMENT
The author wishes thanks to management of Maulana Azad College of Arts, Science and Commerce for providing excellent research facilities.

AUTHORS CONTRIBUTIONS
All the authors have contributed equally.

CONFLICT OF INTERESTS
Declared none