Working standards of pharmaceutical-grade DOX were obtained as a generous gift from Cipla Pharmaceuticals Pvt. Ltd., Mumbai, India. The purity of DOX was found to be 99.9% on a dried basis. Restavit tablet label claim: each tablet contains 25 mg of DOX used for the study. Sodium hydroxide, hydrochloric acid, and hydrogen peroxide were purchased from Qualigens Fine Chemicals (Glaxo Ltd.) for the study. HPLC grade acetonitrile, water, phosphate buffer, and methanol were used in the study and purchased from Merck (Darmstadt, Germany).
Instruments
The modular HPLC system is equipped with Waters 510 HPLC pump (Waters Chromatography Division, Milford, MA, USA), a Rheodyne injector (20 μl), and solvent degasser with PDA 6000 LP detector. The chromatographic partition was accomplished on a Kromasil C18 (250 mm × 4.6 mm and 5-μm particle size) column. The eluent was monitored using a photodiode array (PDA) detector. The ultrasonic bath (Biomedica, India) was used for the extraction of the drug from the tablets. A precision water bath equipped with an MV controller (i-therm, Biomedica, India) was used to execute preferred reactions in solution over stress degradation study. A thermal stability study was implemented in a dry air oven (Biotechnics BTI–20D, Mumbai, India). Other equipments used were sonicator (Biomedica, India), analytical balance (Shimadzu AUX 220, Japan), and auto pipettes (Eppendorf, Hamburg, Germany).
Preparation of standard solution
To prepare a stock solution of DOX, weigh exactly 50 mg of DOX and transfer into a separate 50-ml volumetric flask containing 25 ml of mobile phase with continuous stirring. The retrieved transparent solution was ultrasonicated for 10 min after that volume was made up with the remaining mobile phase to get a stock solution of 1000 μg/ml. From this stock solution, sample aliquots (0.1, 0.2, 0.3, 0.4, 0.5 ml) were withdrawn and diluted in a 10-ml volumetric flask separately and made up the volume up to the mark with mobile phase to achieve the final concentration (10, 20, 30, 40, and 50 μg/ml) for DOX. All the volumetric flask used for analysis was covered with an aluminum foil against light preservation. Furthermore, the solution was kept at 4°C for 1-week condition and found stable. Additionally, the marketed formulation was transferred to a 50-ml volumetric flask containing a small volume of mobile phase (10 ml) and stirred to extract out drug; further volume of the flask was made up with mobile phase to get a final concentration of 500 μg/ml. Subsequently, from the groomed stock solution, aliquots (0.2, 0.4, 0.6, 0.8, and 1 ml) were withdrawn and transferred to a 10-ml volumetric flask to attain a closing concentration.
Forced degradation studies on API and marketed formulation
A stress degradation study of marketed formulation and API was executed to arbitrate the stability of the developed method under different conditions using placebo and blank samples. All stress experiments were performed at 25-μg/ml concentration subjected to suitable dilution at periodical withdrawn followed by filtration of the sample before injection in the chromatographic system.
Degradation study
For acid, alkali, neutral oxidation, 25 μg/ml analyte in combination with methanolic solution, 2 M hydrochloric acid, and 2 M NaOH; water; and 6% H2O2 (20:80) were prepared in three replicates and solution reflux for 8 h at 70°C.
Photolytic degradation
For photolytic degradation analysis, 50 mg marketed formulation (tablets) and API were transferred in a petri dish and kept in a UV cabinet by exposing the intense UV radiation of shorter and longer wavelength for 48 h. After the closure time interval, stressed samples were diluted with mobile phase to obtain a final concentration of 25 μg/ml and then a volume of 20-μl solution was injected into the system.
Development of the analytical method
A chromatographic separation of API and tablet formulation from its degradation products was achieved using an isocratic mobile phase consisting of phosphate buffer (pH 3.5) and methanol in the ratio 45:55 v/v followed by filtration (0.45-μm nylon) and degassed before use. All determination at 20-μl volume with constant 1-ml flow rate was performed and eluents were measured using a PDA detector at 262 nm.
Assay
Twenty tablets were weighed and tablet powder equivalent to 12.5 mg of DOX was diluted with 15ml of mobile phase in a volumetric flask and sonicated for 20 min for complete dissolution. Then, the volume was made up to 50 ml after filtration. Further, the 1-ml filtrate was diluted to 10ml with mobile phase in a volumetric flask and then filtered through a 0.45-μm nylon filter to yield the final concentration (25 μg/ml).
Isolation of alkaline degradation product
A weight equivalent to 100 mg of DOX was transferred to a volumetric flask containing 20 ml methanol and stir to get a clear solution. The final volume of 100 ml was achieved by subsequent addition of 2.0 M NaOH. The reflux assembly of the round bottom flask containing the resultant mixture was maintained at respective temperature along with a precision-controlled water bath at 70°C for 5 h. Under the alkaline set degradation condition, DOX was studied and evaluated by the proposed method wherein the majority of alkaline degradant isolated employing preparative column chromatographic technique. The scheme of qualitative estimation of DOX and alkaline degradant is accomplished under the alkaline stress condition shown in Fig. 2.
Validation of the developed analytical method
The recommended analytical approach was validated conferred to ICH guidelines. The system was checked for any obstructive substance or backdrop noises at the retention time of DOX found establish to a tolerable range of < 20%. A standard linearity plot was constructed by making the appropriate concentration of 10–50 μg/ml range. The precision study was performed with six replicate injections of the same concentration considering system error as the parameter of the precision experiment. All the retrieved findings were revealed as percent relative standard deviation (% RSD). The interday and intraday experiment was executed for three replicates for the same and on a different day. The accuracy of the current method was assessed through fortifying marketed formulation of three concentrations. Later, to estimate method’s specificity, the resolution factor amidst peak area and nearest resolving peaks were considered. The peak purity of the entire peaks along with degradant using diode array (DAD) detector was checked for its selectivity (www.ich.org/fileadmin/public_web_site/ich_products/guidelines/quality/q1a_r2/step4/q1a_r2_guideline.pdf).