Instruments
Chromatographic analysis of DEH and DEC was performed employing “Waters HPLC system” furnished with quaternary pump, autosampler, column oven and degasser. UV spectrometric detection operated at 265 nm was used for DEH and DEC detection and quantitation. Hardware regulation, data acquisition and management were done using “Waters Empower 2” software.
Pure samples
Reference standards of DEH (99.8% purity) and DEC (98.4% purity) were gifted by “Rainbow Pharma Training Labs”, Hyderabad.
Formulations
Colicare Tablets (“Omega Pharmaceuticals Pvt Ltd”, Chennai) with labelled content of DEH 10 mg and DEC 40 mg were analysed employing the developed and authenticated SILC method.
Chemicals and reagents
K2HPO4, H2O2, HCl and NaOH of analytical grade were from “Finar Chemicals Limited”, Ahmedabad; acetonitrile and Millipore water of chromatography grade were purchased from “Merck India limited”, Mumbai, and “Loba chemicals limited”, Mumbai, respectively.
SILC method conditions
“XTerra C18 column (250 × 4.6 mm, 5 μm)” was applied for chromatographic separation of DEH and DEC using 0.1 N K2HPO4 (55% vol. ratio; pH 4.5) and acetonitrile (45% vol. ratio) in an isocratic elution mode. Separation of DEH and DEC was done at a stream rate of 1.0 mL/min with an injection volume of 10 μL with column warmth of 27°C.
Stock DEH and DEC solution
Quantities equal to 40 mg DEC and 10 mg DEH reference standards were appropriately measured and properly transferred to the 100-mL flask, and 40 mL mobile phase was introduced to the same flask. The solution was well shaken and flask volume was completed with the same mobile phase to achieve 400 μg/mL DEC and 100 μg/mL DEH.
Working DEH and DEC solution
The working DEH and DEC solutions were prepared by appropriate dilution of their formerly prepared stock DEH and DEC solution (400 μg/mL DEC and 100 μg/mL DEH) with the mobile phase solvent to obtain a 10-mL working solution of 40 μg/mL DEC and 10 μg/mL DEH.
Calibration curve
Quantity ranges covering 5 to 15 μg/mL for DEH and 20 to 60 μg/mL for DEC were made from stock DEH and DEC solution (400 μg/mL DEC and 100 μg/mL DEH) using the mobile phase solvent. The solutions were evaluated using the SILC technique proposed. Calibration curves were prepared with a regression line equation to map each analyte’s peak area against its concentrations.
DEH and DEC content evaluation in tablets
Ten Colicare tablets containing a specific of DEH (10 mg/tablet) and DEC (40 mg/tablet) were taken and were crushed to a finer powder with the pestle and mortar. A weighed quantity of the tablet formulation crushed powder, corresponding to 10 mg of DEH and 40 mg of DEC, was transferred properly to a 100-mL flask. Forty milliliters of mobile phase was introduced to the same flask and was sonicated for nearly 20 min and then filtrated using Whatman no. 1 paper. The flask volume was completed with the same mobile phase to achieve 40 μg/mL DEC and 10 μg/mL DEH. The sample for analysing Colicare tablets was made ready by diluting an aliquot (1 mL) of tablet formulation sample to 10 mL with the mobile phase. The tablet formulation test solution was evaluated using the SILC technique proposed.
Forced degradation study
Forced degradation tests were made on tablet formulation samples of 400 μg/mL DEC and 100 μg/mL DEH concentrations under “ICH Q1A (R2)” given conditions [26].
Sonicating tablet formulation samples (10 mL) at 27 °C in 0.1 N HCl (10 mL) for 30 min and in 0.1 N NaOH (10 mL) for 30 min were used for degradation tests in acid and base stresses, respectively. Peroxide facilitated oxidation was studied by exposing the tablet formulation sample (10 mL) to 30% H2O2 (10 mL) for 30 min at 27 °C in the dark. Photolytic degradation was executed by exposing the tablet formulation crushed powder, corresponding to 10 mg of DEH and 40 mg of DEC, to the sunlight directly for 24 h. Similarly, for thermal stress, the tablet formulation crushed powder was exposed at 105°C for 30 min in the oven.
Each sample was properly diluted by the mobile phase at the end of exposures to acid, H2O2 and base, to achieve an ultimate amount of 40 μg/mL DEC and 10 μg/mL DEH. The solutions from the tablet formulation crushed powder exposed to sunlight and dry heat were made as portrayed in the section “DEH and DEC content evaluation in tablets”. All the sample solutions were evaluated using the SILC technique proposed.