Chemicals
Vortioxetine reference substance was supplied from Lundbeck Pharm. Ind. The purity of the drug is 98.87%. A commercial preparation, Brintellix® film-coated tablets (produced by Lundbeck Pharm. Ind., Turkey, containing 10.0 mg of VRT per coated tablet) was assayed. Methanol, potassium dihydrogen phosphate, ammonium acetate, and acetonitrile of HPLC grade were received from Merck Chem. Ind. All reagents employed in this study were of analytical-reagent grade.
Apparatus
Instrument used in the study was Dionex Ultimate focused 3000-9 equipped with auto sampler and DAD. The wavelength of the UV detector was set at 225.0 nm. Peak areas were integrated by using the computer Chromeleon Client software program. Absorbance measurements were measured on a double beam UV-VIS spectrophotometer model UV-1601 by using quartz cells with 10.0 mm path length.
Chromatographic conditions
An isocratic mobile phase consisted of potassium dihydrogen phosphate buffer (pH:3.0 ± 0.05):methanol in the ratio (30:70 volumetric ratio) filtered and degassed through a membrane filter of 0.45 μm porosity before use. The analytical column was a Waters Symmetry RP-18 (3.5 μm, 100 cm × 4.6 mm) column. The flow rate of 1.3 mL min−1 and the detector was set at 225.0 nm. All determinations were made at 50 °C and the injection volume was 20.0 μL. The working solutions and mobile phases were prepared daily.
Construction of the calibration graph and linearity
For the HPLC method, the standard stock solution was prepared separately by dissolving VRT and caffeine (CAF) (internal standard) in the degassed mobile phase, respectively. Based on these stock solutions, the working standard solution, VRT, was prepared in a linear working range of 10.0-70.0 μg mL−1 and caffeine as internal standard at the amount of 20.0 μg mL−1 into 10.0 mL volumetric flasks using the mobile phase. Five replicate measurements were made for all concentrations. The injection volume of each sample is 20 μL. To calculate the concentration of VRT, the ratio of VRT to IS peak area was calculated. The linear working graph was constructed and the calibration equal was then stored. In the first derivative spectrophotometric method, Beer’s law range, linear regression equations, and correlation coefficients determined.
Validation parameters
Accuracy as recovery
The accuracy of the suggested method has quantified the percentage of analytical recovery of VRT by using the standard addition method. The recovery test was performed at three levels: 30.0, 40.0, and 50.0 μg mL−1 of the pure sample. Five replicate experiments were performed and recoveries (%), RSD (%) were found.
Precision
So as to control the precision experiment, intraday and inter-day experiments were evaluated. The method was found to be in precision at three different independent (low, medium, and high) amounts during the same day, and 5 different days. The relative standard deviations were found.
Sensitivity
The LOD and LOQ parameters were estimated as described at ICH guidelines by the formulas given below; where Sb is the standard deviation of the intercept of the linear line and a is the slope of the regression equation [22].
$$ LOD= 3.3\ {S}_b/a\kern1.25em ;\kern1em LOQ= 10\ {S}_b/a $$
Robustness
The robustness of the proposed RP-HPLC method was carried out by altering the experimental conditions such as analytical column, column temperature, flow rate, changing pH, and mobile phase. The robustness of the derivative spectrophotometric method was established by introducing small changes in experimental conditions like wavelength ± 1 nm. Making a deliberate change in wavelength was taken place and RSD of absorbance found to be less than 2, specify that the method was robust.
Degradation studies
Degradation studies were conducted to determine specificity and stability-indicating properties of the suggested method. That these stress conditions were performed under the following acidic, alkaline, oxidative, and photodegradation conditions. VRT (20.0 mg) and CAF (20.0 mg) were exactly weighed. The weighed active ingredients were taken into 100.0 mL volumetric flasks and diluted with the mobile phase and made up to its volume. The prepared stock solutions were used of forced degradation experiments.
For acidic or alkaline degradation studies, 1.0 mL of standard stock VRT solution was transferred to a 10.0-mL volumetric flask in the presence of IS. A total of 2.0 mL of 0.1 M HCl or 0.1 M NaOH solution was added and stirred. The volumetric flask was heated in the water bath at 80 °C for 1 h, cooled to room temperature, and neutralized with 0.1 M NaOH or 0.1 M HCl to pH 7.0. The above solution has diluted the volume with the mobile phase.
For the oxidation degradation study, 1.0 mL of standard stock solution of VRT in the presence of IS was transferred into a 10.0-mL volumetric flask. A total of 2.0 mL of 3.0 H2O2 solution was added and mixed well. The volumetric flask was heated in the water bath at 80 °C for 1 h, cooled to room temperature. The above solution has diluted the volume with the mobile phase.
For photodegradation study, analytically pure sample of VRT and CAF were transferred into a petri dish and exposed into the sunlight for 48 h. The solids were dissolved with the mobile phase.
Filter the degradation solutions with 0.45-μm syringe filters and place in vials of the RP-HPLC system for all degradation studies.
Application to pharmaceutical formulation
At first, ten tablets of Brintellix were weighed and finely powdered in a mortar. The contents were diluted with the mobile phase. Quantity of one tablet weigh was weighed and diluted with the mobile phase in a 100.0-mL volumetric flask. The volumetric flask was shaken for 20.0 min by an ultrasonic shaker. The excipient was separated by filtration. After the filtration process, the appropriate volume of filtered solution and IS was transferred into a 100.0-mL volumetric flask and completed with the mobile phase. All solutions were filtered through 0.45 μm Millipore membrane filters before injections into the HPLC system. The concentration of VRT was calculated by calibration graph prepared with stock solution.