Chemicals and reagents
Efonidipine hydrochloride was provided as a gift sample by Bajaj Healthcare Ltd. Eudragit EPO was gifted by Evonik Industries (India). HPLC grade acetonitrile was purchased from Merck, Germany, and orthoboric acid was purchased from Avantor pharma, Mumbai. Before use, phosphate buffer solution (pH 2.5) was prepared using deionized water (Milli-Q®, Moslheim, France). Other chemicals like analytical grade citric acid were purchased from SD Fine Chemicals Ltd.
Preparation of solid dispersions
Solid dispersions of EFO were prepared using 30% of the drug, 60% Eudragit EPO, 10% CA (SD1), and the other without CA (SD2). The premixed drug and polymeric carrier were sifted through #60 and blended with a weighed amount of plasticizer. The prepared mixtures were processed at a temperature of 120 ± 2 °C and speed of 100 rpm on a twin screw hot-melt extruder (Thermo Scientific HAAKE MiniCTW, Germany). The extrudes of 0.5 mm diameter die were collected, dried, powdered, and subjected for further studies.
Method development
Instrumentation
The chromatographic separation was accomplished on a Jasco AS-2055 plus series intelligent sampler (Jasco, Tokyo, Japan) equipped with an automatic sample injector, Jasco MD-2018Plus Intelligent HPLC pump, photodiode array (PDA Detector), rheodyne injection valve, online degasser, and Agilent Eclipsed XDB-C18 column (4.6 × 250 mm) packed with 5 μm particles. Milli-Q water was obtained by the Millipore system (Moslheim, France). Sonicator was from Enertech fast ultrasonicate cleaner, Mumbai, and vacuum pump model no TID-15 of Conformite Europeenne with a vacuum of 22 mmHg.
Chromatographic conditions
The optimized composition of the mobile phase was set in the ratio 85:15 (v/v) of HPLC grade acetonitrile and phosphate buffer (pH 2.5), respectively, under the gradient method of the elution process. The flow rate of the mobile phase was 1.2 ml/min with an injection volume of 50 μl, and the detection wavelength was kept at 252.0 nm as the λmax. The column equilibration was carried out for 15 min before the start of the analysis. All experiments were conducted at a temperature of 25 ± 2 °C. The total peak area of the drug was used to estimate the concentration of EFO. Chromatograms were recorded using ChromNAV software. These conditions were inspected to achieve an easy to use, cost-effective, and reliable procedure with a better peak resolution for symmetry, tailing factor, and early retention time.
Preparation of stock solution and standard solutions
One hundred micrograms/milliliter of the stock solution was prepared by dissolving 10 mg of EFO in 50 ml of HPLC grade acetonitrile in a 100-ml volumetric flask. This drug solution was sonicated for 15 min to solubilize the remaining drug particles. The final volume of the solution was made up of HPLC grade acetonitrile. The working standard solutions were prepared by diluting the stock solution of concentration 100 μg/ml using HPLC grade acetonitrile in the range of 2.5–100 μg/ml.
Method validation
The developed chromatographic method was validated according to the International Council for Harmonization (ICH) guidelines Q2 (R1) [14].
System suitability
System suitability test is performed to demonstrate the suitability of the system for the analysis. The fresh standard stock solution of EFO was prepared to check for the suitability of the method. The sample solution of concentration 100 μg/ml was injected six times at regular short time intervals. The peak area, retention time, resolution, theoretical plate, and tailing factor were recorded as the system suitability parameters using the software under optimized chromatographic conditions to examine the suitability of the system. The results were analyzed by determining the standard deviation of all the parameters of the system suitability study. The acceptance criteria and the limits for RSD were in concordance to USP limits [15,16,17].
Linearity and range
The method was validated for linearity by injecting the sample solutions of concentration 2.5, 5, 10, 25, 50, and 100 μg/ml for analysis. Each sample solution was injected three times. The peak area of each solution was recorded at λmax of 252.0 nm. The peak area corresponding to each concentration was determined from the recorded chromatograms. The graph of peak area on the y-axis versus concentration on the x-axis was plotted. The results were recorded for the regression coefficient (R2), the slope of the regression line, and the y-intercept. The R2 > 0.999 was set as the acceptance criteria for the linearity of the developed method [16].
Method precision
The intermediate precision (inter-day precision) and repeatability (intra-day precision) of sample solutions were examined to check whether the developed method shows precise results on repeated samplings at any point in time. Standard solutions of EFO of concentrations 2.5, 5, 10, 25, 50, and 100 μg/ml were prepared and injected into the HPLC system. The intra-day precision study was carried by injecting each concentration of the sample solutions of EFO in triplicates on the same day. The inter-day precision study was carried by injecting each concentration of the sample solutions of EFO on three consecutive days over 2 weeks. The peak area of all six solutions, detected at a λmax of 252.0 nm, and % relative standard deviation (RSD) was calculated from the recorded chromatograms. The acceptance criteria for the %RSD was kept as ≤ 2% [16, 18].
Robustness
Robustness is the measure of sustainability, i.e., to remain unaffected by deliberate variations in the essential parameters of the chromatographic conditions and also provide knowledge on the implications of its variations. In the current robustness study, the two parameters mobile phase ratio and flow rate were considered for minute deliberate changes in the method, to check whether the changes majorly affect the peak areas and the retention time. The selected two parameters were studied at three different levels, i.e., 85 ± 2:15 ± 2 v/v and 1.2 ± 0.2 ml/min for mobile phase ratio and flow rate, respectively, to verify the robustness of the proposed HPLC method. The constant standard solutions of 10 μg/ml were prepared and injected for the evaluation of the parameters. The standard deviation of the peak areas and the retention time were calculated from the recorded chromatograms.
Accuracy (percentage recovery)
Accuracy is defined as the closeness of the test value of the method in comparison with the expected value. It is calculated in terms of percentage recovery of the analyte using the following equation [16].
$$ \mathrm{Percentage}\ \mathrm{recovery}=\frac{\mathrm{Recovered}\ \mathrm{concentration}\ }{\mathrm{Injected}\ \mathrm{concentration}} \times 100 $$
The percentage recovery of the EFO was determined by spiking a known amount of the drug into the sample solution. The known concentration solutions of 20 μg/ml and 45 μg/ml were added to the sample solution of 5 μg/ml to obtain the final concentrations of 25 μg/ml and 50 μg/ml. The amount of EFO was estimated by measuring the peak area from the recorded chromatograms. Each sample concentration was injected in triplicate to estimate the percentage recovery of the EFO in the spiked samples. The acceptance criteria for the percentage recovery is 98–102%.
Limits of detection (LOD) and limits of quantitation (LOQ)
The lowest concentration of an analyte, which can be identified specifically from the background noise levels, is known as the limit of detection (LOD). The minimum amount of the analyte, which can be quantified effectively with precision and accuracy using the developed method, is known as the limit of quantification (LOQ). LOD and LOQ were determined from the standard deviation of the intercept and the slope of the calibration curve. The following equation as per ICH guidelines was then applied to determine the values:
$$ \mathrm{LOD}=3.3\times \sigma /\mathrm{S};\mathrm{LOQ}=10\times \sigma /S $$
where σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve [18].
Specificity
Accurate determination of the drug in the presence of other excipients without any interference is termed as specificity. The excipient interferences in SD formulations were evaluated during the analysis, and the results were compared with the placebo batches to determine the specificity of the developed method for EFO.
Assay
The prepared SD of EFO was examined for the drug content using the developed and validated HPLC method. The formulation equivalent to 10 mg of EFO was weighed accurately in the volumetric flask. A solution equivalent to 10 μg/ml concentration was prepared and quantified using the developed HPLC method. The experiments were performed in triplicate.
Stability of the analyte solution
The drug in the stock solution was evaluated by preparing 100 μg/ml solutions in acetonitrile. The test solutions were kept in tightly sealed vials at room temperature for 24 h. The solutions were analyzed by using the developed HPLC method for drug stability. The measured values were computed to determine the %RSD values.
Characterization of the SD
In vitro dissolution study
The dissolution experiments were performed in the 6.8 pH phosphate buffer at a temperature of 37 °C and the paddle speed of 75 rpm in USP apparatus II. The dissolution experiment was conducted on the developed SDs and EFO. The SDs equivalent to 20 mg drug and the pure drug were weighed accurately and added to each vessel. The dissolution behavior of each sample was investigated in triplicates. Samples withdrawn at predetermined intervals were filtered through a 0.45-μm Whatman filter. The amount of drug dissolved from the pure form of the drug and the SD was determined using the developed HPLC method.
Powder X-ray diffractometry (PXRD)
Powder X-ray diffraction patterns for EFO and the selected SD was studied using X-ray diffractometer (XRD) Shimadzu XRD 6100 (Japan). Cu-Kα X-ray source was used with λ = 1.54 °A, and the scanning rate was monitored at 2°/min. The samples were measured with a voltage of 40 kV and a current of 20 mA. Each sample was scanned over a diffraction range of 2° to 60°.
Differential scanning calorimetry (DSC)
The thermal analysis and the drug-polymer interactions of the pure EFO and selected SD formulation, respectively, were carried out using DSC-PYRIS-1, Perkin Elmer, USA. The experiments were performed in a dry nitrogen atmosphere. The accurately weighed 5-mg samples were heated at the rate of 10 °C/min under nitrogen purge (20 ml/min) from 30 to 300 °C/min. An empty crimped aluminum pan was used as a reference cell.
