Materials and reagents
BQF was purchased from Clearsynth labs, Mumbai, India, for the study purpose. HPLC grade methanol and analytical grade acetic acid, hydrochloric acid (HCl) and sodium hydroxide (NaOH) were used in the study and purchased from Merck India Ltd. Ammonium acetate was obtained from Himedia, Mumbai, India. Triple distilled water was obtained from in house Milli-Q water purifier system (Merck, Prague, Czech Republic) which was used for the preparation of mobile phase and other solutions.
Instrumentation
The liquid chromatographic system used in the present work was consisted of Waters 1525 binary pump system with inline degasser, Waters 2707 auto sampler, column oven, Waters 2998 Photodiode Array (PDA) detector and the EMPOWER 2.0 software for data acquisition and processing (all Waters Corporation, Milford, MA, USA). The analysis was performed at 40 °C column oven temperature on a Sunfire C18 column (250 mm × 4.6 mm, 5 μm particle size). A Sunfire C18 analytical guard column (12.5 mm × 4.6 mm, 5 μm particle size) packed with the same sorbent was connected before the analytical column. The isocratic mode was employed for all the experiments.
Chromatographic conditions
The mobile phase consisted of 10 mM ammonium acetate buffer as aqueous phase (A) and methanol as organic phase (B). The aqueous phase was maintained at pH 4.5, adjusting with acetic acid and filtered through 0.45 μ membrane filter by applying vacuum. Both the phases were degassed prior to use by applying ultrasonication for 10 min. Isocratic mode was employed for the elution of analyte (BQF) in the ratio of 15:85 v/v (A:B), and flow rate was set at 1.2 ml/min. Elution was performed on Sunfire C18 column with dimensions of 250 × 4.5 mm and 5 μm particle size. The column oven temperature was set at 40 °C. The analyte detection was carried out using PDA detector with spectral range of 210 to 400 nm, and chromatogram was extracted at 226-nm wavelength. The injection volume was set to a constant volume of 10 μl for each injection.
Preparation of standard stock solution and calibration curve
Stock solution of 1 mg/mL was prepared by solubilising 6.1 mg of BQF (equivalent to 5.0 mg of free base) in 5 ml of HPLC methanol. The prepared stock was kept in a refrigerator at 2 to 8 °C till it is being used. The standard solutions were prepared by diluting the stock solution with methanol to reach concentration range 10–100 μg/mL for BQF. These standard samples were considered at eight different levels, i.e. 10%, 25%, 50%, 75%, 100%, 125%, 150% and 200% of the target concentration (50 μg/mL).
Method validation
The method validation protocol was set on the basis of recommendations provided by the USFDA and ICH guidelines.
System suitability
The verification of the resolution and reproducibility was evaluated by performing the system suitability test. The standard concentration of 10 μg/mL in methanol was injected in six replicates (n = 6). The RSD of retention time and the area was determined and compared with the ICH criteria for the system suitability.
Linearity
The linearity of the BQF was obtained by making the calibration standard curve in HPLC grade methanol. The calibration standards were prepared as mentioned previously in four replicates (n = 4), duplicate on day 1 and duplicate on day 2 in order to investigate the interday variability. The samples were injected to HPLC covering the range of 10 to 100 μg/mL. The peak area obtained from each standard was plotted against the concentration, and linearity equation along with regression coefficient (r2) was obtained. The linearity was evaluated by linear regression analysis.
Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ are denoting ability of the method to reliably detect and quantitate the smallest amount of analyte, respectively. The LOD and LOQ of the proposed method were calculated by using standard deviation of response and slope of regression line by using the following equations.
where σ is the standard deviation of response and s is the slope of regression line.
Accuracy
Accuracy of the developed analytical method was evaluated by injecting the analyte at three different levels of target concentration (80%, 100% and 120%) as per the ICH guideline. The samples at each level were prepared in six replicates (n = 6), and recovery was calculated from the obtained peak area using linearity equation.
Precision
The repeatability and intermediate precision were determined by considering the injection of analyte at three levels (low, medium and high, i.e. 10, 50 and 100 μg/mL, (n = 3)) on three different days. The concentrations were calculated from standard curve, and results were noted as relative standard deviation (RSD).
Specificity
The specificity of the method was established by injecting the blank sample without analyte, and the interference at the retention time (tR) of analyte was investigated. The blank methanol was injected in triplicate to evaluate whether there are any other interfering substances eluting at the tR of analyte.
Robustness
The robustness of the method was studied by deliberately changing in previously mentioned parameters. The standard concentration of 10 μg/mL was injected in triplicate, and the change in RSD was observed.
Mobile phase composition
The effect of changes in the mobile phase composition was evaluated by changing the composition by ± 3% from the actual composition (15:85% v/v of A:B).
Column temperature
The robustness of the method to the change in column temperature was evaluated to see its effect on the peak retention and peak area. The temperature was varied at points includes 1 °C above and 1 °C below fixed method temperature (40 °C). The variation in room temperature and the area was reported as RSD.
pH of mobile phase
The method sensitivity for change in pH of the mobile phase was evaluated to check the robustness of the developed method. The pH of the mobile phase was varied by ± 0.1 from the actual method pH (4.5).
Flow rate
The method sensitivity for change in flow rate was also evaluated. For this purpose, the flow rate was varied by 0.1 ml/min above and below the method actual flow rate (1.2 ml/min). The results are reported as the RSD in peak tR and peak area.
Accelerated degradation studies
All degradation experiments were performed in solution state at a drug concentration of 50 μg/mL, and injections were made to HPLC (10 × dilution). For acid-alkali degradation, 5 ml of stock solution of 50 μg/mL analyte in 0.5 N HCl and 0.5 N NaOH was prepared in three replicates and sonicated for 2 h followed by injection in HPLC (after dilution in mobile phase). For photo and thermal decomposition of analyte, 50 μg/mL stock solutions was prepared 0.01 N HCl (official dissolution media) and in methanol in triplicates. The stocks were exposed to direct sunlight for 12 h for photo stability and to 80 °C for thermal stability evaluation for 4 h, respectively. The percent degradation of BQF was calculated from the linear equation.
Stability study
The short-term stability study was performed to evaluate the stability of analyte in official dissolution media (0.01 N HCl) and in methanol up to 72 h at room temperature. The photo and thermal stability were also investigated in the same solvents by keeping the samples protected from normal daylight and keeping sample at refrigerator temperature (2 to 8 °C) respectively. For this, three stocks of 50 μg/ml were prepared in 0.01 N HCl and in methanol (normal daylight exposed, protected from daylight and freeze sample) directly from the 1 mg/ml methanolic stock solution (n = 3). The samples were withdrawn from each stock at a predetermined time period (1 h, 2 h, 4 h, 8 h, 24 h, 28 h and 72 h), diluted in HPLC grade methanol and injected in HPLC for quantification.