Chemicals and reagents
All chemicals unless otherwise stated were mentioned in a previous published article [8]. Masitinib and bosutinib were procured from LC Laboratories (Woburn, MA, USA). Preparation of RLMs was done in-house using Sprague Dowely rats [9].
Chromatographic conditions
Chromatographic separation for the MST (analyte) and bosutinib (IS) was done using Agilent LC–MS/MS (6410 QqQ) which has been described in detail elsewhere [8]. Reversed phase liquid chromatography was used for separation of MST and bosutinib (IS) using C18 (50 mm × 2.1 mm, 1.8 μm). Binary solvent system consisted of 35% solvent A (0.1% formic acid in H2O, pH: 3.2) and 65% solvent B (acetonitrile) used as mobile phase at flow rate of 0.25 mL with a total run time of 5 min. Injection volume was 5 µL. Temperature of the column was fixed at 22 °C. Mass spectrometric parameters and chromatographic conditions for MST and bosutinib were optimized to accomplish the best resolution in a very short time and the highest response. Nitrogen generator was adjusted to generate low purity nitrogen for electrospray ionization (ESI) source (drying gas) at a flow rate of 12 L/min and high purity nitrogen for collision cell (collision gas) at a pressure of 50 psi. ESI source temperature and capillary voltage were adjusted at 350 °C and 4000 V respectively. Agilent triple quadrupole was controlled by Mass Hunter software. Multiple reaction monitoring (MRM) of the transition 499 → 399 for MST and 530 → 113 and 530 → 141 for bosutinib (IS) data was used for quantification. Fragmentor voltage was set to 135 V with collision energy 20 for MST and 135 and 140 V with collision energy of 15, 20 for bosutinib.
Preparation of working standard solutions
Concentration of 1.0 mg/mL of MST was dissolved in DMSO then 1 mL of this solution was diluted ten fold using mobile phase to prepare working standard 1 (WS1, 100 µg/mL). One milliliter WS1 was diluted tenfold using mobile phase to prepare working standard 2 (WS2, 10 µg/mL). Concentration of 0.1 mg/mL of IS was prepared in DMSO then 200 µL of this solution was diluted 50-fold using mobile phase to prepare working IS (WI, 2 µg/mL).
Calibration curve and sample preparation
Proper volumes of MST WS2 (10 µg/mL) were diluted with a suitable volume of RLMs matrix to prepare eight concentrations: 5, 10, 15, 20, 50, 100, 150 and 200 ng/mL in a final volume of 1 mL. Fifteen, fifty and one hundred fifty were chosen as low quality control (LQC), medium quality control (MQC) and high quality control (HQC), respectively. Two milliliter of ACN were added for protein precipitation. Centrifugation (14,000 rpm, 12 min and 4 °C) was done to remove precipitated proteins. Supernatants were detached and filtered using 0.22 µm syringe filters. Fifty microliter of WI solution was added to 1 mL of calibration standards. Blank samples were prepared in the same procedure without adding MST. Blank samples were tested to confirm the absence of any interference with MST and IS at their retention times. Calibration curve was created for spiked RLMs samples by plotting the peak area ratio of MST to IS (y axis) against MST nominal concentrations (x axis).
Validation of the method
Validation of the current method was done following the guidelines of FDA [10] and the general recommendations of ICH [11, 12].
Specificity
Six separate blank RLMs matrix samples were treated with the previously mentioned extraction technique. Chromatograms were screened for any interferences peaks at retention times of MST or IS, MRM mode in the mass detector was used to raise the specificity of the current method. Post time (2 min.) and injector washing were used to minimize carryover effects.
Linearity and sensitivity
Evaluation of the linearity and sensitivity of the current method was done using six calibration curves. Statistical least square method was used to analyze the data. ICH guidelines were used for calculation of LOD and LOQ [11], based on the intercept standard deviation and slope of the calibration plot through the following equation.
$$ LOQ\;OR\;LOD = mS{\big/}n$$
where m equals 3.3 for LOD and 10 for LOQ, S is the standard deviation of the intercept, and n is the slope.
Precision and accuracy
The basis of calculation of intra-day and inter-day precisions and accuracies was the analysis of RLMs matrix samples’ spiked with MST at QC levels in 1 day and three consecutive days, respectively. Percentages error and percentages relative standard deviation were utilized for expressing accuracy and precision. The equation of calculations were mentioned Percentages RSD = (SD/Mean) ×100 and percentages error = [(average measured concentration − expected concentration) / expected concentration] ×100].
Stability
For assessing MST stability in RLMs matrix, six replicates of MST QC samples were analyzed under different storage circumstances. Freshly prepared RLMs calibration curves were used for computing of accuracy and precision values were carried out using. Eight hours of MST QC samples storage at room temperature was used to evaluate MST bench-top stability. Three freeze–thaw cycles were used to estimate MST stability of spiked QC samples after freezing them at – 80 °C and thawing them at ambient temperature. Evaluation of MST stability was done by analyzing the spiked QC samples that were kept at 4 °C for 1 day and stored − 20 °C for 1 month.
Metabolic stability of MST
After incubation with RLMs matrix, tracking the decrease in MST concentration was performed utilizing the current method. Incubations were done for 1 µM MST with 1 mg/mL microsomal proteins, and 1 mM NADPH in phosphate buffer (pH 7.4) containing 3.3 mM MgCl2 in 1 mL. NADPH was used to initiate the metabolic reaction and 2 mL of ACN was used to terminate it. Metabolic reaction was terminated at specific time intervals (0, 2.5, 5, 10, 15, 20, 40 and 50 min). Centrifugation (14,000 rpm for 12 min at 4 °C) was done to remove precipitated proteins. Supernatants were filtered using 0.22 µm syringe filter. The filtered samples were diluted twofold with mobile phase to make the final conc. of MST in the linear dynamic range of the current method. One milliliter of the diluted solution was transferred to HPLC vial then IS WI (50 µL) was added. Injection volume was 5 µL into the LC–MS/MS system. Concentrations of MST in RLMs matrix were computed from the regression equation of freshly constructed calibration curve using peak area ratios of MST and IS.
Rate of MST excretion in rat urine
The current method was also applied to measure MST in rat urine at specific time intervals after single oral dose of 33.3 mg/Kg using oral gavage. Six Male Sprague–Dawley rats were brought from college of pharmacy animal house, King Saud University (Riyadh, KSA). Rats were housed individually in special purpose cages. Masitinib was dissolved in (4% DMSO, 30% PEG 300, 5% Tween 80, HPLC H2O) for oral dosing of rats. Dose of masitinib in rats were calculated using a specific conversion equation of drugs between animals [13,14,15]. Kinavet-CA1 dose in dogs was 10 mg/kg. So the dose for rat were 33.3 mg/Kg. Blank urine was collected before MST administration. Urine samples were collected at 6, 12, 18, 24, 48, 72 and 96 h. following masitinib dosing and then filtered over 0.45 µm syringe filters for removal of particulate matters in the urine. Two milliliter were taken from each sample, equal amount of ACN was added and then strongly shaken for 1 min and the mixture was stored at 4 °C overnight. Two solvent layers were formed, an upper ACN layer and a lower aqueous layer. Upper ACN layer was diluted twenty timed with mobile phase to make the final conc. of MST lied in the linearity range. IS (50 µL) was added to 1 mL of the diluted solution. Five microliter of this solution was injected into the LC–MS/MS system. Control urine samples obtained from rats prior to drug dosing were prepared in a similar way to the above mentioned method.
