LC–MS/MS method for the quantification of masitinib in RLMs matrix and rat urine: application to metabolic stability and excretion rate

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].

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 [13–15]. 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.

Optimization of chromatographic and mass spectrometric parameters

Several experiments were performed to attain the best mass response by optimizing all chromatographic and mass spectrometric parameters so as to enhance the resolution and sensitivity. Mobile phase system consisted of ACN and 0.1% formic acid in HPLC H₂O (pH ~ 3.2) with a ratio of (35:65, v/v) flowing at 0.25 mL/min. MST and IS were detected at retention times of 1.9 and 3.3 min, respectively. The run time of the current method was 5 min. MST and IS peaks were well resolved, with no carryover in any blank matrix (RLMs) sample or MST-free standard (blank + internal standard). A representative chromatogram of standard solutions of a calibration curve is shown in Fig. 2.

The positive MS scan showed molecular ion peaks at m/z 499 and at m/z 530 for MST and IS, respectively. Fragmentation of MST at m/z 499 gave one daughter ion of at m/z 399. Similarly, fragmentation of IS ion at m/z 530 gave daughter ions at m/z 113 and at m/z 141. Those ions were chosen for the MRM mode of MST and IS in the current method (Fig. 3).

Method validation

Metabolic stability study

After quenching the metabolic reaction using ACN at specific time intervals, concentrations of MST in RLMs matrix were computed. Plotting the ln of the % remaining MST (with respect to zero time) against incubation time was done as shown in Fig. 4. The first linear part of the curve showed a regression equation of Y = − 0.01378*X + 4.579 that was used for computing of in vitro t_1/2 [16] (where in vitro t_1/2 = ln2/slope). The slope was 0.01378 and therefore in vitro t_1/2 was found to be 50.38 min. Consequently, CL_int was computed following in vitro t_1/2 method [17] and found to be 3.11 ± 0.2 as shown in the following equation.

$$ CL_{int, app} = \,\frac{0.693}{{in vitro t _{1/2} }} . \frac{\text{mL incubation}}{\text{mg microsomes}} .\frac{{45\, {\text{mg microsome}}}}{\text{g liver}} .\frac{{20\, {\text{g liver}}}}{\text{kg per body weight}} $$

$$ CL_{int, app} = \frac{0.693}{50.4} . \frac{1}{1} .\frac{45}{12.5} .\frac{20 }{0.33} $$

$$ CL_{int, app} = 3 \,{\text{ml}}/{ \hbox{min} }/{\text{kg}} . $$

Excretion of masitinib in rat urine

Masitinib was found in rat urine after 6 h of dosing and began to decrease by time until almost disappeared after 96 h (Fig. 5). These concentrations equal to the % MST with respect to 6 h time (which represents 100%). The % MST in urine was plotted against collection time as shown in Fig. 5.