2.1. Chemicals and solvents
Sunitinib was supplied by Dr. M.V. Reddy (Fels Institute for Cancer Research, Temple University, Philadelphia, PA, USA). Ammonium hydroxide (~ 5N), ammonium acetate, acetic acid, camptothecin and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). HPLC-grade acetonitrile, methanol and MTBE were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Deionized water (~18MΜ) (Nanopure deionization system, Barnstead/Thermolyne, Dubuque, IA, USA) was used for all aqueous solutions.
2.2. Preparation of stock solution, calibration standards and quality control samples
Stock solutions of sunitinib and camptothecin (the internal standard (IS)) were prepared separately in DMSO at a target concentration of 1 mg/mL as free base and diluted in methanol to create stock working solutions of sunitinib at a concentration of 0.4 mg/ml and the IS at concentrations of 20 and 1000 ng/ml. The stock working solution of sunitinib was then used to prepare calibration standards and quality control (QC) samples in individual biological matrices.
Blank plasma, normal brain and brain tumor samples were obtained from untreated nude mice bearing intracerebral U87 human glioma xenografts. To each gram of normal brain and brain tumor tissue was added 3 and 5 mL of deionized water, respectively. Tissue homogenization was carried out using a Polytron PT2100 homogenizer. The same matrix from all untreated animals was pooled and used as the control matrix for preparation of standard curves and QCs.
Calibration standards were prepared by spiking mouse plasma, normal brain and brain tumor homogenate with the stock standard working solution, which was further diluted with the matched-matrix to give seven calibration standards in the concentration range of 1.37 - 1000 ng/mL for plasma, and six calibration standards in the range of 4.12 - 1000 ng/g for the normal brain and brain tumor. Similar to calibration standards, QC samples were prepared in replicates (n = 3 and 5 for the intra-day and inter-day validation, respectively) at three concentration levels representing the entire range of concentrations (1.37, 111.1 and 1000 ng/ml for plasma 4.12, 111.1 and 1000 ng/g for normal brain and brain tumor).
2.3. Extraction procedure
Sunitinib and the IS were isolated from plasma using protein precipitation. To 10 μL of plasma sample aliquots were added 10 μL of the IS solution (20 ng/mL of camptothecin in methanol) and 20 μL of methanol containing 0.1 % acetic acid. The samples were vortex-mixed for approximately 15 s and centrifuged for 10 min at 22,000 × g. An aliquot of the clear supernatant ( ~ 30 μL) was transferred to an amber glass autosampler vial with a 250-μL plastic insert and subject to the LC-MS/MS assay. The injection volume was 10 μL.
2.3.2. Brain tumor
To 0.2 mL of the brain tumor homogenate were added 2 μL of the IS solution (1000 ng/mL of camptothecin in methanol) and 1 mL of MTBE. The mixture was vortex-mixed for 5 min. After centrifugation for 5 min at 22,000 × g, the supernatant organic layer was transferred into a clean micro-centrifuge tube. This solvent extraction procedure was repeated once more by adding 50 μL of 0.05 N NH4OH and 1 ml of MTBE to the aqueous layer. The two organic layer fractions were combined and evaporated to dryness under a gentle stream of nitrogen at 45 °C. The dried residue was then reconstituted with 50 μL of the mobile phase (see section 2.4) and 10 μL was injected into the LC-MS/MS system.
2.3.3. Normal brain
To 0.2 mL of the normal brain homogenate were added 4 μL of the IS solution (1000 ng/mL of camptothecin in methanol) and 0.2 mL of methanol. The mixture was vortex-mixed for 5 min and centrifuged at 22,000 × g for 5 min. The supernatant was subject to the solid-phase extraction (SPE). The analytes were extracted from the normal brain homogenate using SPE cartridges with C8 sorbent (50 mg/1mL Bond Elut C8; Varian Inc., Lake Forest, CA, USA). Sorbent was conditioned with 2.0 mL of methanol and equilibrated with 2.0 mL of water. The cartridge was washed with 1.0 mL of water followed by 1.0 mL of 20% methanol. The analytes were eluted with 0.4 mL of methanol containing 0.1% acetic acid. The eluent was evaporated to dryness with nitrogen gas at 45°C and the sample reconstituted with 50μL of mobile phase and 10 μL was injected into the LC-MS/MS system.
2.4. LC-MS/MS conditions
The LC-MS/MS configuration was analogous for plasma, normal brain and brain tumor samples. The LC-MS/MS assay was carried out using an Agilent series 1100 high-performance liquid chromatography system equipped with a binary pump, autosampler and degasser coupled to an API 4000 triple-quadrupole tandem mass spectrometer from Applied Biosystems/MDS SCIEX with ESI source operated in the positive ion mode. Analyst software version 1.4.2 (Applied Biosystems/MDS SCIEX) was used for instrument control, data acquisition and data processing for both chromatography and mass spectrometry.
Separation was achieved on a 50 × 2.0 mm Luna 3 μm C8 column (Phenomenex Inc., Torrance, CA, USA) with a pre-column of the same material. The sample solutions (10 μL) were injected and the analytes were eluted using acetonitrile/1 mM ammonium acetate containing 0.1% acetic acid (28: 72, v/v) at a flow rate fixed at 0.3 mL/min. The isocratic separation run was completed within 3.2 min at 30°C.
The ESI instrumental settings were optimized for the analysis and the appropriate MRM transitions and MS/MS parameters were determined for individual compounds by direct infusion into the mass spectrometer. The optimized tandem mass spectrometry conditions are summarized in . Nitrogen was used as the curtain, collision and ion source gas.
Optimized ESI-MS/MS operating, MRM and MS/MS parameters for sunitinib and the internal standard
2.5. Validation study
The validation study was carried out for sunitinib in three biological matrices of mouse: plasma, normal brain and brain tumor. Linearity of the method was evaluated in five sets of matrix-matched calibration standards. It was considered satisfactory when coefficients of determination (R2) were higher than 0.99.
The extraction efficiency of the analyte was determined in triplicate at three concentration levels for each biological matrix by comparing the peak areas of the extracted QC samples (N = 20 for each concentration in each biomatrix) with those spiked in the reconstituted blank extracts after extraction.
Intra-day accuracy and precision were determined in sextuplicate by analyzing QC samples at low, medium and high concentrations across the linear range. Inter-day accuracy and precision were evaluated on five separate days. Precision was expressed as the relative standard deviation of the determined concentrations. Accuracy was calculated using the following equation: [(mean measured concentration - nominal concentration) /nominal concentration]×100. Precision less than 15% and accuracy within ± 15% were accepted.
The stability of sunitinib in spiked mouse plasma, normal brain and brain tumor homogenate after freeze/thaw cycles from -80°C to ambient temperature was assessed in triplicates by comparing the freshly prepared QC samples with those being frozen and thawed three times with each freeze cycle lasting at least 24 hours.
2.6. Sunitinib treatment and sample collection
Male NIH Swiss nude mice (nu/nu, 8-10 weeks old) were purchased from Taconic Farms (Germantown, NY, USA). All animal experiments were approved by the Institutional Animal Care and Use Committee and performed according to the guidelines of the National Institutes of Health. The orthotopic murine model of human glioma was established by stereotactically injecting U87MG human glioma cells into the left caudate putamen as previously described [12
]. A single dose of 20 mg/kg sunitinib was given as a 20-min IV infusion to the tumor-bearing animals exhibiting a total weight loss of 2 grams over 2 consecutive days. Serial blood samples were taken from the cannulated carotid artery prior to infusion and at 5, 10 and 30 min, and 1, 2, 4, 6, 8 and 12 h after end of infusion. Blood samples were immediately separated by centrifugation and the plasma was stored at -80 °C until the LC-MS/MS analysis. Immediately after the last time point of blood sampling, animals were anesthetized and sacrificed by terminal blood sampling from the vena cava. Brain tumor and normal brain tissues were snap frozen on dry ice and stored at -80°C until the LC-MS/MS analysis. PK data analysis was performed using the software package WinNonlin Version 5.1.2 (Pharsight, Mountain View, CA). Systemic pharmacokinetics of sunitinib were analyzed by standard noncompartmental methods that yielded individual animal estimates of total clearance, the volume of distribution at steady-state, the elimination half-life, as well as the area under the sunitinib plasma concentration-time curves from time 0 to infinity (AUC0-∞