2.1 Chemicals and standards
ON01910 and ON012380, the internal standard (), were synthesized in-house. Details on other chemicals, solvents and preparation of stock solutions, standards and quality control (QC) samples are presented in the Supplementary Information
Structure of internal standard, ON012380
2.2 Chromatographic and mass-spectroscopic conditions
Method development and validation were performed with an LC/MS/MS (Agilent 1100 series HPLC, AB SCIEX API 4000 tandem mass spectrometer, Foster City, CA) using an ESI interface and operated in positive ion mode. Instrument control, data acquisition and processing for both chromatography and mass spectrometry were performed using the Analyst Software v 1.4.2 (AB SCIEX). Separation of the analyte was achieved at 40°C using Luna 3 μm C18 column (50 mm×2.0 mm i.d., Phenomenex, Inc., Torrance, CA, USA) protected by a C18 guard column. The mobile phase used for the chromatographic separation was composed of acetonitrile: ammonium formate (10 mM) (30:70, v/v) that was delivered isocratically at a flow rate of 0.3 ml/min, and enabled a run time of 3 min.
Drug quantitation was performed by ESI-SRM using ion transitions of m/z 469.00 → 284.00 for ON01910 and 466.00 → 208.00 for the internal standard. The optimal declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) for ON01910 were 41 V, 5 V, 18 V and 9 V, respectively. The final DP, EP, CE and CXP of the internal standard used were 79 V, 5 V, 27 V and 6 V, respectively. Nitrogen was used as the nebulizer, curtain gas and collision gas.
2.3 Sample preparation
A protein precipitation sample preparation method was used for the analysis of ON01910 in mouse plasma, urine, feces, normal brain and brain tumor matrices. To aliquots of plasma (10 μl), urine (50 μl), feces (30 μl of a 5% homogenate), normal brain (20 μl of a 10% homogenate) and brain tumor (50 μl of a 10% homogenate), three times the volume of IS (internal standard) working stock solution (500 ng/ml of IS in methanol) was added and vortexed for 1 min followed by centrifugation for 5 min (plasma) or 15 min (rest of the matrices) at 15,000 rpm with 5 μl of the resultant supernatant injected into the LC/MS/MS.
2.4 Method validation
Validation of the method in different matrices involved determining intra-day and inter-day variability as measured by linearity of the standard curve and accuracy and precision. Linearity of the method in each biological matrix was determined in five sets of calibration standards whereby a correlation coefficient (R2) of ≥0.99 was considered satisfactory. Intra-day and inter-day variability in accuracy and precision was determined by analyzing replicates of QC samples prepared over a high to low concentration range either on the same day or separate days, respectively. Precision was expressed as the relative standard deviation of the determined concentrations while accuracy was expressed as percent bias [% Bias = ((mean of the measured concentration-added concentration)/added concentration) × 100]. Extraction efficiency or percentage recovery [(peak area sample/peak area recovery control) × 100] of the protein precipitation method was determined at multiple concentrations in triplicates by comparing the peak area of extracted QC samples to that of control samples prepared from methanolic supernatants spiked with analyte and IS.
2.5 ON01910 treatment and sampling
All animal studies were approved by the Institutional Animal Care and Use Committee. For systemic pharmacokinetic (PK) evaluation of ON01910, a single dose of 50 mg/kg ON01910 was given to healthy nude mice as a 10 min tail vein infusion followed by the collection of serial blood samples (20 μl) from a carotid artery cannula over a period of 24 hr. A second steady-state pharmacokinetic study was conducted in mice bearing intracerebral tumors for the determination of brain and brain tumor disposition of ON01910. Detailed methodology on the systemic and brain and brain tumor pharmacokinetic studies is provided in the Supplementary Information