Development and validation of LC-MS/MS methods for the quantification of TMS-007, a member of the SMTP congeners, in rat plasma and brain: application to a preclinical pharmacokinetic study

Abstract

TMS-007 is a member of the SMTP congeners for the treatment of acute ischemic stroke. To support its preclinical study, LC-MS/MS methods for the quantification of TMS-007 in rat plasma and brain were developed. The samples were prepared by protein precipitation. A UPLC HSS T3 column (2.1 mm × 50 mm, 1.8 µm) was used to achieve chromatographic separation. An acetonitrile–water mixture containing ammonium acetate was used as the mobile phase. The methods were validated with regard to selectivity, calibration curve and lower limit of quantitation, accuracy and precision, matrix effect, extraction recovery, carryover, dilution integrity, and stability. The calibration ranges of TMS-007 in rat plasma and brain homogenate were 10.0–10 000 ng mL⁻¹. There was no endogenous or cross interference in the biological matrices. Across these matrices, the intra- and inter-batch coefficients of variation and accuracy deviations for all QC samples met the acceptance criteria. The inter-batch coefficients of variation of the QC samples were ≤11.7% for plasma and ≤15.0% for brain. The inter-batch accuracy ranged from 97.8% to 104.1% for plasma and 97.1% to 102.3% for brain. No significant matrix effect was observed from the matrices (from 102.9% to 111.0% for plasma and from 114.3% to 118.9% for brain). The extraction recoveries of the methods at different concentrations were consistent and reproducible. For plasma, the coefficients of variation were ≤9.9%, and for brain, the coefficients of variation were ≤4.6%. The analyte was stable in different matrices under various storage conditions (room temperature for 8 h, 4 °C in the autosampler for 3 days, 3 freeze–thaw cycles and 7 days at −70 °C). The methods were successfully applied to a preclinical study in a transient middle cerebral artery occlusion rat model after single dose administration. The pharmacokinetic results of the study drug laid a foundation for its further development.