Solid phase extraction (SPE) or a combined protein precipitation SPE permitted an efficient analyte recovery and sample clean-up. Multiple reaction monitoring (MRM) was used to track both GF449 and its internal standard (IS), MM189. GF449 was determined and quantitated to nanomolar concentrations in both plasma and brain matrix (LOQs = 0.0025 nmol/mL). Specificity was ensured using three further MRM qualifier transitions for both analyte and IS. Linearity was found in the range of 0.0025 nmol/mL to 1.00 nmol/mL (R2 = 0.9965) and from 0.0025 nmol/mL to 50 nmol/mL (R2 = 0.9999) for plasma and brain respectively. Intraday trueness ranged from 94.0% to 117.5% for brain and from 93.7% to 108.1% for plasma, while precision values were within 3.0% - 6.7% and 2.5% - 9.2% for plasma and brain respectively. The interday trueness of plasma ranged from 89.6% to 107.7% and the precision values (CV%) ranged from 4.6% to 7.5%. Interday trueness and precision (CV%) of the brain ranged from 94.3% to 101.2% and from 1.6% to 11.5% respectively.
The method was validated in accordance with the EMEA guidelines and was successfully applied to plasma and brain samples obtained from rats treated with a 10 mg/kg single oral dose of GF449, thus demonstrating its applicability to preclinical pharmacokinetic studies.