Temozolomide concentrations were quantified in acidified sodium heparin plasma and acidified brain interstitial dialysate. For plasma, 15 µl of 8.5% phosphoric acid was added per 0.5 ml of plasma. For microdialysis fluid, 1 µl of glacial acetic acid was added for every 10 µl of the dialysate. Perfusion fluid CNS (artificial CSF) was used as a surrogate matrix for dialysate standards and QCs. Temozolomide was extracted from samples (50 µl of acidified plasma or 20 µl of acidified microdialysis fluid) by adding 600 µl of ethyl acetate containing IS (20 ng/ml of caffeine-13C3) (Sigma–Aldrich, St. Louis, MO). Samples were vortex-mixed and centrifuged at 2000 rpm for 10 min. The top layer was transferred to a clean glass tube and dried under a nitrogen air stream. Samples were reconstituted with 200 µl of 0.5% formic acid in water and stored in the autosampler at 5 °C for LC–MS/MS analysis.