A Validated UPLC-MS/MS Method for Monitoring Temozolomide and Perillyl Alcohol Metabolites in Biological Matrices: Insights From a Preclinical Pharmacokinetic Study.

Glioblastoma multiforme (GBM) is among the most aggressive brain tumors, largely due to the restrictive blood-brain barrier (BBB) and limited drug bioavailability. Temozolomide (TMZ), an alkylating agent, and perillyl alcohol (POH), a monoterpene with antitumor properties, have shown promise in GBM therapy. This study developed and validated a UPLC-MS/MS method for the simultaneous quantification of TMZ, its active metabolite 5-aminoimidazole-4-carboxamide (AIC), and perillic acid (PA), the primary POH metabolite, in rat plasma and tissues. The method met FDA and EMA validation criteria, showing high selectivity, linearity (R2 > 0.995), accuracy (80%-120%), precision (RSD < 15%), and matrix-specific stability across plasma, brain, liver, kidney, spleen, and lung. Following single oral doses of TMZ (2 mg/kg) and POH (47 mg/kg), pharmacokinetic analysis revealed that TMZ had the highest systemic exposure (AUC0-24h: 8173.64 ng·h/mL) and Cmax (1448.64 ng/mL), while PA showed the fastest absorption (tmax: 0.5 h). AIC levels confirmed efficient TMZ metabolism. Biodistribution analysis showed TMZ accumulation in the brain (~1000 ng/mL), supporting its CNS efficacy. PA was mainly distributed to liver and kidneys, with limited brain penetration. The validated method enables preclinical pharmacokinetic and tissue distribution studies, offering valuable insights into TMZ and POH behavior for GBM treatment.