HPLC–UV method for temozolomide determination in complex biological matrices: Application for in vitro,ex vivo and in vivo studies

A high‐performance liquid chromatography method for temozolomide (TMZ) determination in complex biological matrices was developed and validated for application in in vitro, ex vivo and in vivo studies of new nanotechnology‐based systems for TMZ nasal delivery. The method was able to quantify TMZ in nanoemulsions, following cellular uptake, in the porcine nasal mucosa and in mouse plasma and brain. Analyses were performed on a C₁₈ column at 35°C, under UV detection at 330 nm. The mobile phase was methanol–acetic acid 0.5% (30:70, v/v), eluted at an isocratic flow rate of 1.1 mL/min. The method was found to be specific, precise, accurate, robust and linear (0.05 to 5 μg/mL) for TMZ determination in all matrices. No interference of TMZ degradation products was found under various stress conditions such as acidic, alkaline, oxidative, light and thermal exposure, demonstrating stability. The method was applied for the quantification of TMZ in different matrices, i.e. the efficiency of nanoemulsions in vitro in increasing TMZ cellular uptake, ex vivo TMZ permeation and retention in the porcine nasal mucosa tissue, and for in vivo TMZ quantification in mouse brain following intranasal nanoemulsion administration compared with free TMZ.