Purpose: Temozolomide (TMZ) is a FDA approved drug for the treatment of high grade brain tumors. Oral administration of TMZ is found to be less effective due to high vascularity and heterogenous permeability of brain tumors and increased dose has lead to systemic toxicity. Hence, to provide targeted drug delivery, the effectiveness of intracerebral brain micro-dialysis is being investigated in mice. In order to support the investigation we have developed a LC-MS/MS method for the quantitation of TMZ in mice brain with improved recovery and negligible matrix effects.
Briefly, frozen 20 mg aliquots of mice brain were acidified with formic acid to prevent degradation of TMZ. The brain tissues were then lysed with 40 μL Proteinase K at 37 oC after spiking with internal standard, Theophylline. Then, 270 μL of ethanol was used to precipitate out proteins which were removed by centrifugation. 50 μL of supernatants were then diluted with mobile phase. Next, the analyte and internal standard were separated on a Waters Symmetry C18 column using a mobile phase gradient consisting of 0.1% formic acid and 10mM ammonium acetate in water and methanol. The flow rate was 0.4 mL/min and run time was 8 minutes. Detection was carried out in positive ESI-MRM mode with m/Z transitions for TMZ and IS set at 195.1 - 138.1 and m/Z 181.1 - 124.1 respectively.
We were able to achieve higher recovery of TMZ by using Proteinase K to lyse the mice brain tissues. Ethanol helped to combat matrix effects by precipitating out the proteins. The method was found to be linear in the range of 1,020 – 176,800 ng/g and was also validated for accuracy, precision, sensitivity, LLOQ and stability according to the FDA guidelines.
In order to overcome the challenges of low recovery and high matrix effects, we have designed an improved method for quantitation of TMZ from mice brain. The LC-MS/MS method developed may be useful for pre-clinical studies to evaluate the effectiveness of intra cerebral brain micro-dialysis in mice.