DETERMINATION OF TEMOZOLOMIDE IN HUMAN PLASMA AND URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AFTER SOLID-PHASE EXTRACTION

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at -20 degrees C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 mu l) On a 100-mg C-18-endcapped cartridge. Matrix components were eliminated with 750 mu l of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 mu l of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 mu l of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4-20 mu g/ml and 2-150 mu g/ml for plasma and urine, respectively. The extraction recovery of temozolomide was 86-90% from plasma and 103-105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37 degrees C. An acidic pH (< 5-6) should be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.