High-performance liquid chromatographic analysis and stability of anti-tumor agent temozolomide in human plasma

Temozolomide (SCH 52365; TEMODAL(TM)) is an antineoplastic agent with activ ity against a broad spectrum of murine tumors. This compound is currently m arketed in the European Union for the treatment of patients with glioblasto ma multiforme and anaplastic astrocytoma, which are serious and aggressive types of brain cancers. It has been postulated that temozolomide exerts its in vivo activity via the decomposition product MTIC, which is believed to alkylate nucleophiles, and in the process is converted to AIC. A high-perfo rmance liquid chromatographic (HPLC) method was developed and validated for the analysis of temozolomide in human plasma. The determination of temozol omide involved extraction with ethyl acetate followed by separation on a re versed phase C-18 column and quantification by UV absorbance at 316 nm. The calibration curve was linear over a concentration range of 0.1-20 mug/ml. The limit of quantitation was 0.1 mug/ml, where the coefficient of variatio n (CV) was 0% and the bias was 10.0%. The method was precise with a coeffic ient of Variation ranging from 2.5 to 6.9% and accurate with a bias ranging from - 5.0 to 10.0%. Temozolomide was unstable at 37 degreesC in human pla sma with a degradation t(1/2) of 15 min; however, it was stable at 4 degree sC for at least 30 min. Temozolomide was stable in acidified human plasma ( pH < 4) for at least 24 h at 25<degrees>C, and for at least 30 days at - 20 degreesC. Moreover, temozolomide was stable in acidified human plasma afte r being subjected to three freeze-thaw cycles. The assay was shown to be sp ecific, accurate, precise, and reliable for use in pharmacokinetic studies. (C) 2001 Elsevier Science B.V. All rights reserved.