Validated high-performance liquid chromatographic assay for simultaneous determination of dacarbazine and the plasma metabolites 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide

Dacarbazine (DTIC) is a prodrug that is clinically effective in the treatme nt of Hodgkin's disease, melanoma and soft tissue sarcoma. To better charac terize the clinical pharmacology of parent drug and reactive metabolites, a reversed-phase HPLC method with UV detection was developed for simultaneou s determination of dacarbazine and the metabolites 5-(3-hydroxymethyl-3-met hyl-1-triazeno)imidazole-4-carboxamide (HMMTIC) and 5-(3-methyl-1-triazeno) imidazole-4-carboxamide (MTIC). Chromatographic separation was achieved wit h a Zorbax SB-CN column and with a mobile phase of 80%, 50 mM ammonium phos phate, pH 6.5, 20% methanol and 0.1% triethylamine. HMMTIC, MTIC and DTIC w ere extracted from plasma with methanol precipitation of the proteins. Reco very of DTIC and the metabolites from whole blood was greater than 92%. Rap id processing of whole blood. methanol extraction and storage at -70 degree sC substantially increased the stability of HMMTIC and MTIC from less than 15 min to 3 days. Precision for HMMTIC, MTIC and DTIC ranged from 3.7 to 16 .3% relative standard deviation. The accuracy ranged from 101 to 114% for a ll three analytes. The validated assay was used to determine the pharmacoki netic data for dacarbazine and its active metabolites for human patients wi th recurrent glioma receiving DTIC intravenously. (C) 2001 Elsevier Science B.V. All rights reserved.