6-Mercaptopurin is antineoplastic drug that included in antimetabolite group and used in acute lymphoblastic leukemia medication. 6-Mercaptopurin is inactive prodrug that will be metabolized into metabolites. One of its metabolites is 6-methylmercaptopurine. This study aimed to optimize the analytical conditions and do validation for the analysis of 6-mercaptopurine and 6-methylmercaptopurine in plasma. Separation was performed using Waters 2996 HPLC, C18 Sunfire^TM column (5μm, 250 x 4.6 mm) with the mobile phase contains water-methanol-acetonitrile bellow gradient elusion condition, and detected with photodiode array detector at 303 nm. 5-Fluorouracil was used as internal standard. Plasma extraction was done by liquid-liquid extraction method using dichloromethane. The method was valid and linear at concentration range of 2.0 – 200.0 ng/mL with r > 0.9991 for 6-mercaptopurine and 20 – 2000 ng/mL with r > 0.9993 for 6-methylmercaptopurine. Accuracy and precision within-run and between-run fulfill the acceptance criteria with %diff and coefficient of variation ≤ 20% (LLOQ) and ≤ 15% (QC samples). 6-Mercaptopurine and 6-methylmercaptopurine was stable in plasma at least for 21 days when stored at -20ºC. This method fulfill the acceptance based on EMEA bioanalytical guideline.

Key words:

5-fluorouracil, 6-mercaptopurine, 6-methylmercaptopurine, HPLC, validation