Simultaneous Analytical Method Development of 6-Mercaptopurine and 6-Methylmercaptopurine in Plasma by High Performance Liquid Chromatography-Photodiode Array

    Published on:March 2017
    Journal of Young Pharmacists, 2017; 9(1s):S29-S34
    Original Article | doi:10.5530/jyp.2017.1s.8

    Yahdiana Harahap1, Nurul Azizah1, Rizka Andalusia2, Supandi1

    1Faculty of Pharmacy Universitas Indonesia, Depok, INDONESIA.

    2Department of Research and Development of Dharmais Hospital National Cancer Center, Jakarta, INDONESIA.


    Objective: 6-Mercaptopurin is antineoplastic drug that is included in antimetabolite group and is used in acute lymphoblastic leukemia medication. 6-Mercaptopurin is inactive pro-drug that will be metabolized into metabolites. One of its metabolites is 6-methylmercaptopurine. This study is aimed to optimize the analytical conditions and perform validation for the analysis of 6-mercaptopurine and 6-methylmercaptopurine in plasma. Method: Separation was performed using Waters 2996 HPLC, C18 SunfireTM column (5μm, 250 x 4.6 mm) with the mobile phase containing water-methanol-acetonitrile with gradient elution, and detected at 303 nm. 5-Fluorouracil was used as internal standard. Plasma extraction was done by liquid-liquid extraction using dichloromethane. Result: The method was 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 % RE and relative standard deviation (% RSD) ≤ 20% (LLOQ) and ≤ 15% (QC samples). 6-Mercaptopurine and 6-methylmercaptopurine was stable in plasma at least for 21 days when stored at -20ºC. Conclusion: The bio-analytical method was sensitive, selective and all the parameters fulfilled the acceptance criteria of the EMA Bio-analytical Method Validation Guideline, 2011.

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

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