SIMULTANEOUS ANALYTICAL METHOD DEVELOPMENT OF 6-MERCAPTO¬PURINE AND 6-METHYLMERCAPTOPURINE IN PLASMA BY HIGH PERFOR¬MANCE LIQUID CHROMATOGRAPHY-PHOTODIODE ARRAY
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.