ANALISIS 6-MERKAPTOPURIN DAN 6-TIOGUANIN DALAM DRIED BLOOD SPOT DENGAN KROMATOGRAFI CAIR-TANDEM SPEKTROMETRI MASSA
Abstract
6-Mercaptopurine (6-MP) is a chemotherapeutic drug that belongs to a class of purine antagonist antimetabolite used for acutelymphoblastic leukemia. 6-Mercaptopurine has to go through the metabolic pathway to become 6-Thioguanin nucleotide(active metabolites). This study aimed to analyzing 6-Mercaptopurine and 6-Thioguanine (6-TG) simultaneously in Dried Blood Spot (DBS) samples using liquid chromatography-tandem mass spectrometry. The quality control and calibration curve solutions were made by spot 60 μL at DBS CAMAG paper and were dried for 3 hours. DBS papers were cut and extracted with methanol containing internal standard 5-fluorouracil (5-FU). Separation performed with Waters Acquity™ UPLC BEH Amide column 1.7 μm (2.1 x 100 mm) with a mobile phase consists of 0.2 % formic acid in water –0.1% formic acid in acetonitrile –methanol with gradient elution and flow rate 0.2 mL/minutes. Mass detection was done using Waters Xevo TQD with positive electrospray ionization (ESI) for 6-MP and 6-TG and negative ESI for 5-FU in Multiple Reaction Monitoring mode. Detection of 6-MP, 6-MMP, 5-FU respectively are 153,09 > 119,09; 168,09 > 107,06; 129,15 > 42,05. This method is linear with the range 25-1000 ng /mL for 6-MP and 6-TG with consecutive r value is ≥ 0,996 and ≥ 0,995. Diff value percentage and coefficient of variation (CV) for accuracy and precision of intra-day and inter-day are not more than ±15% and not more than ±20% at a concentration LLOQ. This method fulfill the requirements of validation that refers to the European Medicines Agency guideline.