Simultaneous UPLC-MS/MS Determination of 6-mercaptopurine, 6-methylmer-captopurine and 6-thioguanine in Plasma: Application to the Pharmacokinetic Evaluation of Novel Dosage forms in Beagle Dogs

Background: 6-Mercaptopurine (6-MP) is widely used to treat pediatric acute lymphoblastic leukemia (ALL). Mini-tablets of 5 mg per tablet were developed for precision individual therapy for children and individuals with poor thiopurine S-methyltransferase (TPMT) or nucleoside diphophate-linked moiety X-type motif 15 (NUDT15) metabolism. This study investigated the pharmacokinetic profiles of mini-tablets and conventional tablets with an improved ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Methods: After giving 8 healthy beagle dogs 50 mg 6-MP in different dosage forms, plasma samples collected at different time points were analyzed for pharmacokinetic evaluation. The samples were precipitated by methanol with 0.05% formic acid and separated on a Waters Atlantis T3 column (2.1 x 150 mm, 3 mu m particles) using 0.1% formic acid in water and methanol at a flow rate of 0.4 mL/min in 4 min. Results: This method showed good linearity, accuracy, precision and stability with a detection range of 5.0-500.0 ng/mL for 6-MP, 6-methylmercaptopurine (6-MMP) and 6-thioguanine (6-TG). The main parameters, half-life of DO!: apparent terminal disposition, maximum observed plasma concentration, total AUC extrapolated to infinity, AUC since initiation of the experiment, mean residence time, distribution volume and clearance were 1.62 +/- 0.87 hours, 90.58 +/- 60.43 ng/mL, 151.20 +/- 94.18 ng.h/mL, 292.06 = 184.02 ng.h(2)/mL, 1.90 +/- 0.92 hours, 864.08 = 538.52 L, and 432.75 +/- 360.64 L/h for conventional tablets and 1.70 +/- 1.10 hours, 84.15 +/- 39.50 ng/mL, 147.70 +/- 51.80 ng-h/mL, 300.92 +/- 124.48 ng.h(2)/mL, 2.07 +/- 0.50 hours, 756.90 +/- 324.00 L, and 340.75 +/- 125.81 L/h for mini-tablets, respectively. Paired Mests showed no significant difference in any of the evaluated pharmacokinetic parameters between the two types tablets (P > 0.05). Conclusion: Two dosage forms showed the same pharmacokinetic characteristics. This developing, novel formulation will help to provide a more accurate and optimal dosing regimen of 6-MP for humans in the future.