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A new solid phase extraction method for rapid high performance liquid chromatography-UV determination of mitragynine in plasma has been developed. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile-ammonium acetate buffer, 50 mM at pH 5.0 (50:50, v/v). The method had limits of detection and quantification of 0.025 and 0.050 microg/mL, respectively. The method was accurate and precise for the quantitative analysis of mitragynine in human and rat plasma with within-day and between-day accuracies between 84.0 and 109.6%, and their precision values were between 1.7 and 16.8%. Additional advantages over known methods are related to the solid phase extraction technique for sample preparation which yields a clean chromatogram, a short total analysis time, requires a smaller amount of plasma samples and has good assay sensitivity for bioanalytical application. The method was successfully applied in pharmacokinetic and stability studies of mitragynine. In the present study, mitragynine was found to be fairly stable during storage and sample preparation. The present study showed for the first time the detailed pharmacokinetic profiles of mitragynine. Following intravenous administration, mitragynine demonstrated a biphasic elimination from plasma. Oral absorption of the drug was slow, prolonged and was incomplete, with a calculated absolute oral bioavailability value of 3.03%. The variations observed in previous pharmacokinetic studies after oral administration of mitragynine could be attributed to its poor bioavailability rather than to the differences in assay method, metabolic saturation or mitragynine dose.

IntroductionMitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).Methods and ResultsThe rapid delayed rectifier potassium current (I Kr), L-type Ca2+ current (I Ca,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant I Kr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed I Kr in hiPSC-CMs by 67% ∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression,and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine.ConclusionsOur study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of I Kr in human cardiomyocytes.