Determination of kynurnine and tryptophan, biomarkers of indoleamine 2,3-dioxygenase by LC-MS/MS in plasma and tumor

Aim: Tryptophan (Trp) and kynurnine (Kyn) are a pair of biomarkers for indoleamine 2,3-dioxygenase which closely related to the tumor immune escape. To evaluate the effect of drugs on the indoleamine 2,3-dioxygenase activity, the specific and accurate LC-MS/MS methods were developed and validated for simultaneous determination Kyn and Trp in mouse plasma and tumor tissues using surrogate analytes Kyn-d4 and Trp-d5 calibrators. Results: Plasma and tumor homogenates samples were pretreated with the solid phase extraction which assured the method having high recovery (>90% in plasma and >80% in tumor) and no matrix effect. The methods were validated for specificity, linearity, accuracy and precision, recovery, matrix effect and stability using surrogate analytes Kyn-d4 and Trp-d5 in authentic matrices. Conclusion: The validated methods have been successfully applied to the pharmacodynamic study of INCB024360 in CT26 tumor bearing mice after single dose and multiple dosing.