1. A validated surrogate analyte LC–MS/MS assay for quantitation of endogenous kynurenine and tryptophan in human plasma
- Author
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Sami Mahrus, Dennis Milanowski, Kari M. Morrissey, Dennis L. Miller, Brian Dean, Stephanie Cape, Drew Dorshorst, Xiaorong Liang, Janine McKnight, and Lei Tan
- Subjects
0301 basic medicine ,Kynurenine pathway ,Clinical Biochemistry ,Endogeny ,01 natural sciences ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Limit of Detection ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,Lc ms ms ,Humans ,Indoleamine-Pyrrole 2,3,-Dioxygenase ,Enzyme Inhibitors ,General Pharmacology, Toxicology and Pharmaceutics ,Kynurenine ,Chromatography ,Surrogate analyte ,Chemistry ,010401 analytical chemistry ,Tryptophan ,General Medicine ,0104 chemical sciences ,Medical Laboratory Technology ,030104 developmental biology ,Human plasma ,Biomarkers ,Blood Chemical Analysis ,Chromatography, Liquid - Abstract
Aim: Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) catalyze the initial and rate-controlling step of tryptophan metabolism through the kynurenine pathway, which plays an important role in mediating immune response. Accurate measurement of tryptophan and kynurenine is critical for monitoring the activity of IDO/TDO. Experimental: Surrogate analytes ([15N2]-Tryptophan and [13C6]-Kynurenine) were used for preparation of calibration standard and quality control. A fit-for-purpose validation using an approach of surrogate analyte and authentic matrix was carried out. Results: Acid precipitation was used in sample preparation, which yielded good recovery without significant matrix effect. Precision and accuracy results were well within the acceptance criteria. The assay demonstrated successful application to a clinical study to confirm a transient depletion of kynurenine upon IDO inhibition. Conclusion: A robust, specific and simple LC–MS/MS method was developed and validated with a fit-for-purpose style for measuring tryptophan and kynurenine in human plasma samples.
- Published
- 2018
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