Ultra-performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan metabolites in human plasma and its application to clinical study

A sensitive, rapid and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to assay tryptophan (TRP) and its nine metabolites, including kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), 3-indolepropionic acid (IPA) and 3-indoleacetic acid (IAA) in human plasma. Tryptophan- d5 (TRP- d5 ) and carbamazepine (CAR) were applied to the method quantification, where TRP- d5 was the corresponding internal standard (IS) for TRP and KYN, and CAR was the corresponding IS for the other analytes. Plasma samples were processed by deproteinisation with acetonitrile , followed by separation on an Acquity UPLC HSS T3 column by using gradient elution with 0.1% ( v / v ) formic acid in water and acetonitrile and detection by electrospray ionisation tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) within a total run time of 5 min. The calibration ranges were 3–600 ng/mL for 3-HK, 1.5–300 ng/mL for 5-HT, 25–5000 ng/mL for KYN, 1–200 ng/mL for XA, 100–20,000 ng/mL for TRP, 5–1000 ng/mL for KYNA, 2–400 ng/mL for 3-HAA, 2.5–500 ng/mL for 5-HIAA and 10–2000 ng/mL for IAA and IPA. All intra- and inter-day analytical variations were acceptable. Matrix effect and recovery evaluation proved that matrix effect can be negligible, and sample preparation approach was effective. The newly developed method can simultaneously determine a panel of TRP metabolites and was successfully applied in the clinical study characterising TRP metabolism in healthy volunteers.