Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

The concentrations of l ‐tryptophan (Trp) and the metabolite l ‐kynurenine (KYN) can be used to evaluate the in‐vivo activity of indoleamine 2,3‐dioxygenase (IDO) and tryptophan 2,3‐dioxygenase (TDO). As such, a novel method involving derivatization of l ‐Trp and l ‐KYN with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS) and separation by high‐performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole‐bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH₃CN/10 mm ammonium formate in H₂O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l ‐Trp and l ‐KYN were approximately 50 and 4.0 pm , respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC‐MS/MS method, l ‐Trp and l ‐KYN were successfully determined in 10 μL human serum using 1‐methyl‐l ‐Trp as an internal standard. The precision and recovery of l ‐Trp were in the ranges 2.85–9.29 and 95.8–113%, respectively, while those of l ‐KYN were 2.51–16.0 and 80.8–98.2%, respectively. The proposed LC‐MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.