Development and validation of a liquid chromatography method using UV/fluorescence detection for the quantitative determination of metabolites of the kynurenine pathway in human urine: Application to patients with heart failure

Abstract

Recent evidence indicates the relevant role of the tryptophan (TRP) metabolites in the pathophysiology of cardiovascular diseases via inflammatory and oxidative stress mechanisms. Therefore, quantification of TRP and its metabolites in biological samples can be a powerful tool to elucidate the disease mechanisms. The aim of this work was to develop and validate a liquid chromatography with ultraviolet (UV) and fluorescence detection (FD) (LCUV/FD) method for the quantification of TRP and its metabolites (L-kynurenine (KYN) and kynurenic acid (KA)) in urine samples from heart failure (HF) patients. Biochemical parameters and inflammatory markers were quantified, and data correlated with urinary concentrations of TRP and its metabolites. Optimized chromatographic conditions were achieved using a Luna® 3 μm PFP(2) analytical column, a mobile phase of 20 mM of ammonium formate in ultra-pure water (with 0.01 % of formic acid), acetonitrile and ethanol (95/2/3, v/v/v), a flow rate of 0.7 mL/min and a column oven temperature set at 25 °C. The method was validated according to the European Medicines Agency (EMA) guidelines and showed to be linear (r2 >0.99), accurate (82–116%) and precise (%RSD below 15 %). The limits of quantification varied between 50 and 125 ng/mL. The method was applied to the quantification of TRP, KYN and KA in healthy volunteers and male HF patients. The results obtained through this pilot study (small group of patients) showed a relationship between biochemical parameters, inflammatory markers and changes in the concentration of TRP, KYN and KA. The KYN/TRP and KA/KYN ratios were calculated. Results support the hypothesis that KYN/TRP ratio is related with enzymatic activity and that KA/KYN ratio can be a good neuroprotection indicator. The potential of the LCUV/FD method for the monitoring of the selected compounds in cardiac patients was also demonstrated.