Rapid quantitative profiling of kynurenine metabolites in human plasma using LC-MS/MS.

Tryptophan is an essential amino acid for the human body, and its metabolic processes are closely related to a variety of physiological and pathological processes, including neurodegenerative diseases, immune modulation, and cardiovascular diseases. Simultaneous detection of tryptophan and its nine metabolites is challenging and previous methods may have shortcomings of poor sensitivity, selectivity, and specificity in complex matrices. The technology of LC-MS/MS, with its characteristics of high sensitivity and selective, provides a powerful tool for the detection of biomarkers. A rapid and effective method for the simultaneous determination of kynurenine metabolites (Xanthurenic acid (XA), DL-Tryptophan (TRP), Kynurenine (KYN), 3-Hydroxyanthranilic acid (3-HAA), Quinolinic acid (QUIA), Kynurenic acid (KYNA), 3-Hydroxy-DL-kynurenine (3HK), 2-Picolinic acid (2PA), 5-Hydroxytryptamine (5-HT), and Anthranilic acid (AA)) was established in human plasma using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technology. Methanol precipitation was used for sample pretreatment, with [2H5]-L-Tryptophan (TRP-D5) as the internal standard. Mass spectrometric detection was performed in positive electrospray ionization mode using multiple reaction monitoring on a triple-quadrupole mass spectrometer. The method’s selectivity, linearity, quantification limit, detection limit, precision, recovery rate, matrix effect, and stability are evaluated. The results show that the linear ranges of 2PA, QUIA, 3HK, 5HT, KYN, 3HAA, TRP, XA, KYNA and AA are 0.25-50, 0.9–225, 1.2–120, 1.4–700, 2-2000, 0.15–150, 180-180000, 0.1–100, 0.1–100, and 0.2–100 ng/mL, respectively; all analytes have good linear relationships with a determination coefficient R2 greater than 0.99. The precision relative standard deviation (RSD) is less than 15%; the spiked recovery rate is 87.54%-107.43%, with an RSD of 2.76%-12.19%. Additionally, the matrix effects for 10 analytes are summarized. At all three QC levels, the matrix effect in plasma is 88.44% to 107.85%, and the RSD is 2.97%-14.62%. These results indicate that there is no significant matrix effect. Stability was evaluated by comparing the measured concentrations after storage with the initial concentrations and expressed as percentages of the initial values. All analytes remained within 85–115% of the initial concentrations under the tested conditions. This study describes the development and validation of a rapid, sensitive, and robust UHPLC–MS/MS method for the simultaneous quantification of ten kynurenine pathway metabolites—including the precursor tryptophan, key intermediates, and neurotoxic/neuroprotective end products-in human plasma. The method employs a simple protein precipitation procedure, a single deuterated internal standard (TRP-D5), and a 13-minute chromatographic run on a C8 column, providing adequate separation of metabolites with diverse physicochemical properties.