A Rapid 2-Minute LC-MS/MS Method with Simple Protein Precipitation for Simultaneous Determination of Tryptophan and its two metabolites Kynurenine and Kynurenic acid in human serum

Dysregulation of the tryptophan (TRP) metabolic pathway is closely linked to the pathophysiology of neuropsychiatric disorders, such as depression. This study aimed to develop and validate a sensitive, rapid, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of TRP and its metabolites, kynurenine (KYN) and kynurenic acid (KYNA), in human serum. Analytes were extracted from 100   µL of serum via simple protein precipitation with acetonitrile. Chromatographic separation was achieved on an Agilent ZORBAX HILIC Plus column (4.6   mm×100   mm, 3.5   µm) using isocratic elution with a mobile phase of methanol: acetonitrile containing 5   mM ammonium formate. Quantification was performed using an electrospray ionization source in positive ion multiple reaction monitoring (MRM) mode, with a total run time of only 2.0   min.The linear ranges were 1- 50   µg/mL for TRP, 0.1-5   µg/mL for KYN, and 1-50   ng/mL for KYNA, covering clinically relevant levels. A weighting factor of 1/x² provided the best fit for calibration curves (R² > 0.99). Extraction recoveries ranged from 88.23% to 99.39%, and mean internal standard-normalized matrix effects were 81%-100%. Accuracy and precision values met bioanalytical acceptance criteria. Stability assessments confirmed that samples were stable at -20°C and -80°C for 31 days and through three freeze-thaw cycles. This validated method was successfully applied to analyze serum samples from 103 adolescent patients with first-episode depression.