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Xinyue Zhang

doi:10.3390/agriculture16151630

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

2026-05-23

0

PRE

AI

H

Hui Yan

H

Haoyang Lu

黄

黄思铭 (Siming Huang)

Y

Yuqing Li

X

Xia, Hui

H

Huang, Wanting

D

Dewei Shang

M

Ming Zhang *

DOI：10.1016/j.jpba.2026.117448

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Abstract

En 中文

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 & micro;L of serum via simple protein precipitation with acetonitrile. Chromatographic separation was achieved on an Agilent ZORBAX HILIC Plus column (4.6 mm & times;100 mm, 3.5 & micro;m) 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 & micro;g/mL for TRP, 0.1-5 & micro;g/mL for KYN, and 1-50 ng/mL for KYNA, covering clinically relevant levels. A weighting factor of 1/x & sup2; provided the best fit for calibration curves (R & sup2; > 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 degrees C and -80 degrees 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.

Keywords:

Tryptophan

Kynurenine

Kynurenic acid

High-performance liquid chromatography

Tandem mass spectrometry (LC-MS/MS)

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

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