Development, validation and application of LC–MS/MS method for quantification of amino acids, kynurenine and serotonin in human plasma

Dávid Virág, Márton Király, László Drahos, Andrea Edit Édes, Kinga Gecse, György Bagdy, Gabriella Juhász, István Antal, Imre Klebovich, Borbála Dalmadi Kiss, Krisztina Ludányi

Research output: Article

1 Citation (Scopus)


Altered serotonergic neurotransmission is a key factor in several neurologic and psychiatric disorders such as migraine. Human and animal studies suggest that chronically low interictal serotonin levels of plasma and brain may facilitate increased activity of the trigeminovascular pathway, and may contribute to development of repeated migraine attacks. However, brain serotonin synthesis is affected by the concentration of tryptophan, its metabolites and a number of amino acids. In this work a simple and robust LC–MS/MS method for the quantitative determination of valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serotonin and kynurenine in human plasma has been developed and validated. Sample preparation was achieved by protein precipitation, using trifluoroacetic acid. Chromatographic separation was carried out on a Supelco Ascentis® Express C18 column (3.0 mm i.d. × 150 mm, 2.7 μm) equipped with an Agilent Zorbax Eclipse XDB C8 guard-column under isocratic conditions at a flow rate of 0.4 mL/min, over a 6.5 min run time. Mobile phase was 0.2% trifluoroacetic acid – acetonitrile (85:15, v/v). The eight analytes and two internal standards were ionized by positive electrospray ionization and detected in multiple reaction monitoring mode. A “fit-for-purpose” validation approach was adopted using surrogate matrix for the preparation of calibration samples. The calibration curves of all analytes showed excellent linearities with a correlation coefficient (r2) of 0.998 or better. Spiked surrogate matrix samples and pooled human plasma were used as quality control samples. Intra-day and inter-day precisions were less than 11.8% and 14.3%, and accuracies were within the ranges of 87.4–114.3% and 87.7–113.3%, respectively. Stability of the components in standard solutions, surrogate matrix, pooled plasma and processed samples were found to be acceptable under all relevant conditions. No significant carryover effect was observed. The surrogate matrix behaved parallel to human plasma when assessed by standard addition method and diluting the authentic matrix with surrogate matrix. The method was successfully applied for analysis of 800 human plasma samples to support a clinical study.

Original languageEnglish
Article number113018
JournalJournal of Pharmaceutical and Biomedical Analysis
Publication statusPublished - febr. 20 2020


ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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