A validated UHPLC-MS method for tryptophan metabolites: Application in the diagnosis of multiple sclerosis

• Published in: Journal of pharmaceutical and biomedical analysis 2020-06, Vol.185, p.113246-113246, Article 113246
• Main Authors: Tömösi, Ferenc, Kecskeméti, Gábor, Cseh, Edina Katalin, Szabó, Elza, Rajda, Cecília, Kormány, Róbert, Szabó, Zoltán, Vécsei, László, Janáky, Tamás
• Format: Article
• Language: English
• Subjects: Adult; Biomarkers - analysis; Biomarkers - metabolism; Calibration; Case-Control Studies; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; Chromatography, High Pressure Liquid - standards; Derivatization; DryLab®4; Female; Humans; Kynurenic Acid - analysis; Kynurenic Acid - metabolism; Liquid chromatography-mass spectrometry; Middle Aged; Multiple sclerosis; Multiple Sclerosis, Relapsing-Remitting - blood; Multiple Sclerosis, Relapsing-Remitting - cerebrospinal fluid; Multiple Sclerosis, Relapsing-Remitting - diagnosis; Picolinic Acids - analysis; Picolinic Acids - metabolism; Quinolinic Acid - analysis; Quinolinic Acid - metabolism; Reference Standards; Tandem Mass Spectrometry - instrumentation; Tandem Mass Spectrometry - methods; Tandem Mass Spectrometry - standards; Tryptophan - analysis; Tryptophan - metabolism; Validation; Young Adult
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2020.113246

[Display omitted] •Simultaneous and robust quantification of tryptophan and its 11 metabolites.•Derivatization; separation of analytes on pentafluorophenyl column.•Optimization of chromatographic separation using DryLab®4 software.•Quantification of analytes in human cerebrospinal fluid and serum.•Reference study for the clinical and scientific research of multiple sclerosis. The simultaneous quantitative estimation of tryptophan (TRP) and its metabolites represents a great challenge because of their diverse chemical properties, e.g., presence of acidic, basic, and nonpolar functional groups and their immensely different concentrations in biological matrices. A short ultra high-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) method was validated for targeted analysis of TRP and its 11 most important metabolites derived via both kynurenine (KYN) and serotonin (SERO) pathways in human serum and cerebrospinal fluid (CSF): SERO, KYN, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, anthranilic acid, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), xanthurenic acid, melatonin, picolinic acid (PICA), and quinolinic acid (QUIN). After selecting the “best” reversed-phase column and organic modifier, DryLab®4 was used to optimize the gradient time and temperature in chromatographic separation. To achieve absolute quantification, deuterium-labeled internal standards were used. Among all compounds, 3 were analyzed in derivatized (butyl ester) forms (3-HK, PICA, and QUIN) and the remaining 9 in underivatized forms. Validation was performed in accordance with the ICH and FDA guidelines to determine the intraday and interday precision, accuracy, sensitivity, and recovery. To demonstrate the applicability of the developed UHPLC–MS/MS method, the aforementioned metabolites were analyzed in serum and CSF samples from patients with multiple sclerosis (multiple sclerosis group) and those with symptomatic or noninflammatory neurological diseases (control group). The concentration of QUIN dramatically increased, whereas that of KYNA slightly decreased in the multiple sclerosis group, resulting in a significantly increased QUIN/KYNA ratio and significantly decreased PICA/QUIN ratio.