A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4′-Nitroacetophenone

Author:

Jankech Timotej12ORCID,Gerhardtova Ivana12ORCID,Majerova Petra1,Piestansky Juraj13ORCID,Fialova Lubica1,Jampilek Josef12ORCID,Kovac Andrej14ORCID

Affiliation:

1. Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska Cesta 9, 845 10 Bratislava, Slovakia

2. Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University Bratislava, Ilkovicova 6, 842 15 Bratislava, Slovakia

3. Department of Galenic Pharmacy, Faculty of Pharmacy, Comenius University Bratislava, Odbojarov 10, 832 32 Bratislava, Slovakia

4. Department of Pharmacology and Toxicology, University of Veterinary Medicine and Pharmacy in Kosice, Komenského 68/73, 041 81 Kosice, Slovakia

Abstract

Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4′-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5–8.2% and 2.3–7.4%, accuracy was in the range of 93.3–109.7% and 94.7–110.1%, limits of detection (LODs) were in the range of 0.15–9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer’s disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

Funder

Slovak Research and Development Agency

Slovak Grant Agency for Science

Publisher

MDPI AG

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