Please cite this article in press as: E. Hényková, et al., Stable isotope dilution ultra-high performance liquid chromatography–tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.02.009 ARTICLE IN PRESS G Model CHROMA-357283; No. of Pages 13 Journal of Chromatography A, xxx (2016) xxx–xxx Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Stable isotope dilution ultra-high performance liquid chromatography–tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid Eva Hényková a , Hana Pˇ rikrylová Vránová b , Petra Amakorová a , Tomᡠs Pospíˇ sil c , Asta ˇ Zukauskait ˙ e a,d , Magdaléna Vlˇ cková a , Lubor Urbánek a , Ondˇ rej Novák a,∗ , Jan Mareˇ s b , Petr Kaˇ novsk ´ y b , Miroslav Strnad a,∗∗ a Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science of Palack´ y University & Institute of Experimental Botany CAS, ˇ Slechtitel˚ u 27, CZ-783 71 Olomouc, Czech Republic b Department of Neurology, University Hospital in Olomouc, I. P. Pavlova 6, CZ-775 20 Olomouc, Czech Republic c Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science of Palack´ y University, ˇ Slechtitel˚ u 27, CZ-783 71 Olomouc, Czech Republic d Department of Biology, Faculty of Medicine and Dentistry, Palack´ y University, Hnˇ evotínská 3, CZ-775 15 Olomouc, Czech Republic a r t i c l e i n f o Article history: Received 8 October 2015 Received in revised form 29 January 2016 Accepted 2 February 2016 Available online xxx Keywords: Tryptophan metabolism UHPLC–MS/MS Neurochemicals Serum Cerebrospinal fluid a b s t r a c t Many compounds related to L-tryptophan (L-TRP) have interesting biological or pharmacological activ- ity, and their abnormal neurotransmission seems to be linked to a wide range of neurodegenerative and psychiatric diseases. A high-throughput method based on ultra-high performance liquid chromatogra- phy connected to electrospray tandem mass spectrometry (UHPLC–ESI-MS/MS) was developed for the quantitative analysis of L-TRP and 16 of its metabolites in human serum and cerebrospinal fluid (CSF), representing both major and minor routes of L-TRP catabolism. The combination of a fast LC gradient with selective tandem mass spectrometry enabled accurate analysis of almost 100 samples in 24 h. The standard isotope dilution method was used for quantitative determination. The method’s lower limits of quantification for serum and cerebrospinal fluid ranged from 0.05 to 15 nmol/L and 0.3 to 45 nmol/L, respectively. Analytical recoveries ranged from 10.4 to 218.1% for serum and 22.1 to 370.0% for CSF. The method’s accuracy ranged from 82.4 to 128.5% for serum matrix and 90.7 to 127.7% for CSF matrix. All intra- and inter-day coefficients of variation were below 15%. These results demonstrate that the new method is capable of quantifying endogenous serum and CSF levels of a heterogeneous group of com- pounds spanning a wide range of concentrations. The method was used to determine the physiological levels of target analytes in serum and CSF samples from 18 individuals, demonstrating its reliability and potential usefulness in large-scale epidemiological studies. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Interest in L-tryptophan (L-TRP) metabolites has increased over the last few decades due to their influence on human health. Many L-TRP metabolites exhibit biological and/or pharmacological ∗ Corresponding author. Fax: +420 585634870. ∗∗ Corresponding author. E-mail addresses: novako@ueb.cas.cz (O. Novák), miroslav.strnad@upol.cz (M. Strnad). activity, including kynurenines, indoleamines and kynuramines. A simplified scheme of tryptophan metabolism is shown in Fig. 1. The major route of L-TRP catabolism is regulated by an enzy- matic cascade known as the kynurenine pathway (KP) [1]. The principal branch of the KP generates quinolinic acid (QA) and nicoti- namide (NAm), while the side branches generate kynurenic acid (KA) and xanthurenic acid (XA). In mammals, L-kynurenine (KYN) is the pivotal metabolite of the KP and represents the predominant degradation product of ingested L-TRP. KYN is further converted to the neuroprotective compound KA via irreversible transamination mediated by kynurenine aminotransferases (KATs). Alternatively, it http://dx.doi.org/10.1016/j.chroma.2016.02.009 0021-9673/© 2016 Elsevier B.V. All rights reserved.