Journal of Pharmaceutical and Biomedical Analysis 107 (2015) 426–431 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur nal ho me page: www.elsevier.com/lo cate/jpba A single-run liquid chromatography mass spectrometry method to quantify neuroactive kynurenine pathway metabolites in rat plasma Laura Orsatti a , Roberto Speziale a , Maria Vittoria Orsale a , Fulvia Caretti a , Maria Veneziano a , Matteo Zini a , Edith Monteagudo a , Kathryn Lyons c , Maria Beconi d , Kelvin Chan e , Todd Herbst b , Leticia Toledo-Sherman b , Ignacio Munoz-Sanjuan b , Fabio Bonelli a , Celia Dominguez b,∗ a IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia (Roma), Italy b CHDI Management/CHDI Foundation, 6080 Center Drive, Los Angeles, CA, USA c Freelance Consultant, P.O. Box 64, Holland, NY, USA d Retrophin Inc. (RTRX), 301 Binney Street, 3rd Floor, Cambridge, MA 02142, USA e Alcon Research, Ltd., 6201 South Freeway, Fort Worth, TX 76134-2099, USA a r t i c l e i n f o Article history: Received 27 October 2014 Accepted 16 January 2015 Available online 24 January 2015 Keywords: Kynurenine Plasma Mass spectrometry Liquid chromatography Tryptophan metabolism a b s t r a c t Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neu- rodegenerative disorders. Tryptophan is transported across the blood–brain barrier and converted via the kynurenine pathway to N-formyl-l-kynurenine, which is further degraded to l-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC–MS/MS method to quantify l-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-l-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical prop- erties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chro- matography, detected by electrospray mass spectrometry. Quantitation range was 0.098–100 ng/ml for 3HK, 9.8–20,000 ng/ml for KYN, 0.49–1000 ng/ml for KYNA and AA. The method was linear (r > 0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%). © 2015 Elsevier B.V. All rights reserved. 1. Introduction Tryptophan metabolism via the kynurenine pathway plays a key role in several physiological processes and its alteration has been implicated in the pathophysiology of a wide range of ∗ Corresponding author. Tel.: +1 310 342 5503; fax: +1 310 342 5519. E-mail addresses: l.orsatti@irbm.it (L. Orsatti), r.speziale@irbm.it (R. Speziale), m.orsale@irbm.it (M.V. Orsale), f.caretti@irbm.it (F. Caretti), m.veneziano@irbm.it (M. Veneziano), m.zini@irbm.it (M. Zini), e.monteagudo@irbm.it (E. Monteagudo), kathyalyons@gmail.com (K. Lyons), Maria.Beconi@retrophin.com (M. Beconi), kelvinchan2014@verizon.net (K. Chan), todd.herbst@chdifoundation.org (T. Herbst), leticia.toledosherman@chdifoundation.org (L. Toledo-Sherman), ignacio.munoz@chdifoundation.org (I. Munoz-Sanjuan), f.bonelli@irbm.it (F. Bonelli), celia.dominguez@chdifoundation.org (C. Dominguez). disorders, including Alzheimer’s disease, amyotrophic lateral scle- rosis, Huntington’s disease, AIDS dementia complex, malaria, cancer, depression and schizophrenia [1]. The kynurenine path- way also plays a key modulatory role in the immune response and mediates interactions between immunological and neuronal func- tions. Study of these (patho)physiological mechanisms, including potential therapeutic approaches, requires accurate quantification of kynurenine and its major degradative metabolites in human and rodent samples. HPLC methods employing ultraviolet (UV) [2,3], fluorescence [4–9], electrochemical [10] and mass spectrometric [11–13] detec- tion have been described. Among these, HPLC–MS proved to be the most valuable for the specificity and sensitivity of the mass spectrometry detector. Moreover, since a single HPLC–MS method can measure more metabolites together, lower sample http://dx.doi.org/10.1016/j.jpba.2015.01.030 0731-7085/© 2015 Elsevier B.V. All rights reserved.