C18 guard column

To confirm and compare unbound drug concentrations, plasma protein and medium protein binding were evaluated for each drug utilizing rapid equilibrium dialysis (RED) as described elsewhere (Wetmore et al., 2012 (link); Waters et al., 2008 (link)). The RED assay was conducted using single use RED inserts (cat. no. 90006, Pierce Biotechnology, Rockford, IL) according to instructions, with protocol modification to incorporate “no protein” equilibrium controls. Equilibrium controls comprising of PBS buffer in both sample and buffer chambers were used to ensure drugs are fully equilibrated within the device in the absence of proteins. All RED assays were completed in triplicate. Drug concentrations were measured using an Agilent (Santa Clara, CA) 6470 triple quadrupole mass spectrometer operating in positive ion mode with a Waters Acquity H class HPLC (Milford, MA). Samples were spiked with a known amount of internal standard sotalol (CAS: 959–24-0) prior to analysis. Chromatographic separation was achieved using a linear acetonitrile gradient on a C18 column (Agilent Zorbex Eclipse Plus C18 3.0 X 50 mm, 1.8 μm) with a C18 guard column (Agilent). The mobile phase consisted of 0.1% formic acid, the flow rate was kept at a constant 0.4 μl/min. Complete mass spec conditions for the three drugs are listed in Table S1¹.

A Glu and KP metabolism assay was performed on the brain tissue and serum of the animals. All metabolisms were using an HPLC-1100 system (Agilent Technologies, USA) equipped with a quaternary pump, and Glu, TRP, and KYN were measured by a UV detector, while QA was fluorescence. The samples were analyzed with an Agilent C18 column (5 micron particle size, L × I.D. 25 cm × 4.6 mm) preceded by a C18 guard column (Agilent Technologies, USA). For TRP and KYN, in the mobile phase, 6% acetonitrile was dissolved in a buffer containing 15 mM acetic acid–sodium acetate (pH 5.3) (Liang et al., 2019 (link)). For Glu, the gradient elution (GQ) system consisted of 0.05 mol/L sodium acetate buffer (pH = 6) and acetonitrile/water (V/V = 50:50). For QA, as mobile phase solvents, acetonitrile formate (0.1%) and formic acid (0.1% v/v) were added to water and methanol (Gibney et al., 2014 (link)).

All samples were carried on an Agilent 1200 series HPLC and interfaced to an Agilent 6410 triple-quadrupole mass spectrometer equipped with an electrospray ionization source (Agilent Corporation, MA, USA). A ZORBAX SB-C18 column (3.5 μm, 2.1×150 mm, I.D. Agilent Corporation, MA, USA) and a C18 guard column (5 μm, 4.0×2.0 mm, Agilent Corporation, MA, USA) was used. Acetonitrile-5 mM ammonium acetate solution (the concentration of acetonitrile remain 30% in 2.0 min, v/v) was used as mobile phase for the gradient elution at a flow rate of 0.3 ml/min. The column temperature was maintained at 30°C. The injection volume was 10 μL and the analysis time was 2.0 per sample. The ESI source in negative mode was chosen.

To understand the impact of stress on KP and its metabolic enzymes, the brain tissue and serum concentrations of tryptophan (TRP) and its IDO-catalyzed metabolite KYN、KYNA、QA were measured by HPLC and individual metabolite peaks detected were collected as HPLC fractions. The analysis was performed on an HPLC-1100 system (Agilent Technologies, USA) equipped with a quaternary pump and a UV detector for TRP and KYN, a fluorescence detection was used for measure KYNA and QA. HPLC analysis of the samples was performed using an Agilent C18 column (5 μm particle size, L × I.D. 25 cm × 4.6 mm) preceded by a C18 guard column (Agilent Technologies, USA). For TRP and KYN, the mobile phase was 15 mM acetic acid-sodium acetate buffer (pH 5.3) containing 6% acetonitrile by volume [37 (link)]. For KYNA and QA, Water and methanol with acetonitrile formate (0.1%) and formic acid (0.1%v/v) were used as mobile phase solvents [38 (link)].