Chromeleon 7.2 chromatography data system

Mice feces was mixed at a ratio of 1:2 (w/v) with 80% methanol/water (v/v) at 4 °C. Mice plasma samples were thawed on ice and mixed at a 1:1 ratio (v/v) with 80% methanol/water (v/v). Sample extraction was performed as described above and 30 µl of the obtained supernatants were injected into the HPLC for analysis. A Dionex Ultimate® 3000 uHPLC (Thermo Scientific) was used for chromatographic separation of D-Trp. This was achieved on an Astec® CHIROBIOTIC® T Chiral HPLC column, 25 cm × 4.6 mm (Sigma-Aldrich™) with 5 µm particle size. The mobile phase had a flow rate of 1 ml/min, comprised methanol (30%), water (70%), and formic acid (0.02%). D-Trp was detected based on comparison with standards, of their respective retention times and UV emission spectra at 205 nm. Chromeleon™ 7.2 Chromatography Data System (Thermo Scientific™ Dionex™) was utilized to analyze the obtained results.

To detect DA content in DA cells and fly heads, cells and fly heads were collected and homogenized in 0.5 N perchloric acid. After centrifugation and filtration, the supernatants are used to undergo HPLC analysis subsequently. To detect DA content in solutions after oxidation, solutions are filtrated and collected for analysis after different time of reaction. The level of DA were measured using a Reversed-phase UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) with an electrochemical detector and a reversed-phase column (DBS HYPERSIL C18, 15 cm × 3.0 mm, 3 μm, 120 Å pore size, Thermo Fisher Scientific) and analyzed under the control of a Chromeleon™ 7.2 Chromatography Data System (Thermo Fisher Scientific). The mobile phase was a mixture of 0.1 M sodium phosphate, 10 mM sodium 1-heptanesulfonate, 0.1 mM EDTA and 8% methanol (v/v), adjusted to pH 2.95 with 85% phosphoric acid. All solutions for HPLC analysis were double filtered through 0.2 μm membranes and degassed before use. The flow rate was 0.5 mL per min. DA peak appears in HPLC chromatograph at about 13.5 min. DA content in solutions was acquired based on the DA peak areas in HPLC chromatography.

For protein precipitation each 300 μl serum sample was treated with 50.6 μl of 72% trichloroacetic acid. Samples were centrifuged at maximum speed for 12 min and the supernatants were frozen overnight at −20 degrees. After thawing the samples, the centrifugation step was repeated and the obtained supernatants were used for HPLC analysis. A Dionex Ultimate^® 3000 uHPLC (Thermo Scientific, Waltham, MA, USA) was used for chromatographic separation of kyn and trp. This was achieved on a reversed phase Accucore™ aQ column (Thermo Scientific™) with 2.6 μm particle size with a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). Kyn and trp were detected based on comparison with standards, their retention times and UV emission spectra at 365 nm and 280 nm, respectively. Results were analyzed using the Chromeleon™ 7.2 Chromatography Data System (Thermo Scientific™ Dionex™).

Analytics data was collected using Chromeleon 7.2 Chromatography Data System (Thermo Fisher Scientific, UK). Data was compiled using Microsoft Excel 2010 which compared samples using used two-tailed, unpaired, parametric student's T-test. Graphical representation used GraphPad Prism 7.

Striatum tissues dissected from mice brain were homogenized in 0.5 N perchloric acid with 100 µM of deferoxamine mesylate and 100 µM of glutathione. The homogenized samples were further sonicated, centrifuged and the supernatants were filtered using 0.1 µm PVDF centrifugal filters before collecting the filtrates for HPLC analysis. A reversed-phase UltiMate 3000 HPLC system (Thermo Fisher Scientific) with an electrochemical detector and a reversed-phase column (Vydac Denali, C18, 4.6 x 250 mm, 5 µm particle size, 120 Å pore size) were used to run the samples. The HPLC run was performed at a flow rate of 0.5 ml per minute with a mobile phase containing 1.3% sodium acetate, 0.01% EDTA (pH8.0), 0.5% sodium 1-heptanesulfonate, 7% acetonitrile (v/v) and 2% methanol (v/v), adjusted to pH 4.0 with acetic acid. All buffers used for HPLC analysis were double filtered through 0.2 µM nylon membranes. Dopamine in the samples was identified by retention time of dopamine standard (around 14 min) and quantified by measuring the area under the peak using the software Chromeleon^TM 7.2 Chromatography Data System (Thermo Fisher Scientific). The areas under the peaks were normalized to their respective tissue weight.

High-performance liquid chromatography (HPLC) measurement of Kyn was performed in cell culture supernatants collected from each treatment type. One mL of supernatant sample was first treated with 168.6 μL of 72% trichloroacetic acid to precipitate out dissolved proteins; subsequently, samples were centrifuged at maximum speed for 10 min, and the resulting protein-free supernatants were transferred into glass HPLC vials for further HPLC analysis. Chromatographic separation of Kyn was achieved on a Dionex Ultimate^® 3000 UHPLC (Thermo Scientific) on a reversed-phase Accucore^TM aQ column (Thermo Scientific^TM) with 2.6-μm particle size. The mobile phase gradient consisted of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). Standards for all analytes were utilized to determine the retention time and UV emission spectrum at 365 nm for Kyn. Analyte concentration in samples was analyzed by comparison with the respective standards using the Chromeleon^TM 7.2 Chromatography Data System (Thermo Scientific^TM Dionex^TM).