Dgu 20a3 prominence degasser

An acidic mobile phase was used for detection of insulin. The mobile phase consisted of 0.2 M sodium sulphate anhydrous (adjusted to pH 2.3 with ortho phosphoric acid) and acetonitrile (74:26). The aqueous solution was filtered through 0.45 µm pore size nylon membrane filter (Whatman international, Maidstone, UK) under vacuum and degassed prior to use. The analysis was run at a flow rate of 1.2 mL/min and sample injection volume of 20 µL. The detector was set a wavelength of 214 nm. The chromatographic analysis was performed using a Shimadzu Prominence HPLC system (Kyoto, Japan) consisting of an in-line DGU-20A3 Prominence degasser, LC-20AD Prominence solvent delivery pump, SIL-20A HT prominence auto sampler, CTO-10AS VP column oven, and SPD-M20A Prominence UV-VIS detector. Data acquisition and analysis were performed using Shimadzu LC solution software Version 1.24 SP1 (Kyoto, Japan).

The stock solution of MPSEs was freshly prepared at 1 mg/mL volume in purified water and was filtrated with a 0.45 μm syringe filter (Whatman's). HPLC profiles of MPSEs were performed using a Shimadzu LC-20AD Prominence Liquid Chromatograph system equipped with a SPD-M20A Prominence Diode Array Detector and with a DGU-20A3 Prominence Degasser (Shimadzu, Japan). The wavelength scanning was done between 190 and 800 nm, while any wavelength occurring at 270 nm was carefully monitored. Notably, 20 μL of each extract (300 μg/mL) was injected into the ZORBAX Eclipse Plus C18 Analytical column (250 × 4.6 mm i.d., 5 μm particle) with an Eclipse Plus-C18 Analytical Guard Column (12.5 × 4.6 mm i.d., 5 μm particle. Successful separation of MPSEs phytochemicals was accomplished using the following mobile phase; mobile A: 0.1% aqueous trifluoroacetic acid and mobile B: 100% acetonitrile. The time program for gradient elution was 0–5 minutes, 5% B; 7–12 minutes, 10% B; 14–19 minutes, 15% B; 21–26 minutes, 20% B; 30–35 minutes, 25% B; 37–45 minutes, 30% B; 50–55 minutes, and 100%B; 60–65 minutes, 5% B.

IDO activity was measured through the quantification of Trp and Kyn using high performance liquid chromatography (HPLC) as detailed earlier (Hall et al. 2016 (link)). The HPLC system consisted of a Shimadzu CBM-20 A Prominence communications bus control module, two Shimadzu LC-20AD UFLC liquid chromatograph pumps fitted with a solvent mixer, a Shimadzu DGU-20A3 Prominence degasser, a Shimadzu SIL-20 A HT UFLC Prominence chilled autosampler module, a Shimadzu CTO-20AC Prominence column oven, a Shimadzu SPD-M20A Prominence Diode array detector, and Lab solutions software. The column used for the analysis of Trp and Kyn was a Phenomenex Gemini (5 μm, 250 × 4.6 mm) reverse phase column (Phenomenex, Lane Cove, Australia) fitted with a Phenomenex Security Guard guard cartridge (Phenomenex).
Trp and Kyn were quantified using an isocratic method run over 10 min using 0.1% GAC (solvent A) and ACN (solvent B) (90:10% v/v) and 10 µL injections of sample were employed to quantify the analytes of interest. The UV absorbance was monitored at 360 nm (Kyn) and 275 nm (Trp).