Prominence lc system

The levels of Trp and γ-aminobutyric acid (GABA) in the hypothalamus and cortex were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by using a Prominence LC system (SHIMADZU, Kyoto, Japan) connected with a QTRAP 5500 mass spectrometer (AB SCIEX, Foster City, CA). A Restek Ultra Aqueous C18 column (3 μm, 100 mm × 2.1 mm, Bellefonte, PA, United States) was used for chromatographic separation. Acetonitrile and water containing 0.1% formic acid constituted the mobile phase at an eluting rate of 0.4 mL/min. The hypothalamus and cortex samples were homogenized with 100 μL water and then the 50 μL homogenate was spiked with 20 μL 500 ng/mL acetaminol as internal standard and 10 μL trifluoroacetic acid to precipitate the protein. The mixture was blended and centrifuged at 4°C, 21,000 ×g for 20 min. Then, the supernatant was collected and a 2 μL aliquot was injected into the system.

Urine sample preparation for LC-MS analysis was performed as we previously reported19 (link). Briefly, the urine samples were thawed at room temperature. 100 μl of each thawed urine sample was precipitated by 100 μl of methanol. The mixture was then centrifuged under 14000 g for 10 minutes at 4 °C, and the supernatant was used for LC-MS analysis.
Each 10 μL aliquot of extract was injected into a Shimadzu Prominence LC system (Shimadzu) coupled online to an LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, MA, USA) set at 30000 resolution (at m/z 400). Both positive and negative ion modes were used for sample analysis. The mass scanning range was 50–1000 m/z and the capillary temperature was 350 °C. Nitrogen sheath gas was set at a flow rate of 30 L/min. Nitrogen auxiliary gas was set at a flow rate of 10 L/min. Spray voltage was set to 4.5 kV and 3.0 kV for positive or negative ion mode, respectively. The LC-MS system was run in binary gradient mode. Solvent A was 0.1% (v/v) formic acid/water and solvent B was 0.1% (v/v) formic acid/methanol. The flow rate was 0.2 ml/min. A C-18 column (150 × 2.1 mm, 3.5 μm, Agilent, USA) was used for all analysis. The linear gradient was as follows: 5% B at 0 min, 5% B at 5 min, 100% B at 8 min, 100% B at 9 min, 5% B at 18 min and 5% B at 20 min.

The liquid chromatography with tandem mass spectrometry (LC-MS/MS) system consisted of a Shimadzu Prominence LC system (controller CBM-20A, pumps LC-30AD, and autosampler SIL-30AC; Shimadzu, Tokyo, Japan) with an ion trap time-of-flight mass spectrometer (Shimadzu, Tokyo, Japan). The column used for the separation was an Ascentis^® Express C18, 2.7 µm (50 × 3.0 mm; Supelco Analytical, Darmstadt, Germany). The LC mobile phases consisted of 50% water in 0.1% formic acid (A) and 50% acetonitrile in 0.1% formic acid (B) at a flow rate of 0.25 mL/min at room temperature. The ion spray voltage was 4.5 kV. Nitrogen was used as the nebulising gas at 1.50 L/min. The curved desolvation line (CDL) and heat block temperatures were set to 200 °C. The mass spectrometer was operated in the positive ion mode in the range of m/z 150–600 with the collision energy at 50%. Data analysis and acquisition were performed using ACD/Labs (Version S05S41).

After evaporation of chloroform/methanol (2:1, v/v) with nitrogen blowdown, the extracted lipids were suspended in 40 µL of ethyl acetate/methanol (1:1, v/v), and 25 µL of the suspension was used for HPLC analysis. In the animal studies samples, halocynthixanthin was quantified from the peak area at 450 nm using the calibration curve of purified halocynthixanthin. The HPLC analysis was performed on a TSKgel ODS-80Ts column (4.6 mm × 250 mm, Tosoh, Tokyo, Japan) using a Prominence LC System (Shimadzu, Kyoto, Japan) with a photodiode array detector SPD-M20A (Shimadzu, Kyoto, Japan). For the binary gradient elution, methanol/Milli-Q water (90:10, v/v) containing 0.1% ammonium acetate was used for mobile phase A, and methanol/ethyl acetate (70:30, v/v) containing 0.1% ammonium was used for mobile phase B using the following gradient program: 0–5 min, 0% B; 5–20 min, 0–100% B; 20–35 min, 100% B; 35–40 min, 100–0% B; 40–45 min, 100% B, at a flow rate of 1.0 mL/min.

Free SA determination in wheat leaves was performed by high-pressure-liquid chromatography (HPLC) (Shimadzu Prominence LC System, France) following the method of Allasia et al. (2018) (link) with slight modification where 0.1 g of the leaf sample was homogenized using liquid nitrogen. Separations by HPLC were performed on a C18 column (250 × 4.6 mm, 5 μm) using a linear aqueous MeOH gradient from 10 to 82% (v/v), at a flow rate of 1 ml min⁻¹, over 30.4 min. Quantify with fluorimetric detection (measured at 305 nm; emission at 407 nm) and determine areas under the corresponding peaks of the standard [2-Methoxybenzoic acid (o-Anisic acid, OAA; internal standard)]. Briefly, stock solution of 152 mg OAA in 10 ml 70% aqueous EtOH (v/v) and diluted 1:1, 000 in ultra-pure water. Using the peak area, the standard curve and linear equation were used to determine the amount of free SA.

Saliva samples were assessed for cortisol and cortisone content by liquid chromatography-mass spectrometry (LC-MS) at the University of Manchester in the Department of Clinical Biochemistry. A Shimadzu Prominence LC system (Shimadzu, Milton Keynes, UK) was used for chromatography. The eluate was injected directly into a Quattro MicroTM tandem mass spectrometer (Waters, Manchester, UK). MassLynx NT 3.5 software was used for system control and data processing. This software used the height of the detected peaks, 1/x weighting and linear least-squares regression to produce a standard curve to derive concentrations.