Prominence liquid chromatography system

Salivary samples were obtained using a Salivette kit (Sarstedt, Nümbrecht, Germany) to measure cortisol and cortisone concentrations. Once collected, samples were stored at 4 °C before being analyzed. Salivary cortisol and cortisone were measured by LC-MS/MS (Prominence liquid chromatography system; Shimadzu, Nakagyo, Japan; and 5500 Qtrap detector, Sciex, Framingham, MA, USA). A liquefying agent (Sputasol; Thermo Fisher Scientific, Waltham, MA, USA) was added to 400 µL of saliva sample, which was then incubated for 30 min at 37 °C. Next, solid-phase extraction with a hydrophilic lipophilic balance (Waters^TM) was performed before injecting the extract into the LC-MS/MS system. The coefficients of variability for the cortisol and cortisone assays were 8 and 9%, respectively.

Chromatographic separation was carried out on a prominence liquid chromatography system (Shimadzu^®, Kyoto, Japan) with an HTS PAL automatic injector (CTC Ana-lytic, Zwingen, Switzerland). Chromatographic analysis was performed on a Zorbax Eclipse C₁₈ analytical column (5 μm, 150 × 4.6 mm i.d.—Agilent Technologies^®, CA, United States) in isocratic mode. The mobile phase consisted of acetonitrile and methanol (80:20, v/v) and an aqueous solution of formic acid 0,1% (65:35, v/v) at a flow rate of 0.8 mL/min. The injection volume was 20 µL. The temperature of the autosampler and column oven were 10 °C and 40 °C, respectively. The run time was 5.50 min (Supplementary Table S1).

One-dimensional proton nuclear magnetic resonance spectra of CDCl₃ were recorded on a JEOL JNM‐ECA‐600 (600 MHz); residual CHCl₃ (δ = 7.26 ppm) was used as an internal reference. High-performance liquid chromatography (HPLC) was performed using a Shimadzu Prominence liquid chromatography system with an SCL-10Avp system controller, an SPD-M20A photodiode array detector (300–800 nm), two LC-20AD pumps, a DGU-20A₃ degasser, and a CTO-20AC column oven. Liquid chromatography–mass spectrometry (LC–MS) was performed using a Shimadzu LCMS-2010EV system, which was based on the atmospheric pressure chemical ionization mode (Hirose et al. 2022 (link)). For all LC(-MS) analyses, the column oven was set to 30 °C.
Flash column chromatography (FCC) and reversed-phase HPLC were performed using silica gels (Merck Kieselgel 60, 0.040–0.063 mm or Wakogel C-300) and a packed octadecylated silica gel column (Cosmosil 5C₁₈-AR-II, Nacalai Tesque), respectively. The sample was dissolved in an HPLC eluent. The solution was filtered using a Cosmonice filter (0.45 μm pore size, Nacalai Tesque), and the filtrate was subjected to HPLC. Methanol and distilled water for HPLC solvents were purchased as HPLC grade from Nacalai Tesque. Acetone for HPLC grade was purchased from FUJIFILM Wako Pure Chemical.

The sugar profile and the content of ethanol and glycerol were examined by means of High-Performance Liquid Chromatography (HPLC) [26 (link)]. Degassed and centrifuged (5500 rpm, 10 min) beer samples were diluted two-fold (1:1) with ultrapure water and filtered through nylon syringe filters with 0.45 µm pore size for chromatographic vials. The samples were then analysed using a Prominence liquid chromatography system (Shimadzu Corp., Kyoto, Japan) equipped with a Rezed ROA-Organic Acid H + column (300 × 4.6 mm) from Phenomex (Torrance, CA, USA). The following measurement parameters were used: sample volume: 20 µL, separation temperature: 60 °C, mobile phase flow rate: 0.6 mL/min, mobile phase: 0.005 M H₂SO₄, detection temperature: 50 °C. The concentration of ethanol, glycerol, dextrins, maltose, glucose and maltotriose was based on five-point calibration curves using Chromax 10.0 software (Pol-Lab, Wilkowice, Poland). All measurements were performed in triplicate.

High-resolution mass spectrometry analyses with electrospray ionization (ESI) were performed using a Bruker micrOTOF–QII mass spectrometer coupled to a Shimadzu Prominence liquid chromatography system, consisting of two analytical pumps model LC-20AD, with SIL-20AHT automatic injector, SPD-20A UV/Vis detector, CTO-20A column oven, CBM-20A controller. Each compound analyzed by this technique was prepared at 1 mg/mL using LCMS-grade methanol. The column used was a Phenomenex Luna C18 (5 µm, 150 × 2 mm). The flow was 0.2 mL/min, and the mobile phase was a mixture of solvents A (0.1% HCOOH in H₂O) and B (0.1% formic acid in MeOH). The gradient started at 5% B (0 min) and was maintained for 2 min. Then, B was incremented to 100% from 5 to 30 min and maintained for 5 min. The oven temperature was 40 °C, and the wavelength was 254 and 280 nm. The ESI interface was operated in positive ion mode with 4.5 kV in the capillary and 0.5 kV in the endplate offset. The pressure of the nebulization gas was 0.4 Bar; the drying gas was maintained at a flow rate of 8 L/min at 200 °C. The collision and the quadrupole energy were set to 12 and 6 eV, respectively. RF1 and RF2 funnels were programmed to 150 and 200 Vpp, respectively. The mass spectra were calibrated using sodium formate.