Accucore c18 column

Lipids were separated using an Accucore C18 column 2.1 × 150 mm, 2.6 μm (Thermo Scientific) and mobile phase solvents consisted in 10 mM ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10 mM ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The gradient profile used was 30% B for 3 min, 30–43% B in 2 min, 43–55% B in 0.1 min, 55–65% B in 10 min, 65–85% B in 6 min, 85–100% B in 2 min and 100% B for 5 min. Lipids were eluted from the column at 0.4 ml/min, the oven temperature was set at 45 °C, and the injection volume was 5 μl. Autosampler temperature was set at 20 °C to prevent lipid aggregation.

Analysis of barbiturates was performed using an Ultimate 3000 UPLC system interfaced with a TSQ Quantum Access MAX triple quadrupole Mass Spectrometry (Thermo Scientific, Waltham, MA, USA). Chromeleon client software (Thermo Scientific, Waltham, MA, USA) was used for LC control, and Xcalibur software (Thermo Scientific, Waltham, MA, USA) was used to control the MS, data acquisition, and data processing. Chromatographic separation was conducted at 45 °C on an Accucore C18 column (2.1 mm × 100 mm, 2.6 μm) (Thermo Scientific, Waltham, MA, USA) with gradient elution. Water containing 0.5% of acetic acid (v/v) was used as mobile phase A, and methanol was used as mobile phase B. Mobile phase B started from 20% for 0.5 min, and then increased to 95% in 1.5 min and 95% was kept for 2 min. At the end, mobile phase B was decreased to 20% within 0.1 min, which was kept for 1.9 min for equilibration. The flow rate was set to 0.4 mL min⁻¹ and the injection volume was 10 μL. Mass spectrometry was performed with an ESI source in the negative-ionization mode with a sheath gas of 40 Arb and aux gas of 10 Arb. The capillary temperature was set at 320 °C and the vaporizer temperature was set at 350 °C. The spray voltage was 3.2 kV. The parameters for the quantification selected reaction monitoring (SRM) transitions are presented in Table 4.

Ginsenosides standards were obtained from Extrasynthese (Lyon, France). The extract was analysed with HPLC-MS after a modified protocol described elsewhere [26 (link),58 (link),59 (link)]. The samples were dissolved in methanol and separated on a Surveyor HPLC (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an Accucore C18 column (150 mm × 3 mm, 2.6 µm; Thermo Fisher Scientific, Waltham, MA, USA) using a gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B): from 80% A to 70% A in 5 min, to 55% A in 15 min, to 25% A in 10 min which was kept for 2 min. The column flow was 500 µL min⁻¹ and the temperature was kept at 40 °C. A LTQ Orbitrap Velos (Thermo Fisher Scientific, Waltham, MA, USA) was used as a detector with an ESI ion source. The instrument was operated in FTMS negative mode with a resolution of 30,000 and a scan range from 200–2000 m/z. For quantitation, the extracted masses of the formiate adduct ions were used (see Table 2). Calibration was carried out with twelve ginsenoside standards in the range from 30 µg L⁻¹ to 130 mg L⁻¹ in 6 different concentrations, all measured in triplicate. The injection volume for all samples was 1 µL.