Lc ms chromasolv

Ginger was obtained from a local Slovenian vendor (Mercator d.d., Ljubljana, Slovenia). The natural material was dried by a lyophilization process (T_{lyophilization}= −30 °C, t_{lyophilization}= 72 h, p = 0.04 mbar vacuum) and then ground using a grinder before extraction procedures. The mean particle size has been determined by sieve analysis and was 0.5 mm.
Chemicals used in this study were ethanol (C₂H₆O) (Sigma-Aldrich, St. Louis, MO, USA, HPLC grade, ≥99.9%), carbon dioxide (CO₂) (Messer; MG, Ruše, Slovenia, purity 2.5), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (SigmaAldrich, Darmstadt, Germany, ≥97.0%), Folin-Ciocalten reagent (FC) (Sigma Aldrich), sodium(V)carbonate (Na₂CO₃) (Sigma Aldrich, ≥99.9%), gallic acid (C7H6O5) (Sigma Aldrich, 97.5–102.5%), and methanol (H₃OH) (Honeywell, Charlotte, NC, USA, LC-MS CHROMASOLV^®, ≥99.9%).

Plasma or RBC samples were resuspended at room temperature, and 100 μL of the samples from each group was mixed with 300 μL methanol (≥ 99.9%, LC‒MS CHROMASOLV) for protein precipitation, vortexed and incubated at − 20 °C for 2 h. The samples were vortexed again and centrifuged at 14,000 rpm for 15 min, and the supernatants were transferred to new glass vials. The supernatants were evaporated in a SpeedVac EZ-2 Plus (GeneVac, Ipswich, UK) at 35–40 °C. To prepare the extracts for liquid chromatography coupled to mass spectrometry (LC‒MS) analysis, they were first resuspended in 200 µl of deionized water containing 0.1% formic acid (LC‒MS CHROMASOLV, Honeywell, Seelze, Germany) and vortexed for 2 min for complete mixing. The extracts were then filtered using a 0.45 µm hydrophilic nylon syringe filter for LC‒MS/MS analysis. Equal volumes (10 µl) of 30 samples from each study group were collected and pooled for quality control (QC) and placed in the autosampler at 4 °C to analyse the reproducibility of the analysis.

LC coupled to mass spectrometry (MS)-grade water and methanol (LC/MS Chromasolv) were supplied by Honeywell Riedel-de Haën™ (Seelze, Germany). Analytical standards, ISTDs, and formic acid were supplied by Sigma Aldrich (St. Louis, MO, USA), Toronto Research Chemicals (Ontario, Canada), TLC PharmaChem (Ontario, Canada), or Santa Cruz Biotechnology (Dallas, TX, USA), depending on availability (details for analytes and ISTDs are presented in Table 1).
The standard and ISTD solutions (commercially obtained or prepared in the laboratory from powder), at approximately 1 mg/mL, were stored at − 20 °C and diluted in methanol/water (4:1 v/v) on the day of analysis for spiking mixtures and calibration curves. Calibration levels ranged over three orders of magnitude for each metabolite, and concentrations were selected based on MS sensitivity of each metabolite and its expected concentrations in the samples.

Quantitative analysis of UAMC-3203 was performed with the LC-MS/MS system that consisted of an Agilent 1290 series liquid chromatograph connected to an Agilent 6495 triple-quadruple mass spectrometer (Agilent Technologies, Inc., USA). The mobile phase consisted of 0.1% formic acid (Fisher chemical, Geel, Belgium), 1 mM ammonium formate (Honeywell, Fluka, Seelze, Germany) in Milli-Q-H2O (eluent A), and acetonitrile (LC-MS Chromasolv, Honeywell, Riedel-de Haen, Seelze, Germany) (eluent B). Gradient elution was used: 0.0 to 3.0 min: 25%→95% B, 3.0 to 3.5 min: 95% B, 3.5 to 3.6 min: 95%→25% B, 3.6 to 5.0 min 25% B. Solvent flow was 0.3 mL/min, and Poroshell 120 SB-C18 column (2.1 mm × 50 mm, 2.7 µm, Agilent Technologies, Inc., USA) was maintained at 60 °C. The injection volume was 2 µL. Nitrogen was used as a drying, nebulizer, and collision gas. The following conditions were used in the electrospray ion source: positive ion mode, sheath gas flow rate 11 L/min at 350 °C, drying gas flow rate 16 L/min at 200 °C, nebulizer gas pressure adjusted to 25 psi, capillary voltage 3500 V (ESI+), fragmentor voltage 380 V, dwell time 150 ms and cell accelerator voltage 5 V. The data were analyzed with Agilent Mass Hunter Quantitative Analyzed software (vB.09.00, build 9.0.647.0, Agilent Technologies, CA, USA). The precursor and fragment ions are presented in Supplementary Table S1.