Nexera x2 uhplc

Polyphenols and amygdalin were analysed on a Shimadzu Nexera X2 UHPLC coupled with mass spectrometer LCMS 8040 (Shimadzu, Japan) as described in Ben-Othman et al. (2021) (link) with slight modifications. A reverse phase column ACE Excel 3 (C18, PFP, 100 × 2.1 mm; from ACE® Advanced Chromatography Technologies Ltd., Scotland) and pre-column (SecurityGuard ULTRA, C18; from Phenomenex, USA) were used at 40 °C for the separation of individual compounds. The flow rate of the mobile phase was 0.4 mL/min; the sample size was 1 μL. The mobile phase consisted of 1% formic acid in Milli-Q water (A) and 1% formic acid in methanol (B). Separation was carried out for 38 min under the following conditions: gradient 0–10 min, 10–25% B; 10–15 min, 25–35% B; 15–27 min, 35%–80% 27–30min 80-95 29–34 min isocratic 95% B, and re-equilibration of the system with 10% B 5 min.
Phenolic compounds were identified by comparing the retention times, UV spectra, and parent/daughter ion masses with those of the standard compounds described in Ben-Othman et al. (2021) (link).

Detection and quantification of mycotoxins were performed with high-performance liquid chromatography coupled with tandem mass-spectrometry (LC/MS/MS). Chromatographic separation was carried out using Nexera X2 UHPLC (Shimadzu, Tokyo, Japan) equipped with 100 × 2.1 mm, 2.6 µm Kinetex C₁₈ column, (Phenomenex, Torrance, CA, USA). The column was maintained at 40 °C and the injection volume was 2 µL. The mobile phase consisted of 2.5 mM ammonium acetate acidified with 0.1% acetic acid (A), and methanol (B). The methanol (B) concentration was raised gradually from 5% to 95% within 8 min, brought back to the initial conditions at 9 min, and allowed to stabilize for 3 min. The mobile phase was delivered at a flow rate of 0.4 mL/min. The LC system was coupled with API 6500 hybrid triple quadrupole/linear ion trap mass spectrometer (Sciex, Concord, ON, Canada), equipped with a turbo-ion electrospray (ESI) ion source. The mass-spectrometer was operated in scheduled multiple reaction monitoring (sMRM) in both positive and negative mode within a single run. Positive polarity was applied for all analytes except for DON and ZEA. Precursor/quantifier/qualifier ions are specified in Table 4. Source temperature was set at 350 °C, ion-spray voltages at −4500 V (negative mode) and 5000 V (positive mode), curtain gas at 35 arbitrary units (au), nebulizer gas at 60 au, and turbo gas at 40 au.

Blood samples for serum analysis were taken by a medical diagnostic professional according to the experimental protocol at three different time points: 24 h before the run, immediately after the run (within 5 min of finishing the run), and 24 h after the run. The blood was collected in Sarstedt S-Monovette tubes (S-Monovette^® Sarstedt AG&Co, Nümbrecht, Germany) containing a coagulation accelerator. The serum was portioned and frozen at −80 °C until analysis.
For the amino acid analysis, we used a modified method developed by Waraksa et al. 2019 [9 (link)]. Serum proteins were first precipitated and derivatized. Quantitative analysis was performed using liquid chromatography–tandem mass spectrometry (Shimadzu Nexera X2 UHPLC (Shimadzu, Japan)) coupled with an 8050 triple-quadruple detector (Shimadzu, Japan). The raw data were collected, processed, and quantified using LabSolutions LCGC.

A liquid chromatograph‐mass spectrometer (LC–MS/MS) was used to analyze 18 distinct phenolic compounds. A Shimadzu Nexera X2 UHPLC (Shimadzu, Japan) liquid chromatograph equipped with an InertSustain Swift C18 (2.1 mm*100 mm, 3 μm) column was used, and the system was coupled to an 8050 triple quadruple detector (Shimadzu, Japan) controlled by LabSolution 5.60 SP2 software. Ultra‐pure water was prepared on a Milli‐Q IQ 7000 system (Millipore Company, USA). Propolis samples (SP1–SP6) were extracted by the Reflux Mechanism.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was undertaken using a Nexera X2 UHPLC coupled to an 8060 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The LC system was equipped with a Shimpack C18 ODS III (75 mm × 2.0 mm, 10 μm) column maintained at 60°C. Mobile phase A consisted of aqueous ammonium formate (5 mM, pH 3.0) and mobile phase B consisted of 0.1% formic acid in ACN. The run time for each sample was 17.5 min with the gradient: 0–2.5 min (A: B 70:30 v/v), 2.5–11 min (A: B 40:60 v/v), 11–12 min (A: B 5:95 v/v) and this was kept constant until 16 min. The mobile phase returned to initial condition for 1.5 min. The flow rate was 0.3 ml/min and an injection volume of 5 μl was used.
Mass spectrometry (MS) data for HC, C and D₄‐HC were acquired in positive electrospray ionisation (ESI+) mode while the quantification of TACA as formate adduct was performed using negative electrospray ionisation (ESI‐) mode, following routine procedures at ARFL. Collision energies were optimised for each of the compounds. The desolvation line and heat block temperature was set to 250 °C and 400 °C, respectively. The nebulising gas flow, heating gas flow and drying gas flow was set to 2.8, 12.0 and 8.0 L/min respectively.