1200 hplc system

Plasma levels of TMAO, TMA, and their metabolites dimethylamine (DMA) were determined using a previously described method [8 (link)]. For the liquid chromatography–mass spectrometry (LC–MS) analysis, an Agilent 6410 Series Triple Quadrupole mass spectrometer (Agilent Technologies, Wilmington, DE, USA) with an electrospray ionization source was applied. We used diethylamine as an internal standard. Using an Agilent Technologies 1200 HPLC system, chromatographic separation was carried out on a SeQuant ZIC-HILIC column (150 × 2.1 mm, 5 μm; Merck KGaA, Darmstadt, Germany) protected by an Ascentis C18 column (2 cm × 4 mm, 5 μm; Merck KGaA). The eluate was monitored for DMA, TMAO, and TMA in multiple-reaction-monitoring mode using characteristic precursor-product ion transitions: m/z 46.1 → 30, m/z 76.1 → 58.1, and m/z 60.1 → 44.1, respectively.

Caffeic and rosmarinic acid quantification was performed on an Agilent Technologies 1200 HPLC system (Waldbronn, Germany) with a C18e (100 × 4.6 mm I.D.,) Chromolith^® HighResolution column (Merck, Darmstadt, Germany) and a Diode Array Detector (DAD). The mobile phase flow rate was set at 1 mL min⁻¹ and two mobile phase components were used (A: 2% acetic acid aqueous solution; B: acetonitrile) with the following mobile phase gradient program: 0–2 min, 92–91% A; 2–4 min, 91–89.8% A; 4–6 min, 89.8–88.8% A; 6–8 min, 88.8–87.5% A; 8–10 min, 87.5–86.5% A; 10–12 min, 86.5–85.2% A; 12–14 min, 85.2–84.1% A; 14–16 min, 84.1–83.0% A; 16–18 min, 83.0–81.2% A; 18–20 min, 81.2–80.9% A; 20–23 min, 80.9–75.0% A; 23–25 min, 75.0–70.0% A; 25–28 min, 70.0–65.0% A; 28–30 min, 65.0–62.0% A.
A 5000 mg L⁻¹ chia extract methanolic solution was injected with an injection volume of 20 µL. The chromatograms were registered at 278 nm and 322 nm. The identification was performed by comparison of the retention time and the absorbance spectra with the respective standard. Quantification was done by means of a caffeic acid (CA) calibration curve (0.1–1 mgL⁻¹; LOD 0.02 mgL⁻¹; LOQ 0.07 mgL⁻¹) and rosmarinic acid (RA) calibration curve (0.5–10 mgL⁻¹; LOD 0.19 mgL⁻¹; LOQ 0.65 mgL⁻¹).

The HCs (chlorogenic acid, ferulic acid, and caffeic acid) were determined using an Agilent Technologies 1200 HPLC system (Waldbronn, Germany) with a C18e (100 × 4.6 mm ID) Chromolith^® HighResolution column (Merck, Darmstadt, Germany) and a Diode Array Detector (DAD). The same two mobile phases and chromatographic conditions described by Zúñiga-López et al. [26 (link)] were used. A total of 20,000 mg/L of broccoli extract was administered using a 20 µL injection volume. The chromatograms were registered at 280 nm and 322 nm. All HCs were identified by contrasting the retention times and absorbance spectra with the appropriate standard. Quantification was performed using an external calibration curve (caffeic acid: 0.1–2 mg/L [limit of detection (LOD) 0.033 mg/L and limit of quantification (LOQ) 0.111 mg/L]; ferulic acid: 0.1–2 mg/L [LOD 0.045 mg/L and LOQ 0.151 mg/L]; and chlorogenic acid: 0.1–2 mg/L [LOD 0.015 mg/L and LOQ 0.052 mg/L]).