Agilent 6120 single quadrupole mass spectrometer

Optical rotation was measured using a Jasco DIP-1000 polarimeter (Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were recorded using a 250-MHz Bruker NMR spectrometer (DMX 250) and 600-MHz Varian NMR spectrometer (VNS-600, Palo Alto, CA, USA) at the Core Research Support Center for Natural Products and Medical Materials (CRCNM). High-resolution electron ionization mass spectrometry (HR–EI–MS) was performed using a JMS-700 instrument (JEOL, Tokyo, Japan) coupled to a 6890 Series gas chromatography MS (GC–MS) system (Agilent Technologies, Santa Clara, CA, USA). Low-resolution electrospray ionization MS (LR–ESI–MS) was performed using an Agilent 6120 single quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column [Phenomenex Luna 3 μm C18(2) 100 Å, new column, 150 × 4.6 mm (Phenomenex, Torrance, CA, USA)]. Isolation of the compounds was carried out using a Waters 1525 binary HPLC pump with a Waters 996 photodiode array (PDA) (Waters Corp., Milford, MA, USA) with a reversed-phase HPLC [RS Tech, Hector-M 5 μm C18, 250 × 4.6 mm (RStech Corp., Cheongju, Republic of Korea)] and an Alltech 301 HPLC pump (Alltech, Lexington, KY, USA) with a Waters 2487 dual wavelength absorbance detector and a chiral HPLC column [CHIRALPAK AD-H 5 μm, 250 × 4.6 mm (Daicel, Osaka, Japan)].

Chromatographic profiling of the most active fractions, EA-a and MC-bg, was performed using an Agilent 1260 Infinity high-performance liquid chromatograph equipped with a reversed-phase C18 column (Phenomenex Luna 3μ C18(2) 100 Å, 4.6 × 150 mm). The solvent system used was as follows: solvent A (95% deionized water, 5% acetonitrile with 0.05% formic acid), solvent B (100% acetonitrile with 0.05% formic acid); A:B = 95:5 → 95:5 (2 min) → 0:100 (12.5 min) → 0:100 (15 min) → 95:5 (16 min) → 95:5 (19 min) at a flow rate of 0.7 mL/min. Compounds were detected using diode array detector (DAD: Agilent 1260 Infinity DAD; Agilent Technologies, Santa Clara, CA, USA) and electrospray ionization-mass spectrometry detector (ESI MS: Agilent 6120 single-quadrupole mass spectrometer; Agilent Technologies, Santa Clara, CA, USA).

The extraction of metabolites was based on the work described previously by our research group [8 (link)]. After extraction, the samples were injected into an Agilent 1200 series HPLC instrument (Agilent Technologies, California, USA) coupled to an Agilent 6120 single quadrupole mass spectrometer with an orthogonal ESI source. In order to avoid potential degradation of metabolites, the samples were prepared shortly before chromatographic analysis. The analysis of metabolites was only carried out with the liver of animals belonging to the control groups (NC and HC) and the groups with high intake of spinach (N5 and H5)