C18 pre column

The polyphenolic compounds were quantified using high-performance liquid chromatography (HPLC) using a Shimadzu Prominence device (Shimadzu, Kyoto, Japan) equipped with LC-20AT binary pumps, CTO-20A thermostats, SIL-20A automatic dispensers, and SPD-20AV UV/Vis detectors. Different wavelengths of light were used to record chromatograms: 280 nm, 320 nm, and 360 nm. Separation was conducted using a Luna C-18 RP column, 5 μm, 250 × 4.6 mm (Phenomenex, Torrance, CA, USA), which was protected by a C18 pre-column, 4 × 30 mm (Phenomenex, Torrance, CA, USA). As the mobile phase, the following solvents were used: A (acetonitrile) and B (1% formic acid) at the flow rate of 1 mL/min and using the following linear gradient: 0–10 min, from 10 to 25% A, then 10–20 min to 60% A, then 20–30 min to 70% A. After the column has been equilibrated to initial conditions, 10% A in 10 min, there is an additional 5 min stabilization period. Filters with a pore size of 0.45 μm were used to filter all samples and solvents before analysis (Millipore, Bedford, MA, USA). The LC Solution Software (Shimadzu, Kyoto, Japan) was used to identify and quantify the peaks obtained.

The identification and quantification of phenolic compounds in A. indica leaf extract were carried out using an Agilent 1200 Series HPLC-MS equipment. The equipment consists of an automatic sample injection system, two high-pressure isocratic pumps, a degasser and a chromatographic oven. The components were separated by a C18 column (100 mm × 2.1 mm, 3.5 m, Zorbax Eclipse, Agilent, Madrid, Spain) connected to a C18 precolumn (4 mm × 2 mm, Phenomenex, Torrance, CA, USA). HPLC-MS conditions were set as follows. The mobile phase was composed of Phase A (ultrapure water acidified with 0.11% formic acid) and phase B (acetonitrile acidified with 0.11% formic acid). Elution gradient corresponded to 0–2 min, 3% B; 25–27 min, 100% B; 28–38 min, 3% B, at the flow rate of 0.2 mL/min. An injection volume of 10 µL was filtered and injected into the analytical C18 column, at 30 °C (column temperature). Different commercial standards were subsequently used to identify the compounds detected by HPLC-MS. The identification of the components was confirmed by matching their retention time (RT) and the fragment ions (m/z) to those of the corresponding authentic standard compounds, in addition to the literature available.

An Agilent 1200 series high performance liquid chromatography system coupled with a Bruker microTOF (time-of-flight mass analyzer) (Bruker Daltonics, HB, Germany) was used to qualitatively detect the adduct of purpurin with GSH. A phenomenex C18 column (50 × 2.1 mm, 1.7 µm) fitted with C18 precolumn (Phenomenex, Torrance, CA, USA)was used. Water with 0.1% formic acid (A) and 100% acetonitrile with 0.1% formic acid (B) were used as the mobile phase at a flow rate of 0.3 mL/min. The elution gradient was same as used in UPLC analysis: 0–9 min, 10–100% B; 9–10 min, 100% B; 10–10.5 min, 100–10% B; 10.5–15 min, 10% B. The injection volume was 10 µL. Mass spectrometric analysis was performed in negative electrospray ionization mode, with an m/z range from 100–1500.

Mucosa extracts gained as aforementioned were diluted with ultra pure water (1:20), and free amino acids were analyzed by HPLC equipped with a fluorescence detector (Series 1260 Infinity II/ 1200, Agilent Technologies, Germany). The HPLC method was adapted from Krömer et al.^{55 (link)}. Briefly, amino acids were separated after automated pre-column derivatization with ortho-phthalaldehyde/3-mercaptopropionic acid and 9-fluorenylmethoxycarbonyl chloride after reaction with 3-mercaptopropionic acid as reducing agent and iodoacetic acid to block sulfhydryl groups. Analyses were carried out at a flow rate of 0.8 ml/min within 45 min on a 250 × 4 mm Hyperclone ODS (C18) 120 Å column protected by a 4 × 3 mm C18 pre-column (Phenomenex, Aschaffenburg, Germany) using a gradient with 40 mM phosphate buffer (pH 7.8) and acetonitrile/methanol/water (v:v:v: 45:45:10) ranging from 6 to 100%. Amino acid concentrations were normalized to the protein concentration of each sample.

The LC-MS/MS measurements were conducted on a Nexera X2 UHPLC (Shimadzu, Kyoto, Japan) coupled to a triple quadrupole mass spectrometer (LC-MS 8050, Shimadzu, Kyoto, Japan) under positive electrospray ionization (ESI) mode. A Hydrosphere C18 column (150 × 3.0 mm, 3 μm, YMC, Dinslaken, Germany) with a C18 pre-column (4 × 2.0 mm, Phenomenex, Aschaffenburg, Germany) served as stationary phase for chromatographic separation. Mobile phases A and B were 0.1% acetic acid in water and 100% methanol, respectively. The acquisition was performed in scheduled multiple reaction monitoring (MRM) mode. The gradient and instrumental conditions were the same as previously reported for the native SIDA method [7 (link)]. The detailed MRM parameters are listed in Table S2, Electronic Supplementary Material. The MRM transitions of OHCbl, AdoCbl, and MeCbl were monitored for the detection of unconverted cobalamins. LabSolutions software (Shimadzu, Kyoto, Japan) was used for system control, data acquisition, and data analysis.

Sample preparation was carried out using a vortex mixer from Scientific Industries (NY, USA), a Vibra cell VCx130 ultrasound probe from Sonics & Materials Inc. (CT, USA) with a 2 mm diameter titanium microtip and a 130 W high-frequency generator at 20 kHz, X50S metal carbide technical nitrogen stream (Barcelona, Spain), and the Eppendorf 5415R microcentrifuge (Hamburg, Germany). Plastic and glass P100 culture plates, 96-well plates, and a CO₂ incubator (MIDI 40) were purchased from Thermo Fisher Scientific (Spain).
Instrumental determination was performed by Agilent Gas Chromatographic Mod. 7890A Series (Agilent Technologies, Madrid, Spain) equipped with a HP 7683B autoinjector, a microelectron capture detector (μECD), a HP 5975C VL MSD mass spectrometry detector, and a polydimethylsiloxane (95%) ZB-5 capillary column (30 m × 0.25 mm I.D., 0.25-μm film thickness); and HPLC Spectra System P 4000 (Thermo Electron Corporation, San Jose, USA) equipped with a programmable fluorescence detector FL 3000 and Hypersil Green PAH columns (150 mm × 3 mm; particle size 3 μm) attached to a Phenomenex C18 pre-column.