Luna omega

Uremic toxins and Indigo were analyzed using LCMS 8040 Triple Quadrupole LC/MS/MS (Shimadzu), as previously described [55 (link)].
Electrospray (ESI, negative mode at 4000 V) was performed for each analysis in LC-MS/MS. The mobile phase was composed of MeCN mixed with 2 mM of ammonium acetate. Nitrogen was employed for the nebulizer and desolvation gas and finally utilized as collision gas for molecule dissociation [28 (link)]. Notably, for Indigo determination, 10% MeCN was used for equilibrating the column (Luna Omega, size 150 × 4.6 mm, Phenomenex). Next, MeCN was raised up to 50% and finally scaled at 10% of MeCN [56 (link)]. The fragmentation voltages and collision energies of analytes for the first and second methods were optimized, as reported in Table 1. The chromatograms and MRM (multiple reaction monitoring) spectrum of IS, pCS, DHTC, and Indigo are shown in Figure 5.

This further analysis was required to confirm the structure of rutin and chlorogenic acid, which were present at low concentrations, and some of their diagnostic ¹H NMR signals overlapped with those of other metabolites.
Flower bud hydroalcoholic extracts at a concentration of 0.5 mg/ml were analyzed using a Xevo G2-XS QTof system (Waters, Milford, MA, USA) equipped with a polar C18 analytical column (Luna Omega, 100 × 3.0 mm, 3 µm particle size, Phenomenex, Torrance, CA, USA). The column was kept at 45°C, while the samples were kept at a constant temperature of 10°C. The mobile phases were H₂O (A) and MeCN (B). The method and gradients used were the following: 95% A for 1 min followed by a gradient reaching 25% B in 2 min, 25% B was kept for 1 min, then the gradient reached 70% B in 3 min, 70% B was kept for 1 min, and then the gradient reached 5% B again in 20 s. The flow rate was 0.4 ml/min, and the injection volume was 2 µl.
Electrospray ionization in positive and negative modes was applied in the mass scan range of 50−1,200 m/z. Electrospray ionization (ESI) source conditions were as follows: capillary = 0.8 kV, cone = 40 V, source temperature = 120°C, desolvation temperature = 600°C, cone gas flow = 50 L/h, and desolvation gas flow = 1,000 L/h.

For LC-MS analysis, we used an LC (Agilent 1100 series) coupled with a mass spectrometer. All samples were passed through a 13 mm nylon syringe filter with a 0.22 μm pore size before injection to ensure the removal of the solid contaminants. Reverse-phase chromatography was used with a Phenomenex Luna Omega (Phenomenex) LC column with the following specifications: 100 × 4.6 mm, 3 µm, polar C18, 100 Å pore size with a flow rate of 0.3 mL min⁻¹. LC eluents include MiliQ-H₂O containing 0.1 % formic acid (15%) and acetonitrile (85%) using isocratic elution. The time of analysis was 20 min. The mass spectrometer (Finnigan LTQ mass spectrometer) was equipped with an electrospray interface (ESI) set in positive electrospray ionization mode for analysis and with 15 kV collision energy.

Chromatographic separation was achieved by gradient elution on the LC system Accela 1200 from Thermo Fisher Scientific (Waltham, MA, USA). The following three different chromatographic columns were tested in our study: Hypercarb 3 μm 100 × 2.1 mm from Thermo Fisher Scientific (Waltham, MA, USA), Luna Omega 1.6 μm Polar C18 100 × 2.1 mm, and Kinetex 1.7 μm HILIC 100 × 2.1 mm from Phenomenex (Torrance, CA, USA). Chromatographic conditions in the test (mobile phases, flow rate, and gradient) were the same for each column except for the composition of the mobile phases, which was for Hypercarb and Luna Omega (the reverse-phase columns) opposite to the HILIC column (normal-phase column). After evaluating the chromatographic separation properties of the tested columns, chromatographic column Kinetex 1.7 μm HILIC 100 × 2.1 mm from Phenomenex (Torrance, CA, USA) was used. The gradient LC system was operated using 0.1% formic acid in water: acetonitrile (95:5, v/v, mobile phase A) and 0.1% formic acid in water: acetonitrile (5:95, v/v mobile phase B) at a flow of 200 μL·min⁻¹. A gradient elution for separation on HILIC column was performed: 0–4 min (0% A, 100% B), 8 min (linear gradient to 30% A), 8–12 min (linear gradient to 100% B), and 12–14 min (0% A, 100% B).

Liquid Chromatograph (Agilent 1100 series) was coupled with a mass spectrometer for LC-MS analysis. Before injection, all samples were passed through a 13 mm nylon syringe filter with a 0.22 μm pore size. Reverse-phase chromatography was used with a Phenomenex Luna Omega (Phenomenex) LC column with the following specifications: 100 × 4.6 mm, 3 µm, polar C18, 100 Å pore size with a flow rate of 0.3 mL min⁻¹. LC eluents include MilliQ-water (solvent A) and acetonitrile (solvent B) using gradient elution (solution A: B composition change with time: 0 min: 95:5, 3 min: 95:5, 15 min: 85:15, 17 min: 90:10, and 20 min 95:5). The mass spectrometer (Finnigan LTQ mass spectrometer) was equipped with an electrospray interface (ESI) set in positive electrospray ionization mode for analyzing the NRTOCl and NRTBCl. The optimized parameters were sheath gas flow rate at 20 arbitrary units, spray voltage set at 4.00 kV, capillary temperature at 350 °C, capillary voltage at 41.0 V, and tube lens set at 125.0 V.

The acquisition of SWATH-MS data from female and male saliva samples was accomplished with the same microESI qQTOF mass spectrometer used for LC–MS/MS (TripleTOF 6600+ system; AB Sciex LLC) operated in swath mode. The six samples (F1, F2, F3, M1, M2, M3) were loaded in a random order to avoid bias in the analysis. A 5-μl aliquot of each digested sample was individually loaded onto a trap column (LC column; 12 nm; 3 µm; Triart-C18; 0.5 × 5.0 mm; YMC Co. Ltd., Kyoto, Japan) and desalted with 0.1% TFA at 10 µl/min for 5 min. The peptides were then loaded onto an analytical column (LC column; Luna Omega; 3 µm; Polar C18; 150 × 0.3 mm; Capillary; Phenomenex, Torrance, CA, USA) equilibrated in 3% ACN, 0.1% FA, and eluted with a linear gradient of 3–35% buffer B (0.1% FA in ACN) in buffer A (0.1% FA in water) for 45 min at a flow rate of 5 μl/min. Samples were ionised in a OptiFlow Turbo Ion Source system (1–50 µl micro), with 4.5 kV applied to the spray emitter; the analysis was carried out in DIA mode. Survey MS1 scans were acquired from 400 to 1250 m/z for 250 ms, followed by 25-ms product ion scans from 100 to 1500 m/z in ‘high sensitivity’ mode throughout 100 overlapping windows covering from 400 to 1250 m/z. The quadrupole resolution was set to ‘UNIT’ for the MS2 experiments, and the total cycle time was 2.79 s.