Luna omega polar c18 column

The protocol for high-resolution Full MS data dependent MS² analyses was adapted from previous reports (Stien et al., 2019 (link), 2020 (link)). Briefly, the UHPLC system was an Ultimate 3000 (Thermo Fisher Scientific) coupled to a Q-Exactive Focus Orbitrap detector and equipped with a Phenomenex Luna Omega polar C-18 column 150 mm × 2.1 mm, 1.6 μm. Here, crude bacterial and culture medium (starch casein broth) extracts were dissolved in MeOH at a concentration of 1.5 mg/ml. Pure methanol injections were used as blanks for metabolomics. In HPLC, the solvent system was a mixture of water (solution A) with increasing proportions of acetonitrile (solution B), both solvents modified with 0.1% formic acid. Here, the gradient was as follows: 5% of solution B for 5 min before injection, then from 1 to 12 min, a linear increase of B up to 100%, followed by 100% of solution B for 8 min.

Intracellular MET and MHA levels were measured simultaneously using a Waters^® Acquity H-Class Plus UHPLC System equipped with a thermostated autosampler and supported with a Waters^® single quadrupole mass detector QDa (Waters). The sample temperature was 6 °C. Chromatographic separation was achieved on a Phenomenex^® Luna Omega Polar C18 column (2.1 mm × 100 mm, i.d. 1.6 μm). The column was operated at 35 °C. The injection volume was 10 μL, and the flow rate was set at 0.4 mL/min. A ternary solvent system was used consisting of (A) pH 2.00 ± 0.02 ultrapure water, (B) acetonitrile and (C) ultrapure water. The mobile phase was filtered through in-line 0.2-μm membrane filters. The following gradient elution was employed: 0–2 min: 100% A; 2.1 min: 99% A, 1% B; 2.10–7 min: 99% A, 1% B; 7.1 min: 1% B, 99% C; 8 min: 60% B, 40% C; 8–11 min: 60% B, 40% C; 11.5 min: 100% A and 11.5–15 min: 100% A. The ionization source was used both in the electrospray (ESI)-negative and -positive modes, using single ion recording (SIR). The optimal cone voltage was set at 15 V and the capillary voltage at 0.5 kV; the source temperature was maintained at 600 °C. MET was monitored in ESI+ at 150 m/z in SIR mode. MHA was monitored in ESI- at 149 m/z in SIR mode.

Liquid chromatography was performed on a Vanquish UHPLC system (Thermo, Les Ulis, France). The compounds were separated on a Luna Omega Polar C18 column (2.1 mm × 100 mm; 1.6 µm, Phenomenex, Le Pecq, France) with an oven temperature set at 50 °C. The flow rate was fixed at 0.4 mL/min. The mobile phase A was a mixture of 2 mM ammonium formate in water and 0.1% formic acid; mobile phase B was methanol. The chromatographic gradient was as follows: 100% A for 1 min, linear gradient to 55% A in 5 min, linear gradient to 50% A in 1 min held for 1 min, follow by a linear gradient to 0% A in 4 min held for 1 min. The column re-equilibration was performed by a linear gradient to 100% A in 0.1 min held for 2 min.

UPLC-DAD was conducted using an Agilent Technologies 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, CA, USA) with an autosampler (G7167B), a flexible pump (G7104A), a multicolumn thermostat (MCT-G7116B) a DAD detector (G7117A), and a Luna Omega Polar C18 column (100 × 2.1 mm, 1.6 μm; Phenomenex, Torrance, CA, USA). The mobile phase was a mixture of 0.2% acetic acid (pH 3.8, solvent A) and acetonitrile (ACN) (solvent B). For ACET, the mobile phase was eluted using the following gradient program: 10 v/v % solvent B for 3.5 min, 10 to 30 v/v % solvent B over 0.5 min, 30 v/v % solvent B for 6 min, and 10 v/v % solvent B for 5 min. For the measurement of HTOL in microsomal study, the mobile phase was eluted using the following gradient program: 25 v/v % solvent B for 5.5 min; 25 to 35 v/v % solvent B over 1 min; 35 v/v % solvent B for 8.5 min; and 25 v/v % solvent B for 5 min. For AG, the mobile phase consisted of 96 v/v % solvent A and 4 v/v % solvent B was elution isocratically for 19 min. ACET, AG, and HTOL were detected at 245, 245, and 230 nm, respectively. Rutin, CARB, and THEO were used as ISs for the analyses of ACET, HTOL, and AG, respectively. The flow rate used was 1 mL/min, and the sample injection volume was 5 μL for all the analytes except AG (10 μL).

An Agilent 1100 HPLC-DAD was used for the chromatographic analysis (Agilent Technologies, California, USA). On a Luna Omega Polar C18 column (5 μm, 100 Å, 4.6 mm × 250 mm) (Phenomenex Inc., Torrance, CA, USA), the separation runs were carried out at 40 °C. A mixture of aqueous acetic acid (0.1% v/v) and methanol (35:65) was used as a mobile phase. The injection volume was 20 μL, and the flow rate was 0.8 mL/min with isocratic elution. The detection UV wavelength was set at 254 nm.
The electrospun shellac fibers loaded with KP extract were successfully powdered. A 100 mg sample powder was precisely weighed into a 1.0 mL dry, clean volumetric flask. To aid in full solubility, the mobile phase was added and ultrasonically mixed. The same mobile phase was used to adjust the volume, and the analysis was performed using the 100 mg/mL sample solution. The validated method was then used to simultaneously measure the amounts of DMF, TMF, and PMF in various electrospun shellac fibers loaded with KP extract.
In the case of sample solutions obtained from an in vitro dissolution study of KP extract-loaded electrospun shellac fibers, each sample from the dissolution vessels was directly injected into the HPLC-DAD system, and the concentrations of methoxyflavones in each sample were calculated using their corresponding calibration curves.

The volume of 110 µL of human blood plasma was treated with 20 µL of a solution containing 1 mM metronidazole (MET, an internal standard). Subsequently, 20 μL of ultrapure water were added to the treated plasma. Plasma samples were mixed with 750 µL of methanol. After separation of the precipitated proteins, 900 µL of supernatant was evaporated to dryness under a stream of nitrogen at 37 °C. The residue was reconstituted in 100 µL of 25 mM ammonium acetate, pH 6.2. An amount of 10 µL of the prepared sample was injected into HPLC.
All measurements were performed with the Prominence LC–20A HPLC system, and an SPD-20A UV–Vis detector (Shimadzu, Kyoto, Japan). Mobile phase A consisted of 25 mM ammonium acetate, pH 6.2 and mobile phase B consisted of methanol/acetonitrile (75:25, v/v); final mobile phase A:B, 79:21, v/v. Flow rate was 0.4 mL/min. Both compounds were separated at 30 °C on Luna^® Omega Polar C18 column (50 × 4.6 mm; 5 µm) purchased from Phenomenex (Torrance, CA, USA). The detection was performed at 272 nm (CEF) and 320 nm (MET).
The bioanalytical assay was performed in accordance with the European Medicine Agency guidelines and has been previously described in more detail [18 (link),19 (link),20 ].