Rc membrane filter

The mobile phase consisted of potassium dihydrogen phosphate buffer 25 mm, pH 2.60–ACN–sodium perchlorate 1 m (878:120:2, v/v/v), filtered (0.45 μm, RC membrane filter‐Sartorius) and degassed for 30 min. The chromatography was performed at ambient temperature (25 °C) for 10 min at a flow rate of 1.2 mL/min. The injection volume was 50 μL and the wavelength of DAD was set at 343 nm for CQ, MCQ and QN.
A system suitability test was performed prior to any sequence by injecting six consecutive aqueous standard solutions. The tolerated variation was assessed on area response and retention time with accepted variation of <2%.

HPLC samples were prepared by mixing 100 µL of fermentation broth with 900 µL of methanol in an e-tube, centrifuging for 10 min at 13,000 rpm, and filtering using a 0.45 μm RC membrane filter (Sartorius, Germany). Quantification of anthranilate, l-tryptophan, 7-Cl-Trp, APRN, and PRN was carried out using Agilent 1260 equipped with a Poroshell 120-EC-C18 column (Agilent 4.6 × 150 mm, 2.7 μm) at a flow rate of 0.5 mL per min. For the mobile phase, 0.1% formic acid in deionized water and 0.1% formic acid in acetonitrile were used with gradient elution. 10 µL of each sample were injected into the system and eluted for 35 min for each sample analysis at 50 °C of column temperature. Anthranilate, putative MDAP, and APRN were detected at 310 nm, whereas l-tryptophan, 7-Cl-Trp, and PRN were detected at 280 nm. Sample-preparing steps for LC-QTOF-MS (Quadrupole time-of-flight mass spectrometry) analysis were the same as HPLC sample-preparing steps. First, the sample was run using Agilent 1290 Infinity equipped with Poroshell 120-SB-C18 (Agilent 2.1 × 100 mm, 2.7 μm) at 0.5 mL/min flow rate in gradient elution. Next, the mass of the sample was analyzed using Agilent 6530 QTOF with positive ionization.

The intensity size distribution, the Z-average (Z-Ave), and PdI of nanoparticles were performed by using dynamic light scattering technique using a Zetasizer (Zetasizer Nano ZS, Malvern, UK) instrument equipped with 4.0 mV He-Ne laser (633 nm). Measurements were carried out at 25 ± 0.1 °C with using 0.8872 cP of viscosity and 1.330 of refractive index for the solutions. The number of runs and run durations were chose as automatically. Electrophoretic light scattering (ELS) is used for zeta potential (ζ) measurement of particles and carried out in the folded capillary cell at 25 ± 0.1 °C. The measurements were performed with the following parameters: viscosity, 0.8872 cP; dielectric constant, 79; f(ka), 1.50 (Smoluchowski). The measurement durations and voltage selections were set to automatic mode.
All samples were prepared by diluting with phosphate buffer saline (PBS), filtered with a 0.20 μm RC-membrane filter (Sartorius) before measurement, and all measurements were performed three times.