1100 hplc system

S. roseosporus and its derivatives were inoculated in 10 ml liquid TSB and cultured for 36 h as seed culture, then 1 ml of seed culture was transferred into a shake flask containing 100 ml ISP-2 medium (4 g l⁻¹yeast extract, 10 g l⁻¹ malt extract, 4 g l⁻¹ glucose). The cultures were incubated at 28°C for different days before fermentation broths were collected by centrifugation. The supernatants were filtered through a Millipore membrane (pore diameter, 0.22 μm) and 10 μl of sample was used for HPLC analysis. Separation of mureidomycins was achieved with an Agilent 1100 HPLC system and a ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm). HPLC conditions were as follows: gradient elution with buffer A (0.1% [vol/vol] methanoic acid in acetonitrile) and buffer B (0.1% [vol/vol] methanoic acid in ddH₂O), flow rate at 1.0 ml/min, ultraviolet detection at wavelength of 260 nm. The elution profile was a hold at 20% buffer A over 2 min, a linear gradient of 20%–100% buffer A over 20 min, a hold at 100% buffer A over 6 min, a linear gradient of 100%–20% buffer A over 2 min and a final hold at 20% buffer A over 5 min.

Sample extraction was performed using a Unique® ultrasound, model USC 5000A, 40 kHz. Chromatographic analyses were performed on the Agilent 1100 HPLC system and a Zorbax® XDB C-18 column (150 mm × 4.6 mm, 3.5 _m Agilent) at 15 °C. Samples (80 mg) were diluted in 60 % ethanol (10 mL) and subjected to sonication (20 min at 30 °C). Following this step, 2 mL of sample were passed through a column containing 200 mg of polyamide, and the eluate was injected into an HPLC system. Separation was achieved using gradient elution of water (0.2 % acetic acid) adjusted to pH 6.9 with triethylamine (A) and acetonitrile (B) at a flow rate of 0.8 mL/min, detection was performed at 245 nm, and the concentrations of alkaloids were calculated as previously described [28 (link)].

The monosaccharide composition of BSP was determined using the 3-methyl-1-phenyl-5-pyrazolone (PMP)-labeling procedure as previously described (Liao et al., 2019 (link)). Briefly, BSP was hydrolyzed with 4 M trifluoroacetic acid at 110°C for 2 h, and subsequently labeled with PMP. PMP-labeled monosaccharides were identified on an Agilent 1100 HPLC system equipped with a ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm), and a G1315B DAD detector (Agilent, Waldbronn, Germany) at a wavelength of 245 nm. The mobile phase was A, acetonitrile and B, 0.1 M phosphate buffer (pH = 6.7) at a flow rate of 0.8 ml/min and a column temperature of 30°C. Mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose were used as standard. The molar ratios of the monosaccharides of BSP were measured on a Dionex ICS-3000 ion chromatography (IC) system with a CarboPac-PA20 column (3 mm × 15 mm), and integrated pulsed amperometry detector (Sunnyvale, CA, United States). The gradient elution conditions were as follows: 0–20 min, 99.2% H₂O (A), 0.8% 0.25 M NaOH (B); 21.1 min, 94.2% A, 0.8% B, 5% 1 M NaAc (C); 30.0 min, 79.2% A, 0.8% B, 20% C; 30.1–50.0 min, 20% A, 80% B. The flow rate was 0.5 ml/min and the injection volume was 20 μl.

BSEPS molecular weight was determined on an Agilent 1100 HPLC system equipped with a refractive index detector and FPl gel particle (5 μm), three columns of pore type (100, 104, and 105 A°) in series, length 7.5 × 300 mm (1000, 5000000) for DMF solvent Styrogel HR-DMF, 3 μm (7.8 × 300 mm) (Waters, Milford, MA, USA). One column (5000–600,000) was used for water solvent (polyethylene oxide/glycol standard) PL aquagel, OH 7.5 mm and 30 μm pore, 8 um particle size. Sample (0.01 g) was dissolved in 2 mL of solvent and filtrated (0.45 mm) and transferred to a gel-permeation chromatography (GPC) device [31 ]. Number average molecular weight (Mn) and number average molecular weight (Mw) were directly calculated according to the definition of Mn and Mw using molecular weight and refractive index signal values at each elution volume [32 (link)].