Lc 20a hplc system

The oligosaccharide and fructose analysis were performed on the Shimadzu LC-20A HPLC system coupled with RID. Agilent ZORBAX NH2 (5 μm, 4.6 mm × 250 mm, i.d.) and column was used. The column was maintained at 35 °C. Isocratic elution with 70% aqueous acetonitrile was used as mobile phase at a flow rate of 1 mL/min for 45 min.

We performed a simultaneous analysis of extract components using a Shimadzu LC-20A HPLC system (Shimadzu, Kyoto, Japan), consisting of a solvent delivery unit, an online degasser, a column oven, an autosampler, and a photodiode array (PDA) detector. A data processor employed LCsolution software (version 1.24). The analytical column used for separation was a Gemini C18 column (250 mm × 4.6 mm, particle size 5 μm; Phenomenex, Torrance, CA, USA) and was maintained at 40 °C. The mobile phases consisted of 1.0 % (v/v) aqueous acetic acid (A) and 1.0 % (v/v) acetic acid in acetonitrile (B). The gradient flow was as follows: (A)/(B) = 85/15 (0 min) → (A)/(B) = 35/65 (35 min) → (A)/(B) = 0/100 (45 min; hold for 5 min) → (A)/(B) = 85/15 (55 min; hold for 15 min). The analysis was conducted at a flow rate of 1.0 mL/min with PDA detection at 230 nm, 254 nm, and 280 nm. The injection volume was 10 μL.

Millipore Milli-Q water purification system (Millipore, USA) and LC-20A HPLC system with UV detector (Shimadzu, Japan) were used.

The monosaccharide compositions were determined by high-performance liquid chromatography (HPLC) after precolumn derivatization. Purified polysaccharide powder (20 mg) was dissolved in trifluoroacetic acid at 2 mol/L and hydrolyzed at 120°C for 6 hours in a sealed tube. After hydrolysis, the excess acid was removed by codistillation with methanol three times to yield dry hydrolysate. Then, the hydrolysate was derivatized with PMP and analyzed by HPLC according to a previously reported method [17 (link)]. HPLC analysis was performed on an LC20A HPLC system (Shimadzu, Japan) equipped with an SPD-20A ultraviolet detector and a C18 column (250 mm×4.6 mm, 5 μm, Shimadzu, Japan). The mobile phase was a mixture of 0.1 mol/L NaH₂PO₄-Na₂HPO₄ buffer (pH 6.7) and acetonitrile (83:17), and a flow rate of 1.0 mL/min was used. The wavelength of detection was 245 nm, and the column temperature was 30°C. The sugars were identified by comparison to reference monosaccharides (mannose, glucose, D-ribose, rhamnose, D-xylose, D-galactose, L-arabinose, and D-fructose). The molar ratios of the monosaccharides were calculated based on the standard curve of each monosaccharide.

HPLC analysis was performed on an LC-20A HPLC system (Shimadzu, Kyoto, Japan) equipped with an Inertsil-ODS3 C18 column (5 μm, 250 mm × 4.6 mm, GL Science, Tokyo, Japan). The mobile phase was composed of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient profile was optimized as follows: 0–30 min, linear gradient 5%–55% (v/v) B; 30–45 min, linear gradient 55%–65% (v/v) B; 45–50 min, linear gradient 65%–100% (v/v) B at a flow rate of 1 mL·min⁻¹. The temperature of the column compartment was maintained at 40 °C. A diode-array detector was used to detect compounds. The spectra of the compounds were recorded between 210 and 800 nm. The compounds were identified by comparing the retention times and UV spectra with those of the standards.

According to Bayrak Kul et al. (10 (link)), furosine was determined with minor modifications. A total of 100 mg of freeze-dried sample was hydrolyzed with 2 mL of 8 N HCl at 110°C for 23 h in a vial. Before sealing, the ampoule bottle was filled with nitrogen for 2 min. The samples were filtered with a 0.22 μm PVDF membrane after cooling at 25°C. Following the recommendation of Delgado-Andrade et al. (11 (link)), a 0.5 mL aliquot of the hydrolyzate was freeze-dried and dissolved in a mixture of 1 mL of water, acetonitrile, and formic acid (95:5:0.2, v:v:v). The analysis was carried out using LC-20A HPLC system (Shimadzu, Japan) with a diode array detector, followed by a separation which was achieved on a C₁₈ column (150 mm × 4.6 mm × 5 μm, Shim-pack GIST) at 40°C. With an injection volume of 10 μL, the detection was carried out at 280 nm. The study adopted a mobile phase A of 0.4% acetic acid. The elution was isocratic with a flow rate of 0.8 mL/min and a retention time of about 5 min. Furosine was quantified by the external standard method. The protein amount was determined according to the AOAC (20 ). The standard curve was plotted with 1, 2, 5, 10, and 30 μg/mL of furosine standard. The obtained results were given in mg/100 g of protein.