Durashell aa analysis kit

Durashell AA analysis kit contained internal standard and 17 kinds of amino acid standard, which was purchased from Agela Technologies (Tianjin, China). Oxalic acid, tartaric acid, lactic acid, citric acid, succinic acid, hydrochloric acid (HCl), disodium hydrogen phosphate dodecahydrate (Na₂HPO₄•12H₂O), potassium dihydrogen phosphate (KH₂PO₄), phosphoric acid (H₃PO₄) (all AR grade), and malic acid (BR grade) were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Guanosine 5′‐monophosphate (5′‐GMP) disodium salt hydrate, 5′‐IMP, 5′‐AMP, and cytidine 5′‐monophosphate (5′‐CMP) were purchased from Sigma‐Aldrich (St. Louis, Mo., U.S.A.). Methanol, sulfosalicylic acid, and acetonitrile (ACN) (all HPLC‐grade) were purchased from Fisher Scientific (Shanghai, China). The ultrapure water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). Buffer salt I (0.05 mol/L KH₂PO₄) was prepared by dissolving 6.80 g KH₂PO₄ in 1 L water. Buffer salt II (0.01 mol/L KH₂PO₄) was prepared by dissolving 1.36 g KH₂PO₄ in 1 L water and then adjusted to pH 2.8 by 1 mol/L H₃PO₄.

The quantitative analysis of amino acids was carried out as described in the literature [23 (link)] with some modifications. A 1 mL sample was added to 1 mL 10% (v/v) 5-sulfosalicylic acid solution and diluted to 25 mL with 0.1 mol/L HCl. Then, it was mixed with an internal standard solution and filtrated through a 0.22 µm nylon filter membrane before HPLC analysis. The chromatography Durashell AA (4.6 mm × 150 mm, i.d. 3 µm) a column (Agela Technologies, Tianjin, China) and the Durashell AA analysis kit (Agela Technologies, Tianjin, China) were used to analyze the free amino acid in WS, PS and PWS. Mobile phase A (4.50 mg/mL Na₂HPO₄ and 4.75 mg/mL Na₂B₄O₇, pH = 8.2, water solution) and mobile phase B (methanol: acetonitrile: water = 9:9:2, v/v/v) were used for the elution solvent. A 1260 Agilent HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) coupled with a diode array detector (DAD) was used for free amino acid analysis. The gradient elution method was 6–10% B for 0–6 min, 10% B for 6–8 min and 10–16% B for 8–10 min; the flow rate of the mobile phase was 1.6 mL/min; the column temperature was 45 °C; and the detection wavelengths were 338 and 262 nm.