1. Chromatographic conditions

   Chromatographic column: Inertsil ODS-3 4.6×250mm  5μm

   Mobile phase: acetonitrile: water: phosphoric acid (23: 77: 0.1, V/V)

   Detection wavelength: 368nm

   Sensitivity: 0.01AUFS

   Flow rate: 1mL/min

   Injection volume: 20μL

   Column Temperature: 30℃

   Methanol is analytically pure

2. Solution Preparation

   1) Comparison solution preparation: weigh accurately about 4-5mg of Lacosanin in a 50mL volumetric flask, dissolve with methanol and set the volume.

   2) Sample solution preparation: weigh 4-5mg of sample (content of about 98%) in a 50mL volumetric flask, add about 50mL of methanol, dissolve with ultrasonic shaking for 30min, cool to room temperature, and then dilute with methanol to the scale, filtered through 0.45μm micropore membrane, then it is obtained.

3. Sample Determination

   Under the above chromatographic conditions, after the instrument is stabilized and the baseline is smooth, the sample is injected for determination, and the retention time of laccasein is about 20 min, and the content is calculated.

   External standard method    Calculation 

   Where: the peak area of the lacinin standard, S0;

   Weighing volume of lacinin standard, C0;

   Peak area of laccasein in the sample, S1;

   Sample weighing volume C1;

   Sample moisture, W percentage.

   98%  Content of control

   Area normalization method   Calculation

   Manually remove the solvent peaks before 4 minutes (impact peaks caused by inconsistency between the mobile phase and sample solvent), the minimum peak area shows more than 10, the total retention of 25 minutes to calculate the percentage of the peak area of Lacin in the peak results table, that is, to obtain the area-normalized content.