Method for purifying and detecting 6-hydroxynobilonine in fresh stems of dendrobium huoshanense

A method for purifying and detecting 6-hydroxynobilonine in fresh stems of Dendrobium huoshanense, wherein the 6-hydroxynobilonine is detected by using a gas chromatography-mass spectrometry (GC-MS) method, a to-be-detected solution of 6-hydroxynobilonine is extracted from fresh stems of Dendrobium huoshanense for detection, and the extraction method is as follows:

• S1, dehydrating fresh stems of Dendrobium huoshanense through a freeze-drying method, smashing and screening to obtain dendrobium dry powder;
• S2, adding water into the dendrobium dry powder from S1 in a ratio of 1g of dendrobium dry powder to 6 mL of water, performing ultrasonic treatment for 20-30 min, then performing enzymolysis for 1.5 h by adding an amount of a composite enzyme equal to 1.5% the mass of the dendrobium dry powder to obtain an enzymolysis solution wherein the composite enzyme comprises a fiber enzyme and pectinase and the composite enzyme is prepared from a fiber enzyme and pectinase in 10000 U/g;
• S3, adding a set amount of acidity alcohol into the enzymolysis solution, leaving the enzymolysis solution for 18-24 h at room temperature, then vacuuming the enzymolysis solution for 1 min under an ultrahigh pressure of 100 MPa, then filtering the residue and removing the filtrate, wherein the acidity alcohol is an aqueous solution comprising 70% ethanol by volume and having a pH value of 3-3.5;
• and
• S4, concentrating the filtrate in vacuum to 5-8 mL, then purifying the filtered concentrate using a mixed-mode cation exchanger (MCX) extraction column, eluting with a methanol-acetonitrile solution, collecting eluent, blowing the eluent with nitrogen to remove the water until no water, and then dissolving with a methanol solution and filtering to obtain the to-be-detected solution containing 6-hydroxynobilonine;
• wherein
• the GC-MS detection includes gas chromatography and mass spectrometry, wherein the specific operation of the gas chromatography in the GC-MS method is as follows:
• a chromatography column is 30 m×0.25 μm×0.25 mm, an initial column temperature is 70° C. and raised to 150° C. at 2° C./min, and then maintained for 1 min, then the temperature is raised to 180° C. at 3° C./min and then maintained for 1 min, then the temperature is raised to 240° C. at 20° C./min and then maintained for 3 min;
• during the gas chromatography operation, a carrier gas is high-purity He and injected at a flow rate of 1.0 ml/min;
• a split ratio is set as 20:1;
• and
• the specific operation of the mass spectrometry in the GC-MS method is as follows:
• an electron impact (EI) ion source;
• ion energy is set as 70 eV;
• a temperature is set as 200° C.;
• a scanning range m/z of a full-scanning range of 40-500 m/z.