Determination of Emodin in Polygonum Cuspidatum by HPLC
Time: 2021-08-17 16:52:50
Abstract: Polygonum cuspidatum is not only for viewing, but also for food. The tender stems are used as vegetables, and the roots are used as cold drinks. It is placed in cold water to cool down. Rice noodles have a special flavor. It is also called "sour soup stick" because of its sour taste.
Polygonum cuspidatum was included in the first edition of the 2000 edition of the Chinese Pharmacopoeia, and the total anthraquinone control content was determined by spectrophotometry. Polygonum cuspidatum contains anthraquinone derivatives, which are mainly free, and contain emodin, emodin methyl ether, chrysophanol and other ingredients. This article uses high performance liquid chromatography to determine the content of emodin in Polygonum cuspidatum. The method is fast, convenient and convenient. reliable.
1 Instruments and reagents
Agilent1100 type combined automatic high performance liquid chromatograph. Polygonum cuspidatum (commercially available), emodin reference substance (China Institute for the Control of Pharmaceutical and Biological Products, for content determination), methanol is chromatographically pure, and other reagents are analytically pure.
2 Methods and results
2.1 Chromatographic conditions
Chromatographic column: Kromasil C18 (250mm×4.6mm, 5μm), mobile phase: methanol-0.1% phosphoric acid solution (85:15), flow rate: 1.0mlmin, column temperature: room temperature, measurement wavelength: 254nm, detected by external standard method, Sample volume: 5.0μL.
2.2 Preparation of test solution
Take about 0.5g of this product powder (pass through No. 3 sieve) (at the same time, take this product powder to measure the loss on drying at 105℃), accurately weigh it, put it in a 100ml volumetric flask, add about 80ml methanol, ultrasonic treatment (power 250W, frequency 30kHz) for 30min, let cool to room temperature, add methanol to the mark, shake well, filter, accurately measure 10ml of the filtrate, place in a 100ml round bottom flask, evaporate methanol, add 20ml of 2.5mol/L sulfuric acid solution, heat to reflux for 1h , Cool, add chloroform 30nIl, continue to reflux for 2h, divide the chloroform layer, extract the acid solution with chloroform 3 times, 5ml each time, combine the chloroform extracts, pass through a funnel with anhydrous sodium sulfate, place in a 50ml volumetric flask, filter Wash with a small amount of chloroform, add chloroform to the filtrate, add chloroform to the mark, shake well, accurately measure 10ml of chloroform solution, evaporate to dryness in a water bath, add an appropriate amount of methanol to the residue to dissolve, put it in a 25ml volumetric flask, add methanol to dilute to the mark, Shake well and use as the test solution.
2.3 Preparation of reference solution
Precisely weigh 9.81 mg of emodin reference substance, put it in a 100ml volumetric flask, add an appropriate amount of methanol to dissolve it, and add methanol to the mark, shake it up, as a reference substance stock solution (concentration of 0.0981mg/ml). Accurately measure 1ml, add methanol and dilute to 25ml, as the reference solution.
2.4 Investigation of linear relationship
"Inject 2.0μL, 4.0μL, 6.0μL, 8.0μL, 10.0μL, 12.0μL of the above-mentioned emodin reference substance solution. Record the peak area, plot the peak area (Y) against the injection volume (X) to obtain the standard curve, and calculate the regression equation: Y=4.0387×103X-0.844, r=0.9999, and the linear range is 7.848×10-3～4.709 ×10-2.
2.5 precision test
Take the same test solution, repeat the injection 6 times, record the peak area, and the result is an RSD of 0.2%, indicating good precision.
2.6 Repeatability test
Take 5 samples of the same batch of Polygonum cuspidatum, and determine the content of emodin according to the above method. The result is that the RSD is 0.5%.
2.7 Solution stability test
Take the test solution under "2.6", inject 5μL at 0, 4, 8, 12, 16, 20, 24h, record the peak area, and the RSD of the peak area is 0.9%, indicating that the test solution is in 24h Both are stable inside.
2.8 Recovery rate test
Accurately weigh an appropriate amount of a sample with a known content of 1.6%, and accurately add 15ml of the emodin reference substance stock solution respectively, and determine according to the above method to calculate the sample recovery rate.
2.9 Sample measurement
Take Polygonum cuspidatum medicinal material and determine the content of emodin in the sample according to the above method. The results are shown in Table 2. The reference substance solution and the test substance solution were injected into sva respectively. According to this method, the sample and the reference substance have a corresponding chromatographic peak at the same retention time position.
Take the emodin reference substance solution and scan in the wavelength range of 200-400nm. The results show that there are maximum absorption peaks at 219nm, 254nm, and 292nm. Since the absorbance at 254nm is strong and is not interfered by solvent peaks, 254nm is selected As the measurement of the absorption wavelength, it is consistent with the provisions of the "Chinese Pharmacopoeia".
Experiments have shown that after this product is hydrolyzed and then extracted with chloroform 4 times, the emodin has been basically extracted completely.
From the test results of linearity, recovery, reproducibility, solution stability, etc., it can be seen that the HPLC method to determine the content of emodin in Polygonum cuspidatum has high accuracy, good reproducibility, and simple and fast operation.