Abstract: This article uses high performance liquid chromatography to determine the content of emodin in rhubarb, which provides a fast and accurate method for the quality control of the preparation.
Compound Maren Pills are made from seven flavored Chinese medicines such as Hemp Seed, Rhubarb, Aloe, White Peony, Zhi Zhi, etc. It has the effects of purging heat, intestines, cooling blood and detoxifying, and removing blood stasis and menstruation. Knot, habitual constipation, etc. are quite effective.
1 Instruments and reagents
P230 high-performance liquid chromatograph, UV230 ultraviolet detector, EC2000 chromatography workstation, Sartorius BP211D electronic analytical balance (Germany Sartorius); KQ100DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Factory); emodin reference substance (China Pharmaceutical Bio Product Inspection Institute); Compound Maren Pills (30g / bottle) and negative samples (Chinese Medicine Preparation Room of our hospital, sample batch number: 020822, 021128, 030410); methanol is chromatographically pure, water is redistilled water, and the remaining reagents are analytically pure .
2 Methods and results
2.1 Chromatographic conditions
Chromatography column: Techsphese ODS-C18 (4.6mm × 250mm, 5um); mobile phase: methanol-water-glacial acetic acid (80: 20: 1); detection wavelength: 254nm; flow rate: 1ml / min; sensitivity: 0.02AUFS; column Temperature: 25 ℃; Injection volume: 20u1; Retention time: 13.5 minutes; Number of theoretical plates: Based on emodin peak calculation, it should not be less than 3500.
2.2 Preparation of test solution
Take 20 capsules of compound Maren Pills, fully grind them, accurately weigh 5g of fine powder, place them in a conical flask, add 3ml of dilute hydrochloric acid to moisten, add 20ml of methanol precisely, close the plug, shake gently, weigh the weight, sonicate Let cool for 30 minutes. Weigh the weight accurately, make up the lost weight with methanol, shake it well, and filter it. Accurately measure 5ml of the continuous filtrate into a 10ml measuring flask, add methanol to the mark to prepare the test solution; take another negative sample lacking rhubarb to prepare a negative control solution in the same way; accurately weigh the emodin dried with phosphorus pentoxide for 24h 12.5mg of the reference substance was placed in a 50ml measuring flask, dissolved and diluted with methanol to the mark, and shaken to make a 250ug reference solution containing emodin per ml.
2.3 Investigation of linear relationship
Precisely absorb 1.0, 2.0, 4.0, 6.0 and 8.Om1 of the emodin reference solution into a 10ml volumetric flask, dilute with methanol to the mark, shake well, and accurately absorb 20u1 each of the above concentration solutions and the reference solution, respectively, into the injection solution The phase chromatograph was measured according to the above chromatographic conditions, and the concentration of emodin peak was linearly regressed to obtain the regression equation A = 5.9754 × 10000C-8.1135X10000. R = 0.9999 (n = 6). The results show that the injection amount of emodin is in a linear relationship with the absorption peak area value in the range of 0.5 ~ 5.0ug.
2.4 Precision test
Accurately draw 20ul of emodin reference substance solution (80ug / ml), and repeat the injection for 5 times respectively according to the above chromatographic conditions. The RSD value of the emodin peak area was measured to be 0.99%.
2.5 Stability test
Precisely draw freshly prepared sample solution and inject 20ul every 2h. Measure the peak area and calculate it. The daily RSD of emodin is 1.5% (n = 12), indicating that the measurement result of the sample solution is stable within 24h.
2.6 Repeatability test
Weigh 6 samples of the same batch number (030410) in parallel, and process according to the method of "2.2" for the preparation of the test solution, inject 20ul according to the above chromatographic conditions, determine and calculate the content, and the average content of emodin is 40.50mg / 100g, RSD is 0.57%.
2.7 Interference test
Take the control solution of the negative sample and measure it according to the above chromatographic conditions. The results show that the sample chromatography and the reference solution have a similar chromatographic peak at the same retention time (tR = 13.5min), but the negative sample does not have this peak, indicating that the compound Maren The other components of the pill do not interfere with the determination of emodin, and the peak of emodin and other components in the sample can reach baseline separation, with a resolution greater than 1.5.
2.8 Sample recovery test
Precisely weigh about 2.5g of compound Maren Pills (Lot No. 030410) with known emodin content, a total of 6 parts, 2 parts in a group, each group is added emodin reference solution (72.6ug / ml, 96.8 ug / ml, 121.0ug / ml) each 10ml, according to "2.2" for the preparation of the test solution, according to the above chromatographic conditions to determine the content of emodin and calculate the recovery rate.
2. 9 Sample determination
Take three batches of samples, and prepare the test solution according to the method under "2.2", inject into the high performance liquid chromatograph according to the above chromatographic conditions, and calculate the content of emodin in the sample by the external standard peak area method.
3 Discussion
3.1 Selection of chromatographic conditions
With reference to relevant literature, scanning the emodin reference solution in the wavelength range of 200-400nm, the result has the maximum absorption at 254nm wavelength and the highest sensitivity, so 254nm is selected as the detection wavelength; after a variety of proportions of mobile phase selection, methanol-water -Glacial acetic acid (80: 20: 1) is the best. Under this condition, interference can be avoided, so that the emodin in the sample is well separated and the peak shape is sharp.
3.2 Research on extraction method
The samples were extracted by heating and reflux extraction, methanol 24h cold immersion, hydrochloric acid-methanol (3:20) ultrasonic treatment and other methods, the results found that hydrochloric acid-methanol (3:20) ultrasonic treatment 30min measured emodin content is higher, and Easy to operate.
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