Abstract: Rhodiola is a family of Crassulaceae, Rhodiola. It is the name of this genus and the general name of this genus, many of which can be used to extract this medicine. Rhodiola rosea extract normalizes the immune system by improving T-cell immunity. It can improve the body's resistance to the poison accumulated by the progressive development of infection.
Objective To establish a method for the determination of salidroside in Rhodiola rosea by high performance liquid chromatography. Methods The chromatography column was Shim-packVP-ODSC18 (250mm × 4.6mm, 5.0μm), the mobile phase was methanol-water (18:82), the detection wavelength was 278nm, the flow rate was 1.0ml / min, and the column temperature was 30 ℃. Results The injection volume of salidroside in the range of 0.072 ～ 1.296μg showed a good linear relationship with the peak area integral value (r = 0.9999), the average recovery rate was 98.90%, and the RSD was 1.32%. Conclusion The method has high sensitivity, simple operation, accurate and reliable results, and can be used for the determination and quality control and evaluation of salidroside in Rhodiola rosea.
1 Instruments and reagents
LC-2010AHT high-performance liquid chromatograph, including SPD-10A UV detector, SIL-20A automatic sampler, LASSVP6.12SP4 chromatography workstation (Shimadzu Corporation, Japan); KQ318 jewelry ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd. Company); BP-211D analytical electronic balance (Sartorius, Germany, 1 / 100,000); ZFQ85A rotary evaporator (Shanghai Medical Machinery Special Machine Factory Co., Ltd.). Salidroside reference substance (China National Institute for the Control of Pharmaceutical and Biological Products, for content determination, batch number); Rhodiola wine (provided by Sichuan Grassland Research Institute, batch number: 060518, 060810, 070312); methanol is chromatographically pure, water is Double distilled water, the remaining reagents are of analytical grade.
2 Methods and results
2.1 Chromatographic conditions The column is Shim-packVP-ODSC18 (250mm × 4.6mm, 5.0μm); the mobile phase is methanol-water (18:82); the detection wavelength is 278nm; the flow rate is 1.0ml / min; the injection volume is 5.0μl; the column Temperature 30 ℃.
2.2 Preparation of reference substance solution Weigh 7.20mg of salidroside reference substance that has been dried to a constant weight, put it in a 50ml volumetric flask, add methanol to dissolve it, dilute to the mark, shake well, and make the mass concentration to 0.144mg / ml reference substance solution.
2.3 Preparation of the solution for the test product Take 20ml of Rhodiola wine accurately, concentrate it under reduced pressure to no ethanol taste, dissolve it with a small amount of methanol and ultrasonically, dissolve and transfer to a 10ml volumetric flask, dilute to the mark with methanol, shake well , Ultrasound for 10 minutes, and let stand. Absorb the supernatant, filter with 0.45μm organic membrane, and take the continuous filtrate as the test solution.
2.4 System suitability test Take the reference solution and the test solution separately, automatically inject 5.0μl, and record the chromatogram. The results are shown in Figure 1. Using the selected chromatographic conditions, salidroside can be completely separated from other substances in the system, with a retention time of 13.592min, and the theoretical plate number is about 5,500 based on salidroside. a-control substance b-test sample 1-salidroside Figure 1 high-performance liquid chromatogram
2.5 Linear relationship investigation Precision pipetting reference solution 0.5, 1.0, 3.0, 5.0, 7.0, 9.0 μl was injected into the liquid chromatograph, the peak area was determined according to the above chromatographic conditions, with the peak area integral value (Y) as the ordinate, Rhodiola rosea Glycoside injection volume (X) is plotted as a standard curve on the abscissa, calculated, the regression equation Y = 1177.9X-3426.4, r = 0.9999. The results showed that the injection volume of salidroside in the range of 0.072 ～ 1.296μg showed a good linear relationship with the peak area integral value.
2.6 Precision experiment Draw 5.0μl of the reference solution accurately, repeat the injection 5 times under the same conditions, and measure according to the above chromatographic conditions to obtain the peak area of ​​salidroside. Results The RSD of salidroside peak area = 0.26 ﹪ (n = 5), indicating that the precision of the instrument is good. 2.7 Stability experiment The same test solution was taken and measured according to the above chromatographic conditions, and 5.0 μl was injected at 0, 2, 4, 8, 12, 16, 24h to determine the peak area of ​​salidroside. Results RSD of salidroside peak area = 0.72%, indicating that the test solution was stable within 24h.
2.8 Repeatability experiment Take 20ml of the same batch number (batch number: 060518) sample, operate according to the method under "2.3", prepare 5 test solution, each sample accurately absorb 5.0μl, according to the chromatographic conditions under "2.1" The peak area of ​​salidroside was obtained. As a result, the average concentration of salidroside was 32.20 μg / ml, RSD = 0.89 ﹪ (n = 5), indicating that the method had good repeatability.
2.9 Sample recovery rate experiment Take 18.0ml of Rhodiola rosea wine with known content of salidroside, respectively add 2.0ml of salidroside reference solution, and operate according to the method under "2.3" to prepare test solution 5 Parts, determine the peak area, and calculate the sample recovery rate. The results are shown in Table 1. Table 1 Experimental results of sample recovery rate
2.10 Determination of sample content Take 3 batches of samples with different batch numbers, take 5 batches in parallel for each batch number, and operate according to the method under item "2.3" to prepare the test solution. Determine according to the chromatographic conditions under item "2.1", inject 5.0μl respectively, determine the peak area of ​​the test product, and calculate the sample content by external standard method. The results are shown in Table 2. Table 2 Sample content determination results
3 Discussion
3.1 Selection of detection wavelength Take appropriate amount of reference substance, add methanol to dissolve it, and determine by UV spectroscopy, salidroside has maximum absorption peaks at 224nm and 278nm, but due to detection at 224nm, the solvent has certain influence, and the red Salidroside and tyrosol cannot be well separated, so 278nm was selected as the detection wavelength.
3.2 Selection of mobile phase This article examined methanol-water 85:15, 90:10, 15:85, 16:84, 18:82, 20:80, with the increase of methanol ratio, the retention time of salidroside decreased, After comprehensive consideration, the choice of methanol-water 18:82, the retention time is shorter than that reported in the general literature [6], and the adjacent peaks get a good separation effect.
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