Determination of Notoginsenoside R1 in Shexiang Jiegu Capsules by HPLC

[Keywords] Determination of Notoginsenoside R1 in Shexiang Jiegu Capsules by HPLC

Determination of Forsythin content in Shexiang Jiegu Jiaonang by HPLC

[Abstract] OBJECTIVE: To set up a method for quality control of Shexiang Jiegu Jiaonang METHOD HPLC method was developed to quantitative determination. COLUMN Agilent Technologies ZORBAX Extend-C18 4.6 × 250mm, 5μm. The mobilic phase was acetonifrile-water (21: 79V /V).The column temperature was 30 ℃, the wavelength for detection was 203nm, flow rate was 1.0ml · min-1. RESULTS The paeonol average of recovery rate was 97.6%, and RSD was 1.0% (n = 6). CONCLUSION The method is simple, accurate and suitable for its assaying.

【Key word】 HPLC; Shexiang Jiegu Jiaonang; Notoginsenoside R1; determination

Shexiang Jiegu Capsules are the varieties listed in the fifth volume of the Ministry of Health (Standard Preparations of Traditional Chinese Medicine). They are composed of 22 traditional Chinese medicines such as red peony root, panax notoginseng, angelica, dipsacus, hematoxylin and musk. , The effect of continuous tendons and bones. It is clinically used for bruises, fractures of tendons, blood stasis and blood coagulation, and flashback. Panax notoginseng is one of the main drugs in the prescription. Panax notoginseng saponin R1 is its main active ingredient. There is no content determination method in the original standard. In order to better control the internal quality of the preparation, this article uses high performance liquid chromatography to determine the content of notoginsenoside R1 in Shexiang Jiegu Capsules, with a view to providing a rapid and accurate method for determining the quality of the preparation. The report is as follows.

1 Instruments and reagents

LC-2010A high-performance liquid chromatograph, CLASS-VP chromatographic workstation, ultraviolet detector; Panax notoginsenoside R1 reference substance (lot number), provided by China National Pharmaceutical and Biological Products for content determination; Musk Jiegu capsule is a commercially available sample (Liaoyuan Yadong Chinese Medicine Co., Ltd., specification 0.3g · grain-1, batch number: 060801, 061102, 060912). Acetonitrile is chromatographically pure; water is ultrapure water; other reagents are analytically pure.

2 Methods and results

2.1 Chromatographic conditions and system suitability test Column: Agilent Technologies ZORBAX Extend-C18 4.6 × 250mm, 5μm; mobile phase: acetonitrile-water (21:79, V / V); detection wavelength: 203nm; flow rate: 1.0ml · min -1; column temperature: 30 ° C. The number of theoretical plates should not be less than 4000 based on the R1 peak of notoginsenoside.

2.2 Solution preparation

2.2.1 Preparation of reference substance solution Weigh the proper amount of panax notoginsenoside R1 reference substance which has been dried under reduced pressure of phosphorus pentoxide for more than 12 hours, add methanol to make a reference substance stock solution of 0.25 mg · ml-1; Appropriate amount of reference stock solution, add methanol to make a reference solution of 0.05mg · ml-1.

2.2.2 Preparation of test solution Take the content of Musk Elder Bone Capsule under the difference of loading volume, grind it, take about 1g, accurately weigh it, put it in a conical flask with a stopper, and add 50ml of methanol to the stopper. Constant weight, ultrasonic treatment (power 250W, frequency 50 kHz) for 1 hour, take out, let cool, weigh again, make up the lost weight with methanol, shake, filter, take 25ml of the continuous filtrate in a precise amount and place it in an evaporating dish Medium, evaporate to dryness, the residue is dissolved with 30ml of n-butanol saturated with water, shake and extract with ammonia test solution 3 times, 15ml each time, combine the extract solution of ammonia test solution, and shake and extract with water-saturated n-butanol twice. 15ml each time, combine with the above n-butanol solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 10ml volumetric flask, dilute with methanol to set the scale, shake well, and filter with a microporous membrane (0.22μm), Take the continuous filtrate, that is [2].

2.2.3 Preparation of negative control solution

Take one-tenth the amount of the remaining medicinal materials in the prescriptions other than Panax notoginseng saponins R1, make capsules according to the method, and then make the negative control solution according to the preparation method of the test product solution.

2.3 Specific test

Accurately draw 10μl of the test solution, negative control solution and control solution into the liquid chromatograph, and record the chromatogram (Figure 1). It can be seen from Figure 1 that in the chromatogram of the test product, there is a chromatographic peak with the same retention time (notoginsenoside R110.971min) at the position corresponding to the chromatogram of the reference product. The negative test has no interference, which proves that this method is feasible.

2.4 Investigation of linear relationship

Precisely absorb the appropriate amount of Panax notoginsenoside R1 reference stock solution (concentration: 0.25mg.ml-1), add methanol to make 6 solutions with concentrations of 0.005, 0.01, 0.05, 0.10, 0.15, 0.25mg · ml-1, Shake well and filter with 0.22μm microporous membrane. Accurately draw 10μl of each continuous filtrate into the liquid chromatograph and measure the peak area. Taking the peak area (A) as the ordinate and the concentration (C) as the abscissa, the regression equation of notoginsenoside R1 is: A = 392250.2 C +8620.1, r = 0.9998 (n = 6). The results show that the peak area of ​​notoginsenoside R1 in the range of 0.005 ～ 0.25mg · ml-1 has a good linear relationship with its concentration.

2.5 Precision test

Accurately draw 10μl of Notoginsenoside R1 reference solution (0.05mg · ml-1), and repeat the sample injection 6 times under the above chromatographic conditions. The relative standard deviation (RSD) of the peak area of ​​Notoginsenoside R1 solution is 0.3% ( n = 6), indicating good precision.

2.6 Stability test

Accurately draw 10μl of notoginsenoside R1 reference solution (0.05mg · ml-1) and measure at 0, 4, 8, 12, 24, and 48h respectively. The results show that notoginsenoside R1 reference solution is stable within 48h, RSD 0.4% (n = 6).

2.7 Repeatability test

Take the test product (batch number 061101), a total of 6 parts, respectively, according to the "2.2.2 test product solution preparation" method to prepare the test product solution, and determine the RSD of Panax notoginsenoside R1 content is 1.6% (n = 6), indicating good reproducibility.

2.8 Sample recovery test

Accurately weigh a sample of known content (batch number 061101, panax notoginsenoside R1 content 0.93mg · g-1, average loading 0.2996g · grain-1), a total of 6 portions, and place them in conical flasks with stoppers, respectively Accurately add 3ml each of Panax notoginseng saponin R1 reference stock solution (0.25mg · ml-1), and operate according to the method of "2.2.2 Preparation of test solution". The recovery test solution is obtained and determined according to law. The results are shown in Table 1. Table 1 Notoginsenoside R1 sample recovery test (omitted)

2.9 Determination of sample content

Accurately draw 10μl each of the test solution and the reference solution into the liquid chromatograph, determine the peak area of ​​notoginsenoside R1 in the three batches of samples, and calculate its content according to the external standard method (n = 5). The results are shown in Table 2. Table 2 3 batches of sample content determination results (omitted)

3 Discussion

3.1 Through the determination of notoginsenoside R1 in the three batches of samples, the results show that the highest content is 0.31mg · grain-1, the lowest is 0.27mg · grain-1, comprehensive consideration is given to determine the limit of each notoginseng saponin R1 should not be less than 0.20 mg.

3.2 Selection of mobile phase In the experiment, different mobile phases methanol-water (25:75) and acetonitrile-water-glacial acetic acid (25: 75: 0.5) were used. The results showed that the mobile phase acetonitrile-water (21:79) chromatography The baseline of the graph is relatively stable.

3.3 The results of this method are accurate, the method is simple, the reproducibility and recovery rate are ideal, and can be used as the quality control method of the preparation.