Simultaneous Determination Of Echinacoside And 2,3,5,4'-tetrahydroxy Stilbene-2-Ο-β-D-glucoside in Congrong Tongbian Oral Liquid By HPLC

Abstract: The objective was to simultaneously determine echinacoside and 2,3,5,4'-tetrahydroxy stilbene-2-Ο-β-D-glucoside in Congrong digestive oral liquid by HPLC. Tech Mate C18-ST (4.6 mm×250.0 mm, 5 μm) column was used with gradient elution by acetonitrile-0.2% formic acid solution; the detection wavelength was 330 nm; the flow rate was 1.0 mL/min. The results showed that echinacoside and 2,3,5,4'-tetrahydroxy stilbene-2-Ο-β-D-glucoside in the sample presented a good resolution from the adjacent chromatographic peaks with a good linear relationship in the range of 0.159 3-0.955 8 μg and 0.385 3-4.623 5 μg. The established method is simple and accurate and can be used to determine echinacoside and stilbene glycoside in Congrong digestive oral liquid.

Keywords: Congrong Degestive oral liquid; echinacoside; 2,3,5,4'-tetrahydroxy stilbene-2-Ο-β-D-glucoside; HPLC

ORGANIC CISTANCHE EXTRACT WITH 30% ECHINACOSIDE AND 12% ACTEOSIDE

The current quality standards of Cistanche Rong digestive Oral Liquid have problems such as simple test items, poor operability, and cumbersome and time-consuming operations. In addition to the general inspection items, the standard only has thin-layer identification items for Polygonum multiflorum and Citrus aurantium, and Polygonum multiflorum and Citrus aurantium. The actual thin-layer chromatography identification method is not rigorous enough and has poor operability. The content determination item uses ultraviolet spectrophotometry to detect 1,8-dihydroxyanthraquinone in Cistanche purpurea oral liquid. Since this method requires multiple reflux extractions, The operation is cumbersome and time-consuming, and the reproducibility and accuracy are poor. The quality of the original medicinal materials cannot be controlled, and the quality control of the king drug Cistanche deserticola is lacking. In this standard research work, the determination of echinaceaside in Cistanche deserticola and stilbene glycoside in Polygonum multiflorum in Cistanche deserticola Oral Liquid was studied. Based on the formula's composition and the process's characteristics, a high-performance liquid phase was established. The chromatographic method was used to determine the echinacoside and stilbene glycosides in Cistanche Rong digestive Oral Liquid, and its content limits were formulated to further improve and improve the quality control standards. The determination method is simple and fast, and the content limits are reasonably formulated. After methodological verification, the accuracy, precision, linearity, durability, and range accuracy are good, and they can be used to determine the content of Cistanche Rong Tongbang Oral Liquid [1-5].

1 Instruments and reagents

1.1 Instruments

Waters high-performance liquid chromatograph (2695 and ARC); 1/100,000 electronic balance (MS205DU), 1/10,000 electronic balance (ME204E, Mettler-Toledo).

1.2 Test drugs and reagents

Echinacea side (batch number 110670-201706, purity 89.7%); stilbene glycoside (batch number 110844-201915, purity 90.7%), all provided by China Institute of Food and Drug Control; Cistanche purpurea oral liquid (batch number 20191204, 20191205, 20191206, 20200404, 20200405, 20200406, 20200407, 20200408, 20200409, 20200410), provided by a pharmaceutical company in Gansu; methanol, etc. are chromatographically pure; water is ultrapure water; other reagents are analytical grade.

2 Methods and results

2.1 Chromatographic conditions

Use Tech Mate C18-ST (4.6 mm × 250.0 mm, 5 μm) as the chromatographic column, use acetonitrile as the mobile phase A, and use 0.2% formic acid solution as the mobile phase B. Carry out gradient elution according to the regulations in Table 1; column temperature The temperature is 35℃; the detection wavelength is 330 nm; the flow rate is 1.0 mL/min; the injection volume of the test sample and reference substance is 10 µL each; the number of theoretical plates should not be less than 4000 based on the echinaceaside peak.

2.2 Preparation of solution

Preparation of reference solution: Take appropriate amounts of echinaceaside reference substance and stilbene glycoside reference substance, weigh them accurately, and add 70% methanol to prepare a mixed solution containing 70 μg of echinaceaside and 150 μg of stilbene glycoside per 1 mL. , that's it. Preparation of the test solution: Shake the product well, take about 0.3 g, weigh it accurately, put it into a 25 mL brown measuring bottle, add an appropriate amount of 70% methanol, sonicate for 30 minutes, let it cool, add 70% methanol to dilute to Scale, shake well, filter, and take the remaining filtrate to get it. Preparation of negative sample solution: Precisely weigh about 0.3 g of the negative sample and prepare it according to the preparation method of the test solution.

Figure 1 HPLC chromatograms of Congrong digestive oral liquid

2.3 Methodological review

2.3.1 Working curve and linear range

Precisely draw the mixed reference solution and add 70% methanol to prepare a series of mixed reference solutions. Precisely draw 10 µL of each of the above series of control solutions, inject it into the liquid chromatograph, and measure the peak area integrated value. Take the concentration of the reference solution (µg/mL) as the ordinate (x) and the peak area integrated value as the abscissa (y). Draw a graph to get a straight line passing through the origin. The regression equation is y1=1.510 4×106x-1.177 2×105 (r=0.999 9), 2=3.644 0×106x-6.3836×105 (r=0.999 9). The results show that echinaceaside and stilbene glycoside have a good linear relationship in the mass range of 0.159 3 to 0.955 8 μg and 0.385 3 to 4.623 5 μg.

Cistanche Tubulosa

2.3.2 Precision test

Precisely draw 10 µL of the mixed reference solution, and repeat the injection 6 times under the "2.1" chromatographic conditions. The RSD of the peak areas of echinacoside and stilbene glycoside were measured to be 0.66% and 0.59% respectively, indicating that the precision of the instrument is good.

2.3.3 Stability test

According to the method under "2.1", take this product (batch number 20191205) to make a test solution, inject samples for measurement at 0 h, 4 h, 8 h, 12 h, 20 h, and 24 h after preparation, and record the peak area. , the relative standard deviation is between 2.37% and 2.79%. The results show that the test solution is stable within 24 hours.

2.3.4 Repeatability test

Take 6 aliquots of the same batch of test samples (batch number 20191205) and inject samples for measurement according to the chromatographic conditions under "2.1". Results The relative standard deviation of echinaceaside was 1.48%, with good repeatability; the relative standard deviation of stilbene glycoside was 1.10%, with good repeatability.

Cistanche Tubulosa

2.3.5 Recovery Rate Determination

Adopt sample addition and recovery to provide 50% of the sample amount (0.3 g) of the test sample and the sampling content of 1.0:0.5, 1.0:1.0, 1.0:1.5, respectively, and accurately absorb the reference substance reserve solution 1 (echinacea glycoside content is 0.431 5 mg/mL) 1.0 mL, 2.0 mL, and 3.0 mL each, and then accurately add reference substance stock solution 2 (stilbene glycoside mass concentration 0.8678 mg/mL) 1.25 mL, 2.50 mL, and 3.75 mL respectively. Add to 25 mL volumetric flasks respectively, using

Blow dry with a nitrogen blower. Then accurately weigh 9 samples of the same batch with known contents (batch number 20191205, echinaceaside content is 7.810 2 mg/g, stilbene glycoside content is 14.232 4 mg/g), and the sampling amount of each sample is The sample amount of the test sample is about 0.15 g. Add it to the above volumetric flask respectively. Prepare it according to the method under the test solution. Measure it according to the law. Calculate the recovery rate. The result is easy.

The average recoveries of stilbene glycosides and stilbene glycosides were 98.28% and 98.43%, respectively, and the RSDs were 1.56% and 0.97%, respectively, indicating good instrument accuracy.

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