Determination of Rhein in Changtong Oral Liquid by HPLC

Changtong Oral Liquid is a Chinese medicine preparation developed by our hospital. It has the functions of benefiting qi and stasis and ventilating qi, and is used for abdominal trauma or peritoneal adhesion after abdominal surgery. Rhubarb is the king medicine in the prescription. Rhein (C15H8O6) has antibacterial, anti-cancer, cathartic and diuretic effects and is one of its effective ingredients. Therefore, the content of this component is determined as the quality control standard.

Adopt high-performance liquid chromatography, use octadecylsilane-bonded silica gel as the filler, the mobile phase is methanol-0.1% phosphoric acid solution 64:36, and the detection wavelength is 230nm. The standard curve was drawn with the concentration of rhein reference substance X as the ordinate and the peak area Y (μg) as the abscissa. The linear regression equation is: Y = 9.534 × 10-6X + 0.1934, and the correlation coefficient r = 1.0000. This method has a good linear relationship. It indicates that the injection volume is in the range of 0.0402-0.804μg, and the concentration of the reference substance is linearly related to the peak area. The average sample recovery rate was 98.84%, and the RDS was 0.86%. The method is simple and reliable, with high precision and good resolution, and can be used for the quality control of Changtong oral liquid.
High performance liquid chromatography

Determination of rhein in Changtong oral liquid

[Abstract] Objective: To establish a method for the determination of rhein in Changtong oral liquid. Method: High performance phase chromatography, using octadecylsilane-bonded silica gel as filler, the mobile phase was methanol-0.1% phosphoric acid solution 64:36, and the detection wavelength was 230nm. Results: This method has a good linear relationship, with an average recovery of 98.84% and an RDS of 0.86%. Conclusion: The method is simple and reliable, with high precision and good resolution, and can be used for the quality control of Changtong oral liquid.

[Key words] Determination of rhein in Changtong oral liquid by high-performance phase chromatography

Changtong Oral Liquid is a Chinese medicine preparation developed by our hospital. It has the functions of benefiting qi and stasis and ventilating qi, and is used for abdominal trauma or peritoneal adhesion after abdominal surgery. Rhubarb is the king medicine in the prescription. Rhein (C15H8O6) has antibacterial, anti-cancer, cathartic and diuretic effects and is one of its effective ingredients. Therefore, the content of this component is determined as the quality control standard.

1 Instruments and reagents

Waters high-performance phase chromatograph (USA), Million32 chromatography data processing software. Methanol (chromatographically pure), water is double distilled water, other reagents are analytically pure. Rhein acid reference substance (for content determination, batch number 0757-9804) is provided by the China National Institute for the Control of Pharmaceutical and Biological Products. The sample Changtong oral liquid was trial-produced by the Pharmacy Department of Nanfang Hospital.

2 Chromatographic conditions

Waters C18 liquid chromatography column (150 × 4.6mm, 5μm), mobile phase is methanol-0.1% phosphoric acid solution 64:36, detection wavelength is 230nm, flow rate is 3-4ml · min-1. Under this condition, the retention time of rhein is about 12 minutes, which is separated from other peaks. The high-performance liquid chromatograms of rhein reference substance and Changtong oral solution are shown in Figures 1 and 2. Fig.1 HPLC chromatogram of rhein standard productFig.2 HPLC chromatogram of Changtong oral solution

3 Investigation of linear relationship Precisely weigh 0.00402g of rhein acid reference substance, place it in a 100ml measuring flask, add methanol to the mark to obtain the correct

According to the stock solution, take the appropriate amount of the stock solution and add the mobile phase to make 1 ml of the reference series solution containing 201, 4.02, 8.04, 12.06, 20.10, and 40.20 μg. Inject 20 μl each according to the law, and measure according to the above chromatographic conditions. The standard curve was drawn with the concentration of rhein reference substance X as the ordinate and the peak area Y (μg) as the abscissa. The linear regression equation is: Y = 9.534 × 10-6X + 0.1934, and the correlation coefficient r = 1.0000. It indicates that the injection volume is in the range of 0.0402-0.804μg, and the concentration of the reference substance is linearly related to the peak area.

3 Preparation of reference substance solution and test article solution Preparation of reference substance solution: Pipette appropriate amount of rhein acid reference substance stock solution, add mobile phase and dilute to a concentration of 8.04 μg / ml reference substance solution.

Preparation of test solution: Precision transfer 10ml of Changtong oral solution, add 1ml of 0.8% phosphoric acid solution, add methanol to volume to 20ml, shake well, centrifuge for 10min, take supernatant with a microporous filter (0.45μm ) After filtering, the continuous filtrate is taken as the test product solution.

Preparation of blank solution: Take 0.2ml of the blank sample of rhubarb deficiency made by the same process to remove the taste of rhubarb, and operate according to the preparation method of the test solution.

4 Precision experiment

Accurately draw 10μl of 8.04μg / ml reference substance solution and inject it into the high-performance liquid chromatograph for 5 consecutive measurements. The average peak area of ​​rhein is 831543, RSD = 0.18% (n = 5). The results show that the precision is good.

5 Stability test

Accurately draw 10μl of the same reference solution stored at room temperature, and inject the sample at a certain time interval for the same method, a total of 5 times. The results show that the test solution has no significant changes within 7 days, indicating that this reference is basically stable, RSD = 0.49%

6 Repeatability test

Take 6 batches of Changtong oral liquid 10ml each and determine according to law. The average value of rhein content was 165.62, RSD = 2.15% (n = 6). It shows that the method has good repeatability.

7 Recovery rate test

Precisely draw 10ml of Changtong Oral Liquid with known content, add 3ml of rhein reference stock solution (every 1ml contains rhein 8.04μg), prepare according to the preparation method of the test article solution, and determine according to the method. The average sample recovery rate is 98.84%, RSD = 0.86% (n = 6)



Table 1 Rhein acid recovery recovery test results in Changtong Oral Liquid Test No. known content measured amount recovery average RDS
/ μg / μg /% /% /%

1 284.52 98.45

2 283.89 97.93

3 284.89 98.76

4 165.79 285.24 100.41 98.84 0.86

5 286.89 99.05

6 284.52 98.45

Note: The amount of control substance added is 120.60μg
9 Determination of samplesPrecisely draw 10μl of the reference solution and the test solution, and then inject it into a high-performance liquid chromatograph for measurement.The sample measurement results are shown in Table 2.

Table 2 Sample measurement results (n = 3)

Serial number lot number rhein content (μg / ml)
1 20010810 3973.39
2 20010904 3151.95
3 20011121 2136.80
10 Discussion Because the chromatogram of the test product has more impurity peaks and requires higher column efficiency, the theoretical plate number should be calculated according to the rhein peak and should not be less than 5000.

The method is simple and reliable, with high precision and good resolution, and can be used for quality control of Changtong oral liquid.

Determination of Rhein
In the CHANGTONG Oral Liquid By HPLC
HOU Lian-bing YANG Xi-xiao

(NanFang hospital pharmacy of The first military medicaluniversity, Guangzhou 510515, China)

Abstract: Object To develop an HPLC method for determining Rhein inthe CHANGTONG Oral Liquid. Method The contents of Rhein was determined by HPLC. Prodigy ODS (3) C18 column was used, withmethanol –0.1% phosphoric acid 64: 36as the mobile phase anddetected at the wavelength of 230nm. Results The average recoveryof Rhein was 98.84%, RDS = 0.86%. Good linearity of Rhein wasobtained (r = 1.0000) within the range of 0.0402-0.804μg. ConclusionThis method was simple and accurate and was not interfered by otherconstituents in the prescription, can be used for quality controlof CHANGTONG oral liquid.

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