Establishment of a Method for the Determination of Emodin in Wudajiangjun Liquor

Wen ZHONG, Zongxi SUN, Yinghong HUANG, Xianyi SHI, Xian PENG, Meiyan QIU, Jiangcun WEI, Wei TIAN, Guodong HUANG

Guangxi International Zhuang Medicine Hospital, Nanning 530201, China

Abstract [Objectives] To establish a method for the determination of emodin in Wudajiangjun liquor, a hospital preparation. [Methods] A high-performance liquid chromatography (HPLC) method for the determination of emodin in Rhizoma Polygontum Cuspidatum in hospital preparation Wudajiangjun liquor was established. Using HPLC method, octadecylsilane chemically bonded silica gel was used as filler for the chromatographic column [Inertsil-C18 chromatographic column (5 μm, 4.6 mm×250 mm)]. Methanol-0.1% phosphoric acid water (76∶24) was used as mobile phase. The flow rate was 1.0 mL/min, the column temperature was 30 ℃, the detection wavelength was 254 nm, and the injection volume was 10 μL. [Results] The content of emodin in Wudajiangjun liquor was determined by reversed-phase high performance liquid chromatography (RP-HPLC). When the injection volume of emodin was in the range of 5.45-54.5 μg/mL, there was a good linear relationship between the injection volume and the peak area. The regression equation is Y=42.952-15.068 (r=0.999 8), the average recovery rate is 98.23%, and RSD=1.45%. [Conclusions] A method for the determination of emodin in Wudajiangjun liquor by HPLC was established. This method can be used as a quality control method for Wudajiangjun liquor with high accuracy and good repeatability, which lays a foundation for the quality control of the medicinal liquor.

Key words Wudajiangjun liquor, Emodin, Content determination

1 Introduction

Wudajiangjun liquor is an ethnic medicine preparation of Guangxi International Zhuang Medicine Hospital. The prescription is made of Rhizoma Polygontum Cuspidatum, mirabilite, Resina draconis, Curcuma zedoary, Radix Cynanchi Paniculati,Ligusticumwallichii, Rhizoma Drynariae,PiperlongumL., frankincense,Panaxnotoginseng,Toddaliaasiatica, Radix Litseae Cubebae,MenthacanadaensisL. It has a unique effect in promoting blood circulation and removing blood stasis, relaxing muscles and activating collaterals, reducing swelling and relieving pain[1-2]. In order to better control the quality standard of Wudajiangjun liquor preparation, it is necessary to improve the quality standard of the preparation, so as to better control the quality of the preparation and ensure the clinical curative effect. According to the clinical drug demand in injury treatment, the quality standard of Wudajiangjun liquor, a national preparation in Guangxi, was studied for improvement.

Wudajiangjun liquor has obtained a large amount of clinical efficacy data, which has a good therapeutic effect in promoting blood circulation and removing blood stasis, relaxing muscles and activating collaterals, reducing swelling and relieving pain. This preparation applies the unique medicinal materials of Guangxi, such as Rhizoma Polygontum Cuspidatum,R.draconis,C.zedoary, Rhizoma Drynariae,P.notoginseng,T.asiaticaand Radix Litseae Cubebae. It has been included in theQualityStandardofZhuangMedicineinGuangxiZhuangAutonomousRegion, which will help its related industries to develop. At the same time, it is expected that this project can be developed into Guangxi characteristic medicine for treating injuries caused by falls in the future, which can stimulate the local economy, build a strong medicine industry chain, and strengthen the advantages of ethnic medicine[2]. This kind of Wudajiangjun liquor with characteristics of ethnic medicine for external use to reduce swelling and relieve pain will certainly add new vitality to Guangxi ethnic medicine preparations, bring new hope to the majority of patients, and play an exemplary role in the development of Chinese herbal medicine with Guangxi characteristics. Therefore, it can bring inestimable social and economic benefits. This product has the advantages of low cost, good curative effect and no adverse reactions. It is predicted that it has good market competitiveness and will produce great social and economic benefits. In order to better ensure the quality of hospital preparations and ensure the clinical efficacy, this study will use HPLC method for the quantitative study of the liquor, to provide higher quality assurance for hospital preparations.

2 Materials

2.1 InstrumentsAgilent high performance liquid chromatography (1260); KQ3200DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); AND electronic balance (Japan, model: GH-252); high speed desktop centrifuge (model: H1650).

2.2 ReagentsEmodin reference substance (No.110756-201913), all purchased from China Institute for Food and Drug Control; Wudajiangjun liquor (No.200501, 200601, 200701, specification: 100 mL/bottle) was provided by the preparation room of Guangxi International Zhuang Medicine Hospital. Chromatographic methanol was provided by Fisher Company of the United States, and the water used in the test is ultra-pure water.

3 Methods and results [3-6]

3.1 Chromatographic conditionsChromatographic column: octadecylsilane chemically bonded silica gel was used as filler [Inertsil-C18chromatographic column (5 μm, 4.6 mm×250 mm)], methanol-0.1% phosphoric acid water (76∶24) was used as mobile phase. Flow rate: 1.0 mL/min, column temperature: 30 ℃. Detection wavelength[1]: 254 nm. The injection volume is 10 μL, and the number of theoretical plates should not be less than 3 000 according to the emodin peak.

3.2 Preparation of reference solutionAppropriate amount of emodin reference substance was taken, and weighed accurately, and mixed with methanol to make the solution containing 0.545 mg per 1 mL, that is, emodin reference solution with the concentration of 0.545 mg/mL.

3.3 Preparation of sample solution3 mL of Wudajiangjun liquor in batch number 200501, 200601, 200701 was put into evaporator for drying. Appropriate amount of methanol was added for ultrasonic treatment, dissolution, filtration, and placed in a 10 mL volumetric flask. The methanol was added to dilute it to the scale, shaken well, and filtered with a microporous membrane (0.45 μum).

Samples of Wudajiangjun liquor withoutP.cuspidatumwere made according to the technology of Wudajiangjun liquor. Samples of Wudajiangjun liquor withoutP.cuspidatumwere taken, and negative sample solution was prepared according to the above preparation method of the sample solution.

3.4 Specificity test10 μL of control solution, 10 μL of test solution and 10 μL of negative sample solution were injected into liquid chromatograph and determined according to the method of Section3.1. Results showed that there was a chromatographic peak between the sample solution and the control substance at the same retention time, while there was no corresponding peak between the negative sample solution and the control substance at the same retention time. The emodin reference substance was well separated and there was no interference from other components, as shown in Fig.1.

3.5 Methodological investigation

3.5.1Investigation of linear relationship. Preparation of emodin reference solution: Appropriate amount of emodin was taken, weighed accurately, and methanol was added to make the solution containing 0.545 mg per 1 mL. Preparation of standard series solutions: 0.1, 0.3, 0.5, 0.8 and 1.0 mL of emodin reference solutions were precisely pipetted, respectively, and put into 10 mL flasks, then methanol was added to the scale and shaken well, which were used as the reference solutions of different concentrations.

Note: A. negative samples without P. cuspidatum; B. samples of Wudajiangjun liquor; C. emodin reference substance.Fig.1 Liquid chromatogram of emodin in Wudajiangjun liquor

10 μL of each of the above different concentrations of reference substance solution was precisely pipetted, and the sample was injected and determined. The standard curve was drawn with the injection volume of the reference substance (μg/mL) as the abscissa and the peak area value as the ordinate. Results showed that when the injection volume of emodin was in the range of 5.45-54.5 μg/mL, there was a good linear relationship between the injection volume and the peak area. The regression equation isY=42.952-15.068 (r=0.999 8), as shown in Fig.2.

Fig.2 Linear equation diagram

3.5.2Precision test. The same sample solution (200701) was injected continuously for 6 times according to the determination method under the quality standard. Results showed that the peak area of emodin determined 6 times was 1 657, 1 596, 1 578, 1 605, 1 639, 1 588, respectively, and the average peak area value was 1 610.5,RSD=1.92%. The results indicated that the precision of this method was good (Table 1).

Table 1 Precision test results of Wudajiangjun liquor (n=6)

3.5.3Replication test. 3 mL of the same batch of sample (No.200701) was precisely weighed, a total of 6 groups. 6 samples of test solution were prepared in parallel according to the method of content determination, and the samples were injected and determined, respectively. Results showed that the emodin content of the 6 samples was 128.60, 123.86, 123.24, 126.50, 127.20, 120.37 μg/mL, and the average content of emodin was 124.96 μg/mL,RSD=2.42%. This showed that the reproducibility of this method was good (Table 2).

Table 2 Replication test results of Wudajiangjun liquor (n=6)

3.5.4Stability test. The same sample solution (No.200701) was taken and determined at 0, 2, 4, 8, 12 and 24 h, respectively, according to the content determination method under the quality standard. The results showed that the peak area of emodin determined at different time was 1 642, 1 567, 1 593, 1 626, 1 572, 1 569, respectively, and the average peak area of emodin was 1 594.8,RSD=2.01%. This showed that the emodin in the sample solution was stable within 24 h (Table 3).

Table 3 Stability test results of Wudajiangjun liquor (n=6)

3.5.5Sample recovery test. A total of 6 portions of Wudajiangjun liquor (1.5 mL) (No.200701) with known content were collected and placed in a stoppered conical bottle. The corresponding emodin reference substance was precisely added, prepared according to the preparation method of the reference substance solution and filtered. The filtrate was centrifuged at high speed of 10 000 r/min for 10 min, and the supernatant was filtered with 0.45 μm microporous membrane and determined according to the content determination method. The average recovery rate andRSDof emodin in Wudajiangjun liquor were calculated. The results are shown in Table 4, indicating that the method is accurate.

Table 4 Sample recovery test results of emodin (n=6)

3.6 Determination of the content of three batches of samples

Each batch of Wudajiangjun liquor (No.200501, 200601, 200701) (3 mL) was precisely taken to prepare test solution according to the above method. 10 μL of solution was accurately injected into high performance liquid chromatograph, and the content of emodin in Wudajiangjun liquor was determined according to the content determination method, which was 76.09, 80.97 and 127.33 μg/mL, respectively.

4 Discussion

The effects of elution systems such as methanol-water, methanol-0.1% glacial acetic acid, methanol-0.3% glacial acetic acid, methanol-0.2% phosphoric acid, methanol-0.1% phosphoric acid, methanol-0.05% phosphoric acid, acetonitrile-water, acetonitrile-0.1% phosphoric acid and acetonitrile-0.2% phosphoric acid on peak time, separation and baseline were investigated and compared. The results showed that using methanol-0.1% phosphoric acid and acetonitrile-0.1% phosphoric acid system, the chromatographic peak separation was good, the baseline was stable, and the proper adjustment of gradient elution procedure can achieve good separation of each peak, and balanced baseline. Considering the cost, this experiment used methanol-0.1% phosphoric acid system for gradient elution.

In this experiment, the effects of 20, 25, 30 and 35 ℃ on the degree of separation, the time of peak and the stability of the baseline were investigated. The experimental results showed that the column temperature had little effect on it, so the effects of different column temperature and flow velocity on the degree of separation, the time of peak and the stability of the baseline were investigated. In this experiment, the column temperature was 30 ℃, and the effects of three different flow rates of 1.2, 1.0 and 0.8 mL/min on the separation were also investigated. The chromatogram of the test results showed that the separation degree was good and the baseline was balanced when the flow rate was 1.0 mL/min, so the flow rate used in this experiment was 1.0 mL/min.