Fengxian ZHAO, Jiabao MA, Jiangcun WEI, Xianxian LIU, Wenli LI, Yong CHEN, Zhengteng YANG*

1. Guangxi University of Chinese Medicine, Nanning 530200, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 3. Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Nanning 530200, China; 4. International Zhuang Medical Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning 530001, China

Abstract [Objectives] To establish the quality control method of Dujieqing pills. [Methods] Tangerine peel and Panax notoginseng in Dujieqing pills were qualitatively identified by thin layer chromatography (TLC). The content of hesperidin and ginsenoside Rb1 was determined by HPLC. The acetonitrile-aqueous solution was used as mobile phase for gradient elution, the detection wavelength was 203 nm, the flow rate was 1.0 mL/min, the column temperature was 25 ℃, and the injection volume was 10 μL. [Results] Under TLC, tangerine peel and P. notoginseng had clear chromatographic spots, good separation effect, and strong specificity and negative samples showed no interference; there was a good linear relationship between hesperidin and ginsenoside Rb1 in the range of 0.838-8.38 μg (R2=0.999 4) and 0.44-4.4 μg (R2=0.999 4), respectively; the average recovery (n=9) was 98.90% (RSD=2.31%) and 98.31% (RSD=2.56%), respectively. [Conclusions] The established quality control method is simple, reproducible, accurate and reliable, and can be used for the quality control of Dujieqing pills.

Key words Dujieqing pill, Quality control, TLC, HPLC

1 Introduction

Dujieqing pill is composed of R. Ranunculi Ternati, tangerine peel,O.japonicum(Thumb)kunze andE.seuSteleophaga,etc., which is made from the preparation of Dujieqing oral liquid (Z01060068) in the First Affiliated Hospital of Guangxi University of Chinese Medicine. It has the effect of resolving phlegm and removing blood stasis, detoxicating and resolving a mass, invigorating the spleen and kidney, replenishingqiand nourishingyin.The original oral liquid dosage has a good therapeutic effect on lung cancer, liver cancer, osteosarcoma and other malignant tumors[1-4], but the production process of water boiling and precipitation with ethanol for oral liquid is tedious and will cause the loss of active components to a certain extent. In order to further tap the anti-tumor potential of the prescription, the research group modified it into pill, which retains a large number of active ingredients of the original prescription, and also is easy to take, preserve and carry. In this study, the quality standard of the pill dosage form was analyzed in order to establish a stable and effective quality control method to ensure the stability of the drug in clinical use.

2 Experimental instruments and reagents

2.1 InstrumentsWaters 2695-2489 high performance liquid chromatograph (Waters Technology Co., Ltd.); Secura225D-1CN electronic balance (Sartorius Scientific Instruments Beijing Co., Ltd.); YQ-520C ultrasonic cleaning instrument (Shanghai Sonic-Electronics Technology Co., Ltd.); IQ 7000 Millipore ultra pure water system (Millipore, USA); HWS-26 thermostat water bath (Shanghai Qixin Scientific Instrument Co., Ltd.).

2.2 Drugs and reagentsTangerine peel control medicinal materials (No.120969), hesperidin reference substance (No.20170428), ginsenoside Rb1reference substance (No.20140127),P.notoginsengsaponins R1(No.110745-200617), ginsenoside Rg1 (No.22427-39-0) were all purchased from National Institutes For Food and Drug Control; Dujieqing pills (No.20190708, 20190718, 20190729, 20190807, 20190819,20190830) were provided by the First Affiliated Hospital of Guangxi University of Chinese Medicine; silica gel G plate (No.20190612, Qingdao Marine Chemical Plant), the reagents used in TLC were all analytically pure, acetonitrile and methanol for HPLC were chromatographically pure (Fisher, USA), and water was ultra-pure.

3 TLC identification

3.1 Identification ofP.notoginseng[5-7]5 g of this product was taken, ground into fine pieces, placed in a conical flask, mixed with 50 mL of methanol, treated with ultrasound for 1 h, and filtered. After the filtrate was dried in a water bath, 1 mL of methanol was added to dissolve the filtrate as the sample solution. The negative control solution was prepared by the same method as the sample solution (lack ofP.notoginseng). In addition, ginsenoside Rg1 and notoginsenoside R1reference substances were mixed with methanol to produce a 2 mg/mL solution, which was used as the control solution. According to the test requirements of TLC (ChinesePharmacopoeia2015), 5 μL of sample solution, control solution and negative control solution were pipetted and dripped onto the silica gel G thin layer plate, respectively. The lower solution of trichloromethane-n-butanol-methanol-water (2∶4∶1∶2) was used as the developer to develop at a distance of 8 cm, and taken out. It was dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105 ℃ until the spots showed clear color. In the chromatogram of the sample, the spots of the same color were shown in the corresponding position of the chromatogram of the reference substance (Fig.1).

Note: 1: negative control; 2-4: three batches of samples (20190708, 20190718, 20190729); 5: notoginsenoside R1; 6: ginsenoside Rg1.

3.2 Identification of tangerine peel[8-9]5 g of this product was taken, ground into fine pieces, placed in a conical flask, mixed with 20 mL of ethyl acetate, treated with ultrasound for 30 min, and filtered. After the filtrate was dried in a water bath, 1 mL of methanol was added to dissolve it as the sample solution. 1 g of tangerine peel was taken and 20 mL of ethyl acetate was added. The control solution was prepared by the same method. The negative control solution was prepared by the same method as the sample solution (lack of tangerine peel). In addition, hesperidin was used as the reference substance and methanol was added to make a 1 mg/mL solution, which was used as the reference solution. According to the test requirements of TLC (ChinesePharmacopoeia2015), 5 μL of test sample solution and 5 μL of negative control solution, 2 μL of control medicine solution and 2 μL of control solution were pipetted, and dripped onto the silica gel G thin layer plate, respectively. The ethyl acetate-methanol-water (100-100-17-15) was used as developer to develop at a distance of 8 cm, and taken out. It was dried, sprayed with 1% AlCl3test solution, heated at 105 ℃ for 3 min, and examined under 365 nm ultraviolet lamp. In the chromatogram of the sample, the fluorescent spots of the same color were shown in the corresponding position of the chromatogram of the control medicine and the reference substance (Fig.2).

4 Content determination

4.1 Preparation of solution

4.1.1Mixed reference solution. Proper amount of hesperidin and ginsenoside Rb1reference substance was precisely weighed. Methanol was added to dissolve it in a 20 mL volumetric flask and passed through a 0.45 μm microporous filter membrane to obtain ginsenoside Rb1with a concentration of 0.419 mg/mL and hesperidin with a concentration of 0.22 mg/mL.

Note: 1: negative control; 2-4: three batches of samples (20190708, 20190718 and 20190729); 5: tangerine peel control medicinal material; 6: hesperidin.

4.1.2Sample solution. 1g of finely ground Dujieqing pill was precisely weighed and placed in a conical flask with a plug, 20 mL of methanol was precisely added, shaken well, and ultrasonic treatment was carried out for 30 min. It was cooled and filtered, the container and filter residue were washed with 5 mL of methanol, the washing solution was combined with the filtrate, and dried. The sample solution was obtained by dissolving it with methanol into a 5 mL volumetric flask and passing through a 0.45 μm microporous membrane. The negative samples were prepared according to the same method as the sample solution.

4.2 Chromatographic conditionsThe chromatographic column was Diamonsil C18(250 mm×4.6 mm, 5 μm). Mobile phase was acetonitrile-aqueous solution, gradient elution. The elution procedure was shown in Table 1, and the detection wavelength was 203 nm. The column temperature was 25 ℃, the flow rate was 1 mL/min, and the injection volume was 10 μL. Hesperidin and ginsenoside Rb1in Dujieqing pills could be well separated, and other components in the sample showed no interference.

Table 1 Gradient elution

4.3 Methodological investigation

4.3.1Specificity test. 10 μL of mixed reference solution, 10 μL of test solution and 10 μL of negative solution were precisely pipetted, respectively, and the samples were analyzed according to the chromatographic condition in Section4.2. Results showed that the negative solution had no peak at the retention time corresponding to hesperidin and ginsenoside Rb1, and the negative control showed no interference.

4.3.2Investigation of linear relationship. 2, 5, 8, 10, 12, 15, 20 μL of mixed reference solution was precisely pipetted and injected into the liquid chromatograph under the chromatographic condition in Section4.3. Taking the content of hesperidin and ginsenoside Rb1(μg) as abscissa (x), and their peak area value as ordinate (y), a linear regression was performed. The regression equation of hesperidin isy=5 221 082x-570 156 (R2=0.999 4), and the regression equation of ginsenoside Rb1wasy=259 989x-54 321 (R2=0.999 4).

4.3.3Precision test. After precise extraction of the above reference solution, the peak area of hesperidin and ginsenoside Rb1was recorded and the peak areaRSD(%) was calculated after 6 consecutive injections of sample. Results showed that theRSDof ginsenoside Rb1was 2.53% and theRSDof hesperidin was 2.51%, indicating that the precision of the method was good.

4.3.4Stability test. This product was taken (No.20190708) to prepare the test solution under conditions in Section4.1.2, and the peak area of the sample was determined according to the chromatographic conditions at 0, 2, 4, 8, 12 and 24 h, respectively. TheRSDvalues of hesperidin and ginsenoside Rb1peak area were 2.28% and 2.58%, respectively. TheRSDvalues were all less than 3.0%, indicating that the solution of the sample was stable within 24 h.

4.3.5Reproducibility test. Six samples of the same batch number (No.20190708) were precisely weighed to prepare the test solution according to the preparation method mentioned above. The peak area of the sample was determined according to the chromatographic conditions, and the average value andRSDvalue of hesperidin and ginsenoside Rb1were calculated. Results showed that the average content of hesperidin and ginsenoside Rb1in this product was 1.356 9 mg/g and 3.01 mg/g, respectively, and theRSDvalues of hesperidin and ginsenoside Rb1were 2.21% and 2.61%, respectively, indicating that the method had good reproducibility.

4.3.6Sample recovery test. A total of nine samples (0.5 g) of Dujieqing pills (No.20190708) with known content were precisely weighed and put in conical bottles with stoppers. Proper amount of reference substance of hesperidin and ginsenoside Rb1was precisely added to prepare the test solution according to the preparation method mentioned above. It was determined according to the chromatographic conditions under Section4.2. The average recovery rate of hesperidin and ginsenoside Rb1in this product was 98.90% and 98.31%, respectively, and the correspondingRSDvalues were 2.31% and 2.56%, respectively, indicating that the method was accurate.

5 Determination of sample content

The samples of 6 different batches were finely ground and accurately weighed, the sample solution was prepared according to the method mentioned above in Section4.1.2, and the sample was injected and analyzed according to the chromatographic condition in Section4.2. The injection volume was 10 μL. The content of hesperidin and ginsenoside Rb1was calculated by external standard method, and the determination results were 1.278 0 mg/g and 2.831 3 mg/g, respectively.

6 Discussion

6.1 Selection of TLC conditionsAccording to the TLC identification method of tangerine peel andP.notoginsengin the 2015 edition of Chinese Pharmacopoeia, hesperidin reference substance and tangerine peel control medicinal materials were selected as the identification control of tangerine peel in the prescription. Ginsenoside Rg1 and notoginsenoside R1were selected as the identification control ofP.notoginsengin the prescription. Different developing solvents for sample solution were investigated. The results showed that when the identification of tangerine peel andP.notoginsengin sample took ethyl acetate-methanol-water (100∶17∶13) and trichloromethane-n-butanol-methanol-water (2∶4∶1∶2) as developing system, the negative control had no interference, the spots were clear and the degree of separation was good, so it can be used as a qualitative identification method for this preparation.

6.2 Selection of detection wavelengthThe reference solution of two detected components in the sample was scanned in the wavelength range of 200-400 nm. The results showed that hesperidin had the maximum absorption at 284 nm and ginsenoside Rb1had the maximum absorption wavelength at 203 nm. When 284 nm was selected as the detection wavelength, the chromatographic peak of hesperidin was low, and the chromatographic peak of ginsenoside Rb1could not be detected. When the wavelength was changed to 203 nm, the chromatographic peak of hesperidin and ginsenoside Rb1could be detected and the chromatographic peak was moderate, so the detection wavelength was determined to be 203 nm in this experiment.

6.3 Selection of mobile phaseReferring to the related report[10-11], the separation effects of methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid and acetonitrile-0.1% phosphoric acid on different components were investigated. When acetonitrile-aqueous solution was used as the mobile phase, the separation of the two detected components in the sample was good, and it could be separated from the impurity peak at the baseline, so the mobile phase was selected for gradient elution. In this study, the sample treatment method was simple, HPLC gradient elution could effectively separate the target peak without the interference of impurity peaks, and the qualitative spectra of tangerine peel andP.notoginsengwere clear and stable. To sum up, the method established by the test is simple, accurate, specific and sensitive, and can be used for the quality control of Dujieqing pills.