Simultaneous Determination of Effective Components of Gegen Qinlian Tablets by HPLC

Lili LI, Yifeng LU, Xueyan ZHU

Guangxi Institute for Food and Drug Control, Nanning 530021, China

Abstract [Objectives] To establish a HPLC method for the simultaneous determination of 14 characteristic components in Gegen Qinlian tablets and the content of Gegen Qinlian tablets in 11 manufacturers. [Methods] A Inertsil ODS-3 (250 mm×4.6 mm, 5 μm) reversed-phase high performance liquid chromatography column was used with acetonitrile-0.02 mol/L ammonium acetate+0.03% triethylamine solution (adjusted with glacial acetic acid to pH of 4.3) as the mobile phase for gradient elution at a flow rate of 1.0 mL/min. The determination wavelength of 8 components (puerarin, daidzin, liquiritin, baicalin, wogonoside, daidzein, ammonium glycyrrhizinate, baicalein) was 250 nm; the determination wavelength of wogonoside was 280 nm; the determination wavelength of 5 components (epierberine, jatrorrhizine hydrochloride, coptisine, palmatine hydrochloride and berberine hydrochloride) was 346 nm. [Results] There was a good linear relationship between the injection volume of puerarin, daidzin, liquiritin, baicalin, wogonoside, epierberine, jatrorrhizine hydrochloride, coptisine, daidzein, palmatine hydrochloride, ammonium glycyrrhizinate, berberine hydrochloride, baicalein and wogonin and peak area (r>0.999 0) in the range of 146.26-585 0.24, 24.13-965.04, 18.45-738.00, 79.18-316 7.32, 9.57-382.80, 4.76-190.40, 2.57-102.80, 13.41-536.40, 10.60-424.00, 11.33-453.22, 12.08-483.20, 46.73-186 9.25, 20.28-811.20, 12.11-484.50 ng, respectively; the average recovery rates (n=6) were 2.18%, 1.79%, 1.81%, 1.68%, 2.27%, 2.13%, 1.96%, 1.07%, 0.93%, 0.61%, 2.92%, 0.77%, 2.79% and 0.62%, respectively; the precision, repeatability and stability were good, and the RSD was less than 3%. This method was used to determine 16 batches of Gegen Qinlian tablets produced by 11 enterprises. Wogonoside was not detected in a lot of samples, but 14 components were detected for other enterprises, but the content was different. [Conclusions] The method was accurate and feasible and could be used for the overall quality control of this variety.

Key words Gegen Qinlian tablets, Content determination, High performance liquid chromatography, Puerarin, Daidzin, Liquiritin, Baicalin, Wogonoside, Epierberine, Jatrorrhizine hydrochloride, Coptisine, Daidzein, Palmatine hydrochloride, Ammonium glycyrrhizinate, Berberine hydrochloride, Baicalein, Wogonin

1 Introduction

Gegen Qinlian preparation comes from Gegen Qinlian Decoction inTreatiseonFebrileDiseases. It can not only clear the heat and induce sweat, but also improve the weakness of the spleen and stomach and treat dysentery. Supplemented with the bitter cold products ofScutellariabaicalensisGeorgi andCoptischinensisFranch., it can clear the dampness of stomach and intestines, clear heat and realize detoxification with licorice, and harmonize the properties of different drugs[1-3]. At present, Gegen Qinlian preparation has four dosage forms, including tablets, pills, capsules and granules, and Gegen Qinlian tablets are the most widely used dosage form in clinical practice.

The chemical basis of the four traditional Chinese medicines in Gegen Qinlian preparation is clear. The main effective components ofRadixPuerariaelobataeare flavonoids and saponins such as daidzin, daidzein, and puerarin. The main effective components ofS.baicalensisGeorgi are baicalein, baicalin, wogonoside, wogonin and other flavonoids. The main effective components ofC.chinensisFranch. are berberine, palmatine, coptisine, jatrorrhizine, epierberine and other alkaloids. The main effective components ofGlycyrrhizauralensisFisch. are glycyrrhizic acid, glycyrrhetinic acid, liquiritin, liquiritigenin and other saponins and flavonoids[2-3].

In recent years, the overall control mode of traditional Chinese medicine[8-9]has received more and more attention. At present, the research on the overall quality control of Gegen Qinlian preparation is mainly focused on Gegen Qinlian Decoction, and many kinds of effective components are determined simultaneously by HPLC. Zhang Junetal.[5]established a 3-wavelength HPLC method for simultaneous determination of 12 effective components in Gegen Qinlian preparation. Mao Yingetal.[6]established a single wavelength HPLC method for 14 effective components in Gegen Qinlian preparation. With regard to the overall quality control of Gegen Qinlian tablets, only Hu Xiaoruetal.[7]established the fingerprint of Gegen Qinlian tablets and identified 8 characteristic peaks.

In this study, the overall control method of Gegen Qinlian tablets was studied by using HPLC method to determine a variety of effective components at the same time. In this paper, the content of characteristic components in 16 batches of Gegen Qinlian tablets from 11 different production enterprises were compared. A total of 14 characteristic components reported in the literature[2-3]were selected. Among them, the index components ofRadixPuerariaelobataewere puerarin, daidzin and daidzein. The index components ofS.baicalensisGeorgi were baicalin, wogonoside, baicalein and wogonin. The index components ofC.chinensisFranch. were epierberine, jatrorrhizine (in terms of jatrorrhizine hydrochloride), coptisine, palmatine (in terms of palmatine hydrochloride) and berberine (in terms of berberine hydrochloride). The index components ofG.uralensisFisch. were liquiritin, glycyrrhizic acid (in terms of ammonium glycyrrhizinate). The three wavelengths of 250, 280 and 346 nm were used for simultaneous determination, which provided a scientific basis for the overall quality control of Gegen Qinlian tablets.

2 Materials

2.1InstrumentsAgilent 1260 high performance liquid chromatograph, DAD detector, UWD ultraviolet detector, German Elma S180H ultrasonic cleaner, MILLI-PROA pure water processor.

2.2ReagentsandsamplesReference substance puerarin (for content determination, batch No.110752-200912, content 96.0%), daidzin (for content determination, batch No.110738-201302, content 91.3%), liquiritin (for content determination, batch No.111610-200604), baicalin (for content determination, batch No.110715-201117, content 91.7%), jatrorrhizine hydrochloride (for content determination, batch No.110733-200806), daidzein (for content determination, batch No.150262), palmatine hydrochloride (for content determination, batch No.110732-200907, content 86.1%), ammonium glycyrrhizinate (for content determination, batch No.110731-200512), berberine hydrochloride (for content determination, batch No.110713-201212, content 86.7%), baicalein (for content determination, batch No.111595-200402), wogonin (batch No.1514-200202),etc. were provided by China Institute for Food and Drug Control.

Reference substance wogonoside (batch No.A0595), epierberine (batch No.A0627), coptisine (batch No.201201RS),etc., were provided by Yunnan Tefate Biotechnology Co., Ltd. Acetonitrile was of chromatographic purity, water was of high purity, and other reagents were of analytical purity.

The samples of Gegen Qinlian tablets were sold in the market, and the samples from 11 different manufacturers were named A-K.Among them, the samples from manufacturer A were used for methodological investigation. The negative control sample was self-made.

3 Methods and results

3.1ChromatographicconditionsChromatographic column: Japanese GL Science Inc. Inertsil ODS-3 (4.6 mm×250 mm, 5 μm); the mobile phase and flow rate are shown in Table 1; column temperature: 35 ℃; the determination wavelength is shown in Table 2; injection volume: 10 μL.

Table1GradientelutionconditionsofmobilephaseforHPLC

TimeminMobile phaseA:acetonitrile%B: 0.02 mol/L ammonium acetate+0.03%triethylamine (pH adjusted to 4.3with glacial acetic acid)∥%Flow ratemL/min015850.51015850.52022780.54026740.55040600.56050500.77065350.77115851.08515851.0

3.2Preparationofreferencesolution

3.2.1Preparation of mixed reference solution. The reference substance was weighed precisely and methanol was added to prepare the mixed reference solution containing 150 μg of puerarin, 25 μg of daidzin, 10 μg of daidzein, 80 μg of baicalin, 40 μg of wogonoside, 10 μg of baicalein, 10 μg of wogonin, 15 μg of palmatine, 50 μg of berberine, 20 μg of liquiritin, 10 μg of ammonium glycyrrhizinate, 15 μg of coptisine, 2.5 μg of jatrorrhizine, and 5 μg of epierberine per 1 mL. The specific data are shown in Table 2.

Table2Preparationofmixedreferencesolutionandwavelengthdetermination

No.ComponentsSamplingquantity∥mgContent∥%Dilution factorMass concentration of mixedreference substance ∥μg/mLDeterminationwavelength∥ nm1Puerarin30.4796.0200146.262502Daidzin10.5791.340024.132503Liquiritin12.30100.0666.6718.452504Baicalin 17.2791.720079.182505Wogonoside9.57100.0100 09.572506Epierberine9.52100.0200 04.763467Jatrorrhizine hydrochloride10.28100.0400 02.573468Coptisine11.92100.0888.8913.413469Daidzein10.60100.0100 010.6025010Palmatine hydrochloride10.5386.180011.3334611Ammonium glycyrrhizinate12.08100.0100 012.0825012Berberine hydrochloride10.7886.720046.7334613Baicalein10.14100.050020.2825014Wogonin9.69100.080012.11280

3.2.2Preparation of test solution. About 0.25 g of ground sample was precisely weighed and placed in a conical bottle with stopper. 25 mL of 70% methanol solution was precisely added and weighed. After heating and refluxing for 30 min, it was cooled and weighed. 70% methanol solution was used to make up for the weight loss, it was shaken well, filtered, and the filtrate was obtained.

3.2.3Preparation of negative sample solution. According to the prescription and preparation method of Gegen Qinlian tablets, the negative samples were prepared withoutRadixPuerariaelobatae,S.baicalensis,C.chinensisFranch. andG.uralensis, respectively. The negative samples free ofRadixPuerariaelobatae,S.baicalensis,C.chinensisFranch. andG.uralensiswere taken respectively for preparation according to the method of Section3.2.2.

3.4Methodologicalinvestigation

3.4.1Exclusivity. The solution under Section3.2was determined according to the condition under Section3.1. Results showed that there were almost no absorption peaks in the corresponding retention time of the components, indicating that other components in the prescription did not interfere with the determination (Fig.1).

Note: 1. puerarin; 2. daidzin; 3. liquiritin; 4. baicalin; 5. wogonoside; 6. epierberine; 7. jatrorrhizine hydrochloride; 8. coptisine; 9. daidzein; 10. palmatine hydrochloride; 11. ammonium glycyrrhizinate; 12. berberine hydrochloride; 13. baicalein; 14. wogonin.

Fig.1HPLCchromatogramofreferencesubstance(A),sample(B),negativesamplefreeofRadixPuerariaelobatae(C),negativesamplefreeofGlycyrrhizauralensis(D),negativesamplefreeofCoptischinensisFranch.(E),negativesamplefreeofRadixScutellariae(F)

3.4.2Investigation of linear relationship. The mixed reference solution under Section3.2.1was injected into the liquid chromatograph, and the injection volume was 1, 5, 10, 20, 30 and 40 μL, respectively. The standard curve was drawn with the reference sample injection as horizontal ordinate (X), peak area as vertical axis (Y). The regression equation and linear range are shown in Table 3.

3.4.3Replication experiment. The same lot of samples (manufactures A, batch No.20120502) were taken and 6 copies were prepared in parallel according to the method under Section3.2.2. It was determined according to the conditions under Section3.1, and the results showed that the repeatability of this method was good, as detailed in Table 3.

3.4.4Precision test. The same solution under Section3.4.2was determined continuously for 6 times according to the conditions under Section3.1. Results showed that theRSDof peak area of each component was less than 3% (n=6). The experiment demonstrated that the precision of this method was good, as shown in Table 3.

3.4.5Stability test. The same solution under Section3.4.3was determined at 0, 6, 11, 17 and 23 h according to the conditions under Section3.1. Results showed that theRSDof peak area of each component was less than 3% (n=6). The experimental results showed that the precision of this method was good, as shown in Table 3.

Table3Dataofmethodologicalinvestigation

No.ComponentsRepeatabilityAverage∥mg/gRSD∥%PrecisionRSD∥%StabilityRSD∥%Linear relationshipEquationrLinear range∥ng1Puerarin40.103 20.682.271.98Y=5.441 4X+752.230.999 3146.26-585 0.242Daidzin5.474 22.121.391.47Y=5.752 3X+137.520.999 424.13-965.043Liquiritin4.848 01.730.950.84Y=0.924 4X+3.091 40.999 718.45-738.004Baicalin18.725 51.882.232.15Y=2.243 4X+16.6110.999 679.18-316 7.325Wogonoside2.441 91.573.001.76Y=2.559 8X+8.982 10.999 89.57-382.806Epierberine3.018 72.411.922.02Y=7.424X+13.7310.999 74.76-190.407Jatrorrhizine hydrochloride0.671 11.652.001.26Y=7.834 8X+6.083 20.999 72.57-102.808Coptisine1.903 81.471.881.77Y=5.592 3X-21.8080.999 613.41-536.409Daidzein2.499 22.451.632.34Y=10.45X-21.2960.999 710.60-424.0010Palmatine hydrochloride2.514 92.141.891.53Y=6.152 5X-15.2481.000 011.33-453.2211Ammonium glycyrrhizinate2.890 61.742.372.04Y=1.258 2X-1.404 50.999 712.08-483.2012Berberine hydrochloride10.471 51.751.811.61Y=6.728 5X-54.1610.999 846.73-186 9.2513Baicalein4.846 42.221.931.88Y=3.409 9X-88.840.999 220.28-811.2014Wogonin3.024 72.551.932.39Y=3.995 3X-21.110.999 012.11-484.50

3.4.6Sample recovery test. About 0.125 g of sample was taken (manufactures A, Batch No.20120502, and the content of each component as shown in Table 4). A total of 6 samples were precisely weighed and placed in a conical bottle with stopper. 25 mL of mixed reference solution under Section3.2.1was added respectively, and then weighed. After heating and refluxing for 30 min, it was cooled and weighed. 70% methanol solution was used to make up for the weight loss, it was shaken well, filtered, and the filtrate was obtained. The sample was injected and determined, and the recovery rate was calculated. The results are shown in Table 4.

Table4Recoveryrateof14components(n=6)

No.ComponentsContent in sample∥mg/gAddition∥mgAverage recovery∥%RSD∥%1Puerarin40.103 23.656 599.362.182Daidzin 5.474 20.603 2100.071.793Liquiritin 4.848 04.612 5100.601.814Baicalin 18.725 51.979 5100.131.685Wogonoside 2.441 92.392 598.042.276Epierberine 0.701 60.011 999.292.137Jatrorrhizine hydrochloride 0.671 10.064 3100.731.968Coptisine 1.903 80.335 2101.521.079Daidzein 2.449 20.265 095.970.9310Palmatine hydrochloride 2.514 90.283 299.440.6111Ammonium glycyrrhizinate 2.890 60.302 098.982.9212Berberine hydrochloride 10.475 11.168 2101.320.7713Baicalein 4.846 40.507 098.172.7914Wogonin 3.024 70.302 899.040.62

3.5Sampledetermination16 lots of samples from 11 production enterprises of Gegen Qinlian tablets were prepared according to the method of Section3.2.2and determined according to the conditions of Section3.1. The results are shown in Table 5.

Table5Resultsofsampledetermination(mg/g,n=6)

ManufacturerBatch No.Components1234567891011121314A2012050240.105.474.8518.732.440.700.671.9002.502.512.8910.484.853.022013060139.676.926.059.031.380.560.220.5002.052.632.179.655.453.70B12120137.266.924.528.912.231.720.953.6701.014.051.3214.442.032.70CA2013010166.9111.9217.2411.081.810.330.070.0193.410.050.9510.400.590.47D12020137.569.444.1517.373.781.060.752.5101.292.561.019.927.101.90E12110136.065.113.7413.432.261.450.603.4501.722.311.269.150.820.81F13092841.727.132.0926.565.621.390.623.4501.482.582.129.111.270.69G12060156.536.514.640.80/0.210.470.4502.490.642.3412.820.15/H2013030257.515.739.491.530.210.050.370.0201.340.280.707.760.080.202013060271.317.7310.582.300.220.050.490.0401.260.242.0010.150.130.422012070159.574.917.131.760.340.300.310.4301.370.961.3013.320.070.22I12090981.5211.5411.343.443.140.180.130.2001.790.501.367.650.070.1913030275.3910.899.753.750.520.870.541.8402.272.251.2711.601.710.48J1212000743.267.724.042.750.451.160.752.1501.033.372.6210.352.651.245512000448.569.142.943.640.511.160.592.4600.892.603.438.463.691.40K2011100145.967.543.276.701.000.340.441.0001.250.730.819.651.640.88

Note: The name corresponding to the component number is shown in Table 4.

3.6Selectionofchromatographicconditions

3.6.1Selection of mobile phase. 5 mobile phases A[acetonitrile: 0.05% potassium dihydrogen phosphate+0.05% triethylamine solution (pH adjusted to 3.0 with phosphoric acid)], B[acetonitrile: 0.5% phosphoric acid solution], C[acetonitrile: methanol: 0.5% phosphoric acid solution], D[acetonitrile: 0.02 mol/L ammonium acetate+0.03% triethylamine solution (pH adjusted to 4.3 with glacial acetic acid)]and E[acetonitrile: 0.02 mol/L ammonium acetate+0.03% triethylamine solution (pH adjusted to 4.3 with glacial acetic acid)]were used for sample injection analysis, respectively. The separation effect was the best using mobile phase E for gradient elution.

3.6.2Selection of determination wavelength. With reference to the 2010 edition ofChinesePharmacopoeiaand literature[4-7, 10-12], three wavelengths of 250,280,346 nm were selected as the test wavelengths to determine the mixed reference solution under Section3.2.1. The determination wavelength of each component was determined by comparison, as shown in Table 2.

3.7Investigationofextractionconditions

3.7.1Investigation of extraction solvent. 6 solvents including 50% ethanol, 70% ethanol, ethanol, 50% methanol, 70% methanol solution and methanol were investigated. The extraction efficiency of 70% methanol solution was the highest for 14 kinds of reference substances.

3.7.2Investigation of extraction method. Ultrasonic treatment (power 320 W, frequency 40 kHz) and heating reflux were investigated, and the heating reflux efficiency was higher in the same time.

3.7.3Investigation of extraction time. The extraction efficiency of heating and refluxing for 15, 30 and 60 min was investigated.

After heating and refluxing for 30 min, all the components in the sample were extracted completely.

3.7.4Investigation on the amount of extraction solvent. It was extracted with 20, 25, 50 mL of 70% methanol solution, respectively.

Results showed that the components were extracted completely when the extraction solvent volume was 25 mL.

3.8IntegrationmodeofunresolvedpeakIn this study, epierberine and jatrorrhizine hydrochloride were not completely separated, and the degree of separation was 1.0. The integration method of vertical separation of Agilent chromatographic workstation was adopted.

4 Discussions

Through the determination of 16 lots of samples from 11 manufacturers, it was found that all the index components inRadixPuerariaelobatae,C.chinensisFranch.,S.baicalensisandG.uralensisFisch. were detected, suggesting that all enterprises added herbal medicines according to the regulations. However, there were some differences among the index components, which further showed that to carry out the overall control of proprietary Chinese medicine, the simultaneous determination of multiple components was more reliable than using a single index component to control the quality of proprietary Chinese medicine.

Because the hydrolysates of baicalin and wogonoside were baicalein and wogonin, the total amount of four components should be used to control the quality ofS.baicalensisGeorgi. From the data of various manufacturers, it was found that the components contained inS.baicalensisGeorgi varied significantly among manufacturers. Take the main component baicalin ofS.baicalensisGeorgi as an example, its highest content was 26.56 mg/g (manufacturer F, batch No.130928), and its lowest content was only 0.80 mg/g (manufacturer G), with a 30-fold difference. There were also significant differences in other components ofS.baicalensisGeorgi like baicalein, wogonin and wogonoside among the samples. The difference of process control in the preparation process might lead to the product difference among enterprises, and it

might also be caused by the difference of reactant. It is traditionally believed that Chengde, Hebei Province is the authentic producing area ofS.baicalensisGeorgi. Nowadays,S.baicalensisGeorgi is produced in Inner Mongolia, Shanxi, Jilin, Yunnan and Sichuan. According to the literature report[13-14], there are significant differences in baicalin, baicalein, wogonin and other effective components among different areas. The effective components are also different between wildS.baicalensisGeorgi and cultivatedS.baicalensisGeorgi. In order to better control the quality of medicine and ensure the safety and effectiveness of medicine, it is suggested to strengthen the content control ofS.baicalensisGeorgi in Gegen Qinlian tablets.