A high performance liquid chromatography method for detecting index components in Qinzhu Liangxue mixture

Abstract

The present invention provides a high performance liquid chromatography (HPLC) detection method for index components in a Qinzhu Liangxue mixture. The HPLC method is used to detect nine components in the traditional Chinese medicine composition, namely, liquiritin, cimicifugin, 5-O-methylvisamin glycoside, liquiritigenin, baicalin, ethyl p-hydroxybenzoate, paeonol, baicalein and wogonin, so as to more comprehensively characterize the quality of the traditional Chinese medicine composition.

Description

Technical Field

[0001] The invention relates to the technical field of detection of effective components of traditional Chinese medicines, in particular to a high performance liquid chromatography detection method for index components in a Qinzhu Liangxue mixture. Background Art

[0002] Quality control measures for compound Chinese herbal preparations primarily rely on qualitative and quantitative analysis, including microscopic identification, inspection, and content determination, of individual raw medicinal materials. However, compound Chinese herbal preparations often include multiple medicinal materials, some even including animal or mineral drugs, and their chemical composition complexity far exceeds that of individual medicinal materials. In fact, a growing number of pharmaceutical researchers are recognizing that microscopic identification, thin-layer chromatography, and quantitative analysis of single indicator components are far from comprehensive and effective in reflecting the quality consistency and effectiveness of compound Chinese herbal preparations. Since the 2015 edition of the Chinese Pharmacopoeia, Volume I, quality control standards for compound Chinese herbal preparations have been significantly improved. For example, different quality control standards for the same preparation have been unified; TLC identification indicators have been added to some TCM compound preparations that do not have TLC identification items (or have fewer TLC identification items, or the TLC identification items are not for the main drug); the content determination of multiple indicator components related to pharmacodynamics has been increased from the original single indicator component determination; the number of preparations for content determination using TLC scanning has been reduced; and the application of gas chromatography to TCM compound preparations containing volatile chemical components has been strengthened. These improvements in quality control methods undoubtedly reflect the continuous improvement of the quality standards and quality control capabilities of TCM compound preparations. However, the quality evaluation and control of TCM compound preparations remains a bottleneck that plagues the development and internationalization of TCM. Research on new quality evaluation and control models and methods for TCM compound preparations is urgent.

[0003] Chinese Pharmacist, 2016, 19(05), 1026-1029, published the "Quality Standard of Qinzhu Liangxue Mixture". Methods: TLC was used to qualitatively identify Scutellaria baicalensis, Glycyrrhiza uralensis and Coix seed in the prescription, and HPLC was used to determine the contents of baicalin and glycyrrhizic acid in the preparation. Results: TLC was able to specifically identify Scutellaria baicalensis, Glycyrrhiza uralensis and Coix seed, and the concentration of baicalin was 0.030-0.971 mg·ml -1 The linear relationship was good within the range of r = 1.0000, the average recovery was 97.74%, and the RSD was 2.76% (n = 9); ammonium glycyrrhizate was in the range of 0.013 to 0.220 mg ml -1The linear relationship was good within the range of 1.0000, with an average recovery of 99.02% and an RSD of 1.79% (n=9). Conclusion: This method is highly specific, simple, and accurate, and can be used as a control method for the determination of baicalin and glycyrrhizic acid in Qinzhu Liangxue Heji. While methods for the determination of baicalin and glycyrrhizic acid in Qinzhu Liangxue Heji have been reported in the literature, there are no reports on the simultaneous determination of multiple components in this prescription.

[0004] Currently, the overall quality control of Qinzhu Liangxue Heji remains limited. Most studies on Qinzhu Liangxue Heji focus on observational and controlled studies of its clinical efficacy, with few methods for component analysis and content determination established. Firstly, different extraction processes or preparation methods employed during the processing of the formula can lead to variations in the types and contents of active ingredients. Secondly, due to the complex composition and difficulty in controlling the quality of traditional Chinese medicine preparations, controlling their quality using single-component content determinations is difficult to characterize overall quality. Therefore, existing research is insufficient to serve as a basis for evaluating the quality of Qinzhu Liangxue Heji. Multi-indicator quality control of the active ingredients in traditional Chinese medicine preparations, or even comprehensive component analysis, is crucial.

[0005] There is currently no report on a high performance liquid chromatography detection method for the index components in the Qinzhu Liangxue mixture of the present invention. Summary of the Invention

[0006] The purpose of the present invention is to provide a high performance liquid chromatography detection method for the index components in Qinzhu Liangxue mixture in view of the deficiencies in the prior art.

[0007] The first aspect of the present invention is a high performance liquid chromatography detection method for the index components in Qinzhu Liangxue mixture, the method comprising the following steps: step a, preparation of a test solution: accurately measuring 50 mL of Qinzhu Liangxue mixture, adding an equal volume of water-saturated n-butanol to extract twice, combining the upper extracts, concentrating under reduced pressure to dryness, adding an organic solvent to dissolve, ultrasonicating for 10 minutes, transferring to a 5 mL volumetric flask to constant volume, filtering with a 0.45 μM organic phase microporous filter membrane, and taking a subsequent filtrate to obtain; step b, preparation of a reference solution: accurately weighing appropriate amounts of liquiritin, cimicifuga, 5-O-methylvisamidoside, liquiritigenin, baicalin, ethyl p-hydroxybenzoate, paeonol, baicalin and wogonin reference substances, adding an organic solvent to dissolve them to form a mixed reference solution; step c, detection: using high performance liquid chromatography, with phosphoric acid water (A)-methanol solution (B) as the mobile phase for gradient elution.

[0008] Preferably, the organic solvent in steps a and b is selected from any one or more of methanol, ethanol, acetone, dichloromethane, and chloroform, preferably a methanol solution.

[0009] Preferably, the concentrations of liquiritin, cimicifuga, 5-O-methylvisamin, liquiritigenin, baicalin, ethyl parahydroxybenzoate, paeonol, baicalein and wogonin in the reference solution of step b are 0.17, 0.13, 0.09, 0.24, 0.16, 0.14, 0.26, 0.18 and 0.22 mg / mL, respectively.

[0010] Preferably, the gradient elution procedure is:

[0011]

[0012] Preferably, the chromatographic column is Agilent ZORBAX ElipseXDB-C18.

[0013] Preferably, the elution flow rate is 1000 mL / min; the column temperature is 30° C.; the detection wavelength is 280 nm; and the injection volume is 10 μL.

[0014] The advantages of the present invention are:

[0015] The present invention uses multiple components such as liquiritin, cimicifugin, 5-O-methylvisamidoside, liquiritigenin, baicalin, ethyl p-hydroxybenzoate, paeonol, baicalein and wogonin in the Qinzhu Liangxue mixture as indicators, and establishes a method for simultaneously determining the contents of the above nine components by high performance liquid chromatography through extraction and separation. Methodological investigation verifies that the method is simple, rapid and accurate, and has good precision, stability and repeatability, providing a scientific basis for the multi-indicator quality control of Qinzhu Liangxue. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Attachment Figure 1 It is the main ingredient in Qinzhu Liangxue Mixture;

[0017] Attachment Figure 2 The HPLC chromatograms of Qinzhu Liangxue mixture (A) and mixed reference substance (B) (1. liquiritin; 2. cimicifugin; 3. 5-O-methylvisamidoside; 4. liquiritigenin; 5. baicalin; 6. ethyl parahydroxybenzoate; 7. paeonol; 8. baicalein; 9. wogonin). DETAILED DESCRIPTION

[0018] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the application's appended claims.

[0019] Example 1

[0020] 1. Materials

[0021] 1.1 Drugs and reagents

[0022] Qinzhu Liangxue Mixture (batch number: 2208002) was provided by the Preparation Room of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine. The reference substances liquiritin (batch number 111610-201908), cimicifugin (batch number 111710-200602), 5-O-methylvisamidoside (batch number 111523-2018011), liquiritigenin (batch number 110715-201912), baicalin (batch number 110715-201821), ethyl p-hydroxybenzoate (batch number 100847-202105), paeonol (batch number 110708-201908), baicalein (batch number 111595-201808) and wogonin (batch number 111514-201706) were purchased from the China Food and Drug Administration. The other components were isolated and structurally identified. Methanol was chromatographically pure (Fisher, USA), water was ultrapure water, and the other reagents were of analytical grade.

[0023] 1.2 Instruments

[0024] High-performance liquid chromatograph (Agilent 1260), Agilent Technologies, USA; electronic balance (CP225D), Sartorius Instrument Systems Co., Ltd., Beijing; ultrasonic cleaner (DL720-A), Shanghai Zhixin Instrument Co., Ltd.; circulating water vacuum pump [SHZ-D(Ⅲ)], Zhengzhou Boco Equipment Co., Ltd.; rotary evaporator (RV-10), IKA, Germany; multifunctional UV analyzer (BD-II), Beijing Qihang Boda Technology Co., Ltd.; electric constant temperature water bath (HWS28), Shanghai Yiheng Scientific Experiment Co., Ltd.; electric constant temperature drying oven (DNG-9140A), Shanghai Jinghong Experiment Equipment Co., Ltd.

[0025] 2. Methods and Results

[0026] 2.1 Compound extraction and separation

[0027] Ten bottles of Qinzhu Liangxue Mixture (batch number: 2208002), 200 mL each, totaling 2000 mL, were extracted three times with water-saturated n-butanol. The combined extracts were concentrated under reduced pressure to dryness to yield 30 g of water-saturated n-butanol. The extract was then rinsed with D101 macroporous resin using varying ratios of ethanol (0%, 20%, 40%, 60%, 80%, and 100%). HPLC analysis revealed the main substance to be located between 40% and 80%. The combined eluates were concentrated to yield 7 g of extract. This extract was fractionated using a glucose gel column using a dichloromethane:methanol ratio of 1:1 to yield six fractions, Fr.A to Fr.F. Fraction F was subjected to a glucose gel column using a dichloromethane:methanol ratio of 1:1 to yield compound 6 (29 mg). Fraction Fr.A was then purified by semi-preparative HPLC (MeOH / H2O, 70:30) to yield compound 1 (9 mg). Fr.B obtained compound 4 (9 mg) by silica gel column (CHCl3 / MeOH, 100:0→70:1). Fr.C obtained compound 2 (17 mg) and compound 3 (23 mg) by semi-preparative HPLC (MeOH / H2O, 75:25) ( Figure 1 Compounds 5, 7-9 were confirmed by HPLC liquid phase analysis retention time.

[0028] 2.2 Structure identification

[0029] Compound 1 is a yellow powder. HRESIMS m / z 435.1200 [M+H]+, molecular formula C 21 H 22 O 10,1H HMR (600MHz, CD3OD): δH7.74 (1H, d, J = 8.8Hz, H-5), 7.45 (2H, d, J = 8.7Hz, H-2', 6'), 7.16 (2H ,d,J=8.7Hz,H-3',5'),6.51(1H,dd,J=8.7,2.2Hz,H-6),6.38(1H,d,J=2.2Hz,H-8),5.47(1 H,dd,J=12.8,3.0Hz,H-2),4.95(1H,d,J=7.60Hz,H-1″),3.91(1H,dd,J=12.1,5.6Hz,H-6a″ ),3.72(1H,dd,J=12.1,5.6Hz,H-6b″),3.18-3.31(4H,m,H-2″~5″),3.05(1H,dd,J=16.9,12. 8Hz, H-3), 2.75 (1H, dd, J = 16.9, 2.8Hz, H-3); 13C-NMR (150MHz, CD3OD): δc80.7 (C-2), 45.0 ( C-3),193.2(C-4),134.5(C-5),111.9(C-6),167.1(C-7),103.9(C-8),165.4(C-9),114.9(C -10), 129.9(C-1'), 128.8(C-2',6'), 117.8(C-3',5'), 159.2(C-4'), 102.2(C-1"),74.9(C-2"),78.0(C-3"),71.4(C-4"),78.2(C-5"),62.5(C-6"). Compound 1 was identified as liquiritin according to relevant literature.

[0030] Compound 2 was obtained as colorless needles (methanol). HRESIMS m / z 306.1145 [M+H]+. 1H NMR (600 MHz, CDCl3): δH 6.43 (1H, s, H-8), 6.22 (1H, s, H-3), 4.70 (1H, t, J = 8.8 Hz, H-3'), 4.46 (2H, s, H-9), 3.91 (3H, s, H-5), 3.25 (2H, dd, J = 8.8, 2.0 Hz H-2'), 1.37 (3H, s, H-5'), 1.24 (3H, s, H-6'); 13C-NMR (150 MHz, CDCl3); δC 165.9(C-2),109.4(C-3),177.8(C-4),112.1(C-4a),164.7(C-5),117.2(C-6),159.5(C-7),93.9(C-8),155.9(C-8a),61.1(C-9),91.5(C-2'),27.9(C-3'),71.7(C-4'),24.5(C-5'),26.3(C-6'),61.1(5-OCH3). According to relevant literature, compound 2 was identified as cimifugin.

[0031] Compound 3 was obtained as colorless needles (methanol). HRESIMS m / z 468.1611 [M+H]+. 1H NMR (600 MHz, CD3OD): δH 6.60 (1H, H-3), 6.04 (1H, d, J = 0.9 Hz H-8), 4.88 (1H, t, J = 9.0 Hz, H-2'), 4.60 (1H, d, J = 7.8 Hz, H-1"), 3.95 (3H, s, 5-OCH3), 3.30-3.68 (8H, overlapped, H-3', 2"-6"), 2.34 (3H, s, H-9), 1.38 (6H, s, 5', 6'-OCH3); 13C-NMR (150 MHz, CD3OD); δC 166.5(C-2),111.5(C-3),179.8(C-4),111.9(C-4a),166.9(C-5),118.5(C-6),161.4(C-7) ,94.4(C-8),157.0(C-8a),19.7(C-9),92.0(C-2'),29.0(C-3'),79.0(C-4'),23.9(C-5'),2 2.5(C-6'),98.9(C-1"),75.1(C-2"),77.5(C-3"),71.4(C-4"),78.1(C-5"),62.3(C-6"),61.1(5-OCH3). According to relevant literature, compound 3 was identified as 5-O-methylvisamminol (4'-O-β-D-glucosyl-5-O-methylvisamminol).

[0032] Compound 4 is a yellow powder. HRESIMS m / z 257.0830 [M+H] + molecular formula C15H12O4, 1H HMR (600MHz, CD3OD): δH 7.75(1H,d,J=8.7Hz,H-5),7.34(2H,d,J=8.7Hz,H-2',6'),6.83(2H,d,J=8.8Hz,H-3',5'),6.52(1H,dd,J=8.7,2.3Hz,H-6),6.37( 1H,d,J=2.3Hz,H-8),5.40(1H,dd,J=13.1,2.9Hz,H-2),3.07(1H,dd,J=16.9,13.1Hz,H-3a),2.71(1H,dd,J=16.9,3.0Hz,H-3b); 13C NMR (150 MHz, CD3OD) δC: 81.1 (C-2), 45.0 (C-3), 193.5 (C-4), 129.9 (C-5), 111.7 (C-6), 165.6 (C-7), 103.8 (C-8), 166.8 (C-9), 116.3 (C-10), 131.4 (C-1′), 129.0 (C-2', 6'), 116.3 (C-3', 5'), 159.0 (C-4'). Compound 4 was identified as liquiritigenin according to relevant literature.

[0033] Compound 6 was obtained as yellow crystals; its molecular formula was C9H10O3. 1H NMR (CDCl3, 600 MHz) δH: 7.94 (2H, d, J = 8.8 Hz, H-3, 5), 6.91 (2H, d, J = 8.8 Hz, H-2, 6), 4.35 (2H, d, J = 7.2 Hz, HO-CH2), 1.38 (3H, t, J = 7.2 Hz, 10-CH3); 13C-NMR (CD3OD, 150 MHz) δ: 122.2 (C-1), 132.0 (C-2, 6), 115.5 (C-3, 5), 160.9 (C-4), 167.6 (-C=O), 61.2 (-OCH2-), 14.4 (-CH2CH3). Compound 6 was identified as ethyl paraben based on relevant literature.

[0034] Compounds 5, 7–9 were identified as baicalin (5, baicalin), paeonol (7, paeonol), baicalein (8, baicalein) and wogonin (9, wogonin) by comparison of their retention times by high performance liquid chromatography.

[0035] 2.3 Content determination

[0036] 2.3.1 Chromatographic conditions

[0037] The chromatographic column was an Agilent ZORBAX ElipseXDB-C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was 0.1% phosphoric acid in water as mobile phase A and methanol as mobile phase B; the elution program was shown in Table 1; the flow rate was 1.000 mL / min; the column temperature was 30°C; the detection wavelength was 280 nm; and the injection volume was 10 μL.

[0038] Table 1 Gradient elution program

[0039]

[0040]

[0041] 2.3.2 Preparation of reference solution

[0042] Accurately weigh appropriate amounts of liquiritin, cimicifugin, 5-O-methylvisamidoside, liquiritigenin, baicalin, ethyl parahydroxybenzoate, paeonol, baicalein and wogonin reference substances, and add appropriate amount of methanol to prepare mixed reference substance solutions with concentrations of 0.17, 0.13, 0.09, 0.24, 0.16, 0.14, 0.26, 0.18 and 0.22 mg / mL, respectively.

[0043] 2.3.3 Preparation of test solution

[0044] Accurately measure 50 mL of Qinzhu Liangxue mixture (batch number: 2208002), add an equal volume of water-saturated n-butanol to extract twice, combine the upper extracts, concentrate under reduced pressure to dryness, add appropriate amount of methanol, ultrasonicate for 10 minutes, transfer to a 5 mL volumetric flask to make up the volume, filter with a 0.45 μM organic phase microporous filter membrane, and take the filtrate to obtain the product.

[0045] 2.3.4 Specificity test

[0046] 10 μL of the mixed reference solution and 5 μL of the test solution were respectively drawn and analyzed according to 2.3.1. The separation coefficients of the 9 active ingredients and their adjacent chromatographic peaks were all greater than 1.5, and the components did not interfere with each other. The theoretical plate number met the requirements and the specificity was good. Figure 2 .

[0047] 2.4 Methodological Investigation

[0048] 2.4.1 Linear Relationship

[0049] Accurately pipette 1.25, 2.5, 5, 10, 12.5, and 15 μL of the mixed reference solution into the liquid chromatograph, and measure and record the peak areas. A standard curve is plotted with the injection volume as the abscissa (X) and the peak area as the ordinate (Y). The linear equation, correlation coefficient, and linear range for each component are determined. The results show that each component exhibits good linearity within its respective concentration range (see Table 2).

[0050] Table 2 Linear relationship

[0051]

[0052]

[0053] 2.4.2 Precision

[0054] Accurately pipette 10 μL of the mixed reference solution under 2.3.2 and repeat the injection 6 times according to the chromatographic conditions under 2.3.1. Record the peak area and calculate the RSD value. The results are shown in Table 3, indicating that the precision of the instrument is good.

[0055] Table 3 Precision

[0056]

[0057] 2.4.3 Repeatability

[0058] According to the method under 2.3.3, 6 test sample solutions were prepared respectively, and 5 μL was injected into each solution. The peak areas of liquiritin, cimicifugin, 5-O-methylvisamin, liquiritigenin, baicalin, ethyl parahydroxybenzoate, paeonol, baicalein and wogonin were recorded, and the RSD values were calculated. The results are shown in Table 4, indicating that the method has good reproducibility.

[0059] Table 4 Repeatability

[0060]

[0061] 2.4.4 Stability

[0062] Prepare the test solution as described in 2.3.3 and incubate at room temperature for 0 h, 6 h, 12 h, 18 h, 24 h, and 30 h. Inject 5 μL of the sample according to the chromatographic conditions in 2.3.1, determine the peak area, and calculate the RSD. The results are shown in Table 5, indicating that the test solution is stable within 30 h.

[0063] Table 5 Stability

[0064]

[0065] 2.4.5 Sample recovery rate

[0066] Six batches of a mixture sample (2208002) with known index component contents were taken. A certain amount of liquiritin, cimicifugo, 5-O-methylvisamidoside, liquiritigenin, baicalin, ethyl p-hydroxybenzoate, paeonol, baicalein, and wogonin reference solution were added within the linear range, for a total of six portions. The contents were determined according to the chromatographic conditions under 2.3.1. The results are shown in Table 6, indicating that the method had a good recovery rate.

[0067] Table 6 Sample recovery

[0068]

[0069]

[0070] 2.5 Sample determination

[0071] Three batches of Qinzhu Liangxue mixture samples (batch numbers 2208001, 2208002, and 2208003) were taken and test sample solutions were prepared according to the method in 2.3.3. The contents of liquiritin, cimicifugin, 5-O-methylvisamidoside, liquiritigenin, baicalin, ethyl parahydroxybenzoate, paeonol, baicalein, and wogonin were determined according to the chromatographic conditions in 2.3.1. The results are shown in Table 7.

[0072] Table 7 Determination results of the contents of 9 ingredients in Qinzhu Liangxue mixture

[0073]

[0074] 3. Conclusion

[0075] The present invention adopts HPLC to carry out qualitative and quantitative analysis of 9 components in Qinzhu Liangxue mixture, including liquiritin, cimicifugin, 5-O-methylvisamidoside, liquiritigenin, baicalin, ethyl p-hydroxybenzoate, paeonol, baicalein and wogonin. The results show that under the chromatographic conditions established in this experiment, the chromatographic peaks of the measured components are well separated from other components, various methodological verifications meet the requirements, the method is simple, and the measurement results are accurate. The method provides a standard for ensuring the safety and effectiveness of the prescription and provides a reliable basis for the research on the pharmacological substance basis of the prescription and the quality control evaluation of its main chemical components.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principles of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A high performance liquid chromatography method for detecting the index components in Qinzhu Liangxue mixture, characterized in that: The method comprises the following steps: step a, preparing a test solution: accurately measuring 50 mL of Qinzhu Liangxue mixture, adding an equal volume of water-saturated n-butanol to extract twice, combining the upper extracts, concentrating under reduced pressure to dryness, adding an organic solvent to dissolve, ultrasonicating for 10 minutes, transferring to a 5 mL volumetric flask to constant volume, filtering with a 0.45 μM organic phase microporous filter membrane, and taking a subsequent filtrate to obtain; step b, preparing a reference solution: accurately weighing appropriate amounts of liquiritin, cimicifuga, 5-O-methylvisamidoside, liquiritigenin, baicalin, ethyl p-hydroxybenzoate, paeonol, baicalein and wogonin reference substances, adding an organic solvent to dissolve them, and preparing a mixed reference solution; step c, detecting: adopting high performance liquid chromatography, using phosphoric acid water (A)-methanol solution (B) as the mobile phase for gradient elution, and the gradient elution program is as follows: The chromatographic column is Agilent ZORBAX ElipseXDB-C18, the elution flow rate is 1 mL / min; the column temperature is 30° C.; the detection wavelength is 280 nm; the injection volume is 10 μL, and the organic solvent in steps a and b is a methanol solution.

2. The method according to claim 1, characterized in that The concentrations of liquiritin, cimicifuga, 5-O-methylvisamin, liquiritigenin, baicalin, ethyl parahydroxybenzoate, paeonol, baicalein and wogonin in the reference solution of step b are 0.17, 0.13, 0.09, 0.24, 0.16, 0.14, 0.26, 0.18 and 0.22 mg / mL, respectively.

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