A method for simultaneously detecting seven active ingredients in Yangyin Qingfei Pills

Abstract

The present invention discloses a method for simultaneously detecting seven effective ingredients in Yangyin Qingfei Pills, and belongs to the technical field of chemical detection. The method for simultaneously detecting seven effective ingredients in Yangyin Qingfei Pills described in the present invention comprises the following steps: injecting the Yangyin Qingfei Pill sample solution into a liquid chromatograph, and using a segmented wavelength conversion method to perform chromatographic detection to achieve qualitative and quantitative detection of the seven effective ingredients. The present invention uses the UPLC segmented wavelength conversion method to achieve simultaneous determination of the contents of harpagine, paeoniflorin, liquiritin, verbascoside, harpagine, paeonol and glycyrrhizic acid in Yangyin Qingfei Pills, and all seven effective ingredients can be well separated without interference from other ingredients. Moreover, each component has a good linear relationship within its own linear range, and the sample recovery rate is between 95% and 105%. The detection method of the present invention is simple, accurate and effective, and can be used for quality control and overall quality evaluation of Yangyin Qingfei Pills.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a method for simultaneously detecting seven effective components in Yangyin Qingfei Pills. Background Art

[0002] Yangyin Qingfei Pills are composed of eight medicinal flavors: Rehmannia root, Ophiopogon japonicus, Scrophularia ningpoensis, Fritillaria cirrhosa, White Peony Root, Paeonia suffruticosa, Licorice, and Mint. They have the effects of nourishing yin and moistening dryness, clearing the lungs and relieving sore throat. They are mainly used clinically for yin deficiency and dry lungs, dry and sore throat, dry cough with little sputum or blood in sputum. This preparation is now included in the 2020 edition of the Chinese Pharmacopoeia, with four specifications: large honey pills, small honey pills, water honey pills, and water pills. In the [Content Determination] item, only paeonol is controlled, which cannot meet the quality control needs well. In the existing studies on Yangyin Qingfei Pills, only paeoniflorin has been investigated and studied. Chinese herbal compound preparations contain many medicinal flavors and complex chemical components. Examining them with only a single component as an indicator cannot achieve the purpose of comprehensively and objectively evaluating their quality. Therefore, a method was established to simultaneously determine seven index components in Yangyin Qingfei Pills, including harpaoside, paeoniflorin, liquiritin, verbascoside, harpaoside, paeonol and glycyrrhizic acid, and the feeding conditions of five medicinal ingredients in Yangyin Qingfei Pills, including white peony root, rehmannia root, Scrophularia ningpoensis, licorice and peony bark, were investigated and studied, which can provide a certain theoretical basis and technical support for improving the quality control standards of Yangyin Qingfei Pills. Summary of the invention

[0003] The present invention adopts the segmented wavelength conversion method, aiming to establish a method for simultaneously determining seven index components in Yangyin Qingfei Pills, namely, harpaoside, paeoniflorin, liquiritin, verbascoside, harpaoside, paeonol and glycyrrhizic acid, so as to detect the feeding conditions of five traditional Chinese medicines in Yangyin Qingfei Pills, namely, white peony root, rehmannia root, Scrophularia ningpoensis, licorice and peony bark.

[0004] The technical solution of the present invention is as follows:

[0005] A method for simultaneously detecting seven active ingredients in Yangyin Qingfei Pills comprises the following steps: injecting a Yangyin Qingfei Pill sample solution into a liquid chromatograph, and performing chromatographic detection by using a segmented wavelength conversion method to achieve qualitative and quantitative detection of the seven active ingredients.

[0006] The above chromatographic detection is selected from the following chromatographic conditions:

[0007] Using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution, and the gradient elution is selected from the following elution conditions: 0 - 3 min, 3% A; 3 - 16 min, 3 - 16% A; 16 - 20 min, 16% A; 20 - 26 min, 16 - 25% A; 26 - 31 min, 25 - 30% A; 31 - 33 min, 30 - 37% A; 33 - 38 min, 37 - 42% A; 38 - 40 min, 42 - 45% A; 40 - 45 min, 45 - 100% A; the column temperature is 30 °C; the injection volume is 4 μL; the flow rate is 1.0 mL / min.

[0008] The above-mentioned segmented wavelength change method is selected from the following conditions:

[0009] 0 - 8 min, wavelength 210 nm, for determining harpagide; 8 - 18 min, wavelength 230 nm, for determining paeoniflorin; 18 - 21 min, wavelength 278 nm, for determining liquiritin; 21 - 27 min, wavelength 332 nm, for determining acteoside; 27 - 33 min, wavelength 278 nm, for determining harpagoside and paeonol; 33 - 45 min, wavelength 252 nm, for determining glycyrrhizic acid.

[0010] In the above detection method, the seven active ingredients are harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol and glycyrrhizic acid.

[0011] The present invention provides the application of the above method in detecting the traditional Chinese medicine input in Yangyin Qingfei Pills.

[0012] In the above application, the traditional Chinese medicines are white peony root, rehmannia root, scrophularia root, licorice root and cortex moutan.

[0013] The present invention provides the application of the above method in the quality control and evaluation of Yangyin Qingfei Pills.

[0014] In the above quality control and evaluation application, the content standards of the active ingredients in Yangyin Qingfei Pills are:

[0015] Calculated as the total amount of harpagide and harpagoside, the established limits are as follows: for the big honeyed pills, it should be not less than 0.30 mg / g; for the small honeyed pills, it should be not less than 0.30 mg / g; for the water-honeyed pills, it should be not less than 0.35 mg / g; for the water pills, it should be not less than 0.50 mg / g. The established limit for liquiritin is as follows: for the big honeyed pills, it should be not less than 70 μg / g; for the small honeyed pills, it should be not less than 70 μg / g; for the water-honeyed pills, it should be not less than 130 μg / g; for the water pills, it should be not less than 150 μg / g. The established limit for glycyrrhizic acid is as follows: for the big honeyed pills, it should be not less than 0.30 mg / g; for the small honeyed pills, it should be not less than 0.30 mg / g; for the water-honeyed pills, it should be not less than 0.35 mg / g; for the water pills, it should be not less than 0.40 mg / g. The established limit for paeoniflorin is as follows: for the big honeyed pills, it should be not less than 0.60 mg / g; for the small honeyed pills, it should be not less than 0.60 mg / g; for the water-honeyed pills, it should be not less than 0.85 mg / g; for the water pills, it should be not less than 1.0 mg / g. The established limit for paeonol is as follows: for the big honeyed pills, it should be not less than 0.50 mg / g; for the small honeyed pills, it should be not less than 0.50 mg / g; for the water-honeyed pills, it should be not less than 0.60 mg / g; for the water pills, it should be not less than 0.70 mg / g.

[0016] The beneficial effects of the present invention are as follows:

[0017] By adopting the UPLC method with segmented wavelength transformation, the present invention realizes the simultaneous determination of the contents of harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol and glycyrrhizic acid in the Yangyin Qingfei Pills. Good separation of the seven active ingredients can be achieved without interference from other components. Moreover, each component has a good linear relationship within its respective linear range, and the recovery rate of sample addition is between 95% and 105%. The detection method of the present invention is simple, accurate and effective, and thus can be used for the quality control and overall quality evaluation of the Yangyin Qingfei Pills. Description of the Drawings

[0018] Figure 1 It is the UPLC chromatogram of the mixed reference substances; among them, 1 is harpagide, 2 is paeoniflorin, 3 is liquiritin, 4 is acteoside, 5 is harpagoside, 6 is paeonol, and 7 is ammonium glycyrrhizate;

[0019] Figure 2 It is the UPLC chromatogram of the water-honeyed pill test sample;

[0020] Figure 3 It is the UPLC chromatogram of the big honeyed pill test sample;

[0021] Figure 4 It is the UPLC chromatogram of the negative sample without white peony root and moutan bark;

[0022] Figure 5 It is the UPLC chromatogram of the negative sample without scrophularia root and rehmannia root;

[0023] Figure 6 It is the UPLC chromatogram of the negative sample without licorice root;

[0024] Figure 7 It is the UPLC chromatogram of the negative sample without Scrophulariae Radix;

[0025] Figure 8 It is the UPLC chromatogram of the negative sample without Paeoniae Radix Alba. Detailed implementation manners

[0026] The test instruments and raw materials adopted in the present invention are as follows:

[0027] Agilent 1290 Infinity rapid high-resolution liquid chromatograph; ME204T / 02 Mettler balance; AS10200BT ultrasonic cleaner (AUTO SCIENCE). Chromatographic column: Agilent SB-C18 (4.6 mm × 100 mm, 2.7 μm).

[0028] Harpagide (batch number: 111729-201707, purity: 96.8%), paeoniflorin (batch number: 110736-201943, purity: 100%), liquiritin (batch number: 111610-201908, purity: 95.0%), acteoside (batch number: 111530-201914, purity: 95.2%), harpagoside (batch number: 111730-201709, purity: 95.9%), paeonol (batch number: 110708-201407, purity: 99.9%), ammonium glycyrrhizinate (batch number: 110731-201720, purity: 97.7%). The above reference substances are all purchased from the National Institutes for Food and Drug Control.

[0029] Through UV spectral detection, the λ max (maximum absorption wavelength) of harpagide, harpagoside, paeoniflorin, acteoside, paeonol, liquiritin, and glycyrrhizic acid are 210, 282, 230, 332, 274, 278, and 252 nm respectively. Detection is carried out at the maximum absorption wavelength, where the response factor of this component on the instrument is the highest. Therefore, considering that each component is at the best detection wavelength, the method of segmentally changing the wavelength is adopted for determination. Since the retention times of the chromatographic peaks of paeonol and harpagoside are close and the maximum wavelengths are also similar, 278 nm is used for the determination of paeonol and harpagoside.

[0030] Acetonitrile (chromatographic grade, Fisher Scientific), and the remaining reagents are all of analytical grade (Sinopharm Chemical Reagent Co., Ltd.), Wahaha purified water.

[0031] The preparation of each sample solution is as follows:

[0032] 1. Reference substance solution

[0033] Weigh appropriate amounts of harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol, and ammonium glycyrrhizinate reference standards accurately. Dissolve them separately in 70% methanol to prepare reference standard stock solutions containing 435, 883.75, 255, 135, 146, 1036.67, and 711 μg per 1 mL respectively. Then, accurately pipette appropriate volumes of each reference standard stock solution and prepare a mixed reference standard solution with an appropriate concentration using 70% methanol. Calculate the weight of glycyrrhizic acid according to "weight of ammonium glycyrrhizinate / 1.0207".

[0034] 2. Test solution

[0035] Take the aqueous honeyed pills or aqueous pills of this product, grind them into fine powder, weigh about 1.5 g accurately; or take the large honeyed pills under weight variation or the small honeyed pills under filling quantity variation, cut them into pieces, weigh about 2.5 g accurately, place them in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 33 kHz) for 30 min, let it cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0036] 3. Negative control solution

[0037] Prepare negative samples without white peony root and moutan bark, without rehmannia root and scrophularia root, without licorice root, without scrophularia root, and without white peony root according to the prescription ratio and process respectively. Prepare the negative control solutions of pills according to the above method for preparing the test solution.

[0038] Other materials used in the present invention, unless otherwise specified, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise stated, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are only for illustrating the present invention and do not limit the scope of the present invention in any way.

[0039] Example

[0040] UPLC detection test:

[0041] Respectively take 4 μL of the reference standard solution, test solution, and negative control solution, inject them into a liquid chromatograph (UPLC), and perform chromatographic detection.

[0042] The chromatographic conditions are as follows:

[0043] Using octadecylsilane chemically bonded silica gel as the filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution: 0 - 3 min, 3% A; 3 - 16 min, 3 - 16% A; 16 - 20 min, 16% A; 20 - 26 min, 16 - 25% A; 26 - 31 min, 25 - 30% A; 31 - 33 min, 30 - 37% A; 33 - 38 min, 37 - 42% A; 38 - 40 min, 42 - 45% A; 40 - 45 min, 45 - 100% A; column temperature 30°C; injection volume 4 μL; flow rate 1.0 mL / min; wavelength determination with segmented change: 0 - 8 min, 210 nm, for determination of harpagide; 8 - 18 min, 230 nm, for determination of paeoniflorin; 18 - 21 min, 278 nm, for determination of liquiritin; 21 - 27 min, 332 nm, for determination of acteoside; 27 - 33 min, 278 nm, for determination of harpagoside and paeonol; 33 - 45 min, 252 nm, for determination of glycyrrhizic acid.

[0044] The chromatogram is as Figures 1-8 shown:

[0045] As can be seen from the above figure, the method of segmented wavelength change is adopted in the present invention, and the seven active ingredients can be determined simultaneously with a clear chromatogram. In addition, there is no absorption peak at the position corresponding to the chromatographic peak of the reference substance in the negative sample, indicating that other components in the prescription have no influence on the determination result.

[0046] (1) Investigation of linear relationship

[0047] Take appropriate amounts of reference substances of harpagide, paeoniflorin, harpagoside, acteoside, liquiritin, paeonol, and ammonium glycyrrhizinate, accurately weigh them, and use 70% methanol to prepare control solutions ① containing 174 μg, 70.71 μg, 29.18 μg, 135 μg, 101 μg, 31 μg, and 72.47 μg of the above 7 components per 1 mL respectively; then take appropriate amounts of reference substances of harpagide, paeoniflorin, harpagoside, acteoside, liquiritin, paeonol, and ammonium glycyrrhizinate, accurately weigh them, and use 70% methanol to prepare control solutions ② containing 1391.6 μg, 3535.4 μg, 145.9 μg, 359.3 μg, 607.9 μg, 3110.5 μg, and 1421.5 μg of the above 7 components per 1 mL respectively.

[0048] Linear concentration ①: Accurately pipette 1 mL of each of the control solutions ① of the above 7 components into 100 mL volumetric flasks;

[0049] Linear concentration ②: Pipette 1 mL of each of the control solutions ① of harpagide, paeoniflorin, harpagoside, paeonol, and ammonium glycyrrhizinate and 0.5 mL of each of the control solutions ① of acteoside and liquiritin into a 10 mL volumetric flask;

[0050] Linear concentration ③: Accurately measure 0.5 mL of each of the control solutions ② of harpagide, paeoniflorin, liquiritin, and paeonol, 0.8 mL of the control solution ② of acteoside, and 1 mL of each of the control solutions ② of harpagoside and ammonium glycyrrhizinate, and place them in a 25-mL volumetric flask;

[0051] Linear concentration ④: Accurately measure 0.5 mL of each of the control solutions ② of the above 7 components, and place them in a 10-mL volumetric flask;

[0052] Linear concentration ⑤: Accurately measure 1 mL of each of the control solutions ② of the above 7 components, and place them in a 100-mL volumetric flask;

[0053] Add 70% methanol to the above volumetric flasks to volume to the mark respectively to prepare a mixed linear control solution (μg / mL) with 5 concentrations.

[0054] Table 1 Concentrations of the linear control solutions of each component

[0055] Chemical composition Linear concentration ① Linear concentration ② Linear concentration ③ Linear concentration ④ Linear concentration ⑤ Harpagoside 1.7 17.4 27.8 69.9 139.2 Paeoniflorin 0.7 7.1 70.7 176.8 353.5 Harpagide 0.3 2.9 5.8 7.3 14.6 Acteoside 1.4 6.8 11.5 18.0 35.9 Glabroside 1.0 5.1 12.2 30.4 60.8 Paeonol 0.3 3.1 62.2 155.5 311.1 Ammonium glycyrrhizinate 0.7 7.3 58.9 71.1 142.2

[0056] Accurately pipette the above mixed linear control solutions respectively, inject them into the liquid chromatograph, and perform linear regression with the control product concentration as the abscissa and the peak area as the ordinate.

[0057] The test results are shown in Table 2, and the linear relationships of each component are good within their respective concentration ranges. The linear relationship in the present invention means that the concentration of the solution injected into the instrument is proportional to the peak area. A good linear relationship indicates that within this concentration range, the concentration of the component to be measured can be calculated using the peak area and the linear equation.

[0058] (II) Detection limit and quantification limit

[0059] Gradually dilute the mixed reference solution of the 7 compounds and then inject it for analysis. Calculate the detection limit (LOD) of the method with the solution concentration at a signal-to-noise ratio S / N = 3; calculate the quantification limit (LOQ) of the method with the solution concentration at S / N = 10. In the present invention, the quantification limit refers to the lowest amount of the analyte in the sample that can be quantitatively determined; the detection limit refers to the lowest detection concentration that can be distinguished from the noise when the sample is extracted, processed, and detected according to the requirements of the analysis method. The test results are shown in Table 2.

[0060] Table 2 Linear relationships, quantification limits, and detection limits of each component

[0061]

[0062] (III) Precision test

[0063] Take the mixed reference substance solution and continuously determine it 6 times according to the liquid chromatography conditions in the examples. Calculate the precision from the peak areas of each component. The results show that the precisions of harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol, and ammonium glycyrrhizinate are 1.48%, 0.14%, 0.27%, 0.17%, 0.26%, 0.16%, and 0.15% respectively, all less than 2.0%, indicating that the precision of the instrument is good.

[0064] (IV) Repeatability test

[0065] Take the water honey pill (17035288) and big honey pill (18010541) samples, and prepare 6 portions of the test solution respectively according to the method in the above test solution, inject for determination, and calculate the RSD of the content of each component. The results show that the repeatabilities of harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol, and glycyrrhizic acid in the water honey pill are 1.10%, 1.51%, 0.65%, 0.77%, 0.49%, 0.98%, and 0.51% respectively, and those in the big honey pill are 1.60%, 1.69%, 0.94%, 0.51%, 0.30%, 0.75%, and 0.12% respectively, all less than 2.0%, indicating that the repeatability of the method is good.

[0066] (V) Stability test

[0067] Take one portion of the test solution of the water honey pill and big honey pill in the above repeatability test respectively, inject for determination at 0h, 2h, 8h, 10h, 12h, and 24h, record the peak areas of each component and calculate the RSD. The results show that the stabilities of harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol, and glycyrrhizic acid in the water honey pill are 1.05%, 0.59%, 0.97%, 1.01%, 1.14%, 0.48%, and 0.55% respectively, and those in the big honey pill are 1.41%, 0.86%, 0.99%, 1.03%, 1.47%, 0.83%, and 0.64% respectively, all less than 2.0%, indicating that each component is basically stable within 24h.

[0068] (VI) Recovery test by standard addition

[0069] Take appropriate amounts of harpagide, paeoniflorin, harpagoside, acteoside, liquiritin, paeonol, and ammonium glycyrrhizinate reference substances, accurately weigh them, and prepare reference solutions containing 347.9 μg, 353.54 μg, 389.16 μg, 359.28 μg, 405.27 μg, 1555.24 μg, and 379.08 μg per 1 mL respectively with 70% methanol.

[0070] Water honey pills: Take about 0.75 g of the powdered water honey pill test sample (17035288), accurately weigh it, and accurately add 1750 μL, 4750 μL, 200 μL, 350 μL, 500 μL, 700 μL, and 1750 μL of the above-mentioned reference substance solutions of harpagide, paeoniflorin, harpagoside, acteoside, liquiritin, paeonol, and ammonium glycyrrhizinate respectively. Prepare 6 test solutions according to the preparation method of the test sample solution above, inject and determine the recovery rate. The results show that the average recovery rates of harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol, and glycyrrhizic acid are 103.05%, 103.51%, 100.61%, 97.81%, 101.06%, 95.67%, and 101.40% respectively; the RSDs are 1.94%, 2.14%, 2.92%, 1.45%, 1.17%, 1.83%, and 1.46% respectively.

[0071] Big honey pills: Take about 1.25 g of the cut big honey pill test sample (18010541), accurately weigh it, and accurately add 1600 μL, 5000 μL, 300 μL, 350 μL, 500 μL, 800 μL, and 1450 μL of the above-mentioned reference substance solutions of harpagide, paeoniflorin, harpagoside, acteoside, liquiritin, paeonol, and ammonium glycyrrhizinate respectively. Prepare 6 test solutions according to the preparation method of the test sample solution above, inject and determine the recovery rate. The results show that the average recovery rates of harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol, and glycyrrhizic acid are 103.46%, 104.24%, 100.17%, 95.97%, 102.65%, 95.41%, and 101.31% respectively; the RSDs are 0.83%, 1.97%, 2.62%, 0.76%, 2.43%, 2.33%, and 2.35% respectively.

[0072] Application example

[0073] The samples collected this time include all 4 specifications of Yangyin Qingfei Pills. Among them, the water honey pills involve 46 batches of samples from 3 enterprises, the big honey pills involve 46 batches of samples from 12 enterprises, the water pills involve 1 batch of samples from 1 enterprise, and the small honey pills involve 3 batches of samples from 1 enterprise. Determine the content of the above-mentioned Yangyin Qingfei Pill samples according to the method of the above-mentioned examples.

[0074] According to the standard limit of the cut crude drugs in Part I of the Chinese Pharmacopoeia (2015 Edition), the content of acteoside in Rehmanniae Radix should not be less than 0.020%. According to the standard limit of the cut crude drugs in Part I of the Chinese Pharmacopoeia (2015 Edition), the content of liquiritin in Glycyrrhizae Radix should not be less than 0.50%, and the content of glycyrrhizic acid should not be less than 2.0%. The total amount of harpagide and harpagoside in Scrophulariae Radix should not be less than 0.45%. The content of paeoniflorin in Paeoniae Radix Alba should not be less than 1.6%. The content of paeonol in Moutan Cortex should not be less than 1.2%.

[0075] According to the preparation process of Yangyin Qingfei Pills, licorice, scrophularia root, and rehmannia root are all used as crude powder. The transfer rate is calculated at 70%. Since the small honey pills manufacturer did not provide the preparation process and the production quantity, the production quantity of small honey pills is temporarily calculated according to that of big honey pills; the production quantities of water-honey pills are 972 g and 1050 g respectively, so the theoretical value of water-honey pills is calculated according to the production quantity of 1000 g; according to the preparation process of big honey pills, 70 - 90 g of refined honey is added to every 100 g of powder, that is, the production quantity of big honey pills should be between 1377 g and 1539 g. The theoretical value is calculated based on the maximum production quantity of 1539 g. The theoretical values of the contents of each component in the preparation are shown in Table 3.

[0076] Table 3 Theoretical values of the contents of each component in the preparation

[0077]

[0078] In the prescription, scrophularia root also contains acteoside. Combining the theoretical value of rehmannia root and the sample determination results, the limit for the content of acteoside is determined as follows: not less than 100.0 μg / g for water-honey pills, not less than 60.0 μg / g for big (small) honey pills, and not less than 120.0 μg / g for water pills.

[0079] Combining the theoretical value and the sample determination situation, taking the total amount of harpagide and harpagoside as the count, the determined limit is: not less than 0.30 mg / g for big (small) honey pills, not less than 0.35 mg / g for water-honey pills, and not less than 0.50 mg / g for water pills. The determined limit for liquiritin is: not less than 70 μg / g for big (small) honey pills, not less than 130 μg / g for water-honey pills, and not less than 150 μg / g for water pills; the determined limit for glycyrrhizic acid (glycyrrhizic acid weight = ammonium glycyrrhizate weight / 1.0207) is: not less than 0.30 mg / g for big (small) honey pills, not less than 0.35 mg / g for water-honey pills, and not less than 0.40 mg / g for water pills; the determined limit for paeoniflorin is: not less than 0.60 mg / g for big (small) honey pills, not less than 0.85 mg / g for water-honey pills, and not less than 1.0 mg / g for water pills; the determined limit for paeonol is: not less than 0.50 mg / g for big (small) honey pills, not less than 0.60 mg / g for water-honey pills, and not less than 0.70 mg / g for water pills;

[0080] The test results are shown in Tables 4 - 6.

[0081] Table 4 Determination results of the contents of each component in Yangyin Qingfei Pills (big honey pills)

[0082]

[0083] Table 5 Determination results of the contents of each component in Yangyin Qingfei Pills (water-honey pills)

[0084]

[0085] Table 6 Determination Results of the Contents of Each Component in Yangyin Qingfei Pills (Small Honey Pills, Water Pills)

[0086]

[0087] Through literature search, harpagide and harpagoside belong to Scrophulariae Radix; paeoniflorin belongs to Paeoniae Radix Alba and Moutan Cortex; paeonol belongs to Moutan Cortex; acteoside belongs to Rehmanniae Radix and Scrophulariae Radix; liquiritin and glycyrrhizic acid belong to Glycyrrhizae Radix.

[0088] After determination, the medicinal materials with relatively poor feeding quality mainly involve Scrophulariae Radix and Rehmanniae Radix. None of the 3 batches of samples from all enterprises of the big honey pills detected harpagide and harpagoside. Among them, 1 batch of samples did not detect acteoside, and the content of acteoside in 1 batch of samples was relatively low (64 μg·g -1 ), and the content of acteoside in 1 batch of samples was at the upper-middle level among all samples (156 μg·g -1 ), suggesting that this enterprise should pay attention to the feeding risks of Scrophulariae Radix and Rehmanniae Radix. None of the 3 batches of samples from all enterprises of 1 manufacturer detected harpagide and the contents of harpagoside were all relatively low (0.074 - 0.085 mg·g -1 ), and the contents of acteoside were all at normal levels. There is research indicating that the contents of harpagide and harpagoside in Scrophulariae Radix from different origins vary greatly. The content of harpagide in most Scrophulariae Radix is higher than that of harpagoside, and there are also some origins where the content of harpagoside in Scrophulariae Radix is higher than that of harpagide, suggesting that this manufacturer needs to pay attention to the feeding risks of Scrophulariae Radix medicinal materials.

[0089] The contents of flavonoids (such as liquiritin) and triterpenoids (such as glycyrrhizic acid) in Glycyrrhizae Radix are relatively large and are the main medicinal components of Glycyrrhizae Radix. There is research showing that the contents of liquiritin and glycyrrhizic acid in Glycyrrhizae Radix are related to the original plant, origin and growth years. For products from different enterprises and different batches of the same enterprise, the contents of liquiritin and glycyrrhizic acid vary greatly, which may be related to the source and growth years of the original medicinal materials used. Among the water-honey pills, 5 batches of samples from 1 manufacturer had relatively low contents of liquiritin (≤140 μg·g -1 ), and the contents of glycyrrhizic acid in 12 batches of samples were all relatively low (≤0.35 mg·g -1 ), suggesting that this enterprise should pay attention to the quality risks of Glycyrrhizae Radix medicinal materials in subsequent production.

[0090] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for simultaneously detecting seven active ingredients in Yangyin Qingfei Pills, characterized in that, The steps are as follows: Inject the sample solution of Yangyin Qingfei Pills into a liquid chromatograph, and use the method of segmentally changing the wavelength for chromatographic detection to achieve the qualitative and quantitative detection of seven active ingredients. The seven active ingredients are harpagide, paeoniflorin, liquiritin, acteoside, harpagoside, paeonol, and glycyrrhizic acid. The chromatographic detection is selected from the following chromatographic conditions: Use octadecylsilane-bonded silica gel as the filler; use acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution. The gradient elution is selected from the following elution conditions: 0 - 3 min, 3% A; 3 - 16 min, 3 - 16% A; 16 - 20 min, 16% A; 20 - 26 min, 16 - 25% A; 26 - 31 min, 25 - 30% A; 31 - 33 min, 30 - 37% A; 33 - 38 min, 37 - 42% A; 38 - 40 min, 42 - 45% A; 40 - 45 min, 45 - 100% A; the column temperature is 30 °C; the injection volume is 4 μL; the flow rate is 1.0 mL / min. The method of segmentally changing the wavelength is selected from the following conditions: 0 - 8 min, wavelength 210 nm, for the determination of harpagide; 8 - 18 min, wavelength 230 nm, for the determination of paeoniflorin; 18 - 21 min, wavelength 278 nm, for the determination of liquiritin; 21 - 27 min, wavelength 332 nm, for the determination of acteoside; 27 - 33 min, wavelength 278 nm, for the determination of harpagoside and paeonol; 33 - 45 min, wavelength 252 nm, for the determination of glycyrrhizic acid.

2. Application of the method according to claim 1 in detecting the traditional Chinese medicine input in Yangyin Qingfei Pills.

3. The application according to claim 2, characterized in that, The traditional Chinese medicine is white peony root, rehmannia root, figwort root, licorice root, and tree peony bark.

4. Application of the method according to claim 1 in the quality control and evaluation of Yangyin Qingfei Pills.

5. The application according to claim 4, wherein The content standards of each active ingredient in the Yangyin Qingfei Pills are as follows: Calculated based on the total amount of harpagide and harpagoside, the specified limits are: for the big honey pills, not less than 0.30 mg / g; for the small honey pills, not less than 0.30 mg / g; for the water-honey pills, not less than 0.35 mg / g; for the water pills, not less than 0.50 mg / g. The specified limit for liquiritin is: for the big honey pills, not less than 70 μg / g; for the small honey pills, not less than 70 μg / g; for the water-honey pills, not less than 130 μg / g; for the water pills, not less than 150 μg / g. The specified limit for glycyrrhizic acid is: for the big honey pills, not less than 0.30 mg / g; for the small honey pills, not less than 0.30 mg / g; for the water-honey pills, not less than 0.35 mg / g; for the water pills, not less than 0.40 mg / g. The specified limit for paeoniflorin is: for the big honey pills, not less than 0.60 mg / g; for the small honey pills, not less than 0.60 mg / g; for the water-honey pills, not less than 0.85 mg / g; for the water pills, not less than 1.0 mg / g. The specified limit for paeonol is: for the big honey pills, not less than 0.50 mg / g; for the small honey pills, not less than 0.50 mg / g; for the water-honey pills, not less than 0.60 mg / g; for the water pills, not less than 0.70 mg / g.

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