Method for simultaneously measuring saccharide content
By combining the HPLC-ELSD method with appropriate mobile phase and chromatographic conditions, the problems of rapidity and separation in the detection of sugar content in the whole Qingjing San formula were solved, and efficient and low-cost sugar content detection was achieved.
Patent Information
- Application Number
- CN202410274833.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-11
- Publication Date
- 2025-09-12
AI Technical Summary
The existing detection methods are unable to quickly and simultaneously detect the sugar content in the entire Qingjing San formula, and the detection time is long, the repeatability is poor, the instrument requirements are high, and the separation is low.
The HPLC-ELSD method was adopted, using Waters XBridge Amide or Rocksil Carbohydrate ES columns, with a mobile phase of 0.05% ammonia and acetonitrile gradient elution, combined with appropriate column temperature, drift tube temperature and gas pressure to achieve simultaneous detection of sugars.
The rapid and accurate detection of the sugar content of the whole Qingjing San formula is achieved with good repeatability, low instrument requirements, high separation and short detection time.
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Figure CN120629381A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for simultaneously determining sugar content. Background Art
[0002] Qingjingsan is a Chinese medicine formula from Fu Qingzhu's Gynecology. It clears heat and cools blood. It treats excessive water and fire in the kidneys, premature menstruation with heavy, dark red or purple menstrual flow, a thick, sticky texture, a red tongue with a yellow coating, and a rapid pulse. It is composed of Paeonia suffruticosa (11.19g), Radix Rehmanniae (18.65g), White Peony Root (11.19g), Rehmannia Glutinosa (11.19g), Artemisia Annua (7.46g), Poria (3.73g), and Phellodendron amurense (1.87g).
[0003] The monosaccharides, disaccharides, and oligosaccharides in Qingjing San are primarily derived from Rehmannia root, with smaller amounts also found in Cortex Moutan and Radix Paeoniae Alba. Existing detection methods are primarily used for Rehmannia root alone and primarily utilize amino columns, which require 2-3 hours of equilibration and another 2-3 hours of column wash time. If equilibrium is not achieved, sample peaks may exhibit poor shape and unstable retention times.
[0004] At present, there is no literature reporting a method for determining the sugar content in the whole formula of Qingjing San. The sugar content can reflect the stability of the preparation process of Qingjing San. Therefore, determining the sugar content in Qingjing San can provide theoretical guidance for its process production. Summary of the Invention
[0005] In order to overcome the problem that the sugar content of the whole Qingjing Powder cannot be simultaneously detected and the detection time is long, the present invention provides a method for simultaneous determination of sugar content. The method can simultaneously detect the sugar content of the whole Qingjing Powder, has a short detection time, good repeatability, low instrument requirements and high separation.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] The present invention provides a method for simultaneously determining the content of sugars, the method comprising the following steps: using HPLC-ELSD to detect a test solution, wherein the test solution contains D-fructose, D-glucose, sucrose, melibiose, raffinose, mannotriose, and stachyose;
[0008] The liquid chromatography detection conditions are as follows: the chromatographic column is Waters XBridge Amide or Rocksil Carbohydrate ES; the mobile phases are mobile phase A and mobile phase B, mobile phase A is 0-0.05% ammonia water, and mobile phase B is 0-0.05% ammonia water-acetonitrile; based on the total volume of the mobile phase as 100%, the gradient elution program is shown in the following table:
[0009] Elution stage Elution time / min Mobile phase A% Mobile phase B% First elution stage 4-7 x1→x2 (100% - x1) → (100% - x2) Second elution stage 7-13 x2→x3 (100% - x2) → (100% - x3) The third elution stage 0-0.5 Keep x3 Keep (100% - x3) Fourth elution stage 5-11 x3→x4 (100% - x3) → (100% - x4) Fifth elution stage 6-12 x4→x5 (100% - x4) → (100% - x5) Sixth elution stage 2-8 x5→x6 (100% -x5) → (100% -x6) Seventh elution stage 1-6 Keep x6 Keep (100% - x6)
[0010] The elution time is the difference between the elution end time and the elution start time of the elution stage:
[0011] x1 is 22%-31% (22%, 30%),
[0012] x2 is 28%-32% (30%, 30.5%),
[0013] x3 is 30%-33% (30.5%, 31.5%),
[0014] x4 is 30%-35% (31.5%, 33%),
[0015] x5 is 35%-39% (36%, 37%),
[0016] x6 is 28%-31% (29%, 30%),
[0017] Among them, x1 <x2<x3<x4<x5,x5> x6.
[0018] In the present invention, mobile phase A being 0% ammonia water means that mobile phase A is water, and mobile phase B being 0% ammonia water-acetonitrile means that mobile phase B is acetonitrile.
[0019] In some embodiments, mobile phase A is 0.05% ammonia water.
[0020] In some embodiments, mobile phase B is 0.05% aqueous ammonia-acetonitrile.
[0021] In some embodiments, the liquid chromatography detection condition has a flow rate of 0.5-0.6 mL / min.
[0022] In some embodiments, the liquid chromatography detection condition is a column temperature of 20-40°C; preferably, the column temperature is 35°C.
[0023] In some embodiments, the ELSD parameters are: gas pressure of 40 psi-60 psi, and drift tube temperature of 80° C.-100° C.
[0024] In some embodiments, the gas pressure is 60 psi.
[0025] In some embodiments, the drift tube temperature is 100°C.
[0026] In some embodiments, the gradient elution procedure is shown in the following table:
[0027] Time / min Flow rate mL / min A / ％ B / ％ 0 0.5 30 70 5 0.5 30.5 69.5 14.5 0.5 31.5 68.5 14.6 0.6 31.5 68.5 22.5 0.6 33 67 31.5 0.6 37 63 36.5 0.6 30 70 39 0.6 30 70 .
[0028] In some embodiments, the test sample is a decoction of Qingjing Powder, and the decoction of Qingjing Powder can be prepared by conventional methods in the art. Preferably, the preparation method of the decoction of Qingjing Powder comprises the following steps: weighing each raw material according to the formula of Qingjing Powder, soaking it in water, boiling it, and filtering it. Further, the preparation method of the decoction of Qingjing Powder comprises the following steps: weighing each raw material according to the formula of Qingjing Powder, adding 5-15 times the amount of water as the raw material, soaking it for 5-10 hours, boiling it and then simmering it for 20-60 minutes (100-300W), filtering it, adding 5-10 times the amount of water as the filter residue as the filter residue, boiling it and then simmering it for 10-60 minutes (100-300W), filtering it, letting it stand and cool, and diluting the decoction to 1000ml.
[0029] In some embodiments, the Qingjing Powder formula includes the following raw materials in parts by weight: 8-12 parts of peony bark, 16-20 parts of rehmannia root bark, 8-12 parts of white peony root wine, 8-12 parts of rehmannia root prepared, 5-8 parts of artemisia annua, 2-4 parts of poria cocos, and 1-2 parts of salted phellodendron.
[0030] In some embodiments, the method for preparing the test solution comprises the following steps: diluting the Qingjingsan decoction with a 0%-50% methanol aqueous solution. Preferably, the test solution Qingjingsan decoction is diluted with a 30% methanol aqueous solution.
[0031] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.
[0032] The reagents and raw materials used in the present invention are commercially available.
[0033] The positive progress effect of the present invention is that the present invention provides a method for simultaneously determining the content of sugars, which can simultaneously detect the content of sugars, has a short detection time, good repeatability, low instrument requirements, and high separation. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the chromatogram of Example 1.
[0035] Figure 2 The HPLC chromatograms of Qingjing Powder with methanol-water solutions of different proportions corresponding to Examples 1, 2, and 3 are shown.
[0036] Figure 3 HPLC chromatograms of Qingjing Powder at different ELSD drift tube temperatures corresponding to Examples 1, 4, and 5.
[0037] Figure 4 The HPLC chromatograms of Qingjing Powder under different ELSD gas pressures corresponding to Examples 1, 6, and 7 are shown.
[0038] Figure 5 HPLC chromatograms of Qingjing Powder under different mobile phases corresponding to Examples 1 and 8.
[0039] Figure 6 HPLC chromatograms of Qingjing Powder at different column temperatures corresponding to Examples 1, 9, 10, and 11.
[0040] Figure 7 The HPLC chromatograms of Examples 1, 12, and 13 are obtained when the chromatographic columns are Waters XBrige Amide, Waters Xselect HSST3, and Rocksil Carbohydrate ES amino columns, respectively. DETAILED DESCRIPTION
[0041] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.
[0042] Example 1, determination method
[0043] 1) Preparation of reference solution
[0044] Weigh appropriate amounts of fructose, glucose, sucrose, melibiose, raffinose, mannotriose, and stachyose respectively, add appropriate amount of 25% methanol to dissolve them, and prepare mixed reference solutions containing 0.2999 mg of fructose, 0.2004 mg of glucose, 0.4495 mg of sucrose, 0.0521 mg of melibiose, 0.0508 mg of raffinose, 0.3922 mg of mannotriose, and 0.4369 mg of stachyose per 1 mL.
[0045] 2) Preparation of test solution
[0046] Quantitatively weigh each medicinal material, weigh 11.19 g of peony bark, 18.65 g of rehmannia root, 11.19 g of white peony root, 11.19 g of prepared rehmannia root, 7.46 g of artemisia annua, 3.73 g of poria, and 1.87 g of salt phellodendron. Add 600 ml of water to the medicinal materials and soak for 7 hours. Boil for the first time over high heat (500 W) and then simmer for 30 minutes over low heat (200 W). Filter while hot through a 300-mesh nylon filter cloth. Add 480 ml of water to the second decoction. Boil over high heat (500 W) and then simmer for 20 minutes over low heat (200 W). Filter while hot through a 300-mesh nylon filter cloth. Let it stand and cool. The decoction is fixed to 1000 ml to obtain the Qingjingsan decoction, which is stored in a refrigerator at 4°C.
[0047] Pipette 3.0 ml of methanol and place it in a 10 ml volumetric flask with a stopper. Add 4.0 ml of pure water. Let it stand and cool, then add 2.0 ml of Qingjingsan decoction. Add pure water to the scale to make up the volume and mix well.
[0048] 3) Content determination
[0049] Accurately pipette 8ul of the reference solution and the test solution into the liquid chromatograph and record the chromatogram.
[0050] Wherein, the chromatographic conditions are:
[0051] A Waters XBridge Amide column (4.6 mm × 250 mm × 3.5 μm) was used. The ELSD detector was turned on, the drift tube temperature was set to 100°C, the gas pressure was set to 60 psi, and the gas flow rate was set to 2.5 mL / min. After the ELSD was stabilized, the mobile phase A consisted of 0.05% aqueous ammonia, and the mobile phase B consisted of 0.05% aqueous ammonia-acetonitrile. The column temperature was set to 35°C, and the gradient was as follows:
[0052] Table 1
[0053] Time / min <![CDATA[Flow rate / mL·min -1 > A / ％ B / ％ 0 0.5 30 70 5 0.5 30.5 69.5 14.5 0.5 31.5 68.5 14.6 0.6 31.5 68.5 22.5 0.6 33 67 31.5 0.6 37 63 36.5 0.6 30 70 39 0.6 30 70
[0054] Experimental results: Chromatogram see Figure 1 , each chromatographic peak has a good shape and a separation degree greater than 1.5, as follows:
[0055]
[0056]
[0057] Example 2
[0058] In this example, the test solution was prepared by pipetting 2.0 ml of Qingjingsan decoction into a 10 ml volumetric flask with a stopper, adding purified water to the mark, and mixing. The remaining parameters and conditions were the same as in Example 1. The peak areas in the chromatographic results obtained were smaller than those in Example 1, as shown below:
[0059] Chromatographic peak Retention time / min Peak area / % Separation fructose 11.022 22.9140 2.80 glucose 12.735 17.5385 6.63 sucrose 15.340 59.9997 3.56 melibiose 19.400 2.9937 4.81 Raffinose 22.415 1.3914 2.73 Mannotriose 29.187 19.6153 4.89 Stachyose 32.475 12.8018 na .
[0060] Example 3
[0061] In this example, the test solution was prepared by taking 5.0 ml of methanol and placing it in a 10 ml stoppered volumetric flask, adding 2.0 ml of pure water, letting it cool, adding 2.0 ml of Qingjingsan decoction, adding pure water to the mark, and mixing. The remaining parameters and conditions were the same as in Example 1. The peak areas in the chromatographic results obtained were smaller than those in Example 1, as shown below:
[0062] Chromatographic peak Retention time / min Peak area / % Separation fructose 11.587 24.1636 2.74 glucose 12.747 17.8225 6.62 sucrose 15.350 61.2111 3.50 melibiose 19.388 1.7784 5.24 Raffinose 22.418 1.3877 2.80 Mannotriose 29.187 19.9757 4.86 Stachyose 32.463 13.0166 na .
[0063] The specific spectra comparison results of Example 1, Example 2 and Example 3 are shown in Figure 2 .
[0064] Example 4
[0065] In this embodiment, the drift tube temperature is 80° C., and the other parameters and conditions are the same as those in Example 1. The chromatographic peak shapes and separations in the obtained chromatographic results are not much different from those in Example 1.
[0066] Example 5
[0067] In this embodiment, the drift tube temperature is 90°C, and the other parameters and conditions are the same as those in Example 1. The chromatographic peak shapes and separations obtained are not much different from those in Example 1. The specific chromatogram comparison results of Example 1, Example 4 and Example 5 are shown in FIG. Figure 3 .
[0068] Example 6
[0069] In this embodiment, the ELSD gas pressure is 40 psi, and the other parameters and conditions are the same as those in Example 1. The chromatographic peak shapes and separation degrees in the obtained chromatographic results are not much different from those in Example 1.
[0070] Example 7
[0071] In this embodiment, the ELSD gas pressure is 50 psi, and the other parameters and conditions are the same as those in Example 1. The peak shapes and separations of the chromatographic results obtained are not much different from those in Example 1. The specific spectra comparison results of Example 1, Example 6 and Example 7 are shown in FIG. Figure 4 .
[0072] Example 8
[0073] In this embodiment, the liquid mobile phase is pure water and pure acetonitrile, and the other parameters and conditions are the same as those in Example 1. The chromatographic peak shapes of glucose and mannotriose in the obtained chromatographic results are less symmetrical than those in Example 1, as shown below:
[0074] Chromatographic peak Retention time / min Peak area / % Separation fructose 11.570 13.2405 2.04 glucose 12.748 9.1585 5.47 sucrose 15.337 49.558 2.73 melibiose 19.593 1.3428 3.05 Raffinose 22.427 0.9917 2.8 Mannotriose 29.420 12.9761 3.27 Stachyose 32.517 26.5309 na
[0075] For detailed spectrum results, see Figure 5 .
[0076] Example 9
[0077] In this embodiment, the column temperature of the chromatographic column was 20° C., and the other parameters and conditions were the same as those in Example 1. The chromatographic peak shape of melibiose in the obtained chromatographic results was less symmetrical than that in Example 1, as shown below:
[0078] Chromatographic peak Retention time / min Peak area / % Separation fructose 13.103 11.0622 2.17 glucose 14.235 8.8599 5.94 sucrose 16.917 42.3871 3.21 melibiose 21.782 1.0902 3.98 Raffinose 24.880 0.7311 8.84 Mannotriose 31.943 11.7486 3.71 Stachyose 34.858 20.0413 na .
[0079] Example 10
[0080] In this embodiment, the column temperature is 30° C., and the other parameters and conditions are the same as those in Example 1. The chromatographic peak shape of fructose in the obtained chromatographic results is not as good as that in Example 1, as shown in the following:
[0081] Chromatographic peak Retention time / min Peak area / % Separation fructose 12.060 15.1209 2.42 glucose 13.218 10.2695 6.19 sucrose 15.863 49.368 3.49 melibiose 20.212 1.2943 4.15 Raffinose 23.325 1.0373 10.33 Mannotriose 30.312 15.1723 4.66 Stachyose 33.500 23.8109 na .
[0082] Example 11
[0083] In this embodiment, the column temperature is 40° C., and the other parameters and conditions are the same as those in Example 1. The chromatographic peak shape of fructose in the obtained chromatographic results is not as good as that in Example 1, as shown in the following:
[0084] Chromatographic peak Retention time / min Peak area / % Separation fructose 11.248 17.7242 2.69 glucose 12.402 11.0741 6.57 sucrose 15.000 50.1994 3.64 melibiose 18.952 1.1047 5.24 Raffinose 21.908 1.2105 2.79 Mannotriose 28.618 17.7694 5.56 Stachyose 31.995 27.2831 na .
[0085] The specific spectra comparison results of Example 1, Example 9, Example 10 and Example 11 are shown in Figure 6 .
[0086] Example 12
[0087] In this embodiment, the chromatographic column was Waters Xselect HSS T3, and the other parameters and conditions were the same as those in Example 1. The chromatographic results obtained showed that no sugar peaks appeared.
[0088] Example 13
[0089] In this embodiment, the chromatographic column is Rocksil Carbohydrate ES, and the other parameters and conditions are the same as those in Example 1. The peak shape and separation of the chromatographic peaks of each sugar in the obtained chromatographic results are not much different from those in Example 1.
[0090] The specific spectra comparison results of Example 1, Example 12 and Example 13 are shown in Figure 7 .
[0091] Effect Example 1 Methodological Investigation
[0092] 1. Linear relationship investigation
[0093] 1 ml of the reference stock solution was aspirated and centrifuged at 12000 r / min for 10 min at room temperature. 800 μl was aspirated and placed in a liquid phase vial. 2, 4, 8, 10, 12, and 15 μl of the reference solution were aspirated respectively to determine the peak areas (A) of D-fructose, D-anhydroglucose, sucrose, melibiose, raffinose, mannotriose, and stachyose. The logarithm of the peak area (log A) was used as the ordinate (y) and the logarithm of the injection volume (m) was used as the abscissa (x). A standard curve was drawn. The seven sugars showed a good linear relationship within a certain range (r>0.998). The regression equation for D-fructose was y=1.6431x+1.263, r=0.9987, and the linear range was 0.60-5.00 μg. The regression equation for D-glucose was y = 1.4542x + 1.3458, r = 0.9998, with a linear range of 0.40-3.00 μg. The regression equation for sucrose was y = 1.3406x + 1.4494, r = 0.9985, with a linear range of 0.90-6.74 μg. The regression equation for melibiose was y = 1.1856x + 1.2702, r = 0.9998, with a linear range of 0.10-0.78 μg. The regression equation for raffinose was y = 1.1771x + 1.314, r = 0.9996, with a linear range of 0.10-0.76 μg. The regression equation for mannotriose was y = 1.3915x + 1.1437, r = 0.9993, with a linear range of 0.78-5.88 μg. The regression equation of stachyose was y=1.3613x+1.3024, r=0.9988, and the linear range was 0.87-6.55 μg.
[0094] 2. Precision inspection
[0095] Accurately pipette 3.0 ml of methanol, add 4 ml of pure water and place it in a 10 ml stoppered volumetric flask. After the solution is allowed to stand and cool, accurately pipette 2.0 ml of Qingjingsan decoction (batch 20231114) which has been taken out of 4°C and placed at 25°C for 1 hour and placed in a volumetric flask, add pure water to the scale, mix well, pipette 1 ml and place it in a 1.5 ml centrifuge tube, centrifuge at 12000 r / min at room temperature for 10 min, and the supernatant is the test solution. According to the above chromatographic conditions and the detection conditions of the evaporative light scattering detector, the sample is injected continuously for 6 times with an injection volume of 8 μl, and the peak area is measured. The results showed that the RSDs of the fructose, glucose, sucrose, melibiose, raffinose, mannotriose and stachyose contents in Qingjingsan were 0.37%, 0.15%, 0.19%, 0.90%, 1.79%, 0.34% and 0.32%, respectively, all less than 2%, indicating that the instrument has good precision.
[0096] 3. Repeatability test
[0097] Six replicates of the same sample batch were prepared according to the test solution preparation method. Samples were injected continuously with an injection volume of 8 μl. The chromatographic conditions and evaporative light scattering detector (ELSD) were identical to those in Example 1. The RSDs for fructose, glucose, sucrose, melibiose, raffinose, mannotriose, and stachyose were 1.36%, 1.01%, 0.53%, 1.22%, 1.38%, 0.78%, and 1.47%, respectively, all less than 1.5%, demonstrating good reproducibility of this method.
[0098] 4. Stability inspection
[0099] The same test solution was taken and measured within 0, 3, 6, 12, 18, and 24 hours according to the chromatographic conditions of Example 1 and the detection conditions of the evaporative light scattering detector. The RSDs of the fructose, glucose, sucrose, melibiose, raffinose, mannotriose, and stachyose contents were 1.58%, 1.11%, 0.73%, 1.32%, 2.03%, 1.27%, and 1.49%, respectively, all of which were less than 2.5%, indicating that the test solution was stable within 24 hours.
[0100] 5. Sample recovery rate
[0101] Accurately pipette 1.5 ml of methanol, add 2 ml of pure water and place it in a 5 ml stoppered measuring flask. After the solution is allowed to stand and cool, accurately pipette 0.5 ml of the sample into a measuring flask. Pipette 0.5 ml of the mixed reference stock solution containing 1.4995 mg of fructose, 0.9960 mg of glucose, 2.2994 mg of sucrose, 0.1784 mg of melibiose, 0.1675 mg of raffinose, 1.9571 mg of mannotriose, and 2.0916 mg of stachyose per 1 mL into the same measuring flask. Repeat 6 times. According to the chromatographic conditions of Example 1 and the detection conditions of the evaporative light scattering detector, the sample was injected and measured, and the recovery was calculated. The fructose recovery rates were 97.2%, 100.7%, 102.0%, 102.6%, 104.6%, 105.2%, with an average of 102.0%; the glucose recovery rates were 98.0%, 99.1%, 99.6%, 99.6%, 100.4%, 101.5%, with an average of 99.7%; the sucrose recovery rates were 102.8%, 101.4%, 99.0%, 101.0%, 100.7%, 99.1%, with an average of 100.7%; the melibiose recovery rates were 94.2%, 100.4%, 95.5%, 10 The recovery rates of raffinose were 103.0%, 98.7%, 96.8%, 102.2%, 96.4%, 102.5%, with an average of 99.9%. The recovery rates of mannotriose were 96.6%, 99.6%, 101.1%, 101.2%, 104.0%, 104.2%, with an average of 100.7%. The recovery rates of stachyose were 99.2%, 99.3%, 101.0%, 102.4%, 102.6%, 103.1%, with an average of 101.3%.
[0102] Comparative Example 1
[0103] In this embodiment, the mobile phase was isocratic, 79% acetonitrile (containing 0.05% ammonia water), and the reaction time was 40 min. The remaining parameters and conditions were the same as those in Example 1. The chromatographic results obtained showed that only fructose, glucose, and sucrose peaks appeared.
[0104] Comparative Example 2
[0105] In this embodiment, the mobile phase B is increased to 81% in the gradient, and the other parameters and conditions are the same as those in Example 1. In the obtained chromatographic results, no stachyose peak is observed.
[0106] The gradient is as follows:
[0107] Time / min <![CDATA[Flow rate / mL·min -1 > A / ％ B / ％ 0 0.8 19 81 6 0.8 21 79 10 0.8 22 78 23 0.8 28 72 32 0.8 37 63 35 0.8 19 81 40 0.8 19 81 .
Claims
1. A method for simultaneous determination of sugar content, characterized in that: The assay method comprises the following steps: using HPLC-ELSD to detect a test solution, wherein the test solution contains D-fructose, D-glucose, sucrose, melibiose, raffinose, mannotriose and stachyose; The liquid chromatography detection conditions are as follows: the chromatographic column is Waters XBridge Amide or Rocksil Carbohydrate ES; the mobile phases are mobile phase A and mobile phase B, mobile phase A is 0-0.05% ammonia water, and mobile phase B is 0-0.05% ammonia water-acetonitrile; based on the total volume of the mobile phase as 100%, the gradient elution program is shown in the following table: The elution time is the difference between the elution end time and the elution start time of the elution stage: x1 is 22%-31% (22%, 30%), x2 is 28%-32% (30%, 30.5%), x3 is 30%-33% (30.5%, 31.5%), x4 is 30%-35% (31.5%, 33%), x5 is 35%-39% (36%, 37%), x6 is 28%-31% (29%, 30%), Among them, x1 <x2<x3<x4<x5,x5> x6.
2. The method for simultaneous determination of carbohydrate content according to claim 1, wherein: The mobile phase A is 0.05% ammonia water; And / or, the mobile phase B is 0.05% ammonia water-acetonitrile.
3. The method for simultaneous determination of carbohydrate content according to claim 1, wherein: The liquid chromatography detection conditions include a flow rate of 0.5-0.6 mL / min; And / or, the column temperature is 20-40°C; preferably, the column temperature is 35°C.
4. The method for simultaneous determination of carbohydrate content according to claim 1, wherein: The ELSD parameters are as follows: gas pressure is 40 psi-60 psi; drift tube temperature is 80° C.-100° C.
5. The method for simultaneous determination of carbohydrate content according to claim 4, wherein: The ELSD parameters were as follows: the gas pressure was 60 psi; the drift tube temperature was 100°C.
6. The method for simultaneous determination of carbohydrate content according to claim 1, wherein: The procedure of the gradient elution is shown in the following table: 。 7. The method for simultaneous determination of carbohydrate content according to claim 1, wherein: The test sample is the water decoction of Qingjing San.
8. The method for simultaneous determination of carbohydrate content according to claim 7, wherein: The preparation method of the water decoction of Qingjing Powder comprises the following steps: weighing each raw material according to the formula of Qingjing Powder, soaking with water, decocting, and filtering. Preferably, the preparation method of the water decoction of Qingjing Powder comprises the following steps: weighing each raw material according to the formula of Qingjing Powder, adding water 5-15 times the amount of the raw material, soaking for 5-10 hours, boiling and then decocting with low heat (100-300W) for 20-60 minutes, filtering, adding water 5-10 times the amount of the filter residue, boiling and then decocting with low heat (100-300W) for 10-60 minutes, filtering, standing and cooling, and diluting the decoction to 1000ml.
9. The method for simultaneous determination of carbohydrate content according to claim 8, characterized in that: The Qingjing Powder comprises the following raw materials in parts by weight: 8-12 parts of peony bark, 16-20 parts of rehmannia root bark, 8-12 parts of white peony root wine, 8-12 parts of prepared rehmannia root, 5-8 parts of artemisia annua, 2-4 parts of poria and 1-2 parts of salted phellodendron.
10. The method for simultaneous determination of carbohydrate content according to claim 7, characterized in that: The preparation method of the test solution comprises the following steps: diluting the Qingjingsan decoction with a 0%-50% methanol aqueous solution. Preferably, the test solution Qingjingsan decoction is diluted with a 30% methanol aqueous solution.