A method for simultaneous determination of 12 active ingredients and 2 illegal additives in Qingre Jianwei powder

Abstract

The present invention relates to a method for simultaneously determining 12 active ingredients and 2 illegal additives in a Qingre Jianwei powder, and belongs to the field of effective ingredient detection in veterinary drugs. The steps are: (1) weighing the standard products of various active ingredients respectively and configuring them with an organic solvent to form a mixed reference stock solution containing all the standard products at the same time; separately preparing a mixed reference stock solution of olaquindox and mequindox (2) to prepare a test solution; (3) using an ultra-high performance liquid chromatograph to detect the mixed reference stock solution and the test solution respectively. The method of the present invention is simple and rapid, has good separation effect, high determination accuracy, good result reproducibility, is easy to observe, has strong specificity, and has a good chromatographic peak shape; in addition, the present invention greatly increases the detection index, improves the inspection efficiency, detection sensitivity and inspection effect.

Description

Technical Field

[0001] The invention relates to the field of detection of active ingredients in veterinary drugs, and in particular to a method for simultaneously determining 12 active ingredients and 2 illegal additives in Qingre Jianwei powder. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Qingre Jianwei Powder is a Chinese veterinary medicine formula, which is included in the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China, Part II: Quality Standards for Qingre Jianwei Powder. In the quality standards for Qingre Jianwei Powder, hawthorn, phellodendron, rhubarb, malt, tangerine peel, magnolia bark, and rhubarb are microscopically identified; rhubarb is identified by thin layer, but there is no content determination item; the pre-treatment operation of rhubarb thin layer identification is very cumbersome. This national standard does not currently have a detection method for illegal additives.

[0004] Furthermore, in the quality standard of Qingre Jianwei Powder, the thin layer chromatography detection method used for rhubarb has complicated preparations in the early stage of thin layer chromatography detection, and multiple steps such as unfolding and drying, checking and checking the results are required, which is time-consuming and laborious; the fluorescent spots of the test results are greatly disturbed by factors such as environmental temperature and humidity, human operation, and edge effect, and the spots are often unclear, the results are difficult to judge, and the reproducibility is poor; and the entire detection process is time-consuming, reagent-consuming, and cumbersome to operate. In addition, in actual inspection work, the pre-treatment solvent and the developing agent used for chromatography are highly toxic, such as ether, petroleum ether, ethyl formate, formic acid, etc., which are relatively harmful to the experimenters. Therefore, a new Qingre Jianwei Powder detection method is urgently needed to overcome the above problems. Summary of the invention

[0005] In view of the problems that the identification and determination process of the active ingredients in Qingre Jianwei Powder in the Chinese Veterinary Pharmacopoeia is time-consuming, reagent-consuming, has poor reproducibility of results, and affects the safety of testers, the present invention proposes a method for simultaneously determining 12 active ingredients and 2 illegal additives in Qingre Jianwei Powder.

[0006] Qingre Jianwei San is a Chinese veterinary medicine formula, which is included in the quality standard of Qingre Jianwei San in the 2020 edition of the Chinese Veterinary Pharmacopoeia. The content of the national standard is as follows:

[0007] Heat-clearing and stomach-strengthening powder:

[0008] [Prescription] Gentian 30g Phellodendron 30g Anemarrhena 20g Tangerine peel 25g Magnolia bark 20g Rhubarb 20g Hawthorn 20g Liushenqu 20g Malt 30g Sodium bicarbonate 50g; [Preparation] The above 10 ingredients, except sodium bicarbonate, are crushed into powder, added with sodium bicarbonate, sieved, and mixed to obtain. [Properties] This product is a light yellow powder; fragrant, bitter taste.

[0009] Microscopic identification:

[0010] 1. Hawthorn: The stone cells in the pericarp are purplish red, red or yellow-brown, round or polygonal, and contain red-brown substances in the cell cavity, with a diameter of about 125μm.

[0011] 2. Phellodendron chinense: The fiber bundles are bright yellow, and the surrounding cells contain prismatic crystals of calcium oxalate, forming crystalline fibers. The walls of the crystalline cells are lignified and thickened.

[0012] 3. Rhubarb: Calcium oxalate clusters are large, with a diameter of 60 to 140 μm.

[0013] 4. Malt: The pericarp cells are arranged in vertical rows, often with one long cell connected alternately with two short cells. The long cell walls are thick, wavy, and lignified.

[0014] 5. Tangerine peel: Calcium oxalate exists in the form of prismatic flakes in the parenchyma.

[0015] 6. Magnolia officinalis: The stone cells are branched, with thick walls and obvious stratification.

[0016] 7. Anemarrhena asphodeloides: Calcium oxalate needles are in bundles or scattered, 26 to 110 μm long.

[0017] Thin layer chromatography identification:

[0018] Take 2g of this product, add 20ml of methanol, soak overnight, filter, take 5ml of filtrate, evaporate to dryness, add 10ml of water to the residue to dissolve, add 1ml of hydrochloric acid, heat in a water bath for 10 minutes, cool immediately, extract with ether twice, 20ml each time, combine the ether solution, evaporate to dryness, add 1ml of chloroform to the residue to dissolve, as the test solution. Take 0.1g of rhubarb control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography test, take 5μl of each of the above two solutions, and spot them on the same silica gel H thin layer plate with sodium carboxymethyl cellulose as the adhesive, and use the upper layer solution of petroleum ether (30-60℃)-ethyl formate-formic acid (15:5:1) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding positions in the chromatogram of the control medicinal material, the same five orange-yellow fluorescent spots were placed in ammonia vapor and then examined under a fluorescent light, and the spots turned red.

[0019]

Function

Indications

[0020] The technical solution of the present invention is as follows:

[0021] A method for simultaneously determining 12 active ingredients and 2 illegal additives in Qingre Jianwei powder comprises the following steps:

[0022] (1) Weighing the standard samples of various active ingredients respectively and preparing them with an organic solvent to prepare a mixed reference stock solution containing all the standard samples; separately preparing a mixed reference stock solution of olaquindox and mequindox;

[0023] Preferably, the organic solvent is any one of methanol, ethanol or chloroform.

[0024] The 12 active ingredients are: chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, palmatine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether, honokiol, and magnolol;

[0025] The two illegal additives are quinolone and mequindox;

[0026] (2) using an extractant to ultrasonically extract the Qingre Jianwei powder, and then centrifuging and filtering to obtain a test solution;

[0027] Preferably, the extractant is any one selected from methanol, anhydrous ethanol, 50% by volume methanol, 50% by volume ethanol or 70% by volume ethanol; more preferably, the extractant is 70% by volume ethanol.

[0028] Preferably, the volume ratio of the Qingre Jianwei powder to the extractant is 1:25-100; the extraction condition is ultrasonic extraction, and the extraction time is 15-25 minutes. The ultrasonic extraction method can effectively improve the extraction efficiency and extraction effect.

[0029] Preferably, the centrifugal conditions are: a rotation speed of 10000-15000 rpm / min, and a centrifugal time of 15-25 min; more preferably, the centrifugal rotation speed is 12000 rpm / min, and the centrifugal time is 20 min.

[0030] (3) using ultra-high performance liquid chromatography to detect the mixed control stock solution and the test solution respectively, qualitatively analyzing the 12 active ingredients in the test solution by chromatogram and retention time, and calculating the contents of the 12 active ingredients in the test solution by chromatographic peak area;

[0031] Chromatographic conditions: mobile phase A is acetonitrile + 0.2% phosphoric acid, mobile phase B is 0.2% phosphoric acid aqueous solution, column temperature is 39-41°C; mobile phase flow rate: 0.35mL / min; injection volume: 0.5-5μL; chromatographic column is waters UPLC HSS T3 Chromatographic columns;

[0032] Detection conditions: PDA detector, 3D scanning wavelength range 190-400nm; detection wavelengths are 225nm, 228nm, 251nm, 240nm, 264nm, 289nm, 292nm, 327nm, 346nm and other multi-channels.

[0033] Preferably, the detection wavelength is 228 nm, 264 nm, or 243 nm.

[0034] Preferably, the column temperature is 40°C and the injection volume is 1-2 μL.

[0035] Furthermore, the gradient elution program is as follows: from 0 to 4 min, the volume fraction of mobile phase A is maintained at 5%; from 4 to 8 min, the volume fraction of mobile phase A is changed from 5% to 10%; from 8 to 12 min, the volume fraction of mobile phase A is maintained at 10%; from 12 to 16 min, the volume fraction of mobile phase A is changed from 10% to 15%; from 16 to 20 min, the volume fraction of mobile phase A is maintained at 15%; from 20 to 25 min, the volume fraction of mobile phase A is changed from 15% to 20%; from 25 to 30 min, the volume fraction of mobile phase A is maintained at 20 %; 30-32min, the volume fraction of mobile phase A changes from 20% to 40%; 32-36min, the volume fraction of mobile phase A changes from 40% to 60%; 36-40min, the volume fraction of mobile phase A is maintained at 60%; 40-43min, the volume fraction of mobile phase A changes from 60% to 85%; 43-45min, the volume fraction of mobile phase A is maintained at 85%; 45-47min, the volume fraction of mobile phase A changes from 85% to 5%; 47-50min, the volume fraction of mobile phase A is maintained at 5%.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The invention uses ultra-high performance liquid chromatography to simultaneously measure multiple main components in six Chinese medicine components, including hawthorn (chlorogenic acid), gentian (gentiopicroside), phellodendron (berberine hydrochloride), tangerine peel (hesperidin), rhubarb (rhubarb, rhein, aloe-rhubarb, chrysophanol, physophanol methyl ether), and magnolia bark (honokiol, magnolol) in Qingre Jianwei powder. The method is simple and rapid, has good separation effect, high determination accuracy, good result reproducibility, is easy to observe, has strong specificity, and has good chromatographic peak shape. The method meets the requirements of the Chinese Pharmacopoeia of Veterinary Medicine and saves time and reagents. In addition, in the detection process of the sample for random inspection, the illegal additives olaquindox and mequindox are simultaneously found through the optimization of gradient elution and the screening of multi-channel detection wavelength.

[0038] The determination method of the present invention can detect high and low responses of various compounds through comparison of various solvents such as methanol, ethanol, 50% methanol, 50% ethanol, 70% ethanol, etc. Since methanol is more toxic than ethanol, 70% ethanol is finally selected as the extraction solvent, which takes into account the solubility characteristics of many compounds and the response values ​​of multiple compounds, extracts the test sample, uses a solvent with low toxicity, and has a moderate peak area response value of the compound. The sample is easy to handle, and the use of a large amount of organic solvents during thin layer detection is reduced. The operation is safe and environmentally friendly, and the harm to humans and the environment is small.

[0039] The determination method of the present invention uses an ultra-high performance liquid chromatograph in conjunction with a PDA detector to present the results in the form of chromatograms and spectrograms, which is convenient and fast, the results are intuitive, easy to judge, and have a low detection limit, which greatly shortens the inspection time (from sample processing to machine inspection and result determination, the inspection can be completed in about 2-3 hours, while thin layer inspection and microscopic inspection methods require about 18-24 hours for one sample), greatly increases the detection indicators, and improves the inspection efficiency, detection sensitivity and inspection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0041] Figure 1 The spectrum of the mixed reference substance in Example 1 of the present invention is as follows: 8.276min chlorogenic acid, 9.978min gentiopicroside, 24.468min hesperidin, 29.925min berberine hydrochloride, 30.520min palmatine hydrochloride, 34.064min aloe-emodin; 34.478min rhein, 36.306min emodin, 36.575min honokiol, 37.526min magnolol, 38.344min chrysophanol, 39.968min physophanol methyl ether;

[0042] Figure 2 The spectrogram of olaquindox and mequindox in Example 1 of the present invention; wherein, olaquindox is 8.715min, and mequindox is 11.682min;

[0043] Figure 3 The spectrogram of olaquindox and mequindox in Example 2 of the present invention; wherein, olaquindox is 8.128min, and mequindox is 11.097min;

[0044] Figure 4The spectrum of the mixed reference substance in Example 2 of the present invention is as follows: 7.918min chlorogenic acid, 9.525min gentiopicroside, 17.373min hesperidin, 24.510min berberine hydrochloride, 24.880min palmatine hydrochloride, 32.742min aloe-emodin; 33.124min rhein, 34.660min emodin, 34.860min honokiol, 35.635min magnolol, 36.255min chrysophanol, 37.089min physophanol methyl ether;

[0045] Figure 5 This is the mixed control chromatogram of Qingre Jianwei Powder at 228 nm in Example 1 of the present invention;

[0046] Figure 6 This is the mixed control chromatogram of Qingre Jianwei Powder at 243 nm in Example 1 of the present invention;

[0047] Figure 7 The mixed control chromatogram of Qingre Jianwei Powder at 264 nm in Example 1 of the present invention;

[0048] Figure 8 The 273nm Qingre Jianwei powder mixed control chromatogram in Example 1 of the present invention;

[0049] Fig. 9 This is the mixed control chromatogram of Qingre Jianwei Powder at 284 nm in Example 1 of the present invention;

[0050] Fig.10 The mixed control chromatogram of Qingre Jianwei Powder at 292 nm in Example 1 of the present invention;

[0051] Fig.11 This is the 327nm Qingre Jianwei powder mixed control chromatogram in Example 1 of the present invention;

[0052] Fig.12 This is the 346nm Qingre Jianwei powder mixed control chromatogram in Example 1 of the present invention;

[0053] Fig.13 The 228 nm chromatogram of the Qingre Jianwei powder sample in Example 1 of the present invention;

[0054] Fig.14 The 243nm chromatogram of the Qingre Jianwei powder sample in Example 1 of the present invention;

[0055] Fig.15 The chromatogram of the Qingre Jianwei powder sample at 264 nm in Example 1 of the present invention;

[0056] Fig.16 The chromatogram of the Qingre Jianwei powder sample at 273 nm in Example 1 of the present invention;

[0057] Fig.17 The chromatogram of the Qingre Jianwei powder sample at 284 nm in Example 1 of the present invention;

[0058] Fig.18 The chromatogram of the Qingre Jianwei powder sample at 292 nm in Example 1 of the present invention;

[0059] Fig.19 The 327nm chromatogram of the Qingre Jianwei powder sample in Example 1 of the present invention;

[0060] Fig. 20 The 346nm chromatogram of the Qingre Jianwei powder sample in Example 1 of the present invention;

[0061] Fig.21 The chromatogram of the Qingre Jianwei powder sample at 289 nm in Example 2 of the present invention;

[0062] Fig. 22 This is the 278nm chromatogram of the Qingre Jianwei Powder sample in Example 2 of the present invention. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0064] Example 1: A method for simultaneously determining 12 active ingredients and two illegal additives in Qingre Jianwei powder

[0065] Test samples: samples submitted for approval by enterprises. The test samples in this embodiment were supplied by Shandong Xundakang Veterinary Drug Co., Ltd.

[0066] Phosphoric acid was of premium grade, ethanol and methanol were of chromatographic grade, and reference substances were mainly purchased from China Veterinary Drug Administration, China Food and Drug Inspection Institute, China Institute of Biological Products, and Dr.E.

[0067] Instruments used: BP211D analytical balance (Sartorius, Germany); Waters Acquity TM Ultra-high performance liquid chromatograph (Waters, USA), PDA detector, chromatographic column: Waters UPLC HSS T 3 Chromatographic column (2.1 mm × 100 mm, 1.8 μm).

[0068] The 12 active ingredients are: chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether, magnolol, magnolol. The two illegal additives are olaquindox and mequindox.

[0069] The determination method comprises the following steps:

[0070] a. Preparation of control stock solution and mixed control stock solution

[0071] Chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether, magnolol, magnolol, olaquindox and mequindox reference substances were taken respectively, dissolved and fixed to volume with appropriate concentrations of ethanol, chloroform or methanol and other suitable solvents, and prepared into respective control stock solutions; appropriate volumes of each control stock solution were mixed to obtain a mixed control stock solution, as shown in Tables 1 and 2.

[0072] b. Prepare the sample solution to be tested: accurately measure 0.2 g of the Qingre Jianwei Powder test sample, place it in a 10 mL volumetric flask, dissolve it with an appropriate solvent and make up the volume to the mark, weigh it, perform ultrasonic extraction for 20 min, weigh it, make up the reduced weight with an appropriate solvent, accurately measure 5 mL of it, centrifuge it at 12000 rpm / min for 20 min, filter it, and use the filtrate for ultra-high performance liquid chromatography determination.

[0073] Table 1 Configuration information of 5 kinds of rhubarb reference substances

[0074]

[0075]

[0076] Table 2 Configuration information of 7 reference substances

[0077] Reference substance name Sample weight / mg content Fixed volume / mL Solvents Concentration of reference solution / μg / mL Gentiopicroside 9.22 0.969 10 Methanol 893.42 Berberine Hydrochloride 11.69 0.867 50 Methanol 202.70 Bamipine Hydrochloride 10.38 0.862 100 Methanol 89.48 Hesperidin 9.55 0.962 50 Methanol 183.74 Magnolia 15.81 0.998 50 Methanol 315.57 Honokiol 12.15 0.988 50 Methanol 240.08 Chlorogenic acid 8.68 0.973 50 Ethanol 168.91

[0078] Take 4 ml of the mixed reference solution of 5 kinds of rhubarb, add appropriate amount of other 7 reference solutions, mix evenly, and obtain the final mixed reference solution, see Table 3; the configuration information of the 2 illegal additive reference solutions is shown in Table 4.

[0079] Table 3 Mixed reference material configuration information

[0080]

[0081] Table 4 Configuration information of two reference substances containing illegal additives

[0082] Reference substance name Sample weight / mg content Fixed volume / mL Solvents Concentration of reference solution / μg / mL Olaquinoxaline 10.32 0.969 50 Methanol 200.0 Mequindox 10.08 0.996 50 Methanol 200.8

[0083] c. The instrument used was an ultra-high performance liquid chromatograph, the detector was a photodiode array detector, and the chromatographic column was waters UPLC HSS T 3 Chromatographic column, column temperature is 39-41°C, 228nm, 264nm, 243nm are optimally selected as the detection wavelengths of 12 active ingredients, mobile phase A is acetonitrile (containing 0.2% phosphoric acid), mobile phase B is 0.2% phosphoric acid aqueous solution; flow rate: 0.35mL / min; injection volume: 0.5-5μL;

[0084] The PDA detector can collect multiple wavelengths at the same time. Through multi-channel scanning, channels such as 225nm, 228nm, 243nm, 264nm, 273nm, 284nm, 292nm, 327nm, 364nm, etc. are selected. After comparing the detection wavelengths, the chromatographic peaks of the 12 compounds at 225nm, 228nm, and 243nm have good response and good separation. The mobile phase gradient elution program is shown in Table 5:

[0085] Table 5 Mobile phase gradient elution program

[0086] Flow rate Time (min) %A phase %B phase curve 0.35ml / min initial 5.0 95.0 6 0.35ml / min 4.00 5.0 95.0 6 0.35ml / min 8.00 10.0 90.0 6 0.35ml / min 12.00 10.0 90.0 6 0.35ml / min 16.00 15.0 85.0 6 0.35ml / min 20.00 15.0 85.0 6 0.35ml / min 25.00 20.0 80.0 6 0.35ml / min 30.00 20.0 80.0 6 0.35ml / min 32.00 40.0 60.0 6 0.35ml / min 36.00 60.0 40.0 6 0.35ml / min 40.00 60.0 40.0 6 0.35ml / min 43.00 85.0 15.0 6 0.35ml / min 45.00 85.0 15.0 6 0.35ml / min 47.00 5.0 95.0 6 0.35ml / min 50.00 5.0 95.0 6

[0087] The gradient elution program was as follows: 0-4min, the volume fraction of mobile phase A was maintained at 5%; 4-8min, the volume fraction of mobile phase A was changed from 5% to 10%; 8-12min, the volume fraction of mobile phase A was maintained at 10%; 12-16min, the volume fraction of mobile phase A was changed from 10% to 15%; 16-20min, the volume fraction of mobile phase A was maintained at 15%; 20-25min, the volume of mobile phase A was changed from 15% to 20%; 25-30min, the volume fraction of mobile phase A was maintained at 20%; 3 0-32min, the volume fraction of mobile phase A changed from 20% to 40%; 32-36min, the volume fraction of mobile phase A changed from 40% to 60%; 36-40min, the volume fraction of mobile phase A was maintained at 60%; 40-43min, the volume fraction of mobile phase A changed from 60% to 85%; 43-45min, the volume fraction of mobile phase A was maintained at 85%; 45-47min, the volume fraction of mobile phase A changed from 85% to 5%; 47-50min, the volume fraction of mobile phase A was maintained at 5%. 12st control, chromatographic data and injection volume are shown in Table 6:

[0088] Table 6 12st control chromatographic data injection volume (1μL)

[0089]

[0090]

[0091]

[0092] Table 7 Chromatographic results of Qingre Jianwei powder sample data Injection volume (1 μL)

[0093]

[0094]

[0095]

[0096]

[0097] d. Comparison of different injection volumes: 0.5 μL, 1 μL, and 2 μL of the mixed control stock solution and the sample to be tested were injected into the ultra-high performance liquid chromatograph. The chromatographic responses were all good. Due to space limitations, only the results of 1-2 μL were listed. Gradient elution and detection were performed under the chromatographic conditions of step c, and qualitative identification was performed based on retention time and spectrum. Figure 1 As shown; the chromatographic parameter information such as reference and sample injection volume, peak area, theoretical plate number, separation degree, etc. are shown in Tables 6 to 7.

[0098] In the early pre-treatment process, the number of chromatographic peaks detected and the area of ​​chromatographic peaks were different when different concentrations of methanol and ethanol solvents were used for extraction. Due to the high toxicity of methanol, 70% ethanol was finally used as the solvent for extraction. From the data, it can be seen that the amount of bamipine hydrochloride detected in the powder was very small and there was no chromatographic response; only 13 compounds could be detected.

[0099] e. Result analysis: Inject the filtrate in step b into an ultra-high performance liquid chromatograph, perform gradient elution and detection under the chromatographic conditions of step c, and measure the peak area of ​​each target in the filtrate, and characterize it by retention time and spectrum. According to the chromatogram, the same chromatographic peak appears at the corresponding position of the chromatogram of the sample to be tested with the chromatogram of the 12 active ingredient reference substances (except bamipine hydrochloride), and calculate the content of the active ingredient. Figure 5-20 As shown in the figure, the peak shape is good and the separation is acceptable, indicating that the target medicinal materials can be detected. In addition, during the spectral screening process, illegal additives quinolone and acetaminophen were found. See the spectrum Figure 2 .

[0100] We take the 243nm chromatographic data as an example to calculate the content of various compounds in the sample to be tested, see Table 8:

[0101] Table 8 Contents of various components in the tested samples of Qingre Jianwei Powder

[0102] Serial number Compound Name Content (μg / g) 1 Chlorogenic acid 193.98 2 Olaquinoxaline Detection, qualitative determination only 3 Gentiopicroside 112.76 4 Mequindox Detection, qualitative determination only 5 Hesperidin 2181.54 6 Berberine Hydrochloride 1048.49 7 Bamipine Hydrochloride 0.00 8 Aloe-emodin 532.30 9 Rhein 74.79 10 Rhein 316.50 11 Honokiol 246.23 12 Magnolia 144.34 13 Chrysophanol 53.68 14 Physcion 87.84

[0103] Embodiment 2:

[0104] The gradient elution procedure is different, and the other chromatographic conditions are consistent with the embodiment. The gradient elution procedure is 0-4min, the volume fraction of mobile phase A is maintained at 5%; 4-7min, the volume fraction of mobile phase A is changed from 5% to 10%; 7-10min, the volume fraction of mobile phase A is maintained at 10%; 10-12min, the volume fraction of mobile phase A is changed from 10% to 18%; 12-20min, the volume fraction of mobile phase A is maintained at 18%; 20-28min, the volume fraction of mobile phase A is maintained at 10%; The volume fraction of mobile phase A changed from 18% to 25% during the 28-30min period; the volume fraction of mobile phase A was maintained at 25% during the 30-32min period; the volume fraction of mobile phase A changed from 25% to 50% during the 32-37min period; the volume fraction of mobile phase A changed from 50% to 75% during the 37-40min period; the volume fraction of mobile phase A changed from 75% to 5% during the 40-43min period; the volume fraction of mobile phase A was maintained at 5% during the 43-45min period. The chromatographic data table of the mixed reference substance is shown in Table 9, the multi-channel chromatographic data table of the active ingredients of the sample is shown in Table 10, and the detection results of illegal additives of olaquindox and mequindox are shown in Table 11:

[0105] Table 9 Mixed reference substance chromatographic data table Injection volume 1 μL

[0106]

[0107]

[0108]

[0109] Table 10 Multi-channel chromatography data of active ingredients in samples Injection volume 2 μL

[0110]

[0111]

[0112]

[0113] Table 11 Detection results of illegal additives of quinolone and mequindox in sample injection 2 μL

[0114]

[0115] In Example 2, the entire running time is shortened and the detection efficiency is improved, but the matrix interference is relatively larger than that in Example 1, and the separation is relatively smaller. If there is matrix interference at the same retention time as the reference substance, we can use the spectrum to determine whether it is the target compound. The spectrum of the illegal additives quinolone and mequindox in this example is as follows: Figure 3 As shown, the spectra of 12 mixed reference substances are as follows Figure 4 As shown in the figure, due to space limitations, some of the samples to be tested are listed as follows Figure 21-22 shown.

[0116] Under the existing 2020 edition of the national veterinary drug quality standards, it is difficult to control the quality of Qingre Jianwei Powder by thin layer determination. The use of chromatographic parameters such as the retention time peak area of ​​the spectrum of the present invention can be used for accurate and rapid qualitative and quantitative determination; the content of various compounds in Qingre Jianwei Powder can be easily quantified by the ratio of the peak area of ​​the reference substance and the sample to be tested. Several high content indicators such as "hesperidin, berberine hydrochloride, aloe-emodin, emodin, and magnolol" can be determined, and fingerprint characteristic peaks can also be specified for identification, laying a solid foundation for the formulation of the 2025 national veterinary pharmacopoeia quality standards.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may be modified and varied in various ways. Any modification, equivalent replacement, improvement, etc. made by slightly adjusting the gradient elution, flow rate, column temperature, detection wavelength, etc., within the spirit and principle of the present invention, using different brands of liquid phase instruments, different brands of chromatographic columns with similar fillers, similar mobile phases, etc., shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining 12 active ingredients and 2 illegal additives in Qingre Jianwei Powder, comprising the following steps: (1) Weigh the standard samples of various active ingredients separately and prepare them with organic solvents to prepare mixed reference stock solutions containing all the standard samples; separately prepare mixed reference stock solutions of olaquindox and mequindox; The 12 active ingredients are: chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, palmatine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether, honokiol, and magnolol; The two illegal additives are quinolone and mequindox; (2) Using an extractant to ultrasonically extract Qingre Jianwei Powder, and then centrifuging and filtering to obtain a test solution; (3) The mixed control stock solution and the test solution were tested by ultra-high performance liquid chromatography, and the 12 active ingredients and 2 illegal additives in the test solution were qualitatively analyzed by chromatogram and retention time. The contents of the 12 active ingredients in the test solution were calculated by chromatographic peak area; The chromatographic conditions were as follows: mobile phase A was acetonitrile + 0.2% phosphoric acid, mobile phase B was 0.2% phosphoric acid aqueous solution, column temperature was 39-41°C; flow rate of mobile phase was 0.35 mL / min; injection volume was 0.5-5 μL; the chromatographic column was a waters UPLC HSS T3 column; Detection conditions: PDA detector, 3D scanning wavelength range 190-400nm; detection wavelengths 228nm, 264nm, 243nm; gradient elution program: From 0 to 4 min, the volume fraction of mobile phase A was maintained at 5%; 4-8min, the volume fraction of mobile phase A changed from 5% to 10%; 8-12min, the volume fraction of mobile phase A was maintained at 10%; 12-16 min, the volume fraction of mobile phase A changed from 10% to 15%; 16-20min, the volume fraction of mobile phase A was maintained at 15%; 20-25min, the volume of mobile phase A was changed from 15% to 20%; 25-30 min, the volume fraction of mobile phase A was maintained at 20%; 30-32 min, the volume fraction of mobile phase A changed from 20% to 40%; 32-36 min, the volume fraction of mobile phase A changed from 40% to 60%; 36-40 min, the volume fraction of mobile phase A was maintained at 60%; At 40-43 min, the volume fraction of mobile phase A changed from 60% to 85%; 43-45min, the volume fraction of mobile phase A was maintained at 85%; At 45-47 min, the volume fraction of mobile phase A changed from 85% to 5%; 47-50min, the volume fraction of mobile phase A was maintained at 5%; In the step (2), the volume ratio of the Qingre Jianwei powder to the extractant is 1:25-100; and the ultrasonic extraction time is 15-25 minutes.

2. The measuring method according to claim 1, characterized in that The organic solvent in step (1) is any one of methanol, ethanol or chloroform.

3. The measuring method according to claim 1, characterized in that In step (2), the extractant is any one selected from methanol, anhydrous ethanol, 50% by volume methanol, 50% by volume ethanol or 70% by volume ethanol.

4. The measuring method according to claim 3, characterized in that The extractant in step (2) is 70% by volume ethanol.

5. The measuring method according to claim 1, characterized in that The centrifugation conditions in step (2) are: rotation speed 10000-15000 rpm, and centrifugation time 15-25 min.

6. The measuring method according to claim 5, characterized in that The centrifugal conditions in step (2) are: the centrifugal speed is 12000 rpm and the centrifugal time is 20 min.

7. The measuring method according to claim 1, characterized in that In step (3), the column temperature is 40°C and the injection volume is 1-2 μL.

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