CN116008429A - Simultaneous determination method for 12 active ingredients and 2 illegal additives in heat-clearing and stomach-invigorating powder - Google Patents
Simultaneous determination method for 12 active ingredients and 2 illegal additives in heat-clearing and stomach-invigorating powder Download PDFInfo
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Abstract
The invention relates to a method for simultaneously measuring 12 active ingredients and 2 illegal additives in heat-clearing and stomach-invigorating powder, belonging to the field of detection of active ingredients in veterinary medicines. The method comprises the following steps: (1) Respectively weighing standard substances of various active ingredients and preparing a mixed control stock solution containing the standard substances by using an organic solvent; in addition, a mixed reference substance stock solution (2) of olaquindox and mequindox is prepared separately to prepare a liquid to be tested; (3) And detecting the mixed control stock solution and the liquid to be detected by adopting an ultra-high performance liquid chromatograph. The method is simple, convenient and quick, has good separation effect, high measurement precision, good result reproducibility, easy observation, strong specificity and good chromatographic peak shape; in addition, the invention greatly increases the detection index, and improves the detection efficiency, the detection sensitivity and the detection effect.
Description
Technical Field
The invention relates to the field of detection of effective components in veterinary medicines, in particular to a simultaneous determination method for 12 active components and 2 illegal additives in heat-clearing and stomach-invigorating powder.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The heat-clearing and stomach-invigorating powder is a Chinese veterinary medicine prescription preparation and is received in the classical two parts of the veterinary medicine of the people's republic of 2020 edition: quality standard of the heat-clearing and stomach-invigorating powder. Microscopic identification is carried out on hawthorn, phellodendron, rhubarb, malt, dried orange peel, magnolia bark and rhizoma anemarrhenae in the quality standard of the heat-clearing and stomach-invigorating powder; carrying out thin-layer identification on rheum officinale, wherein no content measurement item exists; thin-layer identification of rheum officinale and very complicated pretreatment operation. The detection method of illegal additives by the national standard is not available for a while.
Furthermore, in the quality standard of the heat-clearing and stomach-invigorating powder, in the thin-layer chromatography detection method adopted for rheum officinale, the early detection stage preparation of the thin-layer chromatography is complex, and a plurality of steps such as unfolding, airing, checking and checking results are needed, so that time and labor are wasted; the fluorescence spots of the detection result are greatly interfered by the factors such as ambient temperature and humidity, manual operation, edge effect and the like, the spots are often unclear, the result is difficult to judge, and the reproducibility is poor; and the whole detection process is time-consuming, reagent-consuming and complex in operation. In addition, the pretreatment solvent and the developing agent for chromatography have high toxicity in practical test work, for example: diethyl ether, petroleum ether, ethyl formate, formic acid, etc., have relatively large damage to laboratory staff. Therefore, a new method for detecting the heat-clearing and stomach-invigorating powder is urgently needed to overcome the problems.
Disclosure of Invention
In view of the problems of time and cost waste reagents, poor result reproducibility, influence on the safety of detection personnel and the like in the identification and measurement process of active ingredients in the heat-clearing and stomach-invigorating powder in Chinese animal pharmacopoeia, the invention provides a simultaneous measurement method of 12 active ingredients and 2 illegal additives in the heat-clearing and stomach-invigorating powder.
The heat-clearing and stomach-invigorating powder is a Chinese veterinary medicine prescription preparation and is carried on the quality standard of the heat-clearing and stomach-invigorating powder in 2020 edition of Chinese animal pharmacopoeia. The national standard content is as follows:
heat-clearing and stomach-invigorating powder:
30g of gentian, 30g of phellodendron bark, 20g of rhizoma anemarrhenae, 25g of dried orange peel, 20g of magnolia officinalis, 20g of rheum officinale, 20g of hawthorn, 20g of medicated leaven, 30g of malt and 50g of sodium bicarbonate; the preparation method comprises pulverizing above 10 materials except sodium bicarbonate, pulverizing other 9 materials such as radix Gentianae into powder, adding sodium bicarbonate, sieving, and mixing. [ PROBLEMS ] the product is pale yellow powder; fragrant smell and bitter taste.
Microscopic identification:
1. hawthorn fruit: the peel stone cells are light purple red, red or yellow brown, round or polygonal, and the cells contain reddish brown matters with the diameter of about 125 mu m.
2. Cortex Phellodendri: the fiber bundle is bright yellow, surrounding cells contain calcium oxalate square crystals, crystal fibers are formed, and the walls of the crystal-containing cells are lignified and thickened.
3. Rhubarb: the calcium oxalate cluster crystal is large and has the diameter of 60-140 mu m.
4. Malt: the pericarp cell lines, usually 1 long cell and 2 short cells are connected, long cell wall thickness, wavy bending and lignification.
5. Dried orange peel: the calcium oxalate square crystal flakes exist in the parenchyma.
6. Cortex Magnoliae officinalis: the stone cells are branched, the wall thickness is thick, and the layering is obvious.
7. Rhizoma anemarrhenae: the calcium oxalate needle crystal is bundled or scattered and has the length of 26-110 mu m.
Identification by thin layer chromatography:
2g of the product is taken, 20ml of methanol is added, soaking overnight, filtering is carried out, 5ml of filtrate is taken, evaporation is carried out, 10ml of water is added into residues to dissolve the residues, 1ml of hydrochloric acid is added, heating is carried out on the residues on a water bath for 10 minutes, immediate cooling is carried out, diethyl ether is used for 2 times of extraction, 20ml of diethyl ether solution is used each time, diethyl ether solution is combined, evaporation is carried out, and 1ml of chloroform is added into residues to dissolve the residues to be used as a test sample solution. And preparing a reference medicinal material of rheum officinale 0.1g by the same method. According to a thin layer chromatography test, 5 mu l of each of the two solutions is absorbed and respectively spotted on a silica gel H thin layer plate with sodium carboxymethylcellulose as an adhesive, and an upper layer solution of petroleum ether (30-60 ℃) ethyl formate-formic acid (15:5:1) is taken as a developing agent for developing, taken out and dried, and then is inspected under an ultraviolet lamp (365 nm). In the chromatogram of the sample, five same orange fluorescent spots are fumigated in ammonia vapor at the corresponding positions of the chromatogram of the reference medicinal material, and the spots turn red when inspected under a fluorescent lamp.
[ FUNCTIONS ] is to clear heat, dry dampness and promote digestion. [ mainly used for treating stomach heat and food retention.
The technical scheme of the invention is as follows:
a method for simultaneously measuring 12 active ingredients and 2 illegal additives in a powder for clearing heat and invigorating stomach comprises the following steps:
(1) Respectively weighing standard substances of various active ingredients and preparing a mixed control stock solution containing the standard substances by using an organic solvent; separately preparing a mixed reference stock solution of olaquindox and mequindox;
preferably, the organic solvent is any one of methanol, ethanol or chloroform.
The 12 active ingredients are as follows: chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, palmatine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physcion, honokiol, magnolol;
the 2 illegal additives are olaquindox and mequindox;
(2) Carrying out ultrasonic extraction on the heat-clearing and stomach-invigorating powder by adopting an extractant, centrifuging and filtering to obtain a liquid to be detected;
preferably, the extractant is any one selected from methanol, absolute ethanol, 50% methanol by volume fraction, 50% ethanol by volume fraction or 70% ethanol by volume fraction; more preferably, the extractant is 70% ethanol by volume.
Preferably, the volume ratio of the heat-clearing and stomach-invigorating powder to the extractant is 1:25-100; the extraction condition is ultrasonic extraction, and the extraction time is 15-25min. The ultrasonic extraction mode can effectively improve the extraction efficiency and the extraction effect.
Preferably, the centrifugation conditions are: the rotation speed is 10000-15000rpm/min, and the centrifugation time is 15-25min; more preferably, the rotational speed of centrifugation is 12000rpm/min and the centrifugation time is 20min.
(3) Detecting the mixed control stock solution and the liquid to be detected by adopting an ultra-high performance liquid chromatograph, qualitatively analyzing 12 active ingredients in the liquid to be detected by using a chromatogram and retention time, and calculating the content of the 12 active ingredients in the liquid to be detected by using a chromatographic peak area;
the chromatographic conditions are as follows: the mobile phase A is acetonitrile plus 0.2 percent phosphoric acid, the mobile phase B is 0.2 percent phosphoric acid water solution, and the column temperature is 39-41 ℃; flow rate of mobile phase: 0.35mL/min; sample injection amount: 0.5-5. Mu.L; the chromatographic column is waters UPLC HSS T 3 A chromatographic column;
detection conditions: a PDA detector, 3D scanning wavelength range 190-400nm; the detection wavelength is 225nm, 228nm, 251nm, 240nm, 264nm,289nm,292nm, 227 nm,346nm and other multi-channels.
Preferably, the detection wavelength is 228nm, 264nm, 243nm.
Preferably, the column temperature is 40 ℃; the sample injection amount is 1-2 mu L.
Further, the gradient elution procedure is 0-4min, and the volume fraction of mobile phase A is maintained at 5%;4-8min, changing the volume fraction of the mobile phase A from 5% to 10%;8-12min, the volume fraction of the mobile phase A is maintained at 10%;12-16min, the volume fraction of the mobile phase A is changed from 10% to 15%;16-20min, the volume fraction of the mobile phase A is maintained at 15%;20-25min, the volume of the mobile phase A is changed from 15% to 20%;25-30min, and maintaining the volume fraction of the mobile phase A at 20%;30-32min, the volume fraction of the mobile phase A is changed from 20% to 40%;32-36min, the volume fraction of the mobile phase A is changed from 40% to 60%;36-40min, the volume fraction of the mobile phase A is maintained at 60%;40-43min, the volume fraction of the mobile phase A is changed from 60% to 85%;43-45min, the volume fraction of the mobile phase A is maintained at 85%;45-47min, the volume fraction of the mobile phase A is changed from 85% to 5%; and the volume fraction of the mobile phase A is maintained at 5% for 47-50 min.
Compared with the prior art, the invention has the following advantages:
the invention simultaneously measures a plurality of main components in 6 traditional Chinese medicine components such as heat-clearing stomach-invigorating hawthorn (chlorogenic acid), gentiopicroside, phellodendron bark (berberine hydrochloride), dried orange peel (hesperidin), rheum officinale (emodin, rhein, aloe-emodin, chrysophanol, physcion), magnolia officinalis (honokiol, magnolol) and the like by an ultra-high performance liquid chromatography, and has the advantages of simple, convenient and quick method, good separation effect, high measurement precision, good result reproducibility, easy observation, strong specificity and good chromatographic peak shape, thereby not only meeting the requirements of Chinese animal pharmacopoeia, but also saving time and reagents. In addition, illegal additives of olaquindox and mequindox are simultaneously discovered through gradient elution optimization and multichannel detection wavelength screening in the detection process of the spot check samples.
According to the determination method, through comparison of various solvents such as methanol, ethanol, 50% methanol, 50% ethanol, 70% ethanol and the like, the high and low uniformity of the responses of the compounds can be detected, and as the methanol toxicity is higher than that of the ethanol, the 70% ethanol is finally preferred as an extraction solvent, the dissolution characteristics of a plurality of compounds are considered, the response values of the compounds are considered, the samples are extracted, the toxicity of the used solvents is low, the peak area response value of the compounds is moderate, the sample treatment is simple and convenient, the use of a large amount of organic solvents in thin-layer detection is reduced, the operation is safe and environment-friendly, and the harm to people and the environment is small.
The determination method of the invention uses the ultra-high performance liquid chromatograph to match with the PDA detector, presents the result in a chromatogram and spectrogram mode, has the advantages of visual result, easy judgment and low detection limit, greatly shortens the detection time (from sample processing to on-machine detection judgment result, detection can be completed in about 2-3 hours, and the thin layer detection and microscopic detection method needs about 18-24 hours), greatly increases the detection index, and improves the detection efficiency, the detection sensitivity and the detection effect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a spectrum diagram of a mixed reference substance in example 1 of the present invention: 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 emodin, 39.968min physcion;
FIG. 2 is a spectrum of olaquindox and mequindox of example 1 of the present invention; wherein 8.715min of olaquindox and 11.682min of mequindox;
FIG. 3 is a spectrum of olaquindox and mequindox of example 2 of the present invention; wherein, 8.128min of olaquindox and 11.097min of mequindox;
FIG. 4 is a spectrum chart of the mixed reference substance in the embodiment 2 of the present invention: 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 emodin, 37.089min physcion;
FIG. 5 is a chromatogram of a 228nm powder mixture for clearing heat and invigorating stomach in example 1 of the present invention;
FIG. 6 is a chromatogram of a 243nm powder mixture for clearing heat and invigorating stomach in example 1 of the present invention;
FIG. 7 is a chromatogram of a 264nm heat-clearing and stomach-invigorating powder mixture in example 1 of the present invention;
FIG. 8 is a graph of a 273nm powder mixture for clearing heat and invigorating stomach according to example 1 of the present invention;
FIG. 9 is a chromatogram of a 284nm mixture of the heat-clearing and stomach-invigorating powder in example 1 of the present invention;
FIG. 10 is a comparative chromatogram of 292nm of the powder mixture for clearing heat and invigorating stomach in example 1 of the present invention;
FIG. 11 is a graph showing a mixed control chromatogram of 327nm heat-clearing and stomach-invigorating powder in example 1 of the present invention;
FIG. 12 is a chromatogram of a 346nm mixture of the powder for clearing heat and invigorating stomach in example 1 of the present invention;
FIG. 13 is a chromatogram of 228nm of the powder for clearing heat and invigorating stomach in example 1 of the present invention;
FIG. 14 is a chromatogram of 243nm powder for clearing heat and invigorating stomach in example 1 of the present invention;
FIG. 15 is a chromatogram of 264nm heat-clearing and stomach-invigorating powder sample in example 1 of the present invention;
FIG. 16 is a chromatogram of 273nm heat-clearing stomach-invigorating powder sample in example 1 of the present invention;
FIG. 17 is a chromatogram of 284nm of the powder for clearing heat and invigorating stomach in example 1 of the present invention;
FIG. 18 is a chromatogram of 292nm heat-clearing and stomach-invigorating powder sample in example 1 of the present invention;
FIG. 19 is a chromatogram of 327nm heat-clearing stomach-invigorating powder sample in example 1 of the present invention;
FIG. 20 is a chromatogram of 346nm heat-clearing stomach-invigorating powder sample in example 1 of the present invention;
FIG. 21 is a chromatogram of 289nm heat-clearing and stomach-invigorating powder sample in example 2 of the present invention;
FIG. 22 is a chromatogram of a 278nm powder sample for clearing heat and invigorating stomach in example 2 of the present invention.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. The embodiments are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1: method for simultaneously measuring 12 active ingredients and two illegal additives in heat-clearing and stomach-invigorating powder
Test article: for enterprise batch samples, the samples in this example were supplied by Shandong Xuedakang veterinary drug Co.
The phosphoric acid is of high-grade purity, ethanol and methanol chromatographic purity, and the reference substance is mainly purchased from China veterinary medicine institute, china food and drug inspection institute, china biological product institute and German Dr.E.
The instrument used is as follows: BP211D analytical balance (saidolis, 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. Times.100 mm,1.8 μm).
The 12 active ingredients are as follows: chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physcion, honokiol, magnolol. Two illegal additives are olaquindox and mequindox.
The measuring method comprises the following steps:
a. preparation of control stock solution and mixing control stock solution
Respectively taking chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physcion, honokiol, magnolol, olaquindox and mequindox reference substances, dissolving with proper solvents such as ethanol, chloroform or methanol with proper concentration, and fixing the volume to prepare respective reference stock solutions; the control stock solutions were mixed in appropriate volumes to give mixed control stock solutions, see in particular tables 1 and 2.
b. Preparing a sample solution to be tested: precisely measuring 0.2g of the heat-clearing and stomach-invigorating powder sample, placing in a 10mL volumetric flask, dissolving with a proper solvent, fixing the volume to a scale, weighing, ultrasonically extracting for 20min, weighing, supplementing the reduced weight with the proper solvent, precisely measuring 5mL of the powder sample from the powder sample, centrifuging for 20min at 12000rpm/min, filtering, and measuring the filtrate by ultra-high performance liquid chromatography.
Table 15 rhubarb reference substance configuration information
Table 27 control configuration information
| Reference sample name | Sample weighing/mg | Content of | Constant volume/mL | Solvent(s) | Control solution concentration/. Mu.g/mL |
| Gentiopicroside | 9.22 | 0.969 | 10 | Methanol | 893.42 |
| Berberine hydrochloride | 11.69 | 0.867 | 50 | Methanol | 202.70 |
| Palmatine hydrochloride | 10.38 | 0.862 | 100 | Methanol | 89.48 |
| Hesperidin | 9.55 | 0.962 | 50 | Methanol | 183.74 |
| Magnolol | 15.81 | 0.998 | 50 | Methanol | 315.57 |
| Honokiol of honokiol | 12.15 | 0.988 | 50 | Methanol | 240.08 |
| Chlorogenic acid | 8.68 | 0.973 | 50 | Ethanol | 168.91 |
Taking 4ml of 5 rheum officinale mixed reference substance solutions, adding a proper amount of other 7 reference substance solutions, and uniformly mixing to obtain a final mixed reference substance solution, wherein the final mixed reference substance solution is shown in Table 3;2 illegal additive control configuration information is shown in Table 4.
Table 3 mix control configuration information
Table 42 illegal additive control configuration information
| Reference sample name | Sample weighing/mg | Content of | Constant volume/mL | Solvent(s) | Control solution concentration/. Mu.g/mL |
| Olaquindox | 10.32 | 0.969 | 50 | Methanol | 200.0 |
| Mequindox | 10.08 | 0.996 | 50 | Methanol | 200.8 |
c. The instrument is ultra-high performance liquid chromatograph, the detector is photodiode array detector, and the chromatographic column is waters UPLC HSS T 3 The chromatographic column has the column temperature of 39-41 ℃, and the optimal detection wavelength of 228nm, 264nm and 243nm is selected as 12 effective components, wherein the mobile phase A is acetonitrile (containing 0.2% phosphoric acid), and the mobile phase B is 0.2% phosphoric acid aqueous solution; flow rate: 0.35mL/min; sample injection amount: 0.5-5. Mu.L;
the PDA detector is selected to collect multiple wavelengths simultaneously, channels of 225nm, 228nm, 243nm, 264nm, 273nm, 284nm, 292nm,327nm, 364nm and the like are selected in a multi-channel scanning mode, the detection wavelengths are compared, and 12 compounds are in 225nm, 228nm and 243nm positions, so that chromatographic peaks have good response and good separation degree. The mobile phase gradient elution procedure is shown in table 5:
TABLE 5 gradient elution procedure table for mobile phases
| Flow rate | Time (min) | % A phase | % B phase | Curve |
| 0.35ml/min | Initial initiation | 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 |
Gradient elution procedure was 0-4min, volume fraction of mobile phase a was maintained at 5%;4-8min, changing the volume fraction of the mobile phase A from 5% to 10%;8-12min, the volume fraction of the mobile phase A is maintained at 10%;12-16min, the volume fraction of the mobile phase A is changed from 10% to 15%;16-20min, the volume fraction of the mobile phase A is maintained at 15%;20-25min, the volume of the mobile phase A is changed from 15% to 20%;25-30min, and maintaining the volume fraction of the mobile phase A at 20%;30-32min, the volume fraction of the mobile phase A is changed from 20% to 40%;32-36min, the volume fraction of the mobile phase A is changed from 40% to 60%;36-40min, the volume fraction of the mobile phase A is maintained at 60%;40-43min, the volume fraction of the mobile phase A is changed from 60% to 85%;43-45min, the volume fraction of the mobile phase A is maintained at 85%;45-47min, the volume fraction of the mobile phase A is changed from 85% to 5%; and the volume fraction of the mobile phase A is maintained at 5% for 47-50 min. 12st control, chromatographic data and sample volume are shown in Table 6:
table 6 12st control chromatographic data sample injection volume (1. Mu.L)
TABLE 7 sample volume for data chromatography results of QINGRE JIANWEI powder (1. Mu.L)
d. Comparison of different sample injection amounts: mixing control stock solution and sample to be tested, taking 0.5 μl,1 μl and 2 μl, injecting into ultra-high performance liquid chromatograph, and performing gradient elution and detection under the chromatographic condition of step c with retention time and spectrogram qualitative identification as shown in figure 1, wherein chromatographic response is good, and the space limitation only lists 1-2 μl results; the information of the chromatographic parameters such as the sample introduction amount, the peak area, the theoretical plate number, the separation degree and the like of the control and the sample are shown in tables 6 to 7.
The pre-treatment is carried out by extracting with methanol and ethanol with different concentrations, and the detected number of chromatographic peaks and the area of chromatographic peaks are different. Because methanol has high toxicity, 70% ethanol is preferably selected as a solvent for extraction, and the data show that the amount of palmatine hydrochloride in the detected powder is very small and no color spectrum response exists; only 13 compounds were detected.
e. Analysis of results: and c, filling the filtrate obtained in the step b into an ultra-high performance liquid chromatograph, performing gradient elution and detection under the chromatographic condition of the step c, and measuring the peak area of each target object in the filtrate so as to obtain retention time and spectrogram characterization. According to the chromatogram, the same chromatographic peak appears at the position corresponding to the chromatogram of 12 active ingredient reference substances (except for palmatine hydrochloride) in the chromatogram of the sample to be detected, and the content of the active ingredient is calculated. As shown in chromatograms 5-20, the peak shape is good, and the separation degree can be achieved, which indicates that the target medicinal material can be detected. In the process of screening the spectrogram, illegal additives of olaquindox and mequindox are found, see the spectrogram 2.
Let us take 243nm chromatographic data as an example, and calculate the content of each compound in the sample to be measured as shown in Table 8:
TABLE 8 content of various ingredients in the samples to be tested of the powder for clearing heat and invigorating stomach
| Sequence number | Names of Compounds | Content (μg/g) |
| 1 | Chlorogenic acid | 193.98 |
| 2 | Olaquindox | Detection, qualitative determination only |
| 3 | Gentiopicroside | 112.76 |
| 4 | Mequindox | Detection, qualitative determination only |
| 5 | Hesperidin | 2181.54 |
| 6 | Berberine hydrochloride | 1048.49 |
| 7 | Palmatine hydrochloride | 0.00 |
| 8 | Aloe-emodin | 532.30 |
| 9 | Rhein | 74.79 |
| 10 | Emodin | 316.50 |
| 11 | Honokiol of honokiol | 246.23 |
| 12 | Magnolol | 144.34 |
| 13 | Chrysophanol | 53.68 |
| 14 | Physcion-containing material | 87.84 |
Example 2:
different gradient elution programs are adopted, other chromatographic conditions are consistent with the embodiment, the gradient elution program is 0-4min, and the volume fraction of the mobile phase A is maintained at 5%;4-7min, changing the volume fraction of the mobile phase A from 5% to 10%;7-10min, and maintaining the volume fraction of the mobile phase A at 10%;10-12min, the volume fraction of the mobile phase A is changed from 10% to 18%;12-20min, the volume fraction of the mobile phase A is maintained at 18%;20-28min, the volume of the mobile phase A is changed from 18% to 25%;28-30min, the volume fraction of the mobile phase A is maintained at 25%;30-32min, the volume fraction of the mobile phase A is changed from 25% to 50%;32-37min, the volume fraction of the mobile phase A is changed from 50% to 75%;37-40min, the volume fraction of mobile phase A is maintained at 75%;40-43min, the volume fraction of the mobile phase A is changed from 75% to 5%; and 43-45min, and the volume fraction of the mobile phase A is maintained at 5%. The chromatographic data of the mixed control is shown in table 9, the multi-channel chromatographic data of the sample active ingredients is shown in table 10, and the detection results of the olaquindox and mequindox illegal additives are shown in table 11:
table 9 chromatographic data of the mixed control sample with a sample size of 1. Mu.L
Table 10 sample active ingredient multichannel chromatography data table sample size 2. Mu.L
TABLE 11 sample injection of 2. Mu.L olaquindox and detection results of illegal addition of mequindox
Example 2, the overall run time was reduced and the detection efficiency was improved, but the matrix disturbance was relatively slightly larger and the degree of separation was relatively smaller than in example 1. If matrix interference exists in the same retention time as that of the control, we can judge whether the target compound is the target compound by means of the spectrogram, in this embodiment, the spectrogram of the illegal additives olaquindox and mequindox is shown in fig. 3, the spectrogram of the 12 mixed control is shown in fig. 4, and the graphs of the partial samples to be tested are listed due to space limitation and are shown in fig. 21-22.
Under the quality standard of the traditional 2020 edition national veterinary drug, the quality of the heat-clearing and stomach-invigorating powder is difficult to control by thin layer measurement, and the accurate and rapid qualitative and quantitative measurement can be carried out by using the chromatographic parameters such as the retention time peak area of the spectrogram and the like; the content of various compounds in the heat-clearing and stomach-invigorating powder can be easily quantified through the ratio of the peak areas of the reference substance and the sample to be measured. Can measure several kinds of content indexes such as 'hesperidin, berberine hydrochloride, aloe-emodin, honokiol' and the like, can also be identified by specifying characteristic peaks of fingerprint spectra, and lays a solid foundation for formulating quality standards of national veterinary drugs of 2025.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. of the gradient elution, the flow rate, the column temperature, the detection wavelength, etc. which are slightly adjusted by adopting liquid phase instruments of different brands, chromatographic columns with similar fillers of different brands and similar mobile phases are included in the protection scope of the invention.
Claims (10)
1. A method for simultaneously measuring 12 active ingredients and 2 illegal additives in a powder for clearing heat and invigorating stomach comprises the following steps:
(1) Respectively weighing standard substances of various active ingredients and preparing a mixed control stock solution containing the standard substances by using an organic solvent; separately preparing a mixed reference stock solution of olaquindox and mequindox;
the 12 active ingredients are as follows: chlorogenic acid, gentiopicroside, hesperidin, berberine hydrochloride, palmatine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol, physcion, honokiol, magnolol;
the 2 illegal additives are olaquindox and mequindox;
(2) Carrying out ultrasonic extraction on the heat-clearing and stomach-invigorating powder by adopting an extractant, centrifuging and filtering to obtain a liquid to be detected;
(3) Detecting the mixed control stock solution and the liquid to be detected by adopting an ultra-high performance liquid chromatograph, qualitatively analyzing 12 activities and 2 illegal additive components in the liquid to be detected by using a chromatogram and retention time, and calculating the content of 12 active components in the liquid to be detected by using the chromatographic peak area;
the chromatographic conditions are as follows: the mobile phase A is acetonitrile plus 0.2 percent phosphoric acid, the mobile phase B is 0.2 percent phosphoric acid water solution, and the column temperature is 39-41 ℃; flow rate of mobile phase: 0.35mL/min; sample injection amount: 0.5-5. Mu.L; the chromatographic column is waters UPLC HSS T 3 A chromatographic column;
detection conditions: a PDA detector, 3D scanning wavelength range 190-400nm; the detection wavelength is 225nm, 228nm, 251nm, 240nm, 264nm,289nm,292nm, 227 nm,346nm and other multi-channels.
2. The method according to claim 1, wherein the organic solvent in the step (1) is any one of methanol, ethanol and chloroform.
3. The method according to claim 1, wherein the extractant in the step (2) is any one selected from methanol, absolute ethanol, 50% by volume methanol, 50% by volume ethanol, and 70% by volume ethanol.
4. The method according to claim 3, wherein the extractant in the step (2) is 70% ethanol by volume.
5. The assay method according to claim 1, wherein the volume ratio of the heat-clearing and stomach-invigorating powder to the extractant in step (2) is 1:25-100; the extraction condition is ultrasonic extraction, and the extraction time is 15-25min. The ultrasonic extraction mode can effectively improve the extraction efficiency and the extraction effect.
6. The method according to claim 1, wherein the centrifugation conditions in the step (2) are: the rotation speed is 10000-15000rpm/min, and the centrifugation time is 15-25min.
7. The method according to claim 6, wherein the centrifugation conditions in the step (2) are: the rotational speed of centrifugation was 12000rpm/min, and the centrifugation time was 20min.
8. The method according to claim 1, wherein the detection wavelength in the step (3) is 228nm, 264nm, 243nm.
9. The method according to claim 1, wherein the column temperature in step (3) is 40 ℃; the sample injection amount is 1-2 mu L.
10. The assay of claim 1, wherein the gradient elution procedure in step (3) is:
0-4min, the volume fraction of the mobile phase A is maintained at 5%;
4-8min, changing the volume fraction of the mobile phase A from 5% to 10%;
8-12min, the volume fraction of the mobile phase A is maintained at 10%;
12-16min, the volume fraction of the mobile phase A is changed from 10% to 15%;
16-20min, the volume fraction of the mobile phase A is maintained at 15%;
20-25min, the volume of the mobile phase A is changed from 15% to 20%;
25-30min, and maintaining the volume fraction of the mobile phase A at 20%;
30-32min, the volume fraction of the mobile phase A is changed from 20% to 40%;
32-36min, the volume fraction of the mobile phase A is changed from 40% to 60%;
36-40min, the volume fraction of the mobile phase A is maintained at 60%;
40-43min, the volume fraction of the mobile phase A is changed from 60% to 85%;
43-45min, the volume fraction of the mobile phase A is maintained at 85%;
45-47min, the volume fraction of the mobile phase A is changed from 85% to 5%;
and the volume fraction of the mobile phase A is maintained at 5% for 47-50 min.
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| CN202310026737.XA CN116008429A (en) | 2023-01-09 | 2023-01-09 | Simultaneous determination method for 12 active ingredients and 2 illegal additives in heat-clearing and stomach-invigorating powder |
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