CN115060812B - Fingerprint of fried haw medicinal preparation and construction method and application thereof - Google Patents
Fingerprint of fried haw medicinal preparation and construction method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and particularly relates to a fingerprint of a fried hawthorn/fructus crataegi medicinal preparation, and further discloses a construction method of the fingerprint and a quality detection method of the fried hawthorn/fructus crataegi. The invention discloses a method for constructing fingerprint of fried hawthorn/hawthorn formula particle, which takes fried hawthorn/hawthorn formula particle as a detection object, establishes a fingerprint method for the medicinal preparation based on high performance liquid chromatography, adopts traditional Chinese medicine chromatographic fingerprint to calibrate 9 common characteristic peaks, and determines the characteristics of the fried hawthorn formula particle. The method for constructing the fingerprint spectrum of the fried hawthorn/fructus crataegi formula particle can comprehensively reflect the internal quality and the medication safety of the fried hawthorn/fructus crataegi medicinal preparation, is beneficial to the comprehensive quality detection and the overall quality control of the fried hawthorn/fructus crataegi medicinal preparation, and has the advantages of high stability, high precision and good repeatability.
Description
Technical Field
The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and particularly relates to a fingerprint of a fried hawthorn/fructus crataegi medicinal preparation, and further discloses a construction method of the fingerprint and a quality detection method of the fried hawthorn/fructus crataegi.
Background
The traditional Chinese medicine has complex components, the effective part is often not a single component, and the quality control index of a certain single component is not suitable for the quality control requirement of the traditional Chinese medicine. Therefore, the fingerprint technique of traditional Chinese medicine has been developed. The traditional Chinese medicine fingerprint spectrum technology is derived from fingerprint identification, and the types and the amounts of chemical components contained in the traditional Chinese medicine are comprehensively reflected by utilizing modern information technology and mass analysis means. For traditional Chinese medicinal materials, the fingerprint can be used for identifying authenticity and judging quality; for the Chinese patent medicine, the fingerprint can identify the authenticity of the product, judge the rationality of the preparation process and effectively control the quality of the product. Most of the active ingredients of the traditional Chinese medicine are not clear at the present stage, the integrity and the ambiguity of the fingerprint of the traditional Chinese medicine are just suitable for the characteristic, and the quality control method of the traditional Chinese medicine has more scientificity and comprehensiveness than the quality control method of single ingredients. At present, the traditional Chinese medicine fingerprint is internationally accepted as a traditional Chinese medicine quality control mode. The high performance liquid chromatography has the advantages of high separation efficiency, high analysis speed and the like, and becomes a main analysis means of the current fingerprint.
Hawthorn is a Chinese medicinal raw material used as both medicine and food, and has the effects of promoting digestion, invigorating stomach, promoting qi circulation, relieving fatigue, eliminating turbid pathogen and reducing blood lipid. Can be used for treating food stagnation, gastric distention, diarrhea, abdominal pain, blood stasis, amenorrhea, puerperal fatigue, thoracalgia, chest pain, hernia pain, and hyperlipidemia. However, no fingerprint is specified in the quality standard of the hawthorn at present, a perfect quality evaluation system is lacking, and effective control of the quality of the hawthorn is difficult to achieve, and particularly, the quality control of the hawthorn medicinal preparation under different processing methods is still blank. Therefore, a method for comprehensively and systematically detecting the fried hawthorn medicinal preparation, in particular a method for establishing the fingerprint of the fried hawthorn medicinal preparation, has important significance for the comprehensive quality detection and the overall quality control of the fried hawthorn medicinal preparation.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a method for constructing the fingerprint spectrum which can comprehensively reflect the internal quality and the medication safety of the fried hawthorn/fructus crataegi medicinal preparation;
the second technical problem to be solved by the invention is to provide a quality detection method of a fried hawthorn/fructus crataegi medicinal preparation.
In order to solve the technical problems, the fingerprint construction method of the fried haw pharmaceutical preparation comprises the step of performing high performance liquid chromatography detection on a test solution of the fried haw pharmaceutical preparation;
the chromatographic conditions include: octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, and a 0.1% phosphoric acid solution is used as a mobile phase B, and gradient elution is carried out according to the following procedure:
0-4min, A: b is 5%:95%;
4-12min, A: b is 5%:95% -11%: 89%;
12-13min, A: b is 11%:89% -13%: 87%;
13-21min, A: b is 13%:87% -14%: 86%;
21-30min, A: b is 14%:86% -25%: 75%.
Specifically, in the step of detecting high performance liquid chromatography, the chromatographic conditions further include: the column temperature is 25-35 ℃, the flow rate is 0.3ml/min, and the detection wavelength is 320nm.
Preferably, in the step of detecting high performance liquid chromatography, the chromatographic conditions further include: the column temperature is 30 ℃, the flow rate is 0.2-0.4ml/min, and the detection wavelength is 320nm.
Specifically, the preparation method of the sample solution comprises the following steps: precisely adding an organic solvent into the sample, performing ultrasonic treatment by sealing, filtering, and collecting the subsequent filtrate.
Specifically, the fingerprint construction method of the fried haw pharmaceutical preparation further comprises the step of preparing a reference substance solution of reference medicinal materials, and specifically comprises the following steps: heating and reflux extracting fructus crataegi reference material with water, collecting extractive solution, filtering, evaporating to dryness, adding organic solvent into residue, sealing, ultrasonic treating, filtering, and collecting filtrate to obtain reference solution.
Specifically, the fingerprint construction method of the fried haw pharmaceutical preparation further comprises the steps of preparing a reference substance solution of a reference substance and constructing the fingerprint of the reference substance based on the high performance liquid chromatography;
specifically, the reference substance comprises at least one of 5-hydroxymethylfurfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, hyperoside, isoquercitrin or vitexin;
preferably, the step of preparing the reference solution for the reference comprises the step of preparing the reference solution for the reference with a selected concentration by precisely adding an organic solvent into the selected reference;
preferably, the concentration of each reference substance in the reference substance solution of the reference substance comprises: each 1ml of the solution contains 30-50 mug of 5-hydroxymethylfurfural, 3-8 mug of neochlorogenic acid, 10-30 mug of chlorogenic acid, 3-8 mug of cryptochlorogenic acid, 3-8 mug of hyperoside, 3-8 mug of isoquercitrin and/or 10-20 mug of vitexin.
More preferably, the concentration of each reference substance in the reference substance solution of the reference substance comprises: each 1ml of the solution contains 40 mug of 5-hydroxymethylfurfural, 20 mug of chlorogenic acid, 5 mug of hyperoside, 5 mug of isoquercitrin and/or 15 mug of vitexin.
According to literature reports, the main chemical components of hawthorn (fructus crataegi) mainly comprise flavonoid, triterpenes, phenolic acids, organic acids, saccharides and the like. The flavonoids mainly comprise saccharide compounds with quercetin, apigenin and kaempferol as aglycones, mainly isoquercitrin, hyperoside and vitexin-glucoside, etc., and also comprise a large amount of procyanidins such as procyanidin B2, procyanidin B5, etc. The triterpene compounds are mainly tetracyclic triterpene and pentacyclic triterpene. The phenolic acid compounds include chlorogenic acid, fructus Rhodotorulae acid, gallic acid, protocatechuic acid, etc. The organic acid component mainly comprises oxalic acid, malic acid, citric acid, tartaric acid, quinic acid, etc. Therefore, the above substances were selected as controls.
Specifically, the fingerprint construction method of the fried haw pharmaceutical preparation comprises the steps that the organic solvent comprises methanol with the volume concentration of 40-60 v/v%; more preferably 50v/v% methanol solution.
The power of the ultrasonic step is 200-300W, and the frequency is 30-40kHz. Preferably, the power of the ultrasonic step is 250W and the frequency is 35kHz.
Specifically, the fried haw medicinal preparation comprises fried haw/hawthorn red formula granules, fried haw/hawthorn red decoction pieces or fried haw/hawthorn red decoction pieces.
The invention also discloses a fingerprint spectrum and/or a contrast fingerprint spectrum of the fried haw medicinal preparation, which is constructed by the method.
The invention also discloses a fingerprint construction method of the fried haw pharmaceutical preparation and/or application of the fingerprint of the fried haw pharmaceutical preparation and/or a reference fingerprint in the field of quality detection of the fried haw pharmaceutical preparation.
The invention also discloses a quality detection method of the fried haw pharmaceutical preparation, which comprises the steps of constructing the fingerprint and the reference fingerprint according to the method, and comparing the fingerprint with the reference fingerprint.
The invention discloses a construction method of a fingerprint of fried hawthorn/hawthorn formula particle, which is characterized in that fried hawthorn/hawthorn formula particle is taken as a detection object, a fingerprint method for the medicinal preparation is established based on high performance liquid chromatography, 9 common characteristic peaks are calibrated by adopting a traditional Chinese medicine chromatographic fingerprint, the characteristics of the fried hawthorn formula particle are determined by confirming that peak 2 is 5-hydroxymethylfurfural, peak 3 is neochlorogenic acid, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 7 is hyperin and peak 8 is isoquercitrin, and peak 4 chlorogenic acid is selected as an internal reference peak. The method for constructing the fingerprint spectrum of the fried hawthorn/fructus crataegi formula particle can comprehensively reflect the internal quality and the medication safety of the fried hawthorn/fructus crataegi medicinal preparation, is beneficial to the comprehensive quality detection and the overall quality control of the fried hawthorn/fructus crataegi medicinal preparation, and has the advantages of high stability, high precision and good repeatability.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,
FIG. 1 is a graph of a comparative profile of the construction of the drug formulation of Crataegus pinnatifida described in example 1;
FIG. 2 is a characteristic spectrum of a test sample constructed by the fried haw pharmaceutical preparation in example 1;
FIG. 3 is a fingerprint spectrum of the fried haw pharmaceutical preparation of the invention;
FIG. 4 is a specific localization chromatogram of the fried haw pharmaceutical formulation of the present invention;
FIG. 5 is a precision chromatogram of the signature;
FIG. 6 is a repetitive chromatogram of the signature;
FIG. 7 is an intermediate precision (personnel) chromatogram of the profile;
FIG. 8 is a chromatogram of a negative formulation of example 6;
FIG. 9 is a stability test chromatogram of the profile;
FIG. 10 is a characteristic spectrum of the fried haw pharmaceutical formulation at different flow rates;
FIG. 11 is a characteristic spectrum of the fried haw pharmaceutical formulation at different column temperatures;
FIG. 12 is a characteristic spectrum of the fried haw drug formulation at different mobile phase phosphoric acid concentrations;
FIG. 13 is a characteristic spectrum of the fried haw pharmaceutical formulation under different chromatographic columns;
FIG. 14 is a spectrum of 3 batches of fried hawthorn (Crataegus pinnatifida) formula particles;
fig. 15 is a characteristic spectrum of the fried hawthorn (hawthorn) and the characteristic spectrum of the 18 batches of the fried hawthorn (hawthorn) medicinal preparation in comparison with the characteristic spectrum of the medicinal materials.
Detailed Description
In the following embodiments of the invention, the apparatus and reagents involved include:
instrument and equipment
Chromatograph 1: waters ACQUITY UPLC H-Class PLUS chromatography system comprising quaternary solvent manager (ACQ-QSM), autosampler (ACQ-FTN), original inlet chromatographic column incubator (ACQ-CM), diode array ultraviolet detector (ACQ-PDA), and Empower chromatography management system;
an electronic balance: METTER TOLEDOO (METTLE Switzerland) ME36S, XS, XSE205, XS205 (parts per million);
ultrasonic instrument: KQ-500DE type digital control ultrasonic device of Kunshan ultrasonic instrument Co., ltd;
chromatographic column: agilent Eclipse Plus Rapid Resolution HD C18, 2.1X100 mm,1.8 μm;
reagent and reagent
Acetonitrile (chromatographic purity of zemoer feishier technologies limited), water as purified water, methanol, phosphoric acid and other reagents as analytical purity;
chlorogenic acid reference substance (batch number: 110753-201817, purchased in China food and drug inspection institute, purity 99.3%);
a reference cryptochlorogenic acid (lot number: G1521001, purchased from adadin);
new chlorogenic acid reference (lot number 18011721, purchased from Shanghai HongYongsheng biotechnology Co., ltd., purity 98.0%);
hyperin reference (lot number 111521-201609, purchased in China food and drug inspection institute, purity 94.9%);
isoquercitrin control (lot number: 111809-201604, purchased in China food and drug inspection institute, purity 97.2%);
5-hydroxymethylfurfural control (lot number: 111626-201711, purchased in China food and drug inspection institute, purity 99.4%);
hawthorn (fructus crataegi) control (lot 121138-201206, purchased from China food and drug inspection institute);
fried hawthorn (fructus crataegi) granule (batch number: 1906001Y, 1906002Y, 1906003Y).
Example 1 construction of characteristic Spectrum method of fried Hawthorn (Shandong Red) formula particle
Preparation of reference solutions
Taking 2g of haw reference medicine, precisely weighing, adding 100ml of water, heating and refluxing for 45 minutes, filtering, evaporating the filtrate to dryness, cooling, adding 25ml of 50% methanol into residues, sealing, performing ultrasonic treatment (power 250W, frequency 35 kHz) for 30 minutes, taking out, cooling, filtering, and taking the subsequent filtrate as a reference substance solution of the reference medicine.
Taking a proper amount of 5-hydroxymethylfurfural reference substance, chlorogenic acid reference substance, hyperin reference substance, isoquercitrin reference substance and vitexin reference substance, precisely weighing, adding 50% methanol to prepare a solution containing 40 mug of 5-hydroxymethylfurfural, 20 mug of chlorogenic acid, 5 mug of hyperin, 5 mug of isoquercitrin and 15 mug of vitexin per 1ml, and taking the solution as a reference substance solution of the reference substance.
Preparation of test solutions
Taking a to-be-detected fried haw particle test sample, grinding, taking 1.0g, precisely weighing, adding 25ml of 50% methanol, sealing, performing ultrasonic treatment (power is 250W, frequency is 35 kHz) for 30 minutes, taking out, cooling, filtering, and taking a subsequent filtrate as a test sample solution.
Chromatographic conditions and system adaptation
Octadecylsilane chemically bonded silica as filler (Eclipse Plus Rapid Resolution HD C, 2.1X100 mm,1.8 μm); acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, and the gradient elution was performed according to the following gradient procedure:
0-4min, A: b is 5%:95%;
4-12min, A: b is 5%:95% -11%: 89%;
12-13min, A: b is 11%:89% -13%: 87%;
13-21min, A: b is 13%:87% -14%: 86%;
21-30min, A: b is 14%:86% -25%: 75%;
the flow rate is 0.3ml per minute;
column temperature is 30 ℃;
the detection wavelength was 320nm.
The theoretical plate number should be not less than 5000 calculated as chlorogenic acid peak.
Assay
2 μl of each of the reference solution and the sample solution is precisely sucked, injected into a liquid chromatograph, measured, and the chromatogram is recorded.
The chromatogram of the reference substance solution of the reference substance constructed in this example is shown in fig. 1, and the corresponding chromatogram data is shown in table 1 below.
TABLE 1 control chromatogram data
| Peak number | Peak name | Retention time | Peak area | Peak height | Peak width | Degree of separation | Symmetry factor | |
| 1 | 5-hydroxymethylfurfural | 2.52 | 83407 | 21479 | 17 | - | 1.027 | 9075 |
| 3 | Chlorogenic acid | 9.937 | 409244 | 78547 | 25.9 | 61.134 | 1.009 | 83600 |
| 5 | Hyperin | 21.093 | 44434 | 5203 | 30.75 | 61.704 | 1.007 | 140480 |
| 6 | Isoquercitrin | 22.173 | 39437 | 4380 | 25.6 | 4.651 | 1.028 | 135802 |
The chromatogram of the sample solution constructed in the embodiment is shown in fig. 2, and the corresponding chromatogram data is shown in table 2 below; wherein, peak 2: 5-hydroxymethylfurfural; peak 3: new chlorogenic acid; peak 4: chlorogenic acid; peak 5: chlorogenic acid of Cryptophan; peak 7: hyperin; peak 8: isoquercitrin.
TABLE 2 chromatogram data for test solutions
| Peak number | Peak name | Retention time | Peak area | Peak height | Peak width | Degree of separation | Symmetry factor | |
| 1 | 1.88 | 77120 | 26482 | 8.9 | - | 1.046 | 8873 | |
| 2 | 5-hydroxymethylfurfural | 2.548 | 54291 | 13454 | 16.15 | 7.07 | 1.012 | 8662 |
| 3 | Chlorogenic acid | 5.682 | 21601 | 2443 | 23.55 | 18.14 | 0.989 | 9194 |
| 4 | Chlorogenic acid | 10.086 | 234394 | 43458 | 23.4 | 23.336 | 1.035 | 81417 |
| 5 | Cryptochlorogenic acid | 11.381 | 29965 | 5681 | 18.25 | 3.516 | 1.052 | 113006 |
| 6 | 16.075 | 12294 | 2382 | 17.15 | 8.816 | 0.998 | 262918 | |
| 7 | Hyperin | 21.339 | 25727 | 2940 | 23.6 | 29.783 | 1.123 | 138853 |
| 8 | Isoquercitrin | 22.449 | 20085 | 2220 | 22.1 | 4.708 | 1.074 | 135950 |
| 9 | 27.241 | 10974 | 2271 | 15.15 | 25.936 | 1.07 | 736178 |
The chart shows that the chromatographic method has good system adaptability of the control solution and the sample solution, and can be used as a detection method of the characteristic spectrum of the fried hawthorn (hawthorn) formula granule.
Characteristic spectrum characteristic peak identification and selection
According to the results of peak identification and reference substance positioning, it was confirmed that peak 2 was 5-hydroxymethylfurfural, peak 3 was neochlorogenic acid, peak 4 was chlorogenic acid, peak 5 was cryptochlorogenic acid, peak 7 was hyperin, and peak 8 was isoquercitrin. Wherein chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid are phenolic acid components, hyperoside and isoquercitrin are flavonoid components. The characteristic map comprises main chemical components of parched fructus crataegi (fructus crataegi).
Example 2 feature spectrogram construction
The detection results of 15 batches of sample fingerprint patterns are analyzed, and a comparison characteristic pattern is generated by adopting fingerprint pattern similarity evaluation software '2012 edition of traditional Chinese medicine chromatographic fingerprint pattern similarity evaluation system' compiled by pharmacopoeia committee, as shown in figure 3.
By identifying and identifying characteristic peaks by HPLC and LC/MS/MS, 9 chromatographic peaks with good HPLC characteristic spectrum separation degree of the obtained parched fructus crataegi (fructus crataegi) granule are confirmed, as shown in figure, the 9 chromatographic peaks are selected as characteristic peaks, and the chromatographic peak numbers 1-9 are arranged according to the sequence of the chromatographic peaks. The reference substance is used for positioning and confirming, wherein the peak 2 is 5-hydroxymethyl furfural, the peak 3 is neochlorogenic acid, the peak 4 is chlorogenic acid, the peak 5 is cryptochlorogenic acid, the peak 7 is hyperin, and the peak 8 is isoquercitrin.
Selection basis of characteristic spectrum characteristic peak S-peak
According to peak identification and peak selection results of characteristic peaks in the fried hawthorn (hawthorn) granules, the chromatographic peaks which are easy to obtain by the reference substances are chlorogenic acid, hyperin and quercetin, wherein the chlorogenic acid has higher response, the chlorogenic acid is phenolic acid components, and the phenolic acid components are main chemical components of hawthorn (hawthorn) decoction pieces, so that the method selects the chlorogenic acid as the index component of the content of the fried hawthorn (hawthorn) granules. In summary, chlorogenic acid was used as the S peak of the feature map. The relative retention time and relative peak area of each characteristic peak were calculated. 9 common peaks were selected as characteristic peaks, and a specific localization chromatogram is shown in fig. 4. Wherein S1 is chromatogram of parched fructus crataegi (fructus crataegi red) granule; s2: 5-hydroxymethylfurfural; s3: new chlorogenic acid; s4: chlorogenic acid; s5: chlorogenic acid of Cryptophan; s6: hyperin; s7: isoquercitrin.
EXAMPLE 3 precision test
Taking the same sample solution (1906001Y) of the fried hawthorn/fructus crataegi formula granule, repeatedly sampling for 6 times according to chromatographic conditions under the item of the fried hawthorn (fructus crataegi) formula granule (characteristic spectrum), and determining the relative retention time and relative peak area of each common peak, wherein the results are shown in tables 3-4 respectively, the precision chromatogram is shown in figure 5, and the precision is 1-6 needles from top to bottom.
Table 3 results of the instrument precision versus retention time test
Table 4 results of the instrument precision versus peak area test
The results show that peaks 2, 4, 7 and 8 respectively correspond to the retention time of the corresponding reference substance peaks of the reference substance; the peak corresponding to the chlorogenic acid reference peak is taken as an S peak, and the relative retention time and RSD of the relative peak area of each characteristic peak and the reference S peak (No. 4 peak) are smaller, so that the instrument precision is good.
Example 4 method repeatability test
The same part of fried hawthorn (hawthorn) formula granule (1906001Y) is taken, 6 parts of the preparation method of the sample of the fried hawthorn (hawthorn) formula granule (characteristic spectrum) are repeatedly prepared, the relative retention time and the relative peak area of each common peak are measured according to chromatographic conditions, the results are respectively shown as 5-6, the repeated chromatogram is shown as figure 6, and renaturation 1-6 samples are shown from top to bottom.
Table 5 method repeatability versus retention time test results
TABLE 6 method repeatability vs. peak area test results
The results show that peaks 2, 4, 7 and 8 respectively correspond to the retention time of the corresponding reference substance peaks of the reference substance; the peak corresponding to the chlorogenic acid reference peak is taken as an S peak, and the relative retention time and RSD of the relative peak area of each characteristic peak and the reference S peak (No. 4 peak) are smaller, so that the repeatability of the method is good.
Example 5 intermediate precision experiment (different operators)
The two inspectors respectively take the same fried hawthorn (hawthorn) formula granule at different times, prepare samples according to the preparation method of the sample of the fried hawthorn (hawthorn) formula granule [ characteristic spectrum ] item, and measure the relative retention time and the relative peak area of each common peak by the same equipment, the results are respectively shown in tables 7-8 below, the chromatogram of the middle precision (personnel) is shown in figure 7, and the samples of different personnel 1-6 are shown from top to bottom.
TABLE 7 results of intermediate precision versus retention time experiments (different operators)
TABLE 8 results of intermediate precision versus peak area test
EXAMPLE 6 specificity
According to the preparation method of the sample solution, the sample solution and the negative particle solution are prepared according to the solution method, HPLC analysis is carried out according to the chromatographic conditions described in the item [ characteristic map ], whether the negative sample of the fried hawthorn (fructus crataegi) formula particle causes interference or not is examined, and the chromatographic map of the negative particle solution is shown in figure 8. Comparing the spectrum with the spectrum of the sample solution shown in fig. 2, it can be seen that the blank control of the sample has no interference to the characteristic spectrum, and can be used as a detection method of the characteristic spectrum of the fried hawthorn (fructus crataegi) formula granule.
EXAMPLE 7 stability investigation
Taking the same sample solution of the fried hawthorn (hawthorn) formula granule, sampling (respectively marked as stability 1-6) according to chromatographic conditions under the items of the fried hawthorn (hawthorn) formula granule [ characteristic spectrum ], measuring the relative retention time and the relative peak area of each common peak, wherein the results are shown in tables 9-10, the stability test chromatogram is shown in figure 9, and the stability test chromatogram is shown from top to bottom and shows the samples at each time point in stability 1-6.
TABLE 9 stability versus retention time test results
Table 10 results of instrument precision versus peak area test
The results showed that the peak corresponding to the chlorogenic acid reference peak was S-peak, and the relative retention time and relative peak area of each characteristic peak and the reference S-peak (peak No. 4) were smaller in RSD. The test solution is stable within 24 hours and meets the measurement requirements.
EXAMPLE 8 investigation of different flow Rate
Taking the same batch of sample solution, and setting the flow rates to be 0.27ml/min, 0.3ml/min and 0.33ml/min respectively according to the measurement method of fried hawthorn (hawthorn) formula particles (characteristic spectrum), examining the relative retention time and relative peak area of each characteristic peak and reference substance S peak (No. 4 peak) when the flow rate is changed, wherein the results are shown in tables 11-12 respectively, and the graphs under different flow rate conditions are shown in fig. 10, wherein (a), (b) and (c) respectively show the spectrograms under the flow rates of 0.27ml/min, 0.3ml/min and 0.33 ml/min.
Table 11 comparison of different flow measurements
TABLE 12 relative peak area results for different flow rates
As a result, the peak 6 separation was affected when the flow rate was changed, and therefore, the method was carried out by selecting a fixed flow rate (0.3 ml/min).
EXAMPLE 9 investigation of different column temperatures
Taking the same batch of sample solution, and setting the column temperature at 27deg.C, 30deg.C and 33deg.C respectively according to the measurement method of parched fructus crataegi (fructus crataegi) granule (characteristic spectrum), examining the relative retention time and relative peak area of each characteristic peak and reference substance S peak (No. 4 peak) when the column temperature changes, and respectively showing the results in tables 13-14, wherein the chromatograms at different column temperatures are shown in figure 11, and (a), (b) and (c) respectively show the spectrograms at 27deg.C, 30deg.C and 33deg.C.
TABLE 13 comparison of results of different column temperature measurements
TABLE 14 Peak area results for different columns Wen Xiangdui
The results show that peak 6 separation is affected when the column temperature is changed, and therefore the method selects a fixed column temperature (30 ℃) for measurement.
EXAMPLE 10 investigation of different mobile phases
Taking the same batch of sample solution, setting the concentration of mobile phase phosphoric acid to be 0.08%, 0.1% and 0.12% respectively according to the measurement method of the standard decoction of fried hawthorn (mountain red) decoction pieces, examining the relative retention time and relative peak area of each characteristic peak and reference substance S peak (No. 4 peak) when phosphoric acid changes, and respectively showing the results in tables 15-16, wherein the spectrograms under different phosphoric acid concentrations are shown in fig. 12, and (a), (b) and (c) respectively show the spectrograms under the concentrations of mobile phase phosphoric acid of 0.08%, 0.1% and 0.12% respectively.
TABLE 15 comparison of the results of the measurements of the different phosphate concentrations
TABLE 16 relative peak area results for different phosphoric acid concentrations
The results show that the separation of peak 6 is affected when the phosphoric acid concentration in the mobile phase is different, and therefore the method selects a fixed phosphoric acid concentration (0.1% phosphoric acid) for the measurement.
EXAMPLE 11 investigation of different chromatography columns
Taking the same batch of sample solution (batch No. 1906001Y), and respectively examining the separation of Agilent Eclipse Plus Rapid Resolution HD C (2.1X100 mm,1.8 μm), ACQUITY BEH C18 (2.1X100 mm,1.7 μm) and YMC Triart C18 (2.0X100 mm,1.9 μm) according to the measurement method of the fried haw (mountain red) formula granule (characteristic spectrum). The results are shown in tables 17-18, respectively, and the separation results for the different columns are shown in FIG. 13, wherein (a), (b), and (C) represent the spectra for Agilent Eclipse Plus Rapid Resolution HD C (2.1X100 mm,1.8 μm), ACQUITY BEH C18 (2.1X100 mm,1.7 μm), YMC Triart C18 (2.0X100 mm,1.9 μm), respectively.
TABLE 17 comparison of the measurement results of different chromatographic columns
TABLE 18 results of relative peak areas for different chromatographic columns
The result shows that under the chromatographic condition, each chromatographic column has a larger difference on the peak time of each chromatographic peak of the fried hawthorn (hawthorn) formula granule and has a certain influence on the separation of characteristic peaks, and the method selects and fixes the chromatographic column Agilent Eclipse Plus Rapid Resolution HD C (2.1 multiplied by 100mm,1.8 mu m) to measure the characteristic spectrum of the fried hawthorn (hawthorn) formula granule.
In summary, as can be seen from the above methodology examination results, the construction method for determining the fried hawthorn (fructus crataegi) formula granule (characteristic map) is specifically as follows:
octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.6mm, and particle diameter is 1.8 μm); acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, elution was performed according to the gradient specified in table 19 below; the flow rate is 0.3ml per minute; column temperature is 30 ℃; the detection wavelength was 320nm. The theoretical plate number should be not less than 5000 calculated as chlorogenic acid peak.
TABLE 19 Mobile phase gradient procedure
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0-4 | 5 | 95 |
| 4-12 | 5→11 | 95→89 |
| 12-13 | 11→13 | 89→87 |
| 13-21 | 13→14 | 87→86 |
| 21-30 | 14→25 | 86→75 |
The sample chromatogram should show 9 characteristic peaks, except for peak 2, corresponding to 8 characteristic peaks in the reference chromatogram of the control. Wherein, the retention time of the peaks 2, 4, 7 and 8 respectively correspond to the retention time of the corresponding reference peak of the reference substance; and calculating the relative retention time of each characteristic peak and the S peak by taking the peak corresponding to the chlorogenic acid reference peak as the S peak, wherein the relative retention time is within +/-10% of a specified value. The relative peak area of peak 1 to the sum of peak areas of peak 7 and peak 8 was calculated to be not lower than 0.50. The relative peak area of the characteristic peak corresponding to vitexin and the sum of peak areas of peak 7 and peak 8 is calculated, and the relative peak area is not more than 0.07, or vitexin is not detected.
Example 12 determination of characteristic Properties of Multi-batch fried Hawthorn (Crataegus pinnatifida) formulation particles
According to the method for characteristic spectrum of fried hawthorn (hawthorn) formula particle, 3 batches of fried hawthorn (hawthorn) formula particles (batch numbers: 1906001Y, 1906002Y and 1906003Y) are measured according to the method, the results are shown in tables 20-21 respectively, the spectrograms of the fried hawthorn (hawthorn) formula particles in each batch are shown in figure 14, and samples of fried hawthorn 1906001Y, 1906002Y and 1906003Y are respectively shown from top to bottom.
Table 20 results of relative retention time test of three batches of parched fructus crataegi (Crataegus pinnatifida) granule formulations
Table 21 three batches of fried hawthorn (Shandong hong) formula granule characteristic spectrum relative peak area
The results show that the relative retention time and the relative peak area of 3 batches of fried hawthorn (fructus crataegi red) formula particles are all within the required range, and no characteristic peak corresponding to the vitexin reference substance is detected.
Example 13 characteristic Profile data of Multi-batch parched Hawthorn (Crataegus pinnatifida) pharmaceutical preparation and fitting
The preparation method is carried out according to the method for determining the characteristic map, wherein 3 batches of fried hawthorn (hawthorn) formula particles (batch numbers: 1906001Y, 1906002Y, 1906003Y) and 15 batches of fried hawthorn (hawthorn) decoction piece standard decoction (freeze-dried powder) (batch numbers: 1903001Y, 1903002Y, 1903003Y, 1904004Y, 1904005Y, 1904006Y, 1904007Y, 1904008Y, 1904009Y, 1904010Y, 1904011Y, 1904012Y, 1904013Y, 1904014Y, 1904015Y) are contained, and the determination results are shown in tables 22-23.
Table 22 determination of characteristic spectrum relative retention time of fried haw (mountain red) medicinal preparation
Table 23 determination result of characteristic map relative peak area of fried haw (mountain red) pharmaceutical preparation
The characteristic patterns of the fried hawthorn (fructus crataegi) and the characteristic patterns of the 15 batches of fried hawthorn (fructus crataegi) decoction piece standard decoction (lyophilized powder) and 3 batches of formula particles are respectively shown in (a), (b) and (c) in figure 15. The matching mode of the control map adopts Mark peak matching.
The measurement result shows that the characteristic spectrum of the 18 batches of fried hawthorn (hawthorn) medicinal preparation has 9 characteristic peaks, except peak 2, the characteristic spectrum corresponds to 8 chromatographic peaks in the chromatogram of the reference substance of the reference medicinal material, the peak corresponding to the peak of the reference substance of chlorogenic acid is an S peak, and the relative retention time of each characteristic peak and the S peak (chlorogenic acid) is within +/-10% of a specified value.
Specified value of characteristic map
From the study results, it was determined that: the characteristic spectrum of the fried hawthorn (fructus crataegi) prescription granule should have 9 characteristic peaks and should correspond to the relative retention time of 8 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, wherein 4 peaks should correspond to the retention time of the peaks of the reference substance of 5-hydroxymethylfurfural, chlorogenic acid, hyperin and isoquercitrin respectively; selecting a peak corresponding to a chlorogenic acid reference peak as an S peak, and determining the relative retention time range of each characteristic peak and the S peak as follows: 10 percent. The calculated specified value is: 0.19 (Peak 1), 0.56 (Peak 3), 1.13 (Peak 5), 1.60 (Peak 6), 2.72 (Peak 9). The results of the obtained control feature patterns with respect to retention time and peak area and the control drug feature patterns are shown in tables 24 to 25 below.
Table 24 relative retention time of the formulation granule of parched fructus crataegi (fructus crataegi) and its characteristic spectrum
Table 25 relative peak area of fried haw (mountain red) prescription granule contrast map and haw (mountain red) contrast medicinal material characteristic map
As shown in fig. 15 (a), the haw (mountain red) is compared with the chromatogram of the medicinal material, peak 2: new chlorogenic acid; peak 3: chlorogenic acid; peak 4: chlorogenic acid of Cryptophan; peak 6: hyperin; peak 7: isoquercitrin.
As shown in fig. 15 (b), the characteristic spectrum of 15 batches of standard decoction of fried hawthorn (fructus crataegi) decoction pieces (freeze-dried powder) sequentially shows the spectrogram results of 1903001Y, 1903002Y, 1903003Y, 1904004Y, 1904005Y, 1904006Y, 1904007Y, 1904008Y, 1904009Y, 1904010Y, 1904011Y, 1904012Y, 1904013Y, 1904014Y and 1904015Y batches from top to bottom. As shown in figure 15 (c), the characteristic patterns of 3 batches of fried hawthorn (Shandong Red) formula particles sequentially show the spectrogram results of 1906001Y, 1906002Y and 1906003Y from bottom to top, and can be compared with the characteristic patterns of fried hawthorn (Shandong Red) formula particles (peak 2:5-hydroxymethylfurfural; peak 3: neochlorogenic acid; peak 4: chlorogenic acid; peak 5: cryptochlorogenic acid; peak 7: hyperoside; peak 8: isoquercitrin)
EXAMPLE 14 analysis of different sources of Hawthorn fruit and confounding products
As the hawthorn is a multi-base source variety, the hawthorn under the Chinese pharmacopoeia 2020 edition is the dried mature fruit of Crataegus pinnatifida Bge.var.major N.E.B. or Crataegus pinnatifida Bge. The invention also collects the dried mature fruits of the crataegus cuneata which are frequently used as confusing products of the crataegus cuneata in the market and are distinguished by confusing products, wherein the crataegus cuneata is the crataegus cuneata Crataegus cuneata Sieb et Zucc.
The invention uses haw (hawthorn) and south haw decoction pieces to prepare fried haw particles, and adopts a characteristic spectrum method of haw (hawthorn) particles to measure characteristic spectrums of 6 batches of haw (hawthorn) and 3 batches of south haw decoction pieces formula particles, and the results are shown in tables 26-28 respectively.
Table 26 measurement results of 6 batches of hawthorns (hawthorns) and 3 batches of south hawthorns decoction pieces
Table 27 measurement results of characteristic patterns of 15 batches of Hawthorn (Shandong Red) decoction pieces
Table 28 15 measurement results of vitexin in decoction piece characteristic spectrum of fried hawthorn (Crataegus pinnatifida)
According to the research results, the chemical components of hawthorn (hawthorn), south hawthorn and hawthorn (hawthorn red) are different, and the main difference is that compared with the hawthorn (hawthorn red), the hawthorn (hawthorn) and the south hawthorn have two groups of chromatographic peaks which are about 15.5 minutes and about 20.5 minutes more. LC-MS analysis and comparison of the control substance prove that the chromatographic peak which appears about 20.5 minutes is vitexin. According to analysis, characteristic peaks corresponding to vitexin in 15 batches of hawthorn (hawthorn) decoction piece characteristic maps are smaller or undetected, detection ranges are respectively hawthorn (hawthorn) decoction pieces (0.0124-0.0281), stir-fried hawthorn (hawthorn) decoction pieces (0.009-0.0570, hawthorn (hawthorn) decoction pieces (0.164-0.575) and south hawthorn decoction pieces (0.466-0.677), different primordia are distinguished for more strict control, and the relative peak areas of the characteristic peaks corresponding to vitexin and the sum of peak areas of peak 7 and peak 8 are specified as not more than 0.07 according to multiple batches of data, or the vitexin is not detected so as to distinguish different primordia.
EXAMPLE 15 Hawthorn processing analysis
The standard decoction of haw (mountain red) decoction pieces (freeze-dried powder) was prepared using the haw (mountain red) decoction pieces and the standard decoction of haw (mountain red) was measured using the characteristic spectrum method of fried haw (mountain red), the results are shown in table 29 below.
Table 29 results of measuring relative peak areas of Hawthorn (Crataegus pinnatifida) pharmaceutical preparations of 15 batches
In the process of processing fructus crataegi (fructus crataegi) decoction pieces into parched fructus crataegi, due to heating factor, the change of substance components is caused, in the process of processing raw materials into parched fructus crataegi, organic acids such as chlorogenic acid and flavonoids such as hyperoside and isoquercitrin are reduced, and in the parching process, peak No. 1 is also obviously increased.
The investigation on 1 and flavonoid components (the total area of hyperin and isoquercitrin) shows that the area ratio of the peak area of the standard decoction peak 1 of haw (hawthorn) decoction pieces to the peak area ratio of flavonoid components is: 0.142-0.424, the ratio of peak area of standard decoction peak 1 of fried haw (hawthorn) decoction pieces to peak area of flavonoid components is: 0.526-1.871, the change is obvious, the processing degree of the fried hawthorn is controlled to be not lower than 0.50 by 95% of the minimum value of the standard decoction of the hawthorn (hawthorn) decoction pieces.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (8)
1. The fingerprint construction method of the fried haw pharmaceutical preparation is characterized by comprising the steps of preparing a reference substance solution of a reference substance, constructing the fingerprint of the reference substance based on high performance liquid chromatography and detecting the sample solution of the fried haw pharmaceutical preparation by high performance liquid chromatography;
the reference substance comprises 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, hyperoside, isoquercitrin and vitexin;
the chromatographic conditions include: octadecylsilane chemically bonded silica is used as filler, and chromatographic column Agilent Eclipse Plus Rapid Resolution HD C, 2.1X100 mm,1.8 μm; acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, and the gradient elution was performed as follows:
0-4min, A: b is 5%:95%;
4-12min, A: b is 5%:95% -11%: 89%;
12-13min, A: b is 11%:89% -13%: 87%;
13-21min, A: b is 13%:87% -14%: 86%;
21-30min, A: b is 14%:86% -25%: 75%.
2. The fingerprint construction method of a fried haw pharmaceutical preparation according to claim 1, wherein in the high performance liquid chromatography detection step, the chromatographic conditions further include: the column temperature is 25-35 ℃, the flow rate is 0.2-0.4ml/min, and the detection wavelength is 320nm.
3. The fingerprint construction method of the fried haw pharmaceutical preparation according to claim 2, wherein the preparation method of the sample solution comprises: precisely adding an organic solvent into the sample, performing ultrasonic treatment by sealing, filtering, and collecting the subsequent filtrate.
4. The fingerprint construction method of a fried haw pharmaceutical preparation according to claim 3, further comprising the step of preparing a reference solution of a reference medicinal material, specifically comprising: heating and reflux extracting fructus crataegi reference material with water, collecting extractive solution, filtering, evaporating to dryness, adding organic solvent into residue, sealing, ultrasonic treating, filtering, and collecting filtrate to obtain reference solution.
5. The fingerprint construction method of the fried haw pharmaceutical preparation according to claim 4, wherein the organic solvent comprises 40-60v/v% methanol;
the power of the ultrasonic treatment step is 200-300W, and the frequency is 30-40kHz.
6. The fingerprint construction method of a fried hawthorn pharmaceutical preparation according to any one of claims 1-5, wherein the fried hawthorn pharmaceutical preparation comprises a fried hawthorn/fructus crataegi formulation granule, a fried hawthorn/fructus crataegi decoction piece or a fried hawthorn/fructus crataegi decoction piece decoction.
7. The application of the fingerprint construction method of the fried haw pharmaceutical preparation in the field of quality detection of the fried haw pharmaceutical preparation.
8. A quality detection method of a fried haw pharmaceutical preparation, characterized by comprising the steps of constructing the fingerprint and a reference fingerprint according to the method of any one of claims 1-6, and comparing the fingerprint with the reference fingerprint.
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| CN112611816A (en) * | 2020-11-30 | 2021-04-06 | 山东宏济堂制药集团股份有限公司 | Hawthorn standard reference substance and preparation method, detection method and application thereof |
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| CN108426959A (en) * | 2018-05-14 | 2018-08-21 | 山东省中医药研究院 | A kind of construction method of haw flavonoids substances UPLC finger-prints |
| CN109521114A (en) * | 2018-11-30 | 2019-03-26 | 贵州百灵企业集团制药股份有限公司 | The detection method of main effects ingredient in Capsule YD |
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