CN110907574B - Quality control method, quality control spectrogram and construction method of traditional Chinese medicine composition - Google Patents

Quality control method, quality control spectrogram and construction method of traditional Chinese medicine composition Download PDF

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CN110907574B
CN110907574B CN201911077339.0A CN201911077339A CN110907574B CN 110907574 B CN110907574 B CN 110907574B CN 201911077339 A CN201911077339 A CN 201911077339A CN 110907574 B CN110907574 B CN 110907574B
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mobile phase
quality control
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chinese medicinal
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CN110907574A (en
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邹节明
孔祥春
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Guilin Sanjin Pharmaceuticals Co Ltd
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Guilin Sanjin Pharmaceuticals Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/047—Standards external

Abstract

The invention discloses a quality control method, a quality control spectrogram and a construction method of a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises watermelon frost, calcined borax, golden cypress, coptis chinensis, subprostrate sophora, blackberry lily, thunberg fritillary bulb, indigo naturalis, borneol, soapberry, rhubarb, scutellaria baicalensis, liquorice and menthol, the quality control method comprises the step of simultaneously detecting the components of the golden cypress, the coptis chinensis, the scutellaria baicalensis, the blackberry lily and the liquorice in the traditional Chinese medicine composition by adopting an HPLC method, and the conditions of the HPLC method are as follows: octadecylsilane chemically bonded silica is used as a filler; taking methanol as a mobile phase A and taking 0.1% phosphoric acid solution as a mobile phase B to carry out gradient elution; the theoretical plate number is not less than 300000 calculated by baicalin peak. The quality control method comprises designing multiple reference substance and reference medicinal material on the basis of the results of the components of the medicine obtained by HPLC detection, obtaining characteristic peak information corresponding to cortex Phellodendri, Coptidis rhizoma, Scutellariae radix, rhizoma Belamcandae and Glycyrrhrizae radix by comparative analysis, and determining the types of the above components in one-time sample injection detection.

Description

Quality control method, quality control spectrogram and construction method of traditional Chinese medicine composition
Technical Field
The invention belongs to the field of medicine quality control, and particularly relates to a quality control method, a quality control spectrogram and a construction method of a traditional Chinese medicine composition.
Background
The invention relates to a traditional Chinese medicine composition which is prepared from fourteen medicinal materials such as watermelon frost, calcined borax, phellodendron, coptis chinensis, subprostrate sophora, blackberry lily, thunberg fritillary bulb, indigo naturalis, borneol, Chinese soapberry fruit, rhubarb, scutellaria, liquorice, menthol and the like. The mode needs to carry out difficult detection on the sample according to the thin layer chromatography for many times, the operation is complex, the workload is large, the method is easily influenced by human, and the stability and the repeatability of the result are not good. Meanwhile, because the components contained in the traditional Chinese medicinal materials are complex, taking phellodendron as an example, and various alkaloids are contained in the traditional Chinese medicinal materials, the characteristic peaks of the traditional Chinese medicinal materials in liquid chromatography are complex, and the specific peaks are represented as miscellaneous peaks and interference peaks, so that the identification and judgment of the corresponding components of the traditional Chinese medicinal materials are influenced.
In addition, the differences of the component properties, the polarity and the acidity and alkalinity of different medicinal materials are large, the simultaneous representation of the different medicinal materials in a chromatographic spectrogram under the same condition is difficult, and the detection of various components is carried out separately, so that the complexity of quality control and the detection cost are increased undoubtedly.
The Chinese patent with the application number of 201110105310.6 discloses a traditional Chinese medicine composition for treating oral diseases, a preparation process thereof and a quality detection method, wherein the detection method respectively adopts thin-layer chromatography and liquid chromatography to identify and analyze components in the medicines, but only asiaticoside/madecassoside is detected in the liquid chromatography, and the identification and analysis are not performed on other medicines in view of the property difference and the detection difficulty of other medicines.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a quality control method, a quality control spectrogram and a construction method of a traditional Chinese medicine composition capable of simultaneously identifying and analyzing a plurality of components.
In order to solve the technical problems, the invention adopts the technical scheme that:
the invention provides a quality control method of a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises watermelon frost, calcined borax, golden cypress, coptis chinensis, subprostrate sophora, blackberry lily, thunberg fritillary bulb, natural indigo, borneol, soapberry, rhubarb, scutellaria baicalensis, liquorice and menthol, the quality control method comprises the step of simultaneously detecting the ingredients of the golden cypress, the scutellaria baicalensis, the blackberry lily and the liquorice in the traditional Chinese medicine composition by adopting an HPLC method, and the chromatographic conditions of the HPLC method are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking methanol as a mobile phase A and taking 0.1% phosphoric acid solution as a mobile phase B to carry out gradient elution; the theoretical plate number is not less than 300000 calculated according to baicalin peak;
the procedure of the gradient elution was as follows:
in the period of 0-9 min, the proportion of the mobile phase A is increased from 18% to 23%, and the proportion of the mobile phase B is decreased from 82% to 77%;
in a period of 9-15 min, the proportion of the mobile phase A is increased from 23% to 25%, and the proportion of the mobile phase B is decreased from 77% to 75%;
in a 15-27 min period, the proportion of the mobile phase A is maintained at 25%, and the proportion of the mobile phase B is maintained at 75%;
in the period of 27-36 min, the proportion of the mobile phase A is increased from 25% to 35%, and the proportion of the mobile phase B is decreased from 75% to 65%;
in a period of 36-50 min, the proportion of the mobile phase A is increased from 35% to 45%, and the proportion of the mobile phase B is decreased from 65% to 55%;
in a period of 50-75 min, the proportion of the mobile phase A is increased from 45% to 80%, and the proportion of the mobile phase B is decreased from 55% to 20%;
in a period of 75-88 min, the proportion of the mobile phase A is maintained at 80%, and the proportion of the mobile phase B is maintained at 20%;
in the period of 88-90 min, the proportion of the mobile phase A is reduced from 80% to 18%, and the proportion of the mobile phase B is increased from 20% to 82%.
Because the components of the five medicinal materials of the phellodendron, the coptis chinensis, the scutellaria baicalensis, the blackberry lily and the liquorice in the traditional Chinese medicine composition are detected and identified, in order to effectively separate various components contained in the medicine to obtain characteristic peaks with better separation degree, the invention explores the elution condition of liquid chromatography, so that 16 peaks with better separation degree can be obtained by single detection.
In the scheme, in order to better separate the components in multiple medicines, particularly berberine hydrochloride and palmatine hydrochloride, related workers of the invention perform experimental analysis on the selection of the mobile phase, respectively select methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid and methanol-acetonitrile-0.1% phosphoric acid as the mobile phase, when acetonitrile-0.1% phosphoric acid is selected for gradient elution, as shown in the attached figure 12 of the specification, the retention time of the berberine hydrochloride and the palmatine hydrochloride is about 33min, chromatographic peaks are not separated, and when methanol-acetonitrile-0.1% phosphoric acid is selected, as shown in the attached figure 13 of the specification, the retention time of the berberine hydrochloride and the palmatine hydrochloride is about 22min, and chromatographic peaks of the two components are overlapped. Therefore, methanol-0.1% phosphoric acid which can delay the retention time of berberine hydrochloride and can be separated from palmatine hydrochloride is selected as a mobile phase.
In the above scheme, further, considering the performances of mobile phases with different proportions and different acidity, methanol-water, methanol-0.05% phosphoric acid, and methanol-0.1% phosphoric acid are compared as mobile phases, i.e. the comparison of different acidity of the mobile phases. Fig. 14 to 16 of the attached drawings of the specification show that chromatograms of methanol-0.05% phosphoric acid shown in fig. 14 and methanol-0.1% phosphoric acid shown in fig. 15 as mobile phases are basically similar, and the chromatogram with methanol-water as a mobile phase is greatly different from that of an acid, wherein berberine hydrochloride and palmatine hydrochloride have very poor peak shapes and great peak retention time difference. Therefore, methanol-0.1% phosphoric acid is selected as a mobile phase and a 0.1% phosphoric acid solution is strictly prepared in the actual quality control activity.
The further scheme of the invention is as follows: the chromatographic condition of the HPLC method also comprises the step of collecting characteristic peak information by using a diode array detector with the detection wavelength of 260nm, so that the absorbance multiple difference between any two corresponding characteristic peaks of the phellodendron, the coptis chinensis, the radix scutellariae, the blackberry lily and the liquorice in the same chromatogram is not more than 20-40 times.
In the scheme, because the test sample has more traditional Chinese medicines and more complex components, a diode array detector (PDA) is adopted to obtain multi-level information, and the wavelength is selected in the ultraviolet region of 210-400 nm according to the spectrogram of a chromatographic peak of each reference substance, reference medicinal material and test sample solution at the same retention time. The attached figures 9-11 of the specification describe the spectrograms of the same Chinese medicinal composition sample solution detected by using three different wavelengths of 260nm, 273nm and 345 nm. It is obvious from the figure that the peak area in the 345nm chromatogram is obviously smaller, and the peak height of the chromatogram after 55min is obviously smaller than that of the chromatogram in the same time period under the wavelength of 260nm and 273 nm; compared with the 273nm chromatogram, the chromatogram peak of baicalin and related components in the 273nm chromatogram is obviously higher, so that the peak height of the ammonium glycyrrhizinate corresponding to the peak 16 is obviously lower, and the peak area 14612833 (baicalin, retention time 56.422min) of the peak 6 is about 100 times of the peak area 158225 (ammonium glycyrrhizinate, retention time 75.414min) of the peak 16. By combining the above, the detection wavelength-260 nm with more uniform peak height contrast is selected. The detection wavelength can enable a plurality of characteristic peaks corresponding to five medicinal materials to have good separation degree and proper display proportion in the same chromatogram, and the multiple difference between optional two characteristic peaks does not exceed 20-40 times.
The further scheme of the invention is as follows: the quality control method also comprises the steps of detecting reference substances corresponding to the golden cypress, the golden thread, the radix scutellariae, the blackberry lily and the liquorice through the chromatographic condition of the HPLC method, comparing and analyzing characteristic peak information in detection results of the test substance and the reference substances of the traditional Chinese medicine composition, constructing a quality control spectrogram of the traditional Chinese medicine composition according to the characteristic peak information, and detecting the traditional Chinese medicine composition product by using the quality control spectrogram.
In the scheme, the reference substance comprises a reference substance solution of a reference medicinal material prepared by using the reference medicinal material, a reference substance solution prepared by using one or more standard substances, a negative sample with one or more medicinal flavors of phellodendron amurense, coptis chinensis, scutellaria baicalensis, blackberry lily and liquorice which are absent on the basis of the traditional Chinese medicine composition, and the like.
The further scheme of the invention is as follows: the method for comparatively analyzing the characteristic peak information in the detection results of the test sample and the reference sample of the traditional Chinese medicine composition and constructing the quality control spectrogram of the traditional Chinese medicine composition according to the characteristic peak information comprises the following steps: comparing the peak area and retention time of each characteristic peak in HPLC chromatogram of the test sample and the reference sample of the Chinese medicinal composition, determining the characteristic peaks of all corresponding Chinese medicinal composition test samples, and constructing quality control chromatogram according to the characteristic peaks; preferably, the characteristic peak information further comprises spectral information collected by an HPLC instrument, and the characteristic peak of the corresponding test sample of the traditional Chinese medicine composition is determined by combining the spectral information on the basis of obtaining the peak area and retention time of the characteristic peak.
The further scheme of the invention is as follows: the step of detecting the traditional Chinese medicine composition product by using the quality control spectrogram comprises the following steps: and (3) comparing and analyzing the HPLC (high performance liquid chromatography) spectrum of the Chinese medicinal composition product to be detected with the quality control spectrum of the Chinese medicinal composition, and determining that the characteristic peak of the Chinese medicinal composition product to be detected is consistent with the characteristic peak in the quality control spectrum, namely the qualified product.
The invention also provides a quality control spectrogram of the traditional Chinese medicine composition, which comprises 16 chromatographic peaks sequentially numbered according to a peak appearance time sequence, wherein 1-5 peaks are from phellodendron and/or coptis chinensis; peaks 6-9, 11 and 13-15 are from scutellaria; peaks 10 and 12 are from belamcanda; peak 16 is from Glycyrrhiza uralensis; preferably, peak 1 is a characteristic peak of phellodendron amurense medicinal material, peak 4 is a characteristic peak of berberine hydrochloride, peak 6 is a characteristic peak of baicalin, peak 12 is a characteristic peak of irisflorentin, and peak 16 is a characteristic peak of ammonium glycyrrhetate; more preferably, in the quality control spectrogram of the traditional Chinese medicine composition, the peak 4 is taken as a first main peak with the relative retention time of 1, the relative retention times of the peaks 2 to 5 are calculated to be 0.87, 0.90, 1.00 and 1.03 in sequence, the peak 6 is taken as a second main peak with the relative retention time of 1, and the relative retention times of the peaks 6 to 11,13 to 15 are calculated to be 1.00, 1.05, 1.09, 1.10, 1.12, 1.18, 1.21, 1.25 and 1.28.
In the scheme, the HPLC method provided by the invention represents 16 characteristic peaks of 5 medicinal materials of coptis chinensis, phellodendron amurense, scutellaria baicalensis, blackberry lily and liquorice, and the 16 characteristic peaks are subjected to component confirmation or peak attribution confirmation through reference analysis of a test sample, a negative sample, a reference substance of a reference medicinal material and a standard reference substance. The figure 17 in the attached figure of the specification shows the positions of 16 peaks in a concise way, wherein peak 1 corresponds to the reference substance of the phellodendron amurense reference medicinal material, peak 4(S1) corresponds to berberine hydrochloride, peak 6(S2) corresponds to baicalin, peak 12 corresponds to sub-irigenin, and peak 16 corresponds to ammonium glycyrrhetate. The peak 1 and the peak 16 are respectively consistent with the retention time and the peak area of the phellodendron reference material and the first standard substance.
The invention also provides a construction method of the quality control spectrogram of the traditional Chinese medicine composition, which comprises the following steps:
(1) preparing a plurality of batches of traditional Chinese medicine compositions into a test solution, respectively preparing reference medicinal material solutions of phellodendron, coptis chinensis, radix scutellariae, blackberry lily and liquorice from reference medicinal materials, respectively preparing reference substance solutions of standard substances corresponding to part of characteristic peaks in a quality control spectrogram of the traditional Chinese medicine compositions, and finally removing a negative sample solution of one or more of the components of the phellodendron, the coptis chinensis, the radix scutellariae, the blackberry lily or the liquorice on the basis of the prescription of the traditional Chinese medicine compositions;
(2) detecting the test solution, the reference solution of the control drug material and the negative sample solution in the step (1) by adopting the HPLC chromatographic conditions as in claim 1 or 2;
(3) analyzing and comparing the HPLC chromatogram obtained in the step (2), constructing an HPLC characteristic spectrum of the traditional Chinese medicine composition, and confirming 16 characteristic peak information corresponding to a test sample of the traditional Chinese medicine composition to obtain the quality control spectrum;
and (4) the characteristic peak information in the step (3) comprises a peak area, a peak height, retention time and a spectral index.
In the scheme, the traditional Chinese medicine composition is selected from Guilin watermelon frost test samples, 20 batches of Guilin watermelon frost test samples are measured to obtain the Guilin watermelon frost test sample spectrum, then reference substances of reference medicinal materials, standard reference substances and negative samples are respectively measured to obtain characteristic peak information of each group of reference samples, the reference substances of the reference medicinal materials, the standard reference substances and the negative samples are respectively compared with the Guilin watermelon frost test sample spectrum to find out each peak with characteristic fingerprint significance in a finished product characteristic spectrum, and chemical components of the peaks are researched and identified, so that the Guilin watermelon frost quality control spectrum with 16 peaks is obtained.
The further scheme of the invention is as follows: the preparation method of the test solution comprises collecting 1g of Chinese medicinal composition product, placing in a conical flask with a plug, adding 25ml of extraction solvent, performing ultrasonic treatment for 40min, taking out, cooling, shaking, filtering, and collecting filtrate; the preparation method of the reference solution comprises collecting 1g of the reference medicinal material, placing in a conical flask with a plug, adding 25ml of extraction solvent, performing ultrasonic treatment for 40min, taking out, cooling, shaking, filtering, and collecting filtrate; preferably, the reference solution of the reference medicinal materials is prepared by respectively taking 1g of each of the reference medicinal materials of phellodendron amurense, coptis chinensis, scutellaria baicalensis, blackberry lily and liquorice, placing the reference medicinal materials in a conical flask with a plug, adding 25ml of an extraction solvent, carrying out ultrasonic treatment for 40 minutes, taking out, cooling, shaking up, filtering and taking out a filtrate.
In the above scheme, since it is preferable to prepare a mixed reference solution of multiple medicinal materials, the relevant staff of the present invention specially considers the selection of the extraction solvent and the extraction method, and selects the commonly used extraction solvents of methanol-hydrochloric acid (volume ratio 100:1), methanol, ethanol, 70% ethanol, methanol-water-phosphoric acid (volume ratio 50:50:0.2) and ethanol-water-phosphoric acid (volume ratio 70:30:0.2) in the prior art for comparison. After the mixed medicinal material reference substance of the multiple medicines is extracted in the solvent for 0h, 24h and 48h, the content detection is carried out, and the baicalin detection index corresponding to the baical skullcap root medicinal material is found to be reduced by more than 50% when the content after 24h is compared with that after 0h when methanol-hydrochloric acid (in a volume ratio of 100:1), methanol, ethanol and 70% ethanol are taken as extraction solvents, then methanol-water-phosphoric acid (in a volume ratio of 50:50:0.2) and ethanol-water-phosphoric acid (in a volume ratio of 70:30:0.2) are continuously selected for comparison, and the detection result after 48h has no obvious change with the peak area of 0h, and the methanol-water-phosphoric acid (in a volume ratio of 50:50:0.2) is similar to the composition of the mobile phase, and then methanol-water-phosphoric acid (in a volume ratio of 50:50:0.2) is selected.
In the specification, the accompanying drawings 1 and 2 are both extracted by using a solvent methanol-hydrochloric acid (volume ratio is 100:1), wherein the accompanying drawing 1 is a chromatogram of 0h, the accompanying drawing 2 is a chromatogram of 24h, peaks with the retention time of 50.287min in the accompanying drawing 1 and 50.280min in the accompanying drawing 2 are baicalin components, and the peak areas are reduced after 24 h.
In the specification, the attached fig. 3 and fig. 4 are both extracted by using 70% ethanol solvent, wherein fig. 1 is a chromatogram of 0h, fig. 2 is a chromatogram of 24h, peaks with the retention time of 55.961min in fig. 1 and 55.945min in fig. 2 are baicalin components, and the peak areas are reduced after 24 h.
In the specification, the solvent methanol extraction is adopted in the attached figures 5 and 6, wherein figure 1 is a chromatogram of 0h, figure 2 is a chromatogram of 24h, peaks with the retention time of 57.175min in figure 1 and 57.041min in figure 2 are baicalin components, and the peak areas are reduced after 24 h.
In the specification, the accompanying drawings 7 and 8 are both extracted by using a solvent of methanol-water-phosphoric acid (volume ratio is 50:50:0.2), wherein the chromatogram is 0h in the figure 1, the chromatogram is 48h in the figure 2, and the peaks with the retention time of 57.062min in the figure 1 and 57.024min in the figure 2 are baicalin components, so that the peak areas of the baicalin components have no obvious change after 48 h.
Therefore, the extraction solution is preferably selected from a methanol-water-phosphoric acid solution in a volume ratio of 50:50: 0.2.
The further scheme of the invention is as follows: the reference substance comprises one or more of berberine hydrochloride, palmatine hydrochloride, baicalin, wogonoside, baicalein, wogonin, irigenin and ammonium glycyrrhizinate; the preparation of the reference substance solution comprises dissolving standard substance ammonium glycyrrhizinate in 70% ethanol to obtain a first standard reference substance solution with concentration of 100 μ g/mL, dissolving standard substances berberine hydrochloride, baicalin and sub-wild tectorigenin in methanol to obtain a second standard reference substance solution with concentration of berberine hydrochloride of 100 μ g/mL, concentration of baicalin of 120 μ g/mL and concentration of sub-wild tectorigenin of 5 μ g/mL; preferably, the kit further comprises a third standard control solution, wherein the third standard control solution is prepared by the following steps: dissolving berberine hydrochloride, palmatine hydrochloride, baicalin, wogonin, baicalein, wogonin and irigenin as standard substances in methanol to obtain berberine hydrochloride and palmatine hydrochloride with concentration of 100 μ g/mL, baicalin, wogonoside, baicalein and wogonin with concentration of 120 μ g/mL, and irigenin with concentration of 5 μ g/mL.
In the above scheme, in order to accurately confirm the 16 characteristic peaks in the quality control spectrogram, in the process of constructing the quality control spectrogram, the reference medicinal materials of the five medicinal materials are respectively adopted to be made into reference medicinal material reference substances, and the information of the 16 characteristic peaks is comprehensively investigated by using the first standard solution and the third standard solution. After the quality control spectrogram is determined, when the traditional Chinese medicine composition product is actually detected, the main characteristic peak in 16 peaks can be identified and measured by adopting the mixed standard of the phellodendron reference medicinal material reference substance and the second standard reference substance solution, and the detection cost can be reduced on the basis of ensuring the detection precision.
The further scheme of the invention is as follows: the negative sample is prepared by removing one or more components of cortex Phellodendri, Coptidis rhizoma, Scutellariae radix, rhizoma Belamcandae or Glycyrrhrizae radix on the basis of the Chinese medicinal composition prescription; preferably, the negative samples include no phellodendron bark negative sample, no coptis root negative sample, no phellodendron bark negative sample, no scutellaria root negative sample, no blackberry lily negative sample and no licorice negative sample.
In the scheme, the phellodendron-free coptis chinensis control in the negative sample is a negative sample prepared by removing phellodendron and coptis chinensis in a traditional Chinese medicine composition prescription, and because phellodendron and coptis chinensis contain more alkaloids and contain berberine hydrochloride together, the control is prepared for conveniently carrying out common investigation on the phellodendron and coptis chinensis.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. in the quality control method of the traditional Chinese medicine composition, the conditions of the liquid chromatogram are explored and improved, so that the phellodendron, the coptis chinensis, the scutellaria baicalensis, the blackberry lily and the liquorice contained in the traditional Chinese medicine composition can be identified and detected in a single detection, and the detection efficiency is greatly improved;
2. according to the quality control method of the traditional Chinese medicine composition, negative sample solutions lacking all components are prepared respectively, and characteristic peaks corresponding to all the components are identified and analyzed by combining the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the test substance solution, so that the precision of quality control standard is improved;
3. in the method for controlling the quality of the traditional Chinese medicine composition, the reference substance group of the reference medicinal materials mixed by the five medicinal materials, the negative sample group lacking the five medicinal materials, and the characteristic peaks corresponding to the components in the reference substance group of the reference substance and the test solution can be identified and analyzed, so that the quality control quality is improved, and the detection efficiency is also improved.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, 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 without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a chromatogram of a sample solution of the present invention taken for 0h with methanol-hydrochloric acid (100:1) as the extraction solvent;
FIG. 2 is a chromatogram of a sample solution of the present invention taken 24 hours after methanol-hydrochloric acid (100:1) is selected as an extraction solvent;
FIG. 3 is a chromatogram of a sample solution of the present invention taken for 0 hour with 70% ethanol as the extraction solvent;
FIG. 4 is a chromatogram of a sample solution of the present invention selected from 70% ethanol as an extraction solvent for 24 hours;
FIG. 5 is a chromatogram of 0h when methanol is selected as the extraction solvent for the test solution of the present invention;
FIG. 6 is a chromatogram of a sample solution of the present invention taken 24 hours with methanol as an extraction solvent;
FIG. 7 is a chromatogram of 0h of a sample solution of the present invention using methanol-water-phosphoric acid (50:50:0.2) as an extraction solvent;
FIG. 8 is a chromatogram of a sample solution of the present invention taken for 48 hours with methanol-water-phosphoric acid (50:50:0.2) as the extraction solvent;
FIG. 9 is a chromatogram of a test solution of the present invention at a detection wavelength of 260 nm;
FIG. 10 is a chromatogram of a test solution of the present invention at a detection wavelength of 273 nm;
FIG. 11 is a chromatogram of a test solution of the present invention at a detection wavelength of 345 nm;
FIG. 12 is a chromatogram of a test solution of the present invention in the presence of acetonitrile-0.1% phosphoric acid as the mobile phase;
FIG. 13 is a chromatogram of a test solution of the present invention in the presence of methanol-acetonitrile-0.1% phosphoric acid as the mobile phase;
FIG. 14 is a chromatogram of a test solution of the present invention in the presence of methanol-0.05% phosphoric acid as the mobile phase;
FIG. 15 is a chromatogram of a test solution of the present invention in the presence of methanol-0.1% phosphoric acid as the mobile phase;
FIG. 16 is a chromatogram of a test solution of the present invention when the mobile phase is methanol-water;
FIG. 17 is a schematic diagram of a chromatogram of 16 characteristic peaks corresponding to five herbs in the present invention;
FIG. 18 is the spectrum information of the Guilin watermelon frost sample of peak 1, no Coptidis rhizoma negative sample and the reference substance of the phellodendron amurense reference in example 1 of the present invention;
FIG. 19 is the spectrum information of the phellodendron-free negative sample, phellodendron-free Coptidis rhizoma negative sample and Coptidis rhizoma control drug reference for Peak 1 in example 1 of the present invention;
FIG. 20 is the spectrum information of the Guilin watermelon frost test sample of peak 2 and peak 3, no phellodendron negative sample and no reference substance of the coptis chinensis control drug in example 1 of the present invention;
FIG. 21 is the spectrum information of the golden thread-free negative sample, golden thread-free negative sample and golden reference drug for peaks 2 and 3 in example 1 of the present invention;
FIG. 22 is the spectral information of the Guilin watermelon frost test sample and the third standard control for peaks 4 and 5 in example 1 of the present invention;
FIG. 23 is the spectral information of the phellodendron-free negative sample, the phellodendron-free negative sample and the phellodendron-free coptis negative sample for peaks 4 and 5 in example 1 of the present invention;
FIG. 24 is the spectrum information of the reference substance of the Coptidis rhizoma control drug and the reference substance of the Phellodendri cortex control drug for peaks 4 and 5 in example 1 of the present invention;
FIG. 25 is the spectrum information of the Guilin watermelon frost test sample, the third standard reference substance and the reference substance of the Scutellaria baicalensis Georgi reference substance for peaks 6 to 8 in example 1 of the present invention;
FIG. 26 is the spectral information of the Scutellaria-free negative samples for peaks 6 to 8 in example 1 of the present invention;
FIG. 27 is the spectrum information of the Guilin watermelon frost test sample, the third standard reference sample and the reference substance of the Scutellaria baicalensis Georgi reference drug for peak 9 and peak 11 in example 1 of the present invention;
FIG. 28 is spectral information of a Scutellaria-free negative sample for peaks 9 and 11 in example 1 of the present invention;
FIG. 29 is the spectrum information of the Guilin watermelon frost test sample, the third standard reference sample and the reference substance of the Scutellaria baicalensis Georgi reference drug for peaks 13 to 15 in example 1 of the present invention;
FIG. 30 is the spectral information of the Scutellaria-free negative sample for peak 13 to peak 15 in example 1 of the present invention;
FIG. 31 is the spectrum information of the Guilin watermelon frost sample, the blackberry lily reference and the blackberry lily-free negative sample for peak 10 in example 1 of the present invention;
FIG. 32 is the spectrum information of the Guilin watermelon frost test sample, the third standard reference sample and the blackberry lily reference drug for peak 12 in example 1 of the present invention;
FIG. 33 is the spectral information of a blackberry-free negative sample for peak 12 in example 1 of the present invention;
FIG. 34 is the spectrum information of the Guilin watermelon frost test sample, the first standard reference sample and the licorice reference drug for peak 16 in example 1 of the present invention;
FIG. 35 is spectral information of a licorice-free negative sample for peak 16 in example 1 of the present invention;
FIG. 36 is the chromatogram information of the negative samples of the reference substance of the mixed control drug, the test sample 180920 and the drug lacking five drugs in example 2 of the present invention;
FIG. 37 is an overlay chromatogram of a test sample 180920 and a negative sample of a five-drug deficient drug substance in example 2 of the present invention;
FIG. 38 is a graph of spectral indices for peak 3 interference peaks in example 2 of the present invention;
FIG. 39 is a graph of the spectral index of a Scutellaria-free negative sample for Peak 6 in example 2 of the present invention;
FIG. 40 is a graph of spectral indices for peak 6 interference peak in example 2 of the present invention;
FIG. 41 is a graph of spectral indices for peak 9 interference peak in example 2 of the present invention;
FIG. 42 is a graph of spectral indices for peak 12 interference peaks in example 2 of the present invention.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
In the embodiment, the traditional Chinese medicine composition is selected from a Guilin watermelon frost test sample containing watermelon frost, calcined borax, golden cypress, coptis chinensis, subprostrate sophora, blackberry lily, thunberg fritillary bulb, natural indigo, borneol, Chinese soapberry, rhubarb, scutellaria baicalensis, liquorice and menthol, and specific characteristic peak information corresponding to the golden cypress, the scutellaria baicalensis, the blackberry lily and the liquorice in the Guilin watermelon frost is examined by combining the test sample, a reference substance of a reference medicinal material, a reference substance of the reference substance and a plurality of negative samples, and the specific method is as follows:
firstly, selecting reference medicinal materials of phellodendron, coptis chinensis, scutellaria baicalensis, blackberry lily and liquorice to respectively prepare reference medicinal material solutions of all components, wherein the preparation method comprises the following steps: taking 1g of the reference medicinal material, placing the reference medicinal material in a conical flask with a plug, adding 25ml of methanol-water-phosphoric acid solution with the volume ratio of 50:50:0.2, carrying out ultrasonic treatment for 40 minutes, taking out, cooling, shaking up, filtering, and taking the filtrate to obtain the traditional Chinese medicine composition;
secondly, selecting a first standard reference substance solution of 100 mu g/mL prepared by dissolving a standard substance ammonium glycyrrhizinate in 70% ethanol, and preparing a third standard reference substance solution of 100 mu g/mL of berberine hydrochloride and palmatine hydrochloride, 120 mu g/mL of baicalin, wogonin, baicalein and wogonin and 5 mu g/mL of irigenin by dissolving standard substances berberine hydrochloride, palmatine hydrochloride, baicalin, wogonoside, wogonin and irigenin in methanol;
then, preparing a Guilin watermelon frost test solution according to the following method: putting 1g of Guilin watermelon crystal into a conical flask with a plug, adding 25ml of methanol-water-phosphoric acid solution with the volume ratio of 50:50:0.2, carrying out ultrasonic treatment for 40 minutes, taking out, cooling, shaking up, filtering, and taking the filtrate to obtain the traditional Chinese medicine composition;
then, on the basis of the test sample solution preparation method, a phellodendron-free negative sample solution, a coptis-free negative sample solution, a phellodendron-free coptis-negative sample solution, a scutellaria-free negative sample solution, a blackberry lily-free negative sample solution and a licorice-free negative sample solution are respectively prepared.
And respectively carrying out chromatographic analysis on the test samples, the reference substance of the reference medicinal material, the reference substance of the reference substance and the negative sample of each group, wherein the chromatographic conditions comprise that octadecylsilane chemically bonded silica is used as a chromatographic column filler, gradient elution is adopted, and a mobile phase comprises a mobile phase A: 0.1 wt% phosphoric acid solution and mobile phase B: methanol and a diode array detector with a detection wavelength of 260nm was used. The gradient elution method is shown in the following table:
Figure BDA0002262885270000061
Figure BDA0002262885270000071
and (3) performing specificity analysis of each characteristic peak according to the chromatogram and the spectrogram of the detection result, wherein the specificity analysis comprises the following steps:
for peak 1, peak 1 is detected in the phellodendron reference medicinal material, coptis chinensis-lacking negative sample and Guilin watermelon sample, but coptis chinensis-lacking phellodendron negative sample is not detected, a small peak is arranged at the corresponding position of peak 1 of phellodendron-lacking negative sample and coptis chinensis reference medicinal material, the peak height and the peak area are small, the similarity is low through software analysis, so that peak 1 is not considered as the common characteristic peak of phellodendron and is judged as the characteristic peak of phellodendron. As the absorption wavelength of the characteristic spectrum is comprehensively considered to be selected to be 260nm, the response value of the peak 1 at 260nm is lower, and the peak height value of the peak area is smaller, the integral parameters can be properly adjusted when the characteristic spectrum of the test sample is obtained, and the judgment can be carried out by combining the spectrum. The attached figure 18 of the specification records the spectral information of a Guilin watermelon frost test sample, a coptis chinensis deficiency negative sample and a golden cypress reference medicinal material, and the attached figure 19 of the specification records the spectral information of a phellodendron deficiency negative sample, a golden cypress deficiency negative sample and a golden cypress reference medicinal material, and can further verify that peak 1 corresponds to the component of the golden cypress.
For peak 2 and peak 3, peak 2 and peak 3 are detected in the reference substance of the coptis chinensis control medicinal material, the negative sample lacking golden cypress and the sample of Guilin watermelon frost, while the negative sample lacking golden cypress and the negative sample lacking golden cypress are not detected, so that the peak 2 and peak 3 can be judged as the characteristic peaks of the golden cypress. The attached figure 20 of the specification records the spectral information of the Guilin watermelon frost test sample, the phellodendron bark lacking negative sample and the coptis chinensis reference material, and the attached figure 21 of the specification records the spectral information of the phellodendron bark lacking negative sample and the phellodendron bark lacking coptis chinensis reference material, and can further verify that the peak 2 and the peak 3 correspond to the coptis chinensis components.
Peak 4 is berberine hydrochloride, peak 4 is detected in a reference substance of a coptis root reference medicinal material, a reference substance of a phellodendron reference medicinal material and a Guilin watermelon frost test sample, and the retention time and the spectrum of the berberine hydrochloride are consistent with those of berberine hydrochloride in a third standard reference substance; the negative sample of phellodendron bark lacking coptis is not detected, so that the peak 4 can be judged as the common characteristic peak of phellodendron bark lacking coptis; peak 5 is palmatine hydrochloride, peak 5 is detected in the reference substance of the coptis chinensis reference medicinal material and the Guilin watermelon frost test sample, and the retention time and the spectrum of the peak 5 are consistent with those of palmatine hydrochloride in a third standard reference substance; the cortex phellodendri reference substance and the golden thread-lacking cortex phellodendri negative sample are not detected, so that the peak 5 can be determined to be mainly from the golden thread reference substance and is a characteristic peak of golden thread. The attached drawing 22 of the specification records the spectral information of a Guilin watermelon frost test sample and a third standard reference sample, the attached drawing 23 of the specification records the spectral information of a phellodendron bark lacking negative sample, a coptis chinensis lacking negative sample and a phellodendron bark lacking negative sample, and the attached drawing 24 of the specification records the spectral information of a coptis chinensis reference material and a phellodendron bark reference material, so that the common components of phellodendron bark and coptis chinensis corresponding to peak 4 and peak 5 can be further verified.
As for the scutellaria baicalensis medicinal material, because the scutellaria baicalensis medicinal material contains more flavonoids and has larger absorption in the ultraviolet region of 270-280 nm, the peak area peak height value is still larger by comprehensively considering the absorption wavelength of a characteristic spectrum and selecting 260nm, 8 characteristic peaks (peak 6 baicalin, peak 7, peak 8, peak 9 wogonin, peak 11 baicalein, peak 13, peak 14 wogonin and peak 15) are marked, and the analysis is carried out.
Peak 6 is baicalin, the maximum ultraviolet absorption wavelength is 277nm, peak 6 is detected in a baical skullcap root reference medicinal material reference substance and a Guilin watermelon frost test sample, the retention time is consistent with that of baicalin in a third standard reference substance, and the spectrum is consistent; the negative sample lacking Scutellariae radix is not detected, so that peak 6 (baicalin) can be determined as characteristic peak of Scutellariae radix. Peaks 7 and 8 are characteristic peaks of Scutellariae radix, peak 7 has ultraviolet maximum absorption wavelength of 280nm, peak 8 has ultraviolet maximum absorption wavelength of 272nm, and peaks 7 and 8 are detected in reference substance of Scutellariae radix and arillus longan; the negative sample lacking the scutellaria baicalensis is not detected, so that the peak 7 and the peak 8 can be judged as the characteristic peaks of the scutellaria baicalensis. The attached figure 25 of the specification records the spectral information of the Guilin watermelon frost test sample, the third standard reference substance and the scutellaria baicalensis reference substance, and the attached figure 26 of the specification records the spectral information of the scutellaria baicalensis-lacking negative sample, and can further verify that the peak 6 to the peak 8 correspond to the scutellaria baicalensis component.
Peak 9 is wogonoside, the maximum ultraviolet absorption wavelength is 274nm, peak 9 is detected in the reference substance of radix Scutellariae reference material and Guilin watermelon frost sample, and the retention time and spectrum of the peak 9 are consistent with those of wogonoside in the third standard reference substance; the small peak of the negative sample without scutellaria at the later position of the retention time of the peak 9 is partially overlapped with the peak 9, the maximum ultraviolet absorption wavelength is 265nm, and the peak area of the peak height in the absorption wavelength chromatogram map at 260nm is about one tenth of the peak area of the peak 9 of the sample, so that the peak 9 (wogonoside) can be judged as the characteristic peak of scutellaria. Peak 11 is baicalein, the maximum ultraviolet absorption wavelength is 276-324 nm, peak 11 is detected in both a baical skullcap root reference medicinal material reference substance and a Guilin watermelon frost test sample, the retention time of the baicalein in a third standard reference substance is consistent, and the spectrum of the baicalein is consistent; the negative sample lacking Scutellariae radix is not detected, so that peak 11 (baicalein) can be determined as the characteristic peak of Scutellariae radix. The attached figure 27 of the specification records the spectral information of the Guilin watermelon frost test sample, the third standard reference substance and the scutellaria baicalensis reference substance, and the attached figure 28 of the specification records the spectral information of the scutellaria baicalensis-lacking negative sample, and can further verify that the peak 9 and the peak 11 correspond to the scutellaria baicalensis components.
Peak 13 and Peak 15 are characteristic peaks of Scutellariae radix, peak 13 has maximum ultraviolet absorption wavelength of 269nm, peak 15 has maximum ultraviolet absorption wavelength of 271nm, and peak 13 and Peak 15 are detected in reference substance of Scutellariae radix and Guilin watermelon frost sample; the negative sample lacking Scutellariae radix is not detected, so that peaks 13 and 15 can be determined as characteristic peaks of Scutellariae radix. Peak 14 is wogonin, the maximum ultraviolet absorption wavelength is 275nm, peak 14 is detected in the reference substance of radix Scutellariae reference material and Guilin watermelon frost sample, and the retention time and spectrum of the peak 14 are consistent with those of wogonin in the third standard reference substance; the negative sample without scutellaria was not detected, so peak 14 (wogonin) was determined to be a characteristic peak of scutellaria. The attached figure 29 of the specification records the spectral information of the Guilin watermelon frost test sample, the third standard reference substance and the scutellaria baicalensis reference substance, and the attached figure 30 of the specification records the spectral information of the scutellaria baicalensis-lacking negative sample, and can further verify that the peak 13 to the peak 15 correspond to the scutellaria baicalensis component.
The peak 10 is the belamcanda chinensis characteristic peak, the maximum ultraviolet absorption wavelength is 265nm, the peak 10 is detected in a reference drug of the belamcanda chinensis and a Guilin watermelon frost test sample, and the spectra are basically consistent; a small peak is partially overlapped with the peak 10 at a later position of the retention time of the belamcanda chinensis negative sample, the maximum ultraviolet absorption wavelength is 256nm, the peak area of the sample at the absorption wavelength of 260nm is about one fifth of the peak area of the sample, the influence on the spectrum of the sample is not great, and therefore the peak 10 can still be judged to be the belamcanda chinensis characteristic peak. Peak 12 is wild tectorigenin, the maximum ultraviolet absorption wavelength is 265nm, peak 12 is detected in reference substance of blackberry lily reference medicinal material and Guilin watermelon frost sample, the retention time is consistent with that of wild tectorigenin in the third standard reference substance, and the spectrum is consistent; and the negative sample of the belamcanda chinensis is not detected, so that the peak 12 (sub wild tectorigenin) can be judged as the characteristic peak of the belamcanda chinensis. The attached figure 31 of the specification records the spectral information of the Guilin watermelon frost test sample, the reference substance of the blackberry lily reference medicinal material and the blackberry lily absent negative sample, and can further verify that the peak 10 corresponds to the blackberry lily component. The attached figure 32 of the specification records the spectral information of a Guilin watermelon frost test sample, a third standard reference substance and a blackberry lily reference medicinal material reference substance, and the attached figure 33 of the specification records the spectral information of a blackberry lily lacking negative sample, and can further verify that the peak 12 corresponds to the blackberry lily component.
Peak 16 is ammonium glycyrrhizinate, the maximum ultraviolet absorption wavelength is 251nm, peak 16 is detected in the reference material of the licorice reference medicinal material and the Guilin watermelon frost test sample, the retention time is consistent with that of the ammonium glycyrrhizinate in the first standard reference sample, and the spectrum is consistent; the negative sample without liquorice is not detected, so that the peak 16 (ammonium glycyrrhizinate) can be judged as the characteristic peak of liquorice. The accompanying drawing 34 of the specification records the spectrum information of the Guilin watermelon frost test sample, the first standard reference substance and the reference substance of the licorice reference medicinal material, and the accompanying drawing 35 of the specification records the spectrum information of the negative sample lacking licorice, which can further verify that the peak 16 corresponds to the licorice component.
Example 2
In the embodiment, the traditional Chinese medicine composition is selected from a Guilin watermelon frost test sample containing watermelon frost, calcined borax, golden cypress, coptis chinensis, subprostrate sophora, blackberry lily, thunberg fritillary bulb, natural indigo, borneol, Chinese soapberry, rhubarb, scutellaria baicalensis, liquorice and menthol, and specific characteristic peak information corresponding to the golden cypress, the scutellaria baicalensis, the blackberry lily and the liquorice in the Guilin watermelon frost is examined by combining the test sample, a reference substance of a reference medicinal material, a reference substance of the reference substance and a plurality of negative samples, and the specific method is as follows:
firstly, selecting a reference medicinal material of phellodendron, coptis chinensis, scutellaria baicalensis, blackberry lily and liquorice to prepare a reference medicinal material reference substance solution of mixed components, wherein the preparation method comprises the following steps: taking 1g of each reference medicinal material, placing into a conical flask with a plug, adding 25ml of methanol-water-phosphoric acid solution with the volume ratio of 50:50:0.2, carrying out ultrasonic treatment for 40 minutes, taking out, cooling, shaking up, filtering, and taking the filtrate to obtain the traditional Chinese medicine composition;
secondly, selecting a first standard reference substance solution of 100 mu g/mL prepared by dissolving a standard substance ammonium glycyrrhizinate in 70% ethanol, and dissolving standard substances berberine hydrochloride, palmatine hydrochloride, baicalin, wogonoside, baicalein, wogonin and irigenin in methanol to prepare a third standard reference substance solution of 100 mu g/mL berberine hydrochloride and palmatine hydrochloride, 120 mu g/mL baicalin, wogonoside, baicalein and wogonin and 5 mu g/mL irigenin;
then, preparing a Guilin watermelon frost test solution according to the following method: putting 1g of Guilin watermelon crystal into a conical flask with a plug, adding 25ml of methanol-water-phosphoric acid solution with the volume ratio of 50:50:0.2, carrying out ultrasonic treatment for 40 minutes, taking out, cooling, shaking up, filtering, and taking the filtrate to obtain the traditional Chinese medicine composition;
then, on the basis of the test sample solution preparation method, a negative sample solution free of phellodendron, coptis, scutellaria, blackberry lily and liquorice is prepared.
And respectively carrying out chromatographic analysis on the test samples, the reference substance of the reference medicinal material, the reference substance of the reference substance and the negative sample of each group, wherein the chromatographic conditions comprise that octadecylsilane chemically bonded silica is used as a chromatographic column filler, gradient elution is adopted, and a mobile phase comprises a mobile phase A: 0.1 wt% phosphoric acid solution and mobile phase B: methanol and a diode array detector with a detection wavelength of 260nm was used. The gradient elution method is shown in the following table:
Figure BDA0002262885270000081
Figure BDA0002262885270000091
and (3) performing specificity analysis of each characteristic peak according to the chromatogram and the spectrogram of the detection result, wherein the specificity analysis comprises the following steps:
the detection result shows that the reference substance of the reference medicinal material prepared by the mixed powder of the five medicinal materials of the phellodendron, the coptis chinensis, the scutellaria baicalensis, the blackberry lily and the liquorice is consistent with the spectrogram of a test sample prepared by Guilin watermelon frost medicines (180920 batches), and 16 chromatographic peaks calibrated in the established standard are detected; small interference peaks are arranged at the positions before and after the retention time of peak 3, peak 6, peak 9, peak 10 and peak 12 in the negative sample spectrogram of the absent five medicinal materials, and the 16 chromatographic peaks can still be calibrated by the spectrogram of a test sample prepared from Guilin watermelon frost medicament (180920 batches) after the interference peaks are subtracted (see the attached figure 36 and figure 37 in the specification); comparing the chromatogram and the spectrogram, the retention time of the interference peak and the calibrated chromatographic peak and the spectrogram are not completely consistent, and the analysis is shown later.
The retention time of the interference peak 3 is inconsistent with that of the calibrated chromatographic peak, the retention time of the interference peak is 41.506, the retention time of the peak 3 of the test sample 180920 is 41.0456, and the comparison of the spectral index diagrams shows that the interference does not exist in the peak 3, so that the interference peak 3 can be judged to be a peak after the retention time of the calibrated peak 3, and the retention time of the calibrated peak 3 is different by about 0.5 minute. The spectral index is shown in FIG. 38, and the retention times and peak areas are shown in the following table:
name (R) Retention time (min) Peak area Peak height
Reference substance of mixed reference medicinal materials 40.956 824082 68030
Test sample 180920 41.045 360307 30832
Lack of five negative samples 41.506 141931 10962
The retention time of interference peak 6 was 56.361, which was similar to that of test sample 180920 peak 6 (retention time 56.368), and the peak area of interference peak 6 was 238128, which was about 2.4% of the peak area of test sample 180920 peak 6 (9956358). From the spectrum index chart 39 of the scutellaria-lacking negative sample, it can be seen that the peak 6 is not interfered, so that the interference of the interference peak 6 is determined to be very small and can be ignored. The spectrum index chart 40 of the mixed reference drug and negative sample is shown in the table below for retention time and peak area:
name (R) Retention time (min) Peak area Peak height
Reference substance of mixed reference medicinal materials 56.416 11541483 852254
Test sample 180920 56.368 9956358 738888
Lack of five negative samples 56.361 238128 19069
The retention time of interference peak 9 was 62.074, which is similar to that of test sample 180920 peak 9 (retention time 62.047), and the peak area of interference peak 9 was 101934, which is about 4.0% of the peak area (2547852) of peak 9 of test sample 180920. The interference is small, and therefore the interference of the interference peak 9 can be determined to be small and negligible. The spectral index is shown in FIG. 41, and the retention times and peak areas are shown in the following table:
name (R) Retention time (min) Peak area Peak height
Reference substance of mixed reference medicinal materials 62.061 2765383 243637
Test sample 180920 62.047 2547852 222679
Lack of five negative samples 62.074 101934 7309
The retention time of the interference peak 10 is 63.293, which is similar to the retention time of the 180920 peak 10 (retention time 63.281) of the sample, the maximum ultraviolet absorption wavelength of the interference peak 10 is 256nm, the peak area (254106) of the absorption wavelength at 260nm is about one third of the peak area of the 180920(662330) of the sample, and the influence on the spectrum of the sample is small, so that the interference peak 10 has certain interference and the peak 10 can be still determined as a characteristic peak. The spectral index is shown in FIG. 41, and the retention times and peak areas are shown in the following table:
Figure BDA0002262885270000092
Figure BDA0002262885270000101
the retention time of the interference peak 12 is inconsistent with that of the calibrated chromatographic peak, the retention time of the interference peak is 67.275, the retention time of the peak 12 of the test sample 180920 is 67.800, and the comparison of the spectral index diagrams shows that the peak 12 is not interfered, so that the interference peak 12 can be judged to be a peak before the retention time of the calibrated peak 12 and has a retention time difference with that of the calibrated peak 12 of about 0.55 minutes. The spectral index is shown in FIG. 42, the retention times and peak areas are shown in the following table:
name (R) Retention time (min) Peak area Peak height
Reference substance of mixed reference medicinal materials 67.829 115149 12251
Test sample 180920 67.800 192831 20026
Lack of five negative samples 67.275 123014 11836
Example 3
In this example, the accuracy and durability of the characteristic spectrum method described in example 1 or 2 were measured.
A. Precision test
(1) Repeatability tests and ranges
Taking a test sample (batch number: 180920) of the same batch number, designing 3 different sample concentrations, namely taking 3 different sample quantities (0.5g, 1g and 1.5g), respectively preparing 3 test sample solutions for determination according to the preparation method of the test sample, calculating the RSD% value of relative retention time, and calculating that the RSD% values of 16 characteristic peaks of the result relative retention time are all less than 1, which indicates that the repeatability meets the requirement. The result shows that the sample sampling quantity can obtain a characteristic map within the range of 0.5-1.5 g, so that the sample sampling quantity is determined as follows: about 1g of this product was collected.
(2) Intermediate precision and stability of test sample
Taking a test sample (batch number: 180920) of the same batch number, preparing more than 6 test sample solutions in parallel for determination, calculating the RSD% value of the relative retention time, and calculating the RSD% values of 16 characteristic peaks of the result relative retention time to be less than 1, which indicates that the intermediate precision meets the requirement. The same sample solution is measured at different times, the result shows that the sample can obtain a consistent spectrogram within 24 hours, and the RSD% values of 16 characteristic peaks relative to the retention time are all less than 1, which shows that the sample solution is stable within 24 hours.
B. Durability test
(1) Concentration of extraction solvent
Taking a sample (batch number: 180920) of the same batch, designing 3 different extraction solvent concentrations, namely methanol-water-phosphoric acid solutions, to be prepared according to a ratio of 45:55:0.2, a ratio of 50:50:0.2 and a ratio of 55:45:0.2 respectively, preparing 3 sample solutions according to the preparation method of the sample according to the concentration of each solvent, measuring, calculating the RSD% value of relative retention time, calculating that the RSD% values of 16 characteristic peaks of the result relative retention time are all smaller than 1, and obtaining consistent spectrograms, which shows that the influence of tiny changes of the concentration of the extraction solvent on the measurement result is small.
(2) Extraction time
Taking a test sample (batch number: 180920) of the same batch number, designing 3 different extraction times (30min, 40min and 50min), respectively preparing 3 test sample solutions at each extraction time according to the test sample preparation method, measuring, calculating the RSD% value of the relative retention time, calculating that the RSD% values of 16 characteristic peaks of the result relative to the retention time are all less than 1, and obtaining consistent spectrograms to show that the influence of the tiny change of the extraction time on the measurement result is small.
(3) Different flow rates
Taking a same batch of test articles (batch number: 180920), designing 3 different flow rates (0.9mL/min, 1.0mL/min and 1.1mL/min), respectively preparing 3 test article solutions at each extraction time according to the test article preparation method, determining, calculating the RSD% value of relative retention time, and obtaining a spectrogram with consistent results, wherein the RSD% values of the remaining 15 characteristic peaks relative retention time are less than 1.5 except that the RSD% value of peak 1 relative retention time is 5.8%, and the obtained spectrogram shows that the influence of small change of the flow rate on the determination result is small.
(4) Different sample introduction volumes
Taking a sample (batch number: 180920) of the same batch number, preparing a sample solution according to a sample preparation method, respectively injecting 2 mu L, 5 mu L and 10 mu L for determination, calculating RSD% values of relative retention time, wherein the RSD% values of 16 characteristic peaks relative to the retention time are all less than 1, and the peak area of the chromatogram peak obtained by injecting 2 mu L and 5 mu L is smaller and is not beneficial to marking of the characteristic peak, so the injection volume is defined as 10 mu L.
(5) Different column temperatures
Taking a test sample (batch number: 180920) of the same batch number, preparing a test sample solution according to a test sample preparation method, respectively measuring at the column temperature conditions of 30 ℃, 35 ℃ and 40 ℃, calculating the RSD% value of relative retention time, and obtaining consistent spectrograms, wherein the RSD% values of the remaining 14 characteristic peaks relative retention time are less than 2 except that the RSD% value of peak 1 relative retention time is 8.5%, the RSD% value of peak 16 relative retention time is 2.3%, and the obtained spectrograms indicate that the column temperature change has certain influence on the measurement result.
(6) Chromatographic column
Three columns from different manufacturers were selected for comparison, and the column type specifications are given in the following table. Preparing a test sample solution according to a test sample preparation method, measuring at the condition of 35 ℃ column temperature, and obtaining the spectrogram with consistent results, wherein the RSD% values of the remaining 15 characteristic peaks relative to the retention time are less than 1.5 except that the RSD% value of the peak 1 relative to the retention time is 4.9%. According to the test results, the chromatographic conditions were determined as: octadecylsilane chemically bonded silica is used as a filling agent; the number of theoretical plates is not less than 300000 calculated according to baicalin peak.
Figure BDA0002262885270000111
Example 4
In this embodiment, the quality control spectrogram constructed in embodiment 1 or 2 is used to detect the Guilin watermelon frost drug, a reference substance of a phellodendron bark reference drug and a second standard reference substance are used to prepare a mixed standard, and meanwhile, the HPLC (high performance liquid chromatography) spectrum of the Guilin watermelon frost drug to be detected and the quality control spectrogram are compared and analyzed by using traditional Chinese medicine chromatography fingerprint similarity evaluation software, so that all characteristic peaks appear in the Guilin watermelon frost product to be detected, and the similarity of all characteristic peaks is greater than 0.90, and the Guilin watermelon frost drug is determined to be a qualified product.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (14)

1. A quality control method of a traditional Chinese medicine composition comprises watermelon frost, calcined borax, golden cypress, golden thread, subprostrate sophora, blackberry lily, thunberg fritillary bulb, indigo naturalis, borneol, soapberry fruit, rhubarb, scutellaria baicalensis, liquorice and menthol, and is characterized in that the quality control method comprises the step of simultaneously detecting the ingredients of the golden cypress, the golden thread, the scutellaria baicalensis, the blackberry lily and the liquorice in the traditional Chinese medicine composition by adopting an HPLC method, wherein the chromatographic conditions of the HPLC method are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking methanol as a mobile phase A and taking 0.1% phosphoric acid solution as a mobile phase B to carry out gradient elution; the theoretical plate number is not less than 300000 calculated according to baicalin peak;
the procedure of the gradient elution was as follows:
in the period of 0-9 min, the proportion of the mobile phase A is increased from 18% to 23%, and the proportion of the mobile phase B is decreased from 82% to 77%;
in a period of 9-15 min, the proportion of the mobile phase A is increased from 23% to 25%, and the proportion of the mobile phase B is decreased from 77% to 75%;
in a 15-27 min period, the proportion of the mobile phase A is maintained at 25%, and the proportion of the mobile phase B is maintained at 75%;
in the period of 27-36 min, the proportion of the mobile phase A is increased from 25% to 35%, and the proportion of the mobile phase B is decreased from 75% to 65%;
in a period of 36-50 min, the proportion of the mobile phase A is increased from 35% to 45%, and the proportion of the mobile phase B is decreased from 65% to 55%;
in a period of 50-75 min, the proportion of the mobile phase A is increased from 45% to 80%, and the proportion of the mobile phase B is decreased from 55% to 20%;
in a period of 75-88 min, the proportion of the mobile phase A is maintained at 80%, and the proportion of the mobile phase B is maintained at 20%;
in the period of 88-90 min, the proportion of the mobile phase A is reduced from 80% to 18%, and the proportion of the mobile phase B is increased from 20% to 82%;
the quality control method also comprises detecting reference substances corresponding to cortex Phellodendri, Coptidis rhizoma, Scutellariae radix, rhizoma Belamcandae and Glycyrrhrizae radix by HPLC chromatography conditions, comparing and analyzing characteristic peak information in test and reference detection results of the Chinese medicinal composition, and constructing Chinese medicinal composition quality control spectrogram, and detecting Chinese medicinal composition product with the quality control spectrogram;
the preparation method of the test solution comprises collecting 1g of Chinese medicinal composition, placing in a conical flask with a plug, adding 25ml of extraction solvent, performing ultrasonic treatment for 40min, taking out, cooling, shaking, filtering, and collecting filtrate;
the extraction solvent is selected from methanol-water-phosphoric acid solution with volume ratio of 50:50: 0.2.
2. The method for quality control of a Chinese medicinal composition according to claim 1, wherein the chromatographic conditions of the HPLC method further comprise collecting characteristic peak information using a diode array detector with a detection wavelength of 260 nm.
3. The method for controlling the quality of a Chinese medicinal composition according to claim 1, wherein the step of comparatively analyzing the characteristic peak information of the test results of the Chinese medicinal composition and the reference samples and constructing the quality control spectrogram of the Chinese medicinal composition based on the characteristic peak information comprises the steps of: comparing the peak area and retention time of each characteristic peak in HPLC chromatogram of the test sample and the reference sample, determining the characteristic peak of all corresponding test samples of the Chinese medicinal composition, and constructing quality control chromatogram based on the characteristic peak.
4. The method for controlling the quality of a Chinese medicinal composition according to claim 3, wherein the characteristic peak information further comprises spectral information collected by an HPLC instrument, and the characteristic peak of the corresponding Chinese medicinal composition sample is determined by combining the spectral information on the basis of obtaining the peak area and retention time of the characteristic peak.
5. The method for quality control of a Chinese medicinal composition according to claim 3, wherein the detecting the Chinese medicinal composition product by using the quality control spectrogram comprises: and (3) comparing and analyzing the HPLC (high performance liquid chromatography) spectrum of the Chinese medicinal composition product to be detected with the quality control spectrum of the Chinese medicinal composition, and determining that the characteristic peak of the Chinese medicinal composition product to be detected is consistent with the characteristic peak in the quality control spectrum, namely the qualified product.
6. A method for constructing a quality control spectrogram of a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises watermelon frost, calcined borax, golden cypress, coptis chinensis, subprostrate sophora, blackberry lily, thunberg fritillary bulb, indigo naturalis, borneol, soapberry fruit, rhubarb, scutellaria baicalensis, liquorice and menthol crystal, and is characterized in that the method comprises the following steps:
(1) preparing a plurality of batches of traditional Chinese medicine compositions into a test solution, respectively preparing reference medicinal material solutions of phellodendron, coptis chinensis, radix scutellariae, blackberry lily and liquorice from reference medicinal materials, respectively preparing reference substance solutions of standard substances corresponding to part of characteristic peaks in a quality control spectrogram of the traditional Chinese medicine compositions, and finally removing a negative sample solution of one or more of the components of the phellodendron, the coptis chinensis, the radix scutellariae, the blackberry lily or the liquorice on the basis of the prescription of the traditional Chinese medicine compositions;
(2) detecting the sample solution, the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the negative sample solution in the step (1) by adopting the chromatographic conditions of the following HPLC methods:
octadecylsilane chemically bonded silica is used as a filling agent; taking methanol as a mobile phase A and taking 0.1% phosphoric acid solution as a mobile phase B to carry out gradient elution; the theoretical plate number is not less than 300000 calculated according to baicalin peak;
the procedure of the gradient elution was as follows:
in the period of 0-9 min, the proportion of the mobile phase A is increased from 18% to 23%, and the proportion of the mobile phase B is decreased from 82% to 77%;
in a period of 9-15 min, the proportion of the mobile phase A is increased from 23% to 25%, and the proportion of the mobile phase B is decreased from 77% to 75%;
in a 15-27 min period, the proportion of the mobile phase A is maintained at 25%, and the proportion of the mobile phase B is maintained at 75%;
in the period of 27-36 min, the proportion of the mobile phase A is increased from 25% to 35%, and the proportion of the mobile phase B is decreased from 75% to 65%;
in a period of 36-50 min, the proportion of the mobile phase A is increased from 35% to 45%, and the proportion of the mobile phase B is decreased from 65% to 55%;
in a period of 50-75 min, the proportion of the mobile phase A is increased from 45% to 80%, and the proportion of the mobile phase B is decreased from 55% to 20%;
in a period of 75-88 min, the proportion of the mobile phase A is maintained at 80%, and the proportion of the mobile phase B is maintained at 20%;
in the period of 88-90 min, the proportion of the mobile phase A is reduced from 80% to 18%, and the proportion of the mobile phase B is increased from 20% to 82%;
(3) analyzing and comparing the HPLC chromatogram obtained in the step (2), constructing an HPLC characteristic spectrum of the traditional Chinese medicine composition, and confirming 16 characteristic peak information corresponding to a test sample of the traditional Chinese medicine composition to obtain the quality control spectrum;
the preparation method of the test solution comprises collecting 1g of Chinese medicinal composition, placing in a conical flask with a plug, adding 25ml of extraction solvent, performing ultrasonic treatment for 40min, taking out, cooling, shaking, filtering, and collecting filtrate;
the extraction solvent is selected from methanol-water-phosphoric acid solution with volume ratio of 50:50: 0.2.
7. The method for constructing the quality control spectrogram of the Chinese medicinal composition according to claim 6, wherein the preparation of the reference solution of the reference medicinal material comprises taking 1g of the reference medicinal material, placing in a conical flask with a plug, adding 25ml of an extraction solvent, performing ultrasonic treatment for 40 minutes, taking out, cooling, shaking up, filtering, and taking the filtrate.
8. The method for constructing quality control spectrogram of Chinese medicinal composition according to claim 7, wherein the preparation of the reference solution of reference medicinal materials comprises respectively taking 1g each of cortex Phellodendri, Coptidis rhizoma, Scutellariae radix, rhizoma Belamcandae and Glycyrrhrizae radix, placing in a conical flask with a plug, adding 25ml of extraction solvent, performing ultrasonic treatment for 40min, taking out, cooling, shaking, filtering, and collecting filtrate.
9. The method for constructing quality control spectrogram of the Chinese medicinal composition according to claim 6, wherein the reference substance comprises berberine hydrochloride, palmatine hydrochloride, baicalin, wogonoside, baicalein, wogonin, irigenin, and ammonium glycyrrhizinate; the reference substance solution of the reference substance is prepared by dissolving ammonium glycyrrhizinate as a standard substance in 70% ethanol to obtain a first reference substance solution with a concentration of 100 μ g/mL, and dissolving berberine hydrochloride, baicalin and irisflorentin as standard substances in methanol to obtain a second reference substance solution with a concentration of berberine hydrochloride of 100 μ g/mL, a concentration of baicalin of 120 μ g/mL and a concentration of irisflorentin of 5 μ g/mL.
10. The method for constructing the quality control spectrogram of the Chinese medicinal composition according to claim 9, further comprising a third standard control solution, wherein the third standard control solution is prepared by the following steps: dissolving standard berberine hydrochloride, palmatine hydrochloride, baicalin, wogonin, baicalein, wogonin and irigenin, into methanol to obtain berberine hydrochloride and palmatine hydrochloride with concentration of 100 μ g/mL, baicalin, wogonoside, baicalein and wogonin with concentration of 120 μ g/mL, and irigenin with concentration of 5 μ g/mL;
the negative sample is prepared by removing one or more components of cortex Phellodendri, Coptidis rhizoma, Scutellariae radix, rhizoma Belamcandae or Glycyrrhrizae radix on the basis of Chinese medicinal composition prescription.
11. The method for constructing the quality control spectrogram of the Chinese medicinal composition according to claim 10, wherein the negative samples comprise no cortex Phellodendri negative sample, no Coptidis rhizoma negative sample, no cortex Phellodendri Coptidis rhizoma negative sample, no Scutellariae radix negative sample, no Belamcanda chinensis negative sample and no Glycyrrhrizae radix negative sample.
12. The method for constructing the quality control spectrogram of the traditional Chinese medicine composition according to any one of claims 6 to 11, wherein the quality control spectrogram constructed according to the construction method comprises 16 chromatographic peaks which are sequentially numbered according to the peak appearance time sequence, wherein the peaks 1 to 5 are from phellodendron amurense and/or coptis chinensis; peaks 6-9, 11 and 13-15 are from scutellaria; peaks 10 and 12 are from belamcanda; peak 16 is from Glycyrrhiza uralensis.
13. The method for constructing a quality control spectrogram of the Chinese medicinal composition according to claim 12, wherein Peak 1 is a characteristic peak of cortex Phellodendri, Peak 4 is a characteristic peak of berberine hydrochloride, Peak 6 is a characteristic peak of baicalin, Peak 12 is a characteristic peak of irigenin, and Peak 16 is a characteristic peak of ammonium glycyrrhizinate.
14. The method for constructing the quality control spectrogram of the traditional Chinese medicine composition according to claim 13, wherein in the quality control spectrogram constructed by the method, the peak 4 is taken as a first main peak with the relative retention time of 1, the relative retention times of the calculated peaks 2-5 are 0.87, 0.90, 1.00 and 1.03 in sequence, the peak 6 is taken as a second main peak with the relative retention time of 1, and the relative retention times of the calculated peaks 6-11, 13-15 are 1.00, 1.05, 1.09, 1.10, 1.12, 1.18, 1.21, 1.25 and 1.28.
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