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

Abstract

The invention discloses a quality control method of a traditional Chinese medicine composition and a construction method of a quality control spectrogram. The traditional Chinese medicine composition comprises mirabilitum praeparatum, calcined borax, golden cypress, coptis chinensis, subprostrate sophora, blackberry lily, thunberg fritillary bulb, indigo naturalis, borneol, soapberry fruit, rheum officinale, scutellaria baicalensis, liquorice and menthol. The ultra-high performance liquid chromatography is used for at least simultaneously detecting scutellaria baicalensis, golden cypress, coptis chinensis, rheum officinale, blackberry lily, liquorice and indigo naturalis in a traditional Chinese medicine composition test sample. The chromatographic conditions are as follows: octadecyl bonded silica gel is used as a filler; acetonitrile is used as a mobile phase A, a 0.1%-0.2% phosphoric acid aqueous solution containing 0.1%-0.2% of triethylamine is used as a mobile phase B, and gradient elution is carried out. According to the quality control method disclosed by the invention, a plurality of reference substances of reference substances and reference substances of reference medicinal materials are designed, at least characteristic peak information corresponding to the ingredients of scutellaria baicalensis, golden cypress, coptis chinensis, rheum officinale, blackberry lily, liquorice and indigo naturalis is obtained through contrastive analysis, and the ingredients can be simultaneously determined in one-time sample injection detection, so that the quality control level of a product is improved.

Description

Technical Field

[0001] This invention belongs to the field of drug quality control, specifically, it relates to a method for quality control of a traditional Chinese medicine composition and a method for constructing a quality control spectrum. Background Technology

[0002] Guilin Watermelon Frost has the effects of clearing heat and detoxifying, reducing swelling and relieving pain. It is mainly used to treat tonsillitis, sore throat, and oral ulcers caused by wind-heat attacking upwards and excessive heat in the lungs and stomach, with symptoms such as sore throat, enlarged tonsils, sores in the mouth and tongue, swollen and bleeding gums; acute and chronic pharyngitis, tonsillitis, stomatitis, oral ulcers, gingivitis with the above symptoms, and minor burns (without broken skin).

[0003] Guilin Watermelon Frost is a traditional Chinese medicine composition, comprising watermelon frost, calcined borax, phellodendron bark, coptis rhizome, sophora root, belamcanda rhizome, fritillaria bulb, indigo naturalis, borneol, soapberry fruit (charred), rhubarb, scutellaria root, licorice root, and menthol. Current drug standards primarily employ thin-layer chromatography (TLC) to identify multiple medicinal materials in a prescription using a single-component or main spot identification method. This method requires multiple arduous tests on the sample using TLC, is complex, labor-intensive, and susceptible to human influence, resulting in poor stability and repeatability. Furthermore, the components contained in traditional Chinese medicinal materials are complex; for example, phellodendron bark contains various alkaloids, leading to complex characteristic peak patterns in liquid chromatography, manifesting as impurity peaks and interfering peaks, which affect the identification and determination of the corresponding components. In addition, different medicinal materials have significant differences in the properties, polarity, and acidity / alkalinity of their components, making it difficult to simultaneously reflect these differences in a chromatogram under the same conditions. Detecting multiple components separately would undoubtedly increase the complexity of quality control and testing costs.

[0004] Chinese patent application No. 201911077339.0 discloses a quality control method, quality control spectrum, and construction method for a traditional Chinese medicine composition. The quality control method includes detecting the components in the traditional Chinese medicine composition using HPLC. The HPLC conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material; methanol is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B for gradient elution; the theoretical plate number, calculated based on the baicalin peak, should not be less than 300,000. Based on the HPLC results of each drug component, the quality control method designs multiple reference standards and reference medicinal materials, and can simultaneously determine multiple components in the traditional Chinese medicine composition in a single injection. This technical solution only detects five components in the traditional Chinese medicine composition. To more accurately detect more components in the traditional Chinese medicine composition and improve the quality control method, the inventors have proposed this invention. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a quality control method for traditional Chinese medicine compositions and a method for constructing quality control spectra. The quality control method of this invention uses ultra-high performance liquid chromatography, which can detect at least Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, and Indigo naturalis in a single injection. This not only has high detection efficiency, but also improves the quality control level of traditional Chinese medicine composition products, which is conducive to improving the quality of traditional Chinese medicine composition products.

[0006] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0007] This invention provides a quality control method for a traditional Chinese medicine composition, comprising watermelon frost, calcined borax, phellodendron bark, coptis root, sophora root, belamcanda rhizome, fritillaria bulb, indigo naturalis, borneol, soapberry fruit, rhubarb, scutellaria root, licorice root, and menthol. The method is characterized by employing ultra-high performance liquid chromatography (UHPLC) to simultaneously detect at least the components of scutellaria root, phellodendron bark, coptis root, rhubarb, belamcanda rhizome, licorice root, and indigo naturalis in the sample. The chromatographic conditions include: using octadecyl-bonded silica gel as the stationary phase; using acetonitrile as mobile phase A and a phosphoric acid aqueous solution containing triethylamine as mobile phase B, performing gradient elution; the theoretical plate number, calculated based on the baicalin peak, should be no less than 5000; the gradient elution process includes:

[0008] During the 0–10 min period, the proportion of mobile phase A increased from 20% to 24%, while the proportion of mobile phase B decreased from 80% to 76%.

[0009] During the 10–23 min period, the proportion of mobile phase A increased from 24% to 40%, while the proportion of mobile phase B decreased from 76% to 60%.

[0010] During the period of 23 to 26 minutes, the proportion of mobile phase A was maintained at 40%, and the proportion of mobile phase B was maintained at 60%.

[0011] During the period of 26–32 min, the proportion of mobile phase A increased from 40% to 75%, while the proportion of mobile phase B decreased from 60% to 25%.

[0012] During the 32–35 min period, the proportion of mobile phase A increased from 75% to 92%, while the proportion of mobile phase B decreased from 25% to 8%.

[0013] During the period of 35 to 43 minutes, the proportion of mobile phase A increased from 92% to 95%, while the proportion of mobile phase B decreased from 8% to 5%.

[0014] The triethylamine-containing phosphoric acid aqueous solution is a 0.1%-0.2% phosphoric acid aqueous solution containing 0.1%-0.2% triethylamine;

[0015] Preferably, the triethylamine-containing phosphoric acid aqueous solution is a 0.2% phosphoric acid aqueous solution containing 0.2% triethylamine.

[0016] The chromatographic conditions of the quality control method of the present invention can simultaneously detect and identify the components of at least seven medicinal materials in a traditional Chinese medicine composition, including Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, and Indigo naturalis. This not only improves the detection efficiency but also enhances the quality control standards for traditional Chinese medicine composition products, thereby improving the quality of these products.

[0017] In order to effectively separate the various components contained in the drug and obtain characteristic peaks with good separation, this invention explored the mobile phase and elution conditions of liquid chromatography, so that at least 12 peaks with good separation can be obtained in a single detection.

[0018] In the mobile phase selection experiments of this invention, acetonitrile-0.1% formic acid, acetonitrile-0.2% phosphoric acid, acetonitrile-0.2% triethylamine, and acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine) were selected as mobile phases. It was found that when gradient elution was performed using acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine), the chromatogram showed the most peaks, better separation of each peak, and more symmetrical peak shapes. Therefore, acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine) was selected as the mobile phase.

[0019] When using ultra-high performance liquid chromatography with the elution gradient described above, the analysis time is short, the chromatogram is further optimized, the peak shape symmetry is good, and the resolution of the chromatographic peaks is the best.

[0020] Preferably, acetonitrile is used as mobile phase A, and 0.2% aqueous solution of phosphoric acid containing 0.2% triethylamine is used as mobile phase B.

[0021] This invention also conducted experimental analysis on the selection of the pH adjustment agent ratio of the mobile phase, examining mobile phases with different pH adjustment agent ratios (0.1% phosphoric acid-0.1% triethylamine, 0.1% phosphoric acid-0.2% triethylamine, 0.15% phosphoric acid-0.2% triethylamine, 0.2% phosphoric acid-0.2% triethylamine). The results showed that 0.1%-0.2% phosphoric acid aqueous solutions containing 0.1%-0.2% triethylamine all met the requirements, but the mobile phase with 0.2% phosphoric acid (containing 0.2% triethylamine) had the highest number of chromatographic peaks, the best separation effect, and the most symmetrical peak shape. Therefore, the preferred mobile phase B is a 0.2% phosphoric acid aqueous solution containing 0.2% triethylamine.

[0022] A further scheme includes chromatographic conditions such as a detection wavelength of 220nm-300nm, preferably 254nm;

[0023] This invention conducted experimental analysis on the detection wavelength. Using a DAD detector, the test sample was scanned across the entire wavelength range. The results showed that when 220nm, 240nm, 254nm, and 300nm were used as detection wavelengths, the test sample could present good chromatograms. The absorbance difference between any two randomly selected characteristic peaks in the chromatogram did not exceed 20 to 40 times. Detection wavelengths of 220nm, 240nm, 254nm, and 300nm were all feasible, but when 254nm was used as the detection wavelength, the peak area was moderate, and the symmetry and separation of each peak were better. Therefore, 254nm was the preferred detection wavelength.

[0024] A further protocol, with chromatographic conditions including a flow rate of 0.2 ml per minute and a column temperature of 30°C.

[0025] A further embodiment of the preparation method of the test sample of the traditional Chinese medicine composition includes: taking an appropriate amount of the traditional Chinese medicine composition, adding an extraction solvent, sonicating, filtering, evaporating the filtrate to dryness, dissolving the residue in water, adding hydrochloric acid, shaking well, extracting with chloroform, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample;

[0026] Preferably, the preparation method of the test sample includes: taking an appropriate amount of the traditional Chinese medicine composition, adding an extraction solvent, sonicating for 20-60 minutes, filtering, evaporating the filtrate to dryness, adding 20-30 ml of water to dissolve the residue, adding 2-8 drops of hydrochloric acid, shaking well, extracting with chloroform 1-3 times, separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate 1-3 times, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample;

[0027] More preferably, the preparation method of the test sample includes: taking 1g of the traditional Chinese medicine composition, adding 50ml of extraction solvent, sonicating for 40 minutes, filtering, evaporating the filtrate to dryness, adding 25ml of water to dissolve the residue, adding 3 drops of hydrochloric acid, shaking well, and extracting three times with 25ml of chloroform each time, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting three times with 25ml of ethyl acetate each time, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a 5ml volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample;

[0028] Preferably, the extraction solvent is 50% methanol or 70% methanol, more preferably 70% methanol.

[0029] In a further embodiment, the quality control method further includes detecting reference standards corresponding to Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Sophora tonkinensis, and Indigo naturalis under the UPLC chromatographic conditions, comparing and analyzing the characteristic peak information in the test sample and reference standard detection results of the traditional Chinese medicine composition, constructing a quality control spectrum of the traditional Chinese medicine composition based on this, and then using the quality control spectrum to detect the traditional Chinese medicine composition product;

[0030] Alternatively, the content of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Sophora tonkinensis or Indigo naturalis can be calculated based on the obtained characteristic peak information, and the content of each component can be determined.

[0031] Preferably, the "detection of traditional Chinese medicine composition products using the quality control spectrum" includes: comparing and analyzing the UPLC spectrum of the traditional Chinese medicine composition product to be tested with the quality control spectrum of the traditional Chinese medicine composition; if the characteristic peaks of the traditional Chinese medicine composition product to be tested match the characteristic peaks in the quality control spectrum, it is a qualified product.

[0032] Preferably, ultra-high performance liquid chromatography (UHPLC) is used to simultaneously detect seven components in the test sample of the traditional Chinese medicine composition: Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, and Indigo naturalis. The characteristic chromatogram of the traditional Chinese medicine composition product should show 12 characteristic peaks. The peak corresponding to the reference peak is called the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value, which is 0.55, 0.58, 0.62, 0.95, 1.00, 1.22, 1.61, 1.64, 1.68, 1.78, 2.04, and 2.37.

[0033] Alternatively, ultra-high performance liquid chromatography (UHPLC) can be used to simultaneously detect eight components in the test sample of the traditional Chinese medicine composition: Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis, and Sophora tonkinensis. The characteristic chromatogram of the traditional Chinese medicine composition product should show 14 characteristic peaks. The peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated. The relative retention time should be within ±10% of the specified value, which is 0.55, 0.58, 0.62, 0.95, 1.00, 1.22, 1.41, 1.61, 1.64, 1.68, 1.78, 2.04, 2.37, and 2.50.

[0034] A further embodiment of the preparation method for the reference solution includes: taking an appropriate amount of baicalin reference standard, accurately weighing it, and adding methanol to prepare a solution; preferably, adding methanol to prepare a solution containing 0.1 mg per 1 ml.

[0035] This invention provides a method for constructing a quality control spectrum of a traditional Chinese medicine composition, the composition comprising watermelon frost, calcined borax, phellodendron bark, coptis rhizome, sophora root, belamcanda rhizome, fritillaria bulb, indigo naturalis, borneol, soapberry fruit, rhubarb, scutellaria root, licorice root, and menthol. The method is characterized by comprising:

[0036] (1) Take multiple batches of Chinese herbal medicine compositions to prepare test solution, take the reference herbs of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis and Sophora tonkinensis to prepare reference herb solution, take the standard of some characteristic peaks in the characteristic spectrum of the corresponding Chinese herbal medicine composition to prepare reference standard solution, and remove one or more of the components of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis and Sophora tonkinensis from the prescription of Chinese herbal medicine composition to obtain negative sample solution;

[0037] (2) Combine the test solution, reference material solution, reference standard solution and negative sample solution described in step (1).

[0038] The solution was detected using ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions included: octadecyl-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and a phosphoric acid aqueous solution containing triethylamine as mobile phase B, with gradient elution; the theoretical plate number, calculated based on the baicalin peak, should be no less than 5000; the gradient elution process included:

[0039] During the 0–10 min period, the proportion of mobile phase A increased from 20% to 24%, while the proportion of mobile phase B decreased from 80% to 76%.

[0040] During the 10–23 min period, the proportion of mobile phase A increased from 24% to 40%, while the proportion of mobile phase B decreased from 76% to 60%.

[0041] During the period of 23 to 26 minutes, the proportion of mobile phase A was maintained at 40%, and the proportion of mobile phase B was maintained at 60%.

[0042] During the period of 26–32 min, the proportion of mobile phase A increased from 40% to 75%, while the proportion of mobile phase B decreased from 60% to 25%.

[0043] During the 32–35 min period, the proportion of mobile phase A increased from 75% to 92%, while the proportion of mobile phase B decreased from 25% to 8%.

[0044] During the period of 35 to 43 minutes, the proportion of mobile phase A increased from 92% to 95%, while the proportion of mobile phase B decreased from 8% to 5%.

[0045] The triethylamine-containing phosphoric acid aqueous solution is a 0.1%-0.2% phosphoric acid aqueous solution containing 0.1%-0.2% triethylamine;

[0046] Preferably, the triethylamine-containing phosphoric acid aqueous solution is a 0.2% phosphoric acid aqueous solution containing 0.2% triethylamine;

[0047] (3) Analyze the chromatograms of multiple batches of test sample solutions to identify common characteristic peaks; assign the characteristic peaks to medicinal materials based on the chromatograms of the reference solution; identify the characteristic peaks based on the characteristic peaks of the reference solution, determine the reference peaks, and obtain the quality control chromatogram of the traditional Chinese medicine composition.

[0048] A further embodiment of the preparation method of the test solution of the traditional Chinese medicine composition includes: taking the traditional Chinese medicine composition, adding an extraction solvent, sonicating, filtering, evaporating the filtrate to dryness, dissolving the residue in water, adding hydrochloric acid, shaking well, extracting with chloroform by shaking, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate by shaking, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test solution;

[0049] Preferably, the preparation method of the test sample includes: taking an appropriate amount of the traditional Chinese medicine composition, adding an extraction solvent, sonicating for 20-60 minutes, filtering, evaporating the filtrate to dryness, adding 20-30 ml of water to dissolve the residue, adding 2-8 drops of hydrochloric acid, shaking well, extracting with chloroform 1-3 times, separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate 1-3 times, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample;

[0050] More preferably, the preparation method includes: taking 1g of the traditional Chinese medicine composition, adding 50ml of extraction solvent, sonicating for 40 minutes, filtering, evaporating the filtrate to dryness, adding 25ml of water to dissolve the residue, adding 3 drops of hydrochloric acid, shaking well, and extracting three times with 25ml of chloroform each time, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting three times with 25ml of ethyl acetate each time, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a 5ml volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the final product;

[0051] Preferably, the extraction solvent is 50% or 70% methanol; more preferably, the extraction solvent is 70% methanol.

[0052] This invention explored the selection of extraction solvents and extraction methods, comparing the use of methanol, 70% methanol, 50% methanol, chloroform, and ethyl acetate as extraction solvents. The content of the herbal composition extracted in these solvents was analyzed. It was found that the samples extracted with chloroform and ethyl acetate showed no chromatographic peaks, the samples extracted with methanol showed a few chromatographic peaks, and the samples extracted with 70% methanol and 50% methanol showed relatively abundant peaks. Therefore, 70% methanol and 50% methanol can be selected as extraction solvents. Among these, the samples extracted with 70% methanol showed the largest peak areas and good symmetry; therefore, 70% methanol is more preferably used as the extraction solvent.

[0053] In addition, the present invention explored different extraction methods and extraction times for the test sample solution, and found that when the test sample solution was prepared using the above preparation method, the chromatographic peaks were the most abundant, the peak areas of each chromatographic peak were the largest, and the symmetry was good.

[0054] A further embodiment of the preparation method of the reference solution of the control medicinal materials includes: taking appropriate amounts of the control medicinal materials Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis, and Sophora tonkinensis, adding extraction solvent, sonicating for 20-60 minutes, filtering, evaporating the filtrate to dryness, adding 20-30 ml of water to dissolve the residue, adding 2-8 drops of hydrochloric acid, shaking well, and extracting with chloroform 1-3 times, separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate 1-3 times, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the final product;

[0055] Preferably, appropriate amounts of the reference medicinal materials Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis, and Sophora tonkinensis are taken respectively, and 50 ml of extraction solvent is added. The mixture is ultrasonically treated for 40 minutes, filtered, and the filtrate is evaporated to dryness. The residue is dissolved in 25 ml of water, 3 drops of hydrochloric acid are added, and the mixture is shaken well. The mixture is then extracted three times with 25 ml of chloroform each time. The chloroform solution is collected and set aside. The upper aqueous solution is taken and extracted three times with 25 ml of ethyl acetate each time. The ethyl acetate solution is collected and combined with the chloroform solution, evaporated to dryness, and the residue is dissolved in methanol. The mixture is transferred to a 5 ml volumetric flask, methanol is added to the mark, shaken well, filtered, and the filtrate is collected to obtain the final product.

[0056] Preferably, the extraction solvent is 50% or 70% methanol; more preferably, the extraction solvent is 70% methanol.

[0057] A further embodiment of the preparation method of the reference solution includes: taking appropriate amounts of baicalin, palmatine hydrochloride, berberine hydrochloride, wogonin, irisin, ammonium glycyrrhizate, wogonin, indirubin, and rhein, and dissolving them in methanol to prepare solutions containing 0.1 mg per ml.

[0058] In a further embodiment, the quality control spectrum of the traditional Chinese medicine composition includes 12 chromatographic peaks numbered sequentially according to their elution time. Peaks 1, 5, 9, and 10 are assigned to Scutellaria baicalensis, peaks 2 and 3 to Coptis chinensis, peak 3 to Phellodendron chinense, peaks 4 and 12 to Rheum palmatum, peaks 6 and 7 to Belamcanda chinensis, peak 8 to Glycyrrhiza uralensis, and peak 11 to Indigo naturalis.

[0059] In the quality control spectrum of the traditional Chinese medicine composition, peak 5 is the S peak with a relative retention time of 1. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​for peaks 1 to 12 are 0.55 (peak 1), 0.58 (peak 2), 0.62 (peak 3), 0.95 (peak 4), 1.22 (peak 6), 1.61 (peak 7), 1.64 (peak 8), 1.68 (peak 9), 1.78 (peak 10), 2.04 (peak 11), and 2.37 (peak 12).

[0060] Alternatively, the quality control spectrum of the traditional Chinese medicine composition includes 14 chromatographic peaks numbered sequentially according to their elution time, wherein peaks 1, 5, 10, and 11 are assigned to Scutellaria baicalensis, peaks 2 and 3 are assigned to Coptis chinensis, peak 3 is assigned to Phellodendron chinense, peaks 4 and 13 are assigned to Rheum palmatum, peaks 6 and 8 are assigned to Belamcanda chinensis, peaks 7 and 14 are assigned to Sophora tonkinensis, peak 9 is assigned to Glycyrrhiza uralensis, and peak 12 is assigned to Indigo naturalis.

[0061] Taking peak 5 as the main S peak with a relative retention time of 1, calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are 0.55 (peak 1), 0.58 (peak 2), 0.62 (peak 3), 0.95 (peak 4), 1.22 (peak 6), 1.41 (peak 7), 1.61 (peak 8), 1.64 (peak 9), 1.68 (peak 10), 1.78 (peak 11), 2.04 (peak 12), 2.37 (peak 13), and 2.50 (peak 14), respectively.

[0062] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0063] 1. The quality control method for traditional Chinese medicine compositions provided by this invention explores and improves the conditions of liquid chromatography, enabling the simultaneous identification and detection of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis, and Sophora tonkinensis contained in the traditional Chinese medicine composition in a single detection. This greatly improves the detection efficiency, enhances the quality control level of the traditional Chinese medicine composition, and is beneficial to improving the quality of traditional Chinese medicine composition products.

[0064] 2. In the quality control method of traditional Chinese medicine composition provided by the present invention, the preparation method of the test solution of traditional Chinese medicine composition has been explored and improved. Combined with chromatographic detection conditions, the obtained chromatogram has many characteristic peaks, large peak area and good symmetry, which lays the foundation for improving the quality control level of the method.

[0065] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0066] The accompanying drawings, as part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0067] Figure 1 This is the chromatogram of the test sample in Example 1 of the present invention (a total of 14 characteristic peaks);

[0068] Figure 2 These are the chromatograms of the test sample and each medicinal material in Embodiment 1 of the present invention;

[0069] Figure 3 These are the chromatograms of the test sample and the reference sample in Embodiment 1 of the present invention;

[0070] Figure 4 This is a comparative feature spectrum of the 14 characteristic peaks in Embodiment 1 of the present invention;

[0071] Figure 5 This is a comparative characteristic spectrum of 12 characteristic peaks in Example 1 of the present invention; wherein, peak 1: baicalin; peak 2: palmatine hydrochloride; peak 3: berberine hydrochloride; peak 5(S): baicalin; peak 7: irisin; peak 8: ammonium glycyrrhizate; peak 9: baicalin; peak 11: indirubin; peak 12: rhein.

[0072] Figure 6 The chromatogram is for acetonitrile-0.1% formic acid as the mobile phase;

[0073] Figure 7 The chromatogram is for acetonitrile-0.2% phosphoric acid as the mobile phase;

[0074] Figure 8 The chromatogram is for acetonitrile-0.2% triethylamine as the mobile phase;

[0075] Figure 9 The chromatogram shows that the mobile phase is acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine);

[0076] Figure 10This is the chromatogram of the test sample when the detection wavelength is 220 nm;

[0077] Figure 11 This is the chromatogram of the test sample when the detection wavelength is 240 nm;

[0078] Figure 12 This is the chromatogram of the test sample when the detection wavelength is 254nm;

[0079] Figure 13 This is the chromatogram of the test sample when the detection wavelength is 300 nm;

[0080] Figure 14 This is the chromatogram of the test sample obtained by ultra-high performance liquid chromatography using 0.1% phosphoric acid-0.2% triethylamine as the mobile phase;

[0081] Figure 15 This is the chromatogram of the test sample obtained by ultra-high performance liquid chromatography using 0.15% phosphoric acid-0.2% triethylamine as the mobile phase;

[0082] Figure 16 This is the chromatogram of the test sample obtained by ultra-high performance liquid chromatography using 0.2% phosphoric acid-0.2% triethylamine as the mobile phase;

[0083] Figure 17 This is the chromatogram of the test sample under the optimal chromatographic conditions for ultra-high performance liquid chromatography;

[0084] Figure 18 This is a chromatogram of the test sample prepared using methanol as the extraction solvent;

[0085] Figure 19 The chromatogram is that of the test sample prepared using 50% methanol as the extraction solvent;

[0086] Figure 20 The chromatogram is that of the test sample prepared using 70% methanol as the extraction solvent;

[0087] Figure 21 This is a chromatogram of the test sample prepared using chloroform as the extraction solvent;

[0088] Figure 22 This is a chromatogram of the test sample prepared using ethyl acetate as the extraction solvent;

[0089] Figure 23 This is the chromatogram of the test solution prepared using Method 1 in the investigation of different purification methods;

[0090] Figure 24 This is the chromatogram of the test solution prepared using method 2 in the investigation of different purification methods;

[0091] Figure 25 This is the chromatogram of the test solution prepared using method 3 in the investigation of different purification methods;

[0092] Figure 26 This is the chromatogram of the test solution prepared using method 4 in the investigation of different purification methods;

[0093] Figure 27 This is the chromatogram of the test solution prepared using method 5 in the investigation of different purification methods;

[0094] Figure 28 This is the chromatogram of the specificity test of Phellodendron bark and Coptis chinensis;

[0095] Figure 29 This is a chromatogram of the specificity test of Sophora tonkinensis.

[0096] Figure 30 This is the chromatogram of the specificity test for Belamcanda chinensis;

[0097] Figure 31 This is the chromatogram of the indigo specificity test;

[0098] Figure 32 This is the chromatogram of rhubarb specificity test;

[0099] Figure 33 This is the chromatogram of the specificity test for Scutellaria baicalensis;

[0100] Figure 34 This is a chromatogram of the licorice specificity test;

[0101] Figure 35 It is a superimposed chromatogram of 20 batches of samples.

[0102] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0103] To make the objectives, 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 accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0104] Experimental instruments: Ultra-high performance liquid chromatograph; High performance liquid chromatograph;

[0105] Experimental reagents: Baicalin; palmatine hydrochloride; berberine hydrochloride; wogonin; irisin; ammonium glycyrrhizate; wogonin; indirubin; rhein; acetonitrile were chromatographic grade, and other reagents were analytical grade. Powders of watermelon frost, phellodendron bark, coptis root, scutellaria root, sophora root, belamcanda rhizome, rhubarb, fritillaria bulb, indigo naturalis, licorice root, and soapberry fruit (charred), negative samples, excipients, and 20 batches of traditional Chinese medicine compositions (Guilin watermelon frost) and the samples used for method investigation were provided by Guilin Sanjin Pharmaceutical Co., Ltd.

[0106] Example 1: Construction of Quality Control Chart

[0107] In this embodiment, the traditional Chinese medicine composition was prepared from 14 medicinal materials, including watermelon frost, calcined borax, phellodendron bark, coptis root, sophora root, belamcanda rhizome, fritillaria bulb, indigo naturalis, borneol, soapberry fruit, rhubarb, scutellaria root, licorice root, and menthol. A quality control spectrum of the traditional Chinese medicine composition was constructed by combining the test sample, reference medicinal materials, reference standards, and various negative samples. The specific method is as follows:

[0108] (1) Select the reference medicinal materials of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis and Sophora tonkinensis to prepare reference solutions of each component. The preparation method is as follows: Take 0.2g of each of the reference medicinal materials Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis and Sophora tonkinensis, place them in a stoppered conical flask, add 50ml of extraction solvent, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, add 3 drops of hydrochloric acid, shake well, add chloroform and shake to extract 3 times, 25ml each time, separate the chloroform solution, take the upper aqueous solution, add ethyl acetate and shake to extract 3 times, 25ml each time, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, take the filtrate to obtain the solution;

[0109] (2) Preparation of reference solutions: Take appropriate amounts of baicalin, palmatine hydrochloride, berberine hydrochloride, wogonin, irisin, ammonium glycyrrhizate, wogonin, indirubin, and rhein, and prepare solutions containing approximately 0.1 mg per ml.

[0110] (3) Prepare the test solution according to the following method: Take 1g of the traditional Chinese medicine composition, place it in a stoppered conical flask, add 50ml of extraction solvent, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, add 3 drops of hydrochloric acid, shake well, add chloroform and shake to extract 3 times, 25ml each time, separate the chloroform solution, take the upper aqueous solution, add ethyl acetate and shake to extract 3 times, 25ml each time, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution;

[0111] (4) Based on the preparation method of the test sample solution, prepare negative sample solutions without Scutellaria baicalensis, negative sample solutions without Phellodendron chinense, negative sample solutions without Coptis chinensis, negative sample solutions without Rheum palmatum, negative sample solutions without Belamcanda chinensis, negative sample solutions without Glycyrrhiza uralensis and negative sample solutions without Indigo naturalis.

[0112] (5) Chromatographic analysis was performed on each of the above groups of test samples, reference medicinal materials, reference standards and negative samples.

[0113] Chromatographic conditions included: octadecyl-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm); acetonitrile as mobile phase A; and 0.2% phosphoric acid (containing 0.2% triethylamine) as mobile phase B, with gradient elution as specified in the table below; a DAD detector at a detection wavelength of 254 nm; a flow rate of 0.2 ml / min; a column temperature of 30 °C; and an injection volume of 1 μl. The theoretical plate number, calculated based on the baicalin peak, should be no less than 5000. Chromatograms were recorded.

[0114] Table 1

[0115]

[0116]

[0117] result:

[0118] Identification of characteristic peaks: Twenty batches of the described traditional Chinese medicine composition samples were taken and analyzed according to the above method. Chromatograms were recorded, and a total of 14 common peaks were identified as characteristic peaks. The chromatograms are shown below. Figure 1 .

[0119] Assignment of characteristic peaks: See the chromatograms of the test samples and each medicinal material. Figure 2 Based on the retention time of the chromatographic peaks, the characteristic peaks of the characteristic chromatograms were assigned to medicinal materials. Peak 3 is a component shared by Phellodendron bark and Coptis chinensis. The 14 characteristic peaks reflect information about 8 medicinal materials: Scutellaria baicalensis, Phellodendron bark, Coptis chinensis, Rheum palmatum, Sophora tonkinensis, Belamcanda chinensis, Glycyrrhiza uralensis, and Indigo naturalis. Peaks 1, 5, 10, and 11 are assigned to Scutellaria baicalensis, peaks 2 and 3 are assigned to Coptis chinensis, peak 3 is assigned to Phellodendron bark, peaks 4 and 13 are assigned to Rheum palmatum, peaks 6 and 8 are assigned to Belamcanda chinensis, peaks 7 and 14 are assigned to Sophora tonkinensis, peak 9 is assigned to Glycyrrhiza uralensis, and peak 12 is assigned to Indigo naturalis.

[0120] Identification of characteristic peaks: See the chromatograms of the test sample and the reference standard. Figure 3 Based on the retention time and absorption spectrum of the chromatographic peaks, peak 1 was identified as baicalin, peak 2 as palmatine hydrochloride, peak 3 as berberine hydrochloride, peak 5 as wogonin, peak 8 as irisin, peak 9 as ammonium glycyrrhizate, peak 10 as wogonin, peak 12 as indirubin, and peak 13 as emodin.

[0121] Judgment method: Peak 5 (baicalin) has a moderate retention time, large peak area, symmetrical peak shape, good separation effect, and no negative interference, and can be used as a reference peak (S peak).

[0122] Twenty batches of Guilin Watermelon Frost samples were collected and analyzed according to the proposed method. Chromatograms were recorded, and the samples were processed using the "Traditional Chinese Medicine Chromatogram Fingerprint Similarity Evaluation System (2012 Edition)" software of the National Pharmacopoeia Commission to generate a reference characteristic chromatogram. Figure 4The chromatogram of the test sample should show 14 characteristic peaks, among which the peak corresponding to the baicalin reference peak is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are 0.55 (peak 1), 0.58 (peak 2), 0.62 (peak 3), 0.95 (peak 4), 1.00 [peak 5 (S)], 1.22 (peak 6), 1.41 (peak 7), 1.61 (peak 8), 1.64 (peak 9), 1.68 (peak 10), 1.78 (peak 11), 2.04 (peak 12), 2.37 (peak 13), and 2.50 (peak 14).

[0123] Furthermore, measurements revealed that peaks 7 and 14 in the 20 batches of samples had relatively small peak areas. Therefore, considering all factors, the remaining 12 peaks (excluding peaks 7 and 14) were selected as the characteristic peaks of the characteristic spectrum. The final selected control characteristic spectrum (i.e., the quality control spectrum) is as follows: Figure 5 As shown. Among them, peak 1: baicalin; peak 2: palmatine hydrochloride; peak 3: berberine hydrochloride; peak 5(S): baicalin; peak 7: irisin; peak 8: ammonium glycyrrhizate; peak 9: baicalin; peak 11: indirubin; peak 12: rhein.

[0124] Example 2: Quality Control Method

[0125] Preparation of the reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per ml.

[0126] Preparation of the test solution: Take 1g of the Chinese herbal medicine composition to be tested, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, add 3 drops of hydrochloric acid, shake well, extract with chloroform 3 times, 25ml each time, separate the chloroform solution and set aside; take the upper aqueous solution, extract with ethyl acetate 3 times, 25ml each time, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0127] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm, Waters ACQUITY UPLC BEH C18). Acetonitrile was used as mobile phase A, and 0.2% phosphoric acid solution (containing 0.2% triethylamine) was used as mobile phase B. Gradient elution was performed according to the specifications in the table below; the flow rate was 0.2 mL / min; the detection wavelength was 254 nm; and the column temperature was 30 °C. The theoretical plate number, calculated based on the baicalin peak, should be no less than 5000.

[0128] Table 2

[0129]

[0130] The assay involves precisely pipetting 1 μl each of the reference solution and the test solution, injecting them into the liquid chromatograph, and measuring to obtain the final result.

[0131] The colorimetric spectrum of the Chinese herbal medicine composition product to be tested was compared with the characteristic spectrum of the control. Figure 5 A comparative analysis was conducted, and the product was considered qualified if the characteristic peaks of the tested traditional Chinese medicine composition matched the characteristic peaks in the quality control spectrum.

[0132] The chromatogram of the test sample should show 12 characteristic peaks. The peak corresponding to the reference peak is called the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are 0.55 (peak 1), 0.58 (peak 2), 0.62 (peak 3), 0.95 (peak 4), 1.00 [peak 5 (S)], 1.22 (peak 6), 1.61 (peak 7), 1.64 (peak 8), 1.68 (peak 9), 1.78 (peak 10), 2.04 (peak 11), and 2.37 (peak 12).

[0133] Investigation of experimental conditions in Experiment Example 1

[0134] 1. Selection of mobile phase in high performance liquid chromatography

[0135] Experimental analysis was conducted on the selection of mobile phases, and mobile phases with different acid-base adjusters were investigated. Specifically, acetonitrile-0.1% formic acid, acetonitrile-0.2% phosphoric acid, acetonitrile-0.2% triethylamine, and acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine) were selected as mobile phases.

[0136] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material, a DAD detector was used, the detection wavelength was 254 nm, the flow rate was 1.0 ml per minute, the column temperature was 30 ℃, the injection volume was 10 μl, and elution was performed.

[0137] Results: The chromatograms of acetonitrile-0.1% formic acid, acetonitrile-0.2% phosphoric acid, acetonitrile-0.2% triethylamine, and acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine) as mobile phases are as follows: Figure 6-9 As shown.

[0138] The results showed that when acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine) was used as the mobile phase for gradient elution, the chromatogram showed the most peaks, better separation of each peak, and more symmetrical peak shapes. Therefore, acetonitrile-0.2% phosphoric acid (containing 0.2% triethylamine) was selected as the mobile phase.

[0139] 2. Selection of detection wavelength in high performance liquid chromatography

[0140] This invention conducted experimental analysis on the selection of detection wavelengths. Using a DAD detector to scan the test sample across the entire wavelength range, the results showed that the test sample exhibited good chromatograms at detection wavelengths of 220 nm, 240 nm, 254 nm, and 300 nm. The absorbance difference between any two randomly selected characteristic peaks in the chromatograms did not exceed 20–40 times. Chromatograms for each detection wavelength are shown below. Figure 10-13 220nm, 240nm, 254nm and 300nm are all feasible detection wavelengths, but when 254nm is used as the detection wavelength, the peak area is moderate and the symmetry and separation of each peak are better. Therefore, 254nm is the preferred detection wavelength.

[0141] 3. Ultra-high performance liquid chromatography

[0142] Since high performance liquid chromatography (HPLC) analysis takes more than 60 minutes, ultra-high performance liquid chromatography (UHPLC) analysis can shorten the analysis time.

[0143] 3.1 Selection of pH adjusters with different proportions

[0144] Using octadecylsilane-bonded silica gel as the packing material, a DAD detector at a detection wavelength of 254 nm, a flow rate of 0.2 mL / min, a column temperature of 30 °C, and an injection volume of 1 μL, the mobile phases with different pH adjuster ratios (0.1% phosphate-0.2% triethylamine, 0.15% phosphate-0.2% triethylamine, and 0.2% phosphate-0.2% triethylamine) were investigated.

[0145] Results: Chromatograms of mobile phases with different pH adjuster ratios are shown below. Figure 14-16 The results showed that using 0.2% phosphoric acid (containing 0.2% triethylamine) as the aqueous mobile phase resulted in the highest number of chromatographic peaks, the best separation effect, and the most symmetrical peak shapes.

[0146] 3.2 Selection of elution conditions

[0147] Using octadecylsilane-bonded silica gel as the packing material, a DAD detector at a detection wavelength of 254 nm, a flow rate of 0.2 ml per minute, a column temperature of 30 °C, and an injection volume of 1 μl, different gradient mobile phases were investigated for elution.

[0148] The results showed that using acetonitrile as mobile phase A and 0.2% phosphoric acid (containing 0.2% triethylamine) as mobile phase B, with the gradient shown in the table below, resulted in the best separation effect, more symmetrical peak shape, and shorter analysis time. The chromatogram is shown below. Figure 17 .

[0149] Table 3

[0150]

[0151]

[0152] 4. Selection of the method for preparing the test solution

[0153] The products described below are all traditional Chinese medicine compositions described in this invention, namely Guilin Watermelon Frost from Guilin Sanjin Pharmaceutical Co., Ltd.

[0154] 4.1 Comparison of different extraction methods

[0155] Ultrasonic extraction: Take 1g of this product, add 70% methanol, sonicate for 40min, cool, shake well, and filter to obtain the product.

[0156] Reflux extraction: Take 1g of this product, add 70% methanol, heat under reflux for 40min, cool, shake well, and filter to obtain the product.

[0157] The results showed that there was almost no difference in the number of chromatographic peaks and peak area of ​​the two extraction methods, so ultrasonic extraction, which is simpler to operate, was chosen as the extraction method.

[0158] 4.2 Selection of different extraction solvents

[0159] The extraction effects of different solvents (50% methanol, 70% methanol, methanol, chloroform, ethyl acetate) were compared.

[0160] The extraction method is as follows: Take 1g of this product, add 50ml of 50% methanol, or 50ml of 70% methanol, or 50ml of methanol, or 50ml of chloroform, or 50ml of ethyl acetate, sonicate for 40 minutes, filter, and the product is obtained.

[0161] The results show that the chromatograms of each extraction solvent are shown in the figure below. Figure 18-22 Of the samples extracted with chloroform and ethyl acetate, no obvious chromatographic peaks were observed. Samples extracted with methanol showed a few peaks. Samples extracted with 70% methanol and 50% methanol showed more abundant peaks. Therefore, 70% methanol and 50% methanol were selected as extraction solvents. However, the samples extracted with 70% methanol exhibited the largest peak areas and best symmetry. Preferably, 70% methanol was chosen as the extraction solvent.

[0162] 4.3 Investigation of different purification methods

[0163] The ultrasonic extraction method using 70% methanol yielded fewer chromatographic peaks, and the peak area ratios were inconsistent (the ratio of the area of ​​the remaining peaks to the largest peak was very small, mostly <5%), which could not reflect the information of multiple medicinal flavors in the traditional Chinese medicine composition.

[0164] 4.3.1 The personnel involved in this invention also investigated different purification methods, specifically:

[0165] Extraction Method 1: Take 1g of this product, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, add 25ml of chloroform and shake to extract, separate the chloroform solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0166] Extraction Method 2: Take 1g of this product, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, add 25ml of ethyl acetate and shake to extract, separate the ethyl acetate solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0167] Extraction Method 3: Take 1g of this product, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25ml of water, add 25ml of chloroform and shake to extract, separate the chloroform solution, take the upper aqueous solution, add 25ml of ethyl acetate and shake to extract, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0168] Extraction Method 4: Take 1g of this product, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25ml of water, add 3 drops of concentrated ammonia solution, shake well, add 25ml of chloroform and shake to extract, separate the chloroform solution, take the upper aqueous solution, add 25ml of ethyl acetate and shake to extract, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0169] Extraction Method 5: Take 1g of this product, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25ml of water, add 3 drops of hydrochloric acid, shake well, add 25ml of chloroform and shake to extract, separate the chloroform solution, take the upper aqueous solution, add 25ml of ethyl acetate and shake to extract, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0170] Results: Chromatograms of the test samples obtained by different purification (extraction) methods are shown in the figure. Figure 23-27 As can be seen from the chromatogram, peaks 1 and 9 are missing in the chloroform extraction; peaks 1, 2 and 9 are missing in the ethyl acetate extraction; peaks 1, 6 and 9 are missing in the fractional extraction after alkalization; and peaks 1, 6 and 9 are missing in the combined solution of chloroform and ethyl acetate extraction after acidification (Method 5). Method 5 has the richest chromatographic peaks and can reflect the most medicinal information.

[0171] Therefore, the extraction and purification method is determined as follows: Take 1g of this product, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, add 3 drops of hydrochloric acid, shake well, add 25ml of chloroform and shake to extract, separate the chloroform solution, take the upper aqueous solution, add 25ml of ethyl acetate and shake to extract, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0172] 4.3.2 Examination of the number of extractions

[0173] This invention investigated the number of extractions and compared different extraction times. The results showed that 1-3 extractions were more effective. After 3 extractions with chloroform and ethyl acetate, the peak areas of each peak hardly increased, indicating complete extraction. Therefore, 3 extractions with chloroform and ethyl acetate were determined as the optimal number of extractions.

[0174] 4.5 Therefore, after comprehensive investigation, the preparation method of the test solution was determined as follows: Take 1g of this product, add 50ml of 70% methanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, add 3 drops of hydrochloric acid, shake well, add chloroform and shake to extract 3 times, 25ml each time, separate the chloroform solution, take the upper aqueous solution, add ethyl acetate and shake to extract 3 times, 25ml each time, separate the ethyl acetate solution, combine with the chloroform solution, evaporate to dryness, add methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0175] 5. Identification and Attribution of Characteristic Peaks

[0176] Reference solutions of each component were prepared by selecting the reference medicinal materials Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, and Indigo naturalis. The preparation method was as follows: 0.2g of each of the reference medicinal materials Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, and Indigo naturalis were placed in a stoppered conical flask, 50ml of extraction solvent was added, and the mixture was sonicated for 40 minutes. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 25ml of water. 3 drops of hydrochloric acid were added, and the mixture was shaken well. 25ml of chloroform was added and the mixture was shaken three times. The chloroform solution was separated, and the supernatant was taken. 25ml of ethyl acetate was added and the mixture was shaken three times. The ethyl acetate solution was separated and combined with the chloroform solution. The mixture was evaporated to dryness, and the residue was dissolved in methanol. The mixture was transferred to a 5ml volumetric flask, methanol was added to the mark, and the mixture was shaken well. The mixture was filtered, and the filtrate was taken as the final product.

[0177] Then, perform the determination according to the chromatographic conditions under section 3.2 and record the chromatogram.

[0178] Experimental Example 2: Methodological Verification

[0179] 1. Specificity test

[0180] Preparation of the test solution: Prepared according to the method in Example 1 or 2.

[0181] Preparation of medicinal material solution: Take appropriate amounts of each medicinal material and prepare it using the same extraction method as the test sample.

[0182] Preparation of negative sample solution: Prepared according to the method in Example 1.

[0183] Determine according to the proposed method and record the chromatogram (see...). Figure 28-34 Peak 3 (berberine hydrochloride) is a common component of Phellodendron bark and Coptis chinensis. None of the negative sample solutions showed a chromatographic peak with the same retention time as their respective herbal solutions.

[0184] 2. Repeatability test

[0185] The traditional Chinese medicine composition was used as a test sample, and six test sample solutions were prepared in parallel according to the proposed method. The samples were injected under the proposed chromatographic conditions, the chromatograms were recorded, and the relative retention times of the characteristic peaks were calculated. The results are shown in Table 4. The relative retention times of each characteristic peak were all within ±10% of the specified value, and the RSD was <3.0%, indicating that the method has good repeatability.

[0186] Table 4. Relative retention times of characteristic peaks in repeatability tests

[0187]

[0188] 3. Stability Test

[0189] Take the test solution of the traditional Chinese medicine composition and determine it at 0h, 4h, 8h, 12h, 18h and 24h according to the planned chromatographic conditions. Record the chromatogram and calculate the relative retention time of the characteristic peak. The results are shown in Table 5. The relative retention time of each characteristic peak is within ±10% of the specified value and RSD < 3.0%, indicating that the test solution is stable within 24 hours.

[0190] Table 5. Relative retention times of characteristic peaks in stability tests

[0191]

[0192] 4. Determination of 20 batches of samples

[0193] Twenty batches of samples were tested according to the proposed method, and characteristic chromatograms were recorded. Results: All 20 batches of samples showed 14 characteristic peaks in their chromatograms, which corresponded to the chromatographic peaks of the baicalin reference compound; the relative retention times were all within ±10% of the specified values. Details of the results are shown in Table 6, and the overlay chromatograms of each sample are shown below. Figure 35 .

[0194] Table 6. Relative retention times of 20 batches of samples

[0195]

[0196]

[0197] 5. Examination of the number of theoretical plates

[0198] The theoretical plate number (TPL) of baicalin peaks from three different batches using octadecyl-bonded silica gel as packing material (column 1 batch: 04593313135140; column 2 batch: 02873631215129; column 3 batch: 019631164257) was investigated. The theoretical TPL for the baicalin peak was 73418 for column 1, 101392 for column 2, and 104368 for column 3. Considering that all three columns are relatively new, the theoretical plate number requirement was lowered, stipulating that the theoretical plate number calculated based on the baicalin peak should not be less than 5000.

[0199] 6. Methods for identifying and determining characteristic peaks

[0200] Measurements revealed that peaks 7 and 14 in the 20 batches of samples had relatively small peak areas. Taking all factors into consideration, the other 12 peaks were selected as characteristic peaks.

[0201] The preferred determination method is as follows: the characteristic chromatogram of the test sample should show 12 characteristic peaks, which should correspond to the retention times of the reference peak. The peak corresponding to the baicalin reference peak is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are 0.55 (peak 1), 0.58 (peak 2), 0.62 (peak 3), 0.95 (peak 4), 1.22 (peak 6), 1.61 (peak 7), 1.64 (peak 8), 1.68 (peak 9), 1.78 (peak 10), 2.04 (peak 11), and 2.37 (peak 12).

[0202] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for quality control of a traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition includes watermelon frost, calcined borax, phellodendron bark, coptis root, sophora root, belamcanda rhizome, fritillaria bulb, indigo naturalis, borneol, soapberry fruit, rhubarb, scutellaria root, licorice root, and menthol. Its characteristic is that ultra-high performance liquid chromatography (UHPLC) is used to simultaneously detect at least the components of scutellaria root, phellodendron bark, coptis root, rhubarb, belamcanda rhizome, licorice root, and indigo naturalis in the sample of the traditional Chinese medicine composition. The chromatographic conditions include: using octadecyl-bonded silica gel as the stationary phase; using acetonitrile as mobile phase A and a phosphoric acid aqueous solution containing triethylamine as mobile phase B, performing gradient elution; the theoretical plate number, calculated based on the baicalin peak, should not be less than 5000; the gradient elution process includes: During the 0–10 min period, the proportion of mobile phase A increased from 20% to 24%, while the proportion of mobile phase B decreased from 80% to 76%. During the 10–23 min period, the proportion of mobile phase A increased from 24% to 40%, while the proportion of mobile phase B decreased from 76% to 60%. During the period of 23 to 26 minutes, the proportion of mobile phase A was maintained at 40%, and the proportion of mobile phase B was maintained at 60%. During the period of 26–32 min, the proportion of mobile phase A increased from 40% to 75%, while the proportion of mobile phase B decreased from 60% to 25%. During the 32–35 min period, the proportion of mobile phase A increased from 75% to 92%, while the proportion of mobile phase B decreased from 25% to 8%. During the period of 35 to 43 minutes, the proportion of mobile phase A increased from 92% to 95%, while the proportion of mobile phase B decreased from 8% to 5%. The triethylamine-containing phosphoric acid aqueous solution is a 0.1%-0.2% phosphoric acid aqueous solution containing 0.1%-0.2% triethylamine; Preferably, the triethylamine-containing phosphoric acid aqueous solution is a 0.2% phosphoric acid aqueous solution containing 0.2% triethylamine.

2. The quality control method for the traditional Chinese medicine composition according to claim 1, characterized in that, Chromatographic conditions include: detection wavelength of 220nm-300nm, preferably 254nm.

3. The quality control method for the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The preparation method of the test sample of the traditional Chinese medicine composition includes: taking an appropriate amount of the traditional Chinese medicine composition, adding an extraction solvent, sonicating, filtering, evaporating the filtrate to dryness, dissolving the residue in water, adding hydrochloric acid, shaking well, extracting with chloroform, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample; Preferably, the preparation method of the test sample includes: taking an appropriate amount of the traditional Chinese medicine composition, adding an extraction solvent, sonicating for 20-60 minutes, filtering, evaporating the filtrate to dryness, adding 20-30 ml of water to dissolve the residue, adding 2-8 drops of hydrochloric acid, shaking well, extracting with chloroform 1-3 times, separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate 1-3 times, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample; More preferably, the preparation method of the test sample includes: taking 1g of the traditional Chinese medicine composition, adding 50ml of extraction solvent, sonicating for 40 minutes, filtering, evaporating the filtrate to dryness, adding 25ml of water to dissolve the residue, adding 3 drops of hydrochloric acid, shaking well, and extracting three times with 25ml of chloroform each time, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting three times with 25ml of ethyl acetate each time, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a 5ml volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample; Preferably, the extraction solvent is 50% methanol or 70% methanol, more preferably 70% methanol.

4. The quality control method for the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, The quality control method further includes detecting the reference standards corresponding to Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Sophora tonkinensis and Indigo naturalis under the ultra-high performance liquid chromatography conditions, comparing and analyzing the characteristic peak information in the test sample and reference standard detection results of the traditional Chinese medicine composition, constructing the quality control spectrum of the traditional Chinese medicine composition based on this, and then using the quality control spectrum to detect the traditional Chinese medicine composition product. Alternatively, the content of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Sophora tonkinensis or Indigo naturalis can be calculated based on the obtained characteristic peak information, and the content of each component can be determined. Preferably, the "detection of traditional Chinese medicine composition products using the quality control spectrum" includes: comparing and analyzing the ultra-high performance liquid chromatography spectrum of the traditional Chinese medicine composition product to be tested with the quality control spectrum of the traditional Chinese medicine composition; if the characteristic peaks of the traditional Chinese medicine composition product to be tested match the characteristic peaks in the quality control spectrum, it is a qualified product. Preferably, ultra-high performance liquid chromatography (UHPLC) is used to simultaneously detect seven components in the traditional Chinese medicine composition test sample: Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, and Indigo naturalis. The characteristic spectrum of the traditional Chinese medicine composition product should show 12 characteristic peaks, and the peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are 0.55, 0.58, 0.62, 0.95, 1.00, 1.22, 1.61, 1.64, 1.68, 1.78, 2.04, and 2.

37. Alternatively, ultra-high performance liquid chromatography (UHPLC) can be used to simultaneously detect eight components in the test sample of the traditional Chinese medicine composition: Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis, and Sophora tonkinensis. The characteristic chromatogram of the traditional Chinese medicine composition product should show 14 characteristic peaks. The peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated. The relative retention time should be within ±10% of the specified value, which is 0.55, 0.58, 0.62, 0.95, 1.00, 1.22, 1.41, 1.61, 1.64, 1.68, 1.78, 2.04, 2.37, and 2.

50.

5. The quality control method for the traditional Chinese medicine composition according to any one of claims 1-4, characterized in that, The preparation method of the reference solution includes: taking an appropriate amount of baicalin reference standard, accurately weighing it, adding methanol to prepare a solution, and thus obtaining the reference solution; Preferably, a solution containing 0.1 mg per 1 ml is prepared by adding methanol.

6. A method for constructing a quality control spectrum of a traditional Chinese medicine composition, the traditional Chinese medicine composition comprising watermelon frost, calcined borax, phellodendron bark, coptis rhizome, sophora root, belamcanda rhizome, fritillaria bulb, indigo naturalis, borneol, soapberry fruit, rhubarb, scutellaria root, licorice root, and menthol, characterized in that, The construction method includes: (1) Take multiple batches of Chinese herbal medicine compositions to prepare test solution, take the reference herbs of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis and Sophora tonkinensis to prepare reference herb solution, take the standard of some characteristic peaks in the characteristic spectrum of the corresponding Chinese herbal medicine composition to prepare reference standard solution, and remove one or more of the components of Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis and Sophora tonkinensis from the prescription of Chinese herbal medicine composition to obtain negative sample solution; (2) The test solution, reference medicinal material solution, reference standard solution and negative sample solution described in step (1) are tested under the chromatographic conditions of the quality control method as described in claim 1 or 2; (3) Analyze the chromatograms of multiple batches of test sample solutions to identify common characteristic peaks; assign the characteristic peaks to medicinal materials based on the chromatograms of the reference solution; identify the characteristic peaks based on the characteristic peaks of the reference solution, determine the reference peaks, and obtain the quality control chromatogram of the traditional Chinese medicine composition.

7. The method for constructing the quality control spectrum of the traditional Chinese medicine composition according to claim 6, characterized in that, The preparation method of the test solution of the traditional Chinese medicine composition includes: taking the traditional Chinese medicine composition, adding an extraction solvent, sonicating, filtering, evaporating the filtrate to dryness, dissolving the residue in water, adding hydrochloric acid, shaking well, extracting with chloroform by shaking, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate by shaking, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test solution; Preferably, the preparation method of the test sample includes: taking an appropriate amount of the traditional Chinese medicine composition, adding an extraction solvent, sonicating for 20-60 minutes, filtering, evaporating the filtrate to dryness, adding 20-30 ml of water to dissolve the residue, adding 2-8 drops of hydrochloric acid, shaking well, extracting with chloroform 1-3 times, separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate 1-3 times, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the test sample; Preferably, the preparation method includes: taking 1g of the traditional Chinese medicine composition, adding 50ml of extraction solvent, sonicating for 40 minutes, filtering, evaporating the filtrate to dryness, adding 25ml of water to dissolve the residue, adding 3 drops of hydrochloric acid, shaking well, and extracting three times with 25ml of chloroform each time, and separating the chloroform solution for later use; taking the upper aqueous solution, extracting three times with 25ml of ethyl acetate each time, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a 5ml volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the final product; Preferably, the extraction solvent is 50% or 70% methanol; more preferably, the extraction solvent is 70% methanol.

8. The method for constructing the quality control spectrum of the traditional Chinese medicine composition according to claim 6 or 7, characterized in that, The preparation method of the reference solution of the reference medicinal materials includes: taking appropriate amounts of the reference medicinal materials Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis, and Sophora tonkinensis, adding extraction solvent, sonicating for 20-60 minutes, filtering, evaporating the filtrate to dryness, adding 20-30 ml of water to dissolve the residue, adding 2-8 drops of hydrochloric acid, shaking well, and extracting with chloroform 1-3 times by shaking, separating the chloroform solution for later use; taking the upper aqueous solution, extracting with ethyl acetate 1-3 times by shaking, separating the ethyl acetate solution, combining it with the chloroform solution, evaporating to dryness, dissolving the residue in methanol, transferring it to a volumetric flask, adding methanol to the mark, shaking well, filtering, and taking the filtrate to obtain the solution; Preferably, appropriate amounts of the reference medicinal materials Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Rheum palmatum, Belamcanda chinensis, Glycyrrhiza uralensis, Indigo naturalis, and Sophora tonkinensis are taken respectively, and 50 ml of extraction solvent is added. The mixture is ultrasonically treated for 40 minutes, filtered, and the filtrate is evaporated to dryness. The residue is dissolved in 25 ml of water, 3 drops of hydrochloric acid are added, and the mixture is shaken well. The mixture is then extracted three times with 25 ml of chloroform each time. The chloroform solution is collected and set aside. The upper aqueous solution is taken and extracted three times with 25 ml of ethyl acetate each time. The ethyl acetate solution is collected and combined with the chloroform solution, evaporated to dryness, and the residue is dissolved in methanol. The mixture is transferred to a 5 ml volumetric flask, methanol is added to the mark, shaken well, filtered, and the filtrate is collected to obtain the final product. Preferably, the extraction solvent is 50% or 70% methanol; more preferably, the extraction solvent is 70% methanol.

9. The method for constructing the quality control spectrum of the traditional Chinese medicine composition according to any one of claims 6-8, characterized in that, The preparation method of the reference solution includes: taking appropriate amounts of baicalin, palmatine hydrochloride, berberine hydrochloride, wogonin, irisin, ammonium glycyrrhizate, wogonin, indirubin, and rhein, and dissolving them in methanol to prepare solutions containing 0.1 mg per ml.

10. The method for constructing the quality control spectrum of the traditional Chinese medicine composition according to any one of claims 6-9, characterized in that, The quality control spectrum of the traditional Chinese medicine composition includes 12 chromatographic peaks numbered sequentially according to their elution time. Peaks 1, 5, 9, and 10 are assigned to Scutellaria baicalensis, peaks 2 and 3 to Coptis chinensis, peak 3 to Phellodendron chinense, peaks 4 and 12 to Rheum palmatum, peaks 6 and 7 to Belamcanda chinensis, peak 8 to Glycyrrhiza uralensis, and peak 11 to Indigo naturalis. Alternatively, the quality control spectrum of the traditional Chinese medicine composition includes 14 chromatographic peaks numbered sequentially according to their elution time, wherein peaks 1, 5, 10, and 11 are assigned to Scutellaria baicalensis, peaks 2 and 3 are assigned to Coptis chinensis, peak 3 is assigned to Phellodendron chinense, peaks 4 and 13 are assigned to Rheum palmatum, peaks 6 and 8 are assigned to Belamcanda chinensis, peaks 7 and 14 are assigned to Sophora tonkinensis, peak 9 is assigned to Glycyrrhiza uralensis, and peak 12 is assigned to Indigo naturalis. Preferably, in the quality control spectrum of the traditional Chinese medicine composition, peak 5 is taken as the S peak with a relative retention time of 1, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value; the specified values ​​for peaks 1 to 12 are 0.55, 0.58, 0.62, 0.95, 1.22, 1.61, 1.64, 1.68, 1.78, 2.04, and 2.

37. Alternatively, taking peak 5 as the S-peak with a relative retention time of 1, calculate the relative retention time of each characteristic peak and the S-peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are 0.55, 0.58, 0.62, 0.95, 1.00, 1.22, 1.41, 1.61, 1.64, 1.68, 1.78, 2.04, 2.37, and 2.50, respectively, according to peaks 1 to 14.

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