A fingerprint spectral determination method for a chicken gizzard and chrysanthemum mixed plant solid beverage

Abstract

This invention relates to a method for establishing and detecting the fingerprint spectrum of the traditional Chinese medicine composition Taohongyin. The raw materials of the traditional Chinese medicine composition include: chicken gizzard lining, papaya, hawthorn, chrysanthemum, tangerine peel, peach kernel, and wolfberry. The fingerprint spectrum establishment method includes the preparation of a reference solution and a test solution; detection of the reference solution and the test solution using high-performance liquid chromatography (HPLC); the mobile phase used in the HPLC is acetonitrile and phosphoric acid aqueous solution, and the elution method is gradient elution.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality analysis technology, specifically relating to a high-performance liquid chromatography (HPLC) fingerprint determination method for a chicken gizzard and chrysanthemum mixed plant solid beverage. Background Technology

[0002] The commercial name of this chicken gizzard and chrysanthemum mixed plant solid beverage is "Tao Hong Yin." It is composed of seven traditional Chinese medicines: chicken gizzard, papaya, hawthorn, chrysanthemum, tangerine peel, peach kernel, and wolfberry. It is believed to have the effects of promoting blood circulation, removing blood stasis, clearing qi and blood, and unblocking meridians. Chicken gizzard enters the spleen, stomach, small intestine, and bladder meridians, and has pharmacological activities such as strengthening the stomach and aiding digestion, astringing essence and stopping seminal emission, and clearing urinary tract stones and dissolving stones. Papaya enters the liver and spleen meridians and can be used to treat dampness-induced arthralgia, and lower back and knee pain. Hawthorn, the dried mature fruit of the Rosaceae plant *Crataegus pinnatifida*, enters the spleen, stomach, and liver meridians and is often used to treat food stagnation, abdominal distension, diarrhea, abdominal pain, and amenorrhea due to blood stasis. Chrysanthemum, the dried capitulum of the Asteraceae plant *Chrysanthemum morifolium*, specifically enters the lung and liver meridians and has the effects of dispelling wind and clearing heat, calming the liver and improving eyesight, and clearing heat and detoxifying. Chlorogenic acid and luteolin are the main active ingredients of chrysanthemum. Orange peel, also known as dried tangerine peel, is the dried, mature peel of the tangerine fruit (Citrus reticulata) and its cultivated varieties, belonging to the Rutaceae family. It enters the lung and spleen meridians and has the effects of regulating qi, strengthening the spleen, and resolving dampness and phlegm. Peach kernel is the dried, mature seed of the peach or wild peach plant (Rosa chinensis), belonging to the Rosaceae family. It enters the heart, liver, and large intestine meridians and has the effects of promoting blood circulation, removing blood stasis, moistening the intestines, relieving constipation, and stopping coughs and asthma. Goji berries are the dried, mature fruit of the wolfberry plant (Lycium barbarum), belonging to the Solanaceae family. They enter the liver and kidney meridians and their main effects are nourishing the liver and kidneys, benefiting essence and improving eyesight. They can be used for weakness and deficiency of essence, lower back and knee pain, dizziness and tinnitus, impotence and seminal emission, and internal heat and thirst.

[0003] Peach Blossom Drink (a mixed plant-based solid beverage containing chicken gizzard and chrysanthemum) is a newly developed product by the applicant. It is made from a combination of various main ingredients, resulting in a complex composition, and there are few methods for its quality testing. Currently, there is a lack of rigorous quality control standards, leading to production processes varying based on actual conditions. This will negatively impact the standardized production of Peach Blossom Drink.

[0004] Traditional Chinese medicine (TCM) compositions contain a variety of plant-based medicinal materials with complex compositions. The efficacy of a TCM composition does not stem from a single active ingredient, but rather from the combined effects of all the raw materials in the entire composition. Differences in the content and proportion of active ingredients within a TCM composition can all influence its efficacy. Given the complexity of TCM compositions, it is difficult to effectively evaluate the quality of any single active ingredient or indicator component. Therefore, when evaluating the quality of TCM compositions and their preparations, appropriate testing methods that provide abundant identification information should be employed.

[0005] Traditional Chinese medicine (TCM) fingerprinting is a comprehensive and quantifiable identification method. Based on systematic research into the chemical components of TCM, it is primarily used to evaluate the authenticity, quality, and stability of raw TCM materials and semi-finished TCM preparations. Specifically, it includes two aspects: (1) the characteristic features of the fingerprint spectrum can effectively identify the authenticity of TCM; and (2) the area and proportion of the main characteristic peaks in the fingerprint spectrum can effectively control the quality of TCM. Establishing TCM fingerprinting will comprehensively reflect the types and quantities of chemical components contained in TCM and its preparations, thereby providing an overall description and evaluation of drug quality.

[0006] Currently, the fingerprinting technology for traditional Chinese medicine involves numerous methods, including chromatographic methods such as thin-layer chromatography (TLCS), high-performance liquid chromatography (HPLC), gas chromatography (GC), and high-performance capillary electrophoresis (HPCE), as well as spectroscopic methods such as ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance (NMR), and X-ray diffraction.

[0007] Taohongyin contains various Chinese medicinal herbs, and dozens, or even more, of active ingredients may be detectable. Identifying and determining each of these active ingredients individually would require extensive screening. Currently, there are no reported technologies for establishing fingerprint spectroscopy for quality detection of Taohongyin, thus posing significant technical obstacles to developing a fingerprint spectroscopy detection method for it.

[0008] Therefore, there is a need for a method to monitor the quality of peach blossom drinks using fingerprinting, as well as a standardized production method based on this quality monitoring method. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a method for constructing or detecting the fingerprint spectrum of a chicken gizzard and chrysanthemum mixed plant solid beverage. The medicinal materials for the chicken gizzard and chrysanthemum mixed plant solid beverage are chicken gizzard, papaya, hawthorn, chrysanthemum, tangerine peel, peach kernel, and wolfberry. The fingerprint spectrum construction method includes the following steps:

[0010] 1) Prepare the reference solution;

[0011] 2) Prepare the test solution;

[0012] 3) Take the reference solution and the test solution for high-performance liquid chromatography (HPLC) detection;

[0013] 4) Prepare fingerprint patterns.

[0014] This method is accurate, reliable, and simple to operate, providing a basis for the quality control of this solid beverage.

[0015] The fingerprint chromatogram detection method for the chicken gizzard and chrysanthemum mixed plant solid beverage of the present invention includes: taking the test sample solution for high performance liquid chromatography (HPLC) detection to obtain the fingerprint chromatogram. The chromatographic conditions used are: C18 column, 0.1% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution. The gradient elution program is as follows:

[0016]

[0017] To ensure more accurate fingerprint chromatograms, the reference solution can be tested under the same liquid chromatography conditions before and after the test sample, and the chromatographic peaks can be identified through the chromatogram of the reference solution. The selection of the reference standard was based on the inventor's previous UPLC-Q-Exactive-Orbitrap-MS detection results on the chicken gizzard and chrysanthemum mixed plant solid beverage, followed by literature review, screening of indicator components of each herb in the chicken gizzard and chrysanthemum mixed plant solid beverage, and experimental testing. Ultimately, the hesperidin peak, which showed good peak shape, no interference from impurity peaks before or after the test sample solution of the chicken gizzard and chrysanthemum mixed plant solid beverage, and complete separation, was selected as the reference peak for the fingerprint chromatogram.

[0018] The preparation method of the reference solution is as follows: dissolve the reference standard hesperidin in a solvent capable of dissolving hesperidin. In this invention, methanol and / or ethanol are preferred, with methanol being the most preferred. There are no special requirements for the concentration; any concentration that meets the upper and lower limit detection requirements of the instrument is acceptable. The concentration given in the embodiments of this invention is 95 μg / mL to 110 μg / mL, specifically 100.05 μg / mL.

[0019] The preparation process of the test solution involves extracting a mixture of chicken gizzard and chrysanthemum plant solid beverages using the following method: The chicken gizzard and chrysanthemum plant solid beverages are taken and extracted with a solvent. The solvent can be any solvent commonly used in the art for the extraction of natural compounds, including but not limited to alcohols or aqueous solutions of alcohols, such as methanol or ethanol. In some embodiments of this invention, methanol or a 60%-80% ethanol aqueous solution is used, specifically methanol. The extraction method can use conventional natural compound extraction methods in the art, such as ultrasonic extraction or reflux extraction. In this embodiment of the invention, ultrasonic extraction was specifically employed, with methanol used for ultrasonic extraction for 1 hour. Comparison of several different extraction methods revealed that ultrasonic extraction resulted in a more stable chromatographic baseline, higher peaks, and better peak shapes, making it the optimal method.

[0020] After extraction, the extract is filtered, the filtrate is evaporated to dryness, and the residue is dissolved in a reconstitution solvent, filtered again, and the filtrate is collected. The reconstitution solvent is preferably the same as the extraction solvent. The specific concentration of the reconstituted solution does not need to be strictly within a certain range; it only needs to be sufficient for the equipment to detect the components. A suitable range can be selected as: weight of chicken gizzard and chrysanthemum mixed plant solid beverage: volume of reconstitution solvent = (0.5~1.5) g: 1 mL, for example, 0.5 g, 1 g, or 1.5 g chicken gizzard and chrysanthemum mixed plant solid beverage / mL.

[0021] Under the above conditions, the fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage had 15 common characteristic peaks, of which peak 10 was hesperidin. When the fingerprint chromatogram was taken as reference (S) with the chromatographic peak of hesperidin at peak 10, the relative retention times of the 14 common characteristic peaks were:

[0022]

[0023] Generally, once the fingerprint spectrum identifies the characteristic peaks in a traditional Chinese medicine product, the presence of the characteristic peaks at their respective positions is sufficient to meet the requirements. However, when higher requirements are placed on the fingerprint spectrum, not only must the position of the characteristic peaks meet the requirements, but the area of ​​the characteristic peaks must also meet certain requirements to indicate that the content of each component in the composition meets certain standards.

[0024] When the fingerprint chromatogram uses the chromatographic peak of hesperidin (peak 10) as a reference (S), the relative peak areas of each common characteristic peak are:

[0025]

[0026] In preparing the fingerprint chromatogram, to achieve greater accuracy, chromatographic peaks can be identified using the chromatogram of a reference standard. Fingerprint chromatograms of the chicken gizzard and chrysanthemum mixed plant solid beverage are prepared using chromatograms from different batches of the beverage. Ideally, at least five batches should be used, preferably at least ten. The fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage to be tested is compared with the fingerprint chromatogram constructed according to this invention. A relative retention time within ±5% of the specified value indicates a qualified product. A similarity evaluation is performed between the fingerprint chromatogram and the test chromatogram; a similarity greater than 0.8, preferably greater than 0.90, indicates a qualified product.

[0027] The fingerprinting method of this invention uses a standard C18 column. For more optimized conditions, a 4.6 mm × 150 mm, 3-5 μm column is used. According to the high-performance liquid chromatography (HPLC) detection method of this invention, the C18 column can be an InertSustain column. ® AQ-C18 or XBridge ®In one embodiment of the present invention, the C18 chromatographic column is an XBridge. ® C18 chromatographic column, for example, C 18 The column (4.6 mm × 150 mm, 5 μm) was used. The theoretical plate number, calculated based on hesperidin, should be no less than 5000. Chromatograms of different batches of chicken gizzard and chrysanthemum mixed plant solid beverages and reference standards were obtained. This invention employed both the InertSustain® AQ-C18 column (4.6 mm × 150 mm, 5 μm) and the XBridge® C18 column (4.6 mm × 150 mm, 5 μm). Results showed that the XBridge® C18 column (4.6 mm × 150 mm, 5 μm) produced better peak shapes and the best resolution for each component. Therefore, it can be considered the preferred column for subsequent studies.

[0028] The wavelength range of 280-290 nm can achieve peak elution of various components, which can meet the requirements for fingerprint spectroscopy preparation. Taking the reference standard of this invention as an example, hesperidin showed the best absorption at 283 nm.

[0029] This invention does not involve temperature-sensitive substances or operations, therefore there are no special limitations on column temperature, and normal detection can usually be achieved under laboratory conditions. Therefore, a high-performance liquid chromatography (HPLC) detection environment of 20–40 °C is recommended; for example, 30 °C or 25 °C can be set in the embodiments of this invention.

[0030] The flow rate of the mobile phase can be adjusted according to equipment performance and operating efficiency, provided that the peaks of each component are well separated. Excessive flow rate leads to high equipment wear and tear, while insufficient flow rate results in prolonged processing time. In this embodiment of the invention, based on the instrument and equipment specifications and operating efficiency, the most commonly used flow rate of 1 ml / min is used. -1 And at a flow rate of 0.8-1.3 ml / min. -1 The range is to be tested.

[0031] There is generally no fixed limit to the injection volume in high-performance liquid chromatography (HPLC), as long as it meets the requirements of ease of operation and the basic detection limit of the equipment. The injection volume can be 5.0-20.0 μL, as is the standard for HPLC methods, such as 5 μL or 10 μL.

[0032] The separation effect can be guaranteed within the gradient elution conditions given above in this invention. The most preferred gradient conditions are:

[0033]

[0034] In this invention, "optional," "optionally," or "optionally exist" means that the event or situation described below may but not necessarily occur, and the description includes both cases where the event or situation occurs and cases where it does not occur.

[0035] In this invention, "comprising" and "including" are both open-ended expressions, meaning they include the content specified in this invention, but do not exclude other aspects. It should be understood that "comprising" and "including" can also encompass a closed meaning, that is, "consisting of".

[0036] The beneficial effects of this invention:

[0037] Traditional Chinese medicine fingerprinting is mainly used to evaluate the stability and reproducibility of the quality of Chinese medicinal materials and their preparations. This invention uses chemical fingerprinting as a quality control method to rapidly and intuitively identify the pharmacodynamic material basis of a chicken gizzard and chrysanthemum mixed plant solid beverage, identifying 15 common peaks, thus laying the foundation for establishing comprehensive quality standards for the chicken gizzard and chrysanthemum mixed plant solid beverage.

[0038] By analyzing 10 batches of chicken gizzard and chrysanthemum mixed plant solid beverages, an HPLC fingerprint determination method for these beverages was established, and their quality was comprehensively evaluated. Precision, repeatability, and stability experiments verified that the method of this invention has high stability and reliability, and therefore can be used as a method for determining the fingerprint spectrum of chicken gizzard and chrysanthemum mixed plant solid beverages. Attached Figure Description

[0039] Figure 1 Chromatogram A is the chromatogram of hesperidin reference standard; Figure 1 B is the fingerprint spectrum of a chicken gizzard and chrysanthemum mixed plant solid beverage; Figure 1 C represents the overlay of HPLC fingerprint chromatograms of 10 batches of chicken gizzard and chrysanthemum mixed plant solid beverage.

[0040] Figure 2 In the chromatogram, A represents the upper limit of gradient elution; Figure 2 In the chromatogram, B represents the lower limit of gradient elution.

[0041] Figure 3 The images show the fingerprints of the chicken gizzard and chrysanthemum mixed plant solid beverage obtained under different elution conditions, where A and B are the fingerprints of the chicken gizzard and chrysanthemum mixed plant solid beverage under elution conditions one and two, respectively.

[0042] Figure 4 The fingerprint chromatograms of chicken gizzard and chrysanthemum mixed plant solid beverage obtained under different chromatographic columns are shown. A is the fingerprint chromatogram of chicken gizzard and chrysanthemum mixed plant solid beverage under XBridge® C18 chromatographic column conditions; B is the fingerprint chromatogram of chicken gizzard and chrysanthemum mixed plant solid beverage under InertSustain® AQ-C18 chromatographic column conditions.

[0043] Figure 5 The images show the fingerprints of the chicken gizzard and chrysanthemum mixed plant solid beverage obtained under different sample extraction methods, where A, B, and C are the fingerprints of the chicken gizzard and chrysanthemum mixed plant solid beverage under extraction methods A, B, and C, respectively. Detailed Implementation

[0044] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0045] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0046] Experimental instruments and reagents:

[0047] 1. Instruments

[0048] High-performance liquid chromatograph (model: LC-20A, Shimadzu Corporation, Japan); magnetically stirred water bath (model: LC-WB-8+, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); XS105 electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); Milli-Q ultrapure water purification system (Beijing Wuzhou Dongfang Technology Co., Ltd.).

[0049] 2. Reagents

[0050] Hesperidin (purity ≥ 98%, batch number 110721-202220, China National Institutes for Food and Drug Control); 10 batches (S1-S10) of chicken gizzard and chrysanthemum mixed plant solid beverage, batch numbers 202111191, 202111192, 202111201, 202111211, 202111291, 202112281, 202202101, 202203241, 202203251, 202204121, source: Shanxi Yabao Jiuhe Pharmaceutical Technology Co., Ltd.; chromatographic grade acetonitrile; chromatographic grade methanol; ultrapure water.

[0051] Example 1: Establishment of a standard fingerprint spectrum for a chicken gizzard and chrysanthemum mixed plant solid beverage

[0052] Fingerprint patterns were prepared under the following conditions; see [link to results]. Figure 1 .

[0053] 1. Chromatographic conditions

[0054] HPLC fingerprint analysis conditions were performed using XBridge.® C18 column (4.6 mm × 150 mm, 5 μm); mobile phase A (0.1% phosphoric acid aqueous solution), mobile phase B (acetonitrile); gradient elution; flow rate 1.0 mL·min -1 Column temperature 30℃; injection volume 5 μL; detection wavelength 283 nm.

[0055] Table 1 Gradient elution method

[0056]

[0057] 2. Preparation of reference solution

[0058] Accurately weigh hesperidin reference standard and add methanol to prepare a reference solution with a concentration of 100.05 μg / ml.

[0059] 3. Preparation of test solution

[0060] Weigh 5.0g of the chicken gizzard and chrysanthemum mixed plant solid beverage granules, place them in a stoppered conical flask, add an appropriate amount of methanol, sonicate for 1 hour, shake well, filter, and evaporate the filtrate to dryness. Redissolve in 5ml of methanol, centrifuge, and take the supernatant for nitrogen blowing until the volume is half of the original volume.

[0061] 4. HPLC fingerprint analysis of chicken gizzard and chrysanthemum mixed plant solid beverage

[0062] 4.1 Precision Test

[0063] A batch (batch number: 202203241) of chicken gizzard and chrysanthemum mixed plant solid beverage was sampled and injected six times consecutively. The retention time and peak area of ​​each common chromatographic peak were recorded. Using the retention time and peak area of ​​hesperidin (peak 10) as a reference, the relative retention time and relative peak area of ​​each common peak were calculated to examine the consistency of the ratio of relative retention time to relative peak area. The results showed that the RSD of the relative retention time of each common peak was less than 1%, and the RSD of the relative peak area was less than 2.5%, indicating that the determination method of the present invention is stable, has good system tightness, and is stable and reliable.

[0064] 4.2 Stability Test

[0065] A batch (batch number: 202203241) of chicken gizzard and chrysanthemum mixed plant solid beverage was tested and analyzed at different time points (0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h). The retention time and peak area of ​​each common chromatographic peak were recorded. Using the retention time and peak area of ​​hesperidin (peak 10) as a reference, the relative retention time and relative peak area of ​​each common peak were calculated to examine the consistency of the ratio of relative retention time to relative peak area. The results showed that the RSD of the relative retention time of each common peak was less than 1%, and the RSD of the relative peak area was less than 2.5%. This indicates that the chicken gizzard and chrysanthemum mixed plant solid beverage sample of the present invention has good stability under experimental conditions and can guarantee the reliability of analytical results within 24 hours.

[0066] 4.3 Repeatability Test

[0067] Six parallel solutions of a chicken gizzard and chrysanthemum mixed plant solid beverage from the same batch (batch number: 202203241) were prepared according to the method described in section "3". These solutions were injected and analyzed separately, and the retention time and peak area of ​​each common chromatographic peak were recorded. Using the retention time and peak area of ​​hesperidin (peak 10) as a reference, the relative retention time and relative peak area of ​​each common peak were calculated to examine the consistency of the ratio of relative retention time to relative peak area. The results showed that the RSD of the relative retention time of each common peak was less than 1%, and the RSD of the relative peak area was less than 2.65%, indicating that the method of this invention is stable, reliable, and suitable for widespread application.

[0068] 4.4 Establishment and Similarity Evaluation of HPLC Fingerprint for Chicken Gizzard and Chrysanthemum Mixed Plant Solid Beverage

[0069] Ten batches of chicken gizzard and chrysanthemum mixed plant solid beverage (batch numbers: 202111191, 202111192, 202111201, 202111211, 202111291, 202112281, 202202101, 202203241, 202203251, 202204121) were prepared as test solutions according to the method in section “3”, and injected for analysis. Chromatograms were recorded for each batch. A representative chromatogram (chicken gizzard and chrysanthemum mixed plant solid beverage batch number 202203241) was selected as the reference fingerprint chromatogram. The “Similarity Evaluation System for Chromatogram Fingerprints of Traditional Chinese Medicine” recommended by the Pharmacopoeia Commission was used for analysis. Chromatogram similarity was used as the fingerprint chromatogram evaluation index, and a common pattern of fingerprint chromatograms was generated by multi-point calibration full-spectrum matching. The similarity between each batch of samples and the control fingerprint spectrum was (0.998, 0.997, 0.996, 0.997, 0.996, 0.996, 0.996, 0.997, 0.999, 0.997).

[0070] 4.5 Identification of common peaks in fingerprint spectra, calculation of relative peak area and relative retention time

[0071] Using a similarity evaluation system, 15 peaks were detected in 10 batches of fingerprint chromatograms. Compared with the mixed reference standard, peak number 10 was identified as hesperidin, with a retention time of 68.625 min. Peak number 10 had a good peak shape, with no interference from impurity peaks before or after it, indicating complete separation; therefore, it was selected as the reference peak for calculating the relative peak area and relative retention time of other common peaks. The results are shown in Tables 2 and 3 below.

[0072] Table 2. Relative peak areas of 15 common peaks in 10 batches of samples (S1~S10)

[0073]

[0074] Table 3. Relative retention times of 15 common peaks in 10 batches of samples (S1~S10)

[0075]

[0076] 4.6 Range of gradient elution conditions

[0077] By adjusting the gradient elution conditions, it can be found that under the currently determined elution conditions, the ratio of mobile phases A and B can be appropriately adjusted, and the upper and lower limits of the elution conditions can be determined through experiments.

[0078] Upper limit of elution conditions:

[0079]

[0080] Lower limit elution conditions:

[0081]

[0082] See results Figure 2 , Figure 2 In this context, A represents the upper limit elution condition of the mobile phase. Figure 2 B represents the lower limit elution condition of the mobile phase. Both conditions must be met to achieve good separation and complete elution.

[0083] Example 2: Comparison of fingerprint spectral preparation methods for chicken gizzard and chrysanthemum mixed plant solid beverage

[0084] 1. Determination of chromatographic conditions and exploration of fingerprinting methods

[0085] The fingerprint chromatograms of Taohong drink were prepared under different high-performance liquid chromatography (HPLC) conditions.

[0086] (1) Elution conditions

[0087] The inventors compared several different elution conditions and proved that the mobile phase elution conditions currently selected in this invention are optimal. Due to the large number of samples, only a brief example is given below: Granules of this product were taken and prepared according to the method for preparing the test solution in Example 1. The following two elution conditions were compared, and the results are shown in [Figure 1]. Figure 3 As can be seen, under the elution conditions below, the peaks of each component are well-defined, indicating good separation. Therefore, elution condition one was selected as the elution condition for the fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage sample.

[0088] Washing condition one:

[0089]

[0090] Washing condition two:

[0091]

[0092] (2) Column type

[0093] Take the granules of this product and analyze them according to the preparation method and chromatographic conditions of the test solution in Example 1, using InertSustain® AQ-C18 (4.6 mm × 150 mm, 5 μm) and XBridge® C18 (4.6 mm × 150 mm, 5 μm) columns, respectively. The results are shown in the figure. Figure 4 It can be seen that both can meet the requirements for fingerprint chromatogram testing and can be used as chromatographic columns for fingerprint chromatogram preparation. The chromatograms obtained using the XBridge® C18 column (4.6 mm × 150 mm, 5 μm) showed good peak shapes and resolution for each component. Therefore, the XBridge® C18 column (4.6 mm × 150 mm, 5 μm) was selected as the chromatographic column for the fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage sample.

[0094] 2. Determination of Sample Extraction Method

[0095] Peach Blossom Drink has a complex composition, and different extraction solvents can affect the extraction efficiency of its active ingredients. Finding the most suitable extraction method is crucial to maximizing the extraction of more components and ensuring that they are reflected as peaks in the HPLC chromatogram, which is one of the technical challenges.

[0096] Samples were extracted using several different methods, A, B, and C, with the following procedures:

[0097] A: Take about 5.0 g of the product granules, accurately weigh them, place them in a stoppered conical flask, add an appropriate amount of methanol, extract by sonication for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0098] B: Take about 5.0g of the granules of this product, accurately weigh them, place them in a stoppered conical flask, add an appropriate amount of methanol, heat in an 80°C water bath under reflux for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5ml of methanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0099] C: Take about 5.0 g of the granules of this product, accurately weigh them, place them in a stoppered conical flask, add an appropriate amount of 70% ethanol, heat in an 80°C water bath under reflux for 1 h, cool, filter, evaporate the filtrate to dryness, add 5 ml of 70% ethanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0100] The test solutions prepared by the above three methods were detected according to the final chromatographic conditions determined above, and the results are as follows: Figure 5 As shown, all three methods meet the requirements for preparing fingerprint chromatograms and can be used as extraction methods for fingerprint chromatogram preparation or content determination. However, after comparison, it was found that the methanol ultrasonic extraction method is the best. This method produces the most stable chromatogram baseline, the best peak separation effect, and the best peak shape.

[0101] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fingerprint chromatographic detection method for a traditional Chinese medicine composition, a chicken gizzard lining and chrysanthemum mixed plant solid beverage, wherein the raw materials of the traditional Chinese medicine composition are chicken gizzard lining, papaya, hawthorn, chrysanthemum, tangerine peel, peach kernel, and wolfberry. The sample solution is subjected to high-performance liquid chromatography (HPLC) to obtain the fingerprint chromatogram. The chromatographic conditions used are: a C18 column, with 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and gradient elution is performed. The gradient elution program is as follows: The preparation method of the test solution is as follows: take chicken gizzard and chrysanthemum mixed plant solid beverage, add extraction solvent to extract, filter the extract, evaporate the filtrate to dryness, add extraction solvent to redissolve, filter, and take the filtrate as the test solution; the extraction solvent is methanol or 60%-80% ethanol aqueous solution.

2. The method as described in claim 1, characterized in that, It also includes a high-performance liquid chromatography (HPLC) step for detecting a reference solution, wherein the reference solution is hesperidin.

3. The method as described in claim 2, characterized in that, The preparation method of the reference solution is as follows: take the reference standard hesperidin, add methanol or ethanol to dissolve it, and the concentration of hesperidin in the reference solution is 95 μg / mL to 110 μg / mL.

4. The method as described in claim 1, characterized in that, The extraction process in the preparation method of the test sample solution is ultrasonic extraction.

5. The method as described in claim 1, characterized in that, The fingerprint spectrum has 15 characteristic peaks, of which peak 10 is hesperidin. Using the chromatographic peak of hesperidin (peak 10) as a reference (S), the relative retention times of the 14 characteristic peaks are: 。 6. The method as described in claim 5, characterized in that, When the fingerprint spectrum uses the chromatographic peak of hesperidin (peak 10) as a reference (S), the relative peak areas of each characteristic peak are: 。 7. The method according to any one of claims 1-6, characterized in that, The detection wavelength of the high-performance liquid chromatography is 280-290 nm.

8. The method as described in claim 7, characterized in that, The detection wavelength of the high-performance liquid chromatography is 283 nm.

9. The method according to any one of claims 1-6, characterized in that, The gradient elution procedure is as follows: 。 10. The use of the method according to any one of claims 1-9 in the quality control of chicken gizzard and chrysanthemum mixed plant solid beverage: the fingerprint spectrum of the chicken gizzard and chrysanthemum mixed plant solid beverage to be tested is compared with the fingerprint spectrum of the standard chicken gizzard and chrysanthemum mixed plant solid beverage. The relative retention time should be within ±5% of the specified value for the product to be qualified. The similarity evaluation with the standard fingerprint spectrum is performed. The similarity is greater than 0.8 for the product to be qualified.

Images