Method for detecting rhizoma atractylodis macrocephalae medicinal material in five-ingredient spleen-tonifying mixture based on UPLC-FLR
By combining UPLC-FLR detection with a fluorescence detector, the accuracy and sensitivity issues of Atractylodes macrocephala in the Five-Flavor Spleen-Strengthening Compound were resolved. This enabled precise qualitative and efficient detection of Atractylodes macrocephala, improved the reliability and ease of detection, reduced costs, and provided an effective solution for the quality control of compound Chinese medicines.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-20
- Publication Date
- 2026-04-07
AI Technical Summary
The existing quality assessment methods for Wuwei Jianpi Compound have limitations, including a single evaluation dimension, weak correlation with efficacy, insufficient process control, insufficient accuracy and reproducibility of detection methods, high detection costs, severe interference from the compound matrix, and difficulty in achieving high specificity and sensitivity detection of Atractylodes macrocephala.
The UPLC-FLR detection method, combined with a fluorescence detector, was used to analyze Atractylodes macrocephala in Wuwei Jianpi Heji by ultra-high performance liquid chromatography. Acetonitrile-phosphoric acid and water-phosphoric acid were used as mobile phases. The excitation wavelength of the fluorescence detector was 270-290 nm and the emission wavelength was 440-460 nm. Combined with a specific chromatographic gradient elution program, accurate qualitative and high-sensitivity detection of Atractylodes macrocephala was achieved.
It effectively avoids matrix interference from non-fluorescent components in compound preparations, achieves accurate characterization of Atractylodes macrocephala, shortens analysis time, reduces solvent consumption, improves detection reliability and ease of operation, reduces detection costs, and provides a quality control solution for complex compound Chinese medicine systems.
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Figure CN121805461A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug quality control technology, specifically relating to a method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR. Background Technology
[0002] Wuwei Jianpi Heji (Five-Ingredient Spleen-Strengthening Compound) is a traditional Chinese medicine preparation composed of Atractylodes macrocephala (fried), Codonopsis pilosula, Shenqu (medicated leaven), Dioscorea opposita, and Glycyrrhiza uralensis (processed). It functions to strengthen the spleen, promote digestion, and eliminate dampness in the stomach, primarily used to promote the growth of broiler chickens. Existing quality assessment methods for Wuwei Jianpi Heji are mainly based on qualitative and quantitative analysis of chemical components. The core method involves determining the content of a few key components (such as atractylodes macrocephala lactone III and glycyrrhizic acid) using high-performance liquid chromatography (HPLC), combined with fingerprint analysis for similarity comparison, while controlling conventional physicochemical indicators (such as relative density and pH). This quality assessment method suffers from significant drawbacks, including a single evaluation dimension, weak correlation with efficacy, and insufficient process control.
[0003] Meanwhile, existing technologies for detecting Atractylodes macrocephala in Wuwei Jianpi Heji (a traditional Chinese medicine formula) mainly rely on chromatographic techniques such as HPLC-UV and LC-MS, using atractylodes lactones as indicators. Although basic analytical methods have been established, severe matrix interference from the compound leads to co-elution and ion inhibition effects. The poor stability and insufficient detection sensitivity of trace lactones limit accurate quantification. Moreover, the expensive reference standards result in high usage costs. These problems collectively restrict the accuracy, reproducibility, and practical application value of the detection methods. Summary of the Invention
[0004] The purpose of this invention is to provide a method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR, thereby overcoming the shortcomings of the prior art and constructing a precise detection scheme with high specificity, high sensitivity and high efficiency, providing a reference for the quality control of complex compound Chinese medicine systems.
[0005] To achieve the above objectives, the technical solution of the present invention is as follows: On one hand, this invention provides a method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR, comprising the following steps: Mix the Five-Flavor Spleen-Strengthening Compound with a solvent, centrifuge, collect the supernatant, dry the solvent in the supernatant in a water bath, add solvent to redissolve, filter, and obtain the test solution. The test solution was analyzed using ultra-high performance liquid chromatography with a fluorescence detector; The presence of Atractylodes macrocephala in the Five-Flavor Spleen-Strengthening Compound was determined by the chromatographic peak with the same retention time as that in the control medicinal material. In the ultra-high performance liquid chromatography, mobile phase A was acetonitrile-phosphoric acid, and mobile phase B was water-phosphoric acid; the excitation wavelength of the fluorescence detector used was 270-290 nm, and the emission wavelength was 440-460 nm.
[0006] In some other embodiments, the Five-Flavor Spleen-Strengthening Compound is composed of the following parts by weight: 180-220 parts of stir-fried Atractylodes macrocephala, 180-220 parts of Codonopsis pilosula, 260-270 parts of Shenqu (medicated leaven), 180-220 parts of Dioscorea opposita, and 130-140 parts of prepared Glycyrrhiza uralensis.
[0007] In some other embodiments, the Five-Flavor Spleen-Strengthening Compound is composed of the following parts by weight: 200 parts of stir-fried Atractylodes macrocephala, 200 parts of Codonopsis pilosula, 267 parts of Shenqu (medicated leaven), 200 parts of Dioscorea opposita, and 133 parts of prepared Glycyrrhiza uralensis.
[0008] In some other embodiments, the preparation method of the Five-Flavor Spleen-Strengthening Compound is as follows: The ingredients of the Five-Flavor Spleen-Strengthening Compound are soaked in water and decocted multiple times. The volatile oil is collected and the decoction is combined. The decoction is filtered and concentrated to obtain a concentrated solution. Then, an acetic acid solution containing chitosan is added to clarify the solution and filtered to obtain a filtrate. The volatile oil and filtrate are mixed and sodium benzoate and water are added to form a mixture. The mixture is then filled and sterilized to obtain the final product.
[0009] In some other embodiments, the decoction is performed twice, with the first decoction lasting 1.5-2.5 hours and the second decoction lasting 0.5-1.5 hours; the volume ratio of the concentrated liquid to the acetic acid solution containing chitosan is (8-12):1, the mass fraction of chitosan in the acetic acid solution containing chitosan is 0.5-1.5%, and the mass fraction of the acetic acid solution is 1-3%; the mass fraction of sodium benzoate in the mixture is 0.2-0.4%.
[0010] In some other embodiments, the decoction is performed twice, with the first decoction lasting 2.0 h and the second decoction lasting 1.0 h; the volume ratio of the concentrated liquid to the acetic acid solution containing chitosan is 10:1, the mass fraction of chitosan in the acetic acid solution containing chitosan is 1.0%, and the mass fraction of acetic acid solution is 2%; the mass fraction of sodium benzoate in the mixture is 0.3%.
[0011] In some other embodiments, the volume ratio of the Five-Flavor Spleen-Strengthening Compound to the solvent is (3-5):1, the solvent is ethanol, and the volume concentration of ethanol is 75%-100%; the water bath drying temperature is 55-65℃, and the pore size of the filter membrane used for filtration is 0.2-0.3μm.
[0012] In some other embodiments, anhydrous ethanol is used as the solvent during the mixing of the Five Flavors Spleen-Strengthening Compound with the solvent; and ethanol with a volume concentration of 75%-80% is used as the solvent for reconstitution.
[0013] In some other embodiments, the ultra-high performance liquid chromatography (UPLC) uses a Waters UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) and a column temperature of 30 °C. + At 2℃, the flow rate was 0.3-0.4 mL / min. Mobile phase A was acetonitrile-0.2% phosphoric acid, and mobile phase B was water-0.2% phosphoric acid. The gradient elution program was as follows: 0-3 min, 10% mobile phase A; 3-5 min, 10%-20% mobile phase A; 5-8 min, 20%-30% mobile phase A; 8-12 min, 30%-50% mobile phase A; 12-16 min, 50% mobile phase A; 12-17 min, 50%-10% mobile phase A; 17-19 min, 10% mobile phase A. The fluorescence detector used had an excitation wavelength of 280 nm, an emission wavelength of 450 nm, and an injection volume of 1 μL.
[0014] On the other hand, the present invention provides the application of the method for detecting Atractylodes macrocephala in Wuwei Jianpi Mixture based on UPLC-FLR as described in the first aspect in the quality control of Wuwei Jianpi Mixture.
[0015] The beneficial effects of this invention are: This invention effectively avoids matrix interference from non-fluorescent components in compound preparations by utilizing the selective excitation mechanism of the fluorescence detector in UPLC-FLR, thus achieving accurate characterization of Atractylodes macrocephala. Combined with the rapid separation capability of UPLC, the analysis time is significantly shortened and solvent consumption is reduced. While improving detection reliability, it also has practical value of being easy to operate and cost-controllable, providing an ideal technical path for the quality control of complex compound traditional Chinese medicine systems. Attached Figure Description
[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0017] Figure 1 This is the chromatogram of the reference solution (Atractylodes macrocephala reference material) in Example 1 of the present invention; Figure 2 The chromatogram of the test solution (Wuwei Jianpi Compound) measured once in Example 1 of the present invention; Figure 3 The chromatograms of the test solution (Wuwei Jianpi Compound) measured twice in Example 1 of the present invention; Figure 4 This is a superimposed chromatogram of the reference solution (Atractylodes macrocephala reference material) and the test solution (Wuwei Jianpi Compound) in Example 1 of the present invention. Figure 5The images show the thin-layer chromatograms of Atractylodes macrocephala as a reference material and the test solution (Wuwei Jianpi Mixture) in Comparative Example 1. In the images, a is the thin-layer chromatogram of Atractylodes macrocephala as a reference material and b is the thin-layer chromatogram of the test solution (Wuwei Jianpi Mixture). Figure 6 The HPLC-PDA chromatogram (225 nm) of Atractylodes macrocephala in Comparative Example 2 is shown. Figure 7 The HPLC-PDA chromatogram (225 nm) of the test solution (Wuwei Jianpi Mixture) in Comparative Example 2 is shown. Figure 8 The HPLC-PDA chromatogram (251 nm) of Atractylodes macrocephala in Comparative Example 2 is shown. Figure 9 The HPLC-PDA chromatogram (251 nm) of the test solution (Wuwei Jianpi Heji) in Comparative Example 2 is shown. Detailed Implementation
[0018] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.
[0019] The specific instruments, equipment, and consumables used are as follows: Waters Acquity™ ultra-high performance liquid chromatograph (Waters Corporation, USA), with a fluorescence detector (in this invention) or a PDA detector (comparative example), electronic balance XPR205 (METTLERTOLEDO), ultrasonic cleaner KQ-300DV (Kunshan Shumei Company), high-precision water bath WNB7 (MEMMERT Company), centrifuge CF16RXII (Hitachi Kogyo Co., Ltd.), evaporating dishes, test tubes, Atractylodes macrocephala reference material (China Institute of Veterinary Drug Control), Wuwei Jianpi Mixture, acetonitrile (chromatographic grade), ethanol (analytical grade), phosphoric acid (analytical grade), ultrapure water, etc.
[0020] Example 1 1. Sample pretreatment: The composition and preparation method of the Five-Flavor Spleen-Strengthening Compound are as follows: Composition: Atractylodes macrocephala (fried) 200 g, Codonopsis pilosula 200 g, Massa fermentata 267 g, Dioscorea opposita 200 g, Glycyrrhiza uralensis (processed) 133 g.
[0021] Preparation method: Soak the above 5 ingredients in water for 2 hours, then decoct twice, the first time for 2 hours and the second time for 1 hour. Collect the volatile oil separately. Combine the two decoctions, filter, and concentrate the filtrate to about 800 mL. Add the filtrate to a 1% chitosan and 2% acetic acid solution at a volume ratio of 10:1, clarify thoroughly, and filter. Mix the filtrate with the volatile oil, add 3 g of sodium benzoate, mix well, add water to 1000 mL, fill into containers, and sterilize to obtain the final product.
[0022] Test solution (Wuwei Jianpi Mixture): Take 20 mL of Wuwei Jianpi Mixture, add 5 mL of anhydrous ethanol, sonicate for 30 min, centrifuge at 12000 rpm for 5 min to collect the supernatant, take 10 mL of the supernatant to an evaporating dish, air dry the solvent in a fume hood at 60 ℃, dissolve in 2 mL of 75% ethanol, then pipette into a syringe, filter through a 0.22 μm filter, and use as the test solution for instrumental analysis.
[0023] Reference solution (Atractylodes macrocephala reference material): Take 1g of Atractylodes macrocephala reference material, add 25mL of anhydrous ethanol, sonicate for 30min, centrifuge at 12000 rpm for 5min to collect the supernatant, take 10mL of the supernatant to an evaporating dish, air dry the solvent in a fume hood at 60℃, dissolve in 2mL of 75% ethanol, then pipette into a syringe, filter through a 0.22μm filter, and use as the reference solution for instrumental analysis.
[0024] 2. Liquid chromatography method: Waters UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: acetonitrile (0.2% phosphoric acid), mobile phase B: water (0.2% phosphoric acid); column temperature 30 °C. + At 2 ℃ and a flow rate of 0.35 mL / min, gradient elution was performed as shown in Table 1.
[0025] Fluorescence detector: excitation wavelength 280 nm, emission wavelength 450 nm; injection 1 μL, injection mode: do not fill the quantitative loop, use needle overflow.
[0026] Strong wash solvent: 90% acetonitrile, strong wash volume 200μL; weak wash solvent: 10% acetonitrile, weak wash volume 600μL.
[0027] Table 1 Gradient elution program for liquid chromatography
[0028] UPLC-FLR analysis was performed on the reference solution (Atractylodes macrocephala reference material) and the test solution (Wuwei Jianpi Compound), with an injection volume of 1.0 μL and ACQUITY FLRChA channel used for both. The chromatographic data obtained for the reference solution (Atractylodes macrocephala reference material) are shown in Table 2. Figure 1As shown in Table 3. The chromatographic data obtained from the test solution (Wuwei Jianpi Heji) are shown in Table 3 and... Figures 2-3 As shown.
[0029] Table 2. Chromatographic data of the reference standard (Atractylodes macrocephala reference material).
[0030] Table 3. Chromatographic data of the test solution (Wuwei Jianpi Compound)
[0031] A comparison of Tables 2 and 3 reveals that the retention times of the samples all fall within the range of 0.95–1.05 seconds, which is the retention time of the reference standard. Figure 4 This is a superimposed chromatogram of the reference herb and the test sample of Atractylodes macrocephala, created by... Figure 4 The characteristic peaks of Atractylodes macrocephala are clearly visible in the test sample. Peaks 1-9 show excellent overlap and a high signal-to-noise ratio (SNR). The theoretical plate number and resolution meet the detection requirements, indicating the detection of the target herb. Peaks 5 and 10 have a low SNR (greater than 3), are detectable, but their SNR is less than 10. Peaks 5 and 10 can be selectively removed, retaining the other eight peaks. The remaining peaks all have SNRs greater than 10 and can be considered characteristic peaks. Peaks 2, 3, 6, and 9 have the highest response values and SNRs exceeding 100. Optimizing these four peaks ensures the detection of the target herb. Furthermore, only 1 μL of the test sample was injected. Increasing the injection volume can improve sensitivity and detection limit. The entire detection process takes only about 100 minutes, improving the efficiency of the inspector and reducing the health impact of organic solvents.
[0032] In addition, compared with thin-layer chromatography, the detection limit is improved by at least 25 times. This is because the sample is diluted 5 times during processing compared with thin-layer chromatography, and the injection volume is one-fifth of the spotting volume. Furthermore, based on the lowest signal-to-noise ratio peak 9 among the four peaks, it can be estimated that the detection limit can be further reduced by 118 / 3, approximately 39 times, to 20 mL / 5 / 5 / 39, approximately 0.0205 mL, which is about 975 times higher than that of thin-layer chromatography. If the injection volume is increased by 5 μL, it is estimated that the detection limit can be improved by 975 × 5 times, approximately 4875 times, compared with thin-layer chromatography.
[0033] Comparative Example 1 Thin-layer chromatography (TLC) from the 2017 Chinese Medicine Volume of Standard Methods was used to detect the Atractylodes macrocephala in Wuwei Jianpi Mixture.
[0034] Take 20 mL of this product and extract twice with petroleum ether (30-60℃), 25 mL each time. Combine the extracts, evaporate to dryness, and dissolve the residue in 1 mL of methanol to prepare the test solution. Separately, take 2 g of Atractylodes macrocephala reference material, add 50 mL of water, decoct for 30 min, cool, and filter. Prepare the reference material solution using the same method as described in the step "extracted twice with petroleum ether (30-60℃)". Perform thin-layer chromatography (Appendix 0502). Apply 5 μl of each of the above two solutions to the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate (7:3) as the developing solvent. Remove the plate, air dry, spray with 5% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.
[0035] Thin-layer chromatograms of Atractylodes macrocephala and the test solution (Wuwei Jianpi Compound) are shown below. Figure 5 As shown, the study found that the detection limit of thin-layer chromatography (TLC) was 20 mL of test sample. However, the sample pretreatment process is complex, involving time-consuming steps such as development, drying, spraying of colorimetric reagent, and heating for color development. This process is also highly susceptible to interference, with spots easily affected by temperature and humidity, resulting in unclear spots, severe edge effects, and making it difficult for the control and sample spots to be on the same parallel line. Figure 5 The results (a and b) in the test are not accurate; in addition, the test takes a long time, at least seven or eight hours, and the solvents such as petroleum ether, cyclohexane, ethyl acetate, and dimethylaminobenzaldehyde are highly toxic.
[0036] Comparative Example 2 Unlike Example 1, the ultra-high performance liquid chromatograph used was not equipped with a fluorescence detector, but with a PDA detector. Other detection steps and chromatographic conditions such as mobile phase were the same as in Example 1, with detection wavelengths of 225 nm and 251 nm.
[0037] Depend on Figure 6 It can be seen that four chromatographic peaks can be detected in the 225nm Atractylodes macrocephala reference material. By optimizing the wavelength to 225nm, however... Figure 7 The four chromatographic peaks in the sample showed poor resolution, with peak times being identical (e.g., peaks 1, 2, and 3), and close to baseline noise. Furthermore, the spectra indicated they were not the same substance. Peak 4 also exhibited very low reactivity, making it difficult to identify and detect the Atractylodes macrocephala reference material. This method is incomparable to fluorescence detection. At 20 mL, it was essentially undetectable.
[0038] Depend on Figure 8 It can be seen that two chromatographic peaks can be detected in the 251nm Atractylodes macrocephala reference material, but Figure 9The four chromatographic peaks in the sample had poor resolution, and the peak times were the same, such as peaks 1 and 3. They were close to the baseline and negligible. Moreover, the spectra showed that they were not the same substance at all. Peaks 2 and 4 were also very close to the baseline noise, making it difficult to determine whether the Atractylodes macrocephala reference material was detected.
[0039] Comparative Example 3 Unlike Example 1, the sample pretreatment process uses organic solvents such as ethyl acetate or petroleum ether as solvents for extraction. These solvents have a pungent odor and may affect the health of testing personnel, so their use is not recommended.
[0040] Comparative Example 4 Unlike Example 1, centrifugation was not used in the sample pretreatment process. The resistance during filtration was too great, causing the filter head to become clogged or the pressure to be too great, resulting in splashing. Centrifugation was necessary to collect the supernatant and filter it before it was put into the machine.
[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR, characterized in that, Includes the following steps: Mix the Five-Flavor Spleen-Strengthening Compound with a solvent, centrifuge, collect the supernatant, dry the solvent in the supernatant in a water bath, add solvent to redissolve, filter, and obtain the test solution. The test solution was detected using an ultra-high performance liquid chromatograph with a fluorescence detector; The presence of Atractylodes macrocephala in Wuwei Jianpi Mixture was determined by the chromatographic peak with the same retention time as that in the reference medicinal material. In the ultra-high performance liquid chromatography, mobile phase A was acetonitrile-phosphoric acid, and mobile phase B was water-phosphoric acid; the excitation wavelength of the fluorescence detector used was 270-290 nm, and the emission wavelength was 440-460 nm.
2. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 1, characterized in that, The Five-Flavor Spleen-Strengthening Compound is composed of the following parts by weight: 180-220 parts of stir-fried Atractylodes macrocephala, 180-220 parts of Codonopsis pilosula, 260-270 parts of Shenqu (medicated leaven), 180-220 parts of Dioscorea opposita, and 130-140 parts of prepared Glycyrrhiza uralensis.
3. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 2, characterized in that, The Five-Flavor Spleen-Strengthening Compound is composed of the following ingredients by weight: 200 parts stir-fried Atractylodes macrocephala, 200 parts Codonopsis pilosula, 267 parts Shenqu (medicated leaven), 200 parts Dioscorea opposita, and 133 parts prepared Glycyrrhiza uralensis.
4. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 2, characterized in that, The preparation method of the Five-Flavor Spleen-Strengthening Compound is as follows: The ingredients of the Five-Flavor Spleen-Strengthening Compound are soaked in water and decocted multiple times. The volatile oil is collected and the decoction is combined. The decoction is filtered and concentrated to obtain a concentrated solution. Then, an acetic acid solution containing chitosan is added to clarify the solution and filtered to obtain a filtrate. The volatile oil and filtrate are mixed and sodium benzoate and water are added to form a mixture. The mixture is then filled and sterilized to obtain the final product.
5. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 4, characterized in that, The decoction is performed twice, with the first decoction lasting 1.5-2.5 hours and the second decoction lasting 0.5-1.5 hours. The volume ratio of the concentrated liquid to the acetic acid solution containing chitosan is (8-12):1, with the mass fraction of chitosan in the acetic acid solution being 0.5-1.5% and the mass fraction of acetic acid solution being 1-3%. The mass fraction of sodium benzoate in the mixture is 0.2-0.4%.
6. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 5, characterized in that, The decoction was performed twice, with the first decoction lasting 2.0 h and the second decoction lasting 1.0 h. The volume ratio of the concentrated liquid to the acetic acid solution containing chitosan was 10:
1. The mass fraction of chitosan in the acetic acid solution containing chitosan was 1.0%, and the mass fraction of acetic acid solution was 2%. The mass fraction of sodium benzoate in the mixture was 0.3%.
7. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 1, characterized in that, The volume ratio of the Five-Flavor Spleen-Strengthening Mixture to the solvent is (3-5):1, the solvent is ethanol, and the volume concentration of ethanol is 75%-100%; the water bath drying temperature is 55-65℃, and the pore size of the filter membrane used for filtration is 0.22μm.
8. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 1, characterized in that, In the process of mixing the Five-Flavor Spleen-Strengthening Mixture with the solvent, anhydrous ethanol is used; the solvent used for reconstitution is ethanol with a volume concentration of 75%-80%.
9. The method for detecting Atractylodes macrocephala in Wuwei Jianpi Compound based on UPLC-FLR according to claim 1, characterized in that, The ultra-high performance liquid chromatography (UPLC) column was a Waters UPLC HSS T3 column, and the column temperature was 30 °C. + At 2℃, the flow rate was 0.3-0.4 mL / min. Mobile phase A was acetonitrile-0.2% phosphoric acid, and mobile phase B was water-0.2% phosphoric acid. The gradient elution program was as follows: 0-3 min, 10% mobile phase A; 3-5 min, 10%-20% mobile phase A; 5-8 min, 20%-30% mobile phase A; 8-12 min, 30%-50% mobile phase A; 12-16 min, 50% mobile phase A; 12-17 min, 50%-10% mobile phase A; 17-19 min, 10% mobile phase A. The fluorescence detector used had an excitation wavelength of 280 nm, an emission wavelength of 450 nm, and an injection volume of 1 μL.
10. The application of the method for detecting Atractylodes macrocephala in Wuwei Jianpi Mixture based on UPLC-FLR as described in any one of claims 1-9 in the quality control of Wuwei Jianpi Mixture.