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CN118225938B - Quality detection method of Chinese herbal compound five-god decoction - Google Patents

Quality detection method of Chinese herbal compound five-god decoction Download PDF

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CN118225938B
CN118225938B CN202410393216.2A CN202410393216A CN118225938B CN 118225938 B CN118225938 B CN 118225938B CN 202410393216 A CN202410393216 A CN 202410393216A CN 118225938 B CN118225938 B CN 118225938B
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decoction
wushen
mobile phase
god
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CN118225938A (en
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高珣
张敏
秦昆明
戴晓
李时羽
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Jiangsu Ocean University
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Abstract

The invention discloses a quality detection method of a traditional Chinese medicine compound five-god decoction, which uses a UPLC-Q-Exactive Orbitrap-MS/MS analysis method to carry out qualitative analysis on chemical components in five medicinal materials of honeysuckle, viola yedoensis makino, poria cocos, plantain seed and achyranthes bidentata; and then the content of total flavonoids and total phenolic acids in the five-god decoction is measured by an ultraviolet spectrophotometry. The UPLC-Q-Exactive Orbitrap-MS/MS analysis method has the advantages of high resolution, good quality precision, strong qualitative capability and the like, and can be used for widely and accurately detecting chemical components in complex traditional Chinese medicine compounds; the ultraviolet spectrophotometry can provide a rapid, accurate and reliable detection result, is simple and easy to operate, passes the methodological verification, has good linear relation, meets the requirements of precision, stability, repeatability, recovery rate and the like, and lays a foundation for establishing the quality standard of the five-god decoction by combining qualitative and quantitative analysis.

Description

Quality detection method of Chinese herbal compound five-god decoction
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a quality detection method of a traditional Chinese medicine compound five-god decoction.
Background
The Wushen decoction is derived from the Chan of Qing dynasty Chen Shiduo, and is mainly used for preventing and treating multiple skin carbuncles caused by damp-heat accumulation and steaming, and consists of five medicines of honeysuckle, achyranthes, plantain seed, poria cocos and herba violae. In the book of syndrome differentiation, poria and semen plantaginis are recorded to promote diuresis, and herba Violae is used to clear heat, and honeysuckle and achyranthes root are used to tonify middle energizer and dispel toxin. The five medicines in the whole prescription mainly have the functions of clearing heat, promoting diuresis and dispelling toxin, and are classical formulas for preventing and treating skin carbuncles.
The five-god decoction is used as a traditional Chinese medicine prescription, and is prepared into a liquid preparation by adopting a proper solvent for extraction, so that the recipe is extracted by taking water as the solvent for clinical use. The main active ingredients of the five-god decoction are derived from honeysuckle, viola yedoensis makino, plantain seed and other medicinal materials, and in order to research the quality level of the five-god decoction, an efficient detection method needs to be established, so that the decoction with the optimal medicinal effect can be screened out.
Disclosure of Invention
The invention aims to provide a quality detection method of a traditional Chinese medicine compound five-god decoction.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a quality detection method of WUSHEN decoction comprises performing qualitative analysis on five medicinal materials including flos Lonicerae, herba Violae, poria, semen plantaginis and Achyranthis radix in WUSHEN decoction by UPLC-Q-Exactive Orbitrap-MS/MS analysis method; and then determining the contents of total flavonoids and total phenolic acids in the five-god decoction by using an ultraviolet spectrophotometry.
Compared with the traditional HPLC and thin-layer chromatography qualitative method, the invention has wider quality detection range, higher sensitivity and peak capacity of the UPLC-Q-Exactive Orbitrap-MS/MS analysis method, and can carry out large-scale qualitative analysis on complex Chinese herbal compound
Compared with single quantitative HPLC and thin layer chromatography quantitative methods, the method is simple, feasible, quick, accurate and reliable, and the absorption of the compound under specific wavelength is utilized to quantitatively analyze the major components of the functional components of the total flavonoids and the total phenolic acids in the five-god decoction.
As a preferred scheme of the quality detection method of the five-god decoction, the UPLC-Q-Exactive Orbitrap-MS/MS analysis method is used for carrying out heating reflux extraction on five medicinal materials including honeysuckle, viola yedoensis makino, poria cocos, plantain seed and achyranthes bidentata in the five-god decoction by using water as a solvent, wherein the extraction parameters are as follows: the feed-liquid ratio is 25 times of water; soaking for 60min; extracting for 105min; extracting for 3 times, mixing filtrates, and concentrating under reduced pressure (60deg.C) to a certain concentration.
As a preferred scheme of the quality detection method of the five-god decoction, the five-god decoction is prepared from five medicinal materials including honeysuckle, viola yedoensis makino, poria cocos, plantain seed and achyranthes bidentata by a UPLC-Q-Exactive Orbitrap-MS/MS analysis method, wherein a chromatographic column is Shim-pack XR-ODS C 18 (75 mm multiplied by 3.0mm,2.2 mu m), acetonitrile (mobile phase B) -water (mobile phase A) is used as a mobile phase, the flow rate is 0.5mL/min, the column temperature is 30 ℃, the sample injection amount is 5 mu L, and gradient elution is carried out. Ionization mode: HESI-II ion source, positive and negative ion mode; the temperature of the ion source is 550 ℃; curtain gas (CUR): 206.84kPa; the atomization gas (gas 1) and the auxiliary gas (gas 2) are high-purity nitrogen, and are 379.21kPa; the primary mass spectrum parent ion scanning range is between 50 and 900 m/z, and the secondary mass spectrum scanning range is between 50 and 800 m/z.
As a preferable scheme of the quality detection method of the five-god decoction, the invention comprises the following steps: the gradient elution is specifically that the mobile phase B is increased from 2% to 8% in 0.01-4 min; during 4-5 min, the mobile phase B is increased from 8% to 10%; 5-9 min, the mobile phase B is raised from 10% to 17%; when 9-12 min, the mobile phase B is raised from 17% to 20%; when 12-16 min, the mobile phase B is reduced from 20% to 23%; when 16-16.5 min, the mobile phase B rises from 23% to 2%; ; 16.5-21 min, the mobile phase B rises from 2% to 2%.
As a preferable scheme of the quality detection method of the five-god decoction, the invention comprises the following steps: the ultraviolet spectrophotometry is used for detecting the content of total flavonoids and total phenolic acids in five-god decoction, wherein the total flavonoids chromogenic method is to add 0.3mL of 5% NaNO 2 solution respectively, shake the solution evenly for 6min, then add 0.3mL of 10% Al (NO 3)3, shake the solution evenly for 6min, finally add 4.0mL of 4% NaOH, fix the volume of distilled water to 10mL, shake the solution evenly for 10min, add 3mL of Fulin reagent respectively, shake the solution evenly for 3min, add 4mL of 7.5% Na 2CO3, fix the volume of distilled water to 10mL, put the solution in dark place for 1h, take distilled water as blank, perform full scanning in the wavelength range of 400-900 nm, and respectively measure the maximum absorption at 510nm and 783 nm.
The following beneficial effects can be obtained through the technical scheme:
The invention uses UPLC-Q-Exactive Orbitrap-MS/MS analysis method to carry out qualitative analysis on the chemical components in five medicinal materials of honeysuckle, viola yedoensis makino, poria cocos, plantain seed and achyranthes bidentata; and then the content of total flavonoids and total phenolic acids in the five-god decoction is measured by an ultraviolet spectrophotometry. The UPLC-Q-Exactive Orbitrap-MS/MS analysis method has the advantages of high resolution, good quality precision, strong qualitative capability and the like, and can be used for widely and accurately detecting chemical components in complex traditional Chinese medicine compounds; the ultraviolet spectrophotometry can provide a rapid, accurate and reliable detection result, is simple and easy to operate, passes the methodological verification, has good linear relation, meets the requirements of precision, stability, repeatability, recovery rate and the like, and lays a foundation for establishing the quality standard of the five-god decoction by combining qualitative and quantitative analysis.
Drawings
FIG. 1 is a total ion flow chromatogram of Wushen decoction in positive (A) and negative (B) ion modes;
FIG. 2 is a full-wave scan of a control and test;
FIG. 3 is the effect of feed to liquid ratio (a), soak time (b), extraction time (c), number of extractions (d) on wushen decoction extraction process;
FIG. 4 is a standard curve of rutin and gallic acid controls.
Detailed Description
The invention is further illustrated in the following by reference to the examples in connection with figures 1-4:
example 1:
A quality detection method of five-god decoction comprises the steps of firstly carrying out qualitative analysis on five medicinal materials of honeysuckle, viola yedoensis makino, poria cocos, plantain seed and achyranthes bidentata in the five-god decoction by using a UPLC-Q-Exactive Orbitrap-MS/MS analysis method; then the ultraviolet spectrophotometry is used for detecting the content of total flavonoids and total phenolic acids in the five-god decoction.
The quality detection range of the UPLC-Q-Exactive Orbitrap-MS/MS analysis method is wider, the sensitivity and the peak capacity are higher, and the complex Chinese herbal compound can be subjected to large-scale qualitative analysis; the method comprises the following steps:
(1) Chromatographic and mass spectral conditions
The column was a Shim-pack XR-ODS C 18 (75 mm. Times.3.0 mm,2.2 μm) and was subjected to gradient elution with acetonitrile (mobile phase B) -water (mobile phase A) as the mobile phase at a flow rate of 0.5mL/min at a column temperature of 30℃and a sample loading of 5. Mu.L. Ionization mode: HESI-II ion source, positive and negative ion mode; the temperature of the ion source is 550 ℃; curtain gas (CUR): 206.84kPa; the atomization gas (gas 1) and the auxiliary gas (gas 2) are high-purity nitrogen, and are 379.21kPa; the primary mass spectrum parent ion scanning range is between 50 and 900 m/z, and the secondary mass spectrum scanning range is between 50 and 800 m/z.
TABLE 1 gradient elution table
(2) Preparation of a mixed control solution:
Respectively taking appropriate amounts of rutin, gallic acid, protocatechuic acid, quinic acid, myricetin, geniposide, caffeic acid, luteolin, chlorogenic acid, quercetin, aesculin, acteoside and beta-ecdysterone reference substances, placing the reference substances in a brown volumetric flask, dissolving and diluting with 80% methanol, and fixing the volume to 25mL to prepare 200 mug/mL reference substance stock solution, taking appropriate amounts of each reference substance stock solution, and diluting to obtain about 5 mug/mL of mixed reference substance solution.
(3) Preparation of test solutions
39G of five medicinal materials of five-god decoction are weighed according to the prescription, 25 times of water is added, soaking is carried out for 60min, extraction is carried out for 105min, extraction is carried out for 3 times, continuous filtrate is combined, and vacuum concentration (60 ℃) is carried out by a rotary evaporator until 250mL. And precisely transferring 0.5mL of concentrated solution, placing in a 10mL volumetric flask, diluting with distilled water, fixing the volume to a scale, and shaking uniformly to obtain a five-god soup sample solution (7.8 mg/mL).
The study adopts UPLC-Q-Exactive Orbitrap-MS/MS liquid chromatography-mass spectrometry technology, and acquires mass spectrum information of wushen Shang Shuidi liquid in positive and negative ion modes, and the total ion flow chromatogram is shown in figure 1. According to a self-built five-god decoction compound database and Xcalibur qualitative software, compound molecular ions and secondary fragment ions are combined to compare 95 compounds in the Chinese herbal compound five-god decoction, wherein the 95 compounds comprise 27 flavonoid compounds, 15 phenolic acid compounds, 11 iridoid compounds, 5 coumarin compounds, 5 alkaloid compounds, 4 phenethyl alcohol glycoside compounds and other compounds. See table 2. Wherein, based on chromatographic peak and mass spectrum data of the control, 14 compounds were accurately identified, including: 5 phenolic acids (gallic acid, protocatechuic acid, caffeic acid, chlorogenic acid, quinic acid), 4 flavonoids (quercetin, rutin, luteolin, myricetin), 2 coumarins (aesculin and aesculin), 1 iridoid (geniposidic acid), 1 phenethyl alcohol glycoside (acteoside) and 1 steroid (beta-ecdysterone). The other 81 compounds were deduced by comparison with the molecular ions, secondary fragment ions of the natural compound database.
TABLE 2 information on chemical composition of Wushen decoction
Example 2a method of preparing a wushen decoction comprising the following:
weighing 39g of five-god decoction pieces according to the prescription, soaking in 25 times of water for 60min, decocting for 105min, extracting for 3 times, and mixing filtrates; concentrating at 60deg.C to about 250mL to obtain WUSHEN decoction concentrate.
The preparation process related researches are as follows:
(1) Orthogonal design experiment of extraction process:
The three levels of each factor are determined by taking different feed liquid ratios A (10, 15, 20, 25, 30 mL), soaking time B (30, 60, 90, 120, 150 min), extraction time C (60, 75, 90, 105, 120 min) and extraction times D (1, 2, 3, 4, 5 min) as investigation factors, carrying out heating reflux extraction, and taking total flavone content (mg/g) and total phenolic acid content (mg/g) as evaluation indexes, wherein the three levels of each factor are shown in Table 3.
Table 3 factors and levels of the five-god decoction extraction Process
Based on a single factor test (fig. 2 and 3), an L 9(34 orthogonal test design is adopted, total flavone content and total phenolic acid are used as comprehensive evaluation indexes (P), and extraction process parameters of the five-god decoction are optimized.
TABLE 4 orthogonal test design and results for Wushen decoction extraction Process
Note that:
As can be seen from visual analysis of Table 4, the average values of the four factors at different levels are A3> A2> A1, B2> B1> B3, C2> C3> C1, D2> D3> D1 in sequence, and the optimal extraction process is A3B2C2D2 as can be seen from R A>RD>RC>RB extremely bad data; and according to analysis of variance (table 5), the size order of the influence of the four factors on the extraction process is consistent with that of the direct differential analysis, namely the feed-liquid ratio is greater than the extraction times is greater than the extraction time is greater than the soaking time, the influence of the factor A and the factor D on the extraction process is remarkable, and the influence of other factors is smaller.
TABLE 5 analysis of variance table of five god decoction extraction process
From the standpoint of ensuring the possible extraction of the active ingredients and combining production energy conservation, the optimal extraction process of the traditional Chinese medicine compound five-god decoction is finally determined to be A 3B2C2D2, namely the liquid-to-liquid ratio is 25 times, the soaking time is 60 minutes, the extraction time is 105 minutes, and the extraction times are 3 times.
(2) Verification of extraction process
In order to reduce the error and the contingency of the extraction process, the parameters of the optimal extraction process are verified, namely 3 parts of decoction pieces of the Chinese herbal medicine compound five-god decoction are weighed in parallel, the feed liquid ratio is 25 times, the soaking time is 60min, the extraction time is 105min, the extraction is carried out for 3 times, the filtrate is combined, eight layers of gauze are filtered, the extracted liquid is decompressed and concentrated to 250mL, and the total flavone content and the total phenolic acid content are measured according to the measuring method of claim 3, and the table 6 is shown. Under the condition of the optimal extraction process, the RSD of the total flavone content and the total phenolic acid content are respectively 1.3 percent and 2.4 percent, which are less than 5 percent, and the extraction process is proved to be stable and reliable.
Table 6 verification result of wushen decoction extraction process (n=3)
Example 3 examination of Linear Range
Preparing a series of reference substance solutions of rutin and gallic acid reference substance respectively, measuring the absorbance of the linear solution according to the color development method of claim 3, and linearly plotting by taking absorbance Abs as an ordinate and solution concentration C (mug/mL) as an abscissa to obtain linear equations of the rutin and gallic acid reference substance solutions of Y=0.0109X-0.0015 (R 2＝0.9994,n＝6)、Y＝0.01399X+0.0077(R2 =0.9988, n=6). The results are shown in FIG. 4, which shows that rutin and gallic acid reference substance solutions have good linear relation with absorbance in the concentration ranges of 0-74 mug/mL and 0-6 mug/mL.
EXAMPLE 4 precision experiments
Precisely transferring 1.0mL of rutin control solution and 2.0mL of sample solution, placing into different 10mL colorimetric tubes with plugs, adding three color developers according to the color development method of claim 3, standing for corresponding time, continuously measuring for 6 times, and calculating to obtain RSD with absorbance of 0.18% and 0.23% respectively. Precisely transferring 1.0mL of gallic acid reference substance solution and 0.5mL of sample solution, placing the gallic acid reference substance solution and the sample solution into different 10mL colorimetric tubes with plugs, adding a color reagent according to the method of claim 3, and continuously measuring for 6 times to obtain RSD with absorbance of 0.48% and 0.79% respectively, which shows that the method has good instrument precision.
Example 5 stability test
Precisely removing 1.0mL of rutin control solution and 2.0mL of sample solution, placing into different 10mL colorimetric tubes with plugs, respectively standing at room temperature for 0, 15, 30, 45 and 60min according to the method of claim 3, and measuring absorbance, wherein RSD values are 0.19% and 0.42% respectively. Precisely removing 1.0mL of gallic acid reference substance solution and 0.5mL of sample solution, placing into different 10mL colorimetric tubes with plugs, adding a color developing agent according to claim 3, respectively standing at room temperature for 0, 15, 30, 45 and 60min, and measuring absorbance, wherein RSD values are respectively 0.67% and 1.90%, which shows that rutin, gallic acid reference substance and sample solution develop color stably within 60 min.
Example 6 repeatability
2.0ML and 0.5mL of the sample solution are respectively and precisely removed, and are placed in different 10mL colorimetric tubes with plugs to prepare 6 parts in parallel, the absorbance of the total flavonoids and the total phenolic acids of the sample is measured according to the claim 3, and the concentrations of the total flavonoids and the total phenolic acids are calculated by substituting a linear equation, and RSD values are respectively 1.72% and 3.36%, so that the repeatability of the method is good.
Example 7 sample recovery test
Precisely transferring 1.0mL of the sample solution with known total flavone content and 1.0mL of the rutin control solution (28.7 mug/mL), 0.5mL of the sample solution with known total phenolic acid content and 1.0mL of the gallic acid control solution (3.28 mug/mL), respectively placing the sample solution and the gallic acid control solution into a 10mL colorimetric tube, preparing 6 parts in parallel, measuring the absorbance of the labeled sample according to the method of claim 3, and substituting the absorbance into a linear equation to calculate the total flavone and total phenolic acid content. The results are shown in Table 7, and the average sample feeding recovery rates of the total flavonoids and the total phenolic acids are 97.72% and 102.37%, respectively, which shows that the accuracy of the method is good.
Example 8 content determination
Weighing 6 samples in parallel according to the five-god decoction prescription, and carrying out the optimal extraction process: the feed liquid ratio is 25 times, soaking for 60min, extracting for 105min, extracting for 3 times, mixing filtrates, filtering with eight layers of gauze, concentrating the extractive solution under reduced pressure to 250mL, measuring absorbance according to the measuring method, and calculating total flavone and total phenolic acid content in 6 five-god decoction samples. The results are shown in Table 8, and the RSD values of the total flavonoids and total phenolic acids in the 6 five-god soup samples are 2.40% and 2.80% respectively.
TABLE 7 sample recovery test results
TABLE 8 results of content determination
The foregoing is a preferred embodiment of the present application, and modifications, obvious to those skilled in the art, of the various equivalent forms of the present application can be made without departing from the principles of the present application, are intended to be within the scope of the appended claims.

Claims (5)

1.一种中药复方五神汤的质量检测方法，其特征在于：中药复方五神汤由金银花、紫花地丁、茯苓、车前子和牛膝五味药材组成，具体检测方法如下：1. A quality detection method for a Chinese medicine compound Wushen Decoction, characterized in that the Chinese medicine compound Wushen Decoction is composed of five medicinal materials: honeysuckle, Viola yedoensis, Poria cocos, Plantago and Achyranthes bidentata, and the specific detection method is as follows: S1：用 UPLC-Q-Exactive Orbitrap-MS/MS 分析方法分别对金银花、紫花地丁、茯苓、车前子和牛膝五味药材进行定性分析；S1: UPLC-Q-Exactive Orbitrap-MS/MS analysis was used to qualitatively analyze five medicinal materials: honeysuckle, Viola yedoensis, Poria cocos, Plantago, and Achyranthes bidentata. S2：利用紫外分光光度法检测五神汤中总黄酮和总酚酸的含量；S2: UV spectrophotometry was used to detect the contents of total flavonoids and total phenolic acids in Wushen Decoction; S1中对五神汤中金银花90g、紫花地丁30g、茯苓30g、车前子30g和牛膝15g五味药材进行定性分析，包括以下过程：S1 conducts qualitative analysis on five medicinal materials in Wushen Decoction, including 90g of honeysuckle, 30g of Viola yedoensis, 30g of Poria cocos, 30g of Plantago seed and 15g of Achyranthes bidentata, including the following processes: 第一步：色谱与质谱条件；Step 1: Chromatographic and mass spectrometric conditions; 第二步：混合对照品溶液的制备：Step 2: Preparation of mixed reference solution: 分别取芦丁、没食子酸、原儿茶酸、奎尼酸、杨梅素、京尼平苷酸、咖啡酸、木犀草苷、绿原酸、槲皮素、秦皮乙素、秦皮甲素、毛蕊花糖苷、β-蜕皮甾酮对照品适量，置于棕色容量瓶中，用80%的甲醇溶解稀释，并定容至25mL，制备成200μg/mL的对照品储备液，取各对照品储备液适量，稀释成5μg/mL的混合对照品溶液；Take appropriate amount of rutin, gallic acid, protocatechuic acid, quinic acid, myricetin, geniposide, caffeic acid, luteolin, chlorogenic acid, quercetin, aesculetin, aesculetin, verbascoside, β -ecdysterone reference substances respectively, place them in a brown volumetric flask, dissolve and dilute with 80% methanol, and make up to 25 mL to prepare a 200 μg/mL reference substance stock solution, take appropriate amount of each reference substance stock solution, and dilute to a 5 μg/mL mixed reference substance solution; 第三步：供试品溶液的制备：Step 3: Preparation of test solution: 按处方称取五神汤中药饮片39g，加入25倍量水，浸泡60min，提取105min，提取3次，合并续滤液，旋蒸仪减压浓缩，温度60℃，至250mL；精密移取0.5mL浓缩液，置于10mL容量瓶中，用蒸馏水稀释并定容至刻度，摇匀，即得五神汤供试品溶液7.8mg/mL；Weigh 39g of Wushen Decoction slices according to the prescription, add 25 times of water, soak for 60min, extract for 105min, extract 3 times, combine the filtrate, and concentrate under reduced pressure on a rotary evaporator at 60°C to 250mL; accurately transfer 0.5mL of the concentrate to a 10mL volumetric flask, dilute with distilled water and make up to the mark, shake well, and obtain a Wushen Decoction test solution of 7.8mg/mL; 确定色谱-质谱条件后，取混合对照品溶液及供试品溶液适量，过0.22μm微孔滤膜，导入UPLC-Q-Exactive Orbitrap-MS/MS色谱-质谱联用仪进行检测，可基于对照品的色谱峰与质谱数据对五神汤中的化合物进行准确鉴定，通过与天然化合物数据库的分子离子、二级碎片离子比对，推测其它化合物。After determining the chromatography-mass spectrometry conditions, take an appropriate amount of the mixed reference solution and the test solution, pass them through a 0.22 μm microporous filter membrane, and introduce them into the UPLC-Q-Exactive Orbitrap-MS/MS chromatography-mass spectrometry instrument for detection. The compounds in Wushen Decoction can be accurately identified based on the chromatographic peaks and mass spectrometry data of the reference, and other compounds can be inferred by comparing with the molecular ions and secondary fragment ions of the natural compound database. 2.根据权利要求1所述的一种中药复方五神汤的质量检测方法，其特征在于：S2中总黄酮显色方法为分别加入0.3mL 5% NaNO2溶液，摇匀放置6min，再加入0.3mL 10% Al(NO3)3，摇匀放置6min，最后加入4.0 mL 4% NaOH，蒸馏水定容至10mL，摇匀放置10min；总酚酸显色方法为分别加入3mL福林试剂，摇匀放置3min，再加入4mL 7.5% Na2CO3，蒸馏水定容至10mL，暗处放置显色1h，以蒸馏水作为空白；在400～900nm波长范围内进行全扫描，分别测得其最大吸收在510nm、783nm处。2. A quality detection method for a traditional Chinese medicine compound Wushen Decoction according to claim 1, characterized in that: the color development method for total flavonoids in S2 is to add 0.3mL 5% NaNO2 solution respectively, shake and place for 6min, then add 0.3mL 10% Al( NO3 ) 3 , shake and place for 6min, finally add 4.0mL 4% NaOH, distilled water is diluted to 10mL, and shake and place for 10min; the color development method for total phenolic acid is to add 3mL Folin reagent respectively, shake and place for 3min, then add 4mL 7.5% Na2CO3 , distilled water is diluted to 10mL, and place in the dark for color development for 1h, with distilled water as blank; full scan is performed in the wavelength range of 400-900nm, and the maximum absorption is measured at 510nm and 783nm respectively. 3.根据权利要求1所述的一种中药复方五神汤的质量检测方法，其特征在于：色谱-质谱联用方法，其色谱柱为Shim-pack XR-ODS C18，以乙腈流动相B-水流动相A为流动相，流速为0.5mL/min，柱温30℃，进样量为5μL，进行梯度洗脱；离子化模式：HESI-II离子源，正、负离子模式；离子源温度550℃；气帘气CUR：206.84kPa；雾化气gas1和辅助气gas2为高纯度氮气，均为379.21kPa；一级质谱母离子扫描范围为m/z50～900，二级质谱扫描范围为m/z50～800。3. The quality detection method of the traditional Chinese medicine compound Wushen Decoction according to claim 1 is characterized by: a chromatography-mass spectrometry method, wherein the chromatographic column is Shim-pack XR-ODS C 18 , acetonitrile mobile phase B-water mobile phase A is used as the mobile phase, the flow rate is 0.5 mL/min, the column temperature is 30°C, the injection volume is 5 μL, and gradient elution is performed; ionization mode: HESI-II ion source, positive and negative ion mode; ion source temperature is 550°C; curtain gas CUR: 206.84 kPa; nebulizing gas gas1 and auxiliary gas gas2 are high-purity nitrogen, both of which are 379.21 kPa; the primary mass spectrometry parent ion scanning range is m/z 50-900, and the secondary mass spectrometry scanning range is m/z 50-800. 4.根据权利要求3所述的一种中药复方五神汤的质量检测方法，其特征在于：所述梯度洗脱具体为，0.01~4min时，流动相B由2%升至8%；4~5min时，流动相B由8%升至10%；5~9min时，流动相B由10%升至17%；9~12min时，流动相B由17%升至20%；12~16min时，流动相B由20%降至23%；16~16.5min时，流动相B由23%降至2%；16.5~21min时，流动相B保持2%。4. The quality detection method of a traditional Chinese medicine compound Wushen Decoction according to claim 3 is characterized in that: the gradient elution is specifically as follows: at 0.01~4min, the mobile phase B is increased from 2% to 8%; at 4~5min, the mobile phase B is increased from 8% to 10%; at 5~9min, the mobile phase B is increased from 10% to 17%; at 9~12min, the mobile phase B is increased from 17% to 20%; at 12~16min, the mobile phase B is reduced from 20% to 23%; at 16~16.5min, the mobile phase B is reduced from 23% to 2%; at 16.5~21min, the mobile phase B is maintained at 2%. 5.根据权利要求1所述的一种中药复方五神汤的质量检测方法，其特征在于：五神汤每1g样品以总黄酮计，有44.38mg；以总酚酸计，有11.83mg。5. The quality detection method of the traditional Chinese medicine compound Wushen Decoction according to claim 1 is characterized in that: every 1g of Wushen Decoction sample contains 44.38mg of total flavonoids and 11.83mg of total phenolic acids.
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