Construction of asparagus medicinal material fingerprint and determination method of index component content

Abstract

The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicine, and in particular to a method for determining the content of an asparagus medicinal material fingerprint, including: taking asparagus medicinal materials to prepare a test solution; accurately drawing the test solution for ultra-high performance liquid chromatography-mass spectrometry detection to obtain a fingerprint of the asparagus medicinal material; wherein, the chromatographic conditions of ultra-high performance liquid chromatography-mass spectrometry detection include: the mobile phase used is: mobile phase A is a methanol: acetonitrile mixed solution, wherein the volume ratio of methanol to acetonitrile is 1: 8~12; mobile phase B is acetic acid water containing 0.04%~0.06% volume concentration; gradient elution mode is adopted; the stationary phase used is: a chromatographic column with octadecylsilane bonded silica gel as a filler. The fingerprint obtained by the method of the present invention can more comprehensively reflect the chemical composition of asparagus medicinal materials, improve the accuracy and comprehensiveness of asparagus medicinal material quality control, so as to better evaluate the overall quality of asparagus medicinal materials.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, and in particular to a method for constructing an asparagus medicinal material fingerprint and determining the content of an index component. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Asparagus is the fresh or dried tender stem of Asparagus officinalis L., a plant of the Liliaceae family. It has the effects of clearing away heat and dampness, promoting blood circulation and dispersing stagnation. Only by using effective technical means to evaluate the quality consistency of asparagus medicinal materials and decoction pieces (hereinafter collectively referred to as asparagus medicinal materials) can the safety, effectiveness and quality control of asparagus medicinal materials be ensured.

[0004] However, there is no control standard for the consistency evaluation of asparagus quality. The 2020 edition of the Chinese Pharmacopoeia has not yet included this variety, and the standards of various provinces only stipulate the nitrogen content of asparagus. Current research work is difficult to effectively control the overall quality of asparagus medicinal materials.

[0005] Therefore, it is urgent to develop a method for constructing a fingerprint of asparagus medicinal materials and a method for determining the content of asparagus index components for application in the quality evaluation of asparagus medicinal materials. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a method for constructing an asparagus medicinal material fingerprint and determining the content of an index component, which are applied to the quality evaluation of asparagus medicinal materials.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a method for constructing a fingerprint of asparagus medicinal materials, comprising the following steps:

[0009] Take asparagus medicinal materials to prepare the test solution;

[0010] Accurately pipetting the test solution for ultra-high performance liquid chromatography-mass spectrometry detection to obtain a fingerprint of the asparagus medicinal material;

[0011] Among them, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection include: the mobile phase used is: mobile phase A is a methanol: acetonitrile mixed solution, wherein the volume ratio of the methanol to acetonitrile is 1:8-12; mobile phase B is acetic acid water containing 0.04% to 0.06% volume concentration; a gradient elution method is used, and the gradient elution method is: 0-4min: the volume fraction of mobile phase A changes from 5% to 10%, and the volume fraction of mobile phase B changes from 95% to 90%; 4-8min: the volume fraction of mobile phase A changes from 10% to 20%, and the volume fraction of mobile phase B changes from 90% to 80%; 8-13min : the volume fraction of mobile phase A changes from 20% to 22%, and the volume fraction of mobile phase B changes from 80% to 78%; 13-20min: the volume fraction of mobile phase A changes from 22% to 55%, and the volume fraction of mobile phase B changes from 78% to 45%; 20-25min: the volume fraction of mobile phase A changes from 55% to 90%, and the volume fraction of mobile phase B changes from 45% to 10%; 25-30min: the volume fraction of mobile phase A changes from 90% to 90%, and the volume fraction of mobile phase B changes from 10% to 10%; the stationary phase used is: a chromatographic column with octadecylsilane bonded silica gel as filler.

[0012] In one or more embodiments, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection further include: the flow rate of the mobile phase is 0.3 to 0.5 mL / min, preferably 0.4 mL / min.

[0013] In one or more embodiments, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection further include: the column temperature of the chromatographic column is 35-45°C, preferably 40°C.

[0014] In one or more embodiments, the chromatographic column is an ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm) chromatographic column.

[0015] In one or more embodiments, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry method include: mobile phase: mobile phase A is a mixed solution of methanol: acetonitrile in a volume ratio of 1:10, and mobile phase B is acetic acid water containing 0.05% volume concentration.

[0016] In one or more embodiments, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry method include: the ion source is ESI; the ionization mode is negative ion mode; the ionization voltage (IS) is -4500V, the ionization temperature (TEM) is 550°C, the sprayer (Gas1) is 55L / min, the curtain gas (CUR) is 30L / min, the collision gas (CAD) is 7L / min, and the auxiliary heating gas (Gas2) is 55L / min; the scanning mode is full scan; the scanning range is m / z 100 to 1100.

[0017] In one or more embodiments, the method for preparing the test solution includes: extracting and filtering asparagus medicinal materials and decoction pieces with an extracting solution to obtain a test solution;

[0018] The extracting solution is a methanol-water solution, the volume fraction of methanol in the methanol-water solution is 30% to 100%, and preferably, the extracting solution is a 100% methanol solution.

[0019] In one or more embodiments, the method for preparing the test solution includes: adding 100% methanol solution to asparagus medicinal materials; ultrasonic extraction for 30 minutes at 100kHZ and 30°C; filtering after weighting with 100% methanol solution; and filtering the filtrate through a 0.22μm filter membrane to obtain the test solution.

[0020] In one or more embodiments, the construction method further comprises: preparing a reference solution from protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin; accurately aspirating the reference solution for ultra-high performance liquid chromatography-mass spectrometry detection to obtain a reference chromatogram, and calibrating the common chromatographic peaks on the fingerprint of the asparagus medicinal material with reference to the reference chromatogram. The conditions for ultra-high performance liquid chromatography-mass spectrometry detection are the same as those for the test solution.

[0021] Preferably, the solvent of the reference solution is methanol.

[0022] In one or more embodiments, the fingerprint contains 20 characteristic peaks, wherein peak 2 is protocatechuic acid, peak 4 is vanillic acid, peak 7 is ferulic acid, peak 8 is rutin, peak 9 is naringin, peak 10 is hesperidin, peak 12 is cinnamic acid, and peak 13 is protodioscin.

[0023] Preferably, peak 12 is taken as the S peak, and the relative retention time ranges of peaks 1 to 11 and peaks 13 to 20 relative to the S peak are: 0.104 to 0.110, 0.192 to 0.199, 0.244 to 0.252, 0.389 to 0.398, 0.409 to 0.420, 0.542 to 0.546, 0.597 to 0.602, 0.646 to 0. 653, 0.776~0.798, 0.827~0.846, 0.932~0.940, 1.097~1.111, 1.205~1.219, 1.334~1.357, 1.449~1.469, 1.508~1.529, 1.554~1.575, 1.630~1.652, 1.665~1.689.

[0024] Preferably, taking peak 12 as the S peak, the relative retention time RSD values ​​of peaks 1 to 11 and peaks 13 to 20 relative to the S peak are: 1.62%, 0.90%, 0.85%, 0.50%, 0.67%, 0.22%, 0.20%, 0.31%, 0.73%, 0.74%, 0.24%, 0.42%, 0.37%, 0.54%, 0.43%, 0.45%, 0.45%, 0.47% and 0.46%, respectively.

[0025] The second aspect of the present invention provides a method for determining the content of an index component in an asparagus medicinal material, comprising: adding an extract to the asparagus medicinal material to be tested to extract to obtain a sample solution to be tested; taking protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin and adding a solvent to prepare a reference solution; accurately weighing the sample solution to be tested and the reference solution to perform ultra-high performance liquid chromatography-mass spectrometry detection respectively, to obtain the test spectrum of the asparagus medicinal material and the decoction piece to be tested and the standard solution A control spectrum of the solution; calculating the contents of the index components protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin in the asparagus medicinal material to be tested according to the concentration of the reference solution, the peak area of ​​the reference solution in the control spectrum, and the peak areas of the components corresponding to the reference solution in the asparagus medicinal material to be tested and the reference solution in the test spectrum; the conditions for the ultra-high performance liquid chromatography-mass spectrometry detection are the same as the detection conditions described in the first aspect, and will not be repeated here.

[0026] In one or more embodiments, the extract is a methanol-water solution, and the volume fraction of methanol in the methanol-water solution is 30% to 100%; the solvent of the reference solution is methanol.

[0027] In one or more embodiments, the concentration of the reference solution and the peak area of ​​the reference solution in the reference spectrum satisfy the following relationship: the first regression equation of the protocatechuic acid is y=25618.77145x+86493.5 (r=0.9998), the linear range is 2.0537-513.4135 ng / mL, and the detection limit is 0.8929 ng / mL (S / N≥3); the second regression equation of the vanillic acid is y=13736.69335x+21663.31103 (r=0.9999), the linear range is 2.0460-511.50 00ng / mL, the detection limit is 0.9161ng / mL (S / N≥3); the third regression equation of ferulic acid is y=40296.0x+233153 (r=0.9994), the linear range is 2.1364-534.1050ng / mL, and the detection limit is 0.3009ng / mL (S / N≥3); the fourth regression equation of rutin is y=12753.05735x+674008 (r=0.9996), the linear range is 18.9452-4736.3050ng / mL, and the detection limit is 0.3333ng / mL (S / N≥3 ); the fifth regression equation of naringin is y=8085.51214x+62163.9 (r=0.9999), the linear range is 1.0939-273.4752 ng / mL, and the detection limit is 0.0957 ng / mL (S / N≥3); the sixth regression equation of hesperidin is y=13379.98453x+595595 (r=0.9996), the linear range is 8.2400-2060.0000 ng / mL, and the detection limit is 0.0772 ng / mL (S / N≥3); the seventh regression equation of cinnamic acid is y=29028.85 577x+403980(r=0.9993), the linear range is 4.2563-1064.0760ng / mL, and the detection limit is 0.6650ng / mL(S / N≥3); the eighth regression equation of the protodioscin is y=1678.98789x+1502000(r=0.9996), the linear range is 390.8000-39080.0000ng / mL, and the detection limit is 0.3457ng / mL(S / N≥3); wherein, in the regression equation, the abscissa is the concentration of the reference solution, and the ordinate is the peak area in the reference spectrum.

[0028] Preferably, the MRM scanning parameters of the indicative components protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin are:

[0029]

[0030]

[0031] The present invention has the following beneficial effects:

[0032] The method for constructing the asparagus medicinal material fingerprint and determining the content of the index components provided by the present invention realizes the effective separation of different types of index components in the asparagus medicinal material and the identification and appraisal of the fingerprint chromatographic peaks through the optimized high performance liquid chromatography conditions, and at the same time establishes a quantitative analysis method for the index components in the asparagus medicinal material. The constructed fingerprint can more comprehensively reflect the chemical components of the asparagus medicinal material, improve the accuracy and comprehensiveness of the quality control of the asparagus medicinal material, so as to better evaluate the overall quality of the asparagus medicinal material.

[0033] The evaluation standards for asparagus medicinal materials have been improved stably and comprehensively, ensuring the effectiveness of the medicinal materials in clinical use and providing a reference for the overall control of the quality of asparagus medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 The UPLC-MS spectrum of the test solution in Example 1 of the present invention when the elution method is elution method 02 is shown;

[0036] Figure 2 The UPLC-MS spectrum of the reference solution in Example 1 of the present invention is shown;

[0037] Figure 3 The UPLC-MS spectrum of the test solution in Example 1 of the present invention is shown;

[0038] Figure 4 The fingerprints of 15 batches of asparagus medicinal materials provided in Example 1 of the present invention are shown;

[0039] Figure 5 The fingerprint comparison map of 15 batches of asparagus medicinal material fingerprints provided in Example 1 of the present invention generated by the average method is shown;

[0040] Figure 6 The UPLC-MS spectrum of the reference solution in Example 2 of the present invention when the elution method is elution method-1 is shown;

[0041] Figure 7 The UPLC-MS spectrum of the reference solution in Example 2 of the present invention when the elution method is elution method-2 is shown;

[0042] Figure 8The UPLC-MS comparison spectrum of the mixed reference solution in Example 2 of the present invention is shown;

[0043] Fig. 9 The UPLC-MS spectrum of the sample solution to be tested in Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0045] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0046] Unless otherwise specified, the raw materials, reagents or devices mentioned in the following examples can be obtained from commercial sources or by known methods.

[0047] The origin information of the asparagus medicinal material used in the present invention is shown in Table 1.

[0048] Table 1: Origin information of 15 batches of asparagus medicinal materials

[0049]

[0050] *Tables S1-S7 are white asparagus slices, S8-S11 are green asparagus slices, S12-S14 are white asparagus medicinal materials, and S15 is green asparagus medicinal materials.

[0051] Embodiment 1

[0052] Method for constructing the fingerprint of asparagus medicinal material:

[0053] This embodiment provides a method for constructing a fingerprint of asparagus medicinal materials, comprising: preparing a test solution from asparagus medicinal materials; preparing a reference solution from protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin; respectively injecting the test solution and the reference solution into an ultra-high performance liquid chromatography-mass spectrometer, and obtaining a fingerprint of asparagus medicinal materials according to an ultra-high performance liquid chromatography-mass spectrometry method. The specific construction method is provided below.

[0054] 1. Preparation of test solution

[0055] Accurately weigh 0.50 g of asparagus powder and pass it through a No. 2 sieve (error less than 0.50 mg), add 25 ml of 100% methanol solution, ultrasonicate at 100 kHz and 30°C for 30 min, filter, and pass the filtrate through a 0.22 μm filter membrane to obtain the UPLC-MS fingerprint sample solution.

[0056] 2. Preparation of reference solution

[0057] Weigh appropriate amounts of protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin reference substances, dissolve them in methanol, and prepare solutions with concentrations of 102.6827 μg / mL, 102.3000 μg / mL, 106.8210 μg / mL, 94.7261 μg / mL, 91.1584 μg / mL, 103.0000 μg / mL, 106.4076 μg / mL, and 97 .7000μg / mL reference stock solution, then take an appropriate amount of reference stock solution and use 50% methanol as the dilution solvent to make mixed standard solutions with concentrations of 0.5134μg / mL, 0.5115μg / mL, 0.5341μg / mL, 12.3144μg / mL, 0.2735μg / mL, 8.2400μg / mL, 1.0641μg / mL, and 39.0800μg / mL, respectively.

[0058] 3. Optimization of ultra-high performance liquid chromatography-mass spectrometry conditions

[0059] Preset amounts of reference solution and test solution were taken and injected into a UPLC-MS liquid spectrometer. Octadecylsilane bonded silica gel was used as filler, mobile phase A was a mixed solution of methanol:acetonitrile (volume ratio 1:10), and 0.05% volume concentration of acetic acid water was used as mobile phase B for gradient elution to construct the UPLC-MS fingerprint of asparagus medicinal materials.

[0060] The mass spectrometry detection conditions were as follows: ion source was ESI; ionization mode was negative ion mode; scanning mode was full scan; scanning range: m / z 100-1100; ionization voltage (IS) was -4500 V, ionization temperature (TEM) was 550 °C, nebulizer (Gas1) was 55 L / min, curtain gas (CUR) was 30 L / min, collision gas (CAD) was 7 L / min, and auxiliary heating gas (Gas2) was 55 L / min.

[0061] 3.1 Optimization of LC elution method

[0062] The initial attempt at liquid phase elution method 01 is:

[0063] 0-4min: the volume fraction of mobile phase B changes from 95% to 90%;

[0064] 4-10min: the volume fraction of mobile phase B changes from 90% to 75%;

[0065] 10-17 min: the volume fraction of mobile phase B changed from 75% to 73%;

[0066] 17-23min: the volume fraction of mobile phase B changed from 73% to 45%;

[0067] 23-27min: the volume fraction of mobile phase B changed from 45% to 10%;

[0068] 27-30 min: the volume fraction of mobile phase B changed from 10% to 10%.

[0069] Under this elution condition, some characteristic peaks could not be separated from the baseline, and the separation degree was poor. Based on this experimental result, elution method 02 was optimized, and the gradient elution method was as follows:

[0070] 0-4min: the volume fraction of mobile phase B changes from 95% to 90%;

[0071] 4-8min: the volume fraction of mobile phase B changes from 90% to 80%;

[0072] 8-13min: the volume fraction of mobile phase B changes from 80% to 78%;

[0073] 13-20min: the volume fraction of mobile phase B changed from 78% to 45%;

[0074] 20-25min: the volume fraction of mobile phase B changes from 45% to 10%;

[0075] 25-30 min: The volume fraction of mobile phase B changed from 10% to 10%.

[0076] Under this elution condition, the peak shape and separation of the common peak are relatively ideal. Figure 1 As shown, Figure 1 The liquid chromatogram of the test sample when the elution method is elution method 02 in Example 1 of the present invention is shown.

[0077] Table 2: UPLC-MS fingerprint gradient elution table

[0078]

[0079] 3.2 Determination of liquid chromatography conditions

[0080] The chromatographic column was ACQUITY UPLC HSS T3 (2.1×100mm, 1.8μm) column, the flow rate was 0.4mL / min, and the column temperature was 40°C. The eluent was a mixed solution of methanol:acetonitrile (volume ratio 1:10) (mobile phase A) and an aqueous solution containing 0.05% volume concentration of acetic acid (mobile phase B). The injection volume was 2μL. Based on 3.1, the optimal elution procedure was selected as follows, and the UPLC-MS spectra of the reference solution and the test solution were obtained as shown in, Figure 2 The UPLC-MS spectrum of the reference solution in Example 1 of the present invention is shown; Figure 3 The UPLC-MS spectrum of the test solution in Example 1 of the present invention is shown.

[0081] 0-4min: the volume fraction of mobile phase A changes from 5% to 10%, and the volume fraction of mobile phase B changes from 95% to 90%;

[0082] 4-8min: the volume fraction of mobile phase A changes from 10% to 20%, and the volume fraction of mobile phase B changes from 90% to 80%;

[0083] 8-13min: the volume fraction of mobile phase A changes from 20% to 22%, and the volume fraction of mobile phase B changes from 80% to 78%;

[0084] 13-20 min: the volume fraction of mobile phase A changes from 22% to 55%, and the volume fraction of mobile phase B changes from 78% to 45%;

[0085] 20-25 min: the volume fraction of mobile phase A changes from 55% to 90%, and the volume fraction of mobile phase B changes from 45% to 10%;

[0086] 25-30 min: the volume fraction of mobile phase A changes from 90% to 90%, and the volume fraction of mobile phase B changes from 10% to 10%.

[0087] 4. UPLC-MS fingerprint identification of chemical components

[0088] The UPLC-MS method was used to detect the reference solution and the test solution. By comparing the chromatographic peak retention time, accurate molecular weight and secondary mass spectrometry fragmentation rules of the compounds, a total of 8 chromatographic peaks corresponding to the compounds were determined, namely protocatechuic acid (peak 2), vanillic acid (peak 4), ferulic acid (peak 7), rutin (peak 8), naringin (peak 9), hesperidin (peak 10), cinnamic acid (peak 12), and protodioscin (peak 13).

[0089] Taking peak 12 as the S peak, the relative retention time ranges of peaks 1 to 11 and peaks 13 to 20 are: 0.104-0.110, 0.192-0.199, 0.244-0.252, 0.389-0.398, 0.409-0.420, 0.542-0.546, 0.597-0.602, 0.646-0.653, 0.776~0.798, 0.827~0.846, 0.932~0.940, 1.097~1.111, 1.205~1.219, 1.334~1.357, 1.449~1.469, 1.508~1.529, 1.554~1.575, 1.630~1.652, 1.665~1.689.

[0090] Taking peak 12 as the S peak, the relative retention time RSD values ​​of peaks 1 to 11 and peaks 13 to 20 were 1.62%, 0.90%, 0.85%, 0.50%, 0.67%, 0.22%, 0.20%, 0.31%, 0.73%, 0.74%, 0.24%, 0.42%, 0.37%, 0.54%, 0.43%, 0.45%, 0.45%, 0.47% and 0.46%, respectively.

[0091] 5. Methodological Validation

[0092] Method validation projects include precision, stability and repeatability

[0093] 5.1 Fingerprint precision

[0094] Take asparagus medicinal material (S3), prepare a test solution according to the method under "Example 1, 1", and inject continuously 6 times according to the chromatographic conditions under "Example 1, 3", record the chromatogram, take peak 12 as the S peak, and the relative retention time RSD values ​​of peaks 1 to 11 and peaks 13 to 20 are 0.93%, 0.49%, 0.61%, 0.23%, 0.20%, 0.20%, 0.35%, 0.13%, 0.49%, 0.16%, 0.12%, 0.23%, 0.22%, respectively. , 0.28%, 0.30%, 1.59%, 0.23%, 0.23%, and 0.26%; the relative peak area RSD values ​​were 1.28%, 0.78%, 1.05%, 1.27%, 1.72%, 1.02%, 0.99%, 0.67%, 1.91%, 0.59%, 2.12%, 0.45%, 0.36%, 0.90%, 0.78%, 2.21%, 1.68%, 1.24%, and 1.71%, respectively, indicating that the instrument has good precision.

[0095] 5.2 Fingerprint stability

[0096] The same sample solution (S3) was taken and measured at room temperature for 0, 2, 4, 8, 12 and 24 h according to the chromatographic conditions under "Example 1, 3". Peak 12 was taken as the S peak. The relative retention time RSD values ​​of peaks 1 to 11 and peaks 13 to 20 were 1.35%, 0.76%, 1.01%, 0.69%, 0.61%, 0.39%, 0.51%, 0.38%, 0.63%, 0.51%, 0.58%, 0.42%, 0.38%, 0.35 %, 0.36%, 0.41%, 0.37%, 0.33%, and 0.43%; the relative peak area RSD values ​​were 2.73%, 1.78%, 2.31%, 2.20%, 1.44%, 2.38%, 1.56%, 2.83%, 2.70%, 2.44%, 1.70%, 1.83%, 1.95%, 0.99%, 2.81%, 0.91%, 2.20%, 1.38%, and 2.60%, respectively, indicating that the test sample is stable at room temperature within 24 hours.

[0097] 5.3 Fingerprint repeatability

[0098] From the same batch of asparagus medicinal materials (S3), 6 test solutions were prepared in parallel according to the method under "Example 1, 1", and the samples were injected respectively according to the chromatographic conditions under "Example 1, 3", with peak 12 as the S peak, and the relative retention time RSD values ​​of peaks 1 to 11 and peaks 13 to 20 were 1.37%, 0.85%, 0.52%, 0.35%, 0.25%, 0.20%, 0.19%, 0.18%, 0.24%, 0.20%, 0.30%, 0.43%, 0.40%, respectively. The relative peak area RSD values ​​were 1.35%, 1.06%, 1.37%, 2.14%, 2.24%, 2.02%, 1.73%, 2.97%, 2.33%, 2.07%, 1.87%, 0.83%, 0.96%, 1.33%, 2.19%, 2.27%, 1.88%, 1.33% and 2.62%, respectively, indicating that the method has good repeatability.

[0099] 6. Fingerprint similarity evaluation

[0100] The ULC-MS ion current patterns of the test solutions of 15 batches of asparagus medicinal materials were imported into the "Chinese Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) software, and the fingerprints of 15 batches of asparagus medicinal materials were established with cinnamic acid (peak 12) as the reference peak. Figure 4 As shown, Figure 4 The fingerprints of 15 batches of asparagus medicinal materials provided in Example 1 of the present invention are shown, and the reference maps are generated by the average method, such as Figure 5 As shown, Figure 5 The fingerprint comparison graphs of 15 batches of asparagus medicinal materials provided in Example 1 of the present invention generated by the average method are shown, and the similarity is calculated. The results show that the similarity of the fingerprints of 15 batches of asparagus medicinal materials is between 0.852 and 0.976, indicating that the fingerprint can be used to evaluate the quality of asparagus medicinal materials relatively well. The specific similarity results are shown in Table 3, where the sample numbers can be found in Table 1.

[0101] Table 3: Similarity of LC-MS fingerprints of 15 batches of asparagus medicinal materials

[0102]

[0103] Embodiment 2

[0104] This example provides a method for determining the content of an index component of an asparagus medicinal material, comprising: taking the asparagus medicinal material to be tested and preparing a sample solution to be tested;

[0105] Protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin were prepared as reference solution;

[0106] The sample solution to be tested and the reference solution are respectively injected into the ultra-high performance liquid chromatography-mass spectrometry instrument, and the test spectrum of the asparagus medicinal material to be tested and the reference spectrum of the reference solution are obtained according to the ultra-high performance liquid chromatography-mass spectrometry method; according to the concentration of the reference solution, the peak area of ​​the reference solution in the reference spectrum, and the peak area of ​​the components corresponding to the asparagus medicinal material to be tested and the reference solution in the test spectrum, the contents of the index components protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin in the asparagus medicinal material to be tested are calculated. The following provides a specific determination method.

[0107] 1. Mass spectrometry multiple reaction monitoring (MRM) detection parameters for reference substances Based on eight reference substances (including protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin), an MRM quantitative method for 15 batches of asparagus samples was established. The MRM parameters are shown in Table 4.

[0108] Table 4: MRM detection parameters of 8 reference substances

[0109]

[0110] 2. Optimization of experimental conditions for quantitative methods

[0111] The initial attempt at liquid phase elution method-1 is:

[0112] 0-6min: the volume fraction of mobile phase B changes from 95% to 70%;

[0113] 6-25min: the volume fraction of mobile phase B changes from 70% to 60%;

[0114] 25-27min: the volume fraction of mobile phase B changed from 60% to 40%;

[0115] 27-30min: the volume fraction of mobile phase B changed from 40% to 10%;

[0116] 30-35 min: the volume fraction of mobile phase B changed from 10% to 10%.

[0117] Under this elution condition, rutin, naringin, and hesperidin were eluted simultaneously, and the baselines could not be separated. Figure 6 As shown, Figure 6 The UPLC-MS spectrum of the reference solution in Example 2 of the present invention when the elution method is elution method-1 is shown; based on this experimental result, elution method-2 is optimized, and the elution gradient is:

[0118] 0-4min: the volume fraction of mobile phase B changes from 95% to 90%;

[0119] 4-8min: the volume fraction of mobile phase B changes from 90% to 80%;

[0120] 8-13min: the volume fraction of mobile phase B changes from 80% to 78%;

[0121] 13-20min: the volume fraction of mobile phase B changed from 78% to 45%;

[0122] 20-25min: the volume fraction of mobile phase B changes from 45% to 10%;

[0123] 25-30 min: The volume fraction of mobile phase B changed from 10% to 10%.

[0124] Under these conditions, each component has good separation. Figure 7 As shown, Figure 7 The UPLC-MS spectrum of the reference solution in Example 2 of the present invention when the elution method is elution method-2 is shown.

[0125] 3. Determination of experimental conditions for quantitative methods

[0126] The UPLC-QQQ-MS conditions were as follows: the chromatographic column was an ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm) column, the flow rate was 0.4 mL / min, and the column temperature was 40°C. The eluent was a mixed solution of methanol:acetonitrile (volume ratio 1:10) (mobile phase A) and an aqueous solution of acetic acid containing 0.05% volume concentration (mobile phase B), the injection volume was 2 μL, and the gradient elution was performed according to the following procedure. The UPLC-MS reference spectrum of the mixed reference solution and the UPLC-MS spectrum of the sample solution to be tested are compared in Figure 8 and Fig. 9 ,in, Figure 8 The UPLC-MS comparison spectrum of the mixed reference solution in Example 2 of the present invention is shown; Fig. 9 The UPLC-MS spectrum of the sample solution to be tested in Example 2 of the present invention is shown.

[0127] 0-4min: the volume fraction of mobile phase A changes from 5% to 10%, and the volume fraction of mobile phase B changes from 95% to 90%;

[0128] 4-8min: the volume fraction of mobile phase A changes from 10% to 20%, and the volume fraction of mobile phase B changes from 90% to 80%;

[0129] 8-13min: the volume fraction of mobile phase A changes from 20% to 22%, and the volume fraction of mobile phase B changes from 80% to 78%;

[0130] 13-20 min: the volume fraction of mobile phase A changes from 22% to 55%, and the volume fraction of mobile phase B changes from 78% to 45%;

[0131] 20-25 min: the volume fraction of mobile phase A changes from 55% to 90%, and the volume fraction of mobile phase B changes from 45% to 10%;

[0132] 25-30 min: the volume fraction of mobile phase A changes from 90% to 90%, and the volume fraction of mobile phase B changes from 10% to 10%.

[0133] Mass spectrometry detection adopted negative ion mode; analytes were determined in MRM mode, with ionization voltage (IS) -4500V, ionization temperature (TEM) 550℃, nebulizer (Gas1) 55L / min, curtain gas (CUR) 30L / min, collision gas (CAD) 7L / min, and auxiliary heating gas (Gas2) 55L / min.

[0134] 4. Preparation of sample solution

[0135] Accurately weigh 0.50 g of asparagus medicinal material powder (passed through a No. 2 sieve), add 25 ml of 100% methanol, ultrasonicate for 30 min (100 kHz, 30° C.), filter, and pass the filtrate through a 0.22 μm filter membrane to obtain the asparagus medicinal material sample solution to be tested.

[0136] 5. Methodological Validation

[0137] Method validation items include detection limit and quantification limit, linear range, precision, accuracy, recovery and stability

[0138] 5.1 Limit of detection, limit of quantification and linear range

[0139] Preparation of standard solutions: Take about 10 mg of protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin reference substances, accurately weigh them, add 50% methanol to dissolve, and prepare standard solutions with mass concentrations of 102.6827 μg / mL, 102.3000 μg / mL, 106.8210 μg / mL, 94.7261 μg / mL, 91.1584 μg / mL, 103.0000 μg / mL, and 106 .4076μg / mL, 97.7000μg / mL control stock solutions, take an appropriate amount of reference stock solution, and use 50% methanol to prepare a mixed standard solution containing 0.5134μg of protocatechuic acid, 0.5115μg of vanillic acid, 0.5341μg of ferulic acid, 12.3144μg of rutin, 0.2735μg of naringin, 8.2400μg of hesperidin, 1.0641μg of cinnamic acid, and 39.0800μg of protodioscin per 1mL.

[0140] The mixed reference solution was diluted in a gradient to prepare a series of linear working solutions. A series of linear working solutions were injected into the ultra-high performance liquid chromatography-mass spectrometry instrument, and linear regression was performed with concentration as the horizontal axis and peak area as the vertical axis to obtain the standard curve, and the regression equation and correlation coefficient were calculated. The linear relationship of the 8 index components is shown in Table 5.

[0141] Table 5: Linear regression analysis of the eight components

[0142]

[0143] 5.2 Precision test

[0144] Take the same mixed reference solution and inject it continuously for 6 times under the conditions of "Example 2, 3" to determine the peak area. The RSDs of the peak areas of protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid and protodioscin were calculated to be 1.37%, 1.25%, 2.79%, 2.79%, 1.61%, 1.81%, 0.66% and 1.44%, respectively, indicating that the instrument has good precision.

[0145] 5.3 Stability test

[0146] The same sample solution (S3) was taken and placed at room temperature for 0, 2, 4, 8, 12 and 24 h according to the conditions under "Example 2, 3". The peak areas were determined, and the RSDs of the peak areas of protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid and protodioscin were calculated to be 1.15%, 1.54%, 1.71%, 0.54%, 1.92%, 2.42%, 2.56% and 0.68%, respectively, indicating that the sample was stable within 24 h at room temperature.

[0147] 5.4 Repeatability test

[0148] Take the same batch of asparagus samples (S3), prepare 6 test solutions in parallel according to the method under "Example II, 4", and analyze them under the conditions under "Example II, 3". The average contents of protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid and protodioscin are calculated to be 1.9644 μg / g, 6.4386 μg / g, 3.3797 μg / g, 2.3397 μg / g, 0.2585 μg / g, 0.3441 μg / g, 52.2118 μg / g and 715.3371 μg / g, respectively, and the RSDs are 3.53%, 1.28%, 1.77%, 2.16%, 3.84%, 9.06%, 1.94% and 0.69%, respectively.

[0149] 5.5 Spike recovery test

[0150] 0.25 g of asparagus sample (S3) with known content of the tested component was accurately weighed, a total of 6 portions, and the sample solution was prepared according to the method under "Example 2, 4". The sample was injected and analyzed under the conditions under "Example 2, 3", and the peak area was determined. The average spiked recoveries of the 8 components were calculated to be 119.35%, 86.58%, 80.23%, 106.36%, 101.88%, 85.11%, 84.34%, and 101.88%, and the RSDs were 0.36%, 2.13%, 1.70%, 0.83%, 3.32%, 1.75%, 0.66%, and 1.43%, respectively. The specific results are shown in Table 6.

[0151] Table 6: Spiked recoveries of 8 components (n=6)

[0152]

[0153]

[0154] 6.15 batches of asparagus medicinal materials index component content determination

[0155] The contents of 8 components in 15 batches of asparagus medicinal materials were determined, wherein the preparation methods of different batches of asparagus medicinal materials refer to the preparation of the sample solution to be tested under "Example 2, 4"; wherein the liquid quality detection parameters refer to the quantitative method under "Example 2, 3"; the components and contents of different batches of asparagus medicinal materials were analyzed according to the liquid quality detection results, and the content of protocatechuic acid in 15 batches of asparagus medicinal materials was 0.0872-2.1154 μg / g, and the content of vanillic acid was 0.9088-11.0452 μg / g The content of ferulic acid was 0.7646-21.6540 μg / g, the content of rutin was 0.0054-527.1102 μg / g, the content of naringin was 0.0809-10.4682 μg / g, the content of hesperidin was 0.0881-245.5533 μg / g, the content of cinnamic acid was 6.9906-84.8560 μg / g, and the content of protodioscin was 0.5949-2384.4689 μg / g. The specific measurement results are shown in Table 7.

[0156] Table 7: Quantitative results of index components in 15 batches of asparagus medicinal materials

[0157]

[0158] In summary, the present invention provides a method for constructing an asparagus medicinal material fingerprint and determining the content of an index component. The method makes up for the problem of imperfect asparagus quality standards before, optimizes the liquid phase elution method, effectively separates the index component compound peaks, and establishes an LC-MS fingerprint on this basis. The similarity of 15 batches of asparagus medicinal materials is between 0.852 and 0.976, indicating that the fingerprint can better evaluate the quality of asparagus medicinal materials. Based on the liquid phase conditions for the separation of index components, a UPLC-QQQ-MS quantitative detection method was established to determine the contents of index components protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin in asparagus medicinal materials. The results showed that the content of protocatechuic acid in asparagus medicinal materials samples from different origins was 0.0872-2.1154 μg / g, the content of vanillic acid was 0.9088-11.0452 μg / g, and the content of ferulic acid was 0.7646-21.65 The content of rutin was 40μg / g, the content of rutin was 0.0054-527.1102μg / g, the content of naringin was 0.0809-10.4682μg / g, the content of hesperidin was 0.0881-245.5533μg / g, the content of cinnamic acid was 6.9906-84.8560μg / g, and the content of protodioscin was 0.5949-2384.4689μg / g. This method improves the evaluation standard of asparagus and provides a reference for the overall and comprehensive quality control of asparagus.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of asparagus medicinal material, characterized in that: include: Take asparagus medicinal materials to prepare the test solution; Accurately pipetting the test solution for ultra-high performance liquid chromatography-mass spectrometry detection to obtain a fingerprint of the asparagus medicinal material; The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection include: the mobile phase used is: mobile phase A is a methanol: acetonitrile mixed solution, wherein the volume ratio of the methanol to acetonitrile is 1:10; the mobile phase B is acetic acid water containing a volume concentration of 0.04% to 0.06%; a gradient elution method is used, and the gradient elution method is as follows: 0 to 4 min: the volume fraction of the mobile phase A changes from 5% to 10%, and the volume fraction of the mobile phase B changes from 95% to 90%; 4 to 8 min: the volume fraction of the mobile phase A changes from 10% to 20%, and the volume fraction of the mobile phase B changes from 90% to 80%; 8 to 13 min: the volume fraction of the mobile phase A changes from 20% to 22%, and the volume fraction of the mobile phase B changes from 80% to 78%; 13 to 20 min: the volume fraction of the mobile phase A changes from 22% to 55%, and the volume fraction of the mobile phase B changes from 78% to 45%; 20 to 25 min: the volume fraction of mobile phase A changes from 55% to 90%, and the volume fraction of mobile phase B changes from 45% to 10%; 25-30 min: the volume fraction of mobile phase A changes from 90% to 90%, and the volume fraction of mobile phase B changes from 10% to 10%; the stationary phase used is: a chromatographic column with octadecylsilane bonded silica gel as the filler; The construction method also includes: Protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin were prepared as reference solution; Accurately aspirate the reference substance solution for ultra-high performance liquid chromatography-mass spectrometry detection to obtain a reference substance chromatogram, and calibrate the common chromatographic peaks on the fingerprint of the asparagus medicinal material with reference to the reference substance chromatogram; The chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column, 2.1×100 mm, 1.8 μm; The method for preparing a test solution by taking asparagus medicinal materials comprises: The asparagus medicinal material is extracted and filtered using the extracting solution to obtain a test solution; The extract is a methanol-water solution, and the volume fraction of methanol in the methanol-water solution is 30% to 100%.

2. The method according to claim 1, characterized in that The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection also include: The flow rate of the mobile phase is 0.3-0.5 mL / min.

3. The method according to claim 1, characterized in that The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection also include: The column temperature of the chromatographic column is 35-45°C.

4. The method according to claim 1, characterized in that: The solvent of the reference solution is methanol.

5. The method according to claim 1, characterized in that The fingerprint spectrum contains 20 characteristic peaks, among which peak 2 is protocatechuic acid, peak 4 is vanillic acid, peak 7 is ferulic acid, peak 8 is rutin, peak 9 is naringin, peak 10 is hesperidin, peak 12 is cinnamic acid, and peak 13 is protodioscin.

6. A method for determining the content of an index component in asparagus medicinal material, characterized in that: include: Adding the extracting solution to the asparagus medicinal material to be tested to extract and obtain a sample solution to be tested; Protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin are added to a solvent to prepare a reference solution; Accurately weighing the sample solution to be tested and the reference solution to be tested respectively, and performing ultra-high performance liquid chromatography-mass spectrometry detection to obtain a test spectrum of the asparagus medicinal material to be tested and a reference spectrum of the reference solution; Calculate the contents of the index components protocatechuic acid, vanillic acid, ferulic acid, rutin, naringin, hesperidin, cinnamic acid, and protodioscin in the asparagus medicinal material to be tested according to the concentration of the reference solution, the peak area of ​​the reference solution in the reference spectrum, and the peak areas of the components of the asparagus medicinal material to be tested corresponding to the reference solution in the test spectrum; The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection include: the mobile phase used is: mobile phase A is a methanol:acetonitrile mixed solution, wherein the volume ratio of the methanol to acetonitrile is 1:10; the mobile phase B is acetic acid water containing a volume concentration of 0.04% to 0.06%; a gradient elution method is used, and the gradient elution method is as follows: 0-4 min: the volume fraction of the mobile phase A changes from 5% to 10%, and the volume fraction of the mobile phase B changes from 95% to 90%; 4-8 min: the volume fraction of the mobile phase A changes from 10% to 20%, and the volume fraction of the mobile phase B changes from 90% to 80%; 8-13 min: the volume fraction of the mobile phase A changes from 20% to 22%, and the volume fraction of the mobile phase B changes from 80% to 78%; 13-20 min: the volume fraction of the mobile phase A changes from 22% to 55%, and the volume fraction of the mobile phase B changes from 78% to 45%; 20-25 min: the volume fraction of mobile phase A changes from 55% to 90%, and the volume fraction of mobile phase B changes from 45% to 10%; 25-30 min: the volume fraction of mobile phase A changes from 90% to 90%, and the volume fraction of mobile phase B changes from 10% to 10%; the stationary phase used is: a chromatographic column with octadecylsilane bonded silica gel as the filler; The chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column, 2.1×100 mm, 1.8 μm; The extract is a methanol-water solution, and the volume fraction of methanol in the methanol-water solution is 30% to 100%.

7. The method according to claim 6, characterized in that The solvent of the reference solution was methanol.

8. The method according to claim 6, characterized in that The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection also include: The flow rate of the mobile phase is 0.3-0.5 mL / min.

9. The method according to claim 6, characterized in that The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry detection also include: The column temperature of the chromatographic column is 35-45°C.

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