CN115290768A - Method for detecting effective components of children's Qixing tea granules - Google Patents

Method for detecting effective components of children's Qixing tea granules Download PDF

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CN115290768A
CN115290768A CN202210780933.1A CN202210780933A CN115290768A CN 115290768 A CN115290768 A CN 115290768A CN 202210780933 A CN202210780933 A CN 202210780933A CN 115290768 A CN115290768 A CN 115290768A
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祝晨蔯
林朝展
刘方乐
谢远远
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Guangzhou University of Traditional Chinese Medicine
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components

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Abstract

The invention relates to a method for detecting effective components of children Qixing tea granules, which comprises the following steps: firstly, preparing a sample solution to be detected and a mixed standard solution, and then detecting according to the following method: according to the sample amount of 1-10 mu L, the water affinity BEH C with the column temperature of 20-40 DEG C 18 Injecting a sample into a chromatographic column, performing gradient elution by using a mobile phase consisting of a phase A and a phase B at a flow rate of 0.10-0.45 mL/min according to a specified program, simultaneously detecting by adopting a tandem flight time mass spectrum to obtain a spectrogram of a standard substance and a detected object, and constructing a common mode fingerprint of the detected object by using a traditional Chinese medicine fingerprint similarity evaluation system; finally, the spectrograms of the detected object and the standard substance are compared, and software is utilizedThe possible structural information of the compound is deduced by the aid of the fragment ion prediction function, and is compared with mass spectrum cracking information provided by a standard substance and a report in the existing literature, so that 31 effective components are further determined.

Description

Method for detecting effective components of children Qixing tea granules
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicine preparations, in particular to a method for detecting chemical components of children's Qixing tea granules.
Background
The children seven-star tea granules are a formulated preparation recorded in Chinese pharmacopoeia (552 of the first part of the 2020 version of the Chinese pharmacopoeia), and are prepared by taking 893g of coix seeds, 893g of rice sprouts, 446g of hawthorn, 670g of lophatherum gracile, 335g of uncaria, 112g of cicada slough and 112g of liquorice as raw materials, extracting, concentrating, preparing, sterilizing and packaging. The function of the children's Qixing tea granules is mainly to stimulate the appetite, remove the stagnation, clear heat and calm the fright. Can be used for treating infantile stagnation, dyspepsia, anorexia, dysphoria, insomnia, constipation, and scanty and brownish urine. However, the Chinese pharmacopoeia only adopts high performance liquid chromatography to detect glycyrrhizic acid (C) in the formulation 42 H 62 O 16 ) The content of the Chinese herbal medicines is regulated, which obviously cannot meet the requirement of comprehensively controlling the quality of the children Qixing tea granules.
At present, qualitative analysis of the children seven-star tea particles is mainly fingerprint analysis, but an established method is long in time consumption, a large number of reagents are consumed, high-throughput analysis is not facilitated, and identification of complex components is not sufficient, for example, students adopt high performance liquid chromatography and perform gradient elution for 9 times to determine three components (liquiritin, chlorogenic acid and apigenin, zhu Huan Xiao Ning, wang Xiu, and the like) in the children seven-star tea particles, namely, fingerprint spectra [ J ] of Chinese experimental prescriptions, 2014,20 (13): 93-96 ], but a set of gradient elution program and balance are added to an initial mobile phase to take 118 minutes, the detection wavelength is 254nm, the separation baseline chromatogram is uneven, and quantitative analysis is difficult to perform subsequently.
In addition, the existing qualitative analysis of the children's Qixing tea granules can not consider various compound types simultaneously, and the ultraviolet is poor in selectivity sometimes, so that more than 30 chemical components in the children's Qixing tea granules can not be detected qualitatively.
Disclosure of Invention
The invention provides a method for detecting effective components of children's Qixing tea granules, which has the advantage of simultaneously carrying out qualitative detection on 31 effective components in the children's Qixing tea granules.
The technical scheme for solving the technical problems is as follows:
a method for detecting effective components of children's Qixing tea granules is to detect various effective components in the children's Qixing tea granules by an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method, and specifically comprises the following steps:
(1) Preparing a detection sample injection solution to be detected: grinding the particles of the children's Qixing tea, dissolving the particles by using an organic solvent, extracting and purifying the particles, finally fixing the volume by using the same organic solvent until the concentration is 0.1-1 mg/mL, and filtering a detection sample solution to be detected;
(2) Preparing a mixed standard solution: respectively and precisely weighing a standard substance N-acetyl dopamine dimer A a 88 μ g N-acetyl dopamine dimer A b 93 μ g, hirsutine 110 μ g, dehydrohirsutine 109 μ g, rhynchophylline 122 μ g, isorhynchophylline 124 μ g, isodehydrorhynchophylline 75 μ g, dehydrorhynchophylline 66.6 μ g, liquiritin 535 μ g, isoliquiritin 570 μ g, liquiritigenin 555 μ g, isoliquiritigenin 520 μ g, glycyrrhizic acid 570 μ g, glycyrrhetinic acid 356.25 μ g, chlorogenic acid 105 μ g, neochlorogenic acid 105 μ g, cryptochlorogenic acid 108 μ g, hyperoside 97.6 μ g, orientin 65.25 μ g, isoorientin 57.32 μ g, apioside glycyrrhizin 105.32 μ g, formononetin 68.68 μ g, isozeatin 81.6 μ g, swertisin 856 μ g, rutin 876 μ g, p-coumaric acid 143 μ g, caffeic 81.2 μ g, citric acid 111.5 μ g, digoxin 96.96 g, luteolin 80.9 μ g and quercetin 80.8 μ g, and methanol 80% of a single stock solution; respectively sucking 50 mu L of single stock standard substance stock solution, dissolving with 80% methanol and diluting to 5mL to obtain the mixed standard substance stock solution;
(3) Detection method and conditions: injecting the sample solution to be detected and the mixed standard solution into an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer according to the following method to obtain a total ion flow spectrogram of the detected object and the standard solution: wherein, the first and the second end of the pipe are connected with each other,
the method for detecting the ultra-high performance liquid chromatography and the chromatographic conditions are as follows: according to the sample injection amount of 1-10 mu L, the column temperature of 20-40 ℃ of Waters Acquity BEH C 18 Injecting sample into chromatographic column, then using mobile phase consisting of 0.01-0.1% formic acid aqueous solution as phase A and acetonitrile as phase B, and adding 0.10E to EPerforming positive and negative ion gradient elution at a flow rate of 0.45mL/min, wherein the procedure of positive and negative ion elution is shown in Table 1 below;
TABLE 1 procedure for positive and negative ion elution
Figure BDA0003727690230000021
The mass spectrum detection method and the mass spectrum conditions are as follows: AB SCIEX using electrospray ion source and AB SCIEX automatic correction system
Figure BDA0003727690230000023
5600+ Mass spectrometer acquisition MS n Performing 8 MS/MS analyses on the mass spectrum data of the mode according to the set IDA condition after each TOF-MS scanning, and simultaneously checking dynamic background deduction options in the sampling process; wherein the MS n The mode is an electrospray positive ion mode and an electrospray negative ion mode; the operating parameters of the mass spectrometer are shown in table 2;
TABLE 2 Mass Spectrometry operating parameters
Figure BDA0003727690230000022
Figure BDA0003727690230000031
(4) Establishing a fingerprint spectrum: preparing 11 batches of detected objects according to the step (1), carrying out sample injection detection according to the step (3) to obtain a total ion flow spectrogram of the detected objects, selecting and opening a sample by adopting an Explorer module in AB SCIEX OS software, selecting a function of basic extract chromatography below a Process in a menu bar, and setting a hybridization half window to be 0.1min; returning to the function of selecting Data and Peak table under Show in the menu bar, exporting Data and Peak Data, exporting and storing the Data and the Peak Data in a text format, wherein the Data and the Peak Data comprise retention time, peak intensity value, mass-to-charge ratio, peak area value and Peak height value; reducing the peak intensity value, the peak area value and the peak height value by 10000 times by using a built-in division formula of Microsoft Excel 2019; importing the fingerprint data of each batch into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, setting the time window width of a reference map to be 0.1min, carrying out multipoint correction, and calculating the similarity to obtain the fingerprint under the common mode of the detected object;
(5) Characterization of chemical composition under consensus mode: introducing fingerprint spectrum mass spectrum data under the common mode of the detected object obtained in the step (4) into AB SCIEX OS software, and controlling the molecular formula with the mass error of less than +/-5 ppm by analyzing the excimer ion peak in the primary mass spectrum; selecting the secondary fragment mass spectrum information of each compound in the mixed standard substance, using AB SCIEX OS software to assist fragment ions to predict the structure information of the compound, then comparing the structure information with the mass spectrum cracking information of the standard substance, and determining that the compound is contained in the children Qixing tea particles when the mass spectrum cracking information is consistent with the structure information of the corresponding compound in the mixed standard substance.
The method qualitatively detects that the effective components in the children Qixing tea granules are the following 31 compounds: n-acetyl dopamine dimer A a N-acetyl dopamine dimer A b The drug comprises the following raw materials of hirsutine, rhynchophylline, isochirsutine, isodehydrorhynchophylline, rhynchophylline, liquiritin, isoliquiritin, isoliquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, hyperoside, orientin, isoorientin, apioside liquiritin, formononetin, neocerin, helioxanthin, rutin, p-coumaric acid, caffeic acid, citric acid, luteolin and quercetin.
In the method of the invention, the optimal high performance liquid chromatography conditions are as follows: sample introduction of 5. Mu.L to Waters Acquity BEH C with a column temperature of 30 ℃ 18 Injecting a chromatographic column, and then carrying out positive and negative ion gradient elution by using a mobile phase consisting of a 0.1% formic acid aqueous solution as a phase A and acetonitrile as a phase B at a flow rate of 0.40 mL/min.
The method for detecting the effective components of the children's Qixing tea has the following beneficial effects:
(1) The fingerprint of the effective components of the children's Qixing tea granules prepared from 7 raw material medicines is established by adopting an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry combined technology for the first time, and is combined with a similarity evaluation system, the similarity value of the fingerprint and a reference fingerprint is calculated, 31 characteristic common peaks can be intuitively and quickly identified, wherein the 31 characteristic common peaks are identified by comparing with a standard product, and the stability and the repeatability are good;
(2) The method combining the liquid chromatography-mass spectrometry technology and the fingerprint spectrum has the advantages of high sensitivity, high scanning speed and high flux, considers various compound types, avoids the ultraviolet discrimination effect, and can quickly and conveniently represent the integral quality level of the children Qixing tea granules;
(3) The elution procedure and the mass spectrum detection method are scientific, reasonable, convenient and quick.
Description of the drawings:
fig. 1 is a fingerprint of 11 batches (S1-S11) of pediatric seven-star tea granules after matching in an embodiment of the method of the present invention.
FIG. 2 is a control profile generated from 11 batches (S1-S11) of pediatric Severe tea granules in an embodiment of the method of the invention.
Fig. 3 is a total ion current chromatogram of 31 common fingerprints of 11 batches (S1-S11) of pediatric saturera chinensis granules in an embodiment of the method of the invention. Wherein, A is positive ion, B is negative ion. Wherein, A is positive ion, B is negative ion; the common peaks of all the characteristics in the figure are as follows: the 1 peak is citric acid, the 2 peak is neochlorogenic acid, the 3 peak is chlorogenic acid, the 4 peak is cryptochlorogenic acid, the 5 peak is New Zealand vitexin, the 6 peak is caffeic acid, the 7 peak is isoorientin, the 8 peak is p-coumaric acid, the 9 peak is orientin, the 10 peak is Nidaxinin, the 11 peak is rutin, the 12 peak is hyperin, the 13 peak is apioside liquiritin, the 14 peak is liquiritin, the 15 peak is liquiritigenin, the 16 peak is N-acetyl dopamine dimer A a The 17 th peak is luteolin, the 18 th peak is N-acetyl dopamine dimer A b Peak 19 is rhynchophylline, peak 20 is isoliquiritin, peak 21 is isorhynchophylline, and peak 22 is rhynchophyllineIs isorhynchophylline, formononetin in peak 23, isoliquiritigenin in peak 24, rhynchophylline in peak 25, luteolin in peak 26, quercetin in peak 27, unhardened hirsutine in peak 28, hirsutine in peak 29, glycyrrhizic acid in peak 30 and glycyrrhetinic acid in peak 31.
FIG. 4 is a graph of the elution gradient generation of Table 7 in the following examples.
FIG. 5 is a graph of the elution gradient generated in Table 8 in the following examples.
FIG. 6 is a graph of the elution gradient generation of Table 9 in the following examples.
FIG. 7 is a sample size review for generating a profile in an embodiment of the method of the present invention.
FIG. 8 is a flow rate profile for a specific embodiment of the method of the present invention.
Detailed Description
Example 1 (detection method)
1. Instrument and reagent
The instrument comprises: shimadzu Nexera X 2 An ultra-high performance liquid chromatograph (Shimadzu corporation, japan) equipped with an LC-30AD high performance liquid chromatograph, an SIL-30AC autosampler, a CTO-20AC column oven, and an SPD-M20A detector; AB SCIEX
Figure BDA0003727690230000051
5600 + Mass spectrometer (AB SCIEX, USA) with electrospray ion source and online analysis TF TM version 1.7 data acquisition and processing system; AB SCIEX OS version 2.0 (AB SCIEX, USA); vortex-Genie 2 vortexer (SI corporation, usa); SIGMA 1-14 high speed centrifuge (SIGMA corporation, germany); KQ5200 ultrasonic cleaning machine (ultrasonic instruments ltd, kunshan); waters Acquity BEH C 18 (2.1X 100mm,1.7 μm) chromatography column (Watts science and technology Co., ltd.).
And (3) standard substance: glycyrrhizin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, hyperoside, new Zealand vitexin, ningkoxanthin, orientin, isoorientin, rutin, p-coumaric acid, caffeic acid, citric acidAcid, hirsutine, dehydrohirsutine, rhynchophylline, isochirsutine, isodehydrorhynchophylline, dehydrorhynchophylline, and N-acetyl dopamine dimer A a N-acetyl dopamine dimer A b Quercetin, luteolin, apioside liquiritin and formononetin.
A sample to be detected: the sample to be detected is the children Qixing tea granules, which are prepared by the children Qixing tea granule item recorded in 2020 edition of pharmacopoeia of the people's republic of China.
Other reagents: methanol and acetonitrile were of LC-MS grade (Honeywell, USA) and formic acid was of LC-MS grade (Fluka, USA).
2. Method of producing a composite material
2.1 preparing a mixed standard solution: accurately weighing standard N-acetyl dopamine dimer A respectively a 88 μ g, N-acetyl dopamine dimer A b 93 μ g, hirsutine 110 μ g, dehydrohirsutine 109 μ g, rhynchophylline 122 μ g, isorhynchophylline 124 μ g, isodehydrorhynchophylline 75 μ g, dehydrorhynchophylline 66.6 μ g, liquiritin 535 μ g, isoliquiritin 570 μ g, liquiritigenin 555 μ g, isoliquiritigenin 520 μ g, glycyrrhizic acid 570 μ g, glycyrrhetinic acid 356.25 μ g, chlorogenic acid 105 μ g, neochlorogenic acid 105 μ g, cryptochlorogenic acid 108 μ g, hyperoside 97.6 μ g, orientin 65.25 μ g, isoorientin 57.32 μ g, apioside glycyrrhizin 105.32 μ g, formononetin 68.68 μ g, isozeatin 81.6 μ g, swertisin 856 μ g, rutin 876 μ g, p-coumaric acid 143 μ g, caffeic 81.2 μ g, citric acid 111.5 μ g, digoxin 96.96 g, luteolin 80.9 μ g and quercetin 80.8 μ g, and methanol 80% of a single stock solution; respectively sucking 50 mu L of single stock standard substance stock solution, dissolving with 80% methanol and diluting to 5mL to obtain the mixed standard substance stock solution.
2.2 preparing a sample solution to be tested: respectively weighing 8.93g of coix seeds, 8.93g of rice buds, 4.46g of hawthorn, 6.70g of lophatherum gracile, 3.35g of uncaria, 1.12g of cicada slough and 1.12g of liquorice according to the proportion of the formula of the children Qixing tea granules by using a TB-25 one-hundred thousand electronic analytical balance; wherein Coicis semen and fructus oryzae Germinatus are decocted with pure water twice, each for 2 hr, the decoctions are filtered, the filtrates are mixed, concentrated to relative density of 1.08 at 55 deg.C, ethanol is added to make ethanol content reach 45 vol%, standing, filtering, recovering ethanol from the filtrate, and concentrating to obtain soft extract; decocting the rest five ingredients of hawthorn, lophatherum gracile, uncaria, cicada slough, liquorice and the like in water twice, each time for 2 hours, filtering decoction, combining filtrates, concentrating the filtrate to a proper amount, combining the filtrate with the thick paste, and carrying out freeze drying under the condition of reducing the pressure to 70-75 ℃ by using a freeze dryer to obtain a sample to be detected;
taking 0.5g of the sample to be detected, adding 10mL of 80% methanol, performing ultrasonic treatment for 30min at 20 ℃ by using a KQ5200 ultrasonic cleaning machine, cooling to room temperature, supplementing the mass of the 80% methanol, performing centrifugation for 10min, sucking 200 mu L of supernatant, and performing constant volume to 10mL by using 80% methanol to obtain the sample solution to be detected.
2.3 injecting the mixed standard solution and the sample solution to be tested into the Shimadzu Nexera X respectively 2 Ultra-high performance liquid chromatograph connected in series with AB SCIEX
Figure BDA0003727690230000063
5600 + A detection system consisting of quadrupole time-of-flight mass spectrometry is used for obtaining a total ion flow spectrogram of a detected object and a standard substance: wherein the content of the first and second substances,
the detection method and chromatographic conditions of the high performance liquid chromatography are as follows: water Acquity BEH C with sample size of 5 mul to filler particle diameter of 1.7 mu m, diameter and length of 2.1mm and 100mm 18 Introducing sample into chromatographic column, controlling column temperature at 40 deg.C, and performing gradient elution with positive and negative ions at flow rate of 0.4mL/min by using mobile phase composed of 0.1% formic acid water solution as phase A and acetonitrile as phase B, wherein the procedure of elution of positive and negative ions is shown in Table 3 below:
TABLE 3
Figure BDA0003727690230000061
The mass spectrum detection method and the mass spectrum conditions are as follows: AB SCIEX adopting electrospray ion source and AB SCIEX automatic correction system
Figure BDA0003727690230000064
5600 + Mass spectrometer acquisition of MS n Performing 8 MS/MS analyses according to the set IDA conditions after each TOF-MS scanning on the mass spectrum data of the mode, and simultaneously checking dynamic background deduction options in the sampling process; wherein the MS n The mode is an electrospray positive ion mode and an electrospray negative ion mode; wherein the mass spectrometry operating parameters are shown in table 4;
table 4 Mass Spectrometry operating parameters
Figure BDA0003727690230000062
Figure BDA0003727690230000071
2.4, establishing a fingerprint: preparing 11 batches of detected substances according to the preparation of sample injection liquid to be detected, and injecting the sample injection liquid to be detected of the children's Qixing tea granules into the Shimadzu Nexera X 2 Ultra-high performance liquid chromatograph connected in series with AB SCIEX
Figure BDA0003727690230000072
5600 + Detecting by a detection system consisting of a quadrupole time-of-flight mass spectrum to obtain a total ion flow spectrogram of a detected object, selecting and opening a sample by adopting an Explorer module in AB SCIEX OS software, selecting a function of a basic extract chromatography below a Process in a menu bar, and setting a hybridization half window to be 0.1min; returning to the menu bar to select the function of Data and Peak table under the Show, exporting the Data and Peak Data, exporting and storing the Data and the Peak Data into a text format, wherein the text format comprises retention time, peak intensity value, mass-to-charge ratio, peak area value and Peak height value; reducing the peak intensity value, the peak area value and the peak height value by 10000 times by using a built-in division formula of Microsoft Excel 2019; introducing the fingerprint data of each batch into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, setting the time window width of the reference spectrum to be 0.1min, performing multipoint correction, and calculating similarity to obtain fingerprint under the common mode of the detected objectAs shown in fig. 1, and the generated control fingerprint is shown in fig. 2.
2.5 characterization of chemical composition under consensus mode: introducing mass spectrum data of the detected object obtained by 11 batches of sample solutions to be detected and mixed standard substance solutions into AB SCIEX OS software, and controlling the molecular formula with the mass error less than +/-5 ppm by analyzing the excimer ion peak in the primary mass spectrogram; selecting secondary fragment mass spectrum information of each compound in the mixed standard substance, using AB SCIEX OS software to assist fragment ions to predict structure information of the compound, then comparing the structure information with mass spectrum cracking information of the standard substance, and determining that the infant Qixing tea particles contain the compound when the mass spectrum cracking information is consistent with the structure information of the corresponding compound in the mixed standard substance, wherein the total ion current chromatogram of 31 common fingerprints of the sample liquid to be detected is shown in figure 3.
3 results
3.1 the similarity of the 11 batches of the pediatric Qixing tea medicines and the generated comparison fingerprint is calculated to be more than 0.94, and the similarity of the 11 batches (S1-S11) of the pediatric Qixing tea medicines is shown in the table 5.
TABLE 5
Figure BDA0003727690230000073
Figure BDA0003727690230000081
3.2 comparing the mass spectrum data of the common characteristic peaks of 11 batches (S1-S11) of the pediatric seven-star tea medicaments with mass spectrum cracking information provided by standard products and reports of the existing documents to identify 89 common characteristic peaks (see Table 6)
TABLE 6
Figure BDA0003727690230000082
Figure BDA0003727690230000091
Note: the "#" mark corresponds to the retention time of the compound and the mass spectrum information of the control.
Example 2 (investigation of the Effect of the conditions of liquid chromatography tandem Mass Spectrometry in the present method on the quality of the chromatogram)
1. Instruments and reagents: the apparatus and reagents used in this example were the same as those used in example 1. The sample to be detected is the children seven-star tea granules, which are prepared by the children seven-star tea medicine recorded in 2020 edition of pharmacopoeia of the people's republic of China.
2. Method of producing a composite material
2.1 preparing a mixed standard solution: same as in example 1.
2.2 preparing the infant Qixing tea granule sample injection to be tested: same as in example 1.
2.3 methods of investigation: as can be seen from the detection method described in example 1 above, the main parameters affecting the profile are: elution gradient, sample size, flow rate of the mobile phase 3 parameters, so in this example the parameter to be studied is set as a variable factor, while the other 2 parameters are set as fixed values (same as in example 1).
2.4 elution gradient vs. profile: injecting the infantile seven-star tea granule into Shimadzu Nexera X by changing elution gradient 2 Ultra-high performance liquid chromatograph connected in series with AB SCIEX
Figure BDA0003727690230000101
5600 + A detection system consisting of quadrupole time-of-flight mass spectrometry wherein the detection method is as in example 1 and elution gradients are injected into the detection system as in tables 7, 8 and 9 and the total ion flux chromatogram is recorded using AB SCIEX OS software.
TABLE 7
Figure BDA0003727690230000102
TABLE 8
Figure BDA0003727690230000103
TABLE 9
Figure BDA0003727690230000105
As a result, as shown in fig. 4, 5 and 6, the peak (fig. 5) obtained by using the elution gradient of table 8 is more informative, and the peak shape and the resolution are better, and therefore table 8 was selected as the optimal elution gradient.
2.6 inspection of the sample size on the map: by changing the level of sample injection, the infant's seven-star tea drug is injected into Shimadzu Nexera X 2 Ultra-high performance liquid chromatograph connected in series with AB SCIEX
Figure BDA0003727690230000106
5600 + The detection system consists of quadrupole time-of-flight mass spectrometry, wherein the detection method refers to example 1, the sample volumes are injected into the detection system according to 0, 1, 3, 5, 6, 8 and 10 mu L, and the AB SCIEX OS software is used for recording a total ion current chromatogram. As shown in FIG. 7, the peak information of the compound gradually increased with the increase of the amount of the sample, and the amount of the sample was not increased at 5. Mu.L, so that 5. Mu.L was selected as the optimum amount of the sample.
2.7 flow Rate inspection of the spectra: by changing the flow rate level, the pediatric seven-star tea drug was injected into the Shimadzu Nexera X 2 Ultra-high performance liquid chromatograph connected in series with AB SCIEX
Figure BDA0003727690230000111
5600 + The detection system consists of quadrupole time-of-flight mass spectrometry, wherein the detection method refers to example 1, the flow rate is injected into the detection system according to 0.1, 0.2, 0.3, 0.4 and 0.5mL/min, and AB SCIEX OS software is used for recording the total ion current chromatogram. As a result, as shown in FIG. 8, the peak information of the compound gradually increased with the increase of the flow rate, and the flow rate was not increased at 4. Mu.L, and therefore 0.4mL/min was selected as the optimum flow rate.

Claims (2)

1. A method for detecting effective components of children Qixing tea granules is characterized by comprising the following steps:
(1) Preparing a detection sample injection solution to be detected: grinding the particles of the children's Qixing tea, dissolving the particles by using an organic solvent, extracting and purifying the particles, finally fixing the volume by using the same organic solvent until the concentration is 0.1-1 mg/mL, and filtering a detection sample solution to be detected;
(2) Preparing a mixed standard solution: accurately weighing standard N-acetyl dopamine dimer A respectively a 88 μ g N-acetyl dopamine dimer A b 93 μ g, 110 μ g of hirsutine, 109 μ g of dehydrohirsutine, 122 μ g of rhynchophylline, 124 μ g of isorhynchophylline, 75 μ g of isodehydrorhynchophylline, 66.6 μ g of dehydrorhynchophylline, 535 μ g of liquiritin, 570 μ g of isoliquiritin, 555 μ g of liquiritin, 520 μ g of isoliquiritigenin, 570 μ g of glycyrrhizic acid, 356.25 μ g of glycyrrhetinic acid, 105 μ g of chlorogenic acid, 105 μ g of neochlorogenic acid, 108 μ g of cryptochlorogenic acid, 97.6 μ g of hyperoside, 65.25 μ g of orientin, 57.32 μ g of isoorientin, 105.32 μ g of apioside, 68.68 μ g of formononetin, 81.6 μ g of neocericidin, 856 μ g of swertisin, 876 μ g of digenin, 143 μ g of p-coumaric acid, 81.2 μ g of caffeic, 111.5 μ g of citric acid, 96 g of digitoxin, 96 g of rutin, 80.8 μ g of luteolin, 9.8 μ g of quercetin and 80% of methanol are respectively dissolved in a standard solution with a volume of 0.80 mL and 80% of luteolin solution; respectively sucking 50 mu L of single stock standard substance stock solution, dissolving with 80% methanol and diluting to 5mL to obtain the mixed standard substance stock solution;
(3) Detection method and conditions: injecting the sample solution to be detected and the mixed standard solution into an ultra-high performance liquid chromatography tandem time-of-flight mass spectrometer respectively according to the following method to obtain a total ion flow spectrogram of the detected object and the standard substance: wherein, the first and the second end of the pipe are connected with each other,
the method for detecting the ultra-high performance liquid chromatography and the chromatographic conditions are as follows: according to the sample amount of 1-10 mu L, the water affinity BEH C with the column temperature of 20-40 DEG C 18 Injecting a sample into a chromatographic column, and then performing positive and negative ion gradient elution by using a mobile phase consisting of 0.01-0.1% formic acid aqueous solution of phase A and acetonitrile of phase B at the flow rate of 0.10-0.45 mL/min, wherein the positive and negative ion elution procedure is shown in the following table 1;
TABLE 1 procedure for positive and negative ion elution
Figure FDA0003727690220000011
The mass spectrum detection method and the mass spectrum conditions are as follows: AB SCIEX using electrospray ion source and AB SCIEX automatic correction system
Figure FDA0003727690220000012
5600 + Mass spectrometer acquisition of MS n Performing 8 MS/MS analyses according to the set IDA conditions after each TOF-MS scanning on the mass spectrum data of the mode, and simultaneously checking dynamic background deduction options in the sampling process; wherein the MS n The mode is an electrospray positive ion mode and an electrospray negative ion mode; the operating parameters of the mass spectrometer are shown in table 2;
TABLE 2
Parameters of mass spectrum Positive ion mode Negative ion mode Spray Voltage (ISVF) 5500V -4500V Ion Source Temperature (TEM) 500℃ 500℃ Atomizing Gas (Gas 1) 55psi 55psi Auxiliary heating Gas (Gas 2) 55psi 55psi Air curtain Gas (GUC) 35psi 35psi Declustering voltage (DP) 100eV -100eV Collision Energy (CE) 35eV -35eV One-level Collision energy expansion (CES) 0 0 Secondary collision energy expansion (CES) 15eV -15eV TOF-MS scanning Range 100～1200Da 100～1200Da TOF-MSMS scanning range 50～1000Da 50～1000Da
(4) Establishing a fingerprint spectrum: preparing 11 batches of detected objects according to the step (1), carrying out sample injection detection according to the step (3) to obtain a total ion flow spectrogram of the detected objects, selecting and opening a sample by adopting an Explorer module in AB SCIEX OS software, selecting a function of a basic extract chromatograph under a Process in a menu bar, and setting a hybridization half window to be 0.1min; returning to the function of selecting Data and Peak table below Show in the menu bar, exporting Data and Peak Data, exporting and storing the Data and the Peak Data into a text format, wherein the Data and the Peak Data comprise retention time, peak intensity value, mass-to-charge ratio, peak area value and Peak height value; reducing the peak intensity value, the peak area value and the peak height value by 10000 times by using a built-in division formula of Microsoft Excel 2019; importing the fingerprint data of each batch into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, setting the time window width of a reference map to be 0.1min, performing multipoint correction, and calculating the similarity to obtain the fingerprint under the common mode of the detected object;
(5) Characterization of chemical composition under consensus mode: introducing fingerprint spectrum mass spectrum data under the common mode of the detected object obtained in the step (4) into AB SCIEX OS software, and controlling the molecular formula with the mass error of less than +/-5 ppm by analyzing the excimer ion peak in the primary mass spectrum; selecting the secondary fragment mass spectrum information of each compound in the mixed standard substance, using AB SCIEX OS software to assist fragment ions to predict the structure information of the compound, then comparing the structure information with the mass spectrum cracking information of the standard substance, and determining that the compound is contained in the children Qixing tea particles when the mass spectrum cracking information is consistent with the structure information of the corresponding compound in the mixed standard substance.
2. The method for detecting the effective components of the children Qixing tea granules as claimed in claim 1, wherein the method and chromatographic conditions for the detection by the ultra high performance liquid chromatography are as follows: according to the sample injection amount of 5 mu L, the sample injection amount of the Waters Acquity BEH C is 20-40 DEG to the column temperature 18 Introducing into chromatographic column, and collecting with a mixture of 0.1% formic acid solution as phase A and BAnd acetonitrile at a flow rate of 0.40 mL/min.
CN202210780933.1A 2022-07-04 2022-07-04 Method for detecting effective components of children's Qixing tea granules Pending CN115290768A (en)

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