CN115078569B - Cough relieving key quality attribute identification method of biological sensing integrated UPLC-MS technology - Google Patents
Cough relieving key quality attribute identification method of biological sensing integrated UPLC-MS technology Download PDFInfo
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- CN115078569B CN115078569B CN202210581117.8A CN202210581117A CN115078569B CN 115078569 B CN115078569 B CN 115078569B CN 202210581117 A CN202210581117 A CN 202210581117A CN 115078569 B CN115078569 B CN 115078569B
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Abstract
The invention provides a method for identifying key quality attributes of traditional Chinese medicines by using a biological sensing chip integrated UPLC-MS technology and application of the method in traditional Chinese medicine such as children's anti-food retention and cough relieving oral liquid. The method specifically comprises the following steps: (1) Constructing a biosensor by taking target proteins as research carriers; (2) Screening the interaction strength of the sample to be tested and the key protein by adopting an electrochemical workstation based on the biosensor in the step 1; (3) Eluting the sample bound to the biosensor device with a non-specific eluent and a specific eluent for the target protein; (4) Enriching the eluent in the step 3, and identifying substances combined with the biological sensing sensitive element in the sample to be detected by adopting a liquid chromatography-mass spectrometry technology; (5) And (3) comparing the detected substance obtained in the step (4) with the whole components of the substance to be detected, and screening the key quality attribute of the substance. The invention forms a set of traditional Chinese medicine key quality attribute identification method based on a biological sensing integrated UPLC-MS technology.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a key quality attribute identification method of a biological sensing integrated UPLC-MS technology and application of the key quality attribute identification method in pediatric anti-food retention and cough relieving oral liquid cough relieving key quality attribute identification.
Background
The international committee for pharmaceutical registration technical requirements, international coordination committee, guidelines for drug development (ICHQ 8), explicitly states that key quality attributes (critical quality attributes, CQAs) of a drug refer to physical, chemical, biological or microbiological properties or characteristics that ensure the quality of the desired product within appropriate limits, ranges or distributions. Wherein, the key quality attribute of efficacy guided by clinical efficacy is the precondition of the quality evaluation and control of the traditional Chinese medicine and the preparation thereof. However, currently, the traditional Chinese medicine industry generally has the difficult problem of pain points in the industry, which is related to lack of quality evaluation and process control indexes and clinical curative effects.
Based on the characteristic of complex traditional Chinese medicine system, when the traditional detection technology is adopted to carry out the identification research of the key quality attribute of efficacy, the defects of higher detection limit, more complex separation means, more interference factors, relatively complex analysis, incapability of being directly combined with clinical efficacy and the like exist. Efficacy-oriented biosensors are used as the current leading edge analysis technology, and key technical support is provided for the identification research of key quality attributes associated with efficacy through a biological identification element in direct contact with a transducer. AlGaAs/GaAs High Electron Mobility Transistor (HEMT) biosensor prepared based on semiconductor material is easy to integrate and mass produce due to high sensitivity and higher detection speed, has outstanding biocompatibility and can be subjected to biological functional modification, is widely applied in the field of molecular recognition, and can reach pg level in detection limit. For substances obtained by screening, the ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has better quantitative and identification capabilities according to the high-efficiency chromatographic separation performance and shorter analysis time, and provides important technical support for the separation and identification of chemical components obtained by biosensing screening.
Disclosure of Invention
The invention aims to provide a cough relieving key quality attribute identification method of a biological sensing integrated UPLC-MS technology.
The invention further aims to provide an application of the key quality attribute identification method of the biological sensing integrated UPLC-MS technology in the key quality attribute identification of the children's anti-food retention and cough relieving oral liquid cough relieving.
In order to overcome the defects in the prior art, the invention firstly provides a method for identifying key quality attributes of potential taste, which comprises the following specific steps:
step 1: constructing a biosensor by taking target proteins as research carriers;
step 2: screening the interaction strength of the sample to be tested and the key protein by adopting an electrochemical workstation based on the biosensor in the step 1;
step 3: eluting the sample bound on the biosensing device with a non-specific eluent and a specific eluent for the target protein;
step 4: enriching the eluent in the step 3, and identifying substances combined with the biosensing sensing element in the sample to be detected by adopting an ultra-high performance liquid chromatography-mass spectrometry combined technology;
step 5: and (3) comparing the detected substance obtained in the step (4) with the whole components of the substance to be detected, and screening the key quality attribute of the substance.
According to some specific embodiments of the invention, a key quality attribute identification method of a biological sensing integrated UPLC-MS technology relates to a key target protein MIF modified HEMT biological sensor, and the specific steps of construction are as follows:
(1) Adhering a clean quartz glass tube on the HEMT device to serve as a sample cell, adding 3-mercaptopropionic acid into the sample cell, soaking for 24 hours at room temperature, and generating an Au-S bond on the surface of the HEMT device to form a self-assembled monolayer;
(2) Washing off the excessive 3-mercaptopropionic acid in the step (1), and adding a volume mixture of 20mM of carboxyl activator carbodiimide hydrochloride and 50mM of N-hydroxysuccinimide to a sample cell to generate a stable amine activation product for activating carboxyl;
(3) And (3) cleaning the HEMT device by using Phosphate Buffer Solution (PBS), adding target protein, and reacting for 2 hours at the temperature of 4 ℃ to obtain the MIF modified HEMT device biosensor.
According to some specific embodiments of the invention, the key quality attribute identification method of the biological sensing integrated UPLC-MS technology relates to the adoption of the constructed biological sensor for detecting and researching the interaction intensity between traditional Chinese medicine and key protein, wherein an electrochemical workstation is CHI-660e, a given constant voltage is 2V, and the current precision is mu A level.
According to some embodiments of the invention, the non-specific eluent in step 3 is PBS, the specific eluent is 5-O-Gasivisamiloride and amygdalin which have strong interaction with MIF and cough relieving efficacy, and each eluent is eluted for 3 times.
According to some embodiments of the invention, the molecular weight in step 4 ranges from 100 to 1500; specific detection conditions are shown below.
The invention further provides an application of the key quality attribute identification method of the biological sensing integrated UPLC-MS technology in the research of the key quality attribute of the children's anti-food retention and cough relieving oral liquid cough relieving, which is characterized by comprising the following specific steps:
(1) Adhering a clean quartz glass tube on the HEMT device to serve as a sample cell, adding 3-mercaptopropionic acid into the sample cell, soaking for 24 hours at room temperature, and generating an Au-S bond on the surface of the HEMT device to form a self-assembled monolayer;
(2) Washing off the excessive 3-mercaptopropionic acid in the step (1), and adding a volume mixture of 20mM of carboxyl activator carbodiimide hydrochloride and 50mM of N-hydroxysuccinimide to a sample cell to generate a stable amine activation product for activating carboxyl;
(3) Cleaning the HEMT device by using Phosphate Buffer Solution (PBS), adding target protein, and reacting for 2 hours at the temperature of 4 ℃ to obtain the MIF modified HEMT device biosensor; determination of I before and after modification of MIF protein using electrochemical workstation DS -V DS As shown in FIG. 1 (a), the results are shown in the graph, and the MIF modified I DS -V DS The curves are obviously changed, which indicates that MIF is successfully modified on the HEMT device;
(4) Based on the MIF-HEMT biosensor in (1), the electrochemical workstation CHI-660e is adopted to determine the I of the children's anti-food retention and cough relieving oral liquid and MIF with different concentrations DS -V DS Calculating interaction between children's anti-food retention and cough relieving oral liquid and MIF-biosensorThe Kd was calculated as 3.981X 10 by intensity -10 g/mL;
(5) Eluting a sample combined on the MIF-HEMT biological sensing device by adopting PBS as a non-specific eluent for 3 times, reacting for 5 minutes each time, and combining the eluents; further, eluting with 5-O-methylvisamiloride for 3 times, reacting for 5 minutes each time, and mixing eluates; finally, the amygdalin is adopted for elution, 3 times of reaction are carried out for 5 minutes each time, and the eluates are combined.
(6) Washing out phosphate in the 3 eluents by adopting a solid phase extraction technology, respectively enriching the eluents in (5), and separating and identifying substances combined with the MIF-HEMT biosensing sensitive element in the sample to be detected by adopting an ultra-high performance liquid chromatography mass spectrometry technology, wherein the specific instrument parameters are as follows:
ultra-high performance liquid phase conditions: chromatographic column: ACQUITY UPLC HSS column (150 mm. Times.2.1 mm,1.7 μm); column temperature: 35 ℃; mobile phase: 0.1% formic acid water (A) -acetonitrile (B).
Gradient elution meter
Mass spectrometry conditions: a chromatographic column electrospray ion source (ESI) positive and negative ion modes; sheath air flow rate: 40arb; auxiliary gas flow rate: 20arb; capillary voltage: -35V; spray voltage: 3kV; tube lens voltage: -110V; capillary temperature: 350 ℃; fourier high resolution scan range m/z 100-1200; first order resolution 30000; the secondary mass spectrum adopts data dependency scanning, and 3 ions with highest primary abundance are selected for CID secondary fragmentation. When the secondary fragment information is incomplete, the acquisition efficiency of the secondary mass spectrum information is improved through an ion list scanning mode.
The total ion flow diagram in the positive and negative ion source modes is shown in figure 3. The analysis of the primary fragments and the secondary fragments of the mass spectrum information is adopted to obtain 10 mass attributes of the pediatric anti-food retention and cough relieving oral liquid cough relieving key, which are specifically as follows: rutin, forsythoside E, eriocitrin, neo-North America eriodictyoside, hesperidin, neohesperidin, poncirin, vitexin glucoside, loniceraside, kaempferol-3-O-rutin. The 7 are compared and analyzed through standard substances respectively, and the specific cracking rule is as follows:
as can be seen from the high-resolution mass spectrum data, in the negative ion mode, the excimer ion peaks of the compounds A-5 and M-3 were 609.14563[ M-H ], respectively] - 、609.14612[M-H] - The retention time was 16.03min and 16.04min, both of which were presumed to be C 27 H 30 O 16 The molecular formula is the same as that of rutin, and the deviation from the theoretical value is 1.016ppm and 1.820ppm respectively. Both ion fragments include M/z447.21[ M-H-C 6 H 10 O 5 ] - 、m/z300.96[M-H-C 12 H 20 O 9 ] - 、m/z270.93[M-H-C 12 H 20 O 9 -CH 2 O] - . The fragment information is consistent with rutin in the literature report, so that the compounds A-5 and M-3 are presumed to be rutin.
In the negative ion mode, the excimer ion peak of Compound M-1 is 461.16595[ M-H ]] - The retention time was 8.57min, the predicted molecular formula was C 20 H 30 O 12 The molecular formula is the same as that of forsythoside E, and the deviation from the theoretical molecular weight is 1.295ppm. The compound ion fragments include M/z315.10[ M-H-C ] 6 H 10 O 4 ] - 、m/z205.01[M-H-C 8 H 10 O 3 -C 4 H 6 O 3 ] - 、m/z162.79[M-H-C 6 H 10 O 4 -C 8 H 10 O 3 ] - 、m/z134.95[M-H-C 6 H 10 O 4 -C 9 H 8 O 4 ] - . The fragment information is consistent with that of forsythoside E in the literature report, so that the compound M-1 is presumed to be forsythoside E.
In negative ion mode, the excimer ion peak of compound M-5 is 595.16589[ M-H ]] - Retention time was 16.52min, supposedly molecular formula C 27 H 32 O 15 The molecular formula is the same as that of eriodictyol and the deviation from the theoretical molecular weight is 0.241ppm. The compound ion fragments include M/z459.05[ M-H-C ] 8 H 8 O 2 ] - 、m/z 287.07[M-H-C 12 H 20 O 9 ] - . The fragment information was consistent with that reported in the literature for eriodictyol, so that it was presumed that compound M-5 was eriodictyol.
In negative ion mode, the excimer ion peak of compound M-6 is 595.16620[ M-H ]] - The retention time was 15.41min, and the molecular formula was C 27 H 32 O 15 The molecular formula is the same as that of the compound M-5, and the deviation of the measured molecular weight from the theoretical molecular weight is 0.762ppm. The compound ion fragment includes m/z 287.06[ M-H-C ] 12 H 20 O 9 ] - . The fragment information was consistent with that reported in the literature for eriocitrin, so that it was presumed that compound M-6 was eriocitrin and was the isomer with compound M-5.
In negative ion mode, the excimer ion peaks of compounds M-7 and M-8 were 609.18237[ M-H ], respectively] - 、609.18195[M-H] - The retention time was 21.14min and 22.57min, both of which were presumed to have molecular formula C 28 H 34 O 15 The molecular weight deviation from the theoretical molecular weight is 1.598ppm and 0.908ppm respectively. Wherein the M-7 ion fragment of the compound comprises M/z 300.99[ M-H-C 12 H 20 O 9 ] - The M-8 ion fragments of the compound include M/z489.22[ M-H-C ] 7 H 4 O 2 ] - 、m/z325.08[M-H-C 16 H 12 O 5 ] - 、m/z301.01[M-H-C 12 H 20 O 9 ] - . The fragment information is consistent with that of hesperidin and neohesperidin in the report of the literature, so that the compound M-7 is presumed to be hesperidin, and the compound M-8 is presumed to be neohesperidin.
In negative ion mode, the excimer ion peak of Compound M-9 is 593.18774[ M-H ]] - The retention time was 28.33min, and the molecular formula was C 28 H 34 O 14 The molecular formula is the same as that of poncirin, and the molecular weight is the same as that of theoretical molecular weightThe deviation of (2) was 2.121ppm. The compound ion fragments include M/z473.13[ M-H-C ] 8 H 8 O] - 、m/z285.04[M-H-C 12 H 20 O 9 ] - . The fragment information is consistent with that of poncirin reported in the literature, so that the compound M-9 is presumed to be poncirin.
The beneficial effects of the invention are as follows:
the invention takes an important target MIF with cough relieving efficacy as a research carrier, develops a MIF functionalized modified biosensor and innovates an integrated liquid chromatography-mass spectrometry technology, and provides a biological sensing integrated liquid chromatography-mass spectrometry method for screening candidate compounds by using target fishing drugs in a traditional Chinese medicine compound preparation.
Drawings
FIG. 1 (a) is a diagram showing the combination of children's anti-food retention and cough relieving oral liquid with MIF at different concentrations DS -V DS A signal change; (b) And (3) linearly fitting the combination of the children anti-food retention and cough relieving oral liquid with MIF at different concentrations.
FIG. 2 (a) shows the TIC diagram (positive ion source mode and negative ion source mode) of amygdalin eluate of the oral liquid for resolving food stagnation and relieving cough in children; (b) The pediatric oral liquid for resolving food stagnation and relieving cough is 5-O-methyl vitamin amiloride eluent TIC chart (positive ion source mode and negative ion source mode).
Fig. 3 is a diagram of 10 key quality attributes EIC.
Detailed Description
Example 1 interaction study of pediatric anti-food retention and cough relieving oral liquid and MIF based on biosensing technology
(1) Preparing a solution to be tested: taking commercial children's oral liquid for resolving food stagnation and relieving cough as mother liquor, diluting the mother liquor to 1.0pg/mL according to ten times gradient, and preparing 11 gradient concentration sample solutions of 5-O-methylvitamin amiloride standard substance;
(2) Blank solution influence assay: preparing MIF-AlGaAs/GaAs HEMT biosensor, using 0.1M PBS solution as blank solution, and recording current between source electrode and drain electrode by electrochemical deviceIntensity (I) DS -V DS ) Taking the signal as a blank signal;
(3) Sequentially adding children's oral liquid for removing food retention and relieving cough into MIF-AlGaAs/GaAs HEMT device from low concentration to high concentration (1.0 pg/mL-1.0 g/mL), and recording I under different concentrations by adopting an electrochemical device DS -V DS As shown in FIG. 1 (a), it can be seen that when MIF protein is modified in self-assembled monolayer, I DS -V DS The curve of the modified PBS is similar to that of the modified MIF protein, which shows that the biosensor is successfully constructed;
(4) Logarithmic (Lg [ Ag ] of children's oral liquid concentration for relieving food retention and cough]) On the abscissa, in terms of the relative value of the current change (I-I 0 )/I 0 Performing linear fitting on the ordinate, and judging the linear range of the biosensor; as shown in FIG. 1 (b), it is clear that the concentration of the oral liquid is 1.0 pg/mL-0.1. Mu.g/mL, and the oral liquid for resolving food stagnation and relieving cough for children
(5) According to the linear range determined in the step (4), the concentration of the oral liquid for removing food retention and relieving cough of children ([ Ag)]) On the abscissa, in concentration ([ Ag)]) Current variation (I-I) 0 Δi) is taken as an ordinate, linear fitting is carried out, and the dissociation constant Kd of interaction between the children's anti-food retention and cough relieving oral liquid and MIF is 3.981 multiplied by 10 calculated according to the formula -10 g/mL。
Example 2 UPLC-MS/MS-based pediatric food retention removing and cough relieving oral liquid cough relieving key quality attribute identification
(1) Apparatus and method
A DIONEX Ultimate 3000 ultra high performance liquid chromatography analysis system (Thermo Fisher, inc. of U.S.A.); LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Co., USA) equipped with electrospray ion source (ESI) and Xcalibur 2.1 workstation; grace PureTM SPE C18-Low solid phase extraction column (500 mg/3 mL); milli-Q Synthesis ultra-pure water purification System (Millipore Co., U.S.A.); R200D type electronic analytical balance (1/10 ten thousand) (Sartorius Corp., germany); formic acid (chromatographic purity, merck, germany); methanol, acetonitrile (mass spectro-pure, thermo Fisher company, usa).
(2) Pretreatment of protein eluent sample
Taking protein eluent as a research carrier, adopting a solid phase extraction technology, and adopting 5mL methanol to flush a solid phase extraction small column with the specification of 1mL for activating the column; loading, namely eluting by adopting 5mL of methanol at a flow rate of 1mL/min, and removing PBS in a sample; adding 5mL of water, eluting at a flow rate of 1mL/min, volatilizing the sample by adopting a centrifugal concentrator, and adding 0.5mL of initial mobile phase, namely 0.1% acetonitrile- (0.1% formic acid), to re-dissolve for later use;
(3) UPLC-MS/MS technology-based protein eluent component identification analysis
Liquid phase conditions: ACQUITY UPLC HSS T3 chromatography column (2.1 mm. Times.100 mm,1.8 μm); mobile phase was 0.1% formic acid-water solution (a) -acetonitrile (B); column temperature 25 ℃; gradient elution was performed as in table 1; flow rate 0.30 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the The sample injection amount was 3. Mu.L.
TABLE 1 gradient elution table
Mass spectrometry conditions: a chromatographic column electrospray ion source (ESI) positive and negative ion modes; sheath air flow rate: 40arb; auxiliary gas flow rate: 20arb; capillary voltage: -35V; spray voltage: 3kV; tube lens voltage: -110V; capillary temperature: 350 ℃; fourier high resolution scan range m/z 100-1200; first order resolution 30000; the secondary mass spectrum adopts data dependency scanning, and 3 ions with highest primary abundance are selected for CID secondary fragmentation; an activation energy unit of 0.25q; the activation time is 30ms; normalized collision energy 35%. When the secondary fragment information is incomplete, the acquisition efficiency of the secondary mass spectrum information is improved through an ion list scanning mode. Mass spectrometry conditions: electrospray ion source (ESI); a negative ion mode; sheath gas flow rate 30arb; an auxiliary air flow rate of 10arb; capillary voltage-35V; spraying voltage is 3kV; tube lens voltage-110V; the capillary temperature is 350 ℃; fourier high resolution scan range m/z 50-800;
total ion flow diagram of the original liquid of the oral liquid for resolving food stagnation and relieving cough for children in positive and negative ion modes, and total ion flow diagram of the two eluents (5-O-methylvisamiloride and amygdalin) in positive and negative ion modes are shown in figure 2. The Xcalibur 2.1 workstation is adopted for data processing, the molecular formula prediction module is adopted for predicting the molecular formulas of all parent ions and fragment ions, and the related parameters are set as follows: c0-20, H0-30, O0-15, N0-3, S0-1, cyclic and unsaturated bond number 0-15, and quality accuracy error within 10. The total identification analysis shows that 10 key quality attributes (figure 2) comprise rutin, forsythoside E, eriocitrin, new eriocitrin, hesperidin, neohesperidin, poncirin, vitexin glucoside, lonicera glycoside and kaempferol-3-O-rutinoside, wherein 7 are compared by standard substances, and the specific cleavage rules are as follows:
as can be seen from the high-resolution mass spectrum data, in the negative ion mode, the excimer ion peaks of the compounds A-5 and M-3 were 609.14563[ M-H ], respectively] - 、609.14612[M-H] - The retention time was 16.03min and 16.04min, both of which were presumed to be C 27 H 30 O 16 The molecular formula is the same as that of rutin, and the deviation from the theoretical value is 1.016ppm and 1.820ppm respectively. Both ion fragments include M/z447.21[ M-H-C 6 H 10 O 5 ] - 、m/z300.96[M-H-C 12 H 20 O 9 ] - 、m/z270.93[M-H-C 12 H 20 O 9 -CH 2 O] - . The fragment information is consistent with rutin in the literature report, so that the compounds A-5 and M-3 are presumed to be rutin.
In the negative ion mode, the excimer ion peak of Compound M-1 is 461.16595[ M-H ]] - The retention time was 8.57min, the predicted molecular formula was C 20 H 30 O 12 The molecular formula is the same as that of forsythoside E, and the deviation from the theoretical molecular weight is 1.295ppm. The compound ion fragments include M/z315.10[ M-H-C ] 6 H 10 O 4 ] - 、m/z205.01[M-H-C 8 H 10 O 3 -C 4 H 6 O 3 ] - 、m/z162.79[M-H-C 6 H 10 O 4 -C 8 H 10 O 3 ] - 、m/z134.95[M-H-C 6 H 10 O 4 -C 9 H 8 O 4 ] - . The fragment information is consistent with forsythoside E in literature report, so that it is pushedThe test compound M-1 is forsythoside E.
In negative ion mode, the excimer ion peak of compound M-5 is 595.16589[ M-H ]] - Retention time was 16.52min, supposedly molecular formula C 27 H 32 O 15 The molecular formula is the same as that of eriodictyol and the deviation from the theoretical molecular weight is 0.241ppm. The compound ion fragments include M/z459.05[ M-H-C ] 8 H 8 O 2 ] - 、m/z 287.07[M-H-C 12 H 20 O 9 ] - . The fragment information was consistent with that reported in the literature for eriodictyol, so that it was presumed that compound M-5 was eriodictyol.
In negative ion mode, the excimer ion peak of compound M-6 is 595.16620[ M-H ]] - The retention time was 15.41min, and the molecular formula was C 27 H 32 O 15 The molecular formula is the same as that of the compound M-5, and the deviation of the measured molecular weight from the theoretical molecular weight is 0.762ppm. The compound ion fragment includes m/z 287.06[ M-H-C ] 12 H 20 O 9 ] - . The fragment information was consistent with that reported in the literature for eriocitrin, so that it was presumed that compound M-6 was eriocitrin and was the isomer with compound M-5.
In negative ion mode, the excimer ion peaks of compounds M-7 and M-8 were 609.18237[ M-H ], respectively] - 、609.18195[M-H] - The retention time was 21.14min and 22.57min, both of which were presumed to have molecular formula C 28 H 34 O 15 The molecular weight deviation from the theoretical molecular weight is 1.598ppm and 0.908ppm respectively. Wherein the M-7 ion fragment of the compound comprises M/z 300.99[ M-H-C 12 H 20 O 9 ] - The M-8 ion fragments of the compound include M/z489.22[ M-H-C ] 7 H 4 O 2 ] - 、m/z325.08[M-H-C 16 H 12 O 5 ] - 、m/z301.01[M-H-C 12 H 20 O 9 ] - . The fragment information is consistent with that of hesperidin and neohesperidin in the report of the literature, so that the compound M-7 is presumed to be hesperidin, and the compound M-8 is presumed to be neohesperidin.
In the negative ion mouldThe excimer ion peak of the compound M-9 is 593.18774[ M-H ]] - The retention time was 28.33min, and the molecular formula was C 28 H 34 O 14 The molecular formula is the same as that of poncirin, and the deviation from the theoretical molecular weight is 2.121ppm. The compound ion fragments include M/z473.13[ M-H-C ] 8 H 8 O] - 、m/z285.04[M-H-C 12 H 20 O 9 ] - . The fragment information is consistent with that of poncirin reported in the literature, so that the compound M-9 is presumed to be poncirin.
Claims (2)
1. The application of the biological sensing integrated ultra-high performance liquid chromatography-mass spectrometry technology in identifying key quality attributes of the children's anti-food retention and cough relieving oral liquid is characterized by comprising the following specific steps:
step 1: taking target protein MIF of anti-inflammatory channel of infantile anti-food retention and cough relieving oral liquid as a research carrier to construct an MIF-HEMT biosensor;
step 2: based on the MIF-HEMT biosensor in step 1, combined with an electrochemical workstation, through I DS -V DS The interaction between the children's anti-food retention and cough relieving oral liquid and the MIF-HEMT biosensor is measured;
step 3: eluting the sample combined on the MIF-HEMT biological sensing device for 3 times by adopting phosphate buffer solution and MIF specific eluents of 5-O-ganylvistin and amygdalin;
step 4: and (3) washing out phosphate in the eluent by adopting a solid phase extraction technology, respectively enriching the eluent in the step (3), and separating and identifying substances combined with the MIF-HEMT biosensing sensitive element in the sample to be detected by adopting an ultra-high performance liquid chromatography-mass spectrometry technology, wherein the specific instrument parameters are as follows:
ultra-high performance liquid phase conditions: chromatographic column: ACQUITY UPLC HSS column, 150mm×2.1mm,1.7 μm; column temperature: 35 ℃; mobile phase: 0.1% formic acid water A-acetonitrile B,
mass spectrometry conditions: a chromatographic column electrospray ion source (ESI) positive and negative ion modes; sheath air flow rate: 40arb; auxiliary gas flow rate: 20arb; capillary voltage: -35V; spray voltage: 3kV; tube lens voltage: -110V; capillary temperature: 350 ℃; fourier high resolution scan range m/z 100-1200; first order resolution 30000; the secondary mass spectrum adopts data dependency scanning, 3 ions with highest primary abundance are selected to carry out CID secondary fragmentation, and when the secondary fragmentation information is incomplete, the acquisition efficiency of the secondary mass spectrum information is improved through an ion list scanning mode;
step 5: comparing the substances detected in the step 4 with all components of the children's anti-food retention and anti-cough oral liquid, and screening the key quality attributes of the children's anti-food retention and anti-cough oral liquid, wherein the key quality attributes of the children's anti-food retention and anti-cough oral liquid are rutin, forsythoside E, eriocitrin, neonorth american eriocitrin, hesperidin, neohesperidin, poncirin, vitexin glucoside, lonicera japonica glycoside and kaempferol-3-O-rutin.
2. The use according to claim 1, wherein in step 2 the electrochemical workstation is che-660 e.
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