CN115078569A - Cough-relieving key mass attribute identification method based on biosensing integrated UPLC-MS technology - Google Patents
Cough-relieving key mass attribute identification method based on biosensing integrated UPLC-MS technology Download PDFInfo
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- CN115078569A CN115078569A CN202210581117.8A CN202210581117A CN115078569A CN 115078569 A CN115078569 A CN 115078569A CN 202210581117 A CN202210581117 A CN 202210581117A CN 115078569 A CN115078569 A CN 115078569A
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Abstract
The invention provides a method for identifying key quality attributes of traditional Chinese medicines by integrating a UPLC-MS technology with a biosensor chip and application of the method in traditional Chinese medicine compounds such as children's oral liquid for removing food retention and relieving cough. The method specifically comprises the following steps: (1) constructing a biosensor by taking target protein as a research carrier; (2) based on the biosensor in the step 1, screening the interaction strength of the sample to be tested and the key protein by adopting an electrochemical workstation; (3) eluting the sample bound on the biosensor by using a nonspecific eluent and a specific eluent of the target protein; (4) enriching the eluent in the step 3, and identifying substances combined with the biosensing sensitive element in the sample to be detected by adopting a liquid chromatography-mass spectrometry technology; (5) and (4) comparing the substance detected in the step (4) with the whole components of the substance to be detected, and screening the key quality attributes of the substance. The invention forms a set of Chinese medicine key quality attribute identification method based on biosensing 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 biosensing integrated UPLC-MS technology and application thereof in identification of key quality attributes of cough relieving oral liquid for removing food stagnation and relieving cough in children.
Background
The technical requirement for registration of drugs for human use international committee for drug development guidelines (ICHQ8) states clearly that the Critical Quality Attributes (CQAs) of drugs refer to physical, chemical, biological or microbiological properties or characteristics that ensure the desired product quality within appropriate limits, ranges or distributions. The key quality attribute of efficacy guided by clinical curative effect is the prerequisite basis for evaluating and controlling the quality of Chinese medicine and its preparation. However, the industry pain problem of the quality evaluation and process control index and the lack of clinical curative effect in the traditional Chinese medicine industry generally exists at present.
Based on the characteristic of complex traditional Chinese medicine system, when the traditional detection technology is adopted to carry out identification research on key quality attributes of efficacy, the method also has the defects of high detection limit, complex separation means, more interference factors, relatively complex analysis, incapability of being directly combined with clinical efficacy and the like. Efficacy-oriented biosensors, as the leading-edge analytical technique today, provide a key technical support for the identification studies of efficacy-associated key mass attributes through a biological recognition element in direct contact with the transducer. The AlGaAs/GaAs High Electron Mobility Transistor (HEMT) biosensor prepared based on the semiconductor material has high sensitivity, higher detection speed, easy integration and batch production, outstanding biocompatibility, capability of performing biological functional modification, wide application in the field of molecular recognition and capability of reaching the pg level of detection limit. For substances obtained by screening, ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has better quantification and identification capabilities due to high-efficiency chromatographic separation performance and shorter analysis time, and provides important technical support for separation and identification of chemical components obtained by biosensing screening.
Disclosure of Invention
The invention aims to provide a cough relieving key mass attribute identification method of a biosensing integrated UPLC-MS technology.
The invention also aims to provide application of the key quality attribute identification method of the biosensing integrated UPLC-MS technology in identification of the key quality attribute of the children's food retention removal and cough relieving oral liquid cough relieving.
In order to overcome the defects of the prior art, the invention firstly provides a method for identifying key quality attributes of potential tastes, which comprises the following specific steps:
step 1: constructing a biosensor by taking target protein as a research carrier;
step 2: based on the biosensor in the step 1, screening the interaction strength of the sample to be tested and the key protein by adopting an electrochemical workstation;
and step 3: eluting the sample combined on the biosensor by adopting nonspecific eluent and specific eluent of the target protein;
and 4, step 4: enriching the eluent in the step 3, and identifying substances combined with the biosensing sensitive element in the sample to be detected by adopting an ultra-high performance liquid chromatography-mass spectrometry combined technology;
and 5: and (4) comparing the substance detected in the step (4) with the whole components of the substance to be detected, and screening the key quality attributes of the substance.
According to some specific embodiments of the present invention, a key quality attribute identification method of a biosensing integrated UPLC-MS technology relates to a key target protein MIF modified HEMT biosensor, 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 mixture of 20mM of carboxyl activator carbonyldiimine hydrochloride and 50mM of N-hydroxysuccinimide in equal volume into a sample cell to generate a stable amine activated 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 embodiments of the present invention, a method for identifying key mass attributes of a biosensor integrated UPLC-MS technology involves using a constructed biosensor for detection and study of interaction strength between traditional Chinese medicine and key protein, wherein an electrochemical workstation is CHI-660e, a constant voltage is given as 2V, and a current precision is μ a level.
According to some embodiments of the present invention, the nonspecific eluent used in step 3 is PBS, and the specific eluent is 5-O-Gauyvermisol glycoside and amygdalin, each eluting 3 times, which strongly interact with MIF and have antitussive effect.
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 biosensing integrated UPLC-MS technology in the research of the key quality attribute of cough relieving of the oral liquid for removing food retention and relieving cough for children, 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 mixture of 20mM of carboxyl activator carbonyldiimine hydrochloride and 50mM of N-hydroxysuccinimide in equal volume into a sample cell to generate a stable amine activated product for activating carboxyl;
(3) washing the HEMT device by Phosphate Buffer Solution (PBS), adding target protein, and reacting for 2 hours at 4 ℃ to obtain the MIF modified HEMT device biosensor; determination of I before and after modification of MIF protein by electrochemical workstation DS -V DS The results are shown in FIG. 1(a), which shows that I is after MIF modification DS -V DS The curve is changed remarkably, which shows that the MIF is successfully modified on the HEMT device;
(4) based on the MIF-HEMT biosensor in the step (1), the I of the infantile indigestion removing and cough relieving oral liquid and the MIF with different concentrations are measured by adopting an electrochemical workstation CHI-660e DS -V DS Calculating the interaction strength between the infantile oral liquid for removing food retention and relieving cough and the MIF-biosensor according to the change curve, and calculating the Kd to be 3.981 multiplied by 10 -10 g/mL；
(5) Eluting the sample combined on the MIF-HEMT biosensor by using PBS as a non-specific eluent for 3 times, reacting for 5 minutes each time, and combining the eluates; further, 5-O-methylvisammioside is adopted for elution, 3 times of reaction are carried out for 5 minutes each time, and the eluates are merged; finally, amygdalin is adopted for elution for 3 times, each time reaction is carried out for 5 minutes, and the eluates are merged.
(6) Washing off phosphate in 3 kinds of eluents by adopting a solid phase extraction technology, respectively enriching the eluents in the step (5), 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 combined technology, wherein the specific instrument parameters are as follows:
ultra-high performance liquid phase conditions: a chromatographic column: ACQUITY UPLC HSS column (150 mm. times.2.1 mm,1.7 μm); column temperature: 35 ℃; mobile phase: 0.1% formic acid (a) -acetonitrile (B).
Gradient elution chart
Mass spectrum conditions: chromatographic column electrospray ion source (ESI) positive and negative ion modes; flow rate of sheath gas: 40 arb; flow rate of auxiliary gas: 20 arb; capillary voltage: -35V; spraying voltage: 3 kV; tube lens voltage: -110V; capillary temperature: 350 ℃; fourier high resolution scanning range m/z 100-; primary resolution 30000; the second-order mass spectrum adopts data-dependent scanning, and 3 ions with the highest first-order abundance are selected for CID second-order fragmentation. When the secondary fragment information is incomplete, the acquisition efficiency of the secondary mass spectrum information is improved in an ion list scanning mode.
The total ion flow diagram under the positive ion source mode and the negative ion source mode is shown in figure 3. Through the analysis of the first-level fragment and the second-level fragment of the mass spectrum information, 10 key cough-relieving quality attributes of the children's food retention removing and cough stopping oral liquid are obtained through analysis, and the method specifically comprises the following steps: rutin, forsythoside E, eriocitrin, neoeriocitrin, hesperidin, neohesperidin, poncirin, vitexin glucoside, loniceraside, and kaempferol-3-O-rutinoside. 7 of the samples were analyzed by standard comparison, and the specific cleavage rules were as follows:
according to high-resolution mass spectrum data, the excimer ion peaks of the compounds A-5 and M-3 are 609.14563[ M-H ] respectively in the negative ion mode] - 、609.14612[M-H] - The retention time was 16.03min and 16.04min, and both molecular formulas were presumed to be C 27 H 30 O 16 The molecular formula of the rutin compound is the same as that of rutin, and the deviation from the theoretical value is 1.016ppm and 1.820ppm respectively. Both ion fragments comprise 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 the compound M-1 is 461.16595[ M-H] - Retention time 8.57min, molecular formula C 20 H 30 O 12 The molecular formula of the forsythoside E is the same as that of forsythoside E, and the deviation from the theoretical molecular weight is 1.295 ppm. The ionic fragment of the compound comprises 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 reports, so that the compound M-1 is presumed to be the forsythoside E.
In the negative ion mode, the excimer ion peak of the compound M-5 is 595.16589[ M-H] - The retention time was 16.52min, and the molecular formula was presumed to be C 27 H 32 O 15 The molecular formula of eriocitrin is the same as that of new North American eriocitrin, and the deviation from the theoretical molecular weight is 0.241 ppm. The ionic fragment of the compound comprises 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 is consistent with the new eriocitrin in the literature report, so the compound M-5 is presumed to be the new eriocitrin.
In the negative ion mode, the excimer ion peak of the compound M-6 is 595.16620[ M-H] - Retention time 15.41min, molecular formula C 27 H 32 O 15 The molecular formula was the same as that of compound M-5, and the deviation of the measured molecular weight from the theoretical molecular weight was 0.762 ppm. The ionic fragment of the compound comprises M/z 287.06[ M-H-C [) 12 H 20 O 9 ] - . The fragment information is consistent with the eriocitrin reported in the literature, so that the compound M-6 is presumed to be eriocitrin and to be an isomer with the compound M-5.
Under the negative ion mode, the peaks of the quasi-molecular ions of the compounds M-7 and M-8 are 609.18237[ M-H ] respectively] - 、609.18195[M-H] - The retention time is 21.14min and 22.57min, and the molecular formulas of both are presumed to be C 28 H 34 O 15 Belong to isomers and have a deviation from the theoretical molecular weight of 1.598ppm and 0.908ppm, respectively. Wherein the ionic fragment of compound M-7 comprises M/z 300.99[ M-H-C 12 H 20 O 9 ] - The ionic fragment of compound M-8 comprises 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 respectively consistent with hesperidin and neohesperidin in literature reports, 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 mode, the excimer ion peak of the compound M-9 is 593.18774[ M-H] - Retention time 28.33min, molecular formula C 28 H 34 O 14 The molecular formula of the compound is the same as that of poncirin, and the deviation from the theoretical molecular weight is 2.121 ppm. The ionic fragment of the compound comprises 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 invention has the beneficial effects that:
the invention takes an important target point MIF of cough-relieving efficacy as a research carrier, develops an MIF functional modified biosensor, develops an innovative integrated liquid chromatography-mass spectrometry technology, and provides a biosensing integrated liquid chromatography-mass spectrometry method for screening candidate compounds of a traditional Chinese medicine compound 'target fishing medicine', wherein the method is applied to a traditional Chinese medicine compound preparation children food retention removing and cough relieving oral liquid, and 10 key quality attributes with potential cough-relieving effects are screened out, and the invention provides method guidance for the quality evaluation and control of the traditional Chinese medicine compound with efficacy as the guide.
Drawings
FIG. 1(a) combination of different concentrations of XIAO' ERXIAOJI ZHIKE oral liquid and MIF DS -V DS A change in signal; (b) the linear fitting result of the combination of the pediatric food-retention-elimination cough-relieving oral liquid with different concentrations and the MIF.
FIG. 2(a) TIC chart of amygdalin eluent (positive ion source mode and negative ion source mode) for XIAO ER XIAO JI ZHI KE ORAL LIQUID; (b) 5-O-methylvisamicoside eluent TIC graph (positive ion source mode and negative ion source mode) of the pediatric food stagnation removing and cough relieving oral liquid.
The diagram 310 is an EIC diagram of key quality attributes.
Detailed Description
Example 1 interaction study of pediatric food retention removing and cough relieving oral liquid and MIF (micro-emulsion aggregation) based on biosensing technology
(1) Preparing a solution to be detected: taking a commercial oral liquid for removing food retention and relieving cough of children as a mother solution, diluting the mother solution to 1.0pg/mL according to a ten-fold gradient, and preparing 11 gradient concentration sample solutions of 5-O-methylvisammioside standard substance;
(2) influence measurement of blank solution: preparing MIF-AlGaAs/GaAs HEMT biosensor, using 0.1M PBS solution as blank solution, and recording current intensity (I) between source and drain by electrochemical device DS -V DS ) Taking the signal as a blank signal;
(3) sequentially adding infantile food retention-eliminating cough-relieving oral liquid to MIF-AlGaAs/GaAs HEMT device according to concentration from low to high (1.0pg/mL-1.0g/mL), and recording I under different concentrations by electrochemical device DS -V DS The results of each response are shown in FIG. 1(a), which shows that when the MIF protein is modified in the self-assembled monolayer, I DS -V DS The curve of the modified PBS is similar to that of the modified MIF protein after the PBS is modified, which indicates that the construction of the biosensor is successful;
(4) the logarithm of the concentration of the oral liquid (Lg [ Ag ]) for removing food retention and relieving cough of children]) As abscissa, with relative value of current change (I-I) 0 )/I 0 Linear fitting is carried out for the ordinate, and the linear range of the biosensor is judged; as shown in FIG. 1(b), it can be seen that the concentrations of the oral liquid and the oral liquid for eliminating infantile food stagnation and relieving cough were in the range of 1.0pg/mL to 0.1. mu.g/mL
(5) According to the linear range determined in (4), the concentration ([ Ag ] of the pediatric food retention eliminating and cough relieving oral liquid is used]) As abscissa, in terms of concentration ([ Ag ]]) Change in Current (I-I) 0 Delta I) is the ordinate, linear fitting is carried out, and the dissociation constant Kd of the interaction between the pediatric food retention removing and cough relieving oral liquid and the MIF is calculated to be 3.981 multiplied by 10 according to the formula -10 g/mL。
Example 2 UPLC-MS/MS-based identification of key quality attributes of cough-relieving oral liquid for removing food retention and relieving cough in children
(1) Apparatus and method
DIONEX Ultimate 3000 ultra high performance liquid chromatography system (Thermo Fisher, USA); an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher, USA) equipped with an electrospray ion source (ESI) and an Xcalibur 2.1 workstation; grace Pure SPE C18-LoW solid phase extraction cartridge (500mg/3 mL); Milli-Q Synthesis ultrapure water purification System (Millipore, USA); model R200D electronic analytical balance (1/10 ten thousand) (Sartorius, germany); formic acid (chromatographically pure, Merck, germany); methanol, acetonitrile (mass spectrometric purity, Thermo Fisher, usa).
(2) Pretreatment of protein eluent sample
Taking protein eluent as a research carrier, adopting a solid phase extraction technology, washing a solid phase extraction small column with the specification of 1mL by adopting 5mL of methanol, and activating the column; sampling, namely eluting by using 5mL of methanol at the 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 using a centrifugal concentrator, and adding 0.5mL of an initial mobile phase, namely 0.1% acetonitrile- (0.1% formic acid), for redissolving for later use;
(3) protein eluent composition identification and analysis based on UPLC-MS/MS technology
Liquid phase conditions: an ACQUITY UPLC HSS T3 column (2.1 mm. times.100 mm, 1.8 μm); the mobile phase was 0.1% formic acid-water solution (a) -acetonitrile (B); the column temperature is 25 ℃; gradient elution was performed as in table 1; flow rate 0.30 mL/min -1 (ii) a The sample size was 3. mu.L.
TABLE 1 gradient elution Table
Mass spectrum conditions: chromatographic column electrospray ion source (ESI) positive and negative ion modes; flow rate of sheath gas: 40 arb; flow rate of auxiliary gas: 20 arb; capillary voltage: -35V; spraying voltage: 3 kV; tube lens voltage: -110V; capillary temperature: 350 ℃; the Fourier high resolution scanning range m/z 100-; primary resolution 30000; the second-order mass spectrum adopts data dependency scanning, and 3 ions with the highest first-order abundance are selected for CID second-order fragmentation; activation energy unit 0.25 q; the activation time is 30 ms; the normalized collision energy is 35%. And when the secondary fragment information is incomplete, the acquisition efficiency of the secondary mass spectrum information is improved in an ion list scanning mode. Mass spectrum conditions: electrospray ion source (ESI); a negative ion mode; sheath gas flow rate 30 arb; an auxiliary airflow rate 10 arb; capillary voltage of-35V; the spraying voltage is 3 kV; the tube lens voltage is-110V; the capillary temperature is 350 ℃; the Fourier high resolution scanning range m/z is 50-800;
the total ion flow diagram of the stock solution of the infantile food retention-eliminating cough-relieving oral liquid in the positive and negative ion mode is shown in figure 2, and the total ion flow diagram of two eluents (5-O-methylvisammol glycoside and amygdalin) in the positive and negative ion mode is shown in figure 2. The Xcaliibur 2.1 workstation is adopted for data processing, a molecular formula prediction module is adopted for predicting the molecular formulas of all the parent ions and the fragment ions, and relevant parameters are set as follows: c0-20, H0-30, O0-15, N0-3, S0-1, ring and unsaturated bond number 0-15, and mass precision error is less than 10. 10 key quality attributes (figure 2) are identified and analyzed, specifically including rutin, forsythoside E, eriocitrin, neoeriocitrin, hesperidin, neohesperidin, poncirin, vitexin glucoside, loniceraside, kaempferol-3-O-rutinoside, and 7 of the above are compared by standard substances, and the specific cracking rule is as follows:
as can be seen from the high-resolution mass spectrometry data, the peaks of the excimer ions of the compounds A-5 and M-3 are 609.14563[ M-H ] respectively in the negative ion mode] - 、609.14612[M-H] - The retention time was 16.03min and 16.04min, and both molecular formulas were presumed to be C 27 H 30 O 16 The molecular formula of the rutin compound is the same as that of rutin, and the deviation from the theoretical value is 1.016ppm and 1.820ppm respectively. Both ion fragments comprise 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 the compound M-1 is 461.16595[ M-H] - Retention time 8.57min, molecular formula C 20 H 30 O 12 The molecular formula of the forsythoside E is the same as that of forsythoside E, and the deviation from the theoretical molecular weight is 1.295 ppm. The compound is isolated fromThe subfragments 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 reported in literature, so that the compound M-1 is presumed to be forsythoside E.
In the negative ion mode, the excimer ion peak of the compound M-5 is 595.16589[ M-H] - The retention time was 16.52min, and the molecular formula was assumed to be C 27 H 32 O 15 The molecular formula of eriocitrin is the same as that of new North American eriocitrin, and the deviation from the theoretical molecular weight is 0.241 ppm. The ionic fragment of the compound comprises 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 is consistent with that of new eriocitrin in the literature report, so that the compound M-5 is presumed to be new eriocitrin.
In the negative ion mode, the excimer ion peak of the compound M-6 is 595.16620[ M-H] - Retention time 15.41min, molecular formula C 27 H 32 O 15 The molecular formula was the same as that of compound M-5, and the deviation of the measured molecular weight from the theoretical molecular weight was 0.762 ppm. The ionic fragment of the compound comprises M/z 287.06[ M-H-C [) 12 H 20 O 9 ] - . The fragment information is consistent with the eriocitrin reported in the literature, so that the compound M-6 is presumed to be eriocitrin and to be an isomer with the compound M-5.
Under the negative ion mode, the peaks of the quasi-molecular ions of the compounds M-7 and M-8 are 609.18237[ M-H ] respectively] - 、609.18195[M-H] - The retention times were 21.14min and 22.57min, and the molecular formulas of both are presumed to be C 28 H 34 O 15 Belong to isomers and have a deviation from the theoretical molecular weight of 1.598ppm and 0.908ppm, respectively. Wherein the ionic fragment of compound M-7 comprises M/z 300.99[ M-H-C 12 H 20 O 9 ] - The ionic fragment of compound M-8 comprises 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 respectively consistent with hesperidin and neohesperidin in literature reports, 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 mode, the excimer ion peak of the compound M-9 is 593.18774[ M-H] - Retention time 28.33min, molecular formula C 28 H 34 O 14 The molecular formula of the compound is the same as that of poncirin, and the deviation from the theoretical molecular weight is 2.121 ppm. The ionic fragment of the compound comprises 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 (10)
1. A key mass attribute identification method of a biosensor integrated ultra-high performance liquid chromatography-mass spectrometry technology is characterized by comprising the following specific steps:
step 1: constructing a target protein functionalized modified biosensor by taking a target protein as a research carrier;
step 2: based on the biosensor in the step 1, screening the interaction strength of the sample to be tested and the key protein by adopting an electrochemical workstation;
and step 3: eluting the sample combined on the biosensor by adopting nonspecific eluent and specific eluent of the target protein;
and 4, step 4: enriching the eluent in the step 3, and identifying substances combined with the biosensing sensitive element in the sample to be detected by adopting an ultra-high performance liquid chromatography-mass spectrometry combined technology;
and 5: and (4) comparing the substance detected in the step (4) with the whole components of the substance to be detected, and screening the key quality attributes of the substance.
2. The method of claim 1, wherein the protein functionally modified by the biosensor in step 1 of claim 1 is a target protein for treating diseases, and the biosensor includes but is not limited to surface plasmon resonance biosensor and high electron mobility field effect transistor biosensor.
3. The method of claim 1, wherein the electrochemical workstation of step 2 of claim 1 comprises current and voltage supply devices not limited to 2400, CHI660e, giving a constant voltage of 2-5V and current accuracy on the order of μ a.
4. The method for identifying key mass attributes of the integrated ultra-high performance liquid chromatography-mass spectrometry technology as claimed in claim 1, wherein the non-specific eluent in step 3 of claim 1 comprises a buffer solution for protein dissolution, the specific eluent comprises a specific inhibitor and an agonist of the target protein, and the elution times are not less than 3.
5. An application of a key quality attribute identification method of a biosensor integrated ultra-high performance liquid chromatography-mass spectrometry combined technology in identification research of key quality attributes of traditional Chinese medicine decoction pieces, traditional Chinese medicine compound, Chinese patent medicines, health products and functional foods.
6. An application of a key quality attribute identification method of a biosensor integrated ultra-high performance liquid chromatography-mass spectrometry combined technology in the research of key quality attributes of an oral liquid for removing food retention and relieving cough for children is characterized by comprising the following specific steps:
step 1: constructing an MIF-HEMT biosensor by taking a target protein MIF of an anti-inflammatory pathway of the pediatric food retention removing and cough relieving oral liquid as a research carrier;
and 2, step: the MIF-HEMT biosensor based on the step 1 of claim 6, in combination with an electrochemical workstation, by I DS -V DS Detecting, determining infantile food retention removing and cough relieving oral liquid and MIF-biographyInteraction between sensors;
and 3, step 3: respectively eluting the samples combined on the MIF-HEMT biosensor for 3-6 times by adopting phosphate buffer solution and MIF specific eluent;
and 4, step 4: washing off 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 biosensor in the sample to be detected by adopting an ultra-high performance liquid chromatography-mass spectrometry combined technology;
and 5: comparing the substance detected in step 4 of claim 6 with the total components of the pediatric food-retention-eliminating and cough-relieving oral liquid, and screening key quality attributes.
7. The key quality attributes of the infantile food retention-eliminating cough-relieving oral liquid include, but are not limited to, synephrine, demethylarecoline, rutin, forsythiaside, eriocitrin, neoeriocitrin, hesperidin, neohesperidin, poncirin, vitexin glucoside (Vitexia-glucoside), lonicerin, kaempferol-3-O-rutinoside.
8. The key quality attribute of the children food retention removing and cough relieving oral liquid is applied to the preparation of anti-inflammatory and children food retention cough treating preparations.
9. The anti-inflammatory and infantile indigestion cough preparation according to claim 9, comprising the key quality attributes of the infantile indigestion cough-relieving oral liquid according to claim 7 and pharmaceutical excipients.
10. The preparation as claimed in claim 9, wherein the preparation is in the form of injection, tablet, capsule, aerosol, suppository, membrane, drop pill, ointment, controlled release agent, sustained release agent or nanometer preparation.
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