CN104897840B - A kind of method of polypeptide (oligopeptides) constituents quality control in SHUXUETONG ZHUSHEYE - Google Patents
A kind of method of polypeptide (oligopeptides) constituents quality control in SHUXUETONG ZHUSHEYE Download PDFInfo
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- CN104897840B CN104897840B CN201510337719.9A CN201510337719A CN104897840B CN 104897840 B CN104897840 B CN 104897840B CN 201510337719 A CN201510337719 A CN 201510337719A CN 104897840 B CN104897840 B CN 104897840B
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Abstract
The present invention relates to a kind of method of quality control of Chinese medicine preparation, specifically separated using a kind of liquid chromatogram (HPLC), mass spectrum (MS) detects, with reference to multiple-reaction monitoring (MRM), the Quality Control Model that the LC MS MRM separation, detection, statistics identification of SHUXUETONG ZHUSHEYE are combined is established, and the quality control of polypeptide (oligopeptides) constituents in SHUXUETONG ZHUSHEYE is carried out with this model.This method can fast and effeciently carry out Qualitative Identification to known activity polypeptide (oligopeptides) component in SHUXUETONG ZHUSHEYE, and method is simple, quick, specificity is strong, favorable reproducibility, be more beneficial for being lifted the quality control method of Shu Xue-tong note parenteral solution.
Description
Technical field
The invention belongs to Analysis of Chinese Traditional Medicine field, is related to a kind of method of quality control of Chinese medicine preparation, specifically, exactly adopts
Separated with a kind of liquid chromatogram (HPLC), mass spectrum (MS) detection, with reference to multiple-reaction monitoring (MRM), establish SHUXUETONG ZHUSHEYE
The Quality Control Model that LC-MS-MRM separation, detection, statistics identification are combined, and carried out with this model in SHUXUETONG ZHUSHEYE
The quality control of polypeptide (oligopeptides) constituents.
Background technology
Traditional Chinese medicine and its formulation chemist composition are various and complicated, exactly the diversity and complexity of its material base, altogether
With the wholistic therapy theory of traditional Chinese medicine and the material base of multiple target effect is explained, in recent years, material foundation of tcm is studied such as
The progress of the fire such as bitter edible plant, but the bottleneck broken through in terms of a small amount of, micro and trace materials research be present, it is generally the case that these things
Matter plays the part of very important role in traditional Chinese medicine prevents and treats disease, how to determine these in Chinese medicine and its preparation it is a small amount of,
Micro and trace materials, and these materials are subjected to quality control using simple, easy and science method, it is traditional Chinese medicine working
Personnel need the problem captured.
SHUXUETONG ZHUSHEYE is the kind new medicine of national Chinese medicine two of friendly medicine company independent research of fighting, by leech, earthworm extraction and essence
System forms, and for domestic first animal compound liquid drugs injection formulation kind, has the effect of promoting blood circulation and removing blood stasis, clearing and activating the channels and collaterals, is mainly used in
Acute period of cerebral infarction caused by obstruction of collaterals by blood stasis, it can effectively improve facial paralysis because cerebral ischemia is triggered, hemiplegia, language
The stuttering symptom such as puckery.The big constituents of SHUXUETONG ZHUSHEYE mainly contain endogenous small molecule base class, free amino acid, free list
Multiple big constituents such as sugar, micromolecule polypeptide (oligopeptides), oligosaccharides or glycopeptide, it is generally recognized that the micromolecule polypeptide in Shu Xue-tong is (few
Peptide) it is probably the active component that Shu Xue-tong works, but because content is little, using HPLC direct injecteds and conventional detector point
Analysis, it is difficult to obtain good signal response, the present SHUXUETONG ZHUSHEYE national drug standards are for wherein polypeptide component
Analysis using routine post before amino acid derivedization HPLC analytic approach indirect determinations, mainly hydrolyzed by determination sample
Total amino acid content deducts the free aminoacid content in sample afterwards, converts polypeptide constituent content therein, tests process
Complexity, it is cumbersome, if misoperation, it is understood that there may be the problem of interference and specificity be not strong, and it is (few to its real polypeptide
Peptide) class component is representational strong.
MS-MRM (multiple reaction monitoring) technology is based on Given information or assumes information setting
Mass Spectrometer Method rule, signal record is carried out to legal ion, remove a large amount of interference for not being inconsistent normally ion signal, from
And obtain a kind of data acquiring mode of Information in Mass Spectra.Specifically, it is according to polypeptide parent ion mass number and fragment ion
Mass number, select parent-daughter ion pair, it is allowed to which the parent ion for meeting setting enters collision cell, and after the completion of collision, only record is set
Stator ion signal, by the selection twice of parent ion and daughter ion, interfering ion is removed, reduces Chemical Background, is improved sensitive
Degree, MS-MRM analyses can obtain the quantitative data with enough statistical significances, for selected parent ion-ion pair, matter
Spectrometer can adjust corresponding peptide fragment fragmentation energies, effectively improve the mass spectrum response signal of fragment ion, improve peptide fragment detection
Sensitivity, and the analysis reached in a mass spectrum sample introduction is analyzed to hundreds of or even thousands of individual target peptide fragments can be realized, improve
Detection efficiency, using MS-M R M technologies specificity, the polypeptide for meeting setting is detected, and further carry out enhancing production
Thing scanning analysis, high-resolution tandem mass spectrum (MS/MS) crumb data is obtained, makes the qualitative results false positive rate during analysis
Substantially reduce.Based on aforementioned techniques means, the present invention intends selecting suitable sample-pretreating method, such as by means of molecular sieve column
Chromatography realizes the desalination of SHUXUETONG ZHUSHEYE, and can realize the enrichment to polypeptide (oligopeptides) class component interested, then passes through
HPLC further separates to polypeptide therein (oligopeptides) class component, by MRM technologies, by MS detectors to meeting setting rule
Target peptide fragment parent ion and daughter ion be scanned analysis, eliminate interfering ion, reduce Chemical Background, it is clever to improve analysis
Sensitivity, can be quickly to Shu Xue-tong so as to establish a kind of identification of means of polypeptide (oligopeptides) class component in SHUXUETONG ZHUSHEYE
Related polypeptide (oligopeptides) class component in parenteral solution carries out identification and analysis, so as to further realize to more in SHUXUETONG ZHUSHEYE
The qualitative and quality control of peptide (oligopeptides) class component.
The content of the invention
1st, goal of the invention
It is an object of the invention to provide a kind of liquid chromatogram (HPLC) separation, mass spectrum (MS) detection, with reference to more reaction prisons
Survey (MRM), establish the Quality Control Model that the LC-MS-MRM separation, detection, statistics identification of SHUXUETONG ZHUSHEYE are combined, and
The quality control of polypeptide (oligopeptides) constituents in SHUXUETONG ZHUSHEYE is carried out with this model.
2nd, concrete technical scheme of the invention
The detection method of polypeptide (oligopeptides) constituents, comprises the following steps in SHUXUETONG ZHUSHEYE：
(1) SHUXUETONG ZHUSHEYE need testing solution is subjected to desalting processing
Desalination can use column chromatography or dialysis treatment, preferred molecular sieve column chromatography or ion-exchange chromatography desalting processing,
Still more preferably molecular sieve column chromatography desalting processing.Molecular sieve column chromatography filler can be sephadex (sephadex G
Or sephacryl S) serial or polyacrylamide gel (BIO-Gel P) series, or Ago-Gel
(Sepharose) series, preferably sephadex series sephadex G10, sephadex G15 or sephadex G25, more
Further preferred sephadex G15 or sephadex G25 fillers, the preferred water of eluant, eluent.
(2) HPLC is separated
After the desalted sample solution high speed centrifugation of collection or 0.2 μm of filter membrane excessively, HPLC separation is carried out.It can use common
Liquid phase chromatogram condition separates, and can also carry out a nanoliter liquid phase chromatogram condition analysis, can also use ultra performance liquid chromatography condition point
Analysis.Further optimize common liquid phase chromatogram condition：Chromatographic column uses C18 as stationary phase, and mobile phase A is that 0.1% formic acid is water-soluble
Liquid, Mobile phase B are that acetonitrile or 0.1% formic acid acetonitrile solution, flow velocity and gradient condition can be adjusted suitably according to chromatographic column used.
When selection nanoliter liquid chromatogram, preferable flow rate is 0.4 μ l/min, preferred gradient elution requirement：0~6min, B be 5% →
8%；6~40min, B are 8% → 30%；40~45min, B are 30% → 60%；45~48min, B are 60% → 80%；48
~56min, B 80%；56~58min, B are 80% → 5%；58~65min, B 5%.When chromatographic column used in selection UHPLC
When, flow velocity preferably 0.1~0.3ml/min, condition of gradient elution is preferably：0~2min, B are 5% → 8%；2~13min, B are
8% → 30%；13~15min, B are 30% → 60%；15~16min, B are 60% → 80%；16~18min, B 80%；
18~19min, B are 80% → 5%；19~22min, B 5%.
(3) MS is analyzed
The peptides component separated by HPLC, sequentially enter mass detector analysis.Its optimize Mass Spectrometry Conditions be：EFI
Mist ion gun (ESI), atomization gas and dry gas are nitrogen.Further it is defined as gathering positive ion mode；One-level scans
Scope：350-2000Da；One-level scanning resolution：70000；Two level scanning range：It is automatic dependent on one-level m z
Selection；Two level collision energy (NCEs)：28%；Two level scanning resolution：17500；Capillary temperature：360℃；Ion gun electricity
Pressure：1800V；Fragmentation mode：HCD.
(4) MRM is monitored
Qualitative analysis is carried out according to the Information in Mass Spectra of target peptide fragment.By selecting multiple particular target peptide fragment components, enter one
Step preferably constitutes polypeptide (oligopeptides) constituents for determining molecular weight between 256-2560 dalton, and further optimization composition is true
Determine polypeptide (oligopeptides) constituents of molecular weight between 1000-2000 dalton, select the parent ion of peptide fragment and specific son
The monitoring of ion, parent ion and specific daughter ion all should have with database preferably match with signal response then think to identify into
Work(.
3rd, beneficial effects of the present invention
Be advantageous to, to SHUXUETONG ZHUSHEYE quality control, further ensure product safety using this method.Existing thin blood
Measure in logical parenteral solution quality standard on polypeptide, it is that sample itself free ammonia amino is deducted based on total amino acid after hydrolysis
Acid, the content of polypeptide component is obtained, hydrolysis stage needs elevated-temperature seal, and hydrolysis time is very long, it usually needs 24h, either
The measure of free amino acid or total amino acid is required for pre-column derivatization, cumbersome and unstable, to the table of polypeptide fractions
Sign also not enough determines.And with analysis method provided by the present invention, can be fast and effeciently to the known work in SHUXUETONG ZHUSHEYE
Property polypeptide (oligopeptides) component carry out Qualitative Identification, while the analysis method established of the present invention has method simple, quick, exclusive
Property strong, high repeatability and other advantages, be more beneficial for the quality control method of further lifting Shu Xue-tong note parenteral solution.
Brief description of the drawings
Fig. 1：Polypeptide YB-001 chooses specific daughter ion fragments chromatogram
Fig. 2：Polypeptide YB-001 chooses daughter ion fragments mass spectrogram and database matching collection of illustrative plates
Fig. 3：Polypeptide YB-002 chooses specific daughter ion fragments chromatogram
Fig. 4：Polypeptide YB-002 chooses daughter ion fragments mass spectrogram and database matching collection of illustrative plates
Fig. 5：Polypeptide YB-003 chooses specific daughter ion fragments chromatogram
Fig. 6：Polypeptide YB-003 chooses daughter ion fragments mass spectrogram and database matching collection of illustrative plates
Embodiment
For the feature of the present invention, technological means and the specific purposes reached, function can be further appreciated that, this hair is parsed
The advantages of bright and spirit, further illustrate the present invention, but be not construed as limiting the invention below by way of specific embodiment.
Embodiment：
Instrument：HPLC models：Eksigent 425nano LC；MS models：AB SCIEX6500；
Shu Xue-tong sample：Mudanjiang Youbo Pharmaceutical Co., Ltd. produces.
Sample treatment：SHUXUETONG ZHUSHEYE 1ml is taken, in careful loading to the good Sephadex G15 chromatographic columns of pre-balance
(chromatographic column specification：1.4cm*8cm), de- and Fractional Collections water elution is washed with deionized water, with 0.1M AgNO3Detection is to going out
Existing white opacity stops collecting, and merges the water elution collected before.As need testing solution, LC-MS-MRM analyses are carried out.
Chromatographiccondition：Make the scapus of nozzle needle one, 75 μ m 15cm by oneself, filler is：Venusil × BP, C18 (L), 5 μm,(Ai Jieer Agela Technologies companies), flow velocity are 0.4 μ l/min, and mobile phase A is 0.1% aqueous formic acid,
Mobile phase B is 0.1% formic acid acetonitrile solution, and condition of gradient elution is as follows：0~6min, B：5% → 8%；6~40min, B：8%
→ 30%；40~45min, B：30% → 60%；45~48min, B：60% → 80%；48~56min, B：80%；56~
58min, B：80% → 5%；58~65min, B：5%；
Mass spectral analysis condition：Using electric spray ion source (ESI), atomization gas and dry gas are nitrogen；MRM is taken to sweep
Retouch.Gather ion mode：Positive ion mode；One-level scanning range：350-2000Da；One-level scanning resolution：70000；Two level
Scanning range：Automatically selected dependent on one-level m z；Two level collision energy (NCEs)：28%；Two level scanning is differentiated
Rate：17500；Capillary temperature：360℃；Ion source voltage：1800V；Fragmentation mode：HCD.
Analysis software：SKYLINE softwares
Analysis method：Meet 3 (polypeptide YB-001~YB- of target peptide fragment of MRM standards with the extraction of SKYLINE softwares
003) suitable transition, is selected to carry out MRM detections.
Analyze and identify result：Compared by peptide fragment parent ion with choosing the Information in Mass Spectra of daughter ion, detect 3 targets
Peptide fragment, and have relatively good signal response, it is good with associated mass spectrometry Data Matching in database, it can be assumed that Shuxuetong injection
Contain corresponding 3 target peptide fragments component (1~accompanying drawing of accompanying drawing 6) in liquid.
Claims (6)
1. a kind of method of polypeptide/oligopeptides composition quality control in SHUXUETONG ZHUSHEYE, it is characterised in that using LC-MS-
The Quality Control model that MRM separation, detection, statistics identification are combined, carries out the quality control of polypeptide/oligopeptides composition in SHUXUETONG ZHUSHEYE
System, the quality control method comprise the following steps：
(1) SHUXUETONG ZHUSHEYE is subjected to desalination pre-treatment using column chromatography or dialysis；
(2) desalted sample solution is carried out into nanoliter liquid chromatogram to separate, separation condition：Flow velocity is 0.4 μ l/min, gradient elution bar
Part：0~6min, B are 5% → 8%；6~40min, B are 8% → 30%；40~45min, B are 30% → 60%；45~
48min, B are 60% → 80%；48~56min, B 80%；56~58min, B are 80% → 5%；58~65min, B are
5%；
(3) sample by liquid chromatogram separation carries out mass spectral analysis, and Mass Spectrometry Conditions are：Electric spray ion source, atomization gas and dry
Pathogenic dryness body is nitrogen；Gather positive ion mode；One-level scanning range：350-2000Da；One-level scanning resolution：70000；Two level
Scanning range：Automatically selected dependent on one-level m z；Two level collision energy：28%；Two level scanning resolution：
17500；Capillary temperature：360℃；Ion source voltage：1800V；Fragmentation mode：HCD；
(4) multiple-reaction monitoring pattern is used, particular target peptide fragment component of the molecular weight between 256-2560 dalton is selected, enters
The monitoring of the specific parent ion of row and daughter ion, parent ion all should have with multiple specific daughter ions with database preferably match and
Signal, which responds, then to be thought to identify successfully, and the corresponding daughter ion data of matching are as shown in the table：
2. according to the method for claim 1, it is characterised in that described that SHUXUETONG ZHUSHEYE is utilized into column chromatography desalting processing
For molecular sieve column chromatography or ion-exchange chromatography desalting processing.
3. according to the method for claim 2, it is characterised in that molecular sieve in the molecular sieve column chromatography desalting processing method
Column chromatography filler can be that sephadex series or polyacrylamide gel are serial, or Ago-Gel system
Row, eluant, eluent is water.
4. according to the method for claim 3, it is characterised in that the sephadex series filler be sephadex G10,
Sephadex G15 or sephadex G25.
5. according to the method for claim 1, it is characterised in that the particular target peptide selected in the multiple-reaction monitoring pattern
Duan Zufen, it is 3 polypeptides/oligopeptides component YB-001~YB-003 in SHUXUETONG ZHUSHEYE.
6. according to the method for claim 1, it is characterised in that polypeptide/oligopeptides component in the SHUXUETONG ZHUSHEYE is
Polypeptide/oligopeptides constituents of the molecular weight between 1000-2000 dalton.
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| CN105949335B (en) * | 2015-12-31 | 2018-02-09 | 牡丹江友搏药业有限责任公司 | A kind of oligosaccharide kind component with platelet aggregation inhibitory activity and preparation method thereof |
| CN110133124A (en) * | 2019-04-29 | 2019-08-16 | 天津中医药大学 | The content assaying method of 18 kinds of amino acid in SHUXUETONG ZHUSHEYE |
| CN112557527A (en) * | 2020-11-17 | 2021-03-26 | 牡丹江友搏药业有限责任公司 | Method for measuring polypeptide content in Shuxuetong injection |
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| CN100462084C (en) * | 2006-01-10 | 2009-02-18 | 复旦大学 | Assaying method of Chinese medicine laying particle active principle and content |
| CN101095697A (en) * | 2006-06-28 | 2008-01-02 | 李振国 | Extractive of bdella and/or lumbricus with the molecular weight below 5800 dalton |
| US7897405B2 (en) * | 2008-02-11 | 2011-03-01 | Thermo Finnigan Llc | Method for identifying the elution time of an analyte |
| US8620595B2 (en) * | 2010-03-26 | 2013-12-31 | University Of Manitoba | Methods for determining the retention of peptides in reverse phase chromatography using linear solvent strength theory |
| CN102507792B (en) * | 2011-11-17 | 2014-04-30 | 牡丹江友搏药业股份有限公司 | Quality detection method for Shuxuetong preparation |
| CN103134860A (en) * | 2011-11-23 | 2013-06-05 | 上海市公共卫生临床中心 | Quantitative determination method for target peptides and proteins |
| CN103592376B (en) * | 2013-07-02 | 2014-12-10 | 牡丹江友搏药业股份有限公司 | Novel quality control method of Shuxuetong injection |
| CN104698097B (en) * | 2015-01-07 | 2016-08-17 | 南京中医药大学 | A kind of Chinese medicine effector substance discrimination method based on selective occluding technique |
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Address after: 157013 Heilongjiang province Mudanjiang City Yangming District Yumin Road No. 288 Applicant after: Mudanjiang Youbo Pharmaceutical Co.,Ltd. Address before: 157013 Heilongjiang province Mudanjiang City Yangming District Yumin Road No. 288 Applicant before: Mudanjiang Youbo Pharmaceutical Co., Ltd. |
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Inventor after: Li Zhenguo Inventor after: Chen Yanming Inventor after: Zhou Jianbo Inventor after: Wu Yongsheng Inventor after: Guo Yafang Inventor before: Li Zhenguo Inventor before: Chen Yanming Inventor before: Zhou Jianbo Inventor before: Wu Yongsheng |
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