CN104931635B - Detect method and the liquid matter data base of left drug in animal-derived food - Google Patents
Detect method and the liquid matter data base of left drug in animal-derived food Download PDFInfo
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Abstract
The present invention relates to a kind of liquid matter data base for detecting left drug in animal-derived food and using method thereof, the preparation of this data base comprises the steps: the preparation of (1) standard working solution；(2) analyze pre-treatment, use the extraction and cleaning technology of rapid enzymolysis+quick Solid-Phase Extraction (SPE) to carry out sample to be tested and analyze pre-treatment work；(3) disposable sample introduction chromatography；(4) liquid mass spectral database is built.
Description
Technical field
The invention belongs to field of biological detection, in particular to a kind of liquid matter data base for detecting left drug in animal-derived food and make
Use method.
Background technology
Animal-derived food accounts for suitable proportion in international agriculture trade, and the regulation for animal-derived food drug residue limits increasingly stricter, with
As a example by " positive list " system that Japan implements in May, 06,236 kinds of veterinary drugs and feed additive have been formulated concrete limit standard by it, and
Domestic food security fields, from " malachite green oxalate, the clenbuterol hydrochloride " finally of " chloromycetin, nitrofuran " in early days, because drug residue causes
Food safety affair takes place frequently and highlights China's food safety Regulation control system technical support scarce capacity, in the urgent need to setting up multiple types residual rapid screening
Method.At present, developed into single type multi-residue analysis by single retention analysis for animal-derived food drug residue detection technique and method, 03
Since Nian, the multi-residue determination standard divided with substance classes circle occupies retention analysis country and more than the 70% of industry standard.Same time, mass spectrum divides
Analysis has become as the technical way of multi-residue determination, with European Union's 2002/657/EC regulation be source technical specification rule also by being held in the world
Recognize and accept.Along with the development of retention analysis technology, multiple types retention analysis has advantage in the many-side such as quantity of information, detection efficiency and becomes and grind
Heat generating spot, and mass spectroscopy device field such as Q-TOF, the tandem mass spectrometer such as Q-Trap practical, the spectrum storehouse comparison based on senior mass-spectrometric technique divides
Analysing gradually maturation also may for the quick technology that provides of analyzing of multiple types residual.But, either in method exploitation, checking and standardization still in method
Advising the aspects such as perfect, detection multiple types remains and all there is more difficulty simultaneously:
(1) variety classes veterinary drug physico-chemical property difference is very big, polarity coverage width, remains form and differ in different substrates, and indivedual veterinary drugs are still
Need derivatization effectively to detect, therefore be difficult to effectively break through on general pre-treating method and chromatograph single injected sampling analysis；
(2) limitation of different veterinary drugs (forbidden drugs and limit the use of medicine) is required and uses regulation to differ by regulation, multiple types residue detection proof rule
The most perfect；
(3) bare substrate that method exploitation needs is difficult to obtain, mixed standard solution preparation difficulty；
(4) analysis ability of instrument.In terms of building liquid mass spectral database, compose storehouse such as AB company legal medical expert's database of poisons (1250 kinds despite commercialization
Target analytes), basic veterinary drug database (139 kinds of target analytes)；The small-molecule drug database of Freiburg university hospital, but exist as follows
Application limitation:
1) MS/MS data are only had, without chromatographic information characteristics；
2) without online information feature；
3) without unified pre-treating method and Instrumental Analysis phase coupling；
4) extraction standard data are lacked.
Therefore, under existence conditions, it is difficult to effectively realize multiple types based on liquid mass spectral database technology and remains quick Screening analysis.
Summary of the invention
In view of at present both at home and abroad present in the animal-derived food multiple types left drug analysis sample universal pre-treatment, instrument quickly analyze and screen
The technological difficulties such as method validation.The present invention, for the purpose of setting up the animal-derived food medium or high risk medicine multiple types quick selective mechanisms system of residual, passes through
Use multi-solvent system piecewise combination extract, set up sample high flux versatility pretreatment technology, complex optimum chromatographic realizes multiobjective analysis thing
Wide polarity scope liquid chromatograph is effectively retained and the liquid chromatography-mass spectrography/mass spectrometric data storehouse separating and being contained by structure chromatography-mass spectroscopy multidimensional information
And foundation laws and regulations requirement and detection practice carry out the research and development of the key technology contents such as screening technique checking, break through multiple types retention analysis technical barrier, build
Vertical one with multiple types residual versatility pretreatment technology, sharp separation system and the liquid mass spectral database quickly analysis platform of the opening as technical characteristic and has
Effect examination detection technique system.
The present invention is directed to 12 kinds of Limited Doses at 1.0 μ g/kg following excessive risk left drug (A class residuals) as detection target, this group medicine
Thing is respectively,
(1) steroid hormone compounds: testosterone (T), prednisolone (PNSL), betamethasone (BTS), dexamethasone (DTS)；
(2) nitroimidazoles medicine and metabolite thereof: metronidazole (MNZ), cough up nitre and rattle away azoles (RNZ), Dimetronidazole (DMZ)；
(3) Beta-receptor stimulating agent class material: Clenbuterol (CLB), Tulobuterol (TUL), cimaterol (CIM)；
(4) dye class material: crystal violet (CV) and leuco crystal violet (LCV)；
Present invention firstly relates to the construction method of a kind of rapid screening liquid mass spectral database detecting animal-derived food medium or high risk medicine multiple types residual, described
Animal-derived food be animal muscle based food, dairy products, specifically include following steps,
(1) preparation of standard working solution；
Steroid hormone compounds, nitroimidazoles medicine and metabolite thereof, Beta-receptor stimulating agent class material: use acetonitrile classification to prepare it
Hybrid standard stock solution (0.01g/L)；
The materials such as dye class use methanol classification to prepare its hybrid standard stock solution (0.01g/L)；
Mixing and standard working solution (0.1mg/L), pipette testosterone, prednisolone, betamethasone, dexamethasone, metronidazole respectively, cough up nitre
Rattle away azoles, Dimetronidazole, Clenbuterol, Tulobuterol, cimaterol, crystal violet and leuco crystal violet Standard Stock solutions is appropriate, joins with acetonitrile
Concentration processed is the mixed standard solution of 0.1mg/L；
(2) analyze pre-treatment, use the extraction and cleaning technology of rapid enzymolysis (release Conjugate polyamines)+quick Solid-Phase Extraction (SPE) to treat
Surveying sample analysis pre-treatment work, concrete extraction and cleaning process is as follows:
1) extract: weigh homogeneous samples 5g, be placed in 50mL politef centrifuge tube, add ammonium acetate buffer (0.2mol/L, pH 5.2)
15mL, β-glucosiduronic acid/sulfatase 50 μ L, vortex mixes, 50 DEG C of water-bath vibration 2h, lets cool to room temperature, in 0 DEG C, 15000rpm
Centrifugal 5min, in transfer supernatant to another clean centrifuge tube, regulates pH to 9.0 by NaOH solution (5mol/L), adds ethyl acetate 20
ML, sodium chloride 6g, vibrate vortex 5min, and in 0 DEG C, 15000rpm is centrifuged 5min, takes upper organic phase 35 DEG C rotation and steams near dry, nitrogen
Drying up, residue uses 5mL 0.1mol/L hydrochloric acid solution to dissolve, and ultrasonic 1min remains column purification；
2) purify: MCX post (Waters Oasis MCX SPE pillar) is installed on solid-phase extraction device that (this device is at utility model patent
CN201020014620.X is documented), use methanol 3mL successively, water 3mL, 3mL 0.1mol/L hydrochloric acid solution activates.To carry
Take solution to be carried on solid-phase extraction column, flow out under gravity, successively use 3mL water, 3mL methanol and 5mL normal hexane drip washing pillar,
Drain 2min, add 6mL eluent (ethyl acetate 50mL, methanol 45mL, ammonia 5mL, mixing) eluting.At eluent 35 DEG C
Nitrogen stream dries up, and uses sample lysate (to measure 0.1% formic acid acetonitrile (V/V) solution 5mL, with ammonium formate (5mmol/L)-formic acid (0.1%)
-aqueous solution 95mL mixes) 0.5mL dissolved residue, ultrasonic 1min, crosses 0.22 μM of microporous filter membrane；Described solid-phase extraction device integrates examination
Agent frame, the function of Nitrogen evaporator and operation pressure are relatively uniform, and it is by reagent rack, operating board, 8 glass storehouses, 8 pear shape bottles and 1 automatic suction
Device of air forms, and can realize the quick SPE process of target analytes.
(3) disposable sample introduction chromatography
Using chromatographic column is Kinetex C18 post, and employing acetonitrile is organic facies, and formic acid optimizes agent as ionizing reinforcing agent, formates as peak shape,
Combining as the flow visualizing optimized using aqueous phase/acidity acetonitrile；
Actual conditions is: chromatographic column: Kinetex C18,2.6 μm, 2.1mm × 100mm i.d.；
Flow velocity: 0.2mL/min；
Sample size: 10 μ L；
Column temperature: 30 DEG C；
Gradient elution program: (A:0.1% formic acid-acetonitrile solution；B: ammonium formate (5mmol/L)-formic acid (0.1%)-aqueous solution)
0～2min:5%A；
～8min:20%A；
～15min:95%A；
～16min:100%A；
～19min:100%A；
20min:5%A；
(4) liquid mass spectral database is built
The senior drainage pattern of employing level Four bar/ion trap tandem mass spectrometry: presetting many reaction detection (sMRM)-information dependency collection (IDA)-
Enhancer ion scan (EPI)；
Mass spectrometry parameters determines that process is as follows: using initial flow the most according to target analyte classification dilution mixed mark stock solution to concentration is 0.2mg/L, so
Rear use constant current syringe pump injects in mass ion source with the flow velocity of 5 μ L/min and carries out parameter optimization,
Use the scan patterns such as Q1MS, Q1Multiple Ions, Product Ion, MRM to determine the parent ion of target analytes respectively, son from
Son, and use Ramp function optimization and determine depolymerization voltage (DP), collision cell entrance potential (EP), collision energy (CE), collision cell outlet electricity
The chemical parameters such as pressure (CXP)；
Mass Spectrometry Conditions (API 4000 and API 4000Q-TRAP):
A) ion source: electric spray ion source；
B) scan mode: cation scans；
C) detection mode: sMRM-IDA-EPI
D) electron spray voltage: 5500V；
E) atomization gas pressure: 40psi；
F) gas curtain atmospheric pressure: 30psi；
G) assist gas pressure power: 45psi；
H) ion source temperature: 475 DEG C；
I) sMRM parameter is arranged: MRM detection window is set to 60s, and object is set to 1.4s sweep time；
J) IDA rule: response lag: 3000cps；Dynamic background is deducted；The strongest ion is chosen as 1 to 3；
K) enhancer ion scan (EPI) parameter is arranged: quality of scanning number scope is 70～1000Da；Scanning speed is 10000
Da/s；Scanning accumulative frequency is 1；Collision energy is 35eV；Extension collision energy is 15eV；
By the mass spectral results for 12 kinds of A class materials, build liquid mass spectral database.
Use in the structure of data base builds the Analyst1.5 that library software is AB SCIEX company, and the standard building storehouse is set as according to mentality of designing:
(1) providing each target analytes uses this research to set the RT value under liquid phase chromatogram condition system；
(2) be given each target analytes related chemistry information (as title, chemical formula, molecular weight, No. CAS, compounds category, ID,
Molecular structural formula etc.)；
(3) be given the EPI spectrogram according to the described collection of 3.2.2 item under at least 5 different conditions of each target analytes, i.e. CE be 20eV,
35eV, 50eV and CE are 35eV, CES 4 EPI spectrograms when being 15eV, additionally, the most also to have one under chromatographic condition CE be
35eV, CES are EPI spectrogram during 15eV；
(4) provide the EPI spectrogram of target analytes in different substrates as far as possible.
First 3 to build library standard be the condition must being fulfilled for, and the 4th article is the optional standard according to practical situation.
Chromatographic and the Mass Spectrometry Conditions selected according to conditions above achieve and comprise the 115 of 12 kinds of high residue A class materials of the present invention
Plant the high efficiency separation of the many residuals of veterinary drug.Close despite individual compound retention time, but it extracts ion flow graph spectrum and all can realize mass spectrum separation, can
To carry out quantitative analysis, additionally, EPI spectrum storehouse target analytes patch information feature, representation compound structure qualitatively " fingerprint " information, can
With the most qualitative to target analytes.The liquid mass spectral database of the thus obtained liquid matter information comprising described A class residuals, it is possible to realize described dynamic
Thing derived food medium or high risk medicine multiple types residual rapid screening.
Under above-mentioned chromatograph and Mass Spectrometry Conditions, in addition to 12 kinds of A class materials, the total ion current figure of B class 103 kinds totally 115 kinds of target analytes,
The data of (CE 35eV, CES 15eV) when typical selection ion flow graph, each target analytes extract ion flow graph and select CES extension
Storehouse spectrogram such as Fig. 1～Fig. 4.
The invention still further relates to be built the rapid screening liquid matter for detecting animal-derived food medium or high risk medicine multiple types residual obtained by said method
Spectrum storehouse, described animal-derived food is animal muscle based food, dairy products；
Described excessive risk medicine be Limited Doses at 1.0 μ g/kg following excessive risk left drug, particularly as follows:
(a) steroid hormone compounds: testosterone (T), prednisolone (PNSL), betamethasone (BTS), dexamethasone (DTS)；
(b) nitroimidazoles medicine and metabolite thereof: metronidazole (MNZ), cough up nitre and rattle away azoles (RNZ), Dimetronidazole (DMZ)；
(c) Beta-receptor stimulating agent class material: Clenbuterol (CLB), Tulobuterol (TUL), cimaterol (CIM)；
(d) dye class material: crystal violet (CV) and leuco crystal violet (LCV).
The invention still further relates to described detection animal-derived food medium or high risk medicine multiple types residual rapid screening liquid mass spectral database in detection animal-derived food
The application of drug residue.
The method that the invention still further relates to use described liquid mass spectral database that the excessive risk drug residue in target animal derived food is carried out qualitative or quantitative analysis,
Described method comprises the steps,
(1) standard solution is prepared,
(2) analyze pre-treatment, use the extraction and cleaning technology of rapid enzymolysis (release Conjugate polyamines)+quick Solid-Phase Extraction (SPE) to treat
Survey sample analysis pre-treatment work,
(3) sample to be tested is carried out chromatograph and liquid matter analysis, it is thus achieved that liquid mass spectrum, contrast described liquid mass spectral database, carry out qualitative or quantitative analysis inspection
Survey.
Described qualitative analysis uses library searching to carry out, and qualitative criteria is as follows:
(1) when in sample, compound extracts the reservation that the retention time of ion stream extracts ion stream with target analytes in standard solution or interpolation sample
Between compare, amplitude of variation be less than 5%.
(2) parent ion/daughter ion (transmission ion pair) of target analytes must occur simultaneously, and the signal to noise ratio (S/N) >=3 of transmission ion pair.
(3) compound EPI spectrogram and close concentration level (same concentration numbers magnitude) standard solution or extraction standard solution in spectrum storehouse in sample
EPI spectrogram is compared, spectrogram coupling purity (Purity value) >=60.
The quantitative approach of described quantitative analysis is as follows:
Use external standard single-point method quantitative, calculate the content of target analytes by formula (1).
In formula (1):
Target analytes content in X-sample, μ g/kg；
The concentration of target analytes in c-sample solution, μ g/L；
The constant volume of V-sample solution, mL；
The quality of m-sample, g；
The R-response rate, %.
Accompanying drawing explanation
Fig. 1 .A class material, the total ion current figure of B class material totally 115 kinds of target analytes.
Fig. 2 .A class material, the typical case of B class material totally 115 kinds of target analytes select ion flow graph.
The target analytes of Fig. 3 .A class material extracts ion flow graph.
The target analytes spectrum database data figure of Fig. 4 .A class material.
Detailed description of the invention
The preparation of embodiment 1. standard working solution
Steroid hormone compounds, nitroimidazoles medicine and metabolite thereof, Beta-receptor stimulating agent class material: use acetonitrile classification to prepare it
Hybrid standard stock solution (0.01g/L), standard of physical storing solution all can be stablized 12 months.
The materials such as dye class use methanol classification to prepare its hybrid standard stock solution (0.01g/L), can stablize 3 months.
Mixing and standard working solution (0.1mg/L), pipette testosterone, prednisolone, betamethasone, dexamethasone, metronidazole respectively, cough up nitre
Rattle away azoles, Dimetronidazole, Clenbuterol, Tulobuterol, cimaterol, crystal violet and leuco crystal violet Standard Stock solutions is appropriate, joins with acetonitrile
Concentration processed is the mixed standard solution of 0.1mg/L, and this solution-20 DEG C can be stablized 1 month.
Embodiment 2. analyzes pre-treating method
12 kinds of target compounds (A group material) are under the jurisdiction of steroid hormone class, nitro glyoxaline, beta-receptor agonist class and dye class etc. 4 respectively
Individual classes of compounds, its perform limitation claimed range at 0.05 μ g/kg～0.8 μ g/kg, be one group the most in the world limitation require most stringent
Medicine.Therefore, consider when pretreatment process designs and purify the step concentrated, to meet strict limitation requirement.For involved by A group material
And classes of compounds, the extraction and cleaning technology of the Solid-Phase Extraction (SPE) of research design rapid enzymolysis (release Conjugate polyamines)+quickly, can be fast
Speed effectively completes the pretreatment process of 12 kinds of ultra-low limit quantity of material.Concrete extraction and cleaning process is as follows:
(1) extract: weigh homogeneous samples 5g (being accurate to 0.01g), be placed in 50mL politef centrifuge tube (as interpolation sample need to be made,
Add the A group material hybrid standard working solution of debita spissitudo in this step, and place 30min in dark place), add ammonium acetate buffer (0.2
Mol/L, pH 5.2) 15mL, β-glucosiduronic acid/sulfatase 50 μ L, vortex mixes, 50 DEG C of water-bath vibration 2h, lets cool to room temperature, in 0 DEG C,
15000rpm is centrifuged 5min, in transfer supernatant to another clean centrifuge tube, regulates pH to 9.0 by NaOH solution (5mol/L), adds
Ethyl acetate 20mL, sodium chloride 6g, vibrate vortex 5min, and in 0 DEG C, 15000rpm is centrifuged 5min, takes upper organic phase 35 DEG C rotation and steams
To near dry, nitrogen dries up, and residue uses 5mL 0.1mol/L hydrochloric acid solution to dissolve, and ultrasonic 1min remains column purification.
(2) purify: (Waters Oasis MCX SPE pillar, this type pillar filling adsorption agent is at HLB pillar N-vinylpyridine to MCX post
-SO is added on the basis of pyrrolidone-DVB copolymer3H ion exchanging function base.) (this device is special in utility model to be installed on solid-phase extraction device
Profit CN201020014620.X is documented) on, using methanol 3mL successively, water 3mL, 3mL 0.1mol/L hydrochloric acid solution activates.
Being carried on solid-phase extraction column by extraction solution, flow out under gravity, use 3mL water successively, 3mL methanol and 5mL normal hexane drip washing are little
Post, drains 2min, adds 6mL eluent (ethyl acetate 50mL, methanol 45mL, ammonia 5mL, mixing) eluting.Eluent 35 DEG C
Lower nitrogen stream dries up, and uses sample lysate (to measure 0.1% formic acid acetonitrile (V/V) solution 5mL, with ammonium formate (5mmol/L)-formic acid (0.1%)
-aqueous solution 95mL mixes) 0.5mL dissolved residue, ultrasonic 1min, crosses 0.22 μM of microporous filter membrane, liquid chromatography tandom mass spectrometry determination.
The step of extraction and cleaning process is described as follows: in A group material, the material such as testosterone, Tulobuterol adheres to hormone and beta-receptor agonist class separately,
Literature survey shows this kind of normal with combined state existence, especially this phenol of Tulobuterol type medicine in animal tissue, therefore need to add β-glucoside
Acid/sulfatase enzymolysis residual tissue conjugates, to discharge free state medicine, adds buffer one and is to provide the pH environment that enzymolysis needs, separately
Outer is also to use as Extraction solvent, and this step is compared existing method and be have employed raising hydrolysis temperature (being promoted to 50 DEG C by the most methodical 36 DEG C)
Mode make the process originally needing overnight (16h) to react shorten to 2h, greatly accelerated enzymolysis process；Crystal violet and Recessive Crystal Violet are through test
Proof can also be extracted in this step；Using freezing ultracentrifugal purpose is precipitation fat and albumen；Extracting solution is alkalized and adds sodium chloride
Purpose is to promote nitro glyoxaline compound and beta-receptor agonist by aqueous phase to the distribution of organic facies.Purifying step is owing to relating to multiple types material
Purification, SPE post have selected the reversed material of versatility.Research compares hydrophilic-lipophilic balance (HLB) post and common C18 post, all fails to realize
Preferably effect.Final research uses Waters Oasis MCX SPE pillar, and this type pillar filling adsorption agent is at HLB pillar N-ethylene
-SO3H ion exchanging function base is added on the basis of base ketopyrrolidine-DVB copolymer.Therefore this SPE post possesses anti-phase mixed with what ion exchanged
Close adsorption function, acid, alkalescence and neutral compound can be adsorbed when low ph value, compared with HLB and C18 post, there is higher detergent power.Eluting
The design of program is reported with reference to pertinent literature and adds the step of normal hexane drip washing in the step of drip washing, disturbs with more preferable place to go lipid, eluting
Solution have employed the mixed solution of ethyl acetate (50%)-methanol (45%)-ammonia (5%) water, and through test, 6mL can complete target analytes
Eluting.
Additionally, during using the detection of SPE post, owing to existing SPE system is difficult to realize fast operating, seminar have developed a kind of novel solid
Phase extraction equipment, it is relatively uniform that this device can integrate reagent rack, the function of Nitrogen evaporator and operation pressure, and it is by reagent rack, operating board, 8 glass
Storehouse, 8 pear shape bottles and 1 automatic getter device composition, can realize the quick SPE process of target analytes.
To sum up, A group material pre-treating method is although with classical solvent extraction/Solid phase extraction pre-treatment flow process, but at rapid enzymolysis and general
Property quick SPE purification process design on all consider multiple types residual and extract and flux processes factor, compare existing method and have fast and convenient excellent
Gesture, and in a pretreatment process, complete effective extraction of ultra-low limit quantity of material in 4 classes 12, meet the requirement of fast Screening analysis.
The disposable sample introduction chromatographic analysis system of embodiment 3. is studied
1. the selection of chromatographic column
The liquid chromatograph of multiobjective analysis thing separates and the most generally considers to use ultramicro powder (particle diameter ＜ 2 μm) chromatographic column, but this research uses routine
HPLC system, it is pressure, and the upper limit is 400Bar, therefore cannot use Ultra Performance Liquid Chromatography post.For realizing the separation purpose of design, it is considered to system pressure
Power limits and controls between 2 μm～3 μm by the particle size range of chromatographic column, and chromatogram column length scope is 100mm～150mm；Additionally, due to compound
Kind is more, and polarity range spans is relatively big, just have selected the C18 chromatographic column being suitable for separating wide polarity scope in terms of chromatographic column type.By upper
Stating restriction, this research have selected the one in every compounds according to polarity (selecting with reference to LogD value), and 4 kinds of materials are as analyte generation altogether
The table examination liquid-phase chromatographic column of 4 types.Screening conditions and the results are shown in Table 2.
Table 2 chromatographic column selectivity experimental design and separation analysis result
As seen from the above table, comparing other chromatographic columns, Kinetex C18 post separating degree and sensitivity are preferable.Although this chromatographic column particle diameter is minimum, but
Its post back pressure is still in the pressure scope of conventional liquid phase.And filler particles have employed the core-shell structure copolymer technology of advanced person, compared with Bio-sil post complete with tradition,
Separating degree and sensitivity can be obviously improved.Determining this type chromatographic column of use is preferable separate post.
2. the selection of flow visualizing and the determination of other chromatographic conditions
To mass spectral analysis conventional flowing phase (water, formic acid-aqueous solution, acetic acid-aqueous solution, methanol, acetonitrile, acidified methanol, acidifying acetonitrile, formic acid
Ammonium buffer, ammonium acetate buffer etc.) and composition use 12 kinds of hybrid standard working solutions screened.Considering separating degree, sensitivity
On the basis of the factor such as analysis time, finally determining that employing acetonitrile is organic facies, formic acid is as ionizing reinforcing agent, and formates is as peak shape optimization
Agent, combining as the flow visualizing optimized using aqueous phase/acidity acetonitrile.
The liquid chromatograph parameters such as sample size, column temperature and flow velocity are optimized, main considerations include chromatographic resolution, sensitivity, repeatability and
Matrix effect etc..By using 12 kinds of object mixed standard solutions to carry out chromatographic isolation examination in screening target concentration levels (0.5 μ g/kg)
Test and determine optimal value.
On the basis of optimizing relevant parameter and comparing test of many times result, the actual conditions of the disposable liquid phase chromatographic isolation system originally determined
For: chromatographic column: Kinetex C18,2.6 μm, 2.1mm × 100mm i.d.；Flow velocity: 0.2mL；Sample size: 10 μ L；Column temperature: 30
℃.Gradient elution program: (A:0.1% formic acid-acetonitrile solution；B: ammonium formate (5mmol/L)-formic acid (0.1%)-aqueous solution) 0-2min:
5%A；8min:20%A；15min:95%A；16-19min:100%A；20-35min:5%A.
Embodiment 4. liquid mass spectral database builds
1. foundation and the liquid mass spectral database of mass spectrum acquisition method builds
(1) foundation of mass spectrum acquisition method
This research have employed the senior drainage pattern of level Four bar/ion trap tandem mass spectrometry: presetting many reaction detection (sMRM)-information dependency gathers
(IDA)-enhancer ion scan (EPI).The foundation of this drainage pattern, first has to determine target analytes retention time in chromatographic, its
Secondary multiple-reaction monitoring (MRM) mass spectrometry method of setting up, then sets and triggers the condition (i.e. IDA setting) that EPI gathers, finally determine that EPI gathers
Condition set.
For setting up corresponding mass spectrum acquisition method, the first mass spectrometry parameters to 12 kinds of target analytes carries out test setting.Research employing is first classified excellent
Change chemical parameters (including parent ion, daughter ion and depolymerization voltage, collision energy etc.), then choose representation compound optimize source parameters (include
Atomization gas pressure, ion source temperature etc.).
1. chemical parameters optimization determines.Basis of design chromatographic isolation result about RT parameter.Mass spectrometry parameters determines that process is as follows: use initial
Flowing the most according to target analyte classification dilution mixed mark stock solution to concentration is 0.2mg/L, but it is noted that has the target of same molecular amount in same category
Analyte individually to prepare (because studied use instrument is standard resolution, it is impossible to distinguish isomers), then uses constant current syringe pump with 5
The flow velocity of μ L/min injects in mass ion source and carries out parameter optimization.Use Q1 MS, Q1 Multiple Ions, Product Ion, MRM respectively
Determine the parent ion of target analytes etc. scan pattern, (it is standby, in follow-up test to transmission ion that every kind of target analytes at least chooses 2 to daughter ion
Disturbed condition in the middle signal to noise ratio according to response and bare substrate chooses a pair ion collection ion as sMRM of optimum), and use Ramp
Function optimization also determines the chemical combination such as depolymerization voltage (DP), collision cell entrance potential (EP), collision energy (CE), collision cell exit potential (CXP)
Thing parameter, 12 kinds of materials compounds parameter optimizations the results are shown in Table 3.
3 12 kinds of target analytes of table optimize Mass Spectrometry Conditions and with reference to retention time
Note: 1. in this table, listed ion pair is the optimization ion pair determined after follow-up test examination matrix effect and signal to noise ratio, parameter of unlisted standby ion pair at this；
2. target analytes retention time (RT) variation in RT sets window (15s) all belongs to normal.
2. the optimization of source parameters determines.Flow Injection Analysis (FIA) is used to optimize, because the Limited Number system optimizing ion pair is originally ground by instrument
Studying carefully employing representation compound to be optimized, representation compound selection principle is to use chemical parameters to respond relatively low target analytes when optimizing,
Through test, source parameters optimum results is as follows: gas curtain atmospheric pressure: 30psi；Spray voltage: 5500V；Ion source temperature: 475 DEG C；Mist
Activating QI pressure: 40psi；Assist gas pressure power: 45psi.
3. IDA condition sets.In IDA condition sets, it is most important that the mensuration of response lag, screening target concentration levels (0.025
μ g/kg) on analyze the extraction standard standard solution of preparation (use " bare substrate " extracting solution), with the 1/2 of minimum response substance responds intensity
Response lag is set for degree.It should be noted that this threshold value has substrate dependency, different substrate threshold values is different, it is generally the case that threshold
Value setting principle is to ensure that all responses meeting examination aimed concn all can trigger EPI and gather as far as possible, improves threshold value to reduce data simultaneously as far as possible
Collection capacity (is prone to analyze).This research is investigated two kinds of animal sources substrate (animal muscle based food, dairy products), has been set as with minimum
3000cps。
Secondly, set kind of instrument dynamic background to be selected a deduction function at IDA, can effectively reduce the generation of invalid data.
4. the setting of EPI parameter.
A) EPI acquisition quality number scope: in this research, the mass number scope of 12 kinds of target analyte molecule quasi-molecular ions is 140Da～940Da,
Fragment ion masses number scope 80Da～880Da, therefore EPI acquisition quality number range set is at 70Da～1000Da；
B) EPI acquisition scans speed: this research use equipment is AB SCIEX 5500Q-Trap mass spectrograph, the total 1000Da/s of EPI collection,
10000Da/s and 20000Da/s 3 notch speed degree, for ensureing the quality of data, selecting 10000Da/s is EPI acquisition scans speed, the most both holds concurrently
Turn round and look at the analysis acquisition rate of 12 kinds of target analytes, in turn ensure that spectrogram quality；
C) EPI gathers DP value and the setting of EP value: for ensureing spectrum database data quality, 12 kinds of target analytes DP values and EP value are carried out segmentation
Statistics, the median taking section at high proportion is final setting value.
Through analyzing, target compound DP value strengthens in 60V～100V section ratio, and taking median 80V is the DP setting value that EPI gathers.
Same, target group compound EP value, at 9V～11V section large percentage, takes the EP setting value that median 10V gathers as EPI.Really
After determining DP and EP setting value, DP value and EP value EPI are gathered, it was demonstrated that the DP value of setting and the EPI data acquisition to this compounds of the EP value
Quality has no significant effect；
D) EPI gathers the setting of collision energy (CE) value, with reference to table 3, the CE value of general common compounds at 20eV～30eV, but individual
The CE value of other compound such as crystal violet, when 45～60eV, just has the crumb data that quality is higher.To this end, first fixing CE value is 35, reset
Extension CE value (CES) is that EPI spectrogram quality are examined or check in 3 kinds of combinations such as 5,10,15.Finally choose CE be 35eV, CES be 15eV (phase
When the cumulative mean of 3 spectrograms when CE is respectively 20eV, 35eV, 50eV) as EPI gather CE set, this setting value can be relatively
Good takes into account high, medium and low collision energy section, can obtain high-quality data spectrogram.
By above method, establish the senior acquisition method of the mass spectrum (sMRM-IDA-EPI) of 12 kinds of target analytes, this pattern can be utilized to sieve
Look into collection (using the collection ion pair of sMRM), then the object meeting examination rule (concentration-response exceedes screening aimed concn) is carried out EPI
Gather, obtain detailed ion information to carry out qualitative analysis.
(2) liquid mass spectral database builds
1. online EPI spectral data gathers.After using 12 kinds of object hybrid standard working solutions to use flowing phase dilution, compound concentration is screening target
The mixed standard solution of concentration level, upper machine analysis gathers online EPI data in sMRM-IDA-EPI mode, uses Analyst1.5 software building
Spectrum storehouse, and improve goal analysis information (such as Chinese and English title, chemical formula, No. CAS, chemical structural drawing etc.).
2. off-line EPI spectral data gathers.Use the standard reserving solution (as apoplexy due to endogenous wind has isomers, need independent sample introduction) of classification preparation
Using flowing phase dilution to 0.2mg/L, direct mass spectrum sample introduction, with reference to online EPI condition collection off-line EPI data, the most individually collection 3
CE level (basic, normal, high) EPI spectrogram, can use, according to compound actual nature, the collision energy optimized.Above-mentioned collection data inputting is composed storehouse.
3. liquid mass spectral database judges to use rule.Library searching is one of maximally effective qualitative tool, but in the matched rule system of liquid mass spectral database retrieval
Determine aspect and there is no any regulation and technical stipulation at present.This research determines the judgement rule of retention time and signal to noise ratio according to European Union and U.S.'s relevant regulations
Then；The determination of the EPI spectrogram comparison matching tolerance factor uses 10 " bare substrate " (each 5 of muscle, milk sample) preparation extraction standard,
Use Analyst 1.5 software to carry out the matching analysis with the standard EPI collection of illustrative plates in EPI spectrum storehouse, obtain Reinheitszahl (purity), obtain meansigma methods
And standard deviation.Through comparison, all object Purity meansigma methodss deduct standard deviation and are all higher than 60, for controlling false negative rate to greatest extent, this
Determine purity value 60 for the matching tolerance factor.To sum up, the library searching rule determined is:
A) in sample, compound extracts retention time and the retention time of target analytes extraction ion stream in standard solution or interpolation sample of ion stream
Comparing, amplitude of variation is less than 5%；
B) in table 3, the parent ion/daughter ion (transmission ion pair) of listed target analytes must occur simultaneously, and the signal to noise ratio (S/N) of transmission ion pair
≥3；
C) spectrogram coupling purity (Purity value) or the matching tolerance factor >=60.
Spectrum storehouse comparison qualitative function is powerful, because its information provided is more.Specifying according to European Union 2002/657/EC, this research drainage pattern can obtain
Confirmation is counted >=5.5 (European Union's most stringent of disabling veterinary drugs require nothing more than confirmation count >=4).If therefore library searching coupling, can be to target
Analyte carries out fast qualitative confirmation.
Claims (7)
1. detect a construction method for the rapid screening liquid mass spectral database of animal-derived food medium or high risk medicine multiple types residual, specifically include following steps,
(1) preparation of standard working solution；
(2) analyze pre-treatment, use the extraction and cleaning technology of rapid enzymolysis release Conjugate polyamines+quick Solid-Phase Extraction to carry out sample to be tested and analyze pre-treatment
Work；
(3) disposable sample introduction chromatography；
(4) liquid mass spectral database is built；
Described animal-derived food is animal muscle based food, dairy products,
Described excessive risk medicine be Limited Doses at 1.0 μ g/kg following excessive risk left drug, particularly as follows:
(a) steroid hormone compounds: testosterone, prednisolone, betamethasone, dexamethasone；
(b) nitroimidazoles medicine and metabolite thereof: metronidazole, cough up nitre and rattle away azoles, Dimetronidazole；
(c) Beta-receptor stimulating agent class material: Clenbuterol, Tulobuterol, cimaterol；
(d) dye class material: crystal violet and leuco crystal violet；
The preparation method of the standard working solution described in step (1) is:
Steroid hormone compounds, nitroimidazoles medicine and metabolite thereof, Beta-receptor stimulating agent class material: use acetonitrile to prepare its mixing mark respectively
Quasi-stock solution；
Dye class material uses methanol to prepare its hybrid standard stock solution respectively；
Hybrid standard working solution, pipette respectively testosterone, prednisolone, betamethasone, dexamethasone, metronidazole, cough up nitre rattle away azoles, Dimetronidazole,
Clenbuterol, Tulobuterol, cimaterol, crystal violet and leuco crystal violet Standard Stock solutions are appropriate, are 0.1mg/L's by acetontrile concentration
Mixed standard solution；
Analysis pre-treating method described in step (2) is as follows,
1) extract:
Weigh homogeneous samples 5g, be placed in 50mL politef centrifuge tube, add ammonium acetate buffer 15mL, β-glucosiduronic acid/sulfatase 50
μ L, vortex mixes, 50 DEG C of water-baths vibration 2h, lets cool to room temperature, and in 0 DEG C, 15000rpm is centrifuged 5min, transfer supernatant to another totally from
In heart pipe, regulating pH to 9.0 by NaOH solution, add ethyl acetate 20mL, sodium chloride 6g, vibrate vortex 5min, in 0 DEG C, 15000
Rpm is centrifuged 5min, takes upper organic phase 35 DEG C rotation and steams near dry, and nitrogen dries up, and residue uses 5mL 0.1mol/L hydrochloric acid solution to dissolve, super
Sound 1min, remains column purification；
2) purify:
MCX post is installed on solid-phase extraction device, uses methanol 3mL, water 3mL, 3mL 0.1mol/L hydrochloric acid solution to activate, will extract molten successively
Liquid is carried on solid-phase extraction column, flows out under gravity, uses 3mL water, 3mL methanol and 5mL normal hexane drip washing pillar successively, takes out
Dry 2min, adds 6mL elution, and at eluent 35 DEG C, nitrogen stream dries up, use sample lysate 0.5mL dissolved residue, ultrasonic 1min,
Cross 0.22 μM of microporous filter membrane；
Described solid-phase extraction device integrates reagent rack, the function of Nitrogen evaporator, and operation pressure is relatively uniform, its by reagent rack, operating board, 5～10
Glass storehouse, 5～10 pear shape bottles and 1～2 automatic getter device composition, can realize the quick SPE process of target analytes.
Method the most according to claim 1, it is characterised in that
In step (1),
Steroid hormone compounds, nitroimidazoles medicine and metabolite thereof, Beta-receptor stimulating agent class material: use acetonitrile to be configured to 0.01g/L respectively
Hybrid standard stock solution；
Dye class material uses methanol to prepare the hybrid standard stock solution of 0.01g/L respectively；
In step (2),
Ammonium acetate buffer is 0.2mol/L, pH 5.2；
NaOH solution is 5mol/L；
MCX post is Waters Oasis MCX SPE pillar；
Eluent preparation method is: ethyl acetate 50mL, methanol 45mL, ammonia 5mL, mixing；
Lysate preparation method is: measure 0.1% formic acid acetonitrile solution 5mL, with 5mmol/L ammonium formate-0.1% formic acid-aqueous solution 95mL, mixing.
Method the most according to claim 1 and 2, it is characterised in that the disposable sample introduction chromatogram analysis method described in step (3) is as follows,
Using chromatographic column is Kinetex C18 post, and employing acetonitrile is organic facies, and formic acid optimizes agent as ionizing reinforcing agent, formates as peak shape, with
The combination of aqueous phase/acidity acetonitrile is as the flow visualizing optimized；
Actual conditions is: chromatographic column: Kinetex C18,2.6 μm, 2.1mm × 100mm i.d.；
Flow velocity: 0.2mL；
Sample size: 10 μ L；
Column temperature: 30 DEG C；
Gradient elution program such as following table:
A eluent is: 0.1% formic acid-acetonitrile solution；
B eluent is: 5mmol/L ammonium formate-0.1% formic acid-aqueous solution.
Method the most according to claim 1, it is characterised in that step (4) described structure liquid mass spectral database method is:
The senior drainage pattern of employing level Four bar/ion trap tandem mass spectrometry: presetting many reaction detection-information dependency collection-enhancer ion scan；
Mass Spectrometry Conditions, API 4000 and API 4000 Q-TRAP:
Ion source: electric spray ion source；
Scan mode: cation scans；
Detection mode: sMRM-IDA-EPI
Electron spray voltage: 5500V；
Atomization gas pressure: 40psi；
Gas curtain atmospheric pressure: 30psi；
Assist gas pressure power: 45psi；
Ion source temperature: 475 DEG C；
SMRM parameter is arranged: MRM detection window is set to 60s, and object is set to 1.4s sweep time；
IDA rule: response lag: 3000cps；Dynamic background is deducted；The strongest ion is chosen as 1 to 3；
Enhancer ion scan parameter is arranged: quality of scanning number scope is 70～1000Da；Scanning speed is 10000Da/s；Scanning accumulative frequency is
1；Collision energy is 35eV；Extension collision energy is 15eV；
By the mass spectral results for 12 kinds of excessive risk medicines, build liquid mass spectral database.
Method the most according to claim 4, it is characterised in that the library software of building used in the structure of data base is AB SCIEX company
Analyst1.5, the standard building storehouse is set as according to mentality of designing:
Step (1) provides each target analytes and uses this research to set the RT value under liquid phase chromatogram condition system；
Step (2) provides the related chemistry information of each target analytes, described related chemistry information include title, chemical formula, molecular weight, No. CAS,
Compounds category, ID, molecular structural formula；
Step (3) provides the EPI spectrogram gathered under at least 5 different conditions of each target analytes.
Method the most according to claim 5, it is characterised in that also include that step (4) provides the EPI spectrogram of target analytes in different substrates.
7. the rapid screening liquid mass spectral database of the detection animal-derived food medium or high risk medicine multiple types residual prepared by the arbitrary described method of claim 1-6
The application of the drug residue in detection animal-derived food, described animal-derived food is animal muscle based food, dairy products；
Described excessive risk medicine be Limited Doses at 1.0 μ g/kg following excessive risk left drug, particularly as follows:
(a) steroid hormone compounds: testosterone, prednisolone, betamethasone, dexamethasone；
(b) nitroimidazoles medicine and metabolite thereof: metronidazole, cough up nitre and rattle away azoles, Dimetronidazole；
(c) Beta-receptor stimulating agent class material: Clenbuterol, Tulobuterol, cimaterol；
(d) dye class material: crystal violet and leuco crystal violet；The application of the drug residue in described detection animal-derived food comprises the steps,
(1) standard solution is prepared,
(2) analyze pre-treatment, use the extraction and cleaning technology of rapid enzymolysis+quick Solid-Phase Extraction to carry out sample to be tested and analyze pre-treatment work,
(3) sample to be tested is carried out chromatograph and liquid matter analysis, it is thus achieved that liquid mass spectrum, contrasts described liquid mass spectral database, carry out qualitative or quantitative analysis detection,
Described qualitative analysis uses library searching to carry out, and qualitative criteria is:
(1) in sample, compound extracts retention time and the retention time phase of target analytes extraction ion stream in standard solution or interpolation sample of ion stream
Ratio, amplitude of variation is less than 5%；
(2) parent ion/daughter ion of target analytes must occur simultaneously, and signal to noise ratio >=3 of transmission ion pair；
(3) in sample, compound EPI spectrogram is compared with close concentration level standard solution in spectrum storehouse or extraction standard solution E PI spectrogram, and spectrogram coupling is pure
Degree >=60；
The method of described quantitative analysis is as follows:
Use external standard single-point method quantitative, calculate the content of target analytes by formula (1),
Formula (1):
Wherein:
Target analytes content in X-sample, μ g/kg；
The concentration of target analytes in c-sample solution, μ g/L；
The constant volume of V-sample solution, mL；
The quality of m-sample, g；
The R-response rate, %.
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