CN104897842B - Liquid-mass database for detecting residual chemicals in animal-derived food and application method thereof - Google Patents
Liquid-mass database for detecting residual chemicals in animal-derived food and application method thereof Download PDFInfo
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a liquid-mass database for detecting residual chemicals in animal-derived food and an application method thereof. The building method of the liquid-mass database includes the steps of firstly, preparing standard working solution; secondly, using the extraction purification technology combining fast enzymolysis and fast solid phase extraction (SPE) to process to-be-detected samples before analyzing; thirdly, performing one-step sample injection chromatographic analysis; fourthly, building the liquid-mass database.
Description
Technical field
The invention belongs to field of biological detection, is used to detect left drug in animal-derived food in particular to a kind of
Liquid matter data base and its using method.
Background technology
Animal-derived food accounts for suitable proportion in international agriculture trade, for the regulation of animal-derived food drug residue
Limit increasingly stricter, by taking " positive list " system that Japanese in May, 06 implements as an example, which is to 236 kinds of veterinary drugs and feedstuff addition
Concrete limit standard has been formulated in agent, and field of food safety at home, from " chloromycetin, the nitrofuran " of early stage finally
" malachite green oxalate, clenbuterol hydrochloride ", because caused by drug residue, food safety affair takes place frequently and highlights China's food safety Regulation control volume
It is technical support scarce capacity, in the urgent need to setting up multiple types residual rapid screening method.At present, for animal-derived food Chinese medicine
Thing residue detection technology and method have developed into single type multi-residue analysis by single retention analysiss, since 03 year, with material kind
The multi-residue determination standard of class circle point occupies more than the 70% of retention analysiss country and industry standard.Same time, mass spectral analyses
The technical way of multi-residue determination is had become, the technical specification rule with European Union's 2002/657/EC regulations as source
To be recognized in the world and being received.With the development of retention analysiss technology, multiple types retention analysiss are in quantity of information, detection efficiency etc.
It is many-sided to become research and development focus with advantage, and the practicality of the tandem mass spectrometer such as mass spectroscopy device field such as Q-TOF, Q-Trap
Change, the spectrum storehouse based on senior mass-spectrometric technique compares analysis, and gradually maturation also provides technology for the quick analysis of multiple types residual
May.However, either in method exploitation, checking and standardization still at the aspect such as regulation is perfect, while detection multiple types are residual
Stay：
(1) variety classes veterinary drug physico-chemical property difference is very big, polarity coverage width, in different substrates remains form not
One, indivedual veterinary drugs still need derivatization can effective detection, it is therefore difficult on general pre-treating method and the analysis of chromatograph single injected sampling
To realize effectively breaking through；
(2) regulation is differed to the limitation requirement of different veterinary drugs (forbidden drugses and limit the use of medicine) and using regulation, multiple types
Residue detection proof rule is not perfect enough；
(3) bare substrate that method exploitation needs is difficult to obtain, and mixed standard solution prepares difficult；
(4) analysis ability of instrument.In terms of liquid mass spectral database is built, storehouse such as AB companies legal medical expert's poisonous substance is composed despite commercialization
Data base's (1250 kinds of target analytes), basic veterinary drug database (139 kinds of target analytes)；The small molecule of Freiburg university hospitals
Drug data base etc., but there is following application limitation：
1) only have MS/MS data, without chromatographic information characteristics；
2) without online information feature；
3) mutually it is coupled with Instrumental Analysis without unified pre-treating method；
4) extraction standard data are lacked.
Therefore, under existence conditionses, it is difficult to effectively realize the multiple types residual rapid screening point based on liquid mass spectral database technology
Analysis.
The content of the invention
In view of process before sample universal present in the analysis of animal-derived food multiple types left drug both at home and abroad at present,
The technological difficulties such as the quick analysis of instrument and screening technique checking.The present invention is setting up animal-derived food medium or high risk medicine multiple types
For the purpose of remaining quick selective mechanisms system, by extracting, setting up sample high flux general using multi-solvent system piecewise combination
Property pretreatment technology, complex optimum chromatographic realize multiobjective analysis thing width polarity scope liquid chromatograph be effectively retained and point
From, and by build liquid chromatography-mass spectrography/mass spectrometric data storehouse for covering chromatography-mass spectroscopy multidimensional information and according to laws and regulations requirement and
Detection practice carries out the research and development of the key technology content such as screening technique checking, breaks through multiple types retention analysiss technical barrier, sets up
One remains the opening of versatility pretreatment technology, sharp separation system and liquid mass spectral database as technical characteristic quickly with multiple types
Analysis platform and effective examination detection technique system.
The present invention is for 12 kinds of Limited Doses in the following excessive risk left drugs of 1.0 μ g/kg (A class residuals) as detection
Target, this group of medicine are respectively,
(1) steroid hormone class compound：Testosterone (T), prednisolone (PNSL), betamethasone (BTS), dexamethasone
(DTS)；
(2) nitroimidazoles medicine and its metabolite：Metronidazole (MNZ), cough up nitre and rattle away azoles (RNZ), Dimetronidazole
(DMZ)；
(3) Beta- receptor stimulating agents 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 a kind of rapid screening liquid matter of detection animal-derived food medium or high risk medicine multiple types residual
The construction method in spectrum storehouse, described animal-derived food is bird egg food or Mel based food, specifically includes following steps,
(1) preparation of standard working solution；
Steroid hormone class compound, nitroimidazoles medicine and its metabolite, Beta- receptor stimulating agent class materials：Adopt
Its hybrid standard stock solution (0.01g/L) is prepared with acetonitrile classification；
The materials such as dye class prepare its hybrid standard stock solution (0.01g/L) using methanol classification；
Mixing and standard working solution (0.1mg/L), pipette respectively testosterone, prednisolone, betamethasone, dexamethasone,
Metronidazole, cough up nitre and rattle away azoles, Dimetronidazole, Clenbuterol, Tulobuterol, cimaterol, crystal violet and leuco crystal violet standard
Appropriate stock solution, with the mixed standard solution that acetontrile concentration is 0.1mg/L；
(2) pre-treatment is analyzed, the extraction using rapid enzymolysis (release Conjugate polyamines)+quick Solid-Phase Extraction (SPE) is net
Change technology carries out sample to be tested analysis pre-treatment work, and specific extraction and cleaning process is as follows：
1) extract：Homogeneous samples 5g is weighed, is placed in 50mL politef centrifuge tubes, add ammonium acetate buffer
(0.2mol/L, pH 5.2) 15mL, 50 μ L of β-glucosiduronic acid/sulfatase, are vortexed and mix, and 2h are vibrated in 50 DEG C of water-baths, let cool to
Room temperature, in 0 DEG C, 15000rpm centrifugation 5min, transfer supernatant are adjusted with NaOH solution (5mol/L) into another clean centrifuge tube
PH adds ethyl acetate 20mL to 9.0, and Sodium Chloride 6g vibrates vortex 5min, and in 0 DEG C, 15000rpm centrifugation 5min take
35 DEG C of organic facies of layer are rotated near and are done, and nitrogen is dried up, and residue is dissolved using 5mL 0.1mol/L hydrochloric acid solutions, and ultrasonic 1min is stayed
Treat column purification；
2) purify：MCX posts (Waters Oasis MCX SPE pillars) are installed on solid-phase extraction device that (device is in reality
With being documented in new patent CN201020014620.X), successively using methanol 3mL, water 3mL, 3mL 0.1mol/L salt
Acid solution is activated.Solution will be extracted to be carried on solid-phase extraction column, flowed out under gravity, successively using 3mL water, 3mL first
Alcohol and 5mL normal hexane drip washing pillars, drain 2min, and (ethyl acetate 50mL, methanol 45mL, ammonia 5mL are mixed to add 6mL eluents
It is even) eluting.At 35 DEG C of eluent, nitrogen stream is dried up, using sample lysate (measure 0.1% formic acid acetonitrile (V/V) solution 5mL,
Mix with ammonium formate (5mmol/L)-formic acid (0.1%)-aqueous solution 95mL) 0.5mL dissolved residues, ultrasonic 1min, 0.22 μM excessively
Microporous filter membrane；Described solid-phase extraction device integrates reagent rack, the function of Nitrogen evaporator and operating pressure is relatively uniform, and which is by reagent
Frame, operating board, 8 glass storehouses, 8 pear shape bottles and 1 automatic getter device composition, are capable of achieving the quick SPE of target analytes
Process.
(3) disposable sample introduction chromatography
The use of chromatographic column being Kinetex C18 posts, acetonitrile being adopted for organic faciess, formic acid is used as ionizing reinforcing agent, formic acid
Salt optimizes agent as peak shape, using the combination of water phase/acidity acetonitrile as the flow visualizing of optimization；
Actual conditions is：Chromatographic column：Kinetex C18,2.6 μm, 2.1mm × 100mm i.d.；
Flow velocity：0.2mL/min；
Sample size：10μL；
Column temperature：30℃；
Gradient elution program：(A：0.1% formic acid-acetonitrile solution；B：Ammonium formate (5mmol/L)-formic acid (0.1%)-water-soluble
Liquid)
0～2min：5%A；
～8min：20%A；
～15min：95%A；
～16min：100%A；
～19min：100%A；
20min：5%A；
(4) build liquid mass spectral database
Using the senior drainage pattern of level Four bar/ion trap tandem mass spectrometry：Presetting many reaction detection (sMRM)-information according to
Bad property collection (IDA)-enhancer ion scan (EPI)；
Mass spectrometry parameters determination process is as follows：Using the mixed mark stock solution of liquid phase according to target analyte classification dilution extremely
Concentration is 0.2mg/L, then carries out parameter optimization in the flow velocity injection mass ion source using constant current syringe pump with 5 μ L/min,
Target analytes are determined using scan patterns such as Q1MS, Q1Multiple Ions, Product Ion, MRM respectively
Parent ion, daughter ion, and using Ramp function optimizations and determine depolymerization voltage (DP), collision cell entrance potential (EP), collision
The chemical parameters such as energy (CE), collision cell exit potential (CXP)；
Mass Spectrometry Conditions (API 4000 and API 4000Q-TRAP)：
A) ion source：Electric spray ion source；
B) scan mode：Cation is scanned；
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℃；
I) sMRM parameter settings：MRM detection windows are set to 60s, and object is set to 1.4s sweep time；
J) IDA is regular：Response lag：3000cps；Dynamic background is deducted；Most strong ion is selected as 1 to 3；
K) enhancer ion scan (EPI) parameter setting：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 A class materials, liquid mass spectral database is built.
The Analyst1.5 that library software is AB SCIEX companies of building used in the structure of data base, build the standard in storehouse according to
Mentality of designing is set as：
(1) providing each target analytes uses this research to set the RT values under liquid phase chromatogram condition system；
(2) the related chemistry information of each target analytes is given (such as title, chemical formula, molecular weight, No. CAS, compound
Classification, ID, molecular structural formula etc.)；
(3) the EPI spectrograms according to the collection of 3.2.2 items under at least 5 different conditions of each target analytes are given,
That is 4 EPI spectrograms when CE is 15eV for 35eV, CES for 20eV, 35eV, 50eV and CE, additionally, at least will also have in color
EPI spectrograms when CE is 15eV for 35eV, CES under spectral condition；
(4) the EPI spectrograms of target analytes in different substrates are given as far as possible.
It is the condition must being fulfilled for that first 3 are built library standard, and the 4th article is optional standard according to practical situation.
The chromatographic and Mass Spectrometry Conditions selected according to conditions above is realized comprising 12 kinds of high residue A of the present invention
The high efficiency separation that class material is remained 115 kinds of interior veterinary drugs more.Despite individual compound retention time be close to, but its extract from
Subflow collection of illustrative plates can realize that mass spectrum is separated, and can carry out quantitative analyses, additionally, EPI spectrums storehouse target analytes patch information is special
Levy, representation compound qualitatively structure " fingerprint " information can be accurately qualitative to target analytes.It is thus obtained comprising described
The liquid mass spectral database of the liquid matter information of A class residuals, can realize residual to the animal-derived food medium or high risk medicine multiple types
Stay rapid screening.
Under above-mentioned chromatograph and Mass Spectrometry Conditions, in addition to 12 kinds of A class materials, 103 kinds of B classes totally 115 kinds of target analytes
Total ion current figure, typical (CE when selecting ion flow graph, each target analytes to extract ion flow graph and extend from CES
35eV, CES 15eV) data base's spectrogram such as Fig. 1～Fig. 4.
The invention further relates to built by said method obtain for detecting animal-derived food medium or high risk medicine multiple types
The rapid screening liquid mass spectral database of residual, described animal-derived food are bird egg food or Mel based food；
Described excessive risk medicine be Limited Doses in the following excessive risk left drugs of 1.0 μ g/kg, specially：
(a) steroid hormone class compound：Testosterone (T), prednisolone (PNSL), betamethasone (BTS), dexamethasone
(DTS)；
(b) nitroimidazoles medicine and its metabolite：Metronidazole (MNZ), cough up nitre and rattle away azoles (RNZ), Dimetronidazole
(DMZ)；
(c) Beta- receptor stimulating agent class materials：Clenbuterol (CLB), Tulobuterol (TUL), cimaterol (CIM)；
(d) dye class material：Crystal violet (CV) and leuco crystal violet (LCV).
The invention further relates to the detection animal-derived food medium or high risk medicine multiple types residual rapid screening liquid mass spectral database
The application of the drug residue in detection animal-derived food.
The invention further relates to carry out determining to the excessive risk drug residue in the food of target animal source using the liquid mass spectral database
Property or the method for quantitative analyses, methods described comprise the steps,
(1) standard solution is prepared,
(2) pre-treatment is analyzed, the extraction using rapid enzymolysis (release Conjugate polyamines)+quick Solid-Phase Extraction (SPE) is net
Change technology carries out sample to be tested analysis pre-treatment work,
(3) chromatograph and the analysis of liquid matter are carried out to sample to be tested, liquid mass spectrum is obtained, is contrasted the liquid mass spectral database, it is fixed to carry out
Property or quantitative analyses detection.
Described qualitative analyses are carried out using library searching, and qualitative criteria is as follows：
(1) in sample, compound extracts the retention time of ion stream and target analytes in standard solution or addition sample are carried
The retention time for taking ion stream is compared, and amplitude of variation is less than 5%.
(2) parent ion/daughter ion (transmission ion pair) of target analytes must occur simultaneously, and transmit the letter of ion pair
Make an uproar than (S/N) >=3.
(3) compound EPI spectrograms and close concentration level (the same concentration order of magnitude) standard solution in spectrum storehouse in sample
Or extraction standard solution EPI spectrogram is compared, spectrogram matching purity (Purity values) >=60.
The quantitative approach of described quantitative analyses is as follows：
Using external standard single-point standard measure, the content of target analytes is calculated by formula (1).
In formula (1)：
Target analytes content in X- samples, μ g/kg；
The concentration of target analytes in c- sample solutions, μ g/L；
The constant volume of V- sample solutions, mL；
The quality of m- samples, g；
The R- response rate, %.
Description of the drawings
The total ion current figure of Fig. 1 .A class materials, B classes material totally 115 kinds of target analytes.
The typical of totally 115 kinds of target analytes selects ion flow graph for Fig. 2 .A class materials, B classes material.
The target analytes of Fig. 3 .A class materials extract ion flow graph.
The target analytes spectrum database data figure of Fig. 4 .A class materials.
Specific embodiment
The preparation of 1. standard working solution of embodiment
Steroid hormone class compound, nitroimidazoles medicine and its metabolite, Beta- receptor stimulating agent class materials：Adopt
Its hybrid standard stock solution (0.01g/L) is prepared with acetonitrile classification, standard of physical storing solution can be stablized 12 months.
The materials such as dye class prepare its hybrid standard stock solution (0.01g/L) using methanol classification, can stablize 3 months.
Mixing and standard working solution (0.1mg/L), pipette respectively testosterone, prednisolone, betamethasone, dexamethasone,
Metronidazole, cough up nitre and rattle away azoles, Dimetronidazole, Clenbuterol, Tulobuterol, cimaterol, crystal violet and leuco crystal violet standard
Appropriate stock solution, with the mixed standard solution that acetontrile concentration is 0.1mg/L, -20 DEG C of the solution can be stablized 1 month.
Embodiment 2. analyzes pre-treating method
12 kinds of target compounds (A group materials) are under the jurisdiction of steroid hormone class, nitro glyoxaline, beta-receptor agonist class respectively
With 4 classes of compounds such as dye class, which performs limitation requirement scope in 0.05 μ g/kg～0.8 μ g/kg, is one group of current state
Border upper limit amount requires most stringent medicine.Therefore, the step of considering purification concentration when pretreatment process is designed, to meet
Strict limitation requirement.For the classes of compounds involved by A group materials, (release combined state is residual for research design rapid enzymolysis
Stay) the extraction and cleaning technology of+quick Solid-Phase Extraction (SPE), the pre-treatment of 12 kinds of ultra-low limit quantity of material can be completed quickly and effectively
Process.Specific extraction and cleaning process is as follows：
(1) extract：Homogeneous samples 5g (being accurate to 0.01g) is weighed, is placed in 50mL politef centrifuge tubes and (need to such as be done
Addition sample, adds the A group material mixing standard working solutions of debita spissitudo in this step, and places 30min in dark place), plus
(0.2mol/L, pH 5.2) 15mL, 50 μ L of β-glucosiduronic acid/sulfatase, are vortexed and mix, 50 DEG C of water-baths to enter ammonium acetate buffer
Vibration 2h, lets cool to room temperature, and in 0 DEG C, 15000rpm centrifugation 5min, transfer supernatant are into another clean centrifuge tube, molten with NaOH
Liquid (5mol/L) adjusts pH to 9.0, adds ethyl acetate 20mL, and Sodium Chloride 6g vibrates vortex 5min, in 0 DEG C, 15000rpm
Centrifugation 5min, takes 35 DEG C of upper organic phase and rotates to closely doing, and nitrogen is dried up, and residue is molten using 5mL 0.1mol/L hydrochloric acid solutions
Solution, ultrasonic 1min remain column purification.
(2) purify：(Waters Oasis MCX SPE pillars, this type pillar filling adsorption agent is in HLB to MCX posts
- SO is added on the basis of pillar NVP-DVB copolymers3H ion exchanging function bases.) it is installed on solid phase extraction
Take on device (device is documented in utility model patent CN201020014620.X), successively using methanol 3mL, water
3mL, 3mL 0.1mol/L hydrochloric acid solutions are activated.Solution will be extracted to be carried on solid-phase extraction column, flowed out under gravity, according to
Secondary use 3mL water, 3mL methanol and 5mL normal hexane drip washing pillars, drain 2min, add 6mL eluents (ethyl acetate 50mL, first
Alcohol 45mL, ammonia 5mL, mix) eluting.At 35 DEG C of eluent, nitrogen stream is dried up, and (measures 0.1% formic acid using sample lysate
Acetonitrile (V/V) solution 5mL, is mixed with ammonium formate (5mmol/L)-formic acid (0.1%)-aqueous solution 95mL) 0.5mL dissolved residues,
Ultrasonic 1min, crosses 0.22 μM of microporous filter membrane, liquid chromatography tandom mass spectrometry determination.
The step of to extraction and cleaning process, is described as follows：In A group materials the material such as testosterone, Tulobuterol adhere to separately hormone and β-
Receptor stimulating agent class, literature survey show this kind of normal with combined state presence, especially this benzene of Tulobuterol in animal tissue
Phenolic medicine, therefore β-glucosiduronic acid/sulfatase enzymolysis residual tissue conjugatess need to be added, to discharge free state medicine, plus
Enter buffer one and be to provide the pH environment that enzymolysis needs, use in addition and as Extraction solvent, the step compares existing side
Method employs the mode of raising hydrolysis temperature (being promoted to 50 DEG C by 36 DEG C of existing method) and makes to need originally overnight (16h) reaction
Process shorten to 2h, greatly accelerated enzymolysis process；Crystal violet and Recessive Crystal Violet tests prove that can also be in this step
It is extracted；It is precipitation fat and albumen using ultracentrifugal purpose is freezed；Purpose that is basified and adding Sodium Chloride will be extracted
It is the distribution for promoting nitro glyoxaline compound and beta-receptor agonist by the opposite organic faciess of water.Purifying step is more due to being related to
The purification of species material, SPE posts have selected the reversed material of versatility.Research compares hydrophilic-lipophilic balance (HLB) post and general
Logical C18 posts, fail to realize preferable effect.Using Waters Oasis MCX SPE pillars, this type is little for final research
Column packing adsorbent is on the basis of HLB pillar NVP-DVB copolymers to add-SO3H ion exchanges
Function base.Therefore the SPE posts possess anti-phase and ion exchange mixing and absorption function, can adsorb acidity, alkalescence in low ph value
And neutral compound, there is higher detergent power compared with HLB and C18 posts.The design of elution program is reported simultaneously with reference to pertinent literature
The step of normal hexane drip washing is increased in the step of drip washing, is disturbed with more preferable place to go lipid, and eluting solution employs acetic acid
The mixed solution of ethyl ester (50%)-methanol (45%)-ammonia (5%) water, Jing tests, 6mL can complete to wash target analytes
It is de-.
Additionally, during using SPE post detections, as existing SPE systems are difficult to realize fast operating that seminar grinds
A kind of new solid-phase extraction device is sent out, the device can integrate reagent rack, the function of Nitrogen evaporator and operating pressure is relatively uniform, its
It is made up of reagent rack, operating board, 8 glass storehouses, 8 pear shape bottles and 1 automatic getter device, is capable of achieving the fast of target analytes
Fast SPE processes.
To sum up, solvent extraction/Solid phase extraction pre-treatment flow process of the A groups material pre-treating method although with classics,
But consider in the design of rapid enzymolysis and the quick SPE purification process of versatility multiple types residual extract and flux process because
Element, compares existing method and has fast and convenient advantage, and ultralow limitation thing in 4 classes 12 is completed in a pretreatment process
Effective extraction of matter, meets the requirement of fast Screening analysis.
3. disposable sample introduction chromatographic analysis system of embodiment is studied
1. the selection of chromatographic column
The liquid chromatograph of multiobjective analysis thing is separated and is generally considered using ultramicro powder (2 μm of particle diameter ＜) chromatographic column at present,
But this research adopts conventional H PLC system, and which is pressure, and the upper limit is 400Bar, therefore cannot use Ultra Performance Liquid Chromatography post.To realize
The separation purpose of design, it is considered to which system pressure is limited by the control of the particle size range of chromatographic column between 2 μm～3 μm, column length
Degree scope is 100mm～150mm；Further, since classes of compounds is more, polarity range spans are larger, in chromatographic column type side
Face just have selected the C18 chromatographic columns for being suitable for separating wide polarity scope.By above-mentioned restriction, this research is according to polarity (reference
LogD values are selected) one kind in every class compound is have selected, 4 kinds of materials represent 4 type of examination as analyte altogether
Liquid-phase chromatographic column.Screening conditions and the results are shown in Table 2.
2 chromatographic column selectivity experimental design of table and separation analysis result
As seen from the above table, other chromatographic columns are compared, Kinetex C18 post separations degree and sensitivity are preferable.Although the color
Spectrum post particle diameter is minimum, but its post back pressure is still in the pressure scope of conventional liquid phase.And filler particles employ advanced core-shell structure copolymer skill
Art, compared with traditional Bio-sil post completely, can be obviously improved separating degree and sensitivity.Determine and using the type chromatographic column be
Preferable separate post.
2. the selection of flow visualizing and the determination of other chromatographic conditions
To mass spectral analyses conventional mobile phase (water, formic acid-aqueous solution, acetic acid-aqueous solution, methanol, acetonitrile, acidified methanol, acid
Change acetonitrile, Ammonium formate buffer, ammonium acetate buffer etc.) and composition screened using 12 kinds of hybrid standard working solutions.
On the basis of considering the factors such as separating degree, sensitivity and analysis time, final determination adopts acetonitrile for organic faciess, and formic acid is made
For ionizing reinforcing agent, formates optimize agent as peak shape, using the combination of water phase/acidity acetonitrile as the mobile phase body of optimization
System.
The liquid chromatograph parameter such as sample size, column temperature and flow velocity is optimized, main considerations include chromatographic resolution,
Sensitivity, repeatability and matrix effect etc..By using 12 kinds of object mixed standard solutions in screening target concentration levels
Chromatographic isolation is carried out on (0.5 μ g/kg) to test and determine optimal value.
On the basis of optimizing relevant parameter and comparing test of many times result, the disposable liquid chromatograph originally determined point
The actual conditions of system is in vitro：Chromatographic column：Kinetex C18,2.6 μm, 2.1mm × 100mm i.d.；Flow velocity：0.2mL；Sample introduction
Amount：10μL；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.
4. liquid mass spectral database of embodiment builds
1. the foundation of mass spectrum acquisition method and liquid mass spectral database build
(1) foundation of mass spectrum acquisition method
This research employs the senior drainage pattern of level Four bar/ion trap tandem mass spectrometry：Presetting many reaction detection
(sMRM)-information dependency collection (IDA)-enhancer ion scan (EPI).The foundation of the drainage pattern, first has to determine mesh
Mark analyte retention time in chromatographic, next sets up multiple-reaction monitoring (MRM) mass spectrometry method, then setting triggering EPI
The condition (i.e. IDA settings) of collection, is finally to determine the condition setting of EPI collections.
To set up corresponding mass spectrum acquisition method, test setting is carried out to the mass spectrometry parameters of 12 kinds of target analytes first.
Research is using first Classified optimization chemical parameters (including parent ion, daughter ion and depolymerization voltage, collision energy etc.), then chooses generation
Table compound optimization source parameters (including atomization gas pressure, ion source temperature etc.).
1. chemical parameters optimization determines.About the basis of design chromatographic isolation result of RT parameters.Mass spectrometry parameters determined
Journey is as follows：The use of the mixed mark stock solution of liquid phase according to target analyte classification dilution is 0.2mg/L to concentration, but it is noted that
The target analytes for having same molecular amount in same category individually to prepare (because studied use instrument is standard resolution, nothing
Method distinguishes isomerss), then enter line parameter in the flow velocity injection mass ion source using constant current syringe pump with 5 μ L/min excellent
Change.Determined using scan patterns such as Q1MS, Q1Multiple Ions, Product Ion, MRM respectively the mother of target analytes from
Son, daughter ion (at least choose 2 pairs and transmit the signal to noise ratio that ions are standby, in follow-up test according to response by every kind of target analytes
A pair of optimum ions are chosen as the collection ion of sMRM with the disturbed condition in bare substrate), and it is excellent using Ramp functions
Change and determine that depolymerization voltage (DP), collision cell entrance potential (EP), collision energy (CE), collision cell exit potential (CXP) etc. are changed
Compound 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 refer to retention time
Note：1. in the table, listed ion pair is the optimization determined after matrix effect and signal to noise ratio are examined or check in follow-up test
Ion pair, the parameter of the unlisted standby ion pair of here；2. target analytes retention time (RT) is in RT settings window (15s)
Change.
2. the optimization of source parameters determines.Optimized using Flow Injection Analysis (FIA), because instrument is to optimizing ion pair
This research of Limited Number system is optimized using representation compound, and representation compound selection principle is optimized using chemical parameters
When respond relatively low target analytes, Jing tests, source parameters optimum results are as follows：Gas curtain atmospheric pressure：30psi；Spray
Mist voltage：5500V；Ion source temperature：475℃；Atomization gas pressure：40psi；Assist gas pressure power：45psi.
3. IDA conditions setting.In the setting of IDA conditions, it is most important that the measure of response lag, in screening aimed concn
Extraction standard (standard solution for using " bare substrate " extracting solution to prepare) is analyzed in level (0.025 μ g/kg), with minimum response
The 1/2 of substance responds intensity is degree setting response lag.It should be noted that the threshold value has substrate dependency, different bases
Matter threshold value is different, it is generally the case that threshold value setting principle is to ensure all responses for meeting examination aimed concn as far as possible
EPI collections are can trigger, while improve threshold value to reduce data acquisition amount (being easy to analysis) as far as possible.Two kinds have been investigated in this research
Animal sources substrate (bird egg food or Mel based food), is set as 3000cps with minimum.
Secondly, to also select instrument dynamic background to deduct function in IDA settings kind, can effectively reduce the product of invalid data
It is raw.
4. the setting of EPI parameters.
A) EPI acquisition qualities 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 sets 70Da～
1000Da；
B) EPI acquisition scans speed：This research is AB SCIEX 5500Q-Trap mass spectrographs using equipment, and EPI collections are altogether
There is 3 notch speed degree of 1000Da/s, 10000Da/s and 20000Da/s, be to ensure the quality of data, select 10000Da/s to gather for EPI
Scanning speed, had so both taken into account the analysis acquisition rate of 12 kinds of target analytes, in turn ensure that spectrogram quality；
C) setting of EPI collection DP values and EP values：To ensure spectrum database data quality, to 12 kinds of target analytes DP values and EP
Value carries out segmentation statistics, and the median for taking section at high proportion is final setting value.
Jing is analyzed, and target compound DP values are increased in 60V～100V sections ratio, takes the DP that median 80V is EPI collections
Setting value.Likewise, target group compound EP values are in 9V～11V section large percentages, take what median 10V was gathered as EPI
EP setting values.After determining DP and EP setting values, to DP values and the collection of EP values EPI, it was demonstrated that the DP values and EP values of setting is to this kind ofization
The EPI data acquisition qualities of compound have no significant effect；
D) EPI gathers the setting of collision energy (CE) value, with reference to table 3, the CE values of general common compounds 20eV～
30eV, but the CE values of individual compound such as crystal violet have the higher crumb data of quality in 45～60eV,.For this purpose, first
Fixed CE values are 35, reset extension CE values (CES) be 5,10,15 etc. 3 kinds combine and examine or check EPI spectrogram quality.CE is chosen finally
For 35eV, CES is that 15eV (cumulative means of 3 spectrograms when being respectively 20eV, 35eV, 50eV equivalent to CE) is gathered as EPI
CE settings, the setting value can preferably take into account high, medium and low collision energy section, can obtain high-quality data spectrogram.
By above method, the senior acquisition method of the mass spectrum (sMRM-IDA-EPI) of 12 kinds of target analytes is established, can
Examination collection (using the collection ion pair of sMRM) is carried out using this pattern, then (concentration-response exceedes to meeting examination rule
Screening aimed concn) object carry out EPI collections, obtain detailed ion information to carry out qualitative analyses.
(2) liquid mass spectral database builds
1. online EPI spectral datas collection.Using 12 kinds of object hybrid standard working solutions using flowing phase dilution after match somebody with somebody
Concentration processed is to screen the mixed standard solution of target concentration levels, and upper machine analysis gathers online EPI in sMRM-IDA-EPI modes
Data, compose storehouse using Analyst1.5 software buildings, and improve goal analysis information (as Chinese and English title, chemical formula, No. CAS,
Chemical structural drawing etc.).
2. offline EPI spectral datas collection.Using classification prepare standard reserving solution (as apoplexy due to endogenous wind has isomerss,
Need independent sample introduction) 0.2mg/L is diluted to using mobile phase, direct mass spectrum sample introduction gathers offline EPI with reference to online EPI conditions
Data, individually gather 3 CE levels (basic, normal, high) EPI spectrograms in addition again, can be according to compound actual nature using optimization
Collision energy.Storehouse is composed in above-mentioned gathered data typing.
3. liquid mass spectral database is judged using rule.Library searching is one of maximally effective qualitative tool, but in liquid mass spectral database
Any regulation and technical stipulation are there is no at present in terms of the matched rule formulation of retrieval.This research is according to European Union and U.S.'s relevant regulations
Determine the decision rule of retention time and signal to noise ratio；EPI spectrograms compare the determination of the matching tolerance factor and adopt 10 " blank bases
Matter " (Mel and egg sample are each 5) prepares extraction standard, and the standard EPI collection of illustrative plates composed with EPI in storehouse uses Analyst 1.5
Software carries out the matching analysis, obtains Reinheitszahl (purity), obtains meansigma methodss and standard deviation.Jing is compared, all objects
Purity meansigma methodss deduct standard deviation and are all higher than 60, are to control false negative rate to greatest extent, originally determine purity values 60
For the matching tolerance factor.To sum up, the library searching rule determined is：
A) in sample, compound extracts the retention time of ion stream and target analytes in standard solution or addition sample are carried
The retention time for taking ion stream is compared, and 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 transmit from
Son to signal to noise ratio (S/N) >=3；
C) spectrogram matching purity (Purity values) or the matching tolerance factor >=60.
Spectrum storehouse compare qualitative function it is powerful, because the information which provides it is more.Specified according to European Union 2002/657/EC, originally ground
Study carefully drainage pattern and can obtain confirmation points >=5.5 (the most stringent of disabling veterinary drug of European Union requires nothing more than confirmation points >=4).So if
If library searching matching, fast qualitative confirmation can be carried out to target analytes.
Claims (5)
1. a kind of construction method of the rapid screening liquid mass spectral database of detection animal-derived food medium or high risk medicine multiple types residual, has
Body comprises the steps,
(1) preparation of standard working solution；
(2) pre-treatment is analyzed, and the extraction and cleaning technology for Conjugate polyamines+quick Solid-Phase Extraction being discharged using rapid enzymolysis is treated
Survey sample analyses pre-treatment work；
(3) disposable sample introduction chromatography；
(4) build liquid mass spectral database；
Described animal-derived food is bird egg food or Mel based food,
Described excessive risk medicine be Limited Doses in the following excessive risk left drugs of 1.0 μ g/kg, specially：
(a) steroid hormone class compound：Testosterone, prednisolone, betamethasone, dexamethasone；
(b) nitroimidazoles medicine and its metabolite：Metronidazole, cough up nitre and rattle away azoles, Dimetronidazole；
(c) Beta- receptor stimulating agent class materials：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 class compound, nitroimidazoles medicine and its metabolite, Beta- receptor stimulating agent class materials：Using second
Hybrid standard stock solution of the nitrile classification compound concentration for 0.01g/L；
Dye class material adopts methanol classification compound concentration for the hybrid standard stock solution of 0.01g/L；
Concentration is 0.1mg/L hybrid standard working solutions, pipettes testosterone, prednisolone, betamethasone, dexamethasone, first respectively
Nitre azoles, cough up nitre rattle away azoles, Dimetronidazole, Clenbuterol, Tulobuterol, cimaterol, crystal violet and leuco crystal violet standard storage
Standby solution is appropriate, with the mixed standard solution that acetontrile concentration is 0.1mg/L；
Analysis pre-treating method described in step (2) is as follows,
1) extract：
Weigh homogeneous samples 5g, be placed in 50mL politef centrifuge tubes, addition concentration be 0.2mol/L, the acetic acid of pH 5.2
Ammonium buffer 15mL, β -50 μ L of glucosiduronic acid/sulfatase, are vortexed and mix, and 2h is vibrated in 50 DEG C of water-baths, lets cool to room temperature, in 0
DEG C, 15000rpm centrifugation 5min, transfer supernatant adjust pH to 9.0 with the NaOH solution of 5mol/L into another clean centrifuge tube,
Ethyl acetate 20mL is added, Sodium Chloride 6g vibrates vortex 5min, in 0 DEG C, 15000rpm centrifugation 5min take upper organic phase
35 DEG C rotate to closely doing, and nitrogen is dried up, and residue is dissolved using 5mL 0.1mol/L hydrochloric acid solutions, and ultrasonic 1min remains upper prop net
Change；
2) purify：
MCX posts are installed on solid-phase extraction device, successively using methanol 3mL, the activation of water 3mL, 3mL 0.1mol/L hydrochloric acid solution；
Solution will be extracted to be carried on solid-phase extraction column, flowed out under gravity, successively using 3mL water, 3mL methanol and 5mL
Normal hexane drip washing pillar, drains 2min, adds 6mL elutions, and at 35 DEG C of eluent, nitrogen stream is dried up, and is dissolved using sample
Liquid 0.5mL dissolved residues, ultrasonic 1min cross 0.22 μm of microporous filter membrane；
Described solid-phase extraction device integrates reagent rack, the function of Nitrogen evaporator, and operating pressure is relatively uniform, its by reagent rack,
Operating board, 5～10 glass storehouses, 5～10 pear shape bottles and 1～2 automatic getter device composition, realize the fast of target analytes
Fast SPE processes；
The 1 of described step (2)) in extraction step, such as sample is eggs sample, then add appropriate kieselguhr；
The 2 of described step (2)) in purifying step,
The collocation method of eluent is：Ethyl acetate 50mL, methanol 45mL, ammonia 5mL are mixed；
The collocation method of sample lysate is：Measure 0.1% formic acid acetonitrile solution 5mL of volume ratio, with the ammonium formate of 5mmol/L-
0.1% formic acid-aqueous solution 95mL is mixed；Disposable sample introduction chromatogram analysis method described in the step (3) is as follows,
The use of chromatographic column being Kinetex C18 posts, acetonitrile being adopted for organic faciess, formic acid is made as ionizing reinforcing agent, formates
Optimize agent for peak shape, using the combination of water phase/acidity acetonitrile as the flow visualizing of optimization；
Actual conditions is：Chromatographic column：Kinetex C18,2.6 μm, 2.1mm × 100mm i.d.；
Flow velocity：0.2mL/min；
Sample size：10μL；
Column temperature：30℃；
Gradient elution program is as follows：
A eluents are：0.1% formic acid-acetonitrile solution；
B eluents are：Formic acid-the aqueous solution of the ammonium formate -0.1% of 5mmol/L.
2. method according to claim 1, it is characterised in that step (4) the liquid mass spectral database method that builds is：
Using the senior drainage pattern of level Four bar/ion trap tandem mass spectrometry：Presetting many reaction detection-information dependency collection-
Enhancer ion scan；
The Mass Spectrometry Conditions of API 4000 and API 4000Q-TRAP systems are：
Ion source：Electric spray ion source；
Scan mode：Cation is scanned；
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℃；
SMRM parameter settings：MRM detection windows are set to 60s, and object is set to 1.4s sweep time；
IDA is regular：Response lag：3000cps；Dynamic background is deducted；Most strong ion is selected as 1 to 3；
Enhancer ion scan parameter setting：Quality of scanning number scope is 70～1000Da；Scanning speed is 10000Da/s；Sweep
Accumulative frequency is retouched for 1；Collision energy is 35eV；Extension collision energy is 15eV；
By the mass spectral results for 12 kinds of excessive risk medicines, liquid mass spectral database is built.
3. method according to claim 2, it is characterised in that the library software of building used in the structure of data base is AB
The Analyst1.5 of SCIEX companies, the standard for building storehouse are set as according to mentality of designing：
Step (1) provides each target analytes and uses this research to set the RT values under liquid phase chromatogram condition system；
Step (2) provides the related chemistry information of each target analytes, and described chemical information includes title, chemical formula, divides
Son amount, No. CAS, compounds category, ID, molecular structural formula；
Step (3) provides the EPI spectrograms gathered under at least 5 different conditions of each target analytes, and the EPI spectrograms include CE
For 20eV, 35eV, 50eV and CE be 35eV, CES be 15eV when 4 EPI spectrograms.
4. method according to claim 3, it is characterised in that also provide target in different substrates as far as possible including step (4)
The EPI spectrograms of analyte.
5. the arbitrary described methods of usage right requirement 1-4 carry out qualitative to the excessive risk drug residue in the food of target animal source
Or the method for quantitative analyses, methods described comprises the steps,
(1) standard solution is prepared,
(2) pre-treatment is analyzed, place before sample to be tested analysis is carried out using the extraction and cleaning technology of rapid enzymolysis+quick Solid-Phase Extraction
Science and engineering is made,
(3) chromatograph and the analysis of liquid matter are carried out to sample to be tested, liquid mass spectrum are obtained, is contrasted the liquid mass spectral database, carry out it is qualitative or
Quantitative analyses are detected；Described excessive risk medicine be Limited Doses in the following excessive risk left drugs of 1.0 μ g/kg, specially：
(a) steroid hormone class compound：Testosterone, prednisolone, betamethasone, dexamethasone；
(b) nitroimidazoles medicine and its metabolite：Metronidazole, cough up nitre and rattle away azoles, Dimetronidazole；
(c) Beta- receptor stimulating agent class materials：Clenbuterol, Tulobuterol, cimaterol；
(d) dye class material：Crystal violet and leuco crystal violet；
Described qualitative analyses are carried out using library searching, and qualitative criteria is：
(1) in sample compound extract target analytes in retention time and the standard solution or addition sample of ion stream extract from
The retention time of subflow is compared, and amplitude of variation is less than 5%；
(2) parent ion/daughter ion of target analytes must occur simultaneously, and transmit signal to noise ratio >=3 of ion pair；
(3) compound EPI spectrograms and close concentration level standard solution or extraction standard solution EPI spectrogram phase in spectrum storehouse in sample
Than spectrogram matching purity >=60；
The method of described quantitative analyses is as follows：
Using external standard single-point standard measure, the content of target analytes is calculated by formula (1),
Formula (1)：
Wherein：
Target analytes content in X- samples, μ g/kg；
The concentration of target analytes in c- sample solutions, μ g/L；
The constant volume of V- sample solutions, mL；
The quality of m- samples, g；
The R- response rate, %.
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