CN106546671A - Method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products - Google Patents
Method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses the method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, it is by preparing sample solution, linear solvent, blank solution and mark-on reclaims test sample solution, using three posts two dimension Liquid Chromatography/Mass Spectrometry and internal standard method, switch liquid chromatography mass spectrometric GC-MS using in terminal, realize the in-line purification purpose of sulfa drug residue, substantially increase detection efficiency, reduce the labour intensity of experimenter, solve the cumbersome pre-treating method of the various sulfa drugs of conventional analytical techniques, the sample detection cycle is long, the impurity interference of manual pretreatment is big, reappearance is difficult to ensure that and various sulfa drugs are while be difficult to meet the problem that medicine to be measured obtains good qualitative, quantitative effect when analyzing, the detectability of residue of veterinary drug can be lifted, advanced technology support is provided for analysis decision.
Description
Technical field
The invention belongs to technical field of food safety detection, and in particular to the survey of sulfa drug residue in a kind of meat products
Determine method, the method that sulfa drugs is remained in being based especially on three posts two dimension HPLC/MS-MS meat products.
Prior art
, only to particular substrate, the scope of application is narrower, determines species for the sample pre-treatments of existing sulfa drug residue detection
It is limited, and many pre-treating methods are cumbersome, need a large amount of poisonous and hazardous reagents, expend the substantial amounts of pre-treatment time.
Prior art typically adopts liquid phase or LC-MS law technology in the majority, from ethyl acetate, methyl alcohol, acetonitrile, high chlorine
After sour equal solvent is extracted, purified with solid-phase extraction columns such as C18, HLB, MCX, pre-treatment is relatively complicated, before sample
Long processing period, is affected greatly by artificial technology.
The content of the invention
For the above-mentioned problems in the prior art, it is an object of the invention to provide based on three posts two dimension LC-MS
Method remains sulfa drugs method in determining meat products.It is cumbersome that it will solve the various sulfa drugs of conventional analytical techniques
Pre-treating method, sample detection cycle length, manual pretreatment impurity interference is big, reappearance is difficult to ensure that and various sulfanilamide (SN)
Class medicine is difficult to meet the problem that medicine to be measured obtains good qualitative, quantitative effect when analyzing simultaneously.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
In comprising the steps：
1）The preparation of hybrid standard product working solution：Precision pipettes each sulfa drugs storing solution of 0.1mL 1.0mg/mL respectively
Into 10mL volumetric flasks, with methanol constant volume, then precision pipettes 1 mL mixed liquors into another 10mL volumetric flasks, with methanol constant volume,
The hybrid standard product working solution of 1 mg/L is made, is saved backup at 4 DEG C；
2）The preparation of Isotopic Internal Standard working solution：Precision pipettes the internal standard storing solution of 0.1 mL 1.0mg/mL to 10 mL capacity
In bottle, first is used with internal standard storing solution in methanol constant volume, then the accurate volumetric flask for pipetting 0.1 mL constant volumes into 50 mL volumetric flasks
Alcohol constant volume, is obtained the internal standard working solution of 20 ng/mL, saves backup at 4 DEG C；
3）The preparation of sample solution：Meat products to be measured is taken, is put in 50 mL high speed centrifugation pipes, it is accurate to add step 2）What is obtained is same
The plain internal standard working solution in position and double solvents, high-speed homogenization 1-3 min, are centrifuged 4-6 min in 3500-4500 r/min, take
15 mL nitrogen of clear liquid is dried up, and residue is redissolved with 10% methanol aqueous solution, sample solution is obtained standby；
4）The preparation of linear solvent：The meat products without sulfa drugs is weighed, is put in 50 mL high speed centrifugation pipes, it is accurate to add
Double solvents, high-speed homogenization 1-3 min are centrifuged 4-6 min in 3500-4500 r/min, take 15 mL nitrogen of supernatant and dry up,
Residue is redissolved with 10% methanol aqueous solution, and vehicle solution is obtained, accurate to draw different amounts of step 1）Obtained hybrid standard
Product working solution and same amount of step 2）Obtained Isotopic Internal Standard working solution, obtains each sulphur by vehicle solution dilution
Drug amine concentration is respectively 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, and internal standard concentration is 2 ng/
The linear criterion solution of mL, for chromatographic determination；
5）The preparation of blank solution：The sample without sulfa drugs is taken, is put in 50 mL high speed centrifugation pipes, it is accurate to add step
2）The Isotopic Internal Standard working solution for obtaining and double solvents, high-speed homogenization 1-3 min are centrifuged 4-6 in 3500-4500 r/min
Min, takes 15 mL nitrogen of supernatant and dries up, and residue is redissolved with 10% methanol aqueous solution, blank solution is obtained, for chromatographic determination；
6）The preparation of mark-on reclaims test sample solution：The sample without sulfa drugs is taken, is put in 50 mL high speed centrifugation pipes,
It is accurate to add different amounts of step 1）Obtained hybrid standard product working solution, obtains detection and is limited to 1ng/mL, is quantitatively limited to
2ng/mL, basic, normal, high varying level concentration are respectively the mark-on test specimen of 5ng/mL, 10ng/mL, 20ng/mL, then to this
It is accurate in mark-on test specimen to add 100 L steps 2）Obtained Isotopic Internal Standard working solution is accurate to add 15-25 mL multiple
Bonding solvent, high-speed homogenization 1-3 min are centrifuged 4-6 min in 3500-4500 r/min, take 15 mL nitrogen of supernatant and dry up, residual
Slag is redissolved with 10% methanol aqueous solution, mark-on reclaims test sample solution is obtained, for chromatographic determination；
7）It is molten with three posts two dimension Liquid Chromatography/Mass Spectrometry determination sample solution, linear solvent, blank solution and mark-on reclaims test specimen
Liquid
A is determined：Sample introduction process：The examination of 5 μ L sample solution, linear solvent, blank solution and mark-on reclaims is drawn respectively by injector
Test in sample solution injecting chromatograph, with three posts two dimension Liquid Chromatography/Mass Spectrometry, target compound peak area in determination sample solution,
Internal standard compound peaks area；Target compound peak area, internal standard compound peaks area in linear solvent；Mark-on reclaims test specimen
Target compound peak area, internal standard compound peaks area in solution, blank solution noiseless, standard at the target compound appearance
In working solution and sample solution, the response of component to be measured all should be within the range of linearity of monitor；
B curvilinear equation slopes, the calculating of curvilinear equation intercept
With the target compound peak area ratio internal standard compound peaks area quantitative in linear solvent, curvilinear equation A=aC+b is obtained,
In formula, C is target compound residual quantity in solution, and unit is ng/mL；A is target compound peak area ratio internal standard compound
Peak area ratio；A is curvilinear equation slope；B is curvilinear equation intercept, in concentration according to linear solvent, the linear solvent measured
Target compound peak area, internal standard compound peaks area, using fitting process, calculate a and b；
C interpretations of result：In sample solution the retention time of non-principal component respectively with linear solvent in target compound same
Retention time in chromatographic column compares, two groups of retention times of certain component and a certain sulfa drugs in linear solvent in sample
Two groups of retention times absolute value in 0.05min, regard as the sulfa drugs, the target compound in sample solution is residual
Allowance concentration C, its unit are ng/mL, and computing formula is as follows：
C=(A-b)/a
In formula：A is the target compound peak area ratio internal standard compound peaks area in sample solution；
A and b are calculated by step b；
D result verifications：Mark-on reclaims test sample solution calculates actual target compound residual quantity concentration C by step c, with
Its theoretical target compound residual quantity concentration C compares, and calculates its average recovery rate, and average recovery rate is 60-120%, it was demonstrated that
The detection method is feasible.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
Include sulfaisodimidine, sulfamerazine, sulfamethizole, sulfamethoxypyridazine, nefrosulfin in each sulfa drugs
Pyridazine, sulfamethoxazole, sulfamonomethoxine, sulfanilamide (SN) benzoyl, sulfadimethoxine and sulfamethoxazole.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
In step 2）In in be designated as sulfamethyldiazine -13C6.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
In step 3）In double solvents be 0.1% formic acid, 80% acetonitrile solution.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
In step 3）In sample solution with 0.22 μm of membrane filtration.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
In when prepared by solution, 2 min of high-speed homogenization is centrifuged 5 min in 4000 r/min, takes 15 mL nitrogen of supernatant and dry up, and residue is used
0.75mL10% methanol aqueous solutions redissolve.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
It is as follows in three posts two dimension Liquid Chromatography/Mass Spectrometry：First using the volume exclusion principle of gel chromatography, the matrix interference thing of macromolecular is made
The not reserved direct wash-out of matter, retention time is longer on a column for the target compound of small molecule, realize target compound with
The separation of matrix, removes matrix interference；After gel aqueous phase post is by the separation of matrix interference thing, Two way chromatograms system is cut
Change, target compound is retained on enriching column, after object is completely transferred to enriching column, Two way chromatograms are once being switched to
Original state, while object carries out separation analysis in the second dimension chromatographic column, by switching liquid phase-mass spectrometry skill in terminal
Art realizes the in-line purification of sulfa drug residue.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
It is as follows in chromatographic condition：
1）One-dimensional liquid-phase condition：
Chromatographic column：5 μm of Agilent Bio SEC-5,150,4.6 × 150mm,
Mobile phase：Phosphate buffer (pH7.4), isocratic elution,
Phosphate buffer takes potassium dihydrogen phosphate 1.36g, plus 0.1mol/L sodium hydroxide solution 79mL, is diluted with water to
200mL is obtained,
Flow velocity：0.35mL/min, enriching column：XBridge C8 Direct Connect HP 10μm；
2）Two-dimentional liquid-phase condition：
Chromatographic column：2.1 × 50 mm of ACQUITY UPLC BEH C18 posts, 1.7 μm,
Mobile phase：A phases：0.1 % formic acid waters, B：0.1 % formic acid methyl alcohol, gradient elution,
Column temperature：25 DEG C, sample size：5 μ L,
3）Mass Spectrometry Conditions：
Mass spectrograph：Triple quadrupole rods tandem mass spectrometry instrument, ion gun：Electric spray ion source,
Scan mode：Cation is scanned, detection mode：Multiple-reaction monitoring,
Desolventizing temperature (DEG C)：500, source temperature (DEG C)：150,
Desolventizing flow velocity (L/hr)：800, taper hole flow velocity (L/hr)：150.
The described method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, its feature exist
It is as follows in sample introduction analysis detailed process：In the presence of quaternary pump, from automatic sampler to one-dimensional chromatographic column sample introduction, using gel
The volume exclusion principle of chromatogram, makes the not reserved direct wash-out of matrix interference material of macromolecular, left switching valve and right switching valve
Original state is kept in 0-5.2min, i.e., left No. 2 positions of switching valve and No. 3 positions communicate, matrix interference thing etc. to waste liquid pool, realized
Target compound is separated with matrix, removes matrix interference, and No. 2 positions of right switching valve are communicated with No. 3 positions, are switched to the shape of waste liquid pool
State；Vavle switching is changed in 5.2-11.2min left cuts to communicate with No. 1 position to position 1, i.e., No. 2 position, target compound is switched to into enrichment
Be enriched with post, changed Vavle switching to position 2 in 11.2min left cuts, binary pump by the target compound on enriching column recoil to
Separated on Two way chromatograms post, right cut changes that valve position is constant, using binary pump mobile phase by the phosphate-buffered on enriching column
Solution is rushed to waste liquid pool, prevents phosphate from causing to damage to mass spectrometer system, and the right cut in 16.2min changes Vavle switching to position 1,
I.e. No. 2 positions are communicated with No. 1 position, are analyzed into mass detector.
By using above-mentioned technology, compared with prior art, beneficial effects of the present invention are as follows：
1）The method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products of the present invention, by adopting
Double solvents is effectively extracted to the residual sulfa drugs in meat products, greatly simplifies pretreatment mode, by adopting three
Post two dimension Liquid Chromatography/Mass Spectrometry, switches liquid phase-mass spectrometric hyphenated technique using in terminal, realizes the in-line purification of sulfa drug residue
Purpose, substantially increases detection efficiency, reduces the labour intensity of experimenter, solves the various sulfamidos of conventional analytical techniques
The loaded down with trivial details pre-treating method of pharmaceutical practice, sample detection cycle length, the impurity of manual pretreatment disturb big, reappearance to be difficult to ensure that
And various sulfa drugs are difficult to meet the problem that medicine to be measured obtains good qualitative, quantitative effect when analyzing simultaneously, together
When as using gel aqueous phase chromatogram, its mobile phase is water, therefore also realize sulfa drug residue detection environmental protection；
2）After gel aqueous phase post is by the separation of matrix interference thing, Two way chromatograms system is switched over the present invention, by target chemical combination
Thing is retained on enriching column, after object is completely transferred to enriching column, Two way chromatograms once switching to original state, while
Object carries out separation analysis in the second dimension chromatographic column.Sulfamido is realized by switching liquid phase-mass spectrometric hyphenated technique in terminal
The in-line purification purpose of medicament residue, substantially increases detection efficiency, reduces the labour intensity of experimenter, simultaneously because adopting
Gel aqueous phase chromatogram is used, its mobile phase is water, therefore also realize the environmental protection of sulfa drug residue detection；
3）Detection by gel chromatography to be applied to sulfa drug residue of the invention, establishes a brand-new quick sulphur
The detection method of sulfonamides residues, can effectively monitor and tackle the food security burst thing of corresponding various sulfa drug residues
Part, it is to avoid and reduce the harm that various sulfa drug residue food safety risks are brought.Meanwhile, as a kind of new quick inspection
Survey technology, not only reduces the contact of poisonous and harmful chemical reagent, reduces the cost of experiment, is also beneficial to experimenter oneself
Body health and safety, reaches the purpose of green test, by the two-dimentional LC-MS for the detection of various sulfa drug residues
The project research of method, can lift the detectability of residue of veterinary drug, and the analysis decision for government administration department provides advanced technology
Support.
Description of the drawings
Fig. 1 is the three posts two dimension LC-MS fundamental diagram of the present invention；
Fig. 2 is reference substance（20ng/mL）Typical chromatogram：
In figure：1- quaternary pumps, 2- automatic samplers, the one-dimensional chromatographic columns of 3-, 4- UV-detectors, the left switching valves of 5-, 6- right cuts are changed
Valve, 7- Two way chromatograms posts, 8- enriching columns, 9- binary pumps, 10- mass detectors.
Specific embodiment
The invention will be further described with reference to embodiments, but protection scope of the present invention is not limited to that：
Embodiment：Meat products in the embodiment of the present invention is chicken
1）Hybrid standard product working solution（1 mg/L）Preparation：Precision pipettes 0.1mL sulfaisodimidines, sulfanilamide (SN) respectively
First pyrimidine, sulfamethizole, sulfamethoxypyridazine, cistosulfa, sulfamethoxazole, sulfamonomethoxine, N'-phenylsulfanilamide first
Acyl, sulfadimethoxine and sulfamethoxazole storing solution（1.0mg/mL）Into 10 mL volumetric flasks, with methanol constant volume,
Precision is pipetted in the mL volumetric flasks of 1 mL to 10, with methanol constant volume, obtains final product hybrid standard product working solution, is preserved at 4 DEG C；
2）Isotopic Internal Standard working solution（20 ng/mL）Preparation：Precision pipettes 0.1 mL internal standard storing solutions（1.0mg/mL）Extremely
In 10 mL volumetric flasks, with methanol constant volume, precision is pipetted in the mL volumetric flasks of 0.1 mL to 50, with methanol constant volume, obtains final product 20 ng/
The internal standard working solution of mL, preserves at 4 DEG C；
3）The preparation of sample solution：Precision weighs the commercially available chicken meat samples of 5g, puts in 50 mL high speed centrifugation pipes, accurate to add 100 L
Step 2）Obtained Isotopic Internal Standard working solution（20 ng/mL）, accurate addition 0.1% formic acid, 80% acetonitrile solution is combined molten
20 mL of agent, 2 min of high-speed homogenization, are centrifuged 5 min in 4 000 r/min, take 15 mL nitrogen of supernatant and dry up, and residue uses 0.75
ML10% methanol aqueous solutions redissolve, and are filtered with 0.22 μm of filter membrane, take subsequent filtrate as sample solution；
4）The preparation of linear solvent：Chicken meat samples of the 5g without sulfa drugs is weighed, is put in 50 mL high speed centrifugation pipes, it is accurate
0.1% formic acid of 20mL, 80% acetonitrile solution double solvents, 2 min of high-speed homogenization is added 5min to be centrifuged in 4000 r/min, takes
15 mL nitrogen of supernatant is dried up, and residue 0.75mL10% methanol aqueous solutions redissolve, and vehicle solution is obtained, accurate to draw not
The step of same amount 1）Obtained hybrid standard product working solution and same amount of step 2）Obtained Isotopic Internal Standard working solution,
By vehicle solution dilution obtain each sulfonamide concentration be respectively 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL,
50 ng/mL, internal standard concentration are the linear criterion solution of 2 ng/mL, for chromatographic determination；
5）The preparation of blank solution：Precision weighs chicken meat samples of the 5g without sulfa drugs, puts in 50 mL high speed centrifugation pipes,
It is accurate to add 100 L steps 2）The Isotopic Internal Standard working solution for obtaining is accurate to add 0.1% formic acid of 20mL, 80% aqueous acetonitrile
Liquid double solvents, 2 min of high-speed homogenization are centrifuged 5min in 4000 r/min, take 15 mL nitrogen of supernatant and dry up, and residue is used
0.75 mL10% methanol aqueous solutions redissolve and blank solution are obtained, for chromatographic determination；
6）The preparation of mark-on reclaims test sample solution：Precision weighs chicken meat samples of the 5g without sulfa drugs, puts 50 mL high
It is in fast centrifuge tube, accurate to add different amounts of step 1）Obtained hybrid standard product working solution, obtain detection be limited to 1ng/mL,
The mark-on test chicken that 2ng/mL, basic, normal, high varying level concentration are respectively 5ng/mL, 10ng/mL, 20ng/mL is limited to quantitatively
Sample, then the 100 L steps 2 of accurate addition in the mark-on test specimen）Obtained Isotopic Internal Standard working solution is accurate to add
200.1% formic acid, 80% acetonitrile solution double solvents, 2 min of high-speed homogenization are centrifuged 5min in 4000 r/min, take supernatant 15
ML nitrogen is dried up, and residue 0.75mL10% methanol aqueous solutions redissolve, and mark-on reclaims test chicken meat sample solution is obtained, for chromatogram
Determine；
7）It is molten with three posts two dimension Liquid Chromatography/Mass Spectrometry determination sample solution, linear solvent, blank solution and mark-on reclaims test specimen
Liquid
A is as shown in figure 1, its sample introduction analysis detailed process is as follows：In the presence of quaternary pump 1, drawn by automatic sampler 2 respectively
5 μ L sample solution, linear solvent, blank solution and mark-on reclaims test sample solution to 3 sample introduction of one-dimensional chromatographic column, using gel
The volume exclusion principle of chromatogram, makes the not reserved direct wash-out of matrix interference material of macromolecular, is as a result examined by UV-detector 4
Survey, left switching valve 5 and right switching valve 6 keep original state in 0-5.2min, that is, be in position 2, No. 2 positions of left switching valve 5
Communicate with No. 3 positions, to waste liquid pool, matrix interference thing etc. realizes that target compound is separated with matrix, remove matrix interference, right cut
It is also that No. 2 positions are communicated with No. 3 positions to change valve, is switched to the state of waste liquid；Position 1, i.e., 2 are switched in the left switching valves of 5.2-11.2min 5
Number position is communicated with No. 1 position, target compound is switched to and is enriched with enriching column 8, is switched in the left switching valves of 11.2min 5
Position 2, binary pump 9 are recoiled the target compound on enriching column 8 to Two way chromatograms post 7 and are separated, 6 position of right switching valve
It is constant, the PBS on enriching column 8 is rushed to waste liquid pool using 9 mobile phase of binary pump, prevent phosphate from confronting
Spectra system causes to damage, and in 16.2min, right switching valve 6 switches to position 1, i.e. No. 2 positions of right switching valve 6 are communicated with No. 1 position,
It is analyzed into mass detector 10, its chromatographic condition is as follows：
The one-dimensional liquid-phase conditions of a1：
Chromatographic column：5 μm of Agilent Bio SEC-5,150,4.6 × 150mm
Mobile phase：Phosphate buffer (pH7.4), during the phosphate buffered saline, takes potassium dihydrogen phosphate 1.36g, plus
0.1mol/L sodium hydroxide solution 79mL, are diluted with water to 200mL and obtain,
Using isocratic elution, flow velocity：0.35mL/min
Enriching column：XBridge C8 Direct Connect HP 10μm
A2 two dimension liquid-phase conditions：
Chromatographic column：2.1 × 50 mm of ACQUITY UPLC BEH C18 posts, 1.7 μm
Mobile phase：A phases：0.1 % formic acid waters, B：0.1 % formic acid methyl alcohol, using gradient elution, its gradient elution process such as 1 institute of table
Show：
1 gradient elution process table of table
Column temperature：25 ℃
Sample size：5 μL
Left switching valve, right switching valve in a3 liquid chromatograies handoff procedure during sample introduction analysis is as shown in table 2：
Vavle switching process table is changed in 2 left switching valve of table, right cut
In table 2, diverse location switching valve stream trend is as follows：
Valve position 1 is changed in left cut：1 ~ 2,3 ~ 4,5 ~ 6
Valve position 2 is changed in left cut：2 ~ 3,4 ~ 5,6 ~ 1
Valve position 1 is changed in right cut：1~2
Valve position 2 is changed in right cut：2~3
A4 mass detector conditions：
Mass spectrograph：Triple quadrupole rods tandem mass spectrometry instrument
Ion gun：Electric spray ion source
Scan mode：Cation is scanned
Detection mode：Multiple-reaction monitoring
Desolventizing flow velocity Temp (DEG C)：500
Source Temp(℃)：150
Desolventizing flow velocity (L/hr)：800
Taper hole flow velocity (L/hr)：150
The qualitative, quantitative ion pair and impact energy scale of each sulfa drugs selected by the present invention by mass spectrometry parameters optimization draw,
As a result it is as shown in table 3：
The qualitative, quantitative ion pair and impact energy scale of 3 each sulfa drugs of table
Remarks：In table 3, * is quota ion
B curvilinear equation slopes, the calculating of curvilinear equation intercept
With the target compound peak area ratio internal standard compound peaks area quantitative in linear solvent, curvilinear equation A=aC+b is obtained,
In formula, C is target compound residual quantity in solution, and unit is ng/mL；A is target compound peak area ratio internal standard compound
Peak area ratio；A is curvilinear equation slope；B is curvilinear equation intercept, in concentration according to linear solvent, the linear solvent measured
Target compound peak area, internal standard compound peaks area, using fitting process, calculate a and b, and methylene sulfonamide of the invention is phonetic
Pyridine -13C6 is used as Isotopic Internal Standard；
C interpretations of result：In sample solution the retention time of non-principal component respectively with linear solvent in target compound same
Retention time in chromatographic column compares, two groups of retention times of certain component and a certain sulfa drugs in linear solvent in sample
Two groups of retention times absolute value in 0.05min, regard as the sulfa drugs, the target compound in sample solution is residual
Allowance concentration C, its unit are ng/mL, and computing formula is as follows：
C=(A-b)/a
In formula：A is the target compound peak area ratio internal standard compound peaks area in sample solution；
A and b are calculated by step b；
D result verifications：Mark-on reclaims test sample solution calculates actual target compound residual quantity concentration C by step c, with
Its theoretical target compound residual quantity concentration C compares, and calculates its average recovery rate, and average recovery rate is 60-120%, it was demonstrated that
The detection method is feasible, when average recovery rate is calculated, has surveyed altogether 12 groups of parallel tests, when precision is calculated, has surveyed altogether 6 groups
Parallel test, its checking data result are as shown in table 4.
In 4 each mark-on reclaims of table test chicken meat sample shown in sulfa drugs checking data result
As can be drawn from Table 4, between 84-97%, precision is between 035-1.36% for its average recovery rate, it was demonstrated that the detection side
Method is feasible.
Calculated by the method, by the target compound peak area in step b calculated a and b data, sample solution
Substitute in C=(A-b)/a formula than internal standard compound peaks area A, try to achieve the value of C, be computed in the chicken meat sample of present invention detection
Without each sulfa drugs composition in this experiment.
Claims (9)
1. based on the method that sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products, it is characterised in that include as
Lower step：
1）The preparation of hybrid standard product working solution：Precision pipettes each sulfa drugs storing solution of 0.1mL 1.0mg/mL respectively
Into 10mL volumetric flasks, with methanol constant volume, then precision pipettes 1 mL mixed liquors into another 10mL volumetric flasks, with methanol constant volume,
The hybrid standard product working solution of 1 mg/L is made, is saved backup at 4 DEG C；
2）The preparation of Isotopic Internal Standard working solution：Precision pipettes the internal standard storing solution of 0.1 mL 1.0mg/mL to 10 mL capacity
In bottle, first is used with internal standard storing solution in methanol constant volume, then the accurate volumetric flask for pipetting 0.1 mL constant volumes into 50 mL volumetric flasks
Alcohol constant volume, is obtained the internal standard working solution of 20 ng/mL, saves backup at 4 DEG C；
3）The preparation of sample solution：Meat products to be measured is taken, is put in 50 mL high speed centrifugation pipes, it is accurate to add step 2）What is obtained is same
The plain internal standard working solution in position and double solvents, high-speed homogenization 1-3 min, are centrifuged 4-6 min in 3500-4500 r/min, take
15 mL nitrogen of clear liquid is dried up, and residue is redissolved with 10% methanol aqueous solution, sample solution is obtained standby；
4）The preparation of linear solvent：The meat products without sulfa drugs is weighed, is put in 50 mL high speed centrifugation pipes, it is accurate to add
Double solvents, high-speed homogenization 1-3 min are centrifuged 4-6 min in 3500-4500 r/min, take 15 mL nitrogen of supernatant and dry up,
Residue is redissolved with 10% methanol aqueous solution, and vehicle solution is obtained, accurate to draw different amounts of step 1）Obtained hybrid standard
Product working solution and same amount of step 2）Obtained Isotopic Internal Standard working solution, obtains each sulphur by vehicle solution dilution
Drug amine concentration is respectively 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, and internal standard concentration is 2 ng/
The linear criterion solution of mL, for chromatographic determination；
5）The preparation of blank solution：The sample without sulfa drugs is taken, is put in 50 mL high speed centrifugation pipes, it is accurate to add step
2）The Isotopic Internal Standard working solution for obtaining and double solvents, high-speed homogenization 1-3 min are centrifuged 4-6 in 3500-4500 r/min
Min, takes 15 mL nitrogen of supernatant and dries up, and residue is redissolved with 10% methanol aqueous solution, blank solution is obtained, for chromatographic determination；
6）The preparation of mark-on reclaims test sample solution：The sample without sulfa drugs is taken, is put in 50 mL high speed centrifugation pipes,
It is accurate to add different amounts of step 1）Obtained hybrid standard product working solution, obtains detection and is limited to 1ng/mL, is quantitatively limited to
2ng/mL, basic, normal, high varying level concentration are respectively the mark-on test specimen of 5ng/mL, 10ng/mL, 20ng/mL, then to this
It is accurate in mark-on test specimen to add 100 L steps 2）Obtained Isotopic Internal Standard working solution is accurate to add 15-25 mL multiple
Bonding solvent, high-speed homogenization 1-3 min are centrifuged 4-6 min in 3500-4500 r/min, take 15 mL nitrogen of supernatant and dry up, residual
Slag is redissolved with 10% methanol aqueous solution, mark-on reclaims test sample solution is obtained, for chromatographic determination；
7）It is molten with three posts two dimension Liquid Chromatography/Mass Spectrometry determination sample solution, linear solvent, blank solution and mark-on reclaims test specimen
Liquid
A is determined：Sample introduction process：5 μ L sample solution, linear solvent, blank solution and mark-on are drawn respectively by automatic sampler to return
In acceptance test sample solution injecting chromatograph, with three posts two dimension Liquid Chromatography/Mass Spectrometry, the target compound peak in determination sample solution
Area, internal standard compound peaks area；Target compound peak area, internal standard compound peaks area in linear solvent；Mark-on reclaims are tried
Target compound peak area in sample solution, internal standard compound peaks area are tested, blank solution is at target compound appearance without dry
Disturb, in standard working solution and sample solution, the response of component to be measured all should be within the range of linearity of monitor；
B curvilinear equation slopes, the calculating of curvilinear equation intercept
With the target compound peak area ratio internal standard compound peaks area quantitative in linear solvent, curvilinear equation A=aC+b is obtained,
In formula, C is target compound residual quantity in solution, and unit is ng/mL；A is target compound peak area ratio internal standard compound
Peak area ratio；A is curvilinear equation slope；B is curvilinear equation intercept, in concentration according to linear solvent, the linear solvent measured
Target compound peak area, internal standard compound peaks area, using fitting process, calculate a and b；
C interpretations of result：In sample solution the retention time of non-principal component respectively with linear solvent in target compound same
Retention time in chromatographic column compares, two groups of retention times of certain component and a certain sulfa drugs in linear solvent in sample
Two groups of retention times absolute value in 0.05min, regard as the sulfa drugs, the target compound in sample solution is residual
Allowance concentration C, its unit are ng/mL, and computing formula is as follows：
C=(A-b)/a
In formula：A is the target compound peak area ratio internal standard compound peaks area in sample solution；
A and b are calculated by step b；
D result verifications：Mark-on reclaims test sample solution calculates actual target compound residual quantity concentration C by step c, with
Its theoretical target compound residual quantity concentration C compares, and calculates its average recovery rate, and average recovery rate is 60-120%, it was demonstrated that
The detection method is feasible.
2. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that each sulfa drugs includes sulfaisodimidine, sulfamerazine, sulfamethizole, kynix
Pyridazine, cistosulfa, sulfamethoxazole, sulfamonomethoxine, sulfanilamide (SN) benzoyl, sulfadimethoxine and sulfalene
Base isoxazole.
3. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that step 2）In in be designated as sulfamethyldiazine -13C6.
4. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that step 3）In double solvents be 0.1% formic acid, 80% acetonitrile solution.
5. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that step 3）In sample solution with 0.22 μm of membrane filtration.
6. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that when prepared by solution, 2 min of high-speed homogenization are centrifuged 5 min in 4000 r/min, take 15 mL nitrogen of supernatant
Dry up, residue 0.75mL10% methanol aqueous solutions redissolve.
7. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that three posts two dimension Liquid Chromatography/Mass Spectrometry is as follows：First using the volume exclusion principle of gel chromatography, macromolecular is made
The not reserved direct wash-out of matrix interference material, retention time is longer on a column for the target compound of small molecule, realizes mesh
Mark compound is separated with matrix, removes matrix interference；After gel aqueous phase post is by the separation of matrix interference thing, Two way chromatograms system
System is switched over, and target compound is retained on enriching column, and after object is completely transferred to enriching column, Two way chromatograms are one
It is secondary to switch to original state, while object carries out separation analysis in the second dimension chromatographic column, by switching liquid phase-matter in terminal
Spectrum GC-MS realizes the in-line purification of sulfa drug residue.
8. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that chromatographic condition is as follows：
1）One-dimensional liquid-phase condition：
Chromatographic column：5 μm of Agilent Bio SEC-5,150,4.6 × 150mm,
Mobile phase：PH is 7.4 phosphate buffer, isocratic elution,
Phosphate buffer takes potassium dihydrogen phosphate 1.36g, plus 0.1mol/L sodium hydroxide solution 79mL, is diluted with water to
200mL is obtained,
Flow velocity：0.35mL/min,
Enriching column：XBridge C8 Direct Connect HP 10μm；
2）Two-dimentional liquid-phase condition：
Chromatographic column：2.1 × 50 mm of ACQUITY UPLC BEH C18 posts, 1.7 μm,
Mobile phase：A phases：0.1 % formic acid waters, B：0.1 % formic acid methyl alcohol, gradient elution,
Column temperature：25 DEG C,
Sample size：5 μ L,
3）Mass Spectrometry Conditions：
Mass spectrograph：Triple quadrupole rods tandem mass spectrometry instrument,
Ion gun：Electric spray ion source,
Scan mode：Cation is scanned,
Detection mode：Multiple-reaction monitoring,
Desolventizing temperature (DEG C)：500,
Source temperature (DEG C)：150,
Desolventizing flow velocity (L/hr)：800,
Taper hole flow velocity (L/hr)：150.
9. the side of sulfa drugs is remained in the two dimension HPLC/MS-MS meat products based on three posts according to claim 1
Method, it is characterised in that sample introduction analysis detailed process is as follows：In quaternary pump（1）In the presence of, by automatic sampler（2）To one-dimensional color
Spectrum post（3）Sample introduction, using the volume exclusion principle of gel chromatography, makes the matrix interference material of macromolecular not reserved directly wash
It is de-, left switching valve（5）With right switching valve（6）Original state is kept in 0-5.2min, i.e., left No. 2 positions of switching valve and No. 3 position phases
Logical, to waste liquid pool, matrix interference thing etc. realizes that target compound is separated with matrix, removes matrix interference, and right switching valve is also 2
Number position is communicated with No. 3 positions, is switched to the state of waste liquid pool；In the left switching valves of 5.2-11.2min（5）Switch to position 1, i.e., No. 2 position
Communicate with No. 1 position, target compound is switched to into enriching column（8）In be enriched with, in the left switching valves of 11.2min（5）Switch to
Position 2, binary pump（9）By enriching column（8）On target compound recoil to Two way chromatograms post（7）On separated, right cut is changed
Valve（6）Position is constant, using binary pump（9）Mobile phase is by enriching column（8）On PBS rush to waste liquid pool, prevent
Only phosphate causes to damage to mass spectrometer system, the right switching valve in 16.2min（6）Position 1, i.e., No. 2 position is switched to No. 1 position phase
It is logical, into mass detector（10）It is analyzed.
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