CN109001334A - The measuring method of benzimidazoles residues residual quantity in a kind of chicken tissues - Google Patents
The measuring method of benzimidazoles residues residual quantity in a kind of chicken tissues Download PDFInfo
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Abstract
The invention discloses a kind of measuring methods of benzimidazoles residues residual quantity in chicken, chicken liver and chicken kidney.The present invention provides the residual quantity using nine kinds of benzimidazoles residues residual markers in hplc simultaneous determination chicken, chicken liver and chicken kidney for the first time, has filled up art technology blank.Regression equation related coefficient of the present invention reaches 0.999 or more, and lower limit of measurement is 50 μ g/kg, and the rate of recovery on different addition concentration levels is 75%~110%, relative standard deviation≤15% in laboratory.The present invention solves while measuring the technical problem of nine kinds of benzimidazoles residues, in the pretreatment technology of sample, there is originality especially on purification techniques, this method quantitative science is accurate, strong antijamming capability, matrix wide adaptation range can satisfy the state-of-the-art technology requirement of China's interested regulatory authorities.
Description
Technical field
The invention belongs to the detection technique fields of food veterinary drug residue, and in particular to benzimidazoles residues in chicken tissues
The measuring method of residual quantity.
Background technique
" the national food safety standard animal food herbal medicine maximum residue limit " of the newest appearance in China (consults on
Original text) in define benzimidazoles residues, including febantel, Fenbendazole, oxfendazole, oxibendazole, thiabendazolum, three
Chlorine parbendazole, albendazole, flubendazole and mebendazol residual marker.In No. 235 bulletins of the Ministry of Agriculture before
Regulation has many differences, is that the type of drug increases, and has standardized title first, has secondly also made very to residual marker
Big modification.Febantel/Fenbendazole/oxfendazole residual marker is Fenbendazole, oxfendazole and oxfendazole
The summation of 2- amino sulfone；The residual marker of oxibendazole is oxibendazole；The residual marker of thiabendazolum is thiabendazolum
With 5- hydroxyl thiabendazolum；The residual marker of Triclabendazole is Triclabendazole ketone；The residual marker of albendazole is
Albendazole -2- amino sulfone；The residual marker of flubendazole is flubendazole；The residual marker of mebendazol is 2- ammonia
Base -5- benzoyl benzimidazole and 5- hydroxy-methylbenzene reach azoles.Modified content and Codex Committee on Food (CAC) and European Union
Benzimidazoles residues maximum residue limit require it is almost the same.
The professional standard and national standard either promulgated, the document still delivered all only include therein one
Kind or certain several drugs, there has been no the methods for completely including this nine kinds of benzimidazoles residues to disclose.Benzimidazoles residues by
In the relationship of its chemical structure, generally in alkalescent, but during wherein the residual marker Triclabendazole ketone of Triclabendazole is
Property compound, it is desirable to while the residual marker of this nine kinds of benzimidazoles residues is detected, technically there are many difficulties, especially
It is that have higher requirement for the purification of sample using high performance liquid chromatography.
The present invention is provided for the first time using nine kinds of benzene in hplc simultaneous determination chicken, chicken liver and chicken kidney
And the residual quantity of imidazoles residual marker, fill up art technology blank.Regression equation related coefficient of the present invention reaches
To 0.999 or more, lower limit of measurement is 50 μ g/kg, and the rate of recovery on different addition concentration levels is 75%~110%, experiment
Indoor relative standard deviation≤15%.The present invention solves while measuring the technical problem of nine kinds of benzimidazoles residues, in sample
The pretreatment technology of product has originality especially on purification techniques, and this method quantitative science is accurate, strong antijamming capability, base
Matter wide adaptation range can satisfy the state-of-the-art technology requirement of China's interested regulatory authorities.
Summary of the invention
The technical issues of in view of the detection of existing veterinary drug, the present invention proposes a kind of by benzo miaow in rapid and accurate determination chicken tissues
The measuring method of azole drug residual quantity.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows.
The measuring method of benzimidazoles residues residual quantity, specific steps in a kind of chicken tissues are as follows:
Sample is prepared through homogeneous, is extracted using acetonitrile, and extracting solution is purified through MCX solid phase extraction column, and gained scavenging solution exists
Rotary evaporation is depressurized under 38 DEG C of water-baths to about 500 μ L, 0.1mol/L hydrochloric acid solution is added to be settled to 1mL, vortex 2min, solution with
10000r/min is centrifuged 5min；Then it is detected using high performance liquid chromatograph, quantified by external standard method；The purification method are as follows: take 5mL
The extracting solution adds 3mL acetonitrile to be saturated n-hexane, vortex oscillation 2min, 4 000r/min centrifugation in 15mL plastic centrifuge tube
5min takes lower layer in 50mL plastic centrifuge tube, adds 10mL 0.1mol/L hydrochloric acid solution, mixes, adds 1mL n-hexane, vortex oscillation
1min, 10 000r/min are centrifuged 5min, discard upper layer n-hexane, subnatant is transferred to by pretreated MCX solid-phase extraction column
In, pass through sample solution all with the flow velocity less than 1mL/min, discards efflux；Successively use 3mL0.1mol/L hydrochloric acid solution
+ methanol, the elution of 5% ammonia spirit of volume ratio 2:1,4mL, leacheate all pass through pillar, discard leacheate, finally use 4mL
The elution of 5% ammonia methanol, eluent all pass through pillar with the flow velocity of 0.5mL/min, and are collected in the concentrate bottle of tool scale, i.e.,
?；The solid phase extraction column are as follows: Oasis MCX, 60mg, 3cc；The preprocess method are as follows: successively use 3mL methanol using preceding
It is activated with 3mL water.
Further, the extracting method are as follows: weigh 5g and add in 50mL centrifuge tube through the sample prepared by homogeneous
Enter 15mL acetonitrile solution and set ultrasound 5min in ultrasound bath, vortex oscillation 15min, 4 000r/min are centrifuged 5min, supernatant
It is transferred in 25mL volumetric flask；Residue adds 10mL acetonitrile solution and sets ultrasound 5min, vortex oscillation in ultrasound bath
10min, 4 000r/min are centrifuged 5min, and supernatant is incorporated into 25mL volumetric flask, and is settled to scale with acetonitrile, shakes up, standby
With.
Further, the benzimidazoles residues include: febantel, Fenbendazole, oxfendazole, oxibendazole,
Thiabendazolum, Triclabendazole, albendazole, flubendazole and mebendazol；The chicken tissues include: chicken, chicken liver and
Chicken kidney.
Further, the residual marker of the benzimidazoles residues includes: oxfendazole, Fenbendazole, Ao Fenda
Azoles -2- amino sulfone, albendazole -2- amino sulfone, thiabendazolum, 5- hydroxyl thiabendazolum, 2- amino -5- benzoyl benzo miaow
Azoles, 5- hydroxy-methylbenzene reach azoles, flubendazole, oxibendazole and Triclabendazole ketone.
Further, the preparation method of the Standard Stock solutions of the external standard method are as follows: accurately weigh by its purity conversion be
Oxfendazole, Fenbendazole, oxfendazole -2- amino sulfone, the albendazole -2- amino sulfone, thiabendazolum, 5- of 100% mass
Hydroxyl thiabendazolum, 2- amino -5- benzoyl benzimidazole, 5- hydroxy-methylbenzene reach azoles, flubendazole, oxibendazole and trichlorine
Each 20mg of parbendazole ketone standard items, first uses 5mLN, dinethylformamide dissolution respectively, and again with methanol is dissolved and is settled to
200mL is made into Standard Stock solutions, concentration 200mg/L；The preparation method of the hybrid standard working solution of the external standard method are as follows:
Standard Stock solutions described in 5mL are measured respectively in 100mL volumetric flask, and with dilution in acetonitrile to scale, being configured to concentration is 10mg/
The hybrid standard working solution of L.
Further, the method for the sample preparation are as follows: it takes the chicken tissues blank or for examination edible tissue, rubs,
And make homogeneous；Test sample after taking homogeneous, as sample of having a try；Blank sample after taking homogeneous, as blank sample；It takes
Blank sample after matter adds the 10mg/L hybrid standard working solution, adds sample as blank；The sample is at -18 DEG C
It saves below.
Further, the measurement standard curve the preparation method comprises the following steps: it is accurate to measure the hybrid standard working solution appropriate,
With -1% acetic acid solution of methanol, 30:70, v/v, dilution, being configured to concentration is 0,0.025,0.05,0.1,0.2,0.5 and
The series standard solution of 1.0mg/L is measured for high performance liquid chromatography；To measure chromatographic peak area as ordinate, corresponding standard
Solution concentration is abscissa, draws standard curve, asks regression equation and related coefficient；The liquid phase chromatogram condition are as follows: chromatography
Column: Atlantis T3, 4.6mm × 250mm, 5 μm of partial size；Mobile phase: A: methanol；B:1% acetic acid solution, gradient are shown in Table
1, gradient is linear change；Flow velocity: 1.0mL/min；Detection wavelength: 292nm；Column temperature: 40 DEG C；Sample volume: 40 μ L.
Further, the qualitative determination method of the measurement are as follows: when test specimen drug content exceeds maximum residue limit
When amount, it need to be confirmed with high performance liquid chromatography-diode array detector；Diode array detector spectral conditions: detection
Start wavelength: 190nm；Terminate wavelength: 400nm；Slit width: 1.2nm；Under same test conditions, object to be detected in sample
Matter and the standard items detected simultaneously retention time having the same, deviation is within ± 2.5%；In spectrogram, determinand is answered
There is identical maximum absorption wavelength with standard items, the deviation of the two is within ± 2nm；In 220nm or more, determinand and standard items
Relative Absorbance >=10% when, the spectrogram of the two should not have apparent difference；The qualitative determination method of the measurement are as follows: take
The sample solution and the corresponding hybrid standard working solution make single-point or standard curve calibration, by external standard method, with peak area
It calculates；The response that benzimidazoles residues described in the hybrid standard working solution and the sample solution remain marker is equal
It should be within the range of linearity that instrument detects；The blank test method are as follows: in addition to sample is not added, using identical step
Carry out operation repetitive.
Further, the result calculating of the measurement and expression method are as follows: the residual of benzimidazoles residues described in sample
The residual quantity of marker is calculated by formula (1):
In formula:
For X --- --- for the residual quantity of determinand in sample of having a try, unit is ng/kg (μ g/kg)；
The peak area of A --- --- determinand in sample solution；
cs--- --- in standard solution determinand concentration, unit be micro- gram per liter (μ g/L)；
The constant volume of V --- --- sample solution, unit are milliliter (mL)；
As--- --- in standard solution determinand peak area；
Sample mass representated by m --- --- sample solution, unit are gram (g).
Further, the sensitivity confirmation method of the measuring method are as follows: the determination of quantitative limit is the value according to signal-to-noise ratio
Come what is determined；Add the benzimidazoles residues residual mark of 50 μ g/kg respectively in blank chicken, chicken liver and chicken kidney
Show object hybrid standard working solution, measure the ratio of its signal and noise, as S/N >=10 and the rate of recovery and relative standard deviation are equal
Meeting concentration when method for detecting residue requirement is quantitative limit；Experimental result: quantifying for this method is limited to 50 μ g/kg；The survey
Determine the accuracy confirmation method of method are as follows: accurately weigh blank sample described in 5.0g in 50mL centrifuge tube, oneself is added and knows concentration
The series standard working solution, be made containing the benzimidazoles residues residual marker concentration be respectively 50,200,500
The tissue sample of μ g/kg, each concentration are done 6 and are measured after handling by sample pretreatment process in parallel, and recovery of standard addition is calculated；
Experimental result: the rate of recovery of this method on 50 μ of μ g/kg~500 g/kg addition concentration level is 75%~110%；The survey
Determine the precision confirmation method of method are as follows: accurately weigh blank sample described in 5.0g in 50mL centrifuge tube, oneself is added and knows concentration
The series standard working solution, be made containing the benzimidazoles residues residual marker concentration be respectively 50,200,500
The tissue sample of μ g/kg, each concentration are done 6 and are measured after handling by sample pretreatment process in parallel, and indoor relative standard is calculated
Deviation；Experimental result: relative standard deviation≤15% in the laboratory of this method.
The invention has the following beneficial effects:
The present invention is provided for the first time using nine kinds of benzene in hplc simultaneous determination chicken, chicken liver and chicken kidney
And the residual quantity of imidazoles residual marker, fill up art technology blank.Creatively use a Solid Phase Extraction
Two classes compound of different nature is applied into two different retention mechanisms on pillar, has reached ideal clean-up effect.For
For compound Triclabendazole ketone, n-vinyl pyrrolidone-divinylbenzene copolymer in solid phase extraction column filler is risen
The effect of reverse phase absorption agent is arrived；N- vinyl pyrrole for remaining ten kinds of compound, in solid phase extraction column filler
Alkanone-divinylbenzene copolymer matrix-SO3H has primarily served ion exchange.The present invention effectively combines the two,
The conditions such as percentage shared by organic solvent in the acid-base property and solution of load solution, eluent solution and elution solution are carried out
Detailed research, obtains satisfied result.Although with national standard GB/T 21324-2007 " in edible animal muscle and liver
Benzimidazoles residues residues detection method " be identical solid phase extraction column, but cross each step not phase of column
Together, if indiscriminately imitating the column method excessively of national standard, one of untested compound Triclabendazole ketone is in lessivation just from pillar
On elute, the rate of recovery zero, be unable to satisfy and meanwhile detect this ten it is a kind of remain marker requirement.Regression equation of the present invention
Related coefficient reaches 0.999 or more, and lower limit of measurement is 50 μ g/kg, and the rate of recovery on different addition concentration levels are 75%~
110%, relative standard deviation≤15% in laboratory.The present invention solves while measuring the 11 of nine kinds of benzimidazoles residues
The technical problem of kind residual marker has originality in the pretreatment technology of sample especially in extraction and purification techniques, should
Method quantitative science is accurate, strong antijamming capability, matrix wide adaptation range, can satisfy the newest skill of China's interested regulatory authorities
Art requirement.
Detailed description of the invention
Fig. 1 standard solution chromatogram (100 μ g/L)
The high-efficient liquid phase chromatogram of Fig. 2 chicken blank sample
The high-efficient liquid phase chromatogram (200 μ g/L) of Fig. 3 chicken blank addition sample
The high-efficient liquid phase chromatogram of Fig. 4 chicken liver blank sample
The high-efficient liquid phase chromatogram (500 μ g/L) of Fig. 5 chicken liver blank addition sample
The high-efficient liquid phase chromatogram of Fig. 6 chicken kidney blank sample
The high-efficient liquid phase chromatogram (100 μ g/L) of Fig. 7 chicken kidney blank addition sample
Specific embodiment
Illustrate technical solution of the present invention to become apparent from, the present invention is carried out below in conjunction with specific embodiments and the drawings
Detailed description, it is mentioned that attached drawing be only applicable in following embodiments, can also be according to this for those having ordinary skill in the art
The method mentioned in invention obtains other accompanying drawings.But protection scope of the present invention is not limited to following embodiments.
Embodiment 1: method for building up
1. reagent and material
Reagent used below is analytical reagents in addition to especially indicating；Water is to meet level-one as defined in GB/T 6682
Water.
Standard items: oxfendazole (Oxfendazole, No. CAS: 53716-50-0), Fenbendazole (Fenbendazole,
No. CAS: 43210-67-9), oxfendazole -2- amino sulfone (Oxfendazole sulphone, No. CAS: 54029-20-8), Ah
Parbendazole -2- amino sulfone (Albendazole-2-aminosulfone, No. CAS: 80983-34-2), thiabendazolum
(Thiabendazole, No. CAS: 148-79-8), 5- hydroxyl thiabendazolum (5-hydroxythiabendazole, No. CAS:
948-71-0), 2- amino -5- benzoyl benzimidazole (Mebendazole-amine, No. CAS: 52329-60-9), 5- hydroxyl
Base mebendazol (5-Hydroxymebendazole, No. CAS: 60254-95-7), flubendazole (Flubendazole, No. CAS
31430-15-6), oxibendazole (Oxibendazole, No. CAS: 20559-55-1) and Triclabendazole ketone
(Ketotriclabendazole, No. CAS: 1201920-88-8), content is 97% or more.
Acetonitrile: chromatographically pure；Methanol: chromatographically pure；Acetic acid: chromatographically pure；Hydrochloric acid；25% ammonium hydroxide；
0.1mol/L hydrochloric acid solution: concentrated hydrochloric acid 8.3mL is measured, 1000mL is diluted with water to；
0.1mol/L hydrochloric acid solution+methanol (2+1, v/v): 200mL 0.1mol/L hydrochloric acid solution and 100mL are measured respectively
Methanol mixes.
5% ammonia spirit: 25% ammonium hydroxide of 5mL is measured, 100mL, prepared before use are diluted with water to.
5% methanolic ammonia solution: 25% ammonium hydroxide of 5mL is measured, with methanol dilution to 100mL, prepared before use.
1% acetic acid solution: 10mL acetic acid is measured, 1000mL, prepared before use are diluted with water to.
Solid-phase extraction column: Oasis MCX, 60mg, 3mL.It is successively activated with 3mL methanol and 3mL water using preceding.
The preparation of Standard Stock solutions: it accurately weighs and is reached by the conversion of its purity for oxfendazole, the fragrant benzene of 100% mass
Azoles, oxfendazole -2- amino sulfone, albendazole -2- amino sulfone, thiabendazolum, 5- hydroxyl thiabendazolum, 2- amino -5- benzene first
Acyl group benzimidazole, 5- hydroxy-methylbenzene reach azoles, flubendazole, oxibendazole and each 20mg of Triclabendazole ketone standard items, respectively
5mLN is first used, 200mL is dissolved and be settled to dinethylformamide dissolution in methanol, being made into Standard Stock solutions concentration is
200mg/L。
The preparation of hybrid standard working solution: measuring 5mL Standard Stock solutions in 100mL volumetric flask respectively, dilute with acetonitrile
It releases to scale, being configured to concentration is 10mg/L hybrid standard working solution.
2. equipment and instrument
High performance liquid chromatograph: LC-20AD matches autosampler, column oven, UV detector and Diode Array Detector
Device, Shimadzu Corporation.
Assay balance: 0.000 01g of sensibility reciprocal, Sartorius company.
Balance: sensibility reciprocal 0.01g, Sartorius company.
Meat tissue's bruisher: 200 type of GM, Retsch company.
Vortex oscillator: MS 3basic, IKA company.
Ultrasound bath: SW 30H, SONO SWISS company.
Centrifuge: 3-30K, SIGMA company.
Depressurize Rotary Evaporators: IKA company.
3. the preparation and preservation of sample
The preparation of 3.1 samples
It takes chicken, chicken liver and chicken kidney blank or for trying edible tissue, rubs, and make homogeneous.
The test sample of --- --- after taking homogeneous, as sample of having a try.
The blank sample of --- --- after taking homogeneous, as blank sample.
The blank sample of --- --- after taking homogeneous, adds the standard working solution of suitable concentration, adds sample as blank.
The preservation of 3.2 samples
- 18 DEG C or less preservations.
4 determination steps
4.1 extracting
5g sample is weighed (accurately 15mL acetonitrile solution to be added and sets in ultrasound bath in 50mL centrifuge tube to 0.01g)
Ultrasonic 5min, vortex oscillation 15min, 4 000r/min are centrifuged 5min, and supernatant is transferred in 25mL volumetric flask；Residue adds
10mL acetonitrile solution sets ultrasound 5min in ultrasound bath, and vortex oscillation 10min, 4 000r/min are centrifuged 5min, and supernatant closes
And it is settled to scale into 25mL volumetric flask, and with acetonitrile, and it shakes up, it is spare.
4.2 purification
5mL said extracted liquid is taken in 15mL plastic centrifuge tube, 3mL acetonitrile is added to be saturated n-hexane, vortex oscillation 2min,
4000r/min is centrifuged 5min, takes lower layer in 50mL plastic centrifuge tube, adds 10mL 0.1mol/L hydrochloric acid solution, mixes, is adding 1mL just
Hexane, vortex oscillation 1min, 10 000r/min centrifugation 5min, discards upper layer n-hexane, subnatant is transferred to by pretreated
In MCX solid-phase extraction column, passes through sample solution all with the flow velocity less than 1mL/min, discard efflux.Successively use 3mL
0.1mol/L hydrochloric acid solution+methanol (2+1, v/v), the elution of 5% ammonia spirit of 4mL, leacheate all pass through pillar, discard leaching
Washing lotion.It is finally eluted with 5% ammonia methanol of 4mL, eluent all passes through pillar with the flow velocity of 0.5mL/min, and is collected in tool and carves
The concentrate bottle of degree depressurizes rotary evaporation to about 500 μ L, adds 0.1mol/L hydrochloric acid solution to be settled to 1mL, be vortexed under 38 DEG C of water-baths
2min, solution are centrifuged 5min with 10 000r/min, for measurement.
The preparation of 4.3 standard curves
Precision measurement hybrid standard working solution is appropriate, is diluted, is configured to dense with -1% acetic acid solution of methanol (30+70, v/v)
Degree is the series standard solution of 0,0.025,0.05,0.1,0.2,0.5 and 1.0mg/L, is measured for high performance liquid chromatography.To measure
Chromatographic peak area is ordinate, and corresponding concentration of standard solution is abscissa, draws standard curve, asks regression equation and phase relation
Number.
4.4 measurement
4.4.1 liquid phase chromatogram condition
Chromatographic column: Atlantis T3(4.6mm × 250mm, 5 μm of partial size).
Mobile phase: A: methanol；B:1% acetic acid solution, gradient are shown in Table 1, and gradient is linear change.
1 gradient elution program of table
Flow velocity: 1.0mL/min；Detection wavelength: 292nm；Column temperature: 40 DEG C；Sample volume: 40 μ L.
4.4.2 measuring method
4.4.2.1 qualitative determination
It, need to be with high performance liquid chromatography-diode array inspection when test specimen drug content exceeds maximum residue limit
Device is surveyed to be confirmed.
Diode array detector spectral conditions: detection starts wavelength: 190nm；Terminate wavelength: 400nm；Slit width:
1.2nm。
Under same test conditions, in sample when substance to be detected reservation having the same with the standard items detected simultaneously
Between, deviation is within ± 2.5%.In spectrogram, determinand should have an identical maximum absorption wavelength with standard items, the two it is inclined
Difference is within ± 2nm.In 220nm or more, when the Relative Absorbance of determinand and standard items >=10%, the spectrogram of the two is not answered
There is apparent difference.
4.4.2.2 quantitative determination
It materialses solution and corresponding standard solution, makees single-point or standard curve calibration, by external standard method, in terms of peak area
It calculates.The linear model that the response of benzimidazoles residues residual marker should all be detected in instrument in standard solution and sample solution
Within enclosing.Under above-mentioned chromatographic condition, the high-efficient liquid phase chromatogram of standard solution, blank sample and addition sample is shown in attached drawing.
4.5 blank test
In addition to sample is not added, operation repetitive is carried out using identical step.
4.6 results calculate and statement
The residual quantity of benzimidazoles residues residual marker is calculated by formula (1) in sample:
In formula:
For X --- --- for the residual quantity of determinand in sample of having a try, unit is ng/kg (μ g/kg)；
The peak area of A --- --- determinand in sample solution；
cs--- --- in standard solution determinand concentration, unit be micro- gram per liter (μ g/L)；
The constant volume of V --- --- sample solution, unit are milliliter (mL)；
As--- --- in standard solution determinand peak area；
Sample mass representated by m --- --- sample solution, unit are gram (g).
4.7 method sensitivity, accuracy and precision
4.7.1 sensitivity
The determination of quantitative limit is determined according to the value of signal-to-noise ratio (S/N).In blank chicken, chicken liver and chicken kidney
The middle benzimidazoles residues residual marker standard solution for adding 50 μ g/kg respectively, measures the ratio of its signal and noise, when
Concentration when S/N >=10 and when the rate of recovery and relative standard deviation meet method for detecting residue requirement is quantitative limit.
Experimental result: quantifying for this method is limited to 50 μ g/kg.
4.7.2 accuracy
5.0g blank sample is accurately weighed in 50mL centrifuge tube, oneself is added and knows the series standard working solution of concentration, is made
The residual marker concentration containing benzimidazoles residues is respectively the tissue sample of 50,200,500 μ g/kg, and each concentration is done 6 and put down
Row, measures after handling by sample pretreatment process, calculates recovery of standard addition.
Experimental result: this method 50 μ of μ g/kg~500 g/kg addition concentration level on the rate of recovery be 75%~
110%.
4.7.3 precision
5.0g blank sample is accurately weighed in 50mL centrifuge tube, oneself is added and knows the series standard working solution of concentration, is made
The residual marker concentration containing benzimidazoles residues is respectively the tissue sample of 50,200,500 μ g/kg, and each concentration is done 6 and put down
Row, measures after handling by sample pretreatment process, calculates indoor relative standard deviation.
Experimental result: relative standard deviation≤15% in the laboratory of this method.
5 examples
This method is used for routine testing, totally 150, chicken, chicken liver and chicken kidney sample has been detected, wherein 1 chicken liver
Albendazole -2- amino sulfone is detected, content is 78 μ g/kg, other are not detected.
Embodiment 2: the selection of Extraction solvent
The present invention compares acetonitrile and GB/T 21324-2007 " benzimidazoles residues in edible animal muscle and liver
Residues detection method " employed in alkaline ethyl acetate, under the conditions of the chromatographic of this method, acetonitrile extracting solution it is dry
It disturbs peak and is far less than alkaline ethyl acetate, in addition, alkaline acetic acid ethyl acetate extract is before next step purifies upper prop, it is necessary to convert molten
Agent, nitrogen flushing concentration or decompression rotary evaporation are concentrated to dryness, then are dissolved with acetonitrile -0.1mol/L hydrochloric acid solution, and concentration process can draw
The degradation of Triclabendazole ketone is played, and does not need then to be concentrated using acetonitrile extracting solution, is added on after 0.1mol/L hydrochloric acid solution
Column.Acetonitrile extracting solution reaches 95% or more to the extraction efficiency of all compounds, therefore the present invention selects acetonitrile as extraction
Solvent.
Embodiment 3: the selection of purification condition
Firstly, the present invention uses reverse phase absorption agent HLB solid phase extraction column, acetonitrile ratio is 10% in upper prop solution
When, oxfendazole -2- amino sulfone and 5- hydroxyl thiabendazolum can lose, so acetonitrile extracting solution must be concentrated, so not
Organic solvent ratio is too low when only will cause the degradation of untested compound, and redissolving solution, and solute effect is poor, will lead to recycling
Rate reduces.
Through comparing, this method uses MCX solid phase extraction column, the N- ethylene for Triclabendazole ketone, in filler
Base pyrrolidones-divinylbenzene copolymer plays the role of reverse phase absorption agent；For remaining ten kinds of compound, in filler
N-vinyl pyrrolidone-divinylbenzene copolymer matrix-SO3H primarily served ion exchange.The two is incorporated in
Together, more conditions can all limit load solution, eluent solution and elution solution, the acid-base property and solution of solution
Percentage shared by middle organic solvent will both be taken into account.According to GB/T 21324-2007 " in edible animal muscle and liver
Benzimidazoles residues residues detection method " in the used column condition of institute handle mark-on sample, Triclabendazole ketone can be with first
Alcohol elutes that one-step elution and gets off, and Triclabendazole ketone is practically free of in final eluent.Second in the upper prop solution of this method
The ratio of nitrile and 0.1mol/L hydrochloric acid solution is 1:2, if ratio is 1:1, Triclabendazole ketone can lose most, not protected by pillar
It stays；The ratio of methanol is no more than 50% in eluent solution 0.1mol/L hydrochloric acid solution+methanol, another 5% ammonium hydroxide of eluent solution
Solution can remove the impurity between two peaks of oxfendazole -2- amino sulfone and 5- hydroxyl thiabendazolum, using 0.1mol/L hydrochloric acid
Solution cannot then remove the impurity；Eluent 10% ammonia acetonitrile used by 5% ammonia methanol ratio GB/T 21324-2007 is more
Properly, if being eluted with ammonia acetonitrile, wherein the ratio of ammonia influences very the eluting power of 2- amino -5- benzoyl benzimidazole
Greatly, 5% ammonia acetonitrile can only elute 50% or so, and 10% ammonia acetonitrile is volatile, and the reduction of ammonia will have a direct impact on 2- amino -5-
The rate of recovery of benzoyl benzimidazole causes the reproducibility of method bad, and the ammonia methanol that we select is then more stable, and
The concentration of ammonia methanol is also more easy.
Claims (10)
1. the measuring method of benzimidazoles residues residual quantity in a kind of chicken tissues, which is characterized in that specific steps are as follows: sample warp
Homogeneous preparation, is extracted, extracting solution is purified through MCX solid phase extraction column, and gained scavenging solution depressurizes under 38 DEG C of water-baths using acetonitrile
Rotary evaporation adds 0.1mol/L hydrochloric acid solution to be settled to 1mL, vortex 2min, solution is centrifuged with 10000r/min to about 500 μ L
Then 5min is detected, quantified by external standard method using high performance liquid chromatograph；The purification method are as follows: take extracting solution described in 5mL in
In 15mL plastic centrifuge tube, 3mL acetonitrile is added to be saturated n-hexane, vortex oscillation 2min, 4 000r/min are centrifuged 5min, take lower layer in
50mL plastic centrifuge tube adds 10mL 0.1mol/L hydrochloric acid solution, mixes, adds 1mL n-hexane, vortex oscillation 1min, 10 000r/
Min is centrifuged 5min, discards upper layer n-hexane, and subnatant is transferred to by pretreated MCX solid-phase extraction column, to be less than 1mL/
The flow velocity of min passes through sample solution all, discards efflux；Successively use 3mL 0.1mol/L hydrochloric acid solution+methanol, volume ratio
For the elution of 5% ammonia spirit of 2:1,4mL, leacheate all passes through pillar, discards leacheate, finally washed with 5% ammonia methanol of 4mL
De-, eluent all passes through pillar with the flow velocity of 0.5mL/min, and be collected in the concentrate bottle of tool scale to get；The solid phase
Extract pillar are as follows: Oasis MCX, 60mg, 3cc；The preprocess method are as follows: using preceding successively living with 3mL methanol and 3mL water
Change.
2. measuring method as described in claim 1, which is characterized in that the extracting method are as follows: weigh what 5g was prepared through homogeneous
The sample is added 15mL acetonitrile solution and sets ultrasound 5min, vortex oscillation 15min in ultrasound bath in 50mL centrifuge tube,
4 000r/min are centrifuged 5min, and supernatant is transferred in 25mL volumetric flask；Residue adds 10mL acetonitrile solution and sets ultrasonic water
Ultrasound 5min in bath, vortex oscillation 10min, 4 000r/min are centrifuged 5min, and supernatant is incorporated into 25mL volumetric flask, and uses second
Nitrile is settled to scale, shakes up, spare.
3. measuring method as described in claim 1, which is characterized in that the benzimidazoles residues include: febantel, sweet smell
Parbendazole, oxfendazole, oxibendazole, thiabendazolum, Triclabendazole, albendazole, flubendazole and mebendazol；It is described
Chicken tissues include: chicken, chicken liver and chicken kidney.
4. measuring method as claimed in claim 3, which is characterized in that the residual marker packet of the benzimidazoles residues
It includes: oxfendazole, Fenbendazole, oxfendazole -2- amino sulfone, albendazole -2- amino sulfone, thiabendazolum, 5- hydroxyl thiophene benzene
Azoles, flubendazole, oxibendazole and Triclabendazole are reached up to azoles, 2- amino -5- benzoyl benzimidazole, 5- hydroxy-methylbenzene
Ketone.
5. measuring method as claimed in claim 4, which is characterized in that the preparation method of the Standard Stock solutions of the external standard method
Are as follows: accurately weigh oxfendazole, Fenbendazole, the oxfendazole -2- amino sulfone, acetysalicylic acid phenobarbital by the conversion of its purity for 100% mass
It is reached up to azoles -2- amino sulfone, thiabendazolum, 5- hydroxyl thiabendazolum, 2- amino -5- benzoyl benzimidazole, 5- hydroxy-methylbenzene
Azoles, flubendazole, oxibendazole and each 20mg of Triclabendazole ketone standard items, first use 5mLN respectively, and dinethylformamide is molten
Solution, again with methanol dissolve and are settled to 200mL, be made into Standard Stock solutions, concentration 200mg/L；The mixing of the external standard method
The preparation method of standard working solution are as follows: measure Standard Stock solutions described in 5mL respectively in 100mL volumetric flask, use dilution in acetonitrile
To scale, it is configured to the hybrid standard working solution that concentration is 10mg/L.
6. measuring method as claimed in claim 5, which is characterized in that the method for the sample preparation are as follows: take the chicken tissues
Blank or for trying edible tissue, rubs, and makes homogeneous；Test sample after taking homogeneous, as sample of having a try；After taking homogeneous
Blank sample, as blank sample；Blank sample after taking homogeneous adds the 10mg/L hybrid standard working solution, as sky
White addition sample；The sample is saved at -18 DEG C or less.
7. measuring method as claimed in claim 5, which is characterized in that the standard curve of the measurement is the preparation method comprises the following steps: precision
It is appropriate to measure the hybrid standard working solution, with -1% acetic acid solution of methanol, 30:70, v/v, dilution, be configured to concentration be 0,
0.025, the series standard solution of 0.05,0.1,0.2,0.5 and 1.0mg/L is measured for high performance liquid chromatography；To measure chromatographic peak
Area is ordinate, and corresponding concentration of standard solution is abscissa, draws standard curve, asks regression equation and related coefficient；Institute
The liquid phase chromatogram condition stated are as follows: chromatographic column: Atlantis T3, 4.6mm × 250mm, 5 μm of partial size；Mobile phase: A: methanol；B:
1% acetic acid solution, gradient are shown in Table 1, and gradient is linear change；
1 gradient elution program of table
Flow velocity: 1.0mL/min；Detection wavelength: 292nm；Column temperature: 40 DEG C；Sample volume: 40 μ L.
8. measuring method as claimed in claim 7, which is characterized in that the qualitative determination method of the measurement are as follows: work as test sample
When product drug content exceeds maximum residue limit, it need to be confirmed with high performance liquid chromatography-diode array detector；Two
Pole pipe array detector spectral conditions: detection starts wavelength: 190nm；Terminate wavelength: 400nm；Slit width: 1.2nm；In phase
With under experimental condition, substance to be detected and the standard items retention time having the same detected simultaneously in sample, deviation ±
Within 2.5%；In spectrogram, determinand should have an identical maximum absorption wavelength with standard items, the deviation of the two ± 2nm it
It is interior；In 220nm or more, when the Relative Absorbance of determinand and standard items >=10%, the spectrogram of the two should not have apparent poor
Not；The qualitative determination method of the measurement are as follows: take the sample solution and the corresponding hybrid standard working solution, make single-point or
Standard curve calibration, by external standard method, with calculated by peak area；Benzo described in the standard working solution and the sample solution
The response that imidazoles remain marker should all be within the range of linearity that instrument detects；The blank test method are as follows:
In addition to sample is not added, operation repetitive is carried out using identical step.
9. measuring method as claimed in claim 8, which is characterized in that the result of the measurement calculates and expression method are as follows: examination
The residual quantity that benzimidazoles residues described in sample remain marker is calculated by formula (1):
In formula:
For X --- --- for the residual quantity of determinand in sample of having a try, unit is ng/kg (μ g/kg)；
The peak area of A --- --- determinand in sample solution；
cs--- --- in standard solution determinand concentration, unit be micro- gram per liter (μ g/L)；
The constant volume of V --- --- sample solution, unit are milliliter (mL)；
As--- --- in standard solution determinand peak area；
Sample mass representated by m --- --- sample solution, unit are gram (g).
10. measuring method as claimed in claim 9, which is characterized in that the sensitivity confirmation method of the measuring method are as follows: fixed
The determination for measuring limit, is determined according to the value of signal-to-noise ratio；Add 50 μ g/ respectively in blank chicken, chicken liver and chicken kidney
The benzimidazoles residues of kg remain marker hybrid standard working solution, measure the ratio of its signal and noise, when S/N >=
Concentration when 10 and when the rate of recovery and relative standard deviation meet method for detecting residue requirement is quantitative limit；Experimental result: this
Quantifying for method is limited to 50 μ g/kg；The accuracy confirmation method of the measuring method are as follows: accurately weigh blank sample described in 5.0g
In 50mL centrifuge tube, oneself is added and knows the series standard working solution of concentration, is made residual containing the benzimidazoles residues
Staying marker concentration is respectively the tissue sample of 50,200,500 μ g/kg, and each concentration does 6 in parallel, by sample pre-treatments mistake
It is measured after journey processing, calculates recovery of standard addition；Experimental result: this method is on 50 μ of μ g/kg~500 g/kg addition concentration level
The rate of recovery be 75%~110%；The precision confirmation method of the measuring method are as follows: accurately weigh blank sample described in 5.0g
In 50mL centrifuge tube, oneself is added and knows the series standard working solution of concentration, is made residual containing the benzimidazoles residues
Staying marker concentration is respectively the tissue sample of 50,200,500 μ g/kg, and each concentration does 6 in parallel, by sample pre-treatments mistake
It is measured after journey processing, calculates indoor relative standard deviation；Experimental result: relative standard deviation in the laboratory of this method≤
15%.
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| CN110343669A (en) * | 2019-07-19 | 2019-10-18 | 江南大学 | One plant of hybridoma cell strain DNC for secreting anti-Triclabendazole monoclonal antibody and its application |
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