CN107202840A - A kind of method for detecting 4 kinds of barbiturates downerns of fresh milk - Google Patents
A kind of method for detecting 4 kinds of barbiturates downerns of fresh milk Download PDFInfo
- Publication number
- CN107202840A CN107202840A CN201710368585.6A CN201710368585A CN107202840A CN 107202840 A CN107202840 A CN 107202840A CN 201710368585 A CN201710368585 A CN 201710368585A CN 107202840 A CN107202840 A CN 107202840A
- Authority
- CN
- China
- Prior art keywords
- fresh milk
- barbiturates
- acetonitrile
- kinds
- downerns
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk, fresh milk to be measured and acetonitrile solution are mixed, concussion, ultrasound, add inorganic salts A, centrifugation；Supernatant nitrogen is dried up, and uses acetonitrile solution constant volume, use to be clean；The acid is formic acid or acetic acid；Methanol and acetonitrile solution prewashing post are first used, constant volume liquid to be clean is crossed after post, ultrapure washing post, vacuum is drained；Dichloromethane eluent, coutroi velocity, eluent is dried up in nitrogen, acetonitrile solution constant volume, treats that machine is used；The fresh milk solution to be measured that preprocess method is obtained carries out high performance liquid chromatography mass spectrometry detection and analysis.Preprocess method in detection method can effectively improve the recovery rate of fresh milk barbiturate, and significantly improve the rate of recovery of the barbiturate in fresh milk, method is easy, economical, reliable, the matrix influence of fresh milk is significantly reduced, sensitivity, repetitive rate and the rate of recovery are good.
Description
Technical field
The invention belongs to field of food, it is related to a kind of safety detection method of food, specifically a kind of detection is fresh
The method of 4 kinds of barbiturates downerns in breast.
Background technology
Barbiturates (Barbiturates) is generality central depressant, including phenobarbital (Phenobarbital),
Amobarbital (Pentobarbital), amytal (Amobarbital), quinalbarbitone (Secobarbital) etc. are pacified
Dormancy downern, veterinary drug clinic is used as sedative and chemical Baoding, and approximate physiological can be caused to sleep, be mainly manifested in maincenter
The suppression of nervous system, moreover it is possible to suppress the respiratory center and vasomotor center of medulla oblongata.Suppress nervous centralis depth, typically with
Dose proportional.Low dose plays sedation, and median dose has syngignoscism, and heavy dose plays anesthetic effect.To circulation when measuring big
System and respiratory system also produce inhibitory action, and overdose easily causes acute death, and the lethal period is general more 10 days after the tablet has been ingested
Left and right, long-term taking is also easy to produce drug accumulation, causes death.People eats the animal tissue for remaining barbiturate, can lead
Cause dizzy, Nausea and vomiting, there may be tolerance and additive for long-term intake.Many countries forbid barbiturate drugs being used for
The mankind, also forbid it as feed addictive, in No. 178 bulletin of the Ministry of Agriculture of China with regard to clear stipulaties forbid by barbital,
Phenobarbital, sodium phenobarbital, amobarbital, yellow Jackets are added in feed and animal drinking water.In addition, veterinary drug is clinical
The off-drug period of barbiturate drugs is defined, such drug accumulation excessive concentration in animal body is prevented and influences eater's safety.
The safe mass issue concerns national economy of milk, the quality control of wherein fresh milk be milk production it is important before
Carry.However, from the point of view of the domestic breeding environment of China, in China's most area, the animal of particularly vast rural area and feeding
The situation using violated veterinary drug is still suffered from material production process.In order to prevent propagation of the barbiturate during commercialization
With accumulation, using fresh milk as a kind of important biological material and its barbiturate is detected for ensure dairy products
Safety is significant.
At present, the disclosed biological material for determining barbiturate residual of prior art is mainly human blood, serum, blood
The other biological tissue such as slurry, feed, urine.Method mainly have thin-layered chromatography, capillary electrophoresis, high performance liquid chromatography,
Gas chromatography, gas chromatography-mass spectrometry, Liquid Chromatography-Mass Spectrometry etc..Wherein, liquid chromatography and liquid phase
Chromatograph-mass spectrometer coupling method prepares for the detection of barbiturate and requires relatively low, as long as can be prepared into barbiturate
After solution and purification, with regard to liquid phase analysis can be used for.In addition, for liquid chromatogram is with respect to gas-chromatography, sensitivity is high, analyst coverage
Extensively, the advantages of analysis time is short, has been widely used in the analysis of the classification veterinary drug compound such as barbital in recent years, and cover
Biological material is also quite varied.In addition, because the matrix feature of different samples is different, preprocess method has very big difference
It is different.However, there is presently no using fresh milk is as biological material and detects that the method for wherein barbiturate is reported.Due to life
Protein and fat are rich in fresh milk, how the barbiturates veterinary drug compound that may wherein contain to be extracted, is purified, carried
High its has become urgent problem to be solved in the rate of recovery of fresh milk and sensitivity.
The content of the invention
For above-mentioned technical problem of the prior art, 4 kinds of barbiturateses of fresh milk are detected the invention provides one kind
The method of downern, the method for described this 4 kinds of barbiturates downerns of detection fresh milk will solve prior art
In the technical problem that detection fresh milk barbiturates downern is difficult.
The invention provides a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk, comprise the following steps：
1) a step of barbiturates downern to fresh milk to be measured carries out preextraction, by fresh milk and acetonitrile
Solution is mixed, concussion, ultrasound, adds inorganic salts A, centrifugation, and described fresh milk, acetonitrile solution, inorganic salts A material ratio are 10
~20mL：5~20g：2~10g；Supernatant is dried up using nitrogen, uses acetonitrile solution constant volume, use to be clean；The inorganic salts A
For any one in anhydrous magnesium sulfate, anhydrous sodium sulfate, anhydrous sodium chloride, natrium carbonicum calcinatum or two or more combinations；
2) the step of purification, first with methanol and acetonitrile solution prewashing post, constant volume liquid to be clean is crossed after post, ultrapure
Post is washed, vacuum is drained；Then dichloromethane eluent is used, coutroi velocity, eluent is dried up in nitrogen, and acetonitrile solution is fixed
Hold, treat that machine is used；Purification post used is SPE posts；
3) solution for obtaining preprocess method carries out HPLC-MS detection and analysis；
The mobile phase of described high performance liquid chromatography detection is：
Mobile phase A：Water；
Mobile phase B：Acetonitrile；
The column temperature of described high performance liquid chromatography detection is 25~35 DEG C；
The flow velocity of the testing sample of described high performance liquid chromatography detection is 0.3~0.6mL/min；
The sampling volume of described high performance liquid chromatography detection is 5~15 μ L；
The condition of the gradient elution of described high performance liquid chromatography detection is：
Described Mass Spectrometer Method condition is：
Ion gun：Electric spray ion source；
Scan pattern：Negative ion mode；
Capillary voltage：3.5kv；
Ion source temperature：100℃；
Remove solvent temperature：350℃.
Further, will in a step of barbiturates downern to fresh milk to be measured carries out preextraction
Fresh milk sample, acetonitrile solution mixing, concussion are mixed, and inorganic salts A, centrifugation are added after ultrasound；Take supernatant 10mL in 15mL from
In heart pipe, 50 DEG C of nitrogen dryings, with 2mL constant volumes containing acetonitrile solution, use to be clean；
Further, in the step of purifying for one, constant volume solution to be clean SPE column purifications in step (1) are first used
Methanol and acetonitrile solution cross post, coutroi velocity 0.5mL/min；2mL liquid to be clean is crossed after post, with ultrapure washing post, vacuum
Drain；With dichloromethane eluent, coutroi velocity, eluent is dried up in 50 DEG C of nitrogen, 1mL acetonitrile solution constant volumes, filtering, is treated
Machine is used.
Further, in step (1), the addition of described acetonitrile adds 10~20mL for every 5~20g testing samples；
In step (1), the addition of the inorganic salts A is 2~10g.
Further, acid is also added in described acetonitrile solution；Described acid is formic acid and/or acetic acid；Described sour adds
Dosage is the 0.1~0.3% of the acetonitrile addition, and the percentage is the percent by volume for accounting for acetonitrile.
Further, in step (1), the time of described concussion is 0.5~3min；
In step (1), described ultrasonic time is 20~40min；
In step (1), the rotating speed of described centrifugation is 5000~10000rpm；
In step (1), described centrifugation time is 5~15min；
Further, the SPE posts specification described in step (2) is 6mL or 150mg；
In step (2), the methanol, acetonitrile water, ultra-pure water are 5~10mL；The methylene chloride volume is 10~30mL；
The acetonitrile solution concentration is 30~50%；The percentage is the percent by volume for accounting for the aqueous solution；
In step (2), described filter method is through organic filtering with microporous membrane by solution.
Further, the SPE posts purchase producer described in step (2) is BESEP,
In step (2), the aperture of described organic miillpore filter is 0.22 μm.
Further, the chromatographic column of described high performance liquid chromatography detection is HPLC CSH C18 posts；Described chromatogram
The specification of post is 2.1 × 100mm, 1.7 μm；
The flow velocity of the testing sample of described high performance liquid chromatography detection is 0.3mL/min；
The μ L of sampling volume 5 of described high performance liquid chromatography detection；The column temperature of described high performance liquid chromatography detection
For 25 DEG C.
Further, during described Mass Spectrometer Method, target detection compound parent ion, daughter ion, impact energy, reservation
Time is：
Wherein, a represents quota ion.
Further, described testing sample may preferably contain phenobarbital and/or amobarbital and/or isoamyl bar
Than appropriate and/or quinalbarbitone fresh milk.Preferably, described fresh milk is the fresh milk for not containing solid particle.
Further, in step (1), described concussion is to beneficial to extraction, make acetonitrile further extract phenobarbital
And/or amobarbital and/or amytal and/or quinalbarbitone.The method and condition of described concussion are that this area is normal
The method and condition of rule, the time of described concussion is preferably 0.5~3min.
Further, in step (1), described ultrasound is to beneficial to extraction, make acetonitrile further extract phenobarbital
And/or amobarbital and/or amytal and/or quinalbarbitone.Described ultrasonic method and condition are that this area is normal
The method and condition of rule, the described ultrasonic time is preferably 30~60min.
In the present invention, the method for described HPLC-MS detection is the conventional method in this area, ability
Field technique personnel know that described HPLC-MS method is High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry side
Method.Described mass spectrographic detection mode is multiple-reaction monitoring pattern.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and produce each preferable reality of the present invention
Example.Agents useful for same and raw material of the present invention are commercially available.
The present invention solves the problem that complex matrices fresh milk barbiturates veterinary drug compound is extracted, purified, designed
Preprocess method preferably fresh milk impurity can be removed, efficiently extract fresh milk barbiturate, with compared with
The good rate of recovery；The detection method has carried out preferable separation to two kinds of barbiturates isomers simultaneously, with efficient, fast
Speed, the advantage such as cost is low, easy to operate, good purification, sensitivity are high, and accuracy and precision meets multi-residue analysis side
The requirement of method.
The inventive method use detection method, can simultaneously efficiently, quickly and accurately simultaneously to fresh milk benzene bar ratio
Appropriate, amobarbital, amytal, 4 kinds of barbiturates veterinary drug compounds of quinalbarbitone are measured, especially to penta bar ratio
Appropriate and two isomers of amytal have higher resolution ratio.
The present invention is compared with prior art, and its technological progress is significant.The present invention makes up existing detection fresh milk mini-bus
Than the missing of method of appropriate class compound, there is provided one kind is sensitive, easy, quick and accurately to determine fresh milk simultaneously a variety of
The method of barbiturates veterinary drug compound.Preprocess method in detection method can effectively improve fresh milk mini-bus ratio
The recovery rate of appropriate class medicine, and significantly improve the rate of recovery of the barbiturate in fresh milk, method is easy, economical, can
Lean on, significantly reduce the matrix influence of fresh milk, sensitivity, repetitive rate and the rate of recovery are good.
Brief description of the drawings
Fig. 1 is that High Performance Liquid Chromatography/Mass Spectrometry detection adds phenobarbital, amobarbital, amytal, quinalbarbitone
Fresh milk chromatogram.
Fig. 2 is the chromatogram that High Performance Liquid Chromatography/Mass Spectrometry detects fresh milk blank sample.
Fig. 3 is that High Performance Liquid Chromatography/Mass Spectrometry detection adds phenobarbital, amobarbital, amytal, quinalbarbitone
Fresh milk chromatogram.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product specification is selected.
Embodiment 1
The preparation of standard curve
Phenobarbital, amobarbital, amytal and quinalbarbitone are configured to 10 μ g/mL mother liquor with methanol,
In -20 DEG C of storages.Face used time each accurate absorption 1mL, the mixed standard solution 10mL that concentration is 1 μ g/mL be configured to methanol,
In -20 DEG C of storages.0 μ L, 50 μ L, 100 μ L, 200 μ L, 500 μ L, 1000 μ L mixed standard solutions, 50 DEG C of nitrogen is weighed before use
Drying, with blank fresh milk sample extracting solution 1mL constant volumes.Sample detection, is made standard curve.
Chromatographic column：ACQUITY CSH C18(2.1×100mm,1.7μm)；
Column temperature：25℃；
Flow velocity is 0.3mL/min；
Sample size：5μL；
Mobile phase A：Water；
Mobile phase B：Acetonitrile；
Condition of gradient elution is as shown in table 1
The liquid chromatogram linear gradient elution condition of table 1
| Time (min) | %A (water) | %B (acetonitrile) |
| 0.00 | 90.00 | 10.00 |
| 0.50 | 90.00 | 10.00 |
| 0.60 | 73.50 | 26.50 |
| 4.00 | 73.50 | 26.50 |
| 4.10 | 75.00 | 25.00 |
| 5.50 | 73.50 | 26.50 |
| 6.00 | 73.50 | 26.50 |
| 9.00 | 20.00 | 80.00 |
| 9.10 | 90.00 | 10.00 |
| 10.00 | 90.00 | 10.00 |
Mass Spectrometer Method condition is：
Ion gun：Electric spray ion source；
Scan pattern：Negative ion mode；
Capillary voltage：3.5kv；
Ion source temperature：100℃；
Remove solvent temperature：350℃；
Detection mode：Multiple-reaction monitoring pattern；
Target detection compound parent ion, daughter ion, impact energy, retention time are as shown in table 2：
24 kinds of barbiturate parent ions of table, daughter ion, impact energy, retention time information
Testing result：Fig. 1 be High Performance Liquid Chromatography/Mass Spectrometry detection add phenobarbital, amobarbital, amytal,
The chromatogram of the fresh milk of quinalbarbitone.
Corresponding compound peak area in Fig. 1 is determined, with concentration C (ng/mL) for abscissa, peak area A is ordinate, is painted
Phenobarbital processed, amobarbital, amytal, the standard liquid working curve of quinalbarbitone, obtain calibration curve equation and
Its coefficient correlation, as shown in table 3.
34 kinds of barbiturate linear equations of table, coefficient correlation and the range of linearity
Embodiment 2
The pretreatment of testing sample
(1) by 10.0g fresh milks (pasture A), it is placed in 50mL polypropylene centrifuge tubes, adds the acetic acid acetonitriles of 20mL 0.1%
Solution, shakes 30s, ultrasonic 20min；2g anhydrous sodium chlorides are added, 3min is shaken；5000rpm, centrifuges 5min；Take 10mL supernatants
Liquid is in 15mL polypropylene centrifuge tubes, and 50 DEG C of nitrogen dryings, 2mL 30% acetonitrile solution constant volume is to be clean.
(2) above-mentioned constant volume liquid is crossed into SPE pillars.First pre-processed with 5mL methanol and the acetonitrile solutions of 5mL 30%.It is above-mentioned fixed
Hold liquid and cross post, coutroi velocity is in 0.5mL/min or so.Then the ultrapure washing posts of 5mL are used, vacuum drains 15min；Use 10mL dichloros
Methane is eluted, and collects eluent, in 50 DEG C of nitrogen dryings；The acetonitrile solution constant volumes of 1mL 30%, cross the 0.22 organic filter membranes of μ L, use
In HPLC-MS/MS analyses.
The detection method such as embodiment 1 of phenobarbital, amobarbital, amytal, quinalbarbitone in testing sample
It is shown, by above-mentioned preprocess method and detection method, it is capable of detecting when phenobarbital in testing sample, amobarbital, different
Amobarbital, quinalbarbitone.
Embodiment 3
The pretreatment of testing sample
(1) by 10.0g fresh milks (pasture B), it is placed in 50mL polypropylene centrifuge tubes, adds the acetic acid acetonitriles of 20mL 0.1%
Solution, shakes 1min, ultrasonic 30min；5g anhydrous sodium chlorides are added, 3min is shaken；8000rpm, centrifuges 10min；Take on 10mL
Clear liquid is in 15mL polypropylene centrifuge tubes, and 50 DEG C of nitrogen dryings, 2mL 30% acetonitrile solution constant volume is to be clean.
(2) above-mentioned constant volume liquid is crossed into SPE pillars.First pre-processed with 8mL methanol and the acetonitrile solutions of 8mL 30%.It is above-mentioned fixed
Hold liquid and cross post, coutroi velocity is in 0.5mL/min or so.Then the ultrapure washing posts of 8mL are used, vacuum drains 15min；Use 20mL dichloros
Methane is eluted, and collects eluent, in 50 DEG C of nitrogen dryings；The acetonitrile solution constant volumes of 1mL 30%, cross the 0.22 organic filter membranes of μ L, use
In HPLC-MS/MS analyses.
The detection method such as embodiment 1 of phenobarbital, amobarbital, amytal, quinalbarbitone in testing sample
It is shown, by above-mentioned preprocess method and detection method, it is capable of detecting when phenobarbital in testing sample, amobarbital, different
Amobarbital, quinalbarbitone.
Embodiment 4
The pretreatment of testing sample
(1) by 10.0g fresh milks (pasture C), it is placed in 50mL polypropylene centrifuge tubes, adds the acetic acid acetonitriles of 20mL 0.1%
Solution, shakes 3min, ultrasonic 40min；10g anhydrous sodium chlorides are added, 3min is shaken；10000rpm, centrifuges 15min；Take 10mL
Supernatant is in 15mL polypropylene centrifuge tubes, and 50 DEG C of nitrogen dryings, 2mL 30% acetonitrile solution constant volume is to be clean.
(2) above-mentioned constant volume liquid is crossed into SPE pillars.First pre-processed with 10mL methanol and the acetonitrile solutions of 10mL 30%.It is above-mentioned
Constant volume liquid crosses post, and coutroi velocity is in 0.5mL/min or so.Then the ultrapure washing posts of 10mL are used, vacuum drains 15min；Use 30mL
Dichloromethane eluent, collects eluent, in 50 DEG C of nitrogen dryings；The acetonitrile solution constant volumes of 1mL 30%, crossing 0.22 μ L has machine filter
Film, for HPLC-MS/MS analyses.
The detection method such as embodiment 1 of phenobarbital, amobarbital, amytal, quinalbarbitone in testing sample
It is shown, by above-mentioned preprocess method and detection method, it is capable of detecting when phenobarbital in testing sample, amobarbital, different
Amobarbital, quinalbarbitone.
Comparative example 1
Fresh milk barbiturate is detected using method in SN/T 2217.
SN/T2217 methods detection fresh milk barbiturate step is as follows：
(1) fresh milk that 2g (being accurate to 0.01g) is free of target determinand after testing is weighed, 50mL polypropylene centrifuges are placed in
Guan Zhong, the hybrid standard working solution for adding 200 μ L1.0 μ g/mL stands 5min.5g anhydrous sodium sulfates are added, are mixed；Add 10mL
0.1% acetic acid acetonitrile solution, vortex mixed 3min, ultrasonic 10min；4000rpm, centrifuges 2min；Supernatant is taken in another 50mL
In polypropylene centrifuge tube, lower floor's solution repeats to extract once with 10mL 0.1% acetic acid acetonitrile, merges supernatant.In extract solution
The n-hexane of middle addition 5mL acetonitrile saturations, vortex mixed, centrifugation discards upper strata n-hexane, and lower floor's acetonitrile nitrogen is dried up, 5mL's
0.1mol/L ammonium acetate buffers：Methanol (90+10, volume ratio) dissolves constant volume, to be clean.
(2) above-mentioned constant volume liquid is crossed into HLB pillars.First pre-processed with 5mL methanol and 5mL0.1mol/L ammonium acetate buffers.
Above-mentioned constant volume liquid crosses post, and coutroi velocity is in 0.5mL/min or so.Then 5mL0.1mol/L ammonium acetate buffers are used：Methanol (90+
10, volume ratio) post is washed, vacuum drains 15min；Then use 5mL n-hexanes and 5mL n-hexanes：Ethyl acetate (95+5, volume ratio)
Elution；Finally use 10mL n-hexanes：Ethyl acetate (50+50, volume ratio) is eluted, nitrogen drying, dissolves residual with 1.0ml formic acid
Slag, crosses 0.45 μm of organic filter membrane, is analyzed for HPLC-MS/MS.Analysis condition such as embodiment 1.Experimental design 3 is parallel.
Experimental result is contrasted by index of the rate of recovery to the methods of SN/T 2217 and the inventive method, the results are shown in Table 5.
As can be seen from Table 5, the inventive method is far above the methods of SN/T 2217 to the rate of recovery of fresh milk barbiturate, says
Bright this method is more applicable for the detection of barbiturate in fresh milk class complex matrices.
Effect example 1
The measure of detection limit
The mixed standard solution prepared in embodiment 1 is shaken up, diluted from high concentration toward low concentration, the μ L of sample introduction 5, by implementation
HPLC chromatogram condition analysis in example 1.Concentration is surveyed during signal to noise ratio S/N=3 as detection limit, surveys dense during signal to noise ratio S/N=10
Degree is used as quantitative limit.Its detection limit and quantitative limit are as shown in table 4.
The detection limit and quantitative limit of 44 kinds of barbiturates of table
Effect example 2
The measure of recovery of standard addition
The fresh milk that 10g is free of target veterinary drug compound after testing is weighed, 1 μ g/mL hybrid standard work is separately added into
Liquid 100 μ L, 200 μ L, 400 μ L, be made 10,20, three Quality Control concentration levels of 40ng/g fresh milks, each concentration level does 6
Parallel sample, sample pre-treatments and HPLC-MS/MS analyses are carried out with the inventive method respectively, and according to standard items chromatographic peak face
Product and addition standard items determine the calculated by peak area rate of recovery after extracting, and the results are shown in Table 5.Fig. 2 detects for High Performance Liquid Chromatography/Mass Spectrometry
The chromatogram of fresh milk blank sample.Fig. 3 is that High Performance Liquid Chromatography/Mass Spectrometry detection adds phenobarbital, amobarbital, isoamyl bar ratio
The chromatogram of the fresh milk of appropriate and quinalbarbitone.Detection obtains phenobarbital, amobarbital, amytal, Si Keba ratios
Appropriate concentration is in error allowed band.As can be seen from Table 5, the rate of recovery of 4 kinds of barbiturates veterinary drug compounds in fresh milk
Between 85.04% and 111.37%, and the rate of recovery of the methods of SN/T 2217 detection fresh milk barbiturate exists
Between 23.65% and 43.24%, well below the inventive method, illustrate that the inventive method is applied to complex matrices fresh milk
The detection of barbiturate.
The rate of recovery testing result of table 5 and contrast
Effect example 3
The measure of precision
By same experimental design in effect example 2, determine and calculate the rate of recovery.Precision is in terms of relative standard deviation (RSD)
Calculate, the relative standard deviation of 4 kinds of barbiturates veterinary drugs of fresh milk is shown in Table 6.It can be seen and known by table 6, relative standard deviation exists in batch
Between 1.09% and 9.85%, relative standard deviation meets the requirement of multi-residue analysis between 3.88% and 7.28% between batch.
The Precision Experiment result of table 6
Result above shows that the preprocess method of this method is a kind of extracted to fresh milk barbiturate and purification
Good method, have in testing sample in phenobarbital, amobarbital, amytal, quinalbarbitone residue detection
Important application value.
Effect example 4
The detection of barbiturate in actual sample
The inventive method is applied to different pastures, in totally 50 blind samples of fresh milk barbiturate detection, specifically
Experimental procedure is carried out (making of standard curve is carried out according to embodiment 1) according to embodiment 3.Specific testing result is as follows：50
In the blind sample of fresh milk, totally 48 negative products, detect 2 positive products.7 (negative findings is unlisted) are the results are shown in Table, this knot
Fruit shows that the present invention can effectively detect fresh milk barbiturate.
The testing result of 4 kinds of barbiturates in the blind sample of actual fresh milk of table 7
Note：ND is represented and not detected.
Claims (9)
1. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk, it is characterised in that comprise the following steps：
1) a step of barbiturates downern to fresh milk to be measured carries out preextraction, by fresh milk and acetonitrile solution
Mixing, concussion, ultrasound adds inorganic salts A, centrifugation, described fresh milk, acetonitrile solution, inorganic salts A material ratio for 10~
20mL：5~20g：2~10g；Supernatant is dried up using nitrogen, uses acetonitrile solution constant volume, use to be clean；The inorganic salts A is
Any one in anhydrous magnesium sulfate, anhydrous sodium sulfate, anhydrous sodium chloride, natrium carbonicum calcinatum or two or more combinations；
2) the step of purification, first with methanol and acetonitrile solution prewashing post, constant volume liquid to be clean is crossed after post, ultrapure washing
Post, vacuum is drained；Then dichloromethane eluent is used, coutroi velocity, eluent is dried up in nitrogen, and acetonitrile solution constant volume is treated
Upper machine is used；Purification post used is SPE posts；
3) solution for obtaining preprocess method carries out HPLC-MS detection and analysis；
The mobile phase of described high performance liquid chromatography detection is：
Mobile phase A：Water；
Mobile phase B：Acetonitrile；
The column temperature of described high performance liquid chromatography detection is 25~35 DEG C；
The flow velocity of the testing sample of described high performance liquid chromatography detection is 0.3~0.6mL/min；
The sampling volume of described high performance liquid chromatography detection is 5~15 μ L；
The condition of the gradient elution of described high performance liquid chromatography detection is：
Described Mass Spectrometer Method condition is：
Ion gun：Electric spray ion source；
Scan pattern：Negative ion mode；
Capillary voltage：3.5kv；
Ion source temperature：100℃；
Remove solvent temperature：350℃.
2. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 1, its feature exists
In：
1) in the step of carrying out preextraction to the barbiturates downern of fresh milk to be measured for one, by fresh milk sample,
Acetonitrile solution is mixed, and concussion is mixed, and inorganic salts A, centrifugation are added after ultrasound；Supernatant 10mL is taken in 15mL centrifuge tubes, 50 DEG C
Nitrogen is dried up, with 2mL constant volumes containing acetonitrile solution, use to be clean；
2) one purify the step of in, by step 1) in constant volume solution to be clean SPE column purifications, first with methanol and acetonitrile water
Solution crosses post, coutroi velocity 0.5mL/min；2mL liquid to be clean is crossed after post, with ultrapure washing post, vacuum is drained；Use dichloromethane
Alkane is eluted, and coutroi velocity, eluent is dried up in 50 DEG C of nitrogen, 1mL acetonitrile solution constant volumes, filtering, treats that machine is used.
3. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 2, its feature exists
In：
In step (1), the addition of described acetonitrile adds 10~20mL for every 5~20g testing samples；
In step (1), the addition of the inorganic salts A is 2~10g.
4. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 1, its feature exists
In：Acid is also added in described acetonitrile solution；Described acid is formic acid and/or acetic acid；The sour addition is the acetonitrile
The 0.1~0.3% of addition, the percentage is the percent by volume for accounting for acetonitrile.
5. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 2, its feature exists
In：In step (1), the time of described concussion is 0.5~3min；
In step (1), described ultrasonic time is 20~40min；
In step (1), the rotating speed of described centrifugation is 5000~10000rpm；
In step (1), described centrifugation time is 5~15min.
6. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 2, its
It is characterised by：SPE posts specification described in step (2) is 6mL or 150mg；
In step (2), the methanol, acetonitrile water, ultra-pure water are 5~10mL；The methylene chloride volume is 10~30mL；It is described
Acetonitrile solution concentration is 30~50%；The percentage is the percent by volume for accounting for the aqueous solution；
In step (2), described filter method is through organic filtering with microporous membrane by solution.
7. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 6, its
It is characterised by：SPE posts purchase producer described in step (2) is BESEP,
In step (2), the aperture of described organic miillpore filter is 0.22 μm.
8. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 1, its
It is characterised by：The chromatographic column of described high performance liquid chromatography detection is HPLC CSH C18 posts；Institute
The specification for the chromatographic column stated is 2.1 × 100mm, 1.7 μm；
The flow velocity of the testing sample of described high performance liquid chromatography detection is 0.3mL/min；
The μ L of sampling volume 5 of described high performance liquid chromatography detection；The column temperature of described high performance liquid chromatography detection is 25
℃。
9. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 1, its
It is characterised by：
During described Mass Spectrometer Method, target detection compound parent ion, daughter ion, impact energy, retention time are：
Wherein, a represents quota ion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710368585.6A CN107202840B (en) | 2017-05-23 | 2017-05-23 | A method of detection 4 kinds of barbiturates downerns of fresh milk |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710368585.6A CN107202840B (en) | 2017-05-23 | 2017-05-23 | A method of detection 4 kinds of barbiturates downerns of fresh milk |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107202840A true CN107202840A (en) | 2017-09-26 |
| CN107202840B CN107202840B (en) | 2019-06-28 |
Family
ID=59906139
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710368585.6A Active CN107202840B (en) | 2017-05-23 | 2017-05-23 | A method of detection 4 kinds of barbiturates downerns of fresh milk |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107202840B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111829852A (en) * | 2020-07-28 | 2020-10-27 | 江西省食品检验检测研究院（江西国家果蔬产品及加工食品质量监督检验中心） | Sample pretreatment method for detecting diazepam residue in aquatic product and application thereof |
| CN112083115A (en) * | 2020-09-25 | 2020-12-15 | 上海市农产品质量安全中心 | Kit for detecting residual quantity of 7 barbiturates in raw fresh milk |
| CN114609310A (en) * | 2022-03-24 | 2022-06-10 | 中国农业科学院农业质量标准与检测技术研究所 | Sample pretreatment method and detection method for detecting sedative residue in milk by chromatography-mass spectrometry |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101398414A (en) * | 2008-10-15 | 2009-04-01 | 上海市公安局刑事侦查总队 | Method for qualitatively screening 242 kinds of compounds by liquid phase chromatography-mass spectra at the same times |
| CN102226794A (en) * | 2011-03-28 | 2011-10-26 | 中华人民共和国连云港出入境检验检疫局 | Liquid chromatography-tandom mass spectrometry detection method of thirty-one drugs in human blood |
| CN102486472A (en) * | 2009-04-20 | 2012-06-06 | 上海和黄药业有限公司 | Method for determining medicine active component in serum |
| CN106404925A (en) * | 2015-07-31 | 2017-02-15 | 复旦大学 | Method for determining six barbiturates in human whole blood |
-
2017
- 2017-05-23 CN CN201710368585.6A patent/CN107202840B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101398414A (en) * | 2008-10-15 | 2009-04-01 | 上海市公安局刑事侦查总队 | Method for qualitatively screening 242 kinds of compounds by liquid phase chromatography-mass spectra at the same times |
| CN102486472A (en) * | 2009-04-20 | 2012-06-06 | 上海和黄药业有限公司 | Method for determining medicine active component in serum |
| CN102226794A (en) * | 2011-03-28 | 2011-10-26 | 中华人民共和国连云港出入境检验检疫局 | Liquid chromatography-tandom mass spectrometry detection method of thirty-one drugs in human blood |
| CN106404925A (en) * | 2015-07-31 | 2017-02-15 | 复旦大学 | Method for determining six barbiturates in human whole blood |
Non-Patent Citations (5)
| Title |
|---|
| SHIMOYAMA, R; OHKUBO, T; SUGAWARA, K: "Characteristics of interaction between barbiturate derivatives and various sorbents on liquid chromatography and determination of phenobarbital in japanese human breast milk", 《JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES》 * |
| 中华人民共和国湖南出入境检验检疫局，岳阳出入境检验检疫局: "《SN/T 2217-2008》", 18 November 2008, 中国标准出版社 * |
| 刘义钊: "SPE-HPLC法同时测定5种抗癫痫药的血药浓度", 《中南药学》 * |
| 孙增先: "固相萃取-高效液相色谱法测定3种抗癫痫药物血药浓度", 《中国医院药学杂志》 * |
| 沈晓洁: "SPE-HPLC法测定血清中苯巴比妥、苯妥英钠和卡马西平浓度", 《江苏药学与临床研究》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111829852A (en) * | 2020-07-28 | 2020-10-27 | 江西省食品检验检测研究院（江西国家果蔬产品及加工食品质量监督检验中心） | Sample pretreatment method for detecting diazepam residue in aquatic product and application thereof |
| CN112083115A (en) * | 2020-09-25 | 2020-12-15 | 上海市农产品质量安全中心 | Kit for detecting residual quantity of 7 barbiturates in raw fresh milk |
| CN114609310A (en) * | 2022-03-24 | 2022-06-10 | 中国农业科学院农业质量标准与检测技术研究所 | Sample pretreatment method and detection method for detecting sedative residue in milk by chromatography-mass spectrometry |
| CN114609310B (en) * | 2022-03-24 | 2023-10-03 | 中国农业科学院农业质量标准与检测技术研究所 | Sample pretreatment method for detecting sedative residue in milk by chromatography-mass spectrometry and detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107202840B (en) | 2019-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107202840A (en) | A kind of method for detecting 4 kinds of barbiturates downerns of fresh milk | |
| CN106770865B (en) | A kind of organic acid content testing method in ginkgo biloba p.e | |
| CN102226794A (en) | Liquid chromatography-tandom mass spectrometry detection method of thirty-one drugs in human blood | |
| CN102141552A (en) | High-sensitivity whole blood tacrolimus quantitative assay kit and preparation method thereof | |
| CN111781290A (en) | Kit and detection method for accurately determining blood concentration of multiple antiepileptic drugs in human serum | |
| CN106610410A (en) | Method for detecting biogenic amines in fish and fish products | |
| CN103235117A (en) | Beta 2-acceptor stimulant ELISA kit and usage method and application thereof | |
| CN102628844A (en) | Content determining method for trichlorfon in dried fish | |
| CN106526033A (en) | Method for simultaneously testing 11 macamides content of maca | |
| CN109828071B (en) | Method for simultaneously detecting 9 water-infused drug residues in pork | |
| CN104034835B (en) | The detection method of multiple biotoxin in fermented wine | |
| CN105116063A (en) | Multi-detection method of residual of cephalo-type drugs in milk product | |
| CN103267820A (en) | Method for simultaneously detecting multiple estrogens in sludge | |
| CN105548433B (en) | The method that liquid chromatography tandem mass spectrometry detects various non-protein nitrogen-containing compound contents in milk simultaneously | |
| Wang et al. | Analysis of strychnine and brucine in postmortem specimens by RP-HPLC: a case report of fatal intoxication | |
| CN107290444A (en) | The method for detecting neopterin and biopterin in human urine | |
| CN103163225A (en) | High-sensitivity quantitative detection kit for cyclosporine A in whole blood and preparation method thereof | |
| CN108828098B (en) | Method for determining melatonin in cotton by high performance liquid chromatography-mass spectrometry | |
| CN103160472B (en) | Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit | |
| CN101639467A (en) | Method for determining amount of residual florfenicol in aquatic product by using high-efficiency liquid chromatogram fluorescence method | |
| CN103217498A (en) | Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method | |
| CN106370736A (en) | Method for simultaneous SMART column on-line purification and HPLC/UVE fluorescent detection of four aflatoxins in peanut product | |
| CN106770765B (en) | The detection method and application of a kind of albendazole and its metabolin | |
| CN102445509B (en) | Reversed phase ion pair HPLC (high performance liquid chromatography) method for rapid trace detection of TTX (tetrodoxin) in fresh puffer fish blood | |
| CN104977383A (en) | Method for rapidly and quantitatively detecting microcystins in spirulina food |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |