CN107202840B - A method of detection 4 kinds of barbiturates downerns of fresh milk - Google Patents
A method of detection 4 kinds of barbiturates downerns of fresh milk Download PDFInfo
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Abstract
The invention discloses a kind of methods for detecting 4 kinds of barbiturates downerns of fresh milk, and fresh milk to be measured and acetonitrile solution are mixed, concussion, ultrasound, and inorganic salts A, centrifugation is added；Supernatant is dried with nitrogen, with acetonitrile solution constant volume, use to be clean；The acid is formic acid or acetic acid；Methanol and acetonitrile solution prewashing column are first used, after constant volume liquid to be clean is crossed column, ultrapure washing column, vacuum is drained；Dichloromethane eluent, coutroi velocity, eluent in being dried with nitrogen, use to upper machine by acetonitrile solution constant volume；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 barbiturate is significantly improved in the rate of recovery of fresh milk, method is easy, economical, reliable, the matrix for significantly reducing fresh milk influences, and sensitivity, repetitive rate and the rate of recovery are good.
Description
Technical field
The invention belongs to field of food, are related to a kind of safety detection method of food, and specifically a kind of detection is fresh
The method of 4 kinds of barbiturates downerns in cream.
Background technique
Barbiturates (Barbiturates) are generality central depressants, including phenobarbital (Phenobarbital),
The peace such as amobarbital (Pentobarbital), amytal (Amobarbital), quinalbarbitone (Secobarbital)
Dormancy downern, veterinary drug clinic are used as sedative and chemical Baoding, approximate physiological can be caused to sleep, be mainly manifested in maincenter
The inhibition of nervous system, moreover it is possible to inhibit the respiratory center and vasomotor center of medulla oblongata.Inhibit nervous centralis depth, generally with
Dose proportional.Low dose plays sedation, and median dose has syngignoscism, and large dosage plays anesthetic effect.To circulation when measuring big
System and respiratory system also generate inhibiting effect, and overdose easily causes acute death, and the lethal period is general 10 days after the tablet has been ingested more
Left and right, long-term use are also easy to produce drug accumulation, lead to death.People eats the animal tissue for remaining barbiturate, can lead
Cause dizzy, Nausea and vomiting, there may be tolerances and additive for long-term intake.Many countries forbid for barbiturate drugs to be used for
The mankind also forbid it as feed addictive, in No. 178 bulletin of the Ministry of Agriculture in 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 for defining barbiturate drugs prevents such drug accumulation excessive concentration in animal body and influences eater's safety.
The safe mass issue concerns national economy of milk, wherein fresh milk quality control be milk production it is important before
It mentions.However, animal and feeding from the point of view of the country, China breeding environment, in China's most area, especially the vast rural areas
Expect that there are still use violated veterinary drug in production process.Propagation of barbiturate during commercialization in order to prevent
With accumulation, fresh milk as a kind of important biological material and detects for ensureing dairy products its barbiturate
Safety is of great significance.
Currently, the disclosed measurement remaining biological material of barbiturate of the prior art is mainly people's blood, serum, blood
The other biologicals 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 for barbiturate detection preparation require it is lower, as long as barbiturate can be prepared into
After solution and purification, liquid phase analysis can be used for.In addition, for liquid chromatogram is with respect to gas-chromatography, high sensitivity, analyst coverage
Extensively, the advantages that analysis time is short, has been widely used in the analysis of the classifications veterinary drug compound such as barbital in recent years, and cover
Biological material is also very extensive.In addition to this, since 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 as biological material and detect wherein barbiturate method report.Due to life
It is rich in protein and fat in fresh milk, how the barbiturates veterinary drug compound that may wherein contain to be extracted, is purified, mentioned
High its in the rate of recovery of fresh milk and sensitivity has become urgent problem to be solved.
Summary of the invention
For above-mentioned technical problem in the prior art, the present invention provides a kind of 4 kinds of barbiturates of detection fresh milk
The method of the method for downern, this 4 kinds of barbiturates downerns of detection fresh milk will solve the prior art
In the technical problem of detection fresh milk barbiturates downern difficulty.
The present invention provides a kind of methods for detecting 4 kinds of barbiturates downerns of fresh milk, include the following steps:
1) a barbiturates downern to fresh milk to be measured carries out the step of preextraction, by fresh milk and acetonitrile
Solution mixing, concussion, ultrasound, be added inorganic salts A, centrifugation, the fresh milk, acetonitrile solution, inorganic salts A material ratio be 10
~20mL:5~20g:2~10g；Supernatant is using being dried with nitrogen, with acetonitrile solution constant volume, use to be clean；The inorganic salts A
For any one or the two or more combinations in anhydrous magnesium sulfate, anhydrous sodium sulfate, anhydrous sodium chloride, natrium carbonicum calcinatum；
2) methanol and acetonitrile solution prewashing column are first used in the step of purification, ultrapure after constant volume liquid to be clean is crossed column
Column is washed, vacuum is drained；Then dichloromethane eluent is used, coutroi velocity, for eluent in being dried with nitrogen, acetonitrile solution is fixed
Hold, is used to upper machine；Purifying column used is SPE column；
3) solution for obtaining preprocess method carries out high performance liquid chromatography-mass spectrometry detection and analysis；
The mobile phase of the high performance liquid chromatography detection are as follows:
Mobile phase A: water；
Mobile phase B: acetonitrile；
The column temperature of the high performance liquid chromatography detection is 25~35 DEG C；
The flow velocity of the sample to be tested of the high performance liquid chromatography detection is 0.3~0.6mL/min；
The sampling volume of the high performance liquid chromatography detection is 5~15 μ L；
The condition of the gradient elution of the high performance liquid chromatography detection are as follows:
The Mass Spectrometer Method condition are as follows:
Ion source: electric spray ion source；
Scan pattern: negative ion mode；
Capillary voltage: 3.5kv；
Ion source temperature: 100 DEG C；
Remove solvent temperature: 350 DEG C.
It further, will in a step of barbiturates downern to fresh milk to be measured carries out preextraction
Fresh milk sample, acetonitrile solution mixing, concussion mix, and inorganic salts A, centrifugation are added after ultrasound；Take supernatant 10mL in 15mL from
In heart pipe, 50 DEG C are dried with nitrogen, with 2mL constant volume containing acetonitrile solution, use to be clean；
Further, in the purification the step of, constant volume solution to be clean SPE column purification in step (1) is first used
Methanol and acetonitrile solution cross column, coutroi velocity 0.5mL/min；After 2mL liquid to be clean is crossed column, with ultrapure washing column, vacuum
It drains；With dichloromethane eluent, coutroi velocity, eluent is dried with nitrogen in 50 DEG C, 1mL acetonitrile solution constant volume, filtering, to upper
Machine is used.
Further, in step (1), the additive amount of the acetonitrile is that every 5~20g sample to be tested adds 10~20mL；
In step (1), the additive amount of the inorganic salts A is 2~10g.
Further, also addition is sour in the acetonitrile solution；The acid is formic acid and/or acetic acid；The acid adds
Dosage is the 0.1~0.3% of the acetonitrile additive amount, and the percentage is the percent by volume for accounting for acetonitrile.
Further, in step (1), the time of the concussion is 0.5~3min；
In step (1), the ultrasonic time is 20~40min；
In step (1), the revolving speed of the centrifugation is 5000~10000rpm；
In step (1), the centrifugation time is 5~15min；
Further, SPE column specification described in step (2) is 6mL or 150mg；
In step (2), the methanol, acetonitrile water, ultrapure 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 aqueous solution；
In step (2), the filter method is by solution through organic filtering with microporous membrane.
Further, the purchase of SPE column described in step (2) producer is BESEP,
In step (2), the aperture of organic miillpore filter is 0.22 μm.
Further, the chromatographic column of high performance liquid chromatography detection is HPLC CSH C18 column；The chromatography
The specification of column be 2.1 × 100mm, 1.7 μm；
The flow velocity of the sample to be tested of the high performance liquid chromatography detection is 0.3mL/min；
The 5 μ L of sampling volume of the high performance liquid chromatography detection；The column temperature of the high performance liquid chromatography detection
It is 25 DEG C.
Further, during the Mass Spectrometer Method, target detection compound parent ion, daughter ion, impact energy, reservation
Time are as follows:
Wherein, a represents quota ion.
Further, the sample to be tested may preferably contain phenobarbital and/or amobarbital and/or isoamyl bar
Than appropriate and/or quinalbarbitone fresh milk.Preferably, the fresh milk is the fresh milk without containing solid particle.
Further, in step (1), the concussion is so that acetonitrile is further extracted phenobarbital in order to be conducive to extract
And/or amobarbital and/or amytal and/or quinalbarbitone.The method and condition of the concussion is that this field is normal
The method and condition of rule, the time of the concussion are preferably 0.5~3min.
Further, in step (1), the ultrasound is so that acetonitrile is further extracted phenobarbital in order to be conducive to extract
And/or amobarbital and/or amytal and/or quinalbarbitone.The method and condition of the ultrasound is that this field is normal
The time of the method and condition of rule, the ultrasound is preferably 30~60min.
In the present invention, the method for the high performance liquid chromatography-mass spectrometry detection is the method for this field routine, ability
Field technique personnel know that the high performance liquid chromatography-mass spectrometry method is High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry side
Method.The mass spectrographic detection mode is multiple-reaction monitoring pattern.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.The reagents and materials used in the present invention are commercially available.
The present invention solves the problems, such as 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, have compared with
The good rate of recovery；The detection method has carried out preferable separation to two kinds of barbiturates isomers simultaneously, has efficiently, fastly
The advantages such as fast, at low cost, easy to operate, good purification, high sensitivity, accuracy and precision meet multi-residue analysis side
The requirement of method.
The detection method that the method for the present invention uses can simultaneously efficiently, quickly and accurately simultaneously compare fresh milk benzene bar
4 kinds of appropriate, amobarbital, amytal, quinalbarbitone barbiturates veterinary drug compounds are measured, and are especially compared penta bar
Appropriate and two isomer resolution ratio with higher of amytal.
The present invention is compared with prior art, and technological progress is significant.The present invention makes up existing detection fresh milk mini-bus
Than the missing of the method for appropriate class compound, provide a kind of sensitive, easy, quick and accurately to measure simultaneously fresh milk 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 drug, and significantly improve barbiturate in the rate of recovery of fresh milk, method is easy, it is economical, can
It leans on, the matrix for significantly reducing fresh milk influences, and sensitivity, repetitive rate and the rate of recovery are good.
Detailed description of the invention
Fig. 1 is that phenobarbital, amobarbital, amytal, quinalbarbitone is added in High Performance Liquid Chromatography/Mass Spectrometry detection
Fresh milk chromatogram.
Fig. 2 is the chromatogram of High Performance Liquid Chromatography/Mass Spectrometry detection fresh milk blank sample.
Fig. 3 is that phenobarbital, amobarbital, amytal, quinalbarbitone is added in High Performance Liquid Chromatography/Mass Spectrometry detection
Fresh milk chromatogram.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Embodiment 1
The preparation of standard curve
Phenobarbital, amobarbital, amytal and quinalbarbitone are configured to the mother liquor of 10 μ g/mL with methanol,
It is stored in -20 DEG C.Face used time each accurate absorption 1mL, be configured to the mixed standard solution 10mL that concentration is 1 μ g/mL with methanol,
It is stored in -20 DEG C.Weigh 0 μ L before use, 50 μ L, 100 μ L, 200 μ L, 500 μ L, 1000 μ L mixed standard solutions, 50 DEG C of nitrogen
Drying, with blank fresh milk sample extracting solution 1mL constant volume.Standard curve is made in sample detection.
Chromatographic column: ACQUITY CSH C18 (2.1 × 100mm, 1.7 μm)；
Column temperature: 25 DEG C；
Flow velocity is 0.3mL/min；
Sample volume: 5 μ L；
Mobile phase A: water；
Mobile phase B: acetonitrile；
Condition of gradient elution is as shown in table 1
1 liquid chromatogram linear gradient elution condition of table
| 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 are as follows:
Ion source: electric spray ion source；
Scan pattern: negative ion mode；
Capillary voltage: 3.5kv；
Ion source temperature: 100 DEG C；
Remove solvent temperature: 350 DEG C；
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 be added phenobarbital, amobarbital, amytal,
The chromatogram of the fresh milk of quinalbarbitone.
Corresponding compound peak area in measurement chart 1, with concentration C (ng/mL) for abscissa, peak area A is ordinate, is drawn
Phenobarbital processed, amobarbital, amytal, quinalbarbitone standard solution working curve, obtain calibration curve equation and
Its related coefficient, as shown in table 3.
34 kinds of barbiturate linear equations of table, related coefficient and the range of linearity
Embodiment 2
The pretreatment of sample to be tested
(1) it by 10.0g fresh milk (pasture A), is placed in 50mL polypropylene centrifuge tube, 0.1% acetic acid acetonitrile of 20mL is added
Solution shakes 30s, ultrasonic 20min；2g anhydrous sodium chloride is added, shakes 3min；5000rpm is centrifuged 5min；Take 10mL supernatant
In 15mL polypropylene centrifuge tube, 50 DEG C are dried with nitrogen liquid, and the acetonitrile solution constant volume of 2mL 30% is to be clean.
(2) above-mentioned constant volume liquid is crossed into SPE pillar.First pre-processed with 5mL methanol and 30% acetonitrile solution of 5mL.It is above-mentioned fixed
Hold liquid and cross column, coutroi velocity is in 0.5mL/min or so.Then the ultrapure washing column of 5mL is used, vacuum drains 15min；With 10mL dichloro
Methane elution, collects eluent, is dried with nitrogen in 50 DEG C；30% acetonitrile solution constant volume of 1mL crosses the 0.22 organic filter membrane of μ L, uses
It is analyzed in HPLC-MS/MS.
The detection method such as embodiment 1 of phenobarbital, amobarbital, amytal, quinalbarbitone in sample to be tested
It is shown, by above-mentioned preprocess method and detection method, it is capable of detecting when phenobarbital in sample to be tested, amobarbital, different
Amobarbital, quinalbarbitone.
Embodiment 3
The pretreatment of sample to be tested
(1) it by 10.0g fresh milk (pasture B), is placed in 50mL polypropylene centrifuge tube, 0.1% acetic acid acetonitrile of 20mL is added
Solution shakes 1min, ultrasonic 30min；5g anhydrous sodium chloride is added, shakes 3min；8000rpm is centrifuged 10min；It takes on 10mL
In 15mL polypropylene centrifuge tube, 50 DEG C are dried with nitrogen clear liquid, and the acetonitrile solution constant volume of 2mL 30% is to be clean.
(2) above-mentioned constant volume liquid is crossed into SPE pillar.First pre-processed with 8mL methanol and 30% acetonitrile solution of 8mL.It is above-mentioned fixed
Hold liquid and cross column, coutroi velocity is in 0.5mL/min or so.Then the ultrapure washing column of 8mL is used, vacuum drains 15min；With 20mL dichloro
Methane elution, collects eluent, is dried with nitrogen in 50 DEG C；30% acetonitrile solution constant volume of 1mL crosses the 0.22 organic filter membrane of μ L, uses
It is analyzed in HPLC-MS/MS.
The detection method such as embodiment 1 of phenobarbital, amobarbital, amytal, quinalbarbitone in sample to be tested
It is shown, by above-mentioned preprocess method and detection method, it is capable of detecting when phenobarbital in sample to be tested, amobarbital, different
Amobarbital, quinalbarbitone.
Embodiment 4
The pretreatment of sample to be tested
(1) it by 10.0g fresh milk (pasture C), is placed in 50mL polypropylene centrifuge tube, 0.1% acetic acid acetonitrile of 20mL is added
Solution shakes 3min, ultrasonic 40min；10g anhydrous sodium chloride is added, shakes 3min；10000rpm is centrifuged 15min；Take 10mL
In 15mL polypropylene centrifuge tube, 50 DEG C are dried with nitrogen supernatant, and the acetonitrile solution constant volume of 2mL 30% is to be clean.
(2) above-mentioned constant volume liquid is crossed into SPE pillar.First pre-processed with 10mL methanol and 30% acetonitrile solution of 10mL.It is above-mentioned
Constant volume liquid crosses column, and coutroi velocity is in 0.5mL/min or so.Then the ultrapure washing column of 10mL is used, vacuum drains 15min；Use 30mL
Dichloromethane eluent is collected eluent, is dried with nitrogen in 50 DEG C；30% acetonitrile solution constant volume of 1mL, crossing 0.22 μ L has machine filter
Film is analyzed for HPLC-MS/MS.
The detection method such as embodiment 1 of phenobarbital, amobarbital, amytal, quinalbarbitone in sample to be tested
It is shown, by above-mentioned preprocess method and detection method, it is capable of detecting when phenobarbital in sample to be tested, amobarbital, different
Amobarbital, quinalbarbitone.
Comparative example 1
Fresh milk barbiturate is detected using method in SN/T 2217.
SN/T2217 method detects fresh milk barbiturate, and steps are as follows:
(1) fresh milk that 2g (being accurate to 0.01g) is free of target determinand through detecting is weighed, 50mL polypropylene centrifuge is placed in
Guan Zhong, the hybrid standard working solution that 200 μ L1.0 μ g/mL are added stand 5min.5g anhydrous sodium sulfate is added, mixes；10mL is added
0.1% acetic acid acetonitrile solution, vortex mixed 3min, ultrasonic 10min；4000rpm is centrifuged 2min；Take supernatant in another 50mL
In polypropylene centrifuge tube, lower layer's solution acetic acid acetonitrile of 10mL 0.1% repeats to extract primary, merging supernatant.In extracting solution
The middle n-hexane that 5mL acetonitrile saturation is added, vortex mixed, centrifugation discard upper layer n-hexane, lower layer's acetonitrile is dried with nitrogen, 5mL's
0.1mol/L ammonium acetate buffer: methanol (90+10, volume ratio) dissolves constant volume, to be clean.
(2) above-mentioned constant volume liquid is crossed into HLB pillar.First pre-processed with 5mL methanol and 5mL0.1mol/L ammonium acetate buffer.
Above-mentioned constant volume liquid crosses column, and coutroi velocity is in 0.5mL/min or so.Then 5mL0.1mol/L ammonium acetate buffer: methanol (90+ is used
10, volume ratio) column is washed, vacuum drains 15min；Then use 5mL n-hexane and 5mL n-hexane: ethyl acetate (95+5, volume ratio)
Elution；Finally use 10mL n-hexane: ethyl acetate (50+50, volume ratio) elution is dried with nitrogen, and is dissolved with 1.0ml formic acid residual
Slag crosses 0.45 μm of organic filter membrane, analyzes for HPLC-MS/MS.Analysis condition such as embodiment 1.Experimental design 3 parallel.
Experimental result compares 2217 method of SN/T and the method for the present invention using the rate of recovery as index, the results are shown in Table 5.
As can be seen from Table 5, the method for the present invention is much higher than 2217 method of SN/T 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 measurement of detection limit
The mixed standard solution prepared in embodiment 1 is shaken up, is diluted from high concentration toward low concentration, 5 μ L of sample introduction, by implementation
HPLC chromatogram condition analysis in example 1.Surveyed concentration is as detection limit when signal-to-noise ratio S/N=3, and when signal-to-noise ratio S/N=10 surveys dense
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 measurement of recovery of standard addition
The fresh milk that 10g is free of target veterinary drug compound through detecting is weighed, the hybrid standard work of 1 μ g/mL is separately added into
Liquid 100 μ L, 200 μ L, 400 μ L, are made 10, three 20,40ng/g fresh milk Quality Control concentration levels, each concentration level does 6
Parallel sample carries out sample pre-treatments and HPLC-MS/MS analysis with the method for the present invention respectively, and according to standard items chromatographic peak face
Product and addition standard items measure the calculated by peak area rate of recovery after extracting, and the results are shown in Table 5.Fig. 2 is High Performance Liquid Chromatography/Mass Spectrometry detection
The chromatogram of fresh milk blank sample.Fig. 3 is that phenobarbital, amobarbital, isoamyl bar ratio is added in High Performance Liquid Chromatography/Mass Spectrometry detection
The chromatogram of appropriate and quinalbarbitone fresh milk.Detection obtains phenobarbital, amobarbital, amytal, Si Keba ratio
Appropriate concentration is in allowable range of error.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 2217 method of SN/T detection fresh milk barbiturate exists
Between 23.65% and 43.24%, well below the method for the present invention, illustrate that the method for the present invention is suitable for complex matrices fresh milk
The detection of barbiturate.
5 rate of recovery testing result of table and comparison
Effect example 3
The measurement of precision
By same experimental design in effect example 2, measures and calculate the rate of recovery.Precision is in terms of relative standard deviation (RSD)
It calculates, the relative standard deviation of 4 kinds of barbiturates veterinary drugs of fresh milk is shown in Table 6.It can be seen and be 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.
6 precision experiment result of table
The above result shows that the preprocess method of this method is that a kind of pair of fresh milk barbiturate is extracted and purified
Good method, in sample to be tested phenobarbital, amobarbital, amytal, have in quinalbarbitone residue detection
Important application value.
Effect example 4
The detection of barbiturate in actual sample
The method of the present invention is applied to different pastures, in totally 50 blind samples of fresh milk barbiturate detection, specifically
Experimental procedure carries out (production 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.It the results are shown in Table 7 (negative findings are unlisted), this knot
Fruit shows that fresh milk barbiturate can be effectively detected in the present invention.
The testing result of 4 kinds of barbiturates in the 7 blind sample of practical fresh milk of table
Note: ND representative is not detected.
Claims (8)
1. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk, it is characterised in that include the following steps:
1) a barbiturates downern to fresh milk to be measured carries out the step of preextraction, and the barbiturates are calm
Drug is any one in phenobarbital, amobarbital, amytal or quinalbarbitone, and fresh milk and acetonitrile is molten
Liquid mixing, concussion, ultrasound, be added inorganic salts A, centrifugation, the fresh milk, acetonitrile solution, inorganic salts A material ratio be 10~
20mL:5~20g:2~10g；Supernatant is using being dried with nitrogen, with acetonitrile solution constant volume, use to be clean；The inorganic salts A is
Anhydrous magnesium sulfate, anhydrous sodium sulfate, anhydrous sodium chloride, any one or two or more combinations in natrium carbonicum calcinatum；
2) methanol and acetonitrile solution prewashing column, after constant volume liquid to be clean is crossed column, ultrapure washing are first used in the step of purification
Column, vacuum are drained；Then dichloromethane eluent is used, coutroi velocity, eluent is in being dried with nitrogen, acetonitrile solution constant volume, to
Upper machine is used；Purifying column used is SPE column；The SPE column purchase producer is BESEP；
3) solution for obtaining preprocess method carries out high performance liquid chromatography-mass spectrometry detection and analysis；
The chromatographic column of the high performance liquid chromatography detection is HPLC CSH C18 column；
The mobile phase of the high performance liquid chromatography detection are as follows:
Mobile phase A: water；
Mobile phase B: acetonitrile；Also addition is sour in the acetonitrile solution；The acid is formic acid and/or acetic acid；The acid adds
Dosage is the 0.1~0.3% of the acetonitrile additive amount, and the percentage is the percent by volume for accounting for acetonitrile；
The column temperature of the high performance liquid chromatography detection is 25~35 DEG C；
The flow velocity of the sample to be tested of the high performance liquid chromatography detection is 0.3~0.6mL/min；
The sampling volume of the high performance liquid chromatography detection is 5~15 μ L；
The condition of the gradient elution of the high performance liquid chromatography detection are as follows:
The Mass Spectrometer Method condition are as follows:
Ion source: electric spray ion source；
Scan pattern: negative ion mode；
Capillary voltage: 3.5kv；
Ion source temperature: 100 DEG C；
Remove solvent temperature: 350 DEG C.
2. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 1, feature exist
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 mixing, concussion mix, and inorganic salts A, centrifugation are added after ultrasound；Take supernatant 10mL in 15mL centrifuge tube, 50 DEG C
It is dried with nitrogen, with 2mL constant volume containing acetonitrile solution, use to be clean；
2) in the purification the step of, by the solution to be clean SPE column purification of constant volume in step 1), methanol and acetonitrile water are first used
Solution crosses column, coutroi velocity 0.5mL/min；After 2mL liquid to be clean is crossed column, with ultrapure washing column, vacuum is drained；Use dichloromethane
Alkane elution, coutroi velocity, eluent are dried with nitrogen in 50 DEG C, 1mL acetonitrile solution constant volume, and filtering is used to upper machine.
3. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 2, feature exist
In:
In step (1), the additive amount of the acetonitrile is that every 5~20g sample to be tested adds 10~20mL；
In step (1), the additive amount 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 2, feature exist
In: in step (1), the time of the concussion is 0.5~3min；
In step (1), the ultrasonic time is 20~40min；
In step (1), the revolving speed of the centrifugation is 5000~10000rpm；
In step (1), the centrifugation time is 5~15min.
5. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 2, feature exist
In: SPE column specification described in step (2) is 6mL or 150mg；
In step (2), the methanol, acetonitrile water, ultrapure 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 aqueous solution；
In step (2), the filter method is by solution through organic filtering with microporous membrane.
6. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 5, feature exist
In: in step (2), the aperture of organic miillpore filter is 0.22 μm.
7. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 1, feature exist
In: the chromatographic column of the high performance liquid chromatography detection is HPLC CSH C18 column；The specification of the chromatographic column be 2.1 ×
100mm, 1.7 μm；
The flow velocity of the sample to be tested of the high performance liquid chromatography detection is 0.3mL/min；
The 5 μ L of sampling volume of the high performance liquid chromatography detection；The column temperature of the high performance liquid chromatography detection is 25
℃。
8. a kind of method for detecting 4 kinds of barbiturates downerns of fresh milk according to claim 1, feature exist
In:
During the Mass Spectrometer Method, target detection compound parent ion, daughter ion, impact energy, retention time are as follows:
Wherein, a represents quota ion.
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| CN112083115B (en) * | 2020-09-25 | 2023-06-13 | 上海市农产品质量安全中心 | Kit for detecting residual quantity of 7 barbiturates in raw milk |
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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 (4)
| Title |
|---|
| Characteristics of interaction between barbiturate derivatives and various sorbents on liquid chromatography and determination of phenobarbital in japanese human breast milk;Shimoyama, R; Ohkubo, T; Sugawara, K;《JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES》;20070206;第23卷(第4期);587-599 |
| SPE-HPLC法同时测定5种抗癫痫药的血药浓度;刘义钊;《中南药学》;20081231;第6卷(第6期);686-690 |
| SPE-HPLC法测定血清中苯巴比妥、苯妥英钠和卡马西平浓度;沈晓洁;《江苏药学与临床研究》;20050131;第13卷(第1期);23-24 |
| 固相萃取-高效液相色谱法测定3种抗癫痫药物血药浓度;孙增先;《中国医院药学杂志》;20040531;第24卷(第5期);275-277 |
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