CN105588898A - Pretreatment method of milk sample for sulfonamides residue detection with liquid chromatography and detection method - Google Patents
Pretreatment method of milk sample for sulfonamides residue detection with liquid chromatography and detection method Download PDFInfo
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Abstract
The invention provides a pretreatment method of a milk sample for sulfonamides residue detection with liquid chromatography. The method comprises the following steps: 1-5g of the milk sample to be detected is taken and added into 10-30mL of acetonitrile, mixing is carried out by a vortex mixer for 1-5 minutes, centrifugation is carried out at 8000-10000 r/min for 3-5 minutes, and a supernatant is taken; after centrifugation, acetonitrile is added into residues for repeating extraction for one time; supernatants obtained in the two steps are combined; rotary evaporation is carried out for the combined supernatants at 40-50 DEG C till supernatants are dry; a rotary evaporation bottle is cleaned with 2-5mL of a 0.1-0.2mol/L aqueous hydrochloric acid solution for two times, and is cleaned with 3-5ml of hexane for two times; cleaning fluid is combined and mixed by the vortex mixer for 30-120 seconds, and centrifugation is carried out; after centrifugation, a hexane layer is thrown away, and a subnatant is taken; the subnatant is purified with a MCX solid phase extraction column, and clean fluid is obtained; the clean fluid is dried with nitrogen at 40-50 DEG C, and is dissolved with 1.0-5mL of a 0.1% formic acid-acetonitrile solution.
Description
Technical field
The present invention relates to a kind of sample-pretreating method and detection method that detects milk drug residue, be particularly useful for the detection of sulfa drugs in milk.
Background technology
At present, in milk, sulfa drug residue detection method is taking the method for GB/T22966-2008 " the mensuration Liquid Chromatography-Tandem Mass Spectrometry of 16 kinds of residual quantity of sulfonamides in milk and milk powder " as main, because its detection needs mass spectrum, expensive, and the level to lab technician is had relatively high expectations, cannot be in general, in small enterprise, promote; The method is used dangerous higher medicine-perchloric acid, because perchloric acid can be combustion-supporting, tool severe corrosive, strong and stimulating, can cause human body and burn, and can bring potential safety hazard to laboratory.
In GB29694-2013 " mensuration-high performance liquid chromatography of 13 kinds of sulphonamides multi-relicts in national food safety standard animal food ", the method for inspection is applicable to the detection of muscle and liver organization, due to the difference of milk and meat chemical composition, while making to be applied in this way milk detection, the rate of recovery cannot meet testing requirement.
Summary of the invention
The object of this invention is to provide the sample-pretreating method that a kind of liquid chromatography detects sulfa drug residue in milk, safe, can more fully extract the sulfa drugs in milk sample.
Another object of the present invention is to provide the method for sulfa drug residue in a kind of liquid chromatography detection milk, and cost is low, safe, and the rate of recovery and the precision of detection are better.
The invention provides the sample-pretreating method that a kind of liquid chromatography detects sulfa drug residue in milk, comprise: get 1 to 5 gram of milk sample to be measured, add 10 to 30mL acetonitriles, vortex mixed 1 to 5 minute, again in 8000 to 10000 revs/min centrifugal 3 to 5 minutes, get supernatant; And in the residue after centrifugal, add 10 to 30 milliliters of acetonitriles, vortex mixed 1 to 5 minute, then in 8000 to 10000 revs/min centrifugal 3 to 5 minutes, get supernatant; Merge the supernatant of twice gained; Supernatant after merging is placed in to revolve and steams bottle, rotary evaporated to dryness at 40 to 50 DEG C; Clean respectively to revolve with 0.1 to 0.2mol/L aqueous hydrochloric acid solution 2 to 5mL, 0.1 to 0.2mol/L aqueous hydrochloric acid solution 2 to 5mL, n-hexane 3 to 5mL and n-hexane 3 to 5mL successively again and steam bottle; Gained cleaning fluid is incorporated in same centrifuge tube, vortex mixed 30 to 120 seconds, then in 8000 to 10000 revs/min centrifugal 3 to 5 minutes; After centrifugal, abandon n-hexane layer, get subnatant; By MCX SPE column purification for subnatant, be purified liquid; And in 40 to 50 DEG C of nitrogen of scavenging solution are dried up, then dissolve with 0.1% formic acid acetonitrile solution 1.0 to 5mL; Wherein the compound method of 0.1% formic acid acetonitrile solution is: get 0.1% aqueous formic acid 830mL, dissolve and be diluted to 1000mL with acetonitrile.
In a kind of exemplary embodiment of sample-pretreating method, by the step of MCX SPE column purification be: first MCX solid-phase extraction column is activated with methyl alcohol 2 to 5mL and 0.1mol/L aqueous hydrochloric acid solution 2 to 5mL successively; By the MCX solid-phase extraction column after subnatant overactivation, coutroi velocity 1 is to 2mL/ minute; Use successively 0.1 to 0.2mol/L aqueous hydrochloric acid solution 1 to 2mL and 50% methanol acetonitrile solution 2 to 4mL drip washing MCX solid-phase extraction columns; The compound method of 50% methanol acetonitrile solution is: get methyl alcohol 50mL, dissolve and be diluted to 100mL with acetonitrile; And finally use 4mL eluent wash-out, the compound method of eluent is: get ammoniacal liquor 5mL, dissolve and be diluted to 100mL with methyl alcohol.
In a kind of exemplary embodiment of sample-pretreating method, sulfa drugs comprises: sulfacetamide, sulfapryidine, sulfamoxole, sulfamethyldiazine, sulfamethazine, sulfamethoxypyridazine, daimeton, cistosulfa, NU-445, Sulfamethoxazole, sulfabenzamide and sulfadimethoxine.
The present invention also provides a kind of liquid chromatography to detect the method for sulfa drug residue in milk, it is characterized in that, first according to above-mentioned sample-pretreating method, milk sample to be measured is carried out to pre-treatment, then detects by liquid chromatography.
Detect in a kind of exemplary embodiment of the method for sulfa drug residue in milk in liquid chromatography, the chromatographic condition of liquid chromatogram is: column oven: 30 to 45 DEG C; UV-detector detects wavelength: 270nm; Sample size: 20 to 50 μ L; Chromatographic column: C18 or C8, specification is 250 × 4.6mm, 5 μ m; Mobile phase: 0.1% aqueous formic acid and acetonitrile; Flow velocity: 1.0 to 1.5mL/ minute; Gradient:
| Time, min | 0.1% aqueous formic acid, % | Acetonitrile, % |
| 0.00 | 78 to 88 | 12 to 22 |
| 5.00 | 78 to 88 | 12 to 22 |
| 10.00 | 75 to 85 | 15 to 25 |
| 22.30 | 55 to 65 | 35 to 45 |
| 22.40 | 5 to 15 | 85 to 95 |
| 30.00 | 5 to 15 | 85 to 95 |
| 31.00 | 78 to 88 | 12 to 22 |
| 48.00 | 78 to 88 | 12 to 22 |
A kind of liquid chromatography provided by the invention detects the sample-pretreating method of sulfa drug residue in milk, and in operating process, without using dangerous higher perchloric acid, security is higher, and can more fully extract the sulfa drugs in milk sample. The liquid chromatography that uses this sample-pretreating method to carry out sulfa drug residue in milk detects, and safe, the rate of recovery and the precision of detection are better. Because detection method of the present invention is without using mass detector, also reduce testing cost.
Detailed description of the invention
For technical characterictic, object and the effect of invention are had more clearly and understood, the existing the specific embodiment of the present invention that illustrates with the following Examples.
Although following examples have adopted the chromatographic system with UV-detector, object is to be convenient to illustrate technical scheme of the present invention, the effect of being convenient to relatively detect, and is not intended to limit the present invention. Those skilled in the art can replace UV-detector with the detector of mass detector or other applicable sulfonamides, also can implement detection method of the present invention.
Embodiment 1.
One, reagent:
0.1% formic acid solution: get formic acid 1mL, water dissolves and is diluted to 1000mL;
0.1% formic acid acetonitrile solution: get 0.1% aqueous formic acid 830mL, dissolve and be diluted to 1000mL with acetonitrile;
Eluent: get ammoniacal liquor 5mL, dissolve and be diluted to 100mL with methyl alcohol;
50% methanol acetonitrile solution: get methyl alcohol 50mL, dissolve and be diluted to 100mL with acetonitrile.
Two, sample-pretreating method:
1, get 1 gram of milk sample to be measured, add 30mL acetonitrile, vortex mixed 5 minutes, then in 10000 revs/min centrifugal 5 minutes, get supernatant;
2, in the residue after centrifugal, add 30 milliliters of acetonitriles, vortex mixed 5 minutes, then in 10000 revs/min centrifugal 5 minutes, get supernatant; Merge the supernatant of twice gained;
3, the supernatant after merging is placed in and revolves steaming bottle, rotary evaporated to dryness at 40 DEG C; Clean respectively to revolve with 0.2mol/L aqueous hydrochloric acid solution 2mL, 0.2mol/L aqueous hydrochloric acid solution 2mL, n-hexane 3mL and n-hexane 3mL successively again and steam bottle; Gained cleaning fluid is incorporated in same centrifuge tube, vortex mixed 30 seconds, more centrifugal 5 minutes in 8000 revs/min; After centrifugal, abandon n-hexane layer, get subnatant;
4, by MCX SPE column purification for subnatant, be purified liquid, concrete steps are as follows:
4.1 first activate MCX solid-phase extraction column successively with methyl alcohol 5mL and 0.1mol/L aqueous hydrochloric acid solution 2mL;
4.2 by the MCX solid-phase extraction column after subnatant overactivation, coutroi velocity 1mL/ minute;
4.3 use 0.2mol/L aqueous hydrochloric acid solution 1mL and 50% methanol acetonitrile solution 2mL drip washing MCX solid-phase extraction column successively;
4.4 is last with 4mL eluent wash-out;
5, scavenging solution is dried up in 40 DEG C of nitrogen, then dissolve with 0.1% formic acid acetonitrile solution 1mL, membrane filtration, obtains liquid to be measured, for high-performance liquid chromatogram determination.
Three, high performance liquid chromatography (HPLC) detects.
Use Dai An company high performance liquid chromatograph, join UV-detector. Setting chromatographic condition is as follows:
Column oven: 30 DEG C;
UV-detector detects wavelength: 270nm;
Sample size: 20 μ L;
Chromatographic column: C8, specification is 250 × 4.6mm, 5 μ m;
Mobile phase: 0.1% aqueous formic acid and acetonitrile;
Flow velocity: 1mL/ minute;
Gradient:
| Time (min) | 0.1% aqueous formic acid (%) | Acetonitrile (%) |
| 0.00 | 83 | 17 |
| 5.00 | 83 | 17 |
| 10.00 | 80 | 20 |
| 22.30 | 60 | 40 |
| 22.40 | 10 | 90 |
| 30.00 | 10 | 90 |
| 31.00 | 83 | 17 |
| 48.00 | 83 | 17 |
Detect target substance: sulfacetamide, sulfapryidine, sulfamoxole, sulfamethyldiazine, sulfamethazine, sulfamethoxypyridazine, daimeton, cistosulfa, NU-445, Sulfamethoxazole, sulfabenzamide and sulfadimethoxine.
Concentration of standard solution gradient: the concentration of 12 kinds of sulfamido standard items is 100 μ g/kg, 200 μ g/kg, 300 μ g/kg, 400 μ g/kg, 500 μ g/kg, retarder thinner is 0.1% formic acid acetonitrile solution.
Utilize HPLC to detect 12 kinds of sulfa drugs standard liquids, work out the calibration curve of corresponding relation between sulfanilamide (SN) concentration and peak area, the R of 12 kinds of sulfa drugs calibration curves2All be greater than 0.999. According to calibration curve and the testing result of liquid to be measured is calculated to the content of 12 kinds of sulfa drugs in milk sample to be measured.
Embodiment 2.
One, reagent:
0.1% formic acid solution: get formic acid 1mL, water dissolves and is diluted to 1000mL;
0.1% formic acid acetonitrile solution: get 0.1% aqueous formic acid 830mL, dissolve and be diluted to 1000mL with acetonitrile;
Eluent: get ammoniacal liquor 5mL, dissolve and be diluted to 100mL with methyl alcohol;
50% methanol acetonitrile solution: get methyl alcohol 50mL, dissolve and be diluted to 100mL with acetonitrile.
Two, sample-pretreating method:
1, get 5 grams of milk samples to be measured, add 10mL acetonitrile, vortex mixed 1 minute, then in 8000 revs/min centrifugal 3 minutes, get supernatant;
2, in the residue after centrifugal, add 10 milliliters of acetonitriles, vortex mixed 1 minute, then in 8000 revs/min centrifugal 3 minutes, get supernatant; Merge the supernatant of twice gained;
3, the supernatant after merging is placed in and revolves steaming bottle, rotary evaporated to dryness at 50 DEG C; Clean respectively to revolve with 0.1mol/L aqueous hydrochloric acid solution 5mL, 0.1mol/L aqueous hydrochloric acid solution 5mL, n-hexane 5mL and n-hexane 5mL successively again and steam bottle; Gained cleaning fluid is incorporated in same centrifuge tube, vortex mixed 120 seconds, more centrifugal 3 minutes in 10000 revs/min; After centrifugal, abandon n-hexane layer, get subnatant;
4, by MCX SPE column purification for subnatant, be purified liquid, concrete steps are as follows:
4.1 first activate MCX solid-phase extraction column successively with methyl alcohol 2mL and 0.1mol/L aqueous hydrochloric acid solution 5mL;
4.2 by the MCX solid-phase extraction column after subnatant overactivation, coutroi velocity 2mL/ minute;
4.3 use 0.1mol/L aqueous hydrochloric acid solution 2mL and 50% methanol acetonitrile solution 4mL drip washing MCX solid-phase extraction column successively;
4.4 is last with 4mL eluent wash-out;
5, scavenging solution is dried up in 50 DEG C of nitrogen, then dissolve with 0.1% formic acid acetonitrile solution 5mL, membrane filtration, obtains liquid to be measured, for high-performance liquid chromatogram determination.
Three, high performance liquid chromatography (HPLC) detects.
Use Dai An company high performance liquid chromatograph, join UV-detector. Setting chromatographic condition is as follows:
Column oven: 45 DEG C;
UV-detector detects wavelength: 270nm;
Sample size: 50 μ L;
Chromatographic column: C18, specification is 250 × 4.6mm, 5 μ m;
Mobile phase: 0.1% aqueous formic acid and acetonitrile;
Flow velocity: 1.5mL/ minute;
Gradient:
| Time (min) | 0.1% aqueous formic acid (%) | Acetonitrile (%) |
| 0.00 | 83 | 17 |
| 5.00 | 83 | 17 |
| 10.00 | 80 | 20 |
| 22.30 | 60 | 40 |
| 22.40 | 10 | 90 |
| 30.00 | 10 | 90 |
| 31.00 | 83 | 17 |
| 48.00 | 83 | 17 |
Detect target substance: sulfacetamide, sulfapryidine, sulfamoxole, sulfamethyldiazine, sulfamethazine, sulfamethoxypyridazine, daimeton, cistosulfa, NU-445, Sulfamethoxazole, sulfabenzamide and sulfadimethoxine.
Concentration of standard solution gradient: the concentration of 12 kinds of sulfamido standard items is 100 μ g/kg, 200 μ g/kg, 300 μ g/kg, 400 μ g/kg, 500 μ g/kg, retarder thinner is 0.1% formic acid acetonitrile solution.
Utilize HPLC to detect 12 kinds of sulfa drugs standard liquids, work out the calibration curve of corresponding relation between sulfanilamide (SN) concentration and peak area, the R of 12 kinds of sulfa drugs calibration curves2All be greater than 0.999. According to calibration curve and the testing result of liquid to be measured is calculated to the content of 12 kinds of sulfa drugs in milk sample to be measured.
Embodiment 3.
One, reagent:
0.1% formic acid solution: get formic acid 1mL, water dissolves and is diluted to 1000mL;
0.1% formic acid acetonitrile solution: get 0.1% aqueous formic acid 830mL, dissolve and be diluted to 1000mL with acetonitrile;
Eluent: get ammoniacal liquor 5mL, dissolve and be diluted to 100mL with methyl alcohol;
50% methanol acetonitrile solution: get methyl alcohol 50mL, dissolve and be diluted to 100mL with acetonitrile.
Two, sample-pretreating method:
1, get 2 grams of milk samples to be measured, add 10mL acetonitrile, vortex mixed 2 minutes, then in 8000 revs/min centrifugal 5 minutes, get supernatant;
2, in the residue after centrifugal, add 10 milliliters of acetonitriles, vortex mixed 2 minutes, then in 8000 revs/min centrifugal 5 minutes, get supernatant; Merge the supernatant of twice gained;
3, the supernatant after merging is placed in and revolves steaming bottle, rotary evaporated to dryness at 50 DEG C; Clean respectively to revolve with 0.1mol/L aqueous hydrochloric acid solution 2mL, 0.1mol/L aqueous hydrochloric acid solution 2mL, n-hexane 5mL and n-hexane 5mL successively again and steam bottle; Gained cleaning fluid is incorporated in same centrifuge tube, vortex mixed 60 seconds, more centrifugal 5 minutes in 8000 revs/min; After centrifugal, abandon n-hexane layer, get subnatant;
4, by MCX SPE column purification for subnatant, be purified liquid, concrete steps are as follows:
4.1 first activate MCX solid-phase extraction column successively with methyl alcohol 3mL and 0.1mol/L aqueous hydrochloric acid solution 4mL;
4.2 by the MCX solid-phase extraction column after subnatant overactivation, coutroi velocity 1mL/ minute;
4.3 use 0.15mol/L aqueous hydrochloric acid solution 1.5mL and 50% methanol acetonitrile solution 3mL drip washing MCX solid-phase extraction column successively;
4.4 is last with 4mL eluent wash-out;
5, scavenging solution is dried up in 50 DEG C of nitrogen, then dissolve with 0.1% formic acid acetonitrile solution 2mL, membrane filtration, obtains liquid to be measured, for high-performance liquid chromatogram determination.
Three, high performance liquid chromatography (HPLC) detects.
Use Dai An company high performance liquid chromatograph, join UV-detector. Setting chromatographic condition is as follows:
Column oven: 40 DEG C;
UV-detector detects wavelength: 270nm;
Sample size: 30 μ L;
Chromatographic column: C8, specification is 250 × 4.6mm, 5 μ m;
Mobile phase: 0.1% aqueous formic acid and acetonitrile;
Flow velocity: 1.2mL/ minute;
Gradient:
| Time (min) | 0.1% aqueous formic acid (%) | Acetonitrile (%) |
| 0.00 | 88 | 12 |
| 5.00 | 88 | 12 |
| 10.00 | 85 | 15 |
| 22.30 | 65 | 35 |
| 22.40 | 15 | 85 |
| 30.00 | 15 | 85 |
| 31.00 | 88 | 12 |
| 48.00 | 88 | 12 |
Detect target substance: sulfacetamide, sulfapryidine, sulfamoxole, sulfamethyldiazine, sulfamethazine, sulfamethoxypyridazine, daimeton, cistosulfa, NU-445, Sulfamethoxazole, sulfabenzamide and sulfadimethoxine.
Concentration of standard solution gradient: the concentration of 12 kinds of sulfamido standard items is 100 μ g/kg, 200 μ g/kg, 300 μ g/kg, 400 μ g/kg, 500 μ g/kg, retarder thinner is 0.1% formic acid acetonitrile solution.
Utilize HPLC to detect 12 kinds of sulfa drugs standard liquids, work out the calibration curve of corresponding relation between sulfanilamide (SN) concentration and peak area, the R of 12 kinds of sulfa drugs calibration curves2All be greater than 0.999. According to calibration curve and the testing result of liquid to be measured is calculated to the content of 12 kinds of sulfa drugs in milk sample to be measured.
Embodiment 4.
One, reagent:
0.1% formic acid solution: get formic acid 1mL, water dissolves and is diluted to 1000mL;
0.1% formic acid acetonitrile solution: get 0.1% aqueous formic acid 830mL, dissolve and be diluted to 1000mL with acetonitrile;
Eluent: get ammoniacal liquor 5mL, dissolve and be diluted to 100mL with methyl alcohol;
50% methanol acetonitrile solution: get methyl alcohol 50mL, dissolve and be diluted to 100mL with acetonitrile.
Two, sample-pretreating method:
1, get 2.5 grams of milk samples to be measured, add 15mL acetonitrile, vortex mixed 2 minutes, then in 10000 revs/min centrifugal 5 minutes, get supernatant;
2, in the residue after centrifugal, add 15 milliliters of acetonitriles, vortex mixed 2 minutes, then in 10000 revs/min centrifugal 5 minutes, get supernatant; Merge the supernatant of twice gained;
3, the supernatant after merging is placed in and revolves steaming bottle, rotary evaporated to dryness at 45 DEG C; Clean respectively to revolve with 0.1mol/L aqueous hydrochloric acid solution 3mL, 0.1mol/L aqueous hydrochloric acid solution 3mL, n-hexane 5mL and n-hexane 5mL successively again and steam bottle; Gained cleaning fluid is incorporated in same centrifuge tube, vortex mixed 60 seconds, more centrifugal 5 minutes in 9000 revs/min; After centrifugal, abandon n-hexane layer, get subnatant;
4, by MCX SPE column purification for subnatant, be purified liquid, concrete steps are as follows:
4.1 first activate MCX solid-phase extraction column successively with methyl alcohol 4mL and 0.1mol/L aqueous hydrochloric acid solution 4mL;
4.2 by the MCX solid-phase extraction column after subnatant overactivation, coutroi velocity 2mL/ minute;
4.3 use 0.15mol/L aqueous hydrochloric acid solution 1.5mL and 50% methanol acetonitrile solution 2mL drip washing MCX solid-phase extraction column successively;
4.4 is last with 4mL eluent wash-out;
5, scavenging solution is dried up in 50 DEG C of nitrogen, then dissolve with 0.1% formic acid acetonitrile solution 5mL, membrane filtration, obtains liquid to be measured, for high-performance liquid chromatogram determination.
Three, high performance liquid chromatography (HPLC) detects.
Use Dai An company high performance liquid chromatograph, join UV-detector. Setting chromatographic condition is as follows:
Column oven: 35 DEG C;
UV-detector detects wavelength: 270nm;
Sample size: 50 μ L;
Chromatographic column: C18, specification is 250 × 4.6mm, 5 μ m;
Mobile phase: 0.1% aqueous formic acid and acetonitrile;
Flow velocity: 1mL/ minute;
Gradient:
| Time (min) | 0.1% aqueous formic acid (%) | Acetonitrile (%) |
| 0.00 | 78 | 22 |
| 5.00 | 78 | 22 |
| 10.00 | 75 | 25 |
| 22.30 | 55 | 45 |
| 22.40 | 5 | 95 |
| 30.00 | 5 | 95 |
| 31.00 | 78 | 22 |
| 48.00 | 78 | 22 |
Detect target substance: sulfacetamide, sulfapryidine, sulfamoxole, sulfamethyldiazine, sulfamethazine, sulfamethoxypyridazine, daimeton, cistosulfa, NU-445, Sulfamethoxazole, sulfabenzamide and sulfadimethoxine.
Concentration of standard solution gradient: the concentration of 12 kinds of sulfamido standard items is 100 μ g/kg, 200 μ g/kg, 300 μ g/kg, 400 μ g/kg, 500 μ g/kg, retarder thinner is 0.1% formic acid acetonitrile solution.
Utilize HPLC to detect 12 kinds of sulfa drugs standard liquids, work out the calibration curve of corresponding relation between sulfanilamide (SN) concentration and peak area, the R of 12 kinds of sulfa drugs calibration curves2All be greater than 0.999. According to calibration curve and the testing result of liquid to be measured is calculated to the content of 12 kinds of sulfa drugs in milk sample to be measured.
Embodiment 5.
One, reagent:
0.1% formic acid solution: get formic acid 1mL, water dissolves and is diluted to 1000mL;
0.1% formic acid acetonitrile solution: get 0.1% aqueous formic acid 830mL, dissolve and be diluted to 1000mL with acetonitrile;
Eluent: get ammoniacal liquor 5mL, dissolve and be diluted to 100mL with methyl alcohol;
50% methanol acetonitrile solution: get methyl alcohol 50mL, dissolve and be diluted to 100mL with acetonitrile.
Two, sample-pretreating method:
1, get 1 gram of milk sample to be measured, add 10mL acetonitrile, vortex mixed 2 minutes, then in 9000 revs/min centrifugal 4 minutes, get supernatant;
2, in the residue after centrifugal, add 10 milliliters of acetonitriles, vortex mixed 2 minutes, then in 9000 revs/min centrifugal 4 minutes, get supernatant; Merge the supernatant of twice gained;
3, the supernatant after merging is placed in and revolves steaming bottle, rotary evaporated to dryness at 45 DEG C; Clean respectively to revolve with 0.15mol/L aqueous hydrochloric acid solution 2mL, 0.15mol/L aqueous hydrochloric acid solution 2mL, n-hexane 4mL and n-hexane 4mL successively again and steam bottle; Gained cleaning fluid is incorporated in same centrifuge tube, vortex mixed 60 seconds, more centrifugal 4 minutes in 10000 revs/min; After centrifugal, abandon n-hexane layer, get subnatant;
4, by MCX SPE column purification for subnatant, be purified liquid, concrete steps are as follows:
4.1 first activate MCX solid-phase extraction column successively with methyl alcohol 5mL and 0.1mol/L aqueous hydrochloric acid solution 3mL;
4.2 by the MCX solid-phase extraction column after subnatant overactivation, coutroi velocity 1.5mL/ minute;
4.3 use 0.15mol/L aqueous hydrochloric acid solution 1.5mL and 50% methanol acetonitrile solution 2mL drip washing MCX solid-phase extraction column successively;
4.4 is last with 4mL eluent wash-out;
5, scavenging solution is dried up in 45 DEG C of nitrogen, then dissolve with 0.1% formic acid acetonitrile solution 4mL, membrane filtration, obtains liquid to be measured, for high-performance liquid chromatogram determination.
Three, high performance liquid chromatography (HPLC) detects.
Use Dai An company high performance liquid chromatograph, join UV-detector. Setting chromatographic condition is as follows:
Column oven: 45 DEG C;
UV-detector detects wavelength: 270nm;
Sample size: 20 μ L;
Chromatographic column: C18, specification is 250 × 4.6mm, 5 μ m;
Mobile phase: 0.1% aqueous formic acid and acetonitrile;
Flow velocity: 1.5mL/ minute;
Gradient:
| Time (min) | 0.1% aqueous formic acid (%) | Acetonitrile (%) |
| 0.00 | 83 | 17 |
| 5.00 | 83 | 17 |
| 10.00 | 80 | 20 |
| 22.30 | 60 | 40 |
| 22.40 | 10 | 90 |
| 30.00 | 10 | 90 |
| 31.00 | 83 | 17 |
| 48.00 | 83 | 17 |
Detect target substance: sulfacetamide, sulfapryidine, sulfamoxole, sulfamethyldiazine, sulfamethazine, sulfamethoxypyridazine, daimeton, cistosulfa, NU-445, Sulfamethoxazole, sulfabenzamide and sulfadimethoxine.
Concentration of standard solution gradient: the concentration of 12 kinds of sulfamido standard items is 100 μ g/kg, 200 μ g/kg, 300 μ g/kg, 400 μ g/kg, 500 μ g/kg, retarder thinner is 0.1% formic acid acetonitrile solution.
Utilize HPLC to detect 12 kinds of sulfa drugs standard liquids, work out the calibration curve of corresponding relation between sulfanilamide (SN) concentration and peak area, the R of 12 kinds of sulfa drugs calibration curves2All be greater than 0.999. According to calibration curve and the testing result of liquid to be measured is calculated to the content of 12 kinds of sulfa drugs in milk sample to be measured.
The rate of recovery, precision and detection limit experiment.
Determination of recovery rates method: add 12 kinds of sulfa drugs standard items in milk sample, make mark-on sample. The mark-on concentration of 12 kinds of sulfa drugs is 50 μ g/kg. Mark-on sample is detected by the method for embodiment 1-5, and calculate recovery of standard addition.
Precision assay method: add 12 kinds of sulfa drugs standard items in milk sample, make mark-on sample. The mark-on concentration of 12 kinds of sulfa drugs is 50 μ g/kg. To mark-on sample duplicate detection 6 times, and calculate the relative standard deviation of testing result by the method for embodiment 1-5.
Detection limit assay method: dummy (not containing the normal milk sample of 12 kinds of sulfanilamide (SN)) is detected by the method for embodiment 1-5, and the corresponding concentration of response of calculating 3 times of noises is detection limit, considers the impact of the rate of recovery simultaneously.
A comparative example is set simultaneously, and concrete grammar is: first according to the method for " 7.1 extract " in GB29694-2013 " mensuration of 13 kinds of sulphonamides multi-relicts in animal food " and " 7.2 purify ", milk sample to be measured is carried out to pre-treatment; Then detect according to embodiment 1 " three, high performance liquid chromatography (HPLC) detect ".
Determination of recovery rates result:
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Comparative example | |
| Sulfacetamide | 105.04% | 79.54% | 104.84% | 86.20% | 102.00% | 42.03% |
| Sulfapryidine | 99.90% | 93.12% | 99.28% | 95.60% | 103.80% | 57.99% |
| Sulfamethoxypyridazine | 87.10% | 80.04% | 82.24% | 82.60% | 92.40% | 55.94% |
| Sulfabenzamide | 85.80% | 66.52% | 68.96% | 67.68% | 90.16% | 42.14% |
| Daimeton | 103.76% | 89.44% | 112.20% | 102.56% | 104.68% | 45.00% |
| Cistosulfa | 65.32% | 78.00% | 68.28% | 120.00% | 85.60% | 28.00% |
| NU-445 | 62.68% | 63.60% | 66.08% | 60.04% | 68.96% | 90.42% |
| Sulfadimethoxine | 86.52% | 79.88% | 75.84% | 71.04% | 84.00% | 17.52% |
| Sulfamethoxazole | 73.92% | 67.88% | 67.36% | 60.88% | 70.60% | 44.70% |
| Sulfamoxole | 78.40% | 71.80% | 71.52% | 66.08% | 75.92% | 64.45% |
| Sulfamethyldiazine | 69.00% | 64.20% | 65.96% | 77.16% | 67.12% | 21.64% |
| Sulfamethazine | 81.32% | 67.86% | 76.32% | 66.08% | 77.80% | 113.65% |
As can be seen here, the rate of recovery of embodiment 1-5 all can meet the requirement in GB/T27404, and between 60%-120%, the comparative example rate of recovery cannot meet the demands, and the method for embodiment 1-5 is better.
Precision measurement result:
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Comparative example | |
| Sulfacetamide | 3.10% | 12.42% | 12.10% | 12.66% | 7.35% | 15.64% |
| Sulfapryidine | 6.57% | 7.78% | 4.42% | 5.57% | 7.34% | 20.45% |
| Sulfamethoxypyridazine | 14.55% | 5.19% | 4.82% | 5.19% | 10.56% | 3.64% |
| Sulfabenzamide | 5.81% | 13.94% | 3.47% | 8.83% | 5.55% | 4.72% |
| Daimeton | 3.18% | 4.97% | 14.46% | 2.42% | 3.78% | 0.89% |
| Cistosulfa | 1.50% | 14.58% | 6.66% | 9.61% | 11.38% | 9.59% |
| NU-445 | 2.45% | 7.24% | 3.76% | 4.66% | 5.72% | 14.62% |
| Sulfadimethoxine | 4.32% | 8.32% | 8.49% | 6.53% | 4.66% | 30.42% |
| Sulfamethoxazole | 5.74% | 1.12% | 8.23% | 7.00% | 9.51% | 12.00% |
| Sulfamoxole | 3.79% | 2.36% | 6.54% | 8.30% | 4.65% | 7.32% |
| Sulfamethyldiazine | 2.70% | 2.34% | 10.78% | 6.88% | 8.23% | 5.88% |
| Sulfamethazine | 2.92% | 11.58% | 11.54% | 5.21% | 10.55% | 4.83% |
As can be seen here, the precision of embodiment 1-5 is in 15%, and in comparative example, to exceed 15%, embodiment 1-5 precision good for sulfacetamide, sulfadimethoxine and sulfapryidine precision.
Detection limit measurement result, unit is μ g/kg:
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Comparative example | |
| Sulfacetamide | 4.80 | 5.91 | 4.45 | 6.20 | 3.25 | 9.60 |
| Sulfapryidine | 6.80 | 7.48 | 7.45 | 5.65 | 7.33 | 13.60 |
| Sulfamethoxypyridazine | 7.10 | 7.62 | 9.89 | 9.97 | 8.56 | 14.20 |
| Sulfabenzamide | 8.55 | 5.75 | 6.82 | 6.97 | 5.09 | 17.10 |
| Daimeton | 8.77 | 8.71 | 5.00 | 8.08 | 9.51 | 17.54 |
| Cistosulfa | 9.46 | 6.32 | 8.60 | 9.43 | 7.08 | 18.92 |
| NU-445 | 10.00 | 6.57 | 9.07 | 6.77 | 6.65 | 20.00 |
| Sulfadimethoxine | 9.85 | 6.48 | 6.28 | 5.33 | 8.35 | 19.70 |
| Sulfamethoxazole | 7.92 | 5.95 | 7.54 | 7.35 | 6.26 | 15.84 |
| Sulfamoxole | 8.01 | 6.28 | 8.56 | 6.21 | 8.43 | 16.02 |
| Sulfamethyldiazine | 8.06 | 6.47 | 7.00 | 10.03 | 9.58 | 16.12 |
| Sulfamethazine | 7.01 | 7.36 | 8.33 | 6.99 | 10.43 | 14.02 |
As can be seen here, in embodiment 1-5, detect and be limited between 3.25-10.03. 12 kinds of sulfonamides that relate to from above-mentioned test data, the detection limit of comparative example is all higher than the detection limit of embodiment of the present invention 1-5, so embodiment 1-5 method has significant progress.
In this article, " schematically " expression " is served as example, example or explanation ", any embodiment that is described in this article " schematically " should be interpreted as a kind of preferred or have more the technical scheme of advantage.
In this article, " equating ", " identical " etc. are not the restriction in strict mathematics and/or geometry meaning, also comprise the error it will be appreciated by those skilled in the art that and production or use etc. allow. Except as otherwise noted, number range herein not only comprises the gamut in two end points, also comprises the some subranges that are contained in wherein.
Be to be understood that, although this description is described according to each embodiment, but be not that each embodiment only comprises an independently technical scheme, this narrating mode of description is only for clarity sake, those skilled in the art should make description as a whole, technical scheme in each embodiment also can, through appropriately combined, form other embodiments that it will be appreciated by those skilled in the art that.
Listed a series of detailed description is above only illustrating for feasibility embodiment of the present invention; they are not in order to limit the scope of the invention; allly do not depart from equivalent embodiment or the change that skill spirit of the present invention is done; as the combination of feature, cut apart or repeat, within all should being included in protection scope of the present invention.
Claims (5)
1. liquid chromatography detects a sample-pretreating method for sulfa drug residue in milk, it is characterized in that, comprising:
Get 1 to 5 gram of milk sample to be measured, add 10 to 30mL acetonitriles, vortex mixed 1 to 5 minute, then in 8000 to 10000 revs/min centrifugal 3 to 5 minutes, get supernatant; And in the residue after centrifugal, add 10 to 30 milliliters of acetonitriles, vortex mixed 1 to 5 minute, then in 8000 to 10000 revs/min centrifugal 3 to 5 minutes, get supernatant; Merge the supernatant of twice gained;
Described supernatant after merging is placed in to revolve and steams bottle, rotary evaporated to dryness at 40 to 50 DEG C; Clean respectively to revolve with 0.1 to 0.2mol/L aqueous hydrochloric acid solution 2 to 5mL, 0.1 to 0.2mol/L aqueous hydrochloric acid solution 2 to 5mL, n-hexane 3 to 5mL and n-hexane 3 to 5mL successively again and steam bottle; Gained cleaning fluid is incorporated in same centrifuge tube, vortex mixed 30 to 120 seconds, then in 8000 to 10000 revs/min centrifugal 3 to 5 minutes; After centrifugal, abandon n-hexane layer, get subnatant;
By MCX SPE column purification for described subnatant, be purified liquid; And
In 40 to 50 DEG C of nitrogen of described scavenging solution are dried up, then dissolve with 0.1% formic acid acetonitrile solution 1.0 to 5mL; The compound method of wherein said 0.1% formic acid acetonitrile solution is: get 0.1% aqueous formic acid 830mL, dissolve and be diluted to 1000mL with acetonitrile.
2. sample-pretreating method as claimed in claim 1, wherein, the described step with MCX SPE column purification is:
First MCX solid-phase extraction column is activated with methyl alcohol 2 to 5mL and 0.1mol/L aqueous hydrochloric acid solution 2 to 5mL successively;
By the MCX solid-phase extraction column after described subnatant overactivation, coutroi velocity 1 is to 2mL/ minute;
Use successively 0.1 to 0.2mol/L aqueous hydrochloric acid solution 1 to 2mL and 50% methanol acetonitrile solution 2 to 4mL drip washing MCX solid-phase extraction columns; The compound method of described 50% methanol acetonitrile solution is: get methyl alcohol 50mL, dissolve and be diluted to 100mL with acetonitrile; And
Finally use 4mL eluent wash-out, the compound method of described eluent is: get ammoniacal liquor 5mL, dissolve and be diluted to 100mL with methyl alcohol.
3. sample-pretreating method as claimed in claim 1; wherein, described sulfa drugs comprises: sulfacetamide, sulfapryidine, sulfamoxole, sulfamethyldiazine, sulfamethazine, sulfamethoxypyridazine, daimeton, cistosulfa, NU-445, Sulfamethoxazole, sulfabenzamide and sulfadimethoxine.
4. liquid chromatography detects a method for sulfa drug residue in milk, it is characterized in that, first sample-pretreating method according to claim 1 carries out pre-treatment to milk sample to be measured, then detects by liquid chromatography.
5. liquid chromatography as claimed in claim 4 detects the method for sulfa drug residue in milk, and wherein, the chromatographic condition of liquid chromatogram is:
Column oven: 30 to 45 DEG C;
UV-detector detects wavelength: 270nm;
Sample size: 20 to 50 μ L;
Chromatographic column: C18 or C8, specification is 250 × 4.6mm, 5 μ m;
Mobile phase: 0.1% aqueous formic acid and acetonitrile;
Flow velocity: 1.0 to 1.5mL/ minute;
Gradient:
。
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