CN101241114A - Animal source food sulfonamide residual medium solid phase dispersion-highly effective liquid phase chromatography determination method - Google Patents
Animal source food sulfonamide residual medium solid phase dispersion-highly effective liquid phase chromatography determination method Download PDFInfo
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Abstract
The present invention relates to 'method for examining solid-phase dispersion -high performance liquid chromatogram of residues substrate of sulfonamides in animal derived food' which belongs to the field of food safety-veterinary drug residues. The method comprises of chromatogram examining steps of substrate solid-phase dispersion and high performance liquid, wherein, the mobile phase of sodium dihydrogen phosphate-acetonitrile is 50mmol/L in high performance liquid chromatogram examining method, the volume ratio of sodium dihydrogen phosphate and acetonitrile is 70:30, because the sodium dihydrogen phosphate solution is buffer solution which has a certain PH buffer capability and makes the PH value stable, the stability of pH value of HPLC mobile phase is ensured, and repeatability and reproducibility of analysis result are ensured also, at the same time, the classical process of extraction, separation, concentration and purification are simplified, the operation is simple, the milk combination phenomenon is eliminated. The method of present invention has the merits of ideal precision and sensitivity, and environment protecting by using the less toxicity organic solvent eluting analyte employed.
Description
Technical field
The invention belongs to a kind of method that sulfa drug residue detects in food security-residue of veterinary drug field, particularly animal derived food.
Background technology
(sulfonamides SAs) is meant the general name of the class medicine with P-aminobenzene-sulfonamide structure to sulfa drugs, is the synthetic antibacterials of a quasi-tradition.It is similar to the chemical constitution of p-aminobenzoic acid (PABA), and the two competes dihydrofolate synthetase, suppresses the synthetic of bacterium dihydrofoilic acid, influences bacterial nucleic acid and generates, and then stoped the growth and breeding of bacterium.The sulfonamide antimicrobial spectrum is wider, and most of gram positive bacterias and some gram-negative bacteria are had inhibiting effect.In the animal feeding process, sulfa drugs is usually used in treating domestic animal hysteritis, respiratory tract and infection of digestive canal, atrophic rhinitis, cholera fowl, typhoid fever, paratyphoid and coccidium infection.Sulfa drugs action time and the metabolism time in animal body is longer, often cause sulfa drug residue is arranged in the edible animal tissue, and the long-term edible food that sulfa drug residue is arranged, just may cause sulfa drugs accumulating gradually in vivo, to human beings'health harmful [Li Junsuo, Qiu Yueming, Wang Chao, residue of veterinary drug is analyzed [M], 2002,228-251; Duan Zhenjuan, Zhang Hongyan, Wang Shuo, sulfa drug residue advances in analysis [J] in the animal food, food research and development, 2007,28 (6): 149-153], mainly show as allergic reaction and allergic reaction, bacterial drug resistance, teratogenesis, mutagenesis, carcinogenesis and hormone-like effect.And residual in the food, feed very easily causes drug-resistance of bacteria and environmental pollution.After antibacterials in the animal excrements and endurance strain were released into environment, with polluted source and soil, bacterium can keep drug resistance for a long time in mud.Simultaneously, sulfa drug residue also is the technical problem underlying of present animal product trade, has had a strong impact on the normal development of animal husbandry.
For security and the validity that ensures animal-use drug, and the security after the human ingestion animal food, many national regulations the maximum residue limit(MRL) of sulfa drugs (maximum residue limits, MRL), Canada, European Union, the U.S. stipulate that the maximum residue limit of sulfa drugs total amount in the animal tissue is 100 μ g/kg, and Japan is 20 μ g/kg.According to " veterinary drug and other chemical substances method for detecting residue in edible animal tissue " that China Ministry of Agriculture formulated in 1998, the permission residue criterion of sulfa drugs in edibility animal tissue is 100 μ g/kg～300 μ g/kg.In decades, comprehensively research has been carried out deeply in residual analysis to SAs both at home and abroad, the method for many detection sulfa drugss occurred.High performance liquid chromatography (HPLC), vapor-phase chromatography (GC), thin-layered chromatography (TLC), high performance capillary electrophoresis (HPCE), enzyme-linked immunosorbent assay (ELISA), microbial method, biology sensor immunoassay (BIA), spectrophotometric method (UV), fluorescence method (FL), gas chromatography-mass spectrography (GC/MS), liquid chromatography-mass spectrography (HPLC/MS) and supercritical fluid chromatography methods such as (SFC) are arranged at present.Study maximum animal foods muscle, liver, kidney, skin and fat are arranged, modal analyte is sulfamethazine (SMZ), sulphadiazine (SDZ), sulfamethyldiazine (SMR) and sulphathiazole (STZ) [Duan Zhenjuan, Zhang Hongyan, Wang Shuo, sulfa drug residue advances in analysis [J] in the animal food, food research and development, 2007,28 (6): 149-153].In many detection methods of sulfa drugs, that the HPLC method has is flexible, general, selectivity is strong, highly sensitive, advantage such as detectability is low.The HPLC method is mainly used C
18And C
8Reversed-phase column is also used the ion coupled columns sometimes.Moving phase commonly used be phosphoric acid-acetonitrile gradient wash-out [next is annotated for Liu Hongcheng, Li Qiwan, etc., HPLC measures diethylstilbestrol, furazolidone, sulfa drug residue [L] in the animal tissue, chemistry circular, 2006, (1): 66-69; Ten thousand spring flowers, Long Zhouxiong, Hu Haishan, etc., the RP-HPLC method is measured 15 kinds of sulfa drug residues [J] in the animal tissue simultaneously, Food Science, 2007,28 (10): 493-496; Cao Fang, Wang Qian, it is red to pass away, sulfa drug residue in the high effective liquid chromatography for measuring chicken [J], Jiangxi feed, 2005, (1): 25-26], and formic acid-acetonitrile gradient wash-out [Chen Zhengui, it is auspicious to account for the spring, Guo Ping, etc., the research of 13 kinds of sulfa drug residues [J] in the hplc simultaneous determination aquatic products, Food Science, 2007,28 (10): 448-451], methyl alcohol-phosphate buffer-triethylamine [Liu Peng rock, Jiang Ning, Wang Hongyu, sulfamido and fluoroquinolones residue of veterinary drug amount [J] in Solid-Phase Extraction-hplc simultaneous determination pork, chemistry circular, 2006, (8): 572-576], methyl alcohol-acetonitrile-water-acetate [Zhang Yan, Wu Yinliang, in Solid-Phase Extraction-high effective liquid chromatography for measuring animal flesh tissue residual [L] of sulfa drugs, chromatogram, 2005,23 (6): 636-638; Lin Hongying, Lu Guiping, Shen Zilong, the high effective liquid chromatography for measuring of sulfamethazine residual quantity [L] in the pork, Jiangsu agricultural journal, 2006,22 (3): 285-288], methyl alcohol-acetate [Song Yanhong, Wang Ran, Liu Tiezheng, five kinds of sulfa drug residues [L] in high effective liquid chromatography for measuring egg white and the yolk, Zhejiang agricultural journal, 2007,19 (1): 42-45]; Methyl alcohol-formic acid gradient elution [Wu Zongxian, Shen Chongyu, Chen Huilan, etc., the high performance liquid chromatography-tandem mass method is measured 17 kinds of sulfa drug residue amounts [L] in the casing, assay office, 2007,26 (5): 96-99] etc.The pH value of moving phase is very big to the retention time influence of sulfa drugs, because contain amino in the sulfa drugs structural formula, has alkalescent, regulate the pH in the moving phase, can suppress the disassociation of weak base, thereby cause the change of retention time, therefore in HPLC detects, must keep the stable of moving phase pH, pH changes the variation that then may cause analysis result and separating effect.[ten thousand spring flowers when for example measuring in the animal tissue 15 kinds of sulfa drug residues simultaneously with the RP-HPLC method, Long Zhouxiong, Hu Haishan, measure 15 kinds of sulfa drug residues [J] in the animal tissue simultaneously Deng the .RP-HPLC method, Food Science, 2007,28 (10): 493-496], the result shows add phosphoric acid in acetonitrile-water, can suppress the chromatographic peak hangover effectively, and help separating of sulfamoxole and sulfadimidine, but it is many to add phosphoric acid, sulfamethoxypyridazine, ayerlucil are difficult to separately.HPLC method detecting device commonly used has ultraviolet-visible detecting device (UV), fluorescence detector (FLD), photodiode array detector (DAD) and mass spectrum (MS).The detection wavelength of ultraviolet-visible is elected 270nm～280nm usually as, is 255nm sometimes.The excitation wavelength of fluoroscopic examination is 405nm～420nm, and emission wavelength is 485nm～495nm.Light diode array selects the wavelength of 267nm, 268nm or 263nm usually.MS has high specific and can prove conclusively analyte.
Before carrying out the HPLC stratographic analysis, need purify and concentrate sample usually, with avoid in the sample substrate complex component infringement chromatographic apparatus and to the interference of target analytes.Method commonly used is to extract medicine with methyl alcohol or acetonitrile, distributes through normal hexane liquid-liquid and removes fat, concentrates the back and crosses the SPE column purification; SPE post commonly used comprises Alumina B SPE, kation MCXSPE post, ENVI-18 SPE post, amino SPE post and C
18SPE post etc.
(Mat rix solid phase dispersion MSPD) is another kind of sample-pretreating method to the dispersion of matrix solid phase.MSPD is a kind of sample preparation technology that was proposed and given theoretical explanation by Barker in 1989 first.MSPD combines the solid phase dispersion technology of routine with inverse bonded filler, tissue homogenate, extraction and purification are finished in same operation, analyzes that link greatly reduces, simplified control, so have advantages such as simple and efficient, sample and solvent load are few.The research data of domestic this respect is fewer, the MSPD that sulfa drugs in the pig muscle tissue is arranged of report purifies and HPLC measures [Zhang Suxia, Li Junsuo, Qian Chuanfan, the MSPD of sulfa drugs purifies and HPLC mensuration [J] in the pig muscle tissue, journal of animal science and veterinary medicine, 1999,30 (6): 531-535], method is with 0.5g musculature and 2g C
18Filler mixes, grind 30s, make the semisolid dress and be taken in the 5mL glass syringe, loading highly is 4.2mL, wash with the 8mL normal hexane, after vacuum was drained, sulfa drugs in the heart bottle, added 0.1mL ethylene glycol with 12mL methylene chloride eluted substance, rotary evaporation is removed methylene chloride, residue changes in the glass centrifuge tube the centrifugal 10min of 2000g over to the dissolving of 0.4mL moving phase; Supernatant advances through C
18Post separates, and 0.017mol/L phosphoric acid-acetonitrile (80:20) is a moving phase, and UV detects under the wavelength 270nm and measures with reversed-phase HPLC.The average recovery rate of 7 kinds of sulfa drugss is 70.6% ± 20.3% in 0.1～0.5mg/kg adds concentration range, detects to be limited to 0.01～0.1mg/kg.Sulfa drug residue has carried out studying [Geng Zhiming in the employing matrix solid phase dispersion-high effective liquid chromatography for measuring flesh of fish such as same Geng Zhi is bright, Li Peng, Chen Ming, Deng, sulfa drug residue [J] in the matrix solid phase dispersion-high effective liquid chromatography for measuring flesh of fish, Jiangsu agricultural journal, 2006,22 (3): 310-312], promptly 0.5 g oppresses sample and 2.0g C
18Filler is put into mortar, grinds 5min gently, makes sample and C
18Filler mixes, and is loaded on the 10mL syringe, and loading highly is about 4mL, and with 8mL normal hexane washing 1 time, vacuum is drained, and uses 8mL methylene chloride wash-out again, and vacuum is drained; Eluent dries up with nitrogen in 35 ℃ of water-baths, and residue dissolves with 1mL 40% (volume ratio) methanol aqueous solution, and 0.45 μ m filter membrane is crossed in the vortex vibration, analyzes for HPLC; Chromatographic column W is ODS-3 C
1835 ℃ of column temperatures, UV detects wavelength 270nm, moving phase is 0.5% acetate-methyl alcohol, adopt gradient elution, be 0～2min 76: 24 (volume ratio), 2～20min changes into 60: 40 by 76: 24 (volume ratio), and (volume ratio), 20～30min are 60: 40 (volume ratio), and flow velocity is 1.0mL/min.In the interpolation concentration range of 0.05～0.20mg/kg, the recovery of sulfa drugs is 70.2%～93.7%, and the coefficient of variation is 2.5%～9.9%, the detectability 0.020mg/kg～0.030mg/kg of sulfa drugs.
The HPLC method is to analyze the internationally recognized comparatively general method of sulfa drug residue, and relevant research is more, but in order to realize simple, quick, safe analysis purpose, also there is following problem in said method:
When 1) HPLC analyzes, moving phase adopts acid (formic acid, acetate, phosphoric acid etc.)-organic solvent more, wherein phosphoric acid, formic acid, acetate etc. are owing to have volatility, placing concentration of a specified duration or content can change, so be difficult to the identical accurate pH value of preparation in the different time, influence different analysis time of inner analysis result's repeatability causes the variation of retention time, even analyte can not be separated from each other.
2) the MSPD wash-out is to adopt the toxic organic solvent methylene chloride, and environment and operator are had harm.
3) the pre-treatment mode of above this extraction-purification often uses poisonous organic solvent (methyl alcohol, acetonitrile) to make extraction agent; Processes such as liquid-liquid distributes, concentrates are arranged again simultaneously, because the sulfa drugs poor heat resistance, these processes all can cause damage, and the recovery is reduced; And loaded down with trivial details, tediously long operation stepss such as this traditional sample treatment comprises extraction, separation, concentrates, purification, need to consume a large amount of solvents, limited the raising of analysis efficiency.
Summary of the invention
The present invention is directed to the defective in the above-mentioned HPLC method, the matrix solid phase dispersion-high-performance liquid chromatogram determination method of sulfa drug residue in a kind of animal derived food has been proposed, this method adopts the moving phase of stable compound sodium dihydrogen phosphate preparation HPLC, because sodium dihydrogen phosphate is a buffer solution, has certain pH surge capability, make pH be difficult for changing, thereby guaranteed the stability of HPLC moving phase pH, the repeatability and the reappearance of analysis result have been guaranteed, simplified simultaneously in the classical sample treatment and extracted, separate, concentrate, purification process, simple to operate, eliminate emulsion, have desirable accuracy and sensitivity, adopt the less organic solvent elution analysis thing of toxicity can make the more environmental protection of MSPD method.
The matrix solid phase dispersion-high-performance liquid chromatogram determination method of sulfa drug residue in a kind of animal derived food, comprise that the matrix solid phase is disperseed and high performance liquid chromatography detects step, it is characterized in that: used moving phase is 50 mmol/L sodium dihydrogen phosphate-acetonitriles in the described high-performance liquid chromatogram determination method, and the volume ratio of sodium dihydrogen phosphate and acetonitrile is 70: 30.
Used chromatographic column is ODS-C in the described high-performance liquid chromatogram determination method
18Post, specification are 4.6mm * 250mm * 5 μ m.
Described matrix solid phase is disperseed to comprise grinding, dress post, is removed sample solution step in fat, eluted substance and the preparation, and described eluted substance adopts ethyl acetate solvent.
Sample solution is 35 ℃ of water-baths in the described preparation, and behind the adding 0.1mL ethylene glycol, rotary evaporation is to 0.2mL-0.3mL in the eluent, and nitrogen blows to 0.1mL, adds 0.9mL moving phase constant volume 1.0mL, and vortex mixed 10s crosses 0.22 μ m miillpore filter and gets final product.
It is the method for the HPLC of moving phase that the present invention has set up with sodium dihydrogen phosphate-acetonitrile, wherein replace acidic components such as phosphoric acid, formic acid, acetate with sodium dihydrogen phosphate, make the preparation of moving phase convenient and accurate, make the easier control of pH value of solution and stable, and this method have better repeatability and reappearance.
Adopt ethyl acetate solvent when eluted substance extracts sulfonamides compound, reduced the application of toxic reagent, during sample solution, under the low temperature water-bath, utilize ethylene glycol to make protective agent in preparation, the high temperature evaporate to dryness is to the influence of sulfonamides compound when preventing rotary evaporation.
Chromatographic column: ODS-C in the HPLC assay method of the present invention
18Post (4.6mm * 250mm * 5 μ m), column temperature is a room temperature; Moving phase: 50mmol/L sodium dihydrogen phosphate-acetonitrile (chromatographically pure) (V/V)=70: 30; Constant current mode; Detecting device: UV-detector, the detection wavelength is 270nm.
The sulfa drugs that detects comprises: sulfamethazine, daimeton, fanasil, Sulfamethoxazole, sulphadiazine, madribon, sulfamethyldiazine, sulfaquinoxaline.Eight kinds of sulfa drugss are in 0.010mg/kg～1.000 μ g/mL scopes, and linearly dependent coefficient is more than 0.9999; The recovery is 76.0%～115.0%; The instrument detecting limit is 0.010 μ g/mL; Method detects and is limited to 0.020 μ g/g.
It is the method for the HPLC of moving phase that the present invention has set up with sodium dihydrogen phosphate-acetonitrile, makes the preparation of moving phase convenient and accurate, makes the easier control of pH value of solution, has better repeatability and reappearance.Adopt the environment-friendly type solvent to replace toxic solvent simultaneously, make more environmental protection of the inventive method.
Description of drawings
Figure 18 plants pharmaceutical standards chromatogram (0.100 μ g/mL)
Fig. 2 oppresses the chromatogram that sample adds concentration 0.200mg/Kg
Fig. 3 pork sample adds the chromatogram of concentration 0.200mg/Kg
Embodiment
The present invention is described in further detail below by example.
Embodiment 1: flesh of fish sample detection
1, key instrument equipment, medicine
Instrument:
1) glass mortar;
2) glass syringe 5mL
3) SPE solid-phase extraction device
4) Nitrogen evaporator of tool thermostat water bath
5) transfer pipet 1mL, 0.5mL
6) volumetric flask 10mL
7) electronic balance (scale division value 0.001g)
8) timer
9) vortex mixer
10) liquid chromatograph
Medicine:
1) standard substance (purity 〉=98.5%): sulphadiazine (sulfadiazine), sulfamethyldiazine (sulfamerazine), sulfamethazine (sulfamethazine), daimeton (sulfamonomethoxine), fanasil (sulfadoxine), Sulfamethoxazole (sulfamethoxazole), sulfaquinoxaline (sulfaquinoxzline), madribon (sulfadimethoxine) standard solution.
2) acetonitrile, ethyl acetate, methyl alcohol (chromatographically pure)
3) normal hexane, sodium dihydrogen phosphate (analyzing pure)
4) anhydrous sodium sulfate (analyzing pure) at 450 ℃ of oven dry 4h, is placed standby before using in the exsiccator.
5) C
18Filler uses preceding normal hexane, ethyl acetate, methyl alcohol (chromatographically pure) prewashing C with double volume
18, drain at last.
6) moving phase: 50mmol/L sodium dihydrogen phosphate aqueous buffer solution and chromatographically pure acetonitrile 70: 30 by volume (V/V) are mixed and obtain.
2, standard solution preparation
Standard reserving solution: accurately take by weighing an amount of sulfa drugs, be made into the standard reserving solution of 1mg/mL respectively with methyl alcohol, be stored in-20 ℃ of refrigerators.Be valid for one year.
Standard operation liquid: is 0.01 μ g/mL～1.0 μ g/mL with storing solution with the moving phase dilution, leaves in 2～8 ℃ of refrigerators standby.The term of validity 6 months.
Hybrid standard working fluid: accurately measure each 0.5mL of sulfa drugs standard reserving solution, use methanol constant volume 10mL, be mixed with the sulfa drugs hybrid standard storing solution of 50 μ g/mL, be diluted to the hybrid standard working fluid of 0.01 μ g/mL～1.0 μ g/mL again with moving phase, leave in 2～8 ℃ of refrigerators standby.The term of validity 6 months.
3, the preparation of flesh of fish sample
2kg is oppressed sample with organizing the pulverizer homogenized, take by weighing the 500g sample, the laboratory is standby, 4 ℃ of storages.
4, MSPD method
Take by weighing the pretreated C of 2g
18Filler is put in the glass mortar, accurately takes by weighing the good flesh of fish sample of 0.50g homogenized in mortar, grinds 30s, mixes.The meat sample that grinds is packed in the glass syringe of 5mL (filter paper of packing in the bottom in advance, and the anhydrous sodium sulfate of 0.1mL column volume is housed), reinstall a slice filter paper above, being pressed to column volume is 4.2mL.With 8mL normal hexane drip washing pillar, further to remove fat, flow velocity is 1/s, and vacuum is drained, and removes normal hexane, discards leacheate.Use 15mL eluent ethyl acetate pillar again, the control flow velocity is 1/s, and vacuum is drained, and collects eluent in test tube.
5, concentrate:
Add 0.1mL ethylene glycol in the eluent, about rotary evaporation 0.2mL, nitrogen blows to 0.1mL in 35 ℃ of water-baths, adds 0.9mL moving phase constant volume 1.0mL, and vortex mixed 10s crosses 0.22 μ m miillpore filter, and is to be measured.
6, C18 filler blank assay
In mortar,, be C according to above-mentioned identical MSPD and method for concentration not with flesh of fish sample
18The filler blank assay.
7, sample blank experiment
In mortar, add the flesh of fish sample that empirical tests does not contain sulfonamide, do the experiment of flesh of fish sample blank according to above-mentioned identical MSPD and method for concentration.
8, sample adds experiment
In mortar, add the flesh of fish sample that empirical tests does not contain sulfonamide, add the mixed standard solution of a certain amount of sulfonamide then, cook flesh of fish sample according to above-mentioned identical MSPD and method for concentration and add experiment.
9, actual sample determination experiment
In mortar, add actual flesh of fish sample, cook flesh of fish actual sample determination experiment according to above-mentioned identical MSPD and method for concentration.
10, HPLC measures
The sample test solution 50 μ L that above-mentioned processing is made inject chromatographic system, under the room temperature, with 50mmol/L sodium dihydrogen phosphate-acetonitrile (chromatographically pure) (V/V)=70: 30 be moving phase, flow velocity is 0.7mL/min, at C
18Separate on (4.6mm * 250mm * 5 μ m) chromatographic column, UV-detector, wavelength is 270nn.(seeing Fig. 1, Fig. 2)
11, experimental result
1) typical curve of method
Typical curve equation and the related coefficient of concentration range when 0.010～1.000 μ g/mL sees Table 1.
The typical curve equation and the related coefficient of table 18 kind of medicine
| Medicine name | Linear equation | Related coefficient |
| Sulphadiazine (sulfadiazine) sulfamethyldiazine (sulfamerazine) sulfamethazine (sulfamethazine) daimeton (sulfamonomethoxine) fanasil (sulfadoxine) | A＝338.69c-0.033 A＝333.74c-0.133 A＝297.78c-0.106 A＝263.21c-0.010 A＝273.47c-0.178 | 0.9999 0.9999 0.9999 0.9999 1.0000 |
| Sulfamethoxazole (sulfamethoxazole) sulfaquinoxaline (sulfaquinoxzline) madribon (sulfadimethoxine) | A＝299.76c-0.016 A＝260.00c-0.138 A＝276.03c-0.506 | 1.0000 0.9999 1.0000 |
The A-chromatographic peak area; C-concentration
2) recovery
The recovery of the flesh of fish sample when interpolation concentration is 0.200 μ g/g and 0.100 μ g/g sees Table 2.
The recovery (n=6) of table 28 kind of medicine
3) detection limit of method
According to the ratio of instrumental response value and noise is 3 to come the detection limit of computing method, and then the instrument detecting limit is 0.010 μ g/mL; Method detects and is limited to 0.020 μ g/g.
From the The above results MSPD/HPLC method that sulphonamides multi-relict is analyzed the flesh of fish set up of this experiment as can be known, adopt the MSPD technology to carry out sample preparation, extracting, purify a step finishes, simplified in the classical sample treatment extract, separate, concentrate, loaded down with trivial details step such as purification, simple to operate, method is stablized, and has desirable accuracy and sensitivity; Adopt low poison solvent ethyl acetate to replace methylene chloride simultaneously, reduced in the experimentation organic solvent the pollution of operator and environment.Adopt 50mmol/L NaH2PO4-acetonitrile (V/V:70: 30) be moving phase simultaneously, under constant current mode, carry out, the summarized chromatogram operation, when selecting for use sodium dihydrogen phosphate to regulate the pH of moving phase, because sodium dihydrogen phosphate is a buffer solution, have certain pH surge capability, pH is difficult for changing, and is the favorable reproducibility of measuring.8 kinds of sulfa drugss of separation detection not only can satisfy the detection of the routine of aquatic products sulfamido residual quantity simultaneously, also can be used for the mensuration of the sulfa drugs in other samples.
Embodiment 2 pork sample detection
Operation steps is the same, and pork sample HPLC testing result is seen Fig. 3.
Under the analysis condition that the present invention set up, obtain the chromatogram (Fig. 3, Fig. 2 and Fig. 1) of pork sample and flesh of fish sample and 8 kinds of sulfonamide standard items, from these chromatogram separating effects as seen, do not have matrix interference to influence the mensuration of medicine, separating effect is very good.
Claims (4)
1, the matrix solid phase dispersion-high-performance liquid chromatogram determination method of sulfa drug residue in a kind of animal derived food, comprise that the matrix solid phase is disperseed and high performance liquid chromatography detects step, it is characterized in that: used moving phase is 50mmol/L sodium dihydrogen phosphate-acetonitrile in the described high-performance liquid chromatogram determination method, and the volume ratio of sodium dihydrogen phosphate and acetonitrile is 70: 30.
2, assay method according to claim 1, used chromatographic column is the ODS-C18 post in the described high-performance liquid chromatogram determination method, specification is 4.6mm * 250mm * 5 μ m.
3, assay method according to claim 1, described matrix solid phase disperse to comprise grinding, dress post, remove sample solution step in fat, eluted substance and the preparation, and described eluted substance adopts ethyl acetate solvent.
4, assay method according to claim 3, sample solution is 35 ℃ of water-baths in the described preparation, after adding 0.1mL ethylene glycol in the eluent, rotary evaporation 0.2mL-0.3mL, nitrogen blows to 0.1mL, add 0.9mL moving phase constant volume 1.0mL, vortex mixed 10s crosses 0.22 μ m miillpore filter and gets final product.
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