CN115128187A - Method for determining antibiotic medicines in liquid Chinese herbal medicine preparation for fishing - Google Patents
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Abstract
The invention discloses a method for determining antibiotic medicines in a liquid Chinese herbal medicine preparation for fishing, belonging to the field of determination of antibiotic medicines, and the method for determining antibiotic medicines in the liquid Chinese herbal medicine preparation for fishing comprises the following steps: s1: adding the internal standard mixture into the sample liquid, performing vortex for 0.4-0.6min, standing for 4-6min, adding acetonitrile, performing vortex for 0.8-1.2min, and performing S2: moving 1/4-1/6 of the volume of the mixed solution out, adding phosphate buffer solution and trichloromethane in turn, and vortexing for 0.8-1.2min to form a phosphate buffer solution/trichloromethane/acetonitrile emulsion system, S3: centrifuging the phosphate buffer solution/chloroform/acetonitrile emulsion system for 2.5-3.5min, extracting a precipitate phase, blow-drying with nitrogen at 38-42 ℃, adding a constant volume solution after blow-drying, then performing ultrasonic dissolution, filtering with a filter membrane after ultrasonic dissolution, and measuring with a high performance liquid chromatography-tandem mass spectrometer. The invention adopts the dispersion liquid-liquid microextraction combined with the liquid chromatography-mass spectrometry technology, and simultaneously determines the residues of quinolones and sulfonamides in the liquid Chinese herbal medicine preparation for fishing.
Description
Technical Field
The invention belongs to the field of determination of antibiotic medicines, and particularly relates to a method for determining antibiotic medicines in a liquid Chinese herbal medicine preparation for fishing.
Background
The Chinese herbal medicine preparation for fishing has the advantages of nature, high efficiency, less toxic and side effects, immunity enhancement, no drug resistance and the like, has dual functions of medicine and nutrition, and is more and more widely applied to aquaculture. Compared with chemical medicines, the Chinese herbal medicines have mild medicinal properties and longer medicinal effect reaction time, and are mainly suitable for preventing diseases of aquaculture animals. In order to improve the effect of the Chinese herbal medicine for fishing on treating the diseases of the aquaculture animals, a certain amount of antibiotic is added into the Chinese herbal medicine preparation for fishing, which is beneficial to treating the diseases of the aquaculture animals. However, excessive antibiotics and illegally added antibiotics can cause the antibiotics to remain in the bodies of the aquaculture animals, so that the aquaculture animals and eaters thereof generate drug resistance, and the human health is indirectly influenced.
Quinolone antibiotics and sulfonamide antibiotics are artificially synthesized broad-spectrum antibiotic drugs and are widely applied to the aquaculture industry. According to the maximum residue limit of veterinary drugs in GB31650-2019 foods, the maximum residue limit of the drugs in fish muscles and skin is as follows: 100 mu g/kg of danofloxacin, 300 mu g/kg of difloxacin, 100 mu g/kg of enrofloxacin (the sum of enrofloxacin and ciprofloxacin), 500 mu g/kg of flumequine, 100 mu g/kg of oxolinic acid, 30 mu g/kg of sarafloxacin and 100 mu g/kg of sulfanilamide total amount. On the other hand, lomefloxacin, pefloxacin, ofloxacin and norfloxacin are prohibited from being used in cultivation because they are not used as medicines according to the regulation of No. 2292 of the Ministry of agriculture. In order to better standardize the production and use of the antibiotic medicines in the Chinese herbal medicine preparation for fishing and investigate potential risk hidden dangers of the Chinese herbal medicine preparation, the establishment of a detection and analysis method for the determination of the antibiotic medicines in the Chinese herbal medicine preparation for fishing is particularly important.
The Chinese herbal medicine preparation for fishing has complex matrix, and part of the medicines sold in the market are liquid, so that the enrichment and impurity removal are further required. The traditional liquid-liquid extraction method and the traditional solid-phase extraction method have the defects of large reagent consumption and tedious operation, and are not suitable for rapid analysis. And the existing national standard is not suitable for the residue determination of antibiotics in the liquid Chinese herbal medicine preparation for fishing, and the residue condition of the antibiotics cannot be effectively monitored.
Disclosure of Invention
The invention aims to provide a method for measuring antibiotic medicines in a liquid Chinese herbal medicine preparation for fishing, which adopts the technology of combining dispersion liquid-liquid microextraction with liquid chromatography-mass spectrometry and simultaneously measures the residues of quinolones and sulfonamides in the liquid Chinese herbal medicine preparation for fishing.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for determining antibiotic medicines in a liquid Chinese herbal medicine preparation for fishing, which comprises the following steps: s1: adding the internal standard mixture into the sample liquid, performing vortex for 0.4-0.6min, standing for 4-6min, adding acetonitrile, performing vortex for 0.8-1.2min to obtain a mixed solution, S2: moving 1/4-1/6 of the volume of the mixed solution out, adding phosphate buffer solution and trichloromethane into the moved mixed solution in sequence, swirling for 0.8-1.2min to form a phosphate buffer solution/trichloromethane/acetonitrile emulsion system, S3: centrifuging the phosphate buffer solution/chloroform/acetonitrile emulsion system for 2.5-3.5min, extracting 1.0-1.5mL of a precipitation phase, drying by blowing nitrogen at 38-42 ℃, adding a constant volume solution after drying, then performing ultrasonic dissolution, filtering by a filter membrane after ultrasonic dissolution, and determining by a high performance liquid chromatography-tandem mass spectrometer.
Preferably, in the phosphate buffer solution/chloroform/acetonitrile emulsion system, the volume ratio of the phosphate buffer solution to the chloroform to the acetonitrile emulsion is 9.8-10.2:1.4-1.6: 1.5-1.8.
Preferably, step S1 specifically includes the following steps: weighing 1.0mL of sample solution into a centrifuge tube, adding 0.5mL of internal standard mixed solution, performing vortex at the rotating speed of 2000r/min for 0.5min, standing for 5min, adding 8.5mL of acetonitrile, and performing vortex at the rotating speed of 2500r/min for 1min to obtain 10mL of mixed solution.
Preferably, step S2 specifically includes the following steps: and transferring 2mL of mixed solution from 10mL of mixed solution into a centrifuge tube, sequentially adding 9.8mL of phosphate buffer solution and 1.5mL of trichloromethane, and vortexing at the rotating speed of 2500r/min for 1min to form a phosphate buffer solution/trichloromethane/acetonitrile emulsion system.
Preferably, step S3 specifically includes the following steps: centrifuging the phosphate buffer solution/chloroform/acetonitrile emulsion system at the rotating speed of 2000r/min for 3min, extracting 1.0-1.5mL of a precipitation phase by using a microsyringe, drying by using nitrogen at 40 ℃, adding 1.0mL of constant volume liquid after drying, then performing ultrasonic dissolution, filtering by using a 0.22 mu m filter membrane after ultrasonic dissolution, and determining by using a high performance liquid chromatography-tandem mass spectrometer.
Preferably, the phosphate buffer solution has a pH of 7.0 and a concentration of 0.05 mol/L.
Preferably, the internal standard mixture is a methanol solution of norfloxacin-D5, enrofloxacin-D5, sulfa-dimethoxypyrimidine-D3 and sulfa-dimethoxypyrimidine-D6, and the concentration is 0.5 mug/mL.
Preferably, in step S3, the chromatographic conditions are: adopts Ultimate XB-C with the specification of 2.1mm multiplied by 150mm multiplied by 5 mu m 18 A chromatographic column with the column temperature of 35 ℃, the flow rate of 0.25mL/min and the sample injection amount of 10 mu L; mobile phase: a is 0.002mol/L ammonium acetate-0.1% formic acid water solution, B is acetonitrile; elution gradient: 0-5.5min, 15% -20% B; 15.5-16min, 20% -40% B; 16-23min, 40% B; 23-23.5min, 40% -15% B; 23.5-28min, 15% B.
Preferably, in step S3, the mass spectrometry conditions are: the electrospray ion source, the positive ion detection mode and the spray voltage are adopted: 3500V, sheath gas pressure 241KPa, auxiliary gas pressure: 2L/min, ion transport capillary temperature: 350 ℃, reaction monitoring mode was selected, Q1 half-peak width: 0.7u, half-width of Q3: 0.7u, collision air pressure: argon, 0.2 Pa.
The invention has the beneficial effects that:
1. considering the characteristic that acetonitrile can be used as an extracting agent and a dispersing agent at the same time, the acetonitrile is firstly adopted to directly extract and enrich antibiotics in the Chinese herbal medicine preparation, then phosphate buffer solution is added, and then the antibiotics dissolved in the acetonitrile enters the phosphate buffer solution by virtue of the characteristic that the acetonitrile and water are mutually soluble and the characteristic of being used as the dispersing agent. And then, by adding an extractant trichloromethane, a phosphate buffer solution/trichloromethane/acetonitrile opacification system is smoothly formed, the antibiotics are quickly extracted to the trichloromethane, and the quick DLLME is realized. Meanwhile, the scheme of diluting part of the extracting solution by phosphate buffer solution also solves the problems that the two phases of the DLLME can not be layered and the substrate inhibition effect of the pigment is caused.
2. The content of antibiotic drugs in the liquid Chinese herbal medicine preparation for fishing can be rapidly and effectively monitored by combining a dispersion liquid-liquid microextraction technology with a liquid chromatography-mass spectrometry technology.
3. By optimizing the volume ratio of the phosphate buffer solution, the trichloromethane and the acetonitrile and regulating and controlling the pH value of the opacification system, the recovery rate of the target analyte is improved, and higher extraction efficiency is obtained.
4. The extraction time is 1.0min, and the reproducibility of the extraction method is improved.
5. The introduction of the internal standard improves the sensitivity reduction of the method caused by the dilution of the phosphate buffer.
Drawings
FIG. 1 is a standard selection reaction monitoring ion flow chromatogram of an example of the present invention.
FIG. 2 is a graph of the effect of pH on target analyte recovery for examples of the invention.
FIG. 3 is a graph of the effect of phosphate buffer volume on target analyte recovery for an example of the present invention.
FIG. 4 is a graph of the effect of an extractant on target analyte recovery for an example of the invention.
FIG. 5 is a graph of the effect of extractant volume on target analyte recovery for an example of the invention.
FIG. 6 is a graph of the effect of dispersant volume on target analyte recovery for examples of the invention.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and detailed description.
Instruments and reagents: TSQ Quantum Ultra high performance liquid chromatography-tandem mass spectrometer (Thermo Fisher, usa), power distribution spray ion source; AB204-E type, PL203 type electronic analytical balance (Mettler Toledo corporation); 3-5W small bench low speed centrifuge (Hennno instruments & Equipment Co., Ltd., Hunan); a vortex mixer type MS3 (IKA, Germany); HSC-24B water bath nitrogen blower (Hengotou technology development Co., Ltd., Tianjin); Milli-Q type ultrapure water instruments (Millipore, USA); 0.22 μm nylon microporous filter membrane (Jinteng laboratory instruments Co., Tianjin).
Norfloxacin (Norfloxacin), Ofloxacin (Ofloxacin), Fleroxacin (Fleroxacin), Danofloxacin mesylate (Danofloxacin mesylate), Enrofloxacin (Enrofloxacin), Difloxacin hydrochloride (Difloxacin hydrochloride), Oxolinic acid (Oxolinic acid), Flumequine (Flumequine), Sulfamethazine (Sulfamerazine), Sulfamethazine (Sulfamethazine), Sulfamethazine (Sulfadoxine), Sulfamethazine (Sulfadimethoxine), and sulfaquinoxaline (Sulfachinoxalin), the standard being more than 97% pure; pefloxacin mesylate (Pefloxacin methanesulfonate) with the purity of the standard substance being more than 92 percent; purchased from dr. ehrenstorfer GmBH, germany. The above standard substances are prepared into standard stock solutions of 100 μ g/mL with methanol, and stored at-20 deg.C in dark place (the quality of the standard substance is corrected according to purity). Then methanol is used for preparing 14 kinds of antibiotic standard mixed liquor with the concentration of 1 mu g/mL, and the mixture is preserved in the dark at the temperature of-20 ℃. Deuterated Norfloxacin (Norfloxacin-D5), deuterated enrofloxacin (Enrofloxacin-D5), deuterated sulfa-dimethoxypyrimidine (Sulfadoxine-D3) and deuterated sulfa-dimethoxypyrimidine (Sulfadimethoxine-D6), wherein the standard product has the purity of 5 mu g/mL and the expansion uncertainty of 0.2 mu g/mL; purchased from agricultural product quality standard research center in rural areas. The mixture is prepared into a standard internal standard mixed solution with 0.5 mu g/mL by methanol and is stored in dark at the temperature of minus 20 ℃.
Methanol, acetonitrile and formic acid are all chromatographically pure and purchased from the company Tidea; other reagents are analytically pure and purchased from chemical reagents of national drug group, Inc.; the experimental water was Milli-Q ultrapure water. 0.05mol/L phosphate buffer solution (pH 7.0): 1.56g of sodium dihydrogen phosphate was added to 79mL of sodium hydroxide solution, mixed, and diluted with water to 200 mL. Volume fixing liquid: taking 15mL of acetonitrile, taking 85mL of 0.1% formic acid aqueous solution (containing 0.002mol/L of ammonium acetate), and uniformly mixing. Graphitized carbon black was purchased from Tianjin Borna Aijiel technologies, Inc.
The method for determining antibiotic drugs in the liquid Chinese herbal medicine preparation for fishing provided by the embodiment comprises the following steps:
s1: weighing 1.0mL of sample solution into a 15mL glass centrifuge tube, adding 0.5. mu.g/mL of standard internal standard mixed solution (methanol solution of norfloxacin-D5, enrofloxacin-D5, sulfa-dimethoxypyrimidine-D3 and sulfa-dimethoxypyrimidine-D6) 0.5mL, and vortexing at 2000r/min for 0.5 min. After standing for 5min, 8.5mL of acetonitrile was added, and the mixture was vortexed at 2500r/min for 1min to obtain 10mL of a mixed solution.
S2: and transferring 2mL of mixed solution from 10mL of mixed solution into a 15mL glass centrifuge tube, sequentially adding 9.8mL of phosphate buffer solution and 1.5mL of trichloromethane, and swirling at the rotating speed of 2500r/min for 1min to form a phosphate buffer solution/trichloromethane/acetonitrile emulsion system.
S3: centrifuging the phosphate buffer solution/chloroform/acetonitrile emulsion system at 2000r/min for 3min, extracting 1.0mL of precipitation phase by using a microsyringe, drying by using nitrogen at 40 ℃, adding 1.0mL of constant volume liquid after drying, then performing ultrasonic dissolution, filtering by using a 0.22 mu m filter membrane after ultrasonic dissolution, and determining by using a high performance liquid chromatography-tandem mass spectrometer.
In this example, the volume ratio of the phosphate buffer solution, chloroform, and acetonitrile emulsion is 9.8:1.5: 1.7.
Wherein the pH value of the phosphate buffer solution is 7.0, and the concentration is 0.05 mol/L. Because the pH value of the phosphate buffer solution is 7.0, the trichloromethane is neutral, and the acetonitrile is extremely weakly alkaline, the phosphate buffer solution/trichloromethane/acetonitrile emulsion system formed by the phosphate buffer solution, the trichloromethane and the acetonitrile is neutral. The carbostyril drugs have carboxyl and piperazinyl, and can form positive ions, negative ions and neutral molecules under different pH values. When the quinolone drugs exist in a neutral molecular state, the distribution coefficient of the quinolone drugs in an organic phase is improved, and the extraction efficiency is increased. The extraction efficiency is higher by adopting a neutral phosphate buffer solution/chloroform/acetonitrile emulsion system.
In step S3, the chromatographic conditions are: adopts Ultimate XB-C with the specification of 2.1mm multiplied by 150mm multiplied by 5 mu m 18 A chromatographic column with the column temperature of 35 ℃, the flow rate of 0.25mL/min and the sample injection amount of 10 mu L; mobile phase: a is 0.002mol/L ammonium acetate-0.1% formic acid water solution, B is acetonitrile; elution gradient: 0-5.5min, 15% -20% B; 15.5-16min, 20% -40% B; 16-23min, 40% B; 23-23.5min, 40% -15% B; 23.5-28min, 15% B.
In step S3, the mass spectrometry conditions are: the electrospray ion source, the positive ion detection mode, the spray voltage: 3500V, sheath gas pressure 241KPa, auxiliary gas pressure: 2L/min, ion transport capillary temperature: 350 ℃, reaction monitoring mode was selected, Q1 half-peak width: 0.7u, half-width of Q3: 0.7u, collision air pressure: argon, 0.2 Pa. The parent, daughter and collision energies are listed in table 1. A schematic ion flow chromatogram for monitoring the reaction for selection of the standard is shown in FIG. 1.
Table 1 selective reaction monitoring of parent ions, daughter ions and collision energy
Note: is the quantitative fragment ion.
The extraction method comprises the following steps:
most of the commercially available Chinese herbal medicine preparations are suspension liquid, and the direct splashing method is adopted in the actual production and use process. Therefore, both the solid in the suspension and the antibiotic in the liquid can affect the cultivation environment. In order to fully extract the antibiotics contained in the Chinese herbal medicine, a direct extraction method is adopted, which is different from the pretreatment of a conventional water sample, and the extraction is carried out after the solid in a commercially available Chinese herbal medicine preparation is removed through a membrane.
Acetonitrile with the highest extraction efficiency is selected as a direct extractant, but the acetonitrile and water are mutually soluble, so that the acetonitrile cannot be concentrated to be dry subsequently, and antibiotics in the Chinese herbal medicine preparation cannot be further enriched. The DLLME is directly adopted, but the Chinese herbal medicine preparation has high pigment content and suspended solid in the liquid, so that two phases are not obviously separated and the stability is poor. Considering the characteristic that acetonitrile can be used as an extracting agent and a dispersing agent at the same time, the acetonitrile is firstly adopted to directly extract and enrich antibiotics in the Chinese herbal medicine preparation, then phosphate buffer solution is added, and then the antibiotics dissolved in the acetonitrile enter the phosphate buffer solution by virtue of the characteristic that the acetonitrile is mutually soluble with water and the characteristic of being used as the dispersing agent. And then, by adding an extractant trichloromethane, a phosphate buffer solution/trichloromethane/acetonitrile opacification system is smoothly formed, the antibiotics are quickly extracted to the trichloromethane, and the quick DLLME is realized. Meanwhile, the scheme of diluting part of the extracting solution by phosphate buffer solution also solves the problems that the two phases of the DLLME can not be layered and the substrate inhibition effect of the pigment is caused. The introduction of the internal standard improves the sensitivity reduction of the method caused by the dilution of the phosphate buffer.
Optimizing the dispersion liquid-liquid microextraction condition:
after the extraction method is determined, optimizing DLLME under various conditions by adopting 10mL of phosphate buffer solution added with standard 100ng, and repeating the calculation for 3 times to obtain an average value.
pH value:
when the target analyte is in a non-dissociated state, the DLLME extraction result is ideal. Therefore, the pH of the solution changes the presence of the antibiotic, which in turn affects the recovery rate. The carbostyril drugs have carboxyl and piperazinyl, and can form positive ions, negative ions and neutral molecules under different pH values. When the quinolone drugs exist in a neutral molecular state, the distribution coefficient of the quinolone drugs in an organic phase is improved, and the extraction efficiency is increased. Also, sulfonamides have a greater solubility in acidic or alkaline solutions, which is detrimental to subsequent organic extraction. As shown in the results of FIG. 2, under the conditions of 0.8mL of acetonitrile dispersant and 0.6mL of chloroform extractant, the distribution coefficient of most of quinolone and sulfonamide antibiotic drugs and the chloroform extractant is the highest, and higher extraction efficiency is shown. Therefore, the pH of the phosphate buffer solution was chosen to be neutral (7.0).
Effect of phosphate buffer volume:
phosphate buffer is the carrier for the target analyte in the method. The volume ratio of the phosphate buffer solution to the volume ratio of the dispersing agent and the extracting agent has important influence on the extraction result. The effect of 10.0mL, 6.0mL phosphate buffer samples on the recovery of the target analyte was examined. As shown in FIG. 3, the results show that under the conditions of 0.8mL acetonitrile dispersant, 0.6mL chloroform extractant and neutral pH of phosphate buffer, 10.0mL phosphate buffer volume can obtain better extraction efficiency.
Optimizing an extracting agent and volume:
the extractant is an important factor influencing the extraction efficiency of DLLME, and the density, the solubility, the viscosity and the extraction capacity are important factors influencing the extraction effect and the reproducibility. (1) The density of the extracting agent is greater than or less than that of the water and the dispersing agent, and the extracting agent is easy to obtain from a mixed solution; (2) the extractant is miscible with the dispersant and insoluble in the sample solution; (3) a stable two-phase system can be formed; (4) the extractant has better extraction capacity for target analytes. Dichloromethane and trichloromethane are respectively selected as extracting agents, and under the condition of neutral sample liquid, 0.8mL of acetonitrile is used as a dispersing agent to investigate the extraction efficiency. As shown in fig. 4, the results indicate that, after the carbon dichloride is mixed with the dispersant, the drop loss is severe, the extraction efficiency is low and the repeatability is poor; chloroform is more effective in extracting target analytes.
Under the same conditions, the influence of the volume of the extractant on the recovery rate of the target analyte is examined. As shown in fig. 5, the results indicate that the recovery of the target analyte increases with increasing volume of the extractant, but that the recovery of the target analyte is essentially constant when the volume is increased to 1.5 mL. This indicates that as the amount of chloroform increases, the target analyte entering the organic phase increases. Therefore, the most optimal extraction conditions for 1.5mL of chloroform were finally selected.
Optimization of the volume of the dispersant:
the dosage of the dispersing agent acetonitrile directly influences the formation of a phosphate buffer solution/trichloromethane/acetonitrile emulsion system, influences the dispersion degree of an extracting agent in water, and further influences the recovery rate of a target analyte. The effect of acetonitrile volumes (0.6, 0.8, 1.2, 1.4, 1.6, 1.8mL) on target analyte recovery was examined. As shown in fig. 6, the results indicate that the target analyte recovery increased with increasing acetonitrile volume, which reached a maximum when the acetonitrile volume was increased to 1.6 mL. When the volume was increased to 1.8mL, there was no significant change in the target analyte recovery. The optimal volume of dispersant acetonitrile was therefore chosen to be 1.6 mL.
Influence of extraction time:
the influence of different extraction times (0.5, 1.0, 2.0, 3.0min) on the extraction efficiency was examined. The results show that the length of the vortex time has little influence on the extraction efficiency, but the reproducibility of the extraction method can be improved. After the above factors are considered comprehensively, the extraction time is selected to be 1.0 min.
Standard curve, linear range, detection limit and quantitation limit:
in order to eliminate quantitative deviation caused by matrix effect, a matrix matching standard curve method is adopted in the experiment. And (3) transferring a proper amount of mixed standard solution, and preparing matrix standard solutions with different mass concentrations by using the blank sample extracting solution respectively, wherein the target concentration is 10, 50, 100, 200, 500 and 1000ng/mL respectively. The concentrations of all the components and the chromatographic peak areas thereof are subjected to linear regression to form a good linear relation, and the correlation coefficients are all larger than 0.99. The Limit of Quantitation (LOQ) was calculated as 10 times the signal-to-noise ratio (S/N), and the specific values are listed in Table 2.
Method accuracy and precision:
a standard addition experiment is carried out by taking a commercially available Chinese herbal medicine preparation for fishing as a research object, a standard addition recovery experiment is carried out by respectively taking three addition levels of low, medium and high, and each concentration level is subjected to 6 times of parallel experiments, so that the accuracy and precision of the method are investigated. Table 2 the results show that: the average recovery rate is 60.0-119.7%, and the relative standard deviation is 1.0-14.7%. Within 30 days, 5 standard addition experiments are carried out under the condition of adding standard concentration of 1 mu g/L, and the precision of the method in the daytime is examined, and the relative standard deviation is 3.0-12.3 percent (see
Table 2). The precision and accuracy of the method can meet the requirement of monitoring the drug residue.
TABLE 2 method accuracy and precision measurements
The dispersion liquid-liquid microextraction (DLLME) technology has the advantages of simple operation, rapidness, high sensitivity, environmental friendliness, low price and the like, and can be combined with the liquid chromatography-mass spectrometry technology to rapidly and effectively monitor the content of antibiotic drugs in the liquid Chinese herbal medicine preparation for fishing.
In the embodiment, the fishery Chinese herbal medicine preparation is taken as a research object, the influence of the extracting agent and the volume, the dispersing agent and the volume and the pH value on the recovery rate of the target antibiotic are respectively considered, and the rapid analysis method for simultaneously measuring the residue of quinolones and sulfonamides in the fishery Chinese herbal medicine preparation by the dispersion liquid-liquid microextraction combined with the LC-MS technology is established. Can accelerate and promote the green high-quality development of the aquaculture industry and provide technical support for the supervision work of the quality safety of the aquatic products.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art; the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. The method for determining the antibiotic medicines in the liquid Chinese herbal medicine preparation for fishing is characterized by comprising the following steps:
s1: adding the internal standard mixture into the sample solution, swirling for 0.4-0.6min, standing for 4-6min, adding acetonitrile, and swirling for 0.8-1.2min to obtain a mixed solution;
s2: removing 1/4-1/6 of the volume of the mixed solution, adding phosphate buffer solution and trichloromethane into the removed mixed solution in sequence, and swirling for 0.8-1.2min to form a phosphate buffer solution/trichloromethane/acetonitrile emulsion system;
s3: centrifuging the phosphate buffer solution/chloroform/acetonitrile emulsion system for 2.5-3.5min, extracting a precipitate phase, blow-drying with nitrogen at 38-42 ℃, adding a constant volume solution after blow-drying, then performing ultrasonic dissolution, filtering with a filter membrane after ultrasonic dissolution, and measuring with a high performance liquid chromatography-tandem mass spectrometer.
2. The method for detecting antibiotic drugs in the liquid Chinese herbal medicine preparation for fishery according to claim 1, which comprises the following steps:
in the phosphate buffer solution/trichloromethane/acetonitrile emulsion system, the volume ratio of the phosphate buffer solution to the trichloromethane to the acetonitrile emulsion is 9.8-10.2:1.4-1.6: 1.5-1.8.
3. The method for determining antibiotic drugs in the liquid Chinese herbal medicine preparation for fishery according to claim 1, wherein the step S1 specifically comprises the following steps:
weighing 1.0mL of sample solution into a centrifuge tube, adding 0.5mL of internal standard mixed solution, performing vortex at a rotation speed of 2000r/min for 0.5min, standing for 5min, adding 8.5mL of acetonitrile, performing vortex at a rotation speed of 2500r/min for 1min, and obtaining 10mL of mixed solution.
4. The method for determining antibiotic drugs in the liquid Chinese herbal medicine preparation for fishery according to claim 1, wherein the step S2 specifically comprises the following steps:
and transferring 2mL of mixed solution from 10mL of mixed solution into a centrifuge tube, sequentially adding 9.8mL of phosphate buffer solution and 1.5mL of trichloromethane, and vortexing at the rotating speed of 2500r/min for 1min to form a phosphate buffer solution/trichloromethane/acetonitrile emulsion system.
5. The method for determining antibiotic drugs in a liquid Chinese herbal medicine preparation for fishery according to claim 1, wherein the step S3 comprises the following steps:
centrifuging the phosphate buffer solution/chloroform/acetonitrile emulsion system at 2000r/min for 3min, extracting a precipitation phase by using a microsyringe, blow-drying by using nitrogen at 40 ℃, adding 1.0mL of constant volume solution after blow-drying, then performing ultrasonic dissolution, filtering by using a 0.22 mu m filter membrane after ultrasonic dissolution, and determining by using a high performance liquid chromatography-tandem mass spectrometer.
6. The method for detecting antibiotic drugs in the liquid Chinese herbal medicine preparation for fishery according to claim 1, which comprises the following steps:
the pH value of the phosphate buffer solution is 7.0, and the concentration is 0.05 mol/L.
7. The method for detecting antibiotic drugs in the liquid Chinese herbal medicine preparation for fishery according to claim 1, which comprises the following steps:
the internal standard mixture is methanol solution of norfloxacin-D5, enrofloxacin-D5, sulfa-dimethoxy pyrimidine-D3 and sulfa-dimethoxy pyrimidine-D6, and the concentration is 0.5 mug/mL.
8. The method for determining antibiotic drugs in a liquid Chinese herbal medicine preparation for fishery according to claim 1, which comprises the following steps:
in step S3, the chromatographic conditions are: adopts Ultimate XB-C with the specification of 2.1mm multiplied by 150mm multiplied by 5 mu m 18 A chromatographic column with the column temperature of 35 ℃, the flow rate of 0.25mL/min and the sample injection amount of 10 mu L; mobile phase: a is 0.002mol/L ammonium acetate-0.1% formic acid water solution, B is acetonitrile; elution gradient: 0-5.5min, 15% -20% B; 15.5-16min, 20% -40% B; 16-23min, 40% B; 23-23.5min, 40% -15% B; 23.5-28min, 15% B.
9. The method for detecting antibiotic drugs in the liquid Chinese herbal medicine preparation for fishery according to claim 1, which comprises the following steps:
in step S3, the mass spectrometry conditions are: the electrospray ion source, the positive ion detection mode, the spray voltage: 3500V, sheath gas pressure 241KPa, auxiliary gas pressure: 2L/min, ion transport capillary temperature: 350 ℃, reaction monitoring mode was selected, Q1 half-peak width: 0.7u, half-width of Q3: 0.7u, collision air pressure: argon, 0.2 Pa.
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