CN110988186A - Liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts - Google Patents
Liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts Download PDFInfo
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Abstract
The invention relates to a liquid chromatogram tandem mass spectrum detection method for detecting harmful substance residues in bean sprouts, which is characterized by comprising the following steps of: s100, extracting bean sprouts to obtain a sample to be detected; s200, detecting the harmful substance residue of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer. The bean sprout safety emergency detection system can efficiently and accurately detect various harmful substance residues in bean sprouts, has larger detection capacity and wider application range, and is more suitable for the rapid emergency disposal work of safety risk emergencies in bean sprout foods.
Description
Technical Field
The invention relates to the technical field of agricultural product quality detection, in particular to a liquid chromatogram tandem mass spectrum detection method for detecting harmful substance residues in bean sprouts.
Background
The quality safety degree of plant agricultural products is closely related to the daily life of people, and the supervision of the quality safety of the plant agricultural products by related departments is also more and more strong. Among them, agricultural products such as vegetables and fruits may have a phenomenon of illicitly using plant growth regulators and antibiotic drugs.
The bean sprouts are also called sprouting vegetables, have rich varieties and comprehensive nutrition, and are common vegetables on dining tables of common people. The plant growth regulator possibly existing or remained in the bean sprouts comprises 4-chlorophenoxyacetic acid, 6-benzyladenine, gibberellin and the like, and the antibiotic drugs possibly existing or remained in the bean sprouts comprise enrofloxacin, norfloxacin, metronidazole, chloramphenicol and the like.
Plant growth regulators are a class of pesticides used to regulate plant growth and development, which promote plant growth and improve crop quality and yield. Antibiotic drugs are mainly used for sterilization and disinfection and prevention of root rot. Abuse of plant growth regulators and antibiotic drugs in the bean sprouts can cause acute and chronic poisoning of people and livestock, easily cause generation of drug-resistant bacteria, and seriously harm health of people.
In the related technology, the method for detecting harmful substances such as plant growth regulators, antibiotics and the like in bean sprouts is complicated in steps, long in time consumption and narrow in detection range, is difficult to efficiently detect multiple harmful substances simultaneously, and cannot meet the requirements of high efficiency, multiple components and the like required by rapid disposal work of food safety emergencies. Therefore, the selection of a proper extraction method and an appropriate analysis means is particularly important for solving the simultaneous determination of the multi-component plant growth regulator and the antibiotic medicines in the bean sprouts.
Disclosure of Invention
The present invention is directed to addressing at least one of the deficiencies in the prior art discussed above.
In view of the above, the present invention provides a liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts. The liquid chromatography tandem mass spectrometry detection method for detecting the residue of harmful substances in bean sprouts comprises the following steps:
s100, extracting bean sprouts to obtain a sample to be detected;
s200, detecting harmful substance residues of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer;
wherein, the liquid chromatogram condition for harmful substance residue detection comprises:
a chromatographic column: agent Eclipse Plus-C18, 2.1mm × 100mm, 1.8 μm;
column temperature: 35-45 ℃;
flow rate: 0.2mL/min-0.6 mL/min;
sample introduction amount: 0.1-10 μ L;
mobile phase: the phase A is any one of water, formic acid aqueous solution and ammonium acetate aqueous solution, and the phase B is any one of methanol or acetonitrile;
gradient elution order: 0min-3min, 90% A; 3min-7min, 90% -70% A; 7min-8min, 70% -10% A; 8min-11min, 10% A; 11min-11.01min, 10% -90% A; 11.01min-15min, 90% A;
the mass spectrum conditions for detecting the harmful substance residues comprise:
spraying voltage: 3kV-4 kV;
the scanning mode is as follows: carrying out multi-reaction monitoring by adopting an electrospray positive ion mode and a negative ion mode;
desolventizing gas temperature: 240-260 ℃;
temperature of the heating block: 380-420 ℃;
flow rate of drying gas: 12L/min-18L/min.
In addition, the technical scheme provided by the invention can also have the following additional technical characteristics:
in the above technical solution, the step S100 specifically includes the following steps:
s101, crushing bean sprouts, adding the crushed bean sprouts into anhydrous sodium sulfate, and uniformly mixing to obtain a first mixture;
s102, adding an extracting solution into the first mixture obtained in the step S101, uniformly mixing, and performing centrifugal treatment to obtain a supernatant;
s103, drying the supernatant obtained in the step S102 at the temperature of 40-50 ℃ in a water bath by adopting nitrogen to obtain dried supernatant;
s104, carrying out constant volume on the dried supernatant obtained in the step S103 by adopting methanol to obtain a constant volume sample;
and S105, filtering the constant volume sample obtained in the step S104 by adopting an organic phase filter membrane to obtain a sample to be detected.
In any one of the above technical solutions, in the step S101, the mass ratio of the bean sprouts to the anhydrous sodium sulfate is bean sprouts: anhydrous sodium sulfate ═ (0.4-0.6): 1.
in any one of the above technical solutions, in the step S102, the mass-to-volume ratio of the bean sprouts and the extracting solution is 0.2g/ml-0.3 g/ml; the extracting solution is one of the following: acetonitrile, acidified acetonitrile, ethyl acetate.
In any of the above technical solutions, after the step S105, the method further includes: s106, purifying the sample to be detected obtained in the step S105; wherein the purification is positive mixing type cation exchange column purification or QuEChERS centrifuge tube purification.
In any of the above technical solutions, in the step S200, the column temperature is 40 ℃; the flow rate is 0.4 mL/min; the sample injection amount is 1 mu L; the spraying voltage is 3.5 kV; the temperature of the desolventizing gas is 250 ℃; the temperature of the heating block is 400 ℃; the drying airflow rate was 15L/min.
In any of the above embodiments, in the step S200, the phase a is any one of a mixed solution of water, a 0.01mol/L formic acid aqueous solution, a 0.02mol/L formic acid aqueous solution, a 0.1mol/L formic acid aqueous solution, a 0.15mol/L ammonium acetate aqueous solution, a 0.01mol/L formic acid aqueous solution, and a 0.15mol/L ammonium acetate aqueous solution.
In any of the above technical solutions, the harmful substance residue includes one or a combination of several of the following: 2, 4-dichlorophenoxyacetic acid sodium salt, forchlorfenuron, gibberellin, 4-chlorophenoxyacetic acid, thidiazuron, 6-benzyl adenine, 1-naphthylacetic acid, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, sulfadiazine and the like, and sulfadimidine, sulfamonomethoxine, sulfadimidine, metronidazole, chloramphenicol, norfloxacin, ofloxacin, pefloxacin, lomefloxacin, ciprofloxacin, enrofloxacin and the like. Sulfadimethoxine, paclobutrazol, uniconazole, 4-fluorophenoxyacetic acid and isopentenyladenine.
In any one of the above technical solutions, in the step S200, the thidiazuron, the sodium 2, 4-dichlorophenoxyacetate, the forchlorfenuron, the gibberellin, the 6-benzyladenine, the 4-chlorophenoxyacetic acid, the 4-fluorophenoxyacetic acid, the isopentenyl adenine, the 1-naphthylacetic acid, and the chloramphenicol are scanned by using a negative ion mode; and/or in said step S200, said metronidazole, said paclobutrazol, said sulfamethazine, said sulfamethoxazole, said sulfaquinoxaline, said norfloxacin, said ofloxacin, said pefloxacin, said lomefloxacin, said ciprofloxacin, said enrofloxacin, said sulfadiazine, said sulfadimidine, said sulfamethazine, said sulfadimethoxine, said sulfamonomethoxine, said uniconazole are scanned with a positive ion pattern.
In any one of the above technical solutions, in the step S200, the mass spectrometry conditions further include that the following parameters are respectively adopted to detect each harmful substance residue:
advantageous effects
1. The method adopts a liquid chromatography-tandem mass spectrometry method to measure 27 growth hormones and antibiotic drugs which are possibly added in the bean sprouts under the same chromatographic mass spectrometry condition. The invention can not only expand the detection range and depth of the plant growth regulator and the antibiotic drugs in the bean sprouts, but also improve the working efficiency of daily detection.
2. The method is rapid, high in specificity and high in sensitivity, and can simultaneously complete the detection of 27 plant growth regulators and antibiotic medicines in the bean sprouts. The plant growth regulator and antibiotic drugs added in the bean sprouts are complex, and the invention can redetermine and expand the detection range of target compounds or harmful residual substances in the bean sprouts.
3. In addition to detecting compounds such as 4-chlorophenoxyacetic acid, 6-benzyladenine, gibberellin, enrofloxacin, ofloxacin, metronidazole and the like in the existing recommended standards, the invention can also detect a thidiazuron compound and a 1-naphthylacetic acid compound.
4. The method can complete qualitative and quantitative determination of 27 target compounds within 15 minutes, and compared with the technical scheme of detecting 11 compounds in the same category at most once by the conventional recommended method, the method has the advantages of larger detection capacity and wider application range, and is more suitable for rapid emergency treatment of safety risk emergencies in bean sprout foods.
Drawings
FIG. 1 is a bar graph of recovery for different extraction solvents.
FIG. 2 is a bar graph of recovery for different purification regimes.
FIG. 3 is a MRM chart of the standard solutions of 27 compounds obtained under the condition of 0.01% formic acid water-methanol as a mobile phase.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of specific embodiments of the present invention. Like reference symbols in the various drawings indicate like elements. It should be noted that the described embodiments are only some embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Example 1
The embodiment of the invention provides a liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts. The liquid chromatography tandem mass spectrometry detection method for detecting the residue of harmful substances in bean sprouts comprises the following steps:
s100, extracting bean sprouts to obtain a sample to be detected;
s200, detecting the harmful substance residue of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer.
Wherein, the step S100 specifically includes the following steps:
s101, crushing bean sprouts, adding the crushed bean sprouts into anhydrous sodium sulfate, and uniformly mixing to obtain a first mixture;
s102, adding an extracting solution into the first mixture obtained in the step S101, uniformly mixing, and performing centrifugal treatment to obtain a supernatant;
s103, drying the supernatant obtained in the step S102 at the temperature of 40-50 ℃ in a water bath by adopting nitrogen to obtain dried supernatant;
s104, carrying out constant volume on the dried supernatant obtained in the step S103 by adopting methanol to obtain a constant volume sample;
and S105, filtering the constant volume sample obtained in the step S104 by adopting an organic phase filter membrane to obtain a sample to be detected.
In this embodiment, in the step S101, the mass ratio of the bean sprouts to the anhydrous sodium sulfate is bean sprouts: anhydrous sodium sulfate ═ (0.4-0.6): 1.
preferably, in the step S101, the mass ratio of the bean sprouts to the anhydrous sodium sulfate is bean sprouts: anhydrous sodium sulfate ═ 0.5: 1
In the step S102, the mass-to-volume ratio of the bean sprouts and the extracting solution is 0.2g/ml-0.3 g/ml; the extracting solution is one of the following: acetonitrile, acidified acetonitrile, ethyl acetate.
Preferably, in the step S102, the mass-to-volume ratio of the bean sprouts and the extracting solution is 0.25 g/ml.
Example 2
The embodiment of the invention provides a liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts. The liquid chromatography tandem mass spectrometry detection method for detecting the residue of harmful substances in bean sprouts comprises the following steps:
s100, extracting bean sprouts to obtain a sample to be detected (the specific steps are shown in example 1);
s200, detecting the harmful substance residue of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer.
Wherein, the step S100 specifically includes the following steps:
s101, crushing bean sprouts, adding the crushed bean sprouts into anhydrous sodium sulfate, and uniformly mixing to obtain a first mixture;
s102, adding an extracting solution into the first mixture obtained in the step S101, uniformly mixing, and performing centrifugal treatment to obtain a supernatant;
s103, drying the supernatant obtained in the step S102 at the temperature of 40-50 ℃ in a water bath by adopting nitrogen to obtain dried supernatant;
s104, carrying out constant volume on the dried supernatant obtained in the step S103 by adopting methanol to obtain a constant volume sample;
s105, filtering the constant volume sample obtained in the step S104 by adopting an organic phase filter membrane to obtain a sample to be detected;
s106, purifying the sample to be detected obtained in the step S105.
In this example, the purification was positive mixing type cation exchange column purification or QuEChERS centrifuge tube purification.
Example 3
The embodiment of the invention provides a liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts. The liquid chromatography tandem mass spectrometry detection method for detecting the residue of harmful substances in bean sprouts comprises the following steps:
s100, extracting bean sprouts to obtain a sample to be detected (the specific steps are shown in example 1);
s200, detecting the harmful substance residue of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer.
Wherein, the liquid chromatogram condition for harmful substance residue detection comprises:
a chromatographic column: agent Eclipse Plus-C18, 2.1mm × 100mm, 1.8 μm;
column temperature: 35 ℃;
flow rate: 0.2 mL/min;
sample introduction amount: 0.1 mu L;
mobile phase: the phase A is formic acid aqueous solution, and the phase B is methanol;
gradient elution order: 0min-3min, 90% A; 3min-7min, 90% -70% A; 7min-8min, 70% -10% A; 8min-11min, 10% A; 11min-11.01min, 10% -90% A; 11.01min-15min, 90% A;
the mass spectrum conditions for detecting the harmful substance residues comprise:
spraying voltage: 3 kV;
the scanning mode is as follows: carrying out multi-reaction monitoring by adopting an electrospray positive ion mode and a negative ion mode;
desolventizing gas temperature: 240 ℃;
temperature of the heating block: 380 ℃;
flow rate of drying gas: 12L/min.
The specific gradient elution order can be seen in table 1.
Example 4
The embodiment of the invention provides a liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts. The liquid chromatography tandem mass spectrometry detection method for detecting the residue of harmful substances in bean sprouts comprises the following steps:
s100, extracting bean sprouts to obtain a sample to be detected (the specific steps are shown in example 1);
s200, detecting the harmful substance residue of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer.
The liquid chromatography conditions for detecting the harmful substance residues comprise:
a chromatographic column: agent Eclipse Plus-C18, 2.1mm × 100mm, 1.8 μm;
column temperature: 45 ℃;
flow rate: 0.6 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase: the phase A is ammonium acetate water solution, and the phase B is acetonitrile;
gradient elution order: 0min-3min, 90% A; 3min-7min, 90% -70% A; 7min-8min, 70% -10% A; 8min-11min, 10% A; 11min-11.01min, 10% -90% A; 11.01min-15min, 90% A;
the mass spectrum conditions for detecting the harmful substance residues comprise:
spraying voltage: 4 kV;
the scanning mode is as follows: carrying out multi-reaction monitoring by adopting an electrospray positive ion mode and a negative ion mode;
desolventizing gas temperature: 260 ℃;
temperature of the heating block: 420 ℃;
flow rate of drying gas: 18L/min.
The specific gradient elution order can be seen in table 1.
Example 5
The embodiment of the invention provides a liquid chromatography tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts. The harmful substance residue comprises one or a combination of several of the following components: 2, 4-dichlorophenoxyacetic acid sodium salt, forchlorfenuron, gibberellin, 4-chlorophenoxyacetic acid, thidiazuron, 6-benzyl adenine, 1-naphthylacetic acid, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, sulfadiazine and the like, and sulfadimidine, sulfamonomethoxine, sulfadimidine, metronidazole, chloramphenicol, norfloxacin, ofloxacin, pefloxacin, lomefloxacin, ciprofloxacin, enrofloxacin and the like. Sulfadimethoxine, paclobutrazol, uniconazole, 4-fluorophenoxyacetic acid and isopentenyladenine.
The liquid chromatography tandem mass spectrometry detection method for detecting the residue of harmful substances in bean sprouts comprises the following steps:
s100, extracting bean sprouts to obtain a sample to be detected (the specific steps are shown in example 1);
s200, detecting the harmful substance residue of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer.
Specifically, in step S100, the sample to be tested is obtained by: cutting bean sprouts, fully crushing and uniformly mixing the bean sprouts by using a tissue crusher, accurately weighing 5.0g of sample, placing the sample in a 50mL centrifuge tube with a plug, adding 10g of anhydrous sodium sulfate, immediately carrying out vortex mixing for 1min, adding 20mL of extraction solution, carrying out vortex mixing for 2min, carrying out ultrasound treatment for 15min, centrifuging the sample for 5min at 12000r/min, precisely transferring 10.0mL of supernatant, blowing the supernatant to be dry in 45 ℃ water bath by nitrogen, carrying out volume metering to 1mL by using methanol, carrying out vortex mixing for 1min, filtering the supernatant by using a 0.22 mu m organic phase filter membrane, taking the subsequent filtrate for subsequent detection, and further diluting the subsequent filtrate according to the situation.
In step S200, the liquid chromatography conditions for the harmful substance residue detection include:
a chromatographic column: agent Eclipse Plus-C18, 2.1mm × 100mm, 1.8 μm;
column temperature: 40 ℃;
flow rate: 0.4 mL/min;
sample introduction amount: 1 mu L of the solution;
mobile phase: the phase A is any one of water, a formic acid aqueous solution with the concentration of 0.01mol/L, a formic acid aqueous solution with the concentration of 0.02mol/L, a formic acid aqueous solution with the concentration of 0.1mol/L, an ammonium acetate aqueous solution with the concentration of 0.15mol/L, a mixed solution of the formic acid aqueous solution with the concentration of 0.01mol/L and the ammonium acetate aqueous solution with the concentration of 0.15mol/L, and the phase B is methanol;
gradient elution order: 0min-3min, 90% A; 3min-7min, 90% -70% A; 7min-8min, 70% -10% A; 8min-11min, 10% A; 11min-11.01min, 10% -90% A; 11.01min-15min, 90% A;
the mass spectrum conditions for detecting the harmful substance residues comprise:
spraying voltage: 3.5 kV;
the scanning mode is as follows: carrying out multi-reaction monitoring by adopting an electrospray positive ion mode and a negative ion mode;
desolventizing gas temperature: 250 ℃;
temperature of the heating block: 400 ℃;
flow rate of drying gas: 15L/min.
In step S200, the harmful substance residues are detected using the parameters in table 2.
Performance testing
1. Preparation of Standard solutions
Precisely weighing 27 standard substances (2, 4-dichlorophenoxyacetic acid sodium acetate, forchlorfenuron, gibberellin, 4-chlorophenoxyacetic acid, thidiazuron, 6-benzyladenine, 1-naphthylacetic acid, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, sulfadiazine and the like, and 20mg of each standard substance such as sulfadimidine, sulfamonomethoxine, sulfadimidine, metronidazole, chloramphenicol, norfloxacin, ofloxacin, pefloxacin, lomefloxacin, ciprofloxacin, enrofloxacin and the like, sulfadimidine, paclobutrazol, uniconazole, 4-fluorophenoxyacetic acid and isopenteneadenine) respectively, placing the standard substances into 10mL measuring bottles, treating the standard substances for 15min by ultrasonic waves (power of 250W and working frequency of 40kHz), cooling the standard substances to room temperature, diluting the standard substances to a scale, and shaking the standard substances to obtain standard substance stock solutions. Precisely measuring the standard stock solution in a proper amount, placing the appropriate amount of the standard stock solution into a measuring flask, diluting the standard stock solution with methanol to prepare a mixed standard solution with the concentration of 10-200 mu g/mL, and gradually diluting the mixed standard solution with an initial mobile phase to the mixed standard solution with the concentration of 10, 25, 50, 100 and 200ng/mL (wherein the concentrations of gibberellin and 1-naphthylacetic acid are 100, 250, 500, 1000 and 2000 ng/mL).
2. Testing the Effect of different extraction solvents on recovery
Considering that the 27 plant growth regulators and the antibiotics need a pretreatment method with universality, rapidness and optimized recovery rate for rapid screening, 5 compounds of 4-chlorophenoxyacetic acid, gibberellin, metronidazole, norfloxacin and sulfamonomethoxine with more typical or poorer recovery rate in the 27 plant growth regulators and the antibiotics are selected as the basis, under the condition that the sample has the same addition amount of 20 mu g/kg, different extracting solutions of acetonitrile, acidified acetonitrile and ethyl acetate are respectively inspected, and the results are shown in figure 1. As shown in FIG. 1, when ethyl acetate was used as the extraction solution, the recovery rates of 4-chlorophenoxyacetic acid, gibberellin, metronidazole, norfloxacin and sulfamonomethoxine were stable, and all of them were 80% or more.
3. Testing the Effect of different purification modes and different extraction solvents on recovery
Under the condition that the same extraction reagent and the same addition amount of 20 mu g/kg are used for samples, 5 compounds with relatively typical or poor recovery rate in 27 plant growth regulators and antibiotic drugs are selected as bases, and the influence of three modes, namely positive mixing type cation exchange column purification, QuEChERS centrifuge tube (300mg of anhydrous magnesium sulfate and 100mg of C18) purification and no purification on the recovery rate of the 5 compounds is respectively examined, and the result is shown in figure 2. Because the bean sprout matrix is cleaner, ethyl acetate is selected as an extraction solvent without purification, the recovery rate of the target compound is over 80 percent, the operation is convenient and quick, the recovery rate of the target compound is improved, and the method is more suitable for quickly screening the 27 plant growth regulators and the antibiotics of the bean sprouts in daily inspection.
4. Testing the effects of different flow versus spike out conditions
In a test of mobile phase selection, the invention compares the influence of two organic phases of methanol and acetonitrile on the separation effect of 27 plant growth regulators and antibiotic drugs, and the results show that: when acetonitrile is used as an organic phase, the separation effect of most compounds is slightly better than that of methanol, but the peak time of some compounds such as metronidazole, sulfadiazine and the like is too early and a bifurcation peak is easy to appear, so that the method adopting methanol as the organic phase is a preferred embodiment of the invention.
In addition, the present invention compares the effect of 6A phases (water, 0.01% formic acid aqueous solution, 0.1% formic acid, 0.02% formic acid aqueous solution, 0.15mmol/L ammonium acetate aqueous solution, 0.01% formic acid-0.15 mmol/L ammonium acetate aqueous solution) on the separation effect of 27 plant growth regulators and antibiotic drugs, and the results show that: when pure water solution is used as phase A, most compounds have the phenomenon of bifurcation and tailing. The 0.01% formic acid aqueous solution can obviously improve the quinolone compounds such as ofloxacin and the like, and along with the increase of the content of formic acid in water, although the peak shape of the quinolone compounds can be further improved, the peak response value of 1-naphthylacetic acid is obviously reduced. The 0.15mmol/L ammonium acetate aqueous solution can obviously improve the peak shapes of metronidazole and paclobutrazol, but causes the peak shapes of quinolone compounds such as oxyfloxacin and the like to be poor, the peak area to be greatly reduced, and simultaneously reduces the peak response value of the compounds such as gibberellin and the like under partial negative ion modes. 0.01 percent formic acid-0.15 mmol/L ammonium acetate is used as a water phase, and gibberellin and sodium 2, 4-dichlorophenoxyacetate have low response. The peak areas of the partial compounds in the different mobile phases are shown in Table 3.
In the present invention, in combination with the overall response of 27 plant growth regulators and antibiotic drugs, the principles of 1-naphthylacetic acid, gibberellin, ofloxacin, and 2, 4-dichlorophenoxyacetic acid sodium, which are greatly affected by formic acid and ammonium acetate mobile phases, are preferably considered, and fig. 3 shows the MRM graph of a standard solution obtained under the condition of 0.01% formic acid water-methanol as the mobile phase. Since the partial peak-off time is relatively close, in order to more clearly show the peak-off condition of each channel, a baseline shift of 7% in the horizontal direction was adopted when data analysis was performed using Labsolutions software (shimadzu corporation), and the final result is shown in fig. 3.
Table 1 mobile phase gradient elution procedure
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 3.00 | 90 | 10 |
| 7.00 | 70 | 30 |
| 8.00 | 10 | 90 |
| 11.00 | 10 | 90 |
| 11.01 | 90 | 10 |
| 15.00 | Stop |
Table 2 quantitative ion, qualitative ion, collision energy and retention time for harmful substance residue detection
*Quantitative ion pair, ESI +: positive ion mode ESI-: negative ion mode
TABLE 3 influence of A on the peak area of the compounds
In conclusion, the invention finds that plant growth regulators and antibiotic drugs added in the bean sprouts are relatively complex by detecting a large amount of bean sprouts, and the range of target compounds is determined and expanded again from relevant documents and practical working experience. Except for the compounds such as 4-chlorophenoxyacetic acid, 6-benzyladenine, gibberellin, enrofloxacin, ofloxacin and metronidazole which are the recommended standards, two compounds such as thidiazuron and 1-naphthylacetic acid are also detected in 259 batches of bean sprouts. The method can complete qualitative and quantitative determination of 27 target compounds within 15min, and compared with the conventional recommended method (detecting 11 compounds in the same category at most at one time), the method has the advantages of larger detection capacity and wider application range, and is more suitable for rapid emergency disposal of safety risk emergencies in bean sprout foods.
It should be noted that: description of the drawings figure 3 is merely an "exemplary value" provided by the present invention and represents one of many "exemplary values" of the present invention, which are presented herein as a form of illustration only and are not meant to be limiting in any way.
Exemplary embodiments of the present invention have been described in detail with reference to the preferred embodiments, however, it will be understood by those skilled in the art that various changes and modifications may be made to the specific embodiments described above and various combinations of the various features and structures proposed by the present invention may be made without departing from the concept of the present invention, the scope of which is defined by the appended claims.
Claims (10)
1. A liquid chromatogram tandem mass spectrum detection method for detecting harmful substance residues in bean sprouts is characterized by comprising the following steps:
s100, extracting bean sprouts to obtain a sample to be detected;
s200, detecting harmful substance residues of the sample to be detected obtained in the step S100 by adopting a liquid chromatogram tandem mass spectrometer;
wherein, the liquid chromatogram condition for harmful substance residue detection comprises:
a chromatographic column: 2.1mm × 100mm, 1.8 μm;
column temperature: 35-45 ℃;
flow rate: 0.2mL/min-0.6 mL/min;
sample introduction amount: 0.1-10 μ L;
mobile phase: the phase A is any one of water, formic acid aqueous solution and ammonium acetate aqueous solution, and the phase B is any one of methanol or acetonitrile;
gradient elution order: 0min-3min, 90% A; 3min-7min, 90% -70% A; 7min-8min, 70% -10% A; 8min-11min, 10% A; 11min-11.01min, 10% -90% A; 11.01min-15min, 90% A;
the mass spectrum conditions for detecting the harmful substance residues comprise:
spraying voltage: 3kV-4 kV;
the scanning mode is as follows: carrying out multi-reaction monitoring by adopting an electrospray positive ion mode and a negative ion mode;
desolventizing gas temperature: 240-260 ℃;
temperature of the heating block: 380-420 ℃;
flow rate of drying gas: 12L/min-18L/min.
2. The method for detecting harmful substance residues in bean sprouts according to claim 1, wherein the step S100 specifically comprises the following steps:
s101, crushing bean sprouts, adding the crushed bean sprouts into anhydrous sodium sulfate, and uniformly mixing to obtain a first mixture;
s102, adding an extracting solution into the first mixture obtained in the step S101, uniformly mixing, and performing centrifugal treatment to obtain a supernatant;
s103, drying the supernatant obtained in the step S102 at the temperature of 40-50 ℃ in a water bath by adopting nitrogen to obtain dried supernatant;
s104, carrying out constant volume on the dried supernatant obtained in the step S103 by adopting methanol to obtain a constant volume sample;
and S105, filtering the constant volume sample obtained in the step S104 by adopting an organic phase filter membrane to obtain a sample to be detected.
3. The liquid chromatography-tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts according to claim 2, wherein,
in the step S101, the mass ratio of the bean sprouts to the anhydrous sodium sulfate is bean sprouts: anhydrous sodium sulfate ═ (0.4-0.6): 1.
4. the liquid chromatography-tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts according to claim 2, wherein,
in the step S102, the mass-to-volume ratio of the bean sprouts and the extracting solution is 0.2g/ml-0.3 g/ml;
the extracting solution is one of the following: acetonitrile, acidified acetonitrile, ethyl acetate.
5. The method for detecting harmful substance residues in bean sprouts according to claim 2, further comprising after the step S105:
s106, purifying the sample to be detected obtained in the step S105;
wherein the purification is positive mixing type cation exchange column purification or centrifugal tube purification.
6. The liquid chromatography-tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts according to claim 1,
in the step S200, the column temperature is 40 ℃; the flow rate is 0.4 mL/min; the sample injection amount is 1 mu L; the spraying voltage is 3.5 kV; the temperature of the desolventizing gas is 250 ℃; the temperature of the heating block is 400 ℃; the drying airflow rate was 15L/min.
7. The liquid chromatography-tandem mass spectrometry detection method for detecting bean sprouts harmful substance residues according to any one of claims 1 to 6,
in the step S200, the phase A is any one of water, a 0.01mol/L formic acid aqueous solution, a 0.02mol/L formic acid aqueous solution, a 0.1mol/L formic acid aqueous solution, a 0.15mol/L ammonium acetate aqueous solution, a 0.01mol/L formic acid aqueous solution and a 0.15mol/L ammonium acetate aqueous solution.
8. The method for detecting the bean sprouts harmful substance residues according to any one of claims 1 to 6, wherein the harmful substance residues comprise one or a combination of the following:
2, 4-dichlorophenoxyacetic acid sodium salt, forchlorfenuron, gibberellin, 4-chlorophenoxyacetic acid, thidiazuron, 6-benzyl adenine, 1-naphthylacetic acid, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, sulfadiazine and the like, and sulfadimidine, sulfamonomethoxine, sulfadimidine, metronidazole, chloramphenicol, norfloxacin, ofloxacin, pefloxacin, lomefloxacin, ciprofloxacin, enrofloxacin and the like. Sulfadimethoxine, paclobutrazol, uniconazole, 4-fluorophenoxyacetic acid and isopentenyladenine.
9. The liquid chromatography-tandem mass spectrometry detection method for detecting harmful substance residues in bean sprouts according to claim 8,
in the step S200, scanning the thidiazuron, the sodium 2, 4-dichlorophenoxyacetate, the forchlorfenuron, the gibberellin, the 6-benzyladenine, the 4-chlorophenoxyacetic acid, the 4-fluorophenoxyacetic acid, the isopentenyladenine, the 1-naphthylacetic acid and the chloramphenicol in a negative ion mode; and/or
In said step S200, said metronidazole, said paclobutrazol, said sulfamethazine, said sulfamethoxazole, said sulfaquinoxaline, said norfloxacin, said ofloxacin, said pefloxacin, said lomefloxacin, said ciprofloxacin, said enrofloxacin, said sulfadiazine, said sulfadimidine, said sulfamethazine, said sulfadimethoxine, said sulfamonomethoxine, said uniconazole are scanned with a positive ion pattern.
10. The method of claim 8, wherein in the step S200, the mass spectrometric conditions further comprise detecting each harmful substance residue by using the following parameters:
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