Method for detecting diazepam residual quantity in aquatic product based on magnetic solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting the residual quantity of diazepam in aquatic products based on magnetic solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry. The method comprises the following steps: (1) pretreating a sample; (2) magnetic solid-phase extraction and purification; and (3) carrying out UPLC-MS / MS analysis. The method for detecting the diazepam residual quantity in the aquatic product based on the magnetic solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry is established, the method is easy to operate, high in sensitivity, good in purification effect and high in precision and accuracy, and the requirements for accurate qualitative and quantitative analysis of the diazepam residual quantity in the aquatic product can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of diazepam detection, and in particular to a method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry. Background Art

[0002] Diazepam, commonly known as diazepam, is a benzodiazepine sedative-hypnotic drug widely used to treat anxiety, sedation, insomnia, convulsions, epilepsy, and central nervous system muscle relaxants. Diazepam has been shown to increase meat yield, reduce animal mobility, and reduce stress. Diazepam is poorly metabolized by the animal body, accumulating in food animals. Long-term consumption of foods containing diazepam and its metabolites poses a health hazard to humans. Animal excrement enters the soil and water, causing ecological and environmental problems and indirectly harming human health. This is detrimental to the sustainable development of the aquatic products industry and poses a serious threat to food safety. Therefore, rapid and efficient detection methods are crucial for monitoring diazepam residues in aquatic products. Rapid and accurate quantification of trace targets in complex aquatic products is a challenging task, necessitating sample pretreatment to adsorb and purify the target compound before quantitative analysis to reduce matrix interference.

[0003] Chinese patent application publication number CN118376708A discloses a method for pretreating aquatic product samples and detecting residual diazepam in aquatic products. The sample is extracted with ethyl acetate alkalized with sodium hydroxide, then purified by vortexing, sonication, centrifugation, and QuEChERS cleanup tubes. The sample is then analyzed by UPLC-MS / MS, using an internal standard method for quantification. This detection method uses ethyl acetate alkalized with sodium hydroxide for extraction. However, ethyl acetate is volatile and highly irritating, and is prone to emulsification when extracting samples containing a high amount of fat-soluble impurities, affecting the extraction efficiency and recovery rate of the target compound. Furthermore, the extract requires multiple purification steps (vortexing, sonication, and solid-liquid separation), making the process cumbersome. Summary of the Invention

[0004] The present invention aims to solve the above-mentioned problems existing in the existing methods for detecting diazepam residues in aquatic products, and provides a method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry, which has a safe process, is environmentally friendly, simple to operate, and has high sensitivity, accuracy and precision.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: A method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry of the present invention comprises the following steps: (1) Sample pretreatment The muscle tissue of the aquatic product was minced and used directly or frozen, then removed and thawed before analysis.

[0006] (2) Magnetic solid phase extraction purification Weigh an appropriate amount of sample into a centrifuge tube, add ammonia acetonitrile solution to obtain a mixture; vortex the mixture, let it stand, add magnesium sulfate and sodium chloride, shake it and centrifuge it, and transfer the upper acetonitrile phase to the medium containing Fe3O4-PSA magnetic nanoparticles and C 18 The powder is purified in a centrifuge tube. After adsorption and oscillation, the sample is magnetically separated in an external magnetic field. The liquid is filtered and used as the reagent for the instrument. Sample purification based on Fe3O4-PSA magnetic nanoparticles simplifies and accelerates sample pretreatment, significantly reducing matrix interference in aquatic samples. Using external standard calibration under a solvent matching curve, quantitative analysis achieves excellent measurement accuracy and precision, reducing testing costs.

[0007] (3) UPLC-MS / MS analysis The solution was transferred to an injection bottle for analysis by UPLC-MS / MS. The chromatographic peak area of diazepam was compared with a diazepam standard curve obtained by the external standard method, and the actual diazepam concentration in the sample was calculated by conversion. The diazepam standard curve can be obtained using conventional standard curve preparation steps. In the present invention, 1, 2.5, 5, 20, and 50 μL of a diazepam standard working solution with a mass concentration of 100 ng / mL were respectively aspirated, and 999, 997.5, 995, 980, and 950 μL of a negative sample solution were added, respectively. After mixing, the solution was filtered through a 0.22 μm filter to prepare a series of calibration solutions with mass concentrations of 0.1, 0.25, 0.5, 2, and 5 ng / mL, respectively. The series of calibration solutions were then used to obtain a diazepam standard curve.

[0008] Preferably, in step (1), the freezing temperature is -18°C.

[0009] Preferably, in step (2), 5 g of sample is weighed into a 50 mL centrifuge tube; 15 mL of 1% ammonia acetonitrile solution is added; 5 g of magnesium sulfate and 1.25 g of sodium chloride are added; the upper acetonitrile phase is transferred to a tube containing 30 mg of Fe3O4-PSA magnetic nanoparticles and 30 mg of C 18The powder was purified in a 2mL centrifuge tube and shaken for 3 minutes; the liquid was filtered through a 0.22μm filter membrane. Diazepam is a fat-soluble substance. The commonly used extraction solvents are mainly acetonitrile and ethyl acetate. Considering that ethyl acetate is volatile and highly irritating, and is prone to emulsification when extracting samples with more fat-soluble impurities, acetonitrile is used as an extraction agent in the present invention to reduce background interference; the inventors further compared acetonitrile, 1.0% (v / v) formic acid acetonitrile and 1.0% (v / v) ammonia water (mass concentration 25% ammonia water) acetonitrile solution as sample extraction solvents to examine the recovery rate of the target object, and found that under the same conditions, the use of 1.0% ammonia water acetonitrile to extract the target object had a good extraction effect and a higher recovery rate. This may be because diazepam is a weakly alkaline fat-soluble chemical. The compound is relatively stable under weak alkaline conditions, and acetonitrile creates a weak alkaline extraction environment by adding ammonia water. Based on the principle of like dissolves like, diazepam has a strong affinity with 1.0% (v / v) ammonia water and acetonitrile. Therefore, the present invention ultimately selects 1.0% (v / v) ammonia water and acetonitrile solution as the sample extraction solvent. After determining the sample extraction solvent, the inventors experimentally verified the extraction effect of 8mL, 10mL, 15mL, 20mL and 25mL of 1.0% ammonia water (25% ammonia water) and acetonitrile to determine the optimal amount of extraction solvent. The experimental results show that with the increase of the extraction solvent volume, the recovery rate is significantly improved (p < 0.001), which may be because diazepam has a high solubility in ammonia and acetonitrile. Increasing the volume of the extraction solvent can dissolve more diazepam, thereby pushing more diazepam into the organic phase, thereby improving the recovery rate; but considering the cost of the extraction solvent and the detection efficiency, the optimal amount of the extraction solvent finally selected in the present invention is 15mL; the matrix of aquatic products is complex and contains a large amount of water, protein, fat and other components. In the detection of drug residues in aquatic products, magnesium sulfate and sodium chloride are the core purifiers of the QuEChERS method. Magnesium sulfate is responsible for dehydration and adsorption of polar impurities, and sodium chloride separates the organic phase and the aqueous phase through salting out and inhibits emulsification. The combination of the two can achieve better The purification effect is good, so the inventors investigated the effects of 4g, 5g, 6g, 7g and 8g of magnesium sulfate on the recovery rate of the target through experiments. The experimental results show that when the dosage of magnesium sulfate is 4g, the recovery rate is slightly higher than 100%, indicating that the purification may not be thorough; when the dosage is 5g, the recovery rate is close to the ideal value, indicating that the purification effect is good; and when the dosage is further increased to 6g, the recovery rate is slightly higher than 100% again, which may be a slight matrix interference caused by the increase in dosage; when the dosage is further increased to 7g and 8g, the effect is equivalent to that of 5g, and the recovery rate tends to be stable, indicating that the dosage of magnesium sulfate has reached saturation, so 5g of magnesium sulfate is finally added; the aquatic product matrix The sample is rich in protein and fat. When extracting the target compound, other substances such as lipids and proteins will also be extracted at the same time, which will interfere with the determination of the sample and affect the accuracy of the determination results. Therefore, the sample extract must be purified. The Fe3O4-PSA magnetic nanoparticles in the present invention are ideal adsorbents with excellent selective adsorption and elution properties. They can adsorb and purify the target to reduce matrix interference and can be separated by magnetic force. The separation is very convenient. The inventors experimentally investigated 0mg, 10mg, 20mg, 30mg, 40mg and 50mg of Fe3O4-PSA magnetic nanoparticles. The effect of dosage on the recovery of the target substance. Experimental data showed that the dosage of Fe3O4-PSANPS had a significant effect on the recovery of diazepam (p<0.001). When Fe3O4-PSA magnetic nanoparticles were not added, the matrix interference was serious and the recovery rate was significantly higher. When the addition amount was 30 mg, the recovery rate reached 99.06% and tended to be stable. This may be because the Fe3O4-PSA magnetic nanoparticles reached the maximum adsorption capacity. Therefore, 30 mg of Fe3O4-PSA magnetic nanoparticles were subsequently added. The investigation of adsorption oscillation time is also a necessary process in the extraction process because it directly affects the contact time between the analyte and the adsorbent. Oscillation time also affects the adsorption of Fe3O4-PSA magnetic nanoparticles. The inventors investigated different oscillation times of 0.5 min, 1 min, 3 min, and 5 min. The results showed that when the oscillation time was 3 min, the recovery rate was the highest (89.86%), indicating that this oscillation time can better balance the release of the target and the extraction efficiency; if the oscillation time is too short (such as 0.5min or 1min), the target is not fully released, resulting in a low recovery rate; if the oscillation time is too long (such as 5min), it may cause degradation or increased adsorption of the target. Therefore, 3min is selected as the optimal oscillation time in this invention.

[0010] Preferably, in step (2), the Fe3O4-PSA magnetic nanoparticles are prepared by the following method: dissolving sodium citrate in ethylene glycol, adding ferric chloride ethylene glycol solution and sodium acetate ethylene glycol solution in sequence, stirring vigorously, placing the mixture in a sealed reactor, heating to 180°C for heat-insulating reaction, cooling to room temperature after the reaction, magnetically separating the product, washing with water and ethanol respectively, and vacuum drying to obtain Fe3O4 nanoparticles; dispersing the Fe3O4 nanoparticles in water, adding to a mixture of hexadecyltrimethylammonium bromide and water, adding ammonia water and stirring vigorously, and then adding the Fe3O4 nanoparticles to obtain Fe3O4 nanoparticles. Stir, add tetraethyl orthosilicate and ethanol dropwise under a nitrogen atmosphere, heat to 60°C for reaction, separate the product magnetically after the reaction, wash with acetone and ethanol respectively, and dry in vacuum to obtain magnetic Fe3O4 / SiO2; mix the magnetic Fe3O4 / SiO2 with anhydrous toluene, add N,N-diethyl-3-(trimethoxysilyl)propylamine, anhydrous toluene and diethylamine under a nitrogen atmosphere, stir at room temperature and heat to 65°C for reaction, separate the product magnetically after the reaction, wash with acetone, ethanol and water respectively, and dry in vacuum to obtain Fe3O4-PSA magnetic nanoparticles.

[0011] Preferably, 0.4 g of sodium citrate is dissolved in 72 mL of ethylene glycol, and 16 mL of ferric chloride ethylene glycol solution and 72 mL of sodium acetate ethylene glycol solution are added in sequence; the mixture is heated to 180° C. and kept warm for 10 hours; 1 g of Fe3O4 nanoparticles is dispersed in 100 mL of water; 65 μL of 25% ammonia water is added and stirred vigorously; 2.5 mL of tetraethyl orthosilicate and 1.4 mL of ethanol are added; 0.6 g of Fe3O4 / SiO2 and 250 mL of anhydrous toluene are mixed; 3 mL of N,N-diethyl-3-(trimethoxysilyl)propylamine, 3 mL of anhydrous toluene and 1 mL of diethylamine are added.

[0012] Preferably, the ferric chloride ethylene glycol solution is prepared by mixing 1g of ferric chloride hexahydrate with 50mL of ethylene glycol; and the sodium acetate ethylene glycol solution is prepared by mixing 10g of sodium acetate with 72mL of ethylene glycol.

[0013] As a preference, in step (3), the chromatographic conditions are: AcquityUPLCBEHC 18Chromatographic column, column size: 2.1 mm × 100 mm, filler particle size: 1.7 μm; column temperature: 40°C; injection volume: 5 μL; 0.1% formic acid-2 mM ammonium acetate solution as mobile phase A, methanol as mobile phase B, flow rate: 0.3 mL / min, gradient elution program: 1 min, 90.0% A, 10.0% B; 3 min, 90.0% A, 10.0% B; 3.5min, 85.0%A, 15.0%B; 6 min, 32.0% A, 68.0% B; 6.2min, 5.0%A, 95.0%B; 7.8min, 5.0%A, 95.0%B; 8 min, 90.0% A, 10.0% B; 10min, 90.0%A, 10.0%B.

[0014] Preferably, in step (3), the mass spectrometry conditions are: electrospray ion source; positive ion scanning; multiple reaction monitoring; capillary voltage: 3.5 kV; ion source temperature: 120°C; desolvation gas temperature: 380°C; cone gas and desolvation gas: nitrogen, purity 99.9%; collision gas: argon, purity 99.999%; cone gas flow rate: 50 L / h; desolvation gas flow rate: 600 L / h.

[0015] Therefore, the present invention has the following beneficial effects: a method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry is established, which has simple operation, high sensitivity, good purification effect, high precision and accuracy, and can meet the requirements for accurate qualitative and quantitative analysis of diazepam residues in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the matrix effect of diazepam in swimming crab, crucian carp, Chinese shrimp and mussel.

[0017] Figure 2 This is the MRM chromatogram of a blank crucian carp sample spiked with diazepam and measured on the analyzer. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0019] 1.1 Materials and Reagents Formic acid, methanol, acetonitrile, ammonium acetate, chromatographic grade, Merck, Germany; anhydrous magnesium sulfate, sodium citrate (analytical grade), ethylene glycol (analytical grade), ethanol (analytical grade), hexadecyltrimethylammonium bromide (analytical grade), ammonia (analytical grade), tetraethyl orthosilicate (analytical grade), acetone (analytical grade), toluene (analytical grade), diethylamine (analytical grade), Shanghai Sinopharm Group; ferric chloride (analytical grade), N,N-diethyl-3-(trimethoxysilyl)propylamine (analytical grade), Shanghai Aladdin Industrial Co., Ltd.

[0020] 1.2 Instruments and Equipment ACQUITY™ Ultra-High Performance Liquid Chromatograph, ACQUITY UPLC BEH C 18 Chromatographic column (2.1 mm × 100 mm, 1.7 μm), Waters, USA; Centrifuge 5810 high-speed centrifuge (Eppendorf, Germany); MX-S vortex mixer, Scilogex, USA; ME204E / 02 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.).

[0021] Chromatographic conditions: Acquity UPLC BEH C 18 Chromatographic column, column size: 2.1 mm × 100 mm, filler particle size: 1.7 μm; column temperature: 40°C; injection volume: 5 μL; 0.1% formic acid-2 mM ammonium acetate solution as mobile phase A, methanol as mobile phase B, flow rate: 0.3 mL / min, gradient elution program: 1 min, 90.0% A, 10.0% B; 3 min, 90.0% A, 10.0% B; 3.5min, 85.0%A, 15.0%B; 6 min, 32.0% A, 68.0% B; 6.2min, 5.0%A, 95.0%B; 7.8min, 5.0%A, 95.0%B; 8 min, 90.0% A, 10.0% B; 10min, 90.0%A, 10.0%B; The mass spectrometry conditions were as follows: electrospray ion source; positive ion scanning; multiple reaction monitoring; capillary voltage: 3.5 kV; ion source temperature: 120°C; desolvation gas temperature: 380°C; cone gas and desolvation gas: nitrogen, purity 99.9%; collision gas: argon, purity 99.999%; cone gas flow rate: 50 L / h; desolvation gas flow rate: 600 L / h.

[0022] 1. Method Validation 1. Matrix Effect Matrix effects primarily arise from sample pretreatment and the ionization phase of detection. Incomplete pretreatment purification can interfere with the ionization efficiency of the analyte, causing an increase or decrease in the analyte signal and, consequently, affecting the accuracy of the assay. Therefore, assessing the impact of matrix effects is essential during experimental validation.

[0023] The determination method and evaluation criteria of matrix effect refer to the methods in the literature, and the calculation formula is as follows: E M =(S m / Ss-1)×100%, where: E M ——Matrix effect; S m ——the slope of the linear equation of the matrix calibration curve; Ss——the slope of the linear equation of the solvent standard curve.

[0024] When the matrix effect is less than 0, it indicates a matrix inhibition effect; when the matrix effect is greater than 0, it indicates a matrix enhancement effect; when the absolute value of the matrix effect is 0% to 20%, it is a weak matrix effect; when the absolute value of the matrix effect is 20% to 50%, it is a moderate matrix effect; and when the absolute value of the matrix effect is greater than 50%, it is a strong matrix effect.

[0025] Since diazepam internal standard is generally expensive, the recovery rate was corrected by external standard method. The matrix effect of swimming crab, crucian carp, Chinese whip shrimp and mussel was evaluated by comparing the slope of the calibration curve in matrix and solvent. Figure 1 As shown in the figure, the matrix effect of diazepam in the four aquatic products ranges from -18.3% to -4.27%, indicating a weak matrix effect. This indicates that this method has a good purification effect on the four matrices and a low degree of matrix interference. The MRM chromatogram of the blank crucian carp sample spiked with diazepam (0.5 μg / kg) is shown in the figure. Figure 2 As shown, the results show that at the analyte peak at 5.66 min, there is no matrix impurity peak contamination, the separation is high and the chromatographic peak shape is sharp.

[0026] 2. Linear range, detection limit and quantification limit Prepare standard solution: Accurately pipette 10.00 mL (100 mg / L) of diazepam standard substance into a 50 mL volumetric flask, dilute to the mark with methanol, and prepare a standard intermediate solution with a mass concentration of 20 μg / mL; take an appropriate amount of the standard intermediate solution and dilute it stepwise with methanol to obtain a diazepam standard working solution with a mass concentration of 100 ng / mL, which is prepared and used immediately.

[0027] Prepare a series of sample matrix calibration solutions: 1, 2.5, 5, 20, and 50 μL of diazepam standard working solution were drawn, and 999, 997.5, 995, 980, and 950 μL of negative sample solution were added, respectively. After mixing, the solution was filtered through a 0.22 μm filter to prepare a series of calibration solutions with mass concentrations of 0.1, 0.25, 0.5, 2, and 5 ng / mL, respectively. The standard curve was drawn based on the peak area (y) and the corresponding concentration (x) of the analyte. The limit of detection (LOD) and limit of quantification (LOQ) of the method were determined using 3 times and 10 times the signal-to-noise ratio, respectively. The results showed that diazepam had good linearity in the linear range of 0.1-10 μg / L. The linear equation was y=45864.4x±4024.07, and the linear correlation coefficient (r 2 ) was 0.9983, the detection limit was 0.25 μg / kg, and the quantification limit was 0.5 μg / kg, which showed that this method had high sensitivity.

[0028] 3. Precision and recovery A spike recovery experiment was conducted with crucian carp, Chinese prawn, swimming crab, and mussel. Diazepam standard solutions at three levels (1.5, 6, and 15 μg / kg) were added to the experimental samples, respectively. Three replicates were set for each spike concentration, and a blank experiment was performed. The recovery rate and relative standard deviation were calculated. The calculation results are shown in Table 1.

[0029] Table 1 Recovery and relative standard deviation of diazepam in aquatic products As can be seen from Table 1, the recoveries of the method were 89.7% to 108.8%, and the relative standard deviations were 2.7% to 11.6%, indicating that the accuracy and precision of the method were good.

[0030] Example 1 (1) Sample pretreatment The muscle tissue of crucian carp (collected from aquaculture farm in Zhoushan, Zhejiang) was minced to obtain a sample.

[0031] (2) Magnetic solid phase extraction purification 5 g of sample was weighed into a 50 mL centrifuge tube, and 15 mL of 1% (V / V) ammonia water (mass concentration 25%) acetonitrile solution was added to obtain a mixture; the mixture was vortexed for 1 min, allowed to stand for 30 min, 5 g of magnesium sulfate and 1.25 g of sodium chloride were added, and after oscillation adsorption for 3 min, centrifuged at 5000 rpm for 3 min, and 2 mL of the upper acetonitrile phase was transferred to a tube containing 30 mg of Fe3O4-PSA magnetic nanoparticles and 30 mg of C 18The powder was purified in a 2mL centrifuge tube. After the sample was shaken for 3 minutes, it was magnetically separated under an external magnetic field. 1.0mL of the upper liquid was aspirated with a syringe and filtered through a 0.22μm filter membrane and transferred to an injection bottle as the upper solution; wherein Fe3O4-PSA magnetic nanoparticles were prepared by the following method: 0.4g of sodium citrate was dissolved in 72mL of ethylene glycol, and 16mL of ferric chloride ethylene glycol solution (mixed in a ratio of 1g of ferric chloride hexahydrate: 50mL of ethylene glycol) and 72mL of sodium acetate ethylene glycol solution (mixed by 10g of sodium acetate and 72mL of ethylene glycol) were added in sequence. After vigorous stirring for 30min, the mixture was placed in a sealed reactor, heated to 180℃ and kept warm for 10h. After the reaction, it was cooled to room temperature, and the product was magnetically separated, washed three times with water and ethanol respectively, and dried in a vacuum at 60℃ to obtain Fe3O4 nanoparticles; 1g of Fe3O4 nanoparticles was dispersed in 1 00mL water was then added to a mixture of hexadecyltrimethylammonium bromide and water (2g hexadecyltrimethylammonium bromide + 200mL water), 65μL of 25% ammonia water was added and stirred vigorously, 2.5mL of tetraethyl orthosilicate and 1.4mL of ethanol were added dropwise under a nitrogen atmosphere, and the mixture was heated to 60℃ and kept warm for 12h. After the reaction, the product was magnetically separated and washed three times with acetone and ethanol respectively, and dried in a vacuum at 60℃ to obtain magnetic Fe3O4 / SiO2; 0.6g of magnetic Fe3O4 / SiO2 was mixed with 250mL of anhydrous toluene, 3mL of N,N-diethyl-3-(trimethoxysilyl)propylamine, 3mL of anhydrous toluene and 1mL of diethylamine were added under a nitrogen atmosphere, stirred at room temperature for 30min, and then heated to 65℃ and kept warm for 6h. After the reaction, the product was magnetically separated and washed three times with acetone, ethanol and water respectively, and dried in a vacuum overnight to obtain Fe3O4-PSA magnetic nanoparticles.

[0032] (3) UPLC-MS / MS analysis The solution was transferred to an injection bottle and analyzed by UPLC-MS / MS. The chromatographic peak area of diazepam was substituted into the linear equation of the diazepam standard curve obtained by the external standard method. The actual concentration of diazepam in the sample was calculated to be 2.25 μg / kg. The chromatographic conditions were: Acquity UPLC BEHC 18 Chromatographic column, column size: 2.1 mm × 100 mm, filler particle size: 1.7 μm; column temperature: 40°C; injection volume: 5 μL; 0.1% formic acid-2 mM ammonium acetate solution as mobile phase A, methanol as mobile phase B, flow rate: 0.3 mL / min, gradient elution program: 1 min, 90.0% A, 10.0% B; 3 min, 90.0% A, 10.0% B; 3.5min, 85.0%A, 15.0%B; 6 min, 32.0% A, 68.0% B; 6.2min, 5.0%A, 95.0%B; 7.8min, 5.0%A, 95.0%B; 8 min, 90.0% A, 10.0% B; 10min, 90.0%A, 10.0%B; The mass spectrometry conditions were as follows: electrospray ion source; positive ion scanning; multiple reaction monitoring; capillary voltage: 3.5 kV; ion source temperature: 120°C; desolvation gas temperature: 380°C; cone gas and desolvation gas: nitrogen, purity 99.9%; collision gas: argon, purity 99.999%; cone gas flow rate: 50 L / h; desolvation gas flow rate: 600 L / h.

[0033] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. A method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry, characterized in that: The following steps are involved: (1) Sample pretreatment The muscle tissue of the aquatic product was minced and used directly or frozen, and then removed and thawed before analysis; (2) Magnetic solid phase extraction purification Weigh an appropriate amount of sample into a centrifuge tube, add ammonia acetonitrile solution to obtain a mixture; vortex the mixture, let it stand, add magnesium sulfate and sodium chloride, shake it and centrifuge it, and transfer the upper acetonitrile phase to the medium containing Fe3O4-PSA magnetic nanoparticles and C 18 Purify the powder in a centrifuge tube, shake the sample, and then perform magnetic separation on the sample under an external magnetic field. Take the liquid and filter it as the solution for the machine; (3) UPLC-MS / MS analysis The solution was transferred to an injection bottle for analysis by UPLC-MS / MS. The obtained chromatographic peak area of diazepam was substituted into the linear equation of the diazepam standard curve obtained by the external standard method, and the actual concentration of diazepam in the sample was obtained by conversion.

2. The method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step (1), the freezing temperature is -18°C.

3. The method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step (2), 5 g of sample was weighed into a 50 mL centrifuge tube; 15 mL of 1% ammonia acetonitrile solution was added; 5 g of magnesium sulfate and 1.25 g of sodium chloride were added; the upper acetonitrile phase was transferred to a tube containing 30 mg of Fe3O4-PSA magnetic nanoparticles and 30 mg of C 18 The powder was purified in a 2 mL centrifuge tube and shaken for 3 min; the liquid was filtered through a 0.22 μm filter membrane.

4. The method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step (2), the Fe3O4-PSA magnetic nanoparticles are prepared by the following method: dissolving sodium citrate in ethylene glycol, adding ferric chloride ethylene glycol solution and sodium acetate ethylene glycol solution in sequence, stirring vigorously, placing the mixture in a sealed reactor, heating to 180°C for heat preservation and reaction, cooling to room temperature after the reaction, magnetically separating the product, washing with water and ethanol respectively, and vacuum drying to obtain Fe3O4 nanoparticles; dispersing the Fe3O4 nanoparticles in water, adding to a mixture of hexadecyltrimethylammonium bromide and water, adding ammonia water and stirring vigorously, and Tetraethyl orthosilicate and ethanol were added dropwise under a nitrogen atmosphere, and the mixture was heated to 60°C for reaction. After the reaction, the product was magnetically separated, washed with acetone and ethanol, respectively, and dried in vacuum to obtain magnetic Fe3O4 / SiO2. After the magnetic Fe3O4 / SiO2 was mixed with anhydrous toluene, N,N-diethyl-3-(trimethoxysilyl)propylamine, anhydrous toluene and diethylamine were added under a nitrogen atmosphere. After stirring at room temperature, the mixture was heated to 65°C for reaction. After the reaction, the product was magnetically separated, washed with acetone, ethanol and water, respectively, and dried in vacuum to obtain Fe3O4-PSA magnetic nanoparticles.

5. The method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry according to claim 4, characterized in that: Dissolve 0.4 g of sodium citrate in 72 mL of ethylene glycol, and add 16 mL of ferric chloride ethylene glycol solution and 72 mL of sodium acetate ethylene glycol solution in sequence; heat to 180°C and keep the reaction for 10 hours; disperse 1 g of Fe3O4 nanoparticles in 100 mL of water; add 65 μL of 25% ammonia water and stir vigorously; add 2.5 mL of tetraethyl orthosilicate and 1.4 mL of ethanol; mix 0.6 g of Fe3O4 / SiO2 and 250 mL of anhydrous toluene; add 3 mL of N,N-diethyl-3-(trimethoxysilyl)propylamine, 3 mL of anhydrous toluene and 1 mL of diethylamine.

6. The method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry according to claim 5, characterized in that: The ferric chloride ethylene glycol solution is prepared by mixing 1 g of ferric chloride hexahydrate with 50 mL of ethylene glycol; the sodium acetate ethylene glycol solution is prepared by mixing 10 g of sodium acetate with 72 mL of ethylene glycol.

7. The method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step (3), the chromatographic conditions are: Acquity UPLC BEH C 18 Chromatographic column, column size 2.1mm×100mm, filler particle size 1.7μm; Column temperature: 40°C; injection volume: 5 μL; 0.1% formic acid-2 mM ammonium acetate solution as mobile phase A, methanol as mobile phase B, flow rate 0.3 mL / min, gradient elution program settings: 1 min, 90.0% A, 10.0% B; 3 min, 90.0% A, 10.0% B; 3.5min, 85.0%A, 15.0%B; 6 min, 32.0% A, 68.0% B; 6.2min, 5.0%A, 95.0%B; 7.8min, 5.0%A, 95.0%B; 8 min, 90.0% A, 10.0% B; 10min, 90.0%A, 10.0%B.

8. The method for detecting diazepam residues in aquatic products based on magnetic solid phase extraction-ultra-performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step (3), the mass spectrometry conditions are: electrospray ionization source; positive ion scanning; multiple reaction monitoring; capillary voltage: 3.5 kV; ion source temperature: 120°C; Desolvation gas temperature: 380°C; cone gas and desolvation gas: nitrogen, purity 99.9%; collision gas: argon, purity 99.999%; cone gas flow rate: 50 L / h; desolvation gas flow rate: 600 L / h.

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