Analysis method for detecting sulfamethoxazole antibiotic residue in fish body

Abstract

The invention provides an analysis method for detecting sulfamethoxazole antibiotic residues in fish bodies. Comprising the following steps: (1) preparing a sample to be tested: mixing a nemipterus virgatus sample with anhydrous sodium sulfate, adding an acidified acetonitrile solution for purification and extraction to obtain a nemipterus virgatus sample dissolved residue, and mixing the nemipterus virgatus sample dissolved residue with a reconstitution fluid to prepare the sample to be tested; and (2) determining the sample to be tested by using a liquid chromatography-tandem mass spectrometer, and the analysis method can significantly improve the recovery rate of the target object and reduce the detection limit of the target object, so that the detection method is more accurate, rapid, efficient and sensitive.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to an analytical method for detecting sulfamethoxazole antibiotic residues in fish. Background Technology

[0002] Sulfamethoxazole (SMZ), also known as sulfamethoxazole, is a representative of sulfonamide antibiotics. Due to its broad antibacterial spectrum and low cost, it is widely used in marine aquaculture for the prevention and treatment of bacterial diseases. However, SMZ has a relatively long half-life in fish muscle and liver (t1 / 2 ≈ 16–30 h). Excessive or prolonged use can easily lead to residues in edible parts, potentially inducing allergic reactions, folic acid metabolism disorders, and the spread of drug resistance genes in humans. Golden threadfin bream, an important economic food fish in southeastern coastal my country and Southeast Asia, plays a vital role in aquaculture and seafood trade due to its tender flesh and rich nutritional value. With the expansion of golden threadfin bream farming, sulfamethoxazole, a broad-spectrum sulfonamide antibiotic, is widely used to prevent and treat bacterial diseases in fish (such as enteritis and gill rot) and to promote growth, becoming a commonly used veterinary drug in the aquaculture process. However, sulfamethoxazole is metabolized slowly in fish. If there are improper operations such as excessive use of the drug or failure to comply with the withdrawal period during the breeding process, the drug can easily remain in the fish tissue.

[0003] Currently, the mainstream technique for detecting SMZ residues in fish is liquid chromatography-tandem mass spectrometry (LC-MS / MS), but existing methods generally have the following drawbacks:

[0004] Large matrix effect: The fat content of golden threadfin bream muscle is ≈3–5%, and the co-extraction of lipids leads to signal inhibition >25%, and the lower limit of quantification can only reach 20–50 ng / g, which is not suitable for "low residue + high oil" samples;

[0005] Unstable recovery rate: The recovery rate of SMZ using traditional C18 or HLB solid phase extraction columns is affected by salinity, batch-to-batch RSD >15%, and pH fluctuations are significant.

[0006] Time-consuming and solvent-intensive: Acetonitrile-n-hexane liquid-liquid partitioning requires multiple centrifugations and solvent transfers, with a pretreatment time of >40 min, which does not meet the high-throughput requirements of ports;

[0007] Insufficient chromatographic resolution: Overlapping mass spectrometry pairs of SMZ and associated sulfonamides (such as sulfadiazine) can lead to a risk of false positives.

[0008] Therefore, there is an urgent need to develop a "highly sensitive, highly lipid-tolerant, rapid and simple" method for the analysis of SMZ residues in golden threadfin bream, achieving a limit of quantification ≤2ng / g, a recovery rate of 80–110%, and a single sample pretreatment time of <15min, in order to meet the increasingly stringent limits for "monitoring of sulfonamides in marine fish" in domestic and international regulations. Summary of the Invention

[0009] In view of this, a highly sensitive analytical method for detecting sulfamethoxazole antibiotic residues in golden threadfin bream is proposed to solve the above problems.

[0010] An analytical method for detecting sulfamethoxazole antibiotic residues in fish includes the following steps:

[0011] (1) Preparation of test samples

[0012] The golden threadfin bream sample was mixed with anhydrous sodium sulfate, and acidified acetonitrile solution was added twice in sequence. The mixture was then sonicated and centrifuged. The supernatant was collected, and the supernatants from the two sonic extractions were combined and dried under nitrogen in a water bath to obtain the golden threadfin bream sample dissolution residue. The golden threadfin bream sample dissolution residue was mixed with the reconstituted solution, sonicated, and filtered to obtain the test sample.

[0013] (2) The test samples were analyzed by liquid chromatography-tandem mass spectrometry.

[0014] Further, in step (1), the mass ratio of the golden threadfin bream sample to anhydrous sodium sulfate is 1:

[0015] 1.8-2.2; The ratio of the sample to the acidified acetonitrile solution was 1g:3.8-4.2mL; The volume ratio of the supernatant to the reconstituted solution was 7-9:1.

[0016] Further, in step (1), the acidified acetonitrile is formic acid acetonitrile with a concentration of 0.8%-1.2%; the reconstituted solution is an aqueous solution of acetonitrile formic acid.

[0017] Furthermore, the acetonitrile-formic acid aqueous solution contains 8%-10% acetonitrile and 0.08%-0.12% formic acid.

[0018] Further, in step (1), the mixing is carried out by vortex mixing, the ultrasonication is carried out by ultrasonic power of 130-170W for 8-12 minutes, and the centrifugation is carried out by centrifugation at 8000-10000r / min for 4-6 minutes.

[0019] Furthermore, in step (1), the test sample also contains sulfamethoxazole.

[0020] Further, the method is characterized by mixing the golden threadfin bream sample with sulfamethoxazole, adding anhydrous sodium sulfate, adding acidified acetonitrile solution twice in sequence, mixing, sonicating, taking the supernatant, combining the supernatants extracted by the two sonications, and drying them with nitrogen in a water bath to obtain the golden threadfin bream sample dissolved residue. The golden threadfin bream sample dissolved residue and the reconstituted solution are mixed, sonicated, and filtered to obtain the test sample.

[0021] Furthermore, based on the mass of the golden threadfin bream sample, the amount of sulfamethoxazole added was 4 ng / g.

[0022] Further, in step (2), the detection parameters measured by the liquid chromatography-tandem mass spectrometry are:

[0023] Ion pair information:

[0024] Name | Mother Ion | Daughter Ion | Residence Time (msec) | Declustering Voltage (V) | Collision Energy (V)

[0025]

[0026] Mobile phase A: 0.08%-0.12% formic acid aqueous solution;

[0027] Mobile phase B: 0.08%-0.12% formic acid acetonitrile;

[0028] The mobile phase elution gradient is:

[0029] time A B 0.01 90 10 3.0 65 35 7.5 5 95 8.0 5 95 8.1 90 10 10.0 90 10

[0030] Column: C18 (2.7 μm, 2.1 × 100 mm);

[0031] Injection volume: 5-15 μL;

[0032] Column temperature: 35-45℃.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] In this invention, based on the characteristics of the golden threadfin bream matrix, anhydrous sodium sulfate and acidified acetonitrile are combined. First, all sulfamethoxazole in the protein, aqueous phase, and matrix-encapsulated state is diverted to the anhydrous acidified acetonitrile. Then, the adsorption traps of SPE and nitrogen blowing are eliminated, thereby improving the recovery rate of the target analyte and lowering the detection limit, achieving a quantitation limit ≤2 ng / g. -1 The recovery rate is >80%, and the single-sample pretreatment time is <15 min, in order to meet the increasingly stringent limits for "monitoring of sulfonamides in marine fish" in domestic and international regulations. Attached Figure Description

[0035] Figure 1 This is the standard correlation curve for Example 1.

[0036] Figure 2 Chromatogram of Example 1

[0037] Figure 3 Chromatogram of Comparative Example 1

[0038] Figure 4 Chromatogram of Comparative Example 2

[0039] Figure 5 Chromatogram of Comparative Example 3 Detailed Implementation

[0040] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0041] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0042] Example 1

[0043] 1. Weigh 5.0g of golden threadfin bream muscle and place it in a 50ml centrifuge tube. Add 20ng of sulfamethoxazole, vortex for 30s, and let stand in the dark for 10min.

[0044] 2. Add 10g of anhydrous sodium sulfate and vortex mix for 1 minute.

[0045] 3. Add 20 ml of 1% formic acid acetonitrile, vortex mix for 10 min, sonicate for 10 min at 150 W ultrasonic power, and then centrifuge at 9000 r / min for 5 min. Collect the supernatant and residue separately.

[0046] 4. Add 1% formic acid acetonitrile to the residue, extract by ultrasonication at 150W for 10 min, then centrifuge at 9000 r / min for 5 min, collect the supernatant, and combine the two supernatants and mix them well.

[0047] 5. Transfer 8 ml of the supernatant to a 15 ml plastic centrifuge tube, dry it under nitrogen in a 40 °C water bath, add 1 ml of reconstitution solution (10% acetonitrile and 0.1% formic acid aqueous solution), vortex to dissolve the residue, centrifuge at 9000 r / min for 5 min, and filter the liquid through a 0.22 μm organic filter membrane to obtain the test sample. The sample is then analyzed by liquid chromatography-tandem mass spectrometry. The detection parameters are shown in the table below.

[0048]

[0049]

[0050] (1) Linear relationship of sulfamethoxazole in the substrate of golden threadfin bream

[0051] See Figure 1A linear regression equation was constructed with the concentration of sulfamethoxazole-1 sample as the x-axis and the peak area of ​​sulfamethoxazole-1 as the y-axis, and a standard correlation curve was plotted. The linear range of sulfamethoxazole-1 was 2.5-220 ng / mL, and the linear regression equation was y = 10853.66426x ± 3026.56876 (r = 0.99680, r0...). 2 =0.99362). Where r 2 A value greater than 0.990 demonstrates that sulfamethoxazole-1 is correlated within the linear range of 2.5–220 ng / mL.

[0052] (2) Recovery rate of sulfamethoxazole in the golden threadfin bream substrate

[0053] The test sample was injected six times, and the recovery rate was calculated.

[0054] Recovery rate (%) = (Spiked sample concentration - Blank sample background concentration) / Spike amount × 100%. In Example 1, the recovery rate of sulfamethoxazole in the golden threadfin matrix was 89.3%, with an RSD of 2.1%. The results indicate that the analytical method of the present invention has a high recovery rate.

[0055] (3) Sensitivity of sulfamethoxazole in the substrate of golden threadfin bream

[0056] Under the sampling volume and fixed volume specified in this method, and calculated based on a signal-to-noise ratio of 3, the detection limit of sulfamethoxazole of this invention is 0.2 ng / g, indicating that the analytical method of this invention has higher sensitivity.

[0057] Comparative Example 1

[0058] 1. Weigh 5.0g of golden threadfin bream muscle and place it in a 50ml centrifuge tube. Add 20ng of sulfamethoxazole, vortex for 30s, and let stand in the dark for 10min.

[0059] 2. Add 20 ml of 1% formic acid acetonitrile, vortex mix for 10 min, sonicate for 10 min at 150 W ultrasonic power, add 10 g of anhydrous sodium sulfate, vortex mix for 10 min, sonicate for 10 min at 150 W ultrasonic power, and then centrifuge at 9000 r / min for 5 min. Collect the supernatant and residue separately.

[0060] 3. Add 1% formic acid acetonitrile to the residue, extract by ultrasonication at 150W for 10 min, then centrifuge at 9000 r / min for 5 min, collect the supernatant, and combine the two supernatants and mix them well.

[0061] 4. Take 8 ml of the supernatant into a 15 ml plastic centrifuge tube, dry it in a 40 °C water bath under nitrogen, add 1 ml of reconstitution solution (10% acetonitrile and 0.1% formic acid aqueous solution), vortex to dissolve the residue, centrifuge at 9000 r / min for 5 min, and filter the liquid through a 0.22 μm organic filter membrane to obtain the test sample.

[0062] The detection method for the test samples is the same as in Example 1.

[0063] The recovery rate of sulfamethoxazole in the golden threadfin bream matrix of Comparative Example 1 was 80.6%, with an RSD of 3.4%.

[0064] Comparative Example 2

[0065] 1. Weigh 5.0g of golden threadfin bream muscle and place it in a 50ml centrifuge tube. Add 20ng of sulfamethoxazole, vortex for 30s, and let stand in the dark for 10min.

[0066] 2. Add 10g of anhydrous sodium sulfate and vortex mix for 1 minute.

[0067] 3. Add 20 ml of 1% formic acid acetonitrile, vortex mix for 10 min, sonicate for 10 min at 150 W ultrasonic power, and then centrifuge at 9000 r / min for 5 min. Collect the supernatant separately.

[0068] 4. Take 8 ml of the supernatant into a 15 ml plastic centrifuge tube, dry it in a 40 °C water bath under nitrogen, add 1 ml of reconstitution solution (10% acetonitrile and 0.1% formic acid aqueous solution), vortex to dissolve the residue, centrifuge at 9000 r / min for 5 min, and filter the liquid through a 0.22 μm organic filter membrane to obtain the test sample.

[0069] The detection method for the test samples is the same as in Example 1.

[0070] The recovery rate of sulfamethoxazole in the golden threadfin bream matrix of Comparative Example 2 was 73.2%, with an RSD of 2.7%.

[0071] Comparative Example 3

[0072] 1. Weigh 5.0g of golden threadfin bream muscle and place it in a 50ml centrifuge tube. Add 20ng of sulfamethoxazole, vortex for 30s, and let stand in the dark for 10min.

[0073] 2. Add 10g of anhydrous sodium sulfate and vortex mix for 1 minute.

[0074] 3. Add 20 ml of 0.6% formic acid acetonitrile, vortex mix for 10 min, sonicate for 10 min at 150 W ultrasonic power, and then centrifuge at 9000 r / min for 5 min. Collect the supernatant and residue separately.

[0075] 4. Add 0.6% formic acid acetonitrile to the residue, extract by ultrasonication at 150W for 10 min, then centrifuge at 9000 r / min for 5 min, collect the supernatant, and combine the two supernatants and mix them well.

[0076] 5. Take 8 ml of supernatant into a 15 ml plastic centrifuge tube, dry it in a 40 °C water bath under nitrogen, add 1 ml of reconstitution solution (10% acetonitrile and 0.1% formic acid aqueous solution), vortex to dissolve the residue, centrifuge at 9000 r / min for 5 min, and filter the liquid through a 0.22 μm organic filter membrane to obtain the test sample.

[0077] The detection method for the test samples is the same as in Example 1.

[0078] The recovery rate of sulfamethoxazole in the golden threadfin bream matrix of Comparative Example 3 was 71.9%, with an RSD of 3.8%.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An analytical method for detecting sulfamethoxazole antibiotic residues in fish, characterized in that, Includes the following steps: (1) Preparation of test samples The golden threadfin bream sample was mixed with anhydrous sodium sulfate, and acidified acetonitrile solution was added twice in sequence. The mixture was then sonicated and centrifuged. The supernatant was collected, and the supernatants from the two sonic extractions were combined and dried under nitrogen in a water bath to obtain the golden threadfin bream sample dissolution residue. The golden threadfin bream sample dissolution residue was mixed with the reconstituted solution, sonicated, and filtered to obtain the test sample. (2) The test samples were analyzed by liquid chromatography-tandem mass spectrometry.

2. The analytical method for detecting sulfamethoxazole antibiotic residues in fish as described in claim 1, characterized in that, In step (1), the mass ratio of the golden threadfin bream sample to anhydrous sodium sulfate is 1:1.8-2.2; the material-to-liquid ratio of the golden threadfin bream sample to the acidified acetonitrile solution is 1g:3.8-4.2mL; and the volume ratio of the supernatant to the reconstituted solution is 7-9:

1.

3. The analytical method for detecting sulfamethoxazole antibiotic residues in fish as described in claim 1, characterized in that, In step (1), the acidified acetonitrile has a concentration of 0.8%-1.2%; the reconstituted solution is an aqueous solution of acetonitrile-formic acid.

4. The highly sensitive analytical method for detecting sulfamethoxazole antibiotic residues in golden threadfin bream as described in claim 3, characterized in that, The acetonitrile-formic acid aqueous solution contains 8%-10% acetonitrile and 0.08%-0.12% formic acid.

5. The analytical method for detecting sulfamethoxazole antibiotic residues in fish as described in claim 1, characterized in that, In step (1), the mixing is carried out by vortex mixing, the ultrasonication is carried out by ultrasonic power of 130-170W for 8-12 minutes, and the centrifugation is carried out by centrifugation at 8000-10000r / min for 4-6 minutes.

6. The highly sensitive analytical method for detecting sulfamethoxazole residues in golden threadfin bream as described in claim 1, characterized in that, In step (1), the test sample also contains sulfamethoxazole.

7. The analytical method for detecting sulfamethoxazole antibiotic residues in fish as described in claim 6, characterized in that, The golden threadfin bream sample and sulfamethoxazole were mixed, anhydrous sodium sulfate was added, and acidified acetonitrile solution was added twice in sequence. The mixture was then sonicated, and the supernatant was collected. The supernatants from the two sonic extractions were combined and dried under nitrogen in a water bath to obtain the dissolved residue of the golden threadfin bream sample. The dissolved residue of the golden threadfin bream sample and the reconstituted solution were mixed, sonicated, and filtered to obtain the test sample.

8. The analytical method for detecting sulfamethoxazole antibiotic residues in fish as described in claim 6, characterized in that, Based on the mass of the golden threadfin bream sample, the amount of sulfamethoxazole added was 4 ng / g.

9. The analytical method for detecting sulfamethoxazole antibiotic residues in fish as described in claim 1, characterized in that, In step (2), the detection parameters measured by the liquid chromatography-tandem mass spectrometry are: Ion pair information: Mobile phase A: 0.08%-0.12% formic acid aqueous solution; Mobile phase B: 0.08%-0.12% formic acid acetonitrile; The mobile phase elution gradient is: Column: C18 (2.7 μm, 2.1 × 100 mm); Injection volume: 5-15 μL; Flow rate: 0.3-0.5 mL / min Column temperature: 35-45℃.

Images