Method for detecting illegally added drugs in aquatic products by using liquid chromatography

Abstract

The invention discloses a method for detecting illegally added drugs in aquatic products by using liquid chromatography. The method at least comprises the following steps: extraction, purification, dilution and measurement. The method comprises the following steps: mixing an aquatic product, an extracting agent and inorganic salt, and carrying out vortex treatment, ultrasonic treatment, low-temperature freezing and centrifugation to obtain a first extracting solution and first extracting residues; adding an extracting agent into the first extraction residue, carrying out vortex centrifugation to obtain a second extraction solution, and combining the extraction solutions to obtain all the extraction solutions; adding a degreasing agent into all the extracting solution, and carrying out vortex, centrifugation and repeating to obtain a degreased extracting solution; according to the method, the content of the illegally added medicine in the aquatic product is obtained by adding a precipitator and a dewatering agent into the degreased extracting solution, carrying out vortex centrifugation, carrying out constant volume dilution, filtering and carrying out sample introduction analysis, and when the adding concentration of the illegally added medicine in the aquatic product is 5-50 micrograms / g, the recovery rate range is 51.4-94.0%, and the RSD range is 0.2-4.8%.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a method for detecting illegally added drugs in aquatic products using liquid chromatography. Background Technology

[0002] The illegal addition of drugs to aquatic products refers to the unauthorized use of prohibited or restricted chemical drugs, antibiotics, hormones, etc. during the breeding, transportation, storage, and processing. This includes the illegal addition of chemical drugs due to efficacy claims to achieve the purpose of preventing and treating diseases, promoting growth, and improving functionality. These drugs have a significant harmful effect on human health and the environment. Commonly used illegal additives include antibiotics, hormones, sedatives / anesthetics, preservatives, and other prohibited drugs.

[0003] Currently, the national standard methods for detecting illegally added drugs mostly employ liquid chromatography-tandem mass spectrometry / mass spectrometry. These methods utilize bulky and expensive equipment with high maintenance costs. Furthermore, existing methods are primarily applicable to health food products such as tablets, powders, pills, and oral liquids. For ordinary foods, they are suitable for simple matrices like coffee, alcohol, tea beverages, and biscuits. For aquatic products, they are only applicable to oyster powder. Given the complex composition of most aquatic products, existing detection methods cannot meet the required standards. Therefore, a universal method for detecting illegally added drugs in aquatic products is urgently needed.

[0004] CN 111812250 A discloses a rapid detection method for drug residues in aquatic products. This method uses ultra-high performance liquid chromatography-mass spectrometry to detect veterinary drug residues in aquatic products during the aquaculture process. However, this method has the problem that the pretreatment is not suitable for detecting drugs illegally added due to efficacy claims. Therefore, it cannot be used to detect anti-fatigue, weight loss, and lipid-lowering drugs illegally added to aquatic products due to efficacy claims. In response, this invention discloses a method for detecting illegally added drugs in aquatic products using liquid chromatography. This method is based on a domestically produced chromatographic column for detecting illegally added drugs in shrimp, fish, and shellfish aquatic products. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a pretreatment method for aquatic product samples and its application in liquid chromatography detection. This method significantly reduces detection costs, shortens operation time and testing cycle, thereby improving detection efficiency.

[0006] According to one aspect of the present invention, a method for detecting illegally added drugs in aquatic products using liquid chromatography is provided, the method comprising at least the following steps: Step 1, Extraction: Mix the aquatic products, extractant, and sodium chloride, vortex, sonicate, freeze at low temperature, and centrifuge to obtain the first extract and the first extraction residue; add the extractant to the first extraction residue, vortex and centrifuge to obtain the second extract, and combine the first extract and the second extract to obtain the total extract after extraction. Step 2, Purification: Add a defatting agent to all the extract obtained in Step 1, vortex, centrifuge, repeat, centrifuge to obtain a defatted extract; add a precipitant and a dehydrating agent to the defatted extract in sequence, vortex, centrifuge to obtain a purified aquatic product extract; Step 3, dilution and measurement: Transfer the purified aquatic product extract obtained in Step 2 to a volumetric flask, dilute to volume, filter, and analyze the filtrate using ultra-high performance liquid chromatography to determine the content of illegally added drugs in the aquatic products.

[0007] Further, the extractant in step 1 is selected from at least one of methanol, acetonitrile, 80% methanol aqueous solution, ammoniated acetonitrile, and acidified acetonitrile; The pH of the extract in step 1 is 4.8 to 6.8.

[0008] Further, the pH of the extract in step 1 is independently selected from any value among 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, and 6.8, or a range between any two of the above points.

[0009] The mass ratio of aquatic products, extractant, and sodium chloride in step 1 is 1~2:15~30:2~4; Furthermore, the mass ratio of the aquatic product, extractant, and sodium chloride is 1:15:2; The ultrasound time in step 1 is 5-8 minutes.

[0010] Furthermore, the cryogenic freezing conditions described in step 1 are as follows: The freezing temperature is -20~-16℃; The freezing time is 20-40 minutes.

[0011] Furthermore, the degreasing agent used in step 2 is selected from at least one of n-hexane, C18, solid phase extraction column MCX, and solid phase extraction column WAX; The precipitant used in step 2 for purification is selected from at least one of lead acetate, anhydrous ethanol, trichloroacetic acid, and zinc acetate-potassium ferrocyanide; The dehydrating agent used in step 2 is magnesium sulfate; In step 2, the mass ratio of the extracted aquatic product, degreasing agent, precipitant, and dehydrating agent used for purification is 1~2:10~20:2~4:2~4.

[0012] Furthermore, in step 2, the mass ratio of the extracted aquatic product, degreasing agent, precipitant, and dehydrating agent used for purification is 1:10:2:3.5; The purification process described in step 2 is repeated 2 to 3 times.

[0013] Furthermore, the vortex time in steps 1 and 2 is 1 to 3 minutes; The centrifugation conditions described in steps 1 and 2 are as follows: The centrifugation temperature is 0~4℃; The centrifugation time is 5-10 minutes; The centrifugation speed is 5000~10000 r / min.

[0014] Furthermore, the liquid chromatography conditions for the determination and analysis described in step 3 are as follows: The chromatographic column used was a SinoChrom ODS-BP C18; The mobile phase used includes phase A and phase B, wherein phase A is an aqueous solution of ammonium acetate with a pH of 5 and a concentration of 0.01 mol / L, and phase B is methanol; The detection wavelengths were simultaneously 222 nm and 237 nm. The column temperature is 25~30℃; Furthermore, the column temperature is 25°C.

[0015] Furthermore, the conditions for the determination and analysis described in step 3 also include the use of a gradient elution procedure; The gradient elution program is as follows: 0–2.0 min, 40% B phase; 2.0–3.0 min, 40%–60% B phase; 3.0–4.0 min, 60%–80% B phase; 4.0–7.0 min, 80% B phase; 7.0–7.5 min, 80%–85% B phase; 7.5–9.5 min, 85% B phase; 9.5–10.0 min, 85%–80% B phase; 10.0–11.0 min, 80%–60% B phase; 11.0–12.0 min, 60%–40% B phase.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The extraction process described in the technical solution disclosed in this invention can maximize the extraction efficiency by adjusting the pH of the extractant, adding inorganic salts and combining ultrasound and low temperature freezing assistance. The recovery rate of the three types of aquatic products ranges from 51.4% to 94.0%.

[0017] (2) The purification process described in the technical solution disclosed in this invention extends the life of the chromatographic column by using n-hexane for multiple degreasing and adding precipitant to remove proteins.

[0018] (3) The purification process described in the technical solution disclosed in this invention is carried out by adding a dehydrating agent to remove water and improve the ability to resist matrix interference.

[0019] (4) In the dilution and testing process described in the technical solution disclosed in this invention, dilution is used to prevent the instrument resolution from being affected. At the same time, the test conditions need to strictly control the pH of the mobile phase to 5, the detection wavelength, the column temperature, and the gradient elution program to improve the separation degree and response value of illegally added drugs in aquatic products, extend the service life of the chromatographic column, reduce the detection cost, and improve the detection efficiency. Attached Figure Description

[0020] Figure 1 The chromatogram of a single standard solution with a concentration of 5.0 μg / mL at 222 nm is shown in Example 1 of this invention. Figure 2 The chromatogram of a single standard solution with a concentration of 5.0 μg / mL at 237 nm is shown in Example 1 of this invention. Figure 3 The above is a liquid chromatogram of seven mixed standard substances with a concentration of 5 μg / mL as described in Example 1 of this invention; Figure 4 The characteristic absorption spectra of the seven illegally added drugs described in Example 1 of this invention; Figure 5 The standard curves for the seven analytes within the linear range of 0.4–0.8 μg / mL described in Example 1 of this invention are as follows; Figure 6 The standard curves for the seven analytes within the linear range of 0.5–3.0 μg / mL described in Example 1 of this invention are as follows; Figure 7 This is the standard curve of seven analytes within the linear range of 2.0 to 10 μg / mL described in Example 1 of the present invention. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0022] Example 1 Step 1, Extraction: Weigh 1.0 g of aquatic product (shrimp) and place it in a 50 mL centrifuge tube. Add 10 mL of 80% pH 4.8 methanol aqueous solution (i.e., 80 mL of methanol diluted to 100 mL with water and pH adjusted to 4.8 with acetic acid) and 2 g of sodium chloride. Vortex at room temperature for 1 min, then sonicate for 5 min. Freeze at -18±2℃ for 20 min. After freezing, centrifuge at 8000 r / min for 5 min at 4℃. Transfer the supernatant to another 50 mL centrifuge tube to obtain the first extract and the first extraction residue. Add 5 mL of 80% pH 4.8 methanol aqueous solution (i.e., 80 mL of methanol diluted to 100 mL with water and pH adjusted to 4.8 with acetic acid) to the first extraction residue. Vortex for 1 min, then centrifuge at 8000 r / min for 5 min at 4℃ to obtain the second extract (supernatant). Combine the first and second extracts to obtain the total extract. Step 2, Purification: Add 5 mL of n-hexane to the entire extract obtained in Step 1, vortex for 1 min, centrifuge at 5000 r / min for 5 min at 4℃, discard the upper n-hexane layer, repeat with 5 mL of n-hexane, centrifuge at 5000 r / min for 5 min at 4℃, repeat 2-3 times to obtain the defatted extract. Add 1 mL of 21.9% zinc acetate solution (i.e., 21.9 g zinc acetate, 3 mL acetic acid, diluted to 100 mL water), 1 mL of 10.6% potassium ferrocyanide solution (i.e., 10.6 g potassium ferrocyanide dissolved in 100 mL water), and 3.5 g anhydrous magnesium sulfate to the defatted extract, vortex for 1 min, and centrifuge at 10000 r / min for 5 min at 4℃ to obtain the purified extract. The purification throughput is 7 target compounds. Step 3, Dilution: Transfer all the purified extract obtained in Step 2 to a 10 mL volumetric flask, and dilute to the mark with methanol to obtain the diluted purified solution. Filter 1.0 mL of the diluted purified solution through a 0.22 μm organic microporous membrane, inject the filtrate into a sample vial, and determine it by ultra-high performance liquid chromatography. Step 4, Analysis: The ultra-high performance liquid chromatography (UHPLC) method described in Step 3 used a SinoChromODS-BP C18 column (4.6 × 150 mm, 5 μm); the mobile phase consisted of phase A and phase B, where phase A was 0.01 mol / L ammonium acetate solution (pH 5) and phase B was methanol; the detection wavelengths were 222 nm and 237 nm; the column temperature was 25℃; the gradient elution program was as follows: 0–2.0 min, methanol 40%; 2.0–3.0 min, methanol 40%–60%; 3.0–4.0 min, methanol 60%–80%; 4.0–7.0 min, methanol 80%; 7.0–7.5 min, methanol 80%–85%; 7.5–9.5 min, methanol 85%; 9.5–10.0 min, methanol 85%–80%; 10.0–11.0 min, methanol 80%–60%; 11.0–12.0 min, methanol... The concentration of methanol was 60%–40%. The content of phenolphthalein in the aquatic products was 73.1–94.0%, amlodipine 62.0–72.4%, sildenafil 51.4–60.8%, nifedipine 53.1–62.1%, nisodipine 56.3–83.5%, felodipine 63.0–73.9%, and sibutramine 78.4–85.7%, totaling seven illegally added drugs. The recoveries and relative standard deviations of the spiked aquatic products (shrimp) are shown in Table 7. The chromatogram and retention time of a single standard solution with a concentration of 5.0 μg / mL at 222 nm are shown in Table 7. Figure 1 The chromatogram and retention time of a single standard solution at 237 nm with a concentration of 5.0 μg / mL are shown below (sildenafil, nisodipine, felodipine, sibutramine names). Figure 2 As shown (phenolphthalein, amlodipine, nifedipine); seven illegal drug standard liquid chromatograms as shown Figure 3 The spectrum of the seven illicit drugs shown is as follows (sildenafil, nisoldipine, felodipine, sibutramine, phenolphthalein, amlodipine, nifedipine). Figure 4 As shown; the standard curves for the seven analytes within the linear range of 0.4–0.8 μg / mL are as follows. Figure 5 As shown; the standard curves for the seven analytes within the linear range of 0.5–3.0 μg / mL are as follows. Figure 6 As shown; the standard curves for the seven analytes within the linear range of 2.0–10 μg / mL are as follows. Figure 7 As shown.

[0023] Example 2 The difference from Example 1 is that the type of aquatic product used is different; fish (cod) was used instead. The rest of the process is the same as in Example 1. The contents of phenolphthalein, amlodipine, sildenafil, nifedipine, nisodipine, felodipine, sibutramine, and cod were measured to be 93.2-95.6%, 65.0-83.8%, 56.1-69.8%, 76.6-84.7%, 54.1-57.5%, 72.0-93.1%, and 82.8-98.3% in the fish aquatic product were found to be illegally added drugs. The spiked recoveries and relative standard deviations of the tested aquatic product (cod) are shown in Table 7.

[0024] Example 3 The difference from Example 1 is that the type of aquatic product used is different; the aquatic product used is shellfish (scallops). The rest of the process is the same as in Example 1. The contents of phenolphthalein, amlodipine, sildenafil, nifedipine, nisodipine, felodipine, sibutramine, and succinate in the aquatic product were measured to be 83.3-99.3%, 78.1-88.1%, 54.7-71.7%, 66.2-84.7%, 73.0-78.4%, 70.2-89.0%, and 52.4-62.0%, respectively. There were a total of 7 illegally added drugs. The spiked recoveries and relative standard deviations of the aquatic product (scallops) are shown in Table 7.

[0025] Example 4 The difference from Example 1 is that the extraction solvents used in step 1 are different. The extraction solvents used are 80% methanol (i.e., 80 mL of methanol diluted with water to 100 mL), acetonitrile, methanol, 1% ammonia-acetonitrile (i.e., 40 mL of 25% ammonia diluted with acetonitrile to 1000 mL), and 1% acetic acid-acetonitrile (i.e., 10 mL of acetic acid diluted with acetonitrile to 1000 mL). The rest of the process is the same as in Example 1. The test results of the drug content in aquatic products (shrimp) are shown in Table 1.

[0026] Table 1 Comparison of recovery rates of different extraction solvents

[0027] Example 5 The difference from Example 1 is that different pH extraction solvents were used in step 1, namely 4.8, 5.8 and 6.8 (i.e., the pH was adjusted to 4.8, 5.8 and 6.8 by acetic acid). The rest of the process was the same as in Example 1. The test results of drug content in aquatic products (shrimp) are shown in Table 2.

[0028] Table 2 Comparison of recovery rates of extraction solvents at different pH values

[0029] Example 6 The difference from Example 1 lies in the purification method used in step 2. The purification methods employed are precipitation, QuEChERS, and solid-phase extraction (C...). 18 The remaining process was the same as in Example 1, and the test results of drug content in aquatic products (shrimp) are shown in Table 3.

[0030] Table 3 Comparison of Recovery Rates of Different Purification Methods

[0031] Example 7 The difference from Example 1 is that in step 2, different precipitants were used, namely lead acetate, anhydrous ethanol, trichloroacetic acid, zinc acetate, and potassium ferrocyanide. The rest of the process was the same as in Example 1. The test results of the drug content in aquatic products (shrimp) are shown in Table 4.

[0032] Table 4 Comparison of recovery rates of different precipitants

[0033] Example 8 The difference from Example 1 is that the amount of dehydrating agent (anhydrous magnesium sulfate) used in step 2 is different. The amounts of dehydrating agent used are 2g, 3g, 3.5g and 4g respectively. The rest of the process is the same as in Example 1. The test results of the drug content in aquatic products (shrimp) are shown in Table 5.

[0034] Table 5 Comparison of recovery rates for different amounts of dehydrating agent

[0035] Table 6 shows the recoveries and relative standard deviations (n=6) of the seven analytes spiked into the three aquatic product matrices described in Examples 1-3.

[0036] Note: RSD represents relative standard deviation. The above description is merely a few embodiments of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications or alterations made by those skilled in the art without departing from the scope of the technical solution of the present invention using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for detecting illegally added drugs in aquatic products using liquid chromatography, characterized in that, The method includes at least the following steps: Step 1, Extraction: Mix the aquatic products, extractant, and sodium chloride, vortex, sonicate, freeze at low temperature, and centrifuge to obtain the first extract and the first extraction residue; add the extractant to the first extraction residue, vortex and centrifuge to obtain the second extract, and combine the first extract and the second extract to obtain the total extract after extraction. Step 2, Purification: Add a defatting agent to all the extract obtained in Step 1, vortex, centrifuge, repeat, centrifuge to obtain a defatted extract; add a precipitant and a dehydrating agent to the defatted extract in sequence, vortex, centrifuge to obtain a purified aquatic product extract; Step 3, dilution and measurement: Transfer the purified aquatic product extract obtained in Step 2 to a volumetric flask, dilute to volume, filter, and analyze the filtrate using ultra-high performance liquid chromatography to determine the content of illegally added drugs in the aquatic products.

2. The method according to claim 1, characterized in that, The extractant mentioned in step 1 is selected from at least one of methanol, acetonitrile, 80% methanol aqueous solution, ammoniated acetonitrile, and acidified acetonitrile; The pH of the extract in step 1 is 4.8~6.8; The mass ratio of aquatic products, extractant, and sodium chloride in step 1 is 1~2:15~30:2~4; Preferably, the mass ratio of the aquatic product, the extractant, and the sodium chloride is 1:15:2; The ultrasound time in step 1 is 5-8 minutes.

3. The method according to claim 1, characterized in that, The cryogenic freezing conditions described in step 1 are as follows: The freezing temperature is -20~-16℃; The freezing time is 20-40 minutes.

4. The method according to claim 1, characterized in that, The degreasing agent used in step 2 is selected from at least one of n-hexane, C18, solid phase extraction column MCX, and solid phase extraction column WAX; The precipitant used in step 2 for purification is selected from at least one of lead acetate, anhydrous ethanol, trichloroacetic acid, and zinc acetate-potassium ferrocyanide; The dehydrating agent used in step 2 is anhydrous magnesium sulfate; In step 2, the mass ratio of the extracted aquatic product, degreasing agent, precipitant, and dehydrating agent used in the purification process is 1~2:10~20:2~4:2~4. Preferably, the mass ratio of the extracted aquatic product, degreasing agent, precipitant, and dehydrating agent used in step 2 for purification is 1:10:2:3.5; The defatting process described in step 2 is repeated 2 to 3 times.

5. The method according to claim 1, characterized in that, The vortex time in steps 1 and 2 is 1 to 3 minutes; The centrifugation conditions described in steps 1 and 2 are as follows: The centrifugation temperature is 0~4℃; The centrifugation time is 5-10 minutes; The centrifugation speed is 5000~10000 r / min.

6. The method according to claim 1, characterized in that, The liquid chromatography conditions for the determination and analysis described in step 3 are as follows: The chromatographic column used was a SinoChrom ODS-BP C18; The mobile phase used includes phase A and phase B, wherein phase A is an aqueous solution of ammonium acetate with a pH of 5 and a concentration of 0.01 mol / L, and phase B is methanol; The detection wavelengths were simultaneously 222 nm and 237 nm. The column temperature is 25~35℃; Preferably, the column temperature is 25°C.

7. The method according to claim 6, wherein the conditions for determination and analysis in step 3 further include a gradient elution procedure; The gradient elution program is as follows: 0–2.0 min, 40% phase B; 2.0–3.0 min, 40%–60% phase B; 3.0–4.0 min, 60%–80% phase B; 4.0–7.0 min, 80% phase B; 7.0–7.5 min, 80%–85% phase B. 7.5–9.5 min, 85% B phase; 9.5–10.0 min, 85%–80% B phase; 10.0–11.0 min, 80%–60% B phase; 11.0–12.0 min, 60%–40% B phase.

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