Method for detecting new receptor agonist drug residues in pig hair

Abstract

The present application relates to the technical field of veterinary drug residue detection, and proposes a detection method for new receptor agonist drug residues in pig hair, S1, the pig hair is sequentially cleaned with water, a solvent and water, and is extracted in formic acid dimethyl sulfoxide solution, primary centrifugation, filtration, the filtrate is blown dry with nitrogen, then methanol is added for dissolution, secondary centrifugation, and a test solution is obtained; S2, the test solution is subjected to mass spectrometry detection; the solvent is a mixed solvent of cyclohexane and n-hexane in a volume ratio of 1:1 to 1:3. Through the above technical solution, the problems of complex process, long analysis time, poor stability and low sensitivity in the prior art are solved.

Description

Detection method for novel receptor agonist drug residues in pig hair Technical Field

[0001] This invention relates to the field of veterinary drug residue detection technology, specifically to a method for detecting novel receptor agonist drug residues in pig hair. Background Technology

[0002] Pork is the primary source of animal protein for the Chinese people and has long dominated meat consumption. In recent years, the safety of pork and other animal-derived agricultural products has been a key focus of routine supervision, with drug residues being a particularly serious issue. Hair follicle testing is an in vitro technique for detecting drugs in biological samples such as body hair and scalp hair. Drugs enter the hair follicle through metabolism and then spread into the hair as it grows. Due to the long growth period of hair, the drugs can remain stably in the hair for an extended period. Compared to other biological samples such as muscle, tissue, blood, urine, and feces, it offers advantages such as a longer monitoring window, non-invasiveness, convenient sampling, and sustainable monitoring. Currently, hair testing is widely used in forensic toxicology and environmental science, but its application in animal-derived agricultural products is relatively limited. Concern over the food safety issue of "lean meat powder" has spurred the rapid development of β2-receptor agonist detection technology, effectively curbing its illegal use in animal husbandry. Meanwhile, α2-receptor agonists, as a novel drug that promotes growth and increases lean meat percentage, are gradually attracting attention, and there is a trend of illegal addition in the feed industry. As early as 2010, the Ministry of Agriculture's Announcement No. 1519 explicitly addressed this issue. Clonidine, cyproheptadine, and other substances are listed in the Ministry of Agriculture's "List of Substances Prohibited for Use in Feed and Animal Drinking Water." α2-receptor agonists have a specific affinity for α2 receptors and are mainly used to treat hypertension in humans. However, α2-receptor agonists have toxic side effects on liver, kidney, and brain functions. Overdose can cause excessive sedation, drowsiness, impaired consciousness, and even life-threatening conditions. Studies have shown that adding 0.5 mg / kg of clonidine to feed can significantly increase the lean meat percentage and improve carcass composition in pigs. Other α2-receptor agonists, such as romifedipine and loratadine, have similar effects.

[0003] Currently, the main detection methods for clonidine, romifedipine, and loratadine, α2-receptor agonists, include gas chromatography, liquid chromatography, and chromatography-mass spectrometry. However, these chromatographic methods suffer from drawbacks such as complex detection processes, long analysis times, poor stability, and low sensitivity. Therefore, it is crucial to develop a simple and efficient method for detecting drug residues in animal-derived agricultural products. Summary of the Invention

[0004] This invention proposes a method for detecting novel receptor agonist drug residues in pig hair, which solves the problems of complex processes, long analysis times, poor stability, and low sensitivity in related technologies.

[0005] The technical solution of the present invention is as follows:

[0006] This invention proposes a method for detecting novel receptor agonist drug residues in porcine hair, comprising the following steps:

[0007] S1. The pig hair was washed with water, solvent and water in sequence, extracted with dimethyl sulfoxide formate solution, centrifuged for the first time, filtered, the filtrate was dried with nitrogen gas, dissolved in methanol, centrifuged a second time to obtain the test solution.

[0008] S2. Perform chromatographic and mass spectrometric analysis on the test solution;

[0009] The solvent is a mixture of cyclohexane and n-hexane in a volume ratio of 1:1 to 1:3.

[0010] As a further technical solution, the extraction time is 5-10 minutes.

[0011] As a further technical solution, the volume fraction of formic acid in the formic acid dimethyl sulfoxide solution is 0.1%~0.3%.

[0012] As a further technical solution, the centrifugal force of the first centrifugation is 9000~11000g / min, the centrifugation temperature is 3~5℃, and the centrifugation time is 1~5min; the centrifugal force of the second centrifugation is 9000~11000g / min, the centrifugation temperature is 3~5℃, and the centrifugation time is 1~3min.

[0013] As a further technical solution, the chromatographic mass spectrometry detection conditions are as follows: chromatographic column: WatersAcquityHSST3, flow rate: 0.3~0.8mL / min, column temperature: 30~35℃, injection volume: 1~3μL, and the mobile phase consists of mobile phase A and mobile phase B, where mobile phase A is a 0.1% formic acid aqueous solution and mobile phase B is methanol.

[0014] As a further technical solution, the chromatographic mass spectrometry detection employs gradient elution. The gradient elution program, expressed as a volume fraction of the mobile phase, is as follows:

[0015] From 0 min to 2 min: keep 10% B constant;

[0016] From 2 min to 4 min: B increased linearly from 10% to 95%;

[0017] For 4 to 5 minutes, maintain 95% B.

[0018] From 5 min to 5.1 min: B decreased linearly from 90% to 10%;

[0019] 5.1 min to 6 min: Keep 10% B constant.

[0020] As a further technical solution, the ion source parameters for the chromatographic mass spectrometry detection are as follows: electrospray ionization source voltage is 3500~4200V, ion source temperature is 350~380℃, sheath gas flow rate is 30~55arb, auxiliary gas flow rate is 5~15arb, capillary transfer tube temperature is 350~380℃, and S-lens RF level is 40~80.

[0021] As a further technical solution, the scanning method for the chromatographic mass spectrometry detection is a first-level mass spectrometry full scan and a second-level mass spectrometry scan.

[0022] As a further technical solution, the scanning conditions for the chromatographic mass spectrometry detection are as follows: positive mode, first-stage mass spectrometry full scan resolution: 70000, scan range: 100~1000m / z, maximum residence time: 50ms, second-stage mass spectrometry scan resolution: 17500, maximum residence time: 50ms, step fragmentation energy values: 20, 40, 60.

[0023] The working principle and beneficial effects of this invention are as follows:

[0024] 1. In this invention, pig hair is extracted with dimethyl sulfoxide formate solution. Before extraction, the pig hair is washed with water, solvent and water in sequence. The pretreatment is simple and fast, which increases the recovery rate of novel receptor agonist drug residues in pig hair to more than 90%, and also improves the detection sensitivity and has good stability and reproducibility.

[0025] 2. In this invention, a mixed solvent of cyclohexane and n-hexane is used to clean pig hair, which further improves the recovery rate of novel receptor agonist drug residues in pig hair, and also improves the detection sensitivity and has good stability and reproducibility.

[0026] 3. In this invention, by controlling the conditions of chromatographic mass spectrometry detection, the sensitivity of the detection of novel receptor agonist drug residues in pig hair is further improved, and it has good stability and reproducibility. Attached Figure Description

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0028] Figure 1 shows the mass spectra of clonidine, romifedipine, and loratadine in Example 1 of the present invention;

[0029] Figure 2 is a blank baseline diagram of clonidine, romifedipine, and loratadine in Example 1 of the present invention;

[0030] Figure 3 is a secondary mass spectrum of clonidine in Example 1 of the present invention;

[0031] Figure 4 shows the second-order mass spectrum of romifinite in Example 1 of the present invention;

[0032] Figure 5 shows the secondary mass spectrum of loratadine in Example 1 of the present invention;

[0033] Figure 6 is a linear relationship diagram of clonidine in the verification of the method of the present invention;

[0034] Figure 7 is a linear relationship diagram of Romi-Finite in the verification of the method of the present invention;

[0035] Figure 8 shows the linear relationship of loratadine in the verification of the method of the present invention. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] The sources of the raw materials used in the following examples and comparative examples are shown in the table below:

[0038] Table 1 Source of Raw Materials

[0039]

[0040] Example 1

[0041] 1. Solution preparation

[0042] (1) Preparation of standard solutions: Transfer 10.0 mg of clonidine, romifedipine and loratadine standard solutions into 10 mL volumetric flasks, add methanol to dilute to the mark, and prepare a mixed stock solution of the three substances with a concentration of 1000 μg / mL. Then take 1 mL from the mixed stock solution and dilute to 10 mL to prepare a mixed stock solution of the three substances with a concentration of 100 μg / mL. Store at 4℃ in a refrigerator for later use as a mixed internal standard working solution.

[0043] (2) Preparation of the test solution:

[0044] S1. Freeze the pig hair at -40℃, seal and store for later use. Take 0.5g of the frozen pig hair sample and wash it in sequence with 50mL of water, 50mL of a mixed solvent of cyclohexane and n-hexane (v / v=1:1), and 50mL of water. Discard the washing solvent, blow dry with nitrogen at room temperature, and cut the sample into 1~2mm small pieces.

[0045] S2. Place the sample obtained in S1 into a liquid nitrogen cryogenic grinder, adjust the oscillation frequency of the liquid nitrogen cryogenic grinder to 12 times / s, grind for 3.5 min, and stabilize for 5 min. Grind the hair into powder. Take 80 mg of hair powder into a 50 mL centrifuge tube, add 4 μL of 1 μg / mL mixed internal standard working solution and 10 mL of 0.1% (v / v) dimethyl sulfoxide formate solution, sonicate for 5 min, centrifuge at 10000 g / min at 4℃ for 3 min, filter with a 0.45 μm filter membrane, and pass all the filtrate directly through an Oasis PRIME HLB column to obtain the filtrate.

[0046] S3. Take 5 mL of the column filtrate into a 10 mL centrifuge tube and dry it under nitrogen in a 60 °C water bath. Add 200 μL of methanol to reconstitute the solution, centrifuge at 10000 g / min at 4 °C for 1 min, and transfer the liquid into a sample vial with a weighing tube to obtain the test solution.

[0047] Preparation of matrix-spiked standard working solutions: Place approximately 80 mg of blank pig hair into 6 centrifuge tubes, and sequentially add a certain amount of 1 μg / mL of the three-substance mixed stock solution to make the added standard amounts reach 0.4 ng, 1.6 ng, 4 ng, 5 ng, 12.5 ng, 25 ng, and 50 ng. Add 10 mL of 0.1% (v / v) dimethyl sulfoxide formate solution, sonicate for 5 min, centrifuge at 10000 g / min at 4℃ for 3 min, filter through a 0.45 μm filter membrane, and pass all the filtrate directly through an Oasis PRIME HLB column. Take 5 mL of the column-passed filtrate into a 10 mL centrifuge tube and dry it in a 60℃ water bath under nitrogen. Redissolve the sample in 200 μL of methanol, centrifuge at 10000 g / min at 4 °C for 1 min, and transfer the liquid into a vial with a weighing tube to obtain matrix spiked standard working solutions with concentrations of 1 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL.

[0048] 2. Testing

[0049] The test solution was detected by high-resolution mass spectrometry using liquid chromatography-quadrupole-electrostatic field orbital trap tandem. The detection conditions were as follows:

[0050] Chromatographic column: Waters Acquity HSST3 column, flow rate: 0.35 mL / min, column temperature: 35℃, injection volume: 2 μL, mobile phase A is 0.1% formic acid aqueous solution (v / v), and mobile phase B is methanol solution;

[0051] Gradient elution program: 0 min to 2 min: keep 10% B constant;

[0052] From 2 min to 4 min: B increased linearly from 10% to 95%;

[0053] For 4 to 5 minutes, maintain 95% B.

[0054] From 5 min to 5.1 min: B decreased linearly from 90% to 10%;

[0055] 5.1 min to 6 min: Keep 10% B constant;

[0056] Ion source parameters: Electrospray ionization source;

[0057] Ion source voltage: 3800V;

[0058] Ion source temperature: 380℃;

[0059] Sheath flow velocity: 45 arb;

[0060] Auxiliary airflow velocity: 10arb;

[0061] Capillary tube temperature: 380℃;

[0062] S-lensRFlevel: 60;

[0063] The scanning methods are primary mass spectrometry full scan and secondary mass spectrometry scan;

[0064] Scan mode: Positive mode;

[0065] Level 1 mass spectrometry full scan resolution: 70,000, scan range: 100~1000 m / z, maximum dwell time: 50 ms;

[0066] Secondary mass spectrometry scanning resolution: 17500, maximum residence time: 50ms, step fragmentation energy values: 20, 40, 60.

[0067] Table 2. Theoretical precise molecular weight, adduct precise molecular weight, and second-order ion precise molecular weight of clonidine, romifedipine, and loratadine

[0068]

[0069] Spectral analysis:

[0070] The mass spectra of clonidine, romifedipine, and loratadine are shown in Figure 1. From top to bottom, they are clonidine, romifedipine, and loratadine.

[0071] Figure 2 shows the blank baseline plots of clonidine, romifedipine, and loratadine. From top to bottom, the plots show clonidine, romifedipine, and loratadine.

[0072] The secondary mass spectra of clonidine, romifedipine, and loratadine are shown in Figures 3-5.

[0073] Example 2

[0074] (1) Preparation of standard solutions: Transfer 10.0 mg of clonidine, romifedipine and loratadine standard solutions into 10 mL volumetric flasks, add methanol to dilute to the mark, and prepare a mixed stock solution of the three substances with a concentration of 1000 μg / mL. Then take 1 mL from the mixed stock solution and dilute to 10 mL to prepare a mixed stock solution of the three substances with a concentration of 100 μg / mL. Store at 4℃ in a refrigerator for later use as a mixed internal standard working solution.

[0075] (2) Preparation of the test solution:

[0076] S1. Freeze the pig hair at -40℃, seal and store for later use. Take 0.5g of the frozen pig hair sample and wash it in sequence with 50mL of water, 50mL of a mixed solvent of cyclohexane and n-hexane (v / v=1:3), and 50mL of water. Discard the washing solvent, blow dry with nitrogen at room temperature, and cut into small samples of 1~2mm.

[0077] S2. Place the sample obtained in S1 into a liquid nitrogen cryogenic grinder, adjust the oscillation frequency of the liquid nitrogen cryogenic grinder to 12 times / s, grind for 3.5 min, and stabilize for 5 min. Grind the hair into powder. Take 80 mg of hair powder into a 50 mL centrifuge tube, add 4 μL of 1 μg / mL mixed internal standard working solution and 10 mL of 0.3% (v / v) dimethyl sulfoxide formate solution, sonicate for 10 min, centrifuge at 11000 g / min at 5 °C for 5 min, filter with a 0.45 μm filter membrane, and pass all the filtrate directly through an Oasis PRIME HLB column to obtain the filtrate.

[0078] S3. Take 5 mL of the column filtrate into a 10 mL centrifuge tube and dry it under nitrogen in a 60 °C water bath. Add 200 μL of methanol to reconstitute the solution, centrifuge at 11000 g / min at 5 °C for 3 min, and transfer the liquid into a sample vial with a weighing tube to obtain the test solution.

[0079] Preparation of matrix-spiked standard working solutions: Place approximately 80 mg of blank pig hair into 6 centrifuge tubes, and sequentially add an appropriate amount of 1 μg / mL of the three-substance mixed stock solution to achieve standard concentrations of 0.4 ng, 1.6 ng, 4 ng, 5 ng, 12.5 ng, 25 ng, and 50 ng. Add 10 mL of 0.3% (v / v) dimethyl sulfoxide formate solution, and extract by sonication for 10 min. Centrifuge at 11000 g / min at 5 °C for 5 min, filter through a 0.45 μm filter membrane, and pass all the filtrate directly through an Oasis PRIME HLB column. Take 5 mL of the column-passed filtrate into a 10 mL centrifuge tube and dry it in a nitrogen bath at 60 °C. The solution was reconstituted with 200 μL of methanol, centrifuged at 11000 g / min at 5 °C for 5 min, and the liquid was transferred into a vial with a weighing tube to obtain matrix spiked standard working solutions with concentrations of 1 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL.

[0080] 2. Testing

[0081] The test solution was detected by high-resolution mass spectrometry using liquid chromatography-quadrupole-electrostatic field orbital trap tandem. The detection conditions were as follows:

[0082] Chromatographic column: Waters Acquity HSST3 column, flow rate: 0.3 mL / min, column temperature: 30℃, injection volume: 2 μL, mobile phase A is 0.1% formic acid aqueous solution (v / v), and mobile phase B is methanol solution;

[0083] Gradient elution program: 0 min to 2 min: keep 10% B constant;

[0084] From 2 min to 4 min: B increased linearly from 10% to 95%;

[0085] For 4 to 5 minutes, maintain 95% B.

[0086] From 5 min to 5.1 min: B decreased linearly from 90% to 10%;

[0087] 5.1 min to 6 min: Keep 10% B constant;

[0088] Ion source parameters: Electrospray ionization source;

[0089] Ion source voltage: 3600V;

[0090] Ion source temperature: 350℃;

[0091] Sheath flow velocity: 35 arb;

[0092] Auxiliary airflow velocity: 12arb;

[0093] Capillary tube temperature: 355℃;

[0094] S-lensRFlevel: 55;

[0095] The scanning methods are primary mass spectrometry full scan and secondary mass spectrometry scan;

[0096] Scan mode: Positive mode;

[0097] Level 1 mass spectrometry full scan resolution: 70,000, scan range: 100~1000 m / z, maximum dwell time: 50 ms;

[0098] Secondary mass spectrometry scanning resolution: 17500, maximum residence time: 50ms, step fragmentation energy values: 20, 40, 60.

[0099] Table 3. Theoretical precise molecular weight, adduct precise molecular weight, and second-order ion precise molecular weight of clonidine, romifedipine, and loratadine

[0100]

[0101] 3. Method Validation

[0102] 3.1 Linear Range

[0103] (1) Test methods

[0104] Following the preparation of the matrix-spiked standard working solutions in Example 1, a series of matrix-spiked standard working solutions of clonidine, romifedipine, and loratadine with concentrations of 1 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 100 ng / mL, and 200 ng / mL were prepared, and 2 μL of each solution was injected.

[0105] (2) Test requirements

[0106] Clonidine, romifedipine, and loratadine showed good linearity in the concentration range of 1 to 200 ng / mL.

[0107] (2) Test results

[0108] Linear regression was performed with the external standard peak area as the ordinate (Y) and the external standard concentration as the abscissa (X). The test results are shown in Table 4 and Figures 6-8:

[0109] Table 4 Linearity Detection Results

[0110]

[0111] 3.2 Method Anti-interference

[0112] (1) Test methods

[0113] Take blank pig hair and add clonidine, romifedipine, and loratadine to obtain the test solution. Specific test method: Wash the pig hair sample sequentially with 50 mL of water, 50 mL of a mixed solvent of cyclohexane and n-hexane (v / v = 1:3), and 50 mL of water. Discard the washing solvent, dry under nitrogen at room temperature, and cut into 1-2 mm pieces. Place the obtained sample in a liquid nitrogen cryogenic grinder, adjust the oscillation frequency of the grinder to 12 times / s, grind for 3.5 min, and allow it to stabilize for 5 min until the hair is powdered. Take 80 mg of the hair powder into a 50 mL centrifuge tube, add 4 μL of a 1 μg / mL mixed internal standard working solution and 10 mL of 0.3% (v / v) dimethyl sulfoxide formate solution, and extract ultrasonically for 10 min. Centrifuge at 11000 g / min at 5℃ for 5 min, filter through a 0.45 μm filter membrane, and pass all the filtrate directly through Oasis PRIME. HLB column chromatography was performed to obtain the filtrate. 5 mL of the column filtrate was transferred to a 10 mL centrifuge tube and dried under nitrogen in a 60°C water bath. 200 μL of methanol was added to reconstitute the solution, and the mixture was centrifuged at 11000 g / min for 3 min at 5°C. The liquid was then transferred to a vial with a weighing tube to obtain the test solution.

[0114] (2) Test requirements

[0115] During the elution time of the target analyte, the blank sample should not show a distinct chromatographic peak.

[0116] (3) Test results

[0117] The blank sample did not show any obvious chromatographic peaks, indicating that there was no interference in the method.

[0118] 3.3 Method Recovery Rate

[0119] (1) Test methods

[0120] Take 80 mg of blank pig hair and add 3.2 μL, 64 μL, and 128 μL of a mixed stock solution of clonidine, romifedipine, and loratadine at a concentration of 100 ng / mL, respectively, to achieve concentrations of 4 μg / kg, 80 μg / kg, and 160 μg / kg. Each level is measured 6 times. The samples are pretreated and analyzed according to Example 1. The recovery rates of the added samples are shown in Table 5.

[0121] Table 5. Average recoveries (%) of clonidine, romifedipine, and loratadine in blank samples (n=6)

[0122]

[0123] 3.4 Method Precision

[0124] (1) Test methods

[0125] Take 80 mg of blank pig hair and add 3.2 μL, 64 μL, and 128 μL of a mixed stock solution of clonidine, romifedipine, and loratadine (100 ng / mL) to achieve concentrations of 4 μg / kg, 80 μg / kg, and 160 μg / kg, respectively. Perform sample pretreatment and analysis as described in Example 1. Repeat the measurement 6 times daily at each level to obtain intra-day precision. Perform the measurement once at each level for 6 consecutive days to obtain inter-day precision. Intra-day and inter-day precision are shown in Table 2.

[0126] (2) Test requirements

[0127] This method has good reproducibility

[0128] (3) Test results

[0129] The results showed that the intraday precision was between 0.6% and 13.1%, and the interday precision was between 1.4% and 15.3%, indicating that the method had good reproducibility.

[0130] Table 6 Intra-day and inter-day precision (%)

[0131]

[0132] 3.5 Method Sensitivity

[0133] (1) Test methods

[0134] Blank pig hair was taken, and clonidine, romifedipine, and loratadine were added. Pretreatment and analysis were performed according to Example 1. The specific experimental method was as follows: the amount of clonidine, romifedipine, and loratadine added was gradually reduced, and pretreatment and analysis were performed according to Example 1. The signal-to-noise ratio (S / N) of the mass spectrometry peaks formed by clonidine, romifedipine, and loratadine was calculated. When the S / N reached 3, the mass concentration of clonidine, romifedipine, and loratadine added was recorded.

[0135] (2) Test requirements

[0136] The signal-to-noise ratio reaches S / N=3.

[0137] (3) Test results

[0138] The detection limit is 2 μg / kg.

[0139] 4. Selection of extraction method

[0140] (1) Test methods

[0141] Take a blank sample, add clonidine, romifedipine, and loratadine, change the extraction solvent while keeping other conditions unchanged. The specific experimental method is as follows: Wash the pig hair sample sequentially with 50 mL of water, 50 mL of a mixed solvent of cyclohexane and n-hexane (v / v=1:3), and 50 mL of water. Discard the washing solvent, dry with nitrogen at room temperature, and cut into 1-2 mm small samples. Place the obtained sample in a liquid nitrogen cryogenic grinder, adjust the oscillation frequency of the liquid nitrogen cryogenic grinder to 12 times / s, grind for 3.5 min, and stabilize for 5 min until the hair is powdered. Take 80 mg of hair powder into a 50 mL centrifuge tube, add 4 μL of a 1 μg / mL mixed internal standard working solution, change the extraction solvent, and extract ultrasonically for 10 min. Centrifuge at 11000 g / min at 5℃ for 5 min, filter through a 0.45 μm filter membrane, and pass all filtrate directly through Oasis PRIME. HLB column chromatography was performed to obtain the filtrate. 5 mL of the column filtrate was transferred to a 10 mL centrifuge tube and dried under nitrogen in a 60°C water bath. 200 μL of methanol was added to reconstitute the solution, and the mixture was centrifuged at 11000 g / min for 3 min at 5°C. The liquid was then transferred to a vial with a weighing tube to obtain the test solution.

[0142] (2) Test results

[0143] Dimethyl sulfoxide (DMSO) showed higher recovery rates as an extraction solvent, while acetonitrile and ethyl acetate showed lower recovery rates.

[0144] Table 7 Recovery rates (%) of clonidine, romifedipine, and loratadine in blank samples (n=3)

[0145]

[0146] 4.1 Selection of Standard Solution Preparation

[0147] (1) Test methods

[0148] Using matrix standardization, a series of standard solutions of clonidine, romifedipine, and loratadine were prepared at concentrations of 4 ng / mL, 80 ng / mL, and 160 ng / mL, respectively. Each concentration point was measured three times. The specific experimental method was as follows: approximately 80 mg of blank pig hair was placed in a centrifuge tube, and appropriate amounts of the three substances mixed stock solution at 100 ng / mL were added sequentially to make the added standard substances 1.6 ng, 32 ng, and 64 ng. 10 mL of 0.3% (v / v) dimethyl sulfoxide formate solution was added, and the mixture was ultrasonically extracted for 10 min. The mixture was centrifuged at 11000 g / min at 5 °C for 5 min, filtered through a 0.45 μm filter membrane, and all the filtrate was directly passed through an Oasis PRIME HLB column. 5 mL of the column-passed filtrate was taken into a 10 mL centrifuge tube and dried under nitrogen in a 60 °C water bath. Add 200 μL of methanol to reconstitute the solution, centrifuge at 11000 g / min at 5 °C for 5 min, and transfer the liquid into a vial with a weighing tube to obtain matrix spiked standard working solutions with concentrations of 4 ng / mL, 80 ng / mL, and 160 ng / mL.

[0149] Prepare standard solutions with pure acetonitrile: Take 4 μL, 80 μL, and 160 μL of 1000 ng / mL mixed standard solution respectively, and dilute to 1 mL with acetonitrile to obtain a series of standard solutions of 4 ng / mL, 80 ng / mL, and 160 ng / mL.

[0150] (2) Test results

[0151] The matrix effect is quite obvious, so matrix spiking is chosen for the marking configuration.

[0152] Table 8. Matrix effect (ratio of peak area at the same concentration for matrix standard and pure acetonitrile standard, %)

[0153]

[0154] 4.2 Selection of Mass Spectrometry Conditions

[0155] By setting the deviation of the precise extraction mass number within 5 ppm, and using secondary characteristic fragment ions for auxiliary qualitative analysis, the mass deviation is minimized, effectively reducing instrument noise interference, impurity interference, and the generation of false positives.

[0156] 5. Stability test

[0157] 5.1 Stability test in pure solution

[0158] (1) Test methods

[0159] Add 4 μL, 80 μL, and 160 μL of a mixed stock solution of the three substances (1 μg / mL) respectively, and bring the volume to 1 mL with acetonitrile. Acetonitrile solutions of clonidine, romifedipine, and loratadine with concentrations of 4 ng / mL, 80 ng / mL, and 160 ng / mL were obtained, with three replicates per solution. These solutions were stored at 4°C in a refrigerator for 0, 7, 14, 21, 28, and 35 days. After these periods, the samples were analyzed as described in section 1, and the differences in peak area were compared.

[0160] (2) Test requirements

[0161] The concentration was not statistically significant (P > 0.05).

[0162] (3) Test results

[0163] This indicates that clonidine, romifedipine, and loratadine have stability for more than one month when stored in a refrigerator at 4°C.

[0164] 5.2 Stability Study of the Matrix Spiked Solution

[0165] (1) Test methods

[0166] Three spiking solutions at levels of 4 ng / mL, 80 ng / mL, and 160 ng / mL were prepared using a blank matrix and stored at -20℃. The specific experimental method was as follows: approximately 80 mg of blank pig hair was placed in a centrifuge tube, and appropriate amounts of the three mixed stock solutions at 100 ng / mL were added sequentially to make the added standard amounts 1.6 ng, 32 ng, and 64 ng. 10 mL of 0.3% (v / v) dimethyl sulfoxide formate solution was added, and the mixture was extracted by sonication for 10 min. The mixture was centrifuged at 11000 g / min at 5℃ for 5 min, filtered through a 0.45 μm filter membrane, and all the filtrate was directly passed through an Oasis PRIME HLB column. 5 mL of the column-passed filtrate was taken into a 10 mL centrifuge tube and dried under nitrogen in a 60℃ water bath. The solution was reconstituted with 200 μL of methanol, centrifuged at 11000 g / min at 5 °C for 5 min, and the liquid was transferred into a vial with a weighing tube to obtain matrix-spiked standard working solutions with concentrations of 4 ng / mL, 80 ng / mL, and 160 ng / mL. After standing for 24 hours, 48 ​​hours, and 72 hours, the samples were pretreated and analyzed as described in Example 2 to examine the differences between the analytical results.

[0167] (2) Test requirements

[0168] The results showed that the concentration was not statistically significant (P > 0.05).

[0169] (3) Test results

[0170] The three substances remained stable in the matrix-spiked solution for 72 hours.

[0171] 6. Application Example 1: Detection of three veterinary drug residues in chicken feathers

[0172] Experimental pigs and pig hair: This experiment was conducted on three healthy male Duroc-Mercedes-British crossbred black pigs (weighing 75 kg ± 5 kg). Two weeks prior to the experiment, all drug intervention was stopped. Pig hair was collected as a blank control before the experiment. 2 mL of clonidine hydrochloride (effective content 100 mg) was injected intramuscularly every other day for a total of five injections, with a 28-day withdrawal period. Hair samples were taken from the pigs' heads on days 5, 10, 17, 24, 31, 38, and 45 of the entire cycle (the entire hair was shaved from the root using a razor) for analysis. The results are shown in the table below.

[0173] Table 9 Comparison of Veterinary Drug Residue Detection Results

[0174]

[0175] The data in the table show that pig hair detection takes longer than blood detection, but the data is more stable. This method can be used to detect veterinary drug residues in livestock.

[0176] Comparative Experiment 1: The effect of extraction solvent on the detection method of novel receptor agonist drug residues in pig hair;

[0177] (1) Test methods

[0178] The experimental and control groups were set up as follows:

[0179] Experimental group: Same as Example 1;

[0180] Control group 1: 80 mg of blank pig hair was taken and 3.2 μL, 64 μL, and 128 μL of a mixed stock solution of clonidine, romifedipine, and loratadine (100 ng / mL) were added respectively to achieve concentrations of 4 μg / kg, 80 μg / kg, and 160 μg / kg. Acetonitrile and ethyl acetate were used as extraction solvents respectively. The samples were pretreated and analyzed as described in Example 1, and other conditions were the same as in Example 1.

[0181] (2) Test results

[0182] Dimethyl sulfoxide (DMSO) has a high recovery rate as an extraction solvent, while acetonitrile and ethyl acetate have low recovery rates as extraction solvents.

[0183] Table 10 Recovery rates (%) of clonidine, romifedipine, and loratadine in blank samples determined by different extraction solvents (n=3)

[0184]

[0185] Comparative Experiment 2: The effect of solid-phase extraction column on the detection method of novel receptor agonist drug residues in pig hair;

[0186] (1) Test methods

[0187] The experimental and control groups were set up as follows:

[0188] Experimental group: Same as Example 1;

[0189] Control group 2: Take 80 mg of blank pig hair and add 3.2 μL, 64 μL and 128 μL of a mixed stock solution of clonidine, romifedipine and loratadine at 100 ng / mL respectively to achieve concentrations of 4 μg / kg, 80 μg / kg and 160 μg / kg. Change the solid phase extraction column, keep other conditions unchanged, and perform sample pretreatment and analysis as described in Example 1.

[0190] (2) Experimental results:

[0191] Prime HLB columns showed higher recovery rates when used as solid-phase extraction columns, while C18 and amino columns showed lower recovery rates.

[0192] Table 11 Recovery rates (%) of clonidine, romifedipine, and loratadine in blank samples (n=3)

[0193]

[0194] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting novel receptor agonist drug residues in pig hair, characterized in that, Includes the following steps: S1. The pig hair was washed sequentially with water, solvent, and water. Extraction was performed in a dimethyl sulfoxide formate solution. After initial centrifugation, the mixture was filtered through a 0.45 μm filter membrane. The entire filtrate was then passed directly through an Oasis PRIME HLB column to obtain the filtrate. The filtrate was dried under nitrogen, dissolved in methanol, and centrifuged a second time to obtain the test solution. S2. The test solution was analyzed by chromatography-mass spectrometry. The solvent was a mixture of cyclohexane and n-hexane in a volume ratio of 1:1 to 1:

3. The receptor agonists were clonidine, romifedipine, and loratadine.

2. The method for detecting novel receptor agonist drug residues in pig hair according to claim 1, characterized in that, The extraction time is 5-10 minutes.

3. The method for detecting novel receptor agonist drug residues in pig hair according to claim 1, characterized in that, The formic acid in the formic acid dimethyl sulfoxide solution has a volume fraction of 0.1% to 0.3%.

4. The method for detecting novel receptor agonist drug residues in pig hair according to claim 1, characterized in that, The initial centrifugation is performed with a centrifugal force of 9000~11000g / min, a centrifugation temperature of 3~5℃, and a centrifugation time of 1~5min; the secondary centrifugation is performed with a centrifugal force of 9000~11000g / min, a centrifugation temperature of 3~5℃, and a centrifugation time of 1~3min.

5. The method for detecting novel receptor agonist drug residues in pig hair according to claim 1, characterized in that, The conditions for the chromatographic mass spectrometry detection were as follows: column: WatersAcquityHSS T3, flow rate: 0.3~0.5mL / min, column temperature: 30~35℃, injection volume: 1~3μL.

6. The method for detecting novel receptor agonist drug residues in pig hair according to claim 5, characterized in that, In the chromatographic mass spectrometry detection, the mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is a 0.1% (v / v) formic acid aqueous solution, and mobile phase B is methanol.

7. The method for detecting novel receptor agonist drug residues in pig hair according to claim 6, characterized in that, The chromatographic mass spectrometry detection uses gradient elution. The gradient elution program, based on the volume fraction of the mobile phase, is as follows: 0 min to 2 min: keep 10% B constant; 2 min to 4 min: B increases linearly from 10% to 95%; 4 min to 5 min: keep 95% B constant; 5 min to 5.1 min: B decreases linearly from 90% to 10%; 5.1 min to 6 min: keep 10% B constant.

8. The method for detecting novel receptor agonist drug residues in pig hair according to claim 1, characterized in that, The ion source parameters for the chromatography-mass spectrometry detection are as follows: electrospray ionization source; ion source voltage: 3500~4200V; ion source temperature: 350~380℃; sheath gas flow rate: 30~55arb; auxiliary gas flow rate: 5~15arb; capillary transfer tube temperature: 350~380℃; S-lens RF level: 40~80.

9. The method for detecting novel receptor agonist drug residues in pig hair according to claim 1, characterized in that, The chromatographic mass spectrometry detection method includes a first-level full-scan mass spectrometry scan and a second-level mass spectrometry scan.

10. The method for detecting novel receptor agonist drug residues in pig hair according to claim 9, characterized in that, The scanning conditions for the chromatographic mass spectrometry detection are as follows: positive mode, full scan resolution of primary mass spectrometry: 70000, scan range: 100~1000m / z, maximum residence time: 50ms, scan resolution of secondary mass spectrometry: 17500, maximum residence time: 50ms, and step fragmentation energy values: 20, 40, and 60.

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