Multi-type veterinary drug mixed standard substance as well as preparation method and application thereof

Abstract

The invention discloses a multi-type veterinary drug mixed standard substance as well as a preparation method and application thereof, and the preparation method comprises the following steps: (1) screening veterinary drug candidates: selecting representative veterinary drugs which are wide in application range, diversified in detection method and easy to cause harm to human bodies, comprising difloxacin, florfenicol, sulfadimidine, trimethoprim, thiamphenicol and fenvalerate. And (2) mixing the selected veterinary drugs in proportion: mixing the difloxacin, the florfenicol, the sulfadimidine, the trimethoprim, the thiamphenicol and the fenvalerate in proportion. The multi-type veterinary drug mixed standard substance developed by the invention has a certain quantity value, can be used for simultaneously measuring multiple veterinary drugs, and can be stably stored at-20 DEG C for 6 months. According to the multi-type veterinary drug mixed standard substance, the detection accuracy of various veterinary drugs under different platforms can be improved; the method can be applied to the detection process of various veterinary drugs, and the accuracy and efficiency of veterinary drug residue detection are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary drug standard substances, and in particular to a multi-type veterinary drug mixed standard substance and a preparation method and application thereof. Background Art

[0002] Veterinary drugs have the functions of preventing infectious diseases in animals and shortening feeding cycles, and are widely used in the animal breeding process. Excessive consumption of veterinary drugs can cause residues in animal-derived foods to exceed safety levels, threatening human health and possibly leading to increased bacterial resistance. The combination of rapid screening technology and quantitative confirmation technology is a universal monitoring model that can effectively improve detection efficiency and ensure food safety. However, there are problems such as performance differences in commercially available rapid screening instruments. In the absence of unified standards, it is difficult to guarantee the accuracy of test results. When the claimed detection limit cannot be reached, problems such as "missed detection" may occur, which is not conducive to the detection of veterinary drug residues in animal foods.

[0003] Moreover, in the process of animal breeding, it is inevitable that a variety of different veterinary drugs are used together. At present, relevant researchers have developed an integrated rapid detection device that can detect multiple veterinary drugs at the same time. However, there is a severe shortage of standard substances containing multiple veterinary drugs on the market, which makes it difficult to meet the needs of integrated rapid detection equipment calibration and on-site detection process quality control.

[0004] Therefore, there is an urgent need in the art to develop a standard substance containing a variety of common veterinary drugs (with different chemical properties) and to establish a quantitative measurement method for multi-component standard substances. Summary of the invention

[0005] The purpose of the present invention is to provide a mixed standard substance of multiple types of veterinary drugs and a preparation method and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a mixed standard substance of multiple types of veterinary drugs comprises the following steps:

[0008] (1) Screening of veterinary drug candidates

[0009] All veterinary drug candidates are from the "National Food Safety Standard Maximum Residue Limits in Food" (GB31650-2019), and the veterinary drugs with approved maximum residue limits in animal foods are divided into alkanes, aromatic hydrocarbons, halogenated hydrocarbons, alcohols, ketones, aldehydes, esters, carboxylic acids and other types according to chemical types. According to the requirements of the national standard and market research, different types of veterinary drugs are statistically analyzed based on applicable animals and tissues, maximum residue limits, commonly used detection methods, commercially available kits, etc., and representative veterinary drugs with a wide range of applications, diverse detection methods and easy to cause harm to humans are selected from different types, including difloxacin, florfenicol, sulfadimethoxine, trimethoprim, thiamphenicol and cypermethrin; the chemical formula is as follows:

[0010]

[0011]

[0012] (2) Mix the selected veterinary drugs according to the proportion

[0013] Difloxacin, florfenicol, sulfamethoxazole, trimethoprim, thiamphenicol and cypermethrin were mixed in proportion, and the concentrations of various veterinary drugs in the mixed solution were as follows:

[0014]

[0015] The mixed solution was shaken thoroughly at room temperature, and no obvious precipitation was found by observation. The multi-type veterinary drug mixed solution was further divided into 2mL ampoules, with each tube containing 1mL, to obtain a multi-type veterinary drug mixed standard substance. The divided multi-type veterinary drug mixed standard substance was stored in a -40℃ refrigerator.

[0016] The method for determining the value of the multi-type veterinary drug mixed standard substances prepared by the present invention is specifically: based on HPLC technology, an HPLC-DAD quantitative method is established, and the excitation wavelengths of the components in the multi-type veterinary drug mixed standard substances are 260, 270, 235, 230, 225, and 245 nm respectively.

[0017] Firstly, acetonitrile-water solution was used as the mobile phase; then, the ratio of methanol to acetonitrile was optimized under the condition of 0.1% formic acid water solution as the mobile phase, and the Agilent TC-C18 chromatographic column was used. Based on the analysis of the recovery rate of each component of veterinary drugs, the working conditions were determined to be 0.1% formic acid water solution as the mobile phase, methanol:acetonitrile = 1:4, flow rate of 0.8mL / min, and injection volume of 20μL;

[0018] Pure veterinary drugs were used to draw standard curves, and 200 μL of gradient-diluted pure veterinary drugs and mixed standard substances of various types of veterinary drugs were placed in sample bottles for determination by HPLC-DAD method.

[0019] Among them, the HPLC gradient elution conditions are:

[0020]

[0021] Wherein, A is 0.1% formic acid aqueous solution; B is methanol:acetonitrile = 1:4.

[0022] The multi-type veterinary drug mixed standard substance prepared by the invention is used in verifying the analytical performance of rapid detection equipment and in veterinary drug detection.

[0023] Compared with the prior art, the outstanding effects of the present invention are:

[0024] (1) The multi-type veterinary drug mixed standard substances with definite values ​​of the present invention can provide a reference for the detection of veterinary drug residues. The scope of application includes but is not limited to: the standard substances can be diluted to the maximum residue amount specified in the national standard to verify the analytical performance of the rapid detection equipment and avoid the occurrence of "false positive" results; the standard substances can be carried to the site to provide a reference for quality control in the detection process of actual samples, avoiding disputes caused by disputes over the accuracy of the results and economic losses caused by repeated measurements.

[0025] (2) The multi-type veterinary drug mixed standard substance of the present invention has a definite value and can be used for simultaneous measurement of multiple veterinary drugs. An HPLC-DAD method for simultaneous measurement has been constructed, and the mixed standard substance can be stably stored at -20°C for 6 months. Compared with single-purity veterinary drugs (which cannot be used for simultaneous detection of mixed residues of multiple veterinary drugs), the multi-type veterinary drug mixed standard substance of the present invention can perform quality control on multiple veterinary drugs at the same time.

[0026] (3) The multi-type veterinary drug mixed standard substance of the present invention can improve the accuracy of detection of various veterinary drugs under different platforms.

[0027] (4) The standard substance and quantitative method prepared by the present invention can be applied to the detection process of various veterinary drugs, thereby improving the accuracy and efficiency of veterinary drug residue detection.

[0028] (5) After a large number of preliminary experiments and explorations, the multi-type veterinary drug mixed standard substances of the present invention finally selected 6 veterinary drugs with different chemical properties and wide applications; and these 6 veterinary drugs do not affect the chemical properties and immunogenicity after mixing, and are most suitable for preparing multi-type veterinary drug mixed standard substances.

[0029] The following is a further description of the multi-type veterinary drug mixed standard substance, preparation method and application of the present invention in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Results were optimized for HPLC separation methods.

[0031] Figure 2 This is the standard curve of sulfamethazine in a mixed standard substance of various types of veterinary drugs.

[0032] Figure 3 This is the standard curve of difloxacin in a mixed standard substance of multiple types of veterinary drugs.

[0033] Figure 4 This is the standard curve of trimethoprim in a mixed standard substance of various types of veterinary drugs.

[0034] Figure 5 This is the standard curve of thiamphenicol in a mixed standard substance of multiple types of veterinary drugs.

[0035] Figure 6 This is the standard curve of florfenicol in a mixed standard substance of multiple types of veterinary drugs.

[0036] Figure 7 This is the standard curve of cypermethrin in a mixed standard substance of multiple types of veterinary drugs.

[0037] Figure 8 These are the short-term stability results of trimethoprim in a mixed reference material of various types of veterinary drugs.

[0038] Fig. 9 These are the short-term stability results of difloxacin in a mixed reference material of multiple types of veterinary drugs.

[0039] Fig.10 These are the short-term stability results of thiamphenicol in a mixed standard substance of various types of veterinary drugs.

[0040] Fig.11 These are the short-term stability results of sulfamethazine in a mixed standard substance of various types of veterinary drugs.

[0041] Fig.12 These are the short-term stability results of florfenicol in a mixed reference material of multiple types of veterinary drugs.

[0042] Fig.13 These are the short-term stability results of cypermethrin in a mixed reference material of various types of veterinary drugs.

[0043] Figure 14-Figure 19 The results of the colloidal gold cross-reaction test of mixed standard substances of various types of veterinary drugs, including difloxacin (A), florfenicol (B), sulfamethoxazole (C), trimethoprim (D), thiamphenicol (E), cypermethrin (F), and multi-component mixed standard substances of veterinary drugs (Mix). Fig.14 It is a sulfadimethoxine colloidal gold test strip. Fig.15 For trimethoprim colloidal gold test strips, Fig.16 It is a difloxacin colloidal gold test strip. Fig.17 It is a florfenicol colloidal gold test strip. Fig.18It is a colloidal gold test strip of thiamphenicol. Fig.19 It is a cypermethrin colloidal gold test strip.

[0044] Fig. 20 This is a photo of the multi-component veterinary drug mixed standard substance prepared according to the present invention. DETAILED DESCRIPTION

[0045] Example 1 Preparation of mixed standard substances of various types of veterinary drugs

[0046] A method for preparing a mixed standard substance of multiple types of veterinary drugs comprises the following steps:

[0047] (1) Screening of veterinary drug candidates

[0048] The candidate veterinary drugs are all from the "National Food Safety Standard Maximum Residue Limits in Food" (GB31650-2019), and the veterinary drugs with approved maximum residue limits in animal foods are divided into alkanes, aromatic hydrocarbons, halogenated hydrocarbons, alcohols, ketones, aldehydes, esters, carboxylic acids and other types according to chemical types. According to the requirements of the national standard and market research, different types of veterinary drugs are statistically analyzed based on applicable animals and tissues, maximum residue limits, commonly used detection methods, commercially available kits, etc., and representative veterinary drugs with a wide range of applications, diverse detection methods and easy to cause harm to humans are selected from different types, including difloxacin, florfenicol, sulfadimethoxine, trimethoprim, thiamphenicol and cypermethrin.

[0049] Table 1 Statistics of pure veterinary drugs

[0050]

[0051] (2) Mix the selected veterinary drugs according to the proportion

[0052] According to the residue limit in the national standard, Difloxacin, Florfenicol, Sulfadimidine, Trimethoprim, Thiamphenicol, and Fenvalerate are mixed in proportion. The concentrations of various veterinary drugs in the mixed solution are as follows:

[0053] Table 2 Mass concentration of each component in multi-component veterinary drug mixed solution

[0054]

[0055] Take 35mL of each of the above solutions and mix them. Shake the mixed solution at room temperature. No obvious precipitation is found by observation. Further, the mixed solution of multiple types of veterinary drugs is divided into 2mL ampoules, each containing 1mL, to obtain a mixed standard substance of multiple types of veterinary drugs. Store the divided mixed standard substances of multiple types of veterinary drugs in a -40℃ refrigerator (such as Fig. 20 shown).

[0056] Example 2 Determination of the value of mixed standard substances of various types of veterinary drugs

[0057] HPLC has excellent separation ability, and DAD can simultaneously record spectral information of multiple wavelengths, ensuring that HPLC-DAD can accurately identify and simultaneously determine multi-component veterinary drugs. First, the existing detection methods for each component in veterinary drug reference materials are summarized (Table 3).

[0058] Table 3 HPLC detection method of each component

[0059]

[0060] Based on HPLC technology, an HPLC-DAD quantitative method was established, and the excitation wavelengths of each component in the mixed standard substances of various types of veterinary drugs were 260, 270, 235, 230, 225, and 245 nm, respectively.

[0061] First, acetonitrile-water solution was used as the mobile phase; then, the ratio of methanol to acetonitrile was optimized under the condition of 0.1% formic acid aqueous solution as the mobile phase (such as Figure 1 As shown), using an Agilent TC-C18 (2) (250 mm × 4.6 mm, 5 μm) column, based on the analysis of the recovery rate of each component veterinary drug, the mobile phase was determined to be 0.1% formic acid aqueous solution, methanol: acetonitrile

[0062] =1:4, flow rate of 0.8mL / min, injection volume of 20μL were the working conditions, which were subsequently used for the determination of standard substances.

[0063] Use pure veterinary drugs to draw a standard curve (such as Figure 2-7 As shown in the figure, 200 μL of gradient diluted pure veterinary drugs and mixed standard substances of various types of veterinary drugs were placed in sample bottles for HPLC-DAD method determination. Under the above optimized working conditions, different components can be clearly distinguished, and the detection time is 24 minutes, which can be used for confirmation after rapid on-site screening results.

[0064] Wherein, the HPLC gradient elution conditions are shown in Table 4.

[0065] Table 4 HPLC gradient elution conditions

[0066]

[0067] Wherein, A is 0.1% formic acid aqueous solution; B is methanol:acetonitrile = 1:4.

[0068] Example 3 Homogeneity Analysis of Standard Substances

[0069] 1. Uniformity test

[0070] (1) The uniformity test was performed using the HPLC-DAD method established in Example 2. According to the technical requirements for the uniformity evaluation of standard substances in the national metrology technical specification JJF 1343-2022 "Value determination and uniformity stability evaluation of standard substances", 11 packing units were randomly selected from various types of veterinary mixed standard substances. The extracted samples were quantified using the HPLC-DAD method, and the measurements were repeated three times from each tube of sample. The data results are shown in Table 5. An F test was performed on the obtained data to compare the differences between component differences and intra-group differences. When the F value is less than the Fa value, it indicates that the standard substance has good uniformity.

[0071] Table 5 Homogeneity test results of mixed standard substances of various veterinary drugs

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] According to formulas (1) and (2), the between-group mean square Mbetween and the between-group degrees of freedom as well as the within-group mean square Mwithin and the within-group degrees of freedom are calculated. According to formula (5), the between-unit standard deviation Sbb is calculated and used as the uncertainty component Ubb introduced for uniformity.

[0082]

[0083] in:

[0084]

[0085]

[0086] Wherein:

[0087]

[0088] 2. Results of homogeneity test

[0089] For the data results determined by HPLC-DAD, the Shapiro-Wilk method was first used to test the normality of the data. After testing, the homogeneity data of all reference materials satisfied the normal distribution. After the F-test, the results showed that for all reference materials, F < F0.05(V1, V2), that is, there was no significant difference in the internal homogeneity of the reference material solution.

[0090] As can be seen from the homogeneity data in Table 5, the results of the homogeneity test of the developed reference materials were F < Fd, indicating that the homogeneity of these reference materials was good.

[0091] Investigation on the stability of the reference material in Example 4

[0092] (1) Investigation on short-term stability

[0093] To determine the stability of the reference material during short-term transportation, the short-term stability test was conducted to ensure the stability of the reference material under transportation conditions. Three temperatures, -20°C, 4°C, and 25°C, were investigated. At 4 time points, the 1st day, 3rd day, 7th day, and 14th day, 3 units were randomly selected respectively. The HPLC-DAD method was used to measure the mass concentration of veterinary drugs in different components. The T-test results showed that compared with the condition of -20°C, under the storage condition of 4°C, the mass concentration of the reference material did not change significantly within 14 days. Therefore, when dry ice is added during transportation or the temperature is controlled not to exceed 4°C during transportation, as long as the transportation time does not exceed 14 days, the quantity value of the reference material can remain stable ( Figure 8-Figure 13 as shown).

[0094] (2) Investigation on long-term stability

[0095] For the investigation on long-term stability, the storage condition of -80°C was selected. The sampling time points followed the principle of being dense first and then sparse. Each time, 3 units were randomly selected for each storage condition to investigate its long-term stability under the condition of -80°C. For the mixed reference material of multiple types of veterinary drugs, the long-term stability was investigated at the 0th month, 1st month, 3rd month, and 6th month respectively.

[0096] The long-term stability analysis results of the multi-type veterinary drug mixed reference material are shown in Table 6. The analysis of the long-term stability data is carried out according to JJF-1343-2022 "Evaluation of the Certification and Homogeneity and Stability of Reference Materials", that is, with time (xi) as the abscissa and mass concentration (yi) as the ordinate, a linear fitting is performed, and the intercept (b0) and slope (b1) of the straight line are recorded respectively. The standard deviation and uncertainty (s(b1)) of the slope are calculated according to Formula (7) and Formula (8). The degree of freedom is n - 2, where n is the number of times of sampling and measurement. According to the t critical value table, the value of t0.95,n - 2 at P = 0.95 (95% confidence level) is looked up. If b1 < s(b1) * t0.95,n - 2, then the reference material is stable under these conditions.

[0097] The long-term stability results (Table 6) show that under the storage condition of -80 °C, for the multi-type veterinary drug mixed reference material, b1 < s(b1) * t0.95,n - 2 within 2 months, and no significant change is found. The quantity value of the reference material is stable under this storage condition.

[0098]

[0099] Table 6 Long-term Stability of Multi-type Veterinary Drug Mixed Reference Material

[0100]

[0101] Example 5 Colloidal Gold Cross-reaction Test

[0102] To verify that the multi-type veterinary drug mixed reference material is applicable to colloidal gold detection, that is, whether each component maintains its original immunogenicity, commercially available colloidal gold test strips are purchased, and the multi-component veterinary drug reference material and pure products are detected respectively. The mixed sample and the pure veterinary drugs of each component are dropped onto the colloidal gold test strips for trimethoprim, difloxacin, sulfamethazine, florfenicol, thiamphenicol, and fenvalerate. If the color of the T line on the colloidal gold test strip is the same as or darker than the C line, it is positive; if the T line has no color or is lighter than the C line, it is negative.

[0103] The results show that: the colloidal gold test strip shows a positive reaction with the corresponding veterinary drug, and the other 5 veterinary drugs are all negative, proving that each component can still maintain its original immunogenicity after mixing ( Figure 14-Figure 19 as shown).

[0104] Example 6 Evaluation of Matrix Effect

[0105] In order to determine the influence of matrix effect on the determination results, the applicability of the standard substance under matrix conditions was determined by adding matrix, extracting and determining the value. Veterinary drugs commonly detected in minced meat (trimethoprim, florfenicol, sulfadimethoxine, thiamphenicol, enrofloxacin, chloramphenicol, clenbuterol) were selected, mixed in methanol, slowly added to the minced meat matrix, and further extracted and purified.

[0106] The specific steps are as follows:

[0107] Accurately weigh 1 g (accurate to 0.01 g) of minced meat into a 15 mL centrifuge tube, add 4 mL of acetonitrile, vortex mix for 5 min, ultrasonically extract for 10 min, centrifuge at 10000 rpm for 10 min at 4 ° C, transfer the supernatant to another centrifuge tube, add 30 mg PSA powder and 30 mg C18 powder, vortex mix for 5 min, centrifuge at 10000 r / min for 10 min, aspirate the supernatant, filter through a 0.45 μM syringe filter, and concentrate to dryness on a concentrating centrifuge.

[0108] The residue was dissolved in 1 mL of methanol and used for HPLC-DAD analysis.

[0109] Table 7

[0110]

[0111] The above results show that this method is suitable for the extraction and purification of veterinary drugs in the matrix and can provide a reference for the detection of multiple types of mixed veterinary drugs in actual samples.

[0112] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a mixed standard substance of multiple types of veterinary drugs, characterized in that: The following steps are involved: (1) Screening of veterinary drug candidates Select representative veterinary drugs with wide application range, diverse detection methods and easy to cause harm to human body, including difloxacin, florfenicol, sulfadimethoxine, trimethoprim, thiamphenicol and cypermethrin; (2) Mix the selected veterinary drugs according to the proportion Difloxacin, florfenicol, sulfamethoxazole, trimethoprim, thiamphenicol and cypermethrin were mixed in proportion, and the concentrations of various veterinary drugs in the mixed solution were as follows:

2. The method for preparing a multi-type veterinary drug mixed standard substance according to claim 1, characterized in that: In the step (2), the mixed solution is placed at room temperature and shaken thoroughly. No obvious precipitation is observed. The mixed solution of multiple types of veterinary drugs is further divided into 2 mL ampoules, each containing 1 mL, to obtain a mixed standard substance of multiple types of veterinary drugs.

3. The method for preparing a multi-type veterinary drug mixed standard substance according to claim 2, characterized in that: In the step (2), the packaged mixed standard substances of various types of veterinary drugs are stored in a -40°C refrigerator.

4. A multi-type veterinary drug mixed standard substance prepared according to the preparation method according to any one of claims 1 to 3.

5. The method for determining the value of a multi-type veterinary drug mixed standard substance according to claim 4, characterized in that: Based on HPLC technology, an HPLC-DAD quantitative method was established, and the excitation wavelengths of each component in the mixed standard substances of various types of veterinary drugs were 260, 270, 235, 230, 225, and 245 nm, respectively.

6. The method for determining the value of a multi-type veterinary drug mixed standard substance according to claim 5, characterized in that: Firstly, acetonitrile-water solution was used as the mobile phase; then, the ratio of methanol to acetonitrile was optimized under the condition of 0.1% formic acid water solution as the mobile phase, and the Agilent TC-C18 chromatographic column was used. Based on the analysis of the recovery rate of each component of veterinary drugs, the working conditions were determined to be 0.1% formic acid water solution as the mobile phase, methanol:acetonitrile = 1:4, flow rate of 0.8mL / min, and injection volume of 20μL; Pure veterinary drugs were used to draw standard curves, and 200 μL of gradient-diluted pure veterinary drugs and mixed standard substances of various types of veterinary drugs were placed in sample bottles for determination by HPLC-DAD method.

7. The method for determining the value of a multi-type veterinary drug mixed standard substance according to claim 6, characterized in that: The HPLC gradient elution conditions were: Wherein, A is 0.1% formic acid aqueous solution; B is methanol:acetonitrile = 1:

4.

8. Use of the multi-type veterinary drug mixed standard substance as described in claim 4 in verifying the analytical performance of rapid detection equipment and in veterinary drug detection.

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