Method for detecting anticoccidial drugs in poultry products by liquid chromatography-mass spectrometry

Abstract

The invention provides a method for detecting anticoccidial drugs in poultry products through liquid chromatography-mass spectrometry, and belongs to the technical field of anticoccidial drug detection methods. The method comprises the following steps: dissolving a poultry product sample in an acetonitrile-formic acid-water extracting solution for ultrasonic extraction, then passing through an HLB (Hydrophile-Lipophile Balance) purification column, leaching and eluting to obtain an eluent, then drying the eluent by nitrogen gas, and finally dissolving with a 10% acetonitrile water solution and filtering to obtain a sample solution; and determining the anticoccidial drugs in the sample solution by adopting a liquid chromatography-tandem mass spectrometer, and obtaining the content of each anticoccidial drug in combination with the linear equation. The detection method disclosed by the invention is high in sensitivity, simple in pretreatment, good in repeatability, capable of quantitatively detecting residues, wide in practicability and capable of rapidly and accurately determining the content of the anticoccidial drugs in poultry products, and has important significance on enhancing safety monitoring of the anticoccidial drugs.

Description

Technical Field

[0001] The invention relates to the technical field of anticoccidial drug detection methods, and in particular to a method for detecting anticoccidial drugs in poultry products by liquid chromatography-mass spectrometry. Background Art

[0002] Coccidiosis mainly parasitizes the host's intestines (chickens, ducks, pigeons, cattle, sheep, etc.), livers (rabbits), kidneys (geese), etc., causing varying degrees of damage to them. It is one of the major diseases in the poultry farming industry, causing hundreds of millions of dollars in losses each year. Coccidiostats are mainly used for the prevention of coccidiosis, which can improve feed utilization, reduce the risk of secondary diseases, and promote growth. It is inevitable that unscrupulous businesses will use this feature to seek more profits, leading to the abuse of coccidiostats. However, coccidiostats are highly toxic and can enter the human body through the food chain, which has also attracted great attention.

[0003] Many drugs in feed are detected by high performance liquid chromatography-tandem mass spectrometry. Due to the complex matrix effect of mass spectrometers, and the high content of raw materials in premixes, they are detected after multiple dilutions, which inevitably causes drug loss in the process, affecting the accuracy of the final test results. In addition, the mass spectrometry detection method is complex and has high requirements for instruments. Ultrasonic extraction, HLB solid phase extraction column purification pretreatment combined with ultra-high performance liquid chromatography tandem mass spectrometry technology UPLC-MS / MS has the advantages of a wide range of applications, high separation efficiency, strong specificity and high sensitivity, and has become one of the important methods for pretreatment and analysis of organic matter.

[0004] However, when the existing ultrasonic extraction, HLB solid phase extraction column purification pretreatment combined with ultra-high performance liquid chromatography tandem mass spectrometry are used for the detection of anticoccidial drugs in livestock and poultry feces, most of them have more or less problems such as unstable test results, many interferences, limited number of detected antibiotics and recovery rate due to inappropriate extraction methods and extraction solvents, or improper selection of solid phase extraction pretreatment conditions, liquid chromatography conditions and / or mass spectrometry conditions.

[0005] Therefore, there is an urgent need for a LC-MS method for detecting anticoccidial drugs in poultry products with stable test results and few interference factors. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting anticoccidial drugs in poultry products by liquid chromatography-mass spectrometry, so as to solve the technical problems of many interference factors and unstable detection results in the prior art.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for detecting anticoccidial drugs in poultry products by liquid chromatography-mass spectrometry, comprising the following steps:

[0009] (1) preparing a mixed standard solution; dissolving each anticoccidial drug in an acetonitrile-formic acid-water extract to obtain a standard stock solution, then dissolving each standard stock solution in an acetonitrile aqueous solution to obtain a mixed standard working solution, and detecting the content of each anticoccidial drug in the mixed standard working solution by liquid chromatography-mass spectrometry to obtain a corresponding linear equation;

[0010] (2) preparing a sample solution: dissolving a poultry product sample in an acetonitrile-formic acid-water extract and performing ultrasonic extraction, then passing the sample through an HLB purification column, washing and eluting to obtain an eluent, then drying the eluent with nitrogen gas, and finally dissolving the eluent with a 10% acetonitrile aqueous solution, and filtering to obtain a sample solution;

[0011] (3) using liquid chromatography-tandem mass spectrometry to determine the anticoccidial drugs in the sample solution, and combining with a linear equation to obtain the content of each anticoccidial drug;

[0012] In step (3), the liquid chromatography conditions are: chromatographic column: C 18 Chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 0.3~1mL / min; injection volume: 2~20μL; column temperature: 30~50℃.

[0013] Furthermore, in step (3), the mass spectrometry conditions are: using an electrospray ion source negative ion scanning mode and a multiple reaction monitoring mode; the ion source temperature is 100-200°C, the temperature of the ionized solvent is 400-600°C, the ionized gas flow rate is 1000L / h, the desolvation gas is nitrogen, the collision gas is argon, the capillary voltage is 1-5kV, the cone voltage is 20-40V, and the cone gas flow rate is 30-60L / h.

[0014] Further, in step (1), the anticoccidial drugs include diclazuril, toltrazuril, toltrazuril sulfone and nicarbazine;

[0015] The poultry products include eggs.

[0016] Furthermore, the mass concentration of each standard stock solution is independently 0.5-1.5 mg / mL; and the mass concentration of the mixed standard solution is 1-500 ng / mL.

[0017] Furthermore, in steps (1) and (2), the acetonitrile-formic acid-water extract independently consists of 30% by mass of acetonitrile, 0.5% by mass of formic acid and the remainder of water.

[0018] Furthermore, in step (2), the ultrasonic extraction time is 10 to 20 minutes.

[0019] Furthermore, in step (2), the washing reagent is a 5% methanol aqueous solution, the eluting reagent is a mixed solution of 90% acetonitrile and 10% methanol, and the flow rate of the eluting reagent is 1-2 mL / min.

[0020] Furthermore, in step (2), the temperature of the nitrogen drying is 35-45°C.

[0021] Furthermore, in step (2), the mass volume ratio of the sample in the sample solution to the acetonitrile-formic acid-water extract is 3-10 g:5-50 mL.

[0022] Furthermore, in step (3), the procedure of gradient elution in the liquid chromatography detection is:

[0023] 0min: 10% methanol;

[0024] 0-2min, excluding 0min: 10-80% methanol;

[0025] 2-6 min, excluding 2 min: 80-95% methanol;

[0026] 6-8min, excluding 6min: 95% methanol;

[0027] 8-9 min, excluding 8 min: 95-10% methanol;

[0028] 9-10min, excluding 9min: 10% methanol.

[0029] Beneficial effects of the present invention:

[0030] The invention provides a method for determining the content of anticoccidial drugs in poultry products by liquid chromatography-tandem mass spectrometry, using acetonitrile-formic acid-water extract as a sample extract, HLB purification column for purification, 0.1% ammonium acetate-0.5% formic acid-water mixed solution as mobile phase A, and methanol as mobile phase B. The detection method of the invention has high sensitivity, simple pretreatment, good repeatability, can quantitatively detect residues, has wide practicality, and can also quickly and accurately determine the content of anticoccidial drugs in poultry products, which is of great significance for strengthening the safety monitoring of anticoccidial drugs. DETAILED DESCRIPTION

[0031] The present invention provides a method for detecting anticoccidial drugs in poultry products by liquid chromatography-mass spectrometry, comprising the following steps:

[0032] (1) preparing a mixed standard solution; dissolving each anticoccidial drug in an acetonitrile-formic acid-water extract to obtain a standard stock solution, then dissolving each standard stock solution in an acetonitrile aqueous solution to obtain a mixed standard working solution, and detecting the content of each anticoccidial drug in the mixed standard working solution by liquid chromatography-mass spectrometry to obtain a corresponding linear equation;

[0033] (2) preparing a sample solution: dissolving a poultry product sample in an acetonitrile-formic acid-water extract and performing ultrasonic extraction, then passing the sample through an HLB purification column, washing and eluting to obtain an eluent, then drying the eluent with nitrogen gas, and finally dissolving the eluent with a 10% acetonitrile aqueous solution, and filtering to obtain a sample solution;

[0034] (3) Liquid chromatography-tandem mass spectrometry was used to determine the anticoccidial drugs in the sample solution, and the content of each anticoccidial drug was obtained by combining a linear equation.

[0035] In the present invention, in step (3), the liquid chromatography conditions are: chromatographic column: C 18 Chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 0.3~1mL / min; injection volume: 2~20μL; column temperature: 30~50℃.

[0036] Preferably, the liquid chromatography conditions are: chromatographic column: C18 chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 0.4~0.9mL / min; injection volume: 3~15μL; column temperature: 32~48℃.

[0037] Further preferably, the liquid chromatography conditions are: chromatographic column: C18 chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 0.5~0.8mL / min; injection volume: 5~10μL; column temperature: 35~45℃.

[0038] In the present invention, in step (3), the mass spectrometry conditions are: using an electrospray ion source negative ion scanning mode and a multiple reaction monitoring mode; the ion source temperature is 100-200°C, the temperature of the ionized solvent is 400-600°C, the ionized gas flow rate is 1000L / h, the desolvation gas is nitrogen, the collision gas is argon, the capillary voltage is 1-5kV, the cone voltage is 20-40V, and the cone gas flow rate is 30-60L / h.

[0039] Preferably, in step (3), the mass spectrometry conditions are: using an electrospray ion source negative ion scanning mode and a multiple reaction monitoring mode; the ion source temperature is 110 to 190°C, the temperature of the ionized solvent is 420 to 580°C, the ionized gas flow rate is 1000 L / h, the desolvation gas is nitrogen, the collision gas is argon, the capillary voltage is 1.2 to 4.5 kV, the cone voltage is 22 to 38 V, and the cone gas flow rate is 32 to 58 L / h.

[0040] Further preferably, in step (3), the mass spectrometry conditions are: using an electrospray ion source negative ion scanning mode and a multiple reaction monitoring mode; the ion source temperature is 120-180°C, the temperature of the ionized solvent is 450-550°C, the ionized gas flow rate is 1000L / h, the desolvation gas is nitrogen, the collision gas is argon, the capillary voltage is 1.5-4kV, the cone voltage is 25-35V, and the cone gas flow rate is 35-55L / h.

[0041] In the present invention, in step (1), the anticoccidial drugs include diclazuril, toltrazuril, toltrazuril sulfone and nicarbazine.

[0042] In the present invention, the poultry product is preferably eggs.

[0043] In the present invention, the mass concentration of each standard stock solution is independently 0.5-1.5 mg / mL, preferably 0.6-1.3 mg / mL, and more preferably 0.8-1.2 mg / mL; the mass concentration of the mixed standard solution is 1-500 ng / mL, preferably 5-450 ng / mL, and more preferably 10-400 ng / mL.

[0044] In the present invention, in steps (1) and (2), the acetonitrile-formic acid-water extract independently consists of 30% by mass of acetonitrile, 0.5% by mass of formic acid and the remainder of water.

[0045] In the present invention, in step (2), the ultrasonic extraction time is 10 to 20 minutes, preferably 12 to 18 minutes, and more preferably 14 to 16 minutes.

[0046] In the present invention, in step (2), the eluting reagent is a 5% methanol aqueous solution, the eluting reagent is a mixed solution of 90% acetonitrile and 10% methanol, and the flow rate of the eluting reagent is 1 to 2 mL / min, preferably 1.2 to 1.8 mL / min, and more preferably 1.4 to 1.6 mL / min.

[0047] In the present invention, in step (2), the temperature of the nitrogen drying is 35 to 45°C, preferably 36 to 43°C, and more preferably 37 to 40°C.

[0048] In the present invention, in step (2), the mass volume ratio of the sample in the sample solution to the acetonitrile-formic acid-water extract is 3-10 g:5-50 mL, preferably 3.5-9 g:7-40 mL, and more preferably 4-8 g:10-30 mL.

[0049] In the present invention, in step (3), the procedure of gradient elution in the liquid chromatography detection is:

[0050] 0min: 10% methanol;

[0051] 0-2min, excluding 0min: 10-80% methanol;

[0052] 2-6 min, excluding 2 min: 80-95% methanol;

[0053] 6-8min, excluding 6min: 95% methanol;

[0054] 8-9 min, excluding 8 min: 95-10% methanol;

[0055] 9-10min, excluding 9min: 10% methanol.

[0056] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0057] Example 1

[0058] Weigh 10.00 mg (accurate to 0.01 mg) of each of the four anticoccidial drugs, dissolve them in acetonitrile-formic acid-water extract (the acetonitrile-formic acid-water extract consists of 30% acetonitrile by mass, 0.5% formic acid by mass, and the remainder is water) and dilute to 10 mL to prepare standard stock solutions with a mass concentration of 1 mg / mL. Accurately pipette 1.0 mL of the standard stock solutions of various anticoccidial drugs into a 100 mL volumetric flask, dilute to the mark with acetonitrile-formic acid-water extract to prepare a 10 μg / mL mixed standard intermediate solution. Use acetonitrile-formic acid-water extract to dilute the mixed standard intermediate solution to obtain 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL mixed standard working solutions, respectively, for standby use;

[0059] Weigh 5.00 g of egg sample (accurate to 0.01 g) in a 50 mL centrifuge tube, add 10 mL of acetonitrile-formic acid-water extract, place in an ultrasonic cleaner and ultrasonicate at a power of 50 kHz for 20 min, pass the supernatant HLB purification column, use 5% methanol aqueous solution for elution, and after the elution, use a mixed solution of 90% acetonitrile and 10% methanol for elution at a flow rate of 1.5 mL / min to obtain an eluent; blow dry the eluent with nitrogen at 40° C., and finally dissolve it with 10% acetonitrile aqueous solution, filter it to obtain a sample solution, and use liquid chromatography-tandem mass spectrometry to determine the content of anticoccidial drugs in the sample solution;

[0060] The determination conditions of the mixed standard working solution and sample solution are:

[0061] Liquid chromatography conditions are: Chromatographic column: C 18 Chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 0.5mL / min; injection volume: 10μL; column temperature: 40℃.

[0062] The mass spectrometry conditions were as follows: electrospray ionization source negative ion scanning mode and multiple reaction monitoring mode; ion source temperature was 150°C, ionization solvent temperature was 500°C, ionization gas flow rate was 1000 L / h, desolvation gas: nitrogen, collision gas: argon, capillary voltage: 3 kV, cone voltage: 30 V, cone gas flow rate: 50 L / h.

[0063] Example 2

[0064] Compared with Example 1, the only difference is that the measurement conditions are different. The measurement conditions of the liquid chromatography in Example 2 are:

[0065] Chromatographic column: C 18 Chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 0.3mL / min; injection volume: 3μL; column temperature: 35°C.

[0066] Example 3

[0067] Compared with Example 1, the only difference is the different measuring conditions. The measuring conditions of the liquid chromatography in Example 3 are:

[0068] Chromatographic column: C 18 Chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 1mL / min; injection volume: 20μL; column temperature: 35℃.

[0069] Comparative Example 1

[0070] Compared with Example 1, other conditions remain unchanged, and the only difference is that the extracting solution is a 30% acetonitrile aqueous solution.

[0071] Comparative Example 2

[0072] Compared with Example 1, other conditions remain unchanged, and the only difference is that the extracting solution is a 0.5% formic acid aqueous solution.

[0073] Comparative Example 3

[0074] Compared with Example 1, other conditions remain unchanged, and the only difference is that the mobile phase A is changed to water.

[0075] Comparative Example 4

[0076] Compared with Example 1, other conditions remain unchanged, and the only difference is that mobile phase B is changed to acetonitrile.

[0077] The extraction efficiency of Example 1 and Comparative Examples 1-2 was tested, and the test results are shown in Table 1.

[0078] Table 1

[0079] Extraction efficiency (%) Example 1 Comparative Example 1 Comparative Example 2 Diclazuril 99.0 84.3 80.1 Toltrazuril 99.4 83.4 79.4 Toltrazuril sulfone 99.2 82.1 79.2 Nicarbazine 99.1 73.3 83.6

[0080] As can be seen from Table 1, the acetonitrile-formic acid-water extract used in the present invention has a good extraction efficiency for the anticoccidial drug. When 30% acetonitrile aqueous solution and 0.5% formic acid aqueous solution are used as extraction solvents for comparison, the extractant of the present invention is higher than 30% acetonitrile aqueous solution and 0.5% formic acid aqueous solution in the extraction of diclazuril, toltrazuril and toltrazuril sulfone, and the 30% acetonitrile aqueous solution extract has a better effect on the retention of various analytes when passing through the HLB purification column in the subsequent retention of the analytes, but the extraction efficiency of nicarbazine is low, and the extracted interfering impurities are many, the matrix effect is large, and the pressure for subsequent purification is increased.

[0081] Example 1 and Comparative Examples 3 to 4 were tested, and the results showed that the mass spectrometric response of the target product was significantly improved and the peak shape was better when a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water was added. Therefore, a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water was finally selected as the mobile phase.

[0082] As can be seen from the above embodiments, the present invention provides a method for detecting anticoccidial drugs in poultry products by liquid chromatography-mass spectrometry, wherein each anticoccidial drug is dissolved in an acetonitrile-formic acid-water extract to obtain a standard stock solution, and then various standard stock solutions are dissolved in an acetonitrile aqueous solution to obtain a mixed standard working solution, and the content of each anticoccidial drug in the mixed standard working solution is detected by liquid chromatography-mass spectrometry to obtain a corresponding linear equation; the poultry product sample is dissolved in an acetonitrile-formic acid-water extract for ultrasonic extraction, and then passed through an HLB purification column, rinsed and eluted to obtain an eluent, and then the eluent is dried with nitrogen, and finally dissolved with a 10% acetonitrile aqueous solution, and filtered to obtain a sample solution; the anticoccidial drugs in the sample solution are determined by liquid chromatography-tandem mass spectrometry, and the content of each anticoccidial drug is obtained by combining the linear equation. The detection method of the present invention has high sensitivity, simple pretreatment, good repeatability, can quantitatively detect residues, has wide practicality, and can also quickly and accurately determine the content of anticoccidial drugs in poultry products, which is of great significance for strengthening the safety monitoring of anticoccidial drugs.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting anticoccidial drugs in poultry products by liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: (1) preparing a mixed standard solution; dissolving each anticoccidial drug in an acetonitrile-formic acid-water extract to obtain a standard stock solution, then dissolving each standard stock solution in an acetonitrile aqueous solution to obtain a mixed standard working solution, and detecting the content of each anticoccidial drug in the mixed standard working solution by liquid chromatography-mass spectrometry to obtain a corresponding linear equation; (2) preparing a sample solution: dissolving a poultry product sample in an acetonitrile-formic acid-water extract and performing ultrasonic extraction, then passing the sample through an HLB purification column, washing and eluting to obtain an eluent, then drying the eluent with nitrogen gas, and finally dissolving the eluent with a 10% acetonitrile aqueous solution, and filtering to obtain a sample solution; (3) using liquid chromatography-tandem mass spectrometry to determine the anticoccidial drugs in the sample solution, and combining with a linear equation to obtain the content of each anticoccidial drug; In step (3), the liquid chromatography conditions are: chromatographic column: C 18 Chromatographic column, 100mm×2.1mm, 1.6μm; mobile phase A: a mixed solution of 0.1% ammonium acetate-0.5% formic acid-water; mobile phase B: methanol; flow rate: 0.3~1mL / min; injection volume: 2~20μL; column temperature: 30~50℃.

2. The method according to claim 1, characterized in that: In step (3), the mass spectrometry conditions are: using an electrospray ion source negative ion scanning mode and a multiple reaction monitoring mode; the ion source temperature is 100-200°C, the temperature of the ionized solvent is 400-600°C, the ionized gas flow rate is 1000L / h, the desolvation gas is nitrogen, the collision gas is argon, the capillary voltage is 1-5kV, the cone voltage is 20-40V, and the cone gas flow rate is 30-60L / h.

3. The method according to claim 2, characterized in that In step (1), the anticoccidial drugs include diclazuril, toltrazuril, toltrazuril sulfone and nicarbazine; The poultry products include eggs.

4. The method according to claim 2 or 3, characterized in that: The mass concentration of each standard stock solution is independently 0.5-1.5 mg / mL; the mass concentration of the mixed standard solution is 1-500 ng / mL.

5. The method according to claim 4, characterized in that In steps (1) and (2), the acetonitrile-formic acid-water extract independently consists of 30% by mass of acetonitrile, 0.5% by mass of formic acid and the remainder of water.

6. The method according to claim 5, characterized in that In step (2), the ultrasonic extraction time is 10 to 20 minutes.

7. The method according to claim 5 or 6, characterized in that: In step (2), the washing reagent is a 5% methanol aqueous solution, the eluting reagent is a mixed solution of 90% acetonitrile and 10% methanol, and the flow rate of the eluting reagent is 1-2 mL / min.

8. The method according to claim 7, characterized in that In step (2), the temperature of the nitrogen drying is 35-45°C.

9. The method according to claim 8, characterized in that In step (2), the mass volume ratio of the sample in the sample solution to the acetonitrile-formic acid-water extract is 3-10 g:5-50 mL.

10. The method according to claim 8 or 9, characterized in that: In step (3), the procedure of gradient elution in the liquid chromatography detection is: 0min: 10% methanol; 0-2min, excluding 0min: 10-80% methanol; 2-6 min, excluding 2 min: 80-95% methanol; 6-8min, excluding 6min: 95% methanol; 8-9 min, excluding 8 min: 95-10% methanol; 9-10min, excluding 9min: 10% methanol.