CN112526020B - Method for detecting residual triazamidine - Google Patents

Abstract

The invention discloses a method for detecting triazamidine residues, which comprises the steps of extracting triazamidine in a substance to be detected by using 80% acetonitrile aqueous solution, and then carrying out quantitative analysis on the triazamidine by using an ultra-high performance liquid chromatography-tandem mass spectrometry. The method uses 80% acetonitrile water solution for extraction, and reduces the volatile hazard caused by extraction with organic reagent. The method has a wide detection range, can be used for detecting the triazamidine not only aiming at animal muscle tissues but also aiming at matrixes such as dairy products, kidneys and the like by adopting liquid substances, and avoids the problem of false positive caused by the influence of the matrixes when liquid phase detection is adopted.

Description

Method for detecting residual triazamidine

Technical Field

The invention relates to the field of veterinary drug residue detection, and particularly relates to a method for detecting triazamidine residues.

Background

Amitraz, which belongs to aromatic diamidine class, is a broad-spectrum anti-hematogen drug traditionally used, and has therapeutic effect on livestock pyriformis, trypanosomes and anaplasma. From the existing clinical reports, the safe range of the amitraz is small, adverse reactions are easy to occur when the amitraz is continuously used or the dosage is large, the amitraz is quickly absorbed and distributed after entering the animal body, is slowly eliminated, is hardly metabolized in the body and is mainly discharged out of the body through the kidney in a prototype mode. Studies have shown that few residues remain in lactating cows on day 21 after administration. In order to reduce the chance that medicines enter human bodies through food chains, china and world health organizations limit the maximum residual quantity of the triazamidine in animal milk and animal muscle tissues, but currently, few national standards and related documents describe the detection of the triazamidine, and the communication with related enterprises is recently found to use the triazamidine, but no related detection technology exists at present, so that a related detection method of the triazamidine is researched.

Disclosure of Invention

In order to solve the technical problem, the invention discloses a method for detecting residual triazamidine, which comprises the steps of extracting the triazamidine in a substance to be detected by using an 80% acetonitrile aqueous solution, and then carrying out quantitative analysis on the triazamidine by using an ultra performance liquid chromatography-tandem mass spectrometry.

As an alternative of the technical scheme of the invention, the object to be measured is animal muscle tissue, animal kidney or animal dairy product, and the using state of the object to be measured is pulpiness.

As an alternative of the technical scheme of the invention, the mass spectrum parameters in the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows,

a chromatographic column: agilent eclipse PlusC18 RRHD (2.1 mm. Times.100mm, 1.8 μm)

Column temperature: 40 deg.C

Flow rate: 0.4mL/min

Sample introduction amount: 2 μ L

Mobile phase: a-0.1% formic acid water +5mmol/L ammonium acetate, B-methanol;

the chromatographic parameters in the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows:

an ion source: ESI +

Ion source temperature: 325 deg.C

Flow rate of atomizing gas: 9L/min

Nozzle pressure: 45psi

Temperature of the auxiliary heater: 350 deg.C

Flow rate of the auxiliary heater: 10L/min.

As an alternative of the technical scheme of the invention, the detection method comprises the following steps,

s1: preparing a standard solution, wherein the standard solution comprises a standard stock solution, a standard working solution and a standard solution, and the standard stock solution is a 1000mg/L solution obtained by diluting 10mg of a triazamidine standard substance by deionized water to a constant volume of 10 ml; the standard working solution is a 1mg/L solution obtained by constant volume of the standard stock solution through a methanol water solution (methanol 50+ water 50); the standard solution is a solution with the volume of 0ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL and 20ng/mL obtained by the standard working solution through methanol water solution (methanol 50+ water 50);

s2, preparing a detection sample, namely accurately weighing 5.00g of an object to be detected, placing the object to be detected into a 50mL centrifuge tube after pretreatment, adding 20mL 80% acetonitrile water solution, and extracting according to the following steps:

s2-1, swirling for 1min to fully and uniformly mix the sample and acetonitrile water;

s2-2, putting the sample liquid after the vortex into ultrasonic equipment for ultrasonic treatment for 10min;

s2-3, centrifuging the sample liquid for 5min, wherein the rotating speed of a centrifuge is 4000rpm/min, and pouring all centrifuged supernatant into another 50ml centrifuge tube;

s2-4, adding 10mL of 80% acetonitrile water solution into the centrifuge tube, extracting again according to the steps of S2-1, S2-2 and S2-3, and collecting final centrifugate;

s2-5, adding 4g of solid sodium chloride and 10mL of saturated n-hexane into the filtrate in the final centrifugate for liquid-liquid distribution, removing an n-hexane layer, transferring an acetonitrile layer into a 100mL heart-shaped bottle, carrying out rotary evaporation at 40 ℃ until the acetonitrile layer is nearly dry, carrying out constant volume treatment to 1mL by using 50% methanol water solution, and filtering through a 0.22 mu m filter membrane to obtain a final detection sample;

s3, drawing a standard curve, sequentially carrying out machine detection on the standard solution according to the concentration from low to high, and drawing the standard curve by taking the peak area of the triazamidine as a vertical coordinate and the concentration as a horizontal coordinate;

and S4, sample determination, namely placing the final detection sample obtained in the S2 in a sample bottle for measurement in an up-loading machine.

The beneficial effects of the invention are:

1. the method uses 80% acetonitrile water solution for extraction, and reduces the volatile hazard caused by extraction with organic reagent.

2. The method has wide detection range, and can be used for not only animal muscle tissues but also substrates such as dairy products, kidneys and the like.

3. The method adopts liquid to detect the triazamidine, thereby avoiding the problem of false positive caused by matrix influence when liquid phase is adopted for detection.

4. The method makes up for the blank of the detection of the triazamidine in the national standard, and plays a reference role for other compounds of the same class.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without inventive labor.

FIG. 1 is a spectrum of the high performance liquid chromatography mass spectrometry of triazamidine, wherein the ordinate is response and the abscissa is m/z.

FIG. 2 measurement of triazamidine retention time with response on the ordinate and retention time on the abscissa.

FIG. 3 quantitative limit determination of triazamidine, where the ordinate is response and the abscissa is retention time, the upper four panels of the figure represent S/N of 3 daughter ions, respectively.

FIG. 4 is a standard curve of triazamidine in which the ordinate represents the peak area response and the abscissa represents the concentration of triazamidine.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

A method for detecting residual triazamidine specifically operates as follows.

1. Preparation of Standard solutions

1.1 preparation of stock solutions

1.1.1 accurately weighing 10mg of triazamidine standard substance, fixing the volume by 10ml by deionized water, preparing 1000mg/L standard stock solution, and storing in a refrigerator;

1.1.2 the standard stock solution is subjected to constant volume by a methanol aqueous solution (methanol 50+ water 50) to prepare 1mg/L standard working solution accurately, and the standard working solution is placed in a refrigerator for storage.

1.1.3 preparation of Standard working Curve Standard solutions

The standard working solution is prepared into standard solutions with the constant volume of 0 mug/L, 1 mug/L, 2 mug/L, 5 mug/L, 10 mug/L and 20 mug/L through methanol water solution (methanol 50+ water 50) and used for drawing a standard curve.

2. Analytical procedure

2.1 preparation of samples

Preparation of a detection sample, namely accurately weighing 5.00g of a substance to be detected, placing the substance to be detected into a 50mL centrifuge tube after pretreatment, adding 20mL of 80% acetonitrile water solution, and extracting according to the following steps:

1) Vortexing for 1min to mix the sample and acetonitrile water;

2) Putting the sample liquid after the vortex into ultrasonic equipment for ultrasonic treatment for 10min;

3) Centrifuging the sample solution for 5min, wherein the rotation speed of a centrifuge is 4000rpm/min, and pouring all centrifuged supernatant into another 50ml centrifuge tube;

4) Adding 10mL of 80% acetonitrile aqueous solution into the centrifuge tube, extracting again according to the steps of S2-1, S2-2 and S2-3, and collecting final centrifugate;

5) And adding 4g of solid sodium chloride and 10mL of saturated n-hexane into the filtrate of the final centrifugate for liquid-liquid distribution, removing the n-hexane layer, transferring the acetonitrile layer into a 100mL heart-shaped bottle, carrying out rotary evaporation at 40 ℃ until the acetonitrile layer is nearly dry, carrying out constant volume treatment to 1mL by using 50% methanol aqueous solution, and filtering the solution through a 0.22 mu m filter membrane to obtain a final detection sample.

2.2 Instrument conditions (Agilent LC-MSMS 1290/6460)

2.2.1 chromatographic parameters:

and (3) chromatographic column: agilent eclipse plus C18 RRHD 1.8 μm,2.1mm 100mm

Column temperature: 40 deg.C

Flow rate: 0.4mL/min

Sample injection amount: 2 μ L

Mobile phase and its gradient:

a-0.1% formic acid water +5mmol/L ammonium acetate

B-methanol, concentration gradient as in Table 1.

TABLE 1 concentration gradient of methanol

2.2.2 Mass Spectrometry parameters:

an ion source: ESI +

Ion source temperature: 325 deg.C

Flow rate of atomizing gas: 9L/min

Nozzle pressure: 45psi

Temperature of the auxiliary heater: 350 deg.C

Flow rate of the auxiliary heater: 10L/min

3. Analysis of results

3.1 specificity

1ppm Triazamidine was loaded onto a computer, as shown in FIG. 1, and 3 ion pairs were obtained by mass spectrometry optimization, with the ordinate being response and the abscissa being m/z.

The details are shown in Table 2.

TABLE 2 Triazamidine Mass Spectrometry optimization of ion Pair parameters

3.2 Retention time

Mu.g/kg of triazamidine were separated by C18 column and the retention time was determined, as shown in FIG. 2.

3.3 detection Limit

The matrix was added with a standard sample containing 1.0. Mu.g/kg, and the signal-to-noise ratio (S/N) =134.0 > 10 was calculated by pre-treatment machine detection, as shown in FIG. 3.

3.4 Linear relationship

Sequentially detecting the standard solution from low concentration to high concentration by using a computer, drawing a standard curve by taking the peak area of the triazamidine as the ordinate and the concentration as the abscissa to obtain a regression equation of the triazamidine of Y = 57969.12X-32482.826 ² =0.9996, the linear relationship is good, wherein X is the peak area and Y is the drug concentration.

3.5 validation of matrices and recovery

For animals, the amitraz is generally used as an injection, and is easy to cause residue in the animal body. The detection of the residual triazamidine in the milk, beef, chicken and chicken livers which are samples sent by clients by using the method disclosed by the application document proves that the recovery rate is between 70 and 90 percent, the result is detailed in a table 3, the recovery rate meets the requirement of pharmacokinetic research, and the method has good stability.

TABLE 3 recovery of each sample

4. Conclusion

The method is characterized in that the method comprises the steps of determining the triazamidine in the animal-derived matrix by using a UPLC-LCMSMS method, determining that the triazamidine is extracted by using an 80% acetonitrile water solution, achieving baseline separation of the triazamidine chromatographic peak, good peak shape, low detection limit, high sensitivity and stable recovery of the method, and establishing a reliable analysis method for determining the residual amount of the triazamidine in the animal-derived matrix.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the realization of those skilled in the art; where combinations of features are mutually inconsistent or impractical, such combinations should not be considered as being absent and not within the scope of the claimed invention.

Claims (1)

1. A method for detecting residual triazamidine is characterized in that after 80% acetonitrile aqueous solution is adopted to extract triazamidine in a substance to be detected, an ultra performance liquid chromatography-tandem mass spectrometry method is adopted to carry out quantitative analysis on the triazamidine; the detection method comprises the following steps of,

s1: preparing a standard solution, wherein the standard solution comprises a standard stock solution, a standard working solution and a standard solution, and the standard stock solution is a 1000mg/L solution obtained by diluting 10mg of a triazamidine standard substance by deionized water to a constant volume of 10 ml; the standard working solution is a 1mg/L solution obtained by carrying out constant volume on the standard stock solution through a methanol water solution; the standard solution is a solution with the volume of 0ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL and 20ng/mL obtained by the standard working solution through methanol water solution;

s2: preparing a detection sample, namely accurately weighing 5.00g of an object to be detected, pre-treating the object to be detected, placing the object to be detected into a 50mL centrifuge tube, adding 20mL 80% acetonitrile water solution, and extracting according to the following steps;

s2-1, vortexing for 1min to fully and uniformly mix the sample and acetonitrile water;

s2-2, putting the sample liquid after the vortex into ultrasonic equipment for ultrasonic treatment for 10min;

s2-3, centrifuging the sample liquid for 5min, wherein the rotating speed of a centrifuge is 4000rpm/min, and pouring all centrifuged supernatant into another 50ml centrifuge tube;

s2-4, adding 10mL of 80% acetonitrile water solution into the centrifuge tube, extracting again according to the steps of S2-1, S2-2 and S2-3, and collecting final centrifugate;

s2-5, adding 4g of solid sodium chloride and 10mL of saturated n-hexane into the filtrate in the final centrifugate for liquid-liquid distribution, removing an n-hexane layer, transferring an acetonitrile layer into a 100mL heart-shaped bottle, carrying out rotary evaporation at 40 ℃ until the acetonitrile layer is nearly dry, carrying out constant volume treatment to 1mL by using 50% methanol water solution, and filtering through a 0.22 mu m filter membrane to obtain a final detection sample;

s3: drawing a standard curve, sequentially performing machine detection on the standard solution according to the concentration from low to high, and drawing the standard curve by taking the peak area of the triazamidine as a vertical coordinate and the concentration as a horizontal coordinate;

s4: sample determination, namely placing the final detection sample obtained in the step S2 in a sample bottle for measurement by an upper machine;

the object to be detected is animal muscle tissue, animal kidney or animal dairy product, and the using state of the object to be detected is pulpiness;

the mass spectrum parameters in the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows:

and (3) chromatographic column: agilent eclipseplus C18 RRHD

Column temperature: 40 deg.C

Flow rate: 0.4mL/min

Sample introduction amount: 2 μ L

Mobile phase: a-0.1% formic acid water +5mmol/L ammonium acetate; b-methanol, concentration gradient as in Table 1,

TABLE 1 concentration gradient of methanol

The chromatographic parameters in the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows:

an ion source: ESI +

Ion source temperature: 325 deg.C

Flow rate of atomizing gas: 9L/min

Nozzle pressure: 45psi

Temperature of the auxiliary heater: 350 deg.C

Flow rate of the auxiliary heater: 10L/min.

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