阅读说明：本技术 一种用于血浆中莠去津的检测方法 (Detection method for atrazine in blood plasma ) 是由 缪峰 陶庭磊 邢溪溪 季金风 季中秋 耿家豪 于 2020-05-13 设计创作，主要内容包括：本发明公开了一种用于血浆中莠去津的检测方法。该方法的步骤包括：将所述血浆经蛋白沉淀法得到的上清液用液相色谱-质谱联用分析方法检测所述血浆中的莠去津；所述液相色谱的检测条件为：流动相A为甲酸与水的混合液,其中,所述甲酸占所述流动相A总体积的0.01％～0.5％；流动相B为甲酸与乙腈的混合液,其中,所述甲酸占所述流动相B的总体积的0.01％～0.5％；采用梯度洗脱方式。本发明的检测方法能够快速、简便的测定莠去津在经口给药或经皮给药后在血浆中的浓度,测试准确率高,稳定性好,回收率高,线性范围大,从而能够满足莠去津在动物体中的药代/毒代动力学的分析需求。(The invention discloses a method for detecting atrazine in blood plasma. The method comprises the following steps: detecting atrazine in the plasma by using a liquid chromatography-mass spectrometry combined analysis method for supernatant obtained by subjecting the plasma to a protein precipitation method; the detection conditions of the liquid chromatogram are as follows: the mobile phase A is a mixed solution of formic acid and water, wherein the formic acid accounts for 0.01-0.5% of the total volume of the mobile phase A; the mobile phase B is a mixed solution of formic acid and acetonitrile, wherein the formic acid accounts for 0.01-0.5% of the total volume of the mobile phase B; gradient elution mode is adopted. The detection method provided by the invention can be used for rapidly, simply and conveniently measuring the concentration of atrazine in plasma after oral administration or transdermal administration, and has the advantages of high test accuracy, good stability, high recovery rate and wide linear range, so that the requirement on the pharmacokinetic/toxicological dynamics analysis of atrazine in animals can be met.)

1. The method for detecting atrazine in blood plasma is characterized by comprising the following steps of:

detecting the supernatant obtained by treating the plasma by a protein precipitation method by using a liquid chromatography-mass spectrometry combined analysis method;

the detection conditions of the liquid chromatogram are as follows:

the mobile phase comprises a mobile phase A and a mobile phase B;

the mobile phase A is a mixed solution of formic acid and water, wherein the formic acid accounts for 0.01-0.5% of the total volume of the mobile phase A;

the mobile phase B is a mixed solution of formic acid and acetonitrile, wherein the formic acid accounts for 0.01-0.5% of the total volume of the mobile phase B;

adopting gradient elution, wherein the total volume of the mobile phase is 100%; at 0min, the volume of the mobile phase a was 65%; the volume of the mobile phase A is reduced from 65% to 10% in 0-2 min; in 2-2.5 min, the volume of the mobile phase A is 10%; the volume of the mobile phase A is increased from 10% to 65% within 2.5-2.51 min; and the volume of the mobile phase A is 65% in 2.51-3 min.

2. The method for detecting atrazine in plasma according to claim 1, wherein the detection of atrazine in plasma is performed by an internal standard method;

preferably, the internal standard method uses an internal standard substance of atrazine-D5;

preferably, dissolving the internal standard substance in methanol, and diluting with acetonitrile to obtain an internal standard working solution;

more preferably, the concentration of the internal standard working solution is 500 ng/mL.

3. The method for detecting atrazine in plasma according to claim 1, characterized in that the protein precipitation method comprises the following steps: adding a precipitator into the plasma, vortexing and centrifuging, sucking supernatant, adding a diluent, vortexing and centrifuging again, and taking supernatant.

4. The method for detecting atrazine in plasma according to claim 3, wherein the precipitating agent is acetonitrile;

and/or the volume ratio of the precipitating agent to the blood plasma is (10-20): 1, preferably 18: 1;

and/or the diluent is a mixed solution of acetonitrile and water, wherein the acetonitrile accounts for 10-50% of the total volume of the diluent; preferably, the acetonitrile accounts for 30% of the total volume of the diluent;

and/or the temperature of the centrifugation is 4 ℃;

and/or the rotation speed of the centrifugation is 4000 rpm;

and/or the centrifugation time is 10 min.

5. The method for detecting atrazine in plasma according to claim 1, wherein the stationary phase of the chromatographic column of the liquid chromatography is octadecyl bonded silica gel;

and/or the model of the chromatographic column of the liquid chromatogram is InfinityLabPoroshell 120 SB-C18;

and/or the inner diameter of the chromatographic column of the liquid chromatogram is 2.1mm, and the length is 50 mm;

and/or the pore diameter of the chromatographic column of the liquid chromatogram is 2.7 μm;

and/or the running time of the liquid chromatography is 3 min;

and/or the mobile phase A is a mixed solution of formic acid and water, wherein the formic acid accounts for 0.05% of the total volume of the mobile phase A;

and/or the mobile phase B is a mixed solution of formic acid and acetonitrile, wherein the formic acid accounts for 0.05 percent of the total volume of the mobile phase B;

and/or the sample injection amount of the liquid chromatography is 1-10 mu L, preferably 5 mu L;

and/or the column temperature of the liquid chromatography is 35-45 ℃, preferably 40 ℃;

and/or the flow rate of the liquid chromatography is 0.4 mL/min.

6. The method for detecting atrazine in plasma as claimed in claim 1, wherein the ion source of mass spectrometry is electrospray ionization (ESI);

and/or, the ionization mode of the mass spectrum is a positive ionization mode;

and/or, the mass spectrum employs a multiple reaction monitoring mode;

preferably, in the multiple reaction monitoring mode, the mass-to-charge ratios of the parent ion and the daughter ion pairs of atrazine are 216.0 and 174.0, respectively; detecting atrazine in plasma by adopting an internal standard method, wherein when the internal standard substance is atrazine-D5, the mass-to-charge ratios of the parent ion and the daughter ion of atrazine-D5 are 220.9 and 179.2 respectively; the scanning interval time is 100 ms.

7. The method for detecting atrazine in plasma according to claim 1, wherein the concentration of atrazine in plasma is obtained by comparing the detection result of the liquid chromatography-mass spectrometry with an atrazine standard curve.

8. The method for detecting atrazine in plasma as claimed in claim 7, wherein the atrazine standard curve is obtained by fitting standard samples with different concentrations through liquid chromatography-mass spectrometry analysis method tests.

9. The method for detecting atrazine in plasma as claimed in claim 8, wherein the liquid chromatography-mass spectrometry combined analysis method is performed by preparing a plurality of standard samples with a plurality of concentration gradients, a multi-reaction monitoring mode chromatogram is extracted, the peak area ratio of the standard sample to the internal standard is taken as the ordinate, and the weight is set to be 1/x²And neglecting the origin, and fitting to obtain the atrazine standard curve.

10. The method for detecting atrazine in plasma according to claim 9, wherein the first step comprises the step of measuring atrazine,

dissolving an atrazine standard substance in methanol to prepare a standard substance stock solution, and diluting the standard substance stock solution by using a mixed solvent of methanol and water to obtain a plurality of standard substance working solutions with a plurality of concentration gradients; wherein, the mixed solvent is preferably 30-70% methanol aqueous solution, such as 50% methanol aqueous solution;

preferably, the concentration gradient of the standard working solution is 40ng/mL, 80ng/mL, 400ng/mL, 2000ng/mL, 4000ng/mL, 16000ng/mL, 32000ng/mL and 40000ng/mL in sequence; blank plasma is respectively added into the standard substance working solution to obtain standard samples, and the concentrations of the standard samples are respectively 2, 4, 20, 100, 200, 800, 1600 and 2000 ng/mL.

Technical Field

The invention relates to a method for detecting atrazine in blood plasma.

Background

China is a big agricultural country, pesticide preparations have a critical effect on agricultural development and can be mainly divided into pesticides, bactericides, herbicides and the like, and atrazine is a triazine herbicide which is commonly used. In 1958, the weeding composition is researched and developed by Geigy company of Switzerland, and is widely applied due to the characteristics of good weeding effect, low price and the like. The use area of atrazine is continuously enlarged along with the development of agriculture from the beginning of the 70 th of the 20 th century in China.

The research shows that the atrazine has the influence on the reproductive system, the immune system, the nervous system and the endocrine system of mammals after being contacted with the atrazine for a long time at a low dose. The animal in the atrazine chronic animal test has abnormal blood biochemical index and mammary gland tumor. Extended exposure of the population to atrazine pesticides may result in potential health damage, particularly to occupational exposure populations. In order to better understand the health risk of atrazine, research on the pharmacokinetics/toxicology of atrazine oral or transdermal pesticides in animals is particularly important, and a quick and accurate atrazine detection method capable of being used for pharmacokinetics/toxicology is urgently needed.

At present, the development of an atrazine detection method mainly focuses on residual quantity in environmental samples, including soil, water quality, agricultural products and the like. In the prior art, a main detection method is a chromatographic analysis method, but the existing atrazine detection method has the defects of insufficient analysis sensitivity, relatively complex treatment process, long instrument analysis time and the like. For example, the pretreatment process of gas chromatography-mass spectrometry (GC-MS) is complicated, and generally requires solid-phase headspace microextraction for a long time. In addition, high performance liquid chromatography (HPLC-UV) has long analysis time and insufficient sensitivity due to the detector.

Disclosure of Invention

The invention aims to solve the technical problems that in the prior art, the method for detecting atrazine has the defects of insufficient analysis sensitivity, relatively complex treatment process, long instrumental analysis time and the like, and provides a method for detecting atrazine in blood plasma. The detection method provided by the invention can be used for rapidly, simply and conveniently measuring the concentration of atrazine in plasma after oral administration or transdermal administration, and has the advantages of high test accuracy, good stability, high recovery rate and wide linear range, so that the requirement on the pharmacokinetic/toxicological dynamics analysis of atrazine in animals can be met.

The invention solves the technical problems through the following technical scheme.

The invention provides a method for detecting atrazine in blood plasma, which comprises the following steps:

detecting the supernatant obtained by treating the plasma by a protein precipitation method by using a liquid chromatography-mass spectrometry combined analysis method;

the detection conditions of the liquid chromatogram are as follows:

the mobile phase comprises a mobile phase A and a mobile phase B;

the mobile phase A is a mixed solution of formic acid and water, wherein the formic acid accounts for 0.01-0.5% of the total volume of the mobile phase A;

the mobile phase B is a mixed solution of formic acid and acetonitrile, wherein the formic acid accounts for 0.01-0.5% of the total volume of the mobile phase B;

adopting gradient elution, wherein the total volume of the mobile phase is 100%; at 0min, the volume of the mobile phase a was 65%; the volume of the mobile phase A is reduced from 65% to 10% in 0-2 min; in 2-2.5 min, the volume of the mobile phase A is 10%; the volume of the mobile phase A is increased from 10% to 65% within 2.5-2.51 min; and the volume of the mobile phase A is 65% in 2.51-3 min.

In the invention, the detection of atrazine in plasma can be carried out by adopting an internal standard method.

Wherein, the internal standard method preferably uses atrazine-D5 as the internal standard substance.

Wherein, preferably, the internal standard substance is dissolved in methanol.

In a preferred embodiment, the internal standard atrazine-D5 is dissolved in an appropriate amount of methanol to prepare an internal standard stock solution with an appropriate concentration, and the internal standard stock solution is diluted by acetonitrile to obtain internal standard working solutions with different concentrations.

In a preferred embodiment, the concentration of the internal standard working solution is 500 ng/mL.

The protein precipitation method adopted in the invention has the advantages of simplicity and quickness. The operation and conditions of the protein precipitation method may be conventional in the art, and preferably include the following steps: adding a precipitating agent into the plasma (such as a matrix effect sample and a recovery rate sample) or a blank matrix (namely blank plasma), whirling and centrifuging, sucking supernatant, adding a diluting agent, whirling and centrifuging again, and taking supernatant for injection detection. And adding internal standard working solution into standard curve, quality control and other samples to be examined, adding a precipitator, performing vortex and centrifugation, sucking supernatant, adding a diluent, performing vortex and centrifugation again, and taking supernatant for sample injection detection.

Wherein, the precipitant is preferably acetonitrile.

Wherein the volume ratio of the precipitant to the plasma is preferably (10-20): 1, more preferably 18: 1.

Preferably, the diluent is a mixed solution of acetonitrile and water, wherein the acetonitrile accounts for 10-50% of the total volume of the diluent; more preferably, the acetonitrile is 30% of the total volume of the diluent.

Wherein, the temperature of the centrifugation is preferably 4 ℃.

Wherein, preferably, the rotation speed of the centrifugation is 4000 rpm.

Wherein, the time of centrifugation is preferably 10 min.

In the invention, a liquid chromatography-mass spectrometry combined analysis method is adopted, and the time required by detection is greatly shortened by screening the liquid chromatography detection conditions such as mobile phase ratio and elution mode. Preferably, the LC-MS/MS instrument is an AB SCIEX Triple Quad 6500 PLUS.

In the present invention, the stationary phase of the chromatographic column of the liquid chromatography is preferably octadecyl bonded silica gel.

Wherein the model of the chromatographic column of the liquid chromatogram is preferably InfinityLabPoroshell 120 SB-C18.

Wherein, preferably, the inner diameter of the chromatographic column of the liquid chromatogram is 2.1mm, and the length is 50 mm.

Wherein, the pore diameter of the chromatographic column of the liquid chromatogram is preferably 2.7 μm.

In the present invention, the running time of the liquid chromatography is preferably 3 min.

In the present invention, preferably, the mobile phase a is a mixed solution of formic acid and water, wherein the formic acid accounts for 0.05% of the total volume of the mobile phase a.

In the present invention, preferably, the mobile phase B is a mixed solution of formic acid and acetonitrile, wherein the formic acid accounts for 0.05% of the total volume of the mobile phase B.

In the present invention, the sample size of the liquid chromatography is preferably 1 to 10 μ L, more preferably 5 μ L, and the sample size is small.

In the present invention, the column temperature of the liquid chromatography is preferably 35 to 45 ℃, more preferably 40 ℃.

In the present invention, the flow rate of the liquid chromatography is preferably 0.4 mL/min.

In the present invention, the operation and detection conditions of the mass spectrometer can be conventional in the art.

Wherein, the ion source of the mass spectrum is preferably electrospray ionization ESI.

Wherein the ionization mode of the mass spectrum is preferably a positive ionization mode.

Wherein, preferably, the mass spectrometry employs Multiple Reaction Monitoring (MRM) mode.

Wherein the mass spectrometer is preferably of the AB SCIEX TRIPLE QUAD type^TM 4500。

Preferably, in the multiple reaction monitoring mode, the mass-to-charge ratios of the parent ion and the daughter ion pairs of atrazine are 216.0 and 174.0, respectively; detecting atrazine in plasma by adopting an internal standard method, wherein when the internal standard substance is atrazine-D5, the mass-to-charge ratios of the parent ion and the daughter ion of atrazine-D5 are 220.9 and 179.2 respectively; the scanning interval time is 100 ms.

Preferably, in the multiple reaction monitoring mode, the instrument parameters are as follows:

in the invention, the detection result of the liquid chromatography-mass spectrometry combined analysis method can be compared with the atrazine standard curve to obtain the concentration of atrazine in the plasma. The atrazine standard curve can be obtained by testing atrazine standard substances with different concentrations by a liquid chromatography-mass spectrometry combined analysis method and fitting. As known by the technical personnel in the field, the method can be used for testing a liquid chromatography-mass spectrometry combined analysis method by preparing a plurality of atrazine standards with a plurality of concentration gradients, extracting a multi-reaction monitoring mode chromatogram, and setting the weight to be 1/x by taking the peak area ratio of the standard sample to the internal standard as a vertical coordinate²Neglecting the origin, fitting an atrazine linear standard curve.

In a preferred embodiment, the atrazine standard substance is dissolved in a proper amount of methanol to prepare a standard substance stock solution with a proper concentration, and the standard substance stock solution is diluted by a mixed solvent of methanol and water to obtain a plurality of standard substance working solutions with a plurality of concentration gradients; the mixed solvent is preferably a 30% to 70% methanol aqueous solution, for example, a 50% methanol aqueous solution.

In a preferred embodiment, the concentration gradient of the standard working solution is 40ng/mL, 80ng/mL, 400ng/mL, 2000ng/mL, 4000ng/mL, 16000ng/mL, 32000ng/mL and 40000ng/mL in sequence; blank plasma is respectively added into the standard substance working solution to obtain standard samples, and the concentrations of the standard samples are respectively 2, 4, 20, 100, 200, 800, 1600 and 2000 ng/mL.

In a preferred embodiment, the atrazine standard substance is dissolved in a proper amount of methanol to prepare a quality control stock solution with a proper concentration, and the quality control stock solution is diluted by a mixed solvent of methanol and water to obtain a plurality of quality control working solutions with a plurality of concentration gradients; the mixed solvent is preferably a 30% to 70% methanol aqueous solution, for example, a 50% methanol aqueous solution.

In a preferred embodiment, the concentration gradient of the quality control working solution is 40ng/mL, 120ng/mL, 2400ng/mL and 30000ng/mL in sequence; and respectively adding blank plasma into the quality control working solution to obtain quality control samples, wherein the concentrations of the quality control samples are respectively 2, 6, 120 and 1500 ng/mL.

The invention has the beneficial effects that:

the method systematically verifies the investigation items according to the Chinese pharmacopoeia and the NMPA non-clinical pharmacokinetic study technical guidance principle. The pretreatment method adopted by the method is a protein precipitation method, and compared with the common extraction method, the method is simpler, short in time consumption and easy to operate; meanwhile, the method of the invention needs small sample input amount and short analysis time; the processing mode developed by the invention ensures that the standard curve is linear and accurate, and the accuracy and the reliability of detection are ensured; therefore, the invention can ensure that more accurate, stable, high-precision and high-sensitivity detection data can be obtained in shorter analysis time, and lays a foundation for tamping the analysis of the pharmacokinetics of the atrazine in the plasma.

Drawings

Figure 1 is a map of the blank matrix at the atrazine ion channel.

FIG. 2 is a graph of a blank matrix at the time of internal standard ion channel.

FIG. 3 is a map of the lower limit of atrazine quantitation (2.000 ng/mL).

FIG. 4 is a graph of internal standard (25ng/mL) in the lower limit sample of quantitation.

Fig. 5 is a graph showing atrazine standard curve.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

In the following examples, "FA" refers to formic acid; ' H₂O "means water," ACN "means acetonitrile; "IPA" refers to isopropanol; "MeOH" refers to methanol.

Example 1

1. Preparation of a reagent:

1.1 Mobile Phase A (MPA): 0.05% FA in H₂O

500. mu.L of FA was added to 1000mL of H₂And O, mixing uniformly. The shelf life is 1 week at room temperature.

1.2 Mobile Phase B (MPB): 0.05% FA in ACN

Add 500. mu.L of FA to 1000ml of CaCN and mix well. The shelf life is 1 week at room temperature.

1.3 Strong Wash Solution (SNW): MeOH ACN IPA H₂O＝1:1:1:1，v/v/v/v

500mL of LEOH, 500mL of ACN, 500mL of IPA, 500mL of H₂And O, and mixing uniformly. The shelf life is 1 month at room temperature.

1.4 Weak Wash solution (WNW): MeOH H₂O＝1:1，v/v

500mL of MeOH and 500mL of H were added₂And O, and mixing uniformly. The shelf life is 1 month at room temperature.

1.5 precipitant & internal standard diluent: ACN

1000ml of LCAN was taken. The shelf life is 1 month at room temperature.

1.6 dilution 1 (dilution 1): MeOH H₂O＝1:1，v/v

Taking 100 mM LEOH, 100mLH₂And O, and mixing uniformly. The shelf life is 1 month at room temperature.

1.7 dilution 2 (dilution 2): ACN H₂O＝3:7，v/v

Take 300mL of ACN, 700mL of H₂And O, and mixing uniformly. The shelf life is 1 month at room temperature.

2. Preparing a standard curve (hereinafter referred to as standard curve) and a quality control sample:

the stock solution and the working solution are both stored in an ultra-low temperature refrigerator (70 ℃ below zero to 90 ℃ below zero).

2.1 preparation of stock solutions of Standard koji

Accurately weighing an appropriate amount of atrazine into a transparent sample bottle, adding an appropriate amount of MeOH, dissolving, shaking up, and preparing into a standard yeast stock solution with the concentration of 1.000 mg/mL. (wherein the volume of methanol required was calculated from the atrazine conversion factor. the mass conversion factor was calculated from the purity of a commercially available standard substance. the mass conversion factor for atrazine was 0.947.)

2.2 Standard music&Preparation of SST working solution (diluent: MeOH: H)₂O＝1:1，v/v)

Standard koji & SST working liquids were obtained using standard koji stock solutions, formulated according to table 1 below:

TABLE 1 atrazine Standard and SST working solution preparation

2.3 preparation of Standard Curve & SST samples

Standard curve & SST samples were obtained using standard curve & SST working fluids, formulated according to table 2 below:

table 2 atrazine standard curve & SST sample preparation

The standard curve samples were stored in an ultra-low temperature freezer (-70 to-90 ℃).

2.4 preparation of quality control stock solution

Accurately weighing an appropriate amount of atrazine into a transparent sample bottle, adding an appropriate amount of MeOH solution for dissolving, shaking up, and preparing into a quality control stock solution with the concentration of 1.000 mg/mL. (wherein the required methanol volume is calculated based on the atrazine conversion factor, and the atrazine quality conversion factor is 0.947.)

2.5 preparation of quality control working solution (Diluent: MeOH: H)₂O＝1:1，v/v)

The quality control working solution was prepared according to the following table 3 using the quality control stock solution:

TABLE 3 preparation of atrazine quality control working solution

2.6 preparation of quality control samples

Using the quality control working solution, preparing according to the following table 4 to obtain a quality control sample:

TABLE 4 preparation of atrazine quality control samples

The quality control samples were stored in an ultra-low temperature freezer (-70 to-90 ℃).

2.7 preparation of stock solutions for internal standards

Accurately weighing an appropriate amount of atrazine-D5 in a transparent sample bottle, adding an appropriate amount of MeOH solution for dissolving, shaking up, and preparing into an internal standard stock solution with the concentration of 1.000 mg/mL. (wherein the required methanol volume is calculated from the atrazine-D5 conversion factor, and the mass conversion factor of atrazine-D5 is 0.9955.)

2.8 preparation of internal standard working solution (Diluent: ACN)

Internal standard working solutions were prepared according to table 5 below using internal standard stock solutions to give internal standard working solutions:

TABLE 5 Atrazine-D5 internal standard working solution preparation

2.9 preparation of pure solution for testing matrix effect and recovery

The matrix effect sample, the matrix effect pure solution sample and the recovery rate sample were prepared according to the following table 6.

TABLE 6 preparation of pure solutions

Note: dilution 1: MeOH H₂O is 1:1, v/v; ② diluent 2: ACN H₂O＝3:7，v/v

3. Sample processing step

The samples in step 2 were subjected to the following treatments:

3.1 vortex the sample (if the sample needs to be thawed, re-vortex after thawing at room temperature).

3.2 the samples were treated according to the following procedures, respectively:

marking the yeast: 20 μ L STD +20 μ L internal standard +360 μ L ACN → 100 μ L supernatant +300 μ L ACN H₂O＝3:7，v/v；

Quality control: 20 μ L QC +20 μ L internal standard +360 μ L ACN → 100 μ L supernatant +300 μ L ACN H₂O＝3:7，v/v；

Double blank DB, Carryover (both DB and Carryover are blank plasma without test substance, without internal standard): 20 μ L plasma +20 μ L ACN +360 μ L ACN → 100 μ L supernatant +300 μ L ACN H₂O＝3:7，v/v；

Blank (Blank is Blank plasma with internal standard): 20 μ L plasma +20 μ L internal standard +360 μ L ACN → 100 μ L supernatant +300 μ L ACN H₂O＝3:7，v/v；

ULOQ: 20 μ L of STD8+20 μ L of ACN +360 μ L of ACN → 100 μ L of supernatant +300 μ L of ACN H₂O＝3:7，v/v；

Matrix effect sample: 20 μ L plasma +20 μ L ACN +360 μ L ACN → 100 μ L supernatant +100 μ L (LQC-IS _ N or HQC-IS _ N) +200 μ L ACN H₂O＝3:7，v/v；

Matrix effect pure solution samples: 20 mu L H₂O + 20. mu.L of ACN + 360. mu.L of ACN → 100. mu.L of supernatant + 100. mu.L (LQC-IS _ N or HQC-IS _ N) + 200. mu.L of ACN H₂O＝3:7，v/v；

And (3) recovering samples: 20 μ L QC +20 μ L ACN +360 μ L ACN → 100 μ L supernatant +100 μ L (IS _ N) +200 μ L ACN H₂O＝3:7，v/v；

Recovery reference sample: 20 μ L of plasma +20 μ L of ACN +360 μ L of ACN → 100 μ L of supernatant +100 μ L (low concentration: LQC-IS _ N; medium concentration: MQC-IS _ N; high concentration: HQC-IS _ N) +200 μ L of ACN: H₂O＝3:7，v/v；

The above "→" represents: centrifugation was carried out at 4000rpm for 10min at 4 ℃.

4. Test procedure

Sample introduction and liquid chromatography tandem mass spectrometry detection:

wherein, the detection conditions of the liquid chromatogram are as follows:

sample introduction amount: 5 μ L

A chromatographic column: InfinityLabPoroshell 120SB-C18, 2.1X 50mm,2.7-Micron, Agilent

Column temperature: 40 deg.C

Mobile phase A: 0.05% formic acid and water mixture

Mobile phase B: 0.05% mixed solution of formic acid and acetonitrile

Operating time: 3.0min

Gradient elution was used, with elution gradients as in table 7:

TABLE 7 gradient of mobile phase

In a needle washing solvent of the liquid chromatogram, a strong washing solvent is a mixed solvent of methanol, acetonitrile, isopropanol and water, and the volume ratio of the methanol to the acetonitrile to the isopropanol to the water is 1:1:1: 1; the weak washing solvent is a mixed solvent of methanol and water, and the volume ratio of the methanol to the water is 1: 1.

The needle washing procedure of the liquid chromatography is as follows:

the type of flush: only the exterior;

a flushing mode: before and after aspiration, immersion time: 2 s;

the flushing pump mode: flush pump, then stop, time: 2 s;

and (3) flushing setting: washing speed: 35 mu L/s;

flush volume: 1000 μ L;

measuring the purging amount of the pipeline: 100 μ L.

Wherein, the mass spectrum detection conditions are as follows:

the instrument model is as follows: AB SCIEX TRIPLE QUADTM 4500

An ion source: ESI

Ionization mode: positive ion

MRM multiple reaction monitoring ion pairs are shown in table 8:

TABLE 8 MRM ion pairs

The instrument parameters are shown in table 9:

TABLE 9 Instrument parameters

5. Analytical batch acceptance criteria and standard curve regression method

5.1 regression method

And (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation.

The measured concentration of the analyte in the sample is calculated from the following regression equation:

y＝ax+b

where y is the peak area ratio of analyte to internal standard

a is the slope of the standard curve

x is the analyte concentration (in ng/mL)

b is the intercept of the standard curve (weight factor 1/x)²)

5.2 analysis of batch acceptance criteria

1. The deviation between the recalculated and plotted values for each concentration point of the standard curve (as shown in FIG. 5) should be within. + -. 15.0% (within. + -. 20.0% at the lower limit of quantitation).

2. At least 75% of the standard curve samples, and at least 50% of the samples per concentration point should meet the acceptance criteria.

3. The correlation coefficient (r2) of the regression equation must be equal to or greater than 0.98.

4. The assay lot was considered acceptable when at least 67% of the quality control sample results (at least 50% per concentration) were within ± 15.0% of their corresponding nominal values.

Example 2: methodology validation

The verification and investigation items show the measurement conditions of each analysis batch, and the measurement conditions are shown in an attached table 10.

1. System applicability

1 sample of system suitability (see SST sample of Table 2 in example 1 for the formulation procedure) was prepared and fed continuously 3 times or more, and the system suitability was evaluated with the last 3 feeds. The batch measurement results are shown in the attached table 11, and all the results accord with the technical guidance principle of Chinese pharmacopoeia and NMPA non-clinical pharmacokinetic research, which indicates that the detection instrument is in a normal operation state during sample injection.

2. Residue is remained

The residue was evaluated by analyzing a double blank sample (i.e., blank plasma; no analyte, no internal standard) after injection of a quantitative upper limit sample (ULOQ sample, formulation see example 1). The investigation results of the residual quantity of each batch are shown in the attached table 12, and all the results accord with the Chinese pharmacopoeia and the NMPA non-clinical pharmacokinetic research technical guide principle and accord with the accepted standard, so that when the method is used for measuring the concentration of atrazine in plasma, no residue exists in a system after a high-concentration sample is injected, and no influence is caused on the measurement of a subsequent low-concentration sample.

3. Selectivity is

3.1 substrate Selectivity

Selectivity is the ability to distinguish between the target analyte and the internal standard from endogenous components of the matrix. By examining 6 different individual plasmas (EDTA-K from different individual SD rats)₂Plasma) were measured separately for blank samples without internal standard and for LLOQ level samples (see example 1 for formulation procedure) to evaluate matrix selectivity.

Wherein, blank samples without internal standard are EDTA-K of 6 different SD rats₂Plasma, processed in the same manner as DB sample in example 1;

6 LLOQ level samples were EDTA-K from 6 different SD rats₂Plasma was processed in the same manner as the LLOQ sample in example 1.

The representative chromatogram is shown in figure (blank matrix chromatogram is shown in figure 1-2, and quantitative lower limit chromatogram is shown in figure 3-4), the determination results are shown in attached table 13-1 and attached table 13-2, and all the results accord with the technical guidance principle of non-clinical pharmacokinetic study in Chinese pharmacopoeia and NMPA. The result shows that the selectivity of the method for measuring the concentration of atrazine in plasma is good, and the plasma matrix has no interference to the measurement.

3.2 interference between analyte and internal standard

Using a single matrix or a mixed matrix (SD rat EDTA-K)₂Plasma), three samples containing only the analyte and no internal standard were prepared, processed and analyzed in parallel, respectively (ULOQ samples, see example 1 for formulation). The interference result of the object to be detected on the internal standard is shown in the attached table 14, and the result shows that the object to be detected has no interference on the internal standard.

Using a single matrix or a mixed matrix (SD rat EDTA-K)₂Plasma), three samples containing only internal standard and no analyte (corresponding to Blank samples in example 1) were prepared, processed and analyzed in parallel, respectively, at concentrations consistent with those of the internal standard used at the time of method validation (all 500 ng/mL). The interference result of the internal standard on the object to be measured is shown in the attached table 14, and the result shows that the internal standard has no interference on the object to be measured.

4. Matrix effect

Respectively taking 6 different individuals (EDTA-K of SD rats of different individuals)₂Plasma) is prepared, one part of the blank matrix is prepared for each individual, and after pretreatment, standard substances and internal standard pure solutions with the concentration equivalent to that of the treated low-concentration and high-concentration quality control samples are respectively added for analysis. The volume can be adjusted appropriately if necessary. Six replicates of pure solution, equivalent in concentration to the low and high concentration quality control samples, without matrix present were analyzed. The volume can be adjusted appropriately if necessary. The results are shown in the attached Table 15. When the concentration of atrazine is 6.000ng/mL, the average matrix effect factor normalized by the internal standard is 1.00 +/-0.02, and the relative standard deviation is 2.0 percent; average matrix effect normalized by internal standard when atrazine concentration is 1500.000ng/mLThe response factor is 1.00 +/-0.01, the relative standard deviation is 1.0 percent and is less than 15.0 percent, and all results accord with the technical guidance principle of non-clinical pharmacokinetic research of Chinese pharmacopoeia and NMPA.

5. Intra/inter-batch accuracy and precision

Four quality control samples (atrazine concentrations of 2.000ng/mL, 6.000ng/mL, 120.000ng/mL and 1500.000ng/mL, respectively) at different concentrations were taken for an intra/inter-batch accuracy and precision study. Each concentration was processed in 6 replicates per assay batch, requiring fresh preparation of accurate precision samples. The accuracy and precision in the batch are evaluated through the quality control sample of each analysis batch; batch-to-batch accuracy and precision were evaluated by quality control samples from three independent analytical batches (performed for at least 2 days).

The accuracy and precision were calculated from the measurement results of the quality control samples, and the results are shown in Table 16. The results show that all the results accord with the guiding principles of Chinese pharmacopoeia and NMPA non-clinical pharmacokinetic research technology. Therefore, the precision and accuracy of the quality control sample in batch and between batches meet the biological sample acceptance standard.

6. Quality control sample

Each analysis batch contains at least 3 concentrations of quality control samples (at least 2 parts per concentration level). The analysis batch containing accuracy precision, and the accuracy and precision samples with low, medium and high concentration are also used as quality control samples. The results of quality control samples from the accuracy and precision analysis batches are shown in the attached Table 17. The results show that all the results accord with the guiding principles of Chinese pharmacopoeia and NMPA non-clinical pharmacokinetic research technology.

7. Recovery rate

7.1 extraction recovery ratio of analyte

Six samples of extraction recovery are prepared in parallel from matrix samples of low, medium and high concentration levels respectively, without adding an internal standard solution. And adding a pure solution containing an internal standard with a proper concentration into the supernatant after extraction for dilution. The volume can be adjusted appropriately if necessary.

Recovery reference sample: the extraction process is the same as described above, and a pure solution containing the analyte and the internal standard at appropriate concentrations is added to the supernatant after extraction for dilution. The final concentration was the same as the theoretical extraction recovery sample for 100% extraction.

For the pretreatment method of protein precipitation, after the recovery rate reference sample is added with the substance to be detected and the internal standard pure solution, the solvent composition in the matrix is changed, and at the moment, the extraction recovery rate sample needs to be supplemented with the corresponding solvent.

Calculating the extraction recovery rate of the substance to be detected according to the following formula:

the results are shown in the attached Table 18. The results show that all the results accord with the guiding principles of Chinese pharmacopoeia and NMPA non-clinical pharmacokinetic research technology.

7.2 extraction recovery of internal standard

Because the stable isotope labeling internal standard is used, the extraction recovery rate of the internal standard is referred to the recovery rate data of the object to be detected, so the extraction recovery rate of the internal standard meets the standard.

8. Dilution reliability

A DQC sample (containing atrazine at a concentration of 15000.000ng/mL) formulated at a higher concentration than ULOQ was used for dilution reliability studies. The sample was diluted 10-fold with the blank rat plasma and 6 portions were diluted in parallel, the concentration of atrazine after 10-fold dilution being 1500.000 ng/mL. The diluted sample is measured with a standard curve sample. The results are shown in the attached Table 19. The results show that all the results accord with the guiding principles of Chinese pharmacopoeia and NMPA non-clinical pharmacokinetic research technology.

9. Stability of

9.1 Standard stock/working solution stability

To examine the stability of the atrazine stock solutions at room temperature (6 hours to 48 hours) and at ultra low temperature of-70 to-90 ℃ (1 month, allowable time error range: + -7 days), the atrazine stock solutions were compared with the control atrazine stock solutions. After diluting the stock solution, the internal standard is added for processing, and 6 parts of the operation is carried out in parallel. The results are shown in attached tables 20 and 21. The result shows that the atrazine stock solution can be stable for at least 48 hours at room temperature and at least 34 days in an ultralow temperature refrigerator (-70 to-90 ℃); .

To examine the stability of the atrazine working solutions (examining the working solutions used to formulate LLOQ and ULOQ) at room temperature (typically 6 hours to 48 hours) and in an ultra low temperature refrigerator of-70 to-90 ℃ (1 month, allowed time error range: ± 7 days), they were compared to the control atrazine working solution. After diluting the working solution, adding an internal standard for treatment, and performing parallel operation on 6 parts. The results are shown in tables 22 and 23. The results show that the atrazine working solution can be stable for at least 27 hours at room temperature and at least 33 days in an ultralow temperature refrigerator (-70 to-90 ℃).

9.2 internal standard stock/working fluid stability

The stability of the internal standard is referred to the stability data of the analyte due to the use of the stable isotope labeled internal standard.

9.3 quality control sample stability

9.3.1 short term stability

The low and high concentration quality control samples were placed at room temperature (4 to 24 hours) and analyzed, and 6 parts were measured in parallel at each concentration level. The results are shown in Table 24. The results show that plasma samples of atrazine were stable for at least 24 hours at room temperature.

9.3.2 freeze thaw stability

And respectively carrying out analysis on the low-concentration and high-concentration quality control samples after 5 times of freeze-thaw (-70 to-90 ℃/room temperature) cycles (at least 24 hours after the first time of freezing and at least 12 hours after the first time of freezing). Each concentration level was measured in 6 parts in parallel. The results are shown in Table 25. The results show that atrazine SD rat EDTA-K2 plasma samples were stable for at least 5 (-70 to-90 ℃/room temperature) freeze-thaw cycles.

9.3.3 stability of the treated samples

The processed samples (which may be samples of low or high concentration levels of accuracy and precision that have been successfully sampled in an assay lot) are placed in an autosampler or in an environment at the same temperature as the autosampler for a period of time (typically 24 to 144 hours) and then analyzed with a freshly prepared standard curve. The results are shown in Table 26. The results show that the treated plasma samples of atrazine were stable in the autosampler for at least 72 hours.

9.3.4 Long term stability

The low and high concentration quality control samples were stored in an ultra-low temperature refrigerator (-70 to-90 deg.C) respectively (1 month, allowable time error range: + -7 days). Each concentration level was measured in 6 parts in parallel. Sample stability should at least cover the period from the first sample collection to the end of the sample analysis. Longer periods may be evaluated if desired. The results are shown in Table 27. The results show that the plasma samples of atrazine were stable for at least 31 days in an ultra-low temperature freezer (-70 to-90 ℃).

10. Whole blood stability

In order to examine the stability of the sample in the collection and processing processes, low and high concentration whole blood quality control samples are respectively prepared. After the preparation is finished, after the whole blood is incubated in a water bath at 37 ℃ for 15 minutes, the whole blood quality control sample with low concentration and the whole blood quality control sample with high concentration are respectively divided into two parts. One portion was immediately centrifuged to prepare a zero time (T0) plasma sample. The other part was left to stand in crushed ice for 2 hours and centrifuged to prepare a plasma sample for stability examination. Plasma samples were assayed in duplicate at each time point for 6 aliquots and analyzed together. The results are shown in Table 28. The results show that the atrazine whole blood sample was stable for at least 2 hours when placed in crushed ice.

11. Hemolytic Effect

The evaluation was performed by preparing low and high concentration quality control samples using a simulated hemolyzed plasma sample (adding 2% hemolyzed whole blood to non-hemolyzed plasma, considered as severe hemolysis). 6 samples were run in parallel for each concentration. And (3) determining the hemolysis quality control sample by using a standard curve sample prepared from normal plasma, and carrying out quality control sample prepared from the normal plasma. The results are shown in Table 29. The results show that all the results accord with the guiding principle of Chinese pharmacopoeia and NMPA non-clinical pharmacokinetic research technology, and hemolysis has no influence on the quantitative determination of atrazine in plasma by the method.

12. Maximum number of samples to be analyzed

To assess the maximum number of samples injected into an analysis lot and the robustness of the method, at least one verification should be made that the number of samples in an analysis lot is close to the number of samples included in an analysis lot that is estimated at the time of sample analysis. The analysis batch can comprise more verification samples, repeated quality control samples or blank matrix added with internal standard. The maximum number of sampling needles in the verification experiment is 117 needles.

In conclusion, the verification items investigated in the method all accord with the technical guide principles of Chinese pharmacopoeia and NMPA non-clinical pharmacokinetic research, and can be used for determining the concentration of atrazine in blood plasma.

TABLE 10 List of batch sample testing

TABLE 11 System applicability

TABLE 12 residue from batch sample testing

TABLE 13-1 Atrazine and Atrazine-D5 (internal standard) substrate Selectivity (batch 2)

TABLE 13-2 Atrazine Selectivity quantitation lower limit sample matrix Selectivity

TABLE 14 interference between test substances and internal standards

Interference of Atrazine to internal standard Atrazine-D5 by substance to be tested

Interference of internal standard Atrazine-D5 on Atrazine to be detected

TABLE 15 matrix Effect

Atrazine matrix effect

TABLE 16 precision and accuracy of in-batch and inter-batch sample testing

TABLE 17 summary of atrazine quality control sample concentration data (except for accuracy precision analysis lot)

TABLE 18 recovery rate of test substance extraction

Extraction recovery rate of atrazine

TABLE 19 dilution reliability

Reliability of dilution of atrazine (10 times)

TABLE 20 short term stability of stock solutions

Atrazine stock short term stability:

TABLE 21 short term stability of working fluids (ULOQ, LLOQ working fluids)

Atrazine working fluid short term stability (ULOQ, LLOQ working fluid):

TABLE 22 Long term stability of stock solutions

Atrazine stock solution long term stability:

TABLE 23 Long term stability of working fluids (ULOQ, LLOQ working fluids)

Atrazine working fluid long term stability (ULOQ, LLOQ working fluid):

TABLE 24 short term stability of quality control samples

Short term stability of atrazine quality control samples

The test conditions are as follows: standing at room temperature for 24 hr

TABLE 25 freeze thaw stability of quality control samples

Atrazine quality control sample freeze-thaw stability

The test conditions are as follows: freezing in ultra-low temperature refrigerator (-70-90 deg.C) for at least 24 hr for the first time, and then for at least 12 hr

TABLE 26 stability of the treated samples

Stability of atrazine treated samples

The test conditions are as follows: stability after 72 hours in an autosampler (4 ℃ C.)

TABLE 27 Long term stability of quality control samples

Long term stability of atrazine quality control samples

The test conditions are as follows: standing at-70-90 ℃ for 31 days

TABLE 28 Whole blood stability

Atrazine Whole blood stability

The test conditions are as follows: stability of Whole blood samples after 2 hours on crushed Ice

TABLE 29 hemolytic Effect

Hemolytic effect of atrazine