阅读说明：本技术 血浆中尼古丁和可替宁的前处理方法、前处理试剂盒及检测方法 (Pretreatment method, pretreatment kit and detection method for nicotine and cotinine in blood plasma ) 是由 林广沅 张丹 程朝柱 陈高明 胡玉梅 于 2021-07-21 设计创作，主要内容包括：本发明提供了血浆中尼古丁和可替宁的前处理方法、前处理试剂盒及检测方法,其中,血浆中尼古丁和可替宁的前处理方法,使用混合型阳离子交换柱对血浆进行前处理,并通过各步骤中溶液的组合,使得最终获得的洗脱液中含有更少量杂质的同时又能保持尼古丁和可替宁的高回收率,从而可以满足后续检测,特别是LC-MS/MS的需要,通过LC-MS/MS检测发现该方法获得的尼古丁和可替宁具有更好的精密度及更低的检出限,同时回收率＞88%,可以满足高精度的LC-MS/MS检测的需要。(The invention provides a pretreatment method, a pretreatment kit and a detection method for nicotine and cotinine in blood plasma, wherein the pretreatment method for nicotine and cotinine in blood plasma uses a mixed type cation exchange column to pretreat blood plasma, and the combination of solutions in each step enables the finally obtained eluent to contain a smaller amount of impurities and keep the high recovery rate of nicotine and cotinine, so that the requirements of subsequent detection, particularly LC-MS/MS, can be met, and the detection of LC-MS/MS finds that the nicotine and cotinine obtained by the method have better precision and lower detection limit, and the recovery rate is more than 88%, so that the requirement of high-precision LC-MS/MS detection can be met.)

1. The pretreatment method of nicotine and cotinine in blood plasma is characterized by comprising the following steps in sequence:

step A: mixing the plasma with the internal standard working solution, adding a phosphoric acid aqueous solution, and uniformly mixing to obtain a first mixed solution;

and B: adding the first mixed solution into an activated mixed type cation exchange column, and enabling the first mixed solution to pass through the mixed type cation exchange column through positive pressure or negative pressure;

and C: sequentially adding a formic acid aqueous solution and acetonitrile into the mixed type cation exchange column, and completely removing the solvent in a column bed of the mixed type cation exchange column under the condition of positive pressure or negative pressure;

step D: eluting the mixed type cation exchange column by using a second mixed solution obtained by mixing ammonia water and acetonitrile to obtain an eluent;

step E: and drying and redissolving the eluent.

2. The method for pretreating nicotine and cotinine in plasma according to claim 1, wherein in the step a, the volume ratio of the plasma to the phosphoric acid aqueous solution is 1: 0.8-1.2, and the phosphoric acid aqueous solution contains 1-5% by volume of phosphoric acid.

3. The method for pretreating nicotine and cotinine in blood plasma according to claim 1, wherein in the step B, the first mixed solution is fed into an activated mixed type cation exchange column, and the first mixed solution is allowed to pass through the mixed type cation exchange column at a flow rate of 1-5 s/d by positive pressure or negative pressure.

4. The method for pretreating nicotine and cotinine in plasma according to claim 1, wherein in step C, the aqueous formic acid solution contains 1-5% by volume of formic acid.

5. The method for pretreating nicotine and cotinine in plasma according to claim 1, wherein in the step D, the volume ratio of the ammonia water to the acetonitrile is 0.8-1.2: 9, and the ammonia water contains ammonia in a mass fraction of 15% or more.

6. The method for pretreating nicotine and cotinine in plasma according to claim 1, wherein the step E comprises drying the eluate at 15-30 ℃ under nitrogen, and then redissolving.

7. A pretreatment kit for nicotine and cotinine in blood plasma is characterized by comprising phosphoric acid or a phosphoric acid aqueous solution, formic acid or a formic acid aqueous solution, ammonia water with the mass fraction of more than or equal to 15%, acetonitrile and a mixed type cation exchange column.

8. A method for detecting nicotine and cotinine in blood plasma, which is characterized in that the blood plasma is subjected to pretreatment, wherein the pretreatment is carried out by the pretreatment method for the nicotine and the cotinine in the blood plasma according to any one of claims 1 to 6 or the pretreatment kit for the nicotine and the cotinine in the blood plasma according to claim 7.

9. The method of claim 8, wherein the plasma is pre-treated and then detected by an LC-MS/MS system.

10. The method for detecting nicotine and cotinine in blood plasma according to claim 9, wherein the detection conditions of the LC-MS/MS system are as follows:

chromatographic conditions are as follows: selecting chromatographic column with HILIC specification of 100 × 2.1mm and 2.7 μm; the column temperature is 40 ℃; mobile phase A: water containing 5mM ammonium formate in a volume fraction of 0.1% formic acid; mobile phase B: 0.1% by volume of methanoic acid solution; sample introduction amount: 5 mu L of the solution; gradient elution; the conditions of the gradient elution are as follows:

0min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

1.5min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

3min, the volume ratio of the mobile phase A to the mobile phase B is 1:9, and the flow rate is 0.3 mL/min;

4.5min, the volume ratio of the mobile phase A to the mobile phase B is 1:9, and the flow rate is 0.3 mL/min;

4.6min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

6min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

mass spectrum conditions: electrospray voltage 3300V; sheath gas pressure: 30 arb; auxiliary gas pressure: 5 arb; ion transfer tube temperature: 365 ℃; temperature of the auxiliary gas: 290 ℃; the collection mode is as follows: monitoring multiple reactions; the quantitative ion pairs M/Z of nicotine, cotinine, nicotine-D4 and cotinine-D3 are 163.1/130.1, 177.2/80.1, 167.2/134.1 and 180.2/80.1 respectively, and the qualitative ion pairs M/Z are 163.1/117.1, 177.2/98.1, 167.2/121.1 and 180.2/101.1 respectively.

Technical Field

The invention relates to the field of medical examination, in particular to a pretreatment method, a pretreatment kit and a detection method for nicotine and cotinine in blood plasma.

Background

Long-term tobacco consumption increases the risk of many diseases, including lung cancer, chronic obstructive pulmonary disease, stroke, heart disease, and respiratory infections. Smoking and health have become one of the most interesting public health problems in the world at present, and scientific and accurate measurement of tobacco exposure is the basis for studying the relevance of tobacco exposure to health. Nicotine and a metabolite cotinine thereof are key biomarkers for evaluating tobacco exposure, and the nicotine and the cotinine in biological materials such as urine, blood, saliva, hair and the like can be measured to evaluate the smoking behavior of a human body, the smoking cessation condition and the exposure degree and dosage of environmental smoke.

The detection method of nicotine and cotinine comprises a Liquid Chromatography (LC) method, a liquid chromatography-mass spectrometry combined method (LC-MS/MS), a Supercritical Fluid Chromatography (SFC) method, a gas chromatography-mass spectrometry combined method (GC-MS/MS) method, a Capillary Electrophoresis (CE) method and the like, and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) has the advantages of rapidness, sensitivity, good reproducibility, high flux and the like, and is widely applied to detection of nicotine and cotinine. Prior art detection of nicotine and cotinine mostly uses LC-MS/MS for urine and saliva samples, and rarely involves plasma samples. This is because the plasma sample has a complicated composition and requires a higher pretreatment. The blood plasma contains a large amount of water, and also contains components such as inorganic salts, fibrinogen, albumin, globulin, enzymes, hormones, various nutrients, metabolites and hormones, for example, 1L of blood plasma contains 900-910 g of water (90-91%), 65-85 g of protein (6.5-8.5%) and 20g of low molecular weight substances (2%). These components tend to interfere with the detection of nicotine and cotinine and must therefore be treated to remove most of the interferents. Patent document CN111983110A discloses that an internal standard extraction solution is added to plasma to precipitate proteins in the plasma, but the internal standard extraction solution simply removes proteins in the plasma, and the treated solution still contains a large amount of inorganic salts and low molecular substances, so that the average precision density (RSD) of nicotine (nicotine) and cotinine is greater than 8%, that is, the precision is poor, and it is difficult to meet the requirement of detection, and because the sample needs to be diluted by 4-10 times, the method is not suitable for some samples with low concentration, so it is necessary to invent a high-precision method for detecting nicotine and cotinine in plasma.

A hybrid cation exchange column comprising a dual retention mode: namely ion exchange and reversed phase retention, commonly used for extracting clenbuterol, melamine and the like, and commercially available products comprise Waters Oasis MCX and Copure MS MCX and the like. The use steps of the mixed type cation exchange column comprise the steps of mixing the sample liquid, the internal standard working solution and the acidic regulator, enabling the mixed liquid to pass through the column, washing, purifying, eluting, drying, and redissolving, wherein the combination of the types of the reagents used in the steps brings obvious influence on the final pretreatment effect. At present, a pretreatment method for simultaneously recovering nicotine and cotinine in blood plasma with high recovery rate and high precision based on a mixed type cation exchange column does not exist.

Disclosure of Invention

The invention provides a pretreatment method of nicotine and cotinine in blood plasma, which comprises the following steps in sequence:

step A: mixing the plasma with the internal standard working solution, adding a phosphoric acid aqueous solution, and uniformly mixing to obtain a first mixed solution;

and B: adding the first mixed solution into an activated mixed type cation exchange column, and enabling the first mixed solution to pass through the mixed type cation exchange column through positive pressure or negative pressure;

and C: sequentially adding aqueous solution of formic acid and acetonitrile into the mixed type cation exchange column, and completely removing the solvent in the column bed of the mixed type cation exchange column under the condition of positive pressure or negative pressure;

step D: eluting the mixed type cation exchange column by using a second mixed solution obtained by mixing ammonia water and acetonitrile to obtain an eluent;

step E: drying the eluent for redissolving.

Compared with the prior art, the method has the advantages that the mixed type cation exchange column is used for pretreating the blood plasma, and the combination of the solutions in the steps enables the finally obtained eluent to contain a smaller amount of impurities and simultaneously maintain the high recovery rate of nicotine and cotinine, so that the requirements of subsequent detection, particularly LC-MS/MS can be met, the nicotine and cotinine obtained by the method are found to have higher precision density through LC-MS/MS detection, the standard recovery rate is more than 88%, and the requirements of high-precision LC-MS/MS detection can be met.

Further, in the step A, the volume ratio of the blood plasma to the phosphoric acid aqueous solution is 1: 0.8-1.2, and the phosphoric acid aqueous solution contains 1-5% by volume of phosphoric acid.

Further, in the step B, the first mixed solution is added into an activated mixed type cation exchange column, and the first mixed solution passes through the mixed type cation exchange column at a flow rate of 1-5 s/d through positive pressure or negative pressure.

Further, in the step C, the formic acid aqueous solution contains 1-5% by volume of formic acid.

Further, in the step D, the volume ratio of the ammonia water to the acetonitrile is 0.8-1.2: 9, and the ammonia water contains ammonia with the mass fraction of more than or equal to 15%. Acetonitrile is used for dissolving nicotine and cotinine, ammonia water is used for removing the adsorption of the mixed type cation exchange column on the nicotine and the cotinine, the volume ratio of the acetonitrile to the ammonia water is controlled, the recovery amount can be ensured, and the blow-drying time can be shortened.

Further, in the step E, the eluent is dried under the condition of nitrogen at 15-30 ℃, and then redissolved.

The invention provides a pretreatment kit for nicotine and cotinine in blood plasma, which comprises phosphoric acid or a phosphoric acid aqueous solution, formic acid or a formic acid aqueous solution, ammonia water with the mass fraction of more than or equal to 15%, acetonitrile and a mixed type cation exchange column.

The pretreatment kit can realize high-recovery and high-precision pretreatment of nicotine and cotinine in blood plasma by matching each solution with the mixed type cation exchange column.

The third aspect of the invention provides a method for detecting nicotine and cotinine in blood plasma, wherein the blood plasma is pretreated, and the pretreatment method of the nicotine and cotinine in the blood plasma is selected as the pretreatment method or the pretreatment kit of the nicotine and cotinine in the blood plasma is used for pretreatment.

The pretreated blood plasma does not lose too much nicotine and cotinine, simultaneously removes a large amount of impurities in the blood plasma, is not limited to protein, and also comprises inorganic salt and low molecular substances.

Further, the plasma is pretreated and then detected by an LC-MS/MS system.

Further, the detection conditions of the LC-MS/MS system are as follows:

chromatographic conditions are as follows: selecting chromatographic column with HILIC specification of 100 × 2.1mm and 2.7 μm; the column temperature is 40 ℃; mobile phase A: water containing 5mM ammonium formate in a volume fraction of 0.1% formic acid; mobile phase B: 0.1% by volume of methanoic acid solution; sample introduction amount: 5 mu L of the solution; gradient elution; the conditions of the gradient elution are as follows:

0min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

1.5min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

3min, the volume ratio of the mobile phase A to the mobile phase B is 1:9, and the flow rate is 0.3 mL/min;

4.5min, the volume ratio of the mobile phase A to the mobile phase B is 1:9, and the flow rate is 0.3 mL/min;

4.6min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

6min, the volume ratio of the mobile phase A to the mobile phase B is 9:1, and the flow rate is 0.3 mL/min;

mass spectrum conditions: electrospray voltage 3300V; sheath gas pressure: 30 arb; auxiliary gas pressure: 5 arb; ion transfer tube temperature: 365 ℃; temperature of auxiliary gas: 290 ℃; the collection mode is as follows: monitoring multiple reactions; the quantitative ion pairs M/Z of nicotine, cotinine, nicotine-D4 and cotinine-D3 are 163.1/130.1, 177.2/80.1, 167.2/134.1 and 180.2/80.1 respectively, and the qualitative ion pairs M/Z are 163.1/117.1, 177.2/98.1, 167.2/121.1 and 180.2/101.1 respectively. After the plasma is subjected to the pretreatment, the LC-MS/MS system is used for separating and detecting nicotine and cotinine, and the efficient separation and detection of the nicotine and the cotinine can be ensured by adjusting the detection conditions of the LC-MS/MS system.

Drawings

FIG. 1 is a graph representing the limits of quantitation of NT and CN in plasma samples and their internal standards.

FIG. 2 shows the results of human blood sample detection.

Detailed Description

In order to explain the technical contents of the present invention in detail, the following description is further made in conjunction with the embodiments. The following instruments and reagents are commercially available unless otherwise specified.

Example 1

A kit for pretreatment of nicotine and cotinine in plasma, comprising: the device comprises a 96-pore plate provided with a mixed type cation exchange column, a 96-pore plate collection plate, a 4% phosphoric acid aqueous solution, an internal standard working solution containing nicotine-D4, an internal standard working solution containing cotinine-D3, methanol, ultrapure water, a 2% formic acid aqueous solution, acetonitrile and concentrated ammonia water with the mass fraction of 25%.

Example 2

Detection of nicotine and cotinine in plasma

1. Pretreatment of plasma samples

1.1 working fluid with internal standard

And transferring 200 mu L of plasma sample into a 1.5 mL centrifuge tube, adding 20 mu L of internal standard working solution, uniformly mixing, adding 200 mu L of 4% phosphoric acid aqueous solution, and uniformly mixing for later use.

1.2 activation of MS MCX plates

MS MCX 96 well plates (purchased from a comma organism) were activated by adding 1 mL of methanol followed by 1 mL of purified water.

1.3 sample treatment

The uniformly mixed sample to be treated is added into an activated MCX 96-well plate, and placed in a 96-well positive pressure extraction device to flow downwards at the speed of 2 s/d.

1.4 Leaching purification

1 mL of a 2% formic acid aqueous solution and 1 mL of acetonitrile were added to a 4.3 sample-filtered MCX 96-well plate, and the plate was placed in a 96-well positive pressure extraction apparatus, and dried under a positive pressure nitrogen atmosphere for 30 seconds to remove the solvent as much as possible from the column bed.

1.5 elution

The target was eluted from the MS MCX 96 well plate into a 96 well sample collection plate using 2 100. mu.L of 25% concentrated ammonia/acetonitrile (10: 90).

1.6 blow drying and redissolving

The collection plate was blow dried under nitrogen at ambient temperature and redissolved with 150 μ L of the initial mobile phase.

2. Detection on machine

2.1 Loading

Taking the supernatant of the redissolved sample, and carrying out sample injection detection.

2.2 instruments and parameters

The instrument comprises the following steps: thermo Scientific TSQ Endura;

chromatographic parameters:

a chromatographic column: HILIC (100 x 2.1mm, 2.7 μm);

column temperature: 40 ℃;

sample introduction amount: 5 mu L of the solution;

mobile phase: a: water (containing 5mM ammonium formate and 0.1% formic acid) B: acetonitrile (0.1% formic acid);

and (3) an elution mode: gradient elution;

gradient of mobile phase: as shown in table 1.

TABLE 1 mobile phase gradient elution conditions

Mass spectrum parameters:

electrospray voltage: 3300V;

sheath gas pressure: 30 arb;

auxiliary gas pressure: 5 arb;

ion transfer tube temperature: 365 ℃;

temperature of the auxiliary gas: 290 ℃;

the collection mode is as follows: monitoring multiple reactions;

parameters of each set of segregant pairs: as shown in table 2:

TABLE 2 ion pair parameters for each component detection

。

3. Results

3.1 limit of quantitation

The quantitative limits of NT and CN in the plasma sample and the representative spectrum of the internal standard substance are shown in figure 1, and as can be seen from figure 1, when the concentration of Nicotine (NT) and Cotinine (CN) is 0.5 mug/L, the separation and detection effects of nicotine and cotinine can still be ensured.

3.2 Standard Curve and Linearity

As shown in Table 3, when the linearity of the samples of the plurality of batches processed on different dates was examined, it was found that the linearity between the batches was good, and the R values were all greater than 0.997, so that the accuracy of the detection result was ensured.

TABLE 3 NT, CN Standard Curve and Linearity

。

3.3 stability test

3.3.1 stability in days

The test was repeated 3 times a day, and each test was performed for 6 blank samples with standard addition to obtain day stability. As shown in Table 4, the results show that the RSD in NT days is less than 6%, and the RSD in CN days is less than 2%, which indicates that the method is stable and reliable, and has good reproducibility.

TABLE 4 NT, CN stability in day

。

3.3.2 stability during day

On different days (at least 5 days apart), 5-day experiments were designed, with 6 blank samples being spiked daily to obtain day-to-day stability. The results are shown in Table 5, where RSD between NT days is less than 6%, and RSD between CN days is less than 5%, which shows that the method is stable and reliable, and has good reproducibility.

TABLE 5 NT, CN stability over the day.

3.4 recovery rate by adding standard

The results of low, medium and high concentration calibration of the samples are shown in Table 6. The verification proves that the standard recovery rates of the three concentrations are all larger than 88%, and the recovery rate parallel determination RSD is small, which shows that the method has accurate determination result and good reproducibility, and can ensure the accuracy of sample determination.

TABLE 6 NT, CN spiking recovery results

。

3.5 actual sample determination

The reliability of the method is verified for the determination of a large number of actual samples, wherein fig. 2 shows the results of the detection of nicotine and cotinine in one of the human blood. As can be seen from FIG. 2, the actual sample measurement can obtain an ideal detection result, and matrix interference and other abnormal interference peaks do not exist, which indicates that the method has high specificity and strong specificity, and is suitable for the detection of the sample.

According to the invention, the plasma is pretreated by using the mixed type cation exchange column, and the combination of solutions in each step enables the finally obtained eluent to contain a smaller amount of impurities and simultaneously maintain the high recovery rate of nicotine and cotinine, so that the requirements of subsequent detection, particularly LC-MS/MS can be met, the detection of LC-MS/MS finds that the nicotine and cotinine obtained by the method have better precision and lower detection limit, and the average recovery rate is more than 88%, so that the requirement of high-precision LC-MS/MS detection can be met. Further, the detection conditions of the LC-MS/MS system provided by the invention can ensure the specificity and sensitivity of the method.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications and alterations to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles disclosed herein.