CN115389651B - A method for rapid determination of nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry - Google Patents

Abstract

The invention discloses a method for rapid determination of nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry, which includes: taking 15 to 100 μL of serum, adding 5 to 50 μL of deuterated isotope internal standard solution, mixing, and then adding 45 μL of serum. ~300 μL methanol, vortex thoroughly, centrifuge, and take the supernatant for gas chromatography-tandem mass spectrometry detection; characterize nicotine and cotinine based on the qualitative ion pair and retention time; compare the peak area of the quantitative ion of nicotine or cotinine The ratio of the peak area to the deuterated isotope internal standard was substituted into the matrix-matched internal standard standard curve of nicotine or cotinine to obtain the concentrations of nicotine and cotinine. This invention only requires a trace amount of serum sample, effectively separates nicotine and cotinine based on a specific capillary column, and uses optimized serial quadrupole monitoring parameters to obtain nicotine and cotinine residual data accurately, sensitively, time-saving and efficiently. , and low cost.

Description

A method for rapid determination of nicotine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry How to use cotinine

Technical field

The invention relates to a method for detecting contaminants and their metabolites in human serum, and specifically relates to a method for rapid determination of nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry.

Background technique

Smoking and health are one of the most concerning public health issues worldwide. The human residues of nicotine (formula I, molecular formula: C ₁₀ H ₁₄ N ₂ ) and its metabolite cotinine (formula II, molecular formula C ₁₀ H ₁₂ N ₂ O) are important markers that reflect an individual's smoking status. Serum is an ideal biological sample for determination of nicotine and cotinine residues, but the sampling volume is limited during sample collection.

Commonly used analysis methods for trace amounts of nicotine and cotinine in serum include gas chromatography, liquid chromatography, and liquid chromatography-tandem mass spectrometry. Gas chromatography and liquid chromatography have low sensitivity and poor qualitative performance. They require a large amount of serum samples (about 1 to 2 mL), and the samples need to undergo complex processes such as protein precipitation, liquid-liquid extraction or solid-phase extraction, and nitrogen concentration. Pretreatment; liquid chromatography-tandem mass spectrometry has high sensitivity and accuracy and requires a small amount of serum, but the instruments and equipment are expensive. The above methods are difficult to popularize in the application of smoking screening.

Contents of the invention

In view of the current lack of efficient, accurate and low-cost methods for detecting nicotine and cotinine in trace amounts of human serum, the present invention provides a method for rapid determination of nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry. The inventive method is very simple in pre-processing, and the detection is fast, accurate, time-saving, efficient and low-cost. It is very suitable for research on smoking and harm assessment of large sample groups, and can also be used for objective evaluation and analysis of tobacco control effectiveness.

The object of the present invention can be achieved through the following technical solutions:

A method for rapid determination of nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry, including: taking 15 to 100 μL of human serum, adding 5 to 50 μL of deuterated isotope internal standard solution, mixing, and then adding 45 to 300 μL of methanol , fully vortex, centrifuge, and take the supernatant for gas chromatography-tandem mass spectrometry detection; characterize nicotine and cotinine based on the qualitative ion pair and retention time; compare the peak area of the quantitative product ion of nicotine or cotinine with deuterium The ratio of the peak area of the quantification product ion of the isotope internal standard is substituted into the matrix-matched internal standard standard curve of nicotine or cotinine to obtain the concentrations of nicotine and cotinine;

Among them, the gas chromatography detection conditions: the chromatographic column is a DB-EUPAH capillary column, the specifications are: column length 15~30m, inner diameter 0.18~0.25mm, film thickness 0.14~0.25μm; injection volume is 1~2μL, injection method is For splitless injection, the inlet temperature is 200~280°C; the initial temperature of the chromatographic column is 35~100°C, hold for 1 minute, increase to 280°C at 20°C/min, and hold for 2 minutes;

Tandem mass spectrometry detection conditions: electron impact ionization source, 70eV; the temperatures of the ion source and transmission line are both 300°C; the detection mode is MS/MS detection mode.

Preferably, take 25 μL of human serum, add 5 μL of deuterated isotope internal standard solution, mix well, then add 70 μL of methanol, vortex thoroughly, centrifuge, and take the supernatant for gas chromatography-tandem mass spectrometry detection.

Preferably, the deuterated isotope internal standard is deuterated acenaphthene; the deuterated isotope internal standard solution is a methanol solution with a concentration of deuterated acenaphthene of 1 μg/mL.

The centrifugation conditions are: centrifugation at a temperature of 4 to 40°C and a rotation speed of 5000 to 12000 rpm/min for 5 to 15 minutes; preferably, centrifugation is at a temperature of 4°C and at 12000 rpm/min for 10 minutes.

Preferably, the gas chromatography detection conditions: the chromatographic column is a DB-EUPAH capillary column, the specifications are: column length 20m, inner diameter 0.18mm, film thickness 0.14μm; injection volume is 2μL, and the injection method is splitless injection. , the injection port temperature is 220°C; the initial temperature of the chromatographic column is 60°C, maintained for 1 minute, raised to 280°C at 20°C/min, and maintained for 2 minutes.

The mass spectrometer is a tandem quadrupole mass spectrometer.

Preferably, the qualitative ion pair of nicotine is 162-84m/z and 162-133m/z; the qualitative ion pair of cotinine is 176-98m/z and 176-68m/z; the qualitative ion pair of deuterated acenaphthene is 162 -160m/z and 162-158m/z. The quantitative ion pairs of nicotine, cotinine and deuterated acenaphthylene are 162-84m/z, 176-98m/z and 162-160m/z respectively, that is, the quantitative ion pairs of nicotine, cotinine and deuterated acenaphthylene are 84m respectively. /z, 98m/z and 160m/z,

The higher the response, the higher the sensitivity and the higher the detection rate of the target substance in the sample. Preferably, the collision energy of ion pair 162-84m/z and 176-98m/z is 10eV; the collision energy of ion pair 162-160m/z is 15eV; the collision energy of ion pair 176-68m/z and 162-158m/z The energy is 20eV; the ion pair 162-133m/z collision energy is 35eV.

The retention time of nicotine is 5.7-6.2min, and the retention time of cotinine is 9.0-9.5min;

Preferably, the retention time of nicotine is 5.92-5.93 min, the retention time of cotinine is 9.26-9.27 min, and the retention time of deuterated acenaphthene is 7.13-7.14 min.

Preferably, the matrix-matched internal standard standard curve of nicotine or cotinine is drawn by the following method: take 25 μL of mixed human serum, mix it with 5 μL of deuterated acenaphthene solution and at least 50% of the nicotine standard solution or cotinine standard solution. Mix one and add methanol to 100 μL to obtain a standard curve solution. Vortex and centrifuge. Take the supernatant for gas chromatography-tandem mass spectrometry detection; take the concentration of nicotine or cotinine as the abscissa, and take the concentration of nicotine or cotinine as the abscissa. The ratio of the calibrated peak area of the quantifier ion to the peak area of the quantifier ion of deuterated acenaphthene is the ordinate, and a matrix-matched internal standard standard curve of nicotine or cotinine is established; wherein, the nicotine or cotinine The calibrated peak area of the quantifier ion is the peak area of the quantifier ion of nicotine or cotinine minus the peak area of the quantifier ion of nicotine or cotinine in the matrix background; the range of the matrix-matched internal standard standard curve of nicotine is 0 ~25ng/mL; the matrix-matched internal standard standard curve range of cotinine is 0~500ng/mL.

The mixed human serum is: randomly draw 50 μL of each of 100 human serums, mix them evenly, and prepare a mixed serum matrix.

The matrix background is as follows: take 25 μL of mixed human serum, mix it with 5 μL of deuterated acenaphthene solution, and adjust the volume to 100 μL with methanol.

The beneficial effects of the present invention are:

This invention only requires a trace amount of serum sample, effectively separates nicotine and cotinine based on a specific capillary column, and uses optimized serial quadrupole monitoring parameters to obtain nicotine and cotinine residual data accurately, sensitively, time-saving and efficiently. , and low cost.

The method of the invention is suitable for research on smoking and harm assessment of large sample groups and for objective evaluation and analysis of tobacco control effectiveness.

Description of the drawings

Figure 1 shows the response effects of cotinine on capillary columns DB-5 and DB-EUPAH respectively.

Figure 2 shows the primary mass spectrum and secondary mass spectrum of nicotine.

Figure 3 shows the primary mass spectrum and secondary mass spectrum of cotinine.

Figure 4 shows the primary mass spectrum and secondary mass spectrum of deuterated acenaphthene.

Figure 5 is an optimization diagram of ion pair collision energy.

Figure 6 shows the standard chromatograms of nicotine, cotinine and deuterated acenaphthene (isotope internal standard).

Figure 7 shows the chromatograms of nicotine, cotinine and deuterated acenaphthene in the same human serum sample.

Figure 8 shows the levels of nicotine and cotinine in the serum of regular smokers (n=10) and non-smokers (n=10).

Detailed ways

The instruments and reagents used in the examples are as follows:

Instruments and reagents: Trace1300 gas chromatograph-TSQ8000 tandem quadrupole mass spectrometer (Thermo Company, USA), methanol (chromatographically pure, Merck, Germany)

Standard materials: nicotine, purchased from Nanjing Wanqing Glass Instrument Co., Ltd., solid powder; cotinine, purchased from AChemtek Company of the United States, as a methanol solution with a concentration of 100 μg/mL; deuterated acenaphthene, purchased from Beijing Bailingwei Technology Co., Ltd. It is a methanol solution with a concentration of 500 μg/mL.

Preparation of standard material solution: Accurately weigh the nicotine solid powder, add methanol (chromatographically pure) to dissolve, and prepare a 1 mg/mL nicotine stock solution. For immediate use, use chromatographically pure methanol to dilute the nicotine stock solution into a nicotine application solution with a concentration of 1 μg/mL. Use chromatographically pure methanol to dilute the purchased cotinine standard solution into a cotinine application solution with a concentration of 1 μg/mL. Use Use chromatographically pure methanol to dilute the purchased deuterated acenaphthene standard solution into a deuterated acenaphthene application solution with a concentration of 1 μg/mL. Serum pretreatment: accurately draw 25 μL of human serum, add 5 μL of deuterated acenaphthene application solution with a concentration of 1 μg/mL, mix thoroughly, then add 70 μL of methanol, vortex, and centrifuge at 12000 rpm/min for 10 min at a temperature of 4°C. Aspirate the supernatant and wait for testing.

Gas chromatography conditions: The chromatographic column is a DB-EUPAH capillary column (specification: 20m×0.18mm×0.14μm), the injection volume is 2μL, splitless injection, the injection port temperature is 220°C, the carrier gas is helium, and the flow rate is 1.0mL/min, programmed temperature rise: initial temperature is 60℃, maintained for 1min; raised to 280℃ at 20℃/min, maintained for 2min.

Mass spectrometry conditions are: electron bombardment ionization source, 70eV. The temperatures of the ion source and transfer line were both 300°C, in MS/MS detection mode.

Example 1

Optimization of instrument conditions

1.1 Selection of capillary separation column

Taking cotinine as an example, the DB-5 capillary column (specifications: length 30m, inner diameter 0.25mm, film thickness 0.25μm) and DB-EUPAH capillary column (specifications: length 20m, inner diameter 0.18mm, film thickness 0.14μm) were investigated respectively. Dissociation and response effects to cotinine.

Cotinine application solution with a concentration of 1 μg/mL was used for testing. The test conditions are as follows:

The gas chromatography conditions used in the capillary column are: injection volume is 2 μL, splitless injection, inlet temperature is 220°C, carrier gas is helium, flow rate is 1.0mL/min, temperature program: initial temperature is 60°C , keep for 1 minute; increase to 280°C at 20°C/min, and keep for 2 minutes.

The mass spectrometry conditions of both capillary columns are: electron impact ionization source, 70eV; the temperature of the ion source and transmission line are both 300°C; one-stage full scan mode, the scanning range is 50-300m/z, and the solvent delay is 3min.

The results are shown in Figure 1, which shows that compared with the DB-5 capillary column, the peak shape of cotinine on the DB-EUPAH capillary column is significantly improved, and the sensitivity is also higher. Therefore, DB-EUPAH capillary column was selected.

1.2 Optimization of mass spectrometry conditions

MS/MS has the characteristics of high sensitivity and strong qualitative ability. In order to meet the requirement of using trace amounts of human serum to detect trace amounts of nicotine and cotinine through simple processing, the test optimized the parameters for MS/MS mode detection, including nicotine, cotinine and deuterated acenaphthene (isotope internal standard). Selection of qualitative and quantitative ion pairs, selection of collision energies.

Nicotine application solution with a concentration of 1 μg/mL, cotinine application solution with a concentration of 1 μg/mL, and deuterated acenaphthene application solution with a concentration of 1 μg/mL were used for testing respectively. The gas chromatography conditions use the gas chromatography conditions of the DB-EUPAH capillary column under "1.1 Selection of Capillary Separation Column". The mass spectrometer uses an electron bombardment ionization source, 70eV; the temperatures of the ion source and transmission line are both 300°C.

When selecting ion pairs, each application liquid is first detected in the full scan mode of the first-level mass spectrometry. The first-level scan range is 50 to 300 m/z. The molecular ions of each standard substance in the obtained first-level mass spectrometry full scan spectrum are used as precursor ions. Secondary product ion scanning is performed based on the selected parent ion. The product ion scanning range is 30-200m/z and the collision energy is 30eV. The primary mass spectrum and secondary mass spectrum of nicotine are shown in Figure 2, the primary mass spectrum and secondary mass spectrum of cotinine are shown in Figure 3, and the primary mass spectrum and secondary mass spectrum of deuterated acenaphthene are shown in Figure 4. Select the one with relatively high response intensity. Both ions serve as product ions. According to the results, it is determined that the nicotine ion pair is 162-84m/z, 162-133m/z; the cotinine ion pair is 176-98m/z, 176-68m/z; the deuterated acenaphthene ion pair is 162-160m/z, 162-158m/z.

As shown in Figure 5, the selected product ions are optimized to the best response through different collision energies. The collision energies of ion pairs 162-84m/z and 176-98m/z are both 10eV; the collision energies of ion pairs 162-160m/z are 15eV; the collision energies of ion pairs 176-68m/z and 162-158m/z are both is 20eV; the collision energy of ion pair 162-133m/z is 35eV. Further, by comparing the responsivity of two pairs of ion pairs of each standard substance, the pair of ion pairs with higher response is selected as the quantitative ion pair. The quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z respectively. , 162-160m/z and 176-98m/z.

Dilute the nicotine application solution with a concentration of 1 μg/mL and the cotinine application solution with a concentration of 1 μg/mL with chromatographically pure methanol into a standard solution with a concentration of 10 ng/mL. Take the above 10 ng/mL nicotine standard solution and 10 ng/mL Mix 50 μL of cotinine standard solution and 50 μL of deuterated acenaphthene application solution with a concentration of 1 μg/mL, and then fill it up to 1 mL with chromatographically pure methanol, that is, prepare a final concentration of nicotine and cotinine of 0.5 ng/mL, deuterated Acenaphthene is a mixed standard solution of 50ng/mL. Use the gas phase conditions of the DB-EUPAH capillary column under "1.1 Selection of Capillary Separation Column" and the optimized mass spectrometry conditions under "1.2 Optimization of Mass Spectrometry Conditions". The results are shown in Figure 6, which shows that using optimized gas chromatography and mass spectrometry conditions, both lower concentration nicotine (retention time 5.92min) and lower concentration cotinine (retention time 9.27min) can effectively emit peaks. The sensitivity is high. The peak time of the isotope internal standard (deuterated acenaphthene) is in the middle of nicotine and cotinine (retention time is 7.13 minutes), and the three peaks are completed within 10 minutes.

Example 2

Method performance inspection

2.1 Examination of instrument detection limits: Examine the instrument detection limits of nicotine and cotinine at a signal-to-noise ratio of 3 times. Nicotine is approximately 0.1ng/mL, and cotinine is approximately 0.2ng/mL.

2.2 Examination of instrument quantitation limit: Examine the instrument quantification limit of nicotine and cotinine at a signal-to-noise ratio of 10 times. Nicotine is approximately 0.25ng/mL, and cotinine is approximately 0.5ng/mL.

2.3 Matrix matching internal standard standard curve: randomly draw 50 μL from each of 100 human serums, mix them evenly, and prepare a mixed serum matrix. The calibration range is formulated based on the contents of the two substances in the small sample test. According to Table 1, measure the mixed serum matrix, deuterated acenaphthene standard solution, nicotine standard solution, cotinine standard solution, and methanol to prepare 8 matrix matching standard curve samples, vortex thoroughly, and rotate at 12,000 rpm at 4°C. /min centrifuge, take the supernatant; use the gas phase conditions of the DB-EUPAH capillary column under "1.1 Selection of Capillary Separation Column", mass spectrometry conditions: tandem quadrupole mass spectrometry, the detection mode is MS/MS mode, and electron bombardment is used Ionization source, 70eV; the temperatures of the ion source and transmission line are both 300°C. The quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z, 162-160m/z and 176-98m/z respectively, and the ion pair collision energies are 162m/z-84m/z and 176-98m/z. Both are 10eV; the collision energy of ion pair 162-160m/z is 15eV.

Table 1. Matrix-matched standard curves for nicotine and cotinine

Note: The concentration of nicotine standard solution measured before mixing: 2 and 3 are 10ng/mL, 4 is 100ng/mL, 5 and 6 are 500ng/mL; the concentration of cotinine standard solution measured before mixing: 2 is 10ng/mL mL, 3 is 100ng/mL, 4 is 400ng/mL, 5 and 6 are 2μg/mL, 7 and 8 are 10μg/mL; the concentration of deuterated acenaphthene standard solution measured before mixing: 1-8 are all 1μg/mL (deuterated acenaphthene application solution). The above-mentioned nicotine standard solution in 2-6 and cotinine standard solution in 2-4 are obtained by diluting the corresponding application solution with chromatographically pure methanol; the cotinine standard solution in 5-8 is obtained by diluting its stock solution with chromatographically pure methanol. .

Taking the concentration of nicotine or cotinine as the abscissa, the ratio of the calibrated peak area of the nicotine or cotinine quantifier ion to the peak area of the deuterated acenaphthene quantifier ion (the peak area of the nicotine or cotinine quantifier ion at each concentration point The area is subtracted from the peak area of the nicotine or cotinine quantum ion in the matrix background, and then compared with the peak area of the deuterated acenaphthylene quantum ion, that is, the peak area of the concentration point No. 1 is subtracted) as the ordinate, respectively Establish a nicotine matrix-matched internal standard standard curve: y=0.0098x+0.0009, R ² =0.9997, and a cotinine matrix-matched internal standard standard curve: y=0.0041x+0.0002, R ² =0.9996. It is found that the correlation coefficients are all greater than 0.99. It has good linearity and can be used for accurate quantification of nicotine and cotinine in samples.

2.4 Method recovery rate and precision:

A certain amount of nicotine, cotinine and deuterated acenaphthene standard solutions were added to the mixed serum matrix to conduct a method recovery test. Use chromatographically pure methanol to dilute the nicotine application solution (1μg/mL) into standard solutions with concentrations of 100ng/mL and 10ng/mL respectively, and then use chromatographically pure methanol to dilute the 10ng/mL nicotine standard solution into 2ng/mL and 2ng/mL. 0.5ng/mL standard solution; cotinine application solution (1μg/mL) was diluted into standard solutions with concentrations of 500ng/mL, 100ng/mL and 10ng/mL respectively, and then 10ng/mL cotinine was diluted with chromatographically pure methanol. The tinine standard solution was diluted to a 2ng/mL standard solution.

The specific spiking test is: a spiking recovery test of three concentrations of nicotine. Take three 25 μL mixed serum matrix, add 5 μL of 10ng/mL nicotine standard solution to the first mixed serum matrix, and add 10ng to the second mixed serum matrix. /mL nicotine standard solution 20μL, add 100ng/mL nicotine standard solution to the third mixed serum matrix, and finally add 70μL, 55μL and 65μL chromatographically pure methanol to make up to 100μL, that is, the nicotine in the three mixed serum matrices The final concentrations were 0.5, 2 and 10ng/mL respectively. For the spike recovery test of four concentrations of cotinine, take four 25 μL mixed serum matrix, add 20 μL of 10ng/mL cotinine standard solution to the first mixed serum matrix, and add 100ng/mL to the second mixed serum matrix. 10 μL of cotinine standard solution, 10 μL of 1 μg/mL cotinine application solution was added to the third mixed serum matrix, 50 μL of 1 μg/mL cotinine application solution was added to the fourth mixed serum matrix, and finally 55 μL was added respectively. , 65μL, 65μL and 25μL of chromatographically pure methanol were added to 100μL, that is, the final concentrations of cotinine in the four mixed serum matrices were 2, 10, 100 and 500ng/mL respectively, and each concentration of nicotine and cotinine was repeated three times. . For a spike recovery test of one concentration of deuterated acenaphthene, take 25 μL of mixed serum matrix, add 5 μL of 1 μg/mL deuterated acenaphthene application solution, and finally add 70 μL of chromatographically pure methanol to make up to 100 μL, that is, the concentration of deuterated acenaphthene in the mixed serum matrix The final concentration was 50ng/mL, and this concentration was repeated three times. Take 25 μL of mixed serum matrix and add 75 μL of chromatography-grade methanol to make it up to 100 μL, that is, no spiked matrix (matrix background) is added.

After the above mixed serum matrix was fully vortexed, it was centrifuged at a temperature of 4°C and a rotation speed of 12000 rpm/min, and the supernatant was taken; using the gas phase conditions of the DB-EUPAH capillary column under "1.1 Selection of Capillary Separation Column", mass spectrometry Conditions: Tandem quadrupole mass spectrometry, detection mode is MS/MS mode, using electron bombardment ionization source, 70eV; the temperatures of the ion source and transmission line are both 300°C. The quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z, 162-160m/z and 176-98m/z respectively, and the ion pair collision energies are 162m/z-84m/z and 176-98m/z. Both are 10eV; the collision energy of ion pair 162-160m/z is 15eV. Calculate the recovery rate according to the formula "% recovery = (peak area of spiked matrix - peak area of matrix background) / peak area of standard solution corresponding to spiked final concentration × 100".

Table 2. Spiked recoveries and relative standard deviations of nicotine, cotinine and deuterated acenaphthene (n=3)

The test results are shown in Table 2, which shows that: the average recovery rate of nicotine spiked with different concentrations is between 84.9 and 120.6%, and the relative standard deviation is less than 7%; the average recovery rate of cotinine spiked with different concentrations is between 89.5 and 102.2%, and the relative standard deviation is less than 7%. The standard deviation is less than 13%; the average recovery rate of deuterated acenaphthene is 92.3%, and the relative standard deviation is 7.4%.

Example 3

Serum samples from 10 male smokers and 10 non-smokers aged 50 to 80 years were selected.

Serum treatment: accurately draw 25 μL of human serum, add 5 μL of deuterated acenaphthene application solution with a concentration of 1 μg/mL, mix thoroughly, then add 70 μL of methanol, vortex, centrifuge at 12000 rpm/min for 10 min at 4°C, and aspirate The supernatant was analyzed by gas chromatography-tandem mass spectrometry.

Gas chromatography detection conditions: The gas chromatography column is a DB-EUPAH column, specifications: 20m×0.18mm×0.14μm, the injection volume is 2μL, the injection port temperature is 220°C, the carrier gas is helium, and the flow rate is 1.0 mL/min, programmed temperature rise: the initial temperature is 60°C, maintained for 1 min, raised to 280°C at 20°C/min, and maintained for 2 min.

Mass spectrometry conditions: tandem quadrupole mass spectrometry, detection mode is MS/MS mode, electron bombardment ionization source is used, 70eV; the temperatures of the ion source and transmission line are both 300°C. The quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z, 162-160m/z and 176-98m/z respectively, and the ion pair collision energies are 162m/z-84m/z and 176-98m/z. Both are 10eV; the collision energy of ion pair 162-160m/z is 15eV.

The collected data were processed using Thermo Xcalibur software. The raw data are characterized based on the compound's retention time and qualitative ion transitions; based on the quantitative ion transitions, the matrix-matched internal standard standard curve is used for accurate quantification.

The test results show that the method of the present invention can sensitively and accurately detect nicotine and cotinine in human serum. The chromatogram of a typical sample is shown in Figure 7. The test results also showed that the serum nicotine and cotinine levels in smokers were significantly higher than those in non-smokers; the serum cotinine levels in smokers were significantly higher than nicotine, while the serum cotinine levels in non-smokers were significantly lower than nicotine (Figure 8), which is consistent with the trend of relevant foreign research reports.

Claims (10)

1. A method for rapidly determining nicotine and cotinine in trace human serum based on gas chromatography-tandem mass spectrometry is characterized in that: comprising the following steps: taking 15-100 mu L of serum, adding 5-50 mu L of deuterated isotope internal standard solution, uniformly mixing, adding 45-300 mu L of methanol, fully vortex, centrifuging, and taking supernatant for gas chromatography-tandem mass spectrometry detection; qualitative characterization of nicotine, cotinine, according to qualitative ion pair and retention time; substituting the ratio of the peak area of the quantitative sub-ions of nicotine or cotinine to the peak area of the standard quantum ions in the deuterated isotope into the matrix of nicotine or cotinine to match an internal standard curve, so as to obtain the concentration of nicotine and cotinine;

wherein, the gas chromatography detection conditions are as follows: the chromatographic column is a DB-EUPAH capillary column with the specification of: the column length is 15-30 m, the inner diameter is 0.18-0.25 mm, and the film thickness is 0.14-0.25 mu m; the sample injection amount is 1-2 mu L, the sample injection mode is not split sample injection, and the temperature of a sample injection port is 200-280 ℃; the initial temperature of the chromatographic column is 35-100 ℃, the chromatographic column is kept for 1min, and the chromatographic column is heated to 280 ℃ at 20 ℃/min and is kept for 2min;

quantitative ion pairs of nicotine, cotinine and acenaphthene are 162-84m/z, 176-98m/z and 162-160m/z, respectively;

ion pairs 162-84m/z and 176-98m/z have collision energies of 10eV; the ion pair 162-160m/z has a collision energy of 15eV.

2. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: 25 mu L of serum is taken, 5 mu L of deuterated isotope internal standard solution is added and mixed uniformly, 70 mu L of methanol is added, vortex and centrifuge are carried out fully, and the supernatant is taken for gas chromatography-tandem mass spectrometry detection.

3. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: the deuterated isotope internal standard is deuterated acenaphthene; the deuterated isotope internal standard solution is methanol solution with the concentration of deuterated acenaphthene of 1 mug/mL.

4. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: the centrifugation conditions are as follows: centrifuging at the temperature of 4-40 ℃ and the rotating speed of 5000-12000 rpm/min for 5-15 min.

5. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: the gas chromatography detection conditions are as follows: the chromatographic column is a DB-EUPAH capillary column with the specification of: the column length is 20m, the inner diameter is 0.18mm, and the film thickness is 0.14 mu m; the sample injection amount is 2 mu L, the sample injection mode is non-split sample injection, and the temperature of the sample injection port is 220 ℃; the initial temperature of the chromatographic column is 60 ℃, the chromatographic column is kept for 1min, and the chromatographic column is raised to 280 ℃ at 20 ℃/min and is kept for 2min.

6. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: tandem mass spectrometry detection conditions: electron bombardment ionization source, 70eV; the temperature of the ion source and the transmission line are 300 ℃; the detection mode is an MS/MS detection mode.

7. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: the qualitative ion pairs of nicotine are 162-84m/z and 162-133m/z; the qualitative ion pairs of cotinine are 176-98m/z and 176-68m/z; the qualitative ion pairs of deuterated acenaphthene are 162-160m/z and 162-158m/z.

8. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 7, wherein the method comprises the steps of: ion pairs 176-68m/z and 162-158m/z have collision energies of 20eV; ion pairs 162-133m/z have an impact energy of 35eV.

9. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: the retention time of the nicotine is 5.7-6.2 min, and the retention time of the cotinine is 9.0-9.5 min.

10. The method for rapidly determining nicotine and cotinine in trace amounts of human serum based on gas chromatography-tandem mass spectrometry of claim 1, wherein the method comprises the steps of: the matrix-matched internal standard curve of nicotine or cotinine is drawn by the following method: mixing 25 μl of mixed human serum with nicotine standard solution, cotinine standard solution and 5 μl of deuterated acenaphthene solution, fixing volume with methanol to 100 μl to obtain standard curve solution, swirling, centrifuging, collecting supernatant, and performing gas chromatography-tandem mass spectrometry detection; establishing a matrix matching internal standard curve of nicotine or cotinine by taking the concentration of nicotine or cotinine as an abscissa and taking the ratio of the corrected peak area of quantitative sub-ions of nicotine or cotinine to the peak area of quantitative sub-ions of deuterated acenaphthene as an ordinate; the corrected peak area of the quantitative sub-ions of the nicotine or the cotinine is the peak area of the quantitative sub-ions of the nicotine or the cotinine subtracted by the peak area of the quantitative sub-ions of the nicotine or the cotinine in the substrate background.