CN115389651B - Method for rapidly determining nicotine and cotinine in trace human serum based on gas chromatography-tandem mass spectrometry - Google Patents
Method for rapidly determining nicotine and cotinine in trace human serum based on gas chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for rapidly determining nicotine and cotinine in trace human serum based on gas chromatography-tandem mass spectrometry, which comprises 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 ion of nicotine or cotinine to the peak area of the deuterated isotope internal standard into the matrix of nicotine or cotinine to match the internal standard curve, and obtaining the concentration of nicotine and cotinine. According to the invention, only a trace amount of serum sample is needed, nicotine and cotinine are effectively separated based on a specific capillary column, and the optimized serial four-level rod monitoring parameters are utilized to accurately, sensitively, time-effectively and efficiently obtain residual data of the nicotine and the cotinine, and the cost is low.
Description
Technical Field
The invention relates to a detection method of pollutants and metabolites thereof in human serum, in particular to a method for rapidly determining nicotine and cotinine in trace human serum based on gas chromatography-tandem mass spectrometry.
Background
Smoking and health are among the most interesting public health problems worldwide. Nicotine (formula I, formula C) 10 H 14 N 2 ) And its metabolite cotinine (formula II, formula C) 10 H 12 N 2 The human body residue of O) is an important marker reflecting the individual smoking status. Serum is an ideal biological sample for determining nicotine and cotinine residues, but the sample collection has limited sampling volume.
Common analytical methods for trace amounts of nicotine and cotinine in serum are gas chromatography, liquid chromatography-tandem mass spectrometry. The gas chromatography and the liquid chromatography have low sensitivity and poor fixed performance, the demand on serum samples is large (about 1-2 mL), and the samples need complex pretreatment such as protein precipitation, liquid-liquid extraction or solid phase extraction, nitrogen blowing concentration and the like; the liquid chromatography-tandem mass spectrometry has high sensitivity and accuracy, and the required serum amount is small, but the instrument and the equipment are expensive. The above methods are difficult to popularize in smoking screening applications.
Disclosure of Invention
In view of the lack of a method for efficiently, accurately and low-cost detection of nicotine and cotinine in trace human serum at present, the invention provides a method for rapidly determining nicotine and cotinine in trace human serum based on gas chromatography-tandem mass spectrometry.
The aim of the invention can be achieved by the following technical scheme:
a method for rapidly determining nicotine and cotinine in trace human serum based on gas chromatography-tandem mass spectrometry, comprising: taking 15-100 mu L of human 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 the nicotine or cotinine to the peak area of the quantitative sub-ions of the deuterated isotope internal standard respectively into the matrix matching internal standard curve of the nicotine or cotinine to obtain the concentration of the nicotine and the 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;
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.
Preferably, 25 mu L of human serum is taken, 5 mu L of deuterium isotope internal standard solution is added for uniform mixing, 70 mu L of methanol is added, vortex is carried out fully, centrifugation is carried out, and the supernatant is taken for gas chromatography-tandem mass spectrometry detection.
Preferably, 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.
The centrifugal conditions are as follows: centrifuging at the temperature of 4-40 ℃ and the rotation speed of 5000-12000 rpm/min for 5-15 min; preferably, it is: centrifugation was carried out at 12000rpm/min for 10min at a temperature of 4 ℃.
Preferably, 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.
The mass spectrum is tandem quadrupole mass spectrum.
Preferably, 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. The quantitative ion pairs of nicotine, cotinine and deuterated acenaphthene are 162-84m/z, 176-98m/z and 162-160m/z respectively, i.e. the quantitative ion pairs of nicotine, cotinine and deuterated acenaphthene are 84m/z, 98m/z and 160m/z respectively,
the higher the response, the higher the sensitivity and the higher the detection rate of the target substance in the sample. Preferably, ion pairs 162-84m/z and 176-98m/z have collision energies of 10eV; ion pairs 162-160m/z have a collision energy of 15eV; 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.
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;
preferably, the retention time of the 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 curve of nicotine or cotinine is drawn by the following method: mixing 25 μl of mixed human serum with 5 μl of at least one of deuterated acenaphthene solution and nicotine standard solution or cotinine standard solution, adding 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; wherein 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; the range of the standard curve of the matrix matching internal standard of the nicotine is 0-25 ng/mL; the range of the matrix matching internal standard curve of cotinine is 0-500 ng/mL.
The mixed human serum is as follows: 50 μl of each of 100 human serum was randomly aspirated, and mixed well to prepare a mixed serum matrix.
The substrate background is as follows: 25 μl of the mixed human serum was mixed with 5 μl of deuterated acenaphthene solution, and the volume was fixed with methanol to 100 μl.
The beneficial effects of the invention are as follows:
according to the invention, only a trace amount of serum sample is needed, nicotine and cotinine are effectively separated based on a specific capillary column, and the optimized serial four-level rod monitoring parameters are utilized to accurately, sensitively, time-effectively and efficiently obtain residual data of the nicotine and the cotinine, and the cost is low.
The method is suitable for research on smoking and hazard assessment of large sample population and objective evaluation and analysis of smoking control effect.
Drawings
FIG. 1 is a graph of the response effect of cotinine on capillary columns DB-5 and DB-EUPAH, respectively.
Figure 2 is a primary and secondary mass spectrum of nicotine.
Fig. 3 is a primary and secondary mass spectrum of cotinine.
Fig. 4 is a primary and secondary mass spectrum of deuterated acenaphthene.
Fig. 5 is an ion versus collision energy optimization graph.
Fig. 6 is a chromatogram of a standard of nicotine, cotinine and deuterated acenaphthene (isotopic internal standard).
FIG. 7 is a chromatogram of nicotine, cotinine and deuterated acenaphthene in the same human serum sample.
Fig. 8 is the nicotine and cotinine content in serum of regular smokers (n=10) and non-smokers (n=10).
Detailed Description
Examples the instruments and reagents used were as follows:
instrument and reagent: trace1300 gas chromatograph-TSQ 8000 tandem quadrupole mass spectrometer (Thermo Co., USA), methanol (chromatographic purity, merck, germany)
Standard substance: nicotine, solid powder from Nanjing late-Qing glass instruments Co., ltd; cotinine, available from AChemtek corporation, usa, is a 100 μg/mL methanol solution; deuterated acenaphthene, available from Beijing carbofuran technologies Inc., is a 500 μg/mL methanol solution.
Preparing a standard substance solution: accurately weighing nicotine solid powder, adding methanol (chromatographic purity) for dissolution, and preparing 1mg/mL nicotine stock solution. In use, the nicotine stock solution is diluted to a nicotine application solution with a concentration of 1 mug/mL by using chromatographic pure methanol, the purchased cotinine standard solution is diluted to a cotinine application solution with a concentration of 1 mug/mL by using chromatographic pure methanol, and the purchased deuterated acenaphthene standard solution is diluted to a deuterated acenaphthene application solution with a concentration of 1 mug/mL by using chromatographic pure methanol. Serum pretreatment: accurately sucking 25 mu L of human serum, adding 5 mu L of deuterated acenaphthene application liquid with the concentration of 1 mu g/mL, fully and uniformly mixing, adding 70 mu L of methanol, swirling, centrifuging at the temperature of 4 ℃ for 10min at the speed of 12000rpm/min, sucking out supernatant, and measuring.
Gas chromatography conditions: the chromatographic column is a DB-EUPAH capillary column (specification: 20m×0.18mm×0.14 μm), the sample injection amount is 2 μL, the sample injection is not split, the sample injection port temperature is 220 ℃, the carrier gas is helium, the flow rate is 1.0mL/min, and the temperature programming is carried out: the initial temperature is 60 ℃, and the temperature is kept for 1min; raising the temperature to 280 ℃ at 20 ℃/min and keeping the temperature for 2min.
The mass spectrum conditions are as follows: electrons bombard the ionization source, 70eV. The ion source and the transmission line are both at 300℃and MS/MS detection mode.
Example 1
Optimization of instrument conditions
1.1 selection of capillary separation column
Taking cotinine as an example, the separation and response effects of DB-5 capillary column (specification: length 30m, inner diameter 0.25mm, film thickness 0.25 μm) and DB-EUPAH capillary column (specification: length 20m, inner diameter 0.18mm, film thickness 0.14 μm) on cotinine were examined respectively.
The test was performed using cotinine application liquid at a concentration of 1. Mu.g/mL under the following conditions:
the gas chromatography conditions used for the capillary column were: the sample injection amount is 2 mu L, sample injection is not split, the temperature of the sample injection port is 220 ℃, the carrier gas is helium, the flow rate is 1.0mL/min, and the temperature programming is carried out: the initial temperature is 60 ℃, and the temperature is kept for 1min; raising the temperature to 280 ℃ at 20 ℃/min and keeping the temperature for 2min.
The mass spectrometry conditions of both capillary columns were: electron bombardment ionization source, 70eV; the temperature of the ion source and the transmission line are 300 ℃; the first-stage full-scanning mode is adopted, the scanning range is 50-300m/z, and the solvent delay is 3min.
The results are shown in FIG. 1, which shows: compared with DB-5 capillary column, cotinine has obvious improvement of the upper peak type of DB-EUPAH capillary column and higher sensitivity. Thus, a DB-EUPAH capillary column was selected.
1.2 optimization of Mass Spectrometry Condition
The MS/MS has the characteristics of high sensitivity and strong fixed capacity. In order to meet the requirement that trace nicotine and cotinine can be detected by using trace human serum through simple treatment, the test optimizes parameters of MS/MS mode detection, including selection of qualitative and quantitative ion pairs of nicotine, cotinine and deuterated acenaphthene (isotope internal standard), and selection of collision energy.
The tests were performed with nicotine application solution at a concentration of 1. Mu.g/mL, cotinine application solution at a concentration of 1. Mu.g/mL, and deuterated acenaphthene application solution at a concentration of 1. Mu.g/mL, respectively. The gas chromatography conditions used were those of the DB-EUPAH capillary column under the "selection of 1.1 capillary separation column". Mass spectrometry uses electron bombardment ionization source, 70eV; the temperature of the ion source and the transmission line were 300 ℃.
When the ion pair is selected, each application liquid is detected by adopting a primary mass spectrum full-scanning mode, wherein the primary scanning range is 50-300m/z, the molecular ions of each standard substance in the obtained primary mass spectrum full-scanning spectrum are used as parent ions, secondary sub-ion scanning is carried out according to the selected parent ions, the ion scanning range is 30-200 m/z, and the collision energy is 30eV. The primary mass spectrum and the secondary mass spectrum of nicotine are shown in figure 2, the primary mass spectrum and the secondary mass spectrum of cotinine are shown in figure 3, the primary mass spectrum and the secondary mass spectrum of deuterated acenaphthene are shown in figure 4, and two ions with relatively high response intensity are selected as sub-ions. And according to the result, determining: 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 and 162-158m/z.
As shown in fig. 5, the selected ions are optimized to the best response through different collision energies. The collision energy of the ion pairs 162-84m/z and 176-98m/z is 10eV; ion pairs 162-160m/z have a collision energy of 15eV; the ion pairs 176-68m/z and 162-158m/z have collision energies of 20eV; the ion pair 162-133m/z has a collision energy of 35eV. Further by comparing the responsivity of the two pairs of each standard substance, a pair of ion pairs with higher response is selected as quantitative ion pairs, and the quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z, 162-160m/z and 176-98m/z respectively.
And respectively diluting the nicotine application solution with the concentration of 1 mug/mL and the cotinine application solution with the concentration of 1 mug/mL with chromatographic pure methanol to obtain standard solutions with the concentration of 10ng/mL, mixing 50 mug of the nicotine standard solution with the concentration of 10ng/mL and 50 mug of the deuterated acenaphthene application solution with the concentration of 1 mug/mL with the standard solution of 10ng/mL, and supplementing the mixture to 1mL with the chromatographic pure methanol to obtain the mixed standard solution with the final concentration of nicotine, 0.5ng/mL and 50ng/mL of deuterated acenaphthene. The gas phase condition of DB-EUPAH capillary column under the item of 1.1 capillary separation column selection and the optimized mass spectrum condition under the item of 1.2 mass spectrum condition optimization are adopted. The results are shown in fig. 6, which shows: the optimized gas chromatography and mass spectrum conditions are adopted, the lower concentration nicotine (retention time is 5.92 min) and the lower concentration cotinine (retention time is 9.27 min) can effectively emit peaks, the sensitivity is high, the peak-emitting time of the isotope internal standard (deuterated acenaphthene) is in the middle of the nicotine and cotinine (retention time is 7.13 min), and the peaks are completed within 10min.
Example 2
Method performance investigation
2.1 investigation of instrument detection limit: the instrument limits for nicotine and cotinine were examined at a 3-fold signal to noise ratio, with nicotine being about 0.1ng/mL and cotinine being about 0.2ng/mL.
2.2 quantitative limit investigation by instrument: the instrument limits for nicotine and cotinine were examined at a 10-fold signal to noise ratio, with nicotine being about 0.25ng/mL and cotinine being about 0.5ng/mL.
2.3 matrix matching internal standard curve: 50 μl of each of 100 human serum was randomly aspirated, and mixed well to prepare a mixed serum matrix. The standard curve range is formulated according to the contents of two substances in a small sample test. Measuring and mixing serum matrix, deuterated acenaphthene standard solution, nicotine standard solution, cotinine standard solution and methanol according to table 1, preparing 8 matrix matching standard curve samples, fully vortex, centrifuging at 12000rpm/min at 4deg.C, and collecting supernatant; gas phase conditions, mass spectrometry conditions using a DB-EUPAH capillary column under "selection of 1.1 capillary separation column" section: tandem quadrupole mass spectrometry, wherein the detection mode is MS/MS mode, and electron bombardment ionization source is used, 70eV; the temperature of the ion source and the transmission line were 300 ℃. Quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z, 162-160m/z and 176-98m/z respectively, and collision energy of the ion pairs is 162m/z-84m/z and 176-98m/z is 10eV; ion pairs 162-160m/z have collision energies of 15eV.
TABLE 1 matrix match standard curve for nicotine and cotinine
Note that: concentration of nicotine standard solution measured before mixing: 2.3 is 10ng/mL,4 is 100ng/mL,5 and 6 are 500ng/mL; concentration of cotinine standard solution measured before mixing: 2 is 10ng/mL,3 is 100ng/mL,4 is 400ng/mL,5, 6 is 2 μg/mL,7, 8 is 10 μg/mL; concentration of standard solution of deuterated acenaphthene measured before mixing: 1-8 are 1 μg/mL (deuterated acenaphthene application liquid). The nicotine standard solution in the 2-6 and the cotinine standard solution in the 2-4 are both obtained by diluting corresponding application liquid by chromatographic pure methanol; 5-8, and diluting the stock solution of the cotinine standard solution with chromatographic pure methanol.
The ratio of the corrected peak area of the quantitative sub-ion of nicotine or cotinine to the peak area of the quantitative sub-ion of deuterated acenaphthene (the peak area of the quantitative sub-ion of nicotine or cotinine in each concentration point is respectively subtracted by the Nigulin the substrate background)Ding Huoke Tining quantitative ion peak area, and then with the deuterated acenaphthene quantitative ion peak area ratio, namely minus 1 concentration point peak area) as the ordinate, respectively establishing nicotine matrix matching internal standard curves: y=0.0098x+0.0009, r 2 =0.9997, cotinine matrix matched internal standard curve: y=0.0041x+0.0002, r 2 = 0.9996, and the correlation coefficients were found to be greater than 0.99, with good linearity, and can be used for accurate quantification of nicotine and cotinine in samples.
2.4 method recovery and precision:
a certain amount of nicotine, cotinine and deuterated acenaphthene standard solution are added into the mixed serum matrix for carrying out a method recovery rate test. Diluting nicotine application solution (1 mu g/mL) with chromatographic pure methanol to form standard solutions with the concentration of 100ng/mL and 10ng/mL respectively, and diluting the 10ng/mL nicotine standard solution with chromatographic pure methanol to form standard solutions with the concentration of 2ng/mL and 0.5ng/mL respectively; cotinine use solution (1 mug/mL) is diluted into standard solutions with the concentration of 500ng/mL,100ng/mL and 10ng/mL respectively, and then the 10ng/mL cotinine standard solution is diluted into the 2ng/mL standard solution by using chromatographic pure methanol.
The labeling test is specifically as follows: and (3) adding standard recovery test of three concentrations of nicotine, namely taking 3 parts of 25 mu L of mixed serum matrix, adding 5 mu L of 10ng/mL of nicotine standard solution into the first part of mixed serum matrix, adding 20 mu L of 10ng/mL of nicotine standard solution into the second part of mixed serum matrix, adding 10 mu L of 100ng/mL of nicotine standard solution into the third part of mixed serum matrix, and finally adding 70 mu L, 55 mu L and 65 mu L of chromatographic pure methanol to complement to 100 mu L respectively, namely, the final concentration of nicotine in the three parts of mixed serum matrix is 0.5, 2 and 10ng/mL respectively. Labeling recovery test of cotinine four concentrations, taking 4 parts of 25 mu L of mixed serum matrix, adding 10ng/mL of cotinine standard solution 20 mu L into the first part of mixed serum matrix, adding 100ng/mL of cotinine standard solution 10 mu L into the second part of mixed serum matrix, adding 1 mu g/mL of cotinine application solution 10 mu L into the third part of mixed serum matrix, adding 1 mu g/mL of cotinine application solution 50 mu L into the fourth part of mixed serum matrix, and finally adding 55 mu L, 65 mu L and 25 mu L of chromatographic pure methanol to make up to 100 mu L respectively, namely, the final concentration of cotinine in the four parts of mixed serum matrix is 2, 10, 100 and 500ng/mL respectively, and nicotine and cotinine are repeated three times each concentration. Labeling recovery test of one concentration of deuterated acenaphthene, taking 25 mu L of mixed serum matrix, adding 5 mu L of deuterated acenaphthene application liquid of 1 mu g/mL, and finally adding 70 mu L of chromatographic pure methanol to complement to 100 mu L, namely, the final concentration of deuterated acenaphthene in the mixed serum matrix is 50ng/mL, and the concentration is repeated for 3 times. 25. Mu.L of the mixed serum matrix was taken and supplemented with 75. Mu.L of chromatographically pure methanol to 100. Mu.L, i.e.without addition of the standard matrix (matrix background).
Centrifuging the mixed serum matrix at 12000rpm/min at 4deg.C after fully swirling, and collecting supernatant; gas phase conditions, mass spectrometry conditions using a DB-EUPAH capillary column under "selection of 1.1 capillary separation column" section: tandem quadrupole mass spectrometry, wherein the detection mode is MS/MS mode, and electron bombardment ionization source is used, 70eV; the temperature of the ion source and the transmission line were 300 ℃. Quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z, 162-160m/z and 176-98m/z respectively, and collision energy of the ion pairs is 162m/z-84m/z and 176-98m/z is 10eV; ion pairs 162-160m/z have collision energies of 15eV. The recovery rate was calculated as%recovery rate = (peak area of labeled substrate-peak area of substrate background)/peak area of standard solution corresponding to labeled final concentration x 100 ".
Table 2, additive recovery of nicotine, cotinine and deuterated acenaphthene with relative standard deviation (n=3)
The detection results are shown in table 2, and indicate that: the average recovery rate of different concentrations of nicotine is between 84.9 and 120.6 percent, and the relative standard deviation is less than 7 percent; the average recovery rate of cotinine with different concentrations added with standard is 89.5-102.2%, and the relative standard deviation is less than 13%; the average standard recovery of deuterated acenaphthene was 92.3% with a relative standard deviation of 7.4%.
Example 3
Serum samples of 10 cases of male smokers and non-smokers aged 50-80 years were selected.
Serum treatment: accurately sucking 25 mu L of human serum, adding 5 mu L of deuterated acenaphthene application liquid with the concentration of 1 mu g/mL, fully and uniformly mixing, adding 70 mu L of methanol, centrifuging for 10min at the speed of 12000rpm/min at the temperature of 4 ℃ after vortex, sucking out supernatant, and carrying out gas chromatography-tandem mass spectrometry detection.
Detection conditions for gas chromatography: the chromatographic column of the gas chromatograph is a DB-EUPAH column with the specification: 20m x 0.18mm x 0.14 mu m, the sample injection amount is 2 mu L, the sample injection port temperature is 220 ℃, the carrier gas is helium, the flow rate is 1.0mL/min, and the temperature programming is carried out: the initial temperature was 60℃for 1min, and at 20℃per min up to 280℃for 2min.
Mass spectrometry conditions: tandem quadrupole mass spectrometry, wherein the detection mode is MS/MS mode, and electron bombardment ionization source is used, 70eV; the temperature of the ion source and the transmission line were 300 ℃. Quantitative ion pairs of nicotine, deuterated acenaphthene and cotinine are 162-84m/z, 162-160m/z and 176-98m/z respectively, and collision energy of the ion pairs is 162m/z-84m/z and 176-98m/z is 10eV; ion pairs 162-160m/z have collision energies of 15eV.
The collected data was processed using Thermo Xcalibur software. The raw data are characterized according to retention time of the compound and qualitative ion pairs; based on the quantitative ion pairs, accurate quantification is performed by matching the matrix with an internal standard curve.
The detection result shows that the method can sensitively and accurately detect the nicotine and cotinine in human serum, and a typical sample chromatogram is shown in fig. 7. The detection result also shows that the contents of nicotine and cotinine in the serum of the smoker are obviously higher than those of the non-smoker; the content of the cotinine in the serum of the smoker is obviously higher than that of the nicotine, and the content of the cotinine in the serum of the non-smoker is obviously lower than that of the nicotine (figure 8), which is consistent with the trend reported by related research abroad.
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.
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| CN102565231A (en) * | 2012-01-09 | 2012-07-11 | 贵州省烟草科学研究所 | Method for determining eight kinds of alkaloid in cured tobacco through gas chromatography-nitrogen chemoluminescence detection method |
| CN106353419A (en) * | 2016-08-16 | 2017-01-25 | 国家烟草质量监督检验中心 | Method for measuring aroma components in main stream smoke of cigarettes |
| CN107490641A (en) * | 2017-08-23 | 2017-12-19 | 国家烟草质量监督检验中心 | A kind of method of 9 kinds of secondary alkaloids in gas-chromatography tandem mass spectrometry measure tobacco juice for electronic smoke |
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| CN102565231A (en) * | 2012-01-09 | 2012-07-11 | 贵州省烟草科学研究所 | Method for determining eight kinds of alkaloid in cured tobacco through gas chromatography-nitrogen chemoluminescence detection method |
| CN106353419A (en) * | 2016-08-16 | 2017-01-25 | 国家烟草质量监督检验中心 | Method for measuring aroma components in main stream smoke of cigarettes |
| CN107490641A (en) * | 2017-08-23 | 2017-12-19 | 国家烟草质量监督检验中心 | A kind of method of 9 kinds of secondary alkaloids in gas-chromatography tandem mass spectrometry measure tobacco juice for electronic smoke |
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