CN110779996A - Method for measuring content of volatile N-nitrosamine compound in plasma by gas chromatography-mass spectrometry - Google Patents
Method for measuring content of volatile N-nitrosamine compound in plasma by gas chromatography-mass spectrometry Download PDFInfo
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- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 21
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract
The invention discloses a method for measuring the content of volatile N-nitrosamine compounds in blood plasma by gas chromatography-mass spectrometry, which comprises the following steps: (1) taking a blood sample, centrifuging and separating plasma; adding dichloromethane and NDPA-D14 internal standard solution into plasma, vortexing in a vortex mixer, and performing ultrasonic extraction after uniform mixing; centrifuging again after finishing; sampling the lower layer liquid; (2) the measurement was carried out by gas chromatography-mass spectrometry. The method for measuring the content of the volatile N-nitrosamine compound in the plasma by the gas chromatography-mass spectrometry can simultaneously measure 9 volatile N-nitrosamine compounds in the human plasma, and the detection limit and the lower limit of the quantification of the 9N-nitrosamine compounds are respectively between 0.01 ng/mL and 2.14ng/mL and 0.02 ng/mL to 7.14ng/mL, so that the method conveniently provides a basis for the research of the load level of the N-nitrosamine compound in the human body and the research of the influence of the exposure of the N-nitrosamine compound on the health of the human body.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for determining the content of volatile N-nitrosamine compounds in blood plasma.
Background
Nitrosamine compounds (NAs) are a group of organic compounds having the basic structure R1(R2) N-N ═ O, formed by the reaction of an amine or its derivative with a nitrating agent such as nitrous acid, nitrite or nitrogen oxide. Can be absorbed into human body through respiratory tract, digestive tract and skin surface. Volatile nitrosamines (VANs) are more toxic than non-volatile nitrosamines (NVANs). The volatile nitrosamines mainly comprise: N-Nitrosodimethylamine (NDMA), N-Nitrosomethylethylamine (NMEA), N-Nitrosodiethylamine (NDEA), N-nitrosodipropylamine (N-nisodimethyl ethyl amine, NMEA), N-nitrosodibutylamine (N-nisodiethyl amine, NDEA), N-nitrosodipropylamine (N-nisodi-N-propylamine, NDPA), N-nitrosodibutylamine (N-nisodibutylamine, NDBA), N-nitrosopiperidinane (N-nisodiperidine, NPIP), N-nitrosopyrrolidine (N-nisoproxiline, NPYR), N-nitrosomorpholine (N-nisodipropylamine, NMOR), N-nitrosodiphenylamine (N-nisodiphenylamine, nda). Etc. (the structure is as follows). According to the classification of International Agency for Research on Cancer (IARC), NDPA, NDBA, NPIP, NPYR, NMOR are 2B carcinogens, NDMA, NDEA are 2A carcinogens, and other N-nitrosamine compounds are not known to be carcinogenic to humans.
Exposure of the human body to N-nitrosamine compounds occurs primarily through two pathways: (1) the N-nitrosamine compound is taken, inhaled and contacted by factors such as diet, drinking water, air and the like; (2) endogenous N-nitrosamine compounds formed in the human stomach. N-nitrosamine compounds are widely present in the environment, and are contained in particular in the exhaust gases directly emitted by the production of polymers in plants and in the atmospheric particulates generated by the combustion of plastic materials. Drinking water can produce disinfection by-products containing N-nitrosamine compounds during disinfection, and organic nitrogen precursors containing chloramines can react to form NDMA, NDEA, NMOR, NPYR, NPIP, and NDPhA during drinking water disinfection. Certain processed foods also contain N-nitrosamine compounds, such as nitrosamine compounds produced by the reaction of amines with nitrites during smoking or pickling of fish and bacon; the smoke generated in the combustion process of tobacco contains three volatile N-nitrosamine compounds (14) of NDMA, NMEA and NPYR, and the smokeless tobacco product also contains N-nitrosamine compounds. Some daily necessities such as hair dyes also contain N-nitrosamine compounds.
Most of N-nitrosamine compounds are considered carcinogenic precursors, and the carbon chain beside the N-nitroso group is hydroxylated to form a carcinogenic derivative α -hydroxy-N-nitrosamine, α -hydroxy-N-nitrosamine has a relatively short half-life, is decomposed into an electrophilic intermediate, reacts with DNA, and causes DNA damage, thereby influencing telomere length, and telomere abnormality is an important characteristic of tumorigenesis.
The current determination of the content of N-nitrosamine compounds is limited to certain foods, such as salted fish and bacon. As for the studies on the content of N-nitrosamine compounds in human body, only the content of N-nitrosamine compounds in gastric juice has been studied, it has been found that the concentration of N-nitrosamine compounds in gastric juice of patients with gastritis is generally lower than that of patients with gastric ulcer and gastric cancer, the highest concentration of N-nitrosamine compounds in gastric juice of patients with gastric cancer is 1.90ng/mL, and the lowest concentration of N-nitrosamine compounds in gastric juice of patients with gastritis is 0.063 ng/mL. In addition, it has been shown that dietary intake of N-nitrosamine compounds is associated with increased risk of gastric and esophageal cancer. The N-nitrosamine compound can be continuously accumulated in the body to cause chronic injury after being contacted and ingested by common people for a long time in a low dose, and the determination of the content of the N-nitrosamine compound in human plasma is very necessary. Therefore, it is very important to establish a rapid and reliable detection method for determining the content of the N-nitrosamine compound in human plasma.
The gas chromatography-mass spectrometry has the advantages of strong identification capability, high analysis speed, high sensitivity and the like, and plays an important role in biological sample analysis. However, no relevant report is found about a method for simultaneously detecting 9N-nitrosamine compounds in a human body by gas chromatography-mass spectrometry.
Disclosure of Invention
The invention aims to explore a method for measuring the content of volatile N-nitrosamine compounds in blood plasma by gas chromatography-mass spectrometry, which can simultaneously measure 9 volatile N-nitrosamine compounds in human blood plasma.
The purpose of the invention is realized as follows: a method for measuring the content of volatile N-nitrosamine compounds in blood plasma by gas chromatography-mass spectrometry comprises the following steps:
(1) taking a blood sample, centrifuging and separating plasma; adding dichloromethane and NDPA-D14 internal standard solution into plasma, vortexing in a vortex mixer, and performing ultrasonic extraction after uniform mixing; centrifuging again after finishing; sampling the lower layer liquid;
(2) the method adopts gas chromatography-mass spectrometry to carry out determination, and the chromatographic conditions are as follows: selecting a DB-WAX chromatographic column; the purging flow of the spacer: 2 mL/min; sample introduction mode: no shunt sampling; carrier gas: helium gas; sample introduction amount: 1 mu L of the solution; mass spectrum conditions: ion source temperature: 230 ℃; interface temperature: 250 ℃; solvent retardation: 4 min; an acquisition mode: full scan and select ion detection mode; scanning mass range: m/z is 40-350.
In the step (1), the blood sample is centrifuged for 2.5-3.5min at 3000r/min and 4 ℃, and plasma is separated.
In the step (1), 1 part of plasma is taken according to the volume fraction, and 0.45-0.55 part of dichloromethane and 0.045-0.055 part of NDPA-d14 internal standard solution with the concentration of 500ug/L are added.
In the step (1), vortex in a vortex mixer for 0.8-1.5min, and ultrasonically extracting for 8-12min after uniformly mixing; 13000r/min after finishing, and centrifuging for 8-12min again at the temperature of 4 ℃.
The chromatographic conditions of the step (2) comprise a temperature rise program: maintaining the initial temperature of the column at 50 deg.C for 4 min; heating to 90 deg.C at 20 deg.C/min; then heating to 180 ℃ at the speed of 8 ℃/min; then heating to 200 ℃ at a speed of 15 ℃/min; then raising the temperature to 230 ℃ at the speed of 2 ℃/min, and keeping the temperature for 2 min; sample inlet temperature: at 250 ℃ to obtain a mixture.
And drawing a working curve and carrying out quantitative calculation.
Drawing the working curve: taking a mixed standard solution containing 9N-nitrosamine compounds NDMA, NMEA, NDEA, NDPA, NDBA, NPIP, NPYR, NMOR and NDPhA, diluting with dichloromethane step by step to prepare N-nitrosamine series mixed standard solutions with the concentration of 20, 50, 100,500, 1000,5000 ng/mL; then taking fetal calf serum, and respectively adding dichloromethane, NDPA-D14 internal standard solution and 9N-nitrosamine series mixed standard solutions, wherein the volume ratio of the fetal calf serum, the dichloromethane, the NDPA-D14 internal standard solution and the 9N-nitrosamine series mixed standard solutions is 1: 0.45-0.55: 0.045-0.055; performing vortex, mixing uniformly, performing ultrasonic extraction, centrifuging, sampling lower-layer liquid, and obtaining 9N-nitrosamine compounds with the concentration of 2, 5, 10, 50, 100,500 ng/mL; performing gas chromatography-mass spectrometry according to the conditions in the step (2); and performing linear regression on the concentration of the target substance by using the peak area of the quantitative ions of the target substance to obtain the linear relation of the 9N-nitrosamine compounds.
The quantitative calculation comprises the following steps: and (3) calculating the content of 9N-nitrosamine compounds in the sample according to a working curve quantitative calculation equation:
C＝y-b/a
in the formula: c: the concentration of 9N-nitrosamine compounds is ng/mL; y: peak area ratios of 9N-nitrosamine compounds to the internal standard; a: the slope of the working curve; b: the intercept of the working curve.
The method for measuring the content of the volatile N-nitrosamine compound in the plasma by the gas chromatography-mass spectrometry can simultaneously measure 9 volatile N-nitrosamine compounds in the human plasma, and the detection limit and the lower limit of the quantification of the 9N-nitrosamine compounds are respectively between 0.03 ng/mL and 2.14ng/mL and 0.02 ng/mL to 7.14ng/mL, so that the method conveniently provides a basis for the research of the load level of the N-nitrosamine compound in the human body and the research of the influence of the exposure of the N-nitrosamine compound on the health of the human body.
Drawings
FIG. 1 is a chromatogram of the total ion current of 9N-nitrosamine compounds (ordinate relative abundance R; abscissa time in minutes), in which: NDMA; NMEA; NDEA; NDPA; NDBA; NPIP; NPYR; NMOR; NDPhA;
FIG. 2 is a mass spectrum of 9N-nitrosamine compounds (concentration on the ordinate in mg/L; time on the abscissa in minutes);
FIG. 3 is a graph showing the difference in detection rate of 5 VANs (n: 3) (concentration in mg/Lm on the ordinate; sex on the abscissa: Female group Female group Male).
Detailed Description
The invention relates to a method for measuring the content of volatile N-nitrosamine compounds in blood plasma by gas chromatography-mass spectrometry, which comprises the following steps:
(1) taking a blood sample, centrifuging and separating plasma; collecting plasma, adding dichloromethane and NDPA-D14 (N-nitrosodi-N-propyl-D)
14Amine) internal standard solution, vortexing in a vortex mixer, mixing uniformly and then carrying out ultrasonic extraction; centrifuging again after finishing; taking down the liquid sample. Preferably, the blood sample is centrifuged at 3000r/min at 4 ℃ for 2.5-3.5min to separate the plasma. Preferably, 1 part of plasma is taken and 0.45-0.55 part (most preferably 0.5 part) of dichloromethane and 0.045-0.055 part (most preferably 0.05 part) of NDPA-D14 internal standard solution with the concentration of 500ug/L are added, namely the volume ratio of the plasma, the dichloromethane and the NDPA-D14 internal standard solution is 1: 0.45-0.55: 0.045-0.055, preferably 1: 0.5: 0.05. Preferably, the mixture is vortexed in a vortex mixer for 0.8 to 1.5min, and is ultrasonically extracted for 8 to 12min after being uniformly mixed; after finishing, 13000r/min at 4 ℃ and centrifuging for 8-12 min.
(2) The measurement was carried out by gas chromatography-mass spectrometry.
The chromatographic conditions are as follows: selecting a DB-WAX chromatographic column; the purging flow of the spacer: 2 mL/min; sample introduction mode: no shunt sampling; carrier gas: helium gas; sample introduction amount: 1 μ L. Preferably, the temperature rise program of the chromatographic conditions is: maintaining the initial temperature of the column at 50 deg.C for 4 min; heating to 90 deg.C at 20 deg.C/min; then heating to 180 ℃ at the speed of 8 ℃/min; then heating to 200 ℃ at a speed of 15 ℃/min; then raising the temperature to 230 ℃ at the speed of 2 ℃/min, and keeping the temperature for 2 min; sample inlet temperature: at 250 ℃ to obtain a mixture.
Mass spectrum conditions: ion source temperature: 230 ℃; interface temperature: 250 ℃; solvent retardation: 4 min; an acquisition mode: full scan and select ion detection mode; scanning mass range: m/z is 40-350.
The method inspects the influence of the temperature of the chromatographic column on the experiment, compares different extraction methods, and finally selects an ultrasonic oscillation method and dichloromethane to extract the sample. The standard curve, detection limit and quantitative lower limit of 9N-nitrosamine compounds were examined. Optimizing an instrument, detecting on a machine by adopting a prepared mixed standard solution, automatically drawing a standard curve by the instrument, wherein the linear relation of each ion is good, and the linear range of the 9N-nitrosamine compounds is 2-500 ng/mL; all compounds tested showed a good linear relationship (R) in the linear range
20.9995-0.9999) (table 2). The limit of detection (LOD) was calculated as 3-fold signal-to-noise ratio and the lower limit of quantitation (LOQ) was calculated as 10-fold signal-to-noise ratio. The detection limit and the lower limit of quantification of the 9N-nitrosamine compounds are respectively 0.01-2.14ng/mL and 0.02-7.14 ng/mL.
1.1 instruments
GCMS-QP2010Plus gas chromatography-mass spectrometer (SHIMADZU, Japan) equipped with an AOC-20i autosampler (SHIMADZU, Japan); adjustable pipette (Nichipet EX plus, Japan); KQ-300DB type digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd., China); model CT15RT high speed refrigerated centrifuge (shanghai tianscience instruments ltd); vortex mixer (kylin medical instruments factory, hamen).
1.2 chemical reagents
Standard solution: 9 mixed standard solutions of N-nitrosamine compounds N-Nitrosodimethylamine (NDMA), N-Nitrosomethylamine (NMEA), N-Nitrosodiethylamine (NDEA), N-nitrosodipropylamine (N-nitrosodiethylamine, NDPA), N-nitrosodibutylamine (N-nitrosodiethylamine, NDBA), N-nitrosopiperidinane (N-nitrosodipiperidine, NPIP), N-nitrosopyrrolidine (N-nitrosopropiolidine, NPYR), N-nitrosomorpholine (N-nitrosomorphine, NMOR), N-nitrosodiphenylamine (N-nitrosodiphenylenamine, NDPA) (concentrations of 2000mg/L) (o2 si; America); deuterium-labelled internal standard solution [ N-nitrosodi-N-propyl-d ]
14-amine(NDPA-D14)](concentration 1000mg/L) (o2 si; American); dichloromethane and formamideDimethyl sulfoxide (DMSO), acetonitrile, acetone, ethyl acetate, n-hexane (all chromatographically pure, Mercklin, germany); fetal bovine serum (Gibco; American).
1.3 preparation of the solution
Accurately transferring a mixed standard solution (2000mg/L) containing 9N-nitrosamine compounds into a 10mL volumetric flask, and diluting the mixed standard solution with dichloromethane step by step to prepare the mixed N-nitrosamine standard solution with the series concentration of 20, 50, 100, 200 and 500 ng/mL. Accurately transferring an internal standard solution [ N-nitrosodi-N-propyl-d ]
14-amine(NDPA-D14)](NDPA-D14)](1000mg/L) in a 10mL volumetric flask, an internal standard solution with a concentration of 500ng/mL was prepared by dilution with dichloromethane. And then taking 6 parts of 0.4mL fetal calf serum, respectively adding 200 mu L of dichloromethane, 20 mu L of NDPA-D14 internal standard solution with the concentration of 500ng/mL and 20 mu L of 9N-nitrosamine series mixed standard solutions, vortexing for 1min, ultrasonically extracting for 10min after uniformly mixing, centrifuging for 10min at 13000r/min at the temperature of 4 ℃, taking 150 mu L of lower-layer liquid for sample injection, and obtaining 9N-nitrosamine compounds with the concentration of 2, 5, 10, 50, 100,500ng/mL on a computer.
1.4 pretreatment of the samples
Sample preparation: 5mL of blood sample is collected and immediately centrifuged for 3min at 3000r/min at 4 ℃, and plasma is separated and stored in a refrigerator at-80 ℃ to be tested.
The sample processing method comprises the following steps: the plasma samples stored in the-80 ℃ refrigerator were removed, placed in the 4 ℃ refrigerator for thawing for 15min, and then the sample plasma was allowed to return to room temperature. Sucking 0.4mL of plasma, adding 200 mu L of dichloromethane and 20 mu L of NDPA-D14 internal standard solution with the concentration of 500ug/L, carrying out vortex mixing in a vortex mixer for 1min, and carrying out ultrasonic extraction for 10min after uniform mixing; after finishing, 13000r/min at the temperature of 4 ℃, and centrifuging for 10 min; about 150. mu.L of the supernatant was injected and the supernatant was filtered through an organic phase syringe filter.
2.1 optimization of the chromatography column
The application investigates the influence of chromatographic columns with different polarities (a nonpolar column DB-5MS (30m x 0.25.25 mm x 0.25um) and a strong polar column DB-WAX (30m x 0.25.25 mm x 0.25 um)) on the separation of 9N-nitrosamine compounds to be detected. The results show that: when a non-polar column is selected, the 9N-nitrosamine compounds to be detected cannot all peak; when the strong polarity column DB-WAX is selected, the obtained chromatographic peak has symmetrical peak shape, good separation effect, accurate quantification and good reproducibility. Therefore, the strong polarity column DB-WAX is selected as the chromatographic column in the experiment.
2.2 optimization of the temperature rise Rate of the chromatographic column
Because the peak-off times of NDBA, NPIP, NPYR and NMOR are relatively close, the temperature-rise rate of the first-stage column needs to be optimized to obtain the optimal separation effect. The initial column temperature of the chromatographic column was selected to be 50 deg.C, and the separation effects at temperature rise rates of 15 deg.C, 12 deg.C, and 5 deg.C/min were compared, respectively. The result shows that when the temperature rising rate is 12 ℃/min, the separation degree R is more than or equal to 1.5, and the effective separation can be realized. When the temperature rise rate is 5 ℃/min, the resolution is not obviously changed, so that the temperature rise rate of 12 ℃/min is selected as the temperature rise rate of the first section of the column. Because NDPhA peak-off time is latest and has a long time interval with NMOR peak-off time, the temperature-rising rate of the second section of the column is optimized, so that the peak pattern is better, the separation effects when the temperature-rising rates are 40, 10, 5 and 2 ℃/min are respectively compared, and the result shows that when the temperature-rising rate is 2 ℃/min, effective separation can be realized, and the peak pattern is the best. Therefore, the final optimized temperature-raising program is: maintaining the initial temperature of the column at 50 deg.C for 4 min; heating to 90 deg.C at 20 deg.C/min; then heating to 180 ℃ at the speed of 8 ℃/min; then heating to 200 ℃ at a speed of 15 ℃/min; then raising the temperature to 230 ℃ at the speed of 2 ℃/min, and keeping the temperature for 2 min; sample inlet temperature: at 250 ℃ to obtain a mixture.
And (3) analyzing the mixed standard solution of 9N-nitrosamine compounds and 1 internal standard solution according to the optimized GC-MS conditions to obtain the retention time and characteristic ions of the standard product shown in the table 1, and the total ion flow chromatogram shown in the table 2.
TABLE 1 characteristic ion Table of each substance to be tested
*Selected quantitative ions
2.3 selection of sample Pre-treatment conditions
2.3.1 optimization of sample extraction method
Adding 9 kinds of N-nitrosamine compound series mixed standard solutions into a plasma sample, respectively selecting an ultrasonic oscillation extraction method and a nitrogen-blowing concentration re-dissolution method to pretreat the sample, and testing on a machine after extraction, wherein experimental results show that: the N-nitrosamine compound is lost more in the nitrogen blowing process, and the ultrasonic oscillation extraction method is more complete in the extraction of the target substance and easier to operate. Therefore, the ultrasonic oscillation extraction method is selected as the sample extraction method.
2.3.2 optimization of sample extraction reagents
Adding 9N-nitrosamine compound series mixed standard solutions into a plasma sample, and extracting the plasma sample with a plurality of solvents including formamide, dimethyl sulfoxide (DMSO), acetonitrile, acetone, ethyl acetate, dichloromethane and N-hexane, wherein the experimental result shows that: the dichloromethane has the highest extraction efficiency on 9 target N-nitrosamine compounds and has a better protein precipitation effect, and the recovery rate result is shown in 2.5.1. None of the other solvents extracted all 9 of the target substances. Thus, the present invention selects dichloromethane as the sample extraction reagent.
2.4 Standard Curve and sensitivity
And measuring the prepared 9N-nitrosamine compound series mixed standard solutions. The peak area (y) of the target quantitative ion was subjected to linear regression on its concentration (x, ng/mL) to obtain the linear relationship of 9N-nitrosamine compounds shown in Table 2. The limit of detection (LOD) was calculated as 3-fold signal-to-noise ratio and the lower limit of quantitation (LOQ) was calculated as 10-fold signal-to-noise ratio. As can be seen from Table 2, the method has a wide linear range and a correlation coefficient R
2>0.9995; the sensitivity is high, the LOD range is 0.01-2.14ng/mL, and the LOQ range is 0.02-7.14 ng/mL. The mass spectrum of the 9N-nitrosamine compounds in SIM mode is shown in FIG. 2.
And calculating the content of the 9N-nitrosamine compounds in the sample according to a working curve quantitative calculation equation.
C＝y-b/a
In the formula: c: the concentration of 9N-nitrosamine compounds is ng/mL; y: peak area ratios of 9N-nitrosamine compounds to the internal standard;
a: the slope of the working curve; b: the intercept of the working curve.
TABLE 2.9 correlation coefficients, regression equation, detection limits, lower limits of quantitation for N-nitrosamine compounds
2.5 recovery and precision
2.5.1 recovery rate by adding standard
In order to examine the recovery rate of the method, 9N-nitrosamine series mixed standard solutions with low concentration, medium concentration and high concentration are respectively taken and added into the serum of a fetal calf, the prepared standard adding concentrations are respectively 5, 50 and 100ng/mL, 5 samples are prepared in parallel for each concentration group, the specific sample preparation method is shown as 1.4, a standard adding recovery experiment is carried out, and the recovery rate and the Relative Standard Deviation (RSD) of the measured value of the 9N-nitrosamine compounds with each concentration are respectively calculated. The recovery rate of the 9N-nitrosamine compounds in the standard range is 88-116%, the RSD is less than 10%, and the results are shown in Table 3. Therefore, the method has good accuracy.
TABLE 39 normalized recovery of N-nitrosamine compounds (N ═ 5)
Precision within 2.6 days
The present invention investigates the precision of the method. In order to prove that the method has good repeatability and precision, the experiment is examined for the intra-day precision and the inter-day precision. Precision in the day: adding 9 series of mixed standard solutions of N-nitrosamine compounds into fetal calf serum to make the standard concentration 50ng/mL, preparing 6 samples in parallel, placing in a refrigerator for cold storage at 4 ℃, measuring one sample at intervals of 4 hours, continuously measuring for 24 hours, respectively calculating the Relative Standard Deviation (RSD) of the measured values of the 9N-nitrosamine compounds, wherein the standard recovery range of the 9N-nitrosamine compounds is 91-104%, and the RSD is less than 5% (see table 4).
Precision in day of table 49N-nitrosamine compounds (N ═ 6)
2.7 day precision
Three series of mixed standard solutions of 9N-nitrosamine compounds with different concentrations are respectively taken and added into fetal calf serum to prepare three groups of samples with standard addition concentrations of 5, 50 and 500ng/mL, 9 samples are prepared in parallel for each concentration group and are placed in a refrigerator at 4 ℃ for cold storage, 3 samples are taken out every day for determination, and the determination is carried out for three days continuously. The Relative Standard Deviation (RSD) of the measurements of the 9N-nitrosamine compounds was calculated separately. The recovery rate of the 9N-nitrosamine compounds ranges from 91 to 118% by adding standard, and RSD is less than 10% (see Table 5).
Precision of table 59N-nitrosamine compounds day by day (N ═ 3 days)
2.8 analysis of adult plasma samples in Guangzhou City
The determination method established by the research is adopted to determine and analyze 9N-nitrosamine compounds in adult blood plasma samples in Guangzhou city.
The preparation of the solution and the pretreatment of the sample were the same as in 1.3 and 1.4 described above.
The conditions for the measurement by gas chromatography-mass spectrometry were as follows:
the chromatographic conditions are as follows: selecting a DB-WAX chromatographic column; the purging flow of the spacer: 2 mL/min; sample introduction mode: no shunt sampling; carrier gas: helium gas; sample introduction amount: 1 μ L. The temperature rise program of the chromatographic conditions is as follows: maintaining the initial temperature of the column at 50 deg.C for 4 min; heating to 90 deg.C at 20 deg.C/min; then heating to 180 ℃ at the speed of 8 ℃/min; then heating to 200 ℃ at a speed of 15 ℃/min; then raising the temperature to 230 ℃ at the speed of 2 ℃/min, and keeping the temperature for 2 min; sample inlet temperature: at 250 ℃ to obtain a mixture.
Mass spectrum conditions: ion source temperature: 230 ℃; interface temperature: 250 ℃; solvent retardation: 4 min; an acquisition mode: full scan and select ion detection mode; scanning mass range: m/z is 40-350.
(1) Drawing a working curve: taking a mixed standard solution containing 9N-nitrosamine compounds NDMA, NMEA, NDEA, NDPA, NDBA, NPIP, NPYR, NMOR and NDPhA, diluting with dichloromethane step by step to prepare a mixed N-nitrosamine standard solution with 20, 50, 100, 200, 500ng/mL series concentration, taking 6 parts of 0.4mL fetal calf serum, respectively adding 200 mu L dichloromethane and 20 mu L NDPA-D14 internal standard solution with 500ng/mL concentration and 20 mu L of 9N-nitrosamine series mixed standard solutions, vortex for 1min, ultrasonically extracting for 10min after uniformly mixing, centrifuging for 10min with 13000r at 4 ℃, taking 150 mu L of lower layer liquid for sample injection, and obtaining 9N-nitrosamine compounds with 2, 5, 10, 50, 100,500ng/mL on-machine mixed standard solution; performing gas chromatography-mass spectrometry according to the conditions; and performing linear regression on the concentration of the target substance by using the peak area of the quantitative ions of the target substance to obtain the linear relation of the 9N-nitrosamine compounds.
(2) Plasma samples were determined by gas chromatography-mass spectrometry under the conditions described above for a total of 31 cases, 14 in males and 17 in females.
(3) And (3) calculating the content of 9N-nitrosamine compounds in the sample according to a working curve quantitative calculation equation:
C＝y-b/a
in the formula: c: the concentration of 9N-nitrosamine compounds is ng/mL; y: peak area ratios of 9N-nitrosamine compounds to the internal standard; a: the slope of the working curve; b: the intercept of the working curve.
The results are shown in Table 6: among the 9N-nitrosamine compounds, NDMA, NDBA, NPIP, NMOR and NDPhA were detected, while NMEA, NDEA, NDPA and NPYR were not detected. The content range is n.d. -31.61ng/mL, wherein the NDBA content is 31.61ng/mL at the highest, and the detection rate of NPIP in the population is 96.8% at the highest. Human acceptable VANs exposure levels are reported in the literature to range from 5 to 10 ug/kg.
Table 6 results for 9N-nitrosamine compounds in guangzhou adult plasma (N ═ 31)
n.d. not detected
The groups were classified according to sex and N-nitrosamine compound content, and it was found that the detection rates of 5 VANs detected in the samples were different among the sex groups (FIG. 3). The detection rates of the NDMA in male and female groups are respectively 92.9% and 88.2%; the detection rates of NDBA and NPIP are not very different between the two groups. NDBA was detected in only one of 14 male subjects, the detection rate was 7.1%, and the detection rate of the female group was 100%, and the detection rates of NDBA were significantly different among the two groups; the detection rate of NPIP in the male and female groups is 92.9 percent and 100 percent respectively; four out of 14 male subjects were detected, only one out of 17 female subjects detected NMOR, and the detection rates in the male and female groups were 28.6% and 5.9%, respectively; NDPhA was detected in five of 17 female subjects, with a detection rate of 29.4%, and was not detected in male subjects.
Claims (8)
1. A method for measuring the content of volatile N-nitrosamine compounds in blood plasma by gas chromatography-mass spectrometry comprises the following steps:
(1) taking a blood sample, centrifuging and separating plasma; adding dichloromethane and NDPA-D14 internal standard solution into plasma, vortexing in a vortex mixer, and performing ultrasonic extraction after uniform mixing; centrifuging again after finishing; sampling the lower layer liquid;
(2) the method adopts gas chromatography-mass spectrometry to carry out determination, and the chromatographic conditions are as follows: selecting a DB-WAX chromatographic column; the purging flow of the spacer: 2 mL/min; sample introduction mode: no shunt sampling; carrier gas: helium gas; sample introduction amount: 1 mu L of the solution; mass spectrum conditions: ion source temperature: 230 ℃; interface temperature: 250 ℃; solvent retardation: 4 min; an acquisition mode: full scan and select ion detection mode; scanning mass range: m/z 40-350.
2. The method of claim 1, wherein: in the step (1), the blood sample is centrifuged for 2.5-3.5min at 3000r/min and 4 ℃, and plasma is separated.
3. The method of claim 1, wherein: in the step (1), 1 part of plasma is taken according to the volume fraction, and 0.45-0.55 part of dichloromethane and 0.045-0.055 part of NDPA-D14 internal standard solution with the concentration of 500ug/L are respectively added.
4. The method of claim 1, wherein: in the step (1), vortex in a vortex mixer for 0.8-1.5min, and ultrasonically extracting for 8-12min after uniformly mixing; 13000r/min after finishing, and centrifuging for 8-12min again at the temperature of 4 ℃.
5. The method of claim 1, wherein: the chromatographic conditions of the step (2) comprise a temperature rise program: maintaining the initial temperature of the column at 50 deg.C for 4 min; heating to 90 deg.C at 20 deg.C/min; then heating to 180 ℃ at the speed of 8 ℃/min; then heating to 200 ℃ at a speed of 15 ℃/min; then raising the temperature to 230 ℃ at the speed of 2 ℃/min, and keeping the temperature for 2 min; sample inlet temperature: at 250 ℃ to obtain a mixture.
6. The method of claim 1, wherein: and drawing a working curve and carrying out quantitative calculation.
7. The method of claim 6, wherein: drawing the working curve: taking a mixed standard solution containing 9N-nitrosamine compounds NDMA, NMEA, NDEA, NDPA, NDBA, NPIP, NPYR, NMOR and NDPhA, diluting with dichloromethane step by step to prepare N-nitrosamine series mixed standard solutions with the concentration of 20, 50, 100,500, 1000,5000 ng/mL; then taking fetal calf serum, and respectively adding dichloromethane, NDPA-D14 internal standard solution and 9N-nitrosamine series mixed standard solutions, wherein the volume ratio of the fetal calf serum, the dichloromethane, the NDPA-D14 internal standard solution and the 9N-nitrosamine series mixed standard solutions is 1: 0.45-0.55: 0.045-0.055; performing vortex, mixing uniformly, performing ultrasonic extraction, centrifuging, sampling lower-layer liquid, and obtaining 9N-nitrosamine compounds with the concentration of 2, 5, 10, 50, 100,500 ng/mL; performing gas chromatography-mass spectrometry according to the conditions in the step (2); and performing linear regression on the concentration of the target substance by using the peak area of the quantitative ions of the target substance to obtain the linear relation of the 9N-nitrosamine compounds.
8. The method of claim 6, wherein: the quantitative calculation comprises the following steps: and (3) calculating the content of 9N-nitrosamine compounds in the sample according to a working curve quantitative calculation equation:
C＝y-b/a
in the formula: c: the concentration of 9N-nitrosamine compounds is ng/mL; y: peak area ratios of 9N-nitrosamine compounds to the internal standard; a: the slope of the working curve; b: the intercept of the working curve.
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