CN105891357B - The analysis method of nicotine and its metabolin in a kind of dynamic tracer monitoring animal body - Google Patents
The analysis method of nicotine and its metabolin in a kind of dynamic tracer monitoring animal body Download PDFInfo
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Abstract
The analysis method of nicotine and its metabolin in a kind of dynamic tracer monitoring animal body, it is characterised in that：Use pyridine ring2The nicotine of H labels（[2H4]‑(±)‑Nicotine）Exposure animal after being mixed with unlabelled nicotine equal proportion, blood, brain dialysate samples are collected respectively using Microdialysis sgstem in microcentrifugal tube, after analysis internal standard short-term low speed centrifugation is added, nicotine metabolism object and its pyridine ring in direct loading ultra performance liquid chromatography orbit trap high resolution mass spectrum analysis detection microdialysis sample2H markers identify nicotine metabolism object according to test result and draw corresponding time-concentration curve carrying out Dynamic Changes Analysis.The advantage of the invention is that：Accurately tracer it can identify a variety of nicotine metabolism objects in animal body, greatly simplifie sample pre-treatments step, the accurate analysis of nicotine metabolism object variation in different system in animal body is realized compared with synchronous, has started a kind of new method studied for nicotine metabolism in animal body.
Description
Technical field
The present invention relates to the methods that nicotine metabolism in animal body is analyzed.This method uses stable isotopic tracer nicotine metabolism
Process utilizes ultra performance liquid chromatography-orbit trap high score by Microdialysis Technique synchronous collection rat cerebral tissue and blood sample
Resolution mass spectrum carries out Tracing detection analysis to nicotine metabolism object in sample, to comprehensively disclose the metabolism of nicotine in animal body
Feature.
Background technology
Nicotine（Nicotine, Nic）It is the important feature ingredient of tobacco, the generation of Nicotine Dependence is with individual to nicotine
Metabolic capability is closely related.Although the internal nicotine of intake can enter brain tissue through blood-brain barrier rapidly, body is to cigarette
The metabolism of alkali is completed in liver.The conversion metabolic process of nicotine includes mainly oxidation, N- demethylations, hydroxylating
And the adduction process with Portugal's glycosides nucleic acid, several stages can be divided into：The nicotine of first stage, 70-80% pass throughCOxidation is converted into
Cotinine（Cotinine, Cot）；Second stage, a least a portion of nicotine and cotinine pass throughNOxidation, demethylation and Portugal
Glycosides nucleination is separately converted to nicotine nitrogen oxides（nicotine-N`-oxide, NNO）, nornicotine（nornicotine,
NorNic）, nicotine glucosides（nicotine-N- glucuronide, Nic-Gluc）With cotinine nitrogen oxides
（cotinine-N-oxide, CNO）, cotinine drops（norcotinine, NorCot）, cotinine glucosides（cotinine-N-glucuronide, Cot-Gluc）.In addition, cotinine can be also converted by hydroxylating generates trans- 3 ' hydroxyl cotinine
（trans-3’-hydroxycotinine, OH-Cot）, and a small amount of trans- 3 ' are further ultimately formed by glycosylation effect
Hydroxyl cotinine glucosides（trans- 3’-hydroxycotinine-O- glucuronide, OH-Cot-Gluc）.In addition, grinding
The person of studying carefully also confirms that the nicotine of fraction can also convert in smoker's body and is formed as pyridyl group carboxylic acid（4-oxo- 4-(3-
Pyridyl)-butanoic acid, OxPyBut）With pyridyl group batanone acid（4-hydroxy-4- (3-pyridyl)-
Butanoic acid, HyPyBut）.In fact, it is reported that nicotine can be converted into more than 20 in vivo plants metabolite, but it is big absolutely
Partial research by the analysis of the measurement of nicotine metabolism product be limited to 10 kinds or so or within because it is relatively conventional to measure these simultaneously
Metabolite can cover calculate intake internal 90% or more nicotine.Nicotine and its metabolite especially cotinine conduct
It weighs human body fume exposure degree and distinguishes the biomarker of smoker and non-smoker, be subjected to the extensive pass of researcher
Note.
The analysis method used at present about nicotine metabolism research has enzyme-linked immunization, gas chromatography-mass spectrum（GC-MS）
Method, high performance liquid chromatography（HPLC）Method, liquid chromatography-mass spectrography（LC-MS）Method, high pressure radio chromatography etc., about adopting for sample
The live body sampling etc. that mode set has the vitro samples such as blood, urine, saliva, brain tissue and combines Microdialysis Technique.However, these
All there is certain deficiency or limitations for research.It is that the metabolite especially in brain tissue is not in vivo for nicotine first
Clear and complete information can be provided so that researcher so far intracerebral not fully aware of nicotine metabolism product whether as many and
It is complicated.[the bibliography 1 such as Paula：Paula L. Vieira-Brock, Eleanor I. Miller, Shannon M.
Nielsen, et al. Simultaneous quantification of nicotine and metabolites in
rat brain by liquid chromatography-tandem mass spectrometry[J]. Journal of
Chromatography B, 2011, 879:3465-3474.] it establishes while measuring using Solid Phase Extraction/Liquid Chromatography/Mass Spectrometry
The method of nicotine metabolism object in rat cerebral tissue, but only give the content letter of nicotine in rat cerebral tissue, Cot and NorNic
Breath；Previous research and utilization labelled with radioisotope [14C-N- methyl] nicotine combination high pressure radio chromatography is in mouse
Intracerebral identify [14C] cotinine and [14C] nicotine nitrogen oxides, but nicotineNDemethylation metabolite（Such as
NorNic and NorCot）It is not detected because of the loss of radioisotope in metabolic process then；[the bibliography 2 such as Omar：
Omar A. Ghosheh, Linda. P. Dwoskin, Dennis. K. Miller, Peter. A. Crooks.
Metabolites of nicotine in rat brain after peripheral nicotine administration
[J]. Drug Metabolism and Disposition, 1997, 25(1):47-54.] have studied hypodermic injection [2 '
-14C] metabolite after nicotine in mouse brain, the content of Cot, NorNic and NorCot in rat brain are found at ng/mL grades, and
And intracerebral still has other metabolins for failing identification；Strong et al. [the bibliography 3 of hair：The such as Mao Jian, Lu Jianqing, Xu Yan are micro-
Analysis-UPLC-MS/MS methods measure in live body rat brain nicotine and its metabolin [J] tobacco science and technology simultaneously, and 2014, (5): 37-
41.] recent studies have shown that there are five kinds of metabolins of Cot, NorNic, NorCot, NNO and OH-Cot in rat brain.Secondly, greatly
The nicotine metabolism product in static measurement triangular web is only paid attention in most researchs, rarely has into dynamic in promoting circulation of blood, brain multisystem
State measures and synchronous comparative analysis.Although Chang et al. [bibliography 3：Yuh-Lih Chang, Pi-Lo Tsai, Yueh-
Ching Chou, et al. Simultaneous determination of nicotine and its metabolite,
cotinine, in rat blood and brain tissue using microdialysis coupled with
liquid chromatography: pharmacokinetic application[J]. Journal of
Chromatography A, 2005, 1088:152-157.] and open patent of invention（Trace cigarette in animal blood, brain sample
The synchronized analyzing method number of patent application 201410257438. 8. of alkali and its major metabolite）It reports and utilizes microdialysis skill
Art combination liquid chromatogram or mass spectrographic method are only limitted to rat serum, intracerebral nicotine into Mobile state measurement and Synchronization Analysis
Provide the variation characteristic information of nicotine and cotinine.
Invention content
The purpose of the present invention is the deficiency for current nicotine metabolism research method, and a kind of dynamic tracer specially developed
Monitor the analysis method of nicotine and its metabolin in animal body.This method uses pyridine ring2The nicotine of H labels（[2H4]-(±)-
Nicotine）Tracer nicotine metabolism conversion process, in conjunction with high resolution mass spec means not only high sensitivity, accurate generation can be obtained
Thank to object molecular mass information, also using pyridine ring2The isotopic peak of H labels occurs in pairs with unlabelled nicotine metabolism object peak
Feature further confirm metabolite information of the nicotine in vivo especially in brain tissue, overcome labelled with radioisotope
The shortcomings of sensitivity is not high, radiated signal is easy to be lost when method detects；In addition, this method also utilizes microdialysis sampling technique, it is real
Showed the direct injection analysis of sample with to nicotine in live body rat serum, synchronous, the dynamic analysis of two system intracellular metabolite of brain.
The purpose of the present invention is achieved through the following technical solutions：In a kind of dynamic tracer monitoring animal body nicotine and
The analysis method of its metabolin exposes animal using nicotine and its pyridine ring isotopic label solution, utilizes microdialysis simultaneously
System synchronization collects animal blood dialyzate and brain tissue dialysate samples in different micro centrifugal pipes, and it is molten that analysis internal standard is added
After the centrifugation of liquid mixing, using the nicotine generation in ultra performance liquid chromatography-orbit trap high resolution mass spectrum analysis detection microdialysis sample
Thank object and its pyridine ring2H markers, according to test result identify nicotine metabolism object and draw corresponding time-concentration curve into
Mobile state monitoring analysis.
This method is as follows：
A. nicotine exposes：The nicotine and pyridine ring mixed using equal proportion2The nicotine solution of H labels is with peripheral injection side
Formula exposes animal.
B. microdialysis sample collection：It synchronizes and the Blood Microdialysis sample after collection animal intake nicotine and brain is micro- respectively
Sample dialyse in different micro centrifugal pipes（The miniature tube can be matched with microdialysis sample collecting system but also and color
Spectrum sample injection bottle matches）So that often sample is 30 μ L in pipe.
C. analysis inner mark solution is prepared：With stable isotope Nic-d3As analysis internal standard compound, accurately weighs 10mg and be placed in
In 100mL volumetric flasks, methanol/water is used（Volume ratio 50/50, similarly hereinafter）Solution is diluted to scale, shakes up, and obtains a concentration of 100 μ g/mL
Internal standard storage solutions；Use again step by step dilution method compound concentration for the internal standard working solution of 20ng/mL.
D. mixed standard solution preparation：Nic, Cot, NorNic, NNO, OH-Cot, NorCot, CNO, Nic- are weighed respectively
Each standard items of Gluc, Cot-Gluc, OH-Cot-Gluc, OxPyBut and HyPyBut prepare the series mixing with concentration gradient
Standard solution, and internal standard working solution is added, make a concentration of 5ng/mL of internal standard compound.Concrete mode is：Accurately weigh standard items
Each 10mg, is respectively placed in 100mL volumetric flasks, and scale is dissolved and be diluted to methanol/water solution, is shaken up, and it is 100 to obtain concentration respectively
The Standard Stock solutions of μ g/mL；The Standard Stock solutions for pipetting 10.0mL respectively are placed in same branch 100mL volumetric flasks, use first
Alcohol/water is diluted to scale, shakes up, and obtains level-one mixed standard solution；The level-one mixed standard solution for pipetting 1.0mL, is placed in another
In 100mL volumetric flasks, it is diluted to scale with methanol/water, is shaken up, obtains a concentration of 100ng/mL two levels mixed standard solution；Accurately
0.05,0.10,0.50,1.00,5.00,10.0 and 50.0mL two level mixed standard solutions are pipetted, different 100mL is respectively placed in
It is each that 25.0mL internal standard working solutions are added in volumetric flask, it is diluted to scale with the ringer's solution of filtration, shakes up, it is equal to obtain concentration
For the serial mixed standard solution of 0.05,0.10,0.50,1.00,5.00,10.0 and 50.0ng/mL.
E. Specification Curve of Increasing：Serial mixed standard solution is subjected to ultra high efficiency liquid phase by concentration sequence from low to high
Chromatography-orbit trap high resolution mass spec analysis, with the ratio between the chromatographic peak area of each target analytes and internal standard peak area（Y）To it
Corresponding concentration（X）Linear regression is carried out, standard curve regression equation is obtained.
F. sample detection and data processing：The internal standard working solution of 10 a concentration of 20ng/mL of μ L is added separately to blood, brain
In dialysate samples, loading is analyzed after 1000r/min low-speed centrifugals 10s after mixing.Utilize the accurate molecular masses peak [M] and isotope
Object in the characterization sample that [M+4] peak match occurs calculates each object in dialysate samples according to calibration curve method
Content, according to result draw respectively nicotine injection after each time point blood, intracerebral metabolin Concentration-time change curve, and
Pharmacokinetic analysis is carried out, metabolic rule of the nicotine in rat body is disclosed.
One aspect of the present invention using the deuterated label of pyridine ring nicotine expose animal, can tracer monitoring nicotine in animal body
On the other hand interior metabolic alterations process also utilizes orbit trap high resolution mass spec, can not only accurately measure point of object
Son amount also has the characteristics that high sensitivity, quantitative analysis results are accurate to realize the other identification of nicotine metabolism species.Profit
It is not only identified with technical scheme of the present invention in animal brain there are 8 kinds of nicotine metabolism objects, achieves apparent progress, also achieve
The synchronization, dynamic monitoring of nicotine metabolism process in periphery and central nervous system have sensitive and accurate, knot compared with prior art
The features such as fruit is reliable has started a kind of new method for nicotine metabolism monitoring in animal body.
Description of the drawings
Fig. 1 be embodiment in rat serum, intracerebral Nic concentration changes with time tendency chart.
Fig. 2 be embodiment in rat serum, intracerebral Cot concentration changes with time tendency chart.
Fig. 3 be embodiment in rat serum, intracerebral NorNic concentration changes with time tendency chart.
Fig. 4 be embodiment in rat serum, intracerebral NNO concentration changes with time tendency chart.
Fig. 5 be embodiment in rat serum, intracerebral NorCot concentration changes with time tendency chart.
Fig. 6 be embodiment in rat serum, intracerebral OH-Cot concentration changes with time tendency chart.
Fig. 7 be embodiment in rat serum, intracerebral CNO concentration changes with time tendency chart.
Fig. 8 be embodiment in rat blood in Nic-Gluc concentration changes with time tendency chart.
Fig. 9 be embodiment in rat blood in Cot-Gluc concentration changes with time tendency chart.
Figure 10 be embodiment in rat blood in OH-Cot-Gluc concentration changes with time tendency chart.
Figure 11 be embodiment in rat blood in OxPyBut concentration changes with time tendency chart.
Figure 12 be embodiment in rat blood in HyPyBut concentration changes with time tendency chart.
Specific implementation mode
The present invention is described further with the following Examples, but is not intended to limit the present invention.
Embodiment 1
Male adult SD normal rat, 10 week old, weight（200 ± 20）G is raised using independent isolating cage for rearing poultry,
Freely intake, standard particle feed.Rat is with 1% amobarbital, by 50 mg/kg dosage intraperitoneal injection of anesthesia.It is fixed by solid
Position instrument positioning, in being embedded to probe casing in rat brain striatum.It is saturating in a subtle way in rat free movement state underthrust of regaining consciousness after 24 h
Analyse probe (CMA/12).For Blood Microdialysis sample collection, microdialysis probe (CMA/20) need to be implanted into Pericardium of Rats
Jugular vein.Using the ringer's solution of filtration（Compound sodium chloride injection）With the speed perfusion of 1.5 μ L/min, 120 min are balanced
After start collect sample in micro centrifugal pipe（It can match with microdialysis sample collecting system but also match with chromatography column feed materials bottle
Set uses）In, each 1 pipe brain of synchronous collection of every 20 min, Hemodialysis liquid at 4 DEG C.At the end of the 4th pipe is collected, cigarette is injected intraperitoneally
Alkali and [2H4]-nicotine mixed solution（Prepared using ringer's solution, nicotine and [2H4]-nicotinic density is 0.5mg/mL, injection
Dosage is 1 mg/kg）, continue to collect dialyzate by every 20 min at 4 DEG C.The often pipe dialysate samples gathered are added 10
The internal standard working solution of a concentration of 20ng/mL of μ L, directly loading UPLC-Q Exactive inspections after 1000r/min low-speed centrifugals 10s
It surveys.
Chromatographic condition
Chromatographic column：Waters Atlantis HILIC Silica （3 ×100 mm, i. d. , 3 μm）.
Column temperature：30 ℃；Mobile phase：A：10 mmol/L ammonium acetate solutions（Containing 0.1% formic acid,）, B：Acetonitrile（Containing 0.1%
Formic acid）；Flow velocity：0.6 mL/min；Condition of gradient elution is shown in Table 1；Sample size：10 µL；The column equilibration time is 2 min.
1 condition of gradient elution of table
| Time（min） | Mobile phase A（%） | Mobile phase B（%） |
| 0 | 5 | 95 |
| 2 | 15 | 85 |
| 5 | 25 | 75 |
| 5.5 | 5 | 95 |
| 8 | 5 | 95 |
Mass Spectrometry Conditions
Ion source：Hot type electron spray（HESI）；Ion source temperature：300 ℃；Electron spray voltage under positive ion mode：
3.5 kV；Sheath gas：206.85 kPa；Secondary air speed：3.33 L/min；Transfer capillary temperature：320 ℃；Scan pattern：
FS/ddMS2（Full scan/data dependence two grades scanning）, level-one full scan（Resolution ratioR=70000, scanning quality range 50 ~
400 m/z）, two level scanning resolutionR=17500。
The foundation of standard curve
Serial mixed standard solution is subjected to UPLC-MS/MS analyses by concentration sequence from low to high, with each object
The ratio between chromatographic peak area and analysis internal standard peak area（Y）To its corresponding concentration（X）Linear regression is carried out, standard curve is obtained and returns
Return equation.
Measurement result
Using high resolution mass spec to nicotine metabolism object in rat dialysate samples and its corresponding pyridine ring2H marks generation
It thanks to object and carries out qualitative detection, it can be in sample by the visible accurate molecular weight provided according to orbit trap high resolution mass spec of table 2
Object is accurately measured.According to object and its pyridine ring2The pairing appearance result of H markers can be seen that rat
It can detect that 11 kinds of whole target metabolites in peripheral blood system, and 8 can be identified in brain tissue in addition to glucosides metabolite
Kind metabolite, the results are shown in Table 3.
2 object of table and its pyridine ring2H marks the protonated ion quality of metabolin
Nicotine metabolism object and pyridine ring in 3 rat dialyzate of table2H marks the qualification result of metabolin
Note："+" expression both can detect, and "-" expression does not occur signal response both.
The object peak area and analysis internal standard peak area ratio that blood, brain dialysis sample are measured, with corresponding working curve
Regression equation is fitted calculating and measures content, then calculates content of each object in rat body by the rate of recovery of dialysing,
And draw concentration and change with time trend curve, carry out pharmacokinetic analysis.
Claims (1)
1. the analysis method of nicotine and its metabolin in a kind of dynamic tracer monitoring animal body, it is characterised in that：Using nicotine with
Its pyridine ring2H isotopic labels solution exposes animal simultaneously, utilizes Microdialysis sgstem synchronous collection animal blood dialyzate and brain
Organize dialysate samples in different micro centrifugal pipes, it is high using ultra performance liquid chromatography-orbit trap after analysis internal standard is added
Nicotine metabolism object and its pyridine ring in Resolution Mass Spectrometry analysis detection sample2H markers identify that nicotine exists according to test result
Metabolome in brain, blood at and carry out quantitative analysis, draw corresponding nicotine metabolism object Concentration-time change curve into action
State monitoring analysis；Specifically include following steps：
A. the nicotine and pyridine ring of equal proportion mixing are used2The nicotine solution of H labels exposes animal with periphery injection system；
B. synchronous and collect the Blood Microdialysis sample after animal intake nicotine and encephalic micro-dialysis sample respectively in different miniature
In centrifuge tube, which can match with microdialysis sample collecting system but also be matched with chromatography column feed materials bottle；
C. analysis inner mark solution is prepared：With stable isotope Nic-d3As analysis internal standard compound, compound concentration is the interior of 20ng/mL
Mark working solution；
D. mixed standard solution preparation：Nic, Cot, NorNic, NNO, OH-Cot, NorCot, CNO, Nic- are accurately weighed respectively
The serial hybrid standard of Gluc, Cot-Gluc, OH-Cot-Gluc, OxPyBut and HyPyBut standard items, compound concentration gradient is molten
Liquid, and internal standard working solution is respectively added, makes a concentration of 5ng/mL of internal standard compound, mixed standard solution a concentration of 0.05,0.10,
0.50,1.00,5.00,10.0 and 50.0ng/mL；
E. Specification Curve of Increasing：Serial mixed standard solution is analyzed by concentration sequence from low to high, with each target point
Analyse the ratio between chromatographic peak area and the internal standard peak area of object（Y）To its corresponding concentration（X）Linear regression is carried out, standard curve is obtained
Regression equation；
F. sample detection and data processing：The step b micro centrifugal pipes collected are placed in chromatography column feed materials bottle, 10 μ L concentration are added
For the mixing internal standard working solution of 20ng/mL, loading is analyzed after short-term low speed centrifugation, and specially 1000r/min centrifuges 10s；Profit
Object in the characterization sample occurred with the accurate molecular masses peak [M] and isotope [M+4] peak match, according to calibration curve method
The content for calculating each object in dialysate samples draws the Concentration-time variation of each metabolin of blood, intracerebral according to result respectively
Curve, and pharmacokinetic analysis is carried out, disclose metabolic rule of the nicotine in rat body；
Chromatographic condition, Mass Spectrometry Conditions are specific as follows：
Chromatographic column：Waters Atlantis HILIC Silica, specification 3 × 100 mm, i. d., 3 μm；
Column temperature：30 ℃；Mobile phase：A：10 mmol/L ammonium acetate solutions contain 0.1% formic acid, B in the aqueous solution：Containing 0.1%
The acetonitrile of formic acid；Flow velocity：0.6 mL/min；Gradient elution；Sample size：10 µL；The column equilibration time is 2 min；
Condition of gradient elution is shown in Table 1；
1 condition of gradient elution of table
Mass Spectrometry Conditions
Ion source：Hot type electron spray（HESI）；Ion source temperature：300 ℃；Electron spray voltage under positive ion mode：3.5
kV；Sheath gas：206.85 kPa；Secondary air speed：3.33 L/min；Transfer capillary temperature：320 ℃；Scan pattern：It sweeps entirely
Retouch/data dependence two level scanning FS/ddMS2, level-one full scan resolution ratioR=70000, two level scanning resolutionR=17500。
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