CN111983110B - Chiral analysis method for nicotine and main metabolites thereof in blood plasma - Google Patents
Chiral analysis method for nicotine and main metabolites thereof in blood plasma Download PDFInfo
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- CN111983110B CN111983110B CN202010914265.8A CN202010914265A CN111983110B CN 111983110 B CN111983110 B CN 111983110B CN 202010914265 A CN202010914265 A CN 202010914265A CN 111983110 B CN111983110 B CN 111983110B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to a chiral analysis method of nicotine and main metabolites thereof in blood plasma, which comprises the following steps: (1) adding plasma into the internal standard extraction solution to precipitate protein, oscillating and centrifuging to obtain supernatant; (2) taking the supernatant obtained in the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis; (3) and (4) quantitatively calculating the content of the target object in the sample by adopting an internal standard curve method. The method realizes simultaneous chiral analysis of nicotine and main metabolites thereof, has the advantages of high flux, good resolution, simple operation, high sensitivity, good recovery rate and repeatability and the like, and is suitable for chiral analysis of nicotine and main metabolites thereof in a plasma sample.
Description
Technical Field
The invention belongs to the technical field of chemical analysis, and particularly relates to a chiral analysis method of nicotine and main metabolites thereof in blood plasma.
Background
Nicotine is a determining factor and a main pharmacological active substance of potential addiction of tobacco and tobacco products, and can be rapidly metabolized into various products in animals and human bodies, and the main metabolic products are cotinine, norcotinine, cotinine nitric oxide and the like. Nicotine, cotinine, norcotinine and cotinine nitroxide all have two enantiomers: s-nicotine, R-nicotine, S-cotinine, R-cotinine, S-norcotinine, R-norcotinine, and S-cotinine oxynitride, R-cotinine oxynitride. Enantiomers, although of the same molecular formula, differ in structure and therefore generally differ greatly in their metabolic mechanisms and biological activities. Chiral analysis of enantiomers in biological samples is a key for researching physiological effects such as pharmacology, toxicity and the like of target compounds, so that the establishment of a high-flux, high-sensitivity and high-selectivity chiral separation method of nicotine and metabolites thereof in plasma samples has important significance for metabolism and accumulation of nicotine and smoking and health research.
At present, chiral separation methods of nicotine and metabolites thereof mainly comprise a Liquid Chromatography (LC) method, a liquid chromatography-mass spectrometry combined method (LC-MS/MS), a Supercritical Fluid Chromatography (SFC) method, a gas chromatography-mass spectrometry combined method (GC-MS) method, a Capillary Electrophoresis (CE) method and the like, and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) has the advantages of rapidness, sensitivity, good reproducibility, high flux and the like, and has wide application prospects in chiral separation of alkaloids. However, the related studies are mainly tobacco and tobacco products, rarely involve biological samples, and do not simultaneously achieve chiral separation of nicotine and its main metabolites cotinine, norcotinine, cotinine nitrogen oxides, etc. in plasma samples (Drug test. anal.2017,9(6),944-948, Talanta 2018,181,132-141. heiyo.2019, 5(5), e01719.)
Disclosure of Invention
The invention aims to establish a chiral analysis method of nicotine and main metabolites thereof in plasma based on the technical defects, and the method uses HPLC-MS/MS technology to realize simultaneous chiral separation of nicotine, cotinine, norcotinine and cotinine nitrogen oxides in a plasma sample for the first time, has the advantages of high flux, good separation degree, simplicity and convenience in operation, high sensitivity, good recovery rate and repeatability and the like, and is suitable for chiral analysis of nicotine and main metabolites thereof in the plasma sample.
The purpose of the invention is realized by the following technical scheme:
a chiral analysis method of nicotine and its major metabolites in blood plasma, comprising the steps of:
(1) taking plasma, adding an internal standard extraction solution to precipitate protein, oscillating, and centrifuging to obtain a supernatant;
(2) taking the supernatant obtained in the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis;
(3) and (3) quantitatively calculating the content of the target in the sample by adopting an internal standard curve method.
Preferably, in step (1), the internal standard extraction solution is a methanol solution of each internal standard.
Preferably, in the step (1), the concentration of the internal standard extraction solution is 20-50 ng/mL.
Preferably, in the step (1), the temperature of the centrifugation is 2-5 ℃.
Preferably, in the step (1), the speed of the centrifugation is 10000-15000 rpm.
Preferably, in the step (2), the chromatographic conditions of the high performance liquid chromatography-tandem mass spectrometry comprise: the chromatographic column is a Chiralpak IG-3 chromatographic column (250X 4.6mm X3 μm) combined with a guard column (10mm X4.0 mm X3 μm).
Preferably, in the step (2), the chromatographic conditions of the hplc-tandem mass spectrometry further include: the column temperature was 25 ℃; the mobile phase is ammonium formate/methanol solution with the mass fraction of 0.2 percent; the flow rate is 0.5 mL/min; the total operation time is 40 min; the injection volume was 10. mu.L.
Preferably, in step (2), the mass spectrometry conditions are: an ion source: an electrospray ion source; an ionization mode: ESI (+); detection mode: an MRM mode; ion source Temperature (TEM): 500 ℃; electrospray voltage (IS): 5500V; collision gas (CAD): 4.8X 104Pa; air curtain gas (CUR): 2.4X 105Pa; ion source gas flow (GS 1): 3.4X 105Pa; ion source gas flow(GS2)：4.1×105Pa; inlet voltage (EP): 10V; emission voltage (CXP): 10V.
Preferably, in step (2), the MRM mass spectrometric detection parameters of the target compound and the internal standard are as follows:
note: "+" indicates the quantitative ion.
Preferably, in step (3), the internal standard curve method is: preparing a series of standard working solutions containing a target object, adding isotope internal standards (nicotine-d 3, cotinine-d 3, cotinine nitrogen oxide-d 3 and norcotinine-d 4), and making a standard working curve by taking the peak area ratio of the quantitative ions of the target object and the internal standards in each standard working solution as a vertical coordinate and the content of the target object in each standard working solution as a horizontal coordinate; and (3) substituting the analysis result of the step (2) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
Preferably, the concentration range of each target in the series of standard working solutions is 0.5-250 ng/mL, and the lower limit of quantification is 0.5 ng/mL.
Preferably, the plasma is human or animal (e.g., rat, mouse, etc.) plasma.
Preferably, the primary nicotine metabolites are cotinine, cotinine nitric oxide and norcotinine.
Preferably, in one embodiment, the present invention provides a chiral analysis method of nicotine and its major metabolites in plasma, comprising the steps of:
(1) precisely absorbing 50-200 mu L of a plasma sample to be detected into a 1.5mL centrifuge tube, adding 4-10 times of volume of internal standard extraction solution to precipitate protein, carrying out vortex oscillation for 1-3 min, centrifuging for 5-10 min at the speed of 10000-15000 rpm under the condition of 2-5 ℃, and taking supernatant for later use;
(2) taking the supernatant of the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis under the following analysis conditions:
chromatographic conditions are as follows: the chromatographic column is a Chiralpak IG-3 chromatographic column (250X 4.6mm X3 μm) and a protective column (10mm X4.0 mm X3 μm), and the column temperature is 25 ℃; the mobile phase is ammonium formate/methanol solution with the mass fraction of 0.2%, the flow rate is 0.5mL/min, and the total operation time is 40 min; the sample injection volume is 10 mu L;
mass spectrum conditions: an ion source: an electrospray ion source; an ionization mode: ESI (+); detection mode: an MRM mode; ion source Temperature (TEM): 500 ℃; electrospray voltage (IS): 5500V; air curtain gas (CUR): 2.4X 105Pa; collision gas (CAD): 4.1X 104Pa; ion source gas flow (GS 1): 3.4X 105Pa; ion source gas flow (GS 2): 4.1X 105Pa; inlet voltage (EP): 10V; emission voltage (CXP): 10V; the MRM mass spectrometric detection parameters of the target compound and the internal standard are as follows:
note: "+" indicates the quantitative ion.
(3) Quantitatively calculating the content of the target object in the sample by adopting an internal standard curve method, wherein the internal standard curve method comprises the following steps: preparing a series of standard working solutions containing a target object, adding internal standards (nicotine-d 3, cotinine-d 3, cotinine nitrogen oxide-d 3 and norcotinine-d 4), and making a standard working curve by taking the quantitative ion peak area ratio of the target object and the internal standards in each standard working solution as a vertical coordinate and the content of the target object in each standard working solution as a horizontal coordinate; and (3) substituting the analysis result of the step (2) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
Compared with the prior art, the invention has at least the following beneficial technical effects:
the method is based on HPLC-MS/MS technology to investigate the selection of chromatographic columns, the composition of a mobile phase and the flow rate of the mobile phase, establishes a chiral analysis method of nicotine and main metabolites thereof in a blood sample, realizes the simultaneous chiral analysis of the nicotine and the main metabolites thereof, has the advantages of high flux, good separation degree, simple and convenient operation, high sensitivity, good recovery rate and repeatability and the like, and is suitable for the chiral analysis of the nicotine and the main metabolites thereof in the blood plasma sample.
Drawings
FIG. 1 is a comparison graph of the separation effect of different chromatographic columns (taking nicotine and cotinine as an example), wherein FIGS. 1A and 1B are chromatograms of NicoShell chromatographic columns for separating enantiomers of nicotine and cotinine, respectively; FIGS. 1C and 1D are chromatograms of a TeicoSchell column for separating the enantiomers of nicotine and cotinine, respectively; FIGS. 1E and 1F are chromatograms of chiral Chiralpak IG-3 column for separation of enantiomers of nicotine and cotinine, respectively;
FIG. 2 is a chromatogram of the separation effect of different mobile phase compositions, wherein FIG. 2A is the mobile phase water and FIG. 2B is the formic acid/methanol solution containing 0.1% volume fraction;
FIG. 3 is a chromatogram of separation effect with different flow rates, where FIG. 3A is 0.8mL/min and FIG. 3B is 0.3 mL/min;
FIG. 4 is a Multiple Reaction Monitoring (MRM) chromatogram of a target and its internal standard in a standard working solution.
Detailed Description
The technical solution of the present invention will be further described below with reference to specific embodiments.
Example 1: chiral analysis method for nicotine and main metabolites thereof in blood plasma
(1) Instruments and reagents
The instrument comprises the following steps: the liquid chromatography-tandem mass spectrometer (HPLC-MS/MS) system consists of an American Agilent 1200 high performance liquid chromatograph (comprising a G1367D autosampler, a G1312B binary solvent pump and a G1316B column incubator) and an AB SCIEX 5500 triple quadrupole mass spectrometer (provided with an ESI ion source), and data acquisition and processing Software is analysis 1.5.1 Software; a small animal anesthesia machine (MSS-100 type); model 3-18KS desk top high speed refrigerated centrifuge (Sigma, Germany); vortex oscillator (Vortex Genie 2, Scientific Industries, usa); an electronic balance (AE163, Mettler, Switzerland, sensory: 0.0001 g).
Reagent consumables: (R, S) -nicotine, S-nicotine, R-nicotine, (R, S) -cotinine, S-cotinine, R-cotinine, (R, S) -norcotinine, S-norcotinine, R-norcotinine, (R, S) -cotinine nitroxide, S-cotinine nitroxide, R-cotinine nitroxide, nicotine-d 3, cotinine-d 3, cotinine nitroxide-d 3, norcotinine-d 4 are all available from TRC, methanol (DUKSAN, chromatographically pure), ammonium formate (Acros, purity > 99%), and the ultrapure water used is obtained from Milli-Q system (Milford, MA, USA).
(2) Working conditions of the apparatus
Chromatographic conditions are as follows: the chromatographic column is a Chiralpak IG-3 chromatographic column (250X 4.6mm X3 μm) and a protective column (10mm X4.0 mm X3 μm), and the column temperature is 25 ℃; the mobile phase is ammonium formate/methanol solution with the mass fraction of 0.2%, the flow rate is 0.5mL/min, and the total operation time is 40 min; the sample injection volume is 10 mu L;
mass spectrum conditions: an ion source: an electrospray ion source; an ionization mode: ESI (+); detection mode: an MRM mode; ion source Temperature (TEM): 500 ℃; electrospray voltage (IS): 5500V; air curtain gas (CUR): 2.4X 105Pa; collision gas (CAD): 4.1X 104Pa; ion source gas flow (GS 1): 3.4X 105Pa; ion source gas flow (GS 2): 4.1X 105Pa; inlet voltage (EP): 10V; emission voltage (CXP): 10V; the MRM mass spectrometric detection parameters of the target compound and the internal standard are as follows:
note: "" indicates a quantitative ion.
(3) Sample pretreatment
Preparation of a plasma sample: male SD rats were taken and fasted for 12h before administration with free water. The preparation is administered by subcutaneous injection at a dose of 1.0mg/kg (nicotine dissolved in physiological saline at a concentration of 1.0mg/mL), anesthetized with isoflurane using an anesthesia machine, 0.4mL of blood is taken from the retroorbital venous plexus of rats 60min after administration, placed in a 1.5mL EP centrifuge tube containing 5. mu.L of heparin sodium solution (10g/L), shaken well, centrifuged at 4000r/min for 10min, plasma is separated, and stored at-40 ℃.
② plasma sample pretreatment: naturally thawing a to-be-detected plasma sample at room temperature, precisely sucking 100 mu L of to-be-detected plasma sample into a 1.5mL centrifuge tube, adding 1mL of methanol extraction solution containing an internal standard to precipitate protein, performing vortex oscillation for 2min, centrifuging at 12000rpm for 5min at 4 ℃, and taking supernatant for later use;
(4) standard working solution preparation
Preparing an internal standard stock solution: dissolving the deuterated internal standard by methanol and fixing the volume to prepare a primary internal standard stock solution with the concentration of about 1.0mg/mL, and diluting the primary internal standard stock solution by 1000 times by using methanol to obtain a secondary internal standard stock solution with the concentration of about 1000 ng/mL.
② preparing an internal standard extraction solution: and gradually diluting the secondary internal standard stock solution with methanol to obtain an internal standard extraction solution with the concentration of about 25 ng/mL.
Preparing a standard stock solution: dissolving the standard substance with methanol, diluting to constant volume, preparing primary standard stock solution with concentration of about 1.0mg/mL, and diluting the primary standard stock solution with methanol by 1000 times to obtain secondary standard stock solution with concentration of about 1000 ng/mL.
Fourthly, preparing a standard working solution: preparing a series of standard working solutions by adopting methanol to obtain working solutions with target object concentrations of 0.50, 1.00, 2.50, 5.00, 20.0, 50.0, 100 and 250ng/mL respectively, wherein the concentrations of internal standards are 20ng/mL, wherein S-nicotine-d 3 in (R, S) -nicotine-d 3 is used as an internal standard of S-nicotine and R-nicotine, R-cotinine-d 3 in (R, S) -cotinine-d 3 is used as an internal standard of S-cotinine and R-cotinine, S-norcotinine-d 4 in (R, S) -norcotinine-d 4 is used as an internal standard of S-norcotinine and R-norcotinine, and S-cotinine nitrogen oxide-d 3 in (R, S) -cotinine-d 3 is used as an internal standard of S-cotinine, R-d 3 is used as an internal standard of S-cotinine, An internal standard of R-cotinine nitroxide.
(5) Sample assay
And (3) respectively carrying out HPLC-MS/MS analysis on the standard working solution obtained in the step (4) and the sample solution obtained in the step (3), wherein the target substance and the internal standard Multiple Reaction Monitoring (MRM) chromatogram thereof in the standard working solution are shown in figure 4. Taking the quantitative ion peak area ratio of the target substance to the internal standard in each standard working solution as a vertical coordinate, and taking the content of the target substance in each standard working solution as a horizontal coordinate to prepare a standard working curve; and (4) substituting the analysis result obtained in the step (3) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
(6) Method verification
According to the lowest-grade standard working solution, the detection limit of the method is calculated according to 3 times of signal-to-noise ratio, the matrix effect and the accuracy are calculated according to 3 concentration levels of 2.50 (low), 10.00 (medium) and 40.00ng/mL (high), 5 parallel samples are added at each level, and the test precision is calculated according to the parallel test results. The linear range, linear coefficient, lower limit of quantitation, matrix effect, average accuracy, and average precision of the method are shown in table 1.
TABLE 1 Linear Range, Linear coefficient, lower limit of quantitation, accuracy and average precision for each target①
(7) Analysis of actual samples
The results of chiral analysis of nicotine and its metabolites in typical rat plasma samples according to the above assay are shown in Table 2 (unit: ng/mL):
TABLE 2 chiral analysis results of nicotine and its metabolites in typical rat plasma samples
Note: sample No. 1 is plasma from rats administered with "S-nicotine" subcutaneously at a dose of 1.0mg/kg, and sample No. 2 is plasma from rats administered with "(R, S) -nicotine" subcutaneously at a dose of 1.0 mg/kg.
Example 2: experiments with different precipitants
The sample pretreatment method for measuring nicotine and metabolites thereof in biological samples by HPLC-MS/MS mainly comprises a protein precipitation method, a liquid-liquid extraction method, a solid-phase extraction method and the like, wherein the recovery rate of a target object by the liquid-liquid extraction method is usually low, and the solid-phase extraction method is time-consuming and labor-consuming and is not suitable for large-batch sample analysis.
In this example, acetonitrile and methanol are respectively used as precipitating agents, and the specific experimental process is shown in example 1, and as a result, the effect of precipitating proteins of the two is not greatly different, and the target does not have matrix interference phenomenon. However, acetonitrile affects the chiral separation effect of the target, and methanol has an ideal chiral separation effect on the target.
Therefore, methanol is selected as the protein precipitant.
Example 3 selection experiment of a chromatography column
NicoShell (100 mm. times.4.6 mm. times.2.7 μm) and TeicoShell (150 mm. times.4.6 mm. times.2.7 μm) from AZYP, and Chiralpak IG-3 chiral chromatography column (250 mm. times.4.6 mm. times.3 μm) from Dailn were selected as the subjects. The specific experimental procedure is shown in example 1.
The research finds that:
first, NicoShell achieved baseline separation of the nicotine enantiomers (resolution R2.22, shown in fig. 1A), but had poor separation of the cotinine enantiomers (resolution R0.88, shown in fig. 1B).
② the nicotine enantiomer is not retained on the teicosell chromatographic column, which is shown in fig. 1C, and the separation effect of cotinine enantiomer is not ideal (the separation degree R is 0.56, which is shown in fig. 1D), and the separation effect is not improved significantly by optimizing the chromatographic conditions.
③ Chiralpak IG-3 chiral chromatographic column has ideal effect, nicotine enantiomer can be completely separated basically (shown in figure 1E), cotinine (shown in figure 1F), norcotinine and cotinine nitrogen oxide enantiomer can also be completely separated.
Therefore, the finally determined chiral analytical column is a Chiralpak IG-3 chiral chromatographic column.
Example 4: selection experiment of mobile phase
Several mobile phases were selected for the study: water, acetonitrile, a formic acid/methanol solution containing 0.1% volume fraction, an ammonium acetate/methanol solution containing 0.2% mass fraction, and an ammonium formate/methanol solution containing 0.2% mass fraction. The rest of the process, except for the mobile phase, is referred to example 1.
As a result, it was found that:
first, when a solvent such as water (nicotine is shown in fig. 2A, for example) or acetonitrile is used in the mobile phase, the peak pattern of the target substance is poor and chiral separation of nicotine and cotinine nitroxide cannot be achieved.
② adding formic acid (formic acid/methanol solution containing 0.1 percent of volume fraction, taking nicotine as an example, shown in figure 2B) or ammonium acetate (ammonium acetate/methanol solution containing 0.2 percent of mass fraction) into the mobile phase methanol can affect the chiral separation effect of the target object, and can not realize the chiral baseline separation of nicotine and cotinine oxynitride.
③ 0.2 percent of ammonium formate/methanol solution by mass fraction, and obtains more ideal effect.
The mobile phase composition finally determined was therefore a 0.2% mass fraction ammonium formate/methanol solution.
Example 5: selection experiment of mobile phase flow rate
In order to examine the influence of the flow velocity of the mobile phase on the detection result, the flow velocity of the mobile phase was examined. The flow rate of the mobile phase was set to 0.3 mL/min; 0.5 mL/min; 0.8 mL/min. The rest of the process is referred to example 1, except for the flow rate of the mobile phase.
The research finds that:
under the high flow rate experiment condition of 0.8mL/min, chiral base line separation of nicotine and cotinine nitrogen oxides cannot be realized (taking nicotine as an example, shown in figure 3).
And secondly, under the low flow rate experiment condition of 0.3mL/min, chiral base line separation of the target compound can be realized, but the target object has a wider peak shape and longer system analysis time.
And thirdly, under the flow velocity of 0.5mL/min, the separation degree, the peak type and the analysis time are considered.
Therefore, the flow rate of the mobile phase was finally determined to be 0.5 mL/min.
Claims (6)
1. A chiral analysis method for nicotine and its major metabolites in plasma, comprising the steps of:
(1) precisely absorbing 50-200 mu L of a plasma sample to be detected into a 1.5mL centrifuge tube, adding 4-10 times of volume of internal standard extraction solution to precipitate protein, carrying out vortex oscillation for 1-3 min, centrifuging for 5-10 min at the speed of 10000-15000 rpm under the condition of 2-5 ℃, and taking supernatant for later use;
(2) taking the supernatant of the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis under the following analysis conditions:
chromatographic conditions are as follows: the chromatographic column is a Chiralpak IG-3 chromatographic column with the length multiplied by the inner diameter multiplied by the particle size of 250 multiplied by 4.6mm multiplied by 3 mu m, and a protective column with the length multiplied by the inner diameter multiplied by the particle size of 10mm multiplied by 4.0mm multiplied by 3 mu m is added, and the temperature of the column is 25 ℃; the mobile phase is ammonium formate/methanol solution with the mass fraction of 0.2%, the flow rate is 0.5mL/min, and the total operation time is 40 min; the sample injection volume is 10 mu L;
mass spectrum conditions: an ion source: an electrospray ion source; an ionization mode: ESI +; detection mode: an MRM mode; ion source Temperature (TEM): 500 ℃; electrospray voltage (IS): 5500V; air curtain gas (CUR): 2.4X 105Pa; collision gas (CAD): 4.1X 104 Pa; ion source gas flow GS 1: 3.4X 105Pa; ion source gas flow GS 2: 4.1X 105Pa; inlet voltage (EP): 10V; emission voltage (CXP): 10V; the MRM mass spectrometric detection parameters of the target compound and the internal standard are as follows:
(3) quantitatively calculating the content of the target object in the sample by adopting an internal standard curve method, wherein the internal standard curve method comprises the following steps: preparing a series of standard working solutions containing a target object, adding internal standards of nicotine-d 3, cotinine-d 3, cotinine nitrogen oxide-d 3 and norcotinine-d 4, taking the quantitative ion peak area ratio of the target object and the internal standard in each standard working solution as a vertical coordinate, and taking the content of the target object in each standard working solution as a horizontal coordinate to prepare a standard working curve; and (3) substituting the analysis result of the step (2) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
2. The method according to claim 1, wherein in step (1), the internal standard extraction solution is a methanol solution of each internal standard.
3. The method according to claim 1, wherein in the step (1), the concentration of the internal standard extraction solution is 20-50 ng/mL.
4. The method according to any one of claims 1 to 3, wherein in step (3), the concentration of each target in the series of standard working solutions ranges from 0.5 to 250ng/mL, and the lower limit of quantitation is 0.5 ng/mL.
5. The method according to any one of claims 1 to 3, wherein the plasma is human or animal plasma.
6. The method of claim 5, wherein the animal plasma is rat or mouse plasma.
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