CN106814049A - A kind of Capillary Electrophoresis vivo detection method of Cobratoxin - Google Patents
A kind of Capillary Electrophoresis vivo detection method of Cobratoxin Download PDFInfo
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- CN106814049A CN106814049A CN201611101710.9A CN201611101710A CN106814049A CN 106814049 A CN106814049 A CN 106814049A CN 201611101710 A CN201611101710 A CN 201611101710A CN 106814049 A CN106814049 A CN 106814049A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
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- G01N1/40—Concentrating samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N2001/4038—Concentrating samples electric methods, e.g. electromigration, electrophoresis, ionisation
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Abstract
The present invention provides a kind of Capillary Electrophoresis analysis in vivo of Cobratoxin, take the test plasma sample containing FITC NT, sample introduction carries out HPCE detection, laser induced fluorescence detector is detected, obtain the electrophoresis pattern of testing sample, then compare with standard curve, calculate the concentration of FITC NT in testing sample；The condition of HPCE is：With CEofixTMMEKC kits are buffer solution, and separation voltage is 20kV, and temperature is 24 DEG C, and Detection wavelength is excitation wavelength 488nm, launch wavelength 520nm.The analysis in vivo of the neurotoxin (FITC NT) of the fluorescence list mark that the present invention is provided, the degree of accuracy is high, precision is high, good stability, neurotoxin in internal blood sample can be detected fast and accurately, detection sensitivity is high, and test limit is very low, being capable of extremely micro FITC NT compositions in accurate quantitative analysis detection blood sample, can be used for the vivo pharmacokinetic research of neurotoxin, supported for it provides accurate data.
Description
(1) technical field
The present invention relates to a kind of Capillary Electrophoresis vivo detection method of Cobratoxin, one kind is related in particular to
The side of Cobratoxin is detected using the Capillary Electrophoresis combination laser induced fluorescence detector of high selectivity, hypersensitivity
Method.
(2) background technology
Cobratoxin (Neurotoxin, NT) ----kind from Chinese cobra (Naja naja atra) venom
In isolated water-soluble alkaline polypeptide (C.M.Barber.Alpha neurotoxins [J] .Toxicon, 2013,66:
47-58.), mainly by the cholinergic recepter with maincenter periaqueductal gray (Periaqueductal Gray, PAG)
With reference to, performance analgesia effect, its potency decades of times higher than the morphine of Isodose, and without gastrointestinal side effect, nothing
Tolerance and additive, is a kind of new substituting analgesic, is mainly used in treatment pain caused by cancer and various chronic, intractable pains
Pain etc., there is now intramuscular injection agent listing, trade name Cobratide parenteral solution.
Used as a kind of toxic medicament, to avoid the side effects such as respiration inhibition, dosage needs strict control to NT, and NT has
Dosage is small, and blood concentration is low and easy the features such as disturbed by endogenous material, faces the research of its In vivo kinetics and much chooses
War：Because NT dosages are small, internal blood concentration is very low, and prior art is difficult to the NT concentration that direct detection goes out in blood sample, or
Even if detecting, detection sensitivity is also very low, it is difficult to carry out follow-up pharmacokinetic studies.Hence set up in the NT bodies of efficient sensitivity
Detection method is significant.Being presently used for the protein and peptide drugs vivo detection method such as NT mainly has immune analysis
Method, Isotope tracer labelling method, LC-MS/MS, existing NT detection methods are asked because security, accuracy, pre-treatment are cumbersome etc.
Topic, can not meet the demand of NT vivo detections etc. to a certain extent, using being restricted (L.Bailly-
Chouriberry.Identification ofα-Cobratoxin in Equine Plasma by LC-MS/MS for
Doping Control[J].Anal.Chem.,2013,85:5219–5225).Because Capillary Electrophoresis has, sensitivity is high, spy
The characteristics of opposite sex is by force and the degree of accuracy is high, and it is particularly suitable for the analysis of protein and peptide drugs, in addition, NT is performed the derivatization,
Detected using laser induced fluorescence detector (LIF), sensitivity can be greatly enhanced.This experiment is intended to set up a kind of height
Selectivity, the neurotoxin Capillary Electrophoresis vivo detection method of hypersensitivity, and it is investigated.
(3) content of the invention
The present invention seeks to the defect for overcoming above-mentioned existing method to exist, to avoid pre-treatment, detection sensitivity and experiment
A kind of influence of personal security sex chromosome mosaicism, there is provided capillary electrophoresis detection side in high selectivity, the neurotoxin body of hypersensitivity
Method, supports for the research of neurotoxin vivo pharmacokinetic provides accurate data.
The technical solution adopted by the present invention is：
A kind of Capillary Electrophoresis analysis in vivo of Cobratoxin, the described method comprises the following steps：
(1) test plasma sample containing FITC-NT is taken, sample introduction carries out HPCE, LIF inspection
Device detection is surveyed, the electrophoresis pattern of testing sample is obtained；The condition of the HPCE is：With CEofixTMMEKC reagents
Box is buffer solution, and separation voltage is 20kV, and temperature is 24 DEG C, and Detection wavelength is excitation wavelength 488nm, launch wavelength 520nm；
(2) calibration curve equation of FITC-NT is set up, it is to be measured according to obtained by its calibration curve equation and step (1)
The peak area of FITC-NT in the electrophoresis pattern of sample, calculates the concentration of FITC-NT in testing sample.
The FITC-NT represents the Cobratoxin of fluorescence list mark, from Zhejiang University of Traditional Chinese Medicine's Chinese medicine preparation
Laboratory, can the method according to disclosed in number of patent application 201410566748.8 prepare.FITC-NT is NT by fluorescence list
Mark is obtained, and 1 molecule FITC-NT is 1 molecule NT of correspondence, and both molar concentrations are consistent, therefore, NT can be replaced with FITC-NT
Vivo detection is carried out as target, neurotoxin vivo pharmacokinetic is studied.
Further, in the step (1), the capillary column used in the HPCE is for effective length
The non-capillary column having coated layer of 55cm, internal diameter 75um.
Further, in the step (1), the HPCE uses hydrodynamic injection, 6.9kPa, 10s.
In the step (1), sample size preferably 20 μ L.
Further, in the step (1), 0.1molL is used after the HPCE start-1Sodium hydroxide solution
Rinse 5min, CEofixTMMEKC kits coating rinses 5min, and CEofix is used before each sample introductionTMMEKC wash buffers hair
Capillary column 3min, 20min is run per pin, and CEofix is used after detection per pinTMMEKC kit buffers rinse 5min, analysis knot
Shu Houyong 0.1molL-1Sodium hydroxide solution and water respectively rinse 5min.
In the step (2), the calibration curve equation of the FITC-NT can be obtained by the following method：Take FITC-NT standards
Stock solution stepwise dilution, prepares a series of standard working solution of various concentrations, and difference is configured to SD rat blank plasmas
The plasma standard addition sample of FITC-NT concentration, is detected according to the method for step (1), obtains plasma standard addition sample
Electrophoresis pattern, with FITC-NT concentration as abscissa, peak area in corresponding electrophoresis pattern is linearly returned for ordinate
Return, draw calibration curve equation.
The Capillary Electrophoresis analysis in vivo of the Cobratoxin that the present invention is provided can be used to carry out neurotoxin
Internal pharmacokinetic, because FITC-NT vivo detection sensitivity is high, test limit is very low, can be accurate in blood plasma
Quantify, and molar concentration and NT are consistent, therefore internal pharmacokinetics research can be carried out instead of NT.Specifically, can enter to rat
The tail vein of row FITC-NT and intramuscular injection, determine the concentration of FITC-NT in rat plasma after being administered, and study the medicine of FITC-NT
For dynamic characteristic.
The present invention also provides the side that a kind of internal pharmacokinetic parameter for carrying out neurotoxin using FITC-NT is detected
Method, the described method comprises the following steps：
(A) vein and/or the intramuscular injection of FITC-NT are carried out to rat, is taken a blood sample in different time after injection, bleeding is centrifuged
Slurry, plasma sample is in -20 DEG C of freezen protectives until test；
(B) plasma sample of different time after medicine is drawn, HPCE, laser is carried out as testing sample sample introduction
Induced fluorescent tester detection, obtains the electrophoresis pattern of testing sample；The condition of the HPCE is：With
CEofixTMMEKC kits are buffer solution, and separation voltage is 20kV, and temperature is 24 DEG C, and Detection wavelength is excitation wavelength 488nm,
Launch wavelength 520nm；
(C) calibration curve equation of FITC-NT is set up, it is to be measured according to obtained by its calibration curve equation and step (B)
The peak area of FITC-NT in the electrophoresis pattern of sample, calculates the concentration of FITC-NT in testing sample；
(D) with the time as abscissa, the FITC-NT concentration in plasma sample is ordinate, when drawing the blood medicine of FITC-NT
Half interval contour, obtains internal pharmacokinetics parameter.
In the step (A), injection dosage must not exceed median lethal dose.
The Nervous toxicity that the present invention is marked with Capillary Electrophoresis combination laser induced fluorescence detector (CE-LIF) to fluorescence list
Plain (FITC-NT) carries out the foundation of analysis in vivo, from the standard curve, degree of accuracy, withinday precision, day to day precision, steady
Qualitative, test limit carries out the investigation of methodology to the analysis in vivo, as a result for：FITC-NT is in 0.010~1.0 μ g
mL-1In the range of be in good linear relationship, detection be limited to 0.8nmolL-1(S/N=3), withinday precision RSD≤2.2%,
Day to day precision RSD≤6.1%, accuracy RR has good stability between 93.3% -106.1%, meets wanting for methodology
Ask.
The analysis in vivo of the neurotoxin (FITC-NT) of the fluorescence list mark that the present invention is provided, the degree of accuracy is high, accurate
Degree is high, good stability, can fast and accurately detect that detection sensitivity is high to the neurotoxin in internal blood sample, and test limit is very
It is low, can extremely micro FITC-NT compositions in accurate quantitative analysis detection blood sample, the internal drug metabolism that can be used for neurotoxin moves
Mechanics study, supports for it provides accurate data.
Brief description of the drawings
Fig. 1 is FITC-NT plasma sample specificity electrophoretograms, and in Fig. 1, (a) curve is blank plasma samples, (b) curve
To add the plasma sample of FITC-NT, (c) curve is to collect the plasma sample after rat administration 1h.
Fig. 2 is FITC-NT plasma sample canonical plottings.
Fig. 3 is FITC-NT plasma samples transit time repeatability scatter diagram.
Fig. 4 is injected intravenously and the Drug-time curve after intramuscular injection FITC-NT, dashed curve representative in figure respectively for rat
Intravenous injection；Block curve represents intramuscular injection.The small figure in the upper right corner is the enlarged drawing in the dotted line frame of big figure in figure.
(4) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：The optimization of each parameter
1 instrument and reagent
1.1 instruments
BECKMAN P/ACE MDQ types efficient capillary electrophoresis apparatus (Beckman companies of the U.S.)；
Non- coating quartz capillary column (75 μ m 50cm, the sharp Feng chromatograms device Co., Ltd of Hebei Yongnian)；
Optima Max hypervelocity refrigerated centrifuge (Beckman companies of the U.S.)；
CP225D types electronic analytical balance (German Sartorius companies).
1.2 reagents
Cobratoxin (FITC-NT) (purity of fluorescence list mark>99.29%), Zhejiang University of Traditional Chinese Medicine's Chinese medicine
Preparation laboratory (number of patent application 20140566748.8)；
CEofixTMMEKC kits (Beijing Bo Siya biochemical technologies research institute) (US Patent no.5,611,903)；
It is pure that remaining reagent is domestic analysis.
2 methods and result
The preparation of 2.1 reference substance stock solutions
Precision weighs FITC-NT reference substance 0.50mg, puts in 10.0mL measuring bottles, is dissolved in water and is diluted to scale, shakes up,
(mass concentration is 50 μ gmL to obtain final product FITC-NT reference substances storing solution-1).2.2 sample pretreatment steps
Extracting vein blood fills sample, is placed in 0.5mL centrifuge tubes (plus anticoagulant heparin), and l0min is centrifuged with rotating speed 3500r/min
Afterwards, separated plasma.It is stored in -80 DEG C of refrigerators.Plasma sample is placed in after thawing under 37 DEG C of water-baths, draws 90ul blood plasma, is added
The FITC-NT solution (50ug/mL) that 10ul is prepared, misfortune rotation takes the analysis of 20uL sample introductions after mixing.
2.3 deposition conditions
Buffer solution：CEofixTMMEKC kits；Separation voltage：20kV；Temperature：24℃；Detection wavelength：Excitation wavelength：
488nm, launch wavelength 520nm.Sampling condition：Using hydrodynamic injection, 6.9kPa, 10s.0.1molL is used after start-1Hydroxide
Sodium solution rinses 5min, CEofixTMMEKC kits coating rinses 5min, and CEofix is used before each sample introductionTMMEKC buffer solutions
Capillary column 3min is rinsed, 20min is run per pin, CEofix is used after the completion of sample detectionTMMEKC kit buffers are rinsed
5min, ultimate analysis uses 0.1molL after terminating-1Sodium hydroxide solution and water respectively rinse 5min.
The investigation of 2.4 specificities
Fig. 1 is FITC-NT plasma sample specificity electrophoretograms, and in Fig. 1, (a) curve is blank plasma samples, (b) curve
To add the plasma sample of FITC-NT, (c) curve is to collect the plasma sample after rat administration 1h.Compare blank plasma and
FITC-NT plasma sample electrophoretograms, it was confirmed that the specificity of the vivo detection method, are not detected by other endogenous in electrophoretogram
The interfering material such as material or metabolite.
2.5 Specification Curve of Increasing and test limit
FITC-NT Standard Reserving Solutions stepwise dilution prepares series of standards working solution, and precision is measured in right amount, uses SD rats
The assembling of blank blood is made the plasma standard addition sample of FITC-NT, after being processed by foregoing preprocess method, carries out CE-LIF points
Analysis, linear regression is carried out to drug concentration (x) with peak area (y), and Fig. 2 is the plasma sample canonical plotting of FITC-NT, is obtained
FITC-NT calibration curve equations are：Y=2481x-0.4254, R2=0.9981, FITC-NT are in 0.010~1.0 μ gmL-1Model
In good linear relationship in enclosing, detection is limited to 0.8nmolL-1(S/N=3).
2.6 study on the stability
Study on the stability includes the investigation of short-term stability and long-time stability.Wherein short-term stability is investigated includes that SD is big
Mouse blood dress standard addition sample room temperature shelf-stability and Frozen-thawed cycled stability, long-time stability are included in -80 DEG C of preservations
The stability of 25 days.The plasma standard addition Quality control samples containing the basic, normal, high three kinds of various concentrations of FITC-NT are prepared, each
Each 6 parts of concentration, operates according to the requirement of different investigation projects, after Instrumental Analysis, calculates the measure concentration of each biological sample, measures
Stability meets the requirement of methodology.FITC-NT plasma samples difference condition of storage stability inferior result of the test is as shown in table 1.
Table 1FITC-NT plasma samples difference condition of storage stability inferior result of the test (n=6)
2.7 preci-sion and accuracies are investigated
Prepare the plasma standard addition biological sample containing the basic, normal, high three kinds of various concentrations of FITC-NT, each concentration each 6
Part, operated by aforementioned sample pre-treatment step, by calibration curve equation, calculate the measure concentration of each biological sample.FITC-NT
Plasma sample preci-sion and accuracy result of the test as shown in table 2, counts to obtain withinday precision RSD≤2.2%, day to day precision
RSD≤6.1% and degree of accuracy RR are between 93.3% -106.1%.The repeatability card of the peak area of transit time and biological sample
Real CEofixTMThe use of MEKC coatings optimizes flushing process and the electric current composition of Capillary Electrophoresis.Fig. 3 is FITC-NT blood plasma
Sample migration time repeatability scatter diagram, it is seen that under different number of injections, detects the peak area data variation of sample less, table
Bright detection is not disturbed by number of injections, and preci-sion and accuracy is good.
Table 2FITC-NT plasma sample preci-sion and accuracies result of the test (n=6)
The pharmacokinetics application of 2.8 detection methods
Using in the studies above set up capillary electrophoresis detection method rat is carried out respectively FITC-NT tail vein and
Intramuscular injection, determines the concentration of FITC-NT in rat plasma after being administered, and studies the Pharmacokinetic Characteristics of FITC-NT.
12 SD male rats (200-250g) are randomly divided into two groups, at least more than fasting 12h before experiment, but can be certainly
By drinking water.FITC-NT solution is diluted with the PBS of pH 7.4,6 SD rat tail veins injection injection dosages are
0.06mg/kg.Remaining 6 SD rat muscles injection injection dosage is 0.12mg/kg, 5min after injection, 10min, 20min,
30min, 1h, 2h, 3h, 6h, 12h, 24h are placed in the centrifuge tube containing a small amount of heparin sodium aqua from rat eye socket blood sampling about 0.5mL
In, and blood plasma plasma samples are centrifuged out immediately in -20 DEG C of freezen protectives until test.Inject the blood plasma sample of FITC-NT rats
Product, using the concentration of FITC-NT in Capillary Electrophoresis plasma sample, and draw Drug-time curve.Fig. 4 is that rat difference is quiet
Arteries and veins is injected and the Drug-time curve after intramuscular injection FITC-NT, dashed curve representative intravenous injection in figure；Block curve represents flesh
Meat is injected.The small figure in the upper right corner is the enlarged drawing in the dotted line frame of big figure in figure.Rat carries out FITC-NT tail veins and intramuscular injection
Pharmacokinetic parameters afterwards are as shown in table 3.
The rat of table 3 carries out the pharmacokinetic parameters (n=6) after FITC-NT tail veins and intramuscular injection
Claims (7)
1. the Capillary Electrophoresis analysis in vivo of a kind of Cobratoxin, it is characterised in that methods described includes following step
Suddenly：
(1) test plasma sample containing FITC-NT is taken, sample introduction carries out HPCE, laser induced fluorescence detector
Detection, obtains the electrophoresis pattern of testing sample；The condition of the HPCE is：With CEofixTMMEKC kits are
Buffer solution, separation voltage is 20kV, and temperature is 24 DEG C, and Detection wavelength is excitation wavelength 488nm, launch wavelength 520nm；It is described
FITC-NT represents the Cobratoxin of fluorescence list mark；
(2) calibration curve equation of FITC-NT is set up, the testing sample according to obtained by its calibration curve equation and step (1)
Electrophoresis pattern in FITC-NT peak area, calculate the concentration of FITC-NT in testing sample.
2. the method for claim 1, it is characterised in that in the step (1), used in the HPCE
Capillary column for effective length for 55cm non-capillary column having coated layer, internal diameter 75um.
3. the method for claim 1, it is characterised in that in the step (1), the HPCE is using pressure
Power sample introduction, 6.9kPa, 10s.
4. the method for claim 1, it is characterised in that in the step (1), the μ L of sample size 20.
5. the method for claim 1, it is characterised in that in the step (1), after the HPCE start
Use 0.1molL-1Sodium hydroxide solution rinses 5min, CEofixTMMEKC kits coating rinses 5min, is used before each sample introduction
CEofixTMMEKC wash buffer capillary column 3min, 20min is run per pin, and CEofix is used after detection per pinTMMEKC reagents
Box wash buffer 5min, analysis uses 0.1molL after terminating-1Sodium hydroxide solution and water respectively rinse 5min.
6. the method for claim 1, it is characterised in that in the step (2), the calibration curve equation of the FITC-NT
Obtain by the following method：FITC-NT Standard Reserving Solution stepwise dilutions are taken, a series of standard working solution of various concentrations is prepared,
The plasma standard for being configured to different FITC-NT concentration with SD rat blank plasmas adds sample, is carried out according to the method for step (1)
Detection, obtains the electrophoresis pattern that plasma standard adds sample, with FITC-NT concentration as abscissa, in corresponding electrophoresis pattern
Peak area carries out linear regression for ordinate, draws calibration curve equation.
7. a kind of method that internal pharmacokinetic parameter for carrying out neurotoxin using FITC-NT is detected, the FITC-NT generations
The Cobratoxin of table fluorescence list mark, the described method comprises the following steps：
(A) vein and/or the intramuscular injection of FITC-NT are carried out to rat, are taken a blood sample in different time after injection, be centrifuged out blood plasma,
Plasma sample is in -20 DEG C of freezen protectives until test；
(B) plasma sample of different time after medicine is drawn, HPCE, induced with laser is carried out as testing sample sample introduction
Fluorescence detector detection, obtains the electrophoresis pattern of testing sample；The condition of the HPCE is：With
CEofixTMMEKC kits are buffer solution, and separation voltage is 20kV, and temperature is 24 DEG C, and Detection wavelength is excitation wavelength 488nm,
Launch wavelength 520nm；
(C) calibration curve equation of FITC-NT is set up, the testing sample according to obtained by its calibration curve equation and step (B)
Electrophoresis pattern in FITC-NT peak area, calculate the concentration of FITC-NT in testing sample；
(D) with the time as abscissa, the FITC-NT concentration in plasma sample is ordinate, and the blood medicine time for drawing FITC-NT is bent
Line, obtains internal pharmacokinetics parameter.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111337468A (en) * | 2020-04-24 | 2020-06-26 | 新乡医学院 | Multi-analysis snake venom mixture analysis fluorescent sensor |
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| CN101638689A (en) * | 2009-09-10 | 2010-02-03 | 中国科学院生态环境研究中心 | Integral genome DNA methylation level analysis based on immune capillary electrophoresis-laser induced fluorescence |
| CN104311651A (en) * | 2014-10-22 | 2015-01-28 | 浙江中医药大学 | Method for preparing fluorescent single-labeled cobra neurotoxin |
| CN104777211A (en) * | 2015-03-26 | 2015-07-15 | 浙江医学高等专科学校 | Method for detecting alpha-cobratoxin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111337468A (en) * | 2020-04-24 | 2020-06-26 | 新乡医学院 | Multi-analysis snake venom mixture analysis fluorescent sensor |
| CN111337468B (en) * | 2020-04-24 | 2022-12-09 | 新乡医学院 | Multi-analysis snake venom mixture analysis fluorescent sensor |
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Application publication date: 20170609 |