CN105738498A - Separation and detection method of pyrimethamine - Google Patents
Separation and detection method of pyrimethamine Download PDFInfo
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- CN105738498A CN105738498A CN201610065160.3A CN201610065160A CN105738498A CN 105738498 A CN105738498 A CN 105738498A CN 201610065160 A CN201610065160 A CN 201610065160A CN 105738498 A CN105738498 A CN 105738498A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a separation and detection method of pyrimethamine. The separation and detection method comprises the following steps: (1) adding acetonitrile into a sample; after shaking and uniformly mixing, centrifuging; taking supernatant and blowing with nitrogen gas; dissolving residues with a solvent to obtain a solution to be detected; (2) adding a magnetic molecularly imprinted polymer into the solution to be detected; after shaking for 20-120 minutes, magnetically separating the magnetic molecularly imprinted polymer; (3) eluting the pyrimethamine in the magnetic molecularly imprinted polymer with an eluting solvent with a certain volume, and collecting an eluent; and (4) determining the concentration of the pyrimethamine in the eluent by utilizing HPLC/UV (High Performance Liquid Chromatography/Ultraviolet), wherein the magnetic molecularly imprinted polymer is prepared by taking the pyrimethamine as a template and amobarbital as a functional monomer. The magnetic molecularly imprinted polymer provided by the invention forms an intermolecular acting matrix through a multiple-hydrogen-bond effect between a template molecule and the functional monomer, so that the effect between the template molecule and the functional monomer is enhanced, and the affinity and selectivity on the pyrimethamine are improved.
Description
Technical field
The present invention relates to organic chemistry and analytical chemistry field, particularly relate to the method for separating and detecting of a kind of pyrimethamine.
Background technology
Pyrimethamine (PYR) is anti-malaria medicine wide at present, it is mainly used in the prevention of malaria, the activity of dihydrofolate reductase in polypide can be suppressed, thus interfere with plasmodial folic acid homergy, the pre-erythrocytic stage of to pernicious malaria and Plasmodium vivax effectively, it is commonly used for causal prophylactics.Also plasmodium growth in mosquito body can be suppressed, therefore can blocking propagation.Clinically for prevention of malaria and the treatment of resting stage anti-recurrence.Annual estimate 500,000,000 clinical cases because of malaria death, be in addition to outside acquired immune deficiency syndrome (AIDS) and pulmonary tuberculosis infectious disease one of disease murdering maximum people, cause the attention of WHO.
Pyrimethamine has a wide range of applications in animal product and aquatic products body, and appropriate use can strengthen the resistance against diseases of animal.But pyrimethamine has the savings of height in animal product body, beyond certain limit, destroy the hemopoietic system of people after people is edible, cause haemolytical anaemia, even have and cause potential carcinogenic possibility, central nervous system is had direct toxic action simultaneously.Therefore, set up effective pyrimethamine detecting method to have very important significance.
Prior art mainly adopts the medicine that high performance liquid chromatography (HPLC) is analyzed in biological sample.HPLC generally uses C18 post, goes to toward the pre-treatment relating to complexity being injected into chromatographic column, degrades as by acid adding or organic solvent deposit, and liquid-liquid extraction and off-line Solid-Phase Extraction.But these processes are complicated and consuming time, and medicine is likely at sample pre-treatments stage yield-loss, so in such cases, it is necessary to produce a kind of to target molecule selectively material.1994, Sellergren reported first used molecularly imprinted polymer as the research of adsorbent in state extracts mutually, and hereafter, molecular engram-solid phase extraction techniques develops rapidly.Target molecule is had " memory " effect by molecularly imprinted polymer, it is possible to the target molecule in highly selective identification complex sample.Relevant result of study shows, it is high that molecularly imprinted polymer extracts adsorbent not only selectivity mutually as state, and has adhesion utilization strong, repeatable and the low advantage of cost.
Currently, molecular engram-Solid-Phase Extraction needs to make solid-phase extraction column with molecularly imprinted polymer for filler in actual applications, not only complex operation, and extraction process center pillar pressure is high, flow velocity is low, and these drawbacks greatly limit further genralrlization and the application of molecular engram-Solid-Phase Extraction.Scientific research personnel prepares magnetic molecularly imprinted polymer with magnetic material for carrier.Can being directly dispersing in solution by magnetic molecularly imprinted polymer and target molecule is adsorbed, absorption uses externally-applied magnetic field to make it separate from substrate after terminating.Magnetic molecularly imprinted-solid phase extraction procedure not only easy to operate, bioaccumulation efficiency is high, and avoids the time consuming process preparing filled column, the shortcoming overcoming conventional molecular trace-Solid-Phase Extraction well.
But, owing to Hyarogen-bonding is more weak, general molecular imprinting only interacts with 1-2 hydrogen bond, the limited sorption capacity to object.Thus template molecule is less with the binding site of function monomer, the selectivity of template molecule is more weak, it is impossible to realize effectively selecting absorption to template molecule.
Summary of the invention
It is an object of the invention to overcome shortcoming and defect of the prior art, it is provided that the method for separating and detecting of a kind of efficient pyrimethamine.
The present invention is achieved by the following technical solutions: the method for separating and detecting of a kind of pyrimethamine, comprises the following steps:
(1) in sample, acetonitrile is added, after concussion mixing, centrifugal, take supernatant and dry up with nitrogen, residue solvent redissolves as liquid to be measured；
(2) in liquid to be measured, magnetic molecularly imprinted polymer is added, after concussion 20～120min, Magnetic Isolation magnetic molecularly imprinted polymer；
(3) with the pyrimethamine in the eluting solvent eluting magnetic molecularly imprinted polymer of certain volume, eluent is collected；
(4) concentration of pyrimethamine in eluent is measured with HPLC/UV；
Magnetic molecularly imprinted polymer in described step (2) is prepare with pyrimethamine for the function monomer between template and pyrimethamine with multiple hydrogen bonding effect.
Relative to prior art, the method for separating and detecting of the pyrimethamine of the present invention, the magnetic molecularly imprinted polymer adopted, utilize the multiple hydrogen bonding effect between template molecule and function monomer, form intermolecular interaction matrix, increase the binding site of template molecule and function monomer, thus the effect strengthened between template molecule and function monomer, improve magnetic molecularly imprinted polymer to the affinity of template molecule pyrimethamine and selectivity.
Further, the function monomer of described magnetic molecularly imprinted polymerization is amobarbital.
Further, in step (4), the testing conditions of described HPLC/UV is: chromatographic column is C18 post；Mobile phase is volume ratio is acetonitrile and the water of 9:1；Described flow velocity is 1.0mL/min；The detection wavelength of described UV is λ=272nm；Described detection temperature is 25 DEG C.
Further, in step (3), described eluting solvent is volume ratio is methanol and the acetic acid mixture of 8:2.
Further, described in step (2), the preparation method of magnetic molecularly imprinted polymer comprises the following steps:
S21: sedimentation method synthesis Fe3O4Magnetic nanoparticle；
S22: sol-gal process prepares the magnetic Nano microsphere of coated silica；
S23: the magnetic Nano microsphere of coated silica is carried out surface modification with MPS, obtains the MPS magnetic Nano microsphere modified；
S24: pyrimethamine, amobarbital are sufficiently mixed in DMSO, it is subsequently adding MPS magnetic Nano microsphere, cross-linking agent, initiator and the disperse medium modified, bring it about polyreaction, obtain the molecular engram polymer magnetic microsphere of Surface coating template molecule；
S25: the magnetic microsphere obtained in washing step S24, after removing the template molecule being wherein coated with, dry, obtain magnetic microsphere molecular imprinted polymer on surface.
Further, in described step S24, the mol ratio of pyrimethamine and amobarbital is 1:1.
In order to be more fully understood that and implement, describe the present invention in detail below in conjunction with accompanying drawing.
Accompanying drawing explanation
Fig. 1 adds the liquid-phase chromatographic analysis of (A) and rear (B) before magnetic molecule imprinted polymer in testing sample
Detailed description of the invention
The method for separating and detecting of a kind of pyrimethamine disclosed by the invention, comprises the following steps:
(1) sample treatment: add acetonitrile in sample, after concussion mixing, centrifugal, take supernatant and dry up with nitrogen, residue solvent redissolves as liquid to be measured.
(2) in liquid to be measured, magnetic molecularly imprinted polymer is added, after concussion 20～120min, Magnetic Isolation magnetic molecularly imprinted polymer.
(3) with the pyrimethamine in the eluting solvent eluting magnetic molecularly imprinted polymer of certain volume, eluent is collected.
(4) concentration of pyrimethamine in eluent is measured with HPLC/UV.
In the present embodiment, for urine sample.In the centrifuge tube of 5mL, add the urine of 1mL, be subsequently adding the acetonitrile of 1mL.After concussion 1～2min, with the rotating speed of 12000r/min, mixed liquor being centrifuged 10～30min, takes supernatant and then dry up with nitrogen, the redissolution of residue 1mL solvent is as the liquid to be measured of urine sample.Described solvent is volume ratio is the mixed liquor of the acetonitrile of 7:3 and water.
In step (2), take the urine sample liquid to be measured of 1mL, in liquid to be measured, add the magnetic molecularly imprinted polymer of 1～10mg, after concussion 20～120min, under outside the action of a magnetic field, separate magnetic molecularly imprinted polymer.In the present embodiment, described magnetic molecularly imprinted polymer is with pyrimethamine for template, and the function monomer between pyrimethamine with multiple hydrogen bonding effect prepares.In the present embodiment, described function monomer is amobarbital.The mol ratio of described template molecule and function monomer amobarbital is 1:1.
In step (3), remove, with the eluting solvent eluting of 1mL, the pyrimethamine that magnetic molecularly imprinted polymer is coated with.Repeatedly, and collect eluent.Pyrimethamine is dissolved in eluting solvent.Described eluting solvent is volume ratio is methanol and the acetic acid mixture of 8:2.
In step (4), measure the concentration of pyrimethamine in eluent with HPLC/UV.Described chromatographic condition is as follows: described chromatographic column is C18 post；Mobile phase is volume ratio is acetonitrile and the water of 9:1；Described flow velocity is 1.0mL/min；The detection wavelength of described UV is λ=272nm；Described detection temperature is 25 DEG C.
Concrete, the present embodiment provides the preparation method of magnetic molecularly imprinted polymer described in a kind of step (2), but is not limited to this.The preparation method of described magnetic molecularly imprinted polymer is as follows:
S21: sedimentation method synthesis Fe3O4Magnetic nanoparticle；
S22: sol-gal process prepares the magnetic Nano microsphere of coated silica；
S23: the magnetic Nano microsphere of coated silica is carried out surface modification with MPS, obtains the MPS magnetic Nano microsphere modified；
S24: pyrimethamine, amobarbital are sufficiently mixed in DMSO, it is subsequently adding MPS magnetic Nano microsphere, cross-linking agent, initiator and the disperse medium modified, bring it about polyreaction, obtain the molecular engram polymer magnetic microsphere of Surface coating template molecule；The mol ratio of described pyrimethamine and amobarbital is 1:1；
S25: the magnetic microsphere obtained in washing step S24, after removing the template molecule being wherein coated with, dry, obtain magnetic microsphere molecular imprinted polymer on surface.
Referring to Fig. 1, it is to add the liquid-phase chromatographic analysis of (A) and rear (B) before magnetic molecule imprinted polymer in testing sample.It can be seen that before adding magnetic molecularly imprinted polymer, be can detect that the peak of stronger pyrimethamine by liquid chromatograph.And after adding magnetic molecularly imprinted polymer, it is substantially not detectable the peak of pyrimethamine by liquid chromatograph.But other peak is held essentially constant, describes magnetic molecularly imprinted polymer and pyrimethamine is had significantly high adsorption selectivity.And measuring the magnetic molecularly imprinted polymer response rate to variable concentrations pyrimethamine, described magnetic molecularly imprinted polymer is to the substantially all recovery of pyrimethamine.After adsorbing thus through Magnetic Isolation imprinted polymer, the content of pyrimethamine in the content of pyrimethamine and known sample liquid to be measured in detection eluent.Described magnetic molecularly imprinted polymer is reusable, and repeatedly remains on the significantly high adsorption selectivity to pyrimethamine afterwards reusing.
Relative to prior art, the method for separating and detecting of the pyrimethamine of the present invention, the magnetic molecularly imprinted polymer adopted, utilize the multiple hydrogen bonding effect between template molecule and function monomer, form intermolecular interaction matrix, increase the binding site of template molecule and function monomer, thus the effect strengthened between template molecule and function monomer, improve magnetic molecularly imprinted polymer to the affinity of template molecule pyrimethamine and selectivity.
The invention is not limited in above-mentioned embodiment, if to the various changes of the present invention or deformation without departing from the spirit and scope of the present invention, if these are changed and deform within the claim and the equivalent technologies scope that belong to the present invention, then the present invention is also intended to comprise these changes and deformation.
Claims (6)
1. the method for separating and detecting of a pyrimethamine, it is characterised in that: comprise the following steps:
(1) in sample, acetonitrile is added, after concussion mixing, centrifugal, take supernatant and dry up with nitrogen, residue solvent redissolves as liquid to be measured；
(2) in liquid to be measured, magnetic molecularly imprinted polymer is added, after concussion 20～120min, Magnetic Isolation magnetic molecularly imprinted polymer；
(3) with the pyrimethamine in the eluting solvent eluting magnetic molecularly imprinted polymer of certain volume, eluent is collected；
(4) concentration of pyrimethamine in eluent is measured with HPLC/UV；
Magnetic molecularly imprinted polymer in described step (2) is prepare with pyrimethamine for the function monomer between template and pyrimethamine with multiple hydrogen bonding effect.
2. the method for separating and detecting of pyrimethamine according to claim 1, it is characterised in that: the function monomer of described magnetic molecularly imprinted polymerization is amobarbital.
3. the method for separating and detecting of pyrimethamine according to claim 1, it is characterised in that: in step (4), the testing conditions of described HPLC/UV is: chromatographic column is C18 post；Mobile phase is volume ratio is acetonitrile and the water of 9:1；Described flow velocity is 1.0mL/min；The detection wavelength of described UV is λ=272nm；Described detection temperature is 25 DEG C.
4. the method for separating and detecting of pyrimethamine according to claim 1, it is characterised in that: in step (3), described eluting solvent is volume ratio is methanol and the acetic acid mixture of 8:2.
5. the method for separating and detecting of the pyrimethamine according to any claim in claim 1-4, it is characterised in that: described in step (2), the preparation method of magnetic molecularly imprinted polymer comprises the following steps:
S21: sedimentation method synthesis Fe3O4Magnetic nanoparticle；
S22: sol-gal process prepares the magnetic Nano microsphere of coated silica；
S23: the magnetic Nano microsphere of coated silica is carried out surface modification with MPS, obtains the MPS magnetic Nano microsphere modified；
S24: pyrimethamine, amobarbital are sufficiently mixed in DMSO, it is subsequently adding MPS magnetic Nano microsphere, cross-linking agent, initiator and the disperse medium modified, bring it about polyreaction, obtain the molecular engram polymer magnetic microsphere of Surface coating template molecule；
S25: the magnetic microsphere obtained in washing step S24, after removing the template molecule being wherein coated with, dry, obtain magnetic microsphere molecular imprinted polymer on surface.
6. the method for separating and detecting of pyrimethamine according to claim 5, it is characterised in that: in described step S24, the mol ratio of pyrimethamine and amobarbital is 1:1.
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Citations (2)
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| WO2008112826A1 (en) * | 2007-03-12 | 2008-09-18 | Board Of Regents, The University Of Texas System | Method and process for the production of multi-coated recognitive and releasing systems |
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Patent Citations (2)
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|---|---|---|---|---|
| WO2008112826A1 (en) * | 2007-03-12 | 2008-09-18 | Board Of Regents, The University Of Texas System | Method and process for the production of multi-coated recognitive and releasing systems |
| CN103713056A (en) * | 2013-11-25 | 2014-04-09 | 宁波出入境检验检疫局检验检疫技术中心 | Method for simultaneously analyzing and detecting residual veterinary drug compositions in animal tissue |
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