CN101713740B - Method for spectrophotometry measuring boron content in BNCT medicament in biological sample - Google Patents
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- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention belongs to the technical field of nuclear chemical analysis and test, and particularly discloses a method for measuring the boron content in BNCT drugs in a biological sample by a spectrophotometry. The method comprises the processes of biological sample digestion, standard curve determination, biological sample determination and the like, wherein the used color developing agent is 3-methoxy-methylene imine H, and the determined maximum detection wavelength is 390-410 nm; the final pH of the measurement solution was 4-6.5. The accuracy and precision of the measurement of the boron content in the biological sample meet the requirements.
Description
Technical field
The invention belongs to nuclear chemistry analysis and testing technology field, be specifically related to a kind of method with boron content in the BNCT medicine in the metric measurement biological sample.
Background technology
(boron neutron capture therapy BNCT) is a kind of novel radiotheraping method to boron neutron capture therapy, and it is with containing
10B medicine administered through oral or injecting method are introduced in the body, and make it optionally to accumulate in the cancer cell, use the neutron irradiation diseased region then, make
10B takes place
10B (n, α)
7The Li nuclear reaction, utilize consequent α particle with
7The Li ion is kill cancer cell in the cell scope.The precondition that this method is achieved success is the accurate measurement of boron concentration in the administration artifact body.At present, boron Determination on content method mainly contains mass spectroscopy, atomic spectroscopy, AAS, electrochemical methods and nuclear reaction analysis method in the biological sample.Mass spectroscopy is most important a kind of analytical approach in boron and the isotope assay thereof; Especially the development of Waters Using ICP-MS (like ICP-MS); Not only overcome the shortcoming of most of this method; And realized the mensuration of boron in the biological sample and isotope ratio thereof developing into the method the most widely of using in the current techniques.But defective is an instrument and equipment to cost an arm and a leg, and needs professional's operation and daily servicing.Atomic spectroscopy such as AES and AAS can measure the multiple trace element that comprises boron simultaneously, but mainly sensitive inadequately with the element that the oxidative ionic form exists to boron, phosphorus etc.The electrode that electrochemical methods uses is subject to pollutant effects in the solution, stability and poor reproducibility.The nuclear reaction analysis method has limitation in practical application.The employed instrument and equipment of AAS is simple, and is easy to operate, is a kind of boron element measuring method that is easy to promote.But use commonplace curcumin method to need anhydrous state at present; The blue method reagent blank of methine is big, and it is big to reduce blank difficulty; The concentrated sulphuric acid that anthraquinone need are a large amount of, and reaction velocity is very slow.
Summary of the invention
(1) goal of the invention
The present invention is directed in the existing metric measurement biological sample the existing deficiency of method of boron content in the BNCT medicine, provide a kind of can be accurate, easy, the AAS of boron content in the BNCT medicine in the fast measuring biological sample.
(2) technical scheme
The method of boron content in the BNCT medicine in a kind of spectrophotometry biological sample; Comprise that biological sample is cleared up, standard curve determination, biological sample measure flow process; Key is: used developer is 3-methoxyl-first imido H, and determined maximum detection wavelength is 390～410nm; Final measurement liquid pH value is 4-6.5.
Usually, in the spectrophotometry biological sample in the BNCT medicine during boron content, the final measurement liquid of being got is long-pending to be 5mL.As example, concrete measuring method is following.
It is to get 0.1～0.2mL blood sample that described biological sample is cleared up, or its hetero-organization of 0.1～0.2g and process the sample of homogenate, places 12～24 hours after adding 0.5～1.0mL red fuming nitric acid (RFNA), subsequent use.
Described standard curve determination is to draw 0～1.0mLBPA standard respectively to use liquid, adds EDTA solution 0.5～0.7mL successively, acetate-ammonium acetate damping fluid 1～1.5mL; Developer 3-methoxyl-azomethine H solution 0.8～1.6mL; Add water and be settled to 5.0mL, the pH value is 4.0～6.5, and mixing is placed 30～120min; Survey absorbance, drawing standard curve at 390～410nm place.
It is to get above-mentioned ready digestion solution 0.1～0.2mL that described biological sample is measured flow process, adds EDTA solution 0.5～0.7mL successively, acetate-ammonium acetate damping fluid 1～1.5mL; Developer 3-methoxyl-azomethine H solution 0.8～1.6mL; Add water and be settled to 5.0mL, the pH value is 4.0～6.5, and mixing is placed 30～120min; Survey absorbance at 390～410nm place, calculate boron content according to typical curve.
(3) implementation result
3-methoxyl-azomethine H is a kind of novel boron developer, and the harsh conditions when it has avoided traditional boron chromogenic reagent such as curcumin make operation become simple, and the reaction time are short, and specificity is strong, and is highly sensitive.Simultaneously, discover that boron and 3-methoxyl-azomethine H developer is a color stability in 4.0～6.5 the solution at pH, and pH crosses and will cause maximum absorption wavelength to move to short wavelength's direction when low, and absorbance diminishes; And developer itself has color, and also increases with the increase absorbance of volume, therefore, in the scope of color stability, selects to try one's best little volume.Therefore, technical scheme provided by the present invention has fast, and is easy, special, and advantages such as sensitivity meet the demands to the accuracy and the precision of boron assay in the biosome sample.
Embodiment
Below in conjunction with embodiment technical scheme provided by the present invention is done further to set forth.
Embodiment 1
Present embodiment is BPA bio distribution experiment in the normal mouse body.Prepare following experiment reagent, experimental apparatus before the experiment, and dispose required solution, processing biological sample.
1. experiment reagent
L-BPA (a kind of BNCT medicine is called for short BPA), SIGMA company; 3-methoxyl-azomethine H (98%), the holy chemical reagent of Shanghai gold Ltd; It is pure that glacial acetic acid, ammonium acetate, EDTA, red fuming nitric acid (RFNA), NaOH, ascorbic acid, hydrochloric acid are analysis, Beijing chemical reagents corporation.
2. experimental apparatus
UV2450 type ultraviolet-visible spectrophotometer, Japanese SHIMADZU company; ICP-MS, Britain Isoprobe GB Instrument company; The TGL-16L hydro-extractor, Anting Scientific Instrument Factory, Shanghai; The BP211D electronic balance, German Sartorius company; 211 type pH meters, Orion Research; 100-1000 μ L pipettor, Socorex 1-5mL pipettor, Finland Finnpipette.
3. solution allocation
1. the BPA standard is used liquid: 5.22mgL-BPA to add the 0.5mL red fuming nitric acid (RFNA) to clear up, and thin up is to 10mL, and then wherein boron element concentration is 25 μ g/mL.
2. 3-methoxyl-azomethine H solution: take by weighing 0.450g reagent, dissolve fully with 0.5mol/LNaOH solution, add 1.000g ascorbic acid (protection imido grpup) again, using HCl to regulate pH is 5.7, is settled in the 50mL polypropylene volumetric flask.
3. acetate-ammonium acetate buffer solution: take by weighing the 100g ammonium acetate and be dissolved in the 400mL distilled water, add the 28mL glacial acetic acid, using pH meter to be adjusted to pH is 5.7, is settled to 500mL.
4. EDTA solution (2%): take by weighing 1.0g EDTA-Na
2, the adding distil water heating for dissolving is settled to 50mL.
4. biological sample disposal route
Get 28 of kunming mices, be divided into 7 groups at random, 4 every group, wherein one group is blank control group; Do not inject BPA solution, all the other 6 groups of every mouse are through tail vein injection BPA-F parenteral solution 100 μ L (being equivalent to the 150mgBPA/kg body weight), respectively at 5,30min; 1,2 and the disconnected neck of 3h put to death animal, get tissues such as the blood and the heart, liver, spleen, lung, kidney, stomach, intestines, brain and scalp, after the homogenate; The amount that takes by weighing each tissue homogenate thing 0.1～0.2g is in PA tube, and blood is measured 0.1～0.2mL, adds 0.5～1.0mL red fuming nitric acid (RFNA) respectively and clears up; Placed 12～18 hours, and made to organize fully and clear up, mixing is subsequent use.
5. the boron content in the metric measurement testing sample
Measurement is carried out according to following two steps.
Step 1, the bioassay standard curve.Draw the BPA standard respectively and use liquid 0,0.1,0.2,0.4,0.8,1.0mL in the polypropylene volumetric flask of band scale, add EDTA solution 0.5mL successively, acetate-ammonium acetate damping fluid 1.0mL; Developer 3-methoxyl-azomethine H solution 1.0mL adds water and is settled to 5.0mL, and the pH value is 5.7; Then boron element concentration is respectively 0,0.5,1.0,2.0,4.0 and 5.0 μ g/mL in each solution; Mixing is placed 40min, surveys absorbance at the 400nm place with the 1cm quartz colorimetric utensil, and the drawing standard curve.
Step 2 is measured the boron content in the testing sample, the BPA that absorbs in unit of account quality organization amount.
Get above-mentioned ready digestion solution 0.15mL in the polypropylene volumetric flask of band scale, add EDTA solution 0.5mL successively, acetate-ammonium acetate damping fluid 1.0mL; Developer 3-methoxyl-azomethine H solution 1.0mL adds water and is settled to 5.0mL, and the pH value is 5.7; Mixing is placed; Respectively 5,30,60,120, during 180min, survey absorbance at the 400nm place with the 1cm quartz colorimetric utensil, and calculate boron content according to the typical curve that step 1 is drawn.The gained result carries out statistical analysis, draws the distribution of BPA in the mouse body.The result is as shown in table 1.
The table 1BPA in the normal mouse body bio distribution (μ gB/g, x ± S.D., n=4)
Embodiment 2
Present embodiment is BPA bio distribution experiment in the lotus human glioma nude mouse.Experiment reagent, the experimental apparatus prepared before the experiment, and dispose required solution, the operation of handling biological sample is with embodiment 1.
1. biological sample disposal route
Get 21 of lotus brain glioblastoma cell U87MG nude mices, be divided into 7 groups at random, 3 every group, wherein one group is blank control group; Do not inject BPA, all the other 6 groups of mouse tail vein injection BPA-F parenteral solution 100 μ L (being equivalent to 150mg BPA/kg) are respectively at 5,15,30min; 1,2,3 and the disconnected neck of 6h put to death animal, get tissues such as blood and tumour, the heart, liver, spleen, lung, kidney, stomach, intestines, brain and scalp, after the homogenate; The amount that takes by weighing each tissue homogenate thing 0.1～0.2g is in PA tube, and blood is measured 0.1～0.2mL, adds 0.5～1.0mL red fuming nitric acid (RFNA) respectively and clears up; Placed 18～24 hours, and made to organize fully and clear up, mixing is subsequent use.
2. the boron content in the metric measurement testing sample
Measurement is carried out according to following two steps.
Step 1, the bioassay standard curve.Draw the BPA standard respectively and use liquid 0,0.1,0.2,0.4,0.8,1.0mL in the polypropylene volumetric flask of band scale, add EDTA solution 0.7mL successively, acetate-ammonium acetate damping fluid 1.5mL; Developer 3-methoxyl-azomethine H solution 0.8mL adds water and is settled to 5.0mL, and the pH value is 4; Then boron element concentration is respectively 0,0.5,1.0,2.0,4.0 and 5.0 μ g/mL in each solution; Mixing is placed 30min, surveys absorbance at the 390nm place with the 1cm quartz colorimetric utensil, and the drawing standard curve.
Step 2 is measured the boron content in the testing sample, the BPA that absorbs in unit of account quality organization amount.
Get digestion solution 0.2mL in the polypropylene volumetric flask of band scale, add EDTA solution 0.7mL successively, acetate-ammonium acetate damping fluid 1.5mL; Developer 3-methoxyl-azomethine H solution 0.8mL adds water and is settled to 5.0mL, and the pH value is 4; Then boron element concentration is respectively 0,0.5,1.0,2.0,4.0 and 5.0 μ g/mL in each solution; Mixing is placed 30min, surveys absorbance at the 390nm place with the 1cm quartz colorimetric utensil, and calculates boron content according to the typical curve that step 1 is drawn.The gained result carries out statistical analysis, draws the distribution results of BPA in the mouse body.Result such as table 2.
The table 2BPA in lotus glioma nude mouse bio distribution (μ gB/g, x ± S.D., n=3)
Embodiment 3
Present embodiment is BPA bio distribution experiment in the normal mouse body.The step of the boron content before the experiment in preliminary work and the metric measurement testing sample is with embodiment 1.Difference is that the pH value of solution value is 6.5, and the developer addition is 1.6mL, and the reaction time is 120min, and maximum detection wavelength is 410nm.
Discover, linear good during boron concentration 0～5.0 μ g/mL.Concrete technical scheme of the present invention is with generally, and finally measuring liquid long-pending is the technical scheme of example recommendation for 5mL.During practical application, can amass according to the final liquid of measuring of actual conditions adjustment, other reagent dosage also should corresponding adjustment.
Obviously those skilled in the art can carry out various modifications and modification and not break away from the spirit and scope of the present invention the present invention.Like this, if of the present invention these revise and modification belongs in the scope of its equivalent technologies of claim of the present invention, then the present invention also is intended to comprise these modifications and modification.
Claims (1)
1. the method for boron content in the BNCT medicine in the spectrophotometry biological sample; Comprise that biological sample is cleared up, standard curve determination, biological sample measure flow process; It is characterized in that: used developer is 3-methoxyl-first imido H; Determined maximum detection wavelength is 390～410nm, finally measures liquid pH value and is 4-6.5;
It is to get 0.1～0.2mL blood sample that described biological sample is cleared up, or its hetero-organization of 0.1～0.2g and process the sample of homogenate, places 12～24 hours after adding 0.5～1.0mL red fuming nitric acid (RFNA), subsequent use as the biological sample digestion solution;
Described standard curve determination is to draw 0～1.0mL BPA standard respectively to use liquid, adds EDTA solution 0.5～0.7mL successively, acetate-ammonium acetate damping fluid 1～1.5mL; Developer 3-methoxyl-azomethine H solution 0.8～1.6mL; Add water and be settled to 5.0mL, the pH value is 4.0～6.5, and mixing is placed 30～120min; Survey absorbance, drawing standard curve at 390～410nm place;
It is to get biological sample digestion solution 0.1～0.2mL that described biological sample is measured, and adds EDTA solution 0.5～0.7mL successively, acetate-ammonium acetate damping fluid 1～1.5mL; Developer 3-methoxyl-azomethine H solution 0.8～1.6mL; Add water and be settled to 5.0mL, the pH value is 4.0～6.5, and mixing is placed 30～120min; Survey absorbance at 390～410nm place, calculate boron content according to typical curve.
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| CN102507465A (en) * | 2011-10-17 | 2012-06-20 | 攀钢集团江油长城特殊钢有限公司 | Novel turmeric direct photometry for determining boron content in steel |
| CN103185715A (en) * | 2011-12-30 | 2013-07-03 | 北京有色金属研究总院 | Analytical method of ferroporphyrin in ore biological leaching liquid |
| CN103604801A (en) * | 2013-10-29 | 2014-02-26 | 中国科学院东北地理与农业生态研究所 | Method for measuring boron content of low-grade boron ore by inductively coupled plasma atomic emission spectrometer |
| CN104297234A (en) * | 2014-10-11 | 2015-01-21 | 北京华益精点生物技术有限公司 | Preparation methods of color developing agent and test paper for testing boric acid and borax |
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