CN101713740A - Method for spectrophotometry measuring boron content in BNCT medicament in biological sample - Google Patents
Method for spectrophotometry measuring boron content in BNCT medicament in biological sample Download PDFInfo
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- 229910052796 boron Inorganic materials 0.000 title claims abstract description 39
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- 238000002798 spectrophotometry method Methods 0.000 title claims abstract description 11
- 238000005259 measurement Methods 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 14
- 239000000523 sample Substances 0.000 claims description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 10
- 238000002835 absorbance Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- OZZOVSQSDIWNIP-UHFFFAOYSA-N acetic acid;azane Chemical compound [NH4+].[NH4+].CC([O-])=O.CC([O-])=O OZZOVSQSDIWNIP-UHFFFAOYSA-N 0.000 claims description 9
- 238000013016 damping Methods 0.000 claims description 8
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 4
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- 210000001519 tissue Anatomy 0.000 description 4
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- 238000005516 engineering process Methods 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
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- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 2
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
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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
- 150000004056 anthraquinones Chemical class 0.000 description 1
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- 208000024055 brain glioblastoma Diseases 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- 229910052737 gold Inorganic materials 0.000 description 1
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- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 230000001590 oxidative effect Effects 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 analysis method has the advantages of rapidness, simplicity, convenience, specificity, sensitivity and the like, and meets the requirements on the accuracy and precision of the measurement of the boron content in the biological sample.
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 to contain
10The B medicine is introduced in the body by oral or injecting method, and makes it optionally to accumulate in the cancer cell, uses the neutron irradiation diseased region then, makes
10B takes place
10B (n, α)
7The Li nuclear reaction, utilize consequent α particle and
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, spectrophotometric method, 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 plasma mass method (as 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 that is most widely used 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 actual applications.The employed instrument and equipment of spectrophotometric method 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 spectrophotometric method 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 as follows.
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 make the sample of homogenate, places 12～24 hours after adding 0.5～1.0mL red fuming nitric acid (RFNA), standby.
Described standard curve determination is to draw 0～1.0mLBPA standard respectively to use liquid, add 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, add 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, selects to try one's best little volume in the scope of color stability.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 further elaborated.
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 Shanghai holy chemical reagent of 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, regulating pH with HCl 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, being adjusted to pH with pH meter 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, every group 4, wherein one group is blank 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 the blood and the heart, liver, spleen, lung, kidney, stomach, intestines, tissue such as brain and scalp, after the homogenate, the amount that takes by weighing each tissue homogenate thing 0.1～0.2g is in polypropylene tube, and blood is measured 0.1～0.2mL, adding 0.5～1.0mL red fuming nitric acid (RFNA) respectively clears up, placed 12～18 hours, and made to organize fully and clear up, mixing is standby.
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.Drawing the BPA standard respectively uses 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, handle the operation of biological sample 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, every group 3, wherein one group is blank 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 blood and tumour, the heart, liver, spleen, lung, kidney, stomach, intestines, tissue such as brain and scalp, after the homogenate, the amount that takes by weighing each tissue homogenate thing 0.1～0.2g is in polypropylene tube, and blood is measured 0.1～0.2mL, adding 0.5～1.0mL red fuming nitric acid (RFNA) respectively clears up, placed 18～24 hours, and made to organize fully and clear up, mixing is standby.
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.Drawing the BPA standard respectively uses 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, it is long-pending finally to measure liquid according to the actual conditions adjustment, and other reagent dosage also should corresponding adjustment.
Obviously those skilled in the art can carry out various modifications and variations 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 (3)
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, and determined maximum detection wavelength is 390～410nm; Final measurement liquid pH value is 4-6.5.
2. the method for boron content in the BNCT medicine in the spectrophotometry biological sample according to claim 1, it is characterized in that: 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 make the sample of homogenate, place 12～24 hours after adding 0.5～1.0mL red fuming nitric acid (RFNA), standby.
3. the method for boron content in the BNCT medicine in the spectrophotometry biological sample according to claim 1, it is characterized in that: 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 make the sample of homogenate, place 12～24 hours after adding 0.5～1.0mL red fuming nitric acid (RFNA), standby.
Described standard curve determination is to draw 0～1.0mLBPA standard respectively to use liquid, add 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, add 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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3768442B2 (en) * | 2001-12-28 | 2006-04-19 | オルガノ株式会社 | Analytical sample preparation method and element quantification method |
| SE528407C2 (en) * | 2005-08-19 | 2006-11-07 | Hammercap Ab | Boron compounds useful at BNCT |
| CN100487426C (en) * | 2005-10-31 | 2009-05-13 | 白莉 | Boron determination solution and colorimetric determination tube therefor |
| CN2840019Y (en) * | 2005-10-31 | 2006-11-22 | 白莉 | Boron colorimetric estimation cylinder |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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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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| CN101713740B (en) | 2012-05-02 |
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