CN104280383A - Rapid detection method for nitrites in blood and urine - Google Patents
Rapid detection method for nitrites in blood and urine Download PDFInfo
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- CN104280383A CN104280383A CN201310274145.6A CN201310274145A CN104280383A CN 104280383 A CN104280383 A CN 104280383A CN 201310274145 A CN201310274145 A CN 201310274145A CN 104280383 A CN104280383 A CN 104280383A
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Abstract
The invention discloses a rapid detection method for nitrites in blood and urine. The method comprises: utilizing a diazotization coupling reaction of nitrites and Griess reagent to form an azo compound, performing cloud point extraction, precipitating the colored azo compound at the bottom of a centrifuge tube, comparing the red color at the tube bottom with standard color gradation, and determining the content of the nitrite according to the color depth. Because the enriching multiple of cloud point extraction is large and blood and urine are subjected to decoloring and protein-removal processing, the interference is substantially eliminated, and the detection limit of nitrites in the system can reach 0.01 mu g/mL. The method has the characteristics of simpleness, rapidness, high sensitivity, on-site detection and the like.
Description
Technical field
The invention belongs to drug disposition detection technique field, be specifically related to the method for quick of a kind of blood and urine nitrite.
Background technology
The biochemistry of nitrogen monoxide (NO), physiology, pathological study extensively launch at world's biology and medical circle.As metastable NO metabolic product, nitrite (NO
2 -) at the nearest focus becoming world medical circle for 10 years, nitrite is inorganic salts common in diet and potable water.It is generally acknowledged that such material of excessive absorption may cause methemoglobinemia, and be transformed into the nitrosamine of carcinogenicity in vivo.By measuring the NO in urine, blood
2 -content come NO content in antimer and the important indicator as the multinomial physical function of reaction.
It is national food safety standard GB 5009.33-2010 that current nitrite detects the most general method, adopt the nitrite in sulfate by ion chromatography varieties of food items and nitrate in this standard first method, comprise various types of samples such as fruit, vegetables, fish, meat, milk.Meanwhile, NO
2 -mensuration also have fluorimetry, high performance liquid chromatography, visible spectrophotometry etc.NO in blood or urine
2 -detection usually use Griess detection kit, but NO in blood or urine
2 -content is low, and matrix is complicated, and usually interference experiment result, is difficult to reach detection demand.Utilize high performance liquid chromatography (HPLC) to detect nitrate and nitrite in blood urine and tissue before research group and carry out more deep research, achieve good effect, apply for patent of invention (201310129291.X), further research finds, by nitrite and Griess reagent reacting, carry out being separated through cloud point extraction (CPE), enrichment, direct visual colorimetric determination, highly sensitive, the simple and quick detection of blood urine nitrite can be carried out, this technology does not need instrument and equipment, can Site Detection, there is more obvious advantage.Set up the nitrous nitrification method in a kind of highly sensitive, high specificity, simple and quick urine, blood, significant to the Diagnosis and Treat of the many diseases relating to nitrogen monoxide signal factor in clinical medicine.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide a kind of simple and quick, sensitivity, can Site Detection, qualitative, the semiquantitative method of urine nitrite.
Object of the present invention is achieved by the following technical programs.
Except as otherwise noted, percentage of the present invention is percent by weight.
A detection method for blood and urine nitrite, comprises the following steps:
(1) nitrite standard color range makes: get the NO that concentration is 10.0 μ g/mL
2 -standard solution 0.01,0.05,0.10,0.30,0.70,1.0mL in graduated centrifuge tube, with distilled water diluting to 5mL, add GriessA reagent 250 μ L, mix; Add GriessB reagent 250 μ L after 1min, mix, add non-ionic surfactant 0.5mL, salt 0.1 ~ 0.5g, vortex mixed 1min, centrifugal phase-splitting, at the bottom of centrifuge tube, occur red droplet color range solution from shallow to deep;
(2) sample detection:
The detection of urine: get 10mL urine, zinc sulfate 0.4 ~ 1.0g, adds decolorant 0.5 ~ 1.0g, mixes, centrifugal decolouring, Filter paper filtering removing decolorant; Get the urine after 5mL decolouring, add GriessA reagent 250 μ L, mix; Add GriessB reagent 250 μ L after 1min, mix, after placing 5min, add non-ionic surfactant 0.5mL, salt 0.1 ~ 0.5g, vortex mixed 1min, centrifugal phase-splitting, compares the color of the droplet formed at the bottom of pipe with step (1) standard color range, judges its content;
The detection of blood: get 5 mL new bloods, put into hydro-extractor centrifugal, get supernatant, add 0.1mol/L sodium hydroxide solution 0.2mL and zinc sulfate 0.2g, mix, add acetonitrile 1mL, centrifuging, get supernatant 2mL and be settled to 5mL with distilled water in 10mL centrifuge tube, add GriessA reagent 250 μ L, mix; Add GriessB reagent 250 μ L after 1min, mix, place 5min, add non-ionic surfactant 0.5mL, salt 0.1 ~ 0.5g, vortex mixed 1min, centrifugal phase-splitting, compares the color of the droplet formed at the bottom of pipe with step (1) standard color range, judges its content;
Wherein, the GriessA reagent described in step (1) (2) is that 50mg sulfanilic acid is dissolved in 10% acetic acid 1.5mL and deionized water 3mL; GriessB reagent is that 4mg N-1-naphthodiamide hydrochloride is dissolved in 10% acetic acid 4mL;
Described decolorant is: the one in activated charcoal, molecular sieve, magnesium silicate, calcium oxide, talcum powder;
Described non-ionic surfactant comprises the one in Triton X-114, Triton X-110, NPE class NP-7 and NP-9, Tergitol TMN-6;
Described salt comprises the one in sodium chloride, sodium sulphate, ammonium sulfate, ammonium chloride.
Described centrifugal condition is centrifugation time 5 ~ 15min, centrifugation rate 3000 ~ 10000r/min.
relative to prior art, the present invention has following remarkable advantage:
1, NO is passed through
2 -the azo compound formed with Griess reagent, one is have color, and two is change NO
2 -the polarity of itself, utilizes cloud point extraction to serve the effect of separation and high enrichment, makes NO
2 -the sensitivity detected improves greatly, reaches 0.01 μ g/mL with visual colorimetric determination sensitivity.
2, the Cloud Points of Nonionic Surfactants adopted is low, substantially can extract at normal temperatures, and operating conditions is gentle.
3, utilize decolorant, de-protein agent carry out urine, the removing of the color of blood, albumen, disturb when eliminating colorimetric, substantially increase the accuracy of detection sensitivity and visual colorimetric determination.
Embodiment
Below in conjunction with embodiment, the present invention is further described, but protection scope of the present invention is not limited to this.
embodiment 1
Utilize the content of human body urine nitrite of the present invention.
1, nitrite standard color range makes: get the NO that concentration is 10 μ g/mL
2 -standard solution 0.01,0.05,0.10,0.30,0.70,1.0mL mL in graduated centrifuge tube, with distilled water diluting to 5mL, add GriessA reagent 250 μ L, mix; Add GriessB reagent 250 μ L after 1min, mix, after placing 5min, add non-ionic surfactant Triton X-114 0.5mL, ammonium sulfate 0.2g, vortex mixed 1min,, at the bottom of centrifuge tube, there is red droplet color range solution from shallow to deep in centrifugal phase-splitting;
2, sample preparation and measurement result
Get 10mL urine in 10mL centrifuge tube, add 1.0g activated charcoal, mix, the centrifugal 10min of 4000r/min, Filter paper filtering removing activated charcoal.Get the urine after 5.0mL decolouring in 10mL centrifuge tube, add GriessA reagent 250 μ L, mix.GriessB reagent 250 μ L is added after 1min, mix, after placing 5min, add non-ionic surfactant Triton X-114 0.5mL, ammonium sulfate 0.2g, vortex mixed 1min, centrifugal phase-splitting, drop at the bottom of centrifuge tube is red between content of nitrite 0.01 μ g/mL and 0.05 μ g/mL.
embodiment 2
Utilize the content of human body determinating nitrite in blood of the present invention.
1. make the standard color range of nitrite by embodiment 1, non-ionic surfactant is Triton X-100, and salt is sodium chloride 0.5g.
2. sample preparation and measurement result
Get 5 mL new bloods, the centrifugal 15min of 4000r/min.Get supernatant in centrifuge tube, add 0.1mol/L sodium hydroxide solution 0.2mL and zinc sulfate 0.2g, mix, add acetonitrile 1mL, centrifuging, get supernatant 2mL and be settled to 5mL with distilled water in 10mL centrifuge tube, add GriessA reagent 250 μ L, mix, after 1min, add GriesssB reagent 250 μ L, mix, add non-ionic surfactant Triton X-100 0.5mL, sodium chloride 0.5g, vortex mixed 1min, centrifugal phase-splitting, drop at the bottom of centrifuge tube is red between content of nitrite 0.30 μ g/mL and 0.70 μ g/mL.
embodiment 3
Utilize the content of human body determinating nitrite in blood of the present invention.
1. make the standard color range of nitrite by embodiment 1, non-ionic surfactant is NP-7, and salt is ammonium chloride 0.3g.
2. sample preparation and measurement result
Get 5mL new blood in centrifuge tube, the centrifugal 8min of 6000r/min, get supernatant in centrifuge tube, add 0.1mol/L sodium hydroxide solution 0.2mL and zinc sulfate 0.2g, mix, add acetonitrile 1mL, centrifuging, get supernatant 2mL and be settled to 5mL with distilled water in 10mL centrifuge tube, add GriessA reagent 250 μ L, mix.Add GriessB reagent 250 μ L after 1min, mix, after placing 5min, add non-ionic surfactant NP-7 0.5mL, ammonium chloride 0.3g, vortex mixed 1min, centrifugal phase-splitting, at the bottom of centrifuge tube, drop is colourless, and nitrite does not detect.
embodiment 4
Utilize the content of human body urine nitrite of the present invention.
1. make the standard color range of nitrite by embodiment 1, non-ionic surfactant is TMN-6, and salt is sodium sulphate 0.5g.
2. sample preparation and measurement result
2, sample preparation and measurement result
Get 10mL urine in 10mL centrifuge tube, add molecular sieve 1.0g, mix, the centrifugal 10min of 5000r/min, Filter paper filtering removing molecular sieve.Get the urine after 5.0mL decolouring in 10mL centrifuge tube, add GriessA reagent 250 μ L, mix.Add GriessB reagent 250 μ L after 1min, mix, after placing 5min, add non-ionic surfactant TMN-6 0.5mL, sodium sulphate 0.5g, vortex mixed 1min, centrifugal phase-splitting, drop at the bottom of centrifuge tube is red between content of nitrite 0.70 μ g/mL and 1.0 μ g/mL.
Claims (6)
1. blood and a urine nitrite method for quick, comprises the following steps:
(1) nitrite standard color range solution is prepared: get the NO that concentration is 10.0 μ g/mL
2 -standard solution 0.01,0.05,0.10,0.30,0.70,1.0mL in graduated centrifuge tube, with distilled water diluting to 5mL, add GriessA reagent 250 μ L, mix; Add GriessB reagent 250 μ L after 1min, mix, add non-ionic surfactant 0.5mL, salt 0.1 ~ 0.5g, vortex mixed 1min, centrifugal phase-splitting, at the bottom of centrifuge tube, occur red droplet color range solution from shallow to deep.
(2) nitrite in urine is detected: get 10mL urine, zinc sulfate 0.4 ~ 1.0g, add decolorant 0.5 ~ 1.0g, mix, centrifugal decolouring, Filter paper filtering removing decolorant; Get the urine after 5mL decolouring, add GriessA reagent 250 μ L, mix; Add GriessB reagent 250 μ L after 1min, mix, after placing 5min, add non-ionic surfactant 0.5mL, salt 0.1 ~ 0.5g, vortex mixed 1min, centrifugal phase-splitting, compares the color of the droplet formed at the bottom of pipe with step (1) standard color range, judges its content.
(3) nitrite in blood is detected: get 5mL new blood, put into hydro-extractor centrifugal, get supernatant, add 0.1mol/L sodium hydroxide solution 0.2mL and zinc sulfate 0.2g, mix, add acetonitrile 1mL, centrifuging, get supernatant 2mL and be settled to 5mL with distilled water in 10mL centrifuge tube, add GriessA reagent 250 μ L, mix; Add GriessB reagent 250 μ L after 1min, mix, place 5min, add non-ionic surfactant 0.5mL, salt 0.1 ~ 0.5g, vortex mixed 1min, centrifugal phase-splitting, compares the color of the droplet formed at the bottom of pipe with step (1) standard color range, judges its content.
2. detection method according to claim 1, is characterized in that: described GriessA reagent is 50mg sulfanilic acid, and is dissolved in 1.5mL10% acetic acid and 3mL deionized water; GriessB reagent is 4mg N-1-naphthodiamide hydrochloride, and is dissolved in 4mL10% acetic acid.
3. detection method according to claim 1, is characterized in that: described non-ionic surfactant is the one in Triton X-114, Triton X-110, NPE class NP-7 and NP-9, Tergitol TMN-6;
4. detection method according to claim 1, is characterized in that: described salt is the one in sodium chloride, sodium sulphate, ammonium sulfate, ammonium chloride;
5. detection method according to claim 1, is characterized in that: decolorant described in the detection of urine is the one in activated charcoal, molecular sieve, magnesium silicate, calcium oxide, talcum powder;
6. detection method according to claim 1, is characterized in that: the centrifugal condition described in sample detection is centrifugation time 10 ~ 30min, centrifugation rate 3000 ~ 10000r/min.
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| CN104407097A (en) * | 2014-11-28 | 2015-03-11 | 苏州东辰林达检测技术有限公司 | Detection reagent and method of nitrite |
| CN106323894A (en) * | 2016-08-01 | 2017-01-11 | 广西中烟工业有限责任公司 | Method for determining nitrite in tobaccos through silver nanoparticle auxiliary cloud point extraction |
| CN106596540A (en) * | 2016-12-20 | 2017-04-26 | 江苏食品药品职业技术学院 | Chromogenic agent and watered cow milk determination method |
| CN109142225A (en) * | 2018-10-22 | 2019-01-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of nitrite in sample |
| CN109387507A (en) * | 2018-09-27 | 2019-02-26 | 长沙都正医学检验有限责任公司 | Trimethylamine oxide rapid detection method and detection kit in a kind of urine |
| CN111665241A (en) * | 2020-06-12 | 2020-09-15 | 苏州良辰生物仪器试剂有限公司 | Tyrosine detection test strip and preparation method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104407097A (en) * | 2014-11-28 | 2015-03-11 | 苏州东辰林达检测技术有限公司 | Detection reagent and method of nitrite |
| CN106323894A (en) * | 2016-08-01 | 2017-01-11 | 广西中烟工业有限责任公司 | Method for determining nitrite in tobaccos through silver nanoparticle auxiliary cloud point extraction |
| CN106596540A (en) * | 2016-12-20 | 2017-04-26 | 江苏食品药品职业技术学院 | Chromogenic agent and watered cow milk determination method |
| CN109387507A (en) * | 2018-09-27 | 2019-02-26 | 长沙都正医学检验有限责任公司 | Trimethylamine oxide rapid detection method and detection kit in a kind of urine |
| CN109142225A (en) * | 2018-10-22 | 2019-01-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of nitrite in sample |
| CN111665241A (en) * | 2020-06-12 | 2020-09-15 | 苏州良辰生物仪器试剂有限公司 | Tyrosine detection test strip and preparation method and application thereof |
| CN111665241B (en) * | 2020-06-12 | 2023-03-28 | 苏州良辰生物仪器试剂有限公司 | Tyrosine detection test strip and preparation method and application thereof |
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