CN104181119A - Anti-interference detection method for glutathione - Google Patents
Anti-interference detection method for glutathione Download PDFInfo
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- CN104181119A CN104181119A CN201410378788.XA CN201410378788A CN104181119A CN 104181119 A CN104181119 A CN 104181119A CN 201410378788 A CN201410378788 A CN 201410378788A CN 104181119 A CN104181119 A CN 104181119A
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Abstract
The invention relates to an anti-interference detection method for glutathione. At present, glutathione is mainly synthesized by biological method, cysteine usually remains in products, and sulfydryl properties are similar and cause interference in detection of glutathione, so that an anti-interference detection technique needs to be invented. According to the anti-interference detection method for glutathione, an interference factor and a compensation factor are introduced to quantify and compensate the influence of the interference on a glutathione detection result, correct a detection error, eliminate the interference of cysteine without separating cysteine, and detect glutathione and quantify cysteine at the same time. The invention fills up the technical blank of detecting glutathione in an interference-containing sample with a spectrophotometric method, realizes a novel spectrophotometric method anti-interference detection, effectively simplifies the glutathione detection method, and is an anti-interference detection method with application values.
Description
technical field
The present invention relates to a kind of glutathione anti interference detection method, can be applicable to medicine production, scientific research detection field.
background technology
At present, the production of glutathione (GSH) mainly relies on biological synthesis process, be that halfcystine (Cys), glutamic acid, glycocoll three seed amino acids form through glutathione synthetases catalysis, so often there is halfcystine in the sample that direct sample is analyzed simultaneously.The detection method of GSH is numerous, but for contain the sample of GSH, Cys simultaneously, because sulfydryl character in two components approaches, therefore can be by capillary column separation except liquid phase chromatography while detecting not Interference Detection, it can misidentification halfcystine be glutathione that great majority be take the method that sulfydryl reaction is principle, cause testing result higher, the accuracy of impact analysis.
Ultraviolet spectrophotometry is widely used in analyzing and testing field, technology maturation, and relatively simple to operate, finding speed is fast, instrument low price, sample preparation are easy.Lambert-Beer's law principle is commonly used to determine the linear relationship of concentration and absorbance in quantitative test.Along with the raising of ultraviolet spectrophotometer instrument performance, promoted the application of derivative spectrophotometry in detection of complex composition.At present to utilize interfering material to absorb in certain wavelength coverage stable for derivative spectrophotometry, and derivative approaches 0 and gets rid of interference, thereby detects the larger composition of derivative.The method is considered not comprehensive on the impact of derivative for interfering material concentration change, often can only qualitative eliminating disturb, and can not disturb the impact on testing result by accurate quantitative analysis, and can not detect the content of interference.
summary of the invention
In order to get rid of halfcystine, glutathione is detected to the interference bringing, the present invention uses anti-interference compensation, and to testing result correction, the method, without separated halfcystine, not only detects glutathione content exactly, and can detect cysteine content by simultaneous quantitative.
The technical solution adopted for the present invention to solve the technical problems is: the material generating under alkali condition with sulfydryl and alloxan is absorbed as basis at ultraviolet-visible pectrophotometer 320nm wavelength place, set up the typical curve of concentration-absorbance, by interference test, quantitatively disturb impact the compensation on detecting, thereby the glutathione detection error that judgement unknown sample disturbed condition compensate for disturbances are brought, the while can quantitatively be detected the content of halfcystine.
In conjunction with Fig. 3, Fig. 4 and Fig. 5 of Fig. 1 and embodiment, detection method step of the present invention is as follows:
1. production standard curve: preparation standard sample concentration is the GSH of 0.05 ~ 0.25 mmol/L, respectively gets 1 mL, adds respectively 1 mL 0.05 ~ 0.50 mol/L alloxan aqueous solution, the NaOH-Na that 2 mL 0.20 ~ 0.50 mol/L pH are 10.0 ~ 10.8
2hPO
4damping fluid, 30 ~ 60 ℃ of water-bath 10 ~ 20 min in super constant temperature trough, are down to room temperature and detect after water-bath completes.After replacing GSH to add alloxan, damping fluid with water, make blank sample, examination criteria sample, at the 320 absorbance A bs320 of nm place, is made the typical curve (Fig. 3) of GSH concentration-absorbance;
2. interference compensation design:
(1) interference test: the compound sample of preparation variable concentrations scaled interference is GSH concentration 0.05 ~ 0.25 mmol/L, and Cys concentration is respectively 0-8 times of GSH concentration, disturbs ratio between 1:0-1:8.Under the condition of step 1, compound sample replaces standard sample to detect respectively its absorbance A bs320 of 320 nm place and derivative K320;
(2) introduce coefficient: the impact of interference shows as the variation of absorbance and derivative, therefore definition " interference coefficient " α=K320/Abs320, represents the interference level size to testing result, makes interference coefficient table (Fig. 4).If only use absorbance and typical curve equation (Fig. 3) can calculate the recovery of each compound sample of interference test, represent the result that disturbing effect GSH detects, as higher in 102.7% expression 2.7%, therefore define this value for " penalty coefficient " β, result can be revised to testing result divided by β, make penalty coefficient table (Fig. 5);
3. sample detection flow process: with reference to Fig. 1, specific as follows
(1) unknown sample detects by step 1, can obtain two numerical value, i.e. absorbance A bs320 and derivative K320;
(2) calculate interference coefficient α=K320/Abs320, by Abs320 and typical curve equation (Fig. 3), can calculate GSH intermediate concentration C
1;
(3) α and C
1substitution interference coefficient table, is close to principle with numerical value, determines and disturbs ratio beta; β and C
1substitution penalty coefficient table, determines penalty coefficient γ;
(4) calculate GSH concentration C
gSH=C
1/ γ, Cys concentration C
cys=C
gSH/ β, so far flow process finishes.
The invention has the beneficial effects as follows: owing to being based on the result of known disturbances test and having water-bath temperature control, thereby favorable reproducibility, can quantitatively disturb the impact of testing result and compensation testing result, be able to the in the situation that of separated halfcystine not, get rid of and disturb, and detect glutathione, cysteine content simultaneously, reach the object of anti-interference and fast detecting.The present invention has filled up the technological gap of spectrophotometry containing disturbed specimen GSH-PX activity, introduce interference coefficient and penalty coefficient with quantitatively and the impact of the interference of compensation variable concentrations ratio on testing result, realize a kind of anti-interference detection of new spectrophotometric method, effectively simplify glutathione detection method, cost is low, accuracy is high, and the detection for polycomponent compound sample simultaneously has certain reference.
accompanying drawing explanation
Fig. 1 is overhaul flow chart of the present invention;
Fig. 2 is the overhaul flow chart of the embodiment of the present invention;
Fig. 3 is the canonical plotting of the embodiment of the present invention;
Fig. 4 is the interference coefficient table of the embodiment of the present invention;
Fig. 5 is the penalty coefficient table of the embodiment of the present invention;
Special symbol explanation in Fig. 1 and Fig. 2: 320nm place first order derivative (K), 320nm place absorbance (Abs), interference coefficient (α), GSH intermediate concentration (C
1), interference ratio (β), penalty coefficient (γ), glutathione concentrations (C
gSH), semicystinol concentration (C
cys).
embodiment
Embodiment operation steps:
1. production standard curve: preparation standard sample concentration is 0.05,0.10,0.15,0.20, and the GSH of 0.25 mmol/L, respectively gets 1 mL, adds respectively 1 mL 0.10 mol/L alloxan aqueous solution, the NaOH-Na that 2 mL 0.40 mol/L pH are 10.50 ± 0.02
2hPO
4damping fluid, 50 ℃ of water-bath 10 min in super constant temperature trough, are down to room temperature and detect after water-bath completes.After replacing GSH to add alloxan, damping fluid with water, do to detect blank sample, by ultraviolet-visible pectrophotometer examination criteria sample 320 nm place absorbances, make the typical curve (Fig. 3) of GSH concentration-absorbance;
2. interference compensation design:
(1) interference test: the compound sample of preparation variable concentrations scaled interference is GSH concentration 0.05,0.10,0.15,0.20,0.25 mmol/L, and Cys concentration is respectively 0.5-5 times of GSH concentration, disturbs ratio beta between 1:0.5-1:5.Under the condition of step 1, compound sample replaces standard sample to detect respectively its absorbance A bs320 of 320 nm place and derivative K320;
(2) introduce coefficient: the impact of interference shows as the variation of absorbance and derivative, therefore definition " interference coefficient " α=K320/Abs320, represents the interference level size to testing result, makes interference coefficient table (Fig. 4).If only can calculate the recovery of each compound sample of interference test with absorbance and typical curve equation, represent the result that disturbing effect GSH detects, as higher in 102.7% expression 2.7%, therefore define this value for " penalty coefficient " γ, result can be revised to testing result divided by penalty coefficient, make penalty coefficient table (Fig. 5);
3. sample detection flow process: with reference to Fig. 2, be described as follows
(1) unknown sample detects by step 1, can obtain two numerical value, i.e. absorbance A bs320 and derivative K320;
(2) calculate interference coefficient α=K320/Abs320, by Abs320 and typical curve equation (Fig. 3), can calculate GSH intermediate concentration C
1;
(3) α and C
1substitution interference coefficient table (Fig. 4), is close to principle with numerical value, determine to disturb ratio beta, somely disturbs excessively, and this sample of drawing a conclusion disturbs and surpasses 5 times, cannot detect, and stops carrying out flow process; If in 5 times, β and C
1substitution penalty coefficient table, determines penalty coefficient γ;
(4) calculate GSH concentration C
gSH=C
1/ γ, Cys concentration C
cys=C
gSH/ β, so far flow process finishes.
Claims (1)
1. an anti interference detection method for glutathione, is characterized in that:
A, production standard curve: preparation standard sample concentration is the GSH of 0.05 ~ 0.25 mmol/L, respectively gets 1 mL, adds respectively 1 mL 0.05 ~ 0.50 mol/L alloxan aqueous solution, the NaOH-Na that 2 mL 0.20 ~ 0.50 mol/L pH are 10.0 ~ 10.8
2hPO
4damping fluid, 30 ~ 60 ℃ of water-bath 10 ~ 20 min in super constant temperature trough, are down to room temperature and detect after water-bath completes; After replacing GSH to add alloxan, damping fluid with water, make blank sample, examination criteria sample, at the 320 absorbance A bs320 of nm place, is made the typical curve of GSH concentration-absorbance;
B, interference compensation design:
(1) interference test: the compound sample of preparation variable concentrations scaled interference is GSH concentration 0.05 ~ 0.25 mmol/L, and Cys concentration is respectively 0-8 times of GSH concentration, disturbs ratio between 1:0-1:8;
Under the condition of step 1, compound sample replaces standard sample to detect respectively its absorbance A bs320 of 320 nm place and derivative K320;
(2) introduce coefficient: the impact of interference shows as the variation of absorbance and derivative, therefore definition " interference coefficient " α=K320/Abs320, represents the interference level size to testing result, makes interference coefficient table; If only can calculate the recovery of each compound sample of interference test with absorbance and typical curve equation, represent the result that disturbing effect GSH detects, as higher in 102.7% expression 2.7%, therefore define this value for " penalty coefficient " β, result can be revised to testing result divided by β, make penalty coefficient table;
C, sample detection flow process are as follows
(1) unknown sample detects by step 1, can obtain two numerical value, i.e. absorbance A bs320 and derivative K320;
(2) calculate interference coefficient α=K320/Abs320, by Abs320 and typical curve equation, can calculate GSH intermediate concentration C
1;
(3) α and C
1substitution interference coefficient table, is close to principle with numerical value, determines and disturbs ratio beta; β and C
1substitution penalty coefficient table, determines penalty coefficient γ;
(4) calculate GSH concentration C
gSH=C
1/ γ, Cys concentration C
cys=C
gSH/ β, so far flow process finishes.
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| CN201410378788.XA CN104181119B (en) | 2014-08-04 | A kind of anti interference detection method of glutathion |
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| CN201410378788.XA CN104181119B (en) | 2014-08-04 | A kind of anti interference detection method of glutathion |
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| CN104181119B CN104181119B (en) | 2016-11-30 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107703072A (en) * | 2017-09-21 | 2018-02-16 | 长春黄金研究院 | The correction assay method of total cyanide in a kind of thiocyanate water quality |
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Patent Citations (4)
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| US5434666A (en) * | 1994-02-25 | 1995-07-18 | Northern Illinois University | Acousto-optic spectral manipulator |
| CN1435692A (en) * | 2002-01-31 | 2003-08-13 | 李勤 | Fluorescent analysis reagent for testing blood homo cysteine, method for preparing same and use thereof |
| CN101220338A (en) * | 2007-12-24 | 2008-07-16 | 南京华锦生物制品有限公司 | Glutathione produced bacterial strain and construction method thereof |
| CN103013493A (en) * | 2012-11-07 | 2013-04-03 | 中国科学院理化技术研究所 | Fluorescence chemical sensor capable of selectively detecting biological sulfhydryl compound, preparation method and application |
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| MOHSEN KEYVANFARD等: "Multiwall carbon nanotube paste electrode with 3,4‐dihydroxy‐cinnamic acid as mediator for the determination of utathione in pharmaceutical and urine samples", 《CHINESE JOURNAL OF CATALYSIS》, no. 10, 31 December 2013 (2013-12-31), pages 1883 - 1889 * |
| 刘娟等: "发酵液中还原型谷胱甘肽三种测定方法的改进及其比较", 《北京化工大学学报》, vol. 31, no. 3, 31 December 2004 (2004-12-31), pages 35 - 38 * |
| 尹翠娟等: "紫外分光光度法测定谷肤甘肤片的含量", 《中国药品标准》, 31 December 2004 (2004-12-31), pages 50 - 51 * |
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| 赵少欣等: "还原型谷胱甘肽简便测定法", 《分析检测》, 31 December 2007 (2007-12-31), pages 219 - 220 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107703072A (en) * | 2017-09-21 | 2018-02-16 | 长春黄金研究院 | The correction assay method of total cyanide in a kind of thiocyanate water quality |
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