CN107907618A - The detection method of homocysteine and cysteine in rat plasma - Google Patents
The detection method of homocysteine and cysteine in rat plasma Download PDFInfo
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- CN107907618A CN107907618A CN201711006838.1A CN201711006838A CN107907618A CN 107907618 A CN107907618 A CN 107907618A CN 201711006838 A CN201711006838 A CN 201711006838A CN 107907618 A CN107907618 A CN 107907618A
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- homocysteine
- cysteine
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- plasma
- detection method
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
-
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
Abstract
The invention discloses the detection method of homocysteine and cysteine in a kind of rat plasma, comprise the following steps：Step 1: the preparation of standard items；Step 2: sample is reduced with sodium borohydride；Step 3: except the protein in plasma sample；Step 4: homocysteine and cysteine in HPLC ECD separation determination plasma samples.Step 5: result calculates.Homocysteine and cysteine detecting method have the advantages that easy step, accuracy in rat plasma provided by the invention and sensitivity is good, it can be achieved that homocysteine and the quick of cysteine detect in sample.
Description
Technical field
The present invention relates to medical chemistry detection field, it is more particularly related to a kind of high half Guang ammonia of rat plasma
The detection method of acid and cysteine.
Background technology
Homocysteine is a kind of amino acid containing sulfydryl, is mainly the following form presence, high half Guang ammonia in vivo
Sour free state, homocysteine bimolecular are gathered into disulphide, and homocysteine is combined with protein, and normal human's blood plasma
In, 70% homocysteine is combined with protein.Homocysteine mostlys come from the methionine absorbed in food, is methionine
With the intermediate product in cysteine metabolic process, itself does not participate in the synthesis of protein.Methionine and homocysteine can
Mutually convert.Homocysteine can be acted on the mesostate glycine betaine of choline, i.e., glycine betaine is high as methyl donor
Cysteine and glycine betaine form methionine under the action of betaine homocysteine methyl transferase.Homocysteine and half
For the content of cystine there are close relationship, cysteine may participate in the synthesis of protein, can also be with glutamic acid, Formation of glycine
Glutathione.Homocysteine is by turning sulfydryl approach, i.e. homocysteine shape under the conditions of serine and cystathionine beta-synthase
Into cystathionie, cystathionie forms cysteine under cystathionine lyase effect.At present, the accumulation of homocysteine is considered as
Cause the independent hazard factor of atherosclerosis.Internal choline, glycine betaine, the shortage of folic acid can cause homocysteine
Accumulation, and the shortage of glycine betaine can cause diabetes, and severe patient is then it is possible that diabetes mellitus encephalopathy.And high half Guang ammonia
The accumulation of acid in vivo is likely to result in cardiovascular damage and apoplexy.Homocysteine may cause human body to produce
Cognitive disorder, it is more serious to cause encephalopathic.Therefore, the quantitative determination to internal homocysteine and cysteine seems all the more
It is important.
At present, have isotope method to the common method of measure of homocysteine and cysteine, but this method it is cumbersome and
With radiocontamination, be not suitable for extensive use.GC-MS can measure many kinds of substance such as homocysteine at the same time, although this method has
The advantages that sensitivity is good, and specificity is good, but instrument and equipment is expensive and cannot promote.HPLC-FLD is also used for measuring high half Guang
Propylhomoserin, since this method needs to be derived with o-phthalaldehyde, and needs to close sulfydryl to derivative influence, the party before derivative
Method operation is excessively cumbersome.HPLC-ECD detection methods are easy to operate, and accuracy is high, can at the same time measure and a variety of contain thiol compound.
The content of the invention
It is an object of the present invention to provide a kind of while detect the side of homocysteine and cysteine in rat plasma
Method.
In order to realize these purposes according to the present invention, there is provided homocysteine and cysteine in a kind of rat plasma
Detection method, comprise the following steps：
Step 1: the preparation of standard items；
Step 2: sample is reduced with sodium borohydride；
Step 3: except the protein in plasma sample；
Step 4: homocysteine and cysteine in HPLC-ECD separation determination plasma samples.
Step 5: result calculates.
Preferably, the preparation specific method in step 1 to standard items is：
With homocysteine, cysteine and vanillic acid are standard items, are configured to respectively with 0.1mol/L perchloric acid solutions
Concentration is the storing solution of 1mg/mL, spare；
Preferably, the specific method of the reduction in step 2 to sample is：
Step a, 1.5mol/L NaoH solution is prepared, appropriate sodium borohydride is weighed and is dissolved in 1.5mol/L NaoH solution,
The concentration for making sodium borohydride is 2mol/L；
Step b, 100 μ L plasma samples are taken, add the sodium borohydride solution of 10 μ L 2mol/L into sample, vortex 1min,
Stand reaction 5min.
Preferably, except the specific method of protein in plasma sample is in step 3：
By sample and perchloric acid 300 μ L 0.4mol/L perchloric acid are added for 1: 3 sample into step 1 by volume
(containing the internal standard, vanillic acid), is vortexed and mixes 1min, 15000 × g, 4 DEG C of centrifugation 15min, take supernatant to be measured for HPLC-ECD.
Preferably, homocysteine and cysteine in HPLC-ECD separation determinations plasma sample in step 4, measure
Condition is as follows：
Detector：Shimadzu ED723 electrochemical detectors；
Chromatographic column：Yi Lite C8 (E2020417,4.6mm × 250mm, 5 μm)；
Mobile phase A：Methanol；
Mobile phase B：30mmol/L sodium acetates, 100 μm of ol/L perfluorooctane sulfonates, pH=5.02,
Isocratic elution, elution program are：A: B=0.1: 99.9；
Column temperature：30℃；
Flow velocity：0.7mL/min；
Detect voltage：900mv；
Sample size：20μL.
Preferably, result calculating is specially in step 5：
The content of homocysteine in sample is calculated by formula (1)；Cysteine in sample is calculated by formula (2) to contain
Amount；
Y=0.8499X+0.0288 (1)
Y=0.5278X-0.0014 (2)
In formula：
Y：The ratio between determinand and the peak area of internal standard substance；
X：The concentration of determinand, unit are micrograms per millilitre.
The present invention includes at least following beneficial effect：
1st, ED723 electrochemical detectors provided by the invention, stability is good, and sensitivity is good.
2nd, the measure of homocysteine and cysteine in rat plasma using the present invention, detection process is simple and convenient,
The quick detection of homocysteine and cysteine in actual sample can be achieved.
Other advantages, target and the feature of the present invention embodies part by following explanation.
Brief description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of homocysteine and cysteine standard product in embodiment of the present invention.Its
In：No. 1 peak is homocysteine, No. 2 peaks are cysteine, No. 3 peaks are vanillic acid.
Fig. 2 is that the high-efficient liquid phase color of homocysteine and cysteine in rat plasma is measured in embodiment of the present invention
Spectrogram.Wherein：No. 1 peak is homocysteine, No. 2 peaks are cysteine, No. 3 peaks are vanillic acid.
Embodiment
With reference to embodiment, the embodiment of the present invention is further described.Following embodiments are used to say
The bright present invention, but it is not limited to the scope of the present invention.
<Embodiment>
1 instrument
High performance liquid chromatograph (Shimadzu)
2 reagents
Water：Ultra-pure water (crosses 0.45 μm of water system miillpore filter).
Methanol：U.S. world HPLC methanol (crosses 0.45 μm of organic system system miillpore filter).
Sodium acetate-octane sulfonate sodium solution：30mmol/L sodium acetates and 100 μm of ol/L perfluorooctane sulfonates are configured to the water of 1L
Solution, pH=5.02 (cross 0.45 μm of water system miillpore filter).
Standard items：Homocysteine (sigma-aldrich), cysteine (Aladdin), vanillic acid (Aladdin).
3 detection methods
(1) preparation of standard items and sample：
1a) the preparation of standard items：
With homocysteine, cysteine and vanillic acid are standard items, are configured to respectively with 0.1mol/L perchloric acid solutions
Concentration be 1mg/mL storing solution, packing, be positioned over -20 DEG C it is spare, face be with 0.1mol/L perchloric acid dilution.
2a) the preparation of sample solution：
Take supernatant to be sub-packed in 1.5mL centrifuge tubes after rat plasma centrifugation, be positioned over -80 DEG C of storages.Take 100 μ L above-mentioned
Supernatant samples, the sodium borohydride solution of 10 μ L 2mol/L are added into sample, vortex 1min, stands reaction 5min.Sample and
Perchloric acid is to add 300 μ L 0.4mol/L perchloric acid (containing the internal standard, 2 μ g/ of vanillic acid in 1: 3 above-mentioned response sample by volume
ML), it is vortexed and mixes 1min, 15000 × g, 4 DEG C of centrifugation 15min, takes supernatant to be measured for HPLC-ECD.
(2) high performance liquid chromatography detection
Chromatographic condition：Detector：Shimadzu electrochemical detector ED723 (GL Sciences Inc., Tokyo, Japan)；
Chromatographic column：Yi Lite C8 (E2020417,4.6mm × 250mm, 5 μm)；Column temperature：30℃；Flow velocity：0.7mL/min；Detect voltage：
900mv；Sample size：20μL；Mobile phase：Methanol is 0.1: 99.9 with sodium acetate-octane sulfonate sodium solution volume ratio.
By standard solution 0.1-4 μ g/mL 6 various concentrations of latitude of formulation standard series, respectively after sample introduction, with
The ratio of determinand and internal standard compound peak area is Y, and linear regression, linear relationship R are carried out by X of the concentration of determinand2≥
0.9991。
Standard items and sample solution are distinguished into sample introduction, according to the chromatography of each component in the qualitative sample of the retention time of standard items
Peak；The content of homocysteine in sample and cysteine is calculated with internal standard method.
4 results calculate
The content of homocysteine in sample is calculated by formula (1)；Cysteine in sample is calculated by formula (2) to contain
Amount；
Y=0.8499X+0.0288 (1)
Y=0.5278X-0.0014 (2)
In formula：
Y：The ratio between determinand and the peak area of internal standard substance；
X：The concentration of determinand, unit are micrograms per millilitre；
5 specificities
Under the measure patent condition for testing homocysteine and cysteine, in the chromatogram of blank solvent, to be measured
At the retention time of thing, peak is had no, show that the method has good specificity.
6 linear and sensitivity
Test homocysteine and cysteine is linear under patent condition, standard curve shows in extensive concentration
(0.1,0.2,1.0,2.0,3.0,4.0 μ g/mL) and the wired sexual intercourse (R of concentration2≥0.9991.Homocysteine and half Guang ammonia
The retention time and the range of linearity of acid, regression equation are shown in Table 1 (test limit signal-to-noise ratio ＞ 3 and quantitative limit signal-to-noise ratio ＞ 10).
The retention time and equation of linear regression of 12 kinds of analytes of table
7 rate of recovery
The standard items that the rate of recovery adds known quantity by measuring into sample substrate are drawn.Add 0.2 μ g/mL, 1.0 μ g/
ML, the standard items of 2 tri- concentration of μ g/mL, each 5 parts of concentration is parallel, is measured in one day, for calculating the rate of recovery and in a few days essence
Density.
8 precision
The standard items that precision adds known quantity by measuring into sample substrate are drawn.Add 0.2 μ g/mL, 1.0 μ g/
ML, the standard items of 2 tri- concentration of μ g/mL, each concentration 3 is parallel, METHOD FOR CONTINUOUS DETERMINATION 3 days, for calculating day to day precision.With phase
Precision is represented to standard deviation.
0.2 μ g/mL are added in rat plasma, 1.0 μ g/mL, the rate of recovery and precision of 2 concentration of μ g/mL tri- are shown in Table 2.
The rate of recovery and precision (mean value ± standard deviation) of 2 plasma sample mark-on of table
9 stability
The standard items that stability adds known quantity by measuring into sample substrate are drawn.Add 0.2 μ g/mL, 1.0 μ g/
ML, the standard items of 2 tri- concentration of μ g/mL, each concentration 3 is parallel, places 0h respectively at 4 DEG C, measures after 24h, 48h, when subtracting
When being less than 10% on a small quantity, it is believed that sample is stablized.0.2 μ g/mL, 1.0 μ g/mL, 2 concentration of μ g/mL tri- are added in rat plasma
Shelf-stability under the conditions of 4 DEG C is shown in Table 3.Actual sample has preferably been surveyed in one day.
The stability of 3 plasma sample mark-on of table
By the embodiment of the present invention it can be seen from above example by homocysteine in rat plasma and cysteine into
Detection is gone, method has higher sensitivity, accuracy and precision.
Claims (6)
1. the detection method of homocysteine and cysteine in a kind of rat plasma, it is characterised in that include the following steps：
Step 1: the preparation of standard items；
Step 2: sample is reduced with sodium borohydride；
Step 3: except the protein in plasma sample；
Step 4: homocysteine and cysteine in HPLC-ECD separation determination plasma samples.
Step 5: result calculates.
2. the detection method of homocysteine and cysteine in rat plasma as claimed in claim 1, it is characterised in that step
Preparation specific method in rapid one to standard items is：
With homocysteine, cysteine and vanillic acid are standard items, are configured to concentration with 0.1mol/L perchloric acid solutions respectively
It is spare for the storing solution of 1mg/mL.
3. the detection method of homocysteine and cysteine in rat plasma as claimed in claim 1, it is characterised in that step
The specific method of reduction in rapid two to sample is：
Step a, 1.5mol/L NaoH solution is prepared, appropriate sodium borohydride is weighed and is dissolved in 1.5mol/L NaoH solution, make boron
The concentration of sodium hydride is 2mol/L；
Step b, 100 μ L plasma samples are taken, the sodium borohydride solution of 10 μ L 2mol/L are added into sample, vortex 1min, stands
React 5min.
4. the detection method of homocysteine and cysteine in rat plasma as claimed in claim 1, it is characterised in that step
Except the specific method of protein in plasma sample is in rapid three：
300 μ L 0.4mol/L perchloric acid are added (containing interior for 1: 3 sample into step 1 by volume by sample and perchloric acid
Mark, 2 μ g/mL of vanillic acid), it is vortexed and mixes 1min, 15000 × g, 4 DEG C of centrifugation 15min, takes supernatant to be measured for HPLC-ECD.
5. the detection method of homocysteine and cysteine in rat plasma as claimed in claim 1, it is characterised in that step
Homocysteine and cysteine, determination condition are as follows in HPLC-ECD separation determinations plasma sample in rapid four：
Detector：Shimadzu ED723 electrochemical detectors；
Chromatographic column：Yi Lite C8 (E2020417,4.6mm × 250mm, 5 μm)；
Mobile phase A：Methanol；
Mobile phase B：30mmol/L sodium acetates, 100 μm of ol/L perfluorooctane sulfonates, pH=5.02,
Isocratic elution, elution program are：A: B=0.1: 99.9；
Column temperature：30℃；
Flow velocity：0.7mL/min；
Detect voltage：900mv；
Sample size：20μL.
6. the detection method of homocysteine and cysteine in rat plasma as claimed in claim 1, it is characterised in that step
Result, which calculates, in rapid five is specially：
The content of homocysteine in sample is calculated by formula (1)；The content of cysteine in sample is calculated by formula (2)；
Y=0.8499X+0.0288 (1)
Y=0.5278X-0.0014 (2)
In formula：
Y：The ratio between determinand and the peak area of internal standard substance；
X：The concentration of determinand, unit are micrograms per millilitre.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112924565A (en) * | 2019-12-05 | 2021-06-08 | 湖北远大生物技术有限公司 | Method for detecting content of serine in serine raw material |
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Application publication date: 20180413 |