CN107015003A - It is a kind of to be used for the kit of quantitatively detection homocysteine, cysteine and methionine - Google Patents
It is a kind of to be used for the kit of quantitatively detection homocysteine, cysteine and methionine Download PDFInfo
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- CN107015003A CN107015003A CN201710128473.3A CN201710128473A CN107015003A CN 107015003 A CN107015003 A CN 107015003A CN 201710128473 A CN201710128473 A CN 201710128473A CN 107015003 A CN107015003 A CN 107015003A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
- G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
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Abstract
A kind of to be used for the kit of quantitatively detection homocysteine, cysteine and methionine, it belongs to biochemical analysis detection field.The kit includes standard working solution, mixing internal standard solution, dilution, reducing agent, precipitating reagent, quality-control product and mobile phase.Kit of the present invention is based on mass-spectrometric technique, using non-derivative method, constructs serum（Slurry）The pre-treatment model of homocysteine, cysteine and methionine in sample, simultaneous quantitative homocysteine, the metabolite of 3 methionine cycles of cysteine and methionine are realized first, easy to operate, sensitivity is high, result is accurate, helps more fully to evaluate danger and the physio-pathological condition of patient's cardiovascular and cerebrovascular disease.
Description
Technical field
The invention belongs to biochemical analysis detection field, and in particular to one kind quantitatively detects serum（Slurry）Homotype in sample
The kit of cysteine, cysteine and methionine.
Background technology
Homocysteine (homocysteine, Hcy), cysteine (cysteine, Cys) and methionine
(methionine, Met, also referred to as methionine) is 3 important products of internal methionine cycle.Under normal circumstances, homotype
Cysteine is converted by two approach：One is to methylate again, about 50% homocysteine urging in methionine synthetase
Under change effect, using folic acid as methyl donor, methylate, recombine methionine.Two be to turn sulfenyl approach, in addition about
50% homocysteine ultimately generates cysteine in the presence of cystathionine beta-synthase[1]。
Total homocysteine level 5 ~ 15 in fasting blood in normal humanμmol/L.When total homocysteine in blood
Level is higher than 15μMol/L, then diagnosable is hyperhomocysteinemiainjury[2].And hyperhomocysteinemiainjury is coronary disease
Disease, palsy, the independent hazard factor of Deep vain thrombosis, plasma homocysteine level often raise 5μMol/L, hat
The risk of Coronary disease increases by 1.6 times, and cranial vascular disease risk increases by 1.8 times[3]。
In recent years, scientific research proves not only homocysteine, and cysteine, the level of methionine are to cardiovascular and cerebrovascular
Disease risk degree also has directive function.2010, Page JH results of study showed the homocysteine and half of high concentration
The occurrence risk that cystine dramatically increases heart infarction is up to 3.3 ~ 3.5 times[4].2011, Xiao Y pointed out the Guang of high concentration homotype half
The joint effect of propylhomoserin and cysteine dramatically increases the occurrence risk of coronary heart disease[5].And work as methionine concentration< 20.6μ
During mol/L, recurrent phlebothrombosis occurrence risk will be dramatically increased and be up to 3.5 times[6]；The methionine and high concentration of low concentration
Homocysteine be central retinal vein occlusion hazards[7].Clinical research above shows the Guang of homotype half in blood
Propylhomoserin, cysteine, the anomalous variation of methionine all have extremely strong related to angiocardiopathy, microvascular disease
Property.Therefore, while detecting the level of homocysteine, cysteine and methionine, the life of thoroughly evaluating patient is contributed to
Manage pathological state.
Clinical conventional detection method and kit focus primarily upon the detection to homocysteine level, such as Shanghai at present
Eat medicine prison tool（It is accurate) word 2013 the 2401487th, the homocysteine detection of Shanghai Foxing Changzheng medical science Co., Ltd
Kit（Enzyme parameters)；Also state's food medicine prison tool (enters) word 2013 the 2402998th, Abbott GmbH ＆ Co. KG companies
ARCHITET Homocysteine Reagent Kit（Chemiluminescence particulate immunodetection）, be all to human serum or
Single homocysteine level is detected in blood plasma.Related detecting method mainly has immunization and high performance liquid chromatography
Two kinds.Immunization sensitivity is low, and testing result deviation is larger, and is disturbed by structure similar medicine S- Adenosyl-Methionines.
High performance liquid chromatography was once to determine a kind of widest method of total homocysteine, with good specificity, precision
And sensitivity, although this method is constantly modified in recent years, but it is in all changeable for the treatment of conditions, chromatography condition and sample measure
It is different to make it be difficult to standardize, while detecting that flux is relatively low[8]。
Therefore, more fully diagnose and evaluate in the urgent need to quick, accurate, the highly sensitive method of one kind carrys out adjuvant clinical
The physio-pathological condition of patient.
LC-MS/MS is a kind of new laboratory conventional detection technology, with high sensitivity, high specific and high accuracy
The characteristics of, it is obtained more concerns in clinical examination.This kit is based on LC-MS/MS technologies, using non-derivative
Method, constructs serum（Slurry）The pre-treatment model of homocysteine, cysteine and methionine, is realized first in sample
3 important metabolins in simultaneous quantitative methionine cycle, pre-treatment is easy, as a result accurately, evaluates comprehensive, with good
Potential applicability in clinical practice.
[1] Welch GN, Loscalzo J. N Engl J Med, 1998, 338(15): 1042-1050.
[2] Tsikas D, Sandmann J, Rossa S, etal. Anal Biochem, 1999, 70: 231-241.
[3] Boushey CJ, Beresford SA, Omenn GS, etal. Probable benefits of
increasing folic acid intakes. JAMA. 1995, 274(13): 1049-1057.
[4] Page JH, et al. Am Heart J. 2010, 159(4): 599-604.
[5] Xiao Y, et al. Lipids Health Dis. 2011, 12, 10: 137.
[6] Keijzer MB, et al. Thromb Haemost. 2006, 96(4): 492-749.
[7] Narayanasamy A, et al. Invest Ophthalmol Vis Sci. 2007, 48(4): 1441-
6.
[8] the conventional detection method of Wang Yu homocysteine in plasma clinic and influence factor laboratory medicines and clinic,
2010, 7 (24): 2808-2810。
The content of the invention
It is an object of the invention to provide a kind of detection serum（Slurry）Middle homocysteine, cysteine, methionine
Kit, simultaneous quantitative detection homocysteine, cysteine, methionine 3 methionine cycles are realized first
Metabolite, while overcoming the technological deficiency of existing detection method.
To achieve the above object, the technical solution adopted in the present invention is：One kind is used to quantitatively detect serum（Slurry）In it is same
Type cysteine, cysteine, the kit of methionine, it is described as follows：
1. kit includes following reagent：
1) standard items working solution：Containing homocysteine, cysteine, methionine hybrid standard product solution.
2) inner mark solution is mixed：Containd 4 - homocysteine,d 2 - cysteine,d 3 The solution of-methionine.
3) dilution：Calf serum solution
4) reducing agent：Dithiothreitol (DTT)（DTT）
5) precipitating reagent：Acetonitrile, water, watery hydrochloric acid/formic acid/acetic acid composition
6) quality-control product：Containing homocysteine, cysteine, methionine mixing quality-control product solution, points high, normal, basic 3 are dense
Degree：QC(L)、QC(M)、QC(H).
7) mobile phase：
A:0.1% aqueous formic acid
B:Acetonitrile
Preferably, above-mentioned 1）In 5 standard working solution concentration such as following tables：
Preferably, above-mentioned 5）In precipitating reagent be acetonitrile, water, the wherein mixed solution of watery hydrochloric acid/formic acid/acetic acid, acetonitrile：Water：
Sour volume ratio is 100:10~30:0.001~0.01；Wherein acid is preferably watery hydrochloric acid.
Preferably, above-mentioned 2）In mixing inner mark solution concentration be：
d 4 - homocysteine： 10μmol/L
d 2 - cysteine：100μmol/L
d 3 - methionine：20μmol/L
Preferably, above-mentioned 6）In quality-control product concentration QC (L), QC (M), QC (H) such as following table
Further, homocysteine, cysteine and methionine of the kit of the present invention suitable for serum or blood plasma
The detection of level.
Described sample is serum or blood plasma.
2. a kind of be used for the application method of quantitatively detection homocysteine, cysteine and methionine kit, bag
Include following steps：
1) configuration of reducing agent：Accurate to add 50 mL purified waters under room temperature condition, be vortexed dissolving.
2) standard curve（Quality-control product）Preparation method：Take 5μL standard working solutions（Quality-control product）In 1.5 mL centrifuge tubes,
Sequentially add 45μL dilutions, 50μL internal standard solutions, 100μL reducing agents, after vortex 30S is mixed, 30 DEG C of water-baths 15
min.Add 600μAfter L precipitating reagents, the min of vortex oscillation 3, high speed centrifugation（14000g）3 min, draw 100μL of supernatant liquid enters
Tandem Mass Spectrometry Analysis.
3) preparation method of sample：Take 50μL sample solutions sequentially add 50 in 1.5 mL centrifuge tubesμL internal standard solutions,
100 μL reducing agents, after vortex 30S is mixed, 30 DEG C of min of water-bath 15.Add 600μL precipitating reagents, the min of vortex oscillation 3
Afterwards, high speed centrifugation（14000g）3 min, draw 100μL of supernatant liquid enters Tandem Mass Spectrometry Analysis.
The beneficial effects of the invention are as follows：The kit includes standard working solution, mixing internal standard solution, dilution, reducing agent, precipitation
Agent, quality-control product and mobile phase.The invention kit is based on mass-spectrometric technique, using the method for non-derivative, constructs serum（Slurry）
The pre-treatment model of homocysteine, cysteine and methionine in sample, easy to operate, sensitivity is high, result is accurate,
The concentration of important metabolite in this 3 methionine cycles of homocysteine, cysteine and methionine is realized first
Detection, a kind of detection method for more fully evaluating the danger of patient's cardiovascular and cerebrovascular disease is provided to be clinical.
Brief description of the drawings
The present invention is further described with reference to the accompanying drawings and examples.
Fig. 1 is the MRM chromatograms of homocysteine, cysteine and methionine standard items and inner mark solution.
Fig. 2 is homocysteine, cysteine and methionine and interior target MRM chromatograms in serum.
Embodiment
It is a kind of to be used for the kit of quantitatively detection homocysteine, cysteine and methionine, use tandem mass spectrum
The treated serum of technology for detection（Slurry）Sample, using high performance liquid chromatography by homocysteine, cysteine, first sulphur ammonia
Acid and impurity are separated, using Isotopically labelled internal standard, using the concentration of standard items as X-axis, standard items and internal standard compound peak face
Product ratio is Y-axis, sets up standard curve, calculates homocysteine, cysteine and methionine concentration.Below in conjunction with specific
Embodiment is illustrated：
Specific embodiment：It is serum sample in embodiment
1. material
1.1 instrument：Waters Xevo TQ-S efficient liquid phases-GC-MS, eppendorf single track range-adjustable pipettors
（Research plus series）, THERMO-SHAKER constant temperature blending instruments MSC-100（1.5mL）, Labnet Vortex
MIXER S0200 centrifuge concussion instrument, Labnet Vortex MIXER S0200 centrifugation concussion instruments, graduated cylinder
1.2 reagents/consumptive material：Chromatographic column, kit
Chromatographic column：Waters Xbridge, 4.6 × 50mm, 3.5μm；
Kit：The compositing formula of the kit such as following table；
2. chromatographic mass spectrometry method：
2.1 chromatographic condition：
Mobile phase：0.1% aqueous formic acid（A）, acetonitrile（B）；
Flow velocity：0.6 mL/min；
Column temperature：40℃；
Sample size：1μL
Condition of gradient elution：
2.2 Mass Spectrometry Conditions：
Ion gun：ESI+
Source temperature：150℃
Precipitation is vented one's spleen temperature：500 ℃；
Desolvention gas velocity：800 L/h；
Capillary voltage：3KV；
Determinand MRM sweep parameters are shown in Table 2
Table 2 Hcy, Cys, Met and interior target MRM sweep parameters
3. pre-treating method：
3.1 standard working solutions/quality-control product pre-treating method：
Take 5μL standard working solutions（Quality-control product）In 1.5 mL centrifuge tubes, 45 are sequentially addedμL dilutions, 50μL internal standard solutions,
100 μL reducing agents, after vortex 30S is mixed, 30 DEG C of min of water-bath 15.Add 600μL precipitating reagents, the min of vortex oscillation 3
Afterwards, high speed centrifugation（14000g）3 min, draw 100μL of supernatant liquid enters chromatography.
3.2 serum sample pre-treating methods：
Take 50μL serum samples solution sequentially adds 50 in 1.5 mL centrifuge tubesμL internal standard solutions, 100μL reducing agents, are vortexed
After 30S is mixed, 30 DEG C of min of water-bath 15.Add 600μAfter L precipitating reagents, the min of vortex oscillation 3, high speed centrifugation
（14000g）3 min, draw 100μL of supernatant liquid enters chromatography.
4. Specification Curve of Increasing method：
The peak area of 5 standard working solutions and internal standard peak area ratio（y）To sign concentration（x）It is weighted（W=1/x2）It is minimum
Square law regressing calculation, obtains regression equation：Y=bx+a, and calculate r（Coefficient correlation）, r should be not less than 0.99.
Table 3 Hcy, Cys and Met linear equation, linearly dependent coefficient and retention time
5. result is calculated：
Record the ratio between object and internal standard compound peak area in sample（y）, standard curve regression equation is substituted into, the Guang ammonia of homotype half is calculated
Acid, cysteine, methionine concentration.
Hcy, Cys and Met concentration in the serum of table 4
Although above-mentioned the embodiment of the present invention is described with reference to accompanying drawing, not to the limit of invention protection domain
System, on the basis of technical scheme, it is each that those skilled in the art need not pay that creative work can make
Plant modification or deform still within the scope of the present invention.
Claims (3)
1. a kind of be used for the kit of quantitatively detection homocysteine, cysteine and methionine, it is characterised in that described
Kit includes following reagent：
1）Standard items working solution：Containing homocysteine, cysteine, methionine hybrid standard product solution S TD-1, STD-
2nd, STD-3, STD-4 and STD-5；
2）Isotope mixing inner mark solution：Containd 4 - homocysteine,d 2 - cysteine,d 3 The solution of-methionine；It is mixed
Close inner mark solution concentration：d 4 - homocysteine is 10μMol/L,d 2 - cysteine is 100μMol/L,d 3 - methionine
For 20μmol/L；
3）Dilution：Calf serum solution；
4）Reducing agent：Dithiothreitol (DTT)；
5）Precipitating reagent：Acetonitrile：Water：Sour volume ratio is 100:10~30:0.001 ~ 0.01, acid is watery hydrochloric acid, formic acid or acetic acid；
6）Quality-control product：Containing homocysteine, cysteine, methionine mixing quality-control product solution, points low middle high 3 are dense
Degree：QC-L、QC-M、QC-H；It is that homotype semicystinol concentration investigating is 20 in the quality-control product QC-LμMol/L, semicystinol concentration are
100μMol/L, methionine concentration are 40μmol/L；Homotype semicystinol concentration investigating is 200 in QC-MμMol/L, cysteine are dense
Spend for 1000μMol/L, methionine concentration are 400μmol/L；Homotype semicystinol concentration investigating is 800 in QC-HμMol/L, half Guang
Propylhomoserin concentration is 4000μMol/L, methionine concentration are 1600μmol/L；
7）Mobile phase：Mobile phase A:0.1% aqueous formic acid, Mobile phase B:Acetonitrile；
Homotype semicystinol concentration investigating is 10 in the STD-1μMol/L, semicystinol concentration are 50μMol/L, methionine concentration
For 20μmol/L；Homotype semicystinol concentration investigating is 25 in STD-2μMol/L, semicystinol concentration are 125μMol/L, methionine
Concentration is 50μmol/L；Homotype semicystinol concentration investigating is 100 in STD-3μMol/L, semicystinol concentration are 500μMol/L, first
Methyllanthionine concentration is 200μmol/L；Homotype semicystinol concentration investigating is 400 in STD-4μMol/L, semicystinol concentration are 2000μ
Mol/L, methionine concentration are 800μmol/L；Homotype semicystinol concentration investigating is 1000 in STD-5μMol/L, cysteine are dense
Spend for 5000μMol/L, methionine concentration are 2000μmol/L。
2. a kind of examination for being used to quantitatively detect homocysteine, cysteine and methionine according to claim 1
The application method of agent box, it is characterised in that：
Using the treated serum of tandem mass spectrum technology for detection or plasma sample, using high performance liquid chromatography by the Guang ammonia of homotype half
Acid, cysteine, methionine and impurity are separated, using Isotopically labelled internal standard, using the concentration of standard items as X-axis,
Standard items and internal standard compound peak area ratio are Y-axis, set up standard curve, and then calculate homocysteine, cysteine and first
Methyllanthionine concentration；
1）The configuration of reducing agent：Accurate to add 50mL purified waters under room temperature condition, be vortexed dissolving；
2）Standard curve preparation method：Take 5μL standard working solutions sequentially add 45 in 1.5 mL centrifuge tubesμL dilutions,
50 μL internal standard solutions, 100μL reducing agents, after vortex 30S is mixed, 30 DEG C of min of water-bath 15；Add 600μL precipitating reagents, whirlpool
After 3 min of rotation vibration, with 14000gThe min of high speed centrifugation 3, draws 100μL of supernatant liquid enters Tandem Mass Spectrometry Analysis；
3）The configuration of quality-control product：Take 5μL quality-control products sequentially add 45 in 1.5 mL centrifuge tubesμL dilutions, 50μL internal standards
Liquid, 100μL reducing agents, after vortex 30S is mixed, 30 DEG C of min of water-bath 15；Add 600μL precipitating reagents, vortex oscillation 3
After min, with 14000gThe min of high speed centrifugation 3, draws 100μL of supernatant liquid enters Tandem Mass Spectrometry Analysis；
4）The preparation method of sample：Take 50μL sample solutions sequentially add 50 in 1.5mL centrifuge tubesμL internal standard solutions, 100μL is also
Former agent, after vortex 30S is mixed, 30 DEG C of water-bath 15min；Add 600μAfter L precipitating reagents, vortex oscillation 3min, high speed centrifugation
（14000 g）3min, draws 100μL of supernatant liquid enters Tandem Mass Spectrometry Analysis.
3. a kind of kit for being used to quantitatively detect homocysteine, cysteine and methionine described in claim 1
Application, it is characterised in that：Kit is used for homocysteine, cysteine and first in quantitatively detection serum or plasma sample
Methyllanthionine.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108362795A (en) * | 2018-02-08 | 2018-08-03 | 杭州佰辰医学检验所有限公司 | Content of homocysteine rapid detection method in dried blood spot |
| CN111983057A (en) * | 2020-07-29 | 2020-11-24 | 上海润达榕嘉生物科技有限公司 | Method and kit for detecting homocysteine in human plasma |
| CN112964808A (en) * | 2019-12-13 | 2021-06-15 | 中国科学院大连化学物理研究所 | Biological body fluid total homocysteine detection kit and detection method |
| CN113009033A (en) * | 2021-03-02 | 2021-06-22 | 广东南芯医疗科技有限公司 | Liquid phase tandem mass spectrum detection kit and detection method for testing folic acid metabolic derivatives of human body |
| CN113341012A (en) * | 2021-06-01 | 2021-09-03 | 山东英盛生物技术有限公司 | Method and kit for simultaneously detecting multiple metabolites on homocysteine metabolic pathway and application of kit |
| CN114280190A (en) * | 2021-12-27 | 2022-04-05 | 无锡市江原实业技贸有限公司 | Kit for detecting related substances of bicysteine |
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| CN106442836A (en) * | 2016-10-09 | 2017-02-22 | 辽宁润生康泰生物医药科技有限公司 | Method for detecting contents of folic acid and sulfur-containing amino acid in plasma |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108362795A (en) * | 2018-02-08 | 2018-08-03 | 杭州佰辰医学检验所有限公司 | Content of homocysteine rapid detection method in dried blood spot |
| CN112964808A (en) * | 2019-12-13 | 2021-06-15 | 中国科学院大连化学物理研究所 | Biological body fluid total homocysteine detection kit and detection method |
| CN111983057A (en) * | 2020-07-29 | 2020-11-24 | 上海润达榕嘉生物科技有限公司 | Method and kit for detecting homocysteine in human plasma |
| CN113009033A (en) * | 2021-03-02 | 2021-06-22 | 广东南芯医疗科技有限公司 | Liquid phase tandem mass spectrum detection kit and detection method for testing folic acid metabolic derivatives of human body |
| CN113341012A (en) * | 2021-06-01 | 2021-09-03 | 山东英盛生物技术有限公司 | Method and kit for simultaneously detecting multiple metabolites on homocysteine metabolic pathway and application of kit |
| CN114280190A (en) * | 2021-12-27 | 2022-04-05 | 无锡市江原实业技贸有限公司 | Kit for detecting related substances of bicysteine |
| CN114280190B (en) * | 2021-12-27 | 2024-05-28 | 无锡市江原实业技贸有限公司 | Kit for detecting related substances of double cysteines |
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