CN102539545A - Method for testing branched chain amino acid content of blood - Google Patents
Method for testing branched chain amino acid content of blood Download PDFInfo
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- CN102539545A CN102539545A CN2010105808115A CN201010580811A CN102539545A CN 102539545 A CN102539545 A CN 102539545A CN 2010105808115 A CN2010105808115 A CN 2010105808115A CN 201010580811 A CN201010580811 A CN 201010580811A CN 102539545 A CN102539545 A CN 102539545A
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Abstract
The invention discloses a method for testing the branched chain amino acid content of blood, and the method comprises the following steps: respectively mixing a standard product and a to-be-tested serum sample with an isotope internal standard solution uniformly; carrying out centrifugalization on the obtained mixtures, taking liquid supernatants, and drying the liquid supernatants; respectively adding a mobile-phase compounded solution into the obtained products, and carrying out ultrasonic dissolving on the obtained mixtures; carrying out centrifugalization on the obtained mixtures, taking liquid supernatants, enabling the liquid supernatants and a mobile phase to flow through a chromatographic column together to carry out liquid chromatogram-mass spectrum analysis, and then quantitatively determining the branched chain amino acid content of the blood. The method for testing the branched chain amino acid content of the blood disclosed by the invention is strong in specificity, high in accuracy and sensitivity, and short in analysis time.
Description
Technical field
The present invention relates to branched-chain amino acid content detecting method in a kind of blood.
Background technology
Branched-chain amino acid comprises leucine (Leucine), isoleucine (Isoleucine) and valine (Valine) three seed amino acids.
Leucine is playing an important role aspect adjusting amino acid and the protein metabolism.It is the amino acid of unique scalable Protein Turnover in skeletal muscle and the cardiac muscle; Can promote the synthetic of skeletal muscle protein.Isoleucine is one of human essential amino acid, and its function mainly is to keep organism balance, the treatment phrenoblabia; Appetitive increase and anti-anemia effect; In a single day human body lacks isoleucine, symptoms such as in the flue, stupor can occur.Valine is a kind of essential amino acid, and its function comprises: to muscle extra energy is provided, to prevent muscle weakness; From the unnecessary nitrogen of hepatic clearance, and the nitrogen of somagenic need is transported to each position; Help to treat disease in the liver and gallbladder; Impel nervous function normal.Branched-chain amino acid plays an important role in vivo, mainly comprises: the oxidation energy supply; Participation muscle is synthetic; Prevention and alleviate central nerve fatigue; Stimulate the secretion of associated hormone: leucine can indirect stimulation insulin, secretion of growth hormone discharges, and has to promote the synthetic and anti-double action of decomposing of muscle protein.
Branched-chain amino acid belongs to essential amino acid, mainly acts on muscle metabolism, account for skeletal muscle protein essential amino acid 35%, can help the loss of rigidity of the body mechanical impedance muscle breakdown, nutrition and increase the muscle anti-pressure ability; Prove that through zoopery " branched-chain amino acid " makes the life of small white mouse prolong 12%; And in close relations with some metabolic diseases, like maple syrup urine disease.Therefore quantitative test branched-amino acid content is significant to research nutrition and internal metabolism process.
Summary of the invention
The purpose of this invention is to provide branched-chain amino acid content detecting method in a kind of high specificity, accuracy and the highly sensitive blood.
The branched-chain amino acid content detecting method comprises the steps: in the blood provided by the present invention
Standard items, test serum sample respectively with isotope inner mark solution mixing; Centrifugal, get supernatant, dry up; Add moving phase redissolution liquid, ultrasonic dissolution; Centrifugal, get supernatant, get into liquid chromatography-mass spectrography analysis, the content of branched-chain amino acid in the quantitative blood with the moving phase chromatographic column of flowing through.
Branched-chain amino acid content detecting method in the blood of the present invention, wherein: said moving phase redissolution liquid and moving phase are acetonitrile solution or the methanol aqueous solution that contains 2-8 ‰ perfluoro enanthic acid.
Branched-chain amino acid content detecting method in the blood of the present invention, wherein: the volume percent content of acetonitrile or methyl alcohol is 20-70% in the said moving phase redissolution liquid.
Branched-chain amino acid content detecting method in the blood of the present invention; High specificity, accuracy and highly sensitive, analysis time short, provides for studying metabolic diseases such as nutrition, internal metabolism process and maple syrup urine disease clinically that accuracy is high, high specificity and highly sensitive detection method.The branched-chain amino acid content detecting method is interior mark with isotope in the blood of the present invention, makes that the identification of target compound is more accurate, has better eliminated systematic error simultaneously, and quantitative result is more accurate.
Description of drawings
Fig. 1 is valine (Val) mass-to-charge ratio characteristic pattern.
Fig. 2 is leucine (Leu) and isoleucine (Ile) mass-to-charge ratio characteristic pattern.
Fig. 3 is Val standard items chromatograms.
Fig. 4 is a mark chromatogram in the Val.
Fig. 5 is Leu and Ile standard items chromatogram.
Fig. 6 is a mark chromatogram in the Leu.
Fig. 7 is Leu and an Ile chromatogram in the serum sample.
Fig. 8 is a Val chromatogram in the serum sample.
Fig. 9 marks chromatogram in the Leu in the serum sample.
Figure 10 marks chromatogram in the Val in the serum sample.
Embodiment
(1) detection method
(1) pipettes in standard items and the isotope with pipettor and be marked in the centrifuge tube vortex appearance mixing; Simultaneously parallel pipetting marked vortex mixing, sealing in sample to be tested and the isotope.
(2) high speed centrifugation is got the supernatant of partial volume, dries up,
(3) add moving phase redissolution liquid (acetonitrile solution or the methanol aqueous solution that contain 2-8 ‰ perfluoro enanthic acid, the volume percent content of organic phase are 20-70%) and redissolve ultrasonic dissolution; Centrifugal, get supernatant, by liquid phase automatic sampler sample introduction, get into the liquid chromatography-mass spectrography analysis, the content of branched-chain amino acid in the final quantitative test serum with the moving phase chromatographic column of flowing through.
Liquid phase chromatogram condition:
On Discovery C18 chromatographic column, separate, moving phase is acetonitrile solution or the methanol aqueous solution that contains 2-8 ‰ perfluoro enanthic acid, adopts the gradient elution mode, and flow velocity is 175 μ l/min.
The tandem mass spectrum condition:
Mass spectrum adopts electric spray ion source (ESI), positive ion mode, choice reaction monitoring (SRM) to carry out quantitative test.Ion gun is the ESI source, and it is 4500V that spray voltage is set; Argon gas is as collision gas, and pressure is 1.5mTorr (1mTorr=0.133Pa), collision energy (CE) 15V; Nitrogen is provided with parameter and is respectively 30psi and 10psi (1psi=6.89KPa) as sheath gas and assist gas; 350 ℃ of heated capillary temperature; Detection mode is a positive ion mode, and scan pattern is SRM, and be 0.5s sweep time.
Branched-chain amino acid comprises valine (Val), leucine (Leu), and isoleucine (Ile) three seed amino acids, its molecular structural formula is as follows:
(2) detect instance:
(1) pipettes 10 μ l standard solutions (leucine: 48-480 μ mol/l with pipettor; Isoleucine 24-240 μ mol/l; Valine 50-600 μ mol/l) and isotope inner mark solution (being 50 μ mol/l) mix in the centrifuge tube mesoscale eddies; Parallel 10 μ l test serums and isotope inner mark solution (50 μ mol/l) the vortex mixing of pipetting of while.
(2) with above-mentioned mixed liquor high speed centrifugation, get the supernatant of partial volume, dry up.
(3) add moving phase redissolution liquid (volume ratio is 1: 1 the water and the mixed liquor of acetonitrile, contains 2-8 13 fluoro enanthic acid) and redissolve ultrasonic dissolution.
(4) centrifugal, get supernatant, by liquid phase automatic sampler sample introduction, get into the liquid chromatography-mass spectrography analysis, the content of branched-chain amino acid in the final quantitative test serum with the moving phase chromatographic column of flowing through.
Liquid phase chromatogram condition:
On Discovery C18 chromatographic column, separate, moving phase is pure water, contains the water of 0.2%-0.8% 13 fluoro enanthic acid and the mixed liquor of acetonitrile that adopt the gradient elution mode, flow velocity is 175 μ l/min.
The tandem mass spectrum condition:
Mass spectrum adopts electric spray ion source (ESI), positive ion mode, choice reaction monitoring (SRM) to carry out quantitative test.Ion gun is the ESI source, and it is 4500V that spray voltage is set; Argon gas is as collision gas, and pressure is 1.5mTorr (1mTorr=0.133Pa), collision energy (CE) 15V; Nitrogen is provided with parameter and is respectively 30psi and 10psi (1psi=6.89KPa) as sheath gas and assist gas; 350 ℃ of heated capillary temperature; Detection mode is a positive ion mode, and scan pattern is SRM, and be 0.5s sweep time.
(Val, Leu is shown in mass-to-charge ratio characteristic Fig. 1 and 2 Ile) for standard items.
Standard items and interior target chromatogram thereof are shown in Fig. 3-6.
Serum sample and interior target chromatogram thereof show high specificity, the accuracy and highly sensitive of this method shown in Fig. 7-10.
Above embodiment describes preferred implementation of the present invention; Be not that scope of the present invention is limited; Design under the prerequisite of spirit not breaking away from the present invention; Various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (3)
1. branched-chain amino acid content detecting method in the blood comprises the steps:
Standard items, test serum sample respectively with isotope inner mark solution mixing; Centrifugal, get supernatant, dry up; Add moving phase redissolution liquid, ultrasonic dissolution; Centrifugal, get supernatant, get into liquid chromatography-mass spectrography analysis, the content of branched-chain amino acid in the quantitative blood with the moving phase chromatographic column of flowing through.
2. detection method according to claim 1 is characterized in that: said moving phase redissolution liquid and moving phase are acetonitrile solution or the methanol aqueous solution that contains 2-8 13 fluoro enanthic acid.
3. detection method according to claim 2 is characterized in that: the volume percent content of acetonitrile or methyl alcohol is 20-70% in the said moving phase redissolution liquid.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104133004A (en) * | 2013-05-03 | 2014-11-05 | 上海美迪西生物医药有限公司 | Determination method of concentration of 2,3-isatin in plasma sample |
| CN110389231A (en) * | 2018-04-19 | 2019-10-29 | 北京市心肺血管疾病研究所 | Branched-chain amino acid detectable substance is preparing the application in dissection of aorta patient's Postoperative determination risk assessment reagent kit |
| CN110806458A (en) * | 2019-11-29 | 2020-02-18 | 北京和合医学诊断技术股份有限公司 | Method for simultaneously detecting leucine, isoleucine and valine contents in blood |
-
2010
- 2010-12-09 CN CN2010105808115A patent/CN102539545A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 《Journal of Chromatography B》 20060104 Mariella Zoppa等 Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC-ESI-MS/MS 第268页第2.3节至第269页第2.4节 2-3 第831卷, * |
| MARIELLA ZOPPA等: "Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC–ESI–MS/MS", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| 张轶雯等: "串联质谱法测定血清中20 种游离氨基酸含量", 《检验医学与临床》 * |
| 王一红等: "液相色谱-质谱/质谱联用技术分析18种游离氨基酸", 《中国卫生校验杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104133004A (en) * | 2013-05-03 | 2014-11-05 | 上海美迪西生物医药有限公司 | Determination method of concentration of 2,3-isatin in plasma sample |
| CN110389231A (en) * | 2018-04-19 | 2019-10-29 | 北京市心肺血管疾病研究所 | Branched-chain amino acid detectable substance is preparing the application in dissection of aorta patient's Postoperative determination risk assessment reagent kit |
| CN110389231B (en) * | 2018-04-19 | 2022-09-06 | 北京市心肺血管疾病研究所 | Application of branched chain amino acid detection substance in preparation of post-operation prognosis risk assessment kit for aortic dissection patient |
| CN110806458A (en) * | 2019-11-29 | 2020-02-18 | 北京和合医学诊断技术股份有限公司 | Method for simultaneously detecting leucine, isoleucine and valine contents in blood |
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Application publication date: 20120704 |