CN108152380A - Tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit - Google Patents
Tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit Download PDFInfo
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- CN108152380A CN108152380A CN201611104706.8A CN201611104706A CN108152380A CN 108152380 A CN108152380 A CN 108152380A CN 201611104706 A CN201611104706 A CN 201611104706A CN 108152380 A CN108152380 A CN 108152380A
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- amino acid
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- 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
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Abstract
The present invention provides a kind of tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit and the methods of this kit of application detection amino acid and carnitine.The kit includes：Isotopic standard product, amino acid carnitine releasing agent and mobile phase, which is characterized in that the amino acid carnitine releasing agent is the mixture of methanol, water and NaCL, wherein, the mass ratio of methanol, water and Nacl is 80:20:0.8；The mobile phase is the mixture of methanol, water and formic acid, wherein, the volume ratio of methanol, water and formic acid is 80:20:0.1.The present invention discharges liquid obtained by amino acid and carnitine from dried blood spot and clarifies, is transparent, makes testing result more accurate, stable while the pollution to tandem mass spectrometer is reduced.
Description
Technical field
The present invention relates to tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit and its applications.
Background technology
Newborn's Inherited Metabolic Disorders screening refers in neonatal period to certain serious congenital heredity metabolic diseases of harm
Group's screening is carried out, infant is made to obtain early diagnosis and therapy, the generation of intelligence deformity is avoided, improves the quality of the population.Such disease
Disease even often lacks specific symptom in neonatal period in the young infant phase, once there is symptom infant center of origin nervous system
Irreversible damage, lose treatment good opportunity.And the indexs such as infant is biochemical in neonatal period blood, metabolism have changed, because
This makes early diagnosis using test in laboratory method.The examination of newborn infant diseases is in one of modern preventive medicine is important
Hold, to prenatal and postnatal care, improve the health of the people with far-reaching and positive meaning and fundamentally reduce inborn defect, improve
One of important measures of quality of the people.
Amino acid and carnitine are the important substances for participating in human physiological metabolism, and this kind of substance eubolism in human body is kept
Certain level.The concentration of this kind of substance changes often prompting human body metabolic pathway and is abnormal.By detecting these substances
Concentration with reference to clinical manifestation, can help the judgement of clinical disease.
Tandem mass spectrum quantitatively detects the principle of amino acid and carnitine：The method replaced using Isotopic Internal Standard, is analysed to
13 atomic substitutions of tritium atom or carbon such as hydrogen atom, carbon atom in object form and molecule consistent with analyte molecules structure
The standard substance of amount difference 1-9.It is admitted in tandem mass spectrometer and detects together with standard substance and the analysans in sample.Divided
The concentration for analysing object is directly proportional to the abundance of ions detected by tandem mass spectrum, according to the abundance of ions of the internal standard product of known concentration
With the abundance of ions of analyte, analyte concentration can obtain.
With increasing for screening kinds of Diseases, the clinical requirement to Inherited Metabolic Disorders diagnosis and differential diagnosis is increasing.Closely
Nian Lai, the research and development of tandem mass spectrometer make tandem mass spectrum technology become the conventional diagnostic tools of Inherited Metabolic Disorders.Series connection matter
Spectral technology has just been applied to neonatal screening in nineteen ninety, has more than 30 a countries and regions so far and is sieved using tandem mass spectrum
Newborn's inherited metabolic disease is looked into, screening coverage rate basically reaches more than 90%.Such as the U.S., by 2007, popularize comprehensively
Liquid phase tandem mass spectrum technology, U.S.'s health resources also disclose the newborn of 29 kinds of primary screenings with service management portion in recent years
Disease, including citrullinemia, maple syrup urine disease, homocysteine urine disease, propionic acidemia etc.；In Britain, Germany, the big profit of Australia
Tandem mass spectrum neonatal screening has also been classified as legal screening project by the countries and regions such as Asia, South Korea, Japan and TaiWan, China, sieve
It looks into coverage rate and basically reaches more than 90%.
Invention content
A kind of tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit are provided based on the present invention.Kit
Amino acid and carnitine content in dried blood spot are detected, it can be as the basis of assessment newborn's Inherited Metabolic Disorders.
This kit includes：Isotopic standard product, amino acid carnitine releasing agent and mobile phase, which is characterized in that the ammonia
Base acid carnitine releasing agent is the mixture of methanol, water and NaCL, wherein, the mass ratio of methanol, water and Nacl is 80:20:0.8；
The mobile phase is the mixture of methanol, water and formic acid, wherein, the volume ratio of methanol, water and formic acid is 80:20:0.1
It is detected the present invention provides a kind of using tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit
The method of a variety of amino acid and carnitine, this method include：(1) work is prepared using isotopic standard product and amino acid carnitine releasing agent
Make solution；(2) dried blood spot punching sample-adding；(3) amino acid carnitine discharges；(4) supernatant is shifted and is centrifuged；(5) machine testing on.Its
The condition of middle amino acid carnitine release is：600-650rpm oscillation incubations 30-40 minutes at 30-35 DEG C.
The kit of the present invention can reach higher preci-sion and accuracy when being detected for tandem mass spectrum.
Specific embodiment
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Test method without specific conditions or according to manufacturer in the following example
Proposed condition.
Unless otherwise defined, it anticipates known to all professional and scientific terms used in text and one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the present invention.It is described in text
Preferred implement methods and materials be for illustrative purposes only.
In the present invention, the isotopic standard product include amino acid isotopic standard product and carnitine isotopic standard product,
Can be standard items commonly used in the art, and commercially available various isotopic standard product may be incorporated for the present invention, such as U.S.
The isotopic standard product that Cambridge laboratory provides, type and concentration are as shown in table 1 below：
Table 1
| Amino acid (AA) | Concentration | Carnitine (AC) | Concentration |
| 15N,2-13C-GLY | 12.50μmol/L | 2H9-C0 | 0.76μmol/L |
| 2H4,13C-ARG | 2.50μmol/L | 2H3-C2 | 0.19μmol/L |
| 2H4-ALA | 2.50μmol/L | 2H3-C3 | 0.038μmol/L |
| 2H2-CIT | 2.50μmol/L | 2H3-C4 | 0.038μmol/L |
| 2H8-MET | 2.50μmol/L | 2H9-C5 | 0.038μmol/L |
| 2H8-VAL | 2.50μmol/L | 2H3-C8 | 0.038μmol/L |
| 2H3-LEU | 2.50μmol/L | 2H3-C14 | 0.038μmol/L |
| 2H6-ORN | 2.50μmol/L | 2H3-C16 | 0.076μmol/L |
| 13C6-PHE | 2.50μmol/L | ||
| 13C6-TYR | 2.50μmol/L |
In the present invention, the amino acid carnitine releasing agent be methanol, water and NaCL mixture, wherein, methanol, water and
The mass ratio of Nacl is 80:20:0.8
In the present invention, the mobile phase is the mixture of methanol, water and formic acid, wherein, the volume of methanol, water and formic acid
Than being 80:20:0.1
In the present invention, the method for detecting a variety of amino acid and carnitine using mentioned reagent box provided includes the following steps
(1) using isotopic standard product and amino acid carnitine releasing agent preparation work solution；
(2) dried blood spot punching sample-adding；
(3) amino acid carnitine discharges；
(4) supernatant is shifted and is centrifuged；
(5) machine testing on.
Wherein amino acid carnitine release condition be：600-650rpm oscillation incubations 30-40 minutes at 30-35 DEG C.
It is done using tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit the detection newborn of the present invention
The accuracy of the concentration of amino acid and carnitine and precision are as shown in table 2 below in Blood piece.
Table 2
| Sample | Average recovery rate | CV% |
| ALA | 110.5% | 10.9% |
| ARG | 93.6% | 9.5% |
| CIT | 96.2% | 9.1% |
| GLY | 95.4% | 10.0% |
| LEU | 118.5% | 4.9% |
| MET | 105.0% | 4.5% |
| ORN | 99.1% | 2.8% |
| PHE | 96.4% | 3.3% |
| TYR | 99.6% | 5.1% |
| VAL | 114.8% | 5.2% |
| C0 | 89.8% | 3.2% |
| C2 | 96.3% | 6.5% |
| C3 | 93.5% | 5.2% |
| C4 | 96.9% | 5.9% |
| C5 | 96.6% | 6.6% |
| C5DC | 99.3% | 7.5% |
| C6 | 105.9% | 9.2% |
| C8 | 85.4% | 8.5% |
| C10 | 104.1% | 8.9% |
| C12 | 96.5% | 7.1% |
| C14 | 93.4% | 7.3% |
| C16 | 96.1% | 4.9% |
| C18 | 99.7% | 5.8% |
Embodiment 1
Step 1：Using isotopic standard product and amino acid carnitine releasing agent preparation work solution, isotopic standard product are used
100 times of amino acid carnitine release dilution agent is working solution.
Step 2：Filter paper Blood piece is punched on dry blood cake using automatic or manual card punch, and the immigration of filter paper Blood piece is carried
The V-type of confession is cut in the plate hole of the clean microwell plate at bottom.The diameter of paper disc should be about 3.2mm.Each plate hole only adds in a filter paper
Piece.It is molten to the routine work that 100 μ L are added in the plate hole each containing filter paper Blood piece to be reversely loaded method using multichannel pipettor
Liquid.Microwell plate is covered with viscous plastic big envelope, it is ensured that volatile quantity is reduced to minimum by good seal.
Step 3：Microwell plate is placed in incubation oscillator, is vibrated 35 minutes under the conditions of 30 DEG C.The speed of microwell plate oscillation
Spend ranging from 600rpm.
Step 4：75 μ L solution are transferred in the microwell plate of V-type bottom from each plate hole, are placed in 96 orifice plate centrifuges
5000rpm is centrifuged 5 minutes.
Step 5：Upper machine testing.Plate is put into autosampler, enables application software, establishes sample list, selection is just
True acquisition method, detection.
According to Mass Spectrometry Conditions of the method on tandem mass spectrum instrument Waters TQD
Liquid phase pump gradient and mass spectrograph condition
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
Claims (3)
1. a kind of tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit, the kit include：Isotope mark
Quasi- product, amino acid carnitine releasing agent and mobile phase, which is characterized in that the amino acid carnitine releasing agent is methanol, water and NaCl
Mixture, wherein, the mass ratio of methanol, water and Nacl is 80:20:0.8；The mobile phase is the mixed of methanol, water and formic acid
Object is closed, wherein, the volume ratio of methanol, water and formic acid is 80:20:0.1.
2. using the method for a variety of amino acid and carnitine in kit detection dried blood spot described in claim 1, this method includes
Following steps：
(1) using isotopic standard product and amino acid carnitine releasing agent preparation work solution；
(2) dried blood spot punching sample-adding；
(3) amino acid carnitine discharges；
(4) supernatant is shifted and is centrifuged
(6) machine testing on.
3. according to the method described in claim 2, wherein, the condition of amino acid carnitine release is：600-650rpm at 30-35 DEG C
Oscillation incubation 30-40 minutes.
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| CN201611104706.8A CN108152380A (en) | 2016-12-05 | 2016-12-05 | Tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit |
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|---|---|---|---|
| CN201611104706.8A CN108152380A (en) | 2016-12-05 | 2016-12-05 | Tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit |
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| CN108152380A true CN108152380A (en) | 2018-06-12 |
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| CN201611104706.8A Pending CN108152380A (en) | 2016-12-05 | 2016-12-05 | Tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752473A (en) * | 2019-03-04 | 2019-05-14 | 北京和合医学诊断技术股份有限公司 | It with amino acid and fatty acyl carnitine is targeting target metabolic group analysis method in blood |
| CN111650314A (en) * | 2020-06-05 | 2020-09-11 | 深圳华大临床检验中心 | Kit and method for detecting genetic metabolic disease related substances by non-derivation method tandem mass spectrometry |
| CN114791465A (en) * | 2021-01-26 | 2022-07-26 | 江苏诺明哲天医学检验实验室有限公司 | Method for detecting derivatization of amino acid and carnitine by tandem mass spectrometry |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030199102A1 (en) * | 1999-12-29 | 2003-10-23 | Perkinelmer Life Sciences, Inc. | Test tray, kit and methods for bodily fluid testing for newborn screening by tandem mass spectrometry |
| CN101788528A (en) * | 2009-12-22 | 2010-07-28 | 浙江大学 | Amino acid and carnitine tandem mass-spectrography detection kit |
| CN201607442U (en) * | 2009-12-22 | 2010-10-13 | 浙江大学 | Detection kit applying amino acid and carnitine tandem mass spectrometry |
| CN103278584A (en) * | 2013-05-28 | 2013-09-04 | 上海市儿童医院 | Detection kit based on tandem mass spectrum non-derivatization method |
| CN104237442A (en) * | 2014-10-11 | 2014-12-24 | 杨绪庆 | Reagent kit, application of reagent kit and multiple amino acid and carnitine joint-detection method |
-
2016
- 2016-12-05 CN CN201611104706.8A patent/CN108152380A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030199102A1 (en) * | 1999-12-29 | 2003-10-23 | Perkinelmer Life Sciences, Inc. | Test tray, kit and methods for bodily fluid testing for newborn screening by tandem mass spectrometry |
| CN101788528A (en) * | 2009-12-22 | 2010-07-28 | 浙江大学 | Amino acid and carnitine tandem mass-spectrography detection kit |
| CN201607442U (en) * | 2009-12-22 | 2010-10-13 | 浙江大学 | Detection kit applying amino acid and carnitine tandem mass spectrometry |
| CN103278584A (en) * | 2013-05-28 | 2013-09-04 | 上海市儿童医院 | Detection kit based on tandem mass spectrum non-derivatization method |
| CN104237442A (en) * | 2014-10-11 | 2014-12-24 | 杨绪庆 | Reagent kit, application of reagent kit and multiple amino acid and carnitine joint-detection method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752473A (en) * | 2019-03-04 | 2019-05-14 | 北京和合医学诊断技术股份有限公司 | It with amino acid and fatty acyl carnitine is targeting target metabolic group analysis method in blood |
| CN109752473B (en) * | 2019-03-04 | 2022-04-01 | 北京和合医学诊断技术股份有限公司 | Metabonomics analysis method taking amino acid and acylcarnitine as target in blood |
| CN111650314A (en) * | 2020-06-05 | 2020-09-11 | 深圳华大临床检验中心 | Kit and method for detecting genetic metabolic disease related substances by non-derivation method tandem mass spectrometry |
| CN114791465A (en) * | 2021-01-26 | 2022-07-26 | 江苏诺明哲天医学检验实验室有限公司 | Method for detecting derivatization of amino acid and carnitine by tandem mass spectrometry |
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Application publication date: 20180612 |