CN110118845A - Detect a variety of amino acid and fatty acyl carnitine, the method for methylmalonic acid and kit - Google Patents

Detect a variety of amino acid and fatty acyl carnitine, the method for methylmalonic acid and kit Download PDF

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Publication number
CN110118845A
CN110118845A CN201810117512.4A CN201810117512A CN110118845A CN 110118845 A CN110118845 A CN 110118845A CN 201810117512 A CN201810117512 A CN 201810117512A CN 110118845 A CN110118845 A CN 110118845A
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acid
mobile phase
standard product
sample
kit
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王岩
付洁
王艳
杨尧
王哲
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Beijing Jinze Medical Laboratory Co ltd
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Beijing Jinze Medical Science And Technology Development Co Ltd
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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
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • 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
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G01N2030/342—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient fluid composition fixed during analysis

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

This application provides tandem mass spectrometry detection kits, include extracting solution and mobile phase, it and optionally include one of quality control standard product, the isotopic standard product of target analytes and derivatization reagent or a variety of, wherein the extracting solution is the methanol aqueous solution that concentration is 85-95 volume %, the mobile phase is the mixture comprising water, acetonitrile and five fluorine propionic acid, and wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 70-90:15-25:0.15-0.25.Present invention also provides the application method of the kit and purposes.

Description

Detect a variety of amino acid and fatty acyl carnitine, the method for methylmalonic acid and kit
Technical field
The application relates generally to the application field of tandem mass spectrum, specifically, this application provides tandem mass spectrometry detections With the application method and purposes of kit and the kit.
Background technique
Inherited metabolic disease (inborn error of metabolism, IEM) be one kind be related to amino acid, organic acid, The disease of many kinds of substance such as fatty acid, urea cycle, carbohydrate, steroids metabolism.Its is many kinds of, is Pediatric Clinic Difficult and complicated cases.The specificity that the diagnosis of inherited metabolic disease relies primarily on laboratory checks.It is born about 22,000,000 every year newly in China Raw youngster, wherein Fetal malformation accounts for 900,000, accounts for the 20% of the world, average one Fetal malformation of birth in every 30 seconds.Only high phenylpropyl alcohol Propylhomoserin mass formed by blood stasis (including phenylketonuria) this kind of disease, it is annual just infant 1600-1800 newly-increased.Liquid phase tandem mass spectrometry Development makes it possible that this kind of disease is carrying out intervening before the onset.That is, certain metabolites go out in vivo after newborn's birth Now extremely, it just clarifies a diagnosis, and carries out timely and effective right in early days when not yet there is clinical symptoms or unobvious symptom There is irreversibility damage to avoid the important organ of infant, and then ensures the anthropometic and intelligence of Children Normal in disease treatment Development.
Liquid Chromatography-tandem Mass (LC-MS/MS) can analyze the concentration of multiple compounds in blood, pass through inspection The mass-to-charge ratio (relative molecular mass) of substance in sample carries out qualitative and quantitative analysis to substance.This field is at present for this Various improvement are being developed and attempted to the application of technology also.
Summary of the invention
It include extracting solution and mobile phase in a first aspect, this application provides tandem mass spectrometry detection kit, wherein institute Stating extracting solution is the methanol aqueous solution that concentration is 85-95 volume %, and the mobile phase is mixed comprising water, acetonitrile and five fluorine propionic acid Object is closed, wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 70-90:15-25:0.15-0.25.
In some embodiments, kit also include quality control standard product, target analytes isotopic standard product and spread out One of biochemical reagents are a variety of.
In some embodiments, derivatization reagent is chloroacetic chloride and n-butanol mixture.In some embodiments, second Acyl chlorides and n-butanol volume ratio are 1:8.5-9.2.
In some embodiments, target analytes are selected from amino acid, fatty acyl carnitine, methylmalonic acid and above-mentioned Any combination.
Second aspect, this application provides the method with target analytes in tandem mass spectrometry test sample, including it is following Step:
(a) the isotopic standard product of the target analytes are diluted with extracting solution, to prepare extraction working solution, wherein described Extracting solution is the methanol aqueous solution that concentration is 85-95 volume %；
(b) sample is handled with the extraction working solution, and be dried；
(c) sample after the extraction process from step (b) is handled with derivatization reagent, and is dried；
(d) with mobile phase redissolve from step (c) derivatization treatment after sample, wherein the mobile phase be comprising The mixture of water, acetonitrile and five fluorine propionic acid, wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 70-90:15-25:0.15- 0.25；And
(e) sample after redissolution of the tandem mass spectrometer detection from step (d).
In some embodiments, derivatization reagent is chloroacetic chloride and n-butanol mixture.In some embodiments, second Acyl chlorides and n-butanol volume ratio are 1:8.5-9.2.
In some embodiments, target analytes are selected from amino acid, fatty acyl carnitine, methylmalonic acid and above-mentioned Any combination.
In some embodiments, this method further includes handling and examining in parallel with the sample in step (b)-(e) Survey quality control standard product.
The third aspect, the tandem mass spectrometry detection this application provides following substances in preparation for Inherited Metabolic Disorders diagnosis With the purposes in kit:
Extracting solution, the extracting solution are the methanol aqueous solution that concentration is 85-95 volume %；With
Mobile phase, the mobile phase are the mixture comprising water, acetonitrile and five fluorine propionic acid, wherein water, acetonitrile and five fluorine third The volume ratio of acid is 70-90:15-25:0.15-0.25.
In some embodiments, kit also include quality control standard product, target analytes isotopic standard product and spread out One of biochemical reagents are a variety of.
In some embodiments, Inherited Metabolic Disorders are selected from methylmalonic acidemia, hyperphenylalaninemia, high phenylpropyl alcohol ammonia Acidaemia, bird amino acid carbamylrtansferase deficiency disease, propionic acidemia, short chain acyl CoA Dehydrogenase Deficiency (SCAD), melon Propylhomoserin mass formed by blood stasis, pole long chain acyl Co A Dehydrogenase Deficiency (VLAD), tyrosinemia, ketothiolase deficiency disease, glutaric acid blood Disease I type (GA-I), isovaleric acidemia and hypermethioninemia.
In some embodiments, derivatization reagent is chloroacetic chloride and n-butanol mixture.In some embodiments, second Acyl chlorides and n-butanol volume ratio are 1:8.5-9.2.
In some embodiments, target analytes are selected from amino acid, fatty acyl carnitine, methylmalonic acid and above-mentioned Any combination.
Detailed description of the Invention
Present inventor is continuous by carrying out to Liquid Chromatography-tandem Mass (LC-MS/MS) detection technique Optimization and improvement trial, obtain the extracting solution being used cooperatively and mobile phase with excellent properties.Such reagent combination With at least one of following advantages:
(1) higher detection specificity and/or accuracy are realized,
(2) when detecting peripheral blood sample, only need a small amount of (such as 3 microlitres) blood sample that can quantify multiple analytes, Including but not limited to a variety of amino acid, carnitine, methylmalonic acid etc. can screen a variety of heredity according to these analyte test results Metabolic disease improves the screening to such disease and diagnosis efficiency.
Unless otherwise defined, the term as used herein is identical as the normally understood meaning of those skilled in the art.
The term as used herein " tandem mass spectrum " refers to Liquid Chromatography-tandem Mass (LC-MS/MS) technology.
Various chemicals reagent mentioned herein meets general provision and the requirement of mass-spectrometric technique.
Unless other is bright, the ratio between component described herein is volume ratio.
It should be appreciated that the term as used herein " about " (for example, in component proportion, reaction temperature and in the reaction time) is with this Field technical staff it can be generally understood that meaning explain.Under normal circumstances, term " about " can be understood as given numerical value Any number in positive and negative 5% range, for example, about X can represent any number in the range of 95%X to 105%X.
It is also understood that the specific value (for example, in component proportion, reaction temperature and in the reaction time) being presented herein It can be used as individual numerical value to understand, it should be also appreciated that providing the endpoint value of a certain range, and can be combined with each other and mention For other ranges.For example, when disclose reaction can carry out 1 it is small when or at 5 hours, reaction, which is also correspondingly disclosed, to be carried out 1-5 hours.
Therefore, in a first aspect, this application provides tandem mass spectrometry detection kit, include extracting solution and mobile phase, Wherein the extracting solution is the methanol aqueous solution that concentration is 85-95 volume %, and the mobile phase is to include water, acetonitrile and five fluorine third The mixture of acid, wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 70-90:15-25:0.15-0.25.
For example, the volumetric concentration percentage of the methanol aqueous solution as extracting solution can for 85,86,87,88,89,90, 91,92,93,94 or 95%；In mobile phase the volume parts of water can for 70,71,72,73,74,75,76,77,78,79,80, 81,82,83,84,85,86,87,88,89 or 90；In mobile phase the volume parts of acetonitrile can for 15,16,17,18,19,20, 21,22,23,24 or 25；In mobile phase the volume parts of five fluorine propionic acid can for 0.15,0.16,0.17,0.18,0.19, 0.20,0.21,0.22,0.23,0.24 or 0.25.
In a specific embodiment, extracting solution is the methanol aqueous solution that concentration is 90 volume %, mobile phase be comprising The mixture of water, acetonitrile and five fluorine propionic acid, wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 80:20:0.2.
In some embodiments, kit also include quality control standard product, target analytes isotopic standard product and spread out One of biochemical reagents are a variety of.
" quality control standard product " refer to the sample containing target analytes known to concentration, for correcting testing result and monitoring inspection Mass metering.In general, quality control standard product can be with sample to be tested synchronization process.
According to the target analytes for wishing to detect, it can choose corresponding isotopic standard product.It has deposited in the art The conventional isotopic standard product of plurality of target analyte are being directed to, and can easily bought.In general, commercially available various isotopes Standard items may be incorporated for the application, the isotopic standard product provided such as the U.S., laboratory, Cambridge.
In some embodiments, target analytes are selected from amino acid, fatty acyl carnitine, methylmalonic acid and above-mentioned Any combination.
As unrestricted example, the example of amino acid includes but is not limited to alanine, arginine, aspartic acid, melon Propylhomoserin, cysteine, glutamine, glutamic acid, glycine, histidine, leucine, isoleucine, lysine, methionine, Ornithine, phenylalanine, proline, serine, threonine, tyrosine, valine.
The unrestricted example of fatty acyl carnitine can be found in the table 3 of the application.
As unrestricted example, the isotopic standard product of amino acid include but is not limited to:15N13C-Gly、d4-Ala、 d4C13-Arg、d3-Asp、d2-Cit、d3-Glu、d3-Leu、d3-Met、d2-Orn、13C6-Phe、13C6-Tyr、d8-Val、L-Ala (13C3,99%；15N, 99%), L-ArgHCl (13C6,99%；15N4,99%), L-Asp (13C4,99%；15N, 99%), L-Cys (13C6,99%；15N2,99%), L-Glu (13C5,99%；15N, 99%), Gly (13C2,99%；15N, 99%), L-HisHClH2O(13C6；15N3,99%), L-Ile (13C6,99%；15N, 99%), L-Leu (13C6, 99%；15N, 99%), L-Lys2HCl (13C6,99%；15N2,99%), L-Met (13C5,99%；15N, 99%), L- Phe (13C9,99%；15N, 99%), L-Pro (13C5,99%；15N, 99%), L-Ser (13C3,99%；15N, 99%), L-Thr (13C4,99%；15N, 99%), L-Tyr (13C9,99%；15N, 99%), L-Val (13C5,99%；15N, 99%).
As unrestricted example, the isotopic standard product of fatty acyl carnitine include but is not limited to: d9- carnitine, d3- C2,d3-C3,d3-C4,d9-C5,d3-C8,d9-C14,d3-C16.As unrestricted example, the same position of methylmalonic acid Plain standard items include but is not limited to D3- methylmalonic acid.
The kit of the application is suitable for derivatization method mass spectrum detection, therefore may include derivatization examination in kit Agent.In some embodiments, derivatization reagent is chloroacetic chloride and n-butanol mixture.In some embodiments, chloroacetic chloride It is 1:8.5-9.2 with n-butanol volume ratio.In a specific embodiment, chloroacetic chloride and n-butanol volume ratio are 1:9.But It is, it should be understood that (including commercially available or voluntarily preparation) can also be provided separately in derivatization reagent, therefore can not be the application examination The component part of agent box.
Consumptive material needed for the kit of the application can further include detection, including but not limited to 96 hole of V-type bottom are micro- Orifice plate (heat resistanceheat resistant), 96 hole microwell plate of U-shaped bottom, aluminium foil, adhesive cover and heat-sealing film etc..
Second aspect, this application provides the method with target analytes in tandem mass spectrometry test sample, including it is following Step:
(a) the isotopic standard product of the target analytes are diluted with extracting solution, to prepare extraction working solution, wherein described Extracting solution is the methanol aqueous solution that concentration is 85-95 volume %；
(b) sample is handled with the extraction working solution, and be dried；
(c) sample after the extraction process from step (b) is handled with derivatization reagent, and is dried；
(d) with mobile phase redissolve from step (c) derivatization treatment after sample, wherein the mobile phase be comprising The mixture of water, acetonitrile and five fluorine propionic acid, wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 70-90:15-25:0.15- 0.25；And
(e) sample after redissolution of the tandem mass spectrometer detection from step (d).
In some embodiments, the treatment process of step (b) includes 25 points of oscillation incubation (720rpm) at 23-32 DEG C Clock.
In some embodiments, the processing of step (c) derivedization includes standing 35-45min at 56-66 DEG C.Some In embodiment, the processing of step (c) derivedization includes standing 30min at 60 DEG C.
Preferably, it includes that (720rpm) 10min is vibrated at 23-32 DEG C that processing is redissolved in step (d).
As more specific non-limiting example, the present processes can be implemented as follows:
1. will such as amino acid isotopic standard product, fatty acyl carnitine isotopic standard product, methylmalonic acid isotopic standard Simultaneously mixed preparing, the standard items after being redissolved with 1:200 ratio with extracting solution dilution extract working solution for the concentrate dilution of product；
2. quality control standard product and sample to be tested (such as the dry blood spot sample for being soaked with peripheral blood) is added in punching；
It volatilizees 3. every hole is added prepared 90 μ l of extraction working solution and is covered with adhesive cover and reduces；
4. 700rpm shakes frequency oscillation incubation 45 minutes at 28 DEG C；
5. throwing off adhesive cover from plate, the heat-resisting microwell plate in V-type bottom of 60 μ l to number is shifted in every hole；
6. evaporation drying；
7. 50 μ l derivatization reagents are added in every hole, are sealed with sealer, prevent from volatilizing；
8. standing 30 minutes under the conditions of 60 DEG C of temperature；
9. throwing off sealer, evaporation drying；
10. 75 μ l mobile phases are added in every hole, with aluminium foil film sealing plate；
11., with the frequency of 550rpm, being vibrated 10 minutes under the conditions of 27 DEG C of temperature；
12. plate is put into autosampler, suitable instrument parameter, upper machine testing are selected.
In some embodiments, derivatization reagent is chloroacetic chloride and n-butanol mixture.In some embodiments, second Acyl chlorides and n-butanol volume ratio are 1:8.5-9.2.
In some embodiments, target analytes are selected from amino acid, fatty acyl carnitine, methylmalonic acid and above-mentioned Any combination.
In some embodiments, this method further includes handling and examining in parallel with the sample in step (b)-(e) Survey quality control standard product.
The third aspect, the tandem mass spectrometry detection this application provides following substances in preparation for Inherited Metabolic Disorders diagnosis With the purposes in kit:
Extracting solution, the extracting solution are the methanol aqueous solution that concentration is 85-95 volume %；With
Mobile phase, the mobile phase are the mixture comprising water, acetonitrile and five fluorine propionic acid, wherein water, acetonitrile and five fluorine third The volume ratio of acid is 70-90:15-25:0.15-0.25.
In some embodiments, kit also include quality control standard product, target analytes isotopic standard product and spread out One of biochemical reagents are a variety of.
In some embodiments, Inherited Metabolic Disorders are selected from methylmalonic acidemia, hyperphenylalaninemia, high phenylpropyl alcohol ammonia Acidaemia, bird amino acid carbamylrtansferase deficiency disease, propionic acidemia, short chain acyl CoA Dehydrogenase Deficiency (SCAD), melon Propylhomoserin mass formed by blood stasis, pole long chain acyl Co A Dehydrogenase Deficiency (VLAD), tyrosinemia, ketothiolase deficiency disease, glutaric acid blood Disease I type (GA-I), isovaleric acidemia and hypermethioninemia.In some embodiments, the kit of the application can mention The sensitivity of high methylmalonic acidemia diagnosis.
In some embodiments, derivatization reagent is chloroacetic chloride and n-butanol mixture.In some embodiments, second Acyl chlorides and n-butanol volume ratio are 1:8.5-9.2.
In some embodiments, target analytes are selected from amino acid, fatty acyl carnitine, methylmalonic acid and above-mentioned Any combination.
The specific embodiment of the application is described in detail above, still, during the application is not limited to the above embodiment Detail.In the range of the technology design of the application, every invention of the application can be variously changed and be modified, Technical solution after these variations also belongs to the protection scope of the application.
It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the application to it is various can No further explanation will be given for the combination of energy.
In addition, any combination can also be carried out between a variety of different embodiments of the application, as long as it is without prejudice to originally The thought of application equally should be considered as content disclosed in the present application.
Embodiment
Following embodiment is provided and further describes the application, and is not limited.
The kit of 1. the application of embodiment is compared with commercial reagents box
In the present embodiment, by the exemplary kit of the application, (extracting solution is that the methanol that concentration is 90 volume % is water-soluble Liquid, mobile phase are the mixture comprising water, acetonitrile and five fluorine propionic acid, and wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 80: 20:0.2) and commercial reagents box (the non-derived kit of PerkinElmer company, the U.S.) compares test, in tandem-mass spectrometry It is carried out on instrument Waters TQD, detecting same dry blood spot Quality Control sample, (clinical examination center provides external quality assurance product, criticizes Number 201622) concentration of amino acid and carnitine, average value, standard deviation and the coefficient of variation of calculated result in.
The use of the kit of the application carries out in accordance with the following methods:
1. by amino acid isotopic standard product, fatty acyl carnitine isotopic standard product, methylmalonic acid isotopic standard product Concentrate dilution and mixed preparing dilute the standard items after redissolving with 1:200 ratio extracting solution and extract working solution；
2. quality control standard product and sample to be tested (such as the dry blood spot sample for being soaked with peripheral blood) is added in punching；
It volatilizees 3. every hole is added prepared 100 μ l of extraction working solution and is covered with adhesive cover and reduces；
700rpm shakes frequency oscillation incubation 45 minutes at 4.35 DEG C；
5. throwing off adhesive cover from plate, the heat-resisting microwell plate in V-type bottom of 80 μ l to number is shifted in every hole；
It is dry under stream of nitrogen gas 6. 50 DEG C of temperature conditions；
7. 50 μ l derivatization reagents (chloroacetic chloride and n-butanol mixture, volume ratio 1:9) are added in every hole, sealed with sealer Mouthful, it prevents from volatilizing；
8. standing 30 minutes under the conditions of 60 DEG C of temperature；
9. throw off sealer, 50 DEG C of temperature conditions are dry under stream of nitrogen gas；
10. 85 μ l mobile phases are added in every hole, with aluminium foil film sealing plate；
11., with the frequency of 550rpm, being vibrated 10 minutes under the conditions of 27 DEG C of temperature；
12. plate is put into autosampler, suitable instrument parameter, upper machine testing are selected.
Commercial reagents box is operated according to its specification.Detection data is shown in Table 1.Compared with commercial reagents box, the application Kit detection effect it is highly desirable, and in the coefficient of variation of certain indexs be better than commercial reagents box.
Table 1
Detection performance test of the kit of 2. the application of embodiment in normal newborn
The kit that the present embodiment uses is with embodiment 1, and detection method is also referring generally to embodiment 1.
1520 normal term newborn blood sample samples are detected, when being born 72 hours and sufficiently lactation Heel blood is acquired, is detected.Amino acid, methylmalonic acid and fatty acyl carnitine concentration reference value range are shown in Table 2 and table 3.For The testing result of whole normal newborns each falls within range of normal value.
Amino acid and methylmalonic acid concentration reference value range in 2 normal newborn blood of table
Fatty acyl carnitine concentration reference value range in 3 normal newborn blood of table
Detection performance test of the kit of 3. the application of embodiment in Inherited Metabolic Disorders patient
The kit that the present embodiment uses is with embodiment 1, and detection method is also referring generally to embodiment 1.
The blood sample of 152 Inherited Metabolic Disorders patients is detected.Table 4 shows Inherited Metabolic Disorders classification, correspondence Patient's number, abnormal index and testing result range.In bird amino acid carbamylrtansferase deficiency disease patient, refer to extremely Mark Cit level can be lower than normal reference value；In other diseases, abnormal index level can be higher than normal reference value.It will be examined in table 4 It is found that obtained testing result meets water of each abnormal index in disease after result is surveyed compared with each index normal reference value Flat, thus the kit of proof the application effect in Inherited Metabolic Disorders detection is good.
4 Inherited Metabolic Disorders detection data of table
Application of 4. kit of embodiment in methylmalonic acidemia patient's antidiastole
The kit that the present embodiment uses is with embodiment 1, and detection method is also referring generally to embodiment 1.
Methylmalonic acidemia and the Testing index of propionic acidemia include usually C3, but the level of C3 cannot distinguish between this Two kinds of diseases, and the sample of some normal individuals also will appear C3 and increase (false positive).In the present embodiment, to from 97 The sample of methylmalonic acidemia patient and 3 propionic acidemia patients and C3 is more than normal value in neonatal screening The sample of 230 healthy individuals samples and the horizontal normal individual of 1520 C3 carries out the detection of methylmalonic acid level, detection knot Fruit summarizes and is shown in Table 5.In methylmalonic acidemia patient, methylmalonic acid level is significantly higher than other samples, and propionic acidemia is suffered from Person and C3 increase the methylmalonic acid level in normal sample in range of normal value.Therefore, the reagent of the application is utilized Box joint-detection C3 and methylmalonic acid help to improve the specificity of methylmalonic acidemia patient.
5 methylmalonic acid testing result of table

Claims (10)

1. tandem mass spectrometry detection kit includes extracting solution and mobile phase, and optionally comprising quality control standard product, target point One of isotopic standard product and derivatization reagent of object or a variety of is analysed, wherein it is 85-95 body that the extracting solution, which is concentration, The methanol aqueous solution of product %, the mobile phase are the mixture comprising water, acetonitrile and five fluorine propionic acid, wherein water, acetonitrile and five fluorine The volume ratio of propionic acid is 70-90:15-25:0.15-0.25.
2. kit as described in claim 1, wherein the derivatization reagent is chloroacetic chloride and n-butanol mixture, preferably Ground, chloroacetic chloride and n-butanol volume ratio are 1:8.5-9.2.
3. kit as claimed in claim 1 or 2, wherein the target analytes are selected from amino acid, fatty acyl carnitine, methyl-prop Diacid and above-mentioned any combination.
4. with the method for target analytes in tandem mass spectrometry test sample, comprising the following steps:
(a) the isotopic standard product of the target analytes are diluted with extracting solution, to prepare extraction working solution, wherein the extraction Liquid is the methanol aqueous solution that concentration is 85-95 volume %；
(b) sample is handled with the extraction working solution, and be dried；
(c) sample after the extraction process from step (b) is handled with derivatization reagent, and is dried；
(d) sample after the derivatization treatment from step (c) is redissolved with mobile phase, wherein the mobile phase is to include water, second The mixture of nitrile and five fluorine propionic acid, wherein the volume ratio of water, acetonitrile and five fluorine propionic acid is 70-90:15-25:0.15-0.25；With And
(e) sample after redissolution of the tandem mass spectrometer detection from step (d).
5. method as claimed in claim 4, wherein the derivatization reagent is chloroacetic chloride and n-butanol mixture, it is preferable that Chloroacetic chloride and n-butanol volume ratio are 1:8.5-9.2.
6. method as described in claim 4 or 5, wherein the target analytes are selected from amino acid, fatty acyl carnitine, methyl-prop two Acid and above-mentioned any combination.
7. the method as described in any one of claim 4-6 further includes parallel with the sample in step (b)-(e) is located in Reason and detection quality control standard product.
8. purposes of the following substances in tandem mass spectrometry detection kit of the preparation for Inherited Metabolic Disorders diagnosis:
Extracting solution, the extracting solution are the methanol aqueous solution that concentration is 85-95 volume %；
Mobile phase, the mobile phase are the mixture comprising water, acetonitrile and five fluorine propionic acid, wherein water, acetonitrile and five fluorine propionic acid Volume ratio is 70-90:15-25:0.15-0.25；With
One of isotopic standard product and derivatization reagent of optional quality control standard product, target analytes are a variety of.
9. purposes as claimed in claim 8, wherein the Inherited Metabolic Disorders are selected from methylmalonic acidemia, hyperphenylalaninemia Disease, hyperphenylalaninemia, bird amino acid carbamylrtansferase deficiency disease, propionic acidemia, short chain acyl CoA dehydrogenase azymia Disease (SCAD), citrullinemia, pole long chain acyl Co A Dehydrogenase Deficiency (VLAD), tyrosinemia, ketothiolase lack Disease, glutaric acidemia I type (GA-I), isovaleric acidemia and hypermethioninemia.
10. purposes as claimed in claim 8 or 9, wherein the derivatization reagent is chloroacetic chloride and n-butanol mixture, preferably Ground, volume ratio 1:9；And/or
Wherein the target analytes are selected from amino acid, fatty acyl carnitine, methylmalonic acid and above-mentioned any combination.
CN201810117512.4A 2018-02-06 2018-02-06 Detect a variety of amino acid and fatty acyl carnitine, the method for methylmalonic acid and kit Pending CN110118845A (en)

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Publication number Priority date Publication date Assignee Title
US20040086521A1 (en) * 2002-10-02 2004-05-06 Harald Kropshofer Method for the identification of antigenic peptides
CN103163226A (en) * 2011-12-14 2013-06-19 刘丽宏 A simultaneous quantitative detection method of 30 amino acids and a preparation method thereof
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
CN106442758A (en) * 2016-08-31 2017-02-22 陈大为 Liquid mass spectrometry method for detecting various amino acids in human blood plasma in underivatized mode
CN107091894A (en) * 2017-06-14 2017-08-25 杭州博圣生物技术有限公司 The method of Liquid Chromatography-Tandem Mass Spectrometry detection methylmalonic acid, methyl citric acid and/or homocysteine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040086521A1 (en) * 2002-10-02 2004-05-06 Harald Kropshofer Method for the identification of antigenic peptides
CN103163226A (en) * 2011-12-14 2013-06-19 刘丽宏 A simultaneous quantitative detection method of 30 amino acids and a preparation method thereof
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
CN106442758A (en) * 2016-08-31 2017-02-22 陈大为 Liquid mass spectrometry method for detecting various amino acids in human blood plasma in underivatized mode
CN107091894A (en) * 2017-06-14 2017-08-25 杭州博圣生物技术有限公司 The method of Liquid Chromatography-Tandem Mass Spectrometry detection methylmalonic acid, methyl citric acid and/or homocysteine

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