CN109324132A - Kit and its application of high throughput detection tryptophan and its metabolite - Google Patents
Kit and its application of high throughput detection tryptophan and its metabolite Download PDFInfo
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- CN109324132A CN109324132A CN201811286339.7A CN201811286339A CN109324132A CN 109324132 A CN109324132 A CN 109324132A CN 201811286339 A CN201811286339 A CN 201811286339A CN 109324132 A CN109324132 A CN 109324132A
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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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention discloses a high-throughput method detection tryptophan and its kits of metabolite, are applicable in the content of tryptophan described in detection sample and its metabolite, comprising the following steps: the preparation of tryptophan metabolism access substance quantitative criterion correction curve；The preparation of sample to be tested；High-throughput liquid chromatogram separating tryptophane and metabolite；Triple level four bars mass spectrographs detect the tryptophan and metabolite；The standard correction curve of the tryptophan and metabolite is obtained, the content of the tryptophan and its metabolite in biological sample is efficiently easily detected.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of high-throughput detection tryptophan and its metabolite
Kit and its application are applied to quickly and efficiently detect the content of tryptophan and its metabolite in biological sample.
Background technique
Tryptophan is in addition to being used to synthesize various protein, mainly in liver brain, Shen Deng tissue metabolism.There are three types of its approach,
First is that most of tryptophans are metabolized through kynurenine pathway, i.e., tryptophan is in indoles amine -2,3- dioxygenase (indoleamine-
2,3-dioxygenase, IDO) under the action of generate formylkynurenine, the latter is rapidly in the effect of formamidase
Lower generation kynurenin, kynurenin further generate kynurenic acid under the action of kynurenine aminotransferase
(kynurenic acid, Kyna), major part tryptophan is metabolized through this approach in vivo.The second is a small amount of tryptophan is through tryptophan hydroxyl
Change enzyme and generate 5-HTP, further decarboxylation generates serotonin and 5 hydroxyindole acetic acids.Thirdly there are also minute quantity tryptophans to pass through
The metabolism of heteroauxin approach.Tryptophan is one of constituent of protein, participates in the synthesis of regulatory protein matter, moreover it is possible to promote stomach
The generation of liquid and pancreatic juice promotes human consumption's process.Serotonin has antidepression, promotes sleep, analgesia, antihypertensive function
Energy.Kynurenin is one of most important mesostate of tryptophan, can be metabolized as a series of have the function of special physiological
Bioactive molecule.In short, tryptophan and its metabolite have important work to maintenance central nervous system normal physiological function
With, and 5-hydroxyindoleacetic acid content can be used as the biomarker of monitoring class cancer patient in blood plasma.
The method of detection tryptophan, kynurenin, serotonin and 5 hydroxyindole acetic acids mainly has bioanalysis, radiation at present
Immunization, radiation enzyme process, fluorimetry etc..The bioanalysis of early stage has not had to；Though and radioimmunology have it is preferable sensitive
Degree and specificity, but isotope labelling is harmful to the human body；Though fluorescence method is using more, troublesome in poeration, need to extract repeatedly, it is right
Detection substance has a certain amount of loss.
In short, existing clinically not yet have the detection method for having the property approved for tryptophan and its metabolite, because
This, detect tryptophan and its metabolite content method with researching and developing a set of convenient and efficient is to be of great significance and influence
Power.
Summary of the invention
The object of the present invention is to provide kit and its application of a high-throughput detection tryptophan and its metabolite,
The tryptophan and its high-throughput liquid chromatography tandem mass spectrometry of metabolite high-flux detection method application are efficiently and rapidly examined
The content of colour examining propylhomoserin and its metabolite.
The object of the present invention is to provide kit and its application of a high-throughput detection tryptophan and its metabolite,
It is described when preparing sample to be tested, using centrifugation twice and the preparation work of supernatant, the purity for preparing sample to be tested is improved, is mentioned
The precision of its high detection.
The object of the present invention is to provide kit and its application of a high-throughput detection tryptophan and its metabolite,
It is described when preparing sample to be tested, using its vortex mixed, its extraction yield is improved, and easy to operate easy to handle.
The purpose of the present invention is to provide kit and its application of a high-throughput detection tryptophan and its metabolite,
Described in tryptophan and its metabolite efficient detection method have extraction processing step simple, it is low in cost, it is easy to operate etc.
Advantage.
The purpose of the present invention is to provide kit and its application of a high-throughput detection tryptophan and its metabolite,
Described in tryptophan and its metabolite efficient detection method detection time it is short, flux is high, and detection sensitivity is high, and specificity is good.
In order to achieve the above object, major technique solution of the invention is to provide high-throughput method detection tryptophan and its metabolism
The kit of product is applicable in tryptophan and its metabolite described in detection sample, which is characterized in that
Detecting metabolite includes: tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid；
The kit includes following reagent:
1. mixing internal standard compound: tryptophan-D5, kynurenin-D4, serotonin-D4,5-hydroxyindoleacetic acid-D5；
2. standard items contain: tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid；
3. eluent: eluent A is 0.1% aqueous formic acid, and eluent B is 0.1% formic acid acetonitrile solution；
4. diluent: methanol solution.
The standard items are prepared into standard items mother liquor by the methanol solution, wherein color ammonia in the standard items mother liquor
Acid, kynurenin, serotonin, 5-hydroxyindoleacetic acid concentration be respectively as follows: 3nmol/L~650nmol/L, 3nmol/L~
650nmol/L, 0.99 μm of o/L~200.00 μm o/L and 0.063 μm of o/L~12.77 μm o/L.
The mixing internal standard compound is prepared into mixing internal standard solution by the methanol solution, wherein color in the mixing internal standard solution
Propylhomoserin-D5, kynurenin-D4, serotonin-D4,5-hydroxyindoleacetic acid-D5 concentration be 35~36nmol/L.
The preparation of the eluent A: 1000 parts by volume ultrapure waters are taken, the water of 1 parts by volume is removed using liquid-transfering gun, is added
The formic acid of 1 parts by volume mixes well spare after ultrasonic 5min；
The preparation of eluent B: taking the chromatography acetonitrile of 1000 parts by volume, removes 1 parts by volume water using liquid-transfering gun, adds 1
The formic acid of parts by volume, it is spare after ultrasonic 5min using mixing well.
The application of the kit, which is characterized in that
It is described that tryptophan and metabolite in plasma sample are detected using high-throughput Liquid Chromatography-Tandem Mass Spectrometry technology, it is first sharp
Tryptophan and metabolite are separated with high-throughput liquid chromatogram, tandem mass spectrum Isotopic Internal Standard sizing technique is recycled, with standard
The concentration value of product is X-axis, and the peak area ratio of standard items and internal standard compound is Y-axis, establishes standard correction curve, calculates above-mentioned tryptophan
And the content of metabolite.
The tryptophan and its metabolite high-flux detection method the following steps are included:
S1: tryptophan metabolism access substance quantitative criterion correction curve: the wherein tryptophan metabolism access class is obtained
Substance quantitative criterion correction curve is implemented as the tryptophan of various concentration and its label color ammonia of metabolite and known concentration
Relational graph between acid and its actual ratio of metabolite；
S2: the preparation of sample to be tested: taking plasma sample, and mixing internal standard solution is added and carries out vortex mixed, then generates albumen
Precipitating, stand etc. sufficiently precipitating after, carry out first time centrifugation, obtain the first supernatant, pipette first supernatant utilize from
After the freeze-drying of heart concentrated frozen system, second of centrifugation after aqueous solution is added, obtains the second supernatant, second supernatant is taken to make
For sample to be tested, it is placed in spare on 96 orifice plates；
S3: high-throughput liquid chromatogram separating tryptophane and metabolite: high-throughput liquid chromatogram separating tryptophane and metabolism
Product utilization ultra performance liquid chromatography and corresponding mobile phase, to tryptophan in sample to be tested and metabolite on analytical column
Chromatography eluant separation is carried out, by controlling elution requirement, isolates tryptophan and metabolite；
S4: tandem mass spectrum detects the tryptophan and metabolite: being supervised using the multiple reaction in triple level four bars mass spectrums
Control mode specifically detects the tryptophan and metabolite, detected value is obtained, wherein it is described that the detected value, which is reacted,
Tryptophan and metabolite and the ratio for marking the tryptophan and metabolite peak area；And
S5: it obtains the content of the propylhomoserin and metabolite: the detected value is substituted into the tryptophan metabolism access class
Substance quantitative criterion correction curve, is calculated the content of tryptophan described in the sample and its metabolite.
The step S1 is further included steps of
S11: the preparation of tryptophan and its metabolite standard solution: by tryptophan and its metabolite known concentration
Methanol solution is diluted to that several various concentrations are spare, obtains the standard solution of various concentration；
The preparation of S12: the first supernatant: take the standard solution of several various concentrations that containing for equivalent is added respectively known
Concentration internal standard solution obtains the first supernatant after high speed centrifugation；
S13: after first supernatant is using the freeze-drying of centrifugal concentrating refrigeration system, water the preparation of standard detection product: is added
After solution again after centrifugal treating, the second supernatant is obtained, takes second supernatant as standard detection product, is placed in 96 orifice plates
It is upper spare；
S14: high-throughput liquid chromatogram separating tryptophane and metabolite: high-throughput liquid chromatogram separating tryptophane and metabolism
Product utilization ultra high efficiency imitates phase chromatography and corresponding mobile phase, to tryptophan in sample to be tested and metabolite on analytical column
Carry out chromatography eluant separation；
S15: tandem mass spectrum detects tryptophan and metabolite: monitoring mould using the multiple reaction in triple level four bars mass spectrums
Formula specifically detects the content of tryptophan and metabolite, according to signal noise ratio be calculated quantitative detection limit, according to than
Value and known standard concentration value draw canonical plotting.
Methanol-water solution containing known concentration mixing internal standard compound and the ammonium sulfate containing 1.58g/L in internal standard solution, it is described water-soluble
Liquid is selected from aqueous formic acid.
Wherein the separation condition control of the high-throughput liquid chromatogram is as follows: analytical column is reversed C18 analytical column, chromatographic column
Column temperature: 25 DEG C, sample volume: 10 μ L；
The wherein centrifugal condition control are as follows: centrifugal force 18000g, centrifuging temperature are 4 DEG C, centrifugation time 5min,
Described in first time centrifugal condition it is identical as second of centrifugal condition.
Wherein the Mass Spectrometer Method condition is as follows: electron spray needle voltage: 2.0kV, and go solvent stream fast: 850L/h is gone molten
Agent temperature degree: 550 DEG C, taper hole gas velocity: 0L/h is detected as positive ion mode, and scanning mode is multiple reaction monitoring, each to
Survey object mass spectrometry parameters:
Detailed description of the invention
Fig. 1 and Fig. 2 is that the chromatography eluant of tryptophan and its metabolite efficient detection method according to the present invention goes out
The absolute retention time schematic diagram at peak,
Fig. 3 to Fig. 6 is that the tryptophan metabolism access substance of the tryptophan and its metabolite efficient detection method is fixed
Measure standard correction curve synoptic diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art's every other embodiment obtained belong to what the present invention protected
Range.
It will be understood by those skilled in the art that in exposure of the invention, term " longitudinal direction ", " transverse direction ", "upper",
The orientation of the instructions such as "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom" "inner", "outside" or position are closed
System is to be based on the orientation or positional relationship shown in the drawings, and is merely for convenience of description of the present invention and simplification of the description, without referring to
Show or imply that signified device or element must have a particular orientation, be constructed and operated in a specific orientation, therefore above-mentioned art
Language is not considered as limiting the invention.
It is understood that term " one " is interpreted as " at least one " or " one or more ", i.e., in one embodiment,
The quantity of one element can be one, and in a further embodiment, the quantity of the element can be it is multiple, term " one " is no
It can be interpreted as the limitation to quantity.
Tryptophan (TRP) is one of the essential amino acid for needing to absorb from food, and humans and animals body itself is
Tryptophan can not be synthesized, although tryptophan is less in humans and animals in-vivo content, its polynary metabolism can also be passed through
Approach and its metabolite play various important function.As shown in Fig. 2, wherein tryptophan in vivo main catabolic way
Diameter: first is that along the metabolic pathway of serotonin (5-HT), wherein 5-hydroxyindoleacetic acid (5-HIAA) is then along serotonin (5-
HT the final product of metabolic pathway)；Second is that in other words, the metabolite includes along the metabolic pathway of kynurenin (KYN)
Therefore these four have also been turned out for serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and kynurenin (KYN)
Substance is that there are the chemical structure of common property and elements.Regulatory protein can not only be participated in by the intracorporal tryptophan of dietary int ake
The synthesis of matter, moreover it is possible to play a significant role, therefore survey in terms of controlling animal appetite, immunological regulation and improving
Human body tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid concentration are determined for understanding immune status and therewith
The diagnosis and treatment of related disease are of great significance, and tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid are all to belong to
In tryptophan metabolism access substance, but for now, clinically mainly there is bioanalysis for this kind of substance detecting method, puts
Immunization, radiation enzyme process, fluorimetry etc. are penetrated, but the bioanalysis of early stage has not had to；Though and radioimmunology has preferably
Sensitivity and specificity, but isotope labelling is harmful to the human body；Though fluorescence method is using more, troublesome in poeration, need to extract repeatedly
It takes, has a certain amount of loss to detection substance.
In order to solve to detect the defect of substance of this kind in the prior art, the present invention provides a kind of high-throughput detection tryptophans
And its kit and its application of metabolite.Wherein same detection is utilized for the general character of its tryptophan and its metabolite
Method detection, and the high-throughput liquid chromatography tandem mass spectrometry of application quickly and efficiently detects tryptophan and its generation in biological sample
Thank to the content of product, step is simple to operation twice for supernatant preparation, detection sensitivity with higher and detection specificity.
The present invention provides a kind of high-throughput kit and its application for detecting tryptophan and its metabolite, can directly quickly
Tryptophan and its metabolite are detected to ground quantitative and qualitative, cardinal principle is first to produce with the tryptophan of known concentration and its metabolism
Object and auxiliary reagent are with certain method acquisition tryptophan and its metabolite quantitative criterion correction curve, then with identical
Method detection test sample obtains a detected value, and the detected value is updated to the tryptophan and its metabolite quantitative criterion
In correction curve, to calculate the content for obtaining the tryptophan and its metabolite.
Specifically, as shown in Fig. 1 to Fig. 6, the kit of high throughput detection tryptophan and its metabolite is applicable in inspection
Tryptophan described in test sample sheet and its metabolite,
Detecting metabolite includes: tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid；
The kit includes following reagent:
1. mixing internal standard compound: tryptophan-D5, kynurenin-D4, serotonin-D4,5-hydroxyindoleacetic acid-D5；
2. standard items contain: tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid；
3. eluent: eluent A is 0.1% aqueous formic acid, and eluent B is 0.1% formic acid acetonitrile solution；
4. diluent: methanol solution；
In addition, the application of kit, is applicable in the content of tryptophan described in detection sample and its metabolite, and it is described
Tryptophan and its metabolite belong to tryptophan metabolism access substance, which is characterized in that the tryptophan and its metabolite
High-flux detection method the following steps are included:
S1: tryptophan metabolism access substance quantitative criterion correction curve is obtained:
Wherein the tryptophan metabolism access substance quantitative criterion correction curve is implemented as test value and various concentration
Tryptophan and its metabolite and known concentration label tryptophan and its metabolite actual ratio between relational graph；
S2: the preparation of sample to be tested: taking 0.15mL plasma sample into 1.5mL EP pipe, and 0.45mL internal standard solution is added and carries out
Albumen precipitation after standing etc. sufficiently precipitates, carries out first time centrifugation, obtains the first supernatant, it is sharp to pipette first supernatant
After the freeze-drying of centrifugal concentrating refrigeration system, second of centrifugation after aqueous solution is added, obtains the second supernatant, takes second supernatant
Liquid is placed in spare on 96 orifice plates as sample to be tested；
S3: high-throughput liquid chromatogram separating tryptophane and metabolite: high-throughput liquid chromatogram separating tryptophane and metabolism
Product utilization ultra performance liquid chromatography and corresponding mobile phase, to tryptophan in sample to be tested and metabolite on analytical column
Chromatography eluant separation is carried out, by controlling elution requirement, isolates tryptophan and metabolite；
S4: tandem mass spectrum detects the tryptophan and metabolite: being supervised using the multiple reaction in triple level four bars mass spectrums
Control mode specifically detects the tryptophan and metabolite, detected value is obtained, wherein it is described that the detected value, which is reacted,
Tryptophan and metabolite and the ratio for marking the tryptophan and metabolite；And
S5: it obtains the content of the propylhomoserin and metabolite: the detected value is substituted into the tryptophan metabolism access class
Substance quantitative criterion correction curve, is calculated the content of tryptophan described in the sample and its metabolite.
Emphasis has carried out the preparation of supernatant twice it is emphasized that in the preparation of the sample to be tested here, wherein
Internal standard is added to it in addition, the plasma sample is put into EP pipe in the namely required sample to be tested of second supernatant
Liquid, it is molten to be implemented as the methanol-water containing target ammonium sulfate containing 1.58g/L in known concentration for the internal standard solution in this embodiment
The methanol-water solution of liquid, the ammonium sulfate containing 1.58g/L is implemented as organic reagent, it is believed that being engaged in the natural of the technology can be bright
White, the plasma sample carries out vortex mixed after the organic reagent of containing the internal standard is added, and carrying out vortex mixed is to improve its extraction
Take rate, generate albumen precipitation after vortex mixed, standing a period of time makes precipitating more abundant, then carry out for the first time from
The heart, it is 18000g that the centrifugal condition of the present embodiment, which is implemented as centrifugal force,；Centrifuging temperature is 4 DEG C；Centrifugation time is 5min；Wherein
The first time centrifugal condition is identical as second of centrifugal condition, after being centrifuged for the first time, the first supernatant is obtained, on first
Clear liquid is implemented as the mixture of organic reagent and water, also contains sample to be tested, and first supernatant is carried out freezing jelly
After dry, a certain amount of aqueous solution is added, aqueous solution is implemented as formic acid-aqueous solution here, then carries out second and is centrifuged, obtains
Second supernatant, second supernatant i.e. sample to be tested.
It is worth it is specifically intended that in some embodiments, tryptophan metabolism access can be directly acquired by various means
Substance quantitative criterion correction curve, in further embodiments, the tryptophan metabolism access substance quantitative criterion correction
Curve needs user of service voluntarily to prepare.But in the reality for directly acquiring tryptophan metabolism access substance quantitative criterion correction curve
It applies in example, it is worth mentioning at this point that, the preparation condition of the tryptophan metabolism access substance quantitative criterion correction curve is answered identical
In the testing conditions of the sample when detecting.
The preparation process of the tryptophan metabolism access substance quantitative criterion correction curve explained in detail below, it is described
Tryptophan metabolism access substance quantitative criterion correction curve is implemented as tryptophan and its metabolism of test value and various concentration
Relational graph between product.
The step S1 is further included steps of
S11: the preparation of tryptophan and its metabolite standard solution: by tryptophan and its metabolite known concentration
It is spare that methanol solution is diluted to several various concentrations；
In a specific embodiment of the present invention, the concentration of the tryptophan and its metabolite standard solution is individually controlled
In 3nmol/L~650nmol/L, 3nmol/L~650nmol/L, 0.99~200.00 μm of o/L and 0.063~12.77 μm of o/L.
In other words, in the preparation of tryptophan and its metabolite standard solution, the concentration 0.99- of the tryptophan standards solution
Between 200.00 μm of o/L, between 0.063-12.77 μm of o/L of concentration of the kynurenin standard solution, the serotonin mark
Between the concentration 3nmol/L-650nmol/L of quasi- solution and the control of the concentration of the 5-hydroxyindoleacetic acid standard solution exists
Between 3nmol/L-650nmol/L.But the people for being familiar with this technology should be understood that the tryptophan and its metabolite standard are molten
The concentration range of liquid is as just citing, and without limitation.
One specific embodiment is shown in detail in the form of operating process figure.The tryptophan and its metabolite standard items
With methanol dilution to various concentration, the tryptophan and its metabolite standard solution of various concentration are placed in different EP reaction tubes
It is interior, it stands with spare.In order to avoid unnecessary factor generates interference to experiment, differential responses pipe is preferably in same reaction conditions
Under reacted.
After the tryptophan and its metabolite standard solution for getting out various concentration, the step S1 is further wrapped
Include following steps:
The preparation of S12: the first supernatant: the standard solution of 6 various concentrations of 0.15mL is taken to manage to 1.5mL EP respectively
In, then it is separately added into the mixing internal standard solution of 0.45mL, the first supernatant is obtained after being sufficiently mixed with vortex mixer, after high speed centrifugation
Liquid；
It is worth noting that, in an embodiment of the present invention, the internal standard solution is implemented as mixing internal standard compound and contains
The mixed solution of the methanol-water solution of 1.58g/L ammonium sulfate, the mixing internal standard concentration are controlled in an embodiment of the present invention
In 35.7nmol/L, calculated here to the standard solution of each various concentration addition equivalent containing known concentration internal standard solution
Ratio value out.
In one embodiment of this invention, the centrifugal condition control are as follows: centrifugal force 18000g, centrifuging temperature are 4 DEG C,
Centrifugation time is 5min.
The step S1 is further included steps of
S13: the preparation of standard detection product:
After first supernatant is using the freeze-drying of centrifugal concentrating refrigeration system, after centrifugal treating again is added after aqueous solution, obtain
The second supernatant is obtained, takes second supernatant as standard detection product, is placed in spare on 96 orifice plates；
It is noted that the step S12 of the invention and step S13 be suitable for each concentration tryptophan and
The subsequent processing of its metabolite standard solution.In other words, it in the step S12, is mixed in a certain proportion respectively different dense
The tryptophan of degree and its internal standard working solution of metabolite standard solution and known concentration, obtain containing various concentration color ammonia
The supernatant of acid and its metabolite.In the step S13, it is added described in aqueous solution and centrifugation freezing processing and contains various concentration
The supernatant of tryptophan and its metabolite obtains the standard detection product containing various concentration tryptophan and its metabolite.
The step S1 is further included steps of
S14: high-throughput liquid chromatogram separating tryptophane and metabolite:
High-throughput liquid chromatogram separating tryptophane and metabolite utilize ultra performance liquid chromatography and corresponding mobile phase,
Chromatography eluant separation is carried out to tryptophan in sample to be tested and metabolite on analytical column；
Specifically, in one embodiment, high-throughput liquid chromatogram separation condition in the step S14 are as follows:
Analytical column: reversed C18 analytical column；Chromatographic column column temperature: 25 DEG C；Sample volume: 10 μ L；Flowing phase composition: A phase is
0.1% formic acid-aqueous solution, B phase are 0.1% formic acid-acetonitrile solution.
Gradient is as follows, and it is as follows to be shown in Table 1:
| Serial number | Time (min) | Flow velocity (mL/min) | A% | B% | Curve values |
| 1 | Initial | 0.35 | 95.0 | 5.0 | Initial |
| 2 | 1.00 | 0.35 | 95.0 | 5.0 | 1 |
| 3 | 1.50 | 0.40 | 75.0 | 25.0 | 6 |
| 4 | 1.80 | 0.50 | 40.0 | 60.0 | 6 |
| 5 | 2.00 | 0.50 | 5.0 | 95.0 | 6 |
| 6 | 2.80 | 0.50 | 5.0 | 95.0 | 1 |
| 7 | 3.30 | 0.50 | 95.0 | 5.0 | 1 |
Table 1
The step S1 is further included steps of
S15: tandem mass spectrum detects tryptophan and metabolite:
Tryptophan and metabolite are specifically detected using the multiple reaction monitoring mode in triple level four bars mass spectrums
Content is calculated quantitative detection limit according to signal noise ratio, is drawn according to ratio and known standard items and internal standard concentration value
Canonical plotting.
Be worth noting is that after obtaining standard curve, can further obtain quantitative correction equation, the quantitative correction equation is anti-
Reflect the reality of the tryptophan of test value and various concentration and its label tryptophan and its metabolite of metabolite and known concentration
Functional relation between the ratio of border.
In a specific embodiment of the invention, in the step S15, the Mass Spectrometer Method condition are as follows:
Electron spray needle voltage: 2.0kV goes solvent stream fast: 850L/h, removes solvent temperature degree: 550 DEG C, taper hole gas velocity:
0L/h is detected as positive ion mode, multiple reaction monitoring, and mass spectrum multiple reaction monitoring parameter is shown in Table 2:
Table 2
It is worth noting that, the accuracy in order to guarantee experimental data, in the step S2, the step S2 waits for test sample
The reaction condition of the preparation of product will be unanimously to the preparation of the first supernatant and the preparation of S13 Plays sample in the S12.
In the same manner, the reaction condition of high-throughput liquid chromatogram separating tryptophane and metabolite is also wanted in the step S3
It is unanimously to the reaction condition of high-throughput liquid chromatogram separating tryptophane and metabolite in S14.
Tandem mass spectrum, which detects the tryptophan and the reaction condition liquid of metabolite, in the step S4 will be unanimously to institute
State the reaction condition that tandem mass spectrum in step S15 detects the tryptophan and metabolite.The above reaction condition, it is no longer superfluous herein
It tells.
The present invention provides a specific embodiment, in this specific embodiment,
Tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid and isotope labelling internal standard (tryptophan-D5,
Kynurenin-D4, serotonin-D4,5-hydroxyindoleacetic acid-D5) it is purchased respectively in Sigma and Toronto Research
Chemicals company.Standard items are dissolved with methanol solution, and then packing is stored in -80 DEG C, need to prepare standard curve
When, above-mentioned standard product are diluted between 3nmol/L~650nmol/L with methanol solution, between 3nmol/L~650nmol/L,
Between 0.99~200.00 μm of o/L, 0.063~12.77 μm of o/L, interior target concentration is 35.7nmol/L.
(1) sample preparation: it is added that a certain amount of (internal standard is isotope labelling containing known concentration internal standard into sample to be tested
Test substance) the ammonium sulfate containing 1.58g/L methanol-water solution, Aspirate supernatant after high speed centrifugation；Supernatant uses low temperature
Centrifuge is centrifuged 5min at 4 DEG C of 18000g centrifugal force and a certain amount of formic acid-aqueous solution is added after the freeze-drying of concentrated frozen system
Afterwards, it reuses refrigerated centrifuge mixed solution is centrifuged at 4 DEG C of 18000g centrifugal force, takes supernatant in 96 orifice plates
It is analyzed for Liquid Chromatography-Tandem Mass Spectrometry loading；
(2) liquid chromatogram separates: ultra performance liquid chromatography and corresponding mobile phase is utilized, on reversed C18 analytical column
Chromatography eluant separation is carried out to tryptophan in sample and metabolite, by controlling elution requirement, isolates tryptophan and metabolism
Product；
Chromatographic column: Waters ACQUITY UPLCC18Column:pore sizeparticle size
1.7μm,2.1mm×50mm；cat.#186002350IVD；Chromatographic column column temperature: 25 DEG C；Sample volume: 10 μ L；Flow phase composition: A
It is mutually 0.1% formic acid-aqueous solution (volume ratio), B phase is 0.1% formic acid-acetonitrile solution (volume ratio)
(3) Mass Spectrometer Method and standard curve processed: the tryptophan and metabolite isolated in liquid chromatogram enter triple
Level four bars mass spectrum is detected, using the multiple reaction monitoring mode in triple level four bars mass spectrums specifically detect tryptophan and
The content of metabolite, and draw canonical plotting processed；
Tryptophan and metabolite after separating in chromatography enter Waters Xevo TQD mass spectrum and are detected, and utilize
Multiple reaction monitoring mode in triple level four bars mass spectrums, setting specifically detect tryptophan and metabolite；Serotonin
Retention time is 0.97min, and deuterated serotonin retention time is 0.97min, and 5-hydroxyindoleacetic acid retention time is
1.90min, deuterated 5-hydroxyindoleacetic acid retention time are 1.90min, and tryptophan retention time is 1.80min, deuterated tryptophan
Retention time is 1.80min, and kynurenin retention time is 1.17min, and deuterated kynurenin retention time is 1.15min.Sample
After product are separated by ultra performance liquid chromatography, tryptophan and metabolite are selected in specific elution time appearance by mass spectrum
Reaction monitoring mode detection is selected to arrive.
Mass spectrum is Waters Xevo TQD IVD (Waters, Milford, MA)；Mass Spectrometer Method condition is as follows: electron spray
Needle voltage: 2.0kV, go solvent stream fast: 850L/h, remove solvent temperature degree: 550 DEG C, taper hole gas velocity: 0L/h, detection are positive
Ion mode, multiple reaction monitoring, the specific reactive ion of each determinand to, residence time, orifice potential, collision energy etc.
It is as shown in the table (different type instrument, collision energy, orifice potential value are different, need independent optimization).
Multiple reaction monitoring by screening twice, i.e., first level four bars carries out specific parent ion screening, second level Four
Bar carries out parent ion fragmentation and generates daughter ion, and third level four bars carry out specific daughter ion screening, has extraordinary detection special
It is anisotropic.The ion stream and corresponding retention time that can be detected by selection reaction monitoring, to determine tryptophan and metabolism
The detection of product recycles the internal standard of addition known quantity to be quantified.
Detection limit is defined as signal-to-noise ratio > 3, and quantitative limit is defined as signal-to-noise ratio > 10, and retention time is the exhausted of chromatography eluant appearance
To retention time, > 0.99, the retention time of every kind of color acid ammonia and metabolite is fixed for the coefficient R 2 of all detection substances
Amount limit, the range of linearity and quantitative correction equation difference are as shown in the figure；It is tested by precision on the three, including basic, normal, high
Three concentration, in a few days day to day precision RSD is respectively less than 15%, shows that testing result is accurate, repeats.
The retention time of color acid ammonia and metabolite, quantitative limit, the range of linearity, linear equation, related coefficient are shown in such as table 3:
Table 3
From the preparation for obtaining sample to be tested in biological sample: taking plasma sample to be put into EP pipe, addition contains known concentration
The methanol-water solution of interior target ammonium sulfate containing 1.58g/L carries out vortex mixed, then generates albumen precipitation, and standing etc. is sufficiently heavy
Behind shallow lake, first time centrifugation is carried out, it is 18000g that centrifugal condition, which is controlled in centrifugal force,；Centrifuging temperature is 4 DEG C；Centrifugation time is
5min obtains the first supernatant, after pipetting first supernatant using the freeze-drying of centrifugal concentrating refrigeration system, after aqueous solution is added
Second is centrifuged, consistent with first time centrifugal condition, obtains the second supernatant, takes second supernatant as sample to be tested,
It is placed in spare on 96 orifice plates.
Liquid Chromatography-Tandem Mass Spectrometry analyzes same step (2) and (3), Mass Spectrometer Method obtain tryptophan and its metabolite and mark
The ratio for remembering tryptophan and its metabolite, is updated to quantitative correction equation for ratio, tryptophan in biological sample is calculated
And its content of metabolite.
The present invention is not limited to above-mentioned preferred forms, anyone can show that other are various under the inspiration of the present invention
The product of form, however, make any variation in its shape or structure, it is all that there is skill identical or similar to the present application
Art scheme, is within the scope of the present invention.
Claims (10)
1. the kit of high-throughput method detection tryptophan and its metabolite, is applicable in tryptophan and its generation described in detection sample
Thank to product, which is characterized in that
Detecting metabolite includes: tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid；
The kit includes following reagent:
1. mixing internal standard compound: tryptophan-D5, kynurenin-D4, serotonin-D4,5-hydroxyindoleacetic acid-D5；
2. standard items contain: tryptophan, kynurenin, serotonin and 5-hydroxyindoleacetic acid；
3. eluent: eluent A is 0.1% aqueous formic acid, and eluent B is 0.1% formic acid acetonitrile solution；
4. diluent: methanol solution.
2. the kit of high throughput method detection tryptophan according to claim 1 and its metabolite, which is characterized in that institute
State standard items and standard items mother liquor be prepared by the methanol solution, wherein tryptophan in the standard items mother liquor, kynurenin,
Serotonin, 5-hydroxyindoleacetic acid concentration be respectively as follows: 3nmol/L~650nmol/L, 3nmol/L~650nmol/L, 0.99 μ
Mo/L~200.00 μm o/L and 0.063 μm of o/L-12.77 μm of o/L.
3. the kit of high throughput method detection tryptophan according to claim 2 and its metabolite, which is characterized in that institute
It states mixing internal standard compound and mixing internal standard solution is prepared by the methanol solution, wherein tryptophan-D5, dog in the mixing internal standard solution
Urinary ammonia acid-D4, serotonin-D4,5-hydroxyindoleacetic acid-D5 concentration be 35~36nmol/L.
4. according to claim 1 to the reagent of the claim 3 any high-throughput method detection tryptophan and its metabolite
Box, which is characterized in that the preparation of eluent A: taking 1000 parts by volume ultrapure waters, removes the water of 1 parts by volume using liquid-transfering gun, then plus
The formic acid for entering 1 parts by volume mixes well spare after ultrasonic 5min；
The preparation of eluent B: taking the chromatography acetonitrile of 1000 parts by volume, removes 1 parts by volume water using liquid-transfering gun, adds 1 volume
Part formic acid, mix well spare after ultrasonic 5min.
5. according to claim 1 to the application of kit as claimed in claim 3, which is characterized in that
Using tryptophan and metabolite in high-throughput Liquid Chromatography-Tandem Mass Spectrometry technology detection plasma sample, first with high throughput
Liquid chromatogram separates tryptophan and metabolite, tandem mass spectrum Isotopic Internal Standard sizing technique is recycled, with the concentration of standard items
Being worth is X-axis, and the peak area ratio of standard items and internal standard compound is Y-axis, establishes standard correction curve, calculates above-mentioned tryptophan and metabolism produces
The content of object.
6. the application of kit according to claim 5, which is characterized in that tryptophan and its detection of metabolite high throughput
Method the following steps are included:
S1: tryptophan metabolism access substance quantitative criterion correction curve: the wherein tryptophan metabolism access substance is obtained
Quantitative criterion correction curve be implemented as the tryptophan of various concentration and its label tryptophan of metabolite and known concentration and
Relational graph between the actual ratio of its metabolite；
S2: the preparation of sample to be tested: taking plasma sample, and mixing internal standard solution is added and carries out vortex mixed, then generates albumen precipitation,
After the sufficiently precipitating such as standing, first time centrifugation is carried out, the first supernatant is obtained, pipetted first supernatant and utilize centrifugal concentrating
After refrigeration system freeze-drying, second of centrifugation after aqueous solution is added, obtains the second supernatant, takes second supernatant as to be measured
Sample is placed in spare on 96 orifice plates；
S3: high-throughput liquid chromatogram separating tryptophane and metabolite: high-throughput liquid chromatogram separating tryptophane and metabolite
Using ultra performance liquid chromatography and corresponding mobile phase, tryptophan in sample to be tested and metabolite are carried out on analytical column
Chromatography eluant separation isolates tryptophan and metabolite by controlling elution requirement；
S4: tandem mass spectrum detects the tryptophan and metabolite: monitoring mould using the multiple reaction in triple level four bars mass spectrums
Formula specifically detects the tryptophan and metabolite, detected value is obtained, wherein the detected value is reacted for the color ammonia
Acid and metabolite and the ratio for marking the tryptophan and metabolite peak area；
S5: it obtains the content of the tryptophan and metabolite: the detected value is substituted into the tryptophan metabolism access class object
Matter quantitative criterion correction curve, is calculated the content of tryptophan described in the sample and its metabolite.
7. the application of kit according to claim 6, which is characterized in that the step S1 further comprises following step
It is rapid:
S11: the preparation of tryptophan and its metabolite standard solution: by tryptophan and its methanol of metabolite known concentration
Solution is diluted to that several various concentrations are spare, obtains the standard solution of various concentration；
The preparation of S12: the first supernatant: take the standard solution addition equivalent of several various concentrations respectively contains known concentration
Internal standard solution obtains the first supernatant after high speed centrifugation；
S13: after first supernatant is using the freeze-drying of centrifugal concentrating refrigeration system, aqueous solution the preparation of standard detection product: is added
Afterwards again after centrifugal treating, the second supernatant is obtained, takes second supernatant as standard detection product, is placed in standby on 96 orifice plates
With；
S14: high-throughput liquid chromatogram separating tryptophane and metabolite: high-throughput liquid chromatogram separating tryptophane and metabolite
Using ultra performance liquid chromatography and corresponding mobile phase, tryptophan in sample to be tested and metabolite are carried out on analytical column
Chromatography eluant separation；
S15: tandem mass spectrum detects tryptophan and metabolite: special using the multiple reaction monitoring mode in triple level four bars mass spectrums
The content for detecting tryptophan and metabolite anisotropicly, according to signal noise ratio be calculated quantitative detection limit, according to ratio with
And known standard items and internal standard concentration value draw canonical plotting.
8. the application of kit according to claim 7, which is characterized in that mixed in the internal standard solution containing known concentration
The methanol-water solution of internal standard compound and the ammonium sulfate containing 1.58g/L, the aqueous solution are selected from aqueous formic acid.
9. the application of kit according to claim 8, which is characterized in that
Wherein the separation condition control of the high-throughput liquid chromatogram is as follows: analytical column is reversed C18 analytical column, chromatographic column column
Temperature: 25 DEG C, sample volume: 10 μ L；
The wherein centrifugal condition control are as follows: centrifugal force 18000g, centrifuging temperature are 4 DEG C, centrifugation time 5min, wherein institute
It is identical as second of centrifugal condition to state first time centrifugal condition.
10. the application of kit according to claim 9, which is characterized in that wherein the Mass Spectrometer Method condition is as follows: electricity
Spray needle voltage: 2.0kV goes solvent stream fast: 850L/h, and remove solvent temperature degree: 550 DEG C, taper hole gas velocity: 0L/h is detected
For positive ion mode, scanning mode is that multiple reaction monitors, each determinand mass spectrometry parameters:
。
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| CN109580864A (en) * | 2019-02-18 | 2019-04-05 | 华南农业大学 | The remaining detection method of Nosiheptide in a kind of animal food |
| CN111007173A (en) * | 2019-12-20 | 2020-04-14 | 辉源生物科技（上海）有限公司 | Screening method of indoleamine 2,3-dioxygenase inhibitor |
| CN111077262A (en) * | 2019-12-30 | 2020-04-28 | 中国农业科学院农业质量标准与检测技术研究所 | Method for identifying milk nutritional quality |
| CN113009030A (en) * | 2021-03-01 | 2021-06-22 | 上海阿趣生物科技有限公司 | Amino acid high-throughput target detection method and application thereof |
| CN113406235A (en) * | 2021-06-18 | 2021-09-17 | 广州市红十字会医院（暨南大学医学院附属广州红十字会医院） | Kit and method for detecting tryptophan and metabolites thereof based on UPLC-MS/MS |
| CN114137098A (en) * | 2021-10-28 | 2022-03-04 | 中科新生命(浙江)生物科技有限公司 | Method for detecting tryptophan in human plasma and metabolite thereof |
| CN114509508A (en) * | 2020-11-17 | 2022-05-17 | 麦特瑞思(无锡)科技有限公司 | Method for detecting tryptophan and metabolite and application |
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|---|---|---|---|---|
| CN109580864A (en) * | 2019-02-18 | 2019-04-05 | 华南农业大学 | The remaining detection method of Nosiheptide in a kind of animal food |
| CN111007173A (en) * | 2019-12-20 | 2020-04-14 | 辉源生物科技（上海）有限公司 | Screening method of indoleamine 2,3-dioxygenase inhibitor |
| CN111077262A (en) * | 2019-12-30 | 2020-04-28 | 中国农业科学院农业质量标准与检测技术研究所 | Method for identifying milk nutritional quality |
| CN114509508A (en) * | 2020-11-17 | 2022-05-17 | 麦特瑞思(无锡)科技有限公司 | Method for detecting tryptophan and metabolite and application |
| CN113009030A (en) * | 2021-03-01 | 2021-06-22 | 上海阿趣生物科技有限公司 | Amino acid high-throughput target detection method and application thereof |
| CN113406235A (en) * | 2021-06-18 | 2021-09-17 | 广州市红十字会医院（暨南大学医学院附属广州红十字会医院） | Kit and method for detecting tryptophan and metabolites thereof based on UPLC-MS/MS |
| CN114137098A (en) * | 2021-10-28 | 2022-03-04 | 中科新生命(浙江)生物科技有限公司 | Method for detecting tryptophan in human plasma and metabolite thereof |
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Application publication date: 20190212 |