CN110398554A - A kind of method and application based on Isotopic Internal Standard evaluation endogenous compound matrix effect - Google Patents
A kind of method and application based on Isotopic Internal Standard evaluation endogenous compound matrix effect Download PDFInfo
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Abstract
The present invention provides a kind of method and application based on Isotopic Internal Standard evaluation endogenous compound matrix effect.When the present invention is with choline in liquid chromatography-tandem mass spectrometry detection Milk Powder Formula For Infants and L-carnitine, Isotopic Internal Standard choline-d4 and L-carnitine-d3 corresponding to target analytes is used to carry out matrix effect evaluation (MEIS) for research object.The present invention is aiming at the problem that endogenous material is without blank sample, matrix effect evaluation is carried out by using Isotopic Internal Standard, the analytical procedure of matrix signal value itself need to be deducted by not only having abandoned additive process after traditional extraction, compensate for the shortcomings that matrix effect evaluation can not being carried out using input method after column because of no bare substrate, more importantly matrix effect evaluation can be carried out to all samples analyzed, the value with good practical application.
Description
Technical field
The invention belongs to food analysis technical fields, and in particular to one kind evaluates endogenous compound based on Isotopic Internal Standard
The method and application of matrix effect.
Background technique
Disclosing the information of the background technology part, it is only intended to increase understanding of the overall background of the invention, without certainty
It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art
Art.
Matrix effect is common problem in HPLC-MS/MS detection.When the ionizing efficiency of target analytes is by altogether
When the influence of eluting compounds, matrix effect (matrix effect, ME) will be generated.Since matrix effect not only influences mesh
The response for marking object, has an effect on the precision and accuracy of method, so matrix effect is also that everybody begs in evaluation of methodology
One emphasis of opinion.In order to reduce matrix effect, report, which mostly uses, to be changed Extraction solvent, increases purifying step, optimization chromatostrip
The different ion source etc. of part, selection, but cannot often completely remove.In order to make up matrix effect, document, which mostly uses, prepares matrix
The calibration curve matched is quantified, or is calibrated by Isotopic Internal Standard.
According to the inventors knowledge, the method for evaluation matrix effect mainly has after extraction input method after addition method and column at present.Its
In, addition method is the target analytes that same concentrations are prepared with bare substrate and pure solvent after extraction, using identical instrument item
Part analysis measurement, compares the difference of the two peak area.It is formulated as ME (%)=(B/A-1) × 100, wherein A is pure molten
The peak area of target analytes in agent, B are the peak area for the target analytes that vehicle solution is prepared.Or it is pure by making
The calibration curve of solvent and matrix matching, by comparing the two slope, to determine matrix effect size.This mode can be with quantitative scoring
Calculate matrix effect.And input method is to pass through liquid while peristaltic pump constantly injects certain density target analytes after column
Phase system injects bare substrate sample, and the signal in the method energy real time inspection spectrogram is to inhibit or enhance, it is considered to be one
Kind dynamic technique, it can intuitively show the matrix effect situation of entire collection process, but cannot provide specific numerical value.
However, both technologies are all evaluated just for target compound at present, it is not suitable for endogenous analyte
Matrix effect evaluation.Since there are the samples of endogenous analyte, and bare substrate is not present, so being added after using extraction
A cumbersome calculating process is needed when method.Using peak area ratio compared with when, the peak area of sample itself need to be cut；According to slope
Compare, when making matrix calibration curve, need to consider sample background values.Due to after column infusing method liquid phase input be bare substrate,
So not being suitable for endogenous compound.It is commented importantly, both methods can only carry out matrix effect to specific sample
Valence, it is impossible to which each sample is assessed.
Summary of the invention
For the above-mentioned prior art, the present invention provides a kind of based on Isotopic Internal Standard evaluation endogenous compound matrix effect
Method and application.Specifically, the present invention is directed to Liquid Chromatography-Tandem Mass Spectrometry endogenous material (gallbladder in detection Milk Powder Formula For Infants
Alkali and L-carnitine) when evaluation matrix effect exist due to negative matrix, analyte content is high the problems such as, cause matrix to be imitated
The problems such as inaccurate, cumbersome should be evaluated, thus propose the above method, the value with good practical application.
The present invention is achieved through the following technical solutions:
The first aspect of the invention provides a kind of side based on Isotopic Internal Standard evaluation endogenous compound matrix effect
Method, when the method includes using Liquid Chromatography-Tandem Mass Spectrometry to detect endogenous compound, using corresponding to target analytes
Isotopic Internal Standard carries out matrix effect evaluation.
Further, the above method specifically includes:
The preparation of pure solvent Isotopic Internal Standard solution: Isotopic Internal Standard is added in pure solvent；
The preparation of matrix matching Isotopic Internal Standard solution: Isotopic Internal Standard is added to premenstrual treated sample substrate
In；
Measurement: above two solution injection Liquid Chromatography-Tandem Mass Spectrometry system is analyzed, the peak of two kinds of solution is obtained
Area；
It is calculated using calculation formula MEIS=(A-B)/A × 100, wherein MEIS is Isotopic Internal Standard matrix effect
(abbreviation of the matrix effect of isotope internal standard), A are Isotopic Internal Standard in pure solvent
Response peak area, B be matrix matching Isotopic Internal Standard solution response peak area；
Further, the method also includes the judgements of MEIS, specifically, -20%≤MEIS≤20%, matrix effect
It is negligible；MEIS > 20%, matrix effect inhibit serious；MEIS < -20%, matrix effect enhancing are serious.
The second aspect of the invention provides application of the above method in evaluation endogenous compound matrix effect；Packet
Include but be not limited to the application that evaluation sample pre-treatments mode influences endogenous compound matrix effect.
The invention has the advantages that:
(1) present invention can be to the institute tested using Isotopic Internal Standard evaluation endogenous compound matrix effect (MEIS)
There is sample to carry out matrix effect evaluation；
(2) after the present invention has abandoned traditional extraction using Isotopic Internal Standard evaluation endogenous compound matrix effect (MEIS)
Additive process need to deduct the analytical procedure of matrix signal value itself；
(3) present invention is compensated for using Isotopic Internal Standard evaluation endogenous compound matrix effect (MEIS) because of no blank base
The shortcomings that matter can not carry out matrix effect evaluation using input method after column；
(4) while the present invention simplifies matrix effect evaluation procedure, matrix effect is quantitatively compensated for also by isotopic dilution
It answers, increases the accuracy and precision of method, therefore the value with good practical application.
Detailed description of the invention
The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.
Fig. 1 is that microwave-assisted hydrolysis and water-bath hydrolyze two ways, the endogenous of different types of milk powder in embodiment 1
Close object Isotopic Internal Standard MEIS figure；Wherein choline-d4 is designated as in Fig. 1 (A) endogenous compound；In Fig. 1 (B) endogenous compound
It is designated as L-carnitine-d3.
Fig. 2 is that two kinds of chromatographic columns of ClickXIon and HILIC, the endogenous of different types of milk powder are used in embodiment 1
Close object Isotopic Internal Standard MEIS figure；Wherein choline-d4 is designated as in Fig. 2 (A) endogenous compound；In Fig. 2 (B) endogenous compound
It is designated as L-carnitine-d3.
Fig. 3 is the optimal conditions figure of different disposal condition in embodiment 1；Wherein, Fig. 3 (A) is microwave-assisted hydrolysis acidity
Optimal conditions figure；Fig. 3 (B) is the optimal conditions figure for the treatment of temperature；Fig. 3 (C) is the optimal conditions figure for handling the time.
Fig. 4 is that the second level of choline and L-carnitine scans spectrogram (20ev) in embodiment 1；Wherein, Fig. 4 (A) is choline
Second level scans spectrogram；Fig. 4 (B) is that the second level of L-carnitine scans spectrogram.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.It should be understood that protection model of the invention
It encloses and is not limited to following specific specific embodiments；It is also understood that term used in the embodiment of the present invention is to retouch
Specific specific embodiment is stated, rather than limiting the scope of protection of the present invention.
As previously mentioned, the method for evaluation matrix effect mainly has after extraction input method after addition method and column at present.However, this
Two kinds of technologies are not suitable for the matrix effect evaluation of endogenous analyte.
In view of this, being provided a kind of based on Isotopic Internal Standard evaluation endogenous in the specific embodiment of the present invention
The method of compound matrix effect when the method includes using Liquid Chromatography-Tandem Mass Spectrometry to detect endogenous compound, uses
Isotopic Internal Standard corresponding to target analytes carries out matrix effect evaluation.
In still another embodiment of the invention, the above method is specifically included:
The preparation of pure solvent Isotopic Internal Standard solution: Isotopic Internal Standard is added in pure solvent；
The preparation of matrix matching Isotopic Internal Standard solution: Isotopic Internal Standard is added to premenstrual treated sample substrate
In；
Measurement: above two solution injection Liquid Chromatography-Tandem Mass Spectrometry system is analyzed, the peak of two kinds of solution is obtained
Area；
It is calculated using calculation formula MEIS=(A-B)/A × 100, wherein MEIS is Isotopic Internal Standard matrix effect
(abbreviation of the matrix effect of isotope internal standard), A are Isotopic Internal Standard in pure solvent
Response peak area, B be matrix matching Isotopic Internal Standard solution response peak area；
In still another embodiment of the invention, the method also includes: the judgement of MEIS: specifically, -20%≤
MEIS≤20%, matrix effect are negligible；MEIS > 20%, matrix effect inhibit serious；MEIS < -20%, matrix effect increase
It is strong serious.
In still another embodiment of the invention, the present invention provides a kind of based on Isotopic Internal Standard evaluation endogenous chemical combination
The method of object matrix effect, the method includes using Liquid Chromatography-Tandem Mass Spectrometry detection milk powder endogenous compound choline and a left side
When revolving carnitine, matrix effect evaluation is carried out using Isotopic Internal Standard choline-d4 and L-carnitine-d3, specifically, method includes:
The preparation of pure solvent Isotopic Internal Standard solution: Isotopic Internal Standard is added in pure solvent；
The preparation of matrix matching Isotopic Internal Standard solution: Isotopic Internal Standard is added to premenstrual treated sample substrate
In；
Measurement: above two solution injection Liquid Chromatography-Tandem Mass Spectrometry system is analyzed, the peak of two kinds of solution is obtained
Area；
Calculation formula: MEIS=(A-B)/A × 100, wherein MEIS is Isotopic Internal Standard matrix effect (the matrix
The abbreviation of effect of isotope internal standard), A is the response peak area of Isotopic Internal Standard in pure solvent,
B is the response peak area of matrix matching Isotopic Internal Standard solution；
MEIS determines: -20%≤MEIS≤20%, it is believed that matrix effect can be ignored；MEIS > 20%, matrix effect suppression
System is serious；MEIS < -20%, matrix effect enhancing are serious.
In still another embodiment of the invention, the milk powder includes but is not limited to milk powder, whole milk powder, defatted milk
Powder and goat milk powder.
In still another embodiment of the invention, mass spectrometry parameters condition: the SRM mode under positive ionization electrospray mode,
Ion transfer tube temperature: 350 DEG C, sheath gas: 45arb, auxiliary gas: 10arb, 5500 DEG C of capillary voltage, CID gas: 1.5m
torr；Mass spectrometry parameters, structural formula and retention time of choline, L-carnitine, choline-d4 and L-carnitine-d3 etc. are as follows:
aQuota ion
In still another embodiment of the invention, chromatographic parameter: chromatographic column is Click XIon or HILIC chromatographic column
(preferably Click XIon)；35 DEG C of column temperature；Sampling volume: 0.35 μ L；Flow velocity 0.35mL/min；Type of elution: gradient elution；
The gradient elution program is as follows:
Wherein, using acetonitrile as mobile phase A, using 0.1% aqueous formic acid (ammonium acetate containing 10mM) as Mobile phase B.
In still another embodiment of the invention, the above method is provided in evaluation endogenous compound matrix effect
Using；Including but not limited to evaluate the application that sample pre-treatments mode or chromatographic column influence endogenous compound matrix effect.
In still another embodiment of the invention, the sample is milk powder, further comprises but is not limited to milk powder, complete
Rouge milk powder, skimmed milk power and goat milk powder.
In still another embodiment of the invention, the sample pre-treatments mode includes but is not limited to carry out water to sample
Bath water solution or microwave-assisted hydrolysis；
Preferably, water-bath hydrolysis specific method include: be added into milk powder be preferably 1.0mol/L hydrochloric acid (milk powder with
Hydrochloric acid mass volume ratio is preferably 1g:10mL), it mixes；70 DEG C water-bath 3 hours, after then cooling to room temperature, be diluted with water.
Preferably, the microwave-assisted hydrolysis specific method includes: that preferably 1.0mol/L hydrochloric acid (cream is added into milk powder
Powder and hydrochloric acid mass volume ratio are preferably 1g:10mL), it mixes；Microwave-assisted hydrolysis program: 10min temperature rises to 100 DEG C, In
15min is kept under 1000W, after then cooling to room temperature, is diluted with water.
In still another embodiment of the invention, the chromatographic column includes but is not limited to Click XIon column, T3 column and
Hilic column；Preferably Click XIon column.
Explanation is further explained to the present invention by the following examples, but is not construed as limiting the invention.It should be understood that
These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
Embodiment 1
1, instrument and material
TSQ Quantiva QTRAP system ultra performance liquid chromatography-tandem mass spectrometer of power & light company is (with electron spray electricity
From source)；Chromatographic column: chromatographic column is ClickXIon column (150mm × 3.0mm i.d., 3 μm, the Dalian Chemistry and Physics Institute develops),
BT 125D electronic balance (German Sartorius company)；MS3 type turbine mixer (German IKA company)；SB-800DTD ultrasound
Cleaning device (NingBo XinZhi Biology Science Co., Ltd)；The preparation of Mili-Q ultrapure water machine.
Choline chloride and L-carnitine (being purchased from TRC company) (Toronto, Canada), choline-D4 internal standard and L-carnitine-
D3 internal standard (is purchased from Canadian C/D/N ISOTopes company).The NIST SRM 1849a Quality Control sample is purchased from American National Standard
With technical research institute (Gaithersburg, the Maryland State, the U.S.).The QC-IP-707 quality-control sample is purchased from national measurement research institute
(China, Beijing)；Hydrochloric acid (analysis is pure), ammonium acetate and formic acid (LC/MS grades), ultrapure water.
2, standard reserving solution and standard intermediate fluid
Accurately weigh 10mg choline-D4, L-carnitine-D3 and L-carnitine, 100 milligrams of choline in 10mL volumetric flask,
Scale is settled to after being dissolved with water.Mixed standard solution is diluted bent to prepare suitable concentration calibration with acetonitrile/water (50:50, V/V)
Line, including choline-D4 (200ng/ml) and carnitine-D3 (20ng/ml).
3, sample pre-treatments
Microwave Assisted Process: weighing babies ' formula milk powder (1g) in micro-wave diminishing pot, the hydrochloric acid of 10mL1mol/L be added,
It mixes.Microwave-assisted hydrolysis program: 10min temperature rises under 100 DEG C, then 1000W and keeps 5min, after then cooling to room temperature,
It is diluted with water to 25 milliliters.Take the sample of 100 μ L, 100 μ L inner mark solutions (200ng/mL choline-D4) and 20ng/ml L- meat
Alkali-D3) into another 10mL graded tube, it is settled to scale with acetonitrile/water (50:50, v/v), after mixing, crosses machine measurement on film,
It is measured for liquid chromatography-tandem mass spectrometry instrument.
Method for hydrolysis: weighing babies ' formula milk powder (1g) in 50mL centrifuge tube, and the hydrochloric acid of 10mL 1mol/L is added, and mixes
It is even.70 DEG C water-bath 3 hours, after then cooling to room temperature, be diluted with water to 25 milliliters.The sample of 100 μ L is taken, 100 μ L internal standards are molten
Liquid (200ng/mL choline-D4) and 20ng/ml l-carnitine-D3) into another 10mL graded tube, with acetonitrile/water (50:50, v/
V) it is settled to scale, after mixing, crosses machine measurement on film.
4, liquid chromatogram and Mass Spectrometry Conditions
4.1 chromatographic conditions: chromatographic column be ClickXIon column (150mm × 3.0mm i.d., 3 μm, the Dalian Chemistry and Physics Institute
It develops)；35 DEG C of column temperature；Sampling volume: 5 μ L；Flow velocity: 0.35mL/min；With 0.1% formic acid water (ammonium acetate solution containing 10mM)
(Mobile phase B) and acetonitrile (mobile phase A) gradient elution, type of elution: gradient elution is shown in Table 1.
1 condition of gradient elution of table
4.2 Mass Spectrometry Conditions: the TSQ Quantiva QTRAP system of power & light company, using under positive ionization electrospray mode
SRM mode, instrument parameter are shown in Table 2.Ion transfer tube temperature: 350 DEG C, sheath gas: 45arb assists gas: 10arb, capillary voltage
5500 DEG C, CID gas: 1.5m torr.
The relevant parameters such as parent ion, daughter ion, the impact energy of 2 choline of table, L-carnitine and its Isotopic Internal Standard
A: quota ion
5, result
5.1 ranges of linearity and quantitative limit
According to the content of choline in milk powder and L-carnitine, suitable calibration curve is made, with ultra performance liquid chromatography-string
Connection mass spectrograph is detected, using the peak area ratio of target analytes and corresponding internal standard compound as ordinate, with standard solution
Concentration be abscissa, draw standard curve.In its concentration range, linear relationship is good for choline and L-carnitine, linear phase
Relationship number is all larger than 0.99.Curve concentration minimum point to be capable of accurate quantitative analysis is shown in Table 3 as the quantitative limit of method.
The 5.2 method rate of recovery and precision
Due to containing endogenic choline and L-carnitine in milk powder, so the scalar quantity of two concentration levels has been selected to comment
The precision and the rate of recovery of valence method.Each concentration level is measured in parallel 6 times, and the rate of recovery and precision, concrete outcome are shown in Table 3.
The result relative standard deviation that two kinds of compound rate of recovery are measured in parallel at 98.1%~103%, 6 time is less than 5%, measurement result
Favorable reproducibility.
The rate of recovery of choline and L-carnitine, precision, quantitative limit, the range of linearity in 3 milk powder of table
Experimental example 1
The matrix effect (MEIS) of two kinds of hydrolysis methods in embodiment 1 is compared, sees Fig. 1.For milk powder, entirely
Rouge milk powder, the MEIS of skimmed milk power choline-d4 under water bath condition are obviously higher than microwave mode MEIS, and L-carnitine-d3 is just
It is good opposite.And the MEIS difference of goat milk powder two kinds of internal standard substances under two kinds of hydrolysis methods is little.
Experimental example 2
Chromatographic column in embodiment 1 is changed to HILIC chromatographic column, compares two kinds of chromatographic columns of Click XIon and HILIC
MEIS is shown in Fig. 2.For selected milk powder, whole milk powder, skimmed milk power and goat milk powder, using Click Xion chromatographic column
When, the MEIS of choline-d4 and L-carnitine-d3 are significantly lower than HILIC chromatographic column, this is possibly due to Click Xion is key
The stationary phase of amphoteric compound has been closed, potential interference object and target analytes can have been separated, to reduce matrix effect.
Experimental example 3
By acid concentration (0.5,1.0,1.5,2,2.5, and 3.0mol/L) in condition microwave-assisted in embodiment 1, hydrolysis
Temperature (70 DEG C, 100 DEG C, 90 DEG C, 100 DEG C, 150 DEG C, 180 DEG C) and hydrolysis time (15,20,30, and 60min) are compared
Compared with seeing Fig. 3.Being ultimately determined to acid concentration is 1.0mol/L, 100 DEG C of hydrolysis temperature and hydrolysis time 15min.With traditional water-bath
Method compares, and the two ways rate of recovery and precision are all fine.But microwave hydrolysis only needs 30min, and water-bath hydrolysis needs 3h.
So the microwave-assisted hydrolysis mode of final choice.
Experimental example 4
Chromatographic column in embodiment 1 is changed to T3 and Hilic, from chromatographic peak profile, response, retention characteristic and matrix effect ratio
Compared with tri- kinds of chromatographic columns of Click XIon column, T3 and Hilic, as a result T3 chromatographic column retains weak, less than 1min with regard to appearance.
Hilic and Click Xion reservation is all fine, but matrix effect Click Xion is significantly lower than Hilic (being detailed in experimental example 2), institute
With final choice Click Xion chromatographic column.
Experimental example 5
Mass Spectrometry Conditions in embodiment 1 are optimized, under cation ESI mode, full scan is carried out and optimizes ionspray
Voltage, ion source temperature, temperature of vaporization chamber, sheath gas, auxiliary agent gas and capillary voltage obtain molecular ion [M+H]+, then into
Row daughter ion scanning optimization impact energy.The lytic pathway of choline and L-carnitine is studied.It was found that choline and left-handed meat
Alkali has a common ion (CH3)3N+(m/z 60) (Fig. 4), therefore this research is qualitative using three daughter ions progress.SRM's
Parameter, the impact energy including target compound and internal standard compound, is shown in Table 2.
It should be noted that above example is only used to illustrate the technical scheme of the present invention rather than is limited.Although ginseng
It is described the invention in detail according to given example, but those skilled in the art can be as needed to this hair
Bright technical solution is modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. a kind of method based on Isotopic Internal Standard evaluation endogenous compound matrix effect, which is characterized in that the method packet
When including using Liquid Chromatography-Tandem Mass Spectrometry detection endogenous compound, carried out using Isotopic Internal Standard corresponding to target analytes
Matrix effect evaluation.
2. the method as described in claim 1 based on Isotopic Internal Standard evaluation endogenous compound matrix effect, feature exist
In method specifically includes:
The preparation of pure solvent Isotopic Internal Standard solution: Isotopic Internal Standard is added in pure solvent；
The preparation of matrix matching Isotopic Internal Standard solution: Isotopic Internal Standard is added in premenstrual treated sample substrate；
Measurement: above two solution injection Liquid Chromatography-Tandem Mass Spectrometry system is analyzed, the peak area of two kinds of solution is obtained；
It is calculated using calculation formula MEIS=(A-B)/A × 100, wherein MEIS is Isotopic Internal Standard matrix effect, and A is pure
The response peak area of Isotopic Internal Standard in solvent, B are the response peak area of matrix matching Isotopic Internal Standard solution.
3. the method as claimed in claim 2 based on Isotopic Internal Standard evaluation endogenous compound matrix effect, feature exist
In the method also includes the judgements of MEIS: specifically, -20%≤MEIS≤20%, matrix effect is negligible；MEIS >
20%, matrix effect inhibits serious；MEIS < -20%, matrix effect enhancing are serious.
4. a kind of method based on Isotopic Internal Standard evaluation endogenous compound matrix effect, which is characterized in that the method packet
When including using Liquid Chromatography-Tandem Mass Spectrometry detection milk powder endogenous compound choline and L-carnitine, Isotopic Internal Standard gallbladder is used
Alkali-d4 and L-carnitine-d3 carries out matrix effect evaluation, specifically, method includes:
The preparation of pure solvent Isotopic Internal Standard solution: Isotopic Internal Standard is added in pure solvent；
The preparation of matrix matching Isotopic Internal Standard solution: Isotopic Internal Standard is added in premenstrual treated sample substrate；
Measurement: above two solution injection Liquid Chromatography-Tandem Mass Spectrometry system is analyzed, the peak area of two kinds of solution is obtained；
Calculation formula: MEIS=(A-B)/A × 100, wherein MEIS is Isotopic Internal Standard matrix effect, and A is same position in pure solvent
Target response peak area in element, B are the response peak area of matrix matching Isotopic Internal Standard solution；
MEIS determines: -20%≤MEIS≤20%, and matrix effect is negligible；MEIS > 20%, matrix effect inhibit serious；
MEIS < -20%, matrix effect enhancing are serious.
5. the method as claimed in claim 4 based on Isotopic Internal Standard evaluation endogenous compound matrix effect, feature exist
In the milk powder includes but is not limited to milk powder, whole milk powder, skimmed milk power and and goat milk powder.
6. the method as claimed in claim 4 based on Isotopic Internal Standard evaluation endogenous compound matrix effect, feature exist
In mass spectrometry parameters condition: the SRM mode under positive ionization electrospray mode, ion transfer tube temperature: 350 DEG C, sheath gas: 45arb,
Auxiliary gas: 10arb, 5500 DEG C of capillary voltage, CID gas: 1.5mtorr.
7. the method as claimed in claim 4 based on Isotopic Internal Standard evaluation endogenous compound matrix effect, feature exist
In chromatographic parameter: chromatographic column is Click XIon or HILIC chromatographic column (preferably Click XIon chromatographic column)；35 DEG C of column temperature；
Sampling volume: 0.35 μ L；Flow velocity 0.35mL/min；Type of elution: gradient elution；
The gradient elution program is as follows:
0~1.0min, mobile phase A 95% → 95%, Mobile phase B 5% → 5%；
1.0~2.0min, mobile phase A 95% → 85%, Mobile phase B 5% → 15%；
2.0~6.0min, mobile phase A 85% → 40%, Mobile phase B 15% → 60%；
6.0~8.0min, mobile phase A 40% → 40%, Mobile phase B 60% → 60%；
8.0~9.5min, mobile phase A 40% → 95%, Mobile phase B 60% → 5%；
9.5~12min, mobile phase A 95% → 95%, Mobile phase B 5% → 5%；
Wherein, using acetonitrile as mobile phase A, using 0.1% aqueous formic acid (ammonium acetate containing 10mM) as Mobile phase B.
8. application of any one of the claim 1-7 the method in evaluation endogenous compound matrix effect；Preferably, described
Endogenous compound matrix effect is influenced using including but is not limited to evaluation sample pre-treatments mode or chromatographic column.
9. application as claimed in claim 8, which is characterized in that the sample is milk powder, and the milk powder preferably includes, but is not limited to
Milk powder, whole milk powder, skimmed milk power and goat milk powder；
Preferably, the sample pre-treatments mode includes but is not limited to carry out water-bath hydrolysis or microwave-assisted hydrolysis to sample.
10. application as claimed in claim 8, which is characterized in that the chromatographic column includes but is not limited to Click XIon column, T3 column
With Hilic column；Preferably Click XIon column.
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