CN109030638A - A method of for measuring selenomethionine in bio-matrix - Google Patents
A method of for measuring selenomethionine in bio-matrix Download PDFInfo
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- CN109030638A CN109030638A CN201710425872.6A CN201710425872A CN109030638A CN 109030638 A CN109030638 A CN 109030638A CN 201710425872 A CN201710425872 A CN 201710425872A CN 109030638 A CN109030638 A CN 109030638A
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- RJFAYQIBOAGBLC-BYPYZUCNSA-N Selenium-L-methionine Chemical compound C[Se]CC[C@H](N)C(O)=O RJFAYQIBOAGBLC-BYPYZUCNSA-N 0.000 title claims abstract description 97
- RJFAYQIBOAGBLC-UHFFFAOYSA-N Selenomethionine Natural products C[Se]CCC(N)C(O)=O RJFAYQIBOAGBLC-UHFFFAOYSA-N 0.000 title claims abstract description 97
- 229960002718 selenomethionine Drugs 0.000 title claims abstract description 97
- 239000011159 matrix material Substances 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 52
- 150000001875 compounds Chemical class 0.000 claims abstract description 26
- XDSSPSLGNGIIHP-VKHMYHEASA-N Se-methyl-L-selenocysteine Chemical compound C[Se]C[C@H]([NH3+])C([O-])=O XDSSPSLGNGIIHP-VKHMYHEASA-N 0.000 claims abstract description 23
- 239000006228 supernatant Substances 0.000 claims abstract description 18
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 11
- 230000006920 protein precipitation Effects 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 8
- 238000001819 mass spectrum Methods 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 238000003556 assay Methods 0.000 claims abstract description 5
- 210000002381 plasma Anatomy 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 19
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000012224 working solution Substances 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 235000013336 milk Nutrition 0.000 claims description 7
- 210000004080 milk Anatomy 0.000 claims description 7
- 239000008267 milk Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 210000000577 adipose tissue Anatomy 0.000 claims description 3
- 210000000941 bile Anatomy 0.000 claims description 3
- 210000004556 brain Anatomy 0.000 claims description 3
- 238000000132 electrospray ionisation Methods 0.000 claims description 3
- 210000002216 heart Anatomy 0.000 claims description 3
- 210000003734 kidney Anatomy 0.000 claims description 3
- 210000004185 liver Anatomy 0.000 claims description 3
- 210000004072 lung Anatomy 0.000 claims description 3
- 210000003205 muscle Anatomy 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 210000000952 spleen Anatomy 0.000 claims description 3
- 210000002784 stomach Anatomy 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 210000002149 gonad Anatomy 0.000 claims description 2
- XTEGARKTQYYJKE-UHFFFAOYSA-N chloric acid Chemical compound OCl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-N 0.000 claims 1
- 229940005991 chloric acid Drugs 0.000 claims 1
- 238000007689 inspection Methods 0.000 claims 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 abstract description 11
- 238000005259 measurement Methods 0.000 abstract description 11
- 238000000605 extraction Methods 0.000 abstract description 5
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 35
- 239000011669 selenium Substances 0.000 description 18
- 241000700159 Rattus Species 0.000 description 13
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 13
- 229910052711 selenium Inorganic materials 0.000 description 13
- 230000000694 effects Effects 0.000 description 10
- 238000011084 recovery Methods 0.000 description 9
- 238000005251 capillar electrophoresis Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000001927 high performance liquid chromatography-inductively coupled plasma mass spectrometry Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 206010039921 Selenium deficiency Diseases 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000013062 quality control Sample Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 102000008114 Selenoproteins Human genes 0.000 description 3
- 108010074686 Selenoproteins Proteins 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 229940065287 selenium compound Drugs 0.000 description 3
- 150000003343 selenium compounds Chemical class 0.000 description 3
- 150000003346 selenoethers Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- FDKWRPBBCBCIGA-REOHCLBHSA-N (2r)-2-azaniumyl-3-$l^{1}-selanylpropanoate Chemical compound [Se]C[C@H](N)C(O)=O FDKWRPBBCBCIGA-REOHCLBHSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FDKWRPBBCBCIGA-UWTATZPHSA-N D-Selenocysteine Natural products [Se]C[C@@H](N)C(O)=O FDKWRPBBCBCIGA-UWTATZPHSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000003672 processing method Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- ZKZBPNGNEQAJSX-UHFFFAOYSA-N selenocysteine Natural products [SeH]CC(N)C(O)=O ZKZBPNGNEQAJSX-UHFFFAOYSA-N 0.000 description 2
- 229940055619 selenocysteine Drugs 0.000 description 2
- 235000016491 selenocysteine Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- 208000019926 Keshan disease Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000004807 desolvation Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000018823 dietary intake Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000009616 inductively coupled plasma Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 125000003748 selenium group Chemical group *[Se]* 0.000 description 1
- 125000001554 selenocysteine group Chemical group [H][Se]C([H])([H])C(N([H])[H])C(=O)O* 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- Physics & Mathematics (AREA)
- 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
The present invention relates to technical field of biological, and in particular to a method of for measuring selenomethionine in bio-matrix.Provided by the present invention for the method for selenomethionine in measurement bio-matrix, it includes the following steps: (1) sample pretreatment: taking Biological matrix, internal standard compound methylselenocysteine is added, then the Biological matrix is handled with precipitation of protein, centrifugation obtains supernatant；(2) assay: using high performance liquid chromatography tandem mass spectrum technology (LC-MS/MS) chromatography, by supernatant injecting chromatograph in step (1), calculates the concentration of selenomethionine in Biological matrix.Bio-matrix of the invention uses precipitation of protein, selenomethionine extraction efficiency is high, and bio-matrix is noiseless to measurement selenomethionine, using the selenomethionine in LC-MS/MS chromatography determination bio-matrix, speed is fast, specificity is good, accuracy is high, favorable reproducibility.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of for measuring selenomethionine in bio-matrix
Method.
Background technique
Selenium is microelement necessary to humans and animals, and selenium is only with selenomethionine (SeMet) and two kinds of selenocysteine
Form is present in selenoprotein, and most selenium is SeMet.Selenium influences the intracorporal Radical Metabolism of machine, antioxygen by selenoprotein
Change function, immune function, reproductive function, natural death of cerebral cells and endocrine hormone etc. and plays its biological action, Keshan disease, big bone
It saves more than the 40 kinds of diseases such as disease, diabetes, AIDS and selenium deficiency is related.Therefore, SeMet content is living in reactant in organism
The important physical signs of property selenium level.
There is the selenium deficiency that a countries and regions are different degrees of more than 40 in the whole world, and the area in China 2/3 belongs to selenium deficiency area,
The 29.0% serious selenium deficiency in area.The daily selenium intake for improving China resident is of great importance for improving national health.Selenium
Form is an important factor for influencing the bioavilability and toxicity of selenium.The assimilation effect of inorganic selenium and safety are much not as good as organic
Selenium.The basic existence form of organic selenium is selenoprotein, and selenium only covalently bind in egg in the form of SeMet and two kinds of selenocysteine
In white matter, SeMet is the highest organic selenides of bioavilability.Therefore, SeMet level is measured in animal derived food to guarantor
It demonstrate,proves dietary int ake selenium amount and best selenium is significant.
SeMet level is lower in organism, therefore need to be measured using more sensitive analysis method.Currently, measurement SeMet
Primary analysis method is high performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS), gas chromatography-mass spectrum
Usage (GC-MS), capillary electrophoresis (CE).But HPLC-ICP-MS method can only quantitative determine Se element, cannot survey
Determine SeMet original shape state, there are when a variety of Se forms, needing SeMet standard items to measure especially in body, and once has it
His selenium compound and SeMet are when there is identical retention time in chromatography, then fubaritic SeMet at all；GC-MS for
The detection of the non-volatile selenides such as SeMet carries out derivative materializing strategy, and there are more difficulties；Capillary electrophoresis with
When ICP-MS is combined, required flow velocity is different, limits its development and application.
The chemical formula of selenomethionine of the present invention is C15H11NO2Se, molecular weight 196.1063, structural formula are as follows:
Summary of the invention
Prior art problem solved by the invention is: using high performance liquid chromatography inductively coupled plasma in the prior art
Constitution spectrometry (HPLC-ICP-MS), combined gas chromatography mass spectrometry (GC-MS), capillary electrophoresis (CE), but with efficient liquid
When phase chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) is measured, quantitative limit can only be carried out to Se element
It is fixed, SeMet original shape state cannot be measured, there are when a variety of Se forms, needing SeMet standard items to measure especially in body, and
And once having other selenium compounds and SeMet when there is identical retention time in chromatography, then fubaritic SeMet at all；GC-
The detection of MS selenides non-volatile for SeMet etc. carries out derivative materializing strategy, and there are more difficulties；Capillary Electrophoresis
For method when being combined with ICP-MS, required flow velocity is different, limits its development and application.
The present invention in Biological matrix by being added internal standard compound, then using precipitation of protein to bio-matrix sample
Product are handled, and are then centrifuged for obtaining supernatant, and supernatant is injected high performance liquid chromatography tandem mass spectrum chromatography (LC-MS/
MS), the concentration of SeMet in Biological matrix is calculated using Internal standard curve method.
Specifically, the invention proposes following technical solutions.
A method of for measuring selenomethionine in bio-matrix, which is characterized in that it includes the following steps:
(1) sample pretreatment: taking Biological matrix, and internal standard compound methylselenocysteine is added, then uses protein
The precipitation method processing Biological matrix, centrifugation obtain supernatant；
(2) assay: using high performance liquid chromatography tandem mass spectrum chromatography, and supernatant in step (1) is injected chromatography
Instrument calculates the concentration of selenomethionine in Biological matrix using Internal standard curve method.
Wherein, the Biological matrix include whole blood, blood plasma, serum, milk, the heart, liver, spleen, lung, kidney, stomach, enteron aisle,
One of gonad, brain, body fat, muscle, bile, urine or excrement.
Wherein, in step (1), the specific method for handling the Biological matrix with precipitation of protein includes
Following step: being added internal standard compound methylselenocysteine in bio-matrix, and perchloric acid is then added and is handled.
Wherein, the mass concentration of the perchloric acid is 5-10%, and the volume ratio of the perchloric acid and Biological matrix is
1:1。
Wherein, the chromatographic condition of the LC-MS/MS are as follows: chromatographic column is AcquityXEVO HSS T3, and mobile phase A is
0.1-0.5% aqueous formic acid, Mobile phase B are that 0.1-0.5% formic acid acetonitrile solution carries out gradient elution；
The Mass Spectrometry Conditions of the LC-MS/MS are as follows: it uses electro-spray ionization ionization source (ESI), positive ion mode detection,
Multiple-reaction monitoring (MRM) carries out second mass analysis.
Wherein, the preparation method of the internal standard compound includes the following steps: to take internal standard compound methylselenocysteine, adds water-soluble
Solution obtains the internal standard compound standard reserving solution that concentration is 1-5mg/ml, then internal standard compound standard reserving solution is diluted with water, and obtains interior
The concentration for marking object is 1-5 μ g/ml.
Wherein, the step of prepared by standard sample includes following processing operations: taking selenomethionine, is dissolved in water, obtains dense
Degree is the standard reserving solution of 1mg/ml, and then plus water is diluted, and obtains a series of concentration that concentration is 20-2000ng/ml and passs
The standard working solution of increasing；Blank Biological matrix is taken, a series of standard working solution of increasing concen-trations is added, obtains a series of marks
Quasi- sample.
Wherein, the concentration range of the series of standards sample is 2-200ng/ml.
Wherein, the bio-matrix is rat bio-matrix.
Wherein, the bio-matrix is the blood plasma for picking up from rat.
Beneficial effect obtained by the present invention is: the present invention takes the extracting method of protein precipitation, and SeMet extracts effect
Rate is high, and recovery rate is up to 80% or more, and bio-matrix is noiseless to measurement SeMet, and specificity is good, and sample pre-treatments were extracted
Journey is easy to operate, is easy to repeat, and without many and diverse processing such as derivatizations, the present invention is combined method (LC-MS/ using liquid chromatography mass
MS SeMet) is measured, the chemical structure of SeMet can be parsed, thus can be distinguished selenium compound has identical retention time
It comes, not needing standard items can Qualitive test SeMet.Meanwhile using SeMet standard items can be with quantitative analysis biological sample
In SeMet, method high sensitivity, the good, high sensitivity of specificity, analysis speed are fast and accurate and reliable.
Detailed description of the invention
Fig. 1 is chromatography-mass spectrogram of selenomethionine and internal standard compounds methyl selenocysteine in embodiment 1；
Fig. 2 is SeMet- time graph in blood plasma after rat intravenous injection SeMet in embodiment 1.
Specific embodiment
Fig. 1 is the chromatography-mass spectroscopy figure of selenomethionine and internal standard compound methylselenocysteine in embodiment 1, from figure
As can be seen that the retention time of selenomethionine and methylselenocysteine is respectively 1.8min and 1.2min, two compounds
It does not interfere with each other, and the measurement of endogenous material not disturbed specimen.
Fig. 2 is SeMet- time graph in blood plasma after rat tail vein injection SeMet in embodiment 1, in different times
Interior collection blood, prepares blood plasma, then handles blood plasma, obtains supernatant injection LC-MS/MS and is analyzed, using interior
The concentration that calibration curve method calculates SeMet is marked, so that the content of SeMet in rat plasma can be obtained in different time.
As described above, the present invention provides a kind of method for measuring selenomethionine in bio-matrix, the present invention is
Internal standard compound methylselenocysteine is added in bio-matrix, then handles the bio-matrix sample with precipitation of protein
Product, centrifugation obtain supernatant, and supernatant injection LC-MS/MS is analyzed.
Wherein, in the preferred embodiment of the present invention, the present invention provides one kind for measuring in bio-matrix
The method of selenomethionine comprising following steps:
(1) sample pretreatment: taking Biological matrix, and internal standard compound methylselenocysteine is added, then uses protein
The precipitation method handle Biological matrix, and centrifugation obtains supernatant；
(2) assay: using high performance liquid chromatography tandem mass spectrum (LC-MS/MS) chromatography, will be upper in step (1)
The concentration of selenomethionine in Biological matrix is calculated in clear liquid injecting chromatograph.
Bio-matrix of the invention includes whole blood, serum, blood plasma, milk, the heart, liver, spleen, lung, kidney, stomach, enteron aisle, reproduction
One of gland, brain, body fat, muscle, bile, urine or excrement.
In another embodiment of the invention, the bio-matrix is blood plasma, the protein precipitation being added
Agent is perchloric acid, and the mass concentration of the perchloric acid is 5-10%, preferably 6.2%, the internal standard compound methyl seleno being added half
The concentration of cystine is 1-5 μ g/ml, preferably 4 μ g/ml.The concrete operation method of its sample are as follows: internal standard compound is added in blood plasma
Methylselenocysteine and protein precipitant perchloric acid, centrifugation obtain supernatant.
(He Mengjie, Zhang Shuanqing, beam is clever, Cui Yajuan, He Mei, Huang vibration force ultra performance liquid chromatography-series connection for documents
Dissociate selenomethionine health research in mass spectrometric determination milk, 2016,45 (1): 65-67+97.) disclose ultra high efficiency liquid
Dissociate selenomethionine in phase chromatography-tandem mass spectrometry measurement milk, in the endogenous material as contained in blood plasma and milk
Contained endogenous material is different, and therefore uses blood plasma as matrix, with methyl that internal standard compound is not added in documents
Selenocysteine is internal standard compound, uses Internal standard curve method with the obtained standard of the concentration of calculated by peak area selenomethionine
Exactness, precision and lower limit of quantitation are all relatively better than using milk as the accuracy of matrix, precision and lower limit of quantitation.
The equipment and analysis that manufacturer to raw material used in the present embodiment and equipment and product analysis below uses
Method is described as follows, wherein the chemical substance do not indicate be conventional reagent the pure rank of chemistry.Wherein, real
The information of raw material used in example and the information of experimental facilities are applied as shown in Tables 1 and 2.
The information of raw material used in 1 present invention of table
The information of experimental facilities used in 2 present invention of table
Embodiment 1
(1) condition and method are checked
1. the preparation of standard sample
It is appropriate that precision weighs SeMet, is dissolved in water, and obtains the SeMet standard reserving solution that concentration is 1mg/ml, and precision measures
SeMet standard reserving solution is appropriate, is serially diluted with water, obtain concentration be 20,50,100,200,800,1000,1500,
2000ng/ml series standard working solution, then accurate to measure 90 μ l of rat blank plasma, the SeMet of various concentration is added in precision
10 μ l of working solution, obtains the standard sample of 2,5,10,20,80,100,150,200ng/ml.
2. the preparation of quality-control product
Precision measures the 10 μ l of SeMet working solution of debita spissitudo, then accurate to measure 90 μ l of rat blank plasma, obtains
The SeMet of 5,80,150ng/ml 3 concentration is Quality Control.
3. sample pretreatment: precision measures plasma sample (standard sample blood plasma, quality-control sample blood plasma or administration sample blood
Slurry) 100 μ l, it is accurate that internal standard methylselenocysteine (4 μ g/ml) 10 μ l are added, the perchloric acid solution of 6.2% (w/v) is added
100 μ l, are vortexed and mix 2min, and 12000g is centrifuged 5min, obtains supernatant；
4. assay: LC-MS/MS chromatography is used, by supernatant injecting chromatograph, using Internal standard curve method meter
Calculate the concentration of selenomethionine in plasma sample.
Wherein, the chromatographic condition of the LC-MS/MS are as follows: mobile phase A is 0.1% aqueous formic acid, Mobile phase B 0.1%
The volume ratio of formic acid acetonitrile solution, the aqueous formic acid and formic acid acetonitrile solution is 99:1, flow velocity 0.3ml/min, column temperature
It is 40 DEG C, sample room temperature is 15 DEG C, and sample volume is 5 μ l, and entire analysis time is 3min.
The Mass Spectrometry Conditions of the LC-MS/MS are as follows: use electro-spray ionization ionization source (ESI), capillary voltage is
3500V, ion source temperature are 120 DEG C, and nitrogen is as desolventizing gas and taper hole blowback air, and desolvation temperature is 350 DEG C, precipitation
Agent throughput is 700l/h, and cone hole backflow airflow amount is 50l/h, and argon gas is as collision gas, flow 0.15ml/min, seleno egg
The orifice potential of propylhomoserin (SeMet) and methylselenocysteine (MSC) is respectively 14V and 12V, the collision of SeMet and MSC
It can be respectively 12eV and 14eV, be detected using positive ion mode, scanning mode is multiple-reaction monitoring (MRM), SeMet and MSC
Ionic reaction for quantitative analysis is respectively m/z198.0 → 181.1, m/z 184.0 → 167.0, sweep time 0.2s.
Wherein, for internal standard MSC concentration be 4 μ g/ml's the preparation method comprises the following steps: precision to weigh MSC appropriate, be dissolved in water, obtain
The MSC standard reserving solution for being 1mg/ml to concentration, precision measurement MSC standard reserving solution is appropriate, is diluted with water, and obtaining concentration is 4 μ
The internal standard working solution of g/ml.
(2) methodology validation
Using testing conditions listed by embodiment 1 and method, methodology validation is carried out to measuring method, in 2-200ng/
In the range of linearity of ml selenomethionine, linear relationship is good (r >=0.9945), lower limit of quantitation 2ng/ml, minimum detectability
For 0.5ng/ml, Quality Control in a few days, day to day precision between 1.5-11.0%, show that (guideline provides RSD to reproducibility very well
Value answers < 15%), accuracy 92.6-101.9% shows that method accuracy is fine, matrix effect the result shows that matrix to sample
The influence of product is stablized, and the rate of recovery is the result shows that the present invention is had using plasma sample of the plasma sample processing method to various concentration
Relatively stable extraction effect, methodology validation is the result shows that established LC-MS/MS energy is quick, sensitive, accurately and reliably examines
Survey the concentration of selenomethionine in blood plasma.
2. the drafting of standard curve
Standard sample is prepared into standard song according to sample treatment described in embodiment 1 and chromatographic process preparation method
Line.Linear regression calculating is carried out with weighting (W=1/X) least square method, is with the ratio between selenomethionine and internal standard compound peak area y
Ordinate obtains 6 representative standard curves, seleno in the blood plasma using the concentration x of standard selenomethionine as abscissa
The range of linearity of methionine is 2-200ng/ml, as shown in table 3.
6 standard curves of the table 3 in rat plasma
It can be seen from upper table 3 within the scope of 2-200ng/ml, linearly dependent coefficient r >=0.9945, linear relationship is good
It is good, lower limit of quantitation 2ng/ml.
3. specificity and sensitivity
As shown in Figure 1, retention time of the SeMet after chromatographic isolation is 1.8min, and the retention time of internal standard compound MSC
For 1.2min, two compounds are not interfered with each other, and the measurement of endogenous material not disturbed specimen, illustrates the method in rat plasma
Specificity is good, and selectivity is high, high sensitivity.
4. preci-sion and accuracy
Precision measures the 10 μ l of SeMet standard working solution of debita spissitudo, and the accurate rat blank plasma measured is then added
90 μ l are configured to the standard plasma sample that the SeMet that concentration is 5,80,150ng/ml 3 concentration is basic, normal, high three kinds of concentration
Product, 5 μ l of sample introduction are analyzed, each concentration point 6 multiple pipes, interior on the same day to carry out according to plasma sample processing method same procedure
Operation is detected, while METHOD FOR CONTINUOUS DETERMINATION 3 days, calculation method in a few days with day to day precision and accuracy, the results are shown in Table 4.
The result (n=6) of 4 selenomethionine accuracy of table and precision
Wherein, accuracy is average measurement concentration obtained percentage compared with standard sample concentration；
Precision is that standard deviation is removed in average measurement concentration, i.e. RSD.
As can be seen from the above table, lower limit of quantitation in a few days accuracy is 103.0%, and accuracy in the daytime is 104.33%, in a few days
RSD is 4.3%, and RSD is 6.2% in the daytime, and quality-control sample in a few days accuracy is between 92.6-100.2%, and accuracy exists in the daytime
Between 94.4-101.9%, in a few days RSD is between 1.5-4.0%, and RSD is between 7.1-11.0% in the daytime, lower limit of quantitation sample
In the daytime, in a few days accuracy is between 80-120%, quality-control sample in the daytime, in a few days accuracy within 85-115%, it is quantitative
Lower limit in a few days, in the daytime RSD within 20%, quality-control sample RSD illustrates the method within 15% in a few days, in the daytime
Accuracy and precision are high, favorable reproducibility.
5. extraction recovery and matrix effect
Matrix effect: taking blank rat plasma, and the HClO of equivalent 6.2% (w/v) is added4Solution is vortexed and mixes 2min,
12000g is centrifuged 5min, takes supernatant spare.90 μ L of supernatant is taken, it is accurate respectively that 50,800 and 1500ng/mL SeMet standard is added
Working solution 10 μ L, vortex 30s, taking 5 μ L, sample introduction is analyzed (A).90 μ L of accurate extract water is accurate respectively that 50,800 and are added
100 μ L of water (water is for preparing SeMet sample solution) is added in 10 μ L of 1500ng/mL SeMet sample, and vortex 30s takes
Sample introduction is analyzed by 5 μ L, obtains peak area (B).SeMet chromatographic peak area in every group is compared, matrix of the SeMet in blood plasma is calculated
Effect (A/B).
Extraction recovery:
It is accurate to draw 90 μ L of blank rat plasma, it is accurate respectively that 50,800 and 1500ng/mL SeMet sample, 10 μ is added
The HClO of 6.2% (w/v) is added in L4100 μ L of solution, vortex 2min, 12000g are centrifuged 5min, and taking 5 μ L of supernatant, sample introduction is analyzed
(A).90 μ L of accurate extract water, it is accurate respectively that 50,800 and 1500ng/mL SeMet sample, 10 μ L is added, add 100 μ L of water
(water is for preparing SeMet sample solution), vortex 30s, taking 5 μ L, sample introduction is analyzed, obtains peak area (B).It will be in every group
SeMet chromatographic peak area is compared, and the rate of recovery (A/B) of the SeMet in blood plasma is calculated.
5 selenomethionine matrix effect of table and rate of recovery result (n=6)
| Concentration (ng/ml) | Matrix effect (%) | RSD (%) | The rate of recovery (%) | RSD (%) |
| 5 | 108.9 | 4.5 | 79.4 | 6.0 |
| 80 | 111.7 | 1.6 | 95.4 | 3.6 |
| 150 | 105.3 | 3.4 | 91.4 | 2.1 |
As can be seen from Table 5, basic, normal, high three concentration matrix effects of selenomethionine are 105.3-111.7%, essence
Density is 1.6-4.5%, average recovery rate 79.4-95.4%, precision 2.1-6.0%.The result shows that matrix is to survey
Determine selenomethionine concentration mensuration not making significant difference, and extraction recovery is higher, extracting method is reliable.
3. zoopery
6 male SD healthy rats are taken, are drawn before medicine after blank blood, in 20 μ g/kgSeMet of tail vein injection, after injection
2min, 5min, 15min, 30min, 45min, 1h, 2h, 4h, 6h, 8h, 12h acquire blood for 24 hours, prepare blood plasma, then according to
The concentration of selenomethionine in the method measurement blood plasma of embodiment 1, as a result as shown in Figure 2.
The above is only the preferred embodiment that the present invention is implemented, and not does limitation in any form to the present invention, all
The modifications, equivalent substitutions and improvements etc. done within the spirit and principles in the present invention are required to be included in protection of the invention
Within the scope of.
Claims (10)
1. a kind of method for measuring selenomethionine in bio-matrix, which is characterized in that it includes the following steps:
(1) sample pretreatment: taking Biological matrix, and internal standard compound methylselenocysteine is added, then uses protein precipitation
The method processing Biological matrix, centrifugation obtain supernatant；
(2) assay: supernatant injecting chromatograph in step (1) is adopted using high performance liquid chromatography tandem mass spectrum chromatography
The concentration of selenomethionine in Biological matrix is calculated with Internal standard curve method.
2. the method according to claim 1 for measuring selenomethionine in bio-matrix, which is characterized in that the life
Object matrix sample include whole blood, blood plasma, serum, milk, the heart, liver, spleen, lung, kidney, stomach, enteron aisle, gonad, brain, body fat, muscle,
One of bile, urine or excrement.
3. the method according to claim 1 or 2 for measuring selenomethionine in bio-matrix, which is characterized in that
In step (1), the method for handling the Biological matrix with precipitation of protein includes the following steps: in biology base
Internal standard compound methylselenocysteine is added in matter, perchloric acid is then added and is handled.
4. the method according to claim 3 for measuring selenomethionine in bio-matrix, which is characterized in that the height
The mass concentration of chloric acid is 5-10%, and the volume ratio of the perchloric acid and Biological matrix is 1:1.
5. the method according to claim 1-4 for measuring selenomethionine in bio-matrix, feature exist
In the chromatographic condition of the high performance liquid chromatography tandem mass spectrum are as follows: chromatographic column is Acquity XEVO HSS T3, and mobile phase A is
0.1-0.5% aqueous formic acid, Mobile phase B are that 0.1-0.5% formic acid acetonitrile solution carries out gradient elution；
The Mass Spectrometry Conditions of the high performance liquid chromatography tandem mass spectrum are as follows: use electro-spray ionization ionization source, positive ion mode inspection
It surveys, multiple-reaction monitoring carries out second mass analysis.
6. the method according to claim 1-5 for measuring selenomethionine in bio-matrix, feature exist
In the preparation method of the internal standard compound includes the following steps: to take internal standard compound methylselenocysteine, is dissolved in water, and obtains dense
Degree is the internal standard compound standard reserving solution of 1-5mg/ml, and then internal standard compound standard reserving solution is diluted with water, obtains the dense of internal standard compound
Degree is 1-5 μ g/ml.
7. the method according to claim 1-6 for measuring selenomethionine in bio-matrix, feature exist
In, further include the steps that standard sample prepare, the step includes following processing operations: taking selenomethionine, is dissolved in water, obtains
The standard reserving solution for being 1mg/ml to concentration, then plus water is diluted, and obtaining concentration is a series of dense of 20-2000ng/ml
The incremental standard working solution of degree；Blank Biological matrix is taken, a series of standard working solution of above-mentioned increasing concen-trations is added, obtains
Series of standards sample.
8. the method according to claim 7 for measuring selenomethionine in bio-matrix, which is characterized in that described one
The concentration range of series standard sample is 2-200ng/ml.
9. the method according to claim 1-8 for measuring selenomethionine in bio-matrix, feature exist
In the bio-matrix is rat bio-matrix.
10. the method according to claim 1-8 for measuring selenomethionine in bio-matrix, feature exist
In the bio-matrix is the blood plasma for picking up from rat.
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