CN102608234A - Detection method of angiotensin converting enzyme (ACE) inhibitory activity of antihypertensive peptide - Google Patents
Detection method of angiotensin converting enzyme (ACE) inhibitory activity of antihypertensive peptide Download PDFInfo
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- CN102608234A CN102608234A CN2012100899070A CN201210089907A CN102608234A CN 102608234 A CN102608234 A CN 102608234A CN 2012100899070 A CN2012100899070 A CN 2012100899070A CN 201210089907 A CN201210089907 A CN 201210089907A CN 102608234 A CN102608234 A CN 102608234A
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Abstract
A detection method of angiotensin converting enzyme (ACE) inhibitory activity of antihypertensive peptide comprises the following steps that: firstly, boric-acid buffer solution is prepared to perform an experiment on ACE activity inhibition of the antihypertensive peptide; secondly, reaction products are detected and analyzed by LC-MC (liquid chromatography-mass spectrometry), and the antihypertensive activity of the antihypertensive peptide is represented by detecting change of peak area of hippuric acid (Hip), wherein the purity of the Hip is ensured through monitoring of an HPLC (high performance liquid chromatography) ultraviolet detector and an electrospray ion source mass spectrum as well as with adoption of a total ion chromatogram and an ultraviolet detection chromatogram, and the peak area of the Hip is calculated by quantitatively analyzing the Hip peak area of an ion extract diagram of m/z180 in an SIM model; and thirdly, the peak area of the Hip of the reaction products in a reaction group and a no-treatment control group are obtained through the first and second steps, thereby obtaining the ACE inhibition ratio of a sample. In the method, the LC-MS technology is applied to screening activity of the antihypertensive peptide, so that the influence of impurities on activity detection is reduced; and the detection method has the characteristics of being high in precision, good in repeatability and high in sensitivity.
Description
Technical field
The ACE (ACE) that the present invention relates to a kind of step-down peptide suppresses active detection method, belongs to biological technical field.
Background technology
Human beings'health in the hypertension serious threat, and China hyperpietic reaches 1.8 hundred million people, and annual number because of hypertension death surpasses 1,000,000 people.Plain – hypertensin system of the booster system body kidney of human body and depressurizing system swash peptide release enzyme – kinin system and are playing the part of important role aspect blood pressure regulating and the cardiovascular function.ACE (ACE) reaches the effect of rising blood pressure through the regulation and control to above-mentioned two systems.The step-down peptide plays hypotensive effect through suppressing the activity of ACE.
Characteristics such as the step-down peptide has high security, has no side effect, effect is obvious become the research emphasis of antihypertensive drugs, health products, have huge market outlook.Therefore, foundation efficient, sensitive, the activity test method of ACE inhibition reliably is very important.Cushman etc. have set up ultraviolet spectrophotometry; ACE is at 37 ℃, and (Hip-His-Leu HHL) produces the analogies hippuric acid (Hip) of angiotensin to the analogies hippuric acid-HIS-LEU of catalytic decomposition angiotensin under the condition of pH8.3; This material has characteristic absorption peak at ultraviolet 228nm place; When adding ace inhibitory peptide, ACE is suppressed the catalyticing decomposition action of HHL, and the growing amount of Hip reduces.Confirm to suppress active through measuring the difference that adds the peptide for inhibiting front and back Hip ultraviolet absorption peak that generates.In the method, the Hip that reaction is generated can not effectively separate with HHL, and all at the 228nm place absorption is arranged, and is prone to produce experimental error.On this basis, many scholars set up high performance liquid chromatography, utilize high performance liquid chromatography to separate Hip, confirm that through measuring the difference that adds the peptide for inhibiting front and back Hip peak area that generates the inhibition of sample is active.High performance liquid chromatography utilizes Hip chromatographic peak and impurity peaks to have different retention times, quantitative test Hip, and accuracy is higher than ultraviolet spectrophotometry.But some sample weak absorption peak occurs or does not have absorption peak in ultraviolet detection, and the ACE that high performance liquid chromatography can't detect these samples suppresses active.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of easy, quick, precision, step-down peptide ACE that accuracy is high to suppress activity test method, for the research and development of antihypertensive drugs, health products provide technical support.
For solving the problems of the technologies described above, technical solution of the present invention is:
A kind of ACE of step-down peptide (ACE) suppresses active detection method, comprises the steps:
(1) the inhibition ACE activity experiment of step-down peptide
A certain amount of step-down peptide is dissolved in pH8.3, in the borate buffer of 0.lmol/L, draws the step-down peptide of 25 μ L, add 75 μ L60mUml
-1ACE in 37 ℃ of insulation 5min, add hippuric acid-HIS-LEU of 112 μ L5.0mmol/L subsequently; The blank group then replaces the step-down peptide with the borate buffer of equal volume, at 37 ℃, is incubated 60min in the water bath with thermostatic control, adds 25 μ L0.1% trifluoroacetic acid stopped reactions then, and under the effect of ACE, it is Hip that hippuric acid-HIS-LEU generates hippuric acid;
(2) utilize high performance liquid chromatography-mass spectrometry LC-MS technology for detection analytical reactions product
The reaction product of LC-MS check and analysis reaction group and blank group, liquid phase chromatogram condition is: reverse-phase chromatographic column, moving phase are acetonitrile: ultrapure water is 20:80; Containing volume ratio is 0.1% trifluoroacetic acid; Flow velocity 1.0mL/min detects wavelength 228nm, sample size 10 μ L; The mass spectrum condition is: electric spray ion source (ESI), ionization voltage 3.5KV, one-level taper hole voltage 35V, secondary taper hole voltage 3.0V; Six grades of bar lens voltage 0.1V, 120 ℃ of source temperatures, 350 ℃ of desolventizing temperature; Desolventizing nitrogen flow rate 500L/h, taper hole nitrogen flow rate 50L/h, ion energy 0.5; Utilize the TIC and the ultraviolet detection chromatogram detection reaction product of reaction product, guarantee Hip purity; Under the quantitative test SIM model
M/zThe Hip peak area of 180 extraction ion figure, the peak area of calculating Hip;
(3) calculating of antihypertensive activity
Obtain the peak area of reaction group and blank group reaction product Hip through said method, it is (A that the ACE of step-down peptide suppresses active IP
C-A
S) * 100/A
C, wherein: IP is the inhibiting rate of sample to ACE; A
CPeak area for Hip in the blank group; A
SPeak area for Hip in the reaction group.
The used reverse-phase chromatographic column of said step (2) is the Ug120-C18 analytical column.
The used electric spray ion source of said step (2) (ESI) is the electron spray ionisation positive ion mode.
The said reaction group reaction of said step (2) product repeats to survey three times, and the relative deviation of peak area (RSD) is 0.1-0.4%.
The described blank group of said step (3) ACE and HHL reaction when not adding the step-down peptide generate the peak area of Hip in LC-MS and represent; Reaction group ACE and HHL reaction when adding the step-down peptide generate the peak area of Hip in LC-MS and represent.
After adopting such scheme, the present invention utilizes LC-MS detection reaction product, through detecting under the SIM model
M/zHippuric acid (Hip) peak area of 180 extraction ion figure changes the antihypertensive activity that characterizes the step-down peptide.Under high performance liquid chromatography UV-detector and the mass spectral monitoring of electric spray ion source (ESI), utilize TIC and ultraviolet detection chromatogram, judge the degree of separation of Hip, guarantee the accuracy of step-down peptide detection method.
The present invention is applied to the screening active ingredients of step-down peptide with the LC-MS technology, compares with in the past AAS, chromatography, has the following advantages:
1, the present invention utilizes the ACE of LC-MS technology (LC-MS) detection step-down peptide to suppress active, through detecting under the SIM model
M/zThe variation of hippuric acid (Hip) peak area of 180 extraction ion figure characterizes the antihypertensive activity of step-down peptide.Utilize high performance liquid chromatogram ultraviolet chromatogram to work in coordination with the mass spectrum TIC; Confirm the purity of Hip; When avoiding traditional liquid phase chromatography absorption peak or ACE of not having an absorption peak sample a little less than detecting the tool ultraviolet to suppress active, the low phenomenon of accuracy rate occurs, improve the accuracy that ACE suppresses activity test method.
2, the present invention utilizes the ACE of LC-MS technology (LC-MS) detection step-down peptide to suppress active, when high performance liquid chromatogram separates, utilizes purity, the peak area of Mass Spectrometer Method reaction product, can practice thrift half the detection time.
Description of drawings
Fig. 1 is the chromatogram that embodiment one LC-MS detects the blank group, and wherein 1-1 is under the SIM model
M/z180 extraction ion figure, 1-2 is a TIC, 1-3 is the ultraviolet chromatogram;
Fig. 2 is the chromatogram of embodiment one LC-MS detection reaction group, and wherein 2-1 is under the SIM model
M/z180 extraction ion figure, 2-2 is a TIC, 2-3 is the ultraviolet chromatogram;
Fig. 3 is the chromatogram of embodiment two LC-MS detection reaction groups, and wherein 3-1 is under the SIM model
M/z180 extraction ion figure, 3-2 is a TIC, 3-3 is the ultraviolet chromatogram;
Fig. 4 is the chromatogram of embodiment three LC-MS detection reaction groups, and wherein 4-1 is under the SIM model
M/z180 extraction ion figure, 4-2 is a TIC, 4-3 is the ultraviolet chromatogram.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is made further detailed description.
Instance one:
1, a certain amount of fish scale step-down peptide is dissolved in pH8.3, in the borate buffer of 0.lmol/L, configuration concentration is the sample solution of 1mg/ml, draws the step-down peptide of 25 μ L, adds 75 μ L 60mUml
-1ACE in 37 ℃ of insulation 5min, add the HHL of 112 μ L5.0mmol/L subsequently, the blank group then with the volume of the borate buffer replacement step-down peptide of equal volume, is incubated 60min in 37 ℃ of waters bath with thermostatic control, add 25 μ L0.1%TFA stopped reactions then.
2, LC-MS detects the check and analysis reaction product, and high-efficient liquid phase chromatogram condition is: Ug120-C18 reverse-phase chromatographic column, moving phase are acetonitrile: ultrapure water=20:80 (contain 0.1%TFA, be volume ratio), flow velocity 1.0mLmin
-1, detect wavelength 228nm, sample size 10 μ L.The mass spectrum condition is: electron spray ionisation positive ion mode, ionization voltage 3.5KV, one-level taper hole voltage 35V, secondary taper hole voltage 3.0V; Six grades of bar lens voltage 0.1V, 120 ℃ of source temperatures, 350 ℃ of desolventizing temperature; Desolventizing nitrogen flow rate 500L/h, taper hole nitrogen flow rate 50L/h, ion energy 0.5.
3, utilize the TIC (Fig. 1-2, Fig. 2-2) of reaction product and ultraviolet chromatogram (Fig. 1-3, Fig. 2-3) to confirm that Hip does not contain other impurity in the reaction product, this moment, the appearance time of Hip was 5.02min.Utilize computed in software under the SIM model
M/zThe Hip peak area of 180 extraction ion figure (Fig. 1-1, Fig. 2-1) carries out quantitative test.The peak area of Hip is 15134.13 in the reaction group reaction product, and the peak area of Hip is 118954.55 in the blank group, and the inhibiting rate of the ACE of fish scale step-down peptide is 87.28%.The peak area replication 3 times of Hip in the reaction group, its RSD is 0.4%.This method precision is high, good reproducibility.
Instance two:
1, a certain amount of peanut step-down peptide is dissolved in pH8.3, in the borate buffer of 0.lmol/L, configuration concentration is the sample solution of 1mg/ml, draws the step-down peptide of 25 μ L, adds 75 μ L60mUml
-1ACE in 37 ℃ of insulation 5min, add the HHL of 112 μ L5.0mmol/L subsequently, the blank group then with the volume of the borate buffer replacement step-down peptide of equal volume, is incubated 60min in 37 ℃ of waters bath with thermostatic control, add 25 μ L0.1%TFA stopped reactions then.
2, LC-MS detects the check and analysis reaction product, and high-efficient liquid phase chromatogram condition is: Ug120-C18 reverse-phase chromatographic column, moving phase are acetonitrile: ultrapure water=20:80 (containing 0.1%TFA) (volume ratio), flow velocity 1.0mLmin
-1, detect wavelength 228nm, sample size 10 μ L.The mass spectrum condition is: electron spray ionisation positive ion mode, ionization voltage 3.5KV, one-level taper hole voltage 35V, secondary taper hole voltage 3.0V; Six grades of bar lens voltage 0.1V, 120 ℃ of source temperatures, 350 ℃ of desolventizing temperature; Desolventizing nitrogen flow rate 500L/h, taper hole nitrogen flow rate 50L/h, ion energy 0.5.
3, utilize the TIC (Fig. 1-2, Fig. 3-2) of reaction product and ultraviolet chromatogram (Fig. 1-3, Fig. 3-3) to confirm that Hip does not contain other impurity in the reaction product, this moment, the appearance time of Hip was 5.02min.Utilize computed in software under the SIM model
M/zThe Hip peak area of 180 extraction ion figure (Fig. 1-1, Fig. 3-1) carries out quantitative test.The peak area of Hip is 15691.29 in the reaction group reaction product, and the peak area of Hip is 118954.55 in the blank group, so the inhibiting rate of peanut step-down peptide ACE is 86.81%.The peak area replication of reaction group reaction product Hip 3 times, its RSD is 0.1%.This method precision is high, good reproducibility.
Instance three:
1,(Gly-Phe) is dissolved in pH8.3 with a certain amount of glycocoll-phenylalanine, and in the borate buffer of 0.lmol/L, configuration concentration is the sample solution of 1mg/ml, draws the step-down peptide of 25 μ L, adds 75 μ L60mUml
-1ACE in 37 ℃ of insulation 5min, add the HHL of 225 μ L5.0mmol/L subsequently, the blank group then with the volume of the borate buffer replacement step-down peptide of equal volume, is incubated 60min in 37 ℃ of waters bath with thermostatic control, add 25 μ L0.1%TFA stopped reactions then.
2,LC-MS detects the check and analysis reaction product, and high-efficient liquid phase chromatogram condition is: Ug120-C18 reverse-phase chromatographic column, moving phase are acetonitrile: ultrapure water=20:80 (containing 0.1%TFA) (volume ratio), flow velocity 1.0mLmin
-1, detect wavelength 228nm, sample size 10 μ L.The mass spectrum condition is: electron spray ionisation positive ion mode, ionization voltage 3.5KV, one-level taper hole voltage 35V, secondary taper hole voltage 3.0V; Six grades of bar lens voltage 0.1V, 120 ℃ of source temperatures, 350 ℃ of desolventizing temperature, desolventizing nitrogen flow rate 500L/h; Taper hole nitrogen flow rate 50L/h, ion energy 0.5
3, utilize the TIC (Fig. 1-2, Fig. 4-2) of reaction product and ultraviolet chromatogram (Fig. 1-3, Fig. 4-3) to confirm that Hip does not contain other impurity in the reaction product, this moment, the appearance time of Hip was 5.02min.Utilize computed in software under the SIM model
M/zThe Hip peak area of 180 extraction ion figure (Fig. 1-1, Fig. 4-1) carries out quantitative test.The peak area of Hip is 19668.16 in the reaction group reaction product, and the peak area of Hip is 118954.55 in the blank group, so the inhibiting rate of the ACE of Gly-Phe step-down peptide is 83.47%, the peak area replication of reaction group Hip 3 times, its RSD are 0.3%.This method precision is high, good reproducibility.
Claims (5)
1. the detection method of the angiotensin converting enzyme inhibition activity of a step-down peptide, ACE is abbreviated as ACE, it is characterized in that comprising the steps:
(1) the inhibition ACE activity experiment of step-down peptide
A certain amount of step-down peptide is dissolved in pH8.3, in the borate buffer of 0.lmol/L, draws the step-down peptide of 25 μ L, add 75 μ L60mUml
-1ACE in 37 ℃ of insulation 5min, add hippuric acid-HIS-LEU of 112 μ L5.0mmol/L subsequently; The blank group then replaces the step-down peptide with the borate buffer of equal volume, at 37 ℃, is incubated 60min in the water bath with thermostatic control, adds 25 μ L0.1% trifluoroacetic acid stopped reactions then, and under the effect of ACE, it is Hip that hippuric acid-HIS-LEU generates hippuric acid;
(2) utilize high performance liquid chromatography-mass spectrometry LC-MS technology for detection analytical reactions product
The reaction product of LC-MS check and analysis reaction group and blank group, liquid phase chromatogram condition is: reverse-phase chromatographic column, moving phase are acetonitrile: ultrapure water is 20:80; Containing volume ratio is 0.1% trifluoroacetic acid; Flow velocity 1.0mL/min detects wavelength 228nm, sample size 10 μ L; The mass spectrum condition is: electric spray ion source, ionization voltage 3.5KV, one-level taper hole voltage 35V, secondary taper hole voltage 3.0V; Six grades of bar lens voltage 0.1V, 120 ℃ of source temperatures, 350 ℃ of desolventizing temperature; Desolventizing nitrogen flow rate 500L/h, taper hole nitrogen flow rate 50L/h, ion energy 0.5; Utilize the TIC and the ultraviolet detection chromatogram detection reaction product of reaction product, guarantee Hip purity; Under the quantitative test SIM model
M/zThe Hip peak area of 180 extraction ion figure, the peak area of calculating Hip;
(3) calculating of antihypertensive activity
Obtain the peak area of reaction group and blank group reaction product Hip through said method, it is (A that the ACE of step-down peptide suppresses active IP
C-A
S) * 100/A
C, wherein: IP is the inhibiting rate of sample to ACE; A
CPeak area for Hip in the blank group; A
SPeak area for Hip in the reaction group.
2. the detection method of the angiotensin converting enzyme inhibition activity of step-down peptide according to claim 1 is characterized in that: the used reverse-phase chromatographic column of said step (2) is the Ug120-C18 analytical column.
3. the detection method of the angiotensin converting enzyme inhibition activity of step-down peptide according to claim 1 is characterized in that: the used electric spray ion source of said step (2) is the electron spray ionisation positive ion mode.
4. the detection method of the angiotensin converting enzyme inhibition activity of step-down peptide according to claim 1 is characterized in that: the said reaction group reaction of said step (2) product repeats to survey three times, and the relative deviation of peak area is 0.1-0.4%.
5. the detection method of the angiotensin converting enzyme inhibition activity of step-down peptide according to claim 1 is characterized in that: the described blank group of said step (3) ACE and HHL reaction when not adding the step-down peptide generate the peak area of Hip in LC-MS and represent; Reaction group ACE and HHL reaction when adding the step-down peptide generate the peak area of Hip in LC-MS and represent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108659099A (en) * | 2018-05-21 | 2018-10-16 | 浙江大学 | A kind of MS probe and its application for hypertensin conversion enzyme activity detection |
| CN109206479A (en) * | 2017-09-18 | 2019-01-15 | 北京中医药大学 | A kind of vinegar beans glutelin source Antihypertensive Peptides and its application |
| CN115047092A (en) * | 2022-04-07 | 2022-09-13 | 济宁医学院 | Method for screening angiotensin transferase II inhibitor |
-
2012
- 2012-03-30 CN CN2012100899070A patent/CN102608234A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 《食品与发酵工业》 20081231 许建香 等 RP-HPLC法测定蜂王浆水溶性蛋白及其相关产物对ACE的抑制作用 第1.1节至2.2节 1-5 第34卷, 第9期 * |
| 戴军 等: "绍兴黄酒中ACE活性抑制肽的分离分析", 《分析测试学报》 * |
| 戴军 等: "绍兴黄酒中一种ACE活性抑制肽的分离和鉴定", 《食品与发酵工业》 * |
| 许建香 等: "RP—HPLC法测定蜂王浆水溶性蛋白及其相关产物对ACE的抑制作用", 《食品与发酵工业》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109206479A (en) * | 2017-09-18 | 2019-01-15 | 北京中医药大学 | A kind of vinegar beans glutelin source Antihypertensive Peptides and its application |
| CN109206479B (en) * | 2017-09-18 | 2021-10-29 | 北京中医药大学 | Vinegar bean gluten source antihypertensive peptide and application thereof |
| CN108659099A (en) * | 2018-05-21 | 2018-10-16 | 浙江大学 | A kind of MS probe and its application for hypertensin conversion enzyme activity detection |
| CN108659099B (en) * | 2018-05-21 | 2021-02-02 | 浙江大学 | Mass spectrum probe for detecting activity of angiotensin converting enzyme and application thereof |
| CN115047092A (en) * | 2022-04-07 | 2022-09-13 | 济宁医学院 | Method for screening angiotensin transferase II inhibitor |
| CN115047092B (en) * | 2022-04-07 | 2023-10-20 | 济宁医学院 | Screening method of angiotensin-transferase II inhibitor |
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Application publication date: 20120725 |