CN102128884A - Method for screening superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with mass spectrum - Google Patents
Method for screening superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with mass spectrum Download PDFInfo
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Abstract
The invention provides a method for screening a superoxide anion scavenging agent by coupling ultrahigh-performance liquid chromatography with a mass spectrum. The method comprises the following steps of: analyzing the scavenging rate of a natural product extract or monomer on superoxide anions and detecting and quantifying the content of superoxide anions by detecting the change of 2-(4-iodbenzene)-3-(4-nitrobenzene)-5-(2,4-disulfobenzoic)-2H-tetrazole sodium salt serving as a trapping agent before and after reaction with superoxide anions. A method for coupling ultrahigh-performance liquid chromatography with the mass spectrum is applied to screening of the superoxide anion scavenging agent, so that the dosage of a sample is small, detection is quick and accurate and a relevant coefficient in a linear equation is up to 0.998; and the mass spectrum is used for detecting in specific to the mass-charge ratio of a compound, so that high accuracy and high specificity are achieved, false positive and false negative results in spectrometry screening are avoided, and the problem of difficulty in detecting caused by short service lives of the superoxide anions in an electronic spin resonance technology is solved. The method is used for screening the superoxide anion scavenging agent and detecting and quantifying the superoxide anions.
Description
Invention field
The invention belongs to the analytical chemistry field, relate to the method for Ultra Performance Liquid Chromatography and mass spectrometry screening superoxide anion scavenger, be specifically related to the method for Ultra Performance Liquid Chromatography and mass spectrometry screening superoxide anion scavenger.
Technical background
Along with the development of free radical medical science, people recognize that multiple disease is relevant with free radical.Body produces ultra-oxygen anion free radical in metabolic process, excessive ultra-oxygen anion free radical such as untimely removing, and accumulation can produce cytotoxicity in vivo, and the 26S Proteasome Structure and Function of disrupting biofilm causes multiple disease.Therefore, anti-oxidation medicine has important effect in anti-ageing and the multiple relevant disease of control.Contain multiple different oxidation-resistant active ingredient in the natural drug, more and more be subjected to domestic and international medical worker's extensive concern.Set up fast, superoxide anion scavenger screening technique can lay the foundation for the screening antiaging agent reliably.
Existing screening superoxide anion scavenger and detection, quantitatively the method for superoxide anion mainly contains the free resonant method of electronics (YASUKO NODA, TAKAO KANEYUKI, AKITANEMORI, AND LESTER PACKER, J.Agric.Food Chem.50 (2002), 166-171), chemoluminescence method (hahanara Banu, Gillian M.Greenway, R.Alan Wheatley, Analytica Chimica Acta 541 (2005) 91-97), uv detection method (Bayer W F, FridovichI, 161 (1987) 559-566), these methods are convenient and swift, can satisfy the screening requirement to a certain extent, but be easy to generate false positive and false negative result in the spectroscopic methodology screening, and electronic self-rotary resonant technology causes detection relatively more difficult owing to the superoxide anion life-span is short when detecting superoxide anion.Mass spectrum detects at the compound quality charge ratio, the degree of accuracy height, specificity is good, simultaneously in conjunction with the superelevation liquid chromatography, can carry out accurate quantitatively (Ying Wang to thing to be detected, Yamin Yao, Rui An, Lisha You, Xinhong Wang, Journal of Chromatography B, 877 (2009) 1820-1826).
Summary of the invention
The method that the purpose of this invention is to provide Ultra Performance Liquid Chromatography and mass spectrometry screening superoxide anion scavenger.The present invention can be used for analyzing natural extracts or monomer to the external clearance rate of superoxide anion and detection, the quantitatively Ultra Performance Liquid Chromatography and the mass spectrometry combination method of superoxide anion, is specifically related to the method for Ultra Performance Liquid Chromatography and mass spectrometry screening superoxide anion scavenger.
The method of Ultra Performance Liquid Chromatography and mass spectrometry screening superoxide anion scavenger, comprise following content: produce system as superoxide anion with xanthine oxidase-xanthine enzymatic reaction system, 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt is the superoxide anion trapping agent, with Ultra Performance Liquid Chromatography and mass spectrometry to trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt carries out quantitatively, go out the clearance rate of scavenger by the change calculations of calculating trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content to superoxide anion.
The method of Ultra Performance Liquid Chromatography provided by the invention and mass spectrometry screening superoxide anion scavenger may further comprise the steps:
Described Ultra Performance Liquid Chromatography is meant: compare with high performance liquid chromatography and have superelevation degree of separation, ultraspeed and hypersensitivity;
Described superoxide anion scavenger is natural extracts or monomer; The preferred natural extracts Radix Glycyrrhizae of described superoxide anion scavenger water extract, catechin, genistin or jamaicin;
(1) superoxide anion produces the preparation of system damping fluid
Enzymatic reaction is to produce one of main source of superoxide anion in the biosome, and the used superoxide anion generation system of the present invention is xanthine oxidase-xanthine enzymatic reaction system, and xanthine oxidase catalytic substrate xanthine produces superoxide anion;
Superoxide anion produces the system damping fluid and comprises 50 mMs/rise trishydroxymethylaminomethane, 7 mMs/rise hydrochloric acid, and 1 mM/rise disodium EDTA, the pH value is 8.9;
(2) preparation of standard items
Water is made into the standard solution that concentration is 1 micromoles per liter, 3 micromoles per liter, 5 micromoles per liter, 7 micromoles per liter or 10 micromoles per liter respectively with standard items, and the standard solution of each concentration all contains the TUDCANa of 1 micromoles per liter as interior mark; Described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, and energy and superoxide anion reaction produce water-soluble formazan dye;
(3) preparation of reference substance, blank sample and sample
The preparation of reference substance: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, the xanthine that adds final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, product in contrast; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
The preparation of blank sample: cumulative volume 200 microlitres, do not add xanthine oxidase and xanthine, 2-(4-iodobenzene)-3-(4-the nitrobenzene)-5-(2 that adds final concentration 25 micromoles per liter, the 4-disulfobenzene)-2H-tetrazole sodium salt, the methyl alcohol that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as blank sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
The preparation of sample: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, adding final concentration is the superoxide anion scavenger of 20 micromoles per liter, the xanthine of final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
(4) detection of standard items, reference substance, blank sample and sample
Standard items are detected: adopting TUDCANa is interior mark; with inner mark method ration; series standard product solution to step (2) carries out Ultra Performance Liquid Chromatography and mass spectrometry detection; from total ion current figure, extract 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2; the 4-disulfobenzene)-chromatogram of 2H-tetrazole sodium salt and TUDCANa; integration is asked peak area respectively; with described standard items concentration is horizontal ordinate; with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2; the 4-disulfobenzene)-2H-tetrazole sodium salt and TUDCANa peak area ratio are ordinate; in described standard items concentration is 1 micromoles per liter to 10 micromoles per liter scope; obtain the linear equation of 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt with the Excel software of Microsoft (Microsoft):
y＝ax+b
In the formula, y is described standard items 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt and interior mark TUDCANa peak area ratio; X is the concentration of standard items, and dimension is a micromoles per liter; A, b are the constant that computed in software obtains; This computed in software obtains coefficient R simultaneously
2
1. the testing conditions of Ultra Performance Liquid Chromatography
Ultra Performance Liquid Chromatography instrument: Waters ACQUITY;
Chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 * 50 millimeters, 1.7 microns);
Moving phase: methyl alcohol (A) and water (B);
The gradient elution program: 0～1 minute, 20%A～60%A; 1～2 minute, 60%A～100%A; 2～3 minutes, 100%A～20%A; 3～5 minutes, 20%A; The number percent of indication is percent by volume;
Flow velocity: 0.2 ml/min;
Sample size: 5 microlitres;
2. mass spectrum condition
Mass spectrometer: Waters Xevo TQ;
Ion gun: electron spray (ESI) ionization source negative ion mode;
Capillary voltage: 2000 volts;
The gas nitrogen temperature desolvates: 350 degrees centigrade;
The gas nitrogen flow rate desolvates: 800 liters/hour;
Taper hole gas nitrogen flow rate: 50 liters/hour;
Collision gas argon gas flow velocity: 0.15 liter/hour;
The low side resolution of first quadrupole rod is: 3.0; High-end resolution is 15.0; Ion energy is: 0.5;
The low side resolution of second quadrupole rod is: 3.0; High-end resolution is 13.5; Ion energy is: 0.5;
The above mass spectrum condition is identical when detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt with interior mark TUDCANa;
Taper hole voltage is detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and 2H-tetrazole sodium salt is different during with interior mark TUDCANa: taper hole voltage is 20 volts when detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt; Taper hole voltage is 30 volts when marking TUDCANa in detecting;
The fragmention of fragmentation energies and selection is detecting 2-(4-iodobenzene)-3-(4-nitrobenzene-5-(2, the 4-disulfobenzene)-and 2H-tetrazole sodium salt also is different during with interior mark TUDCANa: detect 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and fragmentation energies is 20 during 2H-tetrazole sodium salt, fragmention is 332.83; Fragmentation energies is 30 when marking TUDCANa in detecting, and fragmention is 124.08;
Reference substance, blank sample and sample detection all detect respectively by above-mentioned Ultra Performance Liquid Chromatography and mass spectrum condition;
(5) the superoxide anion scavenger is to the clearance rate of superoxide anion
Thereby the superoxide anion scavenger is removed superoxide anion and is caused and 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-amount of the superoxide anion of 2H-tetrazole sodium salt reaction reduces, cause trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and the increase of 2H-tetrazole sodium salt surplus, the amount of the water-soluble formazan dye of reduzate reduces; So the variation that the superoxide anion scavenger can pass through trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content or product water dissolubility formazan dye content to the clearance rate of superoxide anion is estimated;
The present invention utilizes the variation of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content to calculate;
In linear equation, try to achieve 2-in reference substance, blank sample and the sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2 according to the peak area ratio that reference substance, blank sample and sample are recorded respectively, the 4-disulfobenzene)-and 2H-tetrazole sodium salt content concentration, the superoxide anion scavenger calculates according to following formula the clearance rate of superoxide anion:
I
Sample=(C
Sample-C
Contrast)/(C
Blank-C
Contrast) * 100%
In the formula, I is the clearance rate of superoxide anion scavenger to superoxide anion;
C
SampleBe 2-in the sample of trying to achieve according to linear equation (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content concentration, dimension is a micromoles per liter;
C
ContrastBe the concentration of 2-in the reference substance of trying to achieve according to linear equation (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content, dimension is a micromoles per liter;
C
Blank(4-nitrobenzene-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content concentration, dimension is a micromoles per liter for 2-in the blank sample of trying to achieve according to linear equation (4-iodobenzene)-3-.
Beneficial effect: the invention provides the method for Ultra Performance Liquid Chromatography and mass spectrometry screening superoxide anion scavenger, described superoxide anion scavenger is natural extracts or monomer.The present invention is with the method for Ultra Performance Liquid Chromatography and mass spectrometry, is used to analyze natural extracts or monomer to the external clearance rate of superoxide anion and detection, quantitative superoxide anion.The present invention is applied to Ultra Performance Liquid Chromatography and mass spectrometry combination method in the screening of superoxide anion scavenger, sample detection fast, accurately, the linear equation related coefficient is higher than 0.998, mass spectrum detects at the compound quality charge ratio, the degree of accuracy height, specificity is good, false positive and false negative result in the spectroscopic methodology screening have been avoided, also overcome simultaneously electronic self-rotary resonant technology when detecting superoxide anion because the short problem that runs into detection difficult of superoxide anion life-span, can be used for the detection, quantitative of screening of superoxide anion scavenger and superoxide anion;
Described superoxide anion scavenger is a catechin, and obtaining 20 micromoles per liter catechins is 67% to the clearance rate I of superoxide anion.
Described superoxide anion scavenger is a genistin, and obtaining 20 micromoles per liter genistins is 48% to the clearance rate I of superoxide anion.
Described superoxide anion scavenger is a jamaicin, and obtaining 20 micromoles per liter jamaicins is 34% to the clearance rate I of superoxide anion.
Described superoxide anion scavenger is the Radix Glycyrrhizae water extract, and obtaining 0.1 mg/ml Radix Glycyrrhizae water extract is 59% to the clearance rate I of superoxide anion.
Description of drawings
Fig. 1 is standard items 2-(4-iodobenzene)-3-(4-the nitrobenzene)-5-(2 of 5 micromoles per liter among the embodiment 1; the 4-disulfobenzene)-and the total ions chromatogram of many reaction detection negative ion mode of the interior mark TUDCANa (peak 2) of 2H-tetrazole sodium salt (peak 1) and 1 micromoles per liter, signal intensity is 2.56 * 10
5
Fig. 2 is standard items 2-(4-iodobenzene)-3-(4-the nitrobenzene)-5-(2 of 5 micromoles per liter among the embodiment 1, the 4-disulfobenzene)-the extraction chromatography figure of many reaction detection negative ion mode of 2H-tetrazole sodium salt, extracting used ion pair is parent ion 627.81 and fragmention 332.83, and signal intensity is 2.53 * 10
5
Fig. 3 is the extraction chromatography figure of many reaction detection negative ion mode of the interior mark TUDCANa of 1 micromoles per liter among the embodiment 1, and extracting used ion pair is parent ion 498.08 and fragmention 124.08, and signal intensity is 1.65 * 10
5
Fig. 4 is the typical curve and the linear formula of variable concentrations 2-among the embodiment 1 (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt standard items.
Fig. 5 is 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2 in the reference substance among the embodiment 1, the 4-disulfobenzene)-the extraction chromatography figure of many reaction detection negative ion mode of 2H-tetrazole sodium salt, extracting used ion pair is parent ion 627.81 and fragmention 332.83, and signal intensity is 7.99 * 10
4
Fig. 6 is the extraction chromatography figure of many reaction detection negative ion mode of the interior mark TUDCANa of 1 micromoles per liter in the reference substance among the embodiment 1, and extracting used ion pair is parent ion 498.08 and fragmention 124.08, and signal intensity is 1.53 * 10
5
Fig. 7 is 2-(4-iodobenzene) in the embodiment 1 empty sample-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-the extraction chromatography figure of many reaction detection negative ion mode of 2H-tetrazole sodium salt, extracting used ion pair is parent ion 627.81 and fragmention 332.83, and signal intensity is 2.60 * 10
5
Fig. 8 is the extraction chromatography figure of many reaction detection negative ion mode of the interior mark TUDCANa of 1 micromoles per liter in the embodiment 1 empty sample, and extracting used ion pair is parent ion 498.08 and fragmention 124.08, and signal intensity is 1.68 * 10
5
Fig. 9 is 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2 in the sample among the embodiment 1, the 4-disulfobenzene)-the extraction chromatography figure of many reaction detection negative ion mode of 2H-tetrazole sodium salt, extracting used ion pair is parent ion 627.81 and fragmention 332.83, and signal intensity is 1.10 * 10
5
Figure 10 is the extraction chromatography figure of many reaction detection negative ion mode of the interior mark TUDCANa of 1 micromoles per liter in the sample among the embodiment 1, extracts used ion pair parent ion 498.08 and fragmention 124.08, and signal intensity is 1.50 * 10
5
Figure 11 is 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2 in the sample among the embodiment 2, the 4-disulfobenzene)-the extraction chromatography figure of many reaction detection negative ion mode of 2H-tetrazole sodium salt, extracting used ion pair is parent ion 627.81 and fragmention 332.83, and signal intensity is 1.33 * 10
5
Figure 12 is the extraction chromatography figure of many reaction detection negative ion mode of the interior mark TUDCANa of 1 micromoles per liter in the sample among the embodiment 2, and extracting used ion pair is parent ion 498.08 and fragmention 124.08, and signal intensity is 1.56 * 10
5
Figure 13 is 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2 in the sample among the embodiment 3, the 4-disulfobenzene)-the extraction chromatography figure of many reaction detection negative ion mode of 2H-tetrazole sodium salt, extracting used ion pair is parent ion 627.81 and fragmention 332.83, and signal intensity is 1.80 * 10
5
Figure 14 is the extraction chromatography figure of many reaction detection negative ion mode of the interior mark TUDCANa of 1 micromoles per liter in the sample among the embodiment 3, and extracting used ion pair is parent ion 498.08 and fragmention 124.08, and signal intensity is 1.62 * 10
5
Figure 15 is 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2 in the sample among the embodiment 4, the 4-disulfobenzene)-the extraction chromatography figure of many reaction detection negative ion mode of 2H-tetrazole sodium salt, extracting used ion pair is parent ion 627.81 and fragmention 332.83, and signal intensity is 1.21 * 10
5
Figure 16 is the extraction chromatography figure of many reaction detection negative ion mode of the interior mark TUDCANa of 1 micromoles per liter in the sample among the embodiment 4, and extracting used ion pair is parent ion 498.08 and fragmention 124.08, and signal intensity is 1.57 * 10
5
Embodiment
Below will invention be described by specific embodiment.In addition, employed material all can be bought by commercial sources except that special instruction is arranged from the market among the embodiment.
The method of Ultra Performance Liquid Chromatography provided by the invention and mass spectrometry screening superoxide anion scavenger, it may further comprise the steps:
Described Ultra Performance Liquid Chromatography is meant: compare with high performance liquid chromatography and have superelevation degree of separation, ultraspeed and hypersensitivity;
Described superoxide anion scavenger is natural extracts or monomer; The described superoxide anion scavenger of present embodiment is a catechin;
(1) superoxide anion produces the preparation of system damping fluid
Enzymatic reaction is to produce one of main source of superoxide anion in the biosome, and the used superoxide anion generation system of the present invention is xanthine oxidase-xanthine enzymatic reaction system, and xanthine oxidase catalytic substrate xanthine produces superoxide anion;
Superoxide anion produces the system damping fluid and comprises 50 mMs/rise trishydroxymethylaminomethane, 7 mMs/rise hydrochloric acid, and 1 mM/rise disodium EDTA, the pH value is 8.9;
(2) preparation of standard items
Water is made into the standard solution that concentration is 1 micromoles per liter, 3 micromoles per liter, 5 micromoles per liter, 7 micromoles per liter or 10 micromoles per liter respectively with standard items, and the standard solution of each concentration all contains the TUDCANa of 1 micromoles per liter as interior mark; Described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, and energy and superoxide anion reaction produce water-soluble formazan dye;
(3) preparation of reference substance, blank sample and sample
The preparation of reference substance: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, the xanthine that adds final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, product in contrast; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
The preparation of blank sample: cumulative volume 200 microlitres, do not add xanthine oxidase and xanthine, 2-(4-iodobenzene)-3-(4-the nitrobenzene)-5-(2 that adds final concentration 25 micromoles per liter, the 4-disulfobenzene)-2H-tetrazole sodium salt, the methyl alcohol that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as blank sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
The preparation of sample: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, adding final concentration is the catechin of 20 micromoles per liter, the xanthine of final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
(4) detection of standard items, reference substance, blank sample and sample
Standard items are detected: adopting TUDCANa is interior mark; with inner mark method ration; series standard product solution to step (2) carries out Ultra Performance Liquid Chromatography and mass spectrometry detection; from total ion current figure, extract 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2; the 4-disulfobenzene)-chromatogram of 2H-tetrazole sodium salt and TUDCANa; integration is asked peak area respectively; with described standard items concentration is horizontal ordinate; with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2; the 4-disulfobenzene)-2H-tetrazole sodium salt and TUDCANa peak area ratio are ordinate; in described standard items concentration is 1 micromoles per liter to 10 micromoles per liter scope; obtain the linear equation of 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt with the Excel software of Microsoft (Microsoft):
y＝ax+b
In the formula, y is described standard items 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt and interior mark TUDCANa peak area ratio; X is the concentration of standard items, and dimension is a micromoles per liter; A, b are the constant that computed in software obtains; This computed in software obtains coefficient R simultaneously
2
1. the testing conditions of Ultra Performance Liquid Chromatography
Ultra Performance Liquid Chromatography instrument: Waters ACQUITY;
Chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 * 50 millimeters, 1.7 microns);
Moving phase: methyl alcohol (A) and water (B);
The gradient elution program: 0～1 minute, 20%A～60%A; 1～2 minute, 60%A～100%A; 2～3 minutes, 100%A～20%A; 3～5 minutes, 20%A; The number percent of indication is percent by volume;
Flow velocity: 0.2 ml/min;
Sample size: 5 microlitres;
2. mass spectrum condition
Mass spectrometer: Waters Xevo TQ;
Ion gun: electron spray (ESI) ionization source negative ion mode;
Capillary voltage: 2000 volts;
The gas nitrogen temperature desolvates: 350 degrees centigrade;
The gas nitrogen flow rate desolvates: 800 liters/hour;
Taper hole gas nitrogen flow rate: 50 liters/hour;
Collision gas argon gas flow velocity: 0.15 liter/hour;
The low side resolution of first quadrupole rod is: 3.0; High-end resolution is 15.0; Ion energy is: 0.5;
The low side resolution of second quadrupole rod is: 3.0; High-end resolution is 13.5; Ion energy is: 0.5;
The above mass spectrum condition is identical when detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt with interior mark TUDCANa;
Taper hole voltage is detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and 2H-tetrazole sodium salt is different during with interior mark TUDCANa: (taper hole voltage is 20 volts during 4-iodobenzene-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt to detect 2-; Taper hole voltage is 30 volts when marking TUDCANa in detecting;
The fragmention of fragmentation energies and selection is detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and 2H-tetrazole sodium salt also is different during with interior mark TUDCANa: detect 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and fragmentation energies is 20 during 2H-tetrazole sodium salt, fragmention is 332.83; Fragmentation energies is 30 when marking TUDCANa in detecting, and fragmention is 124.08;
Reference substance, blank sample and sample detection all detect respectively by above-mentioned Ultra Performance Liquid Chromatography and mass spectrum condition;
(5) the superoxide anion scavenger is to the clearance rate of superoxide anion
Thereby superoxide anion scavenger natural extracts or monomer are removed superoxide anion and are caused and 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-amount of the superoxide anion of 2H-tetrazole sodium salt reaction reduces, cause trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and the increase of 2H-tetrazole sodium salt surplus, the amount of the water-soluble formazan dye of reduzate reduces; So the variation that the superoxide anion scavenger can pass through trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content or product water dissolubility formazan dye content to the clearance rate of superoxide anion is estimated; The present invention utilizes the variation of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content to calculate;
In linear equation, try to achieve 2-in reference substance, blank sample and the sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2 according to the peak area ratio that reference substance, blank sample and sample are recorded respectively, the 4-disulfobenzene)-and 2H-tetrazole sodium salt content concentration, the superoxide anion scavenger calculates according to following formula the clearance rate of superoxide anion:
I
Sample=(C
Sample-C
Contrast)/(C
Blank-C
Contrast) * 100%
In the formula, I is the clearance rate of superoxide anion scavenger to superoxide anion;
C
SampleFor 2-in the sample of trying to achieve (4-iodobenzene)-3-according to linear equation (4-nitrobenzene-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content concentration, dimension is a micromoles per liter;
C
ContrastFor 2-in the reference substance of trying to achieve (4-iodobenzene)-3-according to linear equation (concentration of 4-nitrobenzene-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content, dimension is a micromoles per liter;
C
BlankBe 2-in the blank sample of trying to achieve according to linear equation (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content concentration, dimension is a micromoles per liter.
Calculating 20 mol catechin standard items is 67% to the clearance rate I of superoxide anion.The result arrives Figure 10 referring to Fig. 1.
The described superoxide anion scavenger of present embodiment is a genistin;
Described sample is as follows: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, adding final concentration is the genistin of 20 micromoles per liter, the xanthine of final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
Remaining is with embodiment 1;
Detecting and calculate 20 micromoles per liter genistin standard items according to the detection step of embodiment 1 is 48% to the clearance rate I of superoxide anion.Figure 11 and Figure 12 are the extraction chromatography figure of sample among the embodiment 2.
The described superoxide anion scavenger of present embodiment is a jamaicin;
Described sample is as follows: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, adding final concentration is the jamaicin of 20 micromoles per liter, the xanthine of final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
Remaining is with embodiment 1;
Detecting and calculate 20 micromoles per liter jamaicin standard items according to the detection step of embodiment 1 is 34% to the clearance rate I of superoxide anion.Figure 13 and Figure 14 are the extraction chromatography figure of sample among the embodiment 3.
The described superoxide anion scavenger of present embodiment is the Radix Glycyrrhizae water extract;
Described sample is as follows: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, adding final concentration is the Radix Glycyrrhizae water extract of 0.1 mg/ml, the xanthine of final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
Remaining is with embodiment 1;
The Radix Glycyrrhizae water extract that detects and calculate 0.1 mg/ml according to the detection step of embodiment 1 is 59% to the clearance rate I of superoxide anion.Figure 15 and Figure 16 are the extraction chromatography figure of sample among the embodiment 4.
Claims (6)
1. the method for Ultra Performance Liquid Chromatography and mass spectrometry screening superoxide anion scavenger is characterized in that, may further comprise the steps:
Described Ultra Performance Liquid Chromatography is meant: compare with high performance liquid chromatography and have superelevation degree of separation, ultraspeed and hypersensitivity;
Described superoxide anion scavenger is natural extracts or monomer;
(1) superoxide anion produces the preparation of system damping fluid
Used superoxide anion generation system is xanthine oxidase-xanthine enzymatic reaction system, and xanthine oxidase catalytic substrate xanthine produces superoxide anion;
Superoxide anion produces the system damping fluid and comprises 50 mMs/rise trishydroxymethylaminomethane, 7 mMs/rise hydrochloric acid, and 1 mM/rise disodium EDTA, the pH value is 8.9;
(2) preparation of standard items
Water is made into the standard solution that concentration is 1 micromoles per liter, 3 micromoles per liter, 5 micromoles per liter, 7 micromoles per liter or 10 micromoles per liter respectively with standard items, and the standard solution of each concentration all contains the TUDCANa of 1 micromoles per liter as interior mark; Described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, and energy and superoxide anion reaction produce water-soluble formazan dye;
(3) preparation of reference substance, blank sample and sample
The preparation of reference substance: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, the xanthine that adds final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, product in contrast; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
The preparation of blank sample: cumulative volume 200 microlitres, do not add xanthine oxidase and xanthine, 2-(4-iodobenzene)-3-(4-the nitrobenzene)-5-(2 that adds final concentration 25 micromoles per liter, the 4-disulfobenzene)-2H-tetrazole sodium salt, the methyl alcohol that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as blank sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
The preparation of sample: reaction cumulative volume 200 microlitres, final concentration 100 nanomoles/liter xanthine oxidase, hatched 30 minutes at 37 ℃, adding final concentration is the superoxide anion scavenger of 20 micromoles per liter, the xanthine of final concentration 100 micromoles per liter, the 2-of final concentration 25 micromoles per liter (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt, 37 ℃ of reactions are after 5 minutes, the methyl alcohol cessation reaction that adds 800 microlitres, adding final concentration is the interior mark TUDCANa of 1 micromoles per liter, as sample; Measure for Ultra Performance Liquid Chromatography and mass spectrometry;
(4) detection of standard items, reference substance, blank sample and sample
Standard items are detected: adopting TUDCANa is interior mark; with inner mark method ration; series standard product solution to step (2) carries out Ultra Performance Liquid Chromatography and mass spectrometry detection; from total ion current figure, extract 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2; the 4-disulfobenzene)-chromatogram of 2H-tetrazole sodium salt and TUDCANa; integration is asked peak area respectively; with described standard items concentration is horizontal ordinate; with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2; the 4-disulfobenzene)-2H-tetrazole sodium salt and TUDCANa peak area ratio are ordinate; in described standard items concentration is 1 micromoles per liter to 10 micromoles per liter scope; obtain the linear equation of 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt with the Excel software of Microsoft (Microsoft):
y＝ax+b
In the formula, y is described standard items 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt and interior mark TUDCANa peak area ratio; X is the concentration of standard items, and dimension is a micromoles per liter; A, b are the constant that computed in software obtains; This computed in software obtains coefficient R simultaneously
2
1. the testing conditions of Ultra Performance Liquid Chromatography
Ultra Performance Liquid Chromatography instrument: Waters ACQUITY;
Chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 * 50 millimeters, 1.7 microns);
Moving phase: methyl alcohol (A) and water (B);
The gradient elution program: 0～1 minute, 20%A～60%A; 1～2 minute, 60%A～100%A; 2～3 minutes, 100%A～20%A; 3～5 minutes, 20%A; The number percent of indication is percent by volume;
Flow velocity: 0.2 ml/min;
Sample size: 5 microlitres;
2. mass spectrum condition
Mass spectrometer: Waters Xevo TQ;
Ion gun: electron spray (ESI) ionization source negative ion mode;
Capillary voltage: 2000 volts;
The gas nitrogen temperature desolvates: 350 degrees centigrade;
The gas nitrogen flow rate desolvates: 800 liters/hour;
Taper hole gas nitrogen flow rate: 50 liters/hour;
Collision gas argon gas flow velocity: 0.15 liter/hour;
The low side resolution of first quadrupole rod is: 3.0; High-end resolution is 15.0; Ion energy is: 0.5;
The low side resolution of second quadrupole rod is: 3.0; High-end resolution is 13.5; Ion energy is: 0.5;
The above mass spectrum condition is identical when detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt with interior mark TUDCANa;
Taper hole voltage is detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and 2H-tetrazole sodium salt is different during with interior mark TUDCANa: taper hole voltage is 20 volts when detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt; Taper hole voltage is 30 volts when marking TUDCANa in detecting;
The fragmention of fragmentation energies and selection is detecting 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and 2H-tetrazole sodium salt also is different during with interior mark TUDCANa: detect 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-and fragmentation energies is 20 during 2H-tetrazole sodium salt, fragmention is 332.83; Fragmentation energies is 30 when marking TUDCANa in detecting, and fragmention is 124.08;
Reference substance, blank sample and sample detection all detect respectively by above-mentioned Ultra Performance Liquid Chromatography and mass spectrum condition;
(5) the superoxide anion scavenger is to the clearance rate of superoxide anion
The superoxide anion scavenger calculates evaluation to the variation that the clearance rate of superoxide anion can be passed through trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content or product water dissolubility formazan dye content;
In linear equation, try to achieve 2-in reference substance, blank sample and the sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2 according to the peak area ratio that reference substance, blank sample and sample are recorded respectively, the 4-disulfobenzene)-and 2H-tetrazole sodium salt content concentration, the superoxide anion scavenger calculates according to following formula the clearance rate of superoxide anion:
I
Sample=(C
Sample-C
Contrast)/(C
Blank-C
Contrast) * 100%
In the formula, I is the clearance rate of superoxide anion scavenger to superoxide anion;
C
SampleBe 2-in the sample of trying to achieve according to linear equation (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content concentration, dimension is a micromoles per liter;
C
ContrastBe the concentration of 2-in the reference substance of trying to achieve according to linear equation (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content, dimension is a micromoles per liter;
C
BlankBe 2-in the blank sample of trying to achieve according to linear equation (4-iodobenzene)-3-(4-nitrobenzene)-5-(2, the 4-disulfobenzene)-2H-tetrazole sodium salt content concentration, dimension is a micromoles per liter.
2. the method for a kind of Ultra Performance Liquid Chromatography as claimed in claim 1 and mass spectrometry screening superoxide anion scavenger, it is characterized in that described superoxide anion scavenger is natural extracts Radix Glycyrrhizae water extract, catechin, genistin or jamaicin.
3. the method for a kind of Ultra Performance Liquid Chromatography as claimed in claim 2 and mass spectrometry screening superoxide anion scavenger is characterized in that described superoxide anion scavenger is a natural extracts Radix Glycyrrhizae water extract.
4. the method for a kind of Ultra Performance Liquid Chromatography as claimed in claim 2 and mass spectrometry screening superoxide anion scavenger is characterized in that described superoxide anion scavenger is a catechin.
5. the method for a kind of Ultra Performance Liquid Chromatography as claimed in claim 2 and mass spectrometry screening superoxide anion scavenger is characterized in that described superoxide anion scavenger is a genistin.
6. the method for a kind of Ultra Performance Liquid Chromatography as claimed in claim 2 and mass spectrometry screening superoxide anion scavenger is characterized in that described superoxide anion scavenger is a jamaicin.
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| CN103674862A (en) * | 2012-09-14 | 2014-03-26 | 中国科学院长春应用化学研究所 | Method for screening superoxide anion scavenging agents |
| CN103674870A (en) * | 2012-09-14 | 2014-03-26 | 中国科学院长春应用化学研究所 | Method for measuring activity of superoxide dismutase |
| CN104792910A (en) * | 2015-04-28 | 2015-07-22 | 中国药科大学 | Method for simultaneously screening superoxide anion remover and xanthine oxidase inhibitor |
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| CN103674862A (en) * | 2012-09-14 | 2014-03-26 | 中国科学院长春应用化学研究所 | Method for screening superoxide anion scavenging agents |
| CN103674870A (en) * | 2012-09-14 | 2014-03-26 | 中国科学院长春应用化学研究所 | Method for measuring activity of superoxide dismutase |
| CN103674870B (en) * | 2012-09-14 | 2015-11-18 | 中国科学院长春应用化学研究所 | A kind of method measuring superoxide dismutase activity |
| CN103674862B (en) * | 2012-09-14 | 2015-11-18 | 中国科学院长春应用化学研究所 | A kind of method of screening superoxide anion scavenger |
| CN104792910A (en) * | 2015-04-28 | 2015-07-22 | 中国药科大学 | Method for simultaneously screening superoxide anion remover and xanthine oxidase inhibitor |
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