CN102445509A - Reversed phase ion pair HPLC (high performance liquid chromatography) method for rapid trace detection of TTX (tetrodoxin) in fresh puffer fish blood - Google Patents
Reversed phase ion pair HPLC (high performance liquid chromatography) method for rapid trace detection of TTX (tetrodoxin) in fresh puffer fish blood Download PDFInfo
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Abstract
The invention which belongs to the technical field of the puffer fish toxin detection concretely relates to a reversed phase ion pair HPLC method for the rapid trace detection of TTX in the fresh puffer fish blood. The method comprises the following steps: 1, acquiring and pretreating fresh puffer fish blood samples; 2, preparing a mobile phase; 3, preparing standard samples; 4, setting chromatography conditions; and 5, carrying out sample introduction detection analysis and result determination. Compared with current national standard methods of the TTX detection, the method of the invention has the characteristics of simple sampling, simple sample preparation, time saving and rapidness, low detection cost, high trace, qualitative and quantitative accuracy, good reappearance, no need of mass spectrums or other expensive devices, and easy popularization and application to base detection centers, provides a new detection means and a scientific basis for the safe fresh puffer eating, and performs an important effect to the promotion of the legal puffer fish eating in China and the development of the puffer fish industry and the puffer fish diet culture.
Description
Technical field
The invention belongs to Puffer fish fugutoxin detection technique field, river, be specifically related to bright fugutoxin trace rapidly and efficiently detection technique and river Puffer fish safe edible technical applications.
Background technology
The edible now river Puffer fish of China all belongs to Fugu (Takifugu); Be the genus of Molidae (Tetrodantidae), kind surplus this genus fish whole world has 20 approximately is during the overwhelming majority is distributed in, day coastal waters; U.S.A of its meat flavour; The good reputation that " king in the fish " arranged, in China coastal cities and ground such as Japan, Korea S very popular, have very high economic worth.Become the important object of propagating artificially of China's Coastal Areas at present.But because of these fish contain " fugutoxin " (Tetrodoxin TTX), but under its delicious temptation, the in vogue and rules and regulations strictness of this edible river Puffer day after day still has because of eating river Puffer poisoning and take place every year.The legend that China is among the people to be had for a long time " defy death and eat the river Puffer "; Particularly have diet river Puffer fish history culture in 1,100, becoming the maximum river Puffer fish area of consumption of China; Middle area is raised in Jiangsu Province with " township of river Puffer " /> good reputation, and also idol is eaten poisonous river Puffer by mistake and the poisoning generation.It is all toxic that Fugu belongs to fish; But its virulence size; Not only with river Puffer fingerling class, individuality and also in the river Puffer fish different tissues organ, toxicity all has very big difference, the ovary of river Puffer fish, liver (particularly breeding period is individual), hematotoxicity are the strongest; Intestines, spermary and skin take second place, the muscle virulence a little less than.Only contain several kinds of important edible Puffer classes that then become high economic worth such as the Fugu rubripes of micro-toxin (< 5mug mouse unit), false eyeball Fugu, fugu obscurus, the yellow Fugu of chrysanthemum because of muscle.Fugutoxin is the very strong neurotoxin of a kind of toxicity, and main the sign is that TTX can cross and the sodium-ion channel receptors bind, blocking voltage dependence sodium channel, thus block action potentials causes relevant with it physiological activity obstacle, mainly is neuromuscular paralysis.Detoxifying function causes nervous centralis in brain stem, kinesitherapy nerve, sensory nerve and automatic nervous system, muscle is neural, cardiovascular and Clinical symptoms such as gastrointestinal function obstacle.Respiratory center is benumbed and caused unexpected breath stopped is one of deadly immediate cause of TTX.Also there is not effective toxinicide at present.The chemical property of tetraodotoxin is stable, and general culinary art means are difficult to destroy.In order to ensure eater's safety, can enjoy river Puffer delicious food again simultaneously, set up the method that fast trace detects TTX in the Puffer of river, the incident that can avoid the edible poisonous river of consumer Puffer to cause poisoning takes place.
Detecting the tetraodotoxin method much respectively has its relative merits, roughly is divided into bioassay method, instrumental method, immuno-chemical method etc., wherein adopts instrument detecting TTX analytic approach maximum.But main at present the employing has fluorescence detector and the national standard " tetraodotoxin is measured liquid chromatography-fluorescence detection in the aquatic products " of liquid chromatograph with the liquid chromatography-quadrupole mass spectrometer of connecting of post-column derivation and the national standard " mensuration of tetraodotoxin in the bright globe fish " of ELISA detection.Because of the detection method of liquid-matter coupling, mass spectroscopy device costliness popularity rate at home is not high, detects cost very high (the 10mg amount just needs 45000 yuan for only required TTX standard items 10mg, 4500 yuan/1mg of import TTX), is difficult to popularization and application.The ELISA high specificity, detect sensitive high, but testing process is numerous and diverse, the cycle long (needing nearly 2 day working day), be difficult to grasp, the kit cost is high, also be difficult to generally apply.Because the detection method of existing employing mainly adopts river Puffer fish muscle and liver sample; Pre-treatment process complicacy, consumptive material consuming time, the TTX recovery are lower; And receive often in analyzing and testing that impurity disturbs in the sample, very easily cause the TTX false positive results, thereby still can not satisfy the demand of practical application.
China has the abundant river Puffer stock of fish, and in order to ensure eater's safety, country forbids the edible of river Puffer fish at present and sells, and only offers minority test-meal point in some areas, thereby has caused the significant wastage of this resource.The price of river Puffer is very high on the international market at present; Therefore more domestic aquatic products departments urgently hope the operation of open river Puffer fish; The simultaneously domestic scale of propagating the river Puffer artificially is also very big; Therefore press for revision, improve original management method, accomplish both to bring into play the effect of river Puffer resource in promoting economic development, ensure that again the consumer's is healthy.Therefore, be badly in need of setting up a kind of advanced person with science, reliable, easy, quick, the economic, practical fugutoxin detection method that is the basis.
Summary of the invention
The present invention has overcome the technical barrier that river Puffer fish blood sample is difficult to gather; Set up reversed phase ion that a kind of new fast trace detects fugutoxin (Tetrodoxin TTX) content in the Puffer blood sample of bright river to high performance liquid chromatography; Utilize this method testing result to judge whether bright river Puffer can safe edible.For the aspects such as safe edible of river Puffer fish provide scientific basis and new detection means.
The objective of the invention is to improve traditional only with river Puffer fish muscle and liver as the method that detects the TTX sample; And with river Puffer fish blood as detecting the TTX sample; Simplify the operation steps of sample pre-treatments consumptive material consuming time; Minimizing detects cost, does not need high checkout equipment, reaches to be applicable to that the efficient fast trace of inspection center of basic unit detects the TTX purpose.For the safe edible of river Puffer fish provides accurate qualitative, quantitative foundation.
Ultimate principle of the present invention: (1) TTX is 0.5mg to the minimum lethal dose of human body.Calculate in theory: i.e. the river Puffer fish of an edible 500g of people surpasses then edible not of 1 μ g/g as long as in the Puffer sample of river, detect TTX content.Utilize river Puffer fish blood TTX content to be higher than the characteristic of muscle; To be lower than 0.5 μ g/mL then safe to eat as in the Puffer blood sample of river, detecting TTX content, comparatively scientific and reasonable with its detection index as bright river Puffer fish safe edible.(2) fugutoxin and sodium heptanesulfonate can detect at the UV200nm place after forming ion pair; (3) fugutoxin is an alkalescent, when with can well separate at reverse-phase chromatographic column after sodium heptanesulfonate forms ion pair; (4) according to the retention time and the peak area that detect the TTX standard items, can be to the accurate qualitative and quantitative analysis of TTX in institute's test sample; (5) can judge according to the inspection result whether river Puffer fish can safe edible.
Fast trace according to the invention detects that the TTX reversed phase ion comprises following 5 steps to the HPLC method in the Puffer blood of bright river: 1. bright river Puffer fish blood sample is gathered and pre-treatment; 2. preparation moving phase; 3. preparation standard model; 4. chromatographiccondition is set; 5. sample detection analysis and result judge.
Said step 1; Bright river Puffer fish blood sample is gathered and pre-treatment: bright river Puffer fish blood sample is gathered and pre-treatment: draw about 50 μ L heparin sodium injections with asepsis injector; Draw blood sample 1mL in the 1.5mL centrifuge tube in the river Puffer arteria coeliaca of dissecting; Get the 0.5mL supernatant behind the centrifugal 10min and cross SupeLcLean LC-18 post, said SupeLcLean LC-18 post is used 3mL methyl alcohol, 3mL 0.2% acetate activation respectively, flow velocity 1mL/min before using; Use 0.75mL 0.2% acetate drip washing C18 pillar then, merge twice and collect liquid centrifugal 10min of 13000rpm/min in the 1.5mL centrifuge tube, it is to be analyzed in the sample bottle of marked to get the 1.0mL supernatant.
Said step 2, preparation moving phase: 50mmol/L ammonium dihydrogen phosphate (ADP), 10mmol/L sodium heptanesulfonate transfer to 4.0 backs with 0.22 μ m membrane filtration with phosphoric acid with pH respectively; Measure the acetonitrile 110mL of ammonium dihydrogen phosphate (ADP) and heptanesulfonic acid sodium solution 890mL and HPLC level respectively, ultrasonic degas behind the merging mixing.
Said step 3, preparation standard model: prepare 100 μ g/mL TTX standard reserving solutions.Get 1mg TTX freeze-drying standard items and add pure water dissolving constant volume to 10mL, 4 ℃ of storages.Get standard reserving solution, add pure water and be mixed with the gradient standard operation liquid that concentration is 0.2 μ g/mL, 0.5 μ g/mL, 1.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 50.0 μ g/mL, 100.0 μ g/mL respectively.
Said step 4, chromatographic condition: chromatographic column: Agilent Poroshell 120 EC-C18 4.6mm * 150mm 2.7 μ m; Moving phase: 11% acetonitrile-89% 50mmol/L ammonium dihydrogen phosphate (ADP)-10mmol/L sodium heptanesulfonate (pH 4.0) (V/V); Sample size 20 μ L; 25 ℃ of column temperatures; Detect wavelength 200nm; Flow velocity 0.6mL/min.
Said step 5, the sample introduction analyzing and testing, the TTX typical curve according to chromatographic work station obtains carries out qualitative and quantitative analysis to target sample, and < 0.5 μ g/>mL judges that river Puffer fish can safe edible to testing result TTX content.
Principal feature of the present invention is embodied in 7 aspects: 1. with bright river Puffer fish blood as the TTX test sample; Complex steps and required time, the consumptive material of sample pre-treatments have significantly been reduced; Not only easy but fast (as detecting by the bright river of routine Puffer fish muscle or liver sample method, 10 samples of pre-treatment only, because of workload big; Operation steps is complicated, just needs 8～10 hours consuming time; Need about 2.5 hour from sampling, sample pre-treatments to accomplishing only to analyze and detect 10 blood samples); 2. in the detection method good linearity, the scope of 0.2 μ g/mL～100 μ g/mL peak area with measure concentration and be good linear relationship, related coefficient
R 2 =0.99999; 3. significantly improve the detection method accuracy, the recovery of TTX reaches more than 95%,
RSD<2.09%; 4. method favorable reproducibility, the retention time of TTX detected peaks
RSD<0.007%, the content of TTX detected peaks
RSD<1.0828; Lowest detection be limited to 0.135 μ gmL (
S/n=3.1); Minimum quantitative limit be about 0.5 μ g/mL (
S/n=11.7) can satisfy the detection demand of river Puffer fish safe edible; 6. compare with existing TTX detection method and need not expensive mass spectrometer or fluorescence detector, post-column derivation device (fluorescence detection; With the condition of 100 ℃ of 4N NaOH TTX being hydrolyzed into C9 alkali detects; Though detection sensitivity is high; But instrument uses under high temperature, strong alkaline condition for a long time, is easy to corrode fluorescence detector), have simple to operate, quick, qualitative, quantitative accurately, be fit to basic unit and detect unit and use; 7. reduce testing staff's workload, saved testing cost.
The present invention provides new detection means for detecting river Puffer fish TTX, and for the bright river of safe edible Puffer fish provides scientific basis, making the consumer is " defy death and eat the river Puffer " worry for enjoying the river Puffer when delicious no longer.To play a significant role for the development of further revising, improving original management method, promote the river Puffer to lift a ban, promote river Puffer economic industry, river Puffer cooking culture simultaneously.
Description of drawings
Fig. 1: detect 7 kinds of concentration linear chromatographies of TTX standard model figure.
Fig. 2: the TTX detection method range of linearity and equation.
Fig. 3: TTX LDL s/n=3.3 concentration 0.135 μ g/mL.
Fig. 4: the minimum quantitative limit s/n=11.7 concentration 0.5 μ g/mL of TTX.
Fig. 5: the yellow Fugu blood of chrysanthemum control sample and the chromatogram that adds blood sample behind three kinds of concentration TTX standard specimens.
Embodiment
It is following that the present invention specifically detects the TTX instance:
1 instrument and material
1.1 instrumentAgilent HPLC 1100 Series (U.S.'s Agilent 1100 high performance liquid chromatographs): QuatPump G1311A (quaternary pump), DEGASSER G1379A (degasser), DAD G1315B (diode array detector), COLCOM G1316A (column temperature controller), ALS G1313A (automatic sampler), Agilent HPLC 1100 chem workstations.Desk centrifuge (U.S. Thermos), circulation ability of swimming are used vacuum pump, electronic balance, vortex oscillator, solid-phase extraction device more.
Consumptive materialSupeLcLean LC-18 SPE Tubes 3mL/500mg (U.S. SUPELCO), the 2mL disposable sterilized syringe.
Medicine and reagentPurity is 85% fugutoxin standard items 1mg (Hebei Provincial Aquatic Products Research Institute); Purity is 98.5% fugutoxin standard items 1mg (Shanghai be in harmony nurse instrument Science and Technology Ltd.); Acetonitrile HPLC level; Ammonium dihydrogen phosphate (ADP) is analyzed pure; Acetate is analyzed pure; Methyl alcohol HPLC level; Heparin sodium injection 2mL 12500 units.
Experiment materialPropagate fugu obscurus, the yellow Fugu of chrysanthemum artificially
2 experimental techniques
2.1 reagent preparation0.2% aqueous acetic acid; Moving phase: 50mmol/L ammonium dihydrogen phosphate (ADP)-10mmol/L sodium heptanesulfonate (pH being transferred to 4.0 backs with 0.22 μ m membrane filtration) with phosphoric acid; 100 μ g/mL TTX standard reserving solutions (mark article TTX 1mg is dissolved in the 10mL pure water, 4 ℃ of preservations); Gradient standard operation liquid (get the TTX storing solution and be mixed with 0.2 μ g/mL, 0.5 μ g/mL, 1.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 50.0 μ g/mL, 100.0 μ g/mL respectively).
Chromatographic conditionChromatographic column: Agilent Poroshell 120 EC-C18 4.6mm * 150mm 2.7 μ m; Moving phase: 11% acetonitrile-89% 50mmol/L ammonium dihydrogen phosphate (ADP)-10mmol/L sodium heptanesulfonate (pH 4.0); Sample size 20 μ L; 25 ℃ of column temperatures; Detect wavelength 200nm; Flow velocity 0.6mL/min.
Sample pre-treatmentsDraw about 50 μ L heparin sodium injections with asepsis injector; Draw blood sample 1mL in the 1.5mL centrifuge tube in the yellow Fugu arteria coeliaca of the chrysanthemum of dissecting; Getting the 0.5mL supernatant behind the centrifugal 10min crosses SupeLcLean LC-18 post and (uses 3mL methyl alcohol, 3mL 0.2% acetate activation before the use respectively; Flow velocity 1mL/min), the back is with 0.75mL 0.2% acetate drip washing C
18Pillar merges twice and collects liquid centrifugal 10min of 13000rpm/min in the 1.5mL centrifuge tube, and it is to be analyzed in the sample bottle of marked to get the 1.0mL supernatant.
Result and analysis
3.1 chromatographic condition is confirmed
3.1.1 confirming of stationary phaseWith 85% purity TTX standard items sample introduction, detect under all constant condition of wavelength and flow velocity in moving phase, column temperature, UV, through 85% purity TTX mark article and separate impurities degree and retention time are contrasted, to Agilent ZORBAX SB-C
184.6mm * 250mm 5 μ m, Beckman ULTRASPHRE-ODS C
184.6mm * 250mm 5 μ m, Waters Symmetry C
184.6mm * 250mm 5 μ m, Agilent Poroshell 120 EC-C
184.6mm four kinds of stationary phase of * 150mm 2.7 μ m are selected.The result shows use Agilent Poroshell 120 EC-C
184.6mm * 150mm 2.7 μ m as stationary phase, can effectively separate 85% purity TTX standard items with adjacent impurity peaks.And the symmetry of chromatographic peak is better.
Confirming of moving phaseWith Agilent Poroshell 120 EC-C
184.6mm * 150mm 2.7 μ m are as solid phase; Detect under all constant condition of wavelength at column temperature, flow velocity, UV, through to acetonitrile-ammonium acetate, acetonitrile-0.1% formic acid, 50mmol/L ammonium dihydrogen phosphate (ADP)-10mmol/L sodium heptanesulfonate, 50mmol/L sodium hydrogen phosphate-5mmol/L sodium dihydrogen phosphate-sodium heptanesulfonate 5mmol/L
[, 0.05mol/L sodium hydrogen phosphate-0.05mol/L sodium dihydrogen phosphate-sodium heptanesulfonate 2.5mmol/L, acetonitrile-ammonium dihydrogen phosphate (ADP)-six kinds of moving phases of sodium heptanesulfonate and pH be to the influence of the degree of separation and the retention time of sample; Confirm that finally with 11% acetonitrile-89% 50mmol/L ammonium dihydrogen phosphate (ADP)-10mmol/L sodium heptanesulfonate (phosphoric acid is transferred pH 4.0) be chromatogram flow phase; Can 85% purity TTX standard items effectively be separated with adjacent impurity peaks, and Han You > in the moving phase; 10% organic phase has the better protect effect to reverse-phase chromatographic column.
Confirming of column temperatureUnder the condition that moving phase, flow velocity and detection wavelength are fixed; Column temperature separates the influence of TTX and impurity effect to chromatographic column when observing 20 ℃, 25 ℃, 30 ℃, and when column temperature rising TTX and the reduction of impurity separating effect, retention time shortens; Column temperature reduces degree of separation and increases, but retention time prolongs.When column temperature was 25 ℃, chromatographic column was separated with impurity better TTX, and post is pressed to about 160bar, and sample analysis time can be accomplished in 10min.
3.1.4 detection wavelength determinationUnder the condition of confirming under stationary phase, moving phase, flow velocity, the column temperature; Relatively 196nm, 198nm, 200nm, 204nm, 205nm detect under the wavelength separating the response influence of TTX respectively; The result shows with 200nm to be that signal to noise ratio (S/N ratio) is better when detecting wavelength, and chromatographic integration is accurate.
Confirming of flow velocityUnder the condition that stationary phase, moving phase, column temperature, detection wavelength are confirmed; Compare 0.5mL/min, 0.6 mL/min, 0.7 mL/min, 0.8 mL/min, 0.9 mL/min flow conditions length respectively down to separating the influence of TTX and impurity degree of separation; The result shows with TTX under the 0.6 mL/min flow conditions and can separate preferably with impurity; Chromatographic integration is accurate, and chromatogram is better when detecting river Puffer sample.
Method is estimated
3.2.1 the method range of linearity and detectabilityGet purity 98.5%TTX standard items are mixed with 0.2 μ g/mL, 0.5 μ g/mL, 1.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 50.0 μ g/mL, 7 kinds of concentration of 100.0 μ g/mL with pure water standard solution; With TTX sample introduction concentration is horizontal ordinate; With the peak area is ordinate, and chem workstation provides area-concentration standard curve.The result be illustrated in the scope of 0.2 μ g/mL～100 μ g/mL peak area with measure concentration be good linear relationship (
Fig. 1), related coefficient
R 2 =0.99999 (
Fig. 2).Use this method to detect the TTX standard items, when concentration was 0.135 μ g/mL, it was 3.3 that chem workstation calculates signal to noise ratio (S/N ratio), and when concentration was 0.5 μ g/mL, it was 11.7 that chem workstation calculates signal to noise ratio (S/N ratio), therefore, the LDL of this law be about 0.135 μ g/mL (
Fig. 3), minimum quantitative limit be about 0.5 μ g/mL (
Fig. 4).
The method reappearancePreparation TTX sample detection liquid: basic, normal, high three kinds of concentration; I.e. 0.2 μ g/mL, 5.0 μ g/mL, three kinds of concentration of 100.0 μ g/mL, each concentration do 3 parallel, use this method; Through continuous five days detection; Statistics detects TTX content and retention time, and basis of calculation deviation, and the result sees table 1.
The result shows that the retention time relative standard deviation of TTX detected peaks is between 0.005～0.007; The content relative standard deviation of TTX detected peaks is between 0.0177～1.0828; Show that the method reappearance that detects TTX content in the Puffer blood of river is better, the retention time of TTX detected peaks and content relative standard deviation equal < 2%.
The method accuracyDraw about 50 μ L heparin sodium injections with asepsis injector, artery is drawn blood sample 1mL in the 1.5mL centrifuge tube from the yellow Fugu liver of chrysanthemum top, adds 1mg/mL TTX titer 10 μ L, 20 μ L, 40 μ L respectively, each concentration do three parallel.Get the 0.5mL supernatant behind the centrifugal 10min and cross SupeLcLean LC-18 post (using 3mL methyl alcohol, 3mL 0.2% acetate activation before the use respectively), the back is with 0.75mL 0.2% acetate drip washing C
18Pillar merges twice and collects liquid centrifugal 10min of 13000rpm/min in the 1.5mL centrifuge tube, gets 1.0mL supernatant sample introduction analysis in the sample bottle of marked (sample concentration after pre-treatment is respectively 4 μ g/mL, 8 μ g/mL, 16 μ g/mL).Each concentration do 3 parallel, do a blank in addition.After the sample detection according to typical curve get TTX content results behind the mark-on (
Fig. 5), see table 2 for details.
Calculate three kinds of concentration average recovery rates according to testing result and be respectively 95.47%, 97.34%, 96.26%.Show that getting river Puffer blood sample detects that the TTX pre-treatment is easy and simple to handle, quick, consumptive material is few, do not need complicated instrument and equipment, and the recovery is high, the method accuracy is high.
Claims (4)
1. a fast trace detects in the Puffer blood of bright river the TTX reversed phase ion to the HPLC method, comprises the following steps: that successively (1) bright river Puffer fish blood sample gathers and pre-treatment; (2) preparation moving phase; (3) preparation standard model; (4) chromatographiccondition is set; (5) sample detection analysis and result judge.
2. detect TTX HPLC method in the Puffer blood of bright river according to the fast trace described in the claim 1; (1) bright river Puffer fish blood sample collection and pre-treatment: draw about 50 μ L heparin sodium injections with asepsis injector; Draw blood sample 1mL in the 1.5mL centrifuge tube in the river Puffer arteria coeliaca of dissecting; Get the 0.5mL supernatant behind the centrifugal 10min and cross SupeLcLean LC-18 post, said SupeLcLean LC-18 post is used 3mL methyl alcohol, 3mL 0.2% acetate activation respectively, flow velocity 1mL/min before using; Use 0.75mL 0.2% acetate drip washing C18 pillar then, merge twice and collect liquid centrifugal 10min of 13000rpm/min in the 1.5mL centrifuge tube, it is to be analyzed in the sample bottle of marked to get the 1.0mL supernatant.
3. detect TTX HPLC method in the Puffer blood of bright river according to the fast trace described in the claim 1, it is characterized in that said step (2) preparation moving phase: 50mmol/L ammonium dihydrogen phosphate (ADP), 10mmol/L sodium heptanesulfonate transfer to 4.0 backs with 0.22 μ m membrane filtration with phosphoric acid with pH respectively; Measure the acetonitrile 110mL of ammonium dihydrogen phosphate (ADP) and heptanesulfonic acid sodium solution 890mL and HPLC level respectively, ultrasonic degas behind the merging mixing.
4. detect TTX HPLC method in the Puffer blood of bright river according to the fast trace described in the claim 1, it is characterized in that step (4) chromatographic condition: chromatographic column: Agilent Poroshell 120 EC-C18 4.6mm * 150mm2.7 μ m; Moving phase: 11% acetonitrile-89% 50mmol/L ammonium dihydrogen phosphate (ADP)-10mmol/L sodium heptanesulfonate (V/V), pH 4.0; Sample size 20 μ L; 25 ℃ of column temperatures; Detect wavelength 200nm; Flow velocity 0.6mL/min.
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