CN109884236A - The efficient liquid phase detection method of curcumin - Google Patents
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Abstract
The present invention discloses a kind of efficient liquid phase detection method of curcumin, using reverse phase C18 chromatographic column, PDA detector, mobile phase A is the mixed solution of formic acid-ammonium acetate aqueous solution and acetonitrile, and Mobile phase B is the mixed solution of formic acid-ammonium acetate acetonitrile solution and water, using gradient elution.This method 3~5ul of sample introduction can effectively detect curcumin and its related substance, and separating degree R can be up to 1.5 or more, and good separating effect, HPLC spectrum baseline is steady, does not drift about；This method detection time is short, only needs 3min that efficient liquid phase detection process can be completed, substantially increases detection efficiency, is suitble to high flux screening；Solvent can be saved, cost is reduced, safe operation is simple, handles convenient and efficient；For the assay of curcumin, there is good linear relationship, reproducibility is high, and accuracy and accuracy are high, has important research value in terms of bulk pharmaceutical chemicals, quality of the pharmaceutical preparations research and related pharmacokinetics.
Description
Technical field
The present invention relates to Pharmaceutical Analysis technical fields, and in particular to a kind of efficient liquid phase detection method of curcumin.
Background technique
Curcumin (Itraconazole) is to separate a kind of low average molecular for the first time from turmeric in 1870 earliest
Quality polyphenol compound illustrates the chemical structure of its curcumin in 1910, then its related physiology, pharmacological action
Research achieve apparent progress.It is increasingly deep with studying curcumin, it has been found that it is with anti-inflammatory, anti-oxidant, tune
The extensive pharmacological activity such as rouge, antiviral, anti-infective, antitumor, anticoagulant, anti-hepatic fibrosis, antiatherosclerosis, and toxicity
It is low, adverse reaction is small.Attract researcher's to be not only curcumin as a kind of non-steroidal anti-inflammatory drug, and because it is had
Some chemoprophylaxis characteristics, curcumin have extensive prevention characteristic to disease.In view of modern medical research has found that human body is numerous
The generation of disease is related with the participation of the formation of free radical, inflammatory reaction, and curcumin antioxidant activity has caused with anti-inflammatory effect
The extensive concern of domestic and foreign scholars, develop the efficient liquid phase detection method of curcumin have for the medicinal study of curcumin it is important
Meaning.
About the assay of curcumin, the prior art is had been reported: thundercloud rosy clouds, Sun Lili, poplar is refined, Wang Jiao, China
Pharmacist .2007.10, detection method are: using Agilent 1100Series high performance liquid chromatograph, Lichrospher
ODS C18 (150mm*4.6mm, 5 μm) chromatographic column, mobile phase are acetonitrile-water (5% glacial acetic acid) (45:55), flow velocity: 1ml/
Min, sample volume 20ul are detected at 420nm wavelength；This method is mainly the assay to curcumin in Yujin slices.Hair
Bright people in this way tests turmeric cellulose content, and discovery preprocessing process is complicated, and sampling volume is larger, compound loss
Greatly, and detection needs 15min or more, and time-consuming.
The present invention is improved and has been optimized for detection method in the prior art, with method phase in the prior art
Good separating effect not only than, detection method of the invention, preci-sion and accuracy is high, and safe operation is simple, and processing is convenient
Fast, it is suitble to high flux screening and precisely quantifies.This method is to the raw material of curcumin, quality of the pharmaceutical preparations research and related pharmacokinetics
Quantitative study etc. all has important research value.
Summary of the invention
A kind of the technical problem to be solved by the invention is to provide detection times short, good separating effect, accuracy and accurate
Spend the efficient liquid phase detection method of higher curcumin.
In order to solve the above technical problems, the efficient liquid phase detection method of curcumin provided by the invention, wherein
Chromatographic column uses reverse phase C18 chromatographic column；
Detector uses PDA detector；
Mobile phase includes mobile phase A and Mobile phase B；Mobile phase A is that the mixing of formic acid-ammonium acetate aqueous solution and acetonitrile is molten
Liquid, formic acid volumetric concentration is 0.01~0.03% in aqueous solution, and acetic acid ammonium concentration is 0.5~1.5mM in aqueous solution, aqueous solution with
The volume ratio of acetonitrile is 100~95:0~5；Mobile phase B is the mixed solution of formic acid-ammonium acetate acetonitrile solution and water, acetonitrile
Formic acid volumetric concentration is 0.01~0.03% in solution, and acetic acid ammonium concentration is 0.5~1.5mM in acetonitrile solution, acetonitrile solution with
The volume ratio of water is 100~95:0~5；
Using gradient elution, gradient elution program is carried out by following procedure: mobile phase A+Mobile phase B=100%, 0~
0.01min, it is 50% that Mobile phase B, which keeps percent by volume,；0.01~2.10min, Mobile phase B percent by volume are incremented by by 50%
To 60%；2.10~2.11min, Mobile phase B percentage by volume are decremented to 50% by 60%；2.11~3.00min, Mobile phase B
Keeping percentage by volume is 50%.
In a preferred embodiment, mobile phase A is that the volume fraction of formic acid-ammonium acetate aqueous solution and acetonitrile is
The mixed solution of 95:5, Mobile phase B are that the volume fraction of formic acid-ammonium acetate acetonitrile solution and water is the mixed solution of 95:5.
In a preferred embodiment, in mobile phase A, formic acid volumetric concentration is 0.025% in aqueous solution；Mobile phase B
In, formic acid volumetric concentration is 0.025% in acetonitrile solution.
In a preferred embodiment, in mobile phase A, acetic acid ammonium concentration is 1mM in aqueous solution；In Mobile phase B, acetonitrile
Acetic acid ammonium concentration is 1mM in solution.
In a preferred embodiment, column's length 100mm.
In a preferred embodiment, flow velocity is 1.0~2.0ml/min, more preferably 1.4ml/min.
In a preferred embodiment, Detection wavelength 250-270nm, more preferably 254nm.
In a preferred embodiment, column temperature control is at 35~40 DEG C, and more preferably 40 DEG C.
In a preferred embodiment, sample volume is 2~8 μ l, more preferably 3~5 μ l.
The present invention also provides a kind of efficient liquid phase methods for measuring turmeric cellulose content, comprising the following steps:
(1) preparation of reference substance solution and test solution: precision weighs appropriate curcumin reference substance, molten with methyl sulfoxide
It solves and constant volume is at multiple reference substance solutions with a certain concentration gradient；Precision weighs appropriate curcumin test sample, with methyl Asia
Simultaneously constant volume obtains test solution for sulfone dissolution；
2) chromatographic condition:
Chromatographic column uses reverse phase C18 chromatographic column；
Detector uses PDA detector；
Mobile phase includes mobile phase A and Mobile phase B；Mobile phase A is that the mixing of formic acid-ammonium acetate aqueous solution and acetonitrile is molten
Liquid, formic acid volumetric concentration is 0.025% in aqueous solution, and acetic acid ammonium concentration is 1mM, the volume ratio of aqueous solution and acetonitrile in aqueous solution
For 95:5；Mobile phase B is the mixed solution of formic acid-ammonium acetate acetonitrile solution and water, and formic acid volumetric concentration is in acetonitrile solution
0.025%, acetic acid ammonium concentration is 1mM in acetonitrile solution, and the volume ratio of acetonitrile solution and water is 95:5；
Using gradient elution, gradient elution program is carried out by following procedure: 0~0.01min, Mobile phase B keep volume hundred
Divide than being 50%；0.01~2.10min, Mobile phase B percent by volume are incremented to 60% by 50%；2.10~2.11min, flowing
Phase B percentage by volume is decremented to 50% by 60%；2.11~3.00min, it is 50% that Mobile phase B, which keeps percentage by volume,.
(3) measuring method:
The multiple reference substance solution and test solution are pressed into described (2) chromatographic condition successively sample introduction, record chromatography
Figure, prepares linear related work curve according to the spectrum data of multiple reference substance solutions and concentration data, substitutes into test sample
Spectrum data calculates and obtains test solution concentration, completes the measurement of turmeric cellulose content.
In a preferred embodiment, the concentration of reference substance solution is followed successively by 6.25,12.5,25,50,100,200 μ g/
Ml, flow velocity 1.4ml/min.Detection wavelength is 254nm, and column temperature is 40 DEG C, and sample volume is 3~5 μ l.
The efficient liquid phase detection method of curcumin provided by the invention it is a technical advantage that:
1. method of the invention can effectively detect curcumin and its related substance, and separating degree R can up to 1.5 or more,
Good separating effect.
2. method detection time of the invention is short, only needs 3min that efficient liquid phase detection process can be completed, substantially increase
Detection efficiency is suitble to high flux screening.
3. method measurement HPLC spectrum baseline of the invention is steady, do not drift about.
4. method of the invention can save solvent, cost is reduced, safe operation is simple, handles convenient and efficient.
5. method of the invention is used for the assay of curcumin, there is good linear relationship, reproducibility is high, realizes
The purpose of quick separating detection has important in terms of bulk pharmaceutical chemicals, quality of the pharmaceutical preparations research and related pharmacokinetics
Researching value.
Detailed description of the invention
Fig. 1 is the HPLC map one using method measurement curcumin test sample of the invention.
Fig. 2 is the HPLC map two using method measurement curcumin test sample of the invention.
Fig. 3 is the HPLC map three using method measurement curcumin test sample of the invention.
The curcumin working curve that Fig. 4 is drawn using method of the invention.
Specific embodiment
Present inventor gropes to have obtained a kind of efficient liquid of curcumin by extensive research and a large amount of experiment
Phase detection method, this method provides curcumin can be efficiently separated and its flow visualizing and gradient elution journey in relation to substance
Sequence, will can be difficult in the prior art the curcumin efficiently separated and its related substance realizes good separating effect, separating degree R
Up to 1.5 or more.
The efficient liquid phase detection method for the curcumin that inventor uses, chromatographic condition are as follows:
Chromatographic column uses reverse phase C18 chromatographic column, such as using octadecylsilane chemically bonded silica as the Waters of filler
XBridge chromatographic column；
Detector uses PDA detector；
Mobile phase includes mobile phase A and Mobile phase B；Mobile phase A is formic acid-ammonium acetate aqueous solution/acetonitrile (100~95/
0~5；V/v mixed solution), formic acid volumetric concentration is 0.01~0.03% in aqueous solution, and acetic acid ammonium concentration is in aqueous solution
0.5~1.5mM；Mobile phase B is formic acid-ammonium acetate acetonitrile solution/water (100~95/0~5；V/v mixed solution), acetonitrile
Formic acid volumetric concentration is 0.01~0.03% in solution, and acetic acid ammonium concentration is 0.5~1.5mM in acetonitrile solution；
Using gradient elution, mobile phase A+Mobile phase B=100%, gradient elution program is carried out by following procedure: 0~
0.01min, it is 50% that Mobile phase B, which keeps percent by volume,；0.01~2.10min, Mobile phase B percent by volume are incremented by by 50%
To 60%；2.10~2.11min, Mobile phase B percentage by volume are decremented to 50% by 60%；2.11~3.00min, Mobile phase B
Keeping percentage by volume is 50%.
In a preferred embodiment of the invention, mobile phase A is the volume point of formic acid-ammonium acetate aqueous solution and acetonitrile
Number is the mixed solutions of 95:5, and Mobile phase B is that the mixing that the volume fraction of formic acid-ammonium acetate acetonitrile solution and water is 95:5 is molten
Liquid.
In a preferred embodiment of the invention, in mobile phase A, formic acid volumetric concentration is 0.025% in aqueous solution；Stream
In dynamic phase B, formic acid volumetric concentration is 0.025% in acetonitrile solution.
In a preferred embodiment of the invention, in mobile phase A, acetic acid ammonium concentration is 1mM in aqueous solution；Mobile phase B
In, acetic acid ammonium concentration is 1mM in acetonitrile solution.
In a preferred embodiment of the invention, column's length 100mm.
In a preferred embodiment of the invention, flow velocity is 1.0~2.0ml/min, more preferably 1.4ml/min.
In a preferred embodiment of the invention, Detection wavelength 250-270nm, more preferable 254nm.
In a preferred embodiment of the invention, column temperature is controlled at 35~40 DEG C, and more preferably 40 DEG C.
In a preferred embodiment of the invention, sample volume is 2~8 μ l, more preferably 3~5 μ l.
Clear, complete description is carried out to technical solution of the present invention below in conjunction with specific embodiment, it is clear that described
Embodiment be a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field
Those of ordinary skill's every other embodiment obtained without making creative work, belongs to guarantor of the present invention
The range of shield.
Experiment condition in the embodiment of the present invention is as follows:
Instrument: high performance liquid chromatograph (LC-20AB)；
Chromatographic column: Waters XBridge C18 chromatographic column, column length 100mm；
Test solution: precision weighs curcumin test sample 10mg, is placed in 10ml volumetric flask, simultaneously with dmso solution
Constant volume obtains test solution；
Flow velocity: 1.4ml/min；
Detection wavelength: 254nm；
Column temperature: 40 DEG C；
Mobile phase: mobile phase A+Mobile phase B=100%；
Gradient elution: 0~0.01min, it is 50% that Mobile phase B, which keeps percent by volume,；0.01~2.10min, Mobile phase B
Percent by volume is incremented to 60% by 50%；2.10~2.11min, Mobile phase B percentage by volume are decremented to 50% by 60%；
2.11~3.00min, it is 50% that Mobile phase B, which keeps percentage by volume,.
Embodiment 1
Mobile phase A is formic acid-ammonium acetate aqueous solution/acetonitrile (95/5；V/v mixed solution), formic acid body in aqueous solution
Product concentration is 0.025%, and acetic acid ammonium concentration is 1mM in aqueous solution；Mobile phase B is formic acid-ammonium acetate acetonitrile solution/water
(95/5；V/v mixed solution), formic acid volumetric concentration is 0.025% in acetonitrile solution, and acetic acid ammonium concentration is in acetonitrile solution
1mM。
Pass through three needle sample introductions under the method, HPLC map such as Fig. 1-3 shows that baseline is steady, and reproducible, 3min can be whole
Appearance, curcumin retention time are 2.183~2.185min, theoretical cam curve 9738；Process impurity retention time is
0.741-0.744min and 1.208-1.209min.
Embodiment 2
Mobile phase A is formic acid-ammonium acetate aqueous solution/acetonitrile (95/5；V/v mixed solution), formic acid body in aqueous solution
Product concentration is 0.01%, and acetic acid ammonium concentration is 0.5mM in aqueous solution；Mobile phase B is formic acid-ammonium acetate acetonitrile solution/water
(95/5；V/v mixed solution), formic acid volumetric concentration is 0.01% in acetonitrile solution, and acetic acid ammonium concentration is in acetonitrile solution
0.5mM。
Baseline is steady under the method, and curcumin retention time is 2.01min or so, and separating degree is 11.466.
Embodiment 3
Mobile phase A is formic acid-ammonium acetate aqueous solution/acetonitrile (95/5；V/v mixed solution), formic acid body in aqueous solution
Product concentration is 0.03%, and acetic acid ammonium concentration is 1.5mM in aqueous solution；Mobile phase B is formic acid-ammonium acetate acetonitrile solution/water
(95/5；V/v mixed solution), formic acid volumetric concentration is 0.03% in acetonitrile solution, and acetic acid ammonium concentration is in acetonitrile solution
1.5mM。
Baseline is steady under the method, and curcumin retention time is 2.21min or so, and separating degree is 11.470.
Embodiment 4
Mobile phase A is formic acid-ammonium acetate aqueous solution, and formic acid volumetric concentration is 0.025% in aqueous solution, vinegar in aqueous solution
Sour ammonium concentration is 1mM；Mobile phase B is formic acid-ammonium acetate acetonitrile solution, and formic acid volumetric concentration is 0.025% in acetonitrile solution,
Acetic acid ammonium concentration is 1mM in acetonitrile solution.
Baseline is steady under the method, and curcumin retention time is 2.11min or so, and separating degree is 11.413.
Embodiment 5
Mobile phase A is formic acid-ammonium acetate aqueous solution, and formic acid volumetric concentration is 0.025% in aqueous solution, vinegar in aqueous solution
Sour ammonium concentration is 1mM；Mobile phase B is formic acid-ammonium acetate acetonitrile solution, and formic acid volumetric concentration is 0.025% in acetonitrile solution,
Acetic acid ammonium concentration is 1mM in acetonitrile solution.
Mobile phase: mobile phase A+Mobile phase B=100%；
Gradient elution: 0~0.01min, it is 40% that Mobile phase B, which keeps percent by volume,；0.01~2.10min, Mobile phase B
Percent by volume is incremented to 50% by 40%；2.10~2.11min, Mobile phase B percentage by volume are decremented to 40% by 50%；
2.11~3.00min, it is 40% that Mobile phase B, which keeps percentage by volume,；
Occur under the method baseline it is unstable, go out peak position it is to the rear, be unable to reach the effect of quick appearance in 3min.
The assay of 6 curcumin of embodiment
Based on above-mentioned detection method, inventor also provide it is a kind of measure turmeric cellulose content efficient liquid phase method, by with
Lower step carries out:
(1) preparation of reference substance solution and test solution: precision weighs appropriate curcumin reference substance, uses dimethyl sulfoxide
It dissolves and constant volume obtains the stock solution of 1mg/ml, it is accurate to draw 50 μ l stock solutions, constant volume is successively diluted with water for injection to be obtained
6.25, the reference substance solution of 12.5,25,50,100,200ug/ml；Precision weighs appropriate curcumin test sample, with dimethyl Asia
Simultaneously constant volume obtains test solution for sulfone dissolution；
(2) chromatographic condition:
Chromatographic column uses reverse phase C18 chromatographic column；
Detector uses PDA detector；
Mobile phase includes mobile phase A and Mobile phase B；Mobile phase A is formic acid-ammonium acetate aqueous solution/acetonitrile (95/5；v/
V) mixed solution, formic acid volumetric concentration is 0.025% in aqueous solution, and acetic acid ammonium concentration is 1mM in aqueous solution；Mobile phase B is
Formic acid-ammonium acetate acetonitrile solution/water (95/5；V/v mixed solution), formic acid volumetric concentration is in acetonitrile solution
0.025%, acetic acid ammonium concentration is 1mM in acetonitrile solution；
Using gradient elution, gradient elution program is carried out by following procedure: 0~0.01min, Mobile phase B keep volume hundred
Divide than being 50%；0.01~2.10min, Mobile phase B percent by volume are incremented to 60% by 50%；2.10~2.11min, flowing
Phase B percentage by volume is decremented to 50% by 60%；2.11~3.00min, it is 50% that Mobile phase B, which keeps percentage by volume,.
(3) measuring method:
6 reference substance solutions and test solution are pressed into the chromatographic condition successively sample introduction, record chromatogram, according to
The curcumin peak area and concentration data of 6 reference substance solutions prepare linear related work curve (as shown in Figure 4), and the work is bent
Line linear relationship is good, R=0.9999994, R2=0.9999988；It substitutes into the curcumin calculated by peak area of test sample and obtains
It is dilute when the test solution concentration and sample introduction of test solution concentration out, the test solution concentration of comparative measurements and constant volume
Multiple is released, the content of curcumin is calculated.
The efficient liquid phase detection method of curcumin of the invention can rapidly and efficiently detect curcumin and its related substance,
And separating degree R can be up to 1.5 or more, and detection method in the prior art is difficult to realize curcumin and its in relation to the effective of substance
Separation；The HPLC of detection method of the invention composes baseline stability, does not drift about；Method detection time of the invention is short, only needs
Efficient liquid phase detection process can be completed in 3min, substantially increases detection efficiency, is suitble to high flux screening；Method energy of the invention
Solvent is saved, cost is reduced, safe operation is simple, handles convenient and efficient；Method of the invention is used for the assay of curcumin,
With good linear relationship, R=0.9999994, R2=0.9999988, reproducibility is high, and accuracy and accuracy are high, in original
Expect that medicine, quality of the pharmaceutical preparations research and related pharmacokinetics quantitative study etc. have important research value.
In conclusion the various embodiments described above and attached drawing are only presently preferred embodiments of the present invention, not to limit this
The protection scope of invention, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done all are answered
It is included within the scope of the present invention.
Claims (12)
1. a kind of efficient liquid phase detection method of curcumin, which is characterized in that as follows using chromatographic condition:
Chromatographic column uses reverse phase C18 chromatographic column；
Detector uses PDA detector；
Mobile phase includes mobile phase A and Mobile phase B；Mobile phase A is the mixed solution of formic acid-ammonium acetate aqueous solution and acetonitrile,
Formic acid volumetric concentration is 0.01~0.03% in aqueous solution, and acetic acid ammonium concentration is 0.5~1.5mM, aqueous solution and second in aqueous solution
The volume ratio of nitrile is 100~95:0~5；Mobile phase B is the mixed solution of formic acid-ammonium acetate acetonitrile solution and water, and acetonitrile is molten
Formic acid volumetric concentration is 0.01~0.03% in liquid, and acetic acid ammonium concentration is 0.5~1.5mM, acetonitrile solution and water in acetonitrile solution
Volume ratio be 100~95:0~5；
Using gradient elution, gradient elution program is carried out by following procedure: mobile phase A+Mobile phase B=100%, 0~
0.01min, it is 50% that Mobile phase B, which keeps percent by volume,；0.01~2.10min, Mobile phase B percent by volume are incremented by by 50%
To 60%；2.10~2.11min, Mobile phase B percentage by volume are decremented to 50% by 60%；2.11~3.00min, Mobile phase B
Keeping percentage by volume is 50%.
2. the method as described in claim 1, which is characterized in that the mobile phase A is formic acid-ammonium acetate aqueous solution and acetonitrile
Volume fraction be 95:5 mixed solution, Mobile phase B is that the volume fraction of formic acid-ammonium acetate acetonitrile solution and water is 95:5
Mixed solution.
3. method according to claim 2, which is characterized in that in the mobile phase A, formic acid volumetric concentration is in aqueous solution
0.025%；In the Mobile phase B, formic acid volumetric concentration is 0.025% in acetonitrile solution.
4. method according to claim 2, which is characterized in that in the mobile phase A, acetic acid ammonium concentration is 1mM in aqueous solution；
In the Mobile phase B, acetic acid ammonium concentration is 1mM in acetonitrile solution.
5. the method as described in claim 1, which is characterized in that the column's length is 100mm.
6. the method as described in claim 1, which is characterized in that flow velocity is 1.0~2.0ml/min.
7. the method as described in claim 1, which is characterized in that Detection wavelength 250-270nm.
8. the method as described in claim 1, which is characterized in that column temperature is controlled at 35~40 DEG C.
9. the method as described in claim 1, which is characterized in that sample volume is 2~8 μ l.
10. a kind of efficient liquid phase method for measuring turmeric cellulose content, which comprises the following steps:
(1) preparation of reference substance solution and test solution: precision weighs appropriate curcumin reference substance, uses dmso solution
And constant volume is at multiple reference substance solutions with a certain concentration gradient；Precision weighs appropriate curcumin test sample, uses methyl sulfoxide
It dissolves and constant volume obtains test solution；
(2) chromatographic condition:
Chromatographic column uses reverse phase C18 chromatographic column；
Detector uses PDA detector；
Mobile phase includes mobile phase A and Mobile phase B；Mobile phase A is the mixed solution of formic acid-ammonium acetate aqueous solution and acetonitrile,
Formic acid volumetric concentration is 0.025% in aqueous solution, and acetic acid ammonium concentration is 1mM in aqueous solution, and the volume ratio of aqueous solution and acetonitrile is
95:5；Mobile phase B is the mixed solution of formic acid-ammonium acetate acetonitrile solution and water, and formic acid volumetric concentration is in acetonitrile solution
0.025%, acetic acid ammonium concentration is 1mM in acetonitrile solution, and the volume ratio of acetonitrile solution and water is 95:5；
Using gradient elution, gradient elution program is carried out by following procedure: 0~0.01min, Mobile phase B keep percent by volume
It is 50%；0.01~2.10min, Mobile phase B percent by volume are incremented to 60% by 50%；2.10~2.11min, Mobile phase B
Percentage by volume is decremented to 50% by 60%；2.11~3.00min, it is 50% that Mobile phase B, which keeps percentage by volume,；
(3) measuring method:
The multiple reference substance solution and test solution are pressed into described (2) chromatographic condition successively sample introduction, record chromatogram, root
Linear related work curve is prepared according to the spectrum data and concentration data of multiple reference substance solutions, substitutes into the map number of test sample
According to calculating and obtaining test solution concentration, the measurement of turmeric cellulose content is completed.
11. method as claimed in claim 10, which is characterized in that the concentration of the reference substance solution is followed successively by 6.25,12.5,
25、50、100、200μg/ml。
12. method as claimed in claim 10, which is characterized in that flow velocity is 1.4ml/min in chromatographic condition, and Detection wavelength is
254nm, column temperature are 40 DEG C, and sample volume is 3~5 μ l.
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