CN103091424B - Methods for detecting impurities in tigecycline - Google Patents
Methods for detecting impurities in tigecycline Download PDFInfo
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Abstract
The invention discloses a method for detecting N-tertbutyl glycyl acyl hydrochloride in tigecycline. The invention further discloses a method for detecting N-tertbutyl glycine hydrochloride in tigecycline. The methods are used for detecting the N-tertbutyl glycyl acyl hydrochloride and the N-tertbutyl glycine hydrochloride in tigecycline, are accurate and reliable in detection result, strong in specificity and short in detection time; and moreover a good separation degree of the N-tertbutyl glycyl acyl hydrochloride and the N-tertbutyl glycine hydrochloride is achieved, so that the possibility in comprehensively detecting the impurities in a tigecycline product, controlling the product quality and ensuring the medicine security is provided.
Description
Technical field
The present invention relates to the detection method of impurity in tigecycline, be specifically related to the detection method of N-tert-butyl group glycyl chloride hydrochloride or N-tert-butyl group glycine hydrochloride.
Background technology
Tigecycline (Tigecycline), chemical name: (4S, 4aS, 5aR; 12aS)-4, two (the dimethylamino)-9-[(tert-butyl groups of 7-are amino) acetamido]-3,10,12; 12a-tetrahydroxy-1,11-dioxo-Isosorbide-5-Nitrae; 4a, 5,5a; 6,11,12a-octahydro aphthacene-2-formamide; claiming again Tigecycline or tigecycline, is the first glycylcycline class microbiotic of getting permission listing, and molecular formula is: C
29h
39n
5o
8; Molecular structural formula is as follows:
Tigecycline be a kind of new, can be at treatment initial stage selective broad-spectrum antibiotic when the cause of disease is not yet clear and definite, and do not need according to impaired renal function situation adjustment dosage.Research shows, tigecycline can overcome two kinds of main resistance mechanisms that a lot of microbiotic of restriction are used: efflux pump and ribosomes protection.Therefore, tigecycline is difficult for producing drug resistance, and the scope of application is wider.And tigecycline long half time, approximately 27 hours single medication half life period, repeatedly approximately 42 hours medication half life period, medication in every 12 hours once, is used more convenient.
Known impurities situation analysis in tigecycline is as following table:
The related substance inspection method of reporting in tigecycline for injection import registration quality standard, only can measure minocycline hydrochloride, 9-amino minocycline ring element hydrochloride, tigecycline epimer and 9-nitro minocycline hydrochloride in above-mentioned known impurities, and N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride can not detect under this determination of related substances condition.Also find no at present the detection method of report N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride.
Summary of the invention
The object of the present invention is to provide a kind ofly in tigecycline, effectively detect the method for N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride.
The invention provides the method that detects N-tert-butyl group glycyl chloride hydrochloride in tigecycline, it adopts high performance liquid chromatography to measure, and operation steps is as follows:
(1) get tigecycline, after extraction, prepare need testing solution;
(2) get N-tert-butyl group glycyl chloride hydrochloride, after dilution, preparation reference substance solution;
(3) need testing solution, reference substance solution are injected respectively to high performance liquid chromatograph, detect; Its chromatographic condition is as follows:
Chromatographic column: take amino bonded silica gel as filling agent;
Mobile phase: methyl alcohol-isopropyl alcohol volume ratio: (85 ~ 95) ︰ (15 ~ 5)
Detect wavelength: 210 ~ 215nm.
Further, the diluting solvent of the extraction solvent of step (1) and step (2) is acetonitrile.
Further, mobile phase is: methyl alcohol-isopropyl alcohol volume ratio: 90 ︰ 10.
Further, detecting wavelength is 210nm, 211nm or 215nm.
Further, detecting wavelength is 215nm.
Further, described chromatographic column is of a size of 250mm * 4.6mm, 5 μ m.
Wherein, detecting column temperature is 25-40 ℃.
Further, detecting column temperature is 30 ℃.
The present invention also provides the method that detects N-tert-butyl group glycine hydrochloride in tigecycline, and it adopts high performance liquid chromatography to measure, and operation steps is as follows:
(1) get tigecycline, after extraction, prepare need testing solution;
(2) get N-tert-butyl group glycine hydrochloride, after dilution, preparation reference substance solution;
(3) need testing solution, reference substance solution are injected respectively to high performance liquid chromatograph, detect; Its chromatographic condition is as follows:
Chromatographic column: take amino bonded silica gel as filling agent;
Mobile phase: methyl alcohol-isopropyl alcohol volume ratio: (85 ~ 95) ︰ (15 ~ 5);
Detect wavelength: 210 ~ 215nm.
Further, the diluting solvent of the extraction solvent of step (1) and step (2) is selected from the described mobile phase of step (3).
Further, mobile phase is: methyl alcohol-isopropyl alcohol volume ratio: 90 ︰ 10.
Further, detecting wavelength is 210nm, 211nm or 215nm.
Further, detecting wavelength is 215nm.
Further, described chromatographic column is of a size of 250mm * 4.6mm, 5 μ m.
Wherein, detecting column temperature is 25-40 ℃.
Further, detecting column temperature is 30 ℃.
Detection method of the present invention is measured N-tert-butyl group glycine hydrochloride and the N-tert-butyl group glycyl chloride hydrochloride in tigecycline, measurement result accurately and reliably, specificity is strong, detection time is shorter, and can make N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride reach good degree of separation, for impurity content in complete detection tigecycline product, control product quality, guaranteeing that drug safety provides may.
Accompanying drawing explanation
Fig. 1 N-tert-butyl group glycine hydrochloride HPLC figure.
Fig. 2 N-tert-butyl group glycyl chloride hydrochloride HPLC figure.
Fig. 3 blank solvent HPLC figure.
Fig. 4 N-tert-butyl group glycine hydrochloride HPLC figure.
Fig. 5 N-tert-butyl group glycyl chloride hydrochloride HPLC figure.
Fig. 6 N-tert-butyl group glycine hydrochloride HPLC figure.
Fig. 7 N-tert-butyl group glycyl chloride hydrochloride HPLC figure.
Fig. 8 tigecycline test sample HPLC figure.
Fig. 9 N-tert-butyl group glycocoll and N-tert-butyl group glycyl chloride mixing reference substance chromatogram.
Figure 10 N-tert-butyl group glycine hydrochloride UV scanning figure.
Figure 11 N-tert-butyl group glycyl chloride hydrochloride UV scanning figure.
Figure 12 N-tert-butyl group glycine hydrochloride linear regression graph.
Figure 13 N-tert-butyl group glycyl chloride hydrochloride linear regression graph.
The application of sample recovery test figure of the N-tert-butyl group glycyl chloride hydrochloride in Figure 14 tigecycline test sample.
Embodiment
The detection of N-tert-butyl group glycyl chloride hydrochloride in embodiment 1 tigecycline
(1) get tigecycline, after extracting, filter with acetonitrile, constant volume, obtains need testing solution;
(2) get N-tert-butyl group glycyl chloride hydrochloride, after dissolving, dilute with acetonitrile, obtain reference substance solution;
(3) need testing solution, reference substance solution are injected respectively to high performance liquid chromatograph, by following chromatographic condition, detect:
Chromatographic column: nh 2 column UltimateXB-NH2(250mm * 4.6mm, 5 μ m);
Mobile phase: methyl alcohol-isopropyl alcohol volume ratio: 90 ︰ 10;
Flow velocity: 1.0ml/min;
Detect wavelength: 215nm.
Testing result is referring to table 1.
The detection of N-tert-butyl group glycine hydrochloride in embodiment 2 tigecyclines
(1) get tigecycline, after extracting, filter with methyl alcohol-isopropyl alcohol (90 ︰ 10v/v), constant volume, obtains need testing solution;
(2) get N-tert-butyl group glycine hydrochloride, after dissolving, dilute with methyl alcohol-isopropyl alcohol (90 ︰ 10v/v), obtain reference substance solution;
(3) need testing solution, reference substance solution are injected respectively to high performance liquid chromatograph, by following chromatographic condition, detect:
Chromatographic column: nh 2 column UltimateXB-NH2(250mm * 4.6mm, 5 μ m);
Mobile phase: methyl alcohol-isopropyl alcohol volume ratio: 90 ︰ 10;
Flow velocity: 1.0ml/min;
Detect wavelength: 215nm.
Testing result is referring to table 1.
Table 1
| Lot number | 110408 | 110409 | 110410 | 20110601 | 20110602 | 20110603 |
| N-tert-butyl group glycyl chloride hydrochloride | Do not detect | Do not detect | Do not detect | Do not detect | Do not detect | Do not detect |
| N-tert-butyl group glycine hydrochloride | Do not detect | Do not detect | Do not detect | Do not detect | Do not detect | Do not detect |
Result shows, in above-mentioned each batch products, the content of N-tert-butyl group glycyl chloride hydrochloride and N-tert-butyl group glycine hydrochloride is all under detectability.
Embodiment 3 testing conditions are investigated
1, chromatography condition
(1) checking of existing chromatographic condition
With reference to determination of related substances method in < < tigecycline for injection import registration quality standard > >, detect N-tert-butyl group glycine hydrochloride and the N-tert-butyl group glycyl chloride hydrochloride in tigecycline:
Lucifuge operation.Get this product appropriate, accurately weighed, solubilizer (is got dipotassium hydrogen phosphate 4.35g and sodium bisulfite 0.5g, put in 1000ml measuring bottle, be dissolved in water and be diluted to scale, with 1mol/L potassium hydroxide solution, regulate pH value to 8.0) dissolve and dilute and make the solution that contains tigecycline 0.5mg in every 1ml, as need testing solution; Precision takes N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride is appropriate respectively, with above-mentioned solvent dilution, becomes every 1ml respectively containing the solution of N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride 1mg, shakes up, in contrast solution.According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010) test, with octadecylsilane chemically bonded silica, be filling agent; With phosphate buffer, (get ADKP 4.35g and disodium ethylene diamine tetraacetate 1.03g, be dissolved in 950ml water, phosphoric acid tune pH6.4)-acetonitrile (950:50) is mobile phase A, with phosphate buffer, (get ADKP 4.35g and disodium ethylene diamine tetraacetate 1.03g, be dissolved in 500ml water, phosphoric acid tune pH6.4)-acetonitrile (500:500) is Mobile phase B, and according to the form below carries out gradient elution; Flow velocity is per minute 1.0ml; Detection wavelength is 248nm; Sample size is 20 μ l.
Get above-mentioned need testing solution, reference substance solution and solvent injection liquid chromatography respectively, in result contrast solution chromatogram, fail to detect the chromatographic peak of N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride; Change detection wavelength into 210nm, get again above-mentioned need testing solution and reference substance solution injection liquid chromatography respectively, the chromatographic peak of still failing to detect N-tert-butyl group glycine hydrochloride (see figure 1) and N-tert-butyl group glycyl chloride hydrochloride (see figure 2) in result contrast solution chromatogram, it is consistent with solvent peak (see figure 3) that chromatogram goes out peak situation.Visible existing chromatographic condition can not be for detection of N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride.Other system mobile phases are changed in consideration or chromatographic column is tested.
(2) selection of chromatographic condition of the present invention
Due to N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride, under the determination of related substances condition in tigecycline for injection import registration quality standard, fail to detect, and N-tert-butyl group glycyl chloride hydrochloride is easily hydrolyzed in water, therefore consider to select methyl alcohol-isopropyl alcohol as mobile phase, select nh 2 column UltimateXB-NH2(250mm * 4.6mm, 5 μ m) as chromatographic column, flow velocity: 1.0ml/min.
N-tert-butyl group glycine hydrochloride is similar with physico-chemical property to N-tert-butyl group glycyl chloride hydrochloride structure, both have similarity at the retention time in chromatographic column, in addition acyl chlorides facile hydrolysis becomes acid, when detecting the purity of said two products, need to both are effectively separated in chromatographic column, therefore, flow matched screens:
When methyl alcohol-isopropyl alcohol ratio is (80:20), N-tert-butyl group glycine hydrochloride retention time is 5.697 minutes (see figure 4)s, N-tert-butyl group glycyl chloride hydrochloride retention time is 6.146 minutes (see figure 5)s, yet, between two peaks retention time be separated by too near, easily for assay causes error.
When in mobile phase, methyl alcohol-isopropyl alcohol ratio is (90:10), N-tert-butyl group glycine hydrochloride retention time was 5.079 minutes (see figure 6)s, N-tert-butyl group glycyl chloride hydrochloride retention time was 6.286 minutes (see figure 7)s, and tigecycline retention time was 19.319 minutes (see figure 8)s.Simultaneously, due to solubility, get a large amount of N-tert-butyl group glycine hydrochloride reference substances and a small amount of N-tert-butyl group glycyl chloride hydrochloride reference substance and be jointly placed in acetonitrile jolting extraction, after filtration, subsequent filtrate detects under this chromatographic condition, both good (see figure 9)s of chromatographic peak degree of separation.
2, the screening of solvent
Through solubleness, investigate N-tert-butyl group glycyl chloride hydrochloride slightly soluble in acetonitrile; N-tert-butyl group glycine hydrochloride is almost insoluble in acetonitrile, easily molten in mobile phase; And N-tert-butyl group glycyl chloride hydrochloride be take alcohols derivative reaction is easily occurred during as solvent, therefore select mobile phase as the solvent of N-tert-butyl group glycine hydrochloride, and select acetonitrile as the solvent of N-tert-butyl group glycyl chloride hydrochloride.
3, detecting wavelength selects
Get N-tert-butyl group glycyl chloride hydrochloride and N-tert-butyl group glycine hydrochloride appropriate, with methyl alcohol, dissolve and dilute respectively the solution of making suitable concentration, according to UV-VIS spectrophotometry (two appendix IVA of Chinese Pharmacopoeia version in 2010) in the interscan of 200 ~ 400nm scope, its UV scanning figure that the results are shown in Table 2, N-tert-butyl group glycyl chloride hydrochloride and N-tert-butyl group glycine hydrochloride is shown in Figure 10 and Figure 11.
Table 2 tigecycline wavelength selection result table
| Sample name | Maximum absorption wavelength |
| N-tert-butyl group glycyl chloride hydrochloride | 211nm |
| N-tert-butyl group glycine hydrochloride | 210nm |
From the above results, N-tert-butyl group glycyl chloride hydrochloride has absorption maximum at 211nm place, N-tert-butyl group glycine hydrochloride has absorption maximum at 210nm place, the maximum absorption wavelength 248nm of comprehensive tigecycline, is therefore decided to be 215nm by the detection wavelength of this product N-tert-butyl group glycyl chloride hydrochloride and N-tert-butyl group glycine hydrochloride.
The methodological study of embodiment 4 detection methods of the present invention
In the present embodiment, various tests all adopt following condition:
Chromatographic column: nh 2 column UltimateXB-NH2(250mm * 4.6mm, 5 μ m);
Mobile phase: methyl alcohol-isopropyl alcohol volume ratio: 90 ︰ 10;
Flow velocity: 1.0ml/min;
Detect wavelength: 215nm.
1, specificity research
Contrast solution 1: get N-tert-butyl group glycine hydrochloride appropriate, add mobile phase and dissolve and be diluted to every 1ml containing the solution of 0.2004mg, obtain.
Contrast solution 2: get N-tert-butyl group glycyl chloride hydrochloride appropriate, add acetonitrile and dissolve and be diluted to every 1ml containing the solution of 10.17 μ g, obtain.
Need testing solution: get tigecycline appropriate, add acetonitrile and dissolve and be diluted to every 1ml containing the solution of 10.35mg, obtain.
Get respectively each 20 μ l of acetonitrile, mobile phase, contrast solution 1, contrast solution 2 and need testing solution, injection liquid chromatography, the solvent peak of result acetonitrile and mobile phase does not all disturb the peak of N-tert-butyl group glycyl chloride hydrochloride, N-tert-butyl group glycine hydrochloride and tigecycline, and the peak of N-tert-butyl group glycyl chloride hydrochloride, N-tert-butyl group glycine hydrochloride and tigecycline is completely separated.The specificity that detection method of the present invention is described is stronger.
2, detect N-tert-butyl group glycine hydrochloride
2.1 linear relationship
It is appropriate that precision takes N-tert-butyl group glycine hydrochloride, dissolves and dilute the solution of making a series of concentration with mobile phase.According to above-mentioned chromatographic condition, precision measures each solution 20 μ l injection liquid chromatographies respectively, records chromatogram, measures respectively peak area.The results are shown in Table 3.
Table 3 linear relationship test findings table
Take solution concentration respectively as horizontal ordinate X, take peak area as ordinate Y, measure linear equation and the correlation coefficient r of N-tert-butyl group glycine hydrochloride.Linear regression graph is shown in Figure 12.
Result shows: the concentration of N-tert-butyl group glycine hydrochloride is good in 0.1086mg/ml ~ 0.4345mg/ml scope internal linear relation, linear equation: Y=123642X-960.88, correlation coefficient r=0.9998.
2.2 detectability
Precision takes N-tert-butyl group glycine hydrochloride, adds mobile phase and dissolves and dilute and make certain density reference substance solution, and precision measures 20 μ l, and injection liquid chromatography, records chromatogram.When concentration is that while containing 1.261 μ g in every 1ml, peak height is about 3 times of baseline noise, equals 3:1 by signal to noise ratio (S/N ratio), the lowest detection that records N-tert-butyl group glycine hydrochloride is limited to 25.22ng.
2.3 recovery test
Precision takes N-tert-butyl group glycine hydrochloride 198.41mg, adds mobile phase and dissolves and dilute the solution of making containing N-tert-butyl group glycine hydrochloride (1.9841mg/ml), in contrast stock solution; Get 9 parts of each about 100mg of tigecycline raw material, put in 10ml measuring bottle, add respectively N-tert-butyl group glycine hydrochloride contrast stock solution 0.8ml, 1.0ml, 1.2ml, add acetonitrile and dissolve and be diluted to scale, shake up, as need testing solution, each concentration is prepared 3 parts.Separately get N-tert-butyl group glycine hydrochloride contrast stock solution 1.0ml, put 10ml measuring bottle, add mobile phase and be diluted to scale, shake up, in contrast solution.Precision measures test sample and each 20 μ l sample introduction mensuration of contrast solution respectively, records chromatogram, calculates the amount of recording and the recovery of mentioned component, and it the results are shown in Table 4.
Table 4N-tert-butyl group glycine hydrochloride recovery test result table
Above test findings shows, in this law working sample, the recovery of N-tert-butyl group glycine hydrochloride salt content is good, and relative standard deviation is little, illustrates that this method is measured the content accuracy of N-tert-butyl group glycine hydrochloride higher.
2.4 solution stability testing
Get N-tert-butyl group glycine hydrochloride appropriate, adding mobile phase dissolves and is diluted to every 1ml containing the solution of 9.6484mg, respectively at 0h, 1h, 2h, 4h, 6h, 8h sample introduction, investigate respectively the situation of change of impurity in its solution, by area normalization method, calculate total impurities, and add up impurity number, the results are shown in Table 5.
Table 5N-tert-butyl group glycine hydrochloride solution stability testing result table
Conclusion: from data above, the mensuration solution inclusion-free of N-tert-butyl group glycine hydrochloride produces in 8 hours, and main peak peak area is without significant change, and known N-tert-butyl group glycine hydrochloride is stable in 8 hours in mobile phase.
3, detect N-tert-butyl group glycyl chloride hydrochloride
3.1 linear relationship
It is appropriate that precision takes N-tert-butyl group glycyl chloride hydrochloride, dissolves and dilute the solution of making a series of concentration with acetonitrile.According to above-mentioned chromatographic condition, precision measures each solution 20 μ l injection liquid chromatographies respectively, records chromatogram, measures respectively peak area.The results are shown in Table 6.
Table 6 linear relationship test findings table
Take solution concentration respectively as horizontal ordinate X, take peak area as ordinate Y, measure linear equation and the correlation coefficient r of N-tert-butyl group glycyl chloride hydrochloride.Linear regression graph is shown in Figure 13.
Result shows: the concentration of N-tert-butyl group glycyl chloride hydrochloride is good in 0.0061mg/ml ~ 0.0245mg/ml scope internal linear relation, linear equation: Y=7241654.62X-13945.91, correlation coefficient r=0.9958.
3.2 detectability
Precision takes N-tert-butyl group glycyl chloride hydrochloride, adds acetonitrile and dissolves and dilute and make certain density reference substance solution, and precision measures 20 μ l, and injection liquid chromatography, records chromatogram.When concentration is that while containing 0.098 μ g in every 1ml, peak height is about 3 times of baseline noise, equals 3:1 by signal to noise ratio (S/N ratio), the lowest detection that records N-tert-butyl group glycyl chloride hydrochloric acid is limited to 1.96ng.
3.3 recovery test
Precision takes N-tert-butyl group glycyl chloride hydrochloride 10.72mg, adds acetonitrile and dissolves and dilute the solution of making containing N-tert-butyl group glycyl chloride hydrochloride (0.1072mg/ml), in contrast stock solution; Get 9 parts of each about 100mg of tigecycline raw material, put in 10ml measuring bottle, add respectively N-tert-butyl group glycyl chloride hydrochloride contrast stock solution 0.8ml, 1.0ml, 1.2ml, adding acetonitrile dissolves and is diluted to scale, shake up, as need testing solution, each concentration is prepared 3 parts.Separately get N-tert-butyl group glycyl chloride hydrochloride contrast stock solution 1.0ml, put 10ml measuring bottle, add dilution in acetonitrile to scale, shake up, in contrast solution.Precision measures each 20 μ l sample introductions of test sample and contrast solution and measures respectively, records chromatogram, calculates the amount of recording and the recovery of mentioned component, and it the results are shown in Table 7, Figure 14.
Table 7N-tert-butyl group glycyl chloride hydrochloride recovery test result table
Above test findings shows, in this law working sample, the recovery of N-tert-butyl group glycyl chloride hydrochloride content is good, and relative standard deviation is little, illustrates that this method is measured the content accuracy of N-tert-butyl group glycyl chloride hydrochloride higher.
3.4 solution stability testing
Get N-tert-butyl group glycyl chloride hydrochloride appropriate, adding acetonitrile dissolves and is diluted to every 1ml containing the solution of 0.4048mg, respectively at 0h, 1h, 2h, 4h, 6h, 8h sample introduction, investigate respectively the situation of change of impurity in its solution, by area normalization method, calculate total impurities, and add up impurity number, the results are shown in Table 8.
Table 8N-tert-butyl group glycyl chloride hydrochloride solution stability testing result table
Conclusion: from data above, the mensuration solution of N-tert-butyl group glycyl chloride hydrochloride produces without new impurity in 8 hours, total impurities and main peak peak area are without significant change, and known N-tert-butyl group glycyl chloride hydrochloride is stable in 8 hours in acetonitrile.
To sum up, detection method of the present invention is measured N-tert-butyl group glycine hydrochloride and the N-tert-butyl group glycyl chloride hydrochloride in tigecycline, measurement result accurately and reliably, specificity is strong, detection time is shorter, and can make N-tert-butyl group glycine hydrochloride and N-tert-butyl group glycyl chloride hydrochloride reach good degree of separation, for impurity content in complete detection tigecycline product, control product quality, guaranteeing that drug safety provides may.
Claims (8)
1. in tigecycline, detect the method for N-tert-butyl group glycine hydrochloride, it is characterized in that: it adopts high performance liquid chromatography to measure, and operation steps is as follows:
(1) get tigecycline, after extraction, prepare need testing solution;
(2) get N-tert-butyl group glycine hydrochloride, after dilution, preparation reference substance solution;
(3) need testing solution, reference substance solution are injected respectively to high performance liquid chromatograph, detect; Its chromatographic condition is as follows:
Chromatographic column: take amino bonded silica gel as filling agent;
Mobile phase: methyl alcohol-isopropyl alcohol volume ratio: (85 ~ 95) ︰ (15 ~ 5);
Detect wavelength: 210 ~ 215nm.
2. detection method according to claim 1, is characterized in that: the diluting solvent of the extraction solvent of step (1) and step (2) is selected from the described mobile phase of step (3).
3. detection method according to claim 1, is characterized in that: mobile phase is: methyl alcohol-isopropyl alcohol volume ratio: 90 ︰ 10.
4. detection method according to claim 1, is characterized in that: detection wavelength is 210nm, 211nm or 215nm.
5. detection method according to claim 4, is characterized in that: detection wavelength is 215nm.
6. detection method according to claim 1, is characterized in that: described chromatographic column is of a size of 250mm * 4.6mm, 5 μ m.
7. detection method according to claim 1, is characterized in that: detection column temperature is 25-40 ℃.
8. detection method according to claim 7, is characterized in that: detecting column temperature is 30 ℃.
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