CN101650345B - Analysis method of content of 2-chloronicotinic acid - Google Patents
Analysis method of content of 2-chloronicotinic acid Download PDFInfo
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- CN101650345B CN101650345B CN2009100182314A CN200910018231A CN101650345B CN 101650345 B CN101650345 B CN 101650345B CN 2009100182314 A CN2009100182314 A CN 2009100182314A CN 200910018231 A CN200910018231 A CN 200910018231A CN 101650345 B CN101650345 B CN 101650345B
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- 238000004458 analytical method Methods 0.000 title claims abstract description 12
- IBRSSZOHCGUTHI-UHFFFAOYSA-N 2-chloropyridine-3-carboxylic acid Chemical compound OC(=O)C1=CC=CN=C1Cl IBRSSZOHCGUTHI-UHFFFAOYSA-N 0.000 title abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 114
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 9
- 229910001220 stainless steel Inorganic materials 0.000 claims abstract description 9
- 239000010935 stainless steel Substances 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims abstract description 3
- 239000000460 chlorine Substances 0.000 claims description 71
- 229910052801 chlorine Inorganic materials 0.000 claims description 71
- 238000012360 testing method Methods 0.000 claims description 28
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 7
- 238000013459 approach Methods 0.000 claims description 6
- 238000003825 pressing Methods 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 238000010812 external standard method Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 239000000575 pesticide Substances 0.000 abstract description 4
- 238000011084 recovery Methods 0.000 abstract description 4
- 229910019142 PO4 Inorganic materials 0.000 abstract description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 3
- 239000010452 phosphate Substances 0.000 abstract description 3
- 239000000543 intermediate Substances 0.000 abstract description 2
- 239000008366 buffered solution Substances 0.000 abstract 1
- 239000012450 pharmaceutical intermediate Substances 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 52
- 238000002360 preparation method Methods 0.000 description 20
- 238000005303 weighing Methods 0.000 description 15
- 239000012071 phase Substances 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 230000010355 oscillation Effects 0.000 description 10
- 239000012488 sample solution Substances 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 238000012545 processing Methods 0.000 description 8
- 238000005070 sampling Methods 0.000 description 8
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- 230000033228 biological regulation Effects 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 239000000470 constituent Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical class OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- JZFPYUNJRRFVQU-UHFFFAOYSA-N Niflumic acid Chemical compound OC(=O)C1=CC=CN=C1NC1=CC=CC(C(F)(F)F)=C1 JZFPYUNJRRFVQU-UHFFFAOYSA-N 0.000 description 1
- TVQZAMVBTVNYLA-UHFFFAOYSA-N Pranoprofen Chemical compound C1=CC=C2CC3=CC(C(C(O)=O)C)=CC=C3OC2=N1 TVQZAMVBTVNYLA-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 229960000916 niflumic acid Drugs 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 229960003101 pranoprofen Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
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Abstract
The invention relates to an analysis method of the content of the pharmaceutical intermediate and particularly provides a analysis method of content of 2-chloronicotinic acid which is a high performance liquid chromatography external standard quantitative analysis method and adopts ZORBAX SB-Phenyl 3.5mu m, 4.6*150mm stainless steel chromatographic column to separate. In the method the temperature of column compartment is 30 DEG C and the detection wavelength is 230-254nm, pure methanol is used as solvent, a mixed system of phosphate buffered solution and methanol with the pH value of 2.4 is used as mobile phase, ideal chromatographic peak shape can be obtained in the range of 230nm to 254nm and the content of 2-chloronicotinic acid in the detected sample can be calculated by comparing the response value of the detected sample with the response value of the standard substance. The method has high accuracy, good precision and high recovery rate, the method is simple and practical and is applicable to the analysis of the intermediate of pesticide and the method can effectively control the quality of 2-chloronicotinic acid.
Description
Technical field
The invention belongs to analysis technical field, relate to content analysis method, particularly a kind of 2-chlorine apellagrin content analysis technology.
Background technology
Along with the development of agricultural chemicals and medical science, nicotinic acid series chemicals more and more receives to be paid close attention to widely and uses in recent years.Wherein the 2-chlorine apellagrin is used to prepare new and effective herbicide, the non-anti-inflammatory medicaments of body now high efficiency anti-inflammatory analgesia niflumic acid, pranoprofen etc. as agricultural and medical intermedium, and at home and abroad supply falls short of demand for these products.
The Chinese of 2-chlorine apellagrin: 2-chlorine apellagrin, 2-chloro-3-pyridine carboxylic acid, English name: 2-chloronicotinic acid, 2-chloro-3-pyridinecarboxylic acid, molecular formula is: C
6H
4ClNO
2, molecular weight is: 157.56, and the 2-chlorine apellagrin is white or colour prime white crystalline powder, fusing point is 176-178 ℃.The 2-chlorine apellagrin is as a kind of important medicine and pesticide intermediate, and it can produce accessory substance 6-chlorine apellagrin in process of production.The 6-chlorine apellagrin is if too high levels is with the quality that has a strong impact on subsequent product, therefore extremely important to 2-chlorine apellagrin assay.
Through consulting relevant document both at home and abroad, do not see relevant 2-chlorine apellagrin analysis on Content report, and, need a kind of 2-chlorine apellagrin content detecting method especially in order to guarantee the real-time control in the 2-chlorine apellagrin production run.
Summary of the invention
To not having in the prior art for 2-chlorine apellagrin analysis on Content method; The invention provides a kind of method that adopts efficient liquid phase chromatographic analysis, 2-chlorine apellagrin content is analyzed, this method specificity is strong; Precision is good; The high performance liquid chromatography that the recovery is high is specially adapted to the quality control of pesticide original medicine intermediate product, thereby satisfies the production requirement of subsequent product.
The analytical approach that the present invention adopted is specific as follows:
Adopt high performance liquid chromatography external standard methods analyst, specifically adopt ZORBAX SB-Phenyl stainless steel packed column, 150 millimeters of column lengths, 4.6 millimeters of column internal diameters, used packing material size are 3.5 microns, the column compartment temperature is 30 ℃;
Through test of long duration; When finding to adopt this chromatographic column to detect, for the 2-chlorine apellagrin, be best, therefore preferably adopted this chromatographic column; This chromatographic column adopted with prior art in the ZORBAX SB-Phenyl stainless steel packed column that adopts identical, can on market, directly buy.
Concrete detection method is following: following moving phase ratio is volume fraction entirely
In order to obtain good separating effect, adopting with the PBS of PH=2-3 and the mixed system of methyl alcohol is moving phase, and the volume fraction of methyl alcohol in moving phase is 20% to 30%; Because phosphate itself is salt of weak acid, therefore, need to regulate the PH=2-3 of whole damping fluid in order to guarantee the sour environment in the testing process; The inventor finally finds when the PH=2.4 of whole damping fluid; Separating effect is ideal, and good stability, so the PH=2.4 of preferred buffer.
Make solvent with pure methyl alcohol and dissolve standard substance and testing sample respectively, obtain standard specimen and sample;
With the detection wavelength set of high performance liquid chromatography is 230nm to 254nm; After the instrument baseline stability, the order of pressing standard specimen, sample, sample, standard specimen is sample introduction successively, obtains four scanning curves; 2-chlorine apellagrin peak area to standard specimen and sample averages respectively, gets its peak area mean value;
Calculate according to the massfraction of following formula the 2-chlorine apellagrin:
In the formula:
A
1--in the standard specimen, the mean value of 2-chlorine apellagrin peak area;
A
2--in the sample, the mean value of 2-chlorine apellagrin peak area;
m
1--the quality of standard specimen, g;
m
2--the quality of sample, g;
P
1--the massfraction of 2-chlorine apellagrin in the standard specimen, %.
Be directed to the 2-chlorine apellagrin, do not have to adopt accordingly the detection method of high performance liquid chromatography in the existing technology, and its detection wavelength also is unknown; And pass through himself Study on Properties; The inventor detects wavelength with it and confirms at 230nm to 254nm, in this wavelength, can adopt high performance liquid chromatography that it is carried out the detection of content; Improve the precision that detects, also filled up blank of the prior art simultaneously.
In order to reach better detection effect and accuracy; After the self check of unlatching machine is passed through, under the operating conditions of regulation, inject number pin mark appearance after the instrument baseline stability continuously; Calculate each pin relative response; Reach adjacent two pin relative responses variation and carried out sample introduction successively again, can effectively reduce the error that causes owing to the instability after the instrument start like this, the accuracy that improves final detection result less than 1.5% o'clock.
The volume fraction of methyl alcohol is 23% in the general control moving phase, and the sample volume of each sample introduction is 20 microlitres; The flow velocity of controlling moving phase simultaneously is 1ml/min, and the detection wavelength of detecting device is at 235nm, and concerning the 2-chlorine apellagrin, this is the most stable uv absorption wavelength.
The PBS that is adopted mainly adopts dihydric phosphate; As can adopt the damping fluid of sodium dihydrogen phosphate or the buffer solution of potassium dihydrogen phosphate; The preferred buffer solution that adopts sodium dihydrogen phosphate; Its preparation method is: take by weighing 3.45 gram sodium dihydrogen phosphates and add 1000 milliliters water, it is for use as 2-3 to use 85% phosphoric acid to transfer to pH value.
In sum, the invention provides a kind of method of brand-new detection 2-chlorine apellagrin massfraction, filled up the technological gap in corresponding field in the prior art; Adopt said method to detect the massfraction of 2-chlorine apellagrin, can realize that impurity separates with main peak fully, chromatographic peak profile is good; Integral and calculating result is accurate, good reproducibility; The credible result degree of gained is high, is specially adapted to the quality control of pesticide original medicine intermediate product, and the quality that guarantees final products is had vital role and realistic meaning.
Description of drawings
Fig. 1 is the chromatogram of 2-chlorine apellagrin under chromatographic condition among the embodiment 1;
The chromatographic peak at 1 place is the peak of 2-chlorine apellagrin among the figure, and its retention time is 6.950 minutes.
Embodiment
1120 liquid chromatographs that the high performance liquid chromatograph that is adopted in the implementation process is produced for Agilent company.
Embodiment 1
Existing a collection of 2-chlorine apellagrin goods, 500 kilograms of quantity, need detect the effective content of 2-chlorine apellagrin in this shipments.
Chromatographic column condition: adopt ZORBAX SB-Phenyl 3.5um; 4.6 * 150mm stainless steel chromatogram post separates; The column compartment temperature is 30 ℃, and with the PBS of PH=2.4: methyl alcohol (volume ratio)=77: 23 is moving phase, and flow velocity is 1.0ml/min; The detection wavelength is 235nm, and sampling volume is 20 microlitres; The PBS that is adopted adopts following method preparation: take by weighing 3.45 gram sodium dihydrogen phosphates and add 1000 milliliters water, using 85% phosphoric acid to transfer to pH value is 2.4.
The preparation of standard specimen solution
Accurately take by weighing 2-chlorine apellagrin standard specimen 0.0509g, place the 100mL volumetric flask, add 30ml methyl alcohol, supersonic oscillations dissolvings, be chilled to room temperature after, be diluted to scale with methyl alcohol, obtain the standard specimen solution for standby.
The sample solution preparation
Accurately take by weighing and contain 2-chlorine apellagrin 0.0518g sample, place the 100mL volumetric flask, add 30ml methyl alcohol, the supersonic oscillations dissolving, be chilled to room temperature after, be diluted to scale with methyl alcohol, it is subsequent use to obtain sample solution.
Test and data processing
After opening instrument self checking and passing through, under the operating conditions of regulation, treat the instrument baseline stability after; Inject number pin mark appearance solution continuously; Calculate each pin relative response, reach adjacent two pin relative responses and change less than 1.5% o'clock, the order of pressing standard specimen, sample, sample, standard specimen is sample introduction successively; Under the 235nm wavelength, detect, it is following to get data:
With the 2 pin sample solutions that record with and front and back 2 pin mark appearance solution in 2-chlorine apellagrin peak area average respectively, represent the content X of 2-chlorine apellagrin with massfraction
1Be calculated as follows
In the formula:
A
1--in the standard specimen, the mean value of 2-chlorine apellagrin peak area;
A
2--in the sample, the mean value of 2-chlorine apellagrin peak area;
m
1--the quality of standard specimen, g;
m
2--the quality of sample, g;
P
1--the massfraction of 2-chlorine apellagrin in the standard specimen, %
The massfraction that calculates derived sample is 98.78%.
Embodiment 2
Existing a collection of 2-chlorine apellagrin goods, 1000 kilograms of quantity, need detect the effective content of 2-chlorine apellagrin in this shipments.
Chromatographic column condition: adopt ZORBAX SB-Phenyl 3.5um; 4.6 * 150mm stainless steel chromatogram post separates; The column compartment temperature is 30 ℃, and with the phosphate buffer solution of PH=2.0: methyl alcohol volume ratio=80: 20 is a moving phase, and flow velocity is 1.0ml/min; The detection wavelength is 254nm, and sampling volume is 20 microlitres; The PBS that is adopted adopts following method preparation: take by weighing 3.45 gram sodium dihydrogen phosphates and add 1000 milliliters water, using 85% phosphoric acid to transfer to pH value is 2.0.
The preparation of standard specimen solution
Accurately take by weighing 2-chlorine apellagrin standard specimen 0.0512g, place the 100mL volumetric flask, add 30ml methyl alcohol, supersonic oscillations dissolvings, be chilled to room temperature after, be diluted to scale with methyl alcohol, obtain the standard specimen solution for standby.
The sample solution preparation
Accurately take by weighing and contain 2-chlorine apellagrin 0.0506g sample, place the 100mL volumetric flask, add 30ml methyl alcohol, the supersonic oscillations dissolving, be chilled to room temperature after, be diluted to scale with methyl alcohol, it is subsequent use to obtain sample solution.
Test and data processing
After opening instrument self checking and passing through, under the operating conditions of regulation, treat the instrument baseline stability after; Inject number pin mark appearance solution continuously; Calculate each pin relative response, reach adjacent two pin relative responses and change less than 1.5% o'clock, the order of pressing standard specimen, sample, sample, standard specimen is sample introduction successively; Under the 254nm wavelength, detect, it is following to get data:
With the 2 pin sample solutions that record with and front and back 2 pin mark appearance solution in 2-chlorine apellagrin peak area average respectively, represent the content X of 2-chlorine apellagrin with massfraction
1Be calculated as follows
In the formula:
A
1--in the standard specimen, the mean value of 2-chlorine apellagrin peak area;
A
2--in the sample, the mean value of 2-chlorine apellagrin peak area;
m
1--the quality of standard specimen, g;
m
2--the quality of sample, g;
P
1--the massfraction of 2-chlorine apellagrin in the standard specimen, %
The massfraction that calculates derived sample is 98.93%.
Existing a collection of 2-chlorine apellagrin goods, 800 kilograms of quantity, need detect the effective content of 2-chlorine apellagrin in this shipments.
Chromatographic column condition: adopt ZORBAX SB-Phenyl 3.5um; 4.6 * 150mm stainless steel chromatogram post separates; The column compartment temperature is 30 ℃, and with the phosphate buffer solution of PH=3.0: methyl alcohol volume ratio=70: 30 is a moving phase, and flow velocity is 1.0ml/min; The detection wavelength is 230nm, and sampling volume is 20 microlitres; The PBS that is adopted adopts following method preparation: take by weighing 3.45 gram sodium dihydrogen phosphates and add 1000 milliliters water, using 85% phosphoric acid to transfer to pH value is 3.0.
The preparation of standard specimen solution
Accurately take by weighing 2-chlorine apellagrin standard specimen 0.0496g, place the 100mL volumetric flask, add 30ml methyl alcohol, supersonic oscillations dissolvings, be chilled to room temperature after, be diluted to scale with methyl alcohol, obtain the standard specimen solution for standby.
The sample solution preparation
Accurately take by weighing and contain 2-chlorine apellagrin 0.0510g sample, place the 100mL volumetric flask, add 30ml methyl alcohol, the supersonic oscillations dissolving, be chilled to room temperature after, be diluted to scale with methyl alcohol, it is subsequent use to obtain sample solution.
Test and data processing
After opening instrument self checking and passing through, under the operating conditions of regulation, treat the instrument baseline stability after; Inject number pin mark appearance solution continuously; Calculate each pin relative response, reach adjacent two pin relative responses and change less than 1.5% o'clock, the order of pressing standard specimen, sample, sample, standard specimen is sample introduction successively; Under the 230nm wavelength, detect, it is following to get data:
With the 2 pin sample solutions that record with and front and back 2 pin mark appearance solution in 2-chlorine apellagrin peak area average respectively, represent the content X of 2-chlorine apellagrin with massfraction
1Be calculated as follows
In the formula:
A
1--in the standard specimen, the mean value of 2-chlorine apellagrin peak area;
A
2--in the sample, the mean value of 2-chlorine apellagrin peak area;
m
1--the quality of standard specimen, g;
m
2--the quality of sample, g;
P
1--the massfraction of 2-chlorine apellagrin in the standard specimen, %
The massfraction that calculates derived sample is 98.51%.
Be the feasibility and the accuracy of the said method of checking foregoing invention, carried out following demonstration test:
Test Example 1
Chromatographic condition: adopt ZORBAX SB-Phenyl 3.5um; 4.6 * 150mm stainless steel chromatogram post separates; The column compartment temperature is 30 ℃, and with the phosphate buffer solution of PH=2.4: methyl alcohol volume ratio=77: 23 is a moving phase, and flow velocity is 1.0ml/min; The detection wavelength is 235nm, and sampling volume is 20 microlitres.
The standard specimen preparation
Accurately take by weighing 2-chlorine apellagrin standard specimen 0.0513g, place the 100mL volumetric flask, add 30ml methyl alcohol, supersonic oscillations dissolvings, be chilled to room temperature after, be diluted to scale with methyl alcohol, it is subsequent use to obtain standard specimen.
The sample preparation
Accurately take by weighing and contain 2-chlorine apellagrin 0.05g (being accurate to 0.0002g) sample, 6 parts place the 100mL volumetric flask respectively, add 30ml methyl alcohol, the supersonic oscillations dissolving, be chilled to room temperature after, be diluted to scale with methyl alcohol, it is subsequent use to obtain parallel sample.
Test and data processing
After the self check of unlatching machine is passed through, under the operating conditions of regulation, after the instrument baseline stability; Inject number pin mark appearance continuously, calculate each pin relative response, reach adjacent two pin relative responses and change less than 1.5% o'clock; The order of pressing standard specimen, sample, sample, standard specimen is sample introduction successively, scans at 235nm, and impurity separates fully; Peak shape is good, and by the active constituent content of formula (1) calculating sample, the result lists in table 1
Table 1
Visible by data in the table, this method experimental result repeatability is good.
Test Example 2
The preparation of chromatographic column condition, standard specimen, test in computing method with Test Example 1, with the phosphate buffer solution of PH=2.0: methyl alcohol (volume ratio)=80: 20 be moving phase, and flow velocity is 1.0ml/min, and the detection wavelength is that 230 sampling volumes are 20 microlitres.
The sample preparation
Take by weighing an amount of 2-chlorine apellagrin respectively, add dissolve with methanol and be diluted to the one group of 2-chlorine apellagrin sample that contains 2-chlorine apellagrin 215ug/ml, 318ug/ml, 432ug/ml, 511ug/ml, 614ug/m, 705ug/ml, 814ug/ml, 916ug/ml subsequent use.
Test and data processing
Measure at 230nm in accordance with the law, to sample concentration, do linear regression, obtain regression equation and do with peak area (A)
y＝1351.2x+484.32
Correlation coefficient r=0.9997
It is thus clear that the 2-chlorine apellagrin is good in 200-900ug/ml scope internal linear relation.
Test Example 3
Chromatographic condition, standard specimen preparation, test and computing method are with Test Example 1, and with the phosphate buffer solution with PH=3.0: methyl alcohol (volume ratio)=70: 30 is moving phase, and flow velocity is 1.0ml/min, and the detection wavelength is 254nm, and sampling volume is 20 microlitres.
The sample preparation
Different personnel accurately take by weighing in the different experiments chamber and contain 6 parts on 2-chlorine apellagrin 0.05g (being accurate to 0.0002g) sample, place the 100ml volumetric flask respectively, add 30ml methyl alcohol; The supersonic oscillations dissolving; After being chilled to room temperature, be diluted to scale with methyl alcohol, it is subsequent use with sample to obtain one group of middle precision test.
Test and data processing
Carry out sweep measuring at 254nm, impurity separates fully in accordance with the law, and peak shape is good, and by the active constituent content of formula (1) calculating sample, the result lists in table 2,
Table 2
Visible by data in the table, precision is good in the middle of this method experimental result.
Test Example 4
Chromatographic column condition, standard specimen preparation, test and computing method are with Test Example 1, and with the phosphate buffer solution of PH=2.4: methyl alcohol (volume ratio)=75: 25 is moving phase, and flow velocity is 1.0ml/min, and the detection wavelength is 230nm, and sampling volume is 20 microlitres.
The sample preparation
Different personnel accurately take by weighing in the different experiments chamber and contain 2-chlorine apellagrin 0.0511g sample, place the 100ml volumetric flask, add 30ml methyl alcohol, the supersonic oscillations dissolving, be chilled to room temperature after, be diluted to scale with methyl alcohol, it is subsequent use to obtain sample.
Test and data processing
Different time carries out sweep measuring at 230nm in accordance with the law, and impurity separates fully, and peak shape is good, and by the active constituent content of formula (1) calculating sample, the result lists in table 3
Table 3
| Time (h) | 0 | 1 | 2 | 3 | 6 | 12 | RSD％ |
| Peak area | 24578911 | 24561246 | 24537893 | 24578921 | 24574563 | 24523897 | 0.09 |
Visible by data in the table, this method experimental result time stability is good.
Test Example 5
Chromatographic column condition, standard specimen preparation, test and computing method are with Test Example 1, and with the phosphate buffer solution of PH=2.4: methyl alcohol (volume ratio)=77: 23 is moving phase, and flow velocity is 1.0ml/min, and the detection wavelength is 235nm, and sampling volume is 20 microlitres.
The sample preparation
2-chlorine apellagrin product with the known quality mark is an original pattern, adds the pure article of 2-chlorine apellagrin, and constant volume is done the mark-on recovery test.
Test and data processing
Carry out sweep measuring at 235nm, impurity separates fully, and peak shape is good, and by the active constituent content of formula (1) calculating sample, the result lists in table 4,
Table 4
Visible by data in the table, recovery of standard addition is good.
Claims (6)
1. 2-chlorine apellagrin analysis on Content method is characterized in that: adopt the analysis of high performance liquid chromatography external standard method, concrete grammar is following:
Make solvent with pure methyl alcohol and dissolve standard substance and testing sample respectively, obtain standard specimen and sample;
With the detection wavelength set of high performance liquid chromatography is 230nm to 254nm; After the instrument baseline stability, the order of pressing standard specimen, sample, sample, standard specimen is sample introduction successively, obtains four scanning curves; 2-chlorine apellagrin peak area to standard specimen and sample averages respectively, gets its peak area mean value;
Calculate according to the massfraction of following formula the 2-chlorine apellagrin:
In the formula:
A
1---in the standard specimen, the mean value of 2-chlorine apellagrin peak area;
A
2---in the sample, the mean value of 2-chlorine apellagrin peak area;
m
1---the quality of standard specimen;
m
2---the quality of sample;
P
1---the massfraction of 2-chlorine apellagrin in the standard specimen;
X
1---the massfraction of 2-chlorine apellagrin in the sample;
Said chromatographiccondition is: adopt ZORBAX SB-Phenyl, 4.6 x 150mm x 3.5um stainless steel chromatogram posts separate, with the PBS of PH=2-3: methyl alcohol (volume ratio)=(70-80): (30-20) be moving phase.
2. analytical approach according to claim 1 is characterized in that: used chromatographic column is a ZORBAX SB-Phenyl stainless steel packed column, 150 millimeters of column lengths, and 4.6 millimeters of column internal diameters, used packing material size are 3.5 microns, the column compartment temperature is 30 ℃.
3. analytical approach according to claim 1 is characterized in that: the volume fraction of methyl alcohol is 23% in the described moving phase.
4. analytical approach according to claim 1 is characterized in that: said PBS is the damping fluid of sodium dihydrogen phosphate or the buffer solution of potassium dihydrogen phosphate.
5. analytical approach according to claim 1 is characterized in that: sample volume 20 microlitres of each sample introduction; The flow velocity of moving phase is 1.0ml/min.
6. analytical approach according to claim 1 is characterized in that: the detection wavelength control of high performance liquid chromatography is at 235nm.
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| CN101650345B true CN101650345B (en) | 2012-05-30 |
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| CS247942B1 (en) * | 1985-07-03 | 1987-01-15 | Karel Benes | Method of 5-chloronicotinic acid determination |
| CN101117332A (en) * | 2006-08-04 | 2008-02-06 | 浙江医药股份有限公司新昌制药厂 | Preparation method of 2-chloronicotinic acid |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CS247942B1 (en) * | 1985-07-03 | 1987-01-15 | Karel Benes | Method of 5-chloronicotinic acid determination |
| CN101117332A (en) * | 2006-08-04 | 2008-02-06 | 浙江医药股份有限公司新昌制药厂 | Preparation method of 2-chloronicotinic acid |
Non-Patent Citations (7)
| Title |
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| ADRIAN, NR |
| ADRIAN, NR;SUFLITA, JM.ANAEROBIC BIODEGRADATION OF HALOGENATED AND NONHALOGENATED N-HETEROCYCLIC, S-HETEROCYCLIC, AND O-HETEROCYCLIC COMPOUNDS IN AQUIFER SLURRIES.《ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY》.1994,第13卷(第10期),1551-1557. * |
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| Liu, HX;Song, JM;Zhang, SS, et al..Analysis of residues of imidacloprid in tobacco by high-performance liquid chromatography with liquid-liquid partition cleanup.《PEST MANAGEMENT SCIENCE》.2005,第61卷(第5期),511-514. * |
| Song, JM |
| SUFLITA, JM.ANAEROBIC BIODEGRADATION OF HALOGENATED AND NONHALOGENATED N-HETEROCYCLIC, S-HETEROCYCLIC, AND O-HETEROCYCLIC COMPOUNDS IN AQUIFER SLURRIES.《ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY》.1994,第13卷(第10期),1551-1557. |
| Zhang, SS, et al..Analysis of residues of imidacloprid in tobacco by high-performance liquid chromatography with liquid-liquid partition cleanup.《PEST MANAGEMENT SCIENCE》.2005,第61卷(第5期),511-514. |
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