CN102721775B - A kind of method that proxymetacaine hydrochloride intermediate IV is separated with the high performance liquid chromatography of other intermediate - Google Patents
A kind of method that proxymetacaine hydrochloride intermediate IV is separated with the high performance liquid chromatography of other intermediate Download PDFInfo
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Abstract
The present invention discloses one<b>by the method that proxymetacaine hydrochloride intermediate IV is separated with the high performance liquid chromatography of other intermediate</b>comprise the following steps: step one, get proxymetacaine hydrochloride sample, with organic solvent dissolution proxymetacaine hydrochloride sample, being mixed with the sample solution of every 1ml containing proxymetacaine hydrochloride 0.5��1.0mg, step 2, arranges the moving phase of liquid chromatograph, flow velocity 0.6��the 1.2mL/min of moving phase, determined wavelength 232 �� 3nm, and the column oven 20 DEG C��40 DEG C of liquid chromatograph chromatographic column is set; Step 3, gets sample solution 5-20 �� l in step one and injects in the liquid chromatograph that step 2 is arranged, and completes being separated of proxymetacaine hydrochloride intermediate IV and other intermediate.
Description
Technical field
The present invention provides a kind of method being separated by proxymetacaine hydrochloride intermediate IV with the high performance liquid chromatography of other intermediate.
Background technology
Proxymetacaine hydrochloride is a kind of surface anaesthetic, for the surface anesthesia of various ophthalmologic operation and eye examination, compared with other narcotic, has quick, efficient, the advantages such as corneal toxic side effect is light. Proxymetacaine hydrochloride intermediate compound IV molecular formula: C13H17NO5, chemistry 3-nitro-4-propoxy benzoic acid propyl ester by name, intermediate compound I, II, III, IV structural formula is as follows respectively:
Intermediate I
Intermediate II
Intermediate III
Intermediate compound IV
In order to accurately control the quality of proxymetacaine hydrochloride intermediate, need effectively to be separated proxymetacaine hydrochloride intermediate IV with other intermediate, but the high-efficient liquid phase isocratic elution mode adopted at present can't realize effectively being separated of proxymetacaine hydrochloride intermediate IV and other intermediate.
Summary of the invention
The present invention provides a kind of method being separated by proxymetacaine hydrochloride intermediate IV with the high performance liquid chromatography of other intermediate, and it can realize the accurate control of the disintegrate-quality of proxymetacaine hydrochloride intermediate IV with other intermediate.
Present invention employs following technical scheme: a kind of method being separated by proxymetacaine hydrochloride intermediate IV with the high performance liquid chromatography of other intermediate, comprise the following steps: step one, get proxymetacaine hydrochloride sample, with organic solvent dissolution proxymetacaine hydrochloride sample, it is mixed with the sample solution of every 1ml containing proxymetacaine hydrochloride 0.5��1.0mg; Step 2, arranges the moving phase of liquid chromatograph, the flow velocity 0.6��1.2mL/min of moving phase, determined wavelength 232 �� 3nm, and arranges the column oven 20 DEG C��40 DEG C of liquid chromatograph chromatographic column;
Step 3, gets sample solution 5-20 �� l in step one and injects the liquid chromatograph arranged through step 2, completes being separated of proxymetacaine hydrochloride intermediate IV and other intermediate.
The organic solvent of step one of the present invention is methyl alcohol or ethanol.
The moving phase of step 2 kind liquid chromatograph of the present invention is: A pump is organic modifiers, and B pump is buffer salt solution, and the gradient elution program arranging liquid chromatograph is as follows:
Time (min) A pump moving phase (%) B pump moving phase (%)
5-100-30100-70
10-2030-6070-40
20-306040
30-5060-040-100
50-600100,
The volume ratio of organic modifiers and buffered soln changes in time and changes, 5 minutes to 10 minutes, the volume of mobile phase A pump changes to 30% from 0% gradually, and the volume of Mobile phase B pump changes to 70%, A pump moving phase from 100% gradually simultaneously: B pump moving phase is 0-30:100-70; 10 minutes to 20 minutes, the volume of mobile phase A pump changed to 60% from 30% gradually, and the volume of Mobile phase B pump changes to 40%, A pump moving phase from 70% gradually simultaneously: B pump moving phase is 30-60:70-40; 20 minutes to 30 minutes, the volume of mobile phase A pump kept 60% constant, and the volume of Mobile phase B pump keeps 40% constant simultaneously, A pump moving phase: B pump moving phase is 60:40; 30 minutes to 50 minutes, the volume of mobile phase A pump changed to 0% from 60% gradually, and the volume of Mobile phase B pump changes to 100%, A pump moving phase from 40% gradually simultaneously: B pump moving phase is 60-0:40-100; 50 minutes to 60 minutes, the volume of mobile phase A pump kept 0% constant, and the volume of Mobile phase B pump keeps 100% constant simultaneously, A pump moving phase: B pump moving phase is 0:100.
Liquid chromatograph in step 2 of the present invention adopts carbon octadecane silane key and silica gel to be the chromatographic column of weighting agent, and the specification of chromatographic column is length 250mm, internal diameter 4.6mm, granularity 5 ��m.
Described organic modifiers is acetonitrile or methyl alcohol. Described buffer salt solution is after 0.05M potassium primary phosphate and 0.02M sodium heptanesulfonate are dissolved in water, then adds triethylamine and adjust the solution that obtained by PH to 5.2.
The present invention has following technique effect: the present invention adopts carbon octadecane silane key and silica gel to be the chromatographic column of weighting agent, can effectively be separated proxymetacaine hydrochloride intermediate IV and other intermediate; Selection methyl alcohol is solvent, it is ensured that sample can dissolve completely; Selecting sampling volume 10 �� L, post temperature is room temperature 25 DEG C, it is to increase the symmetry of chromatographic peak; Select flow velocity 1.0mL/min, resolution can be improved; A pump is selected to be organic modifiers, organic modifiers is methyl alcohol, B pump buffer salt solution is 0.05M potassium primary phosphate, 0.02M sodium heptanesulfonate, the solution of PH to 5.2 adjusted by triethylamine, adopt gradient elution to realize being separated completely of proxymetacaine hydrochloride intermediate IV and other intermediate, it is achieved that the detection of proxymetacaine hydrochloride intermediate purity, in the quality control of proxymetacaine hydrochloride pharmaceutical synthesis and formulation manufacturing processes, there is realistic meaning to realizing proxymetacaine hydrochloride intermediate.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of embodiment one.
Fig. 2 is the high-efficient liquid phase chromatogram of embodiment two.
Fig. 3 is the high-efficient liquid phase chromatogram of embodiment three.
Embodiment
By following examples, the present invention is described further.
Embodiment one
Adopting Agilent LC-1200 model high performance liquid chromatograph, Apollo brand carbon 18 silane key and silica gel to be the chromatographic column of weighting agent, the specification of chromatographic column is length 250mm, internal diameter 4.6mm, granularity 5 ��m.
Step one, gets proxymetacaine hydrochloride sample respectively, intermediate IV in sample and intermediate I, II, III each 5mg, it is placed in 10mL volumetric flask, adds dissolve with methanol and be diluted to scale, shake even, it is mixed with the sample solution of every 1ml containing proxymetacaine hydrochloride 0.5��1.0mg, as need testing solution;
Step 2, arranges the moving phase of liquid chromatograph, and namely A pump is organic modifiers, and B pump is buffer salt solution, and the gradient elution program arranging liquid chromatograph is as follows:
Gradient one: time (min) A pump moving phase (%) B pump moving phase (%)
5-101090
10-204060
20-406040
40-607030
601090,
When 5-10 minute, A pump moving phase: B pump moving phase is 10:90, when 10-20 minute, A pump moving phase: B pump moving phase is 40:60, when 20-40 minute, A pump moving phase: B pump moving phase is 60:40, when 40-60 minute, A pump moving phase: B pump moving phase is 70:30, when 60 minutes, A pump moving phase: B pump moving phase is 10:90, arranges flow rate of mobile phase 0.6mL/min, the determined wavelength 230nm of liquid chromatograph, and arranges the column oven 20 DEG C of liquid chromatograph chromatographic column;
Step 3, the need testing solution 20 �� l getting step one injects the liquid chromatograph arranged through step 2, completes being separated of proxymetacaine hydrochloride intermediate IV and other intermediate, and record color atlas, the results are shown in accompanying drawing 1.
In FIG, retention time be the chromatographic peak of 5.577min, 21.500min, 29.475min be respectively intermediate II, I, the chromatographic peak of III, retention time is 33.798min is the chromatographic peak of proxymetacaine hydrochloride intermediate IV. Intermediate III reaches 25.69 with the resolution of intermediate IV, meets " 2005 editions Chinese Pharmacopoeias " resolution completely and is not less than the regulation of 1.5, shows that proxymetacaine hydrochloride intermediate IV can be issued at same chromatographic condition with other intermediate and be well separated.
Embodiment two
Adopting Agilent LC-1200 model high performance liquid chromatograph, Apollo brand carbon octadecane silane key and silica gel to be the chromatographic column of weighting agent, the specification of chromatographic column is length 250mm, internal diameter 4.6mm, granularity 5 ��m.
Step one, get proxymetacaine hydrochloride sample respectively, intermediate IV in sample and intermediate I, II, III each 10mg, it is placed in 10mL volumetric flask, add dissolve with methanol and it is diluted to scale, shake even, it is mixed with the sample solution of every 1ml containing proxymetacaine hydrochloride 0.5��1.0mg, as need testing solution;
Step 2, arranges the moving phase of liquid chromatograph, and namely A pump is organic modifiers, and B pump is buffered soln, and the gradient elution program arranging liquid chromatograph is as follows:
Gradient two: time (min) A pump moving phase (%) B pump moving phase (%)
5-100100
10-203070
20-307030
30-507030
50-601090
601090,
When 5-10 minute, A pump moving phase: B pump moving phase is 0:100, when 10-20 minute, A pump moving phase: B pump moving phase is 30:70, when 20-30 minute, A pump moving phase: B pump moving phase is 70:30, when 30-50 minute, A pump moving phase: B pump moving phase is 70:30, when 50 60 minutes, A pump moving phase: B pump moving phase is 10:90, when 60 minutes, A pump moving phase: B pump moving phase is 10:90, the flow rate of mobile phase 1.2mL/min of liquid chromatograph is set, determined wavelength 237nm, and the column oven 40 DEG C of liquid chromatograph chromatographic column is set,
Step 3, the need testing solution 5 �� l getting step one injects the liquid chromatograph arranged through step 2, completes being separated of proxymetacaine hydrochloride intermediate IV and other intermediate, and record color atlas, is shown in Fig. 2.
In fig. 2, retention time be the chromatographic peak of 5.667min, 16.374min, 22.059min be respectively intermediate II, I, the chromatographic peak of III, retention time is 28.384min is the chromatographic peak of proxymetacaine hydrochloride intermediate IV, intermediate III reaches 25.78 with the resolution of intermediate IV, meet " 2005 editions Chinese Pharmacopoeias " resolution completely and it is not less than the regulation of 1.5, show that proxymetacaine hydrochloride intermediate IV can be issued at same chromatographic condition with other intermediate and be well separated.
Embodiment three
Adopting Agilent LC-1200 model high performance liquid chromatograph, Apollo brand carbon octadecane silane key and silica gel to be the chromatographic column of weighting agent, the specification of chromatographic column is length 250mm, internal diameter 4.6mm, granularity 5 ��m.
Step one, get proxymetacaine hydrochloride sample respectively, intermediate IV in sample and intermediate I, II, III, IV each 8mg, it is placed in 10mL volumetric flask, add dissolve with methanol and it is diluted to scale, shake even, it is mixed with the sample solution of every 1ml containing proxymetacaine hydrochloride 0.5��1.0mg, as need testing solution;
Step 2, arranges the moving phase of liquid chromatograph, and namely A pump is organic modifiers, and B pump is buffered soln, and the gradient elution program arranging liquid chromatograph is as follows:
Gradient three: time (min) A pump moving phase (%) B pump moving phase (%)
5-100100
10-203070
20-306040
30-506040
50-600100
600100,
When 5-10 minute, A pump moving phase: B pump moving phase is 0:100, when 10-20 minute, A pump moving phase: B pump moving phase is 30:70, when 20-30 minute, A pump moving phase: B pump moving phase is 60:40, when 30-50 minute, A pump moving phase: B pump moving phase is 60:40, when 50 60 minutes, A pump moving phase: B pump moving phase is 0:100, when 60 minutes, A pump moving phase: B pump moving phase is 0:100, the flow rate of mobile phase 1.0mL/min of liquid chromatograph is set, determined wavelength 232nm, and the column oven 25 DEG C of liquid chromatograph chromatographic column is set,
Step 3, the need testing solution 10 �� l getting step one injects the liquid chromatograph arranged through step 2, completes being separated of proxymetacaine hydrochloride intermediate IV and other intermediate, and record color atlas, the results are shown in accompanying drawing 3.
In figure 3, retention time be the chromatographic peak of 3.580min, 12.858min, 21.715min be respectively intermediate II, I, the chromatographic peak of III, retention time is 27.501min is the chromatographic peak of proxymetacaine hydrochloride intermediate IV, intermediate III reaches 27.26 with the resolution of intermediate IV, meet " 2005 editions Chinese Pharmacopoeias " resolution completely and it is not less than the regulation of 1.5, show that proxymetacaine hydrochloride intermediate IV can be issued at same chromatographic condition with other intermediate and be well separated.
Claims (1)
1. the method being separated with the high performance liquid chromatography of other intermediate by proxymetacaine hydrochloride intermediate IV, comprises the following steps:
Step one, gets proxymetacaine hydrochloride sample, with organic solvent dissolution proxymetacaine hydrochloride sample, is mixed with the sample solution of every 1ml containing proxymetacaine hydrochloride 0.5��1.0mg, and the organic solvent of step one is methyl alcohol or ethanol;
Step 2, the moving phase of liquid chromatograph is set, flow velocity 0.6��the 1.2mL/min of moving phase, determined wavelength 232 �� 3nm, and the column oven 20 DEG C��40 DEG C of liquid chromatograph chromatographic column is set, the moving phase of step 2 kind liquid chromatograph is: A pump is organic modifiers, B pump is buffer salt solution, and the gradient elution program arranging liquid chromatograph is as follows:
Time (min) A pump moving phase (%) B pump moving phase (%)
5-100-30100-70
10-2030-6070-40
20-306040
30-5060-040-100
50-600100,
The volume ratio of organic modifiers and buffered soln changes in time and changes, 5 minutes to 10 minutes, the volume of mobile phase A pump changes to 30% from 0% gradually, and the volume of Mobile phase B pump changes to 70%, A pump moving phase from 100% gradually simultaneously: B pump moving phase is 0-30:100-70; 10 minutes to 20 minutes, the volume of mobile phase A pump changed to 60% from 30% gradually, and the volume of Mobile phase B pump changes to 40%, A pump moving phase from 70% gradually simultaneously: B pump moving phase is 30-60:70-40; 20 minutes to 30 minutes, the volume of mobile phase A pump kept 60% constant, and the volume of Mobile phase B pump keeps 40% constant simultaneously, A pump moving phase: B pump moving phase is 60:40; 30 minutes to 50 minutes, the volume of mobile phase A pump changed to 0% from 60% gradually, and the volume of Mobile phase B pump changes to 100%, A pump moving phase from 40% gradually simultaneously: B pump moving phase is 60-0:40-100; 50 minutes to 60 minutes, the volume of mobile phase A pump keeps 0% constant, simultaneously the volume of Mobile phase B pump keeps 100% constant, A pump moving phase: B pump moving phase is 0:100, liquid chromatograph in step 2 adopts carbon octadecane silane key and silica gel to be the chromatographic column of weighting agent, the specification of chromatographic column is length 250mm, internal diameter 4.6mm, granularity 5 ��m, described organic modifiers is acetonitrile or methyl alcohol, described buffer salt solution is after 0.05M potassium primary phosphate and 0.02M sodium heptanesulfonate are dissolved in water, then adds triethylamine and adjust the solution that obtained by PH to 5.2;
Step 3, gets the sample solution 5-20 �� l in step one and injects the liquid chromatograph arranged through step 2, complete proxymetacaine hydrochloride intermediate IVWith other intermediate: intermediate I, intermediate II, intermediate IIISeparation.
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| US1317250A (en) * | 1919-09-30 | A cobporatiojet | ||
| US3775464A (en) * | 1970-04-22 | 1973-11-27 | Isf Spa | Process for preparing 2-dialkylaminoethyl 4-alkoxy-3-aminobenzoates |
| CN101104591A (en) * | 2006-07-14 | 2008-01-16 | 海南盛科生命科学研究院 | Polymorph of proparacaine hydrochloride and preparation method thereof |
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2012
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US1317250A (en) * | 1919-09-30 | A cobporatiojet | ||
| US3775464A (en) * | 1970-04-22 | 1973-11-27 | Isf Spa | Process for preparing 2-dialkylaminoethyl 4-alkoxy-3-aminobenzoates |
| CN101104591A (en) * | 2006-07-14 | 2008-01-16 | 海南盛科生命科学研究院 | Polymorph of proparacaine hydrochloride and preparation method thereof |
Non-Patent Citations (5)
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| An Experimental Design Approach to Selecting the Optimum LC Conditions for the Determination of Local Anaesthetics;A. Dincel et al;《Chromatographia 》;20070930;第66卷(第1期);81-85 * |
| Derivatives of 4-Amino-2-hydroxybenzoic Acid. II;R. O. Clinton et al;《J. Am. Chem. Soc》;19520205;第74卷(第3期);592–598 * |
| Development and Validation of an HPLC–UV–Vis Method for the Determination of Proparacaine in Human Aqueous Humour;Dincel A et al;《Chromatographia》;20070523;第66卷(第1期);51-56 * |
| 爱尔卡因滴眼液开启时间与菌落数及表麻后房水浓度影响的研究;王正玲 等;《中国医药指南》;20101010;第8卷(第28期);32-34 * |
| 盐酸丙美卡因对于混合痔术后创面肉芽组织的影响;孙化中 等;《中国药物与临床》;20111115;第11卷(第11期);1346-1347 * |
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