CN104977372B - Method for determining content of sulfonamide-phenylhydrazine hydrochloride in celecoxib raw medicine through high performance liquid chromatography - Google Patents
Method for determining content of sulfonamide-phenylhydrazine hydrochloride in celecoxib raw medicine through high performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for determining the content of sulfonamide-phenylhydrazine hydrochloride in a celecoxib raw medicine through high performance liquid chromatography. The method comprises the following steps: preparing a reference substance solution; determining by adopting octadecyl-bonded graphite carbon as the stationary phase of a high performance liquid chromatograph, 10mmol/l aqueous acetic acid solution-methanol (with a ratio of 45:55-55:45) as a mobile phone A and methanol as a mobile phone B, wherein the column temperature is 30-40DEG C, and the detection wavelength is 218-222nm; sucking 20[mu]l of the reference substance solution, a test sample solution and a blank solution, respectively injecting the solutions into the liquid chromatograph, and recording chromatograms; and calculating the concentrations X of the reference substance solution and corresponding peak areas Y, and carrying out linear fitting to obtain a linear regression equation with the linear range of 99.897-599.382ng/ml. The method for determining sulfonamide-phenylhydrazine hydrochloride in the celecoxib raw medicine through high performance liquid chromatography has the advantages of high separation efficiency, fast analysis speed and high detection sensitivity, and the quality of the celecoxib raw medicine can be well controlled by detecting the content of sulfonamide-phenylhydrazine hydrochloride in the celecoxib raw medicine in order to well detect sulfonamide-phenylhydrazine hydrochloride impurities possibly existing in the celecoxib raw medicine.
Description
Technical field
The present invention relates to high-efficient liquid phase chromatogram technology field is and in particular to a kind of high-performance liquid chromatogram determination Celecoxib is former
Method to sulfoamido phenyl hydrazine hydrochloride salt content in material medicine.
Background technology
There may be during Celecoxib production of raw medicine to sulfoamido hydrazinobenzene hydrochloride salt intermediate.This compound is tied
Structure has similarity with known poison impurity, need to carry out limit handling to it.Take according to the human body that ich specifies gene poison impurity
Amount not can exceed that 1.5 g for each person every day, and therefore we establish the hplc-uv in Celecoxib crude drug to sulfoamido phenylhydrazine
Algoscopy, can preferably control the quality of Celecoxib crude drug, preferably to that may be present in Celecoxib crude drug
Sulfoamido phenyl hydrazine hydrochloride salt impurity is detected.
Content of the invention
The purpose of the present invention is set up one and is measured in Celecoxib crude drug to sulfoamido phenyl hydrazine hydrochloride salt content
Method, can preferably control the quality of Celecoxib crude drug, preferably to that may be present right in Celecoxib crude drug
Sulfoamido phenyl hydrazine hydrochloride salt impurity is detected.
The technical scheme is that in high-performance liquid chromatogram determination Celecoxib crude drug to sulfoamido phenyl hydrazine hydrochloride
The method of salt content, it comprises the steps:
(1) preparation of reference substance solution: weigh to sulfoamido hydrazinobenzene hydrochloride salt reference substance, plus methanol, make every 1ml
Containing 500ng to sulfoamido phenyl hydrazine hydrochloride saline solution；
(2) preparation of need testing solution: take Celecoxib raw material plus methanol to make every 1ml and contain 150mg Celecoxib
Solution；
(3) measure: the fixing phase of high performance liquid chromatograph is bonded graphitic carbon for octadecyl, mobile phase a is 10mmol/
L ammonium acetate solution-methanol=45:55～55:45, mobile phase b is methanol, and column temperature is 30 DEG C～40 DEG C, mobile phase a initial flow
Speed is 0.78～0.82ml/min, and Detection wavelength draws reference substance solution and need testing solution each 15 respectively for 218nm～222nm
～25 l, inject high performance liquid chromatograph, read data.
The detector of described high performance liquid chromatograph is ultraviolet absorption detector, and Detection wavelength is 220nm.
Described mobile phase a is 10mmol/l ammonium acetate solution-methanol=50:50, and mobile phase b is methanol.It runs gradient
Mode is shown in Table 1:
The invention has the beneficial effects as follows: present invention application high performance liquid chromatography is surveyed in Celecoxib crude drug to sulfoamido
Phenyl hydrazine hydrochloride salt content, separation efficiency is high, analyze speed is fast, detection sensitivity is high, right in Celecoxib crude drug by detecting
The hydrochloride content of sulfoamido phenylhydrazine, controlling must not mistake to sulfoamido hydrazinobenzene hydrochloride salt in every gram of Celecoxib crude drug
3.75 g, are beneficial to the more preferable quality controlling Celecoxib crude drug, preferably to that may be present in Celecoxib crude drug
Sulfoamido phenyl hydrazine hydrochloride salt impurity is detected.
Brief description: Fig. 1 is blank solvent methanol chromatogram；
Fig. 2 is contrast solution chromatogram；
Fig. 3 is Celecoxib raw material chromatography space figure；
Fig. 4 is the linear regression graph to sulfoamido hydrazinobenzene hydrochloride salt；
Fig. 5 is change in flow test result comparison diagram；
Fig. 6 is that mobile phase a ratio changes test result comparison diagram；
Fig. 7 changes test result comparison diagram for sample size；
Fig. 8 changes test result comparison diagram for Detection wavelength；
Fig. 9 changes test result comparison diagram for column temperature.
Form is described in further detail to present disclosure more by the following examples, but should not be interpreted as this with regard to this
Invent in above-mentioned subject area and be only limitted to following examples.Under the premise of without departing from the above-mentioned technology of the present invention, general according to this area
Corresponding replacement or the modification of change that logical technological know-how and customary means are made, are included within the scope of the present invention.
The determination of embodiment 1 chromatographic column
Instrument: high performance liquid chromatograph thermo ultimate3000, UV-detector
Chromatographic column: waters xterra rp, 150mm × 4.6mm × 3.50 m, octadecyl silane is filler
Flow velocity: 0.7ml/min；
Sample size: 20 l；
Column temperature: 35 DEG C；
Wavelength: 247nm；
Mobile phase: 10mmol/l ammonium acetate solution-methanol=70:30；
The preparation of contrast solution: precision weighs to sulfoamido hydrazinobenzene hydrochloride salt 2mg, as in 200ml volumetric flask, plus first
Alcohol dissolves and is diluted to scale, shakes up and obtains final product.Precision measures above-mentioned solution 0.5ml as in 10ml volumetric flask, plus methanol dilution
To scale, shake up and obtain final product.
Take mobile phase, to sulfoamido hydrazinobenzene hydrochloride salt 50ng/ml solution, sample introduction record chromatogram respectively.
Solvent peak has less absorption at 4.0min, to sulfoamido hydrazinobenzene hydrochloride salt at 3.8min appearance.
Conclusion: under the process conditions, mobile phase has interference as solvent to the mensure of sulfoamido hydrazinobenzene hydrochloride salt.
The determination of embodiment 2 chromatographic column
Instrument: high performance liquid chromatograph thermo ultimate3000, UV-detector
Chromatographic column: inertsil ods-3,150mm × 4.6mm × 3.00 m, octadecyl silane is filler.
Flow velocity: 0.7ml/min；
Sample size: 20 l；
Column temperature: 35 DEG C；
Wavelength: 247nm；
Mobile phase: 10mmol/l ammonium acetate solution-methanol=70:30；
The preparation of contrast solution: precision weighs to sulfoamido hydrazinobenzene hydrochloride salt 2mg, as in 200ml volumetric flask, plus first
Alcohol dissolves and is diluted to scale, shakes up and obtains final product.Precision measures above-mentioned solution 0.5ml as in 10ml volumetric flask, plus methanol dilution
To scale, shake up and obtain final product.
The preparation of test solution: precision weighs to Celecoxib 1.5g, to 10ml volumetric flask, plus methanol dissolves and dilutes
To scale, shake up and obtain final product.
Take mobile phase, to sulfoamido hydrazinobenzene hydrochloride salt 50ng/ml solution, Celecoxib 150mg/ml solution sample introduction respectively
Record chromatogram.
Solvent peak has less absorption at 4.0min, to sulfoamido hydrazinobenzene hydrochloride salt in 3.89min in contrast solution
Place appearance, for examination in sulfoamido hydrazinobenzene hydrochloride salt at 3.92min appearance,
Conclusion: under the process conditions, mobile phase as solvent in Celecoxib to sulfoamido hydrazinobenzene hydrochloride salt
Mensure has interference.
The determination of embodiment 3 chromatographic column and Detection wavelength
Instrument: high performance liquid chromatograph waters e2695-2998, diode array detector.
Chromatographic column: thermo hypercarb, 150mm × 4.6mm × 3.00 m, octadecyl bonding graphitic carbon is to fill out
Material.
Flow velocity: 0.8ml/min；
Sample size: 20 l；
Column temperature: 35 DEG C；
Wavelength: 190 ~ 400nm；
Mobile phase: 10mmol/l ammonium acetate solution-methanol=70:30；
The preparation of contrast solution: precision weighs to sulfoamido hydrazinobenzene hydrochloride salt 2mg, as in 200ml volumetric flask, plus first
Alcohol dissolves and is diluted to scale, shakes up and obtains final product.Precision measures above-mentioned solution 5ml as in 10ml volumetric flask, plus methanol dilution is extremely
Scale, shakes up and obtains final product.
Take methanol, to sulfoamido hydrazinobenzene hydrochloride salt 500ng/ml solution, sample introduction record chromatogram respectively.
Solvent peak has less solvent peak at 3.0min, in contrast solution, sulfoamido hydrazinobenzene hydrochloride salt is existed
Appearance at 11.93min, has at 197nm, 217nm, 264nm respectively to sulfoamido hydrazinobenzene hydrochloride salt in contrast solution
Big absorption.
Conclusion: be bonded, using octadecyl, the chromatographic column that graphitic carbon is filler under the process conditions, methanol is as solvent
Noiseless to the mensure of sulfoamido hydrazinobenzene hydrochloride salt, maximum to sulfoamido hydrazinobenzene hydrochloride salt uv absorption at 217nm, therefore
Using the wavelength detecting near 217nm.
Embodiment 4 detector and the determination of chromatographic column.
Instrument: high performance liquid chromatograph waters 515-2475, fluorescence detector.
Chromatographic column: thermo hypercarb, 250mm × 4.6mm × 5.00 m, octadecyl bonding graphitic carbon is to fill out
Material.
Sample size: 20 l；
Column temperature: 35 DEG C；
Excitation wavelength: 210-260nm；Launch wavelength: 280nm；
Mobile phase: 10mmol/l ammonium acetate solution-methanol (50:50)；
In excitation wavelength: 210-260nm；Launch wavelength: 280nm；Sample introduction under wavelength condition
To sulfoamido hydrazinobenzene hydrochloride salt 10 μ g/ml solution, record chromatogram.
Solvent peak has larger absorption at 2.3min, to sulfoamido hydrazinobenzene hydrochloride salt in 11.93min in contrast solution
Place's appearance, has absorption maximum to sulfoamido hydrazinobenzene hydrochloride salt in contrast solution at excitation wavelength 220nm.
In excitation wavelength: 220nm；Launch wavelength: 260-360nm；Sample introduction under wavelength condition.
To sulfoamido hydrazinobenzene hydrochloride salt 10 μ g/ml solution, record chromatogram.
Solvent peak has larger absorption at 2.3min, to sulfoamido hydrazinobenzene hydrochloride salt in 11.80min in contrast solution
Place's appearance, has absorption maximum to sulfoamido hydrazinobenzene hydrochloride salt in contrast solution at launch wavelength 280nm.
In excitation wavelength: 220nm；Launch wavelength: 280nm；Sample introduction under wavelength condition.
To sulfoamido hydrazinobenzene hydrochloride salt 10 μ g/ml solution, record chromatogram.
Solvent peak has larger absorption at 2.1min, to sulfoamido hydrazinobenzene hydrochloride salt in 12.2min in contrast solution
Place's appearance, solvent peak does not interfere with to the detection of sulfoamido hydrazinobenzene hydrochloride salt.
In excitation wavelength: 220nm；Launch wavelength: 280nm；Sample introduction under wavelength condition.
To sulfoamido hydrazinobenzene hydrochloride salt 500ng/ml solution, record chromatogram.
Solvent peak has larger absorption at 2.1min, and sulfoamido hydrazinobenzene hydrochloride salt is less than in 500ng/ml concentration
Fluorescence detector test limit, fluorescence detector can't detect.
The determination of embodiment 5 eluent gradient mode.
Instrument: high performance liquid chromatograph waters2695-2998, diode array detector
Chromatographic column: thermo hypercarb, 150mm × 4.6mm × 3.00 m, octadecyl bonding graphitic carbon is to fill out
Material.
Flow velocity: 0.8ml/min；
Sample size: 20 l；
Column temperature: 35 DEG C；
Wavelength: 220nm；
Run time: 80min；
Mobile phase, mobile phase a:10mmol/l ammonium acetate solution-methanol=50:50；Mobile phase b: methanol.
Gradient table is shown in Table 1
The preparation of reference substance storing solution: precision weighs to sulfoamido hydrazinobenzene hydrochloride salt 5mg, as in 500ml volumetric flask,
Plus methanol dissolves and is diluted to scale, shake up and obtain final product.
The preparation of contrast solution: precision measures reference substance storing solution 0.5ml as in 10ml volumetric flask, plus methanol dilution is extremely
Scale, shakes up and obtains final product.
Material solution is prepared: take Celecoxib raw material (lot number 20110712) about 1.5g, accurately weighed, as 10ml measuring bottle
In, plus methanol dissolves and is diluted to scale, shakes up and obtains final product.
Take above-mentioned solution sample introduction result, Celecoxib raw material appearance after 20min respectively, sulfoamido phenylhydrazine is existed
9.6min appearance, Celecoxib and sulfoamido phenylhydrazine can be kept completely separate.
Experimental example 6 system suitability is tested
Instrument: high performance liquid chromatograph Shimadzu lc-20ad.
Chromatographic column: thermo hypercarb, 150mm × 4.6mm × 3.00 m, octadecyl bonding graphitic carbon is to fill out
Material.
Flow velocity: 0.8ml/min；
Sample size: 20 l；
Column temperature: 35 DEG C；
Wavelength: 220nm；
Run time: 80min；
Mobile phase, mobile phase a:10mmol/l ammonium acetate solution-methanol (50:50)；Mobile phase b: methanol；
Gradient table is shown in Table 1
The preparation of reference substance storing solution: precision weighs to sulfoamido hydrazinobenzene hydrochloride salt 5mg, as in 500ml volumetric flask,
Plus methanol dissolves and is diluted to scale, shake up and obtain final product.
The preparation of contrast solution: precision measures reference substance storing solution 0.5ml as in 10ml volumetric flask, plus methanol dilution is extremely
Scale, shakes up and obtains final product.
Sample solution is prepared: takes this product about 1.5g, accurately weighed, as in 10ml measuring bottle, plus methanol dissolves and is diluted to
Scale, shakes up and obtains final product.
Take blank solvent methanol, contrast solution, Celecoxib material sample solution sample introduction respectively, record chromatogram.Test
Result is shown in Fig. 1, Fig. 2, Fig. 3.
Result shows, the retention time of solvent peak methanol is 2.7min, the retention time to sulfoamido hydrazinobenzene hydrochloride salt
For 9.5min, after the retention time of Celecoxib is 32min, blank solvent does not disturb the mensure of determinand.
Experimental example 7 system suitability is tested.
Take contrast solution 20 l sample introduction, record chromatogram to 80min, continuous sample introduction 6 times, calculate each determinand of each sample introduction
Peak area, try to achieve relative standard deviation and should be not more than 5.0%, the results are shown in Table 2.
Table 2 Precision Experiment tables of data.
Conclusion: test shows that this chromatographic system precision is good.
Experimental example 8 is linear and scope.
Take concentration to be 99.897 ng/ml, 199.794 ng/ml, 299.691 ng/ml, 399.588 ng/ml,
499.485 ng/ml, 599.382 ng/ml to sulfoamido hydrazinobenzene hydrochloride salt, respectively as 20%, 40%, 60%, 80%,
100%th, the reference substance solution of 120% limit.Measure respectively by said determination method, record chromatogram, measure peak area, result is shown in
Table.Carry out linear regression with peak area value a, to concentration c (ng/ml), obtain line always and the results are shown in Table 3, Fig. 4.
Table 3 is to sulfoamido hydrazinobenzene hydrochloride salt Linear regression data
Regression equation y=37.908x -634.356 to sulfoamido hydrazinobenzene hydrochloride salt, r=0.9982.
Result shows, to sulfoamido phenyl hydrazine hydrochloride salinity sample introduction in the range of 99.897 ~ 599.382ng/ml respectively
Concentration and peak area value have good linear relationship.
Experimental example 9 test limit and quantitative limit.
According to test limit computing formula lod=3.3 × sd/s, (slope of s: calibration trace, sd: standard curve
The standard deviation of y intercept) calculate.Calculated by Linear regression data and to sulfoamido hydrazinobenzene hydrochloride salt test limit concentration be
7.6ng/ml.According to quantitative limit computing formula loq=10 × sd/s, (slope of s: calibration trace, sd: standard curve
The standard deviation of y intercept) calculate.Calculated by Linear regression data and to sulfoamido hydrazinobenzene hydrochloride salt quantitative limit concentration be
23.1ng/ml.Sulfoamido hydrazinobenzene hydrochloride salt will be diluted to 11.988 ng/ml, 35.963ng/ml concentration sample introductions, tested
Card record chromatogram, the results are shown in Table 4.
Table 4 test limit and quantitative limit
Conclusion: to sulfoamido hydrazinobenzene hydrochloride salt test limit concentration be 11.988 ng/ml.Will be to sulfoamido phenylhydrazine salt
Hydrochlorate is diluted to above-mentioned concentration sample introduction, and rsd value is less than 10% for 8.58%, rsd value.
Conclusion: to sulfoamido hydrazinobenzene hydrochloride salt quantitative limit concentration be 35.963ng/ml.Will be to sulfoamido phenyl hydrazine hydrochloride
Salt is diluted to above-mentioned concentration sample introduction, and rsd value is less than 5% for 4.75%, rsd value.
Experimental example 10 replica test.
By detection method, to same batch sample (lot number 20110712), 5 parts are measured, and try to achieve relative standard deviation, examination
Test result and show that this method repeatability is good, the results are shown in Table 5
Table 5 replica test
Experimental example 11 recovery test.
Weigh weight respectively and be about 9 parts of the Celecoxib sample (lot number 20110712) of 1.5g in 10ml measuring bottle, be divided into 3
Group, every group 3 parts.Dilute to sulfoamido hydrazinobenzene hydrochloride salt methanol solution with containing 300ng/ml, 400ng/ml, 500ng/ml respectively
Release to scale, be obtained and be equivalent to the average recovery solution containing gene poison impurity 60%, 80%, 100% limit level, will prepare
Good above-mentioned solution presses the method for text and above-mentioned chromatographic condition measures.It is calculated as follows the response rate.
Response rate computing formula
Test shows that the accuracy of this method is good, the results are shown in Table 6.
Table 6 is to sulfoamido hydrazinobenzene hydrochloride salt recovery test
Experimental example 12 stability of solution
Take standard solution sample introduction, record chromatogram to 80min, the complete appearance of above each solvent.Respectively at 0h, 4h, 6h,
2d, 4d, 6d sample introduction, calculates the peak area of each solvent of each sample introduction, tries to achieve relative standard deviation, should be not more than 5.0%, data is shown in
Table 7.
Table 7 stability of solution is investigated
Conclusion: test shows that reference substance solution is stable in 6 days.
Experimental example 13 liquid phase chromatogram condition ruggedness, the impact to separating degree for the change in flow.
Original flow velocity is 0.8ml/min, and change in flow is 0.78ml/min, 0.82ml/min.Take containing to sulfonamide
The mixed solution of base hydrazinobenzene hydrochloride salt 500ng/ml and Celecoxib 150mg/ml difference sample introduction, test result after system stability
See Fig. 5.
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect it is seen that flow velocity is in 0.78ml/min
In the range of～0.82ml/min, the change of chromatographic condition does not affect on separating on sulfoamido hydrazinobenzene hydrochloride salt.
Experimental example 14 liquid phase chromatogram condition ruggedness, the impact to separating degree for the mobile phase ratio change.
Original mobile phase a:10mmol/l ammonium acetate solution-methanol=50:50, mobile phase a is 10mmol/l ammonium acetate
Aqueous solution-methanol: 45:55,10mmol/l ammonium acetate solution-methanol 55:45.Take containing to sulfoamido hydrazinobenzene hydrochloride salt
The mixed solution of 500ng/ml and Celecoxib 150mg/ml difference sample introduction after system stability, test result is shown in Fig. 6.
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect it is seen that mobile phase a ratio is 45:
In 55～55:45 allowed band, change does not affect on separating on sulfoamido hydrazinobenzene hydrochloride salt sample.
Experimental example 15 liquid phase chromatogram condition ruggedness, the impact to separating degree for the sample size change.
Original sample size 20 μ l, sample size is become and turns to 15 μ l, 25 μ l.Take containing to sulfoamido hydrazinobenzene hydrochloride salt
The mixed solution of 500ng/ml and Celecoxib 150mg/ml difference sample introduction after system stability, test result is shown in Fig. 7.
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect it is seen that sample size is in 15 μ l～25
In μ l allowed band, change does not affect on separating on sulfoamido hydrazinobenzene hydrochloride salt.
Experimental example 16 liquid phase chromatogram condition ruggedness, the impact to separating degree for the Detection wavelength change.
Original Detection wavelength: 220nm, Detection wavelength is become and turns to, 218nm, 222nm.Take containing to sulfoamido benzene
The mixed solution of hydrazine hydrochloride 500ng/ml and Celecoxib 150mg/ml difference sample introduction after system stability, test result is shown in figure
8.
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect it is seen that Detection wavelength is in 218nm
In～222nm allowed band, change does not affect on separating on sulfoamido hydrazinobenzene hydrochloride salt.
Experimental example 17 liquid phase chromatogram condition ruggedness, the impact to separating degree for the column temperature change.
Original column temperature: 35 DEG C, column temperature is become and turns to 30 DEG C, 40 DEG C.Take containing to sulfoamido hydrazinobenzene hydrochloride salt 500ng/
The mixed solution of ml and Celecoxib 150mg/ml after system stability respectively sample introduction test result see Fig. 9.
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect it is seen that column temperature is at 30 DEG C～40 DEG C
In allowed band, change does not affect on separating on sulfoamido hydrazinobenzene hydrochloride salt.
In above-described embodiment, sulfoamido hydrazinobenzene hydrochloride salt reference substance is purchased from Japanese tokyo chemical
industry co ；ltd.
Claims (4)
1. the method to sulfoamido phenyl hydrazine hydrochloride salt content in high-performance liquid chromatogram determination Celecoxib crude drug, its feature exists
In it comprises the steps:
(1) preparation of reference substance solution: weigh to sulfoamido hydrazinobenzene hydrochloride salt reference substance, plus methanol, make every 1ml and contain
500ng to sulfoamido phenyl hydrazine hydrochloride saline solution；
(2) preparation of need testing solution: take Celecoxib crude drug to add methanol and make the solution that every 1ml contains 150mg Celecoxib；
(3) preparation of blank solution: methanol；
(4) measure: the chromatographic column of high performance liquid chromatograph is thermo hypercarb, mobile phase a is 10mmol/l ammonium acetate
Aqueous solution-methanol=45:55～55:45, mobile phase b is methanol, and column temperature is 30 DEG C～40 DEG C, and mobile phase a initial flow rate is 0.78
～0.82ml/min, Detection wavelength is 218nm～222nm, draws reference substance solution and each 15～25 l of need testing solution respectively,
Injection high performance liquid chromatograph, reads data；
(5) value of reference substance solution concentration and the equation of linear regression of corresponding peak area value are calculated, correlation coefficient should be not less than
0.99, reference substance solution peak shape is symmetrical, and theoretical cam curve more than 2000, if any to sulfoamido benzene in need testing solution chromatogram
Hydrazine hydrochloride, should be consistent to sulfoamido hydrazinobenzene hydrochloride salt retention time with reference substance solution, blank solution chromatogram, no with
Sulfoamido hydrazinobenzene hydrochloride salt reference substance identical peak is occurred, that is, blank solution is noiseless；
(6) method to sulfoamido phenyl hydrazine hydrochloride salt content, described stream in high-performance liquid chromatogram determination Celecoxib crude drug
The operation ratio change procedure of dynamic phase a and mobile phase b is shown in Table 1 gradient table；
(7) 3.75 g be must not exceed to sulfoamido hydrazinobenzene hydrochloride salt, to sulfoamido phenylhydrazine in every gram of Celecoxib crude drug
Hydrochloride concentration scope is 99.897 ~ 599.382ng/ml.
2. to sulfoamido phenyl hydrazine hydrochloride salt content in the high-performance liquid chromatogram determination Celecoxib crude drug described in claim 1
Method it is characterised in that it comprises the steps:
(1) preparation of reference substance solution: weigh to sulfoamido hydrazinobenzene hydrochloride salt reference substance, plus methanol, make every 1ml and contain
500ng to sulfoamido phenyl hydrazine hydrochloride saline solution；
(2) preparation of need testing solution: take Celecoxib crude drug to add methanol and make the solution that every 1ml contains 150mg Celecoxib；
(3) preparation of blank solution: methanol；
(4) measure: the chromatographic column of high performance liquid chromatograph is thermo hypercarb, mobile phase a is 10mmol/l ammonium acetate
Aqueous solution-methanol=50:50, mobile phase b is methanol, and column temperature is 35 DEG C, and mobile phase a initial flow rate is 0.80ml/min, detects ripple
A length of 220nm, draws reference substance solution and each 20 l of need testing solution respectively, injects high performance liquid chromatograph, reads data；
(5) value of reference substance solution concentration and the equation of linear regression of corresponding peak area value are calculated, correlation coefficient should be not less than
0.99, reference substance solution peak shape is symmetrical, and theoretical cam curve more than 2000, if any to sulfoamido benzene in need testing solution chromatogram
Hydrazine hydrochloride, should be consistent to sulfoamido hydrazinobenzene hydrochloride salt retention time with reference substance solution, blank solution chromatogram, no with
Sulfoamido hydrazinobenzene hydrochloride salt reference substance identical peak is occurred, that is, blank solution is noiseless；
(6) method to sulfoamido phenyl hydrazine hydrochloride salt content, described stream in high-performance liquid chromatogram determination Celecoxib crude drug
The operation ratio change procedure of dynamic phase a and mobile phase b is shown in Table 1 gradient table；
(7) 3.75 g be must not exceed to sulfoamido hydrazinobenzene hydrochloride salt, to sulfoamido phenylhydrazine in every gram of Celecoxib crude drug
Hydrochloride concentration scope is 99.897 ~ 599.382ng/ml.
3. to sulfoamido phenyl hydrazine hydrochloride salt content in the high-performance liquid chromatogram determination Celecoxib crude drug described in claim 1
Method it is characterised in that it comprises the steps:
(1) preparation of reference substance solution: weigh to sulfoamido hydrazinobenzene hydrochloride salt reference substance, plus methanol, make every 1ml and contain
500ng to sulfoamido phenyl hydrazine hydrochloride saline solution；
(2) preparation of need testing solution: take Celecoxib crude drug to add methanol and make the solution that every 1ml contains 150mg Celecoxib；
(3) preparation of blank solution: methanol；
(4) measure: the chromatographic column of high performance liquid chromatograph is thermo hypercarb, mobile phase a is 10mmol/l ammonium acetate
Aqueous solution-methanol=45:55, mobile phase b is methanol, and column temperature is 30 DEG C, and mobile phase a initial flow rate is 0.78ml/min, detects ripple
A length of 218nm, draws reference substance solution and each 15 l of need testing solution respectively, injects high performance liquid chromatograph, reads data；
(5) value of reference substance solution concentration and the equation of linear regression of corresponding peak area value are calculated, correlation coefficient should be not less than
0.99, reference substance solution peak shape is symmetrical, and theoretical cam curve more than 2000, if any to sulfoamido benzene in need testing solution chromatogram
Hydrazine hydrochloride, should be consistent to sulfoamido hydrazinobenzene hydrochloride salt retention time with reference substance solution, blank solution chromatogram, no with
Sulfoamido hydrazinobenzene hydrochloride salt reference substance identical peak is occurred, that is, blank solution is noiseless；
(6) method to sulfoamido phenyl hydrazine hydrochloride salt content, described stream in high-performance liquid chromatogram determination Celecoxib crude drug
The operation ratio change procedure of dynamic phase a and mobile phase b is shown in Table 1 gradient table；
(7) 3.75 g be must not exceed to sulfoamido hydrazinobenzene hydrochloride salt, to sulfoamido phenylhydrazine in every gram of Celecoxib crude drug
Hydrochloride concentration scope is 99.897 ~ 599.382ng/ml.
4. to sulfoamido phenyl hydrazine hydrochloride salt content in the high-performance liquid chromatogram determination Celecoxib crude drug described in claim 1
Method it is characterised in that it comprises the steps:
(1) preparation of reference substance solution: weigh to sulfoamido hydrazinobenzene hydrochloride salt reference substance, plus methanol, make every 1ml and contain
500ng to sulfoamido phenyl hydrazine hydrochloride saline solution；
(2) preparation of need testing solution: take Celecoxib crude drug to add methanol and make the solution that every 1ml contains 150mg Celecoxib；
(3) preparation of blank solution: methanol；
(4) measure: the chromatographic column of high performance liquid chromatograph is thermo hypercarb, mobile phase a is 10mmol/l ammonium acetate
Aqueous solution-methanol=55:45, mobile phase b is methanol, and column temperature is 40 DEG C, and mobile phase a initial flow rate is 0.82ml/min, detects ripple
A length of 222nm, draws reference substance solution and each 25 l of need testing solution respectively, injects high performance liquid chromatograph, reads data；
(5) value of reference substance solution concentration and the equation of linear regression of corresponding peak area value are calculated, correlation coefficient should be not less than
0.99, reference substance solution peak shape is symmetrical, and theoretical cam curve more than 2000, if any to sulfoamido benzene in need testing solution chromatogram
Hydrazine hydrochloride, should be consistent to sulfoamido hydrazinobenzene hydrochloride salt retention time with reference substance solution, blank solution chromatogram, no with
Sulfoamido hydrazinobenzene hydrochloride salt reference substance identical peak is occurred, that is, blank solution is noiseless；
(6) method to sulfoamido phenyl hydrazine hydrochloride salt content, described stream in high-performance liquid chromatogram determination Celecoxib crude drug
The operation ratio change procedure of dynamic phase a and mobile phase b is shown in Table 1 gradient table；
(7) 3.75 g be must not exceed to sulfoamido phenylhydrazine, to sulfoamido hydrazinobenzene hydrochloride salt in every gram of Celecoxib crude drug
Concentration range is 99.897 ~ 599.382ng/ml.
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