CN106198778B - It is a kind of while measuring Gefitinib and its method in relation to substance - Google Patents
It is a kind of while measuring Gefitinib and its method in relation to substance Download PDFInfo
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- CN106198778B CN106198778B CN201610475130.XA CN201610475130A CN106198778B CN 106198778 B CN106198778 B CN 106198778B CN 201610475130 A CN201610475130 A CN 201610475130A CN 106198778 B CN106198778 B CN 106198778B
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Abstract
The present invention provides a kind of while measuring Gefitinib and its analyzing detecting method in relation to substance, by high performance liquid chromatograph, with Inert Sustain C18For chromatographic column, mobile phase A：0.5% triethylamine solution/acetonitrile/methanol volume ratio is 90:5:5, Mobile phase B：0.5% triethylamine solution/acetonitrile/methanol volume ratio is 10:85:5, gradient elution, Detection wavelength be 240~260nm, 40~60 DEG C of column temperature.Analysis method specificity provided by the invention is good, separating degree >=3.57 of each absorption peak, and detection method repeatability is good, RSD≤1.18%, high sensitivity, and the detection of each impurity is limited to 0.05ng/mL, and accuracy and durability are good.
Description
Technical field
The invention belongs to Pharmaceutical Analysis fields, use high efficiency liquid chromatography for separating and determining Gefitinib more particularly to a kind of
And its method in relation to substance.
Background technique
Gefitinib is for treatment of solid tumors by first of Astrazeneca AB's exploitation for EGFR tyrosine-kinase
The small molecule tyrosine kinase inhibitors of enzyme are listed in Japan for 2002 for the first time, treat that other chemotherapy are invalid or unsuitable chemotherapy
Locally Advanced or recurrence non-small cell lung cancer, be approved again in the U.S. and Australia single as three lines in May, 2003
Therapeutic agent is used for advanced Non-small cell lung, it is the first small molecule junket for specific target spot for being used for treatment of solid tumors
Histidine kinase inhibitor.In Discussion on Chinese Listed, previously received chemotherapy suitable for treating or be unsuitable for chemotherapy within 2 months 2005
Locally Advanced or Metastatic Nsclc, structural formula are：
For Gefitinib due to complex process, impurity is varied, and different process routes introduces different impurity, therefore
Suitable detection method must be selected to Gefitinib impurity analysis, synthesis process is monitored, guarantee product quality.
Starting material, intermediate, condensate, the side reaction product mainly brought into process of production in relation to substance, with
And catabolite in storage etc..Based on the considerations of safety and production actual conditions, allow to contain a threshold quantity
Harmless or hypotoxicity related substance, but to being more toxic, it is detrimental to health, related object that is invalid or influencing medicine stability
Matter must be strictly controlled.Therefore, the detection in relation to substance is the key index for controlling drug quality.
CN103755648A discloses the synthetic route of Gefitinib and its new impurity, is broadly divided into condensation and Dimroth
Two steps are reset, my company uses the patent route to prepare Gefitinib bulk pharmaceutical chemicals, and carrying out Feed Discovery through analysing impurity mainly has 8
Kind impurity, it is specific as follows：
《Pharmacopoeia of India》Disclose the content assaying method of principal component in Gefitinib drug, chromatographic column：C18Alkyl silica gel
Column, mobile phase 1%w/v ammonium acetate solution and acetonitrile, volume ratio 40:60, Detection wavelength 254nm, but this method is not to specific
Related substance is studied, therefore when detecting in our techniques Gefitinib and its 8 kinds of impurity with this method, main peak and impurity point
From poor.
《Analysis test journal》Reversed phase liquid chromatography measures containing for Gefitinib in bulk pharmaceutical chemicals and its related substance simultaneously
Amount, volume 2016,35, the 1st phase, p35-41 discloses a kind of test Gefitinib and its method in relation to substance, with acetonitrile-
0.15% ammonium acetate buffer solution (pH8.5) is mobile phase, Detection wavelength 255nm.Due to impurity difference, this method is simultaneously not suitable for
In the related substance for detecting Gefitinib in our techniques.
Due to the difference of synthesis technology, the type and content of Gefitinib relative substance is caused to differ greatly, it is therefore necessary to
Corresponding quality control standard is established to monitor synthesis process, guarantee product quality.
Summary of the invention
To overcome prior art defect, chromatographic isolation behavior of the present invention to Gefitinib and its in relation to substance is ground
Study carefully, by being screened to many conditions such as Detection wavelength, mobile phase selection, mobile phase ratio, buffer salinity, pH, it is determined that a kind of
Gefitinib and its analysis method in relation to substance can be measured simultaneously, this method can be used for Gefitinib bulk pharmaceutical chemicals and its related
The detection and analysis of substance.
The present invention provides a kind of new detection method, this method can detect Gefitinib and its 8 kinds of related objects simultaneously
Matter, specificity is good, realizes efficiently separating for impurity, separating degree >=3.57 of each absorption peak, detection side using condition of gradient elution
Method repeatability is good, RSD≤1.18%, high sensitivity, and the detection of each impurity is limited to 0.05ng/mL, and accuracy and durability are good.
Gefitinib is measured and its in relation to the method for substance simultaneously it is an object of that present invention to provide a kind of：
Detecting instrument：High performance liquid chromatograph；
Chromatographic column：Inert Sustain C18, 100mm × 3mm, 3 μm；
Mobile phase presses following gradient：
| Time (min) | Mobile phase A (v/v) | Mobile phase B (v/v) |
| 0 | 80 | 20 |
| 30 | 60 | 40 |
| 45 | 40 | 60 |
| 50 | 0 | 100 |
| 52 | 0 | 100 |
Detector：UV detector, 240~260nm of Detection wavelength；
Flow velocity：0.4mL/min~1.0mL/min；
Column temperature：40~60 DEG C；
Sample volume：The 5 μ L of μ L~20, wherein the mobile phase is molten for the mixing of 0.5% triethylamine solution, acetonitrile and methanol
Liquid, 0.5% triethylamine solution/acetonitrile/methanol volume ratio is 90 in mobile phase A:5:5,0.5% triethylamine is molten in Mobile phase B
Liquid/acetonitrile/methanol volume ratio is 10:85:5.
The preferred solution of the invention, the pH of the mobile phase are 6~8, and the pH value of more preferable mobile phase is 7.0.
The preferred solution of the invention, the Detection wavelength are 248~252nm, and more preferable Detection wavelength is 250nm.
The preferred solution of the invention, the flow velocity are 0.4~0.6mL/min, and more preferable flow velocity is 0.5mL/min.
The preferred solution of the invention, the column temperature are 45~55 DEG C, and more preferable column temperature is 50 DEG C.
The preferred solution of the invention, the sample volume are 5 μ L or 10 μ L, and more preferable sample volume is 5 μ L.
The preferred solution of the invention, the related substance are selected from following impurity：
In some embodiments, method defects inspecting provided by the invention is limited to 0.05ng/mL.
In some embodiments, it be volume ratio is 1 that the present invention, which prepares diluent used in reference substance or test solution,:1
0.2% trifluoroacetic acid/methanol mixed solution.
Method of the invention compared with prior art, has the advantages that：
(1) method of the invention can under the same high-efficient liquid phase chromatogram condition with when separation Gefitinib and its 8 kinds it is miscellaneous
Matter avoids in the detection frequently replacement liquid-phase condition, improves working efficiency, is suitble to the requirement of industry mass production；
(2) method instrument equipment of the invention and reagent are conventional articles, and experiment parameter is without harsh conditions, cost
It is low；
(3) detection method specificity of the invention is good, reaches between each impurity and Gefitinib peak and efficiently separates, each to inhale
Separating degree >=3.57 at peak are received, detection method repeatability of the invention is good, RSD≤1.18%；
(4) detection method high sensitivity, the detection of each impurity are limited to 0.05ng/mL, and impurity is in 0.1~2 μ g/mL
Peak area and concentration linear relationship are good in range, and especially impurity I is linear good within the scope of 0.1019~2.038 μ g/mL,
Impurity II is linear good within the scope of 0.1001~2.002 μ g/mL, and impurity III is in 0.1051~2.102 μ g/mL range interior lines
Property it is good, impurity IV is linear good within the scope of 0.1002~2.004 μ g/mL, and impurity V is in 0.1031~2.062 μ g/mL range
Interior linear good, impurity VI is linear good within the scope of 0.1016~2.032 μ g/mL, and impurity VII is in 0.1008~2.016 μ g/
Linear good within the scope of mL, impurity VIII is linear good within the scope of 0.1021~2.042 μ g/mL；
(5) in 86.61%~103.52% range, rate of recovery RSD value exists the average recovery rate of the method for the present invention
Within 4.76%, the accuracy of this method is good；
(6) the method for the present invention durability is good, flow velocity 0.4mL/min~0.6mL/min, flowing phase pH value 6.0~
8.0, column temperature changes between 248nm~252nm in chromatographic column, the Detection wavelength of 45 DEG C~55 DEG C, the different lot numbers of selection, to Ji
The non-detection for Buddhist nun and its impurity is without significant change.
" v/v " refers to volume ratio；
" about " refer in the present invention within ± the 10% of the numerical value；
" 0.5% triethylamine solution " refers to the percent by volume of triethylamine solution, i.e., contains 0.5mL in 100mL solution
Triethylamine.
" 0.2% trifluoroacetic acid " refers to the percent by volume of trifluoroacetic acid solution, i.e., contains 0.2mL in 100mL solution
Trifluoroacetic acid.
Detailed description of the invention
Chromatogram of 1 blank solution of attached drawing under 250nm Detection wavelength
Chromatogram of the 2 impurity I reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 3 impurity II reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 4 impurity III reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 5 impurity IV reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 6 impurity V reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 7 impurity VI reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 8 impurity VII reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 9 impurity VIII reference substance of attached drawing under 250nm Detection wavelength
Chromatogram of the 10 test sample mixed solution of attached drawing under 250nm Detection wavelength
11 comparative example of attached drawing, 1 method chromatogram
12 comparative example of attached drawing, 2 method chromatogram
13 impurity I peak area of attached drawing is to actual concentrations linear regression curves map
14 impurity II peak area of attached drawing is to actual concentrations linear regression curves map
15 impurity III peak area of attached drawing is to actual concentrations linear regression curves map
16 impurity IV peak area of attached drawing is to actual concentrations linear regression curves map
17 impurity V peak area of attached drawing is to actual concentrations linear regression curves map
18 impurity VI peak area of attached drawing is to actual concentrations linear regression curves map
19 impurity VII peak area of attached drawing is to actual concentrations linear regression curves map
20 impurity VIII peak area of attached drawing is to actual concentrations linear regression curves map
21 Gefitinib peak area of attached drawing is to actual concentrations linear regression curves map
Specific embodiment
The present invention is explained further and illustrated below by embodiment.The embodiment of the present invention is only for illustrating this hair
It is bright, rather than limiting the invention, therefore it is equal to simple modifications of the invention or replacement on the basis of method of the invention
Belong to the scope of protection of present invention.
Detection is synthesized with reference to CN103755648A method by laboratory with Gefitinib in following embodiment, remaining impurity
It is synthesized by laboratory, corresponding lot number is as follows：
Gefitinib bulk pharmaceutical chemicals lot number 20120201
Gefitinib reference substance lot number 20111101
Impurity I reference substance lot number 20111009
Impurity II reference substance lot number 20111011
Impurity III reference substance lot number 20111019
Impurity IV reference substance lot number 20111102
Impurity V reference substance lot number 20111107
Impurity VI reference substance lot number 20111116
Impurity VII reference substance lot number 20111125
Impurity VIII reference substance lot number 20111128
1 chromatography condition of embodiment
1, the determination of Detection wavelength
The preparation of diluent：0.2% trifluoroacetic acid solution 500mL is measured, methanol 500mL is stirred and evenly mixed, and what is obtained is molten
Liquid is also blank solution, with ultraviolet specrophotometer measurement and Gefitinib reference substance and its related substance in 200~400nm wave
The absorbing wavelength of long range, as a result, it has been found that Gefitinib reference substance has larger absorption under 218nm, 250nm and 340nm wavelength,
But impurity II and impurity VII have larger absorption at 250nm~260nm, comprehensively considering determining wavelength is 250nm.
2, mobile phase solvent selects
Use flow visualizing for acetonitrile and ammonium acetate buffer solution in the prior art, Gefitinib and impurity separating effect
It is poor.The present invention replaces flow visualizing, selects the mixed solution of acetonitrile and methanol as organic phase solvent, impurity VIII and impurity
I separation reaches baseline requirement, and since peak shape is poor, hangover is serious, therefore triethylamine is added and makees tailingsuppressing reagent.But remaining impurity is not
It separates, still needs to advanced optimize mobile phase solvent ratio and elution program.
In analysis field, gradient elution program can play the separating degree for optimizing component to be measured, increase eluotropic strength etc. and make
With, since mobile phase composition is to change over time and carry out component rate of change, the selection of mobile phase after mobile phase composition variation
Property etc. can also change therewith, generated chromatographic isolation effect is unpredictable, need test of many times trial could obtain.
2.1 triethylamine concentration
With mobile phase A：Mobile phase B volume ratio 40:60 isocratic elutions, to triethylamine concentration (1%, 8%, 5% and 3%) into
Row screening, the results are shown in Table 1
The selection of 1 triethylamine concentration of table
As the result is shown：The concentration of triethylamine is higher, and the separating degree of impurity III and impurity II are higher, and separating effect is better, but
It is excessively high to allow for triethylamine concentration, alkalinity is consequently increased, and column performance and service life will receive influence.Work as triethylamine concentration
The separating degree of impurity III and impurity II are greater than 1.5 when being 0.5%, meet the requirements, therefore select 0.5% triethylamine/acetonitrile/first
Alcohol carries out chromatographic isolation, but the separating degree of impurity IV is less than 1.5, still need to adjustment mobile phase composition to impurity II and impurity IV make into
The separation of one step.
2.2 mobile phase As and Mobile phase B component selection
Using 0.5% triethylamine/acetonitrile/methanol as eluant, eluent, mobile phase A：Mobile phase B volume ratio 40:60 isocratic elutions, it is right
The ratio of each component is screened in Mobile phase B, and the results are shown in Table 2.
The selection of 2 Mobile phase B each component ratio of table
As the result is shown：As the ratio of acetonitrile in Mobile phase B reduces, impurity II and impurity IV separating degree are gradually mentioned
Height, impurity V appearance time also postpone.When 0.5% triethylamine/acetonitrile/methanol volume ratio is 5 in Mobile phase B:90:Impurity IV when 5
Separating degree is undesirable less than 1.5；When 0.5% triethylamine/acetonitrile/methanol volume ratio is 15 in Mobile phase B：80：5 and/or
10：75：5, for impurity V appearance after 47min, operation program is longer；When 0.5% triethylamine/acetonitrile/methanol body in Mobile phase B
Product is than being 10:85:When 5, impurity II and impurity IV separating degree are greater than 1.5, and impurity V appearance time is in 29min or so.Comprehensively consider
Separating degree and appearance time, 0.5% triethylamine/acetonitrile/methanol volume ratio of selective flow phase A are 90:5:5, Mobile phase B
0.5% triethylamine/acetonitrile/methanol volume ratio is 10:85:5, but the separating degree of condition impurity IV is only 1.69, still need into
One step reaches better separating effect to the optimization of mobile phase gradient.
The selection of 2.3 gradients
0.5% triethylamine/acetonitrile/methanol volume ratio is 90 in mobile phase A:5:5,0.5% triethylamine/second in Mobile phase B
Nitrile/methanol volume ratio is 10:85:5, best gradient elution program is selected, the results are shown in Table 3.
The selection of 3 condition of gradient elution of table
As can be seen from the above table, condition a, b, c, d and e, impurity IV separating degree are all larger than 1.5 and meet the requirements, and with stream
The increase of dynamic phase B, i.e., the increase of organic Phase Proportion, impurity appearance time are more and more early.But in condition a, b and c, impurity V's
After 40min, program is run too long appearance time；With the increase of Mobile phase B ratio, in condition e, when impurity V appearance
Although 28min or so is advanceed between, impurity separating effect has been deteriorated, and the peak impurity VI and VII partly overlaps；And condition
In d, for all impurity separating degrees 3.57 or more, impurity separating degree is preferable, and all impurity appearance times reach before 35min
To a balance of impurity separating degree and appearance time, as optimal selection, it is thus determined that condition d is as final gradient journey
Sequence.
Below by system suitability experiment, degradation experiment, detection limit, range of linearity investigation, repeated experiment, recycling
The above method is verified in the experiment such as rate, tolerance.
The experiment of 2 system suitability of embodiment
Chromatographic condition：Shimadzu LC-20A wears peace U3000 type highly effective liquid phase chromatographic system and work station；Automatic sampling；With
Inert Sustain C18(100mm × 3mm, 3 μm) is splitter；Ultraviolet detection wavelength：250nm；Mobile phase A：0.5% 3 second
Amine aqueous solution/acetonitrile/methanol volume ratio is 90:5:5, Mobile phase B：0.5% triethylamine solution/acetonitrile/methanol volume ratio is 10:
85:5,50 DEG C of column temperature, sampling volume 5 μ L, flow velocity 0.5mL/min；
Gradient program
| Time (min) | Mobile phase A (v/v) | Mobile phase B (v/v) |
| 0 | 80 | 20 |
| 30 | 60 | 40 |
| 45 | 40 | 60 |
| 50 | 0 | 100 |
| 52 | 0 | 100 |
| 52.1 | 80 | 20 |
| 60 | 80 | 20 |
Take Gefitinib impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII and impurity VIII
Reference substance uses diluent (0.2% trifluoroacetic acid/methanol, volume ratio 1 respectively:1) it dissolves and quantifies dilution and every 1mL is made containing 10
The solution of μ g positions solution as each impurity；
Each impurity positioning solution of accurate measurement is appropriate respectively, Gefitinib reference substance is appropriate, and diluent is added quantitatively to dilute system
At every 1mL 1mg containing Gefitinib, impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII and impurity
The poly-doped impurity reference substance solution of each 1 μ g of VIII reference substance tests impurity and separates situation as test sample mixed solution.
It is fixed that precision measures impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII and impurity VIII
Position solution and each 5 μ L of test sample mixed solution are injected separately into liquid chromatography instrument, record chromatogram.Blank solution map is shown in Fig. 1, fixed
The map of position solution is shown in that Fig. 2~Fig. 9, the map of test solution are shown in Figure 10, and this method separates the related substance of Gefitinib
Situation is shown in Table 4.
The separation situation of 4 Gefitinib of table and each impurity
| Title | Retention time/min | Concentration (μ g/mL) | Separating degree |
| Gefitinib | 29.047 | 1008 | 15.10 |
| Impurity I | 4.554 | 1.002 | 14.47 |
| Impurity II | 9.893 | 1.005 | 3.57 |
| Impurity III | 8.697 | 1.007 | 16.69 |
| Impurity IV | 12.404 | 1.002 | 6.99 |
| Impurity V | 32.044 | 1.006 | 5.98 |
| Impurity VI | 17.887 | 1.004 | 12.80 |
| Impurity VII | 21.997 | 1.003 | 8.52 |
| Impurity VIII | 2.152 | 1.006 | / |
Conclusion：Reach between each impurity and Gefitinib peak and efficiently separate, separating degree >=3.57, peak shape is preferable, blank
Solvent is noiseless, illustrates that this method is suitable for measurement Gefitinib and its related substance.
Comparative example 1
According to《Pharmacopoeia of India》Detection method, chromatographic condition：C18Alkyl silicon rubber column gel column, column temperature：60 DEG C, mobile phase 1%w/v vinegar
Acid ammonium solution and acetonitrile, volume ratio 40:60, Detection wavelength 254nm take the test sample mixed solution in 5 μ L embodiments 2 to be examined
It surveys, records chromatogram, be specifically shown in attached drawing 11.
Comparative example 2
According to《Analysis test journal》Reversed phase liquid chromatography measures Gefitinib and its related substance in bulk pharmaceutical chemicals simultaneously
Content, volume 2016,35, the 1st phase, the disclosed detection Gefitinib of p35-41 and its method in relation to substance, with acetonitrile-
0.15% ammonium acetate buffer solution is mobile phase, column temperature：40 DEG C of Detection wavelength 255nm take the test sample in 5 μ L embodiments 2 mixed
It closes solution to be detected, records chromatogram, be specifically shown in attached drawing 12.
Interpretation of result：It is miscellaneous from Figure 11 and 12 it can be seen that the detection method appearance time of comparative example 1 and comparative example 2 is too early
Matter separation is poor, and impurity detected level is few, and this method cannot efficiently separate the Gefitinib and its correlation of our technique generations of detection
Impurity.
The experiment of 3 forced degradation of embodiment
Forced degradation experiment is under the conditions of intensity is biggish, with the side such as strong illumination, high temperature, acid and alkali hydrolysis and oxidation
Method accelerates destruction to Gefitinib sample, it is therefore an objective to by the catabolite and main peak and known impurities of investigating sample
Situation is separated, whether material balances, the efficiency and applicability of analysis and assessment method.
Non- breaking test：Gefitinib bulk pharmaceutical chemicals about 10mg is taken, it is accurately weighed, it sets in 10mL measuring bottle, diluent is added to dissolve
And it is diluted to scale, it shakes up, filters, take subsequent filtrate.
Sour breaking test：Gefitinib bulk pharmaceutical chemicals about 10mg is taken, it is accurately weighed, it sets in 10mL measuring bottle, 1mol/L salt is added
Acid solution 1mL, 60 DEG C of heating 4h of water-bath, be added equivalent 1mol/L sodium hydroxide solution neutralize, then plus diluent be diluted to quarter
Degree, shakes up, and filters, takes subsequent filtrate.
Alkali breaking test：Gefitinib bulk pharmaceutical chemicals about 10mg is taken, it is accurately weighed, it sets in 10mL measuring bottle, 1mol/L hydrogen is added
Sodium hydroxide solution 1mL, 60 DEG C of heating 4h of water-bath, be added equivalent 1mol/L hydrochloric acid solution neutralize, then plus diluent be diluted to quarter
Degree, shakes up, and filters, takes subsequent filtrate.
Strong photo damage experiment：It is taken at the Gefitinib bulk pharmaceutical chemicals about 10mg that 48h is irradiated under the strong light of 4500 ± 500Lx, it is accurate
It is weighed, it sets in 10mL measuring bottle, adds diluent to dissolve and be diluted to scale, shake up, filter, take subsequent filtrate.
High temperature experiment：Gefitinib bulk pharmaceutical chemicals about 10mg is taken, it is accurately weighed, it sets in 10mL measuring bottle, heating water bath 6h,
It lets cool, diluent is added to be diluted to scale, shake up, filter, take subsequent filtrate.
Oxidative demage experiment：Gefitinib bulk pharmaceutical chemicals about 10mg is taken, it is accurately weighed, it sets in 10ml measuring bottle, is added 30% pair
Oxygen water 1mL adds diluent to be diluted to scale, shakes up after placing 4h, filters, takes subsequent filtrate.
Precision measures above-mentioned subsequent filtrate and each 5 μ L of blank solution, is injected separately into liquid chromatograph, the results are shown in Table 5 and table 6.
Table 5 is harsh to destroy degradation impurity spectrum analysis
6 forced degradation of table tests material balance result
Conclusion：Gefitinib bulk pharmaceutical chemicals are more stable under the conditions of strong acid, highly basic, high temperature, illumination failure test, and impurity contains
Amount is without significant change.Unstable under oxidative conditions, selected chromatographic condition can efficiently separate each degradation impurity with main peak；This product
Failure test material is in a basic balance, and the impurity that this product generates can be detected under the chromatographic condition, separating degree is good, and this method is special
Attribute is good.
The detection limit of embodiment 4
The impurity reference substance stock solution of known concentration is diluted to low concentration, the signal of the signal and blank solvent measured
(baseline noise) is compared, and calculates the amount that can be reliably detected.With signal-to-noise ratio about 10:1,3:1 to calculate separately Gefitinib each
The quantitative limit of impurity and detection limit, and the results are shown in Table 7.
Quantitative limit, the detection limit result of 7 Gefitinib impurity of table
As can be seen from Table 7, detection method high sensitivity of the invention, the detection of impurity are limited to 0.05ng/mL.
5 range of linearity of embodiment is investigated
To Gefitinib and its related substance, in quantitative limit to taking 9 concentration in the range of being not less than 150% index concentration
Point is studied.Peak area of the linear relationship to measure carries out line with least square method to the function construction of analyte concentration
Property return, 5~Figure 21 of the result is shown in Figure 1 and 8~table of table 16.
8 impurity I linear determination result of table
9 impurity II linear determination result of table
10 impurity III linear determination result of table
11 impurity IV linear determination result of table
12 impurity V linear determination result of table
13 impurity VI linear determination result of table
14 impurity VII linear determination result of table
15 impurity VIII linear determination result of table
16 Gefitinib linear determination result of table
Conclusion：When analysis method provided by the invention detection Gefitinib and its related substance, detectable concentration about 0.1~
Have good linear relationship, especially impurity I linearly good within the scope of 0.1019~2.038 μ g/mL within the scope of 2 μ g/mL, it is miscellaneous
Matter II is linear good within the scope of 0.1001~2.002 μ g/mL, and impurity III is linear within the scope of 0.1051~2.102 μ g/mL
Well, impurity IV is linear good within the scope of 0.1002~2.004 μ g/mL, and impurity V is within the scope of 0.1031~2.062 μ g/mL
Linear good, impurity VI is linear good within the scope of 0.1016~2.032 μ g/mL, and impurity VII is in 0.1008~2.016 μ g/mL
Linear good in range, impurity VIII is linear good within the scope of 0.1021~2.042 μ g/mL, Gefitinib 0.02262~
It is linear good within the scope of 2.262 μ g/mL.
Test 6 repeated experiments
Repeatability is surveyed under the same conditions by preparing 6 parts of test solutions containing each limit of impurities concentration
Examination, calculates the relative standard deviation of each impurity content in 6 parts of test solutions.
Take Gefitinib impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII and impurity VIII
Each 10mg is set in 100mL measuring bottle, is added diluent shaking to make to dissolve and be diluted to scale, is shaken up, stores as impurity reference substance
Liquid；Precision measures 1mL, is placed in 100mL measuring bottle, diluent is added to be settled to scale, shake up, molten as poly-doped impurity reference substance
Liquid.
Gefitinib bulk pharmaceutical chemicals about 100mg is taken, it is accurately weighed, it is placed in 100mL measuring bottle, impurity reference substance stock solution is added
1mL is diluted to scale with diluent, shakes up, as test solution, 6 parts of configured in parallel.Precision measures above-mentioned each 5 μ L of solution,
Liquid chromatograph is injected, chromatogram is recorded, the results are shown in Table 17.
17 repeated experiment result of table
Conclusion：Relative standard deviation RSD≤1.18% of 6 measurement results of each impurity, shows that this method is reproducible.
7 rate of recovery of embodiment
Accuracy is each miscellaneous by the way that 80%, 100% and 120% 3 various concentration of standard limits is added in test sample
Obtained by the rate of recovery that matter measures.
Take Gefitinib impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII and impurity VIII
Each 10mg, it is accurately weighed, it sets in the measuring bottle of 100mL, adds diluent shaking to make to dissolve and be diluted to scale, shake up, as impurity
Reference substance stock solution, precision measure 1mL, are placed in 100mL measuring bottle, diluent is added to be settled to scale, shake up, as poly-doped impurity
Reference substance solution.
Gefitinib bulk pharmaceutical chemicals 10mg is taken, it is accurately weighed, it sets in 10mL measuring bottle, adds diluent shaking to make to dissolve, and dilute
It to scale, shakes up, as test solution.Gefitinib bulk pharmaceutical chemicals about 100mg is taken, it is accurately weighed, it is respectively placed in 100mL measuring bottle
In, it is accurate respectively to measure impurity reference substance stock solution 0.8mL, 1.0mL, 1.2mL, it is added separately in corresponding measuring bottle, adds dilution
Appropriate agent, shaking makes to dissolve, and is diluted to scale, shakes up, as 80%, 100%, 120% mark-on test solution, Mei Genong
3 parts of configured in parallel of degree.Precision measures above-mentioned each 5 μ L of solution, injects liquid chromatograph, records chromatogram, calculates returning for each impurity
Yield the results are shown in Table 18.
18 rate of recovery experimental result of table
Conclusion：The result shows that the average recovery rate of basic, normal, high level is in 86.61%~103.52% range, the rate of recovery
RSD value shows that the accuracy of this method is good within 4.76%.
The experiment of 8 durability of embodiment
To investigate method itself for the anti-interference ability of variable experimental factor, the durability of method is investigated.
Take Gefitinib, impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII and impurity VIII
Appropriate reference substance, it is accurately weighed, add diluent to dissolve and dilute and is made in every 1mL containing about Gefitinib 1mg and impurity I, impurity
The solution of II, impurity III, impurity IV, impurity V, impurity VI, impurity VII and each 1 μ g of impurity VIII, it is molten as mixing reference substance
Liquid.
Precision measures above-mentioned 5 μ L of solution, when investigating different in flow rate, pH value, column temperature, batch-wise chromatography column and Detection wavelength, is
System applicability solution in main peak retention time and separate situation with impurity.Variation condition：Flow velocity (1.0mL/min, 0.5mL/
Min, 1.2ml/min), mobile phase pH (6.0,7.0,8.0), column temperature (45 DEG C, 50 DEG C, 55 DEG C), different batches chromatographic column
(3AR41105,3CR41213,3CR41212), Detection wavelength (228nm, 226nm, 230nm), the results are shown in Table 19~table 23.
The test result different in flow rate of table 19
Conclusion：Flow velocity changes between 0.4mL/min~0.6mL/min, influences less, to show this method to measurement result
Good tolerance.
The test result of the different pH value mobile phases of table 20
Conclusion：Flowing phase pH value changes between 6.0~8.0, influences less, to show the durable of this method to measurement result
Property is good.
The test result of the different column temperatures of table 21
Conclusion：Column temperature changes between 45 DEG C~55 DEG C, influences less, to show that the durability of this method is good to measurement result
It is good.
The test result of the different lot number chromatographic columns of table 22
Conclusion：The chromatographic column for selecting different lot numbers influences measurement result less, to show the good tolerance of this method.
The test result of the different Detection wavelengths of table 23
Conclusion：Detection wavelength changes between 248nm~252nm, influences less, to show the resistance to of this method to measurement result
It is good with property.
Claims (10)
1. a kind of measure Gefitinib and its method in relation to substance simultaneously, it is characterized in that：
Instrument：High performance liquid chromatograph；
Chromatographic column：Inert Sustain C18Chromatographic column, 100mm × 3mm, 3 μm；
Mobile phase presses following gradient：
Detector：UV detector, 240~260nm of Detection wavelength；
Flow velocity：0.4mL/min~1.0mL/min；
Column temperature：40~60 DEG C；
Sample volume：The 5 μ L of μ L~20, wherein the mobile phase is the mixed solution of 0.5% triethylamine solution, acetonitrile and methanol,
0.5% triethylamine solution/acetonitrile/methanol volume ratio is 90 in mobile phase A:5:5,0.5% triethylamine solution in Mobile phase B/
The volume ratio of acetonitrile/methanol is 10:85:5.
2. according to the method described in claim 1, wherein the pH of the mobile phase is 6~8.
3. according to the method described in claim 1, wherein the Detection wavelength is 248~252nm.
4. according to the method described in claim 3, wherein the Detection wavelength is 250nm.
5. according to the method described in claim 1, wherein the flow velocity is 0.4~0.6mL/min.
6. according to the method described in claim 1, wherein the column temperature is 45~55 DEG C.
7. according to the method described in claim 1, wherein the sample volume is 5 μ L.
8. according to the method described in claim 1, wherein the related substance is selected from following impurity：
9. method according to any one of claims 1 to 7, which is characterized in that the detection of impurity is limited to 0.05ng/mL.
10. method according to any one of claims 1 to 7, which is characterized in that prepare reference substance or test solution institute
Diluent is that volume ratio is 1:1 0.2% trifluoroacetic acid/methanol mixed solution.
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