CN109342603A - A kind of detection method of the pyrrole Lun Panai piece in relation to substance - Google Patents

A kind of detection method of the pyrrole Lun Panai piece in relation to substance Download PDF

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CN109342603A
CN109342603A CN201811390541.4A CN201811390541A CN109342603A CN 109342603 A CN109342603 A CN 109342603A CN 201811390541 A CN201811390541 A CN 201811390541A CN 109342603 A CN109342603 A CN 109342603A
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impurity
mobile phase
detection method
piece
relation
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CN109342603B (en
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杨雨菲
朱峰妍
孙莉莉
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Nanjing Healthnice Pharmaceutical Co ltd
Nanjing Healthnice Pharmaceutical Technology Co ltd
Nanjing Yinuo Medicine Technology Co ltd
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Nanjing Tech University
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The present invention relates to a kind of detection method of the pyrrole Lun Panai piece in relation to substance, belong to Pharmaceutical Analysis technical field.Detection method of the invention uses mobile phase A mutually to carry out gradient elution with Mobile phase B for mixed flow, and mobile phase A is water phase, and Mobile phase B is organic phase；The initial proportion of mobile phase A and Mobile phase B is 25~35:75~65 or 70:30 during gradient elution；In 0-8 minutes, the volume ratio of mobile phase A and Mobile phase B keeps initial proportion constant；In 8-30 minutes, the volume ratio of mobile phase A and Mobile phase B is by initial proportion at the uniform velocity gradual change to 40:60；In 30-40 minutes, the volume ratio of mobile phase A and Mobile phase B keeps 40:60 constant；In 40-50 minutes, the volume ratio of mobile phase A and Mobile phase B is by 40:60 at the uniform velocity gradual change to 70:30；In 50-60 minutes, the volume ratio of mobile phase A and Mobile phase B keeps 70:30 constant.

Description

A kind of detection method of the pyrrole Lun Panai piece in relation to substance
Technical field
The invention belongs to Pharmaceutical Analysis technical fields, and in particular to a kind of detection method of the pyrrole Lun Panai piece in relation to substance.
Background technique
Pyrrole Lun Panai is the drug of Japanese Wei Cai company research and development, passes through the group of Noncompetition inhibition AMPA type glutamate receptor Effect, for treating the partial seizures of 12 years old or more epileptic.The medicine is the first anti-with the mechanism of action of FDA approval Epilepsy drugs.
Related substance is the starting material brought into pharmaceutical synthesis production process, intermediate, side reaction product and degradation Impurity etc., to related substance detect can quality to drug and safety control.At present for pyrrole Lun Panai piece Detection method in relation to substance does not have complete expression.
Summary of the invention
The purpose of the present invention is on the basis of existing technology, provide a kind of detection side of the pyrrole Lun Panai piece in relation to substance Method.
Technical scheme is as follows:
A kind of detection method of the pyrrole Lun Panai piece in relation to substance, the detection method is using high performance liquid chromatography to pyrrole Lun Panai Pyrrole Lun Panai and related substance carry out quantitative detection in tablet, and high-efficient liquid phase chromatogram condition includes: using mobile phase A and flowing Phase B is that mixed flow mutually carries out gradient elution, and mobile phase A is water phase, and Mobile phase B is organic phase；It is flowed during gradient elution Dynamic phase A and the initial proportion of Mobile phase B are 25~35:75~65 or 70:30；In 0-8 minutes, mobile phase A and Mobile phase B Volume ratio keeps initial proportion constant；In 8-30 minutes, the volume ratio of mobile phase A and Mobile phase B by initial proportion at the uniform velocity gradually Fade to 40:60；In 30-40 minutes, the volume ratio of mobile phase A and Mobile phase B keeps 40:60 constant；In 40-50 minutes, The volume ratio of mobile phase A and Mobile phase B is by 40:60 at the uniform velocity gradual change to 70:30；In 50-60 minutes, mobile phase A and Mobile phase B Volume ratio keep 70:30 it is constant.
For example, specific gradient elution process is as follows when the initial proportion of mobile phase A and Mobile phase B is 70:30:
When the present invention is detected using high performance liquid chromatography, gradient is mutually carried out as mixed flow with organic phase using water phase Elution, in the case where not influencing effect of the present invention, water phase is preferably potassium dihydrogen phosphate aqueous solution, and organic phase is preferably acetonitrile.
In a kind of scheme, mobile phase A is 9~11mmol/L potassium dihydrogen phosphate aqueous solution, is adjusted with phosphoric acid or triethylamine PH to 3.05~6.25 adjusts pH value to 6.25 with triethylamine with phosphorus acid for adjusting pH value to 3.05.
In a preferred embodiment, mobile phase A be 9~11mmol/L potassium dihydrogen phosphate aqueous solution, pH value 4.6-5.2, With phosphorus acid for adjusting pH value to 4.2, pH value is adjusted to 5.2 with triethylamine.
In a kind of more preferable scheme, 10mmol/L potassium dihydrogen phosphate aqueous solution is prepared, is not required to adjust, pH value 4.61.
When the present invention is detected using high performance liquid chromatography, chromatographic column selects Shimadzu Wondasil C18- WR or Dikma diamonsil C18；Using octadecylsilane chemically bonded silica as filler.In the case where not influencing detection effect, preferred color of choice The length for composing column is 250mm, and diameter 4.6mm, packing material size is 5 μm.Such as: Shimadzu Wondasil C18- WR column (250x4.6mm, 5 μm) or Dikma diamonsil C18 column (250x4.6mm, 5 μm).
Further, it is 200~250nm, preferably 220nm that high-efficient liquid phase chromatogram condition, which includes: Detection wavelength,；
Further, column temperature is 25 DEG C~35 DEG C, preferably 30 DEG C.
The present invention can according to need, and select suitable sample volume sample introduction, and sample volume is 10~50 μ l；It is preferred that 20 μ l.Example Such as: sample volume is 10 μ l, 20 μ l or 50 μ l.
Related substance in the pyrrole Lun Panai bulk pharmaceutical chemicals that the present invention refers to, including following substance: impurity 2: phenyl boric acid (SMB)； Impurity 3:2- cyanophenylboronic acid -1,3- propanediol cyclic ester (SMC)；Impurity 4:1- phenyl -5- (2- pyridyl group) -1,2- dihydro pyrrole Pyridine -2- ketone (intermediate 1)；Impurity 5:1- phenyl -3- bromo- 5- (2- pyridyl group) -1,2- dihydropyridine -2- ketone (intermediate 2)；It is miscellaneous Matter 6:1,5- diphenyl-[2,3'- bipyridyl] -6'(1'H) -one；Impurity 7:3- (6- oxo -1- phenyl -1,6- dihydro-[2,3- Bipyridyl] -5- base) benzonitrile；Impurity 8:4- (6- oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzonitrile；Impurity 9:2- (6- oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzoic acid；Impurity 10:5- (2- cyano-phenyl) -6- Oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -1- oxygen.
The present invention uses high performance liquid chromatography, is screened, is optimized from chromatographic condition etc. respectively, drafts related object Matter method, carry out methodology validation, to impurity 2 (SMB), impurity 3 (SMC), impurity 4 (intermediate 1), impurity 5 (intermediate 2), Impurity 6~10 (process impurity and degradation impurity) has carried out quantitative study, and provides complete proof scheme, easy to operate, surely Qualitative and reproducibility is good.Wherein, the correction factor of impurity 2 is 1.06, and the correction factor of impurity 3 is 2.45, the correction of impurity 4 The factor is 1.24, and the correction factor of impurity 5 is 1.89, and the correction factor of impurity 6 is 1.29, and the correction factor of impurity 7 is 0.85, The correction factor of impurity 8 is 1.26, and the correction factor of impurity 9 is 1.13, and the correction factor of impurity 10 is 0.65.
HPLC detection method the present invention provides pyrrole Lun Panai in relation to substance, including following operating procedure:
(1) solution is prepared:
Take in prescription that auxiliary material lactose monohydrate adds 70% acetonitrile to dissolve and is diluted to about 20mg/ml, PVP K30 adds respectively 70% acetonitrile dissolves and is diluted to about 0.5mg/ml, magnesium stearate adds 70% acetonitrile to dissolve and is diluted to about 1.0mg/ml, low takes It is dissolved for hydroxypropyl cellulose plus 70% acetonitrile and is diluted to about 4.0mg/ml, coating powder adds 70% acetonitrile to dissolve and is diluted to about 10mg/ml, prescription mixing blank auxiliary add 70% acetonitrile to dissolve and are diluted to about 25mg/ml, take pyrrole Lun Panai slice lapping to powder End adds 70% acetonitrile to dissolve and is diluted to about 25mg/ml, and test solution is used as after 0.45 μm of organic membrane filtration.Respectively Impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10 plus 70% acetonitrile is taken to dissolve and dilute To about 5 μ g/ml.Above-mentioned solution is taken to mix to obtain mixing contrast solution with 70% acetonitrile as solvents respectively in right amount.
(2) test solution and impurity reference substance are injected separately into liquid chromatograph, chromatogram are recorded, by external standard method with peak Areal calculation, chromatographic condition are as follows:
Chromatographic column: Shimadzu Wondasil C18- WR column (250x4.6mm, 5 μm) or Dikma diamonsil C18；, 18 Alkyl silane bonded silica gel is filler；
Detection wavelength: Detection wavelength is 200~250nm, preferably 220nm；
Mobile phase A: 10mmol/L potassium dihydrogen phosphate aqueous solution, pH are 3.05~6.25, preferable ph 4.2-5.2；More Preferable ph is 4.61；Mobile phase B: acetonitrile；Mixed flow phase flow velocity is 1mL/min；Mobile phase A and stream during gradient elution The initial proportion of dynamic phase B is 25~35:75~65 or 70:30；In 0-8 minutes, the volume ratio of mobile phase A and Mobile phase B is protected It is constant to hold initial proportion；In 8-30 minutes, the volume ratio of mobile phase A and Mobile phase B is by initial proportion at the uniform velocity gradual change to 40: 60；In 30-40 minutes, the volume ratio of mobile phase A and Mobile phase B keeps 40:60 constant；In 40-50 minutes, mobile phase A Volume ratio with Mobile phase B is by 40:60 at the uniform velocity gradual change to 70:30；In 50-60 minutes, the volume of mobile phase A and Mobile phase B Than keeping 70:30 constant.
Column temperature is 25 DEG C~35 DEG C, preferably 30 DEG C.
(3) measuring method: precision measures test sample and each 20 μ l of impurity reference substance solution, is injected separately into liquid chromatograph, Record chromatogram.
Using technical solution of the present invention, advantage is as follows:
On the basis of detection method of the invention is the Related substance method in pyrrole Lun Panai bulk pharmaceutical chemicals, for auxiliary material The interference that PVP K30 impurity generates when detecting further changes the optimization of gradient ratio and chromatographic condition, and carries out Correlation technique verifying, is presented above-mentioned impurity 2~10 in a chromatographic behavior in the content in pyrrole Lun Panai sample, just The control of product quality, simple and easy in production process, and accuracy and precision are high.The favorable reproducibility of this detection method, energy Meet testing requirements of the pyrrole Lun Panai piece in relation to substance, can be used for the quality control of pyrrole Lun Panai piece.
Detailed description of the invention
Fig. 1 is blank auxiliary map；
Fig. 2 is PVP K30 map；
Fig. 3 is lactose monohydrate map；
Fig. 4 is low-substituted hydroxypropyl cellulose map；
Fig. 5 is magnesium stearate map；
Fig. 6 is coating powder map；
Fig. 7 is the map that pyrrole Lun Panai sample adds impurity；
Wherein, respectively impurity 3 (retention time 6.221min, Area%0.438%), impurity 2 (when reservation from left to right Between 7.046min, Area%0.682%), impurity 10 (retention time 12.212min, Area%0.949%), impurity 4 (retain Time 15.566min, Area%0.947%), impurity 9 (retention time 19.890min, Area%0.857%), impurity 5 (protect Stay time 27.146min, Area%0.487%), it is pyrrole Lun Panai (retention time 31.070min, Area%92.898%), miscellaneous Matter 7 and impurity 8 (isomer each other, retention time 34.191min, Area%1.746%), 6 (retention time of impurity 35.422min Area%0.926%)；
Fig. 8 is pyrrole Lun Panai (140301 batches of samples) map；
Wherein, respectively pyrrole Lun Panai (retention time 31.313min, Area%99.968%), other lists from left to right Miscellaneous 1 (retention time 33.533min, Area%0.005%), impurity 6 (retention time 35.110min, Area%0.018%), Other single miscellaneous 2 (retention time 35.632min, Area%0.002%), other single miscellaneous 3 (retention time 36.897min, Area% 0.006%), wherein only detecting impurity 6 0.018%, latent gene poison impurity is not detected in other single miscellaneous total amounts 0.013% 10；
Fig. 9 is 2 linear graph of impurity；
Figure 10 is 3 linear graph of impurity；
Figure 11 is 4 linear graph of impurity；
Figure 12 is 5 linear graph of impurity；
Figure 13 is 6 linear graph of impurity；
Figure 14 is 7 linear graph of impurity；
Figure 15 is 8 linear graph of impurity；
Figure 16 is 9 linear graph of impurity；
Figure 17 is 10 linear graph of impurity；
Figure 18 is pyrrole Lun Panai linear graph.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this Invention is further elaborated.It should be appreciated that specific example described herein is only used to explain the present invention, and do not have to It is of the invention in limiting.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, side Method and equipment.
A kind of embodiment: HPLC detection method of the pyrrole Lun Panai in relation to substance
One, experimental material and instrument
1. drug and reagent: (impurity 2 is praised for pyrrole Lun Panai piece (Eisai R&D Management Co.Ltd), phenyl boric acid Emerging city Ai Sen Chemical Co., Ltd.), (impurity 3, it is limited that Nan Jinghai receives medical sci-tech to 2- cyanophenylboronic acid -1,3-PD cyclic ester Company), 1- phenyl -5- (2- pyridyl group) -1,2- dihydropyridine -2- ketone (impurity 4, Nanjing Healthnice Medical Technology Co., Ltd.), The bromo- 5- of 1- phenyl -3- (2- pyridyl group) -1,2- dihydropyridine -2- ketone (impurity 5, Nanjing Healthnice Medical Technology Co., Ltd.), 1, 5- diphenyl-[2,3'- bipyridyl] -6'(1'H) -one (impurity 6, Nanjing Healthnice Medical Technology Co., Ltd.), 3- (6- oxo - 1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzonitrile (impurity 7, Nanjing Healthnice Medical Technology Co., Ltd.), 4- (6- Oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzonitrile (impurity 8, Nanjing Healthnice Medical Technology Co., Ltd.), (impurity 9, it is limited that Nan Jinghai receives medical sci-tech to 2- (6- oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzoic acid Company), (impurity 10, Nan Jinghai receives [2,3- bipyridyl] -1- oxygen 5- (2- cyano-phenyl) -6- oxo -1- phenyl -1,6- dihydro - Pharmaceutical Technology Co., Ltd), acetonitrile (chromatographically pure, DIKMA), potassium dihydrogen phosphate (analyze pure, the limited public affairs of Chinese medicines group chemical reagent Department), phosphoric acid (analyze pure, Sinopharm Chemical Reagent Co., Ltd.), (analysis is pure, and Chinese medicines group chemical reagent is limited for triethylamine Company), ultrapure water (self-control, Millipore).
2. instrument: the title and specification of specific instrument see the table below 1.
The title and specification of the specific instrument of table 1
Two, liquid phase chromatogram condition
Weighing auxiliary material lactose monohydrate 195.57mg in prescription adds the dissolution of 70% acetonitrile to be settled to 10.0ml, povidone K3011.25mg adds 70% acetonitrile to dissolve and is settled to 25.0ml, magnesium stearate 12.50mg adds 70% acetonitrile to dissolve and is settled to 10.0ml, low-substituted hydroxypropyl cellulose 41.50mg add 70% acetonitrile to dissolve and are settled to 10.0ml, coating powder 18.75mg adds 70% acetonitrile dissolves and is settled to 25.0ml, prescription mixing blank auxiliary 645.88mg adds 70% acetonitrile to dissolve and is settled to 25.0ml, ultrasonic 5min, it is spare through 0.45 μm of organic membrane filtration.It takes pyrrole Lun Panai slice lapping to powder, weighs 252.3mg and add 70% acetonitrile dissolves and is settled to 10.0ml, and test solution is used as after 0.45 μm of organic membrane filtration.Chromatographic column uses island Saliva Wondasil C18- WR column (250x4.6mm, 5 μm)；Using 10mmol/L potassium dihydrogen phosphate aqueous solution as mobile phase A, with acetonitrile For Mobile phase B, according to the form below carries out gradient elution；Flow velocity is 1.0ml/min.30 DEG C of column temperature, Detection wavelength 220nm, precision amount 20 μ l of test solution is taken, liquid chromatograph is injected, records chromatogram.
Three, experimentation
1. methodology validation
1.1 specificity
Weighing auxiliary material lactose monohydrate 195.57mg in prescription adds the dissolution of 70% acetonitrile to be settled to 10.0ml, povidone K3011.25mg adds 70% acetonitrile to dissolve and is settled to 25.0ml, magnesium stearate 12.50mg adds 70% acetonitrile to dissolve and is settled to 10.0ml, low-substituted hydroxypropyl cellulose 41.50mg add 70% acetonitrile to dissolve and are settled to 10.0ml, coating powder 18.75mg adds 70% acetonitrile dissolves and is settled to 25.0ml, prescription mixing blank auxiliary 645.88mg adds 70% acetonitrile to dissolve and is settled to 25.0ml, ultrasonic 5min, it is spare through 0.45 μm of organic membrane filtration.It takes pyrrole Lun Panai piece (140301 batches) to be ground to powder, claims It takes 252.3mg that 70% acetonitrile is added to dissolve and is settled to 10.0ml, test solution is used as after 0.45 μm of organic membrane filtration.Point Also known as take impurity 2 (phenyl boric acid) 12.23mg, impurity 3 (2- cyanophenylboronic acid -1,3- propanediol cyclic ester) 9.88mg, 4 (1- of impurity Phenyl -5- (2- pyridyl group) -1,2- dihydropyridine -2- ketone) 10.58mg, impurity 5 (the bromo- 5- of 1- phenyl -3- (2- pyridyl group) -1, 2- dihydropyridine -2- ketone) 12.11mg, impurity 6 (1,5- diphenyl-[2,3'- bipyridyl] -6'(1'H) -one) it is 10.71mg, miscellaneous Matter 7 (3- (6- oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzonitrile) 10.35mg, (4- (the 6- oxo-of impurity 8 1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzonitrile) 9.85mg, the (2- (6- oxo -1- phenyl -1,6- two of impurity 9 Hydrogen-[2,3- bipyridyl] -5- base) benzoic acid) 10.12mg, (5- (2- the cyano-phenyl) -6- oxo -1- phenyl -1,6- of impurity 10 Dihydro-[2,3- bipyridyl] -1- oxygen) 9.91mg, pyrrole Lun Panai compare 12.52mg to 25.0ml volumetric flask, and it is fixed with 70% acetonitrile Hold to graduation mark.It takes blank solvent and above-mentioned solution to distinguish sample introduction respectively, records chromatogram, be detailed in attached drawing 1~8.
Test result is shown: under the chromatographic condition, baseline is steady, each auxiliary material to this product measure it is noiseless, impurity 7 with it is miscellaneous 8 retention time of matter is consistent, in 34min or so；Point of pyrrole Lun Panai piece main peak theoretical cam curve 67929, impurity 5 and principal component It is 6.362 from degree, principal component and principal component rear impurity separating degree are 4.613, and each impurity separating degree meets regulation.
1.2 failure test
The issuable catabolite of pyrrole Lun Panai piece can be detected under selected chromatographic condition to investigate, and used respectively The drastic conditions such as high temperature, acid, alkali, oxidation, illumination destroy this product, and the sample after destruction is dissolved with 70% acetonitrile and is prepared It is accurate respectively to measure above-mentioned each 20 μ l of solution at test solution, liquid chromatograph is injected, chromatogram is recorded, the results are shown in Table 2.
2 pyrrole Lun Panai piece failure test result of table
This product has different degrees of degradation under oxidation and high light conditions in alkali, acid, high temperature, wherein be illuminated by the light, be sour, Alkali, Oxidative demage are affected, each condition destroy caused by catabolite can be detected, main peak peak purity is good.Respectively The separating degree of catabolite and main peak is good.
1.3 quantitative limits, detection limit
It takes each impurity to compare respectively and certain density sample is prepared in pyrrole Lun Panai control, 20 μ l of sample introduction after gradually diluting, With signal-to-noise ratio S/N=3, S/N=10 measurement, it the results are shown in Table 3.
The detection of table 3 limit and quantitative limit result
Title Quantitative limit (ng) Detection limit (ng)
Pyrrole Lun Panai 1.0 0.3
Impurity 2 2.9 0.9
Impurity 3 1.6 0.5
Impurity 4 1.3 0.4
Impurity 5 1.6 0.6
Impurity 6 1.7 0.6
Impurity 7 1.5 0.5
Impurity 8 1.5 0.4
Impurity 9 1.6 0.4
Impurity 10 1.0 0.3
1.4 sample solutions and reference substance solution stability test
It takes this product appropriate, adds 70% acetonitrile to dissolve and dilute solution of every 1ml containing about pyrrole Lun Panai 0.5mg is made, respectively It 0 after preparation, measures within 2,4,6,8,10,12 hours, investigates the stability of sample solution.
Mixing contrast solution is taken, the stabilization of reference substance solution is investigated in 0,2,4,6,8,10,12 after preparation hour measurement Property, it the results are shown in Table 4~5.
4 impurity contrast solution stability test result of table
Time (hour) 0 2 4 6 8 10 12 RSD (%)
2 peak area of impurity 35957 38873 37190 39799 37876 39595 38576 3.6
3 peak area of impurity 22649 22290 22580 23677 21694 23324 22281 3.0
4 peak area of impurity 42906 42746 42420 41070 41586 42347 41496 1.7
5 peak area of impurity 22965 21311 21444 21758 21458 20910 21105 3.1
6 peak area of impurity 40404 41203 39929 39989 39773 39896 40015 1.2
Impurity 7,8 peak areas 87502 92152 90899 89699 89823 83121 82914 4.2
9 peak area of impurity 24001 25518 25266 25194 24213 24482 25153 2.4
10 peak area of impurity 8420 8398 8632 8437 7860 8710 8160 3.4
Pyrrole Lun Panai 433196 433410 434612 433964 430643 433090 433690 0.3
5 sample solution solution stability testing of table
The result shows that sample solution, impurity contrast solution are in 12 hours internal stabilities of room temperature condition under this determination condition Preferably.
1.5 linear
1.5.1 impurity stock solution is prepared
It takes impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9 appropriate respectively, acetonitrile is added to prepare At the solution of about 5 μ g/ml of concentration, storeed as impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9 Liquid；It separately takes impurity 10 appropriate, adds acetonitrile to be configured to the solution of about 0.625 μ g/ml of concentration, as 10 stock solution of impurity, cold It places at place.
1.5.2 linear
Each impurity control stock solution of accurate absorption is set in right amount in 10ml measuring bottle respectively, with 70% acetonitrile constant volume, is shaken up, is obtained A series of solution of concentration.Precision draws above-mentioned each 20 μ l of series of concentrations solution, and successively sample introduction is analyzed from low concentration to high concentration, Chromatogram is recorded, with impurity reference substance solution concentration C (μ g/ml) for abscissa, impurity reference substance peak area is ordinate, is carried out Linear regression simultaneously finds out regression equation, the results are shown in Table 6 and Fig. 9~Figure 18.
Table 6 linearly investigates result
1.6 sample introduction precision tests
4 solution of linear term lower linear is taken, continuous sample introduction measures 6 times, investigates the situation of change of peak area and retention time, knot Fruit is shown in Table 7.
7 sample introduction Precision test result of table
The result shows that instrument precision is good.
1.7 repetitive test
It is each appropriate that parallel precision weighs 6 parts of 140301 batches of samples, adds 70% acetonitrile to dissolve and is respectively prepared in every 1ml and contains The solution of 0.5mg pyrrole Lun Panai is as test solution.
It takes impurity 2,3,4,5,6,7,8,9,10 each appropriate respectively, 70% acetonitrile is added to be configured to impure 2~9 concentration about 0.5 μ g/ml, impure 10 be about the solution of 0.0625 μ g/ml, as reference substance solution.
Precision measures above-mentioned each 20 μ l of solution, liquid chromatograph is injected separately into, by external standard method in calculated by peak area this product The content of each impurity, measurement result are shown in Table 8.
8 repetitive test result of table
Serial number 1 2 3 4 5 6 It is average RSD (%)
Impurity 6 0.008 0.011 0.010 0.009 0.007 0.008 0.009 13.8
Total miscellaneous (%) 0.045 0.040 0.052 0.040 0.042 0.044 0.044 10.2
Other known impurities are not detected.
The test of 1.8 sample recovery rates
It is each appropriate that parallel precision weighs 9 parts of 140301 batches of samples, is separately added into the limitation of impurity 2,3,4,5,6,7,8,9,10 80%, 100%, 120%, add 70% dilution in acetonitrile to concentration to be measured, it is accurate respectively to measure 20 μ l, inject liquid chromatograph, Sample recovery rate measurement is carried out, the results are shown in Table 9~table 17.
9 impurity of table, 2 recovery test result
10 impurity of table, 3 recovery test result
11 impurity of table, 4 recovery test result
12 impurity of table, 5 recovery test result
13 impurity of table, 6 recovery test result
14 impurity of table, 7 recovery test result
15 impurity of table, 8 recovery test result
16 impurity of table, 9 recovery test result
17 impurity of table, 10 recovery test result
The test of 1.9 Intermediate precisions
It is each appropriate that 6 parts of 140301 batches of samples are weighed in parallel, it is accurately weighed, add 70% acetonitrile to dissolve and every 1ml is respectively prepared In the Lun Panai of pyrrole containing 0.5mg solution as test solution.
It takes impurity 2,3,4,5,6,7,8,9,10 each appropriate respectively, 70% acetonitrile is added to be configured to impure 2~9 concentration about 0.5 μ g/ml, impure 10 be about the solution of 0.0625 μ g/ml, as reference substance solution.
Precision measures above-mentioned each 20 μ l of solution, liquid chromatograph is injected separately into, by external standard method in calculated by peak area this product The content of each impurity, measurement result are shown in Table 18-1,18-2.
6 Intermediate precision test result of table 18-1 impurity
The total miscellaneous Intermediate precision test result of table 18-2
Note: instrument 1: Shimadzu LC-20A high performance liquid chromatograph number: 100106
Instrument 2: Shimadzu LC-10A high performance liquid chromatograph number: 100107
Test result shows: this method Intermediate precision is good.
1.10 correction factor measures
Calculated result shows under linear test item: the correction factor of impurity 2 is 1.06, and the correction factor of impurity 3 is 2.29, The correction factor of impurity 4 is 1.21, and the correction factor of impurity 5 is 1.91, and the correction factor of impurity 6 is 1.24, the correction of impurity 7 The factor is 0.84, and the correction factor of impurity 8 is 1.28, and the correction factor of impurity 9 is 1.12, and the correction factor of impurity 10 is 0.68.Each impurity correction factor is consistent under substance-measuring method related with raw material.
1.11 durability is investigated
The present invention further inquires into the tolerance degree of the detection method when minor change occurs for chromatographic condition, carries out durability Test, test solution are mixing contrast solution and sample solution (140301 batches), and project of investigating includes starting mobile phase ratio, Column temperature, different flowing phase pH values and different chromatographic columns, to mix contrast solution peak number, main peak and separating degree, the master of adjacent peak The number of theoretical plate of principal component, principal component retention time and normalization purity are inspection target in peak tailing factor, sample solution, right The durability of this method is investigated, and measurement result is shown in Table 19~22.
1.11.1 the variation of starting aqueous phase ratio
As hereinbefore, starting aqueous phase ratio is 25% to other high-efficient liquid phase chromatogram conditions during gradient elution, 30%, separating degree, the main peak tailing factor, sample solution of contrast solution peak number, main peak and adjacent peak are mixed under the conditions of 35% The situation of change of number of theoretical plate, principal component retention time and the normalization purity of middle principal component etc..The results showed that initial water When Phase Proportion changes in 25%~35% range, in chromatographic behavior in addition to the separating degree of main peak and adjacent peak differs greatly, His parameter includes returning of measuring of mixing control peak number, retention time, tailing factor, principal component number of theoretical plate and sample One changes purity without too many differences, and measurement result is shown in Table 19.
19 durability of table investigates (initial watr-proportion)
1.11.2 the variation of flowing phase pH value
Other high-efficient liquid phase chromatogram conditions in aqueous pH values are respectively 4.2 during gradient elution as hereinbefore, Mixed under the conditions of 4.7 and 5.2 contrast solution peak number, the separating degree of main peak and adjacent peak, main peak tailing factor, in sample solution The situation of change of number of theoretical plate, principal component retention time and the normalization purity of principal component etc..The results showed that aqueous pH values When changing in 4.2~5.2 ranges, each parameter is influenced less, measurement result is shown in Table 20.
20 durability of table investigates (aqueous pH values)
1.11.3 the variation of column temperature
Other high-efficient liquid phase chromatogram conditions as hereinbefore, mix contrast solution when column temperature is 25 DEG C, 30 DEG C and 35 DEG C Peak number, the separating degree of main peak and adjacent peak, main peak tailing factor, the number of theoretical plate of principal component, principal component are protected in sample solution Stay the situation of change of time and normalization purity etc..The results showed that when column temperature changes within the scope of 25 DEG C~35 DEG C, to master The separating degree of peak and adjacent peak has an impact, other each parameters influence less, and measurement result is shown in Table 21.
21 durability of table investigates (column temperature)
1.11.4 the variation of chromatographic column
Other high-efficient liquid phase chromatogram conditions as hereinbefore, investigate different chromatographic columns to mixing contrast solution peak number, master The separating degree of peak and adjacent peak, main peak tailing factor the number of theoretical plate of principal component, principal component retention time and are returned in sample solution One changes the situation of change of purity etc..The results showed that being influenced on each parameter little using different chromatographic columns.Measurement result is shown in Table 22.
22 durability of table investigates (different chromatographic columns)
2. sample measures
According to the above method, related substance is carried out to three batches of samples and listing product and is calculated, the results are shown in Table 23.
23 pilot scale Related substances separation result of table
The present invention studies pyrrole Lun Panai piece impurity that may be present using high performance liquid chromatography.
It using high performance liquid chromatography (HPLC), screened, optimized from chromatographic condition etc. respectively, draft related substance Method, carry out methodology validation, and to impurity 2 (SMB), impurity 3 (SMC), impurity 4 (intermediate 1), impurity 5 (intermediate 2), Impurity 6~10 (process impurity and degradation impurity) has carried out quantitative study.Detection method of the invention, pyrrole Lun Panai and related object Matter linear relationship is good, and accuracy and precision are good, and specificity is strong, and stability is high.The favorable reproducibility of this detection method, energy Meet testing requirements of the pyrrole Lun Panai piece in relation to substance, can be used for the quality control of pyrrole Lun Panai piece.

Claims (10)

1. a kind of detection method of the pyrrole Lun Panai piece in relation to substance, which is characterized in that the detection method uses high-efficient liquid phase color Spectrum carries out quantitative detection to pyrrole Lun Panai in pyrrole Lun Panai tablet and related substance, and high-efficient liquid phase chromatogram condition includes: use Mobile phase A is that mixed flow mutually carries out gradient elution with Mobile phase B, and the mobile phase A is water phase, and the Mobile phase B is organic Phase；The initial proportion of mobile phase A and Mobile phase B is 25~35:75~65 or 70:30 during the gradient elution；At 0-8 points In clock, the volume ratio of mobile phase A and Mobile phase B keeps initial proportion constant；In 8-30 minutes, mobile phase A and Mobile phase B Volume ratio is by initial proportion at the uniform velocity gradual change to 40:60；In 30-40 minutes, the volume ratio of mobile phase A and Mobile phase B keeps 40: 60 is constant；In 40-50 minutes, the volume ratio of mobile phase A and Mobile phase B is by 40:60 at the uniform velocity gradual change to 70:30；At 50-60 points In clock, the volume ratio of mobile phase A and Mobile phase B keeps 70:30 constant.
2. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 1, which is characterized in that the mobile phase A For potassium dihydrogen phosphate aqueous solution, the Mobile phase B is acetonitrile.
3. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 2, which is characterized in that the mobile phase A For 9~11mmol/L potassium dihydrogen phosphate aqueous solution.
4. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 3, which is characterized in that the mobile phase A PH to 3.05~6.25 is adjusted with phosphoric acid or triethylamine；Preferable ph is 4.2-5.2；More preferable pH value is 4.61.
5. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 1, which is characterized in that the efficient liquid phase Chromatographic condition includes: that chromatographic column is Shimadzu Wondasil C18- WR or Dikma diamonsil C18；It is preferred that the length of chromatographic column Degree is 250mm, and diameter 4.6mm, packing material size is 5 μm.
6. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 1, which is characterized in that the efficient liquid phase Chromatographic condition includes: that Detection wavelength is 200~250nm, preferably 220nm.
7. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 1, which is characterized in that the efficient liquid phase Chromatographic condition includes: that column temperature is 25 DEG C~35 DEG C, preferably 30 DEG C.
8. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 1, which is characterized in that the efficient liquid phase Chromatographic condition includes: that sample volume is 10~50 μ l；It is preferred that 20 μ l.
9. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 1, which is characterized in that the related substance Including following substance: impurity 2: phenyl boric acid；Impurity 3:2- cyanophenylboronic acid -1,3- propanediol cyclic ester；Impurity 4:1- phenyl -5- (2- pyridyl group) -1,2- dihydropyridine -2- ketone；Impurity 5:1- phenyl -3- bromo- 5- (2- pyridyl group) -1,2- dihydropyridine -2- Ketone；Impurity 6:1,5- diphenyl-[2,3'- bipyridyl] -6'(1'H) -one；Impurity 7:3- (6- oxo -1- phenyl -1,6- dihydro - [2,3- bipyridyl] -5- base) benzonitrile；Impurity 8:4- (6- oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzyl Nitrile；Impurity 9:2- (6- oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -5- base) benzoic acid；Impurity 10:5- (2- cyano Phenyl) -6- oxo -1- phenyl -1,6- dihydro-[2,3- bipyridyl] -1- oxygen.
10. detection method of the pyrrole Lun Panai piece in relation to substance according to claim 1, which is characterized in that the correction of impurity 2 The factor is 1.06, and the correction factor of impurity 3 is 2.45, and the correction factor of impurity 4 is 1.24, and the correction factor of impurity 5 is 1.89, The correction factor of impurity 6 is 1.29, and the correction factor of impurity 7 is 0.85, and the correction factor of impurity 8 is 1.26, the correction of impurity 9 The factor is 1.13, and the correction factor of impurity 10 is 0.65.
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CN109799298A (en) * 2019-02-20 2019-05-24 南京海纳医药科技股份有限公司 Detection method in relation to substance in a kind of pyrrole Lun Panai bulk pharmaceutical chemicals
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CN113984944A (en) * 2021-11-15 2022-01-28 南京华威医药科技集团有限公司 Detection method of Perampanel genotoxic impurities

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