CN105738492A - Method for detecting impurity content in lapatinib through combination of LC-MS and MS - Google Patents
Method for detecting impurity content in lapatinib through combination of LC-MS and MS Download PDFInfo
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- 229960004891 lapatinib Drugs 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 43
- 239000012535 impurity Substances 0.000 title claims abstract description 26
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 title claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 47
- AYPFEYDGZDPAPE-UHFFFAOYSA-N 3-chloro-4-[(3-fluorophenyl)methoxy]aniline Chemical compound ClC1=CC(N)=CC=C1OCC1=CC=CC(F)=C1 AYPFEYDGZDPAPE-UHFFFAOYSA-N 0.000 claims abstract description 37
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention provides a method for detecting 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride impurity contents in lapatinib through combination of LC-MS and MS. According to the method, 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride impurities in a lapatinib bulk drug can be quickly and effectively detected, and the two impurity contents are subjected to quantitative detection and analysis. Through application of the method, the detection result is accurate and reliable. The method is simple to operate, is fast, and is high in specialization, good in repeatability, and is free of residual effect. The method can be used for quality control of production of a lapatinib medicine.
Description
Technical field
The present invention relates to pharmaceutical analysis technical field, in particular it relates to the method for impurity content in LC-MS/MS coupling detection antineoplastic agent Lapatinib.
Background technology
Lapatinib is that one can be simultaneously targeting human epidermal growth factor acceptor (EGFR) and the small molecule kinase inhibitors of human epidermal growth factor receptor-2 (HER2), developed by GSK company, U.S. FDA approval is obtained in March, 2007, for drug combination: share late period or the metastatic breast cancer of capecitabine treatment overexpression HER2, share the metastatic breast cancer menopausal women of letrozole treatment overexpression HER2, hormone receptor positive.
4-(3-fluorobenzyloxy) 3-chloroaniline (compound 4) and 2-(methylsulfonyl) ethylamine hydrochloride (compound 9) are the two kinds of production intermediate feed (both structures are shown in following formula) preparing Lapatinib crude drug respectively, Lapatinib finished product crude drug usually finally can contain a small amount of compound 4 and compound 9 impurity, in order to improve quality and the stability of Lapatinib, and reduce the poisonous side effect of medicine for clinical Lapatinib, therefore need aborning compound 4 and compound 9 are carried out content analysis mensuration, and in Lapatinib crude drug, the content of compound 4 and compound 9 impurity is limited the quantity.
In prior art, the method only carrying out Light absorbing impurty for compound 4, but it is seen in report currently without the method carrying out assay for compound 4 and compound 9 simultaneously, thus, the impurity content analysis determining method about Lapatinib still haves much room for improvement.
Summary of the invention
It is contemplated that one of technical problem solved at least to a certain extent in correlation technique.For this, it is an object of the present invention to propose a kind of result accurately and reliably, easy, quickly, specificity is strong, reproducible, can LC-MS/MS (liquid chromatography-mass spectrography/mass spectrum) the coupling detection method of content of Simultaneous Quantitative Analysis compound 4 and compound 9, for the quality control of Lapatinib drug manufacture.
The present invention is based on the following discovery of inventor and completes:
Inventor is in the drug standard formulating Lapatinib crude drug, the content limit of compound 4 impurity is not higher than 4 μ g/1g (i.e. 4ppm), the content limit of compound 9 impurity is not higher than 0.02%, the detection limit of common high performance liquid chromatography detection compound 4 is very low, can only measure 0.1% and above impurity.Compound 9 is because of without uv absorption, it is impossible to measure by liquid phase.Although inventor's trial evaporat light scattering can detect, but its detection limit is very low, can only measure 2% and above impurity.Inventor is in research process, use C8 (sunfireC850*4.6mm, 5 μm) chromatographic column optimization analyzes Lapatinib crude drug and compound 4, the chromatographic condition of compound 9, with methanol (B)-water (A) as mobile phase, sampling volume is 10.0 μ L, experimentation finds, in gradient condition as shown in table 1, and in such chromatographic column, the reservation of compound 4 and Lapatinib crude drug is better, but compound 9 does not show good retention behavior, and (compound 9 goes out peak in dead time position substantially, dead volume under this condition is about 0.5mL, and the appearance time of compound 9 is 1.5min).
The gradient condition of table 1:sunfireC8
| Time (min) | Mobile phase B (%) | Flow velocity (ml/min) |
| 0 | 30 | 0.3 |
| 0.4 | 30 | 0.3 |
| 2.0 | 60 | —— |
| 6.0 | 95 | 0.8 |
| 9.0 | 95 | 0.8 |
| 9.1 | 30 | 0.3 |
| 11 | 30 | 0.3 |
In LC-MS/MS analysis process, the reservation of compound is too weak, it is easy to by the interference of the big compound of other polarity, thus affecting the accuracy that compound is quantitative.In order to simultaneously quantitative analysis compound 4 and compound 9 accurately, the reservation of compound 9 need to be strengthened.So for the reservation strengthening compound 9, this research carries out method exploitation under existing experiment condition, it is intended to setting up a simplicity, quickly, specificity is strong, it is adaptable in Lapatinib, the Light absorbing impurty of compound 4 and compound 9 analyzes method.
In research process, through a large amount of repetition tests, inventor have found that when employing AquasilC18 chromatographic column, Mobile phase B (organic facies) use acetonitrile, effect is preferably.
Further, in order to make compound 4 and compound 9 have certain reservation on a column, and (namely residual can there is chromatographic peak at corresponding appearance time in the blank sample (solvent) after injection enriched sample not to have residual effect.In quantitative analysis process, if compound has residual can affect quantitative accuracy, general requirement remains less than the 20% of lower limit of quantitation), the present inventor is through extremely hard and bitter effort and experimentation, it is surprised to find that employing AquasilC18 chromatographic column, use the gradient condition shown in table 2, organic facies (B) is acetonitrile, aqueous phase (A) is water (containing 5mM ammonium formate), when sampling volume is 5.00 μ L, effect is preferably, typical chromatogram is as shown in Figure 1, Lapatinib, compound 4, the appearance time respectively 2.09min of compound 9 and lignocaine (interior mark), 0.54min, 0.81min, 1.21min.Owing to the concentration of crude drug is higher, so being switched it to waste liquid bottle from 1.5 minutes by six-way valve, in order to avoid affecting mass spectrum.
The gradient condition of table 2:AquasilC18
| Time (min) | Mobile phase B (%) | Flow velocity (ml/min) |
| 0 | 55 | 0.6 |
| 1.0 | 55 | 0.6 |
| 1.5 | 95 | 0.6 |
| 2.5 | 95 | 0.6 |
| 2.6 | 55 | 0.6 |
| 3.6 | 55 | 0.6 |
Therefore, the invention provides a kind of method of 4-(3-fluorobenzyloxy) 3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride impurity content in detection Lapatinib.According to embodiments of the invention, the method utilizes LC-MS/MS coupling detection method to carry out.
According to embodiments of the invention, wherein chromatographic condition is: chromatographic column is with octadecyl silane for filler；Mobile phase is made up of mobile phase A and Mobile phase B, and wherein, described mobile phase A is ammonium formate aqueous solution, and described Mobile phase B is acetonitrile；Type of elution is gradient elution.Thus, the compound 4 in Lapatinib crude drug and compound 9 can be carried out detection by quantitative, compound 4 and compound 9 all has good reservation simultaneously, and simple to operate, convenient and swift.
According to embodiments of the invention, wherein chromatographic condition is: with octadecyl silane for filler；Mobile phase is made up of mobile phase A and Mobile phase B, and wherein, described mobile phase A is concentration is the ammonium formate aqueous solution of 5 mM/ls, and described Mobile phase B is acetonitrile；Flow velocity 0.5ml/min～0.7ml/min；Column temperature 25 degrees Celsius～35 degrees Celsius；Sample size 5 μ l, detects wavelength 261nm, and type of elution is gradient elution.Inventor have found that, utilize the method for the present invention, it is possible to the impurity in detection Lapatinib crude drug fast and effectively, and can Simultaneous Quantitative Analysis compound 4 and compound 9, testing result is accurately and reliably, easy and simple to handle, and quickly, specificity is strong, reproducible.
According to embodiments of the invention, wherein Mass Spectrometry Conditions is: ion source is ESI source, adopts positive ion mode detection, multiple reaction monitoring MRM pattern；The detection ion pair of Lapatinib is m/z581 365；The detection ion pair of 4-(3-fluorobenzyloxy) 3-chloroaniline is m/z252 143；The detection ion pair of 2-(methylsulfonyl) ethamine is m/z124 79；And the detection ion pair of lignocaine is m/z235 86；Source parameters is: gas curtain gas (CUR) 30psi；Ion injection electric (IS) 5500V；Temperature (TEM) 550 DEG C；Ion source GS150psi；Ion source GS2260psi.Thereby, it is possible to accurate quantitative analysis detects the content of the impurity compound 4 in Lapatinib crude drug and compound 9, and it being limited the quantity, highly sensitive, accuracy is good, reproducible.
According to embodiments of the invention, the column temperature of chromatographic column is 25 degrees Celsius～35 degrees Celsius.Thus, being conducive to Simultaneous Quantitative Analysis compound 4 and compound 9, Detection results too high or too low for temperature is all undesirable.
According to embodiments of the invention, the flow velocity of described mobile phase is 0.5ml/min～0.7ml/min.Thus, the separating effect of compound 4, compound 9 and Lapatinib is desirable, and the detection time is short, and detection efficiency is high.
According to embodiments of the invention, in described mobile phase A, the concentration of described ammonium formate is 5 mM/ls.Thus, the good separating effect of compound 4, compound 9 and Lapatinib, specificity is strong, and accuracy in detection is high.
According to embodiments of the invention, the condition of described gradient elution is:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 40～50 | 60～50 |
| 1.0 | 40～50 | 60～50 |
| 1.5 | 2～8 | 98～92 |
| 2.5 | 2～8 | 98～92 |
| 2.6 | 40～50 | 60～50 |
| 3.6 | 40～50 | 60～50 |
Thus, compound 4, compound 9 and Lapatinib do not have residual effect, and testing result is accurately good, highly sensitive.
According to embodiments of the invention, described chromatographic column is C18 chromatographic column, it is preferable that described chromatographic column is ThermoaquasilC18 (50*2.1mm, 5 μm) HPLC chromatogram post.Thus, compound 4 and compound 9 all have good reservation, it is possible to effectively compound 4 and compound 9 are carried out quantitative analysis, and simple to operate simultaneously, and accuracy is high, reproducible.
According to embodiments of the invention, method of the present invention comprises the following steps:
(1) chromatographic condition: with octadecyl silane for filler；Described mobile phase A is concentration is the ammonium formate aqueous solution of 5 mM/ls, and described Mobile phase B is acetonitrile；Flow velocity 0.5ml/min～0.7ml/min；Column temperature 25 degrees Celsius～35 degrees Celsius；Sample size 5 μ l, detects wavelength 261nm, carries out gradient elution according to following table:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 40～50 | 60～50 |
| 1.0 | 40～50 | 60～50 |
| 1.5 | 2～8 | 98～92 |
| 2.5 | 2～8 | 98～92 |
| 2.6 | 40～50 | 60～50 |
| 3.6 | 40～50 | 60～50 |
(2) Mass Spectrometry Conditions: ion source is ESI source, adopts positive ion mode detection, multiple reaction monitoring MRM pattern；The detection ion pair of 4-(3-fluorobenzyloxy)-3-chloroaniline is m/z:252 143；The detection ion pair of 2-(methylsulfonyl) ethamine is m/z:124 79；In lignocaine, target detection ion pair is m/z:235 86；
Source parameters is:
Gas curtain gas 30psi；
Ion injection electric 5500V；
Temperature 550 DEG C；
Ion source GS150psi；
Ion source GS260psi,
(3) mark in Lapatinib crude drug and lignocaine is weighed respectively, with acetonitrile: it is the storing solution of 5.00mg/mL that water (volume ratio 4:1) is configured to Lapatinib crude drug concentration, again with acetonitrile: described storing solution is diluted to described need testing solution by water (volume ratio 1:1), in described need testing solution, the concentration of described Lapatinib crude drug is 0.500mg/mL, and in described lignocaine, target concentration is 1.00ng/mL；
(4) drawing described need testing solution 5 μ l and inject LC-MS LC-MS instrument, chromatographic condition and Mass Spectrometry Conditions described in (1) and (2) detect, and record chromatogram；
(5) according to chromatogram obtained in step (4), standard curve method is utilized to calculate the content of 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride in described Lapatinib crude drug.
Inventor have found that, utilize the method for the present invention, it is possible to compound 4 and compound 9 are carried out quantitative analysis simultaneously, and then the impurity in Lapatinib crude drug is limited the quantity, and simple to operate, highly sensitive, reproducible, specificity is strong.
According to embodiments of the invention, described standard curve method comprises the following steps:
A 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride are dissolved in the acetonitrile-aqueous solution that volume ratio is 4:1 by () respectively, the concentration of the first reference substance storing solution that concentration is 1.00mg/mL and 2-(methylsulfonyl) ethylamine hydrochloride that obtain 4-(3-fluorobenzyloxy)-3-chloroaniline is the second reference substance storing solution with 2.00mg/mL；
B () utilizes the acetonitrile-aqueous solution that volume ratio is 1:1 described first reference substance storing solution and described second reference substance storing solution to be diluted, obtain the mixing intermediate solution 1 containing 10.0 μ g/mL4-(3-fluorobenzyloxy)-3-chloroaniline and 500 μ g/mL2-(methylsulfonyl) ethylamine hydrochlorides；
C described mixing intermediate solution 1 is diluted 100 times by (), obtain mixing intermediate solution 2；
D described mixing intermediate solution 2 is diluted 10 times by (), obtain mixing intermediate solution 3；
E mark in lignocaine is dissolved in the acetonitrile-aqueous solution that volume ratio is 1:1 by (), obtaining mark concentration in lignocaine is the interior mark storing solution of 0.200mg/mL, is more also diluted in lignocaine and marks the interior mark intermediate solution that concentration is 20.0ng/mL by described interior mark deposit；
F () utilizes described mixing intermediate solution 2, described mixed intermediate solution 3 and described interior mark intermediate solution, prepare standard curve sample according to following table:
G () is drawn described standard curve sample solution 5 μ l processed and is injected separately into LC-MS LC-MS instrument, chromatographic condition and Mass Spectrometry Conditions described in (1) and (2) detect, and records chromatogram；
H () is with the ratio of 4-(3-fluorobenzyloxy)-3-chloroaniline and interior mark peak area for vertical coordinate, the concentration of 4-(3-fluorobenzyloxy)-3-chloroaniline is that abscissa carries out linear regression, make the first standard curve, with the ratio of 2-(methylsulfonyl) ethylamine hydrochloride and interior mark peak area for vertical coordinate, the concentration of 2-(methylsulfonyl) ethylamine hydrochloride is that abscissa carries out linear regression, makes the second standard curve；
I target peak area in target lignocaine in 4-(3-fluorobenzyloxy)-3-chloroaniline, 2-(methylsulfonyl) ethylamine hydrochloride and lignocaine in chromatogram obtained in step (4) is substituted into described first standard curve and described second standard curve by () respectively, calculate and obtain the content of 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride in described Lapatinib crude drug.
Utilize above-mentioned standard curve method, it is possible to fast and accurately compound 4 and compound 9 are carried out quantitatively, and accurately and reliably, simple and convenient.
Beneficial effects of the present invention:
The method of impurity in antineoplastic agent Lapatinib is detected by LC-MS/MS coupling of the present invention, can fast and effeciently detect the compound 4 in Lapatinib crude drug and compound 9 impurity, and can the content of simultaneous quantitative detection analysis of compounds 4 and compound 9.Applying LC-MS/MS coupling determination method of the present invention, testing result is accurately and reliably, easy and simple to handle, and quickly, specificity is strong, reproducible, and noresidue effect.LC-MS/MS coupling of the present invention measures the method for impurity content in Lapatinib, can be used for the quality control of Lapatinib drug manufacture.
Accompanying drawing explanation
Fig. 1 shows Lapatinib, compound 4, the typical chromatogram of compound 9 and interior mark lignocaine appearance time；
Fig. 2 shows according to embodiments of the invention, compound 4, the full scan mass spectrum of compound 9 and Lapatinib；
Fig. 3 shows according to embodiments of the invention, the second order ms figure of compound 4；
Fig. 4 shows according to embodiments of the invention, the second order ms figure of compound 9；
Fig. 5 shows according to embodiments of the invention, the second order ms figure of Lapatinib；
Fig. 6 shows that the specificity of compound 4 (10.0ng/ml) investigates chromatogram according to embodiments of the invention；
Fig. 7 shows that the specificity of compound 9 (500ng/ml) investigates chromatogram according to embodiments of the invention；
Fig. 8 shows that the specificity of interior mark lignocaine (1.00ng/ml) investigates chromatogram according to embodiments of the invention；
Fig. 9 shows according to embodiments of the invention, the residual effect chromatogram of compound 4 and compound 9.
Detailed description of the invention
Embodiments of the invention are described below in detail.The embodiments described below is illustrative of, and is only used for explaining the present invention, and is not considered as limiting the invention.Unreceipted concrete technology or condition in embodiment, technology or condition described by the document in this area or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can pass through city available from conventional products.
Embodiment 1
Utilize mass spectrograph (ABSciexQ-Trap4500) respectively to compound 4, compound 9 and Lapatinib do full scan (Q1Scan) in the positive-ion mode, find corresponding parent ion, parent ion is done corresponding two grades again, the higher daughter ion of selection intensity is as final detection ion, and Fig. 2 to Fig. 5 is shown in by collection of illustrative plates.The ion pair that last each compound selection intensity is higher, and optimize corresponding parameter (source parameters, the parameter relevant to compound), result is in Table 3 and table 4.
Table 3: source parameters
| Polarity | Positive pole |
| Scan type | MRM |
| Gas curtain gas (CUR) | 30psi |
| Ion source (GS1) | 50psi |
| Ion source (GS2) | 60psi |
| Ion injection electric | 5500V |
| Temperature | 550℃ |
Table 4: the parameter that compound is relevant
Embodiment 2
Utilize chromatographic column (SynergiHydro-RP, 50*3.0,4 μm of 5 kinds of types；SynergiPolar-RP50*3.0,4 μm；AquasilC1850*2.1mm, 5 μm；Kromasilsilica50*3.0mm, 5 μm；Betasilsilica-10050*2.1mm, 5 μm), with acetonitrile for Mobile phase B, with water (containing 5mM ammonium formate) for mobile phase A, respectively compound 4, compound 9 are carried out LC-MS/MS detection, and record chromatogram.
Result:
When adopting Hydro-RP chromatographic column and Polar-RP chromatographic column, compound 9 is substantially free of reservation in Hydro-RP, Polar-RP chromatographic column.
When adopting AquasilC18 chromatographic column, when using low organic facies (20% acetonitrile isocratic elution), compound 9 does not retain, and compound 9 has certain reservation when high organic facies (95% acetonitrile isocratic elution).
When adopting Betasilsilica-100 chromatographic column and Kromasilsilica chromatographic column, the reservation of compound 9 is relatively strong, but compound 4 does not retain.
Therefore present invention preferably employs AquasilC18 chromatographic column.
Embodiment 3: methodological study and sample analysis
Limit according to compound 4 is not higher than 4ug/1g, and the limit of compound 9 is not higher than 0.02%, the mark song scope respectively 0.200ng/ml 10.0ng/ml (compound 4) of fixed two compounds, 10ng/ml 500ng/ml (compound 9).Instrument and reagent
Reagent: acetonitrile (Fisher), water, ammonium formate, Lapatinib crude drug (14 batches), 4-(3-fluorobenzyloxy)-3-chloroaniline (lot number: 120814), 2-(methylsulfonyl) ethylamine hydrochloride (lot number: 20120521CMQA-1-MZP-01-014).
Instrument: mass spectrum: ABSciexQ-Trap4500,
Efficient liquid phase: Shimadzu20ACXR
(1) preparation of reference substance storing solution: take reference substance (compound 4, compound 9) appropriate, dissolves with solvent (acetonitrile: water=4:1), makes the storing solution of 1.00mg/ml.Then with acetonitrile: compound 4 is diluted to the intermediate solution of 10000ng/ml, 100ng/ml, 10.0ng/ml by water (1:1), and compound 9 is diluted to the intermediate solution of 10000ng/ml, 5000ng/ml, 500ng/ml.
(2) preparation of inner mark solution: take mark in lignocaine appropriate, dissolve with (acetonitrile: water=1:1), make the storing solution of 0.200mg/ml, then be diluted to the intermediate solution of 20.0ng/ml.
(3) preparation of standard curve sample: prepare standard curve sample by table 5.
Table 5: standard curve sample
(4) preparation of quality-control sample (QC): prepare quality-control sample by table 6.
Table 6: quality-control sample
(5) sample preparation
Precision weighs a certain amount of Lapatinib crude drug, with acetonitrile: water (volume ratio 4:1) dissolves the storing solution making 1.00mg/ml, again with acetonitrile: water (volume ratio 1:1) dilutes 2.5 times, centrifugal 5 minutes (13000 revs/min), take supernatant, obtaining, sample message is in Table 7.
Table 7: sample message
(6) range of linearity
Preparing standard curve sample by table 5, with the ratio of the peak area of analyte and interior target peak area for vertical coordinate, the concentration of analyte is abscissa, carries out linear regression operation.Compound 4 and compound 9 the two compound are good in corresponding scope internal linear relation.
Regression equation is respectively as follows:
Compound 4:y=0.0416x+0.00195 (r=0.9986)；
Compound 9:y=0.00416x+0.00175 (r=0.9986).
It is shown that compound 4 is good in 0.200 10.0ng/mL scope internal linear relation, compound 9 is good in 10.0 500ng/mL scope internal linear relations.
(7) veracity and precision (in a few days) is investigated
Preparing quality-control sample by table 6, each concentration prepares 3 parts, investigates its accuracy and precision (in a few days), and result is in Table 8.Showing according to result, the accuracy of compound 4 and compound 9 is between 85% to 115%, and precision RSD is in ± 15% scope.
Table 8: accuracy and precision
(8) specificity is investigated
Specificity means under other compositions condition that may be present, and the method energy Accurate Determining adopted goes out the characteristic of measured object.The method measures compound 4 and compound 9 simultaneously, according to Fig. 6 to Fig. 8 it can be seen that compound 4 (10.0ng/ml), compound 9 (500ng/ml) and not interfering between interior mark lignocaine (1.00ng/ml).
(9) residual effect is investigated
In this experimentation, after having injected enriched sample (such as upper limit of quantification, ULOQ), two blank samples of injection detect residual effect continuously.As it is shown in figure 9, compound 4 and compound 9 do not remain under this chromatographic condition.
(9) sample analysis
After table 7 preparation pressed by sample, sample introduction analysis, the peak area of the compound 4 measured and compound 9 is substituted in standard curve with interior mark peak area ratio and calculates, obtain the concentration of compound 4 and compound 9 in sample (crude drug).Then two impurity content in each batch of crude drug is calculated.According to the rules, compound 4 is less than 4 μ g/1g, and compound 9 is less than 0.02%, and result of calculation is in Table 9.It is shown that the content of compound 4 all limits not less than 4 μ g/1g in the sample of 14 batches, the content of compound 9 also equal limit without departing from 0.02%.In the Lapatinib crude drug product of 14 batches, compound 4 and compound 9 are all in limit.
Table 9: sample analysis result table
Embodiment 4
1. instrument and reagent
Reagent: acetonitrile (Fisher), water, ammonium formate, Lapatinib crude drug (3 batches), 4-(3-fluorobenzyloxy)-3-chloroaniline (compound 4 lot number: CM1101-130501-F1), 2-(methylsulfonyl) ethylamine hydrochloride (compound 9 lot number:
CM1101-130801-F1)。
Instrument: mass spectrum: ABSciexQ-Trap4500, efficient liquid phase: Shimadzu20ACXR
2. experiment content
2.1 chromatographs and Mass Spectrometry Conditions
Chromatographic condition and system suitability are with octadecyl silane for filler；Mobile phase is made up of mobile phase A and Mobile phase B, and wherein, described mobile phase A is concentration is the ammonium formate aqueous solution of 5 mM/ls, and described Mobile phase B is acetonitrile；Flow velocity 0.6ml/min；Column temperature 30 degrees Celsius；Sample size 5 μ l, detects wavelength 261nm, and type of elution is gradient elution.Gradient elution is carried out according to table 10 below:
Table 10
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 45 | 55 |
| 1.0 | 45 | 55 |
| 1.5 | 5 | 95 |
| 2.5 | 5 | 95 |
| 2.6 | 45 | 55 |
| 3.6 | 45 | 55 |
Connect with triple level Four bar mass spectrographs and test, adopt multiple reaction monitoring MRM pattern: the detection ion pair of impurity 4-to be measured (3-fluorobenzyloxy)-3-chloroaniline is m/z:252 143；The detection ion pair of 2-(methylsulfonyl) ethamine is m/z:124 79；In lignocaine, target detection ion pair is m/z:235 86；Typical mass detector parameter is as follows:
Source parameters:
CUR:30psi,
IS:5500V,
TEM:550 DEG C,
GS1:50psi,
GS2:60psi.
2.2 sample preparations
Limit according to compound 4 is not higher than 4 μ g/1g, the limit of compound 9 is not higher than 0.02%, the mark song scope respectively 0.200ng/mL 10.0ng/mL (compound 4) of fixed two compounds, 10.0ng/mL 500ng/mL (compound 9).
(1) preparation of reference substance storing solution: take reference substance (compound 4, compound 9) appropriate, dissolves with solvent (acetonitrile: water=4:1), makes the storing solution of 1.00mg/mL, 2.00mg/mL；
(2) preparation of intermediate solution 1 is mixed: with acetonitrile: storing solution is diluted to the mixing intermediate solution 1 containing 10.0 μ g/mL compound 4,500 μ g/mL compounds 9 by water (1:1)；
(3) preparation of intermediate solution 2 is mixed: the mixing intermediate solution 1 in (2) is diluted 100 times, makes into 100ng/mL compound 4,5000ng/mL compound mixing intermediate solution 2 just；
(4) preparation of intermediate solution 3 is mixed: the mixing intermediate solution 2 in (3) is diluted 10 times, makes into the mixing intermediate solution 3 of 10.0ng/mL compound 4,500ng/mL compound 9.
(5) preparation of inner mark solution: take lignocaine reference substance appropriate, dissolves with (acetonitrile: water=1:1), makes the storing solution of 0.200mg/mL, then be diluted to the intermediate solution of 20.0ng/mL.
(6) preparation of standard curve sample: prepare standard curve sample by table 11.
Table 11: standard curve sample preparation table
(7) test sample preparation: weigh this product appropriate (lot number is respectively as follows: LT20140902), accurately weighed, with acetonitrile: water (4:1) is configured to 5.00mg/mL storing solution, then with acetonitrile: water (1:1) is diluted to 0.500mg/mL solution to be measured (containing the internal standard 1.00ng/mL).
2.3 methodological studies
2.3.1 linearly investigate: by condition preparation standard curve sample under " 2.2 " item.Compound 4 and compound 9 are carried out linear regression.It is shown that compound 4 is good in 0.200 10.0ng/mL scope internal linear relation, compound 9 is good in 10.0 500ng/mL scope internal linear relations.
2.4 sample analysis
Test sample is pressed under " 2.2 " item after preparation, sample introduction analysis, is substituted in standard curve with interior mark peak area ratio by the peak area of the compound 4 measured and compound 9 and calculates, obtains the concentration of compound 4 and compound 9 in sample (crude drug).Then two impurity content in each batch of crude drug is calculated.According to the rules, compound 4 is less than 4 μ g/1g, and compound 9 is less than 0.02%, and result of calculation is in Table 12.In the Lapatinib crude drug product of 3 batches, compound 4 and compound 9 are all in limit.
Table 12: sample analysis result table
Embodiment 5
1. instrument and reagent
Reagent: acetonitrile (Fisher), water, ammonium formate, Lapatinib crude drug (3 batches), 4-(3-fluorobenzyloxy)-3-chloroaniline (compound 4 lot number: CM1101-130701-F2), 2-(methylsulfonyl) ethylamine hydrochloride (compound 9 lot number: CM1101-130901-F2).
Instrument: mass spectrum: ABSciexQ-Trap4500, efficient liquid phase: Shimadzu20ACXR
2. experiment content
2.1 chromatographs and Mass Spectrometry Conditions
Chromatographic condition and system suitability are with octadecyl silane for filler；Mobile phase is made up of mobile phase A and Mobile phase B, and wherein, described mobile phase A is concentration is the ammonium formate aqueous solution of 5 mM/ls, and described Mobile phase B is acetonitrile；Flow velocity 0.5ml/min；Column temperature 25 degrees Celsius；Sample size 5 μ l, detects wavelength 261nm, and type of elution is gradient elution.Gradient elution is carried out according to table 13 below:
Table 13
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 40 | 60 |
| 1.0 | 40 | 60 |
| 1.5 | 2 | 98 |
| 2.5 | 2 | 98 |
| 2.6 | 40 | 60 |
[0160]
| 3.6 | 40 | 60 |
Connect with triple level Four bar mass spectrographs and test, adopt multiple reaction monitoring MRM pattern: the detection ion pair of impurity 4-to be measured (3-fluorobenzyloxy)-3-chloroaniline is m/z:252 143；The detection ion pair of 2-(methylsulfonyl) ethamine is m/z:124 79；In lignocaine, target detection ion pair is m/z:235 86；Typical mass detector parameter is as follows:
Source parameters:
CUR:30psi,
IS:5500V,
TEM:550 DEG C,
GS1:50psi,
GS2:60psi.
2.2 sample preparation methods, with embodiment 4.
2.3 linearly investigate
It is shown that compound 4 is good in 0.200 10.0ng/mL scope internal linear relation, compound 9 is good in 10.0 500ng/mL scope internal linear relations.
2.4 sample analysis
The sample analysis result of 3 batches, in Table 14.Result shows that in the Lapatinib crude drug product of 3 batches, compound 4 and compound 9 are all in limit.
Table 14: sample analysis result table
Embodiment 6
1. instrument and reagent
Reagent: acetonitrile (Fisher), water, ammonium formate, Lapatinib crude drug (4 batches), 4-(3-fluorobenzyloxy)-3-chloroaniline (compound 4 lot number: CM1101-130701-F2), 2-(methylsulfonyl) ethylamine hydrochloride (compound 9 lot number:
CM1101-130901-F2)。
Instrument: mass spectrum: ABSciexQ-Trap4500, efficient liquid phase: Shimadzu20ACXR
2. experiment content
2.1 chromatographs and Mass Spectrometry Conditions
Chromatographic condition and system suitability are with octadecyl silane for filler；Mobile phase is made up of mobile phase A and Mobile phase B, and wherein, described mobile phase A is concentration is the ammonium formate aqueous solution of 5 mM/ls, and described Mobile phase B is acetonitrile；Flow velocity 0.7ml/min；Column temperature 35 degrees Celsius；Sample size 5 μ l, detects wavelength 261nm, and type of elution is gradient elution.Gradient elution is carried out according to table 15 below:
Table 15
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 1.0 | 50 | 50 |
| 1.5 | 8 | 92 |
| 2.5 | 8 | 92 |
| 2.6 | 50 | 50 |
| 3.6 | 50 | 50 |
Connect with triple level Four bar mass spectrographs and test, adopt multiple reaction monitoring MRM pattern: the detection ion pair of impurity 4-to be measured (3-fluorobenzyloxy)-3-chloroaniline is m/z:252 143；The detection ion pair of 2-(methylsulfonyl) ethamine is m/z:124 79；In lignocaine, target detection ion pair is m/z:235 86；Typical mass detector parameter is as follows:
Source parameters:
CUR:30psi,
IS:5500V,
TEM:550 DEG C,
GS1:50psi,
GS2:60psi.
2.2 sample preparation methods, with embodiment 4.
2.3 linearly investigate
It is shown that compound 4 is good in 0.200 10.0ng/mL scope internal linear relation, compound 9 is good in 10.0 500ng/mL scope internal linear relations.
2.4 sample analysis
The sample analysis result of 4 batches, in Table 16.Result shows that in the Lapatinib crude drug product of 4 batches, compound 4 and compound 9 are all in limit.
Table 16: sample analysis result table
In the description of this specification, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means in conjunction with this embodiment or example describe are contained at least one embodiment or the example of the present invention.In this manual, the schematic representation of above-mentioned term is necessarily directed to identical embodiment or example.And, the specific features of description, structure, material or feature can combine in one or more embodiments in office or example in an appropriate manner.Additionally, when not conflicting, the feature of the different embodiments described in this specification or example and different embodiment or example can be carried out combining and combining by those skilled in the art.
Although above it has been shown and described that embodiments of the invention, it is understandable that, above-described embodiment is illustrative of, it is impossible to be interpreted as limitation of the present invention, and above-described embodiment can be changed, revises, replace and modification by those of ordinary skill in the art within the scope of the invention.
Claims (9)
1. the method for 4-(3-fluorobenzyloxy) 3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride impurity content in a detection Lapatinib, it is characterised in that described method utilizes LC-MS/MS coupling detection method to carry out.
2. method according to claim 1, it is characterised in that wherein,
Chromatographic condition is:
Chromatographic column is with octadecyl silane for filler；
Mobile phase is made up of mobile phase A and Mobile phase B, and wherein, described mobile phase A is ammonium formate aqueous solution, and described Mobile phase B is acetonitrile；
Type of elution is gradient elution,
Mass Spectrometry Conditions is:
Ion source is ESI source, adopts positive ion mode detection, multiple reaction monitoring MRM pattern；
The detection ion pair of Lapatinib is m/z581 365；The detection ion pair of 4-(3-fluorobenzyloxy) 3-chloroaniline is m/z252 143；The detection ion pair of 2-(methylsulfonyl) ethamine is m/z124 79；And the detection ion pair of lignocaine is m/z235 86；
Source parameters is:
Gas curtain gas 30psi；
Ion injection electric 5500V；
Temperature 550 DEG C；
Ion source GS150psi；
Ion source GS260psi.
3. method according to claim 1, it is characterised in that the column temperature of described chromatographic column is 25 degrees Celsius～35 degrees Celsius.
4. method according to claim 1, it is characterised in that the flow velocity of described mobile phase is 0.5ml/min～0.7ml/min.
5. method according to claim 1, it is characterised in that in described mobile phase A, the concentration of described ammonium formate is 5 mM/ls.
6. method according to claim 1, it is characterised in that the condition of described gradient elution is:
7. method according to claim 1, it is characterised in that described chromatographic column is C18 chromatographic column.
8. method according to claim 1, it is characterised in that including:
(1) chromatographic condition: with octadecyl silane for filler；Described mobile phase A is concentration is the ammonium formate aqueous solution of 5 mM/ls, and described Mobile phase B is acetonitrile；Flow velocity 0.5ml/min～0.7ml/min；Column temperature 25 degrees Celsius～35 degrees Celsius；Sample size 5 μ l, detects wavelength 261nm, carries out gradient elution according to following table:
(2) Mass Spectrometry Conditions: ion source is ESI source, adopts positive ion mode detection, multiple reaction monitoring MRM pattern；The detection ion pair of 4-(3-fluorobenzyloxy)-3-chloroaniline is m/z:252 143；The detection ion pair of 2-(methylsulfonyl) ethamine is m/z:124 79；In lignocaine, target detection ion pair is m/z:235 86；
Source parameters is:
Gas curtain gas 30psi；
Ion injection electric 5500V；
Temperature 550 DEG C；
Ion source GS150psi；
Ion source GS260psi,
(3) mark in Lapatinib crude drug and lignocaine is weighed respectively, being configured to Lapatinib crude drug concentration with the acetonitrile-aqueous solution that volume ratio is 4:1 is the storing solution of 5.00mg/mL, with the acetonitrile-aqueous solution that volume ratio is 1:1, described storing solution is diluted to described need testing solution again, in described need testing solution, the concentration of described Lapatinib crude drug is 0.500mg/mL, and in described lignocaine, target concentration is 1.00ng/mL；
(4) drawing described need testing solution 5 μ l and inject LC-MS LC-MS instrument, chromatographic condition and Mass Spectrometry Conditions described in (1) and (2) detect, and record chromatogram；
(5) according to chromatogram obtained in step (4), standard curve method is utilized to calculate the content of 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride in described Lapatinib crude drug.
9. method according to claim 8, it is characterised in that described standard curve method comprises the following steps:
A 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride are dissolved in the acetonitrile-aqueous solution that volume ratio is 4:1 by () respectively, the concentration of the first reference substance storing solution that concentration is 1.00mg/mL and 2-(methylsulfonyl) ethylamine hydrochloride that obtain 4-(3-fluorobenzyloxy)-3-chloroaniline is the second reference substance storing solution with 2.00mg/mL；
B () utilizes the acetonitrile-aqueous solution that volume ratio is 1:1 described first reference substance storing solution and described second reference substance storing solution to be diluted, obtain the mixing intermediate solution 1 containing 10.0 μ g/mL4-(3-fluorobenzyloxy)-3-chloroaniline and 500 μ g/mL2-(methylsulfonyl) ethylamine hydrochlorides；
C described mixing intermediate solution 1 is diluted 100 times by (), obtain mixing intermediate solution 2；
D described mixing intermediate solution 2 is diluted 10 times by (), obtain mixing intermediate solution 3；
E mark in lignocaine is dissolved in the acetonitrile-aqueous solution that volume ratio is 1:1 by (), obtaining mark concentration in lignocaine is the interior mark storing solution of 0.200mg/mL, then is diluted to by described interior mark storing solution in lignocaine and marks the interior mark intermediate solution that concentration is 20.0ng/mL；
F () utilizes described mixing intermediate solution 2, described mixed intermediate solution 3 and described interior mark intermediate solution, prepare standard curve sample according to following table:
G () is drawn described standard curve sample solution 5 μ l and is injected separately into LC-MS LC-MS instrument, chromatographic condition and Mass Spectrometry Conditions described in (1) and (2) detect, and records chromatogram；
H () is with the ratio of mark peak area in 4-(3-fluorobenzyloxy)-3-chloroaniline and lignocaine for vertical coordinate, the concentration of 4-(3-fluorobenzyloxy)-3-chloroaniline is that abscissa carries out linear regression, make the first standard curve, in 2-(methylsulfonyl) ethylamine hydrochloride and lignocaine, the ratio of mark peak area is for vertical coordinate, the concentration of 2-(methylsulfonyl) ethylamine hydrochloride is that abscissa carries out linear regression, makes the second standard curve；
I target peak area in 4-(3-fluorobenzyloxy)-3-chloroaniline, 2-(methylsulfonyl) ethylamine hydrochloride and lignocaine in chromatogram obtained in step (4) is substituted into described first standard curve and described second standard curve by () respectively, calculate and obtain the content of 4-(3-fluorobenzyloxy)-3-chloroaniline and 2-(methylsulfonyl) ethylamine hydrochloride in described Lapatinib crude drug.
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| CN114594192A (en) * | 2022-03-07 | 2022-06-07 | 山东大学 | Method for detecting chloroaniline and chloronitroaniline isomers in posaconazole starting material |
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| SAADAT, EBRAHIM; KELISHADY, POOYA DEHGHAN; RAVAR, FATEMEH: "Development and Validation of Rapid Stability-Indicating RP-HPLC-DAD Method for the Quantification of Lapatinib and Mass Spectrometry Analysis of Degraded Products", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 * |
| 仲艳: "HPLC法测定二甲苯磺酸拉帕替尼含量及其有关物质", 《中国药师》 * |
| 刘俊华: "高效液相色谱法测定二甲苯磺酸拉帕替尼片有关物质", 《中国药学杂志》 * |
| 徐家根: "HPLC法测定二甲苯磺酸拉帕替尼片中拉帕替尼的含量", 《中国药房》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106932522A (en) * | 2015-12-31 | 2017-07-07 | 上海奥博生物医药技术有限公司 | The assay method of impurity compound I contents in a kind of nilotinib |
| CN114594192A (en) * | 2022-03-07 | 2022-06-07 | 山东大学 | Method for detecting chloroaniline and chloronitroaniline isomers in posaconazole starting material |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2016090730A1 (en) | 2016-06-16 |
| CN105738492B (en) | 2017-12-19 |
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