CN108008024A - The detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals - Google Patents
The detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals Download PDFInfo
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- CN108008024A CN108008024A CN201710771674.5A CN201710771674A CN108008024A CN 108008024 A CN108008024 A CN 108008024A CN 201710771674 A CN201710771674 A CN 201710771674A CN 108008024 A CN108008024 A CN 108008024A
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- G—PHYSICS
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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 Pharmaceutical Analysis technical field, and in particular to the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals, the detection method are measured using high performance liquid chromatography, and specific detection method is as follows：Chromatography column packing is octadecylsilane key and silica gel, and mobile phase is water and the mixed solvent gradient elution of acetonitrile, and column temperature is 25~35 DEG C, and flow rate of mobile phase is 0.5~1.5mL/min, and Detection wavelength is 210~230nm；Doxofylline bulk pharmaceutical chemicals are configured to the solution of 5~15mg/mL；Sample size is 30~60 μ L, records chromatogram.Detection method provided by the invention can efficiently separate out the genotoxicity impurity peaks in doxofylline bulk pharmaceutical chemicals, this method greatly improves the detection sensitivity of genotoxicity impurity, can simply, it is quick, stablize detect doxofylline bulk pharmaceutical chemicals genotoxicity impurity.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, and in particular to the detection of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals
Method.
Background technology
Doxofylline is the derivative of methyl xanthine.English name Doxofylline, chemical name 1,3- dimethyl-
7- (1,3- dioxy cyclopenta -2- bases) methyl -3,7- dihydro -1H- purine -2,6- diketone, molecular formula C11H14N4O4, molecule
Measure as 266.25, chemical structural formula：
Doxofylline is the derivative of methyl xanthine, it is a kind of bronchodilator, can be done directly on bronchus,
Relaxation bronchial smooth muscle is played by suppressing the phosphodiesterase in smooth muscle cell, suppresses the effect of asthma.Doxofylline
The effect of relaxation bronchial muscular spasm is 10-15 times strong compared with aminophylline, and has the unexistent antitussive effect of theophylline.More rope tea
Alkali is without adenosine receptor blocking effect, less to cause the bad of the lung external system such as maincenter, intestines and stomach and angiocarpy therefore compared with theophylline
Reaction, but heavy dose of administration can still cause blood pressure decline etc..In addition, in vivo and in vitro, which confirms that this medicine also has, suppresses blood platelet work
Change the bronchoconstriction of the factor (PAF) induction and the effect of secondary thromboxane A2 generation.Found in rat In vivo study
This medicine can inhibit pleurisy and the exudation of PAF inductions, may also suppress the generation of leukotriene C.Therefore doxofylline validity and
Security higher, clinical value bigger.
Doxofylline uses p-methyl benzenesulfonic acid and ethylene glycol in the synthesis process, it is possible to can react generation to toluene sulphur
Sour second diester and p-methyl benzenesulfonic acid diethylester.The structure contains p-methyl benzenesulfonic acid ester, is latent gene toxic impurities.According to the medicine
The usage and dosage of preparation, said gene toxic impurities limit are difficult to reach in 2ppm, general liquid phase process separating degree and sensitivity
It is required that.At present it is not yet found that closing the document report of genotoxicity defects inspecting in doxofylline bulk pharmaceutical chemicals.
Therefore, there is an urgent need for developing a kind of rational chromatographic system, gene in simple, sensitive doxofylline bulk pharmaceutical chemicals is made
The detection method of toxic impurities, to control the quality of doxofylline bulk pharmaceutical chemicals.
The content of the invention
In order to solve existing in the prior art to lack asking for genotoxicity method for detecting impurities in doxofylline bulk pharmaceutical chemicals
Topic, the present invention provides a kind of detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals, the detection method is using high
Effect liquid phase chromatogram method is measured, and specific detection method is as follows：
(1) chromatographic condition
Filler is octadecylsilane key and silica gel,
Mobile phase is the mixed solvent of water and acetonitrile, gradient elution,
Column temperature is 25~35 DEG C,
Flow rate of mobile phase is 0.5~1.5mL/min,
Detection wavelength is 210~230nm；
(2) sample preparation
Doxofylline bulk pharmaceutical chemicals are weighed, it is 5~15mg/ to be configured to concentration containing doxofylline with the mixed solvent of water and acetonitrile
The solution of mL；
(3) detection method
Precision measures 30~60 μ L of test solution, injects in high performance liquid chromatograph, records chromatogram.
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, step (1) color
The volume ratio of mobile phase water and acetonitrile is 70 in spectral condition：30~75：25.
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, step (1) color
The mobile phase of gradient elution is specially in spectral condition：
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, step (1) color
In spectral condition, the column temperature is arranged to 30 DEG C.
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, step (1) color
In spectral condition, the flow velocity of the mobile phase is 0.8~1.2mL/min.
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, step (1) color
In spectral condition, the Detection wavelength is 226nm.
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, step (2) sample
During product are prepared, bulk pharmaceutical chemicals are taken, are 70 with water and acetonitrile volume ratio:30 mixed solvent is configured to the 10mg/mL's containing doxofylline
Solution.
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, step (3) inspection
In survey method, precision measures 50 μ L of test solution, injects in high performance liquid chromatograph, records chromatogram.
Further, in doxofylline bulk pharmaceutical chemicals of the present invention genotoxicity impurity detection method, the gene
Toxic impurities are p-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid ester) and/or methyl tosylates.
Compared with prior art, beneficial effects of the present invention：
The detection method of genotoxicity impurity, can efficiently separate more ropes in doxofylline bulk pharmaceutical chemicals provided by the invention
Theophylline principal component peak and genotoxicity impurity peaks, at the same can efficiently separate genotoxicity impurity peaks with and other impurity peaks, should
Method greatly improves the detection sensitivity of genotoxicity impurity, so as to detection doxofylline that is simple, quick, stablizing
Genotoxicity impurity in bulk pharmaceutical chemicals, effectively to control the quality of doxofylline bulk pharmaceutical chemicals, avoids objectionable impurities and does not detect
The clinical risk that may be brought.
The present invention detects genotoxicity impurity in doxofylline bulk pharmaceutical chemicals by high performance liquid chromatography, using octadecyl
Silane group silica gel is filler, and this chromatographic column is more common in liquid chromatogram, adds the acetonitrile of proper proportion, Ke Yibao
The stability and service life of chromatographic column are protected, improves the durability of chromatographic column.
Mobile phase ratio is to genotoxicity impurity in result of the test, doxofylline in the detection method of the present invention and other are miscellaneous
Separating degree between mass peak has a significant impact, by further study show that, through setting different proportion mobile phase carry out ladder
Degree elution, can reach being precisely separating for other impurities in genotoxicity impurity and doxofylline and doxofylline, finally confirms
In 0 to 20 minutes, mobile phase ratio is that water than acetonitrile is 70：30~75：25, at 20 minutes to 30 minutes, water and acetonitrile ratio
Example is adjusted to 40:60, kept for 15 minutes, eluant, eluent ratio is then recalled to 70：30~75：25, progress gradient is washed under the conditions of this
It is de-, to genotoxicity impurity p-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid esters) and methyl tosylate
Separating degree is all higher than 30, meets the requirements.And this method can reach 2ppm to the test limit of genotoxicity impurity, have higher
Sensitivity, can strictly control genotoxicity impurity with high quality.
There is the sensitivity of Detection wavelength and sample size to genotoxicity impurity in testing result in the detection method of the present invention
Very big influence, by study find, scanned by diode array, find genotoxicity impurity have at 210~230nm compared with
Big to absorb, three kinds of genotoxicity impurity have maximum absorption wavelength wherein at 226nm, final to confirm that Detection wavelength is 226nm, into
Sample amount is 50 μ L, and under this condition, the quantitative limit of genotoxicity impurity can reach 0.5ppm.
Brief description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram obtained according to the progress doxofylline bulk pharmaceutical chemicals defects inspecting of embodiment 1.
Embodiment
Further detailed description is done to the present invention with reference to specific embodiment, but embodiments of the present invention are not limited to
This.The embodiment of the present invention is merely to illustrate the present invention and provides, rather than limitation of the present invention, so, the present invention's
Any improvement in the present invention belongs to the scope of protection of the invention under the premise of method.
Acetonitrile is trifluoroacetic acid aqueous solution in the mobile phase of efficient liquid phase in following embodiments.It is real without specified otherwise in embodiment
It is design temperature to apply the temperature in example, it is allowed to which temperature error is ± 3 DEG C.
This detection method is the external standard method in Pharmaceutical Analysis field, the mode of operation in following experimentation and test method,
It is not described in detail, is prepared using Standard Operating Procedure, such as solution, the operating method of system suitability.
Embodiment 1：The detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals
(1) chromatographic condition
Instrument：1260 high performance liquid chromatograph of Agilent；
Chromatographic column：Octadecylsilane chemically bonded silica (250mm*4.6mm*5 μm)
Mobile phase：Using pure water as mobile phase A, the acetonitrile of chromatographically pure is Mobile phase B, and gradient elution is carried out according to table 1 below；
Table 1：The condition of gradient elution program
Column temperature：30℃；
The flow velocity 1.0mL/min of mobile phase；
Detection wavelength：226nm.
(2) sample preparation
Contrast solution is prepared：Reference substance is respectively：P-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid ester) and
Methyl tosylate；It is appropriate that three kinds of genotoxicity contamination levels product are weighed respectively, are 70 with water and acetonitrile volume ratio:30 it is mixed
Bonding solvent dissolves and is diluted to the reference substance solution containing each impurity 20ng/mL..
Sample solution is prepared：It is 70 with water and acetonitrile volume ratio:30 mixed solvent is by doxofylline bulk pharmaceutical chemicals to be measured
The solution for being about 10mg/mL containing doxofylline is configured to, accurately weighed preparation more ropes to be detected are carried out according to bulk pharmaceutical chemicals weight
Theophylline raw material medicine solution, general doxofylline concentration is in 5~15mg/mL.
(3) detection method
Precision measures 50 μ L of test solution, injects in high performance liquid chromatograph, chromatogram is as shown in Figure 1.
(4) calculate
According to external standard method, using impurity peak area in contrast solution as external standard, impurity content in sample solution is calculated.
(5) experimental result
Impurity determines：
According to the comparative analysis in relation to material present situation and impurity reference substance in doxofylline, it may be determined that doxofylline raw material
Impurity peaks 8,9 and 10 in medicine are genotoxicity impurity, and specific impurity title is as shown in table 2.
Table 2：Impurity information table
| Impurity title | No. CAS | Chemical name |
| Impurity 8 | 42772-85-0 | P-methyl benzenesulfonic acid hydroxyl ethyl ester |
| Impurity 10 | 7460-82-4 | Diethylene glycol is double (p-methyl benzenesulfonic acid ester) |
| Impurity 9 | 80-48-8 | Methyl tosylate |
Chromatogram result：
Obtained according to above-mentioned chromatographic condition, the chromatographic peak of gene impurity has chromatogram as shown in Figure 1.
Analysis result：
Impurity peaks in Fig. 1 chromatograms are analyzed, the results are shown in Table 3.
Table 3：The analysis result of genotoxicity impurity in embodiment 1HPLC chromatograms
From table 3 it can be seen that can effectively it be detected with doxofylline genotoxicity impurity in the chromatographic system, three gene poison
Retention time difference is obvious between property impurity, can efficiently separate, three genotoxicity impurity and doxofylline and more rope tea
Other impurities in alkali can also efficiently separate, and signal-to-noise ratio is more than 36, meets the requirements.Signal-to-noise ratio is more than under the chromatographic condition
40, impurity signal is not easy to be disturbed, and sensitivity is higher；It can detect that genotoxicity impurity of the content in 2ppm illustrates the present invention
Technical solution can effectively, sensitively detect genotoxicity impurity in doxofylline bulk pharmaceutical chemicals.
Embodiment 2：The detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals
(1) chromatographic condition
Instrument：1260 high performance liquid chromatograph of Agilent；
Chromatographic column：Octadecylsilane chemically bonded silica (250mm*4.6mm*5 μm)
Mobile phase：Using pure water as mobile phase A, the acetonitrile of chromatographically pure is Mobile phase B, and gradient elution is carried out according to table 4；
Table 4：The condition of gradient elution program
Column temperature：25℃；
The flow velocity 0.5mL/min of mobile phase；
Detection wavelength：210nm.
(2) sample preparation
Contrast solution is prepared：Reference substance is respectively：P-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid ester) and
Methyl tosylate；It is appropriate that three kinds of genotoxicity contamination levels product are weighed respectively, are 70 with water and acetonitrile volume ratio:30 it is mixed
Bonding solvent dissolves and is diluted to the reference substance solution containing each gene impurity 20ng/mL.
Sample solution is prepared：It is 70 with water and acetonitrile volume ratio:30 mixed solvent is by doxofylline bulk pharmaceutical chemicals to be measured
It is configured to the solution for being about 10mg/mL containing doxofylline.
(3) detection method
Precision measures 30 μ L of test solution, injects in high performance liquid chromatograph.
(4) calculate
According to external standard method, using impurity peak area in contrast solution as external standard, impurity content in sample solution is calculated.
(5) experimental result
Impurity determines：
According to the comparative analysis in relation to material present situation and impurity reference substance in doxofylline, it may be determined that doxofylline raw material
Impurity peaks 8,9 and 10 in medicine are genotoxicity impurity, and specific impurity title is as shown in the table 2 in embodiment 1.
Impurity peaks in chromatogram and main peak are analyzed, the results are shown in Table 5, testing result show genotoxicity impurity and
Other impurities separation is good in doxofylline and doxofylline.
Table 5：The analysis result of genotoxicity impurity in embodiment 2HPLC chromatograms
As can be seen from Table 5, can effectively be detected with doxofylline genotoxicity impurity in the chromatographic system, three gene poison
Retention time difference is obvious between property impurity, can efficiently separate, three genotoxicity impurity and doxofylline and more rope tea
Other impurities in alkali can also efficiently separate, and signal-to-noise ratio is more than 37, meets the requirements.Signal-to-noise ratio is more than under the chromatographic condition
36, impurity signal is not easy to be disturbed, and sensitivity is higher；It can detect that genotoxicity impurity of the content in 2ppm illustrates the present invention
Technical solution can effectively, sensitively detect genotoxicity impurity in doxofylline bulk pharmaceutical chemicals.
Embodiment 3：The detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals
(1) chromatographic condition
Instrument：1260 high performance liquid chromatograph of Agilent；
Chromatographic column：Octadecylsilane chemically bonded silica (250mm*4.6mm*5 μm)
Mobile phase：Using pure water as mobile phase A, the acetonitrile of chromatographically pure is Mobile phase B, and gradient elution is carried out according to table 6；
Table 6：The condition of gradient elution program
Column temperature：35℃；
The flow velocity 1.5mL/min of mobile phase；
Detection wavelength：230nm.
(2) sample preparation
Contrast solution is prepared：Reference substance is respectively：P-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid ester) and
Methyl tosylate；It is appropriate that three kinds of genotoxicity contamination levels product are weighed respectively, with water and acetonitrile ratio 70:30 mixing is molten
Agent is dissolved and is diluted to containing each impurity 20ng/mL.
Sample solution is prepared：It is 70 with water and acetonitrile volume ratio:30 mixed solvent is by doxofylline bulk pharmaceutical chemicals to be measured
It is configured to the solution for being about 10mg/mL containing doxofylline.
(3) detection method
Precision measures 60 μ L of test solution, injects in high performance liquid chromatograph.
(4) calculate
According to external standard method, using impurity peak area in contrast solution as external standard, impurity content in sample solution is calculated.
Impurity determines：
According to the comparative analysis in relation to material present situation and impurity reference substance in doxofylline, it may be determined that doxofylline raw material
Impurity peaks 8,9 and 10 in medicine are genotoxicity impurity, and specific impurity title is as shown in table 2 in embodiment 1.
Analysis result：
Impurity peaks in chromatogram and main peak are analyzed, the results are shown in Table 7, testing result show genotoxicity impurity and
Other impurities separation is good in doxofylline and doxofylline.
Table 7：The analysis result of genotoxicity impurity in HPLC chromatogram
As can be seen from Table 7, can effectively be detected with doxofylline genotoxicity impurity in the chromatographic system, three gene poison
Retention time difference is obvious between property impurity, can efficiently separate, three genotoxicity impurity and doxofylline and more rope tea
Other impurities in alkali can also efficiently separate, and signal-to-noise ratio is more than 36, meets the requirements.Signal-to-noise ratio is more than under the chromatographic condition
38, impurity signal is not easy to be disturbed, and sensitivity is higher；It can detect that genotoxicity impurity of the content in 2ppm illustrates the present invention
Technical solution can effectively, sensitively detect genotoxicity impurity in doxofylline bulk pharmaceutical chemicals.
Embodiment 4：The detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals
(1) chromatographic condition
Instrument：1260 high performance liquid chromatograph of Agilent；
Chromatographic column：Octadecylsilane chemically bonded silica (250mm*4.6mm*5 μm)
Mobile phase：Using pure water as mobile phase A, the acetonitrile of chromatographically pure is Mobile phase B, and gradient elution is carried out according to table 8；
Table 8：Eluent gradient elutes table
Column temperature：30℃；
The flow velocity 1mL/min of mobile phase；
Detection wavelength：226nm.
(2) sample preparation
Contrast solution is prepared：Reference substance is respectively：P-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid ester) and
Methyl tosylate；It is appropriate that three kinds of genotoxicity contamination levels product are weighed respectively, with water and acetonitrile ratio 70:30 mixing is molten
Agent is dissolved and is diluted to containing each impurity 20ng/mL.
Sample solution is prepared：With water and acetonitrile ratio 70:30 mixed solvent prepares doxofylline bulk pharmaceutical chemicals to be measured
Into the solution for being about 10mg/mL containing doxofylline.
(3) detection method
Precision measures 50 μ L of test solution, injects in high performance liquid chromatograph.
(4) calculate
According to external standard method, using impurity peak area in contrast solution as external standard, impurity content in sample solution is calculated.
Impurity determines：
According to the comparative analysis in relation to material present situation and impurity reference substance in doxofylline, it may be determined that doxofylline raw material
Impurity peaks 8,9 and 10 in medicine are genotoxicity impurity, and specific impurity title is as shown in table 2 in embodiment 1.
Analysis result：
Impurity peaks in chromatogram and main peak are analyzed, the results are shown in Table 9.
Table 9：The analysis result of genotoxicity impurity in HPLC chromatogram
As can be seen from Table 9, can effectively be detected with doxofylline genotoxicity impurity in the chromatographic system, three gene poison
Retention time difference is obvious between property impurity, can efficiently separate, three genotoxicity impurity and doxofylline and more rope tea
Other impurities in alkali can also efficiently separate, and signal-to-noise ratio is more than 38, meets the requirements.Signal-to-noise ratio is more than under the chromatographic condition
42, impurity signal is not easy to be disturbed, and sensitivity is higher；It can detect that genotoxicity impurity of the content in 2ppm illustrates the present invention
Technical solution can effectively, sensitively detect genotoxicity impurity in doxofylline bulk pharmaceutical chemicals.
Embodiment 5：The detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals
(1) chromatographic condition
Instrument：1260 high performance liquid chromatograph of Agilent；
Chromatographic column：Octadecylsilane chemically bonded silica (250mm*4.6mm*5 μm)
Mobile phase：Using pure water as mobile phase A, the acetonitrile of chromatographically pure is Mobile phase B, and gradient elution is carried out according to table 10；
Table 10：Mobile phase gradient table
Column temperature：30℃；
The flow velocity 1mL/min of mobile phase；
Detection wavelength：226nm.
(2) sample preparation
Contrast solution is prepared：It is appropriate that three kinds of genotoxicity contamination levels product are weighed respectively, with water and acetonitrile ratio 70:30
Mixed solvent is dissolved and is diluted to containing each impurity 20ng/mL.
Sample solution is prepared：With water and acetonitrile ratio 70:30 mixed solvent prepares doxofylline bulk pharmaceutical chemicals to be measured
Into the solution for being about 10mg/mL containing doxofylline.
(3) detection method
Precision measures 50 μ L of test solution, injects in high performance liquid chromatograph.
(4) calculate
According to external standard method, using impurity peak area in contrast solution as external standard, impurity content in sample solution is calculated.
Impurity determines：
According to the comparative analysis in relation to material present situation and impurity reference substance in doxofylline, it may be determined that doxofylline raw material
Impurity peaks 8,9 and 10 in medicine are genotoxicity impurity, and specific impurity title is as shown in table 2 in embodiment 1.
Analysis result：
Impurity peaks in chromatogram and main peak are analyzed, the results are shown in Table 11.
Table 11：The analysis result of genotoxicity impurity in HPLC chromatogram
As can be seen from Table 11, can effectively be detected with doxofylline genotoxicity impurity in the chromatographic system, three genes
Retention time difference is obvious between toxic impurities, can efficiently separate, three genotoxicity impurity and doxofylline and more ropes
Other impurities in theophylline can also efficiently separate, and signal-to-noise ratio is more than 33, meets the requirements.Signal-to-noise ratio is more than under the chromatographic condition
58, impurity signal is not easy to be disturbed, and sensitivity is higher；It can detect that genotoxicity impurity of the content in 2ppm illustrates the present invention
Technical solution can effectively, sensitively detect genotoxicity impurity in doxofylline bulk pharmaceutical chemicals.
Found by studying, the mobile phase by setting different proportion carries out gradient elution, can reach genotoxicity impurity
It is precisely separating with other impurities in doxofylline and doxofylline, in 0 to 20 minutes, mobile phase ratio compares acetonitrile for water
For 70：30~75：25, at 20 minutes to 30 minutes, water and acetonitrile ratio were adjusted to 40:60, kept for 15 minutes, then will elution
Agent ratio recalls to 70：30~75：25, gradient elution is carried out under the conditions of this, to genotoxicity impurity p-methyl benzenesulfonic acid hydroxyl ethyl ester, two
Ethylene glycol double (p-methyl benzenesulfonic acid esters) and the separating degree of methyl tosylate are all higher than 30, meet the requirements.And this method pair
The test limit of genotoxicity impurity can reach 2ppm, have higher sensitivity, can strictly control genotoxicity miscellaneous with high quality
Matter.
There is the sensitivity of Detection wavelength and sample size to genotoxicity impurity in testing result in the detection method of the present invention
Very big influence, by study find, scanned by diode array, find genotoxicity impurity have at 210~230nm compared with
Big to absorb, three kinds of genotoxicity impurity have maximum absorption wavelength wherein at 226nm, final to confirm that Detection wavelength is 226nm, into
Sample amount is 50 μ L, and under this condition, the quantitative limit of genotoxicity impurity can reach 0.5ppm.
Methodological study：
It is steady that line, linear, repeatability, solution are detected to the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals
The methods of qualitative, which is learned, to be investigated, and mainly investigates the test limit of the detection method, the definite assay to three kinds of genotoxicity impurity
Linearly interval, prepare impurity quantitation curves, the repeatability and stability of solution of the method for inspection.
Specifically investigation method is：
Detection line investigates method：It is appropriate that three kinds of genotoxicity contamination levels product are weighed respectively, with water and acetonitrile ratio 70:30
Mixed solvent dissolve and be diluted to debita spissitudo, take solution to detect, according to each peak signal-to-noise ratio in test map, then to solution into
Row dilution, is detection line when signal-to-noise ratio is 3, is quantitative limit when signal-to-noise ratio is 10.
It is linear to investigate method：It is appropriate to weigh three kinds of genotoxicity contamination levels product respectively, dissolve and be diluted to 2ng/mL~
6 different solutions of 50ng/mL, according to each 3 pin of solution sample introduction of chromatographic condition, record chromatogram.
Repeated investigation method：Three kinds of genotoxicity contamination levels product are weighed respectively and doxofylline bulk pharmaceutical chemicals are appropriate, are matched somebody with somebody
The solution containing each impurity 20ng/mL, doxofylline bulk pharmaceutical chemicals 10mg/mL respectively is made, 6 parts are prepared with method, according to chromatographic condition
Each 1 pin of solution sample introduction, records chromatogram.
Stability of solution investigates method：Take repeated solution respectively when 0,2,4,8,12,18,24 is small, according to chromatostrip
Part sample introduction, records chromatogram.
Concrete outcome see the table below 12.
Table 12：Methodological study result
From above-mentioned table 12, the detection method of the embodiment of the present invention 1~5, can be used for controlling bulk pharmaceutical chemicals doxofylline
Middle genotoxicity impurity p-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid ester) and/or methyl tosylates.
2ng/mL is limited to for the detection of impurity p-methyl benzenesulfonic acid hydroxyl ethyl ester, equivalent to 0.2ppm；Quantitatively it is limited to 5ng/mL, phase
When in 0.5ppm；Repeated RSD is 2.2%≤3%, is met the requirements, and repeatability is good；Solution interior stabilization when 24 is small.
2ng/mL is limited to for impurity diethylene glycol double (p-methyl benzenesulfonic acid esters) detection, equivalent to 0.2ppm；Quantitatively it is limited to
5ng/mL, equivalent to 0.5ppm；Repeated RSD is 1.8%≤3%, is met the requirements, and repeatability is good；Solution is interior when 24 is small
Stablize.
2ng/mL is limited to for the detection of impurity methyl tosylate, equivalent to 0.2ppm；5ng/mL is quantitatively limited to, quite
In 0.5ppm；Repeated RSD is 3%, is met the requirements, and repeatability is good；Solution interior stabilization when 24 is small.
The detection method of genotoxicity impurity, can efficiently separate more ropes in doxofylline bulk pharmaceutical chemicals provided by the invention
Theophylline principal component peak and genotoxicity impurity peaks, this method greatly improve the detection sensitivity of genotoxicity impurity, so that
Can simply, genotoxicity impurity in quick, the detection doxofylline bulk pharmaceutical chemicals stablized, effectively to control doxofylline former
Expect the quality of medicine, avoid objectionable impurities and do not detect the clinical risk that may be brought.This method is to three kinds of genotoxicity impurity
Detection is limited to 2ng/mL, equivalent to 0.2ppm；5ng/mL is quantitatively limited to, equivalent to 0.5ppm；Repeated RSD, which is respectively less than, to be equal to
3%, meet the requirements, repeatability is good；Solution interior stabilization when 24 is small, is suitable for the application of in bulk pharmaceutical chemicals quality control.
Above content is that a further detailed description of the present invention in conjunction with specific preferred embodiments, it is impossible to is assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (9)
1. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals, the detection method using high performance liquid chromatography into
Row measure, it is characterised in that specific detection method is as follows：
(1) chromatographic condition
Filler is octadecylsilane key and silica gel,
Mobile phase is the mixed solvent of water and acetonitrile, gradient elution,
Column temperature is 25~35 DEG C,
Flow rate of mobile phase is 0.5~1.5mL/min,
Detection wavelength is 210~230nm；
(2) sample preparation
Doxofylline bulk pharmaceutical chemicals are weighed, the molten of the 5~15mg/mL of concentration containing doxofylline is configured to the mixed solvent of water and acetonitrile
Liquid；
(3) detection method
Precision measures 30~60 μ L of test solution, injects in high performance liquid chromatograph, records chromatogram.
2. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 1, it is characterised in that step
Suddenly the volume ratio of mobile phase water and acetonitrile is 70 in (1) chromatographic condition：30~75：25.
3. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 1, it is characterised in that step
Suddenly the mobile phase of gradient elution is specially in (1) chromatographic condition：
4. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 2, it is characterised in that step
Suddenly in (1) chromatographic condition, the column temperature is arranged to 30 DEG C.
5. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 2, it is characterised in that step
Suddenly in (1) chromatographic condition, the flow velocity of the mobile phase is 0.8~1.2mL/min.
6. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 2, it is characterised in that step
Suddenly in (1) chromatographic condition, the Detection wavelength is 226nm.
7. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 2, it is characterised in that step
Suddenly in (2) sample preparation, bulk pharmaceutical chemicals are taken, are 70 with water and acetonitrile volume ratio:30 mixed solvent is configured to contain doxofylline
The solution of 10mg/mL.
8. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 2, it is characterised in that step
Suddenly in (3) detection method, precision measures 50 μ L of test solution, injects in high performance liquid chromatograph, records chromatogram.
9. the detection method of genotoxicity impurity in doxofylline bulk pharmaceutical chemicals according to claim 1, it is characterised in that institute
Genotoxicity impurity is stated as p-methyl benzenesulfonic acid hydroxyl ethyl ester, diethylene glycol double (p-methyl benzenesulfonic acid ester) and/or methyl tosylates.
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| CN114487141A (en) * | 2020-10-28 | 2022-05-13 | 杭州中美华东制药有限公司 | Method for detecting genotoxic impurities in indobufen bulk drug |
| CN114527224A (en) * | 2022-02-11 | 2022-05-24 | 四川邈济生物医药科技有限公司 | Dihydroxypropyltheophylline bulk drug and detection method of related substances in preparation thereof |
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