CN107037153B - The method that high performance liquid chromatography detects genotoxicity impurity in AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation - Google Patents
The method that high performance liquid chromatography detects genotoxicity impurity in AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation Download PDFInfo
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- G—PHYSICS
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- 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
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Abstract
The present invention relates to the methods of genotoxicity impurity in high performance liquid chromatography detection AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation, for methyl tosylate (MPTS) and ethyl p-toluenesulfonate (EPTS), first by sample pre-treatments, high performance liquid chromatography is recycled to be measured: chromatographic column is C18 chromatographic column；Detection wavelength is 225nm；Mobile phase is acetonitrile-phosphate aqueous solution；Flow velocity is 0.8~1.2mL/min；Column temperature: 30~40 DEG C；Sample volume is 20 μ L；Elution time is 18min；The result shows that methyl tosylate detection is limited to 0.3ppm, it is quantitatively limited to 1ppm；Ethyl p-toluenesulfonate detection is limited to 0.5ppm, is quantitatively limited to 1.4ppm.This method specificity is strong, easy to operate, high sensitivity, result are accurate, the measurement of methyl tosylate and ethyl p-toluenesulfonate suitable for AL58805.
Description
Technical field
The present invention relates to a kind of analysis methods of genotoxicity impurity in AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation, especially relate to
And a kind of use high performance liquid chromatography is to genotoxicity impurity methyl tosylate and ethyl p-toluenesulfonate in AL58805
The method for being separated and being quantitative determined.
Background technique
AL58805 is the kind new medicine researched and developed by Nanjing Aidecheng Pharmaceutical Technology Co., Ltd, is to pass through computer aided manufacturing
A kind of novel bis- targeted kinase inhibitors of PI3K/mTor that drug design techniques screen are helped, and there is entirely autonomous knowledge
Property right.The result of study of early period shows that AL58805 is a kind of outstanding anti-tumor drug, and action target spot is unique, antitumor action
Obviously, toxic side effect is small, has had been found apparent in gastric cancer, kidney, osteosarcoma, colon cancer Model in Nude Mice
Anti-tumor activity, therefore, exploitation in clinical treatment tumour at will have unique advantage after new drug.
Genotoxicity impurity: in internal/in vitro test using DNA reactive material as main study subject, them are found
There is potential destructiveness to DNA.For current regulation, (internal) genotoxicity substance is in any intake level on pair
DNA has potential destructiveness, this to destroy the generation that may cause tumour, so must be strictly controlled.
Methyl tosylate and ethyl p-toluenesulfonate are the by-products generated when synthesizing AL58805 bulk pharmaceutical chemicals, are belonged to
Latent gene toxic impurities.For the content of strict control methyl tosylate and ethyl p-toluenesulfonate, one kind need to be sought
Simply, fast, specificity is strong, analysis method of high sensitivity seems still be important.
Do not have also at home and abroad about the method for methyl tosylate in AL58805 and ethyl p-toluenesulfonate assay
It has been reported that.Now, only about the document report of p-methyl benzenesulfonic acid esters gene poison impurity determination in other medicines, in report often
Detection method is high performance liquid chromatography, Liquid Chromatography/Mass Spectrometry, such as methyl tosylate in vinpocetine and to toluene sulphur
The LC-MS/MS method of acetoacetic ester measures[1], LC-MS/MS measurement oxalic acid Escitalopram in p-methyl benzenesulfonic acid esters genotoxicity
The content of impurity[2], HPLC method measurement citalopram hydrobromate in genotoxicity impurity ethyl p-toluenesulfonate[3].Document[1]
And document[2]The range of linearity and quantitative limit concentration of genotoxicity impurity are similar, document[1]Middle methyl tosylate, to first
The quantitative limit of ethyl benzenesulfonat is 2.5ng/mL (2.5ppm), and the range of linearity is 2.5~50ng/mL, document[3]In to toluene
Quantifying for sulfonic acid is limited to 25.6ng/mL (5.12ppm), and the range of linearity is 25.6~187.5ng/mL.Although moreover, mass spectrum
Detector is a kind of all-purpose detector of high sensitivity, however fancy price limits its popularization and use.
[1] Zhang Wei, Chen Yi are spent, the LC-MS/ of methyl tosylate and ethyl p-toluenesulfonate in Zheng Feng vinpocetine
MS method measures [J], Chinese Journal of Pharmaceuticals, 2015,46 (12): 1334-1336.
[2] Liang Jianmou, Fu Cong, Chen Yue .LC-MS/MS measure p-methyl benzenesulfonic acid esters gene in oxalic acid Escitalopram
The content [J] of toxic impurities, Chinese Journal of Modern Applied Pharmacy, 2016,33 (11): 1436-1440.
[3] the genotoxicity impurity ethyl p-toluenesulfonate [J] in Sun Piao .HPLC method measurement citalopram hydrobromate, medicine
Learn research, 2015,34 (11): 637-639.
Summary of the invention
For shortcoming and deficiency existing in the prior art, the object of the present invention is to provide a kind of inspections of high performance liquid chromatography
Survey the side of genotoxicity impurity (methyl tosylate and ethyl p-toluenesulfonate) in AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation
Method.
By high performance liquid chromatography to the conditions repeated screening such as sample pre-treatments, chromatographic column, mobile phase, it is suitable to have explored
Analysis condition.For the separation requirement for taking into account principal component and impurity, the ratio of suitable buffer solution system, mobile phase has been selected
Deng crucial testing conditions；To reduce interference of the test solution to impurity determination, to test sample concentration, pH in sample pre-treatments
Value, crystallization temperature, time of repose, filter type etc. are screened, and finally established method has reached foregoing invention purpose.
The technical solution adopted by the present invention to solve the technical problems is:
The present invention is using gene in pharmaceutical preparation of the high performance liquid chromatography to AL58805 bulk pharmaceutical chemicals or comprising AL58805
The method of toxic impurities methyl tosylate and ethyl p-toluenesulfonate quantitative analysis, specific step is as follows for this method:
(1) preparation of standard working solution
It is appropriate that methyl tosylate, ethyl p-toluenesulfonate reference substance are weighed respectively, are added acetonitrile to dissolve and dilute and are made
Stock solution in every 1mL containing about 1mg is gradually diluted with acetonitrile-water (50:50V/V) be made in every 1mL containing about to toluene sulphur afterwards
Sour methyl esters, ethyl p-toluenesulfonate be each 5,30,60,100,150, the solution of 200ng is as standard working solution.
(2) sample pre-treatments
It takes AL58805 test sample appropriate (being approximately equivalent to AL58805 100mg), sets in 10mL measuring bottle, add appropriate acetonitrile-water
After (50:50V/V) dissolution, pH to 8.5~9.5 is adjusted with 1mol/L sodium hydroxide solution, acetonitrile-water (50:50V/V) is added to dilute
It to scale, shakes up, ultrasound 10 minutes, in 0 DEG C after crystallization 30 minutes, takes out strength and shake, after standing 0.5~1.5 hour, use is micro-
Hole membrane filtration, takes filtrate to measure.
Preferably, in sample pre-treatments: adjusting pH to 9 with 1mol/L sodium hydroxide solution, time of repose is 1 hour, is used
0.45 μm of filtering with microporous membrane.
(3) liquid chromatographic detection condition
Mobile phase: acetonitrile-phosphate aqueous solution；Flow velocity: 0.8~1.2mL/min；Column temperature: 30~40 DEG C；Detection wavelength:
225nm；Sample volume: 20 μ L；Octadecyl silane chromatographic column (C18 chromatographic column), elution time 18min；Detector is
UV detector.
Further, the volume ratio of the mobile phase acetonitrile-phosphate aqueous solution is 50:50.
Further, the concentration of the phosphate aqueous solution is 5mmol/L-50mmol/L, preferably 15mmol/L.
Further, the flow velocity is 0.8~1.2mL/min, preferably 1.0mL/min.
Further, the column temperature is 30~40 DEG C, preferably 35 DEG C.
Further, the specification of the C18 chromatographic column be 4.6mm × 250mm, 5 μm.
(4) it takes in step (1) that methyl tosylate and ethyl p-toluenesulfonate standard reserving solution are appropriate, uses acetonitrile-water
(50:50V/V) is gradually diluted in every 1mL containing about the solution conduct of methyl tosylate, each 100ng of ethyl p-toluenesulfonate
Reference substance solution；Precision weighs AL58805 test sample in right amount (being approximately equivalent to AL58805 100mg), sets in 10mL measuring bottle, by step
Suddenly the method pre-treatment of (2) is as test solution.Precision measures 20 μ L of reference substance solution, injects liquid chromatograph, records color
Spectrogram, with calculated by peak area, the relative standard deviation of 6 sample introductions is not greater than 2.0%；It is accurate again to measure 20 μ of test solution
L injects liquid chromatograph, records chromatogram.It is calculated as follows by external standard method with peak area, calculation formula:
In formula: As: the average peak area of impurity in reference substance solution；Cs: the concentration (ng/mL) of impurity in reference substance solution；
At: the peak area of impurity in test solution；Ct: the concentration (mg/mL) of test solution.
The present invention to using high performance liquid chromatography detection AL58805 in genotoxicity impurity methyl tosylate and
The chromatographic condition of ethyl p-toluenesulfonate content is optimized, and achieves optimum chromatogram condition, then carries out for this condition
Methyl tosylate and the verifying of ethyl p-toluenesulfonate content assaying method.
The present invention establishes methyl tosylate in AL58805, ethyl p-toluenesulfonate using high performance liquid chromatography
The method of content detection after sample is using acetonitrile-water (50:50V/V) dissolution, makes its crystallization by adjusting test sample pH value, from
And test sample concentration is reduced, the method interfered genotoxicity impurity determination is reduced, the effect and advantage of the method for the present invention are:
1, the present invention can efficiently separate out AL58805 and genotoxicity impurity (methyl tosylate and p-methyl benzenesulfonic acid
Ethyl ester), solution dilution blanks do not interfere the detection of gene impurity, and method specificity is strong, very quickly.
2, the present invention can quantify genotoxicity impurity methyl tosylate and ethyl p-toluenesulfonate, and have compared with
High sensitivity, methyl tosylate detection are limited to 0.3ppm, are quantitatively limited to 1ppm；Ethyl p-toluenesulfonate detection is limited to
0.5ppm is quantitatively limited to 1.4ppm, and high sensitivity is in bibliography[1]Middle LC-MS/MS method.
3, detection method testing cost is cheap, economical and practical, specificity is strong, simple and fast, high sensitivity, can use
The quality control of latent gene toxic impurities in AL58805 and pharmaceutical preparation containing AL58805.
4, the method for the present invention passes through methodology validation, as a result meets the requirements, it was demonstrated that the present invention can be used for base in AL58805
Because of the content detection of toxic impurities methyl tosylate and ethyl p-toluenesulfonate.
5, the present invention is compared with p-methyl benzenesulfonic acid esters gene poison method for detecting impurities in other medicines, before increasing sample
Processing step (dissolution, crystallization, filtering etc.) thus greatly reduces test sample concentration in sample introduction solution, reduces high concentration for examination
Interference of the product solution to impurity determination and the injury to chromatographic column, and make experimental result more accurate.
Detailed description of the invention
The chromatogram of methyl tosylate (MPTS), ethyl p-toluenesulfonate (EPTS) under Fig. 1 optimum chromatogram condition；
Methyl tosylate (MPTS), ethyl p-toluenesulfonate are added under Fig. 2 optimum chromatogram condition in AL58805
(EPTS) chromatogram；
Fig. 3 is the canonical plotting of genotoxicity impurity: (a) standard curve of methyl tosylate；(b) to toluene sulphur
The standard curve of acetoacetic ester；
The stability schematic diagram of Fig. 4 methyl tosylate and ethyl p-toluenesulfonate under room temperature.
Specific embodiment
Further description of the technical solution of the present invention with reference to the accompanying drawings and examples, and however, it is not limited to this,
It is all that modifying or equivalently replacing the technical solution of the present invention, without departing from the spirit and scope of the technical solution of the present invention,
It should all cover within the protection scope of the present invention.
Embodiment 1:
One, the preparation of standard reserving solution
It is appropriate to weigh methyl tosylate, ethyl p-toluenesulfonate reference substance for precision respectively, and acetonitrile is added to dissolve and dilute
The solution in every 1mL containing about 1mg is made as Standard Stock solutions, needed for being gradually diluted to acetonitrile-water (50:50V/V) afterwards
Concentration.
Two, liquid phase chromatogram condition
Chromatographic column is InertSustain C18 (4.6 × 250mm, 5 μm)；Mobile phase is 15mmol/L phosphate aqueous solution-
Acetonitrile
(50:50V/V)；Detection wavelength is 225nm；Flow velocity is 1mL/min；Column temperature is 35 DEG C；20 μ L of sample volume；When elution
Between be 18min.
Three, sample-pretreating method
AL58805 test sample about 100mg is taken, is set in 10mL measuring bottle, after adding appropriate acetonitrile-water (50:50V/V) to dissolve, is used
1mol/L sodium hydroxide solution adjusts pH to 9, adds acetonitrile-water (50:50V/V) to be diluted to scale, shakes up, ultrasound 10 minutes, in 0
DEG C after crystallization 30 minutes, takes out strength and shake, after standing 1 hour, with 0.45 μm of filtering with microporous membrane, filtrate is taken to measure.
Four, the verifying of methodology
Specificity is investigated: solution dilution blanks do not interfere the detection of methyl tosylate, ethyl p-toluenesulfonate, specificity
Well.
Detection limited amount limit is investigated: under this experimental condition, preparing the methyl tosylate of various concentration, to toluene sulphur
Acetoacetic ester reference substance, detection limit is in terms of S/N >=3, and quantitative limit is in terms of S/N >=10, and sample introduction determines methyl tosylate, right respectively
The detection limit and quantitative limit of toluenesulfonic acid ethyl ester, experimental result is shown in the following table 1.
Table 1
Reference substance solution study on the stability: methyl tosylate, ethyl p-toluenesulfonate mixed reference substance solution are taken
(100ng/mL) carries out solution stability testing, the peak area after measurement is placed 0,3,7,10,17,24 hour respectively.Measurement knot
Fruit see the table below 2.
Table 2
Reference substance solution is placed at room temperature for 24 hours, and the peak area average value of methyl tosylate is that 6.96, RSD is
3.8%；The peak area average value of ethyl p-toluenesulfonate is 6.46, RSD 3.7%, shows that reference substance solution is placed at room temperature for 24
Stablize in hour.
It is linear to investigate: to take Standard Stock solutions appropriate, it is mixed that following standard series is made with acetonitrile-water (50:50V/V) dilution
Close solution.Precision measures each 20 μ L of standard series mixed solution, is injected separately into liquid chromatograph, records chromatogram.It is with concentration
Abscissa, peak area are ordinate, carry out linear regression, specifically as shown in table 3 and figure 3.
Table 3
Methyl tosylate equation of linear regression is y=0.0638x+0.0004, and regression coefficient r is 0.9999, explanation
This product is linear good within the scope of 3.320ng/mL~199.2ng/mL；Ethyl p-toluenesulfonate equation of linear regression is y=
0.0623x-0.0444, regression coefficient r are 0.9997, illustrate that this product is linear good within the scope of 4.550ng/mL~202.0ng/mL.
Accuracy: weighing AL58805 test sample in right amount (being approximately equivalent to AL58805 100mg), set in 10mL measuring bottle, as
Rate of recovery matrix sample；Accurate measurement methyl tosylate, ethyl p-toluenesulfonate Standard Stock solutions 0.1mL are set respectively
In 10mL measuring bottle, use acetonitrile-water (50:50V/V) dilution that mixed solution in every 1mL containing about 10 μ g is made as dirt solution.
With the content (0.001% matrix sample concentration) of gene impurity for benchmark 100%, precision measurement dirt solution 0.07mL,
0.10mL, 0.13mL are respectively placed in the 10mL measuring bottle for filling matrix sample, are mixed, and with acetonitrile-water (50:50V/V) constant volume, are prepared
At impure 70%, 100%, 130% solution, parallel 3 times with method, as test solution.Specific preparation method see the table below 4.
Table 4
Precision measures above-mentioned each 20 μ L of solution, is injected separately into liquid chromatograph, records chromatogram, measures peak area (its
Methyl tosylate in middle test sample, ethyl p-toluenesulfonate peak area need to deduct p-methyl benzenesulfonic acid first in sample substrate
The peak area of ester, ethyl p-toluenesulfonate), the rate of recovery is calculated, experimental result is shown in the following table 5 and table 6.
Rate of recovery calculation formula:
Actual amount recovered=AU×FS×CS
In formula: WS: the quality (mg) of impurity reference substance；Cs: the content (%) of impurity reference substance；Au: in test solution
The peak area (deducting impurity peak area in matrix) of impurity；Fs: impurity concentration and peak area ratio.
Table 5
Table 6
Can be seen that methyl tosylate measurement average recovery rate from above-mentioned series of tables is 92.1%, RSD 2.8%
(n=9), it is 2.7% (n=9) that ethyl p-toluenesulfonate measurement average recovery rate, which is 101.4%, RSD, shows that this method detects
Methyl tosylate, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 2:
1, standard reserving solution prepare it is same as Example 1；
2, in liquid phase chromatogram condition: mobile phase is 5mmol/L phosphate aqueous solution-acetonitrile (50:50V/V)；Remaining and implementation
Example 1 is identical.
3, sample pre-treatments: same as Example 1.
4, methodology validation and embodiment 1 are identical, the results showed that, (the pharmacopeia acceptance of the bid of methyl tosylate average recovery rate
Standard is 80-115%) it is 89.0%, RSD 1.6%, (pharmacopeia Plays are 80- to ethyl p-toluenesulfonate average recovery rate
It 115%) is 101.7%, RSD 2.3%, this method detection methyl tosylate, the accuracy of ethyl p-toluenesulfonate are good
It is good.
Embodiment 3:
1, standard reserving solution prepare it is same as Example 1；
2, mobile phase is 50mmol/L phosphate aqueous solution-acetonitrile (50:50V/V) in liquid phase chromatogram condition；Remaining and implementation
Example 1 is identical.
3, sample pre-treatments: same as Example 1.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate average recovery rate is 93.1%,
RSD is 1.7%, and ethyl p-toluenesulfonate average recovery rate is 102.1%, RSD 2.7%, and this method detects p-methyl benzenesulfonic acid
Methyl esters, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 4:
1, standard reserving solution prepare it is same as Example 1；
2, mobile phase is 30mmol/L phosphate aqueous solution-acetonitrile (50:50V/V) in liquid phase chromatogram condition；Remaining and implementation
Example 1 is identical.
3, sample pre-treatments: same as Example 1.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate average recovery rate is 94.1%,
RSD is 2.7%, and ethyl p-toluenesulfonate average recovery rate is 99.5%, RSD 2.2%, and this method detects p-methyl benzenesulfonic acid first
Ester, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 5:
1, standard reserving solution prepare it is same as Example 1；
2, liquid phase chromatogram condition is same as Example 1.
3, sample pre-treatments: remaining step and embodiment 1 are identical, only with 1mol/L sodium hydroxide solution adjust pH to
8.5。
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate measurement average recovery rate is
99.0%, RSD 2.3%, it is 105.8%, RSD 1.2%, this method detection that ethyl p-toluenesulfonate, which measures average recovery rate,
Methyl tosylate, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 6:
1, standard reserving solution prepare it is same as Example 1；
2, liquid phase chromatogram condition is same as Example 1.
3, sample pre-treatments: remaining step and embodiment 1 are identical, only with 1mol/L sodium hydroxide solution adjust pH to
9.5。
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate measurement average recovery rate is
86.1%, RSD 2.3%, it is 107.2%, RSD 0.8%, this method detection that ethyl p-toluenesulfonate, which measures average recovery rate,
Methyl tosylate, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 7:
1, standard reserving solution prepare it is same as Example 1；
2, liquid phase chromatogram condition is same as Example 1.
3, sample pre-treatments: remaining step and embodiment 1 are identical, after only taking out strength shaking, stand 0.5 hour.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate measurement average recovery rate is
91.5%, RSD 3.2%, it is 102.8%, RSD 2.3%, this method detection that ethyl p-toluenesulfonate, which measures average recovery rate,
Methyl tosylate, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 8:
1, standard reserving solution prepare it is same as Example 1；
2, liquid phase chromatogram condition is same as Example 1.
3, sample pre-treatments: remaining step and embodiment 1 are identical, after only taking out strength shaking, stand 1.5 hours.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate measurement average recovery rate is
95.5%, RSD 2.1%, it is 95.8%, RSD 2.9%, this method detection that ethyl p-toluenesulfonate, which measures average recovery rate,
Methyl tosylate, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 9:
1, standard reserving solution prepare it is same as Example 1；
2, column temperature is 30 DEG C in liquid phase chromatogram condition, remaining is same as Example 1.
3, sample pre-treatments: same as Example 1.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate average recovery rate is 93.2%,
RSD is 1.8%, and ethyl p-toluenesulfonate average recovery rate is 102.6%, RSD 2.6%, and this method detects p-methyl benzenesulfonic acid
Methyl esters, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 10:
1, standard reserving solution prepare it is same as Example 1；
2, column temperature is 40 DEG C in liquid phase chromatogram condition, remaining is same as Example 1.
3, sample pre-treatments: same as Example 1.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate average recovery rate is 90.4%,
RSD is 3.5%, and ethyl p-toluenesulfonate average recovery rate is 90.6%, RSD 1.9%, and this method detects p-methyl benzenesulfonic acid first
Ester, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 11:
1, standard reserving solution prepare it is same as Example 1；
2, flow velocity is 0.8mL/min in liquid phase chromatogram condition, remaining is same as Example 1.
3, sample pre-treatments: same as Example 1.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate average recovery rate is 101.5%,
RSD is 3.1%, and ethyl p-toluenesulfonate average recovery rate is 106.5%, RSD 3.0%, and this method detects p-methyl benzenesulfonic acid
Methyl esters, the accuracy of ethyl p-toluenesulfonate are good.
Embodiment 12:
1, standard reserving solution prepare it is same as Example 1；
2, flow velocity is 1.2mL/min in liquid phase chromatogram condition, remaining is same as Example 1.
3, sample pre-treatments: same as Example 1.
4, methodology validation and embodiment 1 are identical, the results showed that, methyl tosylate average recovery rate is 92.6%,
RSD is 3.5%, and ethyl p-toluenesulfonate average recovery rate is 107.2%, RSD 2.0%, and this method detects p-methyl benzenesulfonic acid
Methyl esters, the accuracy of ethyl p-toluenesulfonate are good.
In conclusion the method for the present invention can efficiently separate methyl tosylate in AL58805 and to toluene sulphur
Acetoacetic ester, can accurately, quickly measure genotoxicity impurity methyl tosylate, ethyl p-toluenesulfonate, this method is simple,
Quickly, accurate and effective, precision is high, and specificity is strong, is measured in AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation comprising AL58805
The ideal method of methyl tosylate, ethyl p-toluenesulfonate.
Claims (7)
1. the method that high performance liquid chromatography detects genotoxicity impurity in AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation, feature exist
In using genotoxicity impurity in pharmaceutical preparation of the high performance liquid chromatography to AL58805 bulk pharmaceutical chemicals or comprising AL58805 to first
Methyl benzene sulfonate and ethyl p-toluenesulfonate carry out quantitative analysis, include the following steps:
(1) preparation of standard reserving solution: precision weighs methyl tosylate reference substance and ethyl p-toluenesulfonate control respectively
Product dissolve with acetonitrile and dilute the standard reserving solution being made containing above-mentioned reference substance 1mg in every 1mL, after be gradually diluted to it is required
Concentration；
(2) sample carries out pre-treatment: weighing AL58805 test sample, after dissolution, adjusts pH to alkali with 1mol/L sodium hydroxide solution
Property, with acetonitrile solution constant volume, in the acetonitrile solution: the volume ratio of acetonitrile and water is 50:50；Ultrasound, crystallization, standing are filled
Fractional precipitation filters after liquid-solid phase separation, filtrate is taken to measure；
(3) it uses high performance liquid chromatography to AL58805 bulk pharmaceutical chemicals or the pharmaceutical preparation comprising AL58805 carries out quality analysis:
The detector of the high performance liquid chromatograph is UV detector, and chromatographic condition is as follows: mobile phase is acetonitrile-phosphate aqueous solution；
By the way of isocratic elution, the isocratic elution: acetonitrile-phosphate aqueous solution volume ratio in mobile phase is 50:50；Stream
Speed: 0.8~1.2mL/min；Column temperature: 30~40 DEG C；Detection wavelength: 225nm；Sample volume: 20 μ L；Octadecyl silane color
Compose column；Elution time is 18min.
2. genotoxicity is miscellaneous in high performance liquid chromatography detection AL58805 bulk pharmaceutical chemicals as described in claim 1 or pharmaceutical preparation
The method of matter, which is characterized in that in the step (1), it is 50 that the diluent being gradually diluted in required concentration, which is volume ratio:
50 acetonitrile-water.
3. genotoxicity is miscellaneous in high performance liquid chromatography detection AL58805 bulk pharmaceutical chemicals as described in claim 1 or pharmaceutical preparation
The method of matter, which is characterized in that in the step (2), dissolve test sample with the acetonitrile-water that volume ratio is 50:50；With
1mol/L sodium hydroxide solution adjusts pH to 8.5~9.5；Ultrasound 10 minutes；In 0 DEG C crystallization 30 minutes；It is small to stand 0.5~1.5
When；Use filtering with microporous membrane.
4. genotoxicity is miscellaneous in high performance liquid chromatography detection AL58805 bulk pharmaceutical chemicals as claimed in claim 3 or pharmaceutical preparation
The method of matter, which is characterized in that in the step (2), adjust pH to 9 with 1mol/L sodium hydroxide solution, time of repose is 1 small
When, with 0.45 μm of filtering with microporous membrane.
5. genotoxicity is miscellaneous in high performance liquid chromatography detection AL58805 bulk pharmaceutical chemicals as described in claim 1 or pharmaceutical preparation
The method of matter, which is characterized in that in the step (3), acetonitrile-phosphate aqueous solution volume ratio in mobile phase is 50:50；Phosphorus
The concentration of aqueous acid is 5mmol/L~50mmol/L.
6. genotoxicity is miscellaneous in high performance liquid chromatography detection AL58805 bulk pharmaceutical chemicals as claimed in claim 5 or pharmaceutical preparation
The method of matter, which is characterized in that in the step (3), phosphate aqueous solution concentration is 15mmol/L.
7. genotoxicity is miscellaneous in high performance liquid chromatography detection AL58805 bulk pharmaceutical chemicals as described in claim 1 or pharmaceutical preparation
The method of matter, which is characterized in that in the step (3), chromatographic column is 4.6mm × 250mm, 5 μm of C18 chromatographic column；Flow velocity is
1.0mL/min；Column temperature: 35 DEG C.
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