CN111707747B - Method for detecting mesylate genotoxic impurities in gemcitabine hydrochloride by GC-MS/MS (gas chromatography-Mass Spectrometry/Mass Spectrometry) method - Google Patents
Method for detecting mesylate genotoxic impurities in gemcitabine hydrochloride by GC-MS/MS (gas chromatography-Mass Spectrometry/Mass Spectrometry) method Download PDFInfo
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Abstract
The invention relates to the technical field of drug analysis, in particular to a method for detecting mesylate genotoxic impurities in gemcitabine hydrochloride by a GC-MS/MS method, which comprises the following steps: 1) adding 1, 2-dichloroethane into the gemcitabine hydrochloride, carrying out ultrasonic treatment, adding 1, 2-dichloroethane for constant volume to obtain an extracting solution, and filtering the extracting solution to obtain a test solution; 2) measuring the test solution in the step 1) by adopting a GC-MS/MS method, and calculating the content of each mesylate genotoxic impurity in the gemcitabine hydrochloride by adopting an external standard method; the gas chromatographic column is VF-624ms chromatographic column; the mesylate genotoxic impurities comprise methyl mesylate, ethyl mesylate, isopropyl mesylate, propyl mesylate and butyl mesylate. The determination method provided by the invention has the characteristics of strong impurity interference resistance, low noise, strong specificity and high sensitivity.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to a method for detecting mesylate genotoxic impurities in gemcitabine hydrochloride by a GC-MS/MS method.
Background
Genotoxic Impurities (GTIs) are Impurities that cause genotoxicity, which may be introduced by starting materials, intermediates, reaction by-products, degradation products, reagents, solvents and catalysts in the synthesis of pharmaceutically active ingredients. Genotoxic impurities cause functional changes in the human DNA structure compared to other impurities in the drug, resulting in human genetic mutations, chromosome breakage, chromosome rearrangement, and carcinogenesis. Therefore, in recent years, various regulatory agencies have been particularly concerned with genotoxic impurities, such as the european pharmaceutical commission, the U.S. food and drug administration. In 11 months 2013, ICH issued the assessment and control of DNA active (mutagenic) impurities in drugs to limit the potential carcinogenic risk M7(R1), suggesting the adoption of a "threshold of toxicological concern" (TTC) as an acceptable limit for genotoxic impurities, in the absence of strong evidence supporting the existence of genotoxic thresholds. Meaning that in a human lifetime (70 years of age) 1.5 μ g of genotoxic impurities are ingested daily, the risk of carcinogenesis is acceptable (<1/10 ten thousand). The TTC concept is used to define acceptable intake of all chemicals not studied, but with carcinogenic risk or other toxic effects.
The problem of residual genotoxic impurities in the medicine becomes a major challenge in the pharmaceutical industry, the nelfinavir mesylate, an anti-AIDS medicine produced by Roche in 2008, is recalled from the market due to the fact that ethyl methanesulfonate is detected to be seriously overproof, N-nitrosodimethylamine genotoxic impurities in valsartan in Wasabartan in Waihai pharmaceutical industry in 2018 are overproof, so that related preparations are also recalled from markets of Europe, America and China, and genotoxic impurities are found to be overproof in three batches of valsartan medicines in Torrent pharmaceutical company in 2018 and 9. The three events sound the alarm clock for various domestic drug manufacturers, and as the ICH is officially added in China, the relevant regulation and policy of the domestic drug research and development gradually draw close to the ICH. Recently, the official network of the national pharmacopoeia committee issued "the public work on the revised contents (fourth lot) of the four general rules of the" Chinese pharmacopoeia "2020 edition", which included the "inspection draft of the control guidelines for genotoxic impurities". In view of the above, the study of genotoxic impurities has become particularly important.
Methanesulfonic acid esters are currently identified as genotoxic impurities, and in 1999, methyl methanesulfonate is listed as a class 2A carcinogen (which is likely to be carcinogenic to humans, has limited evidence of carcinogenicity to humans and sufficient evidence of carcinogenicity to experimental animals) by the world health organization international agency for research on cancer, and ethyl methanesulfonate is listed as a class 2B carcinogen in 1987 (which is likely to be carcinogenic to humans, has limited evidence of carcinogenicity to humans and insufficient evidence of carcinogenicity to experimental animals, or has insufficient evidence of carcinogenicity to humans and sufficient evidence of carcinogenicity to experimental animals). It has been shown that the methanesulfonic acid esters can act on purine group of DNA molecule, produce alkylated purine, destroy space conformation and stability of DNA double strand, induce body gene mutation and cancer. In accordance with the guidelines issued by the European Committee for drug administration for GTI control, and the associated requirements issued by ICH for genetic impurities (ICH M7), mesylate was based on the maximum daily drug intake (MDD) with a toxicological threshold of 1.5 μ g/day.
During the production process of the medicine, alcohol substances such as methanol, ethanol and isopropanol are commonly used as solvents for medicine crystallization or purification to participate in medicine synthesis, and methanesulfonic acid is commonly used as a counter ion reagent to form a stable conjugate salt substance with active ingredients of the medicine so as to improve the physical and chemical properties of the medicine, such as solubility, absorbability and the like. During the synthesis of the medicine, methanesulfonic acid or methanesulfonyl chloride can generate side reaction with alcohol substances to generate genotoxic methanesulfonic acid ester substances, and the substances are generally difficult to be completely removed from a synthesis system. Gemcitabine hydrochloride is a novel artificially synthesized difluoro nucleoside antimetabolite antitumor drug, and according to a synthetic route provided by an enterprise and a literature report, solvents such as methanesulfonyl chloride, methanol, ethanol, isopropanol and the like are used in the synthesis process of gemcitabine hydrochloride, so that possibility of generating mesylate genotoxic impurities exists; such as methanesulfonate drugs, also because methanesulfonic acid and various common alcohols are used in the synthesis of drugs, there is a possibility of generating genotoxic impurities of methanesulfonate esters. Therefore, in the research and development process, daily inspection and detection of medicines in the circulation link of raw material medicines, qualitative and quantitative detection of the mesylate genotoxic impurities in the medicines (particularly the mesylate medicines) at a trace level is very important.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for detecting genotoxic methanesulfonic acid ester impurities in gemcitabine hydrochloride by a GC-MS/MS method, which can carry out quantitative detection on genotoxic impurities such as methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate and butyl methanesulfonate in gemcitabine hydrochloride bulk drug or gemcitabine hydrochloride injection on a micro-level.
In order to achieve the above purpose of the invention, the following technical scheme is adopted:
a GC-MS/MS method for detecting mesylate genotoxic impurities in gemcitabine hydrochloride comprises the following steps:
1) adding 1, 2-dichloroethane into the gemcitabine hydrochloride, performing ultrasonic treatment, adding 1, 2-dichloroethane for constant volume to obtain an extracting solution, filtering the extracting solution, and taking a subsequent filtrate to obtain a test solution;
2) measuring the test solution in the step 1) by adopting a GC-MS/MS method, and calculating the content of each mesylate genotoxic impurity in the gemcitabine hydrochloride by adopting an external standard method;
the gas chromatographic column is VF-624ms chromatographic column;
the mesylate genotoxic impurities comprise methyl mesylate, ethyl mesylate, isopropyl mesylate, propyl mesylate and butyl mesylate.
Further, the mass volume ratio of gemcitabine hydrochloride to 1, 2-dichloroethane in the extracting solution in the step 1) is 0.022 to 0.026 g/ml; preferably, the mass-to-volume ratio of gemcitabine hydrochloride to 1, 2-dichloroethane in the extracting solution of step 1) is 0.024 g/ml.
The meaning of the mass to volume ratio in the above-described method is exemplified as follows:
for example, when preparing a test solution, gemcitabine hydrochloride is weighed to have a mass of 1.2g and a volumetric flask to have a mass-to-volume ratio of 50ml, and the mass-to-volume ratio is calculated by: 0.024g/ml for 1.2g/50 ml.
1.2g described herein is a numerical value obtained after rounding the second digit after decimal point.
In other embodiments provided by the present invention, the weighed gemcitabine hydrochloride may also have a mass of 1.1g or 1.3g, and when the volumetric flask is 50ml, the corresponding mass-to-volume ratios of gemcitabine hydrochloride in the obtained extract are 0.022g/ml and 0.026g/ml, respectively.
The quality of the gemcitabine hydrochloride refers to the quality of a gemcitabine hydrochloride raw material sample, and when the gemcitabine hydrochloride is gemcitabine hydrochloride for injection, the quality of the gemcitabine hydrochloride is calculated according to the gemcitabine hydrochloride raw material added in gemcitabine hydrochloride shores for injection.
Further, the frequency of the ultrasound in the step 1) is 40000 Hz.
Further, the time of the ultrasonic treatment is 25-35min, preferably 30 min.
Further, the air conditioner is provided with a fan,
when gemcitabine hydrochloride is used as a raw material drug, the step 1) adopts the following specific steps:
precisely weighing about 1.2g gemcitabine hydrochloride, placing the gemcitabine hydrochloride into a 50ml volumetric flask, adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30min, cooling, diluting to a scale with 1, 2-dichloroethane, shaking up, filtering, and taking subsequent filtrate to obtain a test sample solution;
when the gemcitabine hydrochloride is gemcitabine hydrochloride for injection, the specific steps adopted in the step 1) are as follows:
taking a gemcitabine hydrochloride 6 bottle for injection (equivalent to 1.2g of gemcitabine hydrochloride), transferring the content of the gemcitabine hydrochloride into a 50ml measuring flask by using 25ml of 1, 2-dichloroethane, adding the 1, 2-dichloroethane for dilution to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain a gemcitabine hydrochloride test solution for injection.
The preparation method of the test solution provided by the invention selects a direct extraction method, can effectively extract five mesylate genotoxic impurities in the gemcitabine hydrochloride, has few extraction operation steps, is convenient and simple, does not need to carry out multiple extraction operations on the sample, and can directly be used as a test sample for sample injection detection without drying and dewatering the extracted sample. Proved by methodology, the method provided by the invention can be used for detecting 5 methanesulfonic acid ester genotoxic substances in gemcitabine hydrochloride bulk drug, and the average recovery rate is 99% -104%.
Further, in step 2), the gas chromatography measurement conditions employed were:
the column length is 60m, the diameter is 0.25mm, and the film thickness is 1.4 μm;
column temperature: adopts the temperature programming with the initial temperature of 120 ℃ and the temperature of 10 ℃ per minute-1Heating to 250 deg.C and maintaining for 5 min;
sample inlet temperature: 250 ℃;
constant flow rate: 1.0 ml/min;
split-flow sample introduction, split-flow ratio: 10: 1;
sample introduction amount: 1 μ L.
Further, in step 2), the mass spectrometry conditions employed are:
EI source temperature: 230 ℃;
energy of electrons: 70 eV;
solvent delay time: 6.5 min.
Further, in performing mass spectrometry, the qualitative and quantitative ion pairs and collision energies used are as follows:
the technical scheme of the invention has the following advantages: the method for detecting the mesylate genotoxic impurities in the gemcitabine hydrochloride by the GC-MS/MS method has the advantages of wide linear range and good linear correlation through methodological verification; the recovery test result shows that the average recovery rate is 93-104% (original n is 9) and RSD (%) < 3.0, which indicates that the method has higher accuracy and can well realize the accurate determination of the residual quantity of the toxic impurities of the mesylate genes; the repeatability and the precision test are good. The quantitative limit of the 5 components to be detected of the methanesulfonic acid esters is 10 ng/ml; the detection limit is 5ng/ml, which indicates that the method has higher sensitivity. The detection method adopts an MRM mode (multiple interaction monitoring, mass spectrum multiple reaction monitoring), selects ions with higher specificity of each component to be detected for monitoring and analysis, and has better specificity and stronger anti-interference capability compared with a GC-MS method.
In conclusion, the method has the characteristics of strong anti-interference capability, low noise, strong specificity and high sensitivity, and can be well applied to daily detection of methyl sulfonate genotoxic impurities such as methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate and butyl methanesulfonate in gemcitabine hydrochloride bulk drug and gemcitabine hydrochloride for injection of production enterprises. Has important significance on the production and daily supervision and inspection of the medicine, is beneficial to improving the quality of the medicine, further reduces the clinical medication risk and ensures the medication safety of patients.
Drawings
FIG. 1 shows the results of a linear relationship test for methyl methanesulfonate;
FIG. 2 shows the results of a linear relationship test of ethyl methanesulfonate;
FIG. 3 shows the results of a linear relationship test of isopropyl methanesulfonate;
FIG. 4 shows the results of a linear relationship test for propyl methanesulfonate;
FIG. 5 shows the results of a linear relationship test for butyl methanesulfonate;
FIG. 6 blank solvent (1, 2-dichloroethane) chromatogram;
FIG. 7 chromatogram of a control solution;
FIG. 8 a limit quantitation chromatogram;
FIG. 9 detection limit chromatogram;
FIG. 10 chromatogram of comparative example 2 with ethyl acetate as solvent.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The following examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
1 instruments and reagents
Agilent 7890B-7000C gas chromatography-tandem mass spectrometer
Ethylene dichloride source: ladder love (shanghai) into industrial development limited; batch number: p1529190;
and (3) ethyl acetate source: LiChrosolv; batch number: 10942649812, respectively; purity: mass spectral scale
Anhydrous sodium sulfate: west longa science, inc; batch number: 1801051, respectively; the content is more than or equal to 99.0 percent; purity: analytical purity
Reference source
Sources of methyl methanesulfonate: SIGMA-ALORICH; batch number: MKCG 1346; the content is as follows: more than 99 percent
Sources of ethyl methanesulfonate: SIGMA-ALORICH; batch number: BCBW 8635;
sources of isopropyl methanesulfonate: sammer Feishel technologies (China) Co., Ltd; batch number: a031422; the content is as follows: more than 98.0 percent
Sources of propyl methanesulfonate: ladder love (shanghai) into industrial development limited; batch number: G6G 6L-OM; the content is as follows: more than 99 percent
Sources of butyl methanesulfonate: beijing Bailingwei Tech Co., Ltd; batch number: LS20S 35; the contents are as follows: is more than 98.5 percent.
The preparation method of the test solution comprises the following steps: taking about 1.2g of gemcitabine hydrochloride raw material, precisely weighing, placing in a 50ml measuring flask, adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30 minutes, cooling, diluting with 1, 2-dichloroethane to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain a raw material test solution.
Taking a gemcitabine hydrochloride 6 bottle for injection (about equivalent to 1.2g of gemcitabine hydrochloride), transferring the content of the gemcitabine hydrochloride into a 50ml measuring flask by using 25ml of 1, 2-dichloroethane, adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30 minutes, cooling, diluting to a scale by using 1, 2-dichloroethane, shaking up, filtering, and taking a subsequent filtrate to obtain the gemcitabine hydrochloride test sample solution for injection.
2 methodology
2.1.1 chromatographic and Mass Spectrometry conditions
A chromatographic column: VF-624MS (60 m. times.0.25 mm. times.1.4 μm) quartz capillary column;
the measurement conditions were as follows:
carrier gas: helium, purity not less than 99.999%, flow rate: 1.0 mL/min; collision gas: the purity of nitrogen is more than or equal to 99.999 percent.
Column temperature: adopts the temperature programming with the initial temperature of 120 ℃ and the temperature of 10 ℃ per minute-1Heating to 250 deg.C and maintaining for 5 min;
sample inlet temperature: 250 ℃; constant flow rate: 1.0 ml/min; split-flow sample introduction, split-flow ratio: 10:1
Sample introduction amount: 1 μ L
An ionization mode: EI ion source, source temperature: 230 ℃, electron energy: 70 eV; solvent delay time: 6.5 min.
Ionizing each methanesulfonate reference substance by ion source, and testing by GC-MS/MS Scanning (SCAN) mode, wherein methyl methanesulfonate selects molecular ion: (
Molecular weight 110) and demethoxy fragment ion thereof: (
Molecular weight 80) as a qualitative and quantitative parent ion; fragment ion (molecular ion) selected from ethylmethanesulfonate, propylmethanesulfonate and butylmethanesulfonate (1
Molecular weight 123) and fragment ions thereof (
Molecular weight 109) as a qualitative and quantitative parent ion; isopropyl methanesulfonate selective fragment ion (molecular ion) ((II))
Molecular weight 123) as a qualitative and quantitative parent ion.
Ion pair and collision energy optimization: and (3) optimizing ion pairs with strong specificity and large abundance by adopting the parent ions of the objects to be tested through an ion scanning mode test. Through the optimization of collision energy, the final qualitative and quantitative ion pairs of each object to be detected and the corresponding collision energy are finally selected, and the details are shown in table 1.
TABLE 1 ion Pair selection and Collision energy information
2.1.2 assay
Precisely measuring 1 μ L of each of the mixed control solutions, respectively injecting into a gas chromatograph, and drawing a standard curve with the measured value as ordinate and the concentration as abscissa. Precisely measuring 1 μ L of the sample solution, respectively injecting into a gas chromatograph, and calculating to obtain corresponding concentration from the standard curve.
2.1.3 Linear relationship
Precisely weighing 10.25mg of methyl methanesulfonate, 10.10mg of ethyl methanesulfonate, 10.15mg of isopropyl methanesulfonate, 10.33mg of propyl methanesulfonate and 10.26mg of butyl methanesulfonate, putting the weighed materials into a same 10ml measuring flask, adding 1, 2-dichloroethane for dissolving and diluting to scale, shaking up, precisely weighing 1ml, putting the weighed materials into a 200ml measuring flask, diluting to scale with 1, 2-dichloroethane, shaking up, and taking the weighed materials as a reference substance mixed stock solution (about 5 mug/ml);
precisely measuring 0.5 ml, 1ml, 2ml, 3 ml, 4 ml, 5ml and 6ml of reference substance mixed stock solution, respectively placing the reference substance mixed stock solution into 100ml measuring bottles, diluting the reference substance mixed stock solution to a scale by using 1, 2-dichloroethane, shaking the reference substance mixed stock solution uniformly to obtain mixed reference substance solutions of (i), (ii), (iii), (iv), (v), (c) and (c); thus obtaining series of reference substance solutions with the concentration of each substance to be detected being about 25, 50, 100, 150, 200, 250 and 300 ng/ml. The results of the linear relationship test of the control solution are shown in Table 2 and FIGS. 1-5, and the chromatograms of the blank solution and the control solution are shown in FIGS. 6-7.
According to the guidelines issued by the European Committee for drug administration for the control of GTI and the relevant requirements issued by ICH for the guidelines for genetic impurities (ICH M7), the threshold for toxicity was 1.5 μ g/day for the mesylate based on the maximum daily drug intake (MDD). The usage amount in the gemcitabine hydrochloride instruction book for injection shows that the maximum injection amount of gemcitabine hydrochloride for injection is 1000mg/m2Average human epidermis area of 1.6m according to adult2The mesylate should not exceed 6.6ppm, calculated as the 1.5. mu.g/d genotoxic impurity limit. The concentration of the gemcitabine hydrochloride test sample in the test is 0.024g/ml, so that the concentration of each mesylate in the test solution should not exceed 158ng/ml, the concentration of the control solution in the linear relation test in the test is 25-300 ng/ml, the concentration point of 150ng/ml is taken as the middle point concentration, the concentration point of 25ng/ml at the lowest concentration point is the quantitative limit concentration of each instrument to be tested and is far lower than 150ng/ml, and the concentration of each control solution in the linear relation test is reasonably set.
TABLE 2 control solutions linearity test results
From the results of FIGS. 1-7 and Table 2 above, it can be seen that: the method has wide linear range and good linear correlation.
2.1.4 recovery test
Precisely measuring 10ml of reference substance mixed stock solution (about 5 mu g/ml), placing the reference substance mixed stock solution into a 100ml measuring flask, diluting the reference substance mixed stock solution to a scale by using 1, 2-dichloroethane, shaking up, and using the reference substance mixed stock solution as a recovery rate test;
the method comprises the steps of taking about 1.2g of gemcitabine hydrochloride bulk drug, precisely weighing 9 parts, respectively placing the gemcitabine hydrochloride bulk drug into 50ml measuring flasks, respectively placing 1, 1.5, 2 and 2ml of mixed stock solution of a precise reference substance (about 5 mu g/ml), adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30 minutes, cooling, diluting the mixed stock solution with 1, 2-dichloroethane to a scale, shaking up the mixed stock solution, filtering, taking subsequent filtrate to obtain a sample solution for a recovery rate test, measuring the sample solution according to a method under the item of '2.1.2 measuring method', calculating the recovery result of each substance to be measured by using a standard curve under the item of '2.1.3 linear relation', and details are shown in table 3.
TABLE 3 recovery test results of the test substance in the crude drug
As can be seen from Table 3: the average recovery rate of the gemcitabine hydrochloride bulk drug by the method is 99-104%, and RSD (%) < 3.0, which shows that the method has high detection accuracy when being used for detecting methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate and butyl methanesulfonate in the gemcitabine hydrochloride bulk drug.
Taking a gemcitabine hydrochloride 6 bottle (about equivalent to 1.2g of gemcitabine hydrochloride), transferring the content into a 50ml measuring flask by using 25ml of 1, 2-dichloroethane, preparing 9 parts by the same method, respectively and precisely adding a reference substance mixed stock solution (about 5 mu g/ml)1, 1.5, 2 and 2ml, adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30 minutes, cooling, diluting with 1, 2-dichloroethane to a scale, shaking up, filtering, taking a subsequent filtrate to obtain a recovery test sample solution, measuring by using a 2.2.2 measuring method, and calculating the recovery result of each substance by using a 2.2.3 linear relation. The recovery test results are detailed in table 4.
TABLE 4 recovery test results of test substances in gemcitabine for injection
As can be seen from Table 4: the method for determining the genotoxic substance in the gemcitabine hydrochloride for injection has the average recovery rate of 93-101% and the RSD (%) < 3.0, and shows that the method is higher in detection accuracy when used for detecting methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate and butyl methanesulfonate in the gemcitabine hydrochloride for injection.
2.1.5 precision test
Taking the item 2.2.3 mixed reference solution, repeatedly injecting sample for 6 times according to the test condition under the item 2.2.1, and recording the test result. The results are detailed in Table 5, indicating good precision of the instrument.
TABLE 5 results of precision test
2.1.6 repeatability test
Taking about 1.2g of gemcitabine hydrochloride bulk drug, precisely weighing 6 parts, respectively placing the gemcitabine hydrochloride bulk drug into 50ml measuring flasks, respectively and precisely adding 1.5ml of reference substance mixed stock solution (about 5 mu g/ml) into each sample, adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30 minutes, cooling, diluting to a scale with 1, 2-dichloroethane, shaking up, filtering, and taking subsequent filtrate to obtain gemcitabine hydrochloride bulk drug repeatability test solution. The results show that the sample has good repeatability, and are detailed in Table 6.
TABLE 6 results of repeatability tests of the test substances in gemcitabine hydrochloride bulk drug
Taking a gemcitabine hydrochloride 6 bottle for injection (about equivalent to 1.2g of gemcitabine hydrochloride), transferring the content of the gemcitabine hydrochloride into a 50ml measuring flask by using 25ml of 1, 2-dichloroethane, preparing 6 parts by the same method, respectively and precisely adding 1.5ml of a control mixed stock solution (about 5 mu g/ml), adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30 minutes, cooling, diluting to a scale by using 1, 2-dichloroethane, shaking up, filtering, taking subsequent filtrate to obtain a gemcitabine hydrochloride repeatability test solution for injection, measuring by using a 2.2.2 measuring method, and calculating the content of each substance to be measured by using a 2.2.3 linear relation. The results are detailed in Table 7, which shows that the sample has good repeatability.
TABLE 7 repeatability test results of the test substances in gemcitabine hydrochloride for injection
2.1.7 stability test
Taking about 1.2g of gemcitabine hydrochloride raw material, precisely weighing, respectively placing the gemcitabine hydrochloride raw material into 50ml measuring flasks, precisely adding 1.5ml of a reference substance mixed stock solution (about 5 mu g/ml) into a sample, adding 1, 2-dichloroethane to about 40ml, carrying out ultrasonic treatment for 30 minutes, cooling, diluting the sample to a scale with the 1, 2-dichloroethane, shaking up, filtering, taking a subsequent filtrate, measuring according to test conditions under the item '2.1.1' and a measuring method under the item '2.1.2' for 0, 2, 4, 6, 8, 10, 12 and 24 hours, and inspecting the stability of the sample solution. The results show that the sample solutions were stable for each test article over 24 hours, as detailed in table 8.
TABLE 8 stability test results
The gemcitabine hydrochloride 6 bottle for injection (about equivalent to 1.2g gemcitabine hydrochloride) is taken, 25ml 1, 2-dichloroethane is used for transferring the content into a 50ml measuring flask, 1.5ml of a mixed stock solution (about 5 mu g/ml) of a control product is precisely added, 1, 2-dichloroethane is added to about 40ml, ultrasonic treatment is carried out for 30 minutes, the mixture is cooled, the mixture is diluted to a scale by the 1, 2-dichloroethane, shaking up and filtering are carried out, and subsequent filtrate is taken and is measured according to the test condition under the item '2.2.1' and the test method under the item '2.2.2' for 0, 2, 4, 6, 8, 10, 12 and 24 hours, and the stability of the sample solution is examined. The results are summarized in Table 9, which shows that the sample solutions are stable for each test object within 24 hours.
TABLE 9 stability test results
2.1.8 detection limit and quantification limit
Taking the reference solution, diluting the reference solution step by step, then testing the reference solution, taking the corresponding mass concentration as the quantitative limit concentration when the signal-to-noise ratio is about 10:1, taking the corresponding mass concentration as the detection limit concentration when the signal-to-noise ratio is about 3:1, and calculating the detection limit and the quantitative limit according to the sample amount. The results are shown in Table 10, and the quantitative limit and detection limit chromatograms are shown in FIGS. 8-9.
TABLE 10 detection limit and quantitation limit results
As can be seen from FIGS. 8-9 and Table 10, the method of the present invention provides high sensitivity.
Example 2
According to the preparation method of the test solution provided in example 1, the gemcitabine hydrochloride bulk drug is changed to 1.1g, and the rest is unchanged, so that the methodological data can achieve the effect of example 1.
Example 3
According to the preparation method of the test solution provided in the embodiment 1, the quality of the gemcitabine hydrochloride raw material drug is changed to 1.3g, and the rest is not changed, so that the methodological data can achieve the effect of the embodiment 1.
Example 4
According to the preparation method of the test solution provided by the embodiment 1, the ultrasonic extraction time is replaced by 25min, and the rest is unchanged, so that the methodological data can achieve the effect of the embodiment 1.
Example 5
According to the preparation method of the test solution provided in the example 1, the ultrasonic extraction time is replaced by 35min, and the rest is unchanged, so that the methodological data can achieve the effect of the example 1.
Comparative example 1
Extracting genotoxic substances in the gemcitabine hydrochloride raw material medicament by using 1, 2-dichloroethane through an extraction method, and detecting the recovery rate, wherein the extraction method and the detection result are as follows:
preparing a recovery solution by an extraction method: taking about 1.2g and 9 parts of gemcitabine hydrochloride raw material, respectively precisely adding 50ml of deionized water into mixed stock solutions (about 5 mu g/ml) of 1, 1.5, 2 and 2ml of gemcitabine hydrochloride raw material, performing ultrasonic treatment (40000Hz) for 10 minutes, cooling, transferring to a separating funnel, respectively and precisely adding 15, 15 and 20ml of 1, 2-dichloroethane, performing shaking extraction for times, collecting 1, 2-dichloroethane layer after 3 times of extraction, placing in a 100ml centrifuge tube, adding 10g of anhydrous sodium sulfate to remove water, filtering, and taking the subsequent filtrate to obtain the extraction method recovery solution. According to the determination method under the item of 'determination method 2.1.2 in example 1', the recovery result of each substance to be detected is calculated by using a standard curve under the item of '2.1.3 linear relation', the result shows that the recovery rate of the extraction method is lower, wherein the average recovery rate of the methyl methanesulfonate is 78.95%; the average recovery rate of ethyl methanesulfonate was 84.36%; the average recovery rate of isopropyl methanesulfonate is 88.71%; the average recovery rate of propyl methanesulfonate was 87.65%; the average recovery of butyl methanesulfonate was 89.27%.
Comparative example 2
The solvent test was carried out using N, N-dimethylformamide, N-dimethylacetamide and ethyl acetate:
preparation of a mixed control solution from N, N-dimethylformamide: precisely weighing 10mg of methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate and butyl methanesulfonate respectively, placing the weighed materials into a same 10ml measuring flask, adding N, N-dimethylformamide to dissolve and dilute the materials to a scale, shaking up, precisely weighing 1ml, placing the weighed materials into a 200ml measuring flask, diluting the weighed materials to the scale with N, N-dimethylformamide, shaking up, precisely weighing 1ml, placing the weighed materials into a 100ml measuring flask, diluting the weighed materials with N, N-dimethylformamide to the scale, and shaking up to obtain a mixed reference substance solution (40ng/ml) prepared from N, N-dimethylformamide.
Preparation of a mixed control solution of N, N-dimethylacetamide: accurately weighing 10mg of methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate and butyl methanesulfonate respectively, placing the weighed materials into a same 10ml measuring flask, adding N, N-dimethylacetamide to dissolve and dilute the materials to a scale, shaking up, accurately weighing 1ml, placing the weighed materials into a 200ml measuring flask, diluting the weighed materials to a scale by using N, N-dimethylacetamide, shaking up, accurately weighing 1ml, placing the weighed materials into a 100ml measuring flask, diluting the weighed materials to a scale by using N, N-dimethylacetamide, and shaking up to obtain a mixed reference substance solution (40ng/ml) prepared from N, N-dimethylacetamide.
Preparation of ethyl acetate-prepared mixed control solution: accurately weighing 10mg of methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate and butyl methanesulfonate respectively, placing the weighed materials into a same 10ml measuring flask, adding ethyl acetate to dissolve and dilute the materials to a scale, shaking up, accurately weighing 1ml, placing the weighed materials into a 200ml measuring flask, diluting the weighed materials with ethyl acetate to a scale, shaking up, accurately weighing 1ml, placing the weighed materials into a 100ml measuring flask, diluting the weighed materials with ethyl acetate to a scale, and shaking up to obtain a mixed reference substance solution (40ng/ml) prepared from ethyl acetate.
The results show that: gemcitabine hydrochloride and toxic impurities of mesylate genes thereof are better dissolved in N, N-dimethylformamide and N, N-dimethylacetamide, but a methyl mesylate peak in a chromatogram of a reference solution cannot be separated from a solvent peak, so that the methyl mesylate cannot be accurately measured; gemcitabine hydrochloride is difficult to dissolve in two solvents of ethyl acetate and 1, 2-dichloroethane, but the solubility of the toxic impurities of the mesylate genes is good, and the detection shows that: ethyl acetate baseline was more noisy, resulting in lower sensitivity to methyl methanesulfonate, see figure 10. In 1, 2-dichloroethane, no solvent influence exists, the baseline noise is low, and the sensitivity of each object to be detected is high.
Comparative example 3
The invention optimizes the chromatographic column for DB-5ms (30m multiplied by 0.25mm multiplied by 0.25 mu m), VF-624ms (60m multiplied by 0.25mm multiplied by 1.40 mu m) and VF-WAXms (60m multiplied by 0.25mm multiplied by 0.25 mu m);
the results show that: DB-5ms (30m 0.25mm 0.25 m) defects are: the first peak of the component to be measured (methyl methanesulfonate) is susceptible to the influence of the solvent, resulting in a low sensitivity.
The VF-WAXms (60 m.times.0.25 mm. times.1.40 μm) defects are: 5 components to be measured can be well separated by adopting a VF-WAXms (60m multiplied by 0.25mm multiplied by 1.40 mu m) capillary chromatographic column and a VF-624ms (60m multiplied by 0.25mm multiplied by 1.40 mu m) capillary chromatographic column, and each component to be measured is not influenced by a solvent. From the evaluation of the degree of separation of each peak of the component to be measured, the degree of separation of each peak of the component to be measured was more preferable by using a VF-624ms (60 m.times.0.25 mm. times.1.40 μm) capillary column.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A GC-MS/MS method for detecting mesylate genotoxic impurities in gemcitabine hydrochloride is characterized by comprising the following steps:
1) adding 1, 2-dichloroethane into the gemcitabine hydrochloride, performing ultrasonic treatment, adding 1, 2-dichloroethane to a constant volume to obtain an extracting solution, filtering the extracting solution, and taking a subsequent filtrate to obtain a test solution;
2) measuring the test solution in the step 1) by adopting a GC-MS/MS method, and calculating the content of each mesylate genotoxic impurity in the gemcitabine hydrochloride by adopting an external standard method;
the gas chromatographic column is VF-624ms chromatographic column; the gas chromatography measurement conditions adopted were:
the column length is 60m, the diameter is 0.25mm, and the film thickness is 1.4 μm;
column temperature: heating by a program at an initial temperature of 120 ℃ to 250 ℃ at 10 ℃ min < -1 > for 5 min;
sample inlet temperature: 250 ℃;
constant flow rate: 1.0 ml/min;
split-flow sample introduction, split-flow ratio: 10: 1;
sample introduction amount: 1 mu L of the solution;
in step 2), the mass spectrometry conditions used were:
EI source temperature: 230 ℃;
electron energy: 70 eV;
solvent delay time: 6.5 min;
the mass spectrum selects an SCAN detection mode, and when mass spectrum operation is carried out, the adopted qualitative and quantitative ion pairs and collision energy are as follows:
the mesylate genotoxic impurities comprise methyl mesylate, ethyl mesylate, isopropyl mesylate, propyl mesylate and butyl mesylate.
2. The method of claim 1, wherein the ratio of gemcitabine hydrochloride to 1, 2-dichloroethane in the step 1) is 0.022-0.026 g/ml.
3. The method of claim 2, wherein the ratio of gemcitabine hydrochloride to 1, 2-dichloroethane in step 1) is 0.024 g/ml.
4. The method for detecting the mesylate genotoxic impurities in the gemcitabine hydrochloride according to claim 1 or 2, wherein the gemcitabine hydrochloride is gemcitabine hydrochloride bulk drug or gemcitabine hydrochloride for injection.
5. The method for detecting the mesylate genotoxic impurities in the gemcitabine hydrochloride according to claim 1 or 2, wherein the ultrasound in step 1) has a frequency of 40000 Hz.
6. The method for detecting the mesylate genotoxic impurities in gemcitabine hydrochloride of claim 1 or 2, wherein the sonication time is 25-35 min.
7. The method of claim 5, wherein the sonication time is 30 min.
8. The method of claim 1 or 2, wherein the GC-MS/MS method is used to detect the toxic impurity of the mesylate gene in the gemcitabine hydrochloride,
when gemcitabine hydrochloride is used as a raw material drug, the step 1) adopts the following specific steps:
precisely weighing 1.2g gemcitabine hydrochloride, placing in a 50ml volumetric flask, adding 1, 2-dichloroethane to about 40ml, performing ultrasonic treatment for 30min, cooling, diluting with 1, 2-dichloroethane to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution;
when the gemcitabine hydrochloride is gemcitabine hydrochloride for injection, the specific steps adopted in the step 1) are as follows:
taking a gemcitabine hydrochloride 6 bottle for injection, equivalent to 1.2g of gemcitabine hydrochloride, transferring the content of the gemcitabine hydrochloride into a 50ml measuring flask by using 25ml of 1, 2-dichloroethane, adding the 1, 2-dichloroethane for dilution to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain a gemcitabine hydrochloride test solution for injection.
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