CN115598230A - Analysis method for determining antioxidant BHT in anticancer drugs by GC-MS - Google Patents

Analysis method for determining antioxidant BHT in anticancer drugs by GC-MS Download PDF

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CN115598230A
CN115598230A CN202210029657.5A CN202210029657A CN115598230A CN 115598230 A CN115598230 A CN 115598230A CN 202210029657 A CN202210029657 A CN 202210029657A CN 115598230 A CN115598230 A CN 115598230A
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solution
sample
antioxidant bht
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郑浩
高长文
秦秋明
焦玲
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Shanghai Microspectrum Chemical Technology Service Co Ltd
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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
    • G01N30/02—Column chromatography
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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
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
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    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
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Abstract

The invention relates to the technical field of G01N30/00, in particular to an analysis method for determining antioxidant BHT in an anticancer medicine by adopting GC-MS. An analytical method for measuring antioxidant BHT in an anticancer drug by GC-MS, comprising: preparing a standard substance solution; preparing a standard curve solution; preparing a sample mother solution; preparing a sample solution, taking a sample mother solution, adding the sample mother solution into an organic solvent, uniformly mixing, adding inorganic salt, and standing; sampling the upper layer solution of the mother solution of the sample, adding inorganic salt, and taking the upper layer solution as the sample solution; detection is carried out by GC-MS; the method for determining the antioxidant BHT in the paclitaxel injection product has the advantages of good specificity, linearity, accuracy, repeatability and durability.

Description

Analysis method for determining antioxidant BHT in anticancer drugs by GC-MS
Technical Field
The invention relates to the technical field of G01N30/00, in particular to an analysis method for determining antioxidant BHT in an anticancer medicine by adopting GC-MS.
Background
Chinese patent No. CN202010384972.0 discloses a method for measuring antioxidant BHT and major transformation products in different food matrices, which uses liquid chromatography to measure the transformation products of antioxidant BHT, but is not suitable for analyzing and detecting small amount of antioxidant BHT contained in anticancer drugs.
The antioxidant BHT is used as an additive in the preparation process of the infusion set, is easy to migrate into the anticancer drug from the infusion set in the using process of the infusion set, in particular to migrate into a paclitaxel injection used as the anticancer drug, and ethanol and polyoxyethylated castor oil are added into the paclitaxel injection in the processing process, so that the antioxidant BHT is favorably separated out; the migration amount of the antioxidant BHT exceeds the limit value of a human body, and the clinical treatment curative effect of the anticancer drug is influenced.
Therefore, it is only a technical problem to be solved to provide a method for rapidly detecting the content of the antioxidant BHT in the anticancer drug.
Disclosure of Invention
In order to solve the above problems, the present invention provides an analytical method for measuring antioxidant BHT in an anticancer drug using GC-MS, comprising:
(1) preparing a standard substance solution, adding an antioxidant BHT into an organic solvent, and diluting to a scale to obtain the standard substance solution;
(2) preparing a standard curve solution, adding an organic solvent into the standard substance solution, and diluting to a scale to obtain the standard curve solution;
(3) preparing a sample mother solution, adding the anti-cancer drugs into water, uniformly mixing, adding inorganic salt, mixing, adding water to a constant volume, and standing to obtain the sample mother solution;
(4) preparing a sample solution, taking a sample mother solution, adding the sample mother solution into an organic solvent, uniformly mixing, adding inorganic salt, and standing; sampling the upper layer solution of the mother solution of the sample, adding inorganic salt, and taking the upper layer solution as the sample solution;
(5) and detecting by GC-MS, wherein the model of a chromatographic column is HP-5MS, carrier gas is He, and the temperature of a sample inlet is 260-295 ℃.
Preferably, the anticancer drug is paclitaxel injection.
The anti-cancer medicine can be used together with normal saline in the application process and is used for clinical treatment, and in the transfusion use process, the antioxidant BHT is used as an additive in the preparation process of a transfusion device and is easy to migrate into the anti-cancer medicine from the transfusion device, particularly to a paclitaxel injection as the anti-cancer medicine, and because ethanol and polyoxyethylated castor oil are added in the processing process of the paclitaxel injection, the separation of the antioxidant BHT is facilitated; however, the migration amount of the antioxidant BHT exceeds the limit of a human body, the clinical treatment efficacy of the anticancer drug is affected, when the antioxidant BHT is detected, the antioxidant BHT is easily interfered by impurities, and the anticancer drug and normal saline are mixed, water and other inorganic salts cannot be directly used as samples to be tested and need to be removed, but the anticancer drug is dissolved in water, the anticancer drug can be lost in the process of removing water, and the test result of the antioxidant BHT is affected, so that the anticancer drug needs to be specially treated, the impurities can interfere with the antioxidant BHT in the test process, the requirement of the test cannot be met by using gas chromatography, the test is carried out by using a gas chromatography-mass spectrometer, and the interference of the impurities with the antioxidant BHT is removed by combining a chromatographic method and a mass spectrometry method, so that the method for analyzing the antioxidant BHT in the anticancer drug with good specificity, accuracy, repeatability and durability is obtained.
Preferably, the organic solvent is selected from one or more of n-hexane, acetone, ethyl acetate, dichloromethane and acetonitrile.
Further, the organic solvent is acetonitrile.
Preferably, the step of preparing the standard substance solution comprises: weighing a certain amount of antioxidant BHT standard substance in a measuring flask, dissolving with acetonitrile, diluting to scale, and shaking; and (3) measuring the solution into a measuring flask, diluting the solution to a scale by using acetonitrile, and shaking up to obtain an antioxidant BHT standard substance solution.
Further, the step of preparing the standard substance solution includes: weighing a certain amount of antioxidant BHT standard substance 41.935mg in a 10ml measuring flask, dissolving with acetonitrile, diluting to scale, and shaking up; measuring 4.80 mu l of the solution into a 20ml measuring flask, diluting the solution to the scale with acetonitrile, and shaking up to obtain an antioxidant BHT standard substance solution with the concentration of 1.00 mu g/ml.
Preferably, the step of preparing a standard curve solution comprises: weighing an antioxidant BHT standard substance solution in a 10ml measuring flask, diluting to scale with acetonitrile, shaking up, and preparing into a standard curve solution; the amount of the antioxidant BHT standard substance solution is 30-1600 μ l, such as 30 μ l, 50 μ l, 100 μ l, 150 μ l, 200 μ l, 800 μ l, and 1600 μ l; the concentration of the antioxidant BHT in the standard curve solution is 3-160. Mu.g/L, and there may be mentioned 3. Mu.g/L, 5. Mu.g/L, 10. Mu.g/L, 15. Mu.g/L, 20. Mu.g/L, 80. Mu.g/L and 160. Mu.g/L.
In a preferred embodiment, preparing a suitability solution for testing repeatability of sample injection comprises: 1ml of the standard curve solution was measured into a sample vial to obtain a suitability solution.
Preferably, the inorganic salt is selected from one or more of sodium chloride, sodium sulfate, potassium sulfate, calcium chloride and zinc chloride.
Further, sodium chloride is selected when the sample mother liquor is prepared.
Further, sodium chloride is selected for mixing when the sample solution is prepared, and then the upper layer solution of the sample mother liquor is sampled and added with sodium sulfate.
Further, the sodium sulfate is anhydrous sodium sulfate.
Preferably, the step of preparing the sample mother liquor comprises: measuring 1.0ml of paclitaxel injection flowing out of the infusion apparatus into a 100ml measuring flask, adding 50ml of purified water, and mixing; and adding sodium chloride, dissolving, uniformly mixing, diluting to a scale with purified water, uniformly mixing, and standing for 1h to obtain a sample mother solution. The amount of sodium chloride added may be, for example, 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g or 15g.
Preferably, the step of preparing the sample solution comprises: taking 10ml of prepared sample mother liquor into an analysis bottle, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, taking the supernatant to obtain a sample solution, and preparing 2 parts of the sample solution in parallel by the same method.
In a preferred embodiment, the step of preparing the sample solution comprises: taking 10ml of prepared sample mother liquor into an analysis bottle, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate, dehydrating until the bottom of the upper acetonitrile organic phase is in a quicksand-like precipitate, taking the supernatant to obtain a sample solution, and preparing 2 parts of the sample solution in parallel by the same method.
In order to further improve the content of antioxidant BHT in the sample solution, the applicant samples the mother solution, adds the mother solution into an organic solvent, uniformly mixes the mother solution, adds inorganic salt and stands the mixture; sampling the upper layer solution of the mother liquor, adding inorganic salt, and taking the upper layer solution as a sample solution, wherein the applicant finds that not all organic solvents are beneficial to improving the content of antioxidant BHT in the sample solution, when methanol is used as the organic solvent, the methanol can dissolve sodium chloride and cannot form layering, when an anticancer drug is matched with physiological saline, the physiological saline contains sodium chloride, and the sample cannot be subjected to layering treatment by the methanol.
The applicant finds that not all inorganic salts are suitable for being added into the mixed solution of the sample mother solution and the acetonitrile, because the anticancer drug is used together with normal saline in the application process, the inorganic salts are selected according to specific application to reduce the influence of additives on the increase of the BHT content of the antioxidant.
The applicant finds that in the process of preparing a sample solution, paclitaxel injection and water are mixed, in order to further remove water in a sample and simultaneously transfer the paclitaxel injection dissolved in the water to acetonitrile, the applicant finds that inorganic salts are added to an upper layer solution of a sample mother solution, and can remove the water in the upper layer solution of the sample mother solution, but not all the inorganic salts are suitable for being added to the upper layer solution of the sample mother solution, and the inorganic salts are required to be combined with the water to remove the water from the solution and simultaneously transfer the paclitaxel injection dissolved in the water to the acetonitrile.
By processing the sample, the content of the antioxidant BHT in the sample solution is improved, and the problem that the content of the antioxidant BHT in the test sample is lost to influence the specificity, accuracy, repeatability and durability of the test is avoided.
In a preferred embodiment, the step of preparing a reagent blank solution for testing the specificity of the sample comprises: taking 10ml of pure water to be put into an analysis bottle, adding 10ml of acetonitrile into the 20ml sample bottle, and uniformly mixing; adding a proper amount of sodium chloride until the bottom of the mixture is in a quicksand shape, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration until the bottom of the acetonitrile organic phase is in a quicksand shape, and taking the supernatant for testing.
In a preferred embodiment, sample solutions of different concentration levels are prepared, including a low concentration level accuracy solution, a medium concentration level accuracy solution, a first high concentration level accuracy solution, and a second high concentration level accuracy solution.
In a preferred embodiment, the preparation of the low concentration level accuracy solution comprises the steps of: measuring 10ml of sample mother solution into an analysis bottle, adding 30 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a low-concentration horizontal accuracy solution; in parallel, 3 total low concentration levels of accuracy solution with a concentration of 3. Mu.g/L were prepared.
In a preferred embodiment, the preparation of the medium level accuracy solution comprises the steps of: measuring 10ml of sample mother solution into an analysis bottle, adding 100 mul of antioxidant BHT standard curve solution with the concentration of 1 mu g/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a medium-concentration horizontal accuracy solution; a total of 3 medium concentration level accuracy solutions with a concentration of 10. Mu.g/L were prepared in parallel.
In a preferred embodiment, the preparation of the first high concentration level accuracy solution comprises the steps of: measuring 10ml of sample mother solution into an analysis bottle, adding 150 mul of antioxidant BHT standard curve solution with the concentration of 1 mu g/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the mixture is in a quicksand-like precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the upper acetonitrile organic phase is in a quicksand-shaped precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a first high-concentration horizontal accuracy solution; in parallel, a total of 3 accuracy solutions of the first high concentration level having a concentration of 15. Mu.g/L were prepared.
In a preferred embodiment, the preparation of the second high concentration level accuracy solution comprises the following steps: measuring 10ml of sample mother solution into an analysis bottle, adding 800 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a second high-concentration horizontal accuracy solution; in parallel, 3 total of accuracy solutions of the second high concentration level having a concentration of 80. Mu.g/L were prepared.
In a preferred embodiment, the preparing a repetitive solution comprises the following steps: measuring 10ml of sample mother solution into an analysis bottle, adding 100 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration until the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, and taking the supernatant to obtain a repetitive solution; a total of 6 replicate solutions were prepared in parallel, with a concentration of 10. Mu.g/L.
In a preferred embodiment, the preparing the precision solution comprises the following steps: prepared by the same analyst on different days and tested on different instruments; measuring 10ml of sample mother solution into an analysis bottle, adding 100 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the mixture is in a quicksand-like precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration until the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, and taking the supernatant to obtain a precision solution; a total of 6 precision solutions were prepared in parallel with the same method, the concentration of the precision solution being 10. Mu.g/L.
Preferably, the detection is performed by GC-MS, the model of the chromatographic column is HP-5MS, the carrier gas is He, and the temperature of the injection port is 270-290 ℃.
Preferably, the detection is carried out by GC-MS, the sample injection purge flow is 40-60ml/min, the sample injection amount is 1 mul, the column flow rate is 1.0ml/min, and the transmission line temperature is 290-310 ℃.
Preferably, the initial temperature of the chromatographic column is 75-85 ℃ and the temperature is raised to 280-300 ℃.
Preferably, the temperature rise rate of the chromatographic column is 25-35 ℃/min.
Preferably, the temperature of the sample inlet is 270-290 DEG C
In order to further improve the accuracy and the repeatability of the test result, the applicant sets the temperature of the sample inlet, the final temperature of the chromatographic column and the temperature of the transmission line in a temperature-rising temperature gradient manner, and finds that when the temperature of the sample inlet, the final temperature of the chromatographic column and the temperature of the transmission line are not set in a temperature-rising manner, substances with high boiling points are condensed in the temperature-falling process, peaks cannot appear in normal time, the accuracy and the repeatability of the test are influenced, more noises are generated, the temperature setting cannot be too high, otherwise, the tolerance temperature of the chromatographic column is exceeded, the accuracy of the test result is influenced, and the service life of the chromatographic column is also shortened.
In order to further reduce the influence of impurities or interfering substances on the antioxidant BHT, applicants found that the impurities or interfering substances in the sample solution can be separated from the antioxidant BHT and the peak accuracy of the antioxidant BHT can be improved when the injection port temperature is set to 270-290 ℃ and the initial temperature of the chromatographic column is set to 75-85 ℃ in the test process, particularly when the initial temperature is set to 80 ℃, possibly because the injection port temperature is high, the sample is gasified, the gasified sample is condensed in the process of cooling, and substances with different boiling points are separated again by the chromatographic column when the temperature is raised again, so that the influence of the impurities in the sample solution on the peak appearance of the antioxidant BHT is reduced, the detection is influenced, but the test influence on the antioxidant BHT cannot be completely removed when the impurity with the boiling point close to that of the antioxidant BHT is subjected to peak area reduction.
In order to further improve the accuracy and the repeatability of the test result of the antioxidant BHT, when the temperature rise rate of a chromatographic column is set to be 25-35 ℃/min in the test, particularly when the temperature rise rate is 30 ℃/min, an impurity close to the boiling point of the antioxidant BHT can be separated, the influence on the peak height or the peak area of the antioxidant BHT is reduced, and the accuracy and the repeatability of the test result are improved, possibly because when the temperature rise rate of the chromatographic column is 30 ℃/min, a sample adopts an HP-5MS chromatographic column in the gasification process, substances with similar but different melting points can be subjected to segmented overflow, and substances with different boiling points are distinguished, but when the temperature rise rate is too low, the peak emergence time of the antioxidant BHT is prolonged, the peak height of the antioxidant BHT in the test result is reduced, the peak shape is widened, and the detection sensitivity of the antioxidant BHT is reduced; when the temperature rise rate is too fast, the distance between the peak position of the interference substance close to the boiling point of the antioxidant BHT and the peak position of the antioxidant BHT is very close, so that the problem of peak coincidence occurs, and the accuracy and the repeatability of the antioxidant BHT are influenced.
Preferably, the GC-MS sets the solvent delay time to be 2-3.5min.
Further, the GC-MS set the solvent delay time to be 3min.
In order to further improve the sensitivity of the test result, the solvent delay time set by the applicant in the test process is 2-3.5min, the purge flow is 40-60ml/min, particularly the solvent delay time set by the GC-MS is 3min, and the purge flow is 50ml/min, so that the noise in the test result can be reduced, the peak emergence of antioxidant BHT is not influenced, the target peak is prevented from being widened, the sensitivity is improved, energy sources can be saved, the service life of the ion source is prolonged, and the possible reason is that the ion source needs to provide continuous energy in the test process of the sample in the GC-MS, but most of the sample solution is acetonitrile, the boiling point of the acetonitrile is lower than that of the antioxidant BHT, the sample solution can be separated through a chromatographic column firstly in the GC-MS test process, but the acetonitrile peak produces noise on the test result, the ionization energy needed to be provided by electron bombardment is increased, the discharge load of the ion source is increased, and the filament is easy to burn out.
Preferably, the quantitative ion setting for the scan mode in the GC-MS is 200-210m/z.
Further, the quantitative ion setting for the scan mode in the GC-MS was 205m/z.
Preferably, the qualitative ion setting of the scan pattern in the GC-MS is 210-230m/z.
Further, the qualitative ion setting for scan mode in the GC-MS was 220m/z.
The applicant unexpectedly finds that the problem of false positive in a quantitative ion test result can be solved by setting the qualitative ions to be 220m/z, and the accuracy of the measurement result can be improved by setting the qualitative ions to be 220m/z and the quantitative ions to be 205m/z.
Advantageous effects
The method for determining the antioxidant BHT in the paclitaxel injection product has the advantages of good specificity, linearity, accuracy, repeatability and durability.
The method disclosed by the invention is used for processing the sample, so that the content of the antioxidant BHT in the sample solution is improved, and the problem that the content of the antioxidant BHT in the test sample is lost to influence the specificity, accuracy, repeatability and durability of the test is avoided.
The method can improve the content of antioxidant BHT in the sample solution, particularly selects acetonitrile as an organic solvent, and adds sodium chloride and anhydrous sodium sulfate into the solution for treatment. According to the method, the final temperature of the sample inlet, the chromatographic column and the temperature of the transmission line are set in a temperature gradient heating mode, so that the accuracy and the repeatability of the test result are further improved.
The initial temperature of the chromatographic column is set to be 75-85 ℃, the peak-appearing accuracy of the antioxidant BHT can be improved, the temperature rising rate of the chromatographic column is set to be 25-35 ℃/min in the test, the accuracy and the repeatability of the antioxidant BHT test result can be improved, the target peak is prevented from being widened and the sensitivity is improved by setting the solvent delay time to be 2-3.5min, meanwhile, the energy can be saved, the service life of the ion source is prolonged, and the problem of false positive in the quantitative ion test result can be solved by setting the qualitative ion to be 220m/z.
By setting the qualitative ions to be 220m/z and the quantitative ions to be 205m/z, the method can improve the accuracy of the measurement result.
Drawings
The figure 1 is a test spectrum of a reagent blank solution.
The figure 2 is a test pattern of the sample solution.
The figure 3 is a test spectrum of a solution of medium concentration level accuracy.
Detailed Description
Example 1
The invention provides an analysis method for determining antioxidant BHT in an anticancer drug by GC-MS, which comprises the following steps:
(1) Preparing an antioxidant BHT standard substance solution: weighing antioxidant BHT standard substance 41.935mg in a 10ml measuring flask, dissolving with acetonitrile, diluting to scale, and shaking; measuring 4.80 mu l of the solution into a 20ml measuring flask, diluting the solution to the scale with acetonitrile, and shaking up to obtain an antioxidant BHT standard substance solution with the concentration of 1.00 mu g/ml.
(2) Preparation of standard curve solutions: respectively measuring antioxidant BHT standard substance solutions in 10ml measuring bottles, diluting to scale with acetonitrile, shaking up to prepare standard curve solutions, wherein the amounts of the antioxidant BHT standard substance solutions are respectively 30 mu L, 50 mu L, 100 mu L, 150 mu L, 200 mu L, 800 mu L and 1600 mu L, and the concentrations of the antioxidant BHT in the prepared standard curve solutions are respectively 3 mu g/L, 5 mu g/L, 10 mu g/L, 15 mu g/L, 20 mu g/L, 80 mu g/L and 160 mu g/L.
(3) Preparing a suitability solution: 1ml of the standard curve solution with the concentration of 10 mug/L is measured in a sample injection vial to obtain the applicability solution.
(4) Preparing a sample mother solution: measuring 1.0ml of paclitaxel injection flowing out of the infusion apparatus into a 100ml measuring flask, adding 50ml of purified water, and mixing; and adding 5g of sodium chloride, dissolving, uniformly mixing, diluting to a scale with purified water, uniformly mixing, and standing for 1h to obtain a sample mother solution.
(5) Preparing a sample solution: taking 10ml of sample mother liquor, adding 10ml of acetonitrile into an analysis bottle, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate, dehydrating until the bottom of the upper acetonitrile organic phase is in a quicksand-like precipitate, taking the supernatant to obtain a sample solution, and preparing 2 parts of the sample solution in parallel by the same method.
(6) Preparation of a reagent blank solution: taking 10ml of pure water to be put into an analysis bottle, adding 10ml of acetonitrile into a 20ml sample bottle, and uniformly mixing; adding a proper amount of sodium chloride until the bottom of the mixture is in a quicksand shape, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration until the bottom of the acetonitrile organic phase is in a quicksand shape, and taking the supernatant for testing.
(7) Preparation of low concentration level accuracy solutions: measuring 10ml of sample mother solution into an analysis bottle, adding 30 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a low-concentration horizontal accuracy solution; in parallel, 3 low concentration levels of accuracy solution with a concentration of 3. Mu.g/L were prepared.
(8) Preparation of medium concentration level accuracy solutions: measuring 10ml of sample mother solution into an analysis bottle, adding 100 mul of antioxidant BHT standard curve solution with the concentration of 1 mu g/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a medium-concentration horizontal accuracy solution; in parallel, a total of 3 medium concentration level of accuracy solution with a concentration of 10. Mu.g/L was prepared.
(9) Preparation of a first high concentration horizontal accuracy solution: measuring 10ml of sample mother solution, adding 150 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml into an analysis bottle, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration till the bottom of the upper acetonitrile organic phase is in a quicksand-shaped precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a first high-concentration horizontal accuracy solution; a total of 3 first high concentration level accuracy solutions were prepared in parallel, the concentration of the first high concentration level accuracy solution being 15. Mu.g/L.
(10) Second high concentration horizontal accuracy solution in preparation: measuring 10ml of sample mother solution into an analysis bottle, adding 800 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate, dehydrating until the bottom of the upper acetonitrile organic phase is in a quicksand-like precipitate, taking about 1ml of supernate, and putting the supernate into a sample injection bottle to obtain a second high-concentration horizontal accuracy solution; in parallel, 3 aliquots of the second high concentration level of the accuracy solution were prepared, which had a concentration of 80. Mu.g/L.
(11) Preparation of a repetitive solution: sampling 10ml of mother solution, putting the mother solution into an analysis bottle, adding 100 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration until the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, and taking the supernatant to obtain a repetitive solution; a total of 6 replicate solutions were prepared in parallel, with a concentration of 10. Mu.g/L.
(12) Preparation of precision solutions: the same analyst prepares once every 5 days and tests on different instruments; measuring 10ml of sample mother solution into an analysis bottle, adding 100 mul of antioxidant BHT standard curve solution with the concentration of 1 mug/ml, adding 10ml of acetonitrile, and uniformly mixing; adding a proper amount of sodium chloride till the bottom of the sodium chloride is in a quicksand-shaped precipitate, shaking for extraction, and standing for 1h; taking the upper acetonitrile organic phase, adding a proper amount of anhydrous sodium sulfate for dehydration until the bottom of the acetonitrile organic phase is in a quicksand-shaped precipitate, and taking the supernatant to obtain a precision solution; a total of 6 precision solutions were prepared in parallel with the same method, the concentration of the precision solution being 10. Mu.g/L.
The parameters of the GC-MS during detection are set as follows: the model of the chromatographic column is HP-5MS, carrier gas is He, the temperature of a sample inlet is 280 ℃, the sample injection purging flow is 50ml/min, the sample injection amount is 1 mul, the flow rate of the column is 1.0ml/min, the temperature of a transmission line is 300 ℃, the initial temperature of the chromatographic column is 80 ℃, the temperature is increased to 290 ℃, the temperature rising rate is 30 ℃/min, the delay time of a solvent is 3min, the setting of quantitative ions is 205m/z, and the setting of qualitative ions is 220m/z.
The purchase manufacturer of the paclitaxel injection is Wuxi yew drug industry Co.
Example 2
Example 2 provides an analytical method for measuring antioxidant BHT in an anticancer drug using GC-MS, which is different from example 1 in that the injection port temperature for GC-MS measurement is 270 ℃.
Example 3
Example 3 provides an analytical method for measuring antioxidant BHT in an anticancer drug using GC-MS, which is different from example 1 in that the GC-MS detects a sample inlet temperature of 290 ℃.
And (3) performance testing:
1. sample injection repeatability and durability test
The applicability solution prepared in example 1-3 was sampled in GC-MS for 6 needles, and the Relative Standard Deviation (RSD) of peak area of the target substance in 6 needles was calculated, and the test results are shown in Table 1.
Relative standard deviation (RSD%) = peak area Standard Deviation (SD) ÷ average of peak areas (x) × 100% Standard Deviation (SD) = [ ∑ (xi-x) 2 ÷ (n-1) ]1/2
xi is the ith data value, x is the mean value
The Relative Standard Deviation (RSD) should not exceed 8%.
TABLE 1 relative standard deviation of antioxidant BHT
Figure BDA0003465786990000111
The test data provided in table 1 show that, when the same sample is tested repeatedly under different test conditions, the difference between the test results is small, the repeatability of the test results is high, and the Relative Standard Deviation (RSD) obtained by testing under different test conditions is within the standard requirement range of the test, and the durability of the test results is high.
2. Line type testing
Taking 7 standard curve solutions with the concentrations of 3. Mu.g/L, 5. Mu.g/L, 10. Mu.g/L, 15. Mu.g/L, 20. Mu.g/L, 80. Mu.g/L and 160. Mu.g/L prepared in example 1, respectively, adding the solutions into GC-MS for measurement, and taking the concentration of the antioxidant BHT as a horizontal coordinate X and the peak area of the antioxidant BHT as a vertical coordinate Y, and carrying out linear regression by using a linear regression equation: y =1685.9769X-16.5773, and a correlation coefficient is obtained, and the correlation coefficient obtained by testing is 0.9999, so that the linear test standard requirement is met.
3. Specificity determination
One part of the reagent blank solution, two parts of the sample solution and three parts of the medium concentration level accuracy solution prepared in example 1 were taken and tested in a GC-MS and the obtained maps are shown in FIGS. 1-3.
The figure 1 is a test pattern of a reagent blank solution.
The figure 2 is a test pattern of the sample solution.
The figure 3 is a test spectrum of a solution of medium concentration level accuracy.
As can be seen from the graphs in FIGS. 1 to 3, the antioxidant BHT has a specific peak position in the map, is not affected by other impurities or interferents, and has high specificity.
4. Determination of accuracy
The low concentration level accuracy solution, the medium concentration level accuracy solution, the first high concentration level accuracy solution, and the second high concentration level accuracy solution prepared in example 1 were each taken in 3 parts and tested in GC-MS, and the results of the antioxidant BHT test in the solutions are shown in table 2.
TABLE 2
Figure BDA0003465786990000121
Figure BDA0003465786990000131
The accuracy calculation formula is as follows:
background (μ g) = sample solution mean value measured (μ g/L) × sample solution volume (ml) ÷ unit conversion factor (1000).
The background is antioxidant BHT.
The amount of addition (μ g) = concentration of addition sample solution (mg/L) × addition volume (μ L) ÷ unit conversion factor (1000).
Measured amount (μ g) = accuracy solution measurement (μ g/L) × sample solution volume (ml) ÷ unit conversion factor (1000).
Recovery = (measured amount (μ g) -antioxidant BHT (μ g))/(μ g) × 100% addition.
From table 4, it can be seen that through testing of the accuracy solutions with different concentration levels, the difference of the recovery rates of the antioxidant BHT is in the range of 1-6%, the recovery rate RSD is between 1% and 3%, and the analysis result shows that the result of the accuracy test meets the standard requirement that the recovery rate is between 70% and 125%, and the RSD of the recovery rate is not greater than 15%.
5. Precision repeatability test
6 parts of the repetitive solution prepared in example 1 were subjected to a test in GC-MS to determine the content of antioxidant BHT, and the test results are shown in Table 3.
TABLE 3
Figure BDA0003465786990000132
It can be seen from table 3 that RSD of the measured value of the antioxidant BHT in 6 parts of the repetitive solutions is 1%, and the test results show that the precision of the test method is relatively high.
6. Solution stability test
6 parts of the repetitive solution and 6 parts of the precise solution prepared in example 1 were allowed to stand at room temperature for 24 hours and then added to GC-MS for measurement, and the measurement results are shown in Table 4.
TABLE 4
Figure BDA0003465786990000141
From table 4, it can be seen that RSD of antioxidant BHT obtained by testing repetitive solutions and precision solutions after being left for 24 hours is 6%, less than 8%, within the range required by the test, thus obtaining the solution stability of the analytical method is high, and the test method has better durability.
In conclusion, the method for measuring the antioxidant BHT in the paclitaxel injection product has certain specificity, linearity, accuracy, repeatability and durability.

Claims (10)

1. An analytical method for measuring antioxidant BHT in an anticancer drug by GC-MS, comprising:
(1) preparing a standard substance solution, adding an antioxidant BHT into an organic solvent, and diluting to a scale to obtain the standard substance solution;
(2) preparing a standard curve solution, adding an organic solvent into the standard substance solution, and diluting to a scale to obtain the standard curve solution;
(3) preparing a sample mother solution, adding the anti-cancer drugs into water, uniformly mixing, adding inorganic salt, mixing, adding water to a constant volume, and standing to obtain the sample mother solution;
(4) preparing a sample solution, taking a sample mother solution, adding the sample mother solution into an organic solvent, uniformly mixing, adding inorganic salt, and standing; sampling the upper layer solution of the mother solution of the sample, adding inorganic salt, and taking the upper layer solution as the sample solution;
(5) and detecting by GC-MS, wherein the model of a chromatographic column is HP-5MS, carrier gas is He, and the temperature of a sample inlet is 260-295 ℃.
2. The analytical method for the determination of antioxidant BHT in an anticancer drug by GC-MS as set forth in claim 1, wherein the anticancer drug is paclitaxel injection.
3. The analytical method for measuring antioxidant BHT in anticancer drugs by GC-MS according to claim 1, wherein the organic solvent is selected from one or more of n-hexane, acetone, ethyl acetate, dichloromethane and acetonitrile.
4. The analytical method for the measurement of antioxidant BHT in an anticancer drug by GC-MS according to any one of claims 1 to 3, wherein said inorganic salt is selected from one or more of sodium chloride, sodium sulfate, potassium sulfate, calcium chloride and zinc chloride.
5. The analytical method for the determination of antioxidant BHT in anticancer drugs by GC-MS as set forth in claim 1, wherein the initial temperature of the chromatographic column is 75-85 ℃ and the temperature is raised to 280-300 ℃.
6. The analytical method for measuring antioxidant BHT in anticancer drugs by GC-MS according to claim 5, wherein the temperature rising rate of the chromatographic column is 25-35 ℃/min.
7. The analytical method for measuring antioxidant BHT in anticancer drugs by GC-MS according to claim 1, wherein the GC-MS is set to have a solvent delay time of 2 to 3.5min.
8. The analytical method for measuring antioxidant BHT in anticancer drugs by GC-MS according to claim 1, wherein the injection port temperature is 270-290 ℃.
9. The analytical method for the determination of antioxidant BHT in an anticancer drug by GC-MS according to claim 1, wherein the quantitative ion setting of the scan pattern in GC-MS is 200 to 210m/z.
10. The analytical method for the determination of antioxidant BHT in an anticancer drug by GC-MS according to claim 1, wherein the qualitative ion setting of the scan pattern in GC-MS is 210 to 230m/z.
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