CN114527203A - Method for detecting residual solvent in pingyangmycin hydrochloride raw material medicine - Google Patents
Method for detecting residual solvent in pingyangmycin hydrochloride raw material medicine Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000013557 residual solvent Substances 0.000 title claims abstract description 28
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N30/68—Flame ionisation detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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Abstract
The invention relates to the field of drug analysis and detection, and provides a method for detecting residual solvent in bleomycin hydrochloride bulk drug. The invention comprises the following steps: and respectively heating and balancing the blank solution, the mixed reference solution and the test solution, injecting the solutions into a gas chromatograph in a headspace sample injection mode, recording a chromatogram, and calculating the content of the residual solvent by peak area according to an external standard method. The method is simple, accurate and high in sensitivity, meets the requirement of residual solvent detection, is suitable for daily detection of residual solvents in pingyangmycin hydrochloride raw material medicines, provides reference and basis for revision of residual solvent inspection items in the quality standard of the product, effectively improves the quality of the raw material medicines, and reduces the clinical medication risk.
Description
Technical Field
The invention belongs to the field of drug analysis and detection, and particularly relates to a method for detecting residual solvent in a pingyangmycin hydrochloride raw material drug.
Background
The pingyangmycin is a bleomycin antitumor antibiotic independently developed by China, and the chemical structure of the bleomycin antitumor antibiotic is a bleomycin A5 component. Mainly treats neck squamous cell carcinoma such as lip cancer, tongue cancer, tooth trace cancer, nasopharyngeal carcinoma, and the like, has lower lung toxicity than bleomycin, does not inhibit the immunologic function of an organism, and has no leukopenia symptoms caused by common anticancer drugs. The Chinese medicine is entered into the first national basic medicine catalogue in 1994, and the quality standard of the Chinese medicine is recorded in Chinese pharmacopoeia.
The antibiotics are mainly produced by a microbial fermentation process or a semi-synthesis process, the components are complex, the crude pingyangmycin hydrochloride is crystallized and purified by using an organic solvent in the production process, if the process parameters are not strictly controlled, the possibility of residual organic solvent in the finished product exists, when the level of the residual solvent in the medicine is higher than a safety value, the antibiotics can cause harm to human bodies or the environment,
3 solvents of methanol, acetone and isopropanol are used in the preparation process of the bulk drug, but no report for measuring the residual quantity of the organic solvent in the pingyangmycin hydrochloride bulk drug exists at home at present.
The 3 solvents have residual quantity limit requirements according to the requirements of international harmonization (ICH) and 2020 edition of general rules 0861 of Chinese pharmacopoeia for human drug registration. In order to effectively control the quality of the pingyangmycin hydrochloride raw material and ensure the medication safety, the method for measuring 3 organic residual solvents in the pingyangmycin hydrochloride by the gas chromatography is established, has high sensitivity and good repeatability, is simple and easy to operate, is suitable for measuring the residual quantity of the 3 organic solvents in the pingyangmycin hydrochloride, and provides reference for the quality control of the raw material medicine. The pingyangmycin hydrochloride is collected in the 'Chinese pharmacopoeia' 2020 edition, no residual solvent inspection item exists in the existing quality standard, and the product quality cannot be comprehensively controlled.
Disclosure of Invention
The invention aims to solve the problems and provides a method for detecting residual solvent in a pingyangmycin hydrochloride raw material medicine, which comprises the following steps:
heating and balancing the blank solution, the mixed reference solution and the test solution respectively, injecting the heated and balanced blank solution, the mixed reference solution and the test solution into a gas chromatograph in a headspace sample injection mode, recording a chromatogram, and calculating the content of the residual solvent by peak area according to an external standard method;
the conditions of the gas chromatography were:
temperature of the column: the initial column temperature was 40 ℃, maintained for 2 minutes, heated to 100 ℃ at a rate of 5 ℃ per minute, maintained for 8 minutes, heated to 200 ℃ at a rate of 20 ℃ per minute, and maintained for 5 minutes;
sample inlet temperature: 200 ℃;
detector temperature: 250 ℃;
headspace bottle equilibrium temperature: 70 ℃, equilibration time: 30 minutes;
the carrier gas is nitrogen, and the flow rate is 4mL/min, or 5mL/min, or 6 mL/min. Preferably, the column is DB-WAX, 30m × 0.32mm × 0.25 μm or DB-624, 30m × 0.53mm × 3 μm or DB-FFAP, 30m × 0.32mm × 0.5 μm).
Preferably, the blank solution is water.
Preferably, the preparation of the test solution: precisely weighing 0.2g of sample, placing in a headspace bottle, precisely adding 2mL of water for dissolving, and sealing to obtain the final product.
Preferably, the preparation of the mixed control solution comprises:
(1) accurately weighing 1.5089g of methanol, 2.5086g of acetone and 2.5078g of isopropanol, placing the weighed materials into a 50mL measuring flask, diluting the weighed materials to a scale with water, shaking the weighed materials evenly, and obtaining mixed reference stock solutions containing 3mg/mL, 5mg/mL and 5mg/mL of methanol, acetone and isopropanol respectively;
(2) precisely measuring 5mL of the mixed reference stock solution, placing the mixed reference stock solution in a 50mL measuring flask, adding water to dilute the mixed reference stock solution to a scale, and shaking the mixed reference stock solution uniformly to obtain the mixed reference stock solution.
Preferably, the calculation formula of the external standard method is
Wherein A is the content of residual solvent, B is the sample weighing of the sample, C is the dilution multiple of the sample, D is the peak area of the solution of the sample, E is the sample weighing of the reference, F is the dilution multiple of the reference, and G is the average peak area of the reference.
Compared with the prior art, the invention has the following beneficial effects:
the method for simultaneously measuring the content of 3 organic solvents in the pingyangmycin hydrochloride bulk drug by gas chromatography-headspace sampling is simple, accurate and high in sensitivity, meets the requirement of residual solvent detection, is suitable for daily detection of the residual solvent in the pingyangmycin hydrochloride bulk drug, provides reference and basis for revising the residual solvent inspection item in the quality standard of the product, effectively improves the quality of the bulk drug and reduces the clinical medication risk.
Drawings
FIG. 1 is a system suitability chromatogram;
FIG. 2 is a blank solution chromatogram;
FIG. 3 is a chromatogram of a sample solution;
FIG. 4 is a linear relationship diagram for methanol;
FIG. 5 is a graph of acetone linearity;
FIG. 6 is a graph of isopropanol linearity;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
1. Instruments and reagents
The instrument comprises the following steps: agilent 7890B gas chromatograph;
a hydrogen Flame Ionization Detector (FID);
pure water hydrogen generators (denhaoyiwei laboratory instruments ltd);
SGK-2LB Low noise air Pump (Beijing Oriental essence park science, Inc.);
an Open LBACDS Chem Station Edition workstation;
german mettler XS205 electron analytical balance.
Reagent: pingyangmycin hydrochloride raw material, batch number: y190401, Y200901, Y200902, 200322;
methanol, acetone and isopropanol are all chromatographically pure.
2. Chromatographic conditions
A chromatographic column: DB-WAX (30 m.times.0.32 mm. times.0.25 μm);
DB-624(30m×0.53mm×3μm)；
DB-FFAP(30m×0.32mm×0.5μm)。
temperature programming: the initial column temperature was 40 ℃, maintained for 2 minutes, heated to 100 ℃ at a rate of 5 ℃ per minute, maintained for 8 minutes, heated to 200 ℃ at a rate of 20 ℃ per minute, and maintained for 5 minutes;
sample inlet temperature: 200 ℃;
detector temperature: 250 ℃;
headspace bottle equilibrium temperature: 70 ℃, equilibration time: 30 minutes;
carrier gas: nitrogen gas; flow rate: 5 ml/min.
3. Solution preparation
Blank solution: water (W)
Mixing a reference stock solution: 1.5089g of methanol, 2.5086g of acetone and 2.5078g of isopropanol are precisely weighed, placed in a 50mL measuring flask, diluted to scale with water and shaken evenly, and mixed reference stock solutions containing 3mg/mL, 5mg/mL and 5mg/mL of methanol, acetone and isopropanol respectively are prepared.
Control solution: precisely measuring 5mL of mixed reference stock solution, placing in a 50mL measuring flask, adding water to dilute to scale, and shaking to obtain mixed reference stock solution
Test solution: precisely weighing 0.2g of sample, placing in a headspace bottle, precisely adding 2mL of water for dissolving, and sealing to obtain the final product.
4. Methodology validation
4.1 specificity test
And (4) respectively injecting the mixed reference substance solution, the blank solution and the test solution into a gas chromatograph, and recording the chromatogram. The results are shown in FIGS. 1-3.
The results show that under the chromatographic conditions, the peak emergence order is as follows: the peak of each component can reach baseline separation, and the separation degree is more than 1.8; the number of theoretical plates is 50507 in terms of acetone peak, the solvent has no interference to sample determination, and other impurity peaks and main peaks are well separated.
4.2 detection limit and quantitation limit test
Precisely measuring the mixed reference substance, and gradually diluting until the signal-to-noise ratio s/n is about 10, thus obtaining the quantitative limiting solution.
Precisely measuring 3ml of quantitative limiting solution, placing the quantitative limiting solution in a 10ml measuring flask, adding water to dilute the quantitative limiting solution to a scale, and shaking up to obtain the detection limiting solution.
And (4) injecting the quantitative limiting solution into a gas chromatograph, continuously measuring for 6 times, and recording a chromatogram.
TABLE 1 limit of quantitation test
From the above test results, it can be seen that: the peak areas RSD of the results of 6 determinations of the limit of quantitation of methanol, acetone and isopropanol are respectively 1.7%, 1.3% and 1.4%, and the limit of quantitation is 18.107 mug, 1.505 mug and 3.494 mug. The detection limits are 6.036. mu.g, 0.501. mu.g and 1.026. mu.g respectively.
4.3 Linear relationship inspection
Precisely sucking and mixing 0.01mL, 1mL, 5mL, 10mL, 20mL and 50mL of the reference stock solutions respectively, placing in a 100mL measuring flask, adding water to dilute to scale, shaking up, and making into series of reference solutions. And (5) carrying out sample injection measurement according to the chromatographic conditions, and recording the peak area.
TABLE 2 methanol Linear relationship test
Taking the peak area as the ordinate and the concentration as the abscissa to perform linear regression, wherein the linear relationship is shown in the attached figure 4:
methanol is in the range of 0.0003mg/ml to 1.5089mg/ml, the linear equation is that y is 1364x-2.149, the correlation coefficient r is 1.0000, and the peak area A and the concentration C (mg/ml) are in a remarkable linear relation.
TABLE 3 acetone Linear relationship test
Linear regression with peak area as ordinate and concentration as abscissa, the linear relationship is shown in figure 5,
the acetone is in the range of 0.0005mg/ml to 2.5086mg/ml, the linear equation is y is 9824x-61.80, the correlation coefficient r is 0.9995, and the peak area A and the concentration C (mg/ml) are in a remarkable linear relation.
TABLE 4 isopropanol linearity test
Linear regression with peak area as ordinate and concentration as abscissa, the linear relationship is shown in figure 6,
the isopropanol is in the range of 0.0005mg/ml to 2.5018mg/ml, the linear equation is Y5518 x-3.563, the correlation coefficient r is 1.0000, and the peak area A and the concentration C (mg/ml) are in a remarkable linear relation.
4.4 accuracy test
Standard addition solution: precisely measuring 2.5ml, 5.0ml and 7.5ml of mixed standard stock solution, placing in a 50ml measuring flask, adding water to dilute to scale, and shaking to obtain standard addition solutions (1), (2) and (3);
test solution: precisely weighing about 0.2g of the product (batch number: Y190401), 9 parts in total, and 3 groups, placing into a headspace bottle, adding 2.0ml of the above mixed control solution, respectively, and sealing. Injecting the solution into a gas chromatograph, and recording a chromatogram; the ratio of the measured amount to the added amount is the recovery rate.
TABLE 5 recovery test
From the above test results, it can be seen that: the recovery rates of the methanol, the acetone and the isopropanol are respectively 100.3-105.6% (RSD%: 2.8), 102.1-102.9% (RSD%: 0.6) and 100.2-102.1% (RSD%: 1.1), and the recovery rate is good, so that the method is proved to have good accuracy and meet the verification requirements.
4.5 precision test
4.5.1 sample introduction precision test
Injecting the reference solution into a gas chromatograph, and recording the chromatogram. Sample introduction is repeated for 6 times, RSD is calculated, and the instrument precision of the method is inspected.
TABLE 6 sample introduction precision test
From the test results, it can be seen that: the methanol retention time RSD is 0.01%, the peak area RSD is 1.0%, the acetone retention time RSD is 0.0%, the peak area RSD is 0.7%, the isopropanol retention time RSD is 0.01%, the peak area RSD is 0.8%, and the sampling precision is good.
4.5.2 repeatability test
Stock solution of pingyangmycin hydrochloride 1 batch (batch number: Y190401) and mixed reference substance is prepared in parallel 6 parts according to a drawing method, and the prepared stock solution is respectively injected into a gas chromatograph and recorded with a chromatogram. The repeatability of the method was examined.
TABLE 7 results of the repeatability tests
And (4) conclusion: the same experimenter repeats the measurement for 6 times, and the results of the 6 times of measurement are consistent compared with the detected amounts of methanol, acetone and isopropanol. The method has good repeatability.
4.5.3 intermediate precision test
Different experimenters operate according to the reference solution and the test solution to prepare 6 parts of the reference solution and 6 parts of the test solution (batch number: Y190401). Injecting into gas chromatograph, and recording chromatogram.
TABLE 8 intermediate precision test results
And (4) conclusion: different experimenters repeat the measurement 6 times, and the results of the 6 times of measurement are compared, so that the detected amounts of the methanol, the acetone and the isopropanol are consistent. The method has good repeatability.
4.6 solution stability test
Preparing according to the preparation method of the reference solution, standing at room temperature for 0, 1, 2, 3, 4, 5 and 6 hours, injecting the solutions with different standing times into a gas chromatograph, and recording the chromatogram.
TABLE 9 stability test results for control and test solutions
From the test results, it can be seen that: after standing at room temperature for 6 hours, the peak areas RSD of methanol, acetone and isopropanol in the control solution were 4.1%, 1.6% and 1.9%, respectively (n ═ 6), and the control solution was stable for 6 hours.
In summary, the patent establishes a method for simultaneously determining the content of 3 organic solvents in a pingyangmycin hydrochloride bulk drug by gas chromatography-headspace sample injection, has the advantages of simplicity, accuracy and high sensitivity, meets the detection requirement of residual solvents, is suitable for daily detection of the residual solvents in the pingyangmycin hydrochloride bulk drug, provides reference and basis for revising the residual solvent examination item in the quality standard of the product, effectively improves the quality of the bulk drug, and reduces the clinical medication risk.
(II) comparative example 1
The flow rate of the carrier gas is different as in the above embodiment. Injecting the control solution into a gas chromatograph, inspecting the chromatographic retention behavior of 3 organic solvents under the conditions of carrier gas flow rate of 4mL/min, 5mL/min and 6mL/min, and taking the separation degree and theoretical plate number of each component as evaluation indexes, wherein the measurement results are shown in Table 10. As a result, baseline separation was achieved for all 3 solvents under each chromatographic condition.
TABLE 10 measurement results of different carrier gas flow rates
(III) comparative example 2
The temperature of the injection port was varied, as was the conditions in the above examples.
Injecting the control solution into a gas chromatograph, inspecting the chromatographic retention behavior of 3 organic solvents at the injection inlet temperature of 200 ℃, 220 ℃ and 240 ℃, and taking the separation degree and the theoretical plate number of each component as evaluation indexes, wherein the measurement results are shown in Table 11. Baseline separation was achieved for all 3 solvents under each chromatographic condition.
TABLE 11 measurement results of temperatures at different injection ports
(IV) comparative example 3
The temperature of the detector is different, as is the condition in the above embodiment.
Injecting the control solution into a gas chromatograph, observing the chromatographic retention behavior of 3 organic solvents at the detector temperature of 230 ℃, 250 ℃ and 270 ℃, and taking the separation degree and the theoretical plate number of each component as evaluation indexes, wherein the measurement results are shown in Table 12. Baseline separation was achieved for all 3 solvents under each chromatographic condition.
TABLE 12 results of temperature measurements of various detectors
(fifth) comparative example 3
The same procedure as in example 1 was repeated except for the dividing ratio.
The control solution was injected into a gas chromatograph, and the chromatographic retention behavior of 3 organic solvents was examined under the conditions of a split ratio of 5:1 and 10:1, and the separation degree and the number of theoretical plates of each component were used as evaluation indices, and the measurement results are shown in table 13. Baseline separation was achieved for all 3 solvents under each chromatographic condition.
TABLE 13 determination of different split ratios
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A method for detecting residual solvents in bleomycin hydrochloride bulk drugs is characterized by comprising the following steps:
heating and balancing the blank solution, the mixed reference solution and the test solution respectively, injecting the heated and balanced blank solution, the mixed reference solution and the test solution into a gas chromatograph in a headspace sample injection mode, recording a chromatogram, and calculating the content of the residual solvent by peak area according to an external standard method;
the conditions of the gas chromatography were:
temperature of the column: the initial column temperature was 40 ℃, maintained for 2 minutes, heated to 100 ℃ at a rate of 5 ℃ per minute, maintained for 8 minutes, heated to 200 ℃ at a rate of 20 ℃ per minute, and maintained for 5 minutes;
sample inlet temperature: 200 ℃;
detector temperature: 250 ℃;
headspace bottle equilibrium temperature: 70 ℃, equilibration time: for 30 minutes.
2. The method for detecting the residual solvent in the pingyangmycin hydrochloride raw material medicine according to claim 1, wherein the chromatographic column is DB-WAX, 30m x 0.32mm x 0.25 μm or DB-624, 30m x 0.53mm x 3 μm or DB-FFAP, 30m x 0.32mm x 0.5 μm.
3. The method for detecting the residual solvent in the pingyangmycin hydrochloride bulk drug according to claim 1, wherein the blank solution is water.
4. The method for detecting the residual solvent in the pingyangmycin hydrochloride bulk drug according to claim 1, wherein the preparation of the test solution: precisely weighing 0.2g of sample, placing in a headspace bottle, precisely adding 2mL of water for dissolving, and sealing to obtain the final product.
5. The method for detecting the residual solvent in the pingyangmycin hydrochloride bulk drug according to claim 1, wherein the preparation of the mixed reference solution comprises the following steps:
(1) accurately weighing 1.5089g of methanol, 2.5086g of acetone and 2.5078g of isopropanol, placing the weighed materials into a 50mL measuring flask, diluting the weighed materials to a scale with water, shaking the weighed materials evenly, and obtaining mixed reference stock solutions containing 3mg/mL, 5mg/mL and 5mg/mL of methanol, acetone and isopropanol respectively;
(2) precisely measuring 5mL of the mixed reference stock solution, placing the mixed reference stock solution in a 50mL measuring flask, adding water to dilute the mixed reference stock solution to a scale, and shaking the mixed reference stock solution uniformly to obtain the mixed reference stock solution.
6. The method for detecting residual solvent in pingyangmycin hydrochloride bulk drug according to claim 1, wherein the calculation formula of the external standard method is
Wherein A is the content of residual solvent, B is the sample weighing of the sample, C is the dilution multiple of the sample, D is the peak area of the solution of the sample, E is the sample weighing of the reference, F is the dilution multiple of the reference, and G is the average peak area of the reference.
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| PCT/CN2022/138249 WO2023124924A1 (en) | 2021-12-31 | 2022-12-10 | Method for detecting residual solvent in pingyangmycin hydrochloride bulk drug |
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| CN115561353A (en) * | 2022-09-22 | 2023-01-03 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Method for measuring amount of ethanol in ergot maleate tablets |
| WO2023124924A1 (en) * | 2021-12-31 | 2023-07-06 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Method for detecting residual solvent in pingyangmycin hydrochloride bulk drug |
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