CN116165319A - Method for detecting diastereoisomers in formoterol fumarate inhalation solution - Google Patents
Method for detecting diastereoisomers in formoterol fumarate inhalation solution Download PDFInfo
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- CN116165319A CN116165319A CN202310281200.8A CN202310281200A CN116165319A CN 116165319 A CN116165319 A CN 116165319A CN 202310281200 A CN202310281200 A CN 202310281200A CN 116165319 A CN116165319 A CN 116165319A
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- formoterol fumarate
- phosphate
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- 229960000193 formoterol fumarate Drugs 0.000 title claims abstract description 242
- 238000000034 method Methods 0.000 title claims abstract description 162
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- 229940041682 inhalant solution Drugs 0.000 title claims description 100
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 44
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- 229960002848 formoterol Drugs 0.000 claims description 25
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- 238000012856 packing Methods 0.000 claims description 19
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
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Images
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a method for detecting diastereoisomers in a formoterol fumarate solution, which comprises the steps of analyzing the formoterol fumarate solution by adopting a high performance liquid chromatography and calculating the content of diastereoisomers in the formoterol fumarate solution according to the peak area of a chromatogram. The method of the invention can realize excellent separation degree, sensitivity and signal-to-noise ratio.
Description
The present application is a divisional application of the patent application of the invention with the application date of 2022, the application number of 05-30, the application name of 202210598363.4 and the name of a method for detecting diastereoisomers in formoterol fumarate inhalation solution.
Technical Field
The invention relates to the field of detection of drug impurities, in particular to a method for detecting diastereoisomers in formoterol fumarate inhalation solution, and more particularly relates to a method for detecting diastereoisomers in formoterol fumarate inhalation solution by using high performance liquid chromatography based on a chromatographic column of octadecyl bonded ethylene bridge hybrid particles.
Background
Formoterol fumarate is a drug against bronchial diseases that produces a powerful and long lasting antiasthmatic effect mainly by acting on the beta 2 receptor. The common formoterol fumarate dosage forms in the market mainly comprise tablets, syrups, inhalation aerosols and some compound inhalation powder aerosols.
The formoterol fumarate solution, especially the inhalation solution, is a novel administration form, and has the main advantages of simple formulation, quick response, small administration dosage, low side effect and the like. In the production of formoterol fumarate solution dosage forms, there is a need for a method of detecting the presence or absence of impurities in the produced formoterol fumarate solution dosage forms, and in particular for the detection of the formoterol fumarate diastereoisomer which may be present in the formoterol fumarate solution dosage forms.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for detecting diastereoisomers in formoterol fumarate solution.
In one aspect, the invention provides a method for detecting diastereoisomers in a formoterol fumarate solution, which is characterized in that the method comprises the steps of analyzing the formoterol fumarate solution by adopting high performance liquid chromatography and calculating the diastereoisomer content in the formoterol fumarate solution according to the peak area of a chromatogram, wherein the detection conditions of the high performance liquid chromatography comprise:
Packing for chromatographic column stationary phase: octadecyl-bonded ethylene bridge hybrid particles;
-mobile phase: a mixed solution of a phosphate solution and acetonitrile, wherein the pH value of the phosphate solution is 10.0-12.5, and the volume ratio (V/V) of the phosphate solution to the acetonitrile is 90/10-75/25;
mobile phase flow rate: 0.5-1.5 ml/min;
column temperature of chromatographic column: 25-60 ℃;
-detection wavelength: 214-230 nm; and
sample introduction amount: 10 mu l to 100 mu l.
In another aspect, the invention provides a method for quality control of formoterol fumarate inhalation solution, which is characterized in that the method comprises the step of measuring the diastereoisomer content in the formoterol fumarate inhalation solution by adopting high performance liquid chromatography, wherein the detection conditions of the high performance liquid chromatography comprise:
-a chromatographic column: waters XBridge Peptide BEH 300 A.C. 18, specification 4.6X105 mm,3.5 μm;
-mobile phase: a mixed solution of a phosphate solution and acetonitrile, wherein the pH value of the phosphate solution is 11.0-12.0, and the volume ratio (V/V) of the phosphate solution to the acetonitrile is 88/12-80/20; preferably, the volume ratio (V/V) of the phosphate solution to the acetonitrile is 85/15;
Mobile phase flow rate: 0.5-1.5 ml/min;
column temperature of chromatographic column: 25-60 ℃;
-detection wavelength: 220-230 nm; preferably, the detection wavelength: 225nm; and
sample introduction amount: 50 mu l to 100 mu l.
In a further aspect, the invention provides a method for controlling the quality of a process for producing formoterol fumarate inhalation solution, which is characterized in that the method comprises the step of determining whether impurities exist in the formoterol fumarate inhalation solution prepared by the process by adopting high performance liquid chromatography,
wherein the impurity comprises a diastereoisomer of formoterol fumarate in solution selected from at least one of:
wherein the high performance liquid chromatography detection conditions include:
-a chromatographic column: waters XBridge Peptide BEH 300A C18, specification 4.6X105 mm,3.5 μm;
-mobile phase: a mixed solution of a phosphate solution and acetonitrile, wherein the pH value of the phosphate solution is 11.0-12.0, and the volume ratio (V/V) of the phosphate solution to the acetonitrile is 88/12-80/20; preferably, the volume ratio (V/V) of the phosphate solution to the acetonitrile is 85/15;
mobile phase flow rate: 0.5-1.5 ml/min;
column temperature of chromatographic column: 25-60 ℃;
-detection wavelength: 220-230 nm; preferably, the detection wavelength: 225nm; and
sample introduction amount: 50 mu l to 100 mu l.
In a further aspect, the present invention provides a method for detecting the presence of an impurity in a formoterol fumarate inhalation solution, characterised in that the method comprises the step of determining the presence of an impurity in the formoterol fumarate inhalation solution by high performance liquid chromatography,
wherein the impurity comprises a diastereoisomer of formoterol fumarate in solution selected from at least one of:
wherein the high performance liquid chromatography adopts octadecyl bonded ethylene bridge hybrid particles as chromatographic columns of stationary phase filling materials; preferably, the high performance liquid chromatography adopts a chromatographic column of Waters XBridge Peptide BEH 300A C18 with the specification of 4.6X105 mm and 3.5 μm; and is also provided with
Selecting suitable detection conditions for high performance liquid chromatography so that the detection sensitivity (quantitative limit) of the high performance liquid chromatography can reach a detection limit of 0.01% (i.e. a signal to noise ratio greater than 10) and as low as 0.00333%; while achieving a degree of separation of greater than 5.0 in most cases.
The method of the invention has the advantages of high detection sensitivity (the sensitivity (quantitative limit) can reach 0.01 percent (namely the signal to noise ratio is more than 10) and is as low as 0.00333 percent of detection limit), excellent separation degree (the separation degree of formoterol and the impurity I is far more than 3.0, which ensures that an unknown peak does not interfere with the accurate quantification of the impurity I even under the accelerated stability condition test), and excellent accuracy, precision and durability (even if different detectors are used, even under the condition of tiny fluctuation of chromatographic conditions or errors caused by human factors, the detection result is hardly influenced). In addition, the invention can also meet the requirement of realizing high detection sensitivity under the condition of lower sample injection quantity. The method can meet the detection requirement of the small-size formoterol fumarate inhalation solution product with the maximum daily intake (40 mug).
Drawings
Fig. 1: determining a chromatogram of diastereoisomers in the formoterol fumarate inhalation solution by adopting a detection method of Chinese pharmacopoeia;
fig. 2: determining the chromatograms of diastereomers in the formoterol fumarate inhalation solution by using the detection methods of the United states pharmacopoeia and European pharmacopoeia;
fig. 3: the chromatograms of diastereomers in the formoterol fumarate inhalation solutions were determined using the method disclosed by the formoterol fumarate drug substance manufacturer (Industriale Chimica s.r.l.);
fig. 4: detecting a chromatogram of a diastereoisomer of a solution to be detected (formoterol fumarate inhalation solution) by adopting the method of the invention;
fig. 5: the chromatogram of the diastereoisomer in the formoterol fumarate inhalation solution (100 μl sample size) was determined using a Waters Xbridge C18.6 x 250mm 5 μm column;
fig. 6: the chromatogram of the diastereoisomer in the formoterol fumarate inhalation solution (100 μl sample size) was determined using a SUPELCO apHera C18.6 x 150mm 5 μm column;
fig. 7: the method provided by the invention is adopted to detect a typical chromatogram obtained by blank solution/blank auxiliary materials;
fig. 8: detecting a typical chromatogram obtained by the separation degree solution by adopting the method;
Fig. 9: the method of the invention is adopted to detect the linear relation diagram of formoterol fumarate; and
fig. 10: the linear relationship of diastereoisomers (impurity I) was detected using the method of the present invention.
Detailed Description
Definition:
unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present application provides definitions.
The terms "comprising," "including," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.
The word "about" as used herein means that those of ordinary skill in the art consider within acceptable standard deviations of the values, such as + -0.05, + -0.1, + -0.2, + -0.3, + -0.5, + -1, + -2, or + -3, etc.
The term "formoterol fumarate (Formoterol Fumarate)" as used herein is a long-acting β2-adrenoreceptor agonist useful in the treatment of dyspnea caused by airway obstruction disorders such as bronchial asthma, chronic tracheitis, asthmatic bronchitis, emphysema, etc., with CAS numbers 43229-80-7. Formoterol fumarate has the structural formula:
The term "diastereoisomer" as used herein refers to a diastereoisomer of formoterol fumarate. Since formoterol molecules have two chiral centers, a total of 4 optical configurations are produced, wherein the two formoterol optical isomers of the R, S and S, R configuration are known as diastereomers of formoterol. The diastereoisomer of formoterol fumarate refers to the isomer of formoterol contained in formoterol fumarate in the R, S and/or S, R configuration. The diastereoisomer of formoterol fumarate is considered as an impurity. In this context, the terms "diastereoisomer of formoterol fumarate", "diastereoisomer impurity", "impurity of formoterol fumarate", and "impurity" have the same meaning and are used interchangeably. The structural formula of the formoterol fumarate diastereoisomer is as follows:
the term "small-sized formoterol fumarate inhalation solution" as used herein refers to a solution containing formoterol fumarate at a concentration of 5 μg/ml to 15 μg/ml. For example, 2ml of 20 μg is an exemplary small gauge formoterol fumarate inhalation solution, which refers to a single dose package of 2ml per sample containing 20 μg of formoterol fumarate at a concentration of 10 μg/ml. The product of this specification can be administered twice daily, so that the maximum daily intake is equivalent to 4ml, namely 40. Mu.g.
The term "limit of detection" as used herein refers to the lowest concentration or the lowest amount of a component to be detected by an analytical method under defined experimental conditions.
The term "limit of quantitation" as used herein refers to the minimum concentration or amount of a component to be measured in a sample that can be quantitatively determined by an analytical method. The lower the limit of quantitation, the higher the sensitivity.
The term "high performance liquid chromatography" as used herein is also referred to by the english name High Performance Liquid Chromatography (HPLC), also known as "high pressure liquid chromatography", "high performance liquid chromatography", and the like. The principle, method of operation and process conditions of high performance liquid chromatography can be found, for example, in: zhang Danya, yang Jixing, high performance liquid chromatography classification and working principle [ J ]. Chemical management, 2017 (20): 113; leng Xiaochen, zhang Ying, hu Juan, etc., laboratory high performance liquid chromatography safety use and notice [ J ]. Guangzhou chemical, 2022,50 (4): 115-117,120.DOI:10.3969/J. Issn.1001-9677.2022.04.038; and Yang Yujuan, maintenance and use precautions for high performance liquid chromatography. Modern foods, 2015 (19): 66-68.DOI:10.3969/j. Issn.1007-3582.2015.19.032. The skilled person will be familiar with the operation of high performance liquid chromatography and the conditions required to achieve chromatographic separation.
The stationary phase in a chromatographic column is usually composed of solid packing, which allows for separation of components by the difference in type of force and strength of action between the sample molecules. Depending on the packing, the chromatographic column can be divided into: (1) silica gel column; (2) a chromatographic column chemically bound to the stationary phase; (3) high molecular ball chromatographic column; (4) polymer-coated chromatographic columns; and (5) a chromatographic column of a particulate porous carbon filler, wherein the chemically bonded stationary phase chromatographic column is further divided into a nonpolar bonded chromatographic column (octadecylsilane chemically bonded silica is the most commonly used nonpolar reversed phase chromatographic column) and a polar bonded chromatographic column (commonly used amino column, cyano column, diol column, etc.). Alternatively, the chromatographic column may be classified into a silica gel packed chromatographic column, a polymer packed chromatographic column and other inorganic packed chromatographic columns according to the difference of the packing, wherein the silica gel packing is mainly based on silica gel, the surface of the silica gel packing can be bonded with various polar or weak polar functional groups (such as C18 (ODS), C8, C4, C6H5 or normal ethane, chlorine, methylene chloride, etc.), and the polymer packing is mainly polyethylene-divinylbenzene or polymethyl propionate, etc.; other inorganic fillers such as graphitized carbon fillers, alumina, zirconia, and the like. The term "octadecyl bonded ethylene bridge hybrid particles" as used herein is a high purity silica gel filler synthesized from two high purity monomers using inorganic, organic hybrid particle technology, which is mainly applied to the analysis of peptides and proteins. The invention relates to a chromatographic column adopting octadecyl bonded ethylene bridge impurity particles, belonging to a silica gel chromatographic column.
The term "external standard method" as used herein refers to a method of quantifying a pure product of a component to be measured against a control substance by comparing the response signals of the component to be measured in a sample, and can be classified into a standard curve method and an external standard one-point method. In the present invention, unless otherwise specified, the external standard method used in the examples of the present invention is specifically an external standard one-point method without adding correction factors. The external standard method can be specifically referred to the rule 0512 of the 2020 edition of Chinese pharmacopoeia.
The term "external standard one-point method without correction factor" as used herein means that when the correction factor of the test substance (specifically referred to as impurity I herein) is in the range of 0.9 to 1.1, it is considered that correction calculation is not necessary, that is, when the content of impurity I in the test substance solution is calculated using formoterol fumarate as a control substance, the peak area of impurity I is not necessary to be corrected, or the correction factor of impurity I is 1.0 by default, that is, the content of impurity I can be calculated from the peak area of formoterol fumarate in the control substance solution.
Determination of diastereoisomers in formoterol fumarate solution:
the invention aims at solving the problem of separation degree of formoterol and diastereoisomers in the detection of formoterol fumarate inhalation solution. The present invention has found that the inhalation solution of formoterol fumarate generates impurity I during storage, and as impurity I grows, a new unknown peak appears between the formoterol and the chromatographic peak of impurity I (as shown in fig. 4), so whether a method with excellent degree of separation can be provided is one of the keys for accurate quantitative determination of impurity I content.
Further, the invention also aims to solve the problem of sensitivity for diastereoisomeric detection in the inhalation solution detection of formoterol fumarate. The present invention has found that for small format formulation products such as formoterol fumarate inhalation solutions (2 ml:20 μg), it is desirable to provide a detection method with a sensitivity of at least 0.1% (i.e. 0.01 μg/ml, a requirement of a maximum dose per day of less than 1g for an impurity reporting limit of 0.1%). If the chromatographic methods currently used for detecting formoterol impurities, including pharmacopoeia methods, are used, the requirements cannot be met even if the sample injection amount is adjusted to a maximum of 100 μl.
In addition, the invention aims to solve the problems of short service life, poor reproducibility, low precision, poor tolerance and the like of a chromatographic column in the detection of formoterol fumarate inhalation solution. The present invention has found that if chromatographic columns commonly used in methods including pharmacopoeia methods (e.g., octadecyl bonded polyvinyl alcohol chromatographic columns) are used, these chromatographic columns have a short service life and poor tolerance, and it is difficult to satisfy the requirements for reproducibility and precision of results.
To this end, in one aspect, the present invention provides a method for detecting diastereomers in a formoterol fumarate solution, comprising the step of determining the content of diastereomers in a formoterol fumarate solution by high performance liquid chromatography.
In another aspect, the present invention provides a method for quality control of a formoterol fumarate inhalation solution comprising the step of determining the content of diastereoisomers in said formoterol fumarate inhalation solution by high performance liquid chromatography.
In yet another aspect, the present invention provides a method for quality control of a process for producing formoterol fumarate inhalation solution, comprising the step of determining the presence of impurities in the formoterol fumarate inhalation solution prepared by said process using high performance liquid chromatography.
In yet another aspect, the present invention provides a method for detecting the presence of an impurity in a formoterol fumarate inhalation solution comprising the step of determining the presence of an impurity in said formoterol fumarate inhalation solution by high performance liquid chromatography.
In some embodiments, the high performance liquid chromatography detection conditions include the use of octadecyl bonded ethylene bridge hybrid particles as packing for the chromatographic column stationary phase.
In some embodiments, the chromatographic column employing octadecyl bonded ethylene bridge hybrid particles as stationary phase packing is selected from one of the following: waters XBridge Peptide BEH 300AC18, YMC Triart C18, YMC Triart ExRs C18 and Phenomenex Titank C18. In some embodiments, the chromatographic column employing octadecyl bonded ethylene bridge hybrid particles as stationary phase packing has a specification of 4.6x150 mm,3.5 μm, and the chromatographic column is selected from one of the following: waters XBridge Peptide BEH 300A C18, YMC Triart C18, YMC Triart ExRs C18 and Phenomenex Titank C18. In some embodiments, the chromatographic column using octadecyl bonded ethylene bridge hybrid particles as stationary phase packing is Waters XBridge Peptide BEH AC18, 4.6x150 mm,3.5 μm in size.
The inventor proves through experiments that whether the testing method in the current pharmacopoeia (HPLC using octadecyl bonded polyvinyl alcohol chromatographic column) or the testing method of the bulk drug manufacturer (HPLC using octadecyl bonded polyvinyl alcohol chromatographic column) is adopted, when the formoterol fumarate solution is tested, the obtained chromatograms show that the main peak of the formoterol fumarate and the diastereoisomer impurity peak thereof cannot reach the baseline separation, and the accurate quantitative detection of the diastereoisomers in the formoterol fumarate solution cannot be realized.
The inventors speculate that the above methods are all methods for testing formoterol fumarate bulk drug and not for diastereoisomers in formoterol fumarate inhalation solutions. The different formoterol fumarate dosage forms have different diastereoisomers, and the requirements on detection limits and detection sensitivity are different. For example, the above methods are all methods for detecting diastereoisomers in formoterol fumarate, and the minimum injection concentration is 0.2 μg/ml. However, for small-sized formoterol fumarate inhalation solutions (e.g. 2ml: 20. Mu.g) products, the minimum sensitivity of 0.01. Mu.g/ml is required to be achieved for accurate determination of the impurity I content, and the two differ by a factor of 20, so that the sensitivity of the method cannot meet the requirement, and even if the sample injection amount is increased, the sensitivity requirement is only marginally met, but the separation degree cannot meet the requirement. For solutions formulated from formoterol fumarate dosage forms of different origin, it may also occur that the method applicable to the detection of one formoterol fumarate solution (e.g. formulated from an inhalant) is not applicable to another formoterol fumarate solution (e.g. formoterol fumarate inhalant solution, 2ml:20 μg), also because of the different requirements of detection limits, detection sensitivity.
The inventors have surprisingly found through experimentation that when octadecyl bonded ethylene bridge hybrid particles, which are commonly used in peptide and protein analysis, are employed as packing material for chromatographic column stationary phases, the HPLC method allows for the detection of diastereomers in formoterol fumarate solutions, and the detection results show that this method can achieve excellent separation, sensitivity and accuracy at the same time. The detection sensitivity (quantitative limit) of the method can reach 0.01% at the lowest and the detection limit can be as low as 0.00333%; and in most cases a degree of separation of greater than 5.0 can be achieved.
In some embodiments, the high performance liquid chromatography detection conditions comprise a high performance liquid chromatograph. In some embodiments, the high performance liquid chromatograph is selected from one of the following: agilent 1260 II, waters e2695, daian Ulitimate3000, shimadzu LC-2050C/2060C or any other type of chromatograph.
In some embodiments, the high performance liquid chromatography detection conditions include using a mixed solution of phosphate solution and acetonitrile as the mobile phase. In some embodiments, the phosphate solution has a pH of 10.0 to 12.5. In some embodiments, the pH of the phosphate solution is selected from the following values: 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4 and 12.5. In some embodiments, the pH of the phosphate solution is selected from the range consisting of any two of the following values: 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4 and 12.5. For example, the pH of the phosphate solution is selected from the range consisting of a value of 11.0 and a value of 12.0, i.e., the pH of the phosphate solution is 11.0 to 12.0. Experiments prove that the mobile phase with high pH value is more beneficial to separating formoterol from impurity I. Thus, in some embodiments, the pH of the phosphate solution is selected from 12.0.
In some embodiments, the volume ratio (V/V) of the phosphate solution to the acetonitrile is from 90/10 to 75/25. In some embodiments, the volume ratio (V/V) of the phosphate solution to the acetonitrile is selected from the following values: 90/10, 89/11, 88/12, 87/13, 86/14, 85/15, 84/16, 83/17, 82/18, 81/19, 80/20, 79/21, 78/22, 77/23, 76/24 and 75/25. In some embodiments, the volume ratio (V/V) of the phosphate solution to the acetonitrile is selected from the range consisting of any two of the following values: 90/10, 89/11, 88/12, 87/13, 86/14, 85/15, 84/16, 83/17, 82/18, 81/19, 80/20, 79/21, 78/22, 77/23, 76/24 and 75/25. For example, the volume ratio (V/V) of the phosphate solution to the acetonitrile is selected from the range consisting of the values 88/12 and 80/20, i.e., the volume ratio (V/V) of the phosphate solution to the acetonitrile is 88/12 to 80/20. In some embodiments, the volume ratio (V/V) of the phosphate solution to the acetonitrile is 85/15.
In some embodiments, the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, and hydrates thereof. In some embodiments, the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, and hydrates thereof. In some embodiments, the phosphate solution is selected from the group consisting of potassium phosphate trihydrate.
In some embodiments, the phosphate solution is prepared by the following method: taking 5.3g of potassium phosphate trihydrate, adding 1000ml of water for ultrasonic dissolution and uniformly mixing, and regulating the pH value to 12.0+/-0.1 by using potassium hydroxide solution or phosphoric acid. In some embodiments, a mixed solution of phosphate solution and acetonitrile is used as the mobile phase, which is prepared by the following method: taking 5.3g of potassium phosphate trihydrate, adding 1000ml of water, ultrasonically dissolving and uniformly mixing, adjusting the pH value to 12.0+/-0.1 by using potassium hydroxide solution or phosphoric acid, and then uniformly mixing according to the volume ratio (V/V) of the phosphate solution to acetonitrile to prepare a mobile phase.
In some embodiments, the high performance liquid chromatography detection conditions include using a mobile phase flow rate of 0.5ml/min to 1.5ml/min. In some embodiments, the mobile phase flow rate is selected from the following values: 0.5ml/min, 0.6ml/min, 0.7ml/min, 0.8ml/min, 0.9ml/min, 1ml/min, 1.1ml/min, 1.2ml/min, 1.3ml/min, 1.4ml/min and 1.5ml/min. In some embodiments, the mobile phase flow rate is selected from the range consisting of any two of the following values: 0.5ml/min, 0.6ml/min, 0.7ml/min, 0.8ml/min, 0.9ml/min, 1ml/min, 1.1ml/min, 1.2ml/min, 1.3ml/min, 1.4ml/min and 1.5ml/min. For example, the mobile phase flow rate is selected from the range consisting of a value of 0.9ml/min and a value of 1.1ml/min, i.e., the mobile phase flow rate is 0.9ml/min to 1.1ml/min. In some embodiments, the mobile phase flow rate is 1.0ml/min.
In some embodiments, the high performance liquid chromatography detection conditions include using a column temperature of 25 ℃ to 60 ℃. In some embodiments, the column temperature is a value selected from the group consisting of: 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃. In some embodiments, the column temperature is selected from the range consisting of any two of the following values: 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃. For example, the column Wen Xuanzi can be comprised of a range of values of 25 ℃ and 35 ℃, i.e., the column temperature can be 25 ℃ to 35 ℃. In some embodiments, the column temperature is 25 ℃ to 35 ℃. In some embodiments, the column temperature is 28 ℃ to 33 ℃. In some embodiments, the column temperature is 30 ℃.
In some embodiments, the high performance liquid chromatography detection conditions include determining wavelength using a UV detector. In some embodiments, the detector is selected from one of the following: VWD detector, DAD detector, PDA detector or any other type of detector. In some embodiments, the detector is a VWD detector.
In some embodiments, the high performance liquid chromatography detection conditions include the use of detection wavelengths from 214nm to 230nm. In some embodiments, the detection wavelength is a value selected from the group consisting of: 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 221nm, 222nm, 223nm, 224nm, 225nm, 226nm, 227nm, 228nm, 229nm and 230nm. In some embodiments, the detection wavelength is selected from the range consisting of any two of the following values: 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 221nm, 222nm, 223nm, 224nm, 225nm, 226nm, 227nm, 228nm, 229nm and 230nm. For example, the detection wavelength is selected from the range consisting of the number 220nm and the number 230nm, i.e., the detection wavelength is 220nm to 230nm. In some embodiments, the detection wavelength is 220nm to 230nm. In some embodiments, the detection wavelength is 225nm.
In some embodiments, the high performance liquid chromatography detection conditions include using an aqueous solution of methanol as the needle wash water. In some embodiments, the needle wash water uses a solution formulated with water-methanol at a volume ratio (V/V) of 50:50.
In some embodiments, the high performance liquid chromatography detection conditions include using a sample size of 10 μl to 100 μl. In some embodiments, the sample loading is a value selected from the group consisting of: 10. Mu.l, 15. Mu.l, 20. Mu.l, 25. Mu.l, 30. Mu.l, 35. Mu.l, 40. Mu.l, 45. Mu.l, 50. Mu.l, 55. Mu.l, 60. Mu.l, 65. Mu.l, 70. Mu.l, 75. Mu.l, 80. Mu.l, 85. Mu.l, 90. Mu.l, 95. Mu.l, and 100. Mu.l. In some embodiments, the sample amount is selected from the range consisting of any two of the following values: 10. Mu.l, 15. Mu.l, 20. Mu.l, 25. Mu.l, 30. Mu.l, 35. Mu.l, 40. Mu.l, 45. Mu.l, 50. Mu.l, 55. Mu.l, 60. Mu.l, 65. Mu.l, 70. Mu.l, 75. Mu.l, 80. Mu.l, 85. Mu.l, 90. Mu.l, 95. Mu.l, and 100. Mu.l. For example, the sample amount is selected from the range consisting of 50. Mu.l and 100. Mu.l, i.e., the sample amount is 50. Mu.l to 100. Mu.l. In some embodiments, the sample is introduced in an amount of 50 μl to 100 μl. In some embodiments, the sample is introduced at 100 μl.
Experiments prove that compared with an HPLC method adopting an octadecylsilane chemically bonded phase chromatographic column and an octadecyl chemically bonded polyvinyl alcohol as a filling agent, the method can use lower sample injection amount under the same condition. This suggests that the octadecyl bonded ethylene bridge hybrid particle chromatographic column adopted by the invention has higher peak response, so that the signal-to-noise ratio is larger and the sensitivity is higher under the condition of the same sample injection amount.
In some embodiments, the high performance liquid chromatography detection conditions include the use of isocratic elution.
In some embodiments, the diastereoisomers or impurities in the formoterol fumarate solution or inhalation solution are selected from at least one of the following:
in some embodiments, the diastereoisomers or impurities in the formoterol fumarate solution or inhalation solution are a mixture of:
in some embodiments, the formoterol fumarate solution is selected from at least one of the following: formoterol fumarate inhalation solutions, formoterol fumarate solutions formulated from formoterol fumarate bulk drugs, formoterol fumarate solutions formulated from formoterol fumarate aerosols, formoterol fumarate solutions formulated from formoterol fumarate tablets, formoterol fumarate solutions formulated from formoterol fumarate dry syrups, formoterol fumarate solutions formulated from formoterol fumarate inhalation powder mists, and formoterol-containing solutions formulated from formoterol and other drug-formulated compound formulation products. In some embodiments, the formoterol fumarate solution is a formoterol fumarate inhalation solution. In some embodiments, the formoterol fumarate solution is a formoterol fumarate solution formulated from a formoterol fumarate drug substance or other dosage form of formoterol fumarate (in addition to a solution formulation). In some embodiments, the formoterol fumarate solution is a formoterol fumarate solution formulated from a formoterol fumarate aerosol.
In some embodiments, the formoterol fumarate solution is a small gauge formoterol fumarate inhalation solution. In some embodiments, the formoterol fumarate inhalation solution is a small gauge formoterol fumarate inhalation solution. In some embodiments, the formoterol fumarate inhalation solution has 2ml: formoterol fumarate of 20 μg specification was inhaled into solution. In some embodiments, the formoterol fumarate solution has a concentration of formoterol fumarate in the range of 5 μg/ml to 15 μg/ml. In some embodiments, the formoterol fumarate solution has a concentration of formoterol fumarate of 10 μg/ml. In some embodiments, the formoterol fumarate inhalation solution has a formoterol fumarate concentration of 10 μg/ml.
In some embodiments, the high performance liquid chromatography detection conditions include the use of one or more of the following conditions, or a combination thereof:
-a chromatography system: agilent 1260 II or Waters e2695 or similar instruments
-a chromatographic column: peptide separation chromatography column with octadecyl bonded as packing material (Waters XBridge Peptide BEH 300AC18 4.6mm.times.150 mm,3.5 μm)
-mobile phase: mixing solution of phosphate solution and acetonitrile with volume ratio (V/V) of 85:15, and isocratic eluting
-a detector: UV (ultraviolet) light
-detection wavelength: 225nm
-flow rate: 1.0ml/min
Column temperature: 30 DEG C
Sample introduction amount: 100 mu L
Run time: 20min
-needle wash: water-methanol (50:50).
In some embodiments, the method for detecting diastereomers in formoterol fumarate solution of the invention, or the method for quality control of formoterol fumarate inhalation solution production process of the invention, or the method for detecting the presence of impurities in formoterol fumarate inhalation solution of the invention further comprises other steps.
In some embodiments, the additional steps include steps of preparing a blank solution, a sensitivity solution, a resolution solution, a control solution, and/or a formoterol fumarate solution. In some embodiments, the blank solution is a mixed solution of citric acid and its salts. In some embodiments, the control solution is prepared by dissolving formoterol fumarate control in a mixture of phosphate solution and acetonitrile and diluting to a certain concentration with a blank solution. In some embodiments, the sensitivity solution is prepared by dilution of the control solution to a concentration. In some embodiments, the resolution solution is prepared from a mixed solution of formoterol fumarate and a diastereomer at a concentration.
In some embodiments, the additional steps further comprise the step of preparing diluent-1, diluent-2, a test sample solution, a control stock solution, a control solution, a sensitivity solution, an impurity I stock solution, and/or a separation degree solution. Methods of formulating these solutions are known in the art. The skilled artisan can select the solution needed for formulation according to the requirements of the HPLC assay.
In some embodiments, diluent-1, a mixed solution of phosphate buffer and acetonitrile, is formulated, wherein the volume ratio of phosphate buffer to acetonitrile (V/V) is 84:16.
In some embodiments, diluent-2 (i.e., blank solution), a mixed solution of citric acid and its salts, is formulated.
In some embodiments, a control stock solution (100 μg/mL) is prepared, i.e., about 5mg of formoterol fumarate control is precisely weighed, and dissolved in a 50mL volumetric flask with the addition of an appropriate amount of diluent-1.
In some embodiments, a control solution (0.1 μg/mL) is prepared by precisely removing 0.1mL of a control stock solution in a 100mL volumetric flask and metering diluent-2.
In some embodiments, a sensitivity solution (0.01. Mu.g/mL) is prepared by taking 5.0mL of the control solution in a 50mL volumetric flask and metering diluent-2.
In some embodiments, an impurity I stock solution (20 μg/mL) is prepared, i.e., about 2mg of impurity I control is taken, and the diluent (water: acetonitrile=90:10) is dissolved and diluted to scale in a 100mL volumetric flask.
In some embodiments, a resolution solution (formoterol fumarate: 10 μg/mL; impurity I:0.1 μg/mL) is prepared by taking 0.5mL of impurity I stock solution and 10mL of control stock solution, respectively, in a 100mL volumetric flask, and diluting agent-2 to volume.
In some embodiments, the additional step comprises the step of feeding one or more solutions selected from the group consisting of: blank solution, sensitivity solution, resolution solution, control solution, and/or formoterol fumarate solution to be tested.
In some embodiments, the other steps include the step of sequentially feeding the following solutions to a high performance liquid chromatograph: blank solution, sensitivity solution, resolution solution, reference solution, formoterol fumarate solution to be tested and reference solution.
In some embodiments, the additional steps include the step of feeding a solution selected from the group consisting of: the method comprises the steps of (1) blank solution (the sampling frequency is more than or equal to 1 needle), (2) sensitivity solution (the sampling frequency is more than or equal to 1 needle), (3) separation degree solution (the sampling frequency is more than or equal to 1 needle), (4) reference substance solution (the sampling frequency is more than or equal to 6 needles), (5) sample solution (namely formoterol fumarate solution to be tested; the sampling frequency is more than or equal to 1 needle; the sample solution needs to be fed into 6 needles of the following reference substance solution), and (6) following reference substance solution (the sampling frequency is more than or equal to 1 needle).
In some embodiments, the other step includes a step of recording a chromatogram.
In some embodiments, the other step comprises the step of determining whether there is a chromatographic peak of a diastereomer in a chromatogram of the formoterol fumarate inhalation solution. In some embodiments, the formoterol fumarate inhalation solution is determined to contain diastereomers as impurities if there are chromatographic peaks of the diastereomers in the chromatogram of the formoterol fumarate inhalation solution.
In some embodiments, the other steps further comprise the step of determining the impurity content of the formoterol fumarate inhalation solution: when the chromatographic peak of diastereoisomer exists in the chromatogram of the formoterol fumarate inhalation solution, determining the content of the impurity I by an external standard method (external standard one-point method); or calculating the content of the impurity I by a standard curve method.
In some embodiments, the other step further comprises calculating the content of diastereoisomers in the formoterol fumarate solution by the following method. In one embodiment, the diastereomeric content of formoterol fumarate solutions can be calculated using the following formula, for example, as disclosed in the pharmacopoeia.
Wherein A is And (3) supplying: peak area of impurity I in the solution to be measured; a is that And (3) pairing: average peak area of formoterol in the control solution; c (C) And (3) pairing: concentration of the control solution; LC (liquid crystal): a labeled amount of formoterol fumarate inhaled solution product.
In some embodiments, the impurity I in the formoterol fumarate inhalation solution is deemed to be non-compliant with the quality control standard if the impurity I content in the formoterol fumarate inhalation solution is greater than 1.0% (i.e. 0.1 μg/ml or 0.1 ppm). In some embodiments, if the chromatogram of the formoterol fumarate inhalation solution has a peak consistent with the retention time of the known impurity I in the resolution solution, it should be no greater than 1.0% (i.e., 0.1 μg/ml or 0.1 ppm) as calculated by the external standard method without the addition of a correction factor; otherwise the impurity I content of the formoterol fumarate inhalation solution is considered to exceed the quality control requirements. Quality control criteria may be specifically formulated by those skilled in the art in accordance with different circumstances. The inventive method can be used to implement high standard quality control standards.
In some embodiments, the detection sensitivity (limit of quantitation) of the methods of the invention is as low as 0.1% and the limit of detection is as low as 0.0333%. In some embodiments, the detection sensitivity (limit of quantitation) of the methods of the invention is as low as 0.01% and the limit of detection is as low as 0.00333%. In some embodiments, the separation degree of the method of the present invention is greater than 3.0. In some embodiments, the separation degree of the method of the present invention is greater than 4.0. In some embodiments, the separation degree of the method of the present invention is greater than 5.0.
The invention establishes a high performance liquid chromatography detection method capable of accurately and quantitatively detecting diastereoisomers in formoterol fumarate inhalation solution by utilizing an HPLC technology based on octadecyl bonded ethylene bridge hybrid particles. A series of experiments prove that the method has the advantages of good separation degree, high sensitivity, low detection limit, good specificity, high accuracy, high precision, strong durability and the like, and is suitable for detecting diastereoisomers in formoterol fumarate inhalation solution and controlling the quality. The method provides basis for reasonable quality standard formulation so as to better control and master the quality of the product and ensure the safety of clinical medication.
Examples
The following examples are only for further illustration of the invention and do not limit the scope of the invention to these examples.
Experimental materials and reagents:
the materials and reagents used in the present invention may be obtained commercially or formulated by the relevant skilled artisan. In addition, unless specifically stated or described, the experimental materials and reagents of the same names mentioned in the examples of the present invention are specifically as follows:
phosphate solution buffer (part of mobile phase): taking 5.3g of potassium phosphate trihydrate, adding 1000ml of water for ultrasonic dissolution and uniformly mixing, and adjusting the pH value to 12.0+/-0.1 by using a potassium hydroxide solution or a phosphoric acid test solution.
Diluent-1: phosphate buffer (sodium dihydrogen phosphate monohydrate 6.10g and disodium hydrogen phosphate dihydrate 1.03g, dissolved in 1000ml of water, pH adjusted to 6.0.+ -. 0.1) with phosphoric acid or sodium hydroxide solution) -acetonitrile (84:16).
Diluent-2 (blank solution): the formoterol fumarate is inhaled into blank auxiliary material solution (mixed solution of citric acid and its salt, dissolved by ultrasonic, mixed uniformly and filtered).
-a control stock solution: taking a proper amount of formoterol fumarate reference substance (Chinese food and drug verification institute), precisely weighing, dissolving in a volumetric flask, and adding a diluent-1 to prepare a stock solution with the formoterol fumarate concentration of 100 mug/ml.
-control solution: precisely measuring a proper amount of reference substance stock solution in a volumetric flask, adding a diluent-2, and preparing into a reference substance solution containing formoterol fumarate at about 0.1 μg/ml.
Sensitivity solution: precisely measuring 1.0ml of reference substance solution in a 10ml volumetric flask, adding diluent-2 to dilute to scale, and shaking to obtain 0.01 μg/ml sensitivity solution.
-impurity I stock solution: weighing an appropriate amount of impurity I control (standard pharm co., ltd.) in a volumetric flask, adding water: acetonitrile=90:10 dissolved and diluted to scale to prepare 20 μg/ml of impurity I stock solution.
-degree of separation solution: the formoterol fumarate reference stock solution and the impurity I stock solution are taken to be respectively and properly placed in the same volumetric flask, diluted by a diluent-2 to prepare mixed solutions respectively containing the formoterol fumarate of 10 mug/ml and the impurity I of 0.1 mug/ml.
Impurity I localization solution: and (3) taking a proper amount of impurity I stock solution into a volumetric flask, adding a diluent-2 to fix the volume, and preparing a positioning solution of 0.1 mug/ml.
-solution to be tested (formoterol fumarate inhalation solution): the commercial package formoterol fumarate inhalation solution (Mylan), 5 pieces/bag, was removed and the contents were mixed well to prepare the solution to be tested.
Example 1-detection of diastereomers of formoterol fumarate inhaled solutions by methods in pharmacopoeia
During the stability study of commercially available packaged formoterol fumarate inhalation solution products, a growing trend of diastereoisomers (impurity I) in the product was found over a period of time (acceleration conditions-25C/60% rh) (zero-no impurity detected; 1 month-impurity I content of 0.22%;2 months of 0.39%). For quality detection of the impurity content of formoterol fumarate inhalation solution products, in particular of the final product, in particular of specification 2ml: the requirement for detection of 20 μg formoterol fumarate inhalation solution products (at least to a sensitivity requirement of 0.1% concentration (0.01 μg/ml)) was first determined for diastereomers in formoterol fumarate inhalation solutions by methods already known in the pharmacopoeia.
The present experiment was therefore aimed at testing whether the method for detecting the diastereoisomers of formoterol fumarate, which has been described in the chinese, us and european pharmacopoeias, can be used for the detection of diastereoisomers (impurity I) in formoterol fumarate inhalation solutions. Meanwhile, the feasibility of the detection method of diastereoisomers (impurity I) disclosed by formoterol fumarate bulk drug manufacturers (Industriale Chimica s.r.l.) was tested together.
The test methods, conditions and reagents were as follows:
the detection method of Chinese pharmacopoeia comprises the following steps:
-HPLC method, octadecyl bound polyvinyl alcohol column (Shodex Asahipak ODP-50 4E,4.6*250mm,5 μm);
mobile phase ph12.0±0.1 phosphate solution: acetonitrile=88:12 (v/v) isocratic elution;
-flow rate: 1.0ml/min; column temperature: 30 ℃; detection wavelength: 225nm; sample injection amount: 100 μl; run time: 35min.
Detection methods of the United states and European pharmacopoeia:
-HPLC method, octadecyl bound polyvinyl alcohol column (Shodex Asahipak ODP-50 4D,4.6*150mm,5 μm);
mobile phase ph12.0±0.1 phosphate solution: acetonitrile=88:12 (v/v) isocratic elution;
-flow rate: 0.5ml/min; column temperature: 30 ℃; detection wavelength: 225nm; sample injection amount: 100 μl; run time: 35min.
The detection method of the raw material medicine manufacturer comprises the following steps:
-HPLC method, octadecyl bound polyvinyl alcohol chromatographic column (SUPELCO apHera C18 Polymer,4.6 x 150mm,5 μm);
mobile phase ph12.0±0.1 phosphate solution: acetonitrile=88:12 (v/v) isocratic elution;
-flow rate: 0.6ml/min; column temperature: 26 ℃; detection wavelength: 225nm; sample injection amount: 100 μl; the run time was 30min.
Detection solution:
the separation degree solution is prepared by taking a mixed solution with formoterol fumarate concentration of 10 mug/ml and impurity I concentration of 0.1 mug/ml, and carrying out sample injection analysis.
The results of the detection are shown in Table 1 below
TABLE 1
Based on the above experiments, whether the test method in the current pharmacopoeia or the test method of the crude drug manufacturer shows that the main peak of formoterol and the diastereomer impurity peak thereof cannot reach the baseline separation, so that the detection of the diastereomers in the formoterol fumarate inhalation solution cannot be realized, and the detection requirement of the small-dose formoterol fumarate inhalation solution (such as the inhalation solution with the maximum daily intake (40 mug)) product cannot be met.
Example 2-detection of diastereomers in formoterol fumarate inhalation solutions by HPLC method based on octadecyl bonded ethylene bridge hybrid particles as stationary phase
In order to develop a method for detecting formoterol fumarate inhalation solution products with high sensitivity and high separation degree, which can meet the maximum daily intake (40 mug), the experiment tests whether the HPLC method adopting octadecyl bonded ethylene bridge hybrid particles as the chromatographic column stationary phase filling material can be used for detecting diastereoisomer (impurity I) in the formoterol fumarate inhalation solution.
The test methods, conditions and reagents were as follows:
-HPLC method, high performance liquid chromatograph of octadecyl bonded ethylenebridge hybrid particle chromatography column (Waters XBridge Peptide BEH 300ac18,4.6 x 150mm,3.5 μm), agilent 1260 ii (VWD detector);
mobile phase ph12.0±0.1 phosphate solution: acetonitrile=85:15 (v/v) isocratic elution;
-flow rate: 1.0ml/min; column temperature: 30 ℃; detection wavelength: 225nm; sample injection amount: 100 μl; the run time was 20min.
The solution to be tested, formoterol fumarate inhalation solution (Mylan) acceleration conditions were 3 months for samples, three batches (lot number: 21AC6, 20T38 and 20T 54) were taken out and the contents were mixed well to prepare the solution to be tested.
And (3) respectively taking a blank solution, a sensitivity solution, a separation degree solution, a reference substance solution and each solution to be detected, carrying out sample injection analysis, and recording a chromatogram. The sample injection process is as follows: the method comprises the steps of (1) blank solution (the sampling frequency is more than or equal to 1 needle), (2) sensitivity solution (the sampling frequency is 1 needle), (3) separation degree solution (the sampling frequency is 1 needle), (4) reference substance solution (the sampling frequency is 6 needles), (5) sample solution (namely formoterol fumarate solution to be tested; the sampling frequency is more than or equal to 1 needle; the sample solution needs to be fed into 6 needles of the sample solution with a following reference substance), and (6) following reference substance solution (the sampling frequency is more than or equal to 1 needle).
Then, the content of impurity I in the sample solution was calculated as peak area according to the external standard method (external standard one point method, see the rule 0512 of the Chinese pharmacopoeia of 2020 edition).
Figure 4 shows a chromatogram of the detection of diastereoisomers of a solution to be tested (formoterol fumarate inhalation solution) using the method of the invention.
The detection results of the impurity I in the three batches of solutions to be detected are shown in Table 2.
TABLE 2 diastereoisomer content in formoterol fumarate inhalation solution
As can be seen from fig. 4, in the HPLC method using octadecyl bonded ethylene bridge hybrid particles as the chromatographic column stationary phase packing material, even if a new unknown peak appears between formoterol and impurity I, the separation degree of formoterol and impurity I is excellent, so that quantitative detection of impurity I is not interfered; from the results in table 2, it can be seen that this method can be used accurately for the detection of diastereoisomers (impurity I) in formoterol fumarate inhalation solutions.
Example 3-diastereoisomer detection method in formoterol fumarate inhalation solution System suitability test
One objective of this experiment was to determine the systematic suitability of the chromatographic column (Waters XBridge Peptide BEH 300AC18,4.6 x 150mm,3.5 μm) with octadecyl bonded ethylene bridged hybrid particles as stationary phase employed in the present invention for the diastereoisomeric detection method of formoterol fumarate inhalation solution.
Another objective of this experiment was to examine whether the separation and sensitivity of the column (Waters XBridge Peptide BEH 300A C18,4.6*150mm,3.5 μm) using octadecyl bonded ethylene bridged hybrid particles as stationary phase used in the present invention was different from other columns (i.e., octadecyl silane bonded phase column and octadecyl bonded polyvinyl alcohol column). In this experiment, three methods were used for the above investigation using a sensitivity solution and a resolution solution, respectively.
The measurement conditions of the method of the invention:
-HPLC method, octadecyl bonded ethylenebridge hybrid particle chromatography column (Waters XBridge Peptide BEH 300ac18,4.6 x 150mm,3.5 μm), waters e2695 (PDA detector) high performance liquid chromatograph;
mobile phase ph12.0±0.1 phosphate solution: acetonitrile=85:15 (v/v) isocratic elution;
-flow rate: 1.0ml/min; column temperature: 30 ℃; detection wavelength: 225nm; sample injection amount: 100 μl; the run time was 20min.
Comparison of the measurement conditions of method 1:
HPLC method, octadecylsilane chemically bound phase chromatographic column (Waters Xbridge C18 (4.6×250mm,5 μm), waters e2695 (PDA detector) high performance liquid chromatograph;
-mobile phase: solution A is tetrabutylammonium hydroxide aqueous solution (0.6 percent, v/v) with pH value adjusted to 8.5 by phosphoric acid, solution B is acetonitrile, and solution A is eluted at isocratic of solution B=80:20 (v/v);
-flow rate: 1.0ml/min; column temperature: 35 ℃; detection wavelength: 225nm; sample injection amount: 50 μl; the run time was 30min.
-HPLC method, column with octadecyl-bonded polyvinyl alcohol as filler (SUPELCO apHera C18Polymer (4.6 x 150mm,5 μm), waters e2695 (PDA detector) HPLC;
mobile phase a: phosphate solution at ph12.0±0.1: acetonitrile=85:15 (v/v); mobile phase B: water: acetonitrile=15:85 (v/v) gradient elution
-flow rate: 1.0ml/min; column temperature: 40 ℃; detection wavelength: 225nm; sample injection amount: 20 μl; the run time was 55.1min.
Gradient elution procedure:
| Time | 0min | 15 min | 35min | 55.1min |
| mobile phase A% | 100 | 100 | 71 | 100 |
| Mobile |
0 | 0 | 29 | 0 |
Taking a blank solution, a sensitivity solution and a separation degree solution respectively, injecting one needle, injecting a reference solution in parallel with 6 needles, and examining the applicability of the system.
The sensitivity and separation effect of the above methods are shown in Table 3:
the results of the detection are shown in Table 3 below
System applicability requirements: the blank solution should be free of interference, and the signal to noise ratio of formoterol in the sensitivity solution should be not less than 10; the separation degree of the formoterol and the impurity I in the separation degree solution is not less than 3.0; the RSD of the formoterol peak area in the 6-needle control solution should be no more than 2.0% and the repeated results of the sample injection using the method of the present invention are shown in table 4 below.
TABLE 4 Table 4
| |
1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Peak area | 38041 | 38041 | 38041 | 38041 | 38041 | 38041 | 0.2％ |
According to the results, the chromatographic column using octadecyl bonded ethylene bridge hybrid particles as a stationary phase can realize the effective separation of formoterol and impurity I in the sample injection amount range of 20-100 μl. In contrast, for detection methods employing octadecylsilane chemically bonded columns and octadecyl chemically bonded columns, effective separation of formoterol from impurity I could not be achieved in the same sample loading range of 20 μl or 50 μl; moreover, when the sample injection amount (100 μl) is increased, the separation of formoterol and impurity I still cannot be realized, and the requirements of detection on the separation degree and sensitivity cannot be met. The result shows that the method of the invention (the chromatographic column (Waters XBridge Peptide BEH 300A C18,4.6*150mm,3.5 μm) with the octadecyl bonded ethylene bridge hybrid particles as the stationary phase) has the advantages of high sensitivity and good separation degree under the condition of the same sample injection amount. In contrast, for detection methods employing octadecylsilane chemically bonded columns and octadecyl chemically bonded polyvinyl alcohol columns, detection of diastereomers in formoterol fumarate inhalation solutions (2 ml:20 μg) was not achieved under essentially identical assay conditions.
Example 4-specificity test of HPLC method based on octadecyl bonded ethylene bridge hybrid particles as stationary phase
The purpose of this experiment was to examine the ability of the HPLC method using octadecyl bonded ethylene bridge hybrid particles as chromatographic column stationary phase (Waters XBridge Peptide BEH AC18,4.6 x 150mm,3.5 μm) to accurately measure the analyte (impurity I) in the possible presence of other components (e.g. blank diluent, adjuvant, impurity, etc.).
The chromatographic conditions for this experiment were the same as in example 2.
Taking blank solution/blank auxiliary material solution, reference substance solution, impurity I locating solution, separation degree solution and sample solution, analyzing by sample injection according to the chromatographic conditions, and recording chromatogram. Fig. 7 shows a typical chromatogram obtained by detecting a blank solution/blank adjuvant using the method of the present invention. As shown in fig. 7, the blank solution/blank adjuvant was undisturbed at the peak of formoterol or impurity I. FIG. 8 shows a typical chromatogram obtained by detecting a resolution solution using the method of the present invention. As shown in fig. 8, the separation degree of formoterol from impurity I in the separation degree solution was 5.4, and the result showed that the specificity was excellent.
Example 5: quantitative limit and detection limit test
The purpose of this experiment was to accurately determine the minimum amount of impurity I that can be quantitatively determined (limit of quantification) and the minimum amount that can be detected (limit of detection) in a test sample by an HPLC analysis method using octadecyl bonded ethylene bridge hybrid particles as a stationary phase chromatographic column (Waters XBridge Peptide BEH 300ac18,4.6 x 150mm,3.5 μm).
Chromatographic conditions were the same as in example 2.
Impurity I stock solution: about 10mg of the impurity I reference substance is taken, precisely weighed, dissolved and diluted to a scale by adding a diluent (water: acetonitrile=90:10) into a 100ml volumetric flask, and prepared into a stock solution of 100 mug/ml.
Control stock solution: about 10mg of formoterol fumarate reference is taken, dissolved in a 100ml volumetric flask with diluent-1 to prepare a stock solution of 100 μg/ml.
LOQ (limit of quantitation) solution (equivalent to 0.1% of product concentration): respectively and precisely transferring 1.0mL of impurity I stock solution and 1.0mL of formoterol reference substance stock solution into a 100mL volumetric flask, fixing the volume by using a diluent-2, shaking uniformly, precisely transferring 1.0mL of the solution into a 100mL volumetric flask, fixing the volume by using the diluent-2, and shaking uniformly.
LOD (limit of detection) solution (equivalent to 0.0333% of product concentration): diluting the quantitative limiting solution 3 times as a detection limiting solution
Taking the solutions for separate sample injection, wherein the signal to noise ratio (S/N) of quantitative limiting solution parallel sample injection for 6 needles is more than 10, and the RSD of peak area is not more than 5.0%; the signal to noise ratio (S/N) of the detection limit solution should be not less than 3. The results are shown in the following table:
table 5 quantitative limit examination results
Table 6 limit of detection results
The results show that: the signal to noise ratio is far greater than 10 at a quantitative concentration limit level of 0.1% of the concentration of the test substance (2 ml of formoterol fumarate inhalation solution: 20 μg product reported limit is 0.1%); the signal to noise ratio is greater than 3 under the detection limit of 0.0333% of the concentration of the sample, which indicates that the method can accurately and quantitatively detect the content of impurity I in formoterol fumarate inhalation solution. This result is quite satisfactory, if on the basis of an acceptable signal-to-noise ratio of 10, the minimum quantitative limit can be deduced to be 0.01%, and correspondingly the minimum detection limit can be deduced to be 0.00333%.
Example 6: linear and range test
The purpose of this experiment was to examine the ability of the HPLC method using octadecyl bonded ethylene bridge hybrid particles as stationary phase chromatographic column (Waters XBridge Peptide BEH 300AC18,4.6 x 150mm,3.5 μm) in the designed range (LOQ-3.0%), the peak area of the measured substance in proportional relation to the concentration, and the accuracy of the impurity I content calculated by external standard method.
Chromatographic conditions were the same as in example 2.
Linear stock solution: 1.0mL of impurity I stock solution (100 mug/mL) and 1.0mL of reference stock solution (100 mug/mL) are respectively and precisely removed, and put into a 100mL volumetric flask, diluent-2 is used for constant volume, and shaking is carried out, thus obtaining a mixed solution of 1.0 mug/mL.
Linear solution: the appropriate volumes of linear stock solutions were respectively transferred into 10ml volumetric flasks as indicated in the following table, and diluent-2 was fixed to volume and shaken well. TABLE 7
Taking the solutions, respectively sampling and recording chromatograms, taking the concentration as an abscissa and the peak area as an ordinate, and performing linear recovery, wherein the correlation coefficient is required to be not less than 0.990; the y-axis intercept should be within 25% of the target concentration; the residual should be no more than 10%. The results are shown in the following table:
TABLE 8 results of linear investigation of formoterol fumarate inhalation solutions
Fig. 9 shows a graph of the linear relationship of formoterol fumarate inhalation solutions tested using the method of the present invention, showing that formoterol fumarate was well-related in the concentration range of 0.01 μg/ml (LOQ) to 0.30 μg/ml.
FIG. 10 shows a linear relationship between the detection of diastereoisomers of formoterol fumarate inhaled solutions (impurity I) using the method of the invention, showing good linearity between impurity I in the concentration range of 0.01 μg/ml (LOQ) to 0.30 μg/ml.
Example 7: accuracy test
The purpose of this experiment was to examine the fact that the HPLC method using octadecyl bonded ethylene bridge hybrid particles as chromatographic column (Waters XBridge Peptide BEH 300AC18,4.6 x 150mm,3.5 μm) added impurity I solutions of different known concentrations to the sample solutions containing the adjuvants, examined whether the added impurity I could be completely extracted, and examined how close the measurement result is to the true value, generally expressed in terms of recovery rate.
Chromatographic conditions were the same as in example 2.
Impurity I stock solution: 1.0mL of impurity I stock solution (100 mug/mL) is precisely removed, put into a 100mL volumetric flask, diluent-2 is fixed in volume, and shaken uniformly to obtain 1.0 mug/mL stock solution.
R0 test solution: and taking a sufficient amount of sample content solution, and uniformly mixing in the same reagent bottle to obtain the sample.
R1 accuracy solution (LOQ-0.1%): accurately transferring 0.1mL of the impurity I stock solution into a 10mL volumetric flask, and preparing three parts in parallel by using an R0 sample solution to fix the volume and shaking the mixture uniformly.
R2 accuracy solution (1.0%): 1.0mL of the impurity I stock solution is precisely removed, and the volume is fixed by using an R0 sample solution in a 10mL volumetric flask, and the three parts are prepared in parallel.
R3 accuracy solution (2.0%): 2.0mL of the impurity I stock solution is precisely removed, and the volume is fixed by using an R0 sample solution in a 10mL volumetric flask, and the three parts are prepared in parallel.
Taking each solution sample for analysis and recording a chromatogram, and calculating the recovery rate of the impurity I according to an external standard method and the peak area, wherein the result is shown in the following table:
TABLE 9 accuracy results of impurity I in formoterol fumarate inhalation solutions
The results show that: the recovery rate and average recovery rate of each sample of the impurity I are between 80% and 120% at each concentration level, and the RSD of 9 parts of recovery rate is less than 10%, which shows that the method has good accuracy, and the impurity I in the sample solution can be completely extracted and accurately quantified.
Example 8: precision test
The purpose of this experiment was to test the proximity between the results of a multiple sampling assay performed under certain conditions using a chromatographic column (Waters XBridge Peptide BEH 300ac18,4.6 x 150mm,3.5 μm) with octadecyl bonded ethylene bridged hybrid particles as the stationary phase.
1.1 repeatability test
Chromatographic conditions were the same as in example 2.
Accurately transferring 1.0mL of impurity I stock solution (1.0 mug/mL) into a 10mL volumetric flask by the same analyst, fixing the volume by using a sample solution (formoterol fumarate suction solution), shaking uniformly, and preparing 6 parts in parallel as a sample solution; and precisely measuring 100 mu l of each of the reference substance solution and the sample solution, carrying out sample injection analysis, recording a chromatogram, and calculating the content of the impurity I according to an external standard method by using the peak area, wherein the result is shown in the following table.
Table 10 repeatability results
The results show that: the same analyst, in 6 sample solutions prepared in parallel, detected less than 10% RSD of impurity I content%, indicating good reproducibility of the method.
1.2 intermediate precision test
The chromatographic conditions were the same as in example 2 except for HPLC.
Another analyst independently set up the system, take the same lot of sample solution as the repeated sample, prepare 6 test sample solutions with the same concentration in the same way, use different instruments to measure on different days, and the results are shown in the table below.
TABLE 11 results of formoterol fumarate inhalation solution precision
The results show that: the RSD of the content percent of the detected impurity I in 12 sample solutions with the intermediate precision and the repeatability is less than 15 percent, which shows that different personnel and different instruments have little influence on the measurement result of the method, and the method has good precision.
Example 9: test article and control article solution stability test
The test aims to examine the stability of the solution to be tested and the reference solution at room temperature and 2-8 ℃ after the preparation of the solution to be tested and the reference solution is completed, so as to support the accuracy and the reliability of data in the test process.
Chromatographic conditions were the same as in example 2.
Sample solutions of formoterol fumarate inhalation solutions are placed at room temperature and 2-8 ℃ for measurement at zero day, 1 day and 2 days respectively, stability of the sample solutions is inspected, and 0.1 mug/mL formoterol fumarate reference solution is taken for inspecting stability of the reference solution under the same conditions and time points, and the results are shown in the following table.
TABLE 12 results of examination of stability of formoterol fumarate inhaled solutions
The results show that: the reference substance solution and the test substance solution are stable in 2 days at 2-8 deg.C or room temperature.
Example 10: durability test
The aim of the experiment is to find the optimal chromatographic parameters and determine the bearing degree of the target parameters, which is not affected by the result, by examining the condition changes of the chromatographic conditions, such as the flow rate of the chromatographic conditions, the pH of the mobile phase, the proportion of the mobile phase, the chromatographic columns of different batches of the same brand, the chromatographic columns of different brands of the same filler and the like. The specific examination items are shown in the following table.
TABLE 13 durability inspection project
Other chromatographic conditions were the same as in example 2. The results of the measurements are shown in the following Table
Table 14 durability test results
The results show that: the results of the impurity I measurements were substantially uniform when the flow rate, mobile phase pH and mobile phase ratio were slightly varied, indicating that the method was well durable. Meanwhile, the octadecyl bonded ethylene bridge hybrid particle chromatographic column is a key point of whether the method can realize the expected detection requirement, and chromatographic columns with different brands but all belonging to the octadecyl bonded ethylene bridge hybrid particles as the filling material of the chromatographic column stationary phase have similar detection effects. In addition, the chromatographic columns of different brands with the same packing have better durability.
Example 11: chromatographic condition reference screening assay
The aim of the experiment is to find the optimum chromatographic parameters, such as pH of the mobile phase and the proportion of the mobile phase, and then find the tolerance degree of the optimum chromatographic parameters, which is not influenced by the sensitivity and the separation degree. The specific examination items are shown in the following table.
TABLE 15 optimal parameter screening results
The results show that: the method of the invention can be adapted to different chromatographic conditions. The results prove that the method can meet the requirements of sensitivity and separation degree under the conditions of pH11-12 and mobile phase ratio 80/20-88/12.
Technical scheme set
Technical scheme group a:
1. a method for quality control of formoterol fumarate inhalation solution, comprising the step of determining the content of diastereoisomers in said formoterol fumarate inhalation solution by high performance liquid chromatography, wherein the conditions for detection by high performance liquid chromatography comprise:
-a chromatographic column: waters XBridge Peptide BEH 300 A.C. 18, specification 4.6X105 mm,3.5 μm;
-mobile phase: a mixed solution of a phosphate solution and acetonitrile, wherein the pH value of the phosphate solution is 11.0-12.0, and the volume ratio (V/V) of the phosphate solution to the acetonitrile is 88/12-80/20; preferably, the volume ratio (V/V) of the phosphate solution to the acetonitrile is 85/15;
Mobile phase flow rate: 0.5-1.5 ml/min;
column temperature of chromatographic column: 25-60 ℃;
-detection wavelength: 220-230 nm; preferably, the detection wavelength: 225nm; and
sample introduction amount: 50 mu l to 100 mu l.
2. The method for quality control according to claim 1, wherein the elution mode of the high performance liquid chromatography is isocratic elution.
3. The method of quality control according to claim 1, wherein the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, and hydrates thereof; preferably, the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, and hydrates thereof; more preferably, the phosphate solution is selected from the group consisting of potassium phosphate trihydrate.
4. The method for quality control according to any one of claims 1 to 3, wherein the high performance liquid chromatograph used in the high performance liquid chromatography is agilent 1260 ii or Waters e2695; and/or
The detector used in the high performance liquid chromatography is a VWD detector.
5. The method of quality control according to any one of claims 1-4, further comprising one or more of the following steps:
(a) Preparing a blank solution, a sensitivity solution, a separation degree solution, a reference substance solution and a formoterol fumarate solution, wherein:
-the blank solution is a mixed solution of citric acid and salts thereof;
-the control solution dissolves formoterol fumarate pairs by a mixed solution of phosphate solution and acetonitrile
Preparing a standard product by diluting the blank solution to a certain concentration;
-the sensitivity solution is prepared by dilution of the control solution to a certain concentration; and
-said resolution solution is composed of a concentration of formoterol fumarate and a mixture of diastereomers
Preparing;
(b) Continuously and sequentially injecting the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate solution into a chromatograph, sequentially analyzing the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate solution by adopting a high performance liquid chromatography, and recording a chromatogram;
(c) The content of diastereoisomers in the formoterol fumarate solution was calculated from the peak area of the chromatogram by an external standard method (standard curve method or external standard point method).
6. The method of quality control according to any one of claims 1 to 5, wherein the diastereoisomer of formoterol fumarate in inhalation solution is selected from at least one of the following:
7. the method of quality control according to any one of claims 1-6, characterized in that the content of diastereomers in the formoterol fumarate solution is considered to be out of compliance with the quality control standard if the content of diastereomers in the formoterol fumarate solution is greater than 1.0% (i.e. 0.1 μg/ml or 0.1 ppm).
8. The method of quality control according to any one of claims 1 to 7, characterized in that the method has a detection sensitivity (limit of quantitation) as low as 0.01% and a detection limit as low as 0.00333%, and a degree of separation of more than 5.0.
Technical scheme group B:
1. a method for controlling the quality of the process for producing the formoterol fumarate inhalation solution is characterized by comprising the step of determining whether impurities exist in the formoterol fumarate inhalation solution prepared by the process by adopting high performance liquid chromatography,
Wherein the impurity comprises a diastereoisomer of formoterol fumarate in solution selected from at least one of:
wherein the high performance liquid chromatography detection conditions include:
-a chromatographic column: waters XBridge Peptide BEH 300 A.C. 18, specification 4.6X105 mm,3.5 μm;
-mobile phase: a mixed solution of a phosphate solution and acetonitrile, wherein the pH value of the phosphate solution is 11.0-12.0, and the volume ratio (V/V) of the phosphate solution to the acetonitrile is 88/12-80/20; preferably, the volume ratio (V/V) of the phosphate solution to the acetonitrile is 85/15;
mobile phase flow rate: 0.5-1.5 ml/min;
column temperature of chromatographic column: 20-40 ℃;
-detection wavelength: 220-230 nm; preferably, the detection wavelength: 225nm; and
sample introduction amount: 50 mu l to 100 mu l.
2. The method of quality control according to claim 1, wherein the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, and hydrates thereof; preferably, the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, and hydrates thereof; more preferably, the phosphate solution is selected from the group consisting of potassium phosphate trihydrate.
3. The method for quality control according to claim 1 or 2, wherein the elution mode of the high performance liquid chromatography is isocratic elution.
4. A quality control method according to any one of claims 1 to 3, characterized in that,
the high performance liquid chromatograph adopted by the high performance liquid chromatograph is selected from one of the following: agilent 1260 II, waters e2695, daian Ulitimate 3000, shimadzu LC-2050C/2060C or any other type of chromatograph; and/or
The detector used for the high performance liquid chromatography is selected from one of the following: VWD detector, DAD detector, PDA detector or any other type of detector.
5. The method of quality control according to any one of claims 1-4, further comprising one or more of the following steps:
(a) Preparing a blank solution, a sensitivity solution, a separation degree solution, a reference substance solution and a formoterol fumarate solution, wherein:
-the blank solution is a mixed solution of citric acid and salts thereof;
-the control solution dissolves formoterol fumarate pairs by a mixed solution of phosphate solution and acetonitrile
Preparing a standard product by diluting the blank solution to a certain concentration;
-the sensitivity solution is prepared by dilution of the control solution to a certain concentration; and
-said resolution solution is composed of a concentration of formoterol fumarate and a mixture of diastereomers
Preparing;
(b) Continuously and sequentially injecting the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate solution into a chromatograph, sequentially analyzing the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate solution by adopting a high performance liquid chromatography, and recording a chromatogram;
(c) If there is a chromatographic peak of a diastereomer in the chromatogram of the formoterol fumarate inhalation solution, it is determined that the formoterol fumarate inhalation solution contains a diastereomer as an impurity.
6. The method of quality control according to any one of claims 1 to 5, further comprising the step of determining the impurity content of the formoterol fumarate inhalation solution, comprising the step of determining that the chromatogram of the formoterol fumarate inhalation solution has a peak of diastereoisomer, and calculating the diastereoisomer content of the formoterol fumarate solution from the peak area of the chromatogram; preferably, the method comprises the step of calculating the content of diastereoisomers in the formoterol fumarate solution by an external standard method (standard curve method or external standard point method) from the peak area of the chromatogram when determining that the chromatogram of the formoterol fumarate inhalation solution has a chromatographic peak of diastereoisomers.
7. The method of quality control according to any one of claims 1-6, characterized in that the content of diastereomers in the formoterol fumarate solution is considered to be out of compliance with the quality control standard if the content of diastereomers in the formoterol fumarate solution is greater than 1.0% (i.e. 0.1 μg/ml or 0.1 ppm).
8. The method of quality control according to any one of claims 1 to 7, characterized in that the method has a detection sensitivity (limit of quantitation) as low as 0.01% and a detection limit as low as 0.00333%, and a degree of separation of more than 5.0.
Technical scheme group C:
1. a method for detecting whether impurity exists in formoterol fumarate inhalation solution is characterized in that the method comprises the step of determining whether impurity exists in the formoterol fumarate inhalation solution by adopting high performance liquid chromatography,
wherein the impurity comprises a diastereoisomer of formoterol fumarate in solution selected from at least one of:
wherein the high performance liquid chromatography adopts octadecyl bonded ethylene bridge hybrid particles as chromatographic columns of stationary phase filling materials; preferably, the high performance liquid chromatography adopts a chromatographic column of Waters XBridge Peptide BEH 300AC18 with the specification of 4.6X105 mm and 3.5 μm; and is also provided with
The suitable high performance liquid chromatography detection conditions are selected such that the high performance liquid chromatography has a detection sensitivity (limit of quantitation) as low as 0.01% and a detection limit as low as 0.00333%, and a separation degree of greater than 5.0.
2. The method according to claim 1, wherein the high performance liquid chromatography detection conditions include:
-mobile phase: a mixed solution of a phosphate solution and acetonitrile, wherein the pH value of the phosphate solution is 10.0-12.5, and the volume ratio (V/V) of the phosphate solution to the acetonitrile is 90/10-75/25;
mobile phase flow rate: 0.5-1.5 ml/min;
column temperature of chromatographic column: 25-60 ℃;
-detection wavelength: 214-230 nm; and
sample introduction amount: 10 mu l to 100 mu l.
3. The detection method according to claim 1 or 2, characterized in that the pH value of the phosphate solution is 11.0 to 12.0; preferably, the pH value of the phosphate solution is 11.0-12.0; and/or
The phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, and hydrates thereof; preferably, the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, and hydrates thereof; more preferably, the phosphate solution is selected from the group consisting of potassium phosphate trihydrate; and/or
The volume ratio (V/V) of the phosphate solution to the acetonitrile is 88/12-80/20; preferably, the volume ratio (V/V) of the phosphate solution to the acetonitrile is 85/15; and/or
The mobile phase flow rate: 0.8-1.2 ml/min; preferably, the mobile phase flow rate: 0.9-1.1 ml/min; more preferably, the mobile phase flow rate: 1.0ml/min; and/or
Column temperature of the chromatographic column: 25-40 ℃; preferably, the column temperature: 25-35 ℃; more preferably, the column temperature: 30 ℃; and/or
The detection wavelength: 220-230 nm; preferably, the detection wavelength: 225nm; and/or
The sample injection amount is as follows: 20-110 mu l; preferably, the sample injection amount is as follows: 50-100 mu l; more preferably, the sample injection amount is as follows: 100 μl.
4. The detection method according to any one of claims 1 to 3, wherein the chromatographic column using octadecyl bonded ethylene bridge hybrid particles as stationary phase packing is selected from one of the following: waters XBridge Peptide BEH 300AC18, YMC triert C18, YMC Triart ExRs C18 and Phenomenex Titank C18;
preferably, the chromatographic column using octadecyl bonded ethylene bridge hybrid particles as stationary phase packing has a specification of 4.6x150mm, 3.5 μm and is selected from one of the following: waters XBridge Peptide BEH 300A C18, YMC Triart C18, YMC Triart ExRs C18 and Phenomenex Titank C18;
More preferably, the chromatographic column using octadecyl bonded ethylene bridge hybrid particles as stationary phase packing is Waters XBridge Peptide BEH 300A C18, specification 4.6X105 mm,3.5 μm.
5. The detection method according to any one of claims 1 to 4, wherein the elution mode of the high performance liquid chromatography is isocratic elution.
6. The detection method according to any one of claims 1 to 5, characterized in that,
the high performance liquid chromatograph adopted by the high performance liquid chromatograph is selected from one of the following: agilent 1260 II, waters e2695, daian Ulitimate 3000, shimadzu LC-2050C/2060C or any other type of chromatograph; and/or
The detector used for the high performance liquid chromatography is selected from one of the following: VWD detector, DAD detector, PDA detector or any other type of detector.
7. The detection method according to any one of claims 1 to 6, characterized in that the method further comprises one or more of the following steps:
(a) Preparing a blank solution, a sensitivity solution, a separation degree solution, a reference substance solution and a formoterol fumarate solution, wherein:
-the blank solution is a mixed solution of citric acid and salts thereof;
-the control solution dissolves formoterol fumarate pairs by a mixed solution of phosphate solution and acetonitrile
Preparing a standard product by diluting the blank solution to a certain concentration;
-the sensitivity solution is prepared by dilution of the control solution to a certain concentration; and
-said resolution solution is composed of a concentration of formoterol fumarate and a mixture of diastereomers
Preparing;
(b) Continuously and sequentially injecting the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate solution into a chromatograph, sequentially analyzing the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate solution by adopting a high performance liquid chromatography, and recording a chromatogram;
(c) If there is a chromatographic peak of a diastereomer in the chromatogram of the formoterol fumarate inhalation solution, it is determined that the formoterol fumarate inhalation solution contains a diastereomer as an impurity.
8. The method according to any one of claims 1 to 7, further comprising the step of determining the impurity content of the formoterol fumarate inhalation solution, comprising the step of determining that the chromatogram of the formoterol fumarate inhalation solution has a peak of diastereoisomer, and calculating the diastereoisomer content of the formoterol fumarate solution from the peak area of the chromatogram; preferably, the method comprises the step of calculating the content of diastereoisomers in the formoterol fumarate solution by an external standard method (standard curve method or external standard point method) from the peak area of the chromatogram when determining that the chromatogram of the formoterol fumarate inhalation solution has a chromatographic peak of diastereoisomers.
Claims (9)
1. A method for quality control of a process for producing a formoterol fumarate inhalation solution or for detecting the presence or absence of impurities in a formoterol fumarate inhalation solution, said method comprising the steps of analysing a formoterol fumarate inhalation solution by high performance liquid chromatography and calculating the content of diastereoisomers in said formoterol fumarate inhalation solution from the peak areas of the chromatogram, wherein the conditions for detection by high performance liquid chromatography comprise:
packing for chromatographic column stationary phase: waters XBridge Peptide BEH 300A C18, specification 4.6X105 mm,3.5 μm;
-mobile phase: a mixed solution of a phosphate solution and acetonitrile, wherein the pH value of the phosphate solution is 11.0-12.0, and the volume ratio of the phosphate solution to the acetonitrile is 88/12-80/20;
the elution mode of high performance liquid chromatography is isocratic elution;
mobile phase flow rate: 0.9-1.1 ml/min;
column temperature of chromatographic column: 25-35 ℃;
-detection wavelength: 214-230 nm; and
sample introduction amount: 10 mu l-100 mu l;
the diastereoisomers of formoterol fumarate in inhalation solutions are as follows:
if the content of diastereoisomers in the formoterol fumarate inhalation solution is greater than 0.1 μg/ml or 0.1ppm, the impurities in the formoterol fumarate inhalation solution are considered to be present and the impurity content is out of specification or the impurities present in the formoterol fumarate inhalation solution do not meet the quality control criteria.
2. The method of claim 1, wherein the phosphate solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, and hydrates thereof.
3. The method of claim 1, wherein the detection wavelength: 220-230 nm.
4. The method of claim 1, wherein the sample injection amount is: 20 mu l to 110 mu l.
5. The method of claim 1, wherein the step of determining the position of the substrate comprises,
the high performance liquid chromatograph adopted by the high performance liquid chromatograph is selected from one of the following: agilent 1260 II, waters e2695, daian Uilimate 3000, shimadzu LC-2050C/2060C; and/or
The detector used for the high performance liquid chromatography is a UV detector.
6. The method of claim 1, wherein the high performance liquid chromatography detection conditions comprise:
-a chromatography system: agilent 1260 ii or Waters e2695;
-a chromatographic column: a peptide separation chromatographic column with octadecyl bonded as filler, the filler being Waters XBridge Peptide BEH 300A C18, specification 4.6X105 mm,3.5 μm;
-mobile phase: the mixed solution of phosphate solution and acetonitrile with the volume ratio of 85:15 is eluted isocratically, and the pH value of the phosphate solution is 11.0-12.0;
-a detector: UV;
-detection wavelength: 225nm;
-flow rate: 1.0ml/min;
column temperature: 30 ℃;
sample introduction amount: 100. Mu.L;
run time: 20min; and
-needle wash: the volume ratio of water to methanol is 50:50.
7. The method according to any one of claims 1-6, further comprising the step of:
(a) Preparing a blank solution, a sensitivity solution, a separation degree solution, a reference substance solution and a formoterol fumarate inhalation solution, wherein:
-the blank solution is a mixed solution of citric acid and salts thereof;
-the control solution is prepared by dissolving formoterol fumarate control in a mixed solution of phosphate solution and acetonitrile and diluting to a certain concentration with the blank solution;
-the sensitivity solution is prepared by dilution of the control solution to a certain concentration; and
-said resolution solution is prepared from a mixed solution of formoterol fumarate and diastereomers at a concentration;
(b) Continuously and sequentially injecting the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate inhalation solution into a chromatograph, sequentially analyzing the blank solution, the sensitivity solution, the separation degree solution, the reference substance solution and the formoterol fumarate inhalation solution by adopting a high performance liquid chromatography, and recording a chromatogram;
(c) The diastereoisomeric content of the formoterol fumarate inhalation solution was calculated by the external standard method from the peak area of the chromatogram.
8. The method according to claim 7, wherein the content of diastereoisomers in the formoterol fumarate solution is calculated by,
wherein impurity I = diastereoisomer; a is that And (3) supplying: peak area of impurity I in the solution to be measured; a is that And (3) pairing: average peak area of formoterol in the control solution; c (C) And (3) pairing: concentration of the control solution; LC (liquid crystal): a labeled amount of formoterol fumarate inhaled into the solution.
9. The method of claim 8, wherein the formoterol fumarate inhalation solution tested is 40 μg of formoterol fumarate inhalation solution at maximum daily intake.
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| CN116908345A (en) * | 2023-07-10 | 2023-10-20 | 南京力成药业有限公司 | Method for detecting content of formoterol fumarate inhalation solution |
| CN116930381A (en) * | 2023-07-04 | 2023-10-24 | 南京力成药业有限公司 | Method for detecting diastereoisomeric impurities in formoterol fumarate inhalation solution |
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