A method for detecting impurities in esmolol hydrochloride injection

Abstract

The present invention discloses a method for detecting impurities of esmolol hydrochloride injection, comprising the following steps: S1, diluting esmolol hydrochloride injection of different manufacturers with water respectively, and preparing a test solution; S2, esmolol hydrochloride injection of different manufacturers is subjected to liquid phase-mass spectrometry detection and analysis, and the relative peak area and retention time of all impurities are obtained; S3, impurities that may be present in esmolol hydrochloride injection are screened out by mass loss technology; S4, multivariate analysis method analyzes the results in S2, distinguishes the difference between different manufacturers and the impurity spectrum of original research drugs, and screens impurity specific markers; S5, the results of multivariate analysis method in S4 are combined with the mass loss results in S3 to obtain specific marker impurities of consistency evaluation of related substances of different manufacturers and original research drugs. The present invention can quickly and accurately find the method for target impurities in esmolol hydrochloride injection, which can be applied to the study of consistency evaluation of generic drugs.

Description

Technical Field

[0001] The invention relates to a method for detecting impurities in esmolol injection, in particular to a method for detecting impurities in esmolol hydrochloride injection. Background Art

[0002] In recent years, with the rapid development of analytical technology, the research on impurities has transitioned from the previous "simple limit control" to the strategy upgrade of "comprehensive impurity spectrum", but the "bottleneck" problem of impurity research cannot be ignored. 1. Impurity reference material problem: Most impurity reference materials are not available or the purchase price is extremely high. One of the important reasons is that the corresponding impurity reference materials are not available. 2. When using relative retention time to locate special impurities, the durability of the method is poor: In order to solve the problem that impurity reference materials cannot be obtained, some of the current standards use relative retention time to locate special impurities, but studies have shown that the diversity of reagent brands used in mobile phases, the differences in the same type of chromatographic column fillers and the variability of the instruments used can all lead to the inability to reproduce relative retention time. What is more serious is that the target impurities are not effectively controlled due to the wrong positioning of the chromatographic peak, thus affecting the safety of the drug. 3. Impurities and trace impurities without UV absorption: The current "impurity spectrum" research still mostly uses UV detectors. Impurities without UV absorption become "invisible" components in this "impurity spectrum". Many trace impurities cannot be effectively evaluated and controlled due to the sensitivity of the detector. 4. Sharing and application of impurity information: Impurity structures reported in the literature or possible genotoxic impurity structures inferred based on warning structures cannot be effectively located and characterized between different laboratories, which seriously affects the sharing and utilization of resources. 5. "False positive" and "false negative" problems in the consistency evaluation of generic drugs: The consistency of related substances between generic drugs and original drugs is evaluated by comparing the "impurity spectrum". The overlap and wrapping of chromatographic peaks in the "impurity spectrum" diagram, especially when the spectral behavior of the target impurity is consistent with that of the interfering substance, can easily lead to "false positive" and "false negative" conclusions due to misjudgment. Therefore, in response to the above problems, it is urgent to find a suitable method to quickly and conveniently solve the corresponding problems and improve and promote the research on the consistency evaluation of drug-related substances. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for detecting impurities in esmolol hydrochloride injection in view of the shortcomings of the existing technology, so as to realize a method for quickly and accurately finding target impurities in esmolol hydrochloride injection, which can be applied to the consistency evaluation research of generic drugs.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for detecting impurities in esmolol hydrochloride injection, comprising the following steps:

[0005] S1. Dilute different batches of esmolol hydrochloride injection with water to prepare test solutions;

[0006] S2. Perform liquid chromatography-mass spectrometry analysis on the test solution in S1 to obtain the relative peak areas and retention times of all impurities;

[0007] The data obtained in S3 and S2 are used to screen out the impurities that may be present in the test solution through the mass defect technique;

[0008] S4. Analyze the results in S2 by multivariate analysis to distinguish the impurity spectra of different batches of esmolol hydrochloride injection and the original drug, and screen for impurity-specific markers;

[0009] S5. Combine the results of the multivariate analysis method in S4 with the quality loss results in S3 to obtain specific marker impurities for the consistency evaluation of different batches of esmolol hydrochloride injection and related substances of the original drug.

[0010] In a preferred embodiment of the present invention, the mass defect technology in S3 includes screening monomer type impurities and dimer type impurities using esmolol, esmolol acid and esmolol dimer as templates in positive ion mode.

[0011] In a preferred embodiment of the present invention, the mass range of the template of the mass defect technique in S3 is the template molecular weight ±100Da, and the mass defect range is the template molecular weight mass defect value ±100mDa.

[0012] In a preferred embodiment of the present invention, the mass range of the template of the mass defect technology in S3 is ±100Da, and the mass defect range is ±100mDa of the template molecular weight mass defect value. When the mass range and mass defect range are set to ±50Da and ±50mDa, fewer screening compounds are likely to miss homologues, and 150Da leads to more screening compounds but is prone to false positive confusion results.

[0013] In a preferred embodiment of the present invention, the liquid chromatography-mass spectrometry detection and analysis in S2 includes collecting data on test solutions of different batches of esmolol hydrochloride injection by ultra-high performance liquid chromatography tandem with high-resolution mass spectrometry, obtaining a detection multi-peak diagram of the full impurity spectrum, performing peak extraction and correction on the chromatographic peaks, and obtaining the relative peak areas and retention times of all impurities.

[0014] In a preferred embodiment of the present invention, in S2, the liquid chromatography-mass spectrometry detection and analysis adopts ultra-high performance liquid chromatography tandem high-resolution mass spectrometry, which is ultra-high performance liquid chromatography quadrupole electrostatic field orbital trap high-resolution mass spectrometry, and the chromatographic column is C18, 1.8μm, 2.1mm×100mm;

[0015] Chromatographic conditions were as follows: mobile phase was 5 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid)-acetonitrile, gradient elution program was 0-20 min, 10-50% acetonitrile; 20-25 min, 50%-80% acetonitrile; 25-28 min, 80%-10% acetonitrile; 28-35 min, 10%-10% acetonitrile; flow rate was 0.26-0.34 mL / min; column temperature was 27-32°C; injection volume was 1.8-2.2 μL; mass spectrometry mainly included: positive ion mode detection, fragmentation voltage was 4.50 kV, auxiliary gas flow rate was 10 mL / min, ion transfer tube temperature was 320°C, auxiliary gas temperature was 350°C; scanning mode: Full MS / dd-MS 2 , Full MS resolution 70000, dd-MS 2 Resolution 17500; mass scanning range m / z 50~750; in MS / MS mode, the positive ion collision energies are 10, 30, and 50 NCE respectively.

[0016] In a preferred embodiment of the present invention, the multivariate analysis method in S4 includes using multivariate statistics to analyze the results in S2, performing principal component analysis, hierarchical cluster analysis, partial least squares discriminant analysis, and orthogonal partial least squares discriminant analysis in sequence, and finally screening differential markers based on variable weight values ​​and P value analysis in t-test to obtain a distinguishing identification model to evaluate the impurity spectra of different manufacturers and original drugs.

[0017] In a preferred embodiment of the present invention, in S4, variable importance>1 and significance level<0.05 are used as threshold values ​​to obtain impurity difference markers.

[0018] In a preferred embodiment of the present invention, in S5, the main impurity difference markers determined are impurity I and impurity II.

[0019]

[0020] Impurity I

[0021]

[0022] Impurity II

[0023] In the mass defect technology screening, the mass range is template molecular weight ± 100Da, and the mass defect value range is template molecular weight mass defect value ± 100mDa. The above parameter range can be selected to better screen esmolol related homologues. If the window range is too narrow, some homologues are likely to be missed, and if the window range is too large, false positive homologues are likely to appear.

[0024] In a preferred embodiment of the present invention, the concentration of the test solution in S1 is 0.1 mg / mL of the test solution.

[0025] In a preferred embodiment of the present invention, different batches of esmolol hydrochloride injection in S1 are esmolol hydrochloride injection produced by different companies.

[0026] The mass spectrometry technology combined with multivariate analysis proposed in recent years is a scientific and rigorous analysis method, which mainly includes scientific data collection and processing, professional multivariate analysis and efficient identification analysis. It is particularly suitable for complex mixtures as research objects, and can simultaneously determine and quantify potential target substances and study their relationships. The research methods widely used by researchers at present are mainly non-targeted and targeted research. Non-targeted research is to explore the change rules of all substances that can be detected from the overall perspective and further find out the differential substances between samples. Targeted research is mainly a method for quantitative or qualitative analysis of specific target substances. This analysis method first pre-processes the data, first converting the spectral signal obtained by monitoring in the mass spectrometer into data information. The processing process includes normalization, peak matching, noise removal, removal of outliers, peak alignment, peak identification, etc., and then multivariate statistical analysis is performed to determine the similarities and differences between different sample sizes. The processing and analysis methods are mostly cluster analysis, principal component analysis, discriminant function analysis and nonlinear regression. The technology of mass spectrometry combined with multivariate analysis has achieved reliable results in many aspects of research: 1. Applied to in vivo metabolomics, including the study of lipid metabolites, drug metabolites, and food metabolites. This technology compares and analyzes the differences in in vivo metabolites caused by various factors, thereby finding the target differential markers, and then interprets the pathways or influencing factors of disease generation through the analysis of marker metabolic pathways; 2. Applied to the study of the adaptability of plants or agricultural products to the growth environment, including the analysis of differential components contained in target samples under various stress tests, cluster analysis of samples from different production sites, and analysis of differential components in different parts of the same sample. 3. Applied to the quality evaluation of traditional Chinese medicine and preparations, including the similarity evaluation of samples of different specifications and batches, and the screening of illegal additives.

[0027] Mass defect filter (MDF) is a filtering technology based on high-resolution mass spectrometry data. It processes and identifies the collected high-resolution mass spectrometry data according to the similar mass defect rules between compounds and analogs, and discovers and identifies components from a complex background through one or a limited number of injections. According to the rules of similar mass defect values, components can be efficiently identified in clusters. At present, mass defect filtering is commonly used in mass spectrometry screening to identify in vivo metabolites of the same type of compounds or monomeric compounds in traditional Chinese medicines, but it is less used in drug impurity screening analysis. The application of mass defect filtering to screen impurities in preparations must also overcome the interference of excipient components.

[0028] In this study, UPLC-QE-MS was used to comprehensively collect high-precision molecular weights of compounds in esmolol hydrochloride injection. The three compounds with confirmed structures were used as the parent structure, ±100Da was used as the mass window, and ±100mDa was used as the mass loss value window. The possibility of impurities generated by the parent compound through oxidation, reduction, hydrolysis, esterification, etc., which may be introduced or removed from the drug molecular structure, is screened. This method is particularly helpful in locating organic impurities with chemical structures similar to or related to the active ingredients, which are commonly referred to as "related substances."

[0029] The following are consistent with the law of quality loss: 1. Oxidation reaction 2. Reduction reaction 3. Hydrolysis reaction 4. Esterification reaction and other chemical reactions that may occur during the preparation and storage of pharmaceutical preparations and affect stability; if the compound is not structurally similar to the parent compound, it can be excluded, for example, if it is introduced into the excipients.

[0030] Difficulty: 1. Correctly and comprehensively select the parent compound; 2. Determine the appropriate mass range and mass loss range. The effect achieved is to quickly and accurately find "related substances".

[0031] Compared with the prior art, the present invention has the following beneficial effects: the present invention innovatively uses liquid chromatography-high-resolution mass spectrometry to collect the impurity spectrum of esmolol hydrochloride injection from different manufacturers, uses multivariate analysis to non-targetedly evaluate the impurity differences of esmolol hydrochloride injection from different manufacturers, and then combines the mass loss filtering technology, takes esmolol, esmolol acid and esmolol dimer as parent compounds, and conducts targeted analysis to screen the parent compounds for related impurities that may be generated by introducing or removing corresponding groups in the drug molecular structure through oxidation, reduction, hydrolysis, esterification, etc. Thus, a method for quickly and accurately finding target impurities in preparations can be realized, which can be applied to the consistency evaluation research of generic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the total ion current chromatogram of the esmolol hydrochloride injection sample UPLC-Q-Exacitive / MS in positive ion mode in Example 1 of the present invention;

[0033] Figure 2 It is the total ion current chromatogram of the esmolol hydrochloride injection sample UPLC-Q-Exacitive / MS in negative ion mode in Example 1 of the present invention;

[0034] Figure 3 The mass range and mass loss range in the mass loss technology in Example 2 of the present invention;

[0035] Figure 4 Spectra of monomer impurities EP1-EP3 and EP14-18 screened out in Example 2 of the present invention;

[0036] Figure 5 Spectra of monomer impurities EP4-5, EP7-8, and EP11-13 screened out in Example 2 of the present invention;

[0037] Figure 6 The result of multivariate analysis in Example 3 of the present invention Figure 1 ;

[0038] Figure 7 The result of multivariate analysis in Example 3 of the present invention Figure 2 ;

[0039] Among them, A: manufacturer 1; B: manufacturer 2; C: manufacturer 3; D: manufacturer 4; E: original drug; F: raw material drug 1; G: raw material drug 2;

[0040] Figure 8 is a schematic diagram of the method of the present invention;

[0041] Fig. 9 This is a schematic diagram of using the secondary mass spectrometry fragmentation pathway to deduce and verify the structure of EP1 in Example 3 of the present invention;

[0042] Fig.10 This is a schematic diagram of using the secondary mass spectrometry fragmentation pathway to deduce and verify the structure of EP2 in Example 3 of the present invention;

[0043] Fig.11 This is a schematic diagram of using the secondary mass spectrometry fragmentation pathway to deduce and verify the structure of EP3 in Example 3 of the present invention;

[0044] Fig.12 This is a schematic diagram of the derivation and verification of the structure of EP18 using the secondary mass spectrometry fragmentation pathway in Example 3 of the present invention;

[0045] Fig.13 This is a schematic diagram of the use of the secondary mass spectrometry fragmentation pathway to deduce and verify the structure of EP10 in Example 3 of the present invention;

[0046] Fig.14 This is a schematic diagram of the derivation and verification of the EP4 structure using the secondary mass spectrometry fragmentation pathway in Example 3 of the present invention;

[0047] Fig.15 This is a schematic diagram of the derivation and verification of the EP7 structure using the secondary mass spectrometry fragmentation pathway in Example 3 of the present invention;

[0048] Fig.16 This is a schematic diagram of the derivation and verification of the EP5 structure using the secondary mass spectrometry fragmentation pathway in Example 3 of the present invention;

[0049] Fig.17 This is a schematic diagram of the use of the secondary mass spectrometry fragmentation pathway to deduce and verify the structure of EP12 in Example 3 of the present invention;

[0050] Fig.18 Chromatograms of system suitability, blank excipient solution, and test solution;

[0051] Among them: 1. Esmolol; 2. Impurity I; 3. Impurity II; A. Blank solvent; B. Blank excipient solution; C. System suitability solution; D. Reference substance solution; E~H. Test sample solutions of related substances from different manufacturers. DETAILED DESCRIPTION

[0052] Example 1

[0053] Liquid chromatography-mass spectrometry technology method: Ultra-high performance liquid chromatography tandem high-resolution mass spectrometry is ultra-high performance liquid chromatography quadrupole electrostatic field orbital trap high-resolution mass spectrometry, the chromatographic column is C18, 1.8μm, 2.1mm×100mm;

[0054] Chromatographic conditions were as follows: the mobile phase in positive ion mode was 5 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid)-acetonitrile, the mobile phase in negative ion mode was 5 mmol / L ammonium formate aqueous solution-acetonitrile, the gradient elution program was 0-20 min, 10-50% acetonitrile; 20-25 min, 50%-80% acetonitrile; 25-28 min, 80%-10% acetonitrile; 28-35 min, 10%-10% acetonitrile; flow rate was 0.26-0.34 mL / min; column temperature was 27-32 °C; injection volume was 1.8-2.2 μL; mass spectrometry mainly included: HESI ion source, positive and negative ion monitoring modes, fragmentation voltage 4.50 kV (positive ion), 3.00 kV (negative ion), auxiliary gas flow rate 10 mL / min, ion transfer tube temperature 320 °C, auxiliary gas temperature 350 °C; scanning mode: Full MS / dd-MS 2 , Full MS resolution 70000, dd-MS 2 Resolution 17500; mass scanning range m / z 50~750. Figure 1-2 As shown in the figure, the positive ion collision energies of the MS / MS mode are 10, 30, and 50 NCE, and the negative ion collision energies are 30, 50, and 70 NCE, respectively. By comparison, esmolol and esmolol impurities have higher sensitivity in the positive ion mode, and the number of impurities detected is significantly more than that in the negative ion mode, so the positive ion mode is selected.

[0055] Example 2

[0056] Mass loss technology: This study applied mass loss filtering technology to the screening of impurities in esmolol hydrochloride injection for the first time. S1. Dilute the esmolol hydrochloride injection with water to an esmolol hydrochloride concentration of 0.1 mg / mL to prepare a test solution; S2. Detection and analysis of esmolol hydrochloride injection: Collect data on the test solution using ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry to obtain a multi-peak detection diagram of the full impurity spectrum, extract and correct the chromatographic peaks, and obtain the relative peak areas and retention times of all impurities; S3. In the positive ion mode, use esmolol, esmolol acid and esmolol dimer as templates to screen monomer-type impurities and dimer-type impurities. The mass range of each MDF template is 100Da, the mass loss range is 100mDa, and the error is 5ppm( Figure 3 ). The molecular weight of esmolol is 295.1778097Da, the mass loss value is 177.8097mDa, the molecular weight screening window range is 195.1778097-395.1778097Da, and the mass loss value screening is 77.8097-277.8097 mDa. The molecular weight of esmolol acid is 281.1626998Da, the mass loss value is 162.6998mDa, the molecular weight screening window range is 181.1626998-381.1626998Da, and the mass loss value screening is 62.6998-262.6998 mDa. Using esmolol and esmolol acid as templates, monomer impurities EP1-EP3 and EP14-18 ( Figure 4 ). The molecular weight of esmolol dimer is 558.330504Da, the mass loss value is 330.504mDa, the molecular weight screening window range is 458.330504-658.330504Da, the mass loss value screening is 230.504-430.504mDa, and the dimer impurities EP4-5, EP7-8, and EP11-13 are screened out ( Figure 5 ).

[0057] The screening templates of this MDF are mainly divided into three categories: (1) Screening of mass and structure of template compounds that undergo oxidation, reduction, dealkylation and other reactions, so that the template compounds add or subtract one or several C / H / O / N, etc.; (2) Screening of small molecule compounds produced by the splitting of template compounds, such as hydrolysis reactions; (3) Screening of compounds produced by the addition of possible excipients to template compounds. The screening results are then analyzed by elemental analysis and mass spectrometry fragmentation rules to eliminate false positive compounds. Drug impurities are usually divided into organic impurities, inorganic impurities, and residual solvents. Among them, organic impurities with chemical structures similar to or related to active ingredients are usually called related substances. Therefore, using mass defect technology to study related substances is a fast and effective method. The key parameters of this technology are (1) the selection of representative parent structures. The present invention selects esmolol, esmolol acid and esmolol dimer with confirmed structures as templates to screen monomeric impurities and dimer impurities; (2) the selection of mass range and mass loss range. The present invention sets the mass range to 50Da, 100Da, and 150Da in sequence, and the mass loss range to 50mDa, 100mDa, and 150mDa, respectively. The screening results are examined, and the number of compounds is comprehensively considered with the elimination of false positive compounds through elemental analysis and mass spectrometry fragmentation rules. It is found that the mass loss range is set to 100Da, and the mass loss range is 100mDa when it is optimal.

[0058] Example 3

[0059] Chemometric analysis: Figure 8 As shown, this study applied chemometric method to screen impurities of esmolol hydrochloride injection for the first time. S1. Esmolol hydrochloride injection of different manufacturers was diluted with water to a concentration of 0.1 mg / mL of esmolol hydrochloride to prepare the test solution;

[0060] S2. Detection and analysis of esmolol hydrochloride injection from different manufacturers: Data of the test solutions of esmolol hydrochloride injection from different manufacturers were collected by ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry to obtain a multi-peak diagram of the full impurity spectrum, extract and correct the chromatographic peaks, and obtain the relative peak areas and retention times of all impurities;

[0061] In S2, the ultra-high performance liquid chromatography tandem high-resolution mass spectrometry is an ultra-high performance liquid chromatography quadrupole electrostatic field orbital trap high-resolution mass spectrometry, and the chromatographic column is C18, 1.8 μm, 2.1 mm×100 mm;

[0062] Chromatographic conditions were as follows: mobile phase was 5 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid)-acetonitrile, gradient elution program was 0-20 min, 10-50% acetonitrile; 20-25 min, 50%-80% acetonitrile; 25-28 min, 80%-10% acetonitrile; 28-35 min, 10%-10% acetonitrile; flow rate was 0.26-0.34 mL / min; column temperature was 27-32°C; injection volume was 1.8-2.2 μL; mass spectrometry mainly included: positive ion mode detection, fragmentation voltage was 4.50 kV, auxiliary gas flow rate was 10 mL / min, ion transfer tube temperature was 320°C, auxiliary gas temperature was 350°C; scanning mode: Full MS / dd-MS 2 , Full MS resolution 70000, dd-MS 2 Resolution 17500; mass scanning range m / z 50~750. The positive ion collision energies in MS / MS mode are 10, 30, and 50 NCE, respectively.

[0063] S3, in positive ion mode, esmolol, esmolol acid and esmolol dimer were used as templates to screen monomeric and dimer impurities. The mass range of each MDF template was 100Da, the mass loss range was 100mDa, and the error was 5ppm ( Figure 3 ). The molecular weight of esmolol is 295.1778097Da, the mass loss value is 177.8097mDa, the molecular weight of esmolol acid is 281.1626998Da, the mass loss value is 162.6998mDa, and the monomer impurities EP1-EP3 and EP14-18 are screened out ( Figure 4 ). The molecular weight of esmolol dimer is 558.330504Da, and the mass defect value is 330.504mDa. Dimer impurities EP4-5, EP7-8, and EP11-13 were screened out ( Figure 5 ).

[0064] S4. Multivariate analysis method is used to distinguish the differences between the impurity spectra of different manufacturers and original research drugs, and to screen impurity-specific markers: multivariate statistical method is used for analysis, and principal component analysis, hierarchical cluster analysis, partial least squares discriminant analysis, orthogonal partial least squares discriminant analysis are performed in sequence. Finally, the difference markers are screened according to the variable weight value and the P value analysis in the t-test, and the distinguishing identification model is obtained to evaluate the impurity spectra of different manufacturers and original research drugs;

[0065] like Figure 6-7 As shown in S4, variable importance>1 and significance level<0.05 were used as threshold values ​​to obtain impurity difference markers.

[0066] S5. Combine the results of multivariate analysis with the quality loss results to obtain specific markers for consistency evaluation of related substances between different manufacturers and original research drugs. Take manufacturer 1 as an example, see Table 1.

[0067] Table 1

[0068]

[0069] Combining quality loss and multivariate analysis, the potential differences in impurities between different manufacturers and original research drugs are shown in Table 1

[0070] Verification of results: Figure 9-17 As shown, the preliminary verification of the impurity-specific marker structure uses the secondary mass spectrometry fragmentation pathway of the analytical reference substance to deduce and verify the structure of each impurity.

[0071] In S5, the main impurity difference markers determined are impurity I and impurity II:

[0072]

[0073] Impurity I

[0074]

[0075] Impurity II

[0076] Determination of methods for related substances: S4. Based on the determination of the contents of the differential markers determined in S3 by HPLC-UV, the quality standards for related substances of esmolol hydrochloride injection are established.

[0077] The application of impurity difference markers impurity Ⅰ and impurity Ⅱ as special impurities for the control of related substances in esmolol hydrochloride injection was established by high performance liquid chromatography determination. Octadecylsilane bonded silica gel was used as filler (CAPCELL PAKC18 MG Ⅲ column, 4.6mm×250mm, 5μm or chromatographic column with equivalent performance). Acetonitrile-methanol-phosphate buffer (4.6g of potassium dihydrogen phosphate was taken and dissolved in 1000mL of water, and the pH was adjusted to 4.0 with dilute phosphoric acid) (12:20:68) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was as follows: 0~15min, 100%A; 15~30min, 100%A→65%A; 30~50min, 65%A; 50~51min, 65%A→100%A; 51~60min, 100%A. The flow rate was 1.0 mL·min-1, the column temperature was 30℃, and the injection volume was 20 μL.

Claims

1. A method for detecting impurities in esmolol hydrochloride injection, characterized in that The following steps are involved: S1. Dilute different batches of esmolol hydrochloride injection with water to prepare test solutions; S2. Perform liquid chromatography-mass spectrometry analysis on the test solution in S1 to obtain the relative peak areas and retention times of all impurities; The data obtained in S3 and S2 are used to screen out the impurities that may be present in the test solution through the mass defect technique; S4. Analyze the results in S2 by multivariate analysis to distinguish the impurity spectra of different batches of esmolol hydrochloride injection and the original drug, and screen for impurity-specific markers; S5. Combine the results of the multivariate analysis in S4 with the quality loss results in S3 to obtain specific marker impurities for consistency evaluation of different batches of esmolol hydrochloride injection and related substances of the original drug; The liquid chromatography-mass spectrometry detection and analysis in S2 adopts ultra-high performance liquid chromatography tandem high-resolution mass spectrometry, wherein the ultra-high performance liquid chromatography tandem high-resolution mass spectrometry is an ultra-high performance liquid chromatography quadrupole electrostatic field orbital trap high-resolution mass spectrometry, and the chromatographic column is C18, 1.8 μm, 2.1 mm×100 mm; Chromatographic conditions are as follows: the mobile phase is 5mmol / L ammonium formate aqueous solution-acetonitrile, the ammonium formate aqueous solution contains 0.1% formic acid, the gradient elution program is 0-20min, 10-50% acetonitrile; 20-25min, 50%-80% acetonitrile; 25-28min, 80%-10% acetonitrile; 28-35min, 10%-10% acetonitrile; flow rate is 0.26-0.34mL / min; column temperature is 27-32°C; injection volume is 1.8-2.2μL; mass spectrometry includes: positive ion mode detection, fragmentation voltage is 4.50kV, auxiliary gas flow rate is 10mL / min, ion transfer tube temperature is 320°C, auxiliary gas temperature is 350°C; scanning mode: Full MS / dd-MS 2 , Full MS resolution 70000, dd-MS 2 Resolution 17500; mass scanning range m / z 50~750; positive ion collision energies in MS / MS mode are 10, 30, and 50 NCE respectively; In S5, the impurity difference markers measured are impurity I and impurity II Impurity I Impurity II.

2. The method for detecting impurities in esmolol hydrochloride injection according to claim 1, characterized in that: The mass defect technology in S3 includes screening monomer-type impurities and dimer-type impurities using esmolol, esmolol acid and esmolol dimer as templates in positive ion mode.

3. The method for detecting impurities in esmolol hydrochloride injection according to claim 2, characterized in that: The mass range of the template for the mass defect technique in S3 is 90-110 Da, and the mass defect range is 90-110 mDa.

4. The method for detecting impurities in esmolol hydrochloride injection according to claim 2, characterized in that: The mass range of the template for the mass defect technique in S3 was 95-105 Da, and the mass defect range was 95-100 mDa.

5. The method for detecting impurities in esmolol hydrochloride injection according to any one of claims 1 to 4, characterized in that: The liquid chromatography-mass spectrometry detection and analysis in S2 includes collecting data on the test solutions of different batches of esmolol hydrochloride injection through ultra-high performance liquid chromatography tandem with high-resolution mass spectrometry, obtaining a multi-peak detection diagram of the entire impurity spectrum, performing peak extraction and correction on the chromatographic peaks, and obtaining the relative peak areas and retention times of all impurities.

6. The method for detecting impurities in esmolol hydrochloride injection according to any one of claims 1 to 4, characterized in that: The multivariate analysis method in S4 includes using multivariate statistical methods to analyze the results in S2, and then performing principal component analysis, hierarchical cluster analysis, partial least squares discriminant analysis, and orthogonal partial least squares discriminant analysis in sequence. Finally, the difference markers are screened according to the variable weight value and the P value analysis in the t-test, and a discrimination model is obtained to evaluate the impurity spectra of different batches of esmolol hydrochloride injection and the original drug.

7. The method for detecting impurities in esmolol hydrochloride injection according to claim 6, characterized in that: In S4, variable importance > 1 and significance level < 0.05 were used as threshold values ​​to obtain impurity difference markers.

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