Methods for detecting impurities E and F in lincomycin hydrochloride injection

Abstract

This invention discloses a method for detecting impurities E and F in lincomycin hydrochloride injection, using liquid chromatography-mass spectrometry (LC-MS / MS). The method includes the following steps: preparing a reference solution, preparing a test solution, and LC-MS / MS detection. The LC-MS / MS detection involves injecting the reference solution and the test solution into the LC-MS / MS instrument for separation and detection. The LC parameters are as follows: separation is performed using a gradient elution method, with mobile phase A at 65%–75% and mobile phase B at 25%–35% during the 0–2 min period. This method is simple to operate, fast in detection, and has the advantages of high sensitivity, high precision, and high repeatability.

Description

Technical Field

[0001] This invention relates to the technical field of biomedical testing, specifically to a method for detecting the content of impurities E and F in lincomycin hydrochloride injection. Background Technology

[0002] Lincomycin hydrochloride is a narrow-spectrum antibiotic with good antibacterial activity against most Gram-positive bacteria and various anaerobic bacteria, but it is resistant to enterococci and aerobic Gram-negative bacteria. It is mainly used to treat diseases caused by Gram-positive cocci, especially anaerobic bacteria, Staphylococcus aureus, and pneumococcal infections, where it has shown good therapeutic effects.

[0003] Impurities E ((2S,4R)-1-methyl-4-propylpyrrolidine-2-carboxylic acid) and F (methyl-6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galactopyranoside) are impurities generated during drug production or storage. Long-term intake of low-dose toxic impurities may lead to organ damage. These impurities may interact with the main component, interfering with drug absorption or metabolism. For example, the pyrrolidine carboxylic acid derivative of impurity E may have neuromodulatory activity (such as GABA receptor activity). When present as an impurity, if it exceeds the ICH Q3A / B limit (usually ≤0.1%), it may cause neurotoxicity such as dizziness and drowsiness. Genotoxicity assessment (such as the AMES test) is required. The carboxylic acid group of the pyrrolidine derivative may also competitively bind to metal ion-dependent enzymes (such as ACE and MMPs), reducing the efficacy of the main drug. The thio structure of impurity F may interfere with mitochondrial function (similar to some antibiotic impurities), and its ototoxic and nephrotoxic potential, especially the cumulative effects with long-term use, needs to be monitored. The anti-enzymatic properties of thioglycosides may inhibit hepatic drug-metabolizing enzymes (such as CYP450), altering the drug's metabolic kinetics and leading to abnormal blood drug concentrations. This can result in irreversible consequences; for example, the pyrrolidine impurity in pioglitazone has caused a hepatotoxicity recall due to an uncontrolled stereoisomer. Therefore, these two impurities must be strictly controlled as potential genotoxic substances (positive warning structures). It is recommended to conduct impurity profile analysis and toxicological threshold (TTC) assessment. If the levels cannot be reduced to ≤0.1%, bacterial reverse mutation assay (AMES) and micronucleus assay are necessary. Therefore, strict control of the content of these two impurities is required.

[0004] To strictly control the content of impurities E and F in lincomycin hydrochloride, ensure the quality of lincomycin hydrochloride, and ensure the safety of patients taking the medication, developing a detection method for lower concentrations of impurities E and F in lincomycin hydrochloride is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0006] This invention provides a method for detecting impurities E and F in lincomycin hydrochloride injection, which uses liquid chromatography-mass spectrometry (LC-MS / MS) for detection and includes the following steps: preparing a reference solution, preparing a test solution, and detection by liquid chromatography-mass spectrometry (LC-MS / MS).

[0007] Detection by liquid chromatography-mass spectrometry (LC-MS / MS): The reference solution and the test solution are injected into the liquid chromatograph-mass spectrometer for separation and detection;

[0008] The parameters for the liquid chromatography are as follows: separation is performed using a mobile phase gradient elution method. The mobile phase gradient elution process is as follows: from 0 min to 2 min, mobile phase A is 65% to 75%, and mobile phase B is 25% to 35%; from 2 min to 6 min, mobile phase A decreases uniformly to 2.5% to 7.5%, and mobile phase B increases uniformly to 92.5% to 97.5%; from 6 min to 8 min, mobile phase A is 2.5% to 7.5%, and mobile phase B is 92.5% to 97.5%.

[0009] From 8.0 to 8.1 min, mobile phase A increases uniformly to 65%–75%, while mobile phase B decreases uniformly to 25%–35%; from 8.1 to 10 min, mobile phase A is 65%–75%, and mobile phase B is 25%–35%; both mobile phase A and mobile phase B are expressed as volume percentages.

[0010] The mobile phase A is a formic acid-water solution, wherein the volume ratio of formic acid to water in the formic acid-water solution is 0.05% to 0.15%; the mobile phase B is acetonitrile.

[0011] The chromatographic column was an InfinityLab Poroshell 120SB-AQ column, 4.6 × 100 mm, 2.7 μm;

[0012] The flow rate was 0.36–0.44 ml / min;

[0013] The parameters for the mass spectrometry are as follows:

[0014] Mass spectrometry conditions

[0015]

[0016] The present invention employs a liquid chromatography-mass spectrometry (LC-MS / MS) method for detecting impurities E and F in lincomycin hydrochloride injection. This method uses liquid chromatography as the separation system for impurities E and F in lincomycin hydrochloride injection, and mass spectrometry as the detection system for impurities E and F. LC-MS / MS combines the high separation efficiency of liquid chromatography with the high sensitivity and strong qualitative ability of mass spectrometry, offering numerous significant advantages in detecting impurity content.

[0017] In liquid chromatography-mass spectrometry (LC-MS), the liquid chromatography component can effectively separate target impurities from matrix components, while the mass spectrometry component can further improve the selectivity for target impurities by selecting specific ions for detection.

[0018] Liquid chromatography (LC) offers strong separation of samples. By selecting appropriate columns, mobile phases, and elution methods, and analyzing the mass-to-charge ratio of impurity ions, fragment ion information, and comparison with known compound databases, the chemical structure of impurities can be determined. This is crucial for investigating the sources of impurities and assessing their impact on product quality and safety. LC-MS / MS can employ various quantitative methods, such as internal and external standard methods, for accurate quantitative analysis of impurities. By selecting appropriate calibration curves and quantitative ions, the content of impurities in samples can be reliably determined, meeting the precision requirements for impurity content detection in various fields. In pharmaceutical quality control, it enables accurate determination of impurity content, ensuring the safety and efficacy of pharmaceuticals.

[0019] This invention provides a method for detecting impurities E and F in lincomycin hydrochloride injection by screening and optimizing the relevant test conditions of liquid chromatography and mass spectrometry. This method has the advantages of low detection limit, high precision, high repeatability and high accuracy.

[0020] Preferably, the parameters for the liquid chromatography are as follows: separation is performed using a mobile phase gradient elution method, wherein the mobile phase gradient elution process is as follows: from 0 min to 2 min, mobile phase A is 70% and mobile phase B is 30%; from 2 min to 6 min, mobile phase A is reduced to 5% at a constant rate and mobile phase B is increased to 95% at a constant rate; from 6 min to 8 min, mobile phase A is 5% and mobile phase B is 95%.

[0021] From 8.0 to 8.1 min, mobile phase A increases to 70% at a constant rate, while mobile phase B decreases to 30% at a constant rate; from 8.1 to 10 min, mobile phase A is 70% and mobile phase B is 30%.

[0022] The flow rate is 0.4 ml / min.

[0023] Preferably, in the formic acid-water solution, the volume ratio of formic acid to water is 0.1%.

[0024] Preferably, in the liquid chromatography detection, the column temperature is 27–33°C.

[0025] Preferably, the injection volume is 1.5 to 2.5 μl.

[0026] Preferably, the detection limit concentration of impurity E is 0.37 ng / ml, and the detection limit concentration of impurity F is 0.38 ng / ml.

[0027] Preferably, the limit of quantitation (LOQ) concentration of impurity E is 0.75 ng / ml, and the limit of quantitation (LOQ) concentration of impurity F is 0.75 ng / ml.

[0028] Preferably, the scanning parameters of the mass spectrometer are as follows:

[0029] MRM scan parameters

[0030]

[0031] Preferably, the preparation method of the test solution includes: taking 0.1 ml of the lincomycin hydrochloride injection, placing it in a 20 ml volumetric flask, diluting it to the mark with blank solution, and shaking well to obtain the first solution; measuring 0.05 ml of the first solution, placing it in a 20 ml volumetric flask, diluting it to the mark with blank solution, and shaking well to obtain the test solution.

[0032] Preferably, the method for preparing the reference solution includes:

[0033] S1: Take 10.68 mg of impurity E, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with methanol solution, shake well to obtain impurity E stock solution I; take 10.11 mg of impurity F, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with ultrapure water, shake well to obtain impurity F stock solution I.

[0034] S2: Take appropriate amounts of the impurity E stock solution I and the impurity F stock solution I, place them in the same 20ml volumetric flask, dilute to the mark with blank solution, shake well, and the mixed reference stock solution I is obtained.

[0035] S3: Measure 0.50 ml of the mixed reference standard stock solution I, place it in a 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the standard curve stock solution;

[0036] S4: Measure 0.05 ml of the standard curve stock solution, place it in a 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the solution.

[0037] Preferably, the blank solution is methanol.

[0038] In summary, the technical solution of the present invention has the following effects:

[0039] According to the detection method of the present invention, an LC-MS / MS method was established to detect the contents of impurities E and F in lincomycin hydrochloride injection, and the method was validated. The limits for both impurities E and F in lincomycin hydrochloride injection are 1%, meaning the sensitivity of the detection method of the present invention should be less than 1%.

[0040] This invention provides a method for detecting impurities E and F in lincomycin hydrochloride injection by screening and optimizing the relevant test conditions of liquid chromatography and mass spectrometry. This method has the advantages of low detection limit, high precision, high repeatability and high accuracy.

[0041] Under the final selected conditions, the system suitability, specificity, linearity and range, limit of detection, limit of quantitation, accuracy, precision, stability and robustness of the method were validated, confirming that the method is suitable for determining the content of impurities E and F in lincomycin hydrochloride injection.

[0042] This invention provides a fast and simple detection method for detecting impurities E and F in lincomycin hydrochloride injection using liquid chromatography-mass spectrometry. Attached Figure Description

[0043] Figure 1 The results of specificity determination of the blank solution in the examples;

[0044] Figure 2 The results of specificity determination of the test solution in the examples are shown.

[0045] Figure 3 The results of specificity determination of the impurity E localization solution in the examples;

[0046] Figure 4 The results of specificity determination of the impurity F localization solution in the examples;

[0047] Figure 5 The results show the specificity determination of the 100% limit concentration reference solution in the examples;

[0048] Figure 6 The results show the specificity determination of the 100% limit concentration spiked test solution in the examples;

[0049] Figure 7 This is a line graph of impurity E in the examples;

[0050] Figure 8 This is a linear graph of impurity F in the examples. Detailed Implementation

[0051] The present invention will be further described in detail below with reference to embodiments, comparative examples and performance testing tests. These embodiments should not be construed as limiting the scope of protection claimed by the present invention.

[0052] According to the limits of the present invention, the limits for impurities E and F in lincomycin hydrochloride are both 1%. Based on the properties of the injection solution and the target analyte, this study established an LC-MS / MS method to detect the content of impurities E and F in lincomycin hydrochloride injection, and validated the method.

[0053] Under the final selected conditions, the system suitability, specificity, linearity and range, limit of detection, limit of quantitation, accuracy, precision, stability and robustness of the method were validated, confirming that the method is suitable for determining the content of impurities E and F in lincomycin hydrochloride injection.

[0054] The main instruments, reagent samples, reagents and reference standards of this invention are shown in Tables 1-4.

[0055] Table 1. Instrument Information Sheet

[0056]

[0057] Note: "NA" indicates not applicable.

[0058] Table 2. Sample Information Table

[0059] name batch number Lincomycin Hydrochloride Injection 220714901

[0060] Table 3. Reagent Information Table

[0061] name Specification level batch number source methanol 4L / bottle HPLC 52901440 Shanghai Anpu Cuishi Formic acid 50ml / bottle HPLC 5953570 Dima Technology Acetonitrile 4L / bottle HPLC 63101440 Shanghai Anpu Cuishi

[0062] Table 4. Reference Standard Information Table

[0063] Compound Name source batch number content(%) Impurity E CATO 0419-RD-0036 98.5 impurity F CATO 0418-RD-0069 95.2

[0064] Example

[0065] Example 1

[0066] Example 1 provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0067] The method for detecting impurities E and F in lincomycin hydrochloride injection in Example 1 specifically includes the following steps:

[0068] 1. Liquid Chromatography Conditions

[0069] Chromatographic column: InfinityLab Poroshell 120SB-AQ column (4.6×100mm, 2.7μm);

[0070] Column number: GBP-L-22-04-001;

[0071] Mobile phase A: 0.1% formic acid-water solution;

[0072] Mobile phase B: Acetonitrile;

[0073] Column temperature: 30℃;

[0074] Injection volume: 2 μl.

[0075] The gradient elution conditions are as follows:

[0076] Table 5. Gradient Elution Table

[0077] T(min) Flow rate (ml / min) A(％) B(％) 0.0 0.40 70 30 2.0 0.40 70 30 6.0 0.40 5 95 8.0 0.40 5 95 8.1 0.40 70 30 10.0 0.40 70 30

[0078] 2. The parameters of the mass spectrometry ion source are as follows:

[0079] Table 6. Mass Spectrometry Conditions

[0080]

[0081] 3. Impurity scanning parameters

[0082] Table 7. MRM Scan Parameters

[0083]

[0084]

[0085] The structure of impurity E is as follows: C9H 17 NO2171.24, chemical name:

[0086] (2S,4R)-1-methyl-4-propylpyrrolidine-2-carboxylic acid. Impurity F has the following structure:

[0087] Its chemical name is: methyl 6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galactopyranoside.

[0088] 4. Solution preparation and acceptance criteria

[0089] 4.1 Solution Preparation

[0090] 4.1.1 Blank solution

[0091] Methanol was used as a blank solution.

[0092] 1% Formic Acid-Methanol Solution: Measure 20 ml of formic acid, place it in a 2000 ml volumetric flask, dilute with methanol solution to the mark, and shake well.

[0093] 4.1.2 Test solution

[0094] Accurately measure approximately 0.1 ml of lincomycin hydrochloride injection into a 20 ml volumetric flask, dilute to the mark with blank solution, and mix well. Then accurately measure 0.05 ml into the same flask, dilute to the mark with blank solution, and mix well.

[0095] 4.1.3 Reference Standard Stock Solution

[0096] 4.1.3.1 Reference Standard Stock Solution I

[0097] Take an appropriate amount of impurity E, accurately weigh it, place it in a 5ml volumetric flask, dissolve it in methanol solution and dilute it to the mark, shake well, and you will get impurity E stock solution I; take an appropriate amount of impurity F, accurately weigh it, place it in a 5ml volumetric flask, dissolve it in ultrapure water and dilute it to the mark, shake well, and you will get impurity F stock solution I, see Table 8 for details.

[0098] Table 8. Reference Standard Stock Solution I

[0099]

[0100] 4.1.3.2 Mixed reference standard stock solution I

[0101] Accurately measure appropriate amounts of impurity E stock solution I and impurity F stock solution I, place them in the same 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain mixed reference stock solution I, as detailed in Table 9.

[0102] Table 9. Mixed Reference Standard Stock Solution I

[0103]

[0104]

[0105] 4.1.3.3 Positioning Solution

[0106] Accurately measure appropriate amounts of impurity E stock solution I and impurity F stock solution I, place them in different 10 ml volumetric flasks, dilute to the mark with blank solution, and shake well to obtain the reference stock solutions II, as detailed in Table 10.

[0107] Table 10. Reference Standard Stock Solution II

[0108] Source solution name Measure the volume (ml) Dilution volume (ml) Concentration (ng / ml) Preparation solution name Impurity E stock solution I 0.04 10 8415.84 Impurity E Stock Solution II Impurity F stock solution I 0.04 10 7699.776 Impurity F stock solution II

[0109] Accurately measure appropriate amounts of impurity E stock solution II and impurity F stock solution II, place them in different 20ml volumetric flasks, dilute to the mark with blank solution, and shake well to obtain the respective positioning solutions, as detailed in Table 11.

[0110] Table 11. Positioning Solutions

[0111] Source solution name Measure the volume (ml) Dilution volume (ml) Concentration (ng / ml) Preparation solution name Impurity E Stock Solution II 0.09 20 37.87 Impurity E positioning solution Impurity F stock solution II 0.10 20 38.50 Impurity F positioning solution

[0112] 4.1.4 Detection Limit Solution

[0113] Accurately measure 0.01 ml of mixed reference standard stock solution I into a 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the detection limit stock solution. See Table 12 for details.

[0114] Table 12. Detection Limit Stock Solution

[0115]

[0116] Accurately measure 0.05 ml of the detection limit stock solution into a 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the detection limit solution. See Table 13 for details.

[0117] Table 13. Detection Limit Solutions

[0118]

[0119] 4.1.5 Standard Curve Solution

[0120] Accurately measure an appropriate amount of mixed reference standard stock solution I and place it in different volumetric flasks. Dilute to the mark with blank solution and shake well to obtain a series of standard curve stock solutions with different concentrations, as detailed in Table 14.

[0121] Table 14. Standard Curve Stock Solution

[0122]

[0123]

[0124] Accurately measure 0.05 ml of the standard curve stock solution into different 20 ml volumetric flasks, dilute to the mark with blank solution, and shake well to obtain a series of standard curve solutions with different concentrations, as detailed in Table 15.

[0125] Table 15. Standard Curve Solutions

[0126]

[0127] 4.1.6 System Suitability Solution

[0128] Take the "100% limit concentration reference solution (STD5 solution)" under "4.1.5" and inject it 6 times consecutively for analysis.

[0129] 4.1.7 Detection Limit and Quantification Limit Solutions

[0130] Detection limit solution: Take the "LOD solution" under section "4.1.4" and inject it three times consecutively for analysis;

[0131] Limit of Quantitation (LOQ) solution: Inject the "LOQ solution" under section "4.1.5" six times consecutively for analysis.

[0132] 4.1.8 Accuracy Solution

[0133] Test solution: Prepare two parallel portions according to the same method as described in section “4.1.2”;

[0134] 50% limit concentration spiked test solution: Accurately measure about 0.1 ml of lincomycin hydrochloride injection into a 20 ml volumetric flask, accurately add 0.5 ml of mixed reference stock solution I under "4.1.3", dilute to the mark with blank solution, shake well, then accurately measure 0.05 ml into a 20 ml volumetric flask, dilute to the mark with blank solution, shake well, and the solution is ready. Prepare 3 portions in the same way.

[0135] 100% limit concentration spiked test solution: Accurately measure about 0.1 ml of lincomycin hydrochloride injection into a 20 ml volumetric flask, accurately add 1.0 ml of mixed reference stock solution I under "4.1.3", dilute to the mark with blank solution, shake well, then accurately measure 0.05 ml into a 20 ml volumetric flask, dilute to the mark with blank solution, shake well, and the solution is ready. Prepare 3 portions in the same way.

[0136] 150% limit concentration spiked test solution: Accurately measure approximately 0.1 ml of lincomycin hydrochloride injection into a 20 ml volumetric flask, accurately add 1.5 ml of the mixed reference stock solution I under section "4.1.3", dilute to the mark with blank solution, and mix well. Then accurately measure 0.05 ml into a 20 ml volumetric flask, dilute to the mark with blank solution, and mix well. Prepare 3 aliquots using the same method. See Table 16 for details.

[0137] Table 16. Accuracy Solutions

[0138]

[0139] Each of the above solutions was injected once for analysis.

[0140] 4.1.9 Specific solutions

[0141] Blank solvent: Take the "blank solution" under "4.1.1";

[0142] Test solution: Take the "Test solution" under "4.1.2";

[0143] Positioning solution: Take the "positioning solution" under "4.1.3.3";

[0144] 100% limit concentration reference solution: Take the "100% limit concentration reference solution (STD5 solution)" under "4.1.5";

[0145] 100% limit concentration spiked test solution: Take the "100% limit concentration spiked test solution" under "4.1.8";

[0146] Each of the above solutions was injected once for analysis.

[0147] 4.1.10 Stable solutions

[0148] Test solution: Take the "Test solution" under "4.1.2";

[0149] 100% limit concentration reference solution: Take the "100% limit concentration reference solution (STD5 solution)" under "4.1.5";

[0150] 100% limit concentration spiked test solution: Take the "100% limit concentration spiked test solution" under "4.1.8";

[0151] The above solutions were incubated at room temperature for different time periods and then injected once for testing.

[0152] 4.1.11 Durable Solution

[0153] Blank solvent: Take the "blank solution" under "4.1.1";

[0154] System suitability solution: Take the "100% limit concentration reference solution (STD5 solution)" under "4.1.5";

[0155] Standard curve solutions: Take the standard curve solutions "STD1" to "STD7" from section "4.1.5";

[0156] Test solution: Take the "Test solution" under "4.1.2";

[0157] 100% limit concentration spiked test solution: Take the "100% limit concentration spiked test solution" under "4.1.8";

[0158] The above solutions were sequentially injected and analyzed under different durability conditions.

[0159] 4.1.12. Precision solution

[0160] 4.1.12.1 Repeatable solutions

[0161] Test solution: Prepare two parallel portions according to the same method as described in section “4.1.2”;

[0162] Repeatability of 100% limit concentration spiked test solution: Take 3 aliquots of 100% limit concentration spiked test solution from section "4.1.8", and prepare another 3 aliquots using the same method, for a total of 6 solutions, and inject each solution once for analysis. See Table 17 for details.

[0163] Table 17. Repeatable solutions

[0164]

[0165] 4.1.12.2 Intermediate Precision Solution

[0166] Blank solution: 1% formic acid-methanol solution.

[0167] Test solution: Prepare two parallel portions according to the same method as described in section “4.1.2”.

[0168] Take an appropriate amount of impurity E, accurately weigh it, place it in a 5ml volumetric flask, add methanol solution to dissolve and dilute to the mark, shake well, and you will get impurity E stock solution i; take an appropriate amount of impurity F, accurately weigh it, place it in a 5ml volumetric flask, add ultrapure water to dissolve and dilute to the mark, shake well, and you will get impurity F stock solution i, see Table 18 for details.

[0169] Table 18. Reference Standard Stock Solution i

[0170] compound Sample weight (mg) content(%) Dilution volume (ml) Concentration (ng / ml) Preparation solution name Impurity E 11.30 98.5 5 2226100 Impurity E stock solution i impurity F 5.287 95.2 5 1006644.8 Impurity F in stock solution i

[0171] Accurately measure appropriate amounts of impurity E stock solution i and impurity F stock solution i, place them in a 10 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain mixed stock solution i, as detailed in Table 19.

[0172] Table 19. Mixed reference standard stock solution i

[0173]

[0174] Accurately measure an appropriate amount of mixed reference standard stock solution i, place it in different volumetric flasks, dilute to the mark with blank solution, and shake well to obtain a series of standard curve stock solutions with different concentrations, as detailed in Table 20.

[0175] Table 20. Standard Curve Stock Solution

[0176]

[0177]

[0178] Accurately measure 0.05 ml of the standard curve stock solution into different 20 ml volumetric flasks, dilute to the mark with blank solution, and shake well to obtain a series of standard curve solutions with different concentrations, as detailed in Table 21.

[0179] Table 21. Standard Curve Solutions

[0180]

[0181] Intermediate precision 100% limit concentration spiked test solution: Take the above "mixed reference stock solution i" and prepare 6 parallel solutions according to the preparation method of "100% limit concentration spiked test solution" under "4.1.8", as detailed in Table 22.

[0182] Table 22. Intermediate Precision Solutions

[0183] serial number Measure the volume (ml) of the mixed reference standard stock solution i. Approximately equivalent to the limit concentration IP-1 1.00 100 IP-2 1.00 100 IP-3 1.00 100 IP-4 1.00 100 IP-5 1.00 100 IP-6 1.00 100

[0184] 5. Verification Results

[0185] 5.1 Methodological Validation Results

[0186] The methodology was validated based on the analytical method, and the results are shown in Table 23.

[0187] Table 23. Summary of Results

[0188]

[0189]

[0190]

[0191] 5.2 System Applicability

[0192] Take the system suitability solution from section “4.1.6” and perform six consecutive injections according to the analytical method from section “3” to examine the instrument precision. The RSD (n=6) of the target peak area in the six consecutive injections of the system suitability solution should not exceed 10%, and the RSD of the retention time should not exceed 1%. The results of the system suitability solution determination are shown in Table 24.

[0193] Table 24. System Applicability Results

[0194]

[0195]

[0196] The results showed that for six consecutive injections of the system suitability solution, the RSDs (n=6) for the peak area and retention time of impurity E were 0.9% and 0.0%, respectively; and the RSDs (n=6) for the peak area and retention time of impurity F were 1.5% and 0.0%, respectively. The system suitability met the requirements.

[0197] 5.3 Specificity

[0198] Take the specific solution from section "4.1.9" and determine it according to the analytical method in section "3". The blank solution chromatogram should show no significant interference at the target peak. If interference is present, the peak area of ​​the interfering peak should not exceed 30% of the average peak area of ​​the target compound's LOQ. In the test solution chromatogram, the resolution between the target peak and adjacent peaks should be greater than 1.5. Positioning solutions should display their respective target peaks. The chromatogram of the 100% limit concentration reference solution should display the target peak. In the chromatogram of the 100% limit concentration spiked test solution, the resolution between the target peak and all adjacent peaks greater than LOQ should be greater than 1.5. The results of the specificity determinations for each solution are shown below. Figures 1-6 .

[0199] The results showed that the target peak was not detected in the blank solution chromatogram and did not interfere with the detection; the target peak was detected in the test solution chromatogram, but no adjacent peaks were detected; the positioning solutions showed their respective target peaks; the target peak was shown in the chromatogram of the 100% limit concentration reference solution; and the target peak was shown in the chromatogram of the 100% limit concentration spiked test solution, and no adjacent peaks with a peak area greater than LOQ were detected. The results met the requirements, and the method has good specificity.

[0200] 5.4 Linearity and Range

[0201] Take the standard curve solution from section "4.1.5" and analyze it according to the analytical method in section "3". The correlation coefficient r of the linear regression equation should be ≥0.990, and the ratio of the absolute value of the y-intercept to the 100% limit concentration response value should not exceed 20%. The results of the linear solution determination are shown in Table 25. Figures 7-8 .

[0202] Table 25. Linearity Results

[0203]

[0204] The results above show the linearity and range results, as shown in Table 26.

[0205] Table 26. Linearity and Range Results

[0206]

[0207]

[0208] The results showed that impurity E, within the range of 0.75 ng / ml to 74.95 ng / ml, approximately equivalent to 2% to 200% of the limit concentration, exhibited good linearity between peak area and concentration, with a correlation coefficient r of 0.99956 and a ratio of the absolute value of the y-intercept to the response value at 100% limit concentration of 0.4%. Similarly, impurity F, within the range of 0.75 ng / ml to 75.07 ng / ml, also approximately equivalent to 2% to 200% of the limit concentration, showed good linearity between peak area and concentration, with a correlation coefficient r of 0.99969 and a ratio of the absolute value of the y-intercept to the response value at 100% limit concentration of 0.4%. The linearity results met the requirements.

[0209] 5.5 Limit of Detection and Limit of Quantification

[0210] Take the "LOD solution" and "LOQ solution" from section "4.1.7" and perform the analytical method from section "3", injecting them consecutively 3 and 6 times respectively, and record the chromatograms. The S / N ratio of the target compound in the limit of detection solution should be no less than 3, and the S / N ratio of the target compound in the limit of quantitation solution should be no less than 10. The RSD (n=6) of the peak area of ​​the target compound in the 6 consecutive limit of quantitation solutions should not exceed 15%. The results of the limit of detection and limit of quantitation solutions are shown in Tables 27 and 28.

[0211] Table 27. Results of Detection Limit Solution

[0212] name Concentration (ng / ml) Approximately equivalent to the limit concentration percentage (%) S / N(1) S / N(2) S / N(3) Impurity E 0.37 1 797.9 697.3 676.0 impurity F 0.38 1 668.6 619.7 556.6

[0213] Table 28. Results for Limit of Quantification Solutions

[0214]

[0215] Note: "NA" indicates that it is not applicable.

[0216] The results showed that in the detection limit solution of three consecutive injections, the concentration of impurity E was 0.37 ng / ml, approximately equivalent to 1% of the limit concentration, with an S / N range of 676.0–797.9; the concentration of impurity F was 0.38 ng / ml, approximately equivalent to 1% of the limit concentration, with an S / N range of 556.6–668.6. In the quantitation limit solution of six consecutive injections, the concentration of impurity E was 0.75 ng / ml, approximately equivalent to 2% of the limit concentration, with an S / N range of 1010.2–1322.9, and the RSD (n=6) of the peak area was 1.7%; the concentration of impurity F was 0.75 ng / ml, approximately equivalent to 2% of the limit concentration, with an S / N range of 1071.3–1310.8, and the RSD (n=6) of the peak area was 2.3%. The detection limit and quantitation limit results of this method met the requirements.

[0217] 5.6 Precision

[0218] 5.6.1 Repeatability

[0219] Take two test solutions and six 100% limit concentration spiked test solutions from section “4.1.12.1”, and determine them according to the analytical method in section “3”. Record the chromatograms. The recovery rate of the target compound in the six 100% limit concentration spiked test solutions should be between 80% and 115%, and the RSD (n=6) of the recovery rate should not be greater than 10%. The results of the repeatability test are shown in Tables 29 and 30.

[0220] Table 29. Repeatability Results of Impurity E

[0221]

[0222] Table 30. Repeatability Results of Impurity F

[0223]

[0224] The results showed that the recovery rate of impurity E in the six 100% limit concentration spiked test solutions ranged from 97.1% to 103.5%, with an RSD (n=6) of 2.5%; the recovery rate of impurity F ranged from 101.1% to 107.1%, with an RSD (n=6) of 2.1%. The method showed good repeatability.

[0225] 5.6.2 Intermediate Precision

[0226] Take two test solutions and six 100% limit concentration spiked test solutions from section “4.1.12.2”, and determine them according to the analytical method in section “3”. Record the chromatograms. The recovery rate of the target compound in the six 100% limit concentration spiked test solutions of experimenter 2 should be between 80% and 115%, and the RSD (n=6) of the recovery rate should not be greater than 10%. The RSD (n=12) of the recovery rate of the target compound in the twelve 100% limit concentration spiked test solutions of the two experimenters should not be greater than 15%. The results of the intermediate precision solution determination are shown in Tables 31 and 32.

[0227] Table 31. Intermediate Precision Results for Impurity E

[0228]

[0229]

[0230] Table 32. Intermediate Precision Results for Impurity F

[0231]

[0232] The results showed that the recovery rate of impurity E in the 6 100% limit concentration spiked test solutions of experimenter 2 ranged from 97.4% to 101.6%, with an RSD (n=6) of 1.9%, and the recovery rate of impurity F ranged from 97.5% to 107.6%, with an RSD (n=6) of 3.5%. In the 12 100% limit concentration spiked test solutions of the two experimenters, the RSD (n=12) of the recovery rate of impurity E was 2.5%, and the RSD (n=12) of the recovery rate of impurity F was 2.9%. The method has good precision.

[0233] 5.7 Accuracy

[0234] Take the accuracy solution from section “4.1.8”, and perform analysis using the analytical method from section “3”, injecting once each solution, recording the chromatogram and calculating the recovery rate. The recovery rate of the target compound in the nine spiked test solutions at 50%, 100%, and 150% limit concentrations should be between 80% and 115%, with an RSD (n=9) of no more than 15%. The results of the accuracy solution determination are shown in Tables 33 and 34.

[0235] Table 33. Accuracy Results of Impurity E

[0236]

[0237] Table 34. Accuracy Results of Impurity F

[0238]

[0239] The results showed that the recovery rate of impurity E in the nine spiked test solutions at 50%, 100%, and 150% limit concentrations ranged from 95.1% to 102.1%, with an RSD (n=9) of 2.4%; the recovery rate of impurity F in the nine spiked test solutions at 50%, 100%, and 150% limit concentrations ranged from 93.6% to 106.1%, with an RSD (n=9) of 4.4%. The accuracy results met the requirements.

[0240] 5.8 Stability

[0241] Take the stability solutions from section "4.1.10" and place them at room temperature for different times. Analyze them according to the analytical method in section "3" and record the chromatograms. At different time points, the ratio of the detected concentration of the target compound in each stability solution to the initial (0h) detected concentration should be between 80% and 115%. If so, the solution is stable within the observation period. The stability test results for each solution are shown in Tables 35 and 36.

[0242] Table 35. Stability Results of Impurity E

[0243]

[0244] Note: "NA" indicates that it is not applicable.

[0245] Table 36. Stability Results of Impurity F

[0246]

[0247] Note: "NA" indicates that it is not applicable.

[0248] The results showed that after being placed at room temperature for 39.5 hours,

[0249] (1) Impurities E and F were detected in the test solution at each stability point. The ratio of the detected concentration of impurity E to the initial (0h) detected concentration was between 97.1% and 110.1%, and the ratio of the detected concentration of impurity F to the initial (0h) detected concentration was between 93.4% and 111.0%.

[0250] (2) The ratio of the detection concentration of impurity E to the initial (0h) detection concentration in the 100% limit concentration reference solution was between 94.2% and 106.4%; the ratio of the detection concentration of impurity F to the initial (0h) detection concentration was between 86.4% and 102.8%.

[0251] (3) The ratio of the detection concentration of impurity E to the initial (0h) detection concentration in the 100% limit concentration spiked test solution is between 97.6% and 106.1%; the ratio of the detection concentration of impurity F to the initial (0h) detection concentration is between 93.6% and 108.6%.

[0252] Therefore, the test solution, the 100% limit concentration reference solution, and the 100% limit concentration spiked test solution are stable at room temperature for at least 39.5 hours.

[0253] 5.9 Durability

[0254] The column temperature was varied between 27℃ and 33℃, the flow rate between 0.36 ml / min and 0.44 ml / min, and the ion source temperature between 450℃ and 550℃. The robustness solution under section "4.1.11" was injected and analyzed, the chromatogram was recorded, and the recovery rate of the target compound in the 100% limit concentration spiked test solution was calculated. Under each robustness condition, the system suitability must meet the acceptance criteria; the correlation coefficient of the standard curve should be ≥0.990; the detection results of the target compound in the test solution should be consistent with the standard conditions (all values ​​should be less than the limit of quantitation, or, if not less than the limit of quantitation, the rate of change compared with the standard conditions should not exceed 30%); the recovery rate of the target compound in the 100% limit concentration spiked test solution should be between 80% and 115%. The robustness test results are shown in Tables 37 to 46.

[0255] Table 37. System Suitability Results under Impurity E Robustness Conditions - Peak Area

[0256]

[0257] Table 38. System Suitability Results under Impurity F Robustness Conditions - Peak Area

[0258]

[0259]

[0260] Table 39. System Suitability Results under Impurity E Durability Conditions - Retention Time

[0261]

[0262] Table 40. System Suitability Results under Impurity F Robustness Conditions - Retention Time

[0263]

[0264] Table 41. Linearity results under robustness conditions for impurity E

[0265]

[0266] Table 42. Linearity results under robustness conditions for impurity F

[0267]

[0268] Table 43. Linearity and Range Results under Durability Conditions for Impurity E

[0269]

[0270] Table 44. Linearity and Range Results under Robustness Conditions for Impurity F

[0271]

[0272] Table 45. Recovery Results of Impurity E under Robustness Conditions

[0273]

[0274]

[0275] Table 46. Recovery Results of Impurity F under Robustness Conditions

[0276]

[0277] The results showed that the column temperature fluctuated within the range of 27℃ to 33℃, the flow rate fluctuated within the range of 0.36 ml / min to 0.44 ml / min, and the ion source temperature fluctuated within the range of 450℃ to 550℃.

[0278] (1) System suitability: The peak area and retention time RSD (n=6) range of impurity E in the solution are 0.6% to 3.9% and 0.0% to 0.2%, respectively; the peak area and retention time RSD (n=6) range of impurity F are 1.3% to 4.8% and 0.0% to 0.3%, respectively.

[0279] (2) The correlation coefficient r of the standard curve of impurity E ranges from 0.99944 to 0.99984; the correlation coefficient r of the standard curve of impurity F ranges from 0.99948 to 0.99982; the peak area of ​​impurity E and impurity F both show a good linear relationship with concentration.

[0280] (3) Impurities E and F were detected in both of the test solutions. Compared with the test results under standard conditions, the change rate of impurity E was 0.0% to 3.5%, and the change rate of impurity F was 0.0% to 1.7%.

[0281] (4) The recovery rate of impurity E in the 100% limit concentration spiked test solution ranged from 94.5% to 102.6%; the recovery rate of impurity F ranged from 99.1% to 103.8%.

[0282] All the above results meet the durability requirements, indicating that this method has good durability.

[0283] 6. Summary

[0284] This experiment established an LC-MS / MS method for the determination of impurities E and F in lincomycin hydrochloride injection. The method was evaluated for its system suitability, specificity, linearity and range, limit of detection and limit of quantitation, precision, accuracy, solution stability and robustness. The results all met the requirements. Therefore, this method can be used to accurately determine the content of impurities E and F in lincomycin hydrochloride injection.

[0285] Example 2

[0286] Example 2 provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0287] The difference between Example 2 and Example 1 is that:

[0288] The parameters for liquid chromatography were as follows: separation was performed using a gradient elution method. The gradient elution process was as follows: from 0 min to 2 min, mobile phase A was 65% and mobile phase B was 35%; from 2 min to 6 min, mobile phase A was decreased to 2.5% at a constant rate and mobile phase B was increased to 97.5% at a constant rate; from 6 min to 8 min, mobile phase A was 2.5% and mobile phase B was 97.5%.

[0289] From 8.0 to 8.1 min, mobile phase A increased to 65% at a constant rate, while mobile phase B decreased to 35% at a constant rate; from 8.1 to 10 min, mobile phase A was 65% and mobile phase B was 35%; both mobile phase A and mobile phase B are expressed as volume percentages.

[0290] Mobile phase A is a formic acid-water solution, wherein the volume ratio of formic acid to water in the formic acid-water solution is 0.05%; mobile phase B is acetonitrile.

[0291] The flow rate was 0.36 ml / min;

[0292] The drying gas temperature in the mass spectrometry parameters is 450℃.

[0293] Specificity detection results:

[0294] The results showed that the target peak was not detected in the blank solution chromatogram and did not interfere with the detection; the target peak was shown in the test solution chromatogram and no adjacent peaks with a peak area greater than LOQ were detected; the target peak was shown in the 100% limit concentration reference solution chromatogram; the target peak was shown in the 100% limit concentration spiked test solution chromatogram and no adjacent peaks with a peak area greater than LOQ were detected; the results met the requirements and the method had good specificity.

[0295] Example 3

[0296] Example 3 provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0297] The difference between Example 3 and Example 1 is that:

[0298] The parameters for liquid chromatography were as follows: separation was performed using a gradient elution method. The gradient elution process was as follows: from 0 min to 2 min, mobile phase A was 75% and mobile phase B was 25%; from 2 min to 6 min, mobile phase A was decreased to 7.5% at a constant rate and mobile phase B was increased to 92.5% at a constant rate; from 6 min to 8 min, mobile phase A was 7.5% and mobile phase B was 92.5%.

[0299] From 8.0 to 8.1 min, mobile phase A increases to 75% at a constant rate, while mobile phase B decreases to 25% at a constant rate; from 8.1 to 10 min, mobile phase A is 75% and mobile phase B is 25%; both mobile phase A and mobile phase B are expressed as volume percentages.

[0300] Mobile phase A is a formic acid-water solution, wherein the volume ratio of formic acid to water in the formic acid-water solution is 0.15%; mobile phase B is acetonitrile.

[0301] The flow rate was 0.44 ml / min;

[0302] The drying gas temperature in the mass spectrometry parameters is 550℃.

[0303] Specificity detection results:

[0304] The results showed that the target peak was not detected in the blank solution chromatogram and did not interfere with the detection; the target peak was shown in the test solution chromatogram and no adjacent peaks with a peak area greater than LOQ were detected; the target peak was shown in the 100% limit concentration reference solution chromatogram; the target peak was shown in the 100% limit concentration spiked test solution chromatogram and no adjacent peaks with a peak area greater than LOQ were detected; the results met the requirements and the method had good specificity.

[0305] Comparative Example

[0306] Comparative Example 1

[0307] Comparative Example 1 provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0308] The difference between Comparative Example 1 and Example 1 is that the volume ratio of formic acid to water in mobile phase A is 0.01%; and mobile phase B is acetonitrile.

[0309] Specific detection results: The blank solution did not interfere with the detection of the target peak. In the spiked solution of the test sample, the recovery rate of impurity F was only 43%, which is low. The test sample interfered with the detection of impurity F.

[0310] The results showed that the test sample interfered with the detection of impurity F in the target peak.

[0311] Comparative Example 2

[0312] Comparative Example 2 provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0313] The difference between Comparative Example 2 and Example 1 is that the volume ratio of formic acid to water in mobile phase A is 0.2%; and mobile phase B is acetonitrile.

[0314] Specificity test results: The blank solution did not interfere with the detection of the target peak; in the spiked solution of the test sample, the limit concentration of impurity E did not appear as a peak.

[0315] The results showed that the test sample interfered with the detection of impurity E in the target peak.

[0316] Comparative Example 3

[0317] Comparative Example 3 provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0318] The difference between Comparative Example 3 and Example 1 is that the liquid chromatography parameters are as follows: separation is performed using a mobile phase gradient elution method, and the mobile phase gradient elution process is as follows: from 0 min to 2 min, mobile phase A is 50% and mobile phase B is 50%; from 2 min to 6 min, mobile phase A is reduced to 15% at a constant rate and mobile phase B is increased to 85% at a constant rate; from 6 min to 8 min, mobile phase A is 15% and mobile phase B is 85%.

[0319] From 8.0 to 8.1 min, mobile phase A was increased to 50% at a constant rate, and mobile phase B was decreased to 50% at a constant rate; from 8.1 to 10 min, mobile phase A was 50% and mobile phase B was 50%; both mobile phase A and mobile phase B are expressed as volume percentages.

[0320] Specificity test results: The blank solution did not interfere with the detection of the target peak, and the separation degree between impurity E and impurity F peaks was 1.2 < 1.5.

[0321] The results showed that the separation degree between impurity E and impurity F was less than 1.5.

[0322] Comparative Example 4

[0323] Comparative Example 4 provides a method for detecting the content of impurities E and F in lincomycin hydrochloride injection.

[0324] The difference between Comparative Example 4 and Example 1 is that the mass spectrometry parameters are different, as shown below.

[0325] The parameters for liquid chromatography were as follows: separation was performed using a gradient elution method. The gradient elution process was as follows: from 0 min to 2 min, mobile phase A was 85% and mobile phase B was 15%; from 2 min to 6 min, mobile phase A was decreased to 15% at a constant rate and mobile phase B was increased to 85% at a constant rate; from 6 min to 8 min, mobile phase A was 15% and mobile phase B was 85%.

[0326] From 8.0 to 8.1 min, mobile phase A increases at a constant rate to 85%, while mobile phase B decreases at a constant rate to 15%; from 8.1 to 10 min, mobile phase A is 85%, and mobile phase B is 15%; both mobile phase A and mobile phase B are expressed as volume percentages.

[0327] Specificity test results: The blank solution did not interfere with the detection of the target peak, and impurities E and F in the spiked solution of the test sample did not appear as peaks.

[0328] The results showed that no target peak was detected in the spiked solution of the test sample.

[0329] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting the content of impurities E and F in lincomycin hydrochloride injection, characterized in that, The detection was performed using liquid chromatography-mass spectrometry (LC-MS / MS), including the following steps: preparation of a reference solution, preparation of a test solution, and detection by liquid chromatography-mass spectrometry (LC-MS / MS). Detection by liquid chromatography-mass spectrometry (LC-MS / MS): The reference solution and the test solution are injected into the liquid chromatograph-mass spectrometer for separation and detection; The parameters for the liquid chromatography are as follows: separation is performed using a mobile phase gradient elution method. The mobile phase gradient elution process is as follows: from 0 min to 2 min, mobile phase A is 65%–75% and mobile phase B is 25%–35%; from 2 min to 6 min, mobile phase A decreases uniformly to 2.5%–7.5% and mobile phase B increases uniformly to 92.5%–97.5%; from 6 min to 8 min, mobile phase A is 2.5%–7.5% and mobile phase B is 92.5%–97.5%. From 8.0 to 8.1 min, mobile phase A increases at a constant rate to 65%–75%, while mobile phase B decreases at a constant rate to 25%–35%; from 8.1 to 10 min, mobile phase A is 65%–75%, and mobile phase B is 25%–35%; both mobile phase A and mobile phase B are expressed as volume percentages. The mobile phase A is a formic acid-water solution, wherein the volume ratio of formic acid to water in the formic acid-water solution is 0.05% to 0.15%; the mobile phase B is acetonitrile. The chromatographic column was an InfinityLab Poroshell 120 SB-AQ column, 4.6 × 100 mm, 2.7 μm; The flow rate was 0.36–0.44 ml / min; The parameters for the mass spectrometry are as follows: The mass spectrometry scanning parameters are as follows: 。 2. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to claim 1, characterized in that, The parameters and conditions for the liquid chromatography are as follows: separation is performed using a mobile phase gradient elution method. The mobile phase gradient elution process is as follows: from 0 min to 2 min, mobile phase A is 70% and mobile phase B is 30%; from 2 min to 6 min, mobile phase A is reduced to 5% at a constant rate and mobile phase B is increased to 95% at a constant rate; from 6 min to 8 min, mobile phase A is 5% and mobile phase B is 95%. From 8.0 to 8.1 min, the mobile phase A increases to 70% at a constant rate, while the mobile phase B decreases to 30% at a constant rate. 8.1-10 min, mobile phase A is 70%, mobile phase B is 30%; The flow rate is 0.4 ml / min.

3. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to claim 1, characterized in that, In the formic acid-water solution, the volume ratio of formic acid to water is 0.1%.

4. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to claim 1, characterized in that, In the liquid chromatography detection, the column temperature is 27–33 °C.

5. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to claim 1, characterized in that, The injection volume is 1.5–2.5 μl.

6. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to claim 1, characterized in that, The detection limit concentration of impurity E is 0.37 ng / ml, and the detection limit concentration of impurity F is 0.38 ng / ml.

7. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to claim 1, characterized in that, The limit of quantitation (LOQ) for impurity E is 0.75 ng / ml, and the limit of quantitation (LOQ) for impurity F is 0.75 ng / ml.

8. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to any one of claims 1-7, characterized in that, The preparation method of the test solution includes: taking 0.1 ml of the lincomycin hydrochloride injection, placing it in a 20 ml volumetric flask, diluting it to the mark with blank solution, and shaking well to obtain the first solution; measuring 0.05 ml of the first solution, placing it in a 20 ml volumetric flask, diluting it to the mark with blank solution, and shaking well to obtain the test solution.

9. The method for detecting the content of impurities E and F in lincomycin hydrochloride injection according to any one of claims 1-7, characterized in that, The method for preparing the reference solution includes: S1: Take 10.68 mg of impurity E, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with methanol solution, shake well to obtain impurity E stock solution I; take 10.11 mg of impurity F, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with ultrapure water, shake well to obtain impurity F stock solution I. S2: Take appropriate amounts of the impurity E stock solution I and the impurity F stock solution I, place them in the same 20ml volumetric flask, dilute to the mark with blank solution, shake well, and the mixed reference stock solution I is obtained. S3: Measure 0.50 ml of the mixed reference standard stock solution I, place it in a 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the standard curve stock solution; S4: Measure 0.05 ml of the standard curve stock solution, place it in a 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the solution.

Images