Method for detecting contents of impurity E and impurity F in lincomycin hydrochloride injection

Abstract

The invention discloses a method for detecting the content of an impurity E and an impurity F. Liquid chromatography-mass spectrometry (LC-MS / MS) is adopted for detection, and the method comprises the following steps: preparing a reference substance solution, preparing a test solution, and performing liquid chromatography-mass spectrometry (LC-MS / MS) detection. Carrying out liquid chromatography-mass spectrometry (LC-MS / MS) detection: injecting the reference substance solution and the test solution into a liquid chromatography-mass spectrometer, and carrying out separation detection; the parameter conditions of the liquid chromatography are as follows: separation is performed by adopting a mobile phase gradient elution method, and the mobile phase gradient elution process is as follows: when the time is 0-2 minutes, the mobile phase A accounts for 65-75%, and the mobile phase B accounts for 25-35%. The method disclosed by the invention is simple to operate and high in detection speed, and has the advantages of high sensitivity, high precision and high repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine detection, and in particular to a method for detecting the contents of impurities E and F in lincomycin hydrochloride injection. Background Art

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

[0003] Impurity E ((2S,4R)-1-methyl-4-propylpyrrolidine-2-carboxylic acid) and impurity F (methyl 6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-pyranogalactoside) are impurities generated during the production or storage of drugs. Long-term intake of low-dose toxic impurities may cause organ damage. The impurities may interact with the main components and interfere with drug absorption or metabolism. For example, the pyrrolidine carboxylic acid derivative of impurity E may have neuromodulatory activity (such as GABA receptor effect). 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 AMES test) is required. The carboxylic acid group of the pyrrolidine derivative may also competitively bind to metal ion-dependent enzymes (such as ACE, 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 attention should be paid to its potential for ototoxicity and nephrotoxicity, especially the cumulative effect during long-term use. The anti-enzymatic properties of thioglycosides may inhibit liver enzymes (such as CYP450), change the metabolic kinetics of the main drug, and lead to abnormal blood drug concentrations. This can lead to irreversible consequences. The pyrrolidine impurity in pioglitazone has caused a liver toxicity recall due to uncontrolled stereoisomers. Therefore, these two impurities need to be strictly controlled as potential genotoxic substances (warning structure positive), and it is recommended to carry out impurity spectrum analysis and toxicological threshold (TTC) assessment; if it cannot be reduced to ≤0.1%, a bacterial reverse mutation test (AMES) and micronucleus test are required. Therefore, the content of the two impurities needs to be strictly controlled.

[0004] In order 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' medication, it is currently an urgent technical problem to develop a method for detecting the content of lower concentrations of impurities E and F in lincomycin hydrochloride. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting the contents of impurity E and impurity F in lincomycin hydrochloride injection.

[0006] The present invention provides a method for detecting the content of impurity E and impurity F in lincomycin hydrochloride injection, which adopts liquid chromatography-mass spectrometry LC-MS / MS for detection, comprising the following steps: preparing a reference solution, preparing a test solution, and detecting by liquid chromatography-mass spectrometry LC-MS / MS;

[0007] Liquid chromatography-mass spectrometry LC-MS / MS detection: Inject the reference solution and the test solution into a liquid chromatography-mass spectrometer for separation and detection;

[0008] The liquid chromatography parameters 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 65% to 75%, and mobile phase B is 25% to 35%; from 2 min to 6 min, mobile phase A is uniformly reduced to 2.5% to 7.5%, and mobile phase B is uniformly increased 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 minutes, mobile phase A is increased to 65% to 75% at a constant rate, and mobile phase B is decreased to 25% to 35% at a constant rate; from 8.1 to 10 minutes, mobile phase A is 65% to 75%, and mobile phase B is 25% to 35%; both mobile phase A and mobile phase B are expressed in 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] Flow rate is 0.36-0.44 ml / min;

[0013] The parameter conditions of the mass spectrometry are:

[0014] Mass spectrometry conditions

[0015]

[0016] The present invention adopts a liquid chromatography-mass spectrometry (LC-MS / MS) method for detecting the contents of impurities E and F in lincomycin hydrochloride injection. This method uses liquid chromatography as a separation system for impurities E and F in lincomycin hydrochloride injection, and mass spectrometry as a detection system for impurities E and F. LC-MS / MS combines the high separation capability of liquid chromatography with the high sensitivity and strong qualitative analysis capability of mass spectrometry, and has many significant advantages in detecting impurity contents.

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

[0018] Liquid chromatography offers high resolution for samples. By selecting an appropriate chromatographic column, mobile phase, and elution method, analyzing the mass-to-charge ratio of impurity ions, fragment ion information, and comparing it with a database of known compounds, the chemical structure of impurities can be determined. This is crucial for studying the sources of impurities and assessing their impact on product quality and safety. LC-MS / MS can employ a variety of quantitative methods, such as internal and external standards, to accurately quantify impurities. By selecting appropriate calibration curves and quantifying ions, the impurity content in a sample can be reliably determined, meeting the precision requirements for impurity content testing in various fields. In pharmaceutical quality control, it can accurately determine impurity content to ensure drug safety and efficacy.

[0019] The present invention screens and optimizes relevant test conditions of liquid chromatography and mass spectrometry in liquid chromatography-mass spectrometry, and provides a method for detecting the contents of impurities E and F in lincomycin hydrochloride injection, which has the advantages of low detection limit, high precision, high repeatability and high accuracy.

[0020] Preferably, the liquid chromatography parameter conditions are: separation is performed using a mobile phase gradient elution method, and the mobile phase gradient elution process is: 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 uniformly reduced to 5%, and mobile phase B is uniformly increased to 95%; from 6 min to 8 min, mobile phase A is 5%, and mobile phase B is 95%;

[0021] From 8.0 to 8.1 minutes, mobile phase A was increased to 70% at a constant rate, and mobile phase B was decreased to 30% at a constant rate; from 8.1 to 10 minutes, mobile phase A was 70% and mobile phase B was 30%;

[0022] The flow rate was 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 the impurity E is 0.37 ng / ml, and the detection limit concentration of the impurity F is 0.38 ng / ml.

[0027] Preferably, the limit of quantification concentration of the impurity E is 0.75 ng / ml, and the limit of quantification concentration of the 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 comprises: taking 0.1 ml of the lincomycin hydrochloride injection, placing it in a 20 ml volumetric flask, adding a blank solution to dilute it to the scale, and shaking it 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 scale with a blank solution, and shaking it to obtain the first solution.

[0032] Preferably, the preparation method of the reference solution comprises:

[0033] S1: Take 10.68 mg of impurity E, place it in a 5 ml volumetric flask, add methanol solution to dissolve and dilute to the scale, shake well to obtain impurity E stock solution I; take 10.11 mg of impurity F, place it in a 5 ml volumetric flask, add ultrapure water to dissolve and dilute to the scale, 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 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the mixed reference substance stock solution I;

[0035] S3: Measure 10.50 ml of the mixed reference stock solution, place it in a 20 ml volumetric flask, dilute it to the mark with the 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 it to the scale with the blank solution, and shake well.

[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 content of impurities E and F in lincomycin hydrochloride injection, and the method was validated. The limits of impurities E and F in lincomycin hydrochloride injection were both 1%, meaning that the sensitivity of the detection method of the present invention should be less than 1%.

[0040] The present invention screens and optimizes relevant test conditions of liquid chromatography and mass spectrometry in liquid chromatography-mass spectrometry, and provides a method for detecting the contents of impurities E and F in lincomycin hydrochloride injection, which 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, detection limit, quantification limit, accuracy, precision, stability and robustness of the method were verified, confirming that the method is suitable for the determination of impurities E and F in lincomycin hydrochloride injection.

[0042] The content of impurity E and impurity F in lincomycin hydrochloride injection is detected by liquid chromatography-mass spectrometry technology, and the present invention provides a detection method with fast detection speed and simple operation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The specificity determination results of the blank solution in the embodiment are shown in FIG.

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

[0045] Figure 3 The specificity test results of the solution for locating impurity E in the embodiment are shown;

[0046] Figure 4 The specificity test results of the impurity F location solution in the example;

[0047] Figure 5 The specificity test results of the 100% limit concentration reference solution in the examples are as follows;

[0048] Figure 6 The specificity test results of the test solution spiked with 100% limit concentration in the example are as follows;

[0049] Figure 7 It is a linear graph of impurity E in the embodiment;

[0050] Figure 8 It is a linear graph of impurity F in the example. DETAILED DESCRIPTION

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

[0052] According to the limit requirements of the present invention, the limits for impurities E and F in lincomycin hydrochloride are both 1%. In this study, based on the properties of the injection and the target compound, 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.

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

[0054] The information of the main instruments, test samples, reagents and reference substances of the present invention are shown in Tables 1-4 respectively.

[0055] Table 1. Instrument information

[0056]

[0057] Note: “NA” means not applicable

[0058] Table 2. Sample information

[0059] name batch number Lincomycin Hydrochloride Injection 220714901

[0060] Table 3. Reagent information

[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 Material Information

[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 contents of impurity E and impurity F in lincomycin hydrochloride injection.

[0067] The method for detecting the contents of impurity E and impurity F in lincomycin hydrochloride injection in Example 1 specifically comprises the following steps in sequence:

[0068] 1. Liquid chromatography conditions

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

[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°C;

[0074] Injection volume: 2 μl.

[0075] Gradient elution conditions were 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. Mass spectrometry ion source parameters are as follows:

[0079] Table 6. Mass spectrometry conditions

[0080]

[0081] 3. Impurity scanning parameters

[0082] Table 7. MRM scan parameters

[0083]

[0084]

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

[0086] (2S,4R)-1-methyl-4-propylpyrrolidine-2-carboxylic acid. The structure of impurity F is as follows

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

[0088] 4. Solution preparation and acceptance criteria

[0089] 4.1 Solution preparation

[0090] 4.1.1 Blank solution

[0091] Methanol was used as blank solution.

[0092] 1% formic acid-methanol solution: Measure 20 ml of formic acid, place it in a 2000 ml volumetric flask, add methanol solution to dilute to the scale, and shake well.

[0093] 4.1.2 Test solution

[0094] Accurately measure approximately 0.1 ml of lincomycin hydrochloride injection and place it in a 20 ml volumetric flask. Add blank solution to dilute to the mark and shake well. Accurately measure 0.05 ml of lincomycin hydrochloride injection and place it in a 20 ml volumetric flask. Add blank solution to dilute to the mark and shake well.

[0095] 4.1.3 Reference substance stock solution

[0096] 4.1.3.1 Reference substance stock solution I

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

[0098] Table 8. Reference substance stock solution I

[0099]

[0100] 4.1.3.2 Mixed reference substance 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 scale with blank solution, and shake well to obtain mixed reference stock solution I. See Table 9 for details.

[0102] Table 9. Mixed reference substance 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 scale with blank solution, and shake well to obtain the reference substance stock solution II. See Table 10 for details.

[0107] Table 10. Reference substance stock solution II

[0108] Source solution name Measured 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 20 ml volumetric flasks, dilute to the scale with blank solution, and shake well to obtain the respective positioning solutions. See Table 11 for details.

[0110] Table 11. Positioning solutions

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

[0112] 4.1.4 Detection limit solution

[0113] Accurately measure 0.01 ml of mixed reference substance 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 Solutions

[0115]

[0116] Accurately measure 0.05 ml of the detection limit stock solution into a 20 ml volumetric flask, dilute to the mark with the 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 appropriate amounts of mixed reference substance stock solution I, place them 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. See Table 14 for details.

[0121] Table 14. Standard Curve Stock Solutions

[0122]

[0123]

[0124] Accurately measure 0.05 ml of the standard curve stock solution and place it in different 20 ml volumetric flasks. Dilute it to the scale with the blank solution and shake well to obtain a series of standard curve solutions with different concentrations. See Table 15 for details.

[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 continuously for analysis.

[0129] 4.1.7 Detection Limit and Quantitation Limit Solutions

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

[0131] Limit of Quantitation Solution: Take the “LOQ Solution” under “4.1.5” and inject it 6 times continuously for analysis.

[0132] 4.1.8 Accuracy Solution

[0133] Test solution: Prepare 2 portions in parallel according to the same method as in 4.1.2.

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

[0135] 100% limit concentration spiked test solution: Accurately measure about 0.1 ml of lincomycin hydrochloride injection and place it in a 20 ml volumetric flask. Accurately add 1.0 ml of mixed reference substance stock solution I under "4.1.3". Dilute to the mark with blank solution and shake well. Accurately measure 0.05 ml and place it in a 20 ml volumetric flask. Dilute to the mark with blank solution and shake well. 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 Mixed Reference Substance Stock Solution I (described in 4.1.3). Dilute to the mark with blank solution and shake well. Accurately measure 0.05 ml of this solution into a 20 ml volumetric flask. Dilute to the mark with blank solution and shake well. Prepare 3 aliquots by the same method. See Table 16 for details.

[0137] Table 16. Accuracy Solutions

[0138]

[0139] The above solutions were 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] The above solutions were injected once for analysis.

[0147] 4.1.10 Stability Solution

[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 placed at room temperature for different periods of time and injected once for detection.

[0152] 4.1.11 Durability 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 solution: Take the standard curve solutions "STD1" to "STD7" under "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 injected and analyzed in sequence under different durability conditions.

[0159] 4.1.12. Precision solution

[0160] 4.1.12.1. Repeatable solution

[0161] Test solution: Prepare 2 portions in parallel according to the same method as in 4.1.2.

[0162] Repeatability 100% Limit Concentration Spiked Test Solution: Prepare three aliquots of the 100% limit concentration spiked test solution from "4.1.8" and three additional aliquots by the same method, for a total of six aliquots. Inject each aliquot once for analysis. See Table 17 for details.

[0163] Table 17. Repeatability Solutions

[0164]

[0165] 4.1.12.2 Intermediate Precision Solution

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

[0167] Test solution: Prepare in the same manner as in 4.1.2, and prepare 2 portions in parallel.

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

[0169] Table 18. Reference substance 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 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 scale with blank solution, and shake well to obtain mixed stock solution i. See Table 19 for details.

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

[0173]

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

[0175] Table 20. Standard Curve Stock Solutions

[0176]

[0177]

[0178] Accurately measure 0.05 ml of the standard curve stock solution and place it in different 20 ml volumetric flasks. Dilute it to the scale with the blank solution and shake well to obtain a series of standard curve solutions with different concentrations. See Table 21 for details.

[0179] Table 21. Standard curve solutions

[0180]

[0181] Intermediate precision 100% limit concentration spiked test solution: Take the above-mentioned "Mixed reference substance stock solution i" and prepare 6 solutions in parallel according to the preparation method of "100% limit concentration spiked test solution" under "4.1.8". See Table 22 for details.

[0182] Table 22. Intermediate Precision Solutions

[0183] serial number Measure the volume of mixed reference substance stock solution i (ml) 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. Verify the results

[0185] 5.1 Methodology Validation Results

[0186] Methodological validation was performed 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 described in "4.1.6" and, following the analytical method described in "3," perform six consecutive injections to assess instrument precision. The RSD (n=6) of the target compound peak area in these six consecutive injections of the system suitability solution should not exceed 10%, and the RSD of the retention time should not exceed 1%. See Table 24 for the results of the system suitability solution analysis.

[0193] Table 24. System suitability results

[0194]

[0195]

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

[0197] 5.3. Exclusivity

[0198] Take the specific solution under "4.1.9" and determine it according to the analytical method under "3". It is required that there is no obvious interference at the target peak in the blank solution chromatogram. If there is interference, the peak area of ​​the interference peak shall not be greater than 30% of the average peak area of ​​the target compound LOQ; the separation degree between the target peak and the adjacent peaks in the test solution chromatogram should be greater than 1.5; the positioning solution should show each target peak; the target peak should be shown in the chromatogram of the 100% limit concentration reference solution; the separation degree between the target peak and all adjacent peaks greater than LOQ in the chromatogram of the 100% limit concentration spiked test solution should be greater than 1.5. The determination results of each specific solution are shown in Figures 1 to 6 .

[0199] The results showed that the target peak was not detected in the blank solution chromatogram, indicating no interference with the assay. The target peak was detected in the test solution chromatogram, but no adjacent peaks were detected. The target peaks were also detected in the calibration solution chromatogram. The target peak was also detected in the 100% limit concentration reference solution chromatogram. The target peak was also detected in the 100% limit concentration spiked test solution chromatogram, and no adjacent peaks with peak areas greater than the LOQ were detected. These results met the requirements, demonstrating good specificity of the method.

[0200] 5.4 Linearity and range

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

[0202] Table 25. Linearity results table

[0203]

[0204] From the above results, the linearity and range results are shown in Table 26.

[0205] Table 26. Linearity and range results

[0206]

[0207]

[0208] The results showed that: the impurity E was in the range of 0.75ng / ml to 74.95ng / ml, which was approximately equivalent to 2% to 200% of the limit concentration. The peak area of ​​impurity E showed good linearity with the concentration, the correlation coefficient r was 0.99956, and the ratio of the absolute value of the y-axis intercept to the 100% limit concentration response value was 0.4%; the impurity F was in the range of 0.75ng / ml to 75.07ng / ml, which was approximately equivalent to 2% to 200% of the limit concentration. The impurity F peak area showed good linearity with the concentration, the correlation coefficient r was 0.99969, and the ratio of the absolute value of the y-axis intercept to the 100% limit concentration response value was 0.4%. The linear results met the requirements.

[0209] 5.5. Limit of Detection and Limit of Quantitation

[0210] Take the "LOD Solution" and "LOQ Solution" under "4.1.7" and perform the analysis described in "3" for three and six consecutive injections, respectively, and record the chromatograms. The S / N ratio of the target compound in the detection limit solution should be no less than 3, the S / N ratio of the target compound in the quantification limit solution should be no less than 10, and the RSD (n=6) of the peak area of ​​the target compound in six consecutive injections of the quantification limit solution should be no greater than 15%. The results of the detection limit and quantification limit solutions are shown in Tables 27 and 28:

[0211] Table 27. Detection limit solution results

[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. Quantitation limit solution results

[0214]

[0215] Note: “NA” means not applicable.

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

[0217] 5.6 Precision

[0218] 5.6.1 Repeatability

[0219] Take 2 portions of the test solution under "4.1.12.1" and 6 portions of the test solution spiked with 100% limit concentration, and measure according to the analytical method under "3". Record the chromatograms. The recovery rate of the target compound in the 6 portions of the test solution spiked with 100% limit concentration 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 solution measurement are shown in Tables 29 and 30.

[0220] Table 29. Impurity E repeatability results

[0221]

[0222] Table 30. Impurity F repeatability results

[0223]

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

[0225] 5.6.2 Intermediate Precision

[0226] Take 2 portions of test solution and 6 portions of 100% limit concentration spiked test solution under item "4.1.12.2", determine according to the analytical method under item "3", record the chromatogram, and require that the recovery rate of the target compound in the 6 portions of 100% limit concentration spiked test solution of experimenter 2 should be between 80% and 115%, and the RSD of the recovery rate (n=6) should not be greater than 10%. The RSD of the recovery rate of the target compound in the 12 portions of 100% limit concentration spiked test solution of the two experimenters (n=12) should not be greater than 15%. The results of the intermediate precision solution determination are shown in Tables 31 and 32.

[0227] Table 31. Impurity E intermediate precision results

[0228]

[0229]

[0230] Table 32. Impurity F intermediate precision results

[0231]

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

[0233] 5.7 Accuracy

[0234] Take the accuracy solution described in "4.1.8" and analyze it once using the analytical method described in "3." Record the chromatogram and calculate the recovery. The recovery of the target compound in nine spiked test solutions at 50%, 100%, and 150% limit concentrations should be between 80% and 115%, with the RSD (n=9) of the recovery not exceeding 15%. See Tables 33 and 34 for the accuracy solution analysis results.

[0235] Table 33. Impurity E Accuracy Results

[0236]

[0237] Table 34. Impurity F Accuracy Results

[0238]

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

[0240] 5.8 Stability

[0241] The stability solutions described in "4.1.10" were stored at room temperature for various periods of time. Samples were then analyzed according to the analytical method described in "3," and the chromatograms were recorded. The ratio of the target compound concentration in each stability solution to the initial (0 h) concentration at various time points should be between 80% and 115%, indicating that the solution was stable within the observed time period. The stability test results for each solution are shown in Tables 35 and 36.

[0242] Table 35. Impurity E stability results

[0243]

[0244] Note: “NA” means not applicable.

[0245] Table 36. Impurity F stability results

[0246]

[0247] Note: “NA” means 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 detection concentration of impurity E to the initial (0 h) detection concentration was between 97.1% and 110.1%, and the ratio of the detection concentration of impurity F to the initial (0 h) detection concentration was between 93.4% and 111.0%.

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

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

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

[0253] 5.9 Durability

[0254] The column temperature was fluctuated between 27°C and 33°C, the flow rate between 0.36ml / min and 0.44ml / min, and the ion source temperature between 450°C and 550°C. The durability solution described in "4.1.11" was injected and analyzed, the chromatograms were recorded, and the recovery of the target compound in the test solution spiked at the 100% limit concentration was calculated. System suitability was required to meet the acceptance criteria under each durability condition; the standard curve correlation coefficient, r, should be ≥ 0.990; the detection results of the target compound in the test solution should be consistent with those under the standard conditions (all results were less than the limit of quantification, or if not less than the limit of quantification, the rate of change compared to the standard conditions should not exceed 30%); and the recovery of the target compound in the test solution spiked at the 100% limit concentration should be between 80% and 115%. The durability test results are shown in Tables 37 to 46.

[0255] Table 37. System suitability results under rugged conditions for impurity E - Peak area

[0256]

[0257] Table 38. System suitability results under rugged conditions for impurity F - Peak area

[0258]

[0259]

[0260] Table 39. System suitability results under rugged conditions for impurity E - retention time

[0261]

[0262] Table 40. System suitability results under rugged conditions for impurity F - retention time

[0263]

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

[0265]

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

[0267]

[0268] Table 43. Linearity and range results under durability conditions for impurity E

[0269]

[0270] Table 44. Linearity and range results under durability conditions for impurity F

[0271]

[0272] Table 45. Recovery results of impurity E under durability conditions

[0273]

[0274]

[0275] Table 46. Recovery results of impurity F under durability conditions

[0276]

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

[0278] (1) The RSDs (n=6) of the peak area and retention time of impurity E in the system suitability solution ranged from 0.6% to 3.9% and 0.0% to 0.2%, respectively; the RSDs (n=6) of the peak area and retention time of impurity F ranged from 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 areas and concentrations of impurities E and F show good linear relationships;

[0280] (3) Impurities E and F were detected in the test solution. 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 of impurity E in the 100% limit concentration spiked test solution ranged from 94.5% to 102.6%; the recovery of impurity F ranged from 99.1% to 103.8%.

[0282] The above results all meet the durability requirements, and this method has good durability.

[0283] 6. Summary

[0284] In this study, an LC-MS / MS method was established to detect the contents of impurities E and F in lincomycin hydrochloride injection. Methodological evaluation was conducted on the system applicability, specificity, linearity and range, detection limit and quantification limit, precision, accuracy, solution stability and durability of the method. The results met the specified requirements. Therefore, this method can be used to accurately detect the contents of impurities E and F in lincomycin hydrochloride injection.

[0285] Example 2

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

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

[0288] The liquid chromatography parameters were as follows: separation was performed using a mobile phase gradient elution method, with the mobile phase gradient elution process being: 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 uniformly reduced to 2.5% and mobile phase B was uniformly increased to 97.5%; 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 minutes, mobile phase A was increased to 65% at a constant rate, and mobile phase B was decreased to 35% at a constant rate; from 8.1 to 10 minutes, mobile phase A was 65% and mobile phase B was 35%; both mobile phase A and mobile phase B are expressed in 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 is 0.36 ml / min;

[0292] The drying gas temperature in the mass spectrometry parameter conditions was 450°C.

[0293] Specificity test results:

[0294] The results showed that the target peak was not detected in the chromatogram of the blank solution and there was no interference with the detection; the target peak was shown in the chromatogram of the test solution, and no adjacent peak with a peak area greater than LOQ was detected; the target peak was shown in the chromatogram of the 100% limit concentration reference solution; the target peak was shown in the chromatogram of the 100% limit concentration spiked test solution, and no adjacent peak with a peak area greater than LOQ was detected; the results were in compliance with the regulations and the method had good specificity.

[0295] Example 3

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

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

[0298] The liquid chromatography parameters were as follows: separation was performed using a mobile phase gradient elution method, with the mobile phase gradient elution process being: 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 uniformly reduced to 7.5% and mobile phase B was uniformly increased to 92.5%; 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 minutes, mobile phase A was increased to 75% at a constant rate, and mobile phase B was decreased to 25% at a constant rate; from 8.1 to 10 minutes, mobile phase A was 75% and mobile phase B was 25%; both mobile phases A and B are expressed in 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 is 0.44 ml / min;

[0302] The drying gas temperature in the mass spectrometry parameter conditions was 550°C.

[0303] Specificity test results:

[0304] The results showed that the target peak was not detected in the chromatogram of the blank solution and there was no interference with the detection; the target peak was shown in the chromatogram of the test solution, and no adjacent peak with a peak area greater than LOQ was detected; the target peak was shown in the chromatogram of the 100% limit concentration reference solution; the target peak was shown in the chromatogram of the 100% limit concentration spiked test solution, and no adjacent peak with a peak area greater than LOQ was detected; the results were in compliance with the regulations and the method had good specificity.

[0305] Comparative Example

[0306] Comparative Example 1

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

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

[0309] Specificity test results: The blank solution does not interfere with the detection of the target peak. The recovery rate of impurity F in the spiked solution of the test sample is only 43%, which is low. The test sample interferes with the detection of impurity F.

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

[0311] Comparative Example 2

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

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

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

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

[0316] Comparative Example 3

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

[0318] Comparative Example 3 differs from Example 1 in 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 uniformly reduced to 15% and mobile phase B is uniformly increased to 85%; from 6 min to 8 min, mobile phase A is 15% and mobile phase B is 85%;

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

[0320] Specificity test results: The blank solution did not interfere with the detection of the target peak, and the separation between the 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 contents of impurity E and impurity F in lincomycin hydrochloride injection.

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

[0325] The liquid chromatography parameters were as follows: separation was performed using a mobile phase gradient elution method, with the mobile phase gradient elution process being: 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 uniformly reduced to 15% and mobile phase B was uniformly increased to 85%; from 6 min to 8 min, mobile phase A was 15% and mobile phase B was 85%;

[0326] From 8.0 to 8.1 minutes, mobile phase A was increased to 85% at a constant rate, and mobile phase B was decreased to 15% at a constant rate; from 8.1 to 10 minutes, mobile phase A was 85% and mobile phase B was 15%; both mobile phase A and mobile phase B were expressed in volume percentages.

[0327] Specificity test results: The blank solution does not interfere with the detection of the target peak, and impurities E and F in the spiked solution of the test sample do not show 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 using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for detecting the content of impurity E and impurity F in lincomycin hydrochloride injection, characterized in that: Liquid chromatography-mass spectrometry LC-MS / MS is used for detection, comprising the following steps: preparing a reference solution, preparing a test solution, and performing liquid chromatography-mass spectrometry LC-MS / MS detection; Liquid chromatography-mass spectrometry LC-MS / MS detection: Inject the reference solution and the test solution into a liquid chromatography-mass spectrometer for separation and detection; The liquid chromatography parameters 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 65% to 75%, and mobile phase B is 25% to 35%; from 2 min to 6 min, mobile phase A is uniformly reduced to 2.5% to 7.5%, and mobile phase B is uniformly increased 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%; From 8.0 to 8.1 minutes, mobile phase A is increased to 65% to 75% at a constant rate, and mobile phase B is decreased to 25% to 35% at a constant rate; from 8.1 to 10 minutes, mobile phase A is 65% to 75%, and mobile phase B is 25% to 35%; both mobile phase A and mobile phase B are expressed in 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; Flow rate is 0.36-0.44 ml / min; The parameter conditions of the mass spectrometry are: Mass spectrometry conditions 。 2. The method for detecting the content of impurity E and impurity F in lincomycin hydrochloride injection according to claim 1, characterized in that: The liquid chromatography parameters 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 uniformly reduced to 5% and mobile phase B is uniformly increased to 95%; 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 was increased to 70% at a constant rate, and the mobile phase B was decreased to 30% at a constant rate; 8.1-10 min, mobile phase A: 70%, mobile phase B: 30%; The flow rate was 0.4 ml / min.

3. The method for detecting the content of impurity E and impurity 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 impurity E and impurity F in lincomycin hydrochloride injection according to claim 1, characterized in that: During the liquid chromatography detection, the column temperature is 27-33°C.

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

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

7. The method for detecting the content of impurity E and impurity F in lincomycin hydrochloride injection according to claim 1, characterized in that: The quantitative limit concentration of the impurity E is 0.75 ng / ml, and the quantitative limit concentration of the impurity F is 0.75 ng / ml.

8. The method for detecting the content of impurity E and impurity F in lincomycin hydrochloride injection according to claim 1, characterized in that: The mass spectrometry scanning parameters are as follows: MRM scan parameters 。 9. The method for detecting the content of impurity E and impurity F in the lincomycin hydrochloride injection according to any one of claims 1 to 8, characterized in that: The preparation method of the test solution comprises: taking 0.1 ml of the lincomycin hydrochloride injection, placing it in a 20 ml volumetric flask, adding a blank solution to dilute it to the scale, and shaking it to obtain a first solution; measuring 0.05 ml of the first solution, placing it in a 20 ml volumetric flask, diluting it to the scale with a blank solution, and shaking it to obtain the first solution.

10. The method for detecting the content of impurity E and impurity F in lincomycin hydrochloride injection according to any one of claims 1 to 8, characterized in that: The preparation method of the reference substance solution comprises: S1: Take 10.68 mg of impurity E, place it in a 5 ml volumetric flask, add methanol solution to dissolve and dilute to the scale, shake well to obtain impurity E stock solution I; take 10.11 mg of impurity F, place it in a 5 ml volumetric flask, add ultrapure water to dissolve and dilute to the scale, 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 20 ml volumetric flask, dilute to the mark with blank solution, and shake well to obtain the mixed reference substance stock solution I; S3: Measure 10.50 ml of the mixed reference stock solution, place it in a 20 ml volumetric flask, dilute it to the mark with the 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 it to the scale with the blank solution, and shake well.

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