Determination method for related substances of lincomycin hydrochloride injection

Abstract

The invention relates to a method for determining related substances in a lincomycin hydrochloride injection, which is characterized in that a high performance liquid chromatography is adopted, an ion pair reagent is used in a mobile phase, and related impurities in the lincomycin hydrochloride injection are separated from lincomycin; the related impurities are one or more of an impurity A, an impurity B, an impurity C, an impurity F, an impurity G, an impurity H and lincomycin B. The determination method disclosed by the invention is high in sensitivity, strong in specificity, good in accuracy and simple, convenient and rapid to operate, perfects the quality standard of determination of the related substances in the lincomycin hydrochloride injection, can strictly control the quality of the related substances in the lincomycin hydrochloride injection, ensures the safety and reliability of the lincomycin hydrochloride injection, and has a wide application prospect. The practical significance is realized.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and specifically relates to a method for determining related substances in lincomycin hydrochloride injection. Background Technology

[0002] Lincomycin hydrochloride is a lincosamine-based basic antibiotic produced by Streptomyces. It has strong antibacterial activity against various anaerobic and aerobic Gram-positive bacteria. Common dosage forms include tablets, capsules, eye drops, injections, and ointments. It is mainly used for respiratory tract infections, skin and soft tissue infections, female reproductive tract infections, pelvic infections, and abdominal infections caused by susceptible Staphylococcus spp., Streptococcus spp., Streptococcus pneumoniae, and anaerobic bacteria. In addition, it is often used as an alternative drug for patients who are allergic to penicillin or cannot use penicillin.

[0003] Impurity levels are one of the important factors in drug safety and efficacy. Therefore, how to accurately determine the impurity content in lincomycin hydrochloride injection has become an urgent problem to be solved.

[0004] Currently, the quality standards for lincomycin hydrochloride injection are included in the 18th edition of the Japanese Pharmacopoeia and the 2020 edition of the Chinese Pharmacopoeia. The Japanese Pharmacopoeia does not control related substances in this preparation, while the 2020 edition of the Chinese Pharmacopoeia specifies that high-performance liquid chromatography (HPLC) should be used to detect related substances in lincomycin hydrochloride injection. Impurities A, B, C, F, G, H, lincomycin B, and the excipient benzyl alcohol may be present in lincomycin hydrochloride injection. Among these, impurities F, G, and H are all degradation impurities, sensitive to factors such as temperature and light, and may increase during long-term drug storage.

[0005] Using the analytical methods for related substances in the Chinese Pharmacopoeia, impurity F exhibits weak retention, eluting at the dead time, and solvent peaks interfere with its detection. Lincomycin B contains other impurities, making accurate quantification impossible. Baseline separation between impurity G and lincomycin B is not achieved. Furthermore, impurity H fails to meet the required resolution with adjacent impurities. Therefore, the analytical methods for related substances in the Chinese Pharmacopoeia are insufficient for effectively detecting degradation impurities and cannot comprehensively guarantee product quality. To effectively analyze drug quality and ensure medication safety, a highly sensitive, specific, and accurate method for detecting related substances needs to be developed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for determining related substances in lincomycin hydrochloride injection. This method can effectively separate lincomycin hydrochloride injection from various impurities. The method has high sensitivity, good specificity, high accuracy and good durability. It can strictly control the quality of related substances in lincomycin hydrochloride injection, ensuring the safety and reliability of lincomycin hydrochloride injection, and has practical significance.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for determining related substances in lincomycin hydrochloride injection, employing high performance liquid chromatography (HPLC) with an ion-pairing reagent in the mobile phase to separate related impurities from lincomycin in the lincomycin hydrochloride injection.

[0009] The relevant impurities are one or more of impurity A, impurity B, impurity C, impurity F, impurity G, impurity H and lincomycin B;

[0010] The specific structural formula of the relevant impurities is as follows:

[0011]

[0012] Preferably, an ultraviolet / diode array detector is used to detect relevant impurities and lincomycin in lincomycin hydrochloride injection.

[0013] Preferably, the mobile phase is a mixture of mobile phase A and mobile phase B, where mobile phase A is a potassium dihydrogen phosphate solution containing ion-pairing reagents and mobile phase B is an acetonitrile solution, and gradient elution is used.

[0014] Preferably, the ion-pairing reagent is a positive ion-pairing reagent or a negative ion-pairing reagent; more preferably, it is a negative ion-pairing reagent; more preferably, it is an alkyl sulfonate; and most preferably, it is a sodium hexanesulfonate solution.

[0015] Preferably, the concentration of the potassium dihydrogen phosphate solution is 0.03–0.07 mol / L, and more preferably 0.05 mol / L.

[0016] Preferably, the concentration of the sodium hexanesulfonate solution is 0.005–0.015 mol / L, and more preferably 0.010 mol / L.

[0017] Preferably, the pH value of mobile phase A is 5.5 to 6.5, more preferably 6.0 to 6.2, and even more preferably 6.1.

[0018] Preferably, the chromatographic column is an octadecylsilane-bonded silica column, and more preferably a Nano ChromCore C18 column.

[0019] Preferably, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a silica gel particle size of 3 μm.

[0020] Preferably, the column temperature of the chromatographic column is 48℃~52℃, and more preferably 50℃.

[0021] Preferably, the flow rate is 0.6 mL / min to 0.8 mL / min, and more preferably 0.7 mL / min.

[0022] Preferably, the injection volume is 10 μL to 50 μL, and more preferably 20 μL.

[0023] Preferably, the detection wavelength is 200nm to 220nm, and more preferably 210nm.

[0024] Preferably, the gradient elution procedure is as follows:

[0026] .

[0027] Result calculation:

[0028] For known impurities (impurity A, impurity B, impurity C, impurity F, impurity G, impurity H and lincomycin B) and unknown impurities (correction factor 1.0), the peak area was calculated using the external standard method with correction factor added for principal component reference standards.

[0029] Due to the adoption of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0030] 1. Compared with the prior art, the present invention uses high performance liquid chromatography combined with ultraviolet / diode array detector. The use of ion-pairing reagents in the mobile phase can improve the retention of impurity F and avoid peak elution at the dead time. Impurity G and lincomycin B can be completely separated. Compared with the related substances method in the Chinese Pharmacopoeia, the chromatographic peak shape of related substances in this technology is more symmetrical. Combined with the raw material synthesis process and the impurity spectrum of lincomycin hydrochloride injection, it can better control known and unknown impurities.

[0031] 2. The method of this invention has high sensitivity, strong specificity, and good accuracy. One method can simultaneously determine the content of the content, related substances, and excipient benzyl alcohol, which improves the quality standard for the determination of related substances in lincomycin hydrochloride injection. It can strictly control the quality of related substances in lincomycin hydrochloride injection, ensuring the safety and reliability of lincomycin hydrochloride injection, and has practical significance. Attached Figure Description

[0032] Figure 1 This is the chromatogram of the blank solution in Example 1 of the present invention;

[0033] Figure 2This is a chromatogram of the system suitability solution in Example 1 of the present invention;

[0034] Figure 3 This is the chromatogram of the test solution in Example 1 of the present invention. Detailed Implementation

[0035] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0036] Experimental methods not specified in the following examples are generally performed under standard experimental conditions.

[0037] A method for determining related substances in lincomycin hydrochloride injection, using high performance liquid chromatography, wherein the mobile phase contains an ion-pairing reagent to separate related impurities in lincomycin hydrochloride injection from lincomycin;

[0038] The relevant impurities are one or more of the following: impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, and lincomycin B;

[0039] The specific structural formulas of the relevant impurities are as follows:

[0040]

[0041]

[0042] In some implementations, an ultraviolet / diode array detector is used to detect relevant impurities and lincomycin in lincomycin hydrochloride injection.

[0043] In some embodiments, the mobile phase is a mixture of mobile phase A and mobile phase B, where mobile phase A is a potassium dihydrogen phosphate solution containing ion-pairing reagents and mobile phase B is an acetonitrile solution, using gradient elution.

[0044] In some embodiments, the ion-pairing reagent is a positive ion-pairing reagent or a negative ion-pairing reagent.

[0045] In some implementations, the ion-pairing reagent is a negative ion-pairing reagent.

[0046] In some embodiments, the ion-pairing reagent is an alkyl sulfonate.

[0047] In some embodiments, the ion-pairing reagent is a sodium hexanesulfonate solution.

[0048] In some embodiments, the concentration of potassium dihydrogen phosphate solution is 0.03–0.07 mol / L.

[0049] In some embodiments, the concentration of the potassium dihydrogen phosphate solution is 0.05 mol / L.

[0050] In some embodiments, the concentration of the sodium hexanesulfonate solution is 0.005–0.015 mol / L.

[0051] In some embodiments, the concentration of the sodium hexanesulfonate solution is 0.010 mol / L.

[0052] In some implementations, the pH of mobile phase A is 5.5 to 6.5.

[0053] In some implementations, the pH of mobile phase A is 6.0 to 6.2.

[0054] In some implementations, the pH of mobile phase A is 6.1.

[0055] In some embodiments, the chromatographic column is an octadecylsilane-bonded silica column.

[0056] In some implementations, the chromatographic column is a Nano ChromCore C18 column.

[0057] In some embodiments, the column is 250 mm long, has an inner diameter of 4.6 mm, and the silica gel particle size is 3 μm.

[0058] In some implementations, the column temperature is 48°C to 52°C.

[0059] In some implementations, the column temperature is 50°C.

[0060] In some embodiments, the flow rate is 0.6 mL / min to 0.8 mL / min.

[0061] In some implementations, the flow rate is 0.7 mL / min.

[0062] In some implementations, the injection volume is 10 μL to 50 μL.

[0063] In some implementations, the injection volume is 20 μL.

[0064] In some implementations, the detection wavelength is 200 nm to 220 nm.

[0065] In some implementations, the detection wavelength is 210 nm.

[0066] In some implementations, the gradient elution procedure is as follows:

[0067]

[0068] In the specific embodiments of this invention, the lincomycin hydrochloride injection, equipment, and reference standard used are all known products. The lincomycin hydrochloride injection is a product prepared by our company, and the equipment and reference standard are obtained by purchasing commercially available products.

[0069] Table 1 Equipment Information

[0070]

[0071] Table 2 Material Information

[0072] name source batch number Lincomycin Hydrochloride Injection Suzhou Pharmaceutical Factory of Jiangsu Wuzhong Pharmaceutical Group Co., Ltd. 2401232

[0073] Table 3. Reference Standard Information

[0074]

[0075]

[0076] Example 1

[0077] Methodological study of the detection and analysis method of the present invention

[0078] The following conditions were used for all experiments in this embodiment:

[0079] Instrument: Agilent 1260II high performance liquid chromatograph;

[0080] Chromatographic column: Octadecylsilane-bonded silica gel as the packing material (Nano ChromCore 120C18, 4.6mm × 250mm, 3μm or equivalent column);

[0081] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate solution (containing 0.01 mol / L sodium hexanesulfonate, pH adjusted to 6.1 with potassium hydroxide solution);

[0082] Mobile phase B: Acetonitrile;

[0083] Flow rate: 0.7 mL / min;

[0084] Column temperature: 50℃;

[0085] Detection wavelength: 210nm;

[0086] Injection volume: 20 μL;

[0087] Diluent: Use acetonitrile-water (10:90) as the diluent;

[0088] Perform linear gradient elution according to the table below:

[0089]

[0090] 1. Specificity test

[0091] Testing steps:

[0092] Blank solution: diluent.

[0093] Reference solution a: Take an appropriate amount of lincomycin reference standard, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing about 2.4 mg of lincomycin per 1 mL as reference solution a.

[0094] Reference solution b: Accurately measure an appropriate amount of reference solution a and dilute it with a solvent to prepare a solution containing approximately 48 μg of lincomycin per 1 mL as reference solution b.

[0095] Reference solution c: Accurately measure an appropriate amount of reference solution b and dilute it with a solvent to prepare a solution containing approximately 4.8 μg of lincomycin per 1 mL as reference solution c.

[0096] System suitability solution: Appropriate amounts of lincomycin reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, lincomycin B reference standard, impurity F reference standard, impurity G reference standard, impurity H reference standard, and benzyl alcohol reference standard are dissolved and diluted with solvent to prepare a solution containing approximately 2.4 mg lincomycin, 12 μg impurity A, 12 μg impurity B, 4.8 μg impurity C, 24 μg impurity F, 24 μg impurity G, 24 μg impurity H, 48 μg lincomycin B, and 75.6 μg benzyl alcohol per 1 mL as the system suitability solution.

[0097] Test solution: Accurately measure an appropriate amount of lincomycin hydrochloride injection, dilute it with solvent to prepare a solution containing approximately 2.4 mg of lincomycin per 1 mL, which is used as the test solution.

[0098] Accurately measure the blank solution, test solution, reference solution b, reference solution c, and system suitability solution, and inject them separately into the high-performance liquid chromatograph (HPLC). Record the chromatograms; the results are shown in Table 4. The chromatograms of the specificity test blank solution, system suitability solution, and test solution are attached. Figures 1-3 As shown.

[0099] Table 4. Results of Specificity Experiment

[0100]

[0101] Note: Impurity B-1 is the first peak of impurity B;

[0102] Impurity B-2 is the second peak of impurity B, and both are peaks of impurity B.

[0103] The results showed that the blank solvent had no interference. In the chromatogram recorded by the system suitability solution, impurities F, benzyl alcohol, impurity G, lincomycin B, impurity H, impurity C, impurity A, lincomycin, and impurity B eluted in sequence. The resolution between the lincomycin peak and the first peak of impurity B was 2.25, the resolution between the lincomycin B peak and the impurity G peak was 4.03, and the resolution between the lincomycin B peak and the impurity H peak was 2.28, all of which were greater than 1.5, indicating that the system suitability met the requirements.

[0104] 2. Linearity and Range

[0105] Testing steps:

[0106] Impurity A stock solution: Weigh approximately 5 mg of impurity A reference standard accurately, place it in a 5 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0107] Impurity B stock solution: Weigh approximately 5.4 mg of impurity B reference standard accurately, place it in a 5 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0108] Impurity C stock solution: Weigh approximately 2.8 mg of impurity C reference standard accurately, place it in a 5 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0109] Impurity mixed stock solution: Weigh approximately 10 mg of lincomycin B reference standard, 5 mg of impurity F reference standard, 5 mg of impurity G reference standard, and 4.8 mg of impurity H reference standard accurately, and place them in a 20 mL volumetric flask. Accurately measure 2 mL of each of the impurity A, impurity B, and impurity C stock solutions, place them in the same 20 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0110] Lincomycin Stock Solution I: Accurately weigh approximately 5.6 mg of lincomycin reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent, and mix well. (Approximately 0.48 mg / mL)

[0111] Lincomycin Stock Solution II: Accurately weigh 2 mL of Lincomycin Stock Solution I and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and mix well. (Approximately 48 μg / mL)

[0112] Prepare linear solutions according to Table 5.

[0113] Table 5. Preparation of linear solutions

[0114]

[0115] Accurately measure 20 μL of each linear solution and inject it into the high performance liquid chromatograph. Record the chromatogram. Plot a standard curve with concentration as the abscissa (x) and peak area as the ordinate (y). Calculate the regression equation. The results are shown in Table 6.

[0116] Table 6 Results of the linear experiment

[0117]

[0118]

[0119]

[0120] According to the experimental results, the linear equation for impurity A is y = 10.4719x - 2.1883, r = 0.9982 (n = 5); the linear equation for impurity B is y = 23.3972 - 3.7635, r = 0.9997 (n = 5); the linear equation for impurity C is y = 10.1132x - 0.1977, r = 0.9978 (n = 5); and the linear equation for impurity F is y = 2.6896x - 0.5433, r = 0.9995 (n = 5). The linear equation for impurity G is y = 15.3268x - 4.9964, r = 0.9996 (n = 5); the linear equation for impurity H is y = 13.2413x - 3.2042, r = 0.9999 (n = 5); the linear equation for lincomycin B is y = 10.8965x + 2.2122, r = 0.9999 (n = 5); and the linear equation for lincomycin is y = 10.5394x + 1.0956, r = 0.9999 (n = 8), showing good linearity.

[0121] 3. Correction factor

[0122] Testing steps:

[0123] The solution preparation and determination methods are the same as linearity and range.

[0124] The linearity of each impurity with lincomycin was determined, and the correction factor for each impurity was calculated. The results of the correction factors are shown in Table 7.

[0125] Table 7. Results of Correction Factor Tests

[0126] name K value Correction factor Propose correction factors Impurity A 10.4719 1.0 1.0 Impurity B 23.3972 0.5 0.5 Impurity C 10.1132 1.0 1.0 impurity F 2.6896 3.9 3.9 Impurity G 15.3268 0.7 0.7 impurity H 13.2413 0.8 0.8 Lincomycin B 10.8965 1.0 1.0 Lincomycin 10.5394 / /

[0127] According to the experimental results, the correction factors for impurities A, C, and lincomycin B are all in the range of 0.9 to 1.1, so no correction for the peak area is required in the calculation. The correction factors for impurities B are 0.5, G is 0.7, H is 0.8, and F is 3.9, all of which are outside the range of 0.9 to 1.1, so the peak area needs to be corrected in the calculation.

[0128] 4. Limit of Detection and Limit of Quantification

[0129] Testing steps:

[0130] Impurity A stock solution: Weigh approximately 5 mg of impurity A reference standard accurately, place it in a 5 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0131] Impurity B stock solution: Weigh approximately 5.4 mg of impurity B reference standard accurately, place it in a 5 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0132] Impurity C stock solution: Weigh approximately 2.8 mg of impurity C reference standard accurately, place it in a 5 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0133] Impurity mixed stock solution: Weigh approximately 10 mg of lincomycin B reference standard, 5 mg of impurity F reference standard, 5 mg of impurity G reference standard, and 4.8 mg of impurity H reference standard accurately, and place them in a 20 mL volumetric flask. Accurately measure 2 mL of each of the impurity A, impurity B, and impurity C stock solutions, place them in the same 20 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0134] Lincomycin Stock Solution I: Accurately weigh approximately 5.6 mg of lincomycin reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent, and mix well. (Approximately 0.48 mg / mL)

[0135] Lincomycin Stock Solution II: Accurately measure 2 mL of Lincomycin Stock Solution I and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and mix well. (Approximately 48 μg / mL)

[0136] Detection limit and quantitation limit solution: Take linear stock solution and dilute it gradually to obtain the solution.

[0137] Detection: Accurately measure 20 μL of each linear solution and inject it into the high performance liquid chromatograph. Record the chromatogram. The results of the detection limit and quantitation limit are shown in Table 8.

[0138] Table 8 Results of Limit of Detection and Limit of Quantification

[0139] Impurity Name Signal-to-noise ratio Limit of quantitation (μg / mL) Signal-to-noise ratio Detection limit concentration (μg / mL) Impurity A 10.54 0.5787 4.25 0.2894 Impurity B 18.07 0.3679 8.37 0.1839 Impurity C 13.40 0.4087 6.88 0.2043 impurity F 21.33 1.146 7.38 0.5732 Impurity G 25.59 0.4636 6.83 0.2318 impurity H 26.53 0.4895 12.08 0.2447 Lincomycin B 30.51 0.6378 12.43 0.3189 Lincomycin 34.48 0.8652 13.54 0.4326

[0140] The results showed that the lowest detection limit for impurities A, B, C, F, G, H, lincomycin B, and lincomycin was 0.1839 μg / mL, and the lowest quantitation limit was 0.3679 μg / mL, indicating that the method was relatively sensitive.

[0141] 5. Precision

[0142] 5.1 Sample injection precision

[0143] Testing steps:

[0144] Reference solution a: Take an appropriate amount of lincomycin reference standard, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing about 2.4 mg of lincomycin per 1 mL as reference solution a.

[0145] Reference solution b: Accurately measure an appropriate amount of reference solution a and dilute it with a solvent to prepare a solution containing approximately 48 μg of lincomycin per 1 mL as reference solution b.

[0146] Reference solution c: Accurately measure an appropriate amount of reference solution b and dilute it with a solvent to prepare a solution containing approximately 4.8 μg of lincomycin per 1 mL as reference solution c.

[0147] According to the detection method of the present invention, reference solution b and reference solution c were precisely measured and injected 5 times consecutively. The injection precision results are shown in Table 9.

[0148] Table 9. Injection Precision Results

[0149] Reference solution b Peak area Retention time / minute Reference solution c Peak area Retention time / minute STD-1 518.73 36.058 STD-1 47.91 36.230 STD-2 519.45 36.149 STD-2 52.41 36.168 STD-3 523.15 36.085 STD-3 49.27 36.271 STD-4 521.73 36.191 STD-4 49.03 36.135 STD-5 515.98 36.264 STD-5 52.56 36.146 average value(%) 519.81 36.149 average value(%) 50.24 36.190 RSD (%) 0.54 0.23 RSD (%) 4.22 0.17

[0150] According to the experimental results, after five consecutive injections of reference solution b, the RSD values ​​of the peak area and retention time of lincomycin were not greater than 10.0%, and after five consecutive injections of reference solution c, the RSD values ​​of the peak area and retention time of lincomycin were not greater than 10.0%, indicating that the method has good injection precision.

[0151] 5.2 Repeatability

[0152] Testing steps:

[0153] Reference solution b: Same as reference solution b under the injection precision section.

[0154] Reference solution c: Same as reference solution c under the injection precision section.

[0155] Test solution: Accurately measure an appropriate amount of lincomycin hydrochloride injection, dilute it with solvent to prepare a solution containing approximately 2.4 mg of lincomycin per 1 mL, and use this solution as the test solution. Prepare 6 parallel solutions.

[0156] According to the detection method of the present invention, reference solution b, reference solution c and test solution were precisely measured and injected into the high performance liquid chromatograph. The repeatability results are shown in Table 10.

[0157] Table 10 Repeatability Results

[0158]

[0159]

[0160] The results above show that the measurement method of the present invention has good repeatability.

[0161] 6. Accuracy

[0162] Testing steps:

[0163] Reference solution b: Same as reference solution b under the injection precision section.

[0164] Reference solution c: Same as reference solution c under the injection precision section.

[0165] Impurity mixed stock solution: same as the impurity mixed stock solution under the linearity and range item.

[0166] Test sample stock solution: Accurately measure an appropriate amount of lincomycin hydrochloride injection, dilute with solvent to prepare a solution containing approximately 12 mg of lincomycin per 1 mL, and shake well.

[0167] Test solution (background): Same as the test solution in the repeatability section.

[0168] 20% recovery solution: Accurately measure 2 mL of the test sample stock solution and place it in a 10 mL volumetric flask. Accurately measure 0.2 mL of the impurity mixed stock solution, then dilute to the mark with solvent and shake well. Prepare 3 parallel solutions using the same method.

[0169] 100% recovery solution: Accurately measure 2 mL of the test sample stock solution and place it in a 10 mL volumetric flask. Accurately measure 1 mL of the impurity mixed stock solution, then dilute to the mark with solvent and shake well. Prepare 3 parallel solutions using the same method.

[0170] 150% recovery solution: Accurately measure 2 mL of the test sample stock solution and place it in a 10 mL volumetric flask. Accurately measure 1.5 mL of the impurity mixed stock solution, then dilute to the mark with solvent and shake well. Prepare 3 parallel solutions using the same method.

[0171] The samples were injected separately for testing. The content of impurities was calculated using the external standard method with a correction factor added to the principal component reference standard. The background amount was deducted, and the measured amount was compared with the added amount to calculate the recovery rate. The results are shown in Table 11.

[0172] Table 11 Accuracy Results

[0173] Impurity A Average recovery rate % Impurity B Average recovery rate % Impurity C Average recovery rate % impurity F Average recovery rate % 20％ 107.8 20％ 107.7 20％ 101.1 20％ 100.3 100％ 102.2 100％ 105.3 100％ 97.2 100％ 98.1 150％ 106.0 150％ 105.4 150％ 96.7 150％ 97.3 Impurity G Average recovery rate % impurity H Average recovery rate % Lincomycin B Average recovery rate % 20％ 100.0 20％ 100.8 20％ 103.4 100％ 98.8 100％ 96.5 100％ 102.4 150％ 97.7 150％ 97.0 150％ 102.7

[0174] The results showed that in solutions with an accuracy of 20%–150%, the recovery rates of each impurity ranged from 80.0% to 120.0%, and the RSDs were all less than 10.0%, which met the accuracy requirements.

[0175] 7. Solution stability

[0176] Test solution: Same as the test solution under the repeatability section.

[0177] Testing: The test solution placed at room temperature was tested at 0 hours, 18 hours and 48 hours according to the testing method of the present invention. The results of related substances were calculated. The stability results of the test solution are shown in Table 12.

[0178] Table 12 Stability results of the test solution

[0179]

[0180] Under room temperature conditions, the maximum deviation of the content of a single impurity in the test solution from 0h is 0.02%, which is not greater than 20% as specified in the standard. The deviation of the total impurities from 0h is not greater than 10.0% as specified in the standard. No new impurities are generated. The test solution is stable within 48 hours under room temperature conditions.

[0181] 8. Durability

[0182] Various chromatographic conditions were changed to examine the system suitability and resolution of each impurity in the solution. This was used to assess the system's tolerance to unaffected resolution of each impurity when the measurement conditions were changed. The measurement results are shown in Table 13.

[0183] Table 13 Durability Test Results

[0184]

[0185] Note: Column 1: ChromCore C18 4.6mm×250mm, 3μm;

[0186] Column 2: Diamonsil Plus C18 4.6mm×250mm, 3μm.

[0187] According to the test results, under various robustness conditions, in the chromatogram of the system suitability solution, impurities F, benzyl alcohol, G, lincomycin B, H, C, A, lincomycin, and B eluted in sequence. The resolution between the lincomycin peak and the first peak of impurity B met the requirements. The resolutions between the lincomycin B peak and the impurity G peak, as well as between the lincomycin B peak and the impurity H peak, also met the requirements. The theoretical plate numbers of the lincomycin and benzyl alcohol peaks were not less than 3000, indicating good method robustness.

[0188] In summary, this invention provides a method for determining related substances in lincomycin hydrochloride injection. This method has good specificity, and baseline separation can be achieved between all known impurity peaks. The system has good applicability, and the method has good robustness, repeatability, and accuracy. It provides a convenient detection method for monitoring related substances in lincomycin hydrochloride injection, ensuring product quality and patient medication safety.

[0189] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for determining related substances in lincomycin hydrochloride injection, characterized in that, High performance liquid chromatography (HPLC) was used, with an ion-pairing reagent in the mobile phase, to separate relevant impurities from lincomycin in lincomycin hydrochloride injection. The relevant impurities are one or more of impurity A, impurity B, impurity C, impurity F, impurity G, impurity H and lincomycin B; The specific structural formula of the relevant impurities is as follows:

2. The method for determining related substances in lincomycin hydrochloride injection according to claim 1, characterized in that, A UV / diode array detector was used to detect relevant impurities and lincomycin in lincomycin hydrochloride injection.

3. The method for determining related substances in lincomycin hydrochloride injection according to any one of claims 1-2, characterized in that, The mobile phase of the high performance liquid chromatography method is a mixture of mobile phase A and mobile phase B. Mobile phase A is a potassium dihydrogen phosphate solution containing ion-pairing reagents, and mobile phase B is an acetonitrile solution, using gradient elution. Preferably, the ion-pairing reagent is a positive ion-pairing reagent or a negative ion-pairing reagent; more preferably, it is a negative ion-pairing reagent; more preferably, it is an alkyl sulfonate; and most preferably, it is a sodium hexanesulfonate solution. Preferably, the concentration of the potassium dihydrogen phosphate solution is 0.03–0.07 mol / L, and more preferably 0.05 mol / L; Preferably, the concentration of the sodium hexanesulfonate solution is 0.005–0.015 mol / L, and more preferably 0.010 mol / L; Preferably, the pH value of the mobile phase A is 5.5 to 6.5, more preferably 6.0 to 6.2, and even more preferably 6.

1.

4. The method for determining related substances in lincomycin hydrochloride injection according to any one of claims 1-3, characterized in that, The chromatographic column used in the high-performance liquid chromatography method is an octadecylsilane-bonded silica column, preferably a NanoChromCore C18 column.

5. The method for determining related substances in lincomycin hydrochloride injection according to claim 4, characterized in that, The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a silica gel particle size of 3 μm.

6. The method for determining related substances in lincomycin hydrochloride injection according to any one of claims 1-5, characterized in that, The column temperature of the chromatographic column in the high-performance liquid chromatography method is 48℃~52℃, preferably 50℃.

7. The method for determining related substances in lincomycin hydrochloride injection according to any one of claims 1-6, characterized in that, The flow rate of the high-performance liquid chromatography is 0.6 mL / min to 0.8 mL / min, preferably 0.7 mL / min.

8. The method for determining related substances in lincomycin hydrochloride injection according to any one of claims 1-7, characterized in that, The injection volume for the high-performance liquid chromatography method is 10 μL to 50 μL, preferably 20 μL.

9. The method for determining related substances in lincomycin hydrochloride injection according to any one of claims 1-8, characterized in that, The detection wavelength of the high-performance liquid chromatography is 200 nm to 220 nm, preferably 210 nm.

10. The method for determining related substances in lincomycin hydrochloride injection according to any one of claims 3-9, characterized in that, The gradient elution procedure for the high-performance liquid chromatography is as follows:

Images