Method for detecting content of chlorobutane in lincomycin hydrochloride bulk drug

Abstract

The invention discloses a method for detecting the content of chlorobutane in a lincomycin hydrochloride bulk drug. The method comprises the following steps: detecting a sample to be detected by adopting gas chromatography-mass spectrometry (GC-MS); the parameter conditions of the gas chromatography are as follows: the temperature of a sample inlet is 145-155 DEG C; the heating procedure is as follows: the initial temperature is 65-75 DEG C, the temperature is kept for 1.5-2.5 min, and the temperature is raised to 95-105 DEG C at the heating rate of 8-12 DEG C / min; the temperature is raised to 235-245 DEG C at the temperature raising speed of 65-75 DEG C / min, and the temperature is kept for 3-5 min; the parameter conditions of the mass spectrum are as follows: the acquisition type is MRM; the ion source is EI; the solvent delay is 1.5 to 2.5 min; the temperature of the transmission line is 245-255 DEG C; the temperature of a quadrupole rod is 145-155 DEG C; and the temperature of the ion source is 225-235 DEG C. 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 content of chlorobutane in a lincomycin hydrochloride raw material. Background Art

[0002] Lincomycin hydrochloride is a narrow-spectrum antibiotic with excellent antibacterial activity against most Gram-positive bacteria and various anaerobic bacteria, but is resistant to enterococci and aerobic Gram-negative bacteria. Clinically, lincomycin hydrochloride is primarily used to treat infections caused by Gram-positive cocci, particularly anaerobic bacteria, Staphylococcus aureus, and pneumococcal infections, with good therapeutic efficacy. Common ailments include pneumonia, meningitis, cellulitis, tonsillitis, endocarditis, furuncle, urethritis, and Staphylococcus aureus osteomyelitis.

[0003] Chlorobutane is a mutagenic impurity in lincomycin hydrochloride, has a carcinogenic risk, and may offset the therapeutic effect of lincomycin hydrochloride. Publication number CN117741034A, a Chinese patent, discloses an analytical method for the simultaneous determination of chloroethane and chlorobutane and its application, specifically disclosing: an analytical method for the simultaneous determination of chloroethane and chlorobutane, characterized by adopting an HS-GC-MS method and detailed parameter settings. Wherein, in this detection method, the limit of chloroethane and chlorobutane in the clindamycin hydrochloride raw material is 0.833 μg / g (0.833 ppm). In addition, the present invention also provides the application of the analytical method for the simultaneous determination of chloroethane and chlorobutane in the control of clindamycin hydrochloride raw material. It should be noted that toxicological studies have shown that the median lethal dose (LD50 rat oral) of chlorobutane is 1500 mg / kg, and the toxicity of its metabolite β-chloroethanol needs to be paid special attention. In its latest Q3C guidance, the FDA set the permitted daily exposure for chlorobutane at 7.1 mg / day and requires at least two stages of condensation recovery in the synthesis process. The application of modern online infrared monitoring technology enables real-time control of chlorobutane residues in the reaction system with an accuracy of 0.1%. In Pfizer's darunavir synthesis process, chlorobutane remains fluid at -15°C, with a viscosity (0.45 cP) only one-third that of THF. This low-temperature fluidity enables efficient multi-step cascade reactions in microreactors. Innovations in the solvent recovery system have reduced the single use of chlorobutane by 70%, with residual detection levels consistently below 5 ppm. In the field of occupational exposure protection, the coordinated use of engineering controls and personal protective equipment (PPE) has become the industry standard. Roche's Suzhou plant's enclosed production line is equipped with a two-stage activated carbon adsorption system, combined with Level A protective clothing and online gas monitoring, to maintain chlorobutane concentrations in the operating area below 0.5 ppm. This comprehensive protection system keeps occupational health risks at ALARP (As Low As Reasonably Practicable) levels. As can be seen, the requirements for the detection limit of chlorobutane in this field are becoming increasingly stringent. The lower the chlorobutane content in pharmaceuticals, solvents, or everyday situations, the better. To ensure the quality of lincomycin hydrochloride and the safety of patients, the chlorobutane content in lincomycin hydrochloride needs to be strictly controlled. Developing a more sensitive method for detecting chlorobutane content in lincomycin hydrochloride is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material, which can sensitively detect the content of chlorobutane in lincomycin hydrochloride raw material.

[0005] The present invention provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material medicine, which adopts gas chromatography-mass spectrometry GC-MS to detect the sample to be tested;

[0006] The gas chromatography parameters are as follows: injection port temperature: 145-155°C; heating program: initial temperature 65-75°C, hold for 1.5-2.5 min, then increase the temperature to 95-105°C at a heating rate of 8-12°C / min; then increase the temperature to 235-245°C at a heating rate of 65-75°C / min, and hold for 3-5 min;

[0007] The mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 1.5-2.5 min; transfer line temperature: 245-255° C.; quadrupole temperature: 145-155° C.; and ion source temperature: 225-235° C.

[0008] The present invention adopts a gas chromatography-mass spectrometry (GC-MS) method for detecting the chlorobutane content in the lincomycin hydrochloride bulk drug. This method uses gas chromatography as the separation system for the chlorobutane in the lincomycin hydrochloride bulk drug and mass spectrometry as the detection system for the chlorobutane. GC-MS combines the high separation ability of gas chromatography with the advantages of mass spectrometry, which has high selectivity, high sensitivity, and the ability to provide relative molecular mass and structural information.

[0009] Gas chromatography offers a high degree of separation for samples. Gas chromatography test conditions and parameters, including column selection, starting column temperature, heating rate, column flow rate, and other parameters, can directly impact the separation effect. Selecting appropriate chromatographic conditions and parameters can effectively improve the separation of chlorobutane and other substances. This not only provides sufficient ions for mass spectrometry analysis, but also allows for the initial separation of impurities that interfere with the mass spectrometry, preventing the impact of interfering impurities on subsequent mass spectrometry. This allows for the acquisition of favorable peak parameters, effectively improving the accuracy and precision of test results.

[0010] The present invention screens and optimizes relevant test conditions of gas chromatography and mass spectrometry in gas chromatography-mass spectrometry (GC-MS). The method for detecting the content of chlorobutane in lincomycin hydrochloride raw material has the advantages of low detection limit, high precision, high repeatability, and high accuracy. For example, the detection limit of the present invention can be 6.24 ng / ml.

[0011] Preferably, the heating program is: the initial temperature is 147-152° C., the temperature is increased to 235-245° C. at a heating rate of 47-52° C. / min, and maintained for 0 min.

[0012] Preferably, the chromatographic column of the gas chromatography condition is VF-624ms, with specifications of 30m×0.25mm and 1.4μm.

[0013] Preferably, the gas chromatography parameter conditions also include: flow rate: 1.0-1.5 ml / min; injection volume: 0.8-1.2 μl; split mode: split, split ratio of 8-12:1; control mode: constant flow; carrier gas: He.

[0014] Preferably, the mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 1.8-2.2 min; transfer line temperature: 248-252° C.; quadrupole temperature: 248-252° C.; and ion source temperature: 228-232° C.

[0015] Preferably, the scanning parameters of the mass spectrometer are:

[0016] Analytes Precursor ion (m / z) Product ion (m / z) Dwell time (ms) CE(eV) Chlorobutane* 56 41 100 15 chlorobutane 41 39 100 10

[0017] Preferably, the detection limit of the chlorobutane is 6.24 ng / ml; the quantification limit of the chlorobutane is 12.49 ng / ml.

[0018] Preferably, the preparation method of the lincomycin hydrochloride bulk drug test solution is as follows: weigh 0.08-0.12 g of lincomycin hydrochloride bulk drug sample, place it in a 10 ml volumetric flask, add blank solution to completely dissolve the lincomycin hydrochloride bulk drug sample and dilute to the scale, and shake well to obtain the solution.

[0019] Preferably, the blank solution is methanol.

[0020] In summary, the technical solution of the present invention has the following effects:

[0021] In this experiment, a GC-MS / MS method was established to detect the content of chlorobutane in lincomycin hydrochloride raw materials. The method was evaluated for system applicability, specificity, linearity and range, detection limit and quantification limit, precision, accuracy, solution stability and durability. The results were in compliance with the requirements of the "9101 Guidelines for Validation of Analytical Methods for Pharmaceutical Quality Standards" in Part IV of the 2020 edition of the "Chinese Pharmacopoeia". Therefore, this method can be used to accurately detect the content of chlorobutane in lincomycin hydrochloride.

[0022] The invention provides a detection method with fast detection speed and simple operation method for detecting the content of chlorobutane in lincomycin hydrochloride raw material and injection by gas chromatography-mass spectrometry technology.

[0023] The present invention screens and optimizes relevant test conditions of gas chromatography and mass spectrometry in gas chromatography-mass spectrometry (GC-MS), and provides a method for detecting the content of chlorobutane in a lincomycin hydrochloride bulk drug, which has the advantages of low detection limit, high precision, high repeatability and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The specificity determination results of the blank solution in the embodiment are shown in FIG.

[0025] Figure 2 The specificity determination results of the test solution in the embodiment are shown in FIG.

[0026] Figure 3 The specificity test results of the 100% limit concentration reference solution in the examples are as follows;

[0027] Figure 4 The specificity test results of the test solution spiked with 100% limit concentration in the example are as follows;

[0028] Figure 5 It is a linear graph of chlorobutane in the examples. DETAILED DESCRIPTION

[0029] 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.

[0030] According to the limit requirements of the present invention, the limit of chlorobutane (hereinafter referred to as chlorobutane) in lincomycin hydrochloride is 170 ppm. Based on the properties of the raw material and the target substance, the present invention establishes a GC-MS method to detect the content of chlorobutane in lincomycin hydrochloride and conducts method validation.

[0031] Under the final selected conditions, the system suitability, specificity, linearity and range, detection limit, quantification limit, accuracy, repeatability and stability of the method were verified, confirming that the method is suitable for the determination of chlorobutane in lincomycin hydrochloride.

[0032] The information of the main instruments, test samples, reagents and reference substances of the present invention are shown in Tables 1-4 respectively.

[0033] Table 1. Main instrument information

[0034]

[0035] Table 2. Sample information

[0036] name batch number Lincomycin hydrochloride* YL-71220401 Lincomycin hydrochloride YL-71220402 Lincomycin hydrochloride YL-71220403

[0037] Note: “*” indicates that this batch of samples is a method validation batch.

[0038] Table 3. Reagent information

[0039] name Specification level batch number source Methanol 4L / bottle HPLC O4571440 Shanghai Anpu Cui Shi

[0040] Table 4. Reference Material Information

[0041] Compound name source batch number content(%) 1-Chlorobutane Beijing Inokai KYGWH02 99

[0042] Example

[0043] Example 1

[0044] Example 1 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material.

[0045] The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug in Example 1 specifically comprises the following steps in sequence:

[0046] 1. Gas chromatography conditions

[0047] Chromatographic column: VF-624ms (30m×0.25mm, 1.4μm); Chromatographic column number: GB-G-21-09-109;

[0048] Inlet temperature: 150°C; flow rate: 1.2 ml / min; injection volume: 1 μl; split mode: split, split ratio 10:1; control mode: constant flow; carrier gas: He; heating program (see Table 5):

[0049] Table 5. Heating schedule

[0050] Rate (℃ / min) Temperature (℃) Holding time (min) / 70 2 10 100 0 60 240 4

[0051] 2. Mass spectrometry ion source parameters: as shown in Table 6.

[0052] Table 6. Mass spectrometry ion source parameters

[0053] Collection Type MRM Ion source EI Solvent delay 2min Transmission line temperature 250℃ Quadrupole temperature 150℃ Ion source temperature 230℃

[0054] 3. Scan parameters: as shown in Table 7.

[0055] Table 7. Scan parameters

[0056] Analytes Precursor ion (m / z) Product ion (m / z) Dwell time (ms) CE(eV) Chlorobutane* 56 41 100 15 chlorobutane 41 39 100 10

[0057] Note: * indicates quantitative ion.

[0058] 4. Solution Preparation and Acceptance Criteria

[0059] 4.1 Solution preparation

[0060] 4.1.1 Blank solution

[0061] Methanol.

[0062] 4.1.2 Test solution

[0063] Weigh about 0.1 g of lincomycin hydrochloride sample, accurately weigh it, place it in a 10 ml centrifuge tube, accurately measure 2 ml of blank solution to completely dissolve it, and shake well to obtain the product.

[0064] 4.1.3 Reference substance stock solution

[0065] 4.1.3.1 Reference Substance Stock Solution I

[0066] Accurately weigh an appropriate amount of chlorobutane standard solution and place it in a 10 ml volumetric flask. Add blank solution to dilute to the scale and shake well to obtain chlorobutane stock solution I, i.e., reference substance stock solution I. See Table 8 for details.

[0067] Table 8. Reference substance stock solution I

[0068]

[0069] 4.1.3.2 Intermediate concentration stock solution

[0070] Accurately measure an appropriate amount of chlorobutane stock solution I and place it in a 10 ml volumetric flask. Add blank solution to dilute to the scale and shake well to obtain intermediate concentration stock solution I. See Table 9 for details.

[0071] Table 9. Intermediate concentration stock solution I

[0072] Source solution name Measured volume (ml) Fixed volume (ml) Concentration (μg / ml) Preparation solution name Chlorobutane stock solution I 0.25 10 107.63775 Intermediate concentration stock solution I

[0073] Accurately measure an appropriate amount of intermediate concentration stock solution I and place it in a 10 ml volumetric flask. Add blank solution to dilute to the scale and shake well to obtain intermediate concentration stock solution II. See Table 10 for details.

[0074] Table 10. Intermediate concentration stock solution II

[0075] Source solution name Measured volume (ml) Fixed volume (ml) Concentration (ng / ml) Preparation solution name Intermediate concentration stock solution I 0.1 10 1076.3775 Intermediate concentration stock solution II

[0076] 4.1.4 Detection limit solution

[0077] Accurately measure an appropriate amount of intermediate concentration stock solution II into a 10 ml volumetric flask, dilute to the scale with blank solution, shake well, and obtain. See Table 11 for details.

[0078] Table 11. Detection limit solutions

[0079]

[0080] 4.1.5 Standard curve solution

[0081] Accurately measure appropriate amounts of intermediate concentration stock solution II into different volumetric flasks, dilute to the scale with blank solution, and shake well to obtain a series of standard curve solutions with different concentrations. See Table 12 for details.

[0082] Table 12. Standard curve solutions

[0083]

[0084] 4.1.6 System suitability solution

[0085] Take the "100% limit concentration reference solution (STD4 solution)" under "4.1.5" and inject it 6 times continuously for analysis. 4.1.7 Detection limit and quantification limit solution

[0086] Detection limit solution: Take the "LOD solution" under "4.1.4" and inject it three times continuously for analysis;

[0087] Limit of Quantitation Solution: Take the “LOQ Solution” under “4.1.5” and inject it 6 times continuously for analysis.

[0088] 4.1.8 Accuracy solution

[0089] Test solution: Take the “test solution” under “4.1.2”;

[0090] 50% limit concentration spiked test solution: Weigh approximately 0.1 g of lincomycin hydrochloride sample accurately, place in a 10 ml centrifuge tube, accurately add 2 ml of STD2 solution under "4.1.5", shake well, and prepare 3 portions in the same way;

[0091] 100% limit concentration spiked test solution: Weigh approximately 0.1 g of lincomycin hydrochloride sample accurately, place in a 10 ml centrifuge tube, accurately add 2 ml of STD4 solution under "4.1.5", shake well, and prepare 3 portions in the same way;

[0092] 150% limit concentration spiked test solution: Weigh approximately 0.1 g of lincomycin hydrochloride sample accurately, place in a 10 ml centrifuge tube, accurately add 2 ml of STD5 solution from "4.1.5", shake well, and prepare 3 aliquots by the same method. See Table 13 for details.

[0093] Table 13. Accuracy Solutions

[0094]

[0095] The above solutions were injected once for analysis.

[0096] 4.1.9. Specific solutions

[0097] Blank solution: Take the “blank solution” under “4.1.1”;

[0098] Test solution: Take the “test solution” under “4.1.2”;

[0099] 100% limit concentration reference solution: Take the "100% limit concentration reference solution (STD4 solution)" under "4.1.5";

[0100] 100% limit concentration spiked test solution: Take the "100% limit concentration spiked test solution" under "4.1.8";

[0101] The above solutions were injected once for analysis.

[0102] 4.1.10 Stability solution

[0103] Test solution: Take the “test solution” under “4.1.2”;

[0104] 100% limit concentration reference solution: Take the "100% limit concentration reference solution (STD4 solution)" under "4.1.5";

[0105] 100% limit concentration spiked test solution: Take the "100% limit concentration spiked test solution" under "4.1.8";

[0106] The above solutions were placed at room temperature for different periods of time and injected once for detection.

[0107] 4.1.12 Reproducible solutions

[0108] Test solution: Take the “test solution” under “4.1.8”.

[0109] 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 14 for details.

[0110] Table 14. Reproducibility of solutions

[0111] serial number Sample weight (g) Measure the volume of STD4 solution (ml) Approximately equivalent to the limit concentration percentage (%) REP-1 0.1019 2 100 REP-2 0.1020 2 100 REP-3 0.1025 2 100 REP-4 0.1003 2 100 REP-5 0.1010 2 100 REP-6 0.1013 2 100

[0112] 4.2 Acceptance Criteria

[0113] Calculated by the standard curve method, the concentration of chlorobutane shall not exceed 1.25 ppm.

[0114] 5. Verify the results

[0115] 5.1 Methodology Validation Results

[0116] Methodological validation was performed based on the analytical method, and the results are shown in Table 15.

[0117] Table 15. Summary of results

[0118]

[0119]

[0120]

[0121] 5.2 System Applicability

[0122] 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 (n=6) of the retention time should not exceed 1%. See Table 16 for the results of the system suitability solution analysis.

[0123] Table 16. System suitability results

[0124]

[0125]

[0126] The results showed that after 6 consecutive injections of system suitability solution, the RSDs (n=6) of the chlorobutane peak area and retention time were 3.4% and 0.1%, respectively, and the system suitability met the requirements.

[0127] 5.3. Exclusivity

[0128] 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 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 integrated area of ​​the LOQ of each target compound; in the chromatogram of the test solution, if the target peak is detected, the separation degree of the target peak and the adjacent peaks shall be greater than 1.5; the target peak shall be displayed in the chromatogram of the 100% limit concentration reference solution; the separation degree of the target peak and all adjacent peaks greater than LOQ in the chromatogram of the 100% limit concentration spiked test solution shall be greater than 1.5. The determination results of each specific solution are shown in Figures 1 to 4 .

[0129] 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 not detected in the chromatogram of the test solution; 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 peaks with peak areas greater than LOQ were detected; the results were in compliance with the regulations and the method had good specificity.

[0130] 5.4 Linearity and range

[0131] 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 17 and Figure 5 .

[0132] Table 17. Linearity results table

[0133] name Concentration (ng / ml) Peak area STD1 12.49 9065 STD2 31.21 19075 STD3 49.94 30935 STD4 62.43 37255 STD5 93.75 55988 STD6 125.08 70482

[0134] From the above results, the linearity and range results are shown in Table 18.

[0135] Table 18. Linearity and range results

[0136]

[0137]

[0138] The results showed that the concentration of chlorobutane was in the range of 12.49 ng / ml to 125.08 ng / ml, which was approximately 20% to 200% of the limit concentration. The peak area of ​​chlorobutane was linear with the concentration, with a correlation coefficient r of 0.9989. The ratio of the absolute value of the y-axis intercept to the 100% limit concentration response value was 6.8%, and the linear results were in compliance with the regulations.

[0139] 5.5. Limit of Detection and Limit of Quantitation

[0140] Prepare the "LOD Solution" and "LOQ Solution" under "4.1.7" and perform the analytical method under "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 19 and 20.

[0141] Table 19. Detection limit solution results

[0142]

[0143] Table 20. Quantitation limit solution results

[0144]

[0145] Note: “NA” means not applicable.

[0146] The results showed that the concentration of chlorobutane in the detection limit solution for three consecutive injections was 6.24 ng / ml, approximately equivalent to 10% of the limit concentration, and the S / N was in the range of 34.6 to 36.9; the concentration of chlorobutane in the quantitative limit solution for six consecutive injections was 12.49 ng / ml, approximately equivalent to 20% of the limit concentration, and the S / N was in the range of 70.0 to 92.6. The RSD of the peak area (n=6) was 12.0%. The detection limit and quantitative limit results of this method met the requirements.

[0147] 5.6 Repeatability

[0148] Take 2 portions of the test solution under item "4.1.12" and 6 portions of the test solution spiked with 100% limit concentration, and measure according to the analytical method under item "3". Record the chromatogram. The recovery rate of the target compound in the 6 portions of the test solution spiked with 100% limit concentration should be between 75% and 120%, 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 Table 21.

[0149] Table 21. Chlorobutane repeatability results

[0150]

[0151] The results showed that the recoveries of chlorobutane in 6 samples spiked with 100% limit concentration ranged from 98.0% to 101.6%, with an RSD (n=6) of 1.4%. The method had good repeatability.

[0152] 5.7 Accuracy

[0153] 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 75% and 120%, with the RSD (n=9) of the recovery not exceeding 15%. See Table 22 for the accuracy solution analysis results.

[0154] Table 22. Chlorobutane accuracy results

[0155]

[0156]

[0157] The results showed that the recovery of chlorobutane in 9 spiked test solutions at 50%, 100% and 150% limit concentrations ranged from 94.3% to 119.4%, with an RSD (n=9) of 7.6%. The accuracy results met the requirements.

[0158] 5.8 Stability

[0159] 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 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 75% and 120%, indicating that the solution was stable within the observed time period. The stability test results for each solution are shown in Table 23.

[0160] Table 23. Chlorobutane stability results

[0161]

[0162] Note: “NA” means not applicable. LOQ is 12.49 ng / ml, or 0.25 ppm.

[0163] The results showed that

[0164] (1) After the test solution was left at room temperature for 2.5 hours, chlorobutane was detected, but the amount was less than the limit of quantification;

[0165] (2) After the 100% limit concentration reference solution was placed at room temperature for 2.5 hours, the ratio of the detected concentration of chlorobutane to the initial (0 hour) detection concentration was 108.5%; after being placed at room temperature for 7.0 hours, the ratio of the detected concentration of chlorobutane to the initial (0 hour) detection concentration was greater than 120%;

[0166] (3) After the 100% limit concentration spiked test solution was placed at room temperature for 2.5 hours, the ratio of the detected concentration of chlorobutane to the initial (0 hour) detection concentration was 107.8%; after being placed at room temperature for 7.0 hours, the ratio of the detected concentration of chlorobutane to the initial (0 hour) detection concentration was greater than 120%;

[0167] Therefore, the test solution, 100% limit concentration reference solution and 100% limit concentration spiked test solution should be prepared immediately before use at room temperature.

[0168] 6. Summary

[0169] In this experiment, a GC-MS / MS method was established to detect the content of chlorobutane in lincomycin hydrochloride. The method was evaluated for system applicability, specificity, linearity and range, detection limit and quantification limit, repeatability, accuracy and solution stability. The results were in compliance with the requirements of the "9101 Guidelines for Validation of Analytical Methods for Pharmaceutical Quality Standards" in Part IV of the 2020 edition of the "Chinese Pharmacopoeia". Therefore, this method can be used to accurately detect the content of chlorobutane in lincomycin hydrochloride.

[0170] 7. Sample testing

[0171] Prepare the corresponding batch of test solution according to the test solution preparation method under "4.1.2" and use the analytical method under "3" for sampling and analysis. The results are shown in Table 24.

[0172] Table 24. Sample test results

[0173]

[0174] Note: The detection limit of chlorobutane is 6.24 ng / ml, i.e. 0.12 ppm (μg / g, converted to test sample concentration), and the LOQ is 12.49 ng / ml, i.e. 0.25 ppm (μg / g, converted to test sample concentration).

[0175] Example 2

[0176] Example 2 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material.

[0177] The difference between Example 2 and Example 1 is that the temperature rise program in the gas chromatography conditions is different, as shown below.

[0178] Gas chromatography conditions are:

[0179] Chromatographic column: VF-624ms, specifications: 30m×0.25mm, 1.3μm;

[0180] Inlet temperature: 145°C; flow rate: 1.0 ml / min; injection volume: 0.8 μl; split mode: split, split ratio 8:1; control mode: constant flow; carrier gas: He; heating program: initial temperature of 65°C, maintained for 1.5 min, and then heated to 95°C at a heating rate of 8°C / min; then heated to 235 at a heating rate of 65°C / min and maintained for 3 min.

[0181] The parameters of the mass spectrometry were as follows: acquisition type: MRM; ion source: EI; solvent delay: 1.5 min; transfer line temperature: 245°C; quadrupole temperature: 145°C; and ion source temperature: 225°C.

[0182] Specificity test results:

[0183] 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.

[0184] Example 3

[0185] Example 3 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride bulk drug.

[0186] The difference between Example 3 and Example 1 is that the temperature rise program in the gas chromatography conditions is different, as shown below.

[0187] Gas chromatography conditions are:

[0188] Chromatographic column: VF-624ms, specifications: 30m×0.25mm, 1.5μm;

[0189] Inlet temperature: 155°C; heating program: initial temperature 75°C, hold for 2.5 min, increase to 105°C at a rate of 12°C / min; then increase to 245°C at a rate of 75°C / min, hold for 3-5 min;

[0190] The parameters of the mass spectrometry were as follows: acquisition type: MRM; ion source: EI; solvent delay: 2.5 min; transfer line temperature: 255°C; quadrupole temperature: 155°C; and ion source temperature: 235°C.

[0191] Specificity test results:

[0192] 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.

[0193] Comparative Example

[0194] Comparative Example 1

[0195] Comparative Example 1 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material.

[0196] The difference between Comparative Example 1 and Example 1 is that the temperature rise program in the gas chromatography conditions is different, as shown below.

[0197] Chromatographic column: VF-624ms (30m×0.25mm, 1.4μm);

[0198] Inlet temperature: 165°C; flow rate: 1.2 ml / min; injection volume: 0.5 μl; split mode: split, split ratio 5:1; control mode: constant flow; carrier gas: He; heating program: initial temperature of 50°C, heating to 120°C at a heating rate of 15°C / min; then heating to 260°C at a heating rate of 90°C / min, and maintaining for 5 minutes.

[0199] Specificity test results:

[0200] The results showed that the test sample interfered with the detection of the target peak.

[0201] Comparative Example 2

[0202] Comparative Example 2 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material.

[0203] The difference between Comparative Example 2 and Example 1 is that the temperature rise program in the gas chromatography conditions is different, as shown below.

[0204] Gas chromatography conditions are:

[0205] Chromatographic column: VF-624ms (30m×0.25mm, 1.4μm);

[0206] Inlet temperature: 120°C; flow rate: 1.0 ml / min; injection volume: 1 μl; split mode: split, split ratio 15:1; control mode: constant flow; carrier gas: He; heating program: initial temperature of 50°C, maintained for 1.5 min, heated to 70°C at a heating rate of 8°C / min; then heated to 200°C at a heating rate of 50°C / min, maintained for 3 min.

[0207] Specificity test results:

[0208] The results showed that the test sample interfered with the detection of the target peak.

[0209] Comparative Example 3

[0210] Comparative Example 3 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material.

[0211] The difference between Comparative Example 3 and Example 1 is that the temperature rise program in the gas chromatography conditions is different, as shown below.

[0212] Gas chromatography conditions are:

[0213] Chromatographic column: VF-624ms (30m×0.25mm, 1.4μm);

[0214] Inlet temperature: 170°C; flow rate: 0.7 ml / min; injection volume: 2.0 μl; split mode: split, split ratio 10:1; control mode: constant flow; carrier gas: He; heating program: initial temperature 80°C, heating to 240°C at a heating rate of 80°C / min, and maintaining for 5 minutes.

[0215] Specificity test results:

[0216] The results showed that the test sample interfered with the detection of the target peak.

[0217] Comparative Example 4

[0218] Comparative Example 4 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material.

[0219] The difference between Comparative Example 4 and Example 1 is that the mass spectrometry parameter conditions are different, as shown below.

[0220] The parameters of the mass spectrometry were as follows: acquisition type: MRM; ion source: EI; solvent delay: 3.5 min; transfer line temperature: 220°C; quadrupole temperature: 100°C; and ion source temperature: 200°C.

[0221] Specificity test results:

[0222] The results showed that no target peak was detected.

[0223] Comparative Example 5

[0224] Comparative Example 5 provides a method for detecting the content of chlorobutane in lincomycin hydrochloride raw material.

[0225] The difference between Comparative Example 5 and Example 1 is that:

[0226] The chromatographic column used was DB-624UI, 30 m × 0.32 mm × 1.8 μm;

[0227] Inlet temperature: 150°C; flow rate: 1.0 ml / min; injection volume: 1 μl; split mode: split, split ratio 30:1; control mode: constant flow; carrier gas: He; temperature program (see Table 25):

[0228] Table 25. Heating schedule

[0229] Rate (℃ / min) Temperature (℃) Holding time (min) / 40 4 20 100 1 35 150 1

[0230] 2. Mass spectrometry ion source parameters: as shown in Table 26.

[0231] Table 26. Mass spectrometry ion source parameters

[0232] Collection Type SIM Ion source EI Solvent delay 1.6min Transmission line temperature 250℃ Quadrupole temperature 150℃ Ion source temperature 230℃

[0233] 3. Scan parameters: as shown in Table 27.

[0234] Table 27. Scan parameters

[0235] Analytes Precursor ion (m / z) Product ion (m / z) Dwell time (ms) CE(eV) Chlorobutane* 56 41 100 15 Chlorobutane 41 39 100 10

[0236] Specificity test results:

[0237] The results showed that no target peak was detected.

[0238] 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 chlorobutane in lincomycin hydrochloride bulk drug, characterized in that: Gas chromatography-mass spectrometry GC-MS was used to detect the samples to be tested; The gas chromatography parameters are as follows: injection port temperature: 145-155°C; heating program: initial temperature 65-75°C, hold for 1.5-2.5 min, increase to 95-105°C at a heating rate of 8-12°C / min; then increase to 235-245°C at a heating rate of 65-75°C / min, hold for 3-5 min; The mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 1.5-2.5 min; transfer line temperature: 245-255°C; quadrupole temperature: 145-155°C; and ion source temperature: 225-235°C.

2. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to claim 1, wherein The heating program is as follows: the initial temperature is 147-152° C., the temperature is increased to 235-245° C. at a heating rate of 47-52° C. / min, and the temperature is maintained for 0 min.

3. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to claim 1, wherein The chromatographic column of the gas chromatography conditions is VF-624ms, with specifications of 30m×0.25mm and 1.4μm.

4. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to claim 1, wherein The gas chromatography parameters are also Including: flow rate: 1.0~1.5ml / min; injection volume: 0.8~1.2μl; split mode: split, split ratio is 8~12:1; control mode: constant flow; carrier gas: He.

5. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to claim 1, wherein The mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 1.8-2.2 min; transfer line temperature: 248-252° C.; quadrupole temperature: 248-252° C.; and ion source temperature: 228-232° C.

6. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to claim 1, wherein The scanning parameters of the mass spectrometer are:

7. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to claim 1, wherein The detection limit of the chlorobutane is 6.24 ng / ml; the quantification limit of the chlorobutane is 12.49 ng / ml.

8. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to any one of claims 1 to 7, characterized in that: The preparation method of the lincomycin hydrochloride bulk drug test solution is as follows: weigh 0.08-0.12 g of the lincomycin hydrochloride bulk drug sample, place it in a 10 ml volumetric flask, add blank solution to completely dissolve the lincomycin hydrochloride bulk drug sample and dilute to the scale, and shake well to obtain the solution.

9. The method for detecting the content of chlorobutane in the lincomycin hydrochloride bulk drug according to claim 8, wherein: The blank solution is methanol.

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