General analysis and detection method for seven sulfonates in medicine

Abstract

The invention discloses a general analysis and detection method for seven sulfonates in a medicine, which comprises sample pretreatment, gas chromatography mass spectrometry (GCMS) analysis and detection, and qualitative and quantitative analysis, and comprises the following specific steps: step 1, dissolving a sample in an acidic aqueous solution, extracting with an organic solvent, and centrifuging to take supernate to form a sample solution; 2, injecting the sample solution and the reference substance solution into a gas chromatograph-mass spectrometer for detection; and 3, qualitative and quantitative analysis. According to the present invention, the seven sulfonic acid esters are subjected to general analysis method development, the seven sulfonic acid esters common in the process are simultaneously and effectively extracted from the sample matrix after the dilution with the acidic aqueous solution, and the prototype compound is detected by using the GCMS, such that the matrix effect can be avoided, and the false positive can be avoided. The detection sensitivity can reach 0.5-1 ppm, the linear range is 100 times, the stability is 48 hours, and the method is high in universality and can be widely applied to different types of raw materials, intermediates and API samples.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection, and relates to the analysis of sulfonate genotoxic impurities in raw materials, and in particular to a method for simultaneously detecting sulfonates using GCMS and a high-sensitivity analysis and detection method, and specifically relates to a universal analysis and detection method for seven sulfonates in a drug. Background Art

[0002] The analysis and control of sulfonate genotoxic impurities is an important part of drug quality control. The currently commonly used sulfonate GCMS method has a prominent sample matrix problem. Due to the high boiling point of sulfonates, headspace injection cannot be used and can only be tested by direct injection. Therefore, high-concentration samples are easily left in the system, affecting the analysis of subsequent samples and causing failure of label insertion and recovery. The European Pharmacopoeia reported the headspace derivatization GCMS method for methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate, but in actual application, it was found that blank samples were easily interfered at the methyl methanesulfonate derivative product iodomethane, affecting the accuracy and sensitivity of the method, and other methyl groups in the sample may also participate in the derivatization reaction, resulting in false positives. Most of the methanesulfonate detection methods currently on the market are direct injection GCMS detection or reference European Pharmacopoeia methods, which are prone to matrix effects and false positives. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for detecting sulfonate compounds in medicines, which is simple in sample pretreatment, can detect prototype compounds, and avoids matrix effects and false positives.

[0004] In order to solve the above technical problems, the specific technical solutions adopted by the present invention are as follows:

[0005] A universal analytical detection method for seven sulfonate esters in a drug, characterized by comprising sample pretreatment, gas chromatography mass spectrometry (GCMS), qualitative and quantitative analysis, and the specific steps are as follows:

[0006] Step 1: dissolve the sample in an acidic aqueous solution, extract with an organic solvent, and centrifuge to obtain the supernatant to form a sample solution;

[0007] Step 2: injecting the sample solution and the reference solution into a gas chromatography-mass spectrometer for detection;

[0008] Step 3: Qualitative and quantitative analysis;

[0009] The seven sulfonates are methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), methyl ethanesulfonate (MES), isopropyl methanesulfonate (IMS), ethyl ethanesulfonate (EES), isopropyl ethanesulfonate (IES), and n-butyl methanesulfonate (BMS).

[0010] In some specific embodiments, the sulfonate ester detected is one or more of the above 7 types.

[0011] In some specific embodiments, the acidic compound of the acidic aqueous solution in step 1 is one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, sulfuric acid, and nitric acid.

[0012] Furthermore, the acidic aqueous solution is a formic acid aqueous solution with a mass percentage of 0.1% to 8%.

[0013] Preferably, the acidic aqueous solution is a 6% by mass formic acid aqueous solution.

[0014] In some specific embodiments, the organic solvent in step 1 is selected from one or more of ethyl acetate, other ester compounds, methyl tert-butyl ether, and n-butanol.

[0015] In some specific embodiments, the sample solution in step 1 and step 2 is selected from one or more of in-process control samples, raw material samples, intermediate samples, and product samples.

[0016] In some specific embodiments, the preparation method of the reference solution in step 2 is: add an organic solvent to dissolve after weighing, mix to volume, then add the acidic aqueous solution and the organic solvent for extraction, centrifuge and take the supernatant; step 2 also includes a reporting limit solution.

[0017] In some specific embodiments, the chromatographic conditions of step 2 are as follows: the chromatographic column is Agilent DB-624, 60m×0.32mm, ID 1.8μm, PN:123-1364; the injection port temperature is 240°C; the chromatographic column temperature program is 100°C for 1 min, the temperature program is 5°C / min to 180°C, the temperature program is 25°C / min to 220°C and then maintained for 2 min, the temperature program is 50°C / min to 250°C and then maintained for 15 min; the flow rate is 2.5mL / min; the split ratio is 5:1; the mass spectrometry conditions of step 2 are as follows: the ion source is EI; the ion source temperature is 230°C; the quadrupole temperature is 150°C; the scanning mode is SIM.

[0018] In some specific embodiments, the calculation formula for the qualitative and quantitative analysis in step 3 is:

[0019]

[0020] Where:

[0021] A STD Represents the average of the sulfonate peak areas in the first three or six STD injections (IPC is the STD peak area);

[0022] ASPL Represents the peak area of ​​sulfonate ester in the sample solution;

[0023] W STD represents the mass of the control solution in mg;

[0024] V STD represents the volume of the control solution in mL;

[0025] W SPL Represents the mass of the sample in mg;

[0026] V SPL Represents the dilution volume of the sample solution in mL.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention develops a universal analytical method for seven sulfonates. After diluting with an acidic aqueous solution, the seven sulfonates commonly used in the process are effectively extracted from the sample matrix at the same time. The prototype compound is detected by GCMS, which can avoid both matrix effects and false positives. The detection sensitivity can reach 0.5-1ppm, the linear range is 100 times, and the stability is 48h. The method has strong versatility and can be widely applied to different types of raw materials, intermediates and API samples.

[0029] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the linear result and range graph of MMS.

[0031] Figure 2 It is the linear result and range graph of EMS.

[0032] Figure 3 It is the linear result and range diagram of MES.

[0033] Figure 4 It is the linearity result and range graph of IMS.

[0034] Figure 5 are the linearity results and range graphs of EES.

[0035] Figure 6 It is the linear result and range diagram of IES.

[0036] Figure 7 Here are the linear results and range graphs for the BMS.

[0037] Figure 8This is an overlay of blank, sensitivity solution, reference solution, sample solution, and sample spike solution for 7 sulfonate esters. BLK represents blank solution, LOQ represents sensitivity solution, STD represents reference solution, SPL represents sample solution, LOQ-REC represents sensitivity spike solution, and STD-REC represents reference spike solution.

[0038] Fig. 9 This is the test result of the sample using the European Pharmacopoeia method. Among them, BLK represents blank solution, STD represents reference solution, and SPL represents sample solution.

[0039] Fig.10 It is a graph of the test results of the sample using the method of the present invention, wherein BLK represents blank solution, STD represents reference solution, and SPL represents sample solution. DETAILED DESCRIPTION

[0040] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0041] It should be noted that the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology, and are not used to limit the conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance. Any adjustments without creative effort, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0042] All kinds of reagents, antibodies, and consumables involved in the examples are commercially available products, and all commercial products can be used.

[0043] A preferred embodiment of the present invention is to dissolve the sample with 6% formic acid aqueous solution, extract with ethyl acetate, and then centrifuge at 10000rpm to take the supernatant for sampling. This pretreatment method enhances the versatility of the present method. The present invention establishes a universal analytical method for detecting 7 sulfonates by gas chromatography-mass spectrometry (GCMS) technology. This technology is applicable to the GCMS quantitative determination of 7 sulfonate genotoxic impurities methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), methyl ethanesulfonate (MES), isopropyl methanesulfonate (IMS), ethyl ethanesulfonate (EES), isopropyl ethanesulfonate (IES), and n-butyl methanesulfonate (BMS) in raw materials, in-process control, intermediates, and products of clinical projects. Its corresponding working range and retention time are listed in Table 1 below:

[0044] Table 1 Detection range and retention time of 7 sulfonates

[0045]

[0046]

[0047] Note: The above retention times are obtained based on Agilent 8890B GC with 5977B mass spectrometer detector. The retention times may vary depending on the system.

[0048] The instruments, equipment, reagents, etc. designed by the present invention are shown in Table 2 below:

[0049] Table 2 Instruments and reagents

[0050]

[0051]

[0052] Example 1 Chromatographic and mass spectrometric conditions

[0053] Set the chromatographic and mass spectrometric conditions as shown in Table 3 and Table 4 below:

[0054] Table 3 Chromatographic conditions

[0055]

[0056] Table 4 Mass spectrometry conditions

[0057]

[0058]

[0059] Example 2

[0060] For samples with alkaline solution pH, the effects of several different mass percentages of formic acid aqueous solutions on the spiked recovery of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate were studied. The reference concentration was 1 μg / ml, and the sample concentration was 200 mg / ml. Other conditions were: the organic solvent extractant was ethyl acetate, and the supernatant was taken for sampling at 10,000 rpm. The experimental results are shown in Table 5 below:

[0061] Table 5 Comparison of recovery rates of reference substances

[0062] Methyl methanesulfonate Ethyl methanesulfonate Isopropyl methanesulfonate 0% formic acid aqueous solution 0％ 101.9％ 121.6％ 4% formic acid aqueous solution 83.0％ 96.2％ 115.8％ 6% formic acid aqueous solution 104.6％ 108.2％ 108.7％

[0063] It can be seen that under the conditions of this embodiment, the 6% formic acid aqueous solution group has a better spike recovery rate.

[0064] Example 3 Solution Preparation

[0065] 1. Diluent

[0066] Diluent 1: Acetonitrile

[0067] Diluent 2: Ethyl acetate

[0068] Diluent 3: 6% formic acid aqueous solution

[0069] 2. Preparation of blank solution

[0070] Pipette 2.0 mL of diluent 3 into a 5-mL centrifuge tube, add 40 μL of diluent 1, then add 2.0 mL of diluent 2, and seal. Vortex at 1000 rpm for 10 min to mix thoroughly; then transfer to a centrifuge and centrifuge at 10,000 rpm for 5 min. Take the supernatant for injection and mark it as blank.

[0071] 3. Preparation of control solution

[0072] The concentration of the reference solution can be determined based on the specification requirements and the concentration of the sample. The selected reference concentration should be within the verified linear range (MES: 0.0001mg / mL to 0.004mg / mL, MMS, EMS, IMS, EES, IES and BMS: 0.00005mg / mL to 0.004mg / mL).

[0073] Table 6 below shows the preparation method of 0.002 mg / mL STD solution. If STD is of other concentrations, the preparation process can be adjusted according to the actual required concentration.

[0074] Table 6 Preparation of reference solution

[0075]

[0076] Note: If there is no special requirement, for product testing: prepare 2 reference solutions in parallel; for raw material, in-process control and intermediate testing: prepare 1 reference solution. *The concentration of sulfonate in STD and LOQ (ppm, w / w) is converted relative to the sample concentration of 100 mg / mL.

[0077] 4. Preparation of reporting limit solution

[0078] The method for preparing the reporting limit solution is shown in Table 7 below:

[0079] Table 7 Reporting limit solution preparation

[0080]

[0081]

[0082] *The concentration of sulfonate esters at LOQ (ppm, w / w) is converted to a sample concentration of 100 mg / mL.

[0083] 5. Sample solution preparation

[0084] The concentration of the sample solution can be determined based on the specification, linear range, LOQ and sample solubility. 100 mg / mL is recommended as the sample concentration.

[0085] Weigh about 200 mg of sample accurately into a 5 mL centrifuge tube, add 2.0 mL of diluent 3 to dissolve the sample, and dissolve and mix by ultrasonic or vortexing if necessary. Add 40 μL of diluent 1, then add 2.0 mL of diluent 2, seal and mix; vortex oscillator at 1000 rpm, vortex for 10 minutes to mix thoroughly; then transfer to a centrifuge for centrifugation at 10000 rpm for 5 minutes. Take the supernatant for sampling.

[0086] Note: If there is no special requirement, the sample solution should be prepared in duplicate. For the in-process control sample, prepare one.

[0087] Example 4 Calculation of test results

[0088]

[0089] Where:

[0090] A1: average peak area of ​​the target peak in the first six injections of STD-1 solution;

[0091] A2: peak area of ​​target peak in STD-2;

[0092] W1: the sample weight of each target impurity in STD-1 (mg);

[0093] W2: the sample weight of each target impurity in STD-2 (mg);

[0094]

[0095] Where:

[0096] A1: average peak area of ​​the target peak in the first 6 injections (intermediate and raw material are three injections) of STD-1 solution;

[0097] A2: Peak area of ​​the target peak in the insert injection.

[0098] Sulfonate Residue Results Calculation:

[0099]

[0100] Where:

[0101] A STD Represents the average of the sulfonate peak areas in the first three or six STD injections (IPC is the STD peak area);

[0102] A SPL Represents the peak area of ​​sulfonate ester in the sample solution;

[0103] W STD represents the mass of the control solution in mg;

[0104] V STD represents the volume of the control solution in mL;

[0105] W SPL Represents the mass of the sample in mg;

[0106] V SPL Represents the dilution volume of the sample solution in mL.

[0107] Example 5 Analysis and detection results

[0108] In addition to special requirements, the result report requirements are shown in Table 8 below:

[0109] Table 8 Requirements for analysis and test results

[0110]

[0111] The results of the method verification of Example 6 are shown in Table 10 below:

[0112] Table 10 Method validation results

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Comparative Example

[0119] A sample was derivatized with sodium iodide in a headspace bottle according to the European Pharmacopoeia method, and the sulfonate was converted into the corresponding iodide for GCMS detection. The results showed that both methyl methanesulfonate and ethyl methanesulfonate were detected (such as Fig. 9 shown).

[0120] The same sample was tested using the method of the present invention, and neither methyl methanesulfonate nor ethyl methanesulfonate was detected (e.g. Fig.10 shown).

[0121] It can be seen that the existing European Pharmacopoeia method has the problem of easily generating false positives.

[0122] It can be seen from the above embodiments and comparative examples that the method of the present invention has the following advantages over the prior art:

[0123] 1. Can simultaneously detect 7 common sulfonates in the process;

[0124] 2. The detection sensitivity can reach 0.5-1ppm;

[0125] 3. The matrix effect has been overcome, the method is highly versatile and can be widely applied to different API samples;

[0126] 4. Detecting prototype compounds is not prone to false positives.

[0127] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A general analytical method for detecting seven sulfonates in medicines, characterized in that: Including sample pretreatment, gas chromatography-mass spectrometry (GCMS), qualitative and quantitative analysis, the specific steps are as follows: Step 1: dissolve the sample in an acidic aqueous solution, extract with an organic solvent, and centrifuge to obtain the supernatant to form a sample solution; Step 2: injecting the sample solution and the reference solution into a gas chromatography-mass spectrometer for detection; Step 3: Qualitative and quantitative analysis; The seven sulfonates are methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), methyl ethanesulfonate (MES), isopropyl methanesulfonate (IMS), ethyl ethanesulfonate (EES), isopropyl ethanesulfonate (IES), and n-butyl methanesulfonate (BMS).

2. The method according to claim 1, characterized in that The acidic compound of the acidic aqueous solution in step 1 is one or more of formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, sulfuric acid, and nitric acid.

3. The method according to claim 2, characterized in that The acidic aqueous solution is a formic acid aqueous solution with a mass percentage of 0.1% to 8%.

4. The method according to claim 3, characterized in that The acidic aqueous solution is a 6% by mass formic acid aqueous solution.

5. The method according to claim 1, characterized in that The organic solvent in step 1 is selected from one or more of ethyl acetate, other ester compounds, methyl tert-butyl ether, and n-butanol.

6. The method according to claim 1, characterized in that The sample solution in step 1 and step 2 is selected from one or more of in-process control samples, raw material samples, intermediate samples, and product samples.

7. The method according to claim 1, characterized in that The preparation method of the reference solution in step 2 is: add an organic solvent to dissolve after weighing, mix to volume, then add the acidic aqueous solution and the organic solvent for extraction, centrifuge and take the supernatant; step 2 also includes a reporting limit solution.

8. The method according to claim 1, characterized in that The chromatographic conditions of step 2 are as follows: the chromatographic column is Agilent DB-624, 60m×0.32mm, ID 1.8μm, PN:123-1364; the injection port temperature is 240°C; the chromatographic column temperature program is 100°C for 1 min, the temperature program is 5°C / min to 180°C, the temperature program is 25°C / min to 220°C and then maintained for 2 min, the temperature program is 50°C / min to 250°C and then maintained for 15 min; the flow rate is 2.5mL / min; the split ratio is 5:1; the mass spectrometry conditions of step 2 are as follows: the ion source is EI; the ion source temperature is 230°C; the quadrupole temperature is 150°C; the scanning mode is SIM.

9. The method according to claim 1, characterized in that: The calculation formulas for the qualitative and quantitative analysis in step 3 are: Where: A STD Represents the average of the sulfonate peak areas in the first three or six STD injections (IPC is the STD peak area); A SPL Represents the peak area of ​​sulfonate ester in the sample solution; W STD represents the mass of the control solution in mg; V STD represents the volume of the control solution in mL; W SPL Represents the mass of the sample in mg; V SPL Represents the dilution volume of the sample solution in mL.

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