Analysis method for detecting residual quantity of chloroethane and 2-chloropropane in 1-cyclohexylpiperazine
The gas chromatography method with specific solvent and temperature programming effectively addresses the low accuracy issues in detecting residual chloroethane and 2-chloropropane in 1-cyclohexylpiperazine, offering a reliable and efficient analysis for quality control in pharmaceutical production.
Patent Information
- Application Number
- CN202311249311.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-26
- Publication Date
- 2025-03-28
AI Technical Summary
Existing methods for detecting residual chloroethane and 2-chloropropane in 1-cyclohexylpiperazine, such as the GC method using DMSO as a solvent, suffer from low accuracy and are not suitable for quality control in pharmaceutical production due to the reactivity of these impurities.
A gas chromatography method using a flame ionization detector, nitrogen as the carrier gas, a capillary column with a 6% cyanopropylphenyl-94% dimethyloxane fixed phase, gradient temperature programming from 45°C to 220°C, and a top-injection technique with a solvent mixture of ice acetic acid and N,N-dimethylacetamide, optimizing the detection of residual chloroethane and 2-chloropropane in 1-cyclohexylpiperazine.
This method provides a simple, rapid, and accurate analysis for residual chloroethane and 2-chloropropane in 1-cyclohexylpiperazine, ensuring quality standards and being applicable in standard laboratory conditions with common equipment and reagents.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug analysis, and in particular to an analytical method for detecting the residual amounts of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine. Background Art
[0002] 1-Cyclohexylpiperazine is a pharmaceutical intermediate or starting material with a boiling point of 287.25°C and a chemical structural formula of:
[0003]
[0004] The synthetic route of 1-cyclohexylpiperazine is as follows:
[0005]
[0006] The ethanol and isopropanol used in the synthesis of 1-cyclohexylpiperazine may produce potential genotoxic impurities such as ethyl chloride and 2-chloropropane under concentrated hydrochloric acid conditions. According to existing preclinical studies, the DNA alkylation of such impurities can lead to mutagenic, carcinogenic and teratogenic effects. To ensure safety, ethyl chloride and 2-chloropropane need to be controlled.
[0007] "Guangzhou Chemistry", June 2021, Volume 46, Issue 3, discloses a gas chromatography (GC) method for detecting potential genotoxic impurities methyl chloride, ethyl chloride, and 2-chloropropane in elamod, but its solvent is DMSO (dimethyl sulfoxide). When the test sample is 1-cyclohexylpiperazine, the accurate recovery rate of ethyl chloride is low, so it is not suitable for detecting the residual amount of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine. Summary of the invention
[0008] In view of the deficiencies of the prior art, the present invention finally obtains an analytical method suitable for detecting the residual amounts of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine by selecting a suitable solvent and examining different injection methods. Specifically, the present invention adopts the following technical solution:
[0009] Provided is an analytical method for detecting the residual amounts of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine, the method comprising:
[0010] An analytical method for detecting the residual amount of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine, the method being gas chromatography;
[0011] Detector: flame ionization detector;
[0012] Carrier gas: nitrogen;
[0013] Flow rate: 0.9ml / min~1.1ml / min;
[0014] Gas chromatography column: capillary column;
[0015] Preferably, the capillary column is selected from a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase;
[0016] Heating method: 45℃~220℃ gradient heating;
[0017] Injection method: headspace injection;
[0018] Inlet temperature: 230℃~250℃;
[0019] Detector temperature: 230℃~250℃;
[0020] Headspace equilibrium temperature: 80℃~100℃;
[0021] Equilibration time: 20-30 minutes;
[0022] The method comprises: taking ethyl chloride, 2-chloropropane reference solution, and 1-cyclohexylpiperazine test solution for sample analysis respectively, recording chromatograms, and calculating the residual amounts of ethyl chloride and 2-chloropropane in the test solution by using an external standard method.
[0023] Furthermore, the test solution is prepared by taking 1-cyclohexylpiperazine as a test sample, adding a solvent to dissolve it, and diluting it; the ethyl chloride and 2-chloropropane reference solution is prepared by taking ethyl chloride and 2-chloropropane, adding a solvent to dissolve it, and diluting it.
[0024] Furthermore, the final concentration of the test solution is 100 mg / ml; the ethyl chloride and 2-chloropropane reference solutions contain 0.5 mg / ml ethyl chloride and 0.2 mg / ml 2-chloropropane.
[0025] Furthermore, the solvent is a mixed solution of glacial acetic acid-N,N-dimethylformamide.
[0026] Further, the solvent is a mixed solution of glacial acetic acid-N,N-dimethylformamide in a volume ratio of 1:6 to 1:9. Preferably, the solvent is a mixed solution of glacial acetic acid-N,N-dimethylformamide in a volume ratio of 1:9.
[0027] Furthermore, the gas chromatography column has a length of 30 m, an inner diameter of 0.32 mm, and a film thickness of 1.8 μm; preferably, the gas chromatography column is Agilent DB-624.
[0028] Furthermore, the heating method is as follows: the starting temperature is 45° C., maintained for 6-10 minutes, and then increased to 220-260° C. at a rate of 25-35° C. per minute, and maintained for 10-20 minutes.
[0029] Furthermore, the injection port temperature is 230°C.
[0030] Furthermore, the detector temperature is 250°C.
[0031] Furthermore, the headspace equilibrium temperature is 80°C.
[0032] Furthermore, the equilibration time is 20 minutes.
[0033] Compared with the prior art, the present invention has the following beneficial effects:
[0034] (1) This method is simple and rapid. It can not only effectively separate ethyl chloride and 2-chloropropane, but also accurately detect the residual amounts of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine. It also provides a basis for the formulation of the quality standard of 1-cyclohexylpiperazine.
[0035] (2) This method has strong specificity and good durability. The instruments, equipment and reagents used are all conventional items, and the test parameters are also conventional parameters. There are no harsh conditions and it can be achieved in general laboratories. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 The chromatogram is a test chromatogram of the 100% spiked test solution containing the unknown impurity in Example 2.
[0037] Figure 2 The chromatogram is the detection chromatogram of the 150% spiked test solution containing the unknown impurity in Example 2.
[0038] Figure 3 This is the solvent detection chromatogram in Example 4.
[0039] Figure 4 This is the chromatogram detected by the reference solution in Example 4.
[0040] Figure 5 This is the chromatogram of the 100% spiked test solution in Example 4. DETAILED DESCRIPTION
[0041] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.
[0042] Example 1
[0043] Solvent: N,N-dimethylformamide.
[0044] Test solution: dilute with solvent to make a solution containing 100 mg / ml of 1-cyclohexylpiperazine per 1 ml.
[0045] Reference solution: dilute with solvent to prepare a mixed solution containing 0.5 mg of ethyl chloride and 0.2 mg of 2-chloropropane per 1 ml.
[0046] Chromatographic column: Agilent DB-624, column length 30m, inner diameter 0.32mm, film thickness 1.8μm.
[0047] The carrier gas was nitrogen and the flow rate was 1.0 ml / min.
[0048] Heating program: the initial temperature was 45°C, maintained for 8 minutes, then increased to 240°C at a rate of 30°C per minute, and maintained for 15 minutes.
[0049] The injection port temperature was 230 °C.
[0050] The detector temperature was 250°C.
[0051] The injection volume was 1 μl.
[0052] Injection method: direct injection.
[0053] Accurately measure the test solution and the reference solution, inject them into the gas chromatograph respectively, record the chromatogram, and the experimental results are shown in Table 1.
[0054] Table 1. Results of injection precision of reference solution
[0055] Serial number Ethyl chloride peak area Peak area change rate 2-Chloropropane peak area Peak area change rate Comparison 1 3.639 NA 1.956 NA Comparison 2 3.471 4.6％ 1.882 3.8％ Comparison 3 3.377 7.2％ 1.815 7.2％ Comparison 4 3.354 7.8％ 1.797 8.1％ Comparison 5 3.193 12.3％ 1.718 12.2％
[0056] It can be seen from the above table that the stability of the reference solution is poor when using direct injection. The reason is that the boiling point of ethyl chloride is 12.3°C, and the boiling point of 2-chloropropane is about 35°C. Both of them have low boiling points and are easy to volatilize. Therefore, headspace injection with better sealing is required.
[0057] Example 2
[0058] Solvent: N,N-dimethylformamide.
[0059] Test solution: dilute with solvent to make a solution containing 100 mg / ml of 1-cyclohexylpiperazine per 1 ml.
[0060] Reference solution: dilute with solvent to prepare a mixed solution containing 0.5 mg of ethyl chloride and 0.2 mg of 2-chloropropane per 1 ml.
[0061] 100% spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 0.5 mg of ethyl chloride, and 0.2 mg of 2-chloropropane per 1 ml.
[0062] 150% spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 0.75 mg of ethyl chloride, and 0.3 mg of 2-chloropropane per 1 ml.
[0063] Chromatographic column: Agilent DB-624, column length 30m, inner diameter 0.32mm, film thickness 1.8μm.
[0064] The carrier gas is nitrogen, and the flow rate is 1.0 ml / min
[0065] Heating program: the initial temperature was 45°C, maintained for 8 minutes, then increased to 240°C at a rate of 30°C per minute, and maintained for 15 minutes.
[0066] The injection port temperature was 230 °C.
[0067] The detector temperature was 250°C.
[0068] The headspace equilibrium temperature was 80°C.
[0069] Equilibration time: 20 minutes.
[0070] Injection method: headspace injection.
[0071] Take the reference solution, test solution, 100% spiked test solution, and 150% spiked test solution, inject them into the gas chromatograph respectively, and record the chromatogram. The 100% spiked test solution and 150% spiked test solution are shown in Figure 1 , Figure 2 .
[0072] from Figure 1 , Figure 2 It can be seen that in the 100% spiked test solution spectrum, there are unknown peaks at RT5.67 minutes and RT17.92 minutes, and the peak areas of these two unknown peaks in the 150% spiked test solution spectrum are increased. Since N,N-dimethylformamide is alkaline, the two unknown impurity peaks are generated by the reaction of ethyl chloride and 1-cyclohexylpiperazine under alkaline conditions.
[0073] Example 3
[0074] Solvent: glacial acetic acid-N,N-dimethylformamide volume ratio is (5:45, v / v).
[0075] Test solution: dilute with solvent to make a solution containing 100 mg / ml of 1-cyclohexylpiperazine per 1 ml.
[0076] Reference solution: dilute with solvent to prepare a mixed solution containing 0.5 mg of ethyl chloride and 0.2 mg of 2-chloropropane per 1 ml.
[0077] Quantitation limit spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 5 μg of ethyl chloride, and 4 μg of 2-chloropropane per 1 ml.
[0078] 100% spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 0.5 mg of ethyl chloride, and 0.2 mg of 2-chloropropane per 1 ml.
[0079] 150% spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 0.75 mg of ethyl chloride, and 0.3 mg of 2-chloropropane per 1 ml.
[0080] Chromatographic column: Agilent DB-624, column length 30m, inner diameter 0.32mm, film thickness 1.8μm.
[0081] The carrier gas was nitrogen and the flow rate was 1.0 ml / min.
[0082] Heating program: the initial temperature was 45°C, maintained for 8 minutes, then increased to 240°C at a rate of 30°C per minute, and maintained for 15 minutes.
[0083] The injection port temperature was 230 °C.
[0084] The detector temperature was 250°C.
[0085] The headspace equilibrium temperature was 80°C.
[0086] Equilibration time: 20 minutes.
[0087] Injection method: headspace injection.
[0088] Take the reference solution, the test solution, the quantitation limit spiked test solution, the 100% spiked test solution, and the 150% spiked test solution, inject them into the gas chromatograph respectively, and record the chromatogram. The accuracy results are shown in Table 2.
[0089] Table 2 Accuracy results.
[0090] Serial number Ethyl chloride recovery rate % 2-Chloropropane recovery rate % Limit of Quantitation 94 87 100％ 43 89 150％ 49 95
[0091] As can be seen from the above table, after adding glacial acetic acid to the solvent to inhibit the reaction of ethyl chloride and 1-cyclohexylpiperazine, the recovery rate of ethyl chloride is still low, and the amount of glacial acetic acid added continues to increase.
[0092] Example 4
[0093] Solvent: glacial acetic acid-N,N-dimethylformamide volume ratio is (7:43, v / v).
[0094] Test solution: dilute with solvent to make a solution containing 100 mg / ml of 1-cyclohexylpiperazine per 1 ml.
[0095] Reference solution: dilute with solvent to prepare a mixed solution containing 0.5 mg of ethyl chloride and 0.2 mg of 2-chloropropane per 1 ml.
[0096] Quantitation limit spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 5 μg of ethyl chloride, and 4 μg of 2-chloropropane per 1 ml.
[0097] 100% spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 0.5 mg of ethyl chloride, and 0.2 mg of 2-chloropropane per 1 ml.
[0098] 150% spiked test sample solution: Take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of the reference substance, and dilute it with solvent to make a mixed solution containing 100 mg / ml of 1-cyclohexylpiperazine, 0.75 mg of ethyl chloride, and 0.3 mg of 2-chloropropane per 1 ml.
[0099] Chromatographic column: Agilent DB-624, column length 30m, inner diameter 0.32mm, film thickness 1.8μm.
[0100] The carrier gas was nitrogen and the flow rate was 1.0 ml / min.
[0101] Heating program: the initial temperature was 45°C, maintained for 8 minutes, then increased to 240°C at a rate of 30°C per minute, and maintained for 15 minutes.
[0102] The injection port temperature was 230 °C.
[0103] The detector temperature was 250°C.
[0104] The headspace equilibrium temperature was 80°C.
[0105] Equilibration time: 20 minutes.
[0106] Injection method: headspace injection.
[0107] Take the reference solution, test solution, quantitation limit spiked test solution, 100% spiked test solution, and 150% spiked test solution, inject them into the gas chromatograph respectively, and record the chromatogram. The solvent, reference solution, and 100% spiked test solution are shown in Figure 3 , Figure 4 , Figure 5 ,The accuracy results are shown in Table III.
[0108] Table 3 Accuracy results
[0109] Serial number Ethyl chloride recovery rate % 2-Chloropropane recovery rate % Limit of Quantitation 84 95 100％ 98 102 150％ 92 94
[0110] As can be seen from the table above, after adding glacial acetic acid to the solvent, the recovery rates of ethyl chloride and 2-chloropropane meet the requirements. Therefore, this method has good specificity and accuracy, and can accurately detect the residual amounts of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine.
[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. An analytical method for detecting the residual amount of ethyl chloride and 2-chloropropane in 1-cyclohexylpiperazine by gas chromatography, characterized in that: The parameters of the method are as follows: Detector: flame ionization detector; Carrier gas: nitrogen; Flow rate: 0.9ml / min~1.1ml / min; Gas chromatography column: capillary column, preferably, the capillary column is selected from a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase; Heating method: 454℃~220℃ gradient heating; Injection method: headspace injection; Inlet temperature: 230℃~250℃; Detector temperature: 230℃~250℃; Headspace equilibrium temperature: 80℃~100℃; Equilibration time: 20-30 minutes; The method comprises: taking ethyl chloride, 2-chloropropane reference solution, and 1-cyclohexylpiperazine test solution for sample analysis respectively, recording chromatograms, and calculating the residual amounts of ethyl chloride and 2-chloropropane in the test solution by using an external standard method.
2. The analysis method according to claim 1, characterized in that: The test solution is prepared by taking 1-cyclohexylpiperazine test sample, adding a solvent to dissolve it, and diluting it; the ethyl chloride and 2-chloropropane reference solution is prepared by taking ethyl chloride and 2-chloropropane, adding a solvent to dissolve it, and diluting it.
3. The analysis method according to claim 2, characterized in that: The final concentration of the test solution is 100 mg / ml; the ethyl chloride and 2-chloropropane reference solutions contain 0.5 mg / ml ethyl chloride and 0.2 mg / ml 2-chloropropane.
4. The analysis method according to claim 1, characterized in that The solvent is a glacial acetic acid-N,N-dimethylformamide mixed solution.
5. The analysis method according to claim 4, characterized in that: The solvent is a mixed solution of glacial acetic acid and N,N-dimethylformamide in a volume ratio of 1:6 to 1:
9. Preferably, the solvent is a mixed solution of glacial acetic acid and N,N-dimethylformamide in a volume ratio of 1:
9.
6. The analysis method according to claim 1, characterized in that: The gas chromatography column has a length of 30 m, an inner diameter of 0.32 mm, and a film thickness of 1.8 μm; preferably, the gas chromatography column is Agilent DB-624.
7. The analysis method according to claim 1, characterized in that: The heating method is as follows: the initial temperature is 45° C., maintained for 6-10 minutes, then increased to 220-260° C. at a rate of 25-35° C. per minute, and maintained for 10-20 minutes.
8. The analysis method according to claim 1, characterized in that: The injection port temperature is 230°C.
9. The analysis method according to claim 1, characterized in that: The detector temperature was 250°C.
10. The analysis method according to claim 1, characterized in that: The headspace equilibrium temperature is 80° C., and the equilibrium time is 20 minutes.