Detection method for potential mutagenic impurities in eptazocine hydrobromide intermediates

Abstract

The invention provides a method for detecting ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate impurities in an eptazocine intermediate (-)-(1S-6S)-2,3,4,5,6,7-hexahydro-1,4-dimethyl-1,6-methylene-1hydro-4-phenylin-10-methoxy ether (compound 2). The method is rapid, accurate and has a low detection limit. The method can effectively control the content of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate that may remain in compound 2, thereby ensuring the safety of the use of an eptazocine hydrobromide preparation product further prepared from an eptazocine hydrobromide raw material.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for detecting potential mutagenic impurities ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate in an eptazocine hydrobromide intermediate. Background Art

[0002] Etazocine hydrobromide was originally developed by Nihon Lyakuhin Kogyo Co., Ltd. and was launched in Japan in 1987. It is mainly used to treat postoperative pain and cancer pain. Its structural formula is as follows (Compound 1):

[0003]

[0004] Eptazocine hydrobromide is a partial agonist of opioid receptors, acts on K receptors, uses selective antagonists to block postsynaptic receptors, and blocks chemical messengers that transmit pain information. In terms of analgesia, the analgesic efficacy of eptazocine is 1-2 times that of pentazocine. Japanese patents JP57046964A, JP59130843A, JP59130872A, JP60109555A, and JP64061447A disclose the synthesis route of eptazocine. Among them, the intermediate (-)-(1S-6S)-2,3,4,5,6,7-hexahydro-1,4-dimethyl-1,6-methylene-1hydro-4-benzin-10-methoxy ether (compound 2, referred to as compound 2) is the raw material for preparing eptazocine, and the reaction formula is as follows.

[0005]

[0006] The synthetic route of intermediate compound 2 (Compound 2) is as follows:

[0007]

[0008] According to the EMEA's "Guidelines on Limits of Genotoxic Impurities", in the above-mentioned synthesis route, the materials ethyl chloroformate, 1,3-bromochloropropane, and the intermediate ethyl carbamate used in the synthesis of the intermediates MPC and CBM are potential mutagenic impurities. Potentially mutagenic impurities refer to a class of impurities that can directly or indirectly act on DNA, thereby causing gene mutations, chromosome breaks or chromosome recombination, and may induce cancer even when exposed to low levels of doses. Potentially mutagenic impurities may be introduced into the final product as starting materials, reactants, catalysts, reagents, solvents, intermediates, by-products in the drug synthesis process, or degradation products during storage and transportation, affecting the quality of the drug and endangering the physical and mental health of the patient. Affecting the quality of the drug and endangering the physical and mental health of the patient, the content of potentially mutagenic impurities in the drug needs to be strictly controlled.

[0009] Intermediate compound 2 (Compound 2) is the starting material for the production of eptazocine hydrobromide API, so the limit of potential mutagenic impurities in Compound 2 is studied according to the limit requirements of the API eptazocine hydrobromide. The specification of eptazocine hydrobromide injection is 15 mg / vial, and the maximum daily dose is 1 vial. Based on ICH M7, the TTC (Threshold of Toxicological Concern) is evaluated at 1.5 μg / day. The limit of any single genotoxic impurity in eptazocine hydrobromide is 70 ppm, which is an extremely low concentration limit. In addition, genotoxic impurities have high reactivity and poor stability, which makes the analysis of such trace impurities in drugs extremely challenging, requiring the analytical method to have high sensitivity and specificity. The detection method of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate in Compound 2 has not been reported in the literature. In order to ensure the safety of etazocine hydrobromide and the efficiency of production detection, it is necessary to develop a detection method with high sensitivity, good specificity and simple operation for controlling the content of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate impurities in compound 2. Summary of the invention

[0010] The invention provides a method for detecting ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate impurities in an eptazocine intermediate (-)-(1S-6S)-2,3,4,5,6,7-hexahydro-1,4-dimethyl-1,6-methylene-1hydro-4-phenylin-10-methoxy ether (compound 2). The method is rapid, accurate and has a low detection limit, and can effectively control the content of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate that may remain in compound 2, thereby ensuring the safety of eptazocine hydrobromide preparation products further prepared from an eptazocine hydrobromide raw material.

[0011] The present invention provides a method for detecting ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate impurities in an eptazocine intermediate compound 2(-)-(1S-6S)-2,3,4,5,6,7-hexahydro-1,4-dimethyl-1,6-methylene-1hydro-4-phenyl-10-methoxy ether, characterized in that the method adopts gas chromatography-mass spectrometry chromatography for determination, wherein the chromatographic column adopted by the gas chromatography is a DB-5ms capillary chromatographic column, the sample dissolving solvent used is ethanol, and the column temperature adopts a program temperature control mode.

[0012] Since the detection limit of the liquid phase method does not meet the requirements and it is impossible to detect substances with extremely low limits, the present invention uses a gas chromatography-mass spectrometry method to detect the possible trace amounts of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate impurities in compound 2. Experimental screening of the chromatographic columns used found that the DB-5ms capillary chromatographic column can simultaneously detect the peaks of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate, and then the chromatographic column is used to study and explore the chromatographic detection conditions of the three impurities in compound 2.

[0013] The detection method of the present invention uses ethanol as the sample dissolving solvent, firstly because compound 2 is easily soluble in ethanol and has stable properties, and also because the target impurity ethyl chloroformate is active and easily decomposed in water, which is not conducive to detection. During the research process, it was unexpectedly discovered that the target impurity ethyl chloroformate can be quickly and completely converted into the detectable component diethyl carbonate after being derivatized with ethanol (see Example 1).

[0014] The detection method of the present invention adopts a test sample concentration of 10 mg / ml for compound 2. Since the limit requirement for a single genotoxic impurity in compound 2 is 70 ppm, when the test sample concentration of compound 2 is 10 mg / ml, the corresponding limit concentrations of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate are 0.7 μg / ml (70 ppm). The sensitivity experimental results of the chromatographic method of the present invention show that the detection limit concentrations of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate are approximately 0.07 μg / ml (7 ppm), and the quantitative limit concentration is approximately 0.175 μg / ml (17.5 ppm). Therefore, at this sensitivity, the test sample of compound 2 at a concentration of 10 mg / ml satisfies the simultaneous quantitative detection of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate.

[0015] The gas chromatography conditions used in the detection method of the present invention also include: the injection port temperature is 280-300°C, preferably 300°C; the split ratio is 5:1, 10:1 or 20:1, preferably 10:1; the carrier gas flow rate is 0.8ml / min-1.5ml / min, preferably 1.0ml / min; the column temperature is programmed, the temperature program is 30-50°C for 2min and then heated, the heating rate is 10°C / min- 30℃ / min, heat to 110-140℃, maintain for 2min, heat up at a rate of 10℃ / min-30℃ / min, heat to 260-290℃ and maintain for 10min; preferably, the initial temperature is 40℃, maintain for 2min and then heat up at a rate of 10℃ / min, heat to 130℃, maintain for 2min, then heat up at a rate of 30℃ / min to 280℃ and maintain for 10 minutes; the injection volume is 1-10μl, preferably 1μl.

[0016] The mass spectrometry conditions adopted in the detection method of the present invention are: an EI ionization source is adopted, the ion source temperature is 200°C to 250°C, preferably 230°C; a single quadrupole mass analyzer is adopted, the quadrupole temperature is 120°C to 150°C, preferably 150°C; the detection mode adopted is a single ion detection mode, and the extracted ions are m / z91.00, m / z103.00, and m / z158.00; the residence time is 50ms, and the solvent delay time is 12min.

[0017] The detection method of the present invention specifically comprises the following steps:

[0018] 1) Preparation of test solution: Weigh 0.1 g of compound 2 sample, dissolve and clarify with 10 ml of ethanol, and make up to volume;

[0019] 2) Preparation of reference solution:

[0020] a. Ethyl chloroformate control solution: accurately weigh 38.11 mg of ethyl chloroformate with a purity of 99.0% into a 10 ml volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a control solution with a concentration of 3.773 mg / ml;

[0021] b. Ethyl methylcarbamate control solution: accurately weigh 40.01 mg of ethyl methylcarbamate with a purity of 98.0% into a 10 ml volumetric flask, dissolve it with ethanol and make up to the mark to obtain a control solution with a concentration of 3.921 mg / ml;

[0022] c. 1,3-bromochloropropane reference solution: accurately weigh 40.03 mg of 1,3-bromochloropropane with a purity of 99.7% into a 10 ml volumetric flask, dissolve it in ethanol and dilute to the mark to obtain a reference solution with a concentration of 3.991 mg / ml;

[0023] 3) Analysis and determination: Inject the test solution and the reference solution into the gas chromatography-mass spectrometer and record the spectra.

[0024] The invention provides a method for detecting genotoxic impurities ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate in an intermediate compound 2 for preparing an eptazocine raw material drug. The method has high detection sensitivity, a detection limit concentration of 0.07 μg / ml (7 ppm), and a quantitative limit concentration of 0.175 μg / ml (17.5 ppm), which are much lower than the limit concentration of 0.7 μg / ml (70 ppm) of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate; the method has good detection specificity, and blank solvent and compound 2 test solution have no interfering chromatographic peaks at the chromatographic peak positions of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate. The detection method of the present invention is accurate and reliable, with a recovery rate between 85% and 115%, and the RSDs of the peak areas of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate are 3.52%, 3.35% and 3.21% respectively; the repeatability is good, and the RSDs of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate are 2.40%, 3.89% and 2.66% respectively. The detection method of the present invention can achieve effective control of genotoxic ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate in compound 2, can reduce the side effects of the preparation products prepared from the eptazocine hydrobromide raw material, and improve the product quality.

[0025] The present invention will be further described below in conjunction with specific implementation examples and accompanying drawings.

[0026] Instruction Manual

[0027] Attached Figure 1 Specificity test blank solution gas chromatography-mass spectrometry

[0028] Attached Figure 2 Specificity Experiment Gas Chromatography-Mass Spectrum of Standard Solutions of Ethyl Chloroformate, 1,3-Bromochloropropane and Ethyl Methylcarbamate Figure 3 Quantitative limit spectra of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate by gas chromatography-mass spectrometry Figure 4 Detection limit spectra of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate by gas chromatography-mass spectrometry Figure 5 Gas chromatography-mass spectrometry of compound 2 (batch 1) DETAILED DESCRIPTION

[0029] Example 1 Conversion rate test of ethyl chloroformate into diethyl carbonate

[0030] To verify that ethyl chloroformate can be quickly and completely converted into diethyl carbonate in ethanol, the conversion rate of ethyl chloroformate in ethanol was quantitatively checked using diethyl carbonate as a reference substance.

[0031] Take an appropriate amount of 99% pure diethyl carbonate reference substance, dissolve it in ethanol and quantitatively dilute it to make a reference substance solution containing 0.7667μg per 1ml, and take an appropriate amount of ethyl chloroformate, dissolve it in ethanol, shake it, and quantitatively dilute it to make a test solution containing 0.7μg per 1ml. Inject them separately.

[0032] Chromatographic conditions: Capillary column: DB-5ms, 30m×0.25mm, 1.0μm; Inlet temperature: 300℃; Split ratio: 10:1; Constant flow mode, flow rate: 1ml / min; Carrier gas: He; Column box temperature: 40℃ (maintain 2min) - 10℃ / min (temperature increase) - 130℃ (maintain 2min) - 30℃ / min (temperature increase) - 280℃ (maintain 10min); Injection volume: 1μl; Auxiliary heating zone: 280℃. The results are shown in the table below:

[0033]

[0034]

[0035] Wherein, A derivatized product represents the mass spectrum peak area of ​​diethyl carbonate obtained by derivatization;

[0036] Cethyl chloroformate indicates the concentration of ethyl chloroformate (μg / ml);

[0037] A reference substance represents the reference mass spectrum peak area of ​​diethyl carbonate;

[0038] C control substance indicates the concentration of diethyl carbonate control substance (μg / ml).

[0039] The test results show that the conversion rate of ethyl chloroformate to diethyl carbonate is 99.6% when ethanol is used as the solvent, and ethyl chloroformate can be quickly and completely converted into the measurable component diethyl carbonate.

[0040] Example 2 Methodology Verification Experiment

[0041] 1. Instrument: Agilent 7890B-5977 GC-MS

[0042] Reagent: Ethanol (chromatographic grade)

[0043] Sample: Compound 2 (Shanghai Jinhe Biological Co., Ltd.), ethyl chloroformate: purity 99.0%, ethyl methylcarbamate: purity 98.0%, 1,3-bromochloropropane: purity 99.7%

[0044] 2. Chromatographic conditions

[0045] Capillary column: DB-5ms, 30m×0.25mm, 1.0μm; injection port temperature: 300℃; split ratio: 10:1; constant flow mode, flow rate: 1ml / min; carrier gas: He; column box temperature: 40℃ (maintain 2min)-10℃ / min (temperature increase)-130℃ (maintain 2min)-30℃ / min (temperature increase)-280℃ (maintain 10min); injection volume: 1μl; auxiliary heating zone: 280℃

[0046] 3. Mass spectrometry conditions

[0047] Solvent delay: 7.2min; Acquisition mode: SIM mode;

[0048] 7.2 min group 1 (mass m / z, dwell time) (45.00, 50), (63.00, 50), (91.00, 50);

[0049] 9.0 min group 2 (mass m / z, dwell time) (41.00, 50), (58.00, 50), (75.00, 50), (77.00, 50), (103.00, 50), (158.00, 50);

[0050] MS ion source temperature: 230°C; MS quadrupole temperature: 150°C.

[0051] (1) Specificity experiment

[0052] Take 1 μl of the mixed standard solution and inject it into the GC-MS, use the full scan (SCAN) mode for acquisition, and obtain the retention time and characteristic fragment ions of ethyl chloroformate, ethyl methylcarbamate, and 1,3-bromochloropropane. Then establish the selected ion (SIM) acquisition method for the three impurities. Take a blank solution, ethyl chloroformate, ethyl methylcarbamate, 1,3-bromochloropropane mixed standard solution, and compound 2 test solution into one injection each to ensure that there is no mutual interference between potential impurities, impurities and impurities, impurities and main peaks, and impurities and solvents. The results are as follows:

[0053]

[0054] Conclusion: The solvent blank, the test solution has no interfering chromatographic peaks at the chromatographic peaks of ethyl chloroformate, 1,3-bromochloropropane, and ethyl methylcarbamate. The analytical method has good specificity and meets the analytical requirements. The chromatograms of the blank solution and the three mixed standard solutions are attached. Figure 1 and attached Figure 2 .

[0055] (2) Detection limit and quantification limit

[0056] Prepare reference solutions of various impurities at different concentrations, and determine the detection limit of each impurity when the S / N of each impurity is about 3 (see Appendix Figure 3 ); When the S / N of each impurity is about 10, determine the quantitative limit of each impurity (see Appendix Figure 4 ), the RSD of the peak area of ​​each impurity reference solution injected 6 times in a row at the quantitative limit concentration is ≤10%.

[0057] Verification results:

[0058] A. Detection limit and quantification limit of ethyl chloroformate: Dilute the ethyl chloroformate standard stock solution 1 step by step. When the concentration of ethyl chloroformate is 0.07μg / ml, the signal-to-noise ratio is 4.3, which meets the signal-to-noise ratio requirement of the detection limit and can be used as the detection limit. When the concentration of ethyl chloroformate is 0.175μg / ml, the signal-to-noise ratio of 6 injections is about 10, which meets the signal-to-noise ratio requirement of the quantification limit. The RSD of the peak area of ​​6 injections of the quantification limit is 3.80%, which can be used as the quantification limit.

[0059] B. Detection limit and quantification limit of methylcarbamate: Dilute the methylcarbamate quasi-stock solution 2 step by step. When the concentration of methylcarbamate is 0.07μg / ml, the signal-to-noise ratio is 4.9, which meets the signal-to-noise ratio requirement of the detection limit and can be used as the detection limit. When the concentration of methylcarbamate is 0.175μg / ml, the signal-to-noise ratio of 6 injections is about 10, which meets the signal-to-noise ratio requirement of the quantification limit. The RSD of the peak area of ​​6 injections of the quantification limit is 5.03%, which can be used as the quantification limit.

[0060] C. Detection limit and quantification limit of 1,3-bromochloropropane: Dilute the 1,3-bromochloropropane standard intermediate solution step by step. When the concentration of 1,3-bromochloropropane is 0.07μg / ml, the signal-to-noise ratio is 4.9, which meets the signal-to-noise ratio requirement of the detection limit and can be used as the detection limit. When the concentration of 1,3-bromochloropropane is 0.175μg / ml, the signal-to-noise ratio of 6 injections is about 10, which meets the signal-to-noise ratio requirement of the quantification limit. The RSD of the peak area of ​​6 injections of the quantification limit is 1.86%, which can be used as the quantification limit. The injection results are as follows:

[0061] Limit of Quantitation of Ethyl Chloroformate Injection Peak Area and Signal-to-Noise Ratio

[0062]

[0063]

[0064] Limit of Quantitation of Ethyl Methylcarbamate Injection Peak Area and Signal-to-Noise Ratio

[0065]

[0066] 1,3-Bromochloropropane Quantitation Limit Injection Peak Area and Signal-to-Noise Ratio

[0067]

[0068] Test results: The quantitative limit of ethyl chloroformate is 0.175 μg / ml, equivalent to 17.5 ppm of the test sample; the detection limit is 0.07 μg / ml, equivalent to 7 ppm of the test sample;

[0069] Test results: The quantitative limit of 1,3-bromochloropropane is 0.175 μg / ml, equivalent to 17.5 ppm of the test sample; the detection limit is 0.07 μg / ml, equivalent to 7 ppm of the test sample;

[0070] Test results: The quantitative limit of ethyl carbamate was 0.175 μg / ml, equivalent to 17.5 ppm of the test sample concentration; the detection limit was 0.07 μg / ml, equivalent to 7 ppm of the test sample concentration.

[0071] (3) Linear

[0072] Prepare 5 concentration points of each impurity reference solution from the limit of quantitation to the 200% limit concentration, inject each concentration once, and perform linear calculation based on concentration and peak area. The r2 should be greater than 0.99.

[0073] Linear solution concentration and peak area of ​​ethyl chloroformate, ethyl methylcarbamate and 1,3-bromochloropropane

[0074]

[0075] Test conclusion:

[0076] The linear equation of ethyl chloroformate is y=260547x–2060.9, R2=0.9999; the linear range is 0.175μg / ml~1.4μg / ml;

[0077] The linear equation of methylcarbamate is y=186419x-14630; R2=0.9996; linear range 0.175μg / ml~1.4μg / ml;

[0078] The linear equation of 1,3-bromochloropropane is y=193356x+2315.2; R2=0.9991; linear range: 0.175μg / ml~1.4μg / ml.

[0079] (4) Injection precision

[0080] Prepare a 100% limit concentration target impurity reference solution, inject 6 times in a row, and count the peak area of ​​each target impurity. The RSD should be less than 5%. The RSD of the retention time of each impurity should be no more than 2%.

[0081] The mixed standard solution of 100% limit concentration of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate was injected continuously for 6 times. The injection results are shown in the table below.

[0082]

[0083]

[0084] Conclusion: After 6 consecutive injections of 100% limit concentration impurity reference solution of ethyl chloroformate, ethyl methylcarbamate and 1,3-bromochloropropane, the RSDs of the peak areas of ethyl chloroformate, ethyl methylcarbamate and 1,3-bromochloropropane were 3.52%, 3.35% and 3.21%, respectively, all less than 5.0%; the RSDs of the retention times of ethyl chloroformate, ethyl methylcarbamate and 1,3-bromochloropropane were 0.024%, 0.016% and 0.010%, respectively, all less than 2.0%, which met the requirements.

[0085] (5) Repeatability

[0086] Prepare a mixed standard solution of ethyl chloroformate, ethyl methylcarbamate, and 1,3-bromochloropropane at a concentration of 0.7 μg / ml. Weigh an appropriate amount of compound 2 and add it to the 100% limit concentration reference solution to prepare 6 portions, and inject each portion once. Count the peak area of ​​each impurity and calculate the recovery rate. The RSD of the recovery rate of the 6 portions of 100% limit concentration spiked test solution should be less than 5.0%.

[0087] Ethyl chloroformate repeatability test results

[0088]

[0089] Results of repeatability test of ethyl carbamate

[0090]

[0091]

[0092] Repeatability test results of 1,3-bromochloropropane

[0093]

[0094] in conclusion:

[0095] The RSDs of the peak areas (recoveries) of ethyl chloroformate, ethyl methylcarbamate and 1,3-bromochloropropane impurities in 6 samples of compound 2 added to the 100% limit concentration impurity reference solution were 2.40%, 3.89% and 2.66%, respectively, all less than 5%, meeting the requirements.

[0096] (6) Accuracy

[0097] Prepare two portions of the test solution, three portions of each spiked sample solution of 80%, 100%, and 120% limit concentrations of ethyl chloroformate, ethyl methylcarbamate, and 1,3-bromochloropropane, inject each portion once, count the peak area of ​​each impurity, and conduct a recovery test. The recovery rate should be between 85% and 115%. The results are as follows.

[0098] Results of accuracy determination of ethyl chloroformate

[0099]

[0100]

[0101] Results of accuracy test of ethyl methylcarbamate

[0102]

[0103] Results of accuracy determination of 1,3-bromochloropropane

[0104]

[0105] Conclusion: The recovery of ethyl chloroformate in the mixed solution of the test sample and the impurity reference sample with limit concentrations of 80%, 100% and 120% was between 87.38% and 96.17%, with an RSD of 3.34%; the recovery of ethyl methylcarbamate was between 95.55% and 106.2%, with an RSD of 4.07%; the recovery of 1,3-bromochloropropane was between 93.77% and 102.8%, with an RSD of 3.56%, all of which met the requirements.

[0106] (7) Durability

[0107] Change the chromatographic conditions, prepare the test solution containing 100% spiked impurity reference substance, modify the chromatographic conditions under standard conditions, and the ratio of the detection result of the component to be tested under durability conditions to the impurity detection result under standard conditions should be between 90.0% and 110.0%.

[0108] Standard conditions: see 2 Chromatographic conditions

[0109] Condition 1: initial temperature 38°C;

[0110] Condition 2: initial temperature 42°C;

[0111] Condition 3: Inlet temperature: 298°C

[0112] Condition 4: Inlet temperature: 302°C

[0113] Prepare 100% spiked test solution: weigh 0.10g of compound 2 sample into a 10ml volumetric flask, then accurately pipette 0.5ml of 14μg / ml mixed standard solution (see specificity experiment) into the volumetric flask, dissolve with ethanol and dilute to scale, mix well, and test. Prepare two copies in parallel, and inject one injection into each copy.

[0114] Ethyl chloroformate durability test results

[0115]

[0116] Results of the durability test of methyl urethane

[0117]

[0118] 1,3-Bromochloropropane Durability Test Results

[0119]

[0120] Conclusion: Compared with the test results under the original conditions, the ratios of the test results under different conditions to the test results under standard conditions for ethyl chloroformate, ethyl methylcarbamate and 1,3-bromochloropropane were all between 90.0% and 110.0%, which met the requirements.

[0121] Example 3 Compound 2 Sample Determination

[0122] 1. Instruments and Reagents

[0123] Instrument: Agilent 7890B-5977 gas chromatography-mass spectrometer;

[0124] Reagents: ethanol (chromatographic grade);

[0125] Samples: Compound 2 (Shanghai Jinhe Biological Co., Ltd.), ethyl chloroformate (purity 99.0%), ethyl methylcarbamate (purity 98.0%), 1,3-bromochloropropane (purity 99.7%);

[0126] Blank solution: ethanol;

[0127] Compound 2 test solution: Weigh 0.1 g of compound 2 sample, dissolve it in 10 ml of ethanol, make up to volume, and set aside.

[0128] Ethyl chloroformate standard stock solution 1: Accurately weigh 33.1.11 mg of ethyl chloroformate with a purity of 99.0% into a 10 ml volumetric flask, dissolve it with ethanol and make up to the mark to obtain a standard stock solution 1 with a concentration of 3.773 mg / ml.

[0129] Ethyl methylcarbamate standard stock solution 2: Accurately weigh 40.01 mg of ethyl methylcarbamate with a purity of 98.0% into a 10 ml volumetric flask, dissolve it with ethanol and make up to the mark to obtain a standard stock solution 2 with a concentration of 3.921 mg / ml.

[0130] 1,3-Bromochloropropane standard stock solution 3: Accurately weigh 40.03 mg of 1,3-bromochloropropane with a purity of 99.7% into a 10 ml volumetric flask, dissolve it in ethanol and dilute to the mark to obtain a standard stock solution 3 with a concentration of 3.991 mg / ml.

[0131] Impurity reference solution: Weigh 0.10 g of compound 2 sample into a 10 ml volumetric flask, then accurately transfer 0.5 ml of a mixed standard solution with a concentration of 14 μg / ml into the flask, add ethanol to dissolve and dilute to the mark.

[0132] 2. Chromatographic conditions

[0133] Capillary column: DB-5ms, 30m×0.25mm, 1.0μm; injection port temperature: 300℃; split ratio: 10:1; constant flow mode, flow rate: 1ml / min; carrier gas: He; column box temperature: 40℃ (maintain 2min)-10℃ / min (temperature increase)-130℃ (maintain 2min)-30℃ / min (temperature increase)-280℃ (maintain 10min); injection volume: 1μl; auxiliary heating zone: 280℃3. Mass spectrometry conditions

[0134] Solvent delay: 7.2min;

[0135] Acquisition mode: SIM mode;

[0136] 7.2 min group 1 (mass m / z, dwell time) (45.00, 50), (63.00, 50), (91.00, 50);

[0137] 9.0 min group 2 (mass m / z, dwell time) (41.00, 50), (58.00, 50), (75.00, 50), (77.00, 50), (103.00, 50), (158.00, 50);

[0138] MS ion source temperature: 230°C

[0139] MS quadrupole temperature: 150 °C;

[0140] 4. Content determination:

[0141] Using the above conditions, the test solution, reference solution and blank solution were sampled for detection and analysis. The detection results of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate residues in three batches of compound 2 samples are as follows.

[0142]

[0143] According to the test results, no ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate were detected in the three batches of compound 2 samples. The spectrum of the first batch of compound 2 is attached. Figure 5 .

Claims

1. A method for detecting impurities of ethyl chloroformate, 1,3-bromochloropropane and ethyl methylcarbamate in the intermediate compound 2(-)-(1S-6S)-2,3,4,5,6,7-hexahydro-1,4-dimethyl-1,6-methylene-1hydro-4-phenyl-10-methoxy ether of eptazocine, characterized in that: The method adopts gas chromatography-mass spectrometry chromatography, wherein the chromatographic column used in the gas chromatography is a DB-5ms capillary chromatographic column, the sample dissolving solvent used is ethanol, the concentration of the compound 2 test sample is 10 mg / ml, the column temperature is programmed, and the temperature rise program of the gas chromatography programmed temperature rise method is to maintain the initial temperature at 30-50°C for 2 minutes and then increase the temperature at a rate of 10°C / min-30°C / min to 110-140°C. Maintain for 2 minutes, heat up at a rate of 10°C / min to 30°C / min, heat to 260 to 290°C and maintain for 10 minutes; the mass spectrometry conditions used in the detection method are: using an EI ionization source, the ion source temperature is 200°C to 250°C; using a single quadrupole mass analyzer, the quadrupole temperature is 120 to 150°C; the detection mode used is a single ion detection mode, and the extracted ions are m / z91.00, m / z103.00, and m / z158.00; The dwell time was 50 ms and the solvent delay time was 12 min.

2. The method according to claim 1, characterized in that: The temperature program of the gas chromatography programmed temperature heating method is as follows: initial temperature 40°C, maintained for 2 minutes and then heated at a heating rate of 10°C / min, heated to 130°C, maintained for 2 minutes, heated to 280°C at a heating rate of 30°C / min and maintained for 10 minutes.

3. The method according to claim 1, characterized in that Other gas chromatography conditions of the detection method include: the injection port temperature is 280-300°C; the split ratio is 5:1, 10:1 or 20:1; the carrier gas flow rate is 0.8ml / min-1.5ml / min; The injection volume is 1 to 10 μl.

4. The method according to claim 3, characterized in that The gas chromatography conditions of the detection method also include: injection port temperature of 300° C.; split ratio of 10:1; carrier gas flow rate of 1.0 ml / min; and injection volume of 1 μl.

5. The method according to claim 1, characterized in that The mass spectrometry conditions used in the detection method include an EI ionization source with an ion source temperature of 230° C. and a single quadrupole mass analyzer with a quadrupole temperature of 150° C.

6. The method according to claim 1, characterized in that The detection method specifically comprises the following steps: 1) Preparation of test solution: Weigh 0.1 g of compound 2 sample, dissolve and clarify with 10 ml of ethanol, and make up to volume; 2) Preparation of reference solution: a. Ethyl chloroformate control solution: accurately weigh 38.11 mg of ethyl chloroformate with a purity of 99.0% into a 10 ml volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a control solution with a concentration of 3.773 mg / ml; b. Ethyl methylcarbamate control solution: accurately weigh 40.01 mg of ethyl methylcarbamate with a purity of 98.0% into a 10 ml volumetric flask, dissolve it with ethanol and make up to the mark to obtain a control solution with a concentration of 3.921 mg / ml; c. 1,3-bromochloropropane reference solution: accurately weigh 40.03 mg of 1,3-bromochloropropane with a purity of 99.7% into a 10 ml volumetric flask, dissolve it in ethanol and dilute to the mark to obtain a reference solution with a concentration of 3.991 mg / ml; 3) Analysis and determination: Inject the test solution and the reference solution into the gas chromatography-mass spectrometer and record the spectra.

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