CN115840012A - Method for detecting potential mutagenic impurities in eptazocine hydrobromide intermediate - Google Patents

Abstract

The invention provides a method for detecting ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate impurities in an eptazocine intermediate (-) - (1S-6S) -2,3,4,5,6, 7-hexahydro-1, 4-dimethyl-1, 6-methylene-1 hydro-4-phenyllin-10-methoxy ether (compound 2), which is rapid and accurate and has low detection limit, can effectively control the content of the ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate possibly remaining in the compound 2, and ensures the safety of the eptazocine hydrobromide preparation product further prepared from eptazocine raw material medicines.

Description

Method for detecting potential mutagenic impurities in eptazocine hydrobromide intermediate

Technical Field

The invention relates to the field of pharmaceutical analysis, in particular to a method for detecting potential mutagenic impurities of ethyl chloroformate, 1, 3-bromochloropropane and ethyl methyl carbamate in an eptazocine hydrobromide intermediate.

Background

Eptazocine hydrobromide, originally developed by japan scientific co (Nihon lyakuhin Kogyo co., ltd.), was marketed in japan in 1987 for the treatment of postoperative pain and cancer pain, and has the following structural formula (compound 1):

eptazocine hydrobromide is a partial agonist of opioid receptors, acts on K receptors, blocks postsynaptic receptors with selective antagonists, and blocks chemical messengers that transmit pain information. In the aspect of analgesia, the analgesic efficacy of eptazocine is 1-2 times that of pentazocine. Japanese patents JP57046964A, JP59130843A, JP59130872A, JP60109555A, JP64061447A disclose synthetic routes to etazocine. Wherein the intermediate (-) - (1S-6S) -2,3,4,5,6, 7-hexahydro-1, 4-dimethyl-1, 6-methylene-1 hydro-4-phenyllin-10-methylether (compound 2, referred to as compound 2 for short) is a raw material for preparing the eptazocine, and the reaction formula is as follows.

The synthetic route of intermediate compound 2 (compound 2) is shown below,

according to the guidelines for limits of genotoxic impurities in EMEA, the materials used in the synthesis of the intermediates MPC and CBM, ethyl chloroformate, 1, 3-bromochloropropane, and the intermediate ethyl methylcarbamate, are potentially mutagenic impurities in the above synthetic route. Potentially mutagenic impurities refer to those impurities that act directly or indirectly on DNA, thereby causing genetic mutations, chromosomal breaks, or chromosomal recombinations, which can induce cancer even when exposed to low levels of doses. The potential mutation-causing impurities can be introduced into final products as initial raw materials, reactants, catalysts, reagents, solvents, intermediates, byproducts or degradation products in the storage and transportation processes in the drug synthesis process, so that the quality of the drugs is influenced, and the physical and psychological health of patients is harmed. The quality of the medicine is affected, and the physical and psychological health of patients is damaged, so the content of potential mutagenic impurities in the medicine needs to be strictly controlled.

The intermediate compound 2 (compound 2) is a starting material for producing eptazocine hydrobromide bulk drugs, so the limit of potential mutagenic impurities in the compound 2 is researched according to the limit requirement of the bulk drug eptazocine hydrobromide, the specification of the eptazocine hydrobromide injection is 15 mg/per unit, the maximum daily dosage is 1 unit, the eptazocine injection is evaluated based on ICH M7 by using TTC (toxicological concern threshold) of 1.5 mu g/day, the limit of any single genotoxic impurity in the eptazocine is 70ppm, the concentration limit is extremely low, the genotoxic impurity has high reaction activity and poor stability, the analysis of the trace impurities in the drug is very challenging, and the analysis method is required to have high sensitivity and specificity. The detection methods of ethyl chloroformate, 1, 3-bromochloropropane and ethyl methylcarbamate in the compound 2 are not reported in the literature. In order to ensure the medication safety of the eptazocine hydrobromide and the production detection efficiency, a detection method with high sensitivity, good specificity and simple operation is necessary to be developed for controlling the content of the ethyl chloroformate, 1, 3-bromochloropropane and ethyl methyl carbamate impurities in the compound 2.

Disclosure of Invention

The invention provides a method for detecting ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate impurities in an eptazocine intermediate (-) - (1S-6S) -2,3,4,5,6, 7-hexahydro-1, 4-dimethyl-1, 6-methylene-1 hydro-4-phenyllin-10-methoxy ether (compound 2), which is rapid and accurate and has low detection limit, can effectively control the content of the ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate possibly remaining in the compound 2, and ensures the safety of the eptazocine hydrobromide preparation product further prepared from eptazocine raw material medicines.

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

Because the detection limit of the liquid phase method can not meet the requirement and can not detect substances with extremely low limit, the method adopts a gas chromatography-mass spectrometry combined method to detect trace ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate impurities possibly existing in the compound 2. Experimental screening of the chromatographic column adopted shows that the capillary chromatographic column with the model of DB-5ms can simultaneously detect the peaks of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate, and further the chromatographic column is adopted to research and grope the chromatographic detection conditions of three impurities in the compound 2.

The detection method adopts ethanol as a sample dissolving solvent, firstly because the compound 2 is easy to dissolve in the ethanol and has stable property, and in addition, because the target impurity ethyl chloroformate has active property, the compound is easy to decompose when meeting water, and is not beneficial to detection. During the course of the study it was unexpectedly found that derivatization of the target impurity ethyl chloroformate with ethanol resulted in a rapid and complete conversion to the measurable component diethyl carbonate (see example 1).

The detection method adopts the compound 2 to be tested with the concentration of 10mg/ml. Since the limit of toxic impurities of a single gene in the compound 2 is required to be 70ppm, when the test sample concentration of the compound 2 is 10mg/ml, the limit concentrations of the corresponding ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate are 0.7 mu g/ml (70 ppm). The sensitivity test result of the chromatographic method of the invention shows that the detection limit concentration of the ethyl chloroformate, the 1, 3-bromochloropropane and the ethyl carbamate is about 0.07 mu g/ml (7 ppm), and the quantification limit concentration is about 0.175 mu g/ml (17.5 ppm). Therefore, at this sensitivity, the test sample of Compound 2 was quantitatively detected at a concentration of 10mg/ml, while satisfying the simultaneous detection of ethyl chloroformate, 1, 3-bromochloropropane and ethyl methylcarbamate.

The gas chromatography conditions adopted by the detection method of the invention also comprise: the temperature of the sample inlet is 280-300 ℃, and 300 ℃ is preferred; the flow dividing ratio is 5; the flow rate of the carrier gas is 0.8 ml/min-1.5 ml/min, and the preferred flow rate is 1.0ml/min; the column temperature adopts a programmed heating mode, the heating program is that the temperature is maintained for 2min at 30-50 ℃, then the temperature is raised, the heating speed is 10-30 ℃/min, the temperature is raised to 110-140 ℃, the temperature is maintained for 2min, the heating speed is 10-30 ℃/min, the temperature is raised to 260-290 ℃ and the temperature is maintained for 10min; preferably, the initial temperature is 40 ℃, the temperature is maintained for 2min and then is increased, the temperature increasing speed is 10 ℃/min, the temperature is increased to 130 ℃, the temperature is maintained for 2min, and then the temperature increasing speed is 30 ℃/min to 280 ℃ and is maintained for 10min; the amount of the sample is 1 to 10. Mu.l, preferably 1. Mu.l.

The mass spectrum conditions adopted by the detection method are as follows: the EI ionization source is adopted, the ion source temperature is 200-250 ℃, and 230 ℃ is preferred; adopting a single-quadrupole mass analyzer, wherein the temperature of a quadrupole is 120-150 ℃, and preferably 150 ℃; the adopted detection mode is a single ion detection mode, and the extracted ions are m/z91.00, m/z103.00 and m/z158.00; the residence time was 50ms and the solvent delay time was 12min.

The detection method specifically comprises the following steps:

1) Preparing a test solution: weighing 0.1g of compound 2 sample, dissolving with 10ml of ethanol, clarifying, and fixing the volume;

2) Preparation of control solutions:

a. ethyl chloroformate control solution: accurately weighing 38.11mg of ethyl chloroformate with the purity of 99.0% into a measuring flask of 10ml, dissolving the ethyl chloroformate with ethanol, and fixing the volume to scale to obtain a control solution with the concentration of 3.773 mg/ml;

b. ethyl carbamate control solution: accurately weighing 40.01mg to 10ml of ethyl carbamate with the purity of 98.0%, dissolving by using ethanol, and fixing the volume to a scale to obtain a control solution with the concentration of 3.921 mg/ml;

c. 1, 3-bromochloropropane control solution: accurately weighing 40.03mg of 1, 3-bromochloropropane with the purity of 99.7% into a 10ml measuring flask, dissolving with ethanol, and fixing the volume to a scale to obtain a control solution with the concentration of 3.991 mg/ml;

3) Analysis and determination: and (3) injecting the test solution and the reference solution into a gas chromatography-mass spectrometer and recording the spectra.

The invention provides a method for detecting genotoxic impurity ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate in an intermediate compound 2 for preparing an eptazocine bulk drug, which has high detection sensitivity, the detection limit concentration is 0.07 mu g/ml (7 ppm), the quantitative limit concentration is 0.175 mu g/ml (17.5 ppm), and the limit concentration is far lower than the limit concentrations of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate, namely 0.7 mu g/ml (70 ppm); the detection specificity is good, and the blank solvent and the compound 2 sample solution do not have interference chromatographic peaks at the chromatographic peak positions of ethyl chloroformate, 1, 3-bromochloropropane and ethyl methyl carbamate. The detection method is accurate and credible, the recovery rate is between 85% and 115%, and the RSD of the peak areas of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate with the injection precision is 3.52%, 3.35% and 3.21% respectively; good repeatability, and RSD of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate is 2.40%, 3.89% and 2.66%, respectively. The detection method can realize effective control of the genotoxic ethyl chloroformate, the 1, 3-bromochloropropane and the ethyl carbamate in the compound 2, reduce the related side effects of the preparation product prepared from the raw material medicine of the eptazocine hydrobromide and improve the product quality.

The invention is further illustrated by the following examples of specific embodiments and the accompanying drawings of the specification.

Drawings

FIG. 1 is a gas chromatogram-mass spectrum of a special experimental blank solution

FIG. 2 is a gas chromatography-mass spectrum diagram of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate standard solution for specific experiments, FIG. 3 is a quantitative limit spectrum diagram of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate gas chromatography-mass spectrum, FIG. 4 is a detection limit spectrum diagram of gas chromatography-mass spectrum of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate, FIG. 5 is a gas chromatography-mass spectrum diagram of Compound 2 (batch No. 1)

Detailed Description

Example 1 conversion of ethyl chloroformate to diethyl carbonate

To verify that ethyl chloroformate was converted rapidly and completely into diethyl carbonate in ethanol. The conversion of ethyl chloroformate in ethanol was quantitatively checked against a diethyl carbonate control.

Taking a proper amount of diethyl carbonate reference substance with the purity of 99%, adding ethanol for dissolving, quantitatively diluting to prepare a reference substance solution containing 0.7667 mu g per 1ml, taking another proper amount of ethyl chloroformate, adding ethanol for dissolving, shaking, and quantitatively diluting to prepare a test substance solution containing 0.7 mu g per 1 ml. And (5) injecting samples respectively.

Chromatographic conditions are as follows: capillary column: DB-5ms, 30mx 0.25mm,1.0 μm; sample inlet temperature: 300 ℃; the split ratio is as follows: 10; constant current mode, flow rate: 1ml/min; carrier gas: he; temperature of the column box: 40 ℃ (maintaining for 2 min) -10 ℃/min (heating) -130 ℃ (maintaining for 2 min) -30 ℃/min (heating) -280 ℃ (maintaining for 10 min); sample introduction amount: 1 mul; auxiliary heating area: 280 ℃. The results are shown in the following table:

wherein, the A derivative product represents the mass spectrum peak area of diethyl carbonate obtained by derivatization;

c ethyl chloroformate represents the ethyl chloroformate concentration (. Mu.g/ml);

the reference A represents the peak area of the reference quality spectrum of diethyl carbonate;

c control represents diethyl carbonate control concentration (. Mu.g/ml).

The test result shows that the conversion rate of ethyl chloroformate into diethyl carbonate is 99.6% by using ethanol as a solvent, and the ethyl chloroformate can be quickly and completely converted into diethyl carbonate which can be used as a measurable component.

Example 2 methodological validation experiment

1. The instrument comprises the following steps: agilent7890B-5977 gas chromatograph-mass spectrometer

Reagent: ethanol (pure chromatography)

Sample preparation: compound 2 (shanghai gold and bio ltd), ethyl chloroformate: purity 99.0% ethyl carbamate: purity 98.0%,1, 3-bromochloropropane: the purity is 99.7 percent

2. Chromatographic conditions

Capillary column: DB-5ms, 30mX0.25mm, 1.0 μm; sample inlet temperature: 300 ℃; the split ratio is as follows: 10; constant current mode, flow rate: 1ml/min; carrier gas: he; temperature of the column box: 40 ℃ (maintaining for 2 min) -10 ℃/min (heating) -130 ℃ (maintaining for 2 min) -30 ℃/min (heating) -280 ℃ (maintaining for 10 min); sample introduction amount: 1 mul; auxiliary heating area: 280 deg.C

3. Conditions of Mass Spectrometry

Solvent retardation: 7.2min; the acquisition mode is SIM mode;

7.2min group 1 (mass number m/z, residence time) (45.00, 50), (63.00, 50), (91.00, 50);

9.0min group 2 (mass number m/z, residence time) (41.00, 50), (58.00, 50), (75.00, 50), (77.00, 50), (103.00, 50), (158.00, 50);

MS ion source temperature: 230 ℃; MS quadrupole temperature: at 150 ℃.

(1) Specificity experiments

Injecting 1 mul of mixed standard solution into a gas chromatograph-mass spectrometer, and collecting by adopting a full SCAN (SCAN) mode to obtain retention time and characteristic fragment ions of the ethyl chloroformate, the ethyl carbamate and the 1, 3-bromochloropropane. Then, a Selective Ion (SIM) collection method of three impurities is established. A blank solution, ethyl chloroformate, ethyl methyl carbamate and 1, 3-bromochloropropane are mixed with a standard use solution and a compound 2 sample solution, and then a needle is added to ensure that potential impurities, impurities and main peaks do not interfere with each other, and impurities and a solvent do not interfere with each other. The results are as follows:

and (4) conclusion: the solvent is blank, the chromatographic peak position of the sample solution in ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate is free from interference, the analytical method has good specificity and meets the analytical requirements. The chromatogram of the blank solution and the solutions used in the three mixing standards are shown in the attached figures 1 and 2.

(2) Detection limit and quantification limit

Preparing reference solutions of different impurities with different concentrations, and determining the detection limit of each impurity when S/N of each impurity is about 3 (see figure 3); when S/N of each impurity is about 10, determining the quantitative limit of each impurity (see figure 4), and the RSD of the peak area of each impurity reference substance solution with the quantitative limit concentration continuously entering 6 needles is less than or equal to 10%.

And (4) verification result:

A. detection limit and quantification limit of ethyl chloroformate: and diluting the standard stock solution 1 of ethyl chloroformate step by step, wherein when the concentration of the ethyl chloroformate is 0.07 mu g/ml, the signal-to-noise ratio is 4.3, and the standard stock solution can meet the signal-to-noise ratio requirement of a detection limit and can be used as the detection limit. When the concentration of the ethyl chloroformate is 0.175 mu g/ml, the signal-to-noise ratio of 6 times of sample injection is about 10, the signal-to-noise ratio requirement of the quantification limit is met, and the RSD of the peak area of 6 times of sample injection of the quantification limit is 3.80 percent and can be used as the quantification limit.

B. Detection limit and quantification limit of ethyl carbamate: and diluting the ethyl carbamate quasi-stock solution 2 step by step, wherein when the concentration of the ethyl carbamate is 0.07 mu g/ml, the signal-to-noise ratio is 4.9, the signal-to-noise ratio requirement of the detection limit is met, and the detection limit can be used. When the concentration of the ethyl carbamate is 0.175 mu g/ml, the signal-to-noise ratio of 6 times of sample injection is about 10, the signal-to-noise ratio requirement of the quantification limit is met, and the RSD of the peak area of 6 times of sample injection of the quantification limit is 5.03 percent and can be used as the quantification limit.

C. Detection limit and quantification limit of 1, 3-bromochloropropane: the 1, 3-bromochloropropane standard intermediate solution is diluted step by step, when the concentration of the 1, 3-bromochloropropane is 0.07 mu g/ml, the signal-to-noise ratio is 4.9, the signal-to-noise ratio requirement of the detection limit is met, and the signal-to-noise ratio can be used as the detection limit. When the concentration of the 1, 3-bromochloropropane is 0.175 mu g/ml, the signal-to-noise ratio of 6 times of sample injection is about 10, the signal-to-noise ratio requirement of the limit of quantitation is met, and the RSD of the peak area of the 6 times of sample injection of the limit of quantitation is 1.86 percent and can be used as the limit of quantitation. The injection results are as follows:

ethyl chloroformate quantitative limit sample injection peak area and signal-to-noise ratio

Ethyl carbamate quantitative limit sample injection peak area and signal-to-noise ratio

Peak area and signal-to-noise ratio of quantitative limit sample introduction of 1, 3-bromochloropropane

And (3) test results: the quantitative limit concentration of the ethyl chloroformate is 0.175 mu g/ml, which is equivalent to 17.5ppm of the concentration of the test sample; the detection limit concentration is 0.07 mu g/ml, which is equivalent to 7ppm of the concentration of the test sample;

and (3) test results: the quantitative limit concentration of the 1, 3-bromochloropropane is 0.175 mu g/ml, which is equivalent to 17.5ppm of the concentration of a test sample; the detection limit concentration is 0.07 mu g/ml, which is equivalent to 7ppm of the concentration of the test sample;

and (3) test results: the quantitative limit concentration of the ethyl carbamate is 0.175 mu g/ml, which is equivalent to 17.5ppm of the concentration of the test sample; the detection limit concentration was 0.07. Mu.g/ml, corresponding to 7ppm of the sample concentration.

(3) Linearity

Preparing each impurity reference substance solution with 5 concentration points from the limit concentration of quantification to 200%, carrying out sample injection once for each concentration, and carrying out linear calculation by using the concentration and the peak area, wherein r2 is more than 0.99.

Concentration and peak area of linear solutions of ethyl chloroformate, ethyl methylcarbamate and 1, 3-bromochloropropane

And (4) test conclusion:

the linear equation for ethyl chloroformate is y =260547x-2060.9, R2=0.9999; the linear range is 0.175 mu g/ml to 1.4 mu g/ml;

the linear equation for ethyl carbamate is y =186419x-14630; r2=0.9996; the linear range is 0.175 mu g/ml to 1.4 mu g/ml;

the linear equation for 1, 3-bromochloropropane is y =193356x +2315.2; r2=0.9991; the linear range is 0.175. Mu.g/ml to 1.4. Mu.g/ml.

(4) Precision of sample introduction

Preparing a target impurity reference substance solution with 100% limit concentration, continuously injecting a sample for 6 needles, and counting the peak area of each target impurity, wherein RSD is less than 5%. The retention time RSD of each impurity should be no more than 2%.

The mixed standard solution of 100% limiting concentration ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate was injected continuously 6 times, and the injection results are shown in the following table.

And (4) conclusion: ethyl chloroformate, ethyl carbamate and 1, 3-bromochloropropane impurity reference substance solutions with 100% limit concentration are continuously injected for 6 needles, and the RSD of the peak areas of the ethyl chloroformate, the ethyl carbamate and the 1, 3-bromochloropropane are respectively 3.52%, 3.35% and 3.21%, and are all less than 5.0%; the RSD of the retention time of the ethyl chloroformate, the ethyl methyl carbamate and the 1, 3-bromochloropropane is respectively 0.024 percent, 0.016 percent and 0.010 percent, which are all less than 2.0 percent and meet the requirement.

(5) Repeatability of

A standard use solution was prepared of ethyl chloroformate, ethyl methylcarbamate, 1, 3-bromochloropropane at a concentration of 0.7. Mu.g/ml. An appropriate amount of compound 2 is weighed and added into a 100% limit concentration reference solution to prepare 6 parts, and each part is injected for 1 time. And (4) counting the peak area of each impurity, calculating the recovery rate, wherein the RSD of the recovery rate of 6 parts of the sample solution with 100% limit concentration and the standard should be less than 5.0%.

Repeatability of ethyl chloroformate

Results of repeated measurement of Ethyl Methocarbamate

Repeatability determination result of 1, 3-bromochloropropane

And (4) conclusion:

RSD of the impurity peak areas (recovery rates) of ethyl chloroformate, ethyl carbamate and 1, 3-bromochloropropane added to the impurity reference substance solution with 100% limit concentration of 6 parts of the compound 2 sample are respectively 2.40%, 3.89% and 2.66%, and the RSD is less than 5%, so that the requirement is met.

(6) Accuracy of

Preparing two portions of sample solution, adding ethyl chloroformate, ethyl methyl carbamate and 1, 3-bromochloropropane with limit concentrations of 80%, 100% and 120% into three portions of sample solution, sampling each portion for 1 time, counting the peak area of each impurity, and performing a recovery rate test, wherein the recovery rate is 85-115%. The results are as follows.

Ethyl chloroformate accuracy measurement

Results of ethyl methyl carbamate accuracy measurement

1, 3-bromochloropropane accuracy measurement result

And (4) conclusion: the recovery rate of ethyl chloroformate in the mixed solution of the test sample and the impurity reference substance with the limit concentration of 80%, 100% and 120% is 87.38-96.17%, and the RSD is 3.34%; the recovery rate of the ethyl methyl carbamate is between 95.55 and 106.2 percent, and the RSD is 4.07 percent; the recovery rate of the 1, 3-bromochloropropane is between 93.77 and 102.8 percent, and the RSD is 3.56 percent, which all meet the requirements.

(7) Durability

Changing chromatographic conditions, preparing a test solution containing 100 percent of standard impurity-added reference substance, changing the chromatographic conditions under the standard conditions, wherein the ratio of the detection result of the component to be detected under the durability condition to the detection result of the impurity under the standard conditions is 90.0-110.0 percent.

Standard conditions: see 2 chromatographic conditions

Condition 1: an initial temperature of 38 ℃;

condition 2: an initial temperature of 42 ℃;

condition 3: sample inlet temperature: 298 deg.C

Condition 4: sample inlet temperature: 302 deg.C

Preparing a 100% standard-added test sample solution: weighing a compound 2 sample 0.10g to 10ml in a measuring flask, accurately transferring 0.5ml of mixed standard solution (shown in a special experiment) with the concentration of 14 mu g/ml into the measuring flask, adding ethanol for dissolving, fixing the volume to a scale, uniformly mixing, and measuring. Two portions were made in parallel, one injection for each portion.

Ethyl chloroformate durability test detection result

Results of testing durability of ethyl carbamate

1, 3-bromochloropropane durability test detection result

And (4) conclusion: compared with the original condition test result, the test result after the condition change has the ratio range of 90.0-110.0% between the test result under different conditions of ethyl chloroformate, ethyl methyl carbamate and 1, 3-bromochloropropane and the test result under the standard condition, and meets the requirement.

EXAMPLE 3 Compound 2 sample assay

1. Instruments and reagents

The instrument comprises the following steps: agilent7890B-5977 gas chromatograph-mass spectrometer;

reagent: ethanol (chromatographically pure);

sample preparation: compound 2 (shanghai gold and bio ltd), ethyl chloroformate (purity 99.0%), ethyl methylcarbamate (purity 98.0%), 1, 3-bromochloropropane (purity 99.7%);

blank solution: ethanol;

compound 2 test solution: 0.1g of compound 2 sample is weighed, dissolved by 10ml of ethanol, and the volume is determined for standby.

Ethyl chloroformate standard stock solution 1: ethyl chloroformate of 99.0% purity (33.1.11mg) was weighed accurately into a 10ml measuring flask, dissolved in ethanol and fixed to a predetermined volume to obtain a standard stock solution 1 of 3.773 mg/ml.

Standard stock solution of ethyl methylcarbamate 2: accurately weighing 40.01mg to 10ml of ethyl carbamate with the purity of 98.0%, dissolving the ethyl carbamate with ethanol, and fixing the volume to a scale to obtain a standard stock solution 2 with the concentration of 3.921 mg/ml.

1, 3-bromochloropropane standard stock solution 3: accurately weighing 40.03mg of 1, 3-bromochloropropane with the purity of 99.7 percent into a 10ml measuring flask, dissolving the measuring flask by using ethanol, and fixing the volume to a scale to obtain a standard stock solution 3 with the concentration of 3.991 mg/ml.

Impurity control solution: weighing 0.10g to 10ml of the compound 2 sample, accurately transferring 0.5ml of mixed standard solution with the concentration of 14 mu g/ml into the measuring flask, adding ethanol for dissolving, and fixing the volume to the scale.

2. Chromatographic conditions

A capillary column: DB-5ms, 30mx 0.25mm,1.0 μm; sample inlet temperature: 300 ℃; the split ratio is as follows: 10, a step of; constant flow mode, flow rate: 1ml/min; carrier gas: he; temperature of the column box: 40 ℃ (maintaining for 2 min) -10 ℃/min (heating) -130 ℃ (maintaining for 2 min) -30 ℃/min (heating) -280 ℃ (maintaining for 10 min); sample injection amount: 1 mul; auxiliary heating area: 280 ℃ 3. Mass Spectrometry conditions

Solvent retardation: 7.2min;

the acquisition mode is SIM mode;

7.2min group 1 (mass number m/z, residence time) (45.00,50), (63.00,50), (91.00,50);

9.0min group 2 (mass number m/z, residence time) (41.00, 50), (58.00, 50), (75.00, 50), (77.00, 50), (103.00, 50), (158.00, 50);

MS ion source temperature: 230 deg.C

MS quadrupole temperature: 150 ℃;

4. content determination:

by adopting the conditions, the sample solution to be tested, the reference solution and the blank solution are subjected to sample injection detection and analysis, and the detection results of the ethyl chloroformate, the 1, 3-bromochloropropane and the ethyl carbamate residues in the three batches of the compound 2 samples are as follows.

According to the detection results, ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate were not detected in the three batches of compound 2 samples. The spectrum of batch 1 of compound 2 is shown in figure 5.

Claims (9)

1. The method for detecting impurities of ethyl chloroformate, 1, 3-bromochloropropane and ethyl carbamate in the eptazocine intermediate compound 2 (-) - (1S-6S) -2,3,4,5,6, 7-hexahydro-1, 4-dimethyl-1, 6-methylene-1 hydro-4-phenyllin-10-methoxy ether is characterized by adopting gas chromatography-mass spectrometry combined chromatography for determination, wherein a chromatographic column adopted by the gas chromatography is a DB-5ms capillary vessel chromatographic column, a sample dissolving solvent is ethanol, and the column temperature is controlled by a program.

2. The method of claim 1, wherein the test compound 2 is present at a concentration of 10mg/ml.

3. The method according to claim 1, wherein the temperature-raising program of the gas chromatography temperature-raising program is to maintain an initial temperature of 30-50 ℃ for 2min, raise the temperature at a speed of 10-30 ℃/min, raise the temperature to 110-140 ℃, maintain the temperature for 2min, raise the temperature at a speed of 10-30 ℃/min, raise the temperature to 260-290 ℃ and maintain the temperature for 10min.

4. The method according to claim 3, wherein the temperature-raising procedure of the gas chromatography temperature-raising procedure is an initial temperature of 40 ℃, a temperature-raising speed of 10 ℃/min, a temperature-raising speed of up to 130 ℃, a temperature-raising speed of 2min, and a preferred temperature-raising speed of 30 ℃/min to 280 ℃ and a temperature-raising speed of 10min.

5. The method of claim 1, wherein the other gas chromatography conditions of the detection method comprise: the temperature of a sample inlet is 280-300 ℃; the flow dividing ratio is 5; the flow rate of the carrier gas is 0.8ml/min to 1.5ml/min;

the sample amount is 1-10 mul.

6. The method of claim 1, wherein the gas chromatography conditions of the detection method further comprise: the temperature of a sample inlet is 300 ℃; the flow division ratio is 10; the flow rate of the carrier gas is 1.0ml/min; the sample volume was 1. Mu.l.

7. The method of claim 1, wherein the detection method employs mass spectrometry conditions of: the EI ionization source is adopted, and the temperature of the ion source is 200-250 ℃; adopting a single-quadrupole mass analyzer, wherein the temperature of a quadrupole is 120-150 ℃; the adopted detection mode is a single ion detection mode, and the extracted ions are m/z91.00, m/z103.00 and m/z158.00; the residence time was 50ms and the solvent delay time was 12min.

8. The method of claim 7, wherein the detection method employs EI ionization source ion source temperature of 230 ℃ in the mass spectrometry conditions employed; the quadrupole temperature was 150 ℃ using a single quadrupole mass analyser.

9. The method according to claim 1, characterized in that the detection method comprises in particular the steps of:

1) Preparation of a test solution: weighing 0.1g of compound 2 sample, dissolving with 10ml of ethanol, clarifying, and fixing the volume;

2) Preparation of control solutions:

a. ethyl chloroformate control solution: accurately weighing 38.11mg of ethyl chloroformate with the purity of 99.0% into a measuring flask of 10ml, dissolving the ethyl chloroformate with ethanol, and fixing the volume to scale to obtain a control solution with the concentration of 3.773 mg/ml;

b. ethyl carbamate control solution: accurately weighing 40.01mg to 10ml of ethyl carbamate with the purity of 98.0%, dissolving by using ethanol, and fixing the volume to a scale to obtain a control solution with the concentration of 3.921 mg/ml;

c. 1, 3-bromochloropropane control solution: accurately weighing 40.03mg of 1, 3-bromochloropropane with the purity of 99.7% into a 10ml measuring flask, dissolving with ethanol, and fixing the volume to a scale to obtain a control solution with the concentration of 3.991 mg/ml;

3) Analysis and determination: and (3) injecting the test solution and the reference solution into a gas chromatography-mass spectrometer and recording the spectra.

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