Method for determining residual solvent amount in estradiol bulk drug

Abstract

The invention discloses a method for determining the residual solvent amount in an estradiol raw material medicine, and relates to the technical field of instrument analysis of chemical pharmacy. According to the method, the residual quantity of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene and ethylene glycol in an estradiol bulk drug sample is calculated by utilizing a gas chromatography and headspace sampling determination. The method has better specificity and higher sensitivity and accuracy, one analysis method can be used for simultaneously determining seven residual solvents, and the method is simple and convenient to operate, simple and easy to implement. Accurate data support is provided for control of the estradiol residual solvent, product quality is improved, and medication safety of patients is guaranteed.

Description

Technical Field

[0001] This invention relates to the field of instrumental analysis technology in chemical pharmaceutical manufacturing, and more specifically, to a method for determining the amount of residual solvent in estradiol raw material. Background Technology

[0002] Various organic solvents are used in the production of estradiol, and accurate determination of residual solvent levels in estradiol is crucial for the quality of estradiol raw materials and patient safety. Because the organic solvents used in the estradiol production process are complex in composition and present in low concentrations, a specific and sensitive assay method is needed to ensure accurate and reliable results, while also being easy and rapid to perform. Currently, there is no clearly defined and easily implemented method for this purpose. There are currently no publicly reported analytical methods, either domestically or internationally, for determining the residual amounts of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene, and ethylene glycol in estradiol raw materials. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for determining the amount of residual solvent in estradiol raw materials, so as to solve the problems existing in the prior art. The method provided by this invention can efficiently, quickly and accurately determine the residual amounts of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene and ethylene glycol in estradiol raw materials with complex solvents, thereby improving product quality and ensuring patient medication safety.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows: A method for determining the amount of residual solvent in estradiol raw material, wherein the residual solvent includes methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene, and ethylene glycol; The method includes: Methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene, and ethylene glycol were diluted to prepare reference solutions of known concentrations. The reference solutions were then injected into a gas chromatograph via a headspace sampler to obtain the chromatogram of the reference solutions. The estradiol raw material to be tested was prepared into a sample solution using a diluent. The sample solution was injected into a gas chromatograph through a headspace sampler to obtain a chromatogram of the sample solution. The amount of residual solvent in the estradiol raw material was calculated by peak area using the external standard method. The chromatographic conditions for the gas chromatograph are as follows: The chromatographic column was a capillary column with a stationary phase of 6% cyanopropylphenyl-94% dimethylpolysiloxane and a specification of 30m × 0.53mm × 3µm. Injector temperature: 190~210℃, detector temperature: 240~260℃; column oven temperature: programmed temperature rise method, initial temperature: 40~60℃, hold for 4~8min; heating rate: 15~25℃ / min, final temperature: 190~210℃, hold for 4~8min; Headspace sampler platform temperature: 90~100℃, quantitative loop temperature: 140~150℃, transfer line temperature: 170~180℃, headspace vial filling pressure: 9~11Psi, equilibration time: 18~25 minutes.

[0005] Furthermore, the headspace sampler has the following parameters: platform temperature: 95°C, quantitative loop temperature: 145°C, transfer line temperature: 175°C, headspace vial filling pressure: 10 Psi, and equilibration time: 20 minutes.

[0006] Furthermore, the gas chromatograph has the following specifications: injection port temperature: 200℃; detector temperature: 250℃; column oven temperature: programmed temperature ramp method, initial temperature: 50℃, hold for 5 min; heating rate: 20℃ / min; final temperature: 200℃, hold for 5 min.

[0007] Furthermore, the carrier gas of the gas chromatograph is nitrogen, and the flow rate is 3.0 ml / min.

[0008] Furthermore, the diluent is N,N-dimethylformamide.

[0009] Further, the preparation method of the reference solution includes: accurately weighing appropriate amounts of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene, and ethylene glycol, and quantitatively diluting them with N,N-dimethylformamide to prepare a mixed solution containing 0.1 mg of methanol, 1.0 mg of ethanol, 2.5 mg of acetone, 0.3 mg of dichloromethane, 0.36 mg of tetrahydrofuran, 0.32 mg of ethylene glycol, and 1 µg of benzene per 1 ml; accurately measuring 5 ml of this solution, placing it in a headspace bottle, sealing it, and using it as the reference solution.

[0010] Furthermore, the method for preparing the sample solution includes: weighing the estradiol raw material to be tested as a sample, accurately weighing it, placing it in an empty bottle, and accurately adding N,N-dimethylformamide to dilute it to a concentration of 0.5 g / mL as the sample solution.

[0011] Further, in the method, the limit of quantitation (LOQ) for methanol is 0.71 ppm, for ethanol it is 0.70 ppm, for acetone it is 0.25 ppm, for dichloromethane it is 0.82 ppm, for tetrahydrofuran it is 0.11 ppm, for benzene it is 0.07 ppm, and for ethylene glycol it is 53 ppm.

[0012] Furthermore, in the method, the separation degree between adjacent residual solvents is greater than 1.5.

[0013] Compared with the prior art, the present invention has the following advantages: This invention demonstrates good specificity, sensitivity, and accuracy for the determination of residual methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene, and ethylene glycol in estradiol raw materials. Specifically, the limit of quantitation (LOQ) for ethylene glycol reaches 53 ppm, and the limit of detection (LOD) reaches 16 ppm, overcoming the challenge in the residual solvent determination method of General Chapter 0861, Part IV, of the 2025 edition of the Chinese Pharmacopoeia that "for Class II solvents such as ethylene glycol with high boiling points, it is difficult to obtain the required sensitivity through headspace sampling." Furthermore, the LQ for benzene reaches 0.07 ppm, which effectively ensures the quality of estradiol raw materials and protects patient safety. Moreover, a single analytical method can simultaneously detect seven target solvents with different polarities, offering simple, rapid, and easy-to-perform analysis. Attached Figure Description

[0014] Figure 1 This is a chromatographic result of a specific mixed solution (100% standard solution) for the method of this invention; Figure 2 This is a chromatogram of the sample detection results of the method of the present invention. Detailed Implementation

[0015] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0017] Unless otherwise specified, all materials and reagents used in this invention are available from commercially available products in the field.

[0018] The solvents used in estradiol raw materials are complex, mainly consisting of methanol, ethanol, and benzene. Except for ethanol, the residual amounts of other solvents are relatively low. The challenge of this invention is how to accurately measure the residual amounts of these solvents in order to better control the quality of the raw materials.

[0019] This invention employs a standard reference method in gas chromatography, preparing reference solutions and sample solutions of a certain concentration, selecting parameters based on the specific characteristics of the solvents to be tested in the sample, and calculating the content of each residual solvent using the external standard method.

[0020] The following is a description using specific embodiments.

[0021] Example 1 1. The chromatographic conditions are selected as follows: Chromatograph: Gas chromatograph; Chromatographic column: DB-624 column, 30m × 0.53mm × 3µm; Inlet temperature: 200℃; Detector (FID): 250℃; Column temperature (programmed temperature): Initial temperature: 50℃ (hold for 5 min), heating rate: 20℃ / min, final temperature: 200℃ (hold for 5 min); Carrier gas: nitrogen, flow rate 3.0 ml / min; Headspace gas sampler: Platform temperature: 95℃; Metering loop temperature: 145℃; Transmission line temperature: 175℃; Balancing time: 20 minutes.

[0022] 2. Reagent selection: Methanol (AR grade); ethanol (AR grade); acetone (AR grade); dichloromethane (AR grade); tetrahydrofuran (AR grade); benzene (AR grade); ethylene glycol (AR grade); DMF (HPLC grade).

[0023] 3. Solution preparation 3.1 Sample solution preparation: Weigh 1.0g of estradiol raw material sample accurately, place it in a headspace vial, add 2.0ml of DMF to dissolve it, and then seal the vial with a pressure cap.

[0024] 3.2 Preparation of standard solutions: Accurately weigh 0.4 g of benzene and place it in a 100 ml volumetric flask. Dilute to the mark with DMF and mix well to prepare the benzene stock solution. Separately weigh 0.1 g of methanol, 1.0 g of ethanol, 2.5 g of acetone, 0.3 g of dichloromethane, 0.36 g of tetrahydrofuran, and 0.32 g of ethylene glycol. Pipette 0.25 ml of each benzene stock solution into a 50 ml volumetric flask and dilute to the mark with DMF. Mix well to prepare the standard stock solution. Accurately transfer 5.0 ml of the standard stock solution into a 100 ml volumetric flask, dilute to the mark with DMF, and mix well to obtain the standard solution. Accurately transfer 2.0 ml of the standard solution into a headspace vial and seal tightly. (This solution concentration is 100% standard solution concentration.) Calculation method: External standard method.

[0025]

[0026] In the formula: Au: Peak area value of the relevant residual solvent in the sample solution; As: The average peak area of ​​each standard solvent in the standard solution; Cs: Concentration of each standard solvent in the standard solution, in µg / ml. m: Sample weight, in g. 2: The volume of the test solution diluted, in ml.

[0027] 4. Methodological Validation 4.1 Specificity Verification: The qualitative solutions (100% standard solution concentration) of the solvents to be tested—methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene, and ethylene glycol—were prepared and injected individually and in combination. The resolution between the peaks of each solvent to be tested was calculated.

[0028] 4.1.1 Results of specific solution determination: The results of the determination of the seven solvents are shown in the table below:

[0029] 4.1.2 Method Review: According to the relevant guidelines for analytical method validation, the resolution between each solvent peak should be ≥1.5. The results show that under the chromatographic conditions set in this method, the resolution between each solvent peak is >1.5, indicating that this method has good specificity.

[0030] 4.2 Limit of Quantification: 4.2.1. Take 2.0 ml of linear standard solution S8 (accurately transfer 0.25 ml of standard stock solution into a 50 ml volumetric flask, dilute to the mark with DMF, and mix well) into six 20 ml headspace vials, inject one sample into each, and record the chromatogram. Calculate the limit of quantitation (LOQ) concentration (concentration at S / N = 10:1) for each solvent based on the injected concentration and signal-to-noise ratio, and calculate the average LQ for each solvent accordingly. (The ppm values ​​in the table below are all ppm values ​​of the solvent in the solution relative to the sample weight.)

[0031] 4.2.2. Based on the average S / N values ​​in the table above, calculate the concentration of each solvent when S / N = 10:1, and use this concentration as the LOQ concentration. Prepare the LOQ solution and perform sample injection and measurement. The results are as follows:

[0032] 4.3 Detection Limit: Accurately transfer 15.0 ml of LOQ solution into a 50 ml volumetric flask, dilute to the mark with DMF to obtain LOD solution. Inject the LOD solution into six syringes according to the method, record the spectrum, and calculate the signal-to-noise ratio (S / N) of each solvent peak. The results are as follows:

[0033] 4.4 Linearity Validation: 4.4.1. Prepare linear solutions with concentrations ranging from the LOQ concentration to 120% of the standard solution concentration (transfer 0.25 ml, 0.5 ml, 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, 5.0 ml, and 6.0 ml of the standard stock solution to 50 ml volumetric flasks, dilute to the mark with DMF, and shake well to obtain linear solutions S8 to S1). Inject each linear solution of different concentrations once, and the results are shown in the table below: Test results of standard solutions of different concentrations:

[0034] Standard curves for six solvents:

[0035] 4.4.2 Method Review: The results show that, under the chromatographic conditions set by this method, the concentration and peak area responses of each analyte solvent exhibit good linearity (correlation coefficients all greater than 0.99), which meets the relevant guidelines for method validation. This indicates that the method can accurately and reliably determine the samples within the expected concentration range (LOQ ~ 120% of standard solution concentration).

[0036] 4.5 Accuracy Verification: 4.5.1. Weigh 18 samples quantitatively (each sample weight is within 1.000±0.005g, and weighing error can be ignored). Add 2.0ml of linear standard solution S1 quantitatively to 6 samples, add 2.0ml of standard solution quantitatively to another 6 samples, and add 2.0ml of LOQ solution quantitatively to the remaining 6 samples to prepare accuracy verification solutions. Use the average detection value of the six samples as a blank control. Calculate the spiked recovery rate of each solvent.

[0037] 4.5.2 Sample solution (recovery rate %) after adding linear standard solution S1:

[0038] 4.5.3 Sample solution (recovery rate after adding standard solution):

[0039] 4.5.4 Sample Solution (Recovery Rate after Adding LOQ Solution):

[0040] Note: Since the sample mainly contains residual ethanol solvent, the recovery rate of ethanol is not considered when adding LOQ to the sample solution.

[0041] 4.5.5 Method Review: The results show that under the chromatographic conditions set in this method, the recoveries of all analyte solvents were good under the three spiking conditions. The recoveries of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, and ethylene glycol were generally between 90% and 105%, all meeting the relevant guidelines for method validation. The recovery rate of benzene was lower than that of the other solvents because the limit for benzene is only 2 ppm, resulting in a relatively small peak response and thus a lower recovery rate. However, the current recovery levels are sufficient for the detection requirements.

[0042] 4.6 Repeatability verification: 4.6.1. Select the same batch of samples, prepare 6 parallel sample solutions, inject each solution and record the chromatogram (one sample's test result is as follows). Figure 2 (As shown), calculate the average content and relative standard deviation (RSD) of each residual solvent in the 6 sample solutions:

[0043] Note: In the sample solution test results, methanol was less than the limit of quantitation (0.71 ppm), ethanol results are shown in the table above, and other solvents were not detected. The repeatability of the sample residue results was good.

[0044] 4.6.2 Method Review: The results show that under the chromatographic conditions set by this method, the residual solvent in the sample solution has good reproducibility, proving that the method has good precision.

[0045] Comparative Example 1 The only difference between this comparative example and Example 1 is that different chromatographic columns were used: DB-624 30m×0.32mm×1.8um and DB-1701 30m×0.32mm×0.25um. Take the 100% standard solution from Example 1 and perform gas chromatography detection according to the steps in Example 1. In the chromatogram of column DB-624 (30m × 0.32mm × 1.8um), the ethanol and acetone peaks could not be effectively separated (resolution < 1.5); in the chromatogram of column DB-1701 (30m × 0.32mm × 0.25um), all target peaks could not be separated (all solvent peaks eluted within 1 minute, interfering with each other), and the benzene peak could not be detected.

[0046] Comparative Example 2 The only difference between this comparative example and Example 1 is that the headspace platform temperature is set to 80°C. Take the 100% standard solution from Example 1 and perform gas chromatography detection according to the steps in Example 1.

[0047] The results showed that the platform heating temperature was too low, the boiling point of ethylene glycol was too high, and the ethylene glycol peak could not be detected in the chromatogram.

[0048] Comparative Example 3 The only difference between this comparative example and Example 1 is that the direct solution injection method is used instead of the headspace injection method for determination.

[0049] Take the sample solution and perform gas chromatography detection according to the steps in Example 1.

[0050] The results showed that there were many impurity peaks in the sample solution, which interfered with the determination of the solvent to be tested. Therefore, headspace sampling method, which has fewer interfering components and is less likely to contaminate the injection port, chromatographic column and detector, was selected for the determination.

[0051] Comparative Example 4 The only difference between this comparative example and Example 1 is that different platform heating times (10 min, 20 min, 30 min) are used.

[0052] Take the 100% standard solution from Example 1 and perform gas chromatography detection according to the steps in Example 1.

[0053] The results showed that gas-liquid equilibrium was reached after heating for 20 minutes, so this experiment selected heating for 20 minutes for measurement.

[0054] In summary, the method for determining residual solvents in estradiol raw materials provided by this invention utilizes the aforementioned gas chromatography with headspace sampling to calculate the residual amounts of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene, and ethylene glycol in the sample. This method exhibits good specificity, high sensitivity, and accuracy. A single analytical method can simultaneously determine seven residual solvents, and it is simple and easy to operate. It provides accurate data support for the control of residual solvents in estradiol, improves product quality, and ensures patient medication safety.

[0055] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0056] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for determining the amount of residual solvent in estradiol drug substance, characterized in that, The residual solvents include methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene and ethylene glycol. The method comprises: The methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, benzene and ethylene glycol are configured into a control solution of a known concentration by a diluent, and then the control solution is injected into a gas chromatograph through a headspace sampler to obtain a control solution chromatogram; The sample solution is prepared by weighing the estradiol raw material to be determined as a sample, precisely weighing, and placing in a headspace bottle, and precisely adding N,N-dimethylformamide to dilute into a solution with a concentration of 0.5 g / mL as a sample solution. The chromatographic conditions of the gas chromatograph are as follows: The chromatographic column is a capillary column with 6% cyano propyl phenyl-94% dimethyl polysiloxane as a stationary liquid, with a specification of 30m*0.53mm*3µm; The injection port temperature is 190-210℃, the detector temperature is 240-260℃, and the column oven temperature is programmed to rise, with an initial temperature of 40-60℃, a holding time of 4-8min, a temperature rising rate of 15-25℃ / min, a final temperature of 190-210℃, and a holding time of 4-8min; The platform temperature of the headspace sampler is 90-100℃, the quantification ring temperature is 140-150℃, the transmission line temperature is 170-180℃, the headspace bottle filling pressure is 9-11Psi, and the equilibrium time is 18-25min.

2. The method of claim 1, wherein, The platform temperature of the headspace sampler is 95℃, the quantification ring temperature is 145℃, the transmission line temperature is 175℃, the headspace bottle filling pressure is 10Psi, and the equilibrium time is 20min.

3. The method of claim 1, wherein, The injection port temperature of the gas chromatograph is 200℃, the detector temperature is 250℃, and the column oven temperature is programmed to rise, with an initial temperature of 50℃, a holding time of 5min, a temperature rising rate of 20℃ / min, a final temperature of 200℃, and a holding time of 5min.

4. The method of claim 1, wherein, The carrier gas of the gas chromatograph is nitrogen, and the flow rate is 3.0ml / min.

5. The method of claim 1, wherein, The diluent is N,N-dimethylformamide.

6. The method of claim 5, wherein, The configuration method of the control solution comprises: taking appropriate amounts of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran and benzene respectively, precisely weighing, and quantitatively diluting with N,N-dimethylformamide to obtain a mixed solution containing 0.1mg of methanol, 1.0mg of ethanol, 2.5mg of acetone, 0.3mg of dichloromethane, 0.36mg of tetrahydrofuran, 1µg of benzene and 0.32mg of ethylene glycol per 1ml, precisely taking 5ml, placing in a headspace bottle, sealing, and serving as a control solution.

7. The method of claim 1, wherein, The configuration method of the sample solution comprises: weighing the estradiol raw material to be determined as a sample, precisely weighing, placing in a headspace bottle, and precisely adding N,N-dimethylformamide to dilute into a solution with a concentration of 0.5g / mL as a sample solution.

8. The method of claim 1, wherein, In the method, the quantitation limit of methanol is 0.71 ppm, the quantitation limit of ethanol is 0.70 ppm, the quantitation limit of acetone is 0.25 ppm, the quantitation limit of dichloromethane is 0.82 ppm, the quantitation limit of tetrahydrofuran is 0.11 ppm, the quantitation limit of benzene is 0.07 ppm, and the quantitation limit of ethylene glycol is 53 ppm.

9. The method of claim 1, wherein, In the method, the separation between adjacent residual solvents is greater than 1.5.

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