A GC method for detecting dimethyl sulfate and diethyl sulfate in the non-neramexane raw material medicine

Abstract

Invention Title: A GC Method for the Determination of Dimethyl Sulfate and Diethyl Sulfate in Fennellone Raw Material Abstract: This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a GC method for the detection of genotoxic impurities—dimethyl sulfate (DMS) and diethyl sulfate (DES)—in fenelone raw material. Dimethyl sulfate and diethyl sulfate pose potential mutagenic and carcinogenic risks and are easily hydrolyzed in water, making conventional analytical methods insufficient for trace detection. To address this issue, this invention employs headspace gas chromatography, optimizing sample dissolution conditions, headspace equilibrium parameters, and column type to achieve efficient separation and accurate quantification of the two target analytes in a fenelone matrix.

Description

Technical Field

[0001] This invention provides a GC method for determining dimethyl sulfate and diethyl sulfate in phenelzine raw material, belonging to the field of pharmaceutical analysis and testing. This method provides efficient and accurate quantitative analysis of dimethyl sulfate and diethyl sulfate in phenelzine, offering strong support for the quality control of phenelzine raw material. Background Technology

[0002] Finerenone is a nonsteroidal, highly selective mineralocorticoid receptor antagonist, primarily used clinically to treat type 2 diabetes-related chronic kidney disease. With the ongoing research into its formulations and active pharmaceutical ingredients, higher demands are being placed on controlling potential impurities in its synthetic process. In existing finelone synthetic routes, methylating or ethylating agents are commonly used to modify the structure of drug intermediates containing phenolic hydroxyl or amino groups. For example, in the synthesis of key intermediates, dimethyl sulfate and / or diethyl sulfate are used as alkylating agents in the chemical reaction. Dimethyl sulfate and diethyl sulfate are not only highly toxic but also typical genotoxic impurities (GTi). They can alkylate DNA in organisms, causing gene mutations and posing potential carcinogenic and teratogenic risks.

[0003] According to ICH M7 guidelines, impurities with warning structures must be strictly controlled according to the Threshold of Toxicological Concern (TTC), with an acceptable daily intake of extremely low levels (typically 1.5 μg / day). However, dimethyl sulfate and diethyl sulfate are extremely unstable in water at room temperature, readily hydrolyzing to form the corresponding monoalkyl sulfate esters and alcohols, thus becoming ineffective, and this decomposition accelerates with increasing temperature. This characteristic poses a significant challenge to trace detection: direct detection of the original drug is problematic due to its extremely low concentration and easy decomposition. Currently, some general methods exist for the detection of dimethyl sulfate and diethyl sulfate, such as headspace in-situ derivatization GC-MS, ion chromatography, or LC-MS, but these methods are costly. Therefore, to ensure the clinical safety of fenelazol, there is an urgent need to develop a specific, sensitive, simple, and cost-effective method for the trace detection of dimethyl sulfate and diethyl sulfate in fenelazol. Summary of the Invention

[0004] Existing methods for detecting dimethyl sulfate and diethyl sulfate, two potential residual reagents, mostly employ headspace in-situ derivatization GC-MS, ion chromatography, or LC-MS, which involve high economic investment and maintenance costs. Therefore, it is necessary to provide a more applicable and economical method for detecting these two impurities in phenelzine.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for detecting dimethyl sulfate and diethyl sulfate in fenelazol raw material, the method employing gas chromatography (GC) and mainly including the following steps:

[0007] (a) Prepare reference solutions of dimethyl sulfate and diethyl sulfate respectively;

[0008] (b) Take the fenelazol test sample, add a suitable solvent to dissolve it, and prepare a test sample solution;

[0009] (c) Inject the reference solution and the test solution into the GC system for detection;

[0010] (d) Obtain chromatograms of the test solution and the reference solution under qualified chromatographic separation conditions, and determine the contents of dimethyl sulfate and diethyl sulfate in feneformin by external standard method;

[0011] Gas chromatography conditions: A capillary column (e.g., DB-624) with a 6% cyanopropylphenyl-94% dimethylpolysiloxane stationary phase was used. Injector temperature was 180–220℃. Nitrogen was used as the carrier gas, in constant flow mode, at a flow rate of 2.0–4.0 mL / min. The split ratio was 10:1, and the injection volume was 1 mL. Temperature program: Initially 40℃, increased to 75℃ at 1–3℃ / min and held for 2 min; then increased to 250℃ at 30–50℃ / min and held for 8 min. Headspace vial equilibration temperature was 60–100℃, and sample equilibration time was 20–40 min.

[0012] Beneficial effects

[0013] Compared with the prior art, the present invention has the following advantages: high specificity: through a specific chromatographic column and temperature program, dimethyl sulfate, diethyl sulfate and the main drug components and other impurities can all achieve baseline separation without interference.

[0014] High sensitivity: Detection is performed using GC, meeting the stringent limits for genotoxic impurities in fenelone raw materials.

[0015] Economical and practical: It adopts general GC detection, avoiding the high cost and maintenance of GC-MS, ion chromatography or LC-MS methods. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope of the present invention.

[0017] Figure 1This is a chromatogram of the reference solution in a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material according to Example 1 of this application.

[0018] Figure 2 This is a chromatogram of the system suitability solution in a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material according to Example 1 of this application.

[0019] Figure 3 This is a chromatogram of the blank solution in a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material according to Example 1 of this application.

[0020] Figure 4 This is a chromatogram of the dimethyl sulfate localization solution in a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material according to Example 1 of this application.

[0021] Figure 5 This is a chromatogram of the diethyl sulfate localization solution in a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material according to Example 1 of this application. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0023] Example 1 This example provides a gas phase detection method for simultaneously determining the content of dimethyl sulfate and diethyl sulfate in phenelzine.

[0024] Instruments and reagents:

[0025] Agilent 7890B gas chromatograph, equipped with an FID detector and headspace autosampler;

[0026] XS105DU electronic balance with a range of 1 / 100,000 (1 / 100,000 electronic balance)

[0027] The chromatographic column was a DB-624 (60m × 0.53mm, 3μm) (Agilent).

[0028] Dimethyl sulfoxide (batch number: 1630250301D, manufacturer: Shanghai Xingke High Purity Reagent Co., Ltd.)

[0029] Methanol (Batch No.: 0234251105D Manufacturer: Shanghai Xingke High Purity Reagent Co., Ltd.)

[0030] Ethanol (Batch No.: 0330250901D Manufacturer: Shanghai Xingke High Purity Reagent Co., Ltd.)

[0031] Dimethyl sulfate (batch number: ZXDKGEKK, manufacturer: Anaiji Chemical)

[0032] Diethyl sulfate (batch number: PBDA5RJA, manufacturer: Anaiji Chemical)

[0033] Fennedone (Batch No.: CP6401-240601 Manufacturer: Xinxiang Shuanglu Pharmaceutical Co., Ltd.)

[0034] Sodium acetate (batch number 20240719, manufacturer: Sinopharm Chemical Reagent Co., Ltd.)

[0035] The specific operating steps are as follows:

[0036] (1) Solution preparation

[0037] Accurately weigh 0.20 g of phenelzine and place it in an empty bottle. Accurately add 0.5 ml of 10% sodium acetate solution and 2 ml of dimethyl sulfoxide, seal the bottle, and obtain the test solution.

[0038] Accurately measure 2 ml of dimethyl sulfoxide into an empty cap, then accurately add... 0.5 ml, sealed, as a blank solution.

[0039] Accurately weigh approximately 75 mg of dimethyl sulfate and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with dimethyl sulfoxide, and shake well. This is the dimethyl sulfate stock solution. (Prepare immediately before use)

[0040] Accurately weigh approximately 75 mg of diethyl sulfate and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with dimethyl sulfoxide, and shake well. This is the diethyl sulfate stock solution. (Prepare immediately before use)

[0041] Accurately measure 1 ml each of dimethyl sulfate stock solution and diethyl sulfate stock solution into 100 ml volumetric flasks, dilute to the mark with dimethyl sulfoxide, and shake well. This is the reference stock solution. (Prepare immediately before use)

[0042] Accurately measure 2 ml of the reference standard stock solution, place it in a headspace bottle, add 0.5 ml of 10% sodium acetate solution, seal, and use as the reference standard solution.

[0043] Accurately weigh 0.20 g of feneline and place it in a top empty bottle. Accurately add 0.5 ml of 10% sodium acetate solution and 2 ml of reference stock solution, seal, and obtain the system suitability solution.

[0044] Accurately measure 2 ml of dimethyl sulfate stock solution, place it in a headspace vial, add 0.5 ml of 10% sodium acetate solution, seal, and use as the dimethyl sulfate positioning solution.

[0045] Accurately measure 2 ml of diethyl sulfate stock solution, place it in a headspace bottle, add 0.5 ml of 10% sodium acetate solution, seal, and use as the diethyl sulfate positioning solution.

[0046] (2) Chromatographic conditions

[0047] Headspace injection conditions: headspace equilibrium temperature 80℃, equilibration time 30 min; injection loop temperature 90℃, transfer line temperature 100℃; injection time 1 min. Gas chromatography conditions: injector temperature 200℃, split ratio 10:1; carrier gas: high-purity nitrogen, flow rate 3.0 mL / min. Column temperature program: initial 40℃, ramp to 75℃ at 2℃ / min and hold for 2 min, then ramp to 250℃ at 40℃ / min and hold for 8 min. FID detector temperature 250℃.

[0048] (3) Sample determination

[0049] Inject blank solution, reference solution, and test solution into the headspace separately and record the peak areas. Quantify using the external standard method.

[0050] The calculation formula is as follows: Impurity content % =

[0051] —The peak area of ​​impurities in the test solution;

[0052] —This represents the peak area in the reference solution;

[0053] —The concentration of the test solution (μg / ml);

[0054] — represents the concentration of the reference solution (μg / ml);

[0055] S-Reference Standard Content

[0056] Methodological validation of chromatographic methods:

[0057] 1. Exclusivity

[0058] Take the blank solution, reference solution, system suitability solution, dimethyl sulfate positioning solution, and diethyl sulfate positioning solution prepared in (1). Perform GC analysis according to the chromatographic conditions in (2). See the results below. Figures 1-5 Experimental results show that the blank solution does not interfere with the determination of the target analyte, and the separation between the dimethyl sulfate peak, the diethyl sulfate peak and their adjacent peaks in the system suitability solution meets the requirements.

[0059] Table 1

[0060]

[0061] 2. Linearity and Range

[0062] Take dimethyl sulfate stock solution and diethyl sulfate stock solution, and prepare a series of reference standard solutions of different concentrations according to the reference solution preparation method in (1). Perform GC according to the chromatographic method in (2), and plot a standard curve with peak area as the ordinate (y) and concentration as the abscissa (x). Figure 2 As shown in Table 2, the peak area of ​​dimethyl sulfate showed a linear relationship with concentration in the concentration range of 2.51 µg / ml to 12.4 µg / ml, with the linear equation being y = 17237.9x - 19263.6; the peak area of ​​diethyl sulfate showed a linear relationship with concentration in the concentration range of 2.4 µg / ml to 11.9 µg / ml, with the linear equation being y = 16279.0x - 16782.2, indicating a good linear relationship.

[0063] Table 2

[0064]

[0065] Table 3

[0066]

[0067] 3. Limit of Detection and Limit of Quantification

[0068] Take the reference solution prepared in (1) and dilute it appropriately. Use the concentration at which the signal-to-noise ratio (S / N) is about 3 as the limit of detection.

[0069] The concentration at which the signal-to-noise ratio (S / N) is approximately 10 is used as the limit of quantitation. GC analysis was performed according to the chromatographic method in (2). The detection limit concentration of dimethyl sulfate was 0.84 μg / mL, equivalent to 0.0008% of the sample concentration, and the quantitation limit concentration was 2.51 μg·mL, equivalent to 0.0025% of the sample concentration; the detection limit concentration of diethyl sulfate was 0.80 μg / mL, equivalent to 0.0008% of the sample concentration, and the quantitation limit concentration was 2.42 μg / mL, equivalent to 0.0024% of the sample concentration.

[0070] 4. Precision and repeatability

[0071] The reference solution prepared in (1) was injected six times consecutively. The RSD of the peak area of ​​dimethyl sulfate was 1.9%, and the RSD of the peak area of ​​diethyl sulfate was 2.3%. This indicates that the instrument precision of this method is good.

[0072] Table 4

[0073]

[0074] Six portions of the system suitability solution (1) and the reference solution prepared in (1) were taken and analyzed by GC according to the chromatographic method in (2). The RSD of dimethyl sulfate content was 3.9%; the RSD of diethyl sulfate content was 3.7%.

[0075] Table 5

[0076]

[0077] 5. Recovery rate

[0078] 80% control solution: Accurately measure 0.8 ml of the reference stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with dimethyl sulfoxide and shake well.

[0079] 100% control solution: Accurately measure 1.0 ml of the reference stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with dimethyl sulfoxide and shake well.

[0080] 120% control solution: Accurately measure 1.2 ml of the reference stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with dimethyl sulfoxide and shake well.

[0081] 80% test solution: Accurately weigh 0.20 g of fenelazol, place it in a headspace bottle, and accurately add 2.0 ml of 80% control solution and... 0.5ml, sealed. (Makes 3 servings)

[0082] 100% test solution: Accurately weigh 0.20 g of fenelazol, place it in a headspace, and accurately add 2.0 ml of 100% control solution and... 0.5ml, sealed. (Makes 3 servings)

[0083] 120% test solution: Accurately weigh 0.20 g of fenelazol, place it in a headspace, and accurately add 2.0 ml of 120% control solution and... 0.5ml, sealed. (Makes 3 servings)

[0084] Take the reference solution, test solution and the above test solutions prepared in (1) and perform GC analysis according to the chromatographic method in (2). The recovery rate of dimethyl sulfate content is 96% to 101%, and the RSD of the recovery rate is 1.8%; the recovery rate of diethyl sulfate content is 93% to 101%, and the RSD of the recovery rate is 2.8%, with good accuracy.

[0085] Table 6 Recovery rates (dimethyl sulfate)

[0086]

[0087] Table 7 Recovery Rate (Diethyl Sulfate)

[0088]

[0089] 6. Solution stability

[0090] The reference solution, system suitability solution, and test solution prepared in (1) were subjected to GC analysis according to the chromatographic method in (2) at 0, 6, 19, 77, and 142 hours, respectively. In the system suitability solution, the resolution between the dimethyl sulfate peak, the diethyl sulfate peak, and their adjacent peaks was greater than 1.5. In the reference solution, the RSD of the dimethyl sulfate peak area was 1.3%, and the RSD of the diethyl sulfate peak area was 2.6%. In the test solution, the content of dimethyl sulfate and diethyl sulfate was not detected. All solutions showed good stability.

[0091] Table 8 System Suitability Solutions

[0092]

[0093] Table 9 Reference Solution

[0094]

[0095] Table 10 Test Solution

[0096]

[0097] Example 3:

[0098] This embodiment provides a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material. The specific steps are the same as in Example 1, except that the flow rate is 2.0~4.0 ml / min.

[0099] Example 4:

[0100] This embodiment provides a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material. The specific steps are the same as in Example 1, except that the injection port temperature is 150~220℃.

[0101] Example 5:

[0102] This embodiment provides a GC method for determining dimethyl sulfate and diethyl sulfate in fenelone raw material. The specific steps are the same as in Example 1, except that the detector temperature is 220~280℃.

[0103] Comparative Example 1:

[0104] This embodiment is basically the same as Example 1, except that 10% sodium acetate is replaced with 10% sodium propionate. The reference solution is prepared using the method in (1) of Example 1, and the chromatographic method in (2) is used for GC analysis. Dimethyl sulfate and diethyl sulfate have low response and low sensitivity.

[0105] The applicant declares that the above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining dimethyl sulfate and diethyl sulfate in fenelazol raw material by GC, characterized in that, Includes the following steps: Provide test samples and standard references; use gas chromatography to detect and obtain chromatographic data of the test samples and standard references; and perform quantitative analysis of the target gaseous impurities based on the chromatographic data.

2. The method according to claim 1, characterized in that, The method uses a capillary column (DB-624 60m×0.53mm, 3μm) with a 6% cyanopropylphenyl-94% dimethylpolysiloxane stationary phase.

3. The method according to claim 1, characterized in that, The test sample is introduced into the gas chromatograph via headspace injection.

4. The method according to claim 1, characterized in that, Quantitative analysis was performed using the external standard method.

5. The method according to claim 1, characterized in that, The target gaseous impurities are dimethyl sulfate and diethyl sulfate in fenelone raw material.

6. The method according to claim 1, characterized in that, The carrier gas flow rate for the gas chromatography detection is 2.0~4.0 ml / min.

7. The method according to claim 1, characterized in that, The injection port temperature for the gas chromatography detection is 150~220℃.

8. The method according to claim 1, characterized in that, The detector temperature for the gas chromatography detection is 220~280℃.

9. A method for determining dimethyl sulfate and diethyl sulfate in fenelazol raw material by GC, characterized in that, The method described in any one of claims 1 to 8 is used for detection, wherein the content of the target gaseous impurity is ≤75ppm.

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