Method for detecting two potential genotoxic impurities in carbon [13C]-urea based on gas chromatography

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and discloses a method for determining two potential genotoxic impurities in carbon [13C]-urea by gas chromatography. Comprising the following steps: (1) preparing a sample solution; (2) preparing a reference solution; (3) taking nitrogen as carrier gas, and detecting by adopting a medium-polarity or weak-polarity chromatographic column separation system; and (4) calculating by a peak area through an external standard method to obtain the accurate contents of methyl carbamate and ethyl carbamate in the carbon [13C]-urea. The method disclosed by the invention is simple to operate, good in specificity, linearity, precision and accuracy and high in sensitivity, and can realize accurate detection of residual potential genotoxic impurities, namely methyl carbamate and ethyl carbamate, in the carbon [13C]-urea.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting carbon [ 13 A method for identifying two potentially genotoxic impurities in urea [C]-. Background Technology

[0002] carbon[ 13 C]-Urea is urea[ 13 C] The active ingredient in the breath test kit has the molecular formula: 13 CH4N2O, with a molecular weight of 61.06, has the following structural formula;

[0003]

[0004] carbon[ 13 C]-urea preparations can be used in breath tests to diagnose Helicobacter pylori infection. Helicobacter pylori is a bacterium that can grow in the stomach and cause diseases such as gastritis and peptic ulcers, and is closely related to the occurrence of gastric cancer. However, in carbon [ 13 C]- Genotoxic impurities, namely methyl carbamate and ethyl carbamate, can be generated during the production of urea.

[0005] Methyl carbamate, abbreviated as MC, has the molecular formula C2H5NO2 and a molecular weight of 75.07. It is also known as urethane or methyl urethane, and has the CAS number 598-55-0. Ethyl carbamate, abbreviated as EC, has the molecular formula C3H7NO2 and a molecular weight of 89.09. It is also known as urethane, and has the CAS number 51-79-6. Both substances are classified as Group 3 carcinogens by the International Agency for Research on Cancer (IARC) of the World Health Organization. Their structural formulas are as follows;

[0006]

[0007] Currently, the main methods for determining the content of methyl carbamate and ethyl carbamate are mass spectrometry and the commonly used gas chromatography. Mass spectrometry instruments are expensive, cumbersome to operate, and have high maintenance costs. Gas chromatography is more commonly used, but the specified carrier gas is helium, which is expensive. The chromatographic column is also specific, resulting in poor selectivity. More importantly, this method has extremely stringent requirements for the injection port liner, requiring the use of highly deactivated glass floc liners and ultra-high inert sand core liners. When using non-highly deactivated glass floc liners or non-ultra-high inert sand core liners, the silanol groups in the liner can catalyze the reaction between methanol and one molecule of ammonia released from the product at high temperatures to form methyl carbamate, interfering with the accurate detection of the product.

[0008] carbon[ 13A major challenge in the production process of urea [C] is the control of genotoxic impurities. Therefore, the risk of excessive residues of methyl carbamate and ethyl carbamate in the product is high. According to the EU Guidelines on the Limits of Genotoxic Impurities and the ICH M7 Guidelines for the Assessment and Control of Genotoxic Impurities, these potentially genotoxic impurities need to be controlled. Therefore, it is necessary to develop a convenient, specific, accurate, and sensitive detection method for quality control carbon [C]. 13 The potential genotoxic impurities in C-urea are of great significance for improving drug quality. Summary of the Invention

[0009] The purpose of this invention is to provide a method for detecting carbon [ 13 A new method for detecting two potentially genotoxic impurities in C]-urea. This method exhibits high specificity, linearity, precision, accuracy, and sensitivity, and can achieve detection of carbon [ 13 Accurate detection of potentially genotoxic impurities (methyl carbamate and ethyl carbamate) remaining in C]-urea.

[0010] The objective of this invention is achieved through the following technical solution: a method for detecting carbon based on gas chromatography. 13 [C]-A method for removing two potentially genotoxic impurities from urea, wherein the two potentially genotoxic impurities are methyl carbamate and ethyl carbamate; the method includes the following steps:

[0011] (1) Preparation of blank solvent: Acetonitrile aqueous solution was used as blank and solvent;

[0012] (2) Preparation of the test solution: using carbon [ 13 C]-urea, using acetonitrile aqueous solution as solvent, to prepare the test solution;

[0013] (3) Preparation of reference solution: Use methyl carbamate and ethyl carbamate, and acetonitrile aqueous solution as solvent to prepare reference solution;

[0014] (4) Using nitrogen as the carrier gas, a DB-624 (30m×0.530mm, 3.00μm) chromatographic column was used to separate the sample using a gradient temperature program and the sample was detected by an FID detector.

[0015] (5) The external standard method calculates carbon based on peak area. 13 The accurate content of methyl carbamate and ethyl carbamate in C]-urea.

[0016] Furthermore, in the acetonitrile aqueous solution, the acetonitrile-water ratio is 70-90:30-10. Preferably, the acetonitrile-water ratio is 70:30. More preferably, the acetonitrile-water ratio is 90:10.

[0017] Further, in step (2), the concentration of the test sample solution is 10–50 mg / ml. Preferably, the concentration of the test sample solution can be, but is not limited to, 10 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, or 50 mg / ml. More preferably, the concentration of the test sample solution is 25 mg / ml. The preparation method is as follows: accurately weigh 250 mg of the test sample into a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0018] Further, in step (3), the concentrations of methyl carbamate and ethyl carbamate are 10–50 μg / ml. Preferably, the concentrations of methyl carbamate and ethyl carbamate can be, but are not limited to, 10 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 40 μg / ml, or 50 μg / ml. More preferably, the concentrations of methyl carbamate and ethyl carbamate are 25 μg / ml. The preparation method is as follows: accurately weigh 25 mg each of methyl carbamate and ethyl carbamate, add solvent to dissolve and dilute to the mark, shake well, accurately measure 1 ml, place in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0019] Furthermore, the detection conditions for the gas chromatography detection are as follows:

[0020] Column: DB-624 (30m × 0.530mm, 3.00μm); or other columns with equivalent performance;

[0021] Heating program: Start at 50℃, hold for 2 minutes, increase to 200℃ at a rate of 15℃ per minute, then increase to 230℃ at a rate of 30℃ per minute and hold for 5 minutes;

[0022] Detector: FID;

[0023] Inlet temperature: 220℃;

[0024] Detector temperature: 250℃;

[0025] Carrier gas: Nitrogen;

[0026] Carrier gas flow rate: 2 ml / min;

[0027] Injection volume: 1 μl.

[0028] The flow rate of the carrier gas is 1 to 3 ml / min, preferably 2 ml / min.

[0029] The present invention has the following beneficial effects:

[0030] (1) Commonly used gas chromatography methods have extremely stringent requirements for the injection "liner". In methanol systems, the silanol groups in the liner will catalyze the reaction between methanol and one molecule of ammonia released from the product after high temperature to form methyl carbamate, which will interfere with the accurate detection of methyl carbamate in the product. Moreover, even when helium is used as the carrier gas and a specific polarity column is used, the specificity and accuracy of the method are still poor. This invention does not require the use of a special "liner" and creatively uses 90% acetonitrile as the solvent and nitrogen as the carrier gas. Ordinary liners can be used to achieve better specificity, accuracy of detection results and high sensitivity.

[0031] (2) The detection method of the present invention is simple to operate, reduces the difficulty of drug detection, and has low detection cost. Method validation shows that this method has strong specificity, good linearity and repeatability, high sensitivity, and high recovery rate, resulting in accurate and reliable detection results. This method provides a basis for the detection of carbon […]. 13 This provides a new, accurate, effective, and reproducible method for detecting the content of potentially genotoxic impurities in urea [C]-, and also provides a new method for detecting urea [C]. 13 The quality research of [C] provides a new detection approach. Attached Figure Description

[0032] Figure 1 : Chromatogram of the blank solvent in Example 1;

[0033] Figure 2 : Chromatogram of the reference solution in Example 1;

[0034] Figure 3 : Chromatogram of the test solution in Example 1;

[0035] Figure 4 Chromatogram of the methyl carbamate localization solution in Example 1;

[0036] Figure 5 Chromatogram of the ethyl carbamate localization solution in Example 1;

[0037] Figure 6 Chromatogram of the spiked test solution in Example 1.

[0038] Figure 7 Linear graph of methyl carbamate in Example 3.

[0039] Figure 8 Linear graph of ethyl carbamate in Example 3. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0041] The equipment, reagents, consumables, etc. used in the specific embodiments of the present invention are all commercially available products, and no special customization or procurement is required.

[0042] The following chromatographic conditions were used in the examples:

[0043] Chromatographic column: DB-624, 30m×0.530mm, 3.00μm or equivalent column (such as AT-624, 30m×0.530mm, 3.00μm);

[0044] Heating program: Start at 50℃, hold for 2 minutes, increase to 200℃ at a rate of 15℃ per minute, then increase to 230℃ at a rate of 30℃ per minute and hold for 5 minutes;

[0045] Detector: FID

[0046] Inlet temperature: 220℃

[0047] Detector temperature: 250℃

[0048] Carrier gas: Nitrogen

[0049] Carrier gas flow rate: 2 ml / min

[0050] Injection volume: 1 μl.

[0051] Example 1: Specificity Test

[0052] a. Blank (solvent): Acetonitrile-water (90:10).

[0053] b. Reference stock solution: Weigh approximately 25 mg each of methyl carbamate and ethyl carbamate reference standards, place them in separate 100 ml volumetric flasks, dissolve and dilute to the mark with solvent, and shake well.

[0054] c. Reference solution: Accurately measure 1 ml of each reference stock solution and place them in the same 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0055] d. Positioning solution: Measure 2 ml of each of the reference standard stock solution and place them in 10 ml volumetric flasks. Dilute to the mark with solvent and shake well.

[0056] e. Spiked test solution: Weigh carbon [ 13 Accurately weigh approximately 250 mg of C-urea and place it in a 10 ml volumetric flask. Dissolve the urea in an appropriate amount of solvent, add 1 ml of the reference stock solution, dilute to the mark with solvent, and shake well.

[0057] Accurately measure 1 μl each of blank solvent, reference solution, impurity localization solution, test solution, and spiked test solution, and inject them separately into the gas chromatograph, recording the chromatograms. The results are shown in Table 1 and... Figures 1-6 ,in, Figure 1 This is the blank solvent chromatogram from Example 1; Figure 2 This is the chromatogram of the reference solution in Example 1; Figure 3 This is the chromatogram of the test solution in Example 1; Figure 4 This is the chromatogram of the methyl carbamate positioning solution in Example 1; Figure 5 This is the chromatogram of the ethyl carbamate positioning solution in Example 1; Figure 6 This is the chromatogram of the spiked test solution from Example 1.

[0058] Table 1 Results of specificity test

[0059]

[0060] From Table 1 and Figures 1-6 As shown, the blank solvent did not interfere with the detection of methyl carbamate and ethyl carbamate. In the chromatogram of the reference solution, the resolution between the peaks of methyl carbamate and ethyl carbamate was greater than 1.5. The retention times of the chromatographic peaks of each positioning solution were consistent with those of the reference solution and the spiked test solution, indicating good method specificity.

[0061] Example 2: Sensitivity Test

[0062] a. Limit of Quantitation Solution: Accurately measure 2 ml of methyl carbamate reference standard stock solution and 1 ml of ethyl carbamate stock solution from the "Specificity" section, place them in the same 20 ml volumetric flask, dilute to the mark with solvent, and shake well. Accurately measure 1 ml of this solution and place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 6 aliquots using the same method.

[0063] b. Limit of Detection Solution: Accurately measure 3 ml and 5 ml of the limit of quantitation solution and place them in 10 ml volumetric flasks respectively. Dilute to the mark with solvent and shake well to prepare the limit of detection solutions for ethyl carbamate and methyl carbamate.

[0064] Accurately measure 1 μl each of the limit of quantitation solution and the limit of detection solution, inject them separately into the gas chromatograph, and record the chromatograms. The results are shown in Tables 2 and 3.

[0065] Table 2 Results of Limit of Quantitation Test

[0066]

[0067] Table 3 Results of the detection limit test

[0068]

[0069]

[0070] The results above show that the limits of quantitation (LOQ) of both methyl carbamate and ethyl carbamate solutions are less than 0.05% of the concentration of the test sample solution, and the limits of detection (LOD) of both solutions are less than 0.02% of the concentration of the test sample solution, indicating good method sensitivity.

[0071] Example 3: Linear

[0072] a. Linear stock solution: Weigh approximately 25 mg each of methyl carbamate and ethyl carbamate reference standards, place them in separate 100 ml volumetric flasks, dissolve and dilute to the mark with solvent, and shake well.

[0073] b. Linear solutions: Accurately measure different volumes of linear stock solution and dilute with solvent to obtain linear solutions of the concentration levels shown in Table 4.

[0074] Table 4

[0075]

[0076] Accurately measure 1 μl of each linear solution and inject it into the gas chromatograph, then record the chromatogram. The results are shown in Table 5.

[0077] Table 5 Results of the linearity test

[0078]

[0079]

[0080] The results above show that methyl carbamate exhibits a linear correlation coefficient (r) greater than 0.990 within the concentration range of 1.0549 μg / ml to 52.7472 μg / ml, and ethyl carbamate exhibits a linear correlation coefficient (r) greater than 0.990 within the concentration range of 0.5235 μg / ml to 52.3512 μg / ml. The response factor (RSD) is less than 10%, and the Y-intercept is within 25% of the 100% response value, indicating a good linear relationship.

[0081] Example 4: Precision Test

[0082] a. Reference stock solution: Weigh approximately 25 mg each of methyl carbamate and ethyl carbamate reference standards, place them in the same 100 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0083] b. Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0084] c. Spiked test solution: Take carbon [ 13 Accurately weigh approximately 250 mg of C-urea and place it in a 10 ml volumetric flask. Add an appropriate amount of solvent to dissolve it, then accurately add 1 ml of the reference stock solution. Dilute to the mark with solvent and shake well. Prepare 6 portions using the same method.

[0085] (1) Repeatability

[0086] Accurately measure 1 μl each of the reference solution and the spiked test solution, and inject them separately into the gas chromatograph. Record the chromatograms. The results are shown in Table 6.

[0087] Table 6 Results of Repeatability Tests

[0088] name 1 2 3 4 5 6 mean RSD (%) Methyl carbamate (%) 0.11 0.11 0.11 0.10 0.11 0.11 0.11 3.8 Ethyl carbamate (%) 0.10 0.11 0.11 0.10 0.10 0.10 0.10 5.0

[0089] In the six spiked test solutions, the RSDs of methyl carbamate and ethyl carbamate were all less than 10.0%, indicating good method repeatability.

[0090] (2) Intermediate precision

[0091] To investigate the effect of random variations on precision, different personnel repeated the repeatability test at different times to verify the accuracy of the method. The results are shown in Table 7.

[0092] Table 7 Results of intermediate precision test

[0093]

[0094] In 12 spiked test solutions, the RSD of methyl carbamate and ethyl carbamate content was less than 10.0%, indicating good intermediate precision of the method.

[0095] Example 5: Recovery Test

[0096] a. Reference stock solution: Weigh approximately 25 mg each of methyl carbamate and ethyl carbamate reference standards, place them in the same 100 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0097] b. Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0098] c. Test solution: Take carbon [ 13 Weigh approximately 250 mg of C-urea accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0099] d. Accuracy solution: Weigh out carbon [as specified in the table below] 13 C]-urea was precisely added to different volumes of reference stock solution, and the solution was diluted with solvent to prepare the accuracy solution according to Table 8.

[0100] Table 8

[0101]

[0102] Accurately measure 1 μl each of the reference solution, test solution, and accuracy solution, and inject them separately into the gas chromatograph, recording the chromatograms. The results are shown in Table 9.

[0103] Table 9 Results of Recovery Rate Test

[0104]

[0105]

[0106] The average recoveries of methyl carbamate and ethyl carbamate at all concentration levels were between 90% and 110%, and the RSDs of the nine recovery data were all less than 10.0%, indicating good method accuracy.

[0107] In summary, this invention provides a novel method for detecting carbon [ 13 A method for detecting the content of potentially genotoxic impurities in urea [C] is described. This method exhibits good specificity, linearity, precision, accuracy, and sensitivity, and can effectively detect carbon [C]. 13 The content of potentially genotoxic impurities in C-urea.

[0108] The above descriptions are merely embodiments of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for detecting carbon based on gas chromatography [ 13 A method for removing two potentially genotoxic impurities from urea [C], characterized in that... The two potentially genotoxic impurities are methyl carbamate and ethyl carbamate; the method includes the following steps: (1) Preparation of blank solvent: Acetonitrile aqueous solution was used as blank and solvent; (2) Preparation of the test solution: Take carbon [ 13 C]-urea, using acetonitrile aqueous solution as solvent, to prepare the test solution; (3) Preparation of reference solution: Take methyl carbamate and ethyl carbamate, and use acetonitrile aqueous solution as solvent to prepare reference solution; (4) Using nitrogen as the carrier gas, a DB-624 chromatographic column was used to separate the sample using a gradient temperature program and gas chromatography detection was performed using an FID detector; (5) The external standard method calculates carbon based on peak area. 13 The accurate content of methyl carbamate and ethyl carbamate in C]-urea.

2. The method according to claim 1, characterized in that, In the acetonitrile aqueous solution, the ratio of acetonitrile to water is 70:

30.

3. The method according to claim 1, characterized in that, In the acetonitrile aqueous solution, the ratio of acetonitrile to water is 90:

10.

4. The method according to claim 1, characterized in that, In step (2), the concentration of the test solution is 10-50 mg / ml.

5. The method according to claim 1, characterized in that, In step (2), the concentration of the test solution is 25 mg / ml.

6. The method according to claim 1, characterized in that, In step (3), the concentration of the reference solution is 10–50 μg / ml.

7. The method according to claim 1, characterized in that, In step (3), the concentration of the reference solution is 25 μg / ml.

8. The method according to claim 1, characterized in that, The detection conditions for the gas chromatography detection are as follows: Column: DB-624 (30m × 0.530mm, 3.00μm); Heating program: Start at 50℃, hold for 2 minutes, increase to 200℃ at a rate of 15℃ per minute, then increase to 230℃ at a rate of 30℃ per minute and hold for 5 minutes; Detector: FID Inlet temperature: 220℃ Detector temperature: 250℃ Carrier gas: Nitrogen Carrier gas flow rate: 2 ml / min Injection volume: 1 μl.

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