Analysis method for determining nitrosamine impurities in sulfadoxine bulk drug

Abstract

The invention provides an analysis method for determining nitrosamine impurities in a sulfadoxine bulk drug, and belongs to the technical field of drug safety control. According to the method, the sensitivity and the accuracy of detection of the nitrosamine impurities in the sulfadoxine raw material medicine are effectively improved, various trace nitrosamine impurities can be efficiently measured at the same time, and the reliability of medicine quality control is remarkably enhanced; the application of the method practically ensures the drug safety, meets the strict industry supervision requirements, and has important practical significance for maintaining the public medication safety.

Description

Technical Field

[0001] This invention relates to the field of drug safety control technology, and in particular to an analytical method for determining nitrosamine impurities in sulfadoxine raw material. Background Technology

[0002] In the field of pharmaceutical manufacturing and quality control, the control of genotoxic impurities (GTIs) in active pharmaceutical ingredients (APIs) and finished drug products is one of the core aspects of ensuring drug safety. Nitrosamines, as a common class of potential genotoxic impurities, are of great concern to global drug regulatory agencies due to their potential carcinogenic risks. The sources of nitrosamine impurities in pharmaceuticals are complex, potentially originating from multiple stages such as manufacturing processes, solvents, reagents, or packaging materials; therefore, establishing strict control strategies is crucial.

[0003] International drug regulatory agencies, such as the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA), have issued guidelines explicitly requiring marketing authorization holders to conduct risk assessments of nitrosamine impurities and establish corresponding detection and control methods to ensure that their levels in drugs are below the prescribed acceptable intake limits. This regulatory trend reflects a proactive and in-depth approach to drug risk control and places extremely high demands on the sensitivity, specificity, and reliability of analytical techniques.

[0004] Against this backdrop, drug quality control protocols not only need to meet routine purity and content determination requirements, but also need to possess the ability to monitor trace levels of high-risk impurities. Traditional detection methods often have limitations in sensitivity, specificity, or the ability to simultaneously detect multiple components, making it difficult to cope with increasingly stringent regulatory standards. Therefore, developing analytical methods that can efficiently, accurately, and reliably detect multiple nitrosamine impurities in complex matrices has become an urgent and significant challenge in the field of drug quality control. Summary of the Invention

[0005] The purpose of this invention is to provide an analytical method for determining nitrosamine impurities in sulfadoxine raw material, which can accurately assess the content of nitrosamine impurities in sulfadoxine raw material, ensuring that the quality of the drug meets safety standards, and is of great significance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an analytical method for determining nitrosamine impurities in sulfadoxine raw material. The method includes the following steps: preparing a diluent, a blank solution, a reference solution, and a test solution; setting the chromatographic conditions and mass spectrometric parameters of a gas chromatography-triple quadrupole tandem mass spectrometer; injecting the sample for analysis and calculating the content of each impurity.

[0008] The reference solution contains five nitrosamine impurities: NDMA, NDEA, NDIPA, NEIPA, and NDBA.

[0009] Preferably, the chromatographic conditions include the use of an Agilent DB-WAX UI column or a polyethylene glycol stationary phase column.

[0010] Preferably, the diluent is a 1 mol / L sodium hydroxide solution.

[0011] Preferably, the preparation of the blank solution includes mixing an equal volume of 0.5-1.5 mol / L sodium hydroxide solution with dichloromethane, vortexing to mix thoroughly, allowing to stand, and then taking the lower layer of liquid.

[0012] Preferably, the preparation of the test solution includes taking sulfadoxine raw material, dissolving it in 0.5-1.5 mol / L sodium hydroxide solution, adding an equal volume of dichloromethane, vortexing to mix, allowing it to stand, and taking the lower layer.

[0013] Preferably, the chromatographic column has a specification of 30m × 0.25mm and a film thickness of 0.25μm.

[0014] Preferably, the flow rate in the chromatographic conditions is 0.8 to 1.2 ml / min.

[0015] Preferably, the injection port temperature in the chromatographic conditions is 240–260°C;

[0016] The temperature program in the chromatographic conditions includes an initial column temperature of 50°C held for 1 minute, increasing the temperature at 10°C per minute to 145°C held for 6 minutes, and then increasing the temperature at 40°C per minute to 240°C held for 5 minutes.

[0017] Preferably, the mass spectrometry parameters include the use of an electron bombardment ion source and a multiple reaction monitoring mode;

[0018] The ion pairs used in the multi-reaction monitoring mode include 74 / 44 and 74 / 42 of NDMA, 102 / 85 and 102 / 44 of NDEA, 130 / 88 and 130 / 42 of NDIPA, 116 / 99 and 71 / 56 of NEIPA, and 116 / 99 and 158 / 99 of NDBA.

[0019] Preferably, the impurity content is calculated using the external standard method, and the calculation formula is as follows:

[0020] In ppb, the target analyte content = (A 供试品 ×g 对照品 (×Reference content×Test sample dilution factor) / (A reference standard×g test sample×Reference standard dilution factor)×10 9 ;

[0021] In the formula, A 供试品 The average peak area of ​​the target analyte in the test solution using the double needle.

[0022] A 对照品 The average peak area of ​​the target analyte in six consecutive injections of the reference solution;

[0023] g 对照品 Weigh the sample amounts of the reference solutions NDMA, NDEA, NDIPA, NEIPA, and NDBA; g 供试品 The sample weight for testing is ***.

[0024] The beneficial effects of this invention are:

[0025] This invention effectively improves the sensitivity and accuracy of detecting nitrosamine impurities in sulfadoxine raw materials, enabling the simultaneous and efficient determination of multiple trace nitrosamine impurities, and significantly enhancing the reliability of drug quality control. The application of this method effectively ensures drug safety, meets stringent industry regulatory requirements, and has important practical significance for safeguarding public drug safety. Attached Figure Description

[0026] Figure 1 The chromatograms are of control solutions containing five nitrosamine impurities. Detailed Implementation

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] The relevant information on the five nitrosamine impurities and sulfadoxine raw material involved in the examples is shown in Table 1.1 below:

[0029] Table 1.15 Information on Nitrosamine Impurities and Sulfadoxine Raw Material

[0030]

[0031]

[0032] The limits for the five nitrosamine impurities are shown in Table 1.2 below:

[0033] Table 1. Limits for 25 Nitrosamine Impurities

[0034]

[0035] Note: AI stands for Acceptable Daily Intake of each nitrosamine impurity. The AI ​​in this table is from the EMA. <Qusetion and answers formarketing authorization holders / applicants on theCHMP Opinion fortheArticle 5(3)ofRegulation(EC)No726 / 2004referred onnitrosamine impurities in human medicinal products> 19July2024Rev.21Appendix1:Accetable intakes established for N-nitorsamine.

[0036] The limit is calculated as follows: Limit = AI ÷ MDD

[0037] As can be seen from the table above, the low limits for these five nitrosamine impurities result in high requirements for instrument sensitivity.

[0038] This invention solves the problem of low sensitivity and difficulty in detecting these five impurities by using gas chromatography-triple quadrupole tandem mass spectrometry in MRM mode and simultaneously extracting impurities. The method is simple, sensitive, and effective, and has good quantitative analysis results. Specific examples are as follows:

[0039] Example

[0040] This invention provides an analytical method for NDMA, NDEA, NDIPA, NEIPA, and NDBA impurities in sulfadoxine raw material, the method being as follows:

[0041] Diluent (1 mol / L sodium hydroxide solution): Weigh 4 g of sodium hydroxide, dilute with water to 100 ml, and shake well.

[0042] Blank solution: Accurately measure 3.0 ml of 1 mol / L sodium hydroxide solution and 3.0 ml of dichloromethane into a 15 ml centrifuge tube, vortex for 60 seconds, let stand, and take the lower layer as the blank solution.

[0043] NDEA, NDIPA, NEIPA, and NDBA reference stock solutions ①: Accurately weigh 10 mg of each of the following reference standards: NDEA, NDIPA, NEIPA, and NDBA. Place them in the same 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. This is the reference stock solution ①. (1000 μg / ml)

[0044] NDEA, NDIPA, NEIPA, and NDBA reference stock solution ②: Accurately transfer 1.0 ml of NDEA, NDIPA, NEIPA, and NDBA reference stock solution ① into a 100 ml volumetric flask, dilute to the mark with acetonitrile, and mix well. This is NDEA, NDIPA, NEIPA, and NDBA reference stock solution ②. (10 μg / ml)

[0045] NDMA Standard Stock Solution ①: Accurately measure 0.375 ml of NDMA standard solution (1 mg / ml) into a 10 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well. This is NDMA Standard Stock Solution ① (37.5 μg / ml).

[0046] NDMA, NDEA, NDIPA, NEIPA, and NDBA reference stock solutions ③: Accurately transfer 0.5 ml of NDEA, NDIPA, NEIPA, and NDBA reference stock solutions ② and 0.5 ml of NDMA reference stock solution ① into 100 ml volumetric flasks, add purified water to the mark, and mix well. This is NDMA, NDEA, NDIPA, NEIPA, and NDBA reference stock solutions ③. (NDMA: 187.5 ng / ml, NDEA, NEIPA, NDIPA, and NDBA: 50 ng / ml)

[0047] NDMA, NDEA, NDIPA, NEIPA, and NDBA reference stock solution ④: Accurately transfer 1.5 ml of NDMA, NDEA, NDIPA, NEIPA, and NDBA reference stock solution ③ into a 25 ml volumetric flask, add diluent to the mark, and mix well. This is NDMA, NDEA, NDIPA, NEIPA, and NDBA reference stock solution ④. (NDMA: 11.25 ng / ml, NDEA, NEIPA, NDIPA, and NDBA: 3 ng / ml)

[0048] Reference solution: Accurately measure 3.0 ml of the NDMA, NDEA, NDIPA, NEIPA and NDBA reference stock solutions and place them in a 15 ml centrifuge tube. Add 3.0 ml of dichloromethane, vortex for 60 s, allow to stand and separate into layers, and take the lower clear liquid as the solution.

[0049] Test solution: Accurately weigh 0.530 g of sulfadoxine API, place it in a stoppered test tube, add 3.0 ml of 1 mol / L sodium hydroxide solution to dissolve it, then add 3.0 ml of dichloromethane, vortex for 60 seconds, let stand, and take the lower layer as the test solution.

[0050] The chromatographic conditions are shown in Table 1.3 below:

[0051] Table 1.3 Chromatographic conditions

[0052]

[0053]

[0054] Determination method:

[0055] 1. The blank solution should not have obvious interfering peaks at the elution positions of NDMA, NDEA, NDIPA, NEIPA, and NDBA. If there are interfering peaks, they should be smaller than the peak areas corresponding to the limits of quantitation solutions of NDMA, NDEA, NDIPA, NEIPA, and NDBA.

[0056] 2. For the reference solution, inject it continuously for 6 injections. The retention time RSD of NDMA, NDEA, NDIPA, NEIPA and NDBA shall not be greater than 1.0%, and the peak area RSD shall not be greater than 15.0%.

[0057] 3.6 The peak area RSD of NDMA, NDEA, NDIPA, NEIPA and NDBA in the reference solution and the retest reference solution shall not exceed 15.0%.

[0058] 4. The test sample shall be calculated according to the external standard method. NDMA shall not exceed 64 ppb, and NDEA, NDIPA, NEIPA and NDBA shall not exceed 17 ppb.

[0059] The contents of NDMA, NDEA, NDIPA, NEIPA, and NDBA are calculated using the following formula:

[0060] Target analyte content (ppb) = (A 供试品 ×g 对照品 × Reference standard content × Test sample dilution factor) / (A 对照 (sample × g, dilution factor of test sample × reference sample) × 10 9

[0061] Among them: A 供试品 Average peak area of ​​the target analytes (NDMA, NDEA, NDIPA, NEIPA, and NDBA) in the test solution using the dual-needle technique;

[0062] A 对照品 The average peak area of ​​the target analytes (NDMA, NDEA, NDIPA, NEIPA, and NDBA) in six consecutive injections of the reference solution;

[0063] g 对照品 Weigh the reference solutions NDMA, NDEA, NDIPA, NEIPA and NDBA, in g.

[0064] g 供试品 : Sample weight, g;

[0065] Experimental Example

[0066] As can be seen from the above embodiments, the method provided by the present invention mainly solves the problem of low sensitivity and difficulty in detecting these five impurities by using gas chromatography-triple quadrupole tandem mass spectrometry in MRM mode and extracting impurities simultaneously. Compounds with low sensitivity are usually undetectable under conventional chromatographic conditions, so gas chromatography-triple quadrupole tandem mass spectrometry is used for quantitative analysis.

[0067] (1) Results of method specificity examination

[0068] The peak elution order in the reference solution of the method of the present invention is NDMA peak, NDEA peak, NEIPA peak, NDIPA peak and NDBA peak, with a resolution greater than 1.5 for each peak.

[0069] In the method of this invention, the interfering peak at the NDMA elution position in the blank solution is smaller than the detection limit peak area (peak area of ​​5% NDMA reference standard); there is no obvious interfering peak at the NDEA elution position; there is no obvious interfering peak at the NEIPA elution position; there is no obvious interfering peak at the NDIPA elution position; and there is no obvious interfering peak at the NDBA elution position.

[0070] (2) Results of System Precision Examination

[0071] The method of this invention produces sharp peaks for each impurity, demonstrating good system precision. The results of six consecutive injections of the impurity reference solution are shown in Table 2.1 below.

[0072] Table 2.1 System precision results

[0073] name Retention time RSD (%) Peak area RSD (%) NDMA 0.1 2.5 NDEA 0.1 2.6 NEIPA 0.1 2.2 NDIPA 0.0 2.4 NDBA 0.1 2.5

[0074] (3) Results of the method detection limit and quantitation limit investigation

[0075] The method of this invention features sharp peaks for each impurity, strong detection capability for each impurity, and high method sensitivity. The results are shown in Table 2.2 below.

[0076] Table 2.2 Results of Limit of Detection and Limit of Quantitation

[0077]

[0078] (4) Results of linearity analysis of the method

[0079] In the method of this invention, the components exhibit good linearity within their respective concentration ranges, and the impurity content calculation using the external standard method yields accurate results. The results are shown in Table 2.3 below.

[0080] Table 2.3 Linear Results

[0081]

[0082]

[0083] (5) Results of the method accuracy evaluation

[0084] The method was validated by determining the recovery rate of the spiked impurity solution. The limit concentration range of 10%–150% was examined, and four concentration levels were measured (relative limit concentration of 10% (limit of quantitation), 50% (relative limit concentration), 100% (relative limit concentration), and 150% (relative limit concentration), with three samples measured at each level. Accuracy at the limit of quantitation level: the target analyte recovery rate was between 50% and 150%; accuracy at other limit levels: the target analyte recovery rate was between 70% and 130%. The RSD of the recovery rates of all 12 test samples was less than 15%, demonstrating the good accuracy of the method. The results are shown in Table 2.4 below.

[0085] Table 2.4 Accuracy Results

[0086]

[0087] (6) Method precision assessment

[0088] Repeatability tests were performed by determining the content of each impurity in six 100% limit spiking solutions. Intermediate precision tests were conducted on different dates by different personnel using the same sample preparation method, and the solutions were then injected and analyzed according to the prescribed method. The results showed that the method had good precision. The results are shown in Table 2.5 below.

[0089] Table 2.5 Method precision results

[0090]

[0091] (7) Sample Measurement Results

[0092] Nine batches of test samples were analyzed using the above method, and none of them were detected, as shown in Table 2.6 below.

[0093] Table 2.6 Sample Measurement Results

[0094]

[0095]

[0096] Note: The detection limit for NDMA is 3.2 ppb, the detection limit for NDEA is 0.85 ppb, the detection limit for NEIPA is 0.85 ppb, the detection limit for NDIPA is 0.85 ppb, and the detection limit for NDBA is 0.85 ppb.

[0097] The method of this invention has been validated and all parameters meet industry standards. It has high sensitivity, short analysis time, strong specificity, good accuracy and precision, and strong repeatability, which can meet the needs of sample detection.

[0098] Figure 1 The chromatograms are for reference solutions of five nitrosamine impurities. Among them, RT=8.25 is NDMA, RT=9.335 is NDEA, RT=9.808 is NEIPA, RT=10.167 is NDIPA, and RT=14.388 is NDBA.

[0099] As can be seen from the above embodiments, the present invention provides an efficient, reliable and practical analytical method for nitrosamine impurities in sulfadoxine raw materials. This method has excellent sensitivity, specificity and repeatability, and can accurately and stably detect multiple trace nitrosamine impurities simultaneously, significantly improving the level of drug quality control. It fully meets the stringent requirements of international drug regulatory agencies for the detection of nitrosamine impurities and provides key technical support for ensuring drug safety.

[0100] 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. An analytical method for determining nitrosamine impurities in sulfamethoxazole drug substance, characterized in that, The method comprises the following steps: preparing a diluent, a blank solution, a control solution and a test solution; setting chromatographic conditions and mass spectrometry parameters of a gas chromatography-triple quadrupole mass spectrometry instrument; analyzing and calculating impurity contents; The control solution comprises five nitrosamine impurities of NDMA, NDEA, NDIPA, NEIPA and NDBA.

2. The analysis method according to claim 1, characterized in that, The chromatographic conditions comprise using an Agilent DB-WAX UI chromatographic column or a polyethylene glycol stationary phase chromatographic column.

3. The analysis method of claim 1, wherein, The diluent is 1 mol / L sodium hydroxide solution.

4. The analysis method of claim 1, wherein, The preparation of the blank solution comprises mixing 0.5-1.5 mol / L sodium hydroxide solution and dichloromethane in equal volumes, vortex mixing and standing, and taking the lower liquid.

5. The analysis method of claim 1, wherein, The preparation of the test solution comprises taking sulfonamides drug substance, adding 0.5-1.5 mol / L sodium hydroxide solution to dissolve, then adding equal volume of dichloromethane, vortex mixing and standing, and taking the lower liquid.

6. The analysis method of claim 2, wherein, The specification of the chromatographic column is 30 m*0.25 mm, and the film thickness is 0.25 μm.

7. The analysis method of claim 1, wherein, The flow rate in the chromatographic conditions is 0.8-1.2 ml / min.

8. The analysis method of claim 1, wherein, The injection port temperature in the chromatographic conditions is 240-260 ℃. The temperature rising program in the chromatographic conditions comprises an initial column temperature of 50 ℃ for 1 minute, rising at 10 ℃ per minute to 145 ℃ for 6 minutes, and then rising at 40 ℃ per minute to 240 ℃ for 5 minutes.

9. The analysis method of claim 1, wherein, The mass spectrometry parameters comprise using an electron impact ion source and a multiple reaction monitoring mode. The ion pairs used in the multiple reaction monitoring mode comprise 74 / 44 and 74 / 42 of NDMA, 102 / 85 and 102 / 44 of NDEA, 130 / 88 and 130 / 42 of NDIPA, 116 / 99 and 71 / 56 of NEIPA, and 116 / 99 and 158 / 99 of NDBA.

10. The analysis method of claim 1, wherein, The calculation of the impurity contents adopts an external standard method, and the calculation formula is: Target concentration in ppb = (A 供试品 × g 对照品 × control concentration x dilution factor of test sample) / (A control x g test sample x dilution factor of control) x 10 9 ; In the formula, the A 供试品 is the average peak area of the double needle target of the test solution; A 对照品 The peak area of the target substance was continuously measured for 6 injections of the control solution. g 对照品 For the control solutions NDMA, NDEA, NDIPA, NEIPA and NDBA, weigh out the amounts; g 供试品 Take sample amount for test product.

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