Method for determining dimethyl sulfate in medicine by headspace in-situ derivatization GC-MS (Gas Chromatography-Mass Spectrometer) method

Abstract

The invention provides a method for determining dimethyl sulfate in a medicine by a headspace in-situ derivatization GC-MS (Gas Chromatography-Mass Spectrometer) method. A derivatization reagent adopted by the method is a sodium propionate aqueous solution, the equilibrium temperature of a headspace bottle is not lower than 45 DEG C, the equilibrium time of the headspace bottle is not less than 15 min, and solvents adopted by a sample solution of the medicine and a dimethyl sulfate reference substance solution are acetonitrile. According to the headspace in-situ derivatization GC-MS method for determining trace dimethyl sulfate in the medicine, sodium propionate which is economical, easy to obtain, stable in property, non-toxic and harmless is adopted as a derivatization reagent, and dimethyl sulfate with high reaction activity is converted into methyl propionate which is stable in property and easy to detect. The derivatization reaction does not need additional addition of inorganic base, the reaction is rapid and efficient, the operation is simple and convenient, the method is good in specificity and high in sensitivity, and a reliable new choice is provided for determination of trace dimethyl sulfate in the raw material medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical detection, and in particular to a method for determining dimethyl sulfate in medicines by a headspace in-situ derivatization GC-MS method. Background Art

[0002] Dimethyl sulfate, a mild methylating agent, is widely used in pharmaceutical synthetic chemistry for the methylation of functional groups such as alcohols, phenols, carboxylic acids, and amines. As scientists delve deeper into the pathogenic mechanisms of genotoxic impurities, dimethyl sulfate can cause chromosomal aberrations and has been included in the list of substances of very high concern. According to the regulations of the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA), the threshold of toxicological concern (TTC) for genotoxic impurities is 1.5 μg·d. −1 Therefore, it is very necessary to detect the content of dimethyl sulfate in medicines.

[0003] For example, sulpiride hydrochloride is a white needle-shaped crystalline powder, an antipsychotic drug, mainly used for chorea, Tourette syndrome and senile psychosis, with the structural formula: According to its synthesis process, O-methylation is an essential key reaction step in the synthesis route of sulphide hydrochloride raw material. The maximum daily dose of sulphide hydrochloride is 600 mg, from which it can be inferred that the limit of dimethyl sulfate in sulphide hydrochloride raw material is 0.00025% (2.5μg·g -1 ).

[0004] The determination of trace levels of dimethyl sulfate in pharmaceuticals has been extensively documented, with analytical methods primarily categorized into two main categories: direct determination and derivatization. Direct determination involves the direct determination of dimethyl sulfate as a solid compound without derivatization. Dimethyl sulfate has a high boiling point, high polarity, lacks sensitive UV-absorbing groups, and is chemically reactive, susceptible to hydrolysis in water, particularly alkaline aqueous solutions. Therefore, direct determination methods struggle to simultaneously meet analytical requirements in terms of sensitivity, accuracy, and reproducibility, and applicable chromatographic techniques are also severely limited.

[0005] Derivatization offers numerous advantages over direct determination. For example, the derivatized products possess more stable structures, higher volatility, lower polarity, or sensitive UV-absorbing groups, significantly expanding applicable chromatographic techniques and detector types. Furthermore, derivatization offers improved sensitivity, accuracy, and reproducibility. However, current derivatization methods not only present challenges in selecting suitable derivatization reagents but also require associated additives, resulting in cumbersome procedures and high costs. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems in the prior art to a certain extent. To this end, one object of the present invention is to provide a method for determining dimethyl sulfate in medicines by headspace in situ derivatization GC-MS.

[0007] In order to solve the problem of derivatization detection of dimethyl sulfate in current drugs, the present invention proposes a method for determining dimethyl sulfate in drugs by headspace in situ derivatization GC-MS. The derivatization reagent used in this method is a sodium propionate aqueous solution, the equilibrium temperature of the headspace bottle is not lower than 45°C, the equilibrium time of the headspace bottle is not less than 15 minutes, and the solvent used for the drug sample solution and the dimethyl sulfate reference solution is acetonitrile.

[0008] The method for determining dimethyl sulfate in drugs provided by the present invention uses a sodium propionate aqueous solution as a derivatization reagent. The inventors have found that sodium propionate and dimethyl sulfate undergo nucleophilic substitution (S N 2) The reaction yields a stable methyl propionate derivative product. The specific reaction process of nucleophilic substitution is as follows: . The derivatization reaction of this method is rapid and does not require additional reaction containers and reaction time. Therefore, the conversion can be quickly completed "in situ" during the headspace equilibration process. The headspace gas chromatography-mass spectrometry method is then used to determine methyl propionate, and finally the determination of the residual amount of dimethyl sulfate in the drug is achieved. Moreover, the acrylic acid used in the present invention is economical, easy to obtain, environmentally friendly, and stable in nature. It completely avoids the use of derivatization reagent adjuvants, and can achieve the determination of dimethyl sulfate in the drug. The equilibrium temperature of the headspace bottle is not less than 45°C to ensure that a sufficient amount of methyl propionate derivatization product is volatilized to ensure the sensitivity of the detection. The equilibration time of the headspace bottle is not less than 15 minutes to ensure that the gas-liquid two phases are fully balanced. The solvents used for the drug sample solution and the dimethyl sulfate reference solution are both acetonitrile, which can improve the stability of dimethyl sulfate in the solvent, and the solvent has little interference with the test results.

[0009] Specifically, in order to further verify the reaction efficiency of sodium propionate and dimethyl sulfate, the present invention used nuclear magnetic resonance hydrogen spectrum to detect the reaction product (see Figure 1 , A: sodium propionate; B: reaction of sodium propionate and dimethyl sulfate (5 min); C: methyl propionate; D: dimethyl sulfate). The results showed that within 5 min at room temperature, the characteristic H signals of sodium propionate (―CH3, ―CH2―) disappeared and converted into the characteristic H signals of methyl propionate, indicating a highly efficient reaction.

[0010] In some embodiments of the present invention, the concentration of the sodium propionate aqueous solution is 100 mg·mL -1 -300mg·mL -1 .

[0011] In some embodiments of the present invention, the temperature of the quantitative loop of the headspace vial is 55°C-80°C, and the temperature of the transfer line is 65°C-90°C.

[0012] In some embodiments of the present invention, the equilibrium temperature of the head space vial is lower than the temperature of the sample loop of the head space vial.

[0013] Preferably, the equilibrium temperature of the headspace bottle is 30°C-70°C, more preferably 40°C-60°C.

[0014] In some embodiments of the present invention, the equilibrium temperature of the headspace bottle is 15 min-40 min, preferably 25 min-35 min.

[0015] In some embodiments of the present invention, the carrier gas flow rate used in the GC is 1.0 mL min −1 , the split ratio is 10:1, and the carrier gas is preferably helium.

[0016] In some embodiments of the present invention, the GC column oven temperature program is: starting temperature 38-42 ° C for 7.5-8.5 min, 5-6 ° C min −1 The heating rate is raised to 60-64℃, maintained for 1-2min, and then increased to 40-44℃·min −1 The temperature was raised to 240 °C at a heating rate of 100 °C and maintained for 3 min. The injection port temperature was 210 °C.

[0017] In some embodiments of the present invention, the MS uses an EI ion source, the ion source temperature is 230°C, and the quadrupole temperature is 150°C.

[0018] In some embodiments of the present invention, the MS acquisition time is 9-12 min; selected ion monitoring mode: m / z 57, 88; quantitative ion: m / z 88.

[0019] In some embodiments of the present invention, the drug comprises siopride hydrochloride.

[0020] The present invention has at least the following beneficial effects: The headspace in-situ derivatization GC-MS method disclosed herein for determining trace amounts of dimethyl sulfate in pharmaceuticals utilizes economically available, stable, and non-toxic sodium propionate as a derivatization reagent to convert highly reactive dimethyl sulfate into stable and easily detectable methyl propionate. This derivatization reaction requires no additional inorganic base and is rapid, efficient, and easy to operate. The headspace injection method not only avoids contamination of the chromatographic system by high-concentration APIs but also provides an "in-situ" reaction site for the derivatization reaction. This method exhibits excellent specificity and high sensitivity, providing a reliable new option for the determination of trace amounts of dimethyl sulfate in APIs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The sodium propionate of the embodiment of the present invention and dimethyl sulfate react 1 H-NMR spectrum; Figure 2 GC-MS analysis results of blank solution, reference solution and test solution of Example 1 of the present invention are shown; Figure 3 is a standard curve diagram of Example 1 of the present invention; Figure 4 2 is a graph showing headspace equilibrium time and peak area in Example 2 of the present invention; Figure 5 is a graph of headspace equilibrium temperature and peak area of Example 3 of the present invention; Figure 6 is a graph showing the concentration of the derivatization reagent and the peak area in Example 4 of the present invention; Figure 7 This is a chromatographic result diagram of the selection of the derivatization reagent solvent in Example 5 of the present invention. DETAILED DESCRIPTION

[0023] Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention. The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0024] The instruments used in the embodiments of the present invention are as follows: Agilent 7890A / 5975 gas chromatograph-mass spectrometer; DB-624UI capillary column (60 m × 0.25 mm, 1.4 μm) (Agilent, USA); Milli-Q water purifier (Merck Millipore, Germany); XA205DU 1 / 100,000 electronic balance (Mettler, Switzerland).

[0025] The drugs and reagents used in the embodiments of the present invention are as follows: Tiapride hydrochloride API was the sample for the national drug random inspection in 2024; Dimethyl sulfate (TCI Reagent Company, batch number: Q2U3F, purity: 99.6%); Sodium propionate (J&K Reagent Company, batch number: L330W09, purity: 100%); Methyl propionate (J&K Reagent Co., Ltd., batch number: L660W03, purity: 99.99%); Acetonitrile was of chromatographic grade, and the water used in the experiment was ultrapure water.

[0026] Example 1 (1) Preparation of solution Sodium propionate solution (derivation reagent): Weigh 100 g of sodium propionate, place it in a 1000 mL volumetric flask, dissolve it in water and dilute to the scale, and shake well.

[0027] Reference solution: Prepare an appropriate amount of dimethyl sulfate reference substance in acetonitrile to a solution containing 0.5 μg per 1 mL. Accurately measure 4.0 mL of the sodium propionate solution and 1.0 mL of the dimethyl sulfate acetonitrile solution described above into a 20 mL headspace vial and seal with a crimp cap.

[0028] Test solution: Take about 0.22 g of sulpride hydrochloride raw material, accurately weigh it, then accurately measure 4.0 mL of sodium propionate solution and 1.0 mL of acetonitrile, place them in the same 20 mL headspace bottle, and seal it with a crimp cap.

[0029] Blank solution: Accurately measure 4.0 mL of sodium propionate solution and 1.0 mL of acetonitrile, place them in the same 20 mL headspace vial, and seal with a crimp cap.

[0030] (2) Chromatographic and mass spectrometry conditions The headspace vial equilibrium temperature was 50°C, the quantitative loop temperature was 60°C, and the transfer line temperature was 70°C; the equilibrium time was 30 min; and the injection volume was 1.0 mL.

[0031] GC used helium (purity ≥99.99%) as carrier gas with a flow rate of 1.0 mL min −1 , the split ratio is 10:1. Column oven heating program: start temperature 40℃ and maintain for 8min, then increase the temperature by 5℃·min −1 The heating rate was raised to 60℃, maintained for 1min, and then increased to 40℃·min −1 The temperature was raised to 240°C at a rate of 1000 ℃ and maintained for 3 min. The injection port temperature was 210°C.

[0032] Mass spectrometry was performed using an EI ion source at 230°C and a quadrupole temperature of 150°C. Mass spectrometry acquisition time was 9–12 min. Selected ion monitoring (SIM) mode was used for the analysis of m / z 57 and 88, and the quantification ion was m / z 88.

[0033] Method validation of chromatographic methods: 1. Exclusivity Take the blank solution, reference solution and test solution prepared in (1) and perform GC-MS analysis according to the chromatographic conditions of (2). The specific results are shown in Figure 2 , where A is the reference solution, B is the test solution, C is the blank solution, D is methyl propionate in the reference solution, and E is methyl propionate in the NIST library. The experimental results show that the blank solution does not interfere with the determination of the target compound, indicating that the detection method has good specificity.

[0034] 2. Linear relationship and range investigation Take an appropriate amount of dimethyl sulfate reference substance and prepare a series of concentration reference substance solutions according to the reference substance solution preparation method in (1). Perform GC-MS analysis according to the chromatographic method in (2). Draw a standard curve (e.g., Figure 3 The specific results are shown in Table 1. The linear equation is y=46985x+330(r=0.9999), which shows that dimethyl sulfate is in the range of 0.03~0.8μg·mL −1 The internal linear relationship is good.

[0035] Table 1

[0036] 3. Limit of detection and limit of quantification The reference solution prepared in (1) was appropriately diluted, and the concentration at a signal-to-noise ratio (S / N) of about 3 was used as the detection limit, and the concentration at a signal-to-noise ratio (S / N) of about 10 was used as the quantification limit. GC-MS analysis was performed according to the chromatographic method in (2), and the detection limit of dimethyl sulfate was determined to be 0.01 μg·mL −1 , equivalent to a sample concentration of 0.05 μg·g −1 The limit of quantification was 0.03 μg·mL −1 , equivalent to a sample concentration of 0.15 μg·g −1 .

[0037] 4. Precision and repeatability The reference solution prepared in (1) was injected six times in succession, and the RSD of the peak area of the derivatized product methyl propionate was 1.1%, indicating that the instrument precision of this method was good.

[0038] Table 2

[0039] Precisely weigh the bulk drug substance of sulphuric acid hydrochloride (batch number: 20240104). Six sample solutions were prepared in parallel according to the sample solution preparation method (1) and the chromatographic method (2) for GC-MS analysis. The dimethyl sulfate content in the six samples was calculated by the external standard method (see Table 3). The RSD was 4.7%, indicating that the determination method has good reproducibility.

[0040] Table 3

[0041] 5. Recovery rate Take an appropriate amount of dimethyl sulfate reference substance and use acetonitrile to make the concentrations of about 0.25, 0.5, and 0.75 μg mL −1 Reference solution. Accurately weigh approximately 220 mg of sulpiride hydrochloride bulk drug substance (batch number: 20240106), add 1.0 mL of the reference solution and 4.0 mL of the sodium propionate solution prepared in (1), place in the same 20 mL headspace bottle, and seal with a roll cap. Prepare three replicates at each of the three concentration levels, as shown in Table 4. GC-MS analysis was performed according to the method in (2), and calculation was performed using the external standard method. The average recovery of dimethyl sulfate was 80.4% (RSD = 1.7%, n = 9), which meets the requirements of the ICH analytical method validation guidelines and indicates that this method has a good recovery rate.

[0042] Table 4

[0043] 6. Stability Six reference solutions were prepared according to the method in (1). Each reference solution was identical and placed at room temperature for 0, 2, 4, 6, 12, and 24 h, respectively. The results were determined according to the chromatographic method in (2). The RSD of the peak area of the derivatized product methyl propionate was 1.3%, as shown in Table 5, indicating that the reaction product had good stability.

[0044] Table 5

[0045] 7. Sample determination Five batches of sulphuric acid hydrochloride bulk drug were subjected to GC-MS analysis according to the preparation method of the test solution in (1) and the chromatographic method in (2). The content of residual dimethyl sulfate in the bulk drug was calculated by the external standard method. The results showed that trace amounts of dimethyl sulfate were detected in all five batches of samples, but the content was far below the prescribed limit of 2.5 μg·g −1 , see Table 6 for details. This shows that the manufacturer of sulphide hydrochloride API has good control over the genotoxic impurity dimethyl sulfate in the production process.

[0046] Table 6

[0047] Example 2 Selection of headspace equilibration time Example 2 screened the headspace equilibrium time, using the reference solution preparation method in Example 1 (1) to prepare the reference solution and the chromatographic method in (2) for GC-MS analysis. The effects of equilibration for 10, 15, 20, 30, and 40 minutes were studied, and the specific results can be seen. Figure 4 The results showed that the target peak area no longer increased significantly after 15 minutes of equilibrium. In order to ensure sufficient equilibrium between the gas and liquid phases, the equilibrium time was finally selected to be 30 minutes.

[0048] Example 3 Selection of headspace equilibrium temperature Example 3 screened the headspace equilibrium temperature. The reference solution was prepared using the preparation method of the reference solution in (1) of Example 1 and the GC-MS analysis was performed using the chromatography method of (2). The effects of equilibrium temperatures of 40°C, 50°C, 60°C, 70°C, and 80°C were studied. The specific results are shown in Figure 5 Considering the requirements of temperature control and sensitivity, when the equilibrium temperature of Example 1 is 50°C, the limit of quantification is low enough. When the temperature is not lower than 45°C, the requirement can be met. Furthermore, 50-70°C is preferred.

[0049] Example 4 Selection of derivatization reagent concentration Example 4 screened the concentration of the derivatization reagent and prepared 1, 10, 20, 50, 100, 150, and 200 mg·mL of sodium propionate solution using the preparation method of (1) in Example 1. -1 Derivatization reagents with different sodium propionate concentrations. Then, different reference solutions were prepared according to the preparation method of the reference solution in (1) of Example 1. Then, GC-MS analysis was performed using the chromatography method of (2) of Example 1. The specific results are shown in Figure 6 When the concentration of sodium propionate is greater than 100 mg·mL -1 There was no significant increase in the target peak area.

[0050] Example 5 Solvent selection for derivatization solution In Example 5, solvents for the derivatization solution were screened, and dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and water were selected as solvents for the derivatization solution, respectively. The derivatization solutions were prepared using the preparation method of the sodium propionate solution in (1) of Example 1, and GC-MS analysis was performed using the chromatography method in (2) of Example 1.

[0051] The experimental results show that when DMSO is used as solvent, the matrix has a great interference. Figure 7 B in the text. Literature research indicates that DMSO can, under certain reaction conditions, serve as a methyl source for methylation of carboxylic acids, amines, and phenolic compounds. However, since sodium propionate is nearly insoluble in DMF, it is not suitable as a solvent for sodium propionate.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for determining dimethyl sulfate in drugs by headspace in situ derivatization GC-MS, characterized in that: The derivatization reagent used is sodium propionate aqueous solution, the equilibrium temperature of the headspace bottle is not lower than 45° C., the equilibrium time of the headspace bottle is not less than 15 minutes, and the solvents used for the drug sample solution and dimethyl sulfate reference solution are both acetonitrile.

2. The method according to claim 1, characterized in that The concentration of the sodium propionate aqueous solution is 100 mg·mL -1 -300mg·mL -1 .

3. The method according to claim 1, characterized in that The temperature of the quantitative loop of the headspace bottle is 55°C-80°C, and the temperature of the transfer line is 65°C-90°C.

4. The method according to claim 1, characterized in that The equilibrium temperature of the headspace bottle is lower than the quantitative loop temperature of the headspace bottle; Preferably, the equilibrium temperature of the headspace bottle is 30°C-70°C, more preferably 40°C-60°C.

5. The method according to claim 1, characterized in that: The equilibrium temperature of the headspace bottle is 15 min-40 min, preferably 25 min-35 min.

6. The method according to any one of claims 1 to 5, characterized in that The carrier gas flow rate used in the GC was 1.0 mL min −1 , the split ratio is 10:1, and the carrier gas is preferably helium.

7. The method according to any one of claims 1 to 5, characterized in that The GC column oven temperature program is: starting temperature 38-42 ° C for 7.5-8.5 min, and then increasing by 5-6 ° C min −1 The heating rate is raised to 60-64℃, maintained for 1-2min, and then increased to 40-44℃·min −1 The temperature was raised to 240 °C at a heating rate of 100 °C and maintained for 3 min. The injection port temperature was 210 °C.

8. The method according to any one of claims 1 to 5, characterized in that The MS used an EI ion source with a temperature of 230° C. and a quadrupole temperature of 150° C.

9. The method according to any one of claims 1 to 5, characterized in that The MS acquisition time was 9-12 min; selected ion monitoring mode: m / z 57, 88; quantitative ion: m / z 88.

10. The method according to any one of claims 1 to 5, characterized in that: The drugs include sulpiride hydrochloride.

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