CN112083088A - GC-MS combined detection method for N-nitrosodimethylamine in metformin hydrochloride sustained-release tablets - Google Patents

Abstract

The invention discloses a GC-MS combined detection method of N-nitrosodimethylamine in metformin hydrochloride sustained-release tablets, which comprises the steps of precisely weighing metformin hydrochloride sustained-release tablet fine powder, placing the metformin hydrochloride sustained-release tablet fine powder in a centrifuge tube, adding 1N hydrochloric acid aqueous solution, placing the metformin hydrochloride sustained-release tablets on a vortex oscillator to vortex, then precisely adding dichloromethane, further vortex, continuing to shake and uniformly mix, then carrying out centrifugal separation, filtering a dichloromethane layer filter membrane, taking filtrate as a sample solution, finally adopting a gas phase mass spectrometer to carry out determination, recording a gas phase mass spectrogram, and calculating the peak area according to a standard curve method if a chromatographic peak which is consistent with the NDMA peak retention time exists in the gas phase mass spectrogram of the sample solution. The method provided by the invention can effectively prevent residual raw material dimethylamine in the sample from generating NDMA (Newcastle disease Virus) by chemical reaction when encountering active oxide in the sample preparation process by adopting the 1N hydrochloric acid aqueous solution to dissolve the sample, thereby improving the detection accuracy. According to the different solubilities of metformin hydrochloride and NDMA in dichloromethane, after a hydrochloric acid solution is adopted to dissolve a sample, the NDMA sample is extracted by adopting dichloromethane, so that the load of an instrument detector is reduced, and the service life of the detector is prolonged. Meanwhile, metformin hydrochloride is effectively separated, the matrix effect is reduced, and the detection accuracy is further improved.

Description

GC-MS combined detection method for N-nitrosodimethylamine in metformin hydrochloride sustained-release tablets

Technical Field

The invention relates to a method for detecting N-nitrosodimethylamine in a metformin hydrochloride sustained-release tablet, in particular to a GC-MS combined detection method of N-nitrosodimethylamine in the metformin hydrochloride sustained-release tablet.

Background

Metformin hydrochloride is a biguanide oral hypoglycemic drug, also known as metformin hydrochloride, glauber and medecan, has weaker hypoglycemic action than phenformin, mainly has the effects of reducing the secretion of glucagon in A cells and promoting the conversion of glucose into glycogen, and has the characteristic of not stimulating insulin secretion of pancreatic beta-cells. Clinically, the metformin hydrochloride is mainly used for treating type II diabetes patients with unsatisfactory diet control, especially obesity and hyperinsulinemia patients, and the metformin hydrochloride not only has the effect of reducing blood sugar, but also has the effects of reducing weight and reducing hyperinsulinemia. Can be used for treating patients with poor curative effect of some sulfonylureas, such as sulfonylureas, small intestine glycosidase inhibitor or thiazolidinedione hypoglycemic agent, which has better effect than single use. Can also be used for patients with insulin therapy to reduce insulin dosage.

Metformin hydrochloride may produce N-Nitrosodimethylamine (NDMA), a potentially genotoxic impurity during the manufacturing, synthesis, and storage and transportation processes. Moreover, the U.S. Food and Drug Administration (FDA) announces that the content of N-nitrosodimethylamine in some metformin sustained-release preparations exceeds the standard, and the health of diabetics is seriously threatened. NDMA has been classified as a class 2A carcinogen, and the FDA set an acceptable daily drug intake limit for NDMA of 96ng per day, based on a Maximum Daily Dose (MDD) of 2550mg per day; the NMDA limit in metformin hydrochloride is 0.048ppm (based on 2000mg MDD).

At present, FDA and Chinese food and drug testing research institute both disclose detection methods for metformin hydrochloride sustained release tablets. The FDA adopts two methods of LC-HRMS and LC-ESI-HRMS, and the method adopts high-resolution liquid-phase mass spectrometry, so the instrument is expensive and is not suitable for small and medium-sized enterprises. As shown in fig. 1 to fig. 3, when the sample is dissolved in methanol, the metformin hydrochloride is completely dissolved in the methanol solution, which easily causes matrix interference, and results in inaccurate measurement results. The detection method comprises the steps of measuring by a gas chromatography (general guidelines in Chinese pharmacopoeia 0521) and a mass spectrometry (general guidelines in Chinese pharmacopoeia 0431) at a Chinese food and drug detection institute, precisely weighing 500mg of metformin hydrochloride, placing the metformin hydrochloride in a 50mL centrifuge tube, precisely adding 10mL of dichloromethane, vortexing for 1min, shaking for 10min at a frequency of 350 times/min, then centrifuging for 10min at a speed of 4000rpm, filtering a dichloromethane layer by a 0.45-micrometer filter membrane, and taking a filtrate as a sample solution. In the detection process of the method, residual dimethylamine serving as a raw material in a sample can generate chemical reaction to generate NDMA when meeting active oxides, and the detection result is higher than the actual result of the sample, so that the detection result is inaccurate.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a GC-MS combined detection method of N-nitrosodimethylamine in a metformin hydrochloride sustained-release tablet. The method has the advantages of small matrix interference, high accuracy, simple and convenient operation and low cost, and is suitable for wide medium and small enterprises.

In order to achieve the above purpose, the solution adopted by the invention is as follows:

accurately weighing metformin hydrochloride sustained-release tablet fine powder, placing the fine powder into a centrifuge tube, adding a hydrochloric acid aqueous solution, placing the fine powder into a vortex oscillator for vortex, then accurately adding dichloromethane, performing vortex again, continuing to shake and mix uniformly, then performing centrifugal separation, filtering with a dichloromethane layer filter membrane, taking filtrate as a sample solution, and finally detecting with a gas phase mass spectrometer, wherein the specific steps are as follows:

s1, preparing a reference substance solution: precisely measuring 100 mu L of NDMA, placing the NDMA in a 100mL measuring flask, adding dichloromethane to dilute the NDMA to a scale, shaking up to obtain an NDMA solution with the concentration of 100 mu g/mL, and adding dichloromethane to quantitatively dilute the NDMA solution to prepare solutions with gradient concentrations of 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL, wherein the solutions serve as reference substance linear series solutions.

S2, preparing a test solution: grinding the metformin hydrochloride sustained-release tablets into powder, precisely weighing fine powder equivalent to 500mg of metformin hydrochloride, placing the fine powder into a 50mL centrifuge tube, adding 5-10mL of 1N hydrochloric acid aqueous solution, placing the mixture on a vortex oscillator, precisely adding 10mL of dichloromethane, carrying out vortex again, continuously shaking and uniformly mixing, then carrying out centrifugal separation, filtering a dichloromethane layer by using a 0.45 mu m filter membrane, and taking filtrate as a test solution.

And S3, respectively measuring the reference solution and the test solution by adopting a gas phase mass spectrometer, recording a gas phase mass spectrogram, and calculating the peak area according to a standard curve method if a chromatographic peak with the retention time consistent with that of the NDMA peak exists in the gas phase mass spectrogram of the test solution.

Further, the frequency of the vortex oscillator is 1000 times/min, and the vortex time is 1 min.

Further, the shaking frequency of the invention is 1200 times/min, and the shaking mixing time is 10 min.

Further, the centrifugation speed of the invention is 4000rpm, and the centrifugation time is 10 min.

Further, the amount of the 1N aqueous hydrochloric acid solution added is 5 mL.

Further, the operating parameters of the gas phase mass spectrometer according to the present invention are shown in table 1.

TABLE 1 gas phase Mass spectrometer operating parameters

Further, the gas phase mass spectrometer of the present invention has the following requirements for systematic applicability when measuring a control linear series solution: and (3) repeatedly injecting the control solution with the concentration of 0.5ng/mL for 6 times, wherein the signal-to-noise ratio of an NDMA peak in the obtained chromatogram is not lower than 10, the relative standard deviation of the main peak area is not more than 10%, a standard curve is drawn by using the concentration to the peak area, and the correlation coefficient of the obtained linear equation is not lower than 0.995.

In order to verify the accuracy of the detection method, NDMA (New Desorption-free maleic anhydride) with a known amount is added into the fine powder of the metformin hydrochloride sustained-release tablet for determination, and the method comprises the following specific steps:

s4, preparing accuracy sample solutions with quantitative limit, 50% limit concentration, 100% limit concentration and 150% limit concentration: respectively and precisely weighing 500mg of metformin hydrochloride sustained-release tablet fine powder equivalent to metformin hydrochloride, respectively placing the fine powder into 50ml of centrifuge tubes, and respectively and precisely adding 5ng, 25ng, 50ng and 75ng of NDMA; subsequent operations repeat steps S2-S3.

The GC-MS combined detection method for N-nitrosodimethylamine in the metformin hydrochloride sustained-release tablets has the beneficial effects that:

the method provided by the invention can effectively prevent residual raw material dimethylamine in the sample from generating NDMA (Newcastle disease Virus) by chemical reaction when encountering active oxide in the sample preparation process by adopting the 1N hydrochloric acid aqueous solution to dissolve the sample, thereby improving the detection accuracy. According to the invention, according to the different solubilities of metformin hydrochloride and NDMA in dichloromethane, after a hydrochloric acid solution is adopted to dissolve a sample, the NDMA sample is extracted by adopting dichloromethane, so that the load of an instrument detector is reduced, and the service life of the detector is prolonged. Meanwhile, metformin hydrochloride is effectively separated, the matrix effect is reduced, and the detection accuracy is further improved. The invention has simple operation, can be detected by adopting the gas-phase mass spectrometer, and can meet the detection requirements of vast middle and small enterprises.

Drawings

FIG. 1 is a liquid phase mass spectrum of NDMA control solution dissolved in methanol by FDA method

FIG. 2 is a liquid phase mass spectrum of a test solution prepared by FDA method using methanol to dissolve metformin hydrochloride

FIG. 3 is a liquid phase mass spectrum of a sample solution of metformin hydrochloride dissolved in methanol + NDMA by FDA method

FIG. 4 is a gas mass spectrum of methylene chloride according to the present invention

FIG. 5 is a gas mass spectrum of a control solution of NDMA dissolved in methylene chloride according to the present invention

FIG. 6 gas mass spectrum of example 3 of the method of the present invention

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described below with reference to specific examples.

Example 1

S1, preparing a reference substance solution: precisely measuring 100 mu L of NDMA, placing the NDMA in a 100mL measuring flask, adding dichloromethane to dilute the NDMA to a scale, shaking up to obtain an NDMA solution with the concentration of 100 mu g/mL, and adding dichloromethane to quantitatively dilute the NDMA solution to prepare solutions with gradient concentrations of 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL, wherein the solutions serve as reference substance linear series solutions.

S2, preparing a test solution: grinding the metformin hydrochloride sustained release tablets into powder, precisely weighing fine powder equivalent to 500mg of metformin hydrochloride, placing the fine powder into a 50mL centrifuge tube, placing the centrifuge tube on a vortex oscillator, carrying out vortex for 1min at the frequency of 1000 times/min, then precisely adding 10mL of dichloromethane, then shaking for 10min at the frequency of 1200 times/min, carrying out vortex again, continuing to shake and mix for 10min at the frequency of 1200 times/min, then centrifuging for 10min at the speed of 4000rpm, taking a dichloromethane layer, filtering with a 0.45 mu m filter membrane, and taking filtrate as a sample solution.

And S3, respectively measuring the reference solution and the test solution by adopting a gas phase mass spectrometer, recording a gas phase mass spectrogram, calculating peak areas according to a standard curve method by using the peak areas if a chromatographic peak with the retention time consistent with that of an NDMA peak exists in the gas phase mass spectrogram of the test solution, and respectively testing each solution for 3 times.

S4, preparing accuracy sample solutions with quantitative limit, 50% limit concentration, 100% limit concentration and 150% limit concentration: respectively and precisely weighing 500mg of metformin hydrochloride sustained-release tablet fine powder equivalent to metformin hydrochloride, respectively placing the fine powder into 50ml of centrifuge tubes, and respectively and precisely adding 5ng, 25ng, 50ng and 75ng of NDMA; subsequent operations repeat steps S2-S3.

Example 2

S1, preparing a reference substance solution: precisely measuring 100 mu L of NDMA, placing the NDMA in a 100mL measuring flask, adding dichloromethane to dilute the NDMA to a scale, shaking up to obtain an NDMA solution with the concentration of 100 mu g/mL, and adding dichloromethane to quantitatively dilute the NDMA solution to prepare solutions with gradient concentrations of 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL, wherein the solutions serve as reference substance linear series solutions.

S2, preparing a test solution: grinding the metformin hydrochloride sustained release tablets into powder, precisely weighing fine powder equivalent to 500mg of metformin hydrochloride, placing the fine powder into a 50mL centrifuge tube, adding 10mL of 1N hydrochloric acid aqueous solution, placing the mixture on a vortex oscillator, carrying out vortex for 1min at the frequency of 1000 times/min, then precisely adding 10mL of dichloromethane, then shaking for 10min at the frequency of 1200 times/min, carrying out vortex again, continuing to shake and mix for 10min at the frequency of 1200 times/min, then centrifuging for 10min at the speed of 4000rpm, taking a dichloromethane layer, filtering with a 0.45 mu m filter membrane, and taking filtrate as a test solution.

And S3, respectively measuring the reference solution and the test solution by adopting a gas phase mass spectrometer, recording a gas phase mass spectrogram, calculating peak areas according to a standard curve method by using the peak areas if a chromatographic peak with the retention time consistent with that of an NDMA peak exists in the gas phase mass spectrogram of the test solution, and respectively testing each solution for 3 times.

S4, preparing accuracy sample solutions with quantitative limit, 50% limit concentration, 100% limit concentration and 150% limit concentration: respectively and precisely weighing 500mg of metformin hydrochloride sustained-release tablet fine powder equivalent to metformin hydrochloride, respectively placing the fine powder into 50ml of centrifuge tubes, and respectively and precisely adding 5ng, 25ng, 50ng and 75ng of NDMA; subsequent operations repeat steps S2-S3.

Example 3

S1, preparing a reference substance solution: precisely measuring 100 mu L of NDMA, placing the NDMA in a 100mL measuring flask, adding dichloromethane to dilute the NDMA to a scale, shaking up to obtain an NDMA solution with the concentration of 100 mu g/mL, and adding dichloromethane to quantitatively dilute the NDMA solution to prepare solutions with gradient concentrations of 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL, wherein the solutions serve as reference substance linear series solutions.

S2, preparing a test solution: grinding the metformin hydrochloride sustained release tablets into powder, precisely weighing fine powder equivalent to 500mg of metformin hydrochloride, placing the fine powder into a 50mL centrifuge tube, adding 5mL of 1N hydrochloric acid aqueous solution, placing the mixture on a vortex oscillator, carrying out vortex for 1min at the frequency of 1000 times/min, then precisely adding 10mL of dichloromethane, then shaking for 10min at the frequency of 1200 times/min, carrying out vortex again, continuing to shake and mix for 10min at the frequency of 1200 times/min, then centrifuging for 10min at the speed of 4000rpm, taking a dichloromethane layer, filtering with a 0.45 mu m filter membrane, and taking filtrate as a test solution.

And S3, respectively measuring the reference solution and the test solution by adopting a gas phase mass spectrometer, recording a gas phase mass spectrogram, calculating peak areas according to a standard curve method by using the peak areas if a chromatographic peak with the retention time consistent with that of an NDMA peak exists in the gas phase mass spectrogram of the test solution, and respectively testing each solution for 3 times.

S4, preparing accuracy sample solutions with quantitative limit, 50% limit concentration, 100% limit concentration and 150% limit concentration: respectively and precisely weighing 500mg of metformin hydrochloride sustained-release tablet fine powder equivalent to metformin hydrochloride, respectively placing the fine powder into 50ml of centrifuge tubes, and respectively and precisely adding 5ng, 25ng, 50ng and 75ng of NDMA; subsequent operations repeat steps S2-S3.

Table 2 examples 1-3 concentration of NDMA in test solutions

Name (R)	Example 1	Example 2	Example 3
				Test solution-1	0.041ppm	ND	ND
Test solution-2	0.003ppm	ND	ND
				Test solution-3	0.021ppm	ND	ND

The test result shows that: in the embodiment 2-3, the 1N hydrochloric acid aqueous solution is adopted to dissolve the sample, so that residual dimethylamine serving as a raw material in the sample can be effectively prevented from generating NDMA (Newcastle disease Virus) through chemical reaction when encountering active oxides in the sample preparation and test processes, and the detection accuracy is improved.

TABLE 3 recovery of examples 1-3

The test result shows that: the recovery rate of the method in the embodiment 3 is between 80.23% and 99.52%, and the standard requirement that the verification guiding principle of the medicine quality standard analysis method in Chinese pharmacopoeia is between 75% and 120% is completely met, which shows that the detection method provided by the invention has high accuracy. Meanwhile, the detection method provided by the invention is shown by combining with the attached figures 4-6, so that the matrix effect is reduced, and the detection accuracy is further improved.

Claims (7)

1. A GC-MS combined detection method of N-nitrosodimethylamine in metformin hydrochloride sustained-release tablets is characterized in that: precisely weighing the metformin hydrochloride sustained-release tablet fine powder, placing the metformin hydrochloride sustained-release tablet fine powder into a centrifuge tube, adding a hydrochloric acid aqueous solution, placing the metformin hydrochloride sustained-release tablet fine powder on a vortex oscillator for vortex, then precisely adding dichloromethane, carrying out vortex again, continuing to shake and mix uniformly, then carrying out centrifugal separation, filtering a dichloromethane layer filter membrane, taking filtrate as a sample solution, and finally detecting by using a gas phase mass spectrometer.

2. The GC-MS combined detection method according to claim 1, wherein: the operation steps are as follows:

s1, preparing a reference substance solution: precisely measuring NDMA 100 mu L, putting the NDMA 100 mu L into a 100mL measuring flask, adding dichloromethane for dilution to a scale, shaking up to obtain an NDMA solution with the concentration of 100 mu g/mL, adding dichloromethane for quantitative dilution to prepare solutions with gradient concentrations of 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL as a reference substance linear series solution,

s2, preparing a test solution: grinding metformin hydrochloride sustained release tablets into powder, precisely weighing fine powder equivalent to 500mg of metformin hydrochloride, placing the fine powder into a 50mL centrifuge tube, adding 5-10mL of 1N hydrochloric acid aqueous solution, placing the mixture on a vortex oscillator, precisely adding 10mL of dichloromethane, carrying out vortex again, continuously shaking and uniformly mixing, then carrying out centrifugal separation, filtering a dichloromethane layer by using a 0.45 mu m filter membrane, taking filtrate as a test solution,

and S3, respectively measuring the reference solution and the test solution by adopting a gas phase mass spectrometer, recording a gas phase mass spectrogram, and calculating the peak area according to a standard curve method if a chromatographic peak with the retention time consistent with that of the NDMA peak exists in the gas phase mass spectrogram of the test solution.

3. The GC-MS combined detection method according to claim 2, wherein: the addition amount of the 1N hydrochloric acid aqueous solution is 5 mL.

4. The GC-MS combined detection method according to any one of claims 1 to 3, wherein: the frequency of the vortex oscillator is 1000 times/min, and the vortex time is 1 min.

5. The GC-MS combined detection method according to any one of claims 1 to 3, wherein: the shaking frequency was 1200 times/min, and the shaking mixing time was 10 min.

6. The GC-MS combined detection method according to any one of claims 1 to 3, wherein: the centrifugation speed is 4000rpm, and the centrifugation time is 10 min.

7. The GC-MS combined detection method according to claim 2, wherein: the determination method also comprises an accuracy verification method, and the operation steps are as follows:

s4, preparing accuracy sample solutions with quantitative limit, 50% limit concentration, 100% limit concentration and 150% limit concentration: respectively and precisely weighing 500mg of metformin hydrochloride sustained-release tablet fine powder equivalent to metformin hydrochloride, respectively placing the fine powder into 50ml of centrifuge tubes, and respectively and precisely adding 5ng, 25ng, 50ng and 75ng of NDMA; subsequent operations repeat steps S2-S3.

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