A method for determining the content of N-nitrosodimethylamine in metformin using two-dimensional liquid chromatography-mass spectrometry

Abstract

The present application discloses a method for determining the content of N-nitrosodimethylamine in metformin using two-dimensional liquid chromatography-mass spectrometry. The detection method comprises the following steps: sample preparation, two-dimensional liquid phase separation of N-nitrosodimethylamine, mass spectrometry detection, and quantitative processing; the use of a mixed filler of strong cation exchange (SCX) and C18 in the first-dimensional liquid phase completely eliminates the interference of incomplete separation and elution of metformin on the detection of N-nitrosodimethylamine in conventional liquid-mass detection methods, thereby reducing contamination of the mass spectrometry detector and extending its service life; the second-dimensional liquid phase can separate the excipients and interfering substance N,N-dimethylformamide (DMF) co-eluted with N-nitrosodimethylamine, thereby avoiding false positives and improving the accuracy of the results.

Description

Technical Field

[0001] The present application relates to the technical field of N-nitrosodimethylamine (NDMA) analysis and testing, and in particular to a method for determining the content of N-nitrosodimethylamine in metformin using two-dimensional liquid chromatography-mass spectrometry. Background Art

[0002] N-nitrosodimethylamine (NDMA) is a type of genotoxic impurity in the N-nitrosamine class. It can cause damage and mutation of genetic material at relatively low concentrations. Studies have shown that it has significant carcinogenicity and teratogenicity, and its content must be accurately controlled for safe drug use.

[0003] The chromatographic columns used in the methods recommended by the FDA and the China Food and Drug Inspection Institutes have a retention time for N-nitrosodimethylamine (NDMA) after that of metformin in chromatographic separations. The main peak of high-concentration metformin and N-nitrosodimethylamine (NDMA) must be completely separated during analysis, placing high demands on the performance of the chromatographic columns used for analysis and detection. Otherwise, incompletely eluted metformin can easily enter the detector through the mass spectrometer valve, causing increased baseline noise and mass spectrum contamination. Literature has reported that in the detection of N-nitrosodimethylamine (NDMA) using tandem mass spectrometry, some samples may contain N,N-dimethylformamide (DMF). The isotopic peak of N,N-dimethylformamide (DMF) has the same 75→43 MRM transition and is very close to the chromatographic retention of N-nitrosodimethylamine (NDMA). If the two are not completely separated, the test results may be biased high, leading to false positives and misjudgments. Currently, there is literature using high-resolution mass spectrometry HRMS for detection, which can distinguish N,N-dimethylformamide DMF from N-nitrosodimethylamine NDMA through high-precision mass numbers. However, this method uses high-cost detection instruments and is not easy to promote and use. In addition, the sensitivity of HRMS is orders of magnitude different from that of a triple quadrupole mass spectrometer, making it unsuitable for the detection of trace amounts of N-nitrosodimethylamine NDMA. Summary of the Invention

[0004] Technical issues solved:

[0005] The technical problems to be solved by this application are the technical problems in the existing technology such as increased baseline noise, mass spectrometry contamination, misjudgment, and high cost of detection instruments. A method for determining the content of N-nitrosodimethylamine (NDMA) in metformin using two-dimensional liquid chromatography-mass spectrometry is provided. The detection results have little interference, can avoid false positives, have high sensitivity, and accurate quantitative results.

[0006] Technical solution:

[0007] A method for determining the content of N-nitrosodimethylamine in metformin using two-dimensional liquid chromatography-mass spectrometry comprises the following steps:

[0008] The first step is to obtain a test solution containing the sample to be tested: the sample to be tested that contains or may contain N-nitrosodimethylamine (NDMA) impurity is dissolved in a diluent to prepare a test solution;

[0009] Step 2: Transfer the prepared test solution to an injection vial, and detect the test solution in the injection vial using two-dimensional liquid chromatography-mass spectrometry to determine the content of N-nitrosodimethylamine (NDMA) in the sample.

[0010] Furthermore, the first step specifically includes the following steps:

[0011] Step 1: When the sample to be tested is metformin hydrochloride API, accurately weigh 0.5 g of the sample to be tested, and the specific mass is recorded as W 称样量 , put it into a 50mL centrifuge tube, accurately add 10mL of purified water, shake for 10min to dissolve, and filter to obtain the test solution;

[0012] Step 2: When the sample to be tested is metformin hydrochloride tablets, metformin hydrochloride enteric-coated tablets, metformin hydrochloride capsules, compound tablets or capsules containing metformin hydrochloride, accurately weigh 10 tablets of the sample to be tested or 10 pellets of the sample to be tested, grind them into powder, and record the specific mass as W. 10 The average tablet weight is recorded as W 平均片重 , where W 平均片重 =W 10 / 10; the weight of metformin contained in each tablet is the labeled amount of the drug, recorded as W 标示量 ; Accurately weigh the sample powder equivalent to 0.5g of metformin hydrochloride, and the specific mass is recorded as W 称样量 , where W 称样量 =0.5 / W 标示量 *W 平均片重 , placed in a 50 mL centrifuge tube, accurately added 10 mL of purified water, vortexed for 2 min to dissolve, 13000 r min -1 Centrifuge for 15 min, take the supernatant, and obtain the test solution;

[0013] Step 3: When the sample to be tested is metformin hydrochloride sustained-release tablets or a compound sustained-release preparation containing metformin hydrochloride, accurately weigh 5 tablets of the sample to be tested, grind them into powder, and record the specific mass as W5 and the average tablet weight as W 平均片重 , where W 平均片重 =W5 / 5; the weight of metformin contained in each tablet is the labeled amount of the drug, recorded as W 标示量 ; Accurately weigh the sample powder equivalent to 0.5g of metformin hydrochloride, and the specific mass is recorded as W 称样量 , where W 称样量 =0.5 / W 标示量 *W 平均片重, placed in a 50 mL centrifuge tube, accurately added 10 mL of methanol, vortexed for 10 min to dissolve, 13000 r min -1 Centrifuge for 15 minutes, accurately remove 5 mL of the supernatant, place it in a 15 mL centrifuge tube, accurately add 5 mL of water, shake and mix, and rotate at 13000 r·min. -1 Centrifuge for 15 min and take the supernatant to obtain the test solution.

[0014] Furthermore, the second step of testing the test solution in the injection vial specifically includes the following steps: injecting the test solution into a two-dimensional liquid chromatography-mass spectrometry instrument for separation and detection, recording the two-dimensional chromatogram of the test solution, obtaining a standard curve of N-nitrosodimethylamine, and calculating the concentration C of N-nitrosodimethylamine based on the peak area of ​​the unknown sample, and calculating the content of N-nitrosodimethylamine in the sample to be tested according to the calculation formula.

[0015] Furthermore, the chromatographic and mass spectrometric conditions of the two-dimensional liquid chromatography-mass spectrometry method are as follows:

[0016] One-dimensional liquid phase conditions: chromatographic column: CAPCELLPAKCR1:4 (150 mm × 2.0 mm I.D., 5.0 μm); flow rate: 0.8 mL min -1 , column temperature was 40°C, injection volume was 20 μL; mobile phase: phase A was 20 mM ammonium acetate aqueous solution, phase B was acetonitrile; gradient elution: 0.00 min (5% B), 1.20 min (5% B), 1.21 min (90% B), 7.00 min (90% B), 8.10 min (5% B), 16.00 min (stop); trapping column: Shim-Pack Velox SPC18 (4.6 mm I.D. × 50 mm L., 2.7 μm); one-dimensional chromatography online trapping time: 0.8 to 1.06 min

[0017] 2D-LC conditions: chromatographic column Shim-Pack GISTC18-AQ (4.6 mm I.D. × 150 mm L., 3.0 μm); flow rate 1.0 mL min -1 Column temperature was 40°C; mobile phase: phase C was 0.1% formic acid in water, phase D was 0.1% formic acid in acetonitrile, gradient elution: 0.00 min (5% D), 7.00 min (10% D), 10.00 min (90% D), 10.10 min (5% D), 16.00 min (stop); time for switching the valve after the second column to the mass spectrometer: 3.5 to 7 min;

[0018] Mass spectrometry conditions: APCI ionization mode; interface voltage 3.5 kV, interface temperature 300 °C; nebulizer gas flow rate 3 L min -1; Desolvation tube temperature 180℃; Drying gas flow rate 5.0L·min -1 ; Scan mode: positive ion mode; Monitoring mode: multiple reaction monitoring (MRM): 75.10→43.10, collision voltage -19 V, Q1 deflection voltage -12 V, Q3 deflection voltage -15 V; 75.10→58.15, collision voltage -16 V, Q1 deflection voltage -25 V, Q3 deflection voltage -21 V.

[0019] Furthermore, the calculation formula for the content of N-nitrosodimethylamine in the sample to be tested is as follows:

[0020] a. When the sample to be tested is metformin hydrochloride API, content (ng / g) = C*10 / W 称样量 ;

[0021] b. When the sample to be tested is metformin hydrochloride tablets, metformin hydrochloride enteric-coated tablets, metformin hydrochloride capsules, compound tablets or capsules containing metformin hydrochloride, content (ng / g) = C*10 / W 称样量 *W 平均片重 / W 标示量 ;

[0022] c. When the sample to be tested is metformin hydrochloride sustained-release tablets or a compound sustained-release preparation containing metformin hydrochloride, content (ng / g) = C*20 / W 称样量 *W 平均片重 / W 标示量 .

[0023] Furthermore, the chromatographic column in the one-dimensional liquid phase condition is an ion exchange type or an ion exchange type composite filler chromatographic column.

[0024] Furthermore, in the one-dimensional liquid phase condition, the trapping column is a C18 or C18 composite filler chromatographic column, and the trapping time bandwidth is 0.2 to 0.5 min.

[0025] Furthermore, the capture time bandwidth is 0.2 to 0.4 min.

[0026] Beneficial effects:

[0027] The method for determining the content of N-nitrosodimethylamine in metformin by two-dimensional liquid chromatography-mass spectrometry described in this application adopts the above technical solution and has the following technical effects compared with the existing technology:

[0028] 1. This application uses two-dimensional liquid chromatography to separate interfering substances. The first dimension of liquid chromatography uses ion exchange chromatography to make N-nitrosodimethylamine (NDMA) elute before metformin. After N-nitrosodimethylamine (NDMA) elutes, the valve is cut to elute metformin, completely eliminating the interference caused by metformin separation and elution tailing residue on N-nitrosodimethylamine (NDMA) detection;

[0029] 2. This application avoids the problem of false positives and improves the accuracy of the test results by re-separating N,N-dimethylformamide (DMF) and impurity components that co-elute in the first dimension in the second dimension liquid phase by N-nitrosodimethylamine NDMA;

[0030] 3. This application uses two-dimensional liquid phase to prevent the main component and co-eluting excipients from entering the mass spectrometer, reducing mass spectrometer contamination, reducing maintenance frequency, and increasing service life;

[0031] 4. The analysis time of this application is short, and the analysis time for one sample is 16 minutes. This is because after N-nitrosodimethylamine (NDMA) in the first dimension is cut into the second dimension, N-nitrosodimethylamine (NDMA) separated in the second dimension and metformin in the first dimension are eluted simultaneously, without interfering with each other, thus saving analysis time.

[0032] 5. The detection method of this application has strong resolution ability and reference significance for false positive samples encountered in conventional liquid quality testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the mass spectrometry chromatogram of NDMA in Sample 1 in Comparative Example 1 of this application;

[0034] Figure 2 This is the mass spectrometry chromatogram of the 1 ng / mL NDMA reference solution in Comparative Example 1 of this application (SN=21);

[0035] Figure 3 Schematic diagram of a two-dimensional liquid chromatography-mass spectrometry system based on a trapping column; 1D represents a one-dimensional liquid phase system, and 2D represents a two-dimensional liquid phase system;

[0036] Figure 4 This is the mass spectrometry chromatogram of NDMA in Sample 1 in Example 1 of the present application;

[0037] Figure 5 This is the mass spectrometry chromatogram of the 1 ng / mL NDMA reference solution in Example 1 of the present application (SN=16);

[0038] Figure 6 This is the mass spectrometry chromatogram of NDMA in sample 2 in comparative example 2 of this application;

[0039] Figure 7 This is the mass spectrometry chromatogram of the 0.5 ng / mL NDMA reference solution in Comparative Example 2 of this application (SN=12);

[0040] Figure 8 This is the mass spectrometry chromatogram of NDMA in sample 2 in Example 2 of the present application;

[0041] Figure 9This is the mass spectrometry chromatogram of the 0.5 ng / mL NDMA reference solution in Example 2 of the present application (SN=10). DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with specific embodiments. The features and advantages of the present invention will become clearer as the description proceeds, but these exemplary embodiments are only used to illustrate the present invention and do not constitute any limitation to the scope of the present invention.

[0043] The methanol, acetonitrile, formic acid and ammonium acetate involved in this application are all chromatographically pure and purchased from Merck; the purified water is commercially available Watsons distilled water; the N-nitrosodimethylamine NDMA reference substance was purchased from the China National Institute for Food and Drug Control at 97.8%, batch number 510166-201902.

[0044] Comparative Example 1 (conventional detection method):

[0045] Chromatographic conditions: Instrument: Agilent liquid chromatography triple quadrupole mass spectrometer (Agilent 6470); chromatographic column: ACE EXCEL3C18-AR (150 mm × 4.6 mm, 3 μm); flow rate: 0.6 mL min -1 Column temperature: 40°C; Injection volume: 10 μL; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol, Gradient elution: 0.00 min (5% B), 6 min (5% B), 7 min (95% B), 10 min (95% B), 10.5 min (5% B), 14 min (stop);

[0046] Mass spectrometry conditions: APCI ionization mode, drying gas temperature: 325°C; drying gas flow rate: 5 L min -1 ; Nebulizer gas pressure: 60 psi; Evaporator temperature: 350°C; Capillary voltage: 1500 V; Corona current: 6 μA; Scan mode: positive ion mode; Monitoring mode: Multiple reaction ion monitoring (MRM); NDMA: m / z 75.1→43.1 (quantitative ion), collision voltage: 12 V; m / z 75.1→58.0 (qualitative ion), collision voltage: 15 V; Fragmentation voltage: 90 V; Collision cell acceleration voltage: 3 V.

[0047] Preparation of standard curve solutions of N-nitrosodimethylamine reference substance: Weigh an appropriate amount of N-nitrosodimethylamine (NDMA) reference substance and dilute it with water to NDMA concentrations of 1, 2, 5, 8, 10, and 20 ng·mL, respectively. -1 , shake well.

[0048] Detection of N-nitrosodimethylamine content in metformin hydrochloride tablets: Take 2 metformin hydrochloride tablets (specification: metformin hydrochloride 0.25g, sample 1), accurately weigh, place in a 50mL centrifuge tube, accurately add 10mL of water, vortex mix for 2min, and spin at 13000r·min -1 Centrifuge for 15 minutes and collect the supernatant as the test solution. The test solution and the NDMA standard curve solution were tested using the HPLC-MS method described above, and the NDMA content in Sample 1 was calculated using the standard curve method. The N-nitrosodimethylamine (NDMA) content in metformin hydrochloride tablets (specification: 0.25g metformin hydrochloride, Sample 1) was 0.044ppm.

[0049] Embodiment 1:

[0050] Chromatographic conditions: Instrument: Shimadzu two-dimensional liquid chromatography triple quadrupole mass spectrometer (LCMS8050); one-dimensional liquid chromatography conditions, chromatographic column: CAPCELLPAKCR1:4 (150 mm × 2.0 mm I.D., 5.0 μm); flow rate: 0.8 mL min -1 Column temperature was 40°C, injection volume was 20 μL; mobile phase: phase A was 20 mM ammonium acetate aqueous solution, phase B was acetonitrile; gradient elution: 0.00 min (5% B), 1.20 min (5% B), 1.21 min (90% B), 7.00 min (90% B), 8.10 min (5% B), 16.00 min (stop); trapping column: Shim-Pack Velox SPC18 (4.6 mm I.D. × 50 mm L., 2.7 μm); first-dimensional chromatography online trapping time: 0.8-1.06 min. Second-dimensional liquid chromatography conditions: chromatographic column: Shim-Pack GISTC18-AQ (4.6 mm I.D. × 150 mm L., 3.0 μm); flow rate: 1.0 mL min -1 ; Column temperature was 40°C; Mobile phase: Phase C was 0.1% formic acid aqueous solution, Phase D was 0.1% formic acid acetonitrile, Gradient elution: 0.00min (5% D), 7.00min (10% D), 10.00min (90% D), 10.10min (5% D), 16.00min (stop); Time for switching the two-dimensional post-column valve into the mass spectrometer: 3.5 to 7min.

[0051] Mass spectrometry conditions: APCI ionization mode; interface voltage 3.5 kV, interface temperature 300 °C; nebulizer gas flow rate 3 L min -1 ; Desolvation tube temperature 180℃; Drying gas flow rate 5.0L·min -1Scan mode: positive ion mode; Monitoring mode: multiple reaction monitoring (MRM): 75.10→43.10, collision voltage -19 V, Q1 deflection voltage -12 V, Q3 deflection voltage -15 V; 75.10→58.15, collision voltage -16 V, Q1 deflection voltage -25 V, Q3 deflection voltage -21 V;

[0052] Preparation of standard curve solutions of N-nitrosodimethylamine (NDMA) reference substance: Weigh an appropriate amount of N-nitrosodimethylamine (NDMA) reference substance and dilute it with water to NDMA concentrations of 1, 2, 5, 8, 10, and 20 ng·mL, respectively. -1 , shake well.

[0053] Detection of N-nitrosodimethylamine (NDMA) content in metformin hydrochloride tablets: Take 2 metformin hydrochloride tablets (specification: 0.25 g metformin hydrochloride, sample 1), accurately weigh, place in a 50 mL centrifuge tube, accurately add 10 mL of water, vortex mix for 2 minutes, and spin at 13000 r min. -1 Centrifuge for 15 minutes, and take the supernatant as the test solution. The test solution and NDMA standard curve solution were detected by the above-mentioned two-dimensional liquid chromatography-triple quadrupole tandem mass spectrometry, and the NDMA content in sample 1 was calculated using the standard curve method. The NDMA content in metformin hydrochloride tablets (specification: metformin hydrochloride 0.25g, sample 1) was not detected.

[0054] Compared with Comparative Example 1 (conventional method), when the sample to be tested contains DMF or other excipients, the present method can effectively avoid false positives, and the sensitivity is consistent with that of the conventional method.

[0055] Comparative Example 2 (conventional detection method):

[0056] Chromatographic conditions: Instrument: Agilent liquid chromatography triple quadrupole mass spectrometer (Agilent 6470); chromatographic column: ACE EXCEL3C18-AR (250 mm × 4.6 mm, 3 μm); flow rate: 0.6 mL min -1 ; Column temperature: 40℃; Injection volume: 15μL; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol, gradient elution: 0.00min (5% B), 10min (5% B), 18min (95% B), 20min (95% B), 20.1min (5% B), 25min (stop).

[0057] Mass spectrometry conditions: APCI ionization mode, drying gas temperature: 325°C; drying gas flow rate: 5 L min -1; Nebulizer gas pressure: 60 psi; Evaporator temperature: 350°C; Capillary voltage: 1500 V; Corona current: 6 μA; Scan mode: positive ion mode; Monitoring mode: Multiple reaction ion monitoring (MRM); NDMA: m / z 75.1→43.1 (quantitative ion), collision voltage: 12 V; m / z 75.1→58.0 (qualitative ion), collision voltage: 15 V; Fragmentation voltage: 90 V; Collision cell acceleration voltage: 3 V.

[0058] Preparation of standard curve solutions of N-nitrosodimethylamine reference substance: Weigh an appropriate amount of N-nitrosodimethylamine reference substance and dilute it with 50% methanol to NDMA concentrations of 0.5, 1, 2.5, 4, 5, and 10 ng·mL, respectively. -1 , shake well.

[0059] Detection of N-nitrosodimethylamine content in metformin hydrochloride sustained-release tablets: Take one metformin hydrochloride sustained-release tablet (specification: metformin hydrochloride 0.5g, sample 2), accurately weigh it, place it in a 50mL centrifuge tube, accurately add 10mL of methanol, vortex for 10min to dissolve it, and spin at 13000r·min -1 Centrifuge for 15 minutes, accurately remove 5 mL of the supernatant, place it in a 15 mL centrifuge tube, accurately add 5 mL of water, shake and mix, and rotate at 13000 r·min. -1 Centrifuge for 15 minutes, collect the supernatant, and obtain the test solution. The test solution and the NDMA standard curve solution were tested using the HPLC-MS method described above, and the NDMA content in Sample 2 was calculated using the standard curve method. The N-nitrosodimethylamine content in metformin hydrochloride sustained-release tablets (specification: metformin hydrochloride 0.5g, Sample 2) was 0.194ppm.

[0060] Example 2:

[0061] Chromatographic conditions: Instrument: Shimadzu two-dimensional liquid chromatography triple quadrupole mass spectrometer (LCMS8050); one-dimensional liquid chromatography conditions, chromatographic column: CAPCELLPAKCR1:4 (150 mm × 2.0 mm I.D., 5.0 μm); flow rate: 0.8 mL min -1Column temperature was 40°C, injection volume was 20 μL; mobile phase: phase A was 20 mM ammonium acetate aqueous solution, phase B was acetonitrile; gradient elution: 0.00 min (5% B), 1.20 min (5% B), 1.21 min (90% B), 7.00 min (90% B), 8.10 min (5% B), 16.00 min (stop); trapping column: Shim-Pack Velox SPC18 (4.6 mm I.D. × 50 mm L., 2.7 μm); first-dimensional chromatography online trapping time: 0.8-1.06 min. Second-dimensional liquid chromatography conditions: chromatographic column: Shim-Pack GISTC18-AQ (4.6 mm I.D. × 150 mm L., 3.0 μm); flow rate: 1.0 mL min -1 ; Column temperature was 40°C; Mobile phase: Phase C was 0.1% formic acid aqueous solution, Phase D was 0.1% formic acid acetonitrile, Gradient elution: 0.00min (5% D), 7.00min (10% D), 10.00min (90% D), 10.10min (5% D), 16.00min (stop); Time for switching the two-dimensional post-column valve into the mass spectrometer: 3.5 to 7min.

[0062] Mass spectrometry conditions: APCI ionization mode; interface voltage 3.5 kV, interface temperature 300 °C; nebulizer gas flow rate 3 L min -1 ; Desolvation tube temperature 180℃; Drying gas flow rate 5.0L·min -1 Scan mode: positive ion mode; Monitoring mode: multiple reaction monitoring (MRM): 75.10→43.10, collision voltage -19 V, Q1 deflection voltage -12 V, Q3 deflection voltage -15 V; 75.10→58.15, collision voltage -16 V, Q1 deflection voltage -25 V, Q3 deflection voltage -21 V;

[0063] Preparation of standard curve solutions of N-nitrosodimethylamine reference substance: Weigh an appropriate amount of N-nitrosodimethylamine (NDMA) reference substance and dilute it with 50% methanol to NDMA concentrations of 0.5, 1, 2.5, 4, 5, and 10 ng·mL, respectively. -1 , shake well.

[0064] Detection of N-nitrosodimethylamine content in metformin hydrochloride sustained-release tablets: Take one metformin hydrochloride sustained-release tablet (specification: metformin hydrochloride 0.5g, sample 2), accurately weigh it, place it in a 50mL centrifuge tube, accurately add 10mL of methanol, vortex for 10min to dissolve it, and spin at 13000r·min -1 Centrifuge for 15 minutes, accurately remove 5 mL of the supernatant, place it in a 15 mL centrifuge tube, accurately add 5 mL of water, shake and mix, and rotate at 13000 r·min. -1Centrifuge for 15 minutes, collect the supernatant, and obtain the test solution. The test solution and the NDMA standard curve solution were detected by the above-mentioned two-dimensional liquid chromatography-triple quadrupole tandem mass spectrometry, and the NDMA content in sample 2 was calculated using the standard curve method. The N-nitrosodimethylamine content in metformin hydrochloride sustained-release tablets (specification: metformin hydrochloride 0.5g, sample 2) was 0.170ppm.

[0065] Compared with Comparative Example 2 (conventional method), when the sample to be tested does not have interference from DMF or other excipients, the measurement results and sensitivity of this method are consistent with those of the conventional method, and the accuracy of this method is reliable.

[0066] Moreover, it can be seen from Comparative Example 2 and Example 2 that the detection method provided by the present invention has a sample analysis time of 16 minutes, which shortens the analysis time by nearly 10 minutes, effectively improving the detection efficiency and achieving high-throughput analysis. It can be seen from the above examples that the detection method provided by the present invention has a linear range and sensitivity that is basically consistent with conventional liquid chromatography-triple quadrupole tandem mass spectrometry. Figure 1 and Figure 4 , Figure 6 and Figure 8 It can be seen that the detection method provided by the present invention has a better separation effect, can effectively avoid the false positive caused by interference from N,N-dimethylformamide (DMF) or other auxiliary materials, and has more accurate detection results and better durability.

[0067] The above descriptions are only partial embodiments of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for determining the content of N-nitrosodimethylamine (NDMA) in metformin by two-dimensional liquid chromatography-mass spectrometry, characterized in that The following steps are involved: The first step is to obtain a test solution containing the sample to be tested: the sample to be tested that contains or may contain N-nitrosodimethylamine (NDMA) impurity is dissolved in a diluent to prepare a test solution; Step 2: The prepared test solution is transferred to a sample injection vial, and the test solution in the sample injection vial is tested by two-dimensional liquid chromatography-mass spectrometry to determine the content of N-nitrosodimethylamine (NDMA) in the sample; The chromatographic and mass spectrometric conditions of the two-dimensional liquid chromatography-mass spectrometry method are as follows: First-dimensional liquid phase conditions: chromatographic column: CAPCELL PAK CR 1:4, 150 mm × 2.0 mm ID, 5.0 μm; flow rate: 0.8 mL min -1 , column temperature was 40 °C, injection volume was 20 μL; mobile phase: phase A was 20 mM ammonium acetate aqueous solution, phase B was acetonitrile; gradient elution: 0.00 min 5% B, 1.20 min 5% B, 1.21 min 90% B, 7.00 min 90% B, 8.10 min 5% B, 16.00 min stop; trapping column: Shim-Pack Velox SP C18, 4.6 mm ID × 50 mm L., 2.7 μm; first-dimensional chromatography online trapping time: 0.8–1.06 min; 2D-LC conditions: Shim-Pack GIST C18-AQ column, 4.6 mm ID × 150 mm L., 3.0 μm; flow rate 1.0 mL min -1 Column temperature was 40 °C; mobile phase: C phase was 0.1% formic acid in water, D phase was 0.1% formic acid in acetonitrile, gradient elution: 0.00 min 5% D, 7.00 min 10% D, 10.00 min 90% D, 10.10 min 5% D, 16.00 min stop; time for switching the two-dimensional post-column valve to the mass spectrometer: 3.5 ~7 min; Mass spectrometry conditions: APCI ionization mode; interface voltage 3.5 KV, interface temperature 300 °C; nebulizer gas flow rate 3 L·min -1 ; Desolvation tube temperature 180 ℃; Drying gas flow rate 5.0 L·min -1 ; Scan mode: positive ion mode; Monitoring mode: multiple reaction monitoring MRM: 75.10→43.10, collision voltage -19 V, Q1 deflection voltage -12 V, Q3 deflection voltage -15 V; 75.10→58.15, collision voltage -16 V, Q1 deflection voltage -25 V, Q3 deflection voltage -21 V.

2. The method for determining the content of N-nitrosodimethylamine (NDMA) in metformin by two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that: The first step specifically includes the following steps: Step 1: When the sample to be tested is metformin hydrochloride raw material, accurately weigh 0.5 g of the sample to be tested, and the specific mass is recorded as W 称样量 , put it into a 50 mL centrifuge tube, accurately add 10 mL of purified water, shake for 10 min to dissolve, and filter to obtain the test solution; Step 2: When the sample to be tested is metformin hydrochloride tablets, metformin hydrochloride enteric-coated tablets, metformin hydrochloride capsules, compound tablets or capsules containing metformin hydrochloride, accurately weigh 10 tablets of the sample to be tested or 10 pellets of the sample to be tested, grind them into powder, and record the specific mass as W. 10 ;W 平均质量 =W 10 / 10; the weight of metformin contained in each tablet or each capsule is the labeled amount of the drug, recorded as W 标示量 ; Accurately weigh the sample powder equivalent to 0.5 g of metformin hydrochloride, and record the specific mass as W 称样量 , where W 称样量 =0.5 / W 标示量 ×W 平均质量 , placed in a 50 mL centrifuge tube, accurately added 10 mL of purified water, vortexed for 2 min to dissolve, and centrifuged at 13000 r·min -1 Centrifuge for 15 min and take the supernatant to obtain the test solution; Step 3: When the sample to be tested is metformin hydrochloride sustained-release tablets or a compound sustained-release preparation containing metformin hydrochloride, accurately weigh 5 tablets of the sample to be tested, grind them into powder, and record the specific mass as W5 and the average tablet weight as W 平均片重 , where W 平均片重 = W5 / 5; the weight of metformin contained in each tablet is the labeled amount of the drug, recorded as W 标示量 ; Weigh the sample powder equivalent to 0.5 g of metformin hydrochloride, and record the specific mass as W 称样量 , where W 称样量 =0.5 / W 标示量 ×W 平均片重 , placed in a 50 mL centrifuge tube, accurately added 10 mL of methanol, vortexed for 10 min to dissolve, and centrifuged at 13000 r·min -1 Centrifuge for 15 min, accurately remove 5 mL of the supernatant, place it in a 15 mL centrifuge tube, accurately add 5 mL of water, shake to mix, and rotate at 13000 r·min. -1 Centrifuge for 15 min and take the supernatant to obtain the test solution.

3. The method for determining the content of N-nitrosodimethylamine (NDMA) in metformin by two-dimensional liquid chromatography-mass spectrometry according to claim 2, characterized in that: The second step of testing the test solution in the injection vial specifically includes the following steps: injecting the test solution into a two-dimensional liquid chromatography-mass spectrometry instrument for separation and detection, recording the two-dimensional chromatogram of the test solution, calculating the concentration C of N-nitrosodimethylamine NDMA based on the standard curve of N-nitrosodimethylamine NDMA and the peak area of ​​the sample to be tested, and calculating the content of N-nitrosodimethylamine NDMA in the sample to be tested according to the calculation formula.

4. The method for determining the content of N-nitrosodimethylamine (NDMA) in metformin by two-dimensional liquid chromatography-mass spectrometry according to claim 3, characterized in that The calculation formula for the content of N-nitrosodimethylamine NDMA in the sample to be tested is as follows: a. When the sample to be tested is metformin hydrochloride API, content = C × 10 / W 称样量 , the unit of content is ng / g; b. When the sample to be tested is metformin hydrochloride tablets, metformin hydrochloride enteric-coated tablets, metformin hydrochloride capsules, or compound tablets or capsules containing metformin hydrochloride, the content is C × 10 / W. 称样量 ×W 平均质量 / W 标示量 , the unit of content is ng / g; c. When the sample to be tested is metformin hydrochloride sustained-release tablets or a compound sustained-release preparation containing metformin hydrochloride, content = C × 20 / W 称样量 ×W 平均片重 / W 标示量 , the unit of content is ng / g.

Images