Detection method of N-nitrosotrimetazidine

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting nitrosamine impurity N-nitrosotrimetazidine in trimetazidine. The invention provides the method for detecting the nitrosamine impurity N-nitroso trimetazidine in the trimetazidine, the detection method has excellent sensitivity, precision, durability and linear relation, and the accurate detection of the N-nitroso trimetazidine in the trimetazidine bulk drug can be met. The method comprises the following steps: detecting a test solution and a reference solution by adopting a liquid chromatography-mass spectrometry method; the gradient elution procedure of the liquid chromatography is as follows: 0.00-2.00 min, and the ratio of a mobile phase A to a mobile phase B is 1: 9; (2.50-3.00) min to (4.00-5.00) min, wherein the ratio of the mobile phase A to the mobile phase B is (3-4.5): (5.5-7); (5.50-6.00) min to (7.00-8.00) min, wherein the ratio of the mobile phase A to the mobile phase B is (4-5): (5-6); and the ratio of the mobile phase A to the mobile phase B is 1: 9.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting nitrosamine impurity N-nitrosotrimetazidine in trimetazidine. Background Art

[0002] Trimetazidine, molecular formula C 14 H 22 N2O3, a commonly used drug for treating heart diseases such as angina pectoris and acute myocardial infarction, has the effects of improving myocardial blood supply, protecting myocardial cells, and improving cardiac function. It has the advantages of significant efficacy, can be used in combination with other drugs, and has few adverse reactions. It is a safe and effective drug for treating coronary heart disease.

[0003] Following the 2018 outbreak of the carcinogenic nitrosamine impurity NDMA (nitrosodimethylamine) in sartans, regulatory agencies such as the FDA and the EMA have been emphasizing the importance of quality control for nitrosamine impurities during the production of both active pharmaceutical ingredients and finished pharmaceuticals. Initially, due to the impact of NDMA contamination in sartans and other drugs, the FDA focused primarily on the management of simple nitrosamine impurities. In a subsequent phase, the FDA and EMA shifted their focus to NDSRIs (nitrosamine drug substance-related impurities). These impurities, unlike simple nitrosamine impurities, refer to drug molecules that participate in the formation of nitrosamine impurities. These drugs typically contain secondary amines (including tertiary amines), which provide amino groups for nitrosamine formation. Compared to simple nitrosamine impurities such as dimethylnitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), the upper limits for these product-related nitrosated impurities are significantly lower.

[0004] In September 2019, EMA published "Information for Marketing Authorization Holders on Nitrosamines" and a Q&A document, assessing the risk of nitrosamine formation or presence during the production of human medicinal products. It provides guidance to marketing authorization holders on avoiding the presence of nitrosamine impurities and lists the acceptable intake established for N-nitrosamines. According to the EMA's February 20, 2025 update, Appendix 1: Acceptable intakes established for N-nitrosamines, the structural formula and AI value (400 ng / day) for N-nitrosotrimetazidine were published. The trimetazidine package insert indicates that its maximum daily intake is 60 mg. Using the limit calculation formula (limit = AI / maximum daily intake), the limit for N-nitrosotrimetazidine is 6.67 ppm.

[0005] The prior art does not yet involve a method for detecting the nitrosamine impurity N-nitrosotrimetazidine in trimetazidine. Therefore, developing a method that can detect N-nitrosotrimetazidine is of great significance for drug safety and quality control of trimetazidine.

[0006] Summary of the Invention

[0007] The present invention provides a method for detecting N-nitrosotrimetazidine, a nitrosamine impurity in trimetazidine. The detection method has excellent sensitivity, precision, durability and linearity, and can meet the requirements for accurate detection of N-nitrosotrimetazidine in trimetazidine raw materials.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A method for detecting N-nitrosotrimetazidine, the method comprising liquid chromatography-mass spectrometry, and at least the following steps:

[0010] Step 1: Prepare test solution and reference solution:

[0011] Preparation of reference solution: Take N-nitrosotrimetazidine sample and prepare reference solution with methanol: 8-12mM (preferably 10mM) ammonium formate aqueous solution = 1:9;

[0012] Preparation of test solution: Take trimetazidine sample and prepare test solution with methanol: 8-12mM (preferably 10mM) ammonium formate aqueous solution = 1:9;

[0013] Step 2: Detecting the test solution and the reference solution using liquid chromatography-mass spectrometry;

[0014] The chromatographic conditions of the liquid chromatography are:

[0015] A SunFire C18 column (4.6 × 50 mm × 5 um) was used, with methanol as mobile phase A and 8-12 mM (preferably 10 mM) ammonium formate aqueous solution as mobile phase B, for gradient elution; the flow rate was 0.35 mL / min to 0.45 mL / min, the column temperature was 20°C to 30°C, and the injection volume was 3 μL.

[0016] The gradient elution program is as follows:

[0017] 0.00~2.00min, mobile phase A:B=1:9;

[0018] (2.50-3.00)~(4.00-5.00)min, mobile phase A:B=3~4.5:5.5~7;

[0019] (5.50-6.00)~(7.00-8.00)min, mobile phase A:B=4~5:5~6;

[0020] 8.00~10.00min, mobile phase A:B=1:9.

[0021] The following gradient elution program is preferred:

[0022] 0.00~2.00min, mobile phase A:B=1:9;

[0023] (2.50-3.00)~5.00min, mobile phase A:B=4~4.5:5.5~6;

[0024] 5.50-8.00 min, mobile phase A:B=5:5;

[0025] 9.00-10.00 min, mobile phase A:B=1:9.

[0026] The following gradient elution program is preferred:

[0027] 0.00~2.00min, mobile phase A:B=1:9;

[0028] 2.50-5.00 min, mobile phase A:B=4:6;

[0029] 5.50-8.00 min, mobile phase A:B=5:5;

[0030] 9.00-10.00 min, mobile phase A:B=1:9;

[0031] or

[0032] 0.00~2.00min, mobile phase A:B=1:9;

[0033] 3.00-5.00 min, mobile phase A:B=4.5:5.5;

[0034] 5.50-8.00 min, mobile phase A:B=5:5;

[0035] 9.00-10.00 min, mobile phase A:B=1:9.

[0036] Furthermore, the present invention preferably uses the following chromatographic conditions:

[0037] A SunFire C18 column (4.6 × 50 mm × 5 um) was used, with methanol as mobile phase A and 10 mM ammonium formate aqueous solution (pH = 4) as mobile phase B, and gradient elution was performed; the flow rate was 0.40 mL / min, the column temperature was 25°C, and the injection volume was 3 μL.

[0038] Gradient elution significantly affects the separation and peak shape of the target. Different gradient elution sequences and mobile phase compositions can affect the separation and peak shape of the target. The gradient elution sequence and mobile phase composition of the present invention not only separate N-nitrosotrimetazidine from trimetazidine with excellent separation, but also allow for simultaneous detection, shortening analysis time and increasing efficiency.

[0039] The mass spectrometer used a triple quadrupole, an ESI ion source, positive ion detection, an SRM mode, an ion source temperature of 330° C., a spray voltage of 3500 V, a sheath gas of 30, and an auxiliary gas of 10.

[0040] The quantification ion for N-nitrosotrimetazidine is 166.1 m / z. This quantification ion enables a higher response value for N-nitrosotrimetazidine under the detection conditions, thereby improving detection accuracy.

[0041] The procedure of the mass spectrometer switching valve is as follows:

[0042] 0.00~3.0min, access; 3.0~5.5min, discharge; 5.5~10.0min, access.

[0043] The peak elution time of trimetazidine API is approximately 4.75 minutes, and the peak elution time of N-nitrosotrimetazidine API is approximately 7.38 minutes. By excluding the retention time of trimetazidine API and inserting the retention time of N-nitrosotrimetazidine, we can avoid instrument damage due to high API concentrations while enabling qualitative and quantitative analysis of N-nitrosotrimetazidine.

[0044] The detection method of N-nitrosotrimetazidine provided by the present invention has the following advantages:

[0045] The present invention provides a method for detecting N-nitrosotrimetazidine, which realizes quantitative and qualitative analysis of N-nitrosotrimetazidine in a trimetazidine raw material. The method has good specificity and a low detection limit. The detection limit of N-nitrosotrimetazidine is 10 ng / mL and the quantification limit is 15 ng / mL, which meets the detection requirements of N-nitrosotrimetazidine in a trimetazidine raw material. Moreover, the method has a good linear relationship and high repeatability. The correlation coefficient within the linear range of the standard curve is greater than 0.999. In addition, the detection method has the advantages of fast analysis time and small sample amount required, which can greatly improve sample analysis efficiency.

[0046] The present invention adopts liquid chromatography-mass spectrometry to detect N-nitrosotrimetazidine in trimetazidine, which has the advantages of simplicity, stability, high precision, good durability, etc., can quickly and accurately detect the impurity N-nitrosotrimetazidine in the trimetazidine raw material, and the entire operation process is reliable and controllable, suitable for practical application and promotion, and has broad application prospects.

[0047] The flow rate and column temperature of the present invention can enable N-nitrosotrimetazidine to produce a peak in a short time, thus reducing analysis time, and have excellent separation effect and a better peak shape.

[0048] In step 1 of the present invention, methanol: 8-12 mM ammonium formate aqueous solution = 1:9 is selected as the solvent, which not only ensures that substances other than N-nitrosotrimetazidine can be extracted as little as possible, but also can extract N-nitrosotrimetazidine in the trimetazidine raw material as much as possible, and has no interference with liquid chromatography and mass spectrometry detection, ensuring that the liquid chromatography-mass spectrometry detection results provided by the present invention are more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention.

[0050] Figure 1 This is the ion chromatogram of trimetazidine in method 1 of Example 1 of the present invention;

[0051] Figure 2 This is the ion flow diagram of N-nitrosotrimetazidine in method 1 of Example 1 of the present invention;

[0052] Figure 3 This is the ion chromatogram of trimetazidine in method 2 of Example 1 of the present invention;

[0053] Figure 4 This is the ion flow diagram of N-nitrosotrimetazidine in method 2 of Example 1 of the present invention;

[0054] Figure 5 This is the ion chromatogram of trimetazidine in method 3 of Example 1 of the present invention;

[0055] Figure 6 This is the ion flow diagram of N-nitrosotrimetazidine in method 3 of Example 1 of the present invention;

[0056] Figure 7 This is the ion chromatogram of trimetazidine in method 4 of Example 1 of the present invention;

[0057] Figure 8 This is the ion chromatogram of N-nitrosotrimetazidine in method 4 of Example 1 of the present invention;

[0058] Figure 9 This is the ion chromatogram of trimetazidine in method 5 of Example 1 of the present invention;

[0059] Figure 10 This is the ion chromatogram of N-nitrosotrimetazidine in method 5 of Example 1 of the present invention;

[0060] Figure 11 This is the ion chromatogram of trimetazidine in method 6 of Example 1 of the present invention;

[0061] Figure 12 This is the ion flow diagram of N-nitrosotrimetazidine in method 6 of Example 1 of the present invention;

[0062] Figure 13 This is the ion chromatogram of trimetazidine in method 7 of Example 1 of the present invention;

[0063] Figure 14 This is the ion chromatogram of N-nitrosotrimetazidine in method 7 of Example 1 of the present invention;

[0064] Figure 15 This is the ion chromatogram of trimetazidine in method 8 of Example 1 of the present invention;

[0065] Figure 16 This is the ion chromatogram of N-nitrosotrimetazidine in method 8 of Example 1 of the present invention;

[0066] Figure 17 This is the ion chromatogram of trimetazidine in method 9 of Example 1 of the present invention;

[0067] Figure 18 This is the ion chromatogram of N-nitrosotrimetazidine in method 9 of Example 1 of the present invention;

[0068] Figure 19 This is the ion flow diagram of N-nitrosotrimetazidine in the final detection method of Example 1 of the present invention;

[0069] Figure 20 This is an ion flow diagram of a blank solution for detecting N-nitrosotrimetazidine according to Example 2 of the present invention;

[0070] Figure 21 This is an ion flow diagram of the test solution for detecting N-nitrosotrimetazidine in Example 2 of the present invention;

[0071] Figure 22 This is an ion flow diagram of the reference solution for detecting N-nitrosotrimetazidine in Example 2 of the present invention;

[0072] Figure 23 This is an ion flow diagram of a 100% test sample spiked solution for detecting N-nitrosotrimetazidine in Example 2 of the present invention;

[0073] Figure 24This is the linear regression curve of N-nitrosotrimetazidine in Example 7 of the present invention. DETAILED DESCRIPTION

[0074] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0075] Example 1

[0076] The embodiments of the present invention provide various schemes for developing a method for detecting nitrosamines in trimetazidine, wherein the method comprises the following steps:

[0077] 1. Influence of liquid chromatography conditions on separation effect

[0078] Method 1: The chromatographic conditions of liquid chromatography are as follows: using a SunFire C18 column with a specification of 4.6×50mm×5um, methanol as mobile phase A, purified water as mobile phase B, mobile phase A:mobile phase B (60:40) isocratic elution, a flow rate of 0.40mL / min, a column temperature of 25℃, and an injection volume of 3μL.

[0079] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0080] Figure 1 This is the ion chromatogram of trimetazidine.

[0081] Figure 2 This is the ion chromatogram of N-nitrosotrimetazidine.

[0082] Method 2: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with specifications of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and purified water as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0083] 0.00~1.00min, mobile phase A:B=5:5;

[0084] 2.00-4.00 min, mobile phase A:B=6:4;

[0085] 6.00-8.00 min, mobile phase A:B=5:5.

[0086] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0087] Figure 3 This is the ion chromatogram of trimetazidine.

[0088] Figure 4 This is the ion chromatogram of N-nitrosotrimetazidine.

[0089] Method 3: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with dimensions of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and aqueous formic acid (pH = 2.8) as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0090] 0.00~2.00min, mobile phase A:B=1:9;

[0091] 3.00-4.00 min, mobile phase A:B=3:7;

[0092] 6.00-7.00 min, mobile phase A:B=4:6;

[0093] 8.00~10.00min, mobile phase A:B=1:9.

[0094] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0095] Figure 5 This is the ion chromatogram of trimetazidine.

[0096] Figure 6 This is the ion chromatogram of N-nitrosotrimetazidine.

[0097] Method 4: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with dimensions of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and aqueous formic acid (pH = 4.0) as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0098] 0.00~2.00min, mobile phase A:B=1:9;

[0099] 3.00-4.00 min, mobile phase A:B=3:7;

[0100] 6.00-7.00 min, mobile phase A:B=4:6;

[0101] 8.00~10.00min, mobile phase A:B=1:9.

[0102] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0103] Figure 7 This is the ion chromatogram of trimetazidine.

[0104] Figure 8 This is the ion chromatogram of N-nitrosotrimetazidine.

[0105] Method 5: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with dimensions of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and 10 mM ammonium formate aqueous solution (pH = 4.0) as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0106] 0.00~2.00min, mobile phase A:B=1:9;

[0107] 3.00-4.00 min, mobile phase A:B=3:7;

[0108] 6.00-7.00 min, mobile phase A:B=4:6;

[0109] 8.00~10.00min, mobile phase A:B=1:9.

[0110] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0111] Figure 9 This is the ion chromatogram of trimetazidine.

[0112] Figure 10 This is the ion chromatogram of N-nitrosotrimetazidine.

[0113] in conclusion:

[0114] According to the chromatographic conditions of method 1, trimetazidine can be completely eluted under this condition and the peak is the first to elute ( Figure 1 ), while N-nitrosotrimetazidine peaks after trimetazidine API ( Figure 2 Based on the peak positions of the two, a rough gradient elution program was set: 0.00-1.00 min, mobile phase A:B = 5:5; 2.00-4.00 min, mobile phase A:B = 6:4; 6.00-8.00 min, mobile phase A:B = 5:5. The proportion of water was increased, and the final gradient was also changed accordingly, adjusting to Method 2.

[0115] According to the chromatographic conditions of method 2, trimetazidine has an absorption peak at 1.19 min ( Figure 3 ), N-nitrosotrimetazidine has an absorption peak near 1.79min ( Figure 4 ), but the two could not be completely separated. Method 2 adjusted the gradient elution program, but still could not be separated, and the retention time was too short, so the pH of mobile phase B was adjusted, and the time program was adjusted to reduce the proportion of methanol in the initial mobile phase and extend the overall time. The 0.00-1.00 min, mobile phase A:B = 5:5 was adjusted to 0.00-2.00 min, mobile phase A:B = 1:9, 3.00-4.00 min, mobile phase A:B = 3:7; 6.00-7.00 min, mobile phase A:B = 4:6; 8.00-10.00 min, mobile phase A:B = 1:9, adjusted to Method 3.

[0116] According to the chromatographic conditions of method 3, trimetazidine has absorption peaks at 1.49min and 4.05min ( Figure 5 ), N-nitrosotrimetazidine has an absorption peak at 4.48min ( Figure 6 ), the two absorption peaks of trimetazidine affected the peak elution and separation of N-nitrosotrimetazidine, so the gradient program was not changed, and the pH value of the mobile phase was adjusted to method 4.

[0117] According to the chromatographic conditions of method 4, trimetazidine has an absorption peak at 4.62min ( Figure 7 ), N-nitrosotrimetazidine has an absorption peak at 6.61min ( Figure 8 ), the trimetazidine absorption peak showed a large tailing, which had a great impact on the peak elution and separation of N-nitrosotrimetazidine. Without changing the gradient program, ammonium formate was added to the aqueous mobile phase to try to reduce the tailing phenomenon, and the method was adjusted to 5.

[0118] According to the chromatographic conditions of method 5, trimetazidine has an absorption peak at 5.56 min ( Figure 7 ), N-nitrosotrimetazidine has an absorption peak at 7.75min ( Figure 8 ), the tailing of the trimetazidine absorption peak was alleviated, but the elution time of the two peaks was later, which can be used as the basic chromatographic conditions for subsequent research on the optimization of the gradient elution program.

[0119] 2. Effects of different liquid phase gradient programs on peak shape and separation in ion chromatograms

[0120] Method 6: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with dimensions of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and 10 mM ammonium formate aqueous solution (pH = 4.0) as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0121] 0.00~2.00min, mobile phase A:B=1:9;

[0122] 3.00-5.00 min, mobile phase A:B=3.5:6.5;

[0123] 6.00-8.00 min, mobile phase A:B=4.5:5.5;

[0124] 9.00~10.00min, mobile phase A:B=1:9.

[0125] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0126] Figure 11 This is the ion chromatogram of trimetazidine.

[0127] Figure 12 This is the ion chromatogram of N-nitrosotrimetazidine.

[0128] Method 7: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with dimensions of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and 10 mM ammonium formate aqueous solution (pH = 4.0) as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0129] 0.00~2.00min, mobile phase A:B=1:9;

[0130] 3.00-5.00 min, mobile phase A:B=4:6;

[0131] 5.50-8.00 min, mobile phase A:B=5:5;

[0132] 9.00~10.00min, mobile phase A:B=1:9.

[0133] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0134] Figure 13 This is the ion chromatogram of trimetazidine.

[0135] Figure 14 This is the ion chromatogram of N-nitrosotrimetazidine.

[0136] Method 8: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with dimensions of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and 10 mM ammonium formate aqueous solution (pH = 4.0) as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0137] 0.00~2.00min, mobile phase A:B=1:9;

[0138] 3.00-5.00 min, mobile phase A:B=4.5:5.5;

[0139] 5.50-8.00 min, mobile phase A:B=5:5;

[0140] 9.00~10.00min, mobile phase A:B=1:9.

[0141] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0142] Figure 15 This is the ion chromatogram of trimetazidine.

[0143] Figure 16This is the ion chromatogram of N-nitrosotrimetazidine.

[0144] Method 9: The chromatographic conditions for liquid chromatography were as follows: a SunFire C18 column with dimensions of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and 10 mM ammonium formate aqueous solution (pH = 4.0) as mobile phase B, gradient elution at a flow rate of 0.40 mL / min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0145] 0.00~2.00min, mobile phase A:B=1:9;

[0146] 2.50-5.00 min, mobile phase A:B=4:6;

[0147] 5.50-8.00 min, mobile phase A:B=5:5;

[0148] 9.00~10.00min, mobile phase A:B=1:9.

[0149] The mass spectrometer employed a triple quadrupole, an ESI ion source, positive ion detection, and SRM mode. The ion source temperature was 330°C, the spray voltage was 3500 V, the sheath gas was 30, and the auxiliary gas was 10. The quantification ion of N-nitrosotrimetazidine was 166.1 m / z, and the qualifier ion of trimetazidine was 135.1 m / z. All solutions were added.

[0150] Figure 17 This is the ion chromatogram of trimetazidine.

[0151] Figure 18 This is the ion chromatogram of N-nitrosotrimetazidine.

[0152] in conclusion:

[0153] Method 5 established the basic chromatographic conditions, revealing that the mass spectrometric peak shape of N-nitrosotrimetazidine was unattractive, with tailing affecting the separation from trimetazidine. Methods 6-8 employed several conditions to adjust the peak shape and separation. By varying the gradient elution time, tailing was minimized and the peak elution times of the two peaks were optimized to maximize separation. A comparison revealed that the conditions of Method 8 produced better peak shape and separation. A slight adjustment to the time in Method 8 resulted in Method 9, which achieved the best separation and good peak shape.

[0154] The optimal chromatographic conditions for liquid chromatography were finally determined to be:

[0155] Methanol was used as mobile phase A, 10 mM ammonium formate aqueous solution (pH = 4.0) was used as mobile phase B, gradient elution was performed, the flow rate was 0.40 mL / min, the column temperature was 25°C, and the injection volume was 3 μL. The gradient elution procedure was as follows:

[0156] 0.00~2.00min, mobile phase A:B=1:9;

[0157] 2.50-5.00 min, mobile phase A:B=4:6;

[0158] 5.50-8.00 min, mobile phase A:B=5:5;

[0159] 9.00~10.00min, mobile phase A:B=1:9.

[0160] Its ion chromatogram is as follows Figure 19 shown.

[0161] Under the above gradient conditions, a liquid chromatography flow rate of 0.35 mL / min to 0.45 mL / min, a column temperature of 20°C to 30°C, or C18 chromatographic columns of different specifications had no significant effect on the separation results.

[0162] Example 2 Detection of N-nitrosotrimetazidine

[0163] The detection method comprises the following steps:

[0164] Step 1: Prepare blank solution, reference solution and test solution:

[0165] The blank solution was mobile phase A:B = 1:9;

[0166] Preparation of reference substance stock solution: Take 0.0050 g of N-nitrosotrimetazidine sample, accurately weigh it, place it in a 5 mL volumetric flask, add mobile phase A:B = 1:9 to dissolve and dilute to the scale, shake well; then transfer 0.05 mL of the above solution to a 5 mL volumetric flask, add mobile phase A:B = 1:9 to dilute to the scale, shake well, and use it as reference substance stock solution a; then transfer 0.5 mL of the above reference substance stock solution a to a 5 mL volumetric flask, add mobile phase A:B = 1:9 to dilute to the scale, shake well, and use it as reference substance stock solution b;

[0167] Reference solution: Pipette 0.02 mL of the reference stock solution a into a 5 mL volumetric flask, add mobile phase A:B = 1:9 to dilute to the mark, shake well, and use as the reference solution;

[0168] Preparation of the test solution: Take 0.0300 g of trimetazidine sample, accurately weigh it, place it in a 5 mL volumetric flask, add mobile phase A:B = 1:9 to dissolve and dilute to the scale, shake well, and use it as the test solution;

[0169] 100% test sample spike solution: Take 0.0300 g of trimetazidine sample, accurately weigh it, place it in a 5 mL volumetric flask, accurately add 0.02 mL of reference substance stock solution a, then add mobile phase A:B = 1:9 to the scale, shake well, and use this as the 100% test sample spike solution;

[0170] Step 2: Detect N-nitrosotrimetazidine in trimetazidine by liquid chromatography-mass spectrometry on the blank solution, the test solution, the reference solution and the 100% test solution spiked with the sample, and record the chromatogram. Figure 20-23 As shown;

[0171] The chromatographic conditions of the above liquid chromatography are:

[0172] A SunFire C18 column with specifications of 4.6 × 50 mm × 5 μm was used, with methanol as mobile phase A and 10 mM ammonium formate aqueous solution (pH = 4) as mobile phase B. Gradient elution was performed at a flow rate of 0.4 mL min, a column temperature of 25°C, and an injection volume of 3 μL. The gradient elution procedure was as follows:

[0173] 0.00~2.00min, mobile phase A:B=1:9;

[0174] 2.50-5.00 min, mobile phase A:B=4:6;

[0175] 5.50-8.00 min, mobile phase A:B=5:5;

[0176] 9.00-10.00 min, mobile phase A:B=1:9.

[0177] The mass spectrometer used a triple quadrupole, an ESI ion source, positive ion detection, SRM mode, an ion source temperature of 330° C., a spray voltage of 3500 V, a sheath gas of 30, and an auxiliary gas of 10. The quantitative ion of N-nitrosotrimetazidine was 166.1 m / z.

[0178] The procedure for switching the valve is as follows:

[0179] 0.00~3.0min, access; 3.0~5.5min, discharge; 5.5~10.0min, access.

[0180] from Figure 22-23 It can be seen that the chromatographic peak of N-nitrosotrimetazidine reference substance appears at a retention time of about 7.38 min, and the chromatographic peak separation is good.

[0181] from Figure 20-23It can be seen that the blank solvent has no interference with the detection of N-nitrosotrimetazidine. N-nitrosotrimetazidine is also detected in the trimetazidine bulk drug, but it does not affect the quantification of N-nitrosotrimetazidine. The liquid chromatography-mass spectrometry method provided by the present invention has good specificity.

[0182] Example 3 Repeatability

[0183] Repeatability of N-nitrosotrimetazidine: Six 100% test sample spiked solutions were prepared in parallel from the same batch of samples. The samples were detected by liquid chromatography-mass spectrometry and the spectra were recorded. The specific conditions of liquid chromatography and mass spectrometry were as described in Example 2. The results are shown in Table 1.

[0184] The preparation method of the 100% test sample spiked solution is as follows: 0.0300 g of trimetazidine sample was accurately weighed and placed in a 5 mL volumetric flask. 0.02 mL of reference stock solution a was accurately added. Then, mobile phase A:B = 1:9 was added to the volume and the volume was adjusted to the mark. The solution was shaken to obtain the 100% test sample spiked solution.

[0185] Table 1 Repeatability results

[0186]

[0187] As can be seen from Table 1, the RSD value of the content of N-nitrosotrimetazidine in the 6 analytical repeatability solutions is 0.94%, which is less than 15.0%, indicating that the detection method provided by the present invention has good repeatability.

[0188] Example 4 Injection Precision

[0189] Precision solution injection: The reference solution of Example 2 is the impurity reference solution (100% concentration), and 6 injections were made consecutively. Detection was performed using liquid chromatography-mass spectrometry, and the spectra were recorded. The specific conditions for liquid chromatography and mass spectrometry were as described in Example 2, and the results are shown in Table 2.

[0190] Table 2 Injection precision results

[0191]

[0192] As can be seen from Table 2, the RSD of the peak area of ​​6 consecutive injections is ≤15%, and the injection time is less than 5%, indicating that the instrument has good injection precision.

[0193] Example 5 Intermediate Precision

[0194] Intermediate precision solution: Two parallel batches of six 100% test sample spiked solutions were prepared from the same sample batch at different times by different personnel. Liquid chromatography-mass spectrometry (LC-MS) was performed in the same laboratory using the same instrument. The specific conditions for liquid chromatography and mass spectrometry were as described in Example 2. The spectra were recorded, and the results are shown in Table 3.

[0195] The preparation method of the above-mentioned 100% test sample spiked solution is as follows: take 0.0300 g of trimetazidine sample, accurately weigh it, place it in a 5 mL volumetric flask, accurately add 0.02 mL of reference substance stock solution a, then add mobile phase A:B = 1:9 to the scale, shake well, and use it as the 100% test sample spiked solution;

[0196] Table 3 Intermediate precision results

[0197]

[0198] As can be seen from Table 3, the RSD of the content of each analyte in the two batches of 6 intermediate precision solutions is ≤15.0%, indicating that the intermediate precision of the detection method provided by the present invention is good.

[0199] Example 6 Limit of Quantitation and Limit of Detection

[0200] Quantitation limit solution: Pipette 0.15 mL of the reference substance stock solution b prepared in Example 2 into a 10 mL volumetric flask, dilute to volume with mobile phase A:B in a ratio of 1:9, and shake well to prepare the prepared solution. This solution was then transferred to the prepared solution as the quantitation limit solution. Liquid chromatography-mass spectrometry was used to analyze the quantitation limit solution six times, with the spectra recorded. The results are shown in Table 4 below. The specific conditions for liquid chromatography and mass spectrometry were as described in Example 2.

[0201] Table 4 Quantitation limit results

[0202]

[0203] Detection limit solution: Pipette 0.1 mL of the reference substance stock solution b prepared in Example 2 into a 10 mL volumetric flask, dilute to volume with mobile phase A:B in a ratio of 1:9, and shake well to prepare the prepared solution. This is the detection limit solution. This detection limit solution was analyzed by liquid chromatography-mass spectrometry with one injection. The spectra were recorded. The results are shown in Table 5 below. The specific conditions for liquid chromatography and mass spectrometry are as described in Example 2.

[0204] Table 5 Detection limit results

[0205]

[0206] The results showed that, in the method of the present invention, the detection limit of N-nitrosotrimetazidine was 10 ng / mL and the quantification limit was 15 ng / mL.

[0207] Example 7 Linear Relationship

[0208] Linearity Solutions: Accurately pipette different volumes of reference stock solution b as shown in Table 6 below into different volumes of containers, dilute to scale with mobile phase A:B in a ratio of 1:9, and shake well to obtain a series of linearity solutions of varying concentrations. The reference stock solutions were prepared using the same method as in Example 2.

[0209] Table 6 Linear solutions

[0210]

[0211] The linear solution prepared above was detected by liquid chromatography-mass spectrometry, and the spectrum was recorded. Each concentration was injected once, wherein the specific conditions of liquid chromatography and mass spectrometry were as described in Example 2, and the spectrum was recorded. The concentration of N-nitrosotrimetazidine (ppb) was used as the abscissa and the peak area was used as the ordinate to draw a standard curve, and the regression equation was calculated. The results are shown in Table 7, and the linear relationship diagram is shown in Figure 24 .

[0212] Table 7 Linearity results

[0213]

[0214] As can be seen from the results in Table 7, N-nitrosotrimetazidine has a good linear relationship in the concentration range of 15 ppb-60 ppb.

[0215] Example 8 Accuracy

[0216] Accuracy solutions: including blank spike solution, 50% spike solution, 100% spike solution and 125% spike solution.

[0217] Blank spike solution: Take 0.0300 g of the test sample, accurately weigh it, place it in a 5 mL volumetric flask, dilute it to the scale with mobile phase A:B = 1:9, shake well, and obtain the blank spike solution.

[0218] 50% spiked solution: Take 0.0300 g of the test sample, accurately weigh it, and place it in a 5 mL volumetric flask. Add 0.1 mL of the reference substance stock solution b prepared in Example 2, and dilute to the scale with mobile phase A:B = 1:9. Shake well to obtain 50%. Prepare three replicates in parallel.

[0219] 100% spiked solution: Take 0.0300 g of the test sample, accurately weigh it, and place it in a 5 mL volumetric flask. Add 0.2 mL of the reference substance stock solution a prepared in Example 2, and dilute to the scale with mobile phase A:B = 1:9. Shake well to obtain the 100% spiked solution. Prepare three replicates in parallel.

[0220] 150% spiked solution: Take 0.0300 g of the test sample, accurately weigh it, and place it in a 5 mL volumetric flask. Add 0.3 mL of the reference substance stock solution a prepared in Example 2, and dilute to the scale with mobile phase A:B = 1:9. Shake well to obtain the 150% spiked solution. Prepare three replicates in parallel.

[0221] The accuracy solution prepared above was tested by liquid chromatography-mass spectrometry, and the spectrum was recorded. The blank spiked solution was injected six times continuously, and the average value was taken as the background value for recovery calculation. The specific conditions of liquid chromatography and mass spectrometry are as described in Example 2. The recovery results are shown in Table 8, and the peak area background value of the blank spiked solution is shown in Table 8-1.

[0222] The recovery rate was calculated according to the following formula:

[0223] Recovery rate (%) = (measured value - background value) / theoretical value × 100%

[0224] Table 8 Accuracy results

[0225]

[0226] Table 8-1 Blank plus sample background value results

[0227]

[0228] As can be seen from Table 8, the single value range of the recovery rate of N-nitrosotrimetazidine is 91.21% to 104.13%, all between 70% and 125%, and the RSD is less than 10%, which shows that the detection method of nitrosamines in trimetazidine provided in this application has good accuracy.

[0229] Example 9 Solution Stability

[0230] The reference solution prepared in Example 2 was injected once at 0 h, 2 h, 5 h, 8 h, 12 h, and 24 h, respectively, and detected by liquid chromatography-mass spectrometry. The spectra were recorded. The specific conditions of liquid chromatography and mass spectrometry were as described in Example 2. The results are shown in Table 9 below.

[0231] Table 9 Solution stability results

[0232]

[0233] As can be seen from Table 9, the ratio of the peak area of ​​each analyte to the 0h area in the reference solution is between 1.01 and 1.07, which indicates that the reference solution provided in this application has good stability.

[0234] Example 10 Durability

[0235] Durability of N-nitrosotrimetazidine: Samples from the same batch were taken to prepare 100% test sample spiked solutions, which were detected by liquid chromatography-mass spectrometry and the spectra were recorded. The specific conditions of liquid chromatography and mass spectrometry were as described in Example 2, with minor adjustments to the liquid chromatography conditions:

[0236] The preparation method of the 100% test sample spiked solution is as follows: 0.0300 g of trimetazidine sample was accurately weighed and placed in a 5 mL volumetric flask. 0.02 mL of reference stock solution a was accurately added. Then, mobile phase A:B = 1:9 was added to the volume and the volume was adjusted to the mark. The solution was shaken to obtain the 100% test sample spiked solution.

[0237] Fine-tuning 1. Flow rate adjustment: The flow rate was 0.35 mL / min, and the other liquid chromatography conditions were the same as those in Example 2. The results are shown in Table 10.

[0238] Fine-tuning 2: Flow rate adjustment: The flow rate was 0.45 mL / min, and the other liquid chromatography conditions were the same as those in Example 2. The results are shown in Table 10.

[0239] Fine-tuning 3: Column temperature adjustment: The column temperature was 30° C., and the other liquid chromatography conditions were the same as those in Example 2. The results are shown in Table 10.

[0240] Fine-tuning 4: Column temperature adjustment: The column temperature was 20° C., and the other liquid chromatography conditions were the same as those in Example 2. The results are shown in Table 10.

[0241] Table 10 Solution durability results

[0242]

[0243]

[0244] The results of LC-MS detection in fine-tuning 1 to fine-tuning 4 were basically the same as the separation effect of Example 2, and the separation degree of the chromatographic peaks was good, which showed that the fine-tuning of the chromatographic conditions had no effect on the detection of nitrosamines in trimetazidine.

[0245] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting N-nitrosotrimetazidine, using liquid chromatography-mass spectrometry, characterized in that: The liquid phase conditions are as follows: A SunFire C18 column was used with methanol as mobile phase A and 8-12 mM ammonium formate aqueous solution as mobile phase B, with gradient elution; the flow rate was 0.35 mL / min to 0.45 mL / min, and the column temperature was 20°C to 30°C; The gradient elution program is as follows: 0.00~2.00min, mobile phase A:B=1:9; (2.50-3.00)~(4.00-5.00)min, mobile phase A:B=3~4.5:5.5~7; (5.50-6.00)~(7.00-8.00)min, mobile phase A:B=4~5:5~6; 8.00~10.00min, mobile phase A:B=1:

9.

2. The method for detecting N-nitrosotrimetazidine according to claim 1, wherein: The gradient elution program is as follows: 0.00~2.00min, mobile phase A:B=1:9; (2.50-3.00)~5.00min, mobile phase A:B=4~4.5:5.5~6; 5.50-8.00 min, mobile phase A:B=5:5; 9.00-10.00 min, mobile phase A:B=1:

9.

3. The method for detecting N-nitrosotrimetazidine according to claim 1, wherein: The gradient elution program is as follows: 0.00~2.00min, mobile phase A:B=1:9; 2.50-5.00 min, mobile phase A:B=4:6; 5.50-8.00 min, mobile phase A:B=5:5; 9.00-10.00 min, mobile phase A:B=1:

9.

4. The method for detecting N-nitrosotrimetazidine according to any one of claims 1 to 3, characterized in that: The gradient elution program is as follows: 0.00~2.00min, mobile phase A:B=1:9; 3.00-5.00 min, mobile phase A:B=4.5:5.5; 5.50-8.00 min, mobile phase A:B=5:5; 9.00-10.00 min, mobile phase A:B=1:

9.

5. The method for detecting N-nitrosotrimetazidine according to any one of claims 1 to 4, characterized in that: The mass spectrometer used a triple quadrupole, an ESI ion source, positive ion detection, an SRM mode, an ion source temperature of 330° C., a spray voltage of 3500 V, a sheath gas of 30, and an auxiliary gas of 10.

6. The method for detecting N-nitrosotrimetazidine according to claim 5, characterized in that: The mass spectrometer switching valve program is as follows: 0.00-3.0 min, access; 3.0-5.5 min, discharge; 5.5~10.0min, access.

7. The method for detecting N-nitrosotrimetazidine according to any one of claims 1 to 6, characterized in that: Methanol was used as mobile phase A, 10 mM ammonium formate aqueous solution was used as mobile phase B, gradient elution was performed; the flow rate was 0.40 mL / min, the column temperature was 25°C, and the injection volume was 3 μL.

8. The method for detecting N-nitrosotrimetazidine according to any one of claims 1 to 7, characterized in that: Before testing, N-nitrosotrimetazidine reference solution and trimetazidine test solution were prepared using methanol: 8-12 mM ammonium formate aqueous solution = 1:

9.

9. The method for detecting N-nitrosotrimetazidine according to claim 1, characterized in that: The specifications of the chromatographic column are 4.6×50mm×5um.

10. Use of the method for detecting N-nitrosotrimetazidine according to any one of claims 1 to 9 in detecting N-nitrosotrimetazidine in trimetazidine.

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