Method for detecting N-nitroso sertraline in sertraline hydrochloride by adopting liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for detecting N-nitroso sertraline in sertraline hydrochloride by liquid chromatography, which takes a phenyl column as a chromatographic column and a 10mM ammonium formate aqueous solution and methanol mixed solvent as a mobile phase, adopts an isocratic elution mode and adopts ESI (+) as an ion source. The method has the advantages of good specificity, stable separation effect and high sensitivity, and is suitable for determination of N-nitroso sertraline in sertraline hydrochloride.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting N-nitrososertaline in sertraline hydrochloride by liquid chromatography-mass spectrometry (LC-MS). Background Art

[0002] Sertraline hydrochloride is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class. By binding to the serotonin transporter, it can inhibit the reuptake of serotonin by neurons and enhance serotonin activity in the central nervous system, and can be used to treat major depressive disorder, social anxiety disorder and many other mental illnesses. N-nitrososertaline is a genotoxic impurity of the nitrosamine type that may be produced during the process of sertraline hydrochloride, and its content needs to be strictly controlled. After consulting the literature and materials, there is no determination method for N-nitrososertaline.

[0003]

[0004] In addition, according to the document issued by the FDA, the daily allowable intake of N-nitrososertaline is 100 ng / day, and the maximum daily dose of sertraline hydrochloride is 200 mg. Therefore, the limit is relatively low (0.5 ppm), and the sensitivity requirements for the method are relatively high. It is difficult to develop a method with satisfactory sensitivity. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting N-nitrososertaline in sertraline hydrochloride by liquid chromatography-mass spectrometry (LC-MS).

[0006] To achieve the purpose of the present invention, the following implementation schemes are provided.

[0007] In some embodiments, a method for detecting N-nitrososertaline in sertraline hydrochloride by liquid chromatography-mass spectrometry (LC-MS) according to the present invention includes: using a Waters XBridge C18 chromatographic column or its equivalent chromatographic column, with mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of 10 mM ammonium formate, and mobile phase B is methanol. The ion source is ESI(+). The characteristic is that the quantitative ion pair in the mass spectrometry parameters is 335>275 in the MRM mode or 335 in the SIM mode.

[0008] Preferably, for the method of the present invention described above, the chromatographic column is Waters XBridge C18, 150×4.6 mm, 3.5 μm.

[0009] Preferably, for the method of the present invention described above, the volume ratio of mobile phase A to mobile phase B is 25:75.

[0010] Preferably, in the method of the present invention described above, the column temperature is 28 - 32 °C, preferably 30 °C, the flow rate is 0.4 - 0.6 ml / min, preferably 0.5 ml / min, and the injection volume is 10 μl.

[0011] Furthermore, in the method of the present invention described above, the mass spectrometry parameters are as follows:

[0012] Furthermore, in the method of the present invention described above, it specifically includes the following process: 1) Reference solution: Weigh an appropriate amount of N-nitrososertaline reference substance accurately, quantitatively dilute it with a solvent to prepare a solution containing about 5 ng of N-nitrososertaline per 1 ml. 2) Test solution: Take about 5 tablets of sertraline hydrochloride tablets (50 mg), weigh them and grind them into fine powder. Accurately weigh about 320 mg of the fine powder (equivalent to 100 mg of the active ingredient), or directly accurately weigh 100 mg of sertraline hydrochloride raw material and place it in a 10 ml volumetric flask. Quantitatively add 10 ml of methanol and sonicate for 20 min, centrifuge at 8000 rpm for 5 min, and then filter through a 0.45 μm filter membrane. Take the subsequent filtrate to prepare a test solution containing about 10 mg of sertraline hydrochloride per 1 ml. 3) Inject the reference solution and the test solvent into the liquid chromatography - mass spectrometry instrument for detection, and record the chromatogram.

[0013] Preferably, in the method of the present invention described above, the quantification method is to calculate the content of N-nitrososertaline in the test solution by the external standard method of the reference substance.

[0014] In a specific embodiment, in some embodiments, a method for detecting N-nitrososertaline in sertraline hydrochloride by the LC-MS method of the present invention includes: The chromatographic column is Waters XBridge C18, 150×4.6 mm, 3.5 μm, mobile phase A is 10 mM ammonium formate aqueous solution, mobile phase B is methanol, the volume ratio of mobile phase A to mobile phase B is 25:75, the column temperature is 30 °C, the flow rate is 0.5 ml / min, the injection volume is 10 μl, isocratic elution is used, the ion source of the mass spectrometry is ESI(+), and the mass spectrometry parameters are as follows:

[0015] The beneficial effects of the method of the present invention: 1) Select a lower ion source voltage and temperature to maintain the structural integrity of the analyte, overcoming the problem of poor stability of N-nitrososertaline. 2) Use a general-purpose chromatographic column to complete the determination of N-nitrososertaline, and the method is highly accessible. 3) It has good specificity, stable separation effect, high sensitivity, and the sensitivity can reach 0.05 ppm, which is suitable for the determination of N-nitrososertaline in sertraline hydrochloride. Description of the Drawings

[0016] Figure 1 It is the chromatogram of the blank solvent in Example 1; Figure 2 It is the chromatogram of the reference solution in Example 1; Figure 3 It is the chromatogram of the test solution in Example 1; Figure 4 It is the chromatogram of the spiked test solution in Example 1; Figure 5 It is the chromatogram of the LOD (limit of detection) in Example 1; Figure 6 It is the chromatogram of the LOQ (limit of quantitation) in Example 1; Figure 7 It is the chromatogram of the test sample A20090801 / JX in Example 2; Figure 8 It is the chromatogram of the test sample A20090801 / JX-fc in Example 2; Figure 9 It is the chromatogram of the blank solvent under the SIM mode of 335 Da in Example 4; Figure 10 It is the chromatogram of the reference solution under the SIM mode of 335 Da in Example 4; Figure 11 It is the chromatogram of the test solution under the SIM mode of 335 Da in Example 4; Figure 12 It is the chromatogram of the blank solvent under the SIM mode of 275 Da in Example 4; Figure 13 It is the chromatogram of the reference solution under the SIM mode of 275 Da in Example 4; Figure 14 It is the chromatogram of the test solution under the SIM mode of 275 Da in Example 4; Figure 15 It is the chromatogram of the reference solution when detecting with mobile phase A being 0.1% formic acid aqueous solution in Example 5; Figure 16 It is the chromatogram of the test solution when detecting with mobile phase A being 0.1% formic acid aqueous solution in Example 5; Figure 17 It is the chromatogram of the reference solution when detecting with mobile phase A being 10 mM ammonium acetate aqueous solution in Example 5; Figure 18 It is the chromatogram of the test solution when detecting with mobile phase A being 10 mM ammonium acetate aqueous solution in Example 5; Figure 19 The chromatogram of the spiked test sample solution when mobile phase A is 10 mM ammonium acetate aqueous solution in Example 5. Detailed implementation manners

[0017] The following examples illustrate the technical solutions of the present invention in detail to help understand the spirit of the present invention, but do not limit the scope of the present invention in any way. Any simple modification or variation of the technical solutions of the present invention within the spirit of the present invention also belongs to the scope of the present invention. Example 1

[0018] Solution preparation: (1) Mobile phase A: 10 mM ammonium formate aqueous solution Precisely measure 500 ml of ultrapure water, add 315 mg of ammonium formate, mix well and sonicate for 15 min to obtain.

[0019] (2) Mobile phase B: Methanol.

[0020] (3) Solvent: Methanol.

[0021] (4) Reference solution: Weigh an appropriate amount of N-nitrososertaline reference substance accurately, quantitatively dilute it with the solvent to prepare a solution containing about 5 ng of N-nitrososertaline per ml.

[0022] (5) Test sample solution: Weigh about 5 tablets of sertraline hydrochloride tablets (50 mg), grind them finely, accurately weigh about 320 mg of the fine powder (equivalent to 100 mg of sertraline hydrochloride) and place it in a 10 ml volumetric flask, or directly accurately weigh 100 mg of sertraline hydrochloride raw material and place it in a 10 ml volumetric flask, quantitatively add 10 ml of methanol and sonicate for 20 min, centrifuge at 8000 rpm for 5 min, then filter through a 0.45 μm filter membrane, and take the subsequent filtrate to prepare a solution containing about 10 mg of sertraline hydrochloride per ml.

[0023] (6) Spiked stock solution: Weigh an appropriate amount of N-nitrososertaline reference substance accurately, quantitatively dilute it with the solvent to prepare a solution containing about 50 ng of N-nitrososertaline per ml.

[0024] (7) Spiked test sample solution: Weigh about 5 tablets of sertraline hydrochloride tablets, grind them finely, accurately weigh about 320 mg of the fine powder (equivalent to 100 mg of sertraline hydrochloride) and place it in a 10 ml volumetric flask, quantitatively add 9 ml of methanol and 1 ml of the spiked stock solution and sonicate for 20 min, centrifuge at 8000 rpm for 5 min, then filter through a 0.45 μm filter membrane, and take the subsequent filtrate to prepare a solution containing about 10 mg of sertraline hydrochloride and about 5 ng of N-nitrososertaline per ml.

[0025] The chromatographic conditions and mass spectrometry parameters are shown in Table 1 below.

[0026] Table 1 Chromatographic Conditions and Mass Spectrometry Parameters

[0027] Quantification method: The content of N-nitrososertaline in the test solution was calculated by the external standard method using the reference substance.

[0028] Inject the blank solvent (methanol), reference solution, test solution, and spiked test solution into the gas chromatography-mass spectrometry for detection, and record the chromatograms.

[0029] The typical chromatograms recorded are shown in Figures 1-6 . The results show that the above chromatographic conditions can achieve the detection of N-nitrososertaline in sertraline hydrochloride.

[0030] Example 2 Repeatability Experiment Using the chromatographic conditions and mass spectrometry conditions of Example 1, parallel detection of two test solutions of Example 1 was carried out, and the chromatograms were recorded. The results are shown in Figures 7-8 and Table 2. The results show that the detection method has good reproducibility.

[0031] Table 2 Samples and Detection Results

[0032] Example 3 Chromatographic Condition Screening 3.1 Flow Rate Screening The preparation of the reference solution and the test solution was the same as in Example 1.

[0033] Chromatographic conditions: The flow rate was 0.6 ml / min and 0.4 ml / min; the other chromatographic conditions were the same as in Example 1.

[0034] Inject the reference solution and the test solution into the gas chromatography-mass spectrometry for detection, and record the chromatograms.

[0035] The detection results are shown in Table 3. The results show that it is feasible to detect within the range of 0.4 - 0.6 ml / min of the flow rate.

[0036] Table 3 Detection Results at Different Flow Rates

[0037] 3.2 Column Temperature Screening The preparation of the reference solution and the test solution was the same as in Example 1.

[0038] Chromatographic conditions: The column temperature was 32 °C and 28 °C; the other chromatographic conditions were the same as in Example 1.

[0039] Inject the reference solution and the test solution into the gas chromatography-mass spectrometry for detection, and record the chromatograms.

[0040] The detection results are shown in Table 4. The results indicate that it is feasible to conduct the detection within the range of column temperature from 32 °C to 28 °C.

[0041] Table 4 Detection Results at Different Column Temperatures

[0042] Example 4 Screening of Mass Spectrometry Acquisition Parameters The blank solvent, reference substance solution, and test solution were prepared in the same manner as in Example 1.

[0043] Mass spectrometry parameters: The acquisition mode was SIM, the acquisition ion pairs were 335 Da or 275 Da, and the remaining chromatographic conditions were the same as in Example 1.

[0044] Inject the blank solvent, reference substance solution, and test solution into the chromatograph-mass spectrometer for detection, and record the chromatograms.

[0045] Typical chromatograms are shown in Figures 9-14 . The results show that under the SIM mode, detection can be achieved when the acquisition ion is 335 Da, but the sensitivity is slightly lower than that of the MRM mode. When the acquisition ion is 275 Da, no peaks appear, the baseline is abnormal, and the specificity is poor, so detection cannot be achieved.

[0046] Example 5 Screening of Mobile Phase (1)The reference substance solution and test solution were prepared in the same manner as in Example 1.

[0047] Chromatographic conditions: Mobile phase A was 0.1% formic acid aqueous solution; the remaining chromatographic conditions were the same as in Example 1.

[0048] Inject the reference substance solution and test solution into the chromatograph-mass spectrometer for detection, and record the chromatograms.

[0049] Typical chromatograms are shown in Figures 15-16 .

[0050] (2)The reference substance solution, test solution, and spiked test solution were prepared in the same manner as in Example 1.

[0051] Chromatographic conditions: Mobile phase A was 10 mM ammonium acetate aqueous solution; the remaining chromatographic conditions were the same as in Example 1.

[0052] Inject the reference substance solution, test solution, and spiked test solution into the chromatograph-mass spectrometer for detection, and record the chromatograms.

[0053] Typical chromatograms are shown in Figures 17-19 .

[0054] The results show that when mobile phase A was 0.1% formic acid aqueous solution, the sample peaks had severe tailing and effective detection could not be achieved; when mobile phase A was 10 mM ammonium acetate aqueous solution, the spiked recovery was abnormal, the method was not feasible, and effective recovery and detection could not be achieved.

Claims

1. A method for detecting N-nitrososertaline in sertraline hydrochloride by liquid chromatography-mass spectrometry. The chromatographic conditions of this method include: Using a Waters XBridge C18 chromatographic column or its equivalent chromatographic column; the mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of 10 mM ammonium formate, and mobile phase B is methanol; the ion source is ESI(+). It is characterized in that the quantitative ion pair in the mass spectrometry parameters is 335>275 in the MRM mode or 335 in the SIM mode.

2. The method according to claim 1, wherein the chromatographic column is Waters XBridge C18, 150×4.6 mm, 3.5 μm.

3. The method according to claim 1, wherein the volume ratio of mobile phase A to mobile phase B is 25:

75.

4. The method according to claim 1, wherein in the chromatographic conditions, the column temperature is 28-32°C, preferably 30°C.

5. The method according to claim 1, wherein in the chromatographic conditions, the flow rate is 0.4-0.6 ml / min, preferably 0.5 ml / min, and the injection volume is 10 μl.

6. The method according to claim 1, wherein the mass spectrometry parameters are as follows:

7. The method according to claim 1, further comprising the following steps: 1) Preparation of the reference solution: Take an appropriate amount of N-nitrososertaline reference substance, accurately weigh it, and quantitatively dilute it with a solvent to prepare a solution containing about 5 ng of N-nitrososertaline per 1 ml; 2) Preparation of the test solution: Take about 5 sertraline hydrochloride tablets (50 mg), weigh them and grind them into fine powder. Accurately weigh about 320 mg of the fine powder (equivalent to 100 mg of the active ingredient), or directly accurately weigh 100 mg of sertraline hydrochloride raw material and place it in a 10 ml volumetric flask. Quantitatively add 10 ml of methanol and sonicate for 20 min, centrifuge at 8000 rpm for 5 min, then filter through a 0.45 μm filter membrane, and take the subsequent filtrate to prepare a test solution containing about 10 mg of sertraline hydrochloride per 1 ml; 3) Inject the reference solution and the test solvent into the liquid chromatography-mass spectrometry instrument for detection, and record the chromatogram.

8. The method according to claim 1, wherein the quantitative method of this method is to calculate the content of N-nitrososertaline in the test solution by the external standard method of the reference substance.

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