Separation and determination method of 1-amino-4-methylpiperazine in rifampicin raw materials

Abstract

The present invention belongs to the technical field of chemical analysis, and in particular to a separation and determination method of 1-amino-4-methylpiperazine in a rifampicin bulk drug. The method is: using carbamoyl bonded silica gel as a chromatographic column filler, using a mixed solution of ammonium acetate aqueous solution and acetonitrile as a mobile phase to separate the 1-amino-4-methylpiperazine in the rifampicin bulk drug by isocratic elution; then using a tandem quadrupole mass spectrometer as a detector, a chromatogram is obtained by detection to achieve qualitative identification of 1-amino-4-methylpiperazine, and finally using an external standard method to calculate the content of 1-amino-4-methylpiperazine in the rifampicin bulk drug by peak area. The present invention solves the baseline drift problem of 1-amino-4-methylpiperazine detection in rifampicin bulk drugs by selecting a chromatographic column and regulating the column temperature, and the detection limit concentration is as low as 0.3ng / ml. The present invention provides technical support for improving the drug safety and quality control of rifampicin.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a method for separating and determining 1-amino-4-methylpiperazine in a rifampicin raw material medicine. Background Art

[0002] Rifampicin (RIFAMPICIN / Rifampin) is a semi-synthetic broad-spectrum antibacterial drug of the rifamycin class. It has antibacterial activity against a variety of pathogenic microorganisms and is widely used in the treatment of tuberculosis. Rifampicin is used in combination with other anti-tuberculosis drugs for the initial and retreatment of various tuberculosis, including the treatment of tuberculous meningitis.

[0003] The existing synthesis scheme of rifampicin bulk drug produces impurity 1-amino-4-methylpiperazine during the preparation process. In order to improve the drug safety of rifampicin, it is necessary to detect and control the content of 1-amino-4-methylpiperazine in rifampicin bulk drug. However, in the determination of 1-amino-4-methylpiperazine, since 1-amino-4-methylpiperazine has a strong polarity and rifampicin is weak, on a common C18 chromatographic column, 1-amino-4-methylpiperazine peaks first, but rifampicin is extremely unstable and can be degraded to produce some extremely trace impurities, thereby causing the baseline to drift upward. Therefore, it is necessary to develop an effective determination method for 1-amino-4-methylpiperazine in rifampicin bulk drug to solve the problem of baseline drift in 1-amino-4-methylpiperazine in rifampicin bulk drug.

[0004] In the prior art, the invention patent with publication number CN114019062A discloses a method for detecting related substances in rifampicin. The patent uses LC / MS method to detect impurities N-methylpiperazine and 1-amino-4-methylpiperazine in rifampicin raw materials, including liquid chromatography using C18 chromatographic column, using formic acid aqueous solution with a volume concentration of 0.8‰-1.2‰ as mobile phase A, acetonitrile as mobile phase B, gradient elution; mass spectrometry using ESI ion source, positive ion detection, SIM mode. However, the detection limit and quantitative limit of this method are relatively high. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a method for separating 1-amino-4-methylpiperazine in rifampicin raw materials by LC-MS / MS. The present invention adopts a special Hilic-Amide chromatographic column and solves the baseline drift problem caused by 1-amino-4-methylpiperazine by lowering the column temperature of the chromatographic column.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for separating 1-amino-4-methylpiperazine in a rifampicin raw material by using an LC-MS / MS method, the method comprising: using carbamoyl bonded silica gel as a chromatographic column filler, and using a mixed solution of an ammonium acetate aqueous solution and acetonitrile as a mobile phase to separate 1-amino-4-methylpiperazine in the rifampicin raw material by isocratic elution; in the mobile phase, the volume ratio of the ammonium acetate aqueous solution to the acetonitrile is 25:75; and the column temperature is 8°C.

[0008] Furthermore, the concentration of the ammonium acetate aqueous solution is 10 mM, and the ammonium acetate aqueous solution contains 0.1% by volume of formic acid (ie, the volume ratio of formic acid to the ammonium acetate aqueous solution is 1:1000).

[0009] Furthermore, the specifications of the chromatographic column are 2.1 mm×100 mm, 5 μm.

[0010] Preferably, the chromatographic column is a Welch Ultimate Hilic-Amide chromatographic column.

[0011] Preferably, the flow rate is 0.8 ml / min and the injection volume is 10 μl.

[0012] Furthermore, the sample to be tested is prepared with 90% by volume acetonitrile solution as solvent.

[0013] Furthermore, the samples to be tested need to be freshly prepared before use.

[0014] The second object of the present invention is to provide a method for qualitatively identifying 1-amino-4-methylpiperazine in rifampicin raw materials, which can detect and identify 1-amino-4-methylpiperazine in rifampicin raw materials at an ultra-low concentration of 0.15 ng / ml.

[0015] To achieve the above object, the present invention adopts the following technical solutions:

[0016] A method for qualitatively identifying 1-amino-4-methylpiperazine in a rifampicin raw material drug, comprising separating 1-amino-4-methylpiperazine in the rifampicin raw material drug by the method described in the first objective; after separation, a tandem quadrupole mass spectrometer is used as a detector for detection to obtain a chromatogram.

[0017] Furthermore, the ion source of the tandem quadrupole mass spectrometer is an ESI ion source, and the detection method is multiple reaction detection in positive ion mode.

[0018] Preferably, the mass spectrometry parameters are: nebulizer gas (GS1): 55 psi, heating auxiliary gas (GS2): 55 psi, curtain gas: 20 psi, collision gas: 9 psi, spray voltage: 5500 V, ion source temperature: 550° C., ion source temperature: 550° C., dwell time: 50 msce; Resolution Q1: Unit, Resolution Q3: Unit.

[0019] Furthermore, a mass-to-charge ratio of 116.0→99.0 was used as the detection ion pair of 1-amino-4-methylpiperazine.

[0020] Furthermore, the compound with a retention time of 3.71±0.5 min was identified as 1-amino-4-methylpiperazine.

[0021] Based on the above retention time, 1-amino-4-methylpiperazine in rifampicin raw materials was qualitatively identified.

[0022] The third object of the present invention is to provide a method for determining the content of 1-amino-4-methylpiperazine in a rifampicin bulk drug, which can achieve the content determination of 1-amino-4-methylpiperazine in a rifampicin bulk drug at an ultra-low concentration of 0.3 ng / ml.

[0023] To achieve the above object, the present invention adopts the following technical solutions:

[0024] The method for determining the content of 1-amino-4-methylpiperazine in rifampicin raw materials comprises the following steps:

[0025] (1) Separation: Separating 1-amino-4-methylpiperazine from the rifampicin API by the aforementioned separation method;

[0026] (2) Detection: After separation, 1-amino-4-methylpiperazine in the rifampicin API is detected using the aforementioned identification method and a chromatogram is obtained;

[0027] (3) Content calculation: Based on the chromatogram obtained in step (2), the content of 1-amino-4-methylpiperazine was calculated by peak area using the external standard method.

[0028] Further, the concentration of the 1-amino-4-methylpiperazine is in the range of 0.3ng / ml-6ng / ml, Y=1.27e+006X-1.03e+005, R=0.9996; wherein Y is the Y axis, representing the peak area, X is the X axis, representing the concentration, and R represents the correlation coefficient.

[0029] Furthermore, the test sample concentration was 60 μg / ml, and the reference sample concentration was 4.8 ng / ml.

[0030] According to the chromatogram measured by the detector, the content of 1-amino-4-methylpiperazine in the rifampicin raw material is calculated by peak area according to the external standard method, specifically including the following:

[0031] 1. Take 600 mg of this product, weigh it accurately, put it in a 50 ml volumetric flask, add 45 ml of acetonitrile to dissolve, then add water to dilute to the scale, shake well, accurately measure an appropriate amount of the solution, add solvent to dilute to make a solution containing about 60 μg per 1 ml, shake well, and use it as the sample solution.

[0032] 2. Take an appropriate amount of 1-amino-4-methylpiperazine impurity reference substance, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 4.8 ng of impurities per 1 ml as the reference substance solution.

[0033] 3. Take the solution of step 1 and step 2, such as 10 μl, and inject it into a high performance liquid chromatography-mass spectrometer, record the chromatogram, and calculate the content of the impurity 1-amino-4-methylpiperazine in the rifampicin raw material by the peak area according to the external standard method.

[0034] The calculation formula is as follows:

[0035]

[0036] Where: M 对 --Weighing of reference substance, mg;

[0037] S 纯 --Purity of reference substance, %;

[0038] L 对 --Dilution multiple of reference solution;

[0039] A 对 --The main peak area of ​​the reference solution;

[0040] M 样 —Weight of test sample, mg;

[0041] L 样 —Dilution multiple of the test solution;

[0042] A 样 —The main peak area of ​​the test solution, mg.

[0043] The beneficial effects of the present invention are:

[0044] 1. The LC-MS / MS method for determining the content of 1-amino-4-methylpiperazine in rifampicin raw materials provided by the present invention has good precision and accuracy, can achieve stable detection of 1-amino-4-methylpiperazine in rifampicin raw materials, and the detection limit concentration of 1-amino-4-methylpiperazine can reach an extremely low concentration of 0.3ng / ml. The present invention is of great significance to the improvement of quality control and drug safety of rifampicin.

[0045] 2. The present invention uses a special Hilic-Amide chromatographic column to avoid the problem of the baseline drifting upward caused by the interfering substances introduced by rifampicin; at the same time, by lowering the column temperature of the chromatographic column, the problem of the baseline of the Hilic-Amide chromatographic column slightly drifting downward is solved, and the qualitative and quantitative detection of 1-amino-4-methylpiperazine in the rifampicin raw material is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the chromatogram of the C18 column with the baseline drifting upward;

[0047] Figure 2 This is the chromatogram of the Hilic-Amide column with the baseline drifting downward;

[0048] Figure 3 is a normal chromatogram;

[0049] Figure 4 is the MS1 pattern of 1-amino-4-methylpiperazine;

[0050] Figure 5 It is the MS2 pattern of 1-amino-4-methylpiperazine;

[0051] Figure 6 is the blank solution chromatogram;

[0052] Figure 7 Chromatography of reference solution;

[0053] Figure 8 is the chromatogram of the test solution;

[0054] Fig. 9 The chromatogram is 0.3ng / ml LOQ;

[0055] Fig.10 The chromatogram is 0.15 ng / ml LOD;

[0056] Fig.11 This is the chromatogram of 100% spiked test solution;

[0057] Fig.12 It is a linear relationship diagram of 1-amino-4-methylpiperazine concentration-peak area in the present invention. DETAILED DESCRIPTION

[0058] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0059] In the embodiments of the present invention, the solutions to be tested are all freshly prepared before use.

[0060] In the embodiment of the present invention, the solvent is an acetonitrile aqueous solution with a volume ratio of acetonitrile to water of 90:10.

[0061] In the embodiment of the present invention, the preparation method of the test solution is as follows: 600 mg of the product is accurately weighed, placed in a 50 ml volumetric flask, 45 ml of acetonitrile is added to dissolve, and then water is added to dilute to the scale, shake well, accurately measure an appropriate amount of the solution, add a solvent to dilute to make a solution containing about 60 μg per 1 ml, shake well, and use it as the test solution.

[0062] In the embodiment of the present invention, the preparation method of the reference solution and the sensitivity solution is as follows: take an appropriate amount of 1-amino-4-methylpiperazine impurity reference substance, dissolve it with a solvent and quantitatively dilute it to make a solution containing about 4.8 ng of impurities per 1 ml, as the reference solution, and then add a solvent to quantitatively dilute it to make a solution containing about 0.3 ng of impurities per 1 ml, as the sensitivity solution.

[0063] In the embodiments of the present invention, the determination was carried out strictly in accordance with the high performance liquid chromatography-mass spectrometry method (General Rules 0512 and General Rules 0431 of Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0064] Example 1

[0065] The content of 1-amino-4-methylpiperazine in rifampicin raw materials was determined by LC-MS / MS method, as shown in Table 1.

[0066] Table 1. Application plan

[0067]

[0068] The specific operation method is as follows: using carbamoyl bonded silica gel as filler (Welch Ultimate Hilic-Amide 2.1mm×100mm, 5μm); using 10mM ammonium acetate aqueous solution (containing 0.1% formic acid)-acetonitrile (25:75) as mobile phase, column temperature is 8°C, flow rate is 0.8ml per minute; using tandem quadrupole mass spectrometry as detector, electrospray ion source (ESI) positive ion mode multiple reaction detection (MRM), using mass-to-charge ratio (m / z) 116.0→99.0 as detection ion pair, and selecting appropriate mass spectrometry conditions to make the signal-to-noise ratio of impurity peak of sensitive solution greater than 10. Take 10μl of reference solution, inject it into high performance liquid chromatography-mass spectrometry, inject it 6 times continuously, the relative standard deviation of impurity peak area should not exceed 15.0%, take 10μl of test solution, inject it into high performance liquid chromatography-mass spectrometry, and record the chromatogram. Calculated by peak area using the external standard method, the content of 1-amino-4-methylpiperazine in the test solution shall not exceed 0.0008%.

[0069] result:

[0070] The MS1 and MS2 patterns of 1-amino-4-methylpiperazine are shown as follows: Figure 4 , Figure 5 As shown; the chromatograms of the reference solution and the test solution are shown Figure 7 , Figure 8 shown.

[0071] (1) The blank solvent and sample matrix do not interfere with the determination of 1-amino-4-methylpiperazine. The blank solution chromatogram is shown in Figure 6 .

[0072] (2) The injection repeatability of the reference solution was good. The results are shown in Table 2.

[0073] Table 2. Repeatability results of injection of reference solution

[0074]

[0075] (3) The quantitative limit test results are shown in Table 3 and Fig. 9 As shown, the quantification limit of 1-amino-4-methylpiperazine is about 0.3 ng / ml, which is equivalent to 5 ppm of impurities in rifampicin can be quantitatively detected, and the signal-to-noise ratio is greater than 10.

[0076] Table 3. Quantitation limit detection results

[0077]

[0078] (4) The detection limit results are shown in Table 4 and Fig.10As shown, the detection limit concentration of 1-amino-4-methylpiperazine is about 0.15 ng / ml, which is equivalent to 2.5 ppm of impurities in rifampicin can be detected, and the signal-to-noise ratio is greater than 3.

[0079] Table 4

[0080]

[0081] (5) Linear relationship: The linear relationship of 1-amino-4-methylpiperazine is good in the range of 0.3 ng / ml to 6 ng / ml (2.5 ppm to 100 ppm), and the correlation coefficient r is greater than 0.99. See Table 5 and Fig.12 .

[0082] Table 5

[0083]

[0084] (6) Precision

[0085] Take 600 mg of this product, prepare it fresh before use, add 45 ml of acetonitrile and place it in a 50 ml volumetric flask to dissolve the sample, dilute it to the scale with water, shake well, and use it as the test sample stock solution I. Accurately measure 0.5 ml of the solution, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, shake well, and use it as the test sample solution (prepare 6 copies in parallel).

[0086] Take an appropriate amount of 1-amino-4-methylpiperazine impurity reference substance, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 4.8 ng of impurities per 1 ml as the reference substance solution.

[0087] Accurately measure 10 μl of the test solution and the reference solution for injection, record the chromatogram, and the results are shown in Table 6.

[0088] Table 6

[0089] Number of measurements Batch number: Y038-1811017 1 <0.0005％ 2 <0.0005％ 3 <0.0005％ 4 <0.0005％ 5 <0.0005％ 6 <0.0005％ Average value (n=6) — RSD (n=6) —

[0090] (7) Precision and accuracy of spiked

[0091] Reference substance stock solution I: Take 50.62 mg of impurity, weigh accurately, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0092] Reference substance stock solution II: Accurately measure 1.0 ml of reference substance stock solution I, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0093] Accuracy stock solution: Accurately measure 5 ml of reference stock solution II, place in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0094] Spiked solution: Accurately pipette 0.96 ml of accuracy stock solution and 0.5 ml of sample stock solution I into a 100 ml volumetric flask, add solvent to dilute to scale, shake well, and obtain (relative impurity content 0.008%, prepare 6 portions in parallel).

[0095] Take an appropriate amount of 1-amino-4-methylpiperazine impurity reference substance, dissolve it in the prepared solution and quantitatively dilute it to make a solution containing about 4.8 ng of impurities per 1 ml, which is used as the reference substance solution.

[0096] Accurately measure 10 μl of the test solution and the reference solution and inject them. Record the chromatogram. The results are shown in Table 7 and Fig.11 .

[0097] Table 7

[0098]

[0099] (8) Solution stability

[0100] The reference solution was placed at room temperature for about 0, 1.5, 3, 4, and 16 hours and injected, and the RSD of the peak area of ​​the sample at each time point was calculated. The test solution was placed at room temperature for about 0, 0.5, 1, 1.5, and 2 hours and injected, and the RSD of the peak area of ​​the sample at each time point was calculated. The results are shown in Table 8. The reference solution was stable at room temperature for 16 hours, and the test solution was unstable, so the test solution needed to be prepared fresh before use.

[0101] Table 8

[0102]

[0103] (9) Selection of chromatographic column and column temperature

[0104] Since 1-amino-4-methylpiperazine has a stronger polarity and rifampicin has a weaker polarity, 1-amino-4-methylpiperazine will elute first on a common C18 column. However, rifampicin is extremely unstable and will degrade to produce some extremely trace impurities, causing the baseline to drift upward. Figure 1 Therefore, the present invention selects a special Hilic-Amide chromatographic column to make rifampicin elute first and 1-amino-4-methylpiperazine elute later, thus avoiding the upward drift of the baseline caused by the interfering substances introduced by rifampicin. However, it is found that the baseline has a slight downward drift, such as Figure 2 Therefore, the present invention has repeatedly found and confirmed that lowering the column temperature of the chromatographic column can solve the problem of the baseline drifting downward, such as Figure 3 shown.

Claims

1. A method for separating 1-amino-4-methylpiperazine from rifampicin raw materials by LC-MS / MS, characterized in that: The method comprises the following steps: using carbamoyl bonded silica gel as a chromatographic column filler, using a mixed solution of ammonium acetate aqueous solution and acetonitrile as a mobile phase to separate 1-amino-4-methylpiperazine in a rifampicin raw material drug through isocratic elution; in the mobile phase, the volume ratio of the ammonium acetate aqueous solution to the acetonitrile is 25:75; and the column temperature is 8°C.

2. The method according to claim 1, characterized in that The concentration of the ammonium acetate aqueous solution is 10 mM, and the ammonium acetate aqueous solution contains 0.1% by volume of formic acid.

3. The method according to claim 1, characterized in that: The specifications of the chromatographic column are 2.1 mm×100 mm, 5 μm.

4. The method according to claim 1, characterized in that: The sample to be tested was prepared with 90% by volume acetonitrile solution as solvent.

5. A method for qualitatively identifying 1-amino-4-methylpiperazine in rifampicin raw materials, characterized in that: The method according to any one of claims 1 to 4 is used to separate 1-amino-4-methylpiperazine in the rifampicin raw material; after separation, a tandem quadrupole mass spectrometer is used as a detector for detection to obtain a chromatogram.

6. The method according to claim 5, characterized in that The ion source of the tandem quadrupole mass spectrometer is an ESI ion source, and the detection method is multiple reaction detection in positive ion mode.

7. The method according to claim 5, characterized in that The mass-to-charge ratio of 116.0→99.0 was used as the detection ion pair for 1-amino-4-methylpiperazine.

8. The method according to claim 5, characterized in that The compound with a retention time of 3.71±0.5min was identified as 1-amino-4-methylpiperazine.

9. A method for determining the content of 1-amino-4-methylpiperazine in a rifampicin bulk drug, characterized in that: The following steps are involved: (1) separation: separating 1-amino-4-methylpiperazine from the rifampicin raw material by the method described in any one of claims 1 to 4; (2) Detection: After separation, the 1-amino-4-methylpiperazine in the rifampicin API is detected using the method described in any one of claims 5 to 8, and a chromatogram is obtained; (3) Content calculation: Based on the chromatogram obtained in step (2), the content of 1-amino-4-methylpiperazine was calculated by peak area using the external standard method.

10. The method according to claim 9, characterized in that The concentration of the 1-amino-4-methylpiperazine is in the range of 0.3ng / ml-6ng / ml, Y=1.27e+006X-1.03e+005, R=0.9996; wherein Y is the Y axis, representing the peak area, X is the X axis, representing the concentration, and R represents the correlation coefficient.

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