CN117092229A - Separation and determination method of 1-amino-4-methylpiperazine in rifampicin crude drug - Google Patents
Separation and determination method of 1-amino-4-methylpiperazine in rifampicin crude drug Download PDFInfo
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- CN117092229A CN117092229A CN202310509628.3A CN202310509628A CN117092229A CN 117092229 A CN117092229 A CN 117092229A CN 202310509628 A CN202310509628 A CN 202310509628A CN 117092229 A CN117092229 A CN 117092229A
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- methylpiperazine
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- RJWLLQWLBMJCFD-UHFFFAOYSA-N 4-methylpiperazin-1-amine Chemical compound CN1CCN(N)CC1 RJWLLQWLBMJCFD-UHFFFAOYSA-N 0.000 title claims abstract description 68
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 title claims abstract description 55
- 229960001225 rifampicin Drugs 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 36
- 239000003814 drug Substances 0.000 title claims abstract description 35
- 229940079593 drug Drugs 0.000 title claims abstract description 31
- 238000000926 separation method Methods 0.000 title claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 40
- 238000001514 detection method Methods 0.000 claims abstract description 24
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 11
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 11
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 11
- 239000007864 aqueous solution Substances 0.000 claims abstract description 9
- 238000001819 mass spectrum Methods 0.000 claims abstract description 7
- 238000010812 external standard method Methods 0.000 claims abstract description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 4
- 239000000741 silica gel Substances 0.000 claims abstract description 4
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 4
- 238000010829 isocratic elution Methods 0.000 claims abstract description 3
- 239000011259 mixed solution Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 36
- 150000002500 ions Chemical class 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 14
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 7
- 239000008186 active pharmaceutical agent Substances 0.000 claims description 6
- 229940088679 drug related substance Drugs 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000001575 tandem quadrupole mass spectrometry Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract description 2
- 230000001276 controlling effect Effects 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000012535 impurity Substances 0.000 description 20
- 239000000523 sample Substances 0.000 description 19
- 239000013558 reference substance Substances 0.000 description 14
- 239000012488 sample solution Substances 0.000 description 9
- 239000011550 stock solution Substances 0.000 description 9
- 239000012085 test solution Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000007865 diluting Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000012490 blank solution Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 201000008827 tuberculosis Diseases 0.000 description 2
- PVOAHINGSUIXLS-UHFFFAOYSA-N 1-Methylpiperazine Chemical compound CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 description 1
- 208000022971 Tuberculous meningitis Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940124976 antitubercular drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 208000001223 meningeal tuberculosis Diseases 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- HJYYPODYNSCCOU-ODRIEIDWSA-N rifamycin SV Chemical class OC1=C(C(O)=C2C)C3=C(O)C=C1NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@H](C)[C@@H](OC)\C=C\O[C@@]1(C)OC2=C3C1=O HJYYPODYNSCCOU-ODRIEIDWSA-N 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 239000000814 tuberculostatic agent Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The application belongs to the technical field of chemical analysis, and particularly relates to a separation and determination method of 1-amino-4-methylpiperazine in rifampicin bulk drug. The method comprises the following steps: separating 1-amino-4-methylpiperazine in rifampicin crude drug by isocratic elution with carbamoyl bonded silica gel as chromatographic column filler and mixed solution of ammonium acetate aqueous solution and acetonitrile as mobile phase; and then adopting a tandem quadrupole mass spectrum as a detector to detect and obtain a chromatogram so as to realize qualitative identification of the 1-amino-4-methylpiperazine, and finally adopting an external standard method to calculate the content of the 1-amino-4-methylpiperazine in the rifampicin crude drug through peak area. The application solves the problem of baseline drift of 1-amino-4 methylpiperazine detection in rifampicin crude drugs by selecting chromatographic columns and regulating and controlling column temperature, and the detection limit concentration is as low as 0.3ng/ml. The application provides technical support for improving the medication safety and quality control of rifampicin.
Description
Technical Field
The application belongs to the technical field of chemical analysis, and particularly relates to a separation and determination method of 1-amino-4-methylpiperazine in rifampicin bulk drug.
Background
Rifampin (RIFAMPICIN/Rifampin), a semi-synthetic broad-spectrum antibacterial agent of the rifamycin class. It has antibacterial activity against various pathogenic microorganisms, and can be widely used for treating tuberculosis. Rifampicin is used in combination with other antitubercular drugs for the primary and secondary treatment of various tuberculosis, including the treatment of tubercular meningitis.
The existing synthesis scheme of rifampicin raw material medicine can generate impurity 1-amino-4-methylpiperazine in the preparation process. In order to improve the medication safety of rifampicin, it is necessary to detect and control the content of 1-amino-4-methylpiperazine in the rifampicin raw material drug. However, in the measurement of 1-amino-4-methylpiperazine, since 1-amino-4-methylpiperazine has a strong polarity, rifampicin is weak, and on a common C18 chromatographic column, 1-amino-4-methylpiperazine comes out of a peak, but rifampicin is extremely unstable and can degrade to generate some extremely trace impurities, so that a base line is caused to drift upwards. Therefore, there is a need to develop an effective determination method for 1-amino-4-methylpiperazine in rifampicin drug substance, which solves the problem of baseline drift in 1-amino-4-methylpiperazine in rifampicin drug substance.
In the prior art, the application patent with publication number of CN114019062A discloses a detection method of related substances in rifampicin. The patent adopts an LC/MS method to detect impurities N-methylpiperazine and 1-amino-4-methylpiperazine in rifampicin bulk drug, comprises adopting a C18 chromatographic column in liquid chromatography, taking formic acid aqueous solution with volume concentration of 0.8 per mill-1.2 per mill as a mobile phase A, acetonitrile as a mobile phase B, and performing gradient elution; the mass spectrum adopts ESI ion source, positive ion detection and SIM mode. However, the detection limit and the quantification limit are relatively high.
Disclosure of Invention
Therefore, one of the purposes of the present application is to provide a method for separating 1-amino-4-methylpiperazine from rifampicin crude drug by LC-MS/MS method, which adopts a special hilc-Amide chromatographic column, and solves the problem of baseline drift caused by 1-amino-4-methylpiperazine by lowering the column temperature of the chromatographic column.
In order to achieve the above purpose, the present application adopts the following technical scheme:
the method for separating 1-amino-4-methylpiperazine in rifampicin bulk drug by utilizing an LC-MS/MS method comprises the following steps: separating 1-amino-4-methylpiperazine in rifampicin crude drug by isocratic elution with carbamoyl bonded silica gel as chromatographic column filler and mixed solution of ammonium acetate aqueous solution and acetonitrile as mobile phase; the volume ratio of the ammonium acetate aqueous solution to the acetonitrile in the mobile phase is 25:75; the column temperature was 8 ℃.
Further, the concentration of the ammonium acetate aqueous solution is 10mM, and the ammonium acetate aqueous solution contains formic acid with a volume percentage of 0.1% (namely, the volume ratio of the formic acid to the ammonium acetate aqueous solution is 1:1000).
Further, the specification of the column was 2.1mm×100mm,5 μm.
Preferably, the chromatographic column is a Welch Ultimate Hilic-Amide chromatographic column.
Preferably, the flow rate is 0.8ml/min and the sample volume is 10. Mu.l.
Further, an acetonitrile solution with the volume fraction of 90% is used as a solvent to prepare a sample to be tested.
Further, the sample to be measured needs to be prepared for temporary use.
The second purpose of the application is to provide a method for qualitatively identifying 1-amino-4-methylpiperazine in rifampicin bulk drug, which can realize detection and identification of 1-amino-4-methylpiperazine in rifampicin bulk drug at ultralow concentration of 0.15 ng/ml.
In order to achieve the above purpose, the present application adopts the following technical scheme:
the method for qualitatively identifying the 1-amino-4-methylpiperazine in the rifampicin bulk drug is characterized in that the method is used for separating the 1-amino-4-methylpiperazine in the rifampicin bulk drug; after separation, tandem quadrupole mass spectrometry is used as a detector for detection, and a chromatogram is obtained.
Further, the ion source of the tandem quadrupole mass spectrum is an ESI ion source, and the detection method is multi-reaction detection in a positive ion mode.
Preferably, the mass spectrum parameters are: atomizing gas (GS 1): 55psi, heating assist gas (GS 2): 55psi, door curtain gas: 20psi, collision gas: spray voltage 9 psi: 5500V, ion source temperature: 550 ℃, ion source temperature: 550 ℃, residence time: 50msce; resolution Q1: unit, resolution Q3: unit.
Further, a mass-to-charge ratio of 116.0.fwdarw.99.0 was employed as the detection ion pair for 1-amino-4-methylpiperazine.
Further, the retention time was judged to be 3.71.+ -. 0.5min as 1-amino-4 methylpiperazine.
And carrying out qualitative identification on the 1-amino-4-methylpiperazine in the rifampicin raw material drug according to the retention time.
The application further aims to provide a method for measuring the content of 1-amino-4-methylpiperazine in the rifampicin crude drug, which can realize the content measurement of 1-amino-4-methylpiperazine in the rifampicin crude drug at the ultralow concentration of 0.3ng/ml.
In order to achieve the above purpose, the present application adopts the following technical scheme:
the method for measuring the content of 1-amino-4-methylpiperazine in rifampicin raw material medicine comprises the following steps:
(1) Separating: isolating 1-amino-4-methylpiperazine in a rifampicin drug substance using the method of any one of claims 1-4;
(2) And (3) detection: after separation, detecting the 1-amino-4-methylpiperazine in the rifampicin crude drug by the method of any one of claims 5 to 8, and obtaining a chromatogram;
(3) And (3) content calculation: and (3) calculating the content of the 1-amino-4-methylpiperazine according to the chromatogram obtained in the step (2) and the peak area by an external standard method.
Further, the concentration of the 1-amino-4 methylpiperazine is in the range of 0.3ng/ml to 6ng/ml, y=1.27e+006x-1.03e+005, r= 0.9996; wherein Y is Y axis, representing peak area, X is X axis, representing concentration, R represents correlation coefficient.
Further, the concentration of the test sample was 60. Mu.g/ml, and the concentration of the control sample was 4.8ng/ml.
According to the chromatogram detected by the detector, calculating the content of 1-amino-4-methylpiperazine in the rifampicin bulk drug according to an external standard method by using the peak area, wherein the method specifically comprises the following steps:
1. 600mg of the product is precisely weighed, placed in a 50ml measuring flask, added with 45ml of acetonitrile for dissolution, then added with water for dilution to a scale, shaken uniformly, precisely measured and measured for proper amount of the solution, and added with solvent for dilution to prepare a solution containing about 60 mug in each 1ml, and shaken uniformly to be used as a sample solution.
2. Taking a proper amount of 1-amino-4-methylpiperazine impurity reference substance, dissolving the reference substance with a solvent, and quantitatively diluting to prepare a solution containing about 4.8ng of impurity in each 1ml serving as the reference substance solution.
3. Taking the solutions in the step 1 and the step 2, such as 10 mul, injecting into a high performance liquid chromatography-mass spectrometer, recording a chromatogram, and calculating the content of the impurity 1-amino-4-methylpiperazine in the rifampicin bulk drug according to an external standard method by using a peak area.
The calculation formula is as follows:
in which M is For a pair of Weighing reference substance, mg;
S pure water -control purity,%;
L for a pair of -dilution of the control solution;
A for a pair of -the main peak area of the control solution;
M sample -weighing the test sample, mg;
L sample -dilution factor of the test solution;
A sample The main peak area of the test solution, mg.
The application has the beneficial effects that:
1. the LC-MS/MS method for determining the content of the 1-amino-4-methylpiperazine in the rifampicin raw material medicine provided by the application has good precision and accuracy, can realize stable detection of the 1-amino-4-methylpiperazine in the rifampicin raw material medicine, and can reach extremely low concentration of 0.3ng/ml in the detection limit concentration of the 1-amino-4-methylpiperazine. The application has important significance for the quality control of rifampicin and the improvement of medication safety.
2. According to the application, a special Hilic-Amide chromatographic column is selected, so that the problem that a base line is upwardly deviated due to interference substances introduced by rifampicin is avoided; meanwhile, the problem that the base line of the Hilic-Amide chromatographic column slightly drifts downwards is solved by reducing the temperature of the chromatographic column, and the qualitative and quantitative detection of the 1-amino-4-methylpiperazine in the rifampicin crude drug is realized.
Drawings
FIG. 1 is a C18 column baseline upward drift chromatogram;
FIG. 2 is a Hilic-Amide chromatographic column baseline downward-drift chromatogram;
FIG. 3 is a normal chromatogram;
FIG. 4 is a MS1 diagram of 1-amino-4 methylpiperazine;
FIG. 5 is a diagram of 1-amino-4 methylpiperazine MS 2;
FIG. 6 is a blank solution chromatogram;
FIG. 7 is a control solution chromatogram;
FIG. 8 is a chromatogram of a test solution;
FIG. 9 is a graph of a LOQ chromatogram of 0.3 ng/ml;
FIG. 10 is a LOD chromatogram of 0.15 ng/ml;
FIG. 11 is a chromatogram of a 100% spiked test solution;
FIG. 12 is a graph showing the concentration of 1-amino-4 methylpiperazine in the present application plotted against the peak area.
Detailed Description
The technical scheme of the present application will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the application based on the embodiments of the present application.
In the embodiment of the application, the solution to be detected is prepared in a new way.
In the embodiment of the application, the volume ratio of acetonitrile to water is 90:10 in acetonitrile.
In the embodiment of the application, the preparation method of the sample solution comprises the following steps: 600mg of the product is precisely weighed, placed in a 50ml measuring flask, added with 45ml of acetonitrile for dissolution, then added with water for dilution to a scale, shaken uniformly, precisely measured and measured for proper amount of the solution, and added with solvent for dilution to prepare a solution containing about 60 mug in each 1ml, and shaken uniformly to be used as a sample solution.
In the embodiment of the application, the preparation methods of the reference substance solution and the sensitivity solution are as follows: taking a proper amount of 1-amino-4-methylpiperazine impurity reference substance, dissolving the reference substance with a solvent, quantitatively diluting the solution to prepare a solution containing about 4.8ng of impurity in each 1ml of the reference substance, and then adding the solvent to quantitatively dilute the solution to prepare a solution containing about 0.3ng of impurity in each 1ml of the reference substance, thereby preparing a sensitivity solution.
In the embodiment of the application, the measurement is strictly carried out according to high performance liquid chromatography-mass spectrometry (the four-part rule 0512 and rule 0431 of the 2020 edition of Chinese pharmacopoeia).
Example 1
The content of 1-amino-4-methylpiperazine in rifampicin crude drug was determined by LC-MS/MS method as shown in table 1.
TABLE 1 scheme of the application
The specific operation method is as follows: carbamoyl bonded silica gel as filler (Welch Ultimate Hilic-amide2.1 mm. Times.100 mm,5 μm); 10mM ammonium acetate in water (0.1% formic acid) -acetonitrile (25:75) as mobile phase, column temperature 8 ℃, flow rate 0.8ml per minute; a tandem quadrupole mass spectrum is adopted as a detector, a multi-reaction detection (MRM) is carried out in an electrospray ion source (ESI) positive ion mode, a mass-to-charge ratio (m/z) of 116.0-99.0 is adopted as a detection ion pair, and proper mass spectrum conditions are adopted, so that the signal-to-noise ratio of an impurity peak of a sensitivity solution is more than 10. Taking 10 μl of reference solution, injecting into high performance liquid chromatography-mass spectrometer, continuously sampling for 6 times, wherein the relative standard deviation of impurity peak area should not exceed 15.0%, taking 10 μl of sample solution, injecting into high performance liquid chromatography-mass spectrometer, and recording chromatogram. The content of 1-amino-4-methylpiperazine in the sample solution is less than 0.0008% calculated by the external standard method according to the peak area.
Results:
MS1 and MS2 of 1-amino-4 methylpiperazine are shown in FIG. 4 and FIG. 5 respectively; the chromatograms of the control solution and the test solution are shown in fig. 7 and 8, respectively.
(1) The blank solvent and sample matrix did not interfere with the determination of 1-amino-4 methylpiperazine, and the blank solution chromatogram is shown in fig. 6.
(2) The control solution was sampled with good reproducibility and the results are shown in table 2.
TABLE 2 comparative sample solution sample injection repeatability results Table
(3) The quantitative limit concentration of 1-amino-4 methylpiperazine was about 0.3ng/ml, and the impurity corresponding to rifampicin of 5ppm was quantitatively detected with a signal to noise ratio of more than 10, as shown in Table 3 and FIG. 9.
TABLE 3 quantitative limit detection results
(4) The detection limit results are shown in Table 4 and FIG. 10, and the detection limit concentration of 1-amino-4 methylpiperazine is about 0.15ng/ml, and impurities corresponding to 2.5ppm of rifampicin can be detected with a signal to noise ratio of greater than 3.
TABLE 4 Table 4
(5) Linear relationship: the 1-amino-4 methylpiperazine concentration was linearly related well in the range of 0.3ng/ml to 6ng/ml (2.5 ppm to 100 ppm), and the correlation coefficient r was more than 0.99, as shown in Table 5 and FIG. 12.
TABLE 5
(6) Precision of
600mg of the product is taken, newly prepared, 45ml of acetonitrile is added, the mixture is placed in a 50ml measuring flask to dissolve a sample, the sample is diluted to a scale by water and is uniformly shaken to serve as a sample stock solution I, 0.5ml of the solution is precisely measured, the sample stock solution I is placed in a 100ml measuring flask, the solution is diluted to the scale by adding a solvent and is uniformly shaken to serve as a sample solution (6 parts are prepared in parallel).
Taking a proper amount of 1-amino-4-methylpiperazine impurity reference substance, dissolving the reference substance with a solvent, and quantitatively diluting to prepare a solution containing about 4.8ng of impurity in each 1ml serving as the reference substance solution.
Sample injection was performed by precisely measuring 10. Mu.l of each of the sample solution and the control solution, and the chromatogram was recorded, and the results are shown in Table 6.
TABLE 6
| Number of measurements | Lot 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) | — |
(7) Accuracy and precision of marking
Control stock solution I: and (5) weighing 50.62mg of impurities, precisely weighing, placing into a 100ml measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain the product.
Control stock solution II: precisely measuring the reference stock solution I1.0ml, placing into a 100ml measuring flask, adding solvent to dilute to scale, and shaking to obtain the final product.
Accuracy stock solution: precisely measuring 5ml of reference stock solution II, placing into a 50ml measuring flask, diluting with solvent to scale, and shaking.
Adding a marking solution: and precisely removing 0.96ml of accurate stock solution and 0.5ml of sample stock solution I, placing into a 100ml measuring flask, adding solvent to dilute to scale, and shaking uniformly to obtain the final product. (relative impurity content 0.008%, 6 parts were prepared in parallel).
Taking a proper amount of 1-amino-4-methylpiperazine impurity reference substance, preparing, dissolving and quantitatively diluting to prepare a solution containing about 4.8ng of impurity in each 1ml serving as the reference substance solution.
Sample solutions were precisely measured at 10. Mu.l each, and chromatograms were recorded, and the results are shown in Table 7 and FIG. 11.
TABLE 7
(8) Solution stability
Taking control solution, placing at room temperature for about 0, 1.5, 3, 4 and 16 hours for sample injection, calculating the RSD of the peak area of the sample at each time point, placing at room temperature for about 0, 0.5, 1, 1.5 and 2 hours for sample injection, and calculating the RSD of the peak area of the sample at each time point. The results are shown in Table 8, and the control solution is stable at room temperature for 16 hours, the test solution is unstable, and the test solution needs to be freshly prepared.
TABLE 8
(9) Selection of chromatographic columns and column temperatures
Since 1-amino-4 methylpiperazine is more polar and rifampicin is weaker, 1-amino-4 methylpiperazine peaks first on a common C18 chromatographic column, but rifampicin is extremely unstable and can degrade to produce some extremely trace impurities, resulting in upward drift of the baseline, see fig. 1. The present application therefore selects a particular Hilic-Amide chromatographic column to peak rifampicin first and 1-amino-4 methylpiperazine later, avoiding upward drift of the baseline due to interference species introduced by rifampicin, but finding a slight downward drift of the baseline, as shown in FIG. 2. Thus, the present application has been repeatedly studied to find and confirm that lowering the column temperature of the chromatographic column can solve the problem of downward drift of the base line, as shown in fig. 3.
Claims (10)
1. The method for separating 1-amino-4-methylpiperazine in rifampicin bulk drug by utilizing an LC-MS/MS method is characterized by comprising the following steps: separating 1-amino-4-methylpiperazine in rifampicin crude drug by isocratic elution with carbamoyl bonded silica gel as chromatographic column filler and mixed solution of ammonium acetate aqueous solution and acetonitrile as mobile phase; the volume ratio of the ammonium acetate aqueous solution to the acetonitrile in the mobile phase is 25:75; the column temperature was 8 ℃.
2. The method of claim 1, wherein the concentration of the aqueous ammonium acetate solution is 10mM, and the aqueous ammonium acetate solution contains 0.1% formic acid by volume.
3. The method of claim 1, wherein the chromatographic column has a specification of 2.1mm x 100mm,5 μm.
4. The method according to claim 1, wherein the sample to be tested is prepared using an acetonitrile solution with a volume fraction of 90% as a solvent.
5. A method for qualitatively identifying 1-amino-4-methylpiperazine in a rifampicin drug substance, characterized in that the method according to any one of claims 1-4 is used for separating 1-amino-4-methylpiperazine in a rifampicin drug substance; after separation, tandem quadrupole mass spectrometry is used as a detector for detection, and a chromatogram is obtained.
6. The method of claim 5, wherein the ion source of the tandem quadrupole mass spectrum is an ESI ion source and the detection method is a multi-reaction detection in positive ion mode.
7. The method according to claim 5, wherein a mass-to-charge ratio of 116.0 to 99.0 is used as the detection ion pair of 1-amino-4-methylpiperazine.
8. The method of claim 5, wherein the determination of retention time of 3.71.+ -. 0.5min is 1-amino-4 methylpiperazine.
9. The method for measuring the content of the 1-amino-4-methylpiperazine in the rifampicin raw material drug is characterized by comprising the following steps:
(1) Separating: isolating 1-amino-4-methylpiperazine in a rifampicin drug substance using the method of any one of claims 1-4;
(2) And (3) detection: after separation, detecting the 1-amino-4-methylpiperazine in the rifampicin crude drug by the method of any one of claims 5 to 8, and obtaining a chromatogram;
(3) And (3) content calculation: and (3) calculating the content of the 1-amino-4-methylpiperazine according to the chromatogram obtained in the step (2) and the peak area by an external standard method.
10. The method of claim 9, wherein the concentration of 1-amino-4 methylpiperazine is in the range of 0.3ng/ml to 6ng/ml, Y = 1.27e+006x-1.03e+005, r = 0.9996; wherein Y is Y axis, representing peak area, X is X axis, representing concentration, R represents correlation coefficient.
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