CN111060641B - Method for detecting 9-nitrominocycline in tigecycline for injection - Google Patents
Method for detecting 9-nitrominocycline in tigecycline for injection Download PDFInfo
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- CN111060641B CN111060641B CN201911380648.5A CN201911380648A CN111060641B CN 111060641 B CN111060641 B CN 111060641B CN 201911380648 A CN201911380648 A CN 201911380648A CN 111060641 B CN111060641 B CN 111060641B
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- 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
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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
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- G01N2030/027—Liquid chromatography
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
Abstract
The invention provides a method for detecting 9-nitrominocycline in tigecycline for injection. The detection method comprises the following steps: step S1, obtaining a standard curve of the high performance liquid chromatography tandem mass spectrum of the 9-nitrominocycline, wherein the ordinate of the standard curve is the characteristic ion peak area of the 9-nitrominocycline, and the abscissa is the mass content of the 9-nitrominocycline; step S2, dissolving tigecycline for injection to form a tigecycline solution, and performing high performance liquid chromatography tandem mass spectrometry detection on the tigecycline solution to obtain a mass chromatogram; and step S3, determining a characteristic ion peak of the 9-nitrominocycline in the mass chromatogram according to the analysis result of the mass chromatogram, and calculating the mass content of the 9-nitrominocycline by taking the peak area of the characteristic ion peak as the basis of a standard curve. The detection method can obtain the accurate mass content of the 9-nitrominocycline, and provides a reliable data basis for effective quality control of the 9-nitrominocycline.
Description
Technical Field
The invention relates to the technical field of tigecycline quality control, in particular to a method for detecting 9-nitrominocycline in tigecycline for injection.
Background
Tigecycline (Tigecycline), developed by the american hui pharmaceutical company, is a novel broad-spectrum active antibiotic for intravenous injection, is also active against methicillin-resistant staphylococcus aureus, is the first drug of glycyltetracyclines, and has the following chemical structure:
known impurities that may be present in tigecycline, according to the tigecycline synthesis process, include epi-minocycline, 9-amino minocycline, 9-nitro minocycline, N-t-butylglycine, epimers, oxides, quinone analogs, tricyclics, and the like, where 9-nitro minocycline is a key intermediate for the production of tigecycline and is a genotoxic impurity. However, in the prior art, the impurities with high content are generally quantitatively analyzed, but the trace amount of 9-nitrominocycline is not quantitatively analyzed, so that the effective quality control of the amount of the 9-nitrominocycline cannot be realized.
Disclosure of Invention
The invention mainly aims to provide a method for detecting 9-nitrominocycline in tigecycline for injection, so as to solve the problem that trace 9-nitrominocycline in tigecycline for injection in the prior art cannot be effectively quantified.
In order to achieve the above object, according to an aspect of the present invention, there is provided a method for detecting 9-nitrominocycline in tigecycline for injection, comprising: step S1, obtaining a standard curve of the high performance liquid chromatography tandem mass spectrum of the 9-nitrominocycline, wherein the ordinate of the standard curve is the characteristic ion peak area of the 9-nitrominocycline, and the abscissa is the mass content of the 9-nitrominocycline; step S2, dissolving tigecycline for injection to form a tigecycline solution, and performing high performance liquid chromatography tandem mass spectrometry detection on the tigecycline solution to obtain a mass chromatogram; and step S3, determining a characteristic ion peak of the 9-nitrominocycline in the mass chromatogram according to the analysis result of the mass chromatogram, and calculating the mass content of the 9-nitrominocycline by taking the peak area of the characteristic ion peak as the basis of a standard curve.
Further, in the step S1, a standard curve is obtained by using solutions of 9-nitrominocycline with different concentrations as standard solutions, and preferably, the solvent for preparing the standard solutions is methanol.
Further, the mass content of the 9-nitrominocycline in the standard solution is 0.0-0.25 mug/mL.
Further, in the step S2, tigecycline for injection is diluted by a diluent to form a tigecycline solution, preferably, the diluent is a mixture of methanol and water, and preferably, the volume ratio of the methanol to the water is 40: 60-60: 40.
Further, the setting of the conditions for the high performance liquid chromatography detection includes: the chromatographic column is a C18 chromatographic column, the column temperature is 30-40 ℃, the flow rate is 0.3-0.8 ml/min, the mobile phase A is formic acid with the volume content of 0.05-0.2%, the mobile phase B is acetonitrile, and the gradient elution process comprises the following steps: eluting with 70-80% of mobile phase A and 20-30% of mobile phase B in 0-2 min, eluting with 1-3% of mobile phase A and 97-99% of mobile phase B in 2-8 min, and eluting with 70-80% of mobile phase A and 20-30% of mobile phase B in 8-15 min.
Further, the setting of the conditions for the high performance liquid chromatography detection includes: the chromatographic column is a Welch XB-C18 chromatographic column, the size of the chromatographic column is 4.6X 50mm and 5 mu m, the temperature of the chromatographic column is 35 ℃, the flow rate is 0.5ml/min, the mobile phase A is formic acid with the volume content of 0.1 percent, the mobile phase B is acetonitrile, and the gradient elution process is as follows: eluting with 75% of mobile phase A and 25% of mobile phase B in 0-2 min, eluting with 2% of mobile phase A and 98% of mobile phase B in 2-8 min, and eluting with 75% of mobile phase A and 25% of mobile phase B in 8-13 min.
Further, the setting of the conditions for mass spectrometry detection includes: the ion source is AJS ESI, the temperature of drying gas is 280-310 ℃, the flow rate of the drying gas is 7-9L/min, the pressure of atomization gas is 30-40 psi, the temperature of sheath flow gas is 330-360 ℃, the flow rate of the sheath flow gas is 10-12L/min, the end voltage of a capillary tube is 3000-4000V, the positive voltage of a nozzle is 450-550V, and the negative voltage of the nozzle is 800-1100V.
Further, the setting of the conditions for mass spectrometry detection includes: the ion source was AJS ESI, the drying gas temperature was 300 ℃, the drying gas flow rate was 8L/min, the atomization gas pressure was 35psi, the sheath flow gas temperature was 350 ℃, the sheath flow gas flow rate was 11L/min, the capillary end voltage was 3500V, the nozzle positive voltage was 500V, and the nozzle negative voltage was 1000V.
Further, the testing conditions of the hplc tandem mass spectrometry in step S1 and step S2 are the same.
By applying the technical scheme of the invention, when the tigecycline for injection is tested by connecting the high performance liquid chromatography and the mass spectrum in series, the high performance liquid chromatography is used for separating all substances in the tigecycline for injection, then the mass spectrum is used for further detecting the separators corresponding to all characteristic peaks to obtain a corresponding mass chromatogram, the mass chromatogram can be used for determining the 9-nitrominocycline and the characteristic ion peak of the 9-nitrominocycline in the corresponding chromatogram, and the mass content of the 9-nitrominocycline can be obtained according to the area of the characteristic ion peak and the standard curve established in the step S1. Therefore, on the basis of realizing the full separation of the trace 9-nitrominocycline by using the high performance liquid chromatography, the mass spectrum data is used for accurately corresponding to the characteristic ion peak of the 9-nitrominocycline in the mass chromatogram, so that the accurate mass content of the 9-nitrominocycline is obtained, and a reliable data basis is provided for the effective mass control of the 9-nitrominocycline.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows a standard graph according to embodiment 1 of the present invention;
FIG. 2 shows a mass chromatogram of a system suitability solution according to example 1 of the present invention;
fig. 3 shows a mass chromatogram of a sample solution according to example 1 of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
As analyzed by the background art of the present application, in the prior art, all impurities in tigecycline are generally quantitatively analyzed, but not 9-nitrominocycline, so that effective quality control of 9-nitrominocycline cannot be performed, and in order to solve the problem, the present application provides a method for detecting 9-nitrominocycline in tigecycline for injection, which comprises: step S1, obtaining a standard curve of the high performance liquid chromatography tandem mass spectrum of the 9-nitrominocycline, wherein the ordinate of the standard curve is the characteristic ion peak area of the 9-nitrominocycline, and the abscissa is the mass content of the 9-nitrominocycline; step S2, dissolving tigecycline for injection to form a tigecycline solution, and performing high performance liquid chromatography tandem mass spectrometry detection on the tigecycline solution to obtain a mass chromatogram; and step S3, determining a characteristic ion peak of the 9-nitrominocycline in the mass chromatogram according to the analysis result of the mass chromatogram, and calculating the mass content of the 9-nitrominocycline by taking the peak area of the characteristic ion peak as the basis of a standard curve.
When the tigecycline for injection is tested by connecting the high performance liquid chromatography and the mass spectrum in series, the high performance liquid chromatography separates various substances in the tigecycline for injection, then the mass spectrum further detects separators corresponding to various characteristic peaks to obtain corresponding mass chromatograms, the mass chromatograms can be used for determining the 9-nitrominocycline and the characteristic ion peaks of the 9-nitrominocycline in the corresponding chromatograms, and the mass content of the 9-nitrominocycline can be obtained according to the area of the characteristic ion peaks and the standard curve established in the step S1. Therefore, on the basis of realizing the full separation of the trace 9-nitrominocycline by using the high performance liquid chromatography, the mass spectrum data is used for accurately corresponding to the characteristic ion peak of the 9-nitrominocycline in the mass chromatogram, so that the accurate mass content of the 9-nitrominocycline is obtained, and a reliable data basis is provided for the effective mass control of the 9-nitrominocycline.
In the present application, reference may be made to a standard curve establishing process commonly used in liquid chromatography tests in the prior art, and in an embodiment, in step S1, solutions of 9-nitrominocycline with different concentrations are used as a standard solution to obtain the standard curve, and preferably, the solvent for preparing the standard solution is methanol.
Because the 9-nitrominocycline exists in the tigecycline for injection as an impurity and has a low content, in order to further ensure the accuracy of a test result, the mass content of the 9-nitrominocycline in the standard solution is preferably 0.05-0.25 mug/mL. The standard curve drawn by using the standard solution with the concentration as a test basis has better reliability.
In order to completely dissolve 9-nitrominocycline in the tigecycline for injection in a short time as much as possible, the tigecycline for injection is diluted by a diluent in the step S2 to form a tigecycline solution, the diluent is preferably a mixture of methanol and water, and the volume ratio of the methanol to the water is preferably 40: 60-60: 40.
When the high performance liquid chromatography detection is carried out on the tigecycline for injection, the detection conditions are deeply researched, especially on the premise of using different chromatographic columns, the composition of a mobile phase and the matching of a gradient elution program are experimentally researched, and the condition setting for finally determining the high performance liquid chromatography detection comprises the following steps: the chromatographic column is a C18 chromatographic column, the column temperature is 30-40 ℃, the flow rate is 0.3-0.8 ml/min, the mobile phase A is formic acid with the volume content of 0.05-0.2%, the mobile phase B is acetonitrile, and the gradient elution process comprises the following steps: eluting with 70-80% of mobile phase A and 20-30% of mobile phase B in 0-2 min, eluting with 1-3% of mobile phase A and 97-99% of mobile phase B in 2-8 min, and eluting with 70-80% of mobile phase A and 20-30% of mobile phase B in 8-15 min. Under the conditions, particularly by adopting the combination of a C18 chromatographic column and the mobile phase A and the mobile phase B, the 9-nitrominocycline is effectively separated from other impurities, and the quality determination of the 9-nitrominocycline is further realized. Meanwhile, the mobile phase A and the mobile phase B are stable in property, isomerization of tigecycline and 9-nitrominocycline cannot be caused, and a detection result is further more reliable.
Further, it is preferable that the setting of the conditions for the high performance liquid chromatography detection include: the chromatographic column is a Welch XB-C18 chromatographic column, the size of the chromatographic column is 4.6X 50mm and 5 mu m, the temperature of the chromatographic column is 35 ℃, the flow rate is 0.5ml/min, the mobile phase A is formic acid with the volume content of 0.1 percent, the mobile phase B is acetonitrile, and the gradient elution process is as follows: eluting with 75% of mobile phase A and 25% of mobile phase B in 0-2 min, eluting with 2% of mobile phase A and 98% of mobile phase B in 2-8 min, and eluting with 75% of mobile phase A and 25% of mobile phase B in 8-13 min.
In order to improve the accuracy of the mass spectrometry detection result, it is preferable that the setting of the mass spectrometry detection condition includes: the ion source is AJS ESI, the temperature of drying gas is 280-310 ℃, the flow rate of the drying gas is 7-9L/min, the pressure of atomization gas is 30-40 psi, the temperature of sheath flow gas is 330-360 ℃, the flow rate of the sheath flow gas is 10-12L/min, the end voltage of a capillary tube is 3000-4000V, the positive voltage of a nozzle is 450-550V, and the negative voltage of the nozzle is 800-1100V.
In a preferred embodiment, the setting of the mass spectrometry detection condition includes: the ion source was AJS ESI, the drying gas temperature was 300 ℃, the drying gas flow rate was 8L/min, the atomization gas pressure was 35psi, the sheath flow gas temperature was 350 ℃, the sheath flow gas flow rate was 11L/min, the capillary end voltage was 3500V, the nozzle positive voltage was 500V, and the nozzle negative voltage was 1000V.
Since the composition of the standard solution during the standard curve establishment process is relatively simple, the conditions for the hplc tandem mass spectrometry test are relatively wide, and in order to enable the standard curve to provide a more reliable calculation basis for the test of step S2, it is preferable that the test conditions for the hplc tandem mass spectrometry in steps S1 and S2 are the same.
The advantageous effects of the present application will be further described below with reference to examples and comparative examples.
The following examples and comparative examples employ the following instruments and reagents.
Instrument for measuring the position of a moving object
Agilent HPLC1260-MSQQQ6460
Reagent
Methanol, chromatographic grade;
formic acid, mass spectrum level;
acetonitrile, chromatographic grade;
and (5) purifying the water.
Example 1
Standard curve establishing process
The standard solution preparation process is as follows:
standard solution i.e. system adaptive solution: 14.1mg (equivalent to 12mg of 9-nitrominocycline) of a 9-nitrominocycline standard sample is gradually diluted by methanol and respectively prepared into single standard solutions with the concentrations of 0.05 mu g/mL and 0.25 mu g/mL, and the detection process of the high performance liquid chromatography tandem mass spectrometry of the standard solutions is as follows:
the conditions for the HPLC detection were set as follows: the chromatographic column is a Welch XB-C18 chromatographic column, the size of the chromatographic column is 4.6X 50mm and 5 mu m, the temperature of the chromatographic column is 35 ℃, the flow rate is 0.5ml/min, the mobile phase A is formic acid with the volume content of 0.1 percent, the mobile phase B is acetonitrile, and the gradient elution process is as follows: eluting with 75% of mobile phase A and 25% of mobile phase B in 0-2 min, eluting with 2% of mobile phase A and 98% of mobile phase B in 2-8 min, and eluting with 75% of mobile phase A and 25% of mobile phase B in 8-13 min.
The conditions for mass spectrometric detection were set as follows: the ion source was AJS ESI, the drying gas temperature was 300 ℃, the drying gas flow rate was 8L/min, the atomization gas pressure was 35psi, the sheath flow gas temperature was 350 ℃, the sheath flow gas flow rate was 11L/min, the capillary end voltage was 3500V, the nozzle positive voltage was 500V, and the nozzle negative voltage was 1000V.
The standard curve created is shown in figure 1.
The detection conditions are the same as those of the standard curve.
The recovery rate and precision are verified by an addition method.
Accuracy stock solution: weighing 14.1mg of 9-nitrominocycline reference substance (equivalent to 12mg of 9-nitrominocycline) and placing in a 50ml volumetric flask, adding methanol, ultrasonically dissolving for 5 minutes, diluting to scale with methanol, and uniformly mixing to obtain stock solution A; precisely transferring 1ml of the stock solution A into a 100ml volumetric flask, adding methanol to dilute to a scale, and uniformly mixing to obtain the accurate stock solution.
Sample solution at 100% accuracy: and transferring 2ml of the accuracy stock solution, adding 2 to 20ml of diluent, and uniformly mixing to obtain an accuracy stock solution 1. And (3) taking a tigecycline 1 bottle for injection, dissolving the tigecycline 1 bottle with the 20ml of accuracy stock solution 1, and uniformly mixing to obtain a 100% accuracy sample solution. Three parts are prepared in parallel.
Sample solution at 20% accuracy: and transferring 0.4ml of the accuracy stock solution, adding 2-20 ml of diluent, and uniformly mixing to obtain an accuracy stock solution 2. And (3) taking a tigecycline 1 bottle for injection, dissolving the tigecycline in the 20ml accuracy stock solution 2, and uniformly mixing to obtain a 20% accuracy sample solution. Three parts are prepared in parallel.
Approval criteria: the average recovery rate of each concentration of three samples is 70-130%. (refer to Chinese pharmacopoeia analysis method verification procedure)
The results are shown in Table 1 below.
TABLE 1
Detection limit and quantification limit verification
(LOQ) quantitative limiting solution: weighing 14.1mg of 9-nitrominocycline reference substance (equivalent to 12mg of 9-nitrominocycline) and placing in a 50ml volumetric flask, adding methanol, ultrasonically dissolving for 5 minutes, diluting with methanol to scale, and uniformly mixing to obtain stock solution A; precisely transferring 1ml of the stock solution A into a 100ml volumetric flask, adding methanol to dilute to a scale, and uniformly mixing to obtain a stock solution B; precisely transferring 1ml of stock solution B, adding diluent 1 to dilute to 10ml, and uniformly mixing to obtain the system adaptive solution. Precisely transferring 2ml of the adaptive solution of the system, diluting the adaptive solution of the system to 10ml by using a diluent 1, and uniformly mixing to obtain a quantitative limiting solution. The diluent 1 is a mixture of 0.1% by volume of ammonia water and acetonitrile in a volume ratio of 1: 1.
(LOD) detection Limit solution: precisely transferring 5ml of the quantitative limiting solution, diluting the quantitative limiting solution to 10ml by using a diluent 1, and uniformly mixing to obtain the detection limiting solution.
Approval criteria:
the signal-to-noise ratio of the 9-nitrominocycline in the 6-needle LOQ solution is more than or equal to 10. (refer to Chinese pharmacopoeia analysis method verification procedures) the RSD of the 9-nitrominocycline peak area in 6-needle LOQ solution is less than or equal to 15 percent. (setting according to empirical value)
The signal-to-noise ratio of the 9-nitrominocycline in the 3-needle LOD solution is more than or equal to 3. (refer to Chinese pharmacopoeia analysis method verification procedure)
The results are shown in Table 2.
TABLE 2
Determination process of 9-nitrominocycline in tigecycline for injection:
sample solution: dissolving tigecycline (specification 50mg) for injection in a bottle 1 by using a diluent 2, diluting to 20ml, and mixing uniformly. The diluent 2 is a mixed solution of methanol and water in a volume ratio of 1: 1.
Chromatographic analysis procedure: after the system is balanced, continuously feeding 6 needles of system adaptive solution, and calculating the RSD of the 9-nitrominocycline peak area to be less than or equal to 10 percent; and (3) taking a 20% limiting solution and injecting a sample into a 1-needle, and checking that the signal-to-noise ratio of the 9-nitrominocycline is more than or equal to 10. Each sample solution was injected into 1 needle. When the sample introduction is not more than 12 needles and the sequence is finished, respectively introducing a needle system adaptive solution, and comparing with the first 6 needle system adaptive solutions subjected to continuous sample introduction, the RSD of the 9-nitrominocycline peak area should be not more than 10%, obtaining a mass chromatogram of the system adaptive solution and the later sample solution, and respectively shown in fig. 2 and fig. 3.
And calculating the peak area of the characteristic ion peak of the tigecycline in the mass chromatogram by taking a standard curve as a basis to obtain the mass content of the 9-nitrominocycline of 4.36 ppm.
Example 2
The detection conditions are specifically as follows, unlike those in example 1.
The conditions for the HPLC detection were set as follows: the chromatographic column is a Welch XB-C18 chromatographic column, the size of the chromatographic column is 4.6X 50mm and 5 mu m, the temperature of the chromatographic column is 30 ℃, the flow rate is 0.3ml/min, the mobile phase A is formic acid with the volume content of 0.2 percent, the mobile phase B is acetonitrile, and the gradient elution process is as follows: eluting with 70% of mobile phase A and 30% of mobile phase B in 0-2 min, eluting with 1% of mobile phase A and 99% of mobile phase B in 2-8 min, and eluting with 70% of mobile phase A and 30% of mobile phase B in 8-13 min.
The conditions for mass spectrometric detection were set as follows: the ion source was AJS ESI, the drying gas temperature was 310 ℃, the drying gas flow rate was 9L/min, the atomization gas pressure was 30psi, the sheath flow gas temperature was 330 ℃, the sheath flow gas flow rate was 10L/min, the capillary end voltage was 3000V, the nozzle positive voltage was 450V, and the nozzle negative voltage was 1100V.
The linear regression equation corresponding to the established standard curve is as follows: y is 40879x +50.64 and the linear correlation coefficient is 0.9996.
Example 3
The detection conditions are specifically as follows, unlike those in example 1.
The conditions for the HPLC detection were set as follows: the chromatographic column is a Welch XB-C18 chromatographic column, the size of the chromatographic column is 4.6X 50mm and 5 mu m, the temperature of the chromatographic column is 40 ℃, the flow rate is 0.8ml/min, the mobile phase A is formic acid with the volume content of 0.05 percent, the mobile phase B is acetonitrile, and the gradient elution process is as follows: eluting with 80% of mobile phase A and 20% of mobile phase B in 0-2 min, eluting with 3% of mobile phase A and 97% of mobile phase B in 2-8 min, and eluting with 80% of mobile phase A and 20% of mobile phase B in 8-13 min.
The conditions for mass spectrometric detection were set as follows: the ion source is AJS ESI, the temperature of the drying gas is 280 ℃, the flow rate of the drying gas is 7L/min, the pressure of the atomizing gas is 40psi, the temperature of the sheath flow gas is 360 ℃, the flow rate of the sheath flow gas is 12L/min, the end voltage of the capillary tube is 4000V, the positive voltage of the nozzle is 550V, and the negative voltage of the nozzle is 800V.
The linear regression equation corresponding to the established standard curve is as follows: y is 40857x +51.93 and the linear correlation coefficient is 0.9995.
Example 4
Different from the composition of the diluent 2 used in the example 1, the volume ratio of methanol to water is 40:60, and the finally calculated mass content of the 9-nitrominocycline is 4.34 ppm.
Example 5
Different from the composition of the diluent 2 used in the example 1, the volume ratio of methanol to water is 60:40, and the finally calculated mass content of the 9-nitrominocycline is 4.36 ppm.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:
when the tigecycline for injection is tested by connecting the high performance liquid chromatography and the mass spectrum in series, the high performance liquid chromatography separates various substances in the tigecycline for injection, then the mass spectrum further detects separators corresponding to various characteristic peaks to obtain corresponding mass chromatograms, the mass chromatograms can be used for determining the 9-nitrominocycline and the characteristic ion peaks of the 9-nitrominocycline in the corresponding chromatograms, and the mass content of the 9-nitrominocycline can be obtained according to the area of the characteristic ion peaks and the standard curve established in the step S1. Therefore, on the basis of realizing the full separation of the trace 9-nitrominocycline by using the high performance liquid chromatography, the mass spectrum data is used for accurately corresponding to the characteristic ion peak of the 9-nitrominocycline in the mass chromatogram, so that the accurate mass content of the 9-nitrominocycline is obtained, and a reliable data basis is provided for the effective mass control of the 9-nitrominocycline.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for detecting 9-nitrominocycline in tigecycline for injection is characterized by comprising the following steps:
step S1, obtaining a standard curve of the high performance liquid chromatography tandem mass spectrum of the 9-nitrominocycline, wherein the ordinate of the standard curve is the characteristic ion peak area of the 9-nitrominocycline, and the abscissa is the mass content of the 9-nitrominocycline;
step S2, dissolving tigecycline for injection to form a tigecycline solution, and carrying out high performance liquid chromatography tandem mass spectrometry detection on the tigecycline solution to obtain a mass chromatogram;
step S3, determining the characteristic ion peak of the 9-nitrominocycline in the mass chromatogram according to the analysis result of the mass chromatogram, calculating the peak area of the characteristic ion peak by taking the standard curve as the basis to obtain the mass content of the 9-nitrominocycline,
the step S1 is to adopt solutions of 9-nitrominocycline with different concentrations as standard solutions to obtain the standard curve, the solvent for preparing the standard solutions is methanol, the mass content of the 9-nitrominocycline in the standard solutions is 0.05-0.25 mug/mL,
the condition setting of the high performance liquid chromatography detection comprises the following steps:
the chromatographic column is a C18 chromatographic column, the column temperature is 30-40 ℃, the flow rate is 0.3-0.8 ml/min, the mobile phase A is formic acid with the volume content of 0.05-0.2%, the mobile phase B is acetonitrile, and the gradient elution process comprises the following steps: eluting with 70-80% of mobile phase A and 20-30% of mobile phase B in 0-2 min, eluting with 1-3% of mobile phase A and 97-99% of mobile phase B in 2-8 min, and eluting with 70-80% of mobile phase A and 20-30% of mobile phase B in 8-15 min.
2. The assay of claim 1, wherein said step S2 comprises diluting said injectable tigecycline with a diluent to form said solution of tigecycline.
3. The detection method according to claim 2, wherein the diluent is a mixture of methanol and water.
4. The detection method according to claim 3, wherein the volume ratio of methanol to water in the diluent is 40:60 to 60: 40.
5. The detection method according to claim 1, wherein the condition setting of the high performance liquid chromatography detection comprises:
the chromatographic column is a Welch XB-C18 chromatographic column, the size of the chromatographic column is 4.6X 50mm and 5 mu m, the temperature of the chromatographic column is 35 ℃, the flow rate is 0.5ml/min, the mobile phase A is formic acid with the volume content of 0.1 percent, the mobile phase B is acetonitrile, and the gradient elution process is as follows: eluting with 75% of mobile phase A and 25% of mobile phase B in 0-2 min, eluting with 2% of mobile phase A and 98% of mobile phase B in 2-8 min, and eluting with 75% of mobile phase A and 25% of mobile phase B in 8-13 min.
6. The detection method according to claim 1, wherein the setting of the conditions for mass spectrometry detection comprises:
the ion source is AJS ESI, the temperature of drying gas is 280-310 ℃, the flow rate of the drying gas is 7-9L/min, the pressure of atomization gas is 30-40 psi, the temperature of sheath flow gas is 330-360 ℃, the flow rate of the sheath flow gas is 10-12L/min, the end voltage of a capillary tube is 3000-4000V, the positive voltage of a nozzle is 450-550V, and the negative voltage of the nozzle is 800-1100V.
7. The detection method according to claim 6, wherein the setting of the conditions for mass spectrometry detection comprises:
the ion source was AJS ESI, the drying gas temperature was 300 ℃, the drying gas flow rate was 8L/min, the atomization gas pressure was 35psi, the sheath flow gas temperature was 350 ℃, the sheath flow gas flow rate was 11L/min, the capillary end voltage was 3500V, the nozzle positive voltage was 500V, and the nozzle negative voltage was 1000V.
8. The detection method according to claim 1, wherein the test conditions of the HPLC tandem mass spectrometry in the step S1 and the step S2 are the same.
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