CN117330687A - Detection for determining genotoxic impurities in metasartan potassium based on liquid chromatography-mass spectrometry technology - Google Patents
Detection for determining genotoxic impurities in metasartan potassium based on liquid chromatography-mass spectrometry technology Download PDFInfo
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- G—PHYSICS
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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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Abstract
The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method for simultaneously determining 8 genotoxic impurities in metasartan potassium based on a liquid chromatography-mass spectrometry technology, which comprises the following steps: the reverse phase chromatographic column using octyl silane bonded silica gel as filler, the mixed solution of acid and organic phase as mobile phase, adopts a gradient elution method, and uses a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer for detection. The method can effectively and accurately measure 8 genotoxic impurities in the losartan potassium, and provides a reliable quantitative method for quality research of the losartan potassium.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method for determining 8 genotoxic impurities in metasartan potassium based on a liquid chromatography-mass spectrometry technology.
Background
Melsartan potassium, a prodrug, is rapidly converted to the active ingredient azilsartan after oral absorption, which blocks the action of angiotensin II in various tissues by selectively blocking its binding to AT1 receptors. The structure is as follows:
melsartan potassium, a prodrug, is rapidly converted to the active ingredient azilsartan after oral absorption, which blocks the action of angiotensin II in various tissues by selectively blocking its binding to AT1 receptors. Angiotensin II is the main boosting substance of RAAS, and has the effects of contracting blood vessels, stimulating aldosterone synthesis and release, cardiac excitation, and reabsorption of sodium by kidneys. Losartan potassium blocks the vasoconstrictor and aldosterone secretion of angiotensin II by selectively blocking the binding of angiotensin II to AT1 receptors in many tissues such as vascular smooth muscle and adrenal glands. Thus, its effect is independent of the angiotensin II synthesis pathway. The affinity of the metartan potassium for the AT1 receptor is more than 10,000 times higher than that for the AT2 receptor.
The genotoxic impurities IN the metartan potassium mainly originate from a reaction reagent (DMAP) IN the synthesis process, impurities (SM 1-A, SM 1-B) introduced by reaction raw materials, impurities (IN 1-D, IN1-E, IN3-B, IN 3-C) introduced by intermediates and byproduct impurities (impurity C).
Table 1 list of genotoxic impurities in 8 of mesartan potassium
The impurity with the genotoxicity warning structure related to the metartan potassium is more, and the prior literature does not report on a method for simultaneously measuring the above 8 genotoxic impurities.
In order to ensure the quality of medicines and the safety and effectiveness of clinical medication, the genetic toxicity impurities in the medicines must be subjected to intensive quantitative research. Because of the greater toxicity of genotoxic impurities and lower limits, accurate quantitative determination is extremely important.
Disclosure of Invention
The invention aims to solve the problem that quantitative determination of genetic toxicity impurities in the losartan potassium is lacking in the prior art, and provides an analysis method which is simple and easy to operate and can quantitatively determine 8 genetic toxicity impurities in the losartan potassium.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a detection method for determining genotoxic impurities in mesartan potassium based on a liquid chromatography-mass spectrometry technology, which comprises the following steps: by using a liquid chromatography-mass spectrometry technology, adopting a reversed phase chromatographic column, taking a water phase as a mobile phase A, taking an organic phase as a mobile phase B, and setting liquid phase conditions and mass spectrum conditions, the detection method can quantitatively detect 8 genotoxic impurities in the potassium metasartan.
According to an embodiment of the present invention, the reverse phase chromatography column is selected from the group consisting of a chromatography column packed with octyl silane bonded silica gel (ZORBAX RX-C8, specification 250 mm. Times.4.6 mm,5 μm), a chromatography column packed with octadecyl silane bonded silica gel (Fei Men Titank C18, specification 50 mm. Times.2.1 mm,3 μm) and a chromatography column packed with octadecyl silane bonded silica gel (Fei Men Titank C18, specification 100 mm. Times.2.1 mm,3 μm).
Preferably, the chromatographic column is selected from a reversed phase chromatographic column packed with octylsilane bonded silica gel (ZORBAX RX-C8, specification 250mm 4.6mm,5 μm).
According to an embodiment of the invention, mobile phase a contains an acid selected from formic acid or acetic acid.
Preferably, mobile phase A is selected from formic acid-water, the volume ratio is selected from 1:1000.
According to an embodiment of the invention, mobile phase B is selected from acetonitrile or methanol.
Preferably, mobile phase B is selected from acetonitrile.
According to the embodiment of the invention, in the liquid chromatography condition disclosed by the invention, the flow rate is selected from 0.4-0.6 ml/min, the temperature of a column Wen Xuanzi-35 ℃ and the temperature of a sample introduction disc are selected from 2-8 ℃, and the sample introduction volume is selected from 5 mu L; gradient elution is selected from: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min.
Preferably, the flow rate is selected from 0.5ml/min, the column temperature is selected from 30 ℃, the sample tray temperature is selected from 6 ℃, and the sample volume is selected from 5 μl.
According to an embodiment of the invention, the mass spectrometry conditions disclosed in the invention are: detecting by using a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, performing electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃; the quantitative ion pairs, declustering voltage (DP), collision voltage (CE) of each impurity were:
the beneficial effects of the invention are as follows: the invention discloses a method for simultaneously and quantitatively determining 8 genotoxic impurities and the content thereof in the losartan potassium by adopting a liquid chromatography-mass spectrometry technology for the first time, and the method is accurate, high in sensitivity and strong in specificity, provides a reliable quantitative method for researching the genotoxic impurities in the losartan potassium, and has positive effects and practical application values.
Drawings
FIG. 1 is a diagram of blank solvents SM1-A, SM 1-B;
FIG. 2 is a diagram of blank solvents IN1-D, IN 1-E;
FIG. 3 is a diagram of blank solvent IN3-B, IN 3-C;
FIG. 4 is a diagram of a blank solvent DMAP, impurity C;
FIG. 5 is a diagram of control solution SM1-A, SM 1-B;
FIG. 6 is a diagram of control solutions IN1-D, IN 1-E;
FIG. 7 is a diagram of control solution IN3-B, IN 3-C;
FIG. 8 is a graph of DMAP and impurity C as control solutions;
FIG. 9 is a diagram of a sample labeling solution SM1-A, SM 1-B;
FIG. 10 is a diagram of a sample addition solution IN1-D, IN 1-E;
FIG. 11 is a diagram of a sample labeling solution IN3-B, IN 3-C;
FIG. 12 is a diagram of sample addition of a labeling solution DMAP, impurity C;
FIG. 13 is a linear diagram of impurity SM 1-A;
FIG. 14 is a linear diagram of impurity SM 1-B;
FIG. 15 is a linear plot of impurities IN 1-D;
FIG. 16 is a linear diagram of impurities IN 1-E;
FIG. 17 is a linear diagram of impurity IN 3-B;
FIG. 18 is a linear diagram of impurity IN 3-C;
FIG. 19 is a linear plot of impurity C;
FIG. 20 is a linear plot of impurity DMAP.
Detailed Description
The invention discloses a detection method for quantitatively determining 8 genotoxic impurities in mesartan potassium based on a liquid chromatography-mass spectrometry technology. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is specifically noted that all similar substitutions and modifications will be apparent to those skilled in the art, and are intended to be included in the present invention. While the detection method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and suitable modifications and combinations of the method applications described herein can be made to practice and use the techniques of the present invention without departing from the spirit or scope of the invention.
Example 1
(1) Sample preparation: taking 10mg of metaxartan potassium, placing into a 10ml measuring flask, adding 8ml of 0.1% formic acid (v/v) solution-acetonitrile (50:50), dissolving by ultrasound for 1min, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking uniformly to obtain the final product;
(2) Control solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. 1ml of the solution is precisely measured, placed in a 20ml measuring flask, diluted to a scale by adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), and shaken well. Precisely weighing 1ml, placing in a 100ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(3) Sensitivity solution: precisely measuring 1ml of the reference substance stock solution of (2), placing in a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking uniformly;
(4) Adding a standard solution into a reference substance: taking 10mg of metaxartan potassium, placing into a 10ml measuring flask, adding 8ml of (2) reference substance solution, dissolving by ultrasonic for 1min, adding (2) reference substance solution to the scale, and shaking uniformly to obtain the medicine;
(5) Brand model of high performance liquid chromatography: agilent 1260;
(6) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(7) Chromatographic column: agilent ZORBAX RX-C8, 250X4.6 mm,5 μm;
(8) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(9) The flow rate is 0.5ml per minute;
(10) The column temperature is 30 ℃;
(11) The temperature of the sample introduction disc is 6 ℃;
(12) Sample volume 5. Mu.l;
(13) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(15) The blank solvents of FIGS. 1, 2, 3 and 4 did not interfere with detection, the control solutions of FIGS. 5, 6, 7 and 8 had a peak retention time of 26.702min for impurity SM1-A, 32.343min for impurity SM1-B, 17.971min for impurity IN1-D, 15.577min for impurity IN1-E, 15.238min for impurity IN3-B, 19.935min for impurity IN3-C, 28.127min for impurity C and 9.671min for impurity DMAP; fig. 9, fig. 10, fig. 11 and fig. 12 show that the peak-out retention time of each impurity is identical to that of the reference solution, indicating that other impurities in the metartan potassium do not interfere with the detection of the target peak.
Example 2
(1) Control solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. 1ml of the solution is precisely measured, placed in a 20ml measuring flask, diluted to a scale by adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), and shaken well. Precisely weighing 1ml, placing in a 100ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(2) Brand model of high performance liquid chromatography: agilent 1260;
(3) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(4) Chromatographic column: agilent ZORBAX RX-C8, 250X4.6 mm,5 μm;
(5) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(6) The flow rate is 0.5ml per minute;
(7) The column temperature is 30 ℃;
(8) The temperature of the sample introduction disc is 6 ℃;
(9) Sample volume 5. Mu.l;
(10) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(11) Taking a reference substance solution for sample injection;
(12) Table 1 shows the results of the precision of the peak area of each impurity in the control solution by 6 needles, and table 2 shows the results of the precision of the peak retention time of each impurity in the control solution by 6 needles, as can be seen from tables 1 and 2, which meet the requirements of the chinese pharmacopoeia.
TABLE 1 continuous sample injection of control solution for 6 needles, peak area precision results for each impurity
TABLE 2 continuous sample introduction of control solution for 6 needles, peak retention time precision results for each impurity
Example 3
(1) Control stock solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. Precisely weighing 1ml, placing into 200ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(2) Linear solution 1: precisely measuring 1ml of the reference substance stock solution, placing into a 100ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(3) Linear solution 2: precisely measuring 3ml of the reference substance stock solution of (1), placing in a 100ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(4) Linear solution 3: precisely measuring 1ml of the reference substance stock solution, placing into a 20ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(5) Linear solution 4: precisely measuring 2ml of the reference substance stock solution of (1), placing into a 20ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(6) Linear solution 5: precisely measuring 3ml of the reference substance stock solution of (1), placing into a 20ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(7) Linear solution 6: precisely measuring 4ml of the reference substance stock solution of (1), placing into a 20ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(8) Brand model of high performance liquid chromatography: agilent 1260;
(9) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(10) Chromatographic column: agilent ZORBAX C8, 250×4.6mm,5 μm;
(11) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(12) The flow rate is 0.5ml per minute;
(13) The column temperature is 30 ℃;
(14) The temperature of the sample introduction disc is 6 ℃;
(15) Sample volume 5. Mu.l;
(16) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(17) Taking linear solution 1, linear solution 2, linear solution 3, linear solution 4, linear solution 5 and linear solution 6 for sample injection;
(18) FIG. 13 impurity SM1-A, FIG. 14 impurity SM1-B, FIG. 15 impurity IN1-D, FIG. 16 impurity IN1-E, FIG. 17 impurity IN3-B, FIG. 18 impurity IN3-C, FIG. 19 impurity C, FIG. 20 impurity DMAP. The graph shows that the linear correlation coefficient R of each impurity is larger than 0.990, and meets the requirements of Chinese pharmacopoeia.
Example 4
(1) Quantitative limiting solution: taking 1.8mg of an impurity SM1-A reference substance, 2.88mg of an SM1-B reference substance, 2.88mg of an IN1-D reference substance, 9.0mg of an IN1-E reference substance, 2.88mg of an IN3-B reference substance, 0.18mg of an IN3-C reference substance, 1.44mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving for 1min by ultrasound, diluting to a scale by adding acetonitrile, and shaking uniformly. 1ml of the solution is precisely measured, placed in a 20ml measuring flask, diluted to a scale by adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), and shaken well. 1ml of the solution is precisely measured, placed in a 50ml measuring flask, diluted to a scale by adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), and shaken well. Precisely weighing 1ml, placing in a 100ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(2) Detection limit solution: precisely measuring 5ml of quantitative limiting solution in the step (1), placing in a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) for dilution to scale, and shaking to obtain the final product;
(3) Brand model of high performance liquid chromatography: agilent 1260;
(4) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(5) Chromatographic column: agilent ZORBAX C8, 250×4.6mm,5 μm;
(6) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(7) The flow rate is 0.5ml per minute;
(8) The column temperature is 30 ℃;
(9) The temperature of the sample introduction disc is 6 ℃;
(10) Sample volume 5. Mu.l;
(11) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(12) Sampling the detection limit and the quantitative limit solution;
(13) Table 3 limits of detection and limits of quantification results. As can be seen from table 3, the detection limit and the quantitative limit of each impurity meet the sensitivity requirement of impurity detection;
TABLE 3 limit of detection and limit of quantification results
Example 5
(1) Sample preparation: taking 10mg of metaxartan potassium, placing into a 10ml measuring flask, adding 8ml of 0.1% formic acid (v/v) solution-acetonitrile (50:50), dissolving by ultrasound for 1min, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking uniformly to obtain the final product;
(2) Control stock solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. Precisely weighing 1ml, placing into 200ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(3) Control solution: precisely measuring 1ml of the reference substance stock solution of (2), placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(4) Recovery rate solution: taking 10mg of metaxartan potassium, placing into a 10ml measuring flask, precisely measuring 1ml of a reference substance storage solution of (2), adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking uniformly to obtain the medicine;
(5) Brand model of high performance liquid chromatography: agilent 1260;
(6) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(7) Chromatographic column: agilent ZORBAX RX-C8, 250X4.6 mm,5 μm;
(8) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(9) The flow rate is 0.5ml per minute;
(10) The column temperature is 30 ℃;
(11) The temperature of the sample introduction disc is 6 ℃;
(12) Sample volume 5. Mu.l;
(13) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(14) Taking a reference substance solution and a recovery rate solution for sample injection;
(15) The recovery rate results in table 4 show that the recovery rate of each impurity is 80% -120%, which meets the requirements of Chinese pharmacopoeia.
TABLE 4 recovery and precision results
Example 6
(1) Control stock solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. Precisely weighing 1ml, placing into 200ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(2) Control solution: precisely measuring 1ml of the reference substance stock solution, placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(3) Sensitivity solution: precisely measuring 1ml of the reference substance stock solution of (2), placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(4) Brand model of high performance liquid chromatography: agilent 1260;
(5) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(6) Chromatographic column: agilent ZORBAX RX-C8, 250X4.6 mm,5 μm;
(7) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(8) The flow rate is 0.4ml per minute;
(9) The column temperature is 30 ℃;
(10) The temperature of the sample introduction disc is 6 ℃;
(11) Sample volume 5. Mu.l;
(12) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(13) Sampling the sensitivity solution.
Example 7
(1) Control stock solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. Precisely weighing 1ml, placing into 200ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(2) Control solution: precisely measuring 1ml of the reference substance stock solution, placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(3) Sensitivity solution: precisely measuring 1ml of the reference substance stock solution of (2), placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(4) Brand model of high performance liquid chromatography: agilent 1260;
(5) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(6) Chromatographic column: agilent ZORBAX RX-C8, 250X4.6 mm,5 μm;
(7) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(8) The flow rate is 0.6ml per minute;
(9) The column temperature is 30 ℃;
(10) The temperature of the sample introduction disc is 6 ℃;
(11) Sample volume 5. Mu.l;
(12) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(13) Sampling the sensitivity solution.
Example 8
(1) Control stock solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. Precisely weighing 1ml, placing into 200ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(2) Control solution: precisely measuring 1ml of the reference substance stock solution, placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(3) Sensitivity solution: precisely measuring 1ml of the reference substance stock solution of (2), placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(4) Brand model of high performance liquid chromatography: agilent 1260;
(5) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(6) Chromatographic column: agilent ZORBAX RX-C8, 250X4.6 mm,5 μm;
(7) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(8) The flow rate is 0.5ml per minute;
(9) The column temperature is 25 ℃;
(10) The temperature of the sample introduction disc is 6 ℃;
(11) Sample volume 5. Mu.l;
(12) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(13) Sampling the sensitivity solution.
Example 9
(1) Control stock solution: taking 1.8mg of an impurity SM1-A reference substance, 1.8mg of an SM1-B reference substance, 1.8mg of an IN1-D reference substance, 1.8mg of an IN1-E reference substance, 1.8mg of an IN3-B reference substance, 1.8mg of an IN3-C reference substance, 1.8mg of an impurity C reference substance and 1.8mg of a DMAP reference substance, placing into a 50ml measuring flask, adding 40ml of acetonitrile, dissolving by ultrasonic for 1min, diluting to a scale by adding acetonitrile, and shaking uniformly. Precisely weighing 1ml, placing into 200ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50), diluting to scale, and shaking to obtain the final product;
(2) Control solution: precisely measuring 1ml of the reference substance stock solution, placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(3) Sensitivity solution: precisely measuring 1ml of the reference substance stock solution of (2), placing into a 10ml measuring flask, adding 0.1% formic acid (v/v) solution-acetonitrile (50:50) to dilute to scale, and shaking to obtain the final product;
(4) Brand model of high performance liquid chromatography: agilent 1260;
(5) Brand model of triple quadrupole mass spectrometer: AB SCIEX API4000;
(6) Chromatographic column: agilent ZORBAX RX-C8, 250X4.6 mm,5 μm;
(7) Mobile phase a: formic acid-water (1:1000), mobile phase B acetonitrile; the elution was performed according to the following gradient: taking 5% of mobile phase B and 95% of mobile phase A as initial mobile phases, gradually increasing mobile phase B to 60% within 10min, decreasing mobile phase A to 40%, continuously gradually increasing mobile phase B, decreasing mobile phase A to 30min, adjusting mobile phase B to 80%, adjusting mobile phase A to 20%, maintaining the mobile phase proportion to 35min, gradually decreasing mobile phase B, increasing mobile phase A to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase A to 95%, and maintaining the mobile phase proportion to 50min;
(8) The flow rate is 0.5ml per minute;
(9) The column temperature is 35 ℃;
(10) The temperature of the sample introduction disc is 6 ℃;
(11) Sample volume 5. Mu.l;
(12) The mass spectrum condition is detection by utilizing a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization and positive ion mode scanning; the air curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃;
(13) Sampling a sensitivity solution;
(14) Table 5 the signal to noise ratio results of each impurity in examples 1, 6, 7, 8 and 9 show that the signal to noise ratio of each impurity in the sensitive solution of the method is greater than 10 at the column temperature of 25-35 ℃ at the flow rate of 0.4-0.5 ml/min, which meets the requirements of Chinese pharmacopoeia.
TABLE 5 SNR results for each impurity
Claims (9)
1. The detection method for determining the genotoxic impurities in the metasartan potassium based on the liquid chromatography-mass spectrometry technology is characterized by comprising the following steps of: by using a liquid chromatography-mass spectrometry technology, a reversed-phase chromatographic column with octyl silane bonded silica gel as a filler, a water phase as a mobile phase A and an organic phase as a mobile phase B is adopted, and liquid phase conditions and mass spectrum conditions are set.
2. The detection method for determining genotoxic impurities in potassium metartan based on the liquid chromatography-mass spectrometry technology according to claim 1, wherein the reversed phase chromatographic column is selected from ZORBAX RX-C8 with the specification of 250mm multiplied by 4.6mm and 5 [ mu ] m.
3. The method for detecting the genotoxic impurities in the potassium metartan based on the liquid chromatography-mass spectrometry technology according to claim 1, wherein the mobile phase A contains an acid, and the acid is selected from formic acid or acetic acid.
4. The method for detecting the genotoxic impurities in the potassium metaxalone based on the liquid chromatography-mass spectrometry technology according to claim 1, wherein the acid is selected from formic acid, and the volume ratio of formic acid to water is selected from 1:1000.
5. The method for detecting the genotoxic impurities in the potassium metartan based on the liquid chromatography-mass spectrometry technology according to claim 1, wherein the mobile phase B is selected from acetonitrile or methanol.
6. The method for detecting genotoxic impurities in potassium metartan based on the liquid chromatography-mass spectrometry technique according to claim 5, wherein the mobile phase B is selected from acetonitrile.
7. The method for detecting the genotoxic impurities in the potassium metartan based on the liquid chromatography-mass spectrometry technology according to claim 1, wherein the liquid phase condition in the liquid chromatography-mass spectrometry method is as follows: the flow rate is selected from 0.4-0.6 ml/min, the temperature of the column Wen Xuanzi-35 ℃ and the sample injection disc is selected from 2-8 ℃, and the sample injection volume is selected from 5 mu L; the gradient elution is selected from the group consisting of, by volume, 5% mobile phase B,95% mobile phase a as the starting mobile phase, gradually increasing mobile phase B to 60%, decreasing mobile phase a to 40%, continuing to gradually increase mobile phase B, decreasing mobile phase a to 30min, adjusting mobile phase B to 80%, adjusting mobile phase a to 20%, maintaining the mobile phase ratio to 35min, gradually decreasing mobile phase B, increasing mobile phase a to 35.5min, adjusting mobile phase B to 5%, adjusting mobile phase a to 95%, and maintaining the mobile phase ratio to 50min.
8. The method for detecting the genotoxic impurities in the potassium metaxalone based on the liquid chromatography-mass spectrometry technology according to claim 7, wherein the liquid phase condition in the liquid chromatography-mass spectrometry method is that the flow rate is selected from 0.5ml/min, the column temperature is selected from 30 ℃, the temperature of a sample introduction disc is selected from 6 ℃, and the sample introduction volume is selected from 5 μl.
9. The method for detecting the genotoxic impurities in the potassium metasartan based on the liquid chromatography-mass spectrometry technology according to any one of claims 1 to 7, wherein the mass spectrometry conditions are as follows: detecting by using a multi-reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, performing electrospray ionization and positive ion mode scanning; the collision gas is 4psi, the gas curtain gas is 35psi, the atomizer 1 is 45psi, the atomizer 2 is 45psi, the voltage of a spray needle is 5500V, and the drying temperature is 550 ℃; the quantitative ion pairs, declustering voltage (DP), collision voltage (CE) of each impurity were:
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