CN111721843B - Method for detecting lobaplatin-related substance - Google Patents
Method for detecting lobaplatin-related substance Download PDFInfo
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Abstract
The invention relates to a method for detecting platinum-related substances. The invention provides a method for detecting a lobaplatin related substance, wherein the lobaplatin related substance comprises compounds with structures shown in formulas I and II, and the structure of the formula I is shown in the specification
The structure of the formula II is
Description
Technical Field
The invention relates to the field of medicines, in particular to a method for detecting substances related to lobaplatin, belonging to the technical field of medicine analysis quality control.
Background
Lobaplatin (Lobaplatin, D19466), also known as Lobaplatin, is a third-generation platinum-based antitumor drug following cisplatin and carboplatin, and its chemical name is: cis- [ trans-1, 2-cyclobutanebis (methylamine) -N, N']- [ (2S) -lactic acid-O1, O2]-platinum (II), formula C9H18N2O3Pt has a molecular weight of 397.34 and a chemical structural formula shown in the following formula (a):
lobaplatin has alkylation function, belongs to an alkylating agent (in a broad sense), and has good antitumor effect, such as good inhibition effect on isolated AH 135-tumor, B16-melanoma, colon cancer 115, and P338 leukemia in mice. Lobaplatin is characterized by strong anticancer activity, low toxicity, no accumulative toxicity and renal toxicity and less toxicity to bone marrow, and currently marketed lobaplatin for injection is mainly used for treating breast cancer, small cell lung cancer and chronic myelogenous leukemia.
Disclosure of Invention
In order to ensure the safety, effectiveness and controllable quality of the medicine, the research on related substances and detection methods of the related substances is very important. Aiming at the drug, due to the existence of three chiral carbons and related substances generated in the preparation process, finding a suitable detection method for controlling the product quality of the drug is a technical problem which is urgently needed to be solved in the field.
The technical problem to be solved by the invention is to provide a new detection method to establish the detection of related substances in the lobaplatin so as to carry out quality control on the lobaplatin compound.
One skilled in the art will recognize that any substance that affects the purity of a drug is collectively referred to as a related substance. Research on related substances is an important part of drug development, and comprises selecting a proper analysis method, accurately distinguishing and determining the content of the related substances, and determining the reasonable limit of the related substances by combining the results of pharmaceutical, toxicological and clinical researches. This study is throughout the entire process of drug development.
Specifically, the present invention is realized by the following technical means.
The invention provides a method for detecting a lobaplatin related substance, wherein the lobaplatin related substance comprises compounds with structures shown in formula I and formula II, and the structure of the formula I is shown in the specification
The structure of the formula II is
Preferably, the detection method is an HPLC-MS method or an HPLC method.
Preferably, in the detection method, the detection conditions of the HPLC-MS method are: octadecylsilane chemically bonded silica is used as a filling agent, 10-12mmol/L ammonium acetate is used as a mobile phase A, and methanol: the volume ratio of acetonitrile is 1, (0.8-1.2) is used as a mobile phase B, and gradient elution is carried out; preferably, the mobile phase A is a 10mmol/L ammonium acetate solution, and the mobile phase B is methanol: the volume ratio of acetonitrile is 1:1.
Preferably, in the detection method, the gradient elution pattern in the HPLC-MS method is as follows:
0-10 minutes: mobile phase a decreased from 95% to 40% by volume, and mobile phase B increased from 5% to 60% by volume;
10-15 minutes: mobile phase a decreased from 40% to 10% by volume and mobile phase B increased from 60% to 90% by volume;
15-16 minutes: mobile phase a increased from 10 to 95 vol%, and mobile phase B decreased from 90 to 5 vol%;
16-24 minutes: 95 vol% mobile phase a: 5 vol% mobile phase B;
wherein, each time range of the gradient elution can be increased by 1-2 minutes or the time range of the gradient elution from 10-15 minutes can be decreased by 1-2 minutes;
for example, the time range corresponding to gradient elution may be 0 to 11 minutes (or 0 to 12 minutes), 11 to 16 minutes (or 12 to 17 minutes), 16 to 17 minutes (or 17 to 18 minutes), 17 to 25 minutes (or 18 to 26 minutes); the time may be 0 to 10 minutes, 9 to 14 minutes (or 8 to 13 minutes), 14 to 15 minutes (or 13 to 14 minutes), or 15 to 23 minutes (or 14 to 22 minutes).
Preferably, in the detection method, an electrospray ion source is used as the MS condition in the HPLC-MS.
Preferably, for the detection method described above, wherein the flow rate is 0.8-1.2ml per minute, preferably 1.0 ml; preferably, the column temperature is 38-42 deg.C, preferably 40 deg.C.
Preferably, in the detection method described above, in which, in a chromatogram of the system suitability test solution, a degree of separation of a peak of the related substance from a peak of the related substance adjacent thereto is not less than 1.5; preferably, the relative standard deviation of the peak areas of the compound with the structure of the formula I and the compound with the structure of the formula II is not more than 10.0 percent.
Preferably, in the detection method, if the mass spectrum of the test solution contains the compounds with the structures of formula i and formula ii, the peak areas of the compounds with the structures of formula i and formula ii are not larger than the peak areas of the compounds with the structures of formula i and formula ii in the control solution.
Preferably, for the detection method described above, wherein said lobaplatin comprises either one or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
The invention has the following beneficial effects:
the invention establishes a method for detecting platinum compounds with structures shown in formulas I and II as related substances in a lobaplatin quality standard so as to establish a lobaplatin quality detection system.
Drawings
FIG. 1 is a typical spectrum of a compound of formula I and a compound of formula II in example 1;
FIG. 2-1A is a total ion chromatogram in a white solution specificity experiment in example 4;
FIG. 2-1B is an MS chromatogram from a white solution specificity experiment in example 4;
FIGS. 2-2A are total ion chromatograms from a positional solution specificity experiment for compounds of formula I in example 4;
FIGS. 2-2B are MS chromatograms of compounds of formula I in example 4 with retention time t ═ 1.616min in a positional solution specificity experiment;
FIGS. 2-3A are total ion chromatograms from a positional solution specificity experiment for compounds of formula II in example 4;
FIGS. 2-3B are MS chromatograms with retention times t 10.984min in a positional solution specificity experiment for compounds of formula II in example 4;
FIGS. 2-4A are total ion chromatograms obtained in experiments specific for the lobaplatin test solutions of example 4;
FIGS. 2-4B are MS chromatograms of the lobaplatin test solution in example 4 with a retention time t of 5.165 min;
Detailed Description
The invention provides a method for detecting a lobaplatin related substance, wherein the lobaplatin related substance comprises compounds with structures shown in formulas I and II, and the structure of the formula I is shown in the specification
The structure of the formula II is
In a preferred embodiment of the present invention, wherein the detection method is HPLC-MS method or HPLC method; preferably, the detection conditions of the HPLC-MS method are as follows: octadecylsilane chemically bonded silica is used as a filling agent, 10-12mmol/L ammonium acetate is used as a mobile phase A, and methanol: the volume ratio of acetonitrile is 1, (0.8-1.2) is used as a mobile phase B, and gradient elution is carried out; preferably, the mobile phase A is a 10mmol/L ammonium acetate solution, and the mobile phase B is methanol: the volume ratio of acetonitrile is 1: 1; preferably, the flow rate is 0.8-1.2ml, preferably 1.0ml per minute; preferably, the column temperature is 38-42 ℃, preferably 40 ℃; preferably, in the chromatogram of the system suitability test solution, the degree of separation of the peak of the substance of interest from its neighboring substances is not less than 1.5; preferably, the relative standard deviation of the peak areas of the compound with the structure of the formula I and the compound with the structure of the formula II is not more than 10.0 percent.
Preferably, for the detection method, if the compound with the structure of formula i and the compound with the structure of formula ii are present in the mass spectrum of the test solution, the peak areas of the compounds should not be larger than the peak areas (0.05%) of the compound with the structure of formula i and the compound with the structure of formula ii in the reference solution, and the 0.05% indicates that the concentration of the compound with the structure of formula i and the compound with the structure of formula ii in the reference solution is 0.05% of that of the test solution.
Herein, in the present invention, any substance affecting the purity of the drug is collectively referred to as "related substance affecting the quality of lobaplatin" or "related substance affecting the quality", and is simply referred to as "related substance", for example, a peak of related substance affecting the quality of lobaplatin appearing in an HPLC chromatogram peak for detecting the quality of lobaplatin, is simply referred to as "related substance peak"; the "related substance" in the present invention is sometimes an "impurity" known to those skilled in the art to affect the purity of the drug, however, the "related substance" in the present invention is not limited to the category of "impurity" but also includes substances having a certain anticancer activity even higher than that of lobaplatin, which belong to the category of substances related to lobaplatin with respect to the active molecule "lobaplatin", and the principles of their anticancer activity or other positive effects and functions in developing new drugs have not been fully studied.
The chemicals described in the examples are not indicated to be chemically pure grades of conventional reagents, wherein,
the compound with the structure shown in the formula I is prepared according to the method disclosed in example 1 of patent No. CN102093226B and is obtained by structure identification and confirmation;
the compound with the structure of the formula II is prepared from the compound with the structure of the formula I, and the preparation method comprises the following steps:
a compound of formula I
The reaction process is as follows:
(1) a compound with a structure shown in a formula I (30.0g,101.9mmol), potassium chloroplatinite (36.0g,86.7mmol), potassium iodide (86.0g,518.1mmol) and potassium hydroxide (24.0g,427.7mmol) are dissolved in 170mL,180mL,87mL and 120mL of purified water respectively to obtain solutions A, B, C and D.
(2) And heating the solution B to 30 ℃. Stirring and scattering the material A.
(3) Adding solution C to solution B, and stirring for 0.5h to obtain solution E.
(4) Adding solution D to solution A, stirring, clarifying, and filtering with 0.45 μm filter membrane to obtain solution F.
(5) The solution F was added to the solution E, and a yellow solid precipitated, and stirring was continued at 30 ℃ for 2 hours.
(6) Filtration and the filter cake washed with purified water (100mL x 6) until no halide ions remained. The filter cake was dried by rotary evaporator to give a crude compound of formula II (34.0g, crop) as a yellow powder.
And (3) carrying out preparation and separation on a crude compound (34.0g) with a structure shown in the formula II according to the following conditions, carrying out rotary evaporation at a temperature of below 40 ℃ until no liquid flows out, filtering, and drying at a temperature of 40 ℃ to constant weight to obtain a compound (15.4g) with a structure shown in the formula II.
TABLE 1 preparative isolation conditions
Example 1-1 test methods (Compound of formula I and Compound of formula II as related substances in lobaplatin in quality control of lobaplatin)
Measured according to mass spectrometry (Chinese pharmacopoeia 2015 year version of the general rules of the four parts 0431)
Chromatographic conditions and System suitability test
The instrument model is as follows: agilent 1260+6130MS, packed with octadecylsilane bonded silica (Waters Xselect CSH 4.6 × 150mm, 3.5 μm), mobile phase a of 10mmol/L ammonium acetate, methanol: the volume ratio of acetonitrile is 1:1, the mobile phase B is used, and gradient elution is carried out according to the following procedure; the flow rate was 1.0ml per minute and the column temperature was 40 ℃.
And (3) detecting by using a single quadrupole tandem mass spectrometer, wherein the ion source is an electrospray ionization (ESI) ion source, a positive ion scanning mode is used, the monitoring mode is selected ion monitoring, the monitored ions are 115 and 580, the acquisition time is 15min, and the outlet voltage of a capillary tube is 70V.
In the chromatogram of the system suitability test solution, it is known that the separation degree of a peak of the lobaplatin-related substance from a peak of an adjacent lobaplatin-related substance should be not less than 1.5; the system applicability test solution is continuously injected for 6 times, and the relative standard deviation of the peak areas of the compound with the structure of the formula I and the compound with the structure of the formula II is not more than 10.0 percent.
TABLE 2 gradient elution
| Time (minutes) | Mobile phase a (% by volume) | Mobile phase B (% by volume) |
| 0 | 95 | 5 |
| 10 | 40 | 60 |
| 15 | 10 | 90 |
| 16 | 95 | 5 |
| 24 | 95 | 5 |
Table 3 selective ion monitoring
| Time (minutes) | |
| 0 | 115 |
| 10 | 580 |
Wherein, the scanning m/z is 115 at 0-9.999min, 115 is diamine ion in the compound with the structure of formula I, the scanning m/z is 580 at 10-15min, and 580 is compound ion with the structure of formula II.
Preparation of System suitability test solution/control solution
Taking about 10mg of the compound reference substance with the structure of the formula I, precisely weighing, placing in a 10ml volumetric flask, adding water, ultrasonically dissolving, diluting to scale, shaking up, and taking the solution as the compound reference substance stock solution (1) with the structure of the formula I; taking a compound reference substance with a structure shown in a formula II, precisely weighing, placing the compound reference substance into a 10ml volumetric flask, adding N, N-dimethylformamide for ultrasonic dissolution, diluting to a scale, shaking up, taking 1ml of compound reference substance storage solution (1) with the structure shown in the formula II as a compound reference substance storage solution (1) with the structure shown in the formula I and 1ml of compound reference substance storage solution (1) with the structure shown in the formula II respectively, placing the compound reference substance storage solution (1) and the compound reference substance storage solution (1) with the structure shown in the formula II into the 10ml volumetric flask, adding water for dilution to the scale, shaking up to serve as a reference substance storage solution (2), precisely taking 100 mu l of the reference substance storage solution (2), placing the compound reference substance storage solution into a 20ml flask, adding water for dilution to the scale, shaking up to serve as a system applicability volumetric solution and simultaneously serve as a reference substance solution.
Preparation of test solution
Taking a lobaplatin sample to be tested (prepared by the method disclosed in example 2 of the specification of patent CN 102020679B and confirmed by structure identification, namely, the lobaplatin trihydrate is added as lobaplatin to be tested in the example, and the content of the lobaplatin in each example is calculated as lobaplatin anhydride), weighing about 10mg precisely, placing the sample into a 10ml volumetric flask, adding water, dissolving by ultrasonic waves, diluting to a scale, shaking uniformly, and using the sample as a test solution.
Assay method
And (3) taking 10 mu l of each of the system applicability test solution and the test sample solution, injecting the solution into a liquid chromatography-mass spectrometer, and recording a mass spectrogram for 15 minutes.
Typical spectra of the compound of formula I and the compound of formula II are shown in FIG. 1.
As can be seen from fig. 1, the retention time of the compound having the structure of formula i is t 1.72min, and the retention time of the compound having the structure of formula ii is t 10.966 min.
If the mass spectrogram of the test solution contains the compound with the structure shown in the formula I and the compound with the structure shown in the formula II, the peak areas of the compound with the structure shown in the formula I and the compound with the structure shown in the formula II in the reference solution are not larger than the peak areas (0.05 percent).
Example 1-2 detection method
Measured according to mass spectrometry (Chinese pharmacopoeia 2015 year version of the general rules of the four parts 0431)
Chromatographic conditions and System suitability test
Octadecylsilane bonded silica gel was used as filler (Waters Xselect CSH 4.6 × 150mm, 3.5 μm), 10mmol/L ammonium acetate as mobile phase a, methanol: the mobile phase B is acetonitrile with the volume ratio of 1:0.8, and gradient elution is carried out according to the following procedure; the flow rate was 0.8ml per minute and the column temperature was 38 ℃. And (3) detecting by using a single quadrupole tandem mass spectrometer, wherein the ion source is an electrospray ionization (ESI) ion source, a positive ion scanning mode is used, the monitoring mode is selected ion monitoring, the monitored ions are 115 and 580, the acquisition time is 15min, and the outlet voltage of a capillary tube is 70V. In the chromatogram of the system applicability test solution, it is known that the separation degree of a peak of a lobaplatin-related substance from a peak of an adjacent lobaplatin-related substance should be not less than 1.5; the system applicability test solution is continuously injected for 6 times, and the relative standard deviation of the peak areas of the compound with the structure of the formula I and the compound with the structure of the formula II is not more than 10.0 percent.
TABLE 4 gradient elution
| Time (minutes) | Mobile phase a (% by volume) | Mobile phase B (% by volume) |
| 0 | 95 | 5 |
| 10 | 40 | 60 |
| 15 | 10 | 90 |
| 16 | 95 | 5 |
| 24 | 95 | 5 |
Table 5 selective ion monitoring
| Time (minutes) | |
| 0 | 115 |
| 10 | 580 |
Preparation of System suitability test solution/control solution
Taking about 10mg of the compound reference substance with the structure of the formula I, precisely weighing, placing in a 10ml volumetric flask, adding water, ultrasonically dissolving, diluting to scale, shaking up, and taking the solution as the compound reference substance stock solution (1) with the structure of the formula I; taking a compound reference substance with a structure shown in a formula II, precisely weighing, placing the compound reference substance into a 10ml volumetric flask, adding N, N-dimethylformamide for ultrasonic dissolution, diluting to a scale, shaking up, taking 1ml of compound reference substance storage solution (1) with the structure shown in the formula II as a compound reference substance storage solution (1) with the structure shown in the formula I and 1ml of compound reference substance storage solution (1) with the structure shown in the formula II respectively, placing the compound reference substance storage solution (1) and the compound reference substance storage solution (1) with the structure shown in the formula II into the 10ml volumetric flask, adding water for dilution to the scale, shaking up to serve as a reference substance storage solution (2), precisely taking 100 mu l of the reference substance storage solution (2), placing the compound reference substance storage solution into a 20ml flask, adding water for dilution to the scale, shaking up to serve as a system applicability volumetric solution and simultaneously serve as a reference substance solution.
Preparation of test solution
About 10mg of lobaplatin sample to be tested (the source is the same as that in example 1-1) is precisely weighed, placed in a 10ml volumetric flask, added with water for ultrasonic dissolution and diluted to a scale, and shaken up to be used as a test solution.
Assay method
And (3) taking 10 mu l of each of the system applicability test solution and the test sample solution, injecting the solution into a liquid chromatography-mass spectrometer, and recording a mass spectrogram for 15 minutes.
The obtained typical pattern was consistent with that of example 1-1.
EXAMPLES 1-3 detection methods
Measured according to mass spectrometry (Chinese pharmacopoeia 2015 year version of the general rules of the four parts 0431)
Chromatographic conditions and System suitability test
Octadecylsilane bonded silica gel was used as filler (Waters Xselect CSH 4.6 × 150mm, 3.5 μm), with 11mmol/L ammonium acetate as mobile phase a, methanol: the mobile phase B is acetonitrile with the volume ratio of 1:1.2, and gradient elution is carried out according to the following procedure; the flow rate was 1.2ml per minute and the column temperature was 42 ℃. And (3) detecting by using a single quadrupole tandem mass spectrometer, wherein the ion source is an electrospray ionization (ESI) ion source, a positive ion scanning mode is used, the monitoring mode is selected ion monitoring, the monitored ions are 115 and 580, the acquisition time is 15min, and the outlet voltage of a capillary tube is 70V. In the chromatogram of the system suitability test solution, it is known that the separation degree of a peak of the lobaplatin-related substance from a peak of an adjacent lobaplatin-related substance should be not less than 1.5; the system applicability test solution is continuously injected for 6 times, and the relative standard deviation of the peak areas of the compound with the structure of the formula I and the compound with the structure of the formula II is not more than 10.0 percent.
TABLE 6 gradient elution
| Time (minutes) | Mobile phase a (% by volume) | Mobile phase B (% by volume) |
| 0 | 95 | 5 |
| 10 | 40 | 60 |
| 15 | 10 | 90 |
| 16 | 95 | 5 |
| 24 | 95 | 5 |
Table 7 selective ion monitoring
| Time (minutes) | |
| 0 | 115 |
| 10 | 580 |
Preparation of System suitability test solution/control solution
Taking about 10mg of the compound reference substance with the structure of the formula I, precisely weighing, placing in a 10ml volumetric flask, adding water, ultrasonically dissolving, diluting to scale, shaking up, and taking the solution as the compound reference substance stock solution (1) with the structure of the formula I; taking a compound reference substance with a structure shown in a formula II, precisely weighing, placing the compound reference substance into a 10ml volumetric flask, adding N, N-dimethylformamide for ultrasonic dissolution, diluting to a scale, shaking up, taking 1ml of compound reference substance storage solution (1) with the structure shown in the formula II as a compound reference substance storage solution (1) with the structure shown in the formula I and 1ml of compound reference substance storage solution (1) with the structure shown in the formula II respectively, placing the compound reference substance storage solution (1) and the compound reference substance storage solution (1) with the structure shown in the formula II into the 10ml volumetric flask, adding water for dilution to the scale, shaking up to serve as a reference substance storage solution (2), precisely taking 100 mu l of the reference substance storage solution (2), placing the compound reference substance storage solution into a 20ml flask, adding water for dilution to the scale, shaking up to serve as a system applicability volumetric solution and simultaneously serve as a reference substance solution.
Preparation of test solution
About 10mg of lobaplatin sample to be tested (the source is the same as that in example 1-1) is precisely weighed, placed in a 10ml volumetric flask, added with water for ultrasonic dissolution and diluted to a scale, and shaken up to be used as a test solution.
Assay method
And (3) taking 10 mu l of each of the system applicability test solution and the test sample solution, injecting the solution into a liquid chromatography-mass spectrometer, and recording a mass spectrogram for 15 minutes.
The obtained typical pattern was consistent with that of example 1-1.
Example 2: methodological validation of detection methods
In order to confirm the utility and accuracy of the detection method of the present invention, the specificity, linear equation, and linear range, detection and quantification limits, correction factors, accuracy (recovery), precision, solution stability, durability, etc. of the detection method are described below:
1. specificity
A blank solution (purified water), a localization solution of a compound of the formula I (ID-cpd1), a localization solution of a compound of the formula II (ID-cpd2), and a lobaplatin test Solution (SPL) were each measured by a precision measuring method at 10. mu.L, and injected into a mass spectrometer for LC/MS.
The preparation method of the ID-cpd1 comprises the following steps: weighing 9.96mg of a compound reference substance with a structure shown in the formula I in a 10mL volumetric flask, dissolving a diluent to a constant volume, and shaking up to obtain a solution with the concentration of the compound with the structure shown in the formula I being 0.996mg/mL, wherein the solution is marked as ID-cpd1 solution;
the preparation method of the ID-cpd2 comprises the following steps: weighing 9.87mg of a compound reference substance with a structure shown in a formula II in a 10mL volumetric flask, dissolving DMF to a constant volume, and shaking up to obtain a solution with the concentration of the compound with the structure shown in the formula II being 0.987mg/mL, wherein the solution is marked as ID-cpd2 solution;
the preparation method of the SPL comprises the following steps: weighing 10.16mg of lobaplatin test sample in a 10mL volumetric flask, dissolving the diluent to a constant volume, and shaking up to obtain a solution with the concentration of the lobaplatin test sample of 1.016mg/mL, which is marked as an SPL solution;
the results are shown in FIG. 2-1A, FIG. 2-1B, FIG. 2-2A, FIG. 2-2B, FIG. 2-3A, FIG. 2-3B, FIG. 2-4A, FIG. 2-4B and Table 8.
TABLE 8 specificity results-1
| Sample (I) | Retention time (min) | Degree of peak separation | Conclusion |
| Blank space | --- | --- | Without interference |
| A compound of formula I | 1.616 | --- | Without interference |
| A compound of the structure of formula II | 10.984 | --- | Without interference |
| Lobaplatin | 5.165 | --- | Without interference |
It can be seen that the blank control baseline was clean and stable, with no interference at the peak positions of the compound of formula I and the compound of formula II.
Then precisely measuring blank solution and forced degradation solution of 10 μ L under each condition, injecting into a mass spectrometer,
the preparation method of the acid destroying solution comprises the following steps: weighing 10.61mg of lobaplatin test sample in a 10mL volumetric flask, adding 2mL of water to dissolve the lobaplatin test sample, adding 0.1mL of 0.01mol/L hydrochloric acid solution to destroy the lobaplatin test sample for 5min, neutralizing the lobaplatin test sample with 0.1mL of 0.01mol/L sodium hydroxide solution after destruction, and diluting the lobaplatin test sample with a diluent to a constant volume to obtain an acid destruction solution.
The preparation method of the alkali destruction solution comprises the following steps: weighing 10.48mg of lobaplatin test sample in a 10mL volumetric flask, adding 1mL of 0.01mol/L sodium hydroxide solution, standing for 10min, neutralizing with 1mL of 0.01mol/L hydrochloric acid solution after destruction, and diluting to constant volume with a diluent to obtain an alkali destruction solution.
The preparation method of the photodisruption solution comprises the following steps: a lobaplatin test article 9.82mg destroyed for 10 days under 45,00lx light intensity was weighed into a 10mL volumetric flask, and the volume was determined by dissolving a diluent to a constant volume to obtain a light destruction solution.
The preparation method of the high-temperature destruction solution comprises the following steps: a10.04 mg sample of lobaplatin after 3 days of destruction at 80 ℃ was weighed in a 10mL volumetric flask, and a diluent was dissolved to a constant volume to prepare a high temperature destruction solution.
The preparation method of the oxidative destruction solution comprises the following steps: weighing 10.83mg of lobaplatin test sample in a 10mL volumetric flask, adding 1mL of 0.01% hydrogen peroxide solution, diluting to constant volume to obtain oxidation destruction solution (standing for 10min), and immediately injecting sample; the results are shown in Table 9:
TABLE 9 specificity results-2
As can be seen from the above table, the material balance is between 90% and 110%, and the expected requirements are met.
2. Sensitivity of the probe
Taking the compound with the structure of the formula I and the compound reference substance solution with the structure of the formula II, gradually diluting, and detecting by taking a signal-to-noise ratio (S/N)3 as a detection limit, wherein the detection limit result is shown in a table 10.
TABLE 10 Limit of detection results
| Name of the Compound | Detection limit concentration (ng/mL) | Corresponding to the percentage of the main component | S/N |
| A compound of formula I | 5.210 | 0.005％ | 3.5 |
| A compound of the structure of formula II | 5.065 | 0.005％ | 5.5 |
As can be seen from Table 10, the detection limit concentration of the compound having the structure of formula I was 5.210ng/mL (corresponding to 0.005% of the main component of lobaplatin), and the detection limit concentration of the compound having the structure of formula II was 5.065ng/mL (corresponding to 0.005% of the main component of lobaplatin), indicating that the method had high sensitivity.
3. Stability of solution
The control solutions were measured and injected at 0h, 0.4h, 1.1h, 1.5h, 1.9h, 3.4h, 4.2h, and 5.4h, respectively, and the peak area changes of the compound of formula I and the compound of formula II were examined, with the results shown in Table 11.
TABLE 11 solution stability results
Remarking: (peak area of each related substance in solution/peak area of each related substance in solution at 0h per time interval) × 100
As can be seen from the above table, the S% of the compound reference substance solution with the structure of formula I is stable within 5.4h between 90% and 110%, and the S% of the compound reference substance solution with the structure of formula II is stable within 1.5h between 90% and 110%.
4. Durability
Taking each control solution and test solution, properly adjusting parameters in the LC-MS system, and inspecting the durability of the method after the system conditions change. It was determined that RSD of the response (peak area/concentration) of the compound of the formula I or the compound of the formula II in Bracket STD-1 and the response of the compound of the formula I or the compound of the formula II in STD-1 of the first 6 needles was not more than 10%, and that the detection result of the compound of the formula I or the compound of the formula II in lobaplatin in the test sample before changing the conditions (the magnitude relationship between the peak area of the compound of the formula I or the compound of the formula II in SPL and the peak area of the compound of the formula I or the compound of the formula II in STD) should not be changed, and the results are shown in Table 12.
TABLE 12 durability results
Wherein, the Bracket STD-1 is a follow control solution, namely a needle of the standard solution STD-1.
As can be seen from table 12, the change in RSD is relatively small after a slight variation in column temperature, indicating that the method is excellent in durability against column temperature.
The foregoing is considered as illustrative and not restrictive in character, and that various modifications, equivalents, and improvements made within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (33)
1. A method for detecting lobaplatin related substances, wherein the lobaplatin related substances comprise compounds with structures shown in formulas I and II, and the structure of the formula I is
The structure of the formula II is
The detection method is a method for detecting substances related to lobaplatin in a lobaplatin sample;
the detection method is an HPLC-MS method;
wherein, the detection conditions of the HPLC-MS method are as follows: octadecylsilane chemically bonded silica is used as a filling agent, 10-12mmol/L ammonium acetate is used as a mobile phase A, and methanol: the volume ratio of acetonitrile =1, (0.8-1.2) is mobile phase B, and gradient elution is carried out;
the gradient elution pattern in the HPLC-MS method is as follows:
0-10 minutes: mobile phase a decreased from 95% to 40% by volume and mobile phase B increased from 5% to 60% by volume;
10-15 minutes: mobile phase a decreased from 40% to 10% by volume and mobile phase B increased from 60% to 90% by volume;
15-16 minutes: mobile phase a increased from 10 vol% to 95 vol%, and mobile phase B decreased from 90 vol% to 5 vol%;
16-24 minutes: 95 vol%, mobile phase a: 5 vol% mobile phase B.
2. The detection method according to claim 1, wherein the detection conditions of the HPLC-MS method are as follows: the mobile phase A is 10mmol/L ammonium acetate solution, and the mobile phase B is methanol: the volume ratio of acetonitrile =1: 1.
3. The detection method according to claim 1, wherein the gradient elution is replaced by any one of the following gradient elution methods:
the gradient elution pattern in the HPLC-MS method is as follows:
0-11 minutes: mobile phase a decreased from 95% to 40% by volume and mobile phase B increased from 5% to 60% by volume;
11-16 minutes: mobile phase a decreased from 40% to 10% by volume and mobile phase B increased from 60% to 90% by volume;
16-17 minutes: mobile phase a increased from 10 vol% to 95 vol%, and mobile phase B decreased from 90 vol% to 5 vol%;
17-25 minutes: 95 vol%, mobile phase a: 5 vol% mobile phase B; or
The gradient elution pattern in the HPLC-MS method is as follows:
0-12 minutes: mobile phase a decreased from 95% to 40% by volume and mobile phase B increased from 5% to 60% by volume;
12-17 minutes: mobile phase a decreased from 40% to 10% by volume and mobile phase B increased from 60% to 90% by volume;
17-18 minutes: mobile phase a increased from 10 vol% to 95 vol%, and mobile phase B decreased from 90 vol% to 5 vol%;
18-26 minutes: 95 vol%, mobile phase a: 5 vol% mobile phase B.
4. The detection method according to claim 2, wherein the gradient elution is replaced by any one of the following gradient elution methods:
the gradient elution pattern in the HPLC-MS method is as follows:
0-11 minutes: mobile phase a decreased from 95% to 40% by volume and mobile phase B increased from 5% to 60% by volume;
11-16 minutes: mobile phase a decreased from 40% to 10% by volume and mobile phase B increased from 60% to 90% by volume;
16-17 minutes: mobile phase a increased from 10 vol% to 95 vol%, and mobile phase B decreased from 90 vol% to 5 vol%;
17-25 minutes: 95 vol%, mobile phase a: 5 vol% mobile phase B; or
The gradient elution pattern in the HPLC-MS method is as follows:
0-12 minutes: mobile phase a decreased from 95% to 40% by volume, and mobile phase B increased from 5% to 60% by volume;
12-17 minutes: mobile phase a decreased from 40% to 10% by volume and mobile phase B increased from 60% to 90% by volume;
17-18 minutes: mobile phase a increased from 10 vol% to 95 vol%, and mobile phase B decreased from 90 vol% to 5 vol%;
18-26 minutes: 95 vol%, mobile phase a: 5 vol% mobile phase B.
5. The detection method of claim 1, wherein the MS condition in HPLC-MS is the use of an electrospray ion source.
6. The detection method of claim 2, wherein the MS condition in HPLC-MS is the use of an electrospray ion source.
7. The detection method of claim 3, wherein the MS condition in HPLC-MS is the use of an electrospray ion source.
8. The detection method according to claim 1, wherein the HPLC-MS method has a flow rate of 0.8 to 1.2ml per minute.
9. The method according to claim 2, wherein the HPLC-MS method is carried out under conditions such that the flow rate is 0.8 to 1.2 ml/minute.
10. The detection method according to claim 3, wherein the HPLC-MS method has a flow rate of 0.8 to 1.2ml per minute.
11. The detection method according to claim 5, wherein the HPLC-MS method has a flow rate of 0.8 to 1.2ml per minute.
12. The detection method according to claim 1, wherein the HPLC-MS method has a flow rate of 1.0ml per minute under the detection conditions.
13. The detection method according to claim 1, wherein the HPLC-MS method is carried out under detection conditions in which the column temperature is 38 to 42 ℃.
14. The detection method according to claim 2, wherein the HPLC-MS method is carried out under detection conditions in which the column temperature is 38 to 42 ℃.
15. The detection method according to claim 3, wherein the HPLC-MS method is carried out under detection conditions in which the column temperature is 38 to 42 ℃.
16. The detection method according to claim 5, wherein the HPLC-MS method is carried out under detection conditions in which the column temperature is 38 to 42 ℃.
17. The detection method according to claim 8, wherein the HPLC-MS method is carried out under detection conditions in which the column temperature is 38 to 42 ℃.
18. The detection method according to claim 1, wherein the HPLC-MS method is carried out under detection conditions in which the column temperature is 40 ℃.
19. The detection method according to any one of claims 1 to 18, wherein a separation degree of a peak of the lobaplatin-related substance from its neighboring related substances is not less than 1.5 in a chromatogram of the system suitability test solution.
20. An assay as claimed in any one of claims 1 to 18 wherein the relative standard deviation of the peak areas of the compound of formula i and the compound of formula ii is no more than 10.0%.
21. The detection method according to claim 19, wherein the relative standard deviation of the peak areas of the compound with the structure of formula I and the compound with the structure of formula II is not more than 10.0%.
22. The detection method according to any one of claims 1 to 18, wherein the mass spectrum of the test solution contains compounds having the structures of formula i and formula ii, and the peak areas of the compounds are not larger than the peak areas of the compounds having the structures of formula i and formula ii in the control solution.
23. The detection method according to claim 19, wherein the mass spectrum of the test solution contains the compounds having the structures of formula i and formula ii, and the peak areas of the compounds having the structures of formula i and formula ii are not larger than those of the reference solution.
24. The detection method according to claim 20, wherein the mass spectrum of the test solution contains the compounds having the structures of formula i and formula ii, and the peak areas of the compounds having the structures of formula i and formula ii are not larger than those of the reference solution.
25. The detection method according to claim 21, wherein the mass spectrum of the test solution contains the compounds having the structures of formula i and formula ii, and the peak areas of the compounds having the structures of formula i and formula ii are not larger than those of the reference solution.
26. The detection method according to any one of claims 1 to 18, wherein the lobaplatin comprises either one or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
27. The detection method of claim 19, wherein said lobaplatin comprises either or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
28. The detection method of claim 20, wherein said lobaplatin comprises either or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
29. The detection method of claim 21, wherein said lobaplatin comprises either or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
30. The detection method of claim 22, wherein said lobaplatin comprises either or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
31. The detection method of claim 23, wherein said lobaplatin comprises either or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
32. The detection method of claim 24, wherein said lobaplatin comprises either or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
33. The detection method of claim 25, wherein said lobaplatin comprises either or both of lobaplatin diastereomer i and lobaplatin diastereomer ii.
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| CN105440083A (en) * | 2014-06-20 | 2016-03-30 | 贵州益佰制药股份有限公司 | Lobaplatin crystal and preparation method and drug application thereof |
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