CN111103373A - Daptomycin detection method - Google Patents
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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
- G01N30/02—Column 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
Abstract
The invention discloses a daptomycin detection method, belongs to the technical field of drug detection and analysis, and adopts ultra-high performance liquid chromatography to detect, and particularly discloses chromatographic conditions for detection. The daptomycin detection method provided by the invention can effectively detect daptomycin and related substances in a sample to be detected, can carry out quantification, has the advantages of good separation degree of main components and impurities, low tail removal factor, low detection period, low detection cost, high sensitivity, high accuracy, high precision, good linearity level, strong specificity, good durability and the like, can be widely popularized and used in production enterprises and detection centers due to the characteristics of short period, low cost and accurate detection, is favorable for realizing the quality control of daptomycin, and can prepare high-purity and high-quality daptomycin.
Description
Technical Field
The invention belongs to the technical field of drug detection and analysis, and particularly relates to a daptomycin detection method.
Background
Daptomycin is an N-decanoyl derivative of a lipopeptide compound A21978C which is a fermentation product of streptomyces roseosporus, contains 13 amino acids, 10 of the amino acids form cyclododecyl peptide, and the other 3 amino acids are connected with decanoyl to form a side chain, and is a second generation glycopeptide antibiotic drug following vancomycin. So far, the detection of the content of the daptomycin and related substances is not seen in a pharmacopoeia collection method, impurities in the quality control of the daptomycin seriously affect the quality of products, so that the establishment of a method for detecting the content of the daptomycin and the related substances is very important, the literature reports on the detection method of the content of the daptomycin and the related substances are less,
there are two main methods currently used. The first method is a daptomycin high performance liquid chromatography detection method, which adopts Agilent 1260 high performance liquid chromatography of Agilent, Phenomenex TB-Sil column C84.6 multiplied by 250mm (5 mu m) as a chromatographic column, ammonium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B, and carries out detection under the conditions of flow rate of 1.0ml/mim, detection wavelength of 223nm and column temperature of 30 ℃. However, the detection method has long detection time when detecting daptomycin, needs 60min for one injection, has large resource consumption, needs 60ml of mobile phase for one injection, and is not beneficial to the high-efficiency and energy-saving policy of enterprises. The second method is the measurement of the plasma concentration UPLC-MS/MS of daptomycin released by the department of medicine of the medical university of subsidiary middle and large hospitals of the southeast university and the medical supplement of the Chinese pharmaceutical university, which uses a Nexera X2 liquid chromatograph-API 140000 mass spectrometer, Phenomenex Kinetex XB-C18(50mm multiplied by 2.1mm, 1.7 mu m) as a chromatographic column, and 0.1% formic acid water solution-acetonitrile as a mobile phase under the conditions of the flow rate of 0.4ml/mim, the detection wavelength of 223nm and the column temperature of 45 ℃. The method is used for detecting the content of daptomycin, but related substances of daptomycin cannot be detected, a liquid chromatography-mass spectrometer used for detection belongs to a high-end instrument in the organic matter analysis market, the instrument price and the later maintenance cost are high, the requirement on professional quality of operators is relatively high, LC/MS is still in a development stage in a pharmaceutical enterprise, and the application is not universal enough.
Compared with a high-efficiency chromatograph, the ultra-high performance liquid chromatograph is more precise and more expensive, and a daptomycin ultra-high performance liquid detection method can not be used for reference at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: the method has the advantages of short detection period, low detection cost, strong specificity, high accuracy, high precision, good durability, simplicity and practicality, and can not only quantify the major ingredients of daptomycin, but also accurately detect related substances of daptomycin.
In order to solve the technical problems, the invention adopts the technical scheme that: a daptomycin detection method adopts ultra-high performance liquid chromatography for detection, and the chromatographic conditions are as follows:
a chromatographic column: 2.1X 150mm, 1.7 μm RP 18;
mobile phase A: anhydrous sodium sulfate solution-acetonitrile (74: 26);
mobile phase B: anhydrous sodium sulfate solution-acetonitrile (50: 50);
flow rate: 0.2 ml/mim;
detection wavelength: 223 nm;
column temperature: 30 ℃;
temperature of the sample pan: 5 ℃ is adopted.
The invention has the beneficial effects that: the daptomycin detection method provided by the invention can effectively detect daptomycin and related substances in a sample to be detected, can carry out quantification, and has the advantages of good separation degree of main components and impurities, low tail removal factor, low detection period, low detection cost, high sensitivity, high accuracy, high precision, good linear level, strong specificity, good durability and the like; due to the characteristics of short period, low cost and accurate detection, the method can be widely popularized and used in production enterprises and detection centers, can quickly detect the content, the impurity types and the content of daptomycin, is favorable for realizing the quality control of daptomycin, and prepares high-purity and high-quality daptomycin.
Drawings
FIG. 1 shows an ultra high performance liquid chromatogram of a solvent of example 3 according to an embodiment of the present invention;
FIG. 2 shows an ultra high performance liquid chromatogram of a control of example 3 according to an embodiment of the present invention;
FIG. 3 is an ultra high performance liquid chromatogram of a test sample of example 3, which is an embodiment of the present invention;
FIG. 4 shows an ultra high performance liquid chromatogram of a standard solution of daptomycin lactone hydrolysate of example 3 in accordance with an embodiment of the present invention;
FIG. 5 is an ultra-high performance liquid chromatogram of a standard solution of daptomycin lactone hydrolysate and a sample to be tested in example 3, which is a specific embodiment of the present invention;
FIG. 6 is an ultra high performance liquid chromatogram of a standard solution of daptomycin β -isomer of example 3, according to an embodiment of the present invention;
FIG. 7 is a ultra high performance liquid chromatogram of a standard solution of daptomycin β -isomer as a sample in example 3, which is an embodiment of the present invention;
FIG. 8 is an ultra high performance liquid chromatogram of a daptomycin hydroxy derivative standard solution of example 3, showing an embodiment of the present invention;
FIG. 9 shows an ultra high performance liquid chromatogram of a daptomycin hydroxy derivative standard solution as a sample to be tested in example 3, in accordance with an embodiment of the present invention;
FIG. 10 is an ultra high performance liquid chromatogram of a standard solution of daptomycin isodecanoyl isomer of example 3, in accordance with an embodiment of the present invention;
FIG. 11 is an ultra-high performance liquid chromatogram of a standard solution of a test substance plus daptomycin isodecanoyl isomer in example 3, which is an embodiment of the present invention;
FIG. 12 is an ultra high performance liquid chromatogram of a standard solution of daptomycin ethyl isomer of example 3, in accordance with an embodiment of the present invention;
FIG. 13 is an ultra high performance liquid chromatogram of a daptomycin ethyl isomer standard solution as a sample to be tested in example 3, which is a specific embodiment of the present invention;
FIG. 14 shows an ultra high performance liquid chromatogram of a dehydrated daptomycin standard solution of example 3 in accordance with an embodiment of the present invention;
FIG. 15 is an ultra-high performance liquid chromatogram of a sample to be tested plus a dehydrated daptomycin standard solution in example 3, which is a specific embodiment of the present invention;
FIG. 16 shows an ultra high performance liquid chromatogram of an LOQ standard solution of example 3 according to an embodiment of the present invention;
FIG. 17 shows an ultra high performance liquid chromatogram of an LOD standard solution of example 3 according to an embodiment of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows:
the invention relates to a daptomycin detection method, which adopts ultra-high performance liquid chromatography for detection, and the chromatographic conditions are as follows:
a chromatographic column: 2.1X 150mm, 1.7 μm RP 18;
mobile phase A: anhydrous sodium sulfate solution-acetonitrile (74: 26);
mobile phase B: anhydrous sodium sulfate solution-acetonitrile (50: 50);
flow rate: 0.2 ml/mim;
detection wavelength: 223 nm;
column temperature: 30 ℃;
temperature of the sample pan: 5 ℃ is adopted.
From the above description, the beneficial effects of the present invention are: the daptomycin detection method provided by the invention can effectively detect daptomycin and related substances in a sample to be detected, can carry out quantification, has the advantages of good separation degree of main components and impurities, low tail removal factor, low detection period, low detection cost, high sensitivity, high accuracy, high precision, good linearity level, strong specificity, good durability and the like, the specific one-needle running time is only 45min, only 9ml of mobile phase is consumed by one detection needle, the detection period is short, and the detection cost is low; in the method, the sample injection concentration of the main component LOQ of the daptomycin is 0.652ug/ml according to the signal-to-noise ratio of 10:1, the sample injection concentration of the LOD is 0.218ug/ml according to the signal-to-noise ratio of 3:1, and the sensitivity is high; different persons use different instruments to detect the content of the same sample at different times and the reproducibility of related substances is high, namely the precision is high; the blank solvent used in the test has no interference to main components and related substances, and the daptomycin sample can achieve mass conservation after being destroyed under the conditions of high temperature, acid, alkali, oxidation and illumination, and has strong specificity; different chromatographic columns, buffer solution pH, mobile phase organic phase proportion, column temperature and flow rate in chromatographic conditions fluctuate in the invention, so that the detection result is not obviously influenced, and the durability is good; the LOQ concentration level standard, the 5% concentration level standard, the 25% concentration level standard, the 50% concentration level standard, the 100% concentration level standard and the 150% concentration level standard are used for preparing a linear curve, r is 1.0000, and the relative corresponding factors of the impurities to the main components are all above 0.8, so that the linearity is good; the recovery rates of the main components and the impurities of 50 percent, 100 percent and 150 percent meet the requirements, and the accuracy is high. The method has the characteristics of short period, low cost and accurate detection, can be widely popularized and used in production enterprises and detection centers, can quickly detect the content, the impurity types and the content of the daptomycin, is favorable for realizing the quality control of the daptomycin, and prepares the high-purity and high-quality daptomycin.
Further, the preparation method of the anhydrous sodium sulfate solution in the mobile phase A and the mobile phase B comprises the following steps: 3.46g of anhydrous sodium sulfate is taken, 1000ml of water is added for dissolution, and the pH value is adjusted to 3.4 by phosphoric acid.
Further, the elution mode in the detection by adopting the ultra-high performance liquid chromatography is as follows: flow A-flow B (66:34) isocratic for 45 min.
Further, preparing a reference substance solution before detecting by adopting the ultra-high performance liquid chromatography; the preparation method of the reference substance solution comprises the following steps: precisely weighing 25mg of daptomycin working standard, placing the daptomycin working standard in a 25ml volumetric flask, adding 10% acetonitrile to dissolve and dilute the daptomycin working standard to a scale, and shaking up to obtain a reference substance solution.
Further, preparing a test solution before detecting by adopting the ultra-high performance liquid chromatography; the preparation method of the test solution comprises the following steps: precisely weighing 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10% acetonitrile to dissolve and dilute the daptomycin to a scale, and shaking up to obtain a test solution.
Example 1:
a daptomycin detection method specifically comprises the following steps:
step 1, precisely weighing about 25mg of daptomycin working standard, placing the daptomycin working standard in a 25ml volumetric flask, adding 10% acetonitrile to dissolve and dilute the daptomycin working standard to a scale, shaking up the solution to prepare a solution containing about 1ml of daptomycin in every 1ml, and using the solution as a reference solution;
precisely weighing about 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10% acetonitrile to dissolve and dilute the daptomycin to a scale, shaking up, and preparing solution containing about 1ml of daptomycin in each 1ml as test solution;
step 2, detecting by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a detection instrument: waterst ACQUITY UPLC H-Class ultra high performance liquid chromatograph;
a chromatographic column: waters ACQUIYT UPLC @ BEH shield RP 181.7 μm 2.1X 150 mm;
mobile phase A: anhydrous sodium sulfate solution (taking 3.46g of anhydrous sodium sulfate, adding 1000ml of water to dissolve, adjusting pH value to 3.4 with phosphoric acid) -acetonitrile (74: 26);
mobile phase B: anhydrous sodium sulfate solution (taking 3.46g of anhydrous sodium sulfate, adding 1000ml of water for dissolution, adjusting pH value to 3.4 with phosphoric acid) -acetonitrile (50: 50);
flow rate: 0.2 ml/mim;
detection wavelength: 223 nm;
column temperature: 30 ℃;
temperature of the sample pan: 5 ℃;
and (3) an elution mode: fluidity A-fluidity B (66:34) isocratic elution for 45 min;
wherein, the anhydrous sodium sulfate grade is chromatographic grade, and the manufacturer is the Aradin; the phosphoric acid grade is AR grade, and the manufacturer is Guangdong Guanghua science and technology limited company; anhydrous sodium sulfate grade is HPLC grade, and the manufacturer is SMA; the daptomycin working standard and the daptomycin to be tested are both produced by Fuxing pharmaceutical Co., Fuzhou, Lizhu, Inc.
Example 2:
a method for detecting daptomycin and related substances thereof specifically comprises the following steps:
1. material
1) Reagent: anhydrous sodium sulfate grade is chromatographic grade, and the manufacturer is alatin; the phosphoric acid grade is AR grade, and the manufacturer is Guangdong Guanghua science and technology limited company; anhydrous sodium sulfate grade is HPLC grade, and the manufacturer is SMA;
2) the test reagents comprise a daptomycin working standard product and a daptomycin to-be-tested product which are all produced by Fuxing medicine of Fuxing of Lizhu group, and daptomycin Lactone hydrolysate standard product (Z1, Lactone hydrolysis product RS), a daptomycin β -Isomer standard product (Z2, β -Isomer RS), a daptomycin hydroxyl derivative standard product (Z3, Impurity 3), a daptomycin isodecanoyl Isomer standard product (Z4, Impurity 4), a daptomycin ethyl Isomer standard product (Z5, Impurity5) and a dehydrated daptomycin standard product (Z6, Anhydro-daptomycin) which are all purchased from Tianjin medicine Kangde New drug development Limited.
2. Solution preparation:
1) solvent: 10% acetonitrile;
2) the standard solution S0 is prepared by precisely weighing 25mg of daptomycin working standard, placing the standard solution in a 25mL volumetric flask, adding a solvent for dissolution, fixing the volume and shaking up;
3) precisely weighing 25mg of daptomycin to be tested in a sample solution S1, placing the sample solution in a 25mL volumetric flask, adding a solvent for dissolving, fixing the volume, and shaking up;
4) precisely weighing 50mg of daptomycin in the sample solution S2, placing the daptomycin in a 50mL volumetric flask, adding a solvent for dissolving, fixing the volume, and shaking up;
5) adding a standard solution S3, precisely weighing 25mg of daptomycin, placing the daptomycin into a 25mL volumetric flask, adding an impurity standard solution to dissolve, fixing the volume, and shaking up;
6) detection limit, quantitative limit
And (4) quantitative limit: finding out the concentration meeting the standard (the signal-to-noise ratio is more than or equal to 10: 1);
detection limit: finding out the concentration meeting the standard (the signal-to-noise ratio is more than or equal to 3: 1);
7) precisely weighing 25mg of daptomycin in a sample solution S4 for a thermal destruction test, putting the daptomycin into a 25mL volumetric flask, adding a solvent to dissolve, fixing the volume, shaking up, heating in a water bath with a proper temperature for a proper time, and then putting the solution in a freezer at 2-8 ℃;
8) the method comprises the following steps of (1) obtaining a test sample solution S5 for the light damage test, taking a proper amount of daptomycin, placing the test sample solution in a light source at a distance of about 10cm and a light intensity of 6.5 x 104lux, precisely weighing 25mg after 10 days of irradiation, placing the test sample solution in a 25mL volumetric flask, adding a solvent for dissolving, fixing the volume, shaking up, and placing the volumetric flask in a freezer at the temperature of 2-8 ℃ for storage;
9) acid breakdown test sample solution S6, precisely weighing 25mg daptomycin, placing in a 25mL volumetric flask, adding 10mL HCl with proper concentration, placing in a freezer at 2-8 ℃ for proper time, adding 10mL NaOH with the same concentration for neutralization, fixing the volume with solvent, shaking up, and placing in a freezer at 2-8 ℃ for storage. Simultaneously performing blank comparison;
10) alkali breakdown test sample solution S7, precisely weighing 25mg daptomycin, placing in a 25mL volumetric flask, adding 10mL NaOH with proper concentration, placing in a freezer at 2-8 ℃ for proper time, adding 10mL HCl with the same concentration for neutralization, adding a solvent to a constant volume, shaking up, and placing in a freezer at 2-8 ℃ for storage. Simultaneously performing blank comparison;
11) the test solution S8 for oxidative destruction test is prepared by precisely weighing 25mg daptomycin, placing the daptomycin into a 25mL volumetric flask, adding 10mL H2O2 with proper concentration, placing the volumetric flask in a 2-8 ℃ freezer for proper time, adding a solvent to a constant volume, shaking up, and placing the volumetric flask in a 2-8 ℃ freezer for storage. Simultaneously performing blank comparison;
12) precisely transferring 5mL of a 25% concentration level test solution S9(250 mu g/mL) into a 20mL volumetric flask, dissolving the test solution in a solvent, fixing the volume, and uniformly mixing;
13) precisely weighing 25mg of daptomycin working standard substance and precisely transferring 25mL of test solution into a 100mL volumetric flask in parallel with 50% concentration horizontal solution S10(500 mug/mL), dissolving with a solvent, fixing the volume, and uniformly mixing;
14) 100% concentration level solution S11(1000 mug/mL), precisely weighing 18.75mg daptomycin working standard, precisely transferring 6.25mL test solution into a 25mL volumetric flask, paralleling three parts, dissolving with solvent, fixing volume, and mixing uniformly;
15) 150% concentration level solution (1500 mug/mL) S12, precisely weighing 25mg daptomycin standard, precisely transferring 5mL test solution into a 20mL volumetric flask, parallelly dissolving with solvent, fixing volume, and mixing;
16) the test solutions S13-S15 are shown in tables 1-4, where the same compounds are present in the same amounts P and the same limits L.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
TABLE 5
Remarking: "+" indicates that the solution was used and "-" indicates that the solution was not used.
3. Content calculation formula of detection method
Wherein:
at: major peak area of daptomycin sample;
as: the average standard peak area of daptomycin 5 needles;
wt: daptomycin sample weight, mg;
ws: daptomycin standard weight, mg;
p: content of daptomycin standard,%.
Single impurity calculation formula:
the total impurity calculation formula is as follows:
wherein:
ai-area of peak of individual impurity;
Σ Ai — peak area of total impurities;
at-peak area of daptomycin.
4. Verification of detection method
1) System adaptability
Injecting 2 mu L of standard solution into UPLC, recording chromatogram, calculating 5 times of sample injection results, wherein RSD of the main peak area of daptomycin is not more than 2.0%, the separation degree between Z3 and the main peak of daptomycin is not less than 1.5, the separation degree between the main peak of daptomycin and Z5 is not less than 1.5, and the tailing factor of daptomycin is not more than 2.0.
The standard is as follows: the RSD of the main peak area of the 5-pin standard and the standard back pin at the end of the sequence is not more than 2.0 percent.
2) Specificity
Solvent: and (4) according to the selected chromatographic conditions, adding a needle solvent, and recording a chromatogram map, wherein the solvent has no interference on the main peak and the impurity peak of the sample.
The standard is as follows: the solvent has no interference to the detection, and the mass is conserved (90-110%).
3) Detection limit and quantification limit
① detection limit
The standard is as follows: the average signal-to-noise ratio is more than or equal to 3:1, the peak area RSD is less than or equal to 20 percent.
② quantitative limit
The standard is as follows: the average signal-to-noise ratio is more than or equal to 10:1, and the peak area RSD is less than or equal to 10 percent.
4) Linearity and range
① daptomycin Linearity
Preparing standard daptomycin standard products into standard solutions with LOQ, 5%, 25%, 50%, 100% and 150% concentration levels respectively, detecting the obtained solution by using an ultra-high performance liquid chromatograph, and drawing a linear curve by using the concentration as x and the peak area as y, wherein the related coefficient r of a linear equation is more than or equal to 0.998.
The standard is as follows: the coefficient r of the linear equation is more than or equal to 0.998, and the ratio of the intercept of the linear equation to the response value when the marked quantity (x is 100%) is less than or equal to 2.0%.
② related to material linearity
Taking daptomycin lactone hydrolysate, β -isomer, Z3, Z4 and Z5 standard substances to prepare mixed standard solutions with LOQ, 25%, 50%, 75%, 100% and 150% concentration levels respectively, taking dehydrated daptomycin standard substances to prepare standard solutions with LOQ, 25%, 50%, 75%, 100% and 150% concentration levels respectively, detecting the obtained solution by using an ultra-high performance liquid chromatograph, drawing a linear curve by taking the concentration as x and the peak area as y, wherein the related coefficient r of a linear equation is more than or equal to 0.998.
The standard is as follows: the coefficient r of the linear equation is more than or equal to 0.998, and the ratio of the intercept of the linear equation to the response value when the marked quantity (x is 100%) is less than or equal to 25%.
5) Relative response factor
Relative response factor is the slope of the linearity of each impurity/the slope of the linearity of daptomycin.
6) Precision degree
① repeatability
The standard is as follows: the same sample is tested for 6 times in parallel, and the RSD between the content results is less than or equal to 2.0 percent.
The standard is as follows: related substances, the limit is less than 0.1 percent, and RSD is less than or equal to 30 percent; the limit is more than or equal to 0.1 percent and less than 0.2 percent, and the RSD is less than or equal to 20 percent; the limit is more than or equal to 0.2 percent and less than 0.5 percent, and the RSD is less than or equal to 10 percent; 0.5% and limit < 5%, and RSD < 5.0%; the limit is more than or equal to 5 percent, and the RSD is less than or equal to 4.0 percent.
② intermediate precision
The standard is as follows: in the same laboratory, different inspectors use different instruments to detect the same test sample on different dates, and the RSD between content results is less than or equal to 2.0 percent.
The standard is as follows: related substances, the limit is less than 0.1 percent, and RSD is less than or equal to 40 percent; the limit is more than or equal to 0.1 percent and less than 0.2 percent, and the RSD is less than or equal to 30 percent; the limit is more than or equal to 0.2 percent and less than 0.5 percent, and the RSD is less than or equal to 15 percent; the limit is more than or equal to 0.5 percent and less than or equal to 5 percent, and the RSD is less than or equal to 7.5 percent; the limit is more than or equal to 5 percent, and the RSD is less than or equal to 4.0 percent.
7) Durability
The column temperature is reduced by 2 ℃, the content and related substances are calculated, and the repeatability result is compared.
The standard is as follows: the RSD between the content results is less than or equal to 2.0 percent.
The column temperature is increased by 2 ℃, the content and related substances are calculated, and the repeatability result is compared.
The standard is as follows: the RSD between the content results is less than or equal to 2.0 percent.
The flow rate was reduced by 10%, the content was calculated with respect to the relevant substances, and the reproducibility results were compared.
The standard is as follows: the RSD between the content results is less than or equal to 2.0 percent.
The flow rate is increased by 10%, the content and related substances are calculated, and the repeatability results are compared.
The standard is as follows: the RSD between the content results is less than or equal to 2.0 percent.
The pH is reduced by 0.1, the content and related substances are calculated, and the repeatability results are compared.
The standard is as follows: the RSD between the content results is less than or equal to 2.0 percent.
The pH is increased by 0.1, the content and related substances are calculated, and the repeatability result is compared.
The standard is as follows: the RSD between the content results is less than or equal to 2.0 percent.
The proportion of the organic phase of the mobile phase A is reduced by 0.5 percent, the content and related substances are calculated,
the standard is as follows: the results of the replicates were compared. The RSD between the content results is less than or equal to 2.0 percent.
The organic phase proportion of the mobile phase A is increased by 0.5 percent, the content and related substances are calculated,
the standard is as follows: the results of the replicates were compared. The RSD between the content results is less than or equal to 2.0 percent.
And (4) replacing different chromatographic columns, calculating the content and related substances, and comparing the repeatability results.
The standard is as follows: the RSD between the content results is less than or equal to 2.0 percent.
8) Accuracy of
① daptomycin content
The recovery rates of daptomycin content at concentration levels of 50%, 100% and 150% were calculated.
The standard is as follows: the content recovery rate of each concentration level is 98.0-102.0%, and the RSD (recovery ratio) is less than or equal to 2.0%; the average recovery rate of daptomycin content is 99.1%, and the RSD is 1.2%.
② related substances
The recovery of each impurity standard solution was calculated at 50%, 100%, 150% concentration levels.
The standard is as follows: the limit is less than 0.5 percent, 80 to 120 percent and the RSD is less than or equal to 10 percent; the limit is more than or equal to 0.5 percent and less than 5 percent, 90 to 110 percent and the RSD is less than or equal to 5.0 percent; the limit is more than or equal to 5 percent, 95 to 105 percent and the RSD is less than or equal to 2.5 percent.
And (3) detection results:
the average recovery rate of lactone hydrolysate was 99.5%, and RSD was 1.4%;
β -average recovery rate of isomer 107.1%, RSD 1.08%;
the average recovery rate of the impurity 3 is 99.3 percent, and the RSD is 3.5 percent;
the average recovery rate of impurity 4 is 101.1%, and RSD is 5.5%;
the average recovery rate of impurity5 is 102.6%, and the RSD is 3.2%;
the average recovery rate of dehydrated daptomycin is 104.6 percent, and the RSD is 2.5 percent.
9) Stability of solution
Standard solution: within a specified time, the method has the characteristic that the peak area RSD of the daptomycin is less than or equal to 2.0 percent through the system adaptability.
As a result: the standard solution daptomycin peak area RSD is 0.22 within 24h, indicating that the standard solution is stable within 24 h.
Test solution: within a specified time, no new chromatographic peak appears, and the peak area RSD of the daptomycin is less than or equal to 2.0 percent. Related substances, the limit is less than 0.1 percent, and RSD is less than or equal to 30 percent; the limit is more than or equal to 0.1 percent and less than 0.2 percent, and the RSD is less than or equal to 20 percent; the limit is more than or equal to 0.2 percent and less than 0.5 percent, and the RSD is less than or equal to 10 percent; 0.5% and limit < 5%, and RSD < 5.0%; the limit is more than or equal to 5 percent, and the RSD is less than or equal to 4.0 percent.
The results showed that the standard solution had a peak area RSD of 0.77% for daptomycin in 12 hours, a content of lactone hydrolysate of 9.8%, a content of β -isomer of 9.07%, a content of impurity 3 of 1.83%, a content of impurity 4 of 2.75%, a content of impurity5 of 1.86%, a content of dehydrated daptomycin of 4.74%, a maximum unknown content of monoimpurity of 7.76%, and a total content of impurity of 1.49%, indicating that the standard solution was stable in 12 hours.
The results of the verification of the above detection method are shown in table 6.
TABLE 6
Example 3:
a daptomycin detection method specifically comprises the following steps:
step 1, selection of materials
1) Reagent: anhydrous sodium sulfate grade is chromatographic grade, and the manufacturer is alatin; the phosphoric acid grade is AR grade, and the manufacturer is Guangdong Guanghua science and technology limited company; anhydrous sodium sulfate grade is HPLC grade, and the manufacturer is SMA;
2) the test reagents comprise a daptomycin working standard product and a daptomycin to-be-tested product which are all produced by Fuxing medicine of Fuxing of Lizhu group, and daptomycin Lactone hydrolysate standard product (Z1, Lactone hydrolysis product RS), a daptomycin β -Isomer standard product (Z2, β -Isomer RS), a daptomycin hydroxyl derivative standard product (Z3, Impurity 3), a daptomycin isodecanoyl Isomer standard product (Z4, Impurity 4), a daptomycin ethyl Isomer standard product (Z5, Impurity5) and a dehydrated daptomycin standard product (Z6, Anhydro-daptomycin) which are all purchased from Tianjin medicine Kangde New drug development Limited.
Step 2, preparing reference substance solution and test solution
1) Precisely weighing about 25mg of daptomycin working standard, placing the daptomycin working standard into a 25ml volumetric flask, adding a solvent (10% acetonitrile) to dissolve and dilute the daptomycin working standard to a scale, shaking up the solution to prepare a solution containing about 1ml of daptomycin working standard per 1ml, and using the solution as a reference solution (standard);
2) precisely weighing about 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding a solvent (10% acetonitrile) to dissolve and dilute the daptomycin to a scale, shaking up, and preparing a solution containing about 1ml of daptomycin in every 1ml as a test solution;
3) a daptomycin lactone hydrolysate standard solution; (see Table 1 for preparation method)
4) Precisely weighing about 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10 mu g/ml of daptomycin lactone hydrolysate (the preparation method is shown in table 1) to dissolve and dilute the daptomycin lactone hydrolysate to scale, shaking up the solution to be used as a daptomycin β -isomer standard solution to be tested;
5) daptomycin β -isomer standard solution (preparation method is shown in Table 1);
6) precisely weighing about 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10 mu g/ml of daptomycin β -isomer standard solution (the preparation method is shown in table 1), dissolving and diluting to scale, shaking up, and taking the solution as the daptomycin β -isomer standard solution to be tested;
7) standard solution of daptomycin hydroxy derivative (preparation method is shown in Table 1)
8) Precisely weighing about 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10 mu g/ml of daptomycin hydroxyl derivative standard solution (the preparation method is shown in table 1), dissolving and diluting to scale, shaking up, and taking the solution as the daptomycin hydroxyl derivative standard solution added to the to-be-tested sample;
9) standard solutions of daptomycin isodecanoyl isomer (see table 1 for formulation);
10) precisely weighing about 25mg of a daptomycin to be detected, placing the daptomycin to be detected in a 25ml volumetric flask, adding 10 mu g/ml of standard solution of the daptomycin isodecanoyl isomer (the preparation method is shown in table 1), dissolving and diluting the solution to scale, shaking up the solution to be detected and adding the standard solution of the daptomycin isodecanoyl isomer;
11) standard solution of daptomycin ethyl isomer (preparation method is shown in table 1);
12) precisely weighing about 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10 mu g/ml of daptomycin ethyl isomer standard solution (the preparation method is shown in table 1), dissolving and diluting to scale, shaking up, and taking the solution as the to-be-tested sample and the daptomycin ethyl isomer standard solution;
13) a dehydrated daptomycin standard solution (preparation method is shown in table 1);
14) precisely weighing about 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10 mu g/ml of dehydrated daptomycin standard solution (the preparation method is shown in table 1), dissolving and diluting to scale, shaking up, and taking the solution as the daptomycin to be tested and adding the dehydrated daptomycin standard solution;
15) LOQ standard solutions (formulation see table 3);
16) LOD standard solution (formulation see Table 3).
And 3, detecting the solvent in the step 2 and the solutions 1) -16) by adopting an ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a detection instrument: waterst ACQUITY UPLC H-Class ultra high performance liquid chromatograph;
a chromatographic column: waters ACQUIYT UPLC @ BEH shield RP 181.7 μm 2.1X 150 mm;
mobile phase A: anhydrous sodium sulfate solution (taking 3.46g of anhydrous sodium sulfate, adding 1000ml of water for dissolution, adjusting the pH value to 3.4 by using phosphoric acid) -acetonitrile (74: 26);
mobile phase B: anhydrous sodium sulfate solution (taking 3.46g of anhydrous sodium sulfate, adding 1000ml of water for dissolution, adjusting the pH value to 3.4 by using phosphoric acid) -acetonitrile (50: 50);
flow rate: 0.2 ml/mim;
detection wavelength: 223 nm;
column temperature: 30 ℃;
temperature of the sample pan: 5 ℃;
sample introduction volume: 2 mu l of the solution;
and (3) an elution mode: fluidity A-fluidity B (66:34) isocratic elution for 45 min;
the results are shown in FIGS. 1-17, which shows that the daptomycin detection method provided by the invention can effectively detect daptomycin and related substances in a sample to be detected and can carry out quantification.
In conclusion, the daptomycin detection method provided by the invention can effectively detect daptomycin and related substances in a sample to be detected, can carry out quantification, has the advantages of good separation degree of main components and impurities, low tail removal factor, low detection period, low detection cost, high sensitivity, high accuracy, high precision, good linearity level, strong specificity, good durability and the like, the specific one-needle running time is only 45min, only 9ml of mobile phase is consumed by one detection needle, the detection period is short, and the detection cost is low; in the method, the sample injection concentration of the main component LOQ of the daptomycin is 0.652ug/ml according to the signal-to-noise ratio of 10:1, the sample injection concentration of the LOD is 0.218ug/ml according to the signal-to-noise ratio of 3:1, and the sensitivity is high; different persons use different instruments to detect the content of the same sample at different times and the reproducibility of related substances is high, namely the precision is high; the blank solvent used in the test has no interference to main components and related substances, and the daptomycin sample can achieve mass conservation after being destroyed under the conditions of high temperature, acid, alkali, oxidation and illumination, and has strong specificity; different chromatographic columns, buffer solution pH, mobile phase organic phase proportion, column temperature and flow rate in chromatographic conditions fluctuate in the invention, so that the detection result is not obviously influenced, and the durability is good; the LOQ concentration level standard, the 5% concentration level standard, the 25% concentration level standard, the 50% concentration level standard, the 100% concentration level standard and the 150% concentration level standard are used for preparing a linear curve, r is 1.0000, and the relative corresponding factors of the impurities to the main components are all above 0.8, so that the linearity is good; the recovery rates of the main components and the impurities of 50 percent, 100 percent and 150 percent meet the requirements, and the accuracy is high. The method has the characteristics of short period, low cost and accurate detection, can be widely popularized and used in production enterprises and detection centers, is beneficial to realizing the quality control of daptomycin, and prepares high-purity and high-quality daptomycin.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (5)
1. A daptomycin detection method is characterized in that ultra-high performance liquid chromatography is adopted for detection, and the chromatographic conditions are as follows:
a chromatographic column: 2.1X 150mm, 1.7 μm RP 18;
mobile phase A: anhydrous sodium sulfate solution-acetonitrile (74: 26);
mobile phase B: anhydrous sodium sulfate solution-acetonitrile (50: 50);
flow rate: 0.2 ml/mim;
detection wavelength: 223 nm;
column temperature: 30 ℃;
temperature of the sample pan: 5 ℃ is adopted.
2. The method for detecting daptomycin according to claim 1, wherein the anhydrous sodium sulfate solution in the mobile phase A and the mobile phase B is prepared by the following steps: 3.46g of anhydrous sodium sulfate is taken, 1000ml of water is added for dissolution, and the pH value is adjusted to 3.4 by phosphoric acid.
3. The method for detecting daptomycin according to claim 1, wherein the elution mode in the detection by ultra high performance liquid chromatography is as follows: flow A-flow B (66:34) isocratic for 45 min.
4. The method for detecting daptomycin according to claim 1, wherein a control solution is prepared before detection by ultra high performance liquid chromatography; the preparation method of the reference substance solution comprises the following steps: precisely weighing 25mg of daptomycin working standard, placing the daptomycin working standard in a 25ml volumetric flask, adding 10% acetonitrile to dissolve and dilute the daptomycin working standard to a scale, and shaking up to obtain a reference substance solution.
5. The method for detecting daptomycin according to claim 1, wherein a test solution is prepared before detection by ultra high performance liquid chromatography; the preparation method of the test solution comprises the following steps: precisely weighing 25mg of daptomycin to be tested, placing the daptomycin to be tested in a 25ml volumetric flask, adding 10% acetonitrile to dissolve and dilute the daptomycin to a scale, and shaking up to obtain a test solution.
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