CN108152435A - The method for measuring epothilone B - Google Patents
The method for measuring epothilone B Download PDFInfo
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- CN108152435A CN108152435A CN201711404783.XA CN201711404783A CN108152435A CN 108152435 A CN108152435 A CN 108152435A CN 201711404783 A CN201711404783 A CN 201711404783A CN 108152435 A CN108152435 A CN 108152435A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses measure epothilone B method, including：Step 1: prepare sample solution；Step 2: setting ultra performance liquid chromatography analysis condition, including：Mobile phase A is the aqueous solution of 0.1~0.5% (V/V) formic acid, and Mobile phase B is methanol or acetonitrile；Using bonded-phase chromatography column；By the way of gradient elution；Step 3: setting flight time Tandem Mass Spectrometry Analysis condition, including：Mode is ionized using ESI；Scan mode Salbutamol Selected Ion Monitoring；Monitor ion m/z 508.27；14 16min of run time；Step 4: the epothilone B standard solution that one group of concentration successively increases is measured, so as to draw out the standard curve of epothilone B；Step 5: being measured to sample solution, the content of epothilone B is calculated from standard curve according to detection data.Detection can be completed in 15min of the present invention；Simple and easy to do, method strong operability, the rate of recovery is high, favorable reproducibility.
Description
Technical field
The invention belongs to medical and health and technical field of analysis and detection more particularly to a kind of methods for measuring epothilone B.
Background technology
Epothilones (epothilone) is a kind of macrolides compound, by German National biotechnology center
(GBF) G.H＆ouml；Fle et al. was reported for the first time in 1993.From the Sorangium cellulosum strain zymotic fluid of slime bacteria suborder
Epothilones can be detached, key component is ebomycin A (Epo A) and epothilone B (Epo B), molecular structure：
The similar taxol tubulin polymerization and the activity of microtubule depolymerization is inhibited to make it a new generation that they have
Anti-mitosis medicine, mechanism of action is similar with taxanes drug, can cause cancer cell suitable with tubulin binding
Profit carries out mitosis, and then cancer cell is made to generate apoptosis.Epothilones is in Antitumor test, antitumor activity, safety, water-soluble
Property and synthetic method etc. are superior to taxol, are expected to develop into antitumor drug more more effective than taxol.At present, angstrom
Rich mycin homologue is used to treat the breast cancer to taxol resistance by Food and Drug Adminstration of the US (FDA) approval.
Ultra performance liquid chromatography (UPLC) technology is the high efficient separation technology that developed recently gets up, and has super-pressure, superelevation
The features such as sensitivity, superelevation separating degree.The characteristics of flight time tandem mass spectrometer (Q-TOF/MS) is highly sensitive, high selection
Property, high quality mass spectrogram and compound accurate molecular quality, UPLC and Q-TOF/MS joint technology (UPLC/Q-TOF/ can be obtained
MS) it is the complicated high efficiency method with trace materials of analysis.
Epothilones is mainly obtained by the method for microbial fermentation, and common detection method is high performance liquid chromatography
(HPLC) analysis method, but have the shortcomings that analysis time is long, amount of samples is larger, solvent-oil ratio is big.
Invention content
It is an object of the invention to solve at least the above, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide one kind.
In order to realize these purposes and other advantages according to the present invention, the method for measuring epothilone B, packet are provided
It includes：
Step 1: prepare epothilone B sample solution；
Step 2: setting ultra performance liquid chromatography analysis condition, including：Mobile phase A is 0.1~0.5% (V/V) formic acid
Aqueous solution, Mobile phase B are methanol or acetonitrile；Using bonded-phase chromatography column；By the way of gradient elution；
Step 3: setting flight time Tandem Mass Spectrometry Analysis condition, including：Mode is ionized using ESI；Scan mode selects
Ion monitoring；Monitor ion m/z 508.27；Run time 14-16min；Wherein, m/z 508.27 is the mass spectrum of epothilone B
Peak；
Step 4: draw standard curve：Under conditions of the step 2 and the step 3 give, using ultra high efficiency liquid
Phase chromatography and flight time tandem mass spectrum are measured the epothilone B standard solution that one group of concentration successively increases, so as to paint
Make the standard curve of epothilone B；
Step 5: determination sample solution：Under conditions of the step 3 and the step 4 give, using ultra high efficiency liquid
Phase chromatography and flight time tandem mass spectrum are measured the sample solution, are fallen into a trap according to detection data from the standard curve
Calculate the content of epothilone B.
Preferably, in the method for the measure epothilone B, in the step 2, ebomycin A and Ai Bo are prepared
The mixed reference substance solution of mycin B；In the step 3, using flight time tandem mass spectrum to the mixed reference substance solution into
Row measures, and the mass spectra peak of epothilone B and ebomycin A is determined in the mass spectral analysis figure, and the mass spectra peak of epothilone B is
M/z 508.27, the mass spectra peak of ebomycin A is m/z 494.00.
Preferably, in the method for the measure epothilone B, in the step 2, when using C18 bonded-phase chromatographies
During column, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 50-60%, and Mobile phase B is in mobile phase
In volumetric concentration be 40-50%, flow velocity 0.2-0.25mL/min；3.5-12min, volume of the mobile phase A in mobile phase
A concentration of 20-30%, volumetric concentration of the Mobile phase B in mobile phase are 70-80%, flow velocity 0.2-0.25mL/min；12.1-
16min, volumetric concentration of the mobile phase A in mobile phase are 50-60%, and volumetric concentration of the Mobile phase B in mobile phase is 40-
50%, flow velocity 0.2-0.25mL/min, wherein, flow velocity remains unchanged in gradient elution program.
Preferably, in the method for the measure epothilone B, in the step 2, when using T3 bonded-phase chromatographies
During column, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 90-100%, and Mobile phase B is flowing
Volumetric concentration in phase is 0-10%, flow velocity 0.25-0.3mL/min；2min, volumetric concentration of the mobile phase A in mobile phase
For 55-65%, volumetric concentration of the Mobile phase B in mobile phase is 35-45%, flow velocity 0.25-0.3mL/min；5min, flowing
Volumetric concentrations of the phase A in mobile phase is 35-45%, and volumetric concentration of the Mobile phase B in mobile phase is 55-65%, and flow velocity is
0.25-0.3mL/min；7min, volumetric concentration of the mobile phase A in mobile phase are 25-35%, and Mobile phase B is in mobile phase
Volumetric concentration is 65-75%, flow velocity 0.25-0.3mL/min；10-13min, volumetric concentration of the mobile phase A in mobile phase are
0-10%, volumetric concentration of the Mobile phase B in mobile phase are 90-100%, flow velocity 0.25-0.3mL/min；15min, flowing
Volumetric concentrations of the phase A in mobile phase is 55-65%, and volumetric concentration of the Mobile phase B in mobile phase is 35-45%, and flow velocity is
0.25-0.3mL/min, wherein, flow velocity remains unchanged in gradient elution program.
Preferably, in the method for the measure epothilone B, in the step 2, the ultra performance liquid chromatography
Analysis condition further includes：Column temperature is 24-30 DEG C；The specification of the bonded-phase chromatography column is：Internal diameter 2.1mm length 50mm, filler
1.8-5 μm of grain size.
Preferably, in the method for the measure epothilone B, in the step 3, the flight time series connection matter
Spectrum analysis condition further includes：250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas is helium, flow 1.2-1.8L/
min；Gas is dried as helium, flow 1.7-2.3L/min；Detect voltage 1.2-1.4kV.
Preferably, in the method for the measure epothilone B, in the step 2, sample solution is prepared, it is specific
Process includes：The reference substance 2-20mg of ebomycin A sum is weighed, is dissolved respectively with methanol, is configured to a concentration of 0.5-2.0mg/
The ebomycin A reference substance solution of mL and epothilone B reference substance solution, draw 1mL respectively, mixing, are configured to mixing control
Product solution.
Preferably, in the method for the measure epothilone B, in the step 1, epothilone B sample is weighed,
It is dissolved with methanol, is configured to the sample solution of a concentration of 1.0-2.0mg/mL.
Preferably, in the method for the measure epothilone B, in the step 2, when using T3 bonded-phase chromatographies
During column, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 90%, and Mobile phase B is in mobile phase
Volumetric concentration for 10%, flow velocity 0.3mL/min；2min, volumetric concentration of the mobile phase A in mobile phase are 55%, flowing
Volumetric concentrations of the phase B in mobile phase is 45%, flow velocity 0.25mL/min；5min, volume of the mobile phase A in mobile phase are dense
It is 35% to spend, and volumetric concentration of the Mobile phase B in mobile phase is 65%, flow velocity 0.25mL/min；7min, mobile phase A are flowing
Volumetric concentration in dynamic phase is 35%, and volumetric concentration of the Mobile phase B in mobile phase is 65%, flow velocity 0.20mL/min；10-
13min, volumetric concentration of the mobile phase A in mobile phase are 10%, and volumetric concentration of the Mobile phase B in mobile phase is 90%, stream
Speed is 0.3mL/min；15min, volumetric concentration of the mobile phase A in mobile phase are 65%, volume of the Mobile phase B in mobile phase
A concentration of 35%, flow velocity 0.3mL/min.
The present invention includes at least following advantageous effect：
(1) present invention is disclosed measures containing for epothilone B using ultra performance liquid chromatography-flight time tandem mass spectrum combination
It measures, detection can be completed in 15min；
(2) assay method of the invention is simple and easy to do, method strong operability, and the rate of recovery is high, favorable reproducibility；
(3) Mass accuracy of epothilone B that measures of the present invention up to 5ppm hereinafter, the high score with liquid chromatogram is from energy
The characteristics of power and mass spectrographic high sensitivity, extremely strong qualitative exclusive specificity.
Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Description of the drawings
Fig. 1 is the flow chart of the method for the present invention for measuring epothilone B in one embodiment；
Fig. 2 is mass spectral analysis figure after the mixed reference substance solution dilution in the embodiment of the present invention one；
Fig. 3 is the ultra performance liquid chromatography analysis chart of the sample solution in the embodiment of the present invention one；
Fig. 4 is the mass spectral analysis figure of the sample solution in the embodiment of the present invention one；
Fig. 5 is the standard curve of the epothilone B in the embodiment of the present invention one.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other element or combinations.
As shown in Figure 1, the present invention provides the method for measuring epothilone B, including：
Step 1: prepare epothilone B sample solution；
Step 2: setting ultra performance liquid chromatography analysis condition, including：Mobile phase A is 0.1~0.5% (V/V) formic acid
Aqueous solution, Mobile phase B are methanol or acetonitrile；Using bonded-phase chromatography column；By the way of gradient elution；
Step 3: setting flight time Tandem Mass Spectrometry Analysis condition, including：Mode is ionized using ESI；Scan mode selects
Ion monitoring；Monitor ion m/z 508.27；Run time 14-16min；Wherein, m/z 508.27 is the mass spectrum of epothilone B
Peak；
Step 4: draw standard curve：Under conditions of the step 2 and the step 3 give, using ultra high efficiency liquid
Phase chromatography and flight time tandem mass spectrum are measured the epothilone B standard solution that one group of concentration successively increases, so as to paint
Make the standard curve of epothilone B；
Step 5: determination sample solution：Under conditions of the step 3 and the step 4 give, using ultra high efficiency liquid
Phase chromatography and flight time tandem mass spectrum are measured the sample solution, are fallen into a trap according to detection data from the standard curve
Calculate the content of epothilone B.
The present invention measures trace epothilone B using ultra performance liquid chromatography and flight time tandem mass spectrum joint technology,
Compared with common HPLC methods, have the characteristics that sample size is small, solvent consumption is few, high sensitivity, qualitative and quantitative accurately carry.
The detection limit of the present invention can reach 0.5ng, quantitatively be limited to 2.0ng, the rate of recovery can reach 97.13%.
Ebomycin A and epothilone B are very close on molecular structure, when being measured to epothilone B, Ai Bo
Mycin A occurs often as impurity.The given ultra performance liquid chromatography analysis condition of the present invention and flight time tandem mass spectrum point
, can be completely separable by ebomycin A and epothilone B under the conditions of analysis, so as to the content of Accurate Determining epothilone B.
In a preferred embodiment, in the method for the measure epothilone B, in the step 2, preparation angstrom
The mixed reference substance solution of rich mycin A and epothilone B；In the step 3, using flight time tandem mass spectrum to described mixed
It closes reference substance solution to be measured, the mass spectra peak of epothilone B and ebomycin A, Ai Bo is determined in the mass spectral analysis figure
The mass spectra peak of mycin B is m/z 508.27, and the mass spectra peak of ebomycin A is m/z 494.00.
The mass spectral analysis condition based on determined by the present invention, can be completely separable by ebomycin A and epothilone B, from
And the content of Accurate Determining epothilone B.
In a preferred embodiment, in the method for the measure epothilone B, in the step 2, work as use
During C18 bonded-phase chromatography columns, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 50-60%, are flowed
Volumetric concentrations of the dynamic phase B in mobile phase is 40-50%, flow velocity 0.2-0.25mL/min；3.5-12min mobile phase A is flowing
Volumetric concentration in dynamic phase is 20-30%, and volumetric concentration of the Mobile phase B in mobile phase is 70-80%, flow velocity 0.2-
0.25mL/min；12.1-16min, volumetric concentration of the mobile phase A in mobile phase are 50-60%, and Mobile phase B is in mobile phase
Volumetric concentration for 40-50%, flow velocity 0.2-0.25mL/min, wherein, flow velocity remains unchanged in gradient elution program.
In a preferred embodiment, in the method for the measure epothilone B, in the step 2, work as use
During T3 bonded-phase chromatography columns, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 90-100%, are flowed
Volumetric concentrations of the dynamic phase B in mobile phase is 0-10%, flow velocity 0.25-0.3mL/min；2min, mobile phase A is in mobile phase
Volumetric concentration for 55-65%, volumetric concentration of the Mobile phase B in mobile phase is 35-45%；5min, mobile phase A is in mobile phase
In volumetric concentration for 35-45%, volumetric concentration of the Mobile phase B in mobile phase is 55-65%, flow velocity 0.25-0.3mL/
min；7min, volumetric concentration of the mobile phase A in mobile phase are 25-35%, and volumetric concentration of the Mobile phase B in mobile phase is
65-75%, flow velocity 0.25-0.3mL/min；10-13min, volumetric concentration of the mobile phase A in mobile phase are 0-10%, are flowed
Volumetric concentrations of the dynamic phase B in mobile phase is 90-100%, flow velocity 0.25-0.3mL/min；15min, mobile phase A are flowing
Volumetric concentration in phase is 55-65%, and volumetric concentration of the Mobile phase B in mobile phase is 35-45%, flow velocity 0.25-
0.3mL/min, wherein, flow velocity remains unchanged in gradient elution program.
In a preferred embodiment, it is described super in the step 2 in the method for the measure epothilone B
Efficient liquid phase chromatographic analysis condition further includes：Column temperature is 24-30 DEG C；The specification of the bonded-phase chromatography column is：Internal diameter 2.1mm long
Spend 50mm, 1.8-5 μm of packing material size.
In a preferred embodiment, it is described to fly in the step 3 in the method for the measure epothilone B
Row time Tandem Mass Spectrometry Analysis condition further includes：250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas is helium,
Flow 1.2-1.8L/min；Gas is dried as helium, flow 1.7-2.3L/min；Detect voltage 1.2-1.4kV.
In a preferred embodiment, in the method for the measure epothilone B, in the step 2, sample is prepared
Product solution, detailed process include：The reference substance 2-20mg of ebomycin A sum is weighed, is dissolved respectively with methanol, is configured to concentration
For the ebomycin A reference substance solution of 0.5-2.0mg/mL and epothilone B reference substance solution, 1mL is drawn respectively, is mixed, is matched
Mixed reference substance solution is made.
In a preferred embodiment, it in the method for described measure epothilone B, in the step 1, weighs angstrom
Rich mycin B samples, are dissolved with methanol, are configured to the sample solution of a concentration of 1.0-2.0mg/mL.
In a preferred embodiment, in the method for the measure epothilone B, in the step 2, work as use
During T3 bonded-phase chromatography columns, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 90%, mobile phase
Volumetric concentrations of the B in mobile phase is 10%, flow velocity 0.3mL/min；2min, volumetric concentration of the mobile phase A in mobile phase
It is 55%, volumetric concentration of the Mobile phase B in mobile phase is 45%, flow velocity 0.25mL/min；5min, mobile phase A are flowing
Volumetric concentration in phase is 35%, and volumetric concentration of the Mobile phase B in mobile phase is 65%, flow velocity 0.25mL/min；7min,
Volumetric concentration of the mobile phase A in mobile phase is 35%, and volumetric concentration of the Mobile phase B in mobile phase is 65%, and flow velocity is
0.20mL/min；10-13min, volumetric concentration of the mobile phase A in mobile phase are 10%, volume of the Mobile phase B in mobile phase
A concentration of 90%, flow velocity 0.3mL/min；15min, volumetric concentration of the mobile phase A in mobile phase are 65%, and Mobile phase B exists
Volumetric concentration in mobile phase is 35%, flow velocity 0.3mL/min.
During gradient elution, change the flow of mobile phase, the efficiency of elution can be further improved, and then improve and survey
Fixed accuracy.
Hereinafter with reference to embodiment, the present invention is described in further detail, however, the present invention is not limited thereto specific implementation
Example.
Embodiment 1
(1) preparation of sample solution：
The preparation of test solution：It is accurately weighed to take Epo B sample 1.0mg, it is dissolved, is configured to a concentration of with methanol
The sample solution of 1.0mg/mL；
(2) preparation of reference substance solution：
Precision weighs the reference substance 2mg of Epo A and Epo B, is dissolved respectively with methanol, is configured to a concentration of 1.0mg/mL's
Reference substance solution draws 1mL, mixed reference substance solution is configured to after mixing respectively.
(3) ultra performance liquid chromatography analysis condition is set
Main Analysis instrument：Ultrahigh pressure liquid phase chromatographic system (1290 ultra performance liquid chromatographies of Agilent), ultra low residual
Autosampler, Diode Array Detector, quadrupole rod-flight time series connection LC-MS instrument (Agilent 6530Q-TOF).
UPLC analysis conditions：Use ultra performance liquid chromatography condition for：Mobile phase：A is the water-soluble of 0.1% (V/V) formic acid
Liquid, B are methanol；Flow velocity is 0.25mL/min；Chromatographic column：Agilent ZORBAX Extended C18 Bonded Phase colors can be used
Compose column (2.1mm × 50mm, 1.8 μm)；Column temperature is 30 DEG C；20 μ L of sample size；Gradient elution program is as shown in the table.
Table 1.C18 bonded-phase chromatography column gradient elution methods
(4) flight time Tandem Mass Spectrometry Analysis condition is set
Mass Spectrometry Conditions：Ionization mode ESI；250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas (He) flow
1.8L/min；Dry gas (He) flow 2.3L/min；Detect voltage 1.4kV；Scan mode Salbutamol Selected Ion Monitoring；Monitor ion m/
z 494.00(A)、508.27(B).Run time 14min；1.0 μ L of sample size.
The mixed reference substance solution of Epo A and Epo B are gradually diluted to the solution that about 1 μ g/mL are made, carry out mass spectrum point
Analysis, sample size are 1.0 μ L.As can be seen from Figure 2 [M-H] of EpoA-It is 492.2430, [M+HCOO]-It is 538.2490, reason
By [M-H] that molecular mass is 493.2498, Epo B-It is 506.259 8, [M+HCOO]-It is 552.2643, Theoretical molecular quality
It is 507.2655.
(5) epothilone B standard curve is drawn：2mgEpo B are weighed, with methanol constant volume in 5ml brown volumetric flasks, -20
DEG C refrigerator is preserved to detection；Then, with 50% methanol dilution, the Epo B marks of 1,5,10,20,50,100 μ g/L are prepared respectively
Quasi- solution；It is measured, obtained by being injected separately into ultra performance liquid chromatography-tandem mass spectrum after 0.45 μm of Teflon filtration film
Epo B standard curves.The detection of the embodiment method is limited to 0.5ng, quantitative limit 2.0ng, and the rate of recovery reaches 97.13%.
(6) measurement result
By step (1) acquired solution by supplying high performance liquid chromatography-tandem mass after 0.6 μm of Teflon filtration film
HPLC-MS/MS is measured.Fig. 3 is the ultra performance liquid chromatography analysis chart of the sample solution in the embodiment of the present invention one, can be seen
Go out, the retention time of Epo B is 4.320min, the retention time of Epo A for 3.807min (since its content is too low, in figure 3
It cannot normally show appearance).Fig. 4 is the mass spectral analysis figure of the sample solution in the embodiment of the present invention one, it can be seen that m/z
508.27 be the mass spectra peak of epothilone B, and m/z 494.00 is the mass spectra peak of ebomycin A.The inspection data obtained passes through
Standard curve (see Fig. 5) obtained by step (5) calculates Epo B contents, and Epo B contents are 98.21%.
Embodiment 2
(1) preparation of sample solution：
The preparation of test solution：It is accurately weighed to take Epo B sample 1.0mg, it is dissolved, is configured to a concentration of with methanol
The sample solution of 2.0mg/mL；
(2) preparation of reference substance solution：
Precision weighs the reference substance 2mg of Epo A and Epo B, is dissolved respectively with methanol, is configured to a concentration of 1.0mg/mL's
Reference substance solution draws 1mL, mixed reference substance solution is configured to after mixing respectively.
(3) ultra performance liquid chromatography analysis condition is set
Main Analysis instrument：Ultrahigh pressure liquid phase chromatographic system (1290 ultra performance liquid chromatographies of Agilent), ultra low residual
Autosampler, Diode Array Detector, quadrupole rod-flight time series connection LC-MS instrument (6530 Q-TOF of Agilent).
UPLC analysis conditions：Use ultra performance liquid chromatography condition for：Mobile phase：A is the water-soluble of 0.5% (V/V) formic acid
Liquid, B are acetonitrile；Flow velocity is 0.2mL/min；Chromatographic column：Agilent ZORBAX Extended C18 bonded-phase chromatographies can be used
Column (2.1mm × 50mm, 5 μm)；Column temperature is 24 DEG C；20 μ L of sample size；Gradient elution program is as shown in the table.
2 C18 bonded-phase chromatography column gradient elution methods of table
(4) flight time Tandem Mass Spectrometry Analysis condition is set
Mass Spectrometry Conditions：Ionization mode ESI；250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas (He) flow
1.2L/min；Dry gas (He) flow 1.7L/min；Detect voltage 1.2kV；Scan mode Salbutamol Selected Ion Monitoring；Monitor ion m/
z 494.00(A)、508.27(B).Run time 16min；1.0 μ L of sample size.
(5) epothilone B standard curve is drawn：2mgEpo B are weighed, with methanol constant volume in 5ml brown volumetric flasks, -20
DEG C refrigerator is preserved to detection；Then, with 50% methanol dilution, the Epo B marks of 1,5,10,20,50,100 μ g/L are prepared respectively
Quasi- solution；It is measured, obtained by being injected separately into ultra performance liquid chromatography-tandem mass spectrum after 0.45 μm of Teflon filtration film
Epo B standard curves.The detection of the embodiment method is limited to 0.5ng, quantitative limit 2.0ng, and the rate of recovery reaches 97.21%.
(6) measurement result
By step (1) acquired solution by supplying high performance liquid chromatography-tandem mass after 0.6 μm of Teflon filtration film
HPLC-MS/MS is measured.The retention time of Epo B is 4.221min, and the retention time of Epo A is 3.813min.m/z 508.27
For the mass spectra peak of epothilone B, m/z 494.00 is the mass spectra peak of ebomycin A.The inspection data obtained passes through step (5)
The standard curve of gained calculates Epo B contents, and Epo B contents are 98.23%.
Embodiment 3
(1) preparation of sample solution：
The preparation of test solution：It is accurately weighed to take Epo B sample 1.0mg, it is dissolved, is configured to a concentration of with methanol
The sample solution of 1.0mg/mL；
(2) preparation of reference substance solution：
Precision weighs the reference substance 2mg of Epo A and Epo B, is dissolved respectively with methanol, is configured to a concentration of 1.0mg/mL's
Reference substance solution draws 1mL, mixed reference substance solution is configured to after mixing respectively.
(3) ultra performance liquid chromatography analysis condition is set
Main Analysis instrument：Ultrahigh pressure liquid phase chromatographic system (1290 ultra performance liquid chromatographies of Agilent), ultra low residual
Autosampler, Diode Array Detector, quadrupole rod-flight time series connection LC-MS instrument (Agilent 6530Q-TOF).
UPLC analysis conditions：Use ultra performance liquid chromatography condition for：Mobile phase：A is the water-soluble of 0.1% (V/V) formic acid
Liquid, B are methanol；Flow velocity is 0.25mL/min；Chromatographic column：T3 bonded-phase chromatographies column (2.1mm × 50mm, 1.8 μm) can be used；Column
Temperature is 30 DEG C；20 μ L of sample size；Gradient elution program is as shown in the table.
3 T3 bonded-phase chromatography column gradient elution methods of table
(4) flight time Tandem Mass Spectrometry Analysis condition is set
Mass Spectrometry Conditions：Ionization mode ESI；250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas (He) flow
1.5L/min；Dry gas (He) flow 2.0L/min；Detect voltage 1.4kV；Scan mode Salbutamol Selected Ion Monitoring；Monitor ion m/
z 494.00(A)、508.27(B).Run time 14min；1.0 μ L of sample size.
(5) epothilone B standard curve is drawn：2mgEpo B are weighed, with methanol constant volume in 5ml brown volumetric flasks, -20
DEG C refrigerator is preserved to detection；Then, with 50% methanol dilution, the Epo B marks of 1,5,10,20,50,100 μ g/L are prepared respectively
Quasi- solution；It is measured, obtained by being injected separately into ultra performance liquid chromatography-tandem mass spectrum after 0.45 μm of Teflon filtration film
Epo B standard curves.The detection of the embodiment method is limited to 0.5ng, quantitative limit 2.0ng, and the rate of recovery reaches 97.22%.
(6) measurement result
By step (1) acquired solution by supplying high performance liquid chromatography-tandem mass after 0.6 μm of Teflon filtration film
HPLC-MS/MS is measured.The retention time of Epo B is 4.227min, and the retention time of Epo A is 3.809min.m/z 508.27
For the mass spectra peak of epothilone B, m/z 494.00 is the mass spectra peak of ebomycin A.The inspection data obtained passes through step (5)
The standard curve of gained calculates Epo B contents, and Epo B contents are 98.26%.
Embodiment 4
(1) preparation of sample solution：
The preparation of test solution：It is accurately weighed to take Epo B sample 1.0mg, it is dissolved, is configured to a concentration of with methanol
The sample solution of 2.0mg/mL；
(2) preparation of reference substance solution：
Precision weighs the reference substance 2mg of Epo A and Epo B, is dissolved respectively with methanol, is configured to a concentration of 1.0mg/mL's
Reference substance solution draws 1mL, mixed reference substance solution is configured to after mixing respectively.
(3) ultra performance liquid chromatography analysis condition is set
Main Analysis instrument：Ultrahigh pressure liquid phase chromatographic system (1290 ultra performance liquid chromatographies of Agilent), ultra low residual
Autosampler, Diode Array Detector, quadrupole rod-flight time series connection LC-MS instrument (Agilent 6530Q-TOF).
UPLC analysis conditions：Use ultra performance liquid chromatography condition for：Mobile phase：A is the water-soluble of 0.5% (V/V) formic acid
Liquid, B are acetonitrile；Flow velocity is 0.2mL/min；Chromatographic column：T3 bonded-phase chromatographies column (2.1mm × 50mm, 5 μm)；Column temperature is 24 DEG C；
20 μ L of sample size；Gradient elution program is as shown in the table.
2 C18 bonded-phase chromatography column gradient elution methods of table
(4) flight time Tandem Mass Spectrometry Analysis condition is set
Mass Spectrometry Conditions：Ionization mode ESI；250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas (He) flow
1.2L/min；Dry gas (He) flow 1.7L/min；Detect voltage 1.2kV；Scan mode Salbutamol Selected Ion Monitoring；Monitor ion m/
z 494.00(A)、508.27(B).Run time 16min；1.0 μ L of sample size.
(5) epothilone B standard curve is drawn：2mgEpo B are weighed, with methanol constant volume in 5ml brown volumetric flasks, -20
DEG C refrigerator is preserved to detection；Then, with 50% methanol dilution, the Epo B marks of 1,5,10,20,50,100 μ g/L are prepared respectively
Quasi- solution；It is measured, obtained by being injected separately into ultra performance liquid chromatography-tandem mass spectrum after 0.45 μm of Teflon filtration film
Epo B standard curves.The detection of the embodiment method is limited to 0.5ng, quantitative limit 2.0ng, and the rate of recovery reaches 97.18%.
(6) measurement result
By step (1) acquired solution by supplying high performance liquid chromatography-tandem mass after 0.6 μm of Teflon filtration film
HPLC-MS/MS is measured.The retention time of Epo B is 4.215min, and the retention time of Epo A is 3.792min.m/z 508.27
For the mass spectra peak of epothilone B, m/z 494.00 is the mass spectra peak of ebomycin A.The inspection data obtained passes through step (5)
The standard curve of gained calculates Epo B contents, and Epo B contents are 98.19%.
Embodiment 5
(1) preparation of sample solution：
The preparation of test solution：It is accurately weighed to take Epo B sample 1.0mg, it is dissolved, is configured to a concentration of with methanol
The sample solution of 1.0mg/mL；
(2) preparation of reference substance solution：
Precision weighs the reference substance 2mg of Epo A and Epo B, is dissolved respectively with methanol, is configured to a concentration of 1.0mg/mL's
Reference substance solution draws 1mL, mixed reference substance solution is configured to after mixing respectively.
(3) ultra performance liquid chromatography analysis condition is set
Main Analysis instrument：Ultrahigh pressure liquid phase chromatographic system (1290 ultra performance liquid chromatographies of Agilent), ultra low residual
Autosampler, Diode Array Detector, quadrupole rod-flight time series connection LC-MS instrument (Agilent 6530Q-TOF).
UPLC analysis conditions：Use ultra performance liquid chromatography condition for：Mobile phase：A is the water-soluble of 0.1% (V/V) formic acid
Liquid, B are methanol；Flow velocity is 0.25mL/min；Chromatographic column：T3 bonded-phase chromatographies column (2.1mm × 50mm, 1.8 μm) can be used；Column
Temperature is 30 DEG C；20 μ L of sample size；Gradient elution program is as shown in the table.
3 T3 bonded-phase chromatography column gradient elution methods of table
(4) flight time Tandem Mass Spectrometry Analysis condition is set
Mass Spectrometry Conditions：Ionization mode ESI；250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas (He) flow
1.5L/min；Dry gas (He) flow 2.0L/min；Detect voltage 1.4kV；Scan mode Salbutamol Selected Ion Monitoring；Monitor ion m/
z 494.00(A)、508.27(B).Run time 14min；1.0 μ L of sample size.
(5) epothilone B standard curve is drawn：2mgEpo B are weighed, with methanol constant volume in 5ml brown volumetric flasks, -20
DEG C refrigerator is preserved to detection；Then, with 50% methanol dilution, the Epo B marks of 1,5,10,20,50,100 μ g/L are prepared respectively
Quasi- solution；It is measured, obtained by being injected separately into ultra performance liquid chromatography-tandem mass spectrum after 0.45 μm of Teflon filtration film
Epo B standard curves.The detection of the embodiment method is limited to 0.5ng, quantitative limit 2.0ng, and the rate of recovery reaches 98.19%.
(6) measurement result
By step (1) acquired solution by supplying high performance liquid chromatography-tandem mass after 0.6 μm of Teflon filtration film
HPLC-MS/MS is measured.The retention time of Epo B is 4.229min, and the retention time of Epo A is 3.841min.m/z 508.27
For the mass spectra peak of epothilone B, m/z 494.00 is the mass spectra peak of ebomycin A.The inspection data obtained passes through step (5)
The standard curve of gained calculates Epo B contents, and Epo B contents are 98.95%.
Embodiment 6
(1) preparation of sample solution：
The preparation of test solution：It is accurately weighed to take Epo B sample 1.0mg, it is dissolved, is configured to a concentration of with methanol
The sample solution of 2.0mg/mL；
(2) preparation of reference substance solution：
Precision weighs the reference substance 2mg of Epo A and Epo B, is dissolved respectively with methanol, is configured to a concentration of 1.0mg/mL's
Reference substance solution draws 1mL, mixed reference substance solution is configured to after mixing respectively.
(3) ultra performance liquid chromatography analysis condition is set
Main Analysis instrument：Ultrahigh pressure liquid phase chromatographic system (1290 ultra performance liquid chromatographies of Agilent), ultra low residual
Autosampler, Diode Array Detector, quadrupole rod-flight time series connection LC-MS instrument (Agilent 6530Q-TOF).
UPLC analysis conditions：Use ultra performance liquid chromatography condition for：Mobile phase：A is the water-soluble of 0.5% (V/V) formic acid
Liquid, B are acetonitrile；Flow velocity is 0.2mL/min；Chromatographic column：T3 bonded-phase chromatographies column (2.1mm × 50mm, 5 μm)；Column temperature is 24 DEG C；
20 μ L of sample size；Gradient elution program is as shown in the table.
2 C18 bonded-phase chromatography column gradient elution methods of table
(4) flight time Tandem Mass Spectrometry Analysis condition is set
Mass Spectrometry Conditions：Ionization mode ESI；250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas (He) flow
1.2L/min；Dry gas (He) flow 1.7L/min；Detect voltage 1.2kV；Scan mode Salbutamol Selected Ion Monitoring；Monitor ion m/
z 494.00(A)、508.27(B).Run time 16min；1.0 μ L of sample size.
(5) epothilone B standard curve is drawn：2mgEpo B are weighed, with methanol constant volume in 5ml brown volumetric flasks, -20
DEG C refrigerator is preserved to detection；Then, with 50% methanol dilution, the Epo B marks of 1,5,10,20,50,100 μ g/L are prepared respectively
Quasi- solution；It is measured, obtained by being injected separately into ultra performance liquid chromatography-tandem mass spectrum after 0.45 μm of Teflon filtration film
Epo B standard curves.The detection of the embodiment method is limited to 0.5ng, quantitative limit 2.0ng, and the rate of recovery reaches 98.18%.
(6) measurement result
By step (1) acquired solution by supplying high performance liquid chromatography-tandem mass after 0.6 μm of Teflon filtration film
HPLC-MS/MS is measured.The retention time of Epo B is 4.217min, and the retention time of Epo A is 3.799.M/z 508.27 is
The mass spectra peak of epothilone B, m/z 494.00 are the mass spectra peak of ebomycin A.The inspection data obtained passes through step (5) institute
The standard curve obtained calculates Epo B contents, and Epo B contents are 98.94%.
Although the embodiments of the present invention have been disclosed as above, but its be not restricted in specification and embodiment it is listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, it is of the invention and unlimited
In specific details and legend shown and described herein.
Claims (9)
1. measure the method for epothilone B, which is characterized in that including：
Step 1: prepare epothilone B sample solution；
Step 2: setting ultra performance liquid chromatography analysis condition, including：Mobile phase A is water-soluble for 0.1~0.5% (V/V) formic acid
Liquid, Mobile phase B are methanol or acetonitrile；Using bonded-phase chromatography column；By the way of gradient elution；
Step 3: setting flight time Tandem Mass Spectrometry Analysis condition, including：Mode is ionized using ESI；Scan mode selects ion
Monitoring；Monitor ion m/z 508.27；Run time 14-16min；Wherein, m/z 508.27 is the mass spectra peak of epothilone B；
Step 4: draw standard curve：Under conditions of the step 2 and the step 3 give, using ultra high efficiency liquid phase color
Spectrum and flight time tandem mass spectrum are measured the epothilone B standard solution that one group of concentration successively increases, so as to draw out
The standard curve of epothilone B；
Step 5: determination sample solution：Under conditions of the step 3 and the step 4 give, using ultra high efficiency liquid phase color
Spectrum and flight time tandem mass spectrum are measured the sample solution, are calculated from the standard curve according to detection data angstrom
The content of rich mycin B.
2. the method for epothilone B is measured as described in claim 1, which is characterized in that in the step 2, preparation angstrom is rich mould
The mixed reference substance solution of plain A and epothilone B；In the step 3, using flight time tandem mass spectrum to the mixing pair
It is measured according to product solution, the mass spectra peak of epothilone B and ebomycin A, epothilone B is determined in the mass spectral analysis figure
Mass spectra peak for m/z 508.27, the mass spectra peak of ebomycin A is m/z 494.00.
3. the method for epothilone B is measured as described in claim 1, which is characterized in that in the step 2, when using C18
During bonded-phase chromatography column, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 50-60%, are flowed
Volumetric concentrations of the phase B in mobile phase is 40-50%, flow velocity 0.2-0.25mL/min；3.5-12min mobile phase A is flowing
Volumetric concentration in phase is 20-30%, and volumetric concentration of the Mobile phase B in mobile phase is 70-80%, flow velocity 0.2-
0.25mL/min；12.1-16min, volumetric concentration of the mobile phase A in mobile phase are 50-60%, and Mobile phase B is in mobile phase
Volumetric concentration for 40-50%, flow velocity 0.2-0.25mL/min, wherein, flow velocity remains unchanged in gradient elution program.
4. the method for epothilone B is measured as described in claim 1, which is characterized in that in the step 2, when using T3 keys
When closing phase chromatographic column, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase be 90-100%, mobile phase
Volumetric concentrations of the B in mobile phase is 0-10%, flow velocity 0.25-0.3mL/min；2min, body of the mobile phase A in mobile phase
A concentration of 55-65% of product, volumetric concentration of the Mobile phase B in mobile phase are 35-45%, flow velocity 0.25-0.3mL/min；
5min, volumetric concentration of the mobile phase A in mobile phase are 35-45%, and volumetric concentration of the Mobile phase B in mobile phase is 55-
65%, flow velocity 0.25-0.3mL/min；7min, volumetric concentration of the mobile phase A in mobile phase are 25-35%, and Mobile phase B exists
Volumetric concentration in mobile phase is 65-75%, flow velocity 0.25-0.3mL/min；10-13min, mobile phase A is in mobile phase
Volumetric concentration is 0-10%, and volumetric concentration of the Mobile phase B in mobile phase is 90-100%, flow velocity 0.25-0.3mL/min；
15min, volumetric concentration of the mobile phase A in mobile phase are 55-65%, and volumetric concentration of the Mobile phase B in mobile phase is 35-
45%, flow velocity 0.25-0.3mL/min, wherein, flow velocity remains unchanged in gradient elution program.
5. the method for the measure epothilone B as described in claim 3 or 4, which is characterized in that described super in the step 2
Efficient liquid phase chromatographic analysis condition further includes：Column temperature is 24-30 DEG C；The specification of the bonded-phase chromatography column is：Internal diameter 2.1mm long
Spend 50mm, 1.8-5 μm of packing material size.
6. the method for epothilone B is measured as claimed in claim 1 or 2, which is characterized in that described to fly in the step 3
Row time Tandem Mass Spectrometry Analysis condition further includes：250 DEG C of desolventizing tube temperature degree；Heat 200 DEG C of deblocking temperature；Atomization gas is helium,
Flow 1.2-1.8L/min；Gas is dried as helium, flow 1.7-2.3L/min；Detect voltage 1.2-1.4kV.
7. the method for epothilone B is measured as described in claim 1, which is characterized in that in the step 2, it is molten to prepare sample
Liquid, detailed process include：The reference substance 2-20mg of ebomycin A sum is weighed, is dissolved respectively with methanol, is configured to a concentration of
The ebomycin A reference substance solution of 0.5-2.0mg/mL and epothilone B reference substance solution, draw 1mL respectively, mix, and prepare
Into mixed reference substance solution.
8. measure the method for epothilone B as described in claim 1, which is characterized in that in the step 1, weigh angstrom win it is mould
Plain B samples, are dissolved with methanol, are configured to the sample solution of a concentration of 1.0-2.0mg/mL.
9. the method for epothilone B is measured as claimed in claim 4, which is characterized in that in the step 2, when using T3 keys
When closing phase chromatographic column, gradient elution program is：0min, volumetric concentration of the mobile phase A in mobile phase are 90%, and Mobile phase B exists
Volumetric concentration in mobile phase is 10%, flow velocity 0.3mL/min；2min, volumetric concentration of the mobile phase A in mobile phase are
55%, volumetric concentration of the Mobile phase B in mobile phase is 45%, flow velocity 0.25mL/min；5min, mobile phase A is in mobile phase
In volumetric concentration for 35%, volumetric concentration of the Mobile phase B in mobile phase is 65%, flow velocity 0.25mL/min；7min, stream
Volumetric concentrations of the dynamic phase A in mobile phase is 35%, and volumetric concentration of the Mobile phase B in mobile phase is 65%, and flow velocity is
0.20mL/min；10-13min, volumetric concentration of the mobile phase A in mobile phase are 10%, volume of the Mobile phase B in mobile phase
A concentration of 90%, flow velocity 0.3mL/min；15min, volumetric concentration of the mobile phase A in mobile phase are 65%, and Mobile phase B exists
Volumetric concentration in mobile phase is 35%, flow velocity 0.3mL/min.
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| CN112630369A (en) * | 2020-12-18 | 2021-04-09 | 卓和药业集团有限公司 | High performance liquid chromatography detection and analysis method for content of epothilone B |
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2017
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Non-Patent Citations (2)
| Title |
|---|
| 王堃 等: "UPLC-Q-TOF/MS分析埃博霉素B", 《中国生化药物杂志》 * |
| 穆雅楠 等: "LC-MS/MS法测定人血中埃博霉素B浓度及其在Ⅰ期临床药代动力学研究中的应用", 《中国药科大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112630369A (en) * | 2020-12-18 | 2021-04-09 | 卓和药业集团有限公司 | High performance liquid chromatography detection and analysis method for content of epothilone B |
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