CN106483235A - The method measuring organic solvent residual content in bortezomib - Google Patents
The method measuring organic solvent residual content in bortezomib Download PDFInfo
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Abstract
The present invention proposes a kind of method measuring organic solvent residual content in bortezomib, and the method includes：By capillary gas chromatography, bortezomib is analyzed, to obtain chromatogram；And it is based on gained chromatogram, determine organic solvent residual content in bortezomib.The proposed by the invention method measuring organic solvent residual content in bortezomib has the characteristics that high efficiency, susceptiveness, specificity and accuracy.
Description
Technical field
The present invention relates to biomedicine field, specifically, the present invention relates in bortezomib Determination of Residual Organic Solvents content inspection
Survey method.
Background technology
Entitled [(the 1R) -3- methyl isophthalic acid-[[(2S) -1- oxygen -3- phenyl -2- [(pyrazinecarboxamide) amino] propyl group] amino] fourth of bortezomib chemistry
Base] boric acid.Molecular formula：C19H25BN4O4, molecular weight：384.24.Its structural formula is as follows：
Bortezomib is applied to the treatment of multiple myeloma patients.
However, the detection method of the organic solvent content of residual still has much room for improvement in current bortezomib.
Content of the invention
It is contemplated that at least solving one of technical problem in correlation technique to a certain extent.The present inventor thinks
The organic solvent methanol of residual, normal hexane, ethyl acetate, oxolane, toluene, dimethylformamide meeting in bortezomib
Impact Drug safety, accordingly, it would be desirable to methanol therein, normal hexane, ethyl acetate, oxolane, toluene, two
Methylformamide is detected.Accordingly it is desirable to the method setting up standard compliant detection related solvents residual, to control its medicine
Quality.For this reason, it is an object of the present invention to proposing a kind of detection meanss of Determination of Residual Organic Solvents in bortezomib.
In a first aspect of the present invention, the present invention proposes a kind of method measuring organic solvent residual content in bortezomib.
According to embodiments of the invention, the method comprises the following steps：By capillary gas chromatography, described bortezomib is carried out
Analysis, to obtain chromatogram；And it is based on chromatogram, determine organic solvent residual content in bortezomib.According to this
In this mensure bortezomib of bright embodiment, the method for organic solvent residual content can be effectively to organic solvent in bortezomib
Residual content is detected.
According to embodiments of the invention, in said determination bortezomib, the method for organic solvent residual content has following additional skill
Art feature：
According to embodiments of the invention, capillary gas chromatography adopts following condition：Detector detects for hydrogen flameionization
Device；Detector temperature is 200～300 DEG C；Injector temperature is 150～250 DEG C；Chromatographic column is the capillary chromatography of middle polarity
Post；Heating schedule is 30～60 DEG C of holding 0～10min, and is maintained to 60～100 DEG C with the ramp of 5～10 DEG C/min
0～10min, then with the ramp of 10～30 DEG C/min to 160～220 DEG C of holding 0～10min, carrier gas is nitrogen or helium；
Flow rate of carrier gas is 0.1～10ml/min.Under the conditions of this detection technique, sample burn in hydrogen flame generation substantial amounts of carbon just from
Son, such that it is able to the residual of organic solvent in higher sensitivity rapid and accurate determination bortezomib.
According to embodiments of the invention, capillary gas chromatography adopts following condition：Detector detects for hydrogen flameionization
Device；Detector temperature is 250 DEG C；Injector temperature is 200 DEG C；Chromatographic column is the capillary chromatographic column of middle polarity；Heat up
Program is 40 DEG C of holding 3min, with the ramp of 5 DEG C/min to 70 DEG C, then with the ramp of 15 DEG C/min to 180 DEG C
Keep 3min；Carrier gas is nitrogen；Flow rate of carrier gas is 1ml/min.According to embodiments of the invention, in this detection technique condition
Under, capillary gas chromatography can be with the residual of organic solvent in higher sensitivity rapid and accurate determination bortezomib.
According to embodiments of the present invention, above-mentioned chromatographic column is with 6%- cyanogen propyl group phenyl -94%- dimethyl polysiloxane copolymer
Capillary chromatographic column for the middle polarity of fixing phase.According to embodiments of the invention, with 6%- cyanogen propyl group phenyl -94%- two
Methyl polysiloxane copolymer be the middle polarity of fixing phase capillary chromatographic column have that separation efficiency is high, analyze speed is fast,
The few feature of amount of samples, such that it is able to efficiently and accurately measure the residual of organic solvent in bortezomib.
According to embodiments of the invention, capillary gas chromatography adopts dimethyl sulphoxide solution as blank liquid, every time
Consumption is 1 microlitre.According to embodiments of the invention, capillary gas chromatography adopt methanol, normal hexane, ethyl acetate,
The mixed solution of oxolane, toluene and dimethylformamide and dimethyl sulfoxide as standard solution, according to the reality of the present invention
Apply example, take methanol 0.7586g, normal hexane 0.0714g, ethyl acetate 1.2562g, oxolane 0.1788g, toluene 0.2264g,
Dimethylformamide 0.2336g, puts in 50ml volumetric flask, is diluted to scale with dimethyl sulfoxide, shake up, as each storing solution,
Precision measures each storing solution 1ml and puts in 100ml volumetric flask respectively, is diluted to scale with dimethyl sulfoxide, shakes up, as each
Standard solution.Meanwhile, according to embodiments of the invention, the concentration of the test solution of capillary gas chromatography is every milliliter
The content of bortezomib is 0.05 gram, and consumption is also 1 microlitre of each sample introduction.The embodiment of the present invention is with quantitation dimethyl sulfoxide
As blank, with the organic solvent solution methanol of residual possible in bortezomib, normal hexane, ethyl acetate, tetrahydrochysene furan
Mutter, toluene and dimethylformamide do standard solution, thus drawing standard control curve, with bortezomib concentration for 0.05 gram
The solution of/milliliter is as test solution.Blank and standard solution selection is dissolubility according to bortezomib and feature is selected
Take, this kind of concentration dose in the range of this technical field normal concentration, such that it is able to quick and precisely be surveyed with higher specificity
Determine the residual of organic solvent in bortezomib.
According to embodiments of the invention, described Elements in Organic Solvents include methanol, normal hexane, ethyl acetate, oxolane,
Toluene and dimethylformamide, in described test sample, methanol concentration is in the range of 14～227 mcg/ml and described
In test sample, the concentration of methanol is to be determined based on following equations：Y=137.208x+607.309, y represent corresponding with methanol
Peak area, x represents the concentration of methanol in described test sample；In described test sample, normal hexane concentration is 5.7～22.8
In the range of mcg/ml, and in described test sample, normal hexane concentration is to be determined based on following equations：
Y=320.53x+132.299, y represent peak area corresponding with normal hexane, and x represents the concentration of normal hexane in described test sample；
In described test sample, ethyl acetate concentration is in the range of 9.3～376 mcg/ml, and acetic acid second in described test sample
Ester concentration is to be determined based on following equations：Y=178.027x+782.959, y represent peak area corresponding with ethyl acetate, x
Represent the concentration of ethyl acetate in described test sample；In described test sample, oxolane concentration 7.2～55 micrograms/
In the range of milliliter, and in described test sample, the concentration of oxolane is to be determined based on following equations：
Y=233.677x+562.3, y represent peak area corresponding with oxolane, and x represents the dense of oxolane in described test sample
Degree；In described test sample, toluene concentration is in the range of 2.7～68 mcg/ml, and toluene in described test sample
Concentration is to be determined based on following equations：Y=470.18x+450.593, y represent peak area corresponding with toluene, and x represents described
The concentration of toluene in test sample；In described test sample, dimethylformamide concentration in the range of 18～74 mcg/ml,
And the concentration of dimethylformamide is to be determined based on following equations in described test sample：Y=147.012x+81.3264, y
Represent peak area corresponding with dimethylformamide, x represents the concentration of dimethylformamide in described test sample.Thus,
The content of residual organic solvent in bortezomib can be more accurately determined further.
In still another aspect of the invention, the present invention proposes a kind of method measuring organic solvent residual content in bortezomib.
According to embodiments of the invention, the method includes：(1) instrument and chromatographic condition：Shimadzu GC-2014 gas chromatograph, adopts
It is the capillary column of fixing phase in order to 6%- cyanogen propyl group phenyl -94%- dimethyl polysiloxane copolymer
(30m*0.32mm*1.80um), using flame ionization ditector (FID), detector temperature is 250 DEG C；Injection port temperature
Spend for 200 DEG C；Heating schedule is 40 DEG C of holding 3min, with the ramp of 5 DEG C/min to 70 DEG C, then with 15 DEG C/min
Ramp to 180 DEG C holding 3min；Carrier gas is nitrogen；Flow rate of carrier gas is 1ml/min.(2) prepare placebo solution：
Measure 1 microlitre of dimethyl sulfoxide as described placebo solution；(3) preparing standard solution：Measure methanol, normal hexane,
Ethyl acetate, oxolane, toluene, appropriate dimethylformamide, plus dimethyl sulfoxide is quantitatively diluted in every 1ml and contains
Described methanol 150 microgram, normal hexane 14.5 microgram, ethyl acetate 250 microgram, oxolane 36 microgram, toluene 44.5
Microgram, the solution of dimethylformamide 44 microgram are as described standard solution；(4) prepare test sample solution：Weigh described
Bortezomib, adds described dmso solution and quantitation is diluted to the solution containing 0.05 gram in every 1ml, as described confession
Test agent solution；And (5) injecting chromatograph, obtain chromatogram, described test sample is obtained according to described chromatogram calculation molten
The species and content of Elements in Organic Solvents in liquid.
Wherein, described Elements in Organic Solvents includes methanol, normal hexane, ethyl acetate, oxolane, toluene and dimethyl methyl
Amide, in described test sample, methanol concentration is in the range of 14～227 mcg/ml, and methanol in described test sample
Concentration be based on following equations determine：Y=137.208x+607.309, y represent peak area corresponding with methanol, and x represents
The concentration of methanol in described test sample；In described test sample, normal hexane concentration in the range of 5.7～22.8 mcg/ml,
And normal hexane concentration is to be determined based on following equations in described test sample：Y=320.53x+132.299, y represent and just own
The corresponding peak area of alkane, x represents the concentration of normal hexane in described test sample；In described test sample, ethyl acetate is dense
Degree is in the range of 9.3～376 mcg/ml, and in described test sample, ethyl acetate concentration is to be determined based on following equations：
Y=178.027x+782.959, y represent peak area corresponding with ethyl acetate, and x represents ethyl acetate in described test sample
Concentration；In described test sample, oxolane concentration in the range of 7.2～55 mcg/ml, and in described test sample
The concentration of oxolane is to be determined based on following equations：Y=233.677x+562.3, y represent peak corresponding with oxolane face
Long-pending, x represents the concentration of oxolane in described test sample；In described test sample, toluene concentration 2.7～68 micrograms/
In the range of milliliter, and in described test sample, the concentration of toluene is to be determined based on following equations：Y=470.18x+450.593,
Y represents peak area corresponding with toluene, and x represents the concentration of toluene in described test sample；In described test sample, diformazan
Base concentration of forma is in the range of 18～74 mcg/ml, and in described test sample, the concentration of dimethylformamide is to be based on
Following equations determine：Y=147.012x+81.3264, y represent peak area corresponding with dimethylformamide, and x represents described
The concentration of dimethylformamide in test sample.
Using said method, can effectively the residual organic solvent content in bortezomib be detected.
According to embodiments of the invention, inventor is entered to the various performances of this assay method by following a series of detection test
Go detection：
According to embodiments of the invention, inventor passes through the employment and suitability test (E ＆ ST) of detection method and specificity is tested to assay method
Reliability is detected.In employment and suitability test (E ＆ ST), there is conventional four parameter：Separating degree, post effect, repeatability and delay
The factor.Wherein separating degree and post effect are two most important parameters.According to embodiments of the invention, accurately weighed methanol
0.7586g, normal hexane 0.0714g, ethyl acetate 1.2562g, oxolane 0.1788g, toluene 0.2264g, dimethyl methyl
Amide 0.2336g, puts in 50ml volumetric flask, is diluted to scale with dimethyl sulfoxide, shake up, as each storing solution, difference
Precision measures 100 microlitres of each storing solution and puts in 10ml volumetric flask, is diluted to scale with dimethyl sulfoxide, shakes up, as each mesh
Mark solvent positioning solution, headspace sampling；Precision measures each storing solution 1ml and puts in same 100ml volumetric flask, sub- with dimethyl
Sulfone is diluted to scale, shakes up, as system suitability solution, headspace sampling；Suitability solution continuous sample introduction 5 pin, analysis
Collection of illustrative plates, it is surprisingly found by the inventors that, methanol, normal hexane, ethyl acetate, oxolane, toluene and dimethylformamide
The relative standard deviation (RSD) of the peak area of continuous sample introduction 5 pin be respectively 1.8%, 1.7%, 0.7%, 0.3%, 0.7%,
1.3% it is seen that the dispersion degree very little that is repeated several times between measurement result, thus, it can be seen that detection according to embodiments of the present invention
Method can be with the residual of organic solvent in higher reliability rapid and accurate determination bortezomib.
In addition, according to embodiments of the invention, inventor tests the susceptiveness to assay method by test limit and is detected.
Test limit refers to can to detect from sample the Cmin of test substance or minimum according to being in the given degree of reliability
One of important indicator that method sensitivity embodies.According to embodiments of the invention, with dimethyl sulfoxide dilution target solvent positioning
Solution, measures test limit, analyzes collection of illustrative plates, and inventor finds, the capillary gas chromatography of the present invention, the minimum inspection of methanol
Going out concentration is 3.79 micrograms/ml, and the concentration limit of normal hexane is 1.71 micrograms/ml, the concentration limit of ethyl acetate
For 2.51 micrograms/ml, the concentration limit of oxolane is 2.15 micrograms/ml, and the minimal detectable concentration of toluene is micro- for 0.91
Gram/ml, the minimal detectable concentration of dimethylformamide is 4.67 micrograms/ml, and this assay method can be fast with higher susceptiveness
The residual of organic solvent in fast Accurate Determining bortezomib.
According to embodiments of the invention, inventor is detected to the range of linearity of this assay method by linear test.Line
Property return be using the regression analyses in mathematical statisticss, to determine complementary quantitative relationship between two or more parameter
A kind of one of statistical analysis technique, be simply linear relationship the most in the dependency relation of variable, if stochastic variable with
There is linear relationship between variable, then the point being obtained by test data, will be dispersed in around a certain straight line.According to the present invention
Embodiment, diluting each storing solution with dimethyl sulfoxide is seven Concentraton gradient, analyzes chromatogram, peak area makees line to concentration
Property return, inventor find, methanol, normal hexane, ethyl acetate, oxolane, toluene and dimethylformamide are certain
Concentration range in be in notable linear relationship with its peak response value, the methanol within the range of falling, normal hexane, ethyl acetate,
The content of oxolane, toluene and dimethylformamide all can be calculated by corresponding linear equation, thus this mensure
Method can be with the residual of organic solvent in Accurate Determining bortezomib.
According to embodiments of the invention, inventor passes through the replica test and Intermediate precision experiment precision to this assay method
Degree is detected.Replica test refers to identical method, same test material, and obtaining at identical conditions is
Consistent degree between row result.Identical condition refers to same operator, same measuring instrument, same place, identical
Process of measurement and repeated measure in the of short duration time.Analysis personnel, analysis date are changed in Intermediate precision test.According to the present invention
Embodiment, take sample about 0.1g, accurately weighed, with standard solution 2ml dissolving as test solution, headspace sampling, even
6 parts of continuous sample introduction, analyzes chromatogram, and inventor finds, continuous six test results RSD are less than 10.0%, and repeatability is good,
Inventor tests the precision to this detection method also by Intermediate precision and is detected, analyzes chromatogram, invention Crinis Carbonisatus
Existing, again smaller than 10.0%, Intermediate precision is good, and this assay method can be with higher for continuous ten second test results RSD
The residual of organic solvent in elaboration rapid and accurate determination bortezomib.
According to embodiments of the invention, inventor is detected to the accuracy of this assay method by recovery test.Return
Yield test is to add a certain amount of standard substance in the sample of detection, tests the response rate of the standard substance adding,
Matrix interference in pre-treatment or test process, the cross-contamination of sample, sample loss, instrument performance etc. can be weighed.Root
According to embodiments of the invention, prepare in every 1ml containing about methanol 150 micrograms/ml, normal hexane 14.5 micrograms/ml, ethyl acetate
250 micrograms/ml, oxolane 36 micrograms/ml, toluene 44.5 micrograms/ml, dimethylformamide 44 micrograms/ml mixing molten
Liquid, as standard solution (100%)；Containing about methanol 120 micrograms/ml, normal hexane 11.6 micrograms/ml, acetic acid in every 1ml
Ethyl ester 200 micrograms/ml, oxolane 28.8 micrograms/ml, toluene 35.6 micrograms/ml, dimethylformamide 35.2 micrograms/ml
Mixed solution, as 80% standard solution；In every 1ml containing about methanol 180 micrograms/ml, normal hexane 17.4 micrograms/ml,
Ethyl acetate 300 micrograms/ml, oxolane 43.2 micrograms/ml, toluene 53.4 micrograms/ml, dimethylformamide 52.8 are micro-
Gram/mixed solution of ml, as 120% standard solution.Respectively with 80% standard solution, 100% standard solution, 120% mark
Quasi- solution sample dissolution, makes the solution of every 1ml about 50mg, as three kinds of sample-adding solution, analyzes chromatogram, inventor
Find, under three concentration, between 80%～120%, RSD is respectively less than 10.0% to the response rate of each solvent, this assay method
The residual of organic solvent in bortezomib can quickly be measured with higher accuracy.
According to embodiments of the invention, above-mentioned capillary gas chromatography detects that the method for organic solvent residual in bortezomib has
High efficiency, susceptiveness, specificity and accuracy, can control effectively to the quality surveying bortezomib.
Brief description
Fig. 1 is the blank chromatogram of system suitability according to embodiments of the present invention and specificity test；
Fig. 2 is the methanol positioning chromatogram of system suitability according to embodiments of the present invention and specificity test；
Fig. 3 is the normal hexane positioning chromatogram of system suitability according to embodiments of the present invention and specificity test；
Fig. 4 is the ethyl acetate positioning chromatogram of system suitability according to embodiments of the present invention and specificity test；
Fig. 5 is the oxolane positioning chromatogram of system suitability according to embodiments of the present invention and specificity test；
Fig. 6 is the toluene positioning chromatogram of system suitability according to embodiments of the present invention and specificity test；
Fig. 7 is the DMF positioning chromatogram of system suitability according to embodiments of the present invention and specificity test；
Fig. 8 is the chromatogram of system suitability according to embodiments of the present invention and the system suitability solution of specificity test；
Fig. 9 is the chromatogram under the organic solvent concentration limit of test limit test according to embodiments of the present invention；
Figure 10 is the chromatogram of the standard solution of replica test according to embodiments of the present invention；
Figure 11 is the chromatogram of the test sample solution of replica test according to embodiments of the present invention；
Figure 12 is the chromatogram of the test solution of replica test according to embodiments of the present invention；
Figure 13 is the chromatogram of the standard solution of recovery test according to embodiments of the present invention；
Figure 14 is the chromatogram of the test sample solution of recovery test according to embodiments of the present invention；
Figure 15 is the chromatogram of 80% sample-adding solution of recovery test according to embodiments of the present invention；
Figure 16 is the chromatogram of 100% sample-adding solution of recovery test according to embodiments of the present invention；And
Figure 17 is the chromatogram of 120% sample-adding solution of recovery test according to embodiments of the present invention.
Specific embodiment
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that following enforcement
Example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or condition in embodiment,
Carry out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument are not noted
Bright production firm person, be can by city available from conventional products.
Conventional method：
Unless expressly stated, in the examples below that chromatography is carried out using following instruments and condition：
Instrument：Shimadzu GC-2014 gas chromatograph
Condition：Adopt the capillary column with 6%- cyanogen propyl group phenyl -94%- dimethyl polysiloxane copolymer as fixing phase
(30m*0.32mm*1.80um), using flame ionization ditector (FID), 250 DEG C of detector temperature, injector temperature
For 200 DEG C, carrier gas is nitrogen, and flow rate of carrier gas is 1ml/min, and heating schedule is：40 DEG C of holding 3min, with 5 DEG C/min's
Ramp, to 70 DEG C, keeps 3min with the ramp of 15 DEG C/min to 180 DEG C.
Embodiment 1 system suitability is tested with specificity
Precision measures 1 microlitre of dimethyl sulfoxide (DMSO) as placebo solution, headspace sampling capillary gas chromatography
Instrument, obtains chromatogram.Chromatogram is as shown in Figure 1.
Respectively accurately weighed methanol 0.7586g, normal hexane 0.0714g, ethyl acetate 1.2562g, oxolane 0.1788g,
Toluene 0.2264g, dimethylformamide (DMF) 0.2336g, are placed in 50ml volumetric flask, use DMSO dissolved dilution
To scale, shake up, as each storing solution.Then, precision measures 100 microlitres of each storing solution and is placed in 10ml volumetric flask respectively,
It is diluted to scale with DMSO, shakes up, position solution, sample introduction chromatograph as each target solvent, obtain chromatogram, chromatogram
As illustrated in figs. 2-7；Precision measures each storing solution 1ml and is placed in same 100ml volumetric flask, is diluted to scale with DMSO,
Shake up, as system suitability solution, sample introduction chromatograph, obtain chromatogram, chromatogram is as shown in Figure 8；System suitability is molten
Liquid continuous sample introduction 5 pin.
Dimethyl sulfoxide and methanol, normal hexane, ethyl acetate, oxolane, toluene, DMF be can be seen that by Fig. 1-8
All can efficiently separate between any two, calculate minimum 3.77 (ethyl acetate and the oxolanes) of separating degree.The statistical system suitability
The spectrum data of solution continuous sample introduction 5 pin is as shown in table 1.
Table 1
| Peak area | std-1 | std-2 | std-3 | std-4 | std-5 | Meansigma methodss | RSD |
| Methanol | 20616 | 20027 | 20100 | 20328 | 19665 | 20147 | 1.8% |
| Normal hexane | 5577 | 5514 | 5649 | 5469 | 5411 | 5524 | 1.7% |
| Ethyl acetate | 48754 | 48911 | 48631 | 48823 | 48001 | 48624 | 0.7% |
| Oxolane | 9493 | 9510 | 9448 | 9501 | 9468 | 9484 | 0.3% |
| Toluene | 22528 | 22705 | 22641 | 22416 | 22299 | 22518 | 0.7% |
| DMF | 6681 | 6669 | 6571 | 6511 | 6503 | 6587 | 1.3% |
As can be seen from Table 1, the RSD of the peak area of system suitability solution continuous sample introduction 5 pin is respectively less than 2%, and this is described
The assay method precision that invention proposes is high, reliability is high.
Embodiment 2 test limit is tested
Target solvent positioning solution in embodiment 1 is diluted with dimethyl sulfoxide, measures test limit, chromatogram such as Fig. 9 institute
Show.
Fig. 9 result shows：In terms of 3 times of noise, the concentration limit recording methanol is 3.79 mcg/ml, normal hexane
Concentration limit be 1.71 mcg/ml, the concentration limit of ethyl acetate is 2.51 mcg/ml, oxolane
Concentration limit be 2.15 mcg/ml, the concentration limit of toluene is 0.91 mcg/ml, the minimum inspection of DMF
Going out concentration is 4.67 mcg/ml.It can be seen that detection method proposed by the present invention is sensitive enough.
Embodiment 3 linear test
Storing solution in Example 1 is appropriate, is diluted to the solution of seven shown in table 2 concentration with dimethyl sulfoxide：
Table 2
Precision measures the solution of each concentration, sample introduction chromatograph respectively, records chromatogram, does linear regression with peak area to concentration.
Test result indicate that, methanol concentration in the range of 14～227 mcg/ml with its peak response value be in notable linear relationship,
Linear equation is：Y=137.208x+607.309, r are 0.9994；Normal hexane concentration in the range of 5.7～22.8 mcg/ml with
Its peak response value is in notable linear relationship, and linear equation is：Y=320.53x+132.299, r are 0.9899；Ethyl acetate concentration
With its peak response value in notable linear relationship in the range of 9.3～376 mcg/ml, linear equation is：
Y=178.027x+782.959, r are 0.9993；Oxolane concentration in the range of 7.2～55 mcg/ml with its peak response value
In notable linear relationship, linear equation is：Y=233.677x+562.3, r are 0.9982；Toluene concentration 2.7～68 micrograms/
With its peak response value in notable linear relationship in the range of milliliter, linear equation is：Y=470.18x+450.593, r are 0.9993；
DMF concentration is in notable linear relationship with its peak response value in the range of 18～74 mcg/ml, and linear equation is：
Y=147.012x+81.3264, r are 0.9976.
Embodiment 4 precision test
The present embodiment is divided into replica test and Intermediate precision to test two parts.
Replica test adopts instrument and method in above-mentioned " conventional method ".Precision measures placebo solution 1ul sample introduction,
Obtain chromatogram；The storing solution 1ml that precision measures in embodiment 1 is placed in same 100ml volumetric flask, dilute with dimethyl sulfoxide
Release to scale, shake up, as standard solution, sample introduction, obtain chromatogram.Chromatogram is as shown in Figure 10.
Weigh sample about 0.1g, with dimethyl sulfoxide 2ml dissolving, as test sample solution, sample introduction, obtain chromatogram.Color
Spectrogram is as shown in figure 11.
Weigh sample about 0.1g, with standard solution 2ml dissolving, as test solution, sample introduction, obtain chromatogram.Chromatogram is such as
Shown in Figure 12.6 parts of continuous sample introduction.
Calculate the content of each target solvent of external source in test solution according to gained chromatograph diagram data, and calculate RSD, experiment knot
Fruit is as shown in table 3：
Table 3
| The response rate | 1 | 2 | 3 | 4 | 5 | 6 | Meansigma methodss | RSD |
| Methanol | 95.3% | 96.7% | 98.9% | 100.5% | 101.9% | 103.1% | 99.4% | 3.0% |
| Normal hexane | 81.1% | 84.3% | 84.0% | 85.0% | 86.9% | 86.2% | 84.6% | 2.4% |
| Ethyl acetate | 93.5% | 96.0% | 97.8% | 97.7% | 99.9% | 100.9% | 97.6% | 2.7% |
| Oxolane | 91.7% | 95.3% | 97.7% | 96.8% | 99.1% | 100.4% | 96.8% | 3.2% |
| Toluene | 94.7% | 97.0% | 99.0% | 98.8% | 101.3% | 102.1% | 98.8% | 2.8% |
| DMF | 87.6% | 89.5% | 89.8% | 96.5% | 95.2% | 98.4% | 92.8% | 4.8% |
Figure 10～Figure 12 and table 3 result show, test result RSD that six parts of continuous sample introduction is less than 10.0%, and the present invention is described
Detection method repeatability good.
Analysis personnel are changed in Intermediate precision test, or change using PE Clarus680 gas chromatograph, and in not same date
Test.
Chromatographic condition is：Adopt the capillary with 6%- cyanogen propyl group phenyl -94%- dimethyl polysiloxane copolymer as fixing phase
Tubing string (30m*0.32mm*1.80um), using flame ionization ditector (FID), 250 DEG C of detector temperature, injection port
Temperature is 200 DEG C, and carrier gas is nitrogen, and flow rate of carrier gas is 1ml/min, and heating schedule is：40 DEG C of holding 3min, with 5 DEG C/min
Ramp to 70 DEG C, with the ramp of 15 DEG C/min to 180 DEG C holding 3min.
Precision measures placebo solution (DMSO) sample introduction, obtains chromatogram.
Accurately weighed methanol 0.7671g, normal hexane 0.0749g, ethyl acetate 1.2674g, oxolane 0.1882g, toluene
0.2315g, DMF0.2254g, put in 50ml volumetric flask, are diluted to scale with dimethyl sulfoxide, shake up, as storing solution,
Precision measures storing solution 1ml and puts in same 100ml volumetric flask, is diluted to scale with dimethyl sulfoxide, shakes up, as standard
Solution, sample introduction, obtain chromatogram.
Take sample about 0.1g, with dimethyl sulfoxide 2ml dissolving, as test sample solution, sample introduction, obtain chromatogram.
Take sample about 0.1g, with standard solution 2ml dissolving, as test solution, sample introduction, 6 parts of continuous sample introduction, obtain chromatograph
Figure.
Calculate the content of each target solvent of external source in test solution according to gained chromatograph diagram data, and six with replica test
Secondary test result calculates RSD together.Experimental result is as shown in table 4.
Table 4
| RSD | Methanol | Normal hexane | Ethyl acetate | Oxolane | Toluene | DMF |
| Replacing personnel | 2.3% | 2.1% | 2.3% | 2.6% | 2.8% | 5.3% |
| Change instrument | 4.8% | 2.7% | 3.3% | 4.1% | 4.2% | 6.4% |
Table 4 result shows, changes personnel and each six test results of instrument and six test results of replica test respectively
Calculate RSD and be respectively less than 10.0%, Intermediate precision is good.
To sum up repeatability and Intermediate precision experiment are as a result, it is possible to prove that this detection method precision is good.Embodiment 5 times
Yield is tested
Precision measures placebo solution DMSO, sample introduction, obtains chromatogram.
Measure the storing solution 0.8ml in embodiment 1, be diluted to 100ml with dimethyl sulfoxide, as 80% standard solution；Amount
Storing solution 1ml in Example 1, is diluted to 100ml with dimethyl sulfoxide, as standard solution；Measure embodiment 1
In storing solution 1.2ml, be diluted to 100ml with dimethyl sulfoxide, as 120% standard solution.
Measure standard solution sample introduction, six parts of parallel sample introduction, obtain chromatogram, chromatogram is as shown in figure 13.
Weigh sample about 0.1g, with dimethyl sulfoxide 2ml dissolving, as test sample solution, sample introduction, obtain chromatogram, color
Spectrogram is as shown in figure 14.
Weigh sample appropriate, respectively with 80% standard solution, 100% standard solution, 120% standard solution dissolving, be made into every
The solution of 1ml about 50mg sample, as three kinds of sample-adding solution, sample introduction respectively.Every kind of three parts of solution allocation of sample-adding.Gained color
Spectrogram is as seen in figs. 15-17.
Calculate the response rate of each target solvent in sample-adding solution according to gained chromatograph diagram data.
Experimental result is as shown in table 5～table 10.
Table 5：Methanol Recovery rate test result
Table 6：Normal hexane response rate test result
Table 7：Ethyl acetate response rate test result
Table 8：Oxolane response rate test result
Table 9：Toluene recovery rate test result
Table 10：DMF recovery rate test result
There is table 5～table 10 can draw：Under three kinds of concentration of standard solution, the response rate of each solvent between 80%～120%,
RSD is respectively less than 10.0%, illustrates that this detection method accuracy is high, the requirement of coincidence detection solvent method.
To sum up embodiment 1～5 can draw, the method for organic solvent residual content in mensure bortezomib proposed by the invention
There is high efficiency, susceptiveness, specificity and accuracy, the quality surveying bortezomib can be control effectively.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. mean specific features with reference to this embodiment or example description, structure, material or
Feature is contained at least one embodiment or the example of the present invention.In this manual, the schematic representation to above-mentioned term
Necessarily it is directed to identical embodiment or example.And, the specific features of description, structure, material or feature are permissible
Any one or more embodiments or example combine in an appropriate manner.Additionally, in the case of not conflicting, ability
The feature of the different embodiments described in this specification or example and different embodiment or example can be entered by the technical staff in domain
Row combines and combines.
Although embodiments of the invention have been shown and described above it is to be understood that above-described embodiment is exemplary,
It is not considered as limiting the invention, those of ordinary skill in the art within the scope of the invention can be to above-described embodiment
It is changed, changes, replacing and modification.
Claims (9)
1. a kind of method measuring organic solvent residual content in bortezomib is it is characterised in that the method includes：
(1) instrument and chromatographic condition
Shimadzu GC-2014 gas chromatograph, adopts and with 6%- cyanogen propyl group phenyl -94%- dimethyl polysiloxane copolymer is
The capillary column (30m*0.32mm*1.80um) of fixing phase, using flame ionization ditector (FID), detector temperature
For 250 DEG C；Injector temperature is 200 DEG C；Heating schedule is 40 DEG C of holding 3min, with the ramp of 5 DEG C/min to 70 DEG C,
Again with the ramp of 15 DEG C/min to 180 DEG C of holding 3min；Carrier gas is nitrogen；Flow rate of carrier gas is 1ml/min,
(2) prepare placebo solution
Measure 1 microlitre of dimethyl sulfoxide as described placebo solution,
(3) preparing standard solution
Measure methanol, normal hexane, ethyl acetate, oxolane, toluene, appropriate dimethylformamide, plus dimethyl sulfoxide
Quantitation is diluted in every 1ml and contains described methanol 150 microgram, normal hexane 14.5 microgram, ethyl acetate 250 microgram, tetrahydrochysene
Furan 36 microgram, toluene 44.5 microgram, the solution of dimethylformamide 44 microgram as described standard solution,
(4) prepare test sample solution
Weigh described bortezomib, add described dmso solution and quantitation is diluted to the solution containing 0.05 gram in every 1ml,
As described test sample solution；And
(5) injecting chromatograph, obtains chromatogram, obtains organic solvent in described need testing solution according to described chromatogram calculation
The species and content of composition,
Wherein, described Elements in Organic Solvents includes methanol, normal hexane, ethyl acetate, oxolane, toluene and dimethyl methyl
Amide,
In described test sample, methanol concentration is in the range of 14～227 mcg/ml, and methanol in described test sample
Concentration is to be determined based on following equations：Y=137.208x+607.309, y represent peak area corresponding with methanol, and x represents institute
State the concentration of methanol in test sample,
In described test sample, normal hexane concentration is in the range of 5.7～22.8 mcg/ml and just own in described test sample
Alkane concentration is to be determined based on following equations：Y=320.53x+132.299, y represent peak area corresponding with normal hexane, and x represents
The concentration of normal hexane in described test sample,
In described test sample, ethyl acetate concentration is in the range of 9.3～376 mcg/ml, and second in described test sample
Acetoacetic ester concentration is to be determined based on following equations：Y=178.027x+782.959, y represent peak area corresponding with ethyl acetate,
X represents the concentration of ethyl acetate in described test sample,
In described test sample, oxolane concentration in the range of 7.2～55 mcg/ml, and four in described test sample
The concentration of hydrogen furan is to be determined based on following equations：Y=233.677x+562.3, y represent peak area corresponding with oxolane,
X represents the concentration of oxolane in described test sample,
In described test sample, toluene concentration is in the range of 2.7～68 mcg/ml, and toluene in described test sample
Concentration is to be determined based on following equations：Y=470.18x+450.593, y represent peak area corresponding with toluene, and x represents described
The concentration of toluene in test sample,
In described test sample, dimethylformamide concentration is in the range of 18～74 mcg/ml, and described test sample
The concentration of middle dimethylformamide is to be determined based on following equations：Y=147.012x+81.3264, y represent and dimethyl formyl
The corresponding peak area of amine, x represents the concentration of dimethylformamide in described test sample.
2. a kind of method measuring organic solvent residual content in bortezomib is it is characterised in that comprise the following steps：
By capillary gas chromatography, described bortezomib is analyzed, to obtain chromatogram；And
Based on described chromatogram, determine organic solvent residual content in described bortezomib.
3. method according to claim 2 is it is characterised in that described capillary gas chromatography adopts following condition：
Detector is flame ionization ditector；
Detector temperature is 200～300 DEG C；
Injector temperature is 150～250 DEG C；
Chromatographic column is the capillary chromatographic column of middle polarity；
Heating schedule is 30～60 DEG C of holding 0～10min, and is maintained to 60～100 DEG C with the ramp of 5～10 DEG C/min
0～10min, then with the ramp of 10～30 DEG C/min to 160～220 DEG C of holding 0～10min,
Carrier gas is nitrogen or helium；
Flow rate of carrier gas is 0.1～10ml/min.
4. method according to claim 3 is it is characterised in that described capillary gas chromatography adopts following condition：
Detector is flame ionization ditector；
Detector temperature is 250 DEG C；
Injector temperature is 200 DEG C；
Chromatographic column is the capillary chromatographic column of middle polarity；
Heating schedule is 40 DEG C of holding 3min, with the ramp of 5 DEG C/min to 70 DEG C, then the speed liter with 15 DEG C/min
Temperature keeps 3min to 180 DEG C；
Carrier gas is nitrogen；
Flow rate of carrier gas is 1ml/min.
5. method according to claim 4 is it is characterised in that described chromatographic column is with 6%- cyanogen propyl group phenyl -94%
- dimethyl polysiloxane copolymer is the capillary chromatographic column of the middle polarity of fixing phase.
6. method according to claim 2 is it is characterised in that described capillary gas chromatography adopts dimethyl sulfoxide
Solution is as blank.
7. method according to claim 2 is it is characterised in that described capillary gas chromatography adopts standard solution,
Described standard solution is dimethyl sulfoxide and methanol, normal hexane, ethyl acetate, oxolane, toluene and dimethylformamide
Mixed solution, and be based on every milliliter of described mixed solution, the amount of methanol is 150 micrograms, the amount of normal hexane is 14.5 micro-
Gram, the amount of ethyl acetate be 250 micrograms, the amount of oxolane be 36 micrograms, the amount of toluene be 44.5 micrograms, dimethyl
The amount of Methanamide is 44 micrograms.
8. method according to claim 2 is it is characterised in that in described capillary gas chromatography method, described boron
Bortezomib is to be provided in the form of test sample solution, and wherein, described test sample solution is that the dimethyl of bortezomib is sub-
Sulfolane solution, and it is based on every milliliter of described test solution, the content of bortezomib is 0.05 gram.
9. method according to claim 8 it is characterised in that described Elements in Organic Solvents include methanol, normal hexane,
Ethyl acetate, oxolane, toluene and dimethylformamide, in described test sample, methanol concentration 14～227 micrograms/
In the range of milliliter, and in described test sample, the concentration of methanol is to be determined based on following equations：Y=137.208x+607.309,
Y represents peak area corresponding with methanol, and x represents the concentration of methanol in described test sample；
In described test sample, normal hexane concentration is in the range of 5.7～22.8 mcg/ml and just own in described test sample
Alkane concentration is to be determined based on following equations：Y=320.53x+132.299, y represent peak area corresponding with normal hexane, and x represents
The concentration of normal hexane in described test sample；
In described test sample, ethyl acetate concentration is in the range of 9.3～376 mcg/ml, and second in described test sample
Acetoacetic ester concentration is to be determined based on following equations：Y=178.027x+782.959, y represent peak area corresponding with ethyl acetate,
X represents the concentration of ethyl acetate in described test sample；
In described test sample, oxolane concentration in the range of 7.2～55 mcg/ml, and four in described test sample
The concentration of hydrogen furan is to be determined based on following equations：Y=233.677x+562.3, y represent peak area corresponding with oxolane,
X represents the concentration of oxolane in described test sample；
In described test sample, toluene concentration is in the range of 2.7～68 mcg/ml, and toluene in described test sample
Concentration is to be determined based on following equations：Y=470.18x+450.593, y represent peak area corresponding with toluene, and x represents described
The concentration of toluene in test sample；
In described test sample, dimethylformamide concentration is in the range of 18～74 mcg/ml, and described test sample
The concentration of middle dimethylformamide is to be determined based on following equations：Y=147.012x+81.3264, y represent and dimethyl formyl
The corresponding peak area of amine, x represents the concentration of dimethylformamide in described test sample.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101406765A (en) * | 2008-10-21 | 2009-04-15 | 浙江大学 | Method for removing organic solvent residual in raw medicine |
| US20130085277A1 (en) * | 2010-02-09 | 2013-04-04 | Ranbaxy Laboratories Limited | Process for the preparation of bortezomib |
| CN103212055A (en) * | 2013-04-19 | 2013-07-24 | 海南锦瑞制药股份有限公司 | Drug composition of bortezomib and preparation method thereof |
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2015
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|---|---|---|---|---|
| CN101406765A (en) * | 2008-10-21 | 2009-04-15 | 浙江大学 | Method for removing organic solvent residual in raw medicine |
| US20130085277A1 (en) * | 2010-02-09 | 2013-04-04 | Ranbaxy Laboratories Limited | Process for the preparation of bortezomib |
| CN103212055A (en) * | 2013-04-19 | 2013-07-24 | 海南锦瑞制药股份有限公司 | Drug composition of bortezomib and preparation method thereof |
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