CN107121505B - A method of measurement Dalbavancin impurity - Google Patents
A method of measurement Dalbavancin impurity Download PDFInfo
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- CN107121505B CN107121505B CN201710238376.XA CN201710238376A CN107121505B CN 107121505 B CN107121505 B CN 107121505B CN 201710238376 A CN201710238376 A CN 201710238376A CN 107121505 B CN107121505 B CN 107121505B
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Abstract
The present invention relates to Pharmaceutical Analysis technical fields, disclose the method for detection Dalbavancin impurity.The present invention is using the 3- dimethylaminopropylamine in direct-injection technique detection Dalbavancin.Method provided by the invention is easy to operate, general, requires gas phase relevant device low；Measuring method detection sensitivity height provided by the invention has high-recovery.Detection method provided by the invention improves the quality and drug safety of Dalbavancin.
Description
Technical field
The invention belongs to medicine analysis technical field, in particular to 3- dimethylaminopropylamine in a kind of measurement Dalbavancin
Analysis method.
Background technique
Dalbavancin (Dalbavancin, structural formula are as follows), also referred to as dalbavancin, it is a kind of novel hemizygous saccharogenesis
Peptide antibiotic is the derivative of teicoplanin analog A40926.It is also the granted use of only one that hydrochloric acid Dalbavancin, which is first,
In the intravenous antibiotics of the two-dose schedule of ABSSSI treatment.Dalbavancin mechanism of action and vancomycin and teicoplanin
It is identical, inhibit the biosynthesis of G+ bacterium cell wall, is widely used as treating the drug of Skin and soft tissue infection.Dalbavancin is
One lipophilic side chain is added to a kind of glycopeptide main chain of enhancing by the second generation, semi-synthetic glycosylated adiponectin polypeptide, and the medicine is in vitro to a variety of
Gram-positive bacteria (including
MRSA and micrococcus scarlatinae) and other streptococcus kind bacteriums show bactericidal activity.Dalbavancin has only
Special pharmacokinetics, can every weekly interval medication.Currently, Dalbavancin is in the relevant Hematogenous infection for the treatment of conduit and skin
Good effect has been achieved with soft tissue infection, has been ideal second with excellent antibacterial activity in vivo and safety
For glycopeptide antibiotic.
Use 3- dimethylaminopropylamine as one of raw material in Dalbavancin synthesis process, to may deposit in final finished
In the residual of 3- dimethylaminopropylamine.
3- dimethylaminopropylamine (abbreviation DMAPA), N, N- dimethyl -1,3- propane diamine, English entitled N, N-Dimethyl-
1,3-propane diamine, CAS registration number are 109-55-7, and molecular formula is C5H16N2, and molecular weight 104.1928 is colourless
Liquid has ammonia taste.DMAPA density 0.8100g/cm2, -70 DEG C of freezing point, 134 DEG C of boiling point, molecular weight 102.DMAPA chemistry
Performance is active, can be dissolved in water and organic solvent, has stronger volatility, toxicity, corrosivity, has stronger thorn to skin and eyes
Swash property, contain primary amine groups simultaneously, has and generate the potentially possible of nitrosamines or nitroso compound.Therefore, it establishes and suitably reaches
3- dimethylaminopropylamine detection method of content carries out strict control to it with regard to very necessary in Ba Wanxing.
A kind of Chinese patent " micro N, the analysis method of N- dimethylated propyl diethylenetriamine in measurement amphoteric surfactant " (application
The micro N in measurement amphoteric surfactant number 20111015766.1) is disclosed, the analysis method of N- dimethylated propyl diethylenetriamine: is adopted
It is analyzed with the chromatography of ions, chromatographic test strip part is as follows: chromatographic column: cation chromatographic column；Mobile phase: contained by volume basis
The organic solvent composition that the inorganic acid or organic acid aqueous acid and volumn concentration that amount is 70-100% are 0-30%, it is described
The molar concentration of inorganic acid or aqueous solutions of organic acids is 1-10mmol/l；Detector: electric conductivity detector is used；Flow rate of mobile phase:
0.5-1.5ml/min.Organic solvent is methanol or acetonitrile.The chromatography of ions disclosed in this patent, using cation separation column,
Electric conductivity detector analyzes the content of 3- dimethylaminopropylamine in amphoteric surfactant, and method discusses 3- dimethylaminopropylamine standard
Linear situation, the linear and recycling to 3- dimethylaminopropylamine in surfactant samples is not studied.Using
Cation separation column and electric conductivity detector are expensive, high operation requirements, and working service is at high cost.It is used using liquid chromatogram
A large amount of acid flux material and organic solvent, testing cost are high and not environmentally friendly enough.The patent is measured using acid flux material sample dissolution
N, N- dimethylated propyl diethylenetriamine, and hydrochloric acid Dalbavancin dissolubility in acid flux material is poor, which can not be suitable for hydrochloric acid
The measurement of N in Dalbavancin, N- dimethylated propyl diethylenetriamine.The present invention finds a kind of gas chromatography for being different from principle, behaviour
Make simply, versatile, system robustness is good, has good stability, and is suitble to 3- dimethylamino third in acid compound Dalbavancin
Amine content detection method, testing cost is low, environmental protection.
Document " measurement of 3- dimethylaminopropylamine in alkylamide propyl-betaine " (summer hero swallow etc., Guangdong, 2013,
40 (19) 152-153) it discloses using solid-phase extraction column pretreatment gas chromatography analysis method.This method is to sample pre-treatments
It is complicated for operation, height is required to operator, poor universality, same amount TIANZHU XINGNAO Capsul difference is not big.In addition, this method to measurement up to bar
The 3- dimethylaminopropylamine of Wan Xingzhong is not applicable, because Dalbavancin dissolubility in adopted ethanol-water system is poor, it is difficult to
Meet measurement to require, and the 3- dimethylaminopropylamine combined in Dalbavancin can not form unhindered amina, cannot effectively extract and
Vaporization.
Because drug is to the high request of final product purity, the residual content of 3- dimethylaminopropylamine is very low, using conventional point
When analysis method determination of acid-basetitration, the sensitivity and accuracy of general titration are not reached requirement.
3- dimethylaminopropylamine has been reported that without UV absorption wavelength using fluorescence derivation liquid chromatogram, fluorescence detector
It is detected, but this method need to carry out fluorescent derivatization processing to 3- dimethylaminopropylamine, complicated for operation, pre-treatment is cumbersome.
The aobvious alkalinity of 3- dimethylaminopropylamine, and Dalbavancin shows stronger acidity, therefore cannot be using conventional neutrality or acid
Property solvent dissolution after, direct injected gas chromatography measurement.Numerous acidity in 3- dimethylaminopropylamine and Dalbavancin structure
Group in conjunction with and the vaporization that cannot dissociate, can not be replaced using triethylamine free.Hydrochloric acid Dalbavancin and its Dalbavancin
Free alkali dissolubility in numerous solvents is poor, limits the selection of solvent.Dalbavancin is soluble in sodium hydroxide solution, and hydrogen-oxygen
Change sodium solution and contain a large amount of inorganic bases, is unfavorable for measuring using gas chromatography solution direct injected.Due to 3- dimethylamino third
Amine has very strong polarity, it is difficult to suitable solvent be selected to realize that effective liquid phase is extracted.
Summary of the invention
The present invention in order to overcome Dalbavancin dissolubility in the prior art is poor, the 3- dimethylaminopropylamine in Dalbavancin without
The effectively free vaporization of method effectively liquid phase the technological deficiencies such as cannot extract, and provide a kind of simple general-purpose, detection sensitivity high detection
The method of 3- dimethylaminopropylamine in Dalbavancin.
Dalbavancin mentioned by the present invention, free alkali containing Dalbavancin and Dalbavancin hydrochloride.
Detection method provided by the invention dissolves acid compound Dalbavancin using strong base solution, and will combine
At the 3- dimethylaminopropylamine displacement release of salt at 3- dimethylaminopropylamine free alkali.Utilize intensive polar solvent acetonitrile and aqueous slkali
The phenomenon that immiscible generation is layered carries out 3- dimethylaminopropylamine highly polar in Dalbavancin strong base solution using acetonitrile
Liquid-liquid extraction adds the extraction effect of the inorganic salts enhancing acetonitrile of saturation, using versatile flame ionization detector,
Acetonitrile layer (upper layer) sample introduction is taken using direct injected gas chromatography, so that highly basic be avoided to enter chromatographic system damage system.
Specifically, the present invention is addressed by following technical proposals:
A, prepared by test solution: precision weighs Dalbavancin, by mass volume ratio 1:5~50 be added concentration for 0.5~
The strong base solution of 5mol/L；Acetonitrile is added after dissolution, acetonitrile and strong base solution volume ratio are 1:0.5~10；Add inorganic salts,
The mass volume ratio of inorganic salts and strong base solution is 1:0.5~10 (g/ml)；Shaking is sufficiently mixed, stratification, draws upper layer
Clear liquid, it is spare as test solution；
B, prepared by reference substance solution: precision weighs 3- dimethylaminopropylamine, and it is molten that the highly basic that concentration is 0.5~5mol/L is added
The solution of 50 μ g of dimethylaminopropylamine containing 3-, 100 μ g, 150 μ g respectively is made in every 1ml in liquid, quantitative dilution；Precision measures above-mentioned
Solution adds with above-mentioned liquor capacity than the acetonitrile for 1:0.5~10, in addition stating solution quality volume ratio is 1:0.5~10 (g/
Ml inorganic salts), shaking are sufficiently mixed, stratification, draw supernatant liquor, spare as reference substance solution (1)~(3)；
C, it is detected with gas chromatography, by calibration curve method with the content of calculated by peak area 3- dimethylaminopropylamine.
Wherein the highly basic is sodium hydroxide and/or potassium hydroxide, preferably sodium hydroxide.The present invention selects highly basic molten
Liquid solves the problems, such as insoluble in other numerous solvents, guarantees the highly dissoluble of Dalbavancin, conducive to high detection spirit is guaranteed
Sensitivity.The alkalinity of highly basic is better than triethylamine, can reach 3- dimethylaminopropylamine forming free alkali, to ensure that 3- diformazan ammonia
Base propylamine can completely vaporize in gas-chromatography.Meet requirement of the rate of recovery between 80%~120%, therefore can accurate quantitative analysis inspection
It out may remaining 3- dimethylaminopropylamine in Dalbavancin.
In order to guarantee that the good dissolution of sample and 3- dimethylaminopropylamine are good free, the concentration of highly basic is selected as 0.5
~5mol/L, preferably 2~3mol/L.If strong base solution concentration is too low, 3- dimethylaminopropylamine cannot dissociate completely, average to return
Yield is low, and being unable to may remaining 3- dimethylaminopropylamine in accurate quantitative analysis detection Dalbavancin.
In the step of wherein described A, the quality volume of Dalbavancin and strong base solution is preferably 1:10.
The step of wherein described A and B in, the volume ratio of acetonitrile and strong base solution is preferably 1:0.5~2, more preferably 1:
1.Present invention employs strong base solution-acetonitrile immiscible systems to extract, and forms the layering of water phase organic phase, solves acetonitrile-water body
The problem of system dissolves each other.The present invention is extracted using acetonitrile, solves the problems, such as that other weak polar solvents cannot be extracted effectively.It returns simultaneously
The problem of strong base solution contains a large amount of inorganic bases and salt, is unfavorable for direct injected is kept away.
Wherein the inorganic salts are that sodium chloride, potassium chloride, anhydrous sodium sulfate are any one or more of.Preferably chlorination
Sodium.
Wherein the mass volume ratio of the inorganic salts and strong base solution of the addition is 1:2.
Wherein inorganic salts are added for first addition acetonitrile or first in the sequence of the acetonitrile and inorganic salts of the addition.
The present invention can use various gas chromatographs, for example model Agilent 7890B, column model be
Agilent DB-624: it can be using -94% dimethyl polysiloxane of 6% cyanogen propyl phenyl as the quartz capillary of fixer
Column, 1.8 μm of column length 30m, internal diameter 0.32mm, the thickness of liquid film of the quartz capillary column.
Test solution and control solution are injected separately into gas chromatograph, the gas-chromatography column condition are as follows:
Column temperature: temperature programming；
Injector temperature: 230~270 DEG C；
Flame ionization detector temperature: 280~320 DEG C；
Carrier gas: nitrogen；
Flow rate of carrier gas: 0.5~2ml/min；
Split ratio: 1:1~30:1,
The direct injected volume for injecting gas chromatograph is 1~3 μ l.
Preferably, gas-chromatography column condition are as follows:
Column temperature: temperature programming method is 60 DEG C of initial temperature and is kept for 8 minutes, is then warming up to 20 DEG C of rate per minute
It 220 DEG C, is kept for 8 minutes；
Injector temperature: 250 DEG C；
Flame ionization detector temperature: 300 DEG C；
Carrier gas: nitrogen；
Flow rate of carrier gas: 1.5ml/min；
Split ratio: 5:1；
Direct injected volume is 2 μ l.
When detecting, it takes reference substance solution continuous sample introduction 5 times, the relative standard deviation of 3- dimethylaminopropylamine peak area is not
2.0% was obtained, to take reference substance solution (1)~(3) concentration-peak area mapping, calculates equation of linear regression, related coefficient is not
It obtains and is lower than 0.995.Gas chromatograph is injected in accurate test solution and control solution (1)~(3) that measure respectively, successively records
Chromatogram.By calibration curve method, with 3- dimethylaminopropylamine peak area, 3- dimethylaminopropylamine contains in calculating test solution
Amount.
Compared with prior art, the present invention selects direct injected to solve headspace sampling instrument system to 3- dimethylamino third
The absorption residue problem of amine.
The measuring method of 3- dimethylaminopropylamine in Dalbavancin, this method need methodology validation, including blank test,
Precision test, detection limit and quantitative limit, linear test and recovery test, wherein linear test is verified in the measuring method,
Include the following steps:
1) sodium hydroxide solution of 2.5mol/L is prepared, it is spare.
2) precision weighs reference substance 3- dimethylaminopropylamine 100mg and is placed in 100ml volumetric flask, the hydrogen described in step 1)
Sodium hydroxide solution is diluted to the solution of 1000 μ g/ml, mixes, spare.
3) pipette 0.1 respectively, 0.25,0.5,1.0,1.5,2.0ml in 10ml measuring bottle, the hydroxide described in step 1)
Sodium solution dilutes constant volume, shakes up, accurate respectively to measure the 1ml solution, sets in tool plug test tube, adds the sodium hydroxide of 2.5mol/L molten
Liquid 1ml shaking makes to dissolve, and adds acetonitrile 1ml, adds sodium chloride 0.5g, and shaking is sufficiently mixed, stratification, draws supernatant liquor, makees
It is spare for solution to be measured.
4) accurate respectively to measure each 2 μ l sample introduction of sample in step 3), record chromatogram.
5) using concentration as x-axis, peak area is y-axis, makees linear regression.
Recovery test is wherein verified in the measuring method, and steps are as follows:
1) it is molten for 80 μ g/ml, 100 μ g/ml, the sodium hydroxide of 120 μ g/ml that the concentration of 3- dimethylaminopropylamine is prepared respectively
Liquid；The concentration of sodium hydroxide solution is 2.5mol/L；The concentration of the 3- dimethylaminopropylamine be 80 μ g/ml, 100 μ g/ml,
The sodium hydroxide solution of 120 μ g/ml is reference substance solution；
2) precision weighs 0.1g Dalbavancin in tool plug test tube, after reference substance solution dissolution in step 1), adds acetonitrile
1ml adds sodium chloride 0.5g, and shaking is sufficiently mixed, stratification, measures 2 μ l sample introduction of supernatant liquor, records chromatogram；
3) reference substance solution that Dalbavancin is not added separately is taken, be measured in the same method, record chromatogram, calculated by calibration curve method
The rate of recovery.
The present invention uses direct-injection technique, easy to operate, low to the requirement of gas phase relevant device, has evaded headspace sampling
Mode bulk pharmaceutical chemicals and the big disadvantage of solvent-oil ratio do not occur direct injected bring appearance complexity yet and damage lacking for pillar
It falls into.Conventional capillary chromatographic column of the present invention and hydrogen flame gas chromatography, simple general-purpose；Survey provided by the invention
It is high to determine method detection sensitivity, concentration limit is up to the 5.67 even lower concentration of μ g/ml；Measuring method tool provided by the invention
There is a high-recovery, the rate of recovery is up to 84.24%.The present invention successfully provides the detection method of 3- dimethylaminopropylamine, to have
Help improve the quality and drug safety of Dalbavancin.
Method provided by the present invention can be used for the detection of acid compound neutral and alkali solvent, such as acetate compound, salt
Phosphate compounds and triethylamine, diethylamine, piperazine, pyridine or other basic solvents in other various acid compounds.
Detailed description of the invention
Fig. 1 is 3- dimethylaminopropylamine reference substance solution chromatogram in embodiment 1.
Fig. 2 is Dalbavancin sample detection chromatogram in embodiment 1.
Fig. 3 is blank solution chromatogram in embodiment 1.
Fig. 4 is 3- dimethylaminopropylamine detection limit chromatogram in embodiment 1.
Fig. 5 is 3- dimethylaminopropylamine quantitative limit chromatogram in embodiment 1.
Fig. 6 is the linear relationship chart of 3- dimethylaminopropylamine in embodiment 1.
Specific embodiment
1, instrument:
Assay balance, model: METTLER TOLEDO ABS135-S；
Gas chromatograph model Agilent 7890B, column model are Agilent DB-624: with 6% cyanogen propyl
- 94% dimethyl polysiloxane of phenyl is the quartz capillary column of fixer, column length 30m, the internal diameter of the quartz capillary column
1.8 μm of 0.32mm, thickness of liquid film.
2, reagent:
3- dimethylaminopropylamine (reagent is pure), sodium hydroxide (analysis is pure), acetonitrile (chromatographically pure).
3, sample:
Dalbavancin, lot number: 20161201,20161202,20161203.
Below with reference to embodiment, the present invention is further elaborated, but these embodiments do not constitute any limit to the present invention
System.
Embodiment 1
Chromatographic condition:
Column temperature: temperature programming: 60 DEG C of initial temperature are kept for 8 minutes, are then warming up to 220 with 20 DEG C of rate per minute
DEG C, it is kept for 8 minutes.
Injector temperature: 250 DEG C；
Flame ionization detector temperature: 300 DEG C；
Carrier gas: nitrogen；
Flow rate of carrier gas: 1.5ml/min；
Split ratio: 5:1.
1, it detects
A, prepared by test solution:
The sodium hydroxide solution of 2.5mol/L is prepared, it is spare；
Dalbavancin about 0.1g is taken, it is accurately weighed, it sets in tool plug test tube, adds prepared sodium hydroxide solution 1ml, shake
Make to dissolve, add acetonitrile 1ml, add sodium chloride 0.5g, shaking is sufficiently mixed, stratification, draws supernatant liquor, molten as test sample
Liquid, it is spare；
B, prepared by reference substance solution: precision weighs 3- dimethylaminopropylamine 100mg, sets in 100ml measuring bottle, adds prepared
Sodium hydroxide solution quantitatively dilutes the solution for being made and containing 1000 μ g in every 1ml respectively, then fixed with prepared sodium hydroxide solution
The solution containing 50 μ g, 100 μ g, 150 μ g respectively is made in every 1ml in amount dilution, and precision measures 1ml, sets in tool plug test tube, add acetonitrile
1ml adds sodium chloride 0.5g, and shaking is sufficiently mixed, stratification, draws supernatant liquor, standby as reference substance solution (1)~(3)
With；
C, detection method: measuring referring to Chinese Pharmacopoeia version general rule 0521 in 2015, using direct injected gas chromatography,
It takes reference substance solution continuous sample introduction 5 times, the relative standard deviation of 3- dimethylaminopropylamine peak area must not cross 2.0%, to take pair
According to product solution (1)~(3) concentration-peak area mapping, equation of linear regression is calculated, related coefficient must not be lower than 0.995.Respectively
Precision measures each 2 μ l in test solution and control solution (1)~(3), injects gas chromatograph, successively records chromatogram, 3-
Dimethylaminopropylamine reference substance solution chromatogram is as shown in Figure 1,0161201 batch of detection chromatogram such as Fig. 2 institute of Dalbavancin sample
Show.The content of 3- dimethylaminopropylamine in test solution is calculated with 3- dimethylaminopropylamine peak area by calibration curve method.
2, calculation formula:
Equation of linear regression Y=AX+B
3- dimethylaminopropylamine residual quantity (%)=(Yi-B)/A × V/W × 100%；
Wherein X:3- dimethylaminopropylamine reference substance solution concentration (mg/ml)；
The main peak area of Y:3- dimethylaminopropylamine reference substance solution；
A: equation of linear regression slope；
B: equation of linear regression intercept；
Yi: the peak area of 3- dimethylaminopropylamine in test solution；
W: the sampling amount (mg) of sample when test solution is prepared；
V: test solution dose volume (ml)；
3, measurement result
Three batches of Dalbavancin test samples are detected according to said determination method, is as a result not detected, is shown in Table 1.
Table 1:
| Lot number | 3- dimethylaminopropylamine content (%) |
| 20161201 | It is not detected |
| 20161202 | It is not detected |
| 20161203 | It is not detected |
3, methodology validation
3.1 blank test
Precision measures above-mentioned prepared sodium hydroxide solution 1ml, sets in tool plug test tube, adds acetonitrile 1ml, add sodium chloride
0.5g, shaking are sufficiently mixed, stratification, draw supernatant liquor, and as blank solution, blank solution is to residual 3- dimethylamino
Propylamine measurement is noiseless.Blank solution chromatogram is as shown in Figure 3.
3.2 precision test
(1) repeated
It is appropriate that precision weighs 3- dimethylaminopropylamine, is made often in addition stating prepared sodium hydroxide solution and quantitatively diluting
The solution of 100 μ g in 1ml, precision measure the 1ml solution, set in tool plug test tube, add acetonitrile 1ml, add sodium chloride 0.5g, shaking is filled
Divide mixing, stratification draws supernatant liquor, as solution to be measured.It tests and measures by above-mentioned chromatographic condition, 6 needle of continuous sample introduction
Measurement, as a result 6 needle RSD are 1.30%, and repeatability is good.3- dimethylaminopropylamine repetitive test the results are shown in Table 2.
Table 2:
| Number | 1# | 2# | 3# | 4# | 5# | 6# | Average value | RSD |
| Peak area | 325.94 | 320.95 | 324.56 | 334.83 | 324.33 | 325.54 | 326.03 | 1.30 |
(2) reproducibility
It is appropriate that precision weighs 3- dimethylaminopropylamine, is made often in addition stating prepared sodium hydroxide solution and quantitatively diluting
Containing about the solution of 100 μ g in 1ml, precision measures 6 parts of 1ml solution, sets in tool plug test tube, adds acetonitrile 1ml, add sodium chloride
0.5g, shaking are sufficiently mixed, stratification, supernatant liquor are drawn, as solution to be measured.It tests and measures by above-mentioned chromatographic condition,
As a result 6 parts of RSD are 2.80%, and reproducibility is good.3- dimethylaminopropylamine reproducible test results is shown in Table 3.
Table 3:
| Number | 1# | 2# | 3# | 4# | 5# | 6# | Average value | RSD |
| Peak area | 300.56 | 309.98 | 316.08 | 313.56 | 305.59 | 328.22 | 312.33 | 2.80 |
3.3 detection limits and quantitative limit
Method: precision weighs 3- dimethylaminopropylamine 100mg, is solvent with above-mentioned prepared sodium hydroxide solution, dilute
It is interpreted into the solution of 7 various concentrations, is shown in Table 4.
It is tested by above-mentioned chromatographic condition, precision measures solution 1ml under each concentration, and it sets in tool plug test tube, adds acetonitrile 1ml,
Add sodium chloride 0.5g, shaking is sufficiently mixed, stratification, supernatant liquor is drawn, as solution to be measured.Precision measures each to be measured molten
2 μ l of liquor is injected separately into gas chromatograph, records chromatogram.
Table 4:
| Number | Concentration (μ g/ml) |
| 1# | 226.8 |
| 2# | 170.1 |
| 3# | 113.4 |
| 4# | 56.70 |
| 5# | 28.35 |
| 6# | 11.34 |
| 7# | 6.804 |
| 8# | 5.670 |
Experimental result:
By reducing concentration method measurement step by step, calculated according to S/N=3, the detection for measuring 3- dimethylaminopropylamine is limited to
5.67 0.0057% amount in the suitable sample of μ g/ml；Detection limit chromatogram is as shown in Figure 4.
By reducing concentration method measurement step by step, is calculated according to S/N=10, measure quantifying for 3- dimethylaminopropylamine and be limited to
0.0068% amount in the suitable sample of 6.80 μ g/ml；Quantitative limit chromatogram is as shown in Figure 5.
3.4 linear test
Precision weighs 3- dimethylaminopropylamine 100mg, sets in 100ml measuring bottle, is diluted with prepared sodium hydroxide solution
The solution of 1000 μ g/ml is made, it is spare.
Pipette 0.1 respectively, 0.25,0.5,1.0,1.5,2.0ml in 10ml measuring bottle, with prepared sodium hydroxide solution
Constant volume is diluted, is shaken up.It is accurate respectively to measure the 1ml solution, it sets in tool plug test tube, prepared sodium hydroxide solution 1ml is added to shake
Shaking makes to dissolve, and adds acetonitrile 1ml, adds sodium chloride 0.5g, and shaking is sufficiently mixed, stratification, supernatant liquor is drawn, as to be measured molten
Liquid.It is shown in Table 5.2 μ l sample introductions are measured respectively, record chromatogram.
Table 5:
| Number | Solution concentration (μ g/ml) | Peak area |
| 1# | 11.34 | 10.20 |
| 2# | 28.35 | 50.11 |
| 3# | 56.70 | 119.61 |
| 4# | 113.4 | 291.30 |
| 5# | 170.1 | 457.84 |
| 6# | 226.8 | 670.34 |
As a result: using concentration as x-axis, peak area is y-axis, makees linear regression.Linear equation is Y=3.0399X-40.8172,
Coefficient R=0.9979,3- dimethylaminopropylamine linear relationship in 11.34~226.8 μ g/ml concentration ranges is good.Line
Sexual intercourse curve graph is as shown in Figure 6.
3.5 recovery test
Take the concentration of 3- dimethylaminopropylamine dense for the sodium hydroxide solution of 100 μ g/ml and its response 80% and 120%
Degree solution is reference substance solution.
Precision measures reference substance solution 1ml, and 0.1g sample is added, and after shaking dissolution, adds acetonitrile 1ml, adds sodium chloride
0.5g, shaking are sufficiently mixed, stratification, draw supernatant liquor, as solution to be measured, are measured 2 μ l and are injected gas chromatograph, note
Record chromatogram.The reference substance solution that sample is not added separately is taken, is measured in the same method, calculates the rate of recovery by calibration curve method.As a result it tests
Average recovery rate is that 84.24%, RSD is 2.10% (n=9), and the rate of recovery is between 80%~120%, and RSD is less than 10%, side
The method rate of recovery is good.It is shown in Table 6.
The table 6:3- dimethylaminopropylamine rate of recovery:
Embodiment 2
Test sample, reference substance solution are prepared referring to embodiment 1.Its concentration of sodium hydroxide solution for being distinguished as the present embodiment is
2.0mol/L；Acetonitrile is 2ml；Inorganic salts are potassium chloride, additional amount 2g.
Chromatographic condition:
Column temperature: temperature programming: 60 DEG C of initial temperature are kept for 8 minutes, are then warming up to 220 with 20 DEG C of rate per minute
DEG C, it is kept for 8 minutes.
Injector temperature: 230 DEG C；
Flame ionization detector temperature: 280 DEG C；
Carrier gas: nitrogen；
Flow rate of carrier gas: 1.0ml/min；
Split ratio: 1:1；
Sampling volume is 1 μ l.
According to above-mentioned condition, three batches of test solutions are detected, do not detect 3- dimethylaminopropylamine peak in chromatogram, are passed through
It calculates, the residual quantity of 3- dimethylaminopropylamine is 0% in Dalbavancin.
Embodiment 3
Test sample, reference substance solution are prepared referring to embodiment 1.Its sodium hydroxide solution 10ml for being distinguished as the present embodiment,
Concentration is 3.0mol/L；Acetonitrile is 1ml；Inorganic salts are anhydrous sodium sulfate, additional amount 1g.
Chromatographic condition:
Column temperature: temperature programming: 60 DEG C of initial temperature are kept for 8 minutes, are then warming up to 220 with 20 DEG C of rate per minute
DEG C, it is kept for 8 minutes.
Injector temperature: 270 DEG C；
Flame ionization detector temperature: 320 DEG C；
Carrier gas: nitrogen；
Flow rate of carrier gas: 2.0ml/min；
Split ratio: 10:1；
Sampling volume is 3 μ l.
According to above-mentioned condition, three batches of test solutions are detected, do not detect 3- dimethylaminopropylamine peak in chromatogram, are passed through
It calculates, the residual quantity of 3- dimethylaminopropylamine is 0% in Dalbavancin.
Embodiment 4
Chromatographic condition is same as Example 1.
Solvent direct injected gas chromatography measurement 3- dimethylaminopropylamine recovery test is made using dimethyl sulfoxide:
Take the dimethyl sulphoxide solution and its response 80% and 120% that the concentration of 3- dimethylaminopropylamine is 100 μ g/ml
Strength solution is reference substance solution.
Precision weighs the Dalbavancin 0.5g of known 3- dimethylaminopropylamine residual quantity in 5ml measuring bottle, with 3- diformazan ammonia
Base propylamine reference substance solution dissolves and is diluted to scale, shakes up, and measures 2 μ l and injects gas chromatograph, records chromatogram.Separately take not
The reference substance solution of sample is added, is measured in the same method, calculates the rate of recovery by calibration curve method.As a result test average recovery rate is 0%,
RSD is 0% (n=9), the results showed that this method 3- dimethylaminopropylamine and Dalbavancin can not effectively dissociate and vaporize at salt, no
It can effectively measure.It is shown in Table 7.
Table 7: the measurement 3- dimethylaminopropylamine rate of recovery examination of solvent direct injected gas chromatography is made using dimethyl sulfoxide
It tests:
Claims (12)
1. a kind of method of the 3- dimethylaminopropylamine in detection Dalbavancin, comprising the following steps:
A, prepared by test solution: precision weighs Dalbavancin, by 1: 5~50g/ml of mass volume ratio be added concentration for 2~
The strong base solution of 3mol/L；Acetonitrile is added after dissolution, acetonitrile and strong base solution volume ratio are 1: 0.5~10；Add inorganic salts,
The mass volume ratio of inorganic salts and strong base solution is 1: 0.5~10g/ml；Shaking is sufficiently mixed, stratification, and it is clear to draw upper layer
Liquid, it is spare as test solution；
B, prepared by reference substance solution: precision weighs 3- dimethylaminopropylamine, and the strong base solution that concentration is 2~3mol/L is added, fixed
The solution of 50 μ g of dimethylaminopropylamine containing 3-, 100 μ g, 150 μ g respectively is made in every 1ml in amount dilution；Precision measures above-mentioned solution,
Add the acetonitrile for being 1: 0.5~10 with above-mentioned liquor capacity ratio, adding above-mentioned solution quality volume ratio is 1: 0.5~10g/ml's
Inorganic salts, shaking are sufficiently mixed, stratification, draw supernatant liquor, spare as reference substance solution (1)~(3)；
C, it is detected with gas chromatography, by calibration curve method with the content of calculated by peak area 3- dimethylaminopropylamine.
2. detection method according to claim 1, it is characterized in that in the step A, Dalbavancin and strong base solution
Mass volume ratio is 1: 10g/ml.
3. detection method according to claim 1, wherein the volume ratio of the acetonitrile and strong base solution is 1: 0.5~2.
4. detection method according to claim 3, wherein the volume ratio of the acetonitrile and strong base solution is 1: 1.
5. detection method according to claim 1, wherein the mass volume ratio of the inorganic salts and strong base solution is 1: 2g/
ml。
6. detection method according to claim 1, it is characterized in that the highly basic is sodium hydroxide and/or potassium hydroxide.
7. detection method according to claim 6, it is characterized in that the highly basic is sodium hydroxide.
8. detection method according to claim 1, it is characterized in that the inorganic salts are sodium chloride, potassium chloride, anhydrous sulphur
Sour sodium is any one or more of.
9. detection method according to claim 8, it is characterized in that the inorganic salts are sodium chloride.
10. detection method according to claim 1, it is characterized in that the gas chromatograph type that the gas chromatography uses
Number be Agilent7890B, column model is Agilent DB-624, which is with 6% cyanogen propyl phenyl -94% two
Methyl polysiloxane is the quartz capillary column of fixer, and column length 30m, internal diameter 0.32mm, the liquid film of the quartz capillary column are thick
1.8 μm of degree.
11. detection method according to claim 1, it is characterized in that the gas chromatography condition are as follows:
Column temperature: temperature programming；
Injector temperature: 230~270 DEG C；
Flame ionization detector temperature: 280~320 DEG C；
Carrier gas: nitrogen；
Flow rate of carrier gas: 0.5~2ml/min；
Split ratio: 1: 1~30: 1；
Direct injected volume is 1~3 μ l.
12. detection method according to claim 1, it is characterized in that the gas chromatography condition are as follows:
Column temperature: temperature programming method is 60 DEG C of initial temperature and is kept for 8 minutes, is then warming up to 220 with 20 DEG C of rate per minute
DEG C, it is kept for 8 minutes；
Injector temperature: 250 DEG C；
Flame ionization detector temperature: 300 DEG C；
Carrier gas: nitrogen；
Flow rate of carrier gas: 1.5ml/min；
Split ratio: 5: 1；
Direct injected volume is 2 μ l.
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Application publication date: 20170901 Assignee: Hangzhou Huadi Pharmaceutical Technology Co.,Ltd. Assignor: Hangzhou Huadong Medicine Group Biopharmaceutical Co.,Ltd. Contract record no.: X2021330000100 Denomination of invention: A method for determination of dabavancin impurities Granted publication date: 20191015 License type: Common License Record date: 20210820 |