CN107121505A - A kind of method for determining Dalbavancin impurity - Google Patents
A kind of method for determining Dalbavancin impurity Download PDFInfo
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- CN107121505A CN107121505A CN201710238376.XA CN201710238376A CN107121505A CN 107121505 A CN107121505 A CN 107121505A CN 201710238376 A CN201710238376 A CN 201710238376A CN 107121505 A CN107121505 A CN 107121505A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention relates to Pharmaceutical Analysis technical field, the method for disclosing detection Dalbavancin impurity.The present invention detects 3 dimethylaminopropylamines in Dalbavancin using direct-injection technique.The method that the present invention is provided is simple to operate, general, requires low to gas phase relevant device；The assay method detection sensitivity height that the present invention is provided has high-recovery.The detection method that the present invention is provided improves the quality and drug safety of Dalbavancin.
Description
Technical field
It is more particularly to a kind of to determine 3- dimethylaminopropylamines in Dalbavancin the invention belongs to medicine analysis technical field
Analysis method.
Background technology
Dalbavancin (Dalbavancin, structural formula is as follows), also referred to as dalbavancin, it is a kind of new hemizygous saccharogenesis
Peptide antibiotic, is teicoplanin analog A40926 derivative.Hydrochloric acid Dalbavancin be it is first be also only one it is granted use
In the intravenous antibiotics of the ABSSSI two-dose schedules treated.Dalbavancin mechanism of action and vancomycin and teicoplanin
It is identical, suppress the biosynthesis of G+ bacterium cell membranes, be widely used as treating the medicine of Skin and soft tissue infection.Dalbavancin is
The second generation, semi-synthetic glycosylated adiponectin polypeptide, are added to a kind of enhanced glycopeptide main chain, the medicine is in vitro to a variety of by a lipophilic side chain
Gram-positive bacteria (including
MRSA and micrococcus scarlatinae) and other streptococcus kind bacteriums show bactericidal activity.Dalbavancin has only
Special pharmacokinetics, can be per weekly interval medication.At present, Dalbavancin is in treatment conduit related Hematogenous infection and skin
Good effect is achieved with soft tissue infection, it has excellent antibacterial activity in vivo and security, is preferable second
For glycopeptide antibiotic.
3- dimethylaminopropylamines are used in Dalbavancin building-up process as one of raw material, so as to may be deposited in final finished
In the residual of 3- dimethylaminopropylamines.
3- dimethylaminopropylamines (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 is 104.1928, colourless
Liquid, with 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, with stronger volatility, toxicity, corrosivity, there is stronger thorn to skin and eyes
Swash property, primary amine groups are contained simultaneously, have and produce the potentially possible of nitrosamines or nitroso compound.Therefore, set up and suitably reach
That strict control is carried out to it is just very necessary for 3- dimethylaminopropylamines detection method of content in Ba Wanxing.
Chinese patent《One kind determines micro N, the analysis method of N- dimethylated propyl diethylenetriamines in amphoteric surfactant》(application
Number 20111015766.1) disclose micro N, the analysis method of N- dimethylated propyl diethylenetriamines in amphoteric surfactant is determined：Adopt
Analyzed with the chromatography of ions, chromatographic test strip part is as follows：Chromatographic column：Cation chromatographic column；Mobile phase：Contained by volume basis
Inorganic acid or organic acid aqueous acid that amount is 70-100% and the organic solvent that volumn concentration is 0-30% composition, it is described
The molar concentration of inorganic acid or aqueous solutions of organic acids is 1-10mmol/l；Detector：Using electric conductivity detector；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 post,
The content of 3- dimethylaminopropylamines in electric conductivity detector analysis amphoteric surfactant, method discusses 3- dimethylaminopropylamine standards
Linear situation, is not studied linear and recovery of the 3- dimethylaminopropylamines in surfactant samples.Using
Cation separation post and electric conductivity detector are expensive, and operation requires high, and working service cost is high.Used using liquid chromatogram
Substantial amounts of acid flux material and organic solvent, testing cost are high and not environmentally friendly enough.The patent is determined using acid flux material sample dissolution
N, N- dimethylated propyl diethylenetriamine, and hydrochloric acid Dalbavancin dissolubility in acid flux material is poor, the patented method can not be applied to hydrochloric acid
The measure of N in Dalbavancin, N- dimethylated propyl diethylenetriamine.The present invention finds a kind of gas chromatography for being different from principle, behaviour
Make simple, highly versatile, system robustness is good, has good stability, be adapted to 3- dimethylaminos third in acid compound Dalbavancin
Amine content detection method, testing cost is low, environmental protection.
Document《The measure of 3- dimethylaminopropylamines in alkylamide propyl-betaine》(summer hero swallow etc., Guangdong, 2013,
40 (19) 152-153) disclose using solid-phase extraction column pretreatment gas chromatography analysis method.This method is to sample pre-treatments
Operating personnel are required high by complex operation, poor universality, and same amount TIANZHU XINGNAO Capsul difference is not big.In addition, this method reaches bar to determining
3- dimethylaminopropylamines in ten thousand stars are not applied to, because Dalbavancin dissolubility in adopted ethanol-water system is poor, it is difficult to
Meet to determine and require, and the 3- dimethylaminopropylamines combined in Dalbavancin can not form unhindered amina, it is impossible to effectively extraction and
Vaporization.
Because of high request of the medicine to final product purity, the residual content of 3- dimethylaminopropylamines is very low, using conventional point
During analysis method determination of acid-basetitration, the sensitivity and the degree of accuracy of general titration do not reach requirement.
3- dimethylaminopropylamines are had been reported that using fluorescence derivation liquid chromatogram, fluorescence detector without UV absorption wavelength
Detected, but this method need to carry out fluorescent derivatization processing to 3- dimethylaminopropylamines, and complex operation, pre-treatment is cumbersome.
The aobvious alkalescence of 3- dimethylaminopropylamines, and the aobvious stronger acidity of Dalbavancin, therefore can not be using conventional neutral or acid
Property solvent dissolving after, direct injected gas chromatography determine.3- dimethylaminopropylamines and acidity numerous in Dalbavancin structure
Group is combined and the vaporization that can not dissociate, and can not also be replaced even if 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, be unfavorable for determining using gas chromatography solution direct injected.Due to 3- dimethylaminos third
Amine has very strong polarity, it is difficult to select suitable solvent to realize that effective liquid phase is extracted.
The content of the invention
The present invention is in order to overcome that Dalbavancin dissolubility in the prior art is poor, the 3- dimethylaminopropylamines in Dalbavancin without
Method effectively dissociate vaporization, can not effectively liquid phase extraction etc. technological deficiency there is provided the high detection of a kind of simple general-purpose, detection sensitivity
The method of 3- dimethylaminopropylamines in Dalbavancin.
Dalbavancin mentioned by the present invention, free alkali containing Dalbavancin and Dalbavancin hydrochloride.
The detection method that the present invention is provided is dissolved using strong base solution to acid compound Dalbavancin, and will be combined
3- dimethylaminopropylamines displacement into salt discharges into 3- dimethylaminopropylamine free alkalis.Utilize intensive polar solvent acetonitrile and aqueous slkali
The immiscible phenomenon for producing layering, is carried out using acetonitrile to 3- dimethylaminopropylamines highly polar in Dalbavancin strong base solution
Liquid-liquid extraction, the inorganic salts of addition saturation strengthen the extraction effect of acetonitrile, using the flame ionization detector of highly versatile,
Acetonitrile layer (upper strata) sample introduction is taken using direct injected gas chromatography, so that avoiding highly basic from entering chromatographic system damages system.
Specifically, the present invention is addressed by following technical proposals：
It is prepared by A, need testing solution：Precision weighs Dalbavancin, by mass volume ratio 1：5~50 add concentration be 0.5~
5mol/L strong base solution；Acetonitrile is added after dissolving, acetonitrile is 1 with strong base solution volume ratio：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 strata
Clear liquid, it is standby as need testing solution；
It is prepared by B, reference substance solution：Precision weighs 3- dimethylaminopropylamines, adds concentration molten for 0.5~5mol/L highly basic
The μ g of dimethylaminopropylamine containing 3- 50,100 μ g, 150 μ g solution respectively is made in every 1ml in liquid, quantitative dilution；Precision measures above-mentioned
Solution, plus with above-mentioned liquor capacity ratio be 1：0.5~10 acetonitrile, it is 1 to add and state solution quality volume ratio：0.5~10 (g/
Ml inorganic salts), shaking is sufficiently mixed, stratification, draws supernatant liquor, standby as reference substance solution (1)~(3)；
C, detected with gas chromatography, by calibration curve method with the content of calculated by peak area 3- dimethylaminopropylamines.
Wherein described highly basic is sodium hydroxide and/or potassium hydroxide, preferably sodium hydroxide.Present invention selection highly basic is molten
The problem of liquid solves insoluble in other numerous solvents, it is ensured that the highly dissoluble of Dalbavancin, it is clever beneficial to high detection is ensured
Sensitivity.The alkalescence of highly basic is better than triethylamine, can reach by 3- dimethylaminopropylamines formation free alkali, so as to ensure that 3- diformazan ammonia
Base propylamine can be completely vaporized in gas-chromatography.Requirement of the rate of recovery between 80%~120% is met, therefore can accurate quantitative analysis inspection
Go out the 3- dimethylaminopropylamines that may be remained in Dalbavancin.
In order to ensure the good dissolving of sample and 3- dimethylaminopropylamines well free, the concentration selection of highly basic is 0.5
~5mol/L, preferably 2~3mol/L.If strong base solution concentration is too low, 3- dimethylaminopropylamines can not dissociate completely, average to return
Yield is low, it is impossible to the 3- dimethylaminopropylamines that may be remained in accurate quantitative analysis detection Dalbavancin.
In wherein described the step of A, the quality volume of Dalbavancin and strong base solution is preferably 1：10.
Wherein described the step of A with B in, the volume ratio of acetonitrile and strong base solution is preferably 1:0.5~2, more preferably 1：
1.Extracted present invention employs strong base solution-acetonitrile immiscible system, form the layering of aqueous phase organic phase, solve acetonitrile-water body
The problem of system dissolves each other.The present invention is extracted using acetonitrile, solves the problem of other weak polar solvents can not be extracted effectively.Return simultaneously
Keep away strong base solution and contain a large amount of inorganic bases and salt, the problem of being unfavorable for direct injected.
Wherein described inorganic salts are any of sodium chloride, potassium chloride, anhydrous sodium sulfate or a variety of.Preferably chlorination
Sodium.
The inorganic salts of wherein described addition and the mass volume ratio of strong base solution are 1：2.
The acetonitrile of wherein described addition and the order of inorganic salts is first add acetonitrile or first add inorganic salts.
The present invention can use various gas chromatographs, such as model Agilent 7890B, chromatographic column model
Agilent DB-624：It can be the quartz capillary using the dimethyl polysiloxane of 6% cyanogen propyl group phenyl -94% as fixer
Post, 1.8 μm of column length 30m, internal diameter 0.32mm, the thickness of liquid film of the quartz capillary column.
Need testing solution and reference substance solution are injected separately into gas chromatograph, and the gas-chromatography column condition is：
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 is：
Column temperature：Temperature programming method is that 60 DEG C of initial temperature is kept for 8 minutes, is then warming up to 20 DEG C per minute of speed
220 DEG C, kept for 8 minutes；
Injector temperature：250℃；
Flame ionization detector temperature：300℃；
Carrier gas：Nitrogen；
Flow rate of carrier gas：1.5ml/min；
Split ratio：5:1；
Direct injected volume is 2 μ l.
In detection, reference substance solution continuous sample introduction is taken 5 times, the relative standard deviation of 3- dimethylaminopropylamine peak areas is not
2.0% must be crossed, to take concentration-peak area of reference substance solution (1)~(3) to map, equation of linear regression is calculated, coefficient correlation is not
0.995 must be less than.Precision measures need testing solution and reference substance solution (1)~(3) injection gas chromatograph respectively, records successively
Chromatogram.By calibration curve method, with 3- dimethylaminopropylamine peak areas, 3- dimethylaminopropylamines contains in calculating need testing solution
Amount.
Compared with prior art, present invention selection direct injected solves headspace sampling instrument system to 3- dimethylaminos third
The absorption residue problem of amine.
The assay method of 3- dimethylaminopropylamines in Dalbavancin, this method needs Method validation, including blank test,
Precision test, detection limit and quantitative limit, linear test and recovery test, wherein linear test is verified in the assay method,
Comprise the following steps：
1) 2.5mol/L sodium hydroxide solution is prepared, it is standby.
2) precision weighs reference substance 3- dimethylaminopropylamines 100mg and is placed in 100ml volumetric flasks, with step 1) described in hydrogen
Sodium hydroxide solution is diluted to 1000 μ g/ml solution, mixes, standby.
3) pipette 0.1 respectively, 0.25,0.5,1.0,1.5,2.0ml in 10ml measuring bottles, with step 1) described in hydroxide
Sodium solution dilutes constant volume, shakes up, precision measures the 1ml solution respectively, puts in tool plug test tube, plus 2.5mol/L sodium hydroxide is molten
Liquid 1ml shakings make dissolving, plus acetonitrile 1ml, plus sodium chloride 0.5g, and shaking is sufficiently mixed, stratification, draw supernatant liquor, make
It is standby for solution to be measured.
4) precision measures step 3 respectively) in each 2 μ l sample introductions of sample, record chromatogram.
5) using concentration as x-axis, peak area is y-axis, makees linear regression.
Verify that recovery test step is as follows in wherein described assay method：
1) it is molten for 80 μ g/ml, 100 μ g/ml, 120 μ g/ml sodium hydroxide that the concentration of 3- dimethylaminopropylamines is prepared respectively
Liquid；The concentration of sodium hydroxide solution is 2.5mol/L；The concentration of the 3- dimethylaminopropylamines be 80 μ g/ml, 100 μ g/ml,
120 μ g/ml sodium hydroxide solution is reference substance solution；
2) precision weighs 0.1g Dalbavancins in tool plug test tube, with step 1) in after reference substance solution dissolving, plus acetonitrile
1ml, plus sodium chloride 0.5g, shaking are sufficiently mixed, stratification, measure the μ l sample introductions of supernatant liquor 2, record chromatogram；
3) reference substance solution for not adding Dalbavancin separately is taken, be measured in the same method, recorded chromatogram, calculated by calibration curve method
The rate of recovery.
The present invention uses direct-injection technique, simple to operate, requires low to gas phase relevant device, has evaded headspace sampling
, there is not appearance that direct injected brings complicated and damage lacking for pillar yet in mode bulk drug and the big shortcoming of solvent-oil ratio
Fall into.Conventional capillary chromatographic column of the present invention and hydrogen flame gas chromatography, simple general-purpose；The survey that the present invention is provided
Determine method detection sensitivity high, concentration limit is up to the 5.67 even lower concentration of μ g/ml；The assay method tool that the present invention is provided
There is a high-recovery, the rate of recovery is up to 84.24%.The detection method of the invention for successfully providing 3- dimethylaminopropylamines, so as 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.
Brief description of the drawings
Fig. 1 is 3- dimethylaminopropylamine reference substance solution chromatograms 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 test limit chromatograms in embodiment 1.
Fig. 5 is 3- dimethylaminopropylamine quantitative limit chromatograms in embodiment 1.
Fig. 6 is the linear relationship chart of 3- dimethylaminopropylamines in embodiment 1.
Embodiment
1st, instrument：
Assay balance, model：METTLER TOLEDO ABS135-S；
Gas chromatograph model Agilent 7890B, chromatographic column model Agilent DB-624：With 6% cyanogen propyl group
The dimethyl polysiloxane of phenyl -94% 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.
2nd, reagent：
3- dimethylaminopropylamines (reagent is pure), sodium hydroxide (analysis is pure), acetonitrile (chromatographically pure).
3rd, sample：
Dalbavancin, lot number：20161201、20161202、20161203.
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 is kept for 8 minutes, is then warming up to 220 with 20 DEG C per minute of speed
DEG C, kept for 8 minutes.
Injector temperature：250℃；
Flame ionization detector temperature：300℃；
Carrier gas：Nitrogen；
Flow rate of carrier gas：1.5ml/min；
Split ratio：5:1.
1st, detect
It is prepared by A, need testing solution：
2.5mol/L sodium hydroxide solution is prepared, it is standby；
Dalbavancin about 0.1g is taken, it is accurately weighed, put in tool plug test tube, plus the sodium hydroxide solution 1ml prepared, shaking
Make dissolving, plus acetonitrile 1ml, plus sodium chloride 0.5g, shaking is sufficiently mixed, stratification, draw supernatant liquor, it is molten as test sample
Liquid, it is standby；
It is prepared by B, reference substance solution：Precision weighs 3- dimethylaminopropylamine 100mg, puts in 100ml measuring bottles, plus prepare
Sodium hydroxide solution quantitatively dilutes the solution for being made and containing 1000 μ g in every 1ml respectively, then fixed with the sodium hydroxide solution prepared
Amount dilution is made in every 1ml contains 50 μ g, 100 μ g, 150 μ g solution respectively, and precision measures 1ml, put in tool plug test tube, plus acetonitrile
1ml, plus sodium chloride 0.5g, shaking are sufficiently mixed, stratification, draw supernatant liquor, standby as reference substance solution (1)~(3)
With；
C, detection method：Determined with reference to Chinese Pharmacopoeia version general rule 0521 in 2015, using direct injected gas chromatography,
Reference substance solution continuous sample introduction is taken 5 times, the relative standard deviation of 3- dimethylaminopropylamine peak areas must not cross 2.0%, to take pair
According to concentration-peak area mapping of product solution (1)~(3), equation of linear regression is calculated, coefficient correlation must not be less than 0.995.Respectively
Precision measures need testing solution and each 2 μ l in reference substance solution (1)~(3), injects gas chromatograph, chromatogram, 3- are recorded successively
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.By calibration curve method, with 3- dimethylaminopropylamine peak areas, the content of 3- dimethylaminopropylamines in need testing solution is calculated.
2nd, calculation formula：
Equation of linear regression Y=AX+B
3- dimethylaminopropylamines residual quantity (%)=(Yi-B)/A × V/W × 100%；
Wherein X：3- dimethylaminopropylamine reference substance solution concentration (mg/ml)；
Y：The main peak area of 3- dimethylaminopropylamine reference substance solutions；
A：Equation of linear regression slope；
B：Equation of linear regression intercept；
Yi：The peak area of 3- dimethylaminopropylamines in need testing solution；
W：The sampling amount (mg) of sample when need testing solution is prepared；
V：Need testing solution dose volume (ml)；
3rd, measurement result
Three batches of Dalbavancin test samples are detected according to said determination method, does not as a result detect, is shown in Table 1.
Table 1：
| Lot number | 3- dimethylaminopropylamines content (%) |
| 20161201 | Do not detect |
| 20161202 | Do not detect |
| 20161203 | Do not detect |
3rd, Method validation
3.1 blank test
Precision measures the above-mentioned sodium hydroxide solution 1ml prepared, puts in tool plug test tube, plus acetonitrile 1ml, plus sodium chloride
0.5g, shaking is sufficiently mixed, stratification, draws supernatant liquor, and as blank solution, blank solution is to residual 3- dimethylaminos
Propylamine determines noiseless.Blank solution chromatogram is as shown in Figure 3.
3.2 precision test
(1) it is repeated
It is appropriate that precision weighs 3- dimethylaminopropylamines, add state the sodium hydroxide solution prepared quantitatively dilute be made it is every
100 μ g solution in 1ml, precision measures the 1ml solution, put in tool plug test tube, plus acetonitrile 1ml, plus sodium chloride 0.5g, and shaking is filled
Divide mixing, stratification draws supernatant liquor, is used as solution to be measured.Test and determine by above-mentioned chromatographic condition, the pin of continuous sample introduction 6
Determine, as a result 6 pin RSD are 1.30%, and repeatability is good.3- dimethylaminopropylamine replica tests the results are shown in Table 2.
Table 2：
| Numbering | 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) reappearance
It is appropriate that precision weighs 3- dimethylaminopropylamines, add state the sodium hydroxide solution prepared quantitatively dilute be made it is every
Containing about 100 μ g solution in 1ml, precision measures 6 parts of 1ml solution, puts in tool plug test tube, plus acetonitrile 1ml, plus sodium chloride
0.5g, shaking is sufficiently mixed, stratification, is drawn supernatant liquor, is used as solution to be measured.Test and determine by above-mentioned chromatographic condition,
As a result 6 parts of RSD are 2.80%, and reappearance is good.3- dimethylaminopropylamine reproducible test results is shown in Table 3.
Table 3：
| Numbering | 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 the above-mentioned sodium hydroxide solution prepared, dilute
The solution of 7 various concentrations is interpreted into, 4 are shown in Table.
Tested by above-mentioned chromatographic condition, precision measures solution 1ml under each concentration, put in tool plug test tube, plus acetonitrile 1ml,
Plus sodium chloride 0.5g, shaking is sufficiently mixed, stratification, draws supernatant liquor, is used as solution to be measured.Precision measures each to be measured molten
The μ l of liquor 2, are injected separately into gas chromatograph, record chromatogram.
Table 4：
| Numbering | 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：
Determined, calculated according to S/N=3, the detection for measuring 3- dimethylaminopropylamines is limited to by reducing concentration method step by step
0.0057% amount in the suitable samples of 5.67 μ g/ml；Test limit chromatogram is as shown in Figure 4.
Determined by reducing concentration method step by step, calculated according to S/N=10, measure quantifying for 3- dimethylaminopropylamines and be limited to
0.0068% amount in the suitable samples of 6.80 μ g/ml；Quantitative limit chromatogram is as shown in Figure 5.
3.4 linear test
Precision weighs 3- dimethylaminopropylamine 100mg, puts in 100ml measuring bottles, is diluted with the sodium hydroxide solution prepared
1000 μ g/ml solution is made, it is standby.
Pipette 0.1 respectively, 0.25,0.5,1.0,1.5,2.0ml in 10ml measuring bottles, with the sodium hydroxide solution prepared
Constant volume is diluted, is shaken up.Precision measures the 1ml solution respectively, puts in tool plug test tube, plus the sodium hydroxide solution 1ml prepared shakes
Shaking makes dissolving, plus acetonitrile 1ml, plus 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：
| Numbering | 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 map is as shown in Figure 6.
3.5 recovery test
It is that 100 μ g/ml sodium hydroxide solution and its response 80% and 120% are dense to take the concentration of 3- dimethylaminopropylamines
Degree solution is reference substance solution.
Precision measures reference substance solution 1ml, and adds 0.1g samples, after shaking dissolving, plus acetonitrile 1ml, plus sodium chloride
0.5g, shaking is sufficiently mixed, stratification, draws supernatant liquor, as solution to be measured, measures 2 μ l injection gas chromatographs, note
Record chromatogram.The reference substance solution for not adding sample separately is taken, is measured in the same method, the rate of recovery is calculated by calibration curve method.As a result test
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.
Table 6：The 3- dimethylaminopropylamine rate of recovery：
Embodiment 2
Test sample, reference substance solution are prepared with reference 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, and addition is 2g.
Chromatographic condition：
Column temperature：Temperature programming：60 DEG C of initial temperature is kept for 8 minutes, is then warming up to 220 with 20 DEG C per minute of speed
DEG C, kept for 8 minutes.
Injector temperature：230℃；
Flame ionization detector temperature：280℃；
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, detect in three batches of need testing solutions, its chromatogram and do not detect 3- dimethylaminopropylamines peak, pass through
Calculate, the residual quantity of 3- dimethylaminopropylamines is 0% in Dalbavancin.
Embodiment 3
Test sample, reference substance solution are prepared with reference to embodiment 1.It is distinguished as the sodium hydroxide solution 10ml of the present embodiment,
Concentration is 3.0mol/L；Acetonitrile is 1ml；Inorganic salts are anhydrous sodium sulfate, and addition is 1g.
Chromatographic condition：
Column temperature：Temperature programming：60 DEG C of initial temperature is kept for 8 minutes, is then warming up to 220 with 20 DEG C per minute of speed
DEG C, kept for 8 minutes.
Injector temperature：270℃；
Flame ionization detector temperature：320℃；
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, detect in three batches of need testing solutions, its chromatogram and do not detect 3- dimethylaminopropylamines peak, pass through
Calculate, the residual quantity of 3- dimethylaminopropylamines is 0% in Dalbavancin.
Embodiment 4
Chromatographic condition is same as Example 1.
Solvent direct injected gas chromatography is made using dimethyl sulfoxide (DMSO) and determines 3- dimethylaminopropylamine recovery tests：
It is 100 μ g/ml dimethyl sulphoxide solution and its response 80% and 120% to take the concentration of 3- dimethylaminopropylamines
Strength solution is reference substance solution.
Precision weighs the Dalbavancin 0.5g of known 3- dimethylaminopropylamine residual quantities in 5ml measuring bottles, with 3- diformazan ammonia
Base propylamine reference substance solution dissolves and is diluted to scale, shakes up, and measures 2 μ l injection gas chromatographs, records chromatogram.Separately take not
The reference substance solution of sample is added, is measured in the same method, the rate of recovery is calculated by calibration curve method.As a result experiment average recovery rate is 0%,
RSD is 0% (n=9), as a result shows that this method 3- dimethylaminopropylamines can not effectively dissociate and vaporize into salt with Dalbavancin, no
Can effectively it determine.It is shown in Table 7.
Table 7：Solvent direct injected gas chromatography is made using dimethyl sulfoxide (DMSO) and determines the examination of the 3- dimethylaminopropylamines rate of recovery
Test：
Claims (10)
1. a kind of method for detecting the 3- dimethylaminopropylamines in Dalbavancin, comprises the following steps：
It is prepared by A, need testing solution：Precision weighs Dalbavancin, by mass volume ratio 1：5~50 add concentration for 0.5~5mol/
L strong base solution；Acetonitrile is added after dissolving, acetonitrile is 1 with strong base solution volume ratio：0.5~10；Inorganic salts are added, it is inorganic
The mass volume ratio of salt and strong base solution is 1：0.5~10；Shaking is sufficiently mixed, stratification, is drawn supernatant liquor, is used as confession
Test sample solution, it is standby；
It is prepared by B, reference substance solution：Precision weighs 3- dimethylaminopropylamines, adds the strong base solution that concentration is 0.5~5mol/L,
The μ g of dimethylaminopropylamine containing 3- 50,100 μ g, 150 μ g solution respectively is made in every 1ml in quantitative dilution；Precision measures above-mentioned molten
Liquid, plus with above-mentioned liquor capacity ratio be 1：0.5~10 acetonitrile, it is 1 to add above-mentioned solution quality volume ratio：0.5~10
Inorganic salts, shaking is sufficiently mixed, stratification, draws supernatant liquor, standby as reference substance solution (1)~(3)；
C, detected with gas chromatography, by calibration curve method with the content of calculated by peak area 3- dimethylaminopropylamines.
2. detection method according to claim 1, it is characterized in that in described step A, Dalbavancin and strong base solution
Quality volume is 1：10.
3. detection method according to claim 1, wherein the volume ratio of the acetonitrile and strong base solution is 1:0.5~2, it is excellent
Elect 1 as：1.
4. detection method according to claim 1, wherein the mass volume ratio 1 of the inorganic salts and strong base solution：2.
5. detection method according to claim 1, it is characterized in that described highly basic is sodium hydroxide and/or potassium hydroxide,
Preferably sodium hydroxide.
6. detection method according to claim 1, it is characterized in that described strong base concentrations are 2~3mol/L.
7. detection method according to claim 1, it is characterized in that described inorganic salts are sodium chloride, potassium chloride, anhydrous sulphur
Any of sour sodium is a variety of.Preferably sodium chloride.
8. detection method according to claim 1, it is characterized in that described gas chromatograph model Agilent
7890B, chromatographic column model Agilent DB-624：It using the dimethyl polysiloxane of 6% cyanogen propyl group phenyl -94% is solid that can be
Determine the quartz capillary column of liquid, 1.8 μm of column length 30m, internal diameter 0.32mm, the thickness of liquid film of the quartz capillary column.
9. detection method according to claim 1, it is characterized in that described gas phase gas-chromatography column condition is：
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.
10. detection method according to claim 1, it is characterized in that described gas-chromatography column condition is：
Column temperature：Temperature programming method is that 60 DEG C of initial temperature is kept for 8 minutes, is then warming up to 220 with 20 DEG C per minute of speed
DEG C, kept for 8 minutes；
Injector temperature：250℃；
Flame ionization detector temperature：300℃；
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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