CN106153740B - A kind of related substance of Tulathromycin, its be enriched with preparation method and application - Google Patents
A kind of related substance of Tulathromycin, its be enriched with preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of related substances of Tulathromycin, its enrichment preparation method and application.The present invention provides a kind of related substance 3 of Tulathromycin or its salt.Enrichment preparation method the present invention also provides Tulathromycin in relation to substance 3 comprising following step: using high performance liquid chromatography, determinand eluted in the chromatography column, collects the related substance 3 of Tulathromycin.The related substance of Tulathromycin of the invention is the necessity that quality control is carried out to Tulathromycin；Enrichment preparation method of the invention can efficiently separate the related substance 3 of Tulathromycin, to control the drug quality of Tulathromycin, have laid a good foundation for the research of Tulathromycin unknown impuritie.
Description
Technical field
The present invention relates to a kind of related substances of Tulathromycin, its enrichment preparation method and application.
Background technique
Tulathromycin (Tulathromycin) is semi-synthetic macrolide antibiotics, in 2004 in USA and EU
Listing.The medicine is mainly used for ox and the pig respiratory infectious disease as caused by sensitive bacteria and causes ox to pass by ox catarrhalis
The prevention and treatment of metachromia keratoconjunctivitis, drug effect are better than widely used macrolide antibiotics tylosin in the market and examine for rice
Star, the prospect of the application in Production of Livestock and Poultry are wide.
Tulathromycin is by isomers A, B (molecular formula C41H79N3O12, molecular weight 806.09) and by 15 yuan of 9:1 composition
Ring macrolide antibiotics, two kinds of isomers can be converted by the formation and fracture of lactone bond between C11 and C13.It is safe
It draws containing there are three Polar Amides group in mycin structure, PKa value belongs to three amine macrolide antibiosis between 8.6~9.6
Element is different from azepine cyclic lactone and ketolide antibiotics.
In order to ensure the safety of animal derived food, strict control, medicament contg must be carried out to animal specific drug quality
Measurement, the Structural Identification of unknown impuritie and Light absorbing impurty are the effective ways of Control of drug quality, and impurity analysis is drug quality
The important content of standard.Tulathromycin is produced by semi-synthetic process, and controllability is lower compared with synthetic drug, therefore impurity spectrum is more
For complexity and it is difficult to predict.Existing European Pharmacopoeia (EP) and United States Pharmacopeia (USP) include veterinary drug wherein, and to related substance
Limitation claims, and European Pharmacopoeia also requires to control specific impurities that (British Pharmacopoeia has included European Pharmacopoeia as usual
Whole monographs, content do not make an amendment generally).VICH (veterinary drug registration technology requires international coordination meeting) guideline requires beast
The impurity reporting limit for curing special raw material medicine is 0.10%, and limit of identification 0.20%, control limit is that 0.50% (guidance is not
Including semisynthetic antibiotics)；EMA (European drug administration) requires the limit of impurities of semi-synthetic veterinary drug that should meet VICH to refer to
Lead the requirement of principle.
Currently, both at home and abroad there is not yet using report of the LC-MS method separation detection Tulathromycin in relation to substance.Therefore, it needs
Tulathromycin degrade to prepare the related substance of degradation, establishes LC-MS separation and detection method of the Tulathromycin in relation to substance, and
The related substance of Tulathromycin is identified.
Summary of the invention
Problem to be solved by this invention is to overcome and lack Tulathromycin in the prior art in synthesis and degradation process
The separation and detection method of the related substance of middle generation, and it cannot be identified, be confirmed the defects of structure, and provide one
The related substance of kind Tulathromycin, its enrichment preparation method and application.The related substance of Tulathromycin of the invention is to Tulathromycin
Carry out the necessity of quality control；Enrichment preparation method of the invention can efficiently separate the related substance 3 of Tulathromycin, to control
The drug quality of Tulathromycin processed is had laid a good foundation for the research of Tulathromycin unknown impuritie.
A kind of separation method the present invention provides Tulathromycin in relation to substance comprising following step: efficient liquid is used
Phase chromatography elutes determinand in the chromatography column；The determinand is that Tulathromycin bulk pharmaceutical chemicals or safe drawing are mould
Plain degradation product；The chromatographic column is that C18 analytical column or C18 prepare column；The mobile phase A of the elution is to adjust pH with ammonium hydroxide
Value be 7~8, volume fraction be 0.1%~0.4% aqueous formic acid, the Mobile phase B of the elution is methanol and acetonitrile
Volume ratio is (1.5~2.0): 1 mixed solvent；
When the chromatographic column is C18 analytical column, the parameter of the elution is as follows: 0min → 15min, A:B=(60
~70): (40~30), 15min → 40min, A:B=(60~70): (40~30) → (25~35): (75~65), 40 →
55min, A:B=(25~35): (75~65)；The A:B refers to the volume ratio of the mobile phase A and the Mobile phase B；
When the chromatographic column be C18 prepare column when, the parameter of the elution is as follows: the mobile phase A with it is described
Mobile phase B volume ratio be (75~85): (25~15).
In the separation method, the Tulathromycin bulk pharmaceutical chemicals can be the Tulathromycin raw material of this field routine
Medicine, preferably Jiangsu reach the clouds pharmaceutcal corporation, Ltd production Tulathromycin bulk pharmaceutical chemicals.
In the separation method, the Tulathromycin degradation product is the object that Tulathromycin is obtained through degradation reaction
Matter；The degradation reaction can be the degradation reaction of this field routine, preferably acid degradation reaction, alkaline degradation reaction, high temperature
Degradation reaction, high humidity degradation reaction, oxidative degradation or illumination degrading reaction；
The oxidative degradation may include following step: in acetonitrile, by Tulathromycin and aqueous hydrogen peroxide solution into
Row oxidative degradation, obtains?；
The described alkaline degradation reaction may include following step: in acetonitrile, by Tulathromycin and sodium hydrate aqueous solution into
The reaction of row alkaline degradation, obtains?.
In the separation method, the method sample introduction of this field routine is can be used in the determinand, preferably with to
Survey the form sample introduction of the acetonitrile solution of object；When the determinand is the substance that Tulathromycin is obtained through oxidative degradation,
Preferably, the reaction solution of the oxidative degradation forms the acetonitrile solution of the determinand after dilution in acetonitrile；Work as institute
When the determinand stated is the substance that Tulathromycin is reacted through alkaline degradation, preferably, the reaction solution that the alkaline degradation reacts
It is neutralized through acid and forms the acetonitrile solution of the determinand after dilution in acetonitrile；When the determinand is Tulathromycin warp
When the substance that acid degradation is reacted, preferably, the reaction solution that the acid degradation is reacted is through alkali neutralization and with after dilution in acetonitrile
Form the acetonitrile solution of the determinand；
The acid that the acid neutralizes can be the acid of this field routine, preferably aqueous hydrochloric acid solution, be more preferably 0.1mol/
The aqueous hydrochloric acid solution of L；The alkali of the alkali neutralization can be the alkali of this field routine, preferably NaOH aqueous solution, more preferably for
The NaOH aqueous solution of 0.1mol/L；
The concentration of the acetonitrile solution of the determinand can be this field routine concentration, preferably 3g/L~50g/L,
It is more preferably 5g/L~20g/L；
The sample volume of the acetonitrile solution of the determinand can be the sample volume of this field routine, when the chromatographic column is
When C18 analytical column, the sample volume is preferably the 5 μ L of μ L~100, is more preferably 20 μ of μ L~50 L；When the chromatographic column
When preparing column for C18, the sample volume is preferably the 50 μ L of μ L~500, is more preferably 50 μ of μ L~300 L.
In the separation method, the C18 analytical column can be the C18 analytical column of this field routine, preferably
XbridgeTM C18(250 × 4.6mm, 5 μm) analytical column；The C18, which prepares column, to prepare column for the C18 of this field routine,
Preferably XbridgeTM C18(19*50mm, 5 μm) prepares column.
In the separation method, the volume ratio of methanol and acetonitrile is preferably 45:25 in the Mobile phase B；When
When the chromatographic column is C18 analytical column, the pH value of the mobile phase A is preferably 7.6~7.99 (such as 7.66), described
The volume fraction of aqueous formic acid of mobile phase A be preferably 0.3%~0.35%, more preferably, to adjust pH value with ammonium hydroxide
For 7.6, aqueous formic acid that volume fraction is 0.35%；When the chromatographic column is that C18 prepares column, the mobile phase
The pH value of A is preferably 7.6~7.8, and the volume fraction of the aqueous formic acid of the mobile phase A is preferably 0.3%~
0.35%.
In the separation method, when the chromatographic column is C18 analytical column, the parameter of the elution is preferably
It is as follows: 0min → 15min, A:B=65:35,15min → 40min, A:B=65:35 → 30:70,40 → 55min, A:B=30:
70；When the chromatographic column be C18 prepare column when, the parameter of the elution is preferably as follows: the mobile phase A with it is described
Mobile phase B volume ratio be 80:20.
In the separation method, the ammonium hydroxide is the ammonium hydroxide of this field routine, and preferably mass fraction is
25%~28% ammonium hydroxide.
In the separation method, the high performance liquid chromatography of this field routine can be used, when the chromatographic column is C18
When analytical column, the Alliance 2695/ZQ liquid chromatograph-mass spectrometer of Waters company is preferably used；When the color
When spectrum column is that C18 prepares column, instrument is preferably purified automatically using 2767 types of Waters company.
In the separation method, the flow velocity of the separation method can be the flow velocity of this field routine；When described
When chromatographic column is C18 analytical column, the flow velocity is preferably 0.7ml/min~1.3ml/min, 0.8ml/min~1.2ml/
Min, such as 1.0ml/min；When the chromatographic column be C18 prepare column when, the flow velocity be preferably 10ml/min~
25ml/min is more preferably 15ml/min~20ml/min, such as 17ml/min.
In the separation method, the processing of this field routine is can be used in the mobile phase A and the Mobile phase B
Method is pre-processed, preferably through 0.22 μm of membrane filtration, and ultrasound 10min.
In the separation method, the column temperature of the separation method can be the column temperature of this field routine, preferably
20 DEG C~40 DEG C, be more preferably 25 DEG C~35 DEG C, such as 30 DEG C.
In the separation method, what the UV absorption wavelength when separation method detects can be conventional for this field
UV absorption wavelength, preferably 200~215nm are more preferably 205~210nm；1/ of fraction after UV detector
5~1/3, which enter mass spectrograph, detects.
The present invention also provides a kind of related substance 1 of Tulathromycin or its salt,
Enrichment preparation method the present invention also provides the Tulathromycin in relation to substance 1, Thailand's drawing as above-mentioned are mould
It is known as the separation method for closing substance, collects the related substance 1 of Tulathromycin.
The present invention also provides the related substance 1 of the Tulathromycin or its salt as miscellaneous in the control of Tulathromycin quality
The application of matter identification.
The present invention also provides a kind of related substance 2 of Tulathromycin or its salt,
Preparation method the present invention also provides the Tulathromycin in relation to substance 2 comprising following step: in acetonitrile
In, Tulathromycin and aqueous hydrogen peroxide solution are subjected to oxidative degradation, obtain the related substance 2 of Tulathromycin.
In the oxidative degradation, the mass volume ratio of the Tulathromycin and the acetonitrile can be ability
The regular quality volume ratio of oxidative degradation in domain, preferably 3g/L~50g/L are more preferably 5g/L~20g/L.
In the oxidative degradation, the aqueous hydrogen peroxide solution can be water-soluble for the hydrogen peroxide of this field routine
Liquid, preferably mass fraction be 0.1%~30% aqueous hydrogen peroxide solution, more preferably for 0.15%~0.5% hydrogen peroxide
Aqueous solution.
In the oxidative degradation, the mass volume ratio of the Tulathromycin and the aqueous hydrogen peroxide solution
Can be the regular quality volume ratio of oxidative degradation in this field, preferably 40g/L~90g/L, more preferably for 50g/L~
75g/L。
In the oxidative degradation, the temperature of the oxidative degradation can be anti-for oxidative degradation in this field
The ordinary temperature answered, preferably 20 DEG C~50 DEG C, be more preferably 20 DEG C~30 DEG C.
In the oxidative degradation, the conventional prison in this field is can be used in the process of the oxidative degradation
Survey method (such as LC-MS) is monitored, and the time of the degradation reaction is preferably 1min~30min, more preferably for
5min~20min.
The oxidative degradation preferably further includes post-processing；The post-processing can be oxidative degradation in this field
The conventional post-processing of reaction, preferably, the reaction solution of the oxidative degradation is according to the above-mentioned related substance of Tulathromycin
Separation method post-processed.
The present invention also provides the related substance 2 of the Tulathromycin or its salt as miscellaneous in the control of Tulathromycin quality
The application of matter identification.
The present invention provides a kind of related substance 3 of Tulathromycin or its salt,
Enrichment preparation method the present invention also provides the Tulathromycin in relation to substance 3, Thailand's drawing as above-mentioned are mould
It is known as the separation method for closing substance, collects the related substance 3 of Tulathromycin.
The present invention also provides the related substance 3 of the Tulathromycin or its salt as miscellaneous in the control of Tulathromycin quality
The application of matter identification.
The present invention also provides a kind of related substance 4 of Tulathromycin or its salt,
Preparation method the present invention also provides the Tulathromycin in relation to substance 4 comprising following step: in acetonitrile
In, Tulathromycin is subjected to alkaline degradation with sodium hydrate aqueous solution and is reacted, the related substance 4 of Tulathromycin is obtained.
In the alkaline degradation reaction, the mass volume ratio of the Tulathromycin and the acetonitrile can be this field
The regular quality volume ratio of middle alkaline degradation reaction, preferably 3g/L~50g/L is more preferably 5g/L~20g/L.
In the alkaline degradation reaction, the sodium hydrate aqueous solution can be water-soluble for the sodium hydroxide of this field routine
Liquid, the preferably sodium hydrate aqueous solution of 0.1mol/L~10mol/L are more preferably the sodium hydroxide of 1mol/L~5mol/L
Aqueous solution.
In the alkaline degradation reaction, the mass volume ratio of the Tulathromycin and the sodium hydrate aqueous solution
Can be the regular quality volume ratio of alkaline degradation reaction in this field, preferably 40g/L~90g/L, more preferably for 50g/L~
75g/L。
In the alkaline degradation reaction, the temperature of the alkaline degradation reaction can react normal for alkaline degradation in this field
Temperature is advised, preferably 50 DEG C~90 DEG C, be more preferably 55 DEG C~70 DEG C, such as 60 DEG C.
In the alkaline degradation reaction, the routine monitoring side in this field is can be used in the process of the alkaline degradation reaction
Method (such as TLC, HPLC or LC-MS) is monitored, and the time of the alkaline degradation reaction is preferably 15min~50min, more
It goodly is 20min~40min, such as 25min.
The alkaline degradation reaction preferably further includes post-processing；The post-processing can be alkaline degradation reaction in this field
Conventional post-processing, preferably, separation of the reaction solution that reacts of the alkaline degradation according to above-mentioned Tulathromycin in relation to substance
Method is post-processed.
The present invention also provides the related substance 4 of the Tulathromycin or its salt as miscellaneous in the control of Tulathromycin quality
The application of matter identification.
Without prejudice to the field on the basis of common sense, above-mentioned each optimum condition, can any combination to get the present invention it is each preferably
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that: the related substance of Tulathromycin of the invention is to carry out quality to Tulathromycin
The necessity of control；Enrichment preparation method of the invention can efficiently separate the related substance 3 of Tulathromycin, so that it is mould to control safe drawing
The drug quality of element is had laid a good foundation for the research of Tulathromycin unknown impuritie.
Detailed description of the invention
Fig. 1 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 1 obtains.
Fig. 2 is the first mass spectrometric figure in relation to substance 1.
Fig. 3 is the second order ms figure in relation to substance 1.
Fig. 4 is the first mass spectrometric figure in relation to substance 3.
Fig. 5 is the second order ms figure in relation to substance 3.
Fig. 6 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 2 obtains.
Fig. 7 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 3 obtains.
Fig. 8 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 4 obtains.
Fig. 9 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 5 obtains.
Figure 10 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 6 obtains.
Figure 11 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 7 obtains.
Figure 12 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 8 obtains.
Figure 13 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 9 obtains.
Figure 14 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that embodiment 10 obtains.
Figure 15 is the mass spectrum total ion chromatogram through analytical column after Tulathromycin bulk pharmaceutical chemicals oxidative degradation.
Figure 16 be Tulathromycin bulk pharmaceutical chemicals oxidative degradation after, prepared the mass spectrum total ion chromatogram of column.
Figure 17 is the first mass spectrometric figure in relation to substance 2.
Figure 18 is the second order ms figure in relation to substance 2.
Figure 19 is the mass spectrum total ion chromatogram through analytical column after the reaction of Tulathromycin bulk pharmaceutical chemicals alkaline degradation.
Figure 20 is the first mass spectrometric figure in relation to substance 4.
Figure 21 is the second order ms figure in relation to substance 4.
Figure 22 is the mass spectrum total ion chromatogram through analytical column after Tulathromycin bulk pharmaceutical chemicals acid degradation reaction.
Figure 23 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that comparative example 1 obtains.
Figure 24 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that comparative example 2 obtains.
Figure 25 is the Tulathromycin bulk pharmaceutical chemicals mass spectrum total ion chromatogram that comparative example 3 obtains.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Reagent used herein is as follows:
Acetonitrile, methanol are chromatographically pure (Thermo Fisher Scientific company)；Formic acid (98%), ammonium hydroxide
(25wt%~28wt%), 30wt% hydrogen peroxide, hydrochloric acid are to analyze pure, sodium hydroxide (the limited public affairs of Chinese medicines group chemicals
Department)；Water is Wahaha Pure Water (crossing 0.22 μm of moisture film)；Tulathromycin bulk pharmaceutical chemicals by Jiangsu reach the clouds pharmaceutcal corporation, Ltd provide.
Instrument used herein is as follows:
(1) 2695 type high performance liquid chromatograph；2487 type UV detector；Micromass ZQ mass spectrograph；Q-Tof
Micro mass spectrograph；Masslynx chromatographic work station (Waters company)
(2) Q-Exactive level four bars orbit trap high resolution mass spectrum (Thermo company)
(3) 2767 types purify instrument automatically
Mass spectrometry method used in the present invention is as follows:
Micromass ZQ: detection pattern ESI (+)；Spray voltage 3kV；Orifice potential 30V；100 DEG C of source temperature；Desolventizing
250 DEG C of temperature；Full scan range m/z100~1500.
Q-Exactive level four bars orbit trap high resolution mass spectrum parameter: HESI spray voltage :+3.0KV/-2.7KV is (positive and negative
Switch while scanning)；Sheath atmospheric pressure: 35arb；Assist gas pressure power: 10arb；Capillary temperature: 300 DEG C；Heating temperature: 300
℃；Scan pattern: Full MS (resolution ratio 7000) and dd-MS2 (resolution ratio 17500)
Embodiment 1
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.35% aqueous formic acid (ammonium hydroxide
Adjust pH value 7.66), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=65:35；15 → 40min,
A:B=65:35 → 30:70；40 → 55min, A:B=30:70, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in Figure 1, each impurity such as table 1
It is shown.When mobile phase salinity is 0.35%, main peak retention time is suitable, and peak shape is good.Can well it divide in relation to substance and main peak
From.It can be seen from the figure that method of the invention can be used for detecting Tulathromycin and its impurity, it is mould that this method can be applied to safe drawing
Plain bulk pharmaceutical chemicals synthesis technology monitoring and quality control.
Each impurity in 1 Fig. 1 of table
Note: Aera% is that area normalization method measures percentage composition.
4 related substances 1 i.e. of the invention in table 1, it is C that high-resolution, which measures molecular formula,40H77N3O12([M+H]+=
792.55829), MS2Fragment is 689.45898 (C35H63NO12)、563.39099(C28H55N2O9)、420.29337
(C21H42NO7)、230.17519(C12H24NO3).Its level-one mass spectrogram as shown in Fig. 2, second order ms figure as shown in figure 3, mass spectrum
Lytic pathway is as follows:
13 related substances 3 i.e. of the invention in table 1, it is C that high-resolution, which measures molecular formula,39H72N2O12([M+H]+=
761.51624), MS2Fragment is 532.34827 (C27H50NO9)、230.17520(C12H24NO3).Its level-one mass spectrogram such as Fig. 4 institute
Show, second order ms figure is as shown in figure 5, MS fragment pathways are as follows:
Embodiment 2
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.3% aqueous formic acid (ammonium hydroxide tune
Save pH value 7.66), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=65:35；15 → 40min, A:
B=65:35 → 30:70；40 → 55min, A:B=30:70, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in Figure 6.Mobile phase salt is dense
When degree is 0.3%, main peak retention time is suitable, and peak shape is good.In relation to substance and main peak energy good separation.
Embodiment 3
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.4% aqueous formic acid (ammonium hydroxide tune
Save pH value 7.66), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=65:35；15 → 40min, A:
B=65:35 → 30:70；40 → 55min, A:B=30:70, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in Figure 7.Mobile phase salt is dense
When degree is 0.4%, main peak retention time is suitable, and peak shape is good.In relation to substance and main peak energy good separation.
Embodiment 4
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.1% aqueous formic acid (ammonium hydroxide tune
Save pH value 7.99), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=65:35；15 → 40min, A:
B=65:35 → 30:70；40 → 55min, A:B=30:70, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in Figure 8.Mobile phase salt is dense
When degree is 0.1%, when pH value is 7.99, main peak retention time is suitable, and peak shape is symmetrical.In relation to substance and main peak energy good separation.
Embodiment 5
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.35% aqueous formic acid (ammonium hydroxide
Adjust pH value 7.6), Mobile phase B: methanol: acetonitrile=1.5:1, gradient elution 0 → 15min, A:B=65:35；15 → 40min,
A:B=65:35 → 30:70；40 → 55min, A:B=30:70, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in Figure 9.First in Mobile phase B
Alcohol: when acetonitrile=1.5:1, main peak retention time is suitable, and peak shape is symmetrical.In relation to substance and main peak energy good separation.
Embodiment 6
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.35% aqueous formic acid (ammonium hydroxide
Adjust pH value 7.6), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=65:35；15 → 40min,
A:B=65:35 → 35:65；40 → 55min, A:B=35:65, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in Figure 10.Eluent gradient
Elution program is 0 → 15min, A:B=65:35；15 → 40min, A:B=65:35 → 35:65；40 → 55min, A:B=35:
When 65, main peak retention time is suitable, and peak shape is symmetrical.In relation to substance and main peak energy good separation.
Embodiment 7
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.35% aqueous formic acid (ammonium hydroxide
Adjust pH value 7.6), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=70:30；15 → 40min,
A:B=70:30 → 30:70；40 → 55min, A:B=30:70, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in figure 11.Eluent gradient
Elution program is 0 → 15min, A:B=70:30；15 → 40min, A:B=70:30 → 30:70；40 → 55min, A:B=30:
When 70, main peak retention time is suitable, and peak shape is symmetrical.In relation to substance and main peak energy good separation.
Embodiment 8
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.35% aqueous formic acid (ammonium hydroxide
Adjust pH value 7.6), Mobile phase B: methanol: acetonitrile=2:1, gradient elution 0 → 15min, A:B=70:30；15 → 40min, A:B
=70:30 → 30:70；40 → 55min, A:B=30:70, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.2ml/min,
HPLC fraction is further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in figure 12.Mobile phase B is methanol:
Acetonitrile=2:1, when flow velocity 1.2ml/min, main peak retention time is suitable, and peak shape is symmetrical.In relation to substance and main peak energy good separation.
Embodiment 9
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.35% aqueous formic acid (ammonium hydroxide
Adjust pH value 7.0), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=70:30；15 → 40min,
A:B=70:30 → 35:65；40 → 55min, A:B=35:65, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in figure 13.Mobile phase A
PH value 7.0, gradient are 0 → 15min, A:B=70:30；15 → 40min, A:B=70:30 → 35:65；40 → 55min, A:B=
When 35:65, main peak retention time is suitable, and peak shape is symmetrical.In relation to substance and main peak energy good separation.
Embodiment 10
5mg Tulathromycin bulk pharmaceutical chemicals are dissolved in 1mL acetonitrile, 20 μ L of sample volume.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) column, mobile phase A: 0.35% aqueous formic acid (ammonium hydroxide
Adjust pH value 8.0), Mobile phase B: methanol: acetonitrile=45:25, gradient elution 0 → 15min, A:B=60:40；15 → 40min,
A:B=60:40 → 25:75；40 → 55min, A:B=25:75, UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/
Min, HPLC fraction are further separated into MS detection after UV detector with 3:1.Mass spectrum total ion current is as shown in figure 14.Mobile phase A
PH value 8.0, gradient are 0 → 15min, A:B=60:40；15 → 40min, A:B=60:40 → 25:75；40 → 55min, A:B=
When 25:75, main peak retention time is suitable, and peak shape is symmetrical.In relation to substance and main peak energy good separation.
11 oxidative degradation of embodiment
It takes about 50mg Tulathromycin bulk pharmaceutical chemicals in 10ml measuring bottle, adds 0.15%H2O2Solution 1ml adds 1ml acetonitrile hydrotropy,
(20 DEG C) placement 5min of room temperature, acetonitrile constant volume, 0.22 μm of membrane filtration are measured, 20 μ l of sample introduction by the chromatographic parameter of embodiment 1.
Mass spectrum total ion current figure is as shown in figure 15.
Reaction solution can be also enriched with according to following chromatographic conditions: XBridge C18 (19*50mm, 5 μm) prepares column；Sample introduction 500
μL；Mobile phase A: 0.35% aqueous formic acid (it is 7.8 that ammonium hydroxide, which adjusts pH value)；Mobile phase B: methanol: acetonitrile=45:25；A:B=
80:20；UV absorption wavelength: 205nm；Flow velocity: 17ml/min, mass spectrum total ion current figure are as shown in figure 16.
The related substance 2 of compound in Figure 15 at 23.32min, that is, of the invention, high-resolution measure molecular formula and are
C41H79N3O13([M+H]+=822.56921), more than in Tulathromycin molecular formula O.MS2Fragment 761.35416
(C39H73N2O12 +)、593.40082(C29H57N2O10)、532.34883(C27H50NO9 +)、230.17529(C12H24NO3 +).One
Grade mass spectrogram is as shown in figure 17, and second order ms figure is as shown in figure 18, and MS fragment pathways are as follows:
The reaction of 12 alkaline degradation of embodiment
It takes about 50mg Tulathromycin bulk pharmaceutical chemicals in 10ml measuring bottle, adds 1M NaOH solution 1ml, add 1ml acetonitrile hydrotropy, 60
DEG C heating water bath 25min, 0.1M HCl solution neutralizes, acetonitrile constant volume, 0.22 μm of membrane filtration, by the chromatographic parameter of embodiment 1
Measurement, 20 μ l of sample introduction.Mass spectrum total ion current figure is as shown in figure 19.
The related substance 4 of the compound at 7.80min in Figure 19, that is, of the invention, high-resolution measure molecular formula and are
C41H81N3O13([M+H]+=824.58527), MS2Fragment is 595.41656 (C29H59N2O10)、550.38651(C27H52NO10 +)、420.29623(C21H42NO7)、402.28582(C21H40NO6)、158.11700(C8H16NO2 +)、230.17590
(C12H24NO3 +).Its level-one mass spectrogram is as shown in figure 20, and second order ms figure is as shown in figure 21, and MS fragment pathways are as follows:
13 acid degradation of embodiment reaction
It takes about 50mg Tulathromycin sample in 10ml measuring bottle, adds 1M HCL solution 1ml, room temperature (20 DEG C) placements 4h, 1M
NaOH solution neutralizes, acetonitrile constant volume, 0.22 μm of membrane filtration, measures by the chromatographic parameter of embodiment 1,20 μ l of sample introduction.Mass spectrum is total
Ion flow graph is as shown in figure 22.
The reaction of 14 high temperature degradation of embodiment
Condition 1 takes Tulathromycin bulk pharmaceutical chemicals appropriate, is placed in 105 DEG C of baking ovens for 24 hours, lets cool, 5mg is taken to be dissolved in 1ml acetonitrile,
After 0.22 μm of membrane filtration, measured by the chromatographic parameter of embodiment 1,20 μ L of sample introduction.
Condition 2 takes about 50mg Tulathromycin sample in 10ml measuring bottle, and acetonitrile constant volume is set 12h in boiling water bath, let cool,
0.22 μm of membrane filtration is measured, 20 μ l of sample introduction by the chromatographic parameter of embodiment 1.
High temperature analysis: Tulathromycin solid sample high temperature has no that new impurity peaks occur compared with bulk pharmaceutical chemicals,
Sample is illustrating that the sample is more stable at high temperature.
The reaction of 15 illumination degrading of embodiment
It takes Tulathromycin appropriate, is placed in lighting box (illumination 45Lx ± 500Lx) illumination 10 days, respectively at the 5th day and the 10th
Its sampling is measured by the chromatographic parameter of embodiment 1, is as a result divided with after the comparison of the sample of non-lighting process discovery sample illumination 5 days
Son amount is that the impurity content of 818 (11 i.e. in table 1) increases, this impurity content increases after illumination 10 days.
16 high humidity degradation reaction of embodiment
KNO is placed in closed container lower part3Saturated solution (25 DEG C, humidity 92.5%), takes Tulathromycin appropriate, in the appearance
Placed 10 days in device, in the 5th day and the 10th day sample, by embodiment 1 chromatographic parameter measure, as a result with the sample of non-high humidity treatment
Product compare, and have no that new impurity peaks occur, illustrate that sample is influenced less by humidity.
Tulathromycin does not find new related substance in high temperature, high humidity, illumination, acid degradation reaction.
1 mobile phase A of comparative example is ammonium acetate
Chromatographic condition: XbridgeTMC18 (250*4.6mm, 5 μm) column；Mobile phase A: 15mM ammonium acetate, ammonium hydroxide adjust pH
Value is 7.8；Mobile phase B: methanol: acetonitrile=45:25；Gradient elution 0 → 15min, A:B=65:35；15 → 40min, A:B=
65:35 → 30:70；40 → 55min, A:B=30:70；UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/min；It removes
Outside above-mentioned parameter, remaining parameter is with embodiment 1, and mass spectrum total ion current figure is as shown in figure 23, and retention time is long, impurity separation
Difference.
2 Mobile phase B of comparative example is acetonitrile
Chromatographic condition: XbridgeTM C18 (250*4.6mm, 5 μm) column；Mobile phase A: (ammonium hydroxide adjusts pH to 0.35% formic acid
Value 7.66)；Mobile phase B: acetonitrile；Gradient elution 0 → 15min, A:B=65:35；15 → 40min, A:B=65:35 → 30:70；
40 → 55min, A:B=30:70；UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/min；In addition to above-mentioned parameter,
Remaining parameter is with embodiment 1, and mass spectrum total ion current figure is as shown in figure 24, and retention time is too short, and impurity separation is poor.
3 Mobile phase B of comparative example is methanol: acetonitrile=1:1
Chromatographic condition: XbridgeTMC18 (250*4.6mm, 5 μm) column；Mobile phase A: (ammonium hydroxide adjusts pH to 0.35% formic acid
Value 7.66)；Mobile phase B: methanol: acetonitrile=1:1；Gradient elution 0 → 15min, A:B=65:35；15 → 40min, A:B=65:
35→30:70；40 → 55min, A:B=30:70；UV absorption wavelength 205nm, 35 DEG C of column temperature, flow velocity 1.0ml/min；Except upper
It states outside parameter, for remaining parameter with embodiment 1, mass spectrum total ion current figure is as shown in figure 25, and impurity separation is poor.
Claims (10)
1. a kind of related substance 3 of Tulathromycin or its salt, structure of the Tulathromycin in relation to substance 3 are as follows:
2. a kind of enrichment preparation method of Tulathromycin as described in claim 1 in relation to substance 3, which is characterized in that including under
It states step: using high performance liquid chromatography, determinand being eluted in the chromatography column, collect the related object of Tulathromycin
Matter 3；The determinand is Tulathromycin bulk pharmaceutical chemicals or Tulathromycin degradation product；The chromatographic column is C18 analytical column
Or C18 prepares column；The mobile phase A of the elution be with ammonium hydroxide adjust pH value be 7~8, volume fraction be 0.1%~
0.4% aqueous formic acid, the Mobile phase B of the elution are that the volume ratio of methanol and acetonitrile is (1.5~2.0): 1 mixing is molten
Agent；
When the chromatographic column is C18 analytical column, the parameter of the elution is as follows: 0min → 15min, A:B=(60~
70): (40~30), 15min → 40min, A:B=(60~70): (40~30) → (25~35): (75~65), 40 →
55min, A:B=(25~35): (75~65)；The A:B refers to the volume ratio of the mobile phase A and the Mobile phase B；
When the chromatographic column is that C18 prepares column, the parameter of the elution is as follows: the mobile phase A and the stream
The volume ratio of dynamic phase B is (75~85): (25~15).
3. enrichment preparation method as claimed in claim 2, which is characterized in that the Tulathromycin degradation product is Tulathromycin
The substance obtained through degradation reaction；The degradation reaction is acid degradation reaction, alkaline degradation reaction, high temperature degradation reaction, high humidity
Degradation reaction, oxidative degradation or illumination degrading reaction；
And/or determinand sample introduction in the form of the acetonitrile solution of determinand.
4. enrichment preparation method as claimed in claim 3, which is characterized in that when the determinand is Tulathromycin through aoxidizing
When the substance that degradation reaction obtains, the reaction solution of the oxidative degradation forms the determinand after dilution in acetonitrile
Acetonitrile solution；
And/or when the determinand is the substance that Tulathromycin is reacted through alkaline degradation, the alkaline degradation reaction
Reaction solution neutralizes through acid and forms the acetonitrile solution of the determinand after dilution in acetonitrile；
And/or when the determinand is the substance that Tulathromycin is reacted through acid degradation, the acid degradation reaction
Reaction solution is through alkali neutralization and forms after dilution in acetonitrile the acetonitrile solution of the determinand；
And/or the concentration of the acetonitrile solution of the determinand is 3g/L~50g/L；
And/or when the chromatographic column is C18 analytical column, the sample volume of the acetonitrile solution of the determinand be 5 μ L~
100μL；
And/or when the chromatographic column is that C18 prepares column, the sample volume of the acetonitrile solution of the determinand be 50 μ L~
500μL。
5. enrichment preparation method as claimed in claim 2, which is characterized in that the body of methanol and acetonitrile in the Mobile phase B
Product is than being 45:25；
And/or when the chromatographic column is C18 analytical column, the pH value of the mobile phase A is 7.6~7.99；
And/or when the chromatographic column is C18 analytical column, the volume fraction of the aqueous formic acid of the mobile phase A is
0.3%~0.35%；
And/or when the chromatographic column is that C18 prepares column, the pH value of the mobile phase A is 7.6~7.8；
And/or when the chromatographic column is that C18 prepares column, the volume fraction of the aqueous formic acid of the mobile phase A is
0.3%~0.35%.
6. enrichment preparation method as claimed in claim 2, which is characterized in that when the chromatographic column is C18 analytical column, institute
The parameter for the elution stated is as follows: 0min → 15min, A:B=65:35；
And/or when the chromatographic column is C18 analytical column, the parameter of the elution is as follows: 15min → 40min, A:B=
65:35→30:70；
And/or when the chromatographic column is C18 analytical column, the parameter of the elution is as follows: 40 → 55min, A:B=30:
70。
7. enrichment preparation method as claimed in claim 2, which is characterized in that when the chromatographic column is that C18 prepares column, institute
The parameter for the elution stated is as follows: the volume ratio of the mobile phase A and the Mobile phase B is 80:20.
8. enrichment preparation method as claimed in claim 2, which is characterized in that when the chromatographic column is C18 analytical column, institute
The flow velocity for the enrichment preparation method stated is 0.7ml/min~1.3ml/min；
And/or when the chromatographic column is that C18 prepares column, the flow velocity of the enrichment preparation method for 10ml/min~
25ml/min。
9. enrichment preparation method as claimed in claim 2, which is characterized in that the mobile phase A and the Mobile phase B warp
0.22 μm of membrane filtration, and ultrasound 10min；
And/or the column temperature of the enrichment preparation method is 20 DEG C~40 DEG C；
And/or UV absorption wavelength when described enrichment preparation method detection is 200~215nm.
10. the related substance 3 of Tulathromycin as described in claim 1 or its salt reflect in the control of Tulathromycin quality as impurity
Fixed application.
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