CN106146571A - A kind of Tulathromycin has related substance, its preparation method and application - Google Patents
A kind of Tulathromycin has related substance, its preparation method and application Download PDFInfo
- Publication number
- CN106146571A CN106146571A CN201510133716.3A CN201510133716A CN106146571A CN 106146571 A CN106146571 A CN 106146571A CN 201510133716 A CN201510133716 A CN 201510133716A CN 106146571 A CN106146571 A CN 106146571A
- Authority
- CN
- China
- Prior art keywords
- tulathromycin
- mobile phase
- acetonitrile
- preparation
- related substance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- DNRHIEFLERNINE-BRJRFNKRSA-N CN(C)C(C1)[C@@H]1O Chemical compound CN(C)C(C1)[C@@H]1O DNRHIEFLERNINE-BRJRFNKRSA-N 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a kind of Tulathromycin and have related substance, its preparation method and application.The invention provides a kind of Tulathromycin and have related substance 4 or its salt.Present invention also offers Tulathromycin and have the preparation method of related substance 4, it comprises the steps: in acetonitrile, and Tulathromycin and sodium hydrate aqueous solution are carried out alkaline degradation reaction, and obtaining Tulathromycin has related substance 4.The Tulathromycin of the present invention has related substance to be the necessary that Tulathromycin carries out quality control；The preparation method of the present invention can prepare and efficiently separate Tulathromycin related substance 4, thus controls the drug quality of Tulathromycin, and the research for Tulathromycin unknown impuritie is had laid a good foundation.
Description
Technical field
The present invention relates to a kind of Tulathromycin and have related substance, its preparation method and application.
Background technology
Tulathromycin (Tulathromycin) is semi-synthetic macrolide antibiotics, in 2004
USA and EU lists.This medicine is mainly used in cattle and pig by sensitive microbial respiratory infectious disease
And the preventing and treating of infectious bovine keratocon junctivitis is caused by cattle catarrhalis, drug effect is better than on market and is widely used
Macrolide antibiotics tylosin and tilmicosin, the prospect of the application in Production of Livestock and Poultry is wide.
Tulathromycin is by isomer A, B (molecular formula C41H79N3O12, molecular weight 806.09) and press 9:1
The fifteen-membered ring macrolide antibiotics of composition, two kinds of isomers can be by lactone between C11 and C13
Formation and the fracture of key are changed.Containing three Polar Amides groups in Tulathromycin structure, PKa value
Between 8.6～9.6, belong to three amine macrolide antibiotics, be different from azacyclo-lactone and ketone lactone
Class antibiotic.
In order to ensure the safety of animal derived food, animal specific drug quality strictly must be controlled,
Medicine assay, the Structural Identification of unknown impuritie and Light absorbing impurty be Control of drug quality have efficacious prescriptions
Method, impurity analysis is the important content of medicine quality standard.Tulathromycin is produced by semi-synthetic process, with
It is lower that synthetic drug compares controllability, and therefore impurity spectrum is increasingly complex and is difficult to predict.Existing European Pharmacopoeia
(EP) all veterinary drug is included wherein with American Pharmacopeia (USP), and to there being related substance limitation to claim,
European Pharmacopoeia also requires to be controlled specific impurities that (British Pharmacopoeia has included the complete of European Pharmacopoeia as usual
Portion's monograph, content does not makes an amendment).VICH (veterinary drug registration technology requires international coordination meeting)
Guideline requires that the impurity reporting limit of veterinary's special raw material medicine is 0.10%, and limit of identification is 0.20%,
Control limit was 0.50% (this guidance does not include semisynthetic antibiotics)；EMA (Europe drug administration)
Require that the limit of impurities of semi-synthetic veterinary drug should meet the requirement of VICH guideline.
At present, there is not yet the report using LC-MS method separation detection Tulathromycin to have related substance both at home and abroad.
Therefore, needing degraded Tulathromycin badly has related substance with what preparation was degraded, and setting up Tulathromycin has related substance
LC-MS separation and detection method, and have related substance to identify Tulathromycin.
Summary of the invention
Problem to be solved by this invention be in order to overcome in prior art lack Tulathromycin synthesis and
The separation and detection method having related substance produced in degradation process, and it can not be synthesized, identify,
The defects such as confirmation structure, and provide a kind of Tulathromycin and have related substance, its preparation method and application.This
The Tulathromycin of invention has related substance to be the necessary that Tulathromycin carries out quality control；The system of the present invention
Preparation Method can prepare and efficiently separate Tulathromycin related substance 4, thus controls the medicine of Tulathromycin
Quality, the research for Tulathromycin unknown impuritie is had laid a good foundation.
The invention provides a kind of Tulathromycin and have the separation method of related substance, it comprises the steps: to adopt
By high performance liquid chromatography, determinand is carried out in the chromatography column eluting,；Described determinand is
Tulathromycin crude drug or Tulathromycin degradation product；Described chromatographic column is C18 analytical column or C18 system
Standby post；The mobile phase A of described eluting be with ammonia regulation pH value be 7～8, volume fraction be
0.1%～0.4% aqueous formic acid, the Mobile phase B of described eluting is methanol and the volume ratio of acetonitrile is
(1.5～2.0): the mixed solvent of 1；
When described chromatographic column is C18 analytical column, the parameter of described eluting 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)；Described A:B refers to described
Mobile phase A and the volume ratio of described Mobile phase B；
When described chromatographic column be C18 prepare post time, the parameter of described eluting is as follows: described flowing
Phase A is (75～85) with the volume ratio of described Mobile phase B: (25～15).
In described separation method, described Tulathromycin crude drug can be that the Thailand of this area routine draws mould
Element crude drug, preferably Jiangsu reach the clouds pharmaceutcal corporation, Ltd produce Tulathromycin crude drug.
In described separation method, described Tulathromycin degradation product is that Tulathromycin obtains through degradation reaction
The material arrived；Described degradation reaction can be the degradation reaction that this area is conventional, and preferably acid degradation is anti-
Should, alkaline degradation reaction, high temperature degradation reaction, high humidity degradation reaction, oxidative degradation or illumination degrading
Reaction；
Described oxidative degradation can comprise the steps: in acetonitrile, by Tulathromycin and hydrogen peroxide
Aqueous solution carries out oxidative degradation, obtains?；
Described alkaline degradation reaction can comprise the steps: in acetonitrile, by Tulathromycin and sodium hydroxide
Aqueous solution carries out alkaline degradation reaction, obtains?.
In described separation method, described determinand can use the method sample introduction that this area is conventional, relatively
Goodly with the form sample introduction of the acetonitrile solution of determinand；When described determinand is the oxidized fall of Tulathromycin
When solving the material that reaction obtains, it is preferred that the reactant liquor of described oxidative degradation is after dilution in acetonitrile
The acetonitrile solution of the determinand described in formation；When described determinand is that Tulathromycin reacts through alkaline degradation
During the material arrived, it is preferred that the reactant liquor of described alkaline degradation reaction neutralizes through acid and uses dilution in acetonitrile
The acetonitrile solution of the determinand described in rear formation；When described determinand is that Tulathromycin reacts through acid degradation
During the material obtained, it is preferred that the reactant liquor of described acid degradation reaction neutralizes and uses acetonitrile dilute through alkali
The acetonitrile solution of described determinand is formed after releasing；
The acid that described acid neutralizes can be the acid that this area is conventional, preferably aqueous hydrochloric acid solution, more preferably
Aqueous hydrochloric acid solution for 0.1mol/L；The alkali that described alkali neutralizes can be the alkali that this area is conventional, preferably
For NaOH aqueous solution, it it is more preferably the NaOH aqueous solution of 0.1mol/L；
The concentration of the acetonitrile solution of described determinand can be the concentration that this area is conventional, preferably
3g/L～50g/L, is more preferably 5g/L～20g/L；
The sample size of the acetonitrile solution of described determinand can be the sample size that this area is conventional, when described
When chromatographic column is C18 analytical column, described sample size is preferably 5 μ L～100 μ L, is more preferably
20 μ L～50 μ L；When described chromatographic column be C18 prepare post time, described sample size is preferably
50 μ L～500 μ L, are more preferably 50 μ L～300 μ L.
In described separation method, described C18 analytical column can be the C18 analysis that this area is conventional
Post, preferably XbridgeTM C18(250 × 4.6mm, 5 μm) analytical column；Described C18 prepares post
Can be that the conventional C18 in this area prepares post, preferably XbridgeTM C18(19*50mm, 5 μm) are made
Standby post.
In described separation method, in described Mobile phase B, methanol is preferably with the volume ratio of acetonitrile
45:25；When described chromatographic column is C18 analytical column, the pH value of described mobile phase A is preferably
Being 7.6～7.99 (such as 7.66), the volume fraction of the aqueous formic acid of described mobile phase A is preferably
Be 0.3%～0.35%, more preferably, for ammonia regulation pH value be 7.6, volume fraction be 0.35%
Aqueous formic acid；When described chromatographic column be C18 prepare post time, the pH value of described mobile phase A
Preferably 7.6～7.8, the volume fraction of the aqueous formic acid of described mobile phase A is preferably
0.3%～0.35%.
In described separation method, when described chromatographic column is C18 analytical column, described eluting
Parameter is as follows: 0min → 15min, A:B=65:35,15min → 40min, A:B=65:35 →
30:70,40 → 55min, A:B=30:70；When described chromatographic column be C18 prepare post time, described
The parameter of eluting is as follows: described mobile phase A with the volume ratio of described Mobile phase B is
80:20。
In described separation method, described ammonia is the ammonia that this area is conventional, preferably quality
Mark is the ammonia of 25%～28%.
In described separation method, the high performance liquid chromatography that this area is conventional can be used, when described color
When spectrum post is C18 analytical column, preferably use the Alliance 2695/ZQ liquid chromatograph of Waters company
-GC-MS；When described chromatographic column be C18 prepare post time, preferably use Waters company
2767 type automatic purification instrument.
In described separation method, the flow velocity of described separation method can be the flow velocity that this area is conventional；
When described chromatographic column is C18 analytical column, described flow velocity is preferably
0.7ml/min～1.3ml/min, 0.8ml/min～1.2ml/min, such as 1.0ml/min；When described chromatograph
Post is C18 when preparing post, and described flow velocity is preferably 10ml/min～25ml/min, is more preferably
15ml/min～20ml/min, such as 17ml/min.
In described separation method, described mobile phase A and described Mobile phase B can use ability
The processing method of territory routine carries out pretreatment, preferably through 0.22 μm membrane filtration, and ultrasonic 10min.
In described separation method, the column temperature of described separation method can be the column temperature that this area is conventional,
Preferably 20 DEG C～40 DEG C, be more preferably 25 DEG C～35 DEG C, such as 30 DEG C.
In described separation method, uv absorption wavelength when described separation method detects can be ability
The uv absorption wavelength that territory is conventional, preferably 200～215nm, be more preferably 205～210nm；Pass through
1/5～the 1/3 entrance mass spectrograph detection of the stream part after UV-detector.
Present invention also offers a kind of Tulathromycin and have related substance 1 or its salt,
Present invention also offers described Tulathromycin and have the enrichment preparation method of related substance 1, it is as above-mentioned
Tulathromycin have the separation method of related substance, the Tulathromycin described in collection has related substance 1.
Present invention also offers described Tulathromycin and have related substance 1 or its salt at Tulathromycin quality control
The middle application identified as impurity.
Present invention also offers a kind of Tulathromycin and have related substance 2 or its salt,
Present invention also offers described Tulathromycin and have the preparation method of related substance 2, it includes following step
Rapid: in acetonitrile, Tulathromycin and aqueous hydrogen peroxide solution are carried out oxidative degradation, obtains described
Tulathromycin has related substance 2.
In described oxidative degradation, described Tulathromycin and the mass volume ratio of described acetonitrile
Can be the regular quality volume ratio of oxidative degradation in this area, preferably 3g/L～50g/L, more preferably
Ground is 5g/L～20g/L.
In described oxidative degradation, described aqueous hydrogen peroxide solution can be the dioxygen that this area is conventional
Water aqueous solution, preferably mass fraction are the aqueous hydrogen peroxide solution of 0.1%～30%, are more preferably
The aqueous hydrogen peroxide solution of 0.15%～0.5%.
In described oxidative degradation, described Tulathromycin and the matter of described aqueous hydrogen peroxide solution
Amount volume ratio can be the regular quality volume ratio of oxidative degradation in this area, preferably
40g/L～90g/L, is more preferably 50g/L～75g/L.
In described oxidative degradation, the temperature of described oxidative degradation can be oxygen in this area
Changing the ordinary temperature of degradation reaction, preferably 20 DEG C～50 DEG C, be more preferably 20 DEG C～30 DEG C.
In described oxidative degradation, the process of described oxidative degradation can use in this area
Routine monitoring method (such as LC-MS) be monitored, the time of described degradation reaction is preferably
For 1min～30min, it is more preferably 5min～20min.
Described oxidative degradation the most also includes post processing；Described post processing can be in this area
The conventional post processing of oxidative degradation, it is preferred that the reactant liquor of described oxidative degradation is according to upper
The Tulathromycin stated has the separation method of related substance to carry out post processing.
Present invention also offers described Tulathromycin and have related substance 2 or its salt at Tulathromycin quality control
The middle application identified as impurity.
The invention provides a kind of Tulathromycin and have related substance 3 or its salt,
Present invention also offers described Tulathromycin and have the enrichment preparation method of related substance 3, it is as above-mentioned
Tulathromycin have the separation method of related substance, the Tulathromycin described in collection has related substance 3.
Present invention also offers described Tulathromycin and have related substance 3 or its salt at Tulathromycin quality control
The middle application identified as impurity.
Present invention also offers a kind of Tulathromycin and have related substance 4 or its salt,
Present invention also offers described Tulathromycin and have the preparation method of related substance 4, it includes following step
Rapid: in acetonitrile, Tulathromycin and sodium hydrate aqueous solution are carried out alkaline degradation reaction, obtains described
Tulathromycin has related substance 4.
In described alkaline degradation reaction, the mass volume ratio of described Tulathromycin and described acetonitrile can
For the regular quality volume ratio of alkaline degradation reaction, preferably 3g/L～50g/L in this area, it is more preferably
5g/L～20g/L.
In described alkaline degradation reaction, described sodium hydrate aqueous solution can be the hydrogen-oxygen that this area is conventional
Change the sodium hydrate aqueous solution of sodium water solution, preferably 0.1mol/L～10mol/L, be more preferably
The sodium hydrate aqueous solution of 1mol/L～5mol/L.
In described alkaline degradation reaction, described Tulathromycin and the matter of described sodium hydrate aqueous solution
Amount volume ratio can be the regular quality volume ratio of alkaline degradation reaction in this area, preferably
40g/L～90g/L, is more preferably 50g/L～75g/L.
In described alkaline degradation reaction, the temperature of described alkaline degradation reaction can be alkaline degradation in this area
The ordinary temperature of reaction, preferably 50 DEG C～90 DEG C, be more preferably 55 DEG C～70 DEG C, such as 60 DEG C.
In described alkaline degradation reaction, it is normal that the process of described alkaline degradation reaction can use in this area
Rule monitoring method (such as TLC, HPLC or LC-MS) are monitored, described alkaline degradation reaction
Time is preferably 15min～50min, is more preferably 20min～40min, such as 25min.
Described alkaline degradation reaction the most also includes post processing；Described post processing can be alkali in this area
The conventional post processing of degradation reaction, it is preferred that the reactant liquor of described alkaline degradation reaction is according to above-mentioned Thailand
Mycin is drawn to have the separation method of related substance to carry out post processing.
Present invention also offers described Tulathromycin and have related substance 4 or its salt at Tulathromycin quality control
The middle application identified as impurity.
Without prejudice to the field on the basis of common sense, above-mentioned each optimum condition, can combination in any, obtain this
Invent each preferred embodiments.
Agents useful for same of the present invention and raw material are the most commercially.
The most progressive effect of the present invention is: the Tulathromycin of the present invention has related substance to be to Tulathromycin
Carry out the necessary of quality control；The preparation method of the present invention can prepare and efficiently separate Tulathromycin to be had
Related substance 4, thus control the drug quality of Tulathromycin, the research for Tulathromycin unknown impuritie is established
Good basis.
Accompanying drawing explanation
Fig. 1 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 1 obtains.
Fig. 2 is the first mass spectrometric figure having related substance 1.
Fig. 3 is the second order ms figure having related substance 1.
Fig. 4 is the first mass spectrometric figure having related substance 3.
Fig. 5 is the second order ms figure having related substance 3.
Fig. 6 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 2 obtains.
Fig. 7 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 3 obtains.
Fig. 8 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 4 obtains.
Fig. 9 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 5 obtains.
Figure 10 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 6 obtains.
Figure 11 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 7 obtains.
Figure 12 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 8 obtains.
Figure 13 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 9 obtains.
Figure 14 is the Tulathromycin crude drug mass spectrum total ions chromatogram that embodiment 10 obtains.
After Figure 15 is Tulathromycin crude drug oxidative degradation, through the mass spectrum total ion current color of analytical column
Spectrogram.
After Figure 16 is Tulathromycin crude drug oxidative degradation, through preparing the mass spectrum total ion current color of post
Spectrogram.
Figure 17 is the first mass spectrometric figure having related substance 2.
Figure 18 is the second order ms figure having related substance 2.
After Figure 19 is the reaction of Tulathromycin crude drug alkaline degradation, through the mass spectrum total ionic chromatographic of analytical column
Figure.
Figure 20 is the first mass spectrometric figure having related substance 4.
Figure 21 is the second order ms figure having related substance 4.
After Figure 22 is Tulathromycin crude drug acid degradation reaction, through the mass spectrum total ionic chromatographic of analytical column
Figure.
Figure 23 is the Tulathromycin crude drug mass spectrum total ions chromatogram that comparative example 1 obtains.
Figure 24 is the Tulathromycin crude drug mass spectrum total ions chromatogram that comparative example 2 obtains.
Figure 25 is the Tulathromycin crude drug mass spectrum total ions chromatogram that comparative example 3 obtains.
Detailed description of the invention
Further illustrate the present invention below by the mode of embodiment, but the most therefore limit the present invention to
Among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to often
Rule method and condition, or select according to catalogue.
Reagent used herein is as follows:
Acetonitrile, methanol are chromatographically pure (Thermo Fisher Scientific company)；Formic acid (98%), ammonia
Water (25wt%～28wt%), 30wt% hydrogen peroxide, hydrochloric acid are analytical pure, sodium hydroxide (traditional Chinese medicines collection
Chemicals company limited of group)；Water is Wahaha Pure Water (crossing 0.22 μm moisture film)；Tulathromycin is former
Material medicine 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 matter
Spectrometer；Q-Tof micro mass spectrograph；Masslynx chromatographic work station (Waters company)
(2) Q-Exactive level Four bar track trap high resolution mass spectrum (Thermo company)
(3) 2767 type automatic purification instrument
Mass spectrometry method used in the present invention is as follows:
Micromass ZQ: detection pattern ESI (+)；Spray voltage 3kV；Taper hole voltage 30V；Source
Temperature 100 DEG C；Desolventizing temperature 250 DEG C；Full scan scope m/z100～1500.
Q-Exactive level Four bar track trap high resolution mass spectrum parameter: HESI spray voltage :+3.0
KV/-2.7KV (positive and negative switching scans simultaneously)；Sheath atmospheric pressure: 35arb；Assist gas pressure power: 10arb；Capillary
Pipe temperature: 300 DEG C；Heating-up temperature: 300 DEG C；Scan pattern: Full MS (resolution 7000) and
Dd-MS2 (resolution 17500)
Embodiment 1
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.35% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as it is shown in figure 1, each miscellaneous
Matter is as shown in table 1.When flowing phase salinity is 0.35%, main peak retention time is suitable, and peak shape is good.
There are related substance and main peak energy good separation.It can be seen that the method for the present invention can be used for detection Thailand
Drawing mycin and impurity thereof, the method can be applicable to the monitoring of Tulathromycin crude drug synthesis technique and quality control
System.
Each impurity in table 1 Fig. 1
Note: Aera% is that area normalization method records percentage composition.
The i.e. present invention in table 14 has related substance 1, and it is C that high-resolution records molecular formula40H77N3O12
([M+H]+=792.55829), MS2Fragment is 689.45898 (C35H63NO12)、563.39099
(C28H55N2O9)、420.29337(C21H42NO7)、230.17519(C12H24NO3).One
Level mass spectrum is as in figure 2 it is shown, second order ms figure is as it is shown on figure 3, MS fragment pathways is as follows:
The i.e. present invention in table 1 13 has related substance 3, and it is C that high-resolution records molecular formula39H72N2O12
([M+H]+=761.51624), MS2Fragment is 532.34827 (C27H50NO9)、230.17520
(C12H24NO3).As shown in Figure 4, second order ms figure is as it is shown in figure 5, mass spectrum splits its one-level mass spectrum
Solution approach is as follows:
Embodiment 2
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.3% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in Figure 6.Flowing
When phase salinity is 0.3%, main peak retention time is suitable, and peak shape is good.There is related substance good with main peak energy
Good separation.
Embodiment 3
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.4% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in Figure 7.Flowing
When phase salinity is 0.4%, main peak retention time is suitable, and peak shape is good.There is related substance good with main peak energy
Good separation.
Embodiment 4
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.1% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in Figure 8.Flowing
When phase salinity is 0.1%, when pH value is 7.99, main peak retention time is suitable, and peak shape is symmetrical.Relevant
Material and main peak energy good separation.
Embodiment 5
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.35% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in Figure 9.Flowing
Methanol in phase B: during acetonitrile=1.5:1, main peak retention time is suitable, and peak shape is symmetrical.There are related substance and main peak
Can good separation.
Embodiment 6
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.35% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in Figure 10.Flowing
Phase gradient elution program is 0 → 15min, A:B=65:35；15 → 40min, A:B=65:35 → 35:65；
When 40 → 55min, A:B=35:65, main peak retention time is suitable, and peak shape is symmetrical.There are related substance and master
Peak energy good separation.
Embodiment 7
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.35% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in figure 11.Flowing
Phase gradient elution program is 0 → 15min, A:B=70:30；15 → 40min, A:B=70:30 → 30:70；
When 40 → 55min, A:B=30:70, main peak retention time is suitable, and peak shape is symmetrical.There are related substance and master
Peak energy good separation.
Embodiment 8
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.35% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.2ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in figure 12.Flowing
Phase B is methanol: acetonitrile=2:1, and during flow velocity 1.2ml/min, main peak retention time is suitable, and peak shape is symmetrical.
There are related substance and main peak energy good separation.
Embodiment 9
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.35% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in figure 13.Flowing
The pH value 7.0 of phase A, gradient is 0 → 15min, A:B=70:30；15 → 40min, A:B=70:30 →
35:65；When 40 → 55min, A:B=35:65, main peak retention time is suitable, and peak shape is symmetrical.Relevant thing
Matter and main peak energy good separation.
Embodiment 10
5mg Tulathromycin crude drug is dissolved in 1mL acetonitrile, sample size 20 μ L.
Chromatographic parameter: XbridgeTM C18(250*4.6mm, 5 μm) post, mobile phase A: 0.35% first
Aqueous acid (ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min, HPLC flow part
It is further separated into MS detection with 3:1 after UV detector.Mass spectrum total ion current is as shown in figure 14.Flowing
The pH value 8.0 of phase A, gradient is 0 → 15min, A:B=60:40；15 → 40min, A:B=60:40 →
25:75；When 40 → 55min, A:B=25:75, main peak retention time is suitable, and peak shape is symmetrical.Relevant thing
Matter and main peak energy good separation.
Embodiment 11 oxidative degradation
Take about 50mg Tulathromycin crude drug in 10ml measuring bottle, add 0.15%H2O2Solution 1ml, adds
1ml acetonitrile hydrotropy, room temperature (20 DEG C) is placed 5min, acetonitrile constant volume, 0.22 μm membrane filtration, is pressed
The chromatographic parameter of embodiment 1 measures, sample introduction 20 μ l.Mass spectrum total ion current figure is as shown in figure 15.
Reactant liquor also can be enriched with according to following chromatographic condition: XBridge C18 (19*50mm, 5 μm) makes
Standby post；Sample introduction 500 μ L；Mobile phase A: 0.35% aqueous formic acid (ammonia regulation pH value is 7.8)；
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 is as shown in figure 16.
In Figure 15, the i.e. present invention's of compound at 23.32min has related substance 2, and high-resolution records molecular formula
For C41H79N3O13([M+H]+=822.56921), many O than in Tulathromycin molecular formula.MS2
Fragment 761.35416 (C39H73N2O12 +)、593.40082(C29H57N2O10)、
532.34883(C27H50NO9 +)、230.17529(C12H24NO3 +).Its one-level mass spectrum as shown in figure 17,
As shown in figure 18, MS fragment pathways is as follows for second order ms figure:
Embodiment 12 alkaline degradation reacts
Take about 50mg Tulathromycin crude drug in 10ml measuring bottle, add 1M NaOH solution 1ml, add
1ml acetonitrile hydrotropy, 60 DEG C of heating in water bath 25min, 0.1M HCl solution neutralizes, acetonitrile constant volume, 0.22 μm
Membrane filtration, is measured by the chromatographic parameter of embodiment 1, sample introduction 20 μ l.Mass spectrum total ion current figure such as figure
Shown in 19.
The i.e. present invention of the compound at 7.80min in Figure 19 has related substance 4, and high-resolution records molecular formula
For 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 one-level mass spectrum as shown in figure 20,
As shown in figure 21, MS fragment pathways is as follows for second order ms figure:
Embodiment 13 acid degradation is reacted
Take about 50mg Tulathromycin sample in 10ml measuring bottle, add 1M HCL solution 1ml, room temperature
(20 DEG C) are placed 4h, 1M NaOH solution and are neutralized, acetonitrile constant volume, 0.22 μm membrane filtration, by real
The chromatographic parameter executing example 1 measures, sample introduction 20 μ l.Mass spectrum total ion current figure is as shown in figure 22.
Embodiment 14 high temperature degradation reacts
It is appropriate that condition 1 takes Tulathromycin crude drug, is placed in 24h in 105 DEG C of baking ovens, lets cool, take 5mg
It is dissolved in 1ml acetonitrile, after 0.22 μm membrane filtration, measures by the chromatographic parameter of embodiment 1, sample introduction
20μL。
Condition 2 takes about 50mg Tulathromycin sample in 10ml measuring bottle, and acetonitrile constant volume puts boiling water bath
Middle 12h, lets cool, 0.22 μm membrane filtration, measures by the chromatographic parameter of embodiment 1, sample introduction 20 μ l.
High temperature is analyzed: Tulathromycin solid sample high temperature, compared with crude drug, has no new miscellaneous
Mass peak occurs, sample is illustrating that this sample is the most stable.
Embodiment 15 illumination degrading reacts
Take Tulathromycin appropriate, be placed in lighting box (illumination 45Lx ± 500Lx) illumination 10 days, respectively
In sampling in the 5th day and the 10th day, measure by the chromatographic parameter of embodiment 1, result and non-photo-irradiation treatment
Sample contrast find sample illumination 5 days after molecular weight be the impurity content of 818 (i.e. 11 in table 1)
Increasing, after illumination 10 days, this impurity content increases.
Embodiment 16 high humidity degradation reaction
KNO is placed in hermetic container bottom3Saturated solution (25 DEG C, humidity 92.5%), takes Tulathromycin
In right amount, place 10 days in this embodiment, in sampling in the 5th day and the 10th day, by the chromatograph of embodiment 1
Parametric measurement, result compares with the sample of non-high humidity treatment, and the impurity peaks having no new occurs, sample is described
Affected not quite by humidity.
Tulathromycin does not finds new have related substance in high temperature, high humidity, illumination, acid degradation reaction.
Comparative example 1 mobile phase A is ammonium acetate
Chromatographic condition: XbridgeTMC18 (250*4.6mm, 5 μm) post；Mobile phase A: 15mM
Ammonium acetate, ammonia regulation 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, column temperature 35 DEG C, flow velocity 1.0ml/min；In addition to above-mentioned parameter,
Remaining parameter is all with embodiment 1, and as shown in figure 23, its retention time is long, impurity for mass spectrum total ion current figure
It is poor to separate.
Comparative example 2 Mobile phase B is acetonitrile
Chromatographic condition: XbridgeTM C18 (250*4.6mm, 5 μm) post；Mobile phase A: 0.35%
Formic acid (ammonia regulation pH 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, column temperature 35 DEG C, flow velocity 1.0ml/min；In addition to above-mentioned parameter, remaining parameter all with embodiment 1,
As shown in figure 24, its retention time is the shortest, and magazins' layout is poor for mass spectrum total ion current figure.
Comparative example 3 Mobile phase B is methanol: acetonitrile=1:1
Chromatographic condition: XbridgeTMC18 (250*4.6mm, 5 μm) post；Mobile phase A: 0.35% first
Acid (ammonia regulation pH 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, column temperature 35 DEG C, flow velocity 1.0ml/min；In addition to above-mentioned parameter,
Remaining parameter is all with embodiment 1, and as shown in figure 25, its magazins' layout is poor for mass spectrum total ion current figure.
Claims (10)
1. Tulathromycin has related substance 4 or its salt, and its structure is as follows:
2. Tulathromycin as claimed in claim 1 has the preparation method of related substance 4, it is characterised in that
Comprise the steps: in acetonitrile, Tulathromycin and sodium hydrate aqueous solution carried out alkaline degradation reaction,
Obtain described Tulathromycin and have related substance 4.
3. preparation method as claimed in claim 2, it is characterised in that described Tulathromycin and institute
The mass volume ratio of the acetonitrile stated is 3g/L～50g/L；
And/or, described sodium hydrate aqueous solution is the sodium hydrate aqueous solution of 0.1mol/L～10mol/L；
And/or, described Tulathromycin with the mass volume ratio of described sodium hydrate aqueous solution is
40g/L～90g/L；
And/or, the temperature of described alkaline degradation reaction is 50 DEG C～90 DEG C；
And/or, the time of described alkaline degradation reaction is 15min～50min.
4. preparation method as claimed in claim 3, it is characterised in that described Tulathromycin and institute
The mass volume ratio of the acetonitrile stated is 5g/L～20g/L；
And/or, described sodium hydrate aqueous solution is the sodium hydrate aqueous solution of 1mol/L～5mol/L；
And/or, described Tulathromycin with the mass volume ratio of described sodium hydrate aqueous solution is
50g/L～75g/L；
And/or, the temperature of described alkaline degradation reaction is 55 DEG C～70 DEG C；
And/or, the time of described alkaline degradation reaction is 20min～40min.
5. preparation method as claimed in claim 2, it is characterised in that described alkaline degradation reaction
Post processing, comprises the steps: to use high performance liquid chromatography, by the acetonitrile solution of determinand in chromatograph
Post carries out eluting,；The acetonitrile solution of described determinand is by the reaction of described alkaline degradation reaction
Liquid prepares after acid neutralization, dilution in acetonitrile；Described chromatographic column is C18 analytical column or C18 prepares post；
The mobile phase A of described eluting be with ammonia regulation pH value be 7～8, volume fraction be 0.1%～0.4%
Aqueous formic acid, the Mobile phase B of described eluting is methanol and the volume ratio of acetonitrile is (1.5～2.0): 1
Mixed solvent；
When described chromatographic column is C18 analytical column, the parameter of described eluting 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)；Described A:B refers to described
Mobile phase A and the volume ratio of described Mobile phase B；
When described chromatographic column be C18 prepare post time, the parameter of described eluting is as follows: described flowing
Phase A is (75～85) with the volume ratio of described Mobile phase B: (25～15).
6. preparation method as claimed in claim 5, it is characterised in that the acetonitrile of described determinand
The concentration of solution is 3g/L～50g/L；
And/or, when described chromatographic column is C18 analytical column, the acetonitrile solution of described determinand
Sample size is 5 μ L～100 μ L；
And/or, when described chromatographic column be C18 prepare post time, the acetonitrile solution of described determinand
Sample size is 50 μ L～500 μ L.
7. preparation method as claimed in claim 5, it is characterised in that first in described Mobile phase B
Alcohol is 45:25 with the volume ratio of acetonitrile；
And/or, when described chromatographic column is C18 analytical column, the pH value of described mobile phase A is
7.6～7.99；
And/or, when described chromatographic column is C18 analytical column, the formic acid of described mobile phase A is water-soluble
The volume fraction of liquid is 0.3%～0.35%；
And/or, when described chromatographic column be C18 prepare post time, the pH value of described mobile phase A is
7.6～7.8；
And/or, when described chromatographic column be C18 prepare post time, the formic acid of described mobile phase A is water-soluble
The volume fraction of liquid is 0.3%～0.35%.
8. preparation method as claimed in claim 5, it is characterised in that when described chromatographic column is C18
During analytical column, the parameter of described eluting is as follows: 0min → 15min, A:B=65:35；
And/or, when described chromatographic column is C18 analytical column, the parameter of described eluting is as follows: 15min
→ 40min, A:B=65:35 → 30:70；
And/or, when described chromatographic column is C18 analytical column, the parameter of described eluting is as follows: 40
→ 55min, A:B=30:70；
And/or, when described chromatographic column be C18 prepare post time, the parameter of described eluting is as follows: institute
The mobile phase A stated is 80:20 with the volume ratio of described Mobile phase B.
9. preparation method as claimed in claim 5, it is characterised in that when described chromatographic column is C18
During analytical column, the flow velocity of described preparation method is 0.7ml/min～1.3ml/min；
And/or, when described chromatographic column be C18 prepare post time, the flow velocity of described preparation method is
10ml/min～25ml/min；
And/or, described mobile phase A and described Mobile phase B are through 0.22 μm membrane filtration and ultrasonic
10min；
And/or, the column temperature of described preparation method is 20 DEG C～40 DEG C；
And/or, uv absorption wavelength when described preparation method detects is 200～215nm.
10. Tulathromycin as claimed in claim 1 has related substance 4 or its salt in Tulathromycin quality control
The application identified as impurity in system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510133716.3A CN106146571B (en) | 2015-03-25 | 2015-03-25 | A kind of related substance of Tulathromycin, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510133716.3A CN106146571B (en) | 2015-03-25 | 2015-03-25 | A kind of related substance of Tulathromycin, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106146571A true CN106146571A (en) | 2016-11-23 |
| CN106146571B CN106146571B (en) | 2019-04-12 |
Family
ID=57340117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510133716.3A Active CN106146571B (en) | 2015-03-25 | 2015-03-25 | A kind of related substance of Tulathromycin, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106146571B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456372A (en) * | 2018-12-19 | 2019-03-12 | 江苏威凌生化科技有限公司 | A kind of separation method of the Tulathromycin in relation to substance |
| CN109535211A (en) * | 2018-12-19 | 2019-03-29 | 江苏威凌生化科技有限公司 | A kind of method of synthesizing and purifying Tulathromycin impurity C |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104119413A (en) * | 2014-07-29 | 2014-10-29 | 华中农业大学 | Synthesis method of tulathromycin residue marker |
-
2015
- 2015-03-25 CN CN201510133716.3A patent/CN106146571B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104119413A (en) * | 2014-07-29 | 2014-10-29 | 华中农业大学 | Synthesis method of tulathromycin residue marker |
Non-Patent Citations (2)
| Title |
|---|
| 许卓妮，等: "阿奇霉素中有关物质的LC-MS法检测", 《中国医药工业杂志》 * |
| 黄珊，等: "大环内酯类抗生素杂质谱研究进展", 《中国医药工业杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456372A (en) * | 2018-12-19 | 2019-03-12 | 江苏威凌生化科技有限公司 | A kind of separation method of the Tulathromycin in relation to substance |
| CN109535211A (en) * | 2018-12-19 | 2019-03-29 | 江苏威凌生化科技有限公司 | A kind of method of synthesizing and purifying Tulathromycin impurity C |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106146571B (en) | 2019-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xia et al. | Quantitation of ursolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its pharmacokinetic study | |
| Sun et al. | A UHPLC–MS/MS method for simultaneous determination of six flavonoids, gallic acid and 5, 8-dihydroxy-1, 4-naphthoquinone in rat plasma and its application to a pharmacokinetic study of Cortex Juglandis Mandshuricae extract | |
| Ma et al. | Determination and pharmacokinetic study of amlodipine in human plasma by ultra performance liquid chromatography–electrospray ionization mass spectrometry | |
| Turnipseed et al. | Analysis of avermectin and moxidectin residues in milk by liquid chromatography–tandem mass spectrometry using an atmospheric pressure chemical ionization/atmospheric pressure photoionization source | |
| Cheng et al. | Pharmacokinetic comparisons of typical constituents in white peony root and sulfur fumigated white peony root after oral administration to mice | |
| Xue et al. | Screening for in vitro metabolites of Abelmoschus manihot extract in intestinal bacteria by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry | |
| Wang et al. | Simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and geniposide in rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study after administration of Reduning injection | |
| CN106146576A (en) | A kind of Tulathromycin has related substance, enrichment preparation method, separation method and application | |
| Guo et al. | Fast and automated characterization of major constituents in rat biofluid after oral administration of Abelmoschus manihot extract using ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry and MetaboLynx | |
| Cao et al. | Characterization of chemical constituents and rats metabolites of an alkaloidal extract of Alstonia scholaris leaves by liquid chromatography coupled with mass spectrometry | |
| CN106146571A (en) | A kind of Tulathromycin has related substance, its preparation method and application | |
| CN105572284B (en) | A kind of quick detection and the analysis method for identifying chemical composition in elegant jessamine | |
| Wang et al. | Identification and elucidation of the structure of in vivo metabolites of diaveridine in chicken | |
| CN106146577A (en) | A kind of Tulathromycin has related substance, its preparation method and application | |
| CN106770775A (en) | A kind of method for detecting antibiotic in rural potable water | |
| Guo et al. | A strategy to discover selective α-glucosidase/acetylcholinesterase inhibitors from five function-similar citrus herbs through LC-Q-TOF-MS, bioassay and virtual screening | |
| Bai et al. | Pharmacokinetics of kakkalide and its main metabolites in rat plasma determined by HPLC-DAD and LC–MSn | |
| CN106153740A (en) | A kind of Tulathromycin have related substance, its be enriched with preparation method and application | |
| Shi et al. | Pharmacokinetics and metabolic profiles of swertiamarin in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry | |
| Savchuk et al. | Chromatographic study of expert and biological samples containing desomorphine | |
| Liu et al. | Tentative identification of new metabolites of epimedin C by liquid chromatography–mass spectrometry | |
| Wang et al. | Liquid chromatographic–tandem mass spectrometric method for the quantitation of huperzine A in dog plasma | |
| Zubaran et al. | Noribogaine generalization to the ibogaine stimulus: correlation with noribogaine concentration in rat brain | |
| Zhong et al. | Identification of the metabolites of roxithromycin in humans | |
| Ying et al. | Determination of vitexin-2 ″-O-rhamnoside in rat plasma by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry and its application to pharmacokinetic study |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |