CN103122013B - Dehydrogenation clindamycin, its analysis preparation method and purposes - Google Patents
Dehydrogenation clindamycin, its analysis preparation method and purposes Download PDFInfo
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Abstract
The present invention provides a kind of dehydrogenation clindamycin, has structure shown in following formula III:
Description
Technical field
The present invention relates to analytical chemistry field, particularly relate to pharmaceutical analysis chemical field, particularly to dehydrogenation clindamycin, its analysis preparation method and purposes.
Background technology
Clindamycin Hydrochloride (Clindamycinhydrochloride) is that lincomycin hydrochloride 7-position hydroxyl is substituted by a chlorine atom both and the semi-synthetic derivant that obtains.Antimicrobial spectrum is identical with lincomycin, and antibacterial activity relatively lincomycin is strong 4-8 times, is widely used in gram-positive cocci and the microbial infection of various anaerobism such as treatment staphylococcus aureus.
There is more untoward reaction in clinical practice in Clindamycin Hydrochloride and injection thereof.Medicine produce in Clinical practice bad should except, outside the Pass having with the pharmacologically active of medicine itself, also having much relations with the impurity existed in medicine.
Any material affecting pharmaceutical purity is referred to as impurity, it is however generally that, impurity refers to other chemical substances beyond the medicine producing and introducing in storage process or produce.Impurity in drug standard refers to according in the medicine that the national regulation technique about drug regulatory department's examination and approval in accordance with the law and regulation supplementary material produce, the impurity brought into by its production technology and supplementary material, or the catabolite produced in storing process through stability experiment confirmation.Impurity in drug standard does not include the new impurity changing production technology or changing supplementary material and produce, and does not include the foreign substance penetrating into or polluting yet.Pharmaceutical producing enterprise changes production technology or supplementary material, and thus brings the revision to proper mass standard of the new impurity into, all should declare to approve to relevant drug regulatory department in accordance with the law.
The impurity of medicine is general relevant with specific medicine, comes from the following aspects:
1. stem from lyase, the catalyst etc. that commonly use in drug production process；
2. the reaction raw materials that reaction not exclusively exists, reacts the materials relevant to building-up process such as initial composite thing, synthetic mesophase product, side-product；
3. the oxidation in storage process, decomposition, hydrolyzate；
4. the optical isomer in chipal compounds；
5. the multiple crystal formation of medicine；
6., in animals and plants drug extract except the little molecules such as effective ingredient alkaloid volatile oil, organic acid, there is also the impurity such as the bigger protein of molecular weight, matter of trampling on, starch, resin；
7. the attenuating material in radiation medicine；
8. the protein of unconventionality expression in bioengineering product；
9. heavy metal and inorganic salt.
Impurity of the drug can be divided into by chemical classes and characteristic: organic impurities, inorganic impurity, organic volatile impurities.By sources can be divided into: have related substance (including the precursor of chemical reaction, intermediate, by-product and catabolite etc.), other impurity and exogenous impurities etc..By structural relation, impurity can be divided into again: other steroidals, related alkaloids, geometric isomer, optical isomer and polymer etc..By toxicity, toxic impurities and common impurities etc. can be divided into again.Common impurities is under amount without the impurity of notable bad biological agent, and toxic impurities is have the impurity of strong bad biological agent.
Defects inspecting is an important step of Drug's control, and the assay in the middle of drug quality refers to the content of main component in crude drug and preparation, has related substance to refer to the organic impurities in the middle of crude drug and preparation.Pass through Related substances separation, understand fully the source of related substance, character, detection method and limitation thereof, it is possible to the factors such as optimum synthesis route, experiment condition, so avoid its have related substance or its be preferably minimized limit, ensure from many aspects and improve drug quality, reducing the untoward reaction of medicine.
Impurity of the drug Inspection and analysis method should be sensitive, exclusive.Along with the progress of science and technology, separating, the improving constantly of analysis means, the detection method of impurity of the drug obtains continuous improvement.The method of detection impurity of the drug is a lot, can be easily separated preferably, identify the impurity of medicine, such as high performance liquid chromatography, gas chromatogram, ultraviolet, infrared spectrum, thin-layer chromatographic analysis, capillary zone electrophoresis, thin layer capillary electrophoresis etc., these analysis methods are widely used in content of drug and measure and defects inspecting.In recent years, mass-spectrometric technique is applied increasingly extensive in impurity of the drug analysis, and gas chromatogram coupling technology, liquid chromatograph multiple techniques have become as the important means that impurity of the drug is analyzed.
Impurity research in exploitation new raw material medicine and novel formulation process, should study in strict accordance with the requirement that the relevant new drug of country is declared, can also study with reference to the text Q3A (impurity in new raw material medicine) and Q3B (impurity in novel formulation) of ICH, and the safety and catabolite to impurity carries out safety evaluatio.Its specific requirement following points:
1. pair in esse impurity and potential impurity in synthesis, purification and storage, should adopt and efficiently separate analysis method and detect；
2. apparent content is given qualitative at the impurity with strong biological agent below 0.1% or toxic impurities at 0.1% and impurity and apparent content above or confirms its structure；
3. pair catabolite occurred in stability test, also should study by above-mentioned requirements；
4. the determination of foreign matter project in new drug quality standard should include after deliberation with study on the stability detection, and the impurity occurred in batch production and catabolite, and include corresponding limit；
5. except catabolite and toxic impurities, the impurity controlled in crude drug, generally no longer control in the formulation；
6. the inorganic impurity in crude drug and preparation, should determine inspection project according to its production technology, initiation material situation, but for toxic inorganic impurity, should specify its check item in quality standard.
In the development and production of imitation medicine, kind as found impurity is different from original development medicine or different with existing legal impurity, new impurity item must be increased and check project, should study in strict accordance with said method, declare new drug standard or proper mass standard is revised, and reporting relevant drug regulatory department to examine.
The isomer coexisted and antibiotic multicomponent are generally not as determination of foreign matter project, as coexisting substances, specify its ratio if desired in quality standard, to ensure the crude drug produced and concordance when declaring registration.But when the material when coexisting is toxic impurities, this material is just no longer regarded as being coexisting substances.Single enantiomer medicine, its can other enantiomer compatible should as determination of foreign matter.Racemic drugs, when the official quality standands of its single enantiomer medicine existing, should set optical rotation in the quality standard of this raceme and check project.
Organic volatile impurities, should according to organic solvent used in production technology and residual condition thereof, it is determined that check project.It is referred to the Chinese Pharmacopoeia requirement about organic volatile impurities, or with reference to ICH text Q3C (residual solvent guideline).Toxic solvents to residual, should specify its inspection project.
In order to ensure drug safety, each impurity in crude drug/preparation must carry out safety evaluation and that is must be set up ensureing the limit of impurities of safety, ICH criterion calls: in medicine, the limit of impurity is 0.1% (drug toxicity limit is lower), should identify higher than all unknown impurities of this level, and the more important thing is, all impurity being higher than 0.1% should study its toxicity." impurity in new raw material medicine " guideline that ICH revised on February 7th, 2007, is divided into crude drug two classes according to maximal dose every day of crude drug, and has formulated the reporting thresholds of impurity respectively, identified threshold value and reasonable limit.Reporting thresholds therein refers in the survey report that all impurity higher than this threshold value and content all should charge to every batch of product, and reacts in declaration material.And identify that threshold value refers to that all impurity higher than this threshold value is all tackled its structure and confirmed.As long as reasonable limit refers to that the limit of impurities formulated in quality standard is not higher than this limit, avoids the need for providing the formulation foundation of this limit, all thinks that this limit is rational.
For novel formulation, ICH have also been made clear stipulaties in " impurity in novel formulation " guideline that on February 5th, 2003 revises, and this guideline has been worked out the reporting thresholds of impurity also according to different dosages, identified threshold value and reasonable limit.
Medicine authorities of European Union require manufacturing enterprise: (1) should set up the limit of unknown impuritie (0.1%) in stability study；(2) the reply limit unknown impuritie more than or equal to 0.1% carries out structure and determines and security verification.Require higher for some antibiotics, for instance fermented product erythromycin, this kind EP records, and the limit ignored of regulation impurity is 0.06%, and any impurity must not exceed 3.0%.The requirement of medicine authorities of European Union, any give structure more than the unknown peak that can ignore limit 0.06% and determines and propose the suggestion of the suitable limit of impurities, namely it is carried out safety evaluatio when impurity reaches this limit.FDA also especially pays close attention to the purity of drug manufacture Chinese medicine and the safety of dosage, it is desirable to impurity is comprehensively analyzed by pharmaceutical production person, and provides more structural information as much as possible.Generally, the impurity more than 0.1% need to be identified out and carry out quantitative analysis by the method that selectivity is good, and the impurity of 0.01%～0.1% is also illustrated that keen interest.
Although the determination of the limit of impurities is extremely important for drug research and development, but the reality of domestic drug research and development does not make us optimistic.Declaring situation analysis from new drug in recent years, there is more problem in assorted Quality Research and limit are determined, main manifestations is: the importance that impurity is studied by Some Drugs research unit does not know much have less understanding；In standard the control of impurity is comprehensive and accurate not；Consider a problem when working out the limit of impurities comprehensive not, seldom consider the impurity harmful effect to drug safety；Even if when the content of impurity substantially exceeds the scope that normal process allows, do not note the optimization that present prescription and technique are carried out necessity, to reduce the limit of impurity yet.
" Chinese Pharmacopoeia ", " American Pharmacopeia ", " European Pharmacopoeia " and " British Pharmacopoeia " all has recording of clindamycin related substances item, and the recording more fully of " British Pharmacopoeia " and " European Pharmacopoeia ".
The total amount of impurity is only defined by " Chinese Pharmacopoeia " by clindamycin, single contaminant research is not concrete, and medicine authorities of European Union and FDA all require content 0.1% apparent in clindamycin crude drug and impurity above, carry out Structural Identification and security verification.
Summary of the invention
Present invention aim at the impurity of clindamycin crude drug is studied, essentially consist in the standard substance that impurity is prepared in separation and the impurity structure identifying in crude drug.Impurity in crude drug is analyzed, prepares and Structural Identification by the method according to the invention, basis can be provided with illustrating untoward reaction mechanism for the toxicologic study of impurity, reference can also be provided for the selection of technique compound experiment condition simultaneously, be conducive to the control of production process Quality Evaluation of Chinese Medicinal amount.
According to the first aspect of the invention, it provides dehydrogenation clindamycin, has structure shown in following formula III:
According to the second aspect of the invention, it provides the analysis preparation method of dehydrogenation clindamycin described in first aspect present invention, it is characterised in that clindamycin raw material is analyzed, and therefrom described clindamycin isomer is prepared in separation, comprises the following steps:
A) measure described clindamycin raw material by LC-MS method, determine the dehydrogenation clindamycin in described raw material according to the relative retention time of analyzed composition and/or molecular weight；
B) relative retention time of the dehydrogenation clindamycin according to step a) and/or molecular weight determine the condition of column chromatography, use purification on normal-phase silica gel column chromatography to be enriched with this relative retention time and/or analyzed composition corresponding to molecular weight；
C) the chromatograph retention behavior shown by the relative retention time of the dehydrogenation clindamycin according to step a) determines the condition preparing liquid phase method, collects, with preparing liquid phase method, the analyzed composition that described retention time is corresponding.
One according to the present invention is preferred embodiment, and in step a), described LC-MS method measures adopted HPLC condition and is:
Mobile phase 18% acetonitrile, 3% oxolane, 79% water and 0.2% formic acid；
PH ammonia is adjusted to 5.43-5.47；
Column temperature 25 DEG C；
Flow velocity 1.0ml/min；
Detection wavelength 210nm；
Chromatographic column DiamonsilODSC18,5 μm, 250 × 4.6mm post.
One according to the present invention preferred embodiment, and in step b), fixing that described purification on normal-phase silica gel column chromatography enrichment adopts is 100～200 order silica gel mutually, and mobile phase is ethyl acetate and water saturation methanol mixed solvent.
One according to the present invention is preferred embodiment, and step b) including:
B1) sample: silica gel=1: 50, is dissolved in methanol by sample, dry method upper prop；
B2) using volume ratio is the ethyl acetate of 9: 1: methanol mixed solvent eluting also collects eluent.
One according to the present invention is preferred embodiment, and in step c), described the adopted condition of liquid phase method of preparing includes:
Mobile phase: 18% acetonitrile, 4.5% oxolane, 77.5% water, 0.2% formic acid；
Ammonia adjusts pH value to be about 5.45；
Chromatographic column: AgilentZORBAXSB-C18 (5 μm, 9.4 × 250mm)；Detection wavelength: 210nm；Flow velocity: 1.0 × 2.25mL/min；
Collecting retention time is the corresponding analyte in the peak at 41.080 places.
Gained analyte preferred embodiment, is purified by one according to the present invention, and condition is as follows: instrument: HP1100, is furnished with Waters2695SeparationModule, Waters2487Dual λ AbsorbanceDetectorWaters；Detection wavelength: 210nm；Flow velocity: 1.5mL/min；Column temperature: 35 DEG C；Mobile phase: 25% acetonitrile, 3% oxolane, 72% water, 0.2% formic acid, ammonia adjusts pH to be about 5.19.
According to the third aspect of the present invention, it provides dehydrogenation clindamycin purposes in preparing medicament for resisting gram-positive bacteria described in first aspect present invention.
One according to the present invention preferred embodiment, and described gram positive bacteria is selected from anti-bacillus subtilis or staphylococcus aureus.
According to the fourth aspect of the invention, it provides the contamination levels product that according to a second aspect of the present invention prepared by described method, it is characterised in that described impurity is structured with formula III:
According to the fifth aspect of the invention, it provides contamination levels product purposes in analyzing clindamycin raw material described in fourth aspect present invention.
Accompanying drawing explanation
In conjunction with accompanying drawing provided herein, other features, purpose and advantage be will be better understood.These accompanying drawings are only used for demonstrating, and the present invention does not constitute any restriction.
Fig. 1 is the LC-MS spectrogram (left figure is UV figure, right figure is TIC figure) of Clindamycin Hydrochloride crude drug；
Fig. 2 is the total ion current figure of three batches of crude drug；
Fig. 3 is the TIC spectrogram of Clindamycin Hydrochloride crude product；
Fig. 4 is the LC spectrogram once preparing gained impurity 3；
Fig. 5 is the LC-MS spectrogram once preparing gained impurity 3；
Fig. 6 is the LC spectrogram that impurity 3 is further purified；
Fig. 7 is that (A:S090701 criticizes crude product for the LC spectrogram of impurity 3 purity detecting after purification；B: impurity 3)；
Fig. 8 is the fungistatic effect to staphylococcus aureus (label 1-4 respectively Clindamycin Hydrochloride, impurity 1, impurity 2, impurity 3)；
Fig. 9 is the fungistatic effect to bacillus subtilis (label 1-4 respectively Clindamycin Hydrochloride, impurity 1, impurity 2, impurity 3)；
Figure 10 is the fungistatic effect to Candida albicans (label 1-4 respectively Clindamycin Hydrochloride, impurity 1, impurity 2, impurity 3).
Detailed description of the invention
In order to be more fully understood that technical scheme, below in conjunction with the specific embodiment of the present invention, technical scheme is described further, but it is not intended to the present invention.
The clindamycin raw material that present embodiment uses all adopt that Zhejiang Province Tiantai Pharmaceutical Co., Ltd provides respectively than crude drug.
Embodiment 1
LC-MS measures clindamycin crude drug and crude product
LC-MS instrument: HPLCWaters2486, MSWatersmicromassZQ4000.Chromatographic column: DiamonsilC18 (5 μ 250 × 4.6mm)；Mobile phase is acetonitrile-oxolane-water-formic acid (18%: 3%: 79%: 0.2%), and ammonia adjusts pH value to be 5.45；Column temperature is room temperature；Detection wavelength 210nm；Flow velocity 1.0mL/min, through being diverted into mass spectrum.Mass Spectrometry Conditions is electron spray ionisation source cation (ESI+) detection mode；Source temperature 80 DEG C；Taper hole voltage 35v.
The LC-MS detection of crude drug
Taking the crude drug mobile phase that lot number is 090303 × 7 and be dissolved into the solution that concentration is 2mg/mL, sample size is 20 μ L.LC-MS testing result is as shown in Figure 1.
Measure in Clindamycin Hydrochloride crude drug except clindamycin six of liquid quality inspection have related substance, related substance 1 (3.95min) is respectively had by retention time size, there is related substance 2 (4.20min), there is related substance 3 (12.39min), there is related substance 4 (21.89min), have related substance 5 (23.25min), clindamycin (28.24min, main constituent), there is related substance 6 (32.62min).
Table 1, Clindamycin Hydrochloride crude drug LC-MS analyze
In order to investigate the situation having related substance in different batches crude drug, taking 081002 × 5 batch, 060901 × 5 batch and 060902 × 5 batch of crude drug respectively, be dissolved into, with mobile phase, the solution that concentration is 2mg/mL, sample size is 20 μ L.Carrying out LC-MS detection, result is as shown in Figure 2.
The LC-MS of 2, three batches of crude drug of table analyzes result
As shown in Table 2, what detect in three batches of crude drug has related substance and 090303 × 7 batch of crude drug identical, and the content being respectively arranged with the content of related substance TIC figure integration and 090303 × 7 batch of crude drug TIC figure integration is close.
Specify according to ICH, it is necessary to content is carried out structure description more than millesimal impurity.Liquid quality inspection measures in crude drug other five content having related substance except having related substance 2 and all exceedes one thousandth, it is necessary to have related substance to carry out Structural Identification to these five.With reference to British Pharmacopoeia[24], there are three structures having related substance it is known that determine by contrasting molecular weight: having related substance 1 is lincomycin, having related substance 3 is clindamycin B.Name has related substance 4 to be impurity 1, and having related substance 5 is impurity 2, and having related substance 6 is impurity 3.Primary study impurity 3 of the present invention, i.e. dehydrogenation clindamycin.
The IC-MS detection of crude product
Due in crude drug other except main constituent to be respectively arranged with related substance content little, it is not easy to target impurity is enriched with, and the sample adopted when therefore target impurity being studied is Clindamycin Hydrochloride crude product (lot number: S090701).With the detection method of crude drug, crude product is carried out the result of LC-MS detection as shown in Figure 3.
The LC-MS of table 3 Clindamycin Hydrochloride crude product analyzes
Except except detecting in crude drug six have related substance in crude product, additionally there is related substance 7 and have related substance 8, it is noted that the two has whether related substance can impact the enrichment of target impurity in research.The content of two of which target impurity slightly improves: impurity 1 content increases to 7.27%, and impurity 2 content increases to 1.26%.The content of impurity 3 is still relatively low.
Dehydrogenation clindamycin shown in normal phase silicagel column chromatographic enrichment target impurity-formula III
In order to target impurity-dehydrogenation clindamycin is carried out preliminary concentration, the construction features according to clindamycin, select purification on normal-phase silica gel column chromatography to study.
The separation principle of silica gel chromatography is different and separated according to material absorption affinity on silica gel, the material that generally polarity is bigger is easily by silica gel adsorption, the more weak material of polarity is not easily by silica gel adsorption, and namely whole chromatography process be Adsorption and desorption, adsorb again, desorption process again.
The ratio of the effect that optimal separation is enriched with can be reached as eluent selection with the ethyl acetate of different proportion and methanol.
Chromatographic column: 5 × 100cm；
Pretreatment: owing to containing multiple hydroxyls in Clindamycin Hydrochloride structure, Nature comparison is active.Therefore silica gel should first make its active site inactivate before the use, and specific practice is: weighs the thick silica gel of 100g100～200 order, industrial grade benzenemethanol soaked overnight, and methanol is drained and is placed on 70 DEG C of water-baths and makes the residual methanol in silica gel volatilize by buchner funnel, fills post afterwards.
Loading: weigh 1g Clindamycin Hydrochloride crude product (lot number: S090701), methanol dissolves, and is added dropwise in the crucible equipped with 1g silica gel, is placed in 60 DEG C of water-baths and mixes sample, dry method upper prop.
Normal phase column chromatography condition is: sample: silica gel=1: 50, eluting order is as follows: (ethyl acetate: methanol 9: 1) 1800mL, (ethyl acetate: methanol 6: 1) 1680mL, (ethyl acetate: methanol 5: 1) 600mL, (ethyl acetate: methanol 4: 1) 600mL, (ethyl acetate: methanol 3: 1) 600mL, (ethyl acetate: methanol 2: 1) 600mL, (ethyl acetate: methanol 1: 1) 600mL, methanol 600mL.What wherein can be enriched with impurity 3 is ethyl acetate: methanol 9: 1 position, and what can be enriched with impurity 2 is ethyl acetate: methanol 6: 1 position, can be enriched with the eluting position for (ethyl acetate: methanol 5: 1)～methanol of impurity 1.
HPLC method separates preparation dehydrogenation clindamycin shown in target impurity-formula III
After three target impurity are carried out preliminary concentration by normal phase column chromatography, utilization is prepared liquid phase method and target impurity is separated purification further, obtains the higher target impurity of purity to carry out Structural Identification.
Prepared by the separation of impurity 3
After impurity 3 is positioned at main component clindamycin peak in liquid chromatogram.Owing to clindamycin absorbability in the chromatography column is stronger, even if showing impurity 3 and main component baseline separation in liquid phase figure, prepare for the first time in the impurity 3 of gained and be still mixed with part main component, but the Contents of Main Components comparing crude product declines a lot, it is thus desirable to be further purified, the main component of residual is removed, thus obtaining purity higher impurity 3 sterling.
Instrument: Waters510 type partly prepares liquid phase, Waters484 detector.Chromatographic column is with AgilentZORBAXSB-C18 (5 μm, 9.4 × 250mm), and detection wavelength is 210nm, and flow velocity is 1.0 × 2.25mL/min.
Mobile phase: 18% acetonitrile, 4.5% oxolane, 77.5% water, 0.2% formic acid, ammonia adjusts pH value to be about 5.45.As shown in Figure 4, impurity 3 and main peak baseline separation, 38.088min is main peak, and 41.080min is impurity 3.
Through once preparing, the impurity 3 obtained and the blending ingredients of main peak, LC-MS testing result is as shown in Figure 5.Contrasting with the LC of crude product, retention time is 27.25min place is clindamycin, and retention time is 29.95min place is impurity 3.Each constituent content is respectively as follows: clindamycin: 57.29%；Impurity 3:42.71%.
The purification of impurity 3
Owing to main component clindamycin absorbability in the chromatography column is stronger, and impurity 3 differs less with main peak retention time, once prepare in the impurity 3 of gained and still contain more main component, it is therefore desirable to the impurity 3 once prepared is purified, in order to main component is removed completely.
Instrument: HP1100, is furnished with Waters2695SeparationModule, Waters2487Dual λ AbsorbanceDetectorWaters；Detection wavelength: 210nm, flow velocity: 1.5mL/min, column temperature: 35 DEG C.
Purification condition is as follows: instrument: HP1100, is furnished with Waters2695SeparationModule, Waters2487Dual λ AbsorbanceDetectorWaters；Detection wavelength: 210nm；Flow velocity: 1.5mL/min；Column temperature: 35 DEG C；Mobile phase: 25% acetonitrile, 3% oxolane, 72% water, 0.2% formic acid, ammonia adjusts pH to be about 5.19.Result is as shown in Figure 6.
The purity detecting of impurity 3
Obtaining impurity 3 after purification, after being placed in 50 DEG C of water-baths vacuum rotary steam and removing mobile phase, LC-MS testing result is as shown in Figure 7.Process compares and Mass Spectrometric Identification with crude product, and impurity 3 is confirmed as at the peak that retention time is 28.17min, and content reaches 95.97%.
Embodiment 2
The Structural Identification of the dehydrogenation clindamycin shown in target impurity-formula III
The sterling of the dehydrogenation clindamycin obtained is determined with high resolution mass spectrum elementary composition respectively, then determines the structure of dehydrogenation clindamycin by proton nmr spectra, carbon spectrum and two-dimensional spectrum DEPT, HMBC, HMQC, COSY and NOESY spectrum.Instrument is MicromassQ-TOF mass spectrograph and U.S.'s Varian nuclear magnetic resonance chemical analyser (400MHz).Following structural formula is the structure of clindamycin, and table 4 is the data of clindamycin NMR (Nuclear Magnetic Resonance) spectrum.
Clindamycin
Table 4, clindamycin1H-NMR spectrum,13C-NMR spectrum, HMQC spectrum, COSY spectrum, NOESY compose ownership
Clindamycin structure has four chiral centres, its configuration respectively 6S, 7S, 1 ' S, 3 ' R.
The Structural Identification of impurity 3
Instrument: MicromassQ-TOF mass spectrograph；U.S.'s Varian nuclear magnetic resonance chemical analyser (400MHz)；
[M+Na] of high resolution mass spectrum checked for impurities 3+Mass-to-charge ratio is 445.1537, elementary composition is defined as C18H31ClN2O5S, this impurity molecule amount is than clindamycin few 2, for the dehydrogenation thing of clindamycin.Syncaryon magnetic resonance complete set spectrum is identified shown in the following formula III of structure of impurity 3.
Table 7, impurity 31H-NMR spectrum,13C-NMR spectrum, HMQC spectrum, HMBC spectrum, COSY spectrum, NOESY compose ownership
Hydrogen four CH of spectrum3Belong to as follows: δH0.94 (3H, t, J=7.2Hz) is attributed to H-8 ', δH1.45 (3H, d, J=6.8Hz) are attributed to H-8, δH2.19 (3H s) is attributed to H-9, δH2.49 (3H, s) is attributed to H-5 ', and corresponding HMQC obtains δC15.7 be attributed to C-8 ', δC24.6 be attributed to C-8, δC15.5 be attributed to C-9, δC42.8 be attributed to C-5 '.
Carbon composes the signal of existing 18 carbon, respectively 18 carbon of corresponding impurity 3.Wherein chemical shift can't be peak in 174.0ppm, DEPT spectrum, should be quaternary carbon, can by δ by mother nucleus structure formulaC174.0 be attributed to C-10.1HNMR does not find the signal of 3 ' position hydrogen atoms in similar clindamycin, and at low field δH5.48ppm place adds an alkene Hydrogen Proton, thus it is speculated that there is a double bond in structure.Carbon spectrum δC135.1 and δC128.2 further demonstrate that the existence of double bond.The bright H-7 ' of HMBC stave and C-3 ', C-6 ', C-8 ' is remotely correlated with, and illustrates that impurity 3 has double bond at 3 ' carbon atoms and 6 ' carbon atom places.
In HMBC spectrum, H-8 and C-6, C-7 are remotely correlated with, corresponding δC55.3 and δC61.0, COSY spectrum display H-8 and H-7 is correlated with, it is known that δH4.67～4.59 are attributed to H-7, HMQC infer δC61.0 be C-7, δC55.3 be C-6, δH4.45 is H-6.Because δH4.32 and δH4.42 couplings, therefore δH4.32 are attributed to H-5, δC72.0 be attributed to C-5.
DEPT spectrum can confirm δC35.8 and δC62.9 be mesomethylene carbon, HMQC display is corresponding two hydrogen respectively.Because C-4 ' is connected with N, two Hydrochemistry non-equivalences on C-4 ' position, COSY spectrum shows mutual coupling and splits point.Two H on C-2 ' are restricted due to chemical bond upset, and chemical displacement value also differs.Chemical displacement value shows δC62.9 be attributed to C-4 ', δC35.8 be attributed to C-2 '.The corresponding hydrogen of C-2 ' is positioned at δH3.02～2.96ppm and δH～2.57ppm, C-4 ' corresponding hydrogen is positioned at δH3.77～3.74ppm and δH3.26～3.23ppm.
In COSY, H-1 and H-2 is correlated with, therefore δH4.13 places are attributed to H-2, δ in corresponding HMQCC70.6 be C-2.In COSY, H-2 and H-3 is correlated with, therefore δH4.13 places are attributed to H-3, δ in corresponding HMQCC70.6 be C-3.δ in COSYH3.87 is relevant to H-2, therefore δH3.87 are attributed in H-4, COSY δH3.51 is relevant to H-2 ' and H-5 ', therefore δH3.506 are attributed to H-1 '.
Therefore, dehydrogenation clindamycin has the mother nucleus structure identical with clindamycin, for the dehydrogenation thing of clindamycin.
Clindamycin structure has four chiral carbon, respectively C-6, C-7, C-1 ' and C-3 ', configuration is 6S respectively, 7S, 1 ' S, 3 ' R.Impurity 3 molecular weight ratio clindamycin is little by 2, NMR data be inferred as clindamycin and occur to eliminate reaction gained at H-3 ' and H-6 '.Owing to C-3 ' is that two the C-H chemical bonds upset being connected on a planar structure, C-2 ' and C-4 ' is restricted for the unsaturated carbon atom of sp2 hydridization, C-2 ', C-3 ' and C-4 ', cause that C-2 ' is different with two H chemical displacement values on C-4 '.Impurity 3 does not have pharmacopeia and document to record.
Embodiment 3
The bacteriostatic experiment of Clindamycin Hydrochloride and impurity of the present invention 3
Test for measuring antibacterials vitro inhibition bacterial growth effect is called bacteriostatic test.In this effects Clindamycin Hydrochloride crude drug, the bacteriostatic activity of the apparent content dehydrogenation clindamycin more than 0.1%, carries out bacteriostatic activity comparison by the sterling of the dehydrogenation clindamycin prepared with Clindamycin Hydrochloride crude drug.
The preparation of test solution
Clindamycin Hydrochloride (090303 × 7 batch, Zhejiang Province Tiantai Pharmaceutical Co., Ltd): 1.091mg, 1mL water dissolution；
Impurity 3:1.139mg, 0.4mL water dissolution.
Experimental strain
Staphylococcus aureus (gram positive bacteria), bacillus subtilis (antibacterial), Candida albicans (fungus)；Thered is provided by biology portion of Shanghai Institute of Pharmaceutical Industry.
The preparation of culture medium
Candida albicans bacterium culture medium (%)
Glucose 0.1 yeast extract 0.25
KCl0.18NaAc0.82
Agar 1.5pH7.0
121 DEG C of sterilizing 30min
Staphylococcus aureus and bacillus subtilis (%)
Peptone 0.6 Carnis Bovis seu Bubali cream 0.15
Yeast extract 0.6 glucose 0.1
Agar 1.5pH6.5
121 DEG C of sterilizing 30min
The preparation of filter paper
Select bibulous high-quality filter paper, break into, with card punch, the circular filter paper sheet that diameter is 6mm, standby after dry heat sterilization.
Experimental technique
Agar diffusion paper disk method[30]: draw on 0.1mL bacterium solution, even spread and M-H agar surface ware.Respectively the Clindamycin Hydrochloride of 10 μ L and the solution of dehydrogenation clindamycin are uniformly added on the scraps of paper of sterilizing, grip the scraps of paper after to be dried with aseptic nipper respectively and be equidistantly placed on the surface plate containing bacterium.By on surface plate lid, take out after being flat in 37 DEG C of incubators to cultivate 24h.Observe fungistatic effect.
Experimental result
Staphylococcus aureus and bacillus subtilis are all had fungistatic effect by Clindamycin Hydrochloride and dehydrogenation clindamycin.To the fungistatic effect of staphylococcus aureus as shown in Figure 8, impurity 3 inhibition zone size is similar to Clindamycin Hydrochloride.For the fungistatic effect of bacillus subtilis as it is shown in figure 9, the inhibition zone of four is all relatively big, the inhibitory action that bacillus subtilis has been had by Clindamycin Hydrochloride and dehydrogenation clindamycin is described.Figure 10 shows do not occur around the filter paper of Clindamycin Hydrochloride and dehydrogenation clindamycin illustrating all candida albicans is not had antibacterial activity by inhibition zone.
Claims (3)
1. the analysis preparation method of following formula dehydrogenation clindamycin,
It is characterized in that comprising the following steps:
A) measure described clindamycin raw material by LC-MS method, determine the dehydrogenation clindamycin in described raw material according to the relative retention time of analyzed composition and/or molecular weight；
B) relative retention time of the dehydrogenation clindamycin according to step a) and/or molecular weight determine the condition of column chromatography, purification on normal-phase silica gel column chromatography is used to be enriched with this relative retention time and/or analyzed composition corresponding to molecular weight, fixing that described purification on normal-phase silica gel column chromatography enrichment adopts is 100～200 order silica gel mutually, mobile phase is ethyl acetate and water saturation methanol mixed solvent, and includes:
B1) sample: silica gel=1: 50, is dissolved in methanol by sample, dry method upper prop；
B2) using volume ratio is the ethyl acetate of 9: 1: methanol mixed solvent eluting also collects eluent；
C) the chromatograph retention behavior shown by the relative retention time of the dehydrogenation clindamycin according to step a) determines the condition preparing liquid phase method, collects, with preparing liquid phase method, the analyzed composition that described retention time is corresponding；Described the adopted condition of liquid phase method of preparing includes:
Mobile phase: 18% acetonitrile, 4.5% oxolane, 77.5% water, 0.2% formic acid；
Ammonia adjusts pH value to be 5.45；
Chromatographic column: AgilentZORBAXSB-C18:5 μm, 9.4 × 250mm；Detection wavelength: 210nm；Flow velocity: 1.0 × 2.25mL/min；
Collecting retention time is the corresponding analyte in the peak at 41.080 places.
2. method according to claim 1, it is characterised in that in step a), described LC-MS method measures adopted HPLC condition and is:
Mobile phase 18% acetonitrile, 3% oxolane, 79% water and 0.2% formic acid；
PH ammonia is adjusted to 5.43-5.47；
Column temperature 25 DEG C；
Flow velocity 1.0ml/min；
Detection wavelength 210nm；
Chromatographic column DiamonsilODSC18,5 μm, 250 × 4.6mm post.
3. method according to claim 1, it is characterised in that gained analyte is purified, and condition is as follows: instrument: HP1100, is furnished with Waters2695SeparationModule, Waters2487Dual λ AbsorbanceDetectorWaters；Detection wavelength: 210nm；Flow velocity: 1.5mL/min；Column temperature: 35 DEG C；Mobile phase: 25% acetonitrile, 3% oxolane, 72% water, 0.2% formic acid, ammonia adjusts pH to be 5.19.
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| CN102060882A (en) * | 2009-11-18 | 2011-05-18 | 上海医药工业研究院 | Dehydrogenated clindamycin phosphate, analysis preparation method and application thereof |
| CN102062758A (en) * | 2009-11-18 | 2011-05-18 | 上海医药工业研究院 | Impurity analysis and preparation method for clindamycin phosphate |
| CN103123342A (en) * | 2011-11-18 | 2013-05-29 | 上海医药工业研究院 | Impurity analysis preparation method for clindamycin |
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| US3555007A (en) * | 1968-07-22 | 1971-01-12 | Upjohn Co | 7-deoxy-7-halo lincomycin d derivatives |
| CN102060882A (en) * | 2009-11-18 | 2011-05-18 | 上海医药工业研究院 | Dehydrogenated clindamycin phosphate, analysis preparation method and application thereof |
| CN102062758A (en) * | 2009-11-18 | 2011-05-18 | 上海医药工业研究院 | Impurity analysis and preparation method for clindamycin phosphate |
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