CN103122014A - Clindamycin isomer, analytical preparation method and application thereof - Google Patents
Clindamycin isomer, analytical preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a clindamycin isomer which has a structure shown in the formula II. The invention further provides an analytical preparation method of the clindamycin isomer. The method is characterized in that the clindamycin raw materials are analyzed and the clindamycin isomer is separated and prepared from the raw materials. The method comprises the following steps of: a) determining the clindamycin raw materials by the LC-MS (liquid chromatography-mass spectrometry) method, and determining the clindamycin isomer in the raw materials according to relative retaining time and/or molecular weight of analyzed components; b) determining conditions of column chromatography according to the relative retaining time and/or molecular weight of the clindamycin isomer, obtained in the step a, and enriching analyzed components corresponding to the relative retaining time and/or molecular weight by positive phase silica gel column chromatography; and c) determining condition of the preparation liquid phase method according to chromatographic retention behavior displayed by the relative retaining time of the clindamycin isomer, obtained in the step a, and collecting the analyzed components corresponding to the relative retaining time by the liquid phase method.
Description
Technical field
The present invention relates to the analytical chemistry field, relate in particular to the pharmaceutical analysis chemical field, particularly clindamycin isomer, it analyzes preparation method and purposes.
Background technology
Dalacina (Clindamycin hydrochloride) be U 10149a 7-position hydroxyl replaced by the chlorine atom and semi-synthetic derivative.Antimicrobial spectrum is identical with lincomycin, and anti-microbial activity than the strong 4-8 of lincomycin doubly is widely used in gram-positive cocci and the microbial infection of various anaerobism such as treatment streptococcus aureus.
More untoward reaction appears in Dalacina and injection liquid thereof in clinical application.Medicine produces in clinical use bad should have except the pharmacologically active with medicine itself outside the Pass, with the impurity that exists in medicine, much relations are arranged also.
Any material that affects pharmaceutical purity all is called impurity, generally speaking, and other chemical substances beyond the medicine that impurity refers to introduce in production and storage process or produce.Impurity in drug standard refers to according to through relevant drug regulatory department of country in accordance with the law in the medicine of the regulation technique of examination and approval and the production of regulation supplementary material, the impurity of being brought into by its production technique and supplementary material, or through the degraded product that produces in storage process of stability experiment conclusive evidence.Impurity in drug standard does not comprise change production technique or change supplementary material and the new impurity that produces does not comprise the foreign matter that infiltrates or pollute yet.Drug manufacturing enterprise's change production technique or supplementary material, and bring thus new impurity into to the revision of proper mass standard, all should declare to approve to relevant drug regulatory department in accordance with the law.
The impurity of medicine is generally relevant with specific medicine, comes from the following aspects:
1. stem from drug production process the lyase that generally uses, catalyzer etc.;
2. react incomplete and reaction raw materials that exist, the materials relevant to building-up process such as reaction initial recombination thing, synthetic mesophase product, byproduct;
3. the oxidation in storage process, decomposition, hydrolysate;
4. the optical isomer in chipal compounds;
5. the multiple crystal formation of medicine;
In animals and plants medicine extract except the small molecules such as effective constituent alkaloid volatile oil, organic acid, also have the impurity such as the larger protein of molecular weight, the matter of trampling on, starch, resin;
7. the decay material in the 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 impurity, inorganic impurity, volatile organic impurity.By sources can be divided into: related substance (precursor, intermediate, by product and the degraded product etc. that comprise chemical reaction), other impurity and tramp material etc.By structural relation, impurity can be divided into again: other steroidals, related alkaloids, geometrical isomer, optical isomer and polymkeric substance etc.By toxicity, can be divided into toxic impurities and common impurities etc. again.Common impurities is under amount the impurity without remarkable bad biological action, and toxic impurities is the impurity with strong bad biological action.
It is the important step that drug quality is controlled that impurity detects, and the assay in the middle of drug quality refers to the content of main component in bulk drug and preparation, and related substance refers to the organic impurity in the middle of bulk drug and preparation.By the related substance inspection, understand fully source, character, the detection method of related substance and limit the quantity of, can optimize the factors such as synthetic route, experiment condition, and then avoid it to produce related substance or it drops to bottom line, guarantee from many aspects and improve drug quality, reducing the untoward reaction of medicine.
Impurity of the drug checks that analytical procedure should be sensitive, exclusive.Along with the progress of science and technology, to separate, the improving constantly of analysis means, the detection method of impurity of the drug has obtained continuous improvement.The method of detection of drugs impurity is a lot, can separate preferably, identify the impurity of medicine, as high performance liquid chromatography, gas-chromatography, ultraviolet, infrared spectra, thin-layer chromatographic analysis, capillary zone electrophoresis, thin layer capillary electrophoresis etc., these analytical procedures are widely used in content of drug mensuration and impurity detects.In recent years, mass-spectrometric technique is used increasingly extensive aspect the impurity of the drug analysis, and gas-chromatography coupling technology, liquid chromatography coupling technique have become the important means that impurity of the drug is analyzed.
Impurity research in exploitation new raw material medicine and new preparation process, should study in strict accordance with the requirement that the relevant new drug of country is declared, also can study with reference to text Q3A (impurity in the new raw material medicine) and the Q3B (impurity in new preparation) of ICH, and security and the degraded product of impurity carried out safety evaluation.Its specific requirement following points:
1. in esse impurity and potential impurity in synthetic, purifying and storage, should adopt effective method for separating and analyzing to detect;
For apparent content 0.1% and above impurity and apparent content giving qualitative or proving conclusively its structure at the impurity with strong biological action below 0.1% or toxic impurities;
3. the degraded product to occurring in stability test, also should study by above-mentioned requirements;
4. the determination of foreign matter project in the new drug quality standard should comprise after deliberation with study on the stability and detecting, and the impurity that occurs in batch production and degraded product, and comprises corresponding limit;
5. except degraded product and toxic impurities, the impurity of having controlled in bulk drug is generally no longer controlled in preparation;
6. the inorganic impurity in bulk drug and preparation, should determine inspection item according to its production technique, starting raw material situation, but for toxic inorganic impurity, should stipulate its check item in quality standard.
In the research and production of imitation medicine, as different from the original development medicine in the kind of finding impurity or different with existing legal impurity, must increase new impurity item inspection item, should study in strict accordance with aforesaid method, declare new drug standard or the proper mass standard is revised, and reporting relevant drug regulatory department to examine.
The isomer that coexists and microbiotic polycomponent as coexisting substances, are stipulated its ratio generally not as the determination of foreign matter project in case of necessity in quality standard, the consistence with the bulk drug that guarantees production use when declaring registration.But when the material when coexisting was toxic impurities, this material was just no longer thought coexisting substances.The single enantiomer medicine, its can compatible other enantiomorphs should be as determination of foreign matter.The racemization medicine when having the official quality standands of its single enantiomer medicine, should be established the specific rotation inspection item in the quality standard of this raceme.
Volatile organic impurity should according to organic solvent used and residual condition thereof in production technique, be determined inspection item.Can be with reference to the requirement of Chinese Pharmacopoeia about volatile organic impurity, or with reference to ICH text Q3C (residual solvent governing principle).To residual toxic solvents, should stipulate its inspection item.
In order to guarantee drug safety, each impurity in bulk drug/preparation must carry out safety evaluation and that is to say the necessary limit of impurities that guarantees security of setting up, the ICH criterion requires: in medicine, the limit of impurity is 0.1% (lower to the drug toxicity limit), all unknown impurities higher than this level should identify, and the more important thing is, all should study its toxicity higher than 0.1% impurity.ICH is in " impurity in the new raw material medicine " governing principle of on February 7th, 2007 revision, according to maximal dose every day of bulk drug, bulk drug is divided into two classes, and formulated respectively impurity the report threshold value, identify threshold value and reasonable limit.Report threshold value wherein refers to that all should charge in the survey report of every batch of product higher than impurity and the content of this threshold value, and reaction is in declaration material.And identify that threshold value refers to that all tackle its structure higher than the impurity of this threshold value and prove conclusively.Reasonable limit refers to think that all this limit is rational as long as the limit of impurities of formulating in quality standard not higher than this limit, does not just need to provide the formulation foundation of this limit.
For new preparation, ICH has also done clear in " impurity in new preparation " governing principle of revision on February 5th, 2003, and this governing principle has been worked out report threshold value, evaluation threshold value and the reasonable limit of impurity equally according to different dosages.
Medicine competent 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 is carried out structure more than or equal to 0.1% unknown impuritie and is determined and security verification.Require for some antibiotics higher, leavened prod erythromycin for example, this kind EP is recorded, and the limit ignored of regulation impurity is 0.06%, and any impurity must not surpass 3.0%.The requirement of medicine competent authorities of European Union, any unknown peak greater than ignoring limit 0.06% give structure to be determined and proposes suitable limit of impurities suggestion, namely it is carried out safety evaluation when impurity reaches this limit.FDA also especially pays close attention to the purity of drug manufacture Chinese traditional medicine and the security of dosage, requires pharmaceutical production person that impurity is comprehensively analyzed, and more structural information is provided as much as possible.Usually, need identify out and carry out quantitative analysis with the good method of selectivity over 0.1% impurity, and 0.01%～0.1% impurity is also represented keen interest.
Although determining of limit of impurities is extremely important for drug research and development, it is optimistic that the reality of domestic drug research and development is not made us.Declare situation analysis from new drug in recent years, exist more problem in the research of impurity and limit aspect determining, main manifestations is: Some Drugs research unit does not know much have less understanding to the importance of impurity research; Control to impurity in standard is comprehensive and accurate not; Consider a problem when working out limit of impurities comprehensive not, consider that seldom impurity is to the detrimentally affect of drug safety; Even when the content of impurity obviously exceeds the scope that normal process allows, do not note present prescription and technique are carried out necessary optimization, to reduce the limit of impurity yet.
" Chinese pharmacopoeia, " American Pharmacopeia ", " European Pharmacopoeia " and " British Pharmacopoeia " all have recording of clindamycin related substances item, and recording of " British Pharmacopoeia " and " European Pharmacopoeia " is more comprehensive.
" in Chinese pharmacopoeia, clindamycin is only limited the total amount of impurity, not concrete to single contaminant research, and medicine competent authorities of European Union and FDA all require apparent content 0.1% and above impurity thereof in the clindamycin bulk drug, carry out Structural Identification and security verification.
Summary of the invention
The object of the invention is the impurity of clindamycin bulk drug is studied, and mainly is to separate the standard substance that prepare impurity and identifies impurity structure in bulk drug.The method according to this invention to the impurity in bulk drug analyze, preparation and Structural Identification, can and illustrate the untoward reaction mechanism for the toxicologic study of impurity the basis is provided, simultaneously also can provide reference for the selection of technique compound experiment condition, be conducive to the control of production process Quality Evaluation of Chinese Medicinal amount.
According to a first aspect of the invention, it provides the clindamycin isomer, has as shown in the formula structure shown in II:
According to a second aspect of the invention, the analysis preparation method that it provides the described clindamycin isomer of first aspect present invention is characterized in that the clindamycin raw material is analyzed, and therefrom separates the described clindamycin isomer of preparation, comprises the following steps:
A) measure described clindamycin raw material with the LC-MS method, determine clindamycin isomer in described raw material according to the relative retention time of analyzed composition and/or molecular weight;
B) relative retention time of the clindamycin isomer described in a) and/or the condition that molecular weight is determined column chromatography according to step, use purification on normal-phase silica gel column chromatography this relative retention time of enrichment and/or analyzed composition corresponding to molecular weight;
C) the shown chromatogram retention behavior of the relative retention time of the clindamycin isomer described in a) is determined the condition of preparation liquid phase method according to step, collects the corresponding analyzed composition of described retention time with the preparation liquid phase method.
According to of the present invention one preferred embodiment, step a) in, described LC-MS method is measured the HPLC condition that adopts and is:
Moving phase 18% acetonitrile, 3% tetrahydrofuran (THF), 79% water and 0.2% formic acid;
PH ammoniacal liquor transfers to 5.43-5.47;
25 ℃ of column temperatures;
Flow velocity 1.0ml/min;
Detect wavelength 210nm;
Chromatographic column Diamonsil ODS C18,5 μ m, 250 * 4.6mm post.
According to of the present invention one preferred embodiment, step b) in, the stationary phase that described purification on normal-phase silica gel column chromatography enrichment is adopted is 100～200 order silica gel, moving phase is ethyl acetate and water saturation methanol mixed solvent.
According to of the present invention one preferred embodiment, step b) comprising:
B 1) sample: silica gel=1: 50 is dissolved in sample in methyl alcohol the dry method upper prop;
B2) use ethyl acetate: the methanol mixed solvent gradient elution, elution order is as follows: (ethyl acetate: methyl alcohol 9: 1) 1800mL, (ethyl acetate: methyl alcohol 6: 1) 1680mL;
B3) merge collection (ethyl acetate: elutriant methyl alcohol 6: 1).
According to of the present invention one preferred embodiment, step c) in, described preparation liquid phase method adopts the secondary preparation, wherein once prepares institute's employing condition and comprises:
Moving phase is 20% acetonitrile, 1.25% tetrahydrofuran (THF), 78.75% water, 0.2% formic acid;
It is 5.58 left and right that ammoniacal liquor is transferred pH;
Chromatographic column μ Bondapak
TMC 18,7.8 * 300mm, the detection wavelength is 210nm, flow velocity is 1.0 * 2.25mL/min;
Collecting retention time is the corresponding assay in peak at 29.525min place;
The gained assay is adopted the secondary preparation of binary pump, the condition of moving phase is: the A pump: 21% acetonitrile, and 3% tetrahydrofuran (THF), 76% water, 0.2% formic acid, pH are 5.20 left and right; B pump: acetonitrile; A: B=95: 5; Chromatographic column Sepax HP-C 18,5 μ m 10.0 * 250mm detect wavelength: 210nm, flow velocity: 1.5mL/min, column temperature: 35 ℃; Collecting retention time is the corresponding assay in peak at 27.68min place, i.e. clindamycin isomer shown in formula II.
According to a third aspect of the present invention, it provides the purposes of the described clindamycin isomer of first aspect present invention in the preparation medicament for resisting gram-positive bacteria.
According to of the present invention one preferred embodiment, described gram-positive microorganism is selected from anti-Bacillus subtilus or streptococcus aureus.
According to a forth aspect of the invention, the impurity standard substance that it provides method preparation according to second aspect present invention is characterized in that, described impurity has following structural formula II:
According to a fifth aspect of the invention, it provides the purposes of the described impurity standard substance of fourth aspect present invention in analyzing the clindamycin raw material.
Description of drawings
Accompanying drawing in conjunction with the application provides will be more readily understood the application's other features, objects and advantages.These accompanying drawings only are used for demonstration, the present invention are not consisted of any restriction.
Fig. 1 is the LC-MS spectrogram (left figure is UV figure, and right figure is TIC figure) of Dalacina bulk drug;
Fig. 2 is the total ion current figure of three batches of bulk drugs;
Fig. 3 is the TIC spectrogram of Dalacina crude product;
Fig. 4 is for once preparing the LC spectrogram of gained impurity 1 and impurity 2;
Fig. 5 is crude product and once prepares gained impurity 1 and the LC-MS spectrogram of impurity 2, and wherein A is that S090701 criticizes crude product, and B is impurity 1 and impurity 2 blending ingredients;
Fig. 6 is the LC spectrogram that secondary prepares gained impurity 1 and impurity 2;
LC spectrogram (the A: crude product of the purity detecting of Fig. 7 impurity 1 and impurity 2; B: impurity 1; C: impurity 2);
Fig. 8 is the fungistatic effect (label 1-4 is respectively Dalacina, impurity 1, impurity 2, impurity 3) to streptococcus aureus;
Fig. 9 is the fungistatic effect (label 1-4 is respectively Dalacina, impurity 1, impurity 2, impurity 3) to subtilis;
Figure 10 is the fungistatic effect (label 1-4 is respectively Dalacina, impurity 1, impurity 2, impurity 3) to Candida albicans.
Embodiment
In order to understand better technical scheme of the present invention, below in conjunction with the specific embodiment of the present invention, technical scheme of the present invention is described further, but it does not limit the present invention.
Each batch bulk drug that the clindamycin raw material that present embodiment is used all adopts Zhejiang Province Tiantai Pharmaceutical Co., Ltd to provide.
Embodiment 1
LC-MS measures clindamycin bulk drug and crude product
LC-MS instrument: HPLC Waters 2486, MS Waters micromass ZQ 4000.Chromatographic column: Diamonsil C 18 (5 μ 250 * 4.6mm); Moving phase is acetonitrile-tetrahydrofuran (THF)-water-formic acid (18%:3%:79%:0.2%), and the ammoniacal liquor adjust pH is 5.45; Column temperature is room temperature; Detect wavelength 210nm; Flow velocity 1.0mL/min flows to mass spectrum through dividing.The mass spectrum condition is electron spray ionisation source positive ion (ESI+) detection mode; 80 ℃ of source temperature; Taper hole voltage 35v.
The LC-MS of bulk drug detects
Get lot number and be 090303 * 7 bulk drug and be dissolved into moving phase the solution that concentration is 2mg/mL, sample size is 20 μ L.The LC-MS detected result as shown in Figure 1.
Six related substances except clindamycin in the Dalacina bulk drug are measured in the liquid quality inspection, be respectively related substance 1 (3.95min) by the retention time size, related substance 2 (4.20min), related substance 3 (12.39min), related substance 4 (21.89min), related substance 5 (23.25min), clindamycin (28.24min, principal constituent), related substance 6 (32.62min).
The LC-MS of table 1, Dalacina bulk drug analyzes
In order to investigate the situation of related substance in the different batches bulk drug, get respectively 081002 * 5 batch, 060901 * 5 batch and 060902 * 5 batch of bulk drug, be dissolved into moving phase the solution that concentration is 2mg/mL, sample size is 20 μ L.Carry out LC-MS and detect, result as shown in Figure 2.
The LC-MS analytical results of table 2, three batches of bulk drugs
As shown in Table 2, the related substance that detects in the three batches of bulk drugs is identical with 090303 * 7 batch of bulk drug, and the content of each related substance TIC figure integration is close with the content of 090303 * 7 batch of bulk drug TIC figure integration.
According to the ICH regulation, need to carry out structrual description greater than millesimal impurity to content.The liquid quality inspection measures in bulk drug that the content of other five related substances all over thousandth, need to carry out Structural Identification to these five related substances except related substance 2.With reference to British Pharmacopoeia
[24], there is the structure of three related substances known, determine by the contrast molecular weight: related substance 1 is lincomycin, and related substance 3 is clindamycin B.Name related substance 4 is impurity 1, and related substance 5 is impurity 2, and related substance 6 is impurity 3.Primary study impurity 2 of the present invention, i.e. clindamycin isomer shown in formula II.
The LC-MS of crude product detects
Due to each its related substances of other except principal constituent in bulk drug seldom, be not easy target impurity is carried out enrichment, the sample that adopts when therefore target impurity being studied is Dalacina crude product (lot number: S090701).With the detection method of bulk drug, crude product is carried out result that LC-MS detects as shown in Figure 3.
The LC-MS of table 3 Dalacina crude product analyzes
Detected six related substances, also have in addition related substance 7 and related substance 8 in crude product in bulk drug, will notice in research whether these two related substances can impact the enrichment of target impurity.Wherein the content of two target impurity slightly is improved: impurity 1 content increases to 7.27%, and impurity 2 content increase to 1.26%.The content of impurity 3 is still lower.
Clindamycin isomer shown in purification on normal-phase silica gel column chromatography enrichment target impurity-Shi II
For the clindamycin isomer shown in target impurity-Shi II is carried out preliminary enrichment, according to the constructional feature of clindamycin isomer, select the purification on normal-phase silica gel column chromatography to study.
The separation principle of silica gel chromatography is that the difference of the adsorptive power on silica gel is separated according to material, the material that generally polarity is larger is easily by silica gel adsorption, the weak material of polarity is difficult for by silica gel adsorption, and whole chromatography process is namely Adsorption and desorption, absorption again, desorption process again.
Select to reach the ratio of the effect of optimal separation enrichment as elutriant with the ethyl acetate of different ratios and methyl alcohol.
Chromatography column: 5 * 100cm;
Pre-treatment: owing to containing a plurality of hydroxyls in the Dalacina structure, character is more active.Therefore silica gel should first make its active site inactivation before using, and specific practice is: take 100g100～200 thick silica gel of order, and the industrial grade benzenemethanol soaked overnight, Büchner funnel is drained methyl alcohol and is placed on 70 ℃ of water-baths the residual methanol in silica gel is volatilized, rear dress post.
Loading: (lot number: S090701), dissolve with methanol is added dropwise in the crucible that 1g silica gel is housed, and is placed in 60 ℃ of water-baths and mixes sample, the dry method upper prop to take 1g Dalacina crude product.
The normal phase column chromatography condition is: sample: silica gel=1: 50, elution order is as follows: (ethyl acetate: methyl alcohol 9: 1) 1800mL, (ethyl acetate: methyl alcohol 6: 1) 1680mL, (ethyl acetate: methyl alcohol 5: 1) 600mL, (ethyl acetate: methyl alcohol 4: 1) 600mL, (ethyl acetate: methyl alcohol 3: 1) 600mL, (ethyl acetate: methyl alcohol 2: 1) 600mL, (ethyl acetate: methyl alcohol 1: 1) 600mL, methyl alcohol 600mL.Wherein can enrichment impurity 3 be ethyl acetate: methyl alcohol 9: 1 positions, can enrichment impurity 2 be ethyl acetate: methyl alcohol 6: 1 positions, energy enrichment impurity 1 be (ethyl acetate: the wash-out position of methyl alcohol 5: 1)～methyl alcohol.
The HPLC method is separated the clindamycin isomer shown in preparation target impurity-Shi II
After through normal phase column chromatography, three target impurity being carried out preliminary enrichment, utilize the preparation liquid phase method with the further separation and purification of target impurity, obtain the higher target impurity of purity in order to carry out Structural Identification.
Impurity 1 and impurity 2 separate preparation
Impurity 1 and impurity 2 are the isomers of main component clindamycin, and both separation conditions require harsher.Due to the impact that is subjected to sample size, consuming time longer by once preparing the sterling that just obtains impurity 1 and impurity 2, cost is higher, therefore adopts the method for secondary preparation, first by making for the first time the mixture of a large amount of impurity 1 and impurity 2, then by preparing for the second time, both are separated fully.The method that experimental results show that secondary preparation is feasible, and the impurity 1 for preparing and impurity 2 purity all reach the standard of Structural Identification.
Preparation condition for the first time
Instrument: Waters 510 types partly prepare liquid phase, Waters 484 detectors, and (7.8 * 300mm), the detection wavelength is 210nm to chromatographic column, and flow velocity is 1.0 * 2.25mL/min with μ BondapakTM C18.
The moving phase condition is 20% acetonitrile, 1.25% tetrahydrofuran (THF), and 78.75% water, 0.2% formic acid, ammoniacal liquor adjust pH are 5.58 left and right.As shown in Figure 4, the peak of collecting the 29.525min place namely gets the blending ingredients of impurity 1 and impurity 2.
Through preparation after a while, the blending ingredients of the impurity 1 that obtains and impurity 2 is carried out LC-MS detect, as shown in Figure 5.With the LC of crude product contrast, retention time is that the 19.62min place is impurity 1, and retention time is that the 20.87min place is impurity 2, and retention time is that the 25.53min place is clindamycin.Three component concentrations are respectively: impurity 1:68.67%, impurity 2:16.39%, clindamycin: 14.94%.
Preparation condition for the second time
Instrument: HP 1100 is furnished with Waters 2695Separation Module, Waters 2487Dual λ Absorbance Detector Waters.Chromatographic column: Sepax HP-C18 (5 μ m 10.0 * 250mm).
Impurity 1 and impurity 2 prepare the employing binary pump for the second time, and the condition of moving phase is defined as: the A pump: 21% acetonitrile, and 3% tetrahydrofuran (THF), 76% water, 0.2% formic acid, pH are 5.20 left and right; B pump: acetonitrile; A: B=95: 5.Detect wavelength: 210nm, flow velocity: 1.5mL/min, column temperature: 35 ℃.Preparation liquid phase figure prepares sample as shown in Figure 6 for preparing for the first time impurity 1 and impurity 2 mixtures of gained.In collection figure, the 25.90min place namely gets impurity 1, collects the 27.68min place and namely gets impurity 2.
The impurity 1 that secondary prepares and impurity 2 purity detecting
After preparing for the second time according to the condition of 3.1.2.2, obtain respectively impurity 1 and impurity 2.Two components are placed in respectively 50 ℃ of water-bath vacuum rotary steams moving phase are steamed, and all obtain white solid (containing ammonium formiate).After dissolve with methanol, carry out LC-MS and detect, result as shown in Figure 7.Through and crude product contrast and Mass Spectrometric Identification, in 3-14B figure, retention time is that impurity 1 is confirmed as at the peak of 18.78min, content is 95.00%; In 3-14C figure, retention time is that impurity 2 is confirmed as at the peak of 21.27min, and content is 94.61%.
The Structural Identification of the clindamycin isomer shown in target impurity-Shi II
The sterling of the clindamycin isomer that obtains is determined respectively elementary composition with high resolution mass spectrum, then determined the structure of clindamycin isomer with proton nmr spectra, carbon spectrum and two-dimensional spectrum DEPT, HMBC, HMQC, COSY and NOESY spectrum.Instrument is Micromass Q-TOF mass spectrograph and U.S. Varian nuclear magnetic resonance spectrometer (400MHz).Following structural formula is the structure of clindamycin, and table 4 is the data of clindamycin NMR (Nuclear Magnetic Resonance) spectrum.
Table 4, clindamycin
1The H-NMR spectrum,
13C-NMR spectrum, HMQC compose, COSY composes,
NOESY composes ownership
Have four chiral centres in the clindamycin structure, its configuration is respectively 6S, 7S, 1 ' S, 3 ' R.
The Structural Identification of the clindamycin isomer shown in impurity 2-formula II
Instrument: Micromass Q-TOF mass spectrograph; U.S. Varian nuclear magnetic resonance spectrometer (400MHz);
[M+Na] of high resolution mass spectrum checked for impurities 2
+Mass-to-charge ratio is 447.1694, the elementary composition C that is defined as
18H
33ClN
2O
5S, this impurity are also the isomers of clindamycin.The a complete set of spectrum of syncaryon mr identifies that the structure of impurity 2 is as shown in the formula shown in II.
Table 6, impurity 2
1The H-NMR spectrum,
13C-NMR spectrum, HMBC spectrum, HMQC spectrum, COSY spectrum, NOESY compose ownership
COSY composes demonstration, H-8 ' and δ
H1.12～1.04 (2H, m) are relevant, are attributed to H-7 '.H-8 and δ
H4.36～4.43 (1H, m) are relevant, are attributed to H-7.According to HMQC spectrum, δ
C20.8 be attributed to C-7 ', δ
C58.3 be attributed to C-7.
With reference to Dalacina and impurity 1, δ
H5.18 (1H, d, J=5.6Hz) is attributed to H-1, δ
C88.1 be attributed to C-1.H-1 and δ in the COSY spectrum
H3.91 (1H, dd, J=5.6,10.2Hz) is relevant, is attributed to H-2.H-2 and δ
H3.44 (1H, dd, J=3.2,10.2Hz) is relevant, is attributed to H-3.H-3 and δ
H3.64 (1H, d, J=3.2Hz) is relevant, is attributed to H-4.Contrast HMQC spectrum, δ
C68.0 be attributed to C-2, δ
C70.6 be attributed to C-3, δ
C68.4 be attributed to C-4.
HMBC composes demonstration, H-8 ' and δ
C20.8 and δ
C36.9 relevant.δ
C20.8 be attributed to C-7 ', and δ
C36.9 be CH on the DEPT spectrum
2Signal, so δ
C36.9 be attributed to C-6 '.Contrast HMQC spectrum, δ
H1.19～1.14 (2H, m) are attributed to H-6 '.H-8 and δ
C52.6 and δ
C58.3 relevant, δ
C58.3 be attributed to C-7, δ
C52.6 be attributed to C-6.Contrast HMQC spectrum, δ
H4.21 (1H, dd, J=10.0,1.6Hz) is attributed to H-6.Can be with δ by coupling constant
H4.10 (1H, d, J=10.4Hz) is attributed to H-5, contrast HMQC spectrum, δ
C69.4 be attributed to C-5.
DEPT composes demonstration, δ
C37.4 and δ
C61.0 be CH
2Fignal center can belong to δ by chemical structure
C37.4 be C-2,, δ
C61.0 be C-4 '.According to the HMQC spectrum, H-2 ' and H-4 ' homolysis are divided into two groups of peaks, δ
H2.33～2.26 (1H, m) and 1.36～1.29 (1H, m) are attributed to H-2 ', δ
H2.77～2.75 (1H, m) and 2.58～2.53 (1H, m) are attributed to H-4 '.H-2 ' and δ in the HMBC spectrum
C69.6 relevant, because the deshielding effect of C-1 ' is better than C-3 ', therefore belong to δ
C69.6 be C-1 '.Last δ
C36.3 be attributed to C-3 '.
NOESY composes demonstration, and the H-1 ' of clindamycin is relevant to H-5 ', and the H-1 ' of impurity 2 and H-5 ' be without relevant peaks, illustrates that in clindamycin, H-1 ' and H-5 ' position are nearer, and in impurity 2, H-1 ' and H-5 ' position are far away.Therefore infer that impurity 2 is mainly different at the carbon atom configuration of 1 ' from the clindamycin difference, 1 ' carbon atom of clindamycin is the S configuration, and 1 ' carbon atom of impurity 2 is the R configuration.
Therefore, target impurity all has the mother nucleus structure identical with clindamycin, and wherein the molecular weight of impurity 2 is identical with clindamycin, is the isomers of clindamycin.
Four chiral carbon are arranged in the clindamycin structure, be respectively C-6, C-7, C-1 ' and C-3 ', configuration is respectively 6S, 7S, 1 ' S, 3 ' R.Impurity 2 and clindamycin
1HNMR and
13The CNMR data are similar, and at 1 ', four chiral carbon configurations of impurity 2 are respectively 6S, 7S, 1 ' R, 3 ' S with the difference of clindamycin structure.Impurity 2 does not have pharmacopeia and document to record.
The bacteriostatic experiment of Dalacina and impurity of the present invention 2
The test that is used for mensuration antibacterials vitro inhibition bacterial growth effect is called bacteriostatic test.This effects in the Dalacina bulk drug apparent content surpass the bacteriostatic activity of 0.1% clindamycin isomer, the sterling of the clindamycin isomer for preparing is carried out the bacteriostatic activity contrast with the Dalacina bulk drug.
The preparation of test solution
Dalacina (090303 * 7 batch, Zhejiang Province Tiantai Pharmaceutical Co., Ltd): 1.091mg, 1mL water dissolution;
Impurity 2:1.200mg, the 0.5mL water dissolution;
Experimental strain
Streptococcus aureus (gram-positive microorganism), subtilis (bacterium), Candida albicans (fungi); Provided by biology section of Shanghai Institute of Pharmaceutical Industry.
The preparation of substratum
Candida albicans substratum (%)
Glucose 0.1 yeast extract paste 0.25
KCl 0.18 NaAc 0.82
Agar 1.5 pH 7.0
121 ℃ of sterilization 30min
Streptococcus aureus and subtilis (%)
Peptone 0.6 extractum carnis 0.15
Yeast extract paste 0.6 glucose 0.1
Agar 1.5 pH 6.5
121 ℃ of sterilization 30min
The preparation of filter paper
Select bibulous high-quality filter paper, break into punch tool the circular filter paper sheet that diameter is 6mm, standby after dry sterilization.
Experimental technique
The agar diffusion paper disk method
[30]: draw 0.1mL bacterium liquid, evenly on coating and M-H agar surface ware.Respectively the Dalacina of 10 μ L and the solution of clindamycin isomer evenly are added on the scraps of paper of sterilization, to be dried rear the gripping scraps of paper are equidistant respectively is positioned on the watch-glass that contains bacterium with aseptic nipper.Watch-glass is covered, be flat in 37 ℃ of incubators and take out after cultivation 24h.Observe fungistatic effect.
Experimental result
Dalacina and clindamycin isomer all have fungistatic effect to streptococcus aureus and subtilis.To the fungistatic effect of streptococcus aureus as shown in Figure 8, the inhibition zone of impurity 2 is less than Dalacina.Therefore the fungistatic effect of impurity 2 is poor than Dalacina.For the fungistatic effect of subtilis as shown in Figure 9, four inhibition zone is all larger, illustrates that Dalacina and clindamycin isomer have good restraining effect to subtilis.Figure 10 shows that the filter paper of Dalacina and clindamycin isomer inhibition zone do not occur on every side, and illustrating does not all have anti-microbial activity to candida albicans.
Claims (10)
1. clindamycin isomer has as shown in the formula structure shown in II:
2. the analysis preparation method of clindamycin isomer as claimed in claim 1, is characterized in that the clindamycin raw material is analyzed, and therefrom separate the described clindamycin isomer of preparation, comprises the following steps:
A) measure described clindamycin raw material with the LC-MS method, determine clindamycin isomer in described raw material according to the relative retention time of analyzed composition and/or molecular weight;
B) relative retention time of the clindamycin isomer described in a) and/or the condition that molecular weight is determined column chromatography according to step, use purification on normal-phase silica gel column chromatography this relative retention time of enrichment and/or analyzed composition corresponding to molecular weight;
C) the shown chromatogram retention behavior of the relative retention time of the clindamycin isomer described in a) is determined the condition of preparation liquid phase method according to step, collects the corresponding analyzed composition of described retention time with the preparation liquid phase method.
3. method according to claim 1, is characterized in that, step a) in, described LC-MS method is measured the HPLC condition that adopts and is:
Moving phase 18% acetonitrile, 3% tetrahydrofuran (THF), 79% water and 0.2% formic acid;
PH ammoniacal liquor transfers to 5.43-5.47;
25 ℃ of column temperatures;
Flow velocity 1.0ml/min;
Detect wavelength 210nm;
Chromatographic column Diamonsil ODS C18,5 μ m, 250 * 4.6mm post.
4. method according to claim 1, is characterized in that, step b) in, the stationary phase that described purification on normal-phase silica gel column chromatography enrichment is adopted is 100～200 order silica gel, moving phase is ethyl acetate and water saturation methanol mixed solvent.
5. method according to claim 1, is characterized in that, step b) comprising:
B 1) sample: silica gel=1: 50 is dissolved in sample in methyl alcohol the dry method upper prop;
B2) use ethyl acetate: the methanol mixed solvent gradient elution, elution order is as follows: (ethyl acetate: methyl alcohol 9: 1) 1800mL, (ethyl acetate: methyl alcohol 6: 1) 1680mL;
B3) merge collection (ethyl acetate: elutriant methyl alcohol 6: 1).
6. method according to claim 1, is characterized in that, step c) in, described preparation liquid phase method adopts the secondary preparation, wherein once prepares institute's employing condition and comprises:
Moving phase is 20% acetonitrile, 1.25% tetrahydrofuran (THF), 78.75% water, 0.2% formic acid;
It is 5.58 left and right that ammoniacal liquor is transferred pH;
Chromatographic column μ Bondapak
TMC18,7.8 * 300mm, the detection wavelength is 210nm, flow velocity is 1.0 * 2.25mL/min;
Collecting retention time is the corresponding assay in peak at 29.525min place;
The gained assay is adopted the secondary preparation of binary pump, the condition of moving phase is: the A pump: 21% acetonitrile, and 3% tetrahydrofuran (THF), 76% water, 0.2% formic acid, pH are 5.20 left and right; B pump: acetonitrile; A: B=95: 5; Chromatographic column Sepax HP-C18,5 μ m 10.0 * 250mm detect wavelength: 210nm, flow velocity: 1.5mL/min, column temperature: 35 ℃; Collecting retention time is the corresponding assay in peak at 27.68min place.
7. clindamycin isomer as claimed in claim 1 is in the purposes of preparation in medicament for resisting gram-positive bacteria.
8. purposes as claimed in claim 7, wherein said gram-positive microorganism is selected from anti-Bacillus subtilus or streptococcus aureus.
9. the impurity standard substance of the described method preparation of any one according to claim 2-5, is characterized in that, described impurity has following structural formula II:
10. the purposes of impurity standard substance according to claim 9 in analyzing the clindamycin raw material.
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| US3856943A (en) * | 1971-06-23 | 1974-12-24 | Upjohn Co | Compositions and process |
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