CN102060882A - Dehydrogenated clindamycin phosphate, analysis preparation method and application thereof - Google Patents
Dehydrogenated clindamycin phosphate, analysis preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses dehydrogenated clindamycin phosphate and an analysis preparation method thereof. The analysis preparation method is used for analyzing raw materials of clindamycin phosphate, and separating and preparing the dehydrogenated clindamycin phosphate from the clindamycin phosphate. The analysis preparation method comprises the following steps of: a) measuring the raw materials of the clindamycin phosphate by a liquid chromatography/mass spectroscopy (LC-MS) method; determining the dehydrogenated clindamycin phosphate in the raw materials according to the relative retention time and/or the molecular weight of analyzed components; and b) determining the conditions of a preparative chromatography method according to chromatographic retention behaviors displayed by the relative retention time of the dehydrogenated clindamycin phosphate in the step a), and collecting the analyzed components corresponding to the relative retention time by the preparative chromatography method.
Description
Technical field
The present invention relates to the analytical chemistry field, relate in particular to the pharmaceutical analysis chemical field, particularly the impurity dehydrogenation Clindamycin Phosphate of Clindamycin Phosphate and analysis and preparation method.
Background technology
Clindamycin Phosphate is in external no antibiotic activity, under the effect of phosphoesterase, be hydrolyzed to clindamycin rapidly after entering in the body, change into N-demethyl clindamycin in vivo with strong anti-microbial activity, mainly act on the 50S subunit of bacterial ribosome, thereby stop the prolongation of peptide chain and disturb the synthetic of DNA of bacteria and bacterioprotein, still can remove the A albumen and the fine hair shape coat of bacterium surface, bacterium is engulfed easily and kill.Clindamycin Phosphate mainly has very strong anti-microbial activity to gram-positive cocci and anerobe, its effect and purposes are similar to clindamycin hydrochloride, but absorb rapidly, and effect is lasting, Plasma Concentration is a Broad spectrum antibiotics that has anaerobe resistant and aerophil effect concurrently than the high twice of hydrochloride.Existing in the market aqueous injection, infusion solution, injection, aseptic subpackaged powder injection, lyophilized injectable powder etc., clinical application is based on injection.
Any material that influences pharmaceutical purity all is called impurity, and generally speaking, impurity is meant other chemical substances beyond the medicine of introducing or producing in production and storage process.Impurity in the drug standard is meant according to through relevant drug regulatory department of country in accordance with the law in the medicine of the regulation technology of examination and approval and the production of regulation supplementary material, the impurity of bringing into by its production technique and supplementary material, or through the degraded product that in storage process, produces of stability experiment conclusive evidence.Impurity in the 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 new impurity thus into to the proper mass standard revise, 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 the drug production process lyase that generally uses, catalyzer etc.;
2. react incomplete and reaction raw materials that exist, the materials relevant such as reaction initial recombination thing, synthetic mesophase product, byproduct with building-up process;
3. the oxidation in the storage process, decomposition, hydrolysate;
4. the optical isomer in the chipal compounds;
5. the multiple crystal formation of medicine;
In the animals and plants medicine extract except that small molecules such as effective constituent alkaloid volatile oil, organic acid, also have impurity such as the bigger 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 the 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, organic volatile 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, other biological alkali, geometrical isomer, optical isomer and polymkeric substance etc.By toxicity, can be divided into toxic impurities and common impurities etc. again.Common impurities is the impurity of no remarkable bad biological action under amount, and toxic impurities is the impurity with strong bad biological action.
It is an important step of drug quality control that impurity detects, and the assay in the middle of the drug quality is meant the content of main component in bulk drug and the preparation, and related substance is meant the organic impurity in the middle of bulk drug and the preparation.By the related substance inspection, understand fully source, character, the detection method of related substance and limit the quantity of, can optimize 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, reduce the untoward reaction of medicine.
Impurity of the drug checks that analytical procedure should be sensitive, exclusive.Along with 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 the new preparation process, should study in strict accordance with the requirement that the relevant new drug of country is declared, also can study, and the security and the degraded product of impurity carried out safety evaluation with reference to text Q3A (impurity in the new raw material medicine) and the Q3B (impurity in the new preparation) of ICH.Its specific requirement following points:
1. in esse impurity and potential impurity in synthetic, purifying and the storage, should adopt effective method for separating and analyzing to detect;
For apparent content 0.1% and above impurity and apparent content giving qualitative in the impurity below 0.1% or toxic impurities or proving conclusively its structure with strong biological action;
3. the degraded product to occurring in stability test also should be studied 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 impurity that occurs in batch process and degraded product, and comprises corresponding limit;
5. except that 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 the preparation should be determined inspection item according to its production technique, starting raw material situation, but for toxic inorganic impurity, should stipulate its inspection item in quality standard.
In the development and production of imitation medicine, as the kind of finding impurity is different with the original development medicine 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 report relevant drug regulatory department to examine.
The isomer of coexistence 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, with the bulk drug that the guarantees production usefulness consistence when declaring registration.But when the material when coexistence 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.
Organic volatile impurity should be determined inspection item according to used organic solvent and residual condition thereof in the production technique.Can be with reference to the requirement of Chinese Pharmacopoeia about organic volatile 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 all 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: the limit of impurity is 0.1% (lower to the drug toxicity limit) in the medicine, all unknown impurities that are higher than this level should be differentiated out, and the more important thing is that all are higher than 0.1% impurity should study its toxicity.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 is meant that impurity and content that all are higher than this threshold value all should charge in the survey report of every batch of product, and is reflected in the declaration material.And identify that threshold value is meant that all impurity that are higher than this threshold value all tackle its structure and prove conclusively.Reasonable limit is meant as long as the limit of impurities of formulating in the quality standard is not higher than this limit, does not just need to provide the formulation foundation of this limit, thinks that all this limit is rational.
For new preparation, ICH has also done clearly regulation in " impurity in the 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 anyly gives structure greater than the unknown peak that can ignore limit 0.06% and determines and propose suitable limit of impurities suggestion, promptly 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, surpass 0.1% impurity and need identify out and carry out quantitative analysis, and 0.01%～0.1% impurity is also represented keen interest with the good method of selectivity.
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, mainly show as: part pharmaceutical research unit does not know much have less understanding to the importance of impurity research; Control to impurity in the standard is comprehensive and accurate inadequately; It is comprehensive inadequately to consider a problem when working out limit of impurities, seldom considers the detrimentally affect of impurity to drug safety; Even when the scope that the content of impurity obviously exceeds normal process and allowed, do not note present prescription and technology 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 Phosphate related substances item, and recording of " British Pharmacopoeia " and " European Pharmacopoeia " is more comprehensive, and " British Pharmacopoeia " and " European Pharmacopoeia " recorded lincomycin, clindamycin B2-phosphoric acid ester, clindamycin 3-phosphoric acid ester, clindamycin 4-phosphoric acid ester and five impurity of clindamycin according to the degradation pathway of production technique and Clindamycin Phosphate at " possible impurity " item.
" in the Chinese pharmacopoeia Clindamycin Phosphate is only limited the total amount of impurity, not concrete to single impurity research, and medicine competent authorities of European Union and FDA all require apparent content 0.1% and above impurity thereof in the Clindamycin Phosphate bulk drug, carry out structure and identify and security verification.
Summary of the invention
The object of the invention is the impurity of Clindamycin Phosphate bulk drug is studied, and mainly is to separate the standard substance that prepare impurity and identifies impurity structure in the bulk drug.The method according to this invention to the impurity in the bulk drug analyze, preparation and structure identify, can and illustrate the untoward reaction mechanism for the toxicologic study of impurity the basis is provided, simultaneously also can provide reference, help the control of drug quality in the production process for the selection of technology compound experiment condition.
According to a first aspect of the invention, it provides as shown in the formula the dehydrogenation Clindamycin Phosphate shown in the I:
According to a second aspect of the invention, it provides analysis and preparation method as the described dehydrogenation Clindamycin Phosphate in first aspect of the present invention, this method is used for the Clindamycin Phosphate raw material is analyzed, and therefrom separate the described dehydrogenation Clindamycin Phosphate of preparation, comprise the following steps:
A) measure described Clindamycin Phosphate raw material with the LC-MS method, determine dehydrogenation Clindamycin Phosphate in the described raw material according to the relative retention time of analyzed composition and/or molecular weight;
B) determine the condition of preparative chromatography according to the shown chromatogram retention behavior of the relative retention time of the dehydrogenation Clindamycin Phosphate described in the step a), collect and the analyzed composition of described relative retention time correspondence with preparative chromatography.
Preferably, to measure the moving phase pH value adopted be 4.00～5.00 to the LC-MS method described in the step a); It is 20 ℃～40 ℃ that described LC-MS method is measured the column temperature that is adopted; Described LC-MS method is measured the moving phase that is adopted and is comprised 15%～25% acetonitrile; Described LC-MS method is measured the moving phase that is adopted and is comprised 0.1%～0.3% ammoniacal liquor.
More preferably, the mensuration of the LC-MS method described in the step a) HPLC condition that adopts is:
Moving phase 20% acetonitrile, 0.1% ammoniacal liquor;
PH formic acid is transferred pH to 4.90;
25 ℃ of column temperatures;
Flow velocity 1.0ml/min;
Detect wavelength 210nm;
Chromatographic column Diamonsil ODS C18,5 μ m, 250 * 4.6mm post.
The method according to this invention, the preparative chromatography institute employing condition described in the step b) comprises: moving phase 15%～25% acetonitrile, 0.1～0.3% ammoniacal liquor; PH formic acid is transferred pH to 4.00～5.00; Column temperature: 20 ℃～40 ℃; Chromatographic column Waters μ Bondapak ODS C18,5 μ m, 7.8mm * 300mm post, perhaps Agela Venusil ODS XBP C18,5 μ m, 10mm * 250mm post.
Preferably, the institute of the preparative chromatography described in step b) employing condition is:
Moving phase 22% acetonitrile, 0.1% ammoniacal liquor;
PH formic acid is transferred pH to 4.00;
Flow velocity 1.0ml/min;
Detect wavelength 210nm;
Chromatographic column Agela Venusil ODS XBP C18 post, 5 μ m, 10mm * 250mm post;
Sample size 120 μ l;
Collecting retention time is the corresponding assay of locating in 20.220 minutes in peak.
According to a third aspect of the present invention, it provides the purposes that is used to prepare medicament for resisting gram-positive bacteria as the described dehydrogenation Clindamycin Phosphate of first aspect present invention.
Preferably, described gram-positive microorganism comprises anti-Bacillus subtilus and streptococcus aureus.
According to a third aspect of the present invention, the impurity standard substance that it provides second described method preparation in aspect according to the present invention is characterized in that described impurity has aforementioned structural formula I.
According to a fourth aspect of the present invention, it provides the purposes that is used to analyze the Clindamycin Phosphate raw material as the described impurity standard substance of third aspect present invention.
Description of drawings
In conjunction with the accompanying drawing that the application provided, the application's other features, objects and advantages will be more readily understood.These accompanying drawings only are used for demonstration, the present invention are not constituted any restriction.
Fig. 1 is for investigating pH value and the resolution graph of a relation under different numerical value in the LC-MS method conditioning process;
Fig. 2 is the column temperature under different numerical value and the graph of a relation of resolution in the investigation LC-MS method conditioning process;
Fig. 3 is for investigating acetonitrile ratio and the main peak retention time graph of a relation under different numerical value in the LC-MS method conditioning process;
Fig. 4 is for investigating ammoniacal liquor ratio and the main peak retention time graph of a relation under different numerical value in the LC-MS method conditioning process;
Fig. 5 is for investigating ammoniacal liquor ratio and the resolution graph of a relation under different numerical value in the LC-MS method conditioning process.
Embodiment
In order to understand technical scheme of the present invention better, 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.
The bulk drug of the same name that the Clindamycin Phosphate raw material that present embodiment is used all adopts Zhejiang Province Tiantai Pharmaceutical Co., Ltd to provide.
LC-MS measures the Clindamycin Phosphate bulk drug
In order to study Clindamycin Phosphate bulk drug impurity, utilize the LC-MS instrument that the major impurity of bulk drug is studied, purpose is in order to determine the molecular weight and the chromatogram retention behavior of impurity.
In the Clindamycin Phosphate molecular structure acidic-group is arranged, basic group is also arranged, belong to the amphoteric material.HPLC detects and all to select ion suppression chromatography (ion suppressionchromatography ISC) analyzes in various countries' pharmacopeia.Because the mass spectrograph that present embodiment is selected is electron spray(ES) (electrospray ionization; ESI) ionization mode; in order to reduce the influence of ion pair inhibitor to measuring, also in order to protect instrument, present embodiment adopts volatile ammoniacal liquor, formic acid buffering salt system to detect.Because do not have conjugated structure in the Clindamycin Phosphate molecule, and contain N, O, S heteroatoms, the probability of valence electron n-σ * transition is higher in the molecule, so the molecular end absorption is more intense, selects 210nm as detecting wavelength.Moving phase is at first selected 20% acetonitrile, investigates the influence of pH value to measuring.
The selection of moving phase pH value
Instrument is HP1100, and chromatographic column Diamonsil ODS C18 (5 μ m, 250 * 4.6mm) posts detect wavelength 210nm, 25 ℃ of column temperatures, and flow velocity 1.0ml/min detects wavelength 210nm.Take by weighing the Clindamycin Phosphate bulk drug and be dissolved in the buffered soln of 20% acetonitrile pH value 4.00, be configured to the solution of 20mg/ml, sample size 20 μ l.Dispose the moving phase of a plurality of pH values, measure respectively.
In the present embodiment, having investigated moving phase pH value is the situation of 4.00,4.50,4.75,4.90,5.00 o'clock HPLC mensuration bulk drugs, when moving phase pH value is increased to 5.00 by 4.00, Clindamycin Phosphate is increased by the ratio that ionic condition converts molecularity to, component solubleness in stationary phase increases, capacity factor increases, and the retention time of main peak increases along with increasing of pH value.During moving phase pH value 4.00, can only measure 4 impurity, lack than the moving phase of other four pH and measure an impurity, therefore, this method is not suitable as the condition of measuring bulk drug.According to retention time ascending with six peaks in the color atlas compile successively be the 1st peak, the 2nd peak ... the 6th peak, wherein main peak is the 5th peak, does not have the 4th peak during moving phase pH value 4.00.
Fig. 1 is pH value and resolution graph of a relation, and as can be seen from the figure, moving phase pH value mainly influences the resolution of the 3rd peak and the 4th peak, the 4th peak and main peak, to having the greatest impact of the resolution of the 4th peak and main peak.Be worth 4.00 o'clock than moving phase pH during moving phase pH value 4.50, a small peak (the 4th peak, retention time 17.307min) appears in the main peak front, but peak shape is very poor, and the chromatographic peak symmetrical factor is near 0.During moving phase pH value 4.75, the retention time at the 4th peak is 18.467min, the peak symmetrical factor be 2.09 and the resolution of main peak be 1.44.During moving phase pH value 4.90, the retention time at the 4th peak is 18.942min, the peak symmetrical factor be 1.264 and the resolution of main peak be 1.55, it is 4.75 o'clock situation that peak shape and resolution are better than moving phase pH.When moving phase pH is 5.00, the retention time at the 4th peak is 19.782min, the peak symmetrical factor is 0.77, with the resolution of main peak be 1.97, it is 5.00 o'clock that resolution is better than moving phase pH, but the resolution at the 3rd peak (retention time 18.640min) and the 4th peak is 0.90, and resolution is too low, and during moving phase pH value 4.90, the resolution at two peaks is 1.53.Therefore, take all factors into consideration two factors of resolution and peak shape after, moving phase pH value when selecting pH4.90 to measure as bulk drug.
The selection of column temperature
Instrument is the same, and moving phase is selected i.e. 20% acetonitrile of above-mentioned moving phase 4,0.1% ammoniacal liquor, and formic acid is transferred pH to 4.90, and flow velocity 1.0ml/min detects wavelength 210nm, and sample configuration and sample size are the same.
Fig. 2 is the graph of a relation of column temperature and resolution, 20 ℃, 25 ℃, 30 ℃ and 40 ℃ of four column temperatures have been selected in the present embodiment, investigated the chromatogram retention behavior of bulk drug under the different column temperature of identical moving phase, ascending according to retention time in the color atlas, it is the 1st peak, the 2nd peak that 6 peaks are compiled successively ... the 6th peak, main peak is the 5th peak, during 40 ℃ of column temperatures, does not occur the 4th peak in the color atlas.In the process that column temperature raises, the molecular thermalmotion aggravation, the hydrophobic association effect between the alkyl of solute molecule and bonding phase weakens, and the retention time of main peak shifts to an earlier date, and the post pressure drop is low.Column temperature does not have the influence of moving phase pH value remarkable to the influence of resolution, during 40 ℃ of column temperatures, has only occurred four impurity in the color atlas, detects an impurity than lacking under other three column temperatures, and therefore, 40 ℃ are not suitable for as the conditions that detect bulk drug.During 20 ℃ of column temperatures, the 4th peak retention time is 19.678min, the symmetrical factor of chromatographic peak be 1.841 and the resolution of main peak be 1.39, peak shape and resolution all not as good as 25 ℃ of column temperatures (25 ℃ the time peak symmetrical factor at the 4th peak be 1.264 and the main peak resolution be 1.55).The resolution of the 4th peak and main peak is 1.62 during 30 ℃ of column temperatures, but the resolution at the peak of the peak of retention time 17.391min and retention time 18.677min has only 1.17.In order to guarantee that all impurity all can be detected, under the prerequisite that guarantees resolution, select 25 ℃ of conditions that detect as HPLC.
The selection of organic phase ratio
Instrument, chromatographic column, flow velocity, detection wavelength, sample size are the same, and 25 ℃ of column temperatures dispose the moving phase of 15% acetonitrile, 18% acetonitrile, 20% acetonitrile, 25% acetonitrile respectively.
Fig. 3 is acetonitrile ratio and main peak retention time graph of a relation.In the process that the ratio of acetonitrile raises, the polarity of moving phase reduces, and elutive power increases, and the k of solute increases, and retention time in advance.As can be seen from the figure, the retention time of main peak shifts to an earlier date along with the raising of moving phase acetonitrile ratio.The moving phase of four ratio acetonitriles all can detect 5 impurity, but when moving phase was 15% acetonitrile, 18% acetonitrile, analysis time was oversize.When moving phase was 25% acetonitrile, the separation at peak was not ideal enough, therefore also was not suitable for as the HPLC condition that detects.Determine that 20% acetonitrile is proper as the condition of moving phase.
The selection of buffering salt ratio
Instrument, chromatographic column, flow velocity, detection wavelength, sample size are the same, 25 ℃ of column temperatures.Be configured to the current downflow phase respectively: 20% acetonitrile, 0.05% ammoniacal liquor, formic acid adjust pH to 4.90; 20% acetonitrile, 0.1% ammoniacal liquor, formic acid adjust pH to 4.90; 20% acetonitrile, 0.2% ammoniacal liquor, formic acid adjust pH to 4.90; 20% acetonitrile, 0.3% ammoniacal liquor, formic acid adjust pH to 4.90.
The same, according to the retention time order, 6 peaks are called after the 1st peak, the 2nd peak successively ... the 6th peak.The ratio of buffering salt mainly influences between the 2nd peak and the 3rd peak, the 3rd peak is in the 4th peak-to-peak resolution.
The result shows that moving phase ammoniacal liquor ratio is, can only detect 4 impurity at 0.3% o'clock, and therefore 0.3% ammoniacal liquor is not suitable for as testing conditions.The ammoniacal liquor ratio is 0.05% and 0.2% o'clock in the moving phase, when the 3rd peak and the 4th peak-to-peak resolution are 0.1% ammoniacal liquor not as good as ammoniacal liquor ratio in the moving phase, is 0.1% as HPLC condition moving phase condition so select the ammoniacal liquor ratio.Referring to ammoniacal liquor ratio shown in Figure 4 and main peak retention time graph of a relation, and ammoniacal liquor ratio shown in Figure 5 and resolution graph of a relation.
More than examined or check the influence of pH value, organic phase ratio, column temperature, buffering salt ratio four factors for the retention time and the resolution of impurity in the bulk drug, the HPLC condition that present embodiment selects HPLC to detect bulk drug in view of the above is: the moving phase condition is 20% acetonitrile, 0.1% ammoniacal liquor, formic acid is transferred pH to 4.90; 25 ℃ of column temperatures; Flow velocity 1.0ml/min; Detect wavelength 210nm; Chromatographic column DiamonsilODS C18 (5 μ m, 250 * 4.6mm) posts.
Those skilled in the art are to be understood that, the combination of above-mentioned every HPLC condition can be changed according to actual needs, parameters or the parameter area determined in aforementioned 4 factor examination processes can carry out the combination of arbitrary form, and these combinations all should be considered as the disclosed content of the present invention.
After having determined that HPLC measures the condition of bulk drug, utilize LC-MS to measure, can determine bulk drug impurity molecule amount.
The LC-MS instrument HPLC Waters 2486 that uses, MS is WatersmicromassQ-TOF.Chromatographic column be Capcell pak ODS C18 post (5 μ m, 4.6 * 250mm), 25 ℃ of column temperatures, flow velocity 1.0ml/min, moving phase is 20% acetonitrile, 0.1% ammoniacal liquor, formic acid adjust pH to 4.90.The mass spectrum condition is an electron spray ionisation source positive ion detection mode; 80 ℃ of source temperature; Taper hole voltage 35v.Bulk drug with after the moving phase dissolving, is injected the LC-MS instrument respectively, sample size 10 μ l.
The LC-MS measurement result shows that the retention time of main peak is 18.94min, and the time of withing a hook at the end in the bulk drug is respectively five impurity of 8.42min, 11.32min, 15.62min, 16.95min, 31.32min.The MS collection of illustrative plates shows, [M+H] of five impurity
+M/z is followed successively by 491,585,503,505 and 425.Wherein the impurity molecule amount at retention time 31.32min place is 424, is Clindamycin Phosphate industry synthetic starting raw material clindamycin.The retention time of remaining four impurity and molecular weight are respectively 8.42min, 490; 11.32min, 584; 15.62min, 502 and 16.95min, 504, be defined as impurity 1, impurity 2, impurity 3 and impurity 4 successively.
HPLC measures each batch crude product
Attribute according to above definite four impurity, in bulk drug the total content 1.5% of 4 unknown impurities less than, it is quite difficult directly separating preparation impurity from bulk drug, therefore utilize HPLC to measure the crude product of bulk drug and five batches, utilize the peak area normalization method to measure the content of target impurity, for the column chromatography of back and preparation mask work provide guide.
The HPLC method is separated the preparation target impurity
According to above condition, collect some pins after, carry out aftertreatment according to ordinary method to collecting liquid, obtain white solid.
The HPLC detected result shows that the retention time of preparation gained sample is consistent with the retention time of impurity 3 in the bulk drug, has confirmed that further the sample that makes is an impurity 3, and it is more than 95% that HPLC measures its purity.
The structure of impurity 3 is identified
High resolution mass spectrum is measured [M+H]
+Mass-to-charge ratio is 503.1386, and it is elementary composition to be C
18H
33N
2O
11PSCl, degree of unsaturation is 4, degree of unsaturation Duos 1 than Clindamycin Phosphate, shown in the associative list 1
1H-NMR (400MHz, D
2O) compose,
13The parsing of C-NMR spectrum, COSY spectrum, hsqc spectrum and HMBC spectrum identifies that its structure is as shown in the formula shown in the I:
Table 1: impurity 3
1The H-NMR spectrum,
13C-NMR spectrum, COSY spectrum, hsqc spectrum and HMBC spectrum ownership
According to
1H-
1HCOSY spectrum, and δ 1.94 (q, 2H, J=7.20Hz) relevant with H-8 ', belong to and be H-7 ', according to HSQC, δ 22.9 is C-7 '.
1H-
1In the H COSY spectrum, and δ 5.58 (d, 1H, J=7.20Hz) relevant with H-7 ', so δ 5.58 ownership are H-6 ', and according to HSQC, δ 129.7 ownership are C-6 ', belonging to according to chemical shift δ 127.8 is C-3 '.In the HMBC spectrum, C-3 ' while and δ 2.67 (m, 1H), δ 3.73 (m, 1H) with δ 4.18 (m, 1H), δ in hsqc spectrum
C60.4 while and δ
H3.73, δ
H4.18 relevant, δ
C32.6 while and δ
H2.67, δ
H3.22 it is relevant.According to chemical shift, δ 3.73 and δ 4.18 ownership are H-4 ', and δ 60.4 ownership are C-4 '; δ
H2.67 and δ
H3.22 ownership is H-2 ', δ
C32.6 ownership is C-2 '.
1H-
1H-2 ' and δ 4.36 in the H COSY spectrum (m, 1H) relevant, so δ 4.36 (m, 1H) ownership is for H-1 ', and according to HSQC, δ 69.2 ownership are C-1 '.
1H-
1In the H COSY spectrum, (m, 1H) relevant, (m, 1H) ownership is H-7 to δ 4.60 for H-8 and δ 4.60.H-7 and H-8, δ 4.41 (q, 1H) relevant, so δ 4.41 (q, 1H) ownership is H-6.H-6 and δ 4.32 (m, 1H) relevant, so δ 4.32 (m, 1H) ownership is H-5.According to HSQC, it is C-5, C-6 and C-7 that δ 69.7,53.9,58.8 belongs to respectively.According to
1H-
1The HCOSY spectrum, (q, 1H) relevant, (q, 1H) ownership is H-2 to δ 4.41 for H-1 and δ 4.41.H-2 while and H-1, δ 3.73 (m, 1H) relevant, so δ
H3.73 (m, 1H) ownership is H-3.H-3 while and H-2, δ 3.85 (d, 1H, J=2.40Hz) relevant, so δ
H3.85 (J=2.40Hz) ownership is H-4 for d, 1H.According to hsqc spectrum, it is C-2, C-3 and C-4 that δ 72.0,70.9,68.7 belongs to respectively.C-2 is because in the phosphate radical that is linked to each other
31The coupling of P has produced splits branch, the swarming that splits of δ 72.1 occurs.
Get impurity 3 samples that prepare in the aforementioned embodiments, because sample adheres on the flask walls, the sample estimated weight that spoon is scraped out adds methanol constant volume in the volumetric flask, about 1mg/ml.
Adopt antibacterial ring test method to gram-positive microorganism, and Bacillus subtilus, streptococcus aureus, Candida albicans, intestinal bacteria, resistant organism malignant bacterias such as (MRSA) have carried out bacteriostatic test.The result shows 3 pairs of gram-positive microorganisms of impurity, and Bacillus subtilus, streptococcus aureus etc. present tangible antibacterial ring.And Candida albicans, intestinal bacteria, resistant organism (MRSA) are not presented antibacterial ring.To gram-positive microorganism, especially Bacillus subtilus, streptococcus aureus have tangible bacteriostatic action external for hence one can see that impurity 3; Candida albicans, intestinal bacteria, resistant organism (MRSA) then there is not resistance.
Claims (13)
1. dehydrogenation Clindamycin Phosphate, its structural formula is as shown in the formula shown in the I:
2. the analyte preparation method of dehydrogenation Clindamycin Phosphate according to claim 1 is characterized in that the Clindamycin Phosphate raw material is analyzed, and therefrom separates the described dehydrogenation Clindamycin Phosphate of preparation, comprises the following steps:
A) measure described Clindamycin Phosphate raw material with the LC-MS method, determine dehydrogenation Clindamycin Phosphate in the described raw material according to the relative retention time of analyzed composition and/or molecular weight;
B) determine the condition of preparative chromatography according to the shown chromatogram retention behavior of the relative retention time of the dehydrogenation Clindamycin Phosphate described in the step a), collect and the analyzed composition of described relative retention time correspondence with preparative chromatography.
3. method according to claim 2 is characterized in that, in the step a), it is 4.00～5.00 that described LC-MS method is measured the moving phase pH value that is adopted.
4. method according to claim 2 is characterized in that, in the step a), it is 20 ℃～40 ℃ that described LC-MS method is measured the column temperature that is adopted.
5. method according to claim 2 is characterized in that, in the step a), described LC-MS method is measured the moving phase that is adopted and comprised 15%～25% acetonitrile.
6. method according to claim 2 is characterized in that, in the step a), described LC-MS method is measured the moving phase that is adopted and comprised 0.1%～0.3% ammoniacal liquor.
7. method according to claim 2 is characterized in that, in the step a), described LC-MS method is measured the HPLC condition that adopts and is:
Moving phase 20% acetonitrile, 0.1% ammoniacal liquor;
PH formic acid is transferred pH to 4.90;
25 ℃ of column temperatures;
Flow velocity 1.0ml/min;
Detect wavelength 210nm;
Chromatographic column Diamonsil ODS C18,5 μ m, 250 * 4.6mm post.
8. method according to claim 2 is characterized in that, in the step b), described preparative chromatography institute employing condition comprises:
Moving phase 15%～25% acetonitrile, 0.1～0.3% ammoniacal liquor;
PH formic acid is transferred pH to 4.00～5.00;
Column temperature: 20 ℃～40 ℃;
Chromatographic column Waters μ Bondapak ODS C18,5 μ m, 7.8mm * 300mm post, perhaps Agela Venusil ODS XBP C18,5 μ m, 10mm * 250mm post.
9. method according to claim 8 is characterized in that, in the step b), described preparative chromatography institute employing condition is:
Moving phase 22% acetonitrile, 0.1% ammoniacal liquor;
PH formic acid is transferred pH to 4.00;
Flow velocity 1.0ml/min;
Detect wavelength 210nm;
Chromatographic column Agela Venusil ODS XBP C18 post, 5 μ m, 10mm * 250mm post;
Sample size 120 μ l;
Collecting retention time is the corresponding assay of locating in 20.220 minutes in peak.
10. dehydrogenation Clindamycin Phosphate as claimed in claim 1 is used to prepare the purposes of medicament for resisting gram-positive bacteria.
11. as purposes as described in the claim 10, wherein said gram-positive microorganism comprises anti-Bacillus subtilus and streptococcus aureus.
12., it is characterized in that described impurity has following structural formula I as the impurity standard substance of method preparation as described in each in the claim 2～9:
13. impurity standard substance as claimed in claim 12 are used to analyze the purposes of Clindamycin Phosphate raw material.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102964401A (en) * | 2012-11-20 | 2013-03-13 | 广州白云山天心制药股份有限公司 | Method for preparing clindamycin phosphate |
| CN103122013A (en) * | 2011-11-18 | 2013-05-29 | 上海医药工业研究院 | Hydrogen-removing clindamycin as well as analysis preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103122013A (en) * | 2011-11-18 | 2013-05-29 | 上海医药工业研究院 | Hydrogen-removing clindamycin as well as analysis preparation method and application thereof |
| CN103122013B (en) * | 2011-11-18 | 2016-07-06 | 上海医药工业研究院 | Dehydrogenation clindamycin, its analysis preparation method and purposes |
| CN102964401A (en) * | 2012-11-20 | 2013-03-13 | 广州白云山天心制药股份有限公司 | Method for preparing clindamycin phosphate |
| CN102964401B (en) * | 2012-11-20 | 2015-08-12 | 广州白云山天心制药股份有限公司 | A kind of preparation method of Clindamycin Phosphate |
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