CN107236012A - A kind of cobamamide crystal formation and preparation method and application - Google Patents
A kind of cobamamide crystal formation and preparation method and application Download PDFInfo
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- CN107236012A CN107236012A CN201710445525.XA CN201710445525A CN107236012A CN 107236012 A CN107236012 A CN 107236012A CN 201710445525 A CN201710445525 A CN 201710445525A CN 107236012 A CN107236012 A CN 107236012A
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- cobamamide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
Abstract
The present invention relates to a kind of cobamamide crystal formation and preparation method and application, using Cu K α target emanations, its X ray powder diffraction is in θ=5.1 ± 0.1 of the angle of diffraction 2,6.3 ± 0.1,7.0 ± 0.1,7.6 ± 0.1,8.0 ± 0.1,8.6 ± 0.1,9.4 ± 0.1,13.2 ± 0.1,13.8 ± 0.1,15.0 ± 0.1,17.5 ± 0.1,19.1 ± 0.1,21.4 ± 0.1, there is characteristic peak at 23.5 ± 0.1,25.3 ± 0.1.The present invention dissolves cobamamide crude product using cosolvent；Cobamamide highly finished product are obtained using anti-solvent recrystallization method；Preparation process reduction impurity effect is obvious；Easy to operate, energy consumption is low, and product purity is higher than 99.5%, and always miscellaneous to be less than 0.5%, yield is more than 98.0%, and product is significantly increased to the stability of light, heat, high humidity；Can industrialized production.
Description
Technical field
The present invention relates to a kind of cobamamide crystal formation and preparation method and application, belong to crystallization technique field.
Background technology
(Cobamamide is also referred to as Adocbl or Coenzyme B to cobamamide12, also known as deoxyadenosyl cobalamin), it is dark
Red colored crystalline or powder, it is great to draw moist, see that light is extremely easy in decomposition.Water is slightly soluble in, it is almost insoluble in ethanol, in acetone or ether
In it is insoluble.Its chemical name is 5,6- dimethylbenzimidazole base -5`- deoxyadenosine base cobalt amine, and molecular formula is
C72H100CoN18O17P, structural formula is as shown in Equation 1.
At present, cobamamide is used as biochemical reagents and experiment reagent in the world, is only used as medicine in China, using many
Year, cobamamide bulk drug and tablet are recorded in Chinese Pharmacopoeia 2010 editions and 2015 editions.Cobamamide early stage is mainly used in treatment
Pernicious anaemia, can also be shared with folic acid, for treating various megaloblastic anemias, anaemia etc. caused by antifolic.In recent years,
Cobamamide accommodation constantly extends, and be used to including ramitis, polyneuritis, peripheral neuritis, ophthalmoplegia,
Hepatopathy, cancer, atrophic gastritis, the field such as genital system diseases treatment is presented significant in process of clinical application
Therapeutic effect (1. Liu Lanbo clinical rational drug uses magazine, 2017,10 (2C):43-44. 2. the auspicious fragrant modern medicines in Hubei Province with it is clinical,
2017,32(1):46-50.), be great prospect a variety of diseases medicine.
Cobamamide is in preparation and storing process, and being also easy to produce the impurity such as cyanocobalamin, hydroxycobalamin, (1. the vast China of Yang Hong is raw
Chemical drug thing magazine, 2015,35 (6):The 157-164. 2. red Strait Pharmaceutical Journals of Yan Min, 2013,25 (8):62-65), these impurity with
Cobamamide structure is similar, belongs to cobalamin class compound.Cobalamin class compound is easily formed law of isomorphism thing (Gruber
GK.Vitamin B12 and B12-Proteins, Wiley-VCH, Wein-heim, 1998, p335ff.), that is to say, that it is miscellaneous
Matter is easy to separate out with cobamamide cocrystallization and be difficult to remove.At present, about cobamamide process for purification more use resin
The method of absorption, and removing cobamamide impurity by recrystallization process has great technical difficulty.
The requirement for the cobamamide bulk drug that China's pharmacopeia 2015 editions is listed is that impurity content must not be higher than 1%, the impurity
When content is used for injection preparation, hence it is evident that higher, quality evaluation of medicine system constantly improve, evaluation criterion are constantly carried in China
Under high overall background, find can reduce impurity, easily to operate, meet green chemical concept process for purification have it is important
Value.
Around the purpose of reduction cobamamide impurity, we have investigated different recrystallization conditions to reducing the shadow of impurity
Ring, new cobamamide crystal formation is found that in research process, further, it was found that the preparation condition of the crystal formation has effectively reduction impurity
Effect.
Cobamamide tool it is strong draw moist, therefore, the combination situation of itself and hydrone is the problem of needing to consider first.Text
Offer investigation (1. Savage H.Acta Crystallographica, 1984,40 (a1):C24-C24.②Bouquiere J
P.Acta Crystallographica,1993,49(1):79-89.③Bouquiere J P.Acta
Crystallographica,Section B:structure science,1994,B50(5):566-578.) confirm, from third
The cobamamide separated out in ketone-water mixed solvent exists in the form of hydrate, and 1 molecule cobamamide can combine 17 moisture
Son, these hydrones exist in two forms, and a kind of form is ordered into water, and this part hydrone ordered arrangement is in solvent pocket
In, another form is unordered water, and this part hydrone is disorderly arranged in solvent channel (shown in Fig. 1).Two kinds of combining forms
Hydrone it is different from the combination of the chemical bond of cobamamide, therefore, cobamamide has the chemistry being dehydrated in two times
Architecture basics.By experiment and TG-DSC detections, it is found that cobamamide sloughs unordered water below 100 DEG C, more than 200 DEG C are sloughed
Orderly water, then occurs to decompose (legend can be referring to accompanying drawing 5) completely.
Present Research about cobamamide crystal structure and crystal formation is as follows：
Document (Mattern CFT.Journal of Biological Chemistry, 1960,235 (2):489-491)
The mono-crystalline structures of cobamamide are reported, the preparation condition of the monocrystalline of the detection is：4 DEG C, for containing 87.5% (volume) acetone
The aqueous solution.Because under X-ray, decomposed occurs for the cobamamide of photaesthesia, therefore the data needs obtained are more
Mathematical treatment, the monocrystalline of acquisition is rhombic system P212121.Then, document (Ouyang L.Inorganic Chemistry
2004,43,1235-1241) mono-crystalline structures of the cobamamide obtained from water and acetone mixed solvent have been redefined, it is brilliant
Body is rhombic system P212121, still, the document does not provide powder diffraction data.We repeat the adenosine of experimentation acquisition
Cobalt amine crystal, its powder diffraction (P-XRD) collection of illustrative plates is shown in Fig. 2, the X-ray powder diffraction of the crystal θ=6.9 of the angle of diffraction 2 ±
0.1,7.9 ± 0.1,9.8 ± 0.1,13.2 ± 0.1,14.3 ± 0.1,16.2 ± 0.1,18.1 ± 0.1,19.0 ± 0.1,20.1
There is characteristic peak at ± 0.1,21.2 ± 0.1.For sake of convenience, we are defined as A crystal formation cobamamides.
Patent JP47022716 is published in 1972, and the patent disclosure is recrystallized more than 17 DEG C and in less than 17 DEG C water
2 crystal formations of the cobamamide of acquisition.The patent provides the X- of the crystal formation obtained in two crystal formations and acetone-water mixed system
Powder diffraction spectrum, still, graph-spectrum quality are poor, are only capable of finding out the feature at stronger peak.As can be seen that third from the patent accompanying drawing
In ketone-water crystallization obtain crystallize θ=6.9 of powder diffraction main peak 2 (intensity 100), it is consistent with crystal formation A highest peaks, 17 DEG C with
The θ=9.1,17.0 of powder diffraction main peak 2 for the crystal formation that upper crystallization is obtained, the powder diffraction master for the crystal formation that 17 DEG C of crystallization below are obtained
The θ=6.6,13.0 of peak 2.Two powder diffraction characteristic peaks of crystal formation for being separated out from single solvent water and present invention difference are obvious,
And be not directed to reduce the effect of impurity, therefore repeat no more.
The relevant cobamamide compounds of patent ZL104130303 and its pharmaceutical composition, refer to a kind of from isopropanol, pyridine
The cobamamide crystal obtained with the in the mixed solvent of ether, the patent specification appends powder diffraction spectrum, same to use
Cu-K α target emanations, its θ=6.1 ± 0.1,11.2 ± 0.1,17.0 ± 0.1,19.1 ± 0.1,21.0 ± 0.1,22.2 of angle of diffraction 2
There is characteristic peak at ± 0.1,23.5 ± 0.1.For sake of convenience, we are defined as B crystal form cobamamide.
In summary, at present, it has been reported that separated out two kinds of cobamamide crystal formations from the mixed solvent, crystal formation A is from third
Separated out in ketone-water mixed solvent, the crystal formation is that people recognize for a long time, but unstable to light, heat, soda acid.Crystal formation B stabilization
Property is improved compared with crystal formation A, but its preparation process (ZL104130303) has three weak points：
(1) solvent is done using pyridine and ether, pyridine tanginess has intense irritation, can anaesthetize central nervous system,
After high concentration suction, serious harm is produced to human body, and pyridine boiling point is higher, is 115 DEG C, it is difficult to remove, solvent can be brought residual
Stay problem.Another solvent ether highly volatile, is also easy to produce peroxide during storage, operating personnel are easily caused when using and produce anesthesia
Symptom, security hidden trouble is serious.
(2) preparation process is needed to be cooled to -15~-10 DEG C in 3~6 minutes, and 10~12 hours are stood at -15 DEG C.Need drop
Gentle low-temperature operation.
(3) when repeating the patented method, it is found that cobamamide raw material is not completely dissolved, experimentation is almost without reduction
The effect of impurity.
Miscellaneous Quality Research and control are to ensure that one of key element of drug safety, are especially injections in drug research and development
The important embodiment of R＆D process risk control consciousness, at present, the cobamamide bulk drug impurity of 2015 editions States Pharmacopoeia specifications of China
Content is no more than 1%, and the impurity content of commercially available cobamamide is generally more than 0.7%, deposits by further refining, reaches
Reduce the space of impurity.We are obtained a kind of crystal formation new with higher yields acquisition and entered by the research of recrystallization technology
The method that one step reduces cobamamide impurity, at present, not yet there is the report of similar approach.
The content of the invention
In order to solve problem of the prior art, first purpose of the invention provides a kind of cobamamide novel crystal forms, obtained
The novel crystal forms stability obtained is improved, and is more beneficial for storage and the follow-up ease of formulation of reduction of product.
Second object of the present invention is to be that offer is a kind of to have higher yields, the preparation side of the novel crystal forms of mild condition
Method, the preparation method can effectively reduce cobamamide bulk drug impurity content, available for the refined of cobamamide.
Preparation method of the present invention obtains a kind of new cobamamide crystal formation, uses Cu-K α target emanations, its monocrystalline
Diffracting spectrum is shown in Fig. 3, is monoclinic system P21.C crystal form is different crystallographic systems, its crystal compared with the monocrystalline configuration of document report
Data comparison is shown in Table 1.
Preparation method of the present invention obtains a kind of cobamamide crystal formation, using Cu-K α target emanations, its X-ray powder
Last diffraction θ=5.1 ± 0.1,6.3 ± 0.1,7.0 ± 0.1,7.6 ± 0.1,8.0 ± 0.1,8.6 ± 0.1,9.4 of the angle of diffraction 2 ±
0.1,13.2 ± 0.1,13.8 ± 0.1,15.0 ± 0.1,17.5 ± 0.1,19.1 ± 0.1,21.4 ± 0.1,23.5 ± 0.1,
There is characteristic peak at 25.3 ± 0.1, as shown in Figure 4.It is named as C crystal form cobamamide.
2 θ angles of the X- powder diffractions of crystal formation prepared by the A of document report, B crystal form and the present invention and relative intensity contrast
It is summarized in table 2.As can be seen from the table, the difference of the 2 θ angles and relative intensity of three kinds of crystal formations is obvious, it can be determined that for difference
Crystal formation.
The preparation method of the cobamamide crystal formation (C crystal form) of the present invention, its step is as follows：
(1) cobamamide crude product is dissolved using cosolvent；
Under red light lighting condition；In room temperature, the water that cobamamide crude product is added to 5-7 times of quality, stir 5-30 minutes
To pasty state, it is kept stirring for, cosolvent is added into above-mentioned pastel, it was observed that in 10min, cobamamide is completely dissolved, obtains
Settled solution；
(2) cobamamide highly finished product are obtained using anti-solvent recrystallization method；
Under red light lighting condition；Anti-solvent is added into above-mentioned solution, suspension is obtained, room temperature is stood overnight, suction filtration,
Isopropanol is washed, at 20~25 DEG C, is dried in vacuo 3~8 hours, is obtained cobamamide crystal formation；We are named as C crystal form.
Cosolvent in described step (1) is water and solvent acetone or the mixed solvent of acetonitrile, preferably acetone.Water and third
The volume ratio of ketone or acetonitrile is 1：0.3~0.5；Preferred volume ratio is water：Acetone=1：0.4.
Anti-solvent in described step (2) is isopropanol or the tert-butyl alcohol, preferably isopropanol.Anti-solvent is disposably added
In system, 1~5 times of the volume for the water that the amount of addition is added when dissolving cobamamide for step (1) is middle；Preferably water：Isopropyl
Alcohol=1：2.6.
The dissolving of cobamamide subtractive process of the present invention and precipitation state, sampling, which is taken pictures, sees Figure 15.
Raw material used in the preparation method of the present invention is commercially available cobamamide, its P-XRD collection of illustrative plates and A crystal formation cobamamides
Unanimously (Fig. 2), its TG-DSC heat analysis collection of illustrative plates is shown in Fig. 5, and Electronic Speculum (SEM) collection of illustrative plates is shown in Fig. 6, and infared spectrum (IR) is shown in Fig. 7, content
(HPLC) collection of illustrative plates is shown in Fig. 8.A crystal formations cobamamide is can be seen that in 206 ± 2 DEG C of appearance, one feature heat release from TG-DSC collection of illustrative plates
Below peak, also, 100 DEG C, occur sloughing with weightlessness about 12% for unordered water, occur sloughing for orderly water at 206 DEG C, then
Decompose.From SEM as can be seen that differing for the commercially available cobamamide granule size for doing raw material, outward appearance is in irregular sheet.
According to 2015 editions detection methods of Chinese Pharmacopoeia, HPLC detection principal component contents are 99.24%, and total impurities is 0.76%.
The preparation-obtained C crystal form cobamamide of the present invention, its TG-DSC collection of illustrative plates as shown in figure 9, SEM spectrum is shown in Figure 10,
Infared spectrum (IR) is shown in Figure 11, and HPLC collection of illustrative plates is shown in Figure 12.Its DSC pattern analysis results shows a spy occur at 210 ± 2 DEG C
Levy endothermic peak.Sloughing and with weightlessness about 12% for unordered water is occurred below at 100 DEG C.From SEM as can be seen that the present invention is made
Standby cobamamide epigranular, outward appearance is block in rule.According to 2015 editions detection methods of Chinese Pharmacopoeia, HPLC detection principal components
Content is 99.51%, and total impurities is 0.49%.
2015 editions pharmacopoeial requirements of China are at 60 DEG C, when being dried under reduced pressure to constant weight, and cobamamide bulk drug loss on drying must not
More than 12%, experiment confirm under this condition, slough for unordered water (or other solvents), therefore, cobamamide bulk drug can
Unordered water (or the other solvents) content lost should be less than 12%.Needing the problem further illustrated is, cobamamide
Exist with hydrate or solvate forms, whether the C crystal form cobamamide of different unordered water (or other solvents) contents has
Identical powder diffraction featureBy changing drying temperature and time, multiple different unordered water (or other solvents) contents are obtained
C crystal form cobamamide sample, carry out TG-DSC and X- powder diffraction analysis, find the inventive method obtained by C crystal form
Its powder diffraction feature of cobamamide is unrelated with aqueous (or other solvents) amount.For convenience of description, representational three are selected
C crystal form cobamamide sample a, b, the c brief description of the drawings of different water cut (or other solvents) amount.Figure 13 is the TG-DSC of three samples
Collection of illustrative plates is analyzed, Figure 14 is the X- powder diffraction spectrums of three samples.From Figure 13-14 as can be seen that three C crystal form cobamamide samples
Product are below 100 DEG C, weightlessness 5.2%, 7.4% and 9.3%, but occur feature endothermic peak, three samples at 210 ± 2 DEG C respectively
The P-XRD of product is completely the same in terms of 2 θ angles and relative intensity.Research is confirmed above, and cobamamide C prepared by this method is brilliant
Unordered water (or other solvents) not occupy-place in structure cell of type, on its P-XRD characteristic peak without influence.That is, institute of the present invention
The cobamamide crystal formation of preparation, with the change of drying condition, when unordered water content changes, the X- powder of its crystal formation
Diffractive features do not change.
The process for purification of cobamamide of the present invention is to use cosolvent-anti-solvent recrystallization method.So-called cosolvent
Refer to medicine in the addition average value that the solubility of in the mixed solvent is usually each single solvent solubility, it is each in the mixed solvent
Solvent is in a certain ratio, and maximum occurs in the solubility ratio of the medicine solubility in each simple solvent, and this phenomenon is referred to as diving
Molten (cosolvency), this solvent is referred to as cosolvent (cosolvent).
From commercially available A crystal formation cobamamides, found by lot of experiments, insoluble drug cobamamide water-
Acetone system, or water-acetonitrile system can form latent molten, table 3 list cobamamide in both systems solubility (in
2015 editions solubility test methods of state's pharmacopeia).As can be seen from the table, cobamamide is slightly molten in water, insoluble in acetone or second
In nitrile, still, when the volume ratio of boiling system, or water-acetonitrile system reaches 1：When 0.3~0.4, now, cobamamide
Solubility ratio solubility in single aqueous solvent increases more than 10 times.
Using latent molten principle, in a small amount of boiling in the mixed solvent, substantial amounts of cobamamide can be dissolved, in conjunction with
Isopropanol can quickly separate out cobamamide C crystal form as the operation of anti-solvent, and yield is more than 98%, and scale can realize single batch
Refined 50 grams of cobamamide, has reached industrial rank.
By experimental verification, the cobamamide C crystal form of acquisition is dissolved in after above-described cosolvent, using acetone to be anti-molten
During agent, A crystal formations can be separated out, i.e., when using acetone as anti-solvent, C crystal form can be converted into and commercially available product identical A crystal formations, but the process
The change of impurity content does not occur, the conversion of crystal formation is only there occurs.That is, when it is bulk drug that must use A crystal formations, can
The purpose of reduction impurity is reached by the preparation of C crystal form, C crystal form is then converted into A crystal formations.
The inventive method has the advantages that：
Cosolvent-anti-solvent recrystallization method be used for insoluble drug cobamamide it is refined when, can with a small amount of low-toxic solvent,
Gentle reaction condition and the yield more than 98% obtain the cobamamide highly finished product of higher degree.Adenosine prepared by the present invention
Cobalt amine C crystal form epigranular, outward appearance is block in rule, carries out content analysis according to the Chinese Pharmacopoeia detection method of 2015 editions, obtains
The principal component content arrived is more than 99.5%, and total impurities is less than 0.5%, is significantly better than before refining, to solve cobalamin class compound
There is provided selectable effective ways for the law of isomorphism problem of presence.
The cobamamide C crystal form that the present invention is obtained, mono-crystalline structures and X- powder diffraction features have no document report.This hair
Bright prepared cobamamide C crystal form stability study data are shown in Table 4-6, from experimental result as can be seen that within 5 hours, city
Rapid degraded occurs for the A crystal formations for selling product, and own product C crystal form only occurs slight content and declined, it is seen that C crystal form stability is compared with business
The A crystal formations of product are significantly improved；C crystal form cobamamide is also significantly greater than A crystal formation cobamamides to the stability of high temperature, high humidity.
The cobamamide C crystal form that the present invention is obtained, solubility is close with A crystal formations, the cosolvent that A crystal formations are used when dissolving
Condition to C crystal form completely be applicable.Namely preparation condition of the invention can be used for the secondary recrystallization of cobamamide, experiment hair
Existing, secondary crystallization has the effect of further reduction impurity.
Table 1A, C crystal form single crystal data
The X- powder diffractions contrast of table 2 cobamamide, three kinds of crystal formations
Solubility (25 DEG C ± 2 DEG C) of the cobamamide raw material of table 3 in boiling, water-acetonitrile
Brief description of the drawings
Fig. 1 cobamamide hydrate hydrone distribution maps；
Fig. 2 A crystal formation cobamamide X- powder diagrams；
Fig. 3 C crystal form cobamamide single crystal diffraction figures；
Fig. 4 C crystal form cobamamide X- powder diagrams；
Fig. 5 A crystal formation cobamamides TG-DSC schemes；
Fig. 6 A crystal formation cobamamides SEM figures (1000 times of amplification)；
Fig. 7 A crystal formation cobamamides HPLC schemes；
Fig. 8 A crystal formation cobamamides IR schemes；
Fig. 9 C crystal form cobamamides TG-DSC schemes；
Figure 10 C crystal form cobamamides SEM figures (1000 times of amplification)；
Figure 11 C crystal form cobamamides IR schemes；
Figure 12 C crystal form cobamamides HPLC schemes；
The unordered water contents of Figure 13 a are schemed for the TG-DSC of 5.2% C crystal form cobamamide；
The unordered water contents of Figure 13 b are schemed for the TG-DSC of 7.4% C crystal form cobamamide；
The unordered water contents of Figure 13 c are schemed for the TG-DSC of 9.3% C crystal form cobamamide；
The X- powder diagrams of the C crystal form cobamamide of the different unordered water contents of Figure 14；
The dissolving of Figure 15 cobamamide subtractive processes is illustrated with separating out.
Embodiment
With reference to specific embodiment, the present invention is further illustrated.
The present invention prepares and detected that the structure and performance of the instrument used in crystal formation are as follows：
Red light steeps for 5W Kang Shuai illumination LED red lights ball, Xiamen Kang Shuai Electronics Co., Ltd.s.
X-ray single crystal diffraction, which is analyzed, uses Rigaku XtalAB PRO MM007X x ray diffractometer xs, Cu-K α targets,
Data are collected under 150K.
X-ray powder diffraction, which is analyzed, uses Bruker D8Advance type X-ray diffractometers, Cu-K α targets, nickel monochromator,
Operating voltage 40kV, operating current 40mA, step-length 0.02o.
TGA-DSC is analyzed, using TA companies of U.S. Q600 thermogravimetric analyzers, heating rate 10K/min.N2 protection with
20ml/min speed passes through DSC cells.
Sem analysis, cold field emission SEM, German ZEISS companies, model ZEISS SUPRA 55 are differentiated
Rate：1.0nm (15kV)/1.7nm (1kV), accelerating potential：0.1KV-30kV.
IR is analyzed, infrared chromatograph, silent winged scientific and technological (China) Co., Ltd of generation that of match, model 5225Verona Rd
Madison, WI.53711.KBr tablettings.
HPLC is analyzed, Agilent companies, model 1260, and chromatographic column is Intertsustain C18, during detection method is
State's pharmacopeia 2015 edition second, cobamamide.
Experimental example 1：
Under red light lighting condition, room temperature.Commercially available cobamamide 1g is added in 7ml water, stirring is protected for 5 minutes to pasty state
Stirring is held, 3.5ml acetone is added into above-mentioned pastel, in 1 minute, cobamamide is completely dissolved, and obtains settled solution.Upwards
Addition 35ml isopropanols in solution are stated, while stirring 5min, stir speed (S.S.) control is in 400r/min, and now system is muddy.Then,
It is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*10ml) with isopropanol, 25 DEG C are dried in vacuo 7 hours, obtain C
Crystal formation cobamamide 0.982g, yield 98.2%.
The X- powder diffraction spectrums of obtained C crystal form cobamamide are consistent with Fig. 4, and TG-DSC collection of illustrative plates is consistent with Fig. 9.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.5%, and total impurities is
0.5%.
Experimental example 2：
Under red light lighting condition, room temperature.Commercially available cobamamide 10g is added in 60ml water, 10 minutes are stirred to pasty state,
It is kept stirring for, 24ml acetone is added into above-mentioned pastel, in 3 minutes, cobamamide is completely dissolved, and obtains settled solution.To
156ml isopropanols are added in above-mentioned solution, while stirring 5min, stir speed (S.S.) control is in 300r/min, and now system is muddy.So
Afterwards, it is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*20ml) with isopropanol, 25 DEG C are dried in vacuo 7 hours, obtain
To C crystal form cobamamide 9.930g, yield 99.3%.
The X- powder diffraction spectrums of obtained C crystal form cobamamide are consistent with Fig. 4, and TG-DSC collection of illustrative plates is consistent with Fig. 9.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.6%, and total impurities is
0.5%.
Experimental example 3：
Under red light lighting condition, room temperature.Commercially available cobamamide 50g is added in 250ml water, stirring is extremely pasted for 30 minutes
Shape, is kept stirring for, and 75ml acetone is added into above-mentioned pastel, in 10 minutes, cobamamide is completely dissolved, and obtains clarification molten
Liquid.250ml isopropanols are added into above-mentioned solution, while stir 1min, stir speed (S.S.) control in 100r/min, now system by
It is gradually muddy.Then, it is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*50ml) with isopropanol, 25 DEG C of vacuum drying
7 hours, obtain C crystal form cobamamide 49.200g, yield 98.4%.
The crystal size obtained under the conditions of this is up to 0.2mm, and available for the detection of X- single crystal diffractions, its single crystal diffraction collection of illustrative plates is shown in
Fig. 3, the X- powder diffraction spectrums for obtaining C crystal form cobamamide are consistent with Fig. 9, and TG-DSC collection of illustrative plates is consistent with Fig. 5.According to China
The detection method that pharmacopeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.5%, and total impurities is 0.5%.
The C crystal form cobamamide that this experimental example is obtained carries out stability study, and experimental result is shown in Table shown in 4-6, from
Experimental result can be seen that within 5 hours, and rapid degraded occurs for the A crystal formations of commercially available product, and own product C crystal form only occurs slightly
Content decline, it is seen that C crystal form stability is significantly improved compared with the A crystal formations of commercialization；C crystal form cobamamide is to high temperature, high humidity
Stability is also significantly greater than A crystal formation cobamamides.
The cobamamide illumination experiment Comparative result of table 4
Note：Sample is shared into culture dish, and thickness is about 4mm, is placed in 5000Lx light cupboard, is sampled according to experimental program
Afterwards, using second cobamamide detection method detection level of Chinese Pharmacopoeia 2015 edition.
The cobamamide high temperature experimental result of table 5 is contrasted
Note：Sample is shared into culture dish, and thickness is about 4mm, is placed in 60 DEG C of thermostatic drying chambers, is taken according to experimental program
After sample, using second cobamamide detection method detection level of Chinese Pharmacopoeia 2015 edition.
The cobamamide high humidity experimental result of table 6 is contrasted
Note：Sample is shared into culture dish, and thickness is about 4mm, is placed under the conditions of high humidity (relative humidity 75%), according to reality
After the sampling of proved recipe case, using second cobamamide detection method detection level of Chinese Pharmacopoeia 2015 edition.
Experimental example 4：
Under red light lighting condition, room temperature.Commercially available cobamamide 2g is added in 14ml water, 10 minutes are stirred to pasty state,
It is kept stirring for, 6ml acetonitriles is added into above-mentioned pastel, in 3 minutes, cobamamide is completely dissolved, and obtains settled solution.Upwards
Addition 35ml isopropanols in solution are stated, while stirring 5min, stir speed (S.S.) control is in 600r/min, and now system is muddy.Then,
It is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*15ml) with isopropanol, 25 DEG C are dried in vacuo 8 hours, obtain C
Crystal formation cobamamide 1.966g, yield 98.3%.
The X- powder diffraction spectrums of obtained C crystal form cobamamide are consistent with Fig. 4, and TG-DSC collection of illustrative plates is consistent with Fig. 9.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.6%, and total impurities is
0.5%.
Experimental example 5：
Under red light lighting condition, room temperature.Commercially available cobamamide 5g is added in 36ml water, 16 minutes are stirred to pasty state,
It is kept stirring for, 15ml acetonitriles is added into above-mentioned pastel, in 10 minutes, cobamamide is completely dissolved, and obtains settled solution.To
85ml normal propyl alcohols are added in above-mentioned solution, while stirring 5min, stir speed (S.S.) control is in 500r/min, and now system is muddy.So
Afterwards, it is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*25ml) with isopropanol, 25 DEG C are dried in vacuo 7 hours, obtain
To C crystal form cobamamide 4.901g, yield 98.0%.
The X- powder diffraction spectrums of obtained C crystal form cobamamide are consistent with Fig. 4, and TG-DSC collection of illustrative plates is consistent with Fig. 9.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.5%, and total impurities is
0.5%.
Experimental example 6：
Under red light lighting condition, room temperature.C crystal form cobamamide 1g prepared by experimental example 2 is added in 7ml water, stirring
To pasty state, it is kept stirring within 15 minutes, 2.6ml acetonitriles is added into above-mentioned pastel, in 5 minutes, cobamamide is completely dissolved, and is obtained
To settled solution.25ml acetone is added into above-mentioned solution, while stirring 5min, stir speed (S.S.) is controlled in 500r/min, now
System is muddy.Then, it is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*10ml) with acetonitrile, 25 DEG C of vacuum drying
7 hours, obtain A crystal formation cobamamide 0.990g, yield 99.0%.
The X- powder diffraction spectrums of obtained A crystal formations cobamamide are consistent with Fig. 2, and TG-DSC collection of illustrative plates is consistent with Fig. 5.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.6%, and total impurities is
0.5%.
Experimental example 7：
Under red light lighting condition, room temperature.C crystal form cobamamide 1g prepared by experimental example 2 is added in 7ml water, stirring
To pasty state, it is kept stirring within 25 minutes, 3ml acetone is added into above-mentioned pastel, in 5 minutes, cobamamide is completely dissolved, and is obtained
Settled solution.25ml acetone is added into above-mentioned solution, while stirring 5min, stir speed (S.S.) is controlled in 500r/min, now body
System is muddy.Then, it is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*10ml) with acetone, 25 DEG C of vacuum drying 7
Hour, obtain A crystal formation cobamamide 0.991g, yield 99.1%.
The X- powder diffraction spectrums of obtained A crystal formations cobamamide are consistent with Fig. 2, and TG-DSC collection of illustrative plates is consistent with Fig. 5.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.6%, and total impurities is
0.5%.
Experimental example 8：
Under red light lighting condition, room temperature.C crystal form cobamamide 1g prepared by experimental example 2 is added in 7ml water, stirring
To pasty state, it is kept stirring within 25 minutes, 2.6ml acetonitriles is added into above-mentioned pastel, in 5 minutes, cobamamide is completely dissolved, and is obtained
To settled solution.19ml isopropanols are added into above-mentioned solution, while stirring 5min, stir speed (S.S.) is controlled in 500r/min, this
When system it is muddy.Then, it is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*10ml) with isopropanol, 25 DEG C of vacuum
Dry 7 hours, obtain C crystal form cobamamide 0.996g, yield 99.6%.
The X- powder diffraction spectrums of obtained C crystal form cobamamide are consistent with Fig. 4, and TG-DSC collection of illustrative plates is consistent with Fig. 9.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.7%, and total impurities is
0.4%.
Experimental example 9：
Under red light lighting condition, room temperature.C crystal form cobamamide 1g prepared by experimental example 2 is added in 7ml water, stirring
To pasty state, it is kept stirring within 25 minutes, 3ml acetone is added into above-mentioned pastel, in 5 minutes, cobamamide is completely dissolved, and is obtained
Settled solution.18ml isopropanols are added into above-mentioned solution, while stirring 5min, stir speed (S.S.) is controlled in 600r/min, now
System is muddy.Then, it is stored at room temperature overnight, the crystal that suction filtration is separated out, filter cake is washed (3*10ml) with isopropanol, 25 DEG C of vacuum are done
Dry 7 hours, obtain C crystal form cobamamide 0.993g, yield 99.3%.
The X- powder diffraction spectrums of obtained C crystal form cobamamide are consistent with Fig. 4, and TG-DSC collection of illustrative plates is consistent with Fig. 9.According to
The detection method that Chinese Pharmacopoeia is 2015 editions carries out content analysis, and obtained cobamamide content is 99.7%, and total impurities is
0.4%.
Cobamamide crystal formation that the present invention is disclosed and proposed and preparation method and application, those skilled in the art can pass through
Present disclosure is used for reference, the appropriate links such as condition route that change are realized, although the method and technology of preparing of the present invention are by preferable
Examples of implementation are described, and person skilled substantially can be not departing from present invention, in spirit and scope to this paper institutes
The methods and techniques route stated is modified or reconfigured, to realize final technology of preparing.In particular, institute
There is similar replacement and change apparent to those skilled in the art, they are considered as being included in the present invention
In spirit, scope and content.
Claims (7)
1. a kind of cobamamide crystal formation, it is characterized in that mono-crystalline structures are monoclinic system P21；X-ray powder diffraction is in the θ of the angle of diffraction 2
=5.1 ± 0.1,6.3 ± 0.1,7.0 ± 0.1,7.6 ± 0.1,8.0 ± 0.1,8.6 ± 0.1,9.4 ± 0.1,13.2 ± 0.1,
There is feature at 13.8 ± 0.1,15.0 ± 0.1,17.5 ± 0.1,19.1 ± 0.1,21.4 ± 0.1,23.5 ± 0.1,25.3 ± 0.1
Peak.
2., it is characterized in that DSC pattern analysis results are shown, there is feature suction at 210 ± 2 DEG C in crystal formation as claimed in claim 1
Thermal spike.
3. the preparation method of cobamamide crystal formation as claimed in claim 1, it is characterised in that use cosolvent-anti-solvent weight
Crystallisation, step is as follows：
(1) cobamamide crude product is dissolved using cosolvent；
Under red light lighting condition；In room temperature, the water that cobamamide crude product is added to 5-7 times of quality, stirring is extremely pasted for 5-30 minutes
Shape, is kept stirring for, and cosolvent is added into above-mentioned pastel, it was observed that in 10min, cobamamide is completely dissolved, is clarified
Solution；
(2) cobamamide highly finished product are obtained using anti-solvent recrystallization method；
Under red light lighting condition；Anti-solvent is added into above-mentioned solution, suspension is obtained, room temperature is stood overnight, suction filtration, isopropyl
Alcohol is washed, at 20~25 DEG C, is dried in vacuo 3~8 hours, is obtained cobamamide crystal.
4. method as claimed in claim 3, it is characterized in that the cosolvent in described step (1) is water and solvent acetone or second
The mixed solvent of nitrile；Water：Volume ratio=1 of acetone：0.3~0.5 or water：Volume ratio=1 of acetonitrile：0.3~0.5.
5. method as claimed in claim 4, it is characterized in that described cosolvent is water and acetone；Water：Acetone volume ratio is 1：
0.4。
6. method as claimed in claim 3, it is characterized in that the anti-solvent in described step (2) is isopropanol, normal propyl alcohol or
The tert-butyl alcohol, anti-solvent is disposably in addition system, and the amount of addition is the volume of the water added in step (1) during dissolving cobamamide
1~5 times.
7. method as claimed in claim 6, it is characterized in that described anti-solvent is isopropanol；The amount of addition is in step (1)
The water and the volume ratio 1 of isopropanol added during dissolving cobamamide：2.6.
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