CN101985481A - Bis[6-oxygen-(-3-m-nitrobenzene sulfonyl-amber acid-1,4 uniester-4)-]-beta-cyclodextrin, preparation method and use - Google Patents
Bis[6-oxygen-(-3-m-nitrobenzene sulfonyl-amber acid-1,4 uniester-4)-]-beta-cyclodextrin, preparation method and use Download PDFInfo
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- CN101985481A CN101985481A CN 201010254105 CN201010254105A CN101985481A CN 101985481 A CN101985481 A CN 101985481A CN 201010254105 CN201010254105 CN 201010254105 CN 201010254105 A CN201010254105 A CN 201010254105A CN 101985481 A CN101985481 A CN 101985481A
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- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
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Abstract
The invention relates to bis[6-oxygen-(-3-m-nitrobenzene sulfonyl-amber acid-1,4 uniester-4)-]-beta-cyclodextrin, preparation, and application as a chiral selector to high performance liquid chromatography (HPLC). The bis[6-oxygen-(-3-m-nitrobenzene sulfonyl-amber acid-1,4 uniester-4)-]-beta-cyclodextrin, the preparation and the application have the advantages that: 1, the bis[6-oxygen-(-3-m-nitrobenzene sulfonyl-amber acid-1,4 uniester-4)-]-beta-cyclodextrin is formed by disubstitution of hydrogen in hydroxyl connected to a carbon atom at position-6 in A and D rings in beta-cyclodextrin with maleic anhydride, addition of m-nitrobenzene sulfonyl groups onto double bonds in maleic and addition of the m-nitrobenzene sulfonyl groups onto the carbon atom at position-3 in a succinic acid, and has the molecular formula of C62H84O49N2S2; and 2, a novel method for obtaining purified D(+)-ticarcillin and L(-)-ticarcillin from a despinner of a medicament can be established by a preparative chiral HPLC column of the derivative, single antimer products of the two substances of alpha-phenylethyl alcohol and 1-phenylpropanol can be obtained, and the novel method for separately testing the single antimer content of the chiral substances of ticarcillin, alpha-phenylethyl alcohol and 1-phenylpropanol can be established by an analytic chiral HPLC column.
Description
Technical field
The present invention relates to a kind of chiral selector---two [6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1, the 4-monoesters-4-)-]-beta-cyclodextrin and preparation method and purposes.
Background technology
The chiral drug separation and purification is one of focus of new drug development, is subjected to extensive concern.It is a kind of effective means of preparation chiral drug single enantiomer that HPLC splits, and in numerous chiral stationary phases, beta-cyclodextrin (CD) and derivative thereof are subjected to the favor of numerous researchers.Chiral drug (chiraldrug) is meant in the molecular structure of medicine and has the chirality factor, the pharmacological action of medicine be by and intravital macromole between strict chiral recognition and coupling realize.Constituting proteinic amino acid all is L-amino acid, and the monose of composition polysaccharide and nucleic acid all is D-monose, and after the racemic modification of medicine was introduced in the body, its enantiomorph molecule was handled with two different molecules by the protein that has chirality in the body, enzyme and acceptor.Because the chirality feature of human body, in many cases, there is significant difference in a pair of enantiomorph of chiral drug pharmacologically active, metabolic process, metabolic rate and toxicity etc. in vivo, also there are differences at aspects such as absorption, distribution and drainages, and also mutual conversion may take place between the enantiomorph.
The chirality drug enantiomer is carried out effective separation and purification, more effectively guarantee drug safety, improve social medical level, become important work of researcher.External activity as two enantiomorphs of beta-receptor blockader thing Proprasylyte differs 98 times; L-DOPA (L-dopa) is the Parkinsonian medicine of treatment, and the compound that therapeutic activity is really arranged is L-Dopamine HCL (L-dopamine), must take the levo form DOPA of enantiomer-pure, could be converted into the L-Dopamine HCL and onset by the specificity enzyme catalysis in human body.If take raceme, dextrorotatory form can be built up in vivo, can be by intravital enzymes metabolism, thus may the health of human body be worked the mischief.In current 1327 chemical synthetic drugs commonly used, 528 of chiral drugs wherein have only 61 with what single enantiomer went on the market, and major part is still sold with the racemic modification form.This situation has caused the potential threat to drug safety undoubtedly, has proposed more urgent requirement also for research and development single enantiomer new drug.The clinical chiral drug of taking single enantiomer can reduce drug use dosage, reduces patient's metabolism burden, improves the amplitude of dosage and widens purposes, to pharmacokinetics better control can be arranged.Amplitude is wideer when dosage setting, reacts less; It is more confident when dosage is selected.For pharmacy corporation, produce the single enantiomer chiral drug and can save resource, reduce the cost that clinical pharmacology and pharmacokinetics research are carried out in the new drug listing, reduce trash discharge, reduce pollution to environment.
Obtain optically pure enantiomorph and be not easy, the chipal compounds that obtains single enantiomer has three kinds of methods: chiral source synthesis method, asymmetric synthesis method, racemic modification Split Method.The racemic modification Split Method is the method that the racemic modification compound is split as pure enantiomeric compounds under the effect of chiral selector (chiral auxiliary(reagent)), is divided into chemical resolution method, enzyme or microbial method, chromatogram Split Method again.Wherein the chromatogram Split Method demonstrates superiority, adopts chromatography separation and purification chiral drug ranolazine, and single enantiomer prepares purity and reaches more than 98%; The omeprazole split result is that S-omeprazole purity is up to 98.71%, and R-omeprazole purity is up to 95.47%.Liquid chromatography just is included in the racemic modification Split Method.Liquid chromatography is considered to measure enantiomeric purity and the best method of separating preparation optical purity single enantiomer.Its wide accommodation, phenomenons such as isolate change of configuration or biological activity be destroyed can not take place in the operational condition gentleness, can obtain high optically pure two kinds of enantiomers simultaneously.
People such as Cramer have found that at first beta-cyclodextrin (CD) has recognition reaction preferably to optical isomer.Behind β-CD derivatize, can near binding site, construct the solid geometry relation, form special chirality site; Carry out three-dimensional space and modify, enlarge in conjunction with cavity or provide the space of geometry in particular, the coupling of adaptation to be arranged with guest molecule; But also can introduce the action site that other can play chiral recognition.β-CD class chiral stationary phase has obtained using widely in chiral separation analysis, and the main effect that improves Chiral Separation is: (1) forms the inclusion complex of different structure and character respectively in conjunction with a pair of enantiomorph of separated solute; (2) hydroxyl of β-CD mainly is C-2, C-3 position secondary hydroxyl, or CD modification group and separated enantiomorph form the complex body of different structure, character with hydrogen bond; (3) the closely filling of appropriateness in the cavity, the stable inclusion complex that forms of short distance model ylid bloom action power.Natural β-CD can improve separation efficiency through derivatize, is applicable to multiple chromatogram clastotype, is subjected to the favor of a lot of researchers.As Xin Wang etc. with seven (6-azido-s-6-deoxidation-2, the 3-2-O-phenylcarbamoyl) β-CD bonded chiral stationary phase fills semipreparative column and has separated β-blocking drugs Proprasylyte with high performance liquid chromatography, and separates in batches with the simulated moving bed chromatography method on this basis and prepared the Proprasylyte enantiomorph; XinWang etc. are stationary phase with full carbanilino β-CD bonded silica gel; in four enantiomorphs of nadolol three have been separated with high performance liquid chromatography; and separated active (the RSR)-nadolol of tool in four enantiomorphs fully, adopt five-region simulated moving bed chromatography in the nadolol enantiomorph, to separate on this basis and obtained highly purified target enantiomorph (RSR)-nadolol.Preparing chiral drug with this type of chiral selector has significant separation and purification effect, but kind is many not enough, also need continue to research and develop applicable surface widely, this type of chiral selector that selectivity is higher prepares the chiral drug of more single enantiomer.
Summary of the invention
The object of the present invention is to provide chiral selector---two [6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1, the 4-monoesters-4-)-]-beta-cyclodextrin and preparation method and purposes, it contains m-nitro alkylsulfonyl (NO as chiral selector
2C
6H
5SO
2) and the beta-cyclodextrin derivative of two kinds of functional groups of succinate (OOCCHCHCOOH), this chiral selector can directly be coated with stain or be bonded on the silica-gel bead or on the quartzy tube wall and be prepared into chiral stationary phase.
For realizing purpose of the present invention; on the one hand; the invention provides a kind of chiral selector of beta-cyclodextrin class; this chiral selector is by behind the hydroxyl hydrogen on 6 carbon on the control reaction conditions two replacement of maleic anhydride beta-cyclodextrin A, the D ring; add on two keys of maleic with the m-nitro alkylsulfonyl again, and the addition of m-nitro alkylsulfonyl is on 3 carbon of Succinic Acid and form.Its molecular formula is: C
62H
84O
49N
2S
2Finally obtain the novel β-CDYan Shengwu of substitution value unanimity: two [6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1, the 4-monoesters-4-)-]-β-CD (abbreviation: β-CD-M
2).Characterize with means such as ultraviolet, infrared, mass spectrum, nuclear-magnetism, XPS, this derivant structure is confirmed.Two [6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-structure of beta-cyclodextrin is:
On the other hand, the present invention also provides the synthetic method for preparing above-mentioned beta-cyclodextrins chiral selecting agent, two [6-oxygen-(3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-preparation method of beta-cyclodextrin, beta-cyclodextrin abbreviates β-CD as, it is characterized in that carrying out according to the following steps: (1), elder generation obtain m-nitrobenzene sulfonyl chloride with 1mol oil of mirbane and 80-150 ℃ of reacting by heating of 1-10mol chlorsulfonic acid; (2), again use 2-30mol MALEIC ANHYDRIDE and 1mol β-CD, 60-120 ℃ of reacting by heating obtains two (6-oxygen-butene dioic acid monoesters)-β-CD, is called for short β-CD-A
2(3), use 1mol β-CD-A then
2With 50-120 ℃ of reacting by heating of 2-30mol m-nitrobenzene sulfonyl chloride obtain two [6-oxygen-(3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-β-CD, be called for short β-CD-M
2Reaction formula is as follows:
The compound of the beta-cyclodextrin structure that the present invention makes, can be by directly being coated with stain or being bonded on the carrier such as silica-gel bead or on the quartzy tube wall, be prepared into chiral stationary phase, also can be used as in the chiral additives adding moving phase and carry out chiral material separation, purifying and new qualitative-and-quantitative method is provided.Chiral selector of the present invention is suitable for high performance liquid chromatography (HPLC), gas-chromatography (GC), HPCE instruments such as (HPCE) as chiral stationary phase.Prepared stationary phase chiral recognition ability is strong, good stability, can be made into analysis mode and preparation type chiral column material to chiral material separate, analysis or purifying prepare single enantiomer.
Compared with prior art, the present invention has following effect: 1, can set up D (+)-ticarcillin of obtaining purifying from the raceme of medicine and L (-)-ticarcillin and methyl phenyl carbinol, two pure product of single enantiomer of 1-phenylpropyl alcohol with preparation type chirality HPLC post.2, can set up the novel method of separation determination ticarcillin, methyl phenyl carbinol, phenylpropyl alcohol single enantiomer content with analysis mode chirality HPLC post, look into new reference retrieval center through Chinese Academy of Sciences's Wuhan science and technology and provide the novelty assessment report demonstration, not see bibliographical information.
Instrument and reagent that the present invention is used are respectively:
PE labmda Bio35 ultraviolet-visible pectrophotometer (U.S.)
Nicolet NEXUS-470 Fourier transformation infrared spectrometer (U.S.)
Varian prostar 210 high performance liquid chromatography (U.S.)
LABCONCO FreeZone Freeze Drying Equipment (U.S.)
API3200 liquid chromatography tandom mass spectrometer (U.S.)
Tecnai G2 20S-TWIN transmission electron microscope (Czech)
JSM-6700F scanning electronic microscope (Japan)
VG Multilab 2000 photoelectron spectrographs (U.S.)
AVANCEIII nuclear magnetic resonance spectrometer (BRUKER)
β-CD Chemical Reagent Co., Ltd., Sinopharm Group
Maleic anhydride Chemical Reagent Co., Ltd., Sinopharm Group
Oil of mirbane Chemical Reagent Co., Ltd., Sinopharm Group
The new chemical reagent of Kingsoft, Phenylsulfonic acid Shanghai booth factory
Two [6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-sign of beta-cyclodextrin:
1, UV scanning collection of illustrative plates
2, infrared scan collection of illustrative plates
Comparison diagram 3 and Fig. 2 are at β-CD-A of Fig. 3
2See obviously on the infrared scan figure that the β-CD infrared scan figure than Fig. 2 has more 1726cm
-1C=O (s, 1726cm
-1) peak; Visible 3096cm on Fig. 4 m-nitrobenzene sulfonyl chloride infrared scan figure
-1(w, C-H), 1534cm
-1, 1352cm
-1(w, NO
2), 1603cm
-1, 755cm
-1(w, phenyl ring); Comparison diagram 5 and Fig. 3 are at β-CD-M of Fig. 5
2On the infrared scan figure than β-CD-A of Fig. 3
2Infrared scan figure has had more 1533cm
-1, 1351cm
-1(w, NO
2), 1611cm
-1, 734cm
-1The peak of (w, phenyl ring) shows that m-nitrobenzene sulfonyl chloride has been bonded to β-CD-A
2On.
3, x-ray photoelectron spectroscopy
Scan β-CD, MALEIC ANHYDRIDE, β-CD-A respectively
2, β-CD-M
2X-ray photoelectron spectroscopy, it is as shown below to obtain the result, the full spectrum of each compound is seen Fig. 6 a, Fig. 6 b, Fig. 6 c, Fig. 6 d, each compound collection of illustrative plates is seen Fig. 7 a, Fig. 7 b, Fig. 8 a, Fig. 8 b, Fig. 9 a, Fig. 9 b, Figure 10 a, Figure 10 b, Figure 10 c, Figure 10 d.
By Fig. 6 a, Fig. 6 b, Fig. 6 c, Fig. 6 d as seen, the C1s bound energy is 284.64eV among β-CD, and the O1s bound energy is 530.33eV; MALEIC ANHYDRIDE C1s bound energy is 284.59eV, and it is the bimodal of 538.4eV and 532.25eV that bound energy has appearred in O1s, has two kinds of of bondings to conform in MALEIC ANHYDRIDE with oxygen; β-CD-A
2Middle C1s bound energy is 284.6eV, and the O1s bound energy is 531.17eV; β-CD-M
2Middle C1s bound energy is 284.59eV, and the O1s bound energy is 530.23eV, and the N1s bound energy is 399.82eV, and the S2p bound energy is 166.6eV.At β-CD-M
2Obviously see on the XPS spectrum figure than β-CD-A
2Have more the XPS spectrum band of N, S, illustrate that the group that contains N, S element has been bonded to β-CD-A
2On.
4, nuclear magnetic spectrum
To reaction raw materials β-CD, β-CD-A
2With product β-CD-M
2Adopt the nucleus magnetic resonance means to carry out structure and identify, comprising:
13C NMR,
1H NMR and HMBC (
1The heteronuclear multikey correlation test that H detects), HSQC (
1The single quantum coherent test of the heteronuclear that H detects) (numbering of H is consistent with the numbering of C).
Figure 11 is β-CD's
13C NMR figure can obtain C1:101.83 C2:73.07 C3:72.06 C4:81.12 C5:71.81 C6:60.28.Figure 12 is β-CD's
1H NMR figure, δ 4.989 is that (hydrogen OH), δ 2.148, δ 3.484-3.591 and δ 3.771-3.912 are C6 (CH to the C6 hydroxyl as can be known
2-) hydrogen on the methylene radical, δ 4.680 is the hydrogen on the CD ring C1 position, δ 3.5-4.0 is C2, C3, C4, the hydrogen on the C5, Figure 14 β-CD-A
2 1H NMR spectrum is than Fig. 2 .12 β-CD
1H NMR spectrum δ 4.989 (hydrogen on the OH) obviously reduces, illustrate C6 among β-CD-the OH key is at β-CD-A
2In formed ehter bond, and new peak δ 6.0-6.5 is arranged is the hydrogen on the two keys of C14=C15 on the side chain.
Figure 13 is β-CD-A
2 13C NMR figure, can obtain C1:101.88 C2:73.15 C3:72.05 C4:81.10 C5:71.83 C6:60.19 64.20,-COOH (C16): 169.54,-C=C-(C14, C15): 131.28, C=O (C13): 166.26, by above data as can be seen, after-C=C-, C=O and reaction produce-groups such as COOH all have embodiment in the drawings, can judge that the maleic anhydride substitution reaction occurs in the C6 position from the comparison of the C6 of C6 and Figure 11 β-CD.
The directly related figure of Figure 15 C-H can find out further that C6:30.35 is directly related with δ 2.142, and C1 is directly related with the hydrogen on the δ 4.680, the hydrogen of δ 3.5-4.0 and C2, and C 3, C4, C5 is directly related.The hydrogen of δ 6.0-6.5 and C14, C15 is directly related; The long-range correlogram of Figure 16 C-H can find out that hydrogen δ 6.0-6.5 is long-range relevant with C1 on two keys, also is long-range relevant with C5.With C14, C15 also is long-range each other relevant.
In sum, raw material 2 (β-CD-A
2) structure is:
Figure 17 is β-CD-M
2 13C NMR nuclear-magnetism figure, cause is 400,000,000 nuclear-magnetisms, instrumental sensitivity is lower, not obvious with the chemical shift of last corresponding 6 C of β-CD, but the C13-C16 on phenyl ring Ar (C7-C12) and the side chain is clearly: C12:134.41, C8:133.10, C10:132.42, C7:130.31, C9, C11:123.97 long-chain C16:166.41; C13:150.16; C14:34.03; C15:40.34;
Figure 18 is β-CD-M
2 1H NMR nuclear-magnetism figure, in conjunction with the directly related figure of two-dimentional nuclear-magnetism, promptly HMBC (
1The heteronuclear multikey correlation test that H detects), Figure 19 can obtain: H has 4 groups of peaks on the phenyl ring, and the H directly related with C7 is δ 8.5, and the H directly related with C9 is that δ 8.3, the H directly related with C11 are that δ 8.1, the H directly related with C10 are δ 7.7.(the H δ 4.989 OH) is still stronger, and that directly related with δ 2.5 is C14:34.03 for branched hydroxy group; C15:40.34;
As shown in Figure 20: because the chair form structure of cyclodextrin and the influence of substituted radical, the C1 on the cyclodextrin ring is long-range relevant with H5, H4, and C2 is long-range relevant with H5.Side chain also has some long-range relevant points.As the hydrogen on C13 and H15, the OH; Hydrogen on C14 and the OH; C7 and H9, H10 on the aromatic ring, H11 are long-range relevant, and C9 is long-range relevant with H7, H11, and C10 is long-range relevant with H7, C11 is long-range relevant with H7, H9, C8 is long-range relevant with H10, H11, and C12 is long-range relevant with H10, H9, and C13 and H7, H9, H10, H11 are long-range relevant.
5, mass spectral characteristi
Adopt and partly prepare HPLC β-CD-M
2Carry out separation and purification, obtain purified product after the lyophilize, carry out mass spectral characteristi.Partly prepare HPLC separation chromatography condition: stationary phase: the C18 semipreparative column; Moving phase: 5%-10% acetonitrile-95%-90% water; Flow velocity: 2mlmin
-1Column temperature: 30 ℃; Detect wavelength: 254nm; Sample concentration: 5 * 10
-3MolL
-1The aqueous solution; Sample size 200 μ l.Mass spectrum experiment condition: curtain Gas:10mlmin
-1IonSpray Voltage:5500V; Ion Source Gas:110mlmin
-1Declustering Potential:70mv.
Can obtain following information by mass spectrum: 1699 are molecular ion peak, are β-CD-M
2(1704) lose result behind the proton.Be β-CD-M below
2Possible fracture mode is if by obtaining 516 fragment after (1) two 258 (254.4 fragment ion peaks are arranged) fracture of fracture mode, have corresponding 515.1 fragment ion peak in the mass spectrum; If press in fracture mode (2) mass spectrum not and 244 or the close fragment of twice mass-to-charge ratio 488, hence one can see that, and the reasonable fracture mode of band aromatic ring group is (1), and promptly the m-nitro alkylsulfonyl answers addition in the C14 position, rather than the C15 position.
Be β-CD-M below
2The third fracture mode:
By β-CD-M
2The third fission process as can be known, two replacements occur on 1,4 ring of β-CD, 1354 (among the figure 1354.2), 350 (be 348.2 among the figure, losing a hydroxyl OH again is 333, is 333.8 among the figure, and this two peak is all very strong) can be found foundation in mass spectrum.If 1,3 ring replaces the fragment 673 (do not have this fragment among the figure, also do not have the fragment of another part 1031) that then should have 4 rings to link to each other, if 1,2 ring replaces the fragment 836 (not having this fragment among the figure) that then should have 5 rings to link to each other.
So structure is
The beta-cyclodextrin derivative that the present invention makes, can be by directly being coated with stain or being bonded on the carrier such as silica-gel bead or on the quartzy tube wall, be prepared into chiral stationary phase, also can be used as in the chiral additives adding moving phase and carry out chiral material separation, purifying and new qualitative-and-quantitative method is provided.Chiral selector of the present invention is suitable for high performance liquid chromatography (HPLC), gas-chromatography (GC), HPCE instruments such as (HPCE) as chiral stationary phase.Prepared stationary phase chiral recognition ability is strong, good stability, can be made into analysis mode and preparation type chiral column material to chiral material separate, analysis or purifying prepare single enantiomer.
Compared with prior art, the present invention has following effect: 1, can set up D (+)-ticarcillin that obtains purifying from the raceme of medicine and the novel method of L (-)-ticarcillin with preparation type chirality HPLC post; Also can obtain the pure product of methyl phenyl carbinol, two kinds of material single enantiomers of 1-phenylpropyl alcohol.2, can set up the novel method of separation determination ticarcillin, methyl phenyl carbinol, 1-phenylpropyl alcohol single enantiomer content with analysis mode chirality HPLC post, look into new reference retrieval center through Chinese Academy of Sciences's Wuhan science and technology and provide the novelty assessment report demonstration, not see bibliographical information.
Description of drawings
Fig. 1 is the UV scanning collection of illustrative plates, a: β-CD; B: β-CD-A
2C: m-nitrobenzene sulfonyl chloride; D: oil of mirbane; E: β-CD-M
2
Fig. 2 is β-CD infrared scan figure.
Fig. 3 is β-CD-A
2Infrared scan figure.
Fig. 4 is m-nitrobenzene sulfonyl chloride infrared scan figure.
Fig. 5 is β-CD-M
2Infrared scan figure.
Fig. 6 a is the x-ray photoelectron spectroscopy (full spectrum, VG Multilab 2000) of β-CD.
Fig. 6 b is the x-ray photoelectron spectroscopy (full spectrum, VG Multilab2000) of MALEIC ANHYDRIDE.
Fig. 6 c is β-CD-A
2X-ray photoelectron spectroscopy (full spectrum, VG Multilab 2000).
Fig. 6 d is β-CD-M
2X-ray photoelectron spectroscopy (full spectrum, VG Multilab 2000).
Fig. 7 a is that β-CD X-ray photoelectric can C spectrum (VG Multilab 2000).
Fig. 7 b is that β-CD X-ray photoelectric can O spectrum (VG Multilab 2000).
Fig. 8 a is that MALEIC ANHYDRIDE X-ray photoelectric can C spectrum (VG Multilab 2000).
Fig. 8 b is that MALEIC ANHYDRIDE X-ray photoelectric can O spectrum (VG Multilab 2000).
Fig. 9 a is β-CD-A
2X-ray photoelectric can C spectrum (VG Multilab 2000).
Fig. 9 b is β-CD-A
2X-ray photoelectric can O spectrum (VG Multilab 2000).
Figure 10 a is β-CD-M
2X-ray photoelectric can C spectrum (VG Multilab 2000).
Figure 10 b is β-CD-M
2X-ray photoelectric can O spectrum (VG Multilab 2000).
Figure 10 c is β-CD-M
2X-ray photoelectric can N spectrum (VG Multilab 2000).
Figure 10 d is β-CD-M
2X-ray photoelectric can S spectrum (VG Multilab 2000).
Figure 11 is β-CD
13C nuclear-magnetism figure (AVANCE
IIINMR D
2O 400MH
Z).
Figure 12 is β-CD
1H nuclear-magnetism figure (AVANCE
IIINMR D
2O 400MH
Z).
Figure 13 is β-CD-A
2 13C nuclear-magnetism figure (AVANCE
IIINMR D
2O 400MH
Z).
Figure 14 is β-CD-A
2 1H nuclear-magnetism figure (AVANCE
IIINMR D
2O 400MH
Z).
Figure 15 is β-CD-A
2HSQC nuclear-magnetism figure (AVANCE
IIINMR D
2O 400MH
Z).
Figure 16 is β-CD-A
2HMBC nuclear-magnetism figure (AVANCE
IIINMR D
2O 400MH
Z).
Figure 17 is β-CD-M
2 13C nuclear-magnetism figure (AVANCE
IIINMR DMSO 400MH
Z).
Figure 18 is β-CD-M
2 1H nuclear-magnetism figure (AVANCE
IIINMR D
2O 400MH
Z).
Figure 19 is β-CD-M
2HSQC nuclear-magnetism figure (AVANCE
IIINMR DMSO 400MH
Z).
Figure 20 is β-CD-M
2HMBC nuclear-magnetism figure (AVANCE
IIINMR DMSO 400MH
Z).
Figure 21 is β-CD-M
2Mass spectrum.
Figure 22 a is a silica bead A stationary phase transmission electron microscope scintigram.
Figure 22 b is β-CD-M
2Bonding silica bead A stationary phase transmission electron microscope scintigram.
Figure 22 c is a silica bead B stationary phase transmission electron microscope scintigram.
Figure 22 d is β-CD-M
2Bonding silica bead B stationary phase transmission electron microscope scintigram.
Figure 23 a is a silica bead A5000 times sem photograph.
Figure 23 b is a silica bead A10000 times sem photograph.
Figure 23 c is a silica bead A50000 times sem photograph.
Figure 23 d is β-CD-M
25000 times of sem photographs of bonding silica bead A.
Figure 23 e is β-CD-M
210000 times of sem photographs of bonding silica bead A.
Figure 23 f is β-CD-M
250000 times of sem photographs of bonding silica bead A.
Figure 24 a is a silica bead B5000 times sem photograph.
Figure 24 b is a silica bead B10000 times sem photograph.
Figure 24 c is a silica bead B50000 times sem photograph.
Figure 24 d is β-CD-M
25000 times of sem photographs of bonding silica bead B.
Figure 24 e is β-CD-M
210000 times of sem photographs of bonding silica bead B.
Figure 24 f is β-CD-M
250000 times of sem photographs of bonding silica bead B.
Figure 25 a is x-ray photoelectron spectroscopy (full spectrum) figure of silica bead A.
Figure 25 b is x-ray photoelectron spectroscopy (full spectrum) figure of silica bead B.
Figure 25 c is β-CD-M
2The x-ray photoelectron spectroscopy of bonding silica bead A (full spectrum) figure.
Figure 25 d is β-CD-M
2The x-ray photoelectron spectroscopy of bonding silica bead B (full spectrum) figure.
Figure 26 a is a silica bead A oxygen spectrum XPS figure spectrogram.
Figure 26 b is a silica bead A silicon spectrum XPS figure spectrogram.
Figure 27 a is a silica bead B oxygen spectrum XPS figure spectrogram.
Figure 27 b is a silica bead B silicon spectrum XPS figure spectrogram.
Figure 28 a is β-CD-M
2Bonding silica bead A carbon spectrum XPS figure spectrogram.
Figure 28 b is β-CD-M
2Bonding silica bead A oxygen spectrum XPS figure spectrogram.
Figure 28 c is β-CD-M
2Bonding silica bead A nitrogen spectrum XPS figure spectrogram.
Figure 28 d is β-CD-M
2Bonding silica bead A sulphur spectrum XPS figure spectrogram.
Figure 28 e is β-CD-M
2Bonding silica bead A silicon spectrum XPS figure spectrogram.
Figure 29 a is β-CD-M
2Bonding silica bead B carbon spectrum XPS figure spectrogram.
Figure 29 b is β-CD-M
2Bonding silica bead B oxygen spectrum XPS figure spectrogram.
Figure 29 c is β-CD-M
2Bonding silica bead B nitrogen spectrum XPS figure spectrogram.
Figure 29 d is β-CD-M
2Bonding silica bead B sulphur spectrum XPS figure spectrogram.
Figure 29 e is β-CD-M
2Bonding silica bead B silicon spectrum XPS figure spectrogram.
Figure 30 is that half preparative chromatography is separated preparation ticarcillin single enantiomer HPLC figure.
31a is (-) forward figure that HPLC ticarcillin single enantiomer is collected the checking of liquid purity.
Figure 31 b is (+) postpeak figure that HPLC ticarcillin single enantiomer is collected the checking of liquid purity.
Figure 32 a is that HPLC ticarcillin single enantiomer is collected liquid purity checking (acetonitrile system) forward figure.
Figure 32 b is that HPLC ticarcillin single enantiomer is collected liquid purity checking (acetonitrile system) back cutting edge of a knife or a sword figure.
Figure 33 a is that ticarcillin separates linear (-) preceding peak area-concentration curve.
Figure 33 b is that ticarcillin separates linear (-) leading peak peak height-concentration curve.
Figure 33 c is that ticarcillin separates linear (+) back peak area-concentration curve.
Figure 33 a is that ticarcillin separates linear (+) leading peak peak height-concentration curve.
Figure 34 is methyl phenyl carbinol chromatographic fractionation figure (moving phase is 99% normal hexane-1% dehydrated alcohol).
Figure 35 is methyl phenyl carbinol chromatographic fractionation figure (column temperature is 27 ℃).
Figure 36 is a 1-phenylpropyl alcohol chromatographic fractionation figure (moving phase: 99% normal hexane-1% methyl alcohol).
Figure 37 is 1-phenylpropyl alcohol chromatographic fractionation figure (column temperature: 30 ℃).
Figure 38 a is flow velocity: 1.0mLmin
-11-phenylpropyl alcohol chromatographic fractionation figure.
Figure 38 b is flow velocity: 0.05mLmin
-11-phenylpropyl alcohol chromatographic fractionation figure.
Figure 39 is the amygdalic acid chromatographic fractionation figure.
Embodiment
Two [6-oxygen-(3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-preparation of beta-cyclodextrin: beta-cyclodextrin abbreviates β-CD as, carries out according to the following steps: (1), obtain m-nitrobenzene sulfonyl chloride with 123.2g oil of mirbane and 100 ℃ of reacting by heating of 350.3g chlorsulfonic acid earlier; (2), again use 22.0g MALEIC ANHYDRIDE and 17.0g β-CD, 80 ℃ of reacting by heating obtain two (6-oxygen-butene dioic acid monoesters)-β-CD, are called for short β-CD-A
2(3), use 1mol β-CD-A then
2With 60 ℃ of reacting by heating of 20mol m-nitrobenzene sulfonyl chloride obtain two [6-oxygen-(3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-β-CD, be called for short β-CD-M
2With the β-CD-M that makes
2Be used for following embodiment.
Embodiment one: the HPLC novel preparation method of ticarcillin single enantiomer
Present embodiment relates to D (+)-ticarcillin that obtains purifying from the raceme of this medicine and the method for L (-)-ticarcillin.Work documents such as the compartment analysis mensuration of this medicine single enantiomer are less, and research work is to carry out in the mode of raceme mostly, and this embodiment has big novelty.
Ticarcillin is a kind of semisynthetic antibiotics, chemistry 6-(2-carboxyl-2-thiene-3-yl-acetyl) aminopenicillanic acid by name, and clinical the use is double sodium salt, C
15H
14N
2Na
2O
6S
2, molecular weight 428.40, for white to buff powder, soluble in water, pH value of water solution is 6-8.Ticarcillin is wide spectrum, efficient anti-pseudomonas aeruginosa penicillin, but not anti-β-Nei Xiananmei, does not orally absorb quiet notes or intramuscular administration.Be mainly used in and treat burn infection, meningitis, osteomyelitis, respiratory tract, urinary tract infection and postoperative prevention etc.Normal and beta-lactamase inhibitor clavulanic acid (clavulanic acid) is made compound preparation, and trade(brand)name " timentin " is also done " Ticarcillin/Clavulanate Acid ".Its enantiomer-specific structure is as follows: (* is a chiral carbon).
The ticarcillin structure
Li Yin-Hua etc. have measured ticarcillin in blood plasma and the urine with HPLC; Watson Ian D etc. has measured clavulanic acid and the ticarcillin in the blood plasma respectively with HPLC; Rice Patrick D
[43]Deng measured ticarcillin with the method for HPLC and the coupling of optically-active detection technique; Kelly James W
[44]Do the isomer of HPLC stationary phase Deng respectively with γ-Huan Hujing and ion-exchange vinyl xylene, ethyl vinyl benzene/divinyl benzene copolymer, poly-(vinylbenzene-Vinylstyrene), carried out Separation Research the β-Nei Xiananleikangshengsu that comprises ticarcillin.Hoogmartens J etc. have separated the side chain isomer of penicillin with HPLC: phenethicillin, propicillin, Rixapen, Gepcillin, ticarcillin, peace benzyl XiLin, amoxycilline Trihydrate bp and azidocillin; Usefulness tlc separation determinations such as Hendrickx S comprise 18 kinds of penicillin of ticarcillin; Usefulness gradient elution liquid phase chromatography such as Annesley simultaneously separation determination comprise 13 kinds of microbiotic of ticarcillin in the blood plasma; Haginaka etc. utilize pre-column with product to be tested and 1,2,4-tripyrrole and mercury chloride carry out the method for derivative reaction, have measured Gepcillin, ticarcillin and sulbenicillin in blood plasma and the urine with HPLC; Haginaka Jun etc. separate fluoroscopic examination with HPLC and have measured clavulanic acid in human plasma and the urine; Degraded HPLC method has been measured ticarcillin behind the usefulness clorox posts such as Haginaka Jun; Nakagawa Terumichi etc. have measured clavulanic acid and ticarcillin in human plasma and the urine respectively with HPLC, have carried out the research of pharmacokinetics; Mopper Barry with liquid chromatography for measuring the ticarcillin in the injection type medicine; Tyczkowska etc. have measured ticarcillin in dog and the horse blood plasma with paired ion chromatography; Wright Jennifer C. etc. has measured ticarcillin and the clavulanic acid in the blood plasma with LC; Usefulness HPLC such as Horimoto Shingo and bromofom are done the atmospheric chemical ionization mass spectrometry of ionization accelerated solvent and have been measured 8 kinds of microbiotic and the 13 kinds of cynnematins that comprise ticarcillin; Pajchel Genowefa etc. have measured ticarcillin and clavulanic acid in the Ticarcillin/Clavulanate Acid (composite antibiotic that ticarcillin and clavulanic acid are formed) simultaneously with micellar electrokinetic chromatography; LiChonghua etc. have measured rabbit blood plasma and ticarcillin and the clavulanic acid organized in the body fluid simultaneously with liquid phase chromatography; Liang Wenqing etc. with the micellar electrokinetic capillary chromatography separation determination amoxycilline Trihydrate bp, Ampicillin Trihydrate, three kinds of penicillin of ticarcillin and clavulanic acid, two kinds of beta-lactamase inhibitors of Sulbactam; Ashraf-Khorassani M etc. with multidimensional liquid chromatography/electronic spraying MALDI-MS separation determination amoxycilline Trihydrate bp, clavulanic acid and the ticarcillin in three kinds of matrix.
Present embodiment provides the novel method of a kind of preparation purifying D (+)-ticarcillin and L (-)-ticarcillin; this method is with the β-CDYan Shengwu of above-mentioned preparation: two [6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-β-CD (is called for short: β-CD-M
2) silica bead of two kinds of specifications of bonding constructs two kinds of HPLC stationary phase, characterizes with transmission electron microscope TEM, scanning electronic microscope SEM, x-ray photoelectron spectroscopy XPS, proves β-CD-M
2Successfully be bonded on the silica bead.
Adopt four kinds of moving phases on stationary phase analytical column of the same race, ticarcillin all reaches baseline separation under suitable proportioning, has set up the HPLC method for splitting of four kinds of corresponding enantiomorphs.At β-CD-M
2Carry out ticarcillin single enantiomer preparation in the stationary phase preparative chromatography post of bonding, the checking result of Fractional Collections liquid is shown, obtained the one-component of ticarcillin enantiomorph, i.e. D (+) type and L (-) type enantiomorph solid salt.Do not see bibliographical information.
1 β-CD-M
2The preparation and the sign of bonded silica gel high performance liquid chromatography stationary phase
1.1 β-CD-M
2The preparation of bonded silica gel high performance liquid chromatography stationary phase
Two-[6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1, the 4-monoesters-4-)-]-beta-cyclodextrin+linking agent
1.2 characterize
1.2.1 silica bead and β-CD-M
2Bonding phase transmission electron microscope scanning result
To specification be respectively: specific surface 60m
2G
-1, pore volume 0.36cm
3G
-1, aperture 30nm (silica bead A) and specific surface 380m
2G
-1, pore volume 0.70cm
3G
-1, two kind of 5 full porous silica bead of μ m and two kinds of silica bead bonding β-CD-M of aperture 10nm (silica bead B)
2The stationary phase of constructing carries out transmission electron microscope scanning, result such as Figure 22 a, Figure 22 b, Figure 22 c, Figure 22 d.
The transmission electron microscope scintigram of comparison diagram 22a, Figure 22 c silica bead A and silica bead B be can't see obvious difference in the transmission scan result; Difference comparison diagram 22a silica bead A and Figure 22 b β-CD-M
2Bonding silica bead A, Figure 22 c silica bead B, Figure 22 d (b
2) β-CD-M
2The high performance liquid chromatography stationary phase that bonding silica bead B constructs can see that the novel substance of silica bead has obviously appearred being different from the silica bead surface behind the bonding, has illustrated that material is bonded to the silica bead surface.
1.2.2 silica bead and β-CD-M
2Bonding scanning phase Electronic Speculum result
Specification is specific surface 60m
2G
-1, pore volume 0.36cm
3G
-1, aperture 30nm (silica bead A) and β-CD-M
2Bonding silica bead A carries out scanning electron microscope scanning, and the scanning result that obtains amplifying 5000 times, 10000 times, 50000 times is shown in Figure 23 a, Figure 23 b, Figure 23 c, Figure 23 d, Figure 23 e, Figure 23 f.Specification is specific surface 380m
2G
-1, pore volume 0.70cm
3G
-1, aperture 10nm (silica bead B) and β-CD-M
2Bonding silica bead B carries out scanning electron microscope scanning, and the scanning result that obtains amplifying 5000 times, 10000 times, 50000 times is shown in Figure 24 a, Figure 24 b, Figure 24 c, Figure 24 d, Figure 24 e, Figure 24 f.Contrast silica bead A, B bonding β-CD-M respectively
2Many pure silicon pearl A that is different from have obviously appearred in the rear surface, and other material of B has illustrated β-CD-M intuitively
2Be bonded on the silica bead B.
1.2.3X-X-ray photoelectron spectroscopy X
Scan silica bead A, silica bead B, β-CD-M respectively
2Bonding silica bead A, β-CD-M
2The x-ray photoelectron spectroscopy of bonding silica bead B obtains result such as Figure 25, Figure 26, Figure 27, and as seen from the figure, the O1s bound energy is 531.48eV among the silica bead A, and Si2p is 103.58eV; The O1s bound energy is 531.81eV among the silica bead B, and Si2p is 103.68eV; β-CD-M
2The XPS collection of illustrative plates of bonding silica bead A shows that the C1s bound energy is 284.62eV, and the O1s bound energy is 530.65eV, and S2p is 153.66eV, and Si2p is 102.91eV; β-CD-M
2The XPS collection of illustrative plates of bonding silica bead B shows that the C1s bound energy is 284.62eV, and the O1s bound energy is 543.32eV, and the N1s peak is because of the less not sign of strength ratio, and bound energy is that 400.85[sees Figure 28 (c)], the S2p bound energy is 166.84eV, Si2p is 115.8eV.β-CD-M is described
2Successfully be bonded to the surface of silica bead A and silica bead B.
Two kinds of silica gel bonded β-CD-M among contrast Figure 28 and Figure 29
2The photoelectron spectrum of stationary phase is at β-CD-M
2The peak area of S is 10734.44 among the bonding silica bead A, and the N peak is not obvious; β-CD-M
2The peak area of S is 19460.42 among the bonding silica bead B, and the N peak area is 798.65.Show silica bead A surface bond β-CD-M
2Bonded amount be less than β-CD-M
2Bonded amount on silica bead B surface.Be β-CD-M
2β-CD-M on the silica bead big in the aperture, that specific surface is little
2Bonded amount be starkly lower than bonded amount on the silica bead little in the aperture, that specific surface is big.
3.2 β-CD-M
2Be used as the HPLC stationary phase and prepare the ticarcillin single enantiomer
Chromatographic condition: chromatographic column: β-CD-M
2Bonding silica bead A silicagel column (Φ 4.6mm * 250mm), ticarcillin disodium aqueous solution 10mgmL
-1, 23 ℃ of column temperatures, flow velocity 1mlmin
-1, detect wavelength 230nm.
3.2.1 β-CD-M
2Be used as the HPLC stationary phase and investigate the ticarcillin separation condition
1, methyl alcohol-5mmolL
-1Phosphate buffered saline buffer is that moving phase is investigated the ticarcillin separation case
With methyl alcohol (A)-5mmolL
-1Potassium primary phosphate+5mmolL
-1Dipotassium hydrogen phosphate (Ph=7) aqueous solution (B) is moving phase, and it is as shown in the table for proportion of mobile phase, flow velocity 1mlmin
-1, 23 ℃ of column temperatures detect wavelength 230nm, and sample size 5 μ L obtain a series of chromatographic separation results.As shown in Table 1, when the methyl alcohol ratio increases on 60%, two enantiomorphs reach baseline separation.
Table 1 methyl alcohol-5mmolL
-1Phosphate buffered saline buffer is that moving phase is separated ticarcillin
2, methyl alcohol-three damping fluid (15mmolL
-1Tetra-n-butyl ammonium bromide-3mmolL
-1Dipotassium hydrogen phosphate-3mmolL
-1Potassium dihydrogen phosphate aqueous solution) separates ticarcillin for moving phase
With methyl alcohol (A)-15mmolL
-1Tetra-n-butyl ammonium bromide (B)-3mmo lL
-1Dipotassium hydrogen phosphate (C)-3mmolL
-1Potassium primary phosphate (D) aqueous solution is moving phase, and it is as shown in the table for proportion of mobile phase, flow velocity 1mlmin
-1, 23 ℃ of column temperatures detect wavelength 230nm, and sample size 10 μ L from table 2 data as can be known, when the volume of methyl alcohol in the moving phase reaches 88%, reach baseline separation.
Table 2 methyl alcohol-three damping fluid is that moving phase is separated ticarcillin
3, methyl alcohol-50mmolL
-1Phosphate aqueous solution is that moving phase is separated ticarcillin
With methyl alcohol (A)-50mmolL
-1Phosphate aqueous solution (B) is a moving phase, and it is as shown in the table for proportion of mobile phase, flow velocity 1mlmin
-1, 23 ℃ of column temperatures detect wavelength 230nm, and sample size 10 μ L are as shown in table 3.When methyl alcohol volume ratio in the moving phase 60% when above, reach baseline separation.
Table 3 methyl alcohol-50mmolL
-1Phosphate aqueous solution is that moving phase is separated ticarcillin
4, acetonitrile-50mmolL
-1Potassium dihydrogen phosphate aqueous solution is that moving phase is separated ticarcillin
With acetonitrile (A)-50mmolL
-1Potassium dihydrogen phosphate aqueous solution (B) is a moving phase, and proportion of mobile phase is as showing shown in (A-B) flow velocity 1mlmin
-1, 23 ℃ of column temperatures detect wavelength 230nm, sample size 10 μ L, by table 4 data as can be known, ticarcillin all reaches baseline separation under above each proportion of mobile phase condition, and along with the increase of acetonitrile volume ratio in the moving phase, peak shape is sharpened, peak width narrows down, and it is big that eluting power becomes, and disengaging time obviously shortens.
Table 4 acetonitrile-0.05molL
-1Potassium dihydrogen phosphate aqueous solution is that moving phase is separated ticarcillin
3.2.2 with β-CD-M
2Partly prepare the HPLC post and prepare the ticarcillin single enantiomer
Adopt methyl alcohol-phosphate aqueous solution can avoid the generation of salting-out phenomenon as moving phase; Acetonitrile-potassium dihydrogen phosphate aqueous solution can be selected lower organic solvent concentration as moving phase.So select above-mentioned two kinds of moving phases to prepare the ticarcillin single enantiomer.
1,70% methyl alcohol-30%50mmolL
-1Phosphate aqueous solution moving phase prepares the ticarcillin single enantiomer
Take all factors into consideration resolution and disengaging time factor, determine that ratio is 70% methyl alcohol-30%50mmolL
-1Phosphate aqueous solution is as preparative chromatography moving phase.Chromatographic condition: chromatographic column: β-CD-M
2Bonding silica bead A silicagel column (Φ 10mm * 150mm), ticarcillin disodium aqueous solution 10mgmL
-1, 23 ℃ of column temperatures, flow velocity 2mlmin
-1, detect wavelength 230nm, sample size 500 μ L.The preparation separating resulting is seen Figure 30.Collect the component between 6.75-9.20min, the 21.31-28.94min respectively, handle the back and measure its opticity, the component opticity is "+" between the 21.31-28.94min, and the forward is (-) ticarcillin as can be known, and postpeak is (+) ticarcillin.
Ticarcillin forward (6.75-9.20min), the postpeak (21.31-28.94min) collected are collected liquid verify that with HPLC obtain result such as Figure 31, the result shows that ticarcillin forward who collects and postpeak all are simple spikes.
The collection liquid that obtains after the separation obtains containing phosphatic ticarcillin single enantiomer solid salt after lyophilize, adopt HPLC and HPCE to detect.Be single HPCE electrophoresis peak or liquid chromatography peak as can be known.
2,21% acetonitrile-79%50mmolL
-1Potassium primary phosphate moving phase prepares the ticarcillin single enantiomer
Take all factors into consideration factors such as the peak type is better, resolution is big, disengaging time is short, determine that ratio is 21% acetonitrile-79%50mmolL
-1Potassium dihydrogen phosphate aqueous solution is as preparative chromatography moving phase.Chromatographic condition: chromatographic column: β-CD-M
2Bonding specific surface 60m
2G
-1, pore volume 0.36cm
3G
-1, aperture 30nm, particle diameter 5 μ m silicagel columns (Φ 10mm * 150mm), ticarcillin disodium aqueous solution 10mgmL
-1, 23 ℃ of column temperatures, flow velocity 2mlmin
-1, detect wavelength 230nm, sample size 500 μ L.Ticarcillin forward (9.76-11.47min), the postpeak (12.60-15.27min) collected are collected liquid and verified with analysis mode HPLC, obtain the result shown in figure 32.The result shows that ticarcillin forward who collects and postpeak all are simple spike (negative peak are the water peak among the figure).
The assay novel method of embodiment two chirality medicine ticarcillin single enantiomers
1, with β-CD-M
2The HPLC post is measured ticarcillin single enantiomer component linearity range
Ticarcillin disodium solution (reference substance, the 98%) solution of preparation different concns, C (mgmL
-1) be respectively: 0.01,0.05,0.1,0.5,1,3,5,10,30,50, with β-CD-M
2Bonding silica bead A is a stationary phase, column type Φ 4.6mm * 250mm, 23 ℃ of column temperatures, 70% methyl alcohol-30%50mmolL
-1Phosphate aqueous solution is a moving phase, flow velocity 1mlL
-1, detect wavelength 230nm, sample size 20 μ L, the separation linearity range of mensuration ticarcillin is investigated peak area-concentration, peak height-concentration standard curve respectively under this chromatographic condition, obtains the result as shown in figure 33, leading peak (-) ticarcillin (t
R=4.72min) peak area-concentration standard curve equation is y=338.00047x, coefficient R=0.99984, and peak height-concentration standard curve equation is y=2.73079x, coefficient R=0.99964; Postpeak (+) ticarcillin (t
R=11.79min) peak area-concentration standard curve equation is y=6230.70769x, coefficient R=0.9999, and peak height-concentration standard curve equation is y=22.63206x, coefficient R=0.99562.
2, with β-CD-M
2Do the HPLC stationary phase and measure ticarcillin single enantiomer component precision
Under above-mentioned chromatographic condition, measure 5mgmL
-1Two component precision in the ticarcillin disodium, sample introduction frequency n=8 serve as to investigate parameter with peak area and peak height respectively, it is as shown in table 5 to obtain the result.
It is 3.85% for (-) ticarcillin peak height relative standard deviation that precision is investigated the result, and (-) ticarcillin peak area relative standard deviation is 5.18%; (+) ticarcillin peak height relative standard deviation is 1.58%, and (+) ticarcillin peak area relative standard deviation is 1.81%.
Each component precision in table 5 ticarcillin
Can also be with β-CD-M
2As chiral additives, adopt HPCE (HPCE) to separate ticarcillin, investigated borax-borate buffer pH, chiral additives concentration, separation voltage, buffer concentration to isolating influence, obtain the optimal separation condition; Ticarcillin disodium concentration is at 0.01-10mgmL
-1Having better linearity to separate in the L scope, is to investigate parameter (n=7) with the peak height, and RSD% is 2.41-4.27%, with the peak area is to investigate parameter (n=7), and RSD% is 3.00%-5.23%.
Adopt HPLC with β-CD-M
2Chiral stationary phase method compartment analysis ticarcillin influence factor compares less, and it is 1.81-5.18 that precision is measured RSD%; Adopt HPCE with β-CD-M
2The chiral additives method is carried out compartment analysis to ticarcillin, and it is 2.41%-5.23% that precision is measured RSD.
The assay novel method of embodiment trinitroaniline positional isomers
At β-CD-M
2Bonding specific surface 60m
2G
-1, pore volume 0.36cm
3G
-1, aperture 30nm, particle diameter 5 μ m silica beads (A post); β-CD-M
2Bonding specific surface 380m
2G
-1, pore volume 0.70cm
3G
-1, aperture 10nm, particle diameter 5 μ m silica beads (B post) go up the separation case of investigating the N-methyl-p-nitroaniline positional isomers.Be that moving phase is separated N-methyl-p-nitroaniline with normal hexane-Virahol, normal hexane-ethanol under the A post positive mode, suitable proportion of mobile phase can make N-methyl-p-nitroaniline reach baseline separation, elution order be adjacent--right, under the rp mode be that moving phase is separated N-methyl-p-nitroaniline with the water-methanol, elution order be between-right-neighbour; Be to be that moving phase all fails to make N-methyl-p-nitroaniline reach baseline separation with the water-methanol under moving phase and the rp mode with normal hexane-Virahol, normal hexane-ethanol under the B post positive mode, under the positive mode elution order be adjacent--right, under the rp mode elution order be between-adjacent-right.It is different to show that different bonding radical confrontation N-methyl-p-nitroanilines separates influence, selects the good A post of separation efficiency to carry out quantitative analysis.
The A post to be adjacent for flowing with 70% normal hexane-30% Virahol,, the linearity range of p-Nitroaniline is: o-Nitraniline is 5 * 10
-6～1 * 10
-2MolL
-1, coefficient R=0.99994; M-nitraniline is 5 * 10
-6～2 * 10
-2MolL
-1, coefficient R=0.9999; P-Nitroaniline is 5 * 10
-5-8 * 10
-2MolL
-1, coefficient R=0.99981.Adjacent in the simulation compound sample,, the precision (n=8) of p-Nitroaniline, with the peak height is to investigate parameter, o-Nitraniline RSD% is 1.25%, m-nitraniline RSD% is 0.89%, p-Nitroaniline RSD% is 1.96%, with the peak area is to investigate parameter, o-Nitraniline RSD% is 1.04%, m-nitraniline RSD% is 1.72%, p-Nitroaniline RSD% is 4.32%, adjacent in the simulation compound sample,, the recovery of standard addition of p-Nitroaniline: p-Nitroaniline can reach 96.08%-102.45%.Adjacent under this chromatographic condition,, the detectability of p-Nitroaniline, adjacent, detecting of m-nitraniline is limited to 1.38 * 10
-8G, detecting of p-Nitroaniline is limited to 1.38 * 10
-7G.
Embodiment four coating-type HPLC post separating chiral compound methyl phenyl carbinols
Utilizing the empty stainless steel column of 150 * 4.6mm, is that the chromatographic column (hereinafter to be referred as post C) that filler loads is come the separating chiral material with the coating-type silica stationary mutually.
(methyl phenyl carbinol)
Investigate the separation case of chromatographic condition to methyl phenyl carbinol
1, changes proportion of mobile phase
25 ℃ of column temperatures, flow velocity are 1ml/min, detect wavelength 254nm, and when moving phase was 100% normal hexane, methyl phenyl carbinol can not separate with this understanding.The chromatographic separation situation of chromatogram this moment has improvement when adding a small amount of dehydrated alcohol, when moving phase is 99% normal hexane-1% dehydrated alcohol, and methyl phenyl carbinol (1.0 * 10
-3M) sample size is 1ul, and (instrument: Tianjin, island LC-14A): methyl phenyl carbinol can reach baseline separation with this understanding as shown in Figure 34 as Figure 34 in separation.
2, change temperature
Select moving phase to consist of 99% normal hexane-1% dehydrated alcohol, column temperature changes to 40 ℃, methyl phenyl carbinol (1.0 * 10 for 25 ℃
-3M) sample size is 1ul.Can find out two Chiral Separation degree R maximums under 27 ℃ of conditions, as shown in figure 35.
The separating chiral compound 1-phenylpropyl alcohol of embodiment five, bonding type HPLC post
With the empty stainless steel column of 250 * 4.6mm, be that the chromatographic column (hereinafter to be referred as post D) that filler loads is come the separating chiral material mutually below with the bonding type silica stationary.Factors such as the proportioning of different flow phase composites, moving phases of investigation, column temperature, flow velocity are to the isolating influence of 1-phenylpropyl alcohol respectively.Chromatographic condition: detect wavelength 225nm, moving phase normal hexane-methyl alcohol, flow velocity 1ml.L
-1, 1-phenylpropyl alcohol concentration is 0.1mol.L
-1, sample size is 5 μ l, column temperature is 23 ℃.Instrument: the LC-7000 of Hitachi.
(1-phenylpropyl alcohol)
1, investigating normal hexane and methyl alcohol ratio influences separation case
By investigating proportion of mobile phase (100%; 99%; 98%; 97%; 96% normal hexane) result as can be seen, 1-phenylpropyl alcohol resolution can reach 2.83 under 99% normal hexane-1% methyl alcohol moving phase.Realized baseline separation.As Figure 36.
2, investigating column temperature influences the chromatogram separation case
The selection chromatographic condition is: 99% normal hexane-1% methyl alcohol moving phase, flow velocity is 1ml.L
-1, detect wavelength 225nm. column temperature and select 24 ℃, 27 ℃, 30 ℃, 33 ℃, 37 ℃, 40 ℃ respectively.Investigate the separation of column temperature to chiral material 1-phenylpropyl alcohol.Moving phase reaches baseline separation substantially to two corresponding isomer in the time of 30 ℃, promptly reaches (resolution R ≈ 1.5) more than 99.7%, so separate better when being chosen in 30 ℃.As shown in figure 37.
3, investigating flow velocity influences the chromatogram separation case
Be chosen in 99% normal hexane-1% methyl alcohol moving phase, detecting wavelength 225nm. column temperature is 23 ℃, the gained result as can be known to change different flow velocity (2.0,1.5,1.0,0.7,0.4,0.1mL/min), along with reducing of flow velocity, retention time increases, two enantiomer retention time differences are also big more, so just can prepare, obtain the single enantiomer component better and create condition.Below be flow velocity be 1.0 and the color atlas of 0.05mL/min respectively as shown in figure 38.
Embodiment six, bonding type HPLC post make up reverse-phase chromatography system separating chiral material amygdalic acid
As moving phase, amygdalic acid has had partly separation with this understanding with methyl alcohol-0.3%TEAA.Instrument: the LC-7000 of Hitachi.
(amygdalic acid), 26 ℃ of column temperatures, the chromatographic fractionation figure of flow velocity 0.5ml/min moving phase 95% methyl alcohol-5%0.3%TEAA pH5.5 damping fluid, as shown in figure 39.
Claims (3)
1. two [6-oxygen-(and 3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-molecular formula of beta-cyclodextrin is: C
62H
84O
49N
2S
2, structure is:
2. two [6-oxygen-(3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-preparation method of beta-cyclodextrin, beta-cyclodextrin abbreviates β-CD as, it is characterized in that carrying out according to the following steps: (1), elder generation obtain m-nitrobenzene sulfonyl chloride with 1mol oil of mirbane and 80-150 ℃ of reacting by heating of 1-10mol chlorsulfonic acid; (2), again use 2-30mol MALEIC ANHYDRIDE and 1mol β-CD, 60-120 ℃ of reacting by heating obtains two (6-oxygen-butene dioic acid monoesters)-β-CD, is called for short β-CD-A
2(3), use 1mol β-CD-A then
2With 50-120 ℃ of reacting by heating of 2-30mol m-nitrobenzene sulfonyl chloride obtain two [6-oxygen-(3-m-nitro alkylsulfonyl-Succinic Acid-1,4-monoesters-4-)-]-β-CD, be called for short β-CD-M
2Reaction formula is as follows:
3. two [6-oxygen-(3-m-nitro alkylsulfonyl-Succinic Acid-1; 4-monoesters-4-)-]-beta-cyclodextrin is used for the preparative high-performance liquid chromatographic column packing; be used for HPLC separation and purification chiral drug ticarcillin, methyl phenyl carbinol or 1-phenylpropyl alcohol single enantiomer, set up the HPLC assay novel method of these medicines.
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| CN 201010254105 CN101985481B (en) | 2010-08-12 | 2010-08-12 | Bis[6-oxygen-(-3-m-nitrobenzene sulfonyl-amber acid-1,4 uniester-4)-]-beta-cyclodextrin, preparation method and use |
| PCT/CN2011/076885 WO2012019499A1 (en) | 2010-08-12 | 2011-07-06 | Bis[-6-oxo-(-3-m-nitrobenzene sulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin, preparation process and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102219793A (en) * | 2011-05-04 | 2011-10-19 | 湖南湘药制药有限公司 | Method for purifying D (-)-sulbenicillin sodium |
| WO2012019499A1 (en) * | 2010-08-12 | 2012-02-16 | 中南民族大学 | Bis[-6-oxo-(-3-m-nitrobenzene sulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin, preparation process and use thereof |
| CN103965484A (en) * | 2014-04-09 | 2014-08-06 | 南昌大学 | Preparation method and application of omega-diamine derivatization beta-cyclodextrin bonded SBA-15 chiral stationary phase |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2003095498A1 (en) * | 2002-05-10 | 2003-11-20 | Yunqing Liu | Complex of organic medicines and beta-cyclodextrin derivatives and its preparing process |
| CN1649904A (en) * | 2002-04-29 | 2005-08-03 | 西巴特殊化学品控股有限公司 | Aqueous liquid compositions of reactive cyclodextrin derivatives and a finishing process using the said composition |
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| CN1649904A (en) * | 2002-04-29 | 2005-08-03 | 西巴特殊化学品控股有限公司 | Aqueous liquid compositions of reactive cyclodextrin derivatives and a finishing process using the said composition |
| WO2003095498A1 (en) * | 2002-05-10 | 2003-11-20 | Yunqing Liu | Complex of organic medicines and beta-cyclodextrin derivatives and its preparing process |
Non-Patent Citations (3)
| Title |
|---|
| 《中南民族大学学报（自然科学版）》 20061230 杨曦等 "双[-6-氧-(-3-间硝基苯磺酰基-丁二酸-1,4-单酯-4-)-]-beta-环糊精的合成及模拟抗坏血酸氧化酶" 28-30 1-2 第25卷, 第4期 2 * |
| 《有机化学》 19980215 任维衡等 "用beta-CD构筑琥珀酸脱氢酶的研究" 66-70 2 , 第1期 2 * |
| 《湖北省化学化工学会第十一届分析化学专业年会论文集》 20071201 杨曦等 "双[-6-氧-(-3-间硝基苯磺酰基-丁二酸-1，4-单酯-4-)-]-beta-环糊精手性HPLC固定相的合成与性能研究" 134-137 1，3 , 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012019499A1 (en) * | 2010-08-12 | 2012-02-16 | 中南民族大学 | Bis[-6-oxo-(-3-m-nitrobenzene sulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin, preparation process and use thereof |
| CN102219793A (en) * | 2011-05-04 | 2011-10-19 | 湖南湘药制药有限公司 | Method for purifying D (-)-sulbenicillin sodium |
| CN102219793B (en) * | 2011-05-04 | 2014-04-09 | 湖南湘药制药有限公司 | Method for purifying D (-)-sulbenicillin sodium |
| CN103965484A (en) * | 2014-04-09 | 2014-08-06 | 南昌大学 | Preparation method and application of omega-diamine derivatization beta-cyclodextrin bonded SBA-15 chiral stationary phase |
| CN103965484B (en) * | 2014-04-09 | 2017-02-22 | 南昌大学 | Preparation method and application of omega-diamine derivatization beta-cyclodextrin bonded SBA-15 chiral stationary phase |
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