WO2012019499A1

WO2012019499A1 - Bis[-6-oxo-(-3-m-nitrobenzene sulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin, preparation process and use thereof

Info

Publication number: WO2012019499A1
Application number: PCT/CN2011/076885
Authority: WO
Inventors: 沈静茹
Original assignee: 中南民族大学
Priority date: 2010-08-12
Filing date: 2011-07-06
Publication date: 2012-02-16
Also published as: CN101985481A; CN101985481B

Abstract

Disclosed in the present invention is bis[-6-oxo-(-3-m-nitrobenzene sulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin and a preparation process thereof and its use as a chiral selector in high performance liquid chromatography (HPLC). The bis[-6-oxo-(-3-m-nitrobenzene sulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin of the present invention is produced by bis-substituting hydrogen in a hydroxyl group on carbon-6 in the A and D rings of β-cyclodextrin by maleic anhydride, and then adding an m-nitrobenzenesulfonyl group to the double bond of cis-butene, wherein the addition of the m-nitrobenzenesulfonyl group is carried out at carbon-3 of succinic acid. Its molecular formula is C₆₂H₈₄O₄₉N₂S₂. A preparative chiral HPLC column using this derivate can establish a new process for obtaining purified D(+)-ticarcillin and L(-)-ticarcillin from a pharmaceutical racemic body; it can also produce two single enantiopure products of α-phenylethanol and 1-phenylpropanol; and a new process for separating and determining the content of a single enantiomer of a chiral substance such as ticarcillin, α-phenylethanol, 1-phenylpropanol, etc. can be established by using an analytical chiral HPLC column.

Description

Bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-P-cyclodextrin

And preparation method and use

The present invention relates to a chiral selector bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin and Preparation methods and uses.

Background technique

Separation and purification of chiral drugs is one of the hot spots in the development of new drugs, and has received extensive attention. HPLC resolution is an effective means of preparing single enantiomers of chiral drugs. Among the many chiral stationary phases, β-cyclodextrin (CD) and its derivatives are favored by many researchers. A chiral drug refers to the presence of a chiral factor in the molecular structure of a drug. The pharmacological action of the drug is achieved by strict chiral recognition and matching with macromolecules in the body. The amino acids constituting the protein are all L-amino acids, and the monosaccharides constituting the polysaccharide and the nucleic acid are D-monosaccharides. After the racemic body of the drug is introduced into the body, the enantiomer molecules are chiral proteins and enzymes in the body. And receptors are treated with two different molecules. Due to the chiral characteristics of the human body, in many cases, there is a significant difference in the pharmacological activity, metabolic processes, metabolic rate and toxicity of a pair of enantiomers of chiral drugs in terms of absorption, distribution and excretion. There are differences, and mutual transformations may occur between enantiomers.

The effective separation and purification of the enantiomers of the chiral drugs can more effectively ensure the safety of medication and improve the level of social medical care, becoming an important task for researchers. For example, the in vitro activities of the two enantiomers of the β-receptor blocking drug propranolol are 98 times different; L-dopa is a drug for treating Parkinson's disease, and the truly therapeutically active compound is L. - L-dopamine, which must be enantiomerically pure L-dopa, is required to be converted into L-dopamine by the specific enzyme in the human body. If the racemate is taken, the right-handed body will accumulate in the body and will not be metabolized by the enzymes in the body, which may cause harm to human health. Among the 1327 chemical synthetic drugs commonly used today, there are 528 chiral drugs, of which only 61 are listed as single enantiomers, and most of them are still sold as racemates. This situation undoubtedly poses a potential threat to the safety of medication, and also puts more urgent requirements for the development of a single enantiomeric new drug. Clinical use of a single enantiomeric chiral drug can reduce the dosage of the drug, reduce the metabolic burden of the patient, increase the dose range and broaden the use, and have better control over pharmacokinetics. The dose is wider and the response is smaller at dose setting; more confidence in dose selection. For pharmaceutical companies, the production of a single enantiomeric chiral drug can save resources, reduce the cost of new drug listings for clinical pharmacology and pharmacokinetic studies, reduce waste emissions, and reduce environmental pollution.

It is not easy to obtain an optically pure enantiomer. There are three methods for obtaining a chiral compound of a single enantiomer: chiral source synthesis, asymmetric synthesis, and racemic resolution. The racemic resolution method is a method for separating a racemic compound into a pure enantiomer compound by a chiral selector (chiral auxiliary), and is further divided into a chemical resolution method, an enzyme or a microorganism. Method, chromatographic resolution. Among them, the chromatographic resolution method showed superiority. The chiral drug ranolazine was separated and purified by chromatography. The purity of single enantiomer was over 98%. The resolution of omeprazole was the highest purity of S-omeprazole to 98.71. %, R-omeprazole has a purity of up to 95.47%. Liquid chromatography is included in the racemic resolution method. Liquid Phase chromatography is considered to be the best method for determining enantiomeric purity and isolating optically pure single enantiomers. It has a wide adaptability and mild operating conditions, and does not cause changes in the configuration of the isolate or destruction of biological activity, and at the same time, two enantiomers with high optical purity can be obtained.

Cramer et al. first discovered that β-cyclodextrin (CD) has a good recognition effect on optical isomers. After β-CD derivatization, a three-dimensional geometric relationship can be constructed near the binding site to form a special chiral site; three-dimensional spatial modification can be performed to expand the cavity or provide a space with a specific geometry to adapt to the guest molecule. Matching; and other action sites that can initiate chiral recognition can also be introduced. The β-CD chiral stationary phase has been widely used in chiral separation analysis. The main functions of improving enantiomeric separation are: (1) combining a pair of enantiomers of separated solutes to form different structures and properties. (2) a hydroxyl group of β-CD, mainly a C-2, a C-position secondary hydroxyl group, or a complex of a CD modification group and a separated enantiomer to form a different structure and property by hydrogen bonding; (3) The tightly packed filling in the cavity and the inclusion of the short-range van der Waals force. The derivatization of natural β-CD can improve the separation efficiency and is suitable for many chromatographic separation modes, which is favored by many researchers. For example, Xin Wang et al. used a seven-(6-azido-6-deoxy-2,3-2-0-phenylcarbamoyl) β-CD bonded chiral stationary phase packed semi-preparative column for separation by high performance liquid chromatography. The β-blocking drug propranolol was prepared, and the propranolol enantiomer was prepared by batch separation using simulated moving bed chromatography. XinWang et al. used perphenoylated β-CD bonded silica gel. The stationary phase, three of the four enantiomers of Nadolol were separated by high performance liquid chromatography, and the most active (RSR)-Nadolol in the four enantiomers was completely separated. A high purity target enantiomer (RSR)-Nadolol was isolated from the Nadolol enantiomer by five-zone simulated moving bed chromatography. The preparation of chiral drugs with such chiral selectors has significant separation and purification effects, but the types are not enough. It is necessary to continue to develop more suitable and more selective chiral selectors to prepare more. A single enantiomeric chiral drug.

Summary of the invention

The object of the present invention is to provide a chiral selector bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin And a preparation method and use thereof, as a chiral selector, a β-cyclodextrin derivative containing two functional groups of m-nitrobenzenesulfonyl (N0 ₂ C ₆ H ₅ S0 ₂ ) and succinate (OOCCHCHCOOH) The chiral selector can be directly coated or bonded to the silica gel beads or the quartz tube wall to prepare a chiral stationary phase.

In order to achieve the object of the present invention, in one aspect, the present invention provides a chiral selector of β-cyclodextrin, which is a disubstituted β-cyclodextrin A with maleic anhydride by controlling the reaction conditions. After the hydroxyl group hydrogen on the 6-position carbon on the D ring, the m-nitrobenzenesulfonyl group is added to the double bond of the butylene group, and the m-nitrobenzenesulfonyl group is added to the 3-position carbon of the succinic acid. And formed. Its molecular formula is: C ₆₂ H ₈₄ 0 ₄₉ N ₂ S ₂ . Finally, a novel β-CD derivative with the same degree of substitution is obtained: bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β- CD (referred to as: P-CD-M ₂ ). The structure of the derivative was confirmed by ultraviolet, infrared, mass spectrometry, nuclear magnetic, XPS and the like. The structure of bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin is: ο/.,·,..,

On the other hand, the present invention also provides a synthesis method for preparing the above β-cyclodextrin chiral selector, bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1, 4-monoester-4-)-]-β-cyclodextrin preparation method, β-cyclodextrin is abbreviated as β-CD, which is characterized by the following steps: (1), first use 1 mol of nitrobenzene and 1-lOmol chlorosulfonic acid 80-150 ° C heating reaction to obtain m-nitrobenzenesulfonyl chloride; (; 2), and then 2-30 mol maleic anhydride and lmoip-CD, 60-120 ° C heating reaction to obtain a double (6-oxo-butenedioic acid monoester) -β-CD, abbreviated as P-CD-A ₂ ; (3), then lmoip-CD-A ₂ and 2-30 mol of nitrobenzenesulfonyl chloride 50-120 Heating at °C to obtain bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-CD, abbreviated as P-CD-M ₂ ; The reaction formula is as follows:

0

β-CD R-CD O-C-CH=CHCOOH).

(referred to as: P-CD-A ₂ )

(referred to as: (HXH Ij)

(3).

The β-cyclodextrin structure compound prepared by the invention can be prepared as a chiral stationary phase by directly coating or bonding on a carrier such as silica gel beads or a quartz tube wall, or can be added as a chiral additive to a mobile phase. Separation, purification and provision of new qualitative and quantitative methods for chiral substances. The chiral selector of the present invention is suitable as a chiral stationary phase for instruments such as high performance liquid chromatography (HPLC), gas chromatography (GC), and high performance capillary electrophoresis (HPCE). The prepared stationary phase has strong chiral recognition ability and good stability, and can be separated, analyzed or purified to prepare a single enantiomer by chiral material of analytical and preparative chiral column materials.

Compared with the prior art, the present invention has the following effects: 1. Purified D ( + ) - ticarcillin and L (-) - tica can be established from the racemic body of the drug by using a preparative chiral HPLC column. Xilin and (X-phenylethyl alcohol, 1-phenylpropanol two single enantiomers pure. 2. Separation and determination of ticarcillin, (X-phenylethyl alcohol, styrene-acrylic) by analytical chiral HPLC column A new method for the content of single enantiomers of alcohol has been published by the Wuhan Science and Technology Research and Search Center of the Chinese Academy of Sciences. No report has been reported in the literature.

The instruments and reagents used in the present invention are:

PE labmda Bio35 UV-Vis Spectrophotometer (USA)

Nicolet NEXUS-470 Fourier Transform Infrared Spectrometer (USA)

Varian rostar 210 High Performance Liquid Chromatography (USA)

LABCONCO FreeZone Freeze Dryer (USA)

API3200 Liquid Chromatography Tandem Mass Spectrometer (USA)

Tecnai G2 20S-TWIN Transmission Electron Microscope (Czech Republic)

JSM-6700F Scanning Electron Microscope (Japan)

VG Multilab 2000 Photoelectron Spectrometer (USA)

AVANCEIII NMR Spectrometer (BRUKER)

β-CD Sinopharm Chemical Reagent Co., Ltd.

Maleic anhydride Sinopharm Chemical Reagent Co., Ltd.

Nitrobenzene Sinopharm Chemical Reagent Co., Ltd.

Benzenesulfonic acid Shanghai Jinshanting New Chemical Reagent Factory

Characterization of bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinate-1,4-monoester-4-)-]-p-cyclodextrin:

1, UV scanning spectrum

Prepare SxlO^mol- ¹ nitrophenylethanol solution (ethanol as reference), SxlO^mol- ¹ nitrobenzenesulfonyl chloride ethanol solution (ethanol as reference), SxlO-Smol'l p-CD aqueous solution (water is ^{reference), 3 l0 "5 mol-} L ^ -CD ^ aqueous solution (water as reference), 3xl0- ⁵ mol_L- -CD-Mz ^ aqueous solution (water as reference) UV scanning as nitrobenzene, m Nitrobenzenesulfonyl chloride is insoluble in water, β-CD derivative is insoluble in ethanol, and different solutions are selected as solvent and reference for scanning. The results are shown in Figure 1. The absorption of nitrobenzene at 260 nm is significantly better than that of nitrobenzene. Strong phenylsulfonyl chloride; β-CD has no obvious UV absorption, P-CD-A ₂ has obvious UV absorption between 200nm and 260nm, indicating that the double bond of maleic anhydride has been attached to β-CD; In the P-CD-M ₂ UV scan, the characteristic absorption of the benzene ring appeared at 259 nm, which is different from the UV absorption peak of P-CD-A ₂ , indicating that the group with a benzene ring has been bonded to P- On CD-A ₂ , a product different from P-CD-A ₂ was produced.

2, infrared scanning map

Comparing Fig. 3 with Fig. 2, it is apparent that on the P-CD-A ₂ infrared scan of Fig. 3, the C=0 (8, 162,601^) peak of 1726 cm- ¹ is more than the β-CD infrared scan of Fig. 2. On the infrared scan of nitrobenzenesulfonyl chloride in Figure 4, 3096 cm" ¹ (w, CH), 1534 cm" ¹ 1352 cm" ¹ (w, N0 ₂ ), 1603 cm, 755 cm" ¹ (w, Benzene ring); comparing Fig. 5 with Fig. 3, the P-CD-M ₂ infrared scan of Fig. 5 is 1533 cm- ¹ and 1351 cm1 more than the P-CD-A ₂ infrared scan of Fig. ³ ( The peak of w, N0 ₂ ), 1611 cm ¹ , 734 cm ¹ (w, benzene ring) indicates that m-nitrobenzenesulfonyl chloride has been bonded to P-CD-A ₂ .

3. X-ray photoelectron spectroscopy The X-ray photoelectron spectroscopy of P-CD, maleic anhydride, P-CD-A ₂ and P-CD-M ₂ was scanned separately, and the results are shown in the figure below. The full spectrum of each compound is shown in Fig. 6a and Fig. 6b. Figure 6c, Figure 6d, each compound map is shown in Figure 7a, Figure 7b, Figure 8a, Figure 8b, Figure 9a, Figure 9b, Figure 10a, Figure 10b, Figure 10c, Figure 10d.

It can be seen from Fig. 6a, Fig. 6b, Fig. 6c and Fig. 6d that the Cls binding energy in β-CD is 284.64 eV, the Ols binding energy is 530.33 eV, and the coupling energy of maleic anhydride Cls is 284.59 eV. The binding energy of Ols is The double peaks of 538.4eV and 532.25 eV are consistent with oxygen in maleic anhydride; the binding energy of Cls in P-CD-A ₂ is 284.6eV and the binding energy of Ols is 531.17eV; P-CD- The binding energy of Cls in M ₂ is 284.59 eV, the binding energy of Ols is 530.23 eV, the binding energy of Nls is 399.82 eV, and the binding energy of S2p is 166.6 eV. It is apparent on the P-CD-M ₂ XPS spectrum that the XPS bands of N and S are more than P-CD-A ₂ , indicating that the group containing the N and S elements has been bonded to P-CD-A _{2 .} on.

4, nuclear magnetic spectrum

The reaction materials P-CD, P-CD-A ₂ and product P-CD-M ₂ were identified by NMR, including: ¹³ C NMR, 'H NMR and HMBC (1H detection of heteronuclear multi-bond correlation test) ), HSQC (Nuclear single quantum coherent test for 1H detection) (H number is the same as C number).

Figure 11 is a ¹³ C NMR chart of β-CD, which can be obtained as CI: 101.83 C2: 73.07 C3: 72.06 C4: 81.12 C5: 71.81 C6: 60.28. Figure 12 is a ¹ H NMR chart of β-CD, showing that δ 4.989 is hydrogen on C6 hydroxyl group (-OH), δ 2.148, S3.484-3.591 and S3.771-3.912 are C6 (-CH ₂ -) Hydrogen on the methylene group, hydrogen at the C1 position of the 54.680 3⁄4 CD ring, δ3.5-4.0 is the hydrogen on the C2, C3, C4, C5, and the ¹ H NMR spectrum of Figure 14p-CD-A ₂ is shown in Figure 2.12P. -CD1H NMR spectrum δ 4.989 (hydrogen on -OH) is significantly reduced, indicating that the -OH bond of C6 in β-CD has formed an ether bond in P-CD-A ₂ with a new peak δ6.0-6.5 It is a hydrogen on the C14=C15 double bond on the branch.

Figure 13 is a ¹³ C NMR chart of P-CD-A ₂ which gives 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=0(C13): 166.26. It can be seen from the above data that groups such as -C=C -, C=0 and -COOH after the reaction are produced are shown in the figure. From the alignment of C6 with C6 of Fig. β-CD, it can be judged that the maleic anhydride substitution reaction occurs at the C6 position.

Figure 15C-H direct correlation diagram can further show that C6: 30.35 is directly related to δ2.142, C1 is directly related to hydrogen at δ4.680, and δ3.5-4.0 hydrogen is directly related to C2, C3, C4, C5. Δ6.0-6.5 hydrogen directly with C14, C15 Figure 16C-H remote correlation diagram shows that hydrogen δ6.0-6.5 on the double bond is remotely related to CI and remotely related to C5. It is also remotely related to C14 and C15.

In summary, the original -CD-A ₂ ) structure is:

Figure 17 is a ¹³ C NMR nuclear magnetic diagram of P-CD-M ₂ . Because of the 400 mega NMR, the instrument sensitivity is low, and the chemical shifts of the corresponding 6 C on β-CD are not obvious, but the benzene ring Ar ( C7- C12) and C13-C16 on the branch are obvious: 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 a 1H NMR nuclear magnetic diagram of β-CD-M ₂ , combined with a two-dimensional nuclear magnetic direct correlation diagram, that is, HMBC (1H detection of heteronuclear multi-bond correlation test), Figure 19 can be obtained: There are 4 sets of peaks on H on the benzene ring. The H directly related to C7 is δ8.5, the 直接 directly related to C9 is δ8.3, the 直接 directly related to C11 is δ8.1, and the H directly related to C10 is δ7.7. The Ηδ4.989 on the branched hydroxyl group (-ΟΗ) is still strong, and the direct correlation with δ2.5 is C14: 34.03; C15: 40.34;

It can be seen from Fig. 20 that C1 on the cyclodextrin ring is remotely related to Η5 and Η4 due to the chair structure of the cyclodextrin and the influence of the substituent group, and C2 is remotely related to Η5. Branches also have some remote related points. Such as C13 and H15, hydrogen on CH; hydrogen on C14 and OH; C7 is remotely related to H9, H10, H11 on the aromatic ring, C9 is remotely related to H7 and H11, C10 is remotely related to H7, C11 is remotely related to H7, H9 Relatedly, C8 is remotely related to H10 and H11, C12 is remotely related to H10 and H9, and C13 is remotely related to H7, H9, H10, and H11.

5, mass spectrometry

P-CD-M ₂ was isolated and purified by semi-preparative HPLC, and the product was purified by freeze-drying and characterized by mass spectrometry. Semi-preparative HPLC separation chromatographic conditions: stationary phase: C18 semi-preparative column; mobile phase: 5% - 10% acetonitrile - 95% - 90% water; flow rate: 2 ml 'min - column temperature: 30 ° C; detection wavelength: 254 nm _; Sample concentration: 5x lO- ³ mol'L- ¹ aqueous solution; injection volume 200μ1. Mass spectrometry conditions: curtain Gas: 10 ml-rnin ¹ ; IonSpray Voltage: 5500V; Ion Source Gas: 110 ml-min"¹; Declustering Potential: 70 mv.

From the mass spectrum, the following information can be obtained: 1699 is the molecular ion peak, which is P-CD-M ₂ ( 1704) after the proton loss the result of. The following is the possible fracture mode of P-CD-M _2. If the two pieces of 258 (with 254.4 fragment ion peak) are broken according to the fracture mode (1), 516 fragments are obtained, and the corresponding 515.1 fragment ion peak is present in the mass spectrum; According to the fracture mode (2), there is no fragment similar to 244 or twice the mass-to-charge ratio 488 in the mass spectrum, so that a reasonable fracture of the aromatic ring group is known.

m/z 1704

The following is the third way of breaking P-CD-M ₂ :

m/z 1354

From the third division process of P-CD-M ₂ , the double substitution occurs on the 1,4 ring of β-CD, 1354 (1354.2 in the figure), 350 (348.2 in the figure, and 3% OH). In the figure, it is 333.8, both of which are very strong.) The basis can be found in the mass spectrum. If the 1, 3 ring is substituted, there should be 4 ring-connected fragments 673 (there is no such fragment in the figure, and there is no other part of the 1031 fragment). If the 1, 2 ring is substituted, there should be 5 ring-connected fragments 836 (Fig. There is no such fragment in it).

Therefore, the structure is

The β-cyclodextrin derivative prepared by the invention can be directly coated or bonded on a carrier such as silica gel or a quartz tube. On the wall, it is prepared as a chiral stationary phase. It can also be added as a chiral additive to the mobile phase for separation and purification of chiral materials and to provide new qualitative and quantitative methods. The chiral selector of the present invention is suitable as a chiral stationary phase for instruments such as high performance liquid chromatography (HPLC), gas chromatography (GC), and high performance capillary electrophoresis (HPCE). The prepared stationary phase has strong chiral recognition ability and good stability, and can be prepared as an analytical and preparative chiral column material for separation, analysis or purification of a single enantiomer.

Compared with the prior art, the present invention has the following effects: 1. Purified D ( + ) - ticarcillin and L (-) - tica can be established from the racemic body of the drug by using a preparative chiral HPLC column. A new method of Xilin; can also obtain (X-phenylethanol, 1-phenylpropanol two substances single enantiomer pure. 2, analytical chiral HPLC column can be established to determine the determination of ticarcillin, (X- A new method for the content of single enantiomers of phenylethyl alcohol and 1-phenylpropanol was published by the Wuhan Science and Technology Research and Search Center of the Chinese Academy of Sciences. No report was found in the literature.

DRAWINGS

Figure 1 is an ultraviolet scanning spectrum, a: β-CD; b: P-CD-A ₂ ; c: m-nitrobenzenesulfonyl chloride; d: nitrobenzene; e: P-CD-M ₂ . .

Figure 2 is a β-CD infrared scan.

Figure 3 is an infrared scan of P-CD-A ₂ .

Figure 4 is an infrared scan of m-nitrobenzenesulfonyl chloride.

Figure 5 is a P-CD-M ₂ infrared scan.

Figure 6 a shows the X-ray photoelectron spectroscopy of β-CD (full spectrum, VG Multilab 2000).

Figure 6b shows the X-ray photoelectron spectroscopy of maleic anhydride (full spectrum, VG Multilab 2000).

Figure 6c shows the X-ray photoelectron spectroscopy of P-CD-A ₂ (full spectrum, VG Multilab 2000).

Figure 6 d is the X-ray photoelectron spectroscopy of P-CD-M ₂ (full spectrum, VG Multilab 2000).

Figure 7a shows the β-CD X-ray photoelectron energy C spectrum (VG Multilab 2000).

Figure 7b shows the β-CD X-ray photoelectron energy spectrum (VG Multilab 2000).

Figure 8a shows the X-ray photoelectron energy C spectrum of maleic anhydride (VG Multilab 2000).

Figure 8b shows the X-ray photoelectron energy spectrum of maleic anhydride (VG Multilab 2000).

Figure 9a shows the P-CD-A ₂ X-ray photoelectron energy C spectrum (VG Multilab 2000).

Figure 9b shows the P-CD-A ₂ X-ray photoelectron spectroscopy (VG Multilab 2000).

Figure 10a is a P-CD-M ₂ X-ray photoelectron energy C spectrum (VG Multilab 2000).

Figure 10b is a P-CD-M ₂ X-ray photoelectron energy 0 spectrum (VG Multilab 2000).

Figure 10c is a P-CD-M ₂ X-ray photoelectron energy N spectrum (VG Multilab 2000).

Figure 10d is a P-CD-M ₂ X-ray photoelectron energy S spectrum (VG Multilab 2000).

Figure 11 is a β-CD ¹³ C nuclear magnetic map (AVANCE ⁱ NMR D ₂ 0 400 MH _Z ).

Figure 12 is a β-CD 1H nuclear magnetic map (AVANCE ⁱ NMR D ₂ 0 400 MH _Z ).

Figure 13 is a P-CD-A ₂ ¹³ C nuclear magnetic map (AVANCE ⁱ NMR D ₂ 0 400 MH _Z ).

Figure 14 is a P-CD-A ₂ 1H nuclear magnetic map (AVANCE ⁱ NMR D ₂ 0 400 MH _Z ). Figure 15 is a nuclear magnetic diagram of β-CD-A ₂ HSQC (AVA CE ⁱ NMR D ₂ 0 400MH _Z ). Figure 16 is a β-CD-A ₂ HMBC nuclear magnetic map (AVA CE ⁱ NMR D ₂ 0 400 MH _Z ). Figure 17 is a P-CD-M ₂ ¹³ C nuclear magnetic map (AVA CE ⁱ NMR DMSO 400 MH _Z ). Figure 18 is a P-CD-M ₂ 1H nuclear magnetic map (AVANCE ⁱ NMR D ₂ 0 400 MH _Z ).

Figure 19 is a P-CD-M ₂ HSQC nuclear magnetic map (AVANCE ⁱ NMR DMSO 400 MH _Z ). Figure 20 is a P-CD-M ₂ HMBC nuclear magnetic map (AVA CE ^m NMR DMSO 400 MH _Z ). Figure 21 is a P-CD-M ₂ mass spectrum.

Figure 22a is a scanning electron micrograph of a stationary phase of a silicon bead.

Figure 22b is a transmission electron micrograph of the stationary phase of P-CD-M ₂ bonded silicon beads A.

Figure 22c is a transmission electron microscopy scan of the stationary phase of the silicon bead B.

Figure 22d is a transmission electron microscopy scan of the stationary phase of P-CD-M ₂ bonded silicon beads B.

Figure 23a shows a 5,000x scanning electron micrograph of a silicon bead.

Figure 23b shows a 10,000x scanning electron micrograph of silicon beads A.

Figure 23c shows a 50000x SEM image of a silicon bead.

Figure 23d is a 5000x scan electron micrograph of P-CD-M ₂ bonded silicon beads A.

Figure 23e is a 10,000-fold scanning electron micrograph of P-CD-M ₂ bonded silicon beads A.

Figure 23f is a 50000x scanning electron micrograph of P-CD-M ₂ bonded silicon beads A.

Figure 24a shows a 5,000x scanning electron micrograph of a silicon bead.

Figure 24b is a 10,000x scanning electron micrograph of silicon beads B.

Figure 24c is a scanning electron micrograph of a silicon bead B50000.

Figure 24d is a 5000x scan electron micrograph of P-CD-M ₂ bonded silicon beads B.

Figure 24e is a 10,000-fold scanning electron micrograph of P-CD-M ₂ bonded silicon beads B.

Figure 24f is a 50000x scanning electron micrograph of P-CD-M ₂ bonded silicon beads B.

Figure 25 a is an X-ray photoelectron spectroscopy (full spectrum) of silicon beads A.

Figure 25b shows the X-ray photoelectron spectroscopy (full spectrum) of the silicon bead B.

Figure 25c is an X-ray photoelectron spectroscopy (full spectrum) diagram of P-CD-M ₂ bonded silicon beads A. Figure 25 d is an X-ray photoelectron spectroscopy (full spectrum) diagram of P-CD-M ₂ bonded silicon beads B. Figure 26a is a plot of the oxygen spectrum XPS of the silica beads.

Figure 26 b is a silicon bead A silicon spectrum XPS map.

Figure 27a is a B-oxygen XPS map of the silicon beads.

Figure 27b is a XPS map of the silicon bead B silicon spectrum.

Figure 28a is a carbon spectrum XPS map of P-CD-M ₂ bonded silicon beads A.

Figure 28b is a P-CD-M ₂ bonded silica bead A oxygen spectrum XPS map.

Figure 28c is a P-CD-M ₂ bonded silica bead A nitrogen spectrum XPS map.

Figure 28 d is an XPS chromatogram of the P-CD-M ₂ bonded silica beads A sulfur spectrum. Figure 28 e is a P-CD-M ₂ bonded silicon bead A silicon spectrum XPS map.

Figure 29a is a carbon spectrum XPS map of P-CD-M ₂ bonded silicon beads B.

Figure 29b is a B-oxygen XPS pattern of P-CD-M ₂ bonded silicon beads.

Figure 29c is a P-CD-M ₂ bonded silicon bead B nitrogen spectrum XPS map.

Figure 29 d is a P-CD-M ₂ bonded silica bead B sulfur spectrum XPS map.

Figure 29 e is a P-CD-M ₂ bonded silicon bead B silicon spectrum XPS map.

Figure 30 is a HPLC diagram of the preparation of the single enantiomer of ticarcillin by semi-preparative chromatography.

31 a is the (-) frontal plot of the purity verification of the HPLC cecascilin single enantiomer collection.

Figure 31 b is a (+) post-peak plot of the purity verification of the HPLC ticarcillin single enantiomer collection.

Figure 32 a shows the purity of the HPLC ticarcillin single enantiomer collection (acetonitrile system).

Figure 32 b shows the purity of the HPLC ticarcillin single enantiomer collection (acetonitrile system).

Figure 33 a is a linear (-) front peak area-concentration plot for ticarcillin.

Figure 33 b is a linear (-) pre-peak peak height-concentration plot for ticarcillin.

Figure 33 c is a linear (+) peak area-concentration plot for ticarcillin separation.

Figure 33 a is a linear (+) pre-peak peak height-concentration plot for ticarcillin.

Figure 34 is a chromatogram of α-phenylethanol chromatography (mobile phase 99% n-hexane - 1% absolute ethanol).

Figure 35 shows (X-phenylethanol chromatogram (column temperature 27 °C).

Figure 36 is a chromatogram of 1-phenylpropanol (mobile phase: 99% n-hexane - 1% methanol).

Figure 37 is a chromatogram of 1-phenylpropanol (column temperature: 30 ° C).

Figure 38a is a chromatogram of the flow rate: l.OmL'min- ¹ of 1-phenylpropanol.

Figure 38b is a chromatogram of 1-phenylpropanol at a flow rate of 0.05 mL-rnin ¹ .

Figure 39 is a chromatogram of mandelic acid chromatogram.

detailed description

Preparation of bis[- ₆ -oxo-(- ₃ -m-nitrobenzenesulfonyl-succinic acid-^4-monoester-4-)-]-β-cyclodextrin: β-cyclodextrin is abbreviated as β -CD, proceed as follows: (1) First, 123.2 g of nitrobenzene and 350.3 g of chlorosulfonic acid are heated at 100 ° C to obtain m-nitrobenzenesulfonyl chloride; then 22.0 g of maleic anhydride and 17.0 gP-CD, heated at 80 °C to give bis(6-oxo-butenedioic acid monoester)-β-CD, abbreviated as P-CD-A _{2 ;} (3), then lmoip-CD-A ₂ and 20 mol The reaction of m-nitrobenzenesulfonyl chloride at 60 ° C gives bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-CD , referred to as P-CD-M ₂ . The obtained P-CD-M _{2 was} used in the following examples.

Example 1: A new method for HPLC preparation of ticarcillin single enantiomer

This example relates to a method for obtaining purified D ( + ) - ticarcillin and L (-) - ticarcillin from the racemate of the drug. There is less work literature on the separation and analysis of single enantiomers of this drug, and most of the research work is carried out in the form of racemates. This embodiment is quite innovative.

Ticarcillin is a semi-synthetic antibiotic known under the chemical name 6-(2-carboxy-2-thiophen-3-ylacetyl)aminopenicillanic acid. It is clinically used as a double sodium salt, C ₁₅ H ₁₄ N ₂ Na ₂ 0 ₆ S ₂ , molecular weight 428.40, white to light yellow powder, soluble Water, aqueous solution pH is 6-8. Ticarcillin is a broad-spectrum, highly effective anti-Pseudomonas aeruginosa penicillin, but not resistant to β-lactamase, orally absorbed, administered intravenously or intramuscularly. Mainly used for the treatment of burn infection, meningitis, osteomyelitis, respiratory tract, urinary tract infection and postoperative prevention. It is often combined with the β-lactamase inhibitor clavulanic acid to make a compound "Tamantine", also known as "Temetine". Its enantiomeric structure is as follows: (* is a chiral carbon).

Ticarcillin structure

Li Yin-Hua et al. determined plasma and urine ticarcillin by HPLC; Watson Ian D et al. determined plasma clavulanic acid and ticarcillin by HPLC; Rice Patrick 0 ^[43] used HPLC and optical rotation detection Technical combination method for the determination of ticarcillin; Kelly James ^ ¥ ^[44] and other γ-cyclodextrin and ion exchange ethyl vinyl benzene / divinyl benzene copolymer, poly (styrene - divinyl Benzene was isolated by HPLC for the isomer of β-lactam antibiotics including ticarcillin. Hoogmartens J et al. separated the side chain isomers of penicillin by HPLC: phenoxyethylpenicillin, phenoxypropylpenicillin, dichloromethanemycin, carbenicillin, ticarcillin, ampicillin, amoxicillin, and Aducillin; Hendrickx S et al. Separated 18 penicillins including ticarcillin by thin-layer chromatography; Atmesley et al. simultaneously separated and determined plasma including ticarcillin by gradient elution liquid chromatography. Antibiotics; Haginaka et al. used a precolumn to derivatize test substances with 1,2,4-tripyrrole and mercuric chloride, and determined the carbobenzylcillin, ticarcillin and sulfonate in plasma and urine by HPLC. Benzilin; Haginaka Jun et al. determined the clavulanic acid in human plasma and urine by HPLC separation; Haginaka Jun et al. determined the ticarcillin by post-column degradative HPLC method; Nakagawa Terumichi et al. determined the human plasma by HPLC. In the urine, clavulanic acid and ticarcillin were studied for pharmacokinetics; Mopper Barry used liquid chromatography to determine ticarcillin in the injectable drug; Tyczkowsk Determination of ticarcillin in canine and horse plasma by ion-pair chromatography; Wright Jennifer C. et al. Determination of ticarcillin and clavulanic acid in plasma by LC; Horimoto Shingo et al. Atmospheric pressure chemical ionization mass spectrometry of solvent was used to determine 8 antibiotics and 13 cephalosporins including ticarcillin; Pajchel Genowefa et al. Simultaneous determination of temacetin and clavulanic acid by micellar electrokinetic chromatography Ticacetin and clavulanic acid in the compound antibiotics; Li Chonghua et al. Simultaneous determination of ticarcillin and clavulanic acid in rabbit plasma and tissue fluid by liquid chromatography; capillary thermoelectrophoresis by Jiang Wenqing et al. Separation of amoxicillin, ampicillin, ticarcillin three penicillins and clavulanic acid, sulbactam two β-lactamase inhibitors; Ashraf-K orassani M and other multi-dimensional liquid chromatography / electron spray ionization mass spectrometry Separation and determination of the three matrices Moxilin, clavulanic acid and ticarcillin.

This example provides a novel method for preparing purified D ( + ) - ticarcillin and L (-) - ticarcillin, which uses the β-CD derivative prepared above: bis [-6-oxy-( -3-m-nitrobenzenesulfonyl-succinic acid-1,4-monoester-4-)-]-P-CD (abbreviation: P-CD-M ₂ ) bonded two kinds of silicon beads to construct two The HPLC stationary phase was characterized by transmission electron microscopy TEM, scanning electron microscope SEM and X-ray photoelectron spectroscopy XPS, which proved that P-CD-M _{2 was} successfully bonded to the silicon beads.

Four mobile phases were used on the same stationary phase analysis column. At the appropriate ratio, ticarcillin reached baseline separation, and HPLC separation methods for four corresponding enantiomers were established. The single enantiomer of ticarcillin was prepared in a P-CD-M ₂ bonded stationary phase preparative column. The verification of the fractionated pool showed that the single component of the ticarcillin enantiomer was obtained. D (+) and L (-) type enantiomer solid salts. No literature has been reported.

Preparation and Characterization of 1 P-CD-M ₂ Bonded Silica High Performance Liquid Chromatography Stationary Phase

1.1 Preparation of P-CD-M ₂ bonded silica gel high performance liquid chromatography stationary phase

Bis-[-6_oxy-(-3_m-nitrobenzenesulfonyl-succinic acid-1,4-monoester _4-)-]-β- +

1.2 Characterization

1.2.1 Transmission results of silicon beads and P-CD-M ₂ bonded phase transmission electron microscopy

The specifications are: specific surface 60m ² _g- volume 0.36cm ³ _g- 30nm (silicon beads A) and specific surface

Transmission electron microscopy of 380 m ² -g ^l , 0.70 cm ³ 'g-10 nm (silicon bead B) of two 5μηι full polysilicon beads and two silicon bead bonded P-CD-M ₂ fixed phases The results are shown in Figures 22a, 22b, 22c, and 22d.

Comparing Fig. 22 a, Fig. 22c, TEM scan of silicon bead A and silicon bead B, no significant difference is seen in the transmission scan results; comparing Fig. 22 a silicon bead A with Fig. 22b β-CD-M ₂ bonded silicon Bead A, Fig. 22c silicon bead B, Fig. 22d (b ₂ ) P-CD-M ₂ bonded silicon bead B can be seen in the high performance liquid chromatography stationary phase, the surface of the bonded silicon bead is obviously different. A new substance of silicon beads, indicating that a substance is bonded to the surface of the silicon bead.

1.2.2 Scanning electron microscopy results of silicon beads and P-CD-M ₂ bonded phase

The specifications are 60m ² -g-capacity 0.36cm ³ -g- ¹ , 30nm (silicon beads A) and B P-CD-M ₂ bonded silicon beads A by scanning electron microscopy scanning, which is magnified 5000 times, 10000 times, The scan result of 50000 times is shown in Fig. 23a, Fig. 23b, Fig. 23c, Fig. 23d, Fig. 23e, Fig. 23f. The specifications are 380 ² -g ^l , 0.70 cm ³ -g - 10 nm (silicon beads B ) and P-CD-M ₂ bonded silicon beads B for scanning electron microscopy scanning, which are magnified 5000 times, 10000 times, 50000 times. The scan results are shown in Figures 24a, 24b, 24c, 24d, 24e, and 24f. Separate After the silicon beads A and B were bonded to the P-CD-M ₂ surface, there were obvious other substances different from the pure silicon beads A and B, which directly indicated that the P-CD-M _{2 was} bonded to the silicon. Bead B.

1.2.3 X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy of silicon beads A, silicon beads B, P-CD-M ₂ bonded silicon beads A, and P-CD-M ₂ bonded silicon beads B were respectively scanned, and the results are shown in Fig. 25 and Fig. 26. 27, it can be seen from the figure that the Ols binding energy in the silicon bead A is 531.48 eV, and the Si2p is 103.58 eV; the Ols binding energy in the silicon bead B is 531.81 eV, and the Si2p is 103.68 eV; P-CD-M ₂ bonded silicon bead A The XPS spectrum showed that the Cls binding energy was 284.62 eV, the Ols binding energy was 530.65 eV, the S2p was 153.66 eV, and the Si2p was 102.91 eV. The XPS spectrum of P-CD-M ₂ bonded silicon beads B showed that the Cls binding energy was 284.62 eV. The binding energy of Ols is 543.32 eV. The Nls peak is unlabeled because of its small intensity. The binding energy is 400.85 [see Figure 28 (c)], the S2p binding energy is 166.84 eV, and the Si2p is 115.8 eV. It is indicated that P-CD-M _{2 is} successfully bonded to the surface of the silicon beads A and the silicon beads B.

Comparing the photoelectron spectroscopy of the two silica-bonded P-CD-M ₂ stationary phases in Fig. 28 and Fig. 29, the peak area of S in P-CD-M ₂ bonded silicon beads A was 10734.44, and the N peak was not obvious; The peak area of S in P-CD-M ₂ bonded silicon beads B was 19,460.42, and the N peak area was 798.65. It is shown that the amount of bonding of the surface-bonded P-CD-M ₂ of the silicon bead A is less than the amount of bonding of the P-CD-M ₂ on the surface of the silicon bead B. That is, the amount of P-CD-M ₂ bonded to P-CD-M ₂ on a large, smaller-surface-sized silicon bead is significantly lower than that on a small, larger-surface-sized silicon bead.

3.2 P-CD-M ₂ used as HPLC stationary phase to prepare ticarcillin single enantiomer

Chromatographic conditions: Column: P-CD-M ₂ bonded silica beads A silica gel column (Φ4.6 mmx250 mm), ticarcillin disodium aqueous solution lOmg'mL- ¹ , column temperature 23 ° C, flow rate lml'min, The detection wavelength was 230 nm.

3.2.1 P-CD-M ₂ used as HPLC stationary phase ticarcillin separation conditions

1, methanol-5mmol_L- ¹ acid solution for mobile phase ticarcillin separation

The methanol (A)-Smmol-L" ¹ acid dihydrogen Smmol'L acid hydrogen di(Ph=7) aqueous solution (B) was used as the mobile phase, and the mobile phase was mixed as shown in the table. The flow rate was 1 ml_min - the column temperature was 23 ° C. The wavelength of 230 nm and the injection amount of 5 L gave a series of chromatographic separation results. As can be seen from Table 1, when the methanol ratio was 60%, the two enantiomers reached baseline separation.

Methanol mmol.L- ¹ acid solution for mobile phase separation of ticarcillin

A-B tRi/min H/AU Wmin H/AU Rs

60% -80% 2.85 0.044 4.48 0.576 1.77

80% -20% 3.07 0.043 8.03 0.319 3.65

2, methanol - three liquid (mmol 'L- ¹ tetra-n-butyl bromide mmol 'L- ¹ acid hydrogen two mmol 'L- ¹ acid dihydrogen aqueous solution) for mobile phase separation ticarcillin

Using methanol (A) - mmol 'L - ¹ tetra-n-butyl bromide (B) mmol 'L - ¹ acid hydrogen di (C) mmol 'L - ¹ acid dihydrogen (D) aqueous solution as mobile phase, mobile phase matching As shown in the table, the flow rate lml_min- column temperature 23 ° C, detection wavelength 230 nm, injection volume ΙΟμΙ ^, from the data in Table 2, when the volume of methanol in the mobile phase reaches 88%, reach Baseline separation.

Liquid phase separation of ticarcillin

A-B-C-D tRi/min H/AU Wmin H/AU Rs

88%-4%-4%-4% 3.92 0.045 14.45 0.164 1.81

3,

Acidic aqueous solution for mobile phase separation of ticarcillin

The methanol (A) -50mmol_L- ¹ acid aqueous solution (B) was used as the mobile phase, and the mobile phase was as shown in the table. The flow rate was lmhnin- ¹ , the column temperature was 23 ° C, the detection wavelength was 230 nm, and the injection amount was ΙΟμΙ^, as shown in Table 3. Show. When the volume ratio of methanol in the mobile phase is above 60%, baseline separation is achieved.

Table 3 methanol--50mmol_L- ¹ acid aqueous solution for mobile phase separation of ticarcillin

A-B tRi/min H/AU Wmin H/AU Rs

60%-40% 4.72 0.063 10.11 0.476 2.23

70%-30% 4.72 0.078 11.79 0.437 2.66

80%-20% 5.47 0.076 17.60 0.316 3.64

90% - 10% 8.19 0.043 45.31 0.148 5.36

4, acetonitrile -50mmol_L - ¹ acid dihydrogen aqueous solution for mobile phase separation of ticarcillin

The acetonitrile (A) -50mmoH ¹ acid dihydrogen aqueous solution (B) is used as the mobile phase, and the mobile phase is as shown in Table (AB). The flow rate is lml_min - the column temperature is 23 ° C, the detection wavelength is 230 nm, and the injection volume is ΙΟμΙ^. 4 data show that ticarcillin reached the baseline separation under the above mobile phase ratio conditions, the acetonitrile volume ratio of the mobile phase, the peak shape change, the peak width changed, the elution intensity became larger, and the separation time was significantly shorter.

Table 4 acetonitrile-0.05mol_L- ¹ acid aqueous dihydrogen solution for mobile phase separation of ticarcillin

A-B tRi/min H/AU Wmin H/AU Rs

3%-97% 19.97 0.015 44.00 0.070 1.62

12%-88% 11.01 0.029 28.61 0.104 1.64

21%-79% 7.81 0.036 22.88 0.165 2.10

30%-70% 6.32 0.046 20.16 0.263 2.53

39%-61% 4.51 0.055 16.43 0.362 2.95

48%-52% 5.01 0.055 18.24 0.471 3.05

57%-43% 3.31 0.074 10.93 0.612 4.58

3.2.2 Preparation of ticarcillin single enantiomer by P-CD-M ₂ semi-preparative HPLC column

The use of methanol-acid aqueous solution as the mobile phase can cause salting out; acetonitrile-acid dihydrogen aqueous solution can be used as the mobile phase to select a lower organic solvent concentration. Therefore, the above two mobile phases were selected to prepare a single enantiomer of ticarcillin.

1. 70% methanol-30% 50mmol_L- ¹ aqueous solution mobile phase preparation of ticarcillin single enantiomer

Comprehensive separation and separation time factors, determine the ratio of 70% methanol-30% 50mmol_L- ¹ acid aqueous solution As a preparative chromatographic mobile phase. Chromatographic conditions: Column: P-CD-M ₂ bonded silica beads A silica gel column (Φ10 mmx 150mm), ticarcillin disodium aqueous solution lOmg 'mL- ¹ , column temperature 23 ° C, flow rate 2ml 'min- ¹ , The detection wavelength was 230 nm, and the injection amount was 500 μ. The separation results are shown in Figure 30. The components between 6.75 - 9.20min and 21.31 -28.94min were collected, and the optical rotation was measured after treatment. The optical rotation of the component was "+" between 21.31 and 28.94min. It can be seen that the front is (-) ticarcillin. The peak is (+) ticarcillin.

The collected ticarcillin forward (6.75-9.20 min) and the post-peak (21.31-28.94 min) were verified by HPLC, and the results are shown in Fig. 31. The results showed that the collected ticarcillin front and the back peak were both Single peak.

The collected solution obtained after separation was freeze-dried to obtain a single-enantiomer solid salt of ticarcillin containing an acid salt, which was detected by HPLC and HPCE. It can be seen that all are single HPCE electrophoresis peaks or liquid chromatography peaks.

2. Preparation of ticarcillin single enantiomer by 21% acetonitrile-79% 50 mmol ^-1 acid dihydrogen mobile phase

The composite peak shape is good, the separation degree is large, and the separation time is short. The ratio of 21% acetonitrile-79% 50mmol_L- ¹ acid dihydrogen solution is determined as the preparative chromatographic mobile phase. Chromatographic conditions: Column: P-CD-M ₂ bonding ratio surface 601^·^, volume 0.36cm ³ 'g-30nm, 5μηι silica gel column (Φ10 mmx 150mm), ticarcillin disodium aqueous solution lOmg'mL- ¹ The column temperature is 23 ° C, the flow rate is Sml 'min - ¹ , the detection wavelength is 230 nm, and the injection amount is 500 μΙ. The collected ticarcillin front (9.76 - 11.47 min) and the post-peak ( 12.60 - 15.27 min) collected liquids were verified by analytical HPLC, and the results are shown in Fig. 32. The results showed that the collected ticarcillin front and the back peak were single peaks (the peak in the figure is the water peak).

A new method for determining the content of a single enantiomer of the second-hand drug ticarcillin

1. Determination of linear range of single enantiomers of ticarcillin by P-CD-M ₂ HPLC column

Prepare different concentrations of ticarcillin disodium solution (pair, 98%) solution, C (mg-mL- ¹ ) are: 0.01, 0.05, 0.1, 0.5, 1, 3, 5, 10, 30, 50, P-CD-M ₂ bonded silicon beads A as the stationary phase, column type O4.6mmx250mm, column temperature 23 ° C, 70%

The aqueous acid solution is a mobile phase, the flow rate is lml'I ¹ , the detection wavelength is 230 nm, and the injection volume is 20 μΙ^. The linear range of separation of ticarcillin is determined under the chromatographic conditions, respectively, peak area-concentration, peak height-concentration curve, The results are shown in Figure 33. The peak (-) ticarcillin (t _R = 4.72 min) peak area-concentration curve equation is y=338.00047x, correlation coefficient R=0.99984, peak height-concentration curve equation is y=2.73079x, correlation coefficient R=0.99964; post-peak (+) ticarcillin (t _R =11.79min) peak area-concentration curve equation is y=6230.70769x, correlation coefficient R=0.9999, peak height-concentration The curve equation is y=22.63206x, and the correlation coefficient is R=0.99562.

2. Determination of the precision of the single enantiomer component of ticarcillin by using P-CD-M ₂ as the HPLC stationary phase

The precision of the two components in Smg.mL- ¹ ticarcillin disodium was determined under the above chromatographic conditions. The number of injections was n=8, and the peak area and peak height were taken as parameters respectively. The results are shown in Table 5.

The precision results were (-) ticarcillin peak height relative error 3.85%, (-) ticarcillin peak area relative standard deviation was 5.18%; ( + ) ticarcillin peak height relative deviation was 1.58%, ( + ) The relative variation of the area of ticarcillin is 1.81%. Table 5 Precision of each component in ticarcillin

tRl tR2 hi/mv h ₂ /mv Ai/mv*s A ₂ /mv*s

1 4.93 11.41 13.563 125.967 2042.83 29086.54

2 4.96 11.49 14.429 125.188 2159.36 29320.06

3 4.93 11.52 14.601 125.789 2340.82 28167.56

4 4.93 11.44 14.735 129.359 2349.60 28266.45

5 4.96 11.47 15.046 127.468 2473.23 29468.94

6 4.93 11.52 15.478 124.240 2412.99 29519.38

7 4.96 11.49 14.900 123.055 2401.78 28652.11

8 4.96 11.47 14.377 124.417 2011.53 28988.44

X 14.641 125.685 2274.02 28933.69

SD 0.563 1.987 117.68 523.26

RSD/% 3.85 1.58 5.18 1.81

P-CD-M ₂ can also be used as a chiral additive. High-performance capillary electrophoresis (HPCE) can be used to separate ticarcillin. The effects of acid pH, chiral additive concentration, separation voltage and liquid concentration on separation are also obtained. Conditions; The concentration of ticarcillin disodium has a good linear separation in the range of 0.01-lOn^ml L, with the peak height as the parameter (n=7) and 180% as 2.41-4.27%, with the peak area as the parameter ( n=7) , RSD% is 3.00% - 5.23%.

The factors affecting the isolation of ticarcillin by P-CD-M ₂ chiral stationary phase were relatively low by HPLC. The precision of 1^0% was 1.81-5.18. The HP-based P-CD-M ₂ chiral additive method was used. For the separation analysis of ticarcillin, the precision RSD was 2.41% - 5.23%.

A new method for determining the content of isomers of trinitroaniline

In the P-CD-M ₂ bonding ratio surface 601 ^ · ^, capacity 0.36cm ³ 'g - 30nm, 5μηι silica beads (A column); P-CD-M ₂ bonding ratio surface 3801 ^ · ^, capacity 0.70 Separation of the upper nitroaniline isomers of αη ³ ·^, 10 nm, 5μηι silica beads (B column). In the normal phase of column A, the nitroaniline was separated by n-hexane-isopropanol and n-hexane-ethanol. The appropriate mobile phase ratio can make the nitroaniline reach the baseline separation, and the elution is a pair. In the phase mode, the nitroaniline was separated by water-methanol as the mobile phase, and the elution was in a pair--; the normal phase mode of the B column was n-hexane-isopropyl alcohol, n-hexane-ethanol as the mobile phase and the reverse phase mode. The nitroaniline was not separated from the baseline by water-methanol as the mobile phase. The elution in the normal phase mode was a pair, and the elution in the reverse phase mode was a pair. It is indicated that different bonding matrices have different effects on the separation of nitroaniline, and the separation column A is selected for quantitative analysis.

The linear range of A column with 70% n-hexane to 30% isopropanol as mobile phase, m-p-nitroaniline is: nitroaniline is 5x 10-6 WO- ² mol-L ¹ , correlation coefficient R=0.99994; The m-nitroaniline is 5χ 10" ⁶ 2χ 10" ² mol-L" ¹ , the correlation coefficient is R = 0.9999, and the p-nitroaniline is 5x 10 - ^{5 -} 8x10 - ² mo L - correlation coefficient R = 0.99981. Simulating the precision of the intermediate, m-nitroaniline (n=8), taking the peak height as the parameter, nitroaniline

1^0% is 1.25%, m-nitroaniline 1^0% is 0.89%, p-nitroaniline 1^0% is 1.96%, with peak area as parameter, nitroaniline 1^0% is 1.04%, The nitroaniline 1 ^ 0% was 1.72%, the p-nitroaniline RSD% was 4.32%, and the spiked yield of the intermediate, m-nitroaniline was simulated: p-nitroaniline up to 96.08% -

102.45%. The detection limit of p-nitroaniline under this chromatographic condition, the detection limit of m-nitroaniline is

1.38x l0- ⁸ g, the detection limit for p-nitroaniline 1.38x l0- ⁷ g.

Example 4 Coating HPLC separation of chiral compound α-phenylethanol

The chiral material was separated by an empty 150 x 4.6 mm column packed with a silica-coated stationary phase as a filler (hereinafter referred to as column C).

( _α -phenylethyl alcohol)

Separation of α-phenylethyl alcohol by chromatographic conditions

1. Change the mobile phase ratio

The column temperature was 25 °C, the flow rate was 1 ml/min, and the detection wavelength was 254 nm. When the mobile phase was 100% n-hexane, α-phenylethyl alcohol could not be separated under this condition. When the addition of a small amount of absolute ethanol, the chromatographic separation at the time of chromatography was changed. When the mobile phase was 99% n-hexane-1% absolute ethanol, the injection amount of α-phenylethanol (1.0x lO- ³ M) was lul. , Separation as shown in Figure 34 (instrument: LC-14A): From Figure 34, it can be seen that α-phenylethanol can reach baseline separation under these conditions.

2, change the temperature

The mobile phase composition was selected to be 99% n-hexane-1% absolute ethanol, the column temperature was changed to 40 ° C at 25 ° C, and the injection amount of α-phenylethanol ( 1.0 x lO" ³ M) was lul. The resolution of the two enantiomers is the largest under the condition of °C, as shown in Figure 35. Example 5. Separation of the chiral compound 1-phenylpropanol from the bonded HPLC column

The chromatographic column (hereinafter referred to as column D) filled with a bonded silica gel stationary phase is used to separate the chiral material by using an empty 250 x 4.6 mm column. The effects of different mobile phase composition, mobile phase ratio, column temperature, flow rate and other factors on the separation of 1-phenylpropanol. Chromatographic conditions: detection wavelength 225nm, mobile phase n-hexane-methanol, flow rate

1 , 1-phenylpropanol concentration is O. l moLI ¹ , the injection volume is 5μ1, and the column temperature is 23 °C. Instrument: Hitachi LC-7000.

(1-phenylpropanol)

1. The effect of the ratio of n-hexane to methanol on the separation

By the mobile phase ratio (100%; 99%; 98%; 97%; 96% hexamethylene), it can be seen that the 1-phenylpropanol separation can reach 2.83 in the 99% n-hexane-1% methanol mobile phase. A baseline separation was achieved. See Figure 36.

2. Effect of column temperature on chromatographic separation

The chromatographic conditions were as follows: 99% n-hexane-1% methanol mobile phase, flow rate was 1 ml 丄^-1 , detection wavelength was 225 nm. Column temperature was 24 °C, 27 °C, 30 °C, 33 °C, 37 °C, respectively. , 40 ° C. Column temperature, the substance of the substance, 1-phenylpropanol Leaving. At 30 ° C, the flow relative to the two corresponding isomers basically reached the baseline separation, that is, more than 99.7% (resolution R-1.5), so the separation was better at 30 ° C. As shown in Figure 37.

3. Influence of flow rate on chromatographic separation

Select 99% n-hexane -1% methanol mobile phase, detection wavelength 225nm. Column temperature is 23 °C, change different flow rates

(2.0, 1.5, 1.0, 0.7, 0.4, O.lmL/min) The results show that the flow rate is reduced, the time is strong, and the time difference between the two enantiomers is larger, so that it can be better prepared. The conditions for obtaining a single enantiomeric component are created. The following are the chromatograms at flow rates of 1.0 and 0.05 mL/min, respectively, as shown in Figure 38.

EXAMPLES, Bonded HPLC Column Construction of Reversed Phase Chromatography System for Separation of Chiral Material Mandelic Acid

As a mobile phase, there is partial separation of mandelic acid under these conditions. Instrument: Hitachi

(mandelic acid), column temperature 26 ° C, flow rate 0.5 ml / min mobile phase 95% methanol - 5%

Chromatographic separation of 0.3% TEAApH 5.5, as shown in Figure 39.

Claims

Claim

1. The molecular formula of bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin is C ₆₂ H ₈

H H

c c\>

2. Preparation method of bis[-6-oxy-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin, β-cyclodextrin The abbreviation is β-CD, which is characterized by the following steps: (1), first use 1 mol of nitrobenzene and 1-lOmol of chlorosulfonic acid

Heating at 80-150 ° C to obtain m-nitrobenzenesulfonyl chloride; (2), using 2-30 mol of maleic anhydride and lmoip_CD,

60-120 Ό heating reaction to obtain bis(6-oxo-butenedioic acid monoester)-β-CD, abbreviated as p_CD-A _{2 ;} (3), then lmolp-CD-A ₂ and 2-30 mol of m-nitrobenzene The sulfonyl chloride 50-120 Ό is heated to obtain bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-CD, abbreviated as p- CD-M _{2 ; the} reaction formula is as follows:

o

i-CD p-CD- O-C-CH=CHCOOH).

U (abbreviation: P-CD-A ₂ ) ( ₂ )

(referred to as: (3"CDH^)

(3)

3. Bis[-6-oxo-(-3-m-nitrophenylsulfonyl-succinic acid-1,4-monoester-4-)-]-β-cyclodextrin for the preparation of high performance liquid chromatography columns Filler, used for HPLC separation and purification of the chiral drug ticarcillin, α-phenylethanol or 1-phenylpropanol single enantiomer, to establish a new HPLC method for the determination of these drugs.

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