CN109444294B - High performance liquid chromatography method for separating linezolid and chiral isomer thereof - Google Patents
High performance liquid chromatography method for separating linezolid and chiral isomer thereof Download PDFInfo
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- CN109444294B CN109444294B CN201811607411.1A CN201811607411A CN109444294B CN 109444294 B CN109444294 B CN 109444294B CN 201811607411 A CN201811607411 A CN 201811607411A CN 109444294 B CN109444294 B CN 109444294B
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- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 title claims abstract description 24
- 229960003907 linezolid Drugs 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 21
- BBGSNKXBKDVHPM-CMPLNLGQSA-N (3s,4r)-4-methyl-1-phenylmethoxycarbonylpyrrolidine-3-carboxylic acid Chemical compound C1[C@@H](C(O)=O)[C@@H](C)CN1C(=O)OCC1=CC=CC=C1 BBGSNKXBKDVHPM-CMPLNLGQSA-N 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000010828 elution Methods 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 8
- 239000012069 chiral reagent Substances 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 12
- TYZROVQLWOKYKF-CYBMUJFWSA-N n-[[(5r)-3-(3-fluoro-4-morpholin-4-ylphenyl)-2-oxo-1,3-oxazolidin-5-yl]methyl]acetamide Chemical compound O=C1O[C@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-CYBMUJFWSA-N 0.000 description 10
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 208000008745 Healthcare-Associated Pneumonia Diseases 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 206010062255 Soft tissue infection Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IZXIZTKNFFYFOF-UHFFFAOYSA-N 2-Oxazolidone Chemical compound O=C1NCCO1 IZXIZTKNFFYFOF-UHFFFAOYSA-N 0.000 description 1
- NFCONHPCIUINPB-SFVWDYPZSA-N FC1N(CCOC1)C1=C(C=CC=C1)N1C(O[C@H](C1)CNC(C)=O)=O Chemical compound FC1N(CCOC1)C1=C(C=CC=C1)N1C(O[C@H](C1)CNC(C)=O)=O NFCONHPCIUINPB-SFVWDYPZSA-N 0.000 description 1
- 241000989913 Gunnera petaloidea Species 0.000 description 1
- 206010041925 Staphylococcal infections Diseases 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000004296 chiral HPLC Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 229940061740 zyvox Drugs 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a high performance liquid chromatography method for separating linezolid and chiral isomers thereof, which comprises the following chromatographic parameters: a chromatographic column: agilent ZORBAX extended-C18 column (4.6 mm. times.250 mm, 5 μm); mobile phase a phase: a water-acetonitrile-pyridine solution at a volume ratio of 82:10:8, containing 20mM (3S,4R) -1-Cbz-4-methylpyrrolidine-3-carboxylic acid; mobile phase B phase: acetonitrile; gradient elution procedure: 0-7 min, 0% B; 7-30 min, 0% -90% B; 30-40 min, 90% -0% B; flow rate: 1.0 mL/min; column temperature: 35 ℃ is carried out. According to the method, the linezolid and the chiral isomer thereof can be effectively separated by adding the chiral reagent (3S,4R) -1-Cbz-4-methylpyrrolidine-3-formic acid and using a conventional C18 chromatographic column, so that the separation effect is excellent, and the separation and detection cost is low.
Description
Technical Field
The invention belongs to the field of analysis and detection, and relates to a high performance liquid chromatography method for separating linezolid and chiral isomers thereof.
Background
Linezolid (linezolide) has the chemical name: (S) -5- (acetamidomethyl) -3- [ (3-fluoro-4-morpholinyl) phenyl ] -1, 3-oxazolidin-2-one, trade name: zyvox, a novel oxazolidinone antibacterial agent developed by Pharmacia & Upjohn, was first marketed in the United states in 4 months in 2000. The composition is clinically used for treating infections caused by gram-positive cocci, including suspected or confirmed Hospital Acquired Pneumonia (HAP), Community Acquired Pneumonia (CAP), complex skin or Skin Soft Tissue Infection (SSTI) and vancomycin-resistant enterococci (VRE) infection caused by MRSA. The linezolid has a chiral center in the molecular structure, a pair of optical isomers (the structural formula is shown as the following) exist, and the linezolid with pharmacological activity is levorotatory and has an S-configuration and is called linezolid; dexlinezolid is its R-isomer, known as (R) -linezolid. According to the relevant technical requirements of Chinese medicine registration, (R) -linezolid is used as a relevant substance to be checked and controlled.
The literature provides a method for separating linezolid and its isomer (R) -linezolid (the literature: Liu Xiao et al, chiral HPLC analysis of R-type isomer in linezolid bulk drug, 2016, 37, volume 4 of foreign pharmaceutical antibiotic Manual), but the method has to rely on an expensive chiral chromatographic column with short service life, and the cost is high.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography method for separating linezolid and chiral isomers thereof.
The technical scheme for realizing the aim of the invention is as follows:
a high performance liquid chromatography method for separating linezolid and chiral isomers thereof comprises the following chromatographic parameters:
a chromatographic column: agilent ZORBAX extended-C18 column (4.6 mm. times.250 mm, 5 μm);
mobile phase a phase: a water-acetonitrile-pyridine solution at a volume ratio of 82:10:8, containing 20mM (3S,4R) -1-Cbz-4-methylpyrrolidine-3-carboxylic acid;
mobile phase B phase: acetonitrile;
gradient elution procedure: 0-7 min, 0% B; 7-30 min, 0% -90% B; 30-40 min, 90% -0% B;
flow rate: 1.0 mL/min;
column temperature: 35 ℃ is carried out.
Preferably, the detection wavelength is 254 nm.
Preferably, the sample size is 10. mu.L.
The invention has the outstanding advantages that:
according to the method, the linezolid and the chiral isomer thereof can be effectively separated by adding the chiral reagent (3S,4R) -1-Cbz-4-methylpyrrolidine-3-formic acid and using a conventional C18 chromatographic column, so that the separation effect is excellent, and the separation and detection cost is low.
Drawings
FIG. 1 is an HPLC chromatogram of a mobile phase containing a chiral reagent (3S,4R) -1-Cbz-4-methylpyrrolidine-3-carboxylic acid;
FIG. 2 is an HPLC chromatogram of mobile phase without chiral reagent (3S,4R) -1-Cbz-4-methylpyrrolidine-3-carboxylic acid.
Detailed Description
The following detailed description of the present invention is provided in connection with the examples, and for reasons of brevity, the description of the experimental procedures is not intended to be exhaustive, and all parts not specifically described in the experiments are routine procedures well known to those skilled in the art.
First, experimental material
Shimadzu LC-20AT high performance liquid chromatograph (equipped with LC-20AT pump, SIL-20A autosampler, and SPD-M20A diode array detector, Shimadzu corporation, Japan).
Agilent ZORBAX extended-C18 column (4.6 mm. times.250 mm, 5 μm) is a product of Agilent corporation.
Linezolid and (R) -linezolid standard are purchased or made by self, and the purity is not lower than 95%.
The chiral reagent (3S,4R) -1-Cbz-4-methylpyrrolidine-3-formic acid is purchased with a purity of not less than 95%.
Acetonitrile and pyridine are both chromatographically pure water produced by Hangzhou child haha group limited company.
Second, Experimental methods and results
1. Solution preparation
Mobile phase a preparation: the water-acetonitrile-pyridine solution with the volume ratio of 82:10:8 contains 20mM (3S,4R) -1-Cbz-4-methylpyrrolidine-3-formic acid, and the specific preparation method comprises the steps of mixing the water, the acetonitrile and the pyridine according to the volume ratio of 82:10:8, adding the chiral reagent (3S,4R) -1-Cbz-4-methylpyrrolidine-3-formic acid to 20mM, dissolving, carrying out suction filtration and carrying out ultrasonic treatment for later use.
Mobile phase B preparation: and (5) carrying out suction filtration and ultrasonic treatment on acetonitrile for later use.
Linezolid control solution: dissolving linezolid with mobile phase A to obtain solution with concentration of 0.1mg/mL, filtering with 0.45 μm filter membrane, and storing at 4 deg.C.
(R) -linezolid control solution: dissolving (R) -linezolid with mobile phase A to obtain a solution with a concentration of 0.1mg/mL, filtering with a 0.45 μm filter membrane, and storing at 4 ℃ for later use.
Mixing the reference solution: dissolving linezolid and (R) -linezolid with mobile phase A to prepare mixed solutions with the concentrations of 0.5mg/mL respectively, filtering the mixed solutions with a 0.45-micron filter membrane, and storing the mixed solutions at 4 ℃ for later use.
2. Chromatographic conditions
A chromatographic column: agilent ZORBAX extended-C18 column (4.6 mm. times.250 mm, 5 μm);
mobile phase a phase: a water-acetonitrile-pyridine solution at a volume ratio of 82:10:8, containing 20mM (3S,4R) -1-Cbz-4-methylpyrrolidine-3-carboxylic acid;
mobile phase B phase: acetonitrile;
gradient elution procedure: 0-7 min, 0% B; 7-30 min, 0% -90% B; 30-40 min, 90% -0% B;
flow rate: 1.0 mL/min;
detection wavelength: 254 nm;
column temperature: 35 ℃;
sample introduction amount: 10 μ L.
3. Sample introduction detection and separation effect
Precisely measuring 10 mu L of linezolid control solution, (R) -linezolid control solution and mixed control solution respectively, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram is shown in figure 1, so that linezolid and (R) -linezolid in the mixed control solution can achieve baseline separation, and the separation effect is excellent. If (3S,4R) -1-Cbz-4-methylpyrrolidine-3-formic acid is not added into the phase A, other chromatographic conditions are not changed, and the separation chromatogram is shown in figure 2, and linezolid and (R) -linezolid cannot be separated.
The foregoing embodiments are provided to illustrate the present invention more fully, but those skilled in the art will appreciate that the scope of the present invention should not be limited to the specific embodiments described above.
Claims (3)
1. A high performance liquid chromatography method for separating linezolid and chiral isomers thereof is characterized in that the chromatographic parameters are as follows:
a chromatographic column: an Agilent ZORBAX extended-C18 chromatographic column with the specification of 4.6mm multiplied by 250mm and 5 μm;
mobile phase a phase: a water-acetonitrile-pyridine solution at a volume ratio of 82:10:8, containing 20mM (3S,4R) -1-Cbz-4-methylpyrrolidine-3-carboxylic acid;
mobile phase B phase: acetonitrile;
gradient elution procedure: 0-7 min, 0% B; 7-30 min, 0% -90% B; 30-40 min, 90-0% B;
flow rate: 1.0 mL/min;
column temperature: 35 ℃ is carried out.
2. The high performance liquid chromatography method of claim 1, wherein: the detection wavelength was 254 nm.
3. The high performance liquid chromatography method of claim 1, wherein: the amount of sample was 10. mu.L.
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| CN115236236A (en) * | 2022-07-26 | 2022-10-25 | 上海市食品药品检验研究院 | Linezolid and separation and analysis method of enantiomers in preparation thereof |
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| US6696412B1 (en) * | 2000-01-20 | 2004-02-24 | Cubist Pharmaceuticals, Inc. | High purity lipopeptides, Lipopeptide micelles and processes for preparing same |
| WO2006091731A2 (en) * | 2005-02-24 | 2006-08-31 | Teva Pharmaceutical Industries Ltd. | Processes for the preparation of linezolid intermediate |
| EP2511844B1 (en) * | 2006-10-10 | 2015-08-12 | XRpro Sciences, Inc. | X-ray microscope |
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| CN102643245A (en) * | 2012-04-10 | 2012-08-22 | 杭州华东医药集团生物工程研究所有限公司 | Linezolid crystal form and preparation method thereof |
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| CN106146332B (en) * | 2015-04-21 | 2020-09-11 | 重庆华邦胜凯制药有限公司 | Method for separating and determining linezolid raw material X3 and process impurity X2 thereof |
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