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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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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| JP2008530028A (en) * | 2005-02-24 | 2008-08-07 | テバ ファーマシューティカル インダストリーズ リミティド | Method for preparing linezolid intermediates |
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