Separation and Determination of Florfenicol Enantiomers Using Ultra-performance Convergence Chromatography
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Abstract:
A new method was established for the separation and determination of two florfenicol enantiomers using ultra-performance convergence chromatography (UPC2). The chromatographic separation conditions were optimized, and the stability of standard solutions of (-)-florfenicol and (+)-florfenicol enantiomers were observed. The separation process was carried out in Waters Acquity Trefoil CEL2 (150×3.0 mm, 2.5 µm) chiral columns. The mobile phase used carbon dioxide and methanol at a flow rate of 1.0 mL/min with gradient elution. The UV detection wavelength was set to 224 nm, the injection volume was 5 µL, the system backpressure was 17.2 MPa, and the column temperature was maintained at 40 ℃. The method validation results indicate that the limit of detection (LOD, S/N=3) for both enantiomers was 2.00 mg/L, and the calibration linear range was between 4.00 and 400.00 mg/L, with correlation coefficients (r2) > 0.9993. Repeated measurements of the peak area using 10 mg/L standard solutions of each florfenicol enantiomer showed relative standard deviations (RSD, n=6) of 0.65 and 0.81%, respectively. This method was then used to separate and measure commercially available standard florfenicol racemates. The results indicate that (+)-florfenicol was not detected in the three standard florfenicol racemates, but (-)-florfenicol (9.20~10.00 mg/L) was detected in all of them. The separation and content determination of the two florfenicol enantiomers were completed within 6.0 min. Therefore, this is a rapid, high-resolution, eco-friendly method that can be used for the separation and determination of florfenicol enantiomers.
