[Separation and determination of clenbuterol enantiomers by ultra-performance convergence chromatography]

Clenbuterol enantiomers differ greatly in their bioactivities. By optimizing the conditions for chromatographic separation and method validation, ultra-performance convergence chromatography (UPC²) was adopted to separate the enantiomers of clenbuterol. Standard solutions of (+)-clenbuterol and (-)-clenbuterol were stored at -18 ℃ for 1, 3, 5, 7, 14, 30, and 60 d, and then, their stability was monitored. The impacts of different chromatographic columns, cosolvents, system backpressure, and chromatographic column temperature on the separation of the two enantiomers were investigated. Acquity Trefoil AMY1 (150 mm×3.0 mm, 2.5 μm) was used for separation, and CO₂-0.5% (v/v) ammonium acetate was used as the mobile phase. Gradient elution at a flow rate of 2.0 mL/min was adopted. The detection wavelength was set to 241 nm, and the injection volume was set to 10 μL. The backpressure was set to 13.8 MPa, and the column temperature was maintained at 40 ℃. The two enantiomers showed good linear relationships in the range of 1.0 to 20.0 mg/L with correlation coefficients greater than 0.9997. The limits of detection (LODs, S/N=3) of (+)-clenbuterol and (-)-clenbuterol were both 0.5 mg/L. The relative standard deviation (RSD, n=6) for the peak area of the 10.0 mg/L mixed standard working solution with six replicate injections ranged from 0.65% to 0.76%. The effectiveness and practicability of this method were demonstrated by using it to detect standard clenbuterol racemate. The (+)-clenbuterol and (-)-clenbuterol contents were 5.6 mg/L and 5.5 mg/L, respectively, in the standard clenbuterol racemates, as determined by the external standard method of quantification. The detection results suggested that the content ratio of (+)-clenbuterol and (-)-clenbuterol was close to 1.02∶1.00, which is consistent with the literature data. The established method has the advantages of rapid analysis, good separation effect, and low consumption of organic solvents, and it is suitable for the separation of clenbuterol enantiomers. This method can also provide technical support for the separation of other chiral drugs, analysis of the effects of chiral drugs, and assessment of product quality.