Quantitative determination of clenbuterol enantiomers in human plasma by high-performance liquid chromatography using the macrocyclic antibiotic chiral stationary phase teicoplanin1

We report a method for the high-performance liquid chromatographic (HPLC) chiral separation of racemic clenbuterol in human plasma. Human plasma was spiked with stock solutions of clenbuterol hydrochloride and practolol as the internal standard. Following a liquid-liquid extraction procedure with 10% (+/-)-2-butanol/isopropyl ether under alkaline conditions, the dried samples were reconstituted in methanol and chromatographed using a macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T(trade mark) (teicoplanin). The mobile phase composition was methanol:acetonitrile (70:30, v/v), containing 0.3% (v/v) acetic acid and 0.2% (v/v) triethylamine. The resulting chromatogram achieved baseline separation for the clenbuterol enantiomers. Calibration curves (peak area ratio vs plasma concentration, n = 10) were constructed for the (-)-R-and (+)-S-clenbuterol enantiomers with a plasma concentration range of 0. 25-10 microM. The correlation coefficient (r) range was 0.99988-0. 99999 (mean = 0.99999). The lowest concentration measured was 0.25 microM. Inter- and intra-assay variation was determined for the lowest, medium and highest plasma concentration (0.25, 2 and 10 microM) by calculating the analytical recoveries with a range of 96-104%. The percentage recoveries for the clenbuterol enantiomers were 88.4-102% over the concentration range used. Detailed methodology is presented.