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Quantitative determination of clenbuterol enantiomers in human plasma by high‐performance liquid chromatography using the macrocyclic antibiotic chiral stationary phase teicoplanin 1
Author(s) -
AboulEnein Hassan Y.,
Serignese Vince
Publication year - 1999
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/(sici)1099-0801(199912)13:8<520::aid-bmc919>3.0.co;2-7
Subject(s) - chemistry , chromatography , clenbuterol , enantiomer , high performance liquid chromatography , triethylamine , methanol , calibration curve , analytical chemistry (journal) , detection limit , organic chemistry
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 ™ (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 µ M . The correlation coefficient ( r ) range was 0.99988–0. (mean = 0.). The lowest concentration measured was 0.25 µ M . Inter‐ and intra‐assay variation was determined for the lowest, medium and highest plasma concentration (0.25, 2 and 10 µ M ) 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. Copyright © 1999 John Wiley & Sons, Ltd.