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

Abstract

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 µM. The correlation coefficient (r) range was 0.99988–0.99999 (mean = 0.99999). The lowest concentration measured was 0.25 µM. Inter‐ and intra‐assay variation was determined for the lowest, medium and highest plasma concentration (0.25, 2 and 10 µM) 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.

Methotrexate determination in pharmaceuticals by enantioselective HPLC

“…The polar organic mode was originally developed as a novel way to obtain difficult enantioselective separations on cyclodextrinbased phases, while suppressing inclusion complex formation [38]. Therefore this mode was approached due to the presence of strong polar groups in the macrocyclic peptides and the presence of more than one functional group in Mtx and FA capable of interacting with the chiral selector (some of these groups are near or on the stereogenic center).…”

“…The concentrations of acid and base were varied from 2.0 to 0.1% in methanol. In fact, above 2% the mobile phase becomes too polar and the system assumes typical reversed phase behaviour, whereas below 0.1% it behaves as in normal phase chromatography [38]. No separation of rac-Mtx enantiomers in the presence of 1/2 acid/base ratio occurred, while increasing the acid percentage, enantioselectivity and enantioresolution of rac-Mtx increased.…”

“…Therefore, a satisfactory separation between FA and the Mtx enantiomers was achieved for the simultaneous determination of both drugs under the above conditions. By using a reversed phase mode on antibiotic stationary phases, it was reported that the nature of organic modifiers affects the selectivity of separation and the presence of buffers increases the efficiency and the resolution [38]. Therefore, the enantioseparation of rac-Mtx and FA with mixtures consisting of 20% methanol and 10% acetonitrile and water or TEA buffer as mobile phases was investigated.…”

Recent applications of stereoselective chromatography

“…The assays have been carried out under normal and reversed-phase conditions. 98 The macrocyclic antibiotic teicoplanin has been used as a chiral selector for the determination of clenbuterol enantiomers in human 99 and rat 100 plasma by HPLC. The teicoplanin analytical column has also been applied in the enantioselective assay of propranolol 94 and albuterol enantiomers.…”

Separation of the enantiomers of substituted dihydrofurocoumarins by HPLC using macrocyclic glycopeptide chiral stationary phases

“…All possible molecular interactions, including ionic interactions, hydrogen bonding, steric, dipole-dipole and π-π interactions, as well as hydrophobic interactions responsible for chiral recognition, are available within their relatively compact structures. This class of CSP is multimodal, which means they can be utilized in any of the known mobile phase modes including normal-phase, reverse-phase and polar organic phase modes [15,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56].…”