Trace analysis of clenbuterol using supercritical fluid extraction and gas chromatographic techniques

Clenbuterol is a veterinary drug approved for use in Europe in horses as a bronchial dilator and tocolytic agent. However, it may be being used in an unapproved manner in the Unites States for growth-promoting purposes in cattle. Therefore, a method is needed to monitor for this illegal use. The use of gas chromatography (GC) with electron-capture detection has been shown to be very effective in quantitating clenbuterol at trace concentrations. The procedure was quantitative down to 0.75 $\eta$g of clenbuterol, and it was determined that three structurally similar compounds; cimaterol, salbutamol, and ractopamine, did not interfere. For confirmation of clenbuterol, two different modes of GC-mass spectrometry (MS) were employed. The first mode, which utilized an electron impact ionization source, produced mass spectra containing a weak molecular ion and four characteristic daughter ions. The second mode employing negative ion chemical ionization (NCI) better identified the molecular ion and characteristic chlorine isotope peaks. The threshold pressures for clenbuterol from bovine liver tissue by supercritical fluid extraction (SFE) were established. When clenbuterol was extracted with pure CO$\sb2$, 95% CO$\sb2$:5% MeOH, and 90% CO$\sb2$:10% MeOH, the threshold pressures were approximately 150, 150, and 100 atm, respectively. The effect of temperature on the extraction efficiency of clenbuterol from bovine liver was investigated from 35$\sp\circ$C to 85$\sp\circ$C, and as temperature was increased, the overall recover of clenbuterol from the tissue increased. Water content was determined to play a critical role in the SFE efficiency of clenbuterol from bovine liver. An unaltered tissue sample when extracted produced a recovery of 90%, as compared to 25% and 12% for heat dried and freeze dried samples, respectively. A two-fraction scheme produced an excellent off-line SFE-GC procedure in which 50 ppb of clenbuterol could be quantitatively extracted from bovine liver with a recovery of 76%. After attempting many different on-line SFE-GC schemes, it was determined that due to the high concentration of endogenous compounds present in the liver, on-line SFE-GC analysis of any trace compound in liver would be extremely difficult, if achievable at all.