Gas/liquid chromatographic analysis of pemoline in biological fluids using electron capture detection.

The study presents a detailed methodology for identifying and quantifying pemoline, a stimulant drug, in the biological fluids of horses, using a gas/liquid chromatographic technique.

Method Overview

• The scientists started by obtaining plasma samples from horses that contained known quantities of pemoline along with an internal standard (IS) which is an analog of the substance under investigation.
• The plasma samples were deproteinized using 5-sulfosalicylic acid, then heated at 80 degrees Celsius, and centrifuged. This process is undertaken to remove proteins from the liquid that may interfere with the chemical analyses.
• Following this, 5-Phenyl-2,4-oxazolidinedione was extracted. It is the breakdown product of pemoline in acidic conditions.
• The extraction was done using dichloromethane (DCM), a commonly used organic solvent for extractions in analytical chemistry. The DCM layer (which contained the compound of interest) was separated and re-extracted with 1% sodium bicarbonate (NaHCO3).
• The aqueous layer obtained was acidified with hydrochloric acid (HCl) and re-extracted using DCM. This ensures that non-acidic impurities were removed from the target compound.

Method Specifics

• The DCM was then evaporated, leaving a residue which was methylated with diazomethane in ether, transforming it into a compound more amenable to gas-liquid chromatography.
• The newly formed methylated dione was then dissolved in benzene and analyzed with a gas/liquid chromatograph that uses a tritiated scandium foil (Sc3H) electron capture detector (ECD). This analytical tool allows for the separation and identification of chemical compounds.
• This process was also carried out on urine samples that had pemoline added to it. The urine samples underwent an additional step of extraction using lead acetate solution before the sodium bicarbonate extraction.
• The sensitivity of the method was found to be 0.05 micrograms/ml for plasma samples and 0.1 micrograms/ml for urine samples when 1.0 ml of the sample was used.

Applications

• This method can be used to detect and quantify pemoline levels in horses, a process especially useful for doping control during international equestrian events.