Improved micro-method for the high-performance liquid chromatographic determination of caffeine and paraxanthine in...

A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in microsamples. A 5-micron reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentrations as low as 80 ng/ml using only 25 microliter of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebrospinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy.