Abstract
This study describes the development and application of sensitive and specific, reverse-phase high performance liquid chromatography procedures for the measurement of caffeine and its metabolites in biological fluids. Satisfactory resolution of these compounds was achieved using Hypersil octadecylsilane columns with elution solvents consisting of buffered aceto-nitrile solutions containing tetrahydrofuran as an ion-pairing reagent. Eluted compounds were detected by monitoring their UV absorption at a wavelength of 280 nm. Conditions for processing body fluids for analysis were optimised using organic extraction techniques with proxyphylline incorporated as an internal standard. Maximum recovery of urine metabolites required the formation of ion-pair complex with tetrabutyl-ammonium. hydrogen sulphate. Serum caffeine levels determined by HPLC and a radioimmunoassay procedure showed good agreement. Furthermore, caffeine concentrations in saliva, following oral ingestion of caffeine, correlated well with corresponding values found in serum. An assessment of the consumption of caffeinated beverages in a local population indicated that the dietary intake of caffeine in the U.K. is significantly higher than that previously reported for American and Canadian populations. Measurement of serum caffeine levels in non-pregnant subjects showed a mean value of 2.42 mg/l; 6% of the values were in excess of 5 mg/l which is comparable to the average peak level found following a 250 mg oral dose of caffeine. Reference ranges are established for serum and salivary pharmacokinetic data and recoveries of metabolites from urine of normal volunteers who received oral doses of caffeine. In addition the electrophysiological and psychopharmacological responses to oral doses of caffeine are reported following a collaborative study with the Institute of Psychiatry, London. Evidence for alterations in the biotransformation of caffeine during pregnancy was obtained by comparing urine metabolite concentrations in pregnant women, receiving a known dietary intake of caffeine, with values obtained for a non-pregnant control group. It is suggested that hormonal influences on the hepatic drug metabolising enzymes may be implicated. Whilst pharmacologically significant concentrations of caffeine and its metabolites were detected in samples of maternal serum, cord blood and breast milk, no evidence was found to relate caffeine levels in the newborn infant with the onset of attacks of jitteriness or neonatal apnoea. In a study of caffeine elimination in chronic liver disease, the clearance of caffeine in compensated cirrhotic patients was similar to that of normal controls. However for patients with decompensated liver disease, significant levels of impairement were found in both serum and salivary caffeine clearance. This change in the disposition of caffeine was also reflected in the profiles of it metabolites excreted in the urine. Since the biotransformation pattern of caffeine in these patients was shown to be normal, this impairement of caffeine clearance could be attributed to a reduction in intrinsic clearance resulting from a reduced mass of viable hepatocytes. Preliminary findings indicated that salivary caffeine clearance measurements might provide an accurate means of assessing the extent of hepato-cellular dysfunction.