Determination of harmane and harmine in human blood using reversed-phased high-performance liquid chromatography and fluorescence detection

A number of tremorogenic beta-carboline alkaloids have been found in common plant-derived foodstuffs, beverages, and inhaled substances. Because of th eir natural presence in the food chain, there is a growing concern regardin g the potential risks of certain essential tremors associated with the long -term, low-level dietary exposure to these alkaloids. The purpose of this s tudy was to develop an effective analytical method to determine blood level s of two major beta-carboline derivatives, harmane and harmine. Human blood was extracted with ethyl acetate and methyl-t-butyl ether (2:98) under an alkaline condition. After evaporation of organic solvent, the samples were reconstructed in methanol. The samples were fractionated on a 250 x 4.6-mm C18 reversed-phase column with an isocratic mobile system consisting of 17. 5 mM potassium phosphate buffer (ph 6.5) and methanol (30: 70), followed by an on-line fluorescence detection. The method had the detection limit to d etermine 206 and 81 pg/ml of harmane and harmine, respectively, in 10 mi of human blood. The intraday precision (C.V.) at 25 ng/ml was less than 6.7 a nd 3.4% for harmane and harmine, respectively. The interday precision was 7 .3% for harmane and 5.4% for harmine. The method has proven sensitive, repr oducible, and thus useful for both laboratory and clinical studies of beta- carboline toxicities. (C) 2000 Academic Press.