HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF GUANIDINO COMPOUNDS BY AUTOMATED PRECOLUMN FLUORESCENCE DERIVATIZATION

An automated pre-column derivatization approach was used to develop an high-performance liquid chromatographic method for the determination of endogenous guanidino compounds. A commercially available autoinjector which was capable of adding and mixing reagent solutions and timing the reaction prior to injection was used to generate a fluorescent product by reaction of guanidino compounds with alkaline ninhydrin. The fluorescent products were separated by reversed-phase chromatography and detected with a fluorometer. Optimization of the pre-column reaction conditions resulted in a simple, highly sensitive and specific analytical method for the determination of guanidino compounds with excellent reproducibility and linearity. Application of this methodology to the determination of methylguanidine in human plasma samples resulted in a limit of quantification of 1 ng/ml (13.7 pmol/ml). The method was successfully employed for the quantification of circulating levels of methylguanidine in normal human subjects and uremic patients. The methodology should be generally applicable to the detection of other guanidino compounds in biological fluids. © 1990.