Evaluation of high pressure liquid chromatography, gas chromatography and fluorometry for quantitation of urinary free catecholamines

Abstract

Analytical methods for the quantitation of urinary free norepinephrine, epinephrine and dopamine are described. High pressure liquid chromatography (HPLC) with conventional ultraviolet (UV) absorbance detection at 54 nm and with electrochemical detection is investigated. Urine eluates for electrochemical detection are obtained from a two-step purification procedure, with incorporation of an internal standard, 3, 4-dihydroxybenzylamine (DHBA) throughout the extraction. Rapid and simple quantitation of norepinephrine, epinephrine and dopamine from a 5-ml urine aliquot is achieved with electrochemical detection. Problems encountered with HPLC analysis are also discussed. TMS-TFA derivatization of standard catecholamine solutions prior to gas chromatographic (GLC) analysis gives poor peak resolution. On-column acetaminde derivatization of catecholamine standards utilizing gas chromatography gives better peak resolutions, with potential application to urinary free catecholamine quantitation. Comparison of precision data from traditional fluorometric estimation of norepinephrine and epinephrine with that for the HPLC method shows the latter to be more precise in most respects. Relative merits of each method are discussed, as well as ease of implementation into the clinical laboratory.