| Description |
The catecholamines: dopamine, norepinephrine, and epinephrine, are naturally occurring amines that function as hormones and neurotransmitters. Excess concentrations of catecholamines have been observed in patients with rare neuroendocrine tumors and are associated with chronic hypertension, stroke, myocardial infarction, and cardiovascular disease. These potential consequences of high catecholamine concentrations emphasize the need for a rapid and accurate analytical measurement method. Catecholamines are often measured using high performance liquid chromatography (HPLC) with electrochemical detection; however, the liquid chromatography tandem mass spectrometry (LC-MS/MS) technique is attractive due to its selectivity and throughput. Derivatization of catecholamines prior to LC-MS/MS analysis of plasma specimens may be used to enhance assay sensitivity. The goal of this study was to evaluate reductive amination derivatization of catecholamines using straight-chain and branched-chain aldehydes as a means to improve the sensitivity of the assay. Derivatization was performed on each catecholamine in triplicate using a series of straight-chain and branched-chain aldehydes. Aqueous catecholamine standards were reacted with an aldehyde in the presence of a buffer (ammonium acetate) and reducing agent (sodium cyanoborohydride) at 37 °C for 30 minutes. Samples were quenched with formic acid at room temperature and injected onto an LC-MS/MS system for analysis. Catecholamine derivatives were identified by individual retention times and mass transitions. Peak area counts were determined for three mass transitions for each derivative. The six-carbon straight-chain aldehyde, hexanal, and the branched-chain aldehyde, hydrocinnamaldehyde, proved to be the most effective derivatizing agents for the catecholamines in plasma assay. A derivatization protocol using hydrocinnamaldehyde was optimized for aldehyde and reducing agent concentrations, and incubation time and temperature. Derivatization with hydrocinnamaldehyde produced single alkylated products for all three catecholamines. Comparison of the derivatization agents showed higher peak area counts for norepinephrine and epinephrine derivatized with hydrocinnamaldehyde; hexanal provided greater sensitivity for dopamine. A method application experiment on patient samples using hydrocinnamaldehyde demonstrated its significant effect on assay sensitivity and supports use in a clinical setting. However, sensitivity for dopamine was inadequate and overall accuracy and precision were unsatisfactory. Further optimization of the derivatization protocol using hydrocinnamaldehyde is required to meet acceptable analytical criteria for this assay. |
