Performance characteristics of an LC-MS/MS method for the determination of plasma metanephrines.

BACKGROUND: Catecholamines and their metabolites, metanephrines, are produced excessively in pheochromocytoma tumors of the chromaffin cells. Increased concentrations of these compounds produce symptoms and allow for clinical evaluation of disease. Historically, screening for such tumors by determination of catecholamines and metabolites in urine yielded false negative results in individuals with a genetic predisposition for the disease and those with paroxysmal hypertension. Analysis of metanephrines in plasma, however, is of decisive diagnostic importance. This test exhibits high sensitivity and specificity for the analytes produced by tumors. METHODS: Plasma proteins are removed by solid phase extraction. Chromatographic isolation of the analytes and stable isotope internal standards is achieved by elution on a HILIC column connected to a UPLC MS/MS system. Metanephrines are measured using multiple reaction monitoring with an electrospray source operating in positive ion mode. RESULTS: The method was validated for linearity, limit of quantification, accuracy, and precision. The method was accurate and correlated well to a comparison HPLC method. Potential interferences were evaluated. CONCLUSIONS: Results from this LC-MS/MS assay enable clinical diagnosis of pheochromocytoma and aid in monitoring treatment outcomes.

Aldose reductase: an aldehyde scavenging enzyme in the intraneuronal metabolism of norepinephrine in human sympathetic ganglia.

The neurotransmitter norepinephrine is metabolized by monoamine oxidase into an aldehyde intermediate that is further metabolized to the stable glycol derivative, 3,4-dihydroxyphenylglycol (DHPG). In this study, the possible role of aldose reductase in reducing this aldehyde intermediate in human sympathetic neurons has been examined. DHPG is formed when norepinephrine is incubated with aldose reductase in the presence of monoamine oxidase. DHPG metabolism is inhibited by the monoamine oxidase inhibitor, pargyline which prevents the deamination of norepinephrine, and by the aldose reductase inhibitor AL 1576, which inhibits DHPG formation without affecting the deamination of norepinephrine. Although similar formation of DHPG was observed with human liver aldehyde reductase, the production of DHPG was more effective with aldose reductase than aldehyde reductase. Two peaks of reductase activity corresponding to aldose reductase and aldehyde reductase were observed when sympathetic ganglia were chromatofocused. Molecular modeling studies indicate that the energy-minimized structure of 3,4-dihydroxymandelaldehyde bound to aldose reductase is similar to that of glyceraldehyde where the 2'-hydroxyl group forms hydrogen bonds with Trp111 and NADPH. These results suggest that aldose reductase may be important in metabolizing the potentially toxic aldehyde intermediate formed from norepinephrine in human sympathetic ganglia.

Simultaneous measurement of total catecholamines and metanephrines in human urine by liquid chromatography with coulometric detection.

A simple routine method is described for simultaneous assay of total urinary norepinephrine, epinephrine, dopamine, normetanephrine and metanephrine. An internal standard of 3,4 dihydroxybenzylamine is added to the diluted urine and acidic hydrolysis of the conjugates is followed by reverse-phase HPLC separation and coulometric detection in the redox mode. The method is rapid and precise and it has a broad linear working range for all substances making it suitable for clinical analysis. Examples are shown of excretion patterns of catecholamines and metanephrines for healthy subjects and depressed patients.