Synthetic calibrators for the analysis of total metanephrines in urine: Revisiting the conditions of hydrolysis

BackgroundThe quantification of total (free + sulfated) metanephrines in urine is recommended to diagnose pheochromocytoma. Urinary metanephrines include metanephrine itself, normetanephrine and methoxytyramine, mainly in the form of sulfate conjugates (60–80%). Their determination requires the hydrolysis of the sulfate ester moiety to allow electrochemical oxidation of the phenolic group. Commercially available urine calibrators and controls contain essentially free, unhydrolysable metanephrines which are not representative of native urines. The lack of appropriate calibrators may lead to uncertainty regarding the completion of the hydrolysis of sulfated metanephrines, resulting in incorrect quantification.MethodsWe used chemically synthesized sulfated metanephrines to establish whether the procedure most frequently recommended for commercial kits (pH 1.0 for 30 min over a boiling water bath) ensures their complete hydrolysis.ResultsWe found that sulfated metanephrines differ in their optimum pH to obtain complete hydrolysis. Highest yields and minimal variance were established for incubation at pH 0.7–0.9 during 20 min.ConclusionUrinary pH should be carefully controlled to ensure an efficient and reproducible hydrolysis of sulfated metanephrines. Synthetic sulfated metanephrines represent the optimal material for calibrators and proficiency testing to improve inter-laboratory accuracy.

► Sulfate metanephrines have been synthesized.
► Optimum pH and time to obtain full hydrolysis of sulfate metanephrines in urine are 0.7–0.9 and 20 min.
► Synthetic sulfate metanephrines are useful for calibration and proficiency testing.