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

Abstract

Background: The quantification of total (free + sulfated) metanephrines in urine is recommended to diagnose pheochromocytoma. Urinary metanephrines include metanephrine itself, normetanephrine and methoxytyra-mine, mainly in the form of sulfate conjugates (60-80%). Their determination requires the hydrolysis of the sul-fate 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 hy-drolysis of sulfated metanephrines, resulting in incorrect quantification. Methods: We used chemically synthesized sulfated metanephrines to establish whether the procedure most fre-quently recommended for commercial kits (pH 1.0 for 30 min over a boiling water bath) ensures their complete hydrolysis. Results: We 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. Conclusion: Urinary pH should be carefully controlled to ensure an efficient and reproducible hydrolysis of sul-fated metanephrines. Synthetic sulfated metanephrines represent the optimal material for calibrators and pro-ficiency testing to improve inter-laboratory accuracy.

Enzyme and acid deconjugation of plasma sulfated metanephrines

“…The data for hydrolysis of NMN-S and MN-S were normally distributed accordingly to the Shapiro-Wilk test, whereas MT-S data were not normally distributed probably reflecting a higher variability for the deconjugation of this analyte. These results are in agreement with our previous work on urines [6], indicating that 30 min is ideal to hydrolyze sulfated metanephrines and that the nature of the matrix does not affect the hydrolysis rate as long as the pH is close to 1.0 (pH at 1.2 in our condition). However, our data are in total discrepancy with the work of Pagliary et al [7], who found a maximum concentration of the deconjugation of plasma S-NMN and S-MN submitted to acid hydrolysis after only 10 min and gradually lower concentrations of deconjugated metanephrines when incubation time was 20 and 30 min.…”

“…The chemical protocols used for the synthesis of each sulfate metanephrines has been previously published [6]. All commercially available reagents and solvents (Fluka/Aldrich, Buchs, CH and Acros, Wohlen, CH) were used without further purification.…”

“…Besides, no extraction procedures have yet been reported for sulfated metanephrines for LC-MS/MS quantification. We have previously reported that urine sulfated metanephrines are entirely deconjugated by a treatment consisting of boiling acid perchloric for 30 min at pH 1.0 [6]. Intriguingly, an early study by Pagliari et al [7] established that by following the same methodology, the hydrolysis time should not last more than 10 min to prevent loss of metanephrines.…”

Laboratory Evaluation of Pheochromocytoma and Paraganglioma

“…Importantly, efficiency of the acid hydrolysis conversion of sulfate conjugated to free metanephrines de-pends on pH, temperature, and time of incubation (21,22 ). This would not be a problem if calibrators and QC samples recapitulated the proportions of free and conjugated metabolites in patient samples.…”

“…This would not be a problem if calibrators and QC samples recapitulated the proportions of free and conjugated metabolites in patient samples. However, commercially available calibrators and control samples used in assays of urine fractionated metanephrines are provided predominantly or entirely in the free form (18,21 ). Thus, unless deconjugation conditions are adequately controlled, concentrations of urine metanephrines in patient samples can suffer underestimation without parallel underestimation of controls (Fig.…”