ESPE Abstracts (2022) 95 P1-214

ESPE2022 Poster Category 1 Adrenals and HPA Axis (52 abstracts)

Development and validation of simultaneous quantification of urinary catecholamines and free metanephrines by UHPLC-MS/MS method

Maria Eugenia Rodriguez , Maria Gabriela Ballerini , Veronica Ambao , Ana Vieites , Gabriela Sanso , Rodolfo Rey , Ignacio Bergada & Maria Gabriela Ropelato


Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE) CONICET – FEI – División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Ciudad de Buenos Aires, Argentina

Background: Neuroendocrine tumors from adrenal and extra-adrenal chromaffin cells are characterized by overproduction of catecholamines (CA), leading to the increase of urinary O-methylated metabolites (uO-Met) excretion, reliable biomarkers for the initial screening, diagnosis and monitoring of patients with pheochromocytoma and paragangliomas (PPGLs).

Objective: To develop and validate a UHPLC-MS/MS method for the simultaneous quantification of Epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT).

Methods: UHPLC-MS/MS system (Shimadzu Nexera 20A-AB Sciex QTRAP®6500 system). 15 minutes run; ESI+. Method performance was validated for recovery, linearity, precision and accuracy, ion suppression and carry over. Lower limit of detection (LLD): signal/noise ratio 3. Lower Limit Of Quantitation (LLOQ): lowest concentration with CV% <20% (recovery between 80 and 120%). We included 24hs urine samples from 6 patients with confirmed tumor [Group 1; 4 PPGLs and 2 neuroblastoma (NB)], and Group 2, 10 patients with clinical suspicion of neural crest cells tumors (5 with molecular diagnosis). Median age and range 20.5 (0.2 to 49) years. Results from patients were evaluated according to reference ranges from Peitzsch et al. (1).

Results: Recoveries ranged from 90 to 110%; intra- and inter-CVs% from 2 to 15%; linearity coefficient >0.9900. Analytical range, LLOD and LLOQ (Relative Standard deviation, RSD%) for each analyte are shown in the table. In Group 1, NMN was elevated in all patients, NE in 4, while EPI and MN was elevated in 1/6. Of note, DA was elevated in 2/6 and 3MT in 5/6. In group 2, CA were within normal range in all patients, however, we observed that uO-Met were elevated in the 4 patients with confirmed molecular diagnosis.

Analyte (mg/l) Analytical range LLOD LLOQ (RSD%)
EPI 0.75–135 0.30 0.75 (8.7)
NE 1.50-270 0.90 1.50 (8.7)
DA 20 – 3600 1.57 20 (9.3)
MN 25 – 650 1.12 25 (2.9)
NMN 31.3 – 812.5 7.4 31.3 (5.4)
3MT 25 – 650 0.64 25 (2.0)

Conclusions: We developed and validated an UHPLC-MS/MS method for the accurate simultaneous measurement of urine endogenous CA as well as their respective free uO-Met metabolites. The preliminary results suggest that our method enables the confirmation of the tumor presence in patients with PPGLs and NB. It would be interesting to explore the usefulness of free uO-Met measurement by our method in a larger cohort of patients with clinical suspicion of neuroendocrine pathologies. (1) Peitzsch et al. doi: 10.1016/j.cca.2012.12.03

Volume 95

60th Annual ESPE (ESPE 2022)

Rome, Italy
15 Sep 2022 - 17 Sep 2022

European Society for Paediatric Endocrinology 

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