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

RODRIGUEZ MARIA EUGENIA; BALLERINI MARÍA GABRIELA; AMBAO VERÓNICA; VIEITES ANA; SANSÓ GABRIELA; BERGADÁ IGNACIO; REY RODOLFO; ROPELATO MARÍA GABRIELA

Congreso; 60th Annual ESPE Meeting; 2022

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. Nowadays, measurement of free uO-Met fraction constitutes a better biomarker for diagnosis and monitoring patients with pheochromocytoma. Besides, this method avoids the requirement of acid hydrolysis step.Objective: To develop and validate a UHPLC-MS/MS method for the simultaneous quantification of both free CA and uO-Met. Methods: UHPLC-MS/MS system (Shimadzu Nexera 20A-AB Sciex QTRAP®6500 system). ESI+ ionization method. Epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT) were simultaneously measured in a 15 minutes run. 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% 0.9900. The table shows the analytical range, LLOD and LLOQ (Relative Standard deviation, RSD%) for each neurotransmitter.Analyte (ug/L)Analytical rangeLLOD LLOQ (RSD%)Patient results (ug/24 hs; median and range)EPI0.75–1350.300.75 (8.7)5.65 (0.43-29.7)NE1.50-2700.901.50 (8.7)50.58 (8.58-955)DA20 – 36001.5720 (9.3)335.3 (171.6-2650)MN25 – 6501.1225 (2.9)19.9 (1.4-71.7)NMN31.3 – 812.57.431.3 (5.4)41.9 (12.7-1109)3MT25 – 6500.6425 (2.0)56.4 (33.54-422)Results from patients were evaluated according to reference ranges from Peitzsch et al. (1): EPI was elevated in 1 and MN in 4/18 Pheo, NE in 4 and NMN in 6 Pheo and 2 NB out of 18 tumors and DA and 3MT were elevated in 2 NB. EPI and NE by ULPC-MS/MS and HPLC methods presented a clinical agreement in 100% of the patients.Conclusions: A UHPLC-MS/MS method was developed and validated for the accurate simultaneous measurement of 6 neurotransmitters in urine samples. Free uO-Met measurement allowed the identification of a higher proportion of patients with confirmed neural crest tumors, thus, demonstrating its applicability for screening of patients with clinical suspicion of neuroendocrine pathologies. (1) Peitzsch et al. doi: 10.1016/j.cca.2012.12.031