IMPE Abstracts

Background: Catecholamines and its O-methylated metabolites in urine are useful biomarkers for diagnosis of Pheochromocytoma and Paraganglioma (PPGL). Recently, liquid chromatography coupled to tandem mass spectrometry (UHPLC-MSMS) has become the technique that allows the development of analytical methodologies for the specific, precise, and simultaneous determination of different analytes.

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

Methods: UHPLC-MS/MS system (Shimadzu Nexera 20A-AB Sciex QTRA P®6500 system). Method performance was validated for recovery, linearity, precision, accuracy, ion suppression and carry over. Lower limit of detection (LLD: signal/noise ratio 3), lower limit of quantitation (LLOQ: the lowest concentration with CV% <20%) and recovery between 80 and 120%. We included 14 patients (19 urine samples) with PPGL and 14 subjects with symptoms of suspected PPGL (Non-PPGL) and 5 with positive molecular diagnosis PPGL without positive imaging test (MD). Kruskal-Wallis test with multiple comparison were performed. Reference values were adapted from Peitzsch et al. (Clin Chim Acta. 2013)

Results: EPI, NE, DA, MN, NMN and 3MT were measured by ESI+ ionization. Recoveries ranged from 90 to 110%; intra- and inter-CVs% from 2 to 15%; linearity coefficient >0.99. LLQ were EPI 4.09, NE: 8.87, DA: 130.57, MN: 5.68, NMN: 40.4, 3MT: 40 nmol/24hs. In PPGL patients, NE was elevated in 14 and NMN in 17/19 samples. Of note, 3MT was elevated in all patients with MD and 10 patients with PPGL. Samples results (nmol/24hs) are shown in the table (mean ± SEM, ^a P<0.01 vs non-PPGL, ^b P<0.05 vs MD).

Conclusions: We developed and validated an UHPLC-MS/MS method for the accurate simultaneous measurement of urine CA and its free metabolites. The preliminary results suggest that our method enables the confirmation of the tumor presence in patients with PPGL. Urinary free O-methylated metabolites appears to be particularly useful to rule out PPGL and for the follow-up of patients with positive molecular diagnosis without evidence of tumor.