B-206 The Determination of Vanillylmandelic Acid (VMA), Homovanillic Acid (HVA) and 5-Hydroxyindolacetic Acid (5-HIAA) by LC-Ms/MS Assay in Urine Samples for Diagnosis of Neuroendocrynes Tumors

Abstract Background The catecholamines dopamine, norepinephrine and epinephrine, as well the neurotransmitter serotonin are biogenic amines that were formed by enzymatic descarboxylation of aminoacids in the body. They realize an important function in central nervous system. Vanillylmandelic acid (VMA) and Homovanillic Acid (HVA) are degradation products of the catecholamines, and 5-Hydroxyindolacetic Acid (5-HIAA) is a serotonine degradation product. They are important to the diagnosis of neuroendocrynes tumors, that produce catecholamines and/or serotonine in excess, resulting in elevated levels of VMA, HVA, and/or 5-HIAA in 24 h collection urine. Objective This study aimed to validate a simple LC-MS/MS method for quantification of VMA, HVA and 5-HIAA in isolated and 24 h urine samples for the diagnosis of neuroendocrines tumors. Methods Using a deep-well microtiter plate, 20 µL of isolated or 24 h urine was diluted in 540 µL of 0.05% of formic acid solution. Then, 20 µL of an internal standard solution containing stable isotopes of the analytes were added (VMA-D3, HVA-13C6,18O and 5-HIAA-13C6). The plate was sealed, homogenized for 30 s at 2500 rpm, and centrifuged for 15 min at 4000 rpm and ambient temperature. Then, 5.0 µL of the supernatant was injected into the LC-MS/MS system. Chromatographic separation was performed on the Waters ACQUITY UPLC system equipped with Kinetex XB-C18 1.7 µm X 50 mm X 2.1 mm column (Phenomenex), and methanol and formic acid 0.2% solution as mobile phase in 3.5 min of gradient mode separation. The detection was performed on a Waters XEVO TQ-S Micro mass spectrometer with electrospray ionization (ESI+). The current method was validated for carryover, linearity, precision, recovery, and limit of quantification, and it was evaluated by comparing the analysis in the developed method and a commercial kit by HCPL-ECD, of 15 real samples from volunteers in the laboratory. Results The method was successfully validated for all the parameters. The linearity was between 0.5–100.0 mg/L. The precision was less than 6.3%, the recovery was between 85.4%–103.4% and the quantification limit was 0.5 mg/L. The comparison with the reference method showed good coefficient correlations (R2 > 0.92) for the three analytes. Conclusion We validate a simple LC-MS/MS method to quantify three metabolites to help the diagnosis of neuroendocrynes tumors. The method represents a simplification of the extraction compared with the commercial kit employing HPLC-ECD, and an important reduction of the per sample production cost, without loss of quality.