A-103 Stability Study of the Biogenic Amines 5-Hydroxyindoleacetic Acid (5-HIAA), Vanillylmandelic Acid (VMA), and Homovanillic Acid (HVA) in Acidified Vs Non-acidified Human Urine Samples
Authors: Junior L F , Paulo B F , Zauli D A 
Published: September 2023

Abstract

Background
Carcinoid tumors and neuroblastomas are disorders in which some neurotransmitters are produced. The first one is characterized by the overproduction of serotonin, which the main metabolite is 5-Hydroxyindoleacetic acid (5-HIAA). In neuroblastomas, approximately 90% of the tumors produce catecholamines, wherein Vanillylmandelic acid (VMA) and Homovanillic acid (HVA) are the main final products of catecholamine metabolism. Due to the great clinical importance of these disorders, our group previously developed a very sensitive method to identify and quantify VMA, HVA, and 5-HIAA by LC-MS/MS. Thus, was performed a study of sample stability to identify the impact of no-acidification of the samples and the optimal storage temperature to perform a high accuracy analysis. The study aimed to evaluate stability of acidified and non-acidified urine samples under refrigerated and frozen conditions to identify and quantify VMA, HVA and 5-HIAA compounds.

Methods
Isolated urine samples were collected from 24 volunteers. For each compound, the stability of non-acidified samples was compared with HCl acidified samples, aiming for a final pH among 1 and 4. Independent aliquots of the samples were made to obtain the following cohort: Baseline (day of collection acidification), 24 h post-collection acidification, 4 days, 8 days and 15 days acidification. Those samples were divided into 4 groups: A—refrigerated storage and non-acidified; B—frozen and non-acidified; C—refrigerated and acidified (default) and D—frozen and acidified. The quantification of the analytes was performed by a previously validated method that employ a simple dilution of the samples followed by the LC-MS/MS analysis. Chromatographic separation and detection were performed on the Waters ACQUITY UPLC system equipped with RP-C18 column and gradient separation and on a Waters XEVO TQ-S Micro mass spectrometer and electrospray ionization (ESI+), respectively.

Results
The comparison between the basal groups (collection day) was identical for the 3 compounds, in the non-acidified groups (A and B) and also in the acidified group (C and D). Pearson’s Correlation was 0.995, 0.993 and 0.995 (P &lt; 0.0001) for VMA,HVA and 5 -HIAA, respectively. Stability for refrigerated samples has been confirmed for up to 7 days and 15 days for compound 5-HIAA. For 15-day samples, one of the differences exceeded the total allowed error (31.3%-Westgard). Frozen samples (non-acidified or acidified) reverse percentage differences when compared to the standard group. In addition, for group B (frozen and non-acidified) the differences exceeded the total allowed error. However, comparing those days after collection and basal groups, it was calculated that the maximum acceptable period to maintain the stability of the samples was up to 7 days in refrigerated condition.

Conclusion
The stability for the urinary measurement of VMA, ,HVA and 5-HIAA was considered acceptable in this study. The maximum period allowed for acceptable stability is up to 7 days in the refrigerated condition.