Quantitative Analysis of 5-hydroxyindoleacetic Acid in Rat Urine by High-performance Liquid Chromatography Method

Abstract

ABSTRACT Background: 5-hydroxyindoleacetic acid (5-HIAA) is a metabolite of serotonin, a neurotransmitter primarily associated with regulating mood, appetite, and sleep. When serotonin is broken down in the body, it forms 5-HIAA, which is then excreted in urine. Quantification of 5-HIAA levels in urine is often used as a diagnostic tool for certain conditions, particularly carcinoid tumors, which can produce excess serotonin. Elevated levels of 5-HIAA in urine can indicate increased serotonin production, which may be indicative of carcinoid tumors or other conditions affecting serotonin metabolism. However, there is limited work on employing high-performance liquid chromatography (HPLC)–ultraviolet to estimate monoamines in biological samples. Aim and Objectives: The present study explores the detection of 5-HIAA in rat urine samples, developing an easy, precise validated HPLC method. Materials and Methods: Mobile phase combination acetonitrile and o-phosphoric acid in the ratio of 30:70% v/v and flow rate 0.5 mL/min was able to separate 5-HIAA distinctly. Results: The detection was observed at λmax 277 nm with better sensitivity. According to the parameters indicated in the international council for harmonisation of technical requirements for pharmaceuticals for human use guidelines (Q2A; Q2B), the procedure was verified. The linearity range was selected from 20 to 45 μg/mL, r 2 = 0.992, limit of detection (4.65 μg/mL), limit of quantification (15.52 μg/mL), retention time (6.3 min). Conclusion: The technique has demonstrated that it is repeatable and recoverable within the given range. Thus can be used for routine analysis of 5-HIAA in urine samples.