Assessment and quantification of homovanillic acid and vanillylmandelic acid in rodent urine samples using a validated high-performance liquid chromatography-ultraviolet method

Abstract

Objective: The purpose of this study was to establish a validated analytical method for estimating the biogenic amine metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA) simultaneously using the high-performance liquid chromatography-ultraviolet (HPLC-UV) method. Materials and Methods: For the analysis of VMA and HVA in artificial urine samples, an HPLC method was devised and validated. The chromatographic separation was achieved on Kromasil C8, 5 μm (125 mm × 4.6 mm) column at an ambient temperature of 25°C, with mobile phase combination of acetonitrile: 0.1% orthophosphoric acid in ratio of 30:70 v/v at a flow rate 0.5 mL/min using Agilent HPLC system. For the separation of these two metabolites, different mobile phases were used on a trial and error basis. In terms of linearity, accuracy, repeatability, precision, and robustness, the developed technique was validated according to ICH guidelines. Results: A high-resolution HPLC method was devised for the separation of VMA and HVA. VMA and HVA were found to be linear over the concentration range of 10 to 35 μg/mL, with coefficients of determination (r2) of 0.955 and 0.963 for both metabolites, respectively. VMA and HVA had detection limits of 1.7 and 1.8 μg/mL, respectively, and quantification limits of 5.2 and 5.3 μg/mL. The results showed a low value of % relative standard deviation for repeatability, intra- and inter-day precision, and robustness studies. Conclusion: A validated HPLC-UV method was developed for estimating VMA and HVA in urine samples.