Objective: The research was carried out to develop a validated analytical method for assessing the biogenic amines metabolites Vanillyl mandelic acid (VMA) and Homovanillic acid (HVA) simultaneously in biological fluids by High performance liquid chromatography- Photodiode Array (HPLC-PDA). Method: An HPLC technique was developed and validated for the measurement of VMA and HVA in artificial urine samples. The chromatographic separation was achieved on a Kromasil C8, 5µm (125 X 4.6 mm) column at 250C with combination of mobile phase of acetonitrile: 0.1 % o-phosphoric acid (30:70 v/v), flow rate of 0.9 mL/min using an Shimadzu HPLC system with LC Solutions liquid chromatography software. Different mobile phases were used on a trial and error basis to separate these two metabolites. Validation of ICH parameters in terms of linearity, accuracy, repeatability, precision, and robustness of methodology was performed. Result: The separation of vanillyl mandelic acid and homovanillic acid was achieved using a highresolution HPLC technique. The linearity range for VMA and HVA was selected from 10 µg/mL to 60 µg/mL, and were found to be linear, with coefficients of determination (r2) of 0.996 and 0.990 for both metabolites, respectively. The detection limits for VMA and HVA were 1.96 µg/mL and 1.31 µg/mL, respectively, with quantification limits of 5.96 µg/mL and 3.97 µg/mL. For repeatability, precision, and robustness parameters, the results demonstrated a low percent RSD value. Conclusion: A validated HPLC-PDA approach was developed for quantifying Vanillyl mandelic acid and Homovanillic acid in urine samples.

Homovanillic acid, Vanillyl mandelic acid, HPLC-PDA, method validation, biogenic metabolites, urine sample, chromatographic separation