#157 Simultaneous detection of biogenic amines metabolites (homovanillic acid and vanillyl mandelic acid) in urine sample of rodents by HPLC-UV method

Abstract

A simple, effective, and sensitive HPLC with UV detector was developed for detection of these biogenic amines metabolites in the urine sample of rodents as biotargets for neurological diseases like Parkinson’s disease, Alzheimer’s disease, Depression. The method includes the mobile phase combination of acetonitrile (ACN) and o-phosphoric acid (pH 2) in a ratio of (30:70 v/v), pH 2.2; the spectroscopic conditions were maintained for separation of analytes Kromosil C8 column 5 µm (125 X 4.6 mm) column at an ambient temperature of 25° C, at a flow rate 0.5 mL/min using EZ Chrome elite, Agilent HPLC system. Different mobile phases were used on a trial and error basis for the separation of these two metabolites. Validation of the developed method was carried out according to ICH guidelines. The calibration curve was linear over the concentration range of 10–35 μg/mL with regression coefficient (r²) 0.998 for HVA and 0.996 for VMA for both metabolite components. The limit of quantitation (LOQ) was 1 ng/mL, and the limit of detection (LOD) was 1 ng/mL. Results were satisfactory in terms of precision and accuracy. Hence, the method is suitable for the determination of homovanillic acid and vanillyl mandelic acid in urine samples.