Abstract

Background: Phenylalanine is a significant biomarker for various diseases like phenylketonuria, gastric cancers, and ischemic stroke according to recent studies.

Method: In the present study; a simple, sensitive, selective and novel analytical method was validated by using an ultrafiltration-based extraction and LC-MS/MS quantification of phenylalanine in human plasma using ¹³C phenylalanine heavy isotope. Amicon® Ultra Centrifugal Filter was used for ultrafiltration. Parameters affecting LC separation and MS/MS detection were investigated and optimized. Chromatographic separation was achieved on a Merck SeQuant ZIC-HILIC (100x4.6 mm, 5 μm) at a column temperature of 40°C using a mobile phase of mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid (50:50 v/v) at a flow rate of 0.35 mL/min. The transitions m/z 167→121 for ¹³C phenylalanine, m/z 166→120 for phenylalanine itself were monitored using the MRM mode.

Result: The assay was linear concentration range of 0.0025 μg/mL to 1.20 μg/mL (R²=0.999). The developed method was validated according to FDA guidelines. The method was found linear, sensitive, precise, accurate, and selective.

Keywords: LC-MS/MS, metabolomics, phenylalanine, quantification, ultrafiltration, blood.