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Ultrafiltration-Based Extraction and LC-MS/MS Quantification of Phenylalanine in Human Blood Sample for Metabolite Target Analysis

Author(s):

Mustafa Çelebier*, Tuba Reçbera, Emirhan Nemutlu and Sedef Kır   Pages 1 - 7 ( 7 )

Abstract:


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

Affiliation:

Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara



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