Method development and validation for analysis of phenylalanine, 4‐hydroxyphenyllactic acid and 4‐hydroxyphenylpyruvic acid in serum and urine

Hughes, Andrew T, Milan, Anna M ORCID: 0000-0002-0452-2338, Shweihdi, Ella, Gallagher, James ORCID: 0000-0002-0852-279X and Ranganath, Lakshminarayan (2022) Method development and validation for analysis of phenylalanine, 4‐hydroxyphenyllactic acid and 4‐hydroxyphenylpyruvic acid in serum and urine. JIMD Reports, 63 (4). pp. 341-350.

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Official URL: http://dx.doi.org/10.1002/jmd2.12287

Abstract

Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine (TYR) metabolism which results in a deficiency of the enzyme homogentisate 1,2-dioxygenase activity. Several studies have reported the metabolic changes in homogentisic acid (HGA) concentrations and subsequent deposition of an ochronotic pigment in connective tissues, especially cartilage. Treatment with nitisinone (NTBC) reduces urinary and circulating HGA, but its mode of action results in hypertyrosinaemia. The effect of NTBC on other metabolites in the TYR pathway has not been reported. Modification of the current reverse phase liquid chromatography tandem mass spectrometry methods for serum and urine to include phenylalanine (PHE), hydroxyphenyllactate (HPLA) and hydroxyphenylpyruvate (HPPA) has been validated. HPPA and HPLA (negative ionisation) eluted at 2.8 and 2.9 min respectively on an Atlantis C18 column with PHE (positive ionisation) eluting earlier at 2.4 min. Intra- and inter-assay accuracy was between 96.3% and 100.3% for PHE; 96.6% and 110.5% for HPLA and 95.0% and 107.8% for HPPA in both urine and serum. Precision, both inter- and intra-assay, was <10% for all analytes in both serum and urine. No significant issues with carry-over, stability or matrix interferences were seen in either the urine or serum assays. Measurement of serum and urine from AKU patients has demonstrated a robust, fully validated assay, appropriate for monitoring of patients with AKU and for demonstrating metabolite changes, following NTBC therapy.

Item Type:	Article
Uncontrolled Keywords:	alkaptonuria, mass spectrometry, metabolites, nitisinone, tyrosine
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences
Depositing User:	Symplectic Admin
Date Deposited:	21 Jun 2022 09:39
Last Modified:	18 Jan 2023 20:57
DOI:	10.1002/jmd2.12287
Open Access URL:	https://doi.org/10.1002/jmd2.12287
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3156881