title: Urine homogentisic acid and tyrosine: simultaneous analysis by liquid chromatography tandem mass spectrometry.
abstract: Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine metabolism. Deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid (HGA) which although excreted in gram quantities in the urine, is deposited as an ochronotic pigment in connective tissues, especially cartilage. Ochronosis leads to a severe, early-onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, Nitisinone, an inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of HGA, resulting in accumulation of the upstream pre-cursor, tyrosine. Using reverse phase LC-MS/MS, a method has been developed to simultaneously quantify urinary HGA and tyrosine. Using matrix-matched calibration standards, two product ion transitions were identified for each compound and their appropriate isotopically labelled internal standards. Validation was performed across the AKU and post-treatment concentrations expected. Intrabatch accuracy for acidified urine was 96-109% for tyrosine and 94-107% for HGA; interbatch accuracy (n=20 across ten assays) was 95-110% for tyrosine and 91-109% for HGA. Precision, both intra- and interbatch was <10% for tyrosine and <5% for HGA. Matrix effects observed with acidified urine (12% decrease, CV 5.6%) were normalised by the internal standard. Tyrosine and HGA were proved stable under various storage conditions and no carryover, was observed. Overall the method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of patients with AKU, pre and post-nitisinone therapy.
authors: Department of Clinical Biochemistry and Metabolic Medicine, Royal Liverpool and Broadgreen University Hospital Trust, Duncan Building, Liverpool L7 8XP, United Kingdom; Bone and Joint Research Group, Musculoskeletal Biology, Sherrington Building, University of Liverpool, Liverpool L69 3GE, United Kingdom.
authors: A T
authors: AT
authors: Hughes
authors: Department of Clinical Biochemistry and Metabolic Medicine, Royal Liverpool and Broadgreen University Hospital Trust, Duncan Building, Liverpool L7 8XP, United Kingdom; Bone and Joint Research Group, Musculoskeletal Biology, Sherrington Building, University of Liverpool, Liverpool L69 3GE, United Kingdom. Electronic address: Anna.Milan@rlbuht.nhs.uk.
authors: A M
authors: AM
authors: Milan
authors: Agilent Technologies, 5500 Lakeside, Cheadle Royal Business Park, Cheadle, Stockport SK8 3GR, United Kingdom.
authors: P
authors: P
authors: Christensen
authors: Agilent Technologies, 5500 Lakeside, Cheadle Royal Business Park, Cheadle, Stockport SK8 3GR, United Kingdom.
authors: G
authors: G
authors: Ross
authors: Department of Clinical Biochemistry and Metabolic Medicine, Royal Liverpool and Broadgreen University Hospital Trust, Duncan Building, Liverpool L7 8XP, United Kingdom.
authors: A S
authors: AS
authors: Davison
authors: Bone and Joint Research Group, Musculoskeletal Biology, Sherrington Building, University of Liverpool, Liverpool L69 3GE, United Kingdom.
authors: J A
authors: JA
authors: Gallagher
authors: Department of Clinical Biochemistry and Metabolic Medicine, Royal Liverpool and Broadgreen University Hospital Trust, Duncan Building, Liverpool L7 8XP, United Kingdom.
authors: J J
authors: JJ
authors: Dutton
authors: Department of Clinical Biochemistry and Metabolic Medicine, Royal Liverpool and Broadgreen University Hospital Trust, Duncan Building, Liverpool L7 8XP, United Kingdom; Bone and Joint Research Group, Musculoskeletal Biology, Sherrington Building, University of Liverpool, Liverpool L69 3GE, United Kingdom.
authors: L R
authors: LR
authors: Ranganath
mesh_terms: D000474:Alkaptonuria / Q000175:diagnosis / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods*; D006713:Homogentisic Acid / Q000652:urine*; D006801:Humans; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014443:Tyrosine / Q000652:urine*
chemical_list: D014443:Tyrosine; D006713:Homogentisic Acid
keywords: Alkaptonuria; Homogentisic acid; Liquid chromatography; Tandem mass spectrometry; Tyrosine