Urine homogentisic acid and tyrosine: Simultaneous analysis by liquid chromatography tandem mass spectrometry

作者：Hughes A T; Milan A M^*; Christensen P; Ross G; Davison A S; Gallagher J A; Dutton J J; Ranganath L R

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2014, 963: 106-112.

DOI：10.1016/j.chromb.2014.06.002

• Alkaptonuria
• Homogentisic acid
• Tyrosine
• Liquid chromatography
• Tandem mass spectrometry

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 meth

• 出版日期2014-7-15