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KMID : 0385520150280050323
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Analytical Science & Technology
2015 Volume.28 No. 5 p.323 ~ p.330
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Determination of homogentisic acid in human plasma by GC-MS for diagnosis of alkaptonuria
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Maheshwor Thapa
Yu Jun-dong
Lee Won-Jae
Md. Fokhrul Islam
Yoon Hye-Ran
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Abstract
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Alkaptonuria, a rare inherited metabolic disease, is characterized by a lack of homogentisate dioxygenase and accumulation of homogentisic acid (HGA), leading to homogentisic aciduria, arthritis, and ochronosis. In this study, a rapid analytical method, without an expensive and tedious solid phase extraction step, was developed to quantify HGA in plasma using GC-MS. HGA-spiked pooled plasma samples were subjected to liquid-liquid extraction (LLE) with ethyl acetate, followed by trimethylsilyl derivatization (TMS) and GC-MS quantification using selected ion monitoring. The formation of TMS derivative of the 1 carboxylic and 2 hydroxyl functional groups was performed by reacting BSTFA (with 10% TMCS) for 5 min at 80 ℃. For selected ion monitoring, quantification and confirmation ions were determined based on specific ions (m/z 384, m/z 341 and m/z 252) of the TMS derivative of HGA. Calibration curves of pooled normal plasma specimens showed a linear relationship in the range of 1-100 ng/μL. The precision and accuracy were within a relative standard deviation (RSD) of 1 to 15% and a bias of -5 to 25%. Recoveries were obtained in the range of 99-125% and 95-115% for intra-day and inter-day assay, respectively, at 2, 20 and 80 ng/μL. The limit of detection (LOD) and limit of quantification (LOQ) were 0.4 ng/μL and 4 ng/μL, respectively. No homogentisic acid was excreted from normal Korean plasma samples. Collectively, the results from the present study suggest that this method could be useful for routine diagnosis and therapeutic monitoring of alkaptonuria patients with excellent sensitivity and rapidity.
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KEYWORD
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Homogentisic acid, Alkaptonuria, Inherited metabolic disease, Gas chromatography-mass spectrometry,
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