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Quantitation of sulfur‐containing amino acids, homocysteine, methionine and cysteine in dried blood spot from newborn baby by HPLC‐fluorescence detection
Author(s) -
Wada Mitsuhiro,
Kuroki Mana,
Minami Yuu,
Ikeda Rie,
Sekitani Yui,
Takamura Noboru,
Kawakami Shigeru,
Kuroda Naotaka,
Nakashima Kenichiro
Publication year - 2014
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3142
Subject(s) - chemistry , dried blood spot , chromatography , derivatization , homocysteine , methionine , high performance liquid chromatography , cysteine , dried blood , detection limit , cystine , homocystinuria , amino acid , biochemistry , enzyme
Sulfur‐containing amino acids (SAAs), homocysteine (Hcy), methionine (Met) and cysteine (Cys) in blood are related to homocystinuria, an inborn error of metabolism. In this study, an assay method with HPLC‐fluorescence detection to quantify the SAAs in a dried blood spot was established and applied to samples from newborn babies ( n = 200). Sample pretreatment involving reduction, derivatization with 4‐( N , N ‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole, and liquid–liquid extraction with ethyl acetate gave the separation of the derivatives with retention times within 12 min. The method was enough sensitive to determine the SAAs in a dried blood spot with 0.04–0.14 µ m as the limit of detection at a signal‐to‐noise ratio of 3. However, the absolute recoveries were very low (5.7% for Hcy, 4.6% for Cys) except for Met (105.4%) owing to inefficient recovery of Hcy and Cys from the blood matrix. Other validation parameters such as accuracy (93.5–106.2%) and intra‐ (≤9.0%) and inter‐day precisions (≤8.7%) were acceptable. The reliability of a dried blood spot as an analytical sample was estimated. Furthermore, the proposed method was successfully applied to dried blood spots prepared from newborn babies. Copyright © 2014 John Wiley & Sons, Ltd.