title: Quantitation of sulfur-containing amino acids, homocysteine, methionine and cysteine in dried blood spot from newborn baby by HPLC-fluorescence detection.
abstract: 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 µm 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.
authors.affiliation: Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, 852-8521, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Wada
authors.forename: Mana
authors.initials: M
authors.lastname: Kuroki
authors.forename: Yuu
authors.initials: Y
authors.lastname: Minami
authors.forename: Rie
authors.initials: R
authors.lastname: Ikeda
authors.forename: Yui
authors.initials: Y
authors.lastname: Sekitani
authors.forename: Noboru
authors.initials: N
authors.lastname: Takamura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kawakami
authors.forename: Naotaka
authors.initials: N
authors.lastname: Kuroda
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Nakashima
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003545:Cysteine / Q000097:blood*; D059788:Dried Blood Spot Testing / Q000379:methods*; D005260:Female; D006710:Homocysteine / Q000097:blood*; D006712:Homocystinuria / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008715:Methionine / Q000097:blood*; D015997:Neonatal Screening / Q000379:methods*
chemical_list: D006710:Homocysteine; D008715:Methionine; D003545:Cysteine
keywords: cysteine; dried blood spot; homocysteine; methionine; newborn baby