title: Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA.
abstract: N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450nm and 560nm, respectively. Both intra- and inter-day (n=6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576fmol to 2.0nmol and 556fmol to 2.0nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.
authors.affiliation: Faculty of Pharma Sciences, Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan.
authors.forename: Tatsuhiro
authors.initials: T
authors.lastname: Ota
authors.forename: Makoto
authors.initials: M
authors.lastname: Yasuda
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Iijima
authors.forename: Satoru
authors.initials: S
authors.lastname: Yui
authors.forename: Tomoko
authors.initials: T
authors.lastname: Fukuuchi
authors.forename: Noriko
authors.initials: N
authors.lastname: Yamaoka
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Mawatari
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Kaneko
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nakagomi
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D005470:Fluorometry / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D010084:Oxidation-Reduction; D012463:Saliva / Q000737:chemistry*; D013400:Sugar Acids / Q000032:analysis*
chemical_list: C522105:4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid; D005456:Fluorescent Dyes; D013400:Sugar Acids; D019158:N-Acetylneuraminic Acid
keywords: 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride; 4-(Acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid; 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole; 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid; 4-N,N-dimethylaminopyridine; ADOA; DBD-ED; DMP; EDC; HILIC; HPLC; N-acetylneuraminic acid; NANA