Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA
| Autor: | Makoto Yasuda, Kiyoko Kaneko, Ken-ichi Mawatari, Tatsuhiro Ota, Kazuya Nakagomi, Noriko Yamaoka, Satoru Yui, Ryosuke Iijima, Tomoko Fukuuchi |
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| Rok vydání: | 2013 |
| Předmět: |
Chromatography
Calibration curve Hydrophilic interaction chromatography Clinical Biochemistry Sugar Acids Cell Biology General Medicine Standard solution Biochemistry Fluorescence High-performance liquid chromatography N-Acetylneuraminic Acid Analytical Chemistry chemistry.chemical_compound chemistry Limit of Detection Yield (chemistry) Humans Fluorometry Saliva Hydrogen peroxide Oxidation-Reduction N-Acetylneuraminic acid Chromatography High Pressure Liquid Fluorescent Dyes |
| Zdroj: | Journal of Chromatography B. 932:152-157 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2013.06.003 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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