Gas phase derivatization of ammonia with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to urease assay.
An ammonia-specific and rapid fluorometric method for determination of ammonia and urease activity was developed. The method is designed to assay ammonia levels or urease activity for the rapid diagnosis of Helicobacter pylori infection. 4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole was used to derivatize ammonia and 4-amino-7-nitrobenzo-2-oxa-1,3-diazole was analysed by high performance liquid chromatography at an excitation wavelength of 455 nm and an emission wavelength of 520 nm. Derivatization was designed to react with ammonia gas produced in a strong alkaline pH sample. The fluorescent intensity was linear in the range of 0.1-10 mM ammonia per tube when the reaction was carried out for 15 min at 37 degrees C. Urease activity, judged as the amount of ammonia production from urea, could be measured at 25 ng per tube (S/N = 1.5) with Jack bean meal urease. Because of its rapidity, this assay is potentially superior to the current standard method in use in clinical settings.
Kobayashi
R
R
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Nakashima
K
K
Sugano
K
K
Nishikawa
M
M
Akiyama
S
S
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000641:Ammonia / Q000032:analysis*; D005453:Fluorescence; D016481:Helicobacter Infections / Q000175:diagnosis / Q000201:enzymology; D016480:Helicobacter pylori / Q000201:enzymology; D014510:Urease / Q000032:analysis*
C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D000641:Ammonia; D014510:Urease; D009327:4-Chloro-7-nitrobenzofurazan