RESUMO
To study the relationship between amounts of nitric oxide (NO) in blood and the development of ischemic cardio-cerebrovascular diseases, trace NO in human blood serum has, for the first time, been determined by use of a 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)difluoroboradiaza-s-indacene (TMDABODIPY)-based HPLC method. The proposed method is simple, rapid, and efficient, owing to its high sensitivity and good selectivity for NO. TMDABODIPY and its NO derivative are separated to baseline in 4 min, with simple separation conditions, on a C(18) column eluted with 50 mmol L(-1) ethanolamine in methanol. The derivative is detected by fluorescence at an emission wavelength of 507 nm with excitation at 498 nm. The response is a linear function of concentration in the range 0.8-800 nmol L(-1) NO. The detection limit can reach 2 x 10(-11) mol L(-1) (signal-to-noise ratio=3). The method has been used to detect NO in the serum of patients with five kinds of ischemic cardio-cerebrovascular disease and two diseases closely connected with ischemic cardio-cerebrovascular diseases. Recoveries of NO from spiked serum samples were between 96.58 and 105.71% and concentrations of NO observed in real samples were at 10(-7) mol L(-1) levels. Our studies indicate that the proposed TMDABODIPY-based HPLC technique can be developed into a sensitive and new method for clinical assay and pathology research.
