High sensitivity HPLC method for analysis of in vivo extracellular GABA using optimized fluorescence parameters for o-phthalaldehyde (OPA)/sulfite derivatives

摘要

Reversed-phase HPLC with derivatization using o-phthalaldehyde (OPA) and sulfite allows electrochemical detection of γ-aminobutyric acid (GABA) in microdialysis samples. However, OPA/sulfite derivatives have been reported to produce lower fluorescent yield than OPA derivatives using organic thiols as the nucleophile. To overcome this limitation we examined excitation and emission spectra, reaction time, pH, and concentration of reagents in the derivatization solution. Optimal detection parameters were determined as λex=220 nm and λem=385 nm for maximal fluorescence. The derivatization reaction occurred immediately and the product was stable up to 24 hours. A pH of 10.4 for the borate buffer used in the derivatization solution was significantly better than lower pH. Increasing the amount of sulfite combined with diluting the derivatization solution in borate buffer resulted in complete separation of the GABA peak from contaminants without any loss in signal. Controlling the temperature of the detector at 15°C significantly improved sensitivity with a detection limit of approximately 1 nM. To validate this assay, we performed microdialysis in the dorsal striatum and ventral tegmental area (VTA) of adult Long Evans rats. GABA concentrations in dialysates were determined using external standards and standard additions, in order to further confirm interfering peaks were not present in biological samples. Within the dorsal striatum (n=4), basal GABA concentrations were 12.9±2.2 and 14.5±2.2 nM (external and additions, respectively). Respective basal GABA concentrations in the VTA (n=3) were 4.6±1.1 and 5.1±0.6 nM. Thus, we have developed a novel, sensitive fluorescence method to determine GABA in microdialysates using HPLC of an OPA/sulfite derivative.