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

Abstract

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.

Reduced Activation of the Synaptic-Type GABAA Receptor Following Prolonged Exposure to Low Concentrations of Agonists: Relationship between Tonic Activity and Desensitization

“…Using 3D Monte Carlo reactiondiffusion simulations, Scimemi (Scimemi, 2014) estimated a steady-state GABA concentration of 0.1-1 µM in the cleft. This is similar to the concentration of measured in the cerebrospinal fluid (de Groote and Linthorst, 2007;Lerma et al, 1986;Zandy et al, 2017). Using the activation and desensitization parameters presented above, we estimate that in the presence of 0.1 µM GABA, 0.05% of receptors are in the active state and 0.2% in the desensitized state.…”

Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell

“…Fractions collected from islet perifusion in the BioRep islet perifusion device or supernatants from static incubations in KRBH buffer were analyzed for GABA content using an EICOM HTEC-500 HPLC-ECD with autosampler, online automated OPA-derivatization, and Eicompak FA-3ODS separation column. This automated detection technique is linearly sensitive for GABA from the nano-molar to milli-molar range 107 . Insulin content in the same sample fraction was determined by ELISA kit (Mercodia).…”

The amino acid derivative reactivity assay with fluorescence detection and its application to multi-constituent substances

“…Thus, the use of fluorescence detection makes it possible to avoid the co-elution that occurs so often with UV detection. There are many examples of the use of fluorescence detection in HPLC analysis, and while there are some that include florescent test chemicals (Cecil and Rutan, 1991), most involve the fluorescence derivatization of amino acids, amino acid derivatives, or other non-fluorescent materials (Purcell et al, 1976;Mashat et al, 2008;Zandy et al, 2017;Muramoto and Kamiyama, 1985;Tapuhi et al, 1981;Imai and Watanabe, 1981). Like ADRA and DPRA, the High Throughput with Dansyl Cysteamine (HTS-DCYA) method is an in chemico test that was developed as an alternative to animal testing for skin sensitization.…”