Minor project

Minor project

           My research question is whether performing analysis techniques of gamma-aminobutyric acid (GABA) between photodiode array and fluorescence detector by high-performance liquid chromatography (HPLC). GABA is a small molecule and has polarity so that a detection of GABA is problematic. Moreover, some research studies, such as food and drugs, want to detect amount of GABA in low and/or sensitive levels, for example in levels of milligram, microgram and nanogram. HPLC coupled with detectors, photodiode array detector and/or fluorescence detector, can be detected a chemical compound in a solution. In addition, amino acids, like GABA, have a small molecule and do not have fluorescent or ultraviolet absorbance itself. Therefore, one of the solutions to resolve an obstruction is the pre-column derivatization. The pre-column derivatization will use an agent providing strong ultraviolet (UV) or fluorescent absorbance combined with the sample having amino acids before used in HPLC. This issue is claimed that some researchers use an UV derivatizing agent and others use a fluorescent derivatizing agent.

            Researchers using an UV derivatizing agent, 2-hydroxynapthaldehyde (HN), are Khuhawar and Rajper and others use a fluorescent derivatizing agent, o-phthaladehyde (OPA), are de Freitas Silva et al. The former reveal that they are the first group to study about GABA derivatized with HN and the latter notice that they have developed the method to quantify the amount of GABA with OPA and 3-mercaptopropionic acid (MPA)   

            Khuhawar and Rajper (2003) argue that they find an amount of GABA derivatized with HN in a sample from central nervous system (CNS) and have a standard curve ranged from 1.12 to 28 μg/ml by a short C₁₈ column. The derivative is stable more than 12 hours.  

            De Freitas Silva et al. (2009) argue that they have modified a method to detect GABA derivatized with OPA and used MPA as a stabilizer in samples from the rodent brain. They use a short C₁₈ column and run with an isocratic system. A standard curve is ranged from 0.1 to 0.75 μg/ml and the derivative is stable approximately 30 minutes.

           

            Debate centers on the basic issue if the comparison between photodiode array detector and fluorescence detector shows an effective analysis of GABA because it has not been studied in one study yet on this point.

            My work will be closer to Khuhawar and Rajper and de Freitas Silva et al. for the season that I will be conducting the two experiments to show whether each of techniques can be interpreted results in terms of sensitivity, reliability and reproducibility.

Hopefully my contribution will be the first study to give an idea of the result between two techniques in the one research. Furthermore, this study could be represented as information to guide other researchers selecting the suitable and sensitive experiment to doing their research in the future.

             

Reference List

Khuhawar, M. Y., & Rajper, A. D. (2003). Liquid chromatographic determination of γ-aminobutyric acid in cerebrospinal fluid using 2-hydroxynaphthaldehyde as derivatizing reagent. Journal of Chromatography B, 788(2), 413-418.

de Freitas Silva, D. M., Ferraz, V. P., & Ribeiro, A. M. (2009). Improved high-performance liquid chromatographic method for GABA and glutamate determination in regions of the rodent brain.  Journal of Neuroscience Methods, 177(2), 289-293.