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.

Assignment 2 : Writing an introduction

The Comparative Determination of Gamma-Aminobutyric Acid (GABA) between Ultraviolet and Fluorescence Detection by High-Performance Liquid Chromatography (HPLC)

                                              Kritchapol  Panrod

Stage 1 : Gamma-aminobutyric acid (GABA) is a non-protein amino acid. GABA is a one of the neurotransmission agents which can inhibit over stimulating of the brain and reduce hypertension from the stress (Zhang et al., 2006). Moreover, GABA generally finds in human such as the cortex, hippocampus, hypothalamus, central nervous system (CNS), plants and animals. 

Thus, an analysis of GABA in the quantitation usually use the high-performance liquid chromatography (HPLC) to measure the amount of GABA in unknown sample. HPLC is the technique that can detect an agent in solution by it’s detector (such as ultraviolet light or fluorescence light) which analysts the agent when it passes through the HPLC column and interprets the results in terms of graphs and area under the peak. However, a detection of GABA has a problem when using HPLC because it has a small molecule and does not has fluorescent or ultraviolet absorbance (Shah et al., 2002). Therefore, one of the technique to resolve that problem is the pre-column derivatisation. The pre-column derivatisation will use an agent providing strong ultraviolet (UV) or fluorescent absorbance mixing with the sample before used in HPLC  (Shah et al., 1999) 

             Stage 2 : In UV detection, GABA is analysed by derivatizing agents including phenylisothiocyanate (PITC) (Jarry et al., 1992), 2-hydroxynapthaldehyde (HN) (Khuhawar and Rajper, 2003; Bor et al., 2009; Jannoey et al., 2010 and Hayat et al., 2014), 4-dimethyl-aminoazobenzene-4-sulfonyl chloride (DABSYL-Cl) (Varanyanond et al., 2005), naproxen acyl chloride (NAC) (Hsieh et al., 2006) and 1-fluoro-2,4-dinitrobenzene (FDNB) (Ishikawa et al., 2009). 

            In fluorescence detection, GABA is commonly analysed by a derivatizing agent which is o-phthaladehyde (OPA). OPA reacts with primary amines in the presence of thiol and generates derivatives which are fluorescent (Devall et al., 2007, Zhang et al., 2005, Fekkes et al., 1995 and Sheng et al., 2005). Unfortunately, the deravatives of OPA are not stable especially in the acid solution so they have developed to improve the stability and more sensitivity of derivatives by added naphthalene-2,3-dicarboxaldehyde (NDA) (Clarke et al., 2007), 2-mercaptoethanol (Iwaki and Kitada, 2007) and 3-mercaptopropionic acid (MPA) (Freitas Silva et al., 2009) 

   

            Stage 3 : Many of papers have a single analysis GABA but none of them has not comparative two techniques in one paper.

            Stage 4 & 5 : Thus, this paper will compare between UV detection and fluorescence detection. The sample of UV derivatizing agent is HN, an inexpensive agent and the fluorescence derivatizing agent is OPA because it normally uses in a detection. Therefore, this paper will point out that each of the technique can present the result in quantitation of GABA, retention time, limit of detection and limit of quantitation.