My major project

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

         For the ultraviolet (UV) detection, Khuhawar and Rajper (2003) conducted a study to determine an amount of GABA derivatized with 2-hydroxynapthaldehyde (HN) in a sample from central nervous system (CNS). The samples were collected from patients at Liaquat Medical University Hospital, Jamshoro. The total number of samples in this study was three, one patient suffering from tuberculous meningitis and two patients suffering from septic meningitis. Before the analysis of GABA, the sample was centrifuged. Then, the sample was mixed with HN and borax buffer pH 8.00. After that, the sample solution was heated on a water bath at 80 C° for 10 min and was allowed to cool at room temperature. The final volume was adjusted to 5 ml with methanol. The solution (5 μl) was injected on short column C₁₈, 5 mm and eluted with methanol: water (62:38 v/v) with a flow-rate 1 ml/min. The detection UV was 330 nm. The results showed that a linear calibration curve was obtained for GABA in the range of 1.12–28.0 mg/ml with a coefficient of correlation (r) = 0.998 and detection limit of 2.8 ng/injection (5 μl). In cerebral spinal fluid (CSF) samples, the result was shown 19.0 to 22.4 mg/ml with coefficient of variation 2.4% . Moreover, the derivative of GABA and HN is stable more than 12 hours. The researchers suggested that this might indicate that the high amount of GABA in CSF by the meningitis patients.

For the fluorescent detection, de Freitas Silva et al. (2009) conducted a study to determine an amount of GABA derivatized with o-phthaladehyde (OPA) in a sample from prefrontal cortex, thalamus hippocampus samples. The samples were collected from male Wistar rats. The total number of samples in this study was twenty. Before the analysis of GABA, the sample was centrifuged. Then, the sample was mixed with OPA, borate buffer pH 9.9 and 3-mercaptopropionic acid (MPA). The resulting solution was vortexed and analyzed after 1-min at room temperature. The solution was injected 10 μl on short column C₁₈, 3 mm and eluted with 0.05 M sodium acetate, tetrahydrofuran and methanol (50:1:49, v/v) adjusted to pH 4.0 with a flow-rate 1 ml/min and the temperature = 25 ± 2◦C. The fluorescent detector was set at an excitation wavelength of 337 nm and an emission wavelength of 454 nm. The results showed that a linear calibration curve was obtained for GABA in the range from 0.1 to 0.75 μg/ml by hippocampus samples of male Wistar rats with a correlation coefficient of not less than 0.990. In addition, the derivative of GABA and OPA is stable less than 30 minutes. The researchers noted that this method might perform successfully developed an isocratic HPLC method coupled to fluorescent detection for the determination of GABA and OPA derivatives.

These studies provide scientific evidence of the quantitative analysis of GABA in brain sample. However, there are some limitations.

For the UV detection

1)         The small group of sample could be distorted the real result in amount of GABA in human CSF so that the researchers should be collecting the data from the more people according to the FDA guideline etc. Moreover, the participants should be a normal people having not a disease because of preventing the result from bias or higher than normal condition of GABA in CSF (Rizzo et al. 1996)

2)         The sensitivity of analysis of a GABA derivative by UV detection was lower than fluorescent detection (de Freitas Silva et al. 2009) so when the study was wanted to detect a GABA in a low level, it should select the method of the fluorescent detection.

           

            For the fluorescent detection

1)         The stability of a GABA and OPA derivative was too low. It could be degraded in thirty minutes. Thus, the good planning lab would help to overcome this problem.

2)         This mobile phase used a buffer system. After the detection was finish, the operator had to always clean the column with water such as ultrapure water or HPLC grade water preventing from salting in column and HPLC.

The strength of these two studies is that the analysis reagents of the two techniques can be easily to purchase and not expensively. Furthermore, HPLC technique used is widely accepted for research in the same area. Another advantage of these two studies is that the results were clearly described with illustrated figures and conclusions were made according to the results in their studies.   

References

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. J Neurosci Methods, 177(2), 289-293.

Rizzo, V., Anesi, A., Montalbetti, L., Bellantoni, G., Trotti, R., & Melzi d'Eril, G. V. (1996). Reference values of neuroactive amino acids in the cerebrospinal fluid by high-performance liquid chromatography with electrochemical and fluorescence detection. Journal of Chromatography A, 729(1–2), 181-188.