DEVELOPMENT OF CAPILLARY ELECTROPHORESIS-FLUORESCENCE
METHOD FOR DETERMINATION OF AMINO ACIDS IN FUNCTIONAL
DRINKING SAMPLES
Natta Wiriyakun*, Ajala Praneedsuranon, Duangjai Nacapricha, Rattikan Chantiwas#
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of
Science, Mahidol University, Rama VI Rd, Rachathewi, Bangkok 10400, Thailand
*e-mail: n.wiriyalun@gmail.com, #e-mail: rattikan.cha@mahidol.ac.th
Abstract
Development of a simple capillary electrophoresis has been investigated to determine amino
acids (Lysine; Lys, Tyrosine; Tyr, Phenylalanine; Phe, Isoleucine; Ile and Glycine; Gly) in
health drink samples. An in-house capillary electrophoresis system with fluorescence
detection was set up and the capillary electrophoresis separation method was investigated.
The optimum capillary electrophoresis separation conditions such as buffer system; buffer
concentration, addition of hydroxyl propyl(methyl) cellulose, injection volume (siphoning
injection time) are presented. Electrophoretic separation conditions were borate buffer (40.0
mM, pH 9.00) containing SDS (20.0 mM) and HPMC (0.1%, w/v) and applied electrical field
strength of 270 V/cm. The linear calibration curves of Lys-FITC, Phe-FITC and Ile-FITC
were 30-250 μM, while Tyr-FITC was linear in range of 20-250 μM, and Gly-FITC was
obtained in range of 50-250 μM. Percentage of relative standard deviation (%RSD) of
migration times and peak area were less than 3.2% and 4.9%, (n=3), respectively. Limit of
detection (LOD) and limit of quantification (LOQ) for all amino acids were less than 29 µM
and 95 µM, respectively. Determination of amino acids in health drink samples was carried
out by developed method. The labeled amount of amino acids contained in the samples and
the measured amounts by CE-FL method were compared. Percentage of difference was
acceptable, it was less than 15%. The reasonable accuracy based on recovery study for Lys,
Ile and Phe was in ranges of 80-115% (n=3).
Keywords: amino acids, capillary electrophoresis, fluorescence, functional drinking samples,
spectrofluorometer
References
1.
Poinsot VC, M.; Bouajila, J.; Gavard, P.; Feurer, B.; Couderc, F. Recent advances in amino acid
analysis by capillary electrophoresis. Electrophoresis. 2012;33:14-35.
2.
Herrero MGa-Ca, V.; Simo, C.; Cifuentes, A. Recent advances in the application of capillary
electromigration methods for food analysis and Foodomics. Electrophoresis. 2010;31:205-18.
3.
Jin LJR, I.; Li, S. Enantiomeric separation of amino acids derivatized with fluoresceine isothiocyanate
isomer I by micellar electrokinetic chromatography using b- and g-cyclodextrins as chiral selectors.
Electrophoresis. 1999;20:1538-45.
4.
Lagane BT, M.; Couderc, F. Capillary electrophoresis: theory, teaching approach and separation of
oligosaccharides using indirest UV detection. Biochemistry and Molecular Biology Education. 2000;28:251-5.
5.
MacTaylor CEE, A.G. Critical review of recent developments in fluorescence detection for capillary
electrophoresis. Electrophoresis. 1997;18:2279-90.
6.
Tezcan FU, S.; Uyar, G.; Öztekin, N.; Erim, F. Determination of amino acids in pomegranate juices
and fingerprint for adulteration with apple juices. Food Chemistry. 2013;141:1187-91.
7.
Chapman SDGaPJ. Measuring Electroosmotic Flow in Microchips and Capillaries. In: Henry CS,
editor. Microchip Capillary Electrophoresis: Methods and Protocols. Totowa, NJ: Humana Press Inc.; 2006. p.
187-201.
8.
Weinberge R. Practical Capillary Electrophoresis. New York: Academic Press; 1995.