DEVELOPMENT OF CAPILLARY ELECTROPHORESIS-FLUORESCENCE
METHOD FOR DETERMINATION OF AMINO ACIDS IN HEALTH DRINK
SAMPLES
Natta Wiriyakun1,*, Ajala Praneedsuranon1, Duangjai Nacapricha1,2, Rattikan Chantiwas1,#
1
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of
Science, Mahidol University, Rama VI Rd, Rachathewi, Bangkok 10400, Thailand
2
Flow Innovation-Research for Science and Technology Laboratories (FIRST Labs), Thailand
*e-mail: n.wiriyakun@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, health drink samples,
spectrofluorometer
Introduction
Amino acids play important role in structural formation of protein that necessary for
the growth and repairing of tissue, red blood cells, enzymes, and other materials in the body
(1). Amino acids have been taken from diets, however, the variety of diets in these day
affects unhealthy human-eating behaviors and consequence the insufficient consumption of
amino acids (2). Therefore, the amino acids were added in several health drink samples in
order to help people consume enough nutrients and increase their market values.
Nowadays, there is no standard method to monitor amino acids in health drink
samples. Analysis of amino acids is challenging because of the lack of UV absorbing,
electroactive groups or fluorescent groups in their native structures. Fluorescence is the most
typically used to detect amino acids due to high sensitivity of detection. Fluorescein
isothiocyanate (FITC) is one of the most commonly used fluorophore that has been applied to
derivatize amino acids for fluorescence detection (3). Due to various amino acids containing
in health drink samples, separation techniques are required to use for measurement of amino
acids. Gas chromatography (GC), high performance liquid chromatography (HPLC) and
capillary electrophoresis (CE) are methods that have been utilized for separation of amino
acids. Among of these techniques, CE has been interested as an effective method. It provides
high separation efficiency, low chemical volume consumption in the level of nanoliter and
the variety in separation mode by modification of buffer system (4). Capillary electrophoresis
is widely applied to biochemical samples such as protein, enzyme and amino acids (5).
In this work, we present the development of capillary electrophoresis-fluorescence
method for determination of amino acids in health drink samples. The CE system was an inhouse set up with fluorescence detection using a spectrofluorometer. The spectrofluorometer
assembled with a fiber optic to probe excitation and emission beams onto capillary window
for detection. Capillary electrophoretic separation conditions such as running buffer systems
and injection method have been investigated for the developed CE method for amino acids
separation. The developed capillary electrophoresis-fluorescence detection (CE-FL) method
was applied to determination of amino acids in health drink samples.
Methodology
Chemicals and materials
Borate buffer solution was prepared from boric acid and sodium hydroxide (Fluka,
Switzerland). Standard solutions of L-Lys, L-Tyr, L-Phe, L-Ile and L-Gly (Fluka,
Switzerland) were prepared in borate buffer. Standard solution of FITC (Acros organics,
USA) was prepared in borate buffer. Sodium dodecyl sulfate (SDS) and hydroxyl
propyl(methyl) cellulose (HPMC) were from Sigma-Aldrich (Germany). All solutions were
prepared using ultra purified water (EZ pure 2 model system, USA) prior to filter through
0.45 µm cellulose acetate filter (Steritech, USA).
Amino acids derivatization
Derivatization of individual standard amino acid using FITC was prepared by mixing
each standard amino acid (1.0 mM) and FITC solution (1.25 mM) in ratio of 1:1. The mixture
solution was gently mixed for 12 hours under a dark room (Temperature = 25°C). Then, the
solution was filtered through 0.45 µm cellulose acetate membrane. All solutions were
degassed by ultrasonication for 5 min before the CE analysis.
Capillary electrophoresis with a fluorescence detection system
The in-house CE instrument with FL detection system is shown in Figure 1.
Figure 1. Schematic of in-house capillary electrophoresis with fluorescence detection system.
Fused-silica capillary (50 µm i.d., 360 µm o.d., Polymicro, USA) was 56.0 cm total
length with effective length of 41.0 cm (inlet end to detection end). A high voltage power
supply (model SL30, Spellman, USA) was used for applying electrical voltage of 15 kV. A
spectrofluorometer (LS55 model PerkinElmer, USA) was used as a fluorescent detector for
CE detection system. The FL detector assembled with an optical fiber with excitation
wavelength and emission wavelength of 488 nm and 520 nm.
Results
Investigation of buffer systems
Borate buffer (pH 9.00) containing 20.0 mM SDS has been typically used as a CE
running buffer for amino acids in CE separation (6), therefore, this buffer condition was
chosen as an initial conditions. Investigation of borate buffer concentrations and an addition
of HPMC concentrations to obtain optimal buffer system is presented as shown in Figure 2
and Table 1.
- Effect of borate buffer concentration
Figure 2. Electrophorograms of amino acid mixture solutions (150 M of Lys, 75 µM of Tyr, Phe and Gly):
(1) Lys-FITC, (2) Tyr-FITC, (3) Phe-FITC, (4) Gly-FITC and (*) residual FITC. Conditions: hydrodynamic
injection (10 cm height for 30 s), separation electrical field strength of 270 V/cm, borate buffer (pH 9.00)
containing SDS (20.0 mM) with (A) borate buffer of 20.0 mM and (B) borate buffer of 40.0 mM.
Figure 2A and 2B show electrophorograms of amino acids separation (Lys-FITC, TyrFITC, Phe-FITC and Gly-FITC) using borate buffer concentrations, 20.0 mM and 40.0 mM,
respectively. Borate buffer concentration was studied to control the electroosmotic flow (EOF)
by increasing of borate buffer concentration from 20.0 mM to 40.0 mM, the EOF increased (7).
The resulting electrophorograms illustrated that these standard amino acids were well-separated
except Tyr-FITC (peak 2) and Phe-FITC (peak 3). It can be seen that peak 2 (Tyr-FITC) and
peak 3 (Phe-FITC) overlapped (see Figure 2A). Resolving of these two peaks slightly
improved when using 40.0 mM borate buffer (see Figure 2B). Higher borate buffer
concentrations than 40.0 mM may improve peaks resolution, however, this has not been carried
out due to electrophoretic current issue. Since the joule heating in the capillary possibly occur
when electrophoretic current is higher than 30 µA (4),(8). Resulting electrophoretic current
obtained when utilizing borate buffer of 40.0 mM containing 20.0 mM SDS was 34 µA which
is very close to tolerate limit electrophoretic current for CE. In this experiment, 40.0 mM
borate buffer concentration was then chosen.
- Addition of hydroxyl propyl(methyl) cellulose (HPMC)
Hydroxyl propyl(methyl) cellulose (HPMC) has been generally added into running
buffer for using in CE separation to suppress the electroosmotic flow (9). To improve peak
separation between Tyr-FITC and Phe-FITC, HPMC was added into borate buffer (40.0 mM
containing 20.0 mM SDS) to improve peaks separation, especially between peak 2 and peak
3. Resolution (Rs) between Tyr-FITC and Phe-FITC was calculated from electrophorograms in
order to illustrate the improvement of separation. Table 1 shows Rs between Tyr-FITC and
Phe-FITC from the electrophorograms obtaining of running buffer without HPMC (0%, w/v)
and with HPMC (0.1%, w/v). Table 1 shows Rs between Tyr-FITC and Phe-FITC was 0.5
when running buffer without HPMC (0.0%, w/v), whereas Rs between Tyr-FITC and PheFITC was 0.8 when running buffer with adding HPMC (0.1%, w/v). Further improvement in
Rs between Tyr-FITC and Phe-FITC was limited when adding HPMC (0.2%, w/v), besides
the irreproducibility in migration times of Tyr-FITC and Phe-FITC peaks were experienced
(data are not shown). Therefore, the HPMC concentration (0.1%, w/v) was selected to add
into borate buffer (40.0 mM, pH 9.00) for higher resolution of separation of separating peaks.
Table 1. Resolution (Rs) between Tyr-FITC and Phe-FITC from the electrophorograms obtaining of running
buffer without HPMC (0%, w/v) and with HPMC (0.1%, w/v).
HPMC concentration, %(w/v)
Resolution (Rs*) between Tyr-FITC and Phe-FITC, n=3
0.0
0.5 ± 0.1
0.1
0.8 ± 0.1
0.2
-
*Rs was calculated from [1.7(tm2 – tm1)/(w1/21+ w1/22)], where tm = migration time and w1/2 = peak width at half height.
Effect of siphoning injection time
Due to the fact that injection volume contributes peak broadening in electrophoresis
separation, injection volume of standard amino acids was investigated. This injection method
based-on siphoning method, it was done by elevating the inlet capillary end (10 cm) while
keeping the outlet end at the same position.
(A) Injection time = 5 s
Rs = 1. 2±0.2
(B) Injection time = 10 s
Rs = 1.2±0.1
(C) Injection time = 30 s
Rs = 0.8±0.1
Figure 3. Electrophorograms of amino acid mixture solutions (150 M of Lys, 125 µM of Tyr, Phe and Gly): (1) LysFITC, (2) Tyr-FITC, (3) Phe-FITC, (4) Gly-FITC, (*) residual FITC and (**) unknown when using the different
hydrodynamic injection times of (A) 5 s, (B) 10 s and (C) 30 s. Separation conditions: 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.
Note: Unknown peaks (**) were observed in the electrophorograms, these may be due to impurities from prepared
amino acids.
Injection volume was calculated from [ρg∆Hd4πt/128ηL], where ρ = buffer density (1
g/cm3), g = gravitational constant (6.67×10−11 m3/kg s2), ∆H = height differential of capillary
inlet and outlet (10 cm), d = capillary inside diameter (50 µm), t = injection time (5 s, 10 s, 30
s), η = buffer viscosity (1 kg/ms) and L = total capillary length (56.0 cm). Siphoning injection
times of 5 s, 10 s and 30 s, were carried out in this experiment, the calculated volumes were
1.5 nL, 2.9 nL and 8.7 nL, respectively. Resulting electrophorograms of the separation of
standard amino acids are shown in Figure 3A, 3B and 3C when applying siphoning times of 5
s, 10 s and 30 s, respectively. Resolutions between peak 2 and peak 3 were given in each
electrophorogram; Rs = 1.2 ± 0.2, 1.2 ± 0.1 and 0.8 ± 0.1 were obtained when injection time
of 5 s, 10 s and 30 s were used, respectively. Therefore, siphoning injection time was selected
at 10 s throughout this experiment.
Summary of CE conditions for the amino acids separation
The investigated CE conditions; borate buffer concentration, addition of HPMC and
siphoning injection time were summarized as shown in Table 2.
Table 2. Summary of CE conditions for amino acids separation
Conditions
Optimum value
Borate buffer concentration
40.0 mM
HPMC concentration
0.1% (w/v)
Siphoning injection time
10 s
The figures of merit of the developed CE-FL method such as linearity range, regression
coefficient (r2), limit of detection (LOD), limit of quantification (LOQ) and reproducibility
were listed as in Table 3. Linearity range of each amino acid is shown with obtaining of linear
coefficient (r2) higher than 0.99. The precision of the method was estimated based on the
reproducibility of peak area and migration time. Overall reproducibility (%RSD) was less than
5% (n=3). Limit of detection (LOD) was less than 29 µM and limit of quantification (LOQ)
was less than 95 µM for all interested amino acids.
Table 3. Linearity, limit of detection (LOD), limit of quantification (LOQ) and reproducibility studies for amino
acids separation (n=3).
1
2
Reproducibility (%RSD)
Linearity range
(µM)
Regression
coefficient (r2)
LOD1
(µM)
LOQ2
(µM)
Migration time
Peak area
Lys
30-250
0.9989
29
93
1.7
3.8
Tyr
20-250
0.9994
4
18
1.7
4.7
Phe
30-250
0.9988
28
94
2.8
4.9
Gly
50-250
0.9995
8
27
1.6
3.7
Ile
30-250
0.9924
28
95
3.2
4.4
Amino acid
Limit of detection (LOD) was calculated from 3[SD/slope].
Limit of quantification (LOQ) was calculated from 10[SD/slope].
Determinations of amino acids in health drink samples
The developed CE method was applied to determine amino acids contents in health
drink samples. The samples (S1, S2, S3, and S4) were bought from the supermarkets in
Bangkok. From the labeled amount of each sample, it is found that there are two samples (S1
and S2) contained Lys whereas Ile and Phe were contained in all samples.
Figure 4. Bar graphs of the amino acid contents in health drink samples determined from the developed CEFL method (n=3), vs from the labeled amounts; (A) Lys (B) Ile (C) Phe.
Figure 4 depicts amino acids contents; (A) Lys, (B) Ile, (C) Phe determined by the
developed CE-FL method and stated on the sample label. It is found that the measured
amount of Ile and Phe were 790 ± 50 µM and 660 ± 60 µM, 800 ± 50 µM and 780 ± 60 µM,
590 ± 30 µM and 900 ± 50 µM, 700 ± 50 µM and 580 ± 30 µM (n=3), for S1, S2, S3, S4,
respectively. Whereas, the measured amount of Lys were 610 ± 30 µM and 770 ± 50 µM
(n=3), for S1 and S2, respectively. The labeled amount of amino acid contents with the
measured amount by CE-FL method was compared with acceptable different percentages of
15%.
The recovery of the developed CE-FL method was studied by spiking standard amino
acids to the sample matrix. The recoveries of Lys-FITC, Ile-FITC and Phe-FITC were in
ranges of 90-110%, 81-110% and 80-115%, (n=3), respectively.
Discussion and Conclusion
The in-house CE system FL detection has been developed for determination of five
amino acids (Lys-FITC, Ile-FITC, Tyr-FITC, Phe-FITC, Gly-FITC) in health drink samples.
Electrophoretic separation conditions such as buffer system; buffer concentration, addition of
hydroxyl propyl(methyl) cellulose; siphoning injection time were investigated to obtain the
optimal conditions for amino acid separation. The optimal CE conditions were borate buffer
(40.0 mM, pH 9.00) containing SDS (20.0 mM) and HPMC (0.1 %, w/v) with siphoning (by
elevating the inlet capillary end (10 cm) while keeping the outlet end at the same position)
injection time of 10 s. Linearity range of each amino acid was obtained in ranges of 20-250
µm with acceptable reproducibility (less than 5%, n=3). The developed CE-FL method was
applied for determination for amino acids (Lys, Ile, Phe) in health drink samples. The
different percentage of labeled amount of amino acid contents compared with the measured
amount by CE-FL method was acceptable (%difference ≤15%) for all samples. The
recoveries of spiked standard amino acids (Lys-FITC, Ile-FITC and Phe-FITC) were
acceptable in ranges of 80-115%. These results indicated that the developed CE-FL system is
a promising method for determination of amino acids content in health drink samples with
good accuracy and precision.
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.
9.
Yasui TM, M.R.; Kaji, N.; Okamoto, Y.; Tokeshi,M.; Baba, Y. Characterization of low viscosity
polymer solutions for microchip electrophoresis of non-denatured proteins on plastic chips. Biomicrofluidics.
2011;5:044114.
10.
Cooper SF, G.; Brown, S.; Nelson, S.; Hesselberth,J;. MacCoss, M.; Fields, S. High-throughput
profiling of amino acids in strains of the Saccharomyces cerevisiae deletion collection. Genome Research.
2010;20:1288-96.
Acknowledgements: The authors would like to thank Mahidol University, the Center of
Excellence for Innovation in Chemistry (PERCH-CIC) and King Rama VII and Queen
Foundation for partial support. Thanks to Center for Instrument Facility (CIF), Faculty of
Science, Mahidol University for providing research facilities.