Determination of amino acids by microemulsion electrokinetic
chromatography-laser-induced fluorescence method
Wei-Cheng Lina‡, Wan-Ling Liua‡, Wei-Ying Tanga‡, Chin-Ping Huangb, Hsi-Ya
Huanga*, Ting-Yu Chinc*
a
Department of Chemistry, Chung Yuan Christian University, 200 Chung Pei Road, Chung-Li, 320,
Taiwan
b
Nanotechnology Research Center at Nano-instrumentation and Application Division, Industrial
Technology Research Institute, Hsinchu, Taiwan.
c
Department of Bioscience Technology at CYCU, Chung Yuan Christian University, Chung-Li, Taiwan.
Table of Contents
1. Material and method:
2. Figure S1 Effect of microwave energy.
3. Figure S2 Comparison of different microwave-assisted irradiation time
4. Figure S3 Effect of FITC concentration
5. Figure S4 Effect of various pH values running buffer with CZE method
6. Figure S5 Determination of 20 kinds of amino acid on CZE or MEKC system
7. Figure S6 Effect of co-surfactant concentration on MEEKC system
8. Figure S7 Effect of oil type on MEEKC system
9. Figure S8 Effect of surfactant concentration on MEEKC system
10. Figure S9 Electropherograms of amino acids by MEEKC with different pH
running buffer.
11. Figure S10 Effect of ACN content on MEEKC system
12. Figure S11 Effect of column length on MEEKC system
13. Table S1 Table S1 Recovery of real samples
14. References
1. Material and method:
Chemicals and reagents
All amino acid standards were purchased from Sigma (Steinheim, Germany).
Boric acid and borax were purchased from Merck (Darmstadt, Germany). Fluorescein
isothiocyanate (FITC) was purchased from Fluka (Steinheim, Germany). All other
chemicals used were of analytical grade and used without further purification.
Apparatus and operating conditions for CE and FASS method
All experiments were performed in a Beckman Coulter MDQ capillary
electrophoresis system equipped with a LIF detection system (Fullerton, CA, USA).
Beckman Coulter MDQ 32 Karat software was used for instrumental control and data
analysis. The excitation light from an argon ion laser (3 mW) was focused on the
capillary window by means of a fiber-optic connection. The 488 and 520 nm
band-pass filters were used as excitation and emission filters, respectively.
Separations were performed in a 50 cm effective length (inlet to detector) of a 60.3
cm total length with 50 μm I.D. uncoated fused-silica capillaries (Polymicro
Technologies, Phoenix, AZ, USA) and the temperature of the capillary was
maintained at 25 oC.
The new capillary was conditioned in sequence with 1.0 M
NaOH solution (20 min, 20 psi), deionized water (20 min, 20 psi), 0.1 M NaOH
solution (10 min, 20 psi) and deionized water (10 min, 20 psi). The temperature of the
capillary was maintained at 25 oC and the LIF detection wavelength was fixed at 488
nm during the entire experiment.
Solutions and sample preparation
Standard stock solution of amino acid with a concentration of 200 mg mL-1 was
prepared by dissolving the analyte in 0.1M HCl. Stock solution of 30 mM FITC was
prepared by dissolving 11.6 mg of FITC in 1mL acetone. The 63 mM borate stock
derivatization buffer was prepared and its pH was 10.0 (unadjusted) and the
microemulsion solution was prepared by mixing cyclohexane (0.6 %, w/w), sodium
dodecyl sulphate (SDS; 2.16%, w/w),1-butanol (6%, w/w) ,acetonitrile (2%, w/w), 30
mM phosphate buffers (87.24% , w/w ,pH 6 unadjusted) were prepared by mixing for
30 minutes and standing for 1 hour.
Derivatization procedures
For standard solution derivatization, 10 μL of 200 mg L-1 standard stock solution
was mixed with 20μL FITC and 20 μL derivatization buffer then the mixture was
vortexed for 1 min. The sample was placed in a beaker containing RT water and then
microwave heated in a domestic microwave oven (900 W, SAMPO RE-1002SM)
while the other set-up was done in a 100 mL Teflon autoclave containing 12 mL
deionized water placed in a microwave reactor (START D, Milestone, maximum
power of 1200 W, 2.45 GHz) and heated for a predetermined time. The preparation of
the samples for derivatization were the same for both water-bath and microwave
methods.
Beverage and food sample preparation
The outer film tablet of food sample was removed and put 0.1g sample in centrifuged
tube then adds 0.1 M HCl solution (1mL) and shakes 60 minutes for extracting amino
acids. After extracted step, centrifuging at 5000 rpm for 30 minutes, taking the upper
layer and filtered by 0.2μm filter.
Rat brain sample preparation
Rat brain was obtained from 5-month old rats. The extraction procedure was
referred to previous papers [1-3] and briefly described herein. Cell metabolites
extraction was performed by adding lysis buffer to release the metabolites of interest.
Then the extract was centrifuged for 10 min and the supernatant containing the amino
acids was used as the real sample to be derivatized using FITC.
The
microwave-assisted derivatization was performed in 600 µL microcentrifuge vial as
follows: 5μL real sample, 19μL derivatization buffer and 1μL 30mM FITC solution.
After mixed step, the microcentrifuge vial was sealed by heat tape and placed in a
domestic microwave oven (900 W) together with a beaker containing 250mL water.
2.
1.5
FITC
Arg
(a) 900W
1.0
a
0.5
a
a
a
RFU
0.0
1.5
(b) 729W
FITC
Arg
1.0
a
0.5
a
a
a
0.0
1.5
(c) 590W
FITC
1.0
Arg
0.5
a
a
a
a
0.0
4
5
6
7
8
Time (min)
Figure S1 Effect
of microwave energy
The sample introduction was 0.5 psi for 3s, in which the concentration of Arg standards is 5 µgL-1.
Derivatization condition: 25 mM boric acid +25 mM borax, pH 9 (derivatization buffer), reaction time,
2 min. CE conditions: capillary, 40 cm×50
m i.d,; separation voltage, 20 kV; running buffer, 50 mM
boric acid was mixed with 40 mM borax (pH 9), detection, LIF, 488/520 nm.
3.
Peak area
500000
400000
300000
0
1
2
3
4
Time (min)
Figure S2 Comparison of different microwave-assisted irradiation time
The sample introduction was 0.5 psi for 3s, in which the concentration of Arg standards is 5 µgL -1.
Derivatization condition: 25 mM boric acid +25 mM borax, pH 9 (derivatization buffer), 15mM FITC.
CE conditions: capillary, 40 cm×50 µm i.d,; separation voltage, 20 kV; running buffer, 50 mM boric
acid was mixed with 40 mM borax (pH 9), detection, LIF, 488/520 nm.
4.
Arg
Ser
Gly
Glu
Asp
Area
1600000
1200000
800000
400000
10
15
20
30
FITC concentration (mM)
Figure S3 Effect
of FITC concentration
The sample introduction was 0.5 psi for 3s, in which the concentration of standards is 2 µgL-1.
Derivatization condition: 25 mM boric acid, 25 mM borax, pH 10 (derivatization buffer), reaction time,
3 min. CE conditions: capillary, 40 cm×50 m i.d,; separation voltage, 20 kV; running buffer, 50 mM
boric acid was mixed with 40 mM borax (pH 9), detection, LIF, 488/520 nm.
5.
1.0
FITC
pH10
3
1
0.5
2
a
0.0
1.0
pH9
2 3
FITC
1
5
4
0.5
4 5
a
RFU
0.0
FITC
2 pH8
2
1
4
a
0
1.0
FITC
pH7
5
3
2
1
0.5
4
a
0.0
0.2
3
1
FITC
pH6
3
2
1
4 5
a
0.0
5
5
10
15
20
25
Time (min)
Figure S4 Effect
of various pH values running buffer with CZE method
The sample introduction was 0.5 psi for 3s, in which the concentration of standards is 2 µgL-1.
Derivatization condition: 25 mM boric acid, 25 mM borax, pH 10 (derivatization buffer), reaction time,
3 min. CE conditions: capillary, 40 cm×50 m i.d,; separation voltage, 20 kV; running buffer, 50 mM
boric acid was mixed with 40 mM borax, detection, LIF, 488/520 nm.
Arg (1), Ser (2), Gly (3), Glu (4), Asp (5) and FITC hydrolyzate (a).
6.
3
(b) MEKC
2
1
1,2
a aa
17
7,8,9,10,11
20
12,13,14
15
3,4
56
16 18,19
RFU
0
8
FITC 4,10,14,13,15,16
5,11,7,3,9
6
(a) CZE
12
4
8
17
6
2
2
19 20
a
a
0
5
10
15
20
25
Time (min)
Figure S5 Determination of
20 kinds of amino acid on CZE or MEKC system
Derivatization condition: 25 mM boric acid +25 mM borax, pH 10 (derivatization buffer), reaction time,
3 min. (a) CE conditions: capillary, the concentration of standards is 2 µgL-1 (0.5 psi, 3s), running
buffer, pH 9, 50 mM boric acid was mixed with 40 mM borax, (b) pH-8 borate buffer (59mM), 2%
SDS, 6% 1-butanol, 40 cm×50 m i.d,; separation voltage, 20 kV; detection, LIF, 488/520 nm.
Lys (1), Arg (2), His (3), Pro (4), Val (5), Leu (6), Ile (7), Phe (8), Trp (9), Tyr (10), Met (11), Thr (12),
Gln (13), Ala (14), Asn (15), Cys (16), Ser (17), Gly (18), Glu (19), Asp (20) and FITC hydrolyzate or
impurity (a).
7.
0.8
(a) 6%
0.6
9,10,11
12
8
0.4
467
3
5
0.2
14
13-2 15,16
1,2
13-1
a
18,19 20
a 17
RFU
0.0
2.0
9,10,11,12
(b) 3%
1.5
1.0
8
5,6
3,4 7
1,2
13-2
0.5
13-1
a
14
15,16
a 18,19
a 20
17
0.0
10
15
20
Time (min)
Figure S6 Effect of co-surfactant concentration on MEEKC system
Running buffer, SDS, 6 % 1-butanol, 4 % ACN, pH6 phosphate buffer (40 mM) and the sample
concentration is 4 µgL-1 (0.5 psi, 3s). Other conditions are the same as Fig.1.
His (1), Arg (2), Gln (3), Pro (4), Tyr (5), Asn (6), Thr (7), Ser (8), Ala (9), Val (10), Met (11), Gly
(12), Ile (13), Leu (14), Phe (15), Trp (16), Cys (17), Glu (18), Lys (19), Asp (20), FITC hydrolyzate or
impurity (a).
8.
0.6
0.4
0.2
1 2
0.0
0.4
9,10,11
12
5 8
4 7
14
3 6
13-2 15
a
13-1
16a
18,19
20
17
9,10,11
12
4
12
a
RFU
(a) heptane
5
3 67
8
13-214
13-1
(b) EA
18,19
15,16
a
20
17
0.0
(c) octane
1.2
0.8
9,10,11
12
8
6
4 7
18,19
13-2 14
3
15,16
5
13-1
20
a 17
1 2
0.4
a
0.0
0.8
9,10,11
12
0.4
1 2
a
0.0
10
46 7
3
5
(d) cyclohexane
8
14
13-2 15,16 18,19
13-1
20
a17
15
20
Time (min)
Figure S7 Effect of oil type on MEEKC system
Running buffer, 2.16 % SDS, 6 % 1-butanol, 4 % ACN, 87.24 %, pH6 phosphate buffer (40 mM) and
the sample concentration is 4 µgL-1 (0.5 psi, 3s). Other conditions are the same as Fig.1.
His (1), Arg (2), Gln (3), Pro (4), Tyr (5), Asn (6), Thr (7), Ser (8), Ala (9), Val (10), Met (11), Gly
(12), Ile (13), Leu (14), Phe (15), Trp (16), Cys (17), Glu (18), Lys (19), Asp (20), FITC hydrolyze or
impurity (a).
9.
0.4
4
8
9,10,11
(a) 2.88%
6
1
0.2
2
3
5
7
12
0.0
0.4
9,10,11,12
0.2
1 2
RFU
0.0
5
FITC
10,11,12
0.2
(c)1.43%
7,8,9
12
3,4
a
0.0
56
1415,16
18,19 20
17
13
0.8
FITC
3,4,5,6,7,8,9,10,11,12
13,14,15,16,17
0.4
12
0.0
(b) 2.16%
68
18,19
4 7 13-2
3
1415,16
20
a
13-1
a17
0.4
1.2
14
13-2 15,16 18,19 20
17
13-1
(d) 0.72%
18,19 20
a
10
15
20
25
Time (min)
Figure S8 Effect of surfactant concentration on MEEKC system
Running buffer, 2.16 % SDS, 6 % 1-butanol, 4 % ACN, pH6 phosphate buffer (40 mM) and the sample
concentration is 4 µgL-1 (0.5 psi, 3s). Other conditions are the same as Fig.1.
His (1), Arg (2), Gln (3), Pro (4), Tyr (5), Asn (6), Thr (7), Ser (8), Ala (9), Val (10), Met (11), Gly
(12), Ile (13), Leu (14), Phe (15), Trp (16), Cys (17), Glu (18), Lys (19), Asp (20), FITC hydrolyzate or
impurity (a).
10.
FITC
2
(a) pH10
1
0
3
FITC
(b) pH9
2
1
0
RFU
3
FITC
(c) pH8
2
1
0
3
FITC
(d) pH7
2
1
0
1.0
0.5
0.0
FITC
(e) pH6
9,10,11 12
8
14
5,6 7
13-2 15,16
1 2 3,4
18,19
a
20
17
13-1
10
20
30
Time (min)
Figure S9 Electropherograms of amino acids by MEEKC with different pH
running buffer.
Running buffer, 2% SDS, 6 % co-surfactant, 0.6% cyclohexane, 91.4 %, pH6~10 phosphate buffer (40
mM) and the sample concentration is 4 µgL-1 (0.5 psi, 3s). Other conditions are the same as Fig. 1.
His (1), Arg (2), Gln (3), Pro (4), Tyr (5), Asn (6), Thr (7), Ser (8), Ala (9), Val (10), Met (11), Gly
(12), Ile (13), Leu (14), Phe (15), Trp (16), Cys (17), Glu (18), Lys (19), Asp (20), FITC hydrolyzate or
impurity (a).
11.
1.2
(a) 6%
4,5,6
0.8
1,2
12
17,18,19,20
10,11
0.4
a
a
a
0.0
1.2
3
89
7
15,16
14
a
13
FITC
RFU
(b) 4%
0.8
9,10,1112
2
0.4
4 8
7
1
a
a
a
0.0
1.2
17,18,19,20
13-215
14
16
3
56
13-1
FITC
(c) 2%
0.8
10,11
5,6
12
2
1
0.4
a
0.0
a
a
a
4 8
7
3 9
13-215
14 17,18,19,20
16
13-1
10
15
20
Time (min)
Figure S10 Effect of ACN content on MEEKC system
Running buffer, 1.43 % SDS, 6 % 1-butanol, ACN, pH6 phosphate buffer (40 mM) and the sample
concentration is 4 µgL-1 (0.5 psi, 3s). Other conditions are the same as Fig.1.
His (1), Arg (2), Gln (3), Pro (4), Tyr (5), Asn (6), Thr (7), Ser (8), Ala (9), Val (10), Met (11), Gly
(12), Ile (13), Leu (14), Phe (15), Trp (16), Cys (17), Glu (18), Lys (19), Asp (20), FITC hydrolyzate or
impurity (a).
12.
1.5
(a)50 cm
1.0
5,610,1112
17
9
8
13-2
47
18,19 20
a
14
15
3
12
16
a a
a
0.5
RFU
0.0
13-1
2.5
FITC
2.0
(b) 40 cm
17
1.5
1.0
0.5
0.0
18,19
10,11
20
12
5,6 13-2
89 1415
47
12
3
a
a
a
16 a
13-1
10
15
20
25
Time (min)
Figure S11 Effect of column length on MEEKC system
Running buffer, 1.43 % SDS, 6 % 1-butanol, 4 % ACN, 87.24 %, pH6 phosphate buffer (40 mM) and
the sample concentration is 4 µgL-1 (0.5 psi, 3s). Other conditions are the same as Fig.1.
His (1), Arg (2), Gln (3), Pro (4), Tyr (5), Asn (6), Thr (7), Ser (8), Ala (9), Val (10), Met (11), Gly
(12), Ile (13), Leu (14), Phe (15), Trp (16), Cys (17), Glu (18), Lys (19), Asp (20), FITC hydrolyzate or
impurity (a).
13. Table S1 Recovery of real samples
Recovery of real samples (%)a
Concentration of real samples (mg/L)
Analytes
a
Beverage
Food
Biological sample
Beverage
(spiked with 4ppb)
Food
(spiked with 4ppb)
Biological sample
(spiked with 2ppb)
His (1)
--b
106.1
1.82
87.5
100.2
87.6
Arg (2)
Gln (3)
Pro (4)
Thr (7)
Ser (8)
Ala (9)
Gly (12)
26.07
2.90
17.14
--b
--b
--b
--b
--b
--b
--b
156.8
24.28
--b
--b
5.25
--b
--b
--b
28.49
--b
--b
105.1
87.6
88.6
92.4
99.2
98.5
94.3
102.5
93.9
94.6
94.8
94.7
86.9
100.6
106.6
92.8
89.5
92.3
105.6
89.9
94.4
Ile (13)
Leu (14)
Phe (15)
Trp (16)
Cys (17)
Asp (20)
--b
--b
49.94
57.53
--b
--b
293.5
334.8
369.4
76.16
--b
--b
--b
0.68
0.77
--b
10.07
19.62
104.3
87.8
100.0
101.1
91.6
90.3
99.4
99.2
107.1
108.3
89.9
86.5
93.8
93.6
86.9
88.1
93.6
104.3
Recovery = (Found concentration of analyte standard obtained by the CE-LIF method / spiked concentration of analyte standard to real sample)
× 100%.
b
non-detection
14 References
[1] Ossola, B., Schendzielorz, N., Chen, S-H., Bird, G. S., Tuominen, R. K., Männistö,
P. T., Hong, J-S., Neuropharmacology 2011, 61, 574-582.
[2] Zhang, F., Qian, L., Flood, P. M., Shi, J.-S., Hong, J.-S., Gao, H.-M., J. Pharmacol.
Exp. Ther. 2010, 333, 822-833.
[3] Zhang, F., Shi, J.-S., Zhou, H., Wilson, B., Hong, J.-S., Gao, H.-M., Mol.
Pharmacol 2010, 78, 466-477.