Download Application Notes No. AD-0044

Application
Notes
UHPLC - Nexera
No. AD-0044
Quantitative Analysis of Twenty Amino Acids by PreColumn Derivatization with PITC Reagent and UHPLCUV Method
Xing Jie, Edwin Ting Zhi Wei*, Sun Zhe, Zhan Zhaoqi,
Customer Support Center, Shimadzu (Asia Pacific) Pte. Ltd. Singapore
* Student from National University of Singapore for ITP
Introduction:
Quantitative determination of amino acids (AA) by liquid chromatography method is an important analysis in a variety of fields like in
food, feed, biochemistry, clinical test and protein chemistry etc. The methods involve derivatization of AA with various reagents such as
o-phthaldehyde (OPA), 9-fluoroenylmethyl chloroformate (FMOC-Cl) or phenylisothiocyanate (PITC) to convert them to derivatized AA
before or after separation for high sensitivity detection by UV-Vis detection or fluorescence detection. Pre-column derivatization with
PITC followed by reversed phase separation on C18 column is one of the choices, which has the advantages of using UV-VIS (254nm)
detection, fast separation and high sensitivity. The analysis can be easily performed on any HPLC system with a UV-VIS detector.
Derivatization of AA with PITC is carried out under alkaline conditions [1-2]. The exceed amount of PITC reagent and high concentration
of triethylamine (TEA) are the main causes of poor reproducibility and short lifetime of column used. This application note shows the
improvement of the pre-column PITC derivatization method for analysis of twenty naturally occurring amino acids (except cystine) using
a UHPLC column on Nexera UHPLC system.
.
Experimental:
Table 1: UHPLC system and conditions for analysis of PITC
derivertized amino acids
A mixed stock solution of 20 amino acids (see Table 2) was
prepared from the single AA standards obtained in powder form
from Sigma-Aldrich. The concentration of each AA in the mixed
stock solution is 500 µmol/L, except L-Glutamic acid and LTyrosine (250 µmol/L). PITC (0.1 M) and triethylamine (1.0 M)
solutions were prepared using acetonitrile as solvent. The
details of PITC deritization is described in Figure 1. The system
used is Nexera UHPLC with a UV-VIS detector (Shimadzu
Corporation). The UHPLC column and conditions are shown in
Table 1.
Instrument
Shimadzu Nexera UHPLC
Column
YMC-Triart C18, 150 x 2.0 mm ID 1.9 um
Mobile Phase
A : 10mM Potassium Phosphate (pH 7.0)
B : Acetonitrile
Gradient Elution
(B Conc.)
5% (0 min)  10% (1 min) 23% (10-12 min)
 95% (15-16.5 min) 5% (16.51-19 min)
Flow Rate
0.40 mL/min
Column Temp
35 ᴼC
Detection
UV 254 nm, Semi-micro flow cell
Injection Vol
1.0 µL
Results and Discussion:
Figure 1: Procedure and conditions of PITC derivertization of
mixed amino acids
Amino acids are very polar molecules and could not be retained
by reversed phase column. Deriverization with PITC converts
amino acids into less polar products, which are suitable for
reversed phase separation. Figure 2 shows the UHPLC
chromatograms of twenty derivertized amino acids at lower and
higher concentrations. Well separation of these derivertized
amino acids were obtained under the UHPLC conditions. The
great advantage of using UHPLC or fast LC [3,4] is the
enhanced speed of separation due to the superior resolution of
UHPLC column. Depending on the amino acid composition, the
fast LC separation could be completed within 4 minutes [3] for
analyzing 17 amino acids and 10 minutes for 23 amino acids [4].
The retentions of some less polar amino acids are very closed,
like Asn and Ser (3.987 min and 4.101 min), Gln and Gly (4.270
min and 4.369 min) etc. Well separation of these amino acids
Application
Notes
AD-0044
Figure 2 Chromatograms of twenty amino acids derivatized with PITC derivatization method
SHIMADZU (Asia Pacific) Pte. Ltd
79 Science Park Drive, #02-01 Cintech IV
Singapore Science Park I, Singapore 118264
www.shimadzu.com.sg
Tel: +65-6778 6280 Fax: +65-6778 2050
Copyright © 2011 SHIMADZU (Asia Pacific) Pte. Ltd.
All rights reserved. No part of this document may be reproduced in
any form or by any means without permission in writing from
SHIMADZU (Asia Pacific) Pte. Ltd.
Application
Notes
AD-0044
requires use of longer column and gradient program. The
current UHPLC method is optimized to analyze the 20
naturally occurring amino acids except cystine (Cys). The
method can be optimized further to shorten the analysis
time if samples contain less number of polar amino acids.
Calibration curves of the 20 deriverized amino acids were
established using mixed standard series of 50, 100, 250
and 500 µmol/L (except Glu and Tyr which concentrations
are half as stated). Excellent linearity was obtained for all
amino acids (Table 2). A few calibration curves are shown
as examples in Figure 3.
Area
100000
Repeatability of the method including derivatization and
UHPLC analysis was evaluated by preparing in parallel four
samples of same concentration. The results for 250 and
500 µmol/L (Table 2) show that the RSD for RT is less than
0.6% and that of peak area is less than 1.6% except Try
(2.12%).
Asn
75000
150000
50000
100000
25000
50000
0
0
100
0
250
Conc.
Area
125000
Ser
Tyr
100000
150000
75000
100000
50000
50000
0
0
200Conc.
Area
200000
The sensitivity of the method is considerably good as high
S/N ratios were obtained for 50 µmol/L mixed standard
sample with 1µL injection.
Area
200000
Glu
25000
0
250
Conc.
0
0
100
200Conc.
Figure 3: Selected calibration curves of PITC derivertized
amino acids by UHPLC method. Glu and Tyr: 25~250
µmol/L; Asn and Ser: 50~500 µmol/L. Injection Vol: 1 µL
Finally, the sample preparation procedure as described in
Figure 1 is proven to be less harmful to the UHPLC column
used. Under the conditions of this work, the YMC column
has not faced any separation problem after 300 runs of the
PITC-derivatized amino acid samples.
Table 2: Evaluation results of 20 amino acids analysis by pre-column PITC deriverization and UHPLC method
Ami no Aci d
Abb Na me
RT (mi n)
Ca l i bra tion (4 l evel s )
RSD % (250 umol /L, n=4)
RSD % (500 umol /L, n=4)
Ra nge
r2
RT
Area
RT
Area
L-Aspartic acid
Asp
1.913
50 - 500
0.9993
0.45
0.45
0.25
0.40
L-Glutamic Acid
Glu
2.246
25 - 250
0.9996
0.58
0.75
0.23
0.27
L-Hydroxy-L-Proline
Hydroxy-Pro
3.358
50 - 500
0.9999
0.40
0.26
0.13
0.20
L-Asparagine
Asn
3.987
50 - 500
0.9988
0.29
0.97
0.15
1.54
L-Serine
Ser
4.101
50 - 500
0.9998
0.27
0.69
0.13
0.37
L-Glutamine
Gln
4.270
50 - 500
0.9998
0.27
0.85
0.15
0.68
Glycine
Gly
4.369
50 - 500
0.9999
0.25
0.67
0.13
0.65
L-Histidine
His
4.650
50 - 500
0.9992
0.25
0.37
0.15
0.18
L-Arginine
Arg
5.164
50 - 500
0.9999
0.24
0.19
0.15
0.20
L-Threonine
The
5.361
50 - 500
0.9999
0.23
0.11
0.14
0.21
L-Alanine
Ala
5.649
50 - 500
0.9997
0.22
0.12
0.14
0.38
L-Proline
Pro
5.847
50 - 500
0.9999
0.22
0.29
0.15
0.16
L-Tyrosine
Tyr
9.135
25 - 250
0.9997
0.14
0.19
0.12
0.31
L-Valine
Val
9.566
50 - 500
0.9994
0.14
0.06
0.12
0.41
L-Methionine
Met
10.467
50 - 500
0.9992
0.11
0.08
0.10
0.29
L-Isoleucine
lle
12.355
50 - 500
0.9996
0.12
0.07
0.12
0.35
L-Leucine
Leu
12.602
50 - 500
0.9997
0.12
0.19
0.12
0.32
L-Phenylalanine
Phe
14.331
50 - 500
0.9999
0.05
0.25
0.03
1.42
L-Tryptophan
Trp
14.482
50 - 500
0.9999
0.04
2.13
0.02
0.98
L-Lysine
Lys
14.636
50 - 500
0.9999
0.04
0.40
0.02
1.08
SHIMADZU (Asia Pacific) Pte. Ltd
79 Science Park Drive, #02-01 Cintech IV
Singapore Science Park I, Singapore 118264
www.shimadzu.com.sg
Tel: +65-6778 6280 Fax: +65-6778 2050
Copyright © 2011 SHIMADZU (Asia Pacific) Pte. Ltd.
All rights reserved. No part of this document may be reproduced in
any form or by any means without permission in writing from
SHIMADZU (Asia Pacific) Pte. Ltd.
Application
Notes
AD-0044
Conclusions:
This work was to set up and evaluate the method of precolumn derivatization with PITC of 20 naturally occurring
amino acids except cystine following by analysis using a
C18 UHPLC column (1.9 µm) and UV-VIS detection
(254nm). The results show that the sample preparation,
separation conditions and performance of the analysis
method are reliable, sensitive and reproducible. The
sample preparation procedure employed is proven to be
less harmful to the UHPLC column. Under the conditions
of this work, the YMC column used has not faced any
separation problem after 300 runs of the PITC-derivatized
amino acid samples.
References:
1.
2.
3.
4.
B. A. Bidlingmeyer, S. A. Cohen, T. L. Tarvin, J.
Chromatorgr. 336 (1984) 93.
D. R, Koop, E. T. Morgan, G. E. Tarr and M. J.
Coon, J. Biol. Chem., 257, pp 8472-80 (1982).
Shimadzu Corporation, Application Data Sheet,
High Performance Liquid Chromatography, No. 7
(LAAN-A-LC-E075), 2007.
Shimadzu Corporation, Application Data Sheet,
High Performance Liquid Chromatography, No. 8
(LAAN-A-LC-E076), 2007.
SHIMADZU (Asia Pacific) Pte. Ltd
79 Science Park Drive, #02-01 Cintech IV
Singapore Science Park I, Singapore 118264
www.shimadzu.com.sg
Tel: +65-6778 6280 Fax: +65-6778 2050
Copyright © 2011 SHIMADZU (Asia Pacific) Pte. Ltd.
All rights reserved. No part of this document may be reproduced in
any form or by any means without permission in writing from
SHIMADZU (Asia Pacific) Pte. Ltd.