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Automated Analysis of Thirty-seven D/L-amino Acids
using Liquid Chromatography with Fluorescence
Detection and Its Application to Liquor Samples
Separation and determination of thirty-seven D/L-amino acids in a short time with simple operation are possible.
The automatic operation reduces the labor and more than an hour of the working time per 20 samples consecutive analyses.
The elapsed time of the derivatization reaction is kept constant, resulting in good reproducibility of determination.
N Iwata
High Performance Liquid Chromatograph Nexera™ X3 RF-20AXS
Introduction
Amino acids have D/L enantiomers that have asymmetric
centers in their molecular structures except for glycine. In
contrast to L-amino acids, there has been limited studies of D-
amino acids. The role of D-amino acids in the palatability,
preservability, and the aroma of food and foodstuff has been
largely unknown until recently. However, fermented foods and
biological samples are known to contain small amounts of D-
amino acids in addition to large amounts of L-amino acids.
Therefore, the demand for D/L separation of amino acids is
increasing.
This article introduces the analyses of fluorescent diastereomers
of the proteinogenic D/L-amino acids using derivatizing
reagents with chiral structures. In addition, it also introduces the
automated analysis including derivatization.
Analytical Conditions and Automated
Analysis
The target compounds are thirty-seven D/L-amino acids
excluding D/L-proline from the proteinogenic amino acids
(Table 1). The background colors of red and blue indicate
OPA/NAC and OPA/NIBC derivatized amino acids, respectively.
Figure 1 shows the flow diagram of the HPLC setup for
automated analysis. In this study, the mobile phases were
prepared automatically using the mobile phase blending
function of the solvent delivery pump, and two sets of analytical
conditions were switched automatically. D/L-amino acids were
derivatized using the automatic pretreatment function of the
autosampler. The differences between the two sets of analytical
conditions shown in Table 2 are the blending ratio of organic
solvents and the gradient profile. The mobile phase blending
function provides the solutions to deliver at the specified
blending ratio just by setting the organic solvents and ultrapure
water in the ports of the low-pressure gradient kit integrated
into the pump. The labor and the working time required for the
mobile phase preparation and the mobile phase replacement
accompanied with switching the analytical conditions were
reduced by this function.
In addition, the automatic pretreatment function is described
below. The derivatizing reagents of OPA/NAC and OPA/NIBC
and the target samples were set in the autosampler. This
derivatization process was completed within the injection
needle and the derivatized diastereomers were introduced into
the column without any exposure to the outside (Table 3 and
Table 4). Figure 2 shows the setup screen of the pretreatment
program on the workstation LabSolutions
TM
. The elapsed time
from starting the derivatization to injection to HPLC was kept
constant using the pretreatment function, resulting in good
reproducibility of determination. In addition, the consumable
cost was reduced because the vials for derivatization were not
required.
Table 5 shows the comparison of the required working time of
the automatic and the manual operations when 20 samples
were analyzed. The automatic operations reduced the labor and
more than an hour of the working time in comparison with the
manual operation.
Table 1 List of Target Compounds
1 D-Asp 8 D-Arg 15 D-Ile 22 L-Ser 29 L-Tyr 36 L-Leu
2 D-Glu 9 D-Ala 16 D-Phe 23 L-Gln 30 L-Val 37 L-Lys
3 D-Asn 10 D-Tyr 17 D-Leu 24 L-His 31 L-Met
4 D-Ser 11 D-Val 18 D-Lys 25 L-Thr 32 L-(Cys)2
5 D-Gln 12 D-Met 19 L-Asp 26 Gly 33 L-Trp
6 D-His 13 D-(Cys)2 20 L-Glu 27 L-Arg 34 L-Ile
7 D-Thr 14 D-Trp 21 L-Asn 28 L-Ala 35 L-Phe
* Background color; Red: OPA/NAC derivatized amino acids, Blue: OPA/NIBC derivatized amino acids
Derivatization for chiral amines
Two chiral thiols, N-acetyl-L-cysteine (NAC) and N-isobutyryl-L-
cysteine (NIBC), were used for derivatization. Fluorescence
derivatization of diastereomers of D/L-amino acids was
performed by the reaction with o-phthalaldehyde (OPA) under
NAC or NIBC.
LC analysis of D/L-amino acids
In general, LC/MS or multi-dimensional LC is used for D/L-amino
acids analyses by HPLC, because it is difficult to separate
proteinogenic D/L-amino acids in a single separation mode.
However, it is known that LC/MS is susceptible to matrix effects
and less quantitative than HPLC. It is also known that the multi-
dimensional LC method requires a long analysis time and very
complicated HPLC setup. Therefore, a simple operational
method that provides good separation for D/L-amino acids in a
short time is required.
In food analysis, a small particle column is used because of the
appropriate separation of small amount of D-amino acids from
large amount of L-amino acids and co-existing contaminants.
Therefore, Nexera X3 with 130 MPa pressure tolerance was used
because the system pressure became high.
No. L592
