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LAAN-A-LM-E121

Application Liquid Chromatograph Mass Spectrometry

News
Developing a Chiral Amino Acid Analysis Method
That Uses Column Switching
No. C149

With the exception of glycine, the 20 types of amino acids that analysis methods for accurate measurement. Furthermore,
make up proteins occur as D and L optical isomers. L-amino conventional optical separation analysis of amino acids
acids occur in large quantities in the body as protein necessitated derivatization and long separation times of the
components and sources of nutrients. As for D-amino acids, amino acids.
despite the fact that they are much less abundant than L-amino This article introduces a rapid analysis method that employs
acids, they are attracting attention in various fields as chiral columns to achieve high separation and high sensitivity
components associated with the component analysis of and that dispenses with derivatization [1]. This system uses
fermented foods, physiological functions in the central nervous two types of chiral columns alternately with high-pressure
system, biomarkers, and even health and beauty. column switching valves (FCV) and allows fully automatic
Analysis of D-amino acids is susceptible to interference by a analysis of a wide range of D- and L-amino acids.
wide variety of peptides and amino compounds, and Y. Uno, T. Hattori
therefore requires high sensitivity and highly selective
Pump A
CR-I (+)
FCV FCV waste

CR-I (−) waste

Pump B
Guard column
Mobile phase
LCMS
Autosampler

Chiral Amino Acid Analysis System That Uses Column Switching

A system capable of analysis by automatically switching between two column types, CR-I (+) and CR-I (−), using two high-pressure
column switching valves (FCV) was configured (Fig. 1). Pump A is connected to CR-I (+) and pump B is connected to CR-I (−). This
means that even if one column is undergoing analysis, the other column can undergo stabilization without stopping mobile phase
delivery.
(×1,000,000) (×1,000,000)
2.5 2.25
CROWNPAK CR-I(+) 2.00 CROWNPAK CR-I(−)
2.0 1.75
1.50
1.5 1.25
1.00
1.0 0.75
0.5 0.50
0.0 0.25
0.00
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

MRM Chromatograms of D- and L-Amino Acids in Standard Mixed Solution (Sample Concentration: 1 ng/1 μL)

Table 1 Analysis Conditions
Column : CROWNPAK CR-I (+) / CR-I (–) (3 mm × 150 mm, 5 μm, DAICEL Corp.)
Mobile phase : Acetonitrile/ethanol/water/TFA = 80/15/5/0.5
Flow rate : 0.6 mL/min
Injection volume : 1 μL
Oven temperature : 20 °C
Ionization mode : ESI (Positive)
Probe voltage : +4.0 kV
Neburizing gas flow : 3.0 L/min
Drying gas flow : 15.0 L/min
Heating gas flow : 5.0 L/min
Interface temperature : 250 °C
DL temperature : 250 °C
Block heater temperature : 300 °C