Page 10 - Shimadzu Journal vol.7 Issue2

Food Development

1-3-1. Analytical Method Development the newly developed method cannot discern a difference between the
mass of L-glutamine (monoisotopic mass of 146.069) and D-lysine
In developing our high-sensitivity LC/MS-MS analytical method for (monoisotopic mass of 146.106). To resolve this problem, we investigated
D-amino acids, we first optimized MRM transitions for target amino using the CROWNPAK CR-I (-) column with a structure complementary
acids then verified conditions for chiral amino acid separation. LC to the CROWNPAK CR-I (+). The binaphthyls used in CROWNPAK CR-I
separation conditions were investigated based on the conditions used (+) and CROWNPAK CR-I (-) are axis chiral enantiomers (CR-I (+):
in the already-reported LC-TOFMS method (Konya et al. 2016). As (S)-18-crown-6-ether, CR-I (-): (R)-18-crown-6-ether) (Fig. 5). Although
described in detail earlier, the LC-TOFMS method uses a column we await detailed elucidation of the mechanism of separation, D- and
(CROWNPAK CR-I (+)) with a chiral selector consisting of a crown L-amino acids are eluted in opposite orders by the two columns. That
ether bound to a binaphthyl skeleton. Interaction between the is, using the CR-I (+) column the new method elutes respective D-amino
stationary phase crown ether and the target amino acid and also steric acids first, and using the CR-I (-) column the new method elutes
hindrance caused by the binaphthyl skeleton are presumed to have a respective L-amino acids first. Based on the above, using CR-I (-) in
substantial effect on optical resolution of D- and L-amino acids (Fig. 4). addition to CR-I (+) allowed the newly developed method to recognize
The LC-TOFMS method achieves optical resolution of almost all target L-glutamine and D-lysine as single peaks rather than a coeluted peak
D- and L-amino acids with very high resolving power (Fig. 4). In contrast (Fig. 6). Similarly, using both columns allowed D-isoleucine and
with the LC-TOFMS method, we found the MS/MS of the newly D-allo-isoleucine to be recognized as single peaks rather than a coeluted
developed method failed to differentiate between L-glutamine and peak. Also, by monitoring for peaks only 0.75 minutes either side of
D-lysine and recognized them as the same compound. This failure was the predicted retention time, we reduced the number of MRM transitions
attributed to the amino acids being eluted at similar retention times that needed to be collected per analysis and were able to configure
and having the same MRM transitions. Because TOFMS can obtain analysis to achieve even better repeatability.
accurate mass data on compounds, the LC-TOFMS method can
achieve separation by MS even when LC has difficulty achieving
separation. By contrast, the mass resolving ability of MS/MS used in

Fig. 5 Structure of the Chiral Selectors in CROWNPAK CR-I (+) and CROWNPAK
CR-I (-)

Fig. 6 Extracted Ion Chromatogram of Glutamine and Lysine Obtained Using
CROWNPAK CR-I (-)
Next, we adjusted the position of the electrospray probe at the ion
source. Ionization efficiency and mobile phase desolvation during
target component ionization are largely dependent on probe
position, hence probe position is considered to have a bearing on
sensitivity improvements and a recent report has even investigated
sensitivity and positional parameters for the ion source in detail
(Janusson et al. 2015). The sensitivity of the newly developed
method improved dramatically when the probe was moved from its
default position further away from the area where ions enter the
mass spectrometer. This improvement was particularly apparent for
glycine and alanine, which are relatively low-molecular-weight
Fig. 4 Chromatogram of 18 Protein-Constituent Amino Acids amino acids. Lastly, we optimized MS interface gas parameters
(nebulizer gas, heating gas, and drying gas), heating parameters
All compounds in the 1 nmol/mL D-/L-amino acid standard solution were eluted
within 8 minutes. (desolvation line temperature and heat block temperature), and
Analysis with the CROWNPAK CR-I (+) column eluted the D-form of each amino interface voltage. We consider the two major features of Shimadzu’s
acid before its L-form. The D- and L-forms of isoleucine and leucine were eluted
in the order of D-isoleucine + D-allo-isoleucine (coelution), D-leucine, LCMS-8060 are that it allows this type of detailed investigation of
L-allo-isoleucine, L-isoleucine, and L-leucine.
MS parameters and that its user interface is easy to use.

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