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Application   No. C149
    News



            „  Analysis of Standard Solution
            This system was employed  to analyze a  standard  mixed   With CR-I (–), D-Gln and L-Lys, L-Ile and L-allo-Ile, and L-Thr
                        13
            solution using  C 6-L-Phe as the internal standard  (Fig. 2).   and L-allo-Thr, which each have the same MRM transition, are
            Approximately equal area ratios were obtained with CR-I (+)   detected with the same peak and therefore cannot be
            and CR-I (–) for the amino acids other than Gln, Lys, Ile, allo-Ile,   separated.
            Thr, and allo-Thr, and this confirmed that the system is capable   However, separation measurement can be performed for
            of separation measurement (Table 2).               these amino acids by utilizing two types of  columns. For
            With CR-I (+), L-Gln and D-Lys, D-Ile and D-allo-Ile, and D-Thr   example, while D-Thr and  D-allo-Thr cannot be separated
            and D-allo-Thr, which each have the same MRM transition, are   with CR-I (+) and L-Thr and L-allo-Thr cannot be separated
            detected with the same peak and therefore cannot be   with CR-I (–), interchanging the column types allows for
            separated.                                         separation measurement (Fig. 3).

                                            Table 2  Analysis Results of Standard Solution
                         CR-I (+)      CR-I (−)       Ratio of            CR-I (+)       CR-I (–)       Ratio of
                      Ratio of           Ratio of      Area             Ratio of          Ratio of      Area
                   RT                RT                              RT                RT
                       Area              Area         (+)/(−)            Area              Area         (+)/(−)
             D-Ala   1.394  0.728     3.894  0.751     0.97   D-Leu   1.107  2.019      3.178  2.400     0.84
             L-Ala   3.908  0.565     1.389  0.632     0.89   L-Leu   3.179  2.929      1.105  3.364     0.87
             D-Arg   0.973  3.999     1.506  3.239     1.23   D-Lys   2.181  4.621  with L-Gln      7.395   1.641      2.82
             L-Arg   1.499  5.633     0.981  6.718     0.84   L-Lys   7.348  1.795    2.161   5.118  with D-Gln   0.35
             D-Asn   1.255  1.018     2.036  1.030     0.99   D-Met   1.259  1.704       4.554   1.859     0.92
             L-Asn   2.036  0.805     1.263  0.911     0.88   L-Met   4.556  0.938       1.25   1.060     0.89
             D-Asp   1.253  0.742     2.039  0.863     0.86   D-Phe   1.101  1.568       2.087   1.974     0.79
             L-Asp   2.036  0.72     1.259  0.775      0.93   L-Phe   2.089  2.175       1.106   2.280     0.95
             D-Cys   1.183  0.405     2.307  0.458     0.89   DL-Pro   0.957  2.756       0.971   3.105     0.89
             L-Cys   2.308  0.789     1.186  0.797     0.99   D-Ser   1.222  0.224       1.756   0.253     0.89
             D-Gln   1.247   2.111        2.161   3.478  with L-Lys   0.61  L-Ser   1.758  0.307       1.226   0.301     1.02
             L-Gln   2.183   4.947  with D-Lys      1.239   3.686     1.34  D-Thr   1.023  1.339  with D-allo-Thr    1.53   0.968     1.38
             D-Glu   1.246  2.972    4.426  3.262      0.91   L-Thr   1.533  0.851    1.033   1.324  with L-allo-Thr   0.64
             L-Glu   4.388  3.506    1.24  3.731       0.94   D-allo-Thr  1.023  1.339  with D-Thr      1.205   0.573     2.34
             Gly   2.827  0.037      2.796  0.039      0.93   L-allo-Thr   1.197  0.480         1.033   1.397  with L-Thr   0.34
             D-His   0.967  2.797    1.099  3.917      0.71   D-Trp   1.105  2.839      1.99  3.344     0.85
             L-His   1.09  3.699     0.977  2.969      1.25   L-Trp   1.988  3.458      1.111  3.510     0.99
             D-Ile   0.988   4.745  with D-allo-Ile  1.446   2.983     1.59  D-Tyr   1.103  1.203      2.016  1.560     0.77
             L-Ile   1.44   2.325    0.998   4.408  with L-allo-Ile   0.53  L-Tyr   2.016  1.448      1.109  1.455     1.00
             D-allo-Ile  0.988   4.745  with D-Ile      1.313   2.926     1.62  D-Val   0.999  1.826      1.337  2.052     0.89
             L-allo-Ile   1.308   1.844   0.998   4.101  with L-Ile   0.45  L-Val   1.331  3.170      1.008  3.251     0.97

                 indicates amino acids that can be separated by one column but not the other.
                                CROWNPAK CR-I (+)                                CROWNPAK CR-I (–)
                       120.10>74.00 (+)                           450000  120.10>74.00 (+)
                 600000
                                        D-Thr + D-allo-Thr        400000                 L-Thr + L-allo-Thr
                 500000                                           350000
                                                                                               D-Thr
                                                                  300000
                 400000                                                                D-allo-Thr
                                       L-allo-Thr   L-Thr         250000
                 300000
                                                                  200000
                 200000                                           150000
                                                                  100000
                 100000
                                                                   50000
                     0                                                0
                       0.5        1.0        1.5                        0.5        1.0        1.5

                                           Analysis Result of D/L-Threonine and D/L-allo-Threonine

            References [1] Nakano,Y., Konya,Y., Taniguchi,M., Fukusaki,E., Journal of Bioscience and Bioengineering, 123, 134-138 (2016)

            The analysis method presented in this edition of Application News  was developed by the Fukusaki Lab in  the School/Graduate School of
            Engineering at Osaka University.

                                                                                                     First Edition: Apr.  2017
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