Application of (S)-(NIFE) as a chiral derivatizing reagent
553
for intra-day precision and 0.06 to 0.20% for inter-day
precision and for (-)-norvaline 0.12 to 0.43 and 0.01 to
0.82% for intra and inter day precision, as diastereomers,
respectively. The recoveries of diastereomers of (?)- and
(-)-norvaline were found to be between 96.51 and
99.43%. For accuracy studies, samples were prepared by
spiking (-)-norvaline with fixed amounts of (?)-nor-
valine in the range 0.001–0.01%. The results indicate
that this method can be applied for the detection of (-)-
norvaline in (?)-norvaline up to 0.003% by HPLC in the
form of diastereomers. The present HPLC method using
(S)-NIFE is capable of detecting 0.02 ng (0.01 ng of
each enantiomer) of norvaline while the LOD for nor-
valine with MR was 57.5 lg (28.75 lg of each enan-
tiomer) (Bhushan and Kumar 2008b).
Table 1 Resolution of diastereomers of racemic amino compounds
by RP-HPLC
S. no. Amino alcohols
Chromatographic data
k2 k1
a
RS
1
2
3
4
5
6
7
8
Homophenylalaninol
Valinol
26.474 24.466 1.082 20.550
19.625 17.619 1.114 21.729
Prolinol
17.791 17.282 1.029
5.164
Leucinol
24.578 22.891 1.074 12.159
Alaninol
14.858 14.358 1.035
18.190 17.952 1.013
4.406
3.599
Phenylglycinol
2-Amino-1-butanol
Phenylalaninol
18.307 16.589 1.104 17.552
20.769 20.463 1.015 2.672
Non-protein amino acids
9
Norvaline
59.979 53.087 1.113 24.765
22.645 20.314 1.115 15.913
10
11
12
13
14
15
2-Phenylglycine
2-Aminooctanoic acid 28.954 26.782 1.081 20.958
Conclusion
Pipecolic acid
20.949 20.052 1.044
8.255
2-Aminobutyric acid
2-Aminoadipic acid
Penicillaminea
23.979 21.472 1.117 19.858
16.632 15.326 1.085 11.586
Application of (S)-NIFE as the CDR does not require
special protection of all synthesis steps from light and the
time required for derivatization is less than the other de-
rivatizing reagents. Overall, it is thus more economical.
Under the reaction conditions no racemization was
observed and the derivatives were stable for several weeks.
The method has thus potential application in quality control
in pharmaceutical formulations of these pharmaceutically
and biologically important compounds.
23.266 22.321 1.042
8.471
Column: Eurospher C18; mobile phase as given in experimental; flow
rate 1.0 mL min-1; load amount, 20 lL; detection at 205 nm. t0 value
was 2.41 min; a is the separation factor
a
Bis derivatives
amino acids seems to be the only factor to affect the inter-
action of the diastereomers with the ODS material of the
column and the separation. Thus, looking to the alkyl side
chain in the structures of analytes as shown in Fig. 1, the
following observation was made: with increasing alkyl
group in the side chain the retention times of the analytes
(Fig. 3) increased, particularly for the following sets:
leucinol[ valinol; homophenyl alaninol [phenylalaninol[
phenyl glycinol[prolinol [ 2-aminobutanol[alaninol;
2-aminooctanoic acid [norvaline [phenylglycine[ pipe-
colic acid[2-aminoadipic acid; 2-aminobutyric acid[
2-aminoadipic acid.
Acknowledgments The authors are thankful to the Ministry of
Human Resources Development, Government of India, New Delhi,
for the award of a research assistantship (to C.A.) and to the Alex-
ander von Humboldt Foundation, Bonn, Germany, for donating
Knauer HPLC equipment and award of a fellowship (to R.B.)
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123