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reported using OPA/3-mercaptopropionic acid (3-MP) for the primary amino acids, followed by FMOC
for the secondary amino acids [87]. FMOC was introduced as a protective reagent for the amino group
during peptide synthesis, FMOC derivatives show excellent reactivity, sensitivity and stability,
furthermore they react with primary and secondary amines. Thus FMOC has been a widely used
derivatizing reagent for the determination of amines and fumonisins, a kind of mycotoxin. The problem
is that FMOC itself fluoresces, and then interferes with the fluorescence determination of FMOC
derivatives of amino acids. Thus the elimination of excess reagent such as pentane extraction [72],
heptylamine addition [74], and alkaline hydroxylamine addition [73] is required after derivatization.
Amino acid analysis with FMOC has another problem. Derivatization of aspartic acid and glutamic acid
with FMOC proceeds slower than that of other amino acids. A prolonged reaction time to complete
derivatization of these two amino acids will cause the formation of more hydrophobic disubstituted
derivatives (di-FMOC-Tyr, and di-FMOC-His) of tyrosine and histidine. To solve this problem,
alkaline treatment of these FMOC disubstituted derivatives was carried out to form each
monosubstituted derivative. As the peak area of monosubstituted derivatives of Tyr and His are 2.3-2.5
fold that of disubstituted derivatives, this transformation will be favorable for the analysis of Tyr and
His. After FMOC derivatization, some FMOC derivatives of other amino acids might be subjected to
decomposition by alkaline treatment. A mixture of hydroxylamine and sodium hydroxide proved
effectiveness in converting both di-FMOC-Tyr and (di-FMOC-His to the monosubstituted derivatives,
because it had little effect on normal N-FMOC groups. The method was applied for the analysis of
white lysozome hydrolysate of eggs with fluorescence detection λex263 nm, λem313 nm) [73], and of
free amino acids and amines in wine, fruit or vegetable juice, fish, and cheese [74].

Dansylation of amino acids has been widely studied and appeared to be suitable for UV (254 nm) or
fluorescence (λex330 nm, λem530 nm) measurement. However, the reaction was complicated by a side
reaction whereby the amino acid was degraded. DNS is a electrophilic reagent and reacts with the
unprotonated form of the amino group, and thus a pH of 9 or greater is required. However, at high pH
this reaction competes with hydrolysis of DNS. Moreover, a second competing reaction takes place
between excess DNS and dansylated amino acid, leading to the loss of dansylated amino acids and the
formation of dansylamide. To minimize this side reaction, it was necessary to closely control reaction
pH, temperature, and duration, as well as the ratio of DNS reagent to amino acids. The addition of
methylamine and ethylamine is also suggested [88]. The use of DNS was applied for the analysis of
hydrolysates of feedstuff [77] and casein [78], taurine in milk and infant formula [79]. For the analysis
of taurine, the pre-column method with DNFB and UV detection at 350 nm was reported [76]. Other
than infant formula, this method was also applied for the measurement of taurine in meat, tuna, and
squid (detection limit 10 pmol).

AQC, a novel derivatization reagent, can react in seconds with all primary and secondary amino acids
without appreciable matrix interference to form single, quantitative, and very stable derivatives. In
addition, the excess reagent was hydrolyzed to 6-aminoquinoline(AMQ) in less than 2 min, thus
preventing any unwanted side reactions and the florescence emission maxima of AMQ and AQC-
derivatized amines are approximately 100 nm apart, allowing for selective detection of the desired
analytes without significant reagent interference. This method was applied for the analysis of
hydrolysate of grain and corn powder [80,81]. All data were generated by fluorescence detection except
for the analysis of tryptophan, whose fluorescence response is weak due to internal quenching, which
used UV detection.

Little is known about the post-column derivatization of amino acids in foods. Ninhydrin was used to
measure taurine in infant formula and milk(VIS 570 nm) [89]. To enhance the sensitivity, the use of
OPA/3-MP instead of ninhydrin was reported for post-column derivatization with fluorescence

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