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3.2.3—
Peptides

3.2.3.1—
General Peptides

Fluorogenic reagents such as OPA (Fig. 3.1A), NDA (Fig. 3.1B) and fluorescamine (Fig. 3.1D) are
used for the sensitive HPLC determination of general peptides. These reagents react with primary
amines, so that peptides containing free αand εamino moieties can be detected by pre or post-column
derivatization methods [100-103]. Using chromatographic methods, the peptides can be determined at
the 0.05-100 pmol levels on-column. However, these reagents are not selective for peptides because
nearly all peptides have a primary amino group. Therefore, the separation conditions with a high
resolution for peptides is required, especially when endogenous peptides in biological matrices are
determined by HPLC.

A pre-column HPLC method with NDA reagent is developed for the determination of enkephalin
peptides [102]. In the method, the column-switching technique is necessary for the separation of the
biogenic enkephalins.

3.2.3.2—
Arginine-containing Peptides

The post-column derivatization HPLC method has been established for the determination of arginine-
containing peptides [104,105]. The method is based on the conversion of the guanidino moiety of an
arginyl residue in an alkaline solution (100 °C, 90 s) with benzoin into their respective fluorescent
derivatives (Fig. 3.25). The fluorescence detection is specific for the arginine-containing peptides, and
this method can detect them down to about 10 pmol on-column (S/N = 2).

The pre-column derivatization HPLC method using benzoin is more sensitive for arginine-containing
peptides and permits possible detection at levels as low as 100 fmol on-column [106].

3.2.4—
Fluorescence Derivatization for CE

Fluorescamine and OPA are reagents for pre and post-capillary derivatization of primary amino acids
and small peptides. Fmoc-Cl, FITC, NDA and CBQA can only be used for the precapillary
derivatization of both primary and secondary amino compounds. For the simultaneous derivatization of
primary and secondary amino compounds, parallel evaluation of more than one amino-selective reagent
have been reported [107]. The fluorescent derivatives from these reagents are usually determined by
various types of LIF detection such as He-Cd, Ar-ion, far-red and near-infrared diode lasers, depending
on the reagents used.

Benzoin and 4-methoxy-1,2-phenylenediamine are specific reagents for the derivatization of arginine
and tyrosine containing peptides, respectively. LIF detection employing a He-Cd laser is well suited to
detect the derivatives from both reagents.

3.3—
Reagents for Organic Acids

Various organic acids (e.g. fatty acids, bile acids, prostaglandins, α-keto acids, sialic acids and ascorbic

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