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Fig. 1.2.7.
Typical high-performance liquid chromatograms of fluorescent derivatives.
Processed food: mixture of fishes, shellfishes and meats:
(A) without oxidation; (B) after oxidation. (Reproduced from ref.
144: J. Food Hyg. Soc Japan, (1990) 31, p. 49, Fig. 4.).

Antioxidants have been used to prevent these reactions.

Ascorbic acid (AA) and erysorbic acid (ErA) are stereoisomers, and used for nutritional enrichment and
antioxidants. AA exists in nature as a food component, while ErA does not exist. Each has a reduction
form and an oxidation form. The oxidation forms of AA and ErA react with OPDA to form fluorescent
derivatives. The use of derivatization with OPDA after oxidation of the reduction form with indophenol
is followed by measurement of total AA and total ErA [144]. This method applied to processed food
includes extraction with metaphosphoric acid, oxidation with indophenol, derivatization with OPDA
and clean-up with SPE (Sep-pak C18). Separation with HPLC employs the alkaline mobile phase and
polymer column regarding separation and detection sensitivity. Detection limits for AA and ErA are
both 0.5 ng (Fig. 1.2.7).

1.2.3.3—
Sweeteners

Typical sweeteners are saccharin, glycyrrhizic acid, aspartame and sorbitol.

Analysis of aspartame (N-L-α-aspartyl-L-phenylalanine 1-methyl ester), dipeptide, uses precolumn
method with fluorescamine to form fluorescent derivatives followed by reversed-phase HPLC and
fluorescent detection (λ-ex390 nm, λem480 nm) [145], and applied for such liquid food as soft drinks
and soy sauce, chewing gum, bean paste(miso) and chocolate. The detection limit for liquid food is 2
ppm, for solid food is 4 ppm. The aromatic phenylalanine moiety of aspartame molecule is transformed
to electrochemically active derivatives with UV (254 nm) irradiation. This reaction is applied for post-
column photolytic derivatization with HPLC separation [146], and the method is used for diet colas and
pharmaceutical products. Preliminary operation requires only dilution with water and the detection limit
is 0.5 ppm as the standard concentration.

The derivatized HPLC of such sweeteners as acesulfame K, cyclamate and saccharin is reported using
post-column ion-pair extraction as described in analysis of fatty acids in ''1.2.2 Analysis of food
nutrients" [99]. The principle involves mixing the analyte, as it elutes from the HPLC column, with an
appropriate counter-ion. The resulting ion-pair is dynamically extracted into an organic phase, which
enters the detector for detection. This method uses methyl violet 2B or crystal violet for dyes of

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