Page 75 - Modern Derivatization Methods for Separation Sciences

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Analysis of sorbic acid employs the pre-column method with 4-bromomethyl-6,7-dimethoxycoumarin
(Br-DMC) to produce fluorophore derivatives followed by separation on reversed-phase HPLC and
fluorescence detection (λex355 nm, λem420 nm) [142]. After extraction with steam distillation,
margarine and butter samples were analysed by this method.

Lysozyme has been used for a drug and a food preservative lysing the cell wall of gram positive
bacteria to destroy growth of bacteria. Measurement of lysozyme activity in food has recently been
developed using the fluorescence technique with synthesis substrate, 4-methylumbelliferyl tri-N-acetyl-
β-chitotrioside (4-MU-(GlcNAc) ) [143]. This substrate is a chemical compound of fluorescent
3
substance, 4-methylumbelliferone (4-MU) and trimer acetylglucosamine. The whole molecule shows
non-fluorescence because of bonding with acetylglucosamine. 4-methylumbelliferone (4-MU)
fluoresces when liberated by hydrolysis by lysozyme. This principle was applied for the precolumn
HPLC method, which includes separation of 4-MU produced by enzyme reaction and 4-MU-(GlcNAc) 3,
substrate, and the use of polystyrene reversed-phase column with pH 12 alkaline buffer as the mobile
phase to yield sufficient fluorescence intensity. The method is employed for the measurement of
lysozyme activity in Japanese sake, Mirin (sweet sake), cheese and custard cream.

1.2.3.2—
Antioxidants

Unsaturated fatty acids in fats and oils are subject to oxidation to produce hydroperoxide, and toxic
oxides are further produced by chain reactions.

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