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overcome some of the drawbacks of the current derivatization methods used for precolumn and post-
column techniques. Pre-column derivatization often needs complicated procedures and cannot handle
unstable reaction compounds. In addition, post-column derivatization requires complicated
instrumentation. However, on-column derivatization also has disadvantages. Available derivatization
reagents are limited because oncolumn derivatization must proceed promptly at the inlet site of the
HPLC column. Optimum analytical conditions of analytes are difficult to set up due to interference with
reaction conditions and HPLC mobile phase conditions. On-column derivatization has been developed
for the analysis of amines in foods and its applications are discussed in the following chapter.
1.2.2—
Analysis of Nutrients

Saccharides, amino acids, fatty acids, organic acids and vitamins are the main constituents of foods and
used as food additives such as nutrition supplements and flavoring. This chapter describes the use of
derivatization methods for these compounds without distinguishing the origin on natural or added.

1.2.2.1—
Carbohydrates

Carbohydrates are structurally classified into monosaccharides, oligosaccharides and polysaccharides.
Monosaccharides and some oligosaccharides taste sweet. Compounds whose aldehyde group in aldose
is substituted by primary alcohol are called sugar alcohol. This chapter describes the analysis of
carbohydrates including sugar alcohol.

Analysis by HPLC

Many liquid chromatographic systems have been described for the analysis of major sugars and
polyhydric alcohols in foods. These systems have usually incorporated refractive index (RI) detection
coupled with ion-exchange or aminophase separation. However, insufficient sensitivity and specificity
(the detection limit is at the µg level) of the RI detector has promoted the improvement of the detection
system or use of the derivatization method. The former includes

pulsed amperometric detection (PAD), the use of an electrochemical detector (ECD) whose glassy
carbon electrode surface was coated with Cu/Cl containing film using CuCl [39] or ECD detection with
2
an ion-paired reagent in the alkaline mobile phase regarding saccharides as weak acid [40].

The use of post-column derivatization with paminobenzoic acid hydrazide followed by detection at
visual-absorption (VIS) 410 nm was reported [41]. The detection limit of the method was 5 ppb. A
variant post-column method with immobilized enzyme reactors which detect indirectly produced
hydrogen peroxide with high sensitivity, was also reported. A fundamental system which determines
glucose uses immobilized glucose oxidase (GOD) to produce hydrogen peroxide followed by ECD
detection [42]. This method was applied for the analysis of such disaccharides as maltose [43] and
sucrose [44,45]. The analysis of maltose uses immobilized glucoamylase (GAM) combined with a
glucose detection system. Maltose was hydrolysed to α-D-glucose and β-D-glucose. βD-glucose was
transferred to gluconolacton and hydrogen peroxide by GOD and then hydrogen peroxide was detected
with ECD. As for the analysis of sucrose, immobilized reactors of invertase (INV) and pyranose
oxidase (PyOD) were connected and the end product of hydrogen peroxide was determined by
chemiluminescence method with luminol [44]. In this case, sucrose was hydrolysed to glucose and
fructose by INV, the glucose produced in the first reactor was then used to produce hydrogen peroxide
by GOD. The PyOD, which is also referred to as glucose 2-oxidase, oxidizes the hydroxyl group at the

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