Page 50 - Modern Derivatization Methods for Separation Sciences

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customarily carried out to extract analytes of interest in foods. SFE, using supercritical CO or CHF as a
2 3
fluid, has been developed in recent years. SFE can extract thermally unstable compounds and easily
control the solubility of solutes by temperature and/or pressure, and consequently the selective
extraction of analytes can be easily adjusted. CO is less polar like nhexane, so a polar solvent like
2
methanol is added in part as a modifier. Application of the method has been reported on a number of
food samples; residue analysis of atrazine and fluazifop-P-butyl [26] in onion; organochlorine
pesticides such as BHC, DDT, HCE [27,28] in chicken fat, lard, vegetables, hamburgers and peanut
butter; carbamates of carbofuran [29] in potatoes, methomyl, methiocarb and eptam [30] in apples. This
method is also used for the analysis of PCB [31] in fish, sulfonamides [32] in chicken liver and
Nnitrosamines [33] in hams. Granted to be a clean analysis, this method uses no dichloromethane and
chloroform for the extraction of analytes of interest. In addition, SFE has served not only for extraction
but also, more recently, in chromatography to separate optical isomers [34].

Solid Phase Microextraction (SPME)

SPME, a new conception different from traditional solvent extraction, has been reported for food
analysis. Extraction of the analytes of interest from the sample matrix is carried out without using a
solvent. Immersed in sample solution, fused silica fiber coated with liquid phase (polydimethylsiloxane,
or polyacrylate or an equivalent) adsorbs analytes of interest based on the principle of partition
equilibrium. The analytes would pass through the SPME/HPLC interface, and then be injected into
HPLC [35]. Not many reports have been published for SPME on food samples so far. Using this
method, analysts have less chance to use solvents. In addition, exhaust fumes and drainage from the
laboratory should seldom contain solvents. Thus, this method is preferable from the standpoint of
environmental and industrial hygiene. SPME fiber and instrument kits are offered by Spelco Co.

1.2.1.2—
Derivatization in Food Analysis

Coupled with a high sensitivity detector, fluorescent derivatization is feasible for the detection trace of
analytes in food materials and has recently been developed with a wide variety of applications
available. The bulk of fluorescent derivatization reactions fall into three general reaction types: (a) a
derivative reagent itself is nonfluorescent or weakly fluorescent, however, derivative compounds of the
reagent and the analyte of concern fluoresce; (b) a fluorogenic reagent reacts with the analyte of interest
(fluorescent labeling); (c) a reagent itself does not react with the analyte, but modifies the moiety of the
chemical structure of the analyte with the consequence of fluorescence or fluorescent intensification
(including oxidation or reduction). Use of (a) or (b) is popular for derivatization in food analysis.
Application of (c) was reported for the analysis of aflatoxin [36], a kind of mycotoxin, and avermectin
[37], an insecticide for hops, using trifluoroacetic acid (TFA) as a reaction reagent. In both cases, the
analytical technique was derivatization, but TFA is not a commonly used derivatization reagent. On the
other hand, a non-fluorescent reagent which has an intermolecular fluorescent site, on being interfered
with by some intermolecular group, is modified by the analyte of interest, and fluoresces. For example,
polysaccharide bound to 2-pyridylamino group is non-fluorescent. When combined with α-amylase
causing severe intermolecular α-1,4glycoside bonding from polysaccharide, the 2pyridylamino group
become fluorescent. This technique is applied for post-column derivatization [38]. The HPLC and CE
methods customarily employ pre-column and post-column derivatization and this chapter describes an
outline of newly developed on-column derivatization.

The principle of on-column derivatization is as follows: after the HPLC column is equilibrated with the
mobile phase in the presence of the derivative reagent, the analyte of interest, introduced from an

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