Page 87 - Modern Derivatization Methods for Separation Sciences

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known as carcinogens. Both natural and synthetic hormones have been regulated and their usage and
residue levels are different in each country. Determination levels of hormones are quite low as little as
ppb-ppt. Because the analytes of interest are animal tissues, analysis of these samples is easily affected
by contaminants and requires a complicated procedure. Therefore, the use of GC/MS is common, and
immunoassay which is a feasible technique for detection of trace analytes with a high sensitivity has
been recently applied for the residue analysis. The HPLC method with derivatization was employed for
the analysis of clenbuterol and progesterone. Belonging to a β-blocker with chemical structure of
phenylethylamine, Clenbuterol is a basic molecule and consists of an aromatic ring bonded with the
amino group. An analytical method for animal tissues such as liver includes extraction with diluted
hydrochloride followed by clean-up with SPE (C18) [201] or enzymic degradation with subtilisine A
followed by liquid-liquid extraction clean-up with Chem Elute (CE1020) [202], post-column
derivatization with diazo-coupling reaction and determination with VIS (500 nm)-HPLC.

Fig. 1.2.11.
High-performance liquid chromatograms of progesterone standard 2 ng (A),
beef (B) and fortified beef as 20 ppb progesterone (C). (Reproduced from
ref. 203: J. Food Hyg. Soc. Japan, (1995) 36, p. 599, Fig. 6.).
As for the analysis of progesterone, the carbonyl group reacts with dansylhydrazine (DNS-H) to form
fluorescent derivatives under acidic catalyst (λex340 nm, λem520 nm) [203]. In this case, progesterone
reacts to form dansylhydrazon on the steroid framework with two positions of the second and twentieth
carbon site, while reaction with the carbonyl group of the third carbon site produces both anti- and syn-
stereoisomers represented by two peaks on the chromatogram (Fig. 1.2.11). Sample pretreatment
includes extraction with acetonitrile, liquid-liquid extraction with dichloromethane, and clean-up with
SPE (Bond Elute DEA), moreover Sephadex LH-20. This method was feasible to detect as low as 2 ppb
progesterone.

1.2.5—
Analysis of Pesticides

Among the analytical methods for pesticides found in foods, GC and GC/MS have been the main
methods in use and the use of HPLC and CE has been seldom reported. However, pesticides which
have large polarity are not easily analysed by GC and some pesticides are unstable to the high

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