Joint International Conference on Long-term Experiments ...

FORMATION OF AFLATOXIN M- TRIFLUOROACETIC ACID DERIVATIVE IN
OPTIMUM CONDITIONS
Lucian Bara
University of Oradea, Faculty of Environmental Protection
26, General Magheru Street, Oradea
ABSTRACT
Because thin-layer chromatographic (TLC) confirmation of identity and reverse-phase
liquid chromatographic (LC) determination with fluorescence detection of aflatoxin M1,
both require the derivative formed in the reactions conditions were studied to obtain
complete derivative formation. Of the various organic solvents tested, the reaction between
M1 and TFA preceded best in the nonpolar solvents hexane and isooctane. Other parameters
investigated were reaction temperature and time, aflatoxin M1 concentration, and solvent
volume. The following procedure is considered optimum: 200 µL each of hexane and
trifluoroacetic acid are mixed with M1 standard in a silylated glass vial or with milk residue
in a regular glass vial with a Teflon-lined screw cap and heated 10 min at 40ºC. The mixture
is evaporated to dryness under N2, and the derivative is saved for TLC or LC. No unreacted
aflatoxin M1 was detected by reverse-phase LC after this procedure was incorporated for
analysis of milk samples.
INTRODUCTION
The hemiacetal derivatives of aflatoxins B1 and G1 (B2a and G2a) have been used for thinlayer
chromatographic (TLC) confirmation of identity since 1971. The derivatives are
highly fluorescent and used extensively for the quantitation of these aflatoxins by reversephase
liquid chromatography (LC), because B1 and G1 are only weakly fluorescent in
aqueous mobile phases. Derivatives B2a and G2a are readily prepared by the trifluoroacetic
acid (TFA)-catalyzed hydration of aflatoxins B1 and G1 are at room temperature. This is not
true with aflatoxin M1 because the reaction rarely goes to completion. The first reversephase
LC method for determining aflatoxin M1 in milk and milk products, which measured
the fluorescence of the M1-TFA derivative (designated M2a), was reported by Beebe and
Takahashi in 1980. No formal evidence has been published that definitely identifies this
derivative as the hemiacetal; however, for consistency with the authors, it is reffered to here
as M2a. Beebe and Takahashi were successful in the reproducible derivatization of aflatoxin
M1 to M2; however, other scientists have encountered incomplete derivatization and have
incorporated different reaction conditions. Consequently, some of these workers have
chosen to quantitate aflatoxin M1 directly, even though M1 is less fluorescent than M2a.
In this report, the reaction parameters for derivatization of aflatoxin M1 or M2a are
examined, and the procedure that produces complete conversion of M1 to the M2a derivative
is given.
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