Joint International Conference on Long-term Experiments ...

MATERIALS AND METHOD
Apparatus and Reagents
(a) Solvents. – All are reagent grade.
(b) Trifluoroacetic acid (TFA). – Purity 99 + %.
(c) Dichlorodimethysilane (DDS). – Prepare 5% (v/v) solution in toluene.
(d) Aflatoxin standard solutions. – Aflatoxin M1 standards were 0.5, 7.04, and 8.37
μg/mL in acetonitrile-benzene (1 + 9). Aflatoxin B1 and G1 standard solution was 8.0
μg each/mL in acetonitrile-benzene (2+98).
(e) Mobile phase. – Mix water-isopropyl alcohol-acetonitrile (80+12+8). Degas with
ultrasonic probe.
(f) Heating block. – Reacti-therm heating module or equivalent.
(g) Silylated vials. – Add 5% DDS solution to 1-1.5 dram vials and heat ca 40 min at
45-55°C. Discard solution and rinse vials 3 times with toluene and then 3 times with
methanol. Heat vials at 75°C for 20-30 min to evaporate methanol. Cap vials (with
Teflon liners) and store.
(h) LC system. – Spectra-Physiscs Model 8700, equipped with injector (Rheodyne
Model 7125) with 2.0 mL loop, recorder/integrator (Spectra-Physics SP4270),
fluorescence detector (Kratos FS970), set at 365 nm for excitation and 418 nm for
emission, and 4.3 mm id X 25 cm Zorbax ODSLC column. Mobile phase, waterisopropyl
alcohol-acetonitrile (80 + 12 + 8), 1.0 mL/min.
Initial Derivative Formation
The following were mixed in a Teflon-lined screw-cap vial: 200 µL solvent, 50 µL TFA,
and 50 µL aflatoxin M1 standard. The mixture was allowed to react at room temperature for
15 min and then was evaporated under nitrogen. The residue was dissolved in 2000 µL
water-acetonitrile (75 + 25) for LC. The injection volume was 50 µL.
RESULTS AND DISCUSSION
This study was initiated by duplicating the reaction conditions of Beebe and Takahashi.
They used n-hexane as a base solvent to dissolve the dairy extract residue containing
aflatoxin M1. Although hexane is an excellent solvent for this purpose, it is a poor solvent
for the polar aflatoxin M1, when present in pure form (e.g., aflatoxin M1 standard).
Therefore, other solvents were substituted for hexane to determine if any improvement
colud be obtained. Solvents were chosen that scanned the elutropic series, with values of -
0.25 to 0.95. The data indicate that the solvents that produced n-hexane, isooctane, and
1,1,2-trichlorotrifluoroethane. Generally, as the solvent polarity increased, the M1,
conversion decreased. Unreacted aflatoxin M1 was detected with all solvents. When water
was added to the reaction mixture, poor yields of M2a were obtained. Even though aflatoxin
M1 is less soluble in nonpolar solvents, the reaction was more complete with them. In this
study, aflatoxin M1 standard was added to the solvent-TFA mixture in 50 µL aliquots. This
technique prevented adsorption of M1 to the glass vial – a problem encountered with
aflatoxin standard solutions and discussed in more detail later.
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