HPLC Determination of Aflatoxins by Post-
Aflatoxins B1, and G1, do not fluoresce under standard reverse phase HPLC conditions unless derivatized by chemical or photochemical means. We will demonstrate the application of Aura's photochemical reactor for enhanced detection "PHRED" for the post-column photochemical derivatization of Aflatoxins.
The apparatus consists of a reactor housing, uv lamp and a PTFE knitted reactor coil. The reactor coil is placed inside the housing and irradiated with uv light while the column effluent flows through the reactor coil and subsequently the fluorescence detector.
The chromatograms of identical mixtures of aflatoxin standards G2, G1, B2and B1, are shown in Figures 1 and 2. Both chromatograms were generated under identical conditions (see below) except that the uv light in the photochemical reactor was off for Figure 1 and on for Figure 2.
Apparatus, Chemicals and Chromatographic Conditions
HPLC Apparatus:
An HPLC apparatus with a fluorescence detector, injector and data handling capability is required. Fluorescence detector settings: excitation 365 nm, emission >415 nm.
An HPLC apparatus with a fluorescence detector, injector and data handling capability is required. Fluorescence detector settings: excitation 365 nm, emission >415 nm.
The photochemical reactor is inserted between the column exit and the detector inlet. The specific knitted reactor used for this determination is the KRC 25-25 (Aura Industries Inc.) consisting of 25 meter of PTFE 1/16 inch OD x 0.25 mm ID tubing.
Column:
A Supelcosil LC-18, 15 cm x 4.7 mm ID. The column was kept at 40°C.
A Supelcosil LC-
Chemicals:
The eluent consists of 63:22:15 HPLC grade water, acetonitrile and methanol and is pumped at 1 ml/min.
The eluent consists of 63:22:15 HPLC grade water, acetonitrile and methanol and is pumped at 1 ml/min.
Standards:
Aflatoxin Mix Kit-M (Supelco) containing 1µg B1, 1µg G1, 0.3 µg B2 and 0.3 µg G2 in one ml of methanol in each ampule. The contents of one ampule was diluted with 4.20 ml water and 1.47 ml acetonitrile in order for the standards to be in a solution equivalent in composition to the eluent. Injection size: 20 µg.
Aflatoxin Mix Kit-
References:
Henry Joshua, Determination of Aflatoxins by Reversed-Phase High Performance Liquid Chromatography with Post-Column In-Line Photochemical Derivatization and Fluorescence Detection, Journal of Chromatography, 654, 247-254 (1993).
Henry Joshua, Determination of Aflatoxins by Reversed-
H. Joshua, Analysis of Aflatoxins in Naturally Contaminated Corn by HPLC, Post-Column Photochemical Derivatization and Fluorescence Detection, Pittsburgh Conference and Exhibition, Chicago, IL, March 1994. Published in American Laboratory, p 36J-36M, April 1995.
B. W. Horn, R. L. Greene, V. S. Sobolev, J. W. Dorner, J. H. Powell and R. C. Layton, Association of morphology and mycotoxin production with vegetative compatibility groups in Asperagillus flavus, A. parasiticus and A. tamarii, Mycologia 88, 574-587 (1996).
* Presented at the Satellite Workshop to the Gordon Conference on Mycotoxins and Phycotoxins "Advances in Detection Methods for Fungal and Algal Toxins." June 17-19, 1999.
For a Bibliography of selected review and application articles related to Aura Industries’ Photochemical Reactor for Enhanced Detection PHRED™ please click here.
For more information about the PHRED™ please click here.
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