Page 56 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E208

Application High Performance Liquid Chromatography

News High Speed, High Resolution Analysis (Part 40)

Analysis of Aflatoxins in Food by Nexera UHPLC with
No.L430 an Immunoaffinity Column for Sample Preparation

Aflatoxins are mycotoxins that are extremely In Application News No. L422, we introduced an
carcinogenic and acutely toxic, and are therefore example of aflatoxins analysis which offered improved
subject to stringent monitoring in food products. In the analysis efficiency, in which we conducted ultra-high-
past, aflatoxin control in Japan applied specifically to speed analysis of the 4 aflatoxins using a combination
aflatoxin B1, but from October, 2011, total aflatoxins of the Prominence RF-20Axs high-sensitivity
(total of aflatoxins B1, B2, G1 and G2) became subject to fluorescence detector and the Nexera Ultra High
1)
this restriction . Performance LC system using direct high-sensitivity
The official notification of this regulation change fluorescence detection without derivatization of the
specifies the use of a multifunctional column or an aflatoxins.
immunoaffinity column for isolation and cleanup of the Here we introduce an example of high-speed analysis
2)
aflatoxins . Of the two, it is believed that an using direct fluorescence detection in which the
immunoaffinity column would be more effective for aflatoxins in processed food and spice were cleaned up
samples like spices and processed foods, which contain using an immunoaffinity column.
many impurity substances.

n Analysis of Food Products – Sample Preparation Using Immunoaffinity Column
Roasted peanuts (processed) and nutmeg (spice) were
*1
subjected to sample preparation according to the 0.70 mV
procedures in Fig. 2 and Fig. 3 on the following page . Roasted Peanuts
*2
For the immunoaffinity column, the AFLAKING (Horiba, 3
Ltd.) was used. Standard solution was added to each 0.50 1
*3
sample to adjust the concentrations of aflatoxins B1 and
G1 to 0.8 μg/kg, and B2 and G2 to 0.2 μg/kg.
Fig. 1 shows the results of analysis of the roasted 4
peanuts and nutmeg, and Table 1 shows the analytical 0.25 2
conditions. Use of this analysis method permits the
analysis time to be shortened to one-fourth that of the
conventional conditions (Application News No. L428). Spiked
Furthermore, since almost no substances were detected 0 Unspiked
aside from the aflatoxins due to sample cleanup using
the immunoaffinity column, even faster analysis can be 0.0 1.0 2.0 3.0 min
expected by further shortening the column. mV
Using this analytical method, direct, high-sensitivity 0.65
detection of aflatoxins B1 and G1 is conducted without Nutmeg
derivatization (conversion to hydroxide) using 0.50
trifluoroacetic acid (TFA). For comparison, the analysis
results obtained following TFA pretreatment are shown 3
in Fig. 4 on the following page. 1
0.25 4
*1: The roasted peanuts and nutmeg samples were provided by the 2
Mycotoxin Research Association.
*2: Part of the experimental method differs from the official method. Spiked
*3: AFLAKING can be obtained from Shimadzu GLC, Ltd. 0 Unspiked

0.0 1.0 2.0 3.0 min
Table 1 Analytical Conditions
■Peaks
Column : Shim-pack XR-ODSⅡ（100 mm L. × 3.0 mm I.D., 2.2 μmʣ 1. Aflatoxin G 2, 2. Aflatoxin G 1,
Mobile Phase : Water / Methanol / Acetonitrile = 6/3/1 (v/v/v) 3. Aflatoxin B 2, 4. Aflatoxin B 1
Flow Rate : 1.0 mL/min
Column Temp. : 50 °C
Injection Volume : 8 μL Fig. 1 Chromatograms of Roasted Peanuts and Nutmeg
Detection : RF-20Axs Ex. at 365 nm, Em. at 450 nm (Upper) Spiked, (Lower) Unspiked
RF Cell : Conventional cell
Cell Temp. : 25 °C