Page 57 - Application Handbook - Liquid Chromatography

Sample 10 g Sample 10 g
(Aflatoxin standard solution) (Aflatoxin standard solution)
40 mL Water / Methanol = 2/8 (v/v) 40 mL Acetonitrile / Water / Methanol = 6/4/1 (v/v)
Let stand in dark at room temperature for 5 min Let stand in dark at room temperature for 5 min

Mix for 30 min Mix for 30 min
Filtration Filtration
Adjust volume from 10 mL to 50 mL with water Adjust volume from 10 mL to 100 mL with 10% Tween 20 aq.

Filtration with glass microfiber filter Filtration with glass microfiber filter
10 mL 10 mL
Clean-up by immunoaffinity column AFLAKING Clean-up by immunoaffinity column AFLAKING
Elute up to 10 mL with acetonitrile Elute up to 10 mL with acetonitrile
Fraction Fraction
(1.5 mL) (1.5 mL) (1.5 mL) (1.5 mL)
Evaporation by N2 gas Evaporation by N2 gas Evaporation by N2 gas Evaporation by N2 gas
0.1 mL TFA 0.1 mL TFA
Let stand in dark at room Let stand in dark at room
temperature for 15 min temperature for 15 min
0.5 mL Water / Acetonitrile 0.4 mL Water / Acetonitrile 0.5 mL Water / Acetonitrile 0.4 mL Water / Acetonitrile
= 9/1 (v/v) = 9/1 (v/v) = 9/1 (v/v) = 9/1 (v/v)
Sample solution for Fig. 1 Sample solution for Fig. 4 Sample solution for Fig. 1 Sample solution for Fig. 4

Fig. 2 Sample Preparation for Roasted Peanuts Fig. 3 Sample Preparation for Nutmeg

n Analysis of Food Sample after Derivatization with Trifluoroacetic Acid
Fig. 4 shows the results of analysis of food samples Table 2 Analytical Conditions
subjected to derivatization using TFA, and Table 2 Column : Shim-pack XR-ODSⅡ100 mm L. × 3.0 mm I.D., 2.2 μm)
shows the analytical conditions used. Each sample Mobile Phase : Water / Methanol / Acetonitrile = 6/3/1 (v/v/v)
shown in Fig. 2 and Fig. 3 was subjected to Flow Rate : 0.9 mL/min
derivatization using TFA following pretreatment with an Column Temp. : 40 °C
immunoaffinity column. Injection Volume: 8 μL
Detection
: RF-20Axs Ex. at 365 nm, Em. at 450 nm
RF Cell : Conventional cell
Cell Temp. : 25 °C

mV mV
Roasted Peanuts Nutmeg
1.5 2 1.0
2
1
1.0 1
5.0
3 4
0.5 3 4
Spiked Spiked
0 0
Unspiked Unspiked
0.0 1.0 2.0 3.0 min 0.0 1.0 2.0 3.0 min
■Peaks 1. Aflatoxin G2a (G1), 2. Aflatoxin B2a (B1), 3. Aflatoxin G2, 4. Aflatoxin B2

Fig. 4 Chromatograms of Roasted Peanuts and Nutmeg Obtained After Derivatization with TFA; (Upper) Spiked, (Lower) Unspiked
[References]
1) Handling of Foods Containing Aflatoxins (Notification 0331, No. 5, issued on March 31, 2011 by the Food Safety Division, Ministry of Health,
Labour and Welfare)
2) Test Method for Total Aflatoxins (Notification 0816, No. 1, issued on August 16, 2011 by the Food Safety Division, Ministry of Health, Labour and
Welfare)
First Edition: January, 2012

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