Page 54 - Application Notebook - Solution for Food Safety

LAAN-A-LC-E282

Application High Performance Liquid Chromatography

News Assay of Aflatoxin M1 in Milk Based on

Notification Test Methodology, Using Prominence-i
No.L506 and the RF-20AXS Fluorescence Detector

Aflatoxin M1 (AFM1) is a mycotoxin suspected of
carcinogenicity in humans that is detected in the milk of 2750 uV
mammals that eat food contaminated with aflatoxin B1.
The notification "Handling of Aflatoxin M1 in Milk" 2500
issued on July 23, 2015 (Notification No. 0723-[1] of 2250 1st
1)
the Department of Food Safety, PFSB, MHLW) sets a 2000
regulatory level for AFM1 in milk of 0.5 µg/kg, and
came into force on January 23, 2016. 1750 2nd
The assay methodology for AFM1 in milk was included 1500
in "Test Methodology for Aflatoxin M1 in Milk" 3rd
(Notification No. 0723-[5] of the Department of Food 1250
2)
Safety, PFSB, MHLW) , which was announced on the 1000
same day and describes two test methodologies. 750 4th
(1) Test method consisting of quantitation by HPLC with
attached fluorescence detector and confirmation by 500 5th
LC/MS or LC/MS/MS. 250
(2) Screening method using an assay kit.
We describe an analysis of commercially available 0 6th
milk that is compliant with test method (1). We
analyzed for AFM 1 in bovine milk using the 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
Prominence-i integrated HPLC and the RF-20AXS Fig. 1 Chromatograms for Standard AFM1 Solution Equivalent
fluorescence detector. Under these conditions we to 1/100th the Regulatory Concentration (0.1 μg/L, Test
were able to measure AFM1 at a concentration of Repeated 6 Times)
1/10th Japan's regulatory level for AFM1 in milk.

n Analysis of Standard Aflatoxin M1 Solutions
Chromatograms obtained after analysis of
standard AFM1 solutions (0.1 µg/L, equivalent to
1/100th the regulatory concentration) are shown
in Fig. 1, and the analytical conditions used are
shown in Table 1. The relative standard deviation
(%RSD) of peak areas after repeating analysis six
times was 3.4 %. Fig. 2 shows the calibration
curve for 0.1 to 20 µg/L. Good linearity was
2
achieved with a contribution ratio R of ≥ 0.9999
within the concentration range. These results
show the RF-20AXS fluorescence detector can be
used to analyze trace quantities of AFM 1 with
high sensitivity and high precision. R ≥ 0.99999
2
When the standard AFM1 solution of 0.1 µg/L is
p r o c es s e d a c c o r d i n g t o t he p r e t r e a t m e n t
procedure shown in Fig. 3, which follows the
notification methodology, it produces a sample
equivalent to 1/100th the regulatory level for
AFM1 in milk (0.005 µg/kg). Fig. 2 AFM1 Calibration Curve (0.1-20 μg/L)

Table 1 HPLC Analytical Conditions
System :Prominence-i
Column :Shim-pack VP-ODS (150 mm L. × 4.6 mm I.D., 5 µm)
Mobile Phase :Water/Acetonitrile = 3/1(v/v)
Flowrate :1.0 mL/min
Column Temp. :40 ˚C
Detection :RF-20AXS, Ex. at 365 nm, Em. at 435 nm
RF Cell. :Conventional Cell
Cell Temp. :25 ˚C
Injection Volume :100 µL