Page 55 - Application Notebook - Solution for Food Safety

Application No.L506
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n Analysis of Aflatoxin M1 in Milk
We analyzed commercially available milk and milk with The percentage recovery calculated according to Eqn.
added AFM 1. AFM 1 was added to make up a 1 shown below was 98 %. We found that using the
concentration of 0.05 µg/kg in milk (1/10th the RF-20AXS fluorescence detector allows for analysis at
regulatory level), and pretreatment was performed concentrations 1/10th the regulatory level with high
according to the notification methodology. The sensitivity and good precision.
2)
pretreatment procedure is shown in Fig. 3. Refer to the A small peak was observed at the AFM1 elution
2)
notification methodology for further details. position when milk with no added AFM 1 was
TM
An AflaStar R* immunoaffinity column from Romer analyzed. Using LC/MS/MS to analyze the milk with
Labs was used to remove contaminant constituents. no added AFM1 suggested this peak was derived
The chromatograms obtained after analysis of these from AFM1, and the concentration of the substance
samples are shown in Fig. 4. (A) is the chromatogram present was below 1/100th Japan's regulatory level.
for milk with added AFM1, and (B) is the chromatogram
for milk with no added AFM1.
The analytical conditions were the same as those used
in Fig. 1, which are shown in Table 1.
* "AflaStar" is a registered trademark of Romer Labs.
The AflaStar R can be purchased from Shimadzu GLC Ltd.
TM
Sample (homogenized milk )
*1
Recovery (Peak area of milk with added standard AFM1) - (Peak area of milk with no added standard AFM1)
rate (%) = Peak area of standard AFM1 sample ×100
(A atoxin M1 standard solution (100 µg/L) 10 µL)
Eqn. 1 Percentage Recovery Equation
Heating up to 37 ˚C

Filtration with Glass lter uV
7000
6500
6000
20.0 g 5500
5000 AFM1
4500
4000
Cleaning-up by immunoaf nity column “A aStar R” 3500
TM
*2
3000
2500
2000
Elution by Acetonitrile (3 mL) 1500 (A)
1000
500 (B)
0
*3
Drying-up by N 2 gas
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
1.0 mL Water/Acetonitrile = 4/1(v/v)
Fig. 4 HPLC Chromatograms for Commercially Available Milk
Vortex (A) With added standard AFM1, (B) with no added
standard AFM1
HPLC *4
Fig. 3 Milk Pretreatment Procedure
*1 A pretreatment centrifugation step is needed for raw milk and References
2)
other milks that are not homogenized. Refer to reference for 1) "Handling of Aflatoxin M1 in Milk" (July 23, 2015, Notification
details. No. 0723-[1] of the Department of Food Safety, PFSB, MHLW)
*2 Refer to the annotations in reference for detailed information on 2) "Test Methodology for Aflatoxin M1 in Milk" (Notification
2)
use of the immunoaffinity column. No. 0723-[5] of the Department of Food Safety, PFSB, MHLW)
*3 AFM1 can adhere to the container during drying, so it is
recommended that silane-treated containers be washed with 20 %
to 30 % aqueous acetonitrile then dried before use.
*4 AFM1 can adhere to glass containers used to hold samples for HPLC
even when these containers have been treated with silane, so it is
recommended that plastic containers be used.
First Edition: Jun. 2016
For Research Use Only. Not for use in diagnostic procedure.
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