LC/MS Analysis of Aflatoxin M1 in Milk on Ascentis® Express HILIC after SPE using Supel™ Tox AflaZea, Quantifying Picogram Concentrations
Materials
analytical column
SPE tube or plate
related product
Syringe PP/PE without needle
luer slip tip, centered, capacity 1 mL, graduated, 0.01 mL, non-sterilestandard
CONDITIONS
sample preparation
SPE (Solid Phase Extraction)
sample/matrix
cow milk
SPE tube/cartridge
Supel™ Tox AlflaZea, 6 mL (55314-U)
extraction process
centrifuge 45 g liquid milk for 60 min at 5000 rpm (RCF 4863 xg), cool at 0 °C for 30 minutes, remove fat layer, transfer middle layer to collection flask,
sample addition
10 mL, 2 drop/second using vacuum
washing
2 x 2 mL acetonitrile (collect with sample)
eluate post-treatment
evaporate to dryness at 70 °C using nitrogen
sample preparation
reconstitut in 500 μL 20:80 acetonitrile:water, filter into 750 μL poly vial using PVDF filter, 0.2 μm
column
Ascentis Express C18, 50 cm x 2.1 mm I.D., 2.7 μm particles (53822-U)
mobile phase
[A] 0.1% (v/v) formic acid; [B] acetonitrile
gradient
90% A for 0.5 min; to 10% A in 0.5 min; held at 10% for 3 min; to 90% A in 0.10 min; held at 90% A for 3 min
flow rate
0.4 mL/min
column temp.
ambient
detector
MS/MS (MRM transitions)
injection
10 μL
Description
Analysis Note
Sample pretreatment: centrifuge 45 g liquid milk for 60 min at 5000 rpm (RCF 4863 xg), cool at 0 °C for 30 minutes, remove fat layer, transfer middle layer to collection flask, add 20 mL acetonitrile to 50 mL centrifuge tube, add 20 mL of sample to acetonitrile, add contents of 1 Supel Q non-buffered tube, immedately disperse salt, quicky place on shaker for 5 minutes, centrifuge at 5000 rpm for 20 min, remove top layer (loading solution)
Aflatoxin M1 is the metabolite of Aflatoxin B1 and can be found in milk products when animals have been fed alatoxin-contminated feeds. Aflatoxins are positively associated with hepataocellular carcinomas. Aflattoxin M1 was detected in milk and quantified at concentrations fo 25 pg/mL. Sample preparation included a protein precipitation with solvent partitionng and sample clean-up using Supel Tox AflaZea SPE tubes. Analysis was perfomed via LC-MS using an Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 μm HPLC column for the separation. Detection and quanitification of aflatoxin M1 at 25 pg/mL satisfies the current regulatory requirements for aflatoxin M1 for EU treaty participants as stated in EC 1881/2006.
Legal Information
Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany
Supel is a trademark of Sigma-Aldrich Co. LLC