Japanese Food Safety Notification (Shoku-An) No.1003001* (October 3, 2006, Ministry of Health, Labour and Welfare (MHLW)) added the “Multi-residue Method for Agricultural Chemicals by LC-MS (Animal and Fishery Products)” to Shoku-An No.0124001 (January 24, 2005), “Analytical Methods for Residual Compositional Substances of Agricultural Chemicals, Feed Additives and Veterinary Drugs in Food, Chapter II Multiresidue Method”. In both Multi-residue Method for Agricultural Chemicals by GC-MS or by LC-MS (Animal and Fishery Products), gel permeation chromatography (GPC clean-up) is employed during the purification process of test solution preparation. In this application, milk and egg samples were treated according to the extraction method described in the “Analytical Methods for Residual Compositional Substances of Agricultural Chemicals, Feed Additives and Veterinary Drugs in Food”. Then, GPC clean-up was performed using CLNpak EV-2000 AC and its guard column CLNpak EV-G AC. 

*The version at the time of the application acquisition.

Extraction method

1. (1)Weigh 20.0 mg of the sample.
2. (2)Add 100 mL of acetonitrile and homogenize. 
3. (3)Centrifuge at 2,500 rpm for 5 min and collect the organic solution layer. 
4. (4)Add 50 mL of acetonitrile to the residue from step (3) and homogenize. 
5. (5)Centrifuge at 2,500 rpm for 5 min and collect the organic solution layer.
6. (6)Combine the organic solutions from steps (3) and (5). Add 10 g of sodium chloride and shake.
7. (7)Allow the mixture to stand and remove the aqueous layer.
8. (8)Add anhydrous sodium sulfate to the acetonitrile layer to remove water. 
9. (9)Filter off the sodium sulfate and concentrate the filtrate at below 40 ℃.
10. (10)After removing the solvent, weigh the residue (extracted fat).
11. (11)Dissolve the residue in acetone/cyclohexane mixture (1:4), adjusting the sample load to the HPLC column to be about 5.0 g.
12. (12)Centrifuge at 3,000 rpm for 5 min, then inject 5 mL of the supernatant into the HPLC.

Sample: 5 mL each
Milk extract
Egg extract
Standard solution (5 mg/L each of Acrinathrin and Tricyclazole in eluent)

1.