The reference procedure was performed according to Associa- tion of Analytical Communities 969.33 procedure (AOAC, 2000). Colostrum samples, used as a test material, were collected from Holstein-Friesian cattle from Märja experimental farm. All col- lected samples were stored in a 50 mL vial at 20 C. FAMEs were
prepared by adding twelve drops of the colostrum (approximately 500 mg) to 8 mL 0.5 M methanolic NaOH and heated at 80 C for 7 min. Thereafter 9 mL methanolic boron trifluoride was added and the mixture was further heated at the same temperature for 2 min. Subsequently 4 mL of heptane was added through con- denser and the mixture was boiled for 1 min. After boiling, the mixture was slightly cooled down by keeping it at room tempera- ture for 2 min, after which 15 mL of saturated sodium chloride (NaCl) solution was added. Then the flask was stoppered and sha- ken vigorously for 15 s, after which saturated NaCl solution was added to the flask to trigger the floating of the heptane solution into neck of the flask. Next, ca 1 mL of upper heptane layer was transferred into test tube and anhydrous sodium sulfate was added to remove water. Obtained concentrate was transferred into a new test tube and dissolved in 2 mL of heptane. Finally, 1 lL of dilution was injected manually in GC for the analysis.