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Robustness:

The robustness of the method has been evaluated on the following critical pa-

rameters; extraction time and saponification time.

Extraction time:

The robustness test has been performed on a milk powder and a pet food con-

taining choline.

The sample is weighed into the extraction bottle, extraction solvents are added,

the bottle is closed and placed on the shaking table at 37°C. At the time t. the

sample is removed from the shaking table, centrifuged and placed on cold stor-

age until saponification.

t / hour

1 2 3 4 5 22

Choline:

From the data (see appendix 4) it is seen that an extraction time of 20 hours is

sufficient to extract all the choline from the sample.

Saponification time:

The robustness test has been performed on raw egg, pet food, milk powder (in-

fant formula), cornflakes and fish feed containing choline.

An aliquot of the extraction solution is transferred to the saponification bottle, po-

tassium hydroxide and water is added and the mixture is saponifyed at a 100°C.

At the time t. the sample is removed from the oven and neutralized with hydro-

chloric acid.

0

500

1000

1500

2000

2500

Choline (mg/kg)

Time (hour)

Ekstraktion 37°C

Pet food

Milk powder

Carn-06 (Choline Validation Report)

FOR ERP USE ONLY

DO NOT DISTRIBUTE