Joint International Conference on Long-term Experiments ...

2. MATERIALS AND METHODS
OCP are liposoluble substances, which leads to their accumulation in fats. For this
reason, in the qualitative and quantitative determination of this kind of toxic the first
phase is the separation of fat from the matrix elements, in this case, cow milk. (Hura
Carmen, 2006) However, for this kind of food product the OCP-kind residues content,
is expresses in mg/kg fat. So in order to be sure of an accurate extraction of the toxic for
the purpose of its quantitative determination is useful to know the efficiency of fat
separation methods. This is the preliminary phase of quantitative determination of
pesticides. (Guidelines on Good Laboratory Practice in Pesticide residue Analysis,
1993)
2.1 Determination of fat content
2.2.1– Reference method
Fat content of tested probes has been determined using as reference method, the etherchlorhydric
method (Weinbull method), applied to 100g of test, working with two types
of whole milk. We have worked with a Soxhlet extractor with four posts. After the
mineralization, the fat was extracted in p.a. oil ether, which has been divided in sand
bath, under the niche. For each test there were three determinations plus the witness test.
2.2.2 Application of alternative methods of milk fat separation
For the extraction of fat from milk, we used the following methods:
- Extraction with AOAC method
- Column extraction
- Partition extraction
Extraction through the AOAC method modified:
(Association of Analytical Communities)
The method has been modified (Sprecht, W. in 1987 and Cuniff, P. in 1997) in the
proportional diminution (1:10) of all reactive quantities used, due to the volume of the
test tubes for centrifugation at our disposal. So, in a 50 ml centrifugation test tube, put
10 lm ethanol, 0.1 g potassium oxalate and 10 ml milk. Mix it very well. Add 5 ml
ethylic ether and agitate it thoroughly for 1 minute, then we add 5 ml oil ether and
agitate it thoroughly for 1 minute. Afterwards, centrifuge it for 5 minutes at 1500
rot/min; afterwards transfer the solvent layer in a 100 ml separation funnel with 50 ml
water and 3 ml solution of saturated sodium chlorine. Extract the residue twice agitating
thoroughly with 5 ml ethylic ether: oil ether 1 : 1 (v/v) Centrifuge it and transfer the
solvent layer in the separation funnel after each extraction. Carefully mix combined
extracts with water. Drain and remove water layer. Wash the solvent layer, twice, with
two portions of water, of 100 ml each, remove water each time. For dehydration, pass
the solvent solution through a column of anhydrous sodium sulphate of 50 mm and
exterior diameter of 25 mm; collect the eluate in a Berzelius glass. Wash the column
with small portions of ether (three times, 5 ml each time). Gather all etheric extracts in a
weighted vase and evaporate the solvent from the combined extracts at steam bath
temperature under a current of air, to obtain the fat.
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