Application Note

No. E-416-E-816-HE-006, V2.0

Fat determination in animal feed by hot extraction

Hydrolysis Unit E-416, Extraction Unit E-816 HE:

Fat determination in animal feed by hot extraction according to European Directive 98/64/EC
1. Introduction

This document introduces a simple and reliable procedure for fat determination in feed
products. The sample is hydrolyzed with the Hydrolysis Unit E-416. The hot extraction is
performed with the Extraction Unit E-816. After the extract has been dried to a constant weight,
the total fat content is measured gravimetrically. This application note follows the official method
EN 98/64/EC from the Official Journal of the European Communities.

The Commission Directive 98/64/EC [1] includes both direct extraction and extraction after acid
hydrolysis. Which of the two procedures is used depends on the nature and composition of the
sample:
- Feed products of animal origin always have to be hydrolyzed.
- Feed materials of plant origin do not require hydrolysis prior to extraction (exception: if
the product has been extruded, flaked or heated; or the oils and fats cannot be
completely extracted without prior hydrolysis). If a higher fat content result is obtained
with hydrolysis prior to extraction, this result shall be accepted as the true value.
In all cases, the determined fat content of the tested samples with prior hydrolysis was higher
than without hydrolysis. Therefore, the procedure including acid hydrolysis is described in this
application note.

2. Equipment

⋅ Extraction Unit E-816 Hot Extraction (HE)

⋅ Hydrolysis Unit E-416
⋅ Mixer B-400
⋅ Analytical balance (accuracy +/- 0.1 mg)
⋅ Microwave oven
⋅ Drying oven / Vacuum drying oven

3. Chemicals and Materials

Chemicals:
⋅ Quartz sand, particle size 0.3-0.9 mm, BUCHI (Order No. 037689)
⋅ Celite 545, Macherey-Nagel (815560)
⋅ Hydrochloric acid 3 mol/L, 3 L HCl 32% (Hänseler, 20-2000-5) are filled to 10 L with
deionized water
⋅ Petroleum ether 40-60°C, analytical grade, Scharlau, EGT (ET 0093)
For a safe handling please pay attention to all corresponding MSDS!

Samples:
⋅ Rabbit feed (nibble sticks with cereal and carrots), declared fat content: 3.0 %
⋅ Cat feed (dry pellets with beef), declared fat content: 8.5 %
⋅ Dog feed (dry sticks with meat), declared fat content: 9.0 %
⋅ Horse feed (dry pellets with cereal), declared fat content: 1.9 %
The samples were mixed twice for 2 s to obtain visual homogeneity.
All samples were purchased at local supermarkets and specialty shops for animal feed.

Application Note E-416-E-816-HE-006, V2.0 October 2014 2/6

4. Procedure

Fat determination by hot extraction according to the European directive includes the following
steps:
1. sample homogenization
2. hydrolysis of the sample with 3 M hydrochloric acid to break up the matrix
3. filtration of the hydrolysis solution to separate the fat
4. drying of the filtered sample
5. hot extraction of the fat
6. drying of the extract
7. weighing of the extract
8. calculation of the fat content
Step 2 – 4 can be omitted if the sample meets the requirements described in the Introduction.
The determination of nitrogen and protein in dairy products includes the following steps:

4.1. Acid hydrolysis

Preparation of the glass sample tubes

Add approx. 50 g of quartz sand to the glass sample tube and compact the sand by tapping it
gently on the table. Add approx. 5 g Celite 545; spread it evenly by shaking the tubes carefully.

The sand and the celite layers should not be mixed together. Otherwise the celite
phase may (1) block the frit or (2) break through the frit and influence the results by
showing a higher recovery.

Hydrolyzing the sample matrix

1
Place 5 g celite and 2.5 g homogeneous sample in the digestion vessel. Note the accurate
weight of the sample. Add 50 mL hydrochloric acid (3 M). Suspend carefully by gently swirling
the tube. Add another 50 mL hydrochloric acid (3 M), making sure to rinse any remaining
sample off the glass wall. Preheat the Hydrolysis Unit for 10 min. Place the samples into the
unit, lower the vessels, connect the aspiration tubes, reduce the heat to level 3 and start the
water jet pump after boiling begins.

Violent foaming can be prevented by adding 3 M hydrochloric acid drop by drop.

The degree of foaming depends on the sample but also on the preheating time of
the unit. Do not preheat the unit for too long.

After boiling occurs, the samples are hydrolyzed for 1 h. At the end of the hydrolysis time, add
100 mL of warm (40-50°C) deionized water to each digestion vessel. Switch off the heating and
lift the digestion vessels to the top position in order to filter the hydrolyzate. Wash each of the
vessels by gradually adding a total of at least 500 mL water until washed neutral. Check the pH
with pH paper. To ensure maximum efficiency, aspirate all hydrolyzates/rinsing water at the
same time.

1
For products low in oils and fats, the test sample may be increased to 5 g

Application Note E-416-E-816-HE-006, V2.0 October 2014 3/6

Stir the celite layers (without touching the sand layer) with a spatula to loosen the pulp.
Carefully wipe off the spatula with a piece of tissue and add the residue on the top of the
sample. Dry the glass sample tubes in a vacuum oven (≤ 4 h at 100°C/200mbar), in a drying
oven (≤ 8 h at 100°C) or in a microwave oven. Using a microwave oven significantly reduces
drying time. However, it is more delicate due to the fact that the temperature in the sample can
easily become too high (> 105 °C) if an inappropriate heat setting is used. The following
suggestion applies when drying six hydrolyzed samples at the same time. First step: 18 min
640 W, second step: 13 min 480 W (the optimal parameters may depend on the model of
microwave).

Faster drying at higher temperatures is not recommended because fat can be

decomposed at temperatures above 105°C. Oxidized fat can result in excessive
recovery.

Allow the glass sample tubes to cool down to ambient temperature in a desiccator. Add another
layer of quartz sand (20 g). This prevents the celite from being re-suspended by the condensed
solvent.

4.2. Fat extraction

Preparation of the beakers
Always use dry and clean beakers for hot extraction. Add 2-3 boiling stones to each beaker and
dry them for at least 30 min at 105°C. Let them cool down to ambient temperature in a
desiccator for at least 1 h. Record the exact weight prior to extraction.
Hot Extraction
Add the correct amount of solvent into the beaker. Put the sample tubes into the beakers. Lower
the rack and close the safety shield.
The following parameters must be selected (Table 1):

Table 1: Parameters for Hot Extraction E-816

Parameter Value
2
Solvent Petroleum ether
Extraction step 360 min (Heater 100 %)
Rinse step 30 min (Heater 100 %)
Drain interval 15 min
Drying step 5 min (Heater 100 %)
3
Solvent volume 80 – 100 mL

Drying of the extract

After the extraction, dry the beakers containing the extract in a drying oven to constant weight at
103°C. Let the beakers cool down to ambient temperature for at least 1 h in a desiccator and
record the weight.

Make sure that the cooling down time of the beakers in the desiccator is the same
before and after extraction. Differences in the beakers’ temperature falsify the
results.

2 Please select the solvent you are using from the menu.
3 Solvent volume depends on the sample (voluminous samples absorb more solvent). Generally 80 mL are suitable for
most applications.

Application Note E-416-E-816-HE-006, V2.0 October 2014 4/6

5. Calculation

The results are calculated as a percentage of fat using equation (1).

(m total - mbeaker ) (1)
% Fat = ⋅ 100%
mSample

%Fat : percentage of fat in the sample

mtotal : beaker weight + extract [g]
mbeaker : empty beaker weight [g]
mSample : sample weight [g]

6. Results

Table 2: Rabbit feed (nibble sticks with cereals and carrots)

mSample mbeaker mtotal % Fat
S1 4.8724 165.9538 166.0936 2.87
S2 5.0016 162.9632 163.1027 2.79
S3 4.8910 162.7516 162.8928 2.89
Average [%] 2.85
rsd [%] 1.83

Table 3: Cat feed (dry pellets with beef)

mSample mbeaker mtotal % Fat
S1 4.7192 166.5005 167.0059 10.71
S2 5.0729 163.0351 163.5753 10.65
S3 4.9483 163.0573 163.5842 10.65
Average [%] 10.67
rsd [%] 0.33

Table 4: Dog feed (dry sticks with meat)

mSample mbeaker mtotal % Fat
S1 4.1307 162.8985 163.3730 11.49
S2 4.2259 166.4342 166.9210 11.52
S3 4.2175 163.9043 164.3885 11.48
Average [%] 11.50
rsd [%] 0.18

Table 5: Horse feed (dry pellets with cereals)

mSample mbeaker mtotal % Fat
S1 4.7702 162.9837 163.1897 4.32
S2 4.8744 160.8767 161.0876 4.33
S3 4.7657 163.0743 163.2786 4.29
Average [%] 4.31
rsd [%] 0.49

Application Note E-416-E-816-HE-006, V2.0 October 2014 5/6

7. Comparison to Standard Method 98/64/EC

Table 6: Comparison
BUCHI European Directive
Parameter Application note 98/64/EC Explanation
Extraction method Hot extraction Soxhlet or Hot For Application Note using
extraction, with > Soxhlet see [2].
10 mL/min solvent Extraction Unit E-816 HE
reflux rate. fulfills the requirement
regarding solvent reflux rate.

8. References

[1] Commission Directive 98/64/EC Analysis for the determination of amino-acids, crude oils
and fats, and olaquindox in feeding stuffs. This directive can be downloaded for free at
www.eur-lex.europa.eu
[2] Application Note Fat determination in animal feed by Weibull Stoldt according to European
Directive 98/64/EC
Operation Manual of Mixer B-400
Operation Manual of Hydrolysis Unit B-411/E-416
Operation Manual of Extraction Unit E-816 HE

Application Note E-416-E-816-HE-006, V2.0 October 2014 6/6