888 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO.
5, 2003
AGRICULTURAL MATERIALS
Crude Fat, Diethyl Ether Extraction, in Feed, Cereal Grain, and
Forage (Randall/Soxtec/Submersion Method): Collaborative
Study
NANCY J. THIEX
South Dakota State University, Oscar E. Olson Biochemistry Laboratories, Box 2170, ASC 151, Brookings, SD 57007
SHIRLEY ANDERSON
Foss North America, 7682 Executive Dr, Eden Prairie, MN 55344
BRYAN GILDEMEISTER
South Dakota State University, Oscar E. Olson Biochemistry Laboratories, Box 2170, ASC 151, Brookings, SD 57007
Collaborators: W. Adcock; J. Boedigheimer; E. Bogren; R. Coffin; K. Conway; A. DeBaker; E. Frankenius; M. Gramse;
P. Hogan; T. Knese; J. MacDonald; J. Mller; R. Royle; M. Russell; F. Shafiee; B. Shreve; J. Sieh; M. Spann; E. Tpler;
M. Watts
A method for determining crude fat in animal feed,
cereal grain, and forage (plant tissue) was col-
laboratively studied. Crude fat was extracted from
the animal feed, cereal grain, or forage material
with diethyl ether by the Randall method, also
called the Soxtec method or the submersion
method. The proposed submersion method con-
siderably decreases the extraction time required to
complete a batch of samples. The increase in
throughput is very desirable in the quest for faster
turnaround times and the greater efficiency in the
use of labor. In addition, this method provides for
reclamation of the solvent as a step of the method.
The submersion method for fat extraction was pre-
viously studied for meat and meat products and
was accepted as AOAC Official Method 991.36.
Fourteen blind samples were sent to 12 collabora-
tors in the United States, Sweden, Canada, and
Germany. The within-laboratory relative standard
deviation (repeatability) ranged from 1.09 to 9.26%
for crude fat. Among-laboratory (including within)
relative standard deviation (reproducibility) ranged
from 1.0 to 21.0%. The method is recommended for
Official First Action.
he Randall (1) or submersion method for fat extraction
T is an AOAC Official Method for meats and meat prod-
ucts (2). Its use is also widespread in feed laboratories
Submitted for publication May 2003.
The recommendation was approved by the Methods Committee on
Feeds, Fertilizers, and Related Agricultural Topics as First Action. See
Official Methods Program Actions, (2003) Inside Laboratory
Management, May/June issue.
Corresponding authors e-mail: nancy_thiex@sdstate.edu.
to determine crude fat in feed, grain, and forage. Approxi-
mately 1/3 of the laboratories reporting crude fat results on an-
imal feed to the Association of American Feed Control Offi-
cials (AAFCO) Check Sample Program are reporting fat val-
ues using this method. It therefore seemed appropriate that
this method should be collaborated for animal feed, cereal
grain, and forage in an attempt to establish the method as an
official method and to bring the AOAC Official Methods of
Analysis current with what is practiced in todays laboratories.
Comparisons of the various fat methods reported in the
AAFCO Check Sample Program were made by George Lati-
mer (Office of the Texas State Chemist, Texas A&M Univer-
sity, personal communication). On data from 90 check sam-
ples, the mean recovery by the proposed method was 96.71%
of AOAC Official Method 920.39 (diethyl ether, traditional
Soxhlet extraction; 3), and median recovery was 98.21%. Re-
gression analysis of the proposed method on AOAC Official
Method 920.39 gave a correlation coefficient of 0.9973, R2 =
99.46%, slope of 1.00062, and an intercept of 0.137367. On
this basis, the methods appeared comparable.
Ruggedness Testing
Ruggedness tests (4) were performed as part of the method
validation process. Variables studied were predry time (2 vs
4 h); boil time (20 vs 40 min); solvent (diethyl ether vs petro-
leum ether); rinse time (30 vs 60 min); test portion weight
(1 vs 3 g); extraction cup dry time (2 vs 4 h); and solvent drip
rate (2 vs 4 drops/s). Ruggedness tests were performed on 3
feed materials in 3 laboratories (Table 1). The method was
found to be rugged in all variables tested, with one exception:
the use of petroleum ether as a solvent produced a consistently
low bias compared to diethyl ether.
The low bias for petroleum ether is consistent with obser-
vations reported by Latimer (AAFCO Check Sample 2000-28
Comments).
 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 889

Table 1. Results of ruggedness tests

Lab 1 Lab 2 Lab 3

Feed type Cattle Swine Mixed Cattle Swine Mixed Cattle Swine Mixed

S 11.61 2.45 10.50 11.82 2.41 11.16 11.99 2.99 10.97

T 11.30 2.10 10.24 11.63 2.35 10.25 11.55 2.49 10.64
U 11.79 2.46 10.60 11.98 2.81 10.84 11.84 2.93 11.05
V 11.02 2.17 9.88 11.76 2.70 10.78 11.57 2.46 10.50
W 10.87 2.60 10.67 11.61 2.52 9.86 11.87 4.13 10.94
X 11.11 2.00 10.00 11.28 2.10 10.28 11.24 2.38 10.43
Y 11.62 2.55 10.60 11.49 2.64 10.53 11.94 2.82 10.91
Z 10.73 1.91 9.82 11.19 2.10 10.01 11.74 2.51 10.73
Average 11.26 2.28 10.29 11.60 2.45 10.46 11.72 2.84 10.77
Predry

2h 11.43 2.29 10.30 11.80 2.57 10.76 11.74 2.72 10.79

4h 11.08 2.26 10.28 11.39 2.34 10.17 11.70 2.96 10.75
Difference 0.34 0.03 0.03 0.41 0.23 0.59 0.04 0.24 0.04
Boil time

20 min 11.22 2.29 10.35 11.59 2.35 10.39 11.66 3.00 10.75
40 min 11.29 2.27 10.23 11.61 2.56 10.54 11.77 2.68 10.80
Difference 0.06 0.02 0.13 0.02 0.22 0.15 0.11 0.32 0.05
Ether

Diethyl 11.47 2.52 10.59 11.73 2.60 10.60 11.91 3.22 10.97
Petroleum 11.04 2.04 9.99 11.47 2.31 10.33 11.53 2.46 10.58
Difference 0.43 0.47 0.61 0.26 0.28 0.27 0.38 0.76 0.39
Rinse time

30 min 11.32 2.25 10.29 11.53 2.38 10.49 11.81 2.70 10.81
60 min 11.20 2.31 10.29 11.66 2.53 10.44 11.63 2.98 10.73
Difference 0.12 0.06 0.00 0.13 0.16 0.05 0.17 0.27 0.08
Test portion weight

1g 11.31 2.20 10.23 11.57 2.36 10.57 11.70 2.70 10.80

3g 11.20 2.35 10.35 11.62 2.55 10.36 11.73 2.98 10.75
Difference 0.11 0.15 0.12 0.06 0.20 0.22 0.03 0.27 0.05
Cup dry

30 min 11.06 2.28 10.22 11.60 2.43 10.45 11.79 3.02 10.79
2h 11.46 2.28 10.36 11.60 2.48 10.48 11.64 2.66 10.76
Difference 0.40 0.01 0.14 0.00 0.04 0.02 0.15 0.37 0.03
Drop rate

2/s 11.34 2.29 10.24 11.59 2.46 10.69 11.69 2.66 10.70
6/s 11.17 2.27 10.33 11.60 2.45 10.24 11.75 3.02 10.84
Difference 0.17 0.02 0.09 0.01 0.02 0.45 0.06 0.35 0.14
Combination or determination No.a

Factor value 1 2 3 4 5 6 7 8
A or a A A A A a a a a
B or b B B b b B B b b
C or c C c C c C c C c
D or d D D d d d d D D
E or e E e E e e E e E
F or f F f f F F f f F
G or g G g g G g G G g
Observed result S T u V W x y Z
a
The parameters chosen are (A) predrying 103C 2 h; (a) predrying 103C 4 h; (B) boil time 20 min; (b) boil time 40 min; (C) diethyl ether; (c) petroleum ether
4060C; (D) rinse time 30 min; (d) rinse time 60 min; (E) test portion weight 1 g; (e) test portion weight 3 g; (F) final drying time 2 h; (f) final drying time 4 h; (G)
drop rate 2 drops/s; (g) drop rate 6 drops/s.
890 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003

Collaborative Study starter medicated, dehydrated alfalfa, medicated goat feed,

The Study Directors and 11 collaborating laboratories con-
ducted the collaborative study. Laboratories represented a va-
riety of types that would routinely use the proposed method,
including research, commercial, industrial, and state regula-
tory laboratories. Samples were sent to 4 laboratories outside
the United States, and results on study samples were received
from 3 of them. Participants received no compensation. Fa-
miliarization samples were sent to each collaborator to be ana-
lyzed and reported to the Study Directors before the test sam-
ples to acquaint them with the method and to ensure that the
laboratory was capable of handling the test samples.
Collaborating laboratories were asked to analyze 14 animal
feed, cereal grains, and forage materials as blind duplicate pairs,
resulting in 28 test samples. A blank material (cellulose) was
also included as a blind duplicate pair and labeled as a sample.
Study materials were chosen to be representative of differ-
ent feed, cereal grain, and forage materials (Table 2). All sam-
ples were natural or real world; none were spiked. Samples
were coded at random with no preselection from order of pre-
sentation. Approximately 20 g of each material was provided,
which was in excess of the amount needed to complete the
study. Two materials (a urea-containing feed and a high-sugar
feed) were identified as requiring a water prewash. Partici-
pants were informed which samples were low or high enough
in fat concentration to require weighing a test portion larger or
smaller than 2 g. Concentration ranges for the sample ranged
from a blank (<0.5% fat) to nearly 100% fat.
Study materials were prepared as follows: The meat
meal/hulls mixture, calf feed medicated, broiler starter, calf
and swine feed were donated by the AAFCO Check Sample
Program. These materials were used without further grinding.
The corn silage was ground in a Foss Tecator Cyclotec Mill
(Foss North America, Eden Prairie, MN) with a forage head to
pass through a 1 mm screen. The birdseed, texturized feed, and
beef concentrate pellets were ground in a Retsch ZM 100 Ultra
Centrifugal mill (Retsch, Haan, Germany) to pass through a
0.75 mm sieve. The high oil corn was ground in a Wiley mill
(Thomas Scientific Corp., Swedesboro, NJ) through a 1.0 mm
screen. The fat and cellulose required no grinding. No further
grinding was necessary by the collaborating laboratories for
any of the study materials. All materials were split in a Fritsch
Rotary Sample Divider Model Laborette 27 (Fritsch, c/o Gilson
Co., Inc., Lewis Center, OH). They were stored in polyethylene
bags in ~20 g quantities.
Uniformity (homogeneity) of the test sample sets was veri-
fied by selecting 3 bags at random for each material and ana-
lyzing them by the proposed method in the Study Directors
laboratory. Relative standard deviations (RSDs) are shown in
Table 2. An acceptance criterion of an RSD of ca 2% was con-
sidered acceptable for the study materials.
Collaborators were asked to report results on an as is ba-
sis, to determine analysis in single for each sample, and to re-
port data to 4 significant figures. In addition, collaborators
were asked to complete a Study Survey. In addition to famil-
iarization and study samples, thimbles and defatted cotton
were provided to collaborators.
Note: The study was conducted concurrently using hex-
anes as the extraction solvent. The concurrent study appears as
a companion article in this issue of J. AOAC Int. (see p. 899).

AOAC Official Method 2003.05

Table 2. Categories represented by study materials
and uniformity testing results
Animal
Material
Meat meal/hulls mixture
Calf feed medicated
Broiler starter
Mixed bird seed
Calf starter medicated
Corn silage
Dehydrated alfalfa
High oil corn
Medicated goat feed
Texturized feed
Feedlot concentrate pellets
Fat supplement
Swine feed
Cellulose blank (Solka-Flok)
Crude Fat in Feeds, Cereal Grains, and Forages
Randall/Soxtec/Extraction-Submersion Method
First Action 2003
Fat,
feed Forage Grain RSD, % [Applicable to the analysis of forages, cereal grains, and
animal feeds other than baked or expanded products, dried
milk or milk products, fishmeal, or oilseeds at concentrations
1.4
from 0.5 to 100% fat. It is applicable to the same matrixes as
0.80
AOAC Official Methods 920.39 (see 4.5.01) and 930.09 (see
1.5 3.5.07).]
0.86
0.49 Caution: Store solvents in metal containers in solvent cabi-
1.1
net or solvent room that conforms to applicable
safety legislation. Ethers and hexanes are ex-
1.2
tremely flammable. Have no open flames in the
1.1
laboratory where the analysis is being performed.
0.29 Avoid inhaling vapors. Use solvents in a properly
1.1 operating hood equipped with explosion-proof
1.9 lighting, wiring, and fan. Diethyl ether has the
0.15 potential to form shock-sensitive, explosive per-
1.2
oxides with age. Check each new container of
ether for peroxides when it is opened. Also check
partial containers of ether that have not been used
for several months before using them again. Do
not use ether that contains peroxides. Dispose as
 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 891

hazardous material. Stabilized ether may be used. B. Apparatus

Ground electrical equipment and maintain in
proper working order. Follow manufacturer rec-
ommendations for installation, operation, and
safety of all extraction equipment. Make sure all
solvent is evaporated from cups before placing
them in the oven to avoid a fire or explosion.
See Tables 2003.05A and 2003.05B for results of the
interlaboratory study supporting acceptance of the method.
A. Principle
The Randall modification of the standard Soxhlet extrac-
tion submerges the test portion in boiling solvent, reducing the
time needed for extraction. The solvent dissolves fats, oils,
pigments, and other soluble substances, collectively termed
crude fat.
A dried, ground test portion is extracted by a 2-step pro-
cess: In the first step, the thimble containing the test portion is
immersed into the boiling solvent. The intermixing of matrix
with hot solvent ensures rapid solubilization of extractables.
The thimble is then raised above the solvent and the test por-
tion is further extracted by a continuous flow of condensed
solvent. The solvent is evaporated and recovered by conden-
sation. The resulting crude fat residue is determined
gravimetrically after drying.
The solubility characteristics of different solvents may re-
sult in slight differences in crude fat results. For this reason,
the report should reflect the solvent used. Example: % Crude
Fat, Ether Extraction; % Crude Fat, Hexanes Extraction.
(a) Solvent extraction system.Multiple position extrac-
tion unit conducting 2-stage Randall extraction process with
solvent recovery cycle, with Viton or Teflon seals compati-
ble with ether or hexanes.
(b) Thimbles and stand.Cellulose thimbles and stand to
hold thimbles.
(c) Extraction cups.Aluminum or glass. (Extraction
temperature settings may differ; consult manufacturers oper-
ating instructions.)
Items (a)(c) are available as Soxtec systems from Foss
Tecator, Hgans, Box 70, SE-263 21, Sweden, from Foss
North America, 7682 Executive Dr, Eden Prairie, MN 55344,
Tel: +1-952-974-9892, Fax: +1-952-974-9823, info@foss
northamerica.com, or from other manufacturers of
Randall-type extraction systems.
C. Reagents
(a) Anhydrous diethyl ether.Purified for fat extraction,
Fisher E492 labeled For Fat Extraction is also stabilized, or
E134-4 (UN1155), or equivalent. To prevent ether from ab-
sorbing water, purchase it in small containers and keep con-
tainers tightly closed. Petroleum ether cannot be substituted
for diethyl ether because it does not dissolve all of the plant
lipid material.
(b) Hexanes.Fisher H291 (UN1208; www.fishersci.com),
or equivalent; boiling range: 40C including 68.7C.
(c) Cotton.Defatted. Soak medical grade cotton in di-
ethyl ether or hexanes for 24 h, agitating several times during

Table 2003.05A. Interlaboratory results for crude fat in animal feed, cereal grain, and forage, diethyl ether extraction
(submersion) method
Material Mean No. of labsa Sr RSDr, % SR RSDR, % HORRAT

Dehydrated alfalfa 4.44 10 0.21 4.62 0.23 5.1 1.59

Corn silage 2.09 9(1) 0.02 1.02 0.06 3.06 0.86
Mixed bird seed 7.19 10 0.19 2.64 0.23 3.17 1.07
Texturized feed 3.20 9(1) 0.1 3.22 0.22 6.77 2.02
Fat supplement 96.38 10 4.82 5.00 4.82 5.00 2.49
Medicated goat feed 1.89 10 0.04 2.09 1.08 4.5 1.24
Feedlot concentrate pellets 1.53 10 0.14 9.26 0.32 21.0 5.60
Cellulose (blank) 0.17 10 0.05 26.1 0.08 48.3 9.3
Calf starter medicated 2.96 8(2) 0.05 1.58 0.06 1.9 0.56
Calf feed medicated 3.42 9(1) 0.05 1.44 0.12 3.50 1.05
Meat meal/hulls mix 4.99 10 0.22 4.33 10.55 11.08 3.53
Swine feed 2.77 10 0.07 2.61 0.15 5.5 1.6
Broiler starter 7.05 10 0.1 1.35 0.16 2.33 0.78
High oil corn 7.69 10 0.1 1.31 0.22 2.93 0.99

a
Number of laboratories retained after eliminating the number of laboratories in parentheses.
Table 2003.05B. Interlaboratory study results for determinations of crude fat in animal feed, cereal grain, and forage, diethyl ether extraction (submersion) method
892

(blind duplicate design)

Crude fat, ether extraction, %

Feedlot Meat
Dehydrated Mixed bird Texturized Fat Medicated concentrate Calf starter Calf feed meal/hulls Broiler
alfalfa Corn silage seed feed supplement goat feed pellets Cellulose medicated medicated mix Swine feed starter High oil corn
Lab
No. 1 22 2 3 4 23 5 15 6 21 7 9 8 28 10 26 11 17 12 19 13 14 16 27 18 20 24 25

a a
1 4.50 4.96 2.17 2.04 7.42 7.63 3.21 3.18 98.40 95.50 1.88 1.75 1.65 1.64 0.19 0.25 3.02 2.96 3.34 3.29 4.92 4.93 2.92 3.07 7.11 7.10 8.10 8.13
2 3.80 4.33 2.13 2.11 7.19 7.09 3.14 3.02 98.63 82.25 1.97 1.94 1.41 1.45 0.21 0.15 2.96 2.89 3.50 3.51 5.53 5.55 2.85 2.60 7.18 7.07 7.52 7.54
b b
3 4.21 4.72 1.97 1.92 7.13 6.79 3.30 3.23 98.53 98.21 1.71 1.75 1.33 1.82 0.06 0.17 2.76 2.62 3.23a 3.77a 5.35 5.65 2.56 2.67 6.80 6.84 8.10 7.78
b b
4 4.42 4.31 2.06 2.05 6.81 7.15 3.50 3.52 98.30 90.30 1.86 1.88 1.88 1.71 0.02 0.08 2.71 2.78 3.23 3.31 5.10 4.90 2.54 2.57 6.84 6.66 7.77 7.70
5 4.56 4.56 2.14 2.13 6.97 7.37 3.23 3.20 99.65 99.56 1.95 1.92 1.34 1.39 0.19 0.15 2.97 3.04 3.55 3.60 5.41 5.30 2.90 2.92 7.15 7.25 7.56 7.65
6 4.53 4.56 2.15 2.15 7.15 6.80 3.23 3.39 99.47 98.17 1.94 2.02 2.13 1.95 0.27 0.29 3.03 3.03 5.66 5.49 2.93 2.95 7.17 7.12 7.43 7.51
7 4.33 4.50 2.04 2.05 7.28 7.26 2.87 2.84 97.18 96.32 1.89 1.85 1.20 1.10 0.18 0.18 2.90 2.91 3.42 3.38 4.41 3.64 2.74 2.77 6.91 7.15 7.71 7.63
a a
8 4.47 4.48 2.12 2.17 7.49 7.32 3.37 2.65 99.60 99.22 1.92 1.91 1.34 1.50 0.16 0.20 2.98 2.90 3.30 3.38 4.51 4.47 2.82 2.87 7.19 7.17 7.69 7.96
c c c c c c c
9 4.73 4.62 2.25 2.26 7.37 7.39 3.37c 3.67c 99.96c 99.81c 2.10c 2.11c 1.67c 1.55c 0.42c 0.32c 3.09c 3.05c 3.71c 3.62c 5.36c 5.50c 2.95c 3.28c 7.16c 7.24c 7.65c 7.72c
10 4.50 4.52 2.13 2.08 7.10 7.43 3.57 3.27 97.87 88.66 2.01 1.98 1.72 1.96 0.05 0.08 2.86 2.96 3.50 3.47 5.03 5.32 2.74 2.78 7.06 6.95 7.40 7.37
12 4.20 4.42 2.08 2.08 7.30 7.17 3.05 2.82 98.93 92.90 1.84 1.80 0.99 1.02 0.23 0.35 2.90 2.97 3.23 3.37 4.13 4.42 2.63 2.60 7.06 7.29 7.60 7.60

a
Data excluded by Cochrans test, p = 2.5% (1-tail).
b
Data excluded by Double Grubbs test, p = 2.5% (2-tail), 1.25% (1-tail).
c
Excluded by Lab Ranking Test (invalid data).
THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 893
this period. Remove and air dry. Commercially available from
Foss Tecator, Part No. 1529-0009.
(d) Sand.Ashed (for ignition boats). EM SX0075-3, or
equivalent (CAS 14808-60-7).
(e) Celite 545.Foss Tecator 1900-0014, or equivalent.
D. Preparation of Analytical Sample
Grind laboratory samples to fineness that gives an RSD
2.0% for 10 successive determinations.
RSD % = (SD/mean) 100
Fineness of 0.751 mm usually achieves this precision with
dry mixed feeds and other nonuniform materials.
E. Determination
Weigh 15 g test portions containing ca 100200 mg fat di-
rectly into tared cellulose thimbles, according to following
scheme:
Crude fat, % Test portion weight, g
<2 5 ture. (Note: Take care not to pick up any debris on bottom of
5 24
10 12
>20 1 Dry extraction cups in a 102 2C oven for 30 min to re-
Record weight to nearest 0.1 mg (S) and thimble number.
Note: If test sample contains large amounts of urea salts (>5%) or
soluble carbohydrates (>15%), glycerol, lactic acid, amino acid
salts (>10%) or other water-soluble components, remove by wa-
ter extraction. Weigh test portion onto filter paper, extract with
five 20 mL portions of water, allowing each portion to drain.
Place filter paper containing washed test portion into thimble and
dry at 102 2C for 2 h. To facilitate filtration, add 12 g ashed,
acid-washed sand, or Celite to bottom of filter or mix in with test
portion before water extraction.
Dry thimbles containing test portions at 102 2C for 2 h.
If dried test portions will not be extracted immediately, store
in desiccator. Both solvent and test materials must be free of
moisture to avoid extraction of water-soluble components
such as carbohydrates, urea, lactic acid, and glycerol, which
will result in false high values.
An absorbent, such as diatomaceous earth (Celite or
Super-Cel), can be added to the test portion when high fat ma-
terials, which melt through the thimble during the predry step,
are present. Alternatively, defatted cotton can be added before
the predry step to absorb the melted fat. If the material melts at
102C, place a pretared extraction cup under the thimble dur-
ing the drying step to catch any melted fat that was unabsorbed
and escaped the thimble.
Place defatted (with same solvent to be used for extraction)
cotton plug on top of test portion to keep material immersed
during the boiling step and prevent any loss of test portion
from top of thimble. Prepare cotton plug large enough to hold
materials in place, yet as small as possible to minimize absorp-
tion of solvent. Adding the cotton plug before the
102 2C/2 h drying step is acceptable.
Place three or four 5 mm glass boiling beads into each cup,
and dry cups for at least 30 min at 102 2C. Transfer to des-
iccator and cool to room temperature. Weigh extraction cups
and record weight to nearest 0.1 mg (T).
Extract, following manufacturers instructions for opera-
tion of extractor. Preheat extractor and turn on condenser
cooling water. Attach thimbles containing dried test portions
to extraction columns. Put sufficient amount of solvent into
each extraction cup to cover test portion when thimbles are in
boiling position. Place cups under extraction columns and se-
cure in place. Make sure that cups are matched to their corre-
sponding thimble. Lower thimbles into solvent and boil for
20 min. Verify proper reflux rate which is critical to the com-
plete extraction of fat. This rate depends upon the equipment
and should be supplied by the manufacturer. A reflux rate of
ca 35 drops/s applies to many extraction systems.
Raise thimbles out of solvent and extract in this position for
40 min. Then evaporate as much solvent as possible from cups
to reclaim solvent and attain apparent dryness.
Remove extraction cups from extractor and place in oper-
ating fume hood to finish evaporating solvent at low tempera-
extraction cup while in hood. Let cups remain in hood until all
traces of solvent are gone.)
move moisture. Excessive drying may oxidize fat and give
high results. Cool in desiccator to room temperature and
weigh to nearest 0.1 mg (F).
F. Calculations
% Crude fat, diethyl ether extract =
F -T
100
S
F -T
% Crude fat, hexanes extract = 100
S
where F = weight of cup + fat residue, g; T = weight of empty
cup, g; S = test portion weight, g.
Refs.: J. AOAC Int. 86, 890893(2003); 902904(2003)
Results and Discussion
Study materials were shipped the last week of September
2001 to 13 laboratories. Results were received from 12 labora-
tories (Table 2003.05B) over a period of 3 months, with the
last set received on December 28, 2001. One of the 13 labora-
tories could not provide data due to internal issues. Labora-
tories were asked to provide the type of instrument used.
Equipment used is described in Table 3.
Early into the study, it became apparent that some collabo-
rators were following in-house methods rather than the
method supplied with the study. Also, a number of questions
were received as to the appropriateness of specific lots of sol-
vents. At this point, a survey was developed to document the
manufacturers of solvents, catalog numbers, and lot numbers;
894 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003

Table 3. Extractor type and laboratory ranking scores soak and rinse times from those specified. Data from this labo-
for collaborating laboratories ratory were evaluated using an XY plot, which confirmed that
Lab Extractor type Lab ranking score data from this laboratory should be removed from the study.
Collaborators Comments
1 Soxtec 2050 70.5
In response to these comments and suggestions received
2 Soxtec HT-6 1043 96
from collaborators concerning extraction of high-fat materi-
3 Soxtec HT-6 1043 105.5
als, a better description of how to handle high-fat materials
4 Soxtec 2050 113.5 was incorporated into the method.
5 Soxtec HT-6 1043 67 A number of comments (both written and verbal) were re-
6 Soxtec HT-6 1043 54 ceived about the necessity of the water wash step and the ne-
7 Soxtec 2050 111.5 cessity of the predry step. These steps are specified in AOAC
8 Soxtec HT-6 1043 76
Official Method 920.39 (3) and were previously documented
a in the literature as a critical step (5, 6); they were therefore in-
9 Soxtec HT-6 1043 29a
corporated without further investigation. A review of litera-
10 Soxtec 2055 89.5 ture did not provide data upon which the recommendations
b b
11 Soxtec 2055 were made. Therefore, the Study Directors decided to perform
12 Soxtec HT-6 1043 111.5 recovery experiments to study the effect of potentially inter-
fering substances. Urea and glucose equivalent to ~2%
a
Results removed on the basis of the lab ranking test. (~0.04 g), ~5% (~0.1 g), ~10% (~0.2 g), and ~15% (~0.3 g)
b
XY plot revealed outlier results. Removed from study performing were added to 2 g test portions of ground shelled corn and ex-
the lab ranking test.
tracted without a water rinse. Test portions of ground corn
spiked at the 15% level were also extracted after the water
wash to check whether any potential interference was re-
and to ensure that final results submitted to the Study Direc- moved. The data are presented in Table 5 and graphical repre-
tors by collaborators were generated following the method as sentations of the results of the experiment are provided in Fig-
supplied. Laboratories that did not follow the method were ures 1 and 2. The effect of added urea was dramatic, and the
asked to retest the study materials. Final results of the survey interference was efficiently removed with the water wash. It is
are shown in Table 4. Laboratory 11 did not follow the method recommended that urea levels above 5% be removed by water
as mailed to collaborators and did not have time to retest the wash before the solvent extraction step. Glucose at levels
materials. Deviations from the method included not perform- <15% showed no interfering effect when the test portions
ing the water wash required on 4 materials, and using different were predried.

Table 4. Study survey results

Predry Instrument Soak Rinse Postdry
Lab Prewash time/temp. Solvent temp. setting, C time, min time, min time/temp. Flow Comments

1 5,8,15,28 100C/2 h EM Science EX 0190-3 41159 115 20 40 100C/0.5 h Steady steam Yes
2 5,8,15,28 102C/2 h Fisher E198-4 014950-12 130 20 40 102C/0.5 h Rapid drip None
3 5,8,15,28 100C/2 h VWR VW 2142-5 41201 exp 110 20 40 100C/0.5 h None
7/31/2003
4 5,8,15,28 102C/2 h Fisher E138-4 011436-15 and 135 20 40 102C/0.5 h 35 drips/s None
010930-15
5 5,8,15,28 102C/2 h Fisher E492-4 011185-36 116 20 40 102C/0.5 h Dribble None
6A 5,8,15,28 102C/2 h Fisher E492-4 011185-36 101.5 20 40 102C/0.5 h Dribble None
7 5,8,15,28 102C/2 h Lab Scan H6509 S 3787/9 105 20 40 102C/0.5 h 2 + 3 drops/s Yes
8 5,8,15,28 102C/2 h EM Science (Merck) EX0190-3 70 20 40 102C/0.5 h Distinct drops 2.9/s None
41110
9 5,8,15,28 102C/2 h Fisher (UN 1155) E492-20 140 20 40 102C/0.5 h Distinct drops 35/s Yes
011185-36
10 5,8,15,28 102C/2 h Fisher E134-4 992548-36 135 20 40 102C/0.5 h 4 drips/s None
11 None 103C/2 h Fisher E199-4 005977-12 102 25 30 102C/0.5 h Yes
12 5,8,15,28 102C/2 h Fisher E138-4 011436-15 141.3 20 40 102C/0.5 h Rapid drip
 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 895

Table 5. Recovery of crude fat, diethyl ether extract, overnight at 55C. Recoveries of crude fat, diethyl ether ex-
from ground corn spiked with urea, glucose, and amino traction, ranged from 82 to 153% (Table 6). This is consistent
acids with the literature (5, 6). Water can decrease the efficiency of
Recovery of solvent in extraction, and/or allow for extraction of water-sol-
Urea spike, % crude fat, % uble nonlipid components. The results confirm that the predry
step is critical in the extraction process. Recoveries of fat from
0.00 100.0 the urea-containing feed (feedlot concentrate pellets) and the
2.01 98.9
molasses-based, high-sugar content feed (texturized feed)
with no predry were 153 and 123%, respectively. High recov-
5.30 102.7
eries were also observed with the corn silage, medicated goat
10.00 104.0 feed, and meat meal/hulls mix. A low recovery (incomplete
14.98 107.8 extraction) was observed with the mixed grain (mixed bird
15.13 98.7a seed).
Recovery of A laboratory ranking by the test described by Youden and
Glucose spike, % crude fat, % Steiner (4) was used to assess bias among laboratories partici-
pating in the collaborative study. Overall, Laboratory 9 ranked
0.00 100.0
lowest (reported the highest crude fat values) with a Lab
2.26 98.9
Ranking Score of 29 (Table 3) and based on this score was re-
5.43 98.7 moved from the statistics.
10.08 98.1 Table 2003.05B summarizes the study data and Ta-
15.24 101.1 ble 2003.05A provides the statistics. The overall within-labo-
14.95 97.5a ratory RSDr ranged from 1.02 to 9.26% for feed, forage, and
cereal materials, and 26% for the cellulose blank. Among-lab-
Lysine and methionine Recovery of
spikes, % crude fat, % oratory RSDR ranged from 1.90 to 21% for feed, forage, and
cereal materials, and 48% for the cellulose blank. The 2 mate-
0.00 100.0 rials with the highest RSD in the collaborative study were
0.499 100.0 among those with relatively high RSDs in the homogeneity test
0.995 101.3 (feedlot concentrate pellets and meat meal/hulls mix). This sug-
1.99 101.5 gests that one of the significant sources of variability in the
4.98 101.2
method is related to sampling to obtain the test portion.
The high fat supplement proved to be a challenge for the
a
With water wash. collaborators. The fat (RSDr = 5.00%, RSDR = 5.00%) melted
during the predry step and foamed during extraction. Collabo-
rators unfamiliar with this type of material may not have ob-
served these potential sources of error. As a result of the com-
Similar experiments were performed to determine the ef- ments received, better instructions have been incorporated
fect of the presence of added amino acids, lysine, and into the method describing how to handle these materials to
methionine. Lysine and methionine equivalent to ~ 0.5% each optimize recovery of crude fat.
(~10 mg each), ~1% each (~20 mg each), ~2% each (~40 mg
each), and ~5% each (~100 mg each) were added to 2 g test
portions of ground shelled corn and extracted without a water
rinse. The data are presented in Table 5 and graphical repre-
sentations of the results of the experiment in Figure 3. Amino
Crude fat, % Crude fat, % after water wash Trendline
acids added at 5% levels showed no interfering effects when
Crude fat, dietyl ether extract, %

the test portions were predried. The Study Directors feel that 4.10
very few complete feed or feed concentrates requiring fat 4.00
analysis would contain >5% added lysine and >5% added
3.90
methionine; therefore, as long as test portions are predried,
this potential interference should not be a practical concern. 3.80

To determine the effect of the presence of water during ex- 3.70

traction on the various feed matrixes, the study materials were 3.60
extracted without drying and results were compared with
3.50
predried data collected in the collaborative study. Because the
0 5 10 15 20
study materials had dried out considerably since they were Urea spike as % of test portion
prepared about 1 year before this experiment, they were
rehydrated by placing the weighed thimbles with test portion Figure 1. Effect of urea on crude fat (diethyl ether
into a desiccator charged with water and allowed to equilibrate extract) recovery.
896 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
Another challenge to the collaborators was the water wash.
This was required for the texturized feed (RSDr = 3.22%,
RSDR = 6.77%) and the feedlot concentrate pellets (RSDr =
9.26%, RSDR = 21.0%). Even though the current AOAC Offi-
cial Method 920.39 requires this step, many laboratories did
not have experience with it before the collaborative study, and
this is reflected in the high RSDs compared to materials that
did not require a water wash. Laboratories that had difficulty
washing/filtering were instructed to include ashed sand with
the test portion to facilitate the wash/filtering. This has been
added to the method write-up, as well as defining a longer dry-
ing step after the wash (2 h rather than 30 min). If materials
that were water-washed are removed from the statistics, the
within-laboratory RSDr ranges from 1.02 to 5.00% for feed,
forage, and cereal materials, and among-laboratory RSDR
ranged from 1.90 to 11%. It is the opinion of the Study Direc-
tors that results on the water wash pretreatment will improve
as laboratories gain practice.
HORRAT values for dehydrated alfalfa, corn silage, mixed
bird seed, texturized feed, medicated goat feed, calf starter
medicated, swine feed, broiler starter, and high oil corn ranged
from 0.56 to 2.02 and are excellent. Three materials had
HORRAT values >2.0: fat supplement, 2.59; meat meal/hulls
mix, 3.53; and feedlot concentrate pellets, 5.60. Challenges
with the fat supplement were previously described, and
clearer directions have been incorporated into the method
write-up. One laboratory had what appeared to be an outlier
value but narrowly escaped removal by the Cochrans test. If
this had been removed, it would have lowered the HORRAT
to 1.69 and the RSDR to 3.4%. The feedlot concentrate pellets
were challenging due to the water wash step, and since the pel-
lets were of a low fat content, the RSDR represents weighing
differences among laboratories on the order of 12 mg (on a
weight of ~60 mg fat residue in an aluminum extraction cup
weighing 25 000 mg or in a glass extraction cup weighing
60 000 mg). The material contained 6.4% urea; therefore, an in-
adequate wash of urea from the 4 g test portion would provide a
Crude fat, % Crude fat, % after water wash
Crude fat, diethyl ether extract, %
4.10
4.00
3.90
3.80
3.70
3.60
3.50
3.40
3.30
0 5 10 15
Glucose spike as % of test portion
Figure 2. Effect of glucose on crude fat (diethyl ether
extract) recovery.
% crude fat, diethyl ether extract
Crude fat, % Trendline
4.1
4
3.9
3.8
3.7
3.6
3.5
3.4
3.3
0 2 4 6
Lysine and methionine spike as % of test portion
Figure 3. Effect of added lysine and methionine on
crude fat (diethyl ether extract) recovery.
potential interference of ~260 mg urea (Table 6). Although the
HORRAT of 5.6 seems excessive, under closer scrutiny it is
easily accounted for, and even though this precision could be
improved upon somewhat with practice, it is doubtful that labo-
ratories can routinely achieve a HORRAT <2 on such a chal-
lenging material.
The meat meal/hulls mix did not have an obvious reason
for the relatively higher HORRAT. The RSD for the crude fat
when used as an AAFCO Check Sample was 6.8% for method
code 3.00 (diethyl ether extraction by AOAC Method 920.39)
and 8.5% for method code 3.09 (diethyl ether extraction by
Soxtec; 7). The variability in the collaborative study was
somewhat greater than experienced with the AAFCO Check
Sample Program results. One laboratory narrowly escaped re-
moval by the Cochrans test and if this laboratory had been re-
moved, the HORRAT and RSDR would have been 2.85 and
8.9%, respectively, which are similar to the variability ob-
served in the AAFCO Check Sample Program.
Crude fat is an empirical method, i.e., it falls into the
Trendline Type 1 Codex Alimentarius Commissions scheme of defini-
tion of method types. A defining method is a method which
determines a value that can only be arrived at in terms of the
method per se (8). As discussed by Horwitz et al. (9),
gravimetric methods have limits of detection and precision
that are related to weighing error, and methods by which the
analyte is empirically defined are traditionally prone to greater
inherent variability than methods that are calibrated against a
reference standard. The HORRAT values observed in this
study are favorable with those reported by Horwitz et al. for
20
fat. In 105 fat assays with a concentration range of 370%,
they report an average RSDR of 14%, and a 90% confidence
interval for RSDR % and HORRAT of 0.565 and 0.212, re-
spectively. In this study with a concentration range of
1.599%, the average RSDR was 3.1%, and RSDR ranged
from 1.9 to 21% and HORRAT values from 0.56 to 5.6. The
 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 897

Table 6. Recovery of crude fat, diethyl ether extract, without predry step on collaborative study samples

Moisture, % average Crude fat, % study mean Crude fat, % diethyl ether
Material after rehydration (from Table 2003.05A) extraction, no predry Recovery, %

Dehydrated alfalfa 15.5 4.44 5.23 118

Corn silage 12.0 2.09 2.92 140
Mixed bird seed 14.8 7.19 5.92 82
Texturized feed 15.1 3.20 3.93 123
Fat supplement 5.5 96.38 96.55 100
Medicated goat feed 13.0 1.89 2.37 125
Feedlot concentrate pellets 24.9 1.53 2.33 153
Cellulose (blank) 10.8 0.17 0.20 117
Calf starter medicated 13.0 2.96 3.40 115
Calf feed medicated 13.9 3.42 3.61 106
Meat meal/hulls mix 25.2 4.99 6.77 136
Swine feed 16.2 2.77 3.07 111
Broiler starter 14.2 7.05 7.09 101
High oil corn 17.1 7.69 7.89 103
Average recovery 116

fact that HORRAT values for a few materials are >2.0 does
not invalidate the method.
Because the analyte is defined by the method, it is crucial to
emphasize that fat methods must be followed exactly. As
Horwitz et al. (9) concluded, analysts attempt to improve a
method of analysis by shortening times and eliminating what
appear to be purposeless steps. This was certainly observed in
this study when some collaborators had to be convinced of the
necessity to perform the predry and the water-wash steps,
which they felt were superfluous or not cost effective. These
steps have obviously been eliminated in many laboratories, and
are a source of variability normally associated with the method.
It is also possible that these steps have not been uniformly prac-
ticed in laboratories due to the lack of a formal/standardized
Randall method for extracting fat from feeds.
Recommendations
On the basis of this study, the Study Directors recommend
that the method for Crude Fat, Diethyl Ether Extraction, in
Feed, Cereal Grain, and Forage (Randall/Soxtec/Submersion
Method) be adopted as Official First Action. Based on the
RSD of the cellulose blank, it is recommended that values be-
low 0.5% crude fat be reported as <0.5%. It is also recom-
mended that materials containing >5% urea be water-washed
before the crude fat extraction, and materials containing >15%
sugars be washed before the extraction.
Acknowledgments
We thank Brian Steinlicht (South Dakota State University,
Olson Biochemistry Laboratories, Brookings, SD) for assis-
tance with preparing, splitting, and sealing familiarization,
and study samples; Foss North America for providing extrac-
tion thimbles to collaborators and for supporting the study
with shipment of study materials; and Fisher Scientific for
providing some solvents for the solvent comparability testing.
We also thank the following collaborators for their participa-
tion in this study:
Wayne Adcock, Alabama Department of Agriculture and
Industries State Chemical Laboratory, Auburn, AL
Roy Coffin, Soil & Feed Testing Laboratory, Department
of Agriculture and Forestry, Charlottetown, PEI, Canada
Amy T. DeBaker, Matthew Gramse, and Fatthieh Shafiee,
Wisconsin Department of Agriculture, Trade, and Consumer
Protection, Madison, WI
Pat Hogan, Sure-Tech Laboratory, Indianapolis, IN
John MacDonald and Tom Knese, Ralston Analytical Lab-
oratories, St. Louis, MO
Jrgen Mller, Elisabet Frankenius, and Eva Bogren, Foss
Tecator AB, Hoganas, Sweden
Randy Royle, Servi-Tech Laboratories, Dodge City, KS
Marcy Russell and Jeff Boedigheimer, Foss North Amer-
ica, Eden Prairie, MN
Brian Shreve and Kelli Conway, Servi-Tech Laboratories,
Hastings, NE
Joel Sieh, CN-Labs, Courtland, MN
898 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
Misti Spann and Mick Watts, Kansas Department of Agri-
culture Laboratory, Topeka, KS
Erika Tpler, Schsischer Landeskontrollverband,
Niederwiesa, Germany
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(3) Official Methods of Analysis (2000) 17th Ed., AOAC IN-
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(5) Methods of Analysis for Nutrient Labeling (1992) D.M.
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