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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; Foss North
America, 7682 Executive Dr, Eden Prairie, MN 55344, Tel:
+1-952-974-9892, Fax: +1-952-974-9823, info@fossnorth
america.com; or other manufacturers of Randall-type extrac-
tion systems.
C. Reagents
(a) Hexanes.Fisher H291 (UN1208; www.fishersci.com),
or equivalent; boiling range: 4C including 68.7C.
(b) Anhydrous diethyl ether.Purified for fat extraction,
Fisher P/N E4492 labeled For Fat Extraction is also stabi-
lized, or E134-4 (UN1155), or equivalent. To prevent ether
fromabsorbing water, purchase it in small containers and keep
containers tightly closed. Petroleum ether cannot be substi-
tuted for diethyl ether because it does not dissolve all of the
plant lipid material.
(c) Cotton.Defatted. Soak medical grade cotton in di-
ethyl ether or hexanes for 24 h, agitating several times during
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 of
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
5 24
10 12
>20 1
Record weight to nearest 0.1 mg (S) and thimble number.
Dry thimbles containing test portions at 102 2Cfor 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.
THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 903
Table 2003.06. Interlaboratory results for crude fat in animal feed, cereal grain, and forage, hexanes extraction
(submersion) method
Material Mean Lab
a
s
r
RSD
r
, % s
R
RSD
R
, % HORRAT
Dehydrated alfalfa 4.34 9(1) 0.14 3.21 0.16 3.75 1.17
Corn silage 1.91 9(1) 0.04 1.97 0.15 5.31 1.46
Mixed bird seed 7.15 9(1) 0.25 3.44 0.25 3.44 1.16
Texturized feed 2.91 10 0.09 3.07 0.18 6.27 1.84
Fat supplement 97.77 9(1) 1.29 1.32 1.84 1.88 0.94
Medicated goat feed 1.54 9(1) 0.03 1.94 0.13 8.48 2.26
Feedlot concentrate pellets 1.30 10 0.08 5.80 0.18 14.1 3.67
Cellulose (blank) 0.12 10 0.06 50.5 0.08 65.4 11.9
Calf starter medicated 2.58 10 0.09 3.52 0.14 5.61 1.62
Calf feed medicated 3.23 10 0.18 5.45 0.21 6.48 1.93
Meat meal/hulls mix 5.76 10 0.12 2.10 0.18 3.19 1.04
Swine feed 2.29 10 0.11 4.96 0.15 6.38 1.81
Broiler starter 5.99 10 0.17 2.83 0.22 3.61 1.18
High oil corn 7.63 9(1) 0.09 1.23 0.16 2.09 0.71
a
Number of laboratories retained after the number of laboratories in parentheses were eliminated.
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 mmglass 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 distill 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-
ture. (Note: Take care not to pick up any debris on bottom of
extraction cup while in hood. Let cups remain in hood until all
traces of solvent are gone.)
Dry extraction cups in 102 2C oven for 30 min to re-
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, hexanes extract =
F T
S
100
-
s
t
e
s
t
,
p
=
2
.
5
%
(
1
-
t
a
i
l
)
.
Table 5. Extractor type and laboratory ranking scores
for collaborating laboratories
Laboratory Extractor type Lab ranking score
1 Soxtec 2050 63.5
2 Soxtec HT 6 1043 55.5
3 Soxtec HT 6 1043 107.5
a
4 Soxtec 2050 91
5 Soxtec HT 6 1043 44.5
a
6
b
Soxtec 2050 107
7 Soxtec 2050 90
9
a
Soxtec HT 6 1043 48
10 Soxtec 2055 88
11
b
Soxtec 2055
b
12 Soxtec HT 6 1043 69
a
Results show bias using the lab ranking test.
b
XY plot revealed outlier results.
lets). This suggests that one of the significant sources of vari-
ability in the method is related to sampling the test portion.
The high fat supplement proved to be a challenge for the
collaborators. The fat (RSD
r
= 1.29%, RSD
R
=1.88%) melted
during the predry step, and foamed during extraction. Collab-
orators unfamiliar with this type of material may not have ob-
served these potential sources of error. As a result of the com-
ments received, better instructions have been incorporated
into the method describing how to handle these materials to
avoid incomplete recovery of crude fat.
Another challenge to the collaborators was the water-wash.
This was required for the texturized feed (RSD
r
=3.07%, RSD
R
=6.27%) and the feedlot concentrate pellets (RSD
r
= 5.80%,
RSD
R
=14.1%). Even though the current AOAC Official
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. If materials which were water-washed
are removed from the statistics, the within-laboratory RSD
r
ranges from 1.23 to 4.96% for feed, forage, and cereal materi-
als, and among-laboratory RSD
R
ranged from 1.88 to 8.48%.
Therefore, based on the recovery studies, the water-wash step is
not necessary when hexanes are used as solvent, and only
serves to add error rather than improve recoveries.
HORRATvalues for dehydrated alfalfa, corn silage, mixed
bird seed, texturized feed, fat supplement, calf starter medi-
cated, calf feed medicated, meat meal/hulls mix, swine feed,
broiler starter, and high oil corn ranged from 0.71 to 1.93 and
are excellent. Two materials had HORRAT values >2.0: the
medicated goat feed (HORRAT = 2.26) and feedlot concen-
trate pellets (HORRAT = 3.67). The feedlot concentrate pel-
lets were challenging because of the water-wash step, and be-
cause the pellets had a low fat content, the RSD
R
represents
weighing differences among laboratories on the order of 8 mg
(on a weight of ~60 mg fat residue in an extraction cup weigh-
ing 25 000 mg for an aluminum extraction cup to 60 000 mg
for a glass extraction cup). Although the HORRAT of 3.67
sounds excessive, under closer scrutiny, it is easily accounted
for and may be improved upon by eliminating the water-wash
step. The HORRAT for medicated goat feed was slightly over
the desired 2.0, and not a real concern to the Study Directors.
One laboratory had an elevated Cochrans score, but escaped
removal by the Cochrans test. If this laboratory had been re-
moved, it would have lowered the HORRAT to 1.68.
906 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
Table 6. Study survey results
Lab Prewash Predry, 2 h Solvent
Instrument temp.
setting, C Soak, min Rinse, min
Postdry,
0.5 h Flow Comments
1 5,8,15,28 100C EM Science HX 0297-1 30183 135 20 40 100C Steady steam Yes
2 5,8,15,28 102C Fisher H303-4 013117 130 20 40 100C Rapid drip None
3 5,8,15,28 102C JT Baker 9308-33 unknown 140 20 40 100C None
4 5,8,15,28 102C Fisher H303-4 013115 135 20 40 100C 35 drips/s None
5 5,8,15,28 102C Fisher H291s-4 010762 145 20 40 100C 45 drips/s (rapid
drips)
Mix sand with
water-wash
6B 5,8,15,28 102C Riedel de Haen 15671 135 20 40 100C
Merck 104368 K29342968 124
7 5,8,15,28 102C Lab Scan C2536 1179/1 155 20 40 100C 2 + 3 drops/s Yes
9 5,8,15,28 102C Fisher (UN1208) H291-2c 001933 170 20 40 100C Distinct drop 35
drips/s
Yes
10 5,8,15,28 102C Fisher E291-4 011065 155 20 40 100C 4 drips/s None
11 None 103C Fisher H303-4 996668 102 25 30 100C Yes
12 5,8,15,28 102C Fisher E292-4 011382 185 20 40 100C Rapid drip Yes
Table 7. Recovery of crude fat, hexanes extract, from
ground corn spiked with urea and glucose
Urea spike, % Recovery, %
0.00 100.0
2.20 101.1
5.03 101.4
10.23 100.3
15.07 100.3
15.22 91.3
a
, 98.2
b
Glucose spike, % Recovery, %
0.00 100.0
2.16 96.5
4.99 98.1
10.30 98.1
15.07 98.4
15.04 90.4
a
, 98.2
b
a
With water-wash compared to unwashed control.
b
With water-wash compared to washed control.
Crude fat is an empirical method, i.e., it falls into the
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 (7). As discussed by Horwitz et al. (8),
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
fat. In 105 fat assays with a concentration range of 370%,
they report an average RSD
R
of 14%, and a 90% confidence
interval for RSD
R
% and HORRAT of 0.565 and 0.212, re-
spectively. In this study with a concentration range of
1.599%, the average RSD
R
was 5.4%and RSD
R
ranged from
1.9 to 14% and HORRAT values from 0.71 to 3.7. The fact
that HORRAT values for 2 materials are >2.0 does not invali-
date the method. In fact, the method appears to be as good as
or better than those currently available as Official Methods,
and the hexanes appear to be as good as or better than diethyl
ether as a solvent.
Because the analyte is defined by the method, it is crucial to
emphasize that fat methods must be followed exactly. As
THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 907
3.30
3.40
3.50
3.60
3.70
3.80
3.90
4.00
4.10
0 5 10 15 20
Urea spike as % of test portion
C
r
u
d
e
f
a
t
,
h
e
x
a
n
e
s
e
x
t
r
a
c
t
,
%
Crude fat, % Crude fat, % after water wash Trendline
Figure 1. Effect of urea on crude fat (hexanes extract)
recovery.
3.30
3.40
3.50
3.60
3.70
3.80
3.90
4.00
4.10
0 5 10 15 20
Glucose spike as % of test portion
C
r
u
d
e
f
a
t
,
h
e
x
a
n
e
s
e
x
t
r
a
c
t
,
%
Crude fat, % Crude fat, % after water wash Trendline
Figure 2. Effect of glucose on crude fat (hexanes
extract) recovery.
Table 8. Recovery of crude fat, hexanes extract, without predry step on collaborative study samples
Material
Moisture, % average after
rehydration
Crude fat, % study mean
(from Table 2003.06)
Crude fat, % hexanes
extraction, no predry Recovery, %
Dehydrated alfalfa 15.5 4.34 4.74 109
Corn silage 12.9 1.91 2.02 106
Mixed bird seed 15.4 7.15 6.87 96
Texturized feed 17.3 2.91 3.41 117
Fat supplement 6.70 97.77 96.76 99
Medicated goat feed 14.3 1.54 1.61 102
Feedlot concentrate pellets 27.8 1.30 1.00 77
Cellulose (blank) 18.5 0.12 0.16 134
Calf starter medicated 15.0 2.58 2.67 103
Calf feed medicated 13.4 3.23 3.33 103
Meat meal/hulls mix 24.5 5.76 6.28 109
Swine feed 17.8 2.29 2.08 91
Broiler starter 17.3 5.99 5.78 97
High oil corn 19.0 7.63 7.73 101
Average recovery 103
Horwitz et al. (8) 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 step, which they felt was su-
perfluous or not cost effective. These steps have obviously
been eliminated in many laboratories, and are a source of vari-
ability normally associated with the method.
Recommendations
On the basis of this study, the Study Directors recommend
that the method for Crude Fat, Hexanes 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%.
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; Fisher Scientific for provid-
ing some solvents for the solvent comparability testing. We
also thank the following collaborators for their participation 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 TomKnese, 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
Misti Spann and Mick Watts, Kansas Department of Agri-
culture Laboratory, Topeka, KS
Erika Tpler, Schsischer Landeskontrollverband,
Niederwiesa, Germany
References
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11651168
(2) Official Methods of Analysis (2000) 17th Ed., AOAC IN-
TERNATIONAL, Gaithersburg, MD, Official Method
991.36
(3) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual of
the AOAC, AOAC, Arlington, VA
(4) Official Methods of Analysis (2000) 17th Ed., AOAC IN-
TERNATIONAL, Gaithersburg, MD, Official Method
920.39
(5) Methods of Analysis for Nutrient Labeling (1993) D.M.
Sullivan & D.E. Carpenter (Eds), AOAC, Arlington, VA,
p. 94
(6) Pomeranz, Y., & Meloan, C.E. (1994) Food Analysis Theory
and Practice, 3rd Ed., Chapman & Hall, New York, NY,
pp 684692
(7) Codex Alimentarius Commission (1986) Procedural Manual,
6th Ed., Food and Agricultural Organization, Rome, Italy,
p. 139
(8) Horwitz, W., Albert, R., Deutsch, M.J., & Thompson, J.N.
(1990) J. Assoc. Off. Anal. Chem. 73, 661680
908 THIEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003