Professional Documents
Culture Documents
Fat Cereal
Fat Cereal
5, 2003
AGRICULTURAL MATERIALS
A method for determining crude fat in animal feed, to determine crude fat in feed, grain, and forage. Approxi-
cereal grain, and forage (plant tissue) was col- mately 1/3 of the laboratories reporting crude fat results on an-
laboratively studied. Crude fat was extracted from imal feed to the Association of American Feed Control Offi-
the animal feed, cereal grain, or forage material cials (AAFCO) Check Sample Program are reporting fat val-
with diethyl ether by the Randall method, also ues using this method. It therefore seemed appropriate that
called the Soxtec method or the submersion this method should be collaborated for animal feed, cereal
method. The proposed submersion method con- grain, and forage in an attempt to establish the method as an
siderably decreases the extraction time required to official method and to bring the AOAC Official Methods of
complete a batch of samples. The increase in Analysis current with what is practiced in today’s laboratories.
throughput is very desirable in the quest for faster Comparisons of the various fat methods reported in the
turnaround times and the greater efficiency in the AAFCO Check Sample Program were made by George Lati-
use of labor. In addition, this method provides for mer (Office of the Texas State Chemist, Texas A&M Univer-
reclamation of the solvent as a step of the method. sity, personal communication). On data from 90 check sam-
The submersion method for fat extraction was pre- ples, the mean recovery by the proposed method was 96.71%
viously studied for meat and meat products and of AOAC Official Method 920.39 (diethyl ether, traditional
was accepted as AOAC Official Method 991.36. Soxhlet extraction; 3), and median recovery was 98.21%. Re-
Fourteen blind samples were sent to 12 collabora- gression analysis of the proposed method on AOAC Official
tors in the United States, Sweden, Canada, and Method 920.39 gave a correlation coefficient of 0.9973, R2 =
Germany. The within-laboratory relative standard 99.46%, slope of 1.00062, and an intercept of –0.137367. On
deviation (repeatability) ranged from 1.09 to 9.26% this basis, the methods appeared comparable.
for crude fat. Among-laboratory (including within)
relative standard deviation (reproducibility) ranged Ruggedness Testing
from 1.0 to 21.0%. The method is recommended for
Official First Action. 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-
he Randall (1) or submersion method for fat extraction leum ether); rinse time (30 vs 60 min); test portion weight
Feed type Cattle Swine Mixed Cattle Swine Mixed Cattle Swine Mixed
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
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 103°C 2 h; (a) predrying 103°C 4 h; (B) boil time 20 min; (b) boil time 40 min; (C) diethyl ether; (c) petroleum ether
40–60°C; (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
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
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
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 Cochran’s 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
this period. Remove and air dry. Commercially available from Place three or four 5 mm glass boiling beads into each cup,
Foss Tecator, Part No. 1529-0009. and dry cups for at least 30 min at 102° ± 2°C. Transfer to des-
(d) Sand.—Ashed (for ignition boats). EM SX0075-3, or iccator and cool to room temperature. Weigh extraction cups
equivalent (CAS 14808-60-7). and record weight to nearest 0.1 mg (T).
(e) Celite 545.—Foss Tecator 1900-0014, or equivalent. Extract, following manufacturer’s instructions for opera-
tion of extractor. Preheat extractor and turn on condenser
D. Preparation of Analytical Sample
cooling water. Attach thimbles containing dried test portions
Grind laboratory samples to fineness that gives an RSD to extraction columns. Put sufficient amount of solvent into
£2.0% for 10 successive determinations. each extraction cup to cover test portion when thimbles are in
boiling position. Place cups under extraction columns and se-
RSD % = (SD/mean) ´ 100 cure in place. Make sure that cups are matched to their corre-
sponding thimble. Lower thimbles into solvent and boil for
Fineness of 0.75–1 mm usually achieves this precision with 20 min. Verify proper reflux rate which is critical to the com-
dry mixed feeds and other nonuniform materials. plete extraction of fat. This rate depends upon the equipment
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.
Collaborator’s 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
in the literature as a critical step (5, 6); they were therefore in-
1 5,8,15,28 100°C/2 h EM Science EX 0190-3 41159 115 20 40 100°C/0.5 h Steady steam Yes
2 5,8,15,28 102°C/2 h Fisher E198-4 014950-12 130 20 40 102°C/0.5 h Rapid drip None
3 5,8,15,28 100°C/2 h VWR VW 2142-5 41201 exp 110 20 40 100°C/0.5 h None
7/31/2003
4 5,8,15,28 102°C/2 h Fisher E138-4 011436-15 and 135 20 40 102°C/0.5 h 3–5 drips/s None
010930-15
5 5,8,15,28 102°C/2 h Fisher E492-4 011185-36 116 20 40 102°C/0.5 h Dribble None
6A 5,8,15,28 102°C/2 h Fisher E492-4 011185-36 101.5 20 40 102°C/0.5 h Dribble None
7 5,8,15,28 102°C/2 h Lab Scan H6509 S 3787/9 105 20 40 102°C/0.5 h 2 + 3 drops/s Yes
8 5,8,15,28 102°C/2 h EM Science (Merck) EX0190-3 70 20 40 102°C/0.5 h Distinct drops 2.9/s None
41110
9 5,8,15,28 102°C/2 h Fisher (UN 1155) E492-20 140 20 40 102°C/0.5 h Distinct drops 3–5/s Yes
011185-36
10 5,8,15,28 102°C/2 h Fisher E134-4 992548-36 135 20 40 102°C/0.5 h 4 drips/s None
11 None 103°C/2 h Fisher E199-4 005977-12 102 25 30 102°C/0.5 h Yes
12 5,8,15,28 102°C/2 h Fisher E138-4 011436-15 141.3 20 40 102°C/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 55°C. 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
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
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, %
fact that HORRAT values for a few materials are >2.0 does Acknowledgments
not invalidate the method.
Because the analyte is defined by the method, it is crucial to We thank Brian Steinlicht (South Dakota State University,
emphasize that fat methods must be followed exactly. As Olson Biochemistry Laboratories, Brookings, SD) for assis-
Horwitz et al. (9) concluded, analysts attempt to improve a tance with preparing, splitting, and sealing familiarization,
and study samples; Foss North America for providing extrac-
method of analysis by shortening times and eliminating what
tion thimbles to collaborators and for supporting the study
appear to be purposeless steps. This was certainly observed in
with shipment of study materials; and Fisher Scientific for
this study when some collaborators had to be convinced of the
providing some solvents for the solvent comparability testing.
necessity to perform the predry and the water-wash steps, We also thank the following collaborators for their participa-
which they felt were superfluous or not cost effective. These tion in this study:
steps have obviously been eliminated in many laboratories, and Wayne Adcock, Alabama Department of Agriculture and
are a source of variability normally associated with the method. Industries State Chemical Laboratory, Auburn, AL
It is also possible that these steps have not been uniformly prac- Roy Coffin, Soil & Feed Testing Laboratory, Department
ticed in laboratories due to the lack of a formal/standardized of Agriculture and Forestry, Charlottetown, PEI, Canada
Randall method for extracting fat from feeds. Amy T. DeBaker, Matthew Gramse, and Fatthieh Shafiee,
Wisconsin Department of Agriculture, Trade, and Consumer
Recommendations Protection, Madison, WI
Pat Hogan, Sure-Tech Laboratory, Indianapolis, IN
John MacDonald and Tom Knese, Ralston Analytical Lab-
On the basis of this study, the Study Directors recommend oratories, St. Louis, MO
that the method for Crude Fat, Diethyl Ether Extraction, in Jürgen Möller, Elisabet Frankenius, and Eva Bogren, Foss
Feed, Cereal Grain, and Forage (Randall/Soxtec/Submersion Tecator AB, Hoganas, Sweden
Method) be adopted as Official First Action. Based on the Randy Royle, Servi-Tech Laboratories, Dodge City, KS
RSD of the cellulose blank, it is recommended that values be- Marcy Russell and Jeff Boedigheimer, Foss North Amer-
low 0.5% crude fat be reported as <0.5%. It is also recom- ica, Eden Prairie, MN
mended that materials containing >5% urea be water-washed Brian Shreve and Kelli Conway, Servi-Tech Laboratories,
before the crude fat extraction, and materials containing >15% Hastings, NE
sugars be washed before the extraction. 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- (4) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual of
culture Laboratory, Topeka, KS the AOAC, AOAC, Arlington, VA
Erika Töpler, Sächsischer Landeskontrollverband, (5) Methods of Analysis for Nutrient Labeling (1992) D.M.
Niederwiesa, Germany Sullivan & D.E. Carpenter (Eds), AOAC, Arlington, VA, p. 94
(6) Pomeranz, Y., & Meloan, C.E. (1994) Food Analysis Theory
References and Practice, 3rd Ed., Chapman & Hall, New York, NY, pp
(1) Randall, E.L. (1974) J. Assoc. Off. Anal. Chem. 57, 684–692
1165–1168 (7) AAFCO (1999) Feed Check Sample No. 199930 Report, As-
(2) Official Methods of Analysis (2000) 17th Ed., AOAC IN- sociation of American Feed Control Officials, Oxford, IN,
TERNATIONAL, Gaithersburg, MD, Official Method November 29
991.36 (8) Codex Alimentarius Commission (1986) Procedural Manual,
(3) Official Methods of Analysis (2000) 17th Ed., AOAC IN- 6th Ed., Food and Agricultural Organization, Rome, Italy, p. 139
TERNATIONAL, Gaithersburg, MD, Official Method (9) Horwitz, W., Albert, R., Deutsch, M.J., & Thompson, J.N.
920.39 (1990) J. Assoc. Off. Anal. Chem. 73, 661–680