crude fat, hexanes extraction, in feed, cereal grain, and forage (randall/soxtec/submersion method):...

8/11/2019 Crude Fat, Hexanes Extraction, in Feed, Cereal Grain, and Forage (Randall/Soxtec/Submersion Method): Collabor

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AGRICULTURAL MATERIALS

Crude Fat, Hexanes Extraction, in Feed, Cereal Grain, and Forage(Randall/Soxtec/Submersion Method): Collaborative Study

NANCYJ. THIEX

South Dakota State University, Oscar E. Olson Biochemistry Laboratories, Box 2170, ASC 151, Brookings, SD 57007

SHIRLEYANDERSON

Foss North America, 7682 Executive Dr, Eden Prairie, MN 55344

BRYANGILDEMEISTER

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 fromthe animal feed, cereal grain, or forage material

with hexanes by the Randall method, also calledthe Soxtec method or the submersion method. Theuse of hexanes provides for an alternative to di-

ethyl ether for fat extractions. The proposed sub-mersion method considerably decreases the ex-

traction time required to complete a batch ofsamples compared to Soxhlet. The increase in

throughput is very desirable in the quest for fasterturnaround times and the greater efficiency in theuse 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 andwas accepted as AOAC Official Method 991.36.

Fourteen blind samples were sent to 14 collabora-tors in the United States, Sweden, Canada, andGermany. The within-laboratory relative standard

deviation (repeatability) ranged from 1.23 to 5.80%for crude fat. Among-laboratory (including within)

relative standard deviation (reproducibility) rangedfrom 1.88 to 14.1%. The method is recommended

for Official First Action.

The Randall (1) or submersion method for fat extraction

is an AOAC Official Method for meats and meat prod-

ucts (2). Its use is also widespread in feed laboratories

to determine crude fat in feed, grain, and forage. Approximately

1/3 of the laboratories reporting crude fat results on animal feedto the Association of American Feed Control Officials

(AAFCO) Check Sample Program are reporting fat values ob-

tained 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 of-

ficial method and to bring the AOAC Official Methods of Anal-

ysiscurrent with what is practiced in todays laboratories.

While seeking collaborators for the concurrent study using

diethyl ether as a solvent (see this issue, p. 888), it became ap-

parent that few laboratories actually use diethyl ether because

of safety considerations with the potential for peroxide forma-

tion in ether. A need was evidentfor an official alternative sol-

vent to diethyl ether for laboratories performing fat extrac-tions. Petroleum ether, which is not an official solvent for

animal feed, was found to be in wide use for fat extractions

(numerous personal communications). Therefore, compara-

bility tests for petroleum ether and other potential solvents

were performed.

Ruggedness Testing

Ruggedness tests (3) 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 (seethe concurrent study in

this issue, p. 888).

Solvent Comparability

Single Laboratory

Petroleum ether, pentanes, and hexanes were tested for

comparability to diethyl ether in an effort to find an alternative

to diethyl ether for laboratories desiring to avoid use of a sol-

THIEX ET AL.: JOURNAL OFAOAC INTERNATIONAL VOL. 86, NO. 5, 2003 899

Submitted for publication May 2003.The recommendation was approved by the Methods Committee on

Feeds, Fertilizers, and Related Agricultural Topics as First Action. SeeOfficial Methods Program Actions, (2003) Inside Laboratory

Management, May/June issue.Corresponding authors e-mail: [email protected].


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vent with the potential for peroxide formation. The solventschosen were petroleum ether, pentanes, and hexanes. Solvents

were chosen for comparison based upon boiling point, boiling

point range, polarity, price, and (in the case of petroleum

ether) current usage. Results of intralaboratory comparability

tests using 10 feed and forage materials are shown in Table 1.

Correlation coefficients, slope, and bias for petroleum ether,

pentanes, and hexanes (compared to diethyl ether) are 0.9998,

0.9947, and 0.3179; 0.9997, 0.9965, and 0.5129; and 0.9999,

0.9852, and 0.1181, respectively. The inclusion of a 100% fat

supplement tended to mask the effect of solvent on lower fat

materials; therefore, the statistics were recalculated omitting

the high fat supplement. Correlation coefficients, slope, and

bias for petroleum ether, pentanes, and hexanes (compared to

diethyl ether and omitting the high fat supplement) were

0.9949, 0.9645, and 0.4662; 0.9938, 1.0216, and 0.3941; and

0.9970, 0.9858, and 0.1661, respectively. From these data,

hexanes appear to be the best match to diethyl ether.

Interlaboratory

Comparability tests described above were repeated using

3 laboratories and 6 feed and forage materials. Results of

interlaboratory comparability tests are shown in Table 2. Cor-

relation coefficients, slope, and bias for petroleum ether, hex-

anes, and pentanes (compared to diethyl ether) are 0.9998,

1.0069, and 0.3168; 0.9999, 1.0139, and 0.1449; and

0.9997, 1.0274, and 0.3236, respectively.

Once again, the inclusion of a 100% fat supplement tended

to mask the effect of solvent on lower fat materials; therefore

the data are repeated in Table 2, omitting the high fat supple-

ment. Correlation coefficients, slope, and bias for petroleum

ether, hexanes, and pentanes (compared to diethyl ether and

omitting the high fat supplement) are 0.9878, 1.0077, and

0.3193; 0.9925, 1.0201, and 0.1672; and 0.9880, 1.0274,

and 0.4141, respectively.

Based upon these results and discussions of these results atAAFCO Laboratory Methods and Services Committee Meet-

ings, it was decided that the most suitable alternative to diethyl

ether is hexanes. Petroleum ether is already in common use as

an alternative, but it shows a low bias for some materials, in-

cluding forages. There are also problems with consistency of

the solvent from manufacturer to manufacturer, lot-to-lot, and

country-to-country, as a fat solvent. Some of this may be due

to the wide rangeof components of the solvent. The wide boil-

ing point range also makes it a poor choice for recovery and

reuse of the solvent.

900 THIEX ET AL.: JOURNAL OFAOAC INTERNATIONAL VOL. 86, NO. 5, 2003

Table 1. Comparability of 3 solvents to diethyl ether (intralaboratory)

Sample description Diethyl ether Petroleum ether Pentanes Hexanes

Crude fat,%

Fat supplement 99.02 99.13 98.94 99.35

Alfalfa hay 1.38 1.06 1.01 1.51

Soybean meal 1.17 1.15 1.00 1.32

Corn silage 2.04 1.94 1.85 2.02

Beet pulp 0.35 0.41 0.23 0.41

Pig starter 8.63 8.42 8.40 8.36

Pig starter, wwa

8.09 7.65 7.15 7.62

Meat and bone meal 10.72 10.39 10.73 11.02

Extruded supplement 18.83 19.34 17.73 19.06

Cattle protein supplement, 7%urea 3.64 3.20 3.21 3.35

Cattle protein supplement, 7%urea, wwa

2.57 2.10 1.98 2.49

Corn 3.31 1.99 1.92 2.60

Summary statistics omitting fat supplement

Correlation R 0.99746 0.99690 0.99851

R2 0.99493 0.99381 0.99702

Slope 0.9645 1.0216 0.9858

y-Intercept 0.4662 0.3941 0.1661

Std. error 0.42 0.46 0.32

Sum 60.73 57.65 55.21 59.75

Average 5.52 5.24 5.02 5.43

a ww = Water-wash.


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Collaborative Study

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 receivedfrom 3 of them. Participants received no compensation. Fa-

miliarization samples were sent to each collaborator to be ana-

lyzed before the test samples to acquaint them with the

method and to ensure that the laboratory was capable of han-

dling 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 3). 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 (


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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 Cen-

trifugal 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 mmscreen. 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 3. 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 cotton were pro-

vided to collaborators.

Note: The study was conducted concurrently using diethyl

ether as the extraction solvent. The concurrent study appears

as a companion article in this issue of J. AOAC Int. (see

p. 888).

AOAC Official Method 2003.06Crude Fat in Feeds, Cereal Grains, and Forages

Randall/Soxtec/Extraction-Submersion Method

First Action 2003

[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

from 0.5 to 100% fat. It is applicable to the same matrixes as

AOAC Official Methods920.39(see4.5.01) and930.09(see3.5.07).]

Caution: Store solvents in metal containers in solvent cabi-

net or solvent room that conforms to applicable

safety legislation. Ethers and hexanes are ex-

tremely flammable. Have no open flames in the

laboratory where the analysis is being performed.

Avoid inhaling vapors. Use solvents in a properly

operating hood equipped with explosion-proof

lighting, wiring, and fan. Diethyl ether has the

potential to form shock-sensitive, explosive per-

oxides with age. Check each new container ofether 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

notuse ether that contains peroxides. Dispose as

hazardous material. Stabilized ether may be used.

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.

SeeTable2003.06for results of interlaboratory study sup-

porting 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 matrixwith 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.


Table 3. Categories represented by study materials

and uniformity testing results

MaterialAnimal

feed Forage GrainFat, %RSD

Meat meal/hulls mixture 1.4

Calf feed medicated 0.80

Broiler starter

1.5Mixed bird seed 0.86

Calf starter medicated 0.49

Corn silage 1.1

Dehydrated alfalfa 1.2

High oil corn 1.1

Medicated goat feed 0.29

Texturized feed 1.1

Feedlot concentrate pellets 1.9

Fat supplement 0.15

Swine feed 1.2

Cellulose blank (Solka-Flok)


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B. Apparatus

(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. (Extractiontemperature 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

from absorbing 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 15g test portions containing ca 100200mg fat di-

rectly into tared cellulose thimbles, according to following

scheme:

Crude fat,% Test portion weight, g

20 1

Record weight to nearest 0.1 mg (S) and thimble number.

Dry thimbles containing test portions at 102 2Cfor2h.

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.


Table 2003.06. Interlaboratory results for crude fat in animal feed, cereal grain, and forage, hexanes extraction

(submersion) method

Material Mean Laba sr RSDr,% sR RSDR,% 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.


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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 solventto 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 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 TS

100

% Crude fat, diethyl ether extract =

F T

S100

where F = weight of cup + fat residue, g; T = weight of empty

cup, g; S = test portion weight, g.

Refs.:J. AOAC Int.86, 902904(2003); 890893(2003)

Results and Discussion

Study materials were shipped the last week of September

2001 to 13 laboratories. Results were received from 12 laborato-

ries (Table 4) over a period of 3 months, with the last set received

on December 28, 2001. One of the 13 laboratories could not pro-

vide data due to in-house issues. Laboratories were asked to pro-

vide the type of instrument used. Equipment used is described in

Table 5.Early into the study, it became apparent that some collabora-

tors were following in-house methods rather than the method

supplied with the study. Also, a number of questions were re-

ceived as to the appropriateness of particular lots of solvents.At

this point,a survey wasdeveloped to documentthe manufactur-

ers of solvents, catalog numbers, and lot numbers; and to assure

the Study Directors that collaborators were following the

method as supplied. Results of the survey are shown in Table 6.

Laboratory 11 did not follow the method as mailed to collabo-

rators and did not have time to retest the materials. Deviations

from the method included not performing the water-wash re-

quired on 4 materials, and using different soak and rinse times

from those specified. Data from this laboratory were evaluated

using an XY plot, which confirmed that data from this labora-

tory should be removed from the study.

Collaborators Comments

In response to these comments and suggestions received

from collaborators concerning extraction of high-fat materi-

als, a better description of how to handle high-fat materials

was incorporated into the method.

A number of comments were received about the necessity

of the water-wash step. This step is specified in AOAC Offi-

cial Method920.39(4); and was therefore incorporated with-

out further investigation. To demonstrate the effect of poten-tially interfering substances, recovery experiments were

performed. Urea and glucose equivalent to ~2% (~0.04 g),

~5% (~0.1 g), ~10% (~0.2 g), and ~15% (~0.3 g) were added

to 2 g test portions of ground shelled corn and extracted with-

out 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 removed. The data are

presented in Table 7 and graphical representations of the re-

sults of the experiment in Figures 1 and 2. Neither glucose nor

urea at levels


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rehydrated by placing the weighed thimbles with test portion

into a desiccator charged with water and allowed to equilibrate

overnight at 55C. Recoveries of crude fat, hexanes extrac-

tion, ranged from 77.0 to 134% (Table 8). This is consistent

with the literature (5, 6). Water can decrease the efficiency of

the solvent in extraction, and/or allow for extraction of wa-

ter-soluble nonlipid components. The results confirm that the

predry step is critical in the extraction process. Recoveries of

fat for the urea-containing feed (feedlot concentrate pellets)and the molasses-based, high-sugar content feed (texturized

feed) were 77 and 117%, respectively, with no predry. High

recoveries were also observed with the corn silage, medicated

goat feed, and meat meal/hulls mix. A low recovery (incom-

plete extraction) was observed with the urea-containing feed

(feedlot concentrate pellets).

A laboratory ranking by the test described by Youden and

Steiner (3) was used to assess bias among laboratories partici-

pating in the collaborative study. One laboratory on the high

and low end showed bias based on the laboratory ranking test.

However, the actual spread among values from all laboratories

was tight. The RSD of the sum of results on all materials for all

laboratories was 1.5%, and the relative difference between the

high- and low-ranking laboratory was


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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 (RSDr= 1.29%, RSDR=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 (RSDr = 3.07%, RSDR=6.27%) and the feedlot concentrate pellets (RSDr = 5.80%,

RSDR =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 RSDrranges from 1.23 to 4.96% for feed, forage, and cereal materi-

als, and among-laboratory RSDRranged 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.

HORRAT values 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 Rrepresents

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.


Table 6. Study survey results

Lab Prewash Predry, 2 h SolventInstrument temp.

setting, C Soak , min Rinse, minPostdry,

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 91.3a, 98.2b

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 90.4a, 98.2b

a With water-wash compared to unwashed control.b With water-wash compared to washed control.


9/10

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 RSDRof 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 5.4%and RSDR 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.

Becausethe analyte is defined by the method, it is crucial to

emphasize that fat methods must be followed exactly. As


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

Crudefat,hexanesextract,%

Crude fat, % Crude fat, % after water wash Trendline

Figure 1. Effect of urea on crude fat (hexanes extract)

recovery.

3.30

3.403.50

3.60

3.70

3.80

3.90

4.00

4.10

0 5 10 15 20

Glucose spike as % of test portionCrudefat,hexanesextract,%

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

MaterialMoisture, %average after

rehydrationCrude fat,%study mean

(from Table2003.06)Crude fat,%hexanesextraction, 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


10/10

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

crude fat, hexanes extraction, in feed, cereal grain, and forage (randall/soxtec/submersion method):...

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