MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO.
6, 2002 1217
AGRICULTURAL MATERIALS
Gravimetric Determination of Amylase-Treated Neutral
Detergent Fiber in Feeds with Refluxing in Beakers or Crucibles:
Collaborative Study
DAVID R. MERTENS
U.S. Department of Agriculture, Agricultural Research Service, U.S. Dairy Forage Research Center, 1925 Linden Dr West,
Madison, WI 53706-1108
Collaborators: M. Allen; J. Carmany; J. Clegg; A. Davidowicz; M. Drouches; K. Frank; D. Gambin; M. Garkie;
B. Gildemeister; D. Jeffress; C-S. Jeon; D. Jones; D. Kaplan; G-N. Kim; S. Kobata; D. Main; X. Moua; B. Paul;
J. Robertson; D. Taysom; N. Thiex; J. Williams; M. Wolf
As an important constituent of animal feeds, fiber
represents the portion of feeds that is bulky and
difficult to digest. The neutral detergent fiber (NDF)
method, developed over 30 years ago, is the
method of choice for measuring total fiber in for-
ages and other feeds. Several modifications that
were made to improve its general applicability to
all feeds and others developed in individual labora-
tories often resulted in variability among laborato-
ries in measuring NDF. The amylase-treated NDF
(aNDF) method, therefore, was developed as an ac-
curate and precise method of measuring total in-
soluble fiber in feeds. A collaborative study was
conducted to evaluate the repeatability and
reproducibility of the aNDF method over the full
range of animal feed materials. Twelve laboratories
representing research, feed company, regulatory,
and commercial feed testing laboratories analyzed
11 materials as blind duplicates. The materials rep-
resented feed matrixes, including animal products;
high-protein, high-fat, and high-pectin feeds; oil
seeds; grains; heated by-product feeds; and le-
gume and grass hays and silages. Materials se-
lected varied in chemical composition and con-
tained 090% aNDF, 116% ash, 120% crude fat,
140% crude protein, and 050% starch. Cor-
recting results for changes in blanks and reporting
results as ash-free aNDF organic matter (aNDFom)
improved the repeatability and reproducibility of
results when aNDF was <25%. The within-labora-
tory repeatability standard deviation (sr) for per-
centage aNDFom in feeds varied from 0.21 to 1.82
Submitted for publication July 2002.
The recommendation was approved by the Methods Committee on
Feeds and Fertilizers and Agricultural Related Products as First Action. See
Official Methods Program Actions, (2002) Inside Laboratory
Management, September/October issue.
Corresponding authors e-mail: davem@dfrc.wisc.edu.
and among-laboratory reproducibility standard de-
viation (sR) varied from 0.37 to 2.24. The HORRAT
was <2 for all materials except feed materials con-
taining >10% fat. However, standard deviations of
repeatability and reproducibility for feeds with
>10% fat were similar to those of other materials. It
is recommended that the aNDF method be ac-
cepted for Official First Action status.
iber is nutritionally important because it represents the
F organic portion of feeds and foods that is the most diffi-
cult to digest; nonfiber fractions of feeds are easily and
almost completely digestible by most animals. There is a need
for a rapid and simple assay to determine the total insoluble fi-
ber content of feeds. Originally fiber was related to the cell
wall fraction of plants; however, because fiber occurs in all
feeds, fiber methods must be applicable to all feeds and foods.
Although neutral detergent (ND) extraction solubilizes some
material that is not digested by mammalian enzymes, neutral
detergent soluble fiber is often fermented by bacteria in the gut
of animals and is digested. Thus, neutral detergent fiber
(NDF) is the insoluble fiber in feeds that is either indigestible
or slowly digested, and occupies space in the digestive tract of
animals.
The NDF method was originally developed by Van Soest
and Wine (1) for the analysis of total fiber in forages, and nu-
merous modifications have been proposed to extend its appli-
cation to grains, concentrated feeds, and human foods (25).
Most of these modifications were based on conceptual im-
provement in total fiber analysis without ruggedness testing to
determine their suitability for all feed matrixes or practical ap-
plication as a routine method. Unfortunately, this resulted in
fiber methods that differed, yet were all called NDF. The diffi-
culty in extracting and washing fibrous residues in some mate-
rials and the variety in modifications of the method have led to
the perception that NDF is difficult to measure precisely.
1218 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
Collaborative Study
Optimization and Ruggedness of NDF Methods
The critical steps in the NDF and amylase-treated NDF
(aNDF) procedures were investigated by the original develop-
ers, who proposed modifications, and the Study Director, who
developed the method evaluated in this study. The following
factors were evaluated:
(1) Time of refluxing.Sixty min of refluxing at boiling
temperatures achieves asymptotic extraction for most materi-
als and was selected as an optimum compromise between
analysis time and complete extraction (1). It should not vary
more than 5 min.
(2) Neutral detergent concentration.Sodium lauryl sul-
fate is used at near saturation concentrations to maximize
solubilization of nonfiber with a minimal volume of liquid
that must be filtered (1).
(3) Neutral detergent pH.Phosphate and borate buffers
are used to maintain pH near 7.0 because alkaline or acid con-
ditions can solubilize fiber. The Study Director observed that
laboratories were not confirming that pH was near 7.0 and
conducted a test to evaluate the effects of pH variation. Re-
sults indicated that the pH should be maintained within the
range of 6.957.05 (6).
(4) Triethylene glycol.The original NDF method used
ethylene glycol monoethylether to remove nonfiber soluble
material from concentrated feeds. This compound is a poten-
tial mutagen and was replaced with triethylene glycol. Re-
search confirmed that changing to triethylene glycol did not
alter results (J.B. Robertson, Cornell University, Ithaca, NY,
personal communication, 1988).
(5) Sodium sulfite.Sodium sulfite was included to re-
move proteinaceous material from fiber residues. When
heat-stable amylase was included as a part of the NDF method,
sodium sulfite was removed because it has the potential to de-
stroy phenolic compounds (2). However, research by the Study
Director indicated that sodium sulfite is critical for removal of
proteinaceous matter in heated or cooked feeds, and it was rein-
troduced in the method that is being evaluated (7).
(6) Heat-stable a-amylase.Starch was not completely
removed by the original NDF method. Numerous modifica-
tions of the method used various amylases to remove starch
before or during ND extraction. Both the amylase source and
the method of using the enzyme alter NDF values, and pre-ex-
traction by incubation has the potential to solubilize fiber.
Heating samples and gelatinizing the starch may improve am-
ylase effectiveness in removing starch contamination. Many
amylase solutions are crude extracts that contain enzymes that
can degrade fiber and must be deactivated by near-boiling
temperatures. Alternative methods of using heat-stable
amylases have been evaluated by the Study Director and oth-
ers (4, 8). The approach used in the proposed method uses 2
additions of heat-stable amylase: one in ND after initiation of
boiling, and one in the first residue washing step.
Unfortunately enzyme sources changed or were discontin-
ued and new specifications for the type and amount of amy-
lase to use had to be rediscovered. The Study Director, there-
fore, developed a visual assessment method for determining
the amount of any heat-stable amylase source that is active un-
der the conditions used in aNDF analysis, and this is included
as part of the method.
(7) Washing method.Rapid rinsing of fibers removes
only surface contamination of detergent and soluble matter
and results in high fiber values. Research by the Study Direc-
tors laboratory indicated that multiple soakings of fibrous
residues are required to obtain consistent results and uncon-
taminated fibrous residues.
(8) Reporting fiber as organic matter.Ashing fiber resi-
dues and reporting results as aNDF organic matter (aNDFom)
eliminates some differences in results associated with inade-
quate washing of fibrous residues. In addition, it allows nutri-
tionists to estimate nonfibrous carbohydrates more accurately
by difference (NFC = 100 crude protein crude fat ash
aNDFom), because a portion of ash is not double-subtracted
as it would be if aNDF were used in the calculation. Calcula-
tions in the proposed method allow results to be reported to
meet the needs of the user.
(9) Sample amount and ratio to ND solution.The origi-
nal NDF method used 100 mL ND with 1 g sample (9).
Halving the amount of sample reduced aNDF by about 1 per-
centage unit. To reduce filtering problems, the same ratio of
sample to ND was maintained, but sample and ND solution
amounts were reduced to 0.5 g in 50 mL.
(10) Sample grinding.In most near infrared reflectance
spectroscopy, samples are ground through a cyclone mill with
1 mm screen. Discrepancies in fiber analysis were observed
when these samples were analyzed by chemical methods. Re-
search by the Study Director confirmed that particle size of the
sample and aNDF results were affected by both screen size and
grinder type. Although samples that are ground finer are ex-
tracted more completely by ND, they are often more difficult to
filter. Thus, the proposed method specifies that materials must be
ground through a 1 mm screen with a cutter mill or equivalent.
(11) Sample drying.Artifact fiber can be created during
sample preparation by drying materials at temperatures
>60C (10). Therefore, the method to be evaluated specifies
that wet samples be dried at temperatures <60C.
(12) Modifications for problem samples.Samples that
are high in starch, pectin, fat, or animal proteins are difficult to
filter. Modifications to the basic method have overcome the
difficulties inherent in these types of samples (11).
As indicated by Horwitz et al. (12), empirical methods of
analysis, such as fiber methods, must be followed exactly. This
suggests that the various modifications of the original NDF
method should have different names because they are different
measures of fiber. The proposed fiber method is called aNDF to
distinguish it from the original method and its modifications.
The objective of this research was to evaluate the repeatability
and reproducibility of the aNDF method in a collaborative
study involving laboratories with different missions and using
materials representing the full scope of feed ingredients.
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1219

Table 1. Characteristics of collaborating laboratories filtration vessels, balances, and drying ovens. Collaborators
Amylase were provided with test samples, heat-stable -amylase, and
Lab No. Type Method apparatus source chemicals, but were required to mix and standardize the neces-
sary reagents. They were asked to respond to several question-
0 Research (Study Director) Reflux beakercrucible A
naires about equipment and routine procedures in their labora-
1 Research Reflux beakercrucible B
tories, provide results of standardizing the amylase working
solution, analyze 5 familiarization samples, and provide aNDF
2 Research (dropped out)
results for 11 collaborative study materials and one blank ana-
3 Research Reflux beakercrucible A
lyzed as blind duplicates. Collaborators were asked to perform
4 Regulatory Reflux in crucible A
singles analyses of each sample, to report all weights to 4 signif-
5 Regulatory Reflux beakercrucible B
icant digits, and to submit data on an as-is basis.
6 Testing Reflux in crucible B
7 Feed company Reflux beakercrucible B Materials
8 Testing Reflux beakercrucible A
9 Testing Reflux beakercrucible A There are no officially recognized reference materials for
10 Testing (dropped out) fiber, and it is not possible to prepare spiked samples. To de-
11 Feed company Reflux beakercrucible B termine the general applicability of the aNDF method, study
12 Feed company Reflux beakercrucible B materials were selected to represent the full range of fiber con-
13 Feed company Reflux in crucible A centrations and types of animal feeds (Table 2). In addition,
14 Regulatory Reflux beakercrucible B materials were included that required the use of each of the
modifications described in the method. Materials representing
feed matrixes from animal products; high-protein, high-fat,
and high-pectin feeds; oil seeds; grains; heated by-product
feeds; legume forages; and grass forages (hays and silages)
Collaborating Laboratories were included in the study. A mixed dairy feed that contained
a mixture of animal and plant supplements with added fat was
The 14 laboratories which agreed to participate in the study used to represent a variety of feed ingredients.
represented 3 feed company laboratories, 5 commercial feed Materials selected for the collaborative study encompassed
testing laboratories, 3 feed regulatory laboratories, and 4 re- a wide range of aNDF concentrations in feeds, varying from 0
search laboratories. Two feed testing and one research labora- to 90%. The materials also varied in concentration of ash
tory failed to complete the study; the Study Directors labora- (116%), fat as measured by ether extract (120%), crude pro-
tory was substituted for the missing research laboratory and a tein (140%), and starch (050%). The diverse chemical com-
volunteer feed company laboratory was substituted for one of position of the materials used as blind duplicates permitted
the feed testing laboratories (Table 1). Participants received evaluation of most, if not all, possible interactions of feed
no compensation. Each laboratory provided reflux apparatus, composition on aNDF analysis. Blanks were included to eval-

Table 2. Materials used in the study, representing full range of fiber concentrations and types of animal feedsa
Material Description DM, % Ash, % EE, % CP, % Starch, % NFC, % ADF, % aNDFom, %

Alfalfa silage Forage, fermented legume 91.0 9.7 3.0 20.1 0.2 20.1 37.4 38.1
Brewers grains Concentrate, heated by-product 92.6 5.1 6.3 24.7 2.8 9.2 20.0 47.2
Citrus and beet pulp Concentrate, pectic by-product 91.4 7.8 3.9 7.3 1.3 45.7 25.4 26.7
Corn grain with cob Concentrate, containing starch 92.4 1.3 4.5 6.4 50.4 59.6 10.6 20.6
Corn silage Forage, grass with starch 92.3 3.8 5.0 6.5 33.4 41.5 22.3 35.4
Corn stalks Forage, high-fiber grass 92.0 7.4 1.0 4.1 0.2 11.2 46.5 68.2
Dairy mixed feed Concentrate, mixture with fat 92.6 16.1 13.5 39.8 3.9 11.3 7.4 11.9
Grass hay Forage, temperate grass 92.0 8.6 5.4 8.5 0.5 14.1 38.1 55.5
Milk replacer Concentrate, animal fat 93.5 7.0 17.6 18.6 0.6 50.2 0.3 0.1
Roasted soybeans Concentrate, plant fat 93.9 5.2 18.0 38.2 2.6 19.5 6.2 13.0
Sawdust Reference, high cellulose 92.5 1.6 0.8 0.6 0.6 0.4 77.6 89.1
Blank No material

a
Dry matter (DM), ash, ether extract (EE), crude protein (CP), starch, nonfibrous carbohydrates (NFC = DM ash EE CP aNDFom), acid
detergent fiber (ADF), and amylase-treated neutral detergent fiber organic matter (aNDFom) of materials used in the collaborative study.
1220 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

Table 3. Homogeneity of aNDFom (blank-corrected) for 4 sample sets for each material analyzeda

Material n Mean, % sr sR RSDr, % RSDR, % 2.8 sr 2.8 sR HORRAT

Alfalfa silage 4 38.1 0.50 0.52 1.31 1.37 1.40 1.46 0.59
Brewers grains 4 47.2 0.46 0.59 0.98 1.24 1.30 1.64 0.55
Citrus and beet pulp 4 26.7 0.36 0.50 1.33 1.87 1.00 1.40 0.77
Corn grain with cob 4 20.6 0.65 0.65 3.17 3.17 1.83 1.83 1.25
Corn silage 4 35.4 0.18 0.34 0.51 0.97 0.51 0.96 0.42
Corn stalks 4 68.2 0.82 0.84 1.20 1.23 2.29 2.35 0.58
Dairy mixed feed 4 11.9 0.30 0.31 2.52 2.57 0.84 0.86 0.93
Grass hay 4 55.5 0.22 0.42 0.39 0.76 0.61 1.18 0.35
Milk replacer 4 0.1 0.29 0.32 373.29 409.78 0.81 0.89 69.70
Roasted soybeans 4 13.0 0.36 0.53 2.76 4.07 1.01 1.49 1.50
Sawdust 4 89.1 0.56 0.60 0.63 0.68 1.58 1.69 0.33
Blank, g 4 0.0009 0.0012 0.0015 0.0035 0.0042

a
sr = Standard deviation of replicated analyses within sample set; sR = standard deviation within and among sample sets; RSDr = repeatability
relative standard deviation; RSDR = reproducibility relative standard deviation.

uate weighing technique and determine if blank-correction
improved precision.
Single batches of homogenous materials were obtained from
commercial feed suppliers or the U.S. Dairy Forage Research
Center (Madison, WI). Materials were dried as needed at 55C
and were ground through a 1 mm screen with a Wiley cutter
mill, and thoroughly mixed. Forty sets of samples containing
about 2 g of each of the 11 materials were prepared. Samples
were measured into glass vials that were sealed in mois-
ture-proof pouches for storage until shipment. All samples were
numbered in the order in which they were to be analyzed.
Homogeneity of sample sets was verified by selecting 4 sets
of samples at random for each material and analyzing them in
duplicate in the Study Directors laboratory to provide an esti-
mate of random variation within and among sets of samples.
The results of the homogeneity study were analyzed by using
the AOAC spreadsheet statistical software recommended for
collaborative studies to determine among-sample set instead of
among-laboratory variation (Table 3). For most materials, the
variation between duplicate analyses within a sample set (sr)
was similar to the variation between samples sets (sR). This
analysis allows variability in homogeneity of sample sets to be
compared directly with the variation among collaborating labo-
ratories. Except for milk replacer (MR), which had an aNDF
concentration near zero, the Horwitz ratio (HORRAT; 12) of
observed versus expected relative standard deviations (RSDs)
among results was below the acceptable threshold of 2.0. For
most samples, the HORRAT was between 0.33 and 0.58.
Within the Study Directors laboratory, the aNDF variation was
similar to that of other AOAC Official Methods. The HORRAT
values for roasted soybeans (RS), corn grain (CG), dairy mixed
feed (DM), and citrus and beet pulp (CB) were between 0.80
and 1.50, suggesting that these materials were less homoge-
neous or more difficult to analyze for aNDF.
Familiarization samples were sent to each collaborator in a
preliminary study to acquaint them with the method and eval-
uate their ability to handle difficult samples. Familiarization
materials included alfalfa silage (AS), barley hay (BH), CG,
meat and bone meal (MB), and RS. The latter 3 materials were
selected to represent materials that were extremely difficult to
analyze for aNDF.
Heat-stable amylase is a critical reagent in the aNDF
method. To verify that alternative sources of amylase can be
standardized and used, 3 amylase stock solutions from
2 sources were standardized by each collaborator during the
familiarization study, and half of the laboratories were as-
signed to use one of the 2 sources during the collaborative
study (Table 4).
Statistical Analyses
The experiment was designed as a randomized complete
block design using blind duplicates of 11 unidentified materi-
als. Although feeds were not identified, milk replacer, dairy
feed, and roasted soybeans were labeled to indicate >10% fat
in the first blind duplicate, but not the second. Only one ana-
lyst from each laboratory was asked to submit results, and
blind duplicates were analyzed on the same day within the
Table 4. Sources of stock heat-stable a-amylases
evaluated and used in the study
Amylase type Source Dilution
Taka-Therm L-340 ANKOM Technology Full strength
Corp. (Fairport, NY)
Termamyl 120 L Novo Nordisk Biochem Full strength;
(Raleigh, NC) one-half strength
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1221
same run. Block was defined as first replicate, second repli-
cate, and rerun analyses using modifications. In the first repli-
cate, all laboratories were asked to analyze one duplicate of
each of the samples in a fixed order of decreasing filtration
ease. In the second replicate, one of 12 random orders of anal-
ysis for the samples was assigned to each laboratory. Ordering
samples in the first replicate was used to determine the effect
of a difficult-to-filter sample on the subsequently analyzed
samples. The statistical model for analysis of variance
(ANOVA) was:
Y = + Material + Lab + Block + Material*Lab + error (1)
where = the mean, Material = variation due to materials, Lab =
variation among laboratories, Block = variation due to replicate
or rerun, Material*Lab = variation due to Material by Laboratory
interaction, and error = random variation within laboratory.
Block was included in the model to test the effect of order of
analysis and was removed from the model when not significant.
There are several procedures in the aNDF method that may
not be followed exactly among laboratories. For example, identi-
fying the proper level of amylase to use when determining aNDF
is an integral part of the method because the source and level of
enzyme is not specified in the method. A portion of the collabo-
rative study was designed to evaluate the effects of amylase
source (A or B) and dose (independently determined by each col-
laborator). The effect of source and dose of amylase was evalu-
ated on all materials and only on materials containing significant
starch [corn silage (CS) and CG] using the ANOVA model:
Y = + Material + Amylase + Dose + Amylase*Dose + error (2)
where Amylase = variation due to source of amylase, Dose
and Amylase*Dose = variation explained by linear regression
of amylase dose for each source, and all other variables as de-
scribed previously.
Because aNDF is an empirical method it is important to
evaluate the effects of factors that are left to the discretion of
laboratories and may differ among them. Each collaborator
submitted answers to a questionnaire about factors that might
affect results. In addition to amylase source and dose, collabo-
rators were asked to describe for each result the apparatus, fil-
tering aid, and pre-extraction technique used, and to describe
the temperature of washing water and the methods for residue
washing and weighing. This information was evaluated using
appropriate ANOVA models.
Initially, outlying results were detected with spreadsheet
statistical software provided by AOAC for a blind duplicates
design using a 2.5% significance level. The Cochran test iden-
tifies replicate results within a laboratory that are suspect, and
the Grubb test determines if the average result of laboratories
deviates from those of all laboratories. Cycles of Cochran, sin-
gle Grubb, and pair Grubb tests were used to identify outliers
until no additional removal was necessary or no more than 2/9
of the laboratories were flagged. The ANOVA identified
which of the 2 blind replicates was the outlier by using a main
effects ANOVA with no interaction terms:
Y = Material + Lab + Block + error (3)
Predicted results were compared with observed results, and
the replicate with the largest residual deviation was identified
as the outlier. Laboratory ranking scores (after removal of in-
dividual replicate outliers) were used to identify laboratories
that were outliers across all materials according to
Wernimont (13). All outlying results were checked for calcu-
lation and data entry errors, and answers to the questionnaire
were scrutinized to determine if the collaborators procedure
differed from the aNDF method that was being evaluated.
Within laboratory repeatability (sr), among laboratory sys-
tematic variation (sL), and total reproducibility variation (sR;
where sR2 = sr2 + sL2) associated with a single analysis were
determined by approaches described by Youden and
Steiner (14) and Wernimont (13) with spreadsheet statistical
software recommended by AOAC. Statistical results were de-
termined for individual materials. In addition, ANOVA using
the interaction model (Model 1) was used to determine if re-
sults from all materials or classes of materials (forage, concen-
trates with <10% fat and concentrates with >10% fat) could be
pooled. If the interaction term in Model 1 was not significant,
it was assumed that the method obtained consistent results
among materials, and pooled precision statistics were calcu-
lated according to Wernimont (13).
True values cannot be determined for fiber because the em-
pirical method defines the fraction. Therefore, there is no direct
measure of systematic bias (accuracy) for fiber, and consensus
values derived from the collaborative study were used as the
point of reference. As suggested by Youden (15), variability in
systematic bias was equated with among-laboratory variability
(sL) that does not include within-laboratory variability. Repeat-
ability (sr) is the within-laboratory precision that was calculated
from variation between blind duplicates within laboratories.
Reproducibility (sR) is the total variation associated with a
single analysis, which is the sum of systematic bias and re-
peatability, i.e., sR2 = sr2 + sL2. Repeatability within a labora-
tory is expected to be 1/22/3 of the total reproducibility varia-
tion among laboratories. Although specific criteria for the
acceptability of a fiber method cannot be imposed, it is ex-
pected that the variation among laboratories will be a function
of aNDF concentration. The ratio (HORRAT) of the observed
reproducibility relative standard deviation (RSDR) divided by
the expected RSD from the Horwitz equation (16) was used to
evaluate the acceptability of the method. A HORRAT <2 was
used as an indicator of acceptability (12).
AOAC Official Method 2002.04
Amylase-Treated Neutral Detergent Fiber in Feeds
Using Refluxing in Beakers or Crucibles
First Action 2002
[This method is applicable for the determination of amy-
lase-treated neutral detergent fiber (aNDF) in forages, grains,
grain by-product feeds, animal by-product feeds, oil seeds,
oilseed meals and mixed feeds that range in concentration
from 1.5 to 100%.]
1222 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
Caution: Enzyme (amylase) preparations can cause allergic
reactions in hypersensitive individuals; avoid in-
haling aerosols or dusts. Cover eyes, nose, mouth,
and exposed skin as needed to prevent irritation.
Sodium lauryl sulfate is mildly irritating to mucous
membranes and may cause allergic reactions in hy-
persensitive individuals. Do not inhale or allow
material to contact skin or eyes. Wear a dust mask
and gloves while handling. Triethylene glycol is
harmful if inhaled, ingested, or absorbed through
the skin. Vapors or mists are irritating to the eyes,
mucous membranes, and upper respiratory tract.
Contact is irritating to the skin. Wear appropriate
clothing and mix in a hood or well-ventilated area.
Acetone is a flammable solvent; do not use near
open flames. Use in an effective fume hood and do
not let vapors accumulate in work area. Avoid in-
haling or contact with skin. Evaporate all traces of
acetone before placing fiber residues into an oven.
Avoid skin contact with hot neutral detergent solu-
tion and reagents. Maintain all electrical equipment
in suitable working order and ensure that it is prop-
erly grounded.
See Table 2002.04 for the results of the interlaboratory
study supporting acceptance of the method.
A. Principle
Fiber in feeds is a nutritionally defined entity that repre-
sents the indigestible and slowly digesting fraction of feeds.
Neutral detergent (ND) solution and heat-stable -amylase
are used to dissolve easily digested proteins, lipids, sugars,
starches, and pectins in feeds, leaving a fibrous residue that is
primarily cell wall components in plant materials (cellulose,
hemicellulose, and lignin) and indigestible nitrogenous matter
in animal products. aNDF is a gravimetric method that best es-
timates the total insoluble fiber, and is inversely related to di-
gestibility and intake potential of a feed. ND soluble matter is
almost completely digested by most animals; however, the di-
gestibility of aNDF is variable among feeds and is related to
lignin and other constituents in fiber.
Sodium lauryl sulfate, an anionic detergent, and sodium sul-
fite are used to solubilize nitrogenous matter; EDTA is used to
chelate calcium and enhance removal of pectins at boiling tem-
peratures; triethylene glycol helps remove some nonfibrous
matter from concentrated feeds; disodium phosphate and so-
dium borate are used as buffers to maintain a neutral pH.
Hot ND solution has limited ability to solubilize starch;
therefore, a heat-stable amylase is used to hydrolyze starch to
saccharides that can be easily removed from fiber by filtration.
Heat-stable amylases are used in hot solutions to inactivate
potential contaminating enzymes in crude amylase extracts
that might degrade fibrous constituents. To ensure that the am-
ylase activity is sufficient to remove most starch and to reduce
filtering difficulties, the amount of any specific amylase
source needed to measure aNDF is determined under the con-
ditions of the aNDF method.
Although boiling ND solution dissolves most proteins, lipids,
and nonfibrous carbohydrates, these constituents are nonviscous
only in water that is near boiling temperature. Therefore, boiling
water is needed for washing fibrous residues and removing
nonfibrous matter. Because fiber is particulate matter, mass-ac-
tion equilibration during soaking is needed to migrate ND and
contaminating soluble matter from the interior of fiber particles.
Fibrous residues must be soaked, instead of being simply rinsed,
in near boiling water to remove nonfibrous matter.
Boiling ND solution solubilizes lipids, and acetone soak-
ing of the residue completes the extraction of lipids and pig-
ments in most materials. However, excessive amounts of
lipids in materials can complex with the detergent and reduce
extraction efficiency. Because extraction of lipids by ND and
acetone may be incomplete when feeds contain >10% lipid,
these materials are pre-extracted to ensure complete removal
of lipid contamination from fiber. Pre-extract materials with
510% lipid to minimize filtration difficulties and avoid
variable aNDF results.
B. Apparatus
(a) Refluxing apparatus.Any conventional apparatus
suitable for crude fiber or acid-detergent fiber determinations.
Test samples should be extracted in 500 or 600 mL beakers
without spouts (Pyrex No. 1040, or equivalent) that are covered
with a round cold-water condenser to minimize evaporation.
Calibrate heating unit setting so that 50 mL water boils within
45 min when cold water condensers are used. Fibertec appara-
tus 2010 or M6 (Foss North America, Eden Prairie, MN 55344)
can be used and should boil 50 mL water within 10 min.
(b) Fritted-disk Gooch crucibles.Coarse porosity (pore
size 4060 m) crucibles, high-form, 4050 mL capacity or
Fibertec USP2 (pore size 4090 m, 2628 mL capacity).
Clean new crucibles and ash at 500C for 1 h. Clean crucibles
after each use by ashing at 500C for 3 h, removing ash, in-
verting in detergent solution, and sonicating for 710 min.
Rinse crucibles in hot water, and soak in room temperature
water for at least 30 min. Back-flush crucibles by connecting
top of each crucible to a No. 9 stopper or a No. 9 stopper fit-
ted with a No. 4 filter adapter (Cat. No. 24035-087; VWR,
West Chester, PA 19380, www.vwr.com) that has a port that
is connected to a trap and vacuum line. Back-flush each cruci-
ble with water by rapidly plunging and removing the bottom
of the crucible into water to create a vigorous rinsing action.
Occasionally test filtration rate as follows: Fill each cruci-
ble with 50 mL distilled water (25 mL for Fibertec USP2 cru-
cibles) and record time required to drain completely without
vacuum (should be 180 60 s for Gooch or 75 30 s for
USP2). If <100 s (or <35 s for USP2), discard crucible. If
<120 s (or <45 s for USP2), check for cracks in fritted disk. If
filtration >240 s (or >105 s for USP2), clean crucibles with
slow filtration rates using acid or alkaline cleaning solutions.
(c) Vacuum filter manifold.Any apparatus similar to that
described by Mertens et al. (17) or suitable alternative that al-
lows adequate soaking of fibrous residues (Figure 2002.04A).
Manifold should provide vacuum-tight seal with crucible to re-
duce foam formation in vacuum lines. Use thick-walled vac-
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1223

Table 2002.04. Interlaboratory study results for amylase-treated neutral detergent fiber in feeds
Feed Fiber Mean, % sr sR RSDr, % RSDR, % ra Rb

Forages aNDFc 52.2 0.84 1.10 1.61 2.10 2.36 3.08

d
Forages aNDFbc 52.2 0.84 1.08 1.61 2.08 2.36 3.03
Forages aNDFome 50.4 0.93 1.18 1.85 2.34 2.61 3.31
f
Forages aNDFombc 50.1 0.93 1.16 1.85 2.32 2.60 3.26
Concentrates <10% fat aNDF 33.5 1.18 1.42 3.51 4.25 3.29 3.98
Concentrates <10% fat aNDFbc 33.2 1.25 1.57 3.76 4.72 3.50 4.38
Concentrates <10% fat aNDFom 32.5 1.16 1.46 3.56 4.48 3.24 4.08
Concentrates <10% fat aNDFombc 32.2 1.14 1.47 3.55 4.56 3.20 4.11
Concentrates >10% fat aNDF 8.7 1.01 1.61 11.66 18.57 2.83 4.50
Concentrates >10% fat aNDFbc 8.7 0.79 1.24 9.06 14.26 2.21 3.47
Concentrates >10% fat aNDFom 8.8 0.58 1.20 6.54 13.68 1.61 3.37
Concentrates >10% fat aNDFombc 8.5 0.53 1.10 6.25 12.94 1.49 3.09
All materials aNDF 38.7 1.05 1.33 2.72 3.44 2.94 3.72
All materials aNDFbc 38.6 1.02 1.28 2.64 3.32 2.86 3.59
All materials aNDFom 37.7 1.02 1.28 2.70 3.39 2.85 3.58
All materials aNDFombc 37.4 1.00 1.24 2.67 3.32 2.80 3.48

a
2.8 sr.
b
2.8 sR.
c
Amylase-treated neutral detergent fiber (as-received basis).
d
Amylase-treated neutral detergent fiber, blank-corrected (as-received basis).
e
Amylase-treated neutral detergent fiber organic matter (as-received basis).
f
Amylase-treated neutral detergent fiber organic matter, blank-corrected (as-received basis).

uum tubing to connect manifold to trap (418 L) and vacuum
source. Place a vacuum reservoir (18 L) between trap and vac-
uum source to ensure adequate vacuum capacity to remove
foam. Fibertec apparatus may be used for filtration.
(d) Boiling water supply.Use continuous boiling water
generator as described by Goering and Van Soest (9) or suit-
able alternative (Figure 2002.04B). Apparatus must be capa-
ble of supplying boiling water (>95C) in quantity sufficient
for all residues to be washed at one time through a nozzle pro-
ducing a fine stream (flow rate, 3540 mL/10 s; a 2.5 mL dis-
posable plastic pipet tip makes an acceptable nozzle). A fine
nozzle minimizes water needed to transfer particles to cruci-
ble, but provides water pressure needed to remove residues at-
tached to side of beaker. It is critical that water is boiling when
added to crucibles, especially for products containing starches,
pectic substances, mucilages, or glycoproteins. (Fibertec users:
Use syringe with cone-spray nozzle to rinse condensers and
60 mL disposable syringe with 12 gauge needle 10 cm long to
dislodge any residues adhering to condensers.)
(e) Wash bottle for ND solution.Rinse down particles
that are carried up the side of beakers by foaming during
refluxing. Use fine nozzle to minimize addition of ND solu-
tion during rinsing (flow rate, 1015 mL in 3 s with moderate
pressure; unclipped nozzle on 500 mL Nalgene Unitary wash
bottle or equivalent is acceptable). (Fibertec users: Use ex-
tended nozzle with larger opening to adequately rinse particles
into ND solution during extraction.)
C. Reagents
(a) Sodium sulfite.Na2SO3 anhydrous.
(b) Dried hominy corn (corn grits, raw).Obtained from
food or grocery store. Must be raw or uncooked grits; pre-
cooked or instant grits are not acceptable. Grind grits to pass
1 mm screen in a Wiley cutter mill.
(c) Burkes iodine solution.2 g KI and 1 g I in 100 mL
water. Store in amber or opaque bottle.
(d) Stock heat-stable a-amylase solution.Termamyl
120 by Novozyme Corp. (Cat. No. A3403, Sigma-Aldrich, St.
Louis, MO 63178), Spezyme FRED by Genecor International
(Cat. No. FAA, Ankom Technology Corp., Fairport, NY
14450) or equivalent liquid heat-stable -amylases or water
extracts of heat-stable -amylase powders.
(e) Working heat-stable a-amylase solution.Standard-
ize heat-stable -amylase stock solution or enzyme powder
extract so that 2 additions of 2 mL will remove raw corn starch
from 0.5 g of corn hominy grits. Assay as follows:
(1) Weigh 0.5 g (0.005 g) ground, dried hominy corn into
each of 6 beakers similar to those used to extract fiber residues
described in B(a).
1224 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

Figure 2002.04A. Schematic drawing of vacuum filter manifold: (A) side view; (B) top view; and (C) end view (ref. 17).
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1225
Figure 2002.04B. Water heating unit. Heating unit on
right consists of a 3 L round flask modified to contain 2
standard taper female joints for cold water condensers
at the top and a glass column (6 cm diameter, 17 cm
length) at the bottom to accommodate a 1000 watt quartz
immersion heater, 455 mm long and 19 mm diameter
(Cat. No. 16790-1L, Vycor sheathed heating element,
Corning Glass, Corning, NY, vycor@corning.com, or
equivalent), inserted through the original joint at top.
Cold water enters through a port at the bottom of the
column and leaves through a port in the upper 2/3 of the
round flask. The tank at the left contains a float valve
adjusted to maintain a water level in the heating unit
about 3/4 full.
(2) Preheat calibrated reflux units, prepare ice bath for
cooling beakers (must contain enough ice to maintain temper-
ature below 1C), and prepare tempering bath [shallow pan
containing enough water at exactly 20C to exceed depth of
solutions in beakers and maintain a final temperature between
19.5 and 20.5C at the end of step (8)].
(3) Add 50 mL ND solution, (f), (do not add sodium sul-
fite), swirl beaker, and place on preheated refluxing apparatus
at 1 min intervals.
(4) After ND begins to boil (ca 5 min), add one of 6 doses
stock or powder extract solution (geometric progression, e.g.,
0, 0.025, 0.05, 0.10, 0.20, and 0.40 mL; exact doses will de-
pend on source of amylase) to beakers in ascending order.
(5) Reflux for 10 min, remove at 1 min intervals; add sec-
ond dose of amylase (matching the first), swirl, and rinse sides
of beaker using minimum of room temperature ND solution.
(6) React 60 s and filter through glass wool or 2 layers of
cheesecloth into 100 mL glass beaker. Prepare blank by add-
ing 2 intermediate doses, e.g., 0.10 and 0.20 mL stock solu-
tion, to 40 mL room temperature ND in 100 mL glass beaker.
(7) Place beakers, except blank, in ice bath. Remove from
ice bath after 5 min (temperature of solutions should be ca
21C) and place all beakers in tempering bath (20C).
(8) After solutions are 20 0.5C (may take 5 min or
more), remove beakers from tempering bath and arrange in or-
der of increasing enzyme doses on white background.
(9) Quickly add 0.5 mL Burkes iodine solution to beak-
ers, and mix.
(10) Do not look at beakers. After 90 s, view through solu-
tions from above and make a quick decision (within 30 s)
about color of each solution, using the following scale: Purple
= not adequate enzyme; pink-amber or amber = not adequate
enzyme; pale yellow = adequate enzyme.
Compare to blank. Brown tint of enzyme solution should
not be confused with pink-amber or amber. If color differ-
ences are unclear, place beakers in tempering bath for 5 min
and repeat steps (8)(10).
(11) After lowest dose that is pale yellow (V2) and next
lowest dose (V1) that is pink-amber or amber are identified us-
ing a geometric progression of doses, do final standardization
using the dose below the pink-amber one (V1) and a linear pro-
gression of doses (0.25*V1) between V1 and V2 (e.g., if
0.05 mL treatment is amber [V1] and 0.10 mL treatment is pale
yellow [V2], use doses of 0.025, 0.05, 0.0625, 0.075, 0.0875,
and 0.10 mL) stock solution in the final standardization.
(12) The lowest dose that is pale yellow (and exceeds next
highest inadequate dose with pink-amber or amber solution)
represents the volume of amylase stock solution or extract
(Vs) used to make amylase working solution.
(13) Record date, batch or lot of amylase, doses tested,
amount of iodine solution used, and color and final tempera-
ture (before iodine addition) of each dose in reagent log book.
(14) Determine number (n) of test samples to be analyzed
in the following 5 days or less. To add 2 mL amylase working
solution twice for each test requires a total volume of amylase
working solution of (n) 4 mL. Mix (n) 2 (Vs) mL amy-
lase stock solution with (n) [4 2 (Vs)] mL water. Store
working amylase solution in refrigerator using stoppered con-
tainer no longer than 5 days.
(15) Confirm adequacy of amylase working solution by re-
peating standardization procedure using 0.5 g corn grits with 0,
2, and 4 mL working solution (each added at boiling and after
removal from refluxing apparatus). If there is no appreciable
difference in color between 2 and 4 mL working solution, then
2 additions of 2 mL are adequate for the aNDF method.
Notes: Time and temperature are critical for proper assess-
ment of adequate amylase dose. The solutions must be 20C
and the decision about color must be made within 90120 s af-
ter addition of iodine solution. Pink-amber or amber colors
fade quickly, and waiting longer than 120 s before making a
decision will result in a dosage that is too low.
Initial doses of stock solution or extract may have to be ad-
justed, and the standardization rerun. If maximum dose
(0.4 mL) results in purple or pink-amber color, extend the geo-
metric progression of doses starting at 0.4 mL, and rerun ini-
tial standardization. If minimum dose (0.025 mL) is yellow,
decrease the geometric progression to between 0 and
0.025 mL, and rerun initial standardization.
Each new source or lot of enzyme should be standardized; a
single lot that is being used over a period of time should be
1226 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
checked every 6 months for activity. Excess enzyme is not ben-
eficial and can be detrimental. Concentrated enzyme solutions
are not recommended as working solutions because a 1 drop er-
ror when dispensing enzyme contains significant activity that
can affect results. Many amylase extracts are crude mixtures
that may contain fibrolytic and proteolytic activities. Heat-sta-
ble amylase solution must be used in hot liquids (>80C) to in-
activate contaminating enzymes and minimize fiber loss.
(f) Neutral-detergent (ND) solution.Measure 990 mL
water. Pour ca half of water into a flask, add 4.0 g NaOH,
14.6 g EDTA, 4.56 g dibasic sodium phosphate (Na2HPO4),
6.81 g sodium borate decahydrate (Na2B4O710H2O), and mix
until dissolved (heat if necessary). Under a hood, add 30 g so-
dium lauryl sulfate and of remaining measured water. Mix
until detergent is dissolved and add 10 mL triethylene glycol
to suppress foam. Add remaining water and thoroughly mix.
Verify that pH is between 6.95 and 7.05, and adjust with con-
centrated HCl or NaOH, as required. If pH is off by >0.5, dis-
card. Store ND solution at room temperature or, if cool storage
causes precipitation, warm to 25C, and mix before use. Re-
cord date ND solution was prepared, pH measurements, and
adjustments in reagent log book.
Note: NaOH and EDTA can be replaced with 18.6 g disod-
ium EDTA.
(g) Filter aid.(1) Silica sand.Sand, cristobalite, acid
purified, 40200 mesh (Fluka, Buchs, Switzerland, Cat. No.
84880 or equivalent). (2) Glass microfiber mats.4.25 cm
Whatman GF/D or equivalent.
(h) Crucible cleaning solutions.(1) Acid.Chromic
acid [see Definition of Terms (13)] or prepare from 2.5 L
H2SO4 and one package Nochromix (Godax Laboratories,
Inc., Takoma Park, MD 20912, www.godax.com), or equiva-
lent inorganic oxidizer. (2) Alkaline.Dissolve 5 g
Na2EDTA2H2O 50 g Na3PO4, and 200 g KOH in 1 L water.
Cleaning procedure.After testing filtration rate of cruci-
bles, place crucibles with slow filtration rates in a shallow
glass or enamel pan and add ca 40 mL acid cleaning solution
to each crucible. Let acid cleaning solution filter through cru-
cible and soak for 1 h. Rinse with water by back flushing and
retest filtration rate. Clean crucibles with slow rates with the
alkaline cleaning solution. Place crucibles in a shallow pan
and add 50 mL alkaline cleaning solution and heat to
7080C. Let alkaline cleaning solution filter through the cru-
cible and then back flush each crucible until it is one-half full
of solution. Let solution filter through crucible and back flush
crucible until it is one-half full. Let solution filter through cru-
cible. Remove from alkaline solution and back flush each cru-
cible with water. After it has cooled, alkaline cleaning solution
can be saved and reused. Use alkaline cleaning solution spar-
ingly because it dissolves glass and weakens the fritted disks
in crucibles. Retest crucible filtration rate; do not use those
with slow rates for aNDF analysis.
D. Test Sample Preparation
Dry wet laboratory samples at <60C to prevent creation of
artifact fiber. Fiber extraction is affected by particle size of test
sample. Grind representative samples to obtain geometric
mean particle size of 220260 mm. A 1 mm screen in a cutter
mill (Wiley) or 2 mm screen in cyclone or centrifugal mill is
generally acceptable. Cyclone or centrifugal mills throw parti-
cles through the screen at an angle, which effectively reduces
the size of the aperture that particles pass through. On material
ground to finer particle size, fiber must be determined by
microfiber filter mats to minimize particle loss; however, re-
sults may have negative bias. Grinding segregates the
material, with highest fiber material passing out of grinder
last. Do not discard material in grinder; combine it with mate-
rial in grinder receptacle. Mix ground material by placing on
creased sheet of paper (ca 40 40 cm). Lift corners of paper to
roll material to diagonal corner, turn paper 90, and roll; repeat
10 times. Transfer material to suitable container.
Ground test samples can be pre-extracted with acetone or
ether to remove fat, but do not dry at >60C. Products contain-
ing >5% fat should be pre-extracted; those with >10% fat must
be pre-extracted to remove fat. To pre-extract with acetone,
weigh test portion into crucible, place on filtering manifold,
extract with 4050 mL acetone 4 times (allow material to soak
at least 5 min, and stir 3 times during each soaking), apply vac-
uum to remove traces of acetone, air-dry for 1015 min, en-
sure that all traces of acetone are removed, and transfer to re-
flux beaker. Use same crucible to collect fiber residue for test
portion after ND extraction. If filtering aid is used, dry and
weigh it with crucible, and then transfer it to a small beaker
before test sample is weighed into crucible. After pre-ex-
tracted residues have been transferred from the crucible into
the refluxing beaker, replace filtering aid in crucible before fil-
tering ND-extracted residues. [Fibertec users: Add silica sand
to USP2 crucible, dry and weigh it before adding test sample;
then pre-extract with 2030 mL acetone 4 times (allow mate-
rial to soak at least 5 min and stir with back pressure 3 times)
using cold filtration device. After removing all traces of ace-
tone, transfer crucible containing silica sand and pre-extracted
test portion to the hot extraction device for ND extraction.]
E. Determination
Dry at 105C for >4 h and weigh empty crucibles (hot di-
rectly from oven or room temperature after desiccation). Re-
cord empty crucible weight for test portions (We) or blanks
(Be) to nearest 0.0001 g. Mix material thoroughly and weigh
0.5 (0.0500) g air-dry feed, or equivalent amount of wet ma-
terial into refluxing beaker. (Fibertec users: Weigh test
portion into dried and preweighed USP2 crucible.) Record fi-
nal weight of test portion to nearest 0.0001 g (S). If results are
to be reported on dry matter basis, weigh a second test portion
at the same time for dry matter determination. Include
in-house reference sample and 2 blanks for first 2030 test
samples in a run and add one reference and one blank for each
additional 2030 test samples.
Preheat calibrated reflux units. Add 0.5 (0.1) g sodium
sulfite using calibrated scoop (EKCO Housewares Pinch
measuring spoon, World Kitchen, Inc., Elmira, NY,
www.worldkitchen.com or equivalent) and 50 (5) mL ND to
each refluxing beaker and swirl (critical for starchy feeds that
stick to bottom during refluxing). (Fibertec users: Use back
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1227
pressure to mix reagents.) Do not add ND and sodium sulfite
to test portions more than 60 min before refluxing. Heat to
boiling within 45 min, add 2 mL working amylase solution,
resuspend particles stuck to bottom or sides, and swirl.
(Fibertec users: Use back pressure to mix amylase with ND
solution and test portion.)
Reflux for 60 min at boiling temperature that creates vigorous
particle movement, but not excessive foaming that carries parti-
cles up the side of the beaker. Mixtures may foam vigorously for
12 min (do not reduce temperature of heating unit). Rinse sides
of beaker with minimum amount of ND using bottle with fine
nozzle 510 min after amylase is added, and rinse as needed to
resuspend particles on side of beaker (twice maximum).
Remove extracted mixture from heating unit and let parti-
cles settle for 3060 s. Before transfer (Fibertec users: Before
initial filtration), observe mixture to determine if lipid globules
are present on surface or if solution is milky, which indicates
that test sample should be rerun after acetone pre-extraction.
Place Teflon stirring rod in crucible and preheat by adding
40 mL boiling water for 3060 s. (Fibertec users: Ignore.)
Remove water with vacuum, and immediately decant top
3040 mL of solution, keeping beaker inverted over crucible.
(Fibertec users: Ignore.) Use minimum vacuum to evacuate ex-
cess liquid, but close vacuum before residue becomes dry.
Note: Excessive vacuum and evacuating to dryness cause some
residues to clog crucible and not wash properly. Rinse all unat-
tached particles into crucible using fine stream of boiling water.
(Fibertec users: Ignore.) Fill crucible half full with hot water.
Add 2 mL working amylase solution and stir. (Fibertec users:
Use back pressure to mix amylase in initial water soak.)
React with amylase for minimum of 4560 s while scrap-
ing remaining particles from bottom and sides of reflux beaker
with rubber policeman. Evacuate amylase solution and trans-
fer any remaining residue from reflux beaker into crucible
with 2030 mL boiling water. Two rinses are usually suffi-
cient. After transferring residues from beaker, fill crucible 3/4
full with boiling water and soak for 13 min. (Fibertec users:
Remove amylase-water soak after minimum reaction of 60 s.
Crucibles can be removed from hot to cold filtration unit for
remaining hot water soaks for residues that are easy to filter.
This allows next set of test samples to begin ND extraction on
hot filtration unit. Residues that are difficult to filter can be
washed on Fibertec heating unit with heat reduced to mini-
mize particle agitation.)
Evacuate water, add 4050 mL boiling water, soak
35 min, and repeat. If residues are difficult to filter after first
soak, add additional 2 mL working amylase solution. If resi-
dues appear translucent and become more difficult to filter
with each additional soaking, eliminate third water soak. If
plugged, crucible can be back-flushed by removing it from fil-
ter manifold and reinserting it. (Fibertec users: Use minimal
back pressure to open crucibles and improve filtration.)
Evacuate water, refill crucible with 4050 mL acetone, stir
to disperse particles, soak 35 min, and repeat, rinsing stir rod
to remove attached fiber particles. (Fibertec users: Move cru-
cibles to cold extraction unit for acetone soaks.) Note: After
the last water soak, do not evacuate fiber residues to dryness,
but remove water to leave a damp or moist residue, before
adding acetone. Excessive evacuation clumps the residues and
makes acetone extraction difficult.
Vacuum residue dry, remove crucible from manifold, and
air dry for 1060 min to remove acetone. Dry crucibles at
105C for minimum of 8 h and weigh (Wf and Bf). Ignite cruci-
ble and fiber in 500C furnace for 5 h or until C-free. Temper in
105C oven for at least 1 h and weigh (Wa and Ba). Weigh cru-
cibles containing fiber or ash residues (hot or desiccated to
room temperature) in same order as empty crucibles.
Modifications for specific types of test samples.(a) If
extracted ND solution appears milky and opaque and filtration
is slow during transfer of residues or after first water soaking,
high starch is suspected. Add additional treatment with 2 mL
amylase during second water soaking. Shorten soaking times to
minimum to keep soaking solutions as hot as possible (>85C).
(b) If residue clogs crucible during transfer and additional
amylase does not improve filtration, feed material may contain
proteinaceous, gum, or mucilage residues (meat products and
some oil seed meals). Preheating crucible with boiling water is
crucial for filtering these materials. The best filter aid for these
materials is 1215 g (68 g for Fibertec USP2) of silica sand,
C(g)(1). The gummy substances in these feed materials will
stick to sand particles which prevents them from clogging the
fritted disk and allows residues to be washed. All filter aids
must be added to crucibles (including blanks) before initial
weights are recorded.
(c) If fiber residue has glossy, translucent sheen and filtra-
tion becomes more difficult with each water soaking, pectic
substances are suspected. Preheat crucible with boiling water
and transfer residues as quickly as possible without settling
when removed from reflux unit. Reduce all soaking times to
minimum to maintain temperature >85C to prevent cooling
and jelling in crucible. Filtering aids may improve filtration (in
order of preference): 1215 g (68 g for Fibertec USP2) silica
sand, 0.25 g (0.15 g for Fibertec USP2) glass wool, and glass
microfiber mats, C(g)(2).
(d) If fat globules are observed floating on surface of ND or
wash water and residue is difficult to filter, or if material is known
to contain >10% fat, pre-extract it with acetone or ether (see D).
(e) If material contains fine particles, flocculant precipitates,
dirt (fine clay), or fecal matter, but not pectic substances or
starch, increase settling time to maximum of 2 min after removal
from refluxing unit and use filter aid in crucible. Filter aids (in or-
der of preference) include glass microfiber mats, ceramic fiber,
1215 g silica sand, and 0.25 g glass wool. Microfiber mats can
be gently scraped to renew surface during filtration.
(f) If all other modifications fail, reduce test portion amount
to 0.3 g and repeat analysis with filter aid in crucible. Reducing
test portion will magnify effects of weighing errors and increase
variation in results. Sometimes reducing test portion amount
and increasing ND to 70100 mL is beneficial. If fiber is
<1.5%, do not reduce test portion amount, and if filtration is not
possible, report results as difficult to analyze, fiber <1.5%.
(g) Do not add acetone before all rinse water has been re-
moved. Although this occasionally improves filtering, it does
not remove detergent or detergent solubles from residues.
1228 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
Adding acetone before water washing is complete will give
inflated fiber values.
F. Calculations
Without blank correction:
% aNDF (as-is basis) = 100 (Wf We)/S
where aNDF is amylase-treated neutral detergent fiber and S
is g as-is test portion weight.
% aNDF (DM basis) = 100 (Wf We)/(S*DM)
where DM is (g oven-dried matter weight/g air-dried or wet
test portion weight).
% aNDFom (as-is basis) = 100 (Wf Wa)/S
where aNDFom is aNDF organic matter.
% aNDFom (DM basis) = 100 (Wf Wa)/(S*DM)
With blank correction (average all blanks before calculat-
ing results):
% aNDF (as-is basis) = 100 (Wf We Bf + Be)/S
where aNDF is amylase-treated neutral detergent fiber, S is g
as-is test portion weight, and Bf and Be are averages of all
blanks in the run.
% aNDF (DM basis) = 100 (Wf We Bf + Be)/(S*DM)
where DM is (g oven-dried matter weight/g air-dried or wet
test portion weight).
% aNDFom (as-is basis) = 100 (Wf Wa Bf + Ba)/S
where aNDFom is aNDF organic matter.
% aNDFom (DM basis) = 100 (Wf Wa Bf + Ba)/(S*DM)
Results should be reported to nearest 0.1%, and results
<1.5% aNDF or aNDFom should be reported as aNDF
<1.5% or aNDFom <1.5%.
When fiber is <25%, blank correction is required.
Crucible weights after ashing (Wa) that are less than empty
crucible weights (We) indicate that crucibles lost weight dur-
ing the aNDF procedure. The crucible weight after ashing
(Wa) is the most accurate estimate of the correct crucible
weight for calculating fiber and must be used to calculate
aNDFom. In this circumstance, aNDFom is a more accurate
estimate of fiber than is aNDF.
If results for in-house reference or quality control samples
are outside of control limits, do not report results; re-analyze
test samples.
G. Quality Assurance
Maintain log of reagent preparation and amylase standard-
ization. Check pH of each batch or lot of ND solution and ad-
just as needed. Determine activity of each amylase source and
lot in hot ND solution and adjust amylase working solution ac-
cordingly. Check activity of stock amylase every 6 months
during storage, and adjust working solutions accordingly.
Include at least one in-house reference or quality control
(QC) sample and 2 blanks for the first 2030 test samples in a
run, and add one QC and one blank for each additional 2030
test samples analyzed. Suitable QC materials include brewers
grains, grass hay, or corn silage that has been dried at <60C.
Each of these materials is sensitive to changes in reagents and
technique, but corn silage is preferred because it contains
starch and grass cell walls. Acceptable standard deviation
(SD) among repeated analyses of the reference material
should be 1.00% aNDF. Plot QC sample results on X-control
chart and examine chart for trends. Results outside of upper or
lower warning limits (2.00 standard deviations) are evidence
of possible problems with the analytical system. Results out-
side of upper or lower control limits of 3.00 SDs indicate loss
of QC, and results of the run should be verified. Two consecu-
tive analyses falling on one side of the mean between warning
limits and control limits also indicate loss of QC.
Include at least one set of duplicates in each run if single
determinations are being made. Duplicates should not be run
consecutively, but one replicate should be spaced at the
begining and end of the run. Acceptable differences among
duplicate analyses ranges from 1.50%-units for materials with
<20% NDF to 3.00%-units for materials with >70% NDF.
Change in weights of blank crucibles should be <0.0100 g
after either ND extraction or ashing. If weights blank crucibles
change by >10 mg, or crucible weights after ashing are less
than empty crucible weights, suspect inadequate cleaning of
crucibles or problems with weighing technique.
Ref.: J. AOAC Int. 85, 12211228(2002)
Results and Discussion
Familiarization Study
In the familiarization study, collaborators were provided
6 materials with a mean value and acceptable range for each.
The mean value and acceptable range were determined in the
Study Directors laboratory based on duplicate or triplicate
analyses of 4 randomly selected sets of materials (Table 5).
Except for MB and RS, variation among sample sets was less
than replication variation within sets, as indicated by the ob-
servation that sR was equal to sr. For RS there was a small, but
insignificant variation among sample sets. The variation
among samples sets of MB was about 40% of the
reproducibility standard deviation (RSDR), indicating a lack
of homogeneity among sample sets. The large sr and
HORRAT for MB and RS indicate that samples of these mate-
rials were less homogeneous and more difficult to analyze for
aNDF than other familiarization materials. Collaborators were
asked to analyze the materials using the aNDF method and to
repeat analyses using modifications given in the method until
they achieved acceptable results. The collaborators results in
Table 5 are the best 2 or 3 results selected from all analyses
they performed. However, several participants did not follow
instructions and only provided one set of duplicate results.
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1229

Table 5. Statistical data for 4 random sample sets of materials analyzed in the Study Directors laboratory and the
best 2 or 3 results for collaborator laboratories on materials in the familiarization study
Sample IDa n Mean, % sr sR RSDr, % RSDR, % rb Rc HORRATd

SDBH 4 54.3 0.78 0.78 1.44 1.44 2.20 2.20 0.66

SDAS 4 43.4 0.36 0.36 0.82 0.82 0.99 0.99 0.36
SDCS 4 38.9 0.20 0.20 0.52 0.52 0.57 0.57 0.23
SDCG 4 8.0 0.18 0.18 2.29 2.29 0.51 0.51 0.78
SDMB 4 19.5 1.50 2.42 7.70 12.39 4.20 6.76 4.84
SDRS 4 19.5 0.81 0.88 4.13 4.49 2.26 2.46 1.76
ColBH 13 54.6 0.69 0.98 1.26 1.79 1.93 2.73 0.82
ColAS 13 43.4 0.79 1.06 1.82 2.45 2.21 2.98 1.08
ColCS 13 39.2 0.73 0.85 1.85 2.17 2.03 2.38 0.94
ColCG 12 8.1 0.41 0.62 5.08 7.60 1.16 1.73 2.60
ColMB 11 22.4 2.77 6.68 12.40 29.85 7.77 18.70 11.91
ColRS 10 20.3 1.15 3.29 5.67 16.19 3.23 9.21 6.37

a
SD = Study Directors laboratory; BH = barley hay; AS = alfalfa silage; CS = corn silage; CG = corn grain; MB = meat and bone meal; RS =
roasted soybeans; Col = collaborators laboratories.
b
2.8 sr.
c
2.8 sR.
d
Ratio of RSDR divided by expected RSD from the Horwitz equation (16).

Two forage samples, BH and AS were selected to represent
feeds that were easy to analyze. Deviations from the results of
the Study Directors laboratory were used to determine if labo-
ratories were following the aNDF procedure exactly. The NDF
method is commonly used for feed analysis, and most of the
participating laboratories were determining NDF routinely.
There was concern that these laboratories would use their
method rather than the aNDF method that was being evaluated.
For BH, only one participant exceeded the Study Directors
mean +2.8 sR, and both the mean and sR of all collaborators
agreed closely with those of the Study Director (Table 5). For
AS, the results of the collaborators were more variable than that
of the Study Director. Two laboratories exceeded the Study Di-
rectors mean +2.8 sR, and 2 were less than the Study Direc-
tors mean 2.8 sR. The sR of all collaborators for AS was
nearly triple that of the Study Director, and their HORRAT ap-
proached 2 (Table 5), suggesting that they were not following
the aNDF method exactly. Comments on a questionnaire ask-
ing about specifics of their technique confirmed that collabora-
tors were deviating from the proposed method, and they were
requested to follow the aNDF method exactly.
Two samples, CS and CG, were included in the familiar-
ization set to determine the ability of collaborating laborato-
ries to analyze feeds containing starch. The mean values for
the collaborators were similar to the Study Directors values,
but the collaborators sR was 34 times higher than the Study
Directors (Table 5). Several laboratories had to analyze the
samples numerous times to achieve acceptable results. Some
participants only analyzed the samples in duplicate one time
whether or not they achieved acceptable results. Thus, 4 of the
14 collaborators had unacceptably high values for CS and 3
were unacceptably high for CG. One participant obtained un-
acceptably low values for CS or CG. The high aNDF results
suggest that starch was not adequately removed from fiber
residues. This raised concern that amylase standardization by
the participants was not adequate. However, from question-
naire responses there did not seem to be a relationship be-
tween high results and the dose of amylase used by collabora-
tors. If high results were not related to amylase, this suggests
that filtering difficulties caused problems or that participants
were not following the aNDF method exactly.
Two samples, MB and RS, were known to be extremely
difficult to analyze and were included in the familiarization
study to test the limits of the collaborators ability to handle
difficult samples. The mean results of the collaborators were
higher than those determined by the Study Directors labora-
tory (Table 5). For MB and RS, 3 participants reported unac-
ceptably high values. For RS, 2 collaborators reported unac-
ceptably low values. The sR of the collaborators was 34 times
greater than that observed by the Study Director. Some partici-
pants analyzed these materials 5 or more times without obtaining
acceptable results. Their inability to match the Study Directors
results for CS, CG, MB, and RS convinced most laboratories that
their method of measuring NDF was inadequate. All collabora-
tors were requested to follow the aNDF method exactly when an-
alyzing the collaborative study samples.
It was evident from both the results of the familiarization
study and comments made by the collaborators that the aNDF
method needed to be modified so that laboratories could ana-
lyze extremely difficult samples. After the familiarization
1230 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

study, the aNDF method was modified to improve the analysis Table 6. Doses of 3 amylase stock solutions
of materials that are difficult to filter. Although glass microfiber determined by collaborating laboratories to provide
GF/D mats were used successfully in the Study Directors labo- adequate enzymatic activity for the aNDF method during
ratory, they filtered slowly and were not an acceptable filter aid the familiarization study and actual doses used in the
collaborative study
for some collaborators. Sand had been tried previously by the
Study Director as a filter aid without success when it was mixed Familiarization study Collaborative study
with the sample. There also were concerns that fine sand parti- doses, L doses, L
cles or celite might plug the pores in the fritted disks of Gooch Lab Amylase A Amylase B Amylase C Amylase A Amylase B
crucibles. However, a source of acid-washed silica sand with
particle size distribution between 40 and 200 mesh was found,
0 100 60 120 100
which cannot penetrate and plug the pores of coarse porosity
Gooch crucibles. The sand was stable to ND extraction and 1 75 500 400 200
ashing, and had a negligible effect on blank values. This sand 3 100 200 400 200
was evaluated as a filter aid for the difficult-to-filter samples in 4 300 500 500 300
the familiarization and collaborative studies (MB, RS, CB, and 5 75 125 150 250
DM) and greatly improved filtration and duplicate standard de- 6 200 150 300 150
viations (SDs). The sand was also evaluated with 6 other mate-
7 50 125 200 175
rials that are extremely difficult to filter. In all cases, filtration
was easy and SDs of duplicate analyses were much smaller 8 30 150 100 31
(pooled SD = 0.44 versus 0.77) than was possible without the 9 200 200 200 100
sand filter aid. The aNDF protocol was modified to include sil- 10 400 88 200
ica sand as a filtering aid. Collaborating laboratories were asked 11 88 88 200 88
to reanalyze MB and RS using sand as a filtering aid. With 12 300 200 400 200
sand, their sr decreased from 1.15 to 0.77 for RS and their sR de-
13 300 200 200 250
creased from 6.68 to 2.68 for MB.
14 200 100 150 125
Amylase Standardization Mean 178 202 262 176 170
SD 119 139 124 110 54
Development of the aNDF method was hampered by
changes in the sources of heat-stable -amylase. Sources
would become unavailable or simply be changed by the pro-
vider, making it difficult to specify a source and level of en-
zyme needed for the aNDF method. To eliminate dependence
of the aNDF method on a specific source of amylase and to The mean and median dose of amylase selected by the par-
comply with AOAC policy for Determination of Equivalency ticipants were higher than those selected in the Study Directors
of Products Used in Official Methods, we developed a proce- laboratory. Most collaborators overestimated the dose of amy-
dure for standardizing the heat-stable amylase. Because many lase needed for the aNDF method. This is acceptable because
feed testing laboratories do not have colorimeters, we devised too much amylase is better than too little, although there is a
a simple visual test for standardizing the amylase. In addition, concern that doses >5 times the minimum dose needed may
we found that ND interferes with measurement of starch in cause retrogradation of starch hydrolysis. Except for Labora-
most colorimetric methods, and concluded that a visual test tories 4, 5, and 8, standardization of amylase for the collabora-
would encourage routine evaluation of the amylase. tive study was much more consistent. Laboratories 4 and 8 may
The procedure for standardizing amylase for the aNDF not have restandardized the amylase for the collaborative study
method is based on the reaction of iodine with starch. A proce- samples and simply used the information they collected during
dure was developed that would ensure that raw corn starch the familiarization study. Laboratories 3, 6, 7, and 1012 ob-
(one of the most difficult starches to remove from fiber) would served that amylase C was half the activity of amylase B.
be solubilized and extracted in hot ND solution. It became evi- Several collaborators commented that amylase standard-
dent that the starch-iodine complex that creates a purple color ization was difficult. The procedure was rewritten to clarify
was unstable in ND solution, making visual detection of resid- the steps. There was some indication that participants could
ual starch difficult and time dependent. The original amylase not discriminate between the pink-amber of unhydrolyzed
standardization procedure was evaluated in the Study Direc- starch and the brown of the stock enzyme solutions. Based on
tors laboratory and gave variable results among technicians. results of the familiarization and collaborative studies, we
Additional research indicated that the procedure was sensitive modified the procedure for standardizing amylase for the
to both time and temperature; therefore, it was extensively re- aNDF method to indicate that a control solution of amylase in
written before the collaborative study began. Collaborating blank ND (no grits) should be run with the test doses to improve
laboratories were asked to evaluate 3 amylase stock solutions discrimination between pink-amber (inadequate amylase) and
during the familiarization study to evaluate the adequacy of brown (amylase solution). Although the range in doses of amy-
the amylase standardization procedure (Table 6). lase used was greater than desired, there was no significant rela-
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1231
tionship in the familiarization study between dose of amylase
and aNDF results across samples and laboratories.
Evaluation of the aNDF Method
Results of aNDF analyses were calculated 4 different ways
to evaluate the effects of blank correction and adjusting fiber
to an organic matter (ash-free) basis (Tables 710). Outliers
were detected by the Cochran and Grubbs tests in AOAC
spreadsheet software and are shown in Tables 710. Suspect
results were identified as having large residuals (>3*root
mean squared error) in an ANOVA using Model 3 (see Statis-
tical Analyses section) with main effects of Material, Lab, and
Rep. These latter results were not removed when AOAC sta-
tistics were calculated. To reduce the effect of single suspect
results on laboratory ranking and means, the results in bold
font were removed. An ANOVA using the main-effects
model (Model 3) was used to calculate least-squared means
for laboratories with all suspect data removed. All outlier data
were double checked to verify data entry and to determine if
collaborators comments could explain the aberrant results.
The differences between blind duplicates for grass hay (GH)
and sawdust (SD) reported by Laboratory 11 were not de-
tected as outliers by ANOVA or AOAC statistical methods.
Laboratory 12 had significant or nearly significant Grubb
tests for several materials and reported the highest results for
most materials. The laboratory ranking test (14) indicated that
Laboratory 12 was a statistical outlier. Although it reported
consistent results for blind duplicates (except for RS), it had
an average bias of +1.99 (1.21) %-units for all materials, and
questionnaire results indicated that it used medium porosity
Gooch crucibles with ceramic fiber as a filter aid. Neither of
these modifications was indicated in the aNDF method for
collaborative study. Based on both statistical and methodolog-
ical reasons, the results of Laboratory 12 were not used in the
statistical evaluation of the aNDF method.
Most outliers occurred in 2 laboratories (8 and 9), and in
each case only one of the blind duplicates was suspect. This
suggests that the aNDF method and its description may not be
at fault, because most remaining collaborators produced ac-
ceptable results; even the 2 laboratories with problems gener-
ated one acceptable result for all materials. Horwitz et al. (12)
reported that the reproducibility of empirical methods such as
fiber is generally much poorer than for analytes such as nitro-
gen, which are well defined and have suitable reference stan-
dards. Because the analyte is defined by the method, it is cru-
cial that fiber methods be followed exactly. As Horwitz et
al. (12) surmized, laboratories often modify methods to fit lab-
oratory conditions, attempt to improve the method by elimi-
nating or changing steps or apparatus, or simply ignore details
of the method that are thought to be superfluous. Although the
familiarization study helped to establish that the aNDF
method should be followed as described, we suspected that
some laboratories used modifications to accommodate their
unique laboratory situation. A questionnaire was used to dis-
cover exactly how each participant measured aNDF and the
responses were used to explain atypical results.
For aNDF, the HORRAT was <2 for all materials except
DM, RS, and MR (Table 11). Horwitz suggested that a
HORRAT of 2 is a reasonable, if not liberal, criterion for ac-
cepting a method. The feeds with HORRAT >2 had mean
aNDF <15%. The smaller means for aNDF of these materials
greatly inflated the observed RSDR. The Horwitz (16) equa-
tion for calculating the expected RSD was developed by re-
gressing sR versus the mean concentration across analytes and
methods. Implicit in this equation is the assumption that limits
of detection (LODs) decrease and precision increases as the
mean of the analyte decreases. In general, instrumental meth-
ods for measuring analytes in parts-per-billion and smaller
concentrations (near zero analyte) have very small LODs and
very good precision. However, within a method, the relation-
ship between precision and mean concentration may differ
from the Horwitz equation, especially if the method is
gravimetric and attempts to measure analytes from 0 to 100%
concentrations.
As discussed by Horwitz et al. (12), gravimetric methods
have LODs and precision that are related to weighing error.
Thus, within the aNDF method, it may be more valid to evalu-
ate acceptability across materials by comparing sR rather than
RSDR. For aNDF, the sR for DM, RS, and MR are similar to
those for other materials, indicating that the method can be
used to analyze feeds with <15% aNDF with precision similar
to those with >15% aNDF.
Blanks may account for systematic weighing variation
among runs. The potential magnitude of blank variation within
a single laboratory is indicated in Tables 3 and 4. Blank varia-
tion among runs in the Study Directors laboratory (2.8 sR) is
about 4 mg. The blank variation among collaborating laborato-
ries was greater with a 2.8 sR of 10 mg (Tables 9 and 10). Be-
cause weights of fiber residues are small, the effect of adjusting
for systematic weighing variation using blanks should be
greater for materials with low aNDF. Assuming a 0.5 g sample
and 25% aNDF in the material yields a fiber residue of 125 mg,
this could result in a range in blank-correction of about 8%. If
the material contains 10% aNDF, the fiber residue would weigh
only 50 mg and the range in blank-correction could be 20%.
When aNDF was blank-corrected (aNDFbc), the average
sR for materials containing <25% aNDF decreased from 1.30
to 1.01 (Table 11). Thus, blank-correction increases analytical
precision for materials with <25% aNDF. For unexplained
reasons the sR for materials containing >25% aNDF increased
from 1.29 to 1.36 due primarily to an increase in sR for
brewers grains (BG). It is difficult to envision how correcting
for blanks could harm the precision of materials with >25%
aNDF, given that the magnitude of blank-correction diminishes
as the fiber residue increases. The HORRAT of aNDFbc was
<2 for all materials except BG, DM, MR, and RS (Table 11).
The HORRAT increased slightly for BG, but decreased for
DM, MR, and RS when aNDF was blank-corrected.
The nutritional definition of fiber, which is the guide for
developing appropriate methods, indicates that fiber is the in-
digestible or slowly digesting fraction of a feed that occupies
space in the digestive tract of animals; therefore, indigestible
ash should be included in fiber. However, fiber traditionally
 1232 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
Table 7. Results of collaborating laboratories for amylase-treated neutral detergent fiber (aNDF) expressed on as-received basis
aNDF, % Collaborating laboratory

Feed Duplicate 0 1 3 4 5 6 7 8 9 11 12a 13 14

Alfalfa silage 1 40.51 41.13 41.11 40.08 42.36 38.43 41.49 41.29 39.19 40.92 44.57 39.60 39.84
2 40.26 40.20 40.82 40.88 41.26 40.66 41.43 42.57 40.71 42.20 45.85 41.40 41.20
Brewers grains 1 49.67 46.55 50.19 49.00 52.76 49.25 49.46 48.60b 46.28 51.41 52.25 52.00 48.87
c b
2 47.11 49.30 50.37 48.25 50.64 49.10 44.92 16.18 46.78 47.05 54.11 50.40 47.98
Citrus and beet pulp 1 29.30 30.13 29.69 28.73 31.42 29.06 27.57 29.33 28.45 29.96 31.91 31.20 30.46
2 28.64 30.77 30.63 31.68 30.05 30.87 29.58 29.17 29.67 28.58 32.73 32.00 28.84
Corn grain with cob 1 21.13 21.91 22.79 21.89 21.79 19.60 21.57 22.20 25.50b 22.08 23.72 19.80 21.92
2 22.03 21.60 23.06 21.69 21.81 19.93 21.62 21.47 20.61b 21.12 25.80 21.40 22.10
Corn silage 1 36.43 36.82 38.38 37.08 37.89 36.81 35.94 37.24 35.98 38.88 39.28 37.20 37.10
2 36.05 35.92 38.03 36.42 37.36 35.52 38.66 38.22 35.54 37.23 39.64 37.60 37.42
Corn stalks 1 71.42 72.68 74.30 70.85 74.00 72.05 73.92 74.20b 72.48 74.56 75.03 74.40 73.04
2 70.83 73.40 74.15 71.93 72.83 72.21 73.38 98.79b 73.22 73.05 76.49 74.00 72.26
Dairy mixed feed 1 12.30 12.40 12.90 11.79 14.22 10.87 11.79 13.27 11.14 13.45 16.26 14.60 12.23
2 11.74 12.78 11.62 9.06 12.17 13.18 13.90 14.44 11.76 12.92 15.86 14.80 12.50
b
Grass hay 1 55.66 57.57 58.06 58.21 57.57 57.75 58.79 57.40 48.46 59.65 59.96 59.60 57.56
2 55.41 57.56 58.90 59.48 58.44 58.21 57.62 59.52 56.87b 55.92 62.16 60.40 58.46
d
Milk replacer 1 0.14 0.52 0.62 0.45 0.14 0.38 1.36 0.66 0.00 0.18 1.37 2.40 0.36
2 0.02 0.12 0.28 0.38 0.12 1.83 0.78 3.72d 0.40 0.12 1.65 0.80 0.30
e b
Roasted soybeans 1 12.45 12.75 13.95 8.59 19.13 11.31 14.59 11.91 15.73 15.09 19.11 12.40 13.54
2 11.96 13.63 15.92 11.93e 12.05b 10.82 16.78 11.74 17.83c 14.69 26.00c 12.20 13.24
Sawdust 1 92.01 88.23 90.54 90.49 90.59 91.01 87.93e 87.93 90.61b 88.01 90.19 90.20 90.60
e
2 89.87 89.33 90.44 90.99 90.48 90.98 83.70 89.79 75.10b 93.46 92.15 92.60 91.32
f
Least-squared means 37.95 38.42 39.40 38.33 39.27 37.97 38.88 38.80 37.99 39.12 41.66 39.30 38.69

a
Outlier detected by laboratory ranking.
b
Outlier detected by Cochran test.
c
Outlier detected by analysis of variance to determine least-squared means, but not removed for AOAC statistics.
d
Outlier in Tables 810.
e
Outlier detected by single Grubb test.
f
Least-squared means calculated without data in bold font.
Table 8. Results of collaborating laboratories for amylase-treated neutral detergent fiber organic matter (aNDFom) expressed on as-received basis
aNDF, % Collaborating laboratory

Feed Duplicate 0 1 3 4 5 6 7 8 9 11 12a 13 14

Alfalfa silage 1 38.70 39.37 39.70 39.96 40.48 37.24 39.73 39.55 38.15 39.66 42.02 40.80 37.88
2 38.25 38.45 39.40 39.46 39.48 40.35 39.23 40.62 39.75 40.41 43.04 39.60 39.28
b
Brewers grains 1 48.20 45.30 49.27 48.74 51.64 48.36 48.10 48.04 45.94 50.26 50.72 53.00 49.20
2 45.51 48.05 49.20 49.60 49.36 48.79 43.52c 16.02b 45.30 46.29 52.34 49.40 47.20
Citrus and beet pulp 1 27.20 27.80 27.54 27.68 28.80 27.92 25.49 26.95 27.11 28.40 28.22 29.40 27.42
2 25.95 28.50 28.90 28.53 27.15 29.25 26.74 27.66 26.93 26.98 29.03 28.60 27.12
b
Corn grain with cob 1 20.88 21.55 22.49 22.71 21.57 20.89 21.23 21.45 25.76 21.71 22.76 21.20 21.40
2 21.32 21.25 23.02 22.16 21.63 21.55 20.96 21.25 20.87b 21.04 24.57 21.60 21.78
Corn silage 1 35.76 36.26 37.72 37.44 37.03 36.61 35.30 36.04 35.94 38.04 37.96 37.40 36.20
2 35.12 35.28 37.53 36.74 36.74 36.58 37.02 37.49 35.24 37.07 38.22 37.00 36.76
b
Corn stalks 1 67.18 68.12 71.46 68.62 69.90 68.75 70.76 70.00 69.03 71.37 70.28 72.40 69.52
b
2 67.07 70.31 70.37 69.29 68.88 70.34 67.96 82.74 69.45 70.08 71.82 71.80 68.62

MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1233

Dairy mixed feed 1 11.46 11.98 12.78 12.46 13.52 11.71 12.01 12.78 11.78 13.39 14.63 13.40 12.93
2 10.69 11.99 11.46 11.06 12.01 13.40 12.70 13.50 11.68 12.86 15.96 12.60 12.48
Grass hay 1 53.94 55.24 55.92 57.49 55.22 55.42 56.41 54.83 48.34b 57.24 56.81 57.40 55.48
2 53.48 55.14 57.05 57.94 56.65 57.24 55.40 57.59 55.68b 53.93 59.29 58.80 56.58
b
Milk replacer 1 0.26 0.52 0.78 0.57 0.14 0.34 0.22 0.58 1.55 0.26 0.64 0.40 0.20
b
2 0.02 0.22 0.67 0.73 0.16 0.10 0.44 2.84 0.36 0.53 0.38 0.60 0.20
Roasted soybeans 1 13.01 12.00 14.03 11.80 18.61b 12.64 14.61 11.22 17.04 15.23 17.95 13.60 13.84
2 12.32 13.19 15.48 13.22 11.75b 11.47 15.54 11.02 17.95c 14.77 24.77c 13.00 13.46
d b
Sawdust 1 91.05 87.15 89.63 90.39 89.40 86.26 86.83 86.29 90.19 86.69 90.09 90.40 89.32
d b
2 88.65 88.10 89.67 90.67 89.50 90.42 82.10 88.62 74.41 92.25 90.94 91.40 90.08
e
Least squared means 36.64 37.08 38.37 38.06 37.94 37.53 37.45 37.41 37.30 38.11 39.70 38.80 37.59

a
Outlier detected by laboratory ranking.
b
Outlier detected by Cochran test.
c
Outlier detected by analysis of variance to determine least-squared means, but not removed for AOAC statistics.
d
Outlier detected by single Grubb test.
e
Least-squared means calculated without data in bold font.
 1234 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
Table 9. Results of collaborating laboratories for amylase-treated neutral detergent fiber blank-corrected (aNDFbc) and expressed on as-received basis
aNDF, % Collaborating laboratory

Feed Duplicate 0 1 3 4 5 6 7 8 9 11 12a 13 14

Alfalfa silage 1 40.58 41.11 40.33 40.31 42.34 39.84 41.23 41.25 39.16 40.06 43.25 40.10 39.64
2 40.32 40.18 40.04 41.11 41.24 42.07 41.17 42.53 40.68 41.34 44.53 41.90 41.00
b
Brewers grains 1 49.74 46.53 49.40 49.22 52.74 50.65 49.20 48.44 46.25 50.55 50.93 52.50 48.75
2 47.17 49.28 49.58 48.46 50.62 50.52 44.66c 16.02b 46.56 46.19 52.79 50.90 47.91
Citrus and beet pulp 1 29.37 30.11 28.91 28.96 31.40 30.48 27.31 29.29 28.42 29.09 30.58 31.70 30.26
2 28.70 30.75 29.85 31.90 30.03 32.28 29.32 29.13 29.64 27.71 31.41 32.50 28.77
b
Corn grain with cob 1 21.21 21.89 22.01 22.13 21.77 21.01 21.31 22.16 25.47 21.22 22.39 20.30 21.72
2 22.10 21.59 22.28 21.91 21.79 21.34 21.36 21.43 20.58b 20.26 24.48 21.90 21.90
Corn silage 1 36.50 36.80 37.59 37.31 37.87 38.21 35.68 37.20 35.95 38.01 37.95 37.70 36.90
2 36.12 35.90 37.25 36.65 37.34 36.94 38.40 38.18 35.51 36.36 38.31 38.10 37.22
Corn stalks 1 71.49 72.66 73.52 71.08 73.98 73.46 73.66 74.17b 72.45 73.70 73.71 74.90 72.84
2 70.90 73.38 73.36 72.16 72.81 73.61 73.12 98.63b 73.19 72.19 75.16 74.50 72.06
Dairy mixed feed 1 12.37 12.38 12.12 12.03 14.20 12.29 11.53 13.23 11.11 12.59 14.94 15.10 12.12
2 11.80 12.76 10.84 12.44 12.35 14.59 13.64 14.40 11.73 12.05 14.54 15.30 12.30
Grass hay 1 55.73 57.55 57.28 58.44 57.55 59.16 58.53 57.36 48.43b 58.78 58.64 60.10 57.36
2 55.47 57.54 58.11 59.71 58.42 59.62 57.36 59.48 56.84b 55.05 60.84 60.90 58.26
b
Milk replacer 1 0.21 0.50 0.17 0.23 0.12 1.04 1.62 0.62 0.03 0.69 0.05 1.90 0.16
b
2 0.09 0.10 0.50 0.60 0.10 0.43 0.52 3.68 0.18 0.74 0.33 0.30 0.10
Roasted soybeans 1 12.90 12.73 13.18 12.04 19.11b 12.72 14.33 11.87 15.70 14.23 17.78 12.90 13.34
2 12.62 13.61 15.14 12.14 12.03b 12.22 16.52 11.70 17.80c 13.83 24.68c 12.70 13.17
d e
Sawdust 1 92.08 88.21 89.76 90.72 90.57 92.41 87.67 87.89 90.58 87.14 88.87 90.70 90.40
2 89.94 89.31 89.66 91.22 90.46 92.39 83.44d 89.75 75.07e 92.60 90.83 93.10 91.12
Least-squared meansf 38.06 38.40 38.62 38.65 39.25 39.38 38.62 38.75 37.93 38.25 40.34 39.80 38.51
Blanks 1 0.0007 0.0004 0.0051 0.0007 0.0001 0.0066 0.0022 0.0003 0.0007 0.0034 0.0056 0.0070 0.0012
2 0.0000 0.0002 0.0028 0.0016 0.0001 0.0076 0.0004 0.0001 0.0010 0.0053 0.0077 0.0020 0.0008

a
Outlier detected by laboratory ranking.
b
Outlier detected by Cochran test.
c
Outlier detected by analysis of variance to determine least-squared means, but not removed for AOAC statistics.
d
Outlier detected by single Grubb test.
e
Outlier in Tables 7, 9, and 10.
f
Least-squared means calculated without data in bold font.
Table 10. Results of collaborating laboratories for amylase-treated neutral detergent fiber organic matter blank-corrected (aNDFombc) and expressed on
as-received basis
aNDF, % Collaborating laboratory

Feed Duplicate 0 1 3 4 5 6 7 8 9 11 12a 13 14

Alfalfa silage 1 38.47 39.24 38.97 38.76 40.43 37.67 39.54 39.54 37.50 38.85 41.58 40.60 37.78
2 38.33 38.32 38.67 38.30 39.43 40.78 39.04 40.61 39.11 39.59 42.60 39.40 39.18
Brewers grains 1 47.97 45.17 48.54 47.60 51.59 48.79 47.91 48.00b 45.30 49.44 50.28 52.80 49.08
2 45.59 47.92 48.46 48.50 49.31 49.22 43.33c 15.98b 45.08 45.48 51.91 49.20 47.13
Citrus and beet pulp 1 26.96 27.67 26.81 26.50 28.75 28.36 25.30 26.94 26.46 27.58 27.78 29.20 27.32
2 26.03 28.37 28.17 27.41 27.10 29.69 26.55 27.65 26.28 26.16 28.59 28.40 27.05
Corn grain with cob 1 20.64 21.42 21.76 21.52 21.52 21.32 21.04 21.44 25.11b 20.89 22.32 21.00 21.30
2 21.40 21.12 22.29 21.00 21.58 21.99 20.77 21.24 20.23b 20.23 24.13 21.40 21.68
Corn silage 1 35.53 36.13 36.99 36.27 36.98 37.05 35.11 36.03 35.29 37.23 37.52 37.20 36.10
2 35.20 35.15 36.80 35.55 36.69 37.02 36.83 37.48 34.60 36.25 37.78 36.80 36.66
Corn stalks 1 66.95 67.99 70.73 67.44 69.85 69.19 70.57 69.99b 68.38 70.56 69.84 72.20 69.42
2 67.15 70.18 69.63 68.11 68.83 70.77 67.77 82.71b 68.80 69.27 71.38 71.60 68.52

MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1235

Dairy mixed feed 1 11.23 11.85 12.05 11.27 13.47 12.15 11.82 12.77 11.13 12.58 14.19 13.20 12.82
2 10.77 11.86 10.73 11.27 11.51 13.84 12.51 13.49 11.03 12.04 15.52 12.40 12.38
Grass hay 1 53.71 55.11 55.19 56.32 55.17 55.86 56.23 54.82 47.69b 56.43 56.37 57.20 55.38
2 53.56 55.01 56.31 56.77 56.60 57.68 55.21 57.58 55.03b 53.11 58.85 58.60 56.48
b
Milk replacer 1 0.03 0.39 0.04 0.58 0.09 0.78 0.03 0.57 0.90 0.56 0.20 0.20 0.10
2 0.10 0.09 0.06 0.43 0.11 0.53 0.25 2.83b 0.14 0.28 0.06 0.40 0.10
Roasted soybeans 1 12.72 11.88 13.30 12.02 18.56b 13.08 14.42 11.21 16.40 14.42 17.51 13.40 13.74
2 12.19 13.06 14.75 12.13 11.70b 11.90 15.35 11.01 17.30c 13.96 24.33c 12.80 13.39
d b
Sawdust 1 90.82 87.02 88.90 89.21 89.35 86.70 86.64 86.28 89.54 85.88 89.65 90.20 89.22
2 88.73 87.97 88.94 89.50 89.45 90.86 81.91d 88.61 73.76b 91.44 90.50 91.20 89.98
Least-squared meanse 36.55 36.95 37.64 37.02 37.87 37.97 37.26 37.40 36.70 37.30 39.25 38.61 37.49
Blanks 1 0.0004 0.0009 0.0035 0.0049 0.0002 0.0047 0.0020 0.0000 0.0047 0.0015 0.0021 0.0020 0.0008
2 0.0004 0.0004 0.0039 0.0068 0.0003 0.0003 0.0001 0.0001 0.0018 0.0067 0.0023 0.0000 0.0002

a
Outlier detected by laboratory ranking.
b
Outlier detected by Cochran test.
c
Outlier detected by analysis of variance to determine least-squared means, but not removed for AOAC statistics.
d
Outlier detected by single Grubb test.
e
Least-squared means calculated without data in bold font.
 1236 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
Table 11. Statistical data for aNDF and aNDFbc results
Single Double
Sample ID Outlier n Mean, % sr sR RSDr, % RSDR, % 2.8 sr 2.8 sR HORRAT Cochran Grubb Grubb

aNDF

Alfalfa silage 12 40.81 0.88 0.97 2.15 2.38 2.46 2.71 1.04 0.269 11.4 19.3
Brewers grains 8 11 48.97 1.68 1.98 3.44 4.05 4.72 5.56 1.82 0.330 13.4 29.4
Citrus and beet pulp 12 29.82 1.05 1.14 3.51 3.81 2.93 3.19 1.59 0.331 20.1 28.2
Corn grain with cob 9 11 21.57 0.49 0.87 2.25 4.04 1.36 2.44 1.60 0.493 29.8 49.3
Corn silage 12 37.07 0.79 0.97 2.13 2.63 2.21 2.73 1.13 0.491 10.2 16.7
Corn stalks 8 11 72.95 0.58 1.13 0.79 1.55 1.62 3.17 0.74 0.312 13.7 32.2
Dairy mixed feed 12 12.58 1.04 1.33 8.23 10.59 2.90 3.73 3.88 0.291 16.8 36.6
Grass hay 9 11 58.08 1.06 1.31 1.82 2.25 2.96 3.65 1.04 0.566 35.3 63.3
a
Milk replacer 8 11 0.13 0.69 0.81 547.15 644.34 1.92 2.27 0.440 31.0 66.1
Roasted soybeans 5 11 13.32 1.08 2.18 8.13 16.36 3.03 6.10 6.04 0.433 12.8 22.0
Sawdust 7 and 9 10 90.44 1.51 1.51 1.67 1.67 4.22 4.22 0.82 0.653 19.8 67.4
aNDFbc

Alfalfa silage 12 40.81 0.88 0.88 2.15 2.15 2.46 2.46 0.94 0.268 13.4 33.8
Brewers grains 8 11 48.19 1.85 2.24 3.83 4.64 5.17 6.26 2.08 0.280 11.9 23.5
Citrus and beet pulp 12 29.83 1.04 1.39 3.48 4.66 2.91 3.89 1.94 0.336 16.6 29.0
Corn grain with cob 9 11 21.57 0.48 0.55 2.25 2.55 1.36 1.54 1.01 0.496 19.3 29.1
Corn silage 12 37.07 0.79 0.86 2.13 2.31 2.21 2.40 1.00 0.493 20.1 30.5
Corn stalks 8 11 72.96 0.58 1.04 0.79 1.43 1.62 2.91 0.68 0.311 16.5 35.9
Dairy mixed feed 12 12.72 0.86 1.23 6.73 9.64 2.40 3.43 3.53 0.301 28.7 38.6
Grass hay 9 11 58.08 1.06 1.49 1.82 2.57 2.96 4.18 1.18 0.567 18.7 40.7
a
Milk replacer 8 11 0.13 0.68 0.68 519.84 519.84 1.92 1.92 0.443 22.9 38.5
Roasted soybeans 5 11 13.52 0.81 1.59 5.99 11.80 2.27 4.46 4.36 0.332 26.9 46.1
Sawdust 7 and 9 10 90.47 1.51 1.61 1.67 1.78 4.23 4.50 0.87 0.653 12.4 24.1

a
= HORRAT could not be calculated because the mean was a negative number.
Table 12. Statistical data for aNDFom and aNDFombc results
Single Double
Sample ID Outlier n Mean, % sr sR RSDr, % RSDR, % 2.8 sr 2.8 sR HORRAT Cochran Grubb Grubb

aNDFom

Alfalfa silage 12 39.40 0.91 0.91 2.32 2.32 2.56 2.56 1.01 0.484 7.0 15.9
Brewers grains 8 11 48.19 1.85 2.24 3.83 4.64 5.17 6.26 2.08 0.280 11.9 23.5
Citrus and beet pulp 12 27.67 0.76 1.00 2.76 3.61 2.14 2.79 1.49 0.195 14.7 26.2
Corn grain with cob 9 11 21.57 0.31 0.58 1.46 2.69 0.88 1.62 1.07 0.204 28.1 65.7
Corn silage 12 36.60 0.61 0.85 1.67 2.33 1.71 2.38 1.00 0.329 8.9 19.8
Corn stalks 8 11 69.60 0.97 1.46 1.40 2.09 2.73 4.08 0.99 0.376 19.5 44.4
Dairy mixed feed 12 12.36 0.70 0.81 5.63 6.54 1.95 2.26 2.39 0.246 18.5 24.3
Grass hay 9 11 56.11 1.17 1.43 2.08 2.55 3.26 4.01 1.17 0.368 22.7 39.9
Milk replacer 8 11 0.42 0.29 0.33 68.35 78.87 0.81 0.93 17.31 0.777 21.9 34.4
Roasted soybeans 5 11 13.66 0.67 1.86 4.90 13.61 1.87 5.20 5.04 0.214 25.6 34.9
Sawdust 7 and 9 10 89.30 1.76 1.76 1.97 1.97 4.93 4.93 0.97 0.498 12.5 33.3
aNDFombc

MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1237

Alfalfa silage 12 39.09 0.91 0.91 2.32 2.32 2.54 2.54 1.01 0.489 8.2 20.1
Brewers grains 8 11 47.88 1.82 2.24 3.80 4.68 5.10 6.27 2.09 0.288 13.0 28.6
Citrus and beet pulp 12 27.36 0.75 1.08 2.73 3.95 2.09 3.03 1.63 0.204 12.6 29.0
Corn grain with cob 9 11 21.30 0.34 0.46 1.58 2.16 0.94 1.29 0.86 0.229 17.6 38.3
Corn silage 12 36.29 0.60 0.82 1.66 2.27 1.68 2.30 0.97 0.339 16.1 28.4
Corn stalks 8 11 69.27 0.98 1.46 1.41 2.10 2.73 4.07 0.99 0.375 22.8 41.1
Dairy mixed feed 12 12.09 0.67 0.89 5.55 7.38 1.88 2.50 2.69 0.358 6.6 15.8
Grass hay 9 11 55.83 1.16 1.38 2.08 2.48 3.25 3.87 1.14 0.370 20.4 42.6
Milk replacer 8 11 0.11 0.21 0.37 191.63 347.64 0.58 1.05 62.15 0.622 14.8 35.4
Roasted soybeans 5 11 13.38 0.59 1.63 4.39 12.17 1.65 4.56 4.49 0.276 27.9 38.6
Sawdust 7 and 9 10 89.01 1.74 1.74 1.96 1.96 4.87 4.87 0.96 0.510 14.6 34.1
1238 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
Figure 1. Change in standard deviation of
reproducibility, s(R), due to ashing of fiber residues.
has been defined as organic matter, and in plant materials it
has been equated with plant cell walls, which contain little ash.
Recently, nutritionists have derived an estimate of the easily
digested or rapidly fermentable carbohydrates by difference.
Nonfiber carbohydrates (NFC) or neutral detergent soluble
carbohydrates (NDSC) are calculated as:
Dry matter crude protein crude fat ash aNDF
If some ash is included in aNDF this results in a double sub-
traction of the ash in fiber. Thus, there are nutritional benefits
to expressing aNDF as ash-free organic matter (aNDFom).
Analytically, ashing fiber residues and expressing results
as aNDFom improves the reproducibility of results for materi-
als with <50% aNDF (Tables 11 and 12). However, it de-
creases analytical reproducibility slightly for materials with
>50% aNDF. The consistent inverse relationship between the
change in sR due to ashing of fiber residues and mean concen-
tration of aNDFom is unexplained (Figure 1). A similar trend
was observed when results were blank-corrected. It was not
related to the amount of ash in the fiber. In general, expressing
fiber as aNDFom decreased the HORRAT. Ashing fiber resi-
dues requires an additional step in the procedure, which incurs
extra time and expense. For routine fiber analysis of forages,
measuring aNDFom may not be cost effective.
The average sR for all materials was 1.29, 1.24, 1.20, and
1.18 for aNDF, aNDFbc, aNDFom, and aNDFombc, respec-
tively. Although adjusting results for both ash and blanks im-
proves precision, the small differences among results do not
indicate a strong preference for one method of expressing the
results over another. Thus, it is suggested that the method for
expressing the data be left to the discretion of the laboratory
and calculations for all 4 results are given in the aNDF
method. The laboratory must state clearly which method for
calculating results was used. The exception to this general rule
of allowing analysts to choose the method of expressing
aNDF results is for feeds containing <25% aNDF. The aver-
age sR for these materials is 1.30, 1.01, 0.90, and 0.84 for
aNDF, aNDFbc, aNDFom, and aNDFombc, respectively.
There is a clear advantage to ash- and blank-correction when
materials contain <25% aNDF, and this is the recommended
method for these feeds and for regulatory laboratories.
Occasionally ash values for fiber residues were negative.
When this occurred, some collaborating laboratories treated
this result as an error, ignored the ash correction, and set
aNDFom = aNDF. Correcting for blanks reduced the incidence
of negative fiber ash results, but did not eliminate them. A nega-
tive ash means that crucible weight after ashing was less than
the initial weight of the crucible and indicates that crucibles lost
weight during the aNDF procedure. The most accurate estimate
of the weight of fiber residues is obtained by using the crucible
weight after ashing, because the initial crucible weight is an
overestimate caused by some contaminant in the crucible that
was lost during the aNDF method, i.e., aNDFom calculated us-
ing a negative ash is a more accurate estimate of fiber than is
aNDF. The aNDF method submitted for First Action was modi-
fied to indicate that aNDFom results must be calculated using
ashed crucible weight when it is less than the initial crucible
weight (negative ash) because in this circumstance the aNDF
result is an underestimate of the correct value.
Sokal and Rohlf (18) suggest that the number of decimal
places for reporting results should be based on the magnitude
of the standard error using the following approach: divide the
standard error by 3 and use the decimal place of the first non-
zero digit to determine the significant digits appropriate for re-
porting results. Based on their rule of thumb, it is recom-
mended that aNDF results be reported to the nearest 0.1%.
Irrespective of the basis for calculating results (aNDF,
aNDFbc, aNDFom, or aNDFombc) it is evident that the RSD in-
creases as the mean concentration of aNDF decreases. The data in
Tables 11 and 12 indicate that sR does not approach zero as mean
concentration of aNDF approaches zero; rather it approaches some
minimum value that can exceed the mean value. This has implica-
2.50
BG
2.00
Horwitz
Standard Deviation
RS
1.50 Horwitz
s - Col
s r - Col
s R - Col
R s r - Col
1.00 s R - SD
s R - SD
0.50
0.00
0 20 40 60 80 100
aNDFombc (%)
Figure 2. Relationship of standard deviation of
reproducibility calculated by the equation of Horwitz
(16) and the standard deviations of repeatability (sr) or
reproductibility (sR) obtained in the collaborative study
(Col) or homogeneity test by the Study Director (SD) to
the mean amylase-treated neutral detergent fiber or-
ganic matter, blank-corrected (aNDFombc) for each
material. Data points for roasted soybeans (RS) and
brewers grains (BG) were omitted from the sR Col re-
gression line. Intercepts of lines were 0.31, 0.45, and
0.39 for sr Col, sR Col, and sR SD, respectively.
 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 1239
tions on the minimum level of applicability for the aNDF method.
Horwitz et al. (12) concluded that the inherent variability in a
gravimetric method in which the analyte is empirically defined
limits its ability to detect low levels of concentration.
Figure 2 illustrates that the minimum sR for aNDFombc is
near 0.4. Regressing the standard deviation versus the mean
concentration permits estimation of the repeatability and
reproducibility of aNDFombc at zero concentration. Using the
data from Table 4, the intercept for reproducibility in the
Study Directors laboratory was 0.39. Because the data in Ta-
ble 4 were generated in one laboratory and assume there were
no differences among sample sets, the sR from Table 4 is com-
parable to the within-laboratory repeatability from data in Ta-
ble 12. For the collaborative study, the intercept for repeatabil-
ity within laboratories (sr) was 0.31 and the intercept for
reproducibility among laboratories (sR) was 0.45 (results for
BG and RS were not included in the regression). This suggests
when the true result is zero concentration, one time in 20 labo-
ratories would observe values <1.3 or >1.3% aNDFombc
(1.3% = 2.8 .45). It is recommended that values <1.5%
aNDF be reported as <1.5%.
Laboratories were provided with 2 different amylase
sources and requested to determine the standard dose for their
source to use in the collaborative study. An ANOVA was per-
formed on the fiber results for all materials and only materials
containing significant starch (CG and CS). There was no dif-
ference in aNDF, aNDFbc, aNDFom, or aNDFombc among
amylase sources or doses for all materials or for those contain-
ing starch. This suggests that the amylase standardization pro-
cedure is adequate for the aNDF method. As indicated in Ta-
ble 6, most collaborators used doses of amylase higher than
those selected by the Study Directors laboratory, which may
explain why removal of starch from fiber was not a problem.
Laboratory 8 used a smaller amylase dose than that selected
by the Study Directors laboratory, but obtained acceptable fi-
ber values for CG and CS.
Questionnaire responses were used to evaluate modifica-
tions or variations of the aNDF method that were used by col-
laborating laboratories. Some variations were made by individ-
ual collaborators and others were related to modifications
allowed in the aNDF method. An ANOVA indicated that
refluxing in crucibles (Fibertec) was not significantly different
from refluxing in beakers and filtering in crucibles. Collaborators
using the Fibertec apparatus had an average bias for the 11 mate-
rials of +0.60% for aNDFbc, aNDFom, and aNDFombc, which
was significant but within the sR of the method. When the aver-
age results for each material determined with the Fibertec appa-
ratus was regressed versus the average results with traditional
refluxing in beakers, the intercepts were not different from zero
and the slopes were not different from 1 for aNDF, aNDFbc,
aNDFom, and aNDFombc. This suggests that the Fibertec appa-
ratus is a suitable alternative for measuring aNDF.
Statistical analysis also indicated that the first replicate in
which materials were ordered from least to most difficult to
filter was not different from the second replicate in which ma-
terials were analyzed in random order. This suggests that the
order of analysis, e.g., preceding an analysis with an easy- or
difficult-to-filter sample, did not affect results.
In the first blind duplicate, materials were labeled as con-
taining >10% fat when appropriate. In the second blind dupli-
cate, the materials were not identified as containing >10% fat to
determine if laboratories could detect unidentified samples that
were high in fat. Only 3 of the 12 collaborators identified some
of the materials in the second blind replicate that were >10%
fat, and no laboratory detected all 3 materials. This may explain
why results for RS and DM were less reproducible than those
for other materials. In most instances, the first blind duplicate
identified as >10% fat was pre-extracted and the second un-
identified blind duplicate was not. Statistical analysis of the re-
sults did not detect a significant difference between samples
that were pre-extracted and those that were not; however, the
reproducibility for these materials was large, making it difficult
to detect differences. Results in the Study Directors laboratory
clearly demonstrate that pre-extraction of materials containing
>10% fat improved repeatability of results and lowered aNDF
values. The inability of collaborators to detect unidentified
samples containing >10% fat and the decreased reproducibility
of these materials suggest that perhaps regulatory laboratories
should pre-extract all materials before aNDF determination un-
less it is known that they contain <10% fat.
An ANOVA using Equation 1 without Block indicated a
significant Lab*Material interaction when the results for all
materials were tested. This indicated that the aNDF method
was not consistent for all laboratories across all materials.
When the results for high-fiber materials and forages (SD,
corn stalks, GH, AS, CS) were combined and tested, the
Lab*Material interaction was not significant. Likewise when
concentrated materials containing <10% fat (BG, CB, CG)
were tested, the Lab*Material interaction was not significant.
These statistical analyses indicate that collaborators could ob-
tain consistent results for forages and concentrated feeds with
<10% fat. However, Lab*Material interaction was significant
for concentrated feeds with >10% fat (DM, RS, MR), which
suggests that collaborators had difficulty obtaining consistent
results for these feeds and confirms the variability associated
with high sR for these feeds. This inconsistency and variability
may be related to the variable pre-extraction of these feeds
within- and among-laboratories. Results were pooled (13)
within categories of feed materials to obtain precision perfor-
mance parameters for the aNDF method (Table 2002.04).
Collaborators Comments
Several collaborators had been measuring NDF prior to the
collaborative study and offered suggestions about their modifi-
cations of the method. They suggested that extra crucibles be
dried and tare weights recorded. When a difficult-to-filter test
sample was encountered, the residue could be split between
2 crucibles so that the analysis could be completed without a re-
run. Although this suggestion allowed results to be generated, it
increased the expected variation in results due to additional er-
ror associated with weighing 2 crucibles to obtain results.
It was suggested that the initial dose of amylase be injected
before placing the beaker on the heating unit instead of waiting
1240 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002
until the solution is boiling (ca 5 min after placing beaker on heat-
ing unit). However, this is not recommended for 2 reasons:
(1) Initial heating of the test sample results in partial
gelatinization of starch and improves hydrolysis by amylase; and
(2) heating the solution tends to deactivate any contaminating
fibrolytic enzymes that may be present in crude amylase extracts.
Some laboratories did not like to use stirring rods to mix
residues in the crucibles, or used glass instead of Teflon stir-
ring rods. Stirring the residues is important to insure contact
between fiber particles and washing solutions. This is espe-
cially important for adequate extraction with acetone at the
end of the method. If fiber residues are damp when acetone is
added the first time, a fine stream of acetone from a squirt bot-
tle can be used to mix residues and obtain adequate extraction.
Teflon rods are recommended because particles stick to them
less readily than to glass rods. Other comments by collabora-
tors were used to improve the method description.
Recommendations
Based on the results of the collaborative study, the Study
Director recommends that Gravimetric Determination of Am-
ylase-Treated Neutral Detergent Fiber in Feeds with
Refluxing in Beakers or Crucibles be adopted as Official First
Action. It is recommended that alternative methods of report-
ing results be allowed, except that materials with <25% aNDF
must be blank-corrected, that materials containing >10% fat
must be pre-extracted with a suitable solvent before aNDF de-
termination, and that ash-corrected values be reported when
negative ash weights are observed.
Acknowledgments
I thank Diane Amundson, Leslie Sims, and Rodney Hintz
(U.S. Dairy Forage Research Center) for assistance in devel-
oping the aNDF method. I also thank Diane Amundson for as-
sistance with preparing and distributing materials for the study
and compiling the results for analysis. The USDA Agricul-
tural Research Service provided funding for the materials used
in the study. I also thank the following collaborators for their
participation in the study:
Jane Clegg and Kenneth Frank, University of Nebraska,
Soil and Plant Analytical Laboratory, Lincoln, NE
Michelle M. Drouches and Dave Taysom, Dairyland Labo-
ratories, Inc., Arcadia, WI
Michelle Garkie and Bruce Paul, MoorMan Feeds, Inc.,
Quincy, IL
Bryan Gildemeister and Nancy Thiex, South Dakota State
University, Brookings, SD
Darryl K. Jones and Darrell Gambin, Ralston Analytical
Laboratory, St. Louis, MO
Deborah Kaplan and Ann Davidowicz, Blue Seal Feeds,
Inc., Londonderry, NH
Gyoung-No Kim and Cheon-Seok Jeon, JE-IL Feed Co.
Chemical Laboratory, Taejeon, Korea
Stanley Kobata and Jack Carmany, California Department
of Food and Agriculture, Sacramento, CA
David G. Main and Michael S. Allen, Michigan State Uni-
versity, East Lansing, MI
Xang Moua and Michael Wolf, JL Analytical Services,
Modesto, CA
James B. Robertson, Cornell University, Ithaca, NY
Jim Williams and David Jeffress, Missouri Department of
Agriculture, Jeffferson City, MO
References
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