74 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO.
1, 2009
SPECIAL SECTION ON FEED ADDITIVES AND CONTAMINANTS
Gravimetric Determination of Acid Detergent Fiber and Lignin in
Feed: Interlaboratory Study
JÜRGEN MÖLLER
FOSS Analytical AB, Pål Anders väg 2, SE-26321 Höganäs, Sweden
Collaborators: J. Arananant; K. Boonnayanont; J. Danier; M. Egert; K. Fütö; H. Horst; L. Hüther; S. Janjira;
R. Kongchuensin; W. Korol; J. Kühl; P. Losittisak; J. Marth-Schill; O. Moreira; J. Petrova; S. Plos; K. Prinze;
V. Rungrojpanich; J. Schönherr; R. Siourai; J. Skomial; S. Titya
On the basis of AOAC Official Method 973.18, a
collaborative study was performed to determine
fractions of acid detergent fiber (ADF) and
lignin (ADL) in animal feed. The study used
6 animal feed and forage samples as blind
duplicates and involved 22 participants from
7 countries. The samples analyzed covered a range
from 3.5 to 70% ADF and from <1 to 20% ADL.
Repeatability standard deviations ranged from 0.27
to 0.95% for ADF and from 0.08 to 0.59% for ADL.
The reproducibility standard deviations ranged
from 0.56 to 2.44% for ADF and from 0.52 to 2.09%
for ADL. Analyzed samples comprised clover and
grass silages, soy meal, cattle feed, wheat, and
sawdust. The studied method has been published
as the EN ISO 13906:2008 standard.
M
ethods for the determination of detergent fibers were
developed by Goering and van Soest (1) in the
1960s. The concept behind detergent fiber analysis
is that plant cells can be divided into less digestible cell walls
(comprising hemicellulose, cellulose, and lignin) and mostly
digestible cell contents (comprising starch and sugars). These
2 components can be separated by the use of 2 detergents: a
neutral detergent and an acid detergent. Neutral detergent
fiber (NDF) is a good indicator of “bulk” and thus feed intake.
Acid detergent fiber (ADF) can be used as an indicator of
digestibility and thus energy intake (2). A harmonized
“global” standard method for the determination of NDF has
recently been published by AOAC (3, 4), and another was
published jointly by the International Organization for
Standardization (ISO; 5) and the European Committee for
Standardization (CEN; 6). However, there was no ISO or
CEN standard for the determination of ADF available. In
2005, the ISO Technical Committee 34/Subcommittee 10
Received August 27, 2008. Accepted by EB November 2, 2008.
Corresponding author's e-mail: jmr@foss.dk
This paper is published as part of a themed collection on emerging feed
issues organized by the Feed Additives and Contaminants Subgroup of the
AOAC Agricultural Materials Community.
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(animal feeding stuff) and the CEN Technical Committee 327
(animal feeding stuff) approved a new working item proposal
for the development of a standard method under the title,
“Animal feeding stuffs—Determination of acid detergent
fiber content and lignin” on the basis of AOAC Official
Method 973.18, “Fiber (acid detergent) and lignin (H2SO4) in
animal feed” (7). However, during the course of the project
the collaborative study material of the AOAC method (8)
could not be transferred to the ISO/CEN format, and a new
collaborative trial had to be performed. This report
summarizes the results of an interlaboratory study performed
during 2006 under Swedish project leadership by the
company FOSS, following the protocol described in
ISO 5725-2 (9).
Interlaboratory Study
Test Materials
Samples of sawdust, wheat, cattle feed, soy meal, clover
silage, and grass silage were selected for the study (Table 1).
In addition, a sample of lignocellulose was selected as a
control sample. All samples were dried and ground using
Retsch or Cyclotec mills with 1 mm screens. In order to
achieve a mean particle size of 0.22–0.26 mm, the ground
samples were sieved using a screen with 0.5 mm aperture.
Material that did not pass through the sieve was reground. No
material was discarded.
Homogeneity
After subdivision, the samples were characterized for their
content of moisture (10), crude fat (11), crude protein
(CP; 12), and ash (13; Table 1). Using crude protein as an
indicator, it was found that the repeatability standard deviation
(n = 6) was significantly below the repeatability standard
deviation given in the method used, indicating that the
samples were sufficiently homogeneous for the study.
Stability
All samples were analyzed before and after the study
period. CP was used as an indicator (Table 2).
CP1 is the protein value on a dry matter basis before, and
CP2 after, the study period (n = 2). Because the differences are
 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 75

6.112
1.099
0.134

1.100
RSD
Control sample
within the repeatability and reproducibility limits of the

0.021
0.004
0.009

0.004
Std
method (9), they have been regarded as not significant and the
samples were assumed to be sufficiently stable for the study.

0.336
0.397
6.876

0.322
Avg.
Study Design

2.053 0.0000 0.000

7.192 0.1287 1.789
2.967 0.0502 1.692

6.885 0.0863 1.253

RSD
A blind duplicate design was chosen for the study. After
Grass silage

homogenization and subdivision into 20–40 g lots, the

samples were packed into plastic bags and sealed into
Std

aluminum foil. Samples were numbered 1–12 without

identifying the type of sample, and sent to the participating
Avg.

laboratories by courier service. As all participating

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laboratories were familiar with the method, no training
samples were used. Instead, each participant received a
0.004 0.199
0.056 0.353
0.151 2.439

0.002 0.023
RSD

control sample of lignocellusose with specified ADF and acid

Clover silage

detergent lignin (ADL) values (74.2% ADF; 25.8% ADL)

Std

determined by a ring test organized in Germany. Each

participating laboratory had to return an application form
confirming their participation and that they would be
2.135
15.808
6.175

9.081
Avg.

adequately prepared for cooperative testing of the method.

Together with the samples, the laboratories received a draft of
the method to be followed and a reporting scheme, as well as
Soy meal, heat treated

1.610 0.0260 1.614

50.019 0.1825 0.365
6.279 0.0437 0.696

6.513 0.0105 0.162

RSD

an analysis scheme (see Figure 1). As the method draft

Sample

describes a method for the ADF determination with

Std

subsequent ADL determination, participants were asked, on a

Table 1. Composition of the samples selected for the interlaboratory study (% “as is”)

voluntary basis, to determine also the ADF only, with

subsequent ashing for correction of the ash content.
Avg.

METHOD
2.706 0.0255 0.944
16.380 0.0526 0.321
7.491 0.0428 0.571

6.986 0.0576 0.824

RSD
Cattle feed

Caution: Using this International Standard may involve

Std

hazardous materials, operations, and equipment. This

International Standard does not purport to address all the
safety risks associated with its use. It is the responsibility of
Avg.

the user of this International Standard to establish appropriate

safety and health practices and to determine the applicability
1.598 0.0482 3.018
13.584 0.0384 0.282
10.149 0.0935 0.922

1.656 0.0327 1.974

RSD

of local regulatory limitations prior to use.

Scope
Wheat

Std

This International Standard specifies a method for the

Avg.

determination of ADF and ADL in all types of feed. It

concerns a gravimetric routine and reference method.
1.302
3.140
0.465

5.048
RSD

Principle
Sawdust

ADF is determined in the first stage of the method.

0.024
0.014
0.025

0.020
Std

Cationic detergent solution is used to remove acid-labile

carbohydrates, protein that is not complexed into Maillard
1.806
0.461
5.428

0.396
Avg.

products (heat-damaged), and fats. The remaining fibrous

residue is primarily cellulose and lignin (plant products) or
Protein, 6.25, %

insoluble protein complexes (animal products and

Ash content, %
Fat, crude, %

heat-damaged feeds). The residue is weighed to determine

Moisture, %
Parameter

ADF. In the second stage, the remaining residue is solubilized

by 72% H2SO4, leaving the lignin (ADL), which is
.

determined gravimetrically.
76 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009

Table 2. Crude protein content (% dry matter) of selected test samples before (CP1) and after (CP2) the
interlaboratory study. In all cases, the differences are lower than the repeatability (r) and reproducibility (R) limits
given by the method (ref. 12)

Mean % CP

Sample % CP1 % CP2 Diff. r R

Sawdust 0.49 0.54 0.06 0.44 0.42

Wheat 15.1 15.22 0.12 0.56 0.61
Cattle feed 17.7 17.78 0.08 0.58 0.64
Soymeal 53.3 53.16 –0.14 0.89 1.09
Clover silage 16.84 16.72 –0.12 0.58 0.63
Grass silage 7.4 7.07 –0.34 0.50 0.51

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Control 0.43 0.37 –0.06 0.44 0.42

Figure 1. Analysis scheme, EN ISO 13906.

 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 77

Table 3. Reported moisture values (% m/m)

Lab Value Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

1 1 5.44 10.03 7.39 6.17 6.06 3.03

2 5.44 10.04 7.51 6.16 6.19 2.82
2 1 5.49 10.08 7.57 6.26 6.36 2.97
2 5.49 10.07 7.51 6.33 6.37 3.00
3 1 4.94 9.22 6.88 5.71 6.74 2.74
2 4.97 9.20 6.82 5.71 6.66 2.72
4 1 5.51 9.82 7.32 6.09 7.90 3.57
2 5.52 9.71 7.30 6.16 7.83 3.66
5 1 5.10 9.00 6.40 5.10 6.40 3.00

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2 4.90 8.90 6.30 5.20 6.10 2.60
6 1 6.00 9.70 7.30 6.30 8.70 5.10
2 6.00 9.70 7.30 6.30 7.90 4.70
7 1 5.36 9.62 7.19 5.77 6.75 2.97
2 5.27 9.44 7.19 6.15 7.52 3.24
8 1 4.88 8.98 6.67 5.45 5.90 2.34
2 4.97 8.94 6.72 5.39 6.05 2.54
9 1 5.66 9.29 7.07 5.81 7.12 3.36
2 5.61 9.24 6.98 5.75 6.88 3.44
10 1 5.27 9.40 7.06 5.82 7.42 3.22
2 5.27 9.51 7.11 5.82 7.26 3.81
11 1 5.06 9.25 6.76 5.64 5.85 2.87
2 5.12 9.35 6.74 5.65 5.77 2.78
12 1 5.70 10.20 7.60 6.20 6.20 4.40
2 5.80 9.80 7.50 6.00 6.90 3.50
13 1 5.80 10.20 7.70 6.50 7.70 3.60
2 5.40 9.90 8.00 6.50 8.00 4.40
14 1 5.60 9.70 7.60 6.30 6.70 3.10
2 5.50 10.10 7.50 6.40 6.80 3.20
15 1 5.14 9.37 6.75 5.77 5.83 2.67
2 5.11 9.46 6.90 5.82 5.62 2.84
16 1 5.06 9.25 6.76 5.64 5.85 2.87
2 5.12 9.35 6.74 5.65 5.77 2.78
17 1 5.36 9.94 7.30 6.16 5.86 3.18
2 5.34 9.80 7.24 6.14 5.98 3.12
18 1 5.49 9.82 7.31 6.15 5.82 2.62
2 5.28 9.75 7.12 6.13 5.73 2.68
19 1 5.19 9.70 7.37 6.17 8.44 4.03
2 5.17 9.69 7.34 6.13 8.42 4.26
20 1 5.40 9.50 7.10 6.00 6.30 3.40
2 5.40 9.80 7.20 6.10 6.00 3.40
21 1 4.90 9.40 6.80 5.80 6.00 3.00
2 5.00 9.30 6.90 5.70 6.00 3.10
22 1 5.1 9.1 6.8 5.6 6.3 2.6
2 5.1 9.1 6.8 5.6 6.4 2.8
78 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009

Table 4. Reported results, ADF content (% dry matter)a

Lab Value Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

1 1 72.30 4.47 14.70 7.26 29.16 30.84

2 72.94 4.44 13.83 6.81 30.05 30.95
2 1 73.22 3.64 13.71 6.91 30.56 32.33
2 73.69 3.35 14.00 7.13 29.72 31.63
3 1 72.30 3.43 13.90 7.33 31.10 31.30
2 74.50 3.67 14.10 7.74 28.70 31.40
4 1 73.45 3.66 14.57 7.56 31.05 30.80
2 73.45 3.99 14.35 7.67 30.27 31.55
6 1 72.66 3.10 13.59 6.62 28.48 30.87

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2 72.23 2.99 13.16 6.62 28.66 31.37
7 1 72.98 4.86 14.04 7.04 31.22 31.17
2 73.89 3.58 13.58 7.10 32.00 31.79
8 1 74.56 4.09 15.45 7.67 31.02 30.87
2 74.08 4.04 15.78 8.21 30.02 30.93
9 1 69.52 7.33 15.70 8.64 30.77 32.13
2 69.43 6.67 14.86 8.92 30.60 32.00
10 1 74.01 4.00 14.55 8.03 31.50 32.89
2 75.78 3.24 14.46 8.09 34.19 34.16
11 1 74.76 3.70 14.80 7.30 30.27 32.30
2 74.75 3.71 14.60 7.30 30.41 31.57
12 1 75.61 4.01 14.72 7.25 31.24 32.74
2 75.90 4.10 14.70 7.34 31.15 32.64
13 1 73.14 <1.5 13.00 6.31 28.60 29.15
2 71.67 3.66 13.48 6.10 28.80 30.92
14 1 67.80 2.10 12.12 7.26 28.08 27.86
2 66.56 2.56 11.89 5.77 26.82 28.82
15 1 78.39 4.03 16.79 8.26 29.94 36.33
2 72.91 4.12 16.20 7.57 35.04 34.79
16 1 74.76 3.70 14.80 7.30 30.27 32.30
2 74.75 3.71 14.60 7.30 30.41 31.57
17 1 71.15 3.49 14.12 6.87 29.18 30.50
2 71.50 3.67 14.01 6.99 28.92 30.51
19 1 67.68 2.48 12.02 7.56 28.41 30.83
2 67.72 2.27 12.53 9.81 27.77 28.62
20 1 74.08 3.34 13.64 7.01 31.39 32.95
2 74.04 3.51 13.94 7.25 31.86 32.04
21 1 73.50 3.53 14.06 7.64 28.83 30.62
2 71.16 2.98 13.43 7.53 29.04 30.13
22 1 69.13 3.08 12.77 5.61 29.88 30.49
2 71.76 2.64 12.98 5.72 29.38 30.56

a
Two laboratories excluded due to noncompliance.
 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 79
Reagents
Use only reagents of recognized analytical grade, unless
otherwise specified, and distilled or deionized water or water
of at least equivalent purity.
(a) Acid-detergent solution.—Add 20 g cetyl trimethyl-
ammonium bromide (technical grade) to 1 L 0.5 M H2SO4,
previously standardized. Agitate to aid solution.
(b) Sulfuric acid.—72% by weight. Standardize reagent
grade H2SO4 to specific gravity 1.634 at 20°C or 12.00 M/L.
Calculate grams acid and water needed to prepare 1000 mL of
solution using the following formula:
100 ´ 98.09 ´ 12
Grams acid needed =
H 2 SO 4 assay (%)
Grams water needed = (1000 ´ 1634
. ) - grams acid used
in which 1.634 is the density of 72% H2SO4.
Weigh water into 1000 mL volumetric flask and add the
calculated amount of H2SO4 slowly with occasional swirling.
The flask must be cooled in water in order to add the required
weight of acid. Cool to 20°C and check to make sure the
volume is correct. If the volume is too large, take out 5 mL
water and add 4.55 mL H2SO4. If the volume is too small, take
out 1.5 mL and add 2.5 mL water. Repeat if necessary.
Meniscus should be within 0.5 cm of the calibration
mark at 20°C.
(c) Filtration aid.—Diatomaceous earth, Celite;
acid-washed, Celite 545 AW; or equivalent.
(d) Acetone.—Technical grade.
(e) Antifoaming agent.—n-Octanol.
Apparatus
Usual laboratory apparatus and, in particular, the
following:
(a) Analytical balance.—Capable of weighing to the
nearest 0.1 mg, with readability to 0.1 mg.
(b) Analytical balance.—With a measuring range up to
2000–2500 g, with a readability to 1 g.
(c) Cyclone mill.—With 2 mm screen, or Cutter mill with
1 mm screen, or rotary mill with sieve containing 1 mm
apertures, or equivalent.
(d) Air ventilated oven.—Capable of operating at 105 ±
2°C and <60 ± 2°C, or vacuum oven set at 70°C, respectively.
(e) Refluxing apparatus with individual heating units and
cold water condensers.—Any conventional apparatus
suitable for crude fiber or NDF determinations. Test portions
should be extracted in 500 or 600 mL beakers without sprouts
(Pyrex No. 1040, or equivalent) that are covered with a round
cold-water condenser to minimize evaporation. Calibrate
heating unit settings so that 50 mL water boils within 4–5 min
when cold water condensers are used. Fibertec apparatus
(www.foss.dk) can be used and should boil 50 mL water
within 10 min. Note: This setting should result in significant
particle movement during refluxing in acid detergent.
(f) Fritted-disk crucibles.—Coarse porosity (pore size
40–60 mm) crucibles, 40–50 mL capacity or Fibertec P2 (pore
size 40–100 mm, 26–28 mL capacity). Clean new crucibles
and ash at 525°C for 1 h. Clean crucibles after each use by
ashing at 525°C for 3 h, removing ash by inverting in a
detergent solution and sonicating for 7–10 min. Rinse
crucibles in hot water, and soak in room temperature H2O for
at least 30 min.
Note: Occasionally test filtration rate as follows. Fill each
crucible with 50 mL distilled water (25 mL for Fibertec P2
crucibles) and record the time required to drain completely
without vacuum (should be 180 ± 60 s for Gooch or 75 ± 30 s
for P2). If drain time is <100 s (or <30 s for P2), discard
crucible. If it is <120 s (or <45 s for P2), check for cracks in
fritted disk. If filtration is >240 s (or >105 s for P2), clean
crucible with acid or alkaline cleaning solution (1). If cleaning
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does not improve filtration rate, discard crucible.
(g) Vacuum filter manifold.—Suitable apparatus (e.g.,
Fibertec-type cold extraction unit; www.foss.dk) that allows
adequate soaking of fibrous residues.
(h) Muffle or incineration furnace.—525 ± 15°C.
Preparation of Test Sample
Reduce laboratory sample to ca 100 g (dry weight
equivalent) and place half in a moisture-tight, sealed container
for total H2O determination. Dry remaining wet (>15%
moisture) materials to <15% moisture in a forced-draft oven at
<60°C. Drying at <60°C is necessary to prevent creation of
artifact fiber and lignin. Grind dry (<15% moisture) test
samples using a mill with a 1 mm screen or any grinder giving
similar particle size (geometric mean size 0.22–0.26 mm).
Grinding segregates materials, with the most fibrous matter
grinding through the screen last. Do not discard material in the
grinder, but add it to the ground test sample. Pre-extraction is
required for test samples containing >10% fat, and is
recommended for test samples containing >5% fat. Weigh test
samples into previously tared, oven-dried crucibles, place
crucible on filtering manifold, extract with 30–40 mL acetone
4 times (allow material to soak in acetone 3–5 min each time),
vacuum to remove all traces of acetone, air dry for 10–15 min,
and transfer residue to a reflux beaker for fiber analysis. Use
the same crucible to collect the fiber residue for each test
portion after acid detergent extraction.
Note 1: For Fibertec-type apparatus, place crucible in cold
extraction unit and fill with 25 mL acetone. Leave for 3–5 min
and filter by applying vacuum. Repeat 3 times. Wash
with water.
Note 2: To simplify filtration, 1.00 g Celite can be added to
the crucible before the sample.
Procedure
(a) ADF.—Dry empty crucibles in an oven at 105 ± 2°C
for >4 h (1 h if moved from the ashing furnace) and record tare
weight (W1).
Conventional-type apparatus.—Weigh (W2) ca 1 g test
portion of dried or as-received material to an accuracy of
± 2 mg (materials with >15% moisture should have the weight
adjusted to provide an equivalent amount of dry matter) into
beakers. At the same time, if conversion of results to dry
80 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009

Table 5. Reported results, ADF “as received” (% m/m)a

Lab Value Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

1 1 68.37 4.02 13.62 6.82 27.39 30.01

2 68.97 3.99 12.79 6.39 29.97 28.19
2 1 69.20 3.27 12.67 6.48 28.62 31.37
2 69.64 3.01 12.95 6.68 27.83 30.68
3 1 68.77 3.11 12.90 6.91 29.01 30.47
2 70.80 3.33 13.13 7.30 26.81 30.50
4 1 69.40 3.30 13.50 7.10 28.60 29.70
2 69.40 3.60 13.30 7.20 27.90 30.40
6 1 68.30 2.80 12.60 6.20 26.00 29.30

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2 67.90 2.70 12.20 6.20 26.40 29.90
7 1 69.07 4.39 13.03 6.63 29.11 30.24
2 70.00 3.24 12.60 6.66 29.59 30.76
8 1 70.92 3.72 14.42 7.25 29.19 30.15
2 70.40 3.68 14.72 7.77 28.20 30.14
9 1 65.59 6.65 14.59 8.14 28.58 31.05
2 65.53 6.06 13.82 8.41 28.49 30.90
10 1 70.11 3.62 13.53 7.56 29.16 31.83
2 71.79 2.93 13.43 7.62 31.71 32.85
11 1 70.98 3.36 13.80 6.89 28.50 31.37
2 70.92 3.36 13.62 6.89 28.66 30.69
12 1 71.30 3.60 13.60 6.80 29.30 31.30
2 71.50 3.70 13.60 6.90 29.00 31.50
13 1 68.90 1.50 12.00 5.90 26.40 28.10
2 67.80 3.30 12.40 5.70 26.50 29.56
14 1 64.00 1.90 11.20 6.80 26.20 27.00
2 62.90 2.30 11.00 5.40 25.00 27.90
15 1 74.36 3.65 15.66 7.78 28.19 35.36
2 69.18 3.73 15.08 7.12 33.07 33.80
16 1 86.45 5.09 21.84 18.12 34.12 35.03
2 74.35 4.45 15.86 24.90 34.43 37.47
17 1 67.34 3.14 13.09 6.45 27.47 29.53
2 67.68 3.31 13.00 6.56 27.19 29.56
19 1 64.17 2.24 11.13 7.09 26.01 29.59
2 64.22 2.05 11.61 9.21 25.43 27.40
20 1 70.08 3.03 12.94 6.59 29.41 31.83
2 70.04 3.16 12.67 6.81 29.95 30.95
21 1 69.90 3.20 13.10 7.20 27.10 29.70
2 67.60 2.70 12.50 7.10 27.30 29.20
22 1 65.60 2.80 11.90 5.30 28.00 29.70
2 68.10 2.40 12.10 5.40 27.50 29.70

a
Two laboratories excluded due to noncompliance.
 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 81

Table 6. Reported results, ADL content (% dry matter)a,b

Lab Value Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

1 1 20.15 1.03 4.45 0.47 3.07 2.48

2 20.04 1.01 4.05 0.35 3.84 2.59
2 1 20.08 0.77 3.78 0.29 3.81 2.62
2 20.12 0.64 4.12 1.24 3.71 2.72
3 1 20.70 0.79 4.12 0.38 4.05 2.66
2 19.50 0.75 3.72 0.51 3.26 2.44
4 1 20.00 1.00 4.42 0.64 4.23 3.21
2 20.22 0.78 4.53 0.53 4.23 3.43
6 1 20.32 1.11 4.31 0.64 3.72 2.63

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2 21.49 1.33 4.53 0.96 3.80 3.04
7 1 20.12 0.77 2.69 0.42 2.97 1.76
2 20.89 0.72 3.31 0.35 3.00 2.19
8 1 19.64 1.04 5.27 1.15 4.59 3.44
2 19.76 1.03 5.20 1.10 4.65 3.78
9 1 19.39 0.48 3.35 0.14 3.60 2.11
2 19.31 0.63 3.23 0.02 3.20 2.16
10 1 19.81 0.97 4.47 2.74 4.56 3.25
2 20.20 0.99 4.43 0.45 5.41 3.80
11 1 20.73 0.77 4.06 0.38 3.73 2.68
2 20.88 0.68 4.07 0.37 3.64 2.69
12 1 18.98 1.00 4.44 0.64 4.26 3.45
2 20.17 1.00 4.43 0.85 4.19 3.11
13 1 20.70 <1.5 4.44 <1.5 3.68 2.90
2 18.71 <1.5 4.46 <1.5 4.02 2.94
14 1 21.29 <1.5 3.79 <1.5 3.00 3.30
2 53.54 0.89 3.78 <1.5 3.65 2.48
15 1 21.39 1.72 5.39 1.15 3.91 4.12
2 21.06 0.88 5.44 0.64 5.30 3.93
16 1 20.73 0.77 4.06 0.38 3.73 2.68
2 20.88 0.68 4.07 0.37 3.64 2.69
17 1 20.03 0.89 4.29 0.48 4.50 3.28
2 20.60 1.03 4.26 0.49 4.31 3.34
18 1 20.21 <1.5 4.21 <1.5 4.03 2.88
2 20.38 <1.5 4.31 <1.5 4.35 2.77
19 1 18.58 0.95 3.67 0.49 4.01 3.40
2 20.37 1.44 3.93 0.76 5.00 3.14
20 1 21.02 0.91 3.93 0.42 3.81 3.33
2 21.29 0.96 3.92 0.70 4.27 3.04
21 1 21.45 <1.5 4.94 <1.5 5.43 4.64
2 21.16 <1.5 5.48 <1.5 5.53 4.85
22 1 20.63 <1.5 4.09 <1.5 4.27 2.77
2 21.65 <1.5 4.29 <1.5 4.27 2.65

a
One laboratory excluded due to noncompliance.
b
In compliance with the method draft, many laboratories reported values lower than 1.5% as <1.5%, while others reported the actual values
found.
82 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009

Table 7. Reported ADL content “as received” (% m/m)a

Lab Value Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

1 1 19.05 0.93 4.12 0.44 2.88 2.41

2 18.95 0.91 3.75 0.33 3.60 2.51
2 1 18.98 0.69 3.49 0.27 3.57 2.54
2 19.02 0.58 3.81 1.16 3.47 2.64
3 1 19.64 0.72 3.84 0.36 3.78 2.59
2 18.52 0.68 3.47 0.48 3.04 2.37
4 1 18.90 0.90 4.10 0.60 3.90 3.10
2 19.10 0.70 4.20 0.50 3.90 3.30
6 1 19.10 1.00 4.00 0.60 3.40 2.50

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2 20.20 1.20 4.20 0.90 3.50 2.90
7 1 19.04 0.70 2.50 0.40 2.77 1.71
2 19.79 0.65 3.07 0.33 2.77 2.12
8 1 18.68 0.95 4.92 1.09 4.32 3.36
2 18.78 0.94 4.85 1.04 4.37 3.68
9 1 18.29 0.44 3.11 0.13 3.34 2.04
2 18.23 0.57 3.00 0.02 2.98 2.09
10 1 18.76 0.88 4.16 2.59 4.22 3.15
2 19.13 0.90 4.11 0.43 5.01 3.66
11 1 19.68 0.70 3.79 0.36 3.51 2.60
2 19.81 0.62 3.80 0.35 3.43 2.62
12 1 17.90 0.90 4.10 0.60 4.00 3.30
2 19.00 0.90 4.10 0.80 3.90 3.00
13 1 19.50 <1.5 4.10 <1.5 3.40 2.80
2 17.70 <1.5 4.10 <1.5 3.70 2.81
14 1 20.10 <1.5 3.50 <1.5 2.80 3.20
2 50.60 0.80 3.50 <1.5 3.40 2.40
15 1 20.29 1.56 5.03 1.08 3.69 4.01
2 19.99 0.80 5.06 0.60 5.00 3.82
16 1 65.64 10.57 7.47 0.88 3.61 2.86
2 18.46 3.17 3.94 0.97 3.57 2.68
17 1 18.96 0.80 3.98 0.45 4.24 3.18
2 19.50 0.93 3.95 0.46 4.05 3.24
18 1 19.10 <1.5 3.90 <1.5 3.80 2.80
2 19.30 <1.5 4.00 <1.5 4.10 2.70
19 1 17.62 0.86 3.40 0.46 3.67 3.26
2 19.32 1.30 3.64 0.71 4.58 3.01
20 1 19.89 0.82 3.65 0.40 3.57 3.22
2 20.14 0.87 3.64 0.66 4.02 2.94
21 1 20.40 <1.5 4.60 <1.5 5.10 4.50
2 20.10 <1.5 5.10 <1.5 5.20 4.70
22 1 19.58 <1.5 3.81 <1.5 4.00 2.70
2 20.55 <1.5 4.00 <1.5 4.00 2.58

a
Two laboratories excluded due to noncompliance.
 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 83

Table 8. Reported results, ADF content (% dry matter), ash correcteda

Lab Value Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

1 1 73.18 3.86 13.00 6.65 28.00 27.60

2 73.12 3.02 13.05 6.67 27.58 27.58
2 1 71.96 3.35 12.58 6.71 28.33 28.80
2 72.48 3.45 12.78 6.55 28.17 28.52
3 1 74.41 3.82 13.56 7.02 29.80 28.96
2 74.32 3.61 13.65 7.05 29.68 29.16
4 1 72.18 3.55 13.81 6.92 30.08 28.41
2 73.45 3.65 13.70 7.35 29.51 29.58
6 1 72.34 3.21 12.51 6.72 28.04 28.77

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2 72.13 3.32 12.73 6.72 27.80 28.75
7 1 72.93 3.35 12.99 6.39 31.06 29.07
2 72.86 3.51 13.82 6.92 30.96 29.33
8 1 74.20 3.64 13.48 7.67 29.00 28.88
2 73.98 3.58 13.52 8.00 29.46 28.90
9 1 69.87 5.71 13.72 8.32 29.67 29.58
2 69.36 5.82 14.49 9.85 31.73 30.08
10 1 76.12 3.66 13.75 8.24 28.96 29.21
2 75.77 3.74 13.41 8.66 28.36 29.86
11 1 74.44 3.42 14.01 7.16 29.11 29.12
2 74.69 3.74 13.80 7.15 29.08 29.10
12 1 74.23 3.34 13.20 6.40 29.21 28.87
2 75.16 3.33 13.30 6.70 29.54 29.74
13 1 72.51 <1.5 12.24 5.88 29.04 28.22
2 71.35 3.33 12.72 5.99 28.91 28.45
14 1 67.69 1.99 12.01 7.15 28.19 27.66
2 66.46 2.45 11.78 5.66 26.72 28.72
15 1 81.94 3.94 16.33 16.86 33.11 32.83
2 79.44 4.07 20.51 11.09 34.49 32.36
16 1 74.44 3.42 14.01 7.16 29.11 29.12
2 74.69 3.74 13.80 7.15 29.08 29.10
17 1 70.60 3.39 12.45 6.66 28.01 27.91
2 71.35 3.25 12.10 6.59 28.79 28.41
19 1 68.74 3.17 11.90 6.92 30.13 27.36
2 66.51 3.36 11.61 7.07 27.10 27.40
20 1 75.60 3.57 13.20 7.12 31.76 31.14
2 74.65 3.51 13.46 6.76 30.23 29.39
21 1 73.19 3.20 14.48 7.96 28.19 30.52
2 71.16 2.98 13.00 7.42 28.40 30.34
22 1 71.02 3.19 12.02 6.25 27.00 27.00
2 71.02 3.41 11.05 6.36 27.67 27.67

a
Two laboratories excluded due to noncompliance.
84 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009

Table 9. Reported results, ADF content “as received” (% m/m), ash correcteda
Lab Value Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

1 1 69.20 3.47 12.04 6.24 26.30 26.82

2 69.14 2.72 12.07 6.26 25.87 27.03
2 1 68.01 3.01 11.63 6.29 26.53 27.94
2 68.50 3.10 11.82 6.14 26.38 27.66
3 1 70.73 3.47 12.63 6.62 27.79 28.17
2 70.63 3.28 12.72 6.65 27.70 28.37
4 1 68.20 3.20 12.80 6.50 27.20 27.40
2 69.40 3.30 12.70 6.90 27.70 28.50
6 1 68.00 2.90 11.60 6.30 25.60 27.30

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2 67.80 3.00 11.80 6.30 25.60 27.40
7 1 69.02 3.03 12.06 6.02 28.96 28.21
2 69.02 3.18 12.83 6.49 28.63 28.38
8 1 70.58 3.31 12.58 7.25 27.29 28.20
2 70.30 3.26 12.61 7.57 27.68 28.17
9 1 65.91 5.18 12.75 7.84 27.56 28.58
2 65.46 5.28 13.48 9.29 29.54 29.05
10 1 72.11 3.32 12.78 7.76 26.81 28.27
2 71.78 3.38 12.46 8.16 26.30 28.72
11 1 70.67 3.10 13.06 6.76 27.41 28.28
2 70.87 3.39 12.87 6.75 27.40 28.29
12 1 70.00 3.00 12.20 6.00 27.40 27.60
2 70.80 3.00 12.30 6.30 27.50 28.70
13 1 68.30 <1.5 11.30 5.50 26.80 27.20
2 67.50 3.00 11.70 5.60 26.60 27.20
14 1 63.90 1.80 11.10 6.70 26.30 26.80
2 62.80 2.20 10.90 5.30 24.90 27.80
15 1 77.72 3.57 15.23 15.88 31.18 31.95
2 75.38 3.69 19.09 10.45 32.56 31.44
16 1 69.20 1.47 8.74 3.10 26.30 26.82
2 69.14 2.72 7.08 6.10 25.87 27.03
17 1 66.82 3.05 11.54 6.25 26.37 27.02
2 67.54 2.93 11.22 6.19 27.07 27.52
19 1 65.17 2.86 11.02 6.49 27.59 26.26
2 63.07 3.03 10.76 6.64 24.82 26.23
20 1 71.52 3.23 12.26 6.69 29.76 30.08
2 70.62 3.17 12.49 6.35 28.42 28.39
21 1 69.60 2.90 13.50 7.50 26.50 29.60
2 67.60 2.70 12.10 7.00 26.70 29.40
22 1 67.40 2.90 11.20 5.90 25.30 26.30
2 67.40 3.10 10.30 6.00 25.90 26.90

a
Two laboratories excluded due to noncompliance.
 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 85

Table 10. Interlaboratory study results for determination of moisture content

Parameter Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

Number of laboratories after elimination of outliers 22 22 22 22 22 22

Average moisture content, % m/m 5.323 9.562 7.186 5.936 6.644 3.228
Repeatability standard deviation (sr), % m/m 0.083 0.120 0.087 0.075 0.225 0.236
Repeatability relative standard deviation, % 1.6 1.3 1.2 1.3 3.4 7.3
Repeatability limit r (r = 2.77 ´ sr), % m/m 0.230 0.332 0.241 0.208 0.623 0.654
HorRat value 0.759 0.668 0.619 0.625 1.705 3.307
Reproducibility standard deviation (sR), % m/m 0.109 0.365 0.395 0.337 0.859 0.619
Reproducibility relative standard deviation, % m/m 2.0 3.8 5.5 5.7 12.9 19.2
Reproducibility limit R (R = 2.77 ´ sR), % m/m 0.302 1.011 1.094 0.933 2.379 1.715

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HorRat value 1.761 1.339 1.851 1.854 4.298 5.722

matter basis is required, weigh a test portion for H2O
determination of the test sample. Allocate 2 blanks (no test
material) for the first set of 24 analyses and 1 additional blank
for each additional 12 analyses within a run. Immediately
before refluxing (do not add acid detergent >10 min before
refluxing) add 100 mL of acid detergent solution at room
temperature. Heat to boiling in 5–10 min and, if necessary,
reduce heat slightly to avoid foaming, but provide moderate
particle agitation. After 5–10 min of refluxing, rinse down
sides of beaker using a fine stream of acid detergent solution
(add <5 mL). Reflux for 60 ± 5 min from the time of onset
of boiling. Remove each beaker from heating unit, swirl, and
filter into crucible. Without inverting beaker, use a fine stream
of boiling water to rinse all particles into the crucible. Remove
acid detergent and rinse water using minimum vacuum. Close
vacuum and fill crucible with ca 40 mL of hot (90–100°C)
water, stir to break up the residue filter mat, and let soak
3–5 min. Repeat water soaking twice and vacuum dry. Rinse
sides and bottom of crucible to be sure that all traces of acid
are removed (any residual acid will be concentrated during
drying and cause charring of residues and low fiber values).
Add 30–40 mL acetone, stir to break up all lumps and expose
all particles to acetone, soak 3–5 min and repeat until no color
is removed (typically 2 acetone soakings are sufficient).
Remove residual acetone with vacuum, dry >3 h, preferably
overnight, at 105°C in a forced-draft oven and weigh (W3).
Cool at least 15 min in desiccator and always weigh crucibles
in the same order. Check balance zero after each weight if
crucibles are warm.
Fibertec-type apparatus.—Weigh 1 g (W2) of sample to an
accuracy of ±2 mg into a predried crucible (W1). To simplify
filtration, 1.00 g Celite can be added to the crucible before the
sample. Place crucible in the Fibertec hot extraction unit and
add 100 mL cold acid detergent solution. Add 2–4 drops
n-octanol to prevent foaming and heat to boiling. Adjust
heater and let boil for 60 min. Boiling time is measured from
the time when the solution has reached the boiling point.
Remove acid detergent solution and wash 3 times with hot
deionized water. Use 30 mL portions of water and vacuum dry
between washings. Place crucible in cold extraction unit and
fill crucible with 25 mL acetone. Filter. Repeat one time.
Evaporate solvent and dry crucibles at 130 ± 2°C for 2 h or at
105 ± 2°C for at least 5 h. Cool to room temperature in
desiccator and weigh to nearest 0.0001 g (W3).
Note: If the ADF is to be determined on the basis of organic
matter, i.e., ash corrected, ignite crucible in 525°C furnace 3 h
or until C-free. Place crucible, while still hot, into 105°C
forced-draft oven 1 h. Transfer to desiccator, cool, and
weigh (W6). In this case, a subsequent determination of the
ADL is not possible.
(b) ADL.—Conventional-type apparatus.—Place crucible
containing fiber in 50 mL beaker for support or arrange
crucibles in shallow enamel pan. Cover contents of crucible
with cooled (15°C) 72% H2SO4 and stir with glass rod to
smooth paste, breaking all lumps. Fill crucible about halfway
with acid and stir. Leave glass rod in crucible; refill with 72%
H2SO4 and stir hourly as acid drains, keeping crucible at
20–23°C (cool if necessary). After 3 h, filter as completely as
possible with vacuum, and wash with hot H2O until acid-free
to pH paper. Rinse sides of crucible and remove stirring rod.
Dry crucible >3 h in 105°C forced-draft oven, cool in
desiccator, and weigh (W4). Ignite crucible in 525°C furnace
3 h or until C-free. Place crucible while still hot into 105°C
forced-draft oven 1 h. Transfer to desiccator, cool, and
weigh (W5).
Fibertec-type apparatus.—Place a glass rod into each
crucible for stirring. Add 25 mL 72% H2SO4 cooled to 15°C.
Stir with glass rod and filter off after 3 h. Stir at hourly
intervals. Alternatively, use reversed pressure for breaking
lumps. Wash with water until free from acid. Evaporate
solvent and dry crucibles at 130 ± 2°C for 2 h or at 105 ± 2°C
for at least 5 h. Cool to room temperature in desiccator and
weigh (W4). Ash the sample in the crucible at 525 ± 15°C for
at least 3 h. Cool to room temperature in a desiccator and
weigh (W5).
86 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009

Table 11. Interlaboratory study results for the determination of ADF (dry matter)

Parameter Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

Number of laboratories after elimination of outliers 19 18 18 17 17 20

Average ADF value, g/100 g 72.69 3.54 14.07 7.24 29.98 31.44
Repeatability standard deviation, sr, g/100 g 0.946 0.303 0.302 0.324 0.56 0.648
Coefficient of variation of repeatability, % 1.3 8.6 2.1 4.5 1.9 2.1
Repeatability limit r (r = 2.8 ´ sr), g/100 g 2.65 0.85 0.85 0.91 1.87 1.81
HorRat value 0.9 3.9 1.2 2.3 1.2 1.3
Reproducibility standard deviation, sR, g/100 g 2.443 0.626 0.853 0.561 1.126 1.571
Coefficient of variation of reproducibility, % 3.4 17.7 6.1 7.7 3.8 5.0
Reproducibility limit R (R = 2.8 ´ sR), g/100 g 6.84 1.75 2.39 1.57 3.15 4.40

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HorRat value 1.6 5.4 2.3 2.6 1.6 2.1

Quality assurance.—Include at least one in-house
reference or quality control (QC) sample and 2 blanks for the
first 20–30 samples in a run and add 1 QC and 1 blank for each
additional 20–30 samples analyzed. 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 beginning and end of the run. Change
in weights of blank crucibles should be <0.0100 g after either
extraction or ashing. If weights of blank crucibles change by
more than 10 mg or crucible weights after ashing are less than
empty crucible weights, suspect inadequate cleaning of
crucibles or problems with weighing technique.
Calculation and Expression of Results
ADFAR, ash corrected = 100 ´ [(W3 – W6) – (B6 – B1)]/W2
For dried materials (laboratory sample is dried to prepare
test sample):
% ADF, dry matter (DM) basis:
ADFDM = 100 ´ [(W3 – W1) – (B3 – B1)]/
(W2 ´ % DM/100)
% ADF, DM basis, ash corrected (Note: Not applicable with
subsequent ADL determination):
ADFDM, ash corrected = 100 ´ [(W3 – W6) – (B6 – B1)]/
(W2 ´ % DM/100)

(a) ADF.—For undried materials (test sample same
moisture basis as laboratory sample):
% ADF, as-received (AR) basis:
ADFAR = 100 ´ [(W3 – W1) – (B3 – B1)]/W2
% ADF, AR basis, ash corrected:
where B1 is the average weight of all blanks after oven drying
before acid detergent extraction; B3 is the average weight of
all blanks after oven drying after acid detergent extraction; B6
is the average weight of all blanks after acid detergent
extraction and ashing; W1 is the crucible weight; W2 is the test
sample weight; W3 is the crucible and residue weight; and W6
is the crucible weight after ashing.

Table 12. Interlaboratory study results for the determination of ADF (“as received”)

Parameter Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

Number of laboratories after elimination of outliers 19 19 20 19 19 20

Average moisture content, % m/m 68.703 3.274 13.047 6.803 27.981 30.321
Repeatability standard deviation (sr), % m/m 1.144 0.284 0.281 0.292 0.776 0.663
Repeatability relative standard deviation, % 1.7 8.7 2.2 4.3 2.8 2.2
Repeatability limit r (r = 2.77 ´ sr), % m/m 3.204 0.796 0.787 0.818 2.173 1.856
HorRat value 1.2 3.9 1.2 2.2 1.7 1.4
Reproducibility standard deviation (sR), % m/m 2.475 0.681 1.053 0.728 1.469 1.631
Reproducibility relative standard deviation, % m/m 3.6 20.8 8.1 10.7 5.3 5.4
Reproducibility limit R (R = 2.77 ´ sR), % m/m 6.929 1.906 2.949 2.038 4.114 4.566
HorRat value 1.7 6.2 3.0 3.6 2.2 2.2
 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 87

Table 13. Interlaboratory study results for the determination of ADL (dry matter)

Parameter Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

Number of laboratories after elimination of outliers 20 14 20 15 18 19

Average ADL value, g/100 g 20.33 0.88 4.29 0.58 4 3
Repeatability standard deviation, sr, g/100 g 0.59 0.08 0.152 0.225 0.334 0.206
Coefficient of variation of repeatability, % 2.9 9.1 3.5 39 8.3 6.9
Repeatability limit r (r = 2.8 ´ sr), g/100 g 1.651 0.224 0.426 0.631 0.931 0.578
HorRat value 1.7 3.4 1.7 13.6 3.9 3.1
Reproducibility standard deviation, sR, g/100 g 0.745 0.185 0.53 0.303 0.515 0.479
Coefficient of variation of reproducibility, % 3.7 21.1 12.3 52.6 12.9 16
Reproducibility limit R (R = 2.8 ´ sR), g/100 g 2.087 0.519 1.484 0.849 1.442 1.34

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HorRat value 1.4 5.2 3.8 12.1 4 4.7

(b) ADL.—For undried materials (test sample same
moisture basis as laboratory sample):
% H2SO4 lignin, AR basis:
ADLAR = 100 ´ [(W4 – W5) – (B4 – B5)]/W2
For dried materials (laboratory sample is dried to prepare
test sample):
% H2SO4 lignin, DM basis:
ADLDM = 100 ´ [(W4 – W5) – (B4 – B5)]/(W2 ´ % dry matter/100)
where B4 is the average weight of all blanks after oven drying
before ashing; B5 is the average weight of all blanks after oven
drying after ashing; W2 is the test sample weight; W4 is the
crucible and residue weight after drying; and W5 is the crucible
weight after ashing.
(c) Expression of results.—Results should be reported to
the nearest 0.1%, and results <1.0% ADF or <1.5% ADL
should be reported as "ADF <1.0%" or "ADL <1.5%."
Results and Discussion
The data submitted for the study are presented in
Tables 3–10.
Statistical Analysis
Of the 22 participating laboratories, 4 used conventional or
other equipment and 18 used the Fibertec system. One
laboratory had to be excluded due to noncompliance with the
drafted method. Another had to be excluded for incorrect
execution of the ADF part of the drafted method. This had no
effect on their results for the ADL part. Among the remaining
laboratories, 11 reported results with no outliers at all. An
analysis of the outliers according to Cochran and Grubbs (9)
showed that 2 laboratories accounted for 48% of all
outlying results.
The statistical analysis was made in accordance with
ISO 5725 (9) using the Excel spreadsheet CLSTD.XLT
Version 4.0 from Ken Mathieson, CSL, York, UK. The results
are summarized in Tables 10–16.

Table 14. Interlaboratory study results for the determination of ADL (“as received”)

Parameter Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

Number of laboratories after elimination of outliers 19 14 20 15 21 21

Average moisture content, % m/m 19.215 0.823 3.936 0.582 3.790 2.943
Repeatability standard deviation (sr), % m/m 0.596 0.109 0.168 0.213 0.352 0.207
Repeatability relative standard deviation, % 3.1 13.3 4.3 36.7 9.3 7.0
Repeatability limit r (r = 2.77 ´ sr), % m/m 1.669 0.306 0.470 0.598 0.985 0.580
HorRat value 1.8 4.9 2.0 12.8 4.3 3.1
Reproducibility standard deviation (sR), % m/m 0.736 0.188 0.568 0.298 0.611 0.619
Reproducibility relative standard deviation, % m/m 3.8 22.8 14.4 51.2 16.1 21.0
Reproducibility limit R (R = 2.77 ´ sR), % m/m 2.062 0.525 1.591 0.834 1.712 1.733
HorRat value 1.5 5.5 4.4 11.8 4.9 6.2
88 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009

Table 15. Interlaboratory study results for the determination of ADF (dry matter), ash-corrected

Parameter Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

Number of laboratories after elimination of outliers 19 17 18 17 18 17

Average ADF value, g/100 g 72.48 3.48 13.16 6.99 29.01 28.86
Repeatability standard deviation, sr, g/100 g 0.665 0.187 0.348 0.212 0.557 0.362
Coefficient of variation of repeatability, % 0.9 5.4 2.6 3.0 1.9 1.3
Repeatability limit r (r = 2.8 ´ sr), g/100 g 1.86 0.53 0.97 0.59 1.56 1.01
HorRat value 0.7 2.5 1.5 1.5 1.2 0.8
Reproducibility standard deviation, sR, g/100 g 2.465 0.256 0.754 0.615 1.195 0.809
Coefficient of variation of reproducibility, % 3.4 7.4 5.7 8.8 4.1 2.8
Reproducibility limit R (R = 2.8 ´ sR), g/100 g 6.90 0.72 2.11 1.73 3.35 2.27

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HorRat value 1.6 2.2 2.1 2.9 1.7 1.2

Moisture
The study samples had a moisture content of 3–9.5%
(m/m), which is outside the validated range of the used
method (10). ISO 6496:1999, applying oven drying for 4 h at
103°C, has been validated in the range of 8–86% with a
repeatability limit of r = 0.55–1.5 and a reproducibility limit of
R = 1.6–3.5. As the results in Table 10 indicate, the
performance of this method at lower moisture contents is
also acceptable.
ADF
ADF had actually been determined in 2 independent trials,
once in combination with the subsequent determination of
ADL (Tables 11 and 12 and Figure 2) and once in combination
with the determination of the ash-corrected value for the ADF
content (Tables 15 and 16). The reason for this was to see
whether improved method performance data could be
obtained, analogous to the NDF study (4), by reporting the
ash-corrected values for the ADF.
The difference of the mean values from both runs was less
than one standard deviation of the repeatability, indicating the
robustness of the method.
The performance data in the form of relative errors
(coefficient of variation of repeatability and reproducibility)
were better than 10%, except for sample 2 (wheat; Figure 2).
These data are comparable with those obtained in the NDF
study (4) and fulfill the demands of the ISO. There were no
significant differences in performance for ADF values “as
received” versus on the basis of dry matter.
Comparable performance data for the AOAC Method
973.18 were not available.
ADL
The results of the statistical evaluation for the
determination of ADL are given in Tables 13 and 14 and
Figure 3.
In compliance with the method draft, many laboratories
reported ADL values lower than 1.5% as <1.5%. These values
have been excluded from the statistical evaluation, which
explains the lower number of laboratories for samples 2

Table 16. Interlaboratory study results for the determination of ADF (“as received”), ash-corrected

Parameter Sawdust Wheat Cattle feed Soy meal Clover silage Grass silage

Number of laboratories after elimination of outliers 20 17 19 19 19 19

Average ADF content after correction for ash, % m/m 68.920 3.139 12.087 6.504 26.957 27.831
Repeatability standard deviation (sr), % m/m 0.705 0.206 0.347 0.335 0.678 0.447
Repeatability relative standard deviation, % 1.0 6.6 2.9 5.1 2.5 1.6
Repeatability limit r (r = 2.77 ´ sr), % m/m 1.974 0.577 0.970 0.937 1.898 1.252
HorRat value 0.7 3.0 1.6 2.6 1.6 1.0
Reproducibility standard deviation (sR), % m/m 2.905 0.249 0.764 0.580 1.166 0.935
Reproducibility relative standard deviation, % m/m 4.2 7.9 6.3 8.9 4.3 3.4
Reproducibility limit R (R = 2.77 sR), % m/m 8.135 0.697 2.140 1.623 3.264 2.617
HorRat value 2.0 2.4 2.3 3.0 1.8 1.4
 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 89

As the performance of the studied method was acceptable,

both ISO and CEN have published the proposed method as a
standard method (15, 16). As the method is based on the
AOAC Official Method 973.18, a globally harmonized
protocol for the determination of ADF and ADL could
be established.

Acknowledgments

We thank Marianne Rydälv (FOSS Analytical AB) for

assistance with preparing, splitting, and sealing the samples,
as well as with the homogenization and stability tests. We also
thank the following collaborators for their participation in
the study:

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Jaroslava Petrova, ÚKZÚZ NRL RO, Praha, Czech
Republic
Jochen Kühl, LUFA, Speyer, Germany
Figure 2. Change in the coefficient of variation of Michael Egert, LUFA Nord-West, Oldenburg, Germany
repeatability and reproducibility as a function of the
Saskia Plos, Institute of Animal Science, Bonn, Germany
ADF content.
Jens Schönherr, LUFA, Leipzig, Germany
Katharina Prinze, LUFA NRW, Münster, Germany
Hartmut Horst, Landesbetrieb Hessisches Landeslabor,
(wheat) and 4 (soy meal). As can be seen, unacceptable high Kassel, Germany
values for the precision have been obtained for sample 4, with Jürgen Danier, Bioanalytik, Weihenstephan, Germany
coefficients of variation for the repeatability and Liane Hüther, Federal Agricultural Research Centre,
reproducibility higher than 50%. It is, therefore, Braunschweig, Germany
recommended to report values lower than 1.5% as <1.5%. Judit Marth-Schill, National Institute for Agricultural
Ash-Corrected Values for ADF Quality Control, Budapest, Hungary
Waldemar Korol, National Feed Laboratory of NRIAP,
On the basis of the experiences from the validation of the Lublin, Poland
NDF method (4), it was anticipated that better performance Jacek Skomial, Kielanowski Institute of Animal
data could be obtained by reporting ash-corrected values. This Physiology and Nutrition of the Polish Academy of Sciences,
is normally not possible because of the subsequent ADL Warzaw, Poland
determination and would demand a separate ADF
determination or an ADF determination only.
As this option was not part of the draft, the participants
were asked, on a voluntary basis, to make a separate ADF
determination with a following ash determination. The results
of the statistical evaluation for the ash-corrected ADF values
are given in Tables 15 and 16. Generally, lower results were
achieved by correction for the ash content after the ADF
extraction. These differences are, with the exception for grass
silage, within the repeatability limits. Also, the performance
parameters of the method could be improved slightly. Again,
these improvements are within the uncertainty of the
determination of these values.

Conclusions

The use of HorRat values in empirical gravimetric methods

for the determination of fiber fractions may lead to spurious
conclusions regarding the method performance. Methods
where the analyte is empirically defined are traditionally
prone to greater inherent variability than methods that are
calibrated against a reference standard (14). Because the Figure 3. Change in the coefficient of variation of
analyte is defined by the method, it is crucial that the method repeatability and reproducibility as a function of the
protocols are followed exactly. ADL content.
90 MÖLLER: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009
Olga Moreira, Animal Nutrition and Feeding Department,
Vale de Santarém, Portugal
Katalin Fütö, FOSS AB, Höganäs, Sweden
S. Janjira, Bangkok Feedmill Co., Ltd, Samutprakarn,
Thailand
Klinsunkon Boonnayanont, Beta Agro Group plc,
Prapradeang, Thailand
Preamwadee Losittisak, Central Food Products Co., Ltd,
Klongluang Patunthani, Thailand
Vanvimol Rungrojpanich, Top Feed Mills Co., Ltd,
Latlumkaeo, Thailand
Ratana Kongchuensin, Thai Feed Mills Co., Ltd,
Samutsakorn, Thailand
Ratchai Siourai, Feed Quality Control Laboratory,
Bangkadi, Muang, Thailand
Jantakarn Arananant, Bureau of Quality Control of
Livestock Products, Pathumthani, Thailand
S. Titya, Charoen Phokphand Foods plc, Samut-Sakorn,
Thailand
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