AOAC Official Method 2011.25 supplies/fisherbrand.

html) or 250 mL polypropylene bottles with

Insoluble, Soluble, and Total Dietary Fiber in Foods polypropylene caps.
Enzymatic-Gravimetric-Liquid Chromatography (c)  Fritted crucible.—Gooch, fritted disk, Pyrex® 50 mL,
First Action 2011 pore size, coarse, ASTM 40–60 µm (Corning No. 32940-50C®
[Applicable to plant material, foods, and food ingredients or equivalent; http://www.labplanet.com/corning-crucible-gooch-
consistent with CODEX Alimentarius Commission high-c-50-ml-32940-50c.html). Prepare four for each sample as
Definition adopted in 2009 and modified slightly in 2010 follows: Ash overnight at 525°C in muffle furnace. Cool furnace to
(ALINORM 09/32/REP and ALINORM 10/33/REP, respectively), 130°C before removing crucibles to minimize breakage. Remove
including naturally occurring, isolated, modified, and synthetic any residual Celite and ash material by using a vacuum. Soak in 2%
polymers meeting that definition.] cleaning solution, C(i), at room temperature for 1 h. Rinse crucibles
See Tables 2011.25A–H for the results of the interlaboratory with water and deionized water. For final rinse, use 15 mL acetone
study supporting acceptance of the method. and air dry. Add approximately 1.0 g Celite to dried crucibles and
A.  Principle dry at 130°C to constant weight. Cool crucible in desiccators for
approximately 1 h and record mass of crucible containing Celite.
A method is described for the measurement of insoluble, (d)  Filtering flask.—Heavy-walled, 1 L with side arm.
soluble, and total dietary fiber (IDF, SDF, and TDF, respectively), (e)  Rubber ring adaptors.—For use to join crucibles with
inclusive of the resistant starch (RS) and the water:alcohol soluble filtering flasks.
nondigestible oligosaccharides and polysaccharides of DP ≥3. The (f)  Vacuum source.—Vacuum pump or aspirator with regulator
method combines the key attributes of AOAC Official Methods of capable of regulating vacuum.
AnalysisSM 985.29 (and its extensions, 991.42 and 993.19), 991.43, (g)  Water bath(s).—Rotary motion, large-capacity (20–24 L)
2001.03, and 2002.02. Duplicate test portions are incubated with with covers; capable of maintaining temperature of 37 ± 1°C and
pancreatic a-amylase and amyloglucosidase (AMG) for 16 h at 60 ± 1°C; equipped with automatic timers for on-off operation or
37°C in sealed 250 mL bottles while mixing with sufficient vigor equivalent (e.g., Grant® OLS 200 shaking incubation bath; http://
to maintain continuous suspension. During this step, nonresistant
www.bioresearchonline.com/product.mvc/Grant-OLS200-Orbital-
starch is solubilized and hydrolyzed to glucose and maltose by the
Shaking-Water-Bath-0001). Ensure that shaking action/sample
combined action of the two enzymes. The reaction is terminated by
agitation in water bath used is sufficient to maintain sample solids
pH adjustment and temporary heating. Protein in the sample is then
in suspension and no residue build-up or rings of sample material
digested with protease. For the measurement of IDF, the digestate is
form in the digestion bottle during the enzymatic digestions. A
filtered and the IDF is determined gravimetrically after correction
linear motion (back and forth shaker) can be used if the bottles are
for any protein or ash in the residue. For the measurement of the
placed at 45° to ensure adequate agitation (if the bottles are vertical
water soluble, but water:alcohol insoluble dietary fiber (SDFP),
or horizontal there will not be sufficient agitation to ensure that
ethanol is added to the filtrate of the IDF; the precipitated SDFP
the sample remains suspended). Alternatively, a 2 mag Mixdrive
is captured by filtration and determined gravimetrically after
15 submersible magnetic stirrer apparatus (http://www.2mag.de/
correction for any protein or ash in the precipitate. Nonprecipitable,
water:alcohol soluble dietary fiber (SDFS) in the filtrate is recovered english/stirrer/multiple/stirrer_multiple_04_mixdrive6_15.html)
by concentrating the filtrate, deionizing through ion exchange can be used in a water bath maintained at 37 ± 1°C with a Julabo
resins, concentrating, and quantitating by LC, or, alternatively, circulating heater (http://www.julabo.de/). Samples are stirred
by concentrating the filtrate and simultaneously deionizing and in digestion bottles, (b), with a 7 × 30 mm stir bar at ~170 rpm
quantitating by LC. (Figure 2011.25A).
(h)  Balance.—0.1 mg readability, accuracy, and precision.
B.  Apparatus
(i)  Ovens.—Two, mechanical convection, set at 105 ± 2°C and
(a)  Grinding mill.—Centrifugal, with 12-tooth rotor and 0.5 mm 130 ± 3°C, respectively.
sieve, or similar device. Alternatively, a cyclone mill can be used (j)  Timer.
for small test laboratory samples provided the mill has sufficient air (k)  Desiccator.—Airtight, with SiO2 or equivalent desiccant.
flow or other cooling to avoid overheating samples Desiccant dried biweekly overnight in 130°C oven.
(b)  Digestion bottles.—250 mL Fisherbrand® soda glass, wide- (l)  pH meter.
mouthed bottles with polyvinyl-lined cap (Cat. No. FB73219, (m)  Pipettors and tips.—Positive displacement 50–200 µL and
equivalent: Cat. No. 02-911-916; Fisher USA, www.fisher.co.uk/ 5 mL capacity.

Table  2011.25A.  Interlaboratory study results for IDF

Sample/parameter A B C D E F G H
No. of labs 14 15 15 15 13 13 12 13
Mean, % 25.74 4.79 9.23 1.05 8.26 11.63 11.52 9.90
Sr 0.52 0.18 0.71 0.13 0.19 0.48 0.33 0.29
SR 1.23 0.47 2.24 0.42 0.48 1.12 1.21 0.77
RSDr 2.03 3.76 7.67 12.25 2.26 4.11 2.87 2.98
RSDR 4.76 9.82 24.35 39.64 5.81 9.65 10.53 7.80
HorRat 1.94 3.11 8.51 9.98 2.00 3.49 3.80 2.75

© 2012 AOAC INTERNATIONAL

Table  2011.25B.  Interlaboratory study results data for SDF
Sample/parameter A B C D E F G H
No. of labs 14 14 15 15 14 13 15 13
Mean, % 3.80 5.58 17.32 10.74 12.94 4.56 11.29 8.30
Sr 0.44 0.28 1.03 0.46 0.50 0.83 0.71 0.51
SR 0.99 0.85 1.30 1.12 1.52 1.66 1.61 1.33
RSDr 11.71 5.10 5.97 4.33 3.86 18.10 6.29 6.18
RSDR 26.20 15.20 7.53 10.46 11.73 36.40 14.25 16.07
HorRat 8.01 4.92 2.89 3.74 4.31 11.44 5.13 5.53

(n)  Dispensers.—(1)  15 ± 0.5 mL for 78% EtOH or industrial column cartridges in series, cation cartridge preceding anion
methylated spirits (IMS), 95% ethanol or IMS, and acetone. (2)  40 cartridge.
± 0.5 mL for buffer. (u)  Liquid chromatograph.—(1)  Pump.—Capable of precision
(o)  Cylinder.—Graduated, 500 mL. metering at 0.5 mL/min at up to 10 000 psi. Arrangement for
(p)  Magnetic stirrers and stirring bars. HPLC of manually desalted samples on Waters Sugar-Pak®
(q)  Rubber spatulas. column (http://www.waters.com/waters/support.htm?locale=?&
(r)  Muffle furnace.—525 ± 5°C. cid=513789&lid=10069619) is shown in Figure 2011.25C. [If
(s)  Polypropylene columns.—(If manual deionization is using Sugar-Pak column and simultaneous on line deionization
carried out.) For the interlaboratory study, Alltech SPE cartridges and quantitation, a second pump capable of precision metering at
[Extract-CleanTM reservoirs (15 mL), Cat. No. 210315; http://www.
0.05 mL/min at up to 10 000 psi is needed to add disodium calcium
discoverysciences.com/uploadedFiles/Home/SPE_Filtration_
ethylenediaminetetracetate (Na2CaEDTA) via zero dead volume
ExtractNUltraClean_p302to306.pdf] were used. Ten grams of
t-fitting to the line between the deionization cartridges and the
cation or anion exchange resins were added to each column. The
cartridges were connected with an Alltech Syringe AdapterTM quantitation column (see Figure 2011.25D).]
(Cat. No. 210705), with cation exchange resin [Ambersep 200C (2)  Filled loop injector.—Equipped with 50 µL injection loop.
(H+)] always at the top. Flow was controlled by pump or with an (3)  LC column.—Waters Sugar-Pak 6.5 × 300 mm (Part No.
Alltech One-Way Stopcock (Cat. No. 211524; Figure 2011.25B). WAT085188) or equivalent preceded by Waters Guard Pak LC
A method for resin regeneration was provided, but reuse of resins precolumn inserts (Part No. WAT015209) or equivalent.
is no longer recommended since resin cost is minimal. However, (4)  Refractive index (RI) detector.—Capable of being maintained
Extract-Clean reservoirs and Syringe Adapters subsequently at 50°C.
received were unsatisfactory. There was some leakage of liquid (5)  Oven.—To maintain a column temperature of 90°C.
due to incomplete sealing. Subsequently, sample deionization has (v)  Data integrator or computer.—For peak area measurement.
been achieved using a mixed bed resin of approximately 4 g cation (w)  Filters for disposable syringe.—Polyvinylidene fluoride
[Ambersep 200C (H+)] and 4 g anion [Amberlite FPA53 (OH-)] in 0.45 µm, 13 or 25 mm.
a Bio-Rad Laboratories Econo-PacTM disposable chromatography (x)  Filters for water.—Polyvinylidene fluoride, 0.45 µm, 47 mm.
column (Cat. No. 732-1010; http://www.bio-rad.com/webroot/ (y)  Filter apparatus.—To hold 47 mm, 0.45 µm filter, (x); to
web/pdf/psd/literature/Bulletin_9103.pdf) with an Alltech One- filter larger volumes of water.
Way Stopcock (Cat. No. 211524; Figure 2011.25B). (z)  Syringes.—10 mL, disposable, plastic.
(t)  Cation and anion exchange guard columns.—(If online (aa)  Syringes.—Hamilton® 100 µL, 710SNR syringe.
simultaneous deionization and quantitation are carried out.)
(bb)  Rotary evaporator.—Heidolph Laborota® 4000 or
Cation and anion exchange guard columns, H+ and CO23– forms,
equivalent (http://www.lasallescientific.com/products/heidolph/
respectively (Bio-Rad Laboratories, Cat. No. 125-0118, includes
BAL_LARO4000_Nov06_en1.pdf).
one cation and one anion cartridge), with guard column holder
(cc)  Thermometer.—Capable of measuring to 110°C.
(Bio-Rad Laboratories, Cat. No. 125-0139) to hold the two guard
(dd)  Freeze drying apparatus.

Table  2011.25C.  Interlaboratory study results for TDF

Sample/parameter A B C D E F G H
No. of labs 15 15 15 15 14 13 14 13
Mean, % 29.90 10.45 26.55 11.79 21.37 16.39 23.71 18.40
Sr 0.86 0.47 1.39 0.49 0.52 1.41 0.87 0.64
SR 2.05 0.95 2.74 1.10 1.72 2.37 3.14 1.56
RSDr 2.88 4.51 5.25 4.17 2.43 8.60 3.65 3.47
RSDR 6.85 9.11 10.31 9.30 8.04 14.48 13.23 8.47
HorRat 2.85 3.24 4.22 3.37 3.19 5.51 5.33 3.28

© 2012 AOAC INTERNATIONAL

Table  2011.25D.  Interlaboratory study results for TDF measured directly
Sample/parameter A B C D E F G H
No. of labs 9 8 9 9 9 7 9 9
Mean, % 28.08 9.97 24.60 11.00 21.21 16.47 21.27 16.47
Sr 1.26 0.41 0.70 0.39 1.11 0.72 1.19 1.16
SR 1.31 1.24 1.95 2.54 2.30 5.27 2.10 2.58
RSDr 4.50 4.16 2.83 3.57 5.23 4.38 5.61 7.04
RSDR 4.67 12.41 7.95 23.11 10.86 32.00 9.89 15.66
HorRat 1.93 4.39 3.22 8.29 4.30 12.20 3.92 5.97

C.  Reagents or G-CEL500, Megazyme International Ireland Ltd., Bray, County

Wicklow, Ireland, http://www.megazyme.com/Dynamic.aspx?
(a)  Ethanol, 95% (v/v) or alternatively use IMS.—Specifications
control=CSViewProduct&categoryName=GeneralChem
for preparation or commercial purchase: ethanol 84.83 (w%), 85.95
icals&productId=G-CEL100; or Sigma-Aldrich Cat. No.
(v%); water 5.66 (w%), 4.52 (v%); 2-propanol 4.91 (w%), 5.00
C8656, http://www.sigmaaldrich.com/catalog/ProductDetail.
(v%); methanol 4.60 (w%), 4.52 (v%). Alternatively, IMS can be
do?D7=0&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&
prepared by mixing five volumes of 2-propanol with 95 volumes
N4=C8656%7CSIGMA&N25=0&QS=ON&F=SPEC).
of denatured ethanol formula SDA-3A (SDA-3A alcohol consists
(i)  Cleaning solution.—Micro-90® (International Products
of 100 volumes of 95% ethanol combined with five volumes of
Corp., Burlington, NJ). Make a 2% solution (w/v) with deionized
methanol). water.
(b)  Ethanol (or IMS), 78% (v/v).—Place 207 mL water into 1 L (j)  Sodium maleate buffer.—50 mM, pH 6.0 plus 2 mM CaCl2
volumetric flask. Dilute to volume with 95% ethanol or IMS. Mix. and 0.02% sodium azide. Dissolve 11.6 g maleic acid in 1600 mL
(c)  Acetone.—Reagent grade. distilled water and adjust pH to 6.0 with 4 M (160 g/L) NaOH
(d) Stock AMG solution.—3300 Units/mL in 50% (v/v) glycerol. solution. Add 0.6 g calcium chloride dihydrate and 0.4 g sodium
Solution is viscous; for dispensing use positive displacement azide [Note: Do not add the sodium azide until the pH has been
dispenser. AMG solution is stable for up to 5 years when stored adjusted. Acidification of sodium azide releases a poisonous gas.
at 4°C. (Note: One unit of enzyme activity is amount of enzyme Handle sodium azide and maleic acid with caution only after
required to release 1 micromole glucose from soluble starch per reviewing MSDS, using appropriate personal protective gear and
minute at 40°C and pH 4.5.) AMG solution should be essentially laboratory hood.] and adjust the volume to 2 L. Stable for >1 year
devoid of b-glucanase and b-xylanase and free of detectable levels at 4°C.
of free glucose (see E). (k)  Trizma base.—0.75 M (Sigma Cat. No. T-1503). Add 90.8 g
(e) Pancreatic α-amylase (50 Units/mL)/AMG (3.4 Units/mL).— Trizma base to approximately 800 mL distilled water and dissolve.
Immediately before use, dissolve 0.10 g purified porcine pancreatic Adjust volume to 1 L. Stable for >1 year at room temperature.
a-amylase (~130 000 Units/g; Ceralpha method; AOAC Method (l)  Acetic acid solution, 2 M.—Add 115 mL glacial acetic acid
2002.01) in 290 mL sodium maleate buffer (50 mM, pH 6.0 plus (Fluka 45731) to a 1 L volumetric flask. Dilute to 1 L with distilled
2  mM CaCl2 and 0.02% sodium azide) and stir for 5 min. Add water.
0.3 mL AMG stock solution (see E). (m)  pH standards.—Buffer solutions at pH 4.0, 7.0, and 10.0.
(f)  Protease (50 mg/mL; ~350 Tyrosine Units/mL) in 50% (n)  Fructooligosaccharides.—From chicory (Sigma Aldrich
(v/v) glycerol.—Solution is viscous; for dispensing use positive Cat. No. F8052).
displacement dispenser. Protease must be devoid of a-amylase and (o)  LC retention time standard.—Standard source having the
essentially devoid of b-glucanase and b-xylanase (see E). Use as distribution of oligosaccharides (DP ≥3) corn syrup solids (DE 16;
supplied. Matsutani Chemical Industry Co., Ltd., Itami City, Hyogo, Japan;
(g)  Deionized water. http://www.matsutani.com/productsframeset.html, maltodextrin
(h)  Celite.—Acid-washed, pre-ashed (Megazyme G-CEL100 TK-16; or Sigma Aldrich Cat. No. 419699), analyzed by LC.

Table  2011.25E.  Interlaboratory study results for SDF as measured using online deionization
Sample/parameter A B C D E F G H
No. of labs 6 6 6 6 6 6 5 6
Mean, % 3.45 4.87 16.29 8.60 11.93 2.95 10.98 7.11
Sr 0.34 0.29 0.58 0.49 0.24 0.16 0.24 0.26
SR 0.64 0.49 1.24 1.99 0.83 0.49 0.40 1.07
RSDr 9.98 5.94 3.55 5.73 2.00 5.28 2.18 3.64
RSDR 18.63 10.12 7.60 23.12 6.96 16.73 3.64 15.08
HorRat 5.61 3.21 2.89 7.99 2.53 4.92 1.31 5.07

© 2012 AOAC INTERNATIONAL

Table  2011.25F.  Interlaboratory study results for TDF (sum of IDF and SDF) as measured using online deionization
Sample/parameter A B C D E F G H
No. of labs 6 6 6 4 6 6 5 6
Mean, % 29.65 9.95 26.96 11.21 20.47 15.06 23.04 17.43
Sr 0.73 0.36 0.73 0.27 0.32 0.43 0.25 0.24
SR 1.29 0.66 2.41 0.37 0.90 1.16 1.71 0.77
RSDr 2.47 3.58 2.70 2.42 1.58 2.87 1.09 1.36
RSDR 4.33 6.62 8.95 3.33 4.40 7.67 7.40 4.41
HorRat 1.81 2.34 3.67 1.20 1.73 2.88 2.97 1.69

Dissolve 2.0 g portion of oligosaccharide mixture and 0.50 g solution consisting of 1 mL of 100 mg/mL internal standard and
maltose in deionized water and transfer to 100 mL volumetric flask. 2.5 mL of 10 mg/mL fructooligosaccharides diluted to 10 mL.
[Alternatively, dissolve 2.5 g LC retention time standard mix from Add 2 mL of this solution to the mixed bed deionization resin
Integrated Total Dietary Fiber Assay Kit (K-INTDF; Megazyme (approximately 8 g) as per I(e). Recovery of the internal standards
International Ireland Ltd.), which contains maltodextrins with and fructooligosaccharides should match that of the solution
added maltose.] Pipet 10 mL of stock internal standard solution, injected directly onto the LC.
(q). Bring to volume with 0.02% sodium azide solution, (u). (q)  D-sorbitol (stock internal standard).—100 mg/mL. [Note:
Transfer solution to 100 mL polypropylene bottle. Stable at room Sorbitol is present in some foods and food products, such as plums
temperature for 1 year. (prunes), pears, apples, and Polydextrose® and may also be added
(p)  Manual deionization.—(1)  Original method.— to foods. If sorbitol is present, use of the internal standard will
Column 1.—10 g Ambersep 200 (H+) resin, ion exchange capacity result in artificially low SDFS quantity. In these cases, the external
(R-H exchange capacity data supplied by manufacturer) 1.6 meq/ standard method of calculation should be used. Addition of the
mL (min), or equivalent (Rohm and Haas, France S.A.S.; see also internal standard is still recommended to use as a retention time
Megazyme G-AMBH). Column 2.—10 g Amberlite FPA53 (OH–) reference.] Weigh 100 g dry D-sorbitol into a beaker, dissolve in
resin, 1.6  meq/mL (min) or equivalent (Rohm and Haas, France water, transfer to a 1 L volumetric flask with water, and dilute to
S.A.S.; see also Megazyme G-AMBOH) packed in separate volume. Transfer to a polypropylene bottle and add 0.2 g sodium
columns [Extract-Clean reservoirs (15 mL), B(s)] for analysis of azide as a preservative. (Note: Handle sodium azide with caution
each test portion. Before connecting the columns, wash the resin only after reviewing MSDS, using appropriate personal protective
in each column with sufficient H2O to obtain a pH value of 4–7 for gear and laboratory hood.) Seal well. Stable at room temperature
column 1 and 7–8 for column 2. for >1 year.
(2)  Updated method.—Mix approximately 4 g Ambersep 200 (r)  D-glucose LC standards (5, 10, 20 mg/mL).—Accurately
(H+) resin or equivalent, 1.6 meq/mL (min) or equivalent (Rohm weigh 0.5, 1.0, and 2.0 g portions of high-purity (>99.5%)
and Haas, France S.A.S.; see also Megazyme G-AMBH) with D-glucose (Sigma Chemical Co.; http://www.sigmaaldrich.com/
approximately 4  g Amberlite FPA53 (OH–) resin, 1.6 meq/mL ireland.html) and transfer to three separate 100 mL volumetric
(min) or equivalent (Rohm and Haas, France S.A.S.; see also flasks respectively. To each flask pipet 10 mL of internal standard,
Megazyme G-AMBOH), ion exchange capacity (R-OH exchange (q). Bring to volume with 0.02% sodium azide solution, (u).
capacity data supplied by manufacturer), in a 100 mL beaker and Transfer solutions to 100 mL polypropylene bottles. Stable at room
slurry with a minimum volume of distilled water. Pour this mixture temperature for 1 year.
into a Bio-Rad Econo-Pac disposable chromatography column (s)  Disodium calcium ethylenediaminetetracetate (Na2CaEDTA;
(Cat. No. 732-1010) fitted with an Alltech One-Way Stopcock (Cat. 50 mg/L; if using Sugar-Pak LC column and manual deionization).—
No. 211524). Place a small wad of cotton wool into the top of the Dissolve 50 mg Na2CaEDTA in about 500 mL deionized water in a
column and wash the resin with 20 mL distilled water. The column 1 L volumetric flask; dilute to volume.
is then ready for use. If alternative resins are used, determine that (t)  Na2CaEDTA (500 mg/L; if using Sugar-Pak LC column and
carbohydrates are not retained by the resin by preparing a test simultaneous online deionization and quantitation). —Dissolve

Table  2011.25G.  Interlaboratory study results for SDF calculated using external standard approach
Sample/parameter A B C D E F G H
No. of labs 11 12 12 12 12 11 11 10
Mean, % 3.94 5.49 16.04 10.06 12.52 3.92 11.34 8.09
Sr 0.45 0.54 1.48 0.81 0.67 0.69 0.44 0.63
SR 1.00 1.11 2.02 1.81 2.10 1.20 0.95 1.99
RSDr 11.42 9.92 9.21 8.06 5.37 17.58 3.93 7.81
RSDR 25.40 20.20 12.62 17.95 16.74 30.58 8.39 24.58
HorRat 7.80 6.53 4.79 6.35 6.12 9.39 3.02 8.42

© 2012 AOAC INTERNATIONAL

Table  2011.25H.  Interlaboratory study results for TDF calculated using external standard approach
Sample/parameter A B C D E F G H
No. of labs 12 12 12 12 12 10 12 10
Mean, % 30.05 10.35 25.52 11.20 20.93 15.58 23.52 18.05
Sr 1.05 0.59 1.52 0.85 0.96 0.85 0.77 0.60
SR 1.95 1.13 2.08 1.71 1.97 1.27 2.61 1.55
RSDr 3.49 5.67 5.95 7.57 4.61 5.49 3.26 3.32
RSDR 6.47 10.90 8.13 15.26 9.41 8.18 11.08 8.61
HorRat 2.70 3.87 3.31 5.49 3.72 3.09 4.46 3.33

500 mg Na2CaEDTA in about 500 mL deionized water in a 1 L of 0.75 M Trizma base solution to terminate the starch digestion
volumetric flask; dilute to volume. reactions. (At the same time, if only one shaker bath is available,
(u)  Sodium azide solution (0.02%, w/v).—Add 0.2 g sodium increase the temperature of the shaking incubation bath to 60°C
azide to 1 L deionized water and dissolve by stirring. (Note: Handle in readiness for the protease incubation step.) Slightly loosen the
sodium azide with caution only after reviewing MSDS, using caps of the sample bottles and place the bottles in a water bath
appropriate personal protective gear and laboratory hood.) Stable (nonshaking) at 95–100°C, and incubate for 20 min with occasional
at room temperature for >1 year. shaking (by hand). Using a thermometer, ensure that the final
Items (d)–(f) and (n) are supplied in K-INTDF (Megazyme temperature of the bottle contents is >90°C (checking of just one
International Ireland Ltd), but preparations of reagents and buffers bottle is adequate).
which meet the criteria as specified in the method above may also (5)  Cool.—Remove all sample bottles from the hot water bath
be used. (use appropriate gloves) and cool to approximately 60°C.
D.  Preparation of Test Samples (6)  Protease treatment.—Add 0.1 mL protease solution, C(f),
using a positive displacement dispenser (solution is viscous).
Collect and prepare samples as intended to be eaten, i.e., baking
Incubate at 60°C for 30 min.
mixes should be prepared and baked, pasta should be cooked, etc.
Defat per AOAC 985.29 if >10% fat. For high-moisture samples (7)  pH adjustment.—Add 4.0 mL of 2 M acetic acid, C(l), to
(>25%), it may be desirable to freeze dry. Grind ca 50 g in a each bottle and mix. This gives a final pH of approximately 4.3.
grinding mill, B(a), to pass a 0.5 mm sieve. Transfer all material (8)  Addition of internal standard.—To each sample, add 1 mL of
to a wide-mouthed plastic jar, seal, and mix well by shaking and 100 mg/mL internal standard solution, C(q).
inversion. Store jar in the presence of a desiccant. (9)  Proceed to G(a) for determination of IDF when IDF and
SDF values are desired. Proceed to O(a) when TDF is desired
E.  Enzyme Purity
without fractionation to IDF and SDF.
To ensure absence of undesirable enzymatic activities and G.  Determination of IDF
effectiveness of desirable enzymatic activities, run standards listed
in Table 985.29 each time enzyme lot changes or at a maximum (a)  Filtration setup.—Tare crucible containing Celite to nearest
6 month interval. 0.1 mg. Wet and redistribute the bed of Celite in the crucible, using
F.  Enzymatic Digestion of Sample

(a)  Blanks.—With each assay, run blanks along with samples to

measure any contribution from reagents to residue.
(b)  Samples.—(1) Weigh duplicate 1.000 ± 0.005 g samples
accurately into 250 mL Fisherbrand bottles, B(b).
(2)  Addition of enzymes.—Wet sample with 1.0 mL ethanol (or
IMS) and add 40 mL pancreatic a-amylase/AMG mixture, C(e), to
each bottle. Cap the bottles. Transfer bottles to the orbital or linear
shaking incubation bath and secure bottles in place with the springs
in the shaker frame. Alternatively, use the submersible magnetic
stirrer apparatus, B(g).
(3)  Incubation with pancreatic α-amylase/AMG.—Incubate the
reaction solutions at 150 revolutions/min (orbital motion) or at the
relevant rate to ensure suspension in a reciprocal shaker at 37°C
for 16 h ± 10 min (e.g., 5:00 pm to 9:00 am). Alternatively, stir the
contents of the Fisherbrand bottles, B(b), on a 2 mag Mixdrive 15
stirring device (with 7 × 30 mm stirrer bar) submerged in a water
bath at 37°C for 16 h ± 10 min (Figure 2011.25A). Figure  2011.25A.  Typical arrangement for sample
(4)  Adjustment of pH to approximately 8.2 (pH 7.9–8.4), mixing using a 2 mag Mixdrive 15 submersible
inactivation of α-amylase and AMG.—After 16 h, remove sample magnetic stirrer in a custom made water bath.
bottles from the shaking water bath and immediately add 3.0 mL

© 2012 AOAC INTERNATIONAL

 temperature) of 95% (v/v) EtOH or IMS, C(a), preheated to 60°C
and mix thoroughly. Allow the SDFP precipitate to form at room
temperature for 60 min.
(b)  Filtration setup.—Tare crucible containing Celite to nearest
0.1 mg. Wet and redistribute the bed of Celite in the crucible, using
15 mL of 78% (v/v) EtOH or IMS, C(b), from wash bottle. Apply
suction to crucible to draw Celite onto fritted glass as an even mat.
Discard this filtrate.
(c)  Filtration.—Using vacuum, filter precipitated SDFP, H(a),
from supernatant through crucible. Using a wash bottle with 78%
(v/v) EtOH or IMS, C(b), quantitatively transfer all remaining
particles to crucible. Retain filtrate and washings, (c) and (d),
respectively, for determination of SDFS, I(d).
(d)  Wash.—Using a vacuum, wash residue successively with
two 15 mL portions of the following: 78% (v/v) EtOH or IMS; 95%
(v/v) EtOH or IMS; acetone. Combine with filtrate of previous step.
Figure  2011.25B.  Column and pump arrangement (e)  Dry crucibles containing residue overnight in 105°C oven.
for deionization of sample extracts: (a) Bio-Rad (f)  Cool crucible in desiccator for approximately 1 h. Weigh
Econo-Pac® disposable chromatography column crucible containing dietary fiber residue and Celite to nearest
(Cat. No. 732-1010); (b) cotton wool plug; (c) a mixture 0.1 mg. To obtain residue mass, subtract tare weight, i.e., weight of
of approximately 4 g Ambersep 200(H+) resin and
dried crucible and Celite.
approximately 4 g Amberlite FPA53 (OH–) resin;
(d) stopcock, one-way tap; (e) Gilson Minipuls® (g)  Protein and ash determination.—Residue from one crucible
Evolution pump; (f) Duran bottle collection containers. of the duplicates is analyzed for protein, and the second residue
of the duplicates is analyzed for ash. Perform protein analysis on
residue using Kjeldahl or combustion methods. (Caution should
15 mL of 78% (v/v) EtOH (or IMS), C(b), from wash bottle. Apply be exercised when using a combustion analyzer for protein in the
suction to crucible to draw Celite onto fritted glass as an even mat. residue. Celite volatilized from the sample can clog the transfer
Discard this filtrate. lines of the unit.) Use 6.25 factor for all cases to calculate mg of
(b)  Filtration.—Using vacuum, filter enzyme digest, F(b)(9), protein. For ash analysis, incinerate the second residue for 5 h at
through crucible. With the aid of a wash bottle use 21.9 mL or less 525°C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract
of deionized water (warmed to 60°C) to quantitatively transfer crucible and Celite weight to determine ash.
remaining particles to crucible and rinse residue. Retain combined (h)  Proceed to I(d).
filtrate and transfer water and set aside for determination of water I.  Determination of SDFS
SDF, H(a).
(c)  Wash.—Using vacuum, wash residue two times each with Chromatography of maltodextrins, maltose, glucose, sorbitol,
15 mL portions of 78% ethanol, 95% ethanol, and acetone. (Note: and glycerol on a Sugar-Pak column is shown in Figure 2011.25E
Delay in washing IDF residues with 78% ethanol, 95% ethanol, and and of water:alcohol soluble dietary fiber (WASDF or SDFS),
acetone may cause inflated IDF values.) Discard these washings. glucose, and sorbitol in Figure 2011.25F. Note: Proper deionization
(d)  Dry crucibles containing residue overnight in 105°C oven. is an essential part of obtaining quality chromatographic data on
(e)  Cool crucible in desiccator for approximately 1 h. Weigh SDFS. For manual deionization, be sure to obtain familiarity
crucible containing IDF residue and Celite to nearest 0.1 mg. To regarding the appearance of salt peaks in the SDFS chromatograms
obtain residue mass, subtract tare weight (i.e., weight of dried which may interfere with dietary fiber determination. In evaluating
crucible and Celite). the column containing approximately 8 g of mixed bed resin,
(f)  Protein and ash determination.—Residue from one crucible dissolve 10 mg sodium chloride into 9 mL deionized water and
of the duplicates is analyzed for protein, and the second residue
of the duplicates is analyzed for ash. Perform protein analysis on
residue using Kjeldahl or combustion methods. (Caution should
be exercised when using a combustion analyzer for protein in the
residue. Celite volatilized from the sample can clog the transfer
lines of the unit.) Use 6.25 factor for all cases to calculate mg of
protein. For ash analysis, incinerate the second residue for 5 h at
525°C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract
crucible and Celite weight to determine ash.
(g)  Proceed to determination of water soluble dietary fiber, H(a).
H.  Determination of Water Soluble Dietary Fiber

(a)  Precipitation of water soluble SDFP.—To each sample

filtrate, add any remaining water from the IDF transfer and Figure  2011.25C.  Apparatus assembly of liquid
chromatography system for Sugar-Pak analytical
rinse, G(b), or additional water if necessary to bring the total
column—manual deionization.
volume to exactly 70 mL, then add 279  mL (measured at room

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 0.05 mL/min.
(4)  System must clearly separate maltose from higher malto-
oligosaccharides (Figure 2011.25E).
(5)  Run time.—30 min to ensure that all materials from the
injection are cleared from the column prior to the next injection.
(6)  RI detector at 50°C.—Note: For samples containing
significant amounts of short-chain polyfructose, it is recommended
that chromatography of the SDFS be carried out on 2-size
exclusion LC columns (TSK-Gel 30 cm × 7.8 mm) connected in
series (Sigma Aldrich Part No. 808020) using conditions described
in AOAC Official MethodSM 2009.01.
Figure  2011.25D.  Apparatus assembly of liquid (b)  Calibrate the area of chromatogram to be measured for
chromatography system for Sugar-Pak analytical
SDFS.—Use a 100 µL LC syringe, B(aa), to fill the 50 µL injection
column—simultaneous online deionization and
quantitation. loop with retention time standard, C(o). Determine demarcation
point between DP 2 and 3 oligosaccharides (disaccharides sucrose
and maltose versus higher oligosaccharides). See Figures 2011.25E
add 1 mL of 100 mg/mL LC internal standard, C(q), and proceed and F.
to I(e) at “transfer 2 mL to the top of the column of ...”. The liquid (c)  Determine response factor (Rf) for D-glucose.—Since
chromatogram of this solution should show no peaks in the time D-glucose provides an LC RI response equivalent to the Rf of
range corresponding to carbohydrates of DP ≥3. For simultaneous the nondigestible polysaccharides that make up SDFS, the LC is
online deionization and quantitation by LC, see Figure 2011.25F calibrated using D-glucose, and the Rf is used for determining the
regarding the change in chromatography which occurs as the ion mass of SDFS. Use a 100 µL LC syringe, B(aa), to fill the 50 µL
exchange capacity of the cartridges is exceeded. injection loop for each internal standard/D-glucose solution, C(r).
(a)  LC system setup.—Set up the LC system choosing between (1)  Internal standard method.—Obtain the values for the peak
the setups shown schematically in Figures 2011.25C and D areas of D-glucose and internal standard from the chromatograms.
depending upon whether manual or online deionization is used. The reciprocal of the slope obtained by comparing the ratio of peak
Sugar-Pak column operating conditions are: (1)  Temperature.— area of D-glucose/peak area of internal standard (y-axis) to the ratio
90°C. of the mass of D-glucose/mass of internal standard (x-axis) is the
(2)  Manual deionization-mobile phase.—Distilled water plus “response factor.” Determine the average Rf (typically 0.91 relative
Na2CaEDTA (50 mg/L); flow rate, 0.5 mL/min. to D-sorbitol internal standard).
(3)  Simultaneous online deionization and quantitation.—
Mobile phase for pump 1, distilled water; flow rate, 0.45 mL/min. Response factor (Rf) =
Mobile phase for pump 2, Na2CaEDTA (500 mg/L); flow rate, (PA-IS)/(PA-Glu) × (Wt-Glu/Wt-IS)

Figure  2011.25E.  Chromatography of a mixture of maltodextrins, maltose, D-glucose, D-sorbitol, and glycerol on a
Waters Sugar-Pak column. Right arrow shows demarcation between DP 2 (maltose) and DP 3 (higher maltodextrins).

© 2012 AOAC INTERNATIONAL

Figure  2011.25F.  Chromatogram of typical SDFS in sample on a Sugar-Pak column analyzed before and after
the capacity of the deionizing cartridges has been expended. Note the absence of the peak at 18.4 min when the
deionizing cartridges are functioning properly, and appearance of the ion peak at 18.4 min indicating the need for
replacement of cartridges.

where PA-IS = peak area internal standard (D-sorbitol); PA-Glu = (e)  Deionization of sample.—In the interlaboratory study, manual
peak area D-glucose; Wt-Glu = mass of D-glucose (mg) contained deionization was performed using separate columns of Ambersep
in 1 mL of standard (5, 10, or 20 mg); Wt-IS = mass of internal 200 (H+) resin and Amberlite FPA53 (OH–) resins (10 g of each) in
standard (mg) contained in 1 mL of standard (10 mg D-sorbitol). Alltech Extract-Clean reservoirs (15 mL) connected with an Alltech
(2)  External standard method.—Obtain the values for the peak Syringe Adapter (Cat. No. 210705). However, with subsequent lots
areas of D-glucose from the three chromatograms. Determine the of columns and adaptors, a perfect connection was not obtained
average Rf: and solutions were lost, so it was decided to use mixed bed resin
[Ambersep 200 (H+) and Amberlite FPA53 (OH–)] (4 g of each) in a
Rf = (Wt-Glu)/(PA-Glu) Bio-Rad Econo-Pac disposable chromatographic column (Cat. No.
732-1010) and to reduce sample size by the same percentage (40%;
where Wt-Glu = mass (mg) of D-glucose contained in 1 mL of i.e., from 5 to 2 mL). Since the ratio of sample and resins remained
standard (5, 10, or 20 mg); PA-Glu = peak area D-glucose. the same, identical results were obtained. Weigh approximately 4 g
(d)  Filtrate recovery, deionization, and LC analysis.—(Set aside Ambersep 200 (H+) and approximately 4 g Amberlite FPA53 (OH–)
the filtrate from one of the sample duplicates, H(c), to use in case resins into a 100 mL beaker and slurry with a minimum volume of
of spills or take it through the procedure if duplicate SDFS data is distilled water. Apply this slurry to a Bio-Rad Econo-Pac column
desired.) Transfer filtrate(s), H(c), of the other sample duplicate to and use distilled water from a wash bottle to transfer all resin to
a 1 L evaporator flask and concentrate with a rotary evaporator to the column. Add a small cotton wool plug to the top of the resin
dryness at 50°C. Dissolve the residue in 10 mL deionized water, and wash the column contents with approximately 20 mL deionized
and transfer to a sealable container and store at 4°C if planning to water. Connect the column to a Gilson Minipuls® Evolution pump
store overnight before deionization. (In subsequent work, half of (http://www.gilson.com/en/Pipette/Products/53.229, or similar).
the filtrate was concentrated to dryness and redissolved in 5 mL Centrifuge or filter the sample solution and transfer 2 mL to the
water. This, as expected gave the same results, but halved the time top of the column of mixed bed resin and allow the column to elute
for rotary evaporation.) For manual deionization, proceed to I(e). at approximately 1 mL/min. Collect the eluate in a 250 mL round-
For simultaneous online LC deionization and quantitation, proceed bottom, rotary evaporator flask. When the liquid level has dropped
to I(f). to the level of the cotton wool plug, rinse the sample in with 2 mL

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distilled water and allow this to percolate into the resin collecting [(R1 + R2)/2 – P – A – B]/(M1 + M2)/2] × 100
the eluate. Then add approximately 20 mL distilled water to the top
of the column and allow this to elute into the 250 mL round-bottom
flask at approximately 1 mL/min. Collect the eluate until the liquid SDFP, % = SDFP (mg/100 g)/1000
level reaches the top of the resin in the column. Evaporate the
solution just to dryness at 50°C. Dissolve the sample to bring it up where R1 = SDFP residue mass 1 from M1 in mg; R2 = SDFP
to 2 mL of volume with distilled water by swirling the flask by hand residue mass 2 from M2 in mg; M1 = test portion mass 1 in g; M2
for approximately 2 min. Transfer the sample into a polypropylene = test portion mass 2 in g; A = ash mass (mg) from R1; P = protein
storage tube using a Pasteur pipet. mass (mg) from R2; B = SDFP blank.
(f)  Preparation of samples for LC analyses.—Transfer the L.  Calculations for SDFS
solutions from either (d) or (e) to a 10 mL disposable syringe, B(z),
and filter through a 0.45 µm filter, B(w). (a)  Internal standard method.—[Note: Sorbitol is present in
(g)  HPLC of sample solutions.—Use a 100 µL LC glass syringe, some foods and food products, such as plums (prunes), pears,
B(aa), to fill the 50 µL injection loop on the LC system, B(u)(2), apples, and polydextrose and may also be added to foods. If sorbitol
with filtered sample extracts. For manually deionized samples from is present, use of the internal standard will result in artificially low
I(e)after filtration step, I(f), apply solutions via the injection loop, SDFS quantity. In these cases, the external standard method of
B(u)(2), directly onto the Waters Sugar-Pak column, B(u)(3), using calculation should be used. Addition of the internal standard is still
the arrangement shown in Figure 2011.25C. For samples to be recommended to use as a retention time reference.]
deionized online from I(d) after filtration step, I(f), apply solutions
to the injection loop and the online desalting guard columns, B(t), SDFS, mg/100 g =
using the arrangement shown in Figure 2011.25D. Rf × (Wt-IS, mg) × (PA-SDFS)/(PA-IS) × 10 × 100/M
(h)  Determine peak area of SDFS (PA-SDFS) and internal
standard if used (PA-IS) in chromatograms of sample extracts.—
Record area of all peaks of DP greater than the DP 2/DP 3 SDFS, % = SDFS (mg/100 g)/1000
demarcation point as PA-SDFS. Record the peak area of internal
standard as PA-IS. where Rf = response factor calculated in I(c); Wt-Glu = mass (mg)
of D-glucose contained in 1 mL of standard (5, 10, or 20 mg);
J.  Calculations for IDF
PA-Glu = peak area D-glucose; Wt-IS is mass of internal standard
(a)  Blank (B, mg) determination. in 1 mL of sample (i.e., 10 mg per mL; final sample volume,
10  mL); PA-SDFS is the peak area of the water:alcohol soluble
B = [(BR1 + BR2)]/2 – PB – AB dietary fiber; PA-IS is the peak area of the internal standard; 10
is factor to convert from 1 to 10 mL (final sample volume); 100
where BR1 and BR2 = blank residue mass (mg) for duplicate IDF is factor to convert to 100 g of sample; M is the test portion mass
blank determinations, respectively, and PB and AB = mass (mg) of (g) M1 or M2 of the sample whose filtrate was concentrated and
protein and ash, respectively, determined on first and second blank analyzed by LC.
residues. (b)  External standard method.
(b)  IDF.
SDFS, mg/100 g = Rf × (PA-SDFS) × 10 × 100/M
IDF, mg/100 g =
[(R1 + R2)/2 – P – A – B]/(M1 + M2)/2] × 100
SDFS, % = SDFS (mg/100 g)/1000

IDF, % = IDF (mg/100 g)/1000 where Rf = (Wt-Glu)/(PA-Glu); Wt-Glu = mass (mg) of D-glucose
contained in 1 mL of standard (5, 10, or 20 mg); PA-Glu = peak
where R1 = IDF residue mass 1 from M1 in mg; R2 = IDF residue area for D-glucose (5, 10, or 20 mg); PA-SDFS is the peak area of
mass 2 from M2 in mg; M1 = test portion mass 1 in g; M2 = test the water:alcohol soluble dietary fiber; 10 is factor to convert from
portion mass 2 in g; A = ash mass (mg) from R1; P = protein mass 1 to 10 mL (final sample volume); 100 is factor to convert to 100 g
(mg) from R2; B = IDF blank. of sample; M is the test portion mass (g) M1 or M2 of the sample
whose filtrate was concentrated and analyzed by LC.
K.  Calculations for SDFP
M.  Calculations for SDF
(a)  Blank (B, mg) determination.

B = [(BR1 + BR2)]/2 – PB – AB SDF, mg/100 g = SDFP (mg/100 g) + SDFS (mg/100 g)

where BR1 and BR2 = blank residue mass (mg) for duplicate SDFP
blank determinations, respectively, and PB and AB = mass (mg) of SDF, % = SDF (mg/100 g)/1000
protein and ash, respectively, determined on first and second blank
residues.
N.  Calculation for TDF
(b)  SDFP.

SDFP, mg/100 g = TDF, % = IDF (%) + SDF (%)

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O.  Determination of TDF Without Fractionation of IDF and SDF lines of the unit.) Use 6.25 factor for all cases to calculate mg of
protein. For ash analysis, incinerate the second residue for 5 h at
(a)  Precipitation of water soluble SDFP in the presence of
525°C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract
IDF.—To each sample enzyme digest, from step F(b)(9), add
crucible and Celite weight to determine ash.
water to bring the total volume to exactly 71 mL. Then add 279 mL
(h)  Proceed to I(d) for determination of SDFS.
(measured at room temperature) of 95% (v/v) EtOH or IMS, C(a),
preheated to 60°C and mix thoroughly. Allow the SDFP precipitate P.  Calculations for SDFP:IDF
to form in the beaker with the IDF at room temperature for 60 min.
(a)  Blank (B, mg) determination.
(b)  Filtration setup.—Tare crucible containing Celite to nearest
0.1 mg. Wet and redistribute the bed of Celite in the crucible, using
B = [(BR1 + BR2)]/2 – PB – AB
15 mL of 78% (v/v) EtOH or IMS, C(b), from wash bottle. Apply
suction to crucible to draw Celite onto fritted glass as an even mat.
where BR1 and BR2 = blank residue mass (mg) for duplicate
Discard this filtrate.
SDFP:IDF blank determinations, respectively, and PB and AB =
(c)  Filtration.—Using vacuum, filter precipitated SDFP:IDF,
mass (mg) of protein and ash, respectively, determined on first and
(a), from supernatant through crucible. Using a wash bottle with
second blank residues.
78% (v/v) EtOH or IMS, C(b), quantitatively transfer all remaining
(b)  SDFP:IDF.
particles to crucible. Retain filtrate and washings, (c) and (d), for
determination of low-molecular-weight SDF, I(d). SDFP:IDF, mg/100 g =
(d)  Wash and retain filtrate.—Using a vacuum, wash residue [(R1 + R2)/2 – P – A – B]/(M1 + M2)/2] × 100
successively with two 15 mL portions of the following: 78% (v/v)
EtOH or IMS, followed by two 15 mL portions of 95% (v/v) EtOH
or IMS, followed by two 15 mL portions of acetone. Combine with SDFP:IDF, % = SDFP:IDF (mg/100 g)/1000
filtrate from step immediately preceding.
(e)  Dry crucibles containing residue overnight in 105°C oven. where R1 = SDFP:IDF residue mass 1 from M1 in mg; R2 =
(f)  Cool and weigh crucible in desiccator for approximately 1 SDFP:IDF residue mass 2 from M2 in mg; M1 = test portion mass
h. Weigh crucible containing dietary fiber residue and Celite to 1 in g; M2 = test portion mass 2 in g; A = ash mass (mg) from R1;
nearest 0.1 mg. To obtain residue mass, subtract tare weight, i.e., P = protein mass (mg) from R2; B = blank.
weight of dried crucible and Celite.
(g)  Protein and ash determination.—Residue from one crucible Q.  Calculation for TDF
of the duplicates is analyzed for protein, and the second residue
of the duplicates is analyzed for ash. Perform protein analysis on TDF, % = SDFP:IDF (%) (from P) + SDFS (%) (from L)
residue using Kjeldahl or combustion methods. (Caution should
be exercised when using a combustion analyzer for protein in the
Reference: J. AOAC Int. (future issue)
residue. Celite volatilized from the sample can clog the transfer

© 2012 AOAC INTERNATIONAL