49.2.29
— C ellu lose or cellu lose nitrate,
A O A C O ffic ia l M e th o d 999.07
A fia to x in B 1 a n d T otal A fla to x in s
in P ea n u t B u tte r, P is ta c h io Paste, Fig Paste,
a n d P a p rika P o w d e r
Im m u no affinity C olu m n Liquid C h ro m ato g rap h y
w ith P ost-C olu m n D erivatization
First A ction 1999
Final A ction 2008
(Applicable to detennination o f aflatoxins B ,, B2, G ,, and G2 in
peanut butter, pistachio paste, fig paste, and paprika powder.)
See Tabic 999.07A for the results o f the interlaboratory study
supporting acceptance o f the method.
A. Principle
Test portion is either extracted with M eO H -H 20 (8 + 2) or
M eO H -H 20 (8 + 2) plus hexane (or cyclohexane). Extract is
filtered, diluted with water to a specified solvent concentration, and
applied to an affinity column containing antibodies specific to
aflatoxins B,, B2, G |, and G2. Aflatoxins are removed from the
affinity column with MeOH and arc quantified by rcvcrscd-phasc
liquid chromatography (LC) with post-colum n derivatization
(PC D ) involvin g bromination. PCD is achieved with either
electro ch em ica lly generated brom ine (K obra c e ll) or with
pyridinium hydrobromide perbromide (PBPB) and determined by
fluorescence detection.
B. Performance Standards for Immuno Affinity Column (IAC)
A ffin ity colum n should contain antibodies raised against
aflatoxins B,, B:, G ,, and G2. Column should have a capacity o f not
less than 100 ng o f afiatoxin B, and should give recovery o f not less
than 80% for aflatoxins B,, B2, and G, and not less than 60% for
afiatoxin G , when applied as an aqueous standard solution in 10%
CH3OH containing 5 ng o f each toxin.
C. Apparatus
(a) Blender.— Explosion proof (minimum 8000 rpm).
(b) Vertical shaker.— Adjustable (for maximum solid-liquid
agitation); holding 500 mL Erlenmeyer flasks.
(c ) Filter paper.— 24 cm diameter, prefolded, retention: 30 pm or
better.
(d) Erlenmeyerflask. — 500 mL, screw top or glass stopper.
(e) Glass microfiber filter paper.— 5 cm diameter, retention:
1.6 pm (or better).
(f) Reservoir — 75 mL with Luer tip connector for affinity column.
(g) Hand pump. 20 mL syringe with Luer lock or rubber stopper.
(h) Volumetric glassware.— 2, 3, 10, and 20 mL (accuracy o f at
least 0.5%).
(i) LC pump.— Suitable for flow rate at 1.000 ± 0.005 mL/min.
(j) Injection system.— Valve with 200 pL loop or equivalent.
(k) Reversed-phase LC column.— 4.6 mm x 25 cm, 5 pm, e.g.,
LC-I8 or ODS-2.
( l) Post-colum n d eriva tiza tio n system .— ( / ) With PBPB
reagent.— Second LC pulseless pump, zero-dead volume T-piece,
reaction tubing minimum dimensions 45 cm x 0.5 mm id PTFE.
(2) With electrochemically generated bromine.— e.g., Kobra cell;
R h o n e D ia g n o s t ic s T e c h n o lo g ie s L td ., L y o n , F ra n ce;
http://www.r-biopharmrhonc.com/pro/cquip 1.html.
(m ) Fluorescence detector. Wavelengths 360 nrn excitation
filter and 420 nm cut-off emission filter, or equivalent.
(n ) D isposable filte r unit.
0.45 pm.
( 0 ) Pipets.— 10 mL.
(p) Analytical balance.— Readability 0.1 mg.
(q) Laboratory balance.—Readability 0.1 g.
( r ) C a li b r a te d m i c r o l it e r s y r in g e ( s ) o r m i c r o lite r
pipet(s).— 25 and 500 pL.
(s) Affinity columns.— Vicam (313 Pleasant St, Watertown, MA
02472, USA; www.vicam.com ) and EASI-EXTRACT columns,
RP71, Rhone-Diagnostics (West o f Scotland Science Park, Unit 306
Kelvin Campus, Glasgow G 20 OSP, UK) have been found to meet
the criteria.
D. Reagents
All reagents are analytical grade.
Water, except where specified, should be produced by single
distillation, deionization, or reverse osmosis.
(a) Phosphate buffered saline solution (PBS).— pH 7.4. Dissolve
0.20 g KC1, 0.20 g KH 2P 0 4, 1.16 g anhydrous Na2l IP 0 4 (or 2.92 g
Na 2H P 0 4 12H20 ) , and 8.00 g NaCI in 900 mL water. Adjust to
pH 7.4 with 0.1M HCI or NaOH and dilute to I L. (Commercial
buffered saline tablets may be used.)
(b) Sodium chloride.
(c ) P yridinium h ydrobrom ide perbrom ide
(PB PB J.-C A S-394 16-48-3.
(d) Potassium bromide.
(e) Acetonitrile.— LC grade.
(f) Methanol.— LC grade.
(g) Methanol.— Technical grade, pure, or distilled.
(h) Water.— LC grade; complying with grade 1 o f ISO 3696.
(1) Extraction solvent.— Methanol-water solution (8 + 2, v/v).
(j) Hexane or cyclohexane.
(k) Nitric acid.—C (H N 0 3) = 4M. Dilute 28.1 mL concentrated
HNO 3 (65%) (26.1 mL 70% HNOj) in water to final volume o f
100 mL.
( l) LC mobile phase solvent (A). Water acetonitrile methanol,
(f), solution (6 + 2 + 3, v/v/v).
( m ) L C m o b i l e p h a s e s o l v e n t ( B ) . — F o r u s e w ith
electrochem ically generated Br: w ater-acetonitrile-m ethanol
solution (6 + 2 + 3, v/v/v). To each liter o f mobile phase, add 350 pL
nitric acid (4M ), (k). and 120 mg potassium bromide, (d), and mix to
dissolve.
(n) Post-column reagent (B). Dissolve 25 mg PBPB in 500 mL
H20 . Solution can be used for up to 4 days if stored in dark at room
temperature.
( 0 ) Toluene-acetonitrile.— 98 +2 (v/v).
(p) Afiatoxin standard solutions fo r LC .— ( / ) Mixed aflatoxins
calibranl solution X fo r LC.— Prepare as in 971.22B -F (see
49.2.03) to contain 1000 n g B ,, 200 n g B 2, 1000 n g G ,.a n d 200 ng
G2/mL toluene-acetonitrile (98 + 2). (2 ) Working calibranl
solutions fo r LC.— Prepare solution by pipetting exactly 2.0 mL
calibrant solution X into 20.0 mL volumetric flask (or 2.5 mL into
25 mL volumetric flask). Dilute to mark with toluene-acetonitrile
solution and shake well. Use this solution for pipetting the volumes
listed in Table 999.07B into a set o f 10.0 mL volumetric flasks.
Evaporate toluene-acetonitrile solution just to dryness under stream
o f N at room temperature. To each flask, add 4 mL methanol, mix,
dilute to 10.0 mL with water, and mix again. Prepare working
solutions daily.
© 2008 AOAC INTERNATIONAL
Table 999.07A. Interlaboratory study results fo r aflatoxin B1 and total aflatoxins in peanut butter, pistachio paste, fig paste, and
paprika pow der

Food Contamin. Avg., ng/g No. labs3 sr RSDr, % SR RSD r, % HorRat

Aflatoxin Bi
Peanut butter Fortified 0.9 15 0.09 10 0.16 19 0.42
Fortified 3.6 13 0.11 3 0.66 18 0.49
Naturally 0.8 15 0.05 6 0.26 32 0.69
Naturally 1.5 14 0.10 6 0.22 14 0.33
Naturally 3.4 14 0.13 4 0.65 19 0.51
Pistachio paste Fortified 0.9 15 0.13 14 0.15 16 0.35
Fortified 3.3 12 0.13 4 1.02 31 0.83
Naturally 0.7 13 0.08 11 0.12 17 0.36
Naturally 1.5 15 0.27 18 0.36 23 0.55
Naturally 2.9 14 0.59 20 0.61 21 0.55
Fig paste Fortified 1.1 15 0.18 17 0.21 19 0.43
Fortified 3.6 15 0.39 11 0.46 13 0.35
Naturally 1.3 16 0.12 10 0.30 23 0.53
Naturally 2.1 14 0.12 6 0.31 15 0.37
Naturally 2.6 16 0.41 16 0.73 29 0.75
Paprika powder Fortified 0.9 14 0.05 6 0.09 10 0.22
Fortified 3.4 15 0.18 5 0.35 10 0.27
Naturally 0.8 15 0.12 14 0.16 19 0.41
Naturally 1.4 15 0.14 10 0.24 17 0.40
Naturally 3.0 14 0.13 4 0.28 9 0.24
Total aflatoxin6
Peanut butter Fortified 1.9 15 0.26 13 0.35 18 0.44
Fortified 7.9 15 0.67 9 1.76 22 0.67
Naturally 1.3 15 0.08 6 0.46 34 0.79
Naturally 2.2 13 0.16 7 0.32 14 0.35
Naturally 5.0 14 0.23 5 0.96 19 0.54
Pistachio paste Fortified 2.0 14 0.24 12 0.36 18 0.45
Fortified 7.8 14 1.82 23 1.82 23 0.70
Naturally 0.8 13 0.10 12 0.17 21 0.45
Naturally 1.7 15 0.31 18 0.42 24 0.58
Naturally 3.3 14 0.66 20 0.72 22 0.59
Fig paste Fortified 2.2 15 0.40 18 0.73 32 0.80
Fortified 7.8 15 1.01 13 1.28 17 0.52
Naturally 2.8 16 0.25 9 0.80 28 0.73
Naturally 3.8 16 0.44 12 1.03 29 0.79
Naturally 5.2 16 0.90 17 1.56 30 0.86
Paprika powder Fortified 1.7 13 0.11 6 0.34 20 0.48
Fortified 7.1 15 0.72 10 1.01 14 0.42
Naturally 0.9 16 0.16 17 0.31 34 0.75
Naturally 2.0 16 0.23 12 0.55 28 0.69
Naturally 4.5 14 0.22 5 0.66 15 0.42
s Number of laboratories that submitted acceptable results (total number of participating laboratories was 16).

6 Total aflatoxin parameter was subject to statistical evaluation after summarization of single aflatoxin results. Acceptance of each single result was not
determined prior to summarization (pre-limitation of single results for further evaluation), thus allowing difference in number of accepted results for aflatoxin B,
and total aflatoxin.

© 2008 AOAC INTERNATIONAL

Table 999.07B. Preparation of working calibration solutions
Final mass concentration of working calibrant, ng/mL
Working
standard calibrant solution, pL B,
1 40 0.4 00
2 120 1.200
3 200 2.0 00
4 280 2.8 00
5 360 3.600
E. Extraction
(a ) Peanut butter and pistachio paste. — Weigh, to nearest 0.1 g,
50 g test portion into 500 mL Erlenmeyer flask, add 5 g NaCl,
300 mL methanol-water extraction solvent, and 100 mL hexane or
cyclohexane. Blend 3 min with high speed blender. Filter and pipet
10.0 mL clear filtrate into reservoir containing 60 mL PBS solution
placed on conditioned immunoaffinity column. Mix with plastic
spatula and rinse residues with 1-2 mL PBS from spatula into
reservoir. Transfer solution to column as described in F.
(b ) Paprika powder.— Weigh, to the nearest 0.1 g, 50 g test portion
into 500 mL Erlenmeyer flask with screw top or glass stopper. Add 5 g
NaCl and 300 mL methanol-water solvent. Shake intensively by hand
for 15-30 s and then for 30 min on a shaker. Filter extract using
prefolded paper. Pipet 10.0 mL clear filtrate into reservoir containing
60 mL PBS solution placed on conditioned immunoaffinity column.
Mix with plastic spatula and rinse residues with 1-2 mL PBS into
reservoir. Apply solution on column as described in F.
(c) Dried figs. — Weigh, to nearest 0.1 g, 50 g test portion into
500 mL Erlenmeyer flask, add 5 g NaCl, 300 mL methanol-water
(ISO 3696, grade 3) extraction solvent. Blend 3 min with high speed
blender. Filter and pipet 10.0 mL clear filtrate into reservoir
containing 60 mL PBS placed on conditioned immunoaffinity
column. Mix with plastic spatula and rinse residues with I -2 mL
PBS from spatula into reservoir. Transfer solution on column as
described in F.
F. Affinity Column Chromatography
Adjust columns to room temperature prior to conditioning. For
conditioning, apply 10 mL PBS solution on top o f column and let
pass at a speed o f 2 -3 mL/min through eolumn by gravity. Make sure
that 0.5 mL o f PBS remains on column until test solution is applied.
[Note: Methods for loading onto affinity columns, washing the
column, and elution vary slightly between column manufacturers,
and specific instructions supplied with columns should be followed
p recisely . In g en eral, procedures in v o lv e extraction w ith
methanol-water, filtration or centrifugation, possible dilution with
PBS or water, loading under pressure onto (possibly prewashed)
column, washing o f column with distilled water, and elution o f
aflatoxins with methanol or acetonitrile.]
Pass filtrate through column at flow rate o f ca 1 drop/s (ca
3 mL/min by gravity). Do not exceed 5 mL/min. Wash column with
15 mL water and dry by applying little vacuum for 5 -1 0 s or passing
air through with a syringe for 10 s.
Elute aflatoxins by adding 0.5 mL methanol on column and let
pass through by gravity. Collect eluate in 3.0 or 5.0 mL calibrated
volumetric flask. Wait I min and apply second portion o f 0.75 mL
methanol. Collect applied elution solvent by pressing air through.
Dilute to mark with water and mix. If solution is clear, it can be used
b2 g2
Gi
0.0 80 0.4 00 0.080
0.2 40 1.200 0.2 40
0.4 00 2.000 0.4 00
0.5 60 2.800 0.560
0.7 20 3.600 0.7 20
directly for LC analysis. If solution is not clear, pass through
disposable filter unit (0.45 pm) before injection on the LC column.
G. LC Determination with Fluorescence Detection and
Post-Column Derivatization
When using PBPB. mount mixing T-piece and reaction tubing,
C(l). Then operate using the following parameters: flow rates,
1.00 mL/min (mobile phase A) and 0.30 mL/min (reagent).
When using clectrochemically generated bromine (Kobra cell),
follow instructions for installation o f cell supplied by manufacturer
and operate using the following parameters: flow rate, 1.00 mL/min
(mobile phase B); current. 100 pA.
Inject 200 pL working standard mixture (covering the range o f
1-4 ng/g for aflatoxin B ,) into injector, following manufacturers
instructions to ensure com plete filling o f the injection loop.
Aflatoxins elute in the order G2, G ,, B2, and B, with retention times
o f ca 6, 8, 9, and 11 min, respectively, and should be baseline
resolved. Prepare calibration curve using calibration solutions
described and check curve for linearity. Inject 200 pL extract into
injector and identify each aflatoxin peak in chromatogram by
comparing retention times with those o f corresponding reference
standards. Determine quantity o f aflatoxin in eluate injected from
standard curve.
H. Calculations
Calculate concentration o f aflatoxin in test sample as follows:
Plot data (concentration o f aflatoxin (ng/mL; v-axis) against peak
area (units: A-axis)] from calibrant solution experiments into a table
and calculate calibration curve using linear regression. U se resulting
function (V = ax + b) to calculate concentration o f aflatoxin in
measured solution.
For linear calibration, the formula describes correlation between
detector signal (*) and the corresponding concentration o f analyte (y ).
This means that (y) is a function o f (.v) [y = (/) a ]. The constant (a)
is the corresponding value o f the slope o f the linear function, while b
is the value where the calibration function intercepts the v-axis o f the
coordinate system.
Wt (g) = test portion taken for analysis; solvent (mL) = solvent
tak en for e x tr a c tio n ; a liq u o t (m L ) = a liq u o t tak en for
immunoaffinity cleanup; elution (mL) = final volume collected after
elution from 1AC; Csmp (ng/mL) = concentration o f aflatoxin
calculated from linear regression; Contain, (ng/g) = contamination
o f test sample with aflatoxin; S ig n a l^ (units) = area o f aflatoxin
peak obtained from the measured solution.
Calculate the contamination (ng/g) from the measured solutions
(ng/mL) according to:
(ng/mL) = a x s ig n a l^ (units) + b
© 2008 AOAC INTERNATIONAL
 „ CaK„ x solvent x elution n e x m L x m L
Contain. = -------------------------------------------------
Wtxaliquot |_ m L x g x m L

N ote that for isolation procedures involving use o f hexane or

cyclohexane, the volum e o f these solvents added for extraction must
not be taken into account for the calculation.
Add mass fractions o f the 4 aflatoxins to obtain a total aflatoxin
m ass fraction.
(Note: Soak all laboratory glassw are in 10% so lu tio n o f
household bleach, w hich generally contains 5.25% N aO C l, before
reusing or discarding. See 990-3 2J (see 4 9 .2 .1 6 ) for further details
on decontam ination.)

Reference: J. AO AC Int. 8.3. 320(2000).

© 2008 AOAC INTERNATIONAL