63 / 112
Ltipo
t,.l'At-.:
JorJrìNÄr,
orr
AOAC
INl't..RNÄr'roNr\t-
Vot-.
96.
No. l.
2013
125
Table
4.
lnternal
LOD, ppm
Replicåte
Krce
Oats
Sorghum
Corn
Buckwheal
Sausage
Cracker
1
2
3
5
6
7
I
I
'10
1...,1.:
r
SD
N4ean
+ 3.3 SD
Overall LOD =
1.10
ppm
0.70
0.40
0.30
0.60
0.80
o.20
0.20
0.50
0.1
0
0.00
c.3 8
0.27
t/ó
0.00
0.20
0.00
0.'í0
0.50
0.40
0.1
0
0.30
0.10
0.50
3.12
0.1
9
086
1.50
0.20
0.00
û.50
0.90
1,00
0.60
1.20
1.00
o.20
j.71,
0.49
)a)
0.30
0.20
0.40
0.1
0
0.50
0.40
0.20
0.30
0.'10
0.20
0.27
0.1 3
0.71
0.00
0.00
0.00
0.û0
0.10
0.00
0_00
0.1 0
0.00
0.00
c.
c2
0,04
0.1
6
1.00
0.20
0.1
0
o.20
1.00
0.70
0.40
0.20
0.10
0.60
0.35
1.62
0.40
0.80
0.50
0.40
0_80
0.60
0.40
0.50
0.40
0.70
0,16
1.09
0.00
0.00
0.30
0_10
0.50
0.30
0.20
0.00
0.30
0.00
0.18
o.75
"
All samples prev¡ously tested gluten-free
bean
I'lour) into
5-100 g
batches. One u,as
spiked
rvith
l0
rn[,
2%CMC
solulion
onl)'f'or
a
0
ppm
gliadin
level.
l'he
rL.nìaining
portions
\\'ere
spiked using
above preparatiort
rvith
ad.justed
concentrations
to
ensure each respectiYe
level
(5.
10.
20.
and
40 pptn)
\vere crealed
b¡'spiking
l0
mL
based
on drv *'eight.
\\¡ater
rias
then added. and each batch
uas
ntixed
according
10
tlre
recipe
and
baked
al
220"C fìrr 22
r¡in.
Samples rvere then
dried
in
a
household
f'ood
deh¡'drator. frozen,
and
ground to
a
tne
particle
size
belore
lhe¡,uere
portioned into
0.25
g
sample
sizt'
in
50 cc pol)propvlene
ccnlrilupe
tubes
and
blind-coded.
Saurples
ucre
held
fË/at
4'C
arraitine
arral¡sis.
Sample Preparation and Extract¡on
All
sanrples
u,ere blind-coded and
stored
at
2-8'C
until
analyzed.
(a)
Prepare
80% ethanol
exlraction solution by
cornbining
eight
parls ethanol
u'ith
t\\'o
parts
(r,/v) distilled
s'ater.
(b)
Prepare sanrple
extract
dilution
solution
(PIIS)
by
dissolr
ing
thc'contents
of'the
packet
in
I
I-
distilled
uater.
(c) \\tighout
0.25
g
sarnple
into
a
50
cc scre\\,cap
centriligc
tube.
(d)
Add
2.5
rnl
renatuling cocktail solution.
(e)
lf'samples contain
lrucku'hcat. chestnut
1'lour. ta¡tnins,
or
phenolic cornpounds.
like
chocolate.
cofTèe
or
cocoa.
add
one
level
scoop
ol'extraction
acfdilive .
Irol all
other
commodil\,
l)'pes. do not add
extraction additive.
(f)
Cap and
vortcx
30
s 1o
honrogenize
cocktail
and
sarnple.
(g)
Incubate 40
rnin
at
50'C
(n'ater
lratlr
or
oven).
(h)
Remove sarnples aud let
cool
5-10
min.
(i)
Add
7.5
mL
80% etlranol
ancl
r.ortex again
for l0-20
s,
(j)
Sltake
(150-200 rpm) l'or
I
h
at
rooln
ternperature
olì
a
rotator
(tr¡be on
its
side).
(k)
Centrihrge
sarnple
I0 niin
at
>2500
rpnr.
(l)
Dilute
the
sarnple
2.3
nrl-
PBS)
(nt)
Saniples
are
re¿rcl-r
(if'
material
does
rlot readil¡- settle)
lor
l:12.5 rvith
PBS
(200
FL
sample
+
lìrr
anall'sis.
Procedural
Notes
(a) llash
buffer.-Prepale
the
wash
bulìèr
solution
bv
diluting
the
rvash
bufïer
concentrate
in
960
mL
distilled
or
deionized
water
iu
au
entpty
I
L confainer. Ensure transfcr
ofall
concentrate
b1'rinsing
the
bottle sevelal titnes rvith
rr,ater. and
su
irl
to cnsure
tllorough
¡uixi¡r_r.1.
(b)
Sttbsf
rale.-K-Blue
substl
ale
is
read¡'
l'or
use.
The
substrate
should
be
clear
to
light
blue: discard
il
it
has
tunted
clark
blue.
Only.
pour the
needed
volurne
of
substrate
into
a
reagent boat.
Do
not
retum
r.urusecl
substrate
to
tlle boltle. Cover
the reagent boat
to
keep the subslrate protected
fiorn light until
needed.
(c)
,4nÍibody,
uel/s.-Keep qells
sealed
in
the
f-oil
pouch
until
needed.
Iìemove
u'ells
liorn
the
loil
pouch
onlv
afier
samples alr: extl'acted and the
te
st
procedure
is set
to bcgin.
Precautions
(a)
Ilthanol
solution
is
highly
fìanlnlable.
Kcep
containcr'
Table
5.
Temperature robustness
Samples,
pÞm
Temperature,
oC
Run
No.1
18
24
30
0.20
0.00
0.00
4.70
4.90
4.60
9.70
9.
s0
97ô
Run No.
2
18
24
30
0.60
0.00
010
4.50
4,50
4.90
9.90
9.40
9.50