9.1.

01
AOAC Of fi cial Method 986.15
Ar senic, Cad mium, Lead, Se le nium,
and Zinc in Hu man and Pet Foods
Multielement Method
First Ac tion 1986
Fi nal Ac tion 1988
Co dex-AdoptedAOAC Method*
A. Prin ci ple
Ma te rial is di gested with HNO
3
in closed sys tem. Cd and Pb are
de ter mined by an odic strip ping voltammetry (ASV). As, Se, and Zn
are de ter mined by atomic ab sorp tion spectrophotometry (AAS)
af ter gen er a tion of metal hy drides (for As and Se).
B. Ap pa ra tus
(a) Po laro graph.With an odic strip ping ac ces so ries. Typ i cal
op er at ing pa ram e ters for Prince ton Ap plied Re search Model 174
(current Model 394; www.princetonappliedresearch.com) with
hang ing drop Hg elec trode are: scan rate, 5 mV/s; scan di rec tion, +;
scan range, 1.5 V; ini tial po ten tial, -0.7 V; mod u la tion am pli tude,
25 mV; op er a tion mode, dif fer en tial pulse; dis play di rec tion, -;
drop time, 0.5 s; low pass fil ter, off; se lec tor, off; pushbutton, ini tial;
out put off set, off; and cur rent range, 510 mA, or as needed.
Other in stru ments and elec trodes such as wax im preg nated
graph ite may be used ac cord ing to man u fac turers di rec tions.
(b) Atomic ab sorp tion spectrophotometer.With Zn, As, and Se
hol low cath ode lamps or As and Se electrodeless dis charge lamps,
3 slot, 10 cm Bolling burner head, airC
2
H
2
and H
2
N
2
en trained air
flames, and deu te rium arc back ground corrector.
(c) De com po si tion ves sel.70 mL. See 974.14A (see 9.2.24).
(d) Hy dride gen er a tor.See Fig ure 986.15A. Con structed from
fol low ing: (1) Flat bot tom flask.Boro sili cate glass, 50 mL
(Corning No. 5160, or equiv a lent). (2) Stop per fit tings.Two-hole
(one through cen ter) No. 9 rub ber stop per, fit ted with gas out let tube
of 100 mm
1
8
in. (3 mm) id poly eth yl ene tub ing through cen ter
hole. Place bot tom of gas out let tube through cut off bot tom 1 in.
(25 mm) seg ment of
5
8
in. (16 mm) poly eth yl ene test tube with hole
in bot tom so that 3 mm of tube pro trudes through test tube. In sert
through sec ond hole 75 mm
1
8
in. (3 mm) id poly eth yl ene tub ing as
N
2
in let tube. Seal bot tom end of tube with burner and then punch
sev eral holes at sealed end with 21 gage nee dle. Al ter na tively,
pre pare sim i larly 500 mm
1
16
in. (1.5 mm) id poly eth yl ene tub ing
and hold in place in stop per with hole-through sep tum. Con nect
other end of tub ing to AA spectrophotometer with 500 mm Tygon
tub ing by cut ting auxiliary line at ca 75 mm from mix ing cham ber
and at tach ing tub ing. (3) Gen er a tor mount.(Op tional.) 64 mm
0.5 in. (13 mm) id pipe se cured to lab o ra tory ring stand by means of
clamp holder. In sert ex ten sion clamp into pipe and at tach an other
clamp to back of clamp to hold clamp in place and to serve as han dle;
clamp is now free to ro tate ca 180. At tach rub ber stop per of hy dride
gen er a tor to ex ten sion clamp with stiff wire and po si tion just at level
of clamp jaws. In op er a tion, place flask of gen er a tor be tween jaws of
ex ten sion clamp, in sert stop per firmly into neck of flask, then
tighten clamp jaws around neck of flask. Unit can be rap idly and
uni formly in verted by ro tat ing han dle on ex ten sion clamp, thus
al low ing test solution and so dium borohydride to mix rap idly and
reproducibly.
(e) Pipets.50 and 100 mL Eppendorf micropipets, or equiv a lent.
C. Re agents
(Use dou ble dis tilled water. Rinse all glass ware with HNO
3
[1 + 1]
fol lowed by thor ough H
2
O rinse. De con tam i nate di ges tion ves sels
by di gest ing with re agents to be used in di ges tion. Rinse thor oughly
with H
2
O. De con tam i na tion is nec es sary to re duce blanks,
es pe cially for Pb, to ac cept able level.)
(a) Acids.(1) Ni tric acid.Re dis tilled. (2) Perchloric
acid.70%, dou ble vac uum dis tilled. (3) Hy dro chlo ric acid.8M.
Di lute 66 mL HCl to 100 mL with H
2
O.
(b) Ni trate so lu tion.Equimolar so lu tion of KNO
3
and
NaNO
3
.Dis solve 54.3 g KNO
3
and 45.7 g NaNO
3
(avail able as
Suprapur

, Nos. 5065 and 6546, re spec tively, EM Sci ence) in H

2
O
in 200 mL vol u met ric flask, di lute to vol ume, and mix. To fur ther
pu rify, add 12 drops NH
4
OH to 25 mL aliquot and ex tract with
2 mL 10 mg dithizone/mL CCl
4
un til lower sol vent layer is col or less.
(c) Mag ne si um so l u t i ons. (1) Mag ne si um chl o ri de
so lu tion.37.5 mg/mL. Dis solve to tal of 3.75 g MgO, USP, by
add ing small amounts at time to 100 mL 8M HCl. (2) Mag ne sium
ni trate so lu tion.75 mg/mL. Mix 3.75 g MgO, USP, with ca 30 mL
H
2
O, slowly add HNO
3
to dis solve (ca 10 mL), cool, and di lute to
50 mL with H
2
O.
(d) So dium borohydride so lu tion.4.0 g NaBH
4
/100 mL 4%
NaOH.
(e) Po tas sium io dide so lu tion.Dis solve 20 g KI in H
2
O and
di lute to 100 mL. Pre pare just be fore use.
(f) Metal pow ders.Pu rity: 99.99 + % Cd, Pb, Zn; 99.99% Se.
( g ) Ca d mi u m s t a n d a r d s o l u t i o n s . ( 1 ) S t o c k
so lu tion.1 mg/mL. Dis solve 1.000 g Cd pow der in 20 mL HNO
3
(1 + 1) in 1 L vol u met ric flask, and di lute to vol ume with H
2
O.
(2) Working so lu tion.2 mg/mL. Pipet 10 mL stock so lu tion into
2005 AOAC IN TER NA TIONAL
Fig ure 986.15A. Hy dride gen er a tor: 1, poly eth yl ene
tub ing; 2, rub ber stop per; 3, flame sealed poly eth yl ene
tub ing with holes punched at one end; 4, re agent cup;
5, so dium borohydride so lu tion; 6, test so lu tion; 7,
ni tro gen in let from auxiliary line of AAS.
2006 AOAC INTERNATIONAL
100 mL vol u met ric flask, and di lute to vol ume with H
2
O. Pipet 2 mL
di luted so lu tion into 100 mL vol u met ric flask and di lute to vol ume
with H
2
O.
(h) Lead stan dard so lu tions.(1) Stock so lu tion.1 mg/mL.
Dis solve 1.000 g Pb pow der in 20 mL HNO
3
(1 + 1) in 1 L vol u met ric
f l ask, and di l ut e t o vol ume wi t h H
2
O. ( 2) Worki ng
so lu tion.5 mg/mL. Pipet 1 mL stock so lu tion into 200 mL
vol u met ric flask and di lute to vol ume with H
2
O.
(i) Zinc stan dard so lu tions.(1) Stock so lu tion.1 mg/mL.
Dis solve 1.000 g Zn pow der in 20 mL HCl (1 + 1) in 1 L vol u met ric
flask, and di lute to vol ume with H
2
O. (2) Working so lu tions.0.2,
0.5, 1.0, and 1.5 mg/mL. Pipet 1 mL stock so lu tion into 100 mL
vol u met ric flask and di lute to vol ume with H
2
O. Pipet 2, 5, 10, and
15 mL di luted so lu tion into sep a rate 100 mL vol u met ric flasks, each
con tain ing 1 mL HClO
4
, and di lute to vol ume with H
2
O.
(j) Ar senic stan dard so lu tions.(1) Stock so lu tion.Dis solve
1.320 g As
2
O
3
in min i mum vol ume 20% NaOH in 1 L vol u met ric
flask, acid ify with HCl (1 + 1), and di lute to vol ume with H
2
O.
(2) Working so lu tions.1, 2, 3, 4, and 5 mg/mL. Pipet 10 mL stock
so lu tion into 100 mL vol u met ric flask, and di lute to vol ume with
H
2
O. Pipet 1, 2, 3, 4, and 5 mL di luted so lu tion into sep a rate 100 mL
vol u met ric flasks, and di lute to vol ume with H
2
O.
( k ) S e l e n i u m s t a n d a r d s o l u t i o n s . ( 1 ) S t o c k
so lu tion.1 mg/mL. Dis solve 1.000 g Se pow der in min i mum
vol ume HNO
3
in 200 mL beaker and evap o rate to dry ness. Add 2 mL
H
2
O and evap o rate to dry ness. Re peat ad di tion of H
2
O and
evap o ra tion to dry ness twice. Dis solve in min i mum vol ume HCl (1
+ 9) in 1 L vol u met ric flask, and di lute to vol ume with HCl (1 + 9).
(2) Working so lu tions.1, 2, 3, 4, and 5 mg/mL. Pipet 10 mL stock
so lu tion into 100 mL vol u met ric flask and di lute to vol ume with
H
2
O. Pipet 1, 2, 3, 4, and 5 mL di luted so lu tion into sep a rate 100 mL
vol u met ric flasks and di lute to vol ume with H
2
O.
D. Closed Sys tem Di ges tion
(Do not ex ceed man u fac turers spec i fi ca tions of 0.3 g sol ids with
70 mL ves sel. Pro ceed cau tiously with new or un tried uses. Let such
test por tions stand with HNO
3
over night or heat on hot plate
cau tiously until any vig or ous re ac tion sub sides. Then pro ceed with
closed ves sel di ges tion. Open ves sel in hood since ni tro gen ox ides are
re leased.)
Weigh 0.3 g test portion (dry ba sis) into de con tam i nated
de com po si tion ves sel, add 5 mL HNO
3
, close ves sel with lid, and
heat in 150C oven 2 h. Cool in hood, re move ves sel from jacket, and
trans fer con tents to 10 mL vol u met ric flask. Add 4 mL H
2
O to
ves sel, cover with lid, and while hold ing lid tightly against rim,
in vert sev eral times, and add rinse to flask. Di lute to vol ume with
H
2
O and mix.
E. An odic Stripping Voltammetry
(For Cd and Pb.)
Pipet aliquot of di gested test so lu tion into de con tam i nated 50 mL
Vycor cru ci ble and add 2 mL ni trate so lu tion, C(b). Con duct re agent
blank si mul ta neously. Heat on hot plate at low heat to dry ness; then
in crease heat to max i mum (ca 375C). Ni trate salts will melt and
di gest or ganic mat ter in 1520 min. Place cru ci bles in 450C
fur nace to ox i dize any re main ing car bo na ceous mat ter (1020 min).
Di ges tion is com plete when melt is clear. Let cool, add 1 mL HNO
3
(1 + 1) to so lid i fied melt, and heat on hot plate to dry ness to ex pel
car bon ates and ni trites and to con trol acid ity. Dis solve in 5.0 mL
HNO
3
(0.5 mL/L), warm ing on hot plate to speed so lu tion. Trans fer
to polarographic cell with 5.0 mL H
2
O. Bub ble O
2
-free N
2
through
so lu tion 5 min; then di rect N
2
over so lu tion.
Set dial for Hg drops at 4 mm di vi sions. Stir so lu tion with
mag netic stir rer at con stant and re pro duc ible rate so Hg drop is not
dis turbed. Slide se lec tor switch to Ext. Cell and mea sure time for
120 s with stop watch. Turn off stir rer and let stand 30 s. Press Scan
but ton to ob tain peaks cor re spond ing to Cd and Pb at ca -0.57 and
-0.43 V, re spec tively, against sat u rated cal o mel elec trode.
Add known vol umes of each stan dard to test so lu tion in cell from
Eppendorf pipet. Amounts added should be ca 1 , 2 , etc. of
amount metal pres ent ini tially in cell, and each ad di tion should not
change orig i nal vol ume sig nif i cantly. Af ter each ad di tion, bub ble N
2
through so lu tion briefly and per form de po si tion and strip ping
op er a tions ex actly as for orig i nal so lu tion. Plot mg metal added on
x-axis against peak height on y-axis. Ex trap o late lin ear line to x-axis
to ob tain mg metal in cell.
mg Metal/g test por tion =

M M -

g test portion

10
mL aliquot taken
where M and M = mg metal from stan dard curve for test solution and
blank, re spec tively.
F. Atomic Ab sorp tion Spectrophotometry
(For As, Se, and Zn.)
(a) Ar senic.Pipet aliquot di gested test so lu tion into
de con tam i nated 50 mL round, flat-bottom boro sili cate flask, and
add 1 mL Mg(NO
3
)
2
so lu tion, (c)(2). Heat on hot plate at low heat to
dry ness; then in crease heat to max i mum (ca 375C). Place flask in
450C fur nace to ox i dize any car bo na ceous mat ter and to
de com pose ex cess Mg(NO
3
)
2
(30 min). Cool, dis solve res i due in
2.0 mL 8M HCl, add 0.1 mL 20% KI to re duce As
5+
to As,
3+
and let
stand 2 min. Con duct re agent blank with test portion.
2005 AOAC IN TER NA TIONAL
Fig ure 986.15B. Hy dride gen er a tor and mount
con nected to auxiliary line of spectrophotometer. Test
tube acid trap con nected be tween gen er a tor and
in stru ment is not in cluded in method.
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Pre pare stan dards as fol lows: To six 50 mL flasks (same type as
used for test portion) add 2.0 mL MgCl
2
so lu tion, (c)(1), and to
5 flasks add 50 mL aliquots of re spec tive work ing stan dard so lu tions
so that se ries will con tain 0, 0.05, 0.1, 0.15, 0.20, and 0.25 mg As.
(Other amounts may be used de pend ing on sen si tiv ity of sys tem.)
Add 0.1 mL 20% KI to each flask, mix, and let stand 2 min.
Con nect gen er a tor to in stru ment as shown in Fig ure 986.15B and
ad just pres sures and flows for H
2
H
2
-en trained air flame as in
Ta ble 986.15. Op er ate in stru ment ac cord ing to man u fac turers
in struc tions, with As lamp in place and re corder set for 20 mm/min.
Add 2.0 mL 4% NaBH
4
so lu tion to re agent dis penser of gen er a tor,
and in sert rub ber stop per tightly into neck of flask con tain ing test
solution or stan dard. With sin gle rapid, smooth mo tion, in vert flask,
let ting so lu tion mix with test solution or stan dard. (This op er a tion
must be per formed reproducibly.) Sharp, nar row A peak will ap pear
im me di ately. When re corder pen re turns to base line, re move stop per
from flask, and rinse re agent dis penser with H
2
O from squeeze
bot tle; then suck out H
2
O. Pro ceed with next test solution or
stan dard. When se ries is com plete, rinse glass ware thor oughly.
Plot cal i bra tion curve of mg As against A, and ob tain mg As in test
solution aliquot from this curve. Cor rect for re agent blank.
(b) Se le nium.Pro ceed as in (a), us ing Se lamp and stan dards,
but omit ad di tion of KI so lu tion. KI will re duce Se to el e men tal state
and cause loss of sig nal. In stead, cover flask with small watch glass
and place on steam bath 10 min, and cool to room tem per a ture.
(c) Zinc.Pipet 1 mL aliquot di gested test so lu tion into
de con tam i nated 25 mL Erlenmeyer, and add 0.1 mL HClO
4
. Heat on
hot plate to white fumes of HClO
4
. Solution should be com pletely
di gested as in di cated by clear, prac ti cally col or less so lu tion. If
solids char, add 0.5 mL por tions HNO
3
and again heat to white
fumes. Finally, heat just to dry ness but do not bake. Cool, and
dis solve res i due in 3.0 mL HClO
4
(1 + 99).
Op er ate in stru ment in ac cor dance with man u fac turers
in struc tions, us ing airC
2
H
2
flame, and mea sure A of test solutions
and stan dards, C(i)(2). Di lute test so lu tions with HClO
4
(1 + 99), if
so lu tion is too con cen trated. Plot cal i bra tion curve of mg Zn against A,
and ob tain mg Zn in test so lu tion aliquot from this curve. Cor rect for
re agent blank.
Ref er ence: JAOAC 63, 485(1980).
CAS-7440-38-2 (ar senic)
CAS-7440-43-9 (cad mium)
CAS-7439-92-1 (lead)
CAS-7782-49-2 (se le nium)
CAS-7440-66-6 (zinc)
Re vised: March 1996
* Adopted as a Co dex Al ter na tive Ap proved Method (Type III) for
atomic ab sorp tion spectrophotometry of ar senic in all foods.
Adopted as a Co dex Al ter na tive Ap proved Method (Type III) for an odic
strip ping voltammetry of cad mium, lead, and zinc in all foods.
Adopted as an Al ter na tive Ap proved Method (Type III) for atomic
ab sorp tion spectrophotometry of cad mium and se le nium in nat u ral
min eral wa ters.
Adopted as a Co dex Ref er ence Method (Type II) for atomic ab sorp tion
spectrophotometry of ar senic in nat u ral min eral wa ters.
Para graph E: Adopted as a Co dex Ref er ence Method (Type II) for
colorimetry, dithizone of lead in choc o late. Also adopted as a Co dex
Method for an odic strip ping voltammetry of lead in co coa pow ders
(co coa) and dry cocoasugar mix tures.
Para graph F: Adopted as a Co dex Al ter na tive Ap proved Method
(Type III) for atomic ab sorp tion spectrophotometry of ar senic in fruit
juices.
2005 AOAC IN TER NA TIONAL
Ta ble 986.15. Flow rates and pres sures for ar senic and
se le nium de ter mi na tions
Gas Tank psi
AA control
box, psi
Perkin-Elmer Model 403
flowmeter, di vi sions
H
2
20 10 20 (4 L/min)
N
2
40 30 25 (10 L/min)
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