9.2.

28
AOAC Official Method 990.04
Mercury (Methyl) in Seafood
Liquid ChromatographicAtomic Absorption
Spectrophotometric Method
First Action 1990
Results of Interlaboratory Study:
s
r
= 0.020.12; s
R
= 0.030.25; RSD
r
= 4.518.2%; RSD
R
=
10.518.2%
A. Principle
LC effluent is heated to produce Hg vapor from organomercury
compounds. Hg vapor, together with vaporized mobile phase, is di-
rected into water-cooled condenser where mobile phase is liquefied.
Hg vapor is swept with nitrogen into absorption cell in light path of
atomic absorption spectrophotometer.
B. Apparatus
Wash all glassware with detergent (Micro Laboratory Cleaner, In-
ternational Products, Trenton, NJ 08601; or equivalent) and rinse
thoroughly with hot tap water followed by distilled or deionized wa-
ter. Rinse with acetone and let dry.
(a) LC/AAS interface.See Figure 990.04. Assemble as de-
scribed in D. (1) Heater.HSP disk heater, 120 V, 200 W, 3.25 in
od 11/32 in thick, Leo C. Pilkus, 170 Worcester St, Wellesley, MA
02181, with magnesia and alumina insulation, 1 in thick. (2) Flow
meter.No. 7908, Alltech Associates, Inc., 2051 Waukegan Rd,
Deerfield, IL 60015. (3) Temperature-indicating device.Pyrome-
ter, series 7000, 01370C, 3.5 in case, part No. 7035K2500; ther-
mocouple, TJ 18-CAIN-116G-6-LUG, Omega Engineering, One
Omega Dr, Stamford, CT 06907. (4) Short condenser.175 mm
jacket length, standard taper 24/40 (Aldrich No. Z11,575-4). (5)
Rubber stopper.No. 5, solid neoprene (No. 14141F, Fisher Sci-
entific Co.). (6) Stainless steel tubing.1/16 in (1.6 mm) od, 0.04 in
(1 mm) id (No. 38-3067, Rainin Instrument Co., Mack Rd, Woburn,
MA 01801). (7) Trap.Test tube, 125 15 mm. (8) Boiling
flask.2-neck, 500 mL (Kontes No. 605000). (9) Stainless steel
tubing.Two 6 in (15 cm) lengths (see 6). (10) Plastic tub-
ing.Spaghetti type, 0.0570.067 in (1.451.7 mm) id
(No. 13-9045-12, Ace Scientific Supply Co., Inc., 40-A Cutter Ln,
East Brunswick, NJ 08816, USA). (11) Plastic tubing.Spaghetti
type, as connector to AAS system (see 10). (12) Electrical connec-
tion.Standard 120 V plug, to variable voltage transformer.
(b) LC pump.Tracor 950, or equivalent, with 100 L fixed
loop injector (Rheodyne, Berkeley, CA).
(c) LCcolumn.ZorbaxODS, 5m, 4.6mm25cm(DuPont).
(d) Guard column.7 cm 2.1 mm id, packed with 2537 m
CO:Pell ODS (Whatman, Inc., Clifton, NJ; or equivalent).
(e) Atomic absorption spectrophotometer.Equipped with Hg
hollow cathode lamp, deuterium background corrector, and gas
flow-through cell with open ends or quartz closed ends (1025 mm
id 100115 mm). Followmanufacturers operating instructions for
Hg determination using wavelength of 253.7 nm and deuterium
background correction. Typical response for injection of 0.100 g
Hg/100Lstandard is ca 0.20 A, using cell 25 mmid 115 mm. Use
recording device set to obtain ca 3050% full scale for injection of
0.100 g Hg/100 L standard. Working range is between ca 0.01
and 0.25 g Hg/100 L injected.
(f) Glass chromatographic columns.25 mm od, 22 mm id
250 mm.
(g) Homogenizer.Polytron (No. 10/35) with PTA-10S head
(Brinkmann Instruments, Westbury, NY), or equivalent.
(h) Variable transformer.0140 V.
C. Reagents
(Use water double-distilled in glass.)
(a) Sodium thiosulfate solution.0.01M. Dissolve 2.5 g
Na
2
S
2
O
3
5H
2
O and dilute to 1 L with water.
(b) Hydrochloric acid solution.1.8M. Dilute 148 mL concen-
trated HCl to 1 L.
(c) Celite 545.Acid-washed (Johns-Manville Products Corp.,
Denver, CO).
(d) Methyl mercuric chloride stock standard solution.100 g
Hg/mL. Weigh 0.125 g CH
3
HgCl (Johnson Matthey Co., Ward Hill,
MA, USA) into 1 Lvolumetric flask. Add 20 mLmethanol, shake to
dissolve CH
3
HgCl, dilute solution to volume with water, and mix.
(Caution: See Appendix B, safety note on mercury salts.)
(e) Ammonium acetate solution.0.05M. Dissolve 3.9 g ammo-
nium acetate and dilute to 1 L with water.
2002 AOAC INTERNATIONAL
Figure 990.04LC/AAS interface. Assemble as de-
scribed in D. (1) Disk heater; (2) flow meter; (3) pyrome-
ter; (4) short condenser; (5) rubber stoper; (6) stainless
steel tubing; (7) trap; (8) boiling flask; (9) stainless steel
tubing; (10) and (11) plastic tubing; (12) electrical con-
nection to variable voltage transformer.
(f) Mobile phase.Methanol0.05M ammonium acetate (3 + 2).
Mix 3 volumes of methanol with 2 volumes of ammonium acetate
solution and adjust pH to 5.7 0.2 with glacial acetic acid. Degas
with He. Add 0.1 mL 2-mercaptoethanol/L immediately before use.
Shake very gently to thoroughly mix, taking care to incorporate as
little air as possible into solution.
D. Instrument Set-Up
Figure 990.04 is a diagramof the LC/AASinterface. Components
are placed inside shop-made box of dimensions shown. Box has
Plexiglas door at front, and back and top are removable. Items 13
are bolted to sides of box. Set up remaining items as follows: Bend
30 in stainless steel tubing (item 6) as shown to provide additional
heating surface. Place bent portion, together with thermocouple ele-
ment, between 2 disks of heater, which are held tightly together by
screw at center of upper disk. Enclose heater assembly in 1 in
(25 mm) thick magnesiaalumina insulation, and secure to alumi-
num plate support by means of aluminum cover and screws.
Push stainless steel tubing from heater outlet through center
of rubber stopper (item 5) so that end of tubing is near con-
structed portion of condenser when stopper is inserted tightly
into top of condenser. Push 2 additional 6 in (15 cm) lengths of
stainless steel tubing through rubber stopper to serve as nitro-
gen inlet and Hg vapor outlet, respectively. Connect these
lengths of tubing to flow meter and to test tube trap, respec-
tively, by means of spaghetti-type tubing. Connect nitrogen
tank to flow meter by means of spaghetti-type tubing and stan-
dard Swagelok fittings and unions.
Connect outlet from LC column to 0.01 in (0.25 mm) id stainless
steel tube, which is connected to inlet of heating tube by standard
1/16 in (1.6 mm) Swagelok fittings and zero dead volume union.
Connect outlet of test tube trap (spaghetti tubing, item11) to AAS
cell by small rubber stopper inserted into side arm of cell.
E. Operating Conditions for LC/AAS Interface
(a) To turn system ON.(1) Adjust mobile phase flow rate to
0.7 mL/min. (2) Introduce water into condenser. (3) Adjust nitrogen
sweep to 0.1 L/min (tank pressure 15 psi (1.04 kPa) and 10.0 setting
on flow meter). (4) Gradually adjust temperature of interface heater
to 550C (transformer setting ca 65). (5) After temperature reaches
550C, check systemstability by injecting several aliquots of methyl
mercury standard solutions. Retention time of methyl mercury is
56 min. Precision between methyl mercury peak heights should be
5%. Inject all standard solutions to check linearity. If these parame-
ters cannot be achieved, check for leaks or, after long use, replace ef-
fluent tubing. (Note: To conserve analytical standard solutions,
another set of standards of same concentration may be prepared by
direct dilution of stock standard solution with 0.01M Na
2
S
2
O
3
. Use
these standards only for instrument checking. To prepare 0.05,
0.100, 0.150, 0.200, and 0.250g Hg/100L, dilute 100g Hg/mL
standard solution with 0.01NNa
2
S
2
O
3
solution as follows: 1, 1, 3, 2,
and 5 mL to 200, 100, 200, 100, and 200 mL, respectively.)
(b) To turn system OFF.(1) Turn off interface heater and let
cool to near roomtemperature. (2) Shut off other components, but do
not shut off mobile phase flowwhile heater is hot. If this is done, car-
bon may deposit and clog effluent tube. For same reason do not
pump neat organic solvents, such as methanol, to clean column
while heater is hot. (3) After heater has cooled to roomtemperature,
pump methanol to rinse column.
F. Preparation of Materials
Use only edible portion of seafood. Homogenize test sample in
blender or food processor. Water (double-distilled in glass) may be
added to aid homogenization, but added water must be accounted for
in final calculation. Weigh 10.0 0.30 g (wet) test portion of homog-
enized test sample into 100 mL beaker. Prepare analytical solution
by adding HCl solution so that mass of analytical portion plus HCl
solution totals 25.00 0.30 g. Blend analytical solution in homoge-
nizer (ca 1 min) to obtain fine suspension. Immediately weight
10.0 g homogenized solution into beaker containing 10 g Celite 545,
and mix well. Quantitatively transfer mixture, with aid of
Scupula, to chromatographic column, B(f), containing pledget of
glass wool at bottom. Compact mixture moderately with tamping
rod to height of ca 8 cm, and place pledget of glass wool on top.
Elute column by adding 20 mL followed by four 5 mL aliquots of
CHCl
3
. Collect first 20 mL eluate in tall 25 mL glass-stopper gradu-
ated cylinder. Add 4.0 mLNa
2
S
2
O
3
solution by pipet, shake mixture
gently 1 min, and let stand 5 min. Using disposable pipet, transfer
upper aqueous layer containing methyl mercurythiosulfate com-
plex together with any emulsion into 25 mLErlenmeyer. Blowmod-
erately strong stream of nitrogen into flask for 12 min to break up
emulsion and expel droplets of CHCl
3
. (Note: To aid in breaking
emulsion, hold and rotate the flask at 45 angle with one hand, and
direct nitrogen stream at thin layer of emulsion that adheres to bot-
tom of flask as it rotates.) (Note: Some species of seafood may pro-
duce cloudy extracts. If this occurs, extract may be filtered through
membrane filter.)
G. Preparation of Reagent Blank Solution
Prepare reagent blank analytical solution by weighing 25.00 g
HCl solution into 100 mL beaker. Proceed as in Preparation of Ma-
terials, F, beginning Immediately weigh 10.00 g . . ..
H. Preparation of Standard Solutions
Prepare 0.050, 0.100, 0.150, 0.200, and 0.250 g Hg/100 L stan-
dard solutions by adding, respectively, 20, 40, 60, 80, and 100 L
aliquots of CH
3
HgCl stock standard solution, C(d), to 20 mLCHCl
3
in
separate 25mLglass-stopper graduatedcylinders. Proceedas inPrepa-
rationof Materials, F, beginningAdd4.0mLNa
2
S
2
O
3
solution. . ..
I. Determination
Inject 100 L aliquot of test solution (0.100 g injected for 10.0 g
analytical portion) into LC/AASsystem. After methyl mercury peak
appears, inject 100 L aliquot of standard solution that produces
peak height equal to or slightly higher than test solution peak height.
Repeat by injecting test solution again followed by selected stan-
dard. If test solution peak height is higher than peak height for high-
est standard, dilute appropriate aliquot of test solution (not less than
1.00 mL, using positive displacement pipets) with Na
2
S
2
O
3
solution.
Account for dilution in final calculation.
J. Calculation
Additional dilutions must be accounted for in final calculation. Do
not vary injection volume.
Measure peak heights above baseline and calculate methyl-bound
mercury concentration in sample, g Hg/g, by comparing average
peak heights of sample solution to average peak heights of standard
solution as follows:
Concentration Hg (mg/kg) = (R/R) (W / W)
2002 AOAC INTERNATIONAL
where R=average peak height of test solution (A); R =average peak
height of standard solution (A); W = amount standard injected (g
Hg); W = amount test portion injected (g), and
W = (D/E) [F (0.100 mL/4.0 mL)]
where D= mass of analytical portion, g; E = mass of analytical solu-
tion, g; and F = mass of analytical solution added to Celite, g.
If necessary, correct Afor test solution for Afor correspondingly
diluted blank solution. Quantitation limit, defined as 10 standard
deviation of reagent blank, is 0.006g Hg/100 Linjected. This cor-
responds to quantitation limit of 0.06 g Hg/g for 10 g test portion
treated according to method.
Reference: JAOAC 72, 926(1989).
CAS-7439-97-6 (mercury)
Revised: March 2002
2002 AOAC INTERNATIONAL