9.2.

27
AOAC Official Method 988.11
Mercury (Methyl) in Fish and Shellfish
Rapid Gas Chromatographic Method
First Action 1988
Final Action 1992
A. Principle
Organic interferences are removed from homogenized seafood
by acetone wash followed by toluene wash. Protein-bound methyl
Hg is released by addition of HCl and extracted into toluene. Tolu-
ene extract is analyzed for CH
3
HgCl by electron capture GC.
B. Reagents
Equivalent reagents may be used.
(a) Solvents.Acetone, toluene, and isopropanol, all distilled
in glass (Burdick & Jackson Laboratories, Inc., or EM Science
OmniSolv

reagents). (Caution: Toluene is harmful if inhaled

and is flammable; conduct all operations with toluene in labora-
tory hood.)
(b) Hydrochloric acid solution (1 + 1).Add concentrated HCl
to equal volume distilled or deionized H
2
O and mix. Use 2 volumes
toluene to extract potential interferences from 1 volume HCl solu-
tion by vigorously shaking mixture 15 s in separator. Discard tolu-
ene extract. Repeat extraction step 4 times. Solution may be mixed in
advance. However, extraction must be performed immediately be-
fore HCl solution is used to avoid formation of electron-capturing
compounds which produce extraneous peaks in chromatograms.
Before beginning analysis, check quality of reagents by
chromatographingblanktakenthroughmethod. Donot use HCl andsol-
vents which produce extraneous peaks at retention time of methyl Hg.
(c) Carrier gas.GC quality ArCH
4
(95 + 5).
(d) Sodium sulfate.Anhydrous reagent grade. Heat overnight
in 600C furnace, let cool, and store in capped bottle. Line cap with
acetone-washed Al foil to prevent contamination from cap. Peaks
appearing at 1415 min may be eliminated by refiring Na
2
SO
4
(600C overnight).
(e) Methyl mercuric chloride standard solutions.Keep
tightly stoppered. Seal stopper with Teflon tape. (1) Stock stan-
dard solution.1000 g Hg/mL. Weigh 0.1252 g CH
3
HgCl
(ICN-K&K Laboratories, Inc., PO Box 28050, Cleveland, OH
44128-0250) into 100 mLvolumetric flask. Dilute to volume with
toluene. (2) High level intermediate standard solution.40 g
Hg/mL. Dilute 10.0 mL stock standard solution to 250.0 mL with
toluene. (3) Low level intermediate standard solution.2.0 g
Hg/mL. Dilute 10.0 mL high level intermediate standard solution
to 200.0 mL with toluene. (4) Working standard solu-
tions.0.0050.10g Hg/mL. Prepare monthly by diluting with
toluene in volumetric flasks as follows: Dilute 10.0 mL of 2.0g
Hg/mL solution to 200.0 mL for 0.10 g Hg/mL. Dilute 20.0 mL
of 0.10 g Hg/mL solution to 25.0 mL, 15.0 mL to 25.0 mL,
10.0 mL to 25.0 mL, 10.0 mL to 50.0 mL, 10.0 mL to 100.0 mL,
and 10.0 mL to 200.0 mL for 0.080, 0.060, 0.040, 0.020, 0.010,
and 0.005 g Hg/mL, respectively.
(f) Mercuric chloride column treatment solution.1000 ppm
(g/g) HgCl
2
. Dissolve 0.1 g HgCl
2
in 100 mL toluene.
(g) Fortification solutions.(1) Stock solution.1000 g
Hg/mL. Weigh 0.1252 g CH
3
HgCl into 100 mL volumetric flask.
Dilute to volume with H
2
O. (2) Working fortification solu -
tion.15 g Hg/mL. Dilute 1500L stock fortification solution to
100.0 mL with H
2
O.
C. Apparatus
Wash all glassware with detergent (Micro Laboratory Cleaner,
International Products, PO Box 70, Burlington, NJ 08601-0070,
USA) and rinse thoroughly with hot tap H
2
Ofollowed by distilled
or deionized H
2
O. Then rinse 3 times with acetone and 3 times
with toluene. Dry in hood.
Equivalent apparatus may be used except use packed column
specified.
(a) Centrifuge.Model IEC CRU-5000 or CR6000 (Interna-
tional Equipment Co.).
(b) Centrifuge tubes.Glass, 50 mL capacity with Teflon-lined
screw caps (Cat. No. 9212-K78, Thomas Scientific).
(c) Graduated cylinders.Glass, class A, 50 mL capacity, with
ground-glass stoppers (Kimble 20036).
(d) Transfer pipets.Disposable glass, Pasteur-type.
(e) Dropping pipets.Glass, 5 mL capacity (No. 13-710B,
Fisher Scientific Co.)
(f) Mechanical shaker.Model S-500 shaker-in-the-round, with
timer (Glas-Col Apparatus Co., 711 Hulman St, POBox 2128, Terre
Haute, IN 47802.)
(g) Gas chromatograph.Hewlett Packard Model 5710A (re-
placed by HP-5890 series II) or equivalent, equipped with linear
63
Ni
electron capture detector and 6 ft 2 mm id silanized glass column
packed with 5%DEGS-PS on 100120 mesh Supelcoport (Supelco,
Inc., No. 1-1870). Pack column no closer than 2.0 cmfrominjection
and detector port nuts and hold packing in place with 2 cmhigh qual-
ity, silanized glass wool at both ends. Install oxygen scrubber and
molecular sieve dryer (No. HGC-145, Analabs, Inc.) between car-
rier gas supply and column. Condition column according to manu-
facturers instructions as follows: Flush column 0.5 h with carrier
gas flowing at 30 mL/min at room temperature. Then heat 1 h at
50C. Next, heat column to 200C at 4C/min and hold at 200C
overnight. Do not connect column to detector during this condition-
ing process. Maintain 30 mL/min carrier gas flowat all times during
conditioning, treatment, and use. Operating conditions: column
155C; injector 200C; detector 300C; carrier gas flow30 mL/min;
recorder chart speed 0.51.0 cm/min. Under these conditions and
with HgCl
2
column treatment procedure described below, CH
3
HgCl
peak will appear 23 min after sample injection.
D. Mercuric Chloride Column Treatment
Columnof 5%DEGS-PS, conditionedaccordingtomanufacturers
instructions, can be used to determine CH
3
HgCl only after treatment
by HgCl
2
solution, B(f). Because column performance degrades
with time, also treat column periodically during use. Perform ap-
propriate HgCl
2
treatment procedures described below.
(a) Following 200C column conditioning and after every
23 days of analyses.If column has just been conditioned accord-
ing to manufacturers instructions or has been used 23 days to ana-
lyze extracts, proceed as follows: Adjust column temperature to
200C and inject 20 L HgCl
2
treatment solution 5 times at
510 min intervals. Maintain 200C temperature overnight.
Chromatogramwill contain large, broad peaks. Adjust column tem-
perature to 155C next morning and inject 20 L HgCl
2
treatment
solution 2 more times. Large, broad chromatographic peaks ap-
pearing at ca 12 h signal completion of treatment process and
that column is ready for use.
2000 AOAC INTERNATIONAL
(b) On day preceding sample extract analysis.If column has
been treated by procedure (a) or used 1 day at 155C to analyze ex-
tracts, column may be treated at end of working day for next days
use as follows: Lower column temperature to 115C and inject
20L HgCl
2
treatment solution 1 time. After large, broad peaks ap-
pear in chromatogram (1120 h), treatment process is complete.
Next working day, increase column temperature to 155Coperating
temperature. When baseline is steady, column is ready for use.
(c) During extract analysis at 155C.If column has been used
at 155C to analyze extracts or if column performance and peak
height have degraded enough to require HgCl
2
treatment, inject two
20L aliquots of HgCl
2
treatment solution. Large, broad peaks will
appear in chromatogram12 h after HgCl
2
injection, signaling com-
pletion of treatment process. Wait for steady baseline; then column
is ready for use.
E. Extraction of Methyl Mercury Chloride
Performall operations, except weighing, in laboratory hood. Take
empty centrifuge tube through all steps for method blank determina-
tion. Accurately weigh 1 g homogenized test portion into 50 mLcen-
trifuge tube. Add 25 mL acetone; tightly cap and vigorously shake
tube by hand 15 s. Loosen cap and centrifuge 5 min at 2000 rpm.
Carefully decant and discard acetone. (Use dropping pipet to remove
acetone, if necessary.) Repeat 25 mL acetone wash step 2 more
times. Break up tissue with glass stirring rod before shaking tube, if
necessary. Add 20 mL toluene; tightly cap and vigorously shake
tube by hand 30 s. Loosen cap and centrifuge 5 min at 2000 rpm.
Carefully decant (or drawoff with dropping pipet) and discard tolu-
ene. Extraneous peaks in final GC chromatogram may indicate that
more vigorous shaking with acetone and toluene is required. In prod-
ucts for which methyl Hg recoveries are to be determined, fortify tis-
sue at this point by adding working fortification solution, B(g), to
centrifuge tubes.
Add 2.5 mLHCl solution, B(b), to centrifuge tube containing ace-
tone- and toluene-washed sample. Break up tissue with glass stirring
rod, if necessary. Extract CH
3
HgCl by adding 20 mL toluene and
shaking gently but thoroughly 5 min on mechanical shaker at set-
ting 5 (2 min by hand). Loosen cap and centrifuge 5 min at 2000 rpm.
If emulsion is present after centrifugation, add 1 mLisopropanol and
gently stir into toluene layer with glass stirring rod to reduce emul-
sion. Do not mix isopropanol with aqueous phase. Add equal
amounts of isopropanol to blank and test solutions. If emulsion is not
present, do not add isopropanol to blank or test solutions. Vigorous
mixing of isopropanol with HCl may produce interfering peaks in
chromatograms. Recentrifuge. With dropping pipet, carefully trans-
fer toluene to graduated cylinder. Rinse walls of centrifuge tube with
12 mL toluene and transfer rinse to graduated cylinder. Repeat ex-
traction step 1 more time. Combine both extracts in graduated cylin-
der, dilute to 50 mL with toluene, stopper, and mix well. Add 10 g
Na
2
SO
4
and mix again. Tightly stoppered extracts (sealed with Tef-
lon tape) may be refrigerated and held overnight at this point. Ana-
lyze by GC.
F. Chromatography
Verify that system is operating properly by injecting 5 L stan-
dard solution containing 0.005 g Hg/mL into GC system. Differ-
ence between CH
3
HgCl peak heights for 2 injections should be 4%.
Check detector linearity by chromatographing all working standard
solutions.
Inject 5 L standard solution with concentration approxi-
mately equal to or slightly greater than concentration of ex-
tract. Immediately after CH
3
HgCl peak appears, inject another
5 L extract. Immediately after CH
3
HgCl and background
peaks for extract appear, inject another 5 L of standard solu-
tion. Because column performance and peak height slowly de-
crease with time, calculate each Hg concentration in each test
sample by comparing peak height for each test extract to aver-
age peak height for standard solutions injected immediately af-
ter test extract.
Correct height of CH
3
HgCl peak for test extract by subtracting
height of peak for method blank obtained at same attenuation and
recorder sensitivity. Calculate methyl-bound Hg content of test
sample expressed as g Hg/g (ppm Hg) by comparing height of
peak from injection of test extract to average height of peak from
duplicate injections of standard solution as follows:
g Hg/g fish = (R/R) (C /C) 50
where R=corrected height of CH
3
Hg Cl peak frominjection of test ex-
tract, R =average height of CH
3
HgCl peakfromduplicate injections of
standard solution, C = weight (g) of test portion, C = concentration
(g/mL) of Hg in standard solution, and 50 = final volume (mL).
Reference: JAOAC 70, 24(1987).
CAS-7439-97-6 (mercury)
2000 AOAC INTERNATIONAL