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M

astovska

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

98, N

o

. 2, 2015 

481

calculations reviewed by a peer, laboratory supervisor, or

manager.

Data Analysis

The Study Directors reviewed and compiled all the

data submitted by the participants. Statistical analysis was

conducted using the AOAC spreadsheet for blind duplicates (4)

to determine mean analyte concentrations, SD (S

r

) and RSD

(RSD

r

) for repeatability (for blind duplicate data), SD (S

R

) and

RSD (RSD

R

) for reproducibility, number of valid data points,

HorRat value (RSD

R

/predicted RSD

R

), and percentage recovery

for all data after removal of outliers (3). The following tests were

used in the AOAC spreadsheet (4) to determine outliers: (

a

)

the

Cochran test for removal of laboratories showing significantly

greater variability among replicate (within-laboratory) analyses

than the other laboratories for a given material, and (

b

)

the

Grubbs’ tests for removal of laboratories with extreme averages.

AOAC Official Method 2014.08

Polycyclic Aromatic Hydrocarbons (PAHs)

in Seafood

Gas Chromatography-Mass Spectrometry

First Action 2014

[Applicable for the determination of the following PAHs

in mussel, oyster, and shrimp: 1,7-dimethylphenanthrene,

1-methylnaphthalene, 1-methylphenanthrene, 2,6-dimethyl-

naphthalene, 3-methylchrysene, anthracene, benz[

a

]anthracene,

benzo[

a

]pyrene, benzo[

b

]fluoranthene, benzo[

g,h,i

]perylene,

benzo[

k

]fluoranthene,

chrysene,

dibenz[

a,h

]anthracene,

fluoranthene, fluorene, indeno[1,2,3-

cd

]pyrene, naphthalene,

phenanthrene, and pyrene. These were representative PAH

analytes selected for the collaborative study. The method

has been single-laboratory validated for 32 PAHs in fish and

shrimp (1), and, therefore, is expected to be applicable to other

GC-amenable PAHs and seafood matrices. The concentration

ranges evaluated within the collaborative study are given in

Table

2014.08A

.]

Caution

:

See Appendix B: Laboratory Safety

. Use appropriate

personal protective equipment such as laboratory coat, safety

glasses or goggles, appropriate chemical-resistant gloves, and

a fume hood. Dispose of solvents and solutions according

to federal, state, and local regulations. Always handle open

containers of solvents inside the fume hood, including the

pouring, mixing, evaporating, and preparing standard solution.

Keep containers covered or closed when not in use.

Hexane and isooctane

.—Highly flammable, liquid irritants.

Harmful if inhaled, swallowed, or absorbed through the skin.

May also cause skin and eye irritation.

Ethyl acetate

.—Highly flammable, liquid irritants. Harmful

if swallowed in quantity. Vapors may cause drowsiness.

Toluene

.—Highly flammable, liquid irritant. Harmful if

inhaled, swallowed, or absorbed through the skin. May also

cause skin and eye irritation. May cause drowsiness. Possible

teratogen.

Dichloromethane

.—Noncombustible,

liquid

irritant.

Harmful if inhaled, swallowed, or absorbed through the skin.

May also cause skin and eye irritation. Asphyxiant. Causes

central nervous system (CNS) depression. Possible carcinogen

and mutagen.

PAHs

.—Carcinogens, respiratory sensitizers, teratogens,

reproductive hazard, mutagens. Harmful if inhaled, swallowed,

or absorbed through the skin. May also cause skin and eye

irritation.

See

Tables

2014.08B

D

for results of the interlaboratory

study supporting acceptance of the method.

A. Principle

Homogenized seafood samples (10 g sample with a 5 µg/kg

addition of

13

C-PAH surrogate mixture) are mixed with 5 mL

water (or 10 mL water in the case of shrimp and other more

viscous samples) and shaken vigorously by hand with 10 mL

ethyl acetate in a 50 mL polypropylene centrifuge tube for

1 min. Subsequently, 4 g anhydrous magnesium sulfate and

2 g sodium chloride are added to the mixture to induce phase

separation and force the analytes into the ethyl acetate layer.

The tube is again shaken by hand for 1 min and then centrifuged

for 10 min at >1500 rcf. A 5 mL aliquot of the ethyl acetate

layer is evaporated, reconstituted in 1 mL hexane, and cleaned

on an SPE column with 1 g silica gel and approximately

0.2 g anhydrous sodium sulfate on the top. The column is

conditioned with 6 mL hexane–dichloromethane (3 + 1, v/v)

Table 2014.08A. PAH and

13

C-PAH concentrations in the calibration standard solutions

Concentration, µg/L

Equivalent concentration, µg/kg

Calibration level

BaP and others

a

Chr and others

b

Naph

c

13

C-PAHs BaP and others Chr and others Naph

13

C-PAHs

1

5

12.5

25

50

0.5

1.25

2.5

5

2

10

25

50

50

1

2.5

5

5

3

20

50

100

50

2

5

10

5

4

50

125

250

50

5

12.5

25

5

5

100

250

500

50

10

25

50

5

6

200

500

1000

50

20

50

100

5

7

500

1250

2500

50

50

125

250

5

8

1000

2500

5000

50

100

250

500

5

a

 Analytes at 10 µg/mL in the mixed stock standard solution.

b

 Analytes at 25 µg/mL in the mixed stock standard solution.

c

 Analytes at 50 µg/mL in the mixed stock standard solution.