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504 

M

astovska

et al

.

:

J

ournal of

AOAC I

nternational

V

ol

. 98, N

o

. 2, 2015

to the blind duplicates) in all seven test samples was 70.5 µg/kg

(RSD = 16.3%,

n

= 56), which corresponds to mean recovery of

88.1%. These results indicate that the mussel and oyster samples

sent to the study participants had good homogeneity. 1,7-DMP

was also added to all shrimp test samples at 20 µg/kg during the

fortification step conducted by the study participants. The mean

concentration value was 22.1 µg/kg (RSD = 19.0%,

n

= 63),

corresponding to mean recovery of 111%. Statistical results for

1,7-DMP obtained in blind duplicate samples are summarized

together with the other analytes in Tables

2014.08B–D

.

Tables

2014.08B–D

provide statistical results obtained for

the studied analytes at three different concentration levels

in shrimp, mussel, and oyster after elimination of statistical

outliers (highlighted in Tables 3–11).

Eight to 10 valid results were obtained for the majority of

determinations. Mean recoveries of all tested analytes at the

total of five different concentration levels were all in the range

of 70–120%: 83.8–115% in shrimp, 77.3–107% in mussel, and

71.6–94.6% in oyster, except for a slightly lower mean recovery

of 68.6% for BaA fortified at 25 µg/kg in oyster (RSD

r

: 5.84%,

RSD

R

: 21.1%) and lower mean recoveries for Ant and BaP

in oyster at all three fortification levels (50.3–56.5% and

48.2–49.7%, respectively).

The lower mean recoveries of Ant and BaP were linked to

degradation of these analytes in oyster samples stored at –20°C

(

see

the discussion about degradation issues below), which also

resulted in lower reproducibility (RSD

R

values in the range

of 44.5–64.7% for Ant and 40.6–43.5% for BaP). However,

the repeatability was good (RSD

r

of 8.78–9.96% for Ant and

6.43–11.9% for BaP), and the HorRat values were acceptable

(1.56–1.94 for Ant and 1.10–1.45 for BaP).

In all other cases, repeatability, reproducibility and HorRat

values were as follows:

(

1

) Shrimp: RSD

r

1.40–26.9%, RSD

R

5.41–29.4%, HorRat

0.22–1.34;

(

2

) Mussel: RSD

r

2.52–17.1%, RSD

R

4.19–32.5%, HorRat

0.17–1.13; and

(

3

) Oyster: RSD

r

3.12–22.7%, RSD

R

8.41–31.8%, HorRat

0.34–1.39.

Overall, the results of the collaborative study demonstrate

that the method is fit-for-purpose to determine PAHs and their

alkyl homologs in seafood samples.

Degradation Issues

The Study Directors reported problems with lower recoveries

for Ant and BaP in oyster samples stored at –20°C to the SPSC

PAH Working Group and the AOAC Methods Committee on

PAHs when they discovered a significant difference between

results for these two analytes obtained in two different

participating laboratories storing the samples at two different

freezer temperatures of –20 and –70°C (

see

recovery results in

Tables 12 and 13, respectively). Due to the limited availability

and cost of oyster samples, it was decided to continue with

the study and not proceed with preparation of new study test

samples that would be fortified by collaborators on the day of

the analysis as was done for shrimp (

Note

: in the case of shrimp,

overall lower recoveries were obtained for all studied PAHs

depending on the shrimp sample storage conditions).

The laboratory storing samples at –20°C analyzed another

set of oyster samples about 1.5 months after the first set

and confirmed the lower recoveries for Ant and BaP. This

collaborator also made another interesting observation related

to the color difference between extracts obtained in the first

set (all dark green) and the second set (the dark green extract

was produced only for the blank sample), whereas all extracts

of fortified samples were yellow-brown. This observation,

which was later confirmed by additional study participants,

indicates matrix changes caused by the presence of PAHs and

accompanied by selective losses of Ant and BaP. In addition to

these two analytes, BaA also showed lower recoveries (around

70%) in oysters when compared to the rest of the studied PAHs.

Furthermore, a closer examination of the results obtained for

Ant, BaP, and BaA in mussel also show somewhat lower mean

recoveries of these three analytes (around 80%) when compared

to the other analytes. These mean results do not include data

obtained for BaP in mussel test samples by Laboratory No. 6,

which analyzed the test samples about a year later than most

other participants. All Laboratory No. 6 BaP results in mussel

test samples showed very good repeatability within the

duplicates but were eliminated as Grubbs’ test outliers because

they were significantly lower than the results obtained by other

laboratories. No other data obtained by Laboratory No. 6 were

identified as outliers using the Cochran or Grubbs’ tests.

Figure 1 provides structures of Ant, BaA, and BaP showing

that these PAHs contain the same moiety in terms of the linear

3-ring (anthracene) structure. This structural commonality

and similar degradation behavior indicate that they could be

substrates for the same enzyme(s). The significant differences in

recoveries obtained for samples stored at –20°C versus –70°C

(Tables 12 and 13, respectively) represent more supporting

evidence for an enzymatic degradation being the most probable

cause for the lower recoveries observed for these analytes in

oyster (and mussel) test samples. To prevent degradation of

these analytes in seafood matrixes during long-term storage,

the Study Directors recommend storing homogenized samples

at –70°C or lower. Unfortunately, this was not a feasible

requirement for this collaborative study because the majority

of the collaborating laboratories did not have this storage

capability.

Acknowledgments

The Study Directors wish to thank the SPSC PAH Working

Group (chaired by Gina Ylitalo from National Oceanic and

Atmospheric Administration), the AOAC Methods Committee

on PAHs (chaired by Tom Phillips, Maryland Department of

Agriculture, Annapolis, MD), and AOAC INTERNATIONAL

staff for the discussions about the study design, analyte

selection, and stability issues as well as for the overall support

provided to the collaborative study. Special acknowledgment

goes to Jack Cochran (Restek Corp., Bellefonte, PA) for useful

discussions about the PAH GC/MS analysis.

John Schmitz and Jack Jabusch (Covance Laboratories,

Madison, WI) and Lucie Drabova and Jana Pulkrabova (ICT,

Prague, Czech Republic) are acknowledged for technical advice

and support during the preparation and the entire course of the

study.

The Study Directors thank the Nutritional Chemistry and

Food Safety business unit at Covance Laboratories for supplying

and preparing PAH standards and spiking solutions; obtaining