Methods Reviewed by OMB in 2015

astovska

et al

ournal of

AOAC I

nternational

98, N

. 2, 2015

479

Collaborative Study

Purpose

The purpose of this study was to evaluate the method’s

intralaboratory and interlaboratory performance and submit the

results to AOAC INTERNATIONAL for adoption as an Official

Method for the determination of PAHs in seafood.

Study Design

This study evaluated the method performance for

determination of 19 selected PAHs, including alkyl homologs

relevant to an oil spill contamination (

see

Table 1), in three

seafood matrixes: shrimp, oysters, and mussels, with five

different levels of BaP ranging from 2 to 50 µg/kg. Each matrix

had a varying mixture of three different BaP levels (“low,”

“mid,” and “high”). The other studied PAHs were added

at varying levels from 2 to 250 µg/kg to mimic typical PAH

patterns (Table 2). The fortified analytes in the three matrixes

were analyzed as blind duplicates at each level of BaP and

corresponding other PAH levels. In addition, a blank with no

added PAHs for each matrix was analyzed singly. The AOAC

official method guidelines for collaborative study procedures (3)

were followed for the preparation of the study and data analysis.

Test Sample Preparation

Blank mussel and oyster samples were homogenized with

liquid nitrogen and tested in duplicate by an independent

laboratory for potential contamination with the target PAHs.

During homogenization, portions of the blank matrixes were

spiked with 1,7-dimethylphenanthrene (1,7-DMP) at 40 and

80 µg/kg in the case of mussel and oyster, respectively. These

were utilized as a homogenization check throughout the

course of the study. The collaborators determined 1,7-DMP

along with the other 18 analytes, which were spiked into 10 g

sample portions placed in polypropylene centrifuge tubes by

the study direction team. Five different spiking levels were

made at varying PAH concentrations (Table 2), resulting in

three different duplicate spiked samples/matrix in addition to

a blank. Participants were supplied with the test samples ready

for analysis labeled with unique identification numbers. All test

samples were shipped frozen on dry ice with a material receipt

document to be returned to the Study Directors. The test samples

had to be stored in a freezer set to maintain at least –20 ± 10°C.

Test samples were to be analyzed after completion of laboratory

qualification and practice sample analysis.

Blank shrimp matrix (peeled, without head and tail, and

uncooked) was homogenized without the use of liquid nitrogen

using a blender. After testing for potential contamination with

the target PAHs, 10 g blank sample portions were placed in

polypropylene centrifuge tubes, which were sent to study

participants together with spiking solutions labeled with unique

identification numbers. Using instructions provided by the

Study Directors, participants fortified the blank shrimp samples

themselves on the day of the analysis.

Three different spiking levels were used at varying PAH

concentrations (Table 2), resulting in three different duplicate

spiked samples in addition to a blank (seven samples altogether).

The blank shrimp samples were shipped frozen on dry ice with a

material receipt document to be returned to the Study Directors.

The test samples had to be stored in a freezer set to maintain at

least –20 ± 10°C. The spiking solutions were to be stored in a

refrigerator set to maintain 5

3°C. (

Note

: This modification

of the shrimp test sample preparation protocol (as compared to

mussel and oyster) was made (after consultations with the SPSC

PAH Working Group and the AOAC Methods Committee on

PAHs ) due to potential stability issues discovered during the

practice sample analysis and follow-up experimentswith fortified

shrimp samples stored at different conditions. 3-Methylchrysene

(3-MC) had to be replaced by 6-methylchrysene (6-MC) in the

spiking and calibration solutions for shrimp samples due to

the unavailability of a 3-MC reference standard at the time of

preparation and shipment of the new set of shrimp samples to

the study participants.)

Laboratory Qualification

During the laboratory qualification phase, the collaborators

conducted the following seven steps. These steps were

necessary because the Study Directors allowed the use of

various GC/MS instruments, GC columns, silica SPE cartridges,

and evaporation techniques and equipment. Therefore,

performance-based criteria were developed to help laboratories

optimize their GC/MS, SPE cleanup, and solvent evaporation

conditions; check and eliminate potential PAH contamination

in their reagent blanks; and become familiar with the method.

Laboratory qualification and practice sample results had to be

approved by the Study Directors before proceeding with the test

sample analysis. Sixteen laboratories entered the qualification

phase, but only 10 of them (listed in the

Acknowledgments

section) completed the qualification successfully and/or

continued in the study.

(

) The first step was a GC separation test where participants

analyzed a composite PAH solution by GC/MS/MS to obtain a

baseline separation of BaP and benzo[

]pyrene (concentration

ratio of 1:5); at least 50% valley separation of anthracene

and phenanthrene (concentration ratio 1:2.5, evaluated for

the anthracene peak); and at least 50% valley separation for

benzo[

]fluoranthene, benzo[

]fluoranthene, and benzo[

]

fluoranthene (concentration ratio of 1:1:1).

(

) The second step was a calibration range test where

participants prepared calibration standards and obtained

normalized calibration curves for the studied PAHs versus

respective labeled internal standards (

C-PAHs). Collaborators

had to determine the linear range, test for carryover by injecting

a solvent blank after the highest standard, and adjust injection

conditions (such as injection volume, number of washes, syringe

size, etc.) to achieve low detection limits, acceptable linearity

for the tested concentration range, and minimum carryover.

Coefficient of determination (r

) values should be 0.990 or

greater, and back-calculated concentrations of the calibration

standards should not exceed ±20% of theoretical. For lower

concentration levels, a limited calibration curve (without

the higher-end concentration points) may be used for better

accuracy. If a well characterized quadratic relationship occurs,

then a best-fitted quadratic curve may be used for calibration.

Otherwise, if the back-calculated concentrations exceed ±20%

of theoretical, normalized signals of the nearest two calibration

Candidates for 2016 Method of the Year

234