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492 

M

astovska

et al

.

:

J

ournal of

AOAC I

nternational

V

ol

. 98, N

o

. 2, 2015

of the instrument. Verify identification of the analyte peaks

by comparing the ion ratios of contemporaneously analyzed

calibration standards, which have been analyzed under the same

conditions.

(

c

)

Injection sequence.

—Bracket the seven test samples with

two sets of calibration standards. Inject solvent blanks after

the calibration level 8 (highest) standard and after the samples.

In addition, analyze a reagent blank with each set of samples.

Inject only once from each vial, thus preventing potential losses

of volatile PAHs and/or contamination.

H. Calculations

Quantification is based on linear least-squares calibration

of analyte signals (

S

PAH

) divided by signals (

S

13C-PAH

)

of corresponding

13

C-labeled internal standards (

see

Table 

2014.08I

) plotted versus analyte concentrations.

Peak areas are generally preferred as signals used for the

quantification, but peak heights should be used for peaks that

are not well resolved, such as in the case of anthracene and

phenanthrene. The analyte concentrations in the final extract

(

c

PAH

, µg/L) are determined from the equation:

c

PAH

= [(

S

PAH

/

S

13C-PAH

) –

b

]/

a

where

a

is the slope of the calibration curve and

b

is the

y

-intercept.

The concentration of PAHs in the sample (

C

, µg/kg) is then

calculated:

C

= (

c

PAH

/

c

13C-PAH

) × (

X

13C-PAH

/

m

)

where

c

13C-PAH

is the concentration of the corresponding

13

C-PAH in the calibration standard solutions (in µg/L);

X

13C-PAH

is the amount of the corresponding

13

C-PAH added to

the sample (in ng); and

m

is the sample weight (in g). Based

on the method procedure and preparation of the calibration

standard solutions,

c

13C-PAH

is 50 µg/L,

X

13C-PAH

is 50 ng, and

m

for the test samples is 10 g.

In the collaborative study, eight concentration levels

were used for the calibration, corresponding to 5, 10, 20, 50,

100, 200, 500, and 1000 µg/L for benzo[

a

]pyrene and other

lower-level PAHs, to 12.5, 25, 50, 125, 250, 500, 1250, and

2500 µg/L for higher-level PAHs, except for naphthalene

that was present at 25, 50, 100, 250, 500, 1000, 2500, and

5000 µg/L. Coefficients of determination (r

2

) 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

standards that enclose the analyte signal in the sample can be

used to interpolate the analyte concentration in the final extract.

Results and Discussion

Laboratory Qualification Phase

The analysis of PAHs poses several difficulties due to their

physicochemical properties and occurrence in the environment

and various materials that can lead to contamination issues.

PAH properties, such as their volatility, polarity, and structure,

affect their GC separation, MS determination/identification,

and recoveries during solvent evaporation and silica SPE steps.

To allow for flexibility and the use of various instruments,

equipment, and columns, the Study Directors did not want to

prescribe the use of a specific GC/MS instrument, GC column

and separation conditions, silica SPE cartridge, and evaporation

technique, equipment, or conditions. For this reason, they

developed performance-based criteria for the GC/MS analysis

(including separation of critical PAH pairs/groups, calibration

range, or carryover), optimum elution volume in the SPE step

(based on the elution profiles of PAHs and fat dependent on

the silica deactivation), and evaporation conditions (to avoid

significant loses of volatile PAHs, mainly naphthalene).

These criteria were part of the laboratory qualification phase

to help laboratories optimize conditions independent of their

instrument/equipment choice or availability. This was also a

very important consideration for the future implementation of

the method in other laboratories.

Another essential step in the laboratory qualification

phase involved check of reagent blanks for potential PAH

contamination. The concentrations of all analytes in the

reagent blanks had to be below the concentrations in the lowest

calibration level standard. For naphthalene, levels below the

second lowest calibration standard (equivalent to 5 ng/g of

naphthalene in the sample) were still acceptable if the source of

contamination could not be eliminated, such as by selection of

a silica gel SPE column from a different vendor (or preparation

of silica gel columns in-house), heating of glassware, addition

of a hydrocarbon trap to the nitrogen lines used for solvent

evaporation, etc. Some laboratories found that their reagent

Table 2014.08I. PAH analytes and corresponding

13

C-PAHs used for PAH signal normalization

Analyte

13

C-PAH used for signal normalization

Anthracene

Anthracene (

13

C

6

)

Benz[

a

]anthracene

Benz[

a

]anthracene (

13

C

6

)

Benzo[

a

]pyrene

Benzo[

a

]pyrene (

13

C

4

)

Benzo[

b

]fluoranthene

Benzo[

b

]fluoranthene (

13

C

6

)

Benzo[

g,h,i

]perylene

Benzo[

g,h,i

]perylene (

13

C

12

)

Benzo[

k

]fluoranthene

Benzo[

k

]fluoranthene (

13

C

6

)

Chrysene

Chrysene (

13

C

6

)

Dibenz[

a,h

]anthracene

Dibenz[

a,h

]anthracene (

13

C

6

)

Fluoranthene

Fluoranthene (

13

C

6

)

Fluorene

Fluorene (

13

C

6

)

Indeno[1,2,3-

cd

]pyrene

Indeno[1,2,3-

cd

]pyrene (

13

C

6

)

Naphthalene

Naphthalene (

13

C

6

)

Phenanthrene

Phenanthrene (

13

C

6

)

Pyrene

Pyrene (

13

C

6

)

1-Methylnaphthalene

Naphthalene (

13

C

6

)

2,6-Dimethylnaphthalene

Phenanthrene (

13

C

6

)

1-Methylphenanthrene

Phenanthrene (

13

C

6

)

1,7-Dimethylphenanthrene

Phenanthrene (

13

C

6

)

3-Methylchrysene

Chrysene (

13

C

6

)