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SECTION F

QUALITY ASSURANCE
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QUALITY ASSURANCE

Quality Assurance for Analyses for Fluorescent Aromatic Compounds

(FACs)

Quality assurance procedures for bile analyses included use of naphthalene

(NPH) and phenanthrene (PHN) calibration standards, a “bile pool” control

material, blank analyses, and replicate analyses to evaluate the performance of the

high performance liquid chromatography fluorescence (HPLC/UV-F) method.

Quality Assurance for Analyses for Aromatic Compounds

Quality control (QC) measures included analyses of method blanks, spiked

blanks, matrix spikes, and National Institute of Standards and Technology (NIST)

Standard Reference Material (SRM) 1974. Where possible, primary standard

solutions were purchased. The recoveries of the following surrogates are reported

as QC information: Naphthalene-d8, acenaphthene-d10, and benzo[a]pyrene-d12.

Analyte concentrations are reported on the basis of the internal standards

(“surrogates”) added at the beginning of the sample extraction. The HPLC internal

standard (used to determine the fraction of total sample extract used in the analysis

for aromatic hydrocarbons) was phenanthrene-d10. Hexamethylbenzene was used

as the gas chromatography (GC) internal standard to calculate the recoveries of the

surrogates. The criterion for the recovery of each surrogate standard was greater

than 50%, but less than 130%, and the relative standard deviations (RSDs) for

surrogate recoveries in a set of samples was less than 25%. When the recovery of

any surrogate was outside these limits, corrective action was taken, including

instrument repair, inlet cleaning, column replacement, and/or reanalysis of the

samples.

The GC calibration standards generally included all surrogates and analytes

of interest, except for some classes of alkylated aromatic hydrocarbons and

dibenzothiophenes. In these cases, the response factor for the corresponding
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unsubstituted compound was used to calculate the analyte concentrations.

Graduated concentrations of GC-calibration standards were used for determining

multilevel response factors. A GC-calibration standard was analyzed after every

six samples to demonstrate the stability of the calibration. The GC/MS was

considered “under control” when the response for each analyte or surrogate in a

GC-calibration standard was reproducible within ± 10% from analysis to analysis.

Target detection limits were <0.5 ng/g (wet weight basis). The instrument

sensitivity, the sample weights for the associated sample set, the low-level

instrument calibration standards, and the standard reference material were used to

determine the detection limits.

One method blank was analyzed with each set of approximately 10 samples.

The aromatic hydrocarbons (except NPH, which was present in the solvents at

trace concentrations) and dibenzothiophenes in the blanks should not be present at

the limit of detection. A matrix spike (containing 50-80 ng/g of each analyte) or a

spiked blank (containing 40-70 ng/g of each analyte) was analyzed with each set of

approximately 10 samples during the first year (about 1/3) of the study. During the

latter part of the study, a sample of NIST Standard Reference Material 1974 was

analyzed with each set of 10 samples.

The percent recovery of the surrogate standards provides a measure of losses

incurred for each sample during the extraction and cleanup steps. Ideally,

recoveries would be near 100%, but realistically, recoveries of approximately 90%

are considered within quality control guidelines because the procedures involve so

many sample handling steps. The mean percent recovery and standard deviation of

the surrogate standards (deuterated naphthalene, deuterated acenaphthene, and

deuterated benzo[a]pyrene) were 92% ± 15%.

Method detection limits are usually defined by a combination of factors

including instrument signal-to-noise ratio, the lowest level of multilevel standard

that can be reliably measured, the original sample weight, and the final volume of

the sample extract. Based on the results for field blanks (Table F1), method blanks

analyzed with each set of samples (Table F2), and results for the analyses of the

NIST SRM 1974 analyzed with each set of samples after the first year (Table F3),

our detection limits ranged from about 0.5 ng/g to about 1 ng/g. One of the
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certified values in the NIST SRM is 0.35 ng/g for dibenz[a,h]anthracene (DBA).

Our results for DBA were 0.3 ng/g ± 0.4 ng/g.

Naphthalene was present in the solvents at trace concentrations and resulted

in concentrations in method blanks and in samples of about 1 ng/g (Table F2).

Generally, the other analytes were not found in the method blanks at the stated

limit of detection.

In the first year of the study, analytes were added to a method blank (also

called a spiked blank) or to a tissue sample (also called a matrix spike) and then

analyzed as a sample with each set of samples. The results were used to show that

the analytes would be found by this method and to show losses due to sample

handling but would not provide any information on extraction efficiency. The

recoveries for the analytes in spiked blanks (Table F4) and in matrix spikes (Table

F5) ranged from 75% to 130%. As soon as SRM 1974 tissue material was

available, a sample of it was analyzed with every set of samples in place of a

spiked blank or a matrix spike. The SRM was far more useful than the matrix

spike for quality control because it was a homogeneous material, 11 analyte values

were certified or noted, and its use afforded a way to monitor extraction efficiency

as well as losses due to sample handling. Also, the SRM tissue contained all of the

analytes we were monitoring in the subsistence samples, thus enabling us to

monitor quality control for all of our analytes.
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QA TABLES
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Table F-1. Detection limits and standard deviations, ng/g (ppb) wet weight, of 

 aromatic compounds (ACs) in field blanks (n=10). The "less than" 

 symbol (<) indicates that the analyte was not detected in concentrations 

 above the stated value.

 

ACs Mean Standard deviation

C1-naphthalenes < 0.5 0.4

C2-naphthalenes < 0.6 1

C3-naphthalenes < 0.4 0.6

C4-naphthalenes < 0.2 0.2

acenaphthylene < 0.2 0.2

acenaphthene < 0.3 0.2

fluorene < 0.2 0.2

C1-fluorenes < 0.2 0.1

C2-fluorenes < 0.2 0.1

C3-fluorenes < 0.2 0.2

phenanthrene < 0.4 0.2

C1-phenanthrenes < 0.3 0.3

C2-phenanthrenes < 0.2 0.2

C3-phenanthrenes < 0.2 0.2

C4-phenanthrenes < 0.1 0.1

dibenzothiophene < 0.2 0.3

C1-dibenzothiophenes < 0.2 0.1

C2-dibenzothiophenes < 0.1 0.1

C3-dibenzothiophenes < 0.1 0.1

fluoranthene < 0.2 0.2

pyrene < 0.2 0.2

C1-fluoranthenes/pyrenes < 0.09 0.06

benz[a]anthracene (BAA) < 0.2 0.2

chrysene < 0.1 0.1

C1-chrysenes/BAAs < 0.08 0.03

C2-chrysenes/BAAs < 0.08 0.03

C3-chrysenes/BAAs < 0.1 0.1

C4-chrysenes/BAAs < 0.08 0.04

benzo[b]fluoranthene < 0.1 0.09

benzo[k]fluoranthene < 0.1 0.07

benzo[a]pyrene < 0.1 0.07

indeno[1,2,3-cd]pyrene < 0.1 0.08

dibenz[a,h]anthracene < 0.2 0.1

benzo[ghi]perylene < 0.1 0.1
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Table F-2. Detection limits and standard deviations, ng/g (ppb) wet weight, of 

 aromatic compounds (ACs) in method blanks (n=165). The "less than" 

 symbol (<) indicates that the analyte was not detected in concentrations 

 above the stated value.

ACs Mean Standard deviation

C1-naphthalenes < 0.8 0.9

C2-naphthalenes < 0.4 0.7

C3-naphthalenes < 0.3 0.3

C4-naphthalenes < 0.2 0.2

acenaphthylene < 0.3 0.8

acenaphthene < 0.3 0.3

fluorene < 0.2 0.2

C1-fluorenes < 0.2 0.2

C2-fluorenes < 0.2 0.2

C3-fluorenes < 0.3 0.3

phenanthrene < 0.4 0.7

C1-phenanthrenes < 0.1 0.2

C2-phenanthrenes < 0.1 0.2

C3-phenanthrenes < 0.1 0.1

C4-phenanthrenes < 0.1 0.1

dibenzothiophene < 0.1 0.1

C1-dibenzothiophenes < 0.2 0.1

C2-dibenzothiophenes < 0.2 0.1

C3-dibenzothiophenes < 0.2 0.1

fluoranthene < 0.2 0.3

pyrene < 0.2 0.2

C1-fluoranthenes/pyrenes < 0.1 0.1

benz[a]anthracene (BAA) < 0.2 0.4

chrysene < 0.2 0.4

C1-chrysenes/BAAs < 0.2 0.4

C2-chrysenes/BAAs < 0.2 0.4

C3-chrysenes/BAAs < 0.2 0.4

C4-chrysenes/BAAs < 0.2 0.4

benzo[b]fluoranthene < 0.2 0.3

benzo[k]fluoranthene < 0.1 0.2

benzo[a]pyrene < 0.2 0.3

indeno[1,2,3-cd]pyrene < 0.2 0.3

dibenz[a,h]anthracene < 0.2 0.3

benzo[ghi]perylene < 0.2 0.2
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Table F-3. Mean concentrations, ng/g (ppb) wet weight, of aromatic compounds 

 (ACs) determined in NIST Standard Reference Material 1974, standard 

 deviation (n=121), and certified values.

ACs Mean σ Certified concentrations

Mean σ

naphthalene 4 1

C1-naphthalenes 3 2 3.2* 0.5

C2-naphthalenes 4 2

C3-naphthalenes 8 3

C4-naphthalenes 24 9

acenaphthylene 0.4 0.6

acenaphthene 0.6 0.6

fluorene 1 0.6 1.5* 0.2

C1-fluorenes 4 1

C2-fluorenes 20 8

C3-fluorenes 23 15

phenanthrene 6 2 5.6 1.4

C1-phenanthrenes 11 4

C2-phenanthrenes 44 15

C3-phenanthrenes 49 19

C4-phenanthrenes 11 8

dibenzothiophene 0.9 0.4

C1-dibenzothiophenes 5 2

C2-dibenzothiophenes 29 10

C3-dibenzothiophenes 34 13

fluoranthene 46 8 33.6 5.8

pyrene 44 7 34.1 3.7

C1-fluoranthenes/pyrenes 25 7

benz[a]anthracene (BAA) 5 1 4.6* 0.4

chrysene 18 3

C1-chrysenes/BAAs 7 2

C2-chrysenes/BAAs 3 2

C3-chrysenes/BAAs 0.6 0.9

C4-chrysenes/BAAs 0.0 0.4

benzo[b]fluoranthene 7 2 6.5 1.2

benzo[k]fluoranthene 4 1

benzo[a]pyrene 2 1 2.29 0.47

indeno[1,2,3-cd]pyrene 2 1.0 1.8 0.3

dibenz[a,h]anthracene 0.3 0.4 0.35* 0.01

benzo[ghi]perylene 2 1.0 2.47 0.28

sample weight, grams: 4 1.0

* Noncertified concentrations reported by NIST.
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Table F-4. Spiked blanks. Mean percent recoveries and relative standard

deviations (RSD) of aromatic compounds (ACs) in spiked

blanks (n=6).

Relative standard Amount

ACs Mean deviation spiked

% % ng/g

naphthalene 100 4 65

acenaphthylene 95 7 63

acenaphthene 98 2 69

fluorene 98 4 66

phenanthrene 100 6 68

dibenzothiophene 110 7 120

benz[a]anthracene 120 18 58

chrysene 110 9 68

benzo[b]fluoranthene 110 16 66

benzo[k] fluoranthene 110 12 65

benzo[a]pyrene 96 4 59

indeno[1,2,3-cd]pyrene 110 15 54

dibenz[a.h]anthracene 120 19 43

benzo[ghi]perylene 100 13 56
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Table F-5. Matrix spikes. Mean percent recoveries and relative standard

 deviations (RSD) of aromatic compounds in matrix spikes (n=39). 

 

Relative standard Amount

ACs Mean deviation spiked

% % ng/g

naphthalene 110 25 65

acenaphthylene 93 16 70

acenaphthene 92 13 63

fluorene 95 21 69

phenanthrene 110 13 68

dibenzothiophene 120 23 120

fluoranthene 130 30

pyrene 120 30

benz[a]anthracene 94 25 58

chrysene 94 24 68

benzo[b]fluoranthene 86 20 66

benzo[k]fluoranthene 82 20 65

benzo[a]pyrene 87 17 59

indeno[1,2,3-cd]pyrene 77 26 54

dibenz[a,h]anthracene 76 33 43

benzo[ghi]perylene 75 24 56