Mt
United btates
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S2-83-127 Jan. 1984
SERA Project Summary
Evaluation of Stable Labeled
Compounds as Internal
Standards for Quantitative
GC/MS Determinations
Bruce N. Colby
During the past 30 years, stable iso-
topically labeled materials have been
used in conjunction with mass spectro-
metric measurements to provide precise
and accurate chemical determinations.
Because of this, an investigation into
the applicability of stable labeled com-
pounds in the quantification of trace
organic environmental pollutants was
undertaken. The study focused on the
fundamental and practical aspects of
priority pollutant quantification in
wastewater.
The availability of labeled compounds
and the costs associated with using
them for routine analyses were evalu-
ated using the volatile, acid, and base/
neutral fraction priority pollutants as a
target component base. About 75 per-
cent of these compounds are available
as appropriate stable isotopically la-
beled analogs. Incorporation of the
stable labeled compounds as part of an
analysis protocol is expected to de-
crease overall analysis costs by 30-70
percent when recommended quality
assurance procedures are employed.
Approaches to measurement parameter
selection were identified which indicate
the best ion fragment masses to meas-
ure, the best labeled compound spiking
concentration, and the most appropriate
data reduction methods.
After the fundamental investigations,
20 industrial effluents were analyzed to
establish the analytical precision and
accuracy of isotope dilution analyses.
The samples were also analyzed using a
conventional internal standard. This
study involved 36 priority pollutants
spiked into the test samples. On the
average, isotope dilution yielded accu-
racies in terms of percent recoveries of
100.0, 99.1, and 98.5 percent for the
base/neutral, acid, and volatile frac-
tions, respectively. These values com-
pare with 56.3,51.6, and 122.6 percent
for the same priority pollutants deter-
mined using the conventional internal
standard quantitation approach. Analyt-
ical precision was also better using
isotope dilution: 12.1, 19.0, and 9.2
percent standard deviations for the
base/neutral, acid, and volatile frac-
tions, respectively, compared with
57.3, 67.5, and 39.5 percent standard
deviation for the conventional internal
standard data.
This Project Summary was developed
by EPA's Industrial Environmental Re-
search Laboratory. Research Triangle
Park, NC, to announce key findings of
the research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering in-
formation at back).
Introduction
The requirement for qualitative and
quantitative determinations of trace or-
ganic materials in effluent streams was
established both by EPA's Environmental
Assessment Programs and its response
to the 1976 Consent Decree. The use of
gas chromatographic/mass spectro-
metric (GC/MS) analysis for this purpose
has received universal acceptance.
One of the most desirable features of
GC/MS analysis is its ability to obtain
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highly specif ic qualitative and quantitative
analytical information in a single analysis.
The qualitative aspects of the technique
are well documented. Quantitatively,
however, GC/MS results have been
disappointing in terms of precision and
accuracy. The cost-effectiveness of quan-
titative GC/MS determinations is also
severely impacted when samples, in
addition to the original one, must be
analyzed to evaluate such things as
compound recovery from the original
sample matrix. This has been a particular
problem in determining priority pollutants
in industrial wastewaters, where recover-
ies have been highly variable. As a
consequence, quality assurance proce-
dures involving multiple sample prepara-
tions and analysis have been instituted to
monitor precision and accuracy. These
procedures have increased the cost per
analysis by about a factor of two, and do
not improve the quality of the data, only
check on it.
Similar problems with qualitative and
quantitative GC/MS analyses have been
encountered mother fields where sample
media are extremely complex. Perhaps
the most notable of these is pharma-
cology. The complexity and variation of
biological samples, like environmental
samples, have resulted in a large number
of GC/MS investigations. Because poor
analytical results can have an immediate
impact on human health in some of these
pharmacological studies, reliable high-
quality data have been essential. A route
to these data has been the use of stable
isotopically labeled analogs of the com-
pounds under investigation (target com-
pounds) to act as internal standards for
quantitation. With this approach, called
isotope dilution, the labeled analog of the
target compound is added to the sample
prior to preparation. Because the labeled
and unlabeled materials are chemically
the same compound, the recovery of one
can be directly inferred by recovery of the
other. Consequently, separate analyses
of spiked samples are not necessary and,
hence, costs are reduced. It has been
demonstrated that this approach to
GC/MS quantitation for biological sam-
ples provides precisions and accuracies
on the order of a few percent over
concentration ranges of up to three orders
of magnitude. A logical extension of this
technology was its application to environ-
mental samples where it was expected to
provide equivalent good results.
Results
Results of the study include: the cost
and availability of stable labeled com-
pounds, the potential for label exchange.
isotope label identification, quality assur-
ance costs, fundamental studies, waste-
water demonstrations, and isotope dilu-
tion analysis protocols.
Cost and Availability of
Stable Labeled Compounds
Because a complete compilation of all
compounds of environmental significance
does not exist, the organic priority pollu-
tants covered by the Consent Decree
were selected as a representative group
of compounds in order to perform a
meaningful appraisal of cost and avail-
ability. These compounds were selected
because:
• Priority pollutants cover a broad range
of compound types and, consequently,
provide a representative base for com-
parison.
• Matrix effects associated with recovery
of priority pollutants during sample
preparation are known to exist and to
result in limitations to accurate quanti-
tation.
• Priority pollutants are under intense
investigation using quantitative GC/
MS measurement techniques.
Availability and material costs were as-
sessed on a per-compound basis through
discussions with major commercial dis-
tributors of stable labeled compounds.
Estimates were based on an assumption
that 100 jug of each extractable compound
would be added to the sample for each
1-liter sample extracted and 150 ng of
each purgeable compound would be
added to the sample for each 5-ml sample
purged. The material cost estimates per
analysis were found to be $4.34 (1979
dollars).
Deuterium Label Exchange
When deuterated compounds are used
as internal standards, the potential for
label exchange (deuterium exchange)
exists and could lead to errant results.
The extent of this potential problem is
expected to vary primarily as a function of
sample pH. Highly basic or acidic samples
would promote deuterium exchange in
the same way that they would promote
proton exchange; neutral solutions would
tend to reduce exchange but not necessar-
ily eliminate it. The potential for exchange
with 13C, 16N, and 180 labeled compounds
is essentially nonexistent because major
molecular bonds would be altered in order
for exchange to take place.
Experimental studies for isotope ex-
change were carried out for the three
nonpesticide priority pollutant fractions
by spiking solutions of DaO with naturally
abundant priority pollutants and evalu-
ating the mass spectra of subsequent
sample extracts for evidence of proton-to-
deuterium exchange. Spiked D2O samples
were stored at 4° and 25°C for 0, 48, and
96 hours before extraction. Also, the pD
(equivalent to pH for H2O) was adjusted to
give storage conditions of pD's of 2, 7, and
12. In this way, more severe conditions
than those encountered in performing a
separatory funnel shakeout or continuous
liquid-liquid extraction were tested. The
results of these experiments indicated
that exchange did take place with 6 of the
70~compounds tested. In each of the six
instances, however, nondeuterated an-
alogs were available or could readily be
synthesized.
Isotope Labeled Detail
Using isotope exchange data and elec-
tron ionization fragmentation pattern
interpretations, it was possible to identify
appropriate labels and label locations for
the priority pollutants. For those mole-
cules without specific exchange data,
recommendations are based on inference
from homologous compounds. For exam-
pie, all polynuclear aromatic compounds
(PNAs) were not available in sufficient
quantity to test but, because no exchange
of aromatic protons was observed in the
PNAs tested, these locations were pre-
sumed not to undergo exchange in the
untested PNAs. Also, when the precise
fragmentation of a molecule was unclear,
such as with isophorone, predeuteration
or 13C labeling was recommended.
Cost Comparison for
Quality Assurance
An estimate of costs associated with
the priority pollutant verification protocol
was made for a "typical" verification
episode. For the 12 data sets associated
with the typical verification episode, 36
sample analyses are performed; 24 of
these are solely for quality assurance
(QA) information. By using isotope dilu-
tion where recovery can be evaluated
from labeled compound data, sample
analyses solely for QA are limited to a
precision evaluation; i.e., two additional
analyses. The cost per analysis, however,
can be expected to increase slightly,
primarily due to the reporting costs which
now are spread over a smaller number of
sample analyses. Consequently, for the
1 2 sets of data using isotope dilution, 14
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sample analyses are performed; 2 of these
are solely for QA information. This would
correspond to an 87 5 percent reduction
in QA costs compared with the existing
QA program.
Fundamental Studies
Fundamental studies were undertaken
to identify: (1) the best two m/z's for
measurement; (2) the best data reduction
methods; (3) ways to evaluate isotopic
purity; and (4) the impact of instrument
variables on quantification. These are
described in detail in the final report and
the open literature.
Wastewater Demonstrations
Because the anticipated costs associ-
ated with performing priority pollutant
analyses using isotope dilution method-
ology were favorable, a variety of waste-
waters and simulated wastewaters were
analyzed by isotope dilution (using con-
ventional internal standard methods) and
the data compared. The most extensive of
these comparisons involved 20 industrial
wastewater samples analyzed by two
different laboratories; each analyzed each
sample spiked and unspiked by both
methods. After analyzing the samples,
the accuracies expressed in terms of
percent recovery and precision were
compared for both methods. Fourteen
priority pollutants were analyzed in the
base/neutral fractions, 8 in the acid
fractions, and 13 in the volatile fraction.
The mean percent recoveries and mean
precisions for these determinations are
shown in Table 1.
Isotope Dilutions Analysis
Protocols
Because of the highly favorable results
generated in the three application studies
involving isotope dilution GC/MS quanti-
tation, analysis protocols for that method-
ology were prepared for the determination
of priority pollutants in water and waste-
water.
Conclusions
The use of stable isotopically labeled
internal standards for quantitative isotope
Table 1. Mean Value for 20 Industries
dilution GC/MS determinations has been
shown to be more accurate and more
precise than conventional internal stan-
dard quantitation approaches for measur-
ing priority pollutants in industrial waste-
waters. The isotope dilution method was
found to be free of matrix effects which
reduce the accuracy of other GC/MS
analysis methods and to be equally
precise and accurate for measurements
in two different laboratories. Finally,
incorporation of labeled analogs of com-
pounds of interest into analysis mixtures
can be expected to reduce overall analysis
cost because quality assurance functions
can be incorporated into each sample
analysis rather than into the analysis of
additional samples.
Recommendations
Although the study clearly demonstra-
ted quantitative isotope dilution GC/MS
of great potential value, several aspects
of the method are still unclear:
• A full interlaboratory testing of the
methods needs to be carried out using
blind samples.
• The long-term stability of labeled com-
pounds, especially when contained in
spiking solution media, should be
investigated.
• The priority pollutant methods should
be expanded to include data for all
base/neutral, acid, and volatile frac-
tion compounds.
• Isotope dilution should be investigated
for potential application to pesticide
determination.
• Some available labeled analogs (e.g.,
methylene chloride-d2, 1,1-dichloro-
ethylene-da, and 2,4,6-tri-chloro-
phenol-d2) are not ideal labeled ana-
logs; alternatives should be identified.
Fraction
Base/Neutral
Acid
Volatile
Mean Calculated Percent Recovery Mean Percent SD
624/5' IDb 624/5 ID
56.3 700.0 57.3
51.6 99.1 67.5
122.6 98.5 39.5
12.1
19.0
9.2
'Conventional internal standard method.
^Isotope dilution.
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Bruce N. Colby is with S-Cubed, La Jo/la, CA 92038-1620.
Judith S. Ford is the EPA Project Officer (see belowj.
The complete report, entitled "Evaluation of Stable Labeled Compounds as
Internal Standards for Quantitative GC/MS Determinations," (Order No. PB
84-127 711; Cost: $16.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, V'A 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Industrial Environmental Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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