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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