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 ------- 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 ------- 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. ------- 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 A US GOVERNMENT PRINTING OfFICE 1984-759-015/7276 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Official Business Penalty for Private Use $300 \ jP*«OT§CTlOM 55S S 60604 ------- |