BIOANALYSISRECOVERY DETERMINATIONS FOR BIOASSAY ANALYSIS:CONSIDERATIONS AND RESULTSDavid J. Brown1, Masafumi Nakamura2, Michael D. Chu 1, Michael S. Denison 3, Hiroshi Murata 2
and George C. Clark 1
1

Xenobiotic Detection Systems, Inc., 1601 East Geer Street, Suite S, Durham, NC 27704, USA
Hiyoshi Corporation, 908 Kitanoshocho, Omihachiman, Shiga, 523-8555, Japan3
Department of Environmental Toxicology, 4241 Meyer Hall, University of California, Davis, CA95616, USA2Introduction
Recovery determination is important for quantitative methods such as high resolution gas
chromatography/high resolution mass spectrometry (HRGC/HRMS) so that it is possible to determine thelosses associated with the sample clean up process. This is true for bioassay screening methods as well,however, it is not possible to use the same methods to determined recoveries as are used for chemical
analysis of dioxins. In HRGC/HRMS, known amounts of isotopically labeled congeners are spiked into
the sample prior to extraction and the recovery of these labeled compounds is determined based on theamount that is detected. The recoveries for the labeled congeners are then used to estimate the original
concentration of the congeners from the sample. This is possible because the mass spectrometer detector
can differentiate between the 13C labeled spike and the unlabeled sample analytes. This method ofdetermining recoveries is not possible in bioassays because they do not differentiate between isotopicallylabeled and unlabeled analytes. Adding a spike to the sample, whether labeled or not will only cause acorresponding increase in the response in the bioassays without providing information on the recovery.
Therefore, we have previously employed a surrogate recovery sample; a duplicate aliquot of the sample
that is spiked then treated in the same way as the unspiked unknown sample1. The recovery is determined
for the spiked compound and assumed to relate to the recovery in the unspiked sample. This method has
obvious difficulties including the increase in the number of samples that must be processed as well as theuncertainty in applying the recovery for one sample to a separate sample. Recently we have investigated
the use of biologically inactive congeners as internal spikes. In this report the results of this research ispresented as well as information collected from both HRGC/HRMS recoveries and radiolabeled spike
experiments on the importance of appropriate extraction and clean up methods on recovery determinationand sample quantification.
Materials and Methods
Extraction recoveries
Samples were spiked with 14C labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) andextracted with various solvents or solvent mixtures. Samples were incubated in an ultrasonic water bathwith four solvent changes and the solvents were pooled. The resulting extracts were submitted toscintillation counting to determine extraction efficiencies.
Active congener separation and recovery
Samples were spiked with high-resolution gas chromatography/high-resolution mass
spectrometry (HRGC/HRMS) recovery standards then submitted to extraction and clean up using

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our patent pending sample clean up method 2. Briefly the clean up method involves an acid silica
column in series with an XCARB column. Following sample addition, the XCARB column isdifferentially eluted to yield a polyhalogenated biphenyl (PCB) and a dioxins/dibenzofuran(PCDD/F) fraction. Following clean up the two fractions were submitted to HRGC/HRMS for
recovery determination.
Surrogate congener recovery
Four nanograms of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) were added to each sample
prior to extraction. The samples were extracted and cleaned up using the dual column (acid silica –XCARB) method and the dioxin/dibenzofuran fraction was concentrated under vacuum, thenresuspended in toluene containing 15 ng/ml of each of four injection standards (2,3’,6-PCB; 2,2’,5,5’PCB; 2,3’,4,5’-PCB and 2,3,4,4’,6-PCB). Samples were applied to a 60 m x 0.25 mm DB-5 column
and separated with a 210 oC to 310 oC ramp in temperature at 4 oC/minutes. Detection was by ECD
using a Hewlett Packard Electron Capture Detector. The areas under the peaks were determined byintegration and standardized based on the injection standards. Recovery was calculated based on thecomparison to the original 1,2,3,4-TCDD spiking solution.
Results and Discussion
Extraction method - considerations
Based on US EPA methods 1613 all samples except tissue samples should be extracted using
toluene 3. In an effort to minimize concentration times, various more volatile solvents were tested forextraction versus toluene. Comparison of recovery levels indicated that solvents such as hexane were
not consistently efficient. Non-tissue samples may contain activated carbon, which will retain theanalytes of interest unless extracted with an aromatic solvent. The wide differences in extraction
efficiency seen when using non-aromatic solvents maybe associated with whether or not activatedcarbon is not present, on a sample to sample basis. Based on these results all solid samples with low
moisture content are extracted first with a 20% methanol solution in toluene followed by three
extractions with toluene. The first 20 % methanol solution extraction assists in the removal of traces ofwater from the sample. Use of other solvents for extraction such as hexane, acetone, or methylene
chloride is reserved for tissue samples that should not have activated charcoal present, which could
retain analytes of interest.
Surrogate congener recovery
At the concentrations of 1,2,3,4-TCDD used for spiking samples the peak for 1,2,3,4-TCDD
recorded by GC/ECD was clear and easily measured (see figure 1). This concentration of 1,2,3,4TCDD did not significantly increase the response of the bioassay to the sample extract. Based onthese results the use of 1,2,3,4-TCDD was considered promising. Further studies comparing
recoveries determined by 1,2,3,4-TCDD with paired samples spiked with 14 C- 2,3,7,8-TCDD
indicated that the recoveries determined by the two methods were similar (table 1). Additional
studies with various sample matrices are planned to further determine the applicability of using
1,2,3,4-TCDD as an internal recovery standard. If determined to be effective, 1,2,3,4-TCDD could
be added to all samples prior to extraction with only those samples found to be near the TEQ
regulation level further analyzed by GC/ECD for recovery determination. For example recoveries
would only be determined for samples greater than 50 % of the regulated TEQ level. Samples
measuring less than 50 % of the regulatory level would be unlikely to surpass the regulatory level
following recovery correction as extraction using appropriate solvent systems generally results inrecoveries of greater than 70 %.

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Figure 1. GC/ECD trace for a sample spiked with 1,2,3,4-TCDD. Peaks for the injections standards(2,3’,6-PCB = 6.09 min.; 2,2’,5,5’-PCB = 7.31 min.; 2,3’,4,5’-PCB = 8.05 min. and 2,3,4,4’,6-PCB=
10.13 min.) and for 1,2,3,4-TCDD = 12.78 min. are identified.

Table 1. Comparison of 1,2,3,4-TCDD recoveries determined by GC/ECD and 14C labeled 2,3,7,8TCDD recoveries determined by scintillations counting.
Sample123414

C 2,3,7,8-TCDD
by scintillation counting

1234-TCDD
by GC/ECD
76.3 %
95.4 %
91.6 %
90.6 %+/+/+/+/-

1.1 %
1.7 %
1.8 %
0.3 %

91.2 %
84.1 %
89.9 %
83.5 %+/+/+/+/-

0.9 %
2.9 %
2.4 %
3.4 %

Active congener separation and recovery
13
C labeled congeners were subjected to our sample clean up and separation method and theresulting fractions were submitted to HRGC/HRMS for recovery determination. The PCDD/F
congeners were found only in the PCDD/F fraction (see table 2), however, two of the PCB congeners(PCB #126 and 169) were not completely eluted in the PCB fraction. These two PCB congeners are
retained by the XCARB column and tail into the PCDD/F fraction. Attempts were made to increase theelution of these compounds by increasing the toluene concentration used to elute the PCB fraction.
This resulted in nearly complete elution (greater than 90 % recovery in the PCB fraction) of these two
PCB congeners, however, it also resulted in the tetrachlorinated dioxin and dibenzofuran being
partially eluted in the PCB fraction (24 % and 27 % of 2,3,7,8-TCDD and 2,3,7,8-TCDF wererecovered in the PCB fraction respectively). Further studies indicated that it was not possible toconsistently and completely separate the two classes of compounds, therefore minor contamination ofthe PCDD/F fraction by PCB 126 and 169 were accepted in order to obtain consistent recovery of thedioxin and dibenzofuran congeners in the PCDD/F fraction.

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Table 2. Congener specific recoveries for sample clean up and separation.
13C

PCDDs

PCDF

non-orthomono-ortho

Recovery (%)

2378-TCDD
12378-PeCDD
123678HxCDD
1234678HpCDD
OCDD
2378TCDF
12378PeCDF
123478HxCDF
1234678HpCDF
OCDF
33’44’-TCB
344’5-TCB
33’44’5-PeCB
33’44’55’-HxCB
2344’5-PeCB
233’44’5-HxCB
233’44’55’HpCB

#77
#81
#126
#169
#114
#157
#189

WHO-TEFPCB Fraction

PCDD/Fs Fraction

0000000100101
96
70
72100106118929996102
97989910698
95531234122110.10.010.0001
0.1
0.05
0.1
0.01
0.00010.00010.00010.1
0.01
0.00050.00050.0001

Conclusions
Because of differences in the way that bioassays and chemical analysis methods detect compounds it
is not possible to directly use extraction and clean up recovery methods from chemical analysis in sample
preparation for bioassay analysis. However, it is necessary to include recovery determination in order toavoid under reporting the TEQ contamination of samples. Ignoring recovery will tend to increase the rateof false negatives, especially for samples that are more difficult to extract. Here we report promisingpreliminary data on the use of a surrogate congener internal spike that is appropriate for bioassays. The
use of an inactive congener as a recovery spike allows for estimation of the recovery efficiency forsamples that are close to the regulated level, while not interfering with the detection of the bioassay.
Dioxin bioassays do not normally allow for differentiation between difference types of compounds,
as they respond to all compounds that can bind to and activate the receptor. However, by coupling thebioassay with a sample clean up method that allows for the separation of PCB and PCDD/F fractions it
is possible to obtain additional information about a sample.
Acknowledgements
This study was funded in part by a Small Business Innovation Research Grant from the National
Institutes of Environmental Health Sciences (ES 08372-03).
References
1. Brown, D.J., Goeyens, L., Van Overmeire, I., Chu, M., Murata, H. and Clark, G. (2001)
Organohalogen Compounds 54: 32–35.
2. Chu, M.D. and Clark, G.C. Patent Application No. 09/872,758.
3. EPA method 1613, US Environmental Protection Agency, 40 CFR Part 136.

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