A COMPARATIVE STUDY OF GC-HRMS AND CALUX™ TEQ
DETERMINATIONS IN FOOD SAMPLES BY THE BELGIAN FEDERALMINISTRIES OF PUBLIC HEALTH AND AGRICULTURE
Ilse Van Overmeire 1, Leo Goeyens 1, Hedwig Beernaert 1, Sarah Srebrnik 1, Geert De Poorter 2,Willy Baeyens 3, George Clark 4, Michael Chu 4, Andrew Chu 4, David Chu 4, Richard Morris 5,and David Brown 41

Scientific Institute of Public Health, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium;
Belgian Federal Ministry of Agriculture, WTC3 Boulevard S. Bolivar 30, 1000 Brussels,Belgium; 3 Vrije Universiteit Brussel, Laboratory for Analytical and Environmental Chemistry,
Pleinlaan 2, 1050 Brussels, Belgium; 4 Xenobiotic Detection Systems Inc., 1601 East Geer St.,
Durham, NC 27704; 5 Analytical Sciences Inc., 2605 Meridian Parkway, Durham, NC 27113

2Introduction
In January of 1999 the Belgian food supply was contaminated with transformer fluids
containing polychlorinated biphenyls (PCBs). The Belgian government public health response tothis contamination incident was detailed by G. De Poorter at the 19 th International Symposium ofHalogenated Environmental Organic Pollutants (Dioxins 99) in Venice, Italy on September 17,
1999. The source of contamination was a storage tank at the Verkest fat-rendering works. PCBtransformer oil was included in fat that was being recycled for a feed additive in composite feed.
The transformer oil contained predominantly Aroclor 1254 and 1260 that was contaminated withtraces of chlorinated dibenzofurans (PCDFs) and chlorinated dibenzo-p-dioxins (PCDDs). The
contamination incident was highlighted by a sudden drop in egg production, reduced egg
hatchability and increased mortality of chicks.
The Belgian government mobilized a major public health effort aimed at: a) identifying thecontaminated feed materials; b) placing quarantine on those farms that were identified as being
contaminated with PCBs; c) undertaking a major analytical effort to identify the extent of thecontamination; d) isolating and destroying animals determined to be contaminated. The extent of
this public health disaster resulted in a commitment from the Belgian government that a system
would be identified to prevent a reoccurrence of contamination of the food supply with these toxic
environmental contaminants.
The Belgian Ministries of Public Health and Agriculture made a public tender for proposals fora screening methodology that would allow for the detection of contamination of fat, feed stuffs,animal fat, milk products, egg products and processed feed stuffs at the level of 5 pg TEQ/g fat.
The competition involved the analysis of 23 samples for contamination on a TEQ basis of PCBs,
and PCDDs/PCDFs. If possible, an analysis of the contribution of TEQ by the PCBs versus
PCDDs/PCDFs was requested.
Described in this report are the results of the CALUX™ analysis, as developed by Xenobiotic
Detection Systems Inc., versus detection using gas chromatography/ high resolution mass
spectrometry (GC-HRMS) provided by various laboratories contracted by the Belgiangovernment.

Materials and Methods
The samples were prepared by the Belgian Ministries of Public Health and Agriculture. They
included 2 animal fat samples, a chicken fat sample, 5 porcine fat samples, 3 samples of feed stuff,
4 samples of eggs, 4 milk samples and 4 serum samples. A subsample of all of these samples andone serum sample were sent to each participating laboratory for analysis.
CALUX™ Analysis: 1 gram aliquots of each sample were weighed out and extracted with amixture of methylene chloride/hexane; “total organic extractable lipids” were quantified bygravimetric analysis. The extract was processed through a patent pending sample preparation
procedure to concentrate contaminating PCBs or PCDDs/PCDFs in the sample.1 The sample
extract in DMSO was applied to the patented genetically engineered cell line which contains thefirefly luciferase gene under trans-activational control of the aryl hydrocarbon receptor for thedetection and relative quantification of PCDDs, PCDFs, and coplanar PCBs.2
GC-HRMS Analysis: GC/HRMS determinations of the contamination of the samples were
performed by standard methods for quantification of isomer specific concentrations in the sample
such as EPA method 8290.3 TEQ contamination of the sample was estimated by multiplying
isomer specific concentrations by the TEF values of the WHO.4
Results and Discussion
CALUXTM versus GC-HRMS data
In Figure 1 the results of the analyses of 19 samples (fat (8), feed stuff (3), eggs (4), and milk(4)) by the CALUXTM method and by GC-HRMS are represented on a lipid adjusted basis. A
value of 0.25 pg TEQ/g lipid (1/2 DL) was assigned to 3 results that were not detected byCALUXTM. The Spearman’s Rank correlation coefficient between the CALUXTM and GC-HRMS
values is 0.8875 (p < 0.0001). This demonstrates that the results of both analysis methods arehighly correlated. It suggests that the CALUX™ analyses are predictive for GC-HRMS results andmay serve as a rapid and sensitive screen for dioxin contamination in feed and food samples.
Figure 1: CALUXTM versus GC-MS (pg TEQ/g lipid)

CALUX (pg TEQ/g lipid)

10000
1000
100
1010,10,1110100

GC-MS (pg TEQ/g lipid)

1000

10000

As seen in Figure 1, CALUXTM estimates of TEQ are often higher than GC-HRMS measurements.
CALUXTM measurements exceed GC-HRMS measurements in 13 of the 19 paired observations.
In 8 of 19 observations the values are very similar. A sign test does not reject the hypothesis thatthe difference between the two measurements made on the same biological sample is as likely tobe positive as it is to be negative (p = 0.167).
Graphically represented in Figure 2 are the Log transformed data comparing CALUX™
determinations with GC-HRMS determinations of TEQ contamination in the samples on a lipid
adjusted basis. The correlation coefficient amounts to 0.85; the Log transformed data follow anormal distribution (Shapiro-Wilk W= 0.94296, p < 0.2979).
Figure 2: Correlation between Log transformed CALUX™ and GC-HRMS TEQ data (CALUXTM
and GC-HRMS in pg TEQ/g lipid)
4.5

3.5Log (GC-MS)

2.5

1.5
y=0.015+0.959x

0.7
0.5

-0.5

-1.5-1.0-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0Log (CALUX)

Figure 3: Correlation CALUX™ and GC-HRMS TEQ data around 5 pg TEQ/g lipid

2log(CALUX)

1,5
10,50-0,5-1-1-0,500,5log(GC-MS)

11,52Figure 3 illustrates 12 measurements forobservations in a region of interest near 5 pg
TEQ/g lipid. Dotted reference lines at 5 pg
TEQ/g lipid are drawn. Of 12 observations,
10 have CALUXTM measurements thatexceed corresponding GC-HRMSmeasurements. Using the reference lines,
decisions based on 5 pg TEQ/g lipid would
be concordant between CALUXTM and GCHRMS in 10 of 12 cases. The remaining 2observations (in the upper left square) would
be labeled as exceeding 5 pg TEQ/g lipid by
CALUXTM but not by GC-HRMS.

Precision of the results
It is generally concluded from the evaluation of standard GC-HRMS procedures that an average
uncertainty of 30% must be taken into consideration.3,5 On the other hand standard deviations
obtained from triplicate CALUXTM analyses, performed for this study, range from 2% to 48%(mean value 21%). These results indicate that CALUXTM and GC-MS results are comparable interms of uncertainty on the results.
Prediction of GC-MS data from CALUXTM data
The regression line, obtained with the data of this study, can be used as a model to predict GCHRMS data with their variability from CALUXTM data. In Figure 2 this is illustrated at a 5 pg
TEQ/g lipid level. The log(CALUX) value of 0.71 is converted in a log(GC-MS) value of 0.7 (5pg TEQ/g lipid). The corresponding variability of the GC-HRMS value ranges from 2.5 to 10.2 at
95% probability; there is a spread of ± 44% on the log value.
Conclusion
Biologically based systems of analysis for TEQ determination of contamination of food andfeed have developed to the point that they are useful for risk assessment determinations of thehazards of exposure to this class of environmental contaminants. Other investigators havedemonstrated the value of reporter gene technology in assessing dioxin and/or PCB contaminationand the risk of exposure to this class of environmental contaminants with less stringent parametriccorrelations.6,7The data in this report showed the usefulness of the CALUXTM method as an alternative
method for GC-HRMS to determine TEQ levels in different matrices. It is a rapid and costeffective analytical system to determine planar PCB- and PCDD/PCDF- levels in various foodproducts. We showed that a good correlation exists between GC-HRMS data and CALUXTM data
and that a minimal amount of false positive results is obtained using the CALUXTM method. Moredata are required to obtain an improved model for predicting GC-HRMS data from CALUXTMresults and to study matrix dependent influences.
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