BIOANALYSISVALIDATION OF THE CALUX BIOASSAY: QUANTITATIVESCREENING APPROACH
I. Van Overmeire, 1 S. Carbonnelle, 1 J. Van Loco, 1 P. Roos, 1 D. Brown, 2 M. Chu, 2 G. Clark, 2 andL. Goeyens1
1Scientific Institute of Public Health, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
Xenobiotic Detection Systems, Inc., 1601 E. Geer Street, Suite S., Durham, NC 27704 USA

2Introduction
Several papers and data demonstrating the suitability of the CALUX bioassay as a screening
method have been published in recent years.1-8
The assay enables the detection of samples having unacceptably high levels of dioxins and dioxinlike PCBs and, consequently, the reduction of the amount of samples, which have to be confirmed by
expensive HRGC-HRMS analysis. Moreover, the data obtained by the CALUX assay are usuallyhighly correlated with the corresponding GC-MS data, indicating that CALUX results are good
estimates of the TEQ contamination of samples. However, until present CALUX TEQ results have notbeen considered as quantitative TEQ results.
Objectives
The objective of this paper is to discuss the required performance characteristics to validate theCALUX method as a quantitative screening method. This implies that CALUX TEQ results areconsidered as quantitative results and that statistical evidence must determine the range of CALUX
results requiring GC-HRMS confirmation. Our aim is to implement the proposed approach, using
preliminary data obtained during the validation of the CALUX assay for liquid milk.
Results & Discussion
Screening approaches
Screening methods are used to detect the presence of a substance or class of substances at the levelof interest. Our interpretation of what is meant by a qualitative screening is that the method determines
whether the TEQ values of the investigated samples exceed the limit value or not.
One way to perform such a screening is to compare the response of samples to that of a reference
sample at the level of interest. The level of the reference sample is such that a sample with a lower
response is considered as negative, and a sample with a higher or equally high response as suspected,
thus requiring GC-HRMS confirmation.2 However, the uncertainty of the result for the reference
sample is not demonstrated.
A quantitative screening method determines a TEQ value for an unknown sample and after
appropriate statistical interpretation it is decided whether the sample should be confirmed by GCHRMS analysis or not. This is the approach that we prefer to follow and which is outlined in thefollowing sections.
Determination of the confirmation range for GC-HRMS
To be able to use the CALUX assay as a quantitative screening method we need to know whichobtained results should be confirmed by GC-HRMS. It is clear that it is impossible to determine aORGANOHALOGEN COMPOUNDS Vol. 58 (2002)

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single CALUX TEQ value above which the result should be confirmed. Rather a range of TEQ values
for which there is insufficient certainty to accept or reject the sample should be determined. This is the‘confirmation range’.
To determine the confirmation range, the concept of CCa and CCb (Figure 1) described in the draft
revision of the EC directive 93/256/CE has been used. 9
For substances with a maximum residue level (MRL, the limit TEQ level for dioxins), the results
beyond the decision limit CCa are significantly larger than the MRL at a confidence level of 1-a.
Although we can decide in the case of an obtained result above the CCa that the TEQ-value of thesample exceeds the MRL, we cannot guaranty with the same probability that samples with a true
concentration larger than the MRL are found above the CCa. This can only be demonstrated for results
above the detection capability (CCb). CCb eliminates the possibility of false negative results at aconfidence level of 1-b.
Assuming that the data are normally distributed and that the variances at both the MRL and samplelevel are equal, CCb is the upper boundary and CCa* the lower boundary of the confirmation range(Figure 1). TEQ values beneath CCa* are significantly lower than the MRL.
Samples with a TEQ value within the confirmation range need to be confirmed. The use of thisrange reduces the likelihood that true negative samples or true positive samples are sent for
confirmation to the GC-HRMS. In this way, expensive and time-consuming work can be avoided.

Figure 1. constructing the confirmation range
The CCa and the CCa* for the CALUX assay for milk are estimated by using CALUX results
obtained for milk samples spiked at the MRL (i.e. 5 pg TEQ/g lipid.). When a=5 %, the values of CCaand CCa* are calculated by respectively adding or subtracting 1.64 times the s MRL to/from the meanvalue (mean = 5.45 pg TEQ/g lipid, sMRL=0.8 pg TEQ/g lipid, n=6). When b=5 %, the CCb is estimatedby adding 1.64 x sSAMPLE to CCa. Given the assumptions mentioned above, sMRL can be substituted bys SAMPLE.
The experimentally determined confirmation range reaches from 4.1 to 8.1 pg TEQ/g lipid. All
samples leading to results within these boundaries need to be confirmed by GC-HRMS.
Determination of the detection limit
To estimate the detection limit (LD) the same methodology with a- and b-error, as described above,
can be applied (assuming normality and a=b=5 %). This methodology has also been described in the354

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BIOANALYSISIUPAC recommendations. 10To calculate the LD the solvent blank has been used as surrogate for real
blank samples since real blank milk samples are not available. The solvent blank is a blank solution,
which is taken through the whole procedure, from the pre-treatment up to the measurement. We alsopreferred the solvent blank and not the DMSO response because for solvent blanks TEQ values can becalculated. (mean=0.32 pg TEQ/well; s bl=0.16 pg TEQ/well, n=25). First the critical value LC iscalculated. It is the critical level or decision limit above which a result may be reliably recognized asdetected. When a=5 % LC is calculated by adding .1.64sbl to the mean blank result which gives LC =0.32+ 1.64 x 0.16 = 0.58 pg TEQ/well.
The LD will be given by the following equation: LD = L C + 1.64 x sSAMPLE .LD= 0.58 + 1.64 x 0.15
=0.83 pg TEQ/well.
When analysing milk samples with a TEQ of 5 pg TEQ/g lipid we measured 0.95 pg TEQ/well (s:0.15, n = 12) considering the quantity of milk and purified extract used). This value is close to thecalculated LD.
By adjusting (increasing) the amount of purified milk extract it is possible for samples at the MRL
to measure well above the LD (results not shown). It needs to be confirmed if and to what extent themean blank value and consequently LC and LD will change when more ‘blank extract’ is used.
It is advisable to calculate the LD in this way for a certain procedure and matrix.
Otherwise, the LD in the CALUX assay could be calculated for each plate and corresponding
standard curve. In this case only the DMSO response can be used as a blank specific for that plate. We
believe that the LD expressed as TEQ is underestimated when the DMSO blank responses are used to
calculate it. Moreover this approach hampers the comparison of calculated LDs between laboratories.
We previously suggested calculating the L D from the standard curve (described by the Hill equation) as
the y-intercept plus 2.5 x sDMSO.11 Application of this formula resulted in LDs less than half of the above
mentioned LD of 0.83 pg TEQ/well.
In any case a calculated L D should experimentally be demonstrated. This is not a simple task since
blank samples are usually not available.
Conclusion
An approach to determine the range of CALUX data that need to be confirmed by GC-HRMS has
been proposed. Secondly, a calculation model for the detection limit of a procedure has been illustrated.
When procedures for analysing other matrices are optimised during validation it must be checkedand confirmed that detection limits fall sufficiently below the limit value for the matrix under study.
Acknowledgements
Financial support by the Office for Scientific, Technological and Cultural Affairs and from theBelgian Ministry of Public Health is gratefully acknowledged.
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