The internal standard procedures which are routinely used with GC-MS are not useful in immunoassay because the EIA result is an aggregate response to all the crossreactive congeners present in a sample. However, some EIA applications may require that the immunoassay tolerate internal standards which have been introduced for GC-MS procedures. The DF1 Kit uses an antibody which recognizes PCDD/Fs based on structure, not mass. Because mass-labeled molecules are structurally identical to native molecules, both are recognized equally by the antibody. Thus, the mass-labeled internal standard protocols used routinely in GC-MS analysis of PCDD/Fs are not compatible with analysis of PCDD/Fs by the DF1 EIA because they include high levels of congeners which are strongly recognized by the antibody.

This potential conflict is shared by all methods which are based on structural recognition, such as immunoassays. If PCDD/F samples are spiked with high levels of mass labeled internal standards and split for analysis by immunoassay, then the signal due to native PCDD/Fs can be overwhelmed by the strong response to the internal standards. However, this apparent conflict does not prevent immunoassay analysis from being integrated into a GC-MS based analysis program. Instead, the integration process requires understanding of the critical technical issues and planning to avoid conflicts. The following two sections describe different approaches to resolving this potential conflict.

Avoidance is the simplest and most cost effective way to prevent conflicts between the EIA and mass labeled internal standards. If samples are analyzed in a screening program such as described in Technical Note TN-002, where sample preparation for the EIA is completely different than for the GC-MS method, then many samples will never be analyzed by GC-MS. For example, the DMF extraction method described in Application Note AN-004 allows the conflict to be avoided entirely.

The conflict can also be avoided if a conventional extraction is performed before the addition of internal standards and if a subsample of that extract is taken for immunoassay specific cleanup and EIA analysis. This is the case for the fly ash procedure described in Application Note AN-002 and would also be true for other applications which use an immunoassay specific cleanup of a conventional extract. For example, an unspiked fly ash extract which was positive in the EIA could still be spiked with mass labeled internal standards for conventional cleanup prior to GC-MS confirmation. However, if the sample is spiked before extraction, avoidance is not possible unless two separate sample preparation paths are followed for spiked and unspiked subsamples. Because this would significantly reduce the cost effectiveness of EIA screening, other strategies should be considered for such cases.

Immunoassay compatible internal standard mixtures can be made by reducing the concentrations of EIA incompatible congeners or removing those congeners entirely. It should be noted that performance based measurement systems (PBMS) allow modification of conventional internal standard mixtures if the critical performance specifications are documented as being met by the alternative procedure. Users of PBMS have the flexibility to design an internal standard mixture which does not interfere with the EIA, but which still provides the QA needed for GC-MS analysis.
Three simple goals apply to any immunoassay compatible internal standard mixture: 1) reduce the EIA signal of the internal standard mixture to an acceptably low level, preferably less than the detection limit of the EIA, 2) include PCDD/F congeners which will be representative of target congener behavior during extraction and cleanup, and 3) maintain the GC-MS signal to noise ratio of each congener at a level acceptable for the planned analysis.

Table 1 lists several congeners which could serve these goals well because of their low EIA crossreactivity. All of these are commercially available as 13C-labeled individual congener standards from Wellington Laboratories (Guelph, Ontario, Canada).