Analysis of alternative samples to urine
Immunoassays for drugs have been widely employed in the analysis of urine samples. Other sample matrices, such as whole blood, serum, hair extracts, saliva and sweat, can be used successfully. Heterogeneous assays are ideally suited to these alternative matrices. Their inherent sensitivity provides the lower limits of detection required for forensic use and the wash step endows the ability to deal with difficult matrix effects. Correct selection of antibody allows the assay system to be targeted towards specific drugs or metabolites.
Immunoassays are generally designed for a particular use and sample type. With testing for drugs of abuse the assay is often designed for urine specimens, whereas therapeutic drug assays are generally designed for plasma or serum. Situations can arise in which the drug of interest is in a different matrix from that for which the kit has been validated, so the onus is on the user to test and validate this new application. Urine and serum immunoassay kits are widely available and assays for other matrices, which include blood, cerebrospinal fluid, finger nails, hair, meconium, saliva, stomach contents and sweat, have been developed successfully. Many assays can be adapted to work with different matrices, although it is advisable to consult the kit manufacturer prior to making kit adaptations. After discussion and advice from the developer or manufacturer of an assay, the next step is to obtain similar samples to the test case; it is essential to try and match these as closely as possible, with special reference to the type of preservative used. Some samples (e.g. bottled beer) are readily available and it should be possible to obtain the exact brand under test, so that the alcohol and other ingredients are matched identically. In cases such as aged, putrefied whole blood, it can prove more difficult to obtain and select a sample matrix. Once a suitable sample matrix has been obtained, known amounts of the drug of interest are added to the sample matrix. The concentrations are usually chosen to fall within the range of the kit. The high sensitivity of immuno assays means it is often possible to introduce a dilution factor without compromising the limit of detection in the sample. For example, if the limit of detection of the confirmatory method is 100 lg/L, use of an EIA with a cut-off of 10 lg/L allows the use of a 1:10 dilution factor. Such a dilution reduces potential interference without compromising overall sensitivity.
Heterogeneous immunoassays are more robust and tolerant of different types of sample matrices than homogeneous assays because of the separation stage of the assay.
A common finding, often described as a ‘matrix effect’, is that the newly created calibration curve is not usable through adverse interference. This can arise from an inherent interference from the sample itself with the signal system or from interference with the anti-body–antigen reaction. Homogeneous immuno assays are more prone to interference since the sample is present during the signal-generation stage. Reducing the sample load by dilution in a suitable buffer or using a smaller test volume should reduce the effect. Unfortunately, the trade-off is that the limit of detection and curve shape might be sacrificed. Liquid- or solid-phase extraction of the sample can be used when direct application of the sample fails.
