Sources of Interference

Endogenous Compounds

The risk of interference from endogenous compounds is greater if the sample used is plasma or serum, but as far as urine is concerned as the sample for drug testing, it can be influenced by the patient’s disease or physical state. Non specific cross reactivity can be caused potentially by nitrates, blood, fungi, bacteria, ketones and proteins in the urine. However, the influence of endogenous components on other types of samples has not been clearly proved, it is evident that the physiological features of oral fluid can vary according to the collection techniques.

Structurally Similar Compounds and Their Interference

Congeners or structurally similar compounds tend to show cross reactivity while the binding of antigen/antibody take place. This depends on the affinity and specificity of the antibodies. Most commonly, congener interference takes place in the reactions regarding the amphetamine assays. Antibodies that are used in assays also have varying range of cross reactivities, especially towards drugs which are derived from phenethylamine like phenylpropanolamine and ephedrine. Even though, reagent manufacturers have worked on the specificity of amphetamine assays by introducing monoclonal antibodies, the high concentration of amphetamine compounds in the samples can still cause a false positive result. Since it is easier to avail the drugs having ephedrine and phenylpropanolamine as many of them are available as OTC – Over the Counter drugs, it is not uncommon to find donor samples with higher urine concentration of these drugs. The laws implemented in US over the retail sales of these drugs to curb the illicit methamphetamine concentration in body might be able to impact this issue after some time.

Unstructurally Related Compounds Showing Cross Reactivity

While development of the assay and its submission to the FDA, manufacturers test for many related and unrelated chemicals so that they do not affect the results. However, despite the efforts, the potential compounds that can appear in a population are not distinguished and tested as their availability might not be possible and so, they are not included in the studies regarding cross reactivity. Table 2.2 shows some of the unrelated compounds that cross react in amphetamine assays.

Those compounds that are not identified in the initial development of the products also tend to show unexpected cross reactivities and be identified, depending on inquiries from the customers and the findings from the test fields. Some such examples are the reactions of o-desmethylvanlafaxine and vanlafaxine with PCP assays, rifampin and quinolones interferences in opiate assays, phenothiazine and verapamil interference in methadone assays, oxaprozin causing interference in benzodiazepine assays and lastly, Sustiva® causing interference in cannabiloid immunoassays.

Other than interferences that yield false-positive results, many interferences can also yield false-negative ones too, which rather have a greater impact on the drug testing programs. This is so because false-positive tests undergo confirmatory tests by spectrographic detection in which they are usually cancelled and confirmed as negative one. But those samples that are false-negative are reported as negative after their initial screening and no further tests or investigations are done on them unless the laboratory is doubtful about interferences. This may prove to have a harsher effect on the workplace drug testing regimes. For instance, EIA’s lesser signal due to aspirin metabolites and the reactions of urinary fragments and compounds interfere with lateral flow immunoassays.


Adulterants are chemicals that are added to the samples in order to cheat on the drug test and can be a common source of interference in the assays. Their purpose is to produce negative result. These chemicals range from simple table salt and bleaches to complicated chemical oxidizers which interfere with the enzymatic activity, binding or analyte structure transformation. In commercial assays, a lot of products are tested which differ in chemical compound, concentration and assay design. Even though, some of these additives can result in false-negatives, others can however produce typical false positive results which can alert the laboratory about the use of adulterants in the given donor sample. For instance, assays which test the presence of cannabinoids often are commonly targeted for adulterants, even thought their effect has been shown on other drugs too. Adulteration and it details are given further in this book.

High Dose Hook Effects

High dose hook or prozone phenomenon is quite likely to happen in sandwich assay designs that were mentioned before. This can lead to decreased signal when high concentration of analyte is present. It is also observed commonly that increased concentration of analyte can saturate captured and detected antibodies and prevent them from forming antibody complexes respectively. This hook effect basically denotes to the shape of the curve of the dose v/s response when the signals are plotted against concentration. However, this phenomenon is not usually observed in workplace drugs-of-abuse testing programs.

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