Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine

Abstract

Background: Laboratory tests for routine drug of abuse and toxicology (DOA/Tox) screening, often used in emergency medicine, generally utilize antibody-based tests (immunoassays) to detect classes of drugs such as amphetamines, barbiturates, benzodiazepines, opiates, and tricyclic antidepressants, or individual drugs such as cocaine, methadone, and phencyclidine. A key factor in assay sensitivity and specificity is the drugs or drug metabolites that were used as antigenic targets to generate the assay antibodies. All DOA/Tox screening immunoassays can be limited by false positives caused by cross-reactivity from structurally related compounds. For immunoassays targeted at a particular class of drugs, there can also be false negatives if there is failure to detect some drugs or their metabolites within that class.

Methods: Molecular similarity analysis, a computational method commonly used in drug discovery, was used to calculate structural similarity of a wide range of clinically relevant compounds (prescription and over-the-counter medications, illicit drugs, and clinically significant metabolites) to the target ('antigenic') molecules of DOA/Tox screening tests. These results were compared with cross-reactivity data in the package inserts of immunoassays marketed for clinical testing. The causes for false positives for phencyclidine and tricyclic antidepressant screening immunoassays were investigated at the authors' medical center using gas chromatography/mass spectrometry as a confirmatory method.

Results: The results illustrate three major challenges for routine DOA/Tox screening immunoassays used in emergency medicine. First, for some classes of drugs, the structural diversity of common drugs within each class has been increasing, thereby making it difficult for a single assay to detect all compounds without compromising specificity. Second, for some screening assays, common 'out-of-class' drugs may be structurally similar to the target compound so that they account for a high frequency of false positives. Illustrating this point, at the authors' medical center, the majority of positive screening results for phencyclidine and tricyclic antidepressants assays were explained by out-of-class drugs. Third, different manufacturers have adopted varying approaches to marketed immunoassays, leading to substantial inter-assay variability.

Conclusion: The expanding structural diversity of drugs presents a difficult challenge for routine DOA/Tox screening that limit the clinical utility of these tests in the emergency medicine setting.

Figure 1

Illustration of structural similarity. Using phencyclidine (PCP) as the target compound, 2D similarity was calculated using MDL public keys and the Tanimoto coefficient to five different compounds, three of which (dextromethorphan, meperidine, and the phencyclidine metabolite 4-phenyl-4-piperidino-cyclohexanol) have been reported to cross-react with at least some marketed PCP immunoassays, and two of which (ketamine and ibuprofen) have not been reported to cross-react with PCP screening assays. PCP has the highest similarity (in descending order) to 4-phenyl-4-piperidino-cyclohexanol, dextromethorphan, and meperidine. PCP has low structural similarity to ketamine (despite having similar pharmacological properties to PCP) and essentially no structural similarity to ibuprofen.

Figure 2

Variability in sensitivity of marketed amphetamine and benzodiazepine screening immunoassays. The plotted circles indicate the concentration of compound that produces an equivalent reaction to 1000 ng/mL d-amphetamine (amphetamine assays) or 200 ng/mL diazepam (benzodiazepine assays). The dashed lines bracket clinically or toxicologically relevant concentrations from studies in the published literature (see text of Results for detailed description). A) Amphetamine assays. With one exception (Roche cobas c assay), marketed amphetamine screening immunoassays detect amphetamine and methamphetamine well but have variable and often low cross-reactivity with MDA, MDMA, MDEA, and phentermine. B) Benzodiazepine assays. Marketed benzodiazepine screening immunoassays generally have higher sensitivity to diazepam, oxazepam, and nordiazepam than to 7-aminoclonazepam (main clonazepam urinary metabolite) or lorazepam glucuronide (main lorazepam urinary metabolite).

Figure 3

Variability in sensitivity of marketed cocaine metabolite and opiate screening immunoassays. The plotted circles indicate the concentration of compound that produces equivalent reaction to 300 ng/mL benzoylecgonine (cocaine metabolite assays) or 300 ng/mL morphine (opiate assays). The dashed lines bracket clinically or toxicologically relevant concentrations from studies in the published literature (see text of Results for detailed description). A) Cocaine metabolite assays. Marketed cocaine metabolite detect benzoylecgonine with high sensitivity but generally have low sensitivity for detection of cocaine (parent drug) and metabolites other than benzoylecgonine. B) Opiate assays. Marketed opiate assays detect morphine, codeine, and hydrocodone well but have variability and often poor sensitivity to oxycodone and oxymorphone.

Figure 4

Variability in sensitivity of marketed PCP and tricyclic antidepressant screening immunoassays. The plotted circles indicate the concentration of compound that produces equivalent reaction to 25 ng/mL PCP or 1000 ng/mL desipramine (tricyclic antidepressant assays). The dashed lines bracket clinically or toxicologically relevant concentrations from studies in the published literature (see text of Results for detailed description). A) PCP assays. Marketed PCP assays have varying degrees of cross-reactivity with dextromethorphan, meperidine, thioridazine, and mesoridazine. The brackets for PCP correspond to urine concentrations observed in patients abusing PCP[54] B) TCA assays. Marketed TCA screening immunoassays have similar cross-reactivities to TCAs but variable cross-reactivity to carbamazepine, phenothiazines (such as prochlorperazine), and quetiapine. The marketed TCA assays include those approved for serum/plasma or urine samples.

Figure 5

Tricyclic antidepressant assays. A) Rank of tricyclic antidepressants, cyclobenzaprine, and quetiapine by total number of prescriptions in the United States in the time period from 1998–2007. TCAs are indicated by closed symbols, while the non-TCAs (cyclobenzaprine and quetiapine) are designated by open circles and squares, respectively. Whereas prescriptions for amitriptyline have remained relatively constant in the last decade, prescriptions for other TCAs are steadily declining, with desipramine no longer ranking in the top 400 most prescribed drugs. Cyclobenzaprine is now prescribed more frequently than amitriptyline in the United States. B) Drugs most likely accounting for positive TCAs immunoassay screens in our medical center sample. Of 124 positive TCA screens (see Additional file 1, tab U for details), the most likely causes were sorted into five categories: cyclobenzaprine, amitripytline +/- nortriptyline, other TCAs (e.g. doxepine, imipramine, and their metabolites), phenothiazines, and other drugs (e.g., carbamazepine and quetiapine).