Applicability of a Chemiluminescence Immunoassay to Screen Postmortem Bile Specimens and Its Agreement with Confirmation Analysis

Abstract

Bile has emerged as an alternative matrix for toxicological investigation of drugs in suspected forensic cases of overdose in adults and intoxications in children. Toxicological investigation consists in screening and, subsequently, confirming the result with specific techniques, such as liquid chromatography with tandem mass spectrometry (LC-MS/MS). As there is no screening test on the market to test postmortem bile specimens, the novelty of this study was in investigating the applicability of a chemiluminescence immunoassay, designed for other matrices and available on the market, on bile and validate its use, testing the agreement with LC-MS/MS analysis. Bile specimens were obtained from 25 forensic cases of suspected death from overdose and intoxication. Sample preparation for bile screening consists simply in centrifugation and dilution. Confirmation analysis allows simultaneous identification of 108 drugs and was validated on bile. Kappa analysis assessed a perfect agreement (0.81–1) between the assays for benzodiazepines, methadone, opiates, cocaine, oxycodone, cannabinoids, buprenorphine and pregabalin; a substantial agreement (0.41–0.6) was reported for barbiturates. No agreement was assessed for amphetamines, due to an abundance of putrefactive amines in postmortem specimens. In conclusion, this fast and easy immunoassay could be used for initial screening of bile specimens, identifying presence of drugs, except amphetamines, with reliability.

Simultaneous multi-targeted forensic toxicological screening in biological matrices by MRM-IDA-EPI mode

“…Interestingly, after evaluating compounds identification in several concentration levels and in two different matrices, the high sensitivity of the proposed method was underlined as the lowest concentration tested, which resulted in the cutoff for most analytes. Different from screening by immunoassays, the proposed method is specific in avoiding false positivity for cross-reactivity mechanisms (Franzin et al 2024;Moody et al 2022;Nieddu et al 2022;Wang et al 2014). Indeed, compounds related to the biological matrix, such as sympathomimetic and putrefactive amines, that usually cross react in immunoassays, do not interfere in the present screening (Bonicelli et al 2022;Broussard 2008;McLaughlin et al 2019).…”

“…To date, the screening methods currently used exploit immunological assays or gas chromatography coupled to mass spectrometry (GC-MS) (McLaughlin et al 2019;Ramoo et al 2016). However, immunological assays can sometimes determine false positives, due to cross-reactivity mechanisms with matrix interferents, and generally succeed in determining only traditional drugs of abuse (Franzin et al 2024;Sotnikov et al 2021). On the other hand, the GC-MS technique requires a complex pre-analytical phase including the sample derivatization, thus inevitably lengthening the times of analysis (Fiehn 2016).…”