Immunoassay screening tests are widely used in clinical and forensic toxicology. However, the emergence of new psychoactive substances (NPS) has challenged the scope and sensitivity of these methods. Consequently, specialized analytical toxicology laboratories are today relying more and more on mass spectrometry based techniques. Ultra-high performance liquid chromatography/ high resolution time-of-flight mass spectrometry (UHPLCHR- TOFMS) is an especially attractive technique for comprehensive drug screening in terms of scope, sensitivity and reliability of identification. The objective of the present study was to compare the performance of these two analytical approaches in urine screening for conventional drugs of abuse and NPS.

A total of 279 post-mortem urine samples were analyzed by both an immunoassay (Emit II Plus) and an UHPLC-HRTOFMS screening method. Sample preparation prior to immunoassay was only a centrifugation step, while UHPLC-HR-TOFMS analysis was preceded by solid-phase extraction, including both acidic/neutral and basic fractions. The instrumentation consisted of a Dionex Ultimate 3000 series UHPLC coupled with a Bruker Daltonics maXis Impact HR-TOFMS. Simultaneous acquisition of MS and broad band collision induced fragmentation (bbCID) data was applied. Compound identification was based on post-targeted database search with preset reporting criteria for mass accuracy, isotopic pattern match, retention time, and abundance criteria for qualifier ions. The immunoassay drug panel included amphetamines, benzodiazepines, buprenorphine, cocaine, opiates, and cannabis. The UHPLC-HR-TOFMS method comprised conventional drugs of abuse, various classes of NPS, such as synthetic cannabinoids and cathinones, and commonly abused prescription drugs such as tramadol and pregabalin.

The UHPLC-HR-TOFMS and immunoassay results were compared with quantitative confirmation analyses by established GC-MS and LC-MS/MS methods. The UHPLC-HR-TOFMS results for amphetamines (n=11), buprenorphine (n=27), cannabis (n=28), opiates (n=31), and benzodiazepines (n=99) showed excellent correlation with confirmation analyses, whereas many false negative immunoassay results existed obviously due to high cut-off concentrations and interfering matrix. Moreover, the wider scope of UHPLC-HR-TOFMS method allowed detection of NPS (n=4) and prescription drugs (n=88). A total of 22 false positive (FP) results were produced by immunoassay. For example, the immunoassay gave ten FP buprenorphine results, for which both UHPLC-HRTOFMS and quantification analyses were negative. The UHPLC-HRTOFMS FP results were mainly due to the interference from early eluting endogenous compounds or due to the ion source cleavage of reduced oxycodone resulting in a structure corresponding to codeine. UHPLC-HR-TOFMS revealed additional low-concentration drug findings. The correspondence between symmetric peaks of precursor and qualifier ions supported these results, which were, however, undetectable by confirmation methods. Simultaneous detection of parent compound and metabolites enhances the reliability of compound identification.

Due to the superior results from UHPLC-HR-TOFMS screening, this method has replaced immunoassay in the authors’ laboratory. The possibility to collect both MS and bbCID data simultaneously in a single run increases the reliability of identification and reduces the need for confirmation analyses. However, the data processing parameters of the software should provide more options for individual reporting criteria, which would notably reduce the number of FP results without compromising sensitivity.