A method for the sensitive targeted screening of synthetic cannabinoids and opioids in whole blood by LC-QTOF with simultaneous suspect screening using HighResNPS.com

Abstract

To develop and validate a qualitative screening method capable of detecting the low concentrations found in cases of synthetic cannabinoid and opioid consumption using a targeted scope, while simultaneously performing efficient suspect screening against a much larger database. Screening for synthetic cannabinoids and opioids in blood samples remains challenging due to the large number of substances available and the constantly changing market, as well as the low limits of detection required for some compounds. Many laboratories employ LC-QQQ when screening for these compound classes due to the ubiquity and sensitivity of those instruments but LC-HRMS instruments are similarly able to detect a large number of pre-defined analytes with high sensitivity and specificity, as well as acquiring full scan data which enables both suspect screening and retrospective screening if required. Analytes were extracted from 0.5 mL of whole blood using alkaline liquid-liquid extraction and data were acquired using data dependent acquisition on an Agilent 6545 QTOF. 28 opioids and 23 cannabinoids were validated for limit of detection, recovery, matrix effects, selectivity, extract stability and carryover. Data processing occurred in two stages; first a targeted screen was performed using an in-house database containing retention times (RT), accurate masses and MS/MS spectra for the validated compounds plus an additional 16 opioids and 34 cannabinoids (101 total compounds). Suspect screening was then performed using a database downloaded from the crowdsourced NPS data website HighResNPS.com which contains mass, consensus MS/MS spectra and laboratory specific predicted retention times for over 1400 compounds. The suspect screening workflow was assessed by determining the accuracy of the retention time prediction and MS/MS library matching against consensus spectra. For this workflow, qualifying parameters for initial presumptive identification included mass accuracy of ±4mDa, MS/MS match score greater than 20 and retention time difference to predicted RT of ±2 min. In practice, results of this suspect screen would be verified following purchase of authentic reference material for comparison. The method was applied to 66 forensic cases where synthetic cannabinoid or opioid screening was requested by the client or their use was suspected due to case information. Limits of detection ranged from 0.03–0.27 μg/L (median, 0.04 μg/L) for the 28 validated opioids and 0.04–0.5 (median, 0.07 μg/L) for the 23 synthetic cannabinoids. Recoveries were greater than 70% for all compounds except MDMB-CHMCZCA (63%) and desomorphine (42%). Synthetic cannabinoids or opioids were detected in seven of the 66 forensic cases. CUMYL-PEGACLONE and etodesnitazine were each detected in two cases and 5F-MDMB-PICA, 4-Cyano-CUMYL-BUTINACA and carfentanil were detected in one case each. These compounds were within the targeted scope of the method but were also detected through the suspect screening workflow. The developed method has similar limits of detections to other published methods using LC-QQQ instruments but has several additional advantages. The suspect screening workflow enables efficient, automated screening of data against the HighResNPS database in the absence of reference materials. The suspect screening parameters adopted were verified through the successful detection of compounds in spiked validation samples, as well as compounds known to be present in authentic samples. The data acquired is also suitable for retrospective screening as new compounds emerge. The dual approach of simultaneous targeted and suspect screening allows laboratories certainty in their coverage of a defined scope, as well as a “best attempt” to cover a much large number of compounds. This provides a good compromise, especially for laboratories where the cost of purchasing hundreds of NPS reference standards is prohibitive. The advantages of testing for these compound classes by HRMS over QQQ mean this instrumentation should be selected preferentially wherever possible when developing new methods.