Rapid Identification and Quantification of Novel Psychoactive Substances in Human Whole Blood Using SWATH® Acquisition

Importance of the topic

The rapid appearance of novel psychoactive substances (NPS) in illicit drug markets poses significant public health and forensic challenges. Traditional targeted assays often fail to detect unexpected compounds, creating an urgent need for comprehensive screening methods that deliver high selectivity, sensitivity and confidence in identification and quantitation of emerging synthetic drugs.

Study objectives and overview

This work presents a workflow for simultaneous identification and quantitation of over 600 NPS and metabolites in human whole blood, focusing on 30 representative compounds from the DEA’s Emerging Threat Report. The goal was to implement a non-targeted, data-independent SWATH® acquisition on the SCIEX TripleTOF® 5600+ system, paired with streamlined data processing in SCIEX OS software, to achieve rapid, accurate forensic screening.

Methodology and instrumentation

A ten-step liquid-liquid extraction (LLE) protocol was optimized to selectively isolate 30 target NPS and 12 deuterated internal standards from whole blood with recoveries above 80%. UHPLC separation was performed on a Phenomenex Kinetex C18 column (50 × 3 mm, 2.6 µm) using a Shimadzu Nexera system with a 15.5-minute gradient. Mass spectrometric analysis employed SWATH acquisition across 50–510 Da on the SCIEX TripleTOF 5600+ instrument, alternating full scan TOF-MS and stepwise Q1 isolation windows for comprehensive MS/MS coverage. Data were processed in SCIEX OS 1.5, enabling simultaneous quantitation (via peak area ratios) and confirmation (via ion ratios and library matching).

Key results and discussion

– Extraction recoveries for all analytes exceeded 80%, supporting sub-ng/mL limits of quantitation in whole blood.
– Calibration curves over 1–100 ng/mL exhibited excellent linearity (R2 > 0.999), accuracy (>95%) and precision (<15%).
– SWATH-generated high-resolution MS/MS spectra allowed confident library matching (scores >80%) alongside ion ratio confirmation.
– The workflow delivered baseline separation of 42 NPS in 15 minutes, with MS scanning speeds up to 100 Hz ensuring detection of low-abundance targets.
– Data processing in SCIEX OS reduced manual review, using MQ4 algorithms to resolve isobaric interferences and reporting ion ratio, mass error, isotope fit and library match criteria together.

Benefits and practical applications

The described method offers comprehensive, high-throughput screening suitable for forensic and toxicology laboratories. It enables retrospective data mining for new threats, supports regulatory compliance, and streamlines confirmation workflows by combining quantitation with spectral library searches in a single analysis.

Future trends and potential applications

Advances may include expansion of spectral libraries to cover emerging NPS classes, integration of machine learning for automated feature detection, adaptation to other QTOF platforms (e.g., SCIEX X500R), and application to additional matrices such as urine and postmortem tissues. Portable high-resolution instruments and further automation of sample preparation could extend field-deployable forensic screening.

Conclusion

A SWATH-based LC-MS/MS workflow on the SCIEX TripleTOF 5600+ platform, combined with SCIEX OS software, provides a robust, flexible solution for rapid identification and quantitation of novel psychoactive substances in whole blood. The method’s sensitivity, selectivity and throughput make it well suited to address evolving forensic toxicology needs.

References

1. Drug Enforcement Administration Special Testing and Research Laboratory. Emerging Threat Report (2017).