Intelligently Designed SWATH® Acquisition for Novel Psychoactive Substances (NPS) Detection in Whole Blood

Importance of the Topic

Novel psychoactive substances (NPS) are increasingly introduced to evade legal controls, creating significant analytical challenges in forensic and clinical laboratories. Their structural diversity, variable potency and limited reference information make rapid, comprehensive screening essential to safeguard public health and support legal investigations.

Objectives and Study Overview

This work evaluates a single-run SWATH® acquisition method on the SCIEX X500R QTOF platform for simultaneous screening and quantification of 54 representative NPS classes, including synthetic cathinones, cannabinoids, benzodiazepines and fentanyl analogs in whole blood. The study compares two sample-preparation techniques (protein precipitation and salting-out liquid-liquid extraction) and validates sensitivity, linearity and identification confidence.

Methodology

Whole blood samples spiked at 0.1–200 ng/mL were processed by:

• Protein precipitation (PP) with cold acetonitrile, centrifugation, evaporation and reconstitution in 80:20 water:methanol.
• Salting-out liquid-liquid extraction (SALLE) using saturated KCl and acetonitrile, followed by drying and reconstitution.

Two reverse-phase LC gradients were employed: a 9.5-min general-purpose run for broad analyte coverage and a 17-min extended run to baseline separate fentanyl isomers. Data were acquired by a TOF-MS survey scan followed by variable-window SWATH MS/MS across 12 m/z segments (100–500 m/z), yielding a complete digital record of precursor and fragment ions.

Instrumentation

• SCIEX X500R QTOF mass spectrometer with SWATH® Acquisition.
• ExionLC™ AC HPLC system.
• Phenomenex Kinetex columns: 50×4.6 mm Phenyl-Hexyl for 9.5-min run; 100×2.1 mm Polar C18 for 17-min separation.

Main Results and Discussion

Limits of detection (LOD) for all 54 NPS ranged from 0.1 to 1 ng/mL, with linear dynamic ranges up to 200 ng/mL and R2 values >0.995. PP and SALLE delivered comparable recoveries and matrix effects, with minor signal enhancements observed for a few cathinones at low levels.

Fragment-ion-based quantitation improved sensitivity by up to tenfold for select analytes (e.g. ADB-FUBICA) when revisiting data. The extended LC gradient fully resolved critical fentanyl isomers that coeluted in the faster method. Variable-window SWATH acquisition notably increased MS/MS spectral specificity and library matching scores, enhancing identification confidence (e.g. carfentanil match improved from 14.7% to 98.6%).

Benefits and Practical Applications of the Method

• Comprehensive digital archive of all detectable compounds enables retrospective data mining without reanalysis.
• Flexible panel expansion by adding new targets without altering acquisition settings.
• High throughput screening with a 9.5-min run and specialized isomer separation in 17 minutes.
• Robust identification using combined mass accuracy, retention time, isotope pattern and MS/MS scoring in SCIEX OS software.

Future Trends and Potential Applications

As the NPS landscape evolves, high-resolution full-scan workflows will be essential to discover emerging analogs. Integration of machine-learning-based spectral interpretation and automated SWATH window optimization may further enhance throughput and reduce false negatives. Expanding libraries with collision cross-section or ion mobility data may improve isomer differentiation.

Conclusion

The presented SWATH® approach on the SCIEX X500R QTOF system offers a powerful, unbiased workflow for rapid identification and quantification of a broad NPS panel in biological matrices. Combining versatile sample preparation, fast LC separation and variable-window SWATH acquisition ensures high sensitivity, reliable isomer resolution and flexible retrospective analysis.

Reference

1. vMethod™ Application – Single-Injection Screening of 664 Forensic Toxicology Compounds on a SCIEX X500R QTOF System.
2. Quantitative Analysis of Fentanyl and Analogues in Human Whole Blood. SCIEX Technical Note RUO-MKT-02-8322-A.