Automated Fast Screening Workflow for Novel Psychoactive Substances by High Resolution Mass Spectrometry

Significance of the topic

The rapid emergence and continual structural diversification of novel psychoactive substances (NPS) pose significant analytical challenges for forensic, clinical, and public-health laboratories. Fast, high-confidence screening methods that minimize analyst workload and turnaround time while preserving sensitivity and specificity are therefore critical. The study presents an automated two-step LC/Q-TOF workflow that couples a rapid MS-only screen with an intelligent, reflex-triggered reinjection using a longer chromatographic method with All Ions fragmentation to improve confidence in NPS identification without unduly increasing instrument time for negative samples.

Objectives and overview of the study

• Demonstrate an automated, reflexive screening workflow for NPS in whole blood that minimizes manual review and unnecessary long injections.
• Evaluate sensitivity, reproducibility, linearity, and identification confidence across a panel of 23 representative NPS using a short MS-only first-pass and a longer All Ions second-pass reinjection.
• Showcase integration of the workflow with MassHunter Quantitative Analysis 12.1 and the LC Screener Tool for automated decision-making and reporting.

Methodology

The workflow uses a two-step chromatographic and acquisition strategy: an initial fast screening run (MSOnly) to detect candidate features, followed by an automated reinjection onto a secondary, longer LC method with All Ions fragmentation for fragment-level confirmation when hits are flagged.

Sample preparation:

• Whole blood samples spiked with 23 NPS targets.
• Protein precipitation using cold acetonitrile:methanol, vortexing and centrifugation.
• Cleanup via Agilent Captiva EMR–Lipid filtration cartridges, rinse with organic/water to enhance recovery, drying under nitrogen (45 °C) and reconstitution in 50:50 methanol:water.

Chromatography and acquisition:

• First-pass: short Phenyl-Hexyl column (Poroshell 120 Phenyl-Hexyl, 2.1 x 50 mm, 1.9 µm) with a fast gradient and MSOnly acquisition for rapid feature detection.
• Second-pass: longer C18 column (Poroshell 120 ECC18, 2.1 x 100 mm, 1.9 µm) with a shallower gradient and All Ions (data-independent) fragmentation to obtain coeluting fragment ions for identification.
• Mobile phases: water + 0.1% formic acid (A) and methanol + 0.1% formic acid (B). Typical column temperature 55 °C, injections 2 µL, flow rates ~0.8 mL/min (short) and ~0.5 mL/min (long).
• Data acquisition on Agilent Revident LC/Q-TOF with All Ions and MSOnly modes; MS parameters tuned for positive electrospray (AJS positive), optimized gas flows and temperatures for stable mass accuracy.

Data processing and decision logic:

• MassHunter Quantitative Analysis 12.1 with the LC Screener Tool and a retention-time organized spectral library (ChemVista) was used to automate hit calling.
• LC Screener outlier thresholds (examples): retention time window ~10%, minimum S/N = 3, mass accuracy limit ~5 ppm, mass-match score minimum ~75; MSOnly required at least one verified ion to flag a hit.
• Intelligent reflex logic: samples flagged as present (green) or questionable (orange) after the MSOnly pass are automatically appended to a reinjection worklist for the All Ions method; blanks and QCs are inserted appropriately.

Instrumentation used

• Agilent 1290 Infinity II LC system.
• Columns: Agilent Poroshell 120 Phenyl-Hexyl (2.1 x 50 mm, 1.9 µm) and Agilent Poroshell 120 ECC18 (2.1 x 100 mm, 1.9 µm).
• Mass spectrometer: Agilent Revident LC/Q-TOF (new detector and temperature-controlled flight tube delivering improved mass stability, accuracy, and dynamic range).
• Software: MassHunter Quantitative Analysis 12.1 with LC Screener Tool and ChemVista-organized spectral library.

Main results and discussion

• Sensitivity: Most of the 23 NPS targets were detectable at a reportable level of 1 ppb, with demonstration EICs shown at 10 ppb. Detection limits were consistent between the short and long LC methods.
• Precision and reproducibility: Retention time relative standard deviations (RSDs) were below 1% for all compounds; over 90% of area RSDs were <10%.
• Linearity: Excellent linear response across the tested range (example 0.5–250 ppb) with R2 values > 0.99 for detected compounds.
• Mass accuracy and identification metrics: Average mass accuracy within ±1 ppm for all compounds; mass match scores (combining mass accuracy, isotopic spacing and abundance) >90/100 for most targets, supporting high-confidence identifications when combining chromatographic separation and All Ions fragment coelution.
• Workflow performance: The LC Screener automated outlier analysis successfully identified hits during the MSOnly pass and triggered reinjections only for relevant samples, reducing unnecessary long-run time and analyst review. Reinjection worklists were clearly marked and could be reviewed via summary or detailed reports.

Benefits and practical applications

• Throughput and efficiency: The reflexive two-step approach reduces instrument time spent on negative samples while ensuring targeted confirmatory data for positives.
• Improved confidence: Combination of targeted chromatography and All Ions fragmentation increases specificity and reduces false positives compared with MSOnly screening alone.
• Operational simplicity: Integration with MassHunter and LC Screener enables largely automated decision-making, worklist appending, and reporting—useful for routine forensic, clinical toxicology, and public-health screening workflows.
• Scalability: The approach can be expanded to larger target panels and adapted to different biological matrices with appropriate sample preparation and library entries.

Future trends and potential applications

• Expansion of spectral and retention-time libraries to cover an increasing number of NPS analogs and metabolites.
• Integration of ion mobility separation to add an orthogonal dimension for isomer discrimination and enhanced confidence in complex mixtures.
• Use of machine learning to refine automated hit-calling thresholds and reduce analyst intervention further while controlling false discovery rates.
• Adoption of similar reflexive workflows in clinical toxicology, emergency-room screening, wastewater-based epidemiology, and large-scale forensic screening programs.
• Coupling with laboratory information management systems (LIMS) to streamline reporting and case management in accredited environments.

Conclusion

An intelligent, automated two-step LC/Q-TOF workflow combining a rapid MSOnly screen with a reflex-triggered longer All Ions reinjection provides a practical balance between speed and identification confidence for NPS screening in whole blood. The Revident LC/Q-TOF platform demonstrated excellent sensitivity, precision, linearity, and mass accuracy across a 23-compound panel, while LC Screener-based decision logic reduced unnecessary long-run analyses and analyst workload. The approach is well suited for routine forensic and clinical laboratories that require rapid screening with the capability for robust secondary confirmation data.

References

• Simmermaker C., Hitchcock J.C., Chevallier O. Automated Fast Screening Workflow for Novel Psychoactive Substances by High Resolution Mass Spectrometry. Poster reprint, ASMS 2026, MP 682. Agilent Technologies, Santa Clara. Published in USA, June 30, 2026.
• Simmermaker C., et al. Intelligent Reflex Targeted Reinjection Workflow for Pesticide Screening in Food Matrices. Agilent Application Note 5994-7312EN. 2024.