Screening to Confirmation: Complete Workflow for Comprehensive Forensic Toxicology Analysis Using a Single Software Platform

Significance of the topic

Forensic toxicology requires rapid, reliable detection and confirmation of a wide range of licit and illicit drugs in complex biological matrices. Traditional workflows combine different instruments and software packages, increasing analysis time, training burden, and risk of inconsistencies. An integrated screening-to-confirmation workflow that combines high-resolution untargeted screening with targeted confirmatory assays and unified data processing can improve laboratory throughput, support retrospective data mining, and increase confidence in identifications—particularly important given the emergence of potent synthetic opioids and other novel psychoactive substances.

Objectives and study overview

This study demonstrates a consolidated forensic toxicology workflow using a single software environment (Shimadzu LabSolutions Insight Explore) to perform LC-QTOF-based untargeted screening, LC-MS/MS (triple quadrupole) targeted quantitation/confirmation, and GC-MS single-quadrupole targeted confirmation. The aim was to show feasibility of combined acquisition modes (scan/DIA, SIM, MRM), unified library searching and review, and rapid reporting across techniques using representative standards and mixed analyte panels.

Methodology

Sample preparation: Biological matrices (urine, blood, etc.) were processed with extraction and cleanup; internal standards were spiked prior to analysis. Screening: An LC-QTOF method using a Shim-pack Scepter PFPP-120 column (1.9 µm, 2.1×50 mm) with water/methanol mobile phases (ammonium formate and formic acid modifiers) at 0.4 mL/min and a 5-minute run time was employed for rapid, full-scan ESI+ acquisition with data-independent acquisition (DIA). Confirmation — LC-MS/MS: A triple-quadrupole method with rapid polarity switching and MRM was used to quantify 46 analytes using 23 internal standards; reported cycle times allowed quantitation in under three minutes. Confirmation — GC-MS: A GC-MS single-quadrupole method (Rtx-5ms column, oven 60→320 °C ramp) combined Scan (45–500 m/z) and SIM events, targeting over 50 common toxicology compounds including barbiturates and benzodiazepines.
Calibration and QA: Quantitation used internal standards with linear curves and 1/C2 weighting; acceptance required three replicates per calibration point within ±20% accuracy. LLOQs varied by analyte (sub-ng/mL to ng/mL range) with most calibration fits showing very high linearity (R2 values typically ≥0.99).

Instrumentation used

• LC-QTOF: Shimadzu LCMS-9050 configured for full-scan/DIA high-resolution accurate-mass screening.
• LC-TQ: Shimadzu LCMS-8050RX (triple-quadrupole) used for MRM confirmation and quantitation.
• GC-SQ: Shimadzu GCMS-QP2020NX used for Scan/SIM targeted confirmation analyses.
• Chromatography: Shim-pack Scepter PFPP-120 column for LC separations; Rtx-5ms column for GC separation.
• Software and libraries: LabSolutions Insight Explore (Analyze and Explore modules) for processing, quantitation and reporting; Cayman Spectral Library and Shimadzu forensic database for MS2 library searching and reverse-search capability.

Data analysis and processing

Data handling used Insight Explore to combine library searching (including reverse search), peak detection, formula finding, hit scoring/flagging and review-by-exception. The platform supports searching MS2 against multiple libraries (up to five) to raise confidence, and integrates scan/DIA and targeted SIM/MRM results for consolidated reporting. Explore also provides quantitation workflows with flagging and retrospective feature detection from DIA data.

Main results and discussion

Chromatographic performance: The LC method separated isobaric isomers such as morphine/hydromorphone and codeine/hydrocodone, and resolved methamphetamine/phentermine. A five-minute LC run allowed rapid screening while maintaining chromatographic resolution of challenging pairs.
Identification and library matching: From an opiate standard mix the QTOF scan/DIA data enabled identification of more than ten components in a single TIC. Library searches produced high similarity scores—for example, a methadone match at ~97% and a tramadol match at ~89%—demonstrating strong MS2-based identification in complex matrices.
Quantitation and sensitivity: The LC‑TQ confirmation method quantified 46 compounds with 23 ISTDs in under three minutes with rapid polarity switching. Calibration curves built from triplicates showed high linearity (most R2 ≥0.99) and LLOQs down to low ng/mL or sub-ng/mL for many analytes. The GC-MS targeted method successfully monitored over 50 compounds using combined Scan/SIM acquisition; SIM traces (e.g., methadone) provided robust confirmation signals.
Untargeted capabilities and retrospective analysis: Data-independent acquisition on the QTOF enabled untargeted feature detection for both MS1 and MS2 data, facilitating detection of low-level or previously unanticipated compounds (authors cited Xylazine, Carfentanil, Metonitazene, Protonitazene as examples at or near LLOQ). The unified software permitted combining untargeted hits with targeted confirmation workflows.
Overall, the integrated workflow produced high-quality qualitative and quantitative data while simplifying downstream review and reporting.

Benefits and practical applications

• Single software environment reduces training requirements, data transfer errors, and review time by consolidating screening and confirmation results.
• Combination of high-resolution full-scan (QTOF) and targeted (TQ, GC-SQ) analyses balances broad non-targeted detection with high-sensitivity confirmatory assays.
• Rapid LC methods and combined acquisition modes support higher sample throughput in forensic and clinical toxicology laboratories.
• Retrospective data mining from DIA enables re-interrogation of data for emergent substances without re-running samples.

Future trends and potential applications

Suggested next steps and likely directions include:

• Validation and matrix recovery studies (authors noted planned urine recovery experiments for the GCMS method).
• Expansion and curation of spectral libraries, especially for novel psychoactive substances and analogs to improve identification confidence.
• Automation of sample preparation and data review workflows, and integration with LIMS for routine casework deployment.
• Application of machine-learning approaches to hit scoring and prioritization to further reduce analyst review time.
• Extending quantitative HRMS workflows and harmonizing cross-platform quantitation strategies to streamline regulatory validation.

Conclusion

The presented work demonstrates the feasibility of a consolidated screening-to-confirmation forensic toxicology workflow using one software platform to manage LC-QTOF untargeted screening, LC-TQ targeted quantitation, and GC-SQ confirmation. The approach provided effective chromatographic resolution of isobars, high-confidence library matches, and sensitive quantitation with rapid run times. Adoption in routine forensic laboratories can improve consistency and throughput, but appropriate method validation in relevant matrices is required prior to use in casework. The materials and applications were reported as Research Use Only.

References

• Hiramatsu Y., Matos Mejías C., Monti S.A., Smith J.P., Wiest L., Gilles C. Screening to Confirmation: Complete Workflow for Comprehensive Forensic Toxicology Analysis Using a Single Software Platform. Shimadzu Scientific Instruments, Inc.; Poster MP725. Authors affiliated with Shimadzu Corporation. Materials reported for Research Use Only.