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

Importance of the topic

Comprehensive forensic toxicology requires rapid, sensitive screening followed by robust confirmatory analyses. Traditional workflows often rely on multiple instruments and disparate software environments, increasing turn-around time, user training burden, and potential for reporting inconsistencies. Integrating high-resolution, untargeted screening with targeted confirmation in a single software platform streamlines data handling, improves traceability from putative hits to confirmed identifications, and supports retrospective review of acquired high-resolution data—important capabilities for modern forensic and clinical toxicology laboratories.

Objectives and study overview

This work demonstrates a consolidated screening-to-confirmation workflow using Shimadzu LabSolutions Insight Explore to process data from LC-QTOF screening, LC-triple quadrupole (TQ) targeted confirmation, and GC-single quadrupole (SQ) targeted confirmation. Key aims were to show rapid untargeted detection with high-resolution QTOF, efficient targeted quantitation by LC-MS/MS, and orthogonal confirmation by GC-MS, all managed within one software environment to simplify review, reporting, and retrospective queries.

Methodology and workflow

Overview of the end-to-end workflow:

• Sample types: Biological matrices (urine, blood) following extraction, cleanup, and addition of internal standards.
• Screening: LC-QTOF (untargeted MS1/MS2, data independent acquisition) to detect features and produce library-searchable spectra.
• Confirmation: Targeted LC-MS/MS (MRM) for high-sensitivity quantitation and GC-MS (Scan/SIM) for orthogonal confirmation of volatile/thermostable targets.
• Data processing: Single-platform tools for peak detection, formula finding, library searching (including reverse search), hit scoring/flagging, and quantitative calibration with review-by-exception.

Used instrumentation

• LC-QTOF: Shimadzu LCMS-9050 used for high-resolution screening with DIA (data independent acquisition) to allow untargeted MS2 collection and retrospective data mining. LC conditions employed a Shim-pack Scepter PFPP-120 column (1.9 µm, 2.1 x 50 mm), water/methanol mobile phases with ammonium formate and formic acid, 0.4 mL/min flow and 5 min run time.
• LC-TQ: Shimadzu LCMS-8050/8050RX (triple quadrupole) for fast targeted quantitation using rapid polarity switching and MRM to quantify 46 compounds with 23 isotopically labeled internal standards in under three minutes.
• GC-SQ: Shimadzu GCMS-QP2020NX for confirmation of >50 commonly encountered toxicology targets using Scan plus SIM acquisitions. Column: Rtx-5ms (30 m x 0.25 mm, 0.25 µm); oven program 60 °C (2 min) to 320 °C at 15 °C/min; split injection at 300 °C.
• Software: LabSolutions Insight Explore (single-platform environment) for combined screening, library searching (compatible with multiple library formats including NIST and vendor libraries), quantitation, review and reporting.

Main results and discussion

Key experimental outcomes:

• Chromatographic separation resolved isobaric/opiate pairs such as morphine/hydromorphone and codeine/hydrocodone, enabling reliable identification and quantitation of critical analytes.
• Rapid LC methods (5 min) and very short targeted LC-MS/MS runs (<3 min) provided high throughput while maintaining excellent linearity (many R2 > 0.995) across relevant concentration ranges.
• Limits of quantitation: Method LLOQs ranged down to low pg/ng per mL levels for several analytes (examples reported LLOQs as low as 0.005 ng/mL for some compounds), supporting sensitive detection of fentanyl analogs and emerging nitazene-class opioids.
• Library matching: QTOF MS2 spectra searched against Cayman Spectral Library and a Shimadzu Forensic Toxicology Database produced high similarity scores (e.g., Methadone 97%, Tramadol 89%), aided by reverse-search functionality for complex matrices.
• Combined Scan and SIM acquisition strategies allowed simultaneous non-targeted detection and targeted monitoring (e.g., methadone SIM chromatogram shown), which simplifies confirmation workflows without separate injections.
• Quantitation approach: Calibration used internal standards with linear curves and 1/x or 1/x2 weighting; acceptance required triplicate agreement within ±20% at each calibration level.

Benefits and practical applications

• Consolidation of screening and confirmation in one software reduces data transfer errors, shortens review times, and centralizes reporting for forensic casework.
• High-resolution QTOF screening with DIA permits retrospective interrogation for new/emerging drugs without re-running samples, an advantage when monitoring novel psychoactive substances.
• Orthogonal confirmation (LC-MS/MS and GC-MS) increases confidence in positive findings, particularly for isobaric or matrix-challenged targets.
• Fast chromatographic methods increase sample throughput for high-volume forensic and clinical toxicology laboratories while retaining required analytical performance.

Future trends and possibilities

• Broader adoption of single-platform solutions that integrate HRMS screening, targeted MS/MS confirmation, library management and automated reporting will likely continue as labs seek efficiency and traceability.
• Expanded spectral libraries and improved reverse-search algorithms will enhance identification confidence in complex biological matrices and for novel substances (e.g., nitazenes, carfentanil analogs).
• Further method validation in authentic matrices (planned urine recovery studies noted by the authors) and robustness testing across instrument generations will be important steps toward routine casework implementation and accreditation.
• Machine-learning assisted flagging and automated review-by-exception could further reduce analyst workload and improve consistency in large-scale screening programs.

Conclusion

This study demonstrates a practical, integrated screening-to-confirmation workflow combining LC-QTOF untargeted screening, LC-TQ targeted quantitation, and GC-SQ targeted confirmation within a single software environment. The approach yields sensitive, high-throughput analyses with robust library matching and streamlined data review. Adoption of this consolidated workflow can reduce operational complexity and enable retrospective HRMS-based investigations; however, full validation in relevant biological matrices is required prior to routine forensic use.

References

1. Hiramatsu Y, Matos Mejías C, Monti SA, Smith JP, Wiest L, Gilles C. Screening to Confirmation: Complete Workflow for Comprehensive Forensic Toxicology Analysis Using a Single Software Platform. Shimadzu Scientific Instruments, Inc.; MP725. Authors affiliated with Shimadzu Scientific Instruments, Inc.