Agilent ASMS 2020 Posters Book

Significance of the topic

Screening biological specimens for a wide range of drugs is critical in clinical, forensic, and toxicological laboratories. Traditional targeted assays based on immunoassays or triple quadrupole MS can miss emerging or unexpected substances. High-resolution quadrupole time-of-flight (Q-TOF) mass spectrometry offers data-independent acquisition that captures both precursor and fragment ions, enabling retrospective analysis for new analytes without re-running samples. This approach enhances flexibility to identify emerging drugs and metabolites in complex matrices such as whole blood.

Study objectives and overview

The aim of this work was to develop a rapid, high-throughput workflow for drug screening in whole blood using an Agilent 6546 LC/Q-TOF system with data-independent acquisition (DIA). The workflow combines automated sample preparation on the Bravo liquid handling platform, supported lipid removal, and a 10-minute reversed-phase LC method. An LC Screener software tool was implemented in MassHunter Quantitative Analysis to automate data review and positive identification reporting.

Methodology and instrumentation

• Sample preparation: Whole blood fortified with 153 drugs and ten unknown case samples underwent solid-phase extraction on Captiva EMR-Lipid 96-well plates with Bravo automation. Lipids and proteins were removed without concentration steps.
• Chromatography: Agilent 1290 Infinity II UHPLC with a 10-minute gradient on a 2.1×50 mm RRHD Eclipse Plus C18 column at 0.5 mL/min.
• Mass spectrometry: Agilent 6546 LC/Q-TOF with AJS ESI source acquired full-scan MS (m/z 40–1000 at 8 Hz) and MS/MS spectra using DIA windows. Reference masses ensured sub-5 ppm accuracy.
• Data analysis: MassHunter Quantitative Analysis 10.1 with the LC Screener tool applied mass accuracy, retention time, signal-to-noise, and fragment coelution criteria to classify identifications and display results in a unified interface.

Main results and discussion

• The EMR-Lipid cleanup produced low matrix effects: 77% of analytes showed <10% ion suppression.
• High recoveries (>70% for 91% of analytes) were achieved across 153 compounds.
• The 10-minute LC method baseline-resolved six pairs of isobaric drugs (e.g., morphine/hydromorphone, codeine/hydrocodone).
• Fast Q-TOF acquisition maintained >30,000 resolving power at m/z 118, yielding >12 data points per peak for robust integration.
• LC Screener automated identification reporting—green for confirmed, orange for review, red for negative—enabled rapid data review of hundreds of analytes.
• Simultaneous quantitation for common drugs was possible using calibration curves within the same workflow.

Benefits and practical applications

• Retrospective mining of data for new or unexpected drugs without re-analysis of stored samples.
• High-throughput processing with automated sample prep and DIA acquisition reduces labor and risk of error.
• Rapid screening of hundreds of compounds in a single 10-minute LC run.
• Unified software platform provides streamlined review, reporting, and quantitation.

Future trends and possibilities

• Expansion of DIA window strategies to cover broader m/z ranges and improve detection of low-level analytes.
• Integration of library updates for novel psychoactive substances and designer drugs to keep workflows current.
• Enhanced machine-learning algorithms for automated flagging of atypical mass spectral patterns and unknowns.
• Coupling with high-throughput autosamplers and robotics for population-scale toxicology studies.

Conclusion

The combination of Bravo automated sample preparation, Captiva EMR-Lipid cleanup, fast UHPLC, and high-resolution Q-TOF DIA acquisition provides a robust, sensitive, and flexible workflow for comprehensive drug screening in whole blood. The LC Screener tool streamlines data analysis, enabling rapid confirmation and quantitation of hundreds of analytes and supporting retrospective identification of emerging compounds.

Reference

Blount BC, et al. Vitamin E Acetate in Bronchoalveolar-Lavage Fluid Associated with EVALI. N Engl J Med. 382(8):697–705. 2020.