A ready-to-run workflow for quantitating 80 drugs of abuse in whole blood with the TSQ Certis Triple Quadrupole MS

Importance of the topic

Rapid, sensitive and reproducible quantitation of drugs of abuse in whole blood is essential for forensic toxicology, workplace testing, clinical toxicology and law enforcement. Methods capable of robustly measuring large panels of analytes at low ng/mL (and sub-ng/mL) levels while supporting high sample throughput reduce case backlogs, improve turnaround times and increase confidence in judicial and clinical decisions. The presented workflow addresses these needs by combining streamlined sample preparation, fast UHPLC separation and optimized triple quadrupole MS detection to meet common regulatory and professional cutoffs (ANSI/ASB) across a broad analyte panel.

Objectives and study overview

The study aimed to develop and demonstrate a ready-to-run workflow for simultaneous confirmation and quantitation of 80 drugs of abuse in human whole blood using the Thermo Scientific TSQ Certis triple quadrupole mass spectrometer. Key performance targets included low limits of quantitation (targeting down to 0.05 ng/mL), wide linear range (up to 500 ng/mL), high recovery, chromatographic resolution of isomers, and the ability to support high daily sample throughput (approx. 320 samples/day) with reproducible results.

Methods

Sample preparation:

• Negative human whole blood was spiked with a mix of 80 target analytes and internal standards. Calibration standards were prepared across 13 levels from 0.05 to 500 ng/mL in 100 µL whole blood; 15 µL of an ISTD stock (labelled analogs) was added to samples.
• Protein precipitation and protein crash: 25 µL of 1% ZnSO4 was added and vortexed (soft crash), followed by 300 µL acetonitrile for protein precipitation; samples were vortexed again.
• Filtration: INTip Filtration using Tip-on-Tip (ToT) technology (DPX Technologies) — aspirate the crashed sample into a regular pipette tip and dispense through a low-porosity filtration tip into a clean tube.
• Evaporation and reconstitution: Samples were dried at 50 °C (~14 min) and reconstituted first with 20 µL MeOH + 0.1% formic acid + 2 mM ammonium formate (to keep THC in solution), then with 80 µL H2O + 0.1% formic acid + 2 mM ammonium formate.

Chromatography:

• Column: Thermo Scientific Accucore Biphenyl (2.1 x 50 mm, 2.6 µm).
• System: Thermo Scientific Vanquish Horizon UHPLC.
• Mobile phases: A = water with 0.1% formic acid and 2 mM ammonium formate; B = methanol with 0.1% formic acid and 2 mM ammonium formate.
• Injection volume: 1 µL. Rapid gradient producing a 4.5-minute cycle that resolves critical isomer pairs (e.g., codeine/hydrocodone, morphine/hydromorphone, methamphetamine/phentermine).

Mass spectrometry:

• Instrument: Thermo Scientific TSQ Certis triple quadrupole operated in SRM mode with two transitions per target (quantitative and confirmatory) and one quantitative transition for internal standards.
• Quadrupole settings: Q1 resolution 0.7 FWHM, Q3 resolution 1.2 FWHM.
• Acquisition performance: high SRM acquisition capability (>900 SRM/s) with fast polarity switching (<5 ms) and short dwell times (example 2 ms/transition) to ensure sufficient points across narrow chromatographic peaks.
• Ion source: OptaMax Plus HESI; higher vaporizer temperatures (up to 500 °C) were evaluated and found to increase peak areas (up to ~2-fold) for many analytes.

Used instrumentation

• Thermo Scientific TSQ Certis triple quadrupole mass spectrometer with OptaMax Plus HESI source.
• Thermo Scientific Vanquish Horizon UHPLC system.
• Thermo Scientific Accucore Biphenyl column (2.1 x 50 mm, 2.6 µm).
• INTip Filtration / Tip-on-Tip (ToT) filtration devices (DPX Technologies) for sample clean-up.

Main results and discussion

• Sensitivity and linearity: Limits of quantitation (LOQs) as low as 0.05 ng/mL were achieved for multiple analytes, with upper limits of linearity (ULOL) up to 500 ng/mL; calibration covered 0.05–500 ng/mL across 13 levels.
• Confirmation criteria: LOQ acceptance required peak area CV <20% for triplicate injections and ion ratio tolerance within ±20% relative to calibrators.
• Chromatography: The 4.5-minute gradient successfully separated important isomeric pairs, enabling reliable identification and quantitation in a short cycle time.
• Extraction recovery: Average recovery across the 80 analytes was 96.92%, with the lowest individual recovery of 82.39%, demonstrating efficient analyte retention through the protein precipitation and INTip filtration workflow.
• Source optimization: Increasing the vaporizer temperature improved signal for many compounds (up to ~2× peak area gain), enhancing sensitivity for challenging analytes.
• Robustness: Over 450 sequential injections of extracted whole blood showed stable peak areas (example: lorazepam within ±20% RSD), supporting high-throughput routine operation.

Benefits and practical applications of the method

• High throughput: 4.5-minute run time supports analysis of approximately 320 samples per instrument per day, suitable for forensic and high-volume clinical labs.
• Broad analyte coverage: Simultaneous quantitation of 80 drugs of abuse, including structurally similar isomers, reduces the need for multiple separate assays.
• Low limits of quantitation: Sub-ng/mL LOQs meet or exceed many regulatory and forensic cutoffs, improving detection of low-level exposure or long elimination-phase metabolites.
• Simple sample preparation: Protein precipitation followed by INTip filtration is fast, uses common reagents and minimizes manual transfer and consumable use compared with traditional SPE approaches.
• Reproducibility and robustness: High recovery, tight peak-area stability over hundreds of injections and clear confirmatory ion-ratio criteria support defensible results in investigative contexts.

Future trends and potential uses

• Panel expansion and multiplexing: Extend the panel to include emerging novel psychoactive substances and metabolites while maintaining throughput.
• Automation: Integration of automated liquid handling with INTip workflows to further increase throughput and reduce operator variability.
• Hybrid workflows: Combine targeted triple quadrupole quantitation with high-resolution MS screening/HRMS confirmation for non-target discovery and retrospective data analysis.
• Data analytics: Use advanced software, machine learning and automated flagging to streamline result review, confirmation and reporting for large datasets.
• Sensitivity improvements: Continued source and ion optics optimization, coupled with improved chromatographic selectivity, can push LOQs lower for demanding applications (e.g., postmortem or low-abundance biomarkers).

Conclusion

The described workflow delivers a rapid, robust and sensitive method for simultaneous quantitation and confirmation of 80 drugs of abuse in whole blood. Combining a brief UHPLC gradient, efficient protein precipitation with INTip filtration and optimized TSQ Certis triple quadrupole acquisition provides LOQs down to 0.05 ng/mL, high recovery (~97% average), and the throughput needed for high-volume forensic and clinical laboratories. Source tuning (higher vaporizer temperatures) and the instrument's fast SRM capability are key enablers for sensitivity and chromatographic peak sampling in this high-density panel.

Reference

Patterson C., Hassell K. (2026). A ready-to-run workflow for quantitating 80 drugs of abuse in whole blood with the TSQ Certis Triple Quadrupole MS. Thermo Fisher Scientific, San Jose, CA, USA.