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

Ready-to-run 4.5-minute workflow for quantitation of 80 drugs of abuse in whole blood using TSQ Certis Triple Quadrupole MS

Significance of the topic:

• Rapid, accurate quantitation of drugs of abuse in whole blood is essential for forensic casework, clinical toxicology and high-throughput screening laboratories.
• Modern forensic laboratories require workflows that combine high sample throughput, low limits of quantitation, robust performance in complex matrices and streamlined data processing to meet caseload and regulatory needs.

Objectives and overview of the study:

• Develop and demonstrate an end-to-end quantitative workflow for 80 commonly encountered drugs of abuse in whole blood.
• Integrate a simple, fast sample clean-up using low-porosity filtration tips with a 4.5-minute UHPLC–SRM MS method on the Thermo Scientific TSQ Certis triple quadrupole system to achieve high throughput (approximately 320 samples/day).
• Show acceptable sensitivity (LOQs down to 0.05 ng/mL), linear dynamic range up to 500 ng/mL for many analytes, and long-run robustness.

Methodology and sample preparation:

• Sample matrix: negative human whole blood. Calibration prepared at 13 levels spanning nominally 0.05–500 ng/mL (100 µL final sample volume).
• Internal standards: mix of isotopically labeled standards (15 µL added to samples).
• Protein precipitation: soft crash with 25 µL 1% ZnSO4 (vortex 15 s), followed by addition of 300 µL acetonitrile and vortexing 60 s.
• Filtration clean-up: INTip Filtration using Tip-on-Tip (ToT) low-porosity filtration tips (DPX Technologies). Crashed sample aspirated with a standard pipette tip and dispensed through the filtration tip into a clean tube.
• Evaporation and reconstitution: samples dried at 50 °C for 14 min; reconstituted first with 20 µL methanol (0.1% formic acid, 2 mM ammonium formate) then with 80 µL water (0.1% formic acid, 2 mM ammonium formate) — the MeOH-first step preserves THC solubility.
• Injection volume: 1 µL.

Used instrumentation:

• UHPLC: Thermo Scientific Vanquish Flex UHPLC system.
• Column: Thermo Scientific Accucore Biphenyl, 2.1 × 50 mm, 2.6 µm.
• Mass spectrometer: Thermo Scientific TSQ Certis Triple Quadrupole MS with OptaMax Plus HESI source.
• Software: Thermo Scientific TraceFinder v5.2 (Method Forge tool used to build master methods automatically from a raw file).

Chromatography and mass spectrometry specifics:

• 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.
• Fast gradient delivering baseline separation of key isomeric pairs (e.g., codeine/hydrocodone, morphine/hydromorphone, methamphetamine/phentermine) within a 4.5-minute run.
• TSQ Certis performance: acquisition capability >900 SRM/s, polarity switching <5 ms, Q1 resolution 0.7 FWHM, Q3 resolution 1.2 FWHM; typical minimum dwell times observed ~2.16 ms.
• Source optimization: vaporizer temperature set to 500 °C (OptaMax Plus HESI) produced up to ~2× increase in MS peak area for several analytes.
• SRM strategy: one quantitative transition plus at least one confirming transition per analyte; transitions and optimized collision energies, RF lens voltages and retention times were defined and stored in the TraceFinder compound database.

Data processing and automation:

• TraceFinder v5.2 used for acquisition, quantitative processing and automation.
• Method Forge tool created a full master method automatically by extracting precursor/product pairs, retention times and MS parameters from a single calibrator raw file—reducing manual entry and facilitating method reuse.
• Acceptance criteria implemented in software: LOQ/ULOL determination, peak-area CV requirements, and ion-ratio tolerances (example tolerances ±20%).

Main results and discussion:

• Panel coverage: 80 target compounds (75 measured in positive polarity, 5 in negative).
• Sensitivity and dynamic range: LOQs as low as 0.05 ng/mL for several analytes; upper limits of linearity (ULOL) up to 500 ng/mL for many targets. Calibration spanned 0.05–500 ng/mL (13 levels).
• Chromatographic performance: successful separation of critical isomers within the 4.5-minute runtime; combined extracted ion chromatogram showed distinct peaks for the panel.
• Repeatability and robustness: long-run study of >450 injections showed stable response (example: lorazepam peak areas remained within ±20% RSD across injections), supporting high-throughput operation.
• Instrument tuning: elevated vaporizer temperature substantially improved response for several analytes, demonstrating the value of source optimization for drugs of abuse assays.
• Representative performance: example LOQ chromatograms and calibration curves were demonstrated for buprenorphine, THC and secobarbital with internal standard %RSDs reported (e.g., ISTD %RSD ~6–10% in examples).

Benefits and practical applications of the workflow:

• High throughput: 4.5-minute runs enable analysis of roughly 320 samples per instrument per day, appropriate for forensic and clinical settings with high sample volumes.
• Broad panel and sensitivity: comprehensive coverage of common drugs of abuse with sub-ng/mL sensitivity for many compounds.
• Streamlined sample prep: protein crash plus INTip low-porosity filtration is simple, rapid, and amenable to routine lab operations without complex SPE workflows.
• Robustness: long-injection sequences demonstrated stable performance, reducing downtime and QC burden.
• Automated method building and processing reduce method development time, minimize transcription errors and support method transfer between labs.

Future trends and potential uses:

• Further automation: integration with robotic sample handlers and plate-based workflows would increase throughput and reproducibility for large forensic labs.
• Expanded panels: adding emerging synthetic opioids, benzodiazepine analogs and novel psychoactive substances as standards become available to address changing drug landscapes.
• Hybrid workflows: coupling targeted SRM panels with periodic high-resolution screening runs for non-targeted discovery could provide both quantitative and screening capabilities in a single laboratory.
• Quantitative performance improvements: ongoing optimization of source conditions, chromatographic selectivity and labeled internal standards will continue to lower LOQs and improve matrix compensation.
• Regulatory and accreditation alignment: validated implementations of this workflow can be extended to accredited forensic laboratories with documented performance characteristics and QC procedures.

Conclusion:

• The presented workflow combines rapid UHPLC separation, an efficient low-porosity filtration clean-up and high-speed SRM acquisition on the TSQ Certis to deliver a practical, high-throughput solution for quantifying 80 drugs of abuse in whole blood.
• Key advantages include sub-ng/mL sensitivity for many targets, wide linear ranges, strong run-to-run stability over hundreds of injections, and automated method generation and data processing that streamline laboratory implementation.

Reference:

• Technical note: Thermo Fisher Scientific application note (Technical note | 004362). Authors: Courtney Patterson and Kerry Hassell. Thermo Fisher Scientific, San Jose, CA, United States. (2026)