Rapid Quantification of Clinically Relevant Compound Classes by Chromatography-Free DART-MS/MS

Rapid Quantification of Clinically Relevant Compound Classes by Chromatography-Free DART-MS/MS — Summary

Importance of the topic:
The accurate and timely measurement of drug concentrations in biological matrices is critical for therapeutic drug monitoring, pharmacokinetic studies, and clinical research. Traditional LC-MS/MS workflows are analytically robust but limited by chromatographic run times, solvent consumption, and instrument maintenance. A validated, chromatography-free approach that maintains quantitative performance while dramatically increasing throughput and reducing consumables can accelerate patient sample turnaround, lower operational costs, and support greener laboratory practices.

Objectives and study overview:
The study evaluated a DART-MS/MS (Direct Analysis in Real Time tandem MS) workflow as a chromatography-free alternative to LC-MS/MS for rapid quantification of multiple clinically relevant drug classes in serum and plasma. Goals included establishing linearity, precision, accuracy (trueness), limits of detection/quantification, matrix performance, carryover, stability on the DART mesh, and cross-correlation against established LC-MS/MS methods across panels such as antiepileptics, mycophenolic acid (MPA), and antifungals (antimycotics). High-throughput capability and reduced resource usage were core performance endpoints.

Methods and instrumentation:
- Sample preparation: A simple liquid–liquid extraction (LLE) was used. Typical sample protocol: 50 µL serum, addition of 20 µL internal standard (ISTD) solution and 100 µL extraction solution; in some workflows 50 µL salt solution was added to aid phase separation. After vortexing and centrifugation, 5 µL of the upper organic layer was spotted onto an HTS96 DART mesh and dried (<5 min) before analysis.
- DART-MS/MS analysis: Measurements were performed on an EVOQ DART-TQ+ triple quadrupole system with a Bruker DART source. MS/MS transitions, collision energies, source temperature, spot size, cone gas and grid voltage were optimized per analyte panel. Typical analysis time was <30 seconds per sample, enabling analysis of 96 samples in under 48 minutes.

Main results and discussion:
- Linearity: Five replicate calibration series in serum with 12 concentration levels per analyte produced excellent linearity across relevant clinical ranges (most R² > 0.99). Linear dynamic ranges covered low µg/L up to hundreds of mg/L depending on analyte.
- Precision and trueness: Intra- and interday precision using matrix-matched QCs at three levels over 10 days (4 timepoints/day) met standard acceptance criteria. Trueness (accuracy) across analytes was reported between approximately 85% and 115% of nominal.
- Limits and sensitivity: LOD and LOQ were sufficient to measure clinically relevant concentrations for all tested compounds; internal standard contribution at LLOQ was <20% for all analytes.
- Carryover and specificity: No carryover was observed between high and low samples. A specificity panel of 144 potentially interfering substances (neuroleptics, antidepressants, benzodiazepines, etc.) showed no interference under the tested conditions.
- Stability: Extracted samples spotted on the DART mesh were stable at room temperature for at least 7 hours, allowing practical plate handling and batch analysis.
- Cross-correlation vs LC-MS/MS: Comparison with LC-MS/MS on clinical and spiked samples (223 and 105 samples in different comparisons) demonstrated strong agreement. Correlation slopes for many analytes were close to unity and R² values were high (e.g., typical R² > 0.98). Average biases across analytes generally ranged from ~3% to ~13%, with certain analytes approaching higher bias values (~17%), indicating acceptable agreement for research-use applications but highlighting analyte-dependent measurement offsets to consider when replacing LC-MS/MS.

Key analytes and panels tested:
- Antiepileptics: topiramate, zonisamide, rufinamide, perampanel, brivaracetam, lacosamide, phenobarbital, stiripentol, tiagabine, etc.
- Immunosuppressant/metabolite: mycophenolic acid (MPA).
- Antimycotics (azole antifungals): fluconazole, itraconazole, OH-itraconazole, isavuconazole, posaconazole, voriconazole, ketoconazole, etc.
These panels represent compounds with high clinical sample volumes in routine monitoring and research contexts.

Benefits and practical applications of the method:
- Throughput: Analysis time <30 s per sample allows very high sample throughput (e.g., 96 samples in <48 min), dramatically shortening total batch times compared with LC-MS/MS.
- Operational savings: Substantial reduction in solvent and consumable usage (reported ~95% reduction), lower maintenance needs (no LC columns or pumps), and reduced troubleshooting associated with chromatography.
- Practical workflow: Simple LLE sample prep with rapid drying and direct DART analysis suits research labs, high-volume screening, and workflows where rapid turnaround is prioritized.
- Green chemistry: Reduced solvent consumption and disposable usage align with sustainability goals.

Limitations and considerations:
- Current status: Methods were demonstrated for research use only and not validated for clinical diagnostic use in this report.
- Analyte-dependent bias: While overall agreement with LC-MS/MS is strong, some analytes show measurable bias; laboratory-specific cross-validation and possible calibration adjustments are advisable before replacing established clinical methods.
- Sample preparation remains required: Although chromatography is removed, LLE and careful control of sample spotting and drying are critical for repeatability.

Used instrumentation:
- EVOQ DART-TQ+ triple quadrupole mass spectrometer (Bruker) with DART ion source.
- HTS96 DART plate/mesh for sample introduction.
- Standard laboratory equipment for LLE (vortex, centrifuge) and pipetting. Instrument-specific parameters (transitions, collision energies, temperatures, gas settings) were optimized per analyte/panel during method development.

Future trends and potential uses:
- Panel expansion: The method is being extended to additional therapeutic classes such as tricyclic antidepressants, neuroleptics, benzodiazepines, vitamins, and antibiotics to broaden clinical and forensic applications.
- Clinical translation: With further validation under regulatory guidelines, DART-MS/MS workflows could complement or, in some high-throughput contexts, partially replace LC-MS/MS for routine therapeutic drug monitoring and research assays.
- Automation and integration: Coupling DART sample handling with automated liquid-handling and plate logistics can further increase throughput and reproducibility.
- Quantification improvements: Development of more comprehensive internal standard strategies and matrix-effect mitigation approaches will improve trueness and reduce analyte-specific biases.

Conclusion:
The evaluated DART-MS/MS approach provides a robust, chromatography-free alternative for rapid quantification of multiple drug classes in serum and plasma for research applications. The workflow delivers excellent linearity, acceptable precision and trueness, low carryover, and strong agreement with LC-MS/MS across many analytes, while offering markedly higher throughput and lower resource consumption. Before clinical diagnostic adoption, targeted validation and method harmonization against established LC-MS/MS assays are recommended.

References:
The source material is a Bruker application/technical study (2026) reporting DART-MS/MS method development and cross-comparison with LC-MS/MS for multiple drug panels. No formal literature references were provided in the supplied text.