Fully automated dried spot analysis for the rapid quantitation of tramadol and its metabolites in various matrices

Significance of the Topic

Dried spot analysis has emerged as a powerful tool in clinical research, therapeutic drug monitoring, forensic toxicology and anti-doping control.
The ability to collect small biological volumes in a minimally invasive manner, combined with enhanced analyte stability and simplified logistics, addresses key challenges in large-scale screening and chain-of-custody requirements.

Study Objectives and Overview

This work demonstrates a fully integrated, high-throughput workflow for the quantitation of tramadol and its main metabolites (O-desmethyltramadol, N-desmethyltramadol) from dried blood, serum, urine and saliva spots.
The primary goal is to showcase the Thermo Scientific™ Transcend™ DSX-1 system, which automates sample desorption, online cleanup, chromatographic separation and tandem mass spectrometric detection in a single platform.

Methodology and Instrumentation

• Sample preparation involved spiking biological matrices at 5–400 ng/mL, applying 6 μL onto dried spot cards and air-drying at room temperature.
• Automated flow-through desorption delivered internal standards and elution solvents directly to each 6 mm spot.
• Online sample cleanup employed TurboFlow™ two-dimensional technology in Focus mode, using a Cyclone™-P trapping column followed by a biphenyl analytical column for separation.
• MS/MS detection was carried out in positive electrospray mode with selected reaction monitoring at optimized collision energies.
• Calibration curves were constructed over the 5–400 ng/mL range with 1/x weighting, achieving R² > 0.99.

Instrumentation

• Thermo Scientific Transcend DSX-1 dried spot autosampler with flow-through desorption (FTD™)
• Thermo Scientific TurboFlow UHPLC system (Cyclone-P and biphenyl columns)
• Thermo Scientific TSQ Altis MD series triple quadrupole mass spectrometer with OptaMax NG HESI probe
• Intelligent Vision Camera (IVC™) for spot recognition and barcode tracking
• TraceFinder™ and Aria™ MX software for instrument control and data processing

Main Results and Discussion

The method delivered robust performance across all matrices with:

• Lower limit of quantitation (LLOQ) at 5 ng/mL and upper limit of quantitation (ULOQ) at 400 ng/mL
• Precision and accuracy within ±15% at all calibration levels
• Internal standard signal RSD below 15% over 70 injections
• Total run time of ~8 min per spot including desorption and analysis
• Compatibility with multiple dried spot card vendors

The elimination of manual punch-out and offline extraction reduces labor and potential for human error, while maintaining chain-of-custody through integrated tracking.

Benefits and Practical Applications

• High throughput analysis for therapeutic drug monitoring and anti-doping programs
• Enhanced sample integrity and traceability via automated barcode scanning and imaging
• Reduced sample preparation time and solvent consumption
• Applicability to remote or in-field sampling with simplified transport

Future Trends and Opportunities

Advances may include expansion to broader panels of drugs and metabolites, direct coupling with high-resolution mass spectrometry, further miniaturization of sampling devices and integration of artificial intelligence for real-time data interpretation.
Field-deployable autosamplers and point-of-care implementations may further democratize access to rapid pharmacokinetic and anti-doping analyses.

Conclusion

The Transcend DSX-1 platform provides a streamlined, fully automated solution for dried spot analysis of tramadol and its metabolites with high sensitivity, reproducibility and throughput.
By consolidating sample preparation, cleanup, separation and detection into a single workflow, the system offers significant gains in efficiency and data integrity for clinical, forensic and sports testing laboratories.

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