A decade of DBS steroid innovation in the German Sports University Cologne

Significance of the topic

Dried blood spot (DBS) sampling offers a minimally invasive, logistically simple, and tamper-resistant alternative to conventional matrices for anti-doping testing. Detecting intact anabolic steroid esters from DBS would provide direct evidence of exogenous administration without needing isotope ratio mass spectrometry (IRMS). However, DBS poses analytical obstacles — tiny sample volumes, complex matrix interferences, rapid ester hydrolysis, and very low endogenous concentrations — that historically limited routine deployment. Advances in targeted mass spectrometric hardware and acquisition strategies are therefore critical to unlock DBS utility in operational anti-doping workflows.

Objectives and study overview

This case study recounts a decade of method development at the German Sport University Cologne culminating in the implementation of the Thermo Scientific Stellar mass spectrometer. The primary aims were to: (1) demonstrate routine detection of native (non-derivatized) steroid esters from a single DBS; (2) achieve sub-ng/mL sensitivity and robust selectivity in realistic DBS matrices; and (3) establish a practical workflow compatible with routine high-throughput anti-doping operations. The study traces the evolution from a 2014 derivatized HRAM PRM approach to a native-ester tMS3 workflow enabled by the Stellar MS in 2025.

Methodology and instrumentation used

The analytical approach moved away from derivatization toward a non-derivatized LC–MSn method optimized for DBS extracts. Key methodological elements included:

• DBS sampling on DMPK and TASSO devices; single-spot volumes around 20 μL of whole blood.
• Multi-step solvent extraction protocols optimized to maximize recovery from cellulose-based DBS cards while limiting matrix retention.
• LC separation tailored for steroid ester chromatographic resolution prior to MSn detection.
• Targeted MSn acquisition using the Stellar mass spectrometer: ion-trap CID at MS2 followed by isolation of a diagnostic product ion and HCD at MS3 (tMS3), with automated ion packet management, automatic gain control (AGC), and normalized collision energy (NCE) settings to simplify multi-target acquisition.
• Post-acquisition extracted ion chromatogram (XIC) evaluation for qualitative and quantitative decision-making and product-ion ratio monitoring consistent with WADA identification criteria.

Instrumentation Used

The study referenced and/or utilized the following mass spectrometric platforms and ancillary hardware:

• Thermo Scientific Stellar Mass Spectrometer — hybrid quadrupole–dual-pressure linear ion trap engineered for ultra-sensitive targeted tMS2/tMS3 workflows with automated AGC and NCE.
• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap — used in earlier HRAM PRM-based methods (2014).
• Complementary LC systems (nanoflow and conventional LC variants reported in the literature) and DBS devices (DMPK and TASSO cards).

Main results and discussion

Implementation of tMS3 on the Stellar MS produced a step-change in capability for native steroid ester detection from DBS. Principal outcomes were:

• Sensitivity: sub-ng/mL detection limits (≤ 0.1 ng/mL) across nearly all evaluated steroid esters, enabling detection from small DBS volumes.
• Selectivity and specificity: tMS3 generated more diagnostic product ions and a markedly cleaner chemical background than tMS2 alone. This improved discrimination of low-abundance esters from endogenous isobaric/background signals in complex DBS matrices.
• Recovery and matrix performance: achieved recoveries in the 43%–85% range despite previously reported strong retention by card cellulose (earlier studies reported 11%–24%).
• Robustness and operational metrics: carryover was <1%; ion ratio stability met WADA IDCR criteria; no interference observed in 0/10 blank cards across TASSO and DMPK formats.
• Throughput and workflow simplification: removal of derivatization reduced turnaround and labor; consolidated MS3 instrument settings allowed a unified multi-target method for 17 native steroid esters.

Discussion points:

• The dominant challenge addressed was matrix-derived background and product-ion ambiguity at MS2 in very low-concentration DBS extracts. MS3 (CID at MS2 followed by isolation and HCD at MS3) produced additional, highly diagnostic fragments improving qualitative assurance and reproducible product-ion ratios necessary for confirmatory screening.
• Conserved MS3 settings per base steroid simplified method expansion across structurally related esters and reduced method development overhead for multi-analyte panels.

Benefits and practical applications of the method

The Stellar MS tMS3 workflow delivers multiple practical advantages for anti-doping laboratories:

• Direct evidence of exogenous steroid ester administration (intact ester detection) without routine IRMS, accelerating case management.
• Lean sample preparation: no derivatization, reduced hands-on time, and compatibility with existing DBS collection devices.
• Unified multi-analyte method supporting high-throughput screening of 17 esters from a single DBS.
• Enhanced logistical flexibility: suitable for out-of-competition testing, remote collection sites, large event screening, and sampling of populations where low-invasiveness is required (e.g., juniors).
• Future-proof platform: hardware and acquisition flexibility support expansion to other small molecules and targeted confirmation assays, including potential application to peptide hormones where MSn structural confirmation is advantageous.

Future trends and potential applications

Anticipated developments and opportunities following this work include:

• Regulatory and operational validation: multi-laboratory intercomparison studies, WADA method harmonization, and formal validation to integrate DBS tMS3 workflows into routine anti-doping practice.
• Method expansion: extension to broader small-molecule panels, isomer differentiation using MS3 diagnostic fragments, and incorporation of peptide hormone assays leveraging ion-trap MSn.
• Automation and scale-up: integration with automated DBS extraction and online sample preparation (e.g., TurboFlow-type workflows) to increase throughput and reproducibility.
• Complementary analytical strategies: combined use of DBS MSn screening with targeted urine IRMS or orthogonal confirmation where regulatory frameworks require carbon isotope evidence.
• Standardization: improved DBS collection, card selection, storage, and handling protocols to minimize matrix effects and maximize analyte stability.

Conclusion

Progress from a derivatized proof-of-concept to a native-ester tMS3 workflow demonstrates that modern MSn hardware and acquisition strategies can overcome the principal analytical barriers of DBS analysis for steroid esters. The Stellar mass spectrometer enabled sub-ng/mL sensitivity, superior selectivity, robust recoveries, and operational metrics compatible with routine anti-doping demands. While DBS is unlikely to fully replace urine-based testing, it provides a powerful complementary route for direct detection of exogenous esters and offers clear logistical and ethical advantages in specific testing scenarios.

References

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