Simultaneous Analysis of Diuretics and Beta-Blockers by Mixed Mode SPE and UPLC-MS/MS for Anti-Doping Analysis

Importance of the Topic

Anti-doping laboratories require fast and reliable methods to detect banned diuretics and beta-blockers in athletes’ urine samples. These compounds differ chemically and ionize under opposite electrospray conditions, traditionally necessitating separate analyses. A unified approach enhances laboratory throughput, reduces instrument wear, and ensures compliance with WADA regulations.

Objectives and Study Overview

This study aimed to develop a single-injection workflow combining mixed-mode solid phase extraction (SPE) with rapid polarity-switching UPLC-MS/MS. The goal was to simultaneously extract and analyze 24 positively ionizing beta-blockers and 18 negatively ionizing diuretics in under five minutes per run, meeting or exceeding WADA’s Minimum Required Performance Levels (MRPL).

Methodology and Instrumentation

Sample Preparation and SPE

• Urine (50 µL) was pretreated 1:1 with 5% ammonia, loaded onto Oasis MAX µElution plates, washed with 5:95 MeOH:H₂O, and eluted with 75:25 MeOH:ACN containing 2% formic acid.
• Eluates were diluted with water prior to analysis.

UPLC-MS/MS Analysis

• Separation on Waters ACQUITY UPLC CSH C18 Column (1.7 µm, 2.1 × 100 mm).
• Mobile phases: 0.01% formic acid in water (MPA) and acetonitrile (MPB).
• Instrumentation: Waters ACQUITY UPLC I-Class System coupled to Xevo TQ-S micro Mass Spectrometer with rapid polarity switching.
• Two to three MRM transitions monitored per analyte, UPLC cycle time of five minutes.

Main Results and Discussion

Extraction Performance

• Beta-blockers and basic diuretics: mean recoveries of 85%, all %RSD < 20%.
• Acidic diuretics: recoveries ranged 65–94%, %RSD < 20% except acetazolamide (26%).

Matrix Effects

• SPE reduced ion suppression compared to 1:5 and 1:10 dilutions, where matrix effects increased up to 90% for some compounds.
• ANOVA confirmed significant reduction of matrix effects for all but five analytes.

Chromatography and Detection

• Twenty-four positive-ESI and eighteen negative-ESI compounds eluted within four minutes; total cycle five minutes.
• Retention time tolerances met WADA requirements; ion ratio criteria satisfied at 20% of MRPL (100 ng/mL for beta-blockers, 200 ng/mL for diuretics).

Benefits and Practical Applications

• Single-injection analysis halves runtime and solvent use compared to sequential methods.
• Mixed-mode SPE ensures broad polarity extraction with minimal matrix interference.
• Rapid polarity switching in the Xevo TQ-S micro maintains sensitivity for both ionization modes.
• Workflow fits high-throughput anti-doping labs requiring swift compliance testing.

Future Trends and Opportunities

• Integration with high-resolution MS for broad-spectrum screening of additional prohibited substances.
• Automation of µElution SPE to reduce hands-on time and variability.
• Application of similar mixed-mode strategies in forensic toxicology and clinical testing.
• Development of greener solvents and microflow techniques to further reduce environmental impact.

Conclusion

The combined Oasis MAX µElution SPE and UPLC-MS/MS workflow allows rapid, sensitive, and reproducible detection of banned diuretics and beta-blockers in a single five-minute injection. This method meets stringent WADA criteria while reducing matrix effects and instrument wear, offering a robust solution for anti-doping laboratories.

References

1. World Anti-Doping Agency. The World Anti-Doping Code: WADA Prohibited List 2019. Montreal, Canada: WADA; 2019.
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3. Murray G, Danaceau J. Simultaneous extraction and screening of diuretics, beta-blockers, selected stimulants and steroids in human urine by HPLC-MS/MS and UPLC-MS/MS. J Chromatogr B. 2009;877(30):3857–3864.
4. Deventer K, Van Eenoo P, Delbeke F. Simultaneous determination of beta-blocking agents and diuretics in doping analysis by liquid chromatography/mass spectrometry with scan-to-scan polarity switching. Rapid Commun Mass Spectrom. 2005;19(2):90–98.
5. World Anti-Doping Agency. WADA Technical Document TD2018MRPL. Montreal, Canada: WADA; 2018.
6. World Anti-Doping Agency. WADA Technical Document TD2015ICR. Montreal, Canada: WADA; 2015.