Using the Waters Forensic Toxicology Screening Application Solution With UNIFI to Determine Diuretics in Urine

Importance of the Topic

The detection of diuretic drugs in urine is critical for clinical toxicology, anti-doping control, and forensic investigations. Diuretics can be abused to manipulate body weight and mask the presence of banned substances, leading to strict regulation by the World Anti-Doping Agency (WADA). A robust screening method capable of identifying and quantifying both positive and negative ionising diuretics at low concentration levels (down to the WADA MRPL of 200 ng/mL) is therefore essential for laboratories ensuring athlete safety and regulatory compliance.

Objectives and Study Overview

This work aimed to extend an existing Forensic Toxicology Screening Application Solution with Waters UNIFI software to include negative-mode electrospray ionisation (ESI-) diuretics. Key goals included:

• Detection and semi-quantitative analysis of 16 negative-ionising diuretics in urine.
• Demonstration of chromatographic separation and accurate mass confirmation using UPLC-QTof-MS in MS(E) mode.
• Validation of sensitivity down to the WADA MRPL and evaluation of linear dynamic range up to 2000 ng/mL.

Methodology and Instrumentation Used

Samples of spiked urine were prepared by protein precipitation with acetonitrile and addition of an internal standard. Chromatographic separation was achieved on an ACQUITY UPLC HSS C18 column (2.1 × 150 mm, 1.8 µm) at 50 °C, using a water/acetonitrile gradient (0.001% formic acid) and a 7.5 min run time. Mass spectrometric detection employed a Xevo G2-S QTof instrument in MS(E) mode with negative electrospray ionisation. Key parameters included:

• Source temp: 150 °C; desolvation temp: 400 °C; desolvation gas: 800 L/h.
• Acquisition range m/z 50–1000; scan time 0.1 s; capillary voltage 1.5 kV; cone voltage 20 V.
• Low-energy collision energy 6 eV; high-energy ramp 10–40 eV; leucine enkephalin as lockmass.

Main Results and Discussion

All 16 target diuretics showed clear chromatographic peaks with retention times spanning 1.7–4.1 min. Identification criteria required retention time within ±0.35 min of standards, precursor mass accuracy within ±5 ppm, and at least one diagnostic fragment in the high-energy trace. Calibration curves from 0 to 2000 ng/mL exhibited quadratic fits with 1/x weighting and correlation coefficients (r2) above 0.99 for all analytes. Quantitative accuracy at the WADA MRPL (200 ng/mL) was demonstrated. High-energy fragment patterns enabled differentiation of isobaric compounds (e.g., metolazone vs. indapamide) and confirmation of spiked analytes by binary comparison.

Analysis of an authentic urine sample confirmed the presence of furosemide and revealed phase I and phase II metabolites (including glucuronide conjugates) through UNIFI’s Met ID workflow, highlighting the solution’s capability for metabolite discovery beyond the predefined library.

Benefits and Practical Application of the Method

This extended screening solution offers:

• Comprehensive detection of negative-ionising diuretics alongside existing positive-mode targets.
• Rapid analysis (<8 min per sample) suitable for high-throughput anti-doping labs.
• Automated data processing with confident identification using accurate mass, retention time, and fragmentation.
• Capability to discover and incorporate novel metabolites into custom libraries.

Future Trends and Opportunities

Future developments may include:

• Integration of larger metabolite libraries and automated spectral deconvolution for broader compound coverage.
• Enhanced data mining with machine learning to improve detection of emerging designer drugs.
• Adoption of higher-resolution instrumentation for even greater selectivity and sensitivity.
• Implementation in clinical toxicology for therapeutic drug monitoring and overdose investigations.

Conclusion

The enhanced Waters Forensic Toxicology Screening Application Solution with UNIFI effectively extends forensic and anti-doping screening to negative-ionising diuretics in urine. It delivers sensitive, rapid, and reliable identification and semi-quantitation down to regulatory thresholds, with added capability for metabolite detection. Full user validation is recommended prior to routine deployment.

References

1. The World Anti-Doping Code: The 2015 Prohibited List, International Standard, WADA.
2. The World Anti-Doping Code: Minimum Required Performance Levels for Detection and Identification of Non-Threshold Substances, Technical Document TD2013MRPL, WADA.
3. Wood M. The Utility of MS(E) for Toxicological Screening. Waters Technology Brief 2010; p/n 720005198EN.
4. Forensic Toxicology Screening Application Solution. Waters Brochure; p/n 720004830EN.
5. Roberts M, Wood M. Analysis of Beta-Blockers using UPLC with Accurate Mass Screening. Waters Application Note 2014; p/n 720005188EN.