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

The goal of this study is to simultaneously identify banned diuretics and beta-blockers in urine samples to support anti-doping laboratories.

The use of mixed-mode SPE and the ACQUITY UPLC I-Class/Xevo TQ-S micro System enabled clean, fast, and efficient extraction and analysis of urinary diuretics and beta-blockers for anti-doping purposes. The Oasis MAX μELution plates enabled efficient and reproducible extraction of 20 beta-blockers and 20 diuretic compounds. Matrix effects were significantly decreased compared to diluted samples. The Xevo TQ-S micro had the speed and sensitivity to accurately identify all analytes in under four minutes, meeting WADA’s strict ion ratio criteria even at 20% of the MRPL. The ability to perform rapid inter-scan polarity switching enabled the analysis in a single injection, saving both time and instrument wear compared to sequential injections of positive and negative ionizing compounds. This will enable rapid, accurate analysis of these compounds, while maintaining a cleaner UPLC-MS/MS system.

Benefits

Solid phase extraction and rapid polarity switching enable clean, fast, and efficient analysis of banned diuretics and beta-blockers. 

Diuretics and beta-blockers are both banned by the World Anti-Doping Agency (WADA).¹ Despite their chemical differences, many anti-doping laboratories prefer to analyze these two classes of molecules together for workflow considerations.^2-4 Beta-blocking agents are bases that ionize under positive electrospray ionization (ESI) and diuretics are mostly acids that ionize under negative ESI conditions. This often necessitates sequential analyses via LC-MS; one in positive mode and a second in negative mode. In addition, these chemical differences make simultaneous extraction a challenge, as any SPE sorbent must be able to accommodate a wide range of polarities and chemotypes.

The challenges described above have been solved using Oasis MAX µElution plates to cleanly and efficiently extract diuretics and beta-blockers from urine samples. This was followed by UPLC-MS/MS analysis on a Waters ACQUITY UPLC I-Class System and Xevo TQ-S micro Mass Spectrometer. Separation was achieved on a Waters ACQUITY UPLC CSH C₁₈ Column (1.7 µm, 2.1 x 100 mm). Mobile phases consisted of 0.01% formic acid (MPA) and acetonitrile (MPB).

The compounds and their retention times are listed in Table 1. All compounds eluted within four minutes and the entire UPLC cycle was five minutes (Figure 1). Two to three MRM transitions were acquired for all compounds. The rapid polarity switching of the Xevo TQ-S micro enabled this fast, yet efficient chromatography, despite simultaneous analysis of 24 positive and 18 negative ionizing compounds.  

Solid phase extraction using the Oasis MAX µElution Plate was performed as follows: 50 µL urine samples were pretreated 1:1 with 5% strong ammonia, then loaded onto the SPE plate, washed with 5:95 MeOH:H2O, eluted with 50 µL of 75:25 MeOH:ACN containing 2% formic acid, and diluted with 200 µL of water. Recovery was efficient for all compounds. Figure 2 shows the average recovery from 12 unique lots of urine. Amiloride, clopamide, and canrenone were grouped with the beta-blockers, as they are bases that ionize by positive ESI. Mean recovery for beta-blockers (and basic diuretics) was 85%, with all but one at 80% or higher; %RSDs were all <20%. Diuretic recovery ranged from 65–94%; all %RSDs <20% with the exception of acetazolamide. The use of the Oasis MAX µElution Plate substantially reduced matrix effects associated with simple sample dilution. As Figure 3 shows, matrix effects, particularly ion suppression, increased from negligible levels to up to over 60% for many of the beta blockers and from 20–40% up to 60‒90% for many of the diuretics, even at a 1:10 dilution. Single-factor Anova analysis revealed that matrix effects were significantly increased for all compounds except for acebutolol, metolazone, bumetanide, probenecid, and ethacrynic acid.

All compounds were readily detectable, even at 20% of WADA’s Minimum Required Performance Level (MRPL) of 100 ng/mL for beta-blockers and 200 ng/mL for the diuretic compounds.⁵ Retention time tolerances were well within WADA requirements as well.⁵ Despite the speed of the analysis and the need for polarity switching, WADA ion ratio criteria for confirmation were met for all compounds, even at 20% of the MRPL.⁶ 

The use of mixed-mode SPE and the ACQUITY UPLC I-Class/Xevo TQ-S micro System enabled clean, fast, and efficient extraction and analysis of urinary diuretics and beta-blockers for anti-doping purposes. The Oasis MAX µELution plates enabled efficient and reproducible extraction of 20 beta-blockers and 20 diuretic compounds. Matrix effects were significantly decreased compared to diluted samples. The Xevo TQ-S micro had the speed and sensitivity to accurately identify all analytes in under four minutes, meeting WADA’s strict ion ratio criteria even at 20% of the MRPL. The ability to perform rapid inter-scan polarity switching enabled the analysis in a single injection, saving both time and instrument wear compared to sequential injections of positive and negative ionizing compounds. This will enable rapid, accurate analysis of these compounds, while maintaining a cleaner UPLC-MS/MS system. 

1. World Anti-Doping Agency, The World Anti-Doping Code: WADA Prohibited List 2019, 2019, WADA: Montreal, Canada. Available at: http://www.wada-ama.org
2. Musenga, A and Cowan, D. Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control. Journal of Chromatography A. 1288: p. 82–95.2013.
3. Murray, G and 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. Journal of Chromatography B. 877(30): p. 3857–3864.2009.
4. Deventer, K, Van Eenoo, P and 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 Communications in Mass Spectrometry. 19(2): p. 90–98.2005.
5. World Anti-Doping Agency, The World Anti-Doping Code: WADA Technical Document TD2018MRPL, World Anti-Doping Agency, Montreal, Canada, 2018, Available at: http://www.wada-ama.org.
6. World Anti-Doping Agency, The World Anti-Doping Code: WADA Technical Document TD2015ICR, World Anti-Doping Agency, Montreal, Canada, 2015, Available at: http://www.wada-ama.org.