High-Throughput and Sensitive Screening by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry of Diuretics and other Doping Agents

Abstract

The reliability of ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC- MS/MS) for high throughput screening in anti-doping control has been tested. A method to screen for the presence of diuretics and other doping agents in urine has been optimised and validated. The extraction procedure consisted of an alkaline extraction (pH 9.5) with ethyl acetate and salting-out effect (sodium chloride). The extracts were analysed by UPLC-MS/MS. Analysis of 34 forbidden drugs and metabolites was achieved in a total run time of 5 min, using a C18 column (100 mm x 2.1 mm i.d., 1.7 microm particle size) and a mobile phase containing deionised water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6 mL min(-1). Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionisation in positive- or negative-ion mode. Precursor and product ions were studied for each compound and cone voltage and collision energy were optimised. Due to the different chemical structure of the compounds under study, extraction recoveries varied from less than 10% to 100% depending on the analyte. The limits of detection ranged from 50 ng mL(-1) to 200 ng mL(-1), and all the compounds comply with the requirements of quality established by the World Anti-doping Agency. Intra-assay precision was evaluated at two concentrations for each compound and, in most cases, a relative standard deviation of the signal ratio lower than 20% was obtained. The method has demonstrated to be reliable when analysing routine samples and the short analysis time resulting from a simple sample preparation and a rapid instrumental analysis allow a fast turn-around time and makes it of great interest for routine anti-doping control purposes.

Mass spectrometric characterization of urinary toremifene metabolites for doping control analyses

“…The extraction of free urinary toremifene metabolites was based on a previously described procedure [45,46]. Briefly, 100 ng mL −1 of methyltestostenone was added to 5 mL aliquots of urine samples and the pH was made alkaline with 100 L of 5.3 M ammonium chloride solution (adjusted to pH 9.5 with ammonia).…”

Simultaneous extraction and screening of diuretics, beta-blockers, selected stimulants and steroids in human urine by HPLC-MS/MS and UPLC-MS/MS

“…This methodology has been largely superseded in recent years by high performance liquid chromatography mass spectrometry (HPLC-MS) techniques, which offer greater versatility and robustness [10][11][12]. Recent methods have utilized new technologies such as ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to increase sample throughput and decrease analysis time [13,14].…”

High-Throughput Screening of Drugs of Abuse in Urine by Supported Liquid–Liquid Extraction and UHPLC Coupled to Tandem MS

“…In the field of doping analysis, UHPLC-MS-MS strategy was employed for determining between 30 and 130 prohibited substances in urine in a single analytical run [16][17][18], using a simple ''dilute and shoot'' as sample preparation. Finally, Berg et al [19] developed and validated an SPE-UHPLC-MS-MS method allowing the determination of six opiates, cocaine and benzoylecgonine in less than 6 min, reequilibration included.…”