Analytica Chimica Acta 585 (2007) 94–102 A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry Marjo Kolmonen a,b, ∗ , Antti Leinonen b , Anna Pelander a , Ilkka Ojanper¨ a a a Forensic Toxicology Division, Department of Forensic Medicine, University of Helsinki, Finland b Doping Control Laboratory, United Laboratories Ltd., Helsinki, Finland Received 16 October 2006; received in revised form 12 December 2006; accepted 14 December 2006 Available online 23 December 2006 Abstract A general screening method based on solid phase extraction (SPE) and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and investigated with 124 different doping agents, including stimulants, -blockers, narcotics,  2 -adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids. Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was based on gradient elution on a C18 column. Ionization of the analytes was achieved with electrospray ionization in the positive mode. Identification by LC/TOFMS was based on retention time, accurate mass and isotopic pattern. Validation of the method consisted of analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was attained to 97 doping agents. The extraction recoveries varied between 33 and 98% and the median was 58%. Mass accuracy was always better than 5 ppm, corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for spiked urine samples, expressed as median of relative standard deviations (RSD%) at concentrations of MRPL and 10 times MRPL, were 14% and 9%, respectively. The suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine samples. © 2006 Elsevier B.V. All rights reserved. Keywords: Solid phase extraction; Liquid chromatography; Electrospray ionization; Time-of-flight mass spectrometry; Doping analysis 1. Introduction Hundreds of chemically and pharmacologically different drugs with minimum required performance limits (MRPL) are currently included in the list of prohibited substances (Table 1), published by World Anti-Doping Agency (WADA) [1,2]. The demanding task of doping control laboratories is to screen for a wide range of drugs in a short period of time and confirm the findings. Doping analysis has to conform to the requirements of International Standards for Laboratories established by WADA including e.g chain of custody, validation of screening and con- firmation methods and criteria for identification [3,4]. The con- stant appearance of new abused drugs challenges the doping laboratories to update their methods. Today, doping analysis re- quires the use of several different chromatographic, mass spec- trometric and immunological methods [5–8]. Consequently, a ∗ Corresponding author at: P.O. Box 40, University of Helsinki, Helsinki FIN- 00014, Finland. Tel.: +358 40 5921396. E-mail address: marjo.kolmonen@helsinki.fi (M. Kolmonen). large number of separate analytical procedures results in a more complex, time-consuming and laborious screening procedure. The prohibited substances are analyzed mostly from urine. In urine many drugs exist as their metabolites, and for some drugs the metabolites are the only detectable compounds. For a long time, conventional gas chromatographic-mass spectro- metric methods (GC/MS) have been used extensively in doping analysis. However, laborious sample preparation for the analy- sis of polar and non-volatile compounds complicates the use of GC/MS in comprehensive screening, while thermolabile com- pounds cannot be analyzed at all by this technique. In the last 5 years, the suitability of liquid chromatography-mass spectrome- try (LC/MS) has been demonstrated for multi-analyte screening of many classes of prohibited substances [9–13]. However, com- monly used quadrupole and ion trap mass spectrometers do not allow simultaneous detection of hundreds of analytes with high specificity and sensitivity. Recently, Pelander et al. [14] described a novel approach for comprehensive toxicological screening of urine samples for drugs, based on liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS). In this method, acidic, neutral and 0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.12.028