Generic Sample Preparation Combined with High-Resolution Liquid Chromatography–Time-Of-Flight Mass Spectrometry for Unificatio
Total Page：16
File Type：pdf, Size：1020Kb
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Wageningen University & Research Publications Anal Bioanal Chem (2010) 396:2583–2598 DOI 10.1007/s00216-010-3484-3 ORIGINAL PAPER Generic sample preparation combined with high-resolution liquid chromatography–time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories R. J. B. Peters & J. E. Oosterink & A. A. M. Stolker & C. Georgakopoulos & M. W. F. Nielen Received: 18 August 2009 /Revised: 21 December 2009 /Accepted: 17 January 2010 /Published online: 16 February 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com Abstract A unification of doping-control screening proce- using positive electrospray ionisation with the exception of the dures of prohibited small molecule substances—including thiazide diuretics for which the best sensitivity was obtained by stimulants, narcotics, steroids, β2-agonists and diuretics— using negative electrospray ionisation. The results show that, is highly urgent in order to free resources for new classes with the exception of clenhexyl, procaterol, and reproterol, all such as banned proteins. Conceptually this may be achieved compounds can be detected below the respective minimum by the use of a combination of one gas chromatography– required performance level and the results for linearity, time-of-flight mass spectrometry method and one liquid repeatability, within-lab reproducibility, and accuracy show chromatography–time-of-flight mass spectrometry method. that the method can be used for quantitative screening. If In this work a quantitative screening method using high- qualitative screening is sufficient the instrumental analysis may resolution liquid chromatography in combination with be limited to positive ionisation, because all analytes including accurate-mass time-of-flight mass spectrometry was devel- the thiazides can be detected at the respective minimum oped and validated for determination of glucocorticosteroids, required levels in the positive mode. The results show that the β2-agonists, thiazide diuretics, and narcotics and stimulants in application of accurate-mass time-of-flight mass spectrometry urine. To enable the simultaneous isolation of all the in combination with generic extraction and purification compounds of interest and the necessary purification of the procedures is suitable for unification and expansion of the resulting extracts, a generic extraction and hydrolysis proce- window of screening methods of doping laboratories. More- dure was combined with a solid-phase extraction modified for over, the full-scan accurate-mass data sets obtained still allow these groups of compounds. All 56 compounds are determined retrospective examination for emerging doping agents, without re-analyzing the samples. R. J. B. Peters (*) : J. E. Oosterink : A. A. M. Stolker : Keywords Doping control Validation M. W. F. Nielen High-resolution liquid chromatography . RIKILT- Institute of Food Safety, Wageningen UR, Accurate-mass time-of-flight mass spectrometry. Akkermaalsbos 2, P.O. Box 230, 6700 AE Wageningen, Quantitative screening . Retrospective data analysis The Netherlands e-mail: [email protected] C. Georgakopoulos Introduction Doping Control Laboratory of Athens, Olympic Athletic Center of Athens, Kifissias 37, The task of doping control laboratories is to screen for a wide 15123 Maroussi, Athens, Greece range of drugs currently included in the list of prohibited substances published by the World Anti-Doping Agency M. W. F. Nielen (WADA) [1]. Today, the cost-effectiveness of analytical Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, procedures is becoming an important issue for all laborato- 6703 HB Wageningen, The Netherlands ries involved in doping control or residue analysis. A way to 2584 R.J.B. Peters et al. improve cost-effectiveness is to maximize the number of analysis of these compounds [17, 18]. Most corticosteroids analytes that may be determined in a single procedure, e.g. to are non-volatile, therefore excluding GC–MS analysis of use multi-compound techniques. However, most methods are urine samples unless lengthy derivatization or oxidation developed for the determination of specific prohibited com- procedures are used prior to GC–MS analysis. Generally, pounds or compound groups. Because resources are needed to LC–MS–MS with different ionization techniques has been determine new classes of doping compounds such as banned used [19–21]. In addition LC–TOFMS has been used for proteins, unification of doping control screening procedures toxicological drug screening of related compounds [22]and for prohibited small molecule substances—including stimu- specifically for doping agents in human urine [23]. The lants, narcotics, steroids, β2-agonists, and diuretics—is highly minimum required performance limit (MRPL) established urgent. Conceptually this may be achieved by the use of a for these compounds, at which all laboratories should be able combination of one gas chromatography–time-of-flight mass to perform [24], is 30 ng mL−1 in urine. The structures and spectrometry (GC–TOFMS) method and one liquid chroma- names of the 19 corticosteroids involved in this study are given tography time-of-flight mass spectrometry (LC–TOFMS) in Fig. 1. method. The aim of this study is to develop and validate a β2-Agonists act on the β2 adrenergic receptor, causing multi-compound LC–TOFMS method for compounds that are muscle relaxation resulting in a widening of the bronchial less suitable for GC–MS methods. Traditionally, capillary gas passages and blood vessels in the muscles, thereby chromatography combined with mass spectrometry (GC–MS) improving the performance of the athletes [25]. β2- has been used in doping analysis including laborious sample Agonists can be divided into short-acting and long-acting preparation for analytes or metabolites with a polar, non- groups with salbutamol and terbutaline as examples of the volatile, or thermolabile nature [2]. As an alternative, liquid first group and salmeterol and clenbuterol as examples of chromatography combined with mass spectrometry (LC–MS) the latter group. β2-Agonists, for example salbutamol, have may be used for those targeted molecules in urine that cannot become a concern in sports, because these drugs, when be covered by standard GC–MS methods. While LC used at high doses, can act as anabolic agents to promote combined with tandem mass spectrometry (LC–MS–MS) weight, mainly in the form of muscle [26]. As of 2006, has excellent sensitivity and selectivity for target analytes in WADA permits the use of selected β2-agonists (salbutamol, doping analysis [3–6], true multi-compound analyses salmeterol, terbutaline, and eformoterol) in athletic competi- requires a sensitive full-scan MS technique, for example tion only by asthmatic athletics. For salbutamol the threshold time-of-flight mass spectrometry (TOFMS) or Orbitrap mass value is 1000 ng mL−1 while for all other β2-agonists the spectrometry [7–9]. These analyzers provide high specificity MRPL is 100 ng mL−1. β2-Agonists in urine have been because of both high mass accuracy and high mass analysed using LC–MS–MS [27]andLC–TOFMS [23], but resolution and allow the reconstruction of highly selective also LC combined with Orbitrap mass spectrometry [28], in accurate mass chromatograms for a theoretically unlimited all cases after hydrolysis and liquid–liquid extraction of the number of compounds in complex matrices. Furthermore, sample. The structures and names of the 22 β2-agonists data can be acquired and reprocessed without any a priori involved in this validation study are given in Fig. 2. knowledge about the presence of specific compounds; that Diuretics increase the urine flow, thereby reducing body is, no analyte-specific information is required before injecting mass and potentially leading to the athlete’s classification in a sample and the presence of newly identified compounds can a lower weight class in some sports. Thiazide diuretics are be confirmed in previously analysed samples simply by also regarded as masking agents, because the increased reprocessing the data. The advantage of TOFMS can be urine flow and volume dilute possible residues of sports further improved by combining it with high-resolution LC doping and for these reasons all diuretics appear on the (HRLC) such as ultra performance LC (UPLC). Recent prohibited list, both in and out of competition with an research has shown that UPLC–TOFMS has significant MRPL of 250 ng mL−1. Diuretics have been determined as advantages concerning selectivity, sensitivity, and speed their methyl derivatives by GC–MS [29, 30] and by liquid [10–14]. This paper describes the development and valida- chromatography combined with tandem mass spectrometry tion of a quantitative screening method based on UPLC– (LC–MS–MS) [31–33]. The latter option does not require TOFMS for analysis of 56 restricted substances that are not derivatization and enables simplified sample preparation very suitable for detection with GC–MS methods, including because of to the compatibility of aqueous samples with the corticosteroids, β2-agonists, and diuretics. Corticosteroids analytical system. Figure 3 gives the structures and names affect the nervous system causing euphoria, alleviate pain, of the thiazide diuretics involved in this study. Narcotics and enhance the athlete’s ability to concentrate in endurance and stimulants can be misused in sports and