Search

To free analytical resources for new classes of doping substances, such as banned proteins, maximization of the number of compounds that can be determined with high sensitivity in a single run is highly urgent. This study demonstrates an application of 'wrong-way-round ionization' for the simultaneous detection of multiple classes of doping substances without the need to switch the polarity. A screening method for the detection of 137 compounds from various classes of prohibited substances (stimulants, diuretics, β(2)-agonists, β-blockers, antiestrogens, glucocorticosteroids and anabolic agents) has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography/orbitrap mass spectrometry with wrong-way-round ionization. Up to 64% of compounds had a 10-fold lower limit of detection (LOD) than the minimum required performance limit. To compare the efficiency of conventional ionization relative to wrong-way-round ionization of doping substances in + ESI, a fortified blank urine sample at the minimum required performance limit was analyzed using two ESI approaches. All compounds were detected with markedly better S/N in a high-pH mobile phase, with the exception of acetazolamide (minimal change in S/N, < 20%).The method was validated by spiking 10 different blank urine samples at five different concentrations. Validation parameters included the LOD, selectivity, ion suppression, extraction recovery and repeatability., (Copyright © 2012 John Wiley & Sons, Ltd.)

Drug testing for sports doping control programs is extensive and includes numerous classes of banned compounds including anabolic androgenic steroids, β2-agonists, hormone antagonists and modulators, diuretics, various peptide hormones, and growth factors. During competition, additional compounds may also be prohibited such as stimulants, narcotics, cannabinoids, glucocorticosteroids, and beta-blockers depending both on the sport and level of competition. Each of these classes of compounds can contain many prohibited substances that must be identified during the testing procedure. Various methods that have been designed to detect a large number of compounds in different drug classes are highly desirable as initial screening tools. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used by anti-doping testing laboratories for this purpose and several rapid methods have been described to simultaneously detect different classes of compounds. Here, we describe a simple urine sample cleanup procedure that can be used to detect numerous anabolic androgenic steroids, β2-agonists, hormone antagonists and modulators, glucocorticosteroids, and beta-blockers by LC-MS/MS.

This paper presents a capillary electrophoresis method, developed for the detection, in human urine, of beta-adrenergic agents and phenolalkylamines. The electrophoretic separation is achieved in less than 10 min and is based on the use of CEofix kit, for the dynamic capillary coating. The effects of accelerator buffer pH and separation voltage were investigated. The optimum buffer pH was found to be 2.5 for beta2-agonists and 6.2 for beta-blockers and phenoalkylamines with a separation voltage of 15 kV. Urine samples spiked with the compounds here studied were treated according to the standard procedure (SPE and evaporation to dryness) and analyzed by CE interfaced with an UV diode-array, set at 195 and 210 nm. The quantitative validation results, obtained analyzing samples at three different concentrations, show a good precision of peak areas that do not exceed 5% for intra-day assays and 10% for inter-day assays. Good linearity (r(2) > 0.995) was obtained within the 50-500 ng/mL concentration range. The qualitative validation data show a relative migration times (MTs) variation lower than 1%. The analytes were clearly distinguishable in urine, with LOD and LOQ in the range of 10-80 and 40-100 ng/mL, respectively.

A previously described method for the screening of 18 diuretics and probenecid was substantially extended with 21 beta-blockers and 8 other diuretics allowing simultaneous determination of diuretics and beta-adrenergic blocking agents in human urine. Analysis was performed using an ion trap instrument with an electrospray ionisation (ESI) interface after liquid/liquid extraction with ethyl acetate. Full-scan MS and full-scan MS2 were applied in combination with scan-to-scan polarity switching. All compounds were separated in less than 22 min. The detection limits for the diuretics were between 5 and 100 ng/mL and for the beta-adrenergic blocking agents were between 5 and 500 ng/mL. The excretion of carvedilol was followed after intake of one tablet of Dimitone. Other doping agents including strychnine, norbuprenorphine and mesocarb hydroxysulfate could also be detected with this method.

We describe a simple and selective method for analysing propranolol and a sensitive test for its control in urine. A flow-through fluorescence optosensor based on on-line immobilization in a non-ionic-exchanger (Amberlite XAD-7) solid support in a continuous flow was used in both cases. Determination was made in 5 mM H(2)PO(4)(-)/HPO(4)(2-) buffer solution at pH 6 at a working temperature of 20 degrees C. Fluorescence intensities were measured at lambda(ex/em) = 300/338 nm with a response time of 80 s, thus obtaining a linear concentration range of between 0 and 250.0 ng ml(-1) with a detection limit of 1.3 ng ml(-1), an analytical sensitivity of 6.0 ng ml(-1) and a standard deviation of 2.40% at a 150 ng ml(-1) concentration level for propranolol. We also propose a test to detect propranolol in urine with a linear concentration range between 0 and 100.0 ng ml(-1), a detection limit of 0.2 ng ml(-1), an analytical sensitivity of 1.0 ng ml(-1), and a standard deviation of 0.84% at a 75 ng ml(-1) concentration level. The effect of proteins presents in urine samples were evaluated. The two proposed methods were satisfactorily applied to commercial formulations and urine samples respectively., (Copyright 2003 Elsevier Science B.V.)

We have developed a general method for the detection of beta-blockers and/or of their metabolites in human urine. The method comprises a pretreatment procedure (enzymatic hydrolysis, liquid/liquid extraction and derivatization by pentafluoropropionic anhydride, PFPA), carried out on an initial aliquot of 2.5-5.0 ml of urine, and the instrumental analysis of the derivatives, performed by GC-MS-MS (ion trap) with electronic impact ionization (EI). The GC-MS-MS analysis allows to isolate and to characterize specific fragments of the original molecular structure, and particularly the fragments originating from parent ion clusters specific for all beta blocking drugs, giving rise to m/z = 366 and 202 ions respectively. MS-MS analysis of the parent ion allows checking for the presence of the above-mentioned peaks in the GC-MS chromatogram. The proposed method is capable of detecting a great variety of known (and possibly also of newly synthesized) beta-blockers, with an average sensitivity limit of 20 ng/ml of drug/metabolite in urine. The method is presently being evaluated as a general screening protocol to be followed by an antidoping laboratory to detect illicit beta-blockers administration to the athletes.

More than 25 years of developing doping control methods have led to comprehensive screening and confirmation procedures for stimulants, narcotics and beta-blockers. Much of this work has been initiated and/or improved by the late Prof. Dr. Manfred Donike. The methodological approach covered in this overview was applied to doping control procedures during the XXV Summer Olympics in Barcelona, Spain, in 1992 and the XVII Winter Olympics in Lillehammer, Norway, in 1994. Urine samples are screened through a combination of two analytical methods that are complementary: (a) gas chromatographic analysis of the parent compound and unconjugated metabolites, following single-step sample extraction and detection by a nitrogen-specific detector based on a retention index identification system and (b) gas chromatographic analysis including also conjugated drugs and metabolites after hydrolysis, solid-phase extraction, derivatisation and mass spectrometric detection. Confirmation and identification is always performed by gas chromatographic separation and full scan mass spectrometric detection. These methods facilitate the rapid screening and confirmation of more than 100 stimulants, narcotic analgesics and beta-blockers in urine for at least 24 h after the intake of a pharmaceutical dose. Application of the methods ensures high quality standards for the unequivocal identification of doping agents as well as a rapid turnaround time for sample analyses.

A comprehensive drug testing program was carried out during the 16 days of the 1988 Olympic Winter Games in Calgary, Canada. State-of-the-art technology was applied, involving high-resolution gas chromatography, high-performance liquid chromatography, gas chromatography/mass spectrometry, fluorescence polarization immunoassay, and radioimmunoassay. Samples from selected athletes were screened for five drug classes: stimulants, narcotic analgesics, anabolic steroids, beta-blockers, and diuretics. In addition, samples were also screened for local anesthetics, corticosteroids, beta-human choriogonadotropin, and cannabinoids. During the 16-day event, 428 urine samples were processed and 3090 screening procedures performed. We describe the methods for analysis at the Olympic Games and present the results.

An analytical procedure for determination of phenolalkylamines, narcotic analgesics, and beta-blockers in urine by gas chromatography/mass spectrometry (GC/MS) is described. The detection of phenolalkylamines, narcotic analgesics, and beta-blockers is based on acid hydrolysis, liquid-liquid extraction, and selective derivatization. For screening of phenolalkylamines the m/e 179 and 267 ions were monitored by GC/MS. With narcotic analgesics, the extracted ion corresponded to the molecular ion (M+) of the drug and two additional characteristic ions. Beta-blockers were analyzed as the selectively derivatized forms of the parent molecule and its metabolites by GC/MS with selected ion monitoring. The ions monitored for screening of beta-blockers containing an isopropylamine group were m/e 284 and 129 ions. The ion at m/e 86 was monitored to characterize the tert-butylamine group of beta-blockers.

The equipment, methods, logistics, and results of doping-control analyses for the 1984 Los Angeles Olympic Games are discussed in this article. Within 15 days, 1510 different urine specimens underwent 9440 screening analyses by a combination of gas chromatography, gas chromatography-mass spectrometry, "high-performance" liquid chromatography, and radioimmunoassay. These tests covered more than 200 different drugs and metabolites, including psychomotor stimulants, sympathomimetic amines, central nervous system stimulants, narcotic analgesics, and anabolic steroids. The results are summarized by class of drug. Less than 2% of the samples were found to contain a banned drug.