Preventive doping control screening analysis of prohibited substances in human urine using rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometry

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, β₂‐agonists, β‐blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single‐step liquid‐liquid extraction of hydrolyzed urine and the use of a rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4‐methyl‐2‐hexanamine, which resulted in re‐reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives. Copyright © 2010 John Wiley & Sons, Ltd.     

Elucidation of artefacts in proton transfer reaction time‐of‐flight mass spectrometers

We present an effective procedure to differentiate instrumental artefacts, such as parasitic ions, memory effects, and real trace impurities contained in inert gases. Three different proton transfer reaction mass spectrometers were used in order to identify instrument‐specific parasitic ions. The methodology reveals new nitrogen‐ and metal‐containing ions that up to date have not been reported. The parasitic ion signal was dominated by [N₂]H⁺ and [NH₃]H⁺ rather than by the common ions NO⁺ and O₂⁺. Under dry conditions in a proton transfer reaction quadrupole interface time‐of‐flight mass spectrometer (PTR‐QiTOF), the ion abundances of [N₂]H⁺ were elevated compared with the signals in the presence of humidity. In contrast, the [NH₃]H⁺ ion did not show a clear humidity dependency. On the other hand, two PTR‐TOF1000 instruments showed no significant contribution of the [N₂]H⁺ ion, which supports the idea of [N₂]H⁺ formation in the quadrupole interface of the PTR‐QiTOF. Many new nitrogen‐containing ions were identified, and three different reaction sequences showing a similar reaction mechanism were established. Additionally, several metal‐containing ions, their oxides, and hydroxides were formed in the three PTR instruments. However, their relative ion abundancies were below 0.03% in all cases. Within the series of metal‐containing ions, the highest contribution under dry conditions was assigned to the [Fe(OH)₂]H⁺ ion. Only in one PTR‐TOF1000 the Fe⁺ ion appeared as dominant species compared with the [Fe(OH)₂]H⁺ ion. The present analysis and the resulting database can be used as a tool for the elucidation of artefacts in mass spectra and, especially in cases, where dilution with inert gases play a significant role, preventing misinterpretations.     

A small high‐irradiance laser ionization time‐of‐flight mass spectrometer

A small high‐irradiance laser ionization time‐of‐flight mass spectrometer (LI‐TOFMS) with orthogonal sample introduction was described. High irradiance of 6 × 10¹⁰ W/cm² at 532 nm from a Nd : YAG laser was applied in the experiment to get a high ionization degree in plasma and to dissociate the interferential polyatomic ions. Meanwhile, the interferential multiply charged ions resulted by high‐irradiance were nearly eliminated in the spectrum by utilizing helium as the buffer gas in the ion source due to three‐body recombination, which resulted in a relatively clean background. Improved signal stability was obtained by automated step moving of the sample stage in short time intervals. By using two sets of Einzel lens in transport system, nearly uniform relative sensitivity coefficients (RSCs) were achieved for most of metal elements including light ions which were detected in extremely low sensitivity in previous hexapole transportation instrument. The resolving power reaches 2200, and the detection limits (DLs) are 10^?6 g/g for metal elements in the steel standard. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

T‐Jump/time‐of‐flight mass spectrometry for time‐resolved analysis of energetic materials

We describe a new T‐Jump/time‐of‐flight (TOF) mass spectrometer for the time‐resolved analysis of rapid pyrolysis chemistry of solids and liquids, with a focus on energetic materials. The instrument employs a thin wire substrate which can be coated with the material of interest, and can be rapidly heated (10⁵ K/s). The T‐Jump probe is inserted within the extraction region of a linear TOF mass spectrometer, which enables multiple spectra to be obtained during a single reaction event. By monitoring the electrical characteristics of the heated wire, the temperature could also be obtained and correlated to the mass spectra. As examples, we present time‐resolved spectra for the ignition of nitrocellulose and RDX. The fidelity of the instrument is demonstrated in the spectra presented which show the temporal formation and decay of several species in both systems. The simultaneous measurement of temperature enables us to extract the ignition temperature and the characteristic reaction time. The time‐resolved mass spectra obtained show that these solid energetic material reactions, under a rapid heating rate, can occur on a time scale of milliseconds or less. While the data sampling rate of 10 000 Hz was used in the present experiments, the instrument is capable of a maximum scanning rate of up to ～30 kHz. The capability of high‐speed time‐resolved measurements offers an additional analytical tool for the characterization of the decomposition, ignition, and combustion of energetic materials. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterisation of oxazepam degradation products by high‐performance liquid chromatography/electrospray ionisation mass spectrometry and electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry

Oxazepam has been subjected to controlled degradation at 100°C for 3 h in 0.5 M HCl and 0.5 M NaOH. Following neutralisation of the degradation mixture and removal of salts by solid‐phase extraction (SPE), isocratic high‐performance liquid chromatography/mass spectrometry (HPLC/MS) using water/methanol (25:75 v/v) as the mobile phase was carried out using a flow diverter to collect fractions prior to their characterisation by electrospray ionisation multi‐stage mass spectrometry (ESI‐MSⁿ) and proposal of the corresponding fragmentation patterns. The elemental compositions of the degradation products and their MS fragments were evaluated using electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry (ESI‐QTOF‐MS/MS) which was then used to support the proposed fragmentation patterns. Copyright © 2010 John Wiley & Sons, Ltd.     

Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time‐of‐flight mass spectrometry

The use of hybrid quadrupole ion mobility spectrometry time‐of‐flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T_d) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent‐excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI‐X, MI‐Y and MI‐Z), inverse mobility and collision cross‐section (CCS). The correlation of T_d with these parameters is presented and discussed. IMS/TOF tandem mass spectrometry experiments (MS² and MS³) were successfully performed on the N‐acetyl‐p‐benzoquinoneimine glutathione (NAPQI‐GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time‐of‐flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave‐enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of isochlorogenic acid A metabolites in rats using high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Isochlorogenic acid A is widely present in fruits, vegetables and herbal medicines, and is characterized by anti‐inflammatory, hepatoprotective and antiviral properties. However, little is known about its metabolic fate and pharmacokinetic properties. This study is thus designed to investigate the metabolic fate of isochlorogenic acid A. An analytical method based on high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (HPLC/Q‐TOF MS) was established to characterize the metabolites of isochlorogenic acid A in the plasma, urine and feces of rats. A total of 32 metabolites were identified. The metabolic pathways mainly include hydrolyzation, dehydroxylation, hydrogenation and conjugation with methyl, glucuronic acid, glycine, sulfate, glutathione and cysteine. Moreover, the pharmacokinetic profiles of all the circulating metabolites were investigated. M11 resulting from hydrolyzation, dehydroxylation and hydrogenation was the dominant circulating metabolite after the intragastric administration of isochlorogenic acid A. The results obtained will be useful for further study of elucidating potential bioactive metabolites which can provide better explanation of the pharmacological and/or toxicological effects of this compound.     

Searching for in silico predicted metabolites and designer modifications of (cortico)steroids in urine by high‐resolution liquid chromatography/time‐of‐flight mass spectrometry

Glucocorticosteroids are a restricted class of substances and appear on the ‘in‐competition’ prohibited list of the World Anti‐Doping Agency (WADA). Analysis of glucocorticosteroids is complicated since they show significant phase 1 and 2 metabolism in the human body and are excreted into urine in concentrations in the µg/L range. Full scan, high‐resolution time‐of‐flight mass spectrometry analysis generates information on all ionisable components in urine, including known and unknown metabolites of steroids and even designer modifications of anabolic steroids. However, evaluation of the data obtained can be difficult and time‐consuming because of the need to differentiate between endogenous components and compounds of interest. MetaboLynx?, a spectral and chromatographic search program, was modified for the determination of in silico predicted metabolites of glucocorticosteroids and designer modifications of anabolic steroids in human urine. Spiked urine samples were successfully screened for known components in a targeted approach and for unknown species in a non‐targeted approach using data filtering to limit potential false‐positives. A simplified combined approach of targeted and untargeted screening was used for the detection of metabolites and designer modifications of existing compounds. This approach proved successful and showed its strength in the detection of tetrahydrogestrinone (THG), a designer modification of gestrinone. THG was positively detected in a spiked urine sample and correctly identified as a twofold hydrogenation of gestrinone. The developed screening method can easily be adapted to specific needs and it is envisaged that a similar approach would be amendable to the discovery of metabolites or designer modifications of other compounds of interest. Copyright © 2009 John Wiley & Sons, Ltd.