Identification and profiling of targeted oxidized linoleic acid metabolites in rat plasma by quadrupole time‐of‐flight mass spectrometry

Linoleic acid (LA) and LA‐esters are the precursors of LA hydroperoxides, which are readily converted to 9‐ and 13‐hydroxy‐?octadecadienoic acid (HODE) and 9‐ and 13‐oxo‐?octadecadienoic acid (oxo ODE) metabolites in vivo. These four oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. Here we present a novel quadrupole time‐of‐flight mass spectrometry (Q‐TOFMS) method for quantitation and identification of target OXLAMs in rat plasma. In this method, the esterified OXLAMs were base‐hydrolyzed and followed by liquid–liquid extraction. Quantitative analyses were based on one‐point standard addition with isotope dilution. The Q‐TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted‐ion chromatograms were generated post‐acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7–35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9‐HODE, 13‐HODE, 9‐oxoODE and 13‐oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9‐HODE, 13‐HODE, 9‐oxoODE and 13‐oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one‐point standard addition. Target metabolites were simultaneously characterized based on the accurate Q‐TOFMS data. This is the first study of secondary LA metabolites using Q‐TOFMS. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Differentiation of 18O‐ and 16O‐rich layers in anodic alumina films by glow discharge time‐of‐flight mass spectroscopy

The performance of glow discharge time‐of‐flight mass spectrometry in isotopic differentiation is revealed using the distribution of oxygen isotopes ¹⁶O and ¹⁸O in barrier‐type anodic alumina films as a focus. Anodic alumina films comprising ¹⁸O‐rich layers of controlled thickness were formed by the appropriate combination of anodising of superpure aluminium in electrolytes enriched with ¹⁸O isotopes and of natural abundance of ¹⁸O isotopes. Analysis of the elemental depth profiles of selected ionic species, i.e. ¹⁶O¹⁸O, allowed determination of the locations of the ¹⁸O‐rich layers and the ¹⁸O/¹⁶O interface. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Improved detection sensitivity of elements in solids via laser postionization in laser desorption time‐of‐flight mass spectrometry

A newly constructed laser desorption (532 nm, 5 ns) and laser postionization (266 nm, 5 ns) time‐of‐flight mass spectrometer (LD‐LPI‐TOFMS) has been applied for improving the detection sensitivity of elements in solid samples. This method affords to acquire the information of the elemental impurities in solid standards as well as limit of detection (LOD) down to 10⁻⁸ g/g for some elements. Neutral atoms of solids are generated by low‐irradiance laser desorption (< 10⁸ W/cm²), followed by high‐irradiance laser postionization (~ 10⁹ W/cm²) of the desorbed atoms, facilitating to decouple the desorption and ionization processes in spatial and temporal domain. This non‐interacting feature overcomes the discrimination between deteriorating spectral resolution at high irradiance (10⁹–10¹¹ W/cm²) and limited detectable elemental species and high LOD at low or medium irradiance (below 10⁹ W/cm²). The utilization of originally “wasted” neutral atoms by laser postionization will help improve atom utilization and instrumental sensitivity. In this work, getting the utmost out of the consumed neutral atoms instead of an increment in sampling amounts is given attention with high priority for achieving high sensitivity and low LOD, which is especially useful on the occasions where very low sample consumption is desired.     

Modern MALDI time‐of‐flight mass spectrometry

This paper focuses on development of time‐of‐flight (TOF) mass spectrometry in response to the invention of matrix‐assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI‐TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high‐performance MALDI‐TOF and TOF‐TOF with off‐line high‐capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC‐MS and MS‐MS. Copyright © 2009 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.     

Metabolic profiling of quercetin in rats using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Quercetin, a kind of major flavonoid found in many traditional chinese medicines, is an effective substance for treatments such as lowering blood lipids. However, the studies on quercetin have been mainly focused on its pharmacological effect; the treatment of diseases on a material basis, particularly the metabolites derived from quercetin in vivo , has not been evaluated. In this study, we determined the levels, distributions and types of quercetin's metabolites in plasma, urine, feces and bile of rats after a single oral administration of quercetin at a dose of 80 mg/kg, using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS). A total of 36 metabolites of quercetin were identified, including 11 metabolites in plasma, 34 metabolites in urine, 12 metabolites in feces and 21 metabolites in bile. The results showed that phase I metabolites were reduction metabolites and phase II metabolites mainly included glucuronidation, sulfation and methylation metabolites. These results provide important information on the metabolism of quercetin, which will be helpful for its further development and utilization.     

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.     

Application of gas and liquid chromatography coupled to time‐of‐flight mass spectrometry in pesticides: Multiresidue analysis

Analysis of pesticide residues in water and food matrices is an active research area closely related to food safety and environmental issues. In this aspect mass spectrometry (MS) coupled to gas chromatography (GC) and liquid chromatography (LC) has been increasingly used in the analysis of pesticide residues in water and food. The increasing interest in application of high‐resolution mass spectrometry with time‐of‐flight (TOF) and hybrid triple quadrupole TOF in pesticide analysis is due to its capability of performing both targeted and nontargeted analysis. This article discusses an overview of the application of GC‐TOF‐MS and LC‐TOF‐MS in water and food matrices.     

Laser ablation of AgSbS2 and cluster analysis by time‐of‐flight mass spectrometry

Thin films of AgSbS₂ are important for phase‐change memory applications. This solid is deposited by various techniques, such as metal organic chemical vapour deposition or laser ablation deposition, and the structure of AgSbS₂(s), as either amorphous or crystalline, is already well characterized. The pulsed laser ablation deposition (PLD) of solid AgSbS₂ is also used as a manufacturing process. However, the processes in plasma have not been well studied. We have studied the laser ablation of synthesized AgSbS₂(s) using a nitrogen laser of 337 nm and the clusters formed in the laser plume were identified. The ablation leads to the formation of various single charged ternary Ag_pSb_qS_r clusters. Negatively charged AgSbS, AgSb₂S, AgSb₂S, AgSb₂S and positively charged ternary AgSbS⁺, AgSb₂S⁺, AgSb₂S, AgSb₂S clusters were identified. The formation of several singly charged Ag⁺, Ag, Ag, Sb, Sb, S ions and binary Ag_pS_r clusters such as AgSb, Ag₃S^?, SbS (r = 1–5), Sb₂S^?, Sb₂S, Sb₃S (r = 1–4) and AgS, SbS⁺, SbS, Sb₂S⁺, Sb₂S, Sb₃S (r = 1–4), AgSb was also observed. The stoichiometry of the clusters was determined via isotopic envelope analysis and computer modeling. The relation of the composition of the clusters to the crystal structure of AgSbS₂ is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Quadrupole‐time‐of‐flight mass spectrometry screening for synthetic cannabinoids in herbal blends

‘Legal highs’ are novel substances which are intended to elicit a psychoactive response. They are sold from ‘head shops’, the internet and from street suppliers and may be possessed without legal restriction. Several months ago, a 19‐year‐old woman came searching for medical treatment as she had health problems caused by smoking legal highs. The substances were sold as herbal blends in plastic bags under four different labels. In this work, samples of these herbal blends have been analysed to investigate the presence of psychoactive substances without any reference standard being available at the laboratory. A screening strategy for a large number of synthetic and natural cannabinoids has been applied based on the use of ultra‐high pressure liquid chromatography coupled to quadrupole‐time of flight mass spectrometry (UHPLC‐QTOF MS) under MS^E mode. A customized home‐made database containing literature‐based exact masses for parent and product ions of around 200 synthetic and natural cannabinoids was compiled. The presence of the (de)protonated molecule measured at its accurate mass was evaluated in the samples. When a peak was detected, collision‐induced dissociation fragments and characteristic isotopic ions were also evaluated and used for tentative identification. After this tentative identification, four synthetic cannabinoids (JWH‐081, JWH‐250, JWH‐203 and JWH‐019) were unequivocally confirmed by subsequent acquisition of reference standards. The presence in the herbal blends of these synthetic cannabinoids might explain the psychotic and catatonic symptoms observed in the patient, as JWH compounds could act as potent agonists of CB₁ and CB₂ receptors located in the Limbic System and Basal ganglia of the human brain. Copyright © 2013 John Wiley & Sons, Ltd.     

Fully automatic and precise data analysis developed for time‐of‐flight mass spectrometry

Scientific objectives of current and future space missions are focused on the investigation of the origin and evolution of the solar system with the particular emphasis on habitability and signatures of past and present life. For in situ measurements of the chemical composition of solid samples on planetary surfaces, the neutral atmospheric gas and the thermal plasma of planetary atmospheres, the application of mass spectrometers making use of time‐of‐flight mass analysers is a technique widely used. However, such investigations imply measurements with good statistics and, thus, a large amount of data to be analysed. Therefore, faster and especially robust automated data analysis with enhanced accuracy is required. In this contribution, an automatic data analysis software, which allows fast and precise quantitative data analysis of time‐of‐flight mass spectrometric data, is presented and discussed in detail. A crucial part of this software is a robust and fast peak finding algorithm with a consecutive numerical integration method allowing precise data analysis. We tested our analysis software with data from different time‐of‐flight mass spectrometers and different measurement campaigns thereof. The quantitative analysis of isotopes, using automatic data analysis, yields results with an accuracy of isotope ratios up to 100 ppm for a signal‐to‐noise ratio (SNR) of 10⁴. We show that the accuracy of isotope ratios is in fact proportional to SNR⁻¹. Furthermore, we observe that the accuracy of isotope ratios is inversely proportional to the mass resolution. Additionally, we show that the accuracy of isotope ratios is depending on the sample width T _s by T _s^0.5. Copyright © 2017 John Wiley & Sons, Ltd.     

Detecting changes in arabidopsis cell wall composition using time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was previously used to characterize lignocellulosic materials, including woody biomass. ToF‐SIMS can acquire both rapid spectral and spatial information about a sample's surface composition. In the present study, ToF‐SIMS was used to characterize the cell walls of stem tissue from the plant model organism, Arabidopsis thaliana. Using principal component analyses, ToF‐SIMS spectra from A. thaliana wild‐type (Col‐0), cellulose mutant (irx3), and lignin mutant (fah1) stem tissues were distinguished using ToF‐SIMS peaks annotated for wood‐derived lignocellulose, where spectra from the irx3 and fah1 were characterized by comparatively low polysaccharide and syringyl lignin content, respectively. Spatial analyses using ToF‐SIMS imaging furthermore differentiated interfascicular fiber and xylem vessels based on differences in the lignin content of corresponding cell walls. These new data support the applicability of ToF‐SIMS peak annotations based on woody biomass for herbaceous plants, including model plant systems like arabidopsis. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H]⁺ as precursor ion in full‐scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N‐dimethyl, N‐desmethyl and N,N‐didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono‐ and di‐hydroxylation, methoxylation, N‐desmethylation, N,N‐didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of multiple pesticide residues in teas by gas chromatography with accurate time‐of‐flight mass spectrometry

In this work, gas chromatography tandem with electron ionization and full‐scan high‐resolution mass spectrometry with a time‐of‐flight mass analyzer was evaluated for analyzing pesticide residues in teas. The relevant aspects for mass spectrometry analysis, including the resolution and mass accuracy, acquisition rate, temperature of ion source, were investigated. Under acquisition condition in 2‐GHz extended dynamic range mode, accurate mass spectral library including 184 gas chromatography detectable pesticides was established and retrieval parameters were optimized. The mass spectra were consistent over a wide concentration range (three orders) with good match values to those of NIST (EI‐quadrupole). The methodology was verified by the validation of 184 pesticides in four tea matrices. A wide linear range (1–1000 μg/kg) was obtained for most compounds in four matrices. Limit of detection, limit of quantification, and limit of identification values acquired in this study could satisfy the requirements for maximum residue levels prescribed by the European Community. Recovery studies were performed at three concentrations (10, 50, and 100 μg/kg). Most of the analytes were recovered at an acceptable range of 70–120% with relative standard deviations ≤ 20% in four matrices. The potential extension of qualitative screening scope makes gas chromatography tandem with electron ionization and mass spectrometry with a time‐of‐flight mass analyzer a more powerful tool compared with gas chromatography with tandem mass spectrometry.     

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.     

Cationization of dendritic macromolecule adsorbates on metals studied by time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was utilized to study dendritic macromolecules with various architectures, such as dendrons, dendrimers and hyperbranched polyesters prepared from bis‐(hydroxymethyl)propionic acid (Bis‐MPA) and a series of hyperbranched polyethers based on 3‐ethyl‐3(hydroxymethyl)oxetane. The measurements were performed on spin‐coated thin films of the branched molecules (D) onto silicon, chemically etched copper foil and silver‐coated wafers. They showed weak signatures of molecular ions by proton capture (D + H)⁺ in the high mass range of the spectra (m/z > 400). On the contrary, cationization of the intact molecules with alkali or transition metal ions such as Na⁺, Cu⁺ or Ag⁺ was observed. High‐intensity quasi‐molecular ions (D + M)⁺ (with M = Na⁺, Cu⁺ or Ag⁺) allowed the studied polymers to be identified. The whole molecular species were observed for Bis‐MPA dendrons and dendrimers up to 3000 Da for hydroxyl or acetonide‐terminated derivatives. The success of the so‐called cationization experiments with metal substrates compared with analysis of molecular adsorbates on silicon is highlighted. The ToF‐SIMS sensitivity appeared useful to provide information about the molecular end‐groups or to highlight incomplete reaction occurring during some deprotection step of the synthesis. Only uncationized fragments of low masses were detected for the hyperbranched polyesters. This result suggested the effect of molecular asymmetry and/or flattening of the molecules on the substrates, which hampered the molecule lift‐off efficiency. Nevertheless, the hyperbranched polyethers were characterized based on the peak distribution of intensities, which allowed estimation of their molecular weight average. This work was intended to illustrate the capabilities of ToF‐SIMS to analyse dendritic polymers on surfaces. Copyright © 2003 John Wiley & Sons, Ltd.     

Potential of monitoring isotopologues by quantitative gas chromatography with time‐of‐flight mass spectrometry for metabolomic assay

Because of the extreme complexity of metabolomic samples, the effectiveness of quantitative gas chromatography with time‐of‐flight mass spectrometry depends substantially on the expansion of the linear dynamic range. Facing the existence of numerous saturated detector signals, a data processing method based on monitoring isotopologues has been developed. The monoisotopic ion kept the high mass spectrometry sensitivity, and the less abundant isotopologue ions extended the linear dynamic range. This alternative method was proved to extend the linear dynamic range to five orders of magnitude successfully and overcome the quantitative problems induced by the ion detector saturation. Finally, to validate the applicability, the method was applied to a metabolomic assay of Alzheimer's disease. Comparing with the traditional monoisotopic method, the use of monitoring isotopologues helped us to discover an additional eight metabolites with significant difference and to conduct a more reliable principal component analysis as well. The results demonstrated that monitoring isotopologues in quantitative gas chromatography with time‐of‐flight mass spectrometry could improve the authenticity of metabolomic analysis.     

New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry (LC‐QTOFMS) for the first time. Liquid–liquid extraction was applied to processing urine samples, and dilute‐shoot analyses of intact metabolites were also presented. In LC‐QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M–H]^? as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography–mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α‐methyl‐5α‐androst‐3‐one‐17β‐sulfate) together with S2 (1α‐methyl‐5α‐androst‐17‐one‐3β‐sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse. Copyright © 2015 John Wiley & Sons, Ltd.