Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography-high-resolution mass spectrometry.

RATIONALE: High-resolution mass spectrometry (HRMS) has been demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for the elucidation of chemical structures. This work describes a method to detect 13 esters of testosterone (T) and 5 biomarkers in 0.1 mL of human serum using gas chromatography (GC) coupled to HRMS. METHODS: Analytes were extracted from serum after deproteinization and liquid-liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full-scan mode was applied with a resolving power of 30 000 at m/z 400. Linearity, lower limit of quantitation, and measurement uncertainty were assessed. Precision and accuracy were assessed at 0.5 and 2 ng/mL, respectively. Mass accuracy (MA) and mass extraction window (MEW) were also evaluated. RESULTS: T esters showed a linear response between 0.25 and 10 ng/mL (except for undecanoate, enanthate, and propionate that showed lineal responses between 0.5 and 10 ng/mL and isocaproate between 2 and 10 ng/mL); detection limits remained between 0.1 and 0.5 ng/mL and accuracy between 81% and 119%. The MA (MEW = 10 ppm) was maintained between -2.4 and 4.8 ppm. The biomarkers (T, androstenedione, dehydroepiandrosterone [DHEA], estradiol, and 17-OH-progesterone) showed a linear response within the evaluated range; quantification limits remained between 0.1 and 0.5 ng/mL (except for DHEA), the accuracy between 88% and 99%, and precision between 3.5% and 10.8%. Measurement uncertainties were found between 5.6% and 17.2%. MA (MEW = 3 ppm) was maintained between -0.47 and 0.12 ppm. CONCLUSIONS: The method to detect T esters and five endogenous biomarkers in serum using GC coupled to HRMS showed linear responses up to 10 ng/mL with adequate precision, accuracy, and uncertainties. It was possible to distinguish cholesterol from T-isocaproate based on the MEW of 10 ppm, preventing false positives. In addition, this method allows searching for other biomarkers and/or unknown metabolites and other ester forms not included here but at a later stage if necessary.

LC-HRMS-based metabolomics workflow: An alternative strategy for metabolite identification in the antidoping field.

RATIONALE: The proposed metabolomic workflow, based on coupling high-resolution mass spectrometry with computational tools, can be an alternative strategy for metabolite detection and identification. This approach allows the extension of the investigation field to chemically different compounds, maximizing the information obtainable from the data and minimizing the time and resources required. METHODS: Urine samples were collected from five healthy volunteers before and after oral administration of 3ß-hydroxyandrost-5-ene-7,17-dione as a model compound and defining three excretion time intervals. Raw data were acquired in both positive and negative ionization modes using an Agilent Technologies 1290 Infinity II series HPLC coupled to a 6545 Accurate-Mass Quadrupole Time-of-Flight. They were then processed to align peak retention times with the same accurate mass, and the resulting data matrix was subjected to multivariate analysis. RESULTS: Multivariate analysis (PCA and PLS-DA models) demonstrated high similarity between samples belonging to the same collection time interval and clear discrimination between different excretion intervals. The blank and long excretion groups were distinguished, suggesting the presence of long excretion markers, which are of remarkable interest in anti-doping analyses. The correspondence of some significant features with metabolites reported in the literature confirmed the rationale and usefulness of the proposed metabolomic approach. CONCLUSIONS: The presented study proposes a metabolomics workflow for the early detection and characterization of drug metabolites by untargeted urinary analysis to reduce the range of substances still excluded from routine screening. Its application has detected minor steroid metabolites, as well as unexpected endogenous alterations, proving to be an alternative strategy that can allow gathering a more complete range of information in the antidoping field.

Quantification of thyroid hormones and analogs by liquid chromatography coupled to mass spectrometry. Preliminary results in athletes and non-athletes serum samples.

This paper aimed to assess a method to measure eight thyroid-related compounds in serum by liquid chromatography-mass spectrometry (LC-MS/MS), to verify the correlation with radioimmunoassay (RIA), to evaluate the possible cross-reactivity, and to observe differences between athletes declaring the consumption of sodium levothyroxine and nonathletes serum samples. Validation was carried out to assess carryover, working range and linearity, limit of detection and limit of quantification, precision, matrix influence, recovery, accuracy, and uncertainty. Comparison between RIA and LC-MS/MS results was done. The assay was applied to serum samples, and comparison with RIA was done for T3 and T4 levels supported by RIA Thyroid-stimulating hormone (TSH) measurements. Validation parameters showed satisfactory results. Correlation between RIA and LC-MS/MS for T3 and T4 showed good results, but a cross-reactivity between T3 and T3AA was observed. Although no significant differences were proved, preliminary comparison between athletes and nonathletes serum samples showed a shift towards high values of TSH and lower for T4 values in the athletes' group. Differences between thyronine and T4AA concentrations and ratios were observed. The trend of T4 values supported by TSH measures might indicate subclinical hypothyroidism in athletes. This represents one of the most controversial thyroid statuses as different criteria about its treatment are described, especially since one of the exogenous causes is inadequate levothyroxine therapy. Because the variation of thyroid hormones and TSH has been extensively studied in high-performance sports, it is worth considering the need to set an adequate reference interval to accurately assess the thyroid status in athletes.

Optimization of a method to detect levothyroxine and related compounds in serum and urine by liquid chromatography coupled to triple quadrupole mass spectrometry.

Thyroid hormones and their derivatives are structurally related to the non-essential amino acid tyrosine (4-hydroxyphenylalanine). However, there are physicochemical differences that make it difficult to apply an analytical method for their simultaneous detection. This work focused on the optimization of a method using liquid chromatography-electrospray ionization mass spectrometry to measure eight compounds related to levothyroxine (T4). In addition, the influence of the additives to the mobile phase, the solvents for liquid-liquid extraction and the influence of the hydrolysis of the conjugated analytes were studied. Optimization of MRM transitions and collision energy for analytes and capillary voltage, nebulizer gas pressure, nozzle voltage, sheath gas flow, sheath gas temperature, drying gas flow and drying gas temperature for ionization source was done. The recovery of analytes was studied using five solvents and six solvent systems to introduce them into the liquid-liquid extraction and matrix cleanup steps. Different additives to the mobile phase were evaluated as well as the effectiveness of enzymatic and chemical hydrolysis. The best MRM transitions and source parameters were settled in order to generate an optimized instrumental method. The addition of ammonium formate, ammonium fluoride, and acetic acid to the mobile phase showed no improvement in responses compared to classic 0.1% formic acid. The use of tert-butyl methyl ether: isopropanol (75: 25, V: V) showed a suitable recovery of analytes to perform a liquid-liquid extraction, and n-hexane might be an appropriate solvent if a cleanup step is needed. The stability of T3, T4 and thyronine, checked after the hydrolysis with extracts of E. coli and H. pomatia showed good results, contrary to chemical hydrolysis that showed a total degradation of T3 and T4.

Metabolomics workflow as a driven tool for rapid detection of metabolites in doping analysis. Development and validation.

RATIONALE: This work demonstrates the high potential of combining high-resolution mass spectrometry with chemometric tools, using metabolomics as a guided tool for anti-doping analysis. The administration of 7-keto-DHEA was studied as a proof-of-concept of the effectiveness of the combination of knowledge-based and machine-learning approaches to differentiate the changes due to the athletic activities from those due to the recourse to doping substances and methods. METHODS: Urine samples were collected from five healthy volunteers before and after an oral administration by identifying three time intervals. Raw data were acquired by injecting less than 1 µL of derivatized samples into a model 8890 gas chromatograph coupled to a model 7250 accurate-mass quadrupole time-of-flight analyzer (both from Agilent Technologies), by using a low-energy electron ionization source; the samples were then preprocessed to align peak retention times with the same accurate mass. The resulting data table was subjected to multivariate analysis. RESULTS: Multivariate analysis showed a high similarity between the samples belonging to the same collection interval and a clear separation between the different excretion intervals. The discrimination between blank and long excretion groups may suggest the presence of long excretion markers, which are particularly significant in anti-doping analysis. Furthermore, matching the most significant features with some of the metabolites reported in the literature data demonstrated the rationality of the proposed metabolomics-based approach. CONCLUSIONS: The application of metabolomics tools as an investigation strategy could reduce the time and resources required to identify and characterize intake markers maximizing the information that can be extracted from the data and extending the research field by avoiding a priori bias. Therefore, metabolic fingerprinting of prohibited substance intakes could be an appropriate analytical approach to reduce the risk of false-positive/negative results, aiding in the interpretation of "abnormal" profiles and discrimination of pseudo-endogenous steroid intake in the anti-doping field.

Low-energy electron ionization optimization for steroidomics analysis using high-resolution mass spectrometry.

RATIONALE: Systematic electron ionization fragmentation studies of steroids have been performed to elucidate and trace their characteristic fragmentation patterns. However, the electron ionization source setting at 70 eV electron energy is much higher than the ionization potential (7-15 eV) of most organic compounds, leading to extensive fragmentation. We present a multifactorial study on optimizing a low-energy electron ionization source to maximize molecular ion formation while minimizing the extent of fragmentation to improve the analytical sensitivity of steroids, especially the more thermolabile ones, while preserving the information that can be extracted from the data. METHODS: Twenty-seven steroid reference materials, chosen to cover four main classes of urinary steroids, were considered; gas chromatography/quadrupole time-of-flight (GC/qTOF) analyses were carried out using an Agilent Technologies model 8890 gas chromatograph coupled to an Agilent Technologies model 7250 accurate-mass quadrupole time-of-flight (GC/qTOF) instrument. The effects of electron energy, emission current, and source temperature, as well as their potential interactions on steroid fragmentation pathways, have been assessed in full factorial experimental designs. RESULTS: Three parameters were specifically evaluated to improve the chromatographic/spectrometric response of the selected steroids: (i) degree of fragmentation; (ii) relative abundance of the molecular ion; and (iii) peak width. The first two were evaluated by screening designs that highlighted collision energy and source temperature as the most influential factors on the analytical responses of the considered steroids, while emission current always showed a non-significant influence. Then, an optimization design was performed to select the final source setting by searching for the combination of factors that minimize peak tailing. CONCLUSIONS: The proposed analytical approach permits a faster selection of optimal experimental conditions for steroidomics analysis using low-energy electron ionization and high-resolution mass spectrometry. The development of these designs of experiments (DoE) in full factorial design (FFD) allowed multiple inputs to be monitored at the same time, highlighting the possible interactions and estimating the effects of a factor in the different levels of the other factors considered.

Arimistane: Degradation product or metabolite of 7-oxo-DHEA?

RATIONALE: The instability of androst-5-ene-3,7-dione structures under acidic conditions is known. The formation of arimistane from 7-oxo-DHEA, influenced by the conditions of sample extraction, and mainly derivatization reaction and gas chromatography (GC) injector temperature, was described earlier, potentially leading to misinterpretation of results. By using a liquid chromatography (LC)-mass spectrometry (MS) (LC-MS) we investigated the stability of the 7-oxo-DHEA in two different solvents (methanol and dimethyl sulfoxide [DMSO]), and the arimistane formation after the application common analytical procedures. Additionally, in vitro and in vivo studies of 7-oxo-DHEA were performed. METHODS: The stability of 7-oxo-DHEA was studied in solutions after 60 days storage at -20°C. In vitro studies were performed by incubating 7-oxo-DHEA with human liver microsomes (HLMs). Healthy volunteers collected urine samples before and after the administration of a single dose of 7-oxo-DHEA. Analyses were performed using high-performance LC (HPLC) coupled to a triple quadrupole mass spectrometer (MS/MS) and GC combustion isotope ratio mass spectrometry (GC-C-IRMS) following HPLC purification. RESULTS: 7-oxo-DHEA was stable after 60 days in DMSO while a protic solvent as methanol promotes the degradation of 7-oxo-DHEA to arimistane. HLM incubations showed no formation of arimistane and the sample preparation only influenced the degradation of 7-oxo-DHEA when solvolysis was applied. After the administration study the presence of arimistane also after the hydrolysis with ß-glucuronidase (Escherichia coli) was observed while using ß-glucuronidase/arylsulfatase (Helix pomatia) showed the presence of arimistane already in blank samples collected before administration. CONCLUSIONS: Our results confirm arimistane as a valuable diagnostic marker of 7-oxo-DHEA administration, but also indicate that its formation is due to degradation processes rather than to metabolic biotransformation reactions.

Coupling high-resolution mass spectrometry and chemometrics for the structural characterization of anabolic-androgenic steroids and the early detection of unknown designer structures.

Predictive models have been developed for the early identification of novel anabolic androgenic steroids and to obtain information on their molecular structure. To this purpose, gas-chromatographic and mass spectrometric characteristic parameters of 136 anabolic androgenic steroids have been specifically considered. Starting from Principal Component Analysis, different chemometric methods were applied, such as classification and clustering techniques, outlining a spectral and structural characterization for each steroid subclass, and considering the contribution of more than 30 variables. Mass spectrometric data on the TMS-derivatives of the target steroids were obtained by gas chromatography coupled to quadrupole-time of flight mass spectrometry using electron ionization. Steroids included in the training set were grouped in 5 subclasses according to their structural similarity, and the experimental data, processed by the chemometric models, allowed the identification of class-specific common fragments and spectral trends. The results of this study, validated on a test set of 21 steroids, have confirmed that the proposed approach allows tracing novel "designer anabolic steroids", including those previously unknown new structures that may have been designed and illicitly synthesized to be invisible to the current anti-doping tests.

Detection of urinary arimistane metabolites in humans using liquid chromatography-mass spectrometry: Complementary results to gas chromatography mass spectrometric data and its application to antidoping analyses.

RATIONALE: The metabolism of arimistane (Arim) was first described in 2015, and androst-3,5-diene-7ß-ol-17-one was proposed as the main metabolite excreted in urine. Recently, a more detailed study describing the findings in urine after the administration of Arim has been published. This study corroborated the previously described metabolite but also described several phase I and II metabolites, analyzing trimethylsilylated urinary extracts using accurate mass spectrometry coupled to gas chromatography (GC/qTOF). The present communication is an extension of this late investigation aiming to implement the results of Arim metabolism using either accurate mass spectrometry and/or triple quadrupole tandem mass spectrometry, both coupled to liquid chromatography (LC/qTOF and LC/QqQ). METHODS: The samples used in this study were the same as previously studied using GC/qTOF. One single oral dose of Arim was administered to three volunteers, and samples collected before and up to 10 h after the Arim administration were analyzed. The unconjugated fraction of urine was removed, and the hydrolysis was performed with ß-glucuronidase from Escherichia coli. The extracts were reconstituted in water:acetonitrile before the LC/qTOF and LC/QqQ analysis. RESULTS: The presence of the proposed metabolites studied using GC was verified by accurate mass measurements. Twelve metabolites not found in the blank urine samples were identified by the accurate mass spectra with acceptable errors between -7.5 and 8.1 ppm: 4 reduced metabolites, 4 monohydroxylated metabolites, and 4 with an additional hydroxylation (bis-hydroxylated metabolites). Unlike in the study carried out using GC/qTOF, Arim itself was found in the samples of the three volunteers. CONCLUSIONS: Twelve metabolites were identified, and specific transitions were proposed. Despite the good results, some limitations remain. As for GC/qTOF, the α- or ß configuration of hydroxy groups, as well as the exact position for some unsaturation, cannot be assigned with certainty. Because certified reference materials of these metabolites are not yet available, the molecular structures were hypothesized considering the previous study using GC.