Identification and synthesis of (Z)-3'-hydroxy clomiphene as a new potential doping-relevant metabolite of clomiphene.

A recent study addressed the possibility of unintentional ingestion of clomiphene through residues in chicken eggs. The method developed here helped distinguish between microdose intake of (E/Z)-clomiphene citrate and consumption of clomiphene-containing eggs by the urinary pattern of four mono-hydroxylated clomiphene metabolites. However, reanalyses of doping-control samples, which showed an adverse analytical finding for clomiphene, revealed a hydroxy clomiphene (HC) isomer that was not found after microdose intake or after consumption of clomiphene-containing eggs and could not be assigned to any of the available reference compounds. The aim of the present follow-up study was to identify this HC isomer and to characterize this metabolite with respect to its potential properties as long-term metabolite in doping controls. METHODS: (E/Z)-3'-HC and (E/Z)-4'-HC were synthesized involving the McMurry reaction. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8 months. Thus, isomer-specific HC elimination profiles could be monitored. RESULTS: The metabolite previously found in doping-control samples was identified as (Z)-3'-HC. The elimination profiles of the different HCs confirmed previous results, with (Z)-3-HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)-3'-HC is excreted at higher relative concentrations only several weeks after drug intake. CONCLUSION: These findings might be of particular importance in sport drug testing as they can assist in the decision-making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.

Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing.

Higenamine is prohibited in sports as a ß2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

Human In Vivo Metabolism and Elimination Behavior of Micro-Dosed Selective Androgen Receptor Modulator RAD140 for Doping Control Purposes.

RAD140 is a selective androgen receptor modulator which has been abused in sporting competitions. Its use is prohibited by the World Anti-Doping Agency (WADA) for athletes at all times. In addition to its illicit use, adverse analytical findings of RAD140 in doping control samples might result from other scenarios, e.g., the ingestion of contaminated dietary supplements. The differentiation between samples resulting from such contamination scenarios and intentional doping presents a considerable challenge, as little is known about the metabolism and elimination behavior of RAD140 in humans. In this study, six micro-dose excretion studies with five adult male volunteers each were conducted, and urine samples were analyzed by means of LC-HRMS/MS. Multiple metabolites, firstly detected in human urine, are described in this study. The sample preparation included an enzymatic hydrolysis step, which facilitated the estimation of RAD140 concentrations in urine. The elimination profiles and detection times for six metabolites as well as the intact drug are presented. The method was extensively characterized and deemed fit-for-purpose. The metabolite ratios were investigated for their predictive power in estimating the dose of RAD140 intake. The presented data will aid in better case result management in future doping cases involving RAD140.

Determination and enantioselective separation of zilpaterol in human urine after mimicking consumption of contaminated meat using high-performance liquid chromatography with tandem mass spectrometry techniques.

RATIONALE: The synthetic ß-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers. METHODS: To provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 µg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5 µg or 3 µg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method and a newly developed chiral high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method. RESULTS: Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5-12.5 h after ingestion, and maximum levels >5 ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3 µg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time. CONCLUSION: This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method was successfully developed and applied for the separation of the zilpaterol enantiomers with mass spectrometric detection.

Assessing human urinary clomiphene metabolites after consumption of eggs from clomiphene-treated laying hens using chromatographic-mass spectrometric approaches.

The anti-estrogen clomiphene is prohibited in sports at all times. Yet, adverse analytical findings (AAFs) have increased since 2011. This is possibly due to improved analytical sensitivity, but also contamination of food of animal origin needs to be taken into consideration as a potential source of drug exposure. For instance, studies with laying hens that received orally administered clomiphene have shown a significantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene. In order to evaluate if the consumption of clomiphene-contaminated eggs can cause an AAF of a doping control sample, eggs obtained from an animal administration study with clomiphene were consumed by human volunteers. Each volunteer ate two eggs, and urine samples were collected and analyzed using routine doping control procedures. Subsequently, additional volunteers received a microdosed clomiphene capsule to compare the excretion profiles. Maximum urinary concentrations of hydroxy-clomiphene (HC) between 80 and 300 pg mL-1 were detected following the consumption of clomiphene-containing eggs, which would constitute AAFs if observed in athletes' doping control samples. In order to support the differentiation of potential routes of drug exposure, a method was developed which allows for the chromatographic separation of (E)-3-, (Z)-3-, (E)-4-, and (Z)-4-HC using a derivatization step. By comparing the peak areas of these metabolites, characteristic relative distribution patterns were found that assist in identifying AAFs resulting from clomiphene ingested via contaminated eggs and, thus, enable to distinguish clomiphene intake via contaminated eggs from the intake of microdoses or therapeutic dosages, e.g. for doping purposes.

Textile Electrodes: Influence of Electrode Construction and Pressure on Stimulation Performance in Neuromuscular Electrical Stimulation (NMES).

The major reason for preventable hospital death is venous thromboembolism (VTE). Non-pharmacological treatment options include electrical stimulation or compression therapy to improve blood flow in the extremities. Textile electrodes offer potential to replace bulky devices commonly used in this field, thereby improving the user compliance. In this work, the performance of dry and wet knitted electrodes in combination with pressure application to the electrode was evaluated in neuromuscular electrical stimulation (NMES). A motor point stimulation on the calf was performed on nine healthy subjects to induce a plantarflexion and the required stimulation intensity as well as the perceived pain were assessed. The performance of the different electrode constructions was compared and the influence of the pressure application was analysed. The results show that wet textile electrodes (0.9 % saline solution) perform significantly better than dry electrodes. However, opportunities were found for improving the performance of dry textile electrodes by using an uneven surface topography in combination with an intermediate to high pressure application to the electrode (> 20 mmHg), e.g. by using a compression stocking. Moreover, the smaller of the two tested electrode areas (16 cm2; 32 cm2) appears to be favourable in terms of stimulation comfort and efficiency.

Textile Electrodes: Influence of Knitting Construction and Pressure on the Contact Impedance.

Textile electrodes, also called textrodes, for biosignal monitoring as well as electrostimulation are central for the emerging research field of smart textiles. However, so far, only the general suitability of textrodes for those areas was investigated, while the influencing parameters on the contact impedance related to the electrode construction and external factors remain rather unknown. Therefore, in this work, six different knitted electrodes, applied both wet and dry, were compared regarding the influence of specific knitting construction parameters on the three-electrode contact impedance measured on a human forearm. Additionally, the influence of applying pressure was investigated in a two-electrode setup using a water-based agar dummy. Further, simulation of an equivalent circuit was used for quantitative evaluation. Indications were found that the preferred electrode construction to achieve the lowest contact impedance includes a square shaped electrode, knitted with a high yarn density and, in the case of dry electrodes, an uneven surface topography consisting of loops, while in wet condition a smooth surface is favorable. Wet electrodes are showing a greatly reduced contact impedance and are therefore to be preferred over dry ones; however, opportunities are seen for improving the electrode performance of dry electrodes by applying pressure to the system, thereby avoiding disadvantages of wet electrodes with fluid administration, drying-out of the electrolyte, and discomfort arising from a "wet feeling".