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Background: Cardiac troponin T (cTnT) is key in diagnosing myocardial infarction (MI) but is also elevated in end-stage renal disease (ESRD) patients. Specific larger cTnT proteoforms were identified for the acute phase of MI, while in serum of ESRD patients solely small cTnT fragments were found. However, others allocated this to a pre-analytic effect due to abundant thrombin generation in serum. Therefore, we investigated the effect of various anticoagulation methods on cTnT composition and concentration and compared the cTnT composition of MI and ESRD patients., Methods: The agreement of cTnT concentrations between simultaneously collected serum, lithium-heparin (LH) plasma, and ethylenediaminetetraacetic acid (EDTA) plasma was studied using the high-sensitivity (hs-)cTnT immunoassay. cTnT proteoform composition was investigated in a standardized time-dependent manner through spike experiments and in simultaneously collected blood matrixes of MI and ESRD patients., Results: Excellent hs-cTnT concentration agreements were observed across all blood matrixes (slopes > 0.98; 95% CI, 0.96-1.04). Time-dependent degradation (40 kDa intact:29 kDa fragment:15 to 18 kDa fragments) was found in LH plasma and EDTA plasma, and serum in ratios (%) of 90:10:0, 0:5:95, and 0:0:100, respectively (48 h after blood collection). Moreover, gel filtration chromatography (GFC) profiles illustrated mainly larger cTnT proteoforms in MI patients, while in ESRD patients mainly 15 to 18 kDa fragments were found for all matrices., Conclusions: The extent of cTnT degradation in vitro is dependent on the (anti)coagulation method, without impacting hs-cTnT concentrations. Furthermore, mainly larger cTnT proteoforms were present in MI patients, while in ESRD patients mainly small 15 to 18 kDa cTnT fragments were found. These insights are essential when developing a novel hs-cTnT assay targeting larger cTnT proteoforms., (© Association for Diagnostics & Laboratory Medicine 2024.)

Background and Objectives: Although physical therapy, in particular exercise therapy, is widely used in nursing home residents with dementia, the literature on this topic is relatively scarce. This systematic review aimed to summarize the literature on the characteristics and effectiveness of exercise interventions supervised by physical therapists in nursing home residents with dementia., Research Design and Methods: Six electronic databases were systematically searched for relevant studies up to August 17, 2022. Randomized controlled trials (RCTs) comparing exercise interventions supervised by a physical therapist to any other form of intervention or usual care in nursing home residents with dementia were selected. Data were narratively analyzed and forest plots visualizing exercise effects were created., Results: From the 1 377 records retrieved and screened, 6 RCTs, reported in 11 papers, met the selection criteria. Included studies used multimodal or aerobic exercise interventions, with the frequency, duration and intensity varying across studies. Three of the 6 studies were at high risk of bias. Due to inconsistency in the findings and variety in outcome measures, results on the effectiveness of the interventions are inconclusive., Discussion and Implications: Our review emphasizes the need for more robust studies to offer understanding of the efficacy of exercise interventions supervised by physical therapists for nursing home residents with dementia., Competing Interests: None., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Gerontological Society of America.)

Aims: We aimed to assess the acute and midterm efficacy of premature ventricular contraction (PVC) ablation guided by multielectrode and point-by-point (PbP) mapping., Methods and Results: This is a retrospective, international multicentre study of consecutive patients referred for PVC ablation in 10 hospital centres from January 2017 to December 2021. Based on the mapping approach, two cohorts were identified: the 'Multipolar group', where a dedicated high-density mapping catheter was employed, and the 'PbP group', where mapping was performed with the ablation catheter. Procedural endpoints, safety, and acute (procedural) and midterm efficacies were assessed. Of the 698 patients included in this study, 592 received activation mapping [46% males, median age of 55 (41-65) years]-248 patients in the Multipolar group and 344 patients in the PbP group. A higher number of activation points [432 (217-843) vs. 95 (42-185), P < 0.001], reduced mapping time (40 ± 38 vs. 61 ± 50 min, P < 0.001), and shorter procedure time (124 ± 60 vs. 143 ± 63 min, P < 0.001) were reported in the Multipolar group. Both groups had high acute success rates (84.7% with Multipolar mapping vs. 81.3% with PbP mapping, P = 0.63), as well as midterm efficacy (83.4% vs. 77.4%, P = 0.08), with no significant differences in the risk of adverse events (6.0% vs. 3.5%, P = 0.24). However, for left-sided PVC ablation specifically, there was a higher midterm efficacy in the Multipolar group (80.7% vs. 69.5%, P = 0.04), with multipolar mapping being an independent predictor of success [adjusted OR = 2.231 (95% CI, 1.476-5.108), P = 0.02]., Conclusion: The acute and midterm efficacies of PVC ablation are high with both multipolar and PbP mapping, although the former allows for quicker procedures and may potentially improve the outcomes of left-sided PVC ablation., Competing Interests: Conflict of interest P.A.S. received consulting fees from Abbott, Biosense Webster, Boston Scientific, and Medtronic. S.B. received training grants from Biosense Webster and Biotronik. N.C.-D. and J.d.S. received travel and consulting fees from Abbott, Biosense Webster Boston Scientific, and Medtronic. M.R.-M. received training grants from Biosense Webster. M.P. is an employee of Biosense Webster. N.A. received consulting fees from MicroPort and Abbott. M.O. received speaker and consulting fees from Medtronic, Biosense Webster, and MicroPort. J.S. received speaker and consultancy fees from Biosense Webster. All other authors reported that they have no relationships relevant to the contents of this paper to disclose., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Atrial fibrillation (AF) is the clinically most prevalent rhythm disorder with large impact on quality of life and increased risk for hospitalizations and mortality in both men and women. In recent years, knowledge regarding epidemiology, risk factors, and patho-physiological mechanisms of AF has greatly increased. Sex differences have been identified in the prevalence, clinical presentation, associated comorbidities, and therapy outcomes of AF. Although it is known that age-related prevalence of AF is lower in women than in men, women have worse and often atypical symptoms and worse quality of life as well as a higher risk for adverse events such as stroke and death associated with AF. In this review, we evaluate what is known about sex differences in AF mechanisms-covering structural, electrophysiological, and hormonal factors-and underscore areas of knowledge gaps for future studies. Increasing our understanding of mechanisms accounting for these sex differences in AF is important both for prognostic purposes and the optimization of (targeted, mechanism-based, and sex-specific) therapeutic approaches., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Background: The knowledge level of atrial fibrillation patients about their arrhythmia, its consequences and treatment is poor. The best strategy to provide education is unknown., Aim: To investigate the effect of reinforced targeted in-person education using the Jessa Atrial fibrillation Knowledge Questionnaire (JAKQ)., Methods: Sixty-seven atrial fibrillation patients were randomized to standard care (including brochures) or targeted education. Follow-up visits were scheduled after one, three, six and 12 months. Targeted education during each visit focused on the knowledge gaps revealed by the JAKQ. Patients completed two questionnaires to assess their quality of life and symptom profile. Adherence to non-vitamin K antagonist oral anticoagulants was measured using electronic monitoring., Results: Sixty-two patients (31 education; 31 standard care) completed follow-up. Median baseline score on the JAKQ was similar in education (62.5%) and standard care group (56.3%; p=0.815). The intervention group scored significantly better over time (one month: 75.0%, 12 months: 87.5%; p<0.001) whereas there was no significant improvement in the control group (one month: 62.5%, 12 months: 62.5%; p=0.085). Providing targeted education after completion of the JAKQ required on average 6.9±4.6 min. Some improvements in quality of life, symptom burden and adherence were shown, without significant differences between the two groups ( p-values between 0.282 and 0.677)., Conclusion: The JAKQ is an effective tool for providing individualized education. A first targeted educational session significantly improved patients' knowledge level. Additional educational sessions maintained and strengthened this effect. A larger scale study is warranted to evaluate the impact on adherence and outcome measures.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.

Background: An altered immune response against Staphylococcus aureus might contribute to inflammation and barrier damage in atopic dermatitis (AD)., Objectives: To profile IgG antibodies against 55 S. aureus antigens in sera of children with mild-to-severe AD and to evaluate the association between IgG levels and disease severity., Methods: In this cross-sectional study, we included children with AD from two interventional study cohorts, the Shared Medical Appointment (SMA) cohort (n = 131) and the older DAVOS cohort (n = 76). AD severity was assessed using the Self-Administered Eczema Area and Severity Index (SA-EASI) and levels of thymus and activation-regulated chemokine (TARC) in serum. IgG antibody levels against 55 S. aureus antigens were quantified simultaneously using a Luminex assay. Pair-wise correlations were calculated between the 55 IgG levels using the Spearman rank correlation test. Linear regression analysis was performed to test for associations between 55 IgG levels and SA-EASI and TARC, adjusting for age, sex and S. aureus colonization., Results: In the SMA cohort, 16 antigens were associated with SA-EASI and 12 with TARC (10 overlapping antigens; P-values 0·001-0·044). The associated IgG antibodies targeted mainly secreted proteins with immunomodulatory functions. In the DAVOS study, IgG levels against only four and one S. aureus antigen(s) were associated with SA-EASI and TARC, respectively (no overlap)., Conclusions: In young children, severity of AD is associated with an IgG response directed against S. aureus antigens with mainly immunomodulatory functions. These findings encourage further evaluation of the role of S. aureus in the pathogenesis of AD., (© 2017 British Association of Dermatologists.)

Background: Because of its high cardiospecificity, cardiac troponin T (cTnT) is one of the first-choice biomarkers to diagnose acute myocardial infarction (AMI). cTnT is extensively fragmented in serum of patients suffering from AMI. However, it is currently unknown whether all cTnT is completely degraded in the body or whether some cTnT fragments can leave the body via urine. The aim of the present study is to develop a method for the detection of cTnT in urine and to examine whether cTnT is detectable in patient urine., Methods: Proteins in urine samples of 20 patients were precipitated using a cTnT-specific immunoprecipitation technique and a nonspecific acetonitrile protein precipitation. After in-solution digestion of the precipitated proteins, the resulting peptides were separated and analyzed using HPLC and mass spectrometry with a targeted selected ion monitoring assay with data-dependent tandem mass spectrometry (t-SIM/dd-MS2)., Results: The t-SIM/dd-MS2 assay was validated using a synthetic peptide standard containing 10 specific cTnT peptides of interest and with purified human intact cTnT spiked in urine from healthy individuals. Using this assay, 6 different cTnT-specific peptides were identified in urine samples from 3 different patients, all suffering from AMI., Conclusions: We show here for the first time that cTnT can be present in the urine of AMI patients using a targeted LC-MS/MS assay. Whether the presence of cTnT in urine reflects a physiological or pathophysiological process still needs to be elucidated., (© 2017 American Association for Clinical Chemistry.)

Aims: To evaluate the effect of telemonitoring on adherence to non-vitamin K antagonist oral anticoagulants (NOACs) in atrial fibrillation (AF) patients., Methods and Results: A randomized, single-blind, crossover, controlled trial in 48 AF patients on once or twice daily (OD or BID) NOAC. The Medication Event Monitoring System tracked NOAC intake during three phases of 3 months each: daily telemonitoring, telemonitoring with immediate telephone feedback in case of intake errors, and an observation phase without daily transmissions. Unprotected days were defined as ≥ 3 or ≥ 1 consecutively missed doses for a BID or OD NOAC, respectively, or excess dose intake. Cost-effectiveness was calculated based on anticipated stroke reduction derived from patients' risk profile and measured intake. Persistence over the entire study was 98%. Telemonitoring-only already led to very high taking and regimen adherence (97.4% respectively 93.8%). Nevertheless, direct feedback further improved both to 99.0% and 96.8%, respectively (P < 0.001 respectively P = 0.002). Observation without daily monitoring resulted in a significant waning of taking adherence (94.3%; P = 0.049). Taking adherence was significantly higher for OD compared to BID NOAC, although unprotected days were similar. Feedback intervention had an incremental cost of €344 289 to prevent one stroke, but this could be as low as €15 488 in high-risk patients with low adherence and optimized technology., Conclusion: Telemonitoring resulted in high NOAC adherence due to the notion of being watched, as evidenced by the rapid decline during the observation period. Feedback further optimized adherence. Telemonitoring with or without feedback may be a cost-effective approach in high-risk patients deemed poorly adherent.

Background: Cardiac troponin T (cTnT) is the preferred biomarker for the diagnosis of acute myocardial infarction (AMI). It has been suggested that cTnT is present predominantly in fragmented forms in human serum following AMI. In this study, we have used a targeted mass spectrometry assay and epitope mapping using Western blotting to confirm this hypothesis., Methods: cTnT was captured from the serum of 12 patients diagnosed with AMI using an immunoprecipitation technique employing the M11.7 catcher antibody and fractionated with SDS-PAGE. Coomassie-stained bands of 4 patients at 37, 29, and 16 kDa were excised from the gel, digested with trypsin, and analyzed on a Q Exactive instrument set on targeted Selected Ion Monitoring mode with data-dependent tandem mass spectrometry (MS/MS) for identification. Western blotting employing 3 different antibodies was used for epitope mapping., Results: Ten cTnT peptides of interest were targeted. By using MS/MS, all of these peptides were identified in the 37-kDa, intact, cTnT band. In the 29- and 16-kDa fragment bands, 8 and 4 cTnT-specific peptides were identified, respectively. Some of these peptides were "semitryptic," meaning that their C-termini were not formed by trypsin cleavage. The C-termini of these semitryptic peptides represent the C-terminal end of the cTnT molecules present in these bands. These results were confirmed independently by epitope mapping., Conclusions: Using LC-MS, we have succeeded in positively identifying the 29- and 16-kDa fragment bands as cTnT-derived products. The amino acid sequences of the 29- and 16-kDa fragments are Ser79-Trp297 and Ser79-Gln199, respectively., (© 2016 American Association for Clinical Chemistry.)

Aims: To determine the usability, accuracy, and cost-effectiveness of two handheld single-lead electrocardiogram (ECG) devices for atrial fibrillation (AF) screening in a hospital population with an increased risk for AF., Methods and Results: Hospitalized patients (n = 445) at cardiological or geriatric wards were screened for AF by two handheld ECG devices (MyDiagnostick and AliveCor). The performance of the automated algorithm of each device was evaluated against a full 12-lead or 6-lead ECG recording. All ECGs and monitor tracings were also independently reviewed in a blinded fashion by two electrophysiologists. Time investments by nurses and physicians were tracked and used to estimate cost-effectiveness of different screening strategies. Handheld recordings were not possible in 7 and 21.4% of cardiology and geriatric patients, respectively, because they were not able to hold the devices properly. Even after the exclusion of patients with an implanted device, sensitivity and specificity of the automated algorithms were suboptimal (Cardiology: 81.8 and 94.2%, respectively, for MyDiagnostick; 54.5 and 97.5%, respectively, for AliveCor; Geriatrics: 89.5 and 95.7%, respectively, for MyDiagnostick; 78.9 and 97.9%, respectively, for AliveCor). A scenario based on automated AliveCor evaluation in patients without AF history and without an implanted device proved to be the most cost-effective method, with a provider cost to identify one new AF patient of €193 and €82 at cardiology and geriatrics, respectively. The cost to detect one preventable stroke per year would be €7535 and €1916, respectively (based on average CHA 2 DS 2 -VASc of 3.9 ± 2.0 and 5.0 ± 1.5, respectively). Manual interpretation increases sensitivity, but decreases specificity, doubling the cost per detected patient, but remains cheaper than sole 12-lead ECG screening., Conclusion: Using AliveCor or MyDiagnostick handheld recorders requires a structured screening strategy to be effective and cost-effective in a hospital setting. It must exclude patients with implanted devices and known AF, and requires targeted additional 12-lead ECGs to optimize specificity. Under these circumstances, the expenses per diagnosed new AF patient and preventable stroke are reasonable., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Objective: To examine the added value of measuring and possibly presenting patient experiences at the department level, in addition to the hospital level, and to explore the possibility that patient experiences differ according to the 'type' of hospital department., Design: Secondary analysis of data from a widely used survey on patient experiences of Dutch inpatient hospital care [Consumer Quality Index (CQI) Inpatient Hospital Care]., Setting: Inpatient hospital care experience survey of patients of 78 Dutch hospitals., Participants: A total of 15 171 randomly selected inpatients from 78 Dutch hospitals, who had at least one night of hospitalization between October 2006 and October 2007., Main Outcome Measures: Explained variance in patient experiences at the department level, compared with the explained variance at the hospital level. Significant differences in patient experiences between types of departments, expressed in regression coefficients. Patient experiences were measured using validated quality indicators, calculated from specific survey items., Results: Adding the department level to the analyses of patient experiences is statistically worthwhile for a number of quality indicators of the CQI Inpatient Hospital Care, and will enable the presentation of more detailed results within hospitals. Furthermore, the results indicated that there are some systematic differences in patient experiences between specific types of hospital departments across hospitals. However, the proportion of variance in experiences explained by both department and hospital is limited (max. 14%)., Conclusions: Analyses of quality information on patient experiences of inpatient hospital care should not only take the hospital level, but also at the more specific department level into account., (© The Author 2014. Published by Oxford University Press in association with the International Society for Quality in Health Care; all rights reserved.)

A simple and rapid method was validated to determine furosemide in whole blood. The experimental work was performed so that all validation parameters are considered simultaneously in a one-day assay protocol. A solid-phase extraction procedure using BondElut-LRC Certify columns was used to extract this compound from blood samples, while ketoprofen was used as an internal standard. The extracts were analyzed by gas chromatography-electron ionization-mass spectrometry after on-column derivatization with trimethylanilinium hydroxide (0.2M in methanol). Calibration curves were prepared daily, between 0.10 and 5.00 microg/mL, and the correlation coefficients were above 0.9910. The calculated limits of detection and quantitation were 0.010 and 0.045 microg/mL, respectively. Control samples at low, medium, and high concentrations (0.30, 0.75, and 3.00 microg/mL) of furosemide of an independent source were measured in the same day. Precision and trueness, calculated in terms of relative standard deviation (%), were less than 15% for all concentration levels. The relative recoveries calculated for the three levels of the control samples were 104%, 89%, and 91%, respectively. In general, a sensitive, specific, and reliable procedure has been developed for the determination of furosemide in whole blood samples and was found suitable for the application in postmortem forensic toxicology routine analysis.

We report on a patient in whom an active-fixation pre-shaped atrial lead caused perforation of the right atrial wall, pericardium and pleura, resulting in pericardial effusion and right-sided pneumothorax. Chest X-ray did not demonstrate protrusion of the atrial lead outside the cardiac silhouette but computed tomography visualized the tip of the helix of the atrial screw-in electrode outside the contours of the right atrial appendage touching the right upper lobe of the lung. The lead was repositioned with resolution of pericardial effusion and pneumothorax. Due to their proximity to the right lung, high anterolaterally positioned atrial screw-in leads carry a small but definite risk for right-sided pneumothorax.

An accurate, reproducible, and validated gas chromatography-mass spectrometry (GC-MS) method for the quantitation of 11 -nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), the major metabolite of delta9-tetrahydrocannabinol, in urine is described. Extraction was performed with n-hexane/ethyl acetate. Deuterated THC-COOH was used as the internal standard. The GC-MS analysis was done by selected ion monitoring. No interferences were detected in 20 blank urine samples of different origin. The calibration curve was found to be linear over the range of 10-100 ng/mL. The calculated limits of detection and quantitation were 1.0 ng/mL and 1.7 ng/mL, respectively. Results of positive findings for cannabis use in doping control in Flanders and Portugal in the period of 1997-2000 are commented.

The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.

An enantioselective gas chromatographic-mass spectrometric assay is developed for alprenolol and its metabolite, 4-hydroxy-alprenolol, in saliva and plasma. The procedure is based on a two-step derivatization technique with N-heptafluorobutyryl-l-prolylchloride and N-methyl-N-trimethylsilyl-trifluoroacetamide, followed by a gas chromatographic separation with mass spectrometric detection of the diastereomeric derivatives. A selected ion chromatogram extracted from full scan data shows that the respective enantiomers of alprenolol, 4-hydroxy-alprenolol, and the internal standard (ions at m/z 481) are well-separated in saliva and plasma. Linear and reproducible calibration curves are obtained over the concentration ranges 1.67-13.33 ng/mL and 2.50-20.00 ng/mL enantiomer in saliva and plasma, respectively. The performance of the method for alprenolol, in terms of accuracy and precision, fits well within the generally accepted criteria for validation. The enantioselective assay is successfully used in a study involving a single oral dose of alprenolol administered to two healthy volunteers. Stereoselective differences are observed in the saliva and plasma concentrations following an oral dose of 50 mg (R,S)-alprenolol hydrochloride.

The use of anabolic steroids and related compounds in sport is forbidden by the International Olympic Committee (IOC). Because danazol (17 alpha-pregna-2,4-dien-20-yno[2,3-D] isoxazol-17 beta-ol) is structurally related to the anabolic steroid stanozolol, its use should be questioned. Therefore, the detection and the significance of danazol in doping analysis are discussed. A urine specimen suspected of containing danazol metabolites was analyzed in order to characterize the metabolites. After isolation and conversion into three different derivatives, the metabolites were subjected to gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. The structure assignment was based on the molecular ions, fragmentation patterns observed for the three different derivatives, and the possible metabolite structures given in the literature. Ethisterone was identified as a nonconjugated metabolite. 2-Hydroxymethylethisterone was observed in two stereoisomeric forms. One stereoisomer was found mainly in the nonconjugated steroid fraction and the other in the conjugated fraction. The results were confirmed by analyzing urine specimens of a volunteer who was known to have taken danazol. Derivatization methods and GC/MS data are given to implement danazol detection in routine screening and confirmation procedures.