Asymmetric and symmetric dimethylarginine as markers of endothelial dysfunction in cerebrovascular disease: A prospective study.

BACKGROUND AND AIM: Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have been proposed as mediators of endothelial dysfunction. In this study, we aimed to investigate the diagnostic and prognostic role of ADMA and SDMA in acute cerebrovascular disease. METHODS AND RESULTS: A prospective case-control study was performed, enrolling 48 patients affected by ischemic stroke with no cardioembolic origin, 20 patients affected by TIA, 40 subjects at high cardiovascular risk and 68 healthy subjects. ADMA levels were significantly lower in high-risk subjects (18.85 [11.78-22.83] µmol/L) than in patients with brain ischemic event, both transient (25.70 [13.15-40.20] µmol/L; p = 0.032) and permanent (24.50 [18.0-41.33] µmol/L; p = 0.001). SDMA levels were different not only between high-risk subjects and ischemic patients, but also between TIA and stroke patients, reaching higher levels in TIA group and lower levels in stroke group (1.15 [0.90-2.0] vs 0.68 [0.30-1.07] µmol/L; p < 0.001). SDMA was also correlated with short-term prognosis, with lower levels in case of adverse clinical course, evaluated by type of discharge (p = 0.009) and need of prolonged rehabilitation (p = 0.042). CONCLUSIONS: The present study highlights the relationship between l-arginine, ADMA, SDMA and acute cerebrovascular events. Therefore, our results suggested a potential role of SDMA as a specific marker of transient ischemic damage and as a short-term positive prognostic marker.

LC-MS/MS measurement of endogenous steroid hormones and phase II metabolites in blood volumetric absorptive microsampling (VAMS) for doping control purposes.

BACKGROUND: Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to traditional sampling methods. The objective of this study was to assess the suitability of 30 µL VAMS for the measurement of endogenous steroid hormones. METHODS: A novel LC-MS/MS method was developed for the quantification of 18 analytes in VAMS samples, including main endogenous free steroids and phase II metabolites of androgens. The method underwent validation in accordance with ISO/IEC 17025:2017 and World Anti-Doping Agency (WADA) requirements. Subsequently, it was applied to authentic VAMS samples obtained from 20 healthy volunteers to assess the stability of target analytes under varying storage conditions. RESULTS: The validation protocol assessed method's selectivity, matrix effect, extraction recovery, quantitative performance, carry-over and robustness. The analysis of authentic samples demonstrated the satisfactory stability of monitored steroids in VAMS stored at room temperature, 4 °C, -20 °C and -80 °C for up to 100 days and subjected to up to 3 freezing-thawing cycles. CONCLUSIONS: The validated LC-MS/MS method demonstrated its suitability for the measurement of steroids in dried blood VAMS. The observed stability of steroidal compounds suggests promising prospects for future applications of VAMS, both in anti-doping contexts and clinical research.

Plasma soluble suppression of tumorigenesis 2 measured in the emergency department for diagnosis and outcome prediction of sepsis: A single-center prospective study.

BACKGROUND AND AIMS: The diagnostic and prognostic performance of soluble Suppression of Tumorigenicity 2 (sST2) in suspected septic patients presenting to the Emergency Department (ED) is largely unknown. MATERIALS AND METHODS: Patients were included in this prospective study if there was high suspicion of sepsis. The plasma level of sST2 was measured during initial ED evaluation. Outcomes were the evaluation of (1) sST2 diagnostic performance (alone and in combination with procalcitonin [PCT]), and (2) sST2 ability to predict 30-day and 90-day all-cause mortality. RESULTS: Among 569 patients included, 481 (84.5 %) had sepsis or septic shock. Plasma sST2 levels were more elevated in septic patients (159 [71-331] vs 50 [31-103] ng/mL, P < 0.001). The AUC of sST2 for sepsis diagnosis was lower than the AUC of PCT (0.76 vs 0.85, P = 0.03). The best cut-off for sST2 was 61.7 ng/mL, with a sensitivity of 79.9 % and a specificity of 70.6 %. sST2 was able to correctly reclassify septic patients with PCT <0.5 (NRI 28.9 % [P = 0.02]). sST2 level was an independent predictor of 30-day mortality in a model including clinical variables (aHR 2.03 [1.24-3.33], C-index 0.69). CONCLUSION: sST2 could be a useful adjunct in diagnosing sepsis and in all-cause mortality prediction.

Predictive performance of aldosterone-to-renin ratio in the diagnosis of primary aldosteronism in patients with resistant hypertension.

Background: The systematic use of confirmatory tests in the diagnosis of primary aldosteronism (PA) increases costs, risks and complexity to the diagnostic work-up. In light of this, some authors proposed aldosterone-to-renin (ARR) cut-offs and/or integrated flow-charts to avoid this step. Patients with resistant hypertension (RH), however, are characterized by a dysregulated renin-angiotensin-aldosterone system, even in the absence of PA. Thus, it is unclear whether these strategies might be applied with the same diagnostic reliability in the setting of RH. Methods: We enrolled 129 consecutive patients diagnosed with RH and no other causes of secondary hypertension. All patients underwent full biochemical assessment for PA, encompassing both basal measurements and a saline infusion test. Results: 34/129 patients (26.4%) were diagnosed with PA. ARR alone provided a moderate-to-high accuracy in predicting the diagnosis of PA (AUC=0.908). Among normokalemic patients, the ARR value that maximized the diagnostic accuracy, as identified by the Youden index, was equal to 41.8 (ng/dL)/(ng/mL/h), and was characterized by a sensitivity and a specificity of 100% and 67%, respectively (AUC=0.882); an ARR > 179.6 (ng/dL)/(ng/mL/h) provided a 100% specificity for the diagnosis of PA, but was associated with a very low sensitivity of 20%. Among hypokalemic patients, the ARR value that maximized the diagnostic accuracy, as identified by the Youden index, was equal to 49.2 (ng/dL)/(ng/mL/h), and was characterized by a sensitivity and a specificity of 100% and 83%, respectively (AUC=0.941); an ARR > 104.0 (ng/dL)/(ng/mL/h) provided a 100% specificity for the diagnosis of PA, with a sensitivity of 64%. Conclusions: Among normokalemic patients, there was a wide overlap in ARR values between those with PA and those with essential RH; the possibility to skip a confirmatory test should thus be considered with caution in this setting. A better discriminating ability could be seen in the presence of hypokalemia; in this case, ARR alone may be sufficient to skip confirmatory tests in a suitable percentage of patients.

Copeptin adaptive response to SGLT2 inhibitors in patients with type 2 diabetes mellitus: The GliRACo study.

Introduction: In type 2 diabetes mellitus (T2DM), the antidiuretic system participates in the adaptation to osmotic diuresis further increasing urinary osmolality by reducing the electrolyte-free water clearance. Sodium glucose co-transporter type 2 inhibitors (SGLT2i) emphasize this mechanism, promoting persistent glycosuria and natriuresis, but also induce a greater reduction of interstitial fluids than traditional diuretics. The preservation of osmotic homeostasis is the main task of the antidiuretic system and, in turn, intracellular dehydration the main drive to vasopressin (AVP) secretion. Copeptin is a stable fragment of the AVP precursor co-secreted with AVP in an equimolar amount. Aim: To investigate the copeptin adaptive response to SGLT2i, as well as the induced changes in body fluid distribution in T2DM patients. Methods: The GliRACo study was a prospective, multicenter, observational research. Twenty-six consecutive adult patients with T2DM were recruited and randomly assigned to empagliflozin or dapagliflozin treatment. Copeptin, plasma renin activity, aldosterone and natriuretic peptides were evaluated at baseline (T0) and then 30 (T30) and 90 days (T90) after SGLT2i starting. Bioelectrical impedance vector analysis (BIVA) and ambulatory blood pressure monitoring were performed at T0 and T90. Results: Among endocrine biomarkers, only copeptin increased at T30, showing subsequent stability (7.5 pmol/L at T0, 9.8 pmol/L at T30, 9.5 pmol/L at T90; p = 0.001). BIVA recorded an overall tendency to dehydration at T90 with a stable proportion between extra- and intracellular fluid volumes. Twelve patients (46.1%) had a BIVA overhydration pattern at baseline and 7 of them (58.3%) resolved this condition at T90. Total body water content, extra and intracellular fluid changes were significantly affected by the underlying overhydration condition (p < 0.001), while copeptin did not. Conclusion: In patients with T2DM, SGLT2i promote the release of AVP, thus compensating for persistent osmotic diuresis. This mainly occurs because of a proportional dehydration process between intra and extracellular fluid (i.e., intracellular dehydration rather than extracellular dehydration). The extent of fluid reduction, but not the copeptin response, is affected by the patient's baseline volume conditions. Clinical trial registration: Clinicaltrials.gov, identifier NCT03917758.

Prevalence of Cortisol Cosecretion in Patients With Primary Aldosteronism: Role of Metanephrine in Adrenal Vein Sampling.

CONTEXT: Adrenal venous sampling (AVS) is the gold standard procedure for subtype diagnosis in patients with primary aldosteronism (PA). Cortisol is usually adopted for the normalization of aldosterone levels in peripheral and adrenal samples. However, asymmetrical cortisol secretion can potentially affect the lateralization index, leading to subtype misdiagnosis. OBJECTIVE: We aimed to assess the prevalence of asymmetrical cortisol secretion in patients undergoing AVS and whether variations in adrenal vein cortisol might influence AVS interpretations. We then evaluated the use of metanephrines for the normalization of aldosterone levels for lateralization index. METHODS: We retrospectively included 101 patients with PA who underwent AVS: 49 patients underwent unstimulated AVS, while 52 patients underwent both unstimulated and cosyntropin-stimulated AVS. Eighty-eight patients had bilateral successful AVS according to metanephrine ratio. We assessed the prevalence of asymmetrical cortisol secretion through the cortisol to metanephrine (C/M) lateralization index (LI). We then evaluated whether the use of aldosterone to metanephrine (A/M) LI can improve the diagnostic accuracy of AVS compared with aldosterone to cortisol (A/C) LI. RESULTS: Asymmetrical cortisol secretion is present in 18% of patients with PA. Diagnosis with A/M LI and A/C LI is discordant in 14% of patients: 9% had a diagnosis of unilateral PA with A/M LI instead of bilateral PA with A/C LI and 5% had a diagnosis of bilateral PA with A/M LI instead of unilateral PA. CONCLUSION: The assessment of metanephrine levels in AVS is useful for the determination of selectivity and lateralization, allowing an accurate diagnosis, especially in patients with asymmetrical cortisol secretion.

Longitudinal Profiling of Endogenous Steroids in Blood Using the Athlete Biological Passport Approach.

CONTEXT: Detection of endogenous anabolic androgenic steroids (EAAS), like testosterone (T), as doping agents has been improved with the launch of the Steroidal Module of the Athlete Biological Passport (ABP) in urine samples. OBJECTIVE: To target doping practices with EAAS, particularly in individuals with low level of biomarkers excreted in urine, by including new target compounds measured in blood. DESIGN: T and T/androstenedione (T/A4) distributions were obtained from 4 years of anti-doping data and applied as priors to analyze individual profiles from 2 T administration studies in female and male subjects. SETTING: Anti-doping laboratory. Elite athletes (n = 823) and male and female clinical trials subjects (n = 19 and 14, respectively). INTERVENTION(S): Two open-label administration studies were carried out. One involved a control phase period followed by patch and then oral T administration in male volunteers and the other followed female volunteers during 3 menstrual cycles with 28 days of daily transdermal T application during the second month. MAIN OUTCOME MEASURE(S): Serum samples were analyzed for T and A4 and the performance of a longitudinal ABP-based approach was evaluated for T and T/A4. RESULTS: An ABP-based approach set at a 99% specificity flagged all female subjects during the transdermal T application period and 44% of subjects 3 days after the treatment. T showed the best sensitivity (74%) in response to transdermal T application in males. CONCLUSIONS: Inclusion of T and T/A4 as markers in the Steroidal Module can improve the performance of the ABP to identify T transdermal application, particularly in females.

Single-run UHPLC-MS/MS method for simultaneous quantification of endogenous steroids and their phase II metabolites in serum for anti-doping purposes.

Anti-doping rule violations related to the abuse of endogenous anabolic androgenic steroids can be currently discovered by the urinary steroidal module of Athlete Biological Passport. Since this powerful tool is still subjected to some limitations due to various confounding factors altering the steroid profile, alternative strategies have been constantly proposed. Among these, the measurement of blood concentrations of endogenous steroid hormones by LC-MS is currently of increasing interest in anti-doping, bringing significant advantages for the detection of testosterone abuse in females and in individuals with deletion of UGT2B17 enzyme. Although various research groups have made significant efforts in method development, there is currently no accepted or harmonized anti-doping method for quantitative analysis of the various testosterone doping markers in blood. In this study we present a UHPLC-MS/MS method for the quantification of major circulating steroid hormones together with an extended panel of glucuro- and sulpho-conjugated phase II metabolites of androgens. Chromatographic setup was optimized by comparing the performance of three different C18 stationary phases and by the careful selection of mobile phases with the aim of separating all the target steroids, including numerous isomeric/isobaric compounds. MS parameters were fine-tuned to obtain the sensitivity needed for measuring the target analytes, that show specific serum concentrations ranging from low pg/mL for less abundant compounds to µg/mL for sulpho-conjugated steroids. Finally, sample preparation protocol was developed for the extraction of steroid hormones from 200 µL of serum and the performance was evaluated in terms of extraction recovery and matrix effect. The final method was then applied to authentic serum samples collected from healthy volunteers (40 males and 40 females) at the Blood Bank of the City of Health and Science University Hospital of Turin. The analysis of these samples allowed to obtain results on serum concentrations of the targeted steroids, with particular emphasis on previously undiscovered phase II metabolites, such as the isomers of 5-androstane-3,17-diol glucuronide. This preliminary application also enabled measuring dihydrotestosterone sulphate in male samples, efficiently separating this analyte from its isomer, epiandrosterone sulphate, which circulates in blood at high concentrations. The promising results of this study are encouraging for the measurement of blood steroid profile markers in serum and plasma samples for Athlete Biological Passport purposes.

A retrospective study on the association between urine metanephrines and cardiometabolic risk in patients with nonfunctioning adrenal incidentaloma.

Several studies argued that cardiovascular evaluation of patients with nonfunctioning adrenal incidentaloma is of particular importance. Therefore, we aimed to evaluate the possibility of stratifying the cardiometabolic risk using metanephrine levels in this setting of patients. A retrospective cross-sectional study was designed, collecting data of metanephrine values in 828 patients with nonfunctioning adrenal incidentaloma, referred to our Division within the University of Turin between 2007 and 2021. The univariate analysis showed associations between urine metanephrines and cardiometabolic variables/parameters, particularly considering the noradrenaline metabolite. At the univariate regression, normetanephrine was associated with metabolic syndrome (OR = 1.13, p = 0.002), hypertensive cardiomyopathy (OR = 1.09, p = 0.026), microalbuminuria (OR = 1.14, p = 0.024), and eGFR < 60 mL/min/1.73 m2 (OR = 1.11, p = 0.013), while metanephrine was associated with microalbuminuria (OR = 1.50, p = 0.008). At multivariate regression, considering all major cardiovascular risk factors as possible confounders, normetanephrine retained a significant association with metabolic syndrome (OR = 1.10, p = 0.037). Moreover, metanephrine retained a significant association with the presence of microalbuminuria (OR = 1.66, p = 0.003). The present study showed a further role for metanephrines in the cardiovascular risk stratification of patients with nonfunctioning adrenal incidentaloma. Individuals with high levels of these indirect markers of sympathetic activity should be carefully monitored and may benefit from an aggressive treatment to reduce their additional cardiometabolic burden.

Primary Aldosteronism and Resistant Hypertension: A Pathophysiological Insight.

Primary aldosteronism (PA) is a pathological condition characterized by an excessive aldosterone secretion; once thought to be rare, PA is now recognized as the most common cause of secondary hypertension. Its prevalence increases with the severity of hypertension, reaching up to 29.1% in patients with resistant hypertension (RH). Both PA and RH are "high-risk phenotypes", associated with increased cardiovascular morbidity and mortality compared to non-PA and non-RH patients. Aldosterone excess, as occurs in PA, can contribute to the development of a RH phenotype through several mechanisms. First, inappropriate aldosterone levels with respect to the hydro-electrolytic status of the individual can cause salt retention and volume expansion by inducing sodium and water reabsorption in the kidney. Moreover, a growing body of evidence has highlighted the detrimental consequences of "non-classical" effects of aldosterone in several target tissues. Aldosterone-induced vascular remodeling, sympathetic overactivity, insulin resistance, and adipose tissue dysfunction can further contribute to the worsening of arterial hypertension and to the development of drug-resistance. In addition, the pro-oxidative, pro-fibrotic, and pro-inflammatory effects of aldosterone may aggravate end-organ damage, thereby perpetuating a vicious cycle that eventually leads to a more severe hypertensive phenotype. Finally, neither the pathophysiological mechanisms mediating aldosterone-driven blood pressure rise, nor those mediating aldosterone-driven end-organ damage, are specifically blocked by standard first-line anti-hypertensive drugs, which might further account for the drug-resistant phenotype that frequently characterizes PA patients.

Testosterone as a Biomarker of Adverse Clinical Outcomes in SARS-CoV-2 Pneumonia.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may affect testicles. Lower testosterone levels have been associated with worse clinical outcomes and higher mortality. Our objective was to evaluate the hypothalamic−pituitary−gonadal axis of men admitted with SARS-CoV-2 pneumonia and its link with the pneumonia-treatment intensification. Short-term changes in hormonal parameters were also assessed. Methods: Men admitted with SARS-CoV-2 pneumonia were recruited in two different hospitals in Piedmont, Italy. In all patients, the assessment of total testosterone (TT), calculated free testosterone (cFT), gonadotropins, inhibin B (InhB), and other biochemical evaluations were performed at admission (T0) and before discharge (T1). Through a review of medical records, clinical history was recorded, including data on pneumonia severity. Results: Thirty-five men (median age 64 [58−74] years) were recruited. Lower TT and cFT levels at T0 were associated with CPAP therapy (p = 0.045 and 0.028, respectively), even after adjusting for age and PaO2/FIO2 ratio in a multivariable analysis. In those discharged alive, lower TT and cFT levels were associated with longer hospital stay (p < 0.01). TT, cFT, and InhB were below the normal range at T0 and significantly increased at T1 (TT 1.98 [1.30−2.72] vs. 2.53 [1.28−3.37] ng/mL, p = 0.038; cFT (0.0441 [0.0256−0.0742] vs. 0.0702 [0.0314−0.0778] ng/mL, p = 0.046; InhB 60.75 [25.35−88.02] vs. 77.05 [51.15−134.50], p < 0.01). Conclusions: Both TT and cFT levels are associated with adverse clinical outcomes in men admitted with SARS-CoV-2 pneumonia. As TT, cFT and InhB levels increase before discharge, short-term functional recovery of steroidogenesis and an indirect improvement of spermatozoa functional status could be hypothesized.

Atrial Fibrillation and Aortic Ectasia as Complications of Primary Aldosteronism: Focus on Pathophysiological Aspects.

Primary aldosteronism (PA) is the most common cause of secondary hypertension. A growing body of evidence has suggested that, beyond its well-known effects on blood pressure and electrolyte balance, aldosterone excess can exert pro-inflammatory, pro-oxidant and pro-fibrotic effects on the kidney, blood vessels and heart, leading to potentially harmful pathophysiological consequences. In clinical studies, PA has been associated with an increased risk of cardiovascular, cerebrovascular, renal and metabolic complication compared to essential hypertension, including atrial fibrillation (AF) and aortic ectasia. An increased prevalence of AF in patients with PA has been demonstrated in several clinical studies. Aldosterone excess seems to be involved in the pathogenesis of AF by inducing cardiac structural and electrical remodeling that in turn predisposes to arrhythmogenicity. The association between PA and aortic ectasia is less established, but several studies have demonstrated an effect of aldosterone on aortic stiffness, vascular smooth muscle cells and media composition that, in turn, might lead to an increased risk of aortic dilation and dissection. In this review, we focus on the current evidence regarding the potential role of aldosterone excess in the pathogenesis of AF and aortic ectasia.

Simultaneous Measurement of Cortisol, Cortisone, Dexamethasone and Additional Exogenous Corticosteroids by Rapid and Sensitive LC-MS/MS Analysis.

The simultaneous measurement of dexamethasone and cortisol has proven the ability to increase the diagnostic performance of the overnight dexamethasone-suppression test. Furthermore, the therapeutic drug monitoring of administered corticosteroid drugs could represent a crucial tool for investigating unexpected variations of steroid hormones' circulating levels. In this work, an LC-MS/MS method for the quantification of cortisol, cortisone, dexamethasone and six additional exogenous corticosteroids in the serum/plasma matrix was developed and validated in compliance with the ISO/IEC requirements. To assess the efficiency of the validated method, serum samples of 75 patients undergoing the dexamethasone-suppression test and 21 plasma samples of patients under immunosuppressive treatment after kidney transplant were analyzed. In all dexamethasone-suppression test samples, it was possible to measure the circulating levels of cortisol, cortisone and dexamethasone. Concentrations of the latter were for all tested patients above the proposed cutoff for the dexamethasone-suppression test's results, and the cortisol concentrations showed good correlation with the ones measured by routine immunometric analysis, therefore confirming the screening outcome for all enrolled patients. Prednisone was detected and quantified in all enrolled patients, confirming the use of such a corticosteroid for immunosuppressive therapy. Thanks to these two applications, we proved the overall performance of the developed LC-MS/MS method for four target analytes. The future implementation of such an analytical tool in the clinical biochemistry laboratory's routine will guarantee a single and versatile tool for simultaneously monitoring dexamethasone-suppression-test results and corticosteroid drugs' administration.

Total Value of Ownership and Overall Equipment Effectiveness analysis to evaluate the impact of automation on time and costs of therapeutic drug monitoring.

Total Value of Ownership (TVO) and Overall Equipment Effectiveness (OEE) analysis are novel tools capable of monitoring and analyzing industrial processes by assessing the efficiency of the entire instrumental equipment and calculating instrument capacity utilization. Such integrated analysis, measuring quality indicators of the testing process, could also provide new perspectives and methodologies for the workflow organization of clinical laboratories. In this study, TVO and OEE were employed for the evaluation of two different configurations of a therapeutic drug monitoring sector, comparing the results obtained for immunosuppressant (ISD) and anti-epileptic drugs (AED) analysis as well as checking their quantitative performance in terms of limit of quantification, accuracy and precision. TVO analysis was performed for ISDs, including the Total Direct Labor Time, Total Cycle Time and Turnaround Time as well as cost of testing. Instruments' performance and workload were assessed using OEE indicator, studying Availability, Performance and Quality factors. Total Cycle Time for a batch was 3.55 h, decreasing of 1.5 h in the new setting where personnel are engaged for 0.98 h, 25% of total testing time. The calculated cost per sample was 6.60 euro. Availability values were significantly higher for automated sample-handling system and ISDs analysis by LC-MS. Higher Performance values were obtained for LC-MS system for AED and other TDM. Quality values were >0.94 for all instruments. TVO and OEE proved to be applicable to clinical laboratory environment, quantifying benefits and costs of newly developed semi-automated therapeutic drug monitoring sector. This novel approach based on an integrated analysis may help activity planning and quality improvement and could be used in the future for benchmarking progress as a product/process comparison tool in other laboratory fields.

Development and validation of an UHPLC-MS/MS method for extended serum steroid profiling in female populations.

Aim: Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC-MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. Results: The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20%). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. Conclusion: The UHPLC-MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.

Steroidomics for highlighting novel serum biomarkers of testosterone doping.

Aim: Quantification of testosterone (T) and 5α-dihydrotestosterone serum concentrations proved to be an efficient alternative to urinary steroid profiling for the detection of T doping. In this context, additional serum markers could be discovered by exploratory untargeted steroidomics studies. Results: Endogenous steroid metabolites were monitored by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry in serum samples collected during a T administration clinical trial. A three-step workflow for accurate review of annotation was used and multifactorial data analysis allowed highlighting promising serum biomarkers. Longitudinal monitoring of selected compounds was performed to assess T abuse detection capabilities. Conclusion: Application of serum steroidomics showed high potential for biomarker discovery of T doping, suggesting longitudinal monitoring of steroid hormones in serum as a significant improvement in detection of endogenous steroids abuse.

UHPLC-HRMS Analysis for Steroid Profiling in Serum (Steroidomics).

The extraction and untargeted UHPLC-HRMS analysis of endogenous steroids in serum samples is described in this protocol. The employed full-scan acquisition mode provides the adequate sensitivity to highlight the main endogenous steroids present in blood, including mineralocorticoids, progestogens , and androgens. Technical aspects for both chromatography and mass spectrometry are discussed in detail, together with a proposition of setup for sample sequence and data analysis. Furthermore, general comments are given to help the assessment of data quality and system performance.

High-resolution mass spectrometry as an alternative detection method to tandem mass spectrometry for the analysis of endogenous steroids in serum.

Recently, steroid hormones quantification in blood showed a promising ability to detect testosterone doping and interesting complementarities with the urinary module of the Athlete Biological Passport (ABP). In this work, an ultra-high pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method was developed for the quantification of eleven endogenous steroids in serum. The performance of the full scan and targeted SIM acquisition modes was evaluated and compared to the performance of tandem mass spectrometry (MS/MS). Passing-Bablok regressions and Bland-Altman plots were assessed for each analyte of interest, and concentration values measured by HRMS showed high correlation with the ones obtained by MS/MS for all target hormones, with low absolute differences in the majority of cases. A slight decrease in terms of sensitivity was observed with HRMS in both acquisition modes, but performing an analysis of variance multiblock orthogonal partial least squares (AMOPLS) on the dataset obtained with all three methods revealed that only 0.8% of the total variance was related to instrumentation and acquisition methods. Moreover, the evaluation of the testosterone administration effect over time highlighted testosterone itself and dihydrotestosterone as the most promising biomarkers of exogenous testosterone administration. This conclusion suggests that HRMS could provide suitable performance for blood steroid analysis in the anti-doping field.

Methods for Doping Detection.

Over the past few years, the World Anti-Doping Agency (WADA) has focused its efforts on detecting not only small prohibited molecules, but also larger endogenous molecules such as hormones, in the view of implementing an endocrinological module in the Athlete Biological Passport (ABP). In this chapter, the detection of two major types of hormones used for doping, growth hormone (GH) and endogenous anabolic androgenic steroids (EAASs), will be discussed: a brief historical background followed by a description of state-of-the-art methods applied by accredited anti-doping laboratories will be provided and then current research trends outlined. In addition, microRNAs (miRNAs) will also be presented as a new class of biomarkers for doping detection.

Liquid chromatography and supercritical fluid chromatography as alternative techniques to gas chromatography for the rapid screening of anabolic agents in urine.

This work describes the development of two methods involving supported liquid extraction (SLE) sample treatment followed by ultra-high performance liquid chromatography or ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS and UHPSFC-MS/MS) for the screening of 43 anabolic agents in human urine. After evaluating different stationary phases, a polar-embedded C18 and a diol columns were selected for UHPLC-MS/MS and UHPSFC-MS/MS, respectively. Sample preparation, mobile phases and MS conditions were also finely tuned to achieve highest selectivity, chromatographic resolution and sensitivity. Then, the performance of these two methods was compared to the reference routine procedure for steroid analyses in anti-doping laboratories, which combines liquid-liquid extraction (LLE) followed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). For this purpose, urine samples spiked with the compounds of interest at five different concentrations were analyzed using the three analytical platforms. The retention and selectivity of the three techniques were very different, ensuring a good complementarity. However, the two new methods displayed numerous advantages. The overall procedure was much faster thanks to high throughput SLE sample treatment using 48-well plates and faster chromatographic analysis. Moreover, the highest sensitivity was attained using UHPLC-MS/MS with 98% of the doping agents detected at the lowest concentration level (0.1ng/mL), against 76% for UHPSFC-MS/MS and only 14% for GC-MS/MS. Finally, the weakest matrix effects were obtained with UHPSFC-MS/MS with 76% of the analytes displaying relative matrix effect between -20 and 20%, while the GC-MS/MS reference method displayed very strong matrix effects (over 100%) for all of the anabolic agents.