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The detection of clostebol misuse in sports has been growing recently, especially in Italy, due to the ample availability of pharmaceutical formulations containing clostebol acetate (Trofodermin®) and the use of more sensitive instrumentation by the antidoping laboratories. Most of these cases have been claimed to be related to a nonconscious use of the drug or through contact with relatives or teammates using it. We have investigated, through the application of the well-known and currently used gas chromatographic mass spectrometric procedures, the likelihood of these allegations and have demonstrated that after a single transdermal administration of 5 mg of clostebol acetate and a transient contact with the application area, it is possible to generate adverse analytical findings in antidoping controls. We have reviewed the Phase I and Phase II clostebol metabolism in order to generate evidences that may help the sport authorities reviewing these cases. The main clostebol metabolite (4-chloro-androst-4-en-3α-ol-17-one, M1) generally used at the screening level as well as other three metabolites (M2-M4) are mainly excreted as glucuronides, whereas M5 (4ζ-chloro-5ζ-androstan-3β-ol-17-one) is predominantly excreted as sulfate. Neither the 5α-reductases activity (impaired by the presence of the chlorine in C4) nor specific sulfotransferases present in the skin allowed a clear distinction of the administration route. Studies with a larger number of volunteers and probably investigating another physiological fluid allowed in antidoping such as blood are needed for a deeper investigation. It is not unreasonable to establish a reporting level for M1, maybe creating some false negatives but excluding nonintentional doping scenarios.

We present a liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 16 endogenous steroids (androgens, estrogens, glucocorticoids and progestogens) in human serum. Samples (250 μl of matrix) were extracted with t-butylmethyl ether prior to LC-MS/MS analysis. The chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. The HPLC was coupled to a triple quadrupole mass spectrometer equipped with an electrospray ionization source with acquisition in multiple reaction monitoring mode. The method was validated using surrogate matrices and human serum samples. The specificity of the method was confirmed for all of the considered steroids; linearity was also assessed (R

Anabolic androgenic steroids (AAS) are prohibited as performance-enhancing drugs in sports. Among them, testosterone and its precursors are often referred to as “pseudoendogenous” AAS, that is, endogenous steroids that are prohibited when administered exogenously. To detect their misuse, among other methods, the World Anti-Doping Agency-accredited laboratories monitor the steroid profile (concentrations and concentration ratios of endogenous steroids, precursors and metabolites) in urine samples collected from athletes in and out of competition. Alterations in steroid profile markers are used as indicators for misuse of anabolic steroids in sports. Therefore, especially their metabolic pathways with possible interactions are crucial to elucidate. As steroid metabolism is very complex, and many enzymes are involved, certain non-prohibited drugs may influence steroid metabolite excretion. One important group of steroid-metabolizing enzymes is aldo–keto reductases (AKRs). An inhibition of them by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, was demonstrated in vitro. Thus, this work aims to investigate the influence of NSAID intake on the urinary steroid profile. Kinetic and inhibitory studies were performed using 5α-dihydrotestosterone as substrate. The results obtained from in vitro experiments show that ibuprofen inhibits AKR1C2 and thus influences steroid biotransformation. For in vivo investigations, urine samples prior, during and postadministration of ibuprofen were analyzed using routine methods to monitor the steroid profile. Changes in markers of the steroid profile of volunteers were observed. The combination of in vitro and in vivo results suggests that monitoring of ibuprofen may be useful in doping control analysis. The presented work illustrates the importance to consider co-administration of (non-prohibited) drugs during antidoping analysis. Intake of multiple substances is likely leading to interfering effects. Divergent results in antidoping analysis may therefore be observed and misinterpretation of analytical data may occur. Similar considerations may be appropriate for other fields of forensic applications.

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail.Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite.The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite.It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

Rationale Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens. Methods The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS). Results Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation. Conclusions When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis.

In this work we have investigated the influence of the intake of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the urinary concentration of endogenous steroids, and on their relative ratios, of doping relevance. Specifically, the concentrations of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstan-3α,17α-diol (5αAdiol), 5β-androstan-3α,17α-diol (5βAdiol), and the ratios T/E, A/T, A/Etio, 5αAdiol/5βAdiol, 5αAdiol/E, were considered, in the framework of the Steroidal Module of the Athlete Biological Passport (ABP). The above set of parameters were complemented by the urinary levels of luteinizing hormone (total LH) and the ratio between T and LH (T/total LH), to assess the possible effects on the biosynthesis of the mentioned steroids. Five healthy Caucasian male volunteers were selected for the study. Urine samples were collected before and during the administration of (i) methoxyisoflavone (Methoxyisoflavone, MyProtein) and (ii) ipriflavone (Osteofix ®, Chiesi Farmaceutici). For the analysis of the urinary steroid profile, after enzymatic hydrolysis with β-glucuronidase from Escherichia Coli (E. Coli) and liquid–liquid extraction with tert-buthylmethyl ether, all samples were analyzed by gas chromatography coupled to tandem mass spectrometry (GC–MS/MS), while for the determination of total LH all urine samples were directly analyzed by a chemiluminescent immunometric assay technique (Siemens Immulite 2000 LH). Our results show that the administration of either methoxyisoflavone or ipriflavone causes an alteration of the urinary concentrations and concentration ratios of the investigated steroids, in the range 55–80% from the baseline values. Furthermore, an oversecretion of LH after the daily intake of methoxyisoflavone or ipriflavone was also recorded in all volunteers, corresponding to an increase in the biosynthesis and excretion of T and some of its metabolites. These changes trigger a disregulation in the pattern of urinary excretion of the steroids included in the Steroidal Module of the ABP, which makes more difficult the interpretation of the longitudinal steroid profile based on the definition of individual normality ranges for each athlete. Our data are also consistent with previous evidence regarding the in vitro effects of natural and synthetic isoflavones, suggesting that their monitoring in doping control routine analysis would be very beneficial for the result management activities.

Metandienone and methyltestosterone are orally available anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. After the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“Oral Turinabol”), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identi-fied following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new hypotheses on the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring and the participation of different enzymes.

Background Over the counter preparations containing Saw palmetto and Pygeum africana extracts are commonly used in the treatment of benign prostatic hyperplasia with lower urinary tract symptoms. Their effects are likely due to their anti-androgenic activity, involving the interaction with type 1 and type 2 isoenzymes of the 5α-reductase family. Hypothesis/purpose For effects similar to the above, the inhibitors of 5α-reductase (e.g., finasteride) are classified as confounding factors in the evaluation of the urinary steroid profile in sport doping. We aimed to verify whether the oral intake of products containing Saw palmetto and Pygeum africana could affect the urinary concentration of a series of steroids, not limited to those included in the “steroidal module” of the Athlete Biological Passport (ABP). Study design We have followed the effects of a five-day administration of Saw palmetto (320 mg) and Pygeum africana (100 mg) to three non-smokers male subjects on the urinary parameters constituting the “steroidal module” of the ABP, and on the concentration of additional steroid markers. In parallel, we have also measured the urinary concentration of β-sitosterol, which is the active component of Saw palmetto and Pygeum Africana. Methods Sample collection was performed five days before starting the controlled administration study to obtain the baseline urinary steroid pattern for each subject. After this period, the three volunteers took a dose of Saw palmetto (320 mg) and Pygeum africana (100 mg) formulation orally, once a day, for five consecutive days. Urine samples were collected at different times of the day and stored at −20 °C until analysis. Samples were analyzed by a previously developed and validated method based on gas chromatography coupled to tandem mass spectrometry (GC–MS/MS). Results Our results show that the intake of a single oral dose of Saw palmetto and Pygeum africana for five consecutive days leads to a broader data dispersion of the target steroids' concentration levels, and, consequently, to a variation of the corresponding individual normality ranges. Conclusion The intake of dietary supplements containing Saw palmetto and Pygeum africana could be a source of interference in the correct longitudinal evaluation of the individual steroid profile, as described in the framework of ABP. We believe their urinary levels should be monitored as it is presently done for other confounding factors of the steroidal module of the ABP.

The detection of the abuse of pseudo-endogenous steroids in sport is articulated in two different levels: an initial testing procedure, based on the longitudinal evaluation of the urinary androgenic steroid profile by gas-chromatography mass spectrometry (GC-MSn), and a confirmation analysis, based on the differentiation between the endogenous and exogenous origin of the pseudo-endogenous steroids by gas-chromatography coupled to isotopic ratio mass spectrometry (GC/C/IRMS). The abuse of pharmaceutical preparations displaying a carbon isotopic composition values within a range similar to those reported for endogenous urinary steroids makes more difficult the application of GC/C/IRMS technique. To overcome this limitation, the direct detection of an intact synthetic anabolic steroid ester in blood matrices (plasma and/or serum) could supply the unequivocal proof of exogenous administration of pseudo-endogenous steroids. Here we are presenting a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of 14 testosterone (T) esters and 2 nandrolone (Nand) esters in human serum. Sample pre-treatment consisted of protein precipitation, liquid-liquid extraction and derivatization. The formation of three different derivatives (oxime derivatives, Girard P and Girard T hydrazones) is considered, in order to guarantee an improvement in the detection capability of the assay with respect to underivatized compounds. Once the most suitable derivative was selected, the method was validated, according to the World Anti-Doping Agency (WADA) criteria, in terms of specificity, linearity, limit of detection (LOD), extraction recovery, matrix effect (ion suppression/enhancement), carry over and autosampler stability. The formation of Girard P hydrazones of T and Nand esters provides the best results compared to the underivatized compounds, oxime and Girard T derivatives, respectively. The presented analytical method is specific for all considered compounds and linear in the range of concentrations investigated (0.25-10 ng/mL). The LODs are between 0.03 and 0.30 ng/mL, the extraction recovery higher than 70 % for all esters and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. Finally, the developed and validate method was applied in the analysis of serum samples collected after the administration of a single dose (40 mg, 1 capsule) of testosterone undecanoate (Andriol ®) demonstrating its applicability.

Background Urinary sulfate fraction of the anabolic androgenic steroids is not analyzed routinely in anti-doping analyses but has demonstrated in the last years an increasing interest among the anti-doping community. Sulfate conjugates are linked to plasma proteins increasing the residence time in the body compared to glucuro-conjugated metabolites, and then their analyses may allow improving the detection time window of specific metabolites. Hydrolysis of sulfates can be made enzymatically or chemically and can be challenging, depending on the strategy selected. Methods Hydrolysis by solvolysis was validated for metabolic studies, focusing on setting a quality control able to assess the hydrolytic step. To the internal standards mixture, androsterone-D4 and etiocholanolone-D5 sulfate were added. The proposed protocol was applied over samples collected after dehydroepiandrosterone (DHEA) administrations. Results The stability of the structures showed good results, and no evident formation of degradation products was observed. Internal standard to monitor the efficiency of hydrolysis, recovery, and retention time was successfully introduced. Additional analytes (4β-hydroxy-DHEA, 5-androstene-3β,17β-diol and 5α-androstane-3β,17β-diol) were found to be affected besides of DHEA and epiandrosterone (epiA) as previously described. Conclusions Results in terms of linearity, precision, and accuracy, showed that the method is suitable to quantify seven analytes in urine in the sulfated fraction. The validated method was successfully applied to urine samples after administration of DHEA to detect this compound in the sulfate fraction and preliminarily to negative samples from athletes of both sexes, to determine Q1 and Q3 inter-quartiles. A quality control assessment for the hydrolysis efficiency was established for every individual sample.

Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s using different techniques, the aim of this study was to investigate the hydroxylation in positions C2, C4 and C6 after in vitro experiments and in vivo excretion studies using gas chromatography time-of-flight (GC/TOF) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The results could be influenced by the mass spectrometric analyser used.Incubations were carried out with human liver microsomes and six enzymes belonging to the cytochrome P450 family using MT as a substrate. The trimethylsilyl derivatives of the samples were analysed using GC/TOF and GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6-hydroxymethyltestosterone (6-OH-MT) to avoid artefact interferences. A urinary excretion study was then performed after the administration of a 10 mg single oral dose of MT to a volunteer.The formation of hydroxylated metabolites of MT in the C6, C4 and C2 positions after both in vitro and in vivo experiments was observed. Sample evaluation using GC/TOF showed an interference for 6-OH-MT that could only be resolved in GC/MS/MS by monitoring specific transitions. The transitory detection of these hydroxylated metabolites in urine agrees with previous investigations that had described this metabolic route as being of little significance.In doping analysis, the formation of 4-hydroxymethyltestosterone (oxymesterone) from MT cannot be underestimated. Although it is only detected as a minor and short-term excretion metabolite, it cannot be overlooked as it was found in both in vitro and in vivo experiments. The use of a combination of different mass spectrometric instruments allowed reliable conclusions to be reached, and it was shown that special attention must be given to artefact formation.

In this work, we present a gas-chromatography tandem mass spectrometry (GC-MS/MS) method for the identification of the sulfo-conjugate metabolites of pseudo-endogenous steroids (endogenous steroids when administered exogenously). We have preliminarily evaluated the performances of different preparations of sulfatases from Pseudomonas aeruginosa and Helix pomatia, characterized by various origins and catalytic activities, and compared the efficacy of the enzymatic hydrolysis with chemical hydrolysis, performed with a mixture of ethyl acetate, methanol, and sulphuric acid. A procedure for the selective isolation of steroid conjugates from the urine matrix has been designed and optimized, based on the "sequential" extraction of the glucuro-conjugated and of the sulfo-conjugated fractions, performed by two different direct methods, i.e. by ion paired extraction or solid-phase extraction. More specifically, the former method is based on the use of N,N-dimethylephedrinium bromide as the ion paired extraction reagent, while the latter on the use of WAX® (weak anion exchange) cartridges. The performance of the newly developed procedure has been assessed by the analysis of real urine excretion samples collected after the oral intake of a single dose of dehydroepiandrosterone (DHEA) or androstenedione (AED), measuring the concentration of epiandrosterone (EpiA) sulfate. Our results have shown the following: (i) although the yields of chemical hydrolysis and enzymatic hydrolysis are in some cases quite similar, the former is generally preferable since it results in the quantitative cleavage of sulfate moiety; (ii) ion paired extraction has been selected as the most reliable method for direct isolation of sulfate steroids from urine matrices; (iii) EpiA sulfate allows to prolong the detectability of DHEA and AED when compared to routinely used steroidal target compounds.

The standard approach to detect misuse with testosterone in sport is based on the determination and evaluation of the urinary steroid profile followed by the confirmation of atypical profiles using isotope ratio mass spectrometry. The detection capacity of these methods can be attenuated by confounding factors or testosterone preparations with endogenous isotopic fingerprints. An alternative detection method for misuse of an endogenous steroid in sports is the direct detection of the administered steroid ester present in most preparations. Thus unambiguous proof for doping misuse can be delivered. In this work, the sensitivity of gas chromatography coupled to a triple quadrupole with chemical ionization (GC-CI-MS/MS) is applied to detect trace levels of 10 testosterone and 2 nandrolone esters in plasma for in human doping analysis. The detection method was developed employing a liquid-liquid extraction and HPLC cleanup step before analysis on the GC-CI-MS/MS. The quantitative method was validated in a linear range of 100-2000 pg/ml and proved to be selective, reproducible and very sensitive with limits of detection as low as to 10 pg/ml. A clinical study with the administration of testosterone undecanoate in 3 volunteers was carried out and the compound was detectable up to 86 days after administration.

Testosterone is one of the most abused pseudo-endogenous anabolic steroids in sport doping. The current method adopted to detect the abuse of testosterone and other pseudo-endogenous steroids (endogenous steroids when administered exogenously) is first based on the longitudinal monitoring of several urinary biomarkers, which constitute the so called “steroidal module” of the Athlete Biological Passport (ABP): atypical samples undergo a confirmation analysis based on the measurement of the 13C/12C isotopic ratio of selected target compounds, to distinguish their endogenous or exogenous origin. At the same time, testosterone administration can be allowed in athletes diagnosed with hypogonadism, provided they are granted a therapeutic use exemption by the relevant medical authority. In this pilot study we have investigated whether the approach based on the preliminary determination of the urinary steroid profile, in the format considered in the steroidal module of the ABP, also integrated with the inclusion of the sulfo-conjugates and of additional target steroids, can retain its validity also in the case of hypogonadal athletes. We have studied the effects of a single low dose (40 mg) of testosterone gel (T-gel) on the urinary concentration of the markers of steroidal module of the ABP, as well as on some additional steroid markers. The study was based on the analysis of urinary samples from 19 non-hospitalized hypogonadal men, 10 of them with late-onset hypogonadism (LOH), collected before, after 4 h and after 24 h the transdermal self-administration of 40 mg of T-gel. None of the patient had any co-morbidities possibly affecting the urinary excretion of the steroidal markers. The steroidal markers were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) after the enzymatic hydrolysis of the respective glucuro-conjugates and the chemical hydrolysis of the respective sulfo-conjugates. Targeted GC-MS/MS analysis was carried out operating in electron impact (EI) ionization mode, with acquisition in multiple reaction monitoring (MRM) mode. Our preliminary results show that, as expected, the treatment with T-gel leads, in all hypogonadal men, to an increase of the urinary concentration of the glucuro-conjugate metabolites of testosterone and its main metabolites, with special relevance to those with 5α-reduction. Furthermore, samples collected from non-LOH hypogonadal men showed an increase also in the levels of epitestosterone glucuronide, testosterone sulfate and epitestosterone sulfate. Apart from their biochemical and pharmacological relevance, these outcomes could be leveraged to refine the analytical strategy currently followed in the antidoping field for the analysis of the urinary steroidal markers, with potential implications also in other forensic and/or clinical investigations.

Processing of semi-crystalline thermoplastic matrix composites for aeronautical applications experienced some problems for the high temperatures required and for the need to control the cooling rate, in order to obtain the correct crystallinity content in the final product. The approach of processing the material in the amorphous state would allow lower processing temperatures, but the crystallization isn’t satisfactory (cold crystallization). A new approach, based on hybrid semicrystalline-amorphous thermoplastic composites has been considered, potentially overcoming the mentioned problem, and the main issues of this concept are described. The approach is presently under investigation in a multinational research program supported by the European Commission (NHYTE).

Anectodical information suggests that flavonoids may be widely used among athletes for their multiple biochemical and pharmacological effects. We have evaluated in vitro the effects of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the catalytic activity of human aromatase (CYP19), the enzyme catalyzing the conversion of androgens (ie, testosterone or androstenedione) to estrogens (ie, estradiol and estrone). The potential inhibitory effect was evaluated by measuring the rate of aromatization of testosterone, monitored by gas chromatography-mass spectrometry (GC-MS), both in the presence and in the absence of methoxyisoflavone or ipriflavone, comparing their effects with those of synthetic aromatase inhibitors (formestane, anastrozole, and aminoglutethimide) presently included in the list of prohibited substances and methods, and of natural flavonoids (chrysin, quercetin, and daidzein), that are known inhibitors of CYP19. The preliminary results of our in vitro study show that methoxyisoflavone and ipriflavone act as competitive inhibitors of aromatase, the degree of inhibition measured in vitro being of the same order of magnitude of that of the aromatase inhibitors commonly used in anti-estrogenic therapies. Our preliminary in vitro results indicate that, in principle, a sufficiently large intake of isoflavones could alter the kinetics of the dynamic equilibria between androgens and estrogens, suggesting their monitoring in doping control routine analysis.

The paper describes a system that it is able of monitoring the health state of a composite structure in real time. The hardware of the system consists of a wire of strain sensors connected to a control unit. The software of the system elaborates the strain data and in real time is able to detect the presence of an eventual damage of the structures monitored with the strain sensors. The algorithm requires as input only the strains of the monitored structured measured on real time, i.e. those strains coming from the deformations of the composite structure due to the working loads. The health monitoring system does not require any additional device to interrogate the structure as often used in the literature, instead it is based on a self-learning procedure. The strain data acquired when the structure is healthy are used to set up the correlations between the strain in different positions of structure by means of neural network. Once the correlations between the strains in different position have been set up, these correlations act as a fingerprint of the healthy structure. In case of damage the correlation between the strains in the position of the structure near the damage will change due to the change of the stiffness of the structure caused by the damage. The developed software is able to recognize the change of the transfer function between the strains and consequently is able to detect the damage.

Blends based on the semicrystalline Poly(ether ether ketone) – PEEK and the amorphous Poly(ether imide) – PEI have been widely investigated for their outstanding mechanical, thermal and chemical-physical properties. The conformation of the PEI monomer affects the miscibility of the blends. Although the large interest in high demanding lightweight applications, foams from PEEK/PEI blend have not been considered until recently. The aim of this work was to prepare foams from PEEK/PEI miscible blends by using supercritical carbon dioxide. The role of blend composition on the density and the morphological parameters of the resulting foams was investigated.

We have investigated the influence of oral miconazole administration on the urinary concentrations of endogenous anabolic androgenic steroids of doping relevance, specifically considering all these compounds routinely monitored in doping control analysis, in the framework of the steroidal module of the “athlete biological passport”, and other steroids, including dehydroepiandrosterone, 5α-dihydrotestosterone, and the hydroxylated metabolites recently proposed as additional markers of the intake of testosterone-related steroids (16α-hydroxy-androsterone, 16α-hydroxy-etiocholanolone, 6β-hydroxy-androsterone, 6β-hydroxy-etiocholanolone, 7α-hydroxy-dehydroepiandrosterone, and 7β-hydroxy-dehydroepiandrosterone). Urinary concentrations of the final metabolic products of the glucocorticoid biosynthetic pathways (11β-hydroxy-androsterone and 11β-hydroxy-etiocholanolone, the formerly used as an endogenous reference compound for the gas chromatography–combustion-isotope ratio mass spectrometry confirmation analysis) were also monitored. Two healthy Caucasian volunteers exhibiting physiologically high testosterone/epitestosterone ratios and elevated concentrations of the main target steroids were selected for the study. Miconazole was administered orally (500 mg/day) for 1 week. Multiple urine samples were collected for 1 week before and during the treatment, and analyzed according to a validated analytical procedure based on gas chromatography–electron ionization-mass spectrometry in selected ion monitoring mode. Our results indicated that oral administration of miconazole decreased the urinary concentrations of androsterone, and to a lesser extent, of etiocholanolone (both detected as the sum of free and glucuronated steroids), and consequently the androsterone/testosterone and androsterone/etiocholanolone ratios. Furthermore, the urinary concentrations of 16α-hydroxy-etiocholanolone, 16α-hydroxy-androsterone, 7β-hydroxy-dehydroepiandrosterone, 6β-hydroxy-etiocholanolone, 7α-hydroxy-dehydroepiandrosterone, 6β-hydroxy-androsterone, 11β-hydroxy-androsterone, and 11β-hydroxy-etiocholanolone were significantly suppressed. This evidence suggests the potential intake of miconazole whenever the urinary steroid profile is characterized by abnormally low concentrations of the above-mentioned steroids.

Blends obtained by mixing high temperature applications thermoplastics have been investigated. Namely the blends considered in this work are made by semi-crystalline thermoplastics PEEK with amorphous PEI. The final goal is to analyse the mechanical, chemical-physical and environmental resistance characteristics of these blends to evaluate their suitability as matrices of carbon reinforced composites for aeronautical structural applications. The first collected results are very promising.

Environmental ageing in composites is a very important issue for the effect induced on material properties. The most common effect is induced by absorption of low molecular weight substances (water or organic solvents), usually controlled by diffusion mechanisms. Some cases of use of hybrid materials, coupling low diffusion plies with "conventional" ones are discussed and potential improvement of composites for structural applications based on such concept is envisaged. Specifically, metal hybrid composites (GLARE), multilayer amorphous-semicrystalline materials and composites modified with layer of nanocharges or nano-reinforcements are considered, and the absorption kinetics are evaluated by a numerical model.

The most important aspects of composite usage in aircraft structures are discussed. Different kinds of fiber reinforcement and matrix are compared. A large part of the article is focused on carbon epoxy prepregs, that is, preimpregnated materials having a continuous fiber reinforcement and an epoxy resin matrix. The major issues of prepreg processing are outlined, with a description of the different phases of the process. Most of the discussion is focused on the lay-up plus autoclave processing, which is the most frequent processing approach. A short comparison between laminate and sandwich structures is also reported. “No prepreg” techniques are also listed, such as resin transfer molding and resin infusion molding. Elements are reported of composite structural properties, including the effect of temperature, aging, and damage tolerance. Some additional information on aging and accelerated aging techniques is also included. The behavior of composites exposed to fire, as well as functional properties (conductivity, acoustic damping, etc.) are also shortly listed. The advantages of lighter structures are considered taking in account also the cost issues, and some specific applications of composites in the aircraft structures are described, stressing the very fast increase of these materials in the aeronautic structural applications. Finally, the future trends in the technological improvement of composite material, processes, and structures for aeronautical application are shortly outlined. Keywords: composite materials; aeronautics; prepregs; carbon fiber; epoxy; aeronautical applications

Cooling rate of semicrystalline thermoplastic (PEEK) based composite parts has been assessed by means of a thermal flow model. Cold tool thermoforming was found suitable for thin parts, but the use of a preheated tool is suggested. Cooling rate is critical for automated lay‐up; consequently the optimization of this technique requires the use of a complex apparatus, able to provide both heating of the part during lay‐up and a control of the cooling rate. Alternatively a modified cooling rate vs. crystallinity behavior of the material must be achieved.

A new approach has been considered for aeronautical composite structures with thermoplastic matrix. The new concept is based on the addition of amorphous PEI films to a semicrystalline thermoplastic PEEK/Carbon Fiber prepreg. The performed chemical-physical analysis demonstrate the good blending between the two resins. The new approach, being presently developed in a project funded by EU, could allow to perform structural parts with affordable costs through the utilization of automated lay-up (ATL and/or AFP) techniques, with a strong cost saving due to the elimination of the autoclave cycle.

Composites world is in continuous evolution and there has been a progressive change in terms of manufacturing processes, passing from standard wet or prepreg manual layup to automated (preforming) technologies, with the objective to increase production rates and make cheaper manufacturing processes. This chapter deals with most recent advancements and applications of thermoplastic composites, focusing on the reasons why for aerospace sector, they are increasingly representing a more viable manufacturing solution for structural components. Starting from thermoplastic polymer structures and difference with respect to thermoset matrix based composites, the standard consolidation processes (autoclave/thermoforming) together with most promising automated and continuous out-of- autoclave manufacturing concepts and processes (Automated Fiber Placement/Automated Tape Laying, In Situ Consolidation, Continuous Compression Molding, Pultrusion), including assembling methods (Fusion/Welding), are illustrated. Furthermore, an overview on different recycling concepts related to thermoplastics and thermosets composites is provided. At last, an overview on the European thermoplastic development roadmap, supported by the EU’s Horizon 2020 Research and Innovation program (2014–2021) for the next generation of aircrafts, is illustrated