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Abstract: New analogues of androgens that had never been available as approved drugs are marketed as “dietary supplement” recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product “1-Androsterone” of the brand name “Advanced Muscle Science” was labeled to contain 100mg of “1-Androstene-3b-ol,17-one” per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC–MS. The steroid was identified by comparison with chemically synthesized 3β-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3β-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17β-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17β-diol, and 5α-androst-1-ene-3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of “1-Androsterone”. Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule. [Copyright &y& Elsevier]

Abstract: Since a few years more and more products have appeared on the market for dietary supplements containing steroids that had never been marketed as approved drugs, mostly without proper labeling of the contents. Syntheses and few data on pharmacological effects are available dated back mainly to the 1950s or 1960s. Only little knowledge exists about effects and side effects of these steroids in humans. The present study reports the identification of Δ6-methyltestosterone in a product named “Jungle Warfare”, which was obtained from a web-based supplement store. The main urinary metabolites, 17α-hydroxy-17β-methylandrosta-4,6-dien-3-one (Δ6-epimethyl-testosterone), 17α-methyl-5β-androstane-3α,17β-diol (3α,5β-THMT), and 17β-methyl-5β-androstane-3α,17α-diol, as well as the parent compound excreted after a single oral administration were monitored by GC–MS/MS. Δ6-Epimethyltestosterone and 3α,5β-THMT served for long-term detection (still present in the 181–189h urine). 17α-Methyltestosterone and its 17-epimer were not detected in the urines (LOD 0.3ng/mL). The highest concentrations were found in the 14–20.5h urine for Δ6-epimethyltestosterone (600ng/mL), and 3α,5β-THMT (240ng/mL) and in the 36–44.5h urine for 17β-methyl-5β-androstane-3α,17α-diol (7ng/mL). For reference methyltestosterone and epimethyltestosterone were dehydrogenated with chloranil. The characterization of the products was performed by GC-MS(/MS) and NMR. [Copyright &y& Elsevier]

Abstract: The product Orastan-A from Gaspari Nutrition was analyzed for its steroid content. According to the labeling, it is supposed to contain “5a-Androstano[2,3-c]furazan-17b-tetrahydropyranol ether”, also called furazadrol-THP ether. The GC–MS analyses of the liberated steroids (after extraction from the capsule matrix and cleavage of the THP ether, TMS-derivative and underivatized) revealed mass spectra of two components, both inconsistent with the labeling. Thus, the steroids were characterized by different analytical techniques such as mass spectrometry, nuclear magnetic resonance spectroscopy and X-ray crystal structure analysis. They were identified as 17β-hydroxyandrostano[3,2-c]isoxazole and -[2,3-d]isoxazole. [Copyright &y& Elsevier]

Abstract: In human sports doping control analysis most of the steroids are analyzed after enzymatic hydrolysis of the glucuronides as per-trimethylsilyl (TMS) derivatives applying gas chromatography–mass spectrometry (GC–MS). According to the recommendations of the World Anti-Doping Agency the identification of analytes should be based on retention time and on mass spectrometric characterization. This study shows that the bis-TMS derivatives of 16 specific C19 steroids, namely the stereoisomers of 5ξ-androst-1-ene-3ξ,17ξ-diol (8 isomers), androst-4-ene-3ξ,17ξ-diol (4 isomers), and 17ξ-hydroxy-5ξ-androstan-3-one (4 isomers), reveal very similar mass spectra. As a rule, when taking the retention times, which are provided as Kovac indices for all these isomers, into account, a restriction to two or three possible isomers is possible. Reliable identification should additionally include a comparison of the retention times of the analytes with the reference compounds measured concomitantly. In some cases standard addition may be appropriate. Due to the limited availability, the above mentioned isomers were synthesized by reduction of the corresponding α,β-unsaturated oxo steroids either with K-Selectride or by catalytic hydrogenation (Pd/C as catalyst). The products of the reactions were identified by means of nuclear magnetic resonance (NMR) characterization and by further reduction to the corresponding 5ξ-androstane-3ξ,17ξ-diols and GC–MS comparison with commercially available reference standards. [Copyright &y& Elsevier]

Abstract: 4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography–mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3β-hydroxy-5α-androstane-4,17-dione (2) and 3α-hydroxy-5β-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3ξ,4ξ-dihydroxy-5ξ-androstan-17-one (4, 6–11) except 3α,4α-dihydroxy-5β-androstan-17-one (5). Out of the 17β-hydroxylated analogs 4-hydroxytestosterone (18), 3β,17β-dihydroxy-5α-androstan-4-one (19), 3α,17β-dihydroxy-5β-androstan-4-one (20), 5α-androstane-3β,4β,17β-triol (21), 5α-androstane-3α,4β,17β-triol (26) and 5α-androstane-3α,4α,17β-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation. [Copyright &y& Elsevier]

Chromatographic modeling software packages are valuable tools during method optimization steps. These are well established for reversed‐phase applications utilizing retention time (RT) prediction to optimize separations and receive robust methods, which is of high interest for the analysis of pharmaceuticals. In contrast to liquid chromatography, the knowledge of RT prediction in supercritical fluid chromatography is limited to a manageable number of studies. This study uses the software DryLab to predict the RTs of the peptides bacitracin (Bac), colistin, tyrothricin (Tyro), and insulin analogs. Gradient time, column temperature, and the ternary composition (terC) of carbon dioxide, methanol (MeOH), and acetonitrile (ACN) in the gradient elution are varied in a feasibility approach using a neutral (Viridis BEH) and an amino‐derivatized aromatic (Torus 2‐PIC) stationary phase. In the second part, chromatographic optimization is performed in silico through gradient adjustments to optimize the separation of the fingerprint of Bac. The final gradient method utilizes a Viridis BEH column (100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of ACN/MeOH/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v). In addition, changes in the retention order of Tyro compounds with the proportion of the terC in combination with a Torus Diol column are investigated. [ABSTRACT FROM AUTHOR]

The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed‐phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 μm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18‐crown‐6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH). [ABSTRACT FROM AUTHOR]

Highlights • Review of recent publications in the field of bioanalysis. • Enantioselective chromatography using supercritical fluids as mobile phase constituents (SFC). • Method development in bioanalytical SFC with strongest impact of chiral stationary phases for success. • Method validation showing fitness for purpose in routine applications. Abstract This review covers literature investigating methods for enantioselective chromatography using supercritical fluids as mobile phase constituents (SFC) in the field of bioanalysis. It provides an overview on method development and screening approaches published in scientific literature 2014-2018. Chiral stationary phases are used to create a chiral environment that allows for discrimination of enantiomers. Especially polysaccharide-based stationary phases are used in methods of recent investigations. In comparison to HPLC chiral SFC separation provides more selective cavity effects of inclusion-type chiral separation phases. Modifier and additive choices as well as further operating conditions like backpressure, temperature and flow rate are summarized and critically discussed. Further on, observed sample pretreatment and possible detection techniques are presented. SFC hyphenated to mass detection was found of major relevance and is therefore further discussed. Coupling of SFC with different detectors allows for straightforward use in bioanalysis. Interfacing MS detectors is generally performed including a make-up pump. Thus, applied make-up conditions were also reviewed. While most of the chiral separations in HPLC are performed in normal phase mode, and thus, challenge MS hyphenation, SFC-MS hyphenation can be easily achieved. This allows for convenient application in chiral trace analyses, often required in bioanalysis. Even worse in enantioseparation than in achiral chromatography, method development in SFC suffers from a lack of knowledge in separation mechanisms and thus approaches are often quite unique and most often achieved by screening using a One-Factor-at-a-Time (OFAT) design. Broad screening experiments with methodical approaches still appear as method of choice for now. [ABSTRACT FROM AUTHOR]

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2‐PIC columns (each 100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed. [ABSTRACT FROM AUTHOR]

• Concomitant enantioseparation of Terbutaline and its metabolite pairs by SFC • Delimited response surface of Terbutaline and its metabolite for maximum robustness. • Multivariate crosstalk between method parameters takes place in SFC separations. • Superiority of systematic multivariate design approach in enantioseparation by SFC. Terbutaline is mainly metabolized by sulfoconjugation stereoselectively, favoring its (S) - (+) enantiomer. Reported chiral separations of Terbutaline enantiomers were achieved by various chromatographic methods. However, the simultaneous enantioseparation of Terbutaline and the monosulfate conjugate metabolites was never reported. This study aims at shedding light on the influential factors and interactions leading to successful enantioseparation of Terbutaline and its monosulfate conjugate pairs by Supercritical Fluid Chromatography (SFC) for the first time within a Quality by Design framework using Design of Experiments. The effect of molarity of mobile phase additive, mobile phase flow rate, column temperature and back pressure were evaluated. Compared to previous reports, the response surface interestingly revealed the favorability of high temperature and high flow rate up to 2.25 ml/min for resolution of the two pairs of enantiomers on polysaccharide chiral stationary phase CHIRALPAK IC. In addition, a switch in the elution order of Terbutaline and the sulfate conjugate peak pairs was observed upon elimination of the mobile phase additive where the sulfate conjugate underwent intra-molecular ionic interactions and the change in elution order was only due to TER behavior. The multifactorial interactions would not have been detected with the common one-factor-at-a-time approach during method development, demonstrating the superiority and importance of the Analytical Quality by Design frame in enantioseparation. [ABSTRACT FROM AUTHOR]

The application area of supercritical fluid chromatography expanded tremendously over the last years and more polar analytes such as biomolecules have become accessible. The growing interest in biopharmaceuticals and associated regulatory requirements demand alternative analytical tools. The orthogonal nature of supercritical fluid chromatography compared to reversed‐phase liquid chromatography meets these needs and makes it a useful option during research and development. In this study, we present a systematic approach for the development of a supercritical fluid chromatography method for fingerprinting of tyrothricin, a complex therapeutic peptide covering a mass range from 1200 to 1900 Da. The substance was chosen due to the presence of cyclic and linear peptides and isomeric or highly similar amino acid sequences. Different column chemistries covering neutral, basic, and zwitterionic functionalities in combination with acidic, basic, and neutral additives were screened. Subsequently, Design‐of‐Experiments principles were utilized to perform optimization of the chromatographic parameters. The final mass spectrometry‐compatible gradient method using a diol stationary phase, carbon dioxide, and a modifier consisting of methanol/water/methanesulfonic acid (100:2:0.1, v:v:v) was found to provide orthogonality and superior resolution to other methods published. Isomeric peptide compounds coeluting in reversed‐phase liquid chromatography were resolved by applying the final method. [ABSTRACT FROM AUTHOR]

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

The role of psychoactive substances in the treatment of mental disorders and the risk of suicide are major public health issues. This cross-sectional study examined the prevalence of antidepressants and antipsychotics detected in toxicological screenings in suicides. Cases from the Institute of Legal Medicine of the Charité-University Medicine Berlin were reviewed over a 4-year-period. All cases (n = 477) with positive toxicology for antidepressants and antipsychotics in blood or organ tissue were included. Frequencies of the detected substances in non-suicide cases (n = 212; male n = 177, 55.2%; female n = 95, 52.5%) and suicide cases (n = 235; male n = 149, 63.4%; female n = 86, 36.6%) were examined. Tricyclic antidepressants (48.1%) were found most frequently in suicides, followed by atypical neuroleptics (37.0%), selective serotonin reuptake inhibitors (28.1%), typical neuroleptics (17.4%), tetracyclic antidepressants (16.2%) and other substances (8.9%). Alcohol was detected in 37.2% of suicides. The leading cause of death was drug poisoning (35.6%) followed by polytrauma (26.8%) and death by hanging (18.5%). A mental disorder (depression, schizophrenia, bipolar disorder, suicidality) was known in 22.9% of suicides. The most common location of death was the person's own house (63.8%) followed by public places (28.1%) and hospitals (8.1%) The five most common substances in the suicide group were doxepin (20%) citalopram (15.3%), mirtazapine (14.9%), quetiapine (13.6%) and amitriptyline (12.3%). Toxicological findings from cross-sectional studies provide insight into how often certain types of antidepressants and antipsychotics are associated with suicide. A complementary approach is valuable for assessing the risk of suicide during medical treatment because the various available approaches (analysis of suicidal behavior/ideation, toxicity of drugs) each have strengths and limitations. [ABSTRACT FROM AUTHOR]

Three nutritional supplements with "1-T matrix" were purchased by telephone order. The supplier of these supplements is a company from the UK, and the manufacturer is from the U.S. Aliquots of 1 g each were analyzed with gas chromatography / mass spectrometry and high pressure liquid chromatography with UV detector. In all supplements analyzed were found the anabolic-androgenic steroid metandienone. Metandienone, only available by prescription, was not declared on the labels. The concentrations of metandienone were 17.30 mg/g, 0.41 mg/g, and 0.96 mg/g for supplements 1, 2, and 3. In supplement 1, the concentrations of metandienone varied from capsule to capsule. Maximum concentrations of metandienone of 28.9 mg/g were detected. In addition to enormous health risks, the use of the analyzed supplements can lead to positive doping results for metandienone. The results of this study show that it is of major importance to improve the surveillance of the production and trade of dietary supplements. The first step should be a warning to be issued to consumers and the withdrawal of dietary supplements containing prescription drugs. [ABSTRACT FROM AUTHOR]

• Universal method for nitrosamine analysis. • Novel SFC approach for simultaneous detection of 16 nitrosamines. • High sensitivity and selectivity within extreme short analysis time (separation of nitrosamines in 4 min; total run time 11.5 min). • Case study for lifecycle management with proof of concept. • Development under Quality-by-Design and validation according to ICH Q2. Since the detection of nitrosamines (NA) in valsartan pharmaceuticals, over two years have passed. At present, the occurrence of NAs can be limited to a few drug substances and drug products, but it is already becoming apparent that the issue appears to be much bigger than initially thought. The impact on the global pharmaceutical market has been tremendous and the problem can be attributed mainly to uncritically adopted approval changes and the lack of suitable, modern analytical methods to detect those impurities in time. We hereby demonstrate how lifecycle management (LCM) can be used to develop and improve suitable and universal analytical methods within short time. The resulting SFC-MS/MS method is intended for a universal nitrosamine investigation in drug substances and drug products. Successful NA analysis was demonstrated for seven sartans, metformin, pioglitazone and ranitidine. Additionally, combination drug products, containing also amlodipine, hydrochlorothiazide, vildagliptin and sitagliptin, were analyzed successfully. The method achieved separation of 16 NAs in 4 min with a total run time of 11.5 min, utilizing a Supel Carbon porous graphitic carbon (PGC) column. Carbon dioxide together with 0.1% TFA in methanol as modifier were used as eluents and 0.35% formic acid in methanol as make-up solvent for mass spectrometric NA detection. By implementing LCM in this case study, development time was reduced and knowledge was implemented fast. At the same time, a high adaptability of this "vital" method was achieved, which makes it possible to implement the constantly changing regulatory requirements within the shortest possible time. Supplemental development data, according to the ICH guidelines Q8, Q12 and the proposed Q14 are disclosed, demonstrating the scientific Quality-by-Design (QbD) development approach, the "fitness for use" and the robustness of the analytical procedure. This method contributes to the still ongoing risk assessment process of the pharmaceutical industry and the regulatory agencies, in order to understand root causes of NA formation, maintain the drug supply and prevent drug shortage. [ABSTRACT FROM AUTHOR]

Cancer treatment often lacks individual dose adaptation, contributing to insufficient efficacy and severe side effects. Thus, personalized approaches are highly desired. Although various analytical techniques are established to determine drug levels in preclinical models, they are limited in the automated real-time acquisition of pharmacokinetic profiles. Therefore, an online UHPLC-MS/MS system for quantitation of drug concentrations within 3D tumor oral mucosa models was generated. The integration of sampling ports into the 3D tumor models and their culture inside the autosampler allowed for real-time pharmacokinetic profiling without additional sample preparation. Docetaxel quantitation was validated according to EMA guidelines. The tumor models recapitulated the morphology of head-and-neck cancer and the dose-dependent tumor reduction following docetaxel treatment. The administration of four different docetaxel concentrations resulted in comparable courses of concentration versus time curves for 96 h. In conclusion, this proof-of-concept study demonstrated the feasibility of real-time monitoring of drug levels in 3D tumor models without any sample preparation. The inclusion of patient-derived tumor cells into our models may further optimize the pharmacotherapy of cancer patients by efficiently delivering personalized data of the target tissue. [ABSTRACT FROM AUTHOR]