Probing the hair detectability of prohibited substances in sports: an in vivo-in silico-clinical approach and analytical implications compared with plasma, urine, and faeces.

Hair analysis is a crucial method in forensic toxicology with potential applications in revealing doping histories in sports. Despite its widespread use, knowledge about detectable substances in hair is limited. This study systematically assessed the detectability of prohibited substances in sports using a multifaceted approach. Initially, an animal model received a subset of 17 model drugs to compare dose dependencies and detection windows across different matrices. Subsequently, hair incorporation data from the animal experiment were extrapolated to all substances on the World Anti-Doping Agency's List through in-silico prediction. The detectability of substances in hair was further validated in a proof-of-concept human study involving the consumption of diuretics and masking agents. Semi-quantitative analysis of substances in specimens was performed using ultra-performance liquid chromatography-tandem mass spectrometry. Results showed plasma had optimal dose dependencies with limited detection windows, while urine, faeces, and hair exhibited a reasonable relationship with the administered dose. Notably, hair displayed the highest detection probability (14 out of 17) for compounds, including anabolic agents, hormones, and diuretics, with beta-2 agonists undetected. Diuretics such as furosemide, canrenone, and hydrochlorothiazide showed the highest hair incorporation. Authentic human hair confirmed diuretic detectability, and their use duration was determined via segmental analysis. Noteworthy is the first-time reporting of canrenone in human hair. Anabolic agents were expected in hair, whereas undetectable compounds, such as peptide hormones and beta-2 agonists, were likely due to large molecular mass or high polarity. This study enhances understanding of hair analysis in doping investigations, providing insights into substance detectability.

Diuretic and Antiurolithic Effect of Garcinia humilis (Vahl) C.D. Adams Leaves, a Medicinal Plant Native to South American Countries.

This study evaluated the diuretic and antiurolithic effect of methanolic extract (MEGHL), dichloromethane (DCM), and ethyl acetate (EtA) fractions obtained from the leaves of Garcinia humilis, a medicinal plant known as achachairu and native to South American countries such as Bolivia, Peru, and Brazil. For the analysis of diuretic effect, the female rats received the treatment with MEGHL (3, 10, and 30 mg/kg), DCM (1, 3 and 10 mg/kg), EtA (1, 3, and 10 mg/kg), hydrochlorothiazide (HCTZ; 10 mg/kg), or vehicle (VEH) after an overload of saline solution. At the end 8 h of the experiment, the urinary parameters were measured. Additionally, the antiurolithic effect was analyzed, in which sodium oxalate was added in synthetic urine in the presence or absence of MEGHL, DCM, and EtA in different concentrations (0.1, 0.3, and 1 mg/mL). MEGHL, DCM, and EtA were able to promote 8-h diuresis in rats. MEGHL treatment at dose 30 mg/kg was accompanied by increased urinary Na+ , K+ and Cl- excretion. Moreover, the DCM and EtA fractions treatment increased K+ and Cl- excretion in the urine, although it does not cause any change in Na+ elimination. All the preparations were able to exert an antiurolithic effect in vitro, decreasing the number of calcium oxalate crystals of the monohydrate and dihydrate types. Taking together, the results presented herein showed that the preparations of G. humilis leaves are promising strategies to induce diuresis and antiurolithic effects.

An improved drop casting electrochemical strategy for furosemide quantification in natural waters exploiting chemically reduced graphene oxide on glassy carbon electrodes.

This work exploits the applicability of a chemically reduced graphene oxide (CRGO) modification on the electrochemical response of a glassy carbon electrode (GCE) for the first-time sensitive determination of furosemide in natural waters. The batch injection analysis (BIA) is proposed as an analytical method, where CRGO-GCE is coupled to a BIA cell for amperometric measurements. Acetate buffer (0.1 µmol L-1, pH 5.2) was used as the background electrolyte. The modification provided an increase in sensitivity (0.024 µA/µmol L-1), low limit of detection (0.7 µmol L-1), RSD (< 4%), and broad linear range (1-600 µmol L-1). Recovery tests performed in two different concentration ranges resulted in values between 89 and 99%. Recovery tests were performed and compared with high-performance liquid chromatography (HPLC) with UV-Vis detection using Student's t test at a 95% significance level, and no significant differences were found, confirming the accuracy of the method. The developed method is proven faster (169 h-1) compared with the HPLC analysis (5 h-1), also comparable with other flow procedures hereby described, offering a low-cost strategy suitable to quantify an emerging pharmaceutical pollutant. Graphical abstract.

Development and validation of a reversed-phase high-performance liquid chromatographic method with solid-phase extraction for the quantification of hydrochlorothiazide in ex vivo permeation studies.

Hydrochlorothiazide (HCT) is a diuretic used to treat hypertension. In order to study its intestinal permeation behavior applying an ex vivo methodology, a rapid, sensitive and selective reversed-phase liquid chromatography (RP-HPLC) method coupled with UV detection (RP-HPLC UV) was developed for the analysis of HCT in TC199 culture medium used as mucosal and serosal solutions in the everted rat intestinal sac model. Also, analytical procedures for the quantification of HCT by RP-HPLC with UV detection required a sample preparation step by solid-phase extraction. The method was validated in the concentration range of 8.05 × 10-7 to 3.22 × 10-5 m for HCT. Chromatographic parameters, namely carry-over, lower limit of quantification (1.4491 × 10-7 m), limit of detection (3.8325 × 10-8 m), selectivity, inter- and intraday precision and extraction recovery, were determined and found to be adequate for the intended purposes. The validated method was successfully used for permeability assays across rat intestinal epithelium applying the ex vivo everted rat gut sac methodology to study the permeation behavior of HCT.

Diuretic and antioxidant activities of the aqueous extract of leaves of Vepris heterophylla (Engl.) R. Let (Rutaceae) in rats.

BACKGROUND: Vepris heterophylla (Rutaceae) is a medicinal plant used empirically in African traditional medicine for many clinical conditions including edematous disorders and hypertension. V. heterophylla aqueous extract has been used in northern part of Cameroon by traditional healers for the treatment of arterial hypertension. The study aim was to assess the putative diuretic and antioxidant properties of V. heterophylla leaves aqueous extract. METHODS: Adult rats were administered with V. heterophylla leaves aqueous extract acutely (24 h) at doses 50, 100, 150, 200 and 250 mg/kg (per os). The two positive control groups received the diuretic drugs furosemide (5 mg/kg) and hydrochlorothiazide (HCTZ, 10 mg/kg), while negative control group received only an equivalent volume of distilled water. Urinary elimination of electrolytes in response to treatments was evaluated, together with changes in concentrations of creatinine, urea, aldosterone, glucose and albumin in urine and plasma. Various urinary indicators of kidney function and plasmatic markers of oxidative stress were also assessed. RESULTS: The findings indicated that the aqueous extract of V. heterophylla at doses ranging from 150 to 250 mg/kg caused a significant and dose-dependent increase of urinary water and electrolytes excretion in normal rats. The aqueous extract of the leaves of V. heterophylla accelerated the elimination of overloaded fluid. At the maximum of diuretic response, urinary osmolarity decreased significantly when compared with controls. Oral administration of aqueous extract at different doses produced a significant diuresis and slight increase in electrolytes (Na+, K+ and Cl-) excretion. The results obtained were compared with standard drug-furosemide (5 mg/kg) and hydrochlorothiazide (10 mg/kg). These effects were observed predominantly at 250 mg/kg dose. CONCLUSIONS: Our findings strongly suggest that V. heterophylla aqueous extract has diuretic and antioxidant activities, and deserves further studies considering the potential for the treatment of hypertension.

Simultaneous determination of valsartan, amlodipine besylate and hydrochlorothiazide using capillary zone electrophoresis (CZE).

A capillary zone electrophoresis method was developed for the simultaneous determination of valsartan (VAL), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) in their combined tablets. Separation was achieved on a fused silica capillary by applying a potential of 15 kV (positive polarity) and a running background electrolyte containing 40 mM phosphate buffer at pH 7.5 with UV detection at 230 nm. The samples were injected hydrodynamically for 3s at 0.5 psi and the temperature of the capillary cartridge was kept at 25 degrees C. Pyrazinoic acid was used as an internal standard. The method was validated according to ICH guidelines regarding specificity, linearity, limits of detection and quantitation, accuracy and precision, (Supplementary materials, Table S2). The method showed satisfactory linearity in the ranges of 10-200, 2-20 and 2-20 µg mL(-1) with LODs of 1.82, 0.39, 0.65 µg mL(-1) and LOQs of 5.51, 1.17, 1.96 µg mL(-1) for VAL, AML and HCZ, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their laboratory prepared mixtures and co-formulated tablets. The results were compared with reported methods and no significant differences were found. The proposed method can be used for quality control of the cited drugs in ordinary laboratories.

The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation.

Dietary supplements (DS) are intended for healthy people to maintain or improve their overall health. Its consumption is widespread in large part of the general population and at all levels of athletes. Nevertheless, DS use can also pose health risks to individuals and, in the case of athletes, may lead to adverse analytical findings (AAFs) due to the possibility of DS contamination or adulteration with doping agents banned by the World Anti-Doping Agency. Although educational initiatives are being performed in Brazil to warn the sports community about inadvertent doping cases, AAFs connected to the DS administration have been increasingly growing. The findings of DS analyzed by the Brazilian Doping Control Laboratory (LBCD), between 2017 and 2022, after Testing Authorities (TAs) analysis requests, showed an alarming number of tainted samples. Diuretics were the most common adulterants found in all supplement types. However, the profile of prohibited substances in manufactured and compounded dietary supplements (MDS and CDS, respectively) were distinct, with stimulants being most prevalent in MDS and anabolic agents in CDS products. Additionally, MDS samples generally presented higher estimated concentrations of banned substances (mg/g) than CDS samples (µg/g). The common practice of DS intake by athletes continues to be of great concern for a doping-free sport, given the high prevalence of prohibited substances detected in the analyzed samples by the LBCD. The current Brazilian scenario reinforces the importance of raising awareness in the sports community of the possible consequences of an unintentional doping case linked to DS use.

Highly sensitive voltammetric determination of hydrochlorothiazide using a glassy carbon electrode modified with Super P carbon black nanoparticles.

Hydrochlorothiazide is an extremely important diuretic that regulates body functions, which can prevent several diseases. However, the abuse of this diuretic is concerning since it does not require a medical prescription, particularly for aesthetic purposes such as weight loss, which can lead to various health problems, including ventricular arrhythmia. The present work aims to use a glassy carbon electrode modified with Super P carbon black (SPCB/GCE) to quantify hydrochlorothiazide through Linear Sweep Adsorptive Stripping Voltammetry (LSAdSV). The modification of the GCE with SPCB significantly improved the response of hydrochlorothiazide. Furthermore, due to the adsorptive nature of charge transport, applying preconcentration time enhanced sensitivity. The optimized system provided a linear range of 0.5 to 30.0 µmol L-1 with a detection limit of 0.083 µmol L-1. Pharmaceutical tablet analyses indicated approximately 25 mg per tablet, which was confirmed by the UV-vis and in agreement with that indicated by the manufacturer. Furthermore, analyses of the tea, synthetic urine, tap water and lake water samples indicated recovery values close to 100%, demonstrating that there was no matrix effect. Therefore, it is possible to infer that the proposed method together with the sensor modified with carbon black nanoparticles presented excellent results, demonstrating that it can be an alternative method of monitoring this drug in different samples.

Role of capillary electrophoresis in the fight against doping in sports.

At present the role of capillary electrophoresis in the detection of doping agents in athletes is, for the most part, nonexistent. More traditional techniques, namely gas and liquid chromatography with mass spectrometric detection, remain the gold standard of antidoping tests. This Feature will investigate the in-roads that capillary electrophoresis has made, the limitations that the technique suffers from, and where the technique may grow into being a key tool for antidoping analysis.

Simultaneous determination of aliskiren and hydrochlorothiazide in tablets and spiked human urine by ion-pair liquid chromatography.

An alternative method for analysis of aliskiren (ALI) and hydrochlorothiazde (HCT) in combined dosage forms by ion-pair reversed phase high performance liquid chromatography was developed and validated. The pharmaceutical preparations were analyzed using a C18 column (250 mm x 4.6 mm, 3 microm) with a mobile phase consisting of 25% methanol, 50% sodium monobasic phosphate aqueous solution containing 6 mM tetrabutylammonium bromide and 25% water at pH 7.2. Isocratic analysis was performed at a flow rate of 1 mL/min and a column temperature of 30 degrees C under direct UV detection at 210 nm. Paracetamol was used as internal standard. The validation was performed according to the ICH guidelines. The proposed method was linear over the concentration range of 0.250 to 60 and 0.1 to 10 microg/mL for ALI and HCT, respectively. The limits of detection and quantitation (LOD and LOQ) were 0.075 and 0.198 microg/mL, respectively, for ALI and 0.04 and 0.062 microg/mL, respectively, for HCT. The method proved to be specific, sensitive, precise and accurate with mean recovery values of 101.1 +/- 0.32% and 100.9 +/- 0.41% for ALI and HCT, respectively. The method robustness was evaluated by means of an experimental design. The proposed method was applied successfully to spiked human urine samples with mean recoveries of 98.8 +/- 0.36% and 98.1 +/- 0.21% for ALI and HCT, respectively.

Sensitivity equation for quantitative analysis with multivariate curve resolution-alternating least-squares: theoretical and experimental approach.

A new equation is derived for estimating the sensitivity when the multivariate curve resolution-alternating least-squares (MCR-ALS) method is applied to second-order multivariate calibration data. The validity of the expression is substantiated by extensive Monte Carlo noise addition simulations. The multivariate selectivity can be derived from the new sensitivity expression. Other important figures of merit, such as limit of detection, limit of quantitation, and concentration uncertainty of MCR-ALS quantitative estimations can be easily estimated from the proposed sensitivity expression and the instrumental noise. An experimental example involving the determination of an analyte in the presence of uncalibrated interfering agents is described in detail, involving second-order time-decaying sensitized lanthanide luminescence excitation spectra. The estimated figures of merit are reasonably correlated with the analytical features of the analyzed experimental system.

PtI2-catalyzed tandem 3,3-rearrangement/Nazarov reaction of arylpropargylic esters: synthesis of indanone derivatives.

An efficient PtI(2)-catalyzed tandem reaction of arylpropargylic esters, involving 3,3-rearrangement and Nazarov reaction, has been developed to produce 3-substituted and 3,3-disubstituted indanone derivatives. This approach provided a pathway to the synthesis of indanone skeletons in natural products.

Reversed-phase ultra-performance liquid chromatographic method development and validation for determination of impurities related to torsemide tablets.

A simple RP-ultra-performance LC method was developed and validated for determination of impurities related to torsemide tablets. The rapid method provided adequate separation of all known related impurities and degradation products. Separation was achieved on a Zorbax SB-C18 column (50 x 4.6 mm id, 1.8 microm particle size) with binary gradient elution, and detection was performed at 288 nm. The drug product was subjected to oxidative, hydrolytic, photolytic, and thermal stress conditions to prove the specificity of the proposed method. The linearity and recovery were investigated for known impurities in the range of 0.025 to 1.0%, with respect to the drug concentration in the prepared sample. The linearity of the calibration curve for each of the impurities and torsemide was found to be very good (r2 > 0.999). Relative response factors for each of the known impurities were established by the slope ratio method from the linearity study.

Identification of furosemide in hair in a post-mortem case by UHPLC-MS/MS with guidance on interpretation.

Testing for drugs in hair raises several difficulties. Among them is the interpretation of the final concentration(s). In a post-mortem case, analyses revealed the presence of furosemide (12 ng/mL) in femoral blood, although it was not part of the victim's treatment. The prosecutor requested our laboratory to undertake an additional analysis in hair to obtain information about the use of furosemide. A specific method was therefore developed and validated to identify and quantify furosemide in hair by UHPLC-MS/MS. After decontamination of 30 mg of hair, incubation in acidic condition, extraction with ethyl acetate, the samples were analyzed by UHPLC-MS/MS. Furosemide was found in the victim's hair at 225 pg/mg. However, it was not possible to interpret this concentration due to the absence of data in the literature. Therefore, the authors performed a controlled study in two parts. In order to establish the basis of interpretation, several volunteers were tested (four after a single 20 mg administration and twenty-four under daily treatment). The first part indicated that a single dose is not detectable in hair using our method. The second part demonstrated concentrations ranging from 5 to 1110 pg/mg with no correlation between dosage and hair concentrations. The decedent's hair result was interpreted as repeated exposures. In the case of furosemide analysis, hair can provide information about its presence but cannot give information about dosage or frequency of use.

Frequency and type of adverse analytical findings in athletics: Differences among disciplines.

Athletics is a highly diverse sport that contains a set of disciplines grouped into jumps, throws, races of varying distances, and combined events. From a physiological standpoint, the physical capabilities linked to success are quite different among disciplines, with varying involvements of muscle strength, muscle power, and endurance. Thus, the use of banned substances in athletics might be dictated by physical dimensions of each discipline. Thus, the aim of this investigation was to analyse the number and distribution of adverse analytical findings per drug class in athletic disciplines. The data included in this investigation were gathered from the Anti-Doping Testing Figure Report made available by the World Anti-Doping Agency (from 2016 to 2018). Interestingly, there were no differences in the frequency of adverse findings (overall,~0.95%, range from 0.77 to 1.70%) among disciplines despite long distance runners having the highest number of samples analysed per year (~9812 samples/year). Sprinters and throwers presented abnormally high proportions of adverse analytical findings within the group of anabolic agents (p < 0.01); middle- and long-distance runners presented atypically high proportions of findings related to peptide hormones and growth factors (p < 0.01); racewalkers presented atypically high proportions of banned diuretics and masking agents (p = 0.05). These results suggest that the proportion of athletes that are using banned substances is similar among the different disciplines of athletics. However, there are substantial differences in the class of drugs more commonly used in each discipline. This information can be used to effectively enhance anti-doping testing protocols in athletics.

Annual banned-substance review: Analytical approaches in human sports drug testing 2019/2020.

Analytical chemistry-based research in sports drug testing has been a dynamic endeavor for several decades, with technology-driven innovations continuously contributing to significant improvements in various regards including analytical sensitivity, comprehensiveness of target analytes, differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds, assessment of alternative matrices for doping control purposes, and so forth. The resulting breadth of tools being investigated and developed by anti-doping researchers has allowed to substantially improve anti-doping programs and data interpretation in general. Additionally, these outcomes have been an extremely valuable pledge for routine doping controls during the unprecedented global health crisis that severely affected established sports drug testing strategies. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2019 and September 2020 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified the World Anti-Doping Agency's 2020 Prohibited List.

Identification of a calcitonin-like diuretic hormone that functions as an intrinsic modulator of the American lobster, Homarus americanus, cardiac neuromuscular system.

In insects, a family of peptides with sequence homology to the vertebrate calcitonins has been implicated in the control of diuresis, a process that includes mixing of the hemolymph. Here, we show that a member of the insect calcitonin-like diuretic hormone (CLDH) family is present in the American lobster, Homarus americanus, serving, at least in part, as a powerful modulator of cardiac output. Specifically, during an ongoing EST project, a transcript encoding a putative H. americanus CLDH precursor was identified; a full-length cDNA was subsequently cloned. In silico analyses of the deduced prepro-hormone predicted the mature structure of the encoded CLDH to be GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH), which is identical to a known Tribolium castaneum peptide. RT-PCR tissue profiling suggests that Homam-CLDH is broadly distributed within the lobster nervous system, including the cardiac ganglion (CG), which controls the movement of the neurogenic heart. RT-PCR analysis conducted on pacemaker neuron- and motor neuron-specific cDNAs suggests that the motor neurons are the source of the CLDH message in the CG. Perfusion of Homam-CLDH through the isolated lobster heart produced dose-dependent increases in both contraction frequency and amplitude and a dose-dependent decrease in contraction duration, with threshold concentrations for all parameters in the range 10(-11) to 10(-10) mol l(-1) or less, among the lowest for any peptide on this system. This report is the first documentation of a decapod CLDH, the first demonstration of CLDH bioactivity outside the Insecta, and the first detection of an intrinsic neuropeptide transcript in the crustacean CG.

Spectrophotometric and spectrofluorimetric determination of certain diuretics through ternary complex formation with eosin and lead (II).

Simple and sensitive spectrophotometric and spectrofluorimetric methods have been developed for the determination of hydrochlorothiazide (I), indapamide (II) and xipamide(III) based on ternary complex formation with eosin and lead (II) in the presence of methylcellulose as surfactant. The methods do not involve solvent extraction. For spectrophotometric method, the ternary complex showed an absorption maximum at 543 nm. The factors affecting the formation of ternary complex were studied and optimized. The method obeys Beer's law over concentration range of 8-40 microg mL(-1). A fluorescence quenching method for the determination of the cited drugs by forming this ternary complex was also investigated for the purpose of enhancing the sensitivity of the determination. The analytical performance of both methods was fully validated, and the results were satisfactory. The methods have been successfully applied for the determination of the studied drugs in their pharmaceutical tablets and the results obtained ware in good agreement with those obtained by the reference method. Common excipients used as additives in tablets do not interfere with the proposed methods.

HPLC-DAD method for the simultaneous determination of zofenopril and hydrochlorothiazide in oral pharmaceutical formulations.

An HPLC method with DAD detection was developed and validated for the simultaneous determination of zofenopril and hydrochlorothiazide in tablets. The separation was carried out through a gradient elution using an Agilent LiChrospher C18 column (250x4.0 mm id, 5 microm) and a mobile phase consisting of (A) water-TFA (99.9:0.1 v/v) and (B) acetonitrile-TFA (99.1:0.1 v/v) delivered at a flow-rate of 1.0 mL/min. 8-Chlorotheophylline was used as internal standard. Calibration curves were found to be linear for the two drugs over the concentration ranges of 5.0-40 and 1.0-20 microg/mL for zofenopril and hydrochlorothiazide, respectively. Linearity, precision, accuracy, specificity and robustness were determined in order to validate the proposed method, which was further applied to the analysis of commercial tablets. The proposed method is simple and rapid, and gives accurate and precise results.

Masking and manipulation.

The list of prohibited substances in sports includes a group of masking agents that are forbidden in both in- and out-of-competition doping tests. This group consists of a series of compounds that are misused in sports to mask the administration of other doping agents, and includes: diuretics, used to reduce the concentration in urine of other doping agents either by increasing the urine volume or by reducing the excretion of basic doping agents by increasing the urinary pH; probenecid, used to reduce the concentration in urine of acidic compounds, such as glucuronoconjugates of some doping agents; 5alpha-reductase inhibitors, used to reduce the formation of 5alpha-reduced metabolites of anabolic androgenic steroids; plasma expanders, used to maintain the plasma volume after misuse of erythropoietin or red blood cells concentrates; and epitestosterone, used to mask the detection of the administration of testosterone. Diuretics may be also misused to achieve acute weight loss before competition in sports with weight categories. In this chapter, pharmacological modes of action, intended pharmacological effects for doping purposes, main routes of biotransformation and analytical procedures used for anti-doping controls to screen and confirm these substances will be reviewed and discussed.