Assessing limits of detection in qualitative methods: A simple implementation of logistic regression in a web-based R Shiny application environment and its potential in toxicology and doping control.

The estimation of limits of detection (LOD) for solely qualitative methods in analytical chemistry may prove challenging because all the approaches with which chemists are familiar require some type of numeric data input. The best model to describe the binary response in these methods (detected/not detected) is a logistic model; however, these models are not easily handled by most of the laboratories and generally demand expensive statistical software packages. In this work, the advantages of applying this approach are discussed and its implementation using commercial spreadsheet software is demonstrated. A free online application based on the R environment using shinyapps was developed and its application was validated and discussed with a dataset of 57 different target compounds analyzed in urine according to the requirements of the World Anti-Doping Agency (WADA). This tool allows free, extremely quick, and easy determinations of LOD in qualitative analyses as well as the determination of the probabilities of detection in any given concentration.

Variable Data Independent Acquisition and Data Mining Exploring Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids in Oral Fluid.

Novel psychoactive substances (NPS) are constantly emerging in the drug market, and synthetic cannabinoids (SCs) are included in this NPS family. Forensic laboratories often struggle with these continually emerging SCs, forcing them to develop an untargeted workflow to incorporate these psychoactive drugs in their procedures. Usually, forensic laboratories select analytical methods based on targeted mass spectrometry (MS) technologies for strictly tracking already known NPS. The appropriate way to tackle unknown substances is to develop pipelines for untargeted analysis that include LC-HRMS analytical methods and data analysis. Once established, this strategy would allow drug testing laboratories to be always one step ahead of the new trends concerning the "designer drugs" market. To address this challenge an untargeted workflow based on mass spectrometry data acquisition and data analysis was developed to detect SCs in oral fluid (OF) samples at a low concentration range. The samples were extracted by mixed-mode solid-phase extraction and analyzed by Liquid Chromatography - High-Resolution Mass Spectrometry (LC-HRMS). Tandem mass spectra (MS2) were recorded performing a variable isolation width across a mass range of all theoretical precursor ions (vDIA) after the chromatographic separation. After raw data processing with the MSDial software, the deconvoluted features were sent to GNPS for Feature-Based Molecular Networking (FBMN) construction for nontargeted data mining. The FBMN analysis created a unique integrated network for most of the SCs assessed in the OF at a low level (20 ng/mL). These results demonstrate the potential of an untargeted approach to detect different derivatives of SCs at trace levels for forensic applications.

Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC-Ion Mobility-HRMS.

The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.

Prevalência de doenças crônicas em idosos atendidos na área de abrangência da estratégia saúde da família no interior do RS / Prevalence of chronic diseases in older adults served in the scope of the family health strategy in the city of Marau/RS

Objetivo: avaliar a prevalência de doenças crônicas em idosos atendidos na Estratégia de Saúde da Família em um município do norte do estado do Rio Grande do Sul. Método: trata-se de um estudo transversal de dados secundários, realizado com indivíduos de 60 anos ou mais, através de prontuários eletrônicos do Sistema de Informação do Sistema Único de Saúde, no período de janeiro a dezembro de 2016. As doenças crônicas investigadas foram diabetes mellitus, hipertensão arterial sistêmica, acidente vascular cerebral, infarto, câncer e ainda a presença de multimorbidade para 2 ou mais doenças associadas. Também foram coletadas informações demográficas, internação nos últimos 12 meses e uso de tabaco. Os dados foram analisados através de software de estatística. Para as variáveis quantitativas foram calculadas as medidas de tendência central e dispersão, e para as variáveis qualitativas foram apresentas as frequências absolutas e relativas simples. Para as associações foi aplicado o Teste exato de Fisher e o Teste Qui-quadrado. Resultados: as doenças crônicas com maior prevalência foram hipertensão arterial sistêmica (37,3%) e diabetes mellitus (10,1%), simultaneamente hipertensão arterial sistêmica e diabetes mellitus 9,1%, e a prevalência de multimorbidade foi de 11%. A média de idade foi de 70,02 anos (+7,93), sendo que a maioria tinha entre 60 a 69 anos (55,2%), eram do sexo feminino (57,7%), não haviam internado nos últimos 12 meses (98,6%) e não faziam uso de tabaco (97,3%). Foi observado maior prevalência de multimorbidade entre os idosos com 80 anos ou mais (p<0,001), e associação entre doenças e sexo, a maior prevalência de Hipertensão Arterial foi no sexo feminino e maior prevalência de Infarto Agudo do Miocárdio no sexo masculino (p<0,05). Conclusão: os dados do estudo mostraram uma baixa prevalência de doenças crônicas e multimorbidade. (AU)

Objective: to evaluate the prevalence of chronic diseases in older adults served in the Family Health Strategy in the city in the north of the state of Rio Grande do Sul. Method: this is a cross-sectional study of secondary data, carried out with individuals aged 60 years or older, using electronic records of the Information System of the Unified Health System, in the period from January to December 2016. Chronic diseases investigated were diabetes mellitus, systemic arterial hypertension, stroke, heart attack, cancer and the presence of multimorbidity for 2 or more associated diseases. We also collected demographic information, hospitalization in the last 12 months and use of tobacco. The data were analyzed with statistical software, for the quantitative variables were calculated the measures of central tendency and dispersion; and for the qualitative variables were presented the absolute frequencies and simple relatives. For associations it was applied the Fisher exact test and Chi-square test. Results: the most prevalent chronic diseases were systemic arterial hypertension (37.3%) and diabetes mellitus (10.1%), simultaneously systemic arterial hypertension and diabetes mellitus (9.1%), and the prevalence of multimorbidity was 11%. The mean age was 70.02 years (+7.93), the majority were between 60 and 69 years old (55.2%), were female (57.7%), did not smoke (97, 3%) and had not hospitalized in the last 12 months (98.6%). Results: the most prevalent chronic diseases were systemic arterial hypertension (37.3%) and diabetes mellitus (10.1%), both systemic arterial hypertension and diabetes mellitus 9.1%, and the prevalence of multimorbidity was 11%. A higher prevalence of multimorbidity was observed among older adults with 80 years or more (p<0,001) and the association between diseases and sex, the highest prevalence of Arterial Hypertension was in females and a higher prevalence of acute myocardial infarction in males (p<0,05). Conclusion: the study data showed a low prevalence of chronic diseases and multimorbidity (AU)

Non-targeted acquisition strategy for screening doping compounds based on GC-EI-hybrid quadrupole-Orbitrap mass spectrometry: A focus on exogenous anabolic steroids.

This is a first look at a non-targeted screening method based on Orbitrap gas chromatography-mass spectrometry (GC-MS) technology for a large number of banned compounds in sports found in urine, including exogenous anabolic steroids, ß-agonists, narcotics, stimulants, hormone modulators, and diuretics. A simple sample preparation was processed in four steps: an enzymatic hydrolysis, liquid-liquid extraction, evaporation, and trimethylsilylation. All compounds were able to meet the World Anti-Doping Agency's sensitivity criteria with mass accuracies less than 1 ppm and with sufficient points across the peak by running the Orbitrap GC-MS in full-scan mode. In addition, we discuss our initial findings of using a full-scan selected ion monitoring-tandem mass spectrometry (SIM-MS/MS) approach as a way to obtain lower detection limits and reach desirable selectivity for some exogenous anabolic steroids.

Conhecimento e Atitudes sobre o Câncer de Próstata no Brasil: Revisão Integrativa / Knowledge and Attitudes about Prostate Cancer in Brazil: Integrative Review / Conocimiento y Actitudes sobre el Cáncer de Próstata en Brasil: Revisión Integrativa

Introdução: O câncer de próstata está entre as doenças crônicas não transmissíveis que mais afetam os idosos, sendo a idade um fator relevante para esse agravo. É o segundo tipo de câncer de maior prevalência na população masculina, tornando-se um problema de saúde pública. Para tanto, a prevenção e o diagnóstico ficam comprometidos pela baixa procura dos homens ao serviço de saúde. Objetivo: Identificar os obstáculos para a realização do exame de próstata entre os homens. Método: Revisão integrativa da literatura realizada nas bases de dados da BVS, Lilacs, Medline e BDENF. Foram critérios de inclusão: artigos científicos que retratam as ações públicas direcionadas ao câncer de próstata, indexados na base de dados da BVS, publicados em língua portuguesa, inglesa e em espanhol, disponíveis on-line na íntegra. Resultados: Foram encontradas 68 publicações na base de dados Medline, 62 na Lilacs e na BDENF 17, totalizando 147 artigos que poderiam ter relação com o tema da pesquisa. Após leitura dos resumos, desse total, 139 artigos não possuíam relação direta com o estudo, estavam indexados em mais de uma base de dados ou estavam duplicados, totalizando oito artigos publicados para análise. Discussão: Após leitura interpretativa, emergiram três categorias: dificuldades políticas organizacionais para a prevenção do câncer de próstata, dificuldades socioculturais para prevenção do câncer de próstata e estratégias para prevenção desse câncer. Conclusão: É necessário desenvolver a prevenção com mais naturalidade, a partir de informações claras e eficazes pelos meios de comunicação.

Introduction: Breast cancer is the most common type among women in the world and in Brazil, it is part of a heterogeneous group of diseases, thus having different signs and behavior. Possible treatments include surgical approaches, chemotherapy, radiotherapy, endocrine therapy, and target therapy. The surgical procedure can lead to several complications and consequences for the patient, so a follow-up process is necessary after this approach, exercises are fundamental to return functionality and quality of life, but there is divergence in the literature regarding the moment of beginning of the execution of the same. Objective: To perform a systematic literature review, present studies and analyze the impact of the release of immediate versus late exercises, after onco-mammary surgeries, without associated plastic intervention. Method: This is a systematic literature review. To perform the same, a search was made in the database of PubMed, PEDro and SciELO databases using the descriptors: rehabilitation, breast neoplasms, prospective period, prospective care, exercise and their respective ones in the Portuguese language, being these, rehabilitation, breast cancer, postoperative period, postoperative care and exercise. Results: For the accomplishment of this study, 48 articles were found, being 12 relevant to the review. Conclusion: The studies are divergent, but a literary update emphasizes immediate postoperative exercises of onco-mammary surgeries can be used without increasing the incidence of complications.

Introducción: El cáncer de mama es el tipo más común entre las mujeres en el mundo y en Brasil, forma parte de un grupo heterogéneo de enfermedades, teniendo así signos y comportamiento distintos. Los tratamientos posibles son abordajes quirúrgicos, quimioterapia, radioterapia, endocrinoterapia y terapia objetivo. El procedimiento quirúrgico puede llevar a diversas complicaciones y consecuencias para la paciente, por lo que es necesario un proceso de seguimiento después de este enfoque, ejercicios son fundamentales para devolver funcionalidad y calidad de vida, pero existe divergencia en la literatura en relación al momento de início de la sua ejecución. Objetivo: Realizar una revisión sistemática de la literatura, presentar estudios y analizar el impacto de la liberación de ejercicios inmediatos versus tardíos, después de cirugías onco-mamarias, sin intervención plástica asociada. Método: Se trata de un estudio de revisión sistemática de la literatura. Para la implementación se trataba de una encuesta en la orilla de las bases de datos PubMed, SciELO, PEDro y utilizando las siguientes palabras clave: rehabilitación, neoplasias de la mama, del período prospectivo, de cuidado prospectivo, de ejercicio y su en portugués, siendo éstas, la rehabilitación, el cáncer de mama, período postoperatorio, cuidados postoperatorios y ejercicio. Resultados: Para la realización de este estudio se encontraron 48 artículos, siendo 12 relevantes a la revisión. Conclusión: Los estudios se presentan divergentes, pero se observa una actualización literaria que enfatiza que los ejercicios inmediatos en el postoperatorio de cirugías onco-mamarias pueden ser empleados sin traer aumento en la incidencia de complicaciones.

Doping control analysis at the Rio 2016 Olympic and Paralympic Games.

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America. Copyright © 2017 John Wiley & Sons, Ltd.

Multimorbidity associated with polypharmacy and negative self-perception of health / Multimorbidade associado à polifarmácia e autopercepção negativa de saúde

Abstract Objective: to verify the association between the multimorbidity of the elderly and sociodemographic variables, self-perception of health and polypharmacy. Method: a cross-sectional study was performed. The research data was collected using the Health, Well-Being and Aging questionnaire. The sample was composed of 676 people aged 60 years or more, who were residents of small towns in the north of the state of Rio Grande do Sul, Brazil. The dependent variable was multimorbidity, that is, the occurrence of two or more chronic non-communicable diseases in the same person. The independent variables were demographic, socioeconomic and health-related characteristics. Poisson's raw and robust regression model was used to analyze the effect of the independent variables in relation to the outcome and p was considered significant when <0.05. Result: among the elderly interviewed, 45% presented multimorbidity, 51.1% reported a self-perception of poor/very poor health and 37.1% used polypharmacy. After the analysis was adjusted to the occurrence of multimorbidity, association with the following variables was found: health perception (regular/poor/very poor) PR=1.15 (CI95%; 1.09 - 1.22) and use of polypharmacy PR=1.29 (CI95%; 1.22 - 1.35). Conclusion: Multimorbidity may interfere negatively in the self-perception of health of the elderly contributing to increased medicine consumption. AU

Resumo Objetivo: verificar a associação entre multimorbidade em idosos e variáveis sociodemográficas, autopercepção de saúde e polifarmácia. Método: Estudo de corte transversal, para a coleta de dados utilizou-se o questionário Saúde, Bem-estar e Envelhecimento. A amostra foi constituída de 676 indivíduos com 60 anos de idade ou mais residentes em municípios de pequeno porte do norte do estado do Rio Grande do Sul, Brasil. A variável dependente foi a multimorbidade, isto é, a ocorrência de duas ou mais doenças crônicas não transmissíveis no mesmo indivíduo. As variáveis independentes foram as características demográficas, socioeconômicas e relativas à saúde. O modelo de regressão de Poisson bruta e ajustada foi utilizado para analisar o efeito das variáveis independentes em relação ao desfecho e p foi considerado significativo quando <0,05. Resultados: Dos idosos entrevistados, 45% apresentam multimorbidade, 51,1% relatam autopercepção de saúde ruim/muito ruim e 37,1% faz uso de polifarmácia. Após análise ajustada à ocorrência de multimorbidade, apresentou-se associação às variáveis: percepção de saúde negativa. RP=1,15; (IC95%; 1,09 - 1,22) e uso de polifarmácia RP=1,29; (IC95%; 1,22 - 1,35). Conclusão: A multimorbidade pode interferir negativamente na autopercepção de saúde dos idosos contribuindo para o aumento do consumo de medicamentos. AU

Prevalência e fatores associados a dependência funcional em idosos longevos / Prevalence and factors associated with functional dependence in the oldest old

Objetivou-se avaliar a prevalência e os fatores associados a dependência funcional de idosos longevos residentes em municípios de pequeno porte. Trata-se de um estudo transversal de base populacional com idosos de 80 anos e mais, recorte dos estudos sobre as condições de saúde de idosos dos municípios de Coxilha-RS e Estação-RS. Os dados foram coletados por meio de questionário adaptado da pesquisa Saúde, Bem-Estar e Envelhecimento (SABE). Participaram 77 idosos longevos (63,3% mulheres). A prevalência de dependência funcional foi 26% (IC95% 16,8-36,4) e dentre as atividades básicas da vida diária (ABVD) deitar e levantar da cama (31,6%) e locomover-se (31,2%) representaram a maior frequência de dependência. Na análise de regressão multivariada mostraram-se associadas à dependência funcional a autopercepção de saúde negativa (RP= 1,20; IC95% 1,05-1,37), não praticar atividade física (RP= 1,20; IC95% 1,05-1,36) e possuir três ou mais doenças crônicas (RP= 1,24; IC95% 1,08-1,41). Nesse sentido, observamos que os idosos longevos com a condição de dependência possuem como razão de prevalência fatores passíveis de prevenção, e sobretudo que essa condição é evitável ou possível de ser postergada.

This study aimed to assess the prevalence and factors associated with functional dependence of the oldest elderly people living in small towns. This is a cross-sectional population-based study with aged, 80 and over, cut out studies on the health conditions of the elderly in the municipalities of Coxilha-RS and Estação-RS. Data were collected through adapted questionnaire Health, Well-Being and aging (SABE). Study participants were 77 oldest elderly (63,3% women). The prevalence of functional dependence was 26% (IC95% 16,8-36,4) and among the basic activities of daily living (ADL) bed and out of bed (31,6%) and getting yourself (31,2%) accounted for the frequency dependence. In multivariate regression analysis were associated with functional dependence negative self-rated health (RP= 1,20; IC95% 1,05-1,36), not physical activity (RP= 1,19; IC95% 1,05-1,36) and have three or more chronic diseases (RP= 1,24; IC95% 1,08-1,41). In this regard, we note that the oldest old with the condition of dependency have as prevalence ratio preventable factors, and above all that this condition is preventable or can be postponed.

Microfabricated Otto chip device for surface plasmon resonance-based optical sensing.

Surface plasmon resonance (SPR) based sensors are usually designed using the Kretschmann prism coupling configuration in which an input beam couples with a surface plasmon through a thin metal film. This is generally preferred by sensor developers for building planar devices instead of the Otto prism coupling configuration, which, for efficient coupling, requires the metal surface to be maintained at a distance on the order of the wavelength from the input prism surface. In this paper, we report on the microfabrication and characterization of an Otto chip device, which is suitable for applications of the SPR effect in gas sensing and biosensing.

Maintaining a stationary laser footprint during angular scan in internal-reflection experiments.

In internal-reflection metrology using prisms, the prism is usually mounted on a rotation/translation stage to enable adjusting angle and location of the laser footprint on the surface. If a visual inspection method is used to find the laser footprint, the task becomes impossible if invisible radiation in the near infrared is employed. In addition, it may be desirable to perform angular scan experiments with a stationary footprint on the surface during scans, or even to automatically probe specific points on an extended prism face for predetermined incidence angles. In this paper, a formulation is developed to determine the required translation along the prism face to allow maintaining the laser footprint stationary under a given rotation. A web-based app developed under the scope of this work demonstrates the applicability of the approach for silica, BK7 and SF2 glasses, in the wavelength range from 500 to 1500 nm and for an arbitrary geometry of the glass prism.

Detection of designer steroid methylstenbolone in "nutritional supplement" using gas chromatography and tandem mass spectrometry: elucidation of its urinary metabolites.

The use of "nutritional supplements" containing unapproved substances has become a regular practice in amateur and professional athletes. This represents a dangerous habit for their health once no data about toxicological or pharmacological effects of these supplements are available. Most of them are freely commercialized online and any person can buy them without medical surveillance. Usually, the steroids intentionally added to the "nutritional supplements" are testosterone analogues with some structural modifications. In this study, the analyzed product was bought online and a new anabolic steroid known as methylstenbolone (2,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one) was detected, as described on label. Generally, anabolic steroids are extensively metabolized, thus in-depth knowledge of their metabolism is mandatory for doping control purposes. For this reason, a human excretion study was carried out with four volunteers after a single oral dose to determine the urinary metabolites of the steroid. Urine samples were submitted to enzymatic hydrolysis of glucuconjugated metabolites followed by liquid-liquid extraction and analysis of the trimethylsilyl derivatives by gas chromatography coupled to tandem mass spectrometry. Mass spectrometric data allowed the proposal of two plausible metabolites: 2,17α-dimethyl-16ξ,17ß-dihydroxy-5α-androst-1-en-3-one (S1), 2,17α-dimethyl-3α,16ξ,17ß-trihydroxy-5α-androst-1-ene (S2). Their electron impact mass spectra are compatible with 16-hydroxylated steroids O-TMS derivatives presenting diagnostic ions such as m/z 231 and m/z 218. These metabolites were detectable after one week post administration while unchanged methylstenbolone was only detectable in a brief period of 45 h.

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry.

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17ß-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.

Detection of new urinary exemestane metabolites by gas chromatography coupled to mass spectrometry.

Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17ß-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) after enzymatic hydrolysis and liquid-liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole.

Analysis of exemestane and 17ß-hydroxyexemestane in human urine by gas chromatography/mass spectrometry: development and validation of a method using MO-TMS derivatives.

Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH(4)I/2-mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the monitoring of exemestane and its main metabolite 17ß-hydroxyexemestane prior to GC-MS analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives. Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-exomethylene) of the compounds into account. Although these artifacts are relevant for investigations regarding the derivatization process and may be of interest in many fields, they are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in terms of the limits of detection (LODs) required. To overcome this issue, a method using an alternative derivatization was proposed: formation of methyloxime-TMS derivatives through double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after administration of exemestane to healthy volunteers were analyzed by the proposed method and detection of both exemestane and its main metabolite was possible. This method showed excellent results for both analytes meeting the LODs required for antiestrogenic agents (50 ng/mL) established by WADA. The method was validated for the main metabolite, it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ of 10 ng/mL and 25 ng/mL, respectively.