Marlice Aparecida Sípoli Marques

Improvement in steroid screening for doping control with special emphasis on stanozolol

The Medical Commission of the International Olympic Committee forbids the use of anabolic androgenic steroids and beta2-agonists to improve athletic performance. In this work we have selected examples of anabolic androgenic compounds and their metabolites to evaluate the GC-MS analysis of some trimethylsilyl derivatives. The aim is to set the best GC conditions to improve the detection within the whole range of analyte elution temperatures. The initial column temperature was changed to 105 or 140 degrees C followed by 40 degrees C min(-1) to 200 degrees C and then 15 degrees C min(-1) to 300 degrees C. Using 140 degrees C as the initial oven temperature it was possible to obtain narrower initial analyte distributions for the compounds that elutes at the beginning of the chromatogram as clenbuterol, mabuterol, epimethylenediol and norandrosterone, without loss of derivatized metabolites signal. Later. eluting analytes, such as the stanozolol metabolites, furazabol and oxandrolone were not affected. Temperatures below 140 degrees C. resulted in partial derivatization for some analytes mainly stanozolol related structures. Therefore evaluation of derivatization conditions as occurring in three steps, the vial, vaporization chamber and capillary column, was thoroughly assessed. The new program temperature improves the signal-to-noise ratio for some compounds and shows adequate resolution for endogenous compounds. Some of the difficult key separations necessary for doping control enforcement were also obtained with the proposed method.

Controle de dopagem de anabolizantes: o perfil esteroidal e suas regulações

The concept of steroid profile is discussed in this paper. The main metabolic routes are presented. The importance of evaluating steroid profiles is demonstrated, with special attention to clinical medicine and sports. Parameters used in the literature for doping control of endogenous steroids are briefly evaluated, as well as the factors responsible for alterations in the normal steroid profile. Special focus is turned to the latter approach.

Improvements in steroid screening in doping control with special emphasis to GC-MS analytical conditions and method validation

A procedure is described for the simultaneous determination of androgenic substances including steroids and b2-agonists. The method involves analysis of hydrolized urinary anabolic compounds using liquid-liquid extraction, with subsequent conversion to trimethylsilylether derivatives for the analysis by GC-MS. Pulse split injection 1/10 of the TMS derivatives at 280 °C into the capillary column, initially maintained at 140 °C then programmed to 180 °C at 40 °C min-1, followed by 3 oC min-1 to 230 oC and then 40 oC min-1 to 300 oC, resulted in good resolution and peak shape for all compounds. The detection limits of most of the steroids were 1 ng mL-1 except for formebolone and trenbolone (25 ng mL-1). When applied to selected urine samples with evidence of bacterial degradation and metabolites from usual medications/vitamins, the method allowed rapid screening for androgens and other substances monitored in routine. The resolution was adequate to evaluate the endogenous steroid profile relevant to doping control and medical applications.

Resíduos e contaminantes químicos em alimentos de origem animal no Brasil: histórico, legislação e atuação da vigilância sanitária e demais sistemas regulatórios

Food safety became a relevant subject due to the increasing search for a better way of life and consciousness of the consumers to stand on ones rights to acquire healthy products. The use of substances in animals destined for human consumption requires from pharmacokinetics to residue depletion studies, with the establishment of limitative values so that do not constitute a risk to health. Beyond the substances used deliberately, others coming from environment contamination or contamination of feeding stuffs consumed by these animals may reach human through the diet. The aims of this paper are to collect and discuss the main federal acts covering chemical residues and contaminants in food of animal origin in Brazil, besides those on measures to control veterinary medicinal products and additives for use in animal nutrition. The chronological presentation of the legal basis intends to facilitate the interpretation of the acts inside respective political and economics scenarios. The actions proposed from the different agents involved into the regulatory systems are discussed from the public health point of view.

DNA Typing: An Accessory Evidence in Doping Control

A clear positive case for anabolic steroids doping was confounded by alleged urine tampering during doping control procedures. Review of the chain of custody showed no flaws, but nevertheless the athlete was adamant that the urine sample should be analyzed for DNA in order to support her contention that she was not the donor of the sample. The results obtained showed that the urine sample that scored positive for steroids contained nuclear DNA that could not be matched to the DNA obtained from the athletes blood. On the other hand, the same urine sample contained mitochondrial DNA whose nucleotide sequences spanning the hyper variable regions HV1 and HV2 proved to be identical to those determined in mitochondrial DNA amplified from the athletes blood. The occurrence of an extraneous genotype is compatible with exogenous nuclear DNA admixture to the athletes urine. Alternatively, taking in consideration the mitochondrial DNA, we could not exclude that a sibling or a maternal relative of the athlete could have acted as a donor of the urine utilized for doping control and DNA analysis. Both situations point to possible tampering of the urine by the athlete. Adjudication at CAS maintained previous national and international federation decision that there was no proof of a chain of custody flaw to justify the athletes allegation of urine substitution after collection.

Validation of the determination of oxymetholone in human plasma analysis using gas chromatography-mass spectrometry. Application to pharmacokinetic studies.

A simple and rapid procedure for extraction of oxymetholone in human plasma using gas chromatography coupled with quadrupole mass spectrometric was evaluated. The method involves analyte extraction with tert.-butylmethylether after alkalinization of the plasma and derivatization with MSTFA-NH(4)I-2-mercaptoethanol before the high resolution gas chromatographic-mass spectrometry separation. Methyltestosterone was used as internal standard. The calibration curves were linear, with typical r(2) values >0.995 and F(table)>F(calculated) (alpha=0.05). Recovery from plasma proved to be more than 70%. The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of oxymetholone for healthy volunteers after oral administration of 50 mg of the compound. The (C(max)) and (T(max)) were 18.8 +/- 0.4 ng/ml and 210 +/- 42.4 min, respectively.

Informações sobre o uso de medicamentos no esporte

Endereço para correspondência: Dr. Eduardo Henrique De Rose E-mail: ehderose@terra.com.br destes Jogos, foi iniciado o controle sistemático de doping em Jogos Olímpicos de Inverno e de Verão. De acordo com a Comissão Médica do COI, a lista de substâncias proibidas circulada em janeiro de 2000 contém cinco classes farmacológicas: estimulantes, narcótico-analgésicos, agentes anabolizantes, diuréticos e hormônios peptídicos. Ademais, inclui três métodos igualmente proibidos: a manipulação física, química e farmacológica de urina, a administração de transportadores artificiais de oxigênio e substitutos de plasma, e ainda transfusão de sangue. Após fevereiro de 1993, o grupo de betabloqueadores, que era previamente proibido, foi transferido à categoria de droga restrita, cabendo às Federações Internacionais de cada modalidade esportiva incluir ou não esta substância no elenco das categorias proibidas. Os betabloqueadores são atualmente proibidos em esportes de precisão e pilotagem, tais como arco e flecha, tiro, pentatlo moderno, esgrima, saltos ornamentais, ginástica e equitação. A lista de classes farmacológicas e métodos proibidos é atualizada a cada ano, no mês de janeiro, em função do que está sendo utilizado ou já não tem mais sentido de ser usado pelos atletas, e a partir de pesquisas realizadas e que permitem um maior conhecimento dos agentes dopantes. Qualquer substância que pertença a classes farmacológicas proibidas não poderá ser usada, mesmo se não estiver listada como exemplo. O termo “compostos correlatos” utilizado no fim de cada classe farmacológica descreve drogas relacionadas esta categoria, seja pela sua estrutura química, seja por seu efeito farmacológico. A lista que vigora este ano, e que será a usada para os Jogos Olímpicos de Sydney, é a seguinte:

Análise de glicocorticosteróides por CG-EM: uma nova abordagem de derivatização para o controle de dopagem no esporte

Glucocorticosteroids (CT) show a powerful anti-inflammatory effect. In view of this, they are extensively used in sports medicine, but their misuse (systemic administration) can lead to severe injuries on athletes and has been restricted by the IOC in recent years. For GC-MS analysis, a derivatization step is necessary in order to convert them to a stable form, thus preventing thermal breakdown with the loss of the ketone side chain. Derivatization of CT is reviewed and a new approach of silylation is presented for screening of CT in urine.

Controle de dopagem no esporte: aspectos químicos e farmacológicos que afetam a detecção de drogas no cabelo

Hair analysis is very well documented in forensic toxicology. It has been employed for differentiation between chronic or occasional consumption of certain drugs. The Society of Hair Testing published rules about hair analysis in sportive doping, which are accepted in most courts of justice, in spite that they had not been incorporated by the International Olympic Committee. Among the substances forbidden by the IOC, the great challenge of hair analysis in doping control is to confirm his validity in the detection of anabolic steroids. Before validation of hair analysis in doping control, the scientific community has to answer at least five critical questions: (1) What is the minimal amount detectable in hair after administration? (2) What is the relationship between the amount of the drug used and the concentration of the drug or its metabolites in hair? (3) What is the influence of hairs color? (4) Is there any racial bias in hair testing? (5) What is the influence of cosmetic treatments? Until now, the limiting factor for hair analysis in doping control is the lack of scientific data. The present article revises published material with special attention to the analytical and pharmacological parameters that may hinder the use of hair analysis in doping control.Hair analysis is very well documented in forensic toxicology. It has been employed for differentiation between chronic or occasional consumption of certain drugs. The Society of Hair Testing published rules about hair analysis in sportive doping, which are accepted in most courts of justice, in spite that they had not been incorporated by the International Olympic Committee. Among the substances forbidden by the IOC, the great challenge of hair analysis in doping control is to confirm his validity in the detection of anabolic steroids. Before validation of hair analysis in doping control, the scientific community has to answer at least five critical questions: (1) What is the minimal amount detectable in hair after administration? (2) What is the relationship between the amount of the drug used and the concentration of the drug or its metabolites in hair? (3) What is the influence of hairs color? (4) Is there any racial bias in hair testing? (5) What is the influence of cosmetic treatments? Until now, the limiting factor for hair analysis in doping control is the lack of scientific data. The present article revises published material with special attention to the analytical and pharmacological parameters that may hinder the use of hair analysis in doping control.

Development of a HPLC/MS/MS methodology for determining 3-O-methyldopa in human plasma and its application in a bioequivalence study

A simple, sensitive and specific HPLC/MS/MS methodology was developed and it was validated for determining 3-O-methyldopa, the major metabolite of dopamine, in human plasma. The separation was achieved on Atlantis T3 C18 analytical column (5 μm; 150 x 4.6 mm i.d.) using a mobile phase consisted of a solution of water and methanol (85:15, v/v) and containing formic acid 0.05%. The extraction from the analyte and the internal standard sample was performed using a simple protein plasma precipitation with perchloric acid. The detection was conducted on a triple quadrupole tandem mass spectrometer with a positive multiple reaction monitoring mode (MRM). The monitored fragmentation transitions were m/z 212.0 - m/z 166.0 for 3-O-methyldopa and m/z 227.10 - m/z 181.0 for carbidopa (internal standard). The calibration curves were linear in the range of 50–4000 ng/mL for 3-O-methyldopa. The methodology presented a good precision and accuracy in accordance to the criteria for biomedical analysis. And it was successfully applied to the bioequivalence study of two formulations levodopa + benserazide (200 + 50 mg) in plasma samples from healthy human volunteers, following the ANVISA guidelines.