Development of an analytical method for the determination of the misuse in sports of boldenone through the analysis of urine by on-line coupling liquid chromatography-gas chromatography-combustion-isotope ratio mass spectrometry.

Boldenone (Bo), androsta-1,4-dien-17ß-ol-3-one, is an anabolic androgenic steroid not clinically approved for human application. Despite this, many cases are reported every year of athletes testing positive for Bo or its main metabolite 5ß-androst-1-en-17ß-ol-3-one (BoM). Recently the capability of different human intestinal bacteria to produce enzymes able to modify endogenous steroids in Bo has been demonstrated. When a urinary concentration of Bo and/or BoM between 5 and 30 ng/mL is measured a complementary analysis by gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) must be carried out to discriminate the endogenous or exogenous origin. In the present work, a novel analytical method that couples LC-GC by means of the TOTAD interface with C-IRMS is described. The method is based on a first RPLC separation of unacetyled steroids, followed by acetylation and automated on-line LC-GC-C-IRMS, which includes a second RPLC clean-up of acetyl Bo and BoM, isolation of the two fractions in a fraction collector and their consecutive analysis by GC-C-IRMS. The method has been applied to the analysis of urine samples fortified at 5 and 10 ng/mL, where it has shown a good performance.

Use of hydrogen as a carrier gas for the analysis of steroids with anabolic activity by gas chromatography-mass spectrometry.

Due to the impact in the media and the requirements of sensitivity and robustness, the detection of the misuse of forbidden substances in sports is a really challenging area for analytical chemistry, where any study focused on enhancing the performance of the analytical methods will be of great interest. The aim of the present study was to evaluate the usefulness of using hydrogen instead of helium as a carrier gas for the analysis of anabolic steroids by gas chromatography-mass spectrometry with electron ionization. There are several drawbacks related with the use of helium as a carrier gas: it is expensive, is a non-renewable resource, and has limited availability in many parts of the world. In contrast, hydrogen is readily available using a hydrogen generator or high-pressure bottled gas, and allows a faster analysis without loss of efficiency; nevertheless it should not be forgotten that due to its explosiveness hydrogen must be handled with caution. Throughout the study the impact of the change of the carrier gas will be evaluated in terms of: performance of the chromatographic system, saving of time and money, impact on the high vacuum in the analyzer, changes in the fragmentation behaviour of the analytes, and finally consequences for the limits of detection achieved with the method.

Automated sample preparation and gas chromatographic-mass spectrometric analysis of urinary androgenic anabolic steroids.

This paper presents an automated method for extracting anabolic agents from urine samples for their GC-MS analysis by selected-ion monitoring. The sample preparation was carried out in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed, extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor. When sample preparation was finished 2 microl of the extract was injected into the gas chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials for handling the sample, parameters like time of hydrolysis, type of shaking, number of extractions and some TMS derivatization parameters had to be adjusted to achieve the best recovery for all of the compounds in the screening. Manual and automated sample preparation schemes were compared in terms of linearity, precision, accuracy, limit of detection and recovery data. When large concentrations were analyzed using the automated method no carry-over effect was observed.

Análisis de anfetamina en orina por medio de microextracción en fase sólida / Analysis of amphetamine in urine by headspace solid phase microextraction

En este trabajo se presenta un método automatizado de extracción y análisis por GC (gas chromatography)-MS (mass spectrometry) para la detección de anfetamina en muestras de orina. El sistema esta constituido por un muestreador automatico, un cromatografo de gases y un espectrometro de masas de trampa de iones. El muestrador esta equipado con una aguja en cuyo interior hay una pequeña fibra retráctil de polidimetil siloxano, que puesta en contacto con la muestra, líquido o gas, absorbe selectivamente los analitos. La técnica es conocida como microextracción en fase sólida, SPME (Solid Phase MicroExtraction). En el estudio se hace un análisis detallado de todos los parámetros que afectan al rendimiento de las fibras, esto es; volumen de muestra, tipo de fibra, temperatura de absorción, tiempo de absorción, pH y fuerza ionica del medio. En la preparación de la muestra, tras ajustar manualmente el pH (pH 12) e incrementar la fuerza ionica de la muestra, 200 ml de orina son dispensados en un vial encapsulado de 2 ml. Tras solocar el vial en el carrusel termostatizado, Tª absorción 60°C, la fibra es introducida en el vial. Pasados 10 minutos de absorción, la fibra se retrae en la aguja y se inserta en el portal de inyección del cromatografo de gases, donde debido a alta temperatura, Ta desorbción 200°C, el analito desorbe de modo inmediato. El límite de detección es de 0.25 mg/l, el coeficiente de correlación lineal es de 0.9916 y la desviación estándar relativa menor del 5 por ciento. La concentración mínima de anfetamina que en la muestra pudo ser confirmada mediante un espectro de masas con índice de correlación mayor del 90 por ciento con su correspondiente espectro de librería, fue de 0.5 mg/l(AU)

Use of ion trap gas chromatography-tandem mass spectrometry for detection and confirmation of anabolic substances at trace levels in doping analysis.

A procedure for detecting and confirming 23 anabolic substances and/or metabolites has been developed using a GC-MS-MS ion trap system in full-scan mode. The process used to select the precursor ion, and the optimization of the system parameters used to obtain the daughter ion spectra, are explained. Urine samples were prepared using solid-phase extraction and enzymatic hydrolysis, and after TMS derivatives had been formed, they were injected into the mass spectrometer. This method permits confirmation of the presence of anabolic substances at low ng ml(-1) levels without the need of further purification procedures on the samples. This procedure has been used on more than 2000 urine samples collected from sporting competitions and has made it possible to confirm more than 45 true positive cases which could not have been confirmed using routine GC-MS methods.

Automated analysis of drugs in urine.

A totally automated procedure has been developed for the preparation and analysis of 34 basic and neutral drugs in urine samples using an integrated HP 7686 PrepStation-HP 6890 gas chromatographic system. The automated preparation of the sample consisted of a liquid-liquid extraction of 250 microliters urine at alkaline pH with 100 microliters of methyl tert.-butyl ether. After phase separation the organic solvent was automatically placed in the injector of the gas chromatograph and analysed. High recoveries of extraction were obtained. The limits of detection of most of the drugs were less than 0.5 microgram/ml. The method, which allows the preparation and analysis of the samples to be completely synchronised, showed good accuracy and precision.