Detection of testosterone, nandrolone and precursors in horse hair.

Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid-liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.

Formation of 19-norsteroids by in situ demethylation of endogenous steroids in stored urine samples.

The formation of 19-norsteroids by demethylation of endogenous steroids in stored urine samples was observed. Suspicious urine samples (i.e. containing trace amounts of 19-norandrosterone and 19-noretiocholanolone) were selected and spiked with deuterated analogues of androsterone and etiocholanolone at concentrations corresponding to high endogenous levels (4 microg/mL). After incubation, respective 19-norsteroids (19-norandrosterone-d4 and 19-noretiocholanolone-d5) were identified in these samples by high-resolution mass spectrometry. The transformation of the 5 beta-isomer (etiocholanolone) yields about three-fold higher concentrations, compared to the 5 alpha-isomer. A significant temperature dependence was observed by comparison of reaction kinetics at room temperature (23+/-2 degrees C) and 37 degrees C. Concentrations of 19-norandrosterone-d4 and 19-noretiocholanolone-d5, respectively, were 2.7 and 3.6 times higher at elevated temperature. The conversion of androsterone-d4 to 19-norandrosterone-d4 did not exceed a relative amount of 0.1%. Incubation of the urine samples with androsterone-d4-glucuronide led to the production of 19-norandrosterone-d4-glucuronoide. A partial stabilization was observed after addition of metabolic inhibitors (e.g. EDTA). The application of the incubation experiments described may contribute to the clarification of adverse analytical findings regarding low levels of 19-norsteroid metabolites.

Screening, confirmation and quantification of diuretics in urine for doping control analysis by high-performance liquid chromatography-atmospheric pressure ionisation tandem mass spectrometry.

A sensitive, selective, robust and fast method to identify 32 diuretics and masking agents in urine is described. The analytical procedure is reduced to a single XAD extraction step for sample preparation, followed by reversed-phase liquid chromatography in combination with atmospheric pressure ionisation/tandem mass spectrometry. This technique is, after minor modifications, suitable for screening analyses and confirmation of identity as well as quantitation of diuretics. Considerations relating to the stability and metabolism of the compounds are given if relevant for routine screening analyses.

Analytical strategy for detecting doping agents in hair.

Lists of banned classes of doping agents are released by the International Olympic Committee, adopted by other sports authorities and updated regularly, including the substance classes stimulants, narcotics, diuretics, anabolic agents, peptide hormones, beta-blockers etc. There are different classes of restriction: anabolic and masking agents (anabolic steroids, diuretics etc.) are always banned for athletes regardless of their topical activity (training or competition) several substances are permitted with certain restrictions (caffeine below a cut-off value, or inhalation of some beta 2 agonists) beta-blockers are prohibited in competitions of certain sports disciplines the majority of the substances (stimulants, narcotics etc.) is prohibited during competitions, so that they do not have to be analysed in out-of-competition samples. A differentiation between training and competition period is impossible by means of hair analysis due to the uncertainty of (especially short-term) kinetic considerations related to hair growth. Therefore, the analytical identification of doping relevant substances in hair is not always a sufficient criterion for a doping offence and the identification of stimulants, beta-blockers etc. in hair would be entirely irrelevant. The most interesting target substances are certainly the anabolic agents, because their desired action (enhanced muscle strength) lasts longer than the excretion, leading to sophisticated procedures to circumvent positive analytical results in competition control. Besides the analysis of out-of-competition control samples, the long term detection of steroids in hair could provide complementary information. An analytical approach to the identification of exogenous steroids in hair requires consideration of the presence of many other steroids in the hair matrix interfering the analysis at trace levels, and of a limited chemical stability. The analysis of endogenous steroids in hair appears to be even more complicated, because the possibility of many biotransformation reactions from (into) other precursors (metabolites) has to be taken into account. Precursor substances of anabolic steroids (especially esters as application forms) are very promising analytical targets of hair analysis, because they can only be detected after an exogenous intake. The quantitative evaluation of active parent compounds like testosterone (which is actively involved in physiological processes of hair growth) in hair is still controversial. Clinical applications under reproducible conditions can be useful, but the biovariability of these parameters will probably prevent the definition of acceptable cut-off levels as a criterion of abuse.

Chromatographic techniques--the basis of doping control.

The principal definition of doping, the groups of banned compounds and the basic analytical problems and strategy of doping analysis are outlined, and the position of chromatography in doping analysis is explained. Examples of the application of GC-MS, especially high-resolution MS. and of LC-thermospray MS to doping problems are given. A practical case is presented briefly, showing the post-analytical problem of evaluating even unequivocal results.

Thin-film characterization using a scanning laser acoustic microscope with surface acoustic waves.

The use of surface acoustic waves in a scanning laser acoustic microscope for the characterization of the mechanical or acoustic properties of thin films deposited on piezoelectric substrates is demonstrated. Quantitative measurements of mass loading effects of 5000-A-thick tungsten films deposited on lithium niobate substrates were obtained using 100-MHz surface acoustic waves. No information about the tungsten film could be obtained using 100-MHz compressional waves. Methods of generating surface waves on nonpiezoelectric materials so that this technique could be used on arbitrary substrates are discussed.

Diffraction tomography using beam waves: z-average reconstruction.

This paper discusses the problem of diffraction tomography, when a general beam wave is used as the excitation and the three-dimensional structure of the object is considered. The special case of a zero-order beam wave with measurements of the average of the scattered field in the z direction is detailed. It is shown that in this case a reconstruction algorithm identical to the two-dimensional geometry yields a z-averaged tomogram of the inhomogeneity.

Some problems associated with optical image formation from acoustic holograms.

A theoretical investigation of the imaging capabilities of ultrasonic holograms recorded by arrays of detectors has been carried out. The capabilities and limitations of this technique are illustrated with experimental results obtained with a closely packed array of detectors, simulated by a piezoelectric large area crystal (Sokolov type).