Improved performance and maintenance in gas chromatography/isotope ratio mass spectrometry by precolumn solvent removal.

The crucial step in current concepts to interface isotope ratio mass spectrometry (IRMS) to gas chromatography (GC) is efficient solvent removal. This is due to the essential postcolumn conversion of the analytes into simple gases, which is performed by either combustion or pyrolysis. The capacity of this step merely suffices to convert the analytes. Already small amounts of solvent present in the respective furnace can cause severe damage. In conventional GC/IRMS interfaces, the solvent is removed after passage of the GC column. Either back-flushing or flow diversion is employed for this purpose. Both techniques necessitate the use of numerous components such as unions, tee pieces, valves, and capillary connections. Often this results in significant deterioration of the chromatographic resolution. In contrast, accurate GC/IRMS measurements require baseline separation of adjacent peaks. Moreover, maintenance of conventional interfaces may be tedious and time consuming, mostly because the numerous connections are prone to leakage. In order to avoid these drawbacks, we propose a concept to efficiently remove the solvent before passage of the GC column. It is based on the use of a cooled injection system operated in solvent vent mode, where the solvent elimination is supported by an auxiliary pump. Most unions and tee pieces thus can be removed. The chromatographic resolution is considerably enhanced. In particular, analysis of high-boiling and polar compounds can be improved. At the same time, the maintenance of the system is significantly facilitated. Under the chosen conditions, partial losses of low-boiling analytes during solvent elimination were not associated with significant isotope fractionation.

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry.

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.

Application of stable carbon isotope analysis to the detection of testosterone administration to cattle.

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.