High-temperature high-performance liquid chromatography on a porous graphitized carbon column coupled to an Orbitrap mass spectrometer with atmospheric pressure photoionization for screening exogenous anabolic steroids in human urine.

RATIONALE: The presence in a urinary matrix of a large number of endogenous steroids and corticosteroids with similar structures can hamper the detection of specific exogenous steroids using liquid chromatography/mass spectrometry (LC/MS) with reversed-phase columns. Therefore, the development of LC/MS methods using alternative columns is of great interest. Porous graphitized carbon is a unique stationary phase for high-performance liquid chromatography (HPLC), with properties differing from traditional silica-based and polymeric stationary phases. METHODS: The new method involves enzymatic hydrolysis, liquid-liquid extraction, and determination by high-temperature HPLC/Orbitrap mass spectrometry (HTLC/Orbitrap MS) with atmospheric pressure photoionization (APPI). To achieve APPI of doping substances, the mobile phase consisted of 0.1% CF3COOH (A) and a mixture of acetonitrile/2-propanol (25:75 v/v), containing 0.1% CF3COOH (B), which was used as an effective proton source. RESULTS: A screening method for the detection of 57 exogenous steroids has been developed. The method was validated by spiking 10 different blank urine samples at different concentration levels. Validation parameters included limit of detection (LOD), selectivity, ion suppression, extraction recovery, and repeatability. All studied compounds had an LOD lower than the minimum required performance level. Of the 57 steroids studied, 55 showed recovery better than 70%. For all of the analytes, the relative retention times proved to be stable between days, with relative standard deviations (RSDs) smaller than 0.3%. In addition, the interday RSDs of the peak area ratios ranged between 0.7% and 14.5%. CONCLUSIONS: The proposed method matches the basic requirements of all methods used to analyze drugs or metabolites in an antidoping laboratory, i.e., sensitivity, selectivity, and specificity. The acquisition of full-scan mass spectra with accurate masses can be a valuable tool in the retrospective evaluation of analyzed samples for anabolic steroids recently added to the prohibited list.

Detection of S-adenosylhomocysteine and methylation index in blood by capillary electrophoresis.

This work proposes an approach to the direct analysis of S-adenosylhomocysteine (SAH) and the methylation index in blood using CE with UV detection (CE-UV). After application of meglumine postinjection, we achieved SAH in-capillary preconcentration in the HClO4 extracts of erythrocytes, which improved the detection limit (S/N = 3) of SAH up to 3 fmol or 180 nmol/L at the injection volume of 50 nL, taking into account the sample dilution rate. CE-UV was carried out in 30 mM glycine and 45 mmol/L HCl (pH ~1.8) at 17 kV in a capillary 48 cm in length and 50 µm id. Accuracy of the technique was 101% and reproducibility was about 12%.