Rapid determination of methandrostenolone in equine urine by isotope dilution liquid chromatography-tandem mass spectrometry.

The researchers developed a fast method for identifying Methandrostenolone in horse urine using isotope dilution liquid chromatography-tandem mass spectrometry. This method establishes a reliable way of detecting doping in horses, with a detection limit of 180 pg/ml of urine and a precision of 3% at the 16 ng/ml level.

Research Methodology

• The researchers began by spiking urine samples with [17-methyl-2H3]methandrostenolone, which served as the internal standard. These samples were then extracted using a combination of dichloromethane and cyclohexane.
• After that, the organic phase was concentrated and injected onto a short octyl-silica column. This column helped in separating methandrostenolone and 17-epimethandrostenolone, which are key elements the scientists were examining.

Mass Spectrometry Process

• The researchers used a Sciex TAGA 6000E triple quadrupole mass spectrometer equipped with an atmospheric pressure ion source. The ion source generated ions through a heated pneumatic nebulizer with corona discharge ionization.
• This process led to the creation of [M + H]+ ions and a weak fragment ion that resulted from loss of water. This crucial element of the methodology allowed for the identification and separation of ions, paving the way for a closer examination.

Quantification and Ions Examination

• The protonated molecular ions at m/z 301 and 304 for methandrostenolone, 17-epimethandrostenolone and the internal standard were transmitted to the second quadrupole for collision-induced dissociation.
• Quantification was obtained by monitoring the selected reaction of three daughter ions. Despite having been separated by liquid chromatography, methandrostenolone and 17-epimethandrostenolone gave identical mass spectra.

Detection Limit and Precision

• The method’s detection limit, or the smallest quantity that can be detected reliably, was 180 pg/ml at a 99% confidence level when a urine extract corresponding to 2.8 ml urine was injected.
• The precision (relative standard deviation) was 3% at the 16 ng/ml level and the linear dynamic range was at least 3 orders of magnitude. This indicates a high degree of accuracy and reliability of the method.

Screening of Unknown Metabolites

• Finally, the researchers achieved a parent ion scan of m/z 121, a fragment that matches the intact A-ring of the steroids. This process allowed screening for unknown metabolites in the urine after administering methandrostenolone to horses and humans.