GC-ion trap-MS in doping control: urinary determination of 19-norandrosterone and 19-noretiocholanolone at subnanogram per millilitre levels

A specific and sensitive method was developed for the quali-quantitative determination in urine of 19- norandrosterone (19-NA) and 19-noretiocholanolone (19-NE), main metabolites of the anabolic androgenic steroid nandrolone as well as of other 19-norsteroids. The total steroid fraction, extracted from urine, after addition of Ds-noretiocholanolone, by solid phase extraction on Cis cartridges, was hydrolysed by b-glucuronidase at pH 7.4. Liquid/liquid extraction was then accomplished with n-pentane at pH 9. Derivatization to produce bis-TMS derivatives was obtained by MSTFA / NH4 / dithyoerythrytol. GC-MS-MS analysis was carried out on a Saturn 2000 ion trap instrument, injecting 2-ul aliquots at 250° C under splitless conditions, using a 30-m Chrompack CP-Sil 8 capillary column with an oven temperature program from 150° C to 300° C and a carrier gas (He) flow of 1 ml/min. The ion trap was operated under EI ionisation, and multiple mass spectrometry experiments were optimized in order to achieve the best selectivity and sensitivity. In particular, MS-MS experiments were performed for both 19-NA and 19-NE on El-produced [M - CH3]+ species at m/z 405 which were selectively stored into the ion trap and collided with helium under resonant conditions. Specific product ions were then produced at m/z 315 and 225, which were monitored in the analytical scan function.