Validation of a method for the determination of 19-norandrosterone in human urine by coupled Gas Chromatography a Ion Trap Mass Spectrometry

Abstract

Nandrolone consumption gives in humans three main metabolites,
19-norandrosterone (19-NA), 19-noretiocolanolone (19-NE) and 19-norepiandrosterone.
Recent studies demonstrated the endogenous production of these compounds
in man at concentrations very close to the threshold of the International
Olympic Committee, this means 2 ng/mL for men and 5 ng/mL for women. The
Target metabolite to diagnostic consumption is 19-NA. This paper deals with the
qualitative validation of analytical method for 19-NA determination in human
urine with methyltestosterone as internal standard, using a solid phase extraction
procedure with DetectabuseÔ cartridges, enzymatic hydrolysis with b-glucuronidase
from E. Coli, liquidliquid extraction, derivatization with N-methyl-
N-trimethylsilyltrifluoroacetamide (MSTFA) to obtain the 19-NA trimethylsilyl
derivative (19-NA bis-O-TMS) and analysis by gas chromatography couple with
tandem mass spectrometry (GC-MS-MS), using an ion trap mass spectrometer.
Ultra HP-1 capillary column was used
(20 m x 0.20 mm X 0.11 µm), operated
with temperature programming ranging
from 181 °C (held for 1 min), to
210 °C (10 °C/min), then to 310 (20 °C/
min) held for 2 min . The acquisition
mode was MS/MS with ionization by
electronic impact and helium collision
induced dissociation using as parent
ion for 19-NA bis-O-TMS the ion with
m/z 405, monitoring the daughter ions
m/z 315 and m/z 225, and the parent ion
for the internal standard (methyltestosterone)
was the ion with m/z 446,
monitoring as daughter ion with m/z
356. Detailed results of the method validation
are shown.