AI-64310-CRFT

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2 Experimental
Administration Samples
Two healthy male volunteers (56 and 61 years of age)
received a single oral dose of 5 mg of stanozolol (Winstrol ®).
Urine samples were collected prior to (blank) and up to
28 days post administration of the drug. The urine
specimens were stored at -20 °C until preparation and
analysis. The study was approved by the local ethical
committee and written consent was obtained from
both participants.
Sample Preparation
Ninety microliters of urine were enriched with 10 µL of
an acetonitrile solution containing the internal standard
methyltestosterone (1 µg/mL). The samples were vortexed
for 10 s and subjected to LC-MS/MS analysis.
Confirmatory analyses were conducted by applying 1 mL
of urine to a solid-phase extraction (SPE) cartridge
preconditioned with 2 mL of water and 2 mL of
methanol. After the sample had passed through, the resin
was washed with 2 mL of water and the analytes eluted
with 2 mL of methanol. The organic phase was evaporated
to dryness and reconstituted in 100 µL of solvents A
(0.1% formic acid) and B (acetonitrile) (1:1, v/v) for
LC-MS/MS analysis.
LC-MS/MS
The analyses were conducted using a Thermo Scientific™
Accela™ 1250 liquid chromatograph interfaced via a
heated electrospray ionization (HESI-II) source to a
The LC was equipped with a Nucleodur® C18 Pyramid
analytical column, 50 x 2 mm, particle size 1.8 µm,
(Macherey-Nagel, Düren, Germany) and a corresponding
precolumn (4 x 2 mm, particle size 3 µm). The mobile
phases 0.1% formic acid (A) and acetonitrile (B) were
used to perform a gradient elution at 200 µL/min
from 99% of A to 100% of B in 7 min, followed by
re-equilibration for 3 min.
The mass spectrometer settings were as follows:
Ionization mode Positive
Spray voltage 4000 V
Source temperature 300 °C
Full Scan
Resolution setting 35,000 (FWHM) at m/z 200
Mass range m/z 100–1000
Targeted Higher Energy Collisional Dissociation (HCD)
Preselected ions m/z 505.25 (for stanozolol- and
17-epistanozolol glucuronide)
m/z 521.25 (for 3’-OH-stanozolol
glucuronide)
Resolution setting 35,000 (FWHM) at m/z 200
Mass ranges m/z 50–535 and 50–550
Automatic gain control 2 x 105
Maximum IT fill time 200 ms
Isolation window 1.2 Da
Applied collision energy 55 and 72 eV
Collision gas Nitrogen
Method Characterization
Due to the lack of certified reference material for the
newly identified conjugates, the specificity (20 blank urine
samples from 10 male and 10 female volunteers), limit of
detection (LOD), and ion suppression/enhancement were
determined with 3’-OH-stanozolol glucuronide only. In
the case of the confirmatory assay, the recovery, linearity,
and intra- and interday precision (at 25, 100, and
200 pg/mL), together with the identification capability,
were also determined with 3’-OH-stanozolol glucuronide.
Results and Discussion
Stanozolol Metabolites
Administration study urine samples were collected after
oral application of 5 mg of stanozolol and subjected to
LC-MS/MS with high-resolution, accurate-mass capability
in both MS and MS/MS modes. In agreement with earlier
initial testing protocols, urine samples were injected into
the LC-MS/MS system without further sample preparation,
except for the addition of the internal standard
methyltestosterone (at 100 ng/mL). Targeted product ion
scan experiments [parallel reaction monitoring (PRM)]
were performed on precursor ions of various different
metabolites, including particularly the glucuronide(s) of
stanozolol and its 17-epimer (precursor ion [M+H]+ at
m/z 505.29) and hydroxylated phase-I-metabolites
(precursor ion [M+H]+ at m/z 521.29). These yielded a
series of signals, which were assigned to stanozolol
metabolites by means of accurate masses of the intact
protonated molecules and the respective aglycons
obtained via collisional activation. A typical post-
administration sample (5 days) and a blank urine
specimen are shown in Figure 2.