ratios of unlabeled-to-labeled compound ranging from 0.5 to 1.5.
The mixtures (324-530 µL) were dried under nitrogen at 45 °C
and reconstituted with 120 µL of methanol containing 1 mL/L
acetic acid for LC/MS/MS analysis. The 19-NA concentration
range of the calibration curve was ∼0.1-0.3 ng/µL (for mass ratio
range of 0.5-1.5).
get an approximate 1:1 ratio of analyte to internal standard, and
the aliquots were processed as described above in the sample
preparation for LC/MS/MS measurement.
LC/MS/MS Analysis. Analysis was performed on an Applied
Biosystems API 4000 (Foster City, CA) equipped with an Agilent
1100 Series LC system (Palo Alto, CA). A Zorbax Eclipse XDB-
Sample Preparation. Two freeze-dried urine materials (SRMs
C18 column was used for the analysis. Aliquots (10 µL) of standards
1
507b and 1511), each fortified with 19-NA glucuronide at three
or sample extracts were separated by LC with a gradient mobile
phase consisting of 0.5 mL/L acetic acid in water-acetonitrile.
The gradient was initially set at water-acetonitrile (66:34 by
volume) for 40 min, ramped to 100% acetonitrile at 40.1 min, and
held for 10 min to wash the column. The flow rate was 0.25 mL/
min. The column temperature was set at 30 °C. The autosampler
tray temperature was set at 10 °C. Electrospray ionization in the
positive ion mode and multiple reaction monitoring (MRM) mode
were used for LC/MS/MS. The transitions at m/z 277 f m/z
different concentrations, were used for this study. For each
material, vials of reconstituted urine samples were combined, and
nine aliquots were fortified with 19-NA glucuronide at three
concentrations, three each with 1.718, 3.437, and 17.142 ng/mL
1
9-NA glucuronide (sodium salt) for concentrations 1, 2, and 3,
respectively (equivalent to 1.001, 2.002, and 9.985 ng/mL 19-NA
for concentrations 1, 2, and 3, respectively). Samples were
prepared in six different sets (3 sets each for SRM 1507b and
SRM 1511). Each set consisted of triplicate 8.0-mL aliquots for
each of concentrations 1 and 2 and triplicate 2.0 mL for concentra-
tion 3. One 8.0-mL aliquot of urine sample without fortification
was also included in each set. Each aliquot was placed into a 50-
2
59 and m/z 281 f m/z 263 were monitored for 19-NA and 19-
NA-d4, respectively. The dwell times were 0.25 s for MRM. The
curtain gas and collision gas were nitrogen at settings of 207 (30
psi) and 21 kPa (3 psi), respectively. The ion source gas 1 and
ion source gas 2 were air at settings of 518 (75 psi) and 310 kPa
mL glass centrifuge tube and an appropriate amount of 19-NA-d
4
(45 psi), respectively. The electrospray voltage was set at 5500 V,
was added to give an ∼1:1 ratio of analyte to internal standard.
The pH of each sample was adjusted to 6.3 ( 0.1 by adding 2 mL
of 0.2 mol/L phosphate buffer (pH 6.0) and a small amount of 0.5
mol/L phosphoric acid (up to 250 µL). To each sample, 400 µL of
â-glucuronidase solution was added to hydrolyze 19-NA glucu-
ronide to free 19-NA. The mixtures were incubated at 50 °C for 1
h. Following hydrolysis, each sample was cooled to room tem-
perature and adjusted to pH 9.8 ( 0.2 with 2 mL of 0.1 g/mL
carbonate buffer (pH 9.8) and a small amount of 5 mol/L
ammonium hydroxide (up to 300 µL). To each sample, 2 g of
sodium chloride was added, and the 19-NA was then extracted
from the urine hydrolyzate with 16 mL of hexane. Each sample
was shaken vigorously for 3 min using a mechanical shaker to
allow complete mixing. After centrifugation of the tube for 10 min
at 2000g, the upper hexane layer was transferred to another 50-
mL centrifuge tube. Hexane extraction was repeated once more
with another 16 mL of hexane. The combined hexane extract was
dried under nitrogen at 45 °C, and the residue was reconstituted
with 100 µL of methanol with 1 mL/L acetic acid to a final
concentration of ∼0.2 ng/µL of 19-NA for LC/MS/MS analysis.
Absolute Recovery of 19-NA. The urine samples from SRM
and the turbo gas temperature was maintained at 450 °C. The
declustering potential, entrance potential, collision energy, and
collision exit potential were set at 61, 10, 13, and 18 V, respectively.
The following measurement protocol was used for LC/MS/
MS analysis. The standards were analyzed first, followed by the
samples, and then by the samples and standards in reverse order.
By combining the data of standards run before and after the
samples, a linear regression was calculated (y ) mx + b model),
which was used to convert the measured intensity ratios of analyte
to mass ratios. The mass ratios were then used along with the
amounts of the internal standard added to calculate analyte
concentrations.
Interference Testing. The metabolites of nandrolone and
other structural analogues of 19-NA having relative molecular
masses close to that of 19-NA were tested for interference using
the LC/MS/MS method described above for 19-NA measurement.
Statistical Treatment. Statistical treatment of the data was
in accord with NIST guidelines,19 which conform with the ISO
2
0
Guide to the Expression of Uncertainty in Measurement. Potential
sources of uncertainty were evaluated, and those factors that could
contribute significantly were used to calculate the standard
uncertainty. The type A uncertainty, calculated from the impreci-
sion of the measurements, was combined quadratically with the
type B uncertainty components to determine the standard uncer-
1507b were used for the study of absolute recovery of 19-NA with
the extraction method. Two groups of samples were prepared.
For the first group, urine samples were spiked with a known
amount of 19-NA before extraction and 19-NA-d
For the second group, both 19-NA and 19-NA-d
4
after extraction.
were spiked
c
tainty u . For type A, an analysis of variance calculation was
4
performed on the measurement data to determine whether set-
to-set differences were statistically significant. This analysis
determined the number of independent measurements n used for
calculating the measurement standard deviation of the mean. The
type B factor contributions used were based on knowledge about
uncertainties in the purity of the reference compound, in the
accuracy of volumetric addition steps, in the completeness of the
hydrolysis step, and on an allowance for unknown systematic
before extraction. The samples were processed as described above
in the sample preparation for LC/MS/MS measurement. The
absolute recovery of 19-NA from urine was calculated from the
comparison of the intensity ratios of the two groups.
Recovery of the Added 19-NA (Accuracy Test). The urine
samples from SRM 1507b were used for this study. Triplicate 8.0-
mL aliquots for each of concentrations 1 and 2 and triplicate 2.0
mL for concentration 3 were taken for a study of the accuracy of
the method. A known amount of 19-NA was added to the nine
aliquots, three each with 1.000, 2.001, and 9.981 ng/mL 19-NA.
(19) Taylor, B. N.; Kuyatt, C. E. NIST Technical Note 1297, 1994 (available at
http://physics.nist.gov/Pubs/guidelines/contents.html).
(20) ISO Guide to the Expression of Uncertainty in Measurement; International
Organization for Standardization: Geneva, Switzerland, 1993.
4
An appropriate amount of 19-NA-d was added to each aliquot to
Analytical Chemistry, Vol. 78, No. 10, May 15, 2006 3395
Tai, Susan S.-C.
