sunitinib N-oxide in blood to clarify the
impact of sunitinib N-oxide on the
expression of ADRs of sunitinib.
To date, many methods using liquid
chromatography with ultraviolet detection
(LC-UV) or liquid chromatography-tandem
mass spectrometry (LC-MS/MS) have been
reported as methods for analyzing sunitinib
and N-desethyl sunitinib [19-21]. However,
since the concentration of sunitinib N-oxide
was estimated to be about 1/10 to 1/20 that
of sunitinib in our previous study [17], an
analytical method that is more sensitive
than that used for quantifying sunitinib is
required to study the effect of sunitinib N-
oxide on the body. Therefore, in this study,
we used a supported liquid extraction (SLE)
pretreatment method to concentrate
thesample and analyzed sunitinib and its
metabolites in blood using LC-MS/MS
2. Materials and Methods
2.1 Materials
Sunitinib (>99% purity) was
purchased from LC Laboratories (Woburn,
MA, USA), sunitinib N-oxide (>99%
purity) and N-desethyl sunitinib (96%
purity) were purchased from Toronto
Research Chemicals (Toronto, Canada) and
voriconazole (>98% purity) was purchased
from Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan). ISOLUTE SLE+ 400 μL
96-well plates (SLE array plates) were
purchased from Biotage Japan Ltd. (Tokyo,
Japan). All other reagents were obtained
from commercial sources and were of
LCMS, HPLC, or special grade.
2.2 Preparation of stock solutions,
working solutions, calibration
samples, and quality control
samples
Primary stock solutions of 1.0 mg/mL
of sunitinib N-oxide, sunitinib, N-desethyl
sunitinib, and voriconazole, used as an
internal standard (ISTD), were separately
prepared in methanol. The primary stock
solution of mixture of sunitinib N-oxide,
sunitinib, and N-desethyl sunitinib was
diluted with 50% methanol to yield
standard working solutions(0.5, 1.25, 2.5,
5, 12.5, 25, 50, 125, 250, 500, and 1250
ng/mL). The primary stock solution of the
ISTD was diluted in 50% methanol to 1
μg/mL or 100 ng/mL. The stock solutions
and other diluted solutions were stored at -
20°C and 4°C, respectively, under dark
conditions. All solutions were equilibrated
to room temperature before use. Calibration
samples and quality control (QC) samples
were prepared by spiking blank pooled
human serum (PHS) with a given volume of
different working solutions. The
concentrations of sunitinib N-oxide for the
calibration sample of sunitinib N-oxide
were set at 0.1, 0.25, 0.5, 1, 2.5, and 5
ng/mL. Calibration samples for sunitinib
and N-desethyl sunitinib were prepared at 7
concentrations: 2.5, 5, 10, 25, 50, 100, and
250 ng/mL. QC samples of sunitinib N-
oxide as the lower limit of quantitation, low
level, middle level, and high level were set
as 0.1, 0.25, 1, and 5 ng/mL, respectively,
and those of sunitinib and N-desethyl
sunitinib were set as 2.5, 5, 50, and 250
ng/mL, respectively.
2.3 Sample preparation
The serum samples for analysis of
sunitinib N-oxide were treated using an
SLE array plate. Briefly, a mixture of 50 μL
of blank PHS, 10 μL of a standard working
solution, 10 μL of the ISTD (100 ng/mL),
and 330 μL of 1% aqueous ammonia was
applied onto an ISOLUTE SLE+ 400 μL
96-well plate (Biotage) sitting on top of a
clean 96-well collection plate. Positive
pressure was applied for 2 s to initiate flow,