Vol 2, Issue 3, 2020 (97-103)
http://journal.unpad.ac.id/idjp
*Corresponding author,
e-mail : tkyaraki@gunma-u.ac.jp (T. Araki)
https://doi.org/10.24198/idjp.v2i3.31586
© 2020 N. Sekizaki et al
Simple and Rapid Method for Determination of Abemaciclib in Human Serum Using
Supported Liquid Extraction Pretreatment and LC-MS/MS Analysis
Naoto Sekizaki
1,2
, Hideaki Yashima
1
, Takuya Araki
1,2
*, Koujirou Yamamoto
1,2
1
Department of Clinical Pharmacology and Therapeutics, Gunma University Graduate School of
Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan
2
Department of Pharmacy, Gunma University Hospital, 3-39-15 Showa-machi, Maebashi 371-8511,
Japan
Received : 01 Sept 2020, Revised : 04 Oct 2020, Accepted : 19 Nov 2020, Published : 5 Dec 2020
ABSTRACT
We developed a simple and rapid method for the determination of abemaciclib in human
serum using supported liquid extraction (SLE) method for pretreatment and LC-MS/MS.
Abemaciclib was extracted using SLE method with methyltert-butyl ether (MTBE) as
elution buffer, and analyzed by LC-QTOF MS system, LCMS9030 (Shimadzu).
Abemaciclib and fluconazole (internal standard) were detected with ESI spray in positive
ionization mode, and the transition were set at 507.3/393.1629 for abemaciclib and
307.1/220.0677 for fluconazole. The retention times of abemaciclib and fluconazole were
2.76 and 2.98 min, respectively, and good linearity was obtained from 20–1,000 ng/mL for
abemaciclib. The regression equation (weight = 1/x2) describing the calibration curve in
human serum was y = 0.0196 x – 0.056 (R2 = 0.999), where y is the peak area ratio of
abemaciclib against the IS and x is the nominal concentration of abemaciclib. The intra-
and inter-assay accuracy varied between -4.3–1.7%, and the precision varied between 0.90–
6.19%. The mean recovery rate of abemaciclib was 87.7 ± 4.3%, and the mean matrix factor
was 1.00 ± 0.083. Our method offers speed and simplicity of sample preparation, which is
one of great advantages in the analysis of clinical specimens. We believe that the present
method will contribute to establishing a methodology for determining the optimal dose of
abemaciclib for individual patients.
Keywords: abemaciclib, SLE, supported liquid extraction, quantification
1. Introduction
Abemaciclib is an orally administered small
molecule inhibitor of cyclin-dependent kinases
(CDKs) 4 and 6 [1]. It has reported to be effective
as a first-line treatment for hormone receptor-
positive, HER2-negative advanced breast cancer
when used in combination with a fulvestrant or
nonsteroidal aromatase inhibitor [2–4]. On the
other hand, hematological toxicity and diarrhea,
common adverse drug reactions (ADRs) of
abemaciclib, are known to results in drug
intolerance in patients [5, 6]. Recently, some
ADRs of abemaciclib have been reported to be
dose dependent [7]. However, the
pharmacokinetics of abemaciclib vary largely
among individual patient [8] and have been
reported to be affected by concomitant use of
drugs such as CYP3A modulators [9]. So, to
develop a methodology to determine the
appropriate dose for each patient, establishment
of method for quantifying abemaciclib is needed.
In 2015, Raub et al. detected abemaciclib
and palbociclib using LC-MS/ MS in the study of
exposure of CDK4 and 6 inhibitors to brain, but
no further details of the methods and their
performance were described [10]. Recently,
N. Sekizaki et al / Indo J Pharm 3 (2020) 97-103
98
Martinez-Chavez et al. also reported the
quantitative method of abemaciclib, palbociclib,
and ribociclib in human and mouse matrices using
LC-MS/MS [11]. However, this method could not
be applicable to the analysis of clinical specimens
directly, because the measurement range of
abemaciclib was set at 2–150 ng/mL in their study
though the clinical concentration of abemaciclib
in the blood is known to vary greatly and be about
50–300 ng/mL [12]. In addition, the protein
precipitation method used in the report is widely
used for sample pretreatment because of
simplicity and ease, however, it is also known to
place a heavy load on an analytical system,
especially MS system. Considering the analysis
of a large number of clinical specimens, methods
should be simple, easy, and has a low load on the
detection system. Thus, a method of measurement
of abemaciclib in clinical specimens is needed to
establish safer and more effective therapy using
abemaciclib.
Recently supported liquid extraction (SLE)
method was developed as a new pretreatment
method to replace the widely used liquid-liquid
extraction (LLE) method, and have been already
used for detection of some drugs and other
substances in biological fluids [13–19]. SLE
method is based on the same chemistry as LLE.
However, instead of using a separation funnel and
partitioning between two immiscible liquids the
extraction is performed on a solid support made
of diatomaceous earth. Compared to ordinary
solid-phase extraction (SPE) or LLE, SLE has
many advantages such as the absence of emulsion
formation, high-extraction efficiency, simplicity
of procedure, and short time required [14-23]. In
addition, since SLE does not need the
deproteinization which is needed for SPE as a
pretreatment, SLE also has the advantage of
avoiding coprecipitation with proteins associated
with the deproteinization process [15].
In this study, to establish safer and more
effective therapy using abemaciclib, we
developed a method for the measurement of
abemaciclib in human blood that can be
applicable for clinical use using SLE method and
LC-MS/MS analysis.
2. Methods
2.1 Materials
Abemaciclib and fluconazole was
purchased from LKT Laboratories, Inc. (St. Paul,
Minnesota, USA). Human serum pool (P/N.
12181201, lot#. BJ14005A) was purchased from
COSMO Bio Co., LTD (Tokyo, Japan).
ISOLUTE SLE+ 400 μL 96-well plate (SLE array
plate) was purchased from Biotage Japan Ltd.
(Tokyo, Japan). All other reagents were obtained
from commercial sources and were LCMS or
HPLC-grade or special-grade reagents.
2.2 Preparation of stock solutions, working
solutions, calibration samples, and quality
control samples
Primary stock solutions of 1.0 mg/mL of
abemaciclib and fluconazole were separately
prepared in ethanol and methanol, respectively.
Fluconazole was used as an ISTD because it was
found to be extracted by same pretreatment
process, and show the similar elution behavior in
LC separation as abemaciclib, and has been rarely
used in combination with abemaciclib due to its
CYP3A4 inhibitory effect. Primary stock solution
of abemaciclib was diluted with the initial mobile
phase (0.1% formic acid in Milli-Q
water:acetonitrile = 15:85) to yield standard
working solutions (0.1, 0.25, 0.5, 1.0, 2.5, and 5.0
μg/mL). The ISTD was diluted in 1% aqueous
ammonia to 50 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 serum with a given volume of different
working solutions. The calibration samples
consisted of six concentrations of abemaciclib:
20, 50, 100, 200, 500, and 1,000 ng/mL as
concentrations in serum. QC samples of
abemaciclib as low level (L), middle level (M),
and high level (H) were set at 20, 200, and 1,000
ng/mL, respectively.
2.3 Sample preparation
The serum sample was treated with SLE
method. A mixture of 100 μL of blank serum, 20
μL of standard solution in initial mobile phase,
N. Sekizaki et al / Indo J Pharm 3 (2020) 97-103
99
and 280 μL of 1% aqueous ammonia containing
50 ng/mL of fluconazole was applied into an
Figure 1. Typical chromatograms of abemaciclib and fluconazole in human serum
A~D: Chromatograms of abemaciclib in blank serum (A), spiked with 20 ng/mL (B), spiked with 200
ng/mL (C), and spiked with 1,000 ng/mL (D)
E: Chromatogram of fluconazole as ISTD in serum sample
ISOLUTE SLE+ 400 μL 96-well plate (SLE array
plate) (Biotage) sitting on top of a clean 96-well
collection plate. Positive pressure was applied for
2 sec to initiate flow, and sample were allowed to
stand for 5 min to absorb to the diatomaceous
earth extraction bed. As elution buffer, 900 μL of
methyltert-butyl ether (MTBE) was added to the
SLE array plate. After waiting for 5 min, a further
aliquot of MTBE (900 μL) was applied and eluted
within around 5 min by standing under natural
gravity. The samples in the collection plate were
evaporated to dryness under reduced pressure at
room temperature. The residue was dissolved in 1
mL of initial mobile phase, and 2 μL of samples
were used for LC-MS/MS analysis.
2.4 Detection of abemaciclib by LC-MS/MS
Quadrupole time-of-flight chromatograph
mass spectrometer was used to detect
abemaciclib. LCMS 9030 (Shimadzu, Kyoto,
Japan) with ESI spray in positive ionization mode
was used with the following parameters: interface
voltage, 1,000V; interface temperature, 300°C;
heat block temperature, 400°C; desolvation line
0
1
2
3
0 1 2 3 4 5
0
2
4
6
0 1 2 3 4 5
0
2
4
6
0 1 2 3 4 5
0
5
10
15
0 1 2 3 4 5
0
1
2
3
0 1 2 3 4 5
intensity (cps x 100)
time (min) time (min)
intensity (cps x 10,000)
time (min) time (min)
intensity (cps x 1,000)
time (min)
intensity (cps x 1,000)
intensity (cps x 100,000)
A B
C D
E
N. Sekizaki et al / Indo J Pharm 3 (2020) 89-100
100
temperature, 250°C; nebulizer gas (N
2
) flow, 3
L/min; heating gas (air) flow, 10 L/min; drying
gas (N
2
) flow, 10 L/min; and collision-induced-
dissociation gas pressure, 230 kPa. The following
transitions were monitored: 507.3/393.1629 for
abemaciclib, and 307.1/220.0677 for fluconazole.
Collision energies were 26.0V, and 16.0V,
respectively. For separation analysis, LC was
performed with a Nexera X2
®
system
(Shimadzu). The system consisted of LC-30AD
as solvent systems, SIL-30AC as an auto sampler,
CTO-20AC as a column heater, and CBM-20A as
a system controller. An ACQUITY UPLC
®
BEH130 C18 column (2.1 mm × 100 mm, 1.7
μm) (Waters, Milford, MA) was used as the LC
column. The LC conditions were as follows:
column temperature, 40°C; mobile phase, 0.1%
formic acid in Milli-Q water (A) and acetonitrile
(B); flow rate, 0.3 mL/min; and gradient program,
15 to 35%B in 5 min, 35 to 80%B in 1min, 80%B
in 1 min, 80 to 15%B in 1min, and 15%B in 3min.
2.5 Assay validation
The method was validated according to
guidance of ICH [23]. Precision was measured as
the coefficient of variation expressed as a
percentage, and accuracy was expressed as the
relative error of the nominal versus the measured
concentration. The intra-assay variability was
tested by measuring three different serum
samples against the same calibration curve. The
inter-assay variability was tested on three
different days and a new calibration curve was
constructed for each day. The matrix effect was
also evaluated quantitatively by measurement of
the matrix factor.
3. Results
3.1 Detection of abemaciclib and fluconazole
The typical chromatograms obtained from
blank pooled human serum (PHS) and from blank
PHS spiked with abemaciclib (20, 200, 1,000
ng/mL as concentrations in serum) and
fluconazole (14 ng/mL as concentration in serum)
were shown in figure 1. The retention times of
abemaciclib and fluconazole were 2.76 and 2.98
min, respectively.
3.2 Linearity, Accuracy and Precision
Good linearity was obtained from 20–1,000
ng/mL for abemaciclib. The regression equation
(weight = 1/x
2
) describing the calibration curve in
PHS was y = 0.0196 x – 0.056 (R
2
= 0.999), where
y is the peak area ratio of abemaciclib against the
IS and x is the nominal concentration of
abemaciclib.
Accuracy and precision were shown in
Table 1. The results were within the accepted
limits and therefore the assay was accurate and
precise.
Table 1. Intra- and inter-assay validation
Nominal concentration
Measured concentration
Accuracy
Precision
(ng/mL)
(ng/mL)
(RE %)
(CV %)
Intraday-assay (n = 3)
20
20.3 ± 0.31
1.70
1.52
50
48.0 ± 1.59
-4.09
3.31
100
96.9 ± 1.99
-3.15
2.06
200
196.5 ± 2.41
-1.73
1.23
500
485.6 ± 16.9
-2.89
3.48
1,000
976.9 ± 26.2
-2.31
2.69
Interday-assay (n = 3)
20
20.3 ± 1.26
1.28
6.19
50
47.9 ± 1.42
-4.30
2.96
N. Sekizaki et al / Indo J Pharm 3 (2020) 89-100
101
100
98.1 ± 1.23
-1.94
1.26
200
198.2 ± 6.04
-0.91
3.05
500
499.3 ± 25.9
-0.13
5.19
1,000
993.2 ± 8.92
-0.68
0.90
Data are presented as mean ± standard deviation
3.3 Extraction recovery
The recovery of abemaciclib was
acceptable and reproducible (Table 2). The
mean recovery was 87.7%.
Table 2. Recovery rate
Concentration
Recovery rate
(ng/mL)
(%)
20
86.0 ± 2.54
200
88.2 ± 4.49
1,000
88.8 ± 6.27
Data are presented as mean ± standard deviation
3.4 Interference and Matrix effect
The RSD was less than 15% and peak of
abemaciclib was not detected from blank PHS
sample. The average of matrix factor was 1.00 ±
0.083 (Table 3), suggesting no significant
matrix effect observed and no adverse impact on
the quality of the data produced.
Table 3. Matrix efficacy
Concentration
(ng/mL)
Matrix factor
20
0.976 ± 0.014
200
1.02 ± 0.034
1,000
1.02 ± 0.037
Data are presented as mean ± standard deviation
4. Discussion and Conclusion
We established a method for measurement
of abemaciclib in human serum, which can be
used directly for the analysis of clinical
specimens, using SLE pretreatment method and
LC/MS/MS analysis for the first time. In our
method, SLE was used for pretreatment of
serum sample. SLE has many advantages such
as the high-extraction efficiency, simplicity of
procedure, and short time required compared to
LLE or SPE [14-23]. In fact, with our method,
the time required for pretreatment was less than
1 hour, a recovery rate of abemaciclib was
around 90%, and almost no matrix effect was
found. These points are very advantageous as an
analytical method used for measurement of
drugs in clinical specimens. Furthermore, since
our method needs simple steps using a column
and simple steps, it is not easily affected by
variations in the operator’s skill. Now, this
method has been used as clinical test in clinical
setting, and the concentrations of abemaciclib in
the blood of patients who received abemaciclib
therapy were successfully measured.
In conclusion, we established a method for
measurement of abemaciclib in human serum,
which can be used directly for the analysis of
clinical specimens, for the first time. Our
method offers speed and simplicity of sample
preparation, which is one of great advantages in
the analysis of clinical specimens. We believe
that the present method will contribute to
establishing a methodology for determining the
optimal dose for individual patients.
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