Journal of Pharmaceutical and Biomedical Analysis
UPLC-MS/MS method for the determination of Lenvatinib in rat plasma
and its application to drug-drug interaction studies
Yanjun Cuia,b
, Ying Lib
, Liju Fana,b
, Jing Anb
, Xiaonan Wanga,b
, Ran Fua,b
, Zhanjun Dongb,⁎
a Graduate School of Hebei Medical University, Shijiazhuang 050017,China b Department of Pharmacy, Hebei General Hospital, No. 348 West Heping Road, Shijiazhuang 050051, China
article info
Article history:
Received 28 May 2021
Received in revised form 29 July 2021
Accepted 31 August 2021
Available online 2 September 2021
abstract
Lenvatinib (LEN) is a multitargeted tyrosine kinase inhibitor registered for the first-line treatment of un￾resectable advanced hepatocellular carcinoma. Wuzhi capsule (WZC) is a traditional Chinese medicine
preparation; it is used to decrease the aminotransferase level of the liver and protect liver function. Thus,
patients with hepatocellular carcinoma (HCC) are potentially treated with a combination of LEN and WZC,
but there is no information about the interaction between the two drugs. We developed a simple, rapid, and
sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method for
the quantitative determination of lenvatinib in rat plasma. Liquid–liquid extraction of plasma samples was
carried out with ethyl acetate. Chromatographic separation of analyte was performed using gradient elution
with acetonitrile and 0.1% formic acid water. The positive ion multi-response monitoring mode was used,
and the target of the parent and daughter ions of LEN and IS were m/z 427.1→370 and m/z 432.1→370,
respectively. All the validation projects were in accordance with the guidelines. Good linearity of
0.2–1000 ng/mL (r > 0.999) was achieved. The lower limit of quantification was 0.2 ng/mL. The precision
and accuracy are acceptable. The method was successfully applied to pharmacokinetics and drug interaction
analysis. The results show that WZC can significantly increase the Cmax (maximum plasma concentration)
and AUC (area under the concentration-time curve) of LEN. An UPLC -MS/MS method that can be used for
studying drug–drug interaction as a valuable tool was developed in this study. Drug-drug interactions were
observed between the WZC and LEN.
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
Primary liver cancer is the sixth most common cancer worldwide
and the third leading cause of cancer mortality. Hepatocellular car￾cinoma (HCC) is the most common form of primary liver cancer
comprising 75–85% of cases, and its main risk factors include he￾patitis virus infection, smoking, heavy alcohol intake, excess body
weight, aflatoxin-contaminated foods, and type 2 diabetes[1]. Most
HCC cases are diagnosed at advanced stages when treatment options
are limited. Systemic treatment including tyrosine kinase inhibitors
has become a good option [2,3]. Sorafenib is the first molecular
targeted tyrosine kinase inhibitor for advanced HCC. In the following
decade, the development of many new drugs was attempted, but
failed [4]. A randomized phase 3 non-inferiority trial [5]indicated
that lenvatinib (LEN) is non-inferior to sorafenib in the overall pri￾mary endpoint survival in untreated advanced HCC in 2018; LEN was
approved for the first-line treatment of advanced unresectable HCC
based on this clinical trial. Furthermore, LEN is better than sorafenib
for secondary endpoints. LEN is an orally administered multitargeted
tyrosine kinase inhibitor, an antiangiogenic agent that can inhibit
VEGFR (vascular endothelial growth factor receptor) 1–3, PDGFR
(platelet-derived growth factor receptor) α, FGFR (fibroblast growth
factor receptor) 1–4, oncogenes RET (rearranged during transfec￾tion), and stem cell factor receptor[6,7]. 14% of patients have ele￾vated aspartate aminotransferase level when LEN is orally
administered in the clinical trial [5]. Patients may need to use drugs
that are hepatoprotective and reduce the transaminase level to re￾duce the adverse effects and prolong the use of LEN, strengthening
the antitumor efficacy.
Wuzhi capsule (WZC) is an ethanol extract preparation of
Schisandra chinensis; the main components are schisantherin A,
deoxyshisandrin(schizandrin A), schisandrin, schisandrol B, and
schisanhenol [8,9]. Some studies have shown that WZC and its active
ingredients are effective in suppressing the drug-induced increases
in serum aminotransferase and total bilirubin levels. WZC has also

https://doi.org/10.1016/j.jpba.2021.114360

0731-7085/© 2021 Elsevier B.V. All rights reserved.
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⁎ Corresponding author.
E-mail address: [email protected] (Z. Dong).
Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114360
antioxidant and anti-inflammatory activities, and it accelerates liver
regeneration to protect the liver function [10,11]. In addition, some
studies showed that its active ingredients such as schisantherin A
and schizandrin A exhibited potent anticancer activity in various
cancer cells [12,13], so patients with HCC are likely to administer
WZC. Thus, patients with HCC are potentially treated with a com￾bination of LEN and WZC to protect the liver function, improve the
efficiency, and reduce the side effects, but the information about
drug–drug interaction is limited. Previous research showed that
WZC and its active ingredients probably can regulate cytochrome
P450A4 (CYP3A4) and P-gp (P-glycoprotein) activity[14,15]. Fur￾thermore, WZC and or its active ingredients can influence the
pharmacokinetics of methotrexate, tacrolimus, and cyclosporin
A[16–18]. Studies showed that CYP3A4 plays a major role in the
metabolism of LEN in vitro. In addition, it is also a substrate for P-gp
(P-glycoprotein) and BCRP (breast cancer resistance protein)[19].
Thus, we speculate that there may be an interaction between the
two drugs, it is necessary to assess the drug-drug interactions be￾tween WZC and LEN.
Several good methods are available to determine LEN in human
plasma, but they have some limitations that are not suitable for
drug–drug interactions studies in rats [20–23]. In previous studies, a
HPLC-UV analytical method was used for the therapeutic drug
monitoring of LEN. However, the analysis time was long, and it
lacked sensitivity [22]. Three LC-MS methods had enough sensitivity,
but they have some limitations. For instance, a long analysis time
and narrow range of calibration curves that are not suitable for high￾throughput sample detection [20,23]. Some analytical methods
[20,21] need a high volume of plasma samples, but it is difficult and
unethical to obtain high volume of rat plasma samples. This study
aimed to establish a simple and sensitive method to determine the
blood concentration of LEN in rats, and to analyze the pharmacoki￾netics and drug interactions about LEN.
2. Materials and methods
2.1. Chemicals and reagents
LEN was kindly supplied by Shijiazhuang Pharmaceutical Group
(Shijiazhuang, China). 2
H5-LEN (internal standard, IS) was purchased
from Shanghai Zhen Zhun Biological Technology CO., Ltd.(Shanghai,
China). WZC was purchased from a local pharmacy (Hezheng
Pharmaceutical Company Chengdu, China). Acetonitrile, ethyl
acetate, methanol, and formic acid were of HPLC-grade, and they
were obtained from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure
water which acts as the mobile phase was purchased from Wahaha
Group Co., Ltd. (Hangzhou, China).
2.2. Instruments and analytical conditions
The UPLC-MS/MS system consisted of a UPLC (LC-30A), equipped
with SIL30AC autosampler, a CTO30A column temperature chamber
(Shimadzu, Kyoto, Japan), and an API 5500 triple quadrupole mass
spectrometer equipped with an ESI (electrospray ionization) inter￾face (Framingham, MA, USA). A ZORBAX SB-C18 column
(2.1 × 100 mm, 3.5 µm, Agilent) was used for chromatographic se￾paration.
The mobile phase consisted of water with 0.1% formic acid (A)
and acetonitrile (B). Gradient elution was applied at a flow rate of
0.3 mL/min. The gradient program was applied as follows: 0–2 min,
20–60% B; 2–3 min, 60% B; 3–3.5 min, 60–20% B; 3.5–4 min, 20% B.
The injection volume was 3 µL, and the injection temperature was
40 ℃. A mass spectrometer was used in the positive-ion multiple
reaction monitoring mode. The targets of the parent and daughter
ions of LEN and IS are as follows: m/z 427.1→370, m/z 432.1→370.
The mass spectra of LEN and IS are shown in Fig. 1. The collision
energy and declustering potential of both are 39 V and 100 V, re￾spectively. Other experimental conditions for mass spectrometry
were: curtain gas (20.0 psi), collision gas (8 kPa), source temperature
(600 ℃), ion Spray Voltage (5500 V), ion source gas1 (60.0 psi) and
ion source gas 2 (65.0 psi).
2.3. Preparation of calibration standards, internal standard (IS), and
quality controls (QCs)
LEN was dissolved in dimethyl sulfoxide, and the concentration
of standard stock solution was 2 mg/mL. The stock solution was di￾luted with methanol to obtain a series of working solutions. The IS
was dissolved in dimethyl sulfoxide at a concentration of 1 mg/mL.
10 µL of working solutions were added to blank rat plasma (190 µL)
and vortex-mixed for 1 min to obtain calibration standards. Standard
mass concentrations of LEN were prepared at 0.2, 1, 5, 10, 20, 50, 100,
200, 500, and 1000 ng/mL. QCs were processed in the same manner
with concentrations of 0.5, 80, and 800 ng/mL. The stock solution of
IS was diluted with methanol to obtain the working solution of
Fig. 1. Fragment ions of (A)LEN and (B)2
H5-lenvatinib (IS).
Y. Cui, Y. Li, L. Fan et al. Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114360
2
50 ng/mL of IS. All the stock and working solutions were stored at
−20 °C until use.
2.4. Plasma sample preparation
Liquid–liquid extraction was used to process the aqueous sam￾ples using ethyl acetate. First, 50 µL of plasma samples combined
with 20 µL of IS working solution and 150 µL of ethyl acetate were
vortex-mixed for 1 min and then centrifuged at 12,000 rpm for
10 min. The supernatant was collected in a centrifuge tube and
evaporated till dry under a stream of nitrogen at room temperature.
The residue was redissolved with 100 µL of 50% methanol in water.
Finally, 100 µL of the obtained solution was transferred into an au￾tosampler vial for analysis.
2.5. UPLC-MS/MS method validation
According to the Bioanalytical Method Validation Guidance for
Industry (UFDA), a method to determine LEN in rat plasma was va￾lidated including the selectivity, linearity, accuracy, precision, matrix
effect, extraction recovery, dilution integrity, and stability.
2.5.1. Selectivity
Selectivity was assessed to determine the substances present in
the plasma that can interfere in the determination of the analyte. It
was evaluated by comparing the chromatograms of blank plasma
samples from six different rats, blank rat plasma samples with
0.2 ng/mL of LEN and IS, and rat plasma samples after the oral ad￾ministration of LEN.
2.5.2. Linearity and LLOQ
For LEN, the linearity of calibration curves for sample simulations
was verified in the concentration range of 0.2–1000 ng/mL.
Weighted least squares method was used for linear regression ana￾lysis. The horizontal coordinate was the concentration of calibration
standards, and the vertical coordinate was the peak area ratios of
analyte to IS.
The lower limit of quantification (LLOQ), the lowest concentra￾tion point of the standard curve, was 0.2 ng/mL, where acceptable
accuracy and precision were obtained.
2.5.3. Accuracy and precision
Precision and accuracy were evaluated by calculating the relative
standard deviation (RSD) and the evaluated relative error (RE) of
LLOQ plasma samples and QC samples of three concentrations for six
replicates. The inter-day precision and accuracy were evaluated by
analyzing the four levels of concentration samples for three con￾secutive days. The accuracy of QC samples for the analytical batch
was within 85–115% of the nominal concentration with acceptable
precision (RSD < 15%). The accuracy and precision of LLOQ plasma
samples should be less than 20%.
2.5.4. Matrix effect and extraction recovery
Low, medium, and high QC samples were used to evaluate the
matrix effect and extraction recovery. Six different blank rats plasma
samples spiked with LEN post-extraction that compared with those
of standard solutions prepared in the specified solvent and having
the same nominal concentrations were used to evaluate matrix ef￾fect. The matrix should not affect the determination of analyte. The
extraction recovery was evaluated by comparing plasma samples
spiked pre-extraction with those spiked post-extraction. The ex￾traction recovery is a parameter that indicates the extraction effi￾ciency of the method to extract the analyte from a matrix.
2.5.5. Dilution integrity
Simulated plasma samples above ULOQ (the upper limit of
quantification) at a concentration of 2000 ng/mL were prepared.
Blank rat plasma was used to dilute the simulated plasma samples.
Dilution of samples should not affect the precision and accuracy of
sample determination.
2.5.6. Stability
The stability of LEN in the rat plasma was assessed by analyzing
six replicates of QC samples at low, medium, and high concentra￾tions. The stability was examined under four conditions including
short-term stability at room temperature for 4 h. The processed
samples were placed in an autosampler for 8 h, and freeze–thaw
stability for 3 times and long-term stability at −20 °C for 30 days
were determined.
2.6. Pharmacokinetic and drug interaction studies
Male Sprague-Dawley (SD) rats weighing 220–280 g were pro￾vided by Beijing Weitong Lihua Experimental Animal Technology
Co., Ltd (Beijing, China). The license number is SCXK (Beijing)
2016–0006. All the animal experimental designs were approved by
the Ethics Committee of Hebei General Hospital (Shijiazhuang,
China). The animals were acclimatized in a suitable environment
(12 h dark-12 h light cycles, temperature at approximately 23 ± 2 °C,
relative humidity of 50 ± 10%) for a week, and adequate food and
water were provided. The rats fasted for 12 h before starting this
study, but allowed adequate water.
To explore the effect of WZC on the pharmacokinetics of LEN, the
male SD rats were randomly divided into three groups, with six rats
in each group. The interventions of each group were conducted as
follows: control group (Group 1), 1.2 mg/kg LEN was applied in￾dividually; low WZC (Group 2), WZC (150 mg/kg) combined with
1.2 mg/kg LEN; high WZC (Group 3), WZC (450 mg/kg) combined
with 1.2 mg/kg LEN. Group 1 was given 0.5% sodium carbox￾ymethylcellulose for seven consecutive days. Groups 2–3 were given
different dosages of WZC for seven consecutive days according to the
experimental design. 15 min after the administration of WZC or 0.5%
sodium carboxymethylcellulose, all the groups were given 1.2 mg/kg
for LEN at the seventh days. Blood samples were collected in cen￾trifuge tubes prefilled with heparin at 0 (before LEN administration),
0.25, 0.5, 1, 2, 3, 4, 8, 12, 24, 48, 72, and 96 h after the LEN admin￾istration. Subsequently, at 3500 rpm all blood samples were cen￾trifuged for 10 min, and the supernatant was placed in a centrifuge
tube. The collected plasma samples were stored in a −20 °C freezer
until analysis.
The pharmacokinetic parameters were calculated based on
plasma drug concentration at the corresponding time. DAS 2.1.1
software (Mathematical Pharmacology Professional Committee of
China, Shanghai, China) was used to calculate pharmacokinetic
parameters including AUC (the area under the concentration-time
curve), Cmax (maximum plasma concentration), Tmax (time to max￾imum plasma concentration), t1/2 (time required to eliminate half of
plasma drug concentration), CL (clearance of drug plasma volume
per time unit), Vz (apparent volume of distribution), MRT (mean
residence time).
2.7. Statistical analysis
The analysis of pharmacokinetics parameters was conducted
using the DAS 2.1.1 Software, with non-compartmental analysis. IBM
SPSS 25.0 software package (SPSS Inc., Chicago, IL, USA) was used for
the statistical analysis of the main pharmacokinetic parameters.
Statistical comparisons of the experimental group and control group
Y. Cui, Y. Li, L. Fan et al. Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114360
3
were conducted using nonparametric rank-sum test or t-test de￾pending on the type of data. A P value of < 0.05 was considered a
statistically significant difference.
3. Results and discussion
3.1. Method development and optimization
Based on previous studies [20,21,23], a new method was devel￾oped. We compared protein precipitation with acetonitrile and
methanol, and the results show that protein precipitation with si￾milar extraction recovery, but it is prone to interference from the
matrix. Liquid–liquid extraction with ethyl acetate was used to
process the samples because of better extraction than methyl tert￾butyl ether.
The liquid chromatography and mass spectrometric conditions
were optimized. The mobile phase was 0.1% formic acid in water and
acetonitrile because it had a better peak shape, and gradient elution
was used to obtain a stable and suitable retention time. 2
H5-LEN was
used as the internal standard. It can eliminate errors due to matrix
interference and differential ionization properties in the analyte
[24]. An acquisition method based on multi-reaction monitoring was
developed to optimize the declustering voltage, collision energy
parameters by using a syringe pump to feed the sample. LEN and IS
were generated in abundance when the ESI method was operated in
the multi-response monitoring and positive ion mode, and the
conditions of product ion mass spectrometry of LEN and IS were
optimized. The precursor–product ion transitions at m/z of 427.1→
370.0 and 432.1→370.0 (Fig. 1) were selected for LEN and IS, re￾spectively, for quantification owing to its adequate reproducibility
and intensity profile.
3.2. Method validation
3.2.1. Selectivity
The retention time of both LEN and IS was 2.05 min. The chro￾matograms of blank plasma, blank plasma spiked with LEN at the
LLOQ level and IS, and rat plasma samples after gavaging LEN ad￾ministration are shown in Fig. 2. No other peaks were observed at
the retention time of the analyte and IS, indicating that the sub￾stances present in the blank plasma cannot interfere in the de￾termination of analyte. The peak area recorded at the retention time
of the blank signal was less than 20% that of the analyte LLOQ
concentration level.
3.2.2. Linearity and LLOQ
The calibration curves of LEN were good with a linear range of
0.2–1000 ng/mL. A typical linear regression equation of the calibra￾tion curves can be expressed as follows: y = 0.05352 x + 0.00506(r
> 0.999). The back-calculated concentrations for all the calibration
standards were 15%, and the LLOQ was 0.2 ng/mL, where precision
and accuracy were acceptable within 20%.
3.2.3. Precision and accuracy
The intra-day and inter-day precision and accuracy of the method
for LLOQ and QC samples are summarized in Table 1. RSD% was used
to evaluate the precision, and RE% was used for accuracy. The results
indicate that the within-run and between-run for precision and
accuracy tests were less than 15%, which were acceptable.
3.2.4. Matrix effect and extraction recovery
The matrix effect and extraction recovery for the three QC sam￾ples of LEN are shown in Table 2. The range of extraction recovery
was from 83.29% to 87.41%, and the range of matrix effects was
94.36–104%. No significant matrix effect towards analyte was ob￾served as well, and it has a high rate of extraction recovery.
3.2.5. Dilution integrity
The results show that when the concentration of LEN was higher
than ULOQ, the plasma samples could be accurately quantified by
dilution with blank plasma after a 2-fold, 5-fold, and 10-fold dilu￾tion. The back-calculated concentrations obtained using the cali￾bration curves were within 4% of the nominal concentrations, and
the RSD was less than 6%.
3.2.6. Stability
The stability of LEN was evaluated under various laboratory and
treatment conditions, including short-term storage stability, long￾term storage stability, freeze-thaw stability. Processed samples were
placed at autosampler for 8 h. The samples were left at room tem￾perature for 4 h. Long-term storage stability at − 20 ℃ and three
times freeze–thaw stability in rat plasma were evaluated. All the
stability results are shown in Table 3.
3.3. Pharmacokinetic interaction studies
An UPLC-MS/MS method was developed and validated for LEN
and successfully applied to pharmacokinetics assays and drug–drug
interaction studies. The mean blood concentration vs. time curves
for LEN was fitted using GraphPad Prism (8.0.1) software package, as
shown in Fig. 3. The main pharmacokinetic parameters for LEN are
shown in Table 4. The results show that low-dose WZC (150 mg/kg)
can cause an increase in the AUC and Cmax of LEN, but no significant
difference was observed (P > 0.05). However, the Vz of LEN sig￾nificantly decreased by 44.6% (P < 0.05). The AUC0−t (the area under
the curve of 0 to t hours) and AUC0-∞ (the area under the curve of 0
to infinity) of LEN after oral WZC (450 mg/kg) increased by 62.7%
(P < 0.01) and 61.6% (P < 0.01), respectively, and the Cmax increased
by 1.69-fold (P < 0.05). The MRT0−t (mean residence time of 0 to t)
was significantly decreased, along with more than 1.5-fold decrease
in the MRT0-∞ (mean residence time of 0 to infinity) (P < 0.01). The
results indicate that the main pharmacokinetics parameters of ad￾ministration of LEN did not change significantly by the oral pre￾treatment with WZC (150 mg/kg) except Vz. However, the main
pharmacokinetics parameters of LEN combined with 450 mg/kg of
WZC were altered compared to those for LEN alone.
The results show that WZC can significantly affect the pharma￾cokinetics of LEN, and drug–drug interaction exists between the two
drugs. The blood concentration of LEN is related to the efficacy and
toxic effects. High LEN exposure may lead to an increase in the in￾cidence of adverse reactions, but when the concentration is too low,
the risk of disease progression increases [25,26]. Thus, it is important
to control the concentration of LEN.
LEN is mainly metabolized by CYP3A4 in vitro; at the same time,
it is also a substrate for P-gp and BCRP [19,27]. The main components
of WZC are Schisandra lignans including schisantherin A, deoxy￾shisandrin, schisandrol B, schisanhenol, and schisandrin. Studies [8]
have shown that t1/2 of the 5 bioactive constituents are 1.17, 3.71,
0.79, 1.64, 0.66 h, respectively. It takes about approximately 5 half￾lives to reach steady-state plasma concentrations when continuous
administration. 7 consecutive days of administration were chosen to
investigate its effect on the pharmacokinetics of LEN based on pre￾vious studies [11,16] and the pharmacokinetic characteristics of
WZC. In vitro and in vivo studies showed that Schisandra lignan
extract may be a stronger inhibitor of P-gp, which can increase the
absorption of P-gp substrates [14]. Some studies showed that WZC
and its active ingredients can increase the blood concentrations of
tacrolimus, probably by inhibiting the activity of CYP450 enzymes,
Y. Cui, Y. Li, L. Fan et al. Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114360
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Fig. 2. Chromatograms of LEN (I) and IS (II) in rat plasma samples. A, blank rat plasma sample; B, rat plasma sample spiked with LEN at the LLOQ level and IS; C, rat plasma sample
after oral administration.
Table 1
Precision and accuracy of LLOQ and QC for LEN in rat plasma.
Concentration
(ng/mL)
Intra-day（n = 6） Inter-day（n = 18）
Mean ± SD RSD（%） RE（%） Mean ± SD RSD（%） RE（%）
0.2 0.21 ± 0.02 7.07 4.17 0.20 ± 0.02 7.84 0.84
0.5 0.51 ± 0.03 5.57 1 0.50 ± 0.03 5.21 -0.34
80 84.20 ± 2.36 2.80 5.26 83.58 ± 7.23 8.66 4.67
800 809.94 ± 22.94 2.84 1.25 791.39 ± 28.53 3.61 -1.08
Y. Cui, Y. Li, L. Fan et al. Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114360
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but also by inhibiting the activity of P-gp [15,17,28,29]. In addition,
WZC and its active ingredients can increase the concentration of
cyclosporin A and paclitaxel [18,30,31]. They are both metabolized
by CYP3A4, and they are also the substrates of P-gp. We speculate
that after WZC was administered for seven consecutive days, an
increase in drug concentration of LEN in the blood because of the
inhibition of P-gp in the intestine increases the drug absorption. On
the other hand, metabolic enzymes are inhibited, decreasing the
metabolism of drugs in the liver. Another study showed that single￾dose rifampicin (an inhibitor of P-gp) elevated the AUC and Cmax of
LEN [32]. Moreover, genetic polymorphism of ABCB1 that codes P-gp
can significantly affect the pharmacokinetics of LEN, indicating that
the activity of P-gp affects the LEN exposure and clearance[33].
However, a study found that ketoconazole, a stronger inhibitor of
CYP3A4, had no significant effect on the pharmacokinetics of LEN
[34]. Therefore, we hypothesize that the increase in LEN exposure is
mainly due to the inhibition of P-gp, and specific mechanisms should
be further elucidated.
Table 2
Matrix effect and extraction recovery of LEN in rat plasma (n = 6).
Concentration (ng/mL) Matrix effect Extraction recovery
Mean ± SD（%） RSD（%） Mean ± SD（%） RSD（%）
0.5 94.36 ± 3.32 3.53 87.41 ± 6.28 7.18
80 104.00 ± 4.10 3.95 84.20 ± 8.63 10.26
800 102.25 ± 6.69 6.55 83.29 ± 7.22 8.67
Table 3
Stability of LEN in rat plasma under various storage conditions (n = 6).
Conditions Concentration
0.5 0.53 ± 0.04 6.88 6.34
80 72.29 ± 1.08 1.50 -9.64
800 822.20 ± 24.93 3.04 2.78
Fig. 3. Plasma concentration-time curves of LEN after oral LEN alone and combined with different doses of WZC. LEN, 1.2 mg/kg for LEN; L-WZC, 1.2 mg/kg for LEN combined with
150 mg/kg WZC; H-WZC, 1.2 mg/kg for LEN combined with 450 mg/kg WZC.
Table 4
Pharmacokinetic parameters of LEN without or with different doses of WZC.
Parameters (unit) Alone
LEN (1.2 mg/kg)
Drug combination group
LEN (1.2 mg/kg)
WZC (150 mg/kg) P-value WZC (450 mg/kg) P-value
AUC0–96 (ug/L*h) 3369.02 ± 976.35 4292.22 ± 671.44 0.085 5481.41 ± 604.67** 0.001
AUC0-∞ (ug/L*h) 3396.73 ± 989.35 4300.16 ± 671.14 0.094 5488.27 ± 604.69** 0.001
MRT0−t (h) 7.61 ± 1.45 6.87 ± 2.24 0.513 5.31 ± 0.68** 0.006
MRT0-∞ (h) 8.53 ± 1.80 7.08 ± 2.32 0.252 5.47 ± 0.68** 0.007
Cmax (ug/L) 490.64 ± 124.20 602.14 ± 212.02 0.292 829.57 ± 290.87* 0.025
Tmax (h) 1.75 ± 0.88 1.67 ± 1.47 0.682 1.42 ± 0.92 0.535
t1/2z (h) 20.23 ± 5.97 14.55 ± 1.37 0.055 17.38 ± 5.97 0.522
CLz (L/h/kg) 0.38 ± 0.12 0.29 ± 0.04 0.115 0.22 ± 0.02* 0.023
Vz (L/kg) 10.83 ± 3.19 6.00 ± 1.23** 0.006 5.53 ± 1.94* 0.016
* P < 0.05 ** P < 0.01, compared with LEN alone, indicating statistically significant difference.
Y. Cui, Y. Li, L. Fan et al. Journal of Pharmaceutical and Biomedical Analysis 206 (2021) 114360
6
4. Conclusions
In this study, we developed and validated a rapid, simple, and
sensitive UPLC-MS/MS method for the determination of LEN and
successfully applied to the detection of LEN in rat plasma. It was
found that different doses of WZC affect the pharmacokinetic of LEN.
The pharmacokinetic analysis showed that high-dose WZC sig￾nificantly affects the pharmacokinetics of LEN, resulting in a sig￾nificant increase in the blood concentration and AUC. Thus, when
simultaneously administering WZC and LEN, we should focus on the
concentration of LEN in the blood, and this method can be used as a
valuable tool for LEN pharmacokinetics and drug interaction studies.
CRediT authorship contribution statement
Yanjun Cui, Ying Li and Zhanjun Dong conceived and designed
the experiments. Yanjun Cui, Xiaonan Wang, Ran Fu, Liju Fan, Ying Li,
and Jing An performed the experiments.Yanjun Cui and Liju Fan, Jing
An analyzed data. Yanjun Cui and Ying Li wrote the manuscript. All
authors read and approved the final manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing fi￾nancial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
We acknowledge the support of Hebei General Hospital Clinical
Research Center, Shijiazhuang, Hebei, China.
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