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Screening of stabilizers for LC–MS/MS

analysis of clevidipine and its primary
metabolite in dog whole blood

Background: Clevidipine is an ester-containing antihypertensive agent that Peng Cao1, Gao Li1,2, Ling
undergoes rapid hydrolysis in blood. A reliable stabilizer cocktail containing citric Huang1, Shouwei Zhao1, Yang
acid and ascorbic acid was established and the LC–MS/MS method was validated for Hu1, Lixia Qin1, Lihui Qiu1,
simultaneous determination of clevidipine and its major metabolite in beagle dog Wenwen Zhu1, Luqin Si1,2
& Jiangeng Huang*,1,2
whole blood. Results: The stabilizer could nearly completely inhibit the esterase 1
Department of Pharmaceutics, School
activity. Both analytes were extracted from whole blood by toluene and detected by of Pharmacy, Tongji Medical College,
MS/MS in positive ESI mode. The linearity range was 0.1–100.0 ng/ml for clevidipine Huazhong University of Science &
and 1.0–1000.0 ng/ml for the primary metabolite. Conclusion: The stabilizer cocktail Technology, Wuhan 430030, PR China
was able to effectively suppress the activity of esterase in blood. The method was
2
Hubei Engineering Research Center for
Novel Drug Delivery Systems, Huazhong
successfully applied to a PK study of clevidipine in beagle dogs.
University of Science & Technology,
Wuhan 430030, PR China
Background still exist critical challenges to overcome *Author for correspondence:
Tel.: +86 278 369 2892
The third generation dihydropyridine cal- instability issues during analytical method
Fax: +86 278 369 2892
cium antagonist clevidipine is an ultrashort- development of such ester-containing jiangenghuang@hust.edu.cn
acting antihypertensive agent for the blood drugs such as clevidipine. The instability of
pressure control in perioperative and inten- analytes in biological matrices can be attrib-
sive care settings [1] . Clevidipine contains uted to various factors including tempera-
an ester group and undergoes hydrolysis to ture, light, pH, oxidation and enzymatic
form the primary metabolite without phar- degradation  [7] . Esterase is the major cause
macological activity (structures shown in in ex vivo degradation of ester-containing
Supplementary Figure 1) [2] . Unlike many cur- drugs via catalyzing the unwanted hydroly-
rent antihypertensive drugs, clevidipine can sis during sample collection, processing and
be rapidly hydrolyzed by esterase existing storage and before sample extraction [8–12] .
in the blood and extravascular tissues with an A number of strategies have been utilized to
initial half-life of nearly 1 min in humans, deal with the instability of such drugs. The
less than 1 min in rats and approximately addition of esterase inhibitors is a com-
12 min in dogs [2,3] . Therefore, clevidipine mon approach to stabilize the analytes that
exhibits rapid onset and offset of antihyper- can be hydrolyzed in blood. That is to say,
tensive action, allowing precise titration to optimization of esterase inhibition strat-
attain desirable blood pressure levels [4] . egy is of great importance for the success of
Clevidipine butyrate emulsion has been quantitative assay development for any labile
approved by the US FDA for the reduction of ester-containing drugs.
blood pressure when oral therapy is not fea- MS-based methods have been widely
sible or not desirable [5,6] . Generic products used in preclinical and clinical PK studies.
of injectable emulsion of clevidipine butyrate In the earlier time, GC–MS and HPLC-FL
were developed by Wuhan Docan Pharma- methods have been developed to determine
ceutical Co. Ltd (Wuhan, China) and pre- clevidipine and its primary metabolite sepa-
clinical PK studies were carried out to com- rately in human whole blood [13] . In this
pare in vivo PK parameters between branded work, hydrolysis of clevidipine was imme-
part of
and generic products. Unfortunately, there diately inhibited by SDS. However, SDS is

10.4155/BIO.15.74 © 2015 Future Science Ltd Bioanalysis (2015) 7(12), 1457–1469 ISSN 1757-6180 1457
Research Article  Cao, Li, Huang et al.
Key terms
Esterase: Enzyme that splits esters into an acid and an
alcohol and exists ubiquitously in blood.
Ester-containing drugs: Drugs that contain ester group
and are easily hydrolyzed by esterase.
Oxidation: Combination of a substance with oxygen and
the chemical reaction involves loss of electrons.
Esterase inhibitors: Chemical entities that inhibit the
hydrolysis by esterase.
incompatible with MS-based assays due to its detri-
mental effect on sensitivity. In addition to two distinct
detectors used for clevidipine and its primary metabo-
lite, different sample volumes and extraction proce-
dures were employed for blood sample cleanup, result-
ing in time-consuming biological sample process and
reduced analysis throughput. Recently, two papers
have been published to report PK study in beagle
dogs. Zhou et al.  [14] reported a LC–MS/MS method
for simultaneous determination of clevidipine and its
major metabolite in dog plasma rather than whole
blood using felodipine as internal standard. Neverthe-
less, the authors did not take extra measures to pre-
vent clevidipine hydrolysis as well as its metabolite
from further oxidation in blood except for lowering
the temperature. Stability issues in blood have also not
received enough attentions. Wei H et al. [15] developed
another LC–MS/MS method to measure clevidipine
in dog blood using methanol as an esterase inhibitor.
A major drawback associated with this method is that
clevidipine might be hydrolyzed prior to the addition
of methanol due to its ultra-short half-life. In addi-
tion, the primary metabolite was not determined in
their assay. Interestingly, both methods were found to
be not sufficient to accurately determinate the concen-
tration of clevidipine because hydrolysis of clevidipine
could not be completely inhibited during sample col-
lection and processing and appropriate isotope-labeled
IS were not employed in their methods. Hence, a more
reliable LC–MS/MS method is required for the simul-
taneous measurement of clevidipine and its primary
metabolite.
In this paper, we reported an integrated screening
approach for selecting the best strategy to stabilize
clevidipine in whole blood. Then, a novel LC–MS/MS
method was validated according to FDA guidelines to
ensure it provided the selectivity, sensitivity, accuracy
and precision required to support the simultaneous
quantitation of clevidipine and its primary metabo-
lite in beagle dog whole blood. Finally, this method
was successfully applied to a PK study in beagle dogs
following intravenous infusion of clevidipine butyrate
emulsion.
1458 Bioanalysis (2015) 7(12)
Experimental
Chemicals & reagents
Clevidipine (99.5% purity) and its primary metabolite
(99.5% purity) were provided by Wuhan Docan Phar-
maceutical Co. Ltd. Clevidipine-d7 (99.6% purity),
served as the IS for clevidipine, was purchased from
TLC PharmaChem, Inc. (ON, Canada). O-desmethyl
felodipine (the metabolite IS, 99.5% purity, mentioned
as MeIS below) was obtained from Toronto Research
Chemicals, Inc. (ON, Canada). The chemical struc-
tures of the two analytes and their corresponding ISs
are shown in Supplementary Figure 1. Phenylmeth-
anesulfonyl fluoride (PMSF), thenoyltrifluoroacetone
(TTFA) and bis-[4-nitrophenyl]-phosphate (BNPP)
were purchased from Sigma-Aldrich (MO, USA).
Sodium fluoride (NaF), citric acid, ascorbic acid, formic
acid (FA) and ammonium formate (AF) were obtained
from Sinopharm Chemical Reagent Co. Ltd (Shang-
hai, China). HPLC grade toluene and acetonitrile were
purchased from Fisher Scientific (NJ, USA). Deionized
water was generated in-house by using Milli-Q Plus
Water Purification System (MA, USA). K 2EDTA-con-
taining vacutainer tubes (5ml) and 22 G IV catheters
(0.9 mm × 25 mm) were obtained from BD Biosciences
(MA, USA). All other reagents were of analytical grade.
LC–MS/MS instruments
A Prominence UFLC system (Shimadzu, Kyoto, Japan)
including two binary pumps (LC-30AD), a vacuum
degasser (DGU-20As), a temperature-controlled col-
umn compartment (CTO-30A), an autosampler
(SIL-30AC) and a system controller (CBM-20A) was
used throughout. The LC system was coupled with an
API 4000 Qtrap triple quadrupole mass spectrometer
(AB SCIEX, CA, USA) equipped with electrospray
ionization source. Data acquiring and processing were
controlled by Analyst 1.6.1 software.
LC–MS/MS conditions
The chromatographic separation was performed on a
Shimadzu Inertsil ODS-3 C18 column (3 μm, 3.0 ×
100 mm) protected by a Phenomenex C18 guard col-
umn (4 × 2.0 mm, 5 μm). The mobile phase consisted
of 2 mM AF in water containing 0.1% FA (A) and aceto-
nitrile (B). The flow rate was 0.6 ml/min. The gradient
program started from 70% B and increased linearly to
90% B in 3 min. After maintaining at 90% B for 1 min,
it was brought back to 70% B within 0.01 min followed
by 1.5 min re-equilibration. The injection volume was
10 μl. The column and the autosampler temperature
were maintained at 40°C and 4°C, respectively.
The MS/MS detection was carried out in positive
ESI mode using SRM mode. The SRM transitions and
their respective mass spectrometric parameters for each
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 Stabilizer screening for LC–MS/MS analysis of clevidipine & its primary metabolite
analyte and IS are listed in Table 1. The source param-
eters were optimized as follows: source temperature
550°C; ionspray voltage 5500 V; curtain gas 30 psi; col-
lision gas high; nebulizer gas (gas 1) 40 psi; auxiliary gas
(gas 2) 40 psi.
Preparation of stock & working solutions,
calibration standards and QC samples & esterase
inhibitor solutions
The stock solutions of clevidipine and clevidipine-d7 were
prepared in acetonitrile at a concentration of 1.0 mg/ml,
whereas 0.5 mg/ml stock solutions were prepared in ace-
tonitrile for the metabolite and MeIS. All stock solutions
were stored at -80°C.
The stock solutions of ISs were further diluted by ace-
tonitrile/water (50:50, v/v) to yield combined IS work-
ing solution containing 500 ng/ml clevidipine-d7 and
5 μg/ml MeIS. Standard working solutions containing
1/10, 2/20, 5/50, 10/100, 20/200, 50/5000, 100/1000,
200/2000, 500/5000 and 1000/10000 ng/ml of clevi-
dipine/metabolite and 500/5000 ng/ml of clevidipine-
d7/MeIS were prepared via serially diluting the stock
solutions with acetonitrile/water (50:50; v/v) and stored
at -20°C.
To prepare calibration standards, 10 μl working
solutions were added to 150 μl mixture of whole blood
and stabilizer cocktails to yield final concentrations of
0.1–100.0 ng/ml for clevidipine and 1.0–1000.0 ng/ml for
the metabolite containing 50 ng/ml clevidipine-d7 and
500 ng/ml MeIS. QC samples were prepared in a similar
manner at four different concentrations of 0.1/1, 0.3/3,
8/80, 80/800 ng/ml for clevidipine and the metabolite,
representing the LLOQ, low QC (LQC), medium QC
(MQC) and high QC (HQC), respectively.
Stock solutions of citric acid and NaF were prepared
in water at a concentration of 1 M; stock solutions of
TTFA, PMSF and BNPP were prepared at the same con-
centration in DMSO. The stock solutions were diluted
to 0.1 and 0.01 M solutions by serially diluting with
corresponding solvents. All solutions were stored at 4°C.
Stability evaluation in beagle dog whole blood
Stability of clevidipine in fresh whole blood in the pres-
ence or absence of various stabilizers was examined. All
Table 1. SRM transitions and optimized MS parameters.
Analyte Transitions  DP
Clevidipine 455.9/337.8 46
Clevidipine-d7 464.8/339.8 86
Metabolite 356.2/323.9 66
MeIS 369.9/323.7 86
CE: Collision energy; CXP:Collision cell exit potential; DP: Declustering potential; EP: Entrance potential.
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Research Article
experiments were carried out in triplicates using three
different lots of beagle dog whole blood. The four tested
esterase inhibitors including NaF, PMSF, BNPP and
TTFA were individually added to 100 μl fresh beagle
dog whole blood to obtain final concentrations of 0.1,
1, 10 or 50 mM of each inhibitor. Likewise, citric acid
was tested with the same set of whole blood at final
concentrations of 10, 20, 30 and 40 mM. After the
whole blood was preincubated with different concen-
trations of esterase inhibitors or citric acid for 10 min,
clevidipine was spiked with pretreated whole blood to
obtain an ultimate concentration of 100 ng/ml. The
mixture was maintained at room temperature (25°C)
or 4°C for 8 h. Then, 500 μl of acetonitrile containing
MeIS (500 ng/ml) was added and the mixture was vor-
texed, centrifuged at 20000 × g for 10 min. Ten micro-
liter resulting supernatant was injected and analyzed
by LC–MS/MS. Fresh whole blood was processed by
adding appropriate amount of acetonitrile containing
both analytes and corresponding esterase inhibitor
as zero time point sample. Stability of clevidipine in
whole blood was assessed by comparing the deviation
of the value from 8 h time point with that from zero
time point.
Sample collection & treatment
To acquire accurate concentration of analytes, the
duration of sample collection should be as short as
possible to minimize the degradation of clevidipine
in vitro. The blood samples were directly drawn to
K 2EDTA-containing vacutainer tubes (5 ml) prefilled
with 1 ml stabilizer cocktails precooled in crushed
ice. To avoid losing vacuum, the tubes were required
to gain negative pressure using a 50 ml syringe with
sharp, thin needle piercing through the septa of the
vacutainer tubes before use. Approximately 2 ml blood
was harvested by the scale on tubes. The mixture was
immediately vortexed to guarantee complete termina-
tion of degradation. All samples were transferred with
dry ice and stored at -80°C until analysis. To get the
exact weight of samples, tubes were weighted before
and after the collection. The actual volume of blood
was calculated by dividing the whole blood mass by
the density of beagle dog whole blood (1.054 g/ml) [13] .
CE EP CXP
17 10 10
17 16 10
17 8 10
39 16 10
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Research Article  Cao, Li, Huang et al.
A LLE procedure was performed for blood sample
cleanup. After the addition of 10 μl combined IS work-
ing solution, 500 μl of toluene was added into 150 μl
stabilizer cocktails containing whole blood. The mix-
ture was vortexed for 10 min, followed by centrifuga-
tion at 20000 × g for 10 min. An aliquot of 400 μl
organic layer was transferred to clean tubes and evapo-
rated to dryness under nitrogen flow. The dried extracts
were reconstituted in 80 μl mobile phase (acetonitrile:
2 mM AF in water containing 0.1% FA = 70:30, v/v)
and votexed for 5 min, followed by 10 min centrifuga-
tion at 20000 × g. Ten microliter aliquot of supernatant
was injected and analyzed by LC–MS/MS.
Method validation
The assay validation was performed according to the
FDA guidance for industry: bioanalytical method
validation  [16] . Specificity, limit of detection, LLOQ,
calibration curve, precision, accuracy, matrix effect,
recovery, carry-over and stability were all investigated.
The specificity and selectivity of the method were
evaluated by analyzing six different lots of blank whole
blood as well as corresponding spiked whole blood
samples at LLOQ levels. The SRM chromatograms
were compared. The response of the endogenous com-
pounds in the whole blood mixture co-eluted should
be within 20% of the response of the LLOQ standards.
The limit of detection was defined as the concentration
that produced a signal three-times above the noise level.
The LLOQ was defined as the lowest concentration
detected with acceptable accuracy and precision.
The calibration curves were assayed in three con-
secutive days to assess the linearity. A ten-point cal-
ibration curve (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 and
100 ng/ml for clevidipine and 1, 2, 5, 10, 20, 50, 100,
200, 500 and 1000 ng/ml for the metabolite) was con-
structed by plotting the peak area ratios of the analyte
to its corresponding IS against the theoretical concen-
trations. The linearity was calculated by 1/x 2 weighting
scheme. The deviations of each calculated concentra-
tions should be within 85–115% of the nominal values
except for LLOQ for which 80–120% was allowed.
The intra-day precision and accuracy were assessed
by analyzing six replicates of QC samples containing
clevidipine and the metabolite at three concentration
levels: LQC (0.3 ng/ml for clevidipine and 3 ng/ml
for the metabolite), MQC (8 ng/ml for clevidipine and
80 ng/ml for the metabolite) and HQC (80 ng/ml for
clevidipine and 800 ng/ml for the metabolite). The
inter-day precision and accuracy were estimated by
analyzing the QC samples on three separated runs.
Precision, expressed as RSD (%RSD), was calculated
by dividing standard deviation by the mean measured
concentration, while accuracy, expressed as percentage
1460 Bioanalysis (2015) 7(12)
of nominal values, was defined as the ratio of the mean
observed concentration and the nominal concentra-
tion. Precision should not exceed 15% and accuracy
within 85–115% was acceptable.
The blank whole blood from six different sources
was processed and postspiked with neat solutions of
both analytes and their corresponding ISs to study the
matrix effect. The degree of matrix effect was deter-
mined by dividing the peak area of each analyte in
whole blood spiked post-extraction by the correspond-
ing analyte peak area from neat samples prepared in
the reconstitution solution. The assay was evaluated
at LQC, MQC, HQC levels. The extraction recovery
was determined from the ratio of the analyte peak areas
through overall extraction procedure compared with
those from whole blood samples spiked post-extraction.
The carry-over effect was tested by injecting one
blank whole blood sample after the analysis of the high-
est concentration of calibration standard (100 ng/ml
for clevidipine with 50 ng/ml IS and 1000 ng/ml for
the metabolite with 500 ng/ml MeIS). The peak areas
of the resulting carry-over peaks with the same reten-
tion time of the analyte and IS should be less than 20%
and 5% of those in the LLOQ samples, respectively.
The stability of clevidipine and its primary metabo-
lite was examined under different conditions at three
QC levels. Four replicates of each QC level samples
were stored at ambient temperature for 4 h and -80°C
for 30 days, respectively. The freeze–thaw stability
was determined after freezing at -80°C and thawing at
room temperature for three cycles. Autosampler stabil-
ity was estimated by analyzing QC samples after 24 h
storage in the autosampler at 4°C. Stability of working
solutions was measured after placing working solution
at ambient temperature for 24 h. Similarly, stock solu-
tion stability was determined at -80°C for 60 days. The
criterion for all stability tests was that the mean deter-
mined concentrations deviated less than ±15% from
the nominal concentrations.
According to the 2013 FDA guidance for industry:
bioanalytical method validation, the total number of
incurred sample reanalysis (ISR) samples should
be at least 7% of the study sample size and adequate
coverage of the PK profile was required. In the present
work, a total of 20 samples, approximately 10% of the
total sample size, were selected around Cmax and in the
elimination phase for ISR to evaluate the reproducibil-
ity of the analytical method. The difference between
the original and ISR data should be within 20% of the
mean value for at least 67% of the repeats.
Application to a PK study
The validated method was applied to a PK study of
clevidipine and its major metabolite in beagle dogs
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 Stabilizer screening for LC–MS/MS analysis of clevidipine & its primary metabolite
following intravenous infusion of butyrate inject-
able emulsion. The study protocol was approved by
the Animal Ethics Committee in Tongji Medical
College, Huazhong University of Science and Tech-
nology (Wuhan, China). The reference formulation
of clevidipine butyrate (Cleviprex®) was purchased
from The Medicines Company (NJ, USA), while the
generic product was manufactured by Wuhan Docan
Pharmaceutical Co. Ltd. Six healthy beagle dogs
(three males/three females, 10.0 ± 2.0 kg, Certificate
No.42000200000034) were purchased from Ruikesen
Experimental Animal Co. Ltd (Wuhan, China). The
dogs were individually housed at a temperature of 25°C
and 50% relative humidity under a 12-h light/dark
cycle. Water and laboratory standard diet were provided
ad libitum. The dogs were randomly assigned into two
groups and intravenously infused with either test or
reference clevidipine butyrate emulsion for 60 min at
an infusion rate of 0.8 mg/kg/h. The dose was chosen
based on a previously reported study performed in dogs
as well as dose translation from a clinical study con-
ducted in healthy volunteers [17,18] . Each treatment was
separated by a 7-day washout period. Blood samples
were drawn from the intravenous catheter as described
above 1, 3, 5, 7, 10, 20, 30, 40, 50, 60 min postdos-
ing and 0.5, 1.5, 3, 5, 7, 10, 15, 30, 45, 60 min and 4,
10, 24 h after the end of infusion. PK parameters were
calculated by noncompartmental model analysis using
DAS 2.1.1 software (Mathematical Pharmacology
Professional Committee of China, Shanghai, China).
Furthermore, the time course of both clevidipine and
its major metabolite concentrations was either fitted to
mono- or biexponential disposition function and the
goodness of fit was assessed by the Akaike Information
Criterion using DAS 2.1.1 software.
Results & discussion
Stabilization of clevidipine in dog whole blood
Ester-containing drugs are susceptible to cleavage by
esterase in blood and therefore usually determined in
whole blood. For instance, remifentanil, which has
an N-substituted methyl propanoate ester group, was
reported to be rapidly cleared in humans (8–20 min)
and dogs (3.8–8.3 min) [19] and always determined in
whole blood [20–22] . Esterases ubiquitously located in
the membrane or the cytosol of the red blood cells are
the major cause for the degradation of clevidipine in
blood because the rate of hydrolysis of clevidipine is
much faster in whole blood than in plasma alone [2] . It
suggested that concentration of analytes in whole blood
rather than plasma is more accurate in quantification.
Due to the ultra-short elimination half-life, it is recom-
mended to collect whole blood samples in tubes pre-
filled with esterase inhibitors to immediately stop the
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Research Article
Key term
Incurred sample reanalysis: Intended to verify the
reliability of the reported subject sample analyte
concentrations.
degradation of clevidipine to avoid underestimation of
clevidipine and overestimation of its primary metabo-
lite [13] . Thus, in this method, we determined the drug
concentration in whole blood rather than plasma [14,15]
using tubes prefilled with stabilizer cocktail.
Since clevidipine is rapidly hydrolyzed by esterase
existing in blood [23] , four commonly used esterase
inhibitors including NaF, BNPP, PMSF and TTFA
were initially screened to improve the stability of clevi-
dipine in whole blood [24] . As shown in Figure 1A , dif-
ferent esterase inhibitors reduced the degradation of
clevidipine to different extent. The results obtained
from the control group without any inhibitor showed
that only 0.23% clevidipine remained after 8 h incu-
bation at 25°C. NaF showed little or faint suppression
of esterase activity regardless of NaF concentration
investigated, while PMSF, TTFA and BNPP showed
a concentration-dependent inhibition behavior. Of the
latter three inhibitors, TTFA was the most efficient sta-
bilizer for clevidipine, followed by BNPP and PMSF,
whereas esterase activity was slightly suppressed when
stabilizer concentration was lower than 10 mM. NaF
is a nonspecific esterase inhibitor, while BNPP inhibits
carboxylesterases and phosphodiesterase; PMSF sup-
presses the activity of carboxylesterases, serine ester-
ase and hydrolase, while TTFA has been reported to
be an inhibitor for carboxylesterases [24] . Expect NaF,
all these traditional esterase inhibitors were found to
significantly reduce the degradation of clevidipine
when 50 mM was used. These results indicated that
carboxylesterase inhibition might play a vital role in
diminishing hydrolysis of clevidipine.
In contrast to four tested esterase inhibitors, citric
acid significantly reduced the degradation of clevidip-
ine at 25°C with a concentration-dependent inhibition
manner. More than 80% clevidipine remained when
the dog whole blood was acidified with citric acid at a
final concentration of 40 mM. It is well known that pH
plays an important role in both enzymatic and nonenzy-
matic reactions. Adjusting pH to a certain level beyond
enzymatic working pH window is a common strategy to
minimize enzymatic degradation of an unstable analyte
during bioanalysis [25] . Furthermore, blood samples were
acidified with citric acid to ensure the mixture pH below
both analyte pKa and brought about improved recover-
ies for clevidipine and its major metabolite. Therefore,
pH adjustment resulted in a significant improvement
of clevidipine stability as well as extraction efficiency of
both analytes of interest in dog whole blood.
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Research Article  Cao, Li, Huang et al.

A B NaF TTFA
NaF TTFA PMSF Citric acid
PMSF Citric acid
BNPP Control
BNPP Control
100
80

Remaining clevidipine (%)

Remaining clevidipine (%)

60 80
40
20 60
4
40
3
2 20
1
0 0
0.1
1
10
50
0.1
1
10
50
0.1
1
10
50
0.1
1
10
50
10
20
30
40

50
100

10
50

10
50

10
50

30
40

Control
Control
Esterase stabilizer (mM) Esterase stabilizer (mM)

Figure 1. Remaining clevidipine in fresh beagle dog whole blood after 8 h incubation in the presence or absence
of esterase stabilizer. Pecentage remaining when incubated at (A) 25°C and (B) 4°C (n = 3).
BNPP: Bis-[4-nitrophenyl]-phosphate; NaF: Sodium fluoride; PMSF: Phenylmethanesulfonyl fluoride;
TTFA: Thenoyltrifluoroacetone. 

Temperature also plays a crucial role in delaying
the conversion of clevidipine to its metabolite in vitro,
which has been confirmed in previous reports [14,23] . As
shown in Figure 1B, the hydrolysis rate was obviously
reduced and around 60% clevidipine remained after
8 h at 4°C. In general, hydrolysis reaction is significantly
slower at lower temperature due to the reduced enzyme
activity. Following the previously reported oversimpli-
fied approach [14] , more than 30% of clevidipine was
found to be degraded due to its ultra-short half-life in
beagle dog blood. A representative chromatogram is
displayed in Figure 2A . Our findings confirmed that a
single strategy by lowering the temperature was found
to be not sufficient to stabilize clevidipine in dog whole
blood. Subsequently, the effect of stabilizers including
enzyme inhibitors and citric acid on esterase activ-
ity was investigated at 4°C. Fourty millimolar citric
acid was found to be the most effective stabilizer with
nearly 100% clevidipine remained. Of four test ester-
ase inhibitors, BNPP and TTFA were not effective
in 25°C but manifested strong inhibitory function in
4°C, demonstrating that temperature plays an impor-
tant role in delaying the conversion of clevidipine to its
metabolite. In contrast, NaF and PMSF demonstrated
relatively lower inhibition effect compared with BNPP
and TTFA (Figure 1B) .
In addition to hydrolysis, clevidipine and its pri-
mary metabolite were both readily oxidated [2] .
Supplementary Figure 2 shows the oxidation degration
pathway for both analytes of interest. In the present
study, 0.1 M (final concentration) ascorbic acid was
added into the samples to prevent further oxidation of
both analytes. Considering that previous LC–MS/MS
methods suffered from substantial degradation of
both analytes due to the ex vivo hydrolysis and oxi-
dation, stabilizer cocktails containing 40 mM cit-
ric acid and 0.1 M ascorbic acid were established in
the present work. A typical chromatogram resulting
from beagle dog whole blood sample processed by the
above-mentioned stabilizer cocktails is displayed in
Figure 2B.
Last but not the least, clevidipine-d7, a stable iso-
tope-labeled IS, provided more dependable data than
structural analogs [14,15] , while a structurally similar
molecule O-desmethyl felodipine was employed for
the metabolite quantitation since the corresponding
isotope-labeled IS is not commercially available. Due
to the similarity in the chemical structure and physico-
chemical properties between the metabolite of interest
and analog IS, O-desmethyl felodipine exerted simi-
lar LC behavior and alike sample processing efficiency
determined by both matrix effect and extraction recov-
ery. Similar to stable isotope-labeled IS, the analog IS
assisted in accurate measurements of the metabolite of
clevidipine in dog whole blood.
Liquid chromatography & mass
spectrometry optimization
Multiple conditions including different types of aque-
ous phases (0.1% FA, AF [2, 5 and 10 mM] and
ammonium acetate [2, 5 and 10 mM]), organic phase
(acetonitrile and methanol) as well as reversed-phase
columns were tested. We found that the combination
of aqueous phase with 2 mM AF and 0.1% FA and
acetonitrile provided the best sensitivity for both clevi-
dipine and its metabolite, Shimadzu Inertsil ODS-3

1462 Bioanalysis (2015) 7(12) future science group

 Stabilizer screening for LC–MS/MS analysis of clevidipine & its primary metabolite Research Article

A
2.4e5

Clevidipine
2.0e5
Intensity (cps)

1.5e5
Metabolite

1.0e5

1.18

5.0e4

0.0
1.0 2.0 3.0 4.0 5.0

Time (min)

B
3.0e5

Clevidipine
2.0e5

2.0e5
Intensity (cps)

1.5e5

1.0e5

5.0e4

0.0
1.0 2.0 3.0 4.0 5.0
Time (min)

Figure 2. Comparison of representative chromatograms obtained from processed samples. Samples were treated
with (A) a previously reported method involving immediate centrifugation at 4°C and (B) newly developed
stabilizer cocktails containing 40 mM citric acid and 0.1 M ascorbic acid.

C18 column (3 μm, 3.0 × 100 mm) provided sufficient
retention for all the analytes. The total run time was
5.5 min. Optimal conditions for LC–MS/MS analysis
were achieved by tuning the mass spectrometer in both
positive and negative ESI mode. Positive ESI mode
gave a higher sensitivity for all chemicals.
Sample treatment
A delicate sample cleanup procedure is required for
whole blood samples prior to LC–MS/MS analysis
due to its multifarious components. In the present
study, both protein precipitation (PPT) and LLE using
appropriate organic solvents were tested for whole
blood sample preparation. Sample extracts obtained
from LLE were cleaner and contained fewer interfer-
ence components than PPT. As a result, LLE-based
sample processing approach showed lower matrix
effect and provided a better S/N than PPT(data not
shown). Thereafter, a number of extraction solvents
including ethyl acetate, ether, methyl-t-butyl ether,

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Research Article  Cao, Li, Huang et al.

A B C
455.9 337.8 455.9 337.8 455.9 337.8
2.37
40
1.07 2.32
767
2.37
5.0e4
Intensity (cps)

Intensity (cps)
Intensity (cps)
1.36 2.65 4.39
600 4.0e4
30 0.96 1.47 2.973.39 4.09 5.005.44
3.0e4
20 400
2.0e4
10 200 1.0e4
2.10
0 4.46 4.98

0 0 0.0
2 4 2 4 2 4
Time (min) Time (min) Time (min)

464.8 339.8 464.8 339.8 464.8 339.8

60
0.58
5
2.33
5.6e4 2.34

6.0e4
Intensity (cps)

Intensity (cps)
Intensity (cps)

4.0e4
40 4.0e4
3
1.38
2.21
2 5.38 2.0e4
20 0
0.81 1
1.49 3.47 3.80 4.39
2.0e4
2.29 4.59
4

0 0.0 0.0
2 4 2 4 2 4
Time (min) Time (min) Time (min)

356.2 323.9 356.2 323.9 356.2 323.9

1.18
147 920 1.20
2.0e5
Intensity (cps)

800
Intensity (cps)

Intensity (cps)
4.07

100 600 1.5e5

400 1.0e5
50 2.86
4.86
0.72
1.20 1.34 1.85
3
3.19
3.60 4.20
4
200 1
1.32 2.34
5.0e4
3.90 4.07

0 0 0.0
2 4 2 4 2 4
Time (min) Time (min) Time (min)

369.9 323.7 369.9 323.7 369.9 323.7

713 0.53
1.4e5 1.32
1.17e5 1.33
Intensity (cps)

Intensity (cps)
Intensity (cps)

600 1.00e5
1.0e5
400
5.0e4 5.00e4
200 1.03
1
1
1.45
0.23 1
1.88 2.62 2.85 3.72 4.55

0 0.0 0.0
2 4 2 4 2 4
Time (min) Time (min) Time (min)

Figure 3. Representative LC–MS/MS chromatograms of blood samples. Chromatograms from (A) blank whole blood; (B) blood sample
at LLOQ level and (C) incurred sample 3 min after cessation of intravenous infusion. The chromatograms monitored clevidipine at m/z
455.9→337.8, clevidipine-d7 at m/z 464.8→339.8, metabolite at m/z 356.2→323.9 and MeIS at m/z 369.9→323.7.

toluene, dichloromethane, n-hexane were investigated was extremely low compared with its major metabolite.
to optimize LLE procedure. Of all the solvents, obvi- However, the extraction recovery for the metabolite
ous matrix effects were observed except for toluene was found to be around 50%. Due to the high concen-
and methyl-t-butyl ether (data not shown). Moreover, tration levels of the metabolite, the recovery was also
toluene was able to provide more consistent extraction found to be sufficient for the analyses. Toluene base
recoveries compared with methyl-t-butyl ether. Thus, LLE demonstrated significant differences in extraction
toluene was chosen as the extraction solvent with the recoveries for clevidipine and its primary metabolite
capability of providing sufficient extraction recovery (100 vs 60%) primarily due to their distinct solubili-
for clevidipine (nearly 100%), whose concentration ties in these two different immiscible liquids (toluene

1464 Bioanalysis (2015) 7(12) future science group

 Stabilizer screening for LC–MS/MS analysis of clevidipine & its primary metabolite Research Article

Table 2. The extraction recovery and matrix effect of clevidipine, clevidipine-d7, metabolite and
MeIS (n = 6).
Analyte Nominal Extraction recovery (n = 6) Matrix effect (n = 6)
concentration (ng/ml) Mean ± SD% RSD% Mean ± SD% RSD%
Clevidipine 0.3 (LQC) 103.39 ± 10.24 9.90 97.43 ± 5.05 5.18
  8 (MQC) 104.05 ± 3.65 3.50 99.96 ± 1.03 1.03
  80 (HQC) 98.25 ± 3.83 3.90 102.42 ± 1.93 1.88
  50 (IS) 103.45 ± 4.43 4.28 103.07 ± 1.96 1.90
Metabolite 3 (LQC) 64.04 ± 2.01 3.14 102.17 ± 5.38 5.26
  80 (MQC) 57.04 ± 4.59 8.05 100.50 ± 7.74 7.70
  800 (HQC) 55.74 ± 2.20 3.95 102.12 ± 7.30 7.15
  500 (IS) 44.06 ± 1.92 4.36 96.27 ± 3.61 3.75

and water). The less polar analyte clevidipine dissolves whole blood, LLOQ (0.1 ng/ml for clevidipine and
preferentially in the nonpolar extraction solvent. Also, 1 ng/ml for metabolite), incurred sample 3 min after ces-
the difference in the ratio of unionized and ionized sation of intravenous infusion are shown in Figure 3. The
forms due to different pKa of both analytes contrib- calibration curves of both analytes were linear over the
uted to the discrepancy in extraction recovery from concentration ranges of 0.1–100 ng/ml for clevidipine
acidified blood samples. and 1–1000 ng/ml for its major metabolite. The correla-
According to sample collection and treatment pro- tion coefficients for all calibration curves were >0.996.
cedure, the ratio between stabilizer and blood is ideally For all batches, the deviations of the back-calculated
1:2 (v/v). However, in the actual samples, this specific concentration from their nominal values were within
value is hard to achieve. Thus, we conducted the fol- 88–112%. The mean intra-day and inter-day accuracy
lowing experiment to confirm the effectiveness of the and precision of both analytes were within acceptable
method. To imitate the practical samples, we tested ranges according to the criteria of the guideline for
the accuracy and matrix effect of stabilizer and blood bioanalytical methods (Supplementary Table 1).
volume ratios (1:1.5, 1:2 and 1:2.5) at three QC levels. A summary of extraction recoveries and matrix effect
The deviation of all samples with different rations was of all the analytes is shown in Table 2. Due to significant
within ±10% and the matrix effect was also acceptable concentration difference, desired extraction recover-
(data not shown). ies were established for clevidipine, while relatively low
but consistent recoveries were obtained for its primary
Method validation metabolite at three QC levels. The extraction recover-
The selectivity and specificity were assessed in six indi- ies of their corresponding IS were 103.45 and 44.06%,
vidual blank whole blood samples. No interfering peaks respectively, which also correlated well with those for
were observed at the retention times of the both analytes the analytes. Furthermore, satisfactory results of matrix
and ISs. Representative SRM chromatograms of blank effect obtained were within the acceptable limit, which
Table 3. Stability of clevidipine and its major metabolite under different conditions (n = 4).
Concentration (ng/ml) Clevidipine (RE%) Metabolite (RE%)
0.3 8 80 3 80 800
Three freeze–thaw cycle stability 0.22 2.50 -0.75 -7.56 -1.75 -14.38
Room temperature 4 h stability 1.42 2.34 2.00 2.00 -0.31 -2.81
20-h autosampler stability 2.00 1.09 0.41 0.58 3.97 -8.78
Long-term stability (-80°C 30 days) -7.67 -8.17 -6.58 4.44 1.58 -3.88
Working solution RT 24 h stability -1.25 -0.91 -1.53 4.00 4.38 -5.34
Stock solution stability (-80°C 60 days)   4.85 †
 –  – -4.74  –  –
†
When stock solution stability was determined, the neat solution of clevidipine (100 ng/ml) and metabolite (1000 ng/ml) were diluted form
stock solution, respectively.
The RE% was calculated by the area of analytes.
RT: Room temperature.

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Research Article  Cao, Li, Huang et al.

A B Metabolite
Clevidipine
100 1000
Male, test
Male, test Male, reference
Male, reference Female, test

Concentration (ng/ml)
Concentration (ng/ml)

Female,, test 100 Female, reference

Female, reference
10

10

1
1

0.1 0.1
0 30 60 80 100 120 0 50 100 150 200 300 600 900 1200 1500
Time (min) Time (min)

Figure 4. Mean blood concentration–time profiles. (A) Clevidipine and (B) the major metabolite in male and female
beagle dogs following 60 min intravenous infusion of clevidipine (test and reference products) at 0.8 mg/kg/h.

manifested that no coeluting substance influenced the ISR results met the FDA criterion that at least two-
ionization of all analytes. Carry-over effect results dem- third of the samples tested passed, although two out
onstrated that no residual peaks were observed in the of the 20 ISR samples failed passing the criterion for
blank samples for both clevidipine and the metabolite. the metabolite (Supplementary Table 2) probably due to
The stability test results (Table 3) showed that no further phase II conjugation of clevidipine metabolite.
obvious fluctuation occurred after three cycles of freez-
ing and thawing. The samples were stable under room Assay application
temperature for 4 h and at -80°C for 30 days. There The validated method was successfully applied to
was also no significant degradation during the storage the analysis of whole blood samples obtained from
of processed samples in autosampler for 24 h. All stock beagle dogs following 60 min intravenous infusion of
and working solutions were found to be stable under 0.8 mg/kg/h clevidipine of test or reference products.
their corresponding storage conditions. As a comple- Figure 4 depicts the mean blood concentration versus
mentary control other than QC sample analysis [16] , time profiles of clevidipine as well as its metabolite in

Table 4. PK parameters of clevidipine and its primary metabolite in male and female beagle dogs
following 60 min intravenous infusion of test and reference clevidipine products at 0.8 mg/kg/h.
Analyte    Parameters  Unit     Test Reference
Male Female Male Female
Clevidipine Cmax ng ml -1
50.5 ± 20.0 38.5 ± 2.1 55.0 ± 17.2 36.3 ± 3.7
  t1/2 min 22.2 ± 8.4 24.7 ± 9.0 20.2 ± 5.9 28.9 ± 10.4
  AUC (0-t) ng ml-1 min 2804 ± 1213 2000 ± 268 2903 ± 947 2028 ± 210
  AUC (0-∞) ng ml min-1
2818 ± 1214 2020 ± 275 2922 ± 944 2056 ± 210
  CL z l min-1kg-1 0.316 ± 0.114 0.402 ± 0.058 0.293 ± 0.093 0.392 ± 0.038
  MRT(0-t) min 34.1 ± 0.7 34.0 ± 0.5 34.9 ± 1.0 34.0 ± 0.7
  MRT(0-∞) min 34.8 ± 0.8 35.3 ± 1.3 35.8 ± 1.5 36.1 ± 1.7
Metabolite Cmax ng ml-1 341.1 ± 78.5 360.4 ± 124.2 370.8 ± 20.3 273.2 ± 72.7
  t1/2 min 183.6 ± 191.6 212.1 ± 68.3 292.5 ± 72.2 374.1 ± 115.1
  AUC (0-t) ng ml min-1
29,310 ± 5207 29,018 ± 10,928 41,139 ± 2348 33,248 ± 12,708
  AUC (0-∞) ng ml-1 min 29,990 ± 5965 29,753 ± 10,916 42,091 ± 1730 36,233 ± 9995
  CL z l min kg
-1 -1
0.027 ± 0.006 0.029 ± 0.009 0.019 ± 0.001 0.023 ± 0.008
  MRT(0-t) min 174.3 ± 80.8 117.9 ± 47.5 199.2 ± 9.1 239.5 ± 100.6
  MRT(0-∞) min 204.0 ± 107.4 143.8 ± 47.4 238.7 ± 25.7 370.0 ± 109.2
CL: Clearance; MRT: Mean residence time.

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 Stabilizer screening for LC–MS/MS analysis of clevidipine & its primary metabolite Research Article

both male and female dogs. With the sufficient LLOQ
of 0.1/1 ng/ml for clevidipine/metabolite, the analytical
method was capable of quantifying concentrations of
clevidipine/metabolite in dog blood up to 2/24 h, while
Zhou Y et al. [14] only monitored the metabolite for 2 h
and the half-life of the metabolite (17.53 ± 4.66 min) was
not precisely estimated. As shown in Figure 4, clevidip-
ine and its major metabolite both exhibited a rapid post-
infusion decline, followed by a relatively slow decrease
of both analyte concentration levels, probably due to a
rate-limiting redistribution phase between tissues and
systemic system. The time course of both clevidipine
and its major metabolite concentrations was fitted with
biexponential disposition functions based on the Akaike
information criterion. The PK parameters obtained from
noncompartmental model analysis are summarized in
Table 4. No gender-specific differences were observed.
With respect to blood concentration versus time pro-
files as well as the PK parameters, it was concluded that
test and reference products were considered to be bio-
equivalent in beagle dogs. The present study provided
important preclinical information for the development
of generic clevidipine products in China.
Conclusion
The validated method for the simultaneous determina-
tion of clevidipine and its metabolite in beagle dog whole
blood demonstrated high selectivity, sensitivity, preci-
sion, accuracy and ruggedness. The developed assay was
suitable for PK studies of clevidipine and its metabolite
in beagle dogs. The optimized stabilizer cocktail was
able to effectively suppress the activity of esterase that
would convert the parent drug to the metabolite.
Future perspective
There exist critical challenges to solve the stability issues
of labile ester-containing drugs during sample collec-
tion, storage, processing as well as LC–MS/MS assay.
An effective screening approach for the stabilization of
clevidipine has been developed. This approach can also
be applied to other ester-containing drugs. Citric acid,
a mild acid, is found to be more effective in stabiliza-
tion of clevidipine compared with four commonly used
esterase inhibitors. Acidification is reported to be an effi-
cient strategy for stabilizing ester-containing analytes in
biological matrices, since adjusting pH to a certain level
beyond enzymatic working pH window greatly reduces
enzymatic activity. Another obvious advantage from
acidification approach is the lack of matrix effect caused
by the addition of high concentration pre-added esterase
stabilizers. In addition, other acids (such as formic acid,
acetic acid) with the potential to inhibit the activity of
esterase can also be added to the screening approach to
select the best stabilizer for a specific analyte.
Supplementary data
To view the supplementary data that accompany this paper
please visit the journal website at: www.future-science.com/
doi/full/10.4155/BIO.15.74
Financial & competing interests disclosure
This project was supported in part by Grand Project of Science
Research for the 12th 5-year plan funded by Ministry of Sci-
ence and Technology of China (2012ZX09102-101-016). The
authors have no other relevant affiliations or financial involve-
ment with any organization or entity with a financial inter-
est in or financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this
manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate institu-
tional review board approval or have followed the principles
outlined in the Declaration of Helsinki for all human or animal
experimental investigations. In addition, for investigations in-
volving human subjects, informed consent has been obtained
from the participants involved.

Executive summary
Aim
• The present study describes an integrated esterase inhibitor screening strategy.
• A robust and accurate LC–MS/MS method was established to overcome the shortcomings of published methods.
Results
• The selected inhibitor strategy and sample treatment process guarantee the accurate simultaneous
determination of both clevidipine and its major metabolite.
• The LC–MS/MS method is able to analyze both analytes of interest with sufficient LLOQ.
• Similar PK behaviors between test and reference preparations were observed after 60 min intravenous infusion
at the dose of 0.8 mg/kg/h in dogs.
Discussion
• This strategy can be also applied to the bioanalysis of other ester-containing drugs.
• This method was more accurate and sensitive than other LC–MS/MS methods without robust inhibition strategy.
• Quantitation of clevidipine with an isotope internal standard clevidipine-d7 is more reliable.

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Research Article  Cao, Li, Huang et al.
References
Papers of special note have been highlighted as:
• of interest; •• of considerable interest.
1 Keating GM. Clevidipine: a review of its use for managing
blood pressure in perioperative and intensive care settings.
Drugs 74(16), 1947–1960 (2014).
2 Nordlander M, Sjoquist PO, Ericsson H, Ryden L.
Pharmacodynamic, pharmacokinetic and clinical effects of
clevidipine, an ultrashort-acting calcium antagonist for rapid
blood pressure control. Cardiovasc. Drug Rev. 22(3), 227–250
(2004).
3 Ericsson H, Tholander B, Bjorkman JA, Nordlander M,
Regardh CG. Pharmacokinetics of new calcium channel
antagonist clevidipine in the rat, rabbit, and dog and
pharmacokinetic/pharmacodynamic relationship in
anesthetized dogs. Drug Metab. Dispos. 27(5), 558–564
(1999).
• Study on the PK of clevidipine and its primary metabolite,
H 152/81, in rats, rabbits and dogs.
4 Levy JH, Mancao MY, Gitter R et al. Clevidipine effectively
and rapidly controls blood pressure preoperatively in
cardiac surgery patients: the results of the randomized,
placebo-controlled efficacy study of clevidipine assessing
its preoperative antihypertensive effect in cardiac surgery-1.
Anesth. Analg. 105(4), 918–925 (2007).
5 Clevidipine (Cleviprex) for IV treatment of severe
hypertension. Med. Lett. Drugs Ther. 50(1295), 73–74
(2008).
6 Thompson CA. FDA approves i.v. calcium-channel blocker.
Am. J. Health Syst. Pharm. 65(18), 1686 (2008).
7 Briscoe CJ, Hage DS. Factors affecting the stability of drugs
and drug metabolites in biological matrices. Bioanalysis 1(1),
205–220 (2009).
8 Zeng J, Onthank D, Crane P et al. Simultaneous
determination of a selective adenosine 2A agonist, BMS-
068645, and its acid metabolite in human plasma by liquid
chromatography-tandem mass spectrometry‐‐evaluation of
the esterase inhibitor, diisopropyl fluorophosphate, in the
stabilization of a labile ester-containing drug. J. Chromatogr.
B. Analyt. Technol. Biomed. Life Sci. 852(1–2), 77–84 (2007).
9 Zhao Y, Liu G, Shen H et al. Bioanalysis of propylparaben
and p-hydroxybenzoic acid, and their sulfate conjugates in rat
plasma by liquid chromatography-tandem mass spectrometry.
J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 947–948,
68–74 (2014).
10 Yuan L, Jian W, Zhang D, Aubry AF, Arnold ME.
Application of a stabilizer cocktail of N-ethylmaleimide and
phenylmethanesulfonyl fluoride to concurrently stabilize the
disulfide and ester containing compounds in a plasma
LC–MS/MS assay. J. Pharm. Biomed. Anal. 88, 552–561
(2014).
11 Cheng Z, Zhang L, Zhang Y, Chen G, Jiang H. Simultaneous
determination of capilliposide B and capilliposide C
in rat plasma by LC–MS/MS and its application to a
pharmacokinetic study. Bioanalysis 6(7), 935–945 (2013).
12 Xue K, Li G, Sun X et al. Simultaneous quantification
of fosinopril and its active metabolite fosinoprilat in rat
1468 Bioanalysis (2015) 7(12)
plasma by UFLC–MS/MS. Application of formic acid in
the stabilization of an ester-containing drug. J. Chromatogr.
B. Analyt. Technol. Biomed. Life Sci. 990, 141–149 (2015).
13 Fakt C, Stenhoff H. Determination of an ultrashort-acting
antihypertensive dihydropyridine, clevidipine, in blood
using capillary gas chromatography-mass spectrometry and
of the primary metabolite using liquid chromatography and
fluorescence detection. J. Chromatogr. B. Biomed. Sci. Appl.
723(1–2), 211–219 (1999).
•• Combination of GC–MS and HPLC–fluorescence
detection methods to separately determine clevidipine
and the primary metabolite in whole blood with
efficient inhibition strategy to prevent hydrolyzation of
clevidipine.
14 Zhou Y, Li H, He X et al. Simultaneous determination
of clevidipine and its primary metabolite in dog plasma
by liquid chromatography-tandem mass spectrometry:
application to PK study. J. Pharm. Biomed. Anal. 100C,
294–299 (2014).
• An LC–MS/MS method for quantitation of clevidipine
and its primary metabolite in dog plasma by lowing
temperature to slow down the degradation of clevidipine.
15 Wei H, Gu Y, Liu Y, Chen Y, Liu C, Si D. Quantitation of
clevidipine in dog blood by liquid chromatography tandem
mass spectrometry: application to a pharmacokinetic study.
J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 971C,
52–57 (2014).
• LC–MS/MS method for quantitation of clevidipine in
dog plasma by protein precipitation with methanol.
16 Us Department of Health and Human Services, US FDA,
Center for Drug Evaluation and Research, Center for
Veterinary Medicine. Guidance for Industry : Bioanalytical
Method Validation. US FDA, MD, USA (2013).
17 Leibman KC. Editorial: drug metabolism and disposition:
the biological fate of chemicals. Drug Metab. Dispos. 1(2),
487–488 (1973).
18 Ericsson H, Bredberg U, Eriksson U, Jolin-Mellgard
A, Nordlander M, Regardh CG. Pharmacokinetics and
arteriovenous differences in clevidipine concentration
following a short- and a long-term intravenous infusion in
healthy volunteers. Anesthesiology 92(4), 993–1001 (2000).
19 Davis PJ, Cladis FP. The use of ultra-short-acting opioids
in paediatric anaesthesia: the role of remifentanil. Clin.
Pharmacokinet. 44(8), 787–796 (2005).
20 Selinger K, Lanzo C, Sekut A. Determination of
remifentanil in human and dog blood by HPLC with UV
detection. J. Pharm. Biomed. Anal. 12(2), 243–248 (1994).
21 Haidar SH, Liang Z, Selinger K, Hamlett L, Eddington
ND. Determination of remifentanil, an ultra-short-acting
opioid anesthetic, in rat blood by high performance liquid
chromatography with ultraviolet detection. J. Pharm.
Biomed. Anal. 14(12), 1727–1732 (1996).
22 Grosse CM, Davis IM, Arrendale RF, Jersey J, Amin J.
Determination of remifentanil in human blood by liquid-
liquid extraction and capillary GC-HRMS-SIM using
a deuterated internal standard. J. Pharm. Biomed. Anal.
12(2), 195–203 (1994).
future science group
 Stabilizer screening for LC–MS/MS analysis of clevidipine & its primary metabolite
23 Ericsson H, Tholander B, Regardh CG. In vitro hydrolysis
rate and protein binding of clevidipine, a new ultrashort-
acting calcium antagonist metabolised by esterases, in
different animal species and man. Eur. J. Pharm. Sci. 8(1),
29–37 (1999).
• Investigatation of the in vitro hydrolysis rate and the
protein binding of clevidipine in the rat, dog and man
in different biological matrices including plasma and
blood from volunteers with deficient pseudocholinesterase
activity.
future science group
Research Article
24 Fung EN, Zheng N, Arnold ME, Zeng J. Effective screening
approach to select esterase inhibitors used for stabilizing
ester-containing prodrugs analyzed by LC–MS/MS.
Bioanalysis 2(4), 733–743 (2010).
•• Effective approach to promptly select suitable esterase
inhibitors for stabilizing ester-containing prodrugs.
25 Li W, Zhang J, Tse FL. Strategies in quantitative LC–MS/
MS analysis of unstable small molecules in biological
matrices. Biomed. Chromatogr. 25(1–2), 258–277 (2011).
www.future-science.com 1469