ISSN: 0975 -8542
Journal of Global Pharma Technology
RESEARCH ARTICLE
©2009-2020, JGPT. All Rights Reserved
668
A Capillary Electrophoretic Method for the Analysis of
Bupivacaine and Its Metabolites
Roza Askarova
1*,
Kirill Polyakov
2,
Iuliia Akulinina
2
1.
Urgench branch of the Tashkent Medical Academy, Urgench, Тhe Republic of Uzbekistan.
2.
I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russian
Federation.
*Corresponding Author: Roza Askarova
Abstract
The article studies a capillary electrophoretic (CE) method for the analysis of urinary extracts of the local
anesthetic, bupivacaine, and its three main metabolites, desbutylbupi vacaine, 3’-hydroxybupivacaine,
and 4’-hydroxybupivacaine, in rat urine. After collection of blank urine, the rats were given a 20 mg/kg
intramuscular injection of bupivacaine, and urine was collected for 12 h after dosing. CE analyses were
performed using the CAPEL®-205 capillary electrophoresis systems. The data was collected using the
Elforan® specialized software. The use of methanol to reduce peak tailing was investigated at different
concentrations, but 20% and 30% v/v were proved to be the most optimal at the preparatory stage of the
experiment. The resolution of 3’-hydroxybupivacaine and 4’-hydroxybupivacaine was 1.09, 0.98, 0.89 and
0.89 at 15, 40, 70 and 110 s, respectively. The initial resolution (Rs) of desbutylbupi vacaine was achieved
with all studied injection periods as Rs = 1.09, 0.97, 0.96 and 0.96 at 15, 40, 70 and 110 s, respectively.
Separation efficiencies for 3’- and 4’-hydroxybupivacaine were
312×10
3
, 257×10
3
, 196×10
3
и 169×10
3
μlat
injection times of15, 40, 70 and 110 s, respectively. The results showed that the mass of bupivacaine,
desbutylbupi vacaine, and 3’- and 4’-hydroxybupivacaine significantly recovered in the rat urine after the
dose was administered. The recoveries as a percent of the dose were 0.04, 0.80, 0.15 and 0.05% for
desbutylbupi vacaine, bupivacaine, 3’-hydroxybupivacaine, and 4’-hydroxybupivacaine, respectively.
Separation of bupivacaine and its metabolites was achieved in 15 min. A particular advantage of this
approach over published HPLC methods is that separation of the two hydroxy positional isomers of
bupivacaine is possible. A number of unknown peaks were also observed in the electropherograms from
the rats dosed with bupivacaine. These did not correspond to any peaks appearing in the blank urine
samples. Characterization of these unknown peaks may prove useful for the further understanding of
bupivacaine metabolism.
Keywords:
Bupivacaine, Capillary electrophoresis, Desbutylbupivacaine, Hydroxybupivacaine,
metabolites.
Introduction
Bupivacaine is one of the most commonly
used
local
anesthetics,
particularly in obstetric
anesthesia.
Bupivacaine
has
a
long-
acting anesthetic effect after subcutaneous or
intravenous injection, compering to other
local anesthetics such as procaine and
lidocaine.
However, the toxic effect is greater for the
cardiovascular and central nervous systems
[1], thus the patient may suffer from heart
failure. Since the effects and associated
toxicity are directly related to the
concentration of local anesthetic in the
systemic circulation, it is very important to
determine the concentration of bupivacaine
in plasma and other biological fluids.
Capillary electrophoresis (CE) is known for
its high efficiency, high resolution and ultra-
small sample volume, and therefore is an
alternative to HPLC.
It has been proven to be anexcellent
separation technique for analytical and
bioanalytical
chemistry
[2].
Optical detection is the
most
used in commercial
CE,
particularly UV
absorbance and fluorescence detection.
Roza Askarova
et. al.
| Journal of Global Pharma Technology | 2020| Vol. 12| Issue 06 |668-676
©2009-2020, JGPT. All Rights Reserved
669
However, the detection sensitivity of UV is
relatively low due to the short optical path
length. Fluorescence detection, namely laser-
induced fluorescence, is highly sensitive, but
quite expensive. Among other sensitive
detection methods, electrochemical detection
(ED) is limited by the poor stability, and
mass spectrometry (MS) is expensive.
However, no reported method in the
literature clearly describes an analytical
procedure for the simultaneous separation
and analysis of bupivacaine and its desbutyl,
3’-hydroxy and 4’-hydroxy metabolites in a
biological matrix.
Therefore, it is important for CE to develop a
highly selective, sensitive, stable and
economical detector, which makes the study
relevant. The study describes the capillary
electrophoretic
(CE)
and
solid-phase
extraction (SPE) conditions necessary for the
simultaneous analysis of bupivacaine and the
aforementioned metabolites in urine from
rats administered a therapeutic dose of
bupivacaine. The results showed that this
method is simple, fast, selective and sensitive
for the determination of bupivacaine in urine.
The development of a CE-MS method
employing
electrospray
ionization
is
currently underway in our laboratory for the
identification of further metabolites of
bupivacaine.
Literature Review
Capillary electrophoresis (CE) has long been
used in the analysis of pharmaceuticals and
their metabolites to a wide range of biological
environments. The separation in CE is based
on the charge-to-size characteristics of the
analyte of interest rather than partitioning
as in liquid chromatography (LC) [3].
Moreover, the separation efficiency of CE is
frequently much higher than for LC.
For these reasons, separations not possible
by LC may be possible by CE. Bupivacaine is
one of the most commonly used local
anesthetics,
particularly in obstetric
anesthesia, but careless and excessive use of
bupivacaine and isoflupredone might lead to
direct or indirect continuous contamination of
food commodities [4].
Indirectly, bupivacaine can be released into
the environment through patient excreta and
hospital waste management. Thus, trace
concentrations can be released into surface,
ground and drinking waterthatlead to
pollution of water and it is potential toxicity
to human health. The infected water can also
be used for agricultural irrigation and animal
production, causing contamination of both
plants and animals such as pigs, cattle and
chicken through residues in muscle tissue,
milk
and
eggs,
respectively.
The
determination of the drug residues in animal
foods is an integral part of food safety, due to
their differences in functions, chemical
structure, and physicochemical properties [5].
Since the effects and associated toxicity are
directly related to the concentration of local
anesthetic in the systemic circulation, it is
very
important
to
determine
the
concentration of bupivacaine in plasma and
other biological fluids.
The development of analytical methods
permitting simultaneous determination of
bupivacaine and its metabolites is important
in understanding its fate and distribution in
tissues and biological fluids after therapeutic
administration. The analysis of bupivacaine
and its metabolites in biological tissues and
fluids has been performed by thin-layer
chromatography (TLC), gas chromatography
(GC),
and
high-performance
liquid
chromatography (HPLC).
The earliest approaches to the analysis of
bupivacaine and its metabolites made use of
TLC and GC methods. The detection of
bupivacaine, 3’-hydroxybupivacaine, and
desbutylbupi vacaine in rat urine by GC-MS
after liquid-liquid extraction has been
previously reported [6].
Using this approach in conjunction with
NMR analysis, three further metabolites,
N
-
butyl-pipecolyl-2-amide and two mono-
hydroxylated bupivacaine metabolites were
identified in the urine samples. The GC-MS
method incorporating selected ion monitoring
was also used to determine bupivacaine and
desbutylbupi vacaine in maternal, fetal and
analysis neonatal plasma following its
administration as an epidural anesthesia for
Caesarean section [7]. The majority of
analytical methods in recent years for the
simultaneous analysis of bupivacaine and its
metabolites in biological matrices have made
use of HPLC in conjunction with liquid-liquid
extraction [8], phase extraction (SPE) [9] or
column switching schemes [10].
The analysis of bupivacaine, desbutylbupi
vacaine and 4’-hydroxybupivacaine in human
Roza Askarova
et. al.
| Journal of Global Pharma Technology | 2020| Vol. 12| Issue 06 |668-676
©2009-2020, JGPT. All Rights Reserved
670
serum, plasma and urine using liquid-liquid
extraction and HPLC, has been reported by
several authors [8, 11]. Using the
chromatographic
conditions
described,
excellent separation of all three compounds
was possible. However, no separation or
quantitation of 3’-hydroxybupivacaine was
reported.
The use of capillary electrophoresis for the
separation and quantitation of bupivacaine
enantiomers in human plasma [12] and
therapeutic monitoring of bupivacaine in
drain fluid collected from patients after
pulmonary surgery [13] has been reported.
However, no reported method in the
literature clearly describes an analytical
procedure for the simultaneous separation
and analysis of bupivacaine and its desbutyl,
3’-hydroxy and 4’-hydroxy metabolites in a
biological matrix.
The
study
describes
the
capillary
electrophoretic
(CE)
and
solid-phase
extraction (SPE) conditions necessary for the
simultaneous analysis of bupivacaine and the
aforementioned metabolites in urine from
rats administered a therapeutic dose of
bupivacaine. The results showed that this
method is simple, fast, selective and sensitive
for the determination of bupivacaine in urine.
Problem Statement
The problem of choosing a method for
studying the properties of pharmacological
drugs remains quite relevant, especially for
anesthetics drugs used in medical practice
during surgery. The widespread use of
bupivacaine as local anesthetics is known,
but no reported method in the literature
clearly describes an analytical procedure for
the simultaneous separation and analysis of
bupivacaine and its desbutyl, 3’-hydroxy and
4’-hydroxy metabolites in a biological matrix.
In this regard, it is important for CE to
develop a highly selective, sensitive, stable
and economical detector, which makes the
study relevant. Therefore, the aim of our
study was to describe the capillary
electrophoretic
(CE)
and
solid-phase
extraction (SPE) conditions necessary for the
simultaneous analysis of bupivacaine and the
aforementioned metabolites in urine from
rats administered a therapeutic dose of
bupivacaine. To achieve goal of the study, the
following tasks were set up:
Analyze the results of using different
methods for studying the properties of
bupivacaine and its metabolites;
Determine the most effective method for
obtaining high-quality results, analysis
conditions,
necessary
reagents
and
available equipment;
Find the most optimal order of migration of
solutions of bupivacaine metabolites during
capillary electrophoresis;
Investigate the injection time and the
resolution efficiency of bupivacaine and its
metabolites (desbutyl, 3’-hydroxy and 4’-
hydroxy);
Determine the performance of extracted
bupivacaine and its three metabolites in rat
urine
within
12 hours
after the
administration of a therapeutic dose of
bupivacaine.
Methods and Materials
Materials and Reagents
Bupivacaine hydrochloride and prilocaine
hydrochloride were obtained from Sigma-
Aldrich (St. Louis, MO). Chloroform, used in
the extraction method, was 99.9% A.C.S.
grade. HPLC grade methanol was used in
both the extraction method and as part of the
CE buffer.
All other chemicals were reagent grade or
better and were used as received.All water
was
purified
by
a
double
distilling and deionizing. Two buffers were
used in this work. The CE buffer consisted of
30% v/v methanol and 70% v/v 214 mM
ammonium acetate, pH 5.0. The extraction
buffer consisted of 300 mM sodium
bicarbonate, pH 10. All solutions were
filtered through a 0.22 pm nylon filter prior
to use.
Laboratory Rat Urine Collection
Female rats weighing 300-350g were hold in
a metabolism cage (Zoon Lab). Urine was
collected over a 12 h period in collection tubes
submerged in dry ice. During the collection
period, the animals were provided with food
and water. After collection of blank urine, the
rats were given a 20 mg/kg intramuscular
injection of bupivacaine, and urine was
collected for 12 h after dosing.
Roza Askarova
et. al.
| Journal of Global Pharma Technology | 2020| Vol. 12| Issue 06 |668-676
©2009-2020, JGPT. All Rights Reserved
671
From rat 1, 11.2 mL urine was collected,
while 8.0 mL was collected from rat 2. All
urine samples were frozen until use, when
they
were
thawed,
extracted,
and
immediately analyzed by CE.
CE apparatus
CE analyses were performed using the
CAPEL®-205
capillary
electrophoresis
system
(Lumex-Marketing
LLC,
St.
Petersburg)
in
accordance
with
the
requirements
of
Technical
Regulations CU TR 004/2011 on the safety of
low-voltage equipment in the framework of
the Customs Union and TR CU 020/2011
“Electromagnetic compatibility of technical
means”, and the Directive 2014/30/EU of the
European Parliament on the harmonisation
of the laws of the Member States relating to
electromagnetic compatibility [14].
The data was collected using the Elforan®
specialized software. A window was made in
the capillary by placing a drop of
concentrated sulfuric acid on the capillary,
heating the droplet, wiping the exposed area
with a tissue, and finally cleaning with
methanol and water. All washings, sample
applications, and sample elutions were
performed at a flow rate of approximately 1
mL/min. The chloroform extract was
evaporated to dryness and reconstituted in
0.200 mL of 0.1% formic acid, 20% v/v
methanol. Reconstitution of the sample was
aided by vortexing for 1 min, after which the
sample was electro kinetically injected into
the CE capillary.
Preparation of Stock and Standard
Solutions
A
stock
solution
of
1.5
m
M
desbutylbupivacaine, bupivacaine, 3’-
hydroxybupivacaine,
and
4’-
hydroxybupivacaine and a stock solution of
1.0 m
M
prilocaine were prepared in water
and stored at 4°C. Standards were prepared
daily by serial dilutions of the two stock
solutions into 20% v/v methanol and 0.1% v/v
formic acid.
Statistical Reliability of Results
Statistical processing of data obtained on
quantitative
determination
of
local
anesthetics of the studied concentrations
displays
positive
repeatability
and
reproducibility of the results within the
recommended analytical area. The coefficient
of variation does not exceed 5%, and the
mean error of the result is 1.42%.
Results
The CE separation of desbutylbupivacaine,
3’-hydroxybupivacaine,
4’-
hydroxybupivacaine,
bupivacaine,
and
prilocaine was investigated at several run
buffer values. The experimental run buffers
for the pH 4.5, pH 5.0, and pH 6.0
separations consisted of 70% v/v 214
m
M
ammonium acetate and 30% v/v
methanol, while run buffer for the pH 7.0
separation consisted of 70 % v/v 171
m
M
potassium phosphate and 30% v/v
methanol.
The order of migration is desbutylbupi
vacaine (1), bupivacaine (2), and 3’-
hydroxybupi vacaine (3) and 4’ hydroxybupi
vacaine (4), in all electropherograms. At pH
4.5, baseline resolution was achieved for all
compounds with an analysis time of 15 min.
With an increasing the pH of the buffer to 5.0
permitted
baseline
separation
of
all
compounds in a reduced separation time of
14 min (Fig. 1).
Figure 1: Electropherograms of separation of desbutylbupivacaine (1), bupivacaine (2), and 3’-hydroxybupivacaine (3)
and 4 ’hydroxybupivacaine (4) by capillary electrophoresis (CE) performed at pH 5.0
Roza Askarova
et. al.
| Journal of Global Pharma Technology | 2020| Vol. 12| Issue 06 |668-676
©2009-2020, JGPT. All Rights Reserved
672
With the use of a pH 6.0 buffer, the analysis
time was further improved to 1 min. This co
migration of hydroxyl bupivacaine isomers
was more pronounced with the use of a pH
7.0 run buffers. As the purpose of this pH
optimization study was to define a set of CE
run buffer conditions permitting baseline
resolution of all compounds in as short an
analysis time as possible, a run buffer pH of
5.0 was chosen for all future work.
It was noted that significant peak tailing
occurred when aqueous buffers were
employed. The use of methanol to reduce
peak tailing was investigated at different
concentrations, but 20% and 30% v/v were
proved to be the most optimal at the
preparatory stage of the experiment. With
the use of 20% v/v methanol in the run buffer,
peak tailing was reduced but not eliminated.
Methanol at 30% v/v was found to eliminate
peak tailing; thus, this concentration of
methanol was used in the run buffer for all
further analyses.
In the initial stages of this investigation,
vacuum, hydrodynamic, and electrokinetic
injection schemes were investigated. As the
injection matrix is acidic, bupivacaine, its
metabolites and prilocaine all exist in their
ionized forms and are amenable to
electrokinetic injection. The selection of
electrokinetic injection for the final method
was based on the better limits of detection
associated with this injection technique
compared to vacuum or hydrodynamic
injection. While hydrodynamic injection was
slightly more reproducible than electrokinetic
injection,
1.6%
versus
2.5%
(
n
=3),
electrokinetic injection provided more than a
12-fold improvement in detection limit. The
effect of the injection time on the sensitivity
and resolution of the CE method was also
studied.It can be seen that the analyte
response increases with increasing injection
time; however, the resolution of 3’-
hydroxybupivacaine
and
4’-
hydroxybupivacaine
decreases
as
the
injection time is increased.
The resolution of 3’-hydroxybupivacaine and
4’-hydroxybupivacaine was 1.09, 0.98, 0.89
and 0.89 at 15, 40, 70 and 110 s, respectively.
The initial resolution (Rs) of desbutylbupi
vacaine was achieved with all studied
injection periods as Rs = 1.09, 0.97, 0.96 and
0.96 at 15, 40, 70 and 110 s, respectively.
Separation efficiencies for 3’- and 4’-
hydroxybupivacaine were
312×10
3
, 257×10
3
,
196×10
3
и 169×10
3
μlat injection times of 15,
40, 70 and 110 s, respectively. Although the
use of longer injection times would result in
improved limits of detection for this CE
method, an injection time of 40 s was chosen
as it permitted good resolution of all
compounds,
particularly
that
of
the
hydroxybupi vacaine isomers.
The detection limit using this 40 s injection
time proved sufficient, when used in
conjunction with SPE, for the analysis of
bupivacaine and its desbutyl and hydroxy
metabolites in urine. To demonstrate the
utility of this method, the CE method was
applied to the analysis of bupivacaine in the
urine of two female rats previously
administered a 20 mg/kg intramuscular dose
of bupivacaine (Fig. 2).
Figure 2: Recoveries of bupivacaine and its metabolites in rat urine 12h after dosing with 20 mg / kg bupivacaine
Roza Askarova
et. al.
| Journal of Global Pharma Technology | 2020| Vol. 12| Issue 06 |668-676
©2009-2020, JGPT. All Rights Reserved
673
The results showed that the mass of
bupivacaine, desbutylbupivacaine, and 3’-
and 4’ hydroxybupivacaine recovered, in
addition to their recovery as a percentage of
the administered dose. The recoveries as a
percent of the dose were 0.04, 0.80, 0.15 and
0.05% for desbutylbupivacaine, bupivacaine,
3’-hydroxybupivacaine,
and
4’-
hydroxybupivacaine, respectively.
Discussion
Bupivacaine is a potent local anesthetic with
unique characteristics from the amide group
of local anesthetics, first discovered in 1957.
Local anesthetics are used in regional
anesthesia, epidural anesthesia, spinal
anesthesia, and local infiltration [15]. It is
known that bupivacaine infiltration in the
preoperative period significantly reduce
postoperative pain in accordance with
placebo at the second and sixth hour after
surgery.
Preoperative injection of bupivacaine is
useful
for
controlling
pain
in
the
postoperative periodfor up to 24 hours [16].
Local
anesthetics
generally block
the
generation of an action potential in nerve
cells by increasing the threshold for electrical
excitation. However, the study of bupivacaine
and
its
metabolitesproperties
remains
relevant, since the amount of bupivacaine
used varies greatly.
Thus, it was shown that the urination time
and restoration of the patient's motor
functions are statistically comparable when
using bupivacaine in a sufficiently high dose
of 12.5 mg [17]. Therefore, "selective spinal
anesthesia" or a combination of adjuvants
technique to speed up the resolution of the
unit for outpatient surgery is required. In the
current study, less overall recovery of
bupivacaine and its metabolites was achieved
than in these studies [18, 19].
This lower recovery is likely due to the fact
that urine was only collected for a 12 h period
as opposed to a 24 h period in the previous
studies.It has been shown that bupivacaine,
lidocaine and azaleptin can be determined in
biological fluids in the presence of
coextracted substances using electrophoretic
spectra and quantitative indicators [20]. The
influence of several parameters, such as the
buffer composition, pH, the ratio of the
concentration of substances on the separation
of the analyte is well known [21], but the
nature of this effect remains poorly studied.
The use of methanol to reduce peak tailing
was investigated at different concentrations,
but 20% and 30% v/v were proved to be the
most optimal at the preparatory stage of the
experiment. The addition of methanol to the
working
buffer
decreases
the
peak
concentration for some compounds [22].
Higher concentrations of methanol were not
investigated because evaporation of the
organic portion of the run buffer at higher
organic
concentrations
can
lead
to
irreproducible peak heights and migration
times [23].
The effect of the injection time on the
sensitivity and resolution of the CE method
has also been studied. It can be seen that the
analyte response increases with increasing
injection time; however, the resolution of 3’-
hydroxybupi
vacaine
and
4’-
hydroxybupivacaine
decreases
as
the
injection time is increased. Lower resolution
of the two hydroxy metabolites using longer
injection times is due to the injection of a
longer analyte band.
A longer analyte band leads to decreased
resolution because the effective length of
capillary available for separation of analytes
is decreased. However, according to the aim
of our study, namely the development of a
method necessary for the simultaneous
analysis
of
bupivacaine
and
the
aforementioned metabolites in urine, it is
clear that the CE can be used in combination
with SPE.
The method offers superior resolution of the
3’- and 4’-hydroxy metabolites than can be
achieved using reverse-phase LC methods [24,
25]. When compared to the use of
radiochemical detection in earlier studies [26,
27] or the analysis of the hydroxy isomers,
the CE method represents a more convenient
and rapid approach for their determination
in urine.
In addition, the CE method can be interfaced
directly to a mass spectrometer (MS) to
provide further identification of unknown
peaks in the electropherograms of the urine
extract, as shown in Fig. 1. However,
equipment problems related to the reliability
of automatic sampling and capillary
contamination should be resolved before
adopting
these
methods
as
routine
methodologies in the laboratory for analysis
of compounds.
Roza Askarova
et. al.
| Journal of Global Pharma Technology | 2020| Vol. 12| Issue 06 |668-676
©2009-2020, JGPT. All Rights Reserved
674
He development of a CE-MS method [28, 29]
employing
electrospray
ionization
is
currently underway in our laboratory for the
identification of further metabolites of
bupivacaine.
Conclusions
The study demonstrated an analytical
method for the determination of the local
anesthetic, bupivacaine, and its metabolites
in rat urine using SPE and CE. The excellent
resolution of the CE method makes this a
viable approach for the
in vivo
analysis of
bupivacaine and its metabolites.The order of
migration
is
desbutylbupivacaine
(1),
bupivacaine (2), and 3’-hydroxybupivacaine
(3) and 4’ hydroxybupivacaine (4), in all
electropherograms.
At pH 4.5, baseline resolution was achieved
for all compounds with an analysis time of 15
min. With an increasing the pH of the buffer
to 5.0 permitted baseline separation of all
compounds in a reduced separation time of
14 min. It was noted that significant peak
tailing occurred when aqueous buffers were
employed.
The use of methanol to reduce peak tailing
was investigated at different concentrations,
but 20% and 30% v/v were proved to be the
most optimal at the preparatory stage of the
experiment.
The
resolution
of
3’-
hydroxybupivacaine
and
4’-
hydroxybupivacaine was 1.09, 0.98, 0.89 and
0.89 at 15, 40, 70 and 110 s, respectively. The
initial resolution (Rs) of desbutylbupi vacaine
was achieved with all studied injection
periods as Rs = 1.09, 0.97, 0.96 and 0.96 at 15,
40, 70 and 110 s, respectively. Separation
efficiencies for 3’- and 4’-hydroxybupivacaine
were
312×10
3
,
257×10
3
,
196×10
3
и
169×10
3
μlat injection times of 15, 40, 70 and
110 s, respectively. The results showed that
the mass of bupivacaine, desbutylbupi
vacaine, and 3’- and 4’-hydroxybupivacaine
significantly recoveredin the rat urine after
the dose was administered. The recoveries as
a percent of the dose were 0.04, 0.80, 0.15
and 0.05% for desbutylbupi vacaine,
bupivacaine, 3’-hydroxybupivacaine, and 4’-
hydroxybupivacaine, respectively.
A particular advantage of this approach over
published HPLC methods is that separation
of the two hydroxy positional isomers of
bupivacaine is possible. A number of
unknown peaks were also observed in the
electropherograms from the rats dosed with
bupivacaine. These did not correspond to any
peaks appearing in the blank urine samples.
Characterization of these unknown peaks
may
prove
useful
for
the
further
understanding of bupivacaine metabolism.
Qualitative determination of these unknowns
by CE-MS is currently being carried out in
our laboratories.
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