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HPLC ANALYSIS OF FUROSEMIDE IN RAT
PLASMA. BIOAVAILABILITY STUDY. NOTE 1
CODRUŢA ŞOICA1*, SILVIA IMRE2, C. VARI2, Á. GYÉRESI2,
CRISTINA DEHELEAN1, MARIA DOGARU2
University of Medicine and Pharmacy “Victor Babeş” Timişoara, 2
Eftimie Murgu, Timişoara, Romania
2
University of Medicine and Pharmacy Târgu-Mureş, 34 Gh. Marinescu,
Târgu-Mureş, Romania
*
corresponding author: codtrutasoica@yahoo.com
1
Abstract
Furosemide is a benzoic acid derivative with a powerful diuretic activity used in
the treatment of edemas and hypertension. Cyclodextrins are toroidal shape
oligosaccharides with a cavity that can accommodate a large number of pharmaceuticals.
In this, article a HPLC method for the separation and identification of
furosemide in rat plasma is presented in order to perform a bioavailability study concerning
inclusion complexes of furosemide and randomly methylated β-cyclodextrin (RAMEB).
Under the mentioned HPLC parameters, the separation of furosemide (FS) and internal
standard (ISTD) was specific to endogenous compounds, without significant interferences
in analytes retention times from endogenous compounds. The method proved to be linear
between 0.020 – 10.00 g/mL FS and the calibration model was accepted.
Rezumat
Furosemidul este un derivat al acidului benzoic cu o puternică activitate diuretică,
utilizată în tratamentul edemelor şi hipertensiunii. Ciclodextrinele sunt oligozaharide toroidale
cu o cavitate care poate include un mare număr de substanţe medicamentoase.
În prezentul articol este prezentată o metodă HPLC de separare şi identificare a
furosemidului în plasma de şobolan cu scopul de a realiza un studiu de biodisponibilitate
privind complecşii de incluziune ai furosemidei cu RAMEB. În condiţiile HPLC utilizate,
separarea furosemidei de compuşii endogeni a fost posibilă, fără interferenţe semnificative
ale altor biomolecule endogene. Metoda s-a dovedit liniară între 0,020 – 10,00 g/mL FS
iar modelul de calibrare a fost acceptat.




furosemide
HPLC
cyclodextrin
inclusion complexation
INTRODUCTION
Furosemide is a benzoic acid derivative [1] with a powerful diuretic
activity used in the treatment of edemas and hypertension [2]. Its solubility
in water being very low [3] it leads to a poor bioavailability [4], which can
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be improved by association with cyclodextrins [5]. Cyclodextrins are
toroidal shape oligosaccharides with a cavity that can accommodate a large
number of pharmaceuticals [6].
In previous papers [7-9] we obtained binary and ternary complexes
of furosemide with randomly methylated β-cyclodextrin (RAMEB) and
polyvinylpyrrolidone (PVP) by specific methods (physical mixture,
kneading, ultrasonication) in molar ratio of 1:1 and 1:2. The binary and
ternary products were analysed by in vitro dissolution tests, differential
scanning calorimetry, X-ray diffraction, 1H NMR and in vitro membrane
diffusion tests.
In the present study we elaborated a HPLC method in order to
analyse furosemide (FS) concentration in rat plasma samples after oral
administration of furosemide and furosemide-RAMEB 1:1 complex, to
Wistar white rats.
MATHERIALS AND METHODS
1. Laboratory animals
Experimental pharmacokinetic determinations were accomplished
using Wistar white rats, both males and females, weighting 200±20 g. The
animals were kept under standard conditions (24  2C, 60% air humidity)
and had free access to water. No food was supplied in the last 18 hours
before the experiment. The experimental part was approved by University
Bioethical Committee.
2. Blood samples
The experimental animals were divided in two groups of 42
individuals: a control group, treated with furosemide 40 mg/kg, and an
experimental group, treated with an equivalent amount of furosemide
complex (furosemide: RAMEB 1:1). For each prelevation time, six animals
were sacrificed.
Doses were established depending on the linearity of
pharmacokinetic parameters in rat and the necessary conditions requested by
the analytical method (40 mg/kg, oral administration, corresponding to a
dose of 8 mg/ animal weighing about 200 g).
An additional animal group was used for blood prelevation in order
to obtain rat blank plasma, necessary for developing the analytical method
and rat plasma standards preparation.
After administration, before each blood prelevation, animal
anaesthesia was induced with ethylic ether, which is not metabolised in the
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rat organism; it is not soluble in plasma or bound by plasmatic proteins and
also does not cause interferences with other metabolic processes. In
addition, ether narcosis was used from ethical considerations concerning
animal experiments, in order to get blood and liver samples and also for
euthanasia of the animals.
Blood samples were taken by cardiac punction, each time being taken
2.5-3.5 mL in tubes using K3EDTA as anticoagulant, at different time
periods, as follows: 0 (blank); 0.5; 1; 2; 3; 4; 6 hours. Right after
prelevation, blood samples were transferred in anticoagulant pre-treated
tubes and centrifuged at 3500 rotations/minute for 10 minutes in a cooling
centrifuge at a constant temperature of 4C. The separated plasma was
transferred in Eppendorf micro tubes and kept at -20C until being analysed.
3. HPLC furosemide analysis in rat plasma
-
Apparatus:
HPLC system 1100 Agilent Technologies, made out of quaternary
pump, degazer, automatic injector, thermostat column, UV detector
balance AB54S (Mettler-Toledo, Switzerland)
water purifying device Direct Q5 (Millipore, France)
ultrasonic bath Transsonic T700/H, Elma (Germany)
rotational vacuum concentrator RVC2-25 (Martin Christ, Germany)
vortex Mix 20 (Falc Instruments, Italy)
sample shaker S20 (CAT, Germany)
centrifuge 2-15 (Sigma, Germany)
-
Reagents and reference substances:
furosemide (FS), nitrazepam (internal standard, ISTD)
methanol, gradient grade (Merck)
acetonitrile, gradient grade, for liquid chromatography (Merck)
solution of perchloric acid 20% (Merck)
dichloromethane, ethylic ether (Merck)
solution of HCl 1 M (Merck)
-
-
Chromatographic conditions:
Lichrospher column C18, 250 x 4 mm, 5 m (Merck), protected by a
pre-column RP18 (Merck)
Mobile phase:
o A: potassium dihydrogenophosphate 10 mM, pH 2.5 (with
phosphoric acid 85%)
o B: acetonitrile
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Composition gradient:
o 0.00-18.00 min 77% A, 23% B
o 18.01-25.00 min 10%A, 90% B
o 25.01-29.00 min 77%A, 23% B
mobile phase flow: 2 mL/min
column temperature: 45 C
wavelength of detection: 230 nm
injection volume: 100 l
-
-
Stock and working solutions:
stock solution FS in methanol 1000 g/mL
stock solution of ISTD 100 g/mL (ISTD) in methanol-water
solution 50% V/V
for plasma spiking, 11 working (W) solutions of FS in methanolwater 50:50 (V:V) were prepared, having the following
concentrations: 0.2; 0.5; 0.6; 1; 5; 10; 40; 50; 70; 80; 100 g/mL
Plasma calibration standards and control samples preparation:
0.5 mL blank plasma were treated with 50 l W solution and 50 l
ISTD and stirred 10 seconds on the vortex. Plasma standard solution
concentrations for the calibration curve were: 0.02; 0.05; 0.1; 0.5; 1; 4; 7; 10
g/mL FS, internal standard concentration being 10 g/mL. Plasma control
samples concentrations (QC) were: 0.06; 5; 8 g/mL FS.
Plasma samples preparation for HPLC analysis:
0.5 mL plasma sample is treated with 50 l water (W for
calibration standards), 50 l ISTD and stirred at vortex for 10 seconds. 100
l HCl 1 M are added and the solution is stirred on vortex for 10 seconds. A
volume of four mL extraction mix of ethylic ether: dichloromethane 3:2
(V:V) is added and shaked for 30 minutes. After 10 minutes of
centrifugation at 6000 rpm, 3.5 mL of organic layer were evaporated at
40C for 30 minutes. The cold residue is treated with 200 l mobile phase
and stirred on vortex; 20 l HClO4 20% is added, homogenised and
centrifuged at 6000 rpm for 10 minutes. After centrifugation, a volume of
100 l supernatant is injected in the HPLC system for future analysis.
Performance of the HPLC method
At least six different plasma blanks were used to assess specificity
with respect to endogenous compounds.
Linearity was verified over the concentration domain 0.02-10
µg/mL FS by applying a calibration model as:
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AreaFS/AreaISTD = a cFS/cISTD + b, weighing factor 1/c
where a – slope of the calibration curve, b – interception. The model was
accepted if the correlation coefficient was greater than 0.99 and a random
distribution of the residuals was observed within ±15% limits, except the
lower limit of quantification where limits of ±20% are accepted.
In order to verify method’s performance, three quality control (QC)
samples, prepared in duplicate at three concentration levels, were used to
verify method’s accuracy together with the calibration curve of the run. A
bias within ±15% values was accepted. Two QC samples could be outside
these limits, but not both having the same concentration.
RESULTS AND DISCUSSION
1. Method specificity
Under the mentioned HPLC parameters, the separation of FS and
ISTD is specific as referred to endogenous compounds, without significant
interferences in analytes retention times from endogenous compounds.
(Figure 1). The method implies washing of the column between 18.01 and
25.00 minutes with 90% acetonitril for eluting retarded endogenous
compounds. Even under these circumstances one can notice a peak from the
previous injection, between 8 and 10 minutes.
mAU
FS
250
200
150
Plasma marcata
ISTD
100
Plasma blanc
50
0
2
4
6
8
10
12
14
16
Figure 1
Chromatograms of blank plasma and FS (7 g/mL)
and ISTD (10 g/mL) marked plasma
18
min
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2. Method’s linearity
The method proved to be linear between 0.020 – 10.00 g/mL FS,
with a typical calibration curve of: Area ratio = 2.722 Concentration ratio –
0.0020, N = 8 calibration points, correlation coefficient > 0.998 (Figure 2).
Figure 2
FS calibration curve
The residuals percentage (relative error of the calculated
concentration from the calibration curve) had a random variation with
concentration (Figure 3) and they are between the acceptance limits, ±20%
at the lower limit of quantification and ±15% at all other concentrations.
The calibration model was accepted [10].
20.00
15.00
10.00
Rez%
5.00
0.00
0
1
2
3
4
5
6
-5.00
-10.00
-15.00
-20.00
c [ug/ml]
Figure 3
Residuals distribution
7
8
9
10
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3. Rat plasma samples analysis
Rat plasma samples were analysed in one series, together with 8
standard calibration samples and six QC samples, two at each concentration
level. Injection sequence was validated, calibration curve being valid
(correlation coefficient > 0.998, residuals in interval ±15%), and QC
samples were in limits of 15%, except two QC samples, at concentration
levels of 5 and 8 g/mL, respectively (Table I). The sequence is considered
valid if maximum two QC samples, with different concentrations, out of six,
there are outside limits of ±15% [11-13].
0.06
0.067
11.7
c [g/mL]
cf FS [g/mL]
Er%
0.06
0.063
5
5
5.21
4.2
Table I
QC samples analysis
8
8
8.1
11.82
1.25
47.8*
5
7.96
59.2*
*values outside admissibility limits (15%);
Er% - relative error
cf – found concentration
Rat plasma samples with a concentration above superior
quantification limit were diluted with blank plasma up to a concentration
within calibration curve and reanalyzed (figures 4, 5).
FS
mAU
100
80
60
ISTD
40
20
0
2
4
6
8
10
12
14
16
18
min
Figure 4
Chromatogram of a rat plasma sample 0.5 hours after an oral dose of furosemide RAMEB complex equivalent to 40 mg/kg body weight furosemide
(cFS= 9.94 g/mL)
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mAU
FS
140
120
100
80
60
ISTD
40
20
0
2
4
6
8
10
12
14
16
18
min
Figure 5
Chromatogram of a rat plasma sample 1 hour after an oral dose of 40 mg/kg body
weight furosemide (cFS= 10.5 g/mL)
CONCLUSIONS
This paper presented a HPLC method for the separation and
identification of furosemide in rat plasma in order to perform a
bioavailability study concerning inclusion complexes of furosemide and
RAMEB. Under the mentioned HPLC parameters, the separation of FS and
ISTD is specific as referred to endogenous compounds, without significant
interferences in analytes retention times from endogenous compounds. The
method proved to be linear between 0.020 – 10.00 g/mL FS and the
calibration model was accepted.
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