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HPLC ANALYSIS OF FUROSEMIDE IN RAT

PLASMA. BIOAVAILABILITY STUDY. NOTE 1
CODRUA OICA1*, SILVIA IMRE2, C. VARI2, . GYRESI2,
CRISTINA DEHELEAN1, MARIA DOGARU2
1

University of Medicine and Pharmacy Victor Babe Timioara, 2

Eftimie Murgu, Timioara, Romania
2
University of Medicine and Pharmacy Trgu-Mure, 34 Gh. Marinescu,
Trgu-Mure, Romania
*
corresponding author: codtrutasoica@yahoo.com
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 -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 puternic activitate diuretic,
utilizat n tratamentul edemelor i hipertensiunii. Ciclodextrinele sunt oligozaharide toroidale
cu o cavitate care poate include un mare numr de substane medicamentoase.
n prezentul articol este prezentat o metod HPLC de separare i identificare a
furosemidului n plasma de obolan cu scopul de a realiza un studiu de biodisponibilitate
privind complecii de incluziune ai furosemidei cu RAMEB. n condiiile HPLC utilizate,
separarea furosemidei de compuii endogeni a fost posibil, fr interferene semnificative
ale altor biomolecule endogene. Metoda s-a dovedit liniar ntre 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

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in water being very low [3] it leads to a poor bioavailability [4], which can
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 -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 20020 g. The
animals were kept under standard conditions (24 2C, 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

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anaesthesia was induced with ethylic ether, which is not metabolised in the
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 4C. The separated plasma was
transferred in Eppendorf micro tubes and kept at -20C 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%)

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o B: acetonitrile
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
40C 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.

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Linearity was verified over the concentration domain 0.02-10

g/mL FS by applying a calibration model as:
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 methods performance, three quality control (QC)
samples, prepared in duplicate at three concentration levels, were used to
verify methods 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

10

12

14

16

18

min

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Figure 1
Chromatograms of blank plasma and FS (7 g/mL)
and ISTD (10 g/mL) marked plasma

2. Methods 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].

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Figure 3
Residuals distribution

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].
c [g/mL]
cf FS [g/mL]
Er%

0.06
0.067
11.7

0.06
0.063
5

5
5.21
4.2

5
7.96
59.2*

Table I
QC samples analysis
8
8
8.1
11.82
1.25
47.8*

*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).

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FS

mAU
100

80

60

ISTD

40

20

0
2

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)
mAU

FS

140

120

100

80

60

ISTD

40

20

0
2

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

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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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1. *** European Pharmacopoeia, 5th ed., Council of Europe,
Strasbourg, 2005
2. Kreaz R.M., Dombi Gy., Kata M, The influence of -cyclodextrins
on the solubility of furosemide, J Incl Phenom, 1998, 31, 189-196
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furosemide with cyclodextrins, Proc. 8th Int. Symp. on
Cyclodextrins, Kluwer Academic Publ., Dordrecht, 1996, 341-344
4. Kreaz R.M., Abu-Eida E.Y., Eros I., Kata M., ., Freeze-Dried
Complexes of Furosemide with beta-Cyclodextrin Derivatives, J
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11. *** U. S. Department of Health and Human Services, Food and

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