Pharmacology & Pharmacy, 2013, 4, 520-527 Published Online October 2013 (
Simple and Rapid Determination of Diuretics by
Luminescent Method
Iuna Tsyrulneva, Olga Zaporozhets
Chemistry Department, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine.
Received August 2nd, 2013; revised August 6th, 2013; accepted September 18th, 2013
Copyright © 2013 Iuna Tsyrulneva, Olga Zaporozhets. This is an open access article distributed under the Creative Commons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Diuretics are drugs widely used in treatment of heart failure and hypertension and as doping agents in sports. Wrong
prescription and excessive abuse can lead to negative side effects. Despite the effectiveness of methods usually used for
the determination of diuretics (gas or liquid chromatography, capillary electrophoresis), they do not always provide
necessary sensitivity. Moreover, sample preparation increases time of analysis. A rapid and sensitive luminescent
method for determination of 5 diuretics (amiloride, bendroflumethiazide, bumetanide, furosemide, triamterene) in
aqueous solutions and amiloride and triamterene in human urine is described. Intrinsic luminescent properties of proto-
lytic forms of diuretics were studied in order to provide highly sensitive analysis. Investigation of interfering influence
of diuretics was carried out to provide selective determination of triamterene, bumetanide and furosemide in aqueous
mixtures of diuretics. Influence of urine at luminescent properties of diuretics was studied. The possibility of determina-
tion of triamterene and amiloride in human urine as individual substances and in mixture was proved. Simple and rapid
technique for their determination in human urine was elaborated. The techniques elaborated for determination of triam-
terene in presence of other diuretics and furosemide in presence of commensurate amount of bumetanide allow enhanc-
ing specifity of analysis. Sufficient selectivity and sensitivity were reached in determination of amiloride and triam-
terene in human urine. The reduction of time of analysis due to avoiding sample preparation merits the techniques pro-
Keywords: Furosemide; Bumetanide; Amiloride; Triamterene; Thiazides; Separation; Luminescence; Human Urine
1. Introduction
Diuretics are drugs that increase the amount of urine pro-
duced and enhance the excretion of electrolytes and
water with urine as a result of disruption of ion transport
in the kidney [1]. They are widely used in clinical prac-
tice in the treatment of hypertension and in different
kinds of edema as well as for the correction of acid-base
balance, in the treatment of intoxication and some in-
juries. Despite their effectiveness, diuretics may cause se-
rious problems in case of wrong administration and
excessive abuse [2]. Thus, it is of primary importance to
control the intake of diuretics in order to avoid negative
side effects.
Diuretics are prohibited for samples taken out of and
in competition according to World Anti-Doping Agency
(WADA) [3]. In sports, athletes misuse diuretics for
several reasons: to reduce body weight in order to qualify
for a lower weight, to reduce the urinary concentration of
other prohibited substances to avoid a positive doping
result and to overcome fluid retention caused by the use
of anabolic steroids. Diuretics need to be detected at or a
lower level than the minimum required performance limit
(MRPL) 250 ng·mL1 [4]. The factors mentioned de-
mand to carry out rapid, multicomponent, accurate and
selective analysis for the determination of pharmaceu-
ticals in urine.
The widely used methods for the determination of
diuretics in urine are liquid and gas chromatography with
mass-spectroscopic detection. These methods provide ne-
cessary selectivity and expressivity [5-13]. The main
disadvantage of the methods is insufficient selectivity
towards the urine components. Necessary selectivity is
achieved by preliminary liquid-liquid or solid phase
extraction. However, it leads to an increase in analysis
Other highly sensitive method of analysis of biological
Copyright © 2013 SciRes. PP
Simple and Rapid Determination of Diuretics by Luminescent Method 521
molecules is luminescent method. Isopotential fluori-
metry and fluorimetry in combination with partialleast
squares multivariative calibration for simultaneous de-
termination of amiloride and triamterene in human urine
are described in [14,15]. Although these methods are
characterized by high specifity and rapidity, high dilution
of urine and low recoveries of diuretics lead to low
sensitivity. Besides, the interfering influence of other
diuretics was not studied.
Sensitive method of determination of furosemide
based on its luminescent properties was proposed by
Ioannou et al. [16], but the selectivity of method was not
reached. Determination of amiloride and furosemide in
urine involves separation of the substances on nylon
membrane [17]. The method of the luminescent deter-
mination of triamterene and its metabolite in urine de-
mands preliminary separation on octadecyl (C18) discs
that consist of glass fibber embedded with C18 bonded
silica, providing a hydrophobic surface for retaining non-
polar compounds, and the method of determination of
triamterene in pharmaceuticals—separation on MP1 (a
mixed phase of nonpolar and strong cation which in-
volves both reversed phase and cation exchange) [18].
Literature data concerning luminescent determination of
other diuretics have not been found yet.
The luminescent properties of some representatives of
different classes of diuretics (loop, thiazide and potas-
sium sparing) in aqueous solutions and in human urine
with the aim to develop method of determination of furo-
semide, bumetanide, chlorthiazide, hydrochlorothiazide,
bendroflumethiazide, triamterene and amiloride were
studied in present work.
2. Materials and Methods
2.1. Instruments
Luminescence spectrometer LS55 (Perkin Elmer, USA)
equipped with a xenon impulse lamp source and 1.0 cm
cell, spectrophotometer UV-VIS Unico UV-2800, pH-
meter with glass electrode, centrifuge OPN-3Y4.2 (Rus-
sia), evaporator in dry nitrogen Liebisch (Germany), ana-
lytical balance KERN ABS (Germany), pipettes of adjust-
able volume Eppendorf (Germany) 2 - 20 µL, 20 - 200
µL, 100 - 1000 µL.
2.2. Chemicals and Reagents
Stock standard solutions were prepared for 7 diuretic
drugs (amiloride, bendroflumethiazide, bumetanide, chlor-
thiazide, furosemide, hydrochlorothiazide, triamterene)
(Merck, Germany) by dissolving 1.0 mg of the diuretic in
10 mL of methanol (Sigma, USA, qualification “HPLC
grade”) to obtain concentration of 0.1 mg·mL1. The
methanol stock solutions of diuretics were stored in a
tightly closed container in a cool and dry place. Working
solutions were obtained by taking an appropriate volume
of standard solution, evaporating the methanol in a ni-
trogen stream and diluting it with water. Standard aque-
ous solution was freshly prepared daily.
The pH was regulated by adding of fixed volume of
acid or alkali. Hydrochloric acid and sodium hydroxide
(both Merck, Germany) were prepared by dilution of the
initial concentrated solution.
Urine samples were collected from 5 volunteers who
did not consume banned substances. Urine samples were
stored in polypropylene bottles at a temperature of 20˚C.
Before conducting the experiment samples were defreez-
ed and centrifuged for 10 min at 3000 rpm. Urine sam-
ples were diluted 10 times and the pH value was meas-
ured. Spiked urine samples were prepared by adding an
aliquot of diuretic and appropriate volume of hydroch-
loric acid to negative urine samples after sample prepara-
tion to reach pH 2.0 or 4.0.
3. Results and Discussion
The diuretics selected for investigation can be classified
as strong acids (bumetanide and furosemide), weak acids
(thiazide derivatives) and basic compounds (triamterene
and amiloride) (Table 1). Considering this fact and that
Table 1. The characteristics of selected diuretics [1].
Name Structure
3.9 -
1.4 3.7
8.5 -
6.7 9.5
7.9 9.2
8.7 -
6.2 -
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Simple and Rapid Determination of Diuretics by Luminescent Method
pH of urine of healthy person is 5.5 - 7.0, it was nece-
ssary to study luminescent properties of their different
protolytic forms.
It is seen that at pH of urine of healthy person fu-
rosemide and bumetanide exist as anions in solutions:
furosemide as monoanionic and bumetanide as dianionic.
The spectra of excitation and emission of molecular and
anionic forms of furosemide and bumetanide are shown
in Figures 1 and 2. It is seen that luminescent properties
of protolytic forms of both diuretics differ appreciably.
The intrinsic luminescence of furosemide at рН > 5
significantly decreases due to the destruction of rigidity
of molecule which can be explained by breach of inter-
molecular hydrogen bonds with carboxylic groups of
other furosemide molecules [17].
In contrast to furosemide the highest intensity of lu-
minescence is peculiar to dianionic form of bumetanide,
Figure 1. Excitation and emission spectra of molecular (1, 2)
and monoanionic (1', 2') forms of furosemide. pH = 2.0 (1,
2), pH = 6.0 (1', 2'),
ex = 270 nm,
em = 410 nm.
Figure 2. Excitation and emission spectra of monoanionic (1,
2) and dianionic (1', 2') forms of bumetanide. pH = 2.0 (1, 2),
pH = 6.0 (1', 2'),
ex = 325 nm,
em = 420 nm.
which can be explained by simultaneous deprotonation of
the benzoic acid and of the anilinium ion with con-
sequent formation of stable structure [19]. Dianionic
form of bumetanide exhibits intensive excitation band at
325 nm with emission at 420 nm. So, at pH of urine
bumetanide is characterized with high luminescence
intensity while furosemide demonstrates low intensity
that is the evidence of possibility to determine amount of
In acidic medium (pH 2) furosemide demonstrates
strong intrinsic luminescence with maxima
em 270/
410 nm in contrast to bumetanide. These properties are
served as the basis for the determination of furosemide in
presence of commensurate amount of bumetanide after
acidifying urine sample till pH 2.
Bendroflumethiazide and hydrochlorothiazide at pH of
urine exist in molecular form, while in case of chloro-
thiazide at pH 5.5 - 7.0 molecular and monoanionic
forms coexist in solution. The spectra of excitation and
emission of bendroflumethiazide, hydrochlorothiazide
and chlorothiazide are shown in Figures 3 and 4. The
highest luminescence intensity of bendroflumethiazide is
peculiar to its molecular form (
em 270/400 nm)
which exists at pH 5.5 - 7.0. Anionic form is charac-
terized by negligible emission. Luminescent properties of
protolytic forms of hydrochlorothiazide and chloro-
thiazide do not differ much. Maxima of excitation and
emission for protolytic forms of both diuretics coincide
which can be explained by their identical molecular
structures. Luminescent intensity of chlorothiazide is 5
times more than of hydrochlorothiazide. However, consi-
derable overlapping of excitation and emission spectra
makes their simultaneous determination impossible. At
the same time, determination of bendroflumethiazide in
presence of commensurate amount of hydrochlorothia-
zide is possible due to low luminescent intensity of the
Figure 3. Excitation and emission spectra of molecular (1, 2)
and anionic (1', 2') forms of bendroflumethiazide. pH = 6.0
(1, 2), pH = 10.0 (1', 2'),
ex = 270 nm,
em = 400 nm.
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Simple and Rapid Determination of Diuretics by Luminescent Method 523
At pH of urine amiloride is present in solution in
protonated form, and triamterene in protonated and mo-
lecular forms. Molecular forms of amiloride and triam-
terene are characterized with intensive luminescence
(Figures 5 and 6). Protonated forms which predominate
at pH of urine have less intensive luminescence. At pH
4.0 insignificant bathochromic shift for excitation (Δλex =
6 nm) and emission maxima (Δλem = 4 nm) is observed.
This fact can be explained by protonation of amino-
groupes [18].
Determination of amiloride in mixture with triam-
terene is impossible due to high luminescent intensity of
the latter. However, triamterene can be determined in
presence of any amount of amiloride. Elaborated method
of determination of triamterene as individual substance in
aqueous solution without pre-concentration demonstrates
Figure 4. Excitation and emission spectra of molecular (1, 1')
and anionic (2, 2') forms of hydrochlorothiazide and of mo-
lecular (3, 3') and anionic (4, 4') forms of chlorothiazide. pH
= 6.0 (1, 3), pH = 10.0 (2, 4),
ex = 295 nm,
em = 405 nm.
Figure 5. Excitation and emission spectra of protonated (1,
2) and molecular (1', 2') forms of amiloride. pH = 6.0 (1, 2),
pH = 10.0 (1', 2'),
ex = 285 nm,
em = 420 nm.
Figure 6. Excitation and emission spectra of protonated (1,
2) and molecular (1', 2') forms of triamterene. pH = 6.0 (1,
2), pH = 10.0 (1', 2'),
ex = 360 nm,
em = 440 nm.
the results competitive with that obtained in [18]. The
same LOD and linearity range were achieved without
solid-phase extraction which decreased time of analysis
and simplified the method of determination.
The excitation and emission intensities as function of
diuretic concentration under optimal conditions were
studied. Limit of Detection was calculated as relation of
threefold standard deviation of noise signal to coefficient
of instrumental sensitivity. The results are shown in
Table 2.
3.1. Interfering Influence
The highest sensitivity is inherent for the methods of
determination of furosemide and triamterene as it can be
seen in Table 2. At pH of urine all diuretics except
furosemide show luminescent properties. Furosemide at
pH > 5 does not interfere with determination of other
diuretics but all diuretics except bumetanide interfere
with its determination. Furosemide and bumetanide can
be determined in one sample by adjusting pH. So, at pH
2.0 bumetanide molecule does not emit light which
allows determination of furosemide. On the contrary, at
pH 6.0 bumetanide is characterized with high lumine-
scence intensity, and furosemide molecule does not emit
light. The possibility of determination of one diuretic in
presence of other in commensurate amount with suffi-
cient sensitivity was proved. Results are shown in Table
Due to overlapping of spectra of all diuretics and to
high emission intensity triamterene at optimal conditions
and at pH of urine impedes determination of studied
diuretics, but diuretics in concentration lower or equal to
concentration of triamterene do not impede its determin-
ation. Thus, for determination of other diuretics in pre-
sence of triamterene its preliminary separation is indis-
pesable. n
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Simple and Rapid Determination of Diuretics by Luminescent Method
Copyright © 2013 SciRes. PP
Table 2. Some analytical figures of merit of determination of diuretics by intrinsic luminescence in aqueous solutions. R2 =
0.998 - 0.999.
I = (a ± Δa) + (b ± Δb) C, µg·mL1
Diuretic pH
emmax (
exmax), nm
I a ± Δa b ± Δb
Linearity range,
Іex 9 ± 4 1988 ± 19 6.0
Furosemide 2.0 410 (270)
Іem 14 ± 6 1980 ± 26 9.0
10 - 2000
Іex 3 ± 4 392 ± 4 30
Bumetanide 6.0 420 (325)
Іem 1 ± 2 395 ± 2 20
50 - 5000
Іex 16 ± 5 988 ± 13 20
Bendroflumethiazide 6.0 400 (270)
Іem 17 ± 6 992 ± 15 20
10 - 5000
Іex 12 ± 7 489 ± 7 40
Amiloride 6.0 420 (285)
Іem 16 ± 6 413 ± 6 40
50 - 5000
Іex 1 ± 1 3805 ± 26 0.8
Triamterene 4.0 440 (360)
Іem 2 ± 1 3802 ± 3 0.8
1 - 1000
Table 3. Determination of furosemide (Fur) and bumetanide (Bum) in aqueous solutions.
Added, µg·L1 Found, µg·L1 Recovery, % RSD, %
Fur Bum Fur Bum Fur Bum Fur Bum
0 200 0 2.8 0 1.4 - 8.8
200 0 198.6 0 99.3 0 3.7 -
200 200 203 0 101.5 0 6.9 -
0 200 0 197 0 98.5 - 4.0
200 0 5.1 0 2.6 0 8.9 -
200 200 0 205 0 102.5 - 3.8
3.2. Urine Analysis
Determination of diuretics in urine is complicated due to
the influence of matrix which contains a variety of
organic substances. Most of these substances exhibit high
absorbance in the ultraviolet region [20] and have strong
background luminescence that interferes with the direct
determination of diuretics. As a result, the urine must be
diluted and the fluorescence intensity should be meas-
ured at the maximum of the highest excitation wave-
length where the urine exhibits low absorbance.
Dilution in 10 times and measuring of intensity signal
versus blank urine was proved to be effective in order to
avoid an interfering influence of matrix and distortion of
the spectra. As it was established earlier the highest
luminescence intensity was inherent to molecular forms
of triamterene and amiloride (pH > 8.0 and 9.5, res-
pectively), but alkalization of urine till pH 9.5 - 10.0
leads to appearance of turbidity. Thus, to avoid addi-
tional step in sample preparation including filtration
determination of these diuretics was realized at pH 4.0 at
which both diuretics exist in protonated forms.
As it can be seen from Figure 7 triamterene in urine is
characterized by high intensive excitation and emission
with maxima at 370 and 440 nm, respectively.
Triamterene as individual substance can be determined
in urine by either excitation or emission intensity with
LOD obtained 5 and 6 µg·L1, respectively. Considering
the dilution of urine in 10 times, LOD is higher than that
in aqueous solutions but is still enough to detect triam-
terene with necessary sensitivity. The linearity range is
Simple and Rapid Determination of Diuretics by Luminescent Method 525
20 - 500 µg·L1.
Amiloride does not exhibit luminescence in urine (Fi-
gure 8) which can be explained by binding with urine
components such as derivatives of kynurenine, xanthure-
nic and folic acids [20]. As individual substance it can be
determined by excitation spectra with LOD 30 µg·L1 and
linearity range 200 - 2000 µg·L1. The presence of
triamterene makes determination of amiloride by excita-
tion spectra impossible since their maxima coincide.
Overlapping leads to the increase of total signal intensity.
However, determination of amiloride in presence of any
amount of triamterene is still manageable by spectro-
photometry, by intrinsic absorbance at 360 nm (Figure
9), in area where other diuretics in urine do not absorb.
The linearity range is 50 - 1000 µg·L1 with LOD 10
µg·L1 obtained. Determination of triamterene in pre-
sence of any amount of amiloride is possible by its emis-
sion spectrum at wavelength 440 nm.
High recoveries of amiloride and triamterene from
Figure 7. Excitation and emission spectra of triamterene in
urine. C = 20 - 500 µg·L1. pH = 4.0.
ex = 360 nm,
em = 440
Figure 8. Excitation spectrum of amiloride in urine С = 200
- 2000 µg·L1. рН = 4.0.
ex = 285 nm,
em = 420 nm.
Figure 9. Spectrum of absorbance of amiloride in urine. C =
50 - 1000 µg·L1. pH = 4.0.
= 360 nm.
human urine and low relative standard deviations (Table
4) are the evidence of accuracy and reproducibility of the
method given. Retaining of necessary specifity on
reduction the analysis time compared with that proposed
in [18] owing to avoiding solid-phase extraction is im-
portant achievement of our work.
Bumetanide, furosemide and bendroflumethiazide were
proved to exhibit high luminescence under optimal con-
ditions in aqueous solutions. The influence of complex
urine matrix leads to disappearance of luminescent pro-
perties of these diuretics at optimal conditions. Lumi-
nescence quenching probably can be explained by their
binding with components of urine, supposedly with ami-
noacids [20]. So, determination of bumetanide, furo-
semide and bendroflumethiazide assume preliminary
sample preparation of urine.
4. Conclusions
Intrinsic luminescent properties of different protolytic
forms of furosemide, bumetanide, bendroflumethiazide,
amiloride and triamterene are taken as a principle of
sensitive determination of the diuretics in aqueous so-
lutions. The simplicity and rapidity of method make it a
useful tool in determination.
Techniques elaborated for determination of triam-
terene in presence of other diuretics and furosemide in
presence of commensurate amount of bumetanide allow
enhancing specifity of analysis. Other studied diuretics
can not be determined in presence of triamterene which
requires its preliminary separation.
Dilution of urine and measuring the signal versus
blank solution allowed avoiding matrix influence in case
of triamterene and amiloride analysis and elaborating
simple, selective and sensitive method for their deter-
mination. Other diuretics suppose to be binded with com-
ponents of urine resulting in luminescence quenching
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Simple and Rapid Determination of Diuretics by Luminescent Method
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Table 4. Analytical recoveries of amiloride and triamterene from human urine.
Amiloride Triamterene
µg·L1 Found*a, µg·L1 Recovery,
% RSD, %
225 227 100.9 8.7 228 101.3 5.7 22.5 22.7 100.9 6.2
400 397 99.2 6.2 402 100.5 6.4 90 88.6 98.4 5.9
1000 998 99.8 6.4 1000 100.0 6.5 250 252 100.8 5.8
1500 1508 100.5 5.3 1510 100.6 5.5 400 396 99.0 7.6
*Average of 3 measurements. aQuantity found by luminescent method. bQuantity found by spectrophometry.
which demands preliminary sample preparation of urine.
Triamterene in urine can be determined in presence of
studied diuretics by emission intensity with high sen-
sitivity. Simple method of amiloride determination in
human urine was proposed. It does not include long sam-
ple preparation but provides high specifity of analysis
with sufficient sensitivity. The reduction of time of ana-
lysis due to avoiding sample preparation merits the tech-
niques proposed.
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