Talanta

ELSEVIER Talanta 42 (1995) 1849-1856

Fluorimetric determination of aminoglycoside antibiotics

using lanthanide probe ion spectroscopy
M. Rizk '~, Y. E1-Shabrawy a,., N.A. Zakhari a, S.S. T o u b a r '~, L.A. Carreira b
Anal Chem. Dept., Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt
Chemistry Dept., University of Georgia, Athens, GA, 30602, USA
Received 20 January 1995; revised 10 May 1995; accepted 12 May 1995

Abstract

The application of probe ion fluorimetry has succeeded in the microdetermination of six aminoglycoside
antibiotics: neomycin, streptomycin, gentamicin, tobramycin, amikacin and kanamycin as sulfate salts in
pure form and in some pharmaceutical preparations. The method is based on the reaction of Eu 3+ ions with
aminoglycosides through amino and hydroxy groups. Such interactions enhance the intensity of the 616 nm
fluorescence emission of the Eu 3+ ion. The fluorescence at 592 nm comes from a non-hypersensitive
transition and is not affected by the ligand which is bound to the probe ions. The intensity ratio R, defined
as I~92/16~6 was used to determine the amount of free and bound europium ions. A linear relationship
between bound europium ions and aminoglycoside was found within the concentration ranges 20-100 ppm
for neomycin, 5-60 ppm for streptomycin, and 10-70 ppm for gentamicin, tobramycin, amikacin, and
kanamycin as sulfate salts. The percentage recoveries ranged from 99.22 to 101.07, with standard deviations
ranging from + 1.5 to +4.38. The relative stability constants ranged from 5 x 103 to 2 x 104. The optimum
reaction conditions were studied and the results obtained compared favourably with the fluorimetric method
using fluorescmine reagent.

Keywords: Probe ion fluorimetry; Aminoglycoside antibiotics; Europium ions, Pharmaceutical analysis

1. Introduction Lanthanide ion probe spectrofluorometry

(LIPS) was introduced in 1979 by Horrocks
Numerous procedures are available for the and Sundnick [23]. This technique employs two
determination of aminoglycoside antibiotics emission lines of Eu 3+ located at 592 nm and
such as spectrophotometry [1-4], densitometry 616 nm. The 616 nm emission line is produced
[5], fluorimetry [6,7], polarography [8], gas by a hypersensitive transition [24], ~Do ~ 7F2,
chromatography [9], radioimmunoassay [10], which is normally a forbidden transition, but
H P L C [11,12], MS [13], and N M R [14]. Many interaction with ligands makes the transition
of these methods have been reviewed [15-19]. allowed and enhances the intensity of its emis-
The official methods usually involve microbio- sion. The fluorescence at 592 nm comes from a
logical procedures [20-22]. non-hypersensitive transition and is not affected
by the ligand which is bound to the probe ion
[25], Thus, the ratio of these intensities can be
* Corresponding author. Present address: Chemistry
Dept., University of Georgia, Athens, Georgia, 30602, used to determine free and ligated Eu 3+.
USA. Fax: (706) 542-9454; e-mail: yaser@sunchem. The applications of probe ion spectroscopy
chem.uga.edu, in pharmaceutical analysis are limited in spite

0039-9140/95/$09.50 1995 Elsevier Science B.V. All rights reserved

SSD I 0039-9140(95)01640-6
1850 M. Rick et al. / Talanta 42 (1995) 1849-1856

of the availability of the method for the deter- (2) I E - 4 M , I E - S M and 2.5E - S M Eu 3
mination of many pharmaceutical compounds. ion solutions were prepared by dissolving
Europium ions have been used for the fluori- an appropriate amount of europium
metric determination of tetracycline [26,27], di- chloride hexahydrate in 11 of deionized
clofanec sodium [28] and theophylline [29] in water, and adjusting to pH 5.5 using hy-
blood serum and in pharmaceutical prepara- drochloric acid solution.
tions. In this article, we describe a spectro- (3) Aqueous solutions of pH 2, 5.5, 8, and
scopic method for the determination of 10 were prepared by serial dilution of
aminoglycoside antibiotics in pure form and in hydrochloric acid and sodium hydroxide
pharmaceutical formulations using the LIPS with deiionized water, and pH adjust-
spectrofluorometric technique. ment hydrochloric acid solution or
sodium hydroxide solution using a
Fisher pH meter.
2. Experimental
2.4. Procedure
2.1. Apparatus A. Calibration lines
An aliquot volume of standard aminogly-
A Lambda Physic E M G 102 XeCI excimer coside antibiotic solution equivalent to 0.2-
laser and a FL 3002 tunable dye laser were 1.0 mg neomycin sulfate, 0.05-0.6 mg
used in tandem to supply a high intensity tun- streptomycin sulfate, and 0.1-0.7 mg gentam-
able source. The average output of the XeC1 icin sulfate, tobramycin sulfate, amikacin sul-
excimer was 1.6W ( 1 0 H z repetition rate) at fate and kanamycin sulfate was transferred into
308 nm. A tunable ultraviolet emitting dye a 10 ml calibrated flask. 1 ml of 2.5E -5 M Eu 3
laser was used to provide an excitation source solution was added to the neomycin sulfate and
o f 394.3 nm, which corresponds to a resonant streptomycin sulfate, 1 ml of 1E -a M Eu 3 so-
absorption transition o f the Eu metal ions. The lution was added to the gentamicin sulfate and
Eu fluorescence was collected and frequency tobramycin sulfate, and 1 ml of 1E - s M Eu 3
analyzed with a Spex 1877 triple monochroma- solution was added to the amikacin sulfate and
tor. The fluorescence was detected by a charge kanamycin sulfate. 2 ml o f a hydrochloric acid
coupled detector (cod 9000 Photometric Ltd.). solution of pH 5.5 was added and brought to
The recorded curves were fitted with Lab Calc volume with deionized water. The fluorescence
(Galactic Software) using a gaussian model. was measured from 576 to 633 nm (excitation at
The areas under the two peaks at 592 nm and 394.3 nm). The curves were integrated using a
616 nm were integrated and the ratio of the gaussian model and the area under the two
two peaks was taken as the R value of the peaks at 592 nm and 616 nm was taken as the
sample. R value of the sample.

2.2. Materials B. For injections

An accurate volume of the mixed contents o f
Neomycin sulfate, streptomycin sulfate, gen- three vials, equivalent to 10 mg of drug, was
tamicin sulfate, tobramycin sulfate, amikacin transferred into a CM (carboxylic acid) ion
sulfate and kanamycin sulfate were obtained exchange resin column [10,31] loaded with wa-
from Sigma and the various dosage forms from ter-hydrochloric acid solution pH 2, and then
commercial sources. Europium(Ill) chloride eluted with 25 ml of water-hydrochloric acid
was from Aldrich. Hydrochloric acid, chloro- solution pH 2. The column was washed with
form and ammonia solutions were Baker ana- 25 ml of pH 7 deionized water. The elution and
lyzed reagents. washing water were discarded. Aminoglycoside
was eluted using 30 ml of aqueous pH 11 solu-
2.3. Reagents tion and 10 ml of deionized water into a 50 ml
calibrated flask. The solution was mixed well
All chemicals were analytical reagent grade. and its pH adjusted to about 5.5 using hy-
The following reagents were used: drochloric acid solution; it was brought to
(1) Stock solutions of aminogycosides were volume with deionized water. 2 ml of this solu-
freshly prepared as 0.5 mg ml -~ in deion- tion was assayed as described in the previous
ized water. section.
 M. Rizk et al. / Talanta 42 (1995) 1849-1856 1851
i

I
.2 2

e-
m
1.5
<
E
tad
1
1
0.5

/~ c).&)()5 0.6Ol
Europium ion cone.,
Fig. I. Effect o f Eu 3 ions on the emission ratio of 1 x 10 - a M neomycin sulfate.

C. For tablets The use of lanthanide ions as fluorescent

Twenty tablets were weighed and powdered. probes has been well documented [32]. The
To the quantity of powder equivalent to europium metal ion has been the lanthanide of
500mg was added 10ml of deionized water, choice in most applications and its spectral
and the mixture sonicated for 20 min. It was features are widely documented [33]. This spe-
then filtered into a 100 ml calibrated flask, the ciation information is uniquely encoded in the
powder washed three times each with 5 ml experimental Eu 3+ profile. Fig. l shows a typi-
water, and the combined filtrate mixed well cal spectral titration plot o f Eu 3+ using
and brought to volume with deionized water. I E -4 M aminoglycoside (neomycin sulfate). In
2 ml o f the filtrate was transferred into a CM the early stages of the titration process there is
ion exchange column and the procedure was only a small amount of fluorescent metal probe
completed as described in the previous section. ion compared to the available binding sites,
and hence the majority of metal ions are bound
D. For ointment to the ligand. Consequently, the hypersensitive
The contents of two bottles o f ointment were emission band at 616nm is larger than the
weighed and mixed. A quantity o f ointment non-hypersensitive band at 592 nm, and the
equivalent to 20 mg of drug was transferred intensity ratio R is small and close to the lower
into a separating funnel, and 50 ml of ether limit value, Ls. As the titration progresses the
was added. Gentamicin was extracted three relative amount of total bound species gradu-
times with pH 8 water. The pH of the extract ally increases. Consequently, the intensity ratio,
was adjusted to pH 5.5 using hydrochloric acid R, gradually increases as more metal ions are
solution and brought to volume (100ml) with added to the system. At the end of titration the
deionized water. 2 ml of this solution was as- ratio R is close to its upper limit value, La, and
sayed as described in Section 2.4 A. the concentration of Eu 3 is much greater than
the concentration of ligand. The optimum con-
centration of Eu 3+ ions in a such study is that
3. Results and discussion required to produce a ratio value R between Ls
and La. A ratio value of around unity has been
3.1. Lanthanide ion probe shown to be the most sensitive for this type of
measurement [25,34]. All the aminoglycoside
In a study of any probe, the most fundamen- antibiotics behave similarly to neomycin sul-
tal and necessary characteristic is the ability to fate, except in the amount of Eu 3+ ions added
determine quantitatively the concentration of which is due to the differences in the molecular
the free and bound metal ions simultaneously. weights.
1852 M. Rizk et al. / Talanta 42 (1995) 1849-1856

2000 -

1500
._=
o

,ooo /f ,!!,,k i:, A \\

,,

500
I~ ."",'!.4~f
~ ,L.-~
0,% ,'//
', ~ "-v~.~':L/
", ",,..\
':',
YL,
,
,
r ,,.,:
I "<'~'-L,
. . . . '! . . . . | . . . . i . . . . I . . . . i . . . . i

580 590 600 6'0 620 630

Wave length
Fig. 2. Emission spectra of 1 ml of 2.5E-5 M of europium ions: (A) without neomycin sulfate; (B) in the presence of
600 lag neomycin sulfate; and (C) in the presence of 900 p.g neomycin sulfate, in a total volume of 10ml.

Suitable Eu 3 ion concentrations for such a titration of 1 ml of I x 1 0 - 4 M neomycin sul-

study were found to be 2.5E -5 M Eu(III) ions fate with 1 x 1 0 - 4 M Eu 3 ions.
for the determination of neomycin sulfate and
streptomycin sulfate, 1E - 5 M Eu 3 for the de- 3.4. Effect of time and temperature
termination of gentamicin sulfate and to-
bramycin sulfate, and I E - 4 M E u 3+ for the The complex formation between Eu 3 ions
determination of amikacin sulfate and and aminoglycoside was almost instantaneous,
kanamycin sulfate. Fig. 2 shows emission spec- as a stable ratio was produced within 5 rain
tra of Eu 3 ions in the presence and absence of and the complexes were stable for at least 24 h.
neomycin sulfate. Temperature had little effect on the complex
formation and its stability, so the reaction was
3.2. Effect of buffer and pH carried out at ambient temperature.

Fig. 3 shows that the complexes formed were 3.5. Effect of ionic strength
pH dependent and must be controlled between
pH 5 and 6. Acidic buffers such as acetate, Ionic strength is a factor that sometimes
phosphate, MOPS, MES and T H A M buffer cannot be neglected. The effect of ionic strength
systems were not suitable in such a study owing on the spectral titration plot was studied by
to the formation of europium salts or com- varying the concentration of the background
plexes which interfered with the measurement; electrolyte, sodium chloride. The choice of
such observations have been made in our labo- sodium chloride was based on the fact that the
ratory [25]. At a pH below 5, the competition Na ion is very weakly bound, if at all, to the
between the proton and Eu 3+ will decrease the aminoglycoside binding sites, and CI - j is al-
formation of Eu-aminoglycoside complex, and ready in the system as a counter ion that comes
in neutral solution Eu(OH) 2+ is formed which from dissolving EuCI3. Three titration plots
interferes with the measurement. Adjusting the correspond to 0.0, 0.01 and 0.1 M sodium chlo-
initial pH of the Eu 3+ solution to pH 5.5 and ride added as background electrolyte. The addi-
using 2 ml of aqueous hydrochloric acid solu- tion of background electrolyte will decrease the
tion at pH 5.5 kept the pH of the mixtures total concentration of bound species of the
between 5 and 6. probe ion. It should be emphasized here that
this decrease is not due to the competition effect
3.3. Molar ratio of sodium ion (similar to proton competition)
to occupy the same set of aminoglycoside sites,
Eu is bound with aminoglycoside in a 1:1 but purely due to electrostatic forces as previ-
molar ratio. Fig. 4 shows the spectroscopic ously reported with humic acid substances [34].
 M. Rizk et al./ Talanta 42 (1995) 1849-1856 1853

1.9
O
"~ 1.8

1.7

-~ 1.6
e-,
.o
.~ 1.5

~ 1.4

1.3

3.5 4 4.5 5.5 6 6.5

pH
Fig. 3. Effect of pH on emission ratio of 50 lagml-J neomycin sulfate and 1.00ml of 2.5E-5 M EuJ+ ions.

4. Performance characteristics Susetyo and co-workers [25,35] have devel-

oped a statistical model for the distribution of
The investigated aminoglycosides possess binding sites in humic acid. This model was
amino and hydroxy groups which can form applied to study the spectral titration plot gen-
complexes with metal ions. Evangelista and erated by lanthanide ion probe spectroscopy.
Schapoval [1] have determined these com- They expressed the free and bound Eu 3+ ions
pounds by spectrophotometric determination as a function of experimental parameters. The
of their blue complexes with copper ions at free Eu 3+ ions could be expressed as
634 nm.
M = C M L a ( R -- Ls) (1)
Aminoglycoside antibiotics complex Eu 3
ions, and such ligation leads to a hypersensi- R(LB Ls)
-

tive transition of Eu 3+ ions, and such ligation where M is the concentration of free metal
leads to a hypersenstive transition o f Eu 3+ ions, CM is the total concentration of metal
ions, 5Do--* 7F2 (616 nm). These hypersensitive added, LB is the ratio of the two Eu peaks at
transitions are specific absorption or emission 592 nm and 616 nm when the concentration of
transitions o f lanthanide ions that are ex- Eu 3+ was very large with respect to ligand
tremely sensitive to ligation. The intensity o f concentration, and Ls is the ratio when the
a hypersensitive transition is enhanced in Eu 3+ concentration was very small with respect
the metal-ligand complex relative to that to the ligand concentration. Table I shows LB
transition in aquo ions. Experimentally, this and Ls for each aminoglycoside. R is the ratio
is observed by making the concentration of 1592/I616 of the free and bound metal species
Eu 3 ions small relative to the aminoglycoside which was quantitatively determined from the
concentration, so that the majority o f the experimentally observed parameters.
metal ions are complexed with aminogly- The bound Eu 3+ ion concentration could be
cosides. Such effects are most obvious from expressed as
the observation o f several emissions of Eu 3
ML = CM- M (2)
ions with neomycin sulfate concentrations
(Fig. 2). The absolute emission intensity is not Calibration lines were calculated by plotting
important here as we are concerned only with M L against the concentration of aminogly-
the change in the integrated emission ratio R coside. It was found that there was a linear
(1592/16~6) due to the aminoglycoside antibi- relation between the concentration of amino-
otics. These R values decrease upon the addi- glycoside and the concentration of bound Eu 3
tion of antibiotics owing to the enhanced ions within the ranges shown in Table l, with
emission at 616 nm relative to the emission at standard deviations from 4.338 to 1.50 ppm.
592 nm. The 592 nm emission is not effected Table l also shows regression equations and
by ligation. correlation coefficients o f aminoglycosides us-
1854 M. Rizk et al./ Talanta 42 (1995) 1849-1856

/ 0 ~ 0
.o_ /
w 0

.o_
~

1.5
2

J
i,i . . . . t i . . , ,. i 1 - - - i

0.5 1 1.5 2 2.5

Molar Ratio
Fig. 4. Spectroscopic titration of 1 ml of 1 10 -4 M neomycin sulfate with 1 x 10 -4 M Eu 3+ ions.

ing the proposed method. This has proved to Table 2 shows the assay results of aminogly-
be an accurate method for the determination of cosides by the proposed method and
aminoglycosides. As compared with conven- fluorescamine fluorimetric methods [6]. The
tional organic fluorescent probes [6,7], fluores- proposed method, when compared to the
cent lanthanide chelates can offer several official microbial assays, offers advantages with
advantages. They have narrow-banded emis- regards to rapidity, simplicity and precision, so
sion lines well distinguished from any dis- we compared these results to the fluorimetric
turbing background. Chelates have method using fluorescamine. The calculated
exceptionally wide Stokes' shifts, over 200 nm, student's t and variance ratio F tests were less
while with fluorescamine it is only 50-70 nm. than the respective tabulated one at P = 0.05,
In addition to spectral resolution, their emis- so the results obtained by the proposed method
sion can be distinguished from any organic have proved to be in good agreement with
background interference owing to the very long those obtained by the reference fluorimetric
fluorescence decay time ( ~ 110 las) associated method.
with Eu 3 fluorescence which makes them an Results of the determination of aminogly-
ideal choice for a fluorimetric system [36]. cosides in different dosage forms by the probe

Table I
Regression data, correlation coefficients, stability constants of Eu-aminoglycoside complexes, concentrations of Eu used
for calibration, and concentration ranges of aminoglycoside antibiotics determined by the proposed spectroscopic probe
ion method

Aminoglycoside Regression data a Cone. b La Ls

sulfate salt range
Co CI r k CM

Neomycin 3.59E - s 4.77E -9 0.99681 2.1E 4 2.5E -'~ 20-100 2.30 0.31
Streptomycin 1.08E -9 1.91E - s 0.99983 1.3E 4 2.5E --~ 5-60 2.30 0.31
Gentamicin 4.78E- 7 2.82E q - s 0.99086 3. I E 3 1.0E-4 10-70 2.55 0.61
Tobramyein 7.12E -7 4.29E - s 0.99892 2.5E ~ 1.0E -4 10-70 2.55 0.61
Amikaein 1.13E -7 2.48E -9 0.99857 3.5E 3 1.0E -5 10-70 2.17 0.47
Kanamyein I . I I E -7 3.52E -9 0.99935 5.7E ~ 1.0E -5 10-70 2.17 0.47

Co is the intercept, c~ is the slope, r is the correlation coefficient, K is the stability constant of the Eu-aminoglyoside
complex, CM is the molar concentration of Eu ~+ ions used for calibration, L a is taken to be the ratio of the two Eu ~+
peaks in the absence of aminoglycoside, Ls is taken to be the ratio when the Eu ~+ concentration was very small with
respect to a large ligand concentration.
"Average of at least seven triplicate determinations within the concentration range.
b Concentration ranges were calculated in ppm.
 M. Ri-.k et aL / Talanta 42 (1995) 1849-1856 1855

Table 2 sUch as additives, stabilizers a n d a n t i o x i d a n t s

Assay results of aminoglycoside antibiotics in bulk using
o f tablets, a m p o u l e s a n d o i n t m e n t s , are re-
the proposed method and the reference method [6]
m o v e d b y this p r o c e d u r e a n d d o n o t interfere
Aminoglycoside % Recovery ~, 4-SD with the r e c o m m e n d e d m e t h o d . T h e y were sep-
sulfate salt a r a t e d from a m i n o g l y c o s i d e s in an ion ex-
Proposed method Reference method b c h a n g e resin ( c a r b o x y l i c acid resin) by a q u e o u s
buffer at p H 2 [30,31]. T h e p r o p o s e d p r o c e d u r e
Neomycin 100.16 + 1.05 100.04 4- 1.48
t=0.175 (2.179) F=1.987 (4.28) is as sensitive as the fluorescamine fluorimetric
m e t h o d [6], a n d gives g o o d a g r e e m e n t with it.
Streptomycin 98.61 + 3.57 99.25 4- 1.86
t=0.421 (2.179) c F=3.684(4.28) T h e p r o p o s e d m e t h o d is simpler a n d m o r e
rapid, a n d the reaction p r o d u c t s are m o r e
Gentamicin 100.82 + 2.30 99.00 4- 1.82 stable t h a n for c o n v e n t i o n a l f l u o r o m e t r i c meth-
t = 1.660 (2.179)c F = 1.689 (4.28)c
ods. M o r e o v e r , the d e t e r m i n a t i o n b o u n d Eu 3
Tobramycin 100.26 + 2.23 99.68 + 2.28 using the relative intensity r a t i o R as a function
t=0.481 (2.179) c F = 1.045 (4.28)
o f the c o n c e n t r a t i o n o f a m i n o g l y c o s i d e antibi-
Amikacin 98.81 4- 4.52 97.82 + 2.66 otics is m o r e reliable, a n d has less e x p e r i m e n t a l
t=0.500 (2.179) c F=2.875 (4.28) c e r r o r ( a b o u t + 5 % ) , t h a n that using the a b s o -
Kanamycin 100.19 + 1.50 99.14 4- 2.49 lute emission intensity in c o n v e n t i o n a l o r g a n i c
t = 0.956 (2.179) c F = 2.756 (4.28) ~ fluorescent m e t h o d s . In c o n v e n t i o n a l o r g a n i c
f l u o r o m e t r i c m e t h o d s the a b s o l u t e emission
a Average of at least seven triplicate determinations.
b Fluorometric determination of antibiotics using value is s t r o n g l y effected b y m a n y systemic
fluorescamine [6]. errors d u e to b l a n k a n d a n a l y t e interferences.
Values in parentheses are theoretical values at P = 0.05. M o r e o v e r , the a b s o l u t e emission intensity is
highly d e p e n d e n t o n the intensity o f the excita-
ion m e t h o d a n d the fluorescamine fluorimetric tion source, the q u a n t u m yield o f the c o m -
m e t h o d are p r e s e n t e d in T a b l e 3. T h e p r o p o s e d plexes which is low c o m p a r e d to the
p r o c e d u r e was selectivity e n h a n c e d by an ion E u 3 - c o m p l e x (-~0.18) [37], a n d the d e t e c t o r
e x c h a n g e resin r e s o l u t i o n o f the interferences, used for the m e a s u r e m e n t r a t h e r t h a n the
since it is well k n o w n that Eu 3+ ions are fluorescence o f the a n a l y t e [30]. Thus, the p r o -
s t r o n g l y b o u n d by c h a r g e d oxygen d o n o r p o s e d p r o c e d u r e can be used for r o u t i n e a n a l y -
a t o m s such as c a r b o x y l i c o r p h e n o l i c g r o u p s sis a n d quality c o n t r o l o f a m i n o g l y c o s i d e s in
[29]. I n g r e d i e n t s o t h e r t h a n a m i n o g l y c o s i d e s , pharmaceutical preparations.

Table 3
Assay results of aminoglycoside antibiotics in some pharmaceutical preparations by the proposed method and the
reference method

Drug % Recovery, +SD ~

Proposed method Reference method b

Neomycin Tablets 500 mg 100.99 + 0.36 98.98 + 2.16

(Memphis Co., Egypt)
Gentamicin Oint., 0.1% 104.74 4- 1.6 97.54 4- 1.98
(Memphis Co., Egypt)
Gentamicin Amp. 20 mg 101.22 4- 2.39 98.23 + 1.71
(Memphis Co., Egypt)
Gentamicin Amp. 80 mg 102.53 4- 1.03 98.93 4- 1.98
(Memphis Co., Egypt)
Nebcin Amp. 20 mg Tobramycin 103.49 4- 0.64 99.68 + 2.28
(Lily France S.A. France)
Amikin Amp. I g Amikacin 101.20 4- 1.66 99.68 4- 0.87
(Briston Co., USA)
Kanamycin vial, 1 g per vial 99.27 + 0.41 99.52 _+ 1.62

"Average of at least three triplicate determinations, calculated relative to nominal content.

b Fluorometric determination of aminoglycoside antibiotics using fluorescamine [6].

TAL ~ IZ,~
1856 M. Rizk et al. / Talanta 42 (1995) 1849-1856

5. Conclusion [17] K. Florrey, Analytical Profiles of Drug Substances,

Academic Press, London, Vol. 9, 1980, p. 295.
[181 K. Florrey, Analytical Profiles of Drug Substances,
From the foregoing discussion, the Eu 3 Academic Press, London, Vol. 12, 1983, p. 37.
probe ion fluorimetric technique has been ap- [19] K. FIorrey, Analytical Profiles of Drug Substances,
plied to the determination of six aminogly- Academic Press, London, Vol. 16, 1987, p. 505.
cosides in pure form and in pharmaceutical [20] The United State Pharmacopeia X X I I - National
Formulary XVll, Rockville, MD, 1990.
preparations. Ion exchange was carried out for
[21] British Pharmacopeia, Her Majesty's Stationary
resolution of aminoglycoside antibiotics from Office, London 1993.
other additives such as EDTA, sodium citrate, [22] European Pharmacopeia, Maisonneuve, S., 57 Sainte-
phenol, and other injection and tablet additives. Ruffin, France, 1975.
[23] W. Dew, Horrocks and D.R. Sundnick, J. Am. Soc.,
101, (1979) 334-340.
[24] W.T.T. Carnall, J.V. Beitz, H. Crosswhite, K. Rajank
References and J. B, Mann, In S.P. Sinha (Ed), Systematic and
Properties of the Lanthanides, D. Reidal, Boston,
[1] R.C. Evangelista and E.E.S. Schapoval, Rev. Cleric. MA, 1983, pp. 389.
Farm., 5 (1983) 21-27. [25] W. Susetyo, J.C. Dobbs, L.A. Carreira, L.A. Azarraga
[2] P.R. Bontchev, P. Papzova, M. Confino and D. Di- and D.M. Grimm, Anal. Chem., 62 (1990) 1251-1221.
mova, Mikrochim. Acta, 111 (1984)459-465. [26] L.M. Hirschy, E.V. Dose and J.D. Winefordner, Anal.
[3] N.M. Alykov, Zh. Anal. Khim., 39 (1984) 1425-1427. Chem. Acta, 147 (1983) 311-316.
[4] Nashaat A. Zakhari, Anal. Lett., 23 (1990) 1843- [27] L.A. Files, L. Hirschy and J.D. Winefordner, J.
1865. Pharm.-Biomed. Anal., 3 (1985) 95-100.
[5] M, Breilnger, H. Paulus and W. Weigrebe, Dtsch. [28] L.A. Carreira, M. Rizk, N.A. Zakhari, S.S. Toubar
Apoth. Zth., 120 (1980) 1699. and Y. EI-Shabrawy, in press.
[6] J. Kusnir and K. Barna, Z. Anal. Chem., 271 (1974) [29] L.M. Perry and J.D. Winefordner, Talanta, 37 (1990)
288. 965-969.
[7] N.M. Alyokov, Zh. Anal. Khim., 36 (1982) 1387. [30] G.H. Ford, M.E. Bergy, A.A. Brooks, E.R. Garrett, J.
[8] M.M. Ayad and M. Yousef, Analyst, 110 (1985) 963. Alberti, J.R. Dyer and H.E. Carter, J. Am. Chem.
[9] K. Tsuiji and J.H. Robertson, Anal. Chem., 42 (1970) Soc., 77 (1955) 5311.
1661. [31] H. Galina and B.N. Kolarz, J. Chromatogr., 179
[10] S.G. Thompson and J.F. Burd, Antimicrob. Agents (1979) 173-175.
Chemother., 18 (1980) 264. [32] W. Dew, Horrocks and D.R. Sundnick, Acc. Chem.
[11] D.M. Barends, J.S. Van der Sandt and A. Hulshoff, J. Res., 14 (1981) 384.
Chromatogr. Biomed. Appl., 8 (1980) 201. [33] J.C. Dobbs, W. Susetyo, F.E. Knight, M.A. Castles,
[12] P. Gambardella, R. Punziano, M. Gionti, C. L.A. Carreira and L.V. Azarraga, Intern. J. Environ.
Guadalupi, G. Mancini and A. Mangia, J. Chro- Anal. Chem., 37 (1989) I.
matogr., 348 (1985) 229-240. [34] W. Susetyo, L.A. Carreira, L.A. Azarraga and D.M.
[13] B.V. Rozynov, Yu.V. Zhdanovich, A.D. Kuzovkov, Grimm, Fresenius" Anal. Chem., 339 (1991) 624-
R.N. Elizabeth and A.S. Tikhonova, Antibiotiki, 25 635.
(1980) 652. [35] J.C. Dobbs, W. Susetyo, F.E. Knight, M.A. Castles,
[14] N.P. Reuter, A.C. Hawke, E. Lewis, T.G. Alexander, L.A. Carreira and L.V. Azarraga, Anal. Chem., 61
E. Mazzola and A. Aszalos, J. Assoc. Off. Anal. (1989) 483-488.
Chem., 65 (1882) 1413. [36] 1. Hemmila, Scand J. Clin. Invest., 48 (1988) 389-400.
[15] K. Florrey, Analytical Profiles of Drug Substances, [37] E. Soini and T. Lougrem CRC Crit. Rev. Anal.
Academic Press, London, Vol. 6, 1977, p. 259. Chem., 18 (1987) 105.
[16] K. Florrey, Analytical Profiles of Drug Substances, [38] J.E. Ingle and S.R. Crouch, Spectrochemical Analysis,
Academic Press, London, Vol. 8, 1979, p. 399. Prentice-Hall, Englewood Cliffs, New Jersey, 1988.