Sei sulla pagina 1di 34

213

CHAPTER 8
SPECTROPHOTOMETRIC DETERMINATION OF CEPHALOSPORINS IN
PHARMACEUTICAL SAMPLES
8.1 INTRODUCTION
8.2 ANALYTICAL CHEMISTRY
8.3 APPARATUS
8.4 REAGENTS AND SOLUTIONS
8.5 PROCEDURES
8.6 RESULTS AND DISCUSSION
8.7 APPLICATIONS
8.8 CONCLUSIONS
8.9 REFERENCES
214
8.1 INTRODUCTION
Cephalosporins are penicillinase-resistant antibiotics with significant activity
against both gram-positive and gram-negative bacteria. The key intermediate for
semisynthetic production of a large number of cephalosporins is
7-aminocephalosporanic acid (7-ACA) [1]. A few thousand semisynthetic
cephalosporins have been described in the scientific literature, but only a small
number of those have shown clinical importance.
Cephalosporin C is an antibiotic isolated in 1956 from a species of
Cephalosporium, possesses greater acid and penicillinase stability than other -lactam
containing antibiotics but much weaker antibacterial action [2-4]. The antimicrobial
activity can be enhanced greatly by N-acylation of 7- aminocephalosporanic acid,
which has been obtained in a low yield by mild acid hydrolysis of cephaiosporin C
[5]. However, 7-ACA has not been sufficiently available to evaluate fully this
interesting class of antibiotics.
Cephalosporin C was measured by a cup agar diffusion assay of Salmonella
gallinarum grown on pH 6 nutrient agar medium containing penicillinase
(1250 mL
-1
) to destroy penicillin N [6]. The cephalosporin structure is well
established as a mono-release prodrug nucleus owing to rapid elimination of the
3-substituent following enzyme-catalyzed scission of the -lactam ring. Examples are
known where antimicrobial (quinolones) [7] and cytotoxic components (melphalan,
doxorubicin) [8] have been incorporated at this position.
Deacetoxycephalosporin C synthase (DAOCS) is an iron- and -ketoglutarate-
dependent oxygenase that catalyzes the ring expansion of penicillin N to
deacetoxycephalosporin C (DAOC) in all cephalosporin producing microorganisms
[9-12]. In bacteria, the subsequent hydroxylation of DAOC to deacetylcephalosporin
C (DAC) is catalyzed by a closely related enzyme deacetylcephalosporin C synthase
(DACS), whereas in the fungus Acremonium chrysogenum (previously
Cephalosporium acremonium), the activities of expandase and hydroxylase reside in a
single functional protein [10].
215
Several research groups have reported on the narrow substrate specificity and
lack of detectable activity of expandase on inexpensive and available penicillins such
as penicillin V and G [9,13,14]. Chemical ring expansion of penicillin G plus
enzymatic removal of the phenylacetyl side chain is currently being used in industry
to obtain 7-aminodeacetoxycephalosporanic acid (7-ADCA) that is used for the
manufacture of semisynthetic cephalosporins. However, this chemical process
requires several steps and is expensive and polluting [15]. A biological route requiring
only two enzymatic steps (ring expansion and deacylation) might replace the chemical
process, thereby reducing costs and environmental problems.
Cefotaxime, ceftriaxone, cefadroxil and cephalexin are -lactam antibiotics
possessing a broad spectrum of antibacterial properties [16,17]. These drugs are found
to be very useful in pre and post operative chemotherapy against infections in
abdominal, pelvic, orthopaedic, cardiac, pulmonary, oesophageal and vascular surgery
[18]. Cefotaxime, ceftriaxone and cefadroxil were also determined in pharmaceutical
preparations [19-23], urine [22,24-27] and human serum [28]. Recently a rapid
development in chromatographic determination methods of pharmaceuticals have
been observed too [29,30].
The hydrolysis of -lactum ring, which is the common feature for
cephalosporins and penicillins, has been achieved by the sodium hydroxide addition.
Major difficulties in the determination of cephalosporins were encountered at the -
lactum ring hydrolysis step [31]. A -lactum enzyme [32] is used for the hydrolysis
of the analyte which reacted with iodate in acid medium and liberates iodine. The
liberated iodine bleaches the violet color species is the basis for the
spectrophotometric determination of the analytes. The reaction mechanism followed
the course similar to the one described for penicillins [33].
8.2 ANALYTICAL CHEMISTRY
Several methods have been reported for the quantitative determination of
cephalosporins. These include fluorimetric [34], polorographic [35], chromatographic
[36-39], isotachophoretic [40] and flow injection chemiluminescence methods [41].
216
Sastry et al. reported spectrophotometric determination of penicillins and
cephalosporins in bulk and in dosage forms [42]. Chloranilic acid formed colored
complexes with penicillins and cephalosporins in dioxane and dioxane-DMF media at
maximum absorption 520 nm.
Issopoulos described cephalosporins, cefaclor, cefazolin, cefotaxime, cefoxitin
and cefamandole nafate in pharmaceuticals and bulk by a spectrophotometric method
[43,44]. The method was based on the reaction with (NH
4
)
6
Mo
7
O
24
and 0.5 M sulfuric
acid and measured the absorbance of the blue colored solution at 810 nm. A linear
relation between the absorbance and anion was observed for all antibiotics studied.
The recoveries and relative standard deviations were 96.7-104.7 % and 0.60-2.08 %
respectively. In the other method molybdophosphoric acid was used as an oxidising
agent for the spectrophotometric determination of 4 cephalosporin derivatives;
cefadroxil (I), cefapirin (II), ceforanide L-lysine (III) and cefuroxime (IV) in pure
form or in pharmaceutical formulations [44]. Beer's law was obeyed up to 100 gmL
-1
for I, up to 60 gmL
-1
for II and IV and up to 80 gmL
-1
for III. The molar
absorptivities were 4.5810
3
, 11.310
3
, 9.810
3
and 10.910
3
Lmol
-1
cm
-1
and the
Sandell sensitivities were 83.3, 39.3, 53.0 and 41.0 ngcm
-2
for I, II, III and IV,
resectively.
Abdel-Razeq described two spectrophotometric procedures for the
determination of three cephalosporins; cefixime trihydrate (I), cefoperazone sodium
(II) and cefotaxime sodium (III) [45]. The first procedure was based on the reduction
of ferric ion into ferrous ion in presence of o-phenanthroline by the mentioned drugs,
which formed a highly stable orange-red ferroin chelate [Fe-(Phen)
3
]
2+
and was
measured at 513 nm. The second procedure was also based on the reduction of
tetrazolium blue in alkaline medium by the above cephalosporins, which formed
purple colored formazan, which was measured at 526 nm. Beer's law was obeyed in
the ranges of 0.4-2.4 and 4-20 gmL
-1
for I, 0.8 - 3.6 and 4 - 24 gmL
-1
for II or 0.4 -
2.4 and 4-16 gmL
-1
for III by ferric-phenanthroline and tetrazolium blue procedures
respectively. The optimum assay conditions and their applicability to the
217
determination of the cited drugs in pharmaceutical formulations were described. The
recoveries of the drugs were 90.7-96.0% from urine and 71.7-78.5% from serum.
Abd El-Sattar et al. reported three simple, rapid and accurate
spectrophotometric methods for the determination of cephalosporins; cefepime
dihydrochloride and cefprozil monohydrate [46]. The 1
st
method was based on the
reaction of the named drugs as n-donors with three acceptors; chloranilic acid (CA),
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and 7,7,8,8-tetracyanoquinodi-
methane (TCNQ), which yielded highly colored radical anions measured at 527 nm,
460 nm and 841 nm, respectively. The 2
nd
method was based on the reaction of each
of the two drugs with ninhydrin in boiling water bath, in presence of pyridine, which
produced a bluish violet product and measured at 566 nm. The 3
rd
method was based
on the reduction of Folin Ciocalteu's reagent (FCR) in alkaline medium by the
investigated drugs into blue colored products and measured at 755 nm. Beer's law was
obeyed for cefepime salt at concentration range of 50-450 gmL
-1
, 20-180 gmL
-1
,
10-60 gmL
-1
, 5-25 gmL
-1
and 10-60 gmL
-1
for CA, DDQ, TCNQ, ninhydrin and
FCR resectively. However, for the cefprozil salt, the concentration range were 50-400
gmL
-1
, 20-140 gmL
-1
, 1-7 gmL
-1
, 2-14 gmL
-1
and 2.5-25 gmL
-1
in the same
order of reagents. The methods were successfully applied to the analysis of the
studied cephalosporins, in either pure form and in pharmaceutical formulations.
Walily et al. reported a spectrophotometric and spectrofluorimetric procedures
for the determination of four penicillins [amoxycillin, bacampicillin, piperacillin and
sultamcillin] and ten cephalosporins [cefadroxil, cefamandole nafate, cefuroxime
axetil or sodium, cefaclor, ceftazidime, ceftizoxime, ceftriaxone, cefoperazone,
cefixime and cefpodoxime proxetil] [47]. Both methods were based on the oxidation
of the antibiotics with cerium(IV) at elevated temperature. The effect of acid
concentration and temperature were studied to optimize the reaction conditions. Each
antibiotic was determined at 317 nm or the cerous inherent fluorescence at 256 and
356 nm for excitation and emission wavelengths respectively. The two procedures
were successfully applied to the assay of these antibiotics in their pharmaceutical
dosage forms.
218
Nabi et al. reported a simple, rapid and sensitive method for the determination
of sodium cefazolin, cefaclor and cefadroxil in pharmaceutical preparations [48]. The
drug -lactam ring was hydrolyzed with sodium hydroxide at 800C for 10-15
minutes. Cooled samples were acidified with 1 M HCl and CCl
4
was added. Aliquots
of each sample were titrated with a standard 0.01 M potassium iodate solution. The
colorless CCl
4
layer gradually developed to a deep red colored as the titration end-
point. The absorbance of the CCl
4
layer was measured at 520 nm. The cephalosporins
were determined in the concentration range of 1-5 mgmL
-1
.
Agbaba et al. described cephalexin, cefixime, ceftriaxone and cefotaxime by
spectrophotometry in bulk and in pharmaceuticals by using the ferrihydroxamate
method [49]. Reaction optimization with respect to reaction time and temperature was
investigated. Using cefotaxime sodium as the model drug with an ester functional
group, it was shown that the method gave equally accurate and precise results even in
the presence of the ester functional group.
Mahrous and Abdel-Khalek described simple, accurate and selective
spectrophotometric method for determination of cephalothin sodium, cefoxitin
sodium and cephaloridine [50]. The methods was based on condensation of
acetaldehyde, vanillin or p-dimethylaminobenzaldehyde with the free thienyl moiety
of these cephalosporins in sulfuric acid medium, which formed colored chromophores
measured at the selected wavelengths. The results obtained are reasonably
reproducible with a coefficient of variation of <0.7 %.
Alwarthan et al. described spectrophotometric assay of certain cephalosporins
based on formation of ethylene blue [51]. The hydrolytic degrdation of antibiotics was
very often used as a preliminary step in the analytical procedures for their
determination. Therefore, a procedure was developed for measuring small amounts of
cefadroxil and cefotaxime in pure samples as well as in formulations. The method was
based on the formation of a vis-absorbing compound with N,N-diethyl-p-
phenylenediamine sulfate (N,N-DPPD) (ethylene blue dye), after the hydrolysis of
cefadroxil and cefotaxime in sodium hydroxide solution, which formed hydrogen
sulfide. The method was selective for cephalosporins, since other -lactam
219
compounds such as penicillins do not give hydrogen sulfide under alkaline hydrolysis.
Variables such as pH, temp, reagent concentration and stability of the colored
products were evaluated. Beer's law was obeyed over the concentration range 0.5-10
gmL
-1
and 0.5-7 gmL
-1
for cefadroxil and cefotaxime resectively. The detection
limit was 0.1 gmL
-1
and 0.05 gmL
-1
for cefadroxil and cefotaxime respectively.
The method was successfully applied to the analysis of some pharmaceutical
formulations.
Sengun and Fedai described a determination of cephalosporins in
pharmaceutical formulations [52]. Cephalothin, cephacetrile, cefamandole,
cefamandole and cefoperazone were determined in pharmaceuticals by treatment with
a Hg(II)-imidazole reagent and measured the absorbances at 325 nm for cephacetrile
and at 345 nm for the other cephalosporins. The relative standard deviation was 0.53-
1.73%, the limit of detection was 8 gmL
-1
for cephalothin and 25.36 gmL
-1
for the
other cephalosporins.
Morelli and Peluso described a spectrometric determination for cephalosporin
[53]. The procedure applied successfully to a wide variety of cephalosporins, also in
pharmaceutical preparations: cephalothin, cefacetrile, cephapirin, cefotaxime,
ceftizoxime, cephaloridine, cefazolin, cefamandole nafate, cephalexin, cefadroxil,
cefoxitin and cefuroxime. The method employed a reaction with ammonium
molybdate in H
2
SO
4
medium. The antibiotic was heated at 91.5C for 15 minutes and
the absorbance of the colored product was measured at 670 nm against a reagent
blank. Beer's law was obeyed up to 125-150 g of cephalosporin in the 5 mL final
solution. The effects of reagent concentration and reaction conditions were discussed.
Abdalla et al. reported a selective spectrophotometric determination of
cephalosporins by alkaline degradation to hydrogen sulphide and the formation of
methylene blue [54]. The method was selective for cephalosporins in the presence of
penicillins.
Abdel Khalek and Mahrous reported a spectrophotometric method for the
determination of some cephalosporins [55]. The drug was boiled with (NH
4
)VO
3
220
solution in H
2
SO
4
for 10 minutes and the absorbance of the color developed was
measured at 750 nm. Beer's law was obeyed in the range 20-100 gmL
1
.
Mahrous and Abdel Khalek presented ninhydrin as a reagent for the
spectrophotometric determination of certain cephalosporins in H
2
SO
4
medium [56].
The method was applied successfully to the analysis of injections. Absorbance was
measured at 458 nm against a reagent blank. Beer's law was valid in the concentration
range of 2.5-30 gmL
1
. The coefficient of variation were <0.88%. Excipients in the
injections did not interfere.
Sengun and Ulas described ceftriaxone in bulk and pharmaceuticals by a
spectrophotometric method based on the reaction with imidazole-HgCl
2
reagent and
measurement of the absorbance was at 370 nm [57].
Sastry et al. described haematoxylin-chloramine-T as a reagent for the
spectrophotometric determination of penicillins and cephalosporins in pure samples
and pharmaceutical preparations [58]. The method was based on acid hydrolysis of
penicillins and cephalosporins with 5M HCl and subsequent treatment with oxidized
haematoxylin. The resulting color exhibited maximum absorption at 555 nm.
Abdalla reported a spectrophotometric method for the determination of some
cephalosporins [59]. The method was based on the hydrolysis of the cephalosporins in
NaOH solution to produce H
2
S and the reaction of the sulfide with N,N-diethyl-p-
phenylenediamine to form ethylene blue. The method was successfully applied to the
pharmaceutical formulations and the results were statistically compared with those
obtained by the official methods and the imidazole and mercury(II) method.
Hosny described a spectrophotometric method for the determination of some
cephalosporins using 2,2'-diphenyl-1-picrylhydrazyl [60]. Beer's law was obeyed in
the range of 5-30, 5-25 and 10-30 gmL
1
for cephalexin, cefadroxil and cephradine
respectively with a maximum absorbance at 520 nm.
221
Helaleh et al. reported a spectrophotometric determination of ceftriaxone and
cephalexin in pharmaceuticals [61]. In this method cephalosporin were treated with
potassium iodate in a moderately acidic medium after the -lactam ring had been
hydrolyzed for 10 minutes with sodium hydroxide at 80C. Beer's law was obeyed in
the concentration range of 20-600 and 20-800 gmL
1
for ceftriaxone and cephalexin
respectively. The correlation coefficients were 1.0000 and 0.9999 respectively.
Al-Momani developed a spectrophotometric determination of selected
cephalosporins in drug formulations using flow injection analysis [62]. In this method
cephalosporins were hydrolyzed for 15 minutes with 0.1M NaOH at 80C and then
oxidized with Fe
3+
in H
2
SO
4
medium, which produced Fe
2+
. The produced Fe
2+
was
then complexed by o-phenanthroline in citrate buffer at pH 4.2 and the red complex
formed exhibited an absorption maximum at 510 nm. The method was successfully
applied to the analysis of pharmaceutical preparations.
Amin and Shama reported vanadophosphoric acid in acidic medium as a
modified reagent for the spectrophotometric determination of cephalexin, cephaprine
sodium, cefazolin sodium and cefotaxime in pure samples and in pharmaceutical
preparations [63]. The method was based on acid hydrolysis of cephalosporins and
subsequent oxidation with vanadophosphoric acid. The resulted solution exhibited
maximum absorption at 516 nm. The effect of reaction conditions were investigated.
Beers law was obeyed over a concentration range of 0.445 gmL
1
. The proposed
method was applied to the determination of the drugs in pharmaceutical formulations.
Buhl and Barbara described a sensitive spectrophotometric method for the
determination of cefotaxime, ceftriaxone and cefradine with leuco crystal violet
presented [64]. The determination was based on the reduction of potassium iodate in
acidic medium, followed by hydrolysis of -lactam ring of cephalosporins with
sodium hydroxide. The formed iodine oxidized with leuco crystal violet to crystal
violet dye of maximum absorption at 588 nm. Its absorbance was measured within pH
range of 4.0-4.2. Beer's law was obeyed in the concentration range: 0.8-4.8, 0.4-1.6
and 0.2-2.0 gmL
1
for cefotaxime, ceftriaxone and cefradine respectively. The molar
absorptivity of the colored compound was 8.410
4
Lmol
-1
cm
-1
for cefotaxime,
222
2.410
5
Lmol
-1
cm
-1
for ceftriaxone, and 1.610
5
Lmol
-1
cm
-1
for cefradine. The
analytical parameters were optimized and the method was successfully applied to the
determination of cefotaxime, ceftriaxone and cefradine in pharmaceuticals.
Alaa and Ragab described a spectrophotometric determination of certain
cephalosporins in pure form and in pharmaceutical formulations with metol-
chromium(VI) reagent [65]. Beer's law was obeyed in the range 0.2-28 gmL
1
at
maximum absorption 520 nm. The molar absorptivity and Sandell sensitivity were
calculated.
Abdel-Ghani et al. developed a spectrophotometric method for determination
of some cephalosporins in presence of each of their acid and alkaline induced
degradation products by two-different spectrophotometric methods [66]. Linear
correlations were obtained in a range 4.0-40.0 gmL
1
. The proposed method was
successfully applied for the determination of the cephalosporins in pure form and in
laboratory prepared mixtures with their acid and alkaline induced degradation
products and in pharmaceutical preparations.
El-Ansary et al. developed a spectrophotometric determination of some
cephalosporins using palladium(II) chloride [67]. This method was based on the
reaction of cephalosporins with palladium(II) chloride in the pH range 2.5-6.0 and
yellow water-soluble complexes formed with maximum absorbance at 337-350 nm.
Beer's law was obeyed in the concentration range of 1.5-12.6, 2.0-14.4 and 3.0-19.2
gmL
1
of cefadroxil, cephradine and cefotaxime respectively. The proposed method
was used for the determination of the above mentioned drugs in their pharmaceutical
preparations.
Vadia and Patel described a spectrophotometric determination of cefetamet
pivoxil hydrochloride in bulk and in pharmaceutical formulation [68]. Two simple
and sensitive colorimetric methods were developed for the analysis of cefetamet
pivoxil hydrochloride in bulk and in pharmaceutical formulations. The colored
complex formed was measured at 645 nm and the calibration curve was linear in the
range of 1-7 gmL
-1
, while in the second method the measurement was at 524 nm and
223
the calibration curve was linear in the range of 2-18 mgmL
-1
. The developed methods
were successfully applied to the pharmaceutical formulations.
Patett and Fischer reported a spectrophotometric assay for quantitative
determination of 7-aminocephalosporanic acids from direct hydrolysis of
cephalosporin C [69]. Nkeoma et al. reported a simple and accurate
spectrophotometric method for the analysis of ceftriaxone, cefotaxime and cefuroxime
in pharmaceutical dosage [70]. The method was based on the formation of Prussian
blue complex. The reaction between the acidic hydrolysis product of the antibiotics
with the mixture of Fe
3+
and hexacyanoferrate(III) ions was evaluated. The maximum
absorbance oI the coloured complex occurred at 700 nm and the molar absorptivity
was 3.010
4
Lmol
-1
cm
-1
. The linear range of the calibration graph was 2-20 gmL
-1
for ceftriaxone and cefotaxime and 2-18 gmL
-1
for cefuroxine. The method was
successfully applied to the determination of the selected antibiotics in bulk drugs and
pharmaceutical formulations.
In the present investigation, a facile and sensitive method has been reported
for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin with a
new reagents variamine blue and thionin. The developed method was successfully
applied for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin in
pharmaceuticals.
224
8.3 APPARATUS
A Systronics 2201 UV-VIS Double Beam Spectrophotometer with 1 cm
quartz cell was used for the absorbance measurements and a WTW pH 330, pH meter
was used.
8.4 REAGENTS AND SOLUTIONS
All chemicals used were of analytical grade and double distilled water was
used for dilution of the reagents and samples. Cefotaxime, ceftriaxone, cefadroxil, and
cephalexin stock solutions (1000 gmL
1
) were prepared by dissolving standard
sodium cefotaxime (Alkem Lab. Ltd. Mumbai) or sodium ceftriaxone (Aristo
Pharmaceuticals Ltd. Mumbai) or standard cefadroxil (Alkem Lab. Ltd. Mumbai) or
standard cephalexin (Ranbaxy, India) in water. These compounds were chosen to
represent cephalosporins. They were prepared freshly, as required, by dissolving an
appropriate amount of each antibiotic in water to provide a 1 gmL
1
solution. The
standard solution must be protected from light. The structures of the cephalosporins
studied are listed in 8A1. Sodium hydroxide (0.1 M), hydrochloric acid (1 M),
potassium iodate (0.1 M) were used.
Taxim (Alkem Lab. Ltd. Mumbai), Monocef (Aristo Pharmaceuticals Ltd.
Mumbai), Cefadrox (Aristo Pharmaceuticals Ltd. Mumbai) and Sporidex (Ranbaxy,
India) were examined. A 0.05% solution of variamine blue (E-Merck Limited,
Mumbai) in (75:25) water-ethanol mixture was used and stored in an amber bottle.
Thionin (S. D. fine Chem Limited, Mumbai) 0.1% was prepared by dissolving 0.1g
of thionin in 25 mL of methanol and made up to 100 mL with distilled water.
8.5 PROCEDURES
8.5.1 Using Variamine Blue as a Reagent
Aliquots of sample solution containing 0.55.8 gmL
1
of cefotaxime, 0.2-7.0
gmL
1
of ceftriaxone, 0.2-5.0 gmL
1
of cefadroxil and 0.5-8.5 gmL
1
of
cephalexin were transferred into a series of 25 mL calibrated flasks, 1 mL of 0.1 M
225
sodium hydroxide were added and the mixture was kept on a water bath (80C) for 10
minutes after being cooled to room temperature (272 C), 1.5 mL of 0.1 M
potassium iodate and 2 mL of 1M hydrochloric acid were added The mixture was
gently shaken until the appearance of yellow color, indicating the liberation of iodine,
1 mL of 0.05 % of variamine blue was then added to it followed by the addition 2 mL
of 1 M of acetate buffer of pH 4 and the reaction mixture was shaken for 2 minutes.
The contents were diluted up to 25 mL with distilled water and mixed well. The
absorbance of the oxidized species variamine blue formed was then measured at 556
nm against the reagent blank prepared in the same manner without the analyte. The
amount of the cefotaxime, ceftriaxone, cefadroxil and cephalexin present in the
volume taken was computed from the calibration graph (Figure VIIIC1).
8.5.2 Using Thionin as a Reagent
Aliquots of sample solution containing 0.56.4 gmL
1
of cefotaxime, 0.45.2
gmL
1
of ceftriaxone, 0.84.2 gmL
1
of cefadroxil and 1.07.5 gmL
1
of
cephalexin were transferred into a series of 25 mL calibrated flasks, 1 mL of 0.1 M
sodium hydroxide were added and the mixture was kept in a water bath (80 C) for 10
minutes after being cooled to room temperature (272 C), 1.5 mL of 0.1 M
potassium iodate and 2 mL of 1 M hydrochloric acid were added The mixture was
gently shaken until the appearance of yellow color, indicating the liberation of iodine,
1 mL of 0.1 % of thionin was then added to it followed by the addition 2 mL of 1 M
of acetate buffer of pH 4 and the reaction mixture was shaken for 2 minutes. The
contents were diluted up to 25 mL with distilled water and mixed well. The
absorbance of the resulting solution was measured at 600 nm against distilled water.
A blank was prepared by replacing the analyte (cefotaxime, ceftriaxone, cefadroxil
and cephalexin) solution with distilled water. The absorbance corresponding to the
bleached color that in turn corresponds to the analyte concentration was obtained by
subtracting the absorbance of the blank solution from that of test solution. The
amount of the cefotaxime, ceftriaxone, cefadroxil and cephalexin present in the
volume taken was computed from the calibration graph (Figure VIIIC2).
226
8.5.3 Analysis of injection solution
An appropriate amount of each antibiotic was dissolved in water so as to
prepare 1 mgmL
1
solution and then the recommended procedure was followed
without modification. The presence of other substances caused no significant
interference with the determination of antibiotics.
8.5.4 Analysis of Formulations
Weighed an amount of the sample equivalent to about 250 mg (0.2503 g) of
cephalosporin and was dissolved in a sufficient amount of distilled water. The
solution was shaken and filtered through whatman No. 1 filter paper and washed with
water. The filtrate was diluted with distilled water and made upto 100 mL. The
general procedure was applied with no modification and the presence of excipients in
the sample such as glucose, fructose, lactose, sucrose and calcium caused no
interference in the determination and process of separation was not required.
8.6 RESULTS AND DISCUSSION
8.6.1 Absorption Spectra
8.6.1.1 Using variamine blue as a reagent
This method is based on the hydrolysis of -lactum ring of the analytes on
heating with sodium hydroxide and the reaction of the hydrolysed product with
potassium iodate in acidic medium. The liberated iodine oxidizes variamine blue to
violet colored species of maximum absorption at 556 nm. Determination of
cefotaxime, ceftriaxone, cefadroxil, and cephalexin are represented in Scheme
VIIIA1. The absorption spectra of colored species of variamine blue are presented in
Figure VIIIA1, the absorption spectra of colored species of variamine blue with
cefotaxime, ceftriaxone, cefadroxil and cephalexin against reagent blank in the range
300800 nm are illustrated in Figure VIIIB1. The maximum absorption is at 556 nm
and reaction systems are presented in Scheme VIIIA3.
8.6.1.2 Using thionin as a reagent
This method involves the liberation of iodine by the hydrolysis of -lactum
ring of the analytes on heating with sodium hydroxide and the reaction of the
227
hydrolysed product with potassium iodate in acidic medium to which liberates iodine.
The liberated iodine bleaches the violet color of thionin and is measured at 600nm.
This decrease in absorbance is directly proportional to the cefotaxime, ceftriaxone,
cefadroxil and cephalexin concentration. Determination of cefotaxime, ceftriaxone,
cefadroxil and cephalexin are represented in Scheme VIIIA2. The absorption
spectrum of colored species of thionin is presented in Figure VIIIA2, the absorption
spectra of colored species of thionin with cefotaxime, ceftriaxone, cefadroxil and
cephalexin against reagent blank in the range 300800 nm are illustrated in Figure
VIIIB2. The maximum absorption is at 600 nm and reaction systems are presented in
Scheme VIIIA4.
8.6.2 Effect of Sodium Hydroxide Concentration
The effect of sodium hydroxide concentration on the absorbance is studied
with 2 gmL
1
of cephalosporins. Volumes from 0.52.0 mL of 0.1 M NaOH
solutions are examined. The investigation showed that 1.01.5 mL of 0.1 M NaOH
solution gave maximum absorbance and 1.0 mL of 0.1 M NaOH solution is chosen
for the procedure.
8.6.3 Effect of Temperature, Time and pH
The effect of different variables such as temperature, time and pH on the
colorization is studied with 2 gmL
1
of cephalosporins. It is observed that the
optimum reaction temperature is 80C90C, lower or higher temperature gives
inaccurate results and the reaction time for complete hydrolysis of -lactum ring is
1015 minutes. Constant and maximum absorbance values are obtained in the
pH=4.04.2 hence the pH of the reaction system is maintained at pH=4.04.2
throughout the study by adding 2 mL of 1 M sodium acetate solution.
8.6.4 Analytical Data
8.6.4.1 Using variamine blue as a reagent
Adherence to Beers law is studied by measuring the absorbance values of
solutions varying cephalosporins(analyte) concentration. A straight line graph is
obtained by plotting absorbance against concentration of analyte. Beers law is
obeyed in the range of 0.55.8 gmL
1
, 0.2-7.0 gmL
1
, 0.2-5.0 gmL
1
and 0.5-8.5
228
gmL
1
of cefotaxime, ceftriaxone, cefadroxil and cephalexin respectively (Figure
VIIIC1). The correlation coefficients for cefotaxime, ceftriaxone, cefadroxil and
cephalexin are found to be 0.9980, 0.9992, 0.9996 and 0.9991 respectively. The
following regression coefficients are calculated: Ior ceIotaxime 0.2239 b-0.014,
Ior ceItriaxone 0.1809 b0.0129, Ior ceIadroxil 0.1622 b0.0065 and Ior
cephalexin 0.1630 b0.0079. The Iollowing relative molar absorption coeIIicients
are obtained: 1.0710
5
Lmol
-1
cm
1
, 1.0210
5
Lmol
-1
cm
1
, 2.6810
4
Lmol
-1
cm
1
and
5.9010
4
Lmol
-1
cm
1
for cefotaxime, ceftriaxone, cefadroxil and cephalexin
respectively.
8.6.4.2 Using thionin as a reagent
Adherence to Beers law is studied by measuring the absorbance values of
solutions varying cephalosporins(analyte) concentration. A straight line graph is
obtained by plotting absorbance against concentration of analyte. Beers law is
obeyed in the range of 0.56.4 gmL
1
, 0.45.2 gmL
1
, 0.84.2 gmL
1
and 1.07.5
gmL
1
of cefotaxime, ceftriaxone, cefadroxil and cephalexin respectively(Figure
VIIIC2). The correlation coefficients for cefotaxime, ceftriaxone, cefadroxil and
cephalexin are found to be 0.9990, 0.9992, 0.9981, and 0.9975 respectively. The
following regression coefficients are calculated: for cefotaxime a=0.1563 b=-0.0016,
for ceftriaxone a=0.2251 b=0.0083, for cefadroxil a=0.1694 b=-0.0013 and for
cephalexin a=0.0966 b=0.0161. The following relative molar absorption coefficients
are obtained: 7.2110
4
Lmol
-1
cm
1
, 1.2310
5
Lmol
-1
cm
1
, 6.9110
4
Lmol
-1
cm
1
and
4.0810
4
Lmol
-1
cm
1
for cefotaxime, ceftriaxone, cefadroxil and cephalexin
respectively.
8.6.5 Effect of Divers Ions
The effect of foreign substances is examined for the proposed method. The
maximum tolerance in the determination of 100 gmL
1
cephalosporins is 54.0 mg
for glucose, 35.5 mg for fructose, 56.5 mg for lactose, 32.4 mg for sucrose and 22.0
mg for calcium. In case of thionin method the maximum tolerance in the
determination of 100 gmL
1
cephalosporins is 47.0 mg for glucose, 32.0 mg for
fructose, 54.3 mg for lactose, 34.2 mg for sucrose and 16.0 mg for calcium. The
results are summarized in Table 8A2.
229
8.7 APPLICATIONS
The proposed method is successfully applied to the determination of studied
antibiotics in pharmaceuticals. Cefotaxime was determined in 1g vials of taxim,
ceftriaxone in 250 mg vials of monocef, cefadroxil in 250 mg tablets of cefadrox and
cephalexin in 125 mg tablets of sporidex. Their contents in the investigated drug
samples are calculated from the calibration curves mentioned above are found to be in
a good agreement with the labelled amounts. The results of the analysis are presented
in Table 8A3 and 8A4, compared favorably with those from a reference method [64].
The precision of the proposed method was evaluated by replicate analysis of 3
samples containing cephalosporins at different concentrations.
8.8 CONCLUSIONS
A simple method for the determination of -lactum antibiotics is described.
The method is based on the reaction of iodate with the hydrolysed product of -
lactum antibiotics which liberates iodine, subsequently oxidizes variamine blue into
violet colored species and measured at 556nm and also bleaches the violet colour
species of thionin and measured at 600 nm. The developed method does not involve
any stringent reaction conditions and offers the advantages of high stability of the
reaction system (4 hours). The reagents have an advantage of high sensitivity,
selectivity, and low absorbance of the reagent blank. The proposed method was
applied to the determination of cephalosporins in pharmaceuticals. A comparison of
the method reported is made with earlier methods and is given in Table 8A5.
230
FIGURE VIIIA1
ABSORPTION SPECTRA OF COLORED SPECIES OF VARIAMINE BLUE
WITH REAGENT BLANK
Wavelength (nm)
200 300 400 500 600 700 800 900
A
b
s
o
r
b
a
n
c
e
0.0
0.2
0.4
0.6
0.8
1.0
1.2
FIGURE VIIIA2
ABSORPTION SPECTRUM OF COLORED SPECIES OF THIONIN
W avelength / nm
200 300 400 500 600 700 800 900
A
b
s
o
r
b
a
n
c
e
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
231
FIGURE VIIIB1
ABSORPTION SPECTRA OF COLORED SPECIES OF VARIAMINE BLUE
WITH CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN
AGAINST REAGENT BLANK: CEPHALOSPORINS = 2 gmL
-1
Wavelength (nm)
200 300 400 500 600 700 800 900
A
b
s
o
r
b
a
n
c
e
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Cefotaxime
Ceftriaxone
Cefadroxil
cephalexin
Reagent blank
232
FIGURE VIIIB2
ABSORPTION SPECTRA OF COLORED SPECIES OF THIONIN WITH
CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN AGAINST
REAGENT BLANK: CEPHALOSPORINS = 2 gmL
-1
Wavelength (nm)
200 300 400 500 600 700 800 900
A
b
s
o
r
b
a
n
c
e
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Cephalexin
Cetriaxone
Cefotaxime
Cefadroxil
233
FIGURE VIIIC1
ADHERENCE TO BEERS LAW FOR THE DETERMINATION OF
CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN USING
VARIAMINE BLUE AS A REAGENT
0 2 4 6 8 10
A
b
s
o
r
b
a
n
c
e
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Cefotaxime
Ceftriaxone
Cefadroxil
Cephalexin
Volume of Cefotaxime, Ceftriaxone, Cefadroxil and Cephalexin (gmL
1
)
234
FIGURE VIIIC2
ADHERENCE TO BEERS LAW FOR THE DETERMINATION OF
CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN USING
THIONIN AS A REAGENT
Volume of Cefotaxime, Cefriaxone, Cefadroxil and Cephalexin (gmL
- 1
)
0 2 4 6 8
A
b
s
o
r
b
a
n
c
e
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Cefotaxime
Ceftriaxone
Cefadroxil
Cephalexin
235
SCHEME VIIIA1
DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND
CEPHALEXIN USING VARIAMINE BLUE AS A REAGENT
To the sample containing
0.5 5.8 gmL
1
0.2-7.0 gmL
1
0.2-5.0 gmL
1
0.58.5 gmL
1
Cefotaxime Ceftriaxone Cefradroxil Cephalexin
+ 1 mL of 0.1 MNaOH
Kept 10 min on water bath at 80C
Cooled to room temperature
+ 1.5 mL of 0.1 Mpotassium Iodate
+ 2 mL of 1 M HCl
Analyte + IO
3
-
+ 6H
+
analyte
oxid.
+ I
2
+ 3H
2
O
+ 1 mL of 0.05% VB
I
2
+ VB VB I
-
(colorless) (colored)
+ 2 mL of 1 M sodium acetate
( pH is 4.0-4.2 )
Quantitatively transfered to the 25 mL volumetric flask,
and diluted to 25 mL with water
Measured the absorbance at 556 nm in the 1cm thick cell against the blank solution
236
SCHEME VIIIA2
DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND
CEPHALEXIN USING THIONIN AS A REAGENT
To the sample containing
0.5-6.4 gmL
1
0.4-5.2 gmL
1
0.8-4.2 gmL
1
1.0-7.5 gmL
1
Cefotaxime Ceftriaxone Cefadroxil Cephalexin
+ 1 mL of 0.1 MNaOH
Kept 10 min on water bath at 80C
Cooled to room temperature
+ 1.5 mL of 0.1 Mpotassium Iodate
+ 2 mL of 1MHCl
Analyte + IO
3
-
+ 6H
+
analyte
oxid.
+ I
2
+ 3H
2
O
+ 1 mL of 0.1% thionin
I
2
+ Thionin Thionin I
-
(coloured) (colourless)
+ 2 mL of 1M sodium acetate
( pH is 4.0-4.2 )
Quantitatively transfered to the 25 mL volumetric flask,
and diluted to 25 mL with water
Measured the absorbance at 600nm in the 1cm thick cell against the blank solution
237
SCHEME VIIIA3
Analyte + IO
3
-
+ 6H
+
analyte
oxid.
+ I
2
+ 3H
2
O
I
2
+ VB VB I
-
(colourless) (coloured)
NH
NH
2
O
CH
3
I
2
+
+
I
-
N
NH
2
O
CH
3
+
1
/
2
VARIAMINE BLUE VARIAMINE BLUE
(LEUCOFORM) (VIOLET COLOR)
SCHEME VIIIA4
I
2
+ Thionin Thionin I
-
(coloured) (colourless)
S
N
NH
2
N H
2
1
/
2
I
2
S
N
H
NH
3
+
N H
2
+
I
-
I
2
H
+
+
THIONIN THIONIN
(VIOLET COLOR) (LEUCOFORM)
238
TABLE 8A1
STRUCTURES OF THE CEPHALOSPORINS STUDIED
S
N
R'
O O
R''
H H
N H
R
O
O
Cephalosporin R R' R"
S
N
N
OCH
3
N H
2 CH
2
OCOCH
3 Na
S
N
N
OCH
3
N H
2
N
N
N
CH
3
O
H
2
CS OH
Na
CH
3
H
1. Cefotaxime
2. Ceftriaxone
4. Cephalexin
CH
2
NH
2
CH
3
H
CH
NH
2
HO
3. Cefadroxil
239
TABLE 8A2
TOLERANCE OF EXCIPIENTS FOR THE DETERMINATION OF
CEPHALOSPORINS USING VARIAMINE BLUE AND THIONIN AS
REAGENTS
Common excipient Tolerance limit (mg) Tolerance limit (mg)
Variamine blue Thionin
Glucose 54.0 47.0
Fructose 35.5 32.0
Lactose 56.5 54.3
Sucrose 32.4 34.2
Calcium 22.0 16.0
240
TABLE 8A3
DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND
CEPHALEXIN IN PHARMACEUTICALS PREPARATIONS USING VARIAMINE
BLUE AS A REAGENT
Pharmaceutical Declared Quantity Found in the sample
a
(gmL
1
) (gmL
1
) S.D
TAXIM 1 2.00 1.984 0.03
TAXIM 2 4.00 3.975 0.02
TAXIM 3 5.50 5.454 0.02
MONOCEF 1 1.00 0.986 0.03
MONOCEF 2 3.00 2.992 0.04
MONOCEF 3
MONOCEF 4
5.00
7.00
4.954 0.02
6.966 0.025
CEFADROX 1 2.00 1.994 0.01
CEFADROX 2 4.00 3.986 0.02
SPORIDEX 1
SPORIDEX 2
SPORIDEX 3
SPORIDEX 4
2.00
4.00
6.00
8.00
1.984 0.015
3.896 0.01
5.982 0.04
7.940 0.08
a
Average of three determinations.
241
TABLE 8A4
DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL, AND
CEPHALEXIN IN PHARMACEUTICALS PREPARATIONS USING THIONIN AS
A REAGENT
Pharmaceutical Declared Quantity Found in the sample
a
(gmL
1
) (gmL
1
) S.D
TAXIM 1 2.00 1.896 0.04
TAXIM 2 4.00 3.936 0.015
TAXIM 3 6.00 5.898 0.03
MONOCEF 1 1.00 0.982 0.04
MONOCEF 2 3.00 3.010 0.02
MONOCEF 3 5.00 4.924 0.025
CEFADROX 1 2.00 1.962 0.015
CEFADROX 2 4.00 3.891 0.02
SPORIDEX 1
SPORIDEX 2
SPORIDEX 3
2.00
4.00
6.00
1.994 0.05
3.985 0.03
5.954 0.02
a
Average of three determinations.
242
TABLE 8A5
COMPARISON OF THE METHOD REPORTED WITH EARLIER METHODS
= Molar absorptivity, ss = Sandells sensitivity
Reagent Method Analyte Beers law
(gmL
-1
)
(Lmol
-1
cm
-1
)
ss (gcm
-2
)

max
(nm)
Ref.
No.
Etylene blue Spectropho-
tometry
cefotaxime
cefadroxil
0.5-7.0
0.5-10
--------
--------
----- 51
2,2-Diphenyl-1-
picryl-hydrazyl
Spectropho-
tometry
cephalexin
cefadroxil
cefradine
5.0-30
5.0-25
10-30
--------
--------
--------
520 60
Leuco crystal
violet
Spectropho-
tometry
cefotaxime
ceftriaxone
cefradine
0.8-4.8
0.4-1.6
0.2-2.0
= 8.4010
4
= 2.4010
5
= 1.6010
5
588 64
Proposed
Method
Variamine blue
Thionin
Spectropho-
tometry
Spectropho-
tometry
cefotaxime
ceftriaxone
cefadroxil
cephalexin
cefotaxime
ceftriaxone
cefadroxil
cephalexin
0.5-5.8
0.2-7.0
0.2-5.0
0.5-8.5
0.5-6.4
0.4-5.2
0.8-4.2
1.0-7.5
= 1.0710
5
= 1.0210
5
= 2.6810
4
= 5.9010
4
= 7.2110
4
= 1.2310
5
= 6.9110
4
= 4.0810
4
556
600
243
8.9 REFERENCES
1. W. O. Foye, Principles of Medicinal Chemistry, Lea and Febiger, Philadelpia,
USA, 1975, p.726
2. E. P. Abraham and G. G. F. Newton, Biochem. J., 79 (1961) 377.
3. D. C. Hodgkin and E. N. Maslem, Biochem. J., 79 (1961) 393.
4. G. G. F. Newton and E. P. Abraham, Biochem. J., 62 (1956) 651.
5. B. Loder, G. G F. Newton and E. P. Abraham, Biochem. J., 79 (1961) 408.
6. E. L. Patterson, J. C. Van Meter and N. Bohonos, J. Med. Chem., 7(5) (1964) 689.
7. H. A. Albrecht, G. Beskid, J. G. Christenson, K. H. Dietcher, H. H.
Georgopapadakou, D. D. Keith, F. M. Konzelmann, D. L. Pruess and C. C. Wei, J.
Med. Chem., 37 (1994) 400.
8. H. P. Svensson, I. S. Frank, K. K. Berry and P. D. Senter, J. Med. Chem., 41 (1998)
1507.
9. M. Kohsaka and A. L. Demain, Biochem. Biophys. Res. Commun., 70 (1976) 465.
10. J. Dotzlaf and W. K. Yeh, J. Bacteriol., 169 (1987) 1611.
11. J. Dotzlaf and W. K. Yeh, J. Biol. Chem., 264 (1989) 10219.
12. A. L. Demain, J. L. Adrio and J. M. Piret, Enzyme Technology for Pharmaceutical
and Biotechnological Applications; Marcel Dekker: New York, 2001.
13. J. Kupka, Y. Q. Shen, S. Wolfe and A. L.Demain, FEMS Microbiol. Lett., 16
(1983) 1.
14. K. Maeda, J. M. Luengo, O. Ferrero, S. Wolfe, M. Y. Lebedev, A. Fang and A. L.
Demain, Enzyme Microb. Tech., 17 (1995) 231.
15. C. A. Bunnell, W. D. Luke and R. M. Perry, Beta-lactam Antibiotics for Clinical
Use; Marcel Dekker, New York, 1986.
16. J. K. Podlewski and P. A. Chwalibogowaka, Drugs of the Modern Therapy, Split
Trading, Warsaw. 1999, pp 45-47, 125-132 (in Polish).
17. C. S. P. Sastry, T. E. Divakar and U. V. Prasad, Chem. Anal., (Warsaw) 32 (1987)
301.
18. L. M. Gerald, Merl A.C. Penicillins, Cephalosporin and other -Lactam
Antibiotics. Goodman and Gilmans the Pharmacological Basis of Therapeutics.
New York, Pargamon Press, (1990) p.1065.
19. M. M. Ayad, A. A. Shalaby, H. E. Abdellatef and H. M. Elsaid, J. Pharm.
Biomed. Anal., 20 (1999) 557.
244
20. A. Shalaby, J. Liq. Chromatrogr. Relat. Tech., 21(1998) 3161.
21. S. S. Zarapkar, S. A. Shivalkar, A. A. Dhanvate, P. M. Deshpande and S. S. Kolte,
Indian Drugs, 32 (1995) 232.
22. M. Hefnawy, Y. El-Shabrawy and F. Belal, J. Pharm. Biomed. Anal., 21 (1999)
703.
23. Y. M. Issa and A. S. Amin, Mikrochim. Acta, 124 (1996) 203.
24. H. Tan, R. Wang, S. Wang, W. Wei and S. Yao, Anal. Lett., 31 (1998) 949.
25. H. Fabre, M. D Blanchin, D. Lerner and B. Mandrou, Analyst, 110 (1985) 775.
26. J. A. Murill, J. M. Lemus and L.F. Garci, Anal. Lett., 27 (1994) 1875.
27. M. Azza and M. Ali, Bioelectrochem. Bioenerg., 33 (1994) 201.
28. P. Izquierdo, M. C Gutierrez, H. A. Gomez and B. D. Perez, Anal. Lett., 23
(1990) 487.
29. H. T. Pan, P. Kumari, J. Lim and C. C. Lin, J. Pharm. Sci., 81 (1992) 663.
30. K. Matsubayashi and H. Tachzawa, J. Chromatogr, 515 (1990) 547.
31. Z. H. Earle, D. T. Hurst and M. Viney, J. Chem. Soc., (1969) C 2093.
32. B. M. Frantz, J. Pharm. Sci., 65 (1976) 887.
33. M. I. H. Halaleh, N. Rahman and R. M. A. Q. Jamhour, Chem. Anal., (Warsaw)
42 (1997) 265.
34. J. Yang, G. Zhou, N. Jie, R. Han, C. Li and J. Hu, Anal. Chim. Acta, 325 (1996)
195.
35. B. Ogoreve, V. Hudnik and S. Gomiseek, Fresenius J. Anal. Chem., 330 (1988)
59.
36. V. Hartman and M. Rodiger, Chromatographica, 9 (1976) 266.
37. V. Coman, V. Avram, C. Soran, R. Grecu, Z. Moldwan and H. Farkas, HPLC
Determination of Some Antibiotics. Bohyai, Special Issue. (2003) p.1-4.
38. S. Eric-Jovanovic, D. Agbaba, D. Zinanov-Stakic and S. Vladimiro, J. Pharm.
Biomedic. Anal. 18 (1998) 893.
39. S. A. Nabi, E. Laig and A. Islam, Acta Chromatogr., 14 (2004) 92.
40. A. Cieslak, E. Gwozdz and W. Holska, Chem. Anal.,(Warsaw) 36 (1991) 363.
41. C. Thongpoon, B. Liawruangrath, S. Liawruangrath, R. A. Wheatley and A.
Townshend, Anal. Chim. Acta, 553 (2005) 123.
42. C. S. P. Sastry, T. E. Divakar and U. V. Prasad, Chem. Anal.,(Warsaw) 32 (1987)
301.
43. P. B. Issopoulos, J. Pharm.Biomed. Anal., 7 (1989) 619.
245
44. P. B. Issopoulos, Analyst, 113 (1988) 1083.
45. S. A. Abdel-Razeq, Bull. Fac. Pharm., 40 (2002) 155.
46. I. Abd El-Sattar, N. M. Osama, El-Abasawy, S. A. Abd El-Razeq, M. M. F. Ismail
and N. S. Rashed, Saudi Pharm. J., 9 (2001) 186.
47. A. F. M. El Walily, A. Abdel Kader Gazy, F. B. Saied and F. K. Essam, Spect.
Lett., 33 (2000) 931.
48. S. A. Nabi, E. S. M. Abu-Nameh and I. H. H. Murad, Chem. Anal.(Warsaw) 42
(1997) 881.
49. D. Agbaba, S. Eric, K. Karlijikovic-Rajic, S. Vladimirov and D. Z. Stakic, Spect.
Lett., 30 (1997) 309.
50. M. S. Mahrous and M. M. Abdel-Khalek, Egyptian J Pharm. Sci., 34 (1993) 47.
51. A. A. Alwarthan, F. H. Metwally and I. S. A. Al-Tim, Anal. Lett., 26 (1993) 2619.
52. F. I. Sengun and I. Fedai, Chim. Acta Turcica, 13 (1985) 205.
53. B. Morelli and P. Peluso, Anal. Lett.,18 (1985) 1113.
54. M. A. Abdalla, A. G. Fogg and C. Burgess, Analyst, 107 (1982) 213.
55. M. M. Abdel-Khalek and M. S. Mahrous, Talanta, 30 (1983) 792.
56. M. S. Mahrous and M. M. Abdel-Khalek, Analyst 109 (1984) 611.
57. F. I. Sengun and K. Ulas, Talanta, 33 (1986) 363.
58. C. S. P. Sastry, P. Satyanarayana, A. R. Rao and S. N. R. Prasad, Mikrochim.
Acta, 1 (1989) 17.
59. M. A. Abdalla, Anal. Lett., 24 (1991) 55.
60. E. Hosny, Bull. Pharm. Sci., 20 (1997) 87.
61. M. I. H. Helaleh, E. S. M. Abu-Nameh and R. M. A. Q. Jamhour, Acta Poloniae
Pharm., 55 (1998) 93.
62. I. F. Al-Momani, J. Pharm. Biomed. Anal., 25 (2001) 751.
63. A. S. Amin and S. A. Shama, Monatsh. Chem., 131 (2000) 313.
64. F. Buhl and B. S. Sroka, Chem. Anal.,(Warsaw) 48 (2003) 145.
65. A. S. Alaa and G. H, Ragab, Spectrochim. Acta, 60 (2004) 2831.
66. M. F. Abdel-Ghani, O. Abdel-Aziz, S. El-Weshahy and L. Abdel-Fattah, Bull.
Fac. Pharm., 41 (2003) 109.
67. A. El-Ansary, F. M. Abdel-Gawad, S. S. Badawy and M. M. Ibrahim, J. Drug
Res., 25 (2004), 7.
68. N. H. Vadia and V. B. Patel, Indian J. Pharm. Sci., 68 (2006) 584.
69. F. Patett and L. Fischer, Anal. Biochem. 350 (2006) 304.
246
70. N. O. Nkeoma, G. I. C. Nwokedi, N. N. Ukwueze and F. B. C. Okoye, Scient. Res.
Essay, 2 (2007) 342.

Potrebbero piacerti anche