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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

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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 β-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 μmL-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 β-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 α-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].

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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 β-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 β-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 β-

lactum ring hydrolysis step [31]. A β-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 (NH4)6Mo7O24 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 μgmL-1

for I, up to 60 μgmL-1 for II and IV and up to 80 μgmL-1 for III. The molar

absorptivities were 4.58×103, 11.3×103, 9.8×103 and 10.9×103 Lmol-1cm-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 μgmL-1 for I, 0.8 - 3.6 and 4 - 24 μgmL-1 for II or 0.4 -

2.4 and 4-16 μgmL-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 1st 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 2nd 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 3rd 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 μgmL-1, 20-180 μgmL-1,

10-60 μgmL-1, 5-25 μgmL-1 and 10-60 μgmL-1 for CA, DDQ, TCNQ, ninhydrin and

FCR resectively. However, for the cefprozil salt, the concentration range were 50-400

μgmL-1, 20-140 μgmL-1, 1-7 μgmL-1, 2-14 μgmL-1 and 2.5-25 μgmL-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 β-lactam ring was hydrolyzed with sodium hydroxide at 800°C for 10-15

minutes. Cooled samples were acidified with 1 M HCl and CCl4 was added. Aliquots

of each sample were titrated with a standard 0.01 M potassium iodate solution. The

colorless CCl4 layer gradually developed to a deep red colored as the titration end-

point. The absorbance of the CCl4 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 β-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

μgmL-1 and 0.5-7 μgmL-1 for cefadroxil and cefotaxime resectively. The detection

limit was 0.1 μgmL-1 and 0.05 μgmL-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 μgmL-1 for cephalothin and 25.36 μgmL-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 H2SO4 medium. The antibiotic was heated at 91.5°C 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 μg 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 (NH4)VO3

220

solution in H2SO4 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 μgmL–1.

Mahrous and Abdel Khalek presented ninhydrin as a reagent for the

spectrophotometric determination of certain cephalosporins in H2SO4 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 μgmL–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-HgCl2 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 H2S 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 μgmL–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 β-lactam ring had been

hydrolyzed for 10 minutes with sodium hydroxide at 80°C. Beer's law was obeyed in

the concentration range of 20-600 and 20-800 μgmL–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 80°C and then

oxidized with Fe3+ in H2SO4 medium, which produced Fe2+. The produced Fe2+ 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.

Beer’s law was obeyed over a concentration range of 0.4–�������. 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 β-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 μgmL–1 for cefotaxime, ceftriaxone and cefradine respectively. The molar

absorptivity of the colored compound was 8.4×104 Lmol-1cm-1 for cefotaxime,

222

2.4×105 Lmol-1cm-1 for ceftriaxone, and 1.6×105 Lmol-1cm-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 μgmL–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 μgmL–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

μgmL–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-������-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 Fe3+ and hexacyanoferrate(III) ions was evaluated. The maximum

� ��� ����������������������������������������������������������������� ������ ��!�

was 3.0×104 Lmol-1cm-1. The linear range of the calibration graph was 2-20 µgmL-1

for ceftriaxone and cefotaxime and 2-18 µgmL-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 μgmL–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 μgmL–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 0.5–5.8 μgmL–1 of cefotaxime, 0.2-7.0

μgmL–1 of ceftriaxone, 0.2-5.0 μgmL–1 of cefadroxil and 0.5-8.5 μgmL–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 (80°C) for 10

minutes after being cooled to room temperature (27±2° 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 0.5–6.4 μgmL–1 of cefotaxime, 0.4–5.2

μgmL–1 of ceftriaxone, 0.8–4.2 μgmL–1 of cefadroxil and 1.0–7.5 μgmL–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 (27±2° 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 solutionAn 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 β-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

300–800 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 β-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 300–800 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 μgmL–1 of cephalosporins. Volumes from 0.5–2.0 mL of 0.1 M NaOH

solutions are examined. The investigation showed that 1.0–1.5 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 μgmL–1 of cephalosporins. It is observed that the

optimum reaction temperature is 80°C–90°C, lower or higher temperature gives

inaccurate results and the reaction time for complete hydrolysis of β-lactum ring is

10–15 minutes. Constant and maximum absorbance values are obtained in the

pH=4.0–4.2 hence the pH of the reaction system is maintained at pH=4.0–4.2

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 Beer’s 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. Beer’s law is

obeyed in the range of 0.5–5.8 μgmL–1, 0.2-7.0 μgmL–1, 0.2-5.0 μgmL–1 and 0.5-8.5

228

μgmL–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 a��� ����������"� ���� �����������#��$%%&'� �-0.014,

���� ������������ #��$(�'� ��$�%')� ���� ����������� #��$*%%� ��$��*�� ���� ����

�����������#��$*&�� ��$���'$�+��������,���������� ��������� ����������������������

are obtained: 1.07×105 Lmol-1cm–1, 1.02×105 Lmol-1cm–1, 2.68×104 Lmol-1cm–1 and

5.90×104 Lmol-1cm–1 for cefotaxime, ceftriaxone, cefadroxil and cephalexin

respectively.

8.6.4.2 Using thionin as a reagent

Adherence to Beer’s 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. Beer’s law is

obeyed in the range of 0.5–6.4 μgmL–1, 0.4–5.2 μgmL–1, 0.8–4.2 μgmL–1 and 1.0–7.5

μgmL–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: 7.21×104 Lmol-1cm–1, 1.23×105 Lmol-1cm–1, 6.91×104 Lmol-1cm–1 and

4.08×104 Lmol-1cm–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 μgmL– 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 μgmL–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 β-lactum antibiotics is described.

The method is based on the reaction of iodate with the hydrolysed product of β-

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

Abs

orba

nce

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

Abs

orba

nce

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 μgmL-1

Wavelength (nm)

200 300 400 500 600 700 800 900

Abso

rban

ce

0.00

0.05

0.10

0.15

0.20

0.25

0.30

CefotaximeCeftriaxoneCefadroxilcephalexinReagent blank

232

FIGURE VIIIB2

ABSORPTION SPECTRA OF COLORED SPECIES OF THIONIN WITH

CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN AGAINST

REAGENT BLANK: CEPHALOSPORINS = 2 μgmL-1

Wavelength (nm)

200 300 400 500 600 700 800 900

Abso

rban

ce

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

CephalexinCetriaxoneCefotaximeCefadroxil

233

FIGURE VIIIC1

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF

CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN USING

VARIAMINE BLUE AS A REAGENT

0 2 4 6 8 10

Abs

orba

nce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

CefotaximeCeftriaxoneCefadroxilCephalexin

Volume of Cefotaxime, Ceftriaxone, Cefadroxil and Cephalexin (μgmL– 1)

234

FIGURE VIIIC2

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF

CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN USING

THIONIN AS A REAGENT

Volume of Cefotaxime, Cefriaxone, Cefadroxil and Cephalexin (µgmL- 1)

0 2 4 6 8

Abso

rban

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

CefotaximeCeftriaxoneCefadroxilCephalexin

235

SCHEME VIIIA1 DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND

CEPHALEXIN USING VARIAMINE BLUE AS A REAGENT

To the sample containing

0.5 –5.8 μgmL–1 0.2-7.0 μgmL–1 0.2-5.0 μgmL–1 0.5—8.5 μgmL–1

Cefotaxime Ceftriaxone Cefradroxil Cephalexin

+ 1 mL of 0.1 M NaOH

Kept 10 min on water bath at 80ºC

Cooled to room temperature

+ 1.5 mL of 0.1 M potassium Iodate + 2 mL of 1 M HCl

Analyte + IO3- + 6H+ -�����!���oxid. + ½I2 + 3H2O

+ 1 mL of 0.05% VB

½ I2 + VB -������./���0��1-

(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 VIIIA2DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND

CEPHALEXIN USING THIONIN AS A REAGENT

To the sample containing

0.5-6.4 μgmL– 1 0.4-5.2 μgmL– 1 0.8-4.2 μgmL– 1 1.0-7.5 μgmL– 1

Cefotaxime Ceftriaxone Cefadroxil Cephalexin

+ 1 mL of 0.1 M NaOH

Kept 10 min on water bath at 80ºC

Cooled to room temperature

+ 1.5 mL of 0.1 M potassium Iodate + 2 mL of 1M HCl

Analyte + IO3- + 6H+ -�����!���oxid. + ½I2 + 3H2O

+ 1 mL of 0.1% thionin

½ I2 + Thionin -����+���������0��1-

(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 + IO3- + 6H+ -�����!���oxid. + ½I2 + 3H2O

½ I2 + VB -����������./�����0������1-

(colourless) (coloured)

NH

NH2O

CH3

I2++ I-

N

NH2O

CH3

+1/2

VARIAMINE BLUE VARIAMINE BLUE

(LEUCOFORM) (VIOLET COLOR)

SCHEME VIIIA4

½ I2 + Thionin -����+���������0��1-

(coloured) (colourless)

S

N

NH2

NH2

1/2 I2

S

NH

NH3+

NH2

+ I-

I2

H+ +

THIONIN THIONIN

(VIOLET COLOR) (LEUCOFORM)

238

TABLE 8A1

STRUCTURES OF THE CEPHALOSPORINS STUDIED

S

NR'

O OR''

HHN H

R

O

O

Cephalosporin R R' R"

S

N

NOCH3

NH2 CH2OCOCH3 Na

S

N

NOCH3

NH2

N

N

N

CH3

O

H2CS OH Na

CH3 H

1. Cefotaxime

2. Ceftriaxone

4. Cephalexin CH2

NH2

CH3 H

CH

NH2

HO3. 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 samplea

(μgmL–1) (μgmL–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 samplea

(μgmL–1) (μgmL–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

ε = Molar absorptivity, ss = Sandell’s sensitivity

Reagent Method Analyte Beer’s law2����-1)

ε (Lmol-1cm-1)���2����-2)

λmax(nm)

Ref. No.

Etylene blue Spectropho-tometry

cefotaximecefadroxil

0.5-7.0 0.5-10

----------------

----- 51

2,2′-Diphenyl-1-picryl-hydrazyl

Spectropho-tometry

cephalexincefadroxilcefradine

5.0-30 5.0-2510-30

------------------------

520 60

Leuco crystal violet

Spectropho-tometry

cefotaximeceftriaxonecefradine

0.8-4.80.4-1.60.2-2.0

ε = 8.40×104

ε = 2.40×105

ε = 1.60×105

588 64

Proposed MethodVariamine blue

Thionin

Spectropho-tometry

Spectropho-tometry

cefotaximeceftriaxonecefadroxilcephalexin

cefotaximeceftriaxonecefadroxilcephalexin

0.5-5.80.2-7.00.2-5.00.5-8.5

0.5-6.40.4-5.20.8-4.21.0-7.5

ε = 1.07×105

ε = 1.02×105

ε = 2.68×104

ε = 5.90×104

ε = 7.21×104

ε = 1.23×105

ε = 6.91×104

ε = 4.08×104

556

600

243

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