Spectophotometric method for determination of certain cephalosporins using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)

doi:10.4236/ns.2010.28104

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Vol.2, No.8, 828-840 (2010) Natural Science

doi:10.4236/ns.2010.28104

Spectophotometric method for determination of certain

cephalosporins using 4-chloro-7-nitrobenzo-2-oxa-1,

3-diazole (NBD-Cl)

Azza H. Rageh*, Salwa R. El-Shaboury, Gamal A. Saleh, Fardous A. Mohamed

Department of Pharmaceutical Analytical Chemistry, Facutly of Pharmacy, Assiut University, Assiut, Egypt; *Corresponding Author:

azhesham@yahoo.com

Received 11 February 2010; revised 23 March 2010; accepted 30 March 2010.

ABSTRACT

A simple, accurate and precise spectropho-

tometric method has been proposed for the de-

termination of eleven cephalosporins, namely;

cefaclor monohydrate, cefadroxil monohydrate,

cefalexin anhydrous, cefradine anhydrous, ce-

fotaxime sodium, cefoperazone sodium, ceftri-

axone sodium, ceftazidime penthydrate, cefa-

zolin sodium, cefixime and cefpodoxime pro-

xetil in bulk drug and in pharmaceutical formu-

lations. The method depends on hydrolysis of

the studied drugs using 0.5M NaOH at 100°C

and subsequent reaction of the formed sulfide

ions with NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1,

3-diazole) to form a yellow-colored chromogen

measured at 390 nm. Different variables affect-

ing the reaction (e.g. NaOH concentration, hy-

drolysis time, NBD-Cl concentration and dilut-

ing solvent) were studied and optimized. Under

the optimum conditions, linear relationships

with good correlation coefficients (0.9990-

0.9999) were found in the range of 5-160 μg mL-1

for all studied drugs. The limits of assay detec-

tion and quantitiation ranged from 0.289 to 5.867

and from 0.878 to 17.778 μg mL-1; respectively.

The accuracy and precision of the proposed

method were satisfactory. The method was suc-

cessfully applied for analysis of the studied

drugs in their pharmaceutical formulations and

the recovery percentages ranged from 96.6 to

103.5%.

Keywords: Spectrophotometry; Cephalo spo rins;

NBD-Cl; Pharmaceutical Analysis

1. INTRODUCTION

Cephalosporins have been used since 1948. These anti-

biotics have assumed a prominent role in modern antim-

icrobial therapy due to enhanced intrinsic microbiologi-

cal activities and favorable safety profile. Chemical

structures of cephalosporins drive from the 7-aminoce-

phalosporanic acid (7-ACA) composed of a β-lactam

ring fused with a dihydrothaizine ring (Figure 1), but

differ in the nature of substituents at the 3- and/or

7-positions of the cephem ring. These substituents affect

either the pharmacokinetic properties (3-position) or the

antibacterial spectrum (7-position) of the cephalosporins

[1,2]. Traditionally, cephalosporins are divided into first-,

second-, third-, and fourth-generation agents. Table 1

shows cephalosporins studied in this work. Several

methods have been reported for cephalosporins deter-

mination. The official procedures in pharmaceutical

preparations utilize high-performance liquid chromatog-

raphy (HPLC) [3] which is expensive. Other reported

procedures include spectrophotometric [4-9], spectro-

fluorimetric [10-13], chemiluminescence [14-16], chro-

matographic [17-20] and electrochemical methods [21-24]

and most of them are lengthy and/or tedious.

The hydrolytic degradation of cephalosporins was

very often used as a preliminary step in the analytical

procedure used for their determinations [25-32]. The

literature reveals that many spectrophotometric methods

were developed for cephalosporins determinations that

based on hydrolysis of these drugs using alkaline degra-

Figure 1. Chemical structure of 7-aminocephalosporanic acid.

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

829

Table 1. Chemical structures of the investigated cephalosporin antibiotics.

NR2

OOR

No. Name R1 R

2 R

3 Generation

1. Cefalexin anhydrous

NH2

-CH3 -H First

2. Cefradine anhydrous

NH2

-CH3 -H First

3. Cefadroxil monohy-

drate

NH2

-CH3 -H First

4. Cefazolin sodium NN

NNCH

SCH3

-Na First

5. Cefaclor monohy-

drate

NH2

-Cl -H Second

6. Cefpodoxime

proxetil

H2N

NOCH3

CH2OCH3 CHOCOCH(CH 3)2

CH3

Third

7. Cefixime

H2NC

NOCH2CO2

HCH2 -H Third

8. Cefoperazone so-

dium

HO H

C2H5

NSC

CH3

-Na Third

9. Cefotaxime sodium

NOCH3

H2NCH2OCCH3

-Na Third

10. Ceftazidime penta-

hydrate

H2N

CCO

2HH3C

CH3

-H Third

11. Ceftriaxone sodium

NOCH3

H2N

H3C

-Na Third

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

830

dation and subsequent reaction of the formed sulfide

ions with chromogenic reagents [26,27].

NBD-Cl (Figure 2) has been reported as fluorogenic

reagent for determination of amines [33] and for spec-

trophotometric determination of many compounds

[34-41]. Thiocompounds have been reported to form

intensely colored products in an alkaline medium with

NBD-Cl which could be used for their colorimetric de-

termination [42]. It is always required to develop ana-

lytical methods using low cost techniques. UV-Vis spec-

trophotometry is still considered a convenient and eco-

nomical technique for routine analysis of drugs in phar-

maceutical formulations. On the basis of the aforemen-

tioned reasons, it was decided to develop a quantitative

method for the determination of the studied cepha-

losporins based on their alkaline hydrolysis and subse-

quent reaction of the resulting hydrolysates with

NBD-Cl, which may be used for their analysis either in

pure forms or in pharmaceutical formulations. This

method is selective for cephalosporins, since other

β-lactam antibiotics such as penicillins do not give sul-

fide ions under the degradation conditions employed

[27,43-45].

2. EXPERIMENTAL

2.1. Apparatus

Shimadzu UV-1700 PC, UV-Visible Spectrophotometer

(Tokyo, Japan), ultrasonic cleaner (Cole-Parmer, Chi-

cago, USA), sartorious handy balance-H51 (Hannover,

Germany) and MLV type thermostatically controlled

water bath (Salvis AG Emmenbruck, Luzern, Germany).

2.2. Materials and Reagents

All solvents used were of analytical-reagent grade, so-

dium hydroxide (El-Nasr Chemical Co. Cairo, Egypt)

0.5 M aqueous solution, hydrochloric acid (El-Nasr

Chemical Co. Cairo, Egypt), 4-cholor-7-nitrobenzo-

furazan [NBD-Cl] (Fluka Chemie AG, Switzerland)

freshly prepared (3 × 10-3 M) equivalent to 0.060% w/v

in acetone, samples of cephalosporins were generously

supplied by their respective manufacturers and were

used as supplied: cefaclor monohydrate and cefradine

NO2

Figure 2. Chemical structure of NBD-Cl.

anhydrous (Sigma Chemical Co., St. Louis, USA), ce-

fadroxil monohydrate (Amoun Pharmaceutical Industries

Co., APIC, Cairo, Egypt), cefalexin anhydrous (Gal-

axoWellcome, S.A.E., El Salam City, Cairo, Egypt), ce-

fotaxime sodium (CID, Cairo, Egypt), cefoperazone so-

dium (Pfizer Co., Egypt), ceftazidime pentahydrate and

ceftriaxone sodium (T3A Pharma Group, Assiut, Egypt),

cefpodoxime proxetil (Hoechst Marion Roussel, S. A. E.,

Cairo, Egypt), cefixime (El-Hekma Co., Cairo, Egypt)

and cefazolin sodium (Bristol-Myers Squibb Pharma-

ceutical Co., Cairo, Egypt) and pharmaceutical formula-

tions containing the studied drugs were purchased from

local market.

2.3. Preparation of Standard Solutions

Stock solutions containing 100 mg mL-1 of each cepha-

losporin were prepared in double distilled water

(methanol was used in case of cefadroxil monohydrate,

cefalexin anhydrous, cefaclor monohydrate, cefradine

anhydrous, cepodoxime proxetil and cefixime). Working

standard solutions containing 0.5-2.5 mg mL-1 (in case

of cefadroxil monohydrate and cefalexin anhydrous), 1-

6 mg mL-1 (in case of cefradine anhydrous), 2-8 mg mL-1

(in case of cefaclor monohydrate, cefazolin sodium, ce-

fotaxime sodium, ceftriaxone sodium and cefpodoxime

proxetil), 2-10 mg mL-1 (in case of cefixime) and 2-

16 mg mL-1 (in case of cefoperazone sodium and cef-

tazidime pentahydrate) were prepared by suitable dilu-

tion of the stock solution with double distilled water (in

case of cefadroxil monohydrate, cefalexin anhydrous,

cefaclor monohydrate, cepodoxime proxetil and ce-

fixime, dilution was made using methanol). The stock

and working standard solutions must be freshly pre-

pared.

2.4. Preparation of Sample Solutions

Tablets and capsules. Twenty tablets or the contents of

20 capsules were weighed, finely powdered and mixed

thoroughly. An accurately weighed amount of the pow-

der obtained from tablets or capsules equivalent to 250 mg

of each drug was transferred into a 25-mL volumetric

flask, dissolved in about 10 mL double distilled water

(10 mL methanol was used in case of cefadroxil mono-

hydrate, cefalexin anhydrous, cefaclor monohydrate,

cefradine anhydrous, cepodoxime proxetil and cefixime),

sonicated for 15 min, diluted to the mark with double

distilled water (in case of cefadroxil monohydrate, ce-

falexin anhydrous, cefaclor monohydrate, cefradine an-

hydrous, cefpodoxime proxetil and cefixime, dilution

was made using methanol), mixed well and filtered; the

first portion of the filtrate was rejected. Further dilutions

with the same solvent were made to obtain sample solu-

tion containing the specified concentration for each drug

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

831

as mentioned under the preparation of standard solutions

and then the general procedure was followed.

Vials and powder for oral suspension. An accurately

weighed amount of powder equivalent to 250 mg of each

drug was transferred into a 25-mL volumetric flask, then

the procedure was followed as under tablets and capsules

beginning from (dissolved in about 10 mL double dis-

tilled water……….).

2.5. General Procedure

Accurately measured one milliliter aliquot volume of the

standard or sample solutions was transferred into 10-mL

volumetric flask. 5 mL of 0.5 M NaOH were added and

the flask was heated in a boiling water-bath for 30 min,

cooled to room temperature and completed to volume

with double distilled water. One milliliter of the resulting

drug hydrolysate was pipetted into 10-mL volumetric

flask, 1.0 mL of 3 × 10-3 M NBD-Cl was added followed

by 1 mL of concentrated HCl. The resulting solution was

mixed well and the flask was completed to volume with

ethanol. The absorbance was measured at 390 nm against

reagent blank treated similarly.

3. RESULTS AND DISCUSSION

3.1. Absorption Spectra

As shown in Figure 3, the absorption spectrum of

NBD-Cl in acetone shows a maximum absorption at

340 nm. All the investigated drugs after alkaline hy-

drolysis give a very weak absorption taking cefalexin

anhydrous hydrolysate as a representative example

which gives a very broad absorption maximum at 350 nm.

The interaction colored product of cefalexin anhydrous

hydrolysate with NBD-Cl shows absorption maximum at

390 nm (Figure 3).

Figure 3. Absorption spectra of (a) NBD-Cl (3 ×

10-3 M), (b) cefalexin anhydrous hydrolysate alone

(20 µg mL-1) and (c) the reaction colored product

between NBD-Cl and cefalexin anhydrous hydro-

lysate.

3.2. Optimization of Reaction Variables

Since the developed method depends on the formation of

colored product by the interaction of NBD-Cl with sul-

fide ions resulted from the alkaline degradation of

cephalosporins so, optimization studies were carried out

extensively to find the optimum conditions for the alka-

line degradation and subsequently the optimum yield of

sulfide ions and the maximum stability of the chromogen

formed taking cefalexin anhydrous (15 μg mL-1) as a

representative example for these studies. These variables

include:

Effect of NaOH concentration. The influence of so-

dium hydroxide concentration on producing the maxi-

mum absorption intensity was investigated using 0.1-

1.0 M NaOH keeping other factors constant. Maximum

absorption readings were obtained upon using 0.5 M

NaOH; above this concentration and up to 1 M NaOH,

the absorbance remains constant. So, this concentration

was selected for further work (Figure 4).

Effect of hydrolysis time. The effect of hydrolysis time

on the absorption intensity was studied using different

heating times in a boiling water bath (at 100°C) starting

from 10 min until 2 hours and the reaction was carried

out as usual. The obtained absorbance readings were

plotted against hydrolysis time. The maximum absorp-

tion intensity was attained after 20 min and remained

stable for at least 100 min. Thirty minutes hydrolysis

time was used in all subsequent experiments as shown in

Figure 5.

Effect of NBD-Cl concentration. The concentration of

NBD-Cl, for the maximum color development was var-

ied in the range of 0.75 × 10-3-4 × 10-3 M. It was found

that 1 mL of 3 × 10-3 M NBD-Cl was the most suitable con-

0.1

0.2

0.3

0.4

0.5

0.6

00.1 0.20.3 0.4 0.5 0.6 0.7 0.80.911.1

NaOH concentration (M)

Absorbance, 390 nm

Figure 4. Effect of NaOH concentration on the absorbance

of the reaction colored product at 390 nm.

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

832

0.1

0.2

0.3

0.4

0.5

0.6

0 20406080100120140

Hydrolysis time (min)

Absorbance, 390 nm

Figure 5. Effect of hydrolysis time on the absorbance of the

reaction colored product at 390 nm.

Figure 6. Effect of NBD-Cl concentration on the ab-

sorbance of the reaction colored product at 390 nm.

centration for determination of the studied drugs as

shown in Figure 6. Owing to the presence of labile chlo-

ride, a daily fresh solution is recommended.

Effect of type and concentration of acid. Different ac-

ids such as sulfuric, hydrochloric, perchloric, nitric and

acetic acids were tested to determine the most suitable

acid for the reaction. One milliliter of concentrated hy-

drochloric acid was selected in this study as it gave the

highest absorbance readings taking cefalexin anhydrous

(15 μg mL-1) as a representative example (Table 2).

Further investigations were carried out in order to find

the most suitable concentration of hydrochloric acid. It

was observed that higher absorbance readings and more

reproducible results were obtained upon increasing hy-

drochloric acid concentration. As a result of these inves-

tigations, 1 mL of concentrated hydrochloric acid was

used for subsequent work.

Effect of reaction time. The reaction between the in-

vestigated drugs hydrolysates and NBD-Cl was very

Table 2. Effect of different acids on the absorbance readings of

the reaction colored product of cefalexin anhydrousa with

NBD-Cl.

Acid (1mL) Absorbanceb

Hydrochloric acid 0.460

Sulfuric acid 0.400

Perchloric acid 0.413

Acetic acid 0.210

Nitric acid 0.315

aCefalexin anhydrous concentration used is 15 μg mL-1; bAverage

of three determinations.

rapid and the interaction colored product can survive

before dilution unchanged for at least 1 hour. However,

measurements were achieved instantaneously.

Effect of diluting solvent. Different solvents were

tested in order to select the most appropriate solvent for

optimum color development. The results given in Table

3 show small shifts in the position of the maximum ab-

sorption peak. The absorption intensities were slightly

influenced. Ethanol was used throughout this work be-

cause it gave the highest absorbance readings and the

most reproducible results.

Stability of the reaction colored product. Stability time

was obtained by following the absorbance readings of

the developed reaction product for 24 hours at room

temperature (25 ± 5°C). It was found that the produced

color was stable for 24 hours for all studied drugs.

3.3. Calibration Curves

Linear relationship was obtained for all studied drugs by

applying the developed method (Table 4). Good linear-

ity of the calibration curves were clearly evident by ex-

cellent correlation coefficients which ranged from

0.9990 to 0.9999 and coefficients of determination

ranged from 0.9978 to 0.9998. This wide variation in the

linearity range may be attributed to the different yields

Table 3. Effect of solvent on λ max and the absorbance of the

formed chromogen between cefalexin anhydrousa and NBD-Cl.

Solvent λmax(nm) Ab

Water 404 0.404

Ethanol 390 0.470

Methanol 391 0.401

Acetone 392 0.427

Acetonitrile 398 0.456

Propan-1-ol 390 0.467

Propan-2-ol 389 0.463

Dimethylformamaide 393 0.425

Dimehtylsulfoxide 401 0.458

aCefalexin anhydrous concentration is 15 μg mL-1; bAverage of 3

determinations.

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

833

Table 4. Summary of quantitative parameters and statistical data using the proposed procedure.

Drug Intercept

(a) ± SDa

Slope

(b) ± SDa

Linear-

ity

Range

(µg

mL-1)

Correlation

coefficient

(r)

Determina-

tion coeffi-

cient

(r2)

LODb

(µg

mL-1)

LOQc

(µg

mL-1)

Cefadroxil monhy-

drate -0.013 ± (3.6 × 10-3) 0.041 ± (0.2 × 10-3)5-25 0.9999 0.9998 0.29 0.88

Cefalexin anhydrous -0.126 ± (5.0 × 10-2) 0.398 ± (1.2 × 10-3)5-25 0.9999 0.9998 0.42 1.26

Cefradine anhydrous 0.076 ± (7.5 × 10-3) 0.013 ± (1.5 × 10-4)10-60 0.9999 0.9998 1.90 5.77

Cefaclor monohy-

drate 0.055 ± (1.3 × 10-2) 0.010 ± (2.1 × 10-4)20-80 0.9996 0.9992 4.29 13.00

Cefazolin sodium 0.016 ± (4.3 × 10-3) 0.009 ± (1.9 × 10-4)20-80 0.9994 0.9988 1.58 4.78

Ceftriaxone sodium 0.033 ± (5.3 × 10-3) 0.010 ± (0.5 ×10-4) 20-80 0.9996 0.9992 1.75 5.30

Cefotaxime sodium 0.056 ± (1.2 × 10-2) 0.010 ± (1.5 × 10-4)20-80 0.9990 0.9980 3.96 12.00

Cefpodoxime proxetil 0.046 ± (1.6 × 10-2) 0.009 ± (1.3 × 10-4)20-80 0.9990 0.9980 5.87 17.78

Cefixime 0.095 ± (3.2 × 10-3) 0.007 ± (0.2 × 10-4)20-100 0.9989 0.9978 1.51 4.57

Cefoperazone sodium 0.019 ± (8.3 × 10-3) 0.005 ± (0.6 × 10-4)20-160 0.9998 0.9996 5.48 16.60

Ceftazidime penta-

hydrate 0.048 ± (7.4 × 10-3) 0.005 ± (0.7 × 10-4)20-160 0.9994 0.9988 4.88 14.80

aAverage of six determinations; b Limit of detection; c Limit of quantitation.

of sulfide ions from the studied cephalosporins [45].

3.4. Method Validation Study

The method was validated according ICH guidelines on

the validation of analytical methods [46] and complied

with USP 31 validation guidelines [3]. All results were

expressed as percentages, where n represents the number

of values. For the statistical analysis Excel 2003 (Mi-

crosoft Office) was used. A 5% significance level was

selected.

LOD and LOQ. The limits of detection and quantita-

tion for all studied drugs ranged from 0.29 to 5.87 and

from 0.88 to 17.78 μg mL-1; respectively which indicate

high sensitivity of the proposed method (Table 4).

Accuracy. The accuracy of the method was deter-

mined by investigating the recovery of each of the stud-

ied drugs at three concentration levels covering the spe-

cified range (six replicates of each concentration). The

results shown in Table 5 depict good accuracy and re-

covery percentage ranged from 98.0 to 102.3%.

Precision. As shown in Table 6, the small values of

SD and % RSD point to high precision of the proposed

method.

Selectivity. The effect of the presence of common ex-

cipients such as; starch, talc, lactose, glucose, sucrose,

magnesium-stearate and gum acacia was studied. It was

found that no interference was introduced by any of

them.

Robustness. Robustness was examined by evaluating

the influence of small variation in the experimental pa-

rameters on the analytical performance of the proposed

method [47]. The studied parameters were: NaOH con-

centration, NBD-Cl concentration, heating temperature

and heating time on the method suitability and sensitivity.

It was found that none of these variables significantly

affects the performance of the method (Table 7) which

indicates the robustness of the proposed method.

3.5. Applications to the Analysis of

Pharmaceutical Dosage Forms

The proposed method was applied successfully for de-

termination of the studied drugs in their pharmaceutical

dosage forms. Six replicate measurements were made in

each case, the results obtained were validated by com-

parison with a previously reported method [48]. No sig-

nificant difference was found by applying t- and F-tests

at 95% confidence level indicating good accuracy and

precision (Table 8). Recovery studies were also carried

out by standard addition method [49]. The results in Ta-

ble 9 indicate good recoveries (96.0 to 103.8%) and

confirm that there is no interference from frequently

encountered excipients or additives.

3.6. Suggested Reaction Mechanism

Cephalosporins were previously reported to produce

sulfide ions upon alkaline degradation and it was found

to be one of their major degradation products [43-45,

50-55]. NBD-Cl is an active halide derivative, which

was considered as a likely target for good nuclophiles,

under alkaline conditions, such as amines, amino acids

and thiocompounds [40-42].

In the proposed method, sulfide ions were allowed to

react with NBD-Cl via SN2 mechanism. The high nu-

cleophilicity of sulfide ions, the presence of Cl– anion as

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

834

Table 5. Accuracy of the proposed method for analysis of the studied drugs at three concentration levels.

Recovery (%) ± SD

Drug 25 µg mL-1 50 µg mL-1 75 µg mL-1

Cefaclor monohydrate 100.6 ± 0.93 101.4 ± 0.75 102.1 ± 0.30

Ceftriaxone sodium 99.3 ± 0.52 100.6 ± 0.96 100.2 ± 0.51

Cefotaxime sodium 99.7 ± 1.35 101.5 ± 0.83 101.3 ± 1.16

Cefixime 98.3 ± 1.24 98.7 ± 0.58 98.6 ± 0.73

Cefazolin sodium 101.1 ± 1.08 98.9 ± 0.60 102.3 ± 0.68

Cefpodoxime proxetil 99.4 ± 0.35 99.4 ± 0.47 99.0 ± 0.29

Recovery (%) ± SD

Drug 10 µg mL-1 15 µg mL-1 20 µg mL-1

Cefadroxil monohydrate 99.9 ± 1.31 100.4 ± 0.83 99.1 ± 0.90

Cefalexin anhydrous 98.6 ± 0.26 102.2 ± 1.29 98.0 ± 0.41

Recovery (%) ± SD

Drug 40 µg mL-1 80 µg mL-1 120 µg mL-1

Ceftazidime pentahydrate 102.3 ± 0.86 98.9 ± 1.25 99.6 ± 0.82

Cefoperazone sodium 98.0 ± 0.70 100.3 ± 1.11 101.4 ± 1.03

Recovery (%) ± SD

Drug 15 µg mL-1 30 µg mL-1 45 µg mL-1

Cefradine anhydrous 98.3 ± 0.51 100.8 ± 0.66 100.7 ± 0.87

aAverage of six replicates.

Table 6. Intra- and inter-day precision of the proposed spectrophotometric method.

Intra-day precision Inter-day precision

Drug Drug Conc. (µg

mL-1) Mean ± SDa % RSD Mean ± SDa % RSD

Cefaclor monohydrate 25 99.6 ± 0.93 0.93 99.4 ± 1.29 1.30

50 99.9 ± 1.65 1.66 98.5 ± 0.90 0.91

75 100.1 ± 1.40 1.40 100.7 ± 1.12 1.12

Cefalexin anhydrous 10 100.3 ± 1.17 1.16 98.9 ± 1.23 1.25

15 100.4 ± 1.53 1.52 99.0 ± 0.97 0.98

20 100.1 ± 1.16 1.16 100.5 ± 0.88 0.88

Cefadroxil monohydrate 10 99.4 ± 0.99 1.00 101.0 ± 1.09 1.08

15 99.9 ± 0.85 0.85 100.5 ± 0.77 0.76

20 100.2 ± 1.37 1.37 98.7 ± 0.68 0.69

Cefradine anhydrous 15 100.1 ± 1.03 1.02 99.5 ± 1.13 1.14

30 100.0 ± 1.15 1.15 98.4 ± 0.85 0.86

45 100.6 ± 1.16 1.15 100.4 ± 1.54 1.54

Cefoperazone sodium 40 99.6 ± 0.93 0.93 101.3 ± 1.43 1.41

80 99.7 ± 0.67 0.67 101.7 ± 1.63 1.61

120 100.3 ± 1.35 1.34 99.1 ± 1.40 1.41

Ceftazidime pentahydrate 40 100.0 ± 1.39 1.39 100.9 ± 0.99 0.98

80 99.6 ± 1.30 1.31 101.3 ± 1.27 1.25

120 99.9 ± 1.04 1.05 98.9 ± 1.12 1.13

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

835

Table 6. (Continued).

Intra-day precision Inter-day precision

Drug Drug Conc. (µg

mL-1) Mean ± SDa % RSD Mean ± SDa % RSD

Ceftriaxone sodium 25 100.4 ± 1.57 1.56 99.2 ± 0.99 1.00

50 101.0 ± 1.27 1.26 100.8 ± 1.15 1.14

75 99.8 ± 1.38 1.39 98.6 ± 1.54 1.57

Cefotaxime sodium 25 99.3 ± 1.05 1.06 99.0 ± 0.77 0.78

50 98.8 ± 0.78 0.79 101.4 ± 1.46 1.44

75 99.4 ± 1.29 1.30 98.5 ± 0.91 0.93

Cefixime 25 99.5 ± 0.81 0.81 100.9 ± 0.99 0.98

50 99.8 ± 1.02 1.03 99.7 ± 1.17 1.17

75 99.6 ± 1.48 1.48 98.3 ± 1.65 1.67

Cefazolin sodium 25 100.5 ± 1.15 1.14 100.6 ± 0.68 0.67

50 100.6 ± 1.36 1.35 99.6 ± 1.35 1.35

75 101.4 ± 0.74 0.73 98.8 ± 1.12 1.14

Cefpodoxime proxetil 25 100.4 ± 0.89 0.89 101.3 ± 0.77 0.76

50 100.5 ± 1.15 1.15 99.7 ± 1.55 1.55

75 100.0 ± 1.70 1.70 100.6 ± 1.63 1.62

aAverage of six determinations.

Table 7. Robustness of the proposed spectrophotometric method.

Recovery (%) ± SDa

Experimental parameter variationCefadroxil

monohydrate

(20 µg mL-1)

Cefalexin

anhydrous

(20 µg mL-1)

Cefradine

anhydrous

(40 µg mL-1)

Cefaclor

monohydrate

(60 µg mL-1)

Cefopera-

zone sodium

(80 µg mL-1)

Ceftazidime

pentahydrate

(80 µg mL-1)

No variationb 99.4 ± 1.21 99.8 ± 0.31 99.5 ± 1.82 99.5 ± 1.13 99.2 ± 0.56 99.5 ± 0.47

1- NaOH concentration

0.45 M

0.55 M

98.3 ± 0.85

98.6 ± 1.19

97.9 ± 1.20

100.9 ± 1.15

101.5 ± 1.32

98.2 ± 0.52

97.5 ± 0.54

101.3 ± 0.77

101.8 ± 1.11

99.4 ± 1.31

97.4 ± 1.12

98.3 ± 1.34

2- NBD-Cl concentration

2.8 × 10-3M

3.2 × 10-3M

102.0 ± 0.25

98.5 ± 1.31

100.3 ± 1.35

100.9 ± 0.92

98.2 ± 1.56

98.0 ± 1.15

102.1 ± 0.35

98.5 ± 0.91

100.9 ± 1.58

98.5 ± 0.83

100.9 ± 1.15

102.0 ± 0.88

3- Heating temperature

95°C

100°C

98.8 ± 0.78

99.6 ± 1.35

97.9 ± 1.20

100.8 ± 1.60

102.4 ± 156

100.4 ± 0.90

102.7 ± 2.21

99.8 ± 1.02

101.0 ± 1.27

101.3 ± 1.27

101.4 ± 2.04

98.0 ± 2.15

4- Heating time

25 min

35 min

100.5 ± 1.23

100.1 ± 1.40

99. 2 ± 0.99

100.4 ± 1.53

98.5 ± 1.57

98.7 ± 0.68

97.5 ± 1.98

99.6 ± 0.93

99.1 ± 1.40

100.9 ± 0.99

99.4 ± 1.29

98.5 ± 0.90

Table 7. (Continued).

Recovery (%) ± SDa

Experimental parameter variationCeftriaxone so-

dium

(60 µg mL-1)

Cefotaxime

sodium

(60 µg mL-1)

Cefixime

(60 µg mL-1)

Cefazolin

sodium

(60 µg mL-1)

Cefpodoxime

proxetil

(60 µg mL-1)

No variationb 99.5 ± 0.66 97.6 ± 1.55 100.7 ± 0.98 99.5 ± 1.23 99.5 ± 1.01

1- NaOH concentration

0.45 M

0.55 M

100.2 ± 1.35

98.7 ± 0.98

99.5 ± 0.67

98.3 ± 0.49

99.6 ± 1.27

98.4 ± 0.73

98.6 ± 0.88

102.4 ± 1.145

98.1 ± 0.60

99.5 ± 1.27

2- NBD-Cl concentration

2.8 × 10-3M

3.2 × 10-3M

99.6 ± 1.15

98.0 ± 0.75

102.0 ± 0.71

102.3 ± 0.58

102.3 ± 1.30

99.5 ± 0.69

99.7 ± 1.35

101.5 ± 0.70

98.4 ± 1.40

99.9 ± 0.49

3- Heating temperature

95°C

100°C

99.0 ± 1.30

97.5 ± 0.68

99.4 ± 0.66

98.4 ± 1.13

98.7 ± 0.66

101.9 ± 1.35

100.6 ± 0.48

97.5 ± 1.44

100.8 ± 0.72

99.1 ± 0.65

4- Heating time

25 min

35 min

101.1 ± 1.44

98.1 ± 0.81

97.8 ± 0.90

101.4 ± 1.41

99.6 ± 0.56

100.8 ± 1.27

99.7 ± 0.70

101.9 ± 0.72

97.8 ± 0.49

98.0 ± 1.34

aAverage of three determinations;

bFollowing the general assay procedure conditions.

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

836

Table 8. Determination of the studied drugs in their pharmaceutical dosage forms.

Recovery % ± SD

Drug Pharmaceutical product

Proposed method

(n = 6)

Reported methodb

(n = 6)

Cefaclor

monohydrate

Ceclor® suspensionc

250 mg of cefaclor monohydrate/5 mL

97.8 ± 0.5, t = 0.382a

F = 1.562a 97.7 ± 0.40

Bacticlor® suspensiond

250 mg of cefaclor anhydrous/5 mL

97.2 ± 0.5, t = 1.913

F = 1.562 96.7 ± 0.40

Cefadroxil

monohydrate

Duricef® tabletse

1 g of cefadroxil monohydrate/tablet

98.7 ± 0.3, t = 2.038

F = 2.250 99.0 ± 0.20

Duricef® suspensione

250 mg of cefadroxil monohydrate/5 mL

96.6 ± 1.3, t = 1.605

F = 2.641 97.6 ± 0.80

Duricef® capsulese

500 mg of cefadroxil monohydrate/capsule

97.9 ± 1.3, t = 1.332

F = 1.032 98.9 ± 1.30

Biodroxil® capsulesf

500 mg of cefadroxil monohydrate/capsule

102.4 ± 1.4, t = 0.930

F = 1.361 101.7 ± 1.20

Biodroxil® suspensionf

250 mg of cefadroxil monohydrate/5 mL

103.1 ± 0.6, t = 1.359

F = 2.250 102.7 ± 0.40

Cefalexin

anhydrous

Ceporex® tabletsg

500 mg of cefalexin anhydrous/tablet

99.3 ± 1.6, t = 0.646

F = 1.778 98.7 ± 1.20

Ceporex® suspensiong

250 mg of cefalexin anhydrous/5 mL

99.0 ± 1.5, t = 0.735

F = 3.516 98.5 ± 0.80

Ospexin® suspensionh

250 mg of cefalexin anhydrous/5 mL

103.5 ± 1.5, t = 0.576

F = 3.516 103.1 ± 0.80

Cefradine

anhydrous

Velosef® capsulese

250 mg of cefradine anhydrous/capsule

97.7 ± 0.5, t = 1.530

F = 1.562 97.3 ± 0.40

Velosef® tabletse

1 g of cefradine anhydrous/tablet

103.3 ± 1.2, t = 1.019

F = 2.250 102.7 ± 0.80

Velosef® suspensione

205 mg of cefradine anhydrous/5mL

99.0 ± 1.5, t = 0.588

F = 3.516 98.5 ± 0.80

Velosef® vialse

1 g of cefradine anhydrous/vial

97.9 ± 1.2, t = 0.267

F = 1.778 97.7 ± 0.90

Cefotax® vialsi

500 mg of cefotaxime sodium/vial

98.2 ± 1.8, t = 0.365,

F = 3.932; 97.9 ± 0.90

Cefotaxime

sodium Claforan® vialsj

500 mg of cefotaxime sodium/vial

97.9 ± 1.2, t = 0.326,

F = 1.778; 97.7 ± 0.90

Ceftazidime

pentahydrate

Fortum® vialsk

500 mg of ceftazidime pentahydrate/vial

98.9 ± 0.6, t = 1.460,

F = 4.001; 98.5 ± 0.30

Cefoperazone

sodium

Cefozon® vialsl

500 mg of cefoperazone sodium/vial

102.3 ± 1.4, t = 1.721,

F = 4.003; 101.2 ± 0.70

Ceftriaxone

sodium

Ceftriaxone® vialsm

500 mg of ceftriaxone sodium/vial

97.7 ± 0.4, t = 1.643,

F = 4.002; 98.0 ± 0.20

Cefixime Xi macef® capsulesn

400 mg of cefixime/capsule

102.1 ± 1.4, t = 0.797

F = 1.361 101.5 ± 1.20

Cefazolin

sodium

Zinol® vialso

500 mg of cefazolin sodium/vial

98.9 ± 1.3, t = 1.493

F = 3.449 98.0 ± 0.70

Cefpodoxime

proxetil

Orelox® tabletsq

100 mg of cefpodoxime proxetil/tablet

98.2 ± 1.8, t = 0.298

F = 3.932 97.9 ± 0.90

a Theoretical value for t and F at 95% confidence limit, t = 2.228 and F = 5.053; b Reference 48; cEgyptian Pharmaceuticals and chemicals in-

dustries Co., S.A.E., Bayad El-Arab, Beni Suef, Egypt; d Pharco Pharmaceuticals, Alexandria under license from Ranbaxy UK; e Bristol-Myers

Squibb Pharmaceutical Co., Cairo, Egypt; f Kahira Pharm. & Chem. Ind. Co. under license from Novartis Pharma S.A.E., Cairo, Egypt;

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

837

g GlaxoSmithKline, S.A.E., El Salam City, Cairo, Egypt; h Pharco Pharmaceuticals, Alexandria under license from Biochemie GmbH., Vienna,

Austria; i T3A Pharma Group, Assiut, Egypt; j Hoechst Orient, S.A.E., Cairo, Egypt ; k GalaxoWellcome, S.A.E., El Salam City, Cairo, Egypt;

lEgyptian International Pharmaceutical Industries Co., El Asher Ramadan City, Cairo, Egypt; m Kahira Pharm. & Chem. Ind. Co. under licence

from Novartis Pharma S.A.E., Cairo, Egypt; n Sigma pharmaceutical industries, S.A.E., Egypt; oPharco Pharmaceuticals, Alexandria,

Egypt; q Aventis, Zeitoun, Cairo, Egypt.

Table 9. Standard addition method for the assay of the studied drugs in their pharmaceutical dosage forms by the proposed method.

Drug Pharmaceutical

formulation

Authentic drug added

(μg mL-1)

Authentic drug found

(μg mL-1)

Recovery (%) ± SDa

10.00 9.97 99.7 ± 1.21

20.00 19.40 97.0 ± 1.80

Ceclor® suspension

30.00 28.83 96.1 ± 1.52

10.00 10.06 100.6 ± 1.10

20.00 19.26 96.3 ± 0.81

Cefaclor

monohydrate

Bacticlor® suspension

30.00 29.40 98.0 ± 0.90

5.00 5.11 102.2 ± 1.41

10.00 9.95 99.5 ± 1.72

Duricef®

tablets 15.00 14.85 99.0 ± 0.50

5.00 4.90 98.0 ± 1.81

10.00 10.02 100.2 ± 1.63

Duricef® suspension

15.00 14.64 97.6 ± 1.12

5.00 4.83 96.6 ± 1.50

10.00 9.60 96.0 ± 1.30

Duricef® capsules

15.00 14.78 98.5 ± 0.71

5.00 4.89 97.8 ± 1.54

10.00 10.36 103.6 ± 0.91

Biodroxil® capsules

15.00 14.98 99.9 ± 0.60

5.00 4.87 97.4 ± 1.91

10.00 9.84 98.4 ± 0.42

Cefadroxil

monohydrate

Biodroxil® suspension

15.00 15.38 102.5 ± 1.20

5.00 4.98 99.6 ± 1.41

10.00 10.07 100.7 ± 1.73

Ceporex® tablets

15.00 14.82 98.8 ± 0.62

5.00 5.05 101.0 ± 1.00

10.00 9.79 97.9 ± 1.81

Ceporex® suspension

15.00 15.06 100.4 ± 0.91

5.00 4.95 99.0 ± 1.30

10.00 9.92 99.2 ± 1.81

Cefalexin anhydrous

Ospexin® suspension

15.00 14.68 97.9 ± 1.94

10.00 9.85 98.5 ± 0.85

20.00 20.09 100.5 ± 0.63 Velosef® capsules

30.00 28.86 96.2 ± 1.11

10.00 9.69 96.9 ± 0.80

20.00 19.85 99.3 ± 0.50

Velosef®

tablets 30.00 30.10 100.3 ± 0.71

10.00 9.72 97.2 ± 0.92

20.00 19.94 99.7 ± 1.50 Velosef® suspension

30.00 31.14 103.8 ± 1.93

10.00 10.16 101.6 ± 1.10

20.00 19.62 98.1 ± 1.63

Cefradine

anhydrous

Velosef®

vials 30.00 29.85 99.5 ± 1.94

20.00 20.34 101.7 ± 1.33

40.00 38.76 96.6 ± 1.63

Ceftazidime

pentahydrate

Fortum®

vial 60.00 61.32 102.2 ± 1.32

10.00 9.66 96.6 ± 0.7

20.00 20.20 101.0 ± 0.9

Cefotax®

vials 30.00 29.30 97.6 ± 1.5

10.00 10.35 103.5 ± 1.8

20.00 19.60 98.0 ± 1.6

Cefotaxime

Sodium Claforan®

vials 30.00 29.19 97.3 ± 0.7

20.00 19.50 97.5 ± 1.8

40.00 38.88 97.2 ± 0.9

Cefoperazone

Sodium

Cefozon®

vials 60.00 59.46 99.1 ± 0.7

10.00 10.08 100.8 ± 1.4

20.00 19.36 96.8 ± 1.5

Ceftriaxone

Sodium Ceftriaxone vials

30.00 30.98 103.3 ± 1.3

A. H. Rageh et al. / Natural Science 2 (2010) 828-840

838

Table 9 (Continued)

Drug Pharmaceutical

formulation

Authentic drug added

(μg mL-1)

Authentic drug found

(μg mL-1)

Recovery (%) ± SDa

15.00 14.76

98.4 ± 0.5

30.00 29.82

99.4 ± 1.1

Cefixime Ximacef® capsules

45.00 44.91

99.8 ± 1.4

10.00 9.78

97.8 ± 0.8

20.00 19.87

99.4 ± 0.7

Cefazolin

Sodium

Zinol®

vials 30.00 30.27

100.9 ± 1.7

10.00 9.84

98.4 ± 0.9

20.00 19.83

99.2 ± 1.5

Cefpodoxime

proxetil

Orelox®

tablets 30.00 29.60

98.6 ± 1.6

a Average of six determinations.

a good leaving group at position 4 in addition to the

presence of nitro group as an electron withdrawing

group at position 7 of the aromatic ring in NBD-Cl result

in replacement of Cl– anion with the attacking sulfide

ions which in turn lead to the formation of a yellow-

colored chromophore (λmax at 390 nm). The reaction

product is stable in strong acidic medium, moreover

acidification could minimize possible competition be-

tween the generated sulfide nucloephile and excess OH–

which may lead to decrease in the chromogen formed.

The proposed reaction mechanism is given in the fol-

lowing scheme:

NO2

0.5M NaOH

100°C/ 30 min

Conc. HCl

NO2

HNC

Sulfide ions

Scheme 1 Suggested reaction mechanism between sul-

fide ions and NBD-Cl

The production of sulfide ions was confirmed by car-

rying out specific qualitative tests such as dilute hydro-

chloric acid, cadmium acetate, sodium nitroprusside and

methyelene blue tests [56] or by comparing λmax of the

formed chromogen with that obtained after applying the

developed method to sodium sulfide and the same results

were obtained.

4. Conclusions

The developed spectrophotometric method is precise,

accurate and sensitive. No interference from the fre-

quently encountered excipients and additives. Statistical

analysis proves that the method could be applied for the

analysis of the studied drugs in their pure forms and in

pharmaceutical formulations.

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