Introduction

1

Introduction

It is worthy to mention that it is vital to determine the purity and

concentration of any therapeutic drug in high accuracy and precision.

One of the most important procedures is the spectrophotometric method

having the advantage of being simple and rabid. In this study, a

spectrophotometric method has been developed and validated for

determination of H2-receptor cefotaxime, ceftazidime and cefepime in

pure and pharmaceutical formulations. The applied method is

characterized by simplicity, selectivity and high sensitivity. In this

chapter, short notes about the physical and chemical characters, mode

of action and use are given. Also, a historical survey on some previous

works concerned the determination of the drugs under investigation is

shown briefly.

Cefotaxime

Cefotaxime has the following chemical structure:

Its IUPAC name is: 6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-

aminothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-zabicyclo

[4.2.0] oct-2-ene-2-carboxylic acid

Cefotaxime is a third-generation cephalosporin antibiotic. Like other

third-generation cephalosporins, it has broad spectrum activity against

Introduction

2

Gram positive and Gram negative bacteria. In most cases, it is

considered to be equivalent to ceftriaxone in terms of safety and

efficacy.

Mechanism of action

Inhibits bacterial cell wall synthesis by binding to one or more of

the penicillin-binding proteins (PBPs) which in turn inhibits the final

transpeptidation step of peptidoglycan synthesis in bacterial cell walls,

thus inhibiting cell wall biosynthesis. Bacteria eventually lyse due to

ongoing activity of cell wall autolytic enzymes (autolysins and murein

hydrolases) while cell wall assembly is arrested.

Cefotaxime, like other β-lactam antibiotics does not only block the

division of bacteria, including cyanobacteria, but also the division of

cyanelles, the photosynthetic organelles of the Glaucophytes, and the

division of chloroplasts of bryophytes. In contrast, it has no effect on the

plastids of the highly developed vascular plants. This is supporting the

endosymbiotic theory and indicates an evolution of plastid division in

land plants.

Clinical use

Cefotaxime is used for infections of the respiratory tract, skin,

bones, joints, urogenital system, meningitis, and septicemia. It generally

has good coverage against most Gram-negative bacteria, with the

notable exception of Pseudomonas. It is also effective against most

Gram-positive cocci except for Enterococcus. It is active against

penicillin-resistant strains of Streptococcus pneumoniae. It has modest

activity against the anaerobic Bacteroides fragilis.

Introduction

3

Literature survey on the microdetermination of cefotaxime

Ni Yn(1) found that Cefuroxime sodium, ceftriaxone sodium,

cefotaxime sodium and cefazolin sodium had absorption in ultraviolet

region, and their absorption spectra are overlapping. So they can not be

determined individually by spectrophotometry without prior separation.

In this paper, the chemometric multivariate calibration method was

applied to the simultaneous determination of these four compounds in a

Britton-Robinson buffer solution (pH 2.09), and the analytical results

were compared with those by classical least squares (CLS), principal

components regression (PCR) and partial least squares (PLS). The

linear ranges of cefuroxime sodium, ceftriaxone sodium, cefotaxime

sodium and cefazolin sodium were 1.0-20.0, 2.0-20.0, 2.0-20.0 and 1.0-

18.0 µg/mL, respectively. The proposed procedure was successfully

applied in the determination of these drugs in rabbit serum, and the

result obtained from spectrophotometry was compared with the one by

HPLC with no significant difference found.

A simple, reliable, and sensitive kinetic spectrophotometric method

was developed for determination of eight cephalosporin antibiotics,

namely, Cefotaxime sodium, Cephapirin sodium, Cephradine dihydrate,

Cephalexin monohydrate, Ceftazidime pentahydrate, Cefazoline

sodium, Ceftriaxone sodium, and Cefuroxime sodium(2). The method

depended on oxidation of each of the studied drugs with alkaline

potassium permanganate. The reaction was followed spectrophot-

ometrically by measuring the rate of change of absorbance at 610 nm.

The initial rate and fixed time (at 3 minutes) methods were utilized for

construction of calibration graphs to determine the concentration of the

studied drugs. The calibration graphs were linear in the concentration

ranges 5–15μg/mL and 5–25μg/mL using the initial rate and fixed time

methods, respectively. The results were validated statistically and

checked through recovery studies. The method had been successfully

Introduction

4

applied for the determination of the studied cephalosporins in

commercial dosage forms. Statistical comparisons of the results with the

reference methods showed the excellent agreement and indicated no

significant difference in accuracy and precision.

Three simple, rapid and sensitive spectrophotometric procedures

were developed for the analysis of cephapirin sodium (1), cefazoline

sodium (2), cephalexin monohydrate (3), cefadroxil monohydrate (4),

cefotaxime sodium (5), cefoperazone sodium (6) and ceftazidime

pentahydrate (7) in pure form as well as in their pharmaceutical

formulations(3). The methods were based on the reaction of these drugs

as n-electron donors with the σ-acceptor iodine, and the π-acceptors:

2,3-dichloro-5,6-dicyano-p-benzo-quinone (DDQ) and 7,7,8,8-tetracy-

anoquinodimethane (TCNQ). Depending on the solvent polarity,

different coloured charge-transfer complexes and radicals were

developed. Different variables and parameters affecting the reactions

were studied and optimized. The obtained charge-transfer complexes

were measured at 364 nm for iodine (in 1,2-dichloroethane), 460 nm for

DDQ (in methanol) and 843 nm for TCNQ (in acetonitrile). Ultraviolet–

visible, infrared and 1H-nuclear magnetic resonance techniques were

used to study the formed complexes. Due to the rapid development of

colours at ambient temperature, the obtained results were used on thin-

layer chromatograms for the detection of the investigated drugs. Beer's

plots were obeyed in a general concentration range of 6–50, 40–300

and 4–24 μg ml−1 with iodine, DDQ and TCNQ, respectively, with

correlation coefficients not less than 0.9989. The proposed procedures

could be applied successfully to the determination of the investigated

drugs in vials, capsules, tablets and suspensions with good recovery;

percent ranged from 96.47 (±1.14) to 98.72 (±1.02) in the iodine

method, 96.35 (±1.62) to 98.51 (±1.30) in the DDQ method, and 95.98

(±0.78) to 98.40 (±0.87) in the TCNQ method. The association

Introduction

5

constants and standard free energy changes using Benesi–Hildebrand

plots were studied.

A spectrophotometric method had been set up to determine

cefotaxime sodium using potassium ferricyanide as the spectroscopic

probe reagent(4). With the presence of potassium ferricyanide, the

degradation product of cefotaxime sodium can reduce Fe3+ to Fe2+ at

pH 3.0, which facilitate the formation of soluble Prussian Blue

(KFeIII[FeII(CN)6]). The absorbance of soluble Prussian blue was

measured at its absorption maximum of 730 nm and the amount of

cefotaxime sodium can be indirectly calculated. Under optimized

conditions, a good linear relationship is obtained in the range of

0.040~24 mg/L of cefotaxime sodium. The linear regression equation is

A=0.05088 + 0.2166ρ (mg/L) with linear correlation coefficient of

0.9986. The detection limit and relative standard deviation are 0.01

mg/L and 1.36%, respectively. The apparent molar absorption

coefficient of indirect determination of cefotaxime sodium was 2.3×105

L/(mol·cm). This method had been successfully applied to the

determination of cefotaxime sodium in pharmaceutical and serum

samples.

A simple spectrophotometric method for the determination of

cefotaxime, ceftriaxone, cefadroxil and cephalexin with variamine blue

was presented(5). The determination was based on the hydrolysis of β-

lactam ring of cephalosporins with sodium hydroxide which

subsequently reacts with iodate to liberate iodine in acidic medium. The

liberated iodine oxidized variamine blue to violet colored species of

maximum absorption at 556 nm. The absorbance was measured within

the pH range of 4.0-4.2. Beer's law is obeyed in the range of 0.5-5.8 µg/

mL, 0.2-7.0 µg/mL, 0.2-5.0 µg/mL and 0.5-8.5 µg/mL for cefotaxime,

ceftriaxone, cefadroxil and cephalexin respectively. The analytical

parameters were optimized and the method was successfully applied for

Introduction

6

the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin

in pharmaceuticals.

The detailed mechanism of the irreversible oxidation process of

Cefotaxime sodium at the glassy carbon electrode in various buffer

systems and at different pH values was described(6). Differential pulse

and square wave voltammetric methods were developed for its

determination in pharmaceutical dosage forms and spiked human

serum samples according to the linear relation between the peak

current and cefotaxime sodium concentration. For analytical purposes, a

very well resolved diffusion controlled voltammetric peak was obtained

in Britton-Robinson buffer at pH 2.0 at 0.87 and 0.89V for differential

pulse and square wave voltammetric techniques, respectively. The

linear response was obtained within the range of 1x10-6 - 6x10-5 M with

a detection limit of 2.83x10-7 M for differential pulse and 2x10-6 - 6x10-5

M with a detection limit of 3.61x10-7 M for square wave voltammetric

techniques. The repeatability and reproducibility of the methods for both

media (supporting electrolyte and serum sample) were determined.

Precision and accuracy of the developed method were used for the

recovery studies. The standard addition method was used for the

recovery studies. No electroactive interferences were found in biological

fluids from the endogenous substances and additives present in

pharmaceutical dosage form.

A simple, accurate and precise spectrophotometric method had

been proposed for the determination of eleven cephalosporins, namely;

cefaclor monohydrate, cefadroxil monohydrate, cefalexin anhydrous,

cefradine anhydrous, cefotaxime sodium, cefoperazone sodium,

ceftriaxone sodium, ceftazidime penthydrate, cefazolin sodium, cefixime

and cefpodoxime pro- xetil in bulk drug and in pharmaceutical

formulations(7). The method depended on hydrolysis of the studied

drugs using 0.5M NaOH at 100°C and subsequent reaction of the

Introduction

7

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 affecting the reaction (e.g. NaOH concentration,

hydrolysis time, NBD-Cl concentration and diluting 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 detection 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 successfully applied for analysis of

the studied drugs in their pharmaceutical formulations and the recovery

percentages ranged from 96.6 to 103.5%.

A simple, precise and accurate kinetic spectro-photometric

method for determination of cefradine anhydrous, cefaclor

monohydrate, cefadroxil monohydrate, cefalexin anhydrous and

cefixime in bulk and in pharmaceutical formulations had been

developed(8). The method based on a kinetic investigation of the

reaction of the free carboxylic acid group of the drug with a mixture of

potassium iodate and potassium iodide at room temperature to form

yellow coloured triiodide ions. The reaction was followed up

spectrophotometrically by measuring the increase in absorbance at 352

nm as a function of time. The initial rate, fixed time, variable time and

rate constant methods were adopted for constructing the calibration

curves but fixed time method had been found to be more applicable.

The analytical performance of the method, in terms of accuracy and

precision, was statistically validated; the results were satisfactory. The

method had been successfully applied to the determination of the

studied drugs in commercial pharmaceutical formulations. Statistical

comparison of the results with a well established reported method

Introduction

8

showed excellent agreement and proved that there is no significant

difference in the accuracy and precision.

A simple and reproducible spectrophotometeric method for the

assay of cefotaxime sodium, cefuroxime sodium, and ceftriaxone

disodium with metol-chromium(VI) reagent had been developed(9). The

procedure was based on direct oxidation of metol by potassium

dichromate in presence of drug in acidic medium and subsequent

formation of ternary complex. Beer’s law was obeyed in the range 0.2–

28 μg ml−1 at λmax 520 nm. For more accurate analysis, Ringbom

optimum concentration range was found to be 0.8–26.5 μg ml−1. The

molar absorptivity and Sandell sensitivity were calculated. Six replicate

analyses of solutions containing seven different concentrations of the

examined drugs were carried out and gave a mean correlation

coefficient ≤0.9996; the factors of the regression line equation for the

three cephalosporins were calculated. The proposed method was

applied to the determination of the examined drugs in pharmaceutical

formulations and the results demonstrated that the method is equally

accurate, precise, and reproducible as the official methods.

A rapid, accurate and sensitive method had been developed and

validated for the quantitative simultaneous determination of four

cephalosporins, cephalexin and cefadroxil (first-generation), cefaclor

(second-generation) and cefotaxim (third-generation), in pharma-

ceuticals as well as in human blood serum and urine(10). A Spherisorb

ODS-2 250×4-mm, 5-μm analytical column was used with an eluting

system consisting of a mixture of acetate buffer (pH 4.0)–CH3OH 78–

22% (v/v) at a flow-rate 1.2 ml/min. Detection was performed with a

variable wavelength UV–Vis detector at 265 nm resulting in limit of

detection of 0.2 ng for cefadroxil and cephalexin, but only 0.1 ng for

cefotaxime and cefaclor per 20-μl injection. Hydrochlorothiazide (HCT)

(6-chloro-3,4-dihydro-7 sulfanyl-2H-1,2,4-benzothiadiazine-1-1-dioxide)

Introduction

9

was used as internal standard at a concentration of 2 ng/μl. A rectilinear

relationship was observed up to 8, 5, 12 and 35 ng/μl for cefadroxil,

cefotaxime, cefaclor, cephalexin, respectively. Analysis time was less

than 7 min. The statistical evaluation of the method was examined by

means of within-day repeatability (n=8) and day-to-day precision (n=9)

and was found to be satisfactory with high accuracy and precision. The

method was applied to the determination of the cephalosporins in

commercial pharmaceuticals and in biological fluids: human blood

serum after solid-phase extraction and urine simply after filtration and

dilution. Recovery of analytes in spiked samples was in the range from

76.3 to 112.0%, over the range of 1–8 ng/μl.

A flow-injection spectrophotometric method was described for the

determination of cefadroxil (I) and cefotaxime (II)(11). The method was

based on the hydrolysis of the cephalosporin with sodium hydroxide

whereby the sulfide ion was produced. The latter was allowed to react

with N,N-diethyl-p-phenylenediamine sulfate (N,N-DPPD) and Fe (III),

and the blue color produced was measured at 670 nm (method A).

Linear calibration graphs were obtained in the range 36.34–109.2 and

95.48–477.4 μg ml−1 for I and II, respectively. The experimental limits of

detection (three times the noise signal) were 0.036 and 0.048 μg ml−1

for I and II, respectively. The total flow-rate was 5.3 ml min−1 for both

drugs. Alternately, the sulfide ion produced was allowed to react with p-

phenylenediamine dihydrochloride (PPDD) and Fe (III), and the violet

color produced was measured at 597 nm (method B). Linear calibration

graphs were obtained in the range 0.5–400 and 0.5–450 μg ml−1 for I

and II, respectively. The limits of detection were 0.4 and 0.2 μg ml−1 for

I and II, respectively. The total flow-rate was 3 ml min−1 for both drugs.

The methods had been successfully applied to the analysis of some

pharmaceutical formulations, particularly of the injection and capsule

types. The relative standard deviation (RSD) (n=10) at the 50 and 100

Introduction

10

μg ml−1 levels of I and II were 0.83–0.77 and 0.9–0.8% with N,N-DPPD

and PPDD as reagents, respectively. Recoveries were quantitative; the

results obtained agreed with those obtained by other reported methods.

Two simple, accurate, sensitive and selective procedures for the

determination of eight cephalosporins were described(12). These

procedures were based on the formation of ion-pair complexes between

the drugs and ammonium reineckate. The formed precipitates were

quantitatively determined either colourimetrically or by atomic

absorption spectrometry. The methods consisted of reacting drugs with

Reinecke's salt in an acidic medium at 25±2°. The first colourimetric

procedure (procedure I) was based on dissolving the formed precipitate

with acetone, the volume was completed quantitatively and the

absorbance of the solution was measured at 525 nm against pure

solvent blank. Also, the formed precipitates on the atomic absorption

spectrometric procedure (procedure II) were quantitatively determined

directly or indirectly through the chromium precipitate formed or the

residual unreacted chromium in the filtrate at 358.6 nm. The optimum

conditions for precipitation had been carefully studied. Beer's law was

obeyed for the studied drugs in the range 5–35 μg ml−1 with correlation

coefficients 0.9989. Both procedures I and II hold well accuracy and

precision when applied to the analysis of the cited cephalosporins in

different dosage forms with good recovery percent ranged from

98.7±0.90 to 100.1±0.74 without interference from additives.

The precision of UV absorbance of acid degraded

cephalosporins, ninhydrin, high performance liquid chromatography and

iodometric methods used for analysis of cefoxitin, cefotaxime,

cephazolin and cephalexin were compared(13). To obtain the calibration

graphs the analytical signal used were: absorbance, first derivative

absorbance, second derivative absorbance and H-point Standard

Additions Method by using absorbance values at two selected

Introduction

11

wavelengths as analytical signal. These methods and calibration graphs

were also used for the determination of cephalexin in pharmaceutical

samples.

A new HPTLC method was developed for the determination of

ceftriaxone, cefixime and cefotaxime, cephalosporins widely used in

clinical practice(14). High performance TLC of cephalosporins was

performed on precoated silica gel HPTLC plates with concentrating

zone (2.5×10 cm) by development in mobile phase ethyl acetate-

acetone-methanol-water (5:2.5:2.5:1.5 v/v/v/v). A TLC scanner set at

270 nm was used for direct evaluation of the chromatograms in

reflectance/absorbance mode. The calibration curves were established

as dependence of peak height (linear and polynomial regression) and

peak area (polynomial regression) versus ng level (125–500 ng for all

cephalosporins investigated). Relative standard deviations obtained

from calibration curves was compared. Precision [RSD: 1.12–2.91%

(peak height versus ng) and RSD: 1.05–2.75% (peak area versus ng)],

and detection limits (ng level) was validated and found to be

satisfactory. The method was found to be reproducible and convenient

for quantitative analysis of ceftriaxone, cefixime and cefotaxime in their

raw materials and their dosage forms.

A sensitive, accurate and rapid flow injection analysis (FIA)

method for the determination of cefotaxime, cefuroxime, ceftriaxone,

cefaclor, cefixime, ceftizoxime, and cephalexin was proposed(15).

Aliquots of each cephalosporin were hydrolyzed for 15 min with 0.1 M

NaOH at 80°C and then oxidized with Fe3+ in sulfuric acid medium to

produce Fe2+. The produced Fe2+ was then complexed by o-

phenanthroline (o-phen) in citrate buffer at pH 4.2 to form the red

complex, Fe(o-phen)32+, which exhibits an absorption maximum at 510

nm. Variables such as acidity, reagent concentrations, flow rate of

reagents and other FI parameters were optimized to produce the most

Introduction

12

sensitive and reproducible results. The method was successfully applied

to the analysis of pharmaceutical preparations. The results were

compared with those obtained using the official methods. Excellent

agreement between the results of the proposed method and the official

methods was obtained.

A simple and reproducible spectrophotometeric method for the

assay of cefotaxime sodium, cefuroxime sodium, and ceftriaxone

disodium with metol-chromium(VI) reagent was developed(16). The

procedure was based on direct oxidation of metol by potassium

dichromate in presence of drug in acidic medium and subsequent

formation of ternary complex. Beer’s law was obeyed in the range 0.2–

28 μg ml−1 at λmax 520 nm. For more accurate analysis, Ringbom

optimum concentration range was found to be 0.8–26.5 μg ml−1. The

molar absorptivity and Sandell sensitivity were calculated. Six replicate

analysis of solutions containing seven different concentrations of the

examined drugs were carried out and gave a mean correlation

coefficient ≤0.9996; the factors of the regression line equation for the

three cephalosporins were calculated. The proposed method was

applied to the determination of the examined drugs in pharmaceutical

formulations and the results demonstrated that the method was equally

accurate, precise, and reproducible as the official methods.

Two sensitive spectrophotometric and atomic absorption

spectrometric procedures were developed for the determination of

certain cephalosporins (cefotaxime sodium and cefuroxime sodium)(17).

The spectrophotometric methods were based on the charge-transfer

complex formation between these drugs as n-donors and 7,7,8,8-

tetracyanoquinodimethane (TCNQ) or p-chloranilic acid (p-CA) as pi-

acceptors to give highly coloured complex species. The coloured

products were measured spectrophotometrically at 838 and 529 nm for

TCNQ and p-CA, respectively. Beer's law was obeyed in a

Introduction

13

concentration range of 7.6-15.2 and 7.1-20.0 µg/ml with TCNQ, 95.0-

427.5 and 89.0-400.5 µg/ml with p-CA for cefotaxime sodium and

cefuroxime sodium, respectively. The atomic absorption spectrometric

methods were based on the reaction of the above cited drugs after their

alkali-hydrolysis with silver nitrate or lead acetate in neutral aqueous

medium. The formed precipitates were quantitatively determined directly

or indirectly through the silver or lead content of the precipitate formed

or the residual unreacted metal in the filtrate by atomic absorption

spectroscopy. The optimum conditions for hydrolysis and precipitation

had been carefully studied. Beer's law was obeyed in a concentration

range of 1.9-11.4 and 1.78-8.90 µg/ml with Ag(I), 14.2-57.0 and 13.3-

53.4 µg/ml with Pb(II) for cefotaxime sodium and cefuroxime sodium,

respectively (for both direct and indirect procedures). The spectrop-

hotometric and the atomic absorption spectrometric procedures hold

well their accuracy and precision when applied to the analysis of

cefotaxime sodium and cefuroxime sodium dosage forms.

Cephalexin, cefixime, ceftriaxone and cefotaxime were deter-

mined spectrophotometrically in the pure form and in pharmaceutical

formulations by using ferrihydroxamate method(18). Reaction optimi-

zation with respect to reaction time and temperature had been

investigated. Influence of the presence of ester functional group on the

determination of cephalosporins as -lactams under conditions optimized

was evaluated. Using cefotaxime sodium as model drug with ester

functional group, it was found that the proposed method gave equally

accurate and precise results even in the presence of ester functional

group.

A simple and sensitive spectrophotometric method was described

for the determination of cefotaxime(19). The method was based on the

degradation of cefotaxime which was carried out in 0.3 mol/L NaOH

solution at 100 o, and can be oxidized by Fe(III) in acidic solution.

Introduction

14

Fe(Ⅱ) can form a complex with o-phenanthroline hydrate, of which the

maximum absorption wavelength is at 508 nm,(ε=1.1×104 lmol-1·cm-1).

Beer′s law was obeyed in the range of 0.4～80 μg/mL for cefotaxime.

The linear regression equation is A=-0.00204+0.01989C (μg/mL), with a

linear correlation coefficient of 0.9998. The detection limit was 0.18

μg/mL. RSD is 1.2%(5.0 μg/mL, n=11),and average recovery is 99%.

The reaction mechanism was studied intensively. The proposed method

was successfully applied to the determination of cefotaxime with

satisfactory results.

Two simple and sensitive spectrophotometric methods (A and B)

in the visible region had been developed for the determination of

cefotaxime sodium (CFTS) in bulk and in dosage forms(20). Method A

was based on the reaction of CFTS with nitrous acid under alkaline

conditions to form a stable violet colored chromogen with absorption

maximum of 560 nm and method B was based on the reaction of CFTS

with 1,10-phenanthroline and ferric chloride to form a red colored

chromogen with the absorption maximum of 520mm. The color obeyed

Beer's law in the concentration range of 100-500 µg/ml for method A

and 1.6-16 µg/ml for method B, respectively. When pharmaceutical

preparations containing CFTS were analysed, the results obtained by

the proposed methods were in good agreement with the labeled

amounts and are comparable with the results obtained using a UV

spectrophotometric method.

An analytical method for detecting and quantifying cefotaxime in

plasma and several tissues was described(21). The method was

developed and validated using plasma and tissues of rats. The samples

were analyzed by reversed phase liquid chromatography (HPLC) with

UV detection (254 nm). Calibration graphs showed a linear correlation

(r > 0.999) over the concentration ranges of 0.5–200 μg/mL and 1.25–

Introduction

15

25μg/g for plasma and tissues, respectively. The recovery of cefotaxime

from plasma standards prepared at the concentrations of 25 μg/mL and

100 μg/mL was 98.5±3.5% and 101.8±2.2%, respectively. The recovery

of cefotaxime from tissue standards of liver, fat and muscle, prepared at

the concentration of 10 μg/g was: 89.8±1.2% (liver), 103.9±6.5% (fat)

and 97.8±2.1% (muscle). The detection (LOD) and quantitation (LOQ)

limits for plasma samples were established at 0.11 μg/mL and 0.49

μg/mL, respectively. The values of these limits for tissues samples were

approximately 2.5 times higher: 0.3 μg/g (LOD) and 1.25 μg/g (LOQ).

For plasma samples, the deviation of the observed concentration from

the nominal concentration was less than 5% and the coefficient of

variation for within-day and between-day assays was less than 6% and

12%, respectively. The method was used in a pharmacokinetic study of

cefotaxime in the rat and the mean values of the pharmacokinetic

parameters are given.

Vanadophosphoric acid in acidic medium was proposed as a

modified reagent for the spectrophotometric determination of

cephalexin, cephaprine sodium, cefazolin sodium, and cefotaxime in

pure samples and in pharmaceutical preparations(22). The method was

based on acid hydrolysis of cephalosporins and subsequent oxidation

with vanadophosphoric acid. The resulting solution exhibited maximum

absorption at about 516nm. The effects of reaction conditions were

investigated. Lambert-Beer’s law was obeyed over a concentration

range of about 0.4–45μg/mL. For more accurate results, Ringbom

optimum concentration ranges were obtained, and the molar

absorptivities and Sandell sensitivities were derived. The proposed

method was applied to the determination of the drugs in pharmaceutical

formulations; the results demonstrated that the proposed method was

as accurate, pecise, and reproducible as the official methods

Introduction

16

Ion-selective electrodes based on ion exchangers of

tetradecylammonium (TDA) with cefotaxime (claforan) anions had been

developed(23). The proposed electrodes were sensitive to cephalo-

sporins in the concentration range from 1×10− 5 to 1×10− 1 M. The time

for establishing a steady-state potential was 1–2 min. The potential drift

did not exceed 2 mV/d. The detection threshold for cefotaxime was

3.6×10− 5 M in the optimum pH range of 4.3–6.5. Comparison of the

main electrochemical characteristics of the ion-selective electrodes

based on TDA associates with cephalosporins showed that the best

parameters were found in electrodes with membranes containing

claforan.

Two methods were developed for determination of intact

ceftazidime (I), cefuroxime sodium (II), and cefotaxime sodium (III) in

the presence of their degradation products(24). In the first method, first

derivative spectrophotometry (D1) was used. The (D1) absorbance was

measured at 268.6, 306, and 228.6 nm for I, II, and III, respectively.

The first proposed method determined I, II, and III in concentration

ranges of 5-50, 5-35, and 5-40 μg/mL, respectively, with corresponding

mean accuracies of 99.7 ± 0.8, 100.1 ± 0.7, and 99.8 ± 0.8%. The

method determined the intact drug in the presence of up to 90%

degradation products for I, and II and up to 80% for III. The second

method depended on the quantitative densitometric evaluation of thin-

layer chromatograms of I, II, and III. It determined I, II, and III in

concentration ranges of 4-16 μg for I and 2-12 μg for II and III, with

mean accuracy's of 99.5 ± 0.8, 99.2 ± 0.7, and 99.7 ± 0.8% for I, II, and

III, respectively. The second method retained its accuracy in the

presence of up to 90% degradation products for the 3 drugs. The results

obtained by applying the proposed methods were statistically analyzed

and compared with those obtained by the official method.

Introduction

17

Three simple, rapid and sensitive spectrophotometric procedures

were developed for the analysis of cephapirin sodium (1), cefazoline

sodium (2), cephalexin monohydrate (3), cefadroxil monohydrate (4),

cefotaxime sodium (5), cefoperazone sodium (6) and ceftazidime

pentahydrate (7) in pure form as well as in their pharmaceutical

formulations(25). The methods were based on the reaction of these drugs

as n-electron donors with the σ-acceptor iodine, and the π-acceptors:

2,3-dichloro-5,6-dicyano-p-benzo-quinone (DDQ) and 7,7,8,8-tetracyan-

oquinodimethane (TCNQ). Depending on the solvent polarity, different

coloured charge-transfer complexes and radicals were developed.

Different variables and parameters affecting the reactions were studied

and optimized. The obtained charge-transfer complexes were measured

at 364 nm for iodine (in 1,2-dichloroethane), 460 nm for DDQ (in

methanol) and 843 nm for TCNQ (in acetonitrile). Ultraviolet–visible,

infrared and 1H-nuclear magnetic resonance techniques were used to

study the formed complexes. Due to the rapid development of colours at

ambient temperature, the obtained results were used on thin-layer

chromatograms for the detection of the investigated drugs. Beer's plots

were obeyed in a general concentration range of 6–50, 40–300 and 4–

24μg ml−1 with iodine, DDQ and TCNQ, respectively, with correlation

coefficients not less than 0.9989. The proposed procedures could be

applied successfully to the determination of the investigated drugs in

vials, capsules, tablets and suspensions with good recovery; percent

ranged from 96.47 (±1.14) to 98.72 (±1.02) in the iodine method, 96.35

(±1.62) to 98.51 (±1.30) in the DDQ method, and 95.98 (±0.78) to 98.40

(±0.87) in the TCNQ method. The association constants and standard

free energy changes using Benesi–Hildebrand plots were studied. The

binding of cephalosporins to proteins in relation to their molar

absorptivities was studied.

A new, simple and sensitive spectrophotometric method for the

determination of valacyclovir and cefotaxime had been developed(26).

The method was based on the condensation of valacyclovir and

Introduction

18

cefotaxime with 1, 2- napthaquinone-4- sulfonic acid sodium (NQS) in

alkaline media to yield orange colored products. Valacyclovir and

cefotaxime showed maximum absorbance at 495nm and 475nm with

linearity observed in the concentration range of 20-120 µg/ml and 20-

140 µg/ml respectively. The relative standard deviations of 0.363% for

valacyclovir and 0.66% for cefotaxime were obtained. The recoveries of

valacyclovir and cefotaxime injections were in the range 96.01±0.52 and

98.12±0.96 respectively. The proposed method is simple, rapid, precise

and convenient for the assay of valacyclovir and cefotaxime in

commercial injection preparations.

An accurate, reliable, specific and sensitive kinetic spectro -

fluorimetric method was developed for the determination of seven

cephalosporin antibiotics namely cefotaxime sodium, cephapirin sodium,

cephradine dihydrate, cephalexin monohydrate, cefazoline sodium,

ceftriaxone sodium and cefuroxime sodium(27). The method was based on

their degradation under an alkaline condition producing fluorescent

products. The factors affecting the degradation and the determination were

studied and optimized. The reaction was followed spectrofluorimetrically by

measuring the rate of change of fluorescence intensity at specified

emission wavelength. The initial rate and fixed time methods were used for

the construction of calibration graphs to determine the concentration of the

studied drugs. The calibration graphs were linear in the concentration

ranges 0.2-1.2 µg/ml and 0.2-2.2 µg/ml using the initial rate and fixed time

methods, respectively. The results were statistically validated and checked

through recovery studies. The method was successfully applied for the

determination of the studied cephalosporins in commercial dosage forms.

The high sensitivity of the proposed method allowed the determination of

investigated cephalosporins in human plasma. The statistical comparisons

of the results with the reference methods showed an excellent agreement

and indicate no significant difference in accuracy and precision.

Introduction

19

Ceftazidime

Ceftazidine has the following chemical structure:

Its IUPAC name is (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-

carboxypropan-2-yloxyimino) acetamido) -8-oxo-3-(pyridinium-1-ylmethyl)-

5-thia-1-aza-bicyclo [4.2.0] oct-2-ene-2-carboxylate

Ceftazidime is a third-generation cephalosporin antibiotic. Like

other third-generation cephalosporins, it has broad spectrum activity

against Gram-positive and Gram-negative bacteria. Unlike most third-

generation agents, it is active against Pseudomonas aeruginosa,

however it has weaker activity against Gram-positive microorganisms

and is not used for such infections.

Clinical use

Ceftazidime is usually reserved for the treatment of infections

caused by Pseudomonas aeruginosa. It is also used in the empirical

therapy of febrile neutropenia, in combination with other antibiotics. It is

usually given IV or IM every 8–12 hours (2 - 3 times a day), with dosage

varying by the indication, infection severity, and/or renal function of the

recipient. Ceftazidine is first line treatment for the rare tropical infection,

melioidosis.

Introduction

20

Chemistry

In addition to the syn-configuration of the imino side chain,

compared to other third-generation cephalosporins, the more complex

moiety (containing two methyl and a carboxylic acid group) confers

extra stability to β-lactamase enzymes produced by many Gram-

negative bacteria. The extra stability to β-lactamases increases the

activity of ceftazidime against otherwise resistant Gram-negative

organisms including Pseudomonas aeruginosa. The charged pyridinum

moiety increases water-solubility.

Literature survey on the microdetermination of ceftazidime:

A high-performance liquid chromatography procedure was

developed to analyze ceftazidime concentrations in plasma(29). The

procedure consisted of solid phase extraction followed by ion-pairing

reverse-phase chromatography. An excellent linear relationship

between ceftazidime peak height measurements and concentrations

was demonstrated over the concentration range of 1-200 µg/ml. The

advantage of this assay is the elimination of interference at the

ceftazidime elution time that has been noted in previous studies. Thus,

this study describes an alternative, simple methodology that is clinically

useful for analyzing ceftazidime in the research setting.

A simple micellar electrokinetic chromatography (MEKC) with UV

detection at 254 nm for analysis of ceftazidime in plasma and in

cerebrospinal fluid (CSF) by direct injection without any sample

pretreatment was described(30). The separation of ceftazidime from

biological matrix was performed at 25 oC using a background electrolyte

consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the

electrolyte solution. Under optimal MEKC condition, good separation

with high efficiency and short analyses time was achieved. Several

parameters affecting the separation of the drug from biological matrix

Introduction

21

were studied, including pH and concentration of the Tris buffer and

SDS. Using cefazolin as an internal standard (IS), the linear ranges of

the method for the determination of ceftazidime in plasma and in CSF

were all over the range of 3-90 µg/ml; the detection limit of the drug in

plasma and in CSF (signal-to-noise ratio = 3; injection 0.5 psi, 5 s) was

2.0 µg/ml. The applicability of the proposed method for determination of

ceftazidime in plasma and CSF collected after intravenous

administration of 2 g ceftazidime in patients with meningitis was

demonstrated.

Two spectrophotometric methods for the determination of

ceftazidime (CFZM) in either pure form or in its pharmaceutical

formulations were described(31) The first method was based on the

reaction of 3-methylbenzothiazolin-2-one hydrazone (MBTH) with

ceftazidime in the presence of ferric chloride in acidic medium. The

resulting blue complex absorbs at lambdamax 628 nm. The second

method described the reaction between the diazotized drug and N-(1-

naphthyl)ethylenediamine dihydrochloride (NEDA) to yield a purple

colored product with λmax at 567 nm. The reaction conditions were

optimized to obtain maximum color intensity. The absorbance was

found to increase linearly with increasing the concentration of CFZM;

the systems obeyed the Beer's law in the range 2-10 and 10-50 µg/ml

for MBTH and NEDA methods, resp. LOD, LOQ and correlation

coefficient values were 0.15, 0.79 and 0.50, 2.61. No interference was

observed from common excipients present in pharmaceutical

formulations. The proposed methods are simple, sensitive, accurate and

suitable for quality control applications.

Two simple and sensitive validated spectrophotometric methods

was described for the assay of ceftazidime in drug formulations(32).

Method A was based on the oxidation of the drug with ferric ion followed

by complex formation reaction with 1,10-phenanthroline (1,10-phen) to

Introduction

22

form orange red colored chromogen exhibiting max at 510nm. Method B

is based on the formation of colored Schiff’s base obtained when

ceftazidime in acidic conditions reacted with anisaldehyde (ANLD) in

ethanol to form yellow colored chromogen exhibiting max at 383nm. The

products were stable for more than 10 and 2 h respectively. Common

excipients used as additives in pharmaceutical preparations do not

interfere in the proposed methods. Both the methods are highly

reproducible and have been applied to a wide variety of pharmaceutical

preparations and the results compare favorably with those of official

method.

A new spectrophotometric method for determination of

ceftazidime was developed(33). The method was based on Fe3+ as the

oxidizer of ceftazidime and phenanthroline as the coloring reagent of

Fe2+ which was produced from Fe3+. Under the optimum conditions, the

relationship between the absorbance and the concentration of

ceftazidime was linear in the range of 0.4—10mg/L, the regression

equation was ΔA=0.1008C(mg/L)+0.07789, the correlation coefficient

was 0.9970.The proposed methods had been applied to the

determination of ceftazidime content in samples with satisfactory

results.

A new method for the determination of ceftazidime by

spectrophotometry was developed(34) based on Fe3+ as the oxidizer of

ceftazidime and 2,2′-dipyridine as the coloring reagent of Fe2+,which

was produced from Fe3+. Under the optimum conditions, the relationship

between the absorbance and the concentration of ceftazidime was

linear in the range of 0.4—10 mg/L, the regression equation was

ΔA=0.079 73ρ(mg/L) +0.045 08,and the relative coefficient was

0.9960.The proposed method has been applied to the determination of

ceftazidime content in samples with satisfactory results.

Introduction

23

A simple and reproducible spectrophotometric method for the assay

of ceftazidime with neocuproin-copper(II) reagent had been

developed(35). The procedure was based on the formation of neocuproin

– drug complex in an acidic medium, subsequent formation of yellow

ternary complex in citrate buffer solution (pH 4.2), and measurement at

454 nm. Beer's law was obeyed in the range 15.0-40.0 µg mL-1 with

correlation coefficient r2 = 0.9995. The procedure holds good accuracy

and precision when applied to the analysis of ceftazidime in powder for

injection with good recovery percent ranging from 100.17±1.0 without

interference from additives.

A simple, accurate and precise spectrophotometric method had

been proposed for the determination of eleven cephalosporins, namely;

cefaclor monohydrate, cefadroxil monohydrate, cefalexin anhydrous,

cefradine anhydrous, cefotaxime sodium, cefoperazone sodium, ceftriaxone

sodium, ceftazidime penthydrate, cefazolin sodium, cefixime and

cefpodoxime pro- xetil in bulk drug and in pharmaceutical formulations(36).

The method depended 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 affecting the reaction

(e.g. NaOH concentration, hydrolysis time, NBD-Cl concentration and

diluting 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 for all studied drugs. The limits of assay

detection 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 successfully applied for analysis

of the studied drugs in their pharmaceutical formulations and the recovery

percentages ranged from 96.6 to 103.5%.

A simple, rapid and sensitive spectrophotometric method had

been developed for the quantitative determination of five drugs of

Introduction

24

pharmaceutical interest; cefepime HCI, cefoperazone Na, ceftazidime

pentahydrate, cefuroxime Na and etamsylate in pure form as well as in

pharmaceuticals(37). The method was based on the reduction of the

chromogenic agent, ammonium molybdate (Mo6+), into molybdenum

blue (Mo5+) by the examined drugs in sulphuric acid medium and by aid

of heating in boiling water bath. The resulting "blue coloured" product

showed a characteristic λmax at 695-716 nm. Beers law was obeyed over

the concentration range of 2-70 pg/ml with molar absorpitivities ranging

from 2.704x103-24.14x103 L.mol-1.cm-1 and Sandell sensitivities ranging

from 1.03x10-3- 5.4x10-3μg cm-2. The proposed method had been

applied successfully for the determination of the examined drugs both in

pure form and in pharmaceutical formulations. The accuracy and

precision of the proposed method were comparable with those of the

reported methods.

Two spectrophotometric methods for the determination of

ceftazidime (CFZM) in either pure form or in its pharmaceutical

formulations were described(38). The first method was based on the

reaction of 3-methylbenzothiazolin-2-one hydrazone (MBTH) with

ceftazidime in the presence of ferric chloride in acidic medium. The

resulting blue complex absorbed at λmax 628 nm. The second method

described the reaction between the diazotized drug and N-(1-naphthyl)

ethylenediamine dihydrochloride (NEDA) to yield a purple colored

product with λmax at 567 nm. The reaction conditions were optimized to

obtain maximum color intensity. The absorbance was found to increase

linearly with increasing the concentration of CFZM; the systems obeyed

the Beer's law in the range 2-10 and 10-50 μg/ml for MBTH and NEDA

methods, resp. LOD, LOQ and correlation coefficient values were 0.15,

0.79 and 0.50, 2.61. No interference was observed from common

excipients present in pharmaceutical formulations. The proposed

methods are simple, sensitive, accurate and suitable for quality control

applications.

Introduction

25

Cefepime

IUPAC name: (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)

acetamido)-3-((1-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza-bicyclo

[4.2.0]oct-2-ene-2-carboxylate

Cefepime is a fourth-generation cephalosporin antibiotic

developed in 1994. Cefepime has an extended spectrum of activity

against Gram-positive and Gram-negative bacteria, with greater activity

against both Gram-negative and Gram-positive organisms than third-

generation agents.

Clinical use

Cefepime is usually reserved to treat severe nosocomial

pneumonia, infections caused by multi-resistant microorganisms (e.g.

Pseudomonas aeruginosa) and empirical treatment of febrile

neutropenia. The use of cefepime might become less common, since it

has been associated to an increase mortality when used for different

types of infections.

Cefepime has good activity against important pathogens including

Pseudomonas aeruginosa, Staphylococcus aureus, and multiple drug

resistant Streptococcus pneumoniae. A particular strength is its activity

against Enterobacteriaceae. Whereas other cephalosporins are

degraded by many plasmid - and chromosome - mediated beta-

lactamases, cefepime is stable and is a front line agent when infection

with Enterobacteriaceae is known or suspected.

Introduction

26

Chemistry

The combination of the syn-configuration of the methoxyimino

moiety and the aminothiazolyl moiety confers extra stability to β-

lactamase enzymes produced by many bacteria. The N-

methylpyrrolidine moiety increases penetration into Gram-negative

bacteria. These factors increases the activity of cefepime against

otherwise resistant organisms including Pseudomonas aeruginosa and

Staphylococcus aureus.Its efficacy in bovine mastitis has to be

evaluated

Literature survey on the microdetermination of cefepime

A simple spectrophotometric assay for the determination of

cefepime and L-arginine in injections was described(39). Since zero-

order spectra showed considerable overlap, second-derivative

spectrophotometry was used to enhance the spectral details. A linear

relationship between second-derivative amplitude and concentration of

each compound was found. Beer's law was obeyed up to 50 and 22

µg/ml of cefepime and arginine, respectively, in the second-derivative

mode. Detection limits were 0.31 and 0.58 µg/ml for cefepime and

arginine, respectively. The method, which was rapid, simple and did not

require any separation step, had been successfully applied to the assay

of commercial injections containing cefepime and arginine.

An isocratic reversed-phase HPLC method was developed to

determine cefepime levels in plasma and vitreous fluid(40). Cefepime and

the internal standard cefadroxil were separated on a Shandon Hypersil

BDS C18 column by using a mobile phase of 25 mM sodium dihydrogen

phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet

detection was carried out at 270 nm. The retention times were 4.80 min

for cefepime and 7.70 min for cefadroxil. This fast procedure which

involved an efficient protein precipitation step (addition of HClO4),

Introduction

27

allowed a quantification limit of 2.52 µg/ml and a detection limit of 0.83

µg/ml. Recoveries and absolute recoveries of cefepime from plasma

were 96.13-99.44% and 94-102.5% respectively. The intra-day and

inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50

µg/ml (n=10). The method was proved to be suitable for determining

cefepime levels in human plasma and was modified to measure vitreous

fluid samples.

A simple, rapid and reproducible high-performance liquid

chromatographic method for the quantitative determination of cefepime

in human plasma was developed(41). Ceftazidime was used as internal

standard. Chromatography was performed on a reversed-phase

encapped column (Hypersil BDS C18). The samples, after protein

precipitation, were eluted with a mobile phase of acetonitrile-acetate

buffer, pH 4 (2.8:97.2, v/v). The detection wavelength was 254 nm. The

limit of quantitation of cefepime was 0.5 µg/ml and only 0.5 ml of plasma

sample was required for the determination. The average cefepime

recoveries over a concentration range of 0.5-500 µg/ml ranged from 98

to 104%. Precision and accuracy did not exceed 5%.

The cephalosporin cefepime had been studied by adsorptive

stripping voltammetric on the hanging mercury drop electrode, followed

by linear sweep voltammetry (staircase)(41). The adsorptive stripping

response was evaluated with respect to preconcentration dependence

and other variables. The drug was strongly adsorbed in acid media, with

maximum adsorption at pH 5.8. The detection limit found was

4.8 × 10−10 M. The relative standard deviation at th 10−7 M level was

0.93%. This method was applied to the determination of cefepime in

human urine and cerebrospinal fluid. Differential pulse polarography had

been applied to determination in human serum.

Introduction

28

A simple micellar electrokinetic chromatography (MEKC) with UV

detection was described for simultaneous analysis of cefepime and L-

arginine(42). The determination of cefepime and L-arginine in

pharmaceutical preparations was performed at 25°C using a

background electrolyte consisting of Tris buffer with sodium dodecyl

sulfate (SDS) as the electrolyte solution. Several parameters affecting

the separation of the drugs were studied, including the pH and

concentrations of the Tris buffer and SDS. Under optimal MEKC

conditions, good separation with high efficiency and short analysis times

was achieved. Using cefazolin as an internal standard, the linear ranges

of the method for the determination of cefepime and L-arginine were

over 5–100 μg/ml; the detection limits of cefepime (signal to noise

ratio= 3; injection 3.45 kPa, 3 s) and L-arginine (signal to noise ratio = 3;

injection 3.45 kPa, 3s) were 2μg/mL and 4μg/mL, respectively.

Applicability of the proposed method for the determination of cefepime

and L-arginine in commercial injections was demonstrated.

Three simple, rapid and accurate spectrophotometric methods were

presented for the determination of two cephalosporins: cefepime

dihydrochloride and cefprozil monohydrate(43). The first method

depended on the reaction of the named drugs as n-donors with three

acceptors: namely, chloranilic acid (CA), 2,3dichloro-5,6-dicyano-1,4-

benzo-quinone (DDQ) and 7,7,8,8-tetracyano-quinodimethane (TCNQ)

to yield highly colored radical anions measured at 527 nm, 460 nm and

841 nm, respectively. The second method depended on the reaction of

each of the two drugs with ninhydrin, in boiling water bath, in presence

of pyridine to yield a bluish violet product measured at 566 nm. The third

method is based on the reduction of Folin Ciocalteu's reagent (FCR) in

alkaline medium by the investigated drugs into blue colored products

measurable at 755 nm. Beer's law is obeyed for cefepime salt at

concentration ranges of 50-450 µg/ml, 20-180µg/ml, 10-60µg/ml and

Introduction

29

10-60 µg/ml for CA, DDQ, TCNQ, ninhydrin and FCR, respectively, on

the other hand, for the cefprozil salt, the concentration ranges are 50-

400µg/ml 20-140µg/ml, 1-7µg/ml, 2-14µg/ml and 2.5-25µg/ml in the

same order of reagents. The proposed methods had been successfully

applied to the analysis of the studied cephalosporins, in either pure form

and in pharmaceutical formulations. The comparison of results with

those of pharmacopoeial method revealed that there is no significant

difference in the accuracy (t-test) and reproducibility (F-test)...

A new simple, sensitive, highly specific and economical UV

spectrophotometric method had been developed for determination of

cefepime hydrochloride in pure and pharmaceutical (parentral form)

formulation using different solvents, 0.001 N HCl and phosphate buffer

(pH 6.8)(44). Cefepime hydrochloride exhibited maximum absorbance

(λmax) at 261.6 nm and 258.4 nm for 0.001 N HCl and phosphate buffer

(pH 6.8), respectively. Beer's law is obeyed over a concentration range

of 5-60μg/ml with correlation coefficient r>0.998. The proposed method

was validated statistically for both the solvents. Recovery study

confirmed the accuracy of the proposed method.

A high performance liquid chromatographic procedure had been

developed for the assay of a cefepime and metronidazole mixture in

aqueous solution(45). The separation and quantitation were achieved on

a phenyl column at ambient temperature using a mobile phase of

94.5:5.5 v/v water-acetonitrile containing 0.015 M pentane sulfonic acid

sodium salt (adjusted to pH 3.4 with glacial acetic acid and then 4.0 with

45% potassium hydroxide) at a flow rate of 1.5 mL/min with detection of

both analytes at 280 nm. The separation was achieved within 10 min

with sensitivity in the ng/mL range for each analyte. The method

showed linearity for cefepime and metronidazole in the 18.77 - 300.2

and 9.39 - 150.1 μg/mL ranges, respectively. Accuracy and precision

were in the 0.52-2.40 and 0.63-2.77% ranges, respectively, for both

Introduction

30

analytes. The limits of detection for cefepime and metronidazole were

125 and 63ng/mL, respectively, based on a signal to noise ratio of 3 and

a 10μL injection.

A new application of microfabricated chip with integrated Pt

microelectrodes, for the electrochemical detection (ECD) of cefepime(46).

In this analysis, electro-oxidation of cefepime was investigated on an

unmodified Pt microelectrode in acetate buffer at pH 4.5. Differential

pulse voltammetry (DPV) along with cyclic voltammetry (CV) was

performed as a major read out technique. With this method, a linear

calibration curve for 25-150μM with a limit of detection of 15 μM was

obtained. This investigation presents a simple methodology and rapid

results that, within the above calibration range, can be obtained without

any pretreatment of the analyte.

The cephalosporin cefepime had been studied by adsorptive

stripping voltammetric on the hanging mercury drop electrode, followed

by linear sweep voltammetry (staircase)(47). The adsorptive stripping

response was evaluated with respect to preconcentration dependence

and other variables. The drug was strongly adsorbed in acid media, with

maximum adsorption at pH 5.8. The detection limit found was

4.8×10−10M, with 120-s preconcentration. The relative standard

deviation at the 10−7 M level was 0.93%. This method was applied to the

determination of cefepime in human urine and cerebrospinal fluid.

Differential pulse polarography has been applied to determination in

human serum.

A simple and sensitive assay method was developed for

simultaneous determination of cefepime and sulbactam sodium in

Supime (a fixed dose combined formulation of cefepime and sulbactam

manufactured by Venus Remedies limited, India) with UV detection at

230 nm(48). Chromatographic separation of two drugs was achieved on a

Introduction

31

Hypersil ODS C-18 column using a binary mixture of acetonitrile and

tetrabutyl ammonium hydroxide as a mobile phase adjusted to pH 5.0

with orthophosphoric acid in ratio 20:80 v/v ratio. The developed liquid

chromatographic method offered good linearity, accuracy and precision

over the concentration range of 125-750 ppm for cefepime and 62.5-375

ppm for sulbactam sodium. This method was successfully applied for

the quality control of formulated products and plasma samples

containing Cefepime and sulbactam. Since, Supime, a fixed dose

combination of cefepime and sulbactam was a research product of

Venus Remedies limited the literature lacks any method of analysis for

such combination, the main motive behind this experiment was to

develop and validate a method which could be used for the quality

control of cefepime and sulbactam in combined dosage form.

A simple, rapid, specific and sensitive high-performance liquid

chromatographic method was developed for the determination of

cefepime in human serum(49). Separation was achieved on a reversed-

phase Ultrasphere XL-ODS column (75×4.6 mm I.D.). The mobile

phase was 7% acetonitrile in 20 mM ammonium acetate (pH 4).

Cefepime eluted in the range of 1.8–2.2 min. Detection was by UV

absorbance at 254 nm. The lower limit of quantitation of cefepime in

plasma was 0.5μg/ml. The average absolute recovery was 106.2±2.1%.

The linear range was from 0.1 to 50 μg/ml, with a correlation coefficient

greater than 0.999. The within-day C.V.s for human samples were 4.9

and 2.3% for 1 and 50μg/ml, respectively. The between-day C.V.s for

human serum samples were 14.5, 7.4 and 6.7 for 1, 25 and 50μg/ml,

respectively. Cefepime was found to be unstable in serum at room

temperature. For delayed assay, samples must be stored at −80°C.

A liquid chromatographic method with UV detection for

simultaneous determination of cefepime, vancomycin and imipenem

had been developed(50). Cefuroxime was used as internal standard.

Introduction

32

After the clean up of samples by plasma protein precipitation, 5 μl of the

extract were injected into the chromatograph and peaks were eluted

from the Sulpelcosil™ LC-18 column using a mobile phase consisting of

0.075 M acetate buffer:acetonitrile (92:8, v/v), pH 5.0 at low rate

(0.8ml/min). The detection wavelength was 230 nm. The limit of

detection was 0.4μg/ml for cefepime and 0.2μg/ml for vancomycin and

imipenem. The method was applied to plasma samples of burn patients,

and only small volumes of plasma were required for the simultaneous

determination of those antimicrobial agents.

A simple, rapid sensitive and accurate method for the

determination of aciclovir,cefepime HCI, etamsylate and metoclo-

pramide HCI in pure form and in pharmaceutical formulations was

developed(51). The method was based on the formation of tris(o-

pnenanthroline) iron(II) complex (Ferroin) upon the reaction of the cited

drugs with iron(III)-o- phenanthroline mixture. The ferroin complex was

colorimetrically measured at λmax 510 nm against a reagent blank.

Optimization of the experimental conditionswas described. Beer's law

was obeyed in the concentration range from 0.25-30 μg /ml with molar

absorpιtivities (ε) ranging from 4.796 x 103-9.512 x 104 l.mol-1.cm-1 and

Sandell sensitivities (S) of 2.129x10-3-34.5x10-3μg cm-2. The developed

method was applied successfully for the determination of the cited drugs

in pure forms and in the corresponding pharmaceutical formulations

without any interferences from common excipients

A simple, rapid and sensitive spectrophotometric method had

been developed for the quantitative determination of five drugs of

pharmaceutical interest; cefepime HCI, cefoperazone Na, ceftazidime

pentahydrate, cefuroxime Na and etamsylate in pure form as well as in

pharmaceuticals(52). The method was based on the reduction of the

chromogenic agent, ammonium molybdate (Mo6+), into molybdenum

blue (Mo5+) by the examined drugs in sulphuric acid medium and by aid

Introduction

33

of heating in boiling water bath. The resulting "blue coloured" product

possess a characteristic λmax at 695-716 nm. Beers law was obeyed

over the concentration range of 2-70 pg /ml with molar absorpitivities

ranging from 2.704x103-24.14x103 l.mol-1.cm-1 and Sandell sensitivities

ranging from 1.03x10-3-5.4x10-3 μg cm-2. The proposed method had

been applied successfully for the determination of the examined drugs

both in pure form and in pharmaceutical formulations. The accuracy and

precision of the proposed method were comparable with those of the

reported methods.

34

Aim of the work

No doubt that, it is critically vital to determine the purity and

concentration of any therapeutic drug in high accuracy and precision.

One of the most important procedures is the spectrophotometric

method.

The aim of the present work is to develop a spectrophotometric

method for determination of cefotaxime, ceftazidime and cefepime in

pure and pharmaceutical formulations. The method is based on the

formation of soluble colored complexes (ion pair association complexes)

between the drugs under investigation and some reagents viz: arsenazo

I, eosin yellowish, eosin bluish, orange G and bromocrysol purple. The

optimum conditions favoring the formation of the colored complexes

must be extensively studied. The applied method is characterized by its

simplicity, selectivity and high sensitivity. It is also suitable for the micro-

determination of these drugs in pure and pharmaceutical formulations.

Experimental

35

Experimental

All chemicals used in this study were of the highest purity

available and used without further purification. Bidistilled water was

used throughout the work.

1- Materials

1-1 Drugs

The pharmaceutical compounds used in the present study are;

Cefotaxime sodiume (CEFO), Ceftazidime pentahydrate (CEFTAZ) and

Cefepime (CEFP). All these pharmaceutical compounds are from

Egyptian International Pharmaceutical Industries Company (EIPICO),

10th of Ramadan City, Egypt. The purity of the samples was found to be

99.8% on the dried bases according to the British pharmacopoeia (BP)

method (53) and were used as received. These compounds have the

following structural formula:

*HCl

Cefotaxime sodium; (6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-aminothiazol-

4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-

2-ene-2- carboxylic acid. (C16H17N5O7S2, M. Wt. 455.47 g/mol).

Experimental

36

Ceftazidime pentahydrate; (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-

carboxypropan-2-yloxyimino)acetamido)-8-oxo-3-(pyridinium-1-ylmethyl)-

5- thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate.(C22H22N6O7S2 ).

M. Wt; 546.58 g/mol)

Cefepime hydrochloride; 7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)

acetamido)-3-((1-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza-

bicycle [4.2.0]oct-2-ene-2-carboxylate (C19H24N6O5S2).

M. Wt.; 480.56 g/mol)

1-2 Drug solutions:

A stock solutions containing 200 µg/ml of the studied drugs were

prepared by dissolving 0.02 g of the pure samples in the least amount of

hot bidistilled water then cooled and transferred to 100 ml measuring

flask and finally diluted to the mark with water. For molar ratio and

continuous variation methods, 10-3 M solution of each drug was

prepared by dissolving 0.0455, 0.0547 and 0.0481 g of cefotaxime,

ceftazidime pentahydrate and cefepime, respectively in least amount of

water then complete to 100 ml in measuring flask.

Experimental

37

1-3 Pharmaceutical dosage forms:

i- Cefotaxime ampoule (Epico – Welcome Egypt; 10th of Ramadan

city, Cairo, Egypt) labeled to contain 1.0 mg CEFO per ampoule .

ii- Claforan (Sanoffi Aventis, Egypt) labeled to contain 50 mg CEFO

per ampoule (2 ml).

iii- Fortaz ampoule (Glaxo – Welcome Egypt, S.A.E. El-Salaam city,

Cairo, Egypt) labeled to contain 1.0 mg Ceftaz per ampoule.

iv- Fourtum ampoule (Glaxo – Welcome Egypt, S.A.E. El-Salaam

city, Cairo, Egypt) labeled to contain 1.0 mg Ceftaz per ampoule.

v- Cefepime ampoule (Pharco pharmaceutical Industries, Cairo,

Egypt) labeled to contain 1.0 mg CEFP per ampoule.

1-4 Reagents:

The analytical reagents used in the present work are arsenazo I,

eosin yellowish, eosin bluish, orange G, and bromocresol purple.They

have the following structural formula:

Arsenazo I

Eosin yellowish

Experimental

38

Eosin bluish

Orange G

Bromocresol purple

Experimental

39

Stock solutions of 1.0x10-3 M of the reagents were prepared by

dissolving an appropriate weight of each reagent initially in 25 ml ethanol

followed by dilution in 100 ml measuring flask by ethanol to the mark.

1-5 Britton – Robinson buffer solution:

A stock acid mixture was prepared by mixing equal volumes of

0.4 M of three acids (phosphoric acid, acetic acid and boric acid). A

series of buffer solutions of pH 2.0 to 12.0 were prepared by adding

appropriate volumes of 1.0 M sodium hydroxide as recommended by

Britton (54). The pH values of the prepared buffer solutions were checked

using pH meter type Orion research model 601 A/ digital Ionalyzer.

2- Apparatus

All absorption measurements were made by using a JASCO 530V

(Tokyo, Japan; UV-Vis) spectrophotometer with a scanning speed of

400 nm/min and a band width of 0.2 nm, equipped with 10 mm matched

quartz cells.

An Orion research model 601A/digital ionalyzer pH meter was used to

check the pH of the buffer solution.

3- Working procedures

3-1 Effect of pH

In order to determine the optimum pH values for the formation of

ion-associate complexes between drugs and reagents under study, a

series of solutions containing 2.0 ml (1.0x10-3 M) of reagent, 1.0 ml

(1.0x10-3 M) of the drug and 3.0 ml buffer solution of different pH values

were prepared. Each solution was completed to 10.0 ml with bidistilled

water. The content of each flask was mixed well, and then the

absorbance was measured against a blank prepared in the same way

without drug. The optimum pH value was determined from the curve of

highest absorbance and chosen for further studies.

Experimental

40

3-2 Determination of λmax of complex species

For determination of maximum wavelength (λmax) at which each

ion-associate complex absorbs, the following spectra were recorded:

A) Spectrum of 2.0 ml of 1x10-3 M reagent solution at the suitable pH

using the same pH as a blank.

B) Spectrum of 2.0 ml of 1x10-3 M reagent solution at the suitable pH

+ 1.0 ml of 1x10-3 M drug using the same pH as a blank.

C) Spectrum of solution (B) using solution (A) as a blank.

The λmax at which the last curve (C) absorbs gives the corresponding

maximum wavelength of the ion – associate complex.

3-3 Effect of reagent concentration

The effect of reagent concentration on the complex formation

between drugs and reagents was studied by keeping the drug

concentration constant (1.0 ml of 1x10-3 M), while that of the reagent is

regularly varied (0.2, 0.5,…4.0 ml of 1x10-3 M). The selected pH (3.0 ml)

is added and the volume is completed with bidistilled water to the mark

in 10 ml measuring flask. The solution is mixed well and the absorbance

of each sample solution is measured at the recommended wavelength

against a blank solution prepared in the same manner without the drug.

The best reagent concentration gave the highest absorbance value.

3-4 Effect of buffer volume

The effect of buffer volume on the reaction between the drug

solution and the reagents is investigated by adding different buffer

volumes of the selected pH (1.0,2.0,… 4.0 ml) to fixed concentrations of

drug and reagent (1.0 ml of 1x10-3 M drug solution + 2.0 ml of 1x10-3 M

reagent solution) and the volume is completed to 10 ml with bidistilled

water. The absorbance of each sample solution is measured against

blank solution of reagent at the same pH. The optimum volume of buffer

is chosen from the high absorbance value.

Experimental

41

3-5 Effect of time and temperature

The effect of time on the reaction between drugs and different

reagents is studied by measuring the absorbance of previously

described sample solution against reagent blank solution at different

time intervals. The highest absorbance value is obtained at the optimum

time.

Also, the effect of temperature is studied for the same sample by

heating both sample and blank at different temperatures (25 – 50oC).

The sample and the blank are cooled to room temperature, then the

absorbance is measured at the recommended wavelength. From the

highest absorbance value, the optimum temperature for the formed

complexes is determined.

3-6 Effect of sequence of additions

The effect of sequence of addition (reagent, buffer and drug) on

the formation of ion-associate complex is studied by measuring the

absorbance of sample solutions prepared using different sequences of

additions against blank solution prepared by the same manner except of

drug. The best sequence of addition is determined from the highest

absorbance value.

4- Determination of the molecular structure

The molecular structure of the formed colored complex is

determined by two spectrophotometric methods (mole ratio and

continuous variation methods). The data obtained from these methods

are used for calculating the stability constants of the colored products.

Experimental

42

In the molar ratio method, described by Yoe and Jones (55), the

concentration of the drug is kept constant at (0.5 ml of 1x10-3 M) while

that of the reagent is varied (0.2, …..2.4 ml of 1x10-3 M), 3.0 ml of the

selected buffer solution is added and the volume is completed to 10 ml

with bidistilled water. The absorbance of the sample solution is

measured against reagent blank at the maximum wavelength. The

absorbance values are then plotted against the molar ratio [reagent

/drug] and the inflection of the straight line obtained shows the molar

ratio of the most stable (drug : reagent) products.

4-2 The continuous variation method

In the present work, the modification of Job's (56) continuous

variation method performed by Vosburgh et.al.(57) is used to investigate

the stoichiometry of the complex formed between drug and reagent. A

series of solutions are prepared by mixing equimolar solution of the

reagent and drug in different preparations keeping the total molar

concentration constant (2.0x10-3 M) in the presence of 3.0 ml of the

selected buffer. A plot of the absorbance of the solution at the maximum

wavelength against the mole fraction of the drug gives the molar ratio of

the most stable formed complex.

The stability constant of the formed complex is calculated from

the data obtained from the molar ratio and continuous variation methods

applying Issa equation (58).

4-1 The mole ratio method

Experimental

43

5- Spectrophotometric determination of drugs

5-1 Procedure for obeyence of Beer's law

To investigate the validity of Beer's law for the reaction of the

drugs cefotaxime, ceftazidime pentahydrate and cefepime with the

reagents under study, a series of colored solutions containing the

optimum amount of reagent (2.0 ml of 1x10-3 M), 3.0 ml of the selected

buffer solution and different concentrations of each drug in µg/ml are

mixed well in 10 ml measuring flask. The volume is completed to the

mark with bidistilled water and the absorbance is measured at the

corresponding wavelength against a blank solution containing the same

ingredients except the drug. By plotting the absorbance against the

concentration of the drug in µg/ml, a straight line is obtained after which

a deviation of Beer's law is observed. The sensitivity of the method is

determined by calculating both the molar absorptivity and Sandell

sensitivity (59)

5-2 Ringbom method

For more accurate analysis, Ringbom(60) optimum concentration

range is determined by plotting log[D] of drug in µg/ml against the

percent transmittance (T%). The linear portion of the sigmoid curve

obtained gives the accurate range of concentration detected by the

method.

5-3 General Procedure

In a 10 ml volumetric flask, an aliquot drug solution containing

2.0-15 µg/ml is added to 2.0 ml of 1x10-3 M reagent solution followed by

3.0ml universal buffer solution at the optimum pH. The mixture was

diluted to volume with bidistilled water and the solution was allowed to

stand for 5.0 min at room temperature (25 ± 2oC). The absorbance was

then measured at the recommended wavelength using a reagent blank

Experimental

44

similarly prepared without drug. The concentration of the drug is then

determined from the calibration curve previously constructed under the

optimum conditions.

6- Statistical analysis:

The following statistical functions are used to give information

about the accuracy and precision of the proposed method:

Mean value N

X

X i

i

)(

Standard deviation SD = )1(

)(

N

XX i

Relative standard deviation RSD = X

SDx )(100

Error % E% = N

SDx )(100

Confidence limit N

xtSDX

)(

Limit of detection (LOD) C1 = )(3.0s

SD

Limit of quantification (LOQ) C2 = 10 (SD/s)

Where N = number of observation, Xi = individual observation and s =

slope.

Experimental

45

Tests of significance

These tests are used to compare the results of the proposed

method with those of an accepted (standard) method. These tests tell if

there is a significant difference between the new method and the

accepted one. These tests are:

1- The F-test:

This test is based on the standard deviations of the two methods.

F is defined in terms of the variance of the two methods, where variance

is the square of the standard deviation:

2

2

2

1

s

sF where 2

1s > 2

2s

if the calculated F-value does not exceed a tabulated F-value at

the selected confidence limit (95%) and at degrees of freedom (N-1),

then there is no significance difference between the two methods.

2- The Students t-test:

This test is used to decide whether there is a statistical difference

between the results obtained by two different procedures. The t-value is

given by:

s

NXt )(

where X is the mean value, µ is the taken value and s is the

standard deviation.

Experimental

46

Linear least square:

It is a better approach to apply statistic to define the most

probable straight-line fit of the data. If a straight line relationship is

assumed, then the data fit the equation:

bmXY

where Y is the dependent variable (absorbance), X is the

independent variable (concentration), m is the slope of the straight line

and b is the intercept on the coordinate (Y axis). The values of m and b

are given as:

]/)[(

/)[(22 NxX

Nyxyxm

ii

iiii

xmyb

where x is the mean of all the values of xi and y is the mean of

all the values of yi and N the number of data points.

The correlation coefficient (r):

It is used as a measure of the correlation between two values, the

value of r is given as:

])(][)([ 2222

iiii

iiii

yyNxxN

yxyxNr

Results and Discussion

47

Results and Discussion

I- Spectrophotometric determination of cefotaxime (CEFO)

Preliminary investigations revealed that cefotaxime reacts directly

with each of the reagents used [eosin bluish (EB), eosin yellowish (EY),

bromocrysol purple (BCP) and orange G (OG),] to produce soluble ion-

associate complexes. This was observed from the decrease in the

absorption spectra of each reagent when scanned with the drug using

buffer as a blank.

The optimum conditions favoring the formation of the ion – pair

complexes were studies considering the following effects:

1- Effect of pH

Various aqueous buffers (acetate, borate, phosphate, and

universal buffers) with different pH values were tested to establish the

best buffer media. Universal buffer solutions at pH 2.04 -12.06 gave the

best results. High and constant absorbance values were obtained at pH

3.30, 3.30,12.0 and 12.30 by using EB, EY, BCP and OG, respectively;

therefore, all subsequent studies were carried out at these pH values at

which the results were highly reproducible. Moreover, the optimum

volume of the universal buffer solution was examined and found to be

3.0 ml in a total volume of 10 ml. Figures (1 - 4) show the effect of pH

on the absorption spectra of cefatoxime with EB, EY, BCP and OG,

respectively.

Results and Discussion

48

2- Determination of λmax of complex species:

To determine the wavelength at which ion–pair complex species

possesses maximum absorbance (λmax), the following spectra were

recorded:

A- Spectrum of pure reagent; 2.0 ml (1x10-3 M) at the optimum pH

value using the same buffer as a blank.

B- Spectrum of solution mixture of reagent (A) and drug (1.0 ml of

1x10-3 M) at the optimum pH value using the same buffer as a

blank.

C- Spectrum of solution (B) against (A) as a blank.

The absorption spectra are shown in Fig.'s (5-8), from which the

values of λmax for each complex were determined and cited in Table (1).

These optimal wavelengths are chosen for further investigation.

3- Effect of time and temperature:

The effect of time on complex formation was studied by

measuring the absorbance of the complexes at optimum pH against a

blank solution of the same pH at various time intervals. Also, the effect

of temperature was studied for the same solution by incubating the

sample and blank in water bath at different temperatures (25 – 50oC).

The absorbance was measured after cooling to room temperature.

The experiments showed that complexes are formed within few

minutes (5 minutes) after mixing drug with reagent in the buffered media

and remain stable for about 6 hours. It was found also that, increasing

the temperature up to 50oC has slight effect on the absorbance, while

boiling destroys the complex.

Results and Discussion

49

4- Effect of sequence of addition:

The effect of sequence of addition on ion– pair complex formation

was studied by measuring the absorbance of solutions prepared by

different sequences of addition against a blank solution prepared in the

same manner. Experiments showed that the sequence of reagent –

buffer – drug is the best one. So, it seems that the buffer action must

change the reagent to the anionic form [R-] making it capable to interact

with the drug in the cationic form [D+] to form the ion – pair association

complex [R-][D+].

5- Effect of reagent concentration:

To study the effect of reagent concentration on the complex

formation between cefotaxime and different reagents under study, the

concentration of the drug was kept constant (1.0 ml of 1x10-3 M) while

that of the reagent was varied regularly (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0ml

of 1x10-3 M). The resulted spectra showed that 2.0 ml of each reagent is

sufficient for complete complexation.

6- Effect of buffer volume:

The effect of buffer volume on the reaction between the drug

solution and the reagents was investigated by adding different buffer

volumes of the selected pH (1.0, 2.0,…. 4.0 ml) to fixed concentrations

of drug and reagent (1.0 ml of 1x10-3 M drug solution+ 2.0 ml of 1x10-3M

reagent solution) and the volume was completed to 10.0 ml with

bidistilled water. The absorbance of each sample solution was

measured against a blank solution of reagent at the same pH. The

optimum volume of buffer was found to be 3.0 ml chosen from the

highest absorbance value. This volume is used for further studies.

Results and Discussion

50

7- Stoichiometry of complexes:

The molecular structure of the formed colored complex was

determined by two spectrophotometric methods (mole ratio and

continuous variation methods). The data obtained from these methods

were used to calculate the stability constants of the colored products.

7-1 The continuous variation method

In the present work, the modification of Job's (56) continuous

variation method performed by Vosburgh et.al.(57) was used to

investigate the stoichiometry of the complex formed between drug and

reagent. A series of solutions were prepared by mixing equimolar

solution of the reagent and drug in different preparations keeping the

total molar concentration constant (2.0x10-3 M) in the presence of 3.0 ml

of the selected buffer. A plot of the absorbance of the solution at the

maximum wavelength against the mole fraction of the drug gives the

molar ratio of the most stable formed complex. Experimental results

revealed that the complexes formed have 1:1 stoichiometric ratio.

7-2 The mole ratio method

In the molar ratio method described by Yoe and Jones (55), the

concentration of the drug was kept constant at (0.5 ml of 1x10-3 M) while

that of the reagent was varied (0.2, …..2.4 ml of 1x10-3 M), 3.0 ml of the

selected buffer solution is added and the volume is completed to 10.0ml

with bidistilled water. The absorbance of the sample solution was

measured against reagent blank at the maximum wavelength. The

absorbance values were then plotted against the molar ratio

[reagent/drug]. The inflection of the straight line obtained shows the

molar ratio of 1:1 (drug : reagent) products. Results obtained from mole

ratio and continuous variation methods are in agreement with each

others.

Results and Discussion

51

8- Stability constants of the complexes:

The stability constants of the formed complex were calculated

using the data obtained from the mole ratio and continuous variation

methods applying the equation of Yeo and Jones (55) as modified by Issa

et al (58).

21

max

max

)]/(1[

)/(

nCAA

AAK

n

R

nn

where:

A : the absorbance at concentration CR

Amax : the maximum absorbance value

n : the stoichiometric ratio of the complex

Kn : the stability constant

Log stability constants calculated from mole ratio and continuous

variation methods are listed in Table (1).

Results and Discussion

52

9- Validity to Beer's law:

Under optimum conditions, mentioned above, different

concentrations of cefotaxime (µg/ml) were transferred to 10.0 ml

measuring flask containing 2.0 ml (1x10-3 M) of reagent and 3.0 ml of

buffer solution of the optimum pH. The volume was completed to the

mark by bidistilled water and the content of the flask was mixed well.

The absorbance was measured at optimum λmax, then plotted against

drug concentration [D] as shown in Fig.'s (9 - 12).

Limits of Beer's law, the molar absorptivity (ε; lmol-1cm-1) and

Sandell sensitivity(59) values were calculated and listed in Table (1).

Regression analysis for the results were as carried out using least

square method. In all cases, Beer's law plots were linear with very small

intercepts (-0.0084 – 0.0113) and good correlation coefficients (0.9884 -

0.9991).

For more accurate analysis, Ringbom (60) optimum concentration

range was determined by calculating the percent transmittance (%T)

from the following equation:

10010% xT A

where A is the absorbance of the complex.

By plotting logarithm of drug concentration; log[D] in µg/ml against

%T as in Fig.'s (13 -16), the linear portion of the sigmoid curve gave the

accurate range of analysis. Results are listed in Table (1).

Results and Discussion

53

10- Accuracy and precision:

To determine the accuracy and precision of the proposed method;

solutions of certain concentration (within the concentration range

optained fom Beer's law and Ringbom methods) were prepared and

analyzed in six replicates. The percentage relative standard deviation

(% RSD) did not exceed 0.552 % indicating high accuracy and

reproducibility of the proposed method (Table 2). The percentage

recovery and the range of error (%) at 95% confidence level indicate the

reasonable accuracy and precision. The results are considered as very

satisfactory for the examined concentration levels.

11- Analytical applications:

The validity of the proposed procedure was tested for determination

of cefotaxime in pharmaceutical preparations manufactured in local

companies such as cefotaxime and claforan ampoules (containing 1.0 and

0.5 mg respectively, of cefotaxime per 2 ml). The standard additions

method was used, in which variable amounts of the pure drug were added

to the previously analyzed portion of the pharmaceutical formulations. The

data, c.f. Table (3), showed that the proposed method is highly sensitive;

therefore, it could be used easily for routine determination of CEFO in its

pure form and in its pharmaceutical formulations.

The performance of the proposed method was judged further by

the Student's t-test for accuracy and F-test for precision. At 95%

confidence level, the calculated t- and F-values did not exceed the

tabulated values (t = 2.57 and F = 5.05) suggesting that the method is

accurate and precise as the reference method.

Results and Discussion

54

II- Spectrophotometric determination of ceftazidime

Preliminary investigations revealed that fourtum reacts readily

with each of the reagents used [eosin bluish (EB), orange G (OG),

bromocrysol purple (BCP) and arsenazo I (ARZ I),] to produce soluble

ion-associate complexes. The importance of utility of such reagents

stems from several points, namely, high selectivity of the reactions, high

solubility of the colored complexes, exact stoichiometric composition

and stability of the colored complexes.

To investigate the optimum conditions favoring the formation of

the colored complexes, the following points were extensively studied:

1- Effect of pH

2- Selection of the suitable wavelength at which complex species

maximally absorb.

3- Effect of time and temperature.

4- Effect of sequence of addition.

5- Effect of reagent concentration.

6- Effect of buffer volume.

1- The effect of pH on the ion – pair complex formation was studied by

recording the absorption spectra of series of solutions containing 2.0 ml

(1.0x10-3 M) of reagent, 3.0 ml universal buffer solution of the pH range

2.60 – 11.62 and 1.0 ml (1.0x10-3 M) of the drug against blank solutions

prepared in the same way without drug at the same pH. The absorption

spectra are shown in Fig.'s (17 - 20). Inspection of the data gathered

from these figures shows that the optimum pH values giving maximum

absorbance are 3.35, 7.81, 12.0 and 12.0 for EB, OG, BCP and ARZ I

respectively. These values are recommended for subsequent studies.

Results and Discussion

55

2- The wavelength at which ion – pair complex species possesses

maximum absorbance (λmax), was determined by recording the

following spectra:

(A) Spectrum of pure reagent (2.0 ml of 1x10-3 M) at the optimum

pH using the same buffer as a blank.

(B) Spectrum of solution mixture of reagent (A) and drug (1.0 ml

of 1x10-3 M) at the optimum pH value using the same buffer as

a blank.

(C) Spectrum of solution (B) against (A) as a blank.

The absorption spectra are shown in Fig.'s (21 - 24), from which

the values of λmax for each complex were determined and cited in Table

(4). These optimal wavelengths are chosen for further investigation.

3- Experiment on the effect of time and temperature on complex

formation showed that complexes are formed within few minutes

(5 minutes) after mixing drug with reagent in the buffered media and

remain stable for about 6 hours. It also showed that, increasing the

temperature up to 50oC has slight effect on the absorbance, while

boiling destroys the complex.

4- The effect of sequence of addition on ion – pair complex formation

was studied as previously discussed where it was found that the best

sequence is reagent–buffer–drug. So, it is clear that the buffer action

must change the reagent to the anionic form [R-] making it capable to

interact with the drug in the cationic form [D+] to form the ion – pair

association complex [R-][D+].

5- The effect of reagent concentration on the complex formation was

studied by recording the absorption spectra of series of solutions

containing different reagent concentration and constant drug

concentration. The resulted spectra showed that 2.0 ml (1x10-3 M) of

each reagent is sufficient for developing complete complexation.

Results and Discussion

56

6- The effect of buffer volume on the reaction between the drug solution

and the reagents is investigated as mentioned early. The optimum

volume of buffer is found to be 3.0 ml, chosen from the highest

absorbance value, and was used for further studies.

Stoichiometryand stability constants of complexes

The molecular structure of the formed colored complex is

determined by both mole ratio and continuous variation methods.

Investigation of molecular structure of EB, OG, BCP and ARZ I

complexes with ceftazidime in the light of the results obtained by the two

methods reveals the formation of 1:1 complexes.

The stability constants of the formed complex were calculated

using the data obtained from the molar ratio and continuous variation

methods. The data listed in Table (4) indicate high stability of the formed

complexes.

Validity to Beer's law

The use of EB, OG, BCP and ARZ I as chromophoric reagents for

the spectrophotometric determination of ceftazidime is checked by the

validity of Beer's law. Series of solutions in which the concentration of

each reagent is kept constant (2.0 ml of 1x10-3 M) while that of the drug

is regularly varied, were prepared at the recommended pH. The

absorbance was then measured at the corresponding wavelength for

each complex and plotted vs concentration of the drug [D; µg/ml], (c.f.

Fig.'s 25 - 28).

Limits of Beer's law, molar absorptivity (ε=3.43–6.31x104 lmol1cm-1)

and Sandell sensitivity (0.036 – 0.072 µg/cm2) values were calculated and

listed in Table (4). Regression analysis for the results was also carried out

using least square method. In all cases, Beer's law plots were linear with

very small intercepts (-0.017 - 0.063) and good correlation coefficients

(0.9984 -0.9993).

Results and Discussion

57

For more accurate analysis, Ringbom optimum concentration

range was determined by plotting logarithm of drug concentration,

log[D], in µg/ml against %T as in Fig.'s (29 - 32). The linear portion of

the sigmoid curve gave the accurate range of analysis. Results are

listed in Table (4).

Accuracy and precision

To determine the accuracy and precision of the proposed method;

solutions of certain concentration (within the concentration range

optained fom Beer's law and Ringbom methods) were prepared and

analyzed in six replicates. The percentage relative standard deviation

(% RSD) did not exceed 0.132 % indicating high accuracy and

reproducibility of the proposed method (Table 5). The percentage

recovery and the range of error (%) at 95 % confidence level indicate

the reasonable accuracy and precision. The results are considered as

very satisfactory for the examined concentration levels.

Analytical applications:

The validity of the proposed procedure was tested for

determination of ceftazidime in two of its pharmaceutical formulations

(fourtum and fortaz, containing 1.0 and 1.0 mg of ceftazidime per

ampoule). The standard additions method was used, in which variable

amounts of the pure drug were added to the previously analyzed portion

of the pharmaceutical formulations. The data, c.f. Table (6), showed that

the proposed method is highly sensitive; therefore, it could be used

easily for routine determination of ceftazidime in its pure form and in its

pharmaceutical formulations.

The performance of the proposed method was judged further by

the Student's t-test for accuracy and F-test for precision. At 95%

confidence level, the calculated t- and F-values did not exceed the

tabulated values (t = 2.57 and F = 5.05) suggesting that the method is

accurate and precise as the reference method.

Results and Discussion

58

III- Spectrophotometric determination of cefepime

Preliminary investigations showed that cefepime reacts directly

with each of the reagents used [eosin yellowish (EY), eosin bluish (EB),

orange G (OG) and arsenazo I (ARZ I)] to produce soluble ion-associate

complexes. This was acertained from styding the absorption spectra of

each reagent (in ethanol as a solvent) compaired with that of the

reagent and cefepime in the same solvent. The decrease in the

maximum absorbance in the later case is taken as an evidence for

complex formation.

The optimum conditions favoring the formation of the ion – pair

complexes between cefepime and the reagents under study were

extensively studied taking into consideration the following effects:

1- Effect of pH

The effect of pH on the ion – associate complex formation

between cefepime and the four reagents under investigation was

studied in universal buffer solutions within the pH range 2.60 – 11.62 as

previously mentioned, illustrative spectra are shown in Fig.'s (33 - 36).

Careful investigation of these spectra shows that the formed ion –

associate complexes absorb maximally at the pH values 3.35, 4.52,

12.30 and 10.21 for EY, EB, OG and ARZ I respectively. These values

are recommended for subsequent studies.

2- Determination of λmax of complex species

The maximum wavelength (λmax) at which each ion – pair complex

species absorbs was determined, as previously mention, by recording

the following spectra:

A- Spectrum of pure reagent; 2.0 ml (1x10-3 M) at the optimum pH

value using the same buffer as a blank.

Results and Discussion

59

B- Spectrum of solution mixture of reagent (A) and drug (1.0 ml of

1x10-3 M) at the optimum pH value using the same buffer as a

blank.

C- Spectrum of solution (B) against (A) as a blank.

The absorption spectra are shown in Fig.'s (37 - 40), from which

the values of λmax for each complex were determined and cited in Table

(7). These optimal wavelengths are chosen for further investigation.

3- Effect of time and temperature

By measuring the absorbance of the complexes at optimum pH

against a blank solution of the same pH at various time intervals, it was

found that complexes are formed within few minutes (5 minutes) after

mixing drug with reagent in the buffered media and remain stable for

about 6 hours.

Also, studying the effect of temperature on complex formation,

showed that increasing the temperature up to 50oC has slight effect on

the absorbance, while boiling destroys the complex.

4- Effect of sequence of addition

Experiments on the effect of sequence of addition showed that

the sequence of reagent – buffer – drug is the best one indicating that

the buffer solution changes the reagent to the anionic form [R-] making it

capable to interact with the drug in the cationic form [D+] to form the

ion– pair association complex [R-][D+].

5- Effect of reagent concentration

Studying the effect of reagent concentration on the complex

formation between cefepime and reagents under study, showed that 2.0

ml of each reagent is sufficient for complete complexation.

Results and Discussion

60

6- Effect of buffer volume

Experiments on the effect of buffer volume on the complex

formation, performed as previously mentioned, showed that the

optimum volume of buffer is 3.0 ml. This volume is used for further

studies.

7- Stoichiometry and stability constant of complexes

The molecular structure of the formed colored complex was

determined by two spectrophotometric methods (mole ratio and

continuous variation methods). The data obtained from these methods

are used to calculate the stability constants of the colored products. The

experimental data showed the formation of (1:1) (drug : reagent) ion –

pair complex.

The stability constants of the formed complex are calculated

using the data obtained from the molar ratio and continuous variation

methods. Log stability constants are listed in Table (7). The values

obtained revealed that the complexes formed are fairly stable.

8- Validity to Beer's law

Under optimum conditions mentioned in the preceding discussion,

different concentrations of cefepime (µg/ml) were transferred into

10.0ml measuring flask containing 2.0 ml (1x10-3 M) of reagent and 3.0

ml of buffer solution of the optimum pH. The volume was completed to

the mark by bidistilled water and the content of the flask was mixed well.

The absorbance was measured at optimum λmax, then plotted against

drug concentration [D] as shown in Fig.'s (41 -44)

Limits of Beer's law, the molar absorptivity (ε; lmol-1cm-1) and

Sandell sensitivity, (µg/cm2) values were calculated and listed in Table

(7). Regression analysis for the results were as carried out using least

square method. In all cases, Beer's law plots were linear with very small

intercepts (-0.011 - 0.018) and good correlation coefficients (0.9978 -

0.9998).

Results and Discussion

61

For more accurate analysis, Ringbom optimum concentration

range was determined by calculating the percent transmittance (%T)

from the following equation:

10010% xT A

where A is the absorbance of the complex.

By plotting logarithm of drug concentration, log[D] in µg/ml against

%T as in Fig.'s (45 - 48), the linear portion of the sigmoid curve gave an

accurate range of analysis. Results are listed in Table (7).

9- Accuracy and precision

To determine the accuracy and precision of the proposed method;

solutions of certain concentration (within the concentration range

obtained from Beer's law and Ringbom methods) were prepared and

analyzed in six replicates. The percentage relative standard deviation

(% RSD) did not exceed 0.247% indicating high accuracy and

reproducibility of the proposed method (Table 8). The percentage

recovery and the range of error (%) at 95% confidence level indicate the

reasonable accuracy and precision. The results are considered as very

satisfactory for the examined concentration levels.

Summary and Conclusion

62

Summary and Conclusion

This thesis consists of three main chapters:

The first chapter (the introduction)

Represents short notes about the structure and action of the three

drugs under study (cefatoxime, ceftazidime and cefepime). It also

includes a literature survey on the previous works carried out on the

different techniques for the determination of these drugs.

The second chapter (the experimental)

Describes the procedures used throughout the study so as to get

the optimum conditions favoring colored complex formation between the

drug and reagent molecules by ion – pair mechanism. This chapter also

describes the instruments used, how to prepare different solutions and

the suggested procedure for determination of drugs either in pure or in

dosage forms.

The third chapter (results and discussion)

Includes the results obtained throughout the work and their

discussion, it is subdivided to three parts:

Part I: Presents optimum conditions that favor the spectrophotometric

determination of cefotaxime using the four reagents eosin bluish

(EB), eosin yellowish (EY), bromocresole purple (BCP) and

orange G (OG). These conditions are summarized as:

i- Britton – Rhobinson universal buffer solution was found to be the

best media for complexation process. This series of buffer

solutions has the advantages of wide range of pH (2 – 12) and

that its components do not interfere with the drug or reagents. It

was found that, maximum tendency for complex formation takes

place at pH 3.35, 3.35, 12.0 and 12.0 for EB, EY,BCP and OG

respectively.

Summary and Conclusion

63

ii- An evidence for complex formation between cefotaxime and the

reagents is observed by determination of the maximum wave-

length (λmax) of the colored complexes. It was found that

complexes of cefotaxime with EB, EY,BCP and OG absorb

maximally at 544, 538, 626 and 536 nm respectively.

iii- Study on the effect of time and temperature showed that the

complexes are formed within 5 minutes and remain stable for

about 6 hours. Also, the obtained complexes are stable to heating

up to 50oC.

iv- The sequence of addition was found to be of significance

importance. The best sequence of addition is reagent – buffer –

drug. Thus, it can be concluded that buffered media are required

to maintain the reagent molecule in the suitable form for complex

formation.

v- The stoichiometry of the complexes formed in solution was

detected using the mole ratio and continuous variation methods. It

was found that, all complexes are of 1:1 stoichiometric ratio. The

stability constants of the formed complexes were calculated from

spectral data of the two methods which indicate that these

complexes are fairly stable.

vi- The optimum concentrations of cefotaxime which can be

successfully determined by the reagents under study were

detected by Beer's law. From the data obtained, it was found that

cefotaxime was successfully determined up to 9.6, 5.8, 10.5 and

7.1 µg/ml on using EB, EY,BCP and OG respectively. The values

of molar absorptivity (ε) lie within the range 6.36 – 4.03 x104 l mol-

1 cm-1 and Sandell sensitivity in the range (0.048 – 0.088 µg/cm2).

Such high values reflect the sensitivity of the proposed method.

Regression analysis for the results were carried out using least

Summary and Conclusion

64

square method. In all cases, Beer's law plots were linear with very

small intercepts (-0.0084 - 0.0113) and good correlation

coefficients (0.9884 -0.9989).

vii- Another way for detecting the lower and higher limits of

concentration was determined using Ringbom method where a

satisfactory agreement between Beer's law and Ringbom methods

was observed.

viii- The accuracy and precision of the proposed method was

determined by analyzing 6 replicate samples within the

concentration range obtained from Beer's law and Ringbom

methods. At these concentrations, the relative standard deviation

(RSD) values are in the range 0.231 - 0.552, the detection limits

are in the range 0.36 – 1.87 µg/ml and the quantification limits are

within the range 4.05 – 8.4 µg/ml.

ix- As an application of the proposed method, the content of

cefotaxime in some local samples was determined. The results

obtained agreed with the label claim and with those of the

reference method. The performance of the proposed method was

judged further by the Student's t-test for accuracy and F-test for

precision. At 95% confidence level, the calculated t- and F-values

did not exceed the tabulated values (t = 2.57 and F = 5.05)

suggesting the accuracy and precision of the method.

x- The accuracy and validity of the proposed method were further

ascertained by performing recovery studies from standard addition

technique using the four reagents EB, EY,BCP and OG. The pre-

analyzed ampoule solutions (cefotaxime amp. And claforn) were

spiked with pure cefotaxime at three levels and the total was

found by the proposed method. Each determination was repeated

three times. The results reveal good recoveries of pure drug

added.

Summary and Conclusion

65

Parts II presents the results obtained when the factors affecting the

complexation of ceftazidime with the four reagents; eosin bluish

(EB), orange G (OG), bromocresole purple (BCP) and arzanazo I

(ARZ I) were studied:

i- The optimum pH values required for complex formation are: 3.35,

7.81, 12.0 and 12.0 for EB, OG, BCP and ARZ I respectively.

the λmax (nm) at which each complex absorbs are 537, 513,

626 and 565 nm for EB, OG, BCP and ARZ I respectively.

ii- 2.0 ml of 1x10-3 M of reagent and 3.0 ml of buffer solution were

found to be sufficient for complex formation.,

iii- Complexes were formed within few minutes and unaffected by

temperature up to 50oC time,

iv- All the formed complexes are of 1:1 stoichiometric ratio as

gathered from mole ratio and continuous variation methods.

v- Using Beer's law, it was found that ceftazidime is successfully

determined up to 15.40, 14.13, 12.32 and 12.11 µg/ml on

using EB, OG, BCP and ARZ I respectively. The values of

molar absorptivity (ε) lie within the range 3.43 - 6.31 x104 l mol-

1 cm-1 and Sandell sensitivity in the range (0.036 – 0.072

µg/cm2). In all cases, Beer's law plots were linear with very

small intercepts (-0.0017-0.063) with good correlation

coefficients (0.9984 - 0.9993).

vi- The accuracy and precision of the proposed method was

determined by analyzing 6 replicate samples within the

concentration range obtained from Beer's law and Ringbom

methods. At these concentrations, the relative standard

deviation (RSD) values are in the range 0.035 – 0.132, the

detection limits are in the range 0.29 – 9.78 µg/ml and the

quantification limits are within the range 0.053 – 0.97 µg/ml.

Summary and Conclusion

66

vii- As an application of the proposed method, the content of

ceftazidime in some local samples (fourtum and fortaz) was

determined. The results obtained agreed with the label claim

and with those of the reference method. The performance of

the proposed method was judged further by the Student's t-

test for accuracy and F-test for precision. At 95% confidence

level, the calculated t- and F-values did not exceed the

tabulated values (t=2.57 and F= 5.05) suggesting the accuracy

and precision of the method.

Part III: Presents the optimum conditions that favor the spectro-

photometric determination of cefepime using the four reagents,

eosin yellowish (EY), eosin bluish (EB), orange G (OG) and

arzanazo I (ARZ I). These conditions are summarized as:

i- Studying the effect of pH on the complex formation between

cefepime and the four reagents it was found that, maximum

tendency for complex formation takes place at pH 5.35, 4.52, 12.30

and 10.21 for EY, EB, OG and ARZ I respectively.

ii- The complexes of cefepime with EY, EB, OG and ARZ I absorb

maximally at 622, 531, 531 and 563 nm respectively.

iii- The complexes are formed within 5 minutes and remain stable for

about 6 hours. Also, the obtained complexes are stable to heating

up to 50oC.

iv- The best sequence of addition is reagent – buffer – drug, indicating

that buffered media are required to maintain the reagent molecule

in the suitable form for complex formation.

v- All complexes are of 1:1 stoichiometric ratio as shown from the data

of mole ratio and continuous variation methods. The stability

constants, calculated from spectral data, indicate that these

complexes are fairly stable.

Summary and Conclusion

67

vi- Using Beer's law, it was found that cefepime is successfully

determined up to 10.42, 11.51, 8.78 and 9.76 µg/ml on using EY,

EB, OG and ARZ I respectively. The values of molar absorptivity (ε)

lie within the range 3.52 – 4.81 x104 l mol-1 cm-1 and Sandell

sensitivity in the range (0.066 – 0.081 µg/cm2). In all cases, Beer's

law plots were linear with very small intercepts (-0.011 – 0.018)

with good correlation coefficients (0.9978 - 0.9998).

vii- The detection of the lower and higher limits of concentration was

determined using Ringbom method where a satisfactory agreement

between Beer's law and Ringbom methods was observed.

viii- The accuracy and precision of the proposed method was

determined by analyzing 6 replicate samples within the

concentration range obtained from Beer's law and Ringbom

methods. At these concentrations, the relative standard deviation

(RSD) values are in the range 0.040 – 0.247, the detection limits

are in the range 0.02 – 0.064 µg/ml and the quantification limits are

within the range 0.02 – 2.12 µg/ml.

References

68

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