Validated Analytical Methods for the Determination of Some ...

Validated Analytical Methods for the

Determination of Some Antimicrobial Drugs.

A Thesis Presented by

Mohamed Badawi Badawi Nour El-Din

B.Sc. Pharm. Sci. 2002, M. Pharm. 2012

Faculty of Pharmacy, Cairo University

Submitted for the Degree of Doctor of Philosophy in Pharmaceutical

Sciences

(Pharmaceutical Chemistry)

Supervised by

Prof. Dr. Ramzia Ismail EL-Bagary Professor of Pharmaceutical Chemistry

Faculty of Pharmacy, Cairo University

Head of Pharmaceutical Chemistry Department

Faculty of Pharmacy, Future University

DR. Nisreen Farouk Abo-talib Assistant Professor of Analytical Chemistry

National Organization for Drug Control and Research (NODCAR)

DR. Maha Medhat EL-Hakeem Lecturer of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University

Faculty of Pharmacy

Cairo University 2018

English Abstract

ion of Some Antimicrobial Validated Analytical Methods for the Determinat

Drugs

Different methods were proposed in this thesis for the simultaneous determination of the

antimicrobial agent Ceftriaxone sodium in its binary mixture with Sulbactam sodium or

Tazobactam sodium in their pure forms, in dosage forms and in the presence of their degradation

products degradation products. Chromatographic part included the drugs simultaneous

determination in presence of their acid, alkaline, oxidative, neutral and photolytic degradation

products by HPLC, Green HPLC and TLC methods.

Spectrophotometric part included three methods for the drugs simultaneous

determination. Derivative spectrophotometry was presented to determine Ceftriaxone sodium

and Sulbactam sodium in presence of their oxidative degradation products and determine

Ceftriaxone sodium and Tazobactam sodium in presence of Ceftriaxone sodium acid degradation

products. Derivative ratio method was adopted to determine Ceftriaxone sodium with Sulbactam

sodium or Tazobactam sodium simultaneously in presence of Ceftriaxone sodium alkaline

degradation products. Also ratio difference technique was established for the simultaneous

determination of Ceftriaxone sodium in its binary mixture with Sulbactam sodium or

Tazobactam sodium in bulk powder and in pharmaceutical dosage forms.

Degradation products of Ceftriaxone sodium, Sulbactam sodium and Tazobactam sodium

were prepared and their structures were verified.

Key words:

Ceftriaxone sodium – Sulbactam sodium – Tazobactam sodium – HPLC – Green chemistry –

Human plasma – TLC – Derivative spectrophotometry – Derivative of the ratio spectra – Ratio

difference –Degradation.

Introduction

Microorganisms of medical importance fall into four categories which are bacteria,

viruses, fungi and parasites. The first broad classification of antimicrobial drugs follows this

classification closely, so that we have antibacterial, antiviral, antifungal and antiprotozoal agents

within each of these major categories, drugs are further categorized by their biochemical

properties. The antimicrobial drugs are classified according to chemical structure and the

mechanism of action [1] as follow:

1) Agents inhibit bacterial cell wall synthesis, including the β-lactam class (e.g. penicillins,

cephalosporines, and carbapenemes) and dissimilar agents such as cycloserine,

vancomycin, and bacitracin.

2) Agents act directly on the cell membrane of the microorganism, increasing permeability

and leading to leakage of intracellular compound, including detergents such as

polymyxin, polyene antifungal agents (e.g. nystatin and amphotericin B).

3) Agents disrupt function of 30S or 50S ribosomal subunits to reversibly inhibit protein

synthesis, which generally are bacteriostatic (e.g., chloramphenicol, the tetracyclines,

erythromycin, clindamycin, streptogramins, and linezolid).

4) Agents bind to the 30S ribosomal subunit and alter protein synthesis, which generally are

bactericidal (e.g., aminoglycosides).

5) Agents affect bacterial nucleic acid metabolism, such as the the rifamycins (e.g., rifampin

and rifabutin), which inhibits RNA polymerase, and the quinolones, which inhibit

topoisomerases.

6) Antimetabolites including trimethoprim and the sulfonamides which block essential

enzymes of folate metabolism.

The use of antimicrobial drugs to treat infection is known as antimicrobial chemotherapy,

while the use of antimicrobial drugs to prevent infection is known as antimicrobial prophylaxis

[2]. Due to high resistance of microbes, a combination therapy of antimicrobial agents is widely

used to overcome the microbial resistance. More than one antibiotic is commonly used to treat

multidrug resistant bacterial infections e.g. sepsis due to carbapenem-resistant

Enterobacteriaceae, bacteremic pneumococcal pneumonia, and patients with multiple organ

failure [3, 4].

https://en.wikipedia.org/wiki/Antimicrobial_chemotherapy

https://en.wikipedia.org/wiki/Antibiotic_prophylaxis

Cephalosporines.

They are antibacterial agents of β-lactam antibiotics similar to penicillin in structure and

mode of action. They form part of the most commonly prescribed and administered antibiotics;

more succinctly, they account for one-third of all antibiotics prescribed and administered by

the National Health Scheme in the United Kingdom [5]. Cephalosporins are used in the

treatment of bacterial infections and diseases arising from Penicillinase-producing,

Methicillin-susceptible Staphylococci and Streptococci, Proteus mirabilis, some Escherichia

coli, Klebsiella pneumonia, Haemophilus influenza, Enterobacter aerogenes and some

Neisseria [6].

Cephalosporins have a variety of side chains that enable them get attached to different

penicillin-binding proteins (PBPs), to penetrate blood brain barrier, resist breakdown by

penicillinase producing bacterial strains and ionize to facilitate entry into Gram-negative

bacterial cells [7].

Mode of action

Most bacterial cells are encased by a rigid layer of peptidoglycan, which protects the cells

in the face of prevailing osmotic pressure consistent with the often-harsh environment and

conditions under which they exist. Peptidoglycan has a degree of cross-linking peptide bonds

called β-(1-4) –N– acetyl Hexosamine [8, 9]. To stay alive, bacteria must synthesize

peptidoglycan by the activity of PBPs which play very pivotal roles by adding disaccharide

pentapeptides to extend the glycan strands of existing peptidoglycan molecule and also cross-link

strands of immature peptidoglycan units [10]. Cephalosporins are able to block the cross-linking

of peptidoglycan units by inhibiting the peptide bond formation catalyzed by PBPs and hence

they inhibit the bacterial cell wall synthesis as shown in Figure (1) [11].

.cephalosporines(1) Mode of action of Figure

http://en.wikipedia.org/wiki/Beta-lactam_antibiotic

Classification

Cephalosporines are subdivided into generations (1st-5th) in accordance to their potency

towards target organism but later versions are increasingly more effective against Gram-negative

pathogens.

1- First generation

They are the first lot of this class of antibiotics that were produced. These have good

activity against Gram-positive bacteria and relatively modest activity against Gram-negative

microorganisms. Most Gram-positive cocci are susceptible and most oral cavity anaerobes are

sensitive [12]. They are used to treat skin and soft tissue infections, uncomplicated respiratory

tract infections and urinary tract infections (staphylococcal and streptococcal infections) [13].

Some drugs of this group are listed in Table (1).

Table (1) First generation cephalosporins.

Generic

name

Trade

name

Structure

Cephradine Velosef ®

N

S

OHO

O

NH

O

HH2N

Cefadroxil Ibidroxil ®

N

S

OHO

O

NH

O OH

HH2N

Cephalexin Ceporex ®

N

S

OO

O

NH

H

O

NH

H H

Cefazoline Zinol ® N N

S S N

N N

N

S

OHO

H

N

N

O

O

H

Cefapirin Cefa-Lak®

SS

O

OHO

H

N

N

O

O

N

O

H

2- Second generation

They differ from the first generation by their enhanced Gram-negative spectra, including

bacteria resistant to the first generation. The increased activity is due to increased affinity for

PBPs and increased penetration through the outer envelope of Gram-negative bacteria [14]. They

are commonly used to treat acute sinusitis, otitis media and upper respiratory tract infections.

Some drugs of this group are listed in Table (2).

Table (2) Second generation cephalosporins.

Generic name Trade name Structure

Cefotetan Maxtetan ®

N

N N

N S

O

S

SS

O

H

NO

O

H

O

N

O H

O

O

NH

H

HH

Cefoxitin Primafoxin ®

N

O

H

HH

SS

O

O

H

NO

O

H

O

N

O

Cefuroxime Zinacef ® O

S

O

OH

NO

O

N O

N

O

CH3

OH2N

H

Cefprozil Cefzil ®

S

OH

NO

O

NH

O

H2N

OH

H

Cefamandole Mandol ®

N

N N

N S

S

O

NO

HO

N

O

HOH

3- Third generation

They are more active against enteric bacteria, stable against β-lactamases and have a

longer serum T1/2 than 1st generation cephalosporins, administered twice/day. They cross the

blood brain barrier and effective against Gram-negative CNS infections [15]. CT is one of the

third generation cephalosporins which has broad spectrum activity against Gram-positive

and Gram-negative bacteria. It has been proved to be effective in treating infections due to other

‘difficult’ organisms such as multi drug-resistant Enterobacteriaceae [16]. Two factors contribute

to the prolonged duration of action of CT, a high fraction of protein binding in the plasma and a

slow urinary excretion [12]. It is commonly used with β-lactamase inhibitors like sulbactam (e.g.

Formic® vial) and tazobactam (e.g. Xone Xp® vial) to augment its activity [17, 18]. Table (3)

shows some drugs of this group.

Table (3) Third generation cephalosporins.


Ceftriaxone Triaxone ®

S

O

NO

HO

NH

NN

N

S

O

O

N

SO

N

O

N

H

H H

H

Cefdinir Dinar ®

S

O

NO

O

N

H

H

H

S

NO

N

O

H

N H

H

Ceftibuten Shatbiotic ®

S

N

O

O

N

O

HH

S

NO

OO

H

N

H

H

H

H

http://en.wikipedia.org/wiki/Gram-positive_bacteria

http://en.wikipedia.org/wiki/Gram-negative_bacteria

http://en.wikipedia.org/wiki/Bacteria

Ceftazidime Fortum ® O

HH

S

N

O N

ON

O

H

H

HN+

S

N

O

O

N

O

-

Cefpodoxime Cepodem ® O

H HS

N

O N

ONH2

O

S

N

HO

O

N

Cefixime Ximaxef ® O

HS

N

N

ON H

H

O

S

N

O

O

N

H

HO

O

H

O

4- Fourth generation

They have an extended spectrum of activity compared with the third generation and

increased stability from hydrolysis by β-lactamases as they are zwitterions that can penetrate the

outer membrane of Gram-negative bacteria. Many can cross the blood brain barrier and used to

treat meningitis. They are useful to treat serious infections in hospitalized patients when

Pseudomonas and Enterobacteriaceae are potential etiologies [19]. Table (4) shows some drugs

of this group.

Table (4) Fourth generation cephalosporins.


Cefepime Maxipime ® O

H N

S

N

O

N

H

HS

N

O

O

N

HO

N+

Cefpirome Piromaxef ®

HCH3

O

N

S

H2N

N

N

O

O

N

S

O O-

N

H H

+

5- Fifth generation

They are the most recent cephalosporins which are bactericidal with extended spectrum

antibiotics. They show excellent activity against a wide range of Gram-positive and Gram-

negative organisms. They are also active against Methicillin-resistant Staphylococcus

aureus infections [20, 21]. Some drugs of this group are listed in Table (5).

Table (5) Fifth generation cephalosporins.


Ceftaroline Teflaro ®

N

N

O

S

N

N

S S

O- O

H HN

S

N

O

N

N

O

P

O

O

OH

H

H

+

Ceftobiprole Zeftera ®

HN

S

N

O

N

N

O

HN N

N

O

S

N

C

O O

H

O H

H

H

H

H

Ceftolozane Zerbaxa ® O

HH N

S

N

O N

ON

O

H

O

NN

S

N

O

O

N

N

N

O

N

N

H

H

H

HH

H

H

H+

-

Pharmacokinetics and metabolism of the cephalosporins

Most cephalosporins have a plasma half-life of 30 to 120 minutes. Only ceftriaxone has a

half-life of about 8 hours, which implicates extended dose intervals [22]. Cephalosporins are

excreted renally. Cefoperazone and ceftriaxone, however, are excreted with the bile up to 30%.

Cefalotin, cefacetril and cefotaxime are metabolized to a significant degree. Only the main-

metabolite of cefotaxime shows mentionable antimicrobial activity. The major active metabolite

of ceftriaxone is ceftizoxime [23]. With regard to renal insufficiency (creatinine clearance

greater than 5 ml/min) dose reduction is not necessary with both cefoperazone and ceftriaxone

due to hepatic excretion and with cefotaxime because of metabolism.

Structure-activity relationship

Figure (2) shows the cephalosporin ring system formed of Beta-Lactam Ring (A) and

Dihydrothiazine Ring (B) [24, 25].

Y

N

COOH

O

HX

NC

O

H

A BZ

Ar

v

v`

re.phoCephalosporin pharmaco(2) Figure

1- Beta-Lactam Ring: It is required for PBPs reactivity and antibacterial activity.

2- Carboxyl Group: It mimics the terminal carboxyl of the D-alanyl-D-alanine moiety in PBPs

normal substrate and therefore it is responsible for activity. It is also responsible for salt

formation and site for prodrug formation.

Various molecular changes in the cephalosporin structure can improve in vitro stability,

antibacterial activity and stability towards β-lactamases.

(a) The addition of an amino and a hydrogen to the v and v`positions, respectively, results in

a basic compound that is protonated under the acidic conditions of the stomach.

(b) The ammonium ion improves the stability of the β-lactam of the cephalosporin, leading to

orally active drugs.

(c) The 7 β- amino group is essential for antimicrobial activity (X=H), where as replacement

of H at C7 with an alkoxy (X=OR) results in the improvement of the antibacterial activity

of the cephalosporin within specific cephalosporin deriveatives.

(d) The addition of a 7 α methoxy also improves the drug stability towards β-lactamases.

(e) The derivatives where Y=S exhibit greater antibacterial activity than if Y=O, but the

reverse is true when stability towards β-lactamases is considered.

(f) The 6 α hydrogen is essential for biologic activity.

(g) The antibacterial activity is improved when Z is a five membered heterocycle versus a six

membered heterocycle.

Aim of the present work

The aim of this work is to develop new accurate and reliable methods for determination

of some antimicrobial agents. In this thesis we presented several analytical techniques such as

chromatography and spectrophotometry to determine the cephalosporin drug ceftriaxone (CT)

simultaneously in its binary mixtures with sulbactam (SB) and also with tazobactam (TZ) as β-

lactamase inhibitors.

These mixtures were analyzed in their bulk powder, commercially used formulations and

in the presence of their acid, alkaline, oxidative, neutral and photolytic degradation products. The

investigated drugs are characterized by their susceptibility for degradation by acid, alkaline,

oxidative, neutral and photolytic conditions into inactive products. This fact encouraged the

author to propose stability-indicating procedures for the determination of the intact drugs in the

presence of their degradation products.

HPLC and TLC techniques are efficient analytical tools to separate and analyze the

pharmaceutical active ingredients. The work plan comprised the utilization of these techniques

for the quantitative analysis of the cited drugs in raw materials and in their pharmaceutical

formulations.

The concept of green chemistry was incorporated into the thesis using HPLC technique to

reduce or eliminate organic solvents, eco-toxic reagents, preservatives, and other chemicals that

are hazardous to human health or the environment [115]. The proposed green analytical HPLC

methods were applied to determine the drugs in their pharmaceutical formulations, in presence of

their degradation products including acid, alkaline, oxidative, neutral and photolytic degradation

products and in human plasma in the presence of ceftizoxime (CZ) which is the active metabolite

of ceftriaxone [116] using cefotaxime (CM) as an internal standard.

Moreover, spectrophotometry was proposed as rapid and simple methodology including

derivative, derivative ratio and ratio difference spectrophotometric methods have been applied to

resolve spectral overlap displayed by the investigated drugs and their degradation products.

In addition, identification of the degradation products, method validation and statistical

comparison between the obtained results of the proposed methods and those of the reported

methods were conducted.

http://en.wikipedia.org/wiki/%CE%92-lactamase_inhibitor


Summary

Different methods for the simultaneous determination of the antimicrobial agent

Ceftriaxone sodium in its binary mixtures with β-lactamase inhibitors (Sulbactam sodium or

Tazobactam sodium) in pure form, in presence of their degradation products and in commercial

dosage forms have been presented in this thesis. The thesis consists of the following six sections:

Section 1 : Introduction.

1) A review about antimicrobial agents and their classification.

2) A review about cephalosporins indications, mechanism of action and their structure-

activity relationship.

3) Literature review about the methods for the quantitative determination of the drugs under

investigation either alone or in combinations.

4) Aim of the present work and the basis on which the proposed methods were chosen.

Section 2 : Experimental and Discussion.

Part 1: Chromatographic Methods which is further subdivided into the following:

Part 1.1. HPLC Methods

Part 1.1.1. Conventional HPLC Methods

1.1.1.1. Stability Indicating HPLC Method for the Simultaneous Determination of

Ceftriaxone Sodium in its binary mixture with either Sulbactam Sodium or Tazobactam

Sodium in Presence of their degradation Products.

The method was based on HPLC separation of Ceftriaxone sodium with Sulbactam

sodium (mixture 1) or Tazobactam sodium (mixture 2) and their acid, alkaline, oxidative, neutral

and photolytic degradation products. The separation was achieved using Thermo BDS Hypersil

C18 Column at ambient temperature with mobile phase consisting of 0.01 M potassium

dihydrogen phosphate buffer adjusted by ortho-phosphoric acid to pH 4.6 – acetonitrile (94 : 6,

v/v) with UV detection at 220 nm at concentration ranges fom 0.50 – 50.00 µg/mL for

Ceftriaxone sodium and Sulbactam sodium in (mixture 1) and from 0.50 – 96.00 µg/mL for

Ceftriaxone sodium and Tazobactam sodium in (mixture 2).

The method was successfully applied for the determination of Ceftriaxone sodium and

Sulbactam sodium (mixture 1) in bulk powder with mean percentage recoveries ± S.D. of 99.88

± 1.43 and 99.59 ± 1.24, respectively. It was also successfully applied for the determination of

Ceftriaxone sodium and Tazobactam sodium (mixture 2) in bulk powder with mean percentage


recoveries ± S.D. of 100.46 ± 1.00 and 99.28 ± 1.89, respectively. The method was easily

applied for the determination of the drugs in laboratory prepared mixture with their acid,

alkaline, oxidative, neutral and photolytic degradation products from 10% up to 90% of

degradate with satisfactory results.


Sulbactam sodium in Formic vial and the validity of the method was assessed by applying the

standard addition technique and acceptable recoveries have been obtained. The mean percentage

recoveries ± S.D. of the labeled Ceftriaxone sodium and Sulbactam sodium in Formic vial were

100.15 ± 0.15 and 99.10 ± 0.36, respectively, while that of the added Ceftriaxone sodium was

99.55 ± 0.68 and the added Sulbactam sodium was 100.71 ± 0.79.

The method was also applied for the determination of Ceftriaxone sodium and

Tazobactam sodium in Xone Xp vial and the validity of the method was assessed by applying the

standard addition technique and good recoveries have been obtained. The mean percentage

recoveries ± S.D. of the labeled Ceftriaxone sodium and Tazobactam sodium in Xone Xp vial

were 99.96 ± 0.16 and 100.17 ± 0.29, respectively, while that of the added Ceftriaxone sodium

was 100.28 ± 1.15 and the added Tazobactam sodium was 98.91 ± 1.58.

Part 1.1.2. Green HPLC Methods

1.1.2.1. Stability Indicating Green HPLC Method for the Simultaneous Determination of


Sodium and its Application in Human Plasma

Ceftriaxone sodium with sulbactam sodium (mixture 1) or tazobactam sodium (mixture

2) are well separated from their acid, alkaline, oxidative, neutral and photolytic degradation

products using Thermo BDS Hypersil C18 Column. The green chemistry principles are adopted

to prepare a mobile phase consisting of 0.005 M potassium dihydrogen phosphate buffer adjusted

by ortho-phosphoric acid to pH 4.6 – ethanol (92 : 8, v/v). The detection was performed by UV

detector at 220 nm at concentration ranges 1.00 to 90.00 µg/mL for drugs in both mixtures.





recoveries ± S.D. of 100.83 ± 0.87 and 100.06 ± 1.08, respectively.

The method was successfully applied for the determination of the drugs in laboratory

prepared mixture with their acid, alkaline, oxidative, neutral and photolytic degradation products

from 10% up to 90% of degradates. The method was successfully applied for the determination

of Ceftriaxone sodium and Sulbactam sodium in Formic vial and the validity of the method was

assessed by applying the standard addition technique and good recoveries have been obtained.

The mean percentage recoveries ± S.D. of the labeled Ceftriaxone sodium and Sulbactam sodium

in Formic vial were 99.86 ± 1.22 and 99.95 ± 0.48, respectively, while that of the added

Ceftriaxone sodium was 99.83 ± 1.11 and the added Sulbactam sodium was 100.11 ± 1.13.







The method was also applied for the simultaneous determination of Ceftriaxone sodium

with either Sulbactam or Tazobactam sodium in spiked human plasma in presence of the active

metabolite of Ceftriaxone sodium which is Ceftizoxime - using cefotaxime as an internal

standard - over concentration ranges 1.00 – 120.00 µg/mL for Ceftriaxone sodium and

concentrations of 1.00 – 100.00 µg/mL for Sulbactam sodium, Tazobactam sodium and

Ceftizoxime. The QC plasma samples of the analytes in (mixture 1) show accuracy within 90.39

– 111.60 % and 90.53 – 111.60 % for intra-day and inter-day studies, respectively. The accuracy

for QC plasma samples of the analytes in (mixture 2) was 93.00 – 111.40 % and 91.00 – 111.20

% for intra-day and inter-day studies, respectively.

Part 1.2. TLC Methods

1.2.1. Stability Indicating TLC Method for the Simultaneous Determination of Ceftriaxone

Sodium in its binary mixture with either Sulbactam Sodium or Tazobactam Sodium in

Presence of their degradation Products.

The separation of the drugs from their acid, alkaline, oxidative, neutral and photolytic

degradation products was performed using HPTLC plates of silica gel GF 20×20 cm, 0.5 mm

thickness, fluorescent at 254 nm at ambient temperature with a developing system consisting of

dichloromethane : methanol : isopropanol : n-butanol : ammonia 33 %: water (22.5 : 22.5 : 20 : 5

: 5 : 2.5, by volume). Quantification was achieved with UV detection at 270 nm at concentration

ranges 0.60 to 12.00 µg/spot for Ceftriaxone sodium, from 1.80 to 6.00 µg/spot for Sulbactam

sodium in (mixture 1) and from 0.60 to 42.00 µg/spot for Ceftriaxone sodium, from 1.80 to 18.00

µg/spot for Tazobactam sodium in (mixture 2).





recoveries ± S.D. of 99.16 ± 0.67 and 100.07 ± 0.73, respectively. The method was successfully

applied for the determination of the drugs in laboratory prepared mixture with their acid,

alkaline, oxidative, neutral and photolytic degradation products from 10% up to 60% of

degradates.






99.97 ± 1.22 and the added Sulbactam sodium was 100.46 ± 1.17. The method was also applied

for the determination of Ceftriaxone sodium and Tazobactam sodium in Xone Xp vial and the

validity of the method was ascertained by applying the standard addition technique and good

recoveries have been obtained. The mean percentage recoveries ± S.D. of the labeled Ceftriaxone

sodium and Tazobactam sodium in Xone Xp vial were 99.96 ± 0.03 and 100.33 ± 0.76,

respectively, while that of the added Ceftriaxone sodium was 100.44 ± 0.53 and the added

Tazobactam sodium was 99.68 ± 0.97.

Part 2: Spectrophotometric Methods

Part 2.1. Derivative Spectrophotometric Methods

2.1.1. Stability Indicating Derivative Spectrophotometric Method for the Simultaneous

Determination of Ceftriaxone Sodium in its binary mixture with either Sulbactam Sodium

or Tazobactam Sodium in Presence of their degradation Products.

The method was proposed for the simultaneous determination of Ceftriaxone sodium in

its binary mixture with Sulbactam sodium (mixture 1) in presence of their oxidative degradation

products or Tazobactam sodium (mixture 2) in presence of Ceftriaxone sodium acid degradation

products. The method was based on measuring the second derivative for Ceftriaxone sodium at

320.2 nm in (mixture 1) and at 320 nm in (mixture 2), the fourth derivative for Sulbactam

sodium at 229.2 nm in (mixture 1) and the third derivative for Tazobactam sodium at 238.8 nm

in (mixture 2). The linearity was observed over the concentration ranges of 2.00 – 20.00 µg/mL

for Ceftriaxone sodium and 4.00 – 20.00 µg/mL for Sulbactam sodium in (mixture 1) and over

the ranges of 8.00 – 30.00 µg/mL for Ceftriaxone sodium and 1.00 – 20.00 µg/mL for

Tazobactam sodium in (mixture 2).





recoveries ± S.D. of 99.66 ± 1.93 and 100.60 ± 1.25, respectively. The method was successfully

applied for the determination of Ceftriaxone sodium and Sulbactam sodium (mixture 1) in

laboratory prepared mixture with their oxidative degradation products from 10% up to 60% of

degradates. It was also applied for the determination of Ceftriaxone sodium and Tazobactam

sodium (mixture 2) in laboratory prepared mixture with Ceftriaxone sodium acid degradation

products from 10% up to 60% of degradates.













Part 2.2. Derivative Ratio Spectrophotometric Methods

2.2.1. Stability Indicating Derivative Ratio Spectrophotometric Method for the

Simultaneous Determination of Ceftriaxone Sodium in its binary mixture with either

Sulbactam Sodium or Tazobactam Sodium in Presence of Ceftriaxone Sodium alkaline

degradation Products.

The method was based on measuring the second derivative of the ratio spectrum for

Ceftriaxone sodium and Sulbactam sodium in (mixture 1) and measuring the first derivative of

the ratio spectrum for Ceftriaxone sodium and the fourth derivative of the ratio spectrum for

Tazobactam sodium in (mixture 2). In (mixture 1), Ceftriaxone sodium can be quantitatively

determined at two wavelengths 304.2 nm and at 324 nm over the concentration range of 2.00 –

20.00 µg/mL without any interference from Sulbactam sodium or Ceftriaxone sodium alkaline

degradation products. Moreover, sulbactam sodium can be quantitatively determined at 240.4 nm

over the range of 4.00 – 20.00 µg/mL without interference from Ceftriaxone sodium or

Ceftriaxone sodium alkaline degradation products.

In (mixture 2), Ceftriaxone sodium can be quantitatively determined at 315.2 nm over the

range of 8.00 – 30.00 µg/mL without any interference from Tazobactam sodium or Ceftriaxone

sodium alkaline degradation products and Tazobactam sodium can be quantitatively determined

at 243.6 over the range of 1.00 – 20.00 µg/mL nm without interference from Ceftriaxone sodium

or Ceftriaxone sodium alkaline degradation products. The method was successfully applied for

the determination of Ceftriaxone sodium and Sulbactam sodium (mixture 1) in bulk powder with

mean percentage recoveries ± S.D. of CT at 304.2 & 324 and SB 99.86 ± 1.36, 101.14 ± 1.03 and

99.25 ± 1.73, respectively. It was also successfully applied for the determination of Ceftriaxone

sodium and Tazobactam sodium (mixture 2) in bulk powder with mean percentage recoveries ±

S.D. of 98.89 ± 1.67 and 98.74 ± 1.05, respectively.


Sulbactam sodium (mixture 1) or Tazobactam sodium (mixture 2) in laboratory prepared mixture

with Ceftriaxone sodium alkaline degradation products from 10% up to 60% of degradates.




recoveries ± S.D. of the labeled Ceftriaxone sodium at 304.2 nm, at 324 nm and Sulbactam

sodium in Formic vial were 100.14 ± 1.76, 100.98 ± 1.17 and 100.48 ± 1.68, respectively, while

that of the added Ceftriaxone sodium at 304.2 nm was 99.64 ± 1.09, at 324 nm was 99.78 ± 1.39

and the added Sulbactam sodium was 98.83 ± 0.74.







Part 2.3. Ratio Difference Spectrophotometric Methods

2.3.1. Ratio Difference Spectrophotometric Method for the Simultaneous Determination of


Sodium.

The method was based on the generation of ratio spectra of one compound in each

mixture using the other as the divisor followed by measurement of the peak-to-peak

amplitudes between two selected wavelengths in the generated ratio spectra for the two

mixtures. In (mixture 1), the spectra of Ceftriaxone sodium was divided by the spectrum of the

Sulbactam sodium divisor and the peak-to-peak amplitudes in Ceftriaxone sodium ratio spectra

between 265.8 and 315 nm were measured. The spectra of Sulbactam sodium was divided by the

spectrum of the Ceftriaxone sodium divisor and the peak-to-peak amplitudes in Sulbactam

sodium ratio spectra between 215 and 240 nm were measured.

In (mixture 2), the spectra of Ceftriaxone sodium was divided by the spectrum of the

Tazobactam sodium divisor and the peak-to-peak amplitudes in Ceftriaxone sodium ratio spectra

between 230 and 250.8 nm were measured. The spectra of Tazobactam sodium was divided by

the spectrum of the Ceftriaxone sodium divisor and the peak-to-peak amplitudes in Tazobactam

sodium ratio spectra between 211.4 and 250 nm were measured. The linearity was over the

concentration range of 2.00 – 20.00 µg/mL for Ceftriaxone sodium and 4.00 – 20.00 µg/mL for

Sulbactam sodium in (mixture 1) and over the range of 8.00 – 30.00 µg/mL for Ceftriaxone

sodium and 1.00 – 20.00 µg/mL for Tazobactam sodium in (mixture 2).


Sulbactam sodium (mixture 1) in bulk powder with mean percentage recoveries ± S.D. of CT

and SB 100.93 ± 0.49 and 99.83 ± 1.21, respectively. It was also successfully applied for the

determination of Ceftriaxone sodium and Tazobactam sodium (mixture 2) in bulk powder with

mean percentage recoveries ± S.D. of 99.85 ± 1.59 and 99.29 ± 0.54, respectively.


Sulbactam sodium (mixture 1) or Tazobactam sodium (mixture 2) in their laboratory prepared

mixtures. The method was successfully applied for the determination of Ceftriaxone sodium and












Section 3 : Identification of Degradation Products.

In this part, the acid, alkaline, oxidative, neutral and photolytic degradation products of

Ceftriaxone sodium, Sulbactam sodium and Tazobactam sodium were prepared. The structures

of these prepared degradation products were identified and confirmed by spectral data such as

HPLC, IR and Mass spectrometry.

Section 4 : English Summary.

This section contains English summary.

Section 5 : References.

This section contains 138 references.

Section 6 : Arabic Summary.

This section contains Arabic summary.

The thesis contains 94 tables and 101 figures.

Conclusion

In this thesis ceftriaxone sodium can be simultaneously determined in its binary mixture

with either sulbactam sodium or tazobactam sodium by different analytical methods in pure form

and in commercial dosage forms without interference from excepients. The drugs are subjected

to different stress conditions guided by ICH (to achieve complete degradation). Most of the

suggested methods can be used as stability indicating method for the determination of the

analytes in presence of their degradation products. The green chemistry is incorporated to

determine ceftriaxone sodium simultaneously with sulbactam sodium or tazobactam sodium in

spiked human plasma with its active metabolite (ceftizoxime).

Moreover, these methods have a potential for application in quality control laboratories,

as they are simple, rapid, don't need complicated instruments and show good accuracy and

precision.

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