Post on 11-Jan-2023
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].
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
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.
Generic name Trade name Structure
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
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.
Generic name Trade name Structure
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.
Generic name Trade name Structure
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.
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.
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 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
Ceftriaxone Sodium in its binary mixture with either Sulbactam Sodium or Tazobactam
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.
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 100.83
± 0.87 and 100.09 ± 1.33, 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 100.83 ± 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 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 99.94 ± 0.70 and 99.22 ± 0.63, respectively, while that of the added Ceftriaxone sodium
was 99.67 ± 1.42 and the added Tazobactam sodium was 100.28 ± 1.03.
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).
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 99.85
± 1.21 and 100.44 ± 1.04, 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 99.16 ± 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.
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
100.05 ± 0.81 and 100.83 ± 1.08, respectively, while that of the added Ceftriaxone sodium was
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).
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 100.08
± 1.51 and 100.02 ± 1.53, 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 99.66 ± 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.
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
101.43 ± 0.59 and 99.22 ± 1.35, respectively, while that of the added Ceftriaxone sodium was
98.59 ± 1.50 and the added Sulbactam sodium was 98.68 ± 1.46.
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 98.74 ± 0.71 and 99.96 ± 1.79, respectively, while that of the added Ceftriaxone sodium
was 100.16 ± 1.26 and the added Tazobactam sodium was 100.13 ± 1.82.
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.
The method was successfully applied for the determination of Ceftriaxone sodium and
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.
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 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.
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 98.19 ± 0.76 and 100.82 ± 0.17, respectively, while that of the added Ceftriaxone sodium
was 100.26 ± 0.53 and the added Tazobactam sodium was 100.47 ± 1.16.
Part 2.3. Ratio Difference Spectrophotometric Methods
2.3.1. Ratio Difference Spectrophotometric Method for the Simultaneous Determination of
Ceftriaxone Sodium in its binary mixture with either Sulbactam Sodium or Tazobactam
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).
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
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.
The method was successfully applied for the determination of Ceftriaxone sodium and
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
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
99.27 ± 0.80 and 99.93 ± 0.24, respectively, while that of the added Ceftriaxone sodium was
100.39 ± 1.77 and the added Sulbactam sodium was 100.12 ± 1.03.
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 98.58 ± 0.52 and 99.62 ± 1.22, respectively, while that of the added Ceftriaxone sodium
was 99.69 ± 1.43 and the added Tazobactam sodium was 101.35 ± 0.61.
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.