academic,doc.

1. 1
Ministry of higher education
& scientific researches
Al-Yarmouk University College

2. 2
By
Ala'a Fadhel Hassan
Ahmed Sa'adi
Ayat Abdulqadir
Supervised by
Dr. ZUHAIR HASHIM AL-RAWI (PhD)

3. 3
‫الـرحـــيـم‬ ‫الـرحمـــن‬ ‫اللــه‬ ‫بـــــسـم‬
‫(ع‬‫ـ‬‫ل‬‫ـ‬‫ـ‬‫ـل‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ع‬‫ي‬ ‫ـم‬‫ل‬‫ـا‬‫ـ‬‫م‬ ‫ـان‬‫ـ‬‫ـ‬‫ـ‬‫س‬‫ـ‬‫ـ‬‫ن‬‫اال‬ ‫م‬)‫ـم‬
‫ـلق‬‫ـ‬‫ع‬‫ـ‬‫ل‬‫ا‬5
‫الـعـظـــيـم‬ ‫اللـــه‬ ‫صـــدق‬

4. 4
Dedication
To:
The department of pharmacy in AL-Yarmouk
University College
Our doctors
Our families

5. 5
Acknowledgment
After praiseworthy to the Almighty ALLAH for enabling
us to complete & present this work, we would like to put
on our sincere gratitude for our honorable supervisor
Dr. ZUHAIR HASHIM AL-RAWI (PhD) for his helpful
guidance & useful advice throughout the course of study
& for his fruitful direct supervision of this work
Also we are like to thank Dr.Shaima'a Alshamari, Dr.
Haidar Al-attar & Dr.Eva Dhia'a for their kind help in
designing, rearrangement & providing of additional
sources for us
Also we are deeply grateful for all those who support &
encourage us duringthe stages of this work

6. 6
List of contents
Title Page number
Introduction 8
Types ofluminescence 8
Definition 9
Principle of fluorometry 10
Structural factors affecting
fluorescence
12
Advantages offluorometry 15
Fluorescence & environment 16
Commonproblems of
fluorescence measurement
17
Application offluorometry 18
Modern tests by fluorometry 19
Cephalosporins as example on
fluorimetric assay
20
Cephalosporins major groups
"generations"
21
Cephalosporins fluorimetric
assay (e.g.)
23
Other methods usedfor
cephalosporins analysis
31
Official methods reportedfor
analysis of cephalosporins
38
Beta-lactam antibiotics
fluorimetric assays (e.g.)
38
Conclusion 39
References 40

7. 7
List of figures
Figure no. Title Page
number
Figure1 Level of energy excitationof
molecules
9
Figure2 Principle of fluorometry 11
Figure3 Curve of fluorescence Vs
concentration
11
Figure4 The typical aromatic molecule
that do not fluoresce & that
fluorescence
14
Figure5 Structure ofcephalosporin&
penicillin
20
Figure6 Classificationofcephalosporins 22
Figure7 Spectrofluorimetric excitation&
emissionspectrafor cephradine,
cephalexin & cephaloglycin
22
Figure8 Rate of formationof a fluorescent
derivative in the absence &
presence of formaldehyde of
cephradine , cephalexin &
cephaloglycin
24
Figure9 Structure ofcefoxitin sodium 25
Figure10 Excitation& emissionspectraof
cefoxitin(sodium) in aqueous
solution
26
Figure11 Fluorimetric assay ofcefoxitin 27
Figure12 Structure ofcefuroxime 28
Figure13 Fluorimetric assay of
cephalosporins
30
Figure14 Other methods for cephalosporin
assay
37

8. 8
Introduction
Luminescence is generally defined as the emissionofphotons
from electronically excitedstate. Luminescence is divided,
depending upon the nature of the groundand the excitedstates.
Types of luminescence
Classificationaccording to the means by which energy is
supplied to excite the luminescent molecule:
Photoluminescence: Molecules are excitedby interaction
with photons of radiation.
*Fluorescence: Prompt fluorescence: S1 S0 +h
The release of electromagnetic energy is immediate or from the
singlet state.
Delayed fluorescence: S1 T1 S1 S0 +h
This results from two intersystem crossings,first from the
singlet to the triplet, then from the triplet to the singlet.
*Phospholuminescence: T1 S0 +h
It's a delayed release ofelectromagnetic energy from the triplet
state.
Chemiluminescence: The excitationenergy is obtained
from the chemical energy of reaction.
Bioluminescence: Chemiluminescence from abiological
system:firefly,sea pansy, jellyfish,bacteria, protozoa&
crustacea.
Triboluminescence: Arelease of energy when certain
crystals suchas sugar,are broken.
Cathodoluminescence: Arelease of energy producedby
exposure to cathode rays

9. 9
Thermoluminescence: Whena material existing in high
vibrational energy levels emits energy at a temperature
belowred heat, after being exposedto small amounts of
thermal energy
Figure 1: level of energy excitationofmolecules
Definition
Photoluminescence is a type ofoptical spectroscopy inwhicha
molecule is promotedto an electronically excitedstate by
absorptionof ultraviolet,visible,or near infrared radiation.The
excitedmolecule then decays back to the ground state,or to a
lower-lying excitedelectronic state,by emissionoflight.
The emittedlight is detected.
Photoluminescence processes are subdividedinto
fluorescence,Chemiluminescence andphosphorescence
For simplicity,we use the term fluorescence to mean both
fluorescence and phosphorescence

10. 10
The key characteristic offluorescence spectrometry is its high
sensitivity.Fluorometry may achieve limits of detecting several
orders ofmagnitude lower than those ofmost other techniques.
This is known as the fluorescence advantage, Useful for the
detection(a single molecule) may be reached. Because ofthe
lowdetection limits,fluorescence is widely used for
quantification oftrace constituents ofbiological &
environmental samples;fluorometry is also usedas a detection
methodin separationtechniques, especially liquid
chromatography and electrophoresis.The use offluorescent
tags to detect nonfluorescent molecules is widespreadand has
numerous applications (suchas DNA sequencing because
photons can travel throughtransparent media over large
distances,fluorescence is applicable to remote analyses.
The spectral range for most molecular fluorescence
measurements is 200 to 1000 nm (10,000 -50,000 cm–1).Hence,
optical materials usedin UV/Vis absorptionspectrometry are
suitable for molecular fluorescence.
Principle of fluorometry
The initial step in a fluorescence measurement is electronic
excitationof an analyte molecule viaabsorption of a photon.
Once formed, an excitedmolecule has available a variety of
decay processes by which it can riditselfof the energy imparted
to it by absorption.In addition to fluorescence (the desired
decay route) to release the energy as a light rather than heat like
in spectrophotometer insome cases,Other sample constituents
may interact with an excitedanalyte molecule in such a way as
to prevent it from fluorescing;such processes are called
quenching while in other cases an electronically excited
molecule may undergo chemical reaction
(photodecomposition).

11. 11
Figure 2: principle offluorometry
Figure 3: curve offluorescence Vs concentration

12. 12
Light sources
a. Gas discharge lamps :( Xenon arc lamp, Highpressure
mercury vapor lamp)
b. Incandescent lamps:tungsten wire filament lamp
c. Laser: tunable dye laser
d. X-ray source for X-ray fluorescence
Wavelength selectiondevices
a. Filters.
b. Monochromators (polarized)
Sample compartment
Detectors
Fluorescence efficiency; quantum yieldof fluorescence:
It is the ratio of the fluorescence radiant power to the
absorbedradiant power where the radiant powers are
expressedin photons per second.
 =(luminescene radiant power) / (absorbed radiant power)
= (number of photons emitted) / (number of photons
absorbed) 1    0
Note:the higher the value of ,the greater the fluorescence ofa
compound.
Note:A non-fluorescent molecule is one whose quantum
efficiency is zero or so close to zero that thee fluorescence is not
measurable.All energy absorbedby such a molecule is rapidly
lost by collisional deactivation.

13. 13
Fluorescence relatedto concentration:
The fluorescence radiant power F is proportional to the
absorbedradiant power.
F = (Po – P)
Where  =fluorescence efficiency, Po = incident power, P =
transmittedpower
The relationshipbetween the absorbedradiant power and
concentrationcan be obtained from Beer’s law.
P/ Po = 10–A = 10–bC
Structural factors affecting fluorescence
Fluorescence is expected in molecules that are aromatic or
multiple conjugateddouble bonds with a high degree of
resonance stability.
Fluorescence is also expected in polycyclic aromatic systems
Substituents suchas –NH3,–OH,–F, – OCH3, – NHCH3,and – N
(CH3)2 groups,often enhance fluorescence.
On the other hand, these groups decrease or quench
fluorescence completely:
–Cl,–Br, –I,–NHCOCH3,– NO2, – COOH.
Molecular rigidity enhances fluorescence.Substances
fluoresce more brightly in a glassy state or viscous solution.
Formationof chelates with metal ions also promotes
fluorescence.However,the introductionofparamagnetic
metal ions gives rise to phosphorescence but not fluorescence
in metal complexes.
Changes in the system pH,if it affects the charge status of
chromophore,may influence fluorescence.

14. 14
Figure 4:
A/Typical aromatic molecules that do not fluoresce
B/The Typical aromatic molecules that fluorescence

15. 15
Table 1:substitutioneffect ofn the fluorescence
Advantage of fluorometry
1. Sensitivity:Limits ofdetection depend to a large extent on the
properties ofthe sample being measured. Detectability to parts
per billionor even parts per trillionis commonfor most
analytes. This extraordinary sensitivity allows the reliable
detectionof fluorescent materials (chlorophyll,aromatic
hydrocarbons,etc.) using small sample sizes.Also, field studies
can be performedin open waters without sample treatment.
Fluorometers achieve 1,000 to 500,000 times better limits of
detectionas compared to spectrophotometers.

16. 16
2. Specificity:Spectrophotometers merely measure absorbed
light.5 Spectrophotometrictechniques are prone to interference
problems because many materials absorblight,making it
difficult to isolate the targetedanalyte in a complex matrix.
Fluorometers are highly specific and less susceptible to
interferences because fewer materials absorbandalso emit
light (fluoresce).And, if non-target compounds do absorband
emit light, it is rare that they will emit the same wavelength of
light as target compounds.
3. Wide ConcentrationRange:Fluorescence output is linear to
sample concentrationover a very broadrange.
4. Simplicity andSpeed.
Fluorescence and environment
1. Temperature: A rise in temperature almost always is
accompaniedby a decrease in fluorescence because the greater
frequency of collisions betweenmolecules increases the
probability for deactivationby internal conversionand
vibrational relaxation.
2. PH: Changes in pH influence the degree of ionization,which,
in turn, may affect the extent ofconjugationor the aromaticity of
the compound.
3. Dissolvedoxygen: Dissolvedoxygenoften decreases
fluorescence dramatically andis interference in many
fluorimetric methods.Molecular oxygenis paramagnetic (has
triplet ground state),which promotes intersystem crossing from
singlet to triplet states in other molecules.The longer life times
of the triplet states increase the opportunity for radiationless
deactivationto occur.Other paramagnetic substances,including
most transitionmetals,exhibit this same effect.

17. 17
4. Solvents: Solvents affect fluorescence throughtheir ability to
stabilize ground and excitedstates differently, thereby changing
the probability and the energy of bothabsorptionand emission.
Common problems of fluorescence measurements
1. Reference materials is as fluorescent as the sample
Contaminating substances,Ramanscattering,Rayleigh
scattering
2. Fluorescence reading is not stable Fogging of the cuvette
when the contents are much colder than the ambient
temperature.
 Drops of liquid on the external faces of the cuvette.
 Light passing through the meniscus ofthe sample.
 Bubbles' forming in the solutionas it warms.
3. Self-quenching: it results when luminescing molecule collide
and lose their excitation energy by radiationless due to presence
of impurities.
4. Absorptionofradiant energy: Absorptioneither of the
exciting or ofthe luminescent radiation reduces the luminescent
signal. Remedies involve (a) dilutionthe sample, (b) viewing the
luminescence near the front surface ofthe cell, and (c) using the
methodof standard additions for evaluating samples.
5. Self-absorption:Attenuation of the exciting radiation as it
passes throughthe cell can be causedby too concentratedan
analyte. The remedy is to dilute the sample and note whether
the luminescence increases or decreases.Ifthe luminescence
increases upon sample dilution, one is working on the high-
concentrationside of the luminescence maximum.This region
should be avoided

18. 18
6. Excimer formation: Formationofa complex betweenthe
excited-state molecule and another molecule in the ground
state, calledan excimer,causes a problem when it dissociates
with the emissionofluminescent radiation at longer
wavelengths than the normal luminescence. Dilutionhelps
lessonthis effect.
Application of fluorometry
Tests of fluorometry generally usedfor:
Measure many type of drug in serum.
Measure catecholamine and its derivative.
Assay many of steroid compounds.
Measure many type of alborverinat
In microbiology detectionofbacteriaand its sensitivity to
antibiotic.
Many of drugs can be assay by fluorometry include:
Determination of pregabalin drug in capsules also to
determine pregabalin in urine
Simultaneous analysis of binary mixture of
chlorzoxazone (CLZ) and ibuprofen (IP) because both
exhibit native fluorescence (method based on
measurement of the synchronous fluorescence intensity
of these drugs in methanol)
spectrofluorimetric determination of rosiglitazone
maleate (ROZ)[ in pure form through complex formation
with Al+3 in acetate buffer of pH 5] in spiked and real
human plasma
Kinetic spectrofluorimetric method for the
individual determination of verapamil
hydrochloride, diltiazem hydrochloride, nicardipine
hydrochloride and flunarizine using water as diluting
solvent, (Method based on oxidation of the drugs
with cerium ammonium sulphate in acidic medium)

19. 19
Fluorimetric method to quantify camptothecin (CPT) in
irinotecan (CPT-11) and in topotecan (TPT) based anti-
cancer drugs.
Spectrofluorimetric methods for the determination of
hydrochlorothiazide, indapamide and xipamide based on
complex formation with eosin and in the presence of
methylcellulose as surfactant.
Also fluorometry can use in other diagnostic test:
Fluorescence spectroscopy is used in biochemical
analysis,( proteinfluorescence may be usedas a diagnostic
of the conformational state ofa protein)(tryptophan
fluorescence can be a very sensitive measurement ofthe
conformational state ofindividual tryptophan residues,
used to estimate the nature of microenvironment ofthe
tryptophan)&( several procedure for enzymatic assay of
ALP and others)
Medical (differentiating malignant, bashful skin tumors
from benign, urosurgery & ophthalmology)
Chemical researchfields (analyzing organic compounds)
[Atomic Fluorescence Spectroscopy (AFS) techniques are
useful in other kinds of analysis/measurement ofa
compoundpresent in air or water, or other media, such as
CVAFS which is used for heavy metals]
Modern tests by fluorometry
 Flow Cytometer:refers to the measurement of physical
properties and /or chemical properties ofcells to expand
the properties ofvital molecule.
 By flow cytometer we can measure several parameter
include size and granulationof cell
 Can measure the content ofDNA and RNA, proportionof
nucleotide (A-T), (G-C) & Structure ofchromatin.
 Total protein & Cellular receptor.
 Polarizationofcellular membrane.
 Antigen as well as Concentrationofcalcium ion

20. 20
 In immunology we can measure T-cell and its
proliferation,degree of alert of T-cell, antigen-antibody
reaction.
 Tumor (warning, diagnosis,and control).
 Also can detect virus and parasits.
 Genetic science.
 Fertility and reproduction.
 Hematology ( RBC , WBC and platelets)
 Urology (hyaline cast , nonsequamous epithelial cells , RBC
, WBC , bacteria
Cephalosporins as example on fluorimetric assay
The cephalosporins structurally relatedto the penicillins,consist
of a beta-lactam ring attachedto a dihydrothiazoline ring with D-α-
aminoadipic acid. Substitutions ofchemical groups result in
varying pharmacologic properties andantimicrobial activities.
Figure 5: chemical structure ofcephalosporin& penicillin

21. 21
THE MECHANISM OF ACTION OF CEPHALOSPORINS IS
ANALOGOUS TO THAT OF THE PENICILLINS:
binding to specific penicillin-binding proteins,
inhibition of cell wall synthesis
activationof autolytic enzymes in the cell wall
RESISTANCE to cephalosporins may be due to poor
permeability ofthe drug into bacteria,lack of penicillin-binding
proteins,or degradationby β-lactamase.
Cephalosporins major groups "generations"
According to their antibacterial activity
First-generationcephalosporins have goodactivity against
aerobic gram-positive organisms andsome community
acquired gram-negative organisms (P mirabilis,Escherichia
coli,and Klebsiellaspecies).Invitro activity includes
coverage ofgram-positive cocci,including viridians
streptococci;groupAhemolytic streptococci,andS aureus.
Anaerobic gram-positive cocci are usually susceptible.
Second-generationdrugs are a heterogeneous groupwith
markedindividual differences in activity, pharmacokinetics,
and toxicity,they are active against gram-negative organisms
inhibited by first-generationdrugs;but they have an
extended gram-negative coverage.Indole-positive Proteus
and Klebsiella(including 1st generationcephalosporin-
resistant strains) as well as M catarrhalis and Neisseria
species are susceptible.
Third-generationcephalosporins are active against many
gram-negative bacteria,most third-and fourth-generation
cephalosporins inhibit most streptococci (ceftazidime is an
exceptionto this rule). Ceftriaxone and cefotaxime offer the
most reliable anti-pneumococcal coverage's,they are
consistently active against Serratiamarcescens,Providencia,
Haemophilus,and Neisseriaincluding β-lactamase–producing
strains.Ceftazidime is unique among all agents because it's

22. 22
active against P aeruginosa, Acinetobacter, Citrobacter,
Enterobacter & nonaeruginosa.
Cefepime considereda fourth -generationagent since it is
more stable against plasmid-mediatedβ-lactamase & has
little or no β-lactamase-inducing capacity. It has improved
coverage against Enterobacter and Citrobacter species.Its
gram-positive coverage approaches that of cefotaxime or
ceftriaxone.Ceftobiprole is another 4th antibiotic with
activity against methicillin-resistant Staphylococcus aureus,
penicillin-resistant Streptococcus pneumoniae,Pseudomonas
aeruginosa& Enterococci.
Fifth-generation ceftaroline is uniquely active against Gram-
positive organisms including methicillin-resistant
Staphylococcus aureus, Streptococcus pneumoniae &
Streptococcus pyogenes; it has comparable gram-negative
spectrum activity as third-generation agents. Ceftobiprole is
the 1st broad spectrum anti-MRSA and has anti-
Pseudomonas activity in spectrum of its coverage labeled as
another 5th generation cephalosporin
Figure6: Classificationof cephalosporins
1st generation
•cephalexin
•cephradine
•cefadroxil
•cefazoline
•cephaloridine
•cephapirin
•cefaloglycin
•cefalonium
•cefalothin
•cefatrizine
•cefazaflur
2nd generation
•cefuroxime
•cefoxitin
•cefotetan
•cefaclor
•cefprozil
•cefonicid
•cefuzonam
•cefmetazole
3rd generation
•cefdinir
•cefixime
•cefpodoxime
•ceftibuten
•ceftriaxone
•cefotaxime
•ceftazidime
•cefcapene
•cefdaloxime
•cefdiloren
•cefetamet
•cefmenoime
•cefodizime
4th generation
•cefepime
•cefpirome
•cefozopran
•cefluprenam
•cefquinome
•cefoselis
5th generation
•ceftaroline
•ceftbioprole

23. 23
Cephalosporin's fluorimetric assays (e.g.)
Cephalexin,whensubjectedto alkalinehydrolysis
(degradation) produces a strongly fluorescent yellow
product (Yamana, Tsuji, Kanayama & Nakano 1974),
similar to that obtainedfrom ampicillinby acidhydrolysis.
The fluorimetric assay forcephalexinhas been developed
by increasing the severity of hydrolysis by treatingwith
sodium hydroxide followed by heat treatment at 100°C in a
boiling water bath (Barbhaiya & Turner
1976)&intensity was measured by using BairdAtomic
Fluoripoint spectrofluorimeter equipped withanXenon
lamp.
Note that Sorensen's sodium citrate buffer (pH 5.0)
containing 2.5% (v/v) formaldehyde was used for
fluorimetric estimationof cephalexin and cephaloglycin
but cephradine was estimated in the same buffer at pH
4.0.
Figure7: Spectrofluorimetric excitation (345nm) & emission
(425nm) spectra for cephradine (__ __ __), cephalexin (. . . . . . .) &
cephaloglycin (________)

24. 24
Procedure:
Inaqueous solution,the tubes being loosely covered with
polythene balls to minimize evaporation.After cooling the tubes
for 10 min the fluorescence intensity wasmeasuredat 425nm while
inplasmaorserum10% (w/v) trichloroacetic acid (TCA) was added for
deproteinizationandvortexedtoproduce thoroughmixing tothis
system 0.2 MSorensen's sodium citrate buffer containing2.5%(v/v-)
formaldehydeat pH5.0
Results:
The fluorescentderivativefromcephaloglycinbeingmost
fluorescent followed by cephalexin and cephradine,the excitation
andemissionwavelengths, the rateofformationofthefluorescent
derivativefrom each of these antibiotics increased with
temperature anddurationof heating.Maximum fluorescence was
achievedby heating the solution under the described
experimental conditions for 30minat 100°C inaboilingwaterbath.
Incorporationof2.5% (v/v)formaldehyde exertedacatalyticeffect
onformationofthe fluorescent derivative (Figure 8).
Figure8: Rate of formulation of a fluorescent derivative in the
absence (•) and presence (0) of 2.5% (v/v) formaldehyde for (a)
cephradine, (b) cephalexin and (c) cephaloglycin

25. 25
Discussion
Partial alkaline degradationfollowedby hydrolysisat 100°Cinthe
presence of formaldehyde produces maximum fluorescence within
30min.The structuralsimilaritiesbetweenside-chains (α-amino
group) ofthese antibiotics and the identical excitation and emission
spectrasuggestthat all three cephalosporins couldbe forming similar
fluorescent derivatives on hydrolysis.
Addition of 2.5% formaldehyde in the buffer catalyzedthe
formationofafluorescentderivativeby reducing the basicityofthe α-
amino grouponthe side-chainofthese antibiotics.It might well be
possible to automate this fluorimetric methodfor these
cephalosporins so that more rapid estimation of large numbers of
samples canbe accurately performed ifthe fluorescent productswere
the same
Cefoxitin(cephamycingroup)assay(inaqueous fluidand
humanurine ,Z.H.AL-Rawi & S.Tabaqchali) requiredacid
hydrolysis to formstable product withsatisfactory
fluorescent properties andhighly alkaline conditionto
exhibit optimum fluorescence,Suchalkaline conditionalso
requiredincephalothinassay
Figure9:structure of
cefoxitin sodium

26. 26
Procedure:
Inaqueous solution, it was carried in the fluorimeter at excitation
385 nm and emission 460 nm, while in serum, 1.0 ml of 10% (w/v)
TCA was added in order to precipitate the proteins, This was
carried out using well technique method and DST agar (Oxoid)
pH 6-8
Figure10: Excitation& emissionspectraofCefoxitin(sodium)
160µg/ml inaqueous solution.
Results:
The optimum pH was found to be 12 which is achieved by the
addition of 6.0 N-NaOH (gave stable fluorescence intensity).
The graphs [Figure 11] show linear relationship between the
antibiotic concentrations and the fluorescence intensity in both
aqueous solution and serum

27. 27
Figure11: Fluorimetric assay ofCefoxitin(a) standardcurve in
aqueous solution,(b) standard curve in serum.Each dot represents
the mean of eight separate tests carriedin duplicate
Discussion:
The optimumexcitationand emissionwavelengthsof
Cefoxitinwere foundtobehigher thanthe corresponding
wavelengthsoftheothercephalosporins & the fact that
Cefoxitin hydrolysisrequiredmuchlessheatingtime(3
min) suggest thatthe formationandthe nature ofthe
fluorescence productsaredifferentdue tothepresenceof
the 7αmethoxy groupwhich may be involved in the
formation ofthe fluorescence products, particularly as
comparedwith cephalothin agent (launched by Eli Lilly
1964) which structure differs from thatofCefoxitinby the
presenceofthe methoxygroupinthe 7α-positionofthe
lactam ring aswell as the substitution ofmethyl groupby
amino groupinthe side chain ofthe Cefoxitin
molecule(Barbhaiya& Turner1977;Yuet al.1977).

28. 28
Cefuroxime assay(Zuhair HashimAL-Rawi & Soad
Tabaqchali 1981),(serum urine & aqueous fluid) in
whichfluorescent productobtainedby additionof
Hydrochloric acid,heating & coolingfollowedby
additionof sodium hydroxide and further heating
at 100°C. The fluorescence intensity of the final
solution was measuredinafluorimeter at an
excitationwavelengthof375nm andan emission
wavelength of 440 nm and related to the
antibiotic
Figure12:structure of
cefuroxime
The relative fluorescence
intensities for aqueous solutions, urine, and serum
were measured at the same sensitivity setting ofthe
fluorimeter,ranging from 0.01to1.0,there was a close
correlation between the results ofthe fluorimetricand
microbiological assays ofsamples from the
pharmacokinetic study
 Fluorimetric method also appeared for cefatrizine in
plasma,serum,andurine samples,methodinvolveacid
hydrolysisandfluorescentproduct formationusing
hydrogenperoxide, usingexcitationandemission
wavelengths of 340 and 420 nm. (Miyazaki K, Ogino O,
Arita T.Chem Pharm Bull (Tokyo).1979)

29. 29
Other assay reported for cephalosporin's
 Rapid sensitive fluorimetric analysis ofcephalosporin
(Yu AB, Nightingale CH,Flanagan DR.J Pharm Sci. 1977
Feb)
 Determinationof cephalosporin-C amidohydrolase
activity
(Reyes F, Martinez MJ, Soliveri J.J Pharm Pharmacol.
1989 Feb)
 Spectrophotometric determinationofcertain
cephalosporins inpure form and in pharmaceutical
formulations
 (Amin, Alaa S.; Ragab, Gamal H. Follow Spectrochimica
Acta Part A: Molecular and Bimolecular Spectroscopy ,
Volume 60 (12)
Elsevier – Oct 1, 2004 )
 Fluorimetric determinationofCephalexinin urine.
(Aikawa R, Nakano M, AritaT.Chem Pharm Bull (Tokyo).
1976 Oct; 24)
 Fluorimetric determinationof cephalexin, cephradine,
and cephatrizine in biological fluids
(Miyazaki K, Ogino O, Arita T.Chem Pharm Bull (Tokyo).
1979 Oct; 27)
 Fluorimetric determinationofcephradine in plasma.
(HealdAF, Ita CE, Schreiber EC.J Pharm Sci. 1976 May)
 [Determinationof cefaloglycine and cefroxadin
in biological mediawith thin layer chromatography
with fluorimetric detection]
(Blanchin MD., Rondot-Dudragne ML.J Chromatogr.
1988 Nov, 18). French

30. 30
Figuer13: Fluorimetric assays of cephalosporins
cephalosporins'
flourimetric
assay
cephalexin,
cephradine &
cephaloglycin
cefoxitin(cepha
mycin)
cefalothin
cephalosporin
c
cefuroxime
cefatrizine&
cefroxadin

31. 31
Other methods used for cephalosporins analysis;
(Simplecomparison)
I.CHROMATOGRAPHIC METHODS
Advantages ofchromatographicmethods:
 Chromatographic techniques are usually
sensitive enough for most antibiotics as they
achieve a limit of quantification (LOQ) of
0.3-0.5ug/ml.
 Sensitivity can be further enhanced by coupling
it with fluorimetric, electrochemical or mass-
spectrometric detectionmethods.
Methods include:
1st Liquid chromatographic method with UV-Visible
detection & Highperformanceliquidchromatography
(HPLC),most frequently applied technique for the
determination of cephalosporins in biological fluids
(blood, plasma, urine, cerebrospinal fluid, etc.), animal
tissues, and food. Methods also include high-
performance thin layer chromatography (HPTLC)

32. 32
Advantages:
 It produces symmetric peak shape, good
resolution and reasonable retention time
 Method is simple(no pretreatment of the
sample) and easy to perform analytical
parameters which include linearity, range,
accuracy, precision and robustness of the
method along with combination of more than
one antibiotic from formulation and
biological fluids
 By using different types of columns and varying
combinations of solvent systems, scope of HPLC
method can be expanded to a wide range of
samples
 can provide valuable tool which generating high
pure compound
 has ability to analyze both volatile and nonvolatile
compounds with ultra trace level may be employed
in clinical research
 Methods are sensitive, simple, fast, ease extraction
procedure and possess excellent linearity and
precision characteristics. These observations made it
possible to anticipate the use of this method as an
official procedure.
 HPTLC method by (V. Jagapathi Raju et al.), has
shows a method for analysis of cephalosporin in
tablets with nano gram level and high precision value,
Some HPLC methods can be used for the multi-

33. 33
component analysis with 7 - 10 cephalosporins at a
time
Usedfor:
 analysis of cefotaxime, cefixme, cefaclor, ceftazidime
and ceftriaxone in pharmaceutical formulations
and biological fluids
 the determination of ceftriaxone in injection
 Cefalexin analysis by HPTLC on silica gel F254 plates.
 analysis ofcefoxitin,its decarbomylmetabolite
Disadvantages:
 Due to the insolubility of these compounds in organic
solvents, normal phase LC was sparingly used
 Most methods employ reversed-phase or ion-pair
reversed-phase LC and chemically bonded packing
materials
2nd Thin-layer Chromatographic Method (TLC)
Used for:
 mixtures of cephradine and cephalothin
(Qureshi et al) ceftazidime, cefuroxime sodium and
cefotaxime sodium and their degradation products
were analyzed by quantitative densitometric TLC
 Some cephalosporins in phosphate buffer of pH 3.6
were spotted on TLC plates coated with silica gel
with a fluorescent indicator or silica gel
 Determination of cefadroxil and cefalexin in
pharmaceutical preparations using quantitative
TLC.
 N-bromo- succinimide assay hydroxylamine
determinations and TLC for (Cephradine,cephalexin
and cephaloglycin)
4th Gas chromatography (GC)

34. 34
Advantages: Fast method
Disadvantages:
 It requires elevated temperature& may cause
thermal degradation of drugs.
 it requires derivatization to improve volatility and
to improve chromatographic behavior
(SO THESE METHODS ARE NOT APPLICABLE FOR
ANTIBIOTICS)
5TH Many antibiotics contain ionizable group can be analyzed
by ion exchange chromatographic methods
6th Polarography
II.SPECTROSCOPIC METHODS
1st full spectrum quantitation (FSQ)
Used for rapid multi-component analysis of complex
biological and pharmaceutical mixtures, the present work
reports on the use of FSQ and HPLC to quantify cefotaxime,
ceftazidime and ceftriaxone in the presence of their alkali-
induced degradation products and in commercial injections
2nd Ultraviolet Spectrophotometric Method
Used for:
 Cefotaxime, ceftriaxone and ceftazidime were
determination in the presence of their alkali-induced
degradation products through spectrophotometric full
spectrum quantitation over the range of 265-230 nm
 Various UV spectrophotometric methods are reported
for the analysis of Ceftazidime alone in presence of
other drugs

35. 35
 UV spe c t rophot ome t ry and di ffe re nc e UV
spectrophotometry were applied to determine
cefalexin in tablets
 Determination of the dissociation constants of cefepime
and cefpirome
 simultaneous determination cefuroxime axetil and
probenecid were in solid dosage forms by UV
spectrophotometric method
 determination of Binary mixtures of cefalotin and
cefoxitin by first-derivative spectrophotometry also
Mixtures of ceftazidime, cefuroxime sodium,
cefotaxime sodium and their degradation products were
analyzed by first-derivative spectrophotometry at 268.6,
306, 228.6 nm
 Spectrophotometric method was reported for the
determination of cefalexin bulk drug and its acid-induced
degradation products & Asimilarmethodusedfor
cefatrizine inserumandurine usedUV detection at 254 nm
(18).
3rd NMR spectrometry also reported
4th UV derivative spectrophotometry was reported for the
determination of cefprozil in pharmaceutical dosage forms in the
presence of its alkali induced degradation products also for
determination of the triethylammonium salt of cefotaxime in the
presence of related compounds resulting from the synthesis
5th Iodometric techniques (Alicino)
6th Differential pulse adsorptive stripping voltammetry

36. 36
III.ASSAY IN BIOLOGICAL FLUIDS
1st Capillary Electrophoretic Methods
cephalosporins were determined using capillary zone
electrophoresis(CZE) after hydrodynamic injection on a fused-
silica capillary and detection was performed at 210 nm ,method
proposed by (Mrestani et al).Cefixme and five of its metabolites
were determined in human digestive tissues by high performance
capillary electrophoresis on afused-silica capillary tube with
detection at 280 nm, CZE also used for the determination of four
cephalosporins in clinical sample e.g. Cefotaxime and its
deacetyl metabolite
2nd Micellar electrokinetic capillary chromatography (MEKC):
Cefuroxime was determined in human serum by MEKC using a
fused-silica capillary,(Yeh et al)., proposed a MEKC method for
determination of ceftazidime in plasma and cerebrospinal fluid,
method also used for determination of cefotaxime and its deacetyl
metabolite using a fused-silica capillary with phosphate buffer
pH 8.0.Finally Cefpirome was estimated in human microdialysis
and plasma samples by MEKC.
Advantages:
Good linearity's were obtained. The proposed method was
successfully applied to the analysis of the studied drugs in their
available pharmaceutical formulations and in biological samples
(serum and urine)
Disadvantages: interference of some amino acids urea, ascorbic
acid with analysis
3rd BiologicalAssay
This assay was reportedforcephalosporinC by (JILLIANM.BOND,R.W.
BRIMBLECOMBET& RC CODNER)on1961(methodofassayingthe
antibioticcephalosporinC inlowconcentrationinculturefluidsby
using astrainofVibrio cholerae)

37. 37
Also biologicmethodreportedfor qualitycontrol guidelinesfor
BAL9141(Ro 63-9141),aninvestigational cephalosporin,when
ReferenceMIC& standardizeddiskdiffusionsusceptibility test method
are used(T.R. Anderegg, R. N. Jones, H. S. Sader,1 and the Quality
Control Working Group2004),as well as in evaluation of PPI-
0903M(T91825)optimization of disk diffusion tests(Ronald N.
Jones, Thomas R. Fritsche, Yigong Ge, KonéKaniga and Helio S.
Sader )
Figure 14: other methods used for cephalosporin assay
chromatography,
sensitive method
•HPLC & HPTLC
simple,easy.accurate &
expand to wide range
sample
(Official in USPXXX &
UP2002)
•TLC
•GC
rapid method of analysis
•polargrphy
(Official for cefamandole
analysis according to
USPXXX)
•ionexchange
chromatogrphy
uesd for agents wih
ionizable groups
spectroscopy
•FSQ
beneficial in
multicompound
complexanalysis
•UV
•NMR
•differntial pulse
adsorptive
voltametry
•iodometrictechnic
biological assay
•MIC & DISC
DIFFUSION
appliedforquality
control &evaluation
•electrophoresis(CZE
& MEKC)
methodswith good
linearity

38. 38
Official Methods reported for analysis of cephalosporins
The United States Pharmacopeia XXX prescribes a
polarographic method for the assay of cefamandole naftate and
HPLC methods for the assay of the other cited
cephalosporins while the European Pharmacopeia 2002
prescribes HPLC methods for their assay. The analysis of
cephalosporins in biological materials from human origin and in
food-producing animals' foods, waters and pharmaceuticals was
performed with liquid chromatographic, capillary Electrophoretic,
spectro-scopic and electrochemical methods.
Some methods found in literature survey are for single
cephalosporin while some methods are available with different
combination. Various methods found are on different instrumental
methods such as, HPLC, HPTLC, GC, CE, TLC, UV
spectrophotometric & electrochemical methods
Beta-lactam antibiotic fluorimetric assay (e.g.)
 simultaneous determinationof penicillin and penicilloic
acids
(Tsuji A, Miyamoto E,Yamana T.J Pharm Pharmacol.1978
Dec; 30)
 Fluorimetric determinationofAmoxicillin
(Miyazaki K, Ogino O, Sato H, Nakano M, AritaT.Chem
Pharm Bull (Tokyo).1977 Feb; 25)
 Relative oral bioavailability of microgranulated
amoxicillinin pigs
(Anfossi P, Zaghini A, Grassigli G, MenottaS, Fedrizzi G.J
Vet Pharmacol Ther.2002 Oct;25)
 Fluorimetric analysis ofampicillinin biological fluids.
(Jusko WJ.J Pharm Sci.1971 May
 Fluorimetric determinationofampicillin
(Miyazaki K, Ogino O, Arita T.Chem Pharm Bull (Tokyo).
1974 Aug; 22)
 Fluorimetric determinationofampicillinand
aminobenzylpenicilloic acid

39. 39
(Miyazaki K, Ogino O, Nakano M, Arita T.Chem Pharm Bull
(Tokyo).1975 Jan;23)
 A simple fluorimetric assay ofampicillinserum.
(Dürr A, SchatzmannHJ.Experientia. 1975 Apr 15)
 [Degradationof ampicillinby urine of patients with
complicatedurinary tract infection]
(AritaT, Miyazaki K, Koyanagi T, Tsuji I, Nishiumi S,
Aikawa R, Murase J.Jpn J Antibiot. 1979 Jun)
 [Problems inthe fluorimetric determinationofampicillin].
(Lampe D, Glende M.Pharmazie. 1983 Mar)
 Modified fluorimetric assay for estimating ampicilloate
concentrations
(Baker WL.Analyst. 1997 May; Erratum in: Analyst 1997
Aug)
 Determinationof ampicillin,amoxicillin,cephalexin, and
cephradine in plasmaby high-performance liquid
chromatography using fluorimetric detection
(Miyazaki K, Ohtani K, Sunada K, Arita T.J Chromatogr.
1983 Sep 9)
Other assays have been reported for
aminoglycosides, quinolones, tetracycline's,
sulfonamides, chloramphenicol, macrolids and
antimycobaterial antibiotics.
Conclusion
Fluorimetric techniques are prior to advent of GLC & HPLC.
Drug samples were analyzedby spectrophotometric methods.
Solvent extraction coupled with spectrophotometric finish can
still provide a much derived simplicity in assay procedure when
the level of sensitivity required is not too low i.e. in µg/ml, this
techniques which depend on physicochemical properties of
drug allow an easier, faster (decrease time consumption) &
more accurate analysis that has an important role in quality
control of the drugs as well as therapeutic drug monitoring &
follow up of antibiotics specially of those with risky adverse
effects.

40. 40
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VoL20,No.I,V.6-4.804/81/070025-05302.00/0
Fluorimetric Determination of.Cefuroxime in Body Fluids
ZUHAIR,HASHIMAL-RAWI ANDSOADTABAQCHALI"
DeparVnent ofMedical Microbiolomv, St.Bartholomew's Hospital,
West Smithfield, London ECM 7BE,

42. 42
172
e
Received26January 1931/Accepted22April 1981
Journal List > AntimicrobAgents Chemother > v.20 (1); Jul 1981
 ReprintedfromJournal ofAntimicrobialChemotherapy(1979)S,
81-86,Fluorimeiric assay ofcefoxitin
Z.H. Al-Ravi and SoadTabatichali
Departmentof_NeckedMicrobiology,SsBartholomew'sHospital,
WestSmIttuield,LondonEC1A7BE,England
 HPLC ANALYSIS OF CEPHALOSPORINS
AND STUDY OF DIFFERENT ANALYTICAL
PARAMETERS P. N. Patil*1 and S. Jacob
 Department of Chemistry, Bharati
Vidyapeeth’s College of Engineering,
Near Chitranagari, Kolhapur,
Maharashtra, India
Department of Pharmaceutics, College of Pharmacy, Gulf
Medical University, Ajman, UAE
 Recent applications of analytical techniques for
quantitative pharmaceutical analysis:a review
RUDY BONFILIO
Departamento de Fármacos e Medicamentos,Faculdade de
Ciências Farmacêuticas
Univ Estadual Paulista (UNESP)
RodoviaAraraquara'Jaú, km 1, CEP 14801'902. Araraquara'SP
BRAZIL.
rudybonfilio@yahoo.com.br www.fcfar.unesp.br
MAGALI BENJAMIM DE ARAÚJO
Faculdade de Ciências Farmacêuticas
Universidade Federal de Alfenas (UNIFAL'MG)
Rua Gabriel Monteiro da Silva,700, 37130'000. Alfenas'MG
BRAZIL.
 Journal of Antimicrobial
Chemotherapy (2005) 56, 1047–
1052 doi:10.1093/jac/dki362
Advance Access publication 20 October 2005

43. 43
Evaluation of PPI-0903M (T91825), a novel cephalosporin:
bactericidal activity, effects of modifying invitrotesting
parameters and optimization of disc diffusion tests
Ronald N. Jones1,2, Thomas R. Fritsche1, Yigong Ge3,
KonéKaniga3 and Helio S. Sader1,4*
JMI Laboratories, Inc., 345 Beaver Kreek Centre, Suite A,
North Liberty, IA 52317, USA;
Tufts University School of Medicine, Boston, MA, USA;
Peninsula Pharmaceuticals, Alameda, CA, USA;
Universidade Federal de São Paulo, São Paulo, Brazil
Received 30 March 2005; returned 16 June 2005; revised 8
September 2005; accepted 12 September 2005
 J Pharm Pharmacol.1976 Oct ;28 (10):791-2 10383 Cit:13
Fluorimetric determinationofcephalexin.
R H Barbhaiya, P Turner
 Chapter e1. Anti-Infective Chemotherapeutic & Antibiotic
Agents
B. JosephGuglielmo,PharmD
 Mcgrawhill's access medicineBr. J. clip. Pharmac. (1977), 4,
427-431
FLUORIMETRICASSAYOFCEPHRADINE,CEPHALEXIN AND
CEPHALOGLYCIN
R.H. BARBHAIYA & P. TURNER
Department ofClinical Pharmacology,St.Bartholomew'sHospital,
London, EC1A7BE
 J AntimicrobChemother 2011; 66 Suppl 3: iii11–iii18
doi:10.1093/jac/dkr095
Ceftaroline fosamil:anewbroad-spectrum cephalosporin
Joseph B. Laudano
Medical Affairs, Forest Research Institute, Harborside Financial
Center, Plaza V, Jersey City, NJ 07311, USA
 J. gen. Microbial.(1962), 27, 11-19,11 PrintedinGreat Britain

44. 44
BiologicalAssayofCephalosporinC
By JILLIAN M.BOND,* R.W.BRIMBLECOMBEt ANDR.C.CODNER:
MedicalResearchCouncilAntibioticsResearchStation,
4EltonRoad,Clevedon,Somerset(Received29March1961)
 ANTIMICROBIAL.AGENTS AND CHEMOTHERAPY, Aug.
1987. p_ 1157-110 Vol. M. No. 8 0066.48.0447)
(081157.071.02..04110
Copyright i91987, AmericanSociety forMicrobiology,MINEREVIEW
RecentAnalyticalMethodsforCephalosporinsinBiologicalFluids
ROGER a TOOTHAKER, D. SCOTT WRIGHT, AND
LAWRENCE A. PACHLA*
DepartmentofPhorona•oicineth's,DrogMeraboirirm.Warner-
LanthertiParke-DariNPirarniacenthyli Re:heard',
Ann Ar&kr. Urcitipan 4814.5
 Ceftobiprole From Wikipedia, the free encyclopedia
 Fluorescence spectroscopy From Wikipedia,the free
encyclopedia
 Ceftobiprole medocaril:the new generationof
cephalosporins
INFECTIOUSDISEASENEWSAPRIL2009
BY KIMBERLYD.BOESER,PHARMD
 PubMed ,US National Library of Medicine National
Institutes ofHealth
 DeepDyve Research
The largest online rental service for professional and scholarly
researcharticles
 USP30-NF25(editionof2007)

45. 45