1

Ministry of higher education

& scientific researches

Al-Yarmouk University College

2

By

Ala'a Fadhel Hassan

Ahmed Sa'adi

Ayat Abdulqadir

Supervised by

Dr. ZUHAIR HASHIM AL-RAWI (PhD)

3

ـيـم ـ ــن الـرحـ ــسـم اللــه الـرحمـ ـ ـ بـ

لــلـ)ع ـم(م االنــســــان مــالـم يعـــــ

5الـعــلق

ـيـم ـ ــه الـعـظـ ــدق اللـ صـ

4

Dedication

To:

The department of pharmacy in AL-Yarmouk

University College

Our doctors

Our families

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 during the stages of this work

6

List of contents

Title Page number Introduction 8 Types of luminescence 8 Definition 9 Principle of fluorometry 10

Structural factors affecting fluorescence

12

Advantages of fluorometry 15 Fluorescence & environment 16 Common problems of fluorescence measurement

17

Application of fluorometry 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 used for cephalosporins analysis

31

Official methods reported for analysis of cephalosporins

38

Beta-lactam antibiotics fluorimetric assays (e.g.)

38

Conclusion 39

References 40

7

List of figures

Figure no. Title Page number

Figure1 Level of energy excitation of 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 of cephalosporin & penicillin

20

Figure6 Classification of cephalosporins 22 Figure7 Spectrofluorimetric excitation &

emission spectra for cephradine, cephalexin & cephaloglycin

22

Figure8 Rate of formation of a fluorescent derivative in the absence & presence of formaldehyde of cephradine , cephalexin & cephaloglycin

24

Figure9 Structure of cefoxitin sodium 25 Figure10 Excitation & emission spectra of

cefoxitin (sodium) in aqueous solution

26

Figure11 Fluorimetric assay of cefoxitin 27 Figure12 Structure of cefuroxime 28 Figure13 Fluorimetric assay of

cephalosporins 30

Figure14 Other methods for cephalosporin assay

37

8

Introduction

Luminescence is generally defined as the emission of photons

from electronically excited state. Luminescence is divided,

depending upon the nature of the ground and the excited states.

Types of luminescence

Classification according to the means by which energy is

supplied to excite the luminescent molecule:

Photoluminescence: Molecules are excited by 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 of electromagnetic energy from the triplet

state.

Chemiluminescence: The excitation energy is obtained

from the chemical energy of reaction.

Bioluminescence: Chemiluminescence from a biological

system: firefly, sea pansy, jellyfish, bacteria, protozoa&

crustacea.

Triboluminescence: A release of energy when certain

crystals such as sugar, are broken.

Cathodoluminescence: A release of energy produced by

exposure to cathode rays

9

Thermoluminescence: When a material existing in high

vibrational energy levels emits energy at a temperature

below red heat, after being exposed to small amounts of

thermal energy

Figure 1: level of energy excitation of molecules

Definition

Photoluminescence is a type of optical spectroscopy in which a molecule is promoted to an electronically excited state by absorption of ultraviolet, visible, or near infrared radiation. The excited molecule then decays back to the ground state, or to a lower-lying excited electronic state, by emission of light. The emitted light is detected. Photoluminescence processes are subdivided into fluorescence, Chemiluminescence and phosphorescence For simplicity, we use the term fluorescence to mean both

fluorescence and phosphorescence

10

The key characteristic of fluorescence spectrometry is its high sensitivity. Fluorometry may achieve limits of detecting several orders of magnitude lower than those of most other techniques. This is known as the fluorescence advantage, Useful for the detection (a single molecule) may be reached. Because of the low detection limits, fluorescence is widely used for quantification of trace constituents of biological & environmental samples; fluorometry is also used as a detection method in separation techniques, especially liquid chromatography and electrophoresis. The use of fluorescent tags to detect nonfluorescent molecules is widespread and has numerous applications (such as DNA sequencing because photons can travel through transparent 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 used in UV/Vis absorption spectrometry are

suitable for molecular fluorescence.

Principle of fluorometry

The initial step in a fluorescence measurement is electronic excitation of an analyte molecule via absorption of a photon. Once formed, an excited molecule has available a variety of decay processes by which it can rid itself of 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 in some cases, Other sample constituents

may interact with an excited analyte 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

Figure 2: principle of fluorometry

Figure 3: curve of fluorescence Vs concentration

12

Light sources

a. Gas discharge lamps :( Xenon arc lamp, High pressure

mercury vapor lamp)

b. Incandescent lamps: tungsten wire filament lamp

c. Laser: tunable dye laser

d. X-ray source for X-ray fluorescence

Wavelength selection devices

a. Filters.

b. Monochromators (polarized)

Sample compartment

Detectors

Fluorescence efficiency; quantum yield of fluorescence:

It is the ratio of the fluorescence radiant power to the

absorbed radiant power where the radiant powers are

expressed in 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 of a

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 absorbed by such a molecule is rapidly

lost by collisional deactivation.

13

Fluorescence related to concentration:

The fluorescence radiant power F is proportional to the

absorbed radiant power.

F = (Po – P)

Where = fluorescence efficiency, Po = incident power, P =

transmitted power

The relationship between the absorbed radiant power and

concentration can 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 conjugated double bonds with a high degree of

resonance stability.

Fluorescence is also expected in polycyclic aromatic systems

Substituents such as –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.

Formation of chelates with metal ions also promotes

fluorescence. However, the introduction of paramagnetic

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

Figure 4:

A/Typical aromatic molecules that do not fluoresce

B/The Typical aromatic molecules that fluorescence

15

Table 1:substitution effect ofn the fluorescence

Advantage of fluorometry

1. Sensitivity: Limits of detection depend to a large extent on the

properties of the sample being measured. Detectability to parts

per billion or even parts per trillion is common for most

analytes. This extraordinary sensitivity allows the reliable

detection of fluorescent materials (chlorophyll, aromatic

hydrocarbons, etc.) using small sample sizes. Also, field studies

can be performed in open waters without sample treatment.

Fluorometers achieve 1,000 to 500,000 times better limits of

detection as compared to spectrophotometers.

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2. Specificity: Spectrophotometers merely measure absorbed

light.5 Spectrophotometric techniques are prone to interference

problems because many materials absorb light, making it

difficult to isolate the targeted analyte in a complex matrix.

Fluorometers are highly specific and less susceptible to

interferences because fewer materials absorb and also emit

light (fluoresce). And, if non-target compounds do absorb and

emit light, it is rare that they will emit the same wavelength of

light as target compounds.

3. Wide Concentration Range: Fluorescence output is linear to

sample concentration over a very broad range.

4. Simplicity and Speed.

Fluorescence and environment

1. Temperature: A rise in temperature almost always is

accompanied by a decrease in fluorescence because the greater

frequency of collisions between molecules increases the

probability for deactivation by internal conversion and

vibrational relaxation.

2. PH: Changes in pH influence the degree of ionization, which,

in turn, may affect the extent of conjugation or the aromaticity of

the compound.

3. Dissolved oxygen: Dissolved oxygen often decreases

fluorescence dramatically and is interference in many

fluorimetric methods. Molecular oxygen is 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

deactivation to occur. Other paramagnetic substances, including

most transition metals, exhibit this same effect.

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4. Solvents: Solvents affect fluorescence through their ability to

stabilize ground and excited states differently, thereby changing

the probability and the energy of both absorption and emission.

Common problems of fluorescence measurements

1. Reference materials is as fluorescent as the sample

Contaminating substances, Raman scattering, 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 of the sample.

Bubbles' forming in the solution as it warms.

3. Self-quenching: it results when luminescing molecule collide

and lose their excitation energy by radiationless due to presence

of impurities.

4. Absorption of radiant energy: Absorption either of the

exciting or of the luminescent radiation reduces the luminescent

signal. Remedies involve (a) dilution the sample, (b) viewing the

luminescence near the front surface of the cell, and (c) using the

method of standard additions for evaluating samples.

5. Self-absorption: Attenuation of the exciting radiation as it

passes through the cell can be caused by too concentrated an

analyte. The remedy is to dilute the sample and note whether

the luminescence increases or decreases. If the luminescence

increases upon sample dilution, one is working on the high-

concentration side of the luminescence maximum. This region

should be avoided

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6. Excimer formation: Formation of a complex between the

excited-state molecule and another molecule in the ground

state, called an excimer, causes a problem when it dissociates

with the emission of luminescent radiation at longer

wavelengths than the normal luminescence. Dilution helps

lesson this effect.

Application of fluorometry

Tests of fluorometry generally used for:

Measure many type of drug in serum.

Measure catecholamine and its derivative.

Assay many of steroid compounds.

Measure many type of alborverinat

In microbiology detection of bacteria and 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)

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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,( protein fluorescence may be used as a diagnostic

of the conformational state of a protein )(tryptophan

fluorescence can be a very sensitive measurement of the

conformational state of individual tryptophan residues,

used to estimate the nature of microenvironment of the

tryptophan)&( several procedure for enzymatic assay of

ALP and others)

Medical (differentiating malignant, bashful skin tumors

from benign, urosurgery & ophthalmology)

Chemical research fields (analyzing organic compounds)

[Atomic Fluorescence Spectroscopy (AFS) techniques are

useful in other kinds of analysis/measurement of a

compound present 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 of cells to expand the properties of vital molecule.

By flow cytometer we can measure several parameter include size and granulation of cell

Can measure the content of DNA and RNA, proportion of nucleotide (A-T), (G-C) & Structure of chromatin.

Total protein & Cellular receptor. Polarization of cellular membrane. Antigen as well as Concentration of calcium ion

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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 related to the penicillins, consist

of a beta-lactam ring attached to a dihydrothiazoline ring with D-α-

aminoadipic acid. Substitutions of chemical groups result in

varying pharmacologic properties and antimicrobial activities.

Figure 5: chemical structure of cephalosporin & penicillin

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THE MECHANISM OF ACTION OF CEPHALOSPORINS IS

ANALOGOUS TO THAT OF THE PENICILLINS:

binding to specific penicillin-binding proteins,

inhibition of cell wall synthesis

activation of autolytic enzymes in the cell wall

RESISTANCE to cephalosporins may be due to poor

permeability of the drug into bacteria, lack of penicillin-binding

proteins, or degradation by β-lactamase.

Cephalosporins major groups "generations"

According to their antibacterial activity

First-generation cephalosporins have good activity against aerobic gram-positive organisms and some community acquired gram-negative organisms (P mirabilis, Escherichia coli, and Klebsiella species). In vitro activity includes coverage of gram-positive cocci, including viridians streptococci; group A hemolytic streptococci, and S aureus. Anaerobic gram-positive cocci are usually susceptible.

Second-generation drugs are a heterogeneous group with marked individual differences in activity, pharmacokinetics, and toxicity, they are active against gram-negative organisms inhibited by first-generation drugs; but they have an extended gram-negative coverage. Indole-positive Proteus and Klebsiella (including 1st generation cephalosporin-resistant strains) as well as M catarrhalis and Neisseria species are susceptible.

Third-generation cephalosporins are active against many gram-negative bacteria, most third-and fourth-generation cephalosporins inhibit most streptococci (ceftazidime is an exception to this rule). Ceftriaxone and cefotaxime offer the most reliable anti-pneumococcal coverage's, they are consistently active against Serratia marcescens, Providencia, Haemophilus, and Neisseria including β-lactamase–producing strains. Ceftazidime is unique among all agents because it's

22

active against P aeruginosa, Acinetobacter, Citrobacter, Enterobacter & nonaeruginosa.

Cefepime considered a fourth -generation agent since it is more stable against plasmid-mediated β-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: Classification of 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

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Cephalosporin's fluorimetric assays (e.g.)

Cephalexin, when subjected to alkaline hydrolysis

(degradation) produces a strongly fluorescent yellow

product (Yamana, Tsuji, Kanayama & Nakano 1974),

similar to that obtained from ampicillin by acid hydrolysis.

The fluorimetric assay for cephalexin has been developed

by increasing the severity of hydrolysis by treating with

sodium hydroxide followed by heat treatment at 100°C in a

boiling water bath (Barbhaiya & Turner

1976)&intensity was measured by using Baird Atomic

Fluoripoint spectrofluorimeter equipped with an Xenon

lamp.

Note that Sorensen's sodium citrate buffer (pH 5.0)

containing 2.5% (v/v) formaldehyde was used for

fluorimetric estimation of 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

Procedure:

In aqueous solution, the tubes being loosely covered with

polythene balls to minimize evaporation. After cooling the tubes

for 10 min the fluorescence intensity was measured at 425 nm while

in plasma or serum 10% (w/v) trichloroacetic acid (TCA) was added for

deproteinization and vortexed to produce thorough mixing to this

system 0.2 M Sorensen's sodium citrate buffer containing 2.5% (v/v-)

formaldehyde at pH 5.0

Results:

The fluorescent derivative from cephaloglycin being most

fluorescent followed by cephalexin and cephradine, the excitation

and emission wavelengths, the rate of formation of the fluorescent

derivative from each of these antibiotics increased with

temperature and duration of heating. Maximum fluorescence was

achieved by heating the solution under the described

experimental conditions for 30 min at 100°C in a boiling waterbath.

Incorporation of 2.5% (v/v) formaldehyde exerted a catalytic effect

on formation of the 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

Discussion

Partial alkaline degradation followed by hydrolysis at 100°C in the

presence of formaldehyde produces maximum fluorescence within

30 min. The structural similarities between side-chains (α-amino

group) of these antibiotics and the identical excitation and emission

spectra suggest that all three cephalosporins could be forming similar

fluorescent derivatives on hydrolysis.

Addition of 2.5% formaldehyde in the buffer catalyzed the formation of a fluorescent derivative by reducing the basicity of the α-amino group on the side-chain of these antibiotics. It might well be possible to automate this fluorimetric method for these cephalosporins so that more rapid estimation of large numbers of samples can be accurately performed if the fluorescent products were the same

Cefoxitin (cephamycin group) assay( in aqueous fluid and human urine ,Z.H.AL-Rawi & S.Tabaqchali) required acid hydrolysis to form stable product with satisfactory fluorescent properties and highly alkaline condition to exhibit optimum fluorescence, Such alkaline condition also required in cephalothin assay

Figure9:structure of

cefoxitin sodium

26

Procedure:

In aqueous 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 & emission spectra of Cefoxitin (sodium)

160µg/ml in aqueous 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

Figure11: Fluorimetric assay of Cefoxitin (a) standard curve in

aqueous solution, (b) standard curve in serum. Each dot represents

the mean of eight separate tests carried in duplicate

Discussion:

The optimum excitation and emission wavelengths of

Cefoxitin were found to be higher than the corresponding

wavelengths of the other cephalosporins & the fact that

Cefoxitin hydrolysis required much less heating time(3

min) suggest that the formation and the nature of the

fluorescence products are different due to the presence of

the 7 αmethoxy group which may be involved in the

formation of the fluorescence products, particularly as

compared with cephalothin agent (launched by Eli Lilly

1964) which structure differs from that of Cefoxitin by the

presence of the methoxy group in the 7 α-position of the

lactam ring as well as the substitution of methyl group by

amino group in the side chain of the Cefoxitin

molecule(Barbhaiya & Turner 1977; Yu et al.1977).

28

Cefuroxime assay (Zuhair Hashim AL-Rawi & Soad

Tabaqchali 1981),(serum urine & aqueous fluid) in

which fluorescent product obtained by addition of

Hydrochloric acid ,heating & cooling followed by

addition of sodium hydroxide and further heating

at 100°C. The fluorescence intensity of the final

solution was measured in a fluorimeter at an

excitation wavelength of 375 nm and an 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 of the

fluorimeter, ranging from 0.01 to 1.0, there was a close

correlation between the results of the fluorimetric and

microbiological assays of samples from the

pharmacokinetic study

Fluorimetric method also appeared for cefatrizine in

plasma, serum, and urine samples, method involve acid

hydrolysis and fluorescent product formation using

hydrogen peroxide, using excitation and emission

wavelengths of 340 and 420 nm. (Miyazaki K, Ogino O,

Arita T.Chem Pharm Bull (Tokyo). 1979)

29

Other assay reported for cephalosporin's

Rapid sensitive fluorimetric analysis of cephalosporin (Yu AB, Nightingale CH, Flanagan DR.J Pharm Sci. 1977 Feb)

Determination of cephalosporin-C amidohydrolase activity (Reyes F, Martinez MJ, Soliveri J.J Pharm Pharmacol. 1989 Feb)

Spectrophotometric determination of certain cephalosporins in pure 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 determination of Cephalexin in urine.

(Aikawa R, Nakano M, Arita T.Chem Pharm Bull (Tokyo).

1976 Oct; 24)

Fluorimetric determination of cephalexin, cephradine,

and cephatrizine in biological fluids

(Miyazaki K, Ogino O, Arita T.Chem Pharm Bull (Tokyo).

1979 Oct; 27)

Fluorimetric determination of cephradine in plasma. (Heald AF, Ita CE, Schreiber EC.J Pharm Sci. 1976 May)

[Determination of cefaloglycine and cefroxadin in biological media with thin layer chromatography with fluorimetric detection] (Blanchin MD., Rondot-Dudragne ML.J Chromatogr. 1988 Nov, 18). French

30

Figuer13: Fluorimetric assays of cephalosporins

cephalosporins'

flourimetric assay

cephalexin,

cephradine & cephaloglycin

cefoxitin(cephamycin)

cefalothincephalosporin

c

cefuroxime

cefatrizine & cefroxadin

31

Other methods used for cephalosporins analysis;

(Simple comparison)

I.CHROMATOGRAPHIC METHODS

Advantages of chromatographic methods:

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 detection methods.

Methods include:

1st Liquid chromatographic method with UV-Visible

detection & High performance liquid chromatography

(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

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

component analysis with 7 - 10 cephalosporins at a

time

Used for:

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 of cefoxitin ,its decarbomyl metabolite

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 cephal othin

(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

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

UV s p e c t r o p h o t o me t r y a n d d i f f e r e n c e U V 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 & A similar method used for

cefatrizine in serum and urine used UV 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

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 a fused-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 Biological Assay

This assay was reported for cephalosporin C by (JILLIAN M.BOND, R.W.

BRIMBLECOMBET & RC CODNER) on 1961(method of assaying the

antibiotic cephalosporin C in low concentration in culture fluids by

using a strain of Vibrio cholerae)

37

Also biologic method reported for quality control guidelines for

BAL9141(Ro 63-9141),an investigational cephalosporin, when

Reference MIC & standardized disk diffusion susceptibility 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 & HPTLCsimple,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)•ion exchange

chromatogrphyuesd for agents wih ionizable groups

spectroscopy

•FSQbeneficial in multicompound complex analysis•UV•NMR•differntial pulse adsorptive voltametry•iodometric technic

biological assay

•MIC & DISC DIFFUSIONapplied for quality control & evaluation•electrophoresis(CZE & MEKC)methods with good linearity

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 determination of penicillin and penicilloic acids (Tsuji A, Miyamoto E, Yamana T.J Pharm Pharmacol. 1978 Dec; 30)

Fluorimetric determination of Amoxicillin (Miyazaki K, Ogino O, Sato H, Nakano M, Arita T.Chem Pharm Bull (Tokyo). 1977 Feb; 25)

Relative oral bioavailability of microgranulated amoxicillin in pigs (Anfossi P, Zaghini A, Grassigli G, Menotta S, Fedrizzi G.J Vet Pharmacol Ther. 2002 Oct;25)

Fluorimetric analysis of ampicillin in biological fluids. (Jusko WJ.J Pharm Sci. 1971 May

Fluorimetric determination of ampicillin (Miyazaki K, Ogino O, Arita T.Chem Pharm Bull (Tokyo). 1974 Aug; 22)

Fluorimetric determination of ampicillin and aminobenzylpenicilloic acid

39

(Miyazaki K, Ogino O, Nakano M, Arita T.Chem Pharm Bull (Tokyo). 1975 Jan;23)

A simple fluorimetric assay of ampicillin serum. (Dürr A, Schatzmann HJ.Experientia. 1975 Apr 15)

[Degradation of ampicillin by urine of patients with complicated urinary tract infection] (Arita T, Miyazaki K, Koyanagi T, Tsuji I, Nishiumi S, Aikawa R, Murase J.Jpn J Antibiot. 1979 Jun)

[Problems in the fluorimetric determination of ampicillin]. (Lampe D, Glende M.Pharmazie. 1983 Mar)

Modified fluorimetric assay for estimating ampicilloate concentrations (Baker WL.Analyst. 1997 May; Erratum in: Analyst 1997 Aug)

Determination of ampicillin, amoxicillin, cephalexin, and cephradine in plasma by 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 analyzed by 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

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VoL 20, No. I,V.6-4.804/81/070025-05302.00/0

Fluorimetric Determination of.Cefuroxime in Body Fluids

ZUHAIR, HASHIM AL-RAWI AND SOAD TABAQCHALI"

DeparVnent of Medical Microbiolomv, St. Bartholomew's Hospital,

West Smithfield, London ECM 7BE,

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172

e

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81-86,Fluorimeiric assay of cefoxitin

Z.H. Al-Ravi and Soad Tabatichali

Department of _Necked Microbiology, Ss Bartholomew's Hospital,

West SmIttuield, London EC1A 7BE, 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) Rodovia Araraquara'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

Evaluation of PPI-0903M (T91825), a novel cephalosporin:

bactericidal activity, effects of modifying in vitro testing

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

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FLUORIMETRIC ASSAY OF CEPHRADINE, CEPHALEXIN AND

CEPHALOGLYCIN

R.H. BARBHAIYA & P. TURNER

Department of Clinical Pharmacology, St. Bartholomew's Hospital,

London, EC1A 7BE

J Antimicrob Chemother 2011; 66 Suppl 3: iii11–iii18

doi:10.1093/jac/dkr095

Ceftaroline fosamil: a new broad-spectrum cephalosporin

Joseph B. Laudano

Medical Affairs, Forest Research Institute, Harborside Financial

Center, Plaza V, Jersey City, NJ 07311, USA

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Biological Assay of Cephalosporin C

By JILLIAN M. BOND,* R. W. BRIMBLECOMBEt AND R. C. CODNER:

Medical Research Council Antibiotics Research Station,

4 Elton Road, Clevedon, Somerset (Received 29 March 1961)

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(081157.071.02..04110

Copyright i9 1987, American Society for Microbiology ,MINEREVIEW

Recent Analytical Methods for Cephalosporins in Biological Fluids

ROGER a TOOTHAKER, D. SCOTT WRIGHT, AND

LAWRENCE A. PACHLA*

Department of Phorona•oicineth's,Drog Meraboirirm. Warner-

LanthertiParke-DariN Pirarniacenthyli 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 generation of

cephalosporins

INFECTIOUS DISEASE NEWS APRIL 2009

BY KIMBERLY D.BOESER,PHARMD

PubMed ,US National Library of Medicine National Institutes of Health

DeepDyve Research The largest online rental service for professional and scholarly research articles

USP30-NF25(edition of 2007)

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