1.

INTRODUCTION

1.1 General

Norfloxacin belongs to the family of quinolones. The quinolones are a family of broad-spectrum

antibiotics. The parent of the group is nalidixic acid. The majority of quinolones in clinical use

belong to the subset of fluoroquinolones, which have a fluoro group attached the central ring system,

typically at the 6-position.

1.2 Antibiotics

Antibiotics are among the most frequently prescribed medications in modern medicine. Antibiotics

cure disease by killing or injuring bacteria. The first antibiotic was penicillin, discovered

accidentally from a mold culture. Today, over 100 different antibiotics are available to doctors to

cure minor discomforts as well as life-threatening infections [1].

Although antibiotics are useful in variety of infections, it is important to realize that antibiotics only

treat bacterial infections. They are useless against viral infections and fungal infections. All

antibiotics share the property of selective toxicity. They are more toxic to an invading organism than

they are to an animal or human host [2].

1.3 Quinolones

The quinolones are a family of broad-spectrum antibiotics. The parent of the group is nalidixic acid.

The majority of quinolones in clinical use belong to the subset of fluoroquinolones, which have a

fluoro group attached the central ring system, typically at the 6-position.Quinolones belongs to the

4th generation of antibiotics [3].

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1.4 Generations

The quinolones are divided into different generations on the basis of their antibacterial spectrum [4].

The earlier generation agents are, in general, more narrow spectrum than the later ones.Norfloxacin

belongs to 2nd generation. This generation includes ciprofloxacin (Ciprobay, Cipro, Ciproxin),

enoxacin (Enroxil, Penetrex), fleroxacin (Megalone) lomefloxacin (withdrawn), (Maxaquin),

nadifloxacin, norfloxacin (Lexinor, Noroxin, Quinabic, Janacin),ofloxcin (Floxin, Oxaldin, Tarivid),

pefloxacin, rufloxacin(Uroflox).

1.5 Fluoroquinolones

Fluoroquinolones are synthetic antibiotics that belong to the family of antibiotics called quinolones.

They are simply modified versions that contain one or more flourines as well as other chemical

modifications at specific sites of the molecule. They can be recognized because their generic name

often contains the root ‘floxacin’. While quinolones are useful mostly against urinary tract infections

involving gram negative aerobic bacteria, fluoroquinoles have a much greater range of antibacterial

ability including multidrug resistant pseudomonas caused respiratory or urinary tract infections and

post exposure prophylaxis and treatment of anthrax. Because of their excellent absorption they can

be administered not only by intravenous but orally as well. All quinolones work by inhibiting two

bacteria enzymes resulting in cell death due to DNA breaks and in interference during cell division.

Quinolones do not affect human cells because they affect one enzyme only found in bacteria and do

not bind to human enzymes. Some common fluoroquinolones used today include Ciprofloxacin,

Levofloxacin, Lomefloxacin, Norfloxacin, Sparfloxacin, Clinafloxacin, Gatifloxacin, Moxifloxacin

Sparfloxacin, and Trovafloxacin. While all of them are effective against some bacteria, each one

may be better suited against specific infections. Although resistance is not a major problem for

2
fluoroquinolones, it does arise and new agents are being developed to counteract resistance to

current agents [5].

1.6 Norfloxacin (NRX)

NRX is an oral broad-spectrum antibiotic used in the treatment of urinary tract infections, including

cystitis and gonorrhea [6]. It works by stopping the life cycle of bacteria. It is used to eliminate

certain bacteria that cause infections in your urinary tract. NRX will not work for colds, flu, or other

viral infections. NRX is available in 400-mg tablets. Each tablet contains the following inactive

ingredients: cellulose, croscarmellose sodium, hydroxypropyl cellulose, hydroxypropyl

methylcellulose, magnesium stearate, and titanium dioxide. NRX, a fluoroquinolone, differs from

non-fluorinated quinolones by having a fluorine atom at the 6 position and a piperazine moiety at the

7 position.

1.6.1 Structure of NRX

O O
F
OH

N N
HN

1 -ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid

3
1.6.2 Properties

NRX is a white to pale yellow crystalline powder with a molecular weight of 319.34 g per mole and

a melting point of about 221°C. It is freely soluble in glacial acetic acid, and very slightly soluble in

ethanol, methanol and water. Its empirical formula is C16H18FN3O3 .

1.6.3 Mechanism of Action

The mechanism of action of NRX involves inhibition of the A subunit of bacterial DNA gyrase, an

enzyme which is essential for DNA replication [7]. The DNA gyrase enyme is actually involved in

supercoiling of bacterial DNA. NRX also inhibite DNA replication, recombination, repair and

transcription resulting in lysis of bacteria [8]. DNA topoisomerase ІV is the second target of NRX.

Topoisomerase IV is involved in ATP dependent relaxation of DNA and evidence suggests that

Topoisomerase IV is the primary target in certain bacteria like Staphylococcus aureus and

Streptococci [9].

1.6.4 Distribution

NRX is found in the liver, gallbladder, gallbladder bile, bile in common bile duct, bile, prostate,

kidney.

1.6.5 Susceptible Bacteria

A broad spectrum of bacteria is susceptible including, but not limited to:

Gram positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis,

staphylococcus saprophyticus, staphylococcus faecalis and Gram negative bacteria including E.coli,

4
E. cloacae, K. oxytoca, K. pneumoniae, P. mirabilis, P. aeroginosa, C. diversus, C. freundii.

Gastrointestinal infection pathogens include Shigella, E. coli, S. typhi, N. gonorrhea.

1.6.6 Resistance

The development of resistance during therapy is uncommon. Those pathogens most likely to develop

resistance include, P. aeruginosa, K. pneumonia, Acinetobactaer sp. enterococci. Cross resistance

between NRX and other classes of antibacterial is uncommon, meaning NRX is often active against

indicated organisms resistant to the aminoglycosides, penicillins, cephalosporins, tetracyclines,

macrolides, sulphonamides.

1.6.7 Pharmacokinetics

In healthy, fasting volunteers, 30 to 40% dose is absorbed as food may decrease absorption. Peak

plasma concentrations are achieved close to one hour after dosing. Steady state concentrations are

obtained after about two days. Effective half life is 3 to 4 hrs. It is 10 to 15% bounded to plasma

protein. Excretion of absorbed drug is predominantly renal. Unabsorbed drug is recovered in faeces

[10].

1.6.8 Uses

NRX is an antibacterial mediation used to treat infections of urinary tract including cystitis

(inflammation of the inner lining of the of bladder caused by a bacterial infection), prostatitis

(inflammation of prostate gland), and certain sexually transmitted diseases such as gonorrhea.

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1.6.9 Dosage

Take NRX with full glass of water one hour before, or two hours after, eating a meal or drinking

milk. Drink plenty of liquid while taking NRX. The elderly and people with kidney problems may

need to use a reduced dosage or have their kidney function monitored. The suggested dose for

Uncomplicated Urinary Tract Infections is 800 mg per day; 400 mg should be taken twice a day for

three to ten days, depending upon the kind of bacteria causing the infection. People with impaired

kidney function may take 400 mg once a day for three to ten days. The suggested dose for

Complicated Urinary Tract Infections is 800 mg per day; 400 mg should be taken twice a day for ten

to twenty one days. The usual daily dose for Prostatitis is 800 mg, divided into two doses of 400 mg

each, taken for twenty eight days. The usual recommended dose for Sexually Transmitted Diseases

(Gonorrhea) is one single dose of 800 mg for one day. The total daily dosage of NRX should not be

more than 800 mg. The effects of NRX during pregnancy have not been adequately studied. Inform

your doctor if you are pregnant or planning a pregnancy. Do not take NRX while breastfeeding.

There is a possibility of harm to the infant [11].

1.6.10 Drug Interaction

Quinolones, including NRX, have been shown in vitro to inhibit CYP1A2.This is an enzyme which

abbreviates for Cytochrome P450 1A2. It is involved in metabolism of xenobiotics. Affiliated use

with drugs metabolized by CYP1A2 (e.g., caffeine, clozapine, ropinirole, tacrine, theophylline,

tizanidine) may result in increased substrate drug concentrations when given in usual doses. Patients

taking any of these drugs concomitantly with NRX should be carefully monitored. Elevated plasma

levels of theophylline have been reported with concomitant quinolone use. There have been reports

of theophylline-related side effects in patients on concomitant therapy with NRX and theophylline.

6
Therefore, monitoring of theophylline plasma levels should be considered and dosage of

theophylline adjusted as required. Elevated serum levels of cyclosporine have been reported with

concomitant use of cyclosporine with NRX. Therefore, cyclosporine serum levels should be

monitored and appropriate cyclosporine dosage adjustments made when these drugs are used

concomitantly. Quinolones, including NRX, may enhance the effects of oral anticoagulants,

including warfarin or its derivatives or similar agents. When these products are administered

concomitantly, prothrombin time or other suitable coagulation tests should be closely monitored. The

concomitant administration of non-steroidal anti-inflammatory drugs (NSAIDS) with a quinolone,

including NRX, may increase the risk of CNS stimulation and convulsive seizures. Therefore, NRX

should be used with caution in individuals receiving NSAIDS concomitantly. Videx® (Didanosine)

chewable/buffered tablets or the pediatric powder for oral solution should not be administered

concomitantly with, or within 2 hours of, the administration of NRX, because these products may

interfere with absorption resulting in lower serum and urine levels of NRX [12].

1.6.11 Side Effects

Nausea, diarrhea, dizziness, lightheadedness, or headache may occur. If any of these effects persist

or worsen, tell your doctor or pharmacist promptly. Tell your doctor immediately if any of these

unlikely but serious side effects occur: mental/mood changes (anxiety, confusion, hallucination,

depression and rare thoughts of suicide), shaking (tremors), sunburn (sun sensitivity). Tell your

doctor immediately if any of these rare but very serious side effects occur: usual bruising/bleeding,

signs of new infection (e.g., new/persistent fever, persistent sour throat), seizures, unusual change in

the amount of urine, signs of liver problems (e.g., unusual tiredness, stomach/abdominal pain,

persisting nausea/vomiting, yellowing eyes/skin, dark urine), vision changes. Seek immediate

7
medical attention if any of these rare but very serious side effects occurs: sever dizziness, fainting,

fast/irregular heartbeat. NRX may rarely cause serious nerve problems that may be reversible

identified and treated early. Alarming symptoms are pain, numbness, burning, tingling, weakness in

any part of the body, changes in how you sense touch, pain, temperature, body position and

vibration. NRX may rarely cause a severe intestinal condition (pseudomembranous colitis) due to a

type of resistant bacteria. This condition may occur during treatment or weeks to months after

treatment have stopped. Do not use anti diarrhea products narcotic pain medications if you have any

of the following symptoms because these products may make them worse. Tell your doctor

immediately if you develop: persistent diarrhea, abdominal or stomach pain/cramping, blood/mucus

in your stool. Use of NRX for prolonged repeated periods may result in oral thrush or new vaginal

yeast infection. Contact your doctor if you notice white patches in your mouth, a change in vaginal

discharge, or other new symptoms. Avery serious allergic reaction to this drug is rare. However, seek

immediate medical attention if you notice any of the following symptoms of a serious allergic

reaction: rash, itching/swelling, severe dizziness, trouble breathing [13].

1.6.12 Storage

Keep your tablets in the blister pack until it is time to take them. If you take the tablets out of the

blister pack, they may not keep well. Keep NRX in a cool dry place where the temperature stays

below 25 °C. Do not store it or any other medicine in the bathroom or near a sink. Do not leave it in

the car or on window sills. Heat and dampness can destroy it. Keep it where children cannot reach it.

A locked cupboard at least one-and-a half meters above the ground is a good place to store

medicines [14].

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1.6.13 Precautions

Before taking NRX, tell your doctor or pharmacist if you are allergic to it or to other quinolone

antibiotics such as CIP, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, or

ofloxacin or if you have any other allergies. Before using NRX, tell your doctor or pharmacist your

medical history, especially of: seizures, brain disorders (e.g., cerebral arteriosclerosis, tumor,

increased intracranial pressure), muscle disease/weakness (e.g., myasthenia gravis), heart problems

(e.g., cardiomyopathy, slow heart rate, torsades de pointes, QTc interval prolongation), kidney

disease, mineral imbalance (e.g., low potassium or magnesium), history of tendonitis/tendon

problems. NRX may make you dizzy or drowsy so use caution engaging in activities requiring

alertness such as driving or using machinery. Limit alcoholic beverages. NRX may make you more

sensitive to the sun. Avoid prolonged sun exposure, tanning booths or sun lamps. Use a sunscreen

and wear protective clothing when outdoors. Caution is advised when using NRX in the elderly

because they may be more sensitive to side effects of the drug, especially tendon damage (e.g.,

tendon rupture). Using corticosteroids (e.g., prednisone) and NRX together may increase the risk of

tendon problems. Caution is advised when using NRX in children because they may be more

sensitive to side effects of the drug (joint/tendon problems). Discuss the risk and benefits with your

doctor. NRX should be used only when clearly needed during pregnancy. NRX may pass into breast

milk and could have undesirable effects on a nursing infant. Therefore, breast-feeding is not

recommended while using NRX. Consult your doctor before breast-feeding [15].

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1.7 Bioequivalence

Bioequivalence is a term in pharmacokinetics used to assess whether the two brands of a drug are

biologically equivalent or not. If two products are said to be bioequivalent it means that they are, in

all respects, the same. Bioequivalence is a term used when comparing brand name and generic drugs.

Before a generic drug can be sold, the manufacturer must prove that it has the same strength as the

brand name version, and effects people the same way within the same time frame. If a generic passes

these tests, it is said to be bioequivalent to the original drug [16].

Birkett defined bioequivalence by stating that, “two pharmaceutical products are bioequivalent if

they are pharmaceutically equivalent and their bioavailability (rate and extent of availability) after

administration in the same molar dose are similar to such a degree that their effects, with respect to

both efficiency and safety, can be expected to be essentially the same. Pharmaceutical equivalence

implies the same amount of the same active substance(s), in the same dosage form, for the same rout

of administration and meeting the same or comparable standards [17].

1.8 Bioavailability

Bioavailability is a measurement of the extent of a therapeutically active drug that reaches the

systemic circulation and is available at the site of action [18].

Bioavailability of a drug is largely determined by the properties of the dosage form (which depend

partly on its design and manufacture), rather than by the drug's physicochemical properties, which

determine absorption potential. Differences in bioavailability among formulations of a given drug

can have clinical significance; thus, knowing whether drug formulations are equivalent is essential

[19]. It is denoted as letter F.

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1.8.1 Absolute bioavailability

Absolute bioavailability compares the bioavailability (estimated as area under the curve, or AUC) of

the active drug in systemic circulation following non-intravenous administration (i.e., after oral,

rectal, transdermal, subcutaneous administration), with the bioavailability of the same drug

following intravenous administration. It is the fraction of the drug absorbed through non-intravenous

administration compared with the corresponding intravenous administration of the same drug. The

comparison must be dose normalized if different doses are used; consequently, each AUC is

corrected by dividing the corresponding dose administered.

In order to determine absolute bioavailability of a drug, a pharmacokinetic study must be done to

obtain a plasma drug concentration vs time plot for the drug after both intravenous (IV) and non-

intravenous administration. The absolute bioavailability is the dose-corrected area under curve

(AUC) non-intravenous divided by AUC intravenous. For example, the formula for calculating F for

a drug administered by the oral route (po) is given below.

F= [AUC] po/ [AUC] IV×doseIV/dose po

Therefore, a drug given by the intravenous route will have an absolute bioavailability of 1 (F=1)

while drugs given by other routes usually have an absolute bioavailability of less than one.

1.8.2 Relative bioavailability

This measures the bioavailability (estimated as area under the curve, or AUC) of a certain drug when

compared with another formulation of the same drug, usually an established standard, or through

administration via a different route. It is calculated as under

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Relative bioavailability = [AUC] A/ [AUC] B ×dose B/dose A

Where A and B are two different formulations.

Relative bioavailability is extremely sensitive to drug formulation. Relative bioavailability is one of

the measures used to assess bioequivalence between two drug products, as it is the ratio of

Test/Reference AUC. The maximum concentration of drug in plasma or serum (C max) is also usually

used to assess bioequivalence [20].

1.8.3 Factors affecting bioavailability

Some factors influencing bioavailability are physical properties of the drug (hydrophobicity, pKa,

solubility),the drug formulation (immediate release, excipients used, manufacturing methods,

modified release - delayed release, extended release, sustained release, etc.), if the drug is

administered in a fed or fasted state, gastric emptying rate, circadean differences, enzyme

induction/inhibition by other drugs/foods, transporters: substrate of an efflux transporter (e.g. P-

glycoprotein), health of the GI tract, enzyme induction/inhibition by other drugs/foods,individual

variation in metabolic differences eg. (age, phenotypic differences, enterohepatic circulation, diet,

gender), disease state.

1.8.4 Causes of low value of bioavailability

When a drug rapidly dissolves from a drug product and readily passes across membranes, absorption

from most sites of administration tends to be complete. This is not always the case for drugs given

orally. Before reaching the vena cava, a drug must move down the alimentary canal and pass through

the gut wall and liver, which are common sites of drug metabolism, thus, the drug may be

metabolized before it can be measured in the general circulation. This cause of a decrease in drug

12
input is called the first-pass effect. A large number of drugs show low bioavailabilities owing to

extensive first-pass metabolism. In many instances, the extraction is so complete that the

bioavailability is virtually zero ( isoproterenol, nor epinephrine, phenacetin, and testosterone).

The two other most frequent causes of low bioavailability are an insufficient time in the

gastrointestinal tract and the presence of competing reactions. Ingested drug is exposed to the entire

GI tract for no more than 1 to 2 days and to the small intestine for only 2 to 4 h, unless gastric

emptying is considerably delayed. If the drug does not dissolve readily or if the drug is incapable of

penetrating the epithelial membrane (highly ionized and polar), there may be insufficient time at the

absorption site. Not only is the bioavailability low in this case, but it tends to be highly variable. In

addition, individual variations in age, sex, activity, genetic phenotype, stress can alter or further

increase in variability in drug bioavailability. Reactions that compete with absorption can reduce

bioavailability - include complex formation; hydrolysis by gastric pH or digestive enzymes;

conjugation in gut wall; adsorption to other drugs and metabolism by luminal microflora [21].

1.9 Pharmacokinetics

It is a Greek word consisting of “pharmacon” meaning drug and “kinetikos” meaning putting in

motion. So by definition; it is a branch of pharmacology dedicated to the determination of fate of

substance administered externally to a living organism.

In practice, this discipline is applied mainly to drug substances , though in principle it concerns itself

with all manner of compounds ingested or otherwise delivered externally to an organism, such as

nutrients, metabolites, hormones, toxins, etc. Pharmacokinetics is often divided into several areas

including, but not limited to, the extent and rate of Absorption, Distribution, Metabolism and

13
Excretion. This sometimes is referred to as the ADME scheme. Absorption is the process of a

substance entering the body, Distribution is the dispersion or dissemination of substances throughout

the fluids and tissues of the body, Metabolism is the irreversible transformation of parent compounds

into daughter metabolites, Excretion is the elimination of the substances from the body. In rare cases,

some drugs irreversibly accumulate in a tissue in the body.

Pharmacokinetics (PK) is often studied in conjunction with pharmacodynamics. So while

pharmacodynamics explores what a drug does to the body, pharmacokinetics explores what the body

does to the drug. Pharmacodynamics studies the actions of drugs within the body. This includes the

routes and mechanisms of absorption and excretion, the rate at which a drug action begins and the

duration of the effect, the biotransformation of the substance in the body and the effects and routes

of excretion of the metabolites of the drugs [22].

Pharmacokinetics describes how the body affects a specific drug after administration.

Pharmacokinetic properties of drugs may be affected by elements such as the site of administration

and the concentration in which the drug is administered. These may affect the absorption rate [23].

1.9.1 Population pharmacokinetics

Population pharmacokinetics is the study of the sources and correlates of variability in drug

concentrations among individuals who are the target patient population receiving clinically relevant

doses of a drug of interest [24, 25].

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1.9.2 Pharmacokinetic parameters

Different pharmacokinetic parameters include, area under the curve ranging from zero to specific

time (AUC0-t), Area under the curve from zero to infinity (AUC 0-∞), Maximum concentration (Cmax),

Time to reach maximum concentration (tmax), Elimination half life (t1/2), Elimination constant (kel)

and Volume of distribution.

1.10 Analysis

Pharmacokinetic analysis is performed by non compartmental (model independent) or

compartmental methods. Non compartmental methods estimate the exposure to a drug by estimating

the area under the curve of a concentration-time graph. Compartmental methods estimate the

concentration-time graph using kinetic models.

1.10.1 Non compartmental analysis

Non compartmental PK analysis is highly dependent on estimation of total drug exposure. Total drug

exposure is most often estimated by Area Under the Curve methods, with the trapezoidal rule

(numerical differential equations) the most common area estimation method. Due to the dependence

of the length of 'x' in the trapezoidal rule, the area estimation is highly dependent on the

blood/plasma sampling schedule. That is, the closer your time points are, the closer the trapezoids

are to the actual shape of the concentration-time curve.

1.10.2 Compartmental analysis

Compartmental PK analysis uses kinetic models to describe and predict the concentration-time

curve. PK compartmental models are often similar to kinetic models used in other scientific

15
disciplines such as chemical kinetics and thermodynamics. The advantage of compartmental to non

compartmental analysis is the ability to predict the concentration at any time. The disadvantage is the

difficulty in developing and validating the proper model. The simplest PK compartmental model is

the one-compartmental PK model with IV bolus administration and first-order elimination [26].

1.11 Bioanalytical methods

Bioanalytical methods are necessary to construct a concentration-time profile. Chemical techniques

are employed to measure the concentration of drugs in biological matrix, most often plasma. Proper

bioanalytical methods should be selective and sensitive.

1.11.1 Mass spectrometry

Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the

matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time

point data. The most common instrumentation used in this application is LC-MS with a triple

quadruple mass spectrometer. Tandem mass spectrometry is usually employed for added specificity.

Standard curves and internal standards are used for quantitation of usually a single pharmaceutical in

the samples. The samples represent different time points as a pharmaceutical is administered and

then metabolized or cleared from the body. Blank or t=0 samples taken before administration are

important in determining background and insuring data integrity with such complex sample

matrices. Much attention is paid to the linearity of the standard curve; however it is not uncommon

to use curve fitting with more complex functions such as quadratics since the response of most mass

spectrometers is less than linear across large concentration ranges [27, 28].

16
There is currently considerable interest in the use of very high sensitivity mass spectrometry for

micro dosing studies, which are seen as a promising alternative to animal experimentation [29].

1.12 High Performance Liquid Chromatography (HPLC)

Chromatography is a separation technique in which the sample mixture is distributed between the

two phases in the chromatographic bed (column or plane). One of the phases is stationary phase

while other passes through the chromatographic bed. The stationary phase is either a solid, porous,

surface active material in small particle form or a thin film of liquid coated on a solid support or

column wall. The mobile phase is a gas or a liquid that passes over the stationary phase [30].

1.12.1 Pumps

Pumps are used to deliver the mobile phase to the column. The pumps, its seals, and all connections

in the chromatographic system must be made up of material that is chemically resistant to the mobile

phase. A degassing unit is needed to remove dissolved gases from the solvent. Types of pumps used

in HPLC are reciprocating piston pumps, syringe type pumps, constant - pressure pumps and pulse

dampers.

1.12.2 Columns

Separation columns are available in different lengths and diameters. To withstand high pressures

involved, columns are constructed of heavy-wall glass lined metal tubing or stainless steel tubing.

Connectors and end fittings must be designed with zero void volume. Column packing is retained by

frits inserted in the ends of the column.

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1.12.3 Stationary Phases

The stationary phase may be a totally porous particle or macro porous polymer, a superficially

porous support (porous-layer beads), or a thin film covering of a solid core (pellicular supports).

Each type may have a polymer bonded to the support surface (bonded-phase supports). Different

types of stationary phases used in HPLC are totally porous particles, macro porous polymers,

porous-layer beads, extra column and effects void volume makers [31].

1.12.4 Detectors

The detector should be able to recognize when a substance zone is eluted out of the column.

Therefore, it has to monitor the change in the mobile phase composition, convert this into an

electrical signal and then convey this to the recorder where it is shown as a deviation from the

baseline. The detector is better considered in terms of concentration or mass sensitivity, selectivity,

noise, detection limit, linear range and cell volume.

Different types of detectors are used in HPLC.

UV/VIS Detectors

UV/Visible detector is the commonly used type of detector as it can be rather sensitive, has a wide

linear range, is relatively unaffected by temperature fluctuations and is also suitable for gradient

elution. It records compounds that absorb ultraviolet or visible light. The degree of absorption

resulting from passage of the light beam through the cell is a function of the molar absorptivity (ε),

the molar concentration (c), of the compound and the length of the cell (d). The product of ε, c and d

is known as the absorbance A:

18
 A= εcd [32].

The basic types of UV/VIS detectors are fixed-wavelength detector, variable-wavelength detector

and scanning – wavelength detectors [33].

Refractive Index Detectors

Refractive index (RI) detectors are non-selective and often used to supplement UV models. They

record all eluting zones, which have a refractive index different to that of the pure mobile phase.

More intense is the signal, greater is the difference between the refractive indices of the sample and

eluent. RI detectors are about 1000 times less sensitive than UV/VIS detectors.

Fluorescence Detectors

Compounds that fluorescence or for which fluorescing derivatives can be obtained are picked up

with high sensitivity and specificity by this detector. The sensitivity may be up to 1000 times greater

then UV detection. Light of suitable wavelength is passed through the cell and higher wavelength

radiation emitted is detected in a right-angled direction.

Electrochemical Detectors

Electrochemistry provides a useful means of detecting traces of readily oxidizable or reducible

organic compounds with great selectivity. The detection limit can be extraordinarily low and the

detectors are both simple and inexpensive [34].

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1.13 Applications for HPLC

1.13.1 Preparative HPLC

It refers to the process of isolation and purification of compounds. Important is the degree of solute

purity and the throughput, which is the amount of compound produced per unit time. This differs

from Analytical HPLC, where the focus is to obtain information about the sample compound. The

information that can be obtained includes identification, quantification, and resolution of a

compound.

1.13.2 Chemical Separations

It can be accomplished using HPLC by utilizing the fact that certain compounds have different

migration rates given a particular column and mobile phase. Thus, the chromatographer can separate

compounds (more on chiral separations) from each other using HPLC; the extent or degree of

separation is mostly determined by the choice of stationary phase and mobile phase.

1.13.3 Purification

It refers to the process of separating or extracting the target compound from other (possibly

structurally related) compounds or contaminants. Each compound should have a characteristic peak

under certain chromatographic conditions. Depending on what needs to be separated and how

closely related the samples are, the chromatographer may choose the conditions, such as the proper

mobile phase, to allow adequate separation in order to collect or extract the desired compound as it

elutes from the stationary phase. The migration of the compounds and contaminants through the

20
column need to differ enough so that the pure desired compound can be collected or extracted

without incurring any other undesired compound.

1.13.4 Identification

Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any

compound by HPLC a detector must first be selected. Once the detector is selected and is set to

optimal detection settings, a separation assay must be developed. The parameters of this assay

should be such that a clean peak of the known sample is observed from the chromatograph. The

identifying peak should have a reasonable retention time and should be well separated from

extraneous peaks at the detection levels which the assay will be performed. To alter the retention

time of a compound, several parameters can be manipulated. The first is the choice of column,

another is the choice of mobile phase, and last is the choice in flow rate. All of these topics are

reviewed in detail in this document.

Identifying a compound by HPLC is accomplished by researching the literature and by trial and

error. A sample of a known compound must be utilized in order to assure identification of the

unknown compound. Identification of compounds can be assured by combining two or more

detection methods.

1.13.5 Quantification

Quantification of compounds by HPLC is the process of determining the unknown concentration of

a compound in a known solution. It involves injecting a series of known concentrations of the

standard compound solution onto the HPLC for detection. The chromatograph of these known

concentrations will give a series of peaks that correlate to the concentration of the compound

21
injected. Area under the peak is noted. Now sample is injected into chromatograph and area of

resulting peak is noted. This data is used to determine unknown concentration of analyte in sample

[35].

1.14 HPLC in Pharmaceutical Analysis

In testing the pre-scale procedure the marketing of drugs and their control in the last ten years, high

performance liquid chromatography replaced numerous spectroscopic methods and gas

chromatography in the quantitative and qualitative analysis. In the first period of HPLC application

it was thought that it would become a complementary method of gas chromatography, however,

today it has nearly completely replaced gas chromatography in pharmaceutical analysis. The

application of liquid mobile phase with the possibility of transformation of mobilized polarity during

chromatography and all other modifications of mobile phase depending upon of characteristics of

substance which are being tested is a great advantage in the process of separation in comparison to

other methods. The greater choice of stationary phase is the next factor, which enables the realization

of good separation. The separation line is connected to specific and sensitive detector system,

spectroflourimeter, diode detector, electrochemical detector as other hyphenated systems High

Performance Liquid Chromatography- Mass Spectrometer (HPLC-NMR), are the basic elements on

the basic elements on which is based such wide and effective application of HPLC method. The

purpose of HPLC analysis of any drugs is to confirm the identity of a drug and provide quantitative

results and also to monitor the progress of the therapy of disease. The analysis of drugs and

metabolites in biological fluids, particularly plasma, serum or urine is one of the most demanding

but one of the most common uses of high performance liquid chromatography. When we are using

high performance liquid chromatography, it requires a good selection of detectors, good stationary

22
phase, eluents and adequate program during separation. UV/VIS detector is most versatile detector

used in high performance chromatography is not always ideal since it is lack of specificity means

high resolution of the analyte that may be required. UV detection is preformed against a single

standard of the drug being determined. Diode array and rapid scanning detector are useful for the

peak identification and monitoring peak purity but they are somewhat less sensitive than single

wavelength detectors [36].

1.15 Literature Review

Al-Rashood et al developed a high performance liquid chromatographic (HPLC) method for

bioequivalence study of two oral formulations of 400 mg norfloxacin (NRX). The study was carried

out in 18 healthy volunteers according to a single dose, two-sequence, cross-over randomized

design. The two formulations were: Uroxin (Julphar, United Arab Emirates) as test and Noroxin

(Merck Sharpe & Dohme, BV, Netherlands) as standard. Both test and reference formulations were

administered to each subject after an overnight fasting on two treatment days separated by a wash

out period of one week. After dosing, blood samples were collected at specific time intervals for a

period of 24 h. Plasma separated from blood, was analysed for NRX by a sensitive, reproducible and

accurate HPLC method. Various pharmacokinetic parameters including area under the curve from

zero to time t ( AUC0-t), area under the curve from zero to infinity (AUC0-∞), maxium concentration

(Cmax), time to reach maximum concentration (tmax), elimination half life (t1/2), and elimination

constant (kel) were determined from plasma concentrations for both the formulations and found to be

in good agreement with reported values. AUC0-t, AUC0-∞, and Cmax were tested for bioequivalence

after log-transformation of data. No significant difference was found based on ANOVA; 90%

confidence interval for test/reference ratio of these parameters were found within a bioequivalence

23
acceptance range of 80-125%. Based on these statistical inferences, it was concluded that Uroxin is

bioequivalent to Noroxin [37].

Seth et al studied the bioavailability comparison of NRX 400 mg in an Indian preparation A

(Torrent) and imported preparation B (Merck Sharp and Dohme (MSD), USA).

Twelve adult healthy volunteers participated on two occasions in a cross-over study with an interval

of 30 days administered as single oral dose. Plasma was separated from the blood and stored at -20
°
C for analysis by HPLC. Time taken to achieve Tmax was 2.00 ± 0.74 h in case of Torrent (A) and

1.70 ± 0.49 h in case of Merck Sharp and Dohme, USA (B). Cmax ranged from 1.60 to 2.87 µg mL-1

in Torrent (A) and 1.18 to 2.28 µg mL in case of MSD (B). AUCO-12 h was 12.70 ± 3.2 µg mL-1 h-1 for

'A' and 14.80 ± 2.80 µg mL-1 h-1 for 'B'. The t1/2 for Torrent (A) was 9.25 ±5.10 h and for MSD (B) it

was 12.05 ± 1.05 h. There was no significant difference in the pharmacokinetic parameters between

the two brands .Increased elimination half life and large bioavailability with both the preparations in

the present study suggested the need to be cautious while treating patients with renal problems and to

use lower doses in Indian population to achieve desirable kinetics of NRX [38].

Park et al studied the pharmacokinetics and tissue distribution comparison of two NRX formulations,

norfloxacin-glycine acetate (NRXGA) and norfloxacin nicotinate (NRXN), after single oral

administration with a dose of 5 mg equivalent NRX base kg-1 of body weight in twenty rabbits. The

pharmacokinetic characteristics of all formulations were fitted by a two-compartment open model.

The t1/2 of NRX was 3.37 ± 1.37 h and was not significant as compared with those of NRXN 3.61 ±

0.65 h and NRXGA 3.93 ± 1.54 h. The absolute bioavailability (F) of NRX, NRXN and NRXGA

was calculated as 29%, 45% and 40% respectively. In addition, tissue distribution of NRXN and

NRXGA did not show any differences of NRX concentrations in liver, kidney, serum and muscle.

24
From these results, it was suggested that NRXN and NRXGA are considered to be bioequivalent

[39].

Sousa et al developed a robust method for the determination of NRX in human plasma, using

reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detector.

The plasma protein were precipitated of with acetonitrile and ciprofloxacin was used as internal

standard (IS). Chromatographic separations were performed on a Synergi MAX-RP 150 x 4.6-mm,

4µm column with mobile phase consisting of a mixture of phosphate buffer-acetonitrile (85:15, v/v).

The calibration curve was linear, in the range of 30 to 3500 ng mL-1. The recoveries at concentrations

of 90, 1400, and 2800 ng mL-1 were 103.5%, 100.2%, and 100.2%, respectively. The quantification

limit for NRX was 30 ng mL-1. Fluorescence detector was used with excitation and emission set at

300 and 450 nm, respectively. The method validation was checked by examining the within-run and

between-run precision and accuracy and ensuring that these were within accepted limits; in

summary, the precision was <8.6% and accuracy ranged from 95.8% to 104.1% for concentration

from 90 to 2800 ng mL-1. The precision and accuracy for the lowest calibration standard 30 ng mL -1

was well within accepted limits for lower limit of quantification. The method was then applied in a

bioequivalence study in healthy volunteers given 400-mg doses of reference and test formulations of

NRX in random order with a washout period of a week [40].

Cordoba et al worked on the development and validation study of a sensitive, rapid, reproducible,

easy and precise RP-HPLC assay for NRX samples from photo stability of solid dosage forms. The

method showed excellent linearity (r2 ≥ 0.999) in the range 1 - 20 μg mL-1 using a Lichrosorb-RP C8

column (10 μm, 20 cm x 4.6 mm) and UV-detection (278 nm) at room temperature. This method

showed good efficiency for the analysis of photodegraded NRX samples, and was applied to study

25
the photo stability of NRX tablets under different conditions (direct sun light, ultraviolet light and

fluorescent light). It was proven that the use of a disintegrant can increase the photo stability of the

norfloxacin in the tablets [41].

Danilo et al developed a simple and accurate method based on HPLC with ultraviolet detection for

the quantification of NRX in human plasma and its application to a bioequivalence study between

two NRX formulations. NRX and the internal standard ciprofloxacin (CIP) were extracted from

plasma using liquid-liquid extraction. Chromatographic separation of NRX, CIP and plasma

interferents was achieved with a C18 column and a mobile phase consisting of 20 mM sodium

hydrogen phosphate buffer pH 3.0 and acetonitrile (88:12, v/v) and quantitation was done at 280 nm.

The method was linear from 25 to 3000 ng mL -1 (r2 > 0.997578), and the average recovery of NRX

and CIP from plasma was 93.9% and 91.2% respectively. The (RSD) of inter-day quality control

samples at the lower limit of quantification was less than 15%. After a single oral dose 400 mg of

NRX administered to healthy human volunteers using a randomized 2x2 crossover design,

pharmacokinetic parameters AUC0-t, AUC0-∞, Cmax, t1/2 were derived from the plasma concentration

curves for both formulations. Pharmacokinetic analysis of the data showed that the two formulations

were bioequivalent [42].

Venkata et al proposed a HPLC method for the analysis of NRX, a new nalidixic acid analog, in

human serum and urine. A statistical evaluation of the assay data showed acceptable accuracy and

precision for 0.1 to 10.0 µg mL-1 of NRX in serum and for 1.0 to 500 µg mL -1 of NRX in urine. NRX

was extracted from serum and urine at pH 7.5 with methylene chloride and was extracted back with

sodium hydroxide solution. Column used for chromatography was an anion-exchange column with

acetonitrile and phosphate buffer as the mobile phase. UV/Visible detector was set at 273 nm [43].

26
Parpia et al evaluated the effect of sucralfate on the bioavailability of NRX after single 400 mg doses

of NRX in eight healthy males. Volunteers received each of the following treatments in random

sequence: (i), NRX, 400 mg alone; (ii) sucralfate, 1 g, concurrently with NRX, 400 mg; and (iii)

sucralfate, 1 g, followed by NRX, 400 mg, 2 h later. Blood samples were collected immediately

before the NRX dose and at 0.25, 0.5, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8, 12, and 24 h after administration.

Urine was collected in divided intervals: from 0 to 12, from 12 to 24, and from 24 to 48 h. NRX

concentrations in plasma and urine were determined by HPLC. Mean area under the plasma

concentration-versus-time curve extrapolated to infinity decreased significantly after NRX was given

with and 2 h after sucralfate. The relative bioavailabilities were 1.8% when NRX was taken with

sucralfate and 56.6% when it was taken 2 h after sucralfate. After NRX was given alone, the mean

NRX concentrations in urine collected during intervals of 0 to 12, 12 to 24, and 24 to 28 h were

118.9 ± 72.3, 18.8 ± 12.5, and 2.4 ± 2.2 µg mL-1, respectively. After NRX was given with sucralfate,

however, the mean norfloxacin concentrations in urine collected during the same time intervals were

6.8 ± 4.7, 1.8 ± 1.4, and 0 ± 0 µg mL-1, respectively. Because of low pH and relatively high

magnesium concentration in urine, susceptibilities of bacteria in urine are 8 to 32 fold lower than in

plasma. This fact, along with the reduced bioavailability of NRX in the presence of sucralfate, is

likely to result in treatment failure [44].

Nix et al developed an HPLC method to evaluate the effect of antacids on the systemic absorption of

oral NRX in 12 healthy volunteers.. Treatments included 400 mg of NRX alone, 400 mg of NRX 5

min after aluminum-magnesium hydroxide (Maalox), Maalox 2 h after 400 mg of NRX, and 400 mg

of NRX 5 min after calcium carbonate (Titralac). Blood samples were collected at predetermined

time intervals for 24 and urine samples for 48 h. NRX concentrations in plasma and urine were

determined by HPLC. The AUC0-∞ versus t0-∞ and urinary recovery were used to compare the relative

27
bioavailability of NRX with antacids with that of NRX alone. NRX bioavailability was markedly

reduced when volunteers received antacid pretreatment. When NRX was given 5 min after Maalox

and Titralac, the bioavailabilities were 9.02 and 37.5%, respectively, relative to that for 400 mg of

NRX alone. When Maalox was given 2 h after NRX, Cmax of NRX in plasma occurred between 1 and

1.5 h postdose, and absorption was reduced to a lesser extent, with a relative bioavailability of

81.31%. NRX concentrations in urine were also reduced as a result of antacid administration.

Antacids containing aluminum and magnesium salts and calcium carbonate should be avoided by

patients taking NRX [45].

Nada et al developed a validated HPLC method to evaluate the bioavailability of NRX from urinary

excretion relative to plasma concentration. Twelve healthy volunteers (22-33 years) participated in

the study. Each received a previously developed (M), a local (L) and a multinational (Noroxin) tablet

(Ref), 400 mg each, according to a random balanced three-way crossover design on 3 different days.

Blood samples were collected over a 12 h period and urine over a 24 h period. NRX concentrations

were analyzed by a validated HPLC method. An initial estimate of bioequivalence of the three

products was obtained using analysis of variance on transformed data and based on confidence

interval calculation. Elimination pharmacokinetic parameters (half-life and renal clearance)

calculated from plasma concentration and urinary excretion data (mean values, n = 36) were

comparable to reported values for NRX. Interproduct differences in elimination parameters (mean

values, n = 12) were statistically insignificant (F values, ANOVA). Strong association was found

between the mean of plasma concentration and urinary excretion rates for many volunteers (F

values, regression analysis). Relative bioavailability values calculated for the local and previously

developed products relative to Noroxin were higher than 85% based on AUC and urinary excretion.

Bioequivalence could not be established among the three tested products based on calculated 90%

28
confidence intervals. Urinary excretion of NRX may be a useful noninvasive tool for bioavailability

assessment of NRX oral formulations [46].

Galaon et al proposed a simple, validated, highly selective and sensitive HPLC method with

flourescene detector for isolation and determination of furosemide and/or NRX in human plasma

samples. Samples were deproteinated by using a simple organic solvent, acetonitrile. One of the two

drug substances plays the internal standard role for the determination of the other. Separation of

analyte and internal standard was achieved in less than 5.3 min (injection to injection) on a

Chromolith Performance RP C18 column, using an aqueous component containing 0.015 mol L-1

sodium heptane-sulfonate and 0.2% triethylamine brought to pH of 2.5 with H3PO4. The composition

of the mobile phase was acetonitrile : methanol : aqueous component (70:15:15 v/v/v) and the flow-

rate was set up to 3 mL min-1. The chromatographic method applied to the determination of

furosemide relies on fluorescent detection parameters of 235 nm for the excitation wavelength, and

402 nm for the emission wavelength. In case of NRX, the excitation wavelength is set up to 268 nm

and the emission wavelength is set up to 445 nm. The overall method leads to quantitation limits of

about 27 ng mL-1 for furosemide, and 19.5 ng mL-1 for norfloxacin, using an injection volume of 250

µL. The method was applied to the bioequivalence study of two furosemide-containing formulations

[47].

Hussain et al developed a rapid, sensitive and reproducible RP-HPLC assay for the determination of

NRX. Following protein precipitation with 10% trichloroacetic acid, NRX and the internal standard

enoxacin were extracted from plasma with chloroform, dried and dissolved in the mobile phase. The

chromatographic separation of norfloxacin and the internal standard enoxacin was achieved on a C 8

column with fluorescence detection set at 280 and 418 nm for excitation and emission, respectively.

29
The peaks with a resolution factor greater than 1.5 were free from interferences. Excellent linearity

(r2 > or = 0.998) was observed over the concentration range 0.025-5.0 µg mL -1 in plasma. The inter-

assay variability was 13.6% or less at all concentrations examined. The suitability of the assay for

pharmacokinetic studies was determined by measuring NRX concentration in rat plasma after

administration of a single intravenous 10 mg kg-1 dose [48].

Mascher et al discribed a method for the determination of NRX in human plasma and urine. Plasma

samples were deproteinized using acetonitrile. The supernatant was analysed by C 18 HPLC.

Fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 450

nm was utilized. The assay was validated in the concentration range of 31 to 2507 ng mL -1 when 0.5

mL aliquots of plasma were handled. The intra-day precision of the spiked quality control samples

ranged from ± 0.37 to ± 4.14% in plasma (concentration range: 70.3 - 2109.2 ng mL -1) and from ±

0.51 to ± 1.56% in urine (concentration range: 7.5 - 299.4 µg mL -1). The intra-day accuracy obtained

for NRX in the quality control samples ranged from 5.18% to 9.47% in plasma and from 10.56% to

5.91% in urine. The assay has been used to support human pharmacokinetic studies [49].

Miseljic et al deviced a gradient RP-HPLC method for the detection and quantification of NRX and

its major impurities in NRX containing pharmaceuticals. Chromatographic separations were

performed under the following experimental conditions: column, Zorbax SB RP C18 (5 µm, 250 x 4.6

mm); injection volume, 20 µL; mobile phase, 0.05 M NaH2PO4 (pH 2.5) and acetonitrile (87 : 13)

for 16 min and (58 : 42) for 9 min (stepwise gradient); and flow rate, 1.3 mL min -1. All analyses were

performed at 25 °C, and the eluate was monitored at 275 nm using a diode array detector. Linearity

(correlation coefficient = 0.999), recovery (99.3 - 101.8%), RSD (0.2 - 0.7%), and quantitation limit

30
(0.12-0.47 µg mL-1) were evaluated and found to be satisfactory. The method is simple, rapid, and

convenient for purity control of NRX in both raw materials and dosage forms [50].

Wallis et al described a rapid and economical HPLC assay for norfloxacin in serum. Samples (100

µL) containing N-ethylnorfloxacin as the internal standard were extracted into 1 mL of chloroform.

Chromatography was performed at 30 °C on a 40 x 3.2 mm I.D. C18 guard cartridge (3 µm spherical

particles) using a mobile phase of 11% (v/v) acetonitrile in 0.01 M phosphate buffer (pH 2.5)

containing 0.001 M triethylamine, and pumped at 1 mLmin-1. Detection was at 279 nm. The

retention times of NRX and internal standard were 1.9 and 2.9 min, respectively. Calibration curves

were linear (r > 0.999) from 0.1 mg L-1 to at least 2.0 mg L-1. Within-day and between-day precision

(CV) were 8.6% or less, and accuracy was 5.3% or less. Absolute assay recovery of NRX was over

70% [51].

Nangia et al described a simple and sensitive method for the determination of fluoroquinolones by

HPLC on a C18 column using fluorescence detection. Using a mobile phase of 25% (v/v) acetonitrile

phosphate buffer (pH 2.0), adequate retention and separation among the solutes NRX, amifloxacin,

enoxacin, and pipemidic acid have been obtained using sodium lauryl sulphate as the pairing ion and

tetrabutylammonium bromide as the counter ion. The chromatographic conditions selected have

been used for the quantitation of NRX, amifloxacin, and enoxacin in human plasma using pipemidic

acid as the internal standard. A simple single-step protein precipitation procedure has been employed

for pretreatment of plasma samples. The detection limits of the assay for enoxacin, amifloxacin, and

NRX are approximately 100 ng mL-1, approximately 10 ng mL-1, and approximately 20 ng mL-1,

respectively. The method has been employed for the determination of amifloxacin in plasma samples

from a healthy volunteer following oral administration of a 400 mg amifloxacin capsule [52].

31
Nageswara et al developed a simple and rapid HPLC method for the separation and determination of

synthetic impurities of NRX. The separation was achieved on a RP C18 column. Mobile phase used

consisted of 0.01 M potassium dihydrogen orthophosphate and acetonitrile (60:40, v/v, pH 3.0)

.Flow rate was maintained at 1.0 mL min-1 .The assay was done at 40 °C using a UV detection

wavelength of 260 nm. The method was used not only for quality assurance but also for monitoring

the chemical reactions during the process development work in the laboratory. It was found to be

specific, precise and reliable for determination of unreacted levels of raw materials, intermediates

and the finished products of NRX [53].

Lagana et al described an HPLC method with fluorimetric detection for the quantitative

determination of NRX in renal and prostatic tissues and in plasma. It consisted of tissue

pretreatment, purification by solid-state extraction and separation and quantification by HPLC on a

C8 RP column. Analytical recoveries ranged from 95.2 to 97.6%. Within day and between days

precision were assessed by analysing serum containing 50ng mL-1 and 500 ng mL-1 NRX. At each

concentration, the within day precision was less than or equal to 3.6% (coefficient of variation; n =

10) and the day to day precision was less than or equal to 5.3% (n = 10). The limit of detection was 1

ng mL-1 [54].

Samanidou et al developed a rapid, accurate and sensitive method for the quantitative determination

of four fluoroquinolone antimicrobial agents, enoxacin, NRX, ofloxacin and CIP. A Kromasil 100 C8

(250 mm×24 mm, 5 µm) analytical column was used. The mobile phase consisted of a mixture of

acetonitrile,methanol and citric acid( 0.4 M ) in a ratio of (7:15:78 %, v/v) respectively. Detection

was performed with a variable wavelength UV/Visible detector at 275 nm resulting in limits of

detection of 0.02 ng per 20 mL injection for enoxacin and 0.01 ng for ofloxacin, NRX and CIP.

32
Hydrochlorothiazide (HCT) was used as internal standard at a concentration of 2 ng mL -1. A

rectilinear relationship was observed up to 2 ng mL-1 for enoxacin, 12 ng mL-1 for ofloxacin, 3 ng

mL-1 for NRX, and 5 ng mL-1 for CIP. Separation was achieved within 10 min. The statistical

evaluation of the method was examined by performing intra day (n=8) and inter day precision assays

(n=8) and was found to be satisfactory with high accuracy and precision. The method was applied to

the direct determination of the four fluoroquinolones in human blood serum. Sample pretreatment

involved only protein precipitation with acetonitrile. Recovery of analytes in spiked samples was 97-

6% over the range 0.1-0.5 ng mL-1 [55].

Najla et al described a validated analytical method for quantitative determination of CIP and NRX in

pharmaceutical preparations. A simple and rapid chromatographic method was developed and

validated for quantitative determination of two fluoroquinolone antibiotics in tablets and injection

preparations. The quinolones were analyzed by using a LiChrospher® 100 RP C18 column(5 μm, 125

x 4 mm) and a mobile phase consisted of water : acetonitrile : triethylamine (80:20:0.3 v/v/v). The

pH of final mixture was adjusted to 3.3 with phosphoric acid. The flow rate was 1.0 mL min-1 and

UV detection was made at 279 nm. The analyses were performed at room temperature (24 ± 2 ºC).

CIP and NRX were eluted within 5 min. The calibration curves were linear (r > 0.9999) over a

concentration range from 4.0 to 24.0 μg mL-1. The RSD was < 1.0% and the mean recovery was

101.85% [56].

Groeneveld et al developed a simple ,sensitive HPLC method for the analysis of CIP, NRX,

ofloxacin and pefloxacin in serum The quinolones were extracted using dichloromethane under

neutral conditions, followed by drying under nitrogen and dissolving in mobile phase before

Chromatographic analysis. The stationary phase consisted of a stainless steel column with Nucleosil

33
C18 (5 µm), and a mobile phase of 0.04M phosphoric acid, tetrabutylammoniumiodide as ion-pairing

reagent and methanol (pH 2.2). UV absorbance was used for detection. The method was shown to be

linear, quantitative and reproducible in the therapeutic range of each of these quinolones. Serum

levels of ofloxacin and CIP were determined and compared to those found by a microbiological

assay. Good correlation was found for the assay of CIP as well as for ofloxacin [57].

Ehab et al evaluated pharmacokinetics and bioequivalence of two NRX oral solutions in healthy

broiler chickens after oral administration according to a single dose, randomized, parallel

experimental design. The two formulations were Vapcotril 10%® (Vapco, Jordan) as a test product

and Mycomas 10%® (Univet, Ireland) as a reference product. The chickens were allotted into 3 equal

groups (8 chickens per group). Chickens of group 1 and 2 were given a single oral dose of Vapcotril

10%® and Mycomas 10%® at a dose level of 16 mg kg-1 body weight respectively after an overnight

fasting. Chickens of group3 were given a single intravenous dose of NRX to calculate the systemic

bioavailability. Blood samples were collected at different time points after drug administration. NRX

concentrations in chicken plasma were determined using a microbiological assay and Klebsiella

pneumoniae ATCC 10031 as a test organism. The pharmacokinetic analysis of the data was

performed using non-compartmental analysis based on statistical moment theory (SMT) with the

help of computerized Win Nonlin program (Version 5.2, Pharsight, CA, USA). The Cmax, tmax, AUC0-

12h and AUC0-∞, t1/2 and systemic bioavailability (F) were 4.94 ± 0.06 and 3.88± 0.07 μg mL -1, 1.0 and

2.0 h, 21.60 ± 0.54 and 20.51 ± 0.39 μg h mL-1, 25.40 ± 0.76 and 23.40 ± 0.69 μg h mL-1,4.49 ± 0.13

and 3.87 ± 0.21 h, 50 and 47.5% for Vapcotril 10% ® and Mycomas 10%®, respectively. The 90%

confidence interval for test reference ratio of the AUC0-12h (99.53 - 111.15), AUC0-∞ (100.9 - 116.72)

and Cmax (122.69 -132.15) was within the bioequivalence acceptance range of 80% – 125% for the

34
AUC and 75 -133 for the Cmax. In conclusion, Vapcotril 10% is bioequivalent to Mycomas 10% and

can be used as interchangeable therapeutic agents in veterinary medicine practice [58].

Chen et al compared the pharmacokinetics and bioequivalence of two NRX, Gentle capsule and

Baccidal tablet, in eight healthy male volunteers. A 400 mg dose of NRX was given orally after an

overnight fasting to volunteers in a balanced two way cross over study. Blood samples were obtained

at 0, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 h after the dosing. NRX concentration in serum was

assayed by an HPLC method using an UV detector. All the data was processed by KMCP computer

software and the pharmacokinetic parameters were calculated, based on one-compartment model.

The results revealed that Cmax of Gentle and Baccidal was 0.96 ± 0.089 and 0.99 ± 0.110, t max was 2.0

± 0.0 for both, kel was 0.101 ± 0.006 and 0.098 ± 0.005 h-1, t1/2 beta was 6.909 ± 0.483 and 7.094 ±

0.350 h, the absorption constant (Ka) was 2.444 ± 0.188 and 2.490 ± 0.096 hr-1; the absorption half

life (t1/2, alpha) was 0.278 ± 0.019 and 0.277 ± 0.010 h, AUC0-12 was 7.106 ± 1.065 and 7.380 ± 1.044

µg h mL-1 and AUC0-∞ was 9.183 ± 1.257 and 9.550 ± 1.300 µg h mL-1 respectively. There were no

significant difference found between the two groups after statistical analysis with two way ANOVA

(p greater than 0.05). A series of statistical parameters including d%, delta, and 95% C.I. were

calculated by bioequivalence test computer software of Yamaoka Simi. After evaluating all the

parameters, there was no significant difference found between the two groups. Therefore, the high

similarity of these two formulations was suggested [59].

Fawaz et al carried out a comparative bioavailability study in rabbits on pure powder of NRX and its

formulations: aqueous solution, polyethyleneglycol 6000 solid dispersions (PEG 6000 SD), beta-

cyclodextrin (β-CD) and hydroxy-propyl-beta cyclodextrin (HP-β-CD) complexes. NRX plasma

concentrations were measured by HPLC method with a fluorimetric detection. Estimation of t1/2 and

35
kel proved that PEG 6000 SD and CD complexes did not modify the elimination characteristics of

NRX. Data from plasma concentration profiles indicated that absorption of NRX from of SD and

inclusion complexes was markedly accelerated when compared with powder of pure drug. The

extent of absorption was significantly smaller with powder of NRX than with its formulations.

Bioavailability was improved and significantly higher with CD and complexes SD than with powder,

but the improvement was lower than expected [60].

Well et al assessed the urinary antibacterial activity and pharmacokinetics of enoxacin, NRX and

CIP in an open, randomised monocentric crossover study in six male and six female healthy

volunteers. Urine was collected up to 6 days, and venous blood samples up to 12 h, after a single oral

dose of 400 mg enoxacin, 400 mg NRX and 500 mg CIP. Enoxacin (250 mg L-1) demonstrated the

highest peak concentration (median) in the urine (0-6 h), followed by CIP (237 mg L -1) and NRX

(157 mg L-1) as determined by the HPLC assay. The total amount (mean) excreted by the kidneys as

parent drugs were as follows: enoxacin 54% of dose, CIP 33% of dose, and NRX 22% of dose. The

mean plasma concentrations decreased from 1 to 4 h after administration for enoxacin from 1.9 to

1.4 mg L-1, for CIP from 2.0 to 0.8 mg L-1 and for NRX from 1.3 to 0.5 mg L-1. The antibacterial

activity in urine was determined as urinary bactericidal titers (UBT), i.e. the highest 2 fold dilution

of urine still bactericidal for the reference organism (E. coli ATCC 25,922) and for five uropathogens

with minimal inhibitory (MIC) and bactericidal (MBC) concentrations ranging from highly

susceptible to resistant cultured from the urine of patients with complicated urinary tract infections

(UTI). For the E. coli ATCC 25,922, the organism with the lowest MIC, median UBTs of CIP were

present for 4 days, decreasing from 1:512 to 1:2, that of enoxacin for 2 days, decreasing from 1:256

to 1:4, and that of NRX for 2 days, decreasing from 1:128 to 1:2. For the five uropathogens (with

increasing MICs: K. pneumoniae, P. mirabilis, E. coli (resistant to nalidixic acid), P. aeruginosa and

36
E. faecalis), the UBTs decreased in general, according to MICs, demonstrating the same relations of

UBTs for CIP (highest) versus enoxacin (medium) versus NRX (lowest) with one exception (P.

mirabilis) for which norfloxacin showed higher UBTs than enoxacin. The minimal urinary

bactericidal concentrations (MUBC), as derived from urinary concentrations, and UBTs showed a

fairly wide inter- and intraindividual range and were generally higher than the corresponding MBCs

as determined in Mueller Hinton broth. In conclusion, according to antibacterial activity in urine

determined as UBTs, a single oral dose of CIP 500 mg generally resulted in the highest and longest-

lasting UBTs followed by that of enoxacin 400 mg and NRX 400 mg. A dose of 400 mg enoxacin

can be expected to be at least equivalent if not superior to that of 400 mg NRX. Only enoxacin and

CIP exhibited urinary bactericidal activity against all test organisms up to 12 h in all individuals.

Therefore, clinical comparison of enoxacin versus CIP in the treatment of complicated UTI could be

worth testing [61].

Wajeeha et al studied the bioavailability and pharmacokinetics of two commercially available

preparations of NRX i.e. A (imported) and B (locally prepared) in six healthy female goats after

single intramuscular administration at 5 mg kg-1 by weight following crossover study design. The

blood samples collected at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8 and 12 h postmedication were also analysed

for drug concentration by microbiological assay. Results revealed that preparation A showed higher

(p<0.05) plasma drug levels than the preparation B at 1, 3, 6 and 8 h after medication. Among

bioavailability parameters AUC (g.h mL-1) and relative bioavailability (F%) were higher for

preparation A than the preparation B, while other parameters did not differ between the two

preparations. Similarly, various pharmacokinetic parameters did not show any statistical difference

between preparation A and B. The study revealed comparable elimination kinetics but different

bioavailability of two commercial preparations of NRX. It was concluded from the study that for

37
optimal dosage regimen of drugs, the bioequivalence studies and kinetic behavior of the drugs are of

paramount importance [62].

Eandi et al studied the pharmacokinetics of NRX in six healthy volunteers, and three patients each

with moderate renal and hepatic damage. A new specific and sensitive HPLC method was set up to

measure plasma and urine concentrations of NRX. The mean urinary concentrations after a single

oral dose of 400 mg NRX exceeded many times the MIC and MBC values of most of the bacterial

strains responsible for urinary tract infections. Results in the patients with hepatic and renal damage

indicated slight and not statistically significant differences in comparison with healthy volunteers

[63].

In one study eight patients aged over 65 years (mean age 81 years), with microbiologically proven

urinary tract infections were treated with 400 mg NRX daily for six days. Blood samples were taken

on day 1 and day 6 to give a concentration-time profile, and on each of the other days samples were

obtained before the first dose of the day. Urine was collected throughout. The mean of the Cmax of

NRX after the first dose was 1.5 mg L-1 (range 1.1 – 1.8 mgL-1) at a mean of 3.2 h (range 1 – 6 h).

After the last dose the Cmax was 2.2 mg L-1 (range 1.6 – 3.7 h) at 3 h (range 1–4 h). t1/2 was 5 h (range

3.7 – 6 h) on day 1 and 5.3 h range (4.4 – 6.2 h) on day 6. Serum pre-dose NRX levels showed no

evidence of accumulation. Mean urinary concentrations varied between 95 and 288 mg L-1 from day 1

to day 6. No significant adverse reactions were noted. No alteration of NRX dose is suggested in the

aged with normal renal function [64].

Vybiralova et al developed new validated bioanalytical HPLC method for the determination of CIP

with NRX as an internal standard for plasma samples.NRX is structural homologue of CIP and

exhibits similar retention properties. The quality of respective peak separation is strongly influenced

38
by amphoteric character of CIP and NRX as well. Gradient elution mode using acetonitril and

phosphate buffer pH 3 on the pentafluorophenylpropyl stationary phase (250×4.6 mm Discovery®

HS F5, 5 μm, Supelco) was carried out. The resolution of 4.1 for CIP and NRX peaks was achieved.

Sample preparation by SPE C18 (Supelclean) with recovery 72% was performed. Fluorescence

detection with excitation wave length 280 nm and emission wave length 446 nm was used. After the

validation, the bioanalytical HPLC method was applied to pharmacokinetic studies [65].

1.16 Aim of the project

The aim of this project was to develop a new, rapid and efficient HPLC method for norfloxacin

determination and to use this method for bioequivalence study of different brands of norfloxacin.

2. EXPERIMENTAL

MATERIALS AND METHODS

2.1 MATERIALS

39
Acetonitrile (HPLC grade), Methanol (HPLC grade), orthophosphoric acid, acetic acid, doubelly

distilled deionized water, Norfloxacin powder (std), tablets, Ciprofloxacin powder (internal std).

2.2 HPLC SYSTEM

The analysis was carried out using HPLC system, made by Schemedzu (Japan), consisting of two

LC-10AT pumps, SPD-10A UV/VIS detector, Eclipse XDB-C18 column (Agilent) with dimension

4.6×150 mm.

2.3 Column Efficiency

The number of theoretical plates (N) determines the column efficiency.

N=16(tR/W)2

N=5.5(tR/Wh) 2

Where tR is the retention time of analyte, W is the peak width at baseline and W h is the width at half

peak width.

2.4 Resolution

The resolution is the ability of column to separate two adjacent peaks. It was determined by the

following equation

R=2(∆tR/W1+W2)

Where ∆tR is the difference of retention time of two peaks, W1 and W2 are widths at the base of

peak 1 and peak 2 respectively and R is the resolution.

40
2.5 Linear Range

Seven solutions of different concentrations were run and chromatograms were taken. A plot between

(conc. and area) was drawn using linear regression method.

2.6 Precision

Replicates of a sample solution were analyzed under suitable chromatographic conditions and

coefficient of variation was determined.

2.7 Accuracy

Accuracy was determined as the closeness of experimental values (in %) to the true one.

2.8 Limit of detection, Limit of quantification and capacity factor

2.8.1 Limit of detection (LOD):

Limit of detection was determined from the following equation

LOD=2S/N

Where S is the signal and N is the noise.

2.8.2 Limit of quantification (LOQ):

It is twice of the LOD and equation is below

LOQ=2LOD

41
2.8.3 Capacity factor (K):

Capacity factor was determined from the following equation

K= (tR-t0/t0)

Where tR is the retention time for analyte, t0 is the dead time for mobile phase and K is the capacity

factor.

2.9 HPLC Method Development

2.9.1 Chromatographic Column

The column used for the present project was a stainless steel Eclipse XDB-C18 column (Agilent)

with stationary phase consisting of Octadecyl group, with particle size of 5µm diameter. Column

dimension was 4.6×150mm. Separation mode was Reverse phase.

2.9.2 Selection of Mobile Phase

Following mobile phases were used:

1. Acetonitrile : 20mM sod. hydrogen phosphate (Na2HPO4 ) buffer (12:88 v/v). The pH of mobile

phase was adjusted at 3 with orthophosphoric acid.

2. Acetonitrile : O-phosphoric acid solution (1mL in 1000mL) in a ratio of (150:850 v/v).

2.9.3 Optimization of Conditions

First set of chromatographic conditions

42
Mobile phase 1 Acetonitrile : 20mM sod. hydrogen phosphate (Na 2HPO4 ) buffer

(12:88 v/v) with pH 3.

Flow rate 1 mL min-1

Injection volume 20 µL

Detection wavelength 280 nm

Injection sequence standard, samples, samples, standard

Second set of chromatographic conditions

Mobile phase 2. Acetonitrile : O-phosphoric acid solution (1mL in 1000 mL)

(150:850v/v)

Flow rate 2 mL min-1

Injection volume 20 µL

Detection wavelength 275 nm

Injection sequence standard, samples, and samples, standard

The moile phase selected and used was Acetonitrile : O-phosphoric acid solution (1mL in 1000 mL)

(150:850v/v). The mobile phase was filtered through 0.45 µm nylon filter and degassed for 5-10

minutes in ultrasonic bath.

2.9.4 Analysis of Norfloxacin

The experiments were performed using mobile phases as described in section 2.9.2. The

chromatographic conditions were used as described in section 2.9.3.

Standard solution preparation: Accuratley weighed amount (100 mg) of norfloxacin std. powder

was dissolved in mobile phase (100 mL). Then the solution was diluted to known concentration of

43
about 0.2 mg mL-1, filtered through 0.45 µm nylon filter and degassed for 5-10 minutes in ultrasonic

bath.

Sample Solution preparation: Accuratley weighed amount (100 mg) of norfloxacin tablet powder,

prepared by grinding 20 tablets in a mortar by piston, was dissolved in mobile phase (100 mL). Then

the solution was diluted to known concentration of about 0.2 mg mL-1, filtered through 0.45 µm

nylon filter and degassed for 5-10 minutes in ultrasonic bath.

Procedure: Equal volumes of about 20 µL of standard solution and sample solution were injected

separately and chromatograms were recorded. The retention times (tR) of standards and samples were

noted. The major peaks of chromatogram obtained with standard and major peaks of chromatogram

obtained with sample were compared.

2.10 Method Validation

2.10.1 Precision

The precision of new HPLC method was determined by injecting the replicates of 20 µL sample size

in the high performance liquid chromatograph.

2.10.2 Accuracy

The accuracy of new HPLC method was determined by measuring the concentration of solution of

analyte in replicates.

2.10.3 Linear Range

44
The linearity of the new HPLC method was determined by preparing seven solutions of different

concentrations of norfloxacin in mobile phase. Then samples of about 20 µL size of each

concentration were injected into the high performance liquid chromatograph. The detector response

was noted for each concentration. A calibration plot (conc. vs peak area) was obtained using linear

regression method.

2.10.4 Specificity

Specificity is the ability to find and quantify the compound of interest even in the presence of other

compounds. The specificity of the method was evaluated by analyzing the peaks of norfloxacin in

the samples kept at accelerated conditions of temperature and moisture.

2.10.5 Repeatability

This is the ability to run a sample for many times with low standard deviation among the results of

sample replicates.

2.10.6 Quality Control

The quality control samples were used to accept or reject the run. The replicate measurements was

made at three concentrations, one at lower limit of quantification, one in the mid range and one

approaching the high end of the range.

2.11 Bioequivalence Study

45
The bioequivalence study of two brands of norfloxacin, Noroxin (MSD) of 400 mg and a local

brand, Ecoflaxin (Technovision Pharmaceuticals, Islamabad) of 400 mg, was carried out using newly

developed method as mentioned above. The bioequivalence study was carried out in six healthy

human volunteers.

2.11.1 Clinical Protocol

Firstly, Noroxin (400 mg) tablet was orally administrated to the volunteers with a glass of water.

Then blood samples were collected at 0.5, 1, 1.25, 1.50, 1.75, 2, 2.5, 3, 4, 6, 8, 10, 14, 18 and 24 h

after drug administration. Same procedure was carried out for Ecoflaxin (400 mg) in the same

volunteers. A washout period of one week was given between each two study days.

2.11.2 Sample Collection

Blood samples of about 3mL were taken with the help of 3 mL BD syringes at specific time interval

as mentioned in section 2.11.1. These samples were immediately centrifuged at 30×100 rpm in

centrifuge machine. The supernatant plasma from each sample was separated with the help of

micropipette. Same procedure was repeated for each sample.

2.11.3 Serum Extraction

About 0.5 mL plasma was taken in test tube and 1 mL of Acetonitrile, 1mL of Acetic acid was

mixed into it. The contents in test tube were thoroughly mixed and centrifuged at a speed as

mentioned in section 2.11.2. The supernatant serum was filtered and saved in serum tubes. Serum

tubes were placed in freezer until analysis. Same procedure was repeated for each sample.

2.11.4 Analysis of Serum Samples

46
About 20µL of serum was taken in syringe and injected into HPLC system. A syringe filter of 0.45

µm pore size was used in order to filter serum samples. Standard in serum (0.2 mg mL -1) and blanks

were prepared and analyzed before sample study. First serum samples containing Noroxin were

analyzed for each volunteer and then serum samples containing Ecoflaxin were analyzed for each

volunteer. The peak area for norfloxacin in each serum sample was noted. A blank serum was also

run and chromatogram was recorded.

2.11.5 Pharmacokinetic Data Analysis

The following pharmacokinetic parameters were recorded or calculated from serum norfloxacin

concentration: Cmax, Tmax, Kel, t1/2, AUC0-12, AUC0-∞, CL,V (Volume of distribution). Different formulae

used to calculate pharmacokinetic parameters are given below,

1. Kel = slope × -2.303

Where slope shows the slope of concentration-time curve.

2. t1/2 = ln 2/Kel

3. AUClast = (t2-t1) × (C1-C2)/ln (C1/C2) (Logrithmic trapezoidal method)

Where t1 and t2 are two time intervals and C1, C2 are concentrations at t1 and t2.

4. AUC∞ = AUClast + Clast/Kel

Where Clast is the last observed concentration.

5. CL = Dose/AUC∞

6. V = CL/Kel

Cmax and Tmax are taken directly from concentration-time graph.

3. RESULTS AND DISCUSSION

47
3.1 Calibrations of HPLC System

All the components of HPLC system were calibrated according to the instructions provided in the

manual of the equipment. The calibrations were checked on quarterly basis or as and when there was

a need after service/repairs. The criterion used for qualification was a specified in the service manual

of the equipment. During these studies no major breakdown of any of the equipment occurred.

However, routine service and maintenance was carried out according to the pre-set schedule. Every

time the results of the calibration were found to be satisfactory.

3.2 Method Development

3.2.1. Selection of mobile phase and optimization of chromatographic conditions:

For the selection of mobile phase for method development in order to carryout bioequivalence study,

two types of mobile phases were used. One mobile phase consisted of Acetonitrile and 20 mM

Disodium hydrogen phosphate buffer (pH-3 with o-phosphoric acid) in a ratio of 12:88 (v/v) while

other consisted of Acetonitrile and o-phosphoric acid solution (1 mL in 1000 mL) in a ratio of

150:850 (v/v). These mobile phases were studied at different wavelengths in order to select suitable

mobile phase. The mobile phase selection was based on good resolution, peak width and retention

time.

By varying the wavelength of detector and flow rate of the mobile phase optimization of conditions

was also carried out. Mobile phase consisting of acetonitrile and 20 mM Na2HPO4 buffer (pH-3 with

o-phosphoric acid) in a ratio of 12:88 (v/v) was studied at 280 nm, resolution obtained was not

satisfactory. Then mobile phase consisting of acetonitrile and solution of o-phosphoric acid (1 mL in

1000 mL) with ratio of 150:850 (v/v) was checked at a flow rate of 2 mL min-1 and detection

48
wavelength of 275 nm and found to be most suitable because of better resolution and short retention

time. These conditions were used for subsequent study.

3.2.2 Analysis of Blank Serum

The analysis of blank serum was performed by using mobile phase and chromatographic conditions

described in section 3.2.1. A typical chromatogram is shown in figure 1.

Figure-1. A typical chromatogram of blank serum

3.2.3 Analysis of Norfloxacin

The analysis of norfloxacin was performed by using mobile phase and chromatographic conditions

described in section 3.2.1. A typical chromatogram is shown in figure 2. The retention time of

norfloxacin was found to be 2.50 minutes.

49
 Figure-2. A typical chromatogram of norfloxacin

3.2.4 Analysis of mixture of Norfloxacin and Ciprofloxacin as internal standard.

The analysis of norfloxacin and ciprofloxacin (internal standard) mixture was carried out using

mobile phase and chromatographic conditions as described in section 3.2.1. A typical chromatogram

is shown in figure 3. The retention time of norfloxacin and ciprofloxacin was found to be 2.54 and

2.88 minutes respectively.

Figure-3. A chromatogram of norfloxacin and ciprofloxacin

50
3.3 Method Validation

In order to check the validity of the developed method, different method validation parameters were

calculated as described below,

3.3.1 Accuracy:

The accuracy was determined as the percentage recovery. The percentage recovery was found to be

100.04%

3.3.2 Precision:

The precision was found as the closeness of the experimental values to the true one. The coefficient

of variation (CV within day) was found to be 0.005.

3.3.3 Limit of Detection (LOD):

This is the smallest amount of analyte which can be detected by the chromatograph. The LOD was

found to be 0.002 µg mL-1.

3.3.4 Limit of Quantification (LOQ):

This is twice of the LOD. The LOQ was calculated to be 0.004 µg mL-1.

3.3.5 Resolution (Rs):

The resolution is the ability of column to separate two adjacent peaks. The resolution was calculated

to be 0.904

3.3.6 Number of Theoretical Plates (N):

The number of theoretical plates was calculated to be 961.69.

3.3.7 Capacity Factor (K):

The capacity factor was calculated to be 0.411.

3.3.8 Tailing Factor (T):

The tailing factor was calculated to be 1.01.

51
3.3.9 Linear Range:

The amount of analyte over which the detector response is directly propotional to the concentration

of analyte. A Linear range plot between concentration of analyte and peak area is given in figure-4.

The table 2 shows solutions of different concentrations of norfloxacin and their corresponding peak

areas.

Table-1 Method validation parameters

Sr. No Parameter Value

1 Accuracy 100.04%
2 Precision 0.005
3 Limit of detection 0.002 µg/mL
4 Limit of quantification 0.004 µg/mL
5 Resolution 0.904
6 No. of theoretical plates 961.69
7 Capacity factor 0.411
8 Tailing factor 1.01
9 Linear range 10-200 µg/mL

Table-2 Different concentrations of norfloxacin

and their corresponding peak areas

52
 Sr. No Concentration Area

(µg/mL)
1 10 1023742
2 30 2335183
3 60 3886142
4 80 5323821
5 100 6783460
6 130 8403560
7 150 9984555
8 200 12004060

Figure-4 A graph between norfloxacin concentration and time

3.4 Pharmacokinetic Data Analysis

53
The following pharmacokinetic parameters were calculated for both the local and multinational

formulations : Cmax, Tmax, t1/2, kel, AUClast, AUC∞ and CL. First norfloxacin concentration at different

time intervals was calculated for both formulations. The different time intervals at which norfloxacin

concentration was calculated are given in section 2.11.1.

Table 3 and table 4 indicate the norfloxacin concentration at different time intervals in multinational

formulation, Noroxin (MSD) and local formulation, Ecoflaxin respectively.

Table-3 Mean norfloxacin concentration at different

54
 time intervals for Noroxin

__________________________________________________________________

Sr. No Time (h) Concentration

(μg/mL)
1 0.5 4.495

2 1 8.175

3 1.25 8.591

4 1.5 8.725

5 1.75 8.985

6 2 8.012

7 2.5 7.672

8 3 6.463

9 4 5.241

10 6 3.135

11 8 2.445

12 10 1.942

13 14 1.112

14 18 0.900

15 24 0.400
__________________________________________________________________

55
 Table-4 Mean norfloxacin concentration at different

time intervals for Ecoflaxin

_____________________________________________________________________

Sr. No Time (h) Concentration

(μg/mL)
1 0.5 4.362
2 1 8.05
3 1.25 8.39
4 1.5 8.631
5 1.75 8.789
6 2 7.998
7 2.5 7.439
8 3 6.221
9 4 5.12
10 6 3.032
11 8 2.321
12 10 1.823
13 14 1.092
14 18 0.850
15 24 0.339

________________________________________________________________

3.4.1 Graphical Representation

56
Graphs between concentration and time have been plotted for both of the formulations in figure 5

and 6. Concentration and time data is the same as tabulated in table 3 and 4. Figure 5 represents

graph for multinational drug while figure 6 represents graph for local formulation.

Figure-5 A graph between norfloxacin concentration

And time (Noroxin)

57
 Figure-6 A graph between norfloxacin concentration

and time(Ecoflaxin)

3.4.2 Pharmacokinetic Parameters

The pharmacokinetic parameters, as mentioned in section 3.4, were calculated both for multinational

and local drugs.

Different pharmacokinetic parameters calculated for both of the drugs are given in following tables:

58
 Table-5 Pharmacokinetic Parameters for Multinational Drug (Noroxin)

_________________________________________________________________

Sr. No Parameter Value

1 AUClast (µg/mL.h) 3.70

2 AUC∞ (µg/mL.h) 5.86

3 Cmax (µg/mL) 8.985

4 Tmax (h) 1.75

5 t1/2 (h) 3.76

6 kel 0.184

7 CL (mL/min/Kg) 17.50

________________________________________________________________

Table-6 Pharmacokinetic Parameters for Local Drug (Ecoflaxin)

________________________________________________________________

Sr. No Parameter Value

1 AUClast (µg/mL.h) 3.32

2 AUC∞ (µg/mL.h) 5.07

3 Cmax (µg/mL) 8.789

4 Tmax (h) 1.75

5 t1/2 (h) 3.55

6 kel 0.195

7 CL (mL/min/Kg) 20.22

_________________________________________________________________

59
3.4.3 Comparison of Pharmacokinetic Parameters

The different pharmacokinetic parameters, calculated for both formulations, were compared

applying F-test. For each parameter results of F-test are given below:

AUClast:

The result of F-test for AUClast for both formulations is 0.578.

AUC∞:

The result of F-test for AUC∞ for both formulations is 0.966.

Cmax:

The result of F-test for Cmax for both formulations is 0.930.

Tmax:

The result of F-test for Tmax for both formulations is 0.409.

t1/2:

The result of F-test for t1/2 for both formulations is 0.769.

kel:

The result of F-test for kel for both formulations is 0.531.

CL:

The result of F-test for CL for both formulations is 0.864.

60
 Table-7 showing results of F-test applied on pharmacokinetic parameters

of Noroxin and Ecoflaxin

------------------------------------------------------------------------------------------------------------------

Sr. No Parameter Noroxin Ecoflaxin F-test Result

1 AUClast 3.70 3.32 0.578

2 AUC∞ 5.86 5.07 0.966

3 Cmax 8.985 8.789 0.930

4 Tmax 1.75 1.75 0.409

5 t1/2 3.76 3.55 0.769

6 kel 0.184 0.195 0.531

7 CL 17.50 20.22 0.864

---------------------------------------------------------------------------------------------------------------------

The results of F-test show that there is no marked difference between the pharmacokinetic

parameters of the two formulations, Noroxin and Ecoflaxin. These two formulations have very close

values of all the pharmacokinetic parameters.

3.5 Discussion

In the current study we investigated the pharmacokinetics and bioequivalence of the two oral

norfloxacin formulations, Noroxin and Ecoflaxin in healthy human volunteers. The maximum

plasma concentration Cmax was 8.985 and 8.789 µg mL-1 for Noroxin and Ecoflaxin respectively. The

Cmax obtained in present study were higher then those reported in healthy volunteers (2.28µg mL -1)

by Seth et al, 1995. The Tmax for both formulations was 1.75h. This was higher then reported by Seth

et al (1995) which was 1.70h. The elimination half life t1/2 calculated was 3.76 and 3.55h for Noroxin

and Ecoflaxin respectively. This value was lower as compared to 5.66h as described by Nada et al,

61
2007. The elimination constant kel was 0.184 and 0.195 for Noroxin and Ecoflaxin respectively. The

renal clearance CL values were 17.50 and 20.22 mL/min/kg for Noroxin and Ecoflaxin respectively.

4. Conclusion

The pharmacokinetic parameters evaluated for the bioequivalence study of the two norfloxacin

formulations, Noroxin and Ecoflaxin, were in close agreement with each other. Hence it was

concluded that the norfloxacin brands, Noroxin (MSD) and Ecoflaxin (Technovision pharmaceutical,

Islamabad) were bioequivalent to each other.

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