1. INTRODUCTION1.1 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]. 1
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(withdrawn), (Maxaquin), nadifloxacin, norfloxacin (Lexinor,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 F N HN 1
-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic
acid N O OH
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 221C. 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 V 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.
5
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].
8
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].
9
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. 10
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 nonintravenous 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] IVdoseIV/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
11
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.
Pglycoprotein), 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].
14
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.
17
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 (), the molar
concentration (c), of the compound and the length of the cell (d).
The product of , c and d is known as the absorbance A:
18
A= cd [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].
19
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 g mL-1 in Torrent (A) and 1.18 to 2.28 g
mL in case of MSD (B). AUCO-12 h was 12.70 3.2 g mL-1 h-1 for 'A'
and 14.80 2.80 g 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, 4m 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 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 g mL-1 of NRX in serum and
for 1.0 to 500 g 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 g
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 g 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 flowrate 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 L. 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 g mL -1 in plasma.
The interassay 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 g 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 m, 250 x 4.6 mm); injection volume, 20 L;
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 g 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 L) 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 m 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 mm24 mm, 5
m) 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 976% 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 m, 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 C). 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 g 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 m), 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,
AUC012h
and AUC0-, t1/2 and systemic bioavailability (F) were 4.94 0.06
and 3.88 0.07 g mL -1, 1.0 and
2.0 h, 21.60 0.54 and 20.51 0.39 g h mL-1, 25.40 0.76 and 23.40
0.69 g 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 g h mL-1 and AUC0- was 9.183 1.257
and 9.550 1.300 g 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), betacyclodextrin (-CD) and
hydroxy-propyl-beta cyclodextrin (HP--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 longestlasting 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