Pharmokinetics

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]. 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includes ciprofloxacin (Ciprobay, Cipro, Ciproxin), enoxacin (Enroxil, Penetrex), fleroxacin (Megalone) lomefloxacin (withdrawn), (Maxaquin), nadifloxacin, norfloxacin (Lexinor, Noroxin, Quinabic, Janacin),ofloxcin (Floxin, Oxaldin, Tarivid), pefloxacin, rufloxacin(Uroflox). 1.5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

O OH

N

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

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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,

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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

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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. 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] 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. 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

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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].

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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:

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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

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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

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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

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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 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 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 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 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].

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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.

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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 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, AUC012h

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), betacyclodextrin (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 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<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

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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.

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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 Injection volume

1 mL min-1 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 7

No. of theoretical plates Capacity factor

961.69 0.411

8 9

Tailing factor Linear range

1.01 10-200 µg/mL

Table-2 Different concentrations of norfloxacin and their corresponding peak areas

52

Sr. No

Concentration Area

1

(µg/mL) 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

1

0.5

(μg/mL) 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (h)

Concentration

0.5 1 1.25 1.5 1.75 2 2.5 3 4 6 8 10 14 18 24

(μg/mL) 4.362 8.05 8.39 8.631 8.789 7.998 7.439 6.221 5.12 3.032 2.321 1.823 1.092 0.850 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

1

AUClast

2

AUC∞

Noroxin

Ecoflaxin

3.70

3.32

5.86

5.07

F-test Result 0.578 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

kel

0.184

0.195

6 7

CL

17.50

20.22

0.531 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.

5. References

1. http://www.emedicinehealth.com/antibiotics/article_em.htm 2. Tintinalli J.E, Krome R.L, Ruiz E, ‘Emergency Medicine: A Comprehensive Study Guide’, 1995, 4th ed. McGraw-Hill. 62

3. Robert Berkow, ‘The Merck Manual Of Medical Information’, 1999, Home Edition, Pock ISBN 0-671-02727-1. 4. Ball P., J. Antimicrob. Chemother., 2000, 46, 17-24. 5. www.healthatoz.com/healthatoz/Atoz/common/standard/transform.jsp 6. http://www.medic8.com/healthguide/articles/norfloxacin.html 7. Goldstein E., Am. J. Med., 1987, 82 (6B), 3–17 8. Carlucci G, J. Chromatogr. A., 1998, 812, 343. 9. http://europa.eu.int/comm./food/fs/sc/scv/out64_en.pdf. 10. http://www.virtualinfectioncentre.com/drugs.asp?drugid=1921 11. http://www.drugs.com/pdr/norfloxacin.html 12. http://www.rxlist.com/cgi/generic/norfloxacin.htm 13. http://www.webmd.com/drugs/drug11054Norfloxacin+Oral.aspx?drugid=11054&drugnameal 14. www.genepharm.com.au/pdfs/NorfloxacinCMI.pdf 15. http://www.medicinenet.com/norfloxacin-oral/article.htm 16. http://bipolar.about.com/od/glossary/g/gl_bioequivalen.htm 17. Birkett D., Aust. Presc., 2003, 26 (4), 85-87 18. Shargel, L.; Yu, A.B, ‘Applied biopharmaceutics & pharmacokinetics’, 1999, 4th ed., New York: McGraw-Hill. 19. http://www.merck.com/mmpe/sec20/ch303/ch303c.html 20. http://www.nottingham.ac.uk/nursing/sonet/rlos/bioproc/metabolism/default.html 21. www.ibpassociation.org 22. Bryant, B., Knights, K., Salerno E, ‘Pharmacology for Health Professionals Mosby’, 2003, Sydney. 23. Y. Hsieh and W.A. Korfmacher, Current Drug Metabolism., 2006, 7(5), 479-489. 24. Sheiner, L.B., Beal S.L., Rosenberg, B. Marathe, V.V, Clin. Pharmacol. Ther., 1979, 26, 294– 305. 63

25. Beal, S.; Sheiner L.B., The American Statistician., 1980, 34,118–119. 26. http://en.wikipedia.org/wiki/Pharmacokinetics 27. Covey TR, Lee ED, Henion JD., Anal. Chem., 1986, 58, 2453–2460. 28. Covey TR., JB Crowther, EA Dewey, JD Henion , Anal. Chem., 1895. 57 (2), 474–81. 29. Sheiner, L.B.; Rosenberg, B., Marathe, V.V, J. Pharmacokin. Biopharm., 1977, 5, 445–479. 30. V. R. Meyer, Practical High Performance Liquid Chromatography, 1999, 3rd Ed, p14. 31. G. J. Shugar, J. A. Dean, The Chemist’s Ready Reference Hand Book, 1989, p3. 32. V. R. Meyer, Practical High Performance Liquid Chromatography, 1999, 3rd Ed, p76. 33. G. J. Shugar, J. A. Dean, The Chemist Ready Reference Hand Book, 1989, p13. 34. V. R. Meyer, Practical High Performance Liquid Chromatography, 1999, 3rd Ed, p84-86. 35. http://kerouac.pharm.uky.edu/asrg/hplc/applications.html 36. B. Nikolin, B. Imamovic, S. Sober and M. Bosn J., Basic Med. Sci., 2004, 4(2), 5. 37. Khalid A., Al-Rashooda, Khalil I., Al-Khamisa, Yoursy M. El-Sayeda, Sulaiman Al-Bellaa, Mohd. A. Al-Yamania, S. Mahmood Alamb, Ruwayda Dhamb, Biopharm. Drug Dispos., 2000, 21, 175–179. 38. Seth SD, Beotra A, Seth S. : J. Assoc. Physicians India, 1995 May, 43 (5), 324-330 39. S C Park, H I Yun, T K Oh, J. Vet. Med. Sci., 1998 May, 60 (5), 661- 664 40. Sousa Maia, Maria Bernadete PhD., Martins, Ismael Leite MSc., Nascimento, Demetrius Fernandes do MSc., Cunha, Adriano Nunes PhD., de Lima, Francisco Evanir Goncalves BSc., Bezerra, Fernando Antonio Frota MSc., Moraes, Manoel Odorico PhD., Moraes, Maria Elisabete Amaral PhD., Therapeutic Drug Monitoring, 2008 June, 30 (3), 341-346 41. Cordoba-Borrego M., Cordoba-Diaz M., Cordoba-Diaz D., Journal of pharmaceutical and biomedical analysis, 1999, 18 (6), 919-926 42. Bedor, Danilo Cesar Galindo, Goncalves, Talita Mota, Bastos, Leila Leal et al., Rev. Bras. Cienc. Farm., 2007, 43 (2), 231-238 43. Venkata K., Boppana, Brian N. Swanson, Antimicrobial agents and chemotherapy, 1982 May, 21 (5), 808-810 64

44. S H Parpia, D E Nix, L G Hejmanowski, H R Goldstein, J H Wilton, and J. J. Schentag, Antimicrob Agents Chemother, 1989 January, 33 (1), 99–102 45. D E Nix, J H Wilton, B Ronald, L Distlerath, V C Williams, and A Norman, Antimicrob Agents Chemother, 1990 March, 34 (3), 432–435 46. Nada AH., Sharaf MA., El Gholmy ZA., Khalafallah NM., Med. Princ. Pract., 2007, 16 (6), 426431. 47. Galaon T, Udrescu S, Sora I, David V, Medvedovici A, Biomed. Chromatogr., 2007 Jan, 21 (1), 40-47 48. Hussain MS, Chukwumaeze-Obiajunwa V, Micetich RG, J. Chromatogr B. Biomed. Appl., 1995 Jan 20, 663 (2), 379-384 49. Mascher HJ., Kikuta C., J. Chromatogr A., 1998 Jul 3, 812 (1-2), 381-386 50. Miseljic B., Popovic G., Agbaba D., Markovic S., Simonovska B., Vovk I., J. AOAC. Int., 2008 Mar-Apr, 91 (2), 332-340 51. Wallis SC., Charles BG., Gahan LR., J. Chromatogr B. Biomed. Appl., 1995 Dec 15, 674 (2), 306-315 52. Nangia A., Lam F., Hung CT., J. Pharm. Sci., 1990 Nov, 79 (11), 988-991 53. Nageswara Rao R., Nagaraju V., J. Pharm. Biomed. Anal., 2004 Mar 10, 34 (5) 1049-10 54. Lagana A., Curini R., D. Ascenzo G., Marino A., Rotatori M., J. Chromatogr. 1987 Jun 5, 417 (1), 135-142 55. V. F. Samanidou, C. E. Demetriou, I. N. Papadoyannis, Analytical and Bioanalytical Chemistry, 2003 March,375 (5), 623-629 56. Najla Mohamad Kassab, Anil Kumar Singh, Erika Rosa Maria Kedor-Hackmam,Maria Ines Rocha Miritello Santoro, Brazilian Journal of Pharmaceutical Sciences, 2005, 41(4) 57. A. J. N. Groeneveld and J. R. B. J. Brouwers, Pharmacy World & Science, 1986 February, 8(1), 79-84 58. Ehab A. Abu - Basha, Saad M. Gharaibeh1, Alaeldein M. Abudabos2, Ahmad F. Shunnaq1, Ahmad M. Al – Majali, International Journal of Poultry Science, 2008, 7(3), 289-293 59. I. J. Chen, J. M. Yang, J. L. Yeh, B. N. Wu, K. P. Hwang, C. H. Chiang, Gaoxiong Yi Xue Ke Xue Za Zhi, 1990 Sep, 6(9), 501-510

65

60. F. Fawaz, F. Bonini, M. Guyot, J. Bildet, M. Maury, A. M. Lagueny, International Journal of Pharmaceutics, 1996 April 30, 132(1-2), 271-275 61. Well M., Naber KG., Kinzig-Schippers M., Sörgel F., Int. J. Antimicrob Agents, 1998 April, 10(1), 31-39 62. Wajeeha, F.H. Khan and I. Javed, Pakistan Veterinary Journal, 2006, 26(1), 14-16 63. M. Eandi , I. Viano, F. Di Nola, L. Leone and E. Genazzani, European Journal of Clinical Microbiology &Infectious Diseases,1983 June, 2(3), 253-259 64. A. P. MacGowan, M. A. Greig, E. A. Clarke, L. O. White, D. S. Reeves, Journal of Antimicrobial Chemotherapy 1988, 22, 721-727 65. Vybiral Z., Nobilis M., Zoulova J., Kvetina J., Peter P., Journal of pharmaceutical and biomedical analysis , 2005, 37(5), 360

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