post marketing surveillance of four brands of norfloxacin

1

The National Ribat University

Faculty of Graduate Students and Scientific research

Post marketing surveillance of four brands of Norfloxacin tablets

marketed in sudan

A thesis submitted in partial fulfillment of the Requirements of Master Degree of

Pharmacy (M.pharm) in Drugs Quality Control

By: Hiba Ismail Abaker Eltaher Supervisor:Professor Elrasheed Ahmed GadKariem

2017

I

Dedicated to my dear mother,

My father,

My sister,

My husband

And my beloved little daughter Mayar.

II

Table of contents Subject Page number

Dedication I

Table of content II-III

Acknowledgements IV

Abbreviations V

Abstract VI-VII

VIII المستخلص

List of figures IX

List of tables X-XI

Chapter one: Introduction

1.1. Introduction

1.2. Chemical and physical properties of Norfloxacin

1.2.1. Color

1.2.2.Melting point

1.2.3. Solubility

1.3. Experimental properties

1.3.1. Dissociation constant

1.3.2. Decomposition

1.4.Activity

1.5. Theoretical background

1.6. literature review

1.7. Research Rationale

1.8.Objectives

1-3

4

4

4

4

4

4

4

4

5-8

9

10

10

Chapter two: Materials and Methods

2.Materials and methods

2.1. Instruments and equipment

2.2. Materials

2.3.Methods

2.3.1. Preparation of stock solution

2.3.2.Sample preparation

2.3.3.Construction of standard curve

2.3.4. First order derivative method

11

11

11-12

13

13

13

13

14

III

2.3.5.Physical tests

2.3.5.1.Weight variation test

2.3.5.2.Diameter test

2.3.5.3.Thickness test

2.3.5.4.Hardness test

2.3.5.5.Friability test

2.3.5.6.Disintegration test

14

14

14

14

14

14

14

Chapter three: Results

3.1.Zero order spectrophotometric method

3.1.1. Standard curve of zero order spectrophotometric

method

3.1.2.Assay of Norfloxacin tablets using zero order

spectrophotometric method

3.1.2.1.Trizolin

3.1.2.2. Anquin

3.1.2.3. Yesnorf

3.1.2.4.Norozal

3.2.first derivative spectrophotometric method

3.2.1. Standard curve of 1st derivative method

3.2.2.Assay of Norfloxacin using first derivative

method

3.4.1.Trizolin

3.4.2.Anquin

3.4.3. Yesnorf

3.4.4. Norozal

3.5. Statistical analysis

3.6.Results of physical tests

15

15

17

17

17

18

18

19

19

20

20

21

21

22

23-24

25

Chapter four : Discussion , Conclusion and

Recommendations

4.1. Discussion

4.2. Conclusion

4.3. Recommendation

References

26-29

30

30

31-34

IV

ACKNOWLEDGEMENTS

Firstly, I would like to express my sincere gratitude to my advisor Prof. Elrasheed

Ahmed GadKariem for the continuous support of my master study and related

research, for his patience, motivation, and immense knowledge. His guidance

helped me in all the time since bachelor study until now. I could not have imagined

having a better advisor and teacher.

Besides my advisor, I would like to thank Dr. Imad Osman Abureid , whose

door is always open for any question.

Thanks also extend to my colleges and technical staff in National Ribat

University, faculty of pharmacy. Special thanks to Mr. Ibrahim Mohamed Ismail

for valuable help.

Last, but not least, I would like to express my most sincere gratitude to my

family, particularly my parents, my husband and daughter for their support and

encouragement throughout my studies.

V

List of abbreviations

NOR Norfloxacin

UV Ultra violet

HPLC High performance liquid chromatography

BP British Pharmacopeia

USP United State Pharmacopeia

APIs Active pharmaceutical ingredients

2D 2 dimension

3D 3 dimension

VI

ABSTRACT

Background: One of the major roles of the pharmaceutical analyst is to ensure

circulation of safe and effective drugs .Post marketing surveillance is normally

conducted out to verify this target.

To ensure this target the use of either known dependable method or developing

simple, accurate and precise method is required.

Objective: 1- Carry out post marketing surveillance for the assay of Norfloxacin

which is an important antibiotic of high frequency use and compare the results

obtained using direct zero order spectrophotometric method with first derivative

method.

2-Compare the quality of Norfloxacin brands with each other.

Method: The physicochemical parameters and assay of the four brands of

Norfloxacin tablets were assessed through the evaluation of uniformity of tablet

weight, friability, hardness, diameter , thickness, disintegration, and assay of active

ingredients according to established methods (BP and USP).

Results:All brands complied with the official specification for uniformity of

weight, friability, hardness, thickness, diameter and disintegration. The zero order

UV Visible spectrophotometric assay of Norfloxacin tablets revealed that three

samples contained over 90% and less than 100% (w/w) of labeled claim (Trizolin

97.6% ± 0.03 , Yesnorf 98.2%±0.43 and Norozal 93.03±0.002%) and one

sample contained more than 100% content (Anquin 101.43%±0.44).

The first derivative UV-Visible spectroscopy showed that three samples contained

over 100%content (Trizolin -103.83%±1.08 ,Anquin 101.71% ±0.016,Yesnorf

106.4%±0.16) and one sample contained less than 100% (Norozal 98.9%±0.03

content).

VII

Despite of these differences all brands pass the limits of assay according to BP (95-

105%) or USP (90-110%) limits.

Conclusion: The results evaluated by statistical analysis showed significant

difference in percentage content between the four brands. However, all the results

for the different brands were within specific BP and USP limits.

The methods are simple and rugged for routine analysis for batch-to-batch and

between brands quality control assessment.

VIII

المستخلص

احد اهم اهداف التحليل الصيدلاني هو مون ت تودا ل اد يون اننون . لون ا النواالون التح ليول ل.ود الخلفية:

التس يق تجوى ع دة لتحقيق هذا الهدفا الهدف ان ات نستخدم طويقن ن.و ن ن.تندة ا تط يو طويقن لسيطن دايقنالمن ت هذا

هو نوت النمو دال الحي يون Norfloxacinعنل التح ليل الصيدلانين ل.د التس يق لفحو عقو و -1 الهدف:

اله نن الش ئ.ن الاستخداما نع ل.مه الل.ضا Norfloxacinنق ونن ج دة ال.لان ل التج وين لد اء -2

نق ونن النت ئج التي تم الحص ل عليهو ل سوتخدام طويقون التحليول الطيفوي للاشو.ن النوئيون و ق اللنفسوجين -3

نع طويقن النشتقن الا لىاتوم تقيينوم نوت خولال Norfloxacinالطويقون النششووال الفيوي ميني ئيون حو الاول.ون عينو ل نوت د اء

القطوو السون, التفمو, حو النم نو ل الف. لون قو للاسو لي تقييم اختلاف الو وت التفتول الصولالن النتل.ناالاول.ن ان اع نت الد اء انتثلل للن اصف ل الوسنين لاختلاف ال وت التفتل الصلالن السون, النتائج:

القطو التفم,اثولا عينو ل تحتو نو يويود اختل و الاش.ن ق اللنفسجين الطيفين النوئين ي النست ى الصوفو يلويت ات

Trizolin 97.6%±0.03 ,Yesnorf وت( نووت النسوولن هووم \% ) وت100% ااوول نووت 90علووى 98.2%±0.43 and Norozal 93.03%±0.002 نوت الحتو ى 100 عينون احودة احتو ل امثوو نوت %

Anquin 101.43%±0.44 . % 100اظهو ا ل نشتق التحليل الطيفي للاش.ن ق اللنفسجين النوئين ات ثلا عين ل تحت امثو نت

Trizolin103.83%±1.08,Anquin101.71%±0.016,Yesnorf 106.4%±0.16 . نحت ى هم

% هي 100 عينن احدة تحت اال نت Norozal 98.9%±0.03ا

يع الاد ين منت الحد د الن م عن نت دست و الاد ين اللويط ني على الوغم نت هذه الاختلا ل جن

%(ا110-90ا الانويمي)\%( 95-105)

النت ئج التي اينل ل اسطن التحليل الاحص ئي لينل اختلا مليوا ي نحت ى الن دة الف. لن ليت الخلاصة:

النص نع داخل حد د دس تيو الد اء النصنع ل اسطن نص نع نختلفن نع ذل, م نل جنيع النت ئج لنختلف

ا الانويميا\الاد ين اللويط ني

الطوق النتل.ن ي الدواسن لسيطن . لن لتقييم الد اء نت د .ن لد .ن ي نفس النصنع ليت النص نع النختلفنا

IX

List of Figures

Figure number Title Page number

1 2D structure of Norfloxacin 3

2 3D structure of Norfloxacin 3

3 Scheme Synthesis of Norfloxacin 3

4 Derivatives Spectra of a Gaussian absorbance band 8

5 Absorption spectrum of standard Norfloxacin in zero

order UV.Visible spectrophotometric method

15

6 Calibration curve of zero order spectrophotometric

method of Norfloxacin

16

7 Absorption spectrum of Norfloxacin using 1st

derivative spectrophotometric method

19

8 Standard curve of Norfloxacin 1st derivative method 20

X

List of Tables Table number Title Page number

1 Brands of NOR and their sources 12

2 Concentrations of standard and corresponding

absorption ( zero order UV.Visible method)

16

3 Calculation steps of percentage content of NOR

in Trizolin tablets using zero order UV.Visible

spectrophotometry

17


in Anquin tablets using zero order UV.Visible

spectrophotometry

17


in Yesnorf tablets using zero order UV.Visible

spectrophotometry

18


in Norozal tablets using zero order UV.Visible

spectrophotometry

18

7 Concentrations of standard and corresponding

absorption(1st derivative UV.Visible method)

19


in Trizolin tablets by 1st derivative UV.Visible

spectrophotometry

20


in Anquin tablets by 1st derivative UV.Visible

spectrophotometry

21

10 Calculation steps of percentage content of NOR 21

XI

in Yesnorf tablets by 1st derivative UV.Visible

spectrophotometry


in Norozal tablets by 1st derivative UV.Visible

spectrophotometry

22

12 ANOVA table of Trizolin and Anquin tablets 23

13 ANOVA table of 1st Yesnorf and Norozal tablets 24

14 Results of physical tests 25

1

CHAPTER ONE

INTRODUCTION and

LITERATURE REVIEW

1

1.1. INTRODUCTION

Post-market surveillance or monitoring involves all undertaken activities to

obtain more data and information about a product after it had been granted

marketing authorization and made available for public use. The data and

information so obtained could be employed for product improvement, development

of standards and regulations.

Regulatory agencies rely on limited information obtained during clinical trials and

to some extent scientific literature as guides to granting marketing authorization of

medicines for public use.

It is therefore imperative to conduct post-market surveillance or monitoring of

approved medicines in order to adequately assess the quality, therapeutic

effectiveness and safety of medicines for the larger public(1) .

Post-market monitoring ought not to be a one off event rather it should be a

continuous event throughout the life of a drug product.

Activities of post market monitoring of a drug have been identified to include:

review of product’s condition of approved study; evaluation and investigation of

reported drug complaints; inspection of manufacturer’s processes and procedures

for production and complaint handling; market surveys of technical and clinical

documentation; review of product claims/labeling ; public access to information

taken and reported to the regulatory agency(ices) ; and in vitro testing of products

for compliance to standards (1)

Counterfeit and substandard medicines are a major cause of morbidity, mortality

and loss of public confidence in drugs and health structures (2).

To reduce the cost of medicines especially for the low income group of

developing countries, the World Health Organization (WHO) has continuously

2

advocated the use of generic brands (3) but this approach has not provided sufficient

evidence for the substitution of one brand for another.

The difference in cost between a branded and generic medicine may be as high as

90%.

To assist in substitution of branded with generics for affordability and at the same

time achieve therapeutic efficacy, bioequivalence studies become paramount (4)

Generic substitution could be considered when a generic copy of a reference drug

contains identical amounts of the same active ingredient in the same dose

formulation and route of administration as well as meet standards for strength,

purity, quality and identity (5).

Assay, Uniformity of weight, disintegration and dissolution are compendial

standards to assess the quality of tablets while hardness and friability are referred

to as non-compendial standards although friability is now included in the United

States Pharmacopeia (6).

Uniformity of weight does serve as a pointer to good manufacturing practices

(GMP) as well as amount of the active pharmaceutical ingredient (API)

Norfloxacin (NOR), chemically 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-

piperazinyl)-3-quinoline carboxylic acid(7) (figure 1)

Norfloxacin is the first synthetic second-generation fluoroquinolone antimicrobial

drug. It was developed for use in human and veterinary medicine (8).

The Chemical structure and synthesis of Norfloxacin are demonstrated in figures (1,2 and 3).

3

Figure (1) 2D Chemical Structure

figure( 2 )3D Chemical Structure

Scheme (1) Synthesis of Norfloxacin

http://www.newdruginfo.com/pharmacopeia/usp28/v28230/uspnf/pub/images/v28230/g-604.gif

4

1.2.Chemical and Physical Properties of Norfloxacin

1.2.1.color

White to light-yellow crystalline powder

1.2.2.Melting point

220-221 deg C

1.2.3.Solubility

In water , 0.28 mg/mL at 25 deg C. Solubility in water is pH dependent, increasing

sharply at pH<5 or pH >10

Solubility at 25 deg C (mg/mL): methanol 0.98; ethanol 1.9; acetone 5.1;

chloroform 5.5; diethyl ether 0.01; benzene 0.15; ethyl acetate 0.94; octyl alcohol

5.1; glacial acetic acid 340

1.3. Experimental Proberties:

1.3.1.Dissociation constant

pKa1 = 6.34; pKa2 = 8.75 (9)

1.3.2.Decomposition

When heated to decomposition it decompose to toxic fumes of hydrogen floride

and nitrogen oxides (10)

1.4.Activity:

Norfloxacin, occasionally used to treat common, as well as complicated urinary

tract infections, exhibits a broad spectrum of activity against Gram-positive and

Gram-negative bacteria (11-13). The mechanism of the bacterial effect of Norfloxacin

is based on the primary target in bacterial enzyme DNA gyrase and topoisomerase

II and IV. Inhibition of the activity of these enzymes disables DNA replication

which in turn, inhibits bacterial replication (14_15).

In addition, good tolerability and a favorable safety profile make the

fluoroquinolones important therapeutic options for the treatment of infections

caused by antibiotic-resistant bacteria (16)

5

1.5.Theoretical background Spectroscopy methods are the branch of science dealing with the study of

interaction between Electromagnetic radiation and matter. It is a most powerful

tool available for the study of atomic and molecular structure/s and is used in the

analysis of wide range of samples. Optical spectroscopy includes the region on

electromagnetic spectrum between 100 Å and 400 μm.(17 -18)

Spectroscopic techniques involve UV-Visible spectroscopy , fluorescence

spectroscopy , atomic absorption – emission spectroscopy , infrared spectroscopy ,

Mass spectroscopy , NMR spectroscopy and Raman spectroscopy .

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis

or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the

ultraviolet-visible spectral region (19).

Principal of UV.Visible spectroscopy that molecules containing π-electrons or non-

bonding electrons (n-electrons) can absorb the energy in the form of ultraviolet or

visible light to excite these electrons to higher anti-bonding molecular orbitals.

The more easily excited the electrons, the longer the wavelength of light it can

absorb(20).

UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative

determination of different analytes, such as transition metal ions, highly conjugated

organic compounds, and biological macromolecules. Spectroscopic analysis is

commonly carried out in solutions but solids and gases may also be studied (21).

The quantitative application of UV.Visible spectroscopy depends on Beer-Lambert

law.

The Beer-Lambert law states that the absorbance of a solution is directly

proportional to the concentration of the absorbing species in the solution and the

path length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to

6

determine the concentration of the absorber in a solution. It is necessary to know

how quickly the absorbance changes with concentration. This can be taken from

references (tables of molar extinction coefficients), or more accurately, determined

from a calibration curve.(21)

The Beer-Lambert law (or Beer's law) is the linear relationship between

absorbance and concentration of an absorbing species. The general Beer-Lambert

law is usually written as:

A = a( ) * b * c

where A is the measured absorbance, a( ) is a wavelength-dependent absorptivity

coefficient, b is the path length, and c is the analyte concentration. When working

in concentration units of molarity, the Beer-Lambert law is written as:

A = * b * c

where is the wavelength-dependent molar absorptivity coefficient. Data are

frequently reported in percent transmission (I/I0 * 100) or in absorbance [A = log

I0/I]. The latter is particularly convenient.

Sometimes the extinction coefficient is given in other units; for example,

A = A1%1cm * b * c

where the concentration C is in gram per 100 ml of solution. This is useful when

the molecular weight of the solute is unknown or uncertain.

Derivative spectrophotometry is an analytical technique of great utility for

extracting both qualitative and quantitative information from spectra composed of

unresolved bands.The derivative method has found application not only in

ultraviolet-visible region spectrophotometry, but also in infrared,atomic-absorption

7

and flame emission spectrometry, and also in fluorimetry (normal’ and

synchronous scanning’)(22).

The use of derivative spectrometry is not restricted to special cases, but may be of

advantage whenever quantitative study of normal spectra is difficult. Its

disadvantage is that the differentiation degrades the signal-to-noise ratio, so that

some form of smoothing is required in conjunction with the differentiation.’(23)

The principals and objectives of derivatives spectroscopy is that correction of

interference by impurities ,conversion of irregular interference to linear one , then

the linear to a constant (the slope of the constant is zero) .

For quantitative purpose , 2nd and 4th derivatives spectra are the most used as they

give more sharp peaks which are more close to the reference peak.4th derivative

gives more slopes hence better detection for impurities or resolution.

The first derivative is the rate of change of absorbance against wavelength. It starts

and finishes at zero, passing through zero at the same wavelength as λmax of the

absorbance band.

This derivative has a positive and a negative band with maximum and minimum at

the same wavelengths as the inflection points in the absorbance band. This bipolar

function is characteristic of all odd-order derivatives. The most distinctive feature

of the second-order derivative is a negative band with minimum at the same

wavelength at the maximum on the zero-order band. This derivative also shows

two positive satellite bands on either side of the main band. The fourth derivative

shows a positive band with a maximum at the same wavelength as the maximum

on the zero order bands. Even-order derivatives show a negative or positive band

with minimum or maximum at the same wavelength as λmax on the absorbance

band (24)

8

figure (4) derivatives spectra of a Gaussian absorbance band

9

1.6.litrerature review

• Research workers in Nigeria reported Quality control assessment of four

brands of Norfloxacin tablets marketed in Nigeria. Predictions from DE

indicated that the brands were all bioequivalent and therefore

interchangeable with the innovator brand(25).

• In USP and BP Norfloxacin is assayed by high performance liquid

chromatography equipped with 275 nm detector and 3.9mm*30 cm column

that contains packing L1 (26)

Some other reported methods include:

• Simultaneous spectrophotometric estimation of Norfloxacin and Ornidazole

in tablet dosage form , using UV.Visiblespectrophotometer at 273 and

318.5 nm (27)

• Simultaneous determination of trace Norfloxacin, Pefloxacin and

Ciprofloxacin by TLC fluorescence Spectrodesitometry(28)

• Bioequivalence of Norfloxacin by HPLC-UV method. A simple, rapid and

convenient high performance liquid chromatographic method has been

developed for the determination and bioequivalence of Norfloxacin in

tablets formulations by using ciprofloxacin as an internal standard(29)

• A simple, accurate, sensitive and precise Ultraviolet specrophotometric

method has been developed for the determination of Norfloxacin in tablet

dosage form, the quantitative determination of the drug was carried at 277

nm.(30)

10

1.7.Research rationale:

Routine laboratory testing of drugs in the market is crucial to protect public health

especially in developing countries where counterfeit and substandard drugs have

become a major challenge to health care services.

There is an increasing need to evaluate the performance of a number of the

available fluoroquinolone antibacterial agents because of the unexplainable pattern

of microbial sensitivity to the members of this class of drugs. (31).

1.8.Objectives:

The Study was done to:

1. Assess the quality of Norfloxacin brand in Sudanese market and to ensure that

the label claim comply with the British pharmacopeia and/or US Pharmacopeia .

2.Apply derivative spectrophotometric method for the assay of all brands of

Norfloxacin marketed in sudan.

3. Compare the quality of the brands with each other.

11

Chapter Two

MATERIALS and METHODS

11

2.Materials and Methods

2.1.Instruments and Equipment:

1.UV.Visible spectrophotometer single beam :UV mini-1240, shimadzu

2.UV.Visible spectrophotometer double beam : SHIMADZU ,japan,model, UV

1800-240 V.

3.Sonicater; seial NO.A1145602050 CD.

4.Electronic balance: AX120. NO D432510585,capacity 120g, shimadzu

5.Friabilatortrommel:D-63150 Heusenstamm /Germany ,ERWEKA

6.Disintegration apparatus: ZT 321 HEUSENSTAMM / Germany

7.Hardness tester

8.Vernia caliper: thickness and diameter tester

9.Glass wares: volumetric flasks, volumetric pipettes, beakers, cylinder , funnel ,

conical flask: analytical grade. ISO lab

10.Filter paper: Whatman filter paper

11.Foil

2.2.Materials:

2.2.1.Chemicals:

1.HCL: 37% w/w, analytical grade ,ACS ,ISO, Germany

2.Distelled water

2.2.2.Standard:

3.Norfloxacin standard powder was supplied by Azal Pharmaceuticals, khartoum,

Sudan.

12

2.2.3.Sample

Brands of Norfloxacin and their sources summarized in table (2)

Table (1) Brands of Norfloxacin and their sources

Item Name Agent Manufacturer Country

Trizolin

400mg tablet Siho trading c Remedica ltd Cyprus

Anquin

400mg tablet Badr drugs

Lyka labs , ltd ,

Ankliahwar India

Yesnorf

400mg tablet

Shanghai,

Sudan

shanghai pharmacutical

co, Sudan

Norozal

400mg tablet

Azal

pharmacutical

azalpharmacuticalidustries

Co. ltd Sudan

13

2.3.Methods:

2.3.1.Preparation of stock solution:

100 mg of NOR was placed in 100 ml volumetric flask and dissolved in 75 ml of

0.1 N HCL and the volume was made up to the mark with 0.1NHCL . 10 ml of the

solution was diluted up to 100 ml with 0.1 N HCL to produce final stock solution

of 100 μg/ml of NOR .

2.3.2.Sample preparation

Twenty tablets were taken, powdered and powder weight equivalent to 100 mg of

NOR was accurately taken and transferred to a 100 ml volumetric flask. 70 ml of

0.1N HCL added and sonicated for 30 min ,the flask was shaken before the volume

was diluted to the mark with the same solvent (solution A). The above solution was

filtered using whatman filter paper no. 1.

10 ml of solution A was transferred to a 100 ml volumetric flask and the volume

was made up to the mark with 0.1NHCL (solution B). 3 ml of solution B was then

transferred to 50 ml volumetric flask and made up to the mark with 0.1 N HCL

(solution C) final concentration of about 6 μg/ml . The absorbance of solution C

was read at 277 nm against blank for all brands using UV.Visible

spectrophotometer.

2.3.3.Construction of standard curve

Serial dilutions from stock solution were made to obtain concentrations of 2 , 4 , 6 ,

8 , 10 , 12 μg/ml .The absorbance of each solution was read at 277 nm wave

length using UV.Visible spectrophotometer… Standard curve was then constructed

for absorbances versus concentrations.

14

2.3.4.Derivative Spectrophotometric method:

The standard stock solution(100 μg/ml). and the sample solution used for the

derivative method were prepared as mentioned under zero order method. The

prepared solutions were scanned using UV.Visible spectrophotometer in the 1st

derivative mode.

2.3.5.Physical tests

2.3.5.1.Weight variation test

Twenty tablets were randomly selected, individually weighed, the

Average weight , standard deviation and weight variation of twenty tablets were

calculated.

2.3.5.2.Diameter

Diameter of the 10 tablets were determined using Vernier caliper and average

value was calculated.

2.3.5.3.Thickness

Thickness of the 10 tablets were determined using Vernier caliper and an average

value was calculated.

2.3.5.4.Hardness

Hardness of the 10 tablets were tested using a Monsanto Hardness tester and an

average value was calculated.

2.3.5.5.Friability

Friability of the tablets was determined in a friabilator (Friabilatortrommel). Ten

tablets were weighed initially (w1) and placed in the friabilator that revolves at a

speed of 25 rpm, dropping those tablets at a distance of six inches height with each

revolution and rotated in the friabilator for 100 revolutions. After completion of

rotations, the tablets were dedusted and weighed (w2).

The percent loss in weight or friability (f) is calculated by

using the formula. f = (w1-w2)/w1 x100

2.3.5.6. Disintegration test

The disintegration test was performed using water as disintegration medium and

the temperature maintained at 37±2°C . The time in minutes required for complete

disintegration with no palpable mass remaining in the apparatus was then recorded.

15

Chapter Three

RESULTS

15

3.Results

The results of these work are summarized in (13) tables and (4) figures

3.1. Zero order spectrophotometric method:

Absorbtion spectrum of standard Norfloxacin shown in figure (5)

Figure (5) Zero order absorption spectrum of standard Norfloxacin

(concentration ..6 μg/ml)

3.1.1.Standard curve of simple spectrophotometric method

The obtained absorbance values at 277nm are shown in table (2)

16

Table (2) Absorbance values for the prepared concentrations at lambda max

277nm:

concentration

(μg/ml) Absorbance

2 0.257

4 0.514

6 0.762

8 1.03

10 1.287

12 1.546

r=0.9999 a= -2*10E-4 b=0.1282

Figure (6) Calibration curve of Norfloxacin standard using zero order

spectrophotometric method (absorbances VS concentrations)

y = 0.129x - 0.0039R² = 0.9999

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10 12 14

Series1

Linear (Series1)

17

3.1.2.Assay of Norfloxacin tablets using zero order

spectrophotometric method:

The % content of tablets were determined using calibration curve . Results

obtained are shown in tables (3- 6).

3.1.2.1.TRIZOLIN

Table (3): Calculation of percentage content of Norfloxacin in Trizolin by zero


3.1.2.2.ANQUIN

Table (4): Calculation of percentage content of Norfloxacin in Anquin by zero


Anquin

weight

taken(gm) absorpance

theoretical

concn

(μg/ml)

practical

concn(μg/ml)

percentage

content

sample1 0.1413 0.738 5.7025 5.7562 101.05%

sample2 0.1488 0.778 6.0052 6.0681 101.04%

sample3 0.1439 0.761 5.8074 5.9356 102.20%

Trizolin weight taken(gm)

Absorbanc theoretical concn

(μg/ml)

practical concentration

(μg/ml) percentage content

sample 1 0.14139 0.717 5.7408 5.5925 97.40%

sample 2 0.1382 0.732 5.844 5.7094 97.70%

sample 3 0.141 0.747 5.9627 5.8264 97.70%

18

3.1.2.3.YESNORF

Table (5): Calculation of percentage content of Norfloxacin in

Yesnorf by zero order UV.Visible spectrophotometric method

Yesnorf

weight

taken(gm)

Absorpance

theoretical

concn(μg/ml)

practical

concn

(μg/ml)

percentage

content

sample

1 0.1685

0.755

5.9561 5.8888 98.80%

sample

2 0.1691

0.752

5.9773 5.8654 98.30%

sample

3 0.1679

0.742

5.9349 5.7874 97.50%

3.1.2.4.NOROZAL

Table (6) Calculation of percentage content of Norfloxacin in

NOROZAL by using zero order UV.Visible spectrophotometric

method

Noroza

l

weight

taken(gm

)

Absorpanc

e

theoretical

concentration(μg/ml

)

Practica

l concn

(μg/ml)

percentag

e content

sample

1 0.1708

0.716

5.999 5.5847 93.09%

sample

2 0.171

0.715

6 5.5832 93.00%

sample

3 0.1712

0.717

6.013 5.5925 93.00%

19

3.2.First derivative spectrophotometric method:

Figure (7); Absorption spectra of Norfloxacin using first derivative

spectrophotometric method (6lμg/ml)

3.2.1.Standard curve of first derevative method

The obtained absorbance values at 277nm are shown in table (7)

Table (7); Absorbance values for the prepared concentrations at lambda max

277nm:

concentration(μg/ml) Absorbance

2 0.011

4 0.022

6 0.034

8 0.046

10 0.057

12 0.069

R=0.9999 a= -8.66*E-4 b= 5.814

20

Figure (8) Standard curve of NOR for first derivative method

3.2.2. Assay of Norfloxacin tablets using first derivative

spectrophotometric method:

The % content of tablets were determined using calibration curve .results obtained

are shown in tables(8-11)

3.2.2.1.Trizolin

Table (8); Calculation of percentage content of Norfloxacin in

Trizolin using first derivative spectrophotometric method.

Trizolin

Weight

taken(gm)

Absorbance

Theoretical

concentration(μg/ml)

practical

concn

(μg/ml)

Percent-

age

content

sample

1 0.1527

0.038

6.4586 6.6846 103.50%

sample

2 0.1507

0.038

6.3552 6.6846 105.00%

sample

3 0.1561

0.039

6.6022 6.8566 103.70%

y = 0.0058x - 0.0009R² = 0.9998

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0 2 4 6 8 10 12 14

Series1

Linear (Series1)

21

3.2.2.2.ANQUIN

Table (9); Calculation of percentage content of Norfloxacin in Anquin using

first derivative spectrophotometric mthod

Anqui

n

weight

acually

taken(gm

)

Absorbanc

e theoretical

concentration(μg/ml

)

Practic-

al

concn

(μg/ml)

Percentag

-e content

sample

1 0.1504

0.035

6.069 6.1687 101.60%

sample

2 0.1668

0.039

6.732 6.8566 101.85%

sample

3 0.1629

0.038

6.574 6.6846 101.68%

3.2.2.3.YESNORF

Table (10); Calculation of percentage content of Norfloxacin in Yesnorf using

first derivative spectrophotometry

Yesnorf

weight acually taken(gm)

Absorbance theoretical concentration(μg/ml)

practical concn (μg/ml)

percentage content

sample 1 0.1685

0.036 5.956122 6.340704 106.40%

sample 2 0.1691

0.036 5.97732 6.340704 106.00%

sample 3 0.1679

0.036 5.9349 6.340704 106.80%

22

3.2.2.4.NOROZAL

Table (11); Calculation of percentage content of Norfloxacin in Norozal using

first derivative spectrophotometric method

norozal

weight

acually

taken

(gm)

Absorbance

theoretical

concentration

(μg/ml)

practical

concn

(μg/ml)

percentage

content

sample

1 0.1716

0.034

6.0144 5.9445 98.80%

sample

2 0.1712

0.034

6.0009 5.9445 99.00%

sample

3 0.171

0.034

5.9935 5.9445 99.17%

23

3.3.statistical analysis

Table (12): ANOVA table for Trizolin and Anquin..for tables (3, 4,8,9))

Anova: Single Factor for Trizolin

SUMMARY

Groups No. of

determinant sum of

% Average Variance

zero order 3 292.8 97.6 0.03

first order 3 312.2 104.0667 0.663333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 62.72667 1 62.72667 180.9423 0.000177 7.708647

Within Groups 1.386667 4 0.346667

Total 64.11333 5

Anova: Single Factor for Anquin

SUMMARY

Groups No. of

determinant sum of

% Average Variance

zero order 3 304.29 101.43 0.4447

first order 3 305.13 101.71 0.0163

ANOVA


Between Groups 0.1176 1 0.1176 0.510195 0.514522 7.708647

Within Groups 0.922 4 0.2305

Total 1.0396 5

24

Table (13): ANOVA table for Yesnorf and Norozal..for tables

(5,6,10,11)

Anova: Single Factor for Yesnorf

SUMMARY

Groups Count Sum Average Variance

zero order 3 294.6 98.2 0.43

first order 3 319.2 106.4 0.16

ANOVA


Between Groups 100.86 1 100.86 341.8983 5.03E-

05 7.708647


Total 102.04 5

Anova: Single Factor for Norozal

SUMMARY

Groups Count Sum Average Variance

zero order 3 279.09 93.03 0.0027

first order 3 296.97 98.99 0.0343

ANOVA


Between Groups 53.2824 1 53.2824 2880.13 7.22E-

07 7.708647


Total 53.3564 5

Ss= sum of squares df= degree of freedom ms=mean square p-value=propability Fcrit= F critical

25

3.4. Results of Physical tests:

Table (14): Results of physical tests.

* n =10 ** n = 20

Brand

weight variation

mean± SD(gm)

**

diameter(mm) *

mean±SD

thickness(mm)*

mean± SD

hardness(kg/cm2)*

mean±SD friability(%)

disintegration

time(minutes)

Trizolin 0.57265±0.005 12.032±0.054 4.978±0.0697 10.6±0.063 0 8

Anquin 0.5902±0.008 13.855±0.064 4.304±0.078 10±0.0267 0 4

Yesnorf 0.6882±0.007 12.616±0.0457 5.813±0.102 8.53±0.080 0.029 3.58

Norozal 0.6964±0.0103 17.961±0.0023 5.298±0.053 11.5±0.035 0.0428 15.27

26

Chapter Four

DISSCUSION ,CONCLUSION and

RECOMMENDATIONS

26

4.1.Discussion:

Pharmaceutical Analysis may be defined as the application of analytical

procedures used to determine the purity, safety and quality of drugs and chemicals.

In recent years, the number of counterfeit drugs has increased dramatically,

including not only "lifestyle" products but also vital medicines. Besides the threat

to public health, the financial and reputational damage to pharmaceutical

companies is substantial. The lack of robust information on the prevalence of fake

drugs is an obstacle in the fight against drug counterfeiting. It is generally accepted

that approximately 10% of drugs worldwide could be counterfeit, but it is also well

known that this number covers very different situations depending on the country,

the places where the drugs are purchased, and the definition of what constitutes a

counterfeit drug. The chemical analysis of drugs suspected to be fake is a crucial

step as counterfeiters are becoming increasingly sophisticated, rendering visual

inspection insufficient to distinguish the genuine products from the counterfeit

ones. Analytical methods employed to control the quality of drug formulations, .a

broad panel of techniques, ranging from simple and cheap in-field ones include:

spectroscopic methods UV/Visible spectrophotometry(zero ,first,second…etc

orders) to more advanced laboratory methods (mass spectrometry, nuclear

magnetic resonance, and vibrational spectroscopies) through chromatographic

methods, which remain the most widely used. (32).

For all these reasons mentioned above it was deemed important to carry post

marketing surveillance for important class of antibiotics ( Flouroquinolones)..the

target drug in these work brands of Norfloxacin.

The results of the conducted post marketing surveillance of Norfloxacin in sudan

are summarized in tables (2-11) and figures(5-8).

As shown in the above mentioned tables and figures , all brands were complying

with specifications of the BP (95-105%) and/or USP(90-110%).

27

Anquin shows the best percentage content with mean percentage = 101.43% ±

0.44(when assayed by zero order UV.Visible spectrophotometry) and 101.71% ±

0.02(when assayed by 1st derivative spectrophotometry).

The results for the other brands were as follow, Trizolin

97.6±0.03 and 103. 8%±1.08, Yesnorf 98.2%±0.43 and 106.4%±0.16 , Norozal

93.03%±0.003 and 98.99%±0.03 for zero order and 1st derivative

spectrophotometry respectively .

Statistical analysis of the results obtained for the four brands (table 12 and 13). The

statistical analysis for Trizolin using Anova single factor revealed that F calculated

=180.94 which more than F tabulated= 7.7 and P-value =0.00017 which less than

0.05 at 95% confidance interval , thus there is significant difference in the results.

For Anquin F calculated = 0.510195 less than Ftabulated= 7.7 and P-value= 0.5145

more than 0.05 , thus there is no significant difference in the results.

For Yesnorf F calculated=341.8 more than F tabulated= 7.7 and P-value = 5.03E-

05 less than 0.05 , thus there is significant difference between results.

For Norozal F calculated = 2880.13 more than F tabulated 7.7 and P value =7.22E-

07 less than 0.05 and hence there is significant difference between results.

These significant difference in % content may be due to that zero order and first

order assay are not carried out in the same day with same sample ,but they done in

different days ,also may be due to inconsistency of the scale in first derivative

spectrophotometry.

The results of the physical test carried( thickness, hardness, friability, weight

variation, uniformity of content and disintegration time) are summarized in table

(14).

Variation between tablet with respect to dose and weight must be reduced to a

minimum through good manufacturing practice (GMP) .Uniformity of weight is a

28

parameter which ensures consistency of dosage units during compression and in

finished product.(33)

As shown in the mentioned above table the weight lies between 0.565 mg -0.581

mg (Trizolin) , 0.574 mg - .612 mg (Anquin) , 0.679 mg – 0.707 mg (Yesnorf) and

0.679 mg – 0.712 mg (Norozal) .However, all the tablets passed weight variation

test as the % weight variation was within the Pharmacopoeial limits. The weight of

all the tablets was found to be comply with the BP and USP standards (uniform,

with low standard deviation values indicating efficient mixing of drug,

superdisintegrants and excipients) the percentage deviatiorn not exceed ± 5%

(tablet weight more than 250 mg in BP and more than 324 mg in USP).(26,34)

Diameters are also important feature in quality testing of tablets. based on the

results obtained and calculated, the diameter lies in the range of 11.97 mm and

12.14 mm for trizolin. 13.81 mm and 14.03 mm for anquin.17.85 mm and 18.01

for norozal.12.59 mm and 12.71 for yesnorf. The percentage deviation from

average not exceeded ± 3%.the four brands comply with the standard that if tablet

diameter more thn 12mm ,the percentage deviation should not exeed ± 3%.(33)

Tablets thickness should be controlled to ensure uniformity in tablets appearance

and fitting into the containers for packaging process. for Anquin 4.3±0.07 ,

Trizolin 4.97±0.06 , Yesnorf 5.8±0.1 and Norozal 5.29±0.05. (33) all brands of

NOR have uniform thickness with low SD .

Hardness is also one of the important parameters measured. Averagely, all the

tablets are strong enough to withstand the pressure applied. The hardness of tablets

is important to withstand the mechanical shocks during the tablets manufacturing,

packaging and transport. If the tablets are too hard, it may not disintegrate in

required period of time and if it is too soft, it may break during the handling of

manufacturing or packaging process.

29

The brand Yesnorf required the least pressure before fracture 8.53±0.08 while

brand Norozal required the most pressure before fracture 11.5±0.0354. A force of

about 4 kg is the minimum requirement for a satisfactory tablet (33)

Friability test is used to determine the physical strength or tablets resistance to

abrasion.All four brands of NOR passed the test with no cracked, cleaved or

broken tablet ,and the weight loss was less than 1%w/w (33)

Disintegration test was done to determine whether tablets disintegrate within the

prescribed time and conditions when placed in a liquid medium.

Disintegration could be directly related to dissolution and subsequent

bioavailability of a drug. A drug incorporated in a tablet is released rapidly as the

tablet disintergrates. This is a crucial step for immediate release dosage forms

because the rate of disintegration affects the dissolution and subsequently the

therapeutic efficacy of the medicine. All the brands complied with the compendial

specifications for disintegration. The BP specification is that uncoated tablets

should disintegrate within 15 min and film coated in 30 min while USP specifies

that uncoated and film coated tablets should disintegrate within 30 min. and if there

is a residue remaining it should be soft mass having no palpably firm core) and

satisfying the criteria of fast dissolving tablets.(26,34)

The four brands of NOR passed the test with the least time for disintegration

showed by Norozal (4 minutes) and longest time for Anquin with (15.2 minutes) .8

minutes for Trizolin and 3.58 minutes for Yesnorf.

30

4.2.Conclusion

This study proved that the four brands (Trizolin , Anquin ,Yesnorf and Norozal )

comply with the pharmacopieal standards in percentage content of APIs (BP and/or

USP)and physical tests including weight uniformity, hardness , friability , diameter

, thickness and disintegration time .

The presented quality control methods (zero order and 1st derivative UV.Visible

spectrophotometry) proved to be useful in monitoring the production consistency

of batch-to-batch product release of each brand of Norfloxacin and in comparing

the quality characteristics of different brands marketed.

4.3.Recommendations:

It is very important to do post marketing surveillance of drugs especially for those

drugs used in chronic diseases and antibiotic ,this is because of the universally

reported counterfeit and substandard drugs.

31

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34

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