prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1260/2/1163S.pdf · ii Certificate This is to certify that Mr. ABDUL RAUF KHASKHELI has carried out his research work on the

Ph. D Thesis

QUANTIFICATION OF ASPIRIN, BRUFEN, DICLOFEN AND PARACETAMOL IN HUMAN BODY FLUIDS BY

VARIOUS ANALYTICAL TECHNIQUES

A THESIS SUBMITTED TOWARDS THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF DOCTOR OF PHILOSOPHY

IN ANALYTICAL CHEMISTRY

ABDUL RAUF KHASKHELI

National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro - PAKISTAN

2011

i

To My Affectionate Supervisors, Beloved Parents and Friends

who’s Prayers, Encouragement and Cooperation Enabled Me for

this Achievement

DEDICATED

ii

Certificate This is to certify that Mr. ABDUL RAUF KHASKHELI has carried out his research

work on the topic “QUANTIFICATION OF ASPIRIN, BRUFEN, DICLOFEN AND

PARACETAMOL IN HUMAN BODY FLUIDS BY VARIOUS ANALYTICAL TECHNIQUES”

under our supervision at the laboratories of National Centre of Excellence in Analytical

chemistry, University of Sindh, Jamshoro, Pakistan and UNESCO laboratory of

Environmental Electrochemistry, Department of Analytical Chemistry, Charles

University, Prague, Czech Republic under the abroad Ph.D split program (IRSIP) of

Higher Education Commission, Pakistan. The work reported in this thesis is original and

distinct. His dissertation is worthy of presentation to the University of Sindh for the award

of degree of Doctor of Philosophy in Analytical Chemistry.

Dr. Sirajuddin Associate Professor Supervisor National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan

Dr. Syed Tufail Hussain Sherazi Associate Professor Co-Supervisor National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan

iii

ACKNOWLEDGEMENTS

All praises be to Almighty Allah (The Most Merciful, The Most Gracious and The Most

Compassionate) Who is the entire and only source of every knowledge, Who guides me in

the obscurity and helps me in difficulties, and His Prophet Hazrat Mohammad Mustafa

(Salallah-o-Alaihe Wasallim) whose teachings provide the spirit of learning the hidden and

unconcealed facts of nature.

I am highly grateful to my supervisor Dr. Sirajuddin (Associate Professor) and Co-

supervisor Dr. Syed Tufail Hussain Sherazi (Associate Professor) for their genius and

superb ideas, support and untiring efforts for my appreciation during my studies that enabled

me to complete my research work successfully and truly made my research very fruitful and

of high quality.

I would like to extend my gratitude to Prof. Dr. Muhammad Iqbal Bhanger Director,

NCEAC University of Sindh Jamshoro, Pakistan for his sincere advices and facilities to

carry out this research work successfully.

I am particularly indebted to my teachers, Prof. Dr. Tasneem Gul Kazi, Dr. Shahabuddin

Memon, Dr. Najma Memon, Dr. Amber Rehana Solangi, Dr. Farah Naz Talpur, Dr.

Aamna Baloch, Dr. Hassan Imran Afridi and Engr. Mairaj Ahmed Noorani who always

offered their professional skills whenever needed. I am grateful for their encouraging

attitude in the solution of problems faced during the course of my research work.

I am extremely grateful to Prof. Dr. Jiri Barek and Dr. Jan Fischer for their kind

supervision, excellent research assistance in completing a part of my Ph.D research work

and in up grading my electrochemical approach during the course of my study in Czech

Republic, at the UNESCO Laboratory of Environmental Electrochemistry, Department

of Analytical Chemistry, Charles University Prague, under IRSIP by the HEC, Pakistan.

I am also very much grateful to Higher Education Commission of Pakistan, for awarding

me the six months scholarship for research training in Czech Republic.

I would extend my sincere and heartily thanks and appreciation to my friends, fellows and

colleagues Dr. Sarfaraz Ahmed Mahesar, Dr. Afzal Shah, Dr. Abdul Niaz, Dr.

Munawar Saeed Qureshi, Sajidullah Abbasi, Kamran Ahmed Abro, Dr. Ghulam

Abbass Kandhro, Ateeq-ur-Rehman Memon, Nazar Hussain Kalwar,

iv

M. Younis Talpur, Imam Bakhsh Solangi, Jameel Ahmed Baig, Zulfiqar Ali Tagar,

Aijaz Ahmed Bhutto, Yawar Latif and Muhammad Ali.

They always encouraged and cooperated with me and made every possible effort to provide

the useful contribution for the improvement of this study.

I would like to place on record sincere thanks to my teachers as my parents especially, Mr.

Abdul Razaque Abro, Prof. Dr. M. Usman Memon, Prof. Dr. Ubedullah M. Abbasi,

Prof. Dr. Mehboob Ali Rind, Mr. Abdul Hakeem Memon and Mr. Ubed-ur- Rehman

Mughal and Mr. Imran-ul-Haq for their assistance and constructive attitude. I am highly

thankful to Dr. Aftab Ahmed Kandhro and Munawar Ali Soomro for their dear and

useful thoughts, and support for editing and printing during this dissertation.

It seems an obligation upon me to record the word of encouragement for my family and

simple words of thankfulness would not cover the genuine affection, absolute support,

remarkable encouragement and profound prayers of my father Muhammad Bakhsh

Khaskheli, brother Abdul Salam Khaskheli and all family members. I am thankful to all of

them, without their co-operation, it would have been impossible for me to devote so much of

time to this study.

I would also like to thank administrative and supportive staff at National Centre of

Excellence in Analytical Chemistry Pir Ziauddin, Nasrullah Kalhoro, Akhtar Vighio, M.

Mudasir Arain, Pir Siraj, Shafiq A. Bhutto, Jawad Ahmed and Junaid Talpur

ABDUL RAUF KHASKHELI

v

ABSTRACT

A very fast, economical and simple direct spectrophotometric method was investigated for

Paracetamol (PC) determination in aqueous medium without using any reagent. The method

is based on the photo activation of the analyte at 243 nm after dissolution in water. The

change in structure of PC after addition of water was studied by comparing the

corresponding FTIR spectra. Optimization studies were conducted by using a 5 µg ml-1

standard solution of the analyte. Various parameters studied include, time for stability and

measurement of spectra, effect of HCl, NaOH, CH3COOH and NH3 for change in

absorbance and shift in spectra, interference by some analgesic drugs and some polar

solvents and temperature effect. After optimization, Beer’s law was obeyed in the range of

0.3–20 µg ml-1 PC solution with a correlation coefficient of 0.9999 and detection limit of 0.1

µg ml-1 for 3/1 S/N ratio. The newly developed method was successfully applied for PC

determination in some locally available tablets and urinary samples. The proposed method is

very useful for quick analysis of various types of solid and liquid samples containing PC.

Another spectrophotometric work describes a simple, sensitive, rapid and economical

analytical procedure for direct spectrophotometric evaluation of Diclofenac Sodium (DS)

using aqueous medium without using a chemical reagent. Parameters like time, temperature,

acidic and basic conditions and interference by analgesic drugs was studied for a 5µg ml-1

solution of DS at 276 nm. Under optimized parameters, a linear working range of 0.1–30 g

ml-1 with regression coefficient of 0.9998 and lower detection limit of 0.01 g ml-1 was

obtained. The method was applied for DS contents in tablets, serum and urine samples.

vi

A new method was developed for the determination of paracetamol by differential pulse

voltammetry (DPV) at carbon film electrode (CFE). The experimental parameters, such as

pH of Britton-Robinson buffer and potentials for regeneration of electrode surface were

optimized. Under optimized conditions in Britton-Robinson buffer pH 4.0 a linear

calibration curve was obtained in the range 0.02–100 μmol L-1. The limit of determination

was 0.034 μmol L-1 which showed high sensitivity of developed method. The method was

applied for the quantitative determination of Paracetamol in pharmaceutical formulations as

well as urine samples.

A rapid, reliable and economical analytical procedure for the estimation of ibuprofen in

pharmaceutical formulations and human urine samples was developed using transmission

Fourier Transform Infrared (FT-IR) spectroscopy. For the determination of ibuprofen, a KBr

window with 500 µm spacer was used to acquire the FT-IR spectra of standards,

pharmaceuticals as well as urine samples. The Partial Least Squares (PLS) calibration model

was developed based on carbonyl region (C=O) from 1807-1461 cm−1 in the range from 10-

1000 ppm. The developed model was checked by cross-validation steps to diminish standard

error of the models, such as root mean square error of calibration (RMSEC), root mean

square error of cross validation (RMSECV) and root mean square error of prediction

(RMSEP). The good coefficient of determination (R2) was achieved 0.999 with minimum

standard errors RMSEC, RMSECV and RMSEP 1.89, 1.956 and 1.38, respectively.

The other method was based on indirect determination of acetylsalicylic acid (aspirin)

utilizing differential pulse voltammetry at carbon film electrode as working electrode. The

theory of indirect determination of ASA is based on the hydrolysis of aspirin in salicylic

vii

acid (SA) for detection. Moreover, we optimized conditions such as pH of Britton-Robinson

buffer, potentials for regeneration and activation of electrode surface, amplitude and scan

rate. Under optimized conditions in Britton-Robinson buffer pH 2.0 a linear calibration

curve was obtained in the range 0.2 – 100 µmol L-1. The limit of determination was 0.15

µmol L-1 which showed high sensitivity of developed method. The method for indirect

determination of ASA was thus developed for the quantification of pharmaceutical

formulations as well in human urine model samples.

viii

List of Contents Dedication i Certificate ii Acknowledgement iii Abstract v List of Contents viii List of Tables xii List of Figures xiv Abbreviations xviii

Chapter - One INTRODUCTION 1 – 14

1.1. Analgesics / NSAIDs -An overview 1 1.1.1. Mechanism of Action (NSAIDs) 2 1.2. Paracetamol 3 1.2.1. Pharmacopoeias 3 1.2.2. Uses and Administration 4

1.2.3. Adverse Effects 4

1.2.4. Overdosage and its Treatment 5 1.2.5. Precautions 5 1.3. Aspirin 6 1.3.1. Pharmacopoeias 6 1.3.2. Uses and Administration 7 1.3.3. Adverse Effects 8 1.3.4. Overdosage and its Treatment 9 1.3.5. Precautions 9 1.4. Diclofen 10 1.4.1. Pharmacopoeias 10 1.4.2. Uses and Administration 10 1.4.3. Adverse Effects 11 1.4.4. Precautions 12 1.5. Brufen 12 1.5.1. Pharmacopoeias 12 1.5.2. Uses and Administration 12 1.5.3. Adverse Effects 13 1.5.4. Overdosage and its Treatment 14

viii

ix

Chapter - Two

LITERATURE REVIEW

15 – 42

2.1. Analytical Techniques for Assessment of NSAIDs 15 2.2. Electrochemical Techniques 16 2.3. Spectroscopic Methods 21 2.4. Chromatographic Methods 25 2.5. Other Techniques 27 2.6. Quantification of Paracetamol in Biological Samples and

Pharmaceuticals 27

2.7. Quantification of Aspirin in Pharmaceutical and Biological Samples

31

2.8. Quantification of Diclofenac Sodium in Pharmaceutical and Biological Samples

35

2.9. Quantification of Ibuprofen in Pharmaceutical and Biological Samples

39

Chapter - Three EXPERIMENTAL 43 – 53

3.1. Material and Methods for Paracetamol Using UV-Visible Spectrophotometry

43

3.1.1. Reagents and Chemicals 43 3.1.2. Instruments and Apparatus 43 3.1.3. Procedure for Determining PC 44 3.1.4. Analysis of PC in Urine Samples 44 3.2. Material and Methods for Diclofenac Sodium Using UV-

Visible Spectrophotometry

45

3.2.1. Apparatus 45 3.2.2. Washing of Glassware 45 3.2.3. Reagents and Solutions 45 3.2.4. Procedure for Determining DS in Tablets 46 3.2.5. Procedure for DS in Serum and Urine Samples 46 3.3. Material and Methods for the Determination of

Paracetamol Using Differential Pulse Voltammetry 47

3.3.1. Reagents and Solutions 47 3.3.2. Apparatus 47 3.3.3. Procedures 48 3.4. Material and Methods for the Analysis of Ibuprofen

Using FT-IR Spectroscopy

49

3.4.1. Reagents and Samples 49 3.4.2. FT-IR Spectral Measurements 49

x

3.4.3. FT-IR Calibrations 50 3.4.4. Sample Preparation Procedure 50 3.4.5. Collection and Preparation of Urine Samples 50 3.5. Material and Methods for Investigation of Aspirin Using

Voltammetry51

3.5.1. Reagents 51 3.5.2. Apparatus 51 3.5.3. Voltammetric Procedure 52 3.5.4. Indirect Determination of ASA 52 3.5.5. Indirect Determination of ASA in Pharmaceutical

Drugs and Urine Samples by DPV at CFE 52

Chapter - Four Result and Discussions 54 - 108

4.1. Simpler Spectrophotometric Assay of PC in Tablets and Urine Samples 54

4.1.1. Effect of Water Addition to PC (FTIR studies)

54

4.1.2. Optimization of Time for Measurement and Stability of Analytical Signal

55

4.1.3. Effect of Temperature 56 4.1.4. Effect of Polar Solvents 57 4.1.5. Interference by Various Analgesic Drugs 58 4.1.6. Effect of Acidic and Basic Solutions 60 4.1.7. Calibration Range 61 4.1.8. Analysis of Tablets 62 4.1.9. Analysis of Urine Samples 63 4.2. Simpler and Faster Spectrophotometric Determination of

Diclofenac Sodium (DS) in Tablets, Serum and Urine Samples

65

4.2.1. Influence of Time 65 4.2.2. Effect of Temperature 66 4.2.3. Interference by Various Analgesic Drugs 67 4.2.4. Effect of Acidic and Alkaline Conditions 68 4.2.5. Calibration Plot 71 4.2.6. Comparison with Other Reported Spectroscopic

Methods 71

4.2.7. Analysis of Tablets 72 4.2.8. Analysis of Urine and Serum Samples 74 4.3. Differential Pulse Voltammetric Determination of PC in

Tablet and Urine Samples at Carbon Film Electrode

77

4.3.1. Influence of pH on PC at DC and DP Voltammetry 77

xi

4.3.2. Optimization of Parameters and Calibration Curve 78 4.3.3. Analysis of Pharmaceutical Drugs 82 4.3.4. Analysis of Urine Samples 84 4.4. Quantification of Ibuprofen in Pharmaceuticals and

Biological Samples by FTIR Transmission Spectroscopy. 86

4.4.1. Analysis of Pharmaceutical Samples

86

4.4.2. Analysis of Urine Samples 89 4.4.3. Limits of Detection and Quantitation 92 4.5. Differential Pulse Voltammetric Determination of

Salicylic acid and Acetylsalicylic acid in Tablet and Urine Samples at Carbon Film Electrode

93

4.5.1. Influence of pH on Salicylic Acid in DC and DP

Voltammetry

93

4.5.2. Reproducibility of Salicylic Acid 94 4.5.3. Linear Calibration Curves of Salicylic Acid 95 4.5.4. Hydrolysis of Acetylsalicylic acid 97 4.5.5. Linear Calibration Curves of Hydrolyzed

Acetylsalicylic Acid at CFE 98

4.5.6. Determination of Salicylic Acid in Different Drugs 100 4.5.7. Determination of Acetylsalicylic Acid from Different

Drugs 101

4.5.8. Determination of Salicylic Acid in Urine Samples at CFE

103

4.5.9. Determination of Acetylsalicylic Acid in Urine at CFE

104

4.5.10. Interference Study 106

4.6. Conclusion 107

References 109-132

xii

LIST OF TABLES

Table. 4.1.1. Effect of different polar solvents on absorbance of PC.

Table. 4.1.2. % interference by various analgesics in different ratios on 5 µg ml-1 PC 59

Table. 4.1.3. Effect of strong and weak acids and bases on PC determination 60

Table. 4.1.4. Determination of PC in tables of various companies by proposed method 63

Table. 4.1.5. Comparative analyses of PC in urine samples. 63 Table. 4.2.1. % interference by various analgesics in different ratios on

5 µg ml-1 DS 67

Table. 4.2.2. Effect of strong and weak acids and bases on DFS determination

70

Table. 4.2.3. Comparison of current method with other spectroscopic methods for determination of DS

72

Table. 4.2.4. Determination of DS in tablets of various companies by proposed method

73

Table. 4.2.5. Concentration of determined DS and its relation with ingested DS by oral administration

76

Table 4.3.1. The influence of pH on DCV and DPV of Paracetamol (c = 100 µmol L-1)

78

Table. 4.3.2. Parameters of the calibration straight lines for the determination of Paracetamol in Britton-Robinson buffer pH 4.0 using DPV at CFE with regeneration potential Ereg1 = –400 mV, and Ereg2 = 1300 mV.

79

Table. 4.3.3. The amount of Paracetamol determined by DPV in tablets of commercial drugs with declare contents 500 mg of Paracetamol.

83

Table. 4.3.4. Parameters of the calibration straight lines for the determination of model samples of Paracetamol in urine, media of Britton-Robinson buffer pH 4.0 using DPV at CFE with regeneration potentials Ereg1 = –400 mV and Ereg2 = 1300 mV.

84

Table. 4.4.1. Results for the ibuprofen concentration found in the tablet samples

87

Table. 4.4.2. Recovery result of ibuprofen from tablet samples after spiking with known concentrations of standards

89

Table. 4.4.3. Recovery results of ibuprofen from urine samples after spiking with known concentrations of standard

90

Table 4.4.4. Recovery results of ibuprofen in urine samples after spiking with known concentrations of standard

91

Table. 4.5.1. The influence of pH on DCV and DPV of Salicylic Acid (c = 100 µmol L-1 )

94

xiii

Table 4.5.2. Parameters of the calibration straight lines for the determination of Salicylic Acid in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

97

Table 4.5.3. Parameters of the calibration straight lines for the determination of hydrolyzed Acetylsalicylic Acid in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

100

Table 4.5.4. The amount of Salicylic Acid determined by DPV in tablets

101

Table 4.5.5. The amount of hydrolyzed Acetylsalicylic Acid determined by DPV in tablets

102

Table 4.5.6. Parameters of the calibration straight lines for the determination of salicylic acid in 0.1 ml urine samples in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

104

Table 4.5.7. Parameters of the calibration straight lines for the determination of hydrolyzed Acetylsalicylic acid in 0.1 ml urine sample in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

106

xiv

LIST OF FIGURES

Figure. 1.1. Structural formula of Paracetamol (PC) 3 Figure. 1.2. Structural formula of Aspirin (ASA) 6 Figure. 1.3. Structural formula of Diclofenac Sodium (DS) 10 Figure. 1.4. Structural formula of Ibuprofen (IBP) 12 Figure. 4.1.1. FTIR spectra of (A), pure PC (B), aqueous PC paste and

(C) aqueous solution of 5 µgml-1 PC. 55

Figure. 4.1.2. Dependence of absorbance of PC on time 56 Figure. 4.1.3. Effect of temperature on absorbance of PC 57 Figure. 4.1.4. Shift of wavelength with pH: (A) 3.3 and (B) 12. 3 for

5µgml-1 PC solution. 61

Figure. 4.1.5. Calibration range of absorbance vs. concentration for PC from 0.3 to 20 µg ml-1

. 61

Figure. 4.1.6. A 1000 times diluted sample of urine (a) with PC and (b) without PC.

64

Figure. 4.2.1. Time effect on absorbance of Diclofenac Sodium 65 Figure. 4.2.2. Temperature effect on UV absorbance of 5 µg ml-1 DS

solution. 66

Figure. 4.2.3. UV spectra of DS at acidic pH, 3.56 (lower) and basic pH, 11.68 (higher).

69

Figure. 4.2.4. Calibration plot of absorbance verses concentration for DS solutions from below to above as 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 and 30 µg ml-1.

71

Figure. 4.2.5. Representative UV-spectrum of expected 5 µg ml-1 DS in (Diclofen) tablet.

73

Figure. 4.2.6. UV spectra showing blank (black), DS containing (blue) and DS spiked (red) urine.

74

Figure. 4.2.7. UV spectra showing blank (black), DS containing (blue) and DS spiked (red) serum.

75

Figure. 4.3.1. DC voltammograms (A) and DP voltammograms (B) of Paracetamol (c = 100 μmol-1.l) at CFE in Britton-Robinson buffer pH 2 to 12 (numbers above curves correspond to given pH) without electrode regeneration. Inset is corresponding dependence of peak potential on the pH.

77

Figure. 4.3.2. Repetitive measurements of 100 µmol L-1 Paracetamol using DPV at CFE in Britton-Robinson buffer pH 4.0 with regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV Inset is corresponding dependence of peak current on the number of scans.

79

Figure. 4.3.3. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 µmol L-1 . Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

80

xv


81

Figure. 4.3.2. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(Paracetamol): 0 (1), 0.2 (2), 0,4 (3), 0,6 (4), 0,8 (5), 1 µmol L-1(6). Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300

mV. Inset is corresponding calibration dependence.

81

Figure. 4.3.6. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 0,02 (3) 0,04, (4) 0,06, (5) 0,08, (6) 0,1 µmol L-1. Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

82

Figure. 4.3.3. Differential pulse voltammograms of Paracetamol 10 µmol L-1 with Ascorbic acid from 10 to 100 µmol L-1 (concentrations are written above curves in plot) at CFE in Britton-Robinson buffer pH 4.0, regeneration potential Ereg1 = –400 mV, and Ereg2 = 1300 mV. Inset is corresponding dependence of peak current of Paracetamol on concentration of Ascorbic acid.

83

Figure. 4.3.4. Differential pulse voltammograms of 0.1 ml urine with model sample of Paracetamol (2 – 10 µmol L-1 (A), and 20 – 100 µmol L-1 (B), concentration is written next to curves in plots) sample at CFE in Britton-Robinson buffer pH 4.0, regeneration potentials Ereg1 = –400 mV, and Ereg2 = 1300 mV. Inset is corresponding calibration dependence.

84

Figure. 4.4.1. Calibration plot in the range of 10-100 ppm for pharmaceutical samples

87

Figure. 4.4.2. Pharmaceutical Tablet sample and spikes of 30 ppm, 50 ppm, 70 ppm ibuprofen

88

Figure. 4.4.3. Blank urine and 3 spikes of 10, 20, 30 ppm ibuprofen 90 Figure. 4.4.5. Group Spectra of Ibuprofen Standards 92 Figure. 4.5.1. A and B. DC & DP voltammograms of Salicylic Acid (c = 100 µmol L-1 ) at CFE in Britton-Robinson buffer pH

2 to 12 (numbers in above curves correspond to given pH). Inset is corresponding dependence of peak potential on the pH

93

Figure. 4.5.2. Measurements of 100 µmol L-1 Salicylic Acid using DPV at CFE in Britton-Robinson Buffer pH 2.0 with activation potential (1) 2000,(2) 1500 and (3) 2200 mV

95

xvi

Figure. 4.5.3. Differential pulse voltammograms of Salicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 8,0 (6) 100 µmol L-1 .

Activation of potential=2200mV for 120 sec Inset is corresponding calibration dependence

96

Figure. 4.5.4. Differential pulse voltammograms of Salicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L-1. Activation of potential=2200mV for 120 sec. Inset is corresponding calibration dependence

96

Figure. 4.5.5. Differential pulse voltammograms of Salicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 µmol L-1. Activation of potential=2200mV for 120 sec. Inset is corresponding calibration dependence

97

Figure.4.5.6. Differential pulse voltammograms of 10 µmol L-1 hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2.0, (1) Electrolyte, (2) 10 µmol L-1 without hydrolysis (3) 10 µmol L-1 Salicylic Acid (4) 10 µmol L-1 after hydrolysis Acetylsalicylic acid. Activation potential=2200mV for 120 sec

98

Figure. 4.5.7. DP voltammograms of hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 µmol L-1. Activation potential=2200mV for 120 sec. Inset is corresponding calibration dependence

99

Figure. 4.5.8. DP voltammograms of hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2. c (ASA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L-1. Activation potential=2200mV for 120 sec. Inset is corresponding calibration dependence

99

Figure. 4.5.9. DP voltammograms of hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 µmol L-1. Activation potential=2200mV for 120 sec. Inset is corresponding calibration dependence

100

Figure.4.5.10.Differential pulse voltammograms of 4 µmol L-1 Duofilm sample with spikes of salicylic acid standard at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) electrolyte, (2) expected 4 μmol L-1 salicylic acid in sample of Duofilm (3) 6 μmol L-1 spike, (4) 8 μmol L-1 spike and (5) 10 μmol L-1 spike. Activation potential 2200 mV

101

Figure. 4.5.11. Differential pulse voltammograms of 4 µmol L-1 Aspirin sample with spikes of hydrolyzed acetylsalicylic acid standard at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) electrolyte, (2) expected 4 μmol L-1 salicylic acid in sample of Aspirin (3) 6 μmol L-1 spike, (4) 8 μmol L-1

spike and (5) 10 μmol L-1 spike. Activation potential 2200 mV

102

xvii

Figure. 4.5.12. Differential pulse voltammograms of 0.1 ml urine sample at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 µmol L-1 . Activation potential 2200 mV. Inset is corresponding calibration

dependence

103

Figure. 4.5.13. Differential pulse voltammograms of 0.1 ml urine sample at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L-1. Activation potential 2200 mV. Inset is corresponding calibration dependence

103

Figure. 4.5.14. Differential pulse voltammograms of 0.1 ml urine sample at CFE in Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 µmol L-1 . Activation potential 2200 mV. Inset is corresponding calibration dependence

105


105

Figure.4.5.16.Differential pulse voltammograms of hydrolyzed acetylsalicylic acid 5 μmolL-1 with ascorbic acid 5-100 µmol L-1 at CFE in Britton-Robinson buffer pH 2.0. Activation potential 2200 mV

106

xviii

List of Abbreviations AdSV Adsorptive Stripping Voltammetry AgA–CFE Silver Amalgam Carbon Film Electrode

AgA–PE Silver Amalgam Paste Electrode AgE Silver Electrode AOAC Association of Official Analytical Chemists ASA Acetylsalicylic Acid ATR Attenuated Total Reflection AuE Gold Electrode CFE Carbon Film Electrode CNS Central Nerves System COX Cyclo-Oxygenase CPE Carbon Paste Electrode CV Cyclic Voltammetry DDP Differential Pulse Polarography DME Dropping Mercury Electrode DPV Differential Pulse Voltammetry DS Diclofenac Sodium E1/2 Half Wave Potential FIA Flow Injection Analysis FT-IR Fourier Transform Infrared Spectroscopy FT-NIR Fourier Transform Near-Infrared GC Gas Chromatography GC– MS Gas Chromatography Mass Spectroscopy GCE Glassy Carbon Electrode GGE Glassy Graphite Electrode HEC Higher Education Commission HMDE Hanging Mercury Drop Electrode HPLC High Performance Liquid Chromatography HT-GLC High-Temperature Gas–Liquid Chromatography Hz Hertz IBP Ibuprofen LOD Limit of Detection LOQ Limit of Quantification LSV Linear Sweep Voltammetry mg/kg Milligram per Kilogram mg/kg /b.w/day Milligram per Kilogram per Body Weight per Day mg/L Milligram per Liter mm Millimeter mol.L-1 Mole per liter mV Milli volt mV s-1 Milli volt per second nA Nano Ampere NIR Near-Infrared Spectroscopy nM Nano Molar NMR Nuclear Magnetic Resonance NPV Normal Pulse Voltammetry NSAIDs Non Steroidal Anti-Inflammatory Drugs PC Paracetamol

xix

PLS Partial Least Square ppb parts per billion ppm parts per million PtSE Platinum Solid Electrode PVC Polyvinyl Chloride RE Reference Electrode RMSEC Root Mean Standard Error of Calibration RMSEP Root Mean Square Error of Prediction RP-HPLC Reserved Phase-High-Performance Liquid Chromatography rpm revolutions per minute RSD Relative Standard Deviation SA Salicylic Acid SPME Solid Phase Microextraction SWV Square Wave Voltammetry TLC Thin Layer Chromatography TOF-MS Time-of-Flight Mass Spectrometry TQ Turbo Quant V/s Volt per second v/v Volume by volume WE Working Electrode WHO World Health Organization μg/kg Microgram per kilogram µg L-1 Microgram per liter µg/g Microgram per gram µL Micro liter µm Micrometer µM Micro mole µmol L-1 Micro mole per liter

List of Contents Dedication i Certificate ii Acknowledgement iii Abstract v List of Contents viii List of Tables xii List of Figures xiv Abbreviations xviii

Chapter - One INTRODUCTION 1 - 14

1.1. Analgesics / NSAIDs -An overview 1 1.1.1. Mechanism of Action (NSAIDs) 2 1.2. Paracetamol 3 1.2.1. Pharmacopoeias 3 1.2.2. Uses and Administration 4

1.2.3. Adverse Effects 4

1.2.4. Overdosage and its Treatment 5 1.2.5. Precautions 5 1.3. Aspirin 6 1.3.1. Pharmacopoeias 6 1.3.2. Uses and Administration 7 1.3.3. Adverse Effects 8 1.3.4. Overdosage and its Treatment 9 1.3.5. Precautions 9 1.4. Diclofen 10 1.4.1. Pharmacopoeias 10 1.4.2. Uses and Administration 10 1.4.3. Adverse Effects 11 1.4.4. Precautions 12 1.5. Brufen 12 1.5.1. Pharmacopoeias 12 1.5.2. Uses and Administration 12 1.5.3. Adverse Effects 13 1.5.4. Overdosage and its Treatment 14

Chapter - Two LITERATURE REVIEW ix 15 - 42

2.1. Analytical Techniques for Assessment of NSAIDs 15 2.2. Electrochemical Techniques 16 2.3. Spectroscopic Methods 21 2.4. Chromatographic Methods 25 2.5. Other Techniques 27 2.6. Quantification of Paracetamol in Biological Samples and

Pharmaceuticals 27

2.7. Quantification of Aspirin in Pharmaceutical and Biological Samples

31

2.8. Quantification of Diclofenac Sodium in Pharmaceutical and Biological Samples

35

2.9. Quantification of Ibuprofen in Pharmaceutical and Biological Samples

39

Chapter - Three EXPERIMENTAL 43 - 53

3.1. Material and Methods for Paracetamol Using UV-Visible Spectrophotometry

43

3.1.1. Reagents and Chemicals 43 3.1.2. Instruments and Apparatus 43 3.1.3. Procedure for Determining PC 44 3.1.4. Analysis of PC in Urine Samples 44 3.2. Material and Methods for Diclofenac Sodium Using UV-

Visible Spectrophotometry 45

3.2.1. Apparatus 45 3.2.2. Washing of Glassware 45 3.2.3. Reagents and Solutions 45 3.2.4. Procedure for Determining DS in Tablets 46 3.2.5. Procedure for DS in Serum and Urine Samples 46 3.3. Material and Methods for the Determination of

Paracetamol Using Differential Pulse Voltammetry 47

3.3.1. Reagents and Solutions 47 3.3.2. Apparatus 47 3.3.3. Procedures 48 3.4. Material and Methods for the Analysis of Ibuprofen

Using FT-IR Spectroscopy

49

3.4.1. Reagents and Samples 49

3.4.2. FT-IR Spectral Measurements 49 3.4.3. FT-IR Calibrations 50 3.4.4. Sample Preparation Procedure 50 3.4.5. Collection and Preparation of Urine Samples 50 3.5. Material and Methods for Investigation of Aspirin Using

Voltammetry51

3.5.1. Reagents 51 3.5.2. Apparatus 51 3.5.3. Voltammetric Procedure 52 3.5.4. Indirect Determination of ASA 52 3.5.5. Indirect Determination of ASA in Pharmaceutical

Drugs and Urine Samples by DPV at CFE 52

Chapter - Four EXPERIMENTAL 54 - 108

4.1. Simpler Spectrophotometric Assay of PC in Tablets and Urine Samples

54

4.1.1. Effect of Water Addition to PC (FTIR studies) 54 4.1.2. Optimization of Time for Measurement and Stability

of Analytical Signal 55

4.1.3. Effect of Temperature 56 4.1.4. Effect of Polar Solvents 57 4.1.5. Interference by Various Analgesic Drugs 58 4.1.6. Effect of Acidic and Basic Solutions 60 4.1.7. Calibration Range 61 4.1.8. Analysis of Tablets 62 4.1.9. Analysis of Urine Samples 63 4.2. Simpler and Faster Spectrophotometric Determination of

Diclofenac Sodium (DS) in Tablets, Serum and Urine Samples

65

4.2.1. Influence of Time 65 4.2.2. Effect of Temperature 66 4.2.3. Interference by Various Analgesic Drugs 67 4.2.4. Effect of Acidic and Alkaline Conditions 68 4.2.5. Calibration Plot 71 4.2.6. Comparison with Other Reported Spectroscopic

Methods 71

4.2.7. Analysis of Tablets 72 4.2.8. Analysis of Urine and Serum Samples 74 4.3. Differential Pulse Voltammetric Determination of PC in 77

Tablet and Urine Samples at Carbon Film Electrode 4.3.1. Influence of pH on PC at DC and DP Voltammetry 77 4.3.2. Optimization of Parameters and Calibration Curve 78 4.3.3. Analysis of Pharmaceutical Drugs 82 4.3.4. Analysis of Urine Samples 84 4.4. Quantification of Ibuprofen in Pharmaceuticals and

Biological Samples by FTIR Transmission Spectroscopy. 86

4.4.1. Analysis of Pharmaceutical Samples 86 4.4.2. Analysis of Urine Samples 89 4.4.3. Limits of Detection and Quantitation 92 4.5. Differential Pulse Voltammetric Determination of

Salicylic acid and Acetylsalicylic acid in Tablet and Urine Samples at Carbon Film Electrode

93

4.5.1. Influence of pH on Salicylic Acid in DC and DP Voltammetry

93

4.5.2. Reproducibility of Salicylic Acid 94 4.5.3. Linear Calibration Curves of Salicylic Acid 95 4.5.4. Hydrolysis of Acetylsalicylic acid 97 4.5.5. Linear Calibration Curves of Hydrolyzed

Acetylsalicylic Acid at CFE 98

4.5.6. Determination of Salicylic Acid in Different Drugs 100 4.5.7. Determination of Acetylsalicylic Acid from Different

Drugs 101

4.5.8. Determination of Salicylic Acid in Urine Samples at CFE

103

4.5.9. Determination of Acetylsalicylic Acid in Urine at CFE

104

4.5.10. Interference Study 106

4.6. Conclusion 107

References 109-132

LIST OF TABLES

Table. 4.1.1. Effect of different polar solvents on absorbance of PC. 58

Table. 4.1.2. % interference by various analgesics in different ratios on 5 µg ml-1 PC 59

Table. 4.1.3. Effect of strong and weak acids and bases on PC determination 60

Table. 4.1.4. Determination of PC in tables of various companies by proposed method 63

Table. 4.1.5. Comparative analyses of PC in urine samples. 63 Table. 4.2.1. % interference by various analgesics in different ratios on

5 µg ml-1 DS 67

Table. 4.2.2. Effect of strong and weak acids and bases on DFS determination

70

Table. 4.2.3. Comparison of current method with other spectroscopic methods for determination of DS

72

Table. 4.2.4. Determination of DS in tablets of various companies by proposed method

73

Table. 4.2.5. Concentration of determined DS and its relation with ingested DS by oral administration

76

Table 4.3.1. The influence of pH on DCV and DPV of Paracetamol (c = 100 µmol L-1)

78

Table. 4.3.2. Parameters of the calibration straight lines for the determination of Paracetamol in Britton-Robinson buffer pH 4.0 using DPV at CFE with regeneration potential Ereg1 = –400 mV, and Ereg2 = 1300 mV.

79

Table. 4.3.3. The amount of Paracetamol determined by DPV in tablets of commercial drugs with declare contents 500 mg of Paracetamol.

83

Table. 4.3.4. Parameters of the calibration straight lines for the determination of model samples of Paracetamol in urine, media of Britton-Robinson buffer pH 4.0 using DPV at CFE with regeneration potentials Ereg1 = –400 mV and Ereg2 = 1300 mV.

84

Table. 4.4.1. Results for the ibuprofen concentration found in the tablet samples

87

Table. 4.4.2. Recovery result of ibuprofen from tablet samples after spiking with known concentrations of standards

89

Table. 4.4.3. Recovery results of ibuprofen from urine samples after spiking with known concentrations of standard

90

Table 4.4.4. Recovery results of ibuprofen in urine samples after

spiking with known concentrations of standard 91

Table. 4.5.1. The influence of pH on DCV and DPV of Salicylic Acid (c = 100 µmol L-1 )

94

Table 4.5.2. Parameters of the calibration straight lines for the determination of Salicylic Acid in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

97


100


101

Table 4.5.5. The amount of hydrolyzed Acetylsalicylic Acid determined by DPV in tablets

102

Table 4.5.6. Parameters of the calibration straight lines for the determination of salicylic acid in 0.1 ml urine samples in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

104

Table 4.5.7. Parameters of the calibration straight lines for the determination of hydrolyzed Acetylsalicylic acid in 0.1 ml urine sample in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

106

LIST OF FIGURES

Figure. 1.1. Structural formula of Paracetamol (PC) 3 Figure. 1.2. Structural formula of Aspirin (ASA) 6 Figure. 1.3. Structural formula of Diclofenac Sodium (DS) 10 Figure. 1.4. Structural formula of Ibuprofen (IBP) 12 Figure. 4.1.1. FTIR spectra of (A), pure PC (B), aqueous PC paste and

(C) aqueous solution of 5 µgml-1 PC. 55

Figure. 4.1.2. Dependence of absorbance of PC on time 56 Figure. 4.1.3. Effect of temperature on absorbance of PC 57 Figure. 4.1.4. Shift of wavelength with pH: (A) 3.3 and (B) 12. 3 for

5µgml-1 PC solution. 61

Figure. 4.1.5. Calibration range of absorbance vs. concentration for PC from 0.3 to 20 µg ml-1

. 61

Figure. 4.1.6. A 1000 times diluted sample of urine (a) with PC and (b) without PC.

64

Figure. 4.2.1. Time effect on absorbance of Diclofenac Sodium 65 Figure. 4.2.2. Temperature effect on UV absorbance of 5 µg ml-1 DS

solution. 66

Figure. 4.2.3. UV spectra of DS at acidic pH, 3.56 (lower) and basic pH, 11.68 (higher).

69

Figure. 4.2.4. Calibration plot of absorbance verses concentration for DS solutions from below to above as 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 and 30 µg ml-1.

71

Figure. 4.2.5. Representative UV-spectrum of expected 5 µg ml-1 DS in (Diclofen) tablet.

73

Figure. 4.2.6. UV spectra showing blank (black), DS containing (blue) and DS spiked (red) urine.

74

Figure. 4.2.7. UV spectra showing blank (black), DS containing (blue) and DS spiked (red) serum.

75

Figure. 4.3.1. DC voltammograms (A) and DP voltammograms (B) of Paracetamol (c = 100 μmol-1.l) at CFE in Britton-Robinson buffer pH 2 to 12 (numbers above curves correspond to given pH) without electrode regeneration. Inset is corresponding dependence of peak potential on the pH.

77

Figure. 4.3.2. Repetitive measurements of 100 µmol L-1 Paracetamol using DPV at CFE in Britton-Robinson buffer pH 4.0 with regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV Inset is corresponding dependence of peak current on the number of scans.

79

Figure. 4.3.3. Differential pulse voltammograms of Paracetamol at CFE

in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 µmol L-1 . Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

80


81

Figure. 4.3.2. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(Paracetamol): 0 (1), 0.2 (2), 0,4 (3), 0,6 (4), 0,8 (5), 1 µmol L-1(6). Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300

mV. Inset is corresponding calibration dependence.

81

Figure. 4.3.6. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 0,02 (3) 0,04, (4) 0,06, (5) 0,08, (6) 0,1 µmol L-1. Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

82

Figure. 4.3.3. Differential pulse voltammograms of Paracetamol 10 µmol L-1 with Ascorbic acid from 10 to 100 µmol L-1 (concentrations are written above curves in plot) at CFE in Britton-Robinson buffer pH 4.0, regeneration potential Ereg1 = –400 mV, and Ereg2 = 1300 mV. Inset is corresponding dependence of peak current of Paracetamol on concentration of Ascorbic acid.

83

Figure. 4.3.4. Differential pulse voltammograms of 0.1 ml urine with model sample of Paracetamol (2 – 10 µmol L-1 (A), and 20 – 100 µmol L-1 (B), concentration is written next to curves in plots) sample at CFE in Britton-Robinson buffer pH 4.0, regeneration potentials Ereg1 = –400 mV, and Ereg2 = 1300 mV. Inset is corresponding calibration dependence.

84

Figure. 4.4.1. Calibration plot in the range of 10-100 ppm for pharmaceutical samples

87

Figure. 4.4.2. Pharmaceutical Tablet sample and spikes of 30 ppm, 50 ppm, 70 ppm ibuprofen

88

Figure. 4.4.3. Blank urine and 3 spikes of 10, 20, 30 ppm ibuprofen 90 Figure. 4.4.5. Group Spectra of Ibuprofen Standards 92

Figure. 4.5.1. A and B. DC & DP voltammograms of Salicylic Acid (c = 100 µmol L-1 ) at CFE in Britton-Robinson buffer pH

2 to 12 (numbers in above curves correspond to given pH). Inset is corresponding dependence of peak potential on the pH

93

Figure. 4.5.2. Measurements of 100 µmol L-1 Salicylic Acid using DPV at CFE in Britton-Robinson Buffer pH 2.0 with activation potential (1) 2000,(2) 1500 and (3) 2200 mV

95

Figure. 4.5.3. Differential pulse voltammograms of Salicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 8,0 (6) 100 µmol L-1 .

Activation of potential=2200mV for 120 sec Inset is corresponding calibration dependence

96

Figure. 4.5.4. Differential pulse voltammograms of Salicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L-1. Activation of potential=2200mV for 120 sec. Inset is corresponding calibration dependence

96

Figure. 4.5.5. Differential pulse voltammograms of Salicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 µmol L-1. Activation of potential=2200mV for 120 sec. Inset is corresponding calibration dependence

97

Figure.4.5.6. Differential pulse voltammograms of 10 µmol L-1 hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2.0, (1) Electrolyte, (2) 10 µmol L-1 without hydrolysis (3) 10 µmol L-1 Salicylic Acid (4) 10 µmol L-1 after hydrolysis Acetylsalicylic acid. Activation potential=2200mV for 120 sec

98

Figure. 4.5.7. DP voltammograms of hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 µmol L-1. Activation potential=2200mV for 120 sec. Inset is corresponding calibration dependence

99

Figure. 4.5.8. DP voltammograms of hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2. c (ASA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L-1. Activation potential=2200mV for 120 sec. Inset is corresponding calibration dependence

99

Figure. 4.5.9. DP voltammograms of hydrolyzed Acetylsalicylic Acid at CFE in Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 µmol L-1. Activation potential=2200mV for 120 sec. Inset is corresponding calibration dependence

100

Figure.4.5.10.Differential pulse voltammograms of 4 µmol L-1 Duofilm

sample with spikes of salicylic acid standard at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) electrolyte, (2) expected 4 μmol L-1 salicylic acid in sample of Duofilm (3) 6 μmol L-1 spike, (4) 8 μmol L-1 spike and (5) 10 μmol L-1 spike. Activation potential 2200 mV

101

Figure. 4.5.11. Differential pulse voltammograms of 4 µmol L-1 Aspirin sample with spikes of hydrolyzed acetylsalicylic acid standard at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) electrolyte, (2) expected 4 μmol L-1 salicylic acid in sample of Aspirin (3) 6 μmol L-1 spike, (4) 8 μmol L-1

spike and (5) 10 μmol L-1 spike. Activation potential 2200 mV

102

Figure. 4.5.12. Differential pulse voltammograms of 0.1 ml urine sample at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 µmol L-1 . Activation potential 2200 mV. Inset is corresponding calibration

dependence

103

Figure. 4.5.13. Differential pulse voltammograms of 0.1 ml urine sample at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L-1. Activation potential 2200 mV. Inset is corresponding calibration dependence

103


105


105

Figure.4.5.16.Differential pulse voltammograms of hydrolyzed acetylsalicylic acid 5 μmolL-1 with ascorbic acid 5-100 µmol L-1 at CFE in Britton-Robinson buffer pH 2.0. Activation potential 2200 mV

106

List of Abbreviations AdSV Adsorptive Stripping Voltammetry AgA–CFE Silver Amalgam Carbon Film Electrode

AgA–PE Silver Amalgam Paste Electrode AgE Silver Electrode AOAC Association of Official Analytical Chemists ASA Acetylsalicylic Acid ATR Attenuated Total Reflection AuE Gold Electrode CFE Carbon Film Electrode CNS Central Nerves System COX Cyclo-Oxygenase CPE Carbon Paste Electrode CV Cyclic Voltammetry DDP Differential Pulse Polarography DME Dropping Mercury Electrode DPV Differential Pulse Voltammetry DS Diclofenac Sodium E1/2 Half Wave Potential FIA Flow Injection Analysis FT-IR Fourier Transform Infrared Spectroscopy FT-NIR Fourier Transform Near-Infrared GC Gas Chromatography GC– MS Gas Chromatography Mass Spectroscopy GCE Glassy Carbon Electrode GGE Glassy Graphite Electrode HEC Higher Education Commission HMDE Hanging Mercury Drop Electrode HPLC High Performance Liquid Chromatography HT-GLC High-Temperature Gas–Liquid Chromatography Hz Hertz IBP Ibuprofen LOD Limit of Detection LOQ Limit of Quantification LSV Linear Sweep Voltammetry mg/kg Milligram per Kilogram mg/kg /b.w/day Milligram per Kilogram per Body Weight per Day mg/L Milligram per Liter mm Millimeter mol.L-1 Mole per liter mV Milli volt mV s-1 Milli volt per second nA Nano Ampere NIR Near-Infrared Spectroscopy

nM Nano Molar NMR Nuclear Magnetic Resonance NPV Normal Pulse Voltammetry NSAIDs Non Steroidal Anti-Inflammatory Drugs PC Paracetamol PLS Partial Least Square ppb parts per billion ppm parts per million PtSE Platinum Solid Electrode PVC Polyvinyl Chloride RE Reference Electrode RMSEC Root Mean Standard Error of Calibration RMSEP Root Mean Square Error of Prediction RP-HPLC Reserved Phase-High-Performance Liquid Chromatography rpm revolutions per minute RSD Relative Standard Deviation SA Salicylic Acid SPME Solid Phase Microextraction SWV Square Wave Voltammetry TLC Thin Layer Chromatography TOF-MS Time-of-Flight Mass Spectrometry TQ Turbo Quant V/s Volt per second v/v Volume by volume WE Working Electrode WHO World Health Organization μg/kg Microgram per kilogram µg L-1 Microgram per liter µg/g Microgram per gram µL Micro liter µm Micrometer µM Micro mole µmol L-1 Micro mole per liter

1

Chapter-01

INTRODUCTION 1.1. Analgesics / NSAIDs -An overview A drug designed to control pain. Analgesic comes from the Greek word an means without

and algesis means sense of pain collectively called without sense of pain. They are the

commonly prescribed and widely consumed (over-the-counter) formulation in the world.

Although analgesics are generally supposed to be safe agents, harmful effect may occur

in the case of chronic abuse and acute overdose (Suzanne & Steven, 1998). Analgesics

are used in the treatment of a broad range of therapeutic situations, they are used in

treatment of both chronic and acute pain symptom such as dysmenorrheal or cephalgia.

Antipyresis is also common sign for the administration of these drugs, mainly in the

children.

Non steroidal anti-inflammatory drugs (NSAIDs) are the chemically heterogeneous group

of compounds, often chemically unrelated (although most of them are organic acids)

which nevertheless share certain therapeutic actions and adverse effects Goodman &

gilmans (2007). It is given as single dose or in short-term alternating therapy; it can

reduce mild to moderate ache. The anti-inflammatory effects become apparent when it

may give up to 21 day. The combined anti-inflammatory and analgesic effects make them

helpful for the symptomatic relief of painful situation including rheumatic disorders such

as rheumatoid arthritis, osteoarthritis, and the spondyloarthropathies, and also in peri-

articular disorders, and soft-tissue rheumatism. Some NSAIDs are used in the

management of postoperative pain (Bidaut-Russell & Gabriel, 2001).

2

Anti-inflammatory activity of drug shows the small differences, between the NSAIDs and

preference is mostly empirical. If one NSAID fail to respond the patient, in that case

another drug may be selected for treatment. However, it has been recommended that

NSAIDs associated with a low risk of gastrointestinal toxicity should generally be

preferred and the lowest effective dose used. NSAIDs are usually given by mouth, with

or after food, although some such as diclofenac, ketorolac, lornoxicam, parecoxib, and

tenoxicam can be given intravenously and intramuscularly. Some NSAIDs are applied

topically or given rectally as suppositories. Several NSAIDs are used in ophthalmic

preparations for the inhibition of intra-operative miosis, control of postoperative ocular

inflammation.

1.1.1. Mechanism of Action (NSAIDs) Cyclo-oxygenases play an important role in the biosynthesis of prostaglandins. NSAIDs

inhibit cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) and it is thought

that inhibition of COX-1 is connected with unfavorable gastrointestinal effects while

inhibition of COX-2 is related with anti-inflammatory activity (Hayllar, et al., 1995,

Richardson and Emery, 1996) hence the interest in preferential or selective inhibitors of

COX-2. COX-2 inhibitors may also have a potential use in other diseases in which COX-

2 might be implicated (Jouzeau, et al., 1997, Hawkey, 1999). There is evidence that

NSAIDs may also have a central mechanism of action that augments the peripheral

mechanism (Gupta & Tarkkila 1998; Cashman, 1996).

In view of the analgesic (Paracetamol, Aspirin, Diclofenac and Brufen) studied during the

current project it is essential to focus on the properties, uses and toxic effects of these

drugs.

3

1.2. Paracetamol

Fig. 1.1. Structural formula of Paracetamol (PC) Chemical name: 4´-Hydroxyacetanilide; N-(4-Hydroxyphenyl)acetamide Molecular formula: C8H9NO2 =151.2

1.2.1. Pharmacopoeias: According to British, European and United States Pharmacopoeias Paracetamol

(Acetaminophen) is a white crystalline odourless powder. It is sparingly soluble in water,

freely in alcohol and sodium hydroxide and very slightly soluble in dichloromethane

(Jacoby, 2000). It should be protected from light and stored in airtight containers.

Paracetamol (N-acetyl-p-aminophenol) is an effective alternative to aspirin as an

analgesic, antipyretic agent and safe up to therapeutic doses. It work as painkiller by

control prostaglandin’s synthesis in the central nervous system and reduced fever by

painkilling hypothalamic heat-regulating center (Goyal and Singh 2006; de los, et al.,

2005; Moreira, et al., 2005; Campanero, et al., 1999). Paracetamol is readily absorbed

after administration and widely distributed throughout most body fluids (Criado, et al.,

2000), as a weak acid (pKa value 9.5), it gets quickly absorbed and distributed after oral

administration and is immediately emitted through urine (Goyal and Singh 2006;

Parojčić, et al., 2003). Generally paracetamol does not shows any unsafe side effects but

hypersensitivity or overdoses in few cases lead to the formation of some liver and

nephrotoxic metabolites (Goyal and Singh 2006; Patel, 1992).

4

1.2.2. Uses and Administration Paracetamol is often self-prescribed and given by mouth or as a rectal suppository for

relief of moderate pain, fever, lumber pain, backache, migraine or non-specific

indications without any medical control (Mcgregir, et al., 2003; Nikles, et al., 2005). It

has been reported as a useful drug in osteoarthritis therapy and in recent years,

postoperative pain as well (Brandt, 2003). After ingestion of an overdose amount of

paracetamol, the accumulation of harmful metabolite may cause severe and occasionally

fatal nephrotoxicity and hepatotoxicity (Xu and Li, 2004; Maria, et al., 2005). However,

the frequent use of paracetamol in late pregnancy may be associated with an increased

risk of persistent wheezing in the infant (Farquhar; 2009; Shaheen, et al., 2002).

Usual doses in children are: under one to five years, 120 to 250 mg and six to twelve

years, 250-500 mg. The normally adult dose by 30 days is 0.5-1 g every four to six hrs up

to a highest of 4 g each day. These doses may be given every four to six hrs when

necessary up to a maximum of four doses in one day (Prescott, 1996).

1.2.3. Adverse Effects

Side-effects of paracetamol are exceptional and typically soft, although haematological

reactions including leucopenia, thrombocytopenia, neutropenia, agranulocytosis and

pancytopenia have been described. Skin rashes and other hypersensitivity reactions occur

rarely (Xu and Li, 2004; Martin & McLean, 1998; KocaoÄŸlu, et al., 1997; Mugford &

Tarloff, 1997; Nagasawa, et al., 1996; Ishida, et al., 1997).

5

1.2.4. Overdosage and its Treatment Ingestion of as little as ten to fifteen g of paracetamol by adults may cause severe

hepatocellular necrosis and less often renal tubular necrosis. However, chronic use of

supratherapeutic doses in children has resulted in unintentional overdoses and severe

hepatotoxicity (Miles, et al., 1999; American Academy of Pediatrics Committee on

Drugs, 2001; Wyszecka-Kaszuba, et al., 2003).

Activated charcoal may be used to reduce gastrointestinal absorption, if it can be given

within 1 hour of the overdose, and if greater than 150 mg/kg of paracetamol has been

consumed. However, if acetylcysteine or methionine is to be given by mouth the charcoal

is best cleared from the stomach to prevent its reducing the absorption of the antidote.

There is little evidence that gastric lavage is of benefit in those who have overdosed

solely with paracetamol.

1.2.5. Precautions Paracetamol should be prescribed with carefully to patients with harmed liver or kidney

and alcohol dependence patients. About 0.01% population of the United state and 0.02%

population of the Australian were appraised in hospital every year because of PC

poisoning (de los et al., 2005; Dargan & Jones, 2003).

6

1.3. Aspirin

Fig. 1.2. Structural formula of Aspirin (ASA) Chemical name: O-Acetylsalicylic acid; 2-Acetoxybenzoic acid Molecular formula: C9H8O4 =180.2

1.3.1. Pharmacopoeias According to British, European and United States Pharmacopoeias Acetylsalicylic Acid

or Aspirin is white crystalline powder, commonly tubular or needle-like, stable in dry, air

has a faint odour or odourless, while in moist air it steadily hydrolyzes to salicylic and

acetic acids. It is slightly soluble in water but freely in alcohol, chloroform and sparingly

soluble in absolute ether (Jacoby, 2000), store in airtight containers.

Acetylsalicylic acid (ASA), shown in Figure 2, more popularly known as aspirin, is one

of the oldest medicines that still plays an important role in modern therapeutics. It is

widely employed in pharmaceutical formulations for the relief of headaches, fever,

muscular pain, and inflammation (Xianwen, et al., 2009). Acetylsalicylic acid or aspirin,

was introduced in the late 1890s (Dreser, 1899). It was first synthesized in 1897, by Felix

Hoffmann, in the Farbenfabrik Freidrich Bayer laboratories, in Elberfeld, Germany

(Hammerschmidt, 1998; Sneader, 2000; Moore, et al., 1995; Kibbey, et al., 1992).

Salicylates, in the form of willow bark, were used as an analgesic during the time of

Hippocrates (Pirker, et al., 2004). This substance is consumed worldwide (Erica et al.,

2010; Elwood, 2001) indicates the importance of the development of new analytical

methods to assess not only the quality but also the authenticity of the product (Erica et

al., 2010).

7

The analgesic and anti-pyretic efficiency of aspirin was promptly comproved, however

only after the years 1940 it began to be employed in higher doses as anti-inflammatory

agent. When its mechanism of action began to be understood the possibility for use

against cardiac and circulatory disturbances became evident (Muralidharan et al., 2008;

Eccles, et al., 1998).

The rate of decomposition of ASA to salicylic acid (SA) and acetic acid (AA) is reliant

on solution pH and temperature (Lu & Tsai, 2010). In the pH range 11-12 ASA is quickly

hydrolyzed, in the pH range 4-8 its hydrolysis rate is slow while highest constancy is

attained at pH 2-3 (Connors, et al., 1979).

1.3.2. Uses and Administration It is used in acute conditions such as arthralgia, headaches, myalgia and other cases

involving mild analgesia. Once ingested, ASA is quickly hydrolysed in the body to

formulate salicylic acid (SA), the compound that is primarily responsible for the

pharmacological activity of ASA (Houshmand, et al., 2008). It is also used for reduce the

excitements of viral or bacterial origin (Houshmand, et al., 2008; Boopathi, et al., 2004,

De Carvalho, et al., 2004). In general, the salicylates action is achieved by the SA

contents, although some of the characteristic properties of the ASA are their capability

for protein acetylation. The esters in the phenolic or carboxylic functional groups alter the

power in the toxicity of salicylates (Torriero, et al., 2004; Hardman, et al., 1996).

Aspirin act as inhibitors of the enzyme cyclo-oxygenase, which outcome in the direct

inhibition of the bio-synthesis of thromboxanes and prostaglandins from arachidonic acid.

8

It has also been used in the management of inflammation and pain in acute and chronic

rheumatic disorders such as juvenile idiopathic arthritis, rheumatoid arthritis, ankylosing

spondylitis and osteoarthritis. In the treatment of minor febrile conditions, such as colds

or influenza, aspirin can reduce temperature and relieve headache and joint and muscular

pains (Vane, et al., 2006, Thun, et al., 2002).

Aspirin is usually taken by mouth. Various dosage forms are available including plain

uncoated tablets, buffered tablets, dispersible tablets, enteric-coated tablets, and

modified-release tablets. In some instances aspirin may be given rectally by suppository.

The usual oral dose of aspirin as an analgesic and antipyretic is 300-900 mg, repeated

every 4 to 6 hours according to clinical needs, to a maximum of 4 g daily. The dose as

suppositories is 600-900 mg every four hrs to a maximum of 3.6 g every day.


The most common adverse effects of therapeutic doses of aspirin are gastrointestinal

disturbances such as nausea, dyspepsia, and vomiting (Dirckx, et al., 2009). This

substance is highly consumed in all over the world, and its remedial act and its toxic

effects make it a drug that is subjected to continuous researches (Majdi, et al., 2007;

Elwood, 2001), and its antipyretic effects have been recognized for more than 200 years

(Stone, 1763). However, the antiplatelet activity of this agent was not recognized until

almost 70 years later (Awtry & Loscalzo, 2000; Wong, et al., 2004). Researchers have

also demonstrated therapeutic benefit of aspirin in a variety of cardiovascular diseases

with its doses of 30 to 1500 mg/d (Awtry & Loscalzo, 2000, Wong, et al., 2004, Patrono,

1994, Fuster et al., 1993, Hennekens, et al., 1997, Manson, et al., 1991).

9

The effect of salicylates on cancer treatment has been studied, too (Thun et al., 2002,

Shen, et al., 2004, Keloff, et al., 2004, Hardwick, et al., 2004, Martnett, 1992). Moreover,

inhibition of growth of bacteria Helicobacter pylori (Wang, et al., 2003) and

Staphyloccocus aureus (Kupferwasser, et al., 2003) has been also described. Kidney

damage related with the therapeutic utilization of aspirin alone appears to be relatively

rare (Dubach, et al., 1991, Perneger, et al., 1994). Aspirin-induced liver injury is

frequently reversible on stopping the drug (Lewis, 1984, Freeland, et al., 1988). Aspirin

burns when ulceration of the mucosal layer of the lips) developed in a 26-year old woman

after getting an aspirin-containing powder for a migraine (Dellinger and Livingston,

1998).

1.3.4. Overdosage and its Treatment In case of acute oral salicylate overdosage, repeated doses of activated charcoal may be

given by mouth if the patient is suspected of ingesting more than 250 mg/kg of salicylate.

Intravenous sodium bicarbonate is given to enhance urinary salicylate excretion if plasma

salicylate concentration exceeds 500 micrograms/mL (350 micrograms/mL in children).

Haemodialysis or haemoperfusion are also effective methods of removing salicylate from

the plasma.

1.3.5. Precautions

Aspirin should not be given to patients with haemophilia or other haemorrhagic disorders

or the patients with a history of sensitivity reactions to aspirin, including those in whom

attacks of asthma, angioedema, urticaria, or rhinitis have been precipitated by such drugs

although low-dose aspirin might be given in some pregnant patients as an analgesic.

10

1.4. Diclofen

Fig. 1.3. Structural formula of Diclofenac Sodium (DS) Chemical name: Sodium 2-[(2, 6-dichlorophenyl) amino] phenyl acetate Molecular formula: C14H10Cl2NNaO2 =318.1

1.4.1. Pharmacopoeias According to British, European and United States Pharmacopoeias diclofenac Sodium is

a white to slightly yellowish (off-white), slightly hygroscopic, crystalline powder. It is

sparingly soluble in water, alcohol, slightly in acetone and freely in methyl alcohol,

practically insoluble in chloroform and in ether (Bahram, 2008). pH of a 1% solution in

water is between 7.0 and 8.5. Store in airtight containers and protect from light (Rients &

Jan, 2008).

1.4.2 Uses and Administration

A phenylacetic acid derivative, Diclofenac is an NSAID (Matin, et al., 2005). It is used

mostly as the sodium salt for the release of inflammation and pain in many situations:

joint disorders and musculoskeletal such as osteoarthritis, rheumatoid arthritis, and

ankylosing spondylitis; peri-articular disarray such as tendinitis and bursitis; soft-tissue

disarray such as strains and sprains; and other painful circumstances such as renal colic,

dysmenorrhoea, migraine and acute gout subsequent surgical processes (Lala, et al.,

2002). Eye drops of diclofenac sodium are used for the prevention of intra-operative

11

miosis for the duration of cataract removal, for the medication of inflammation following

surgery or laser treatment of the eye, for pain in corneal epithelial defects following

surgery or accidental trauma, and for the relief of ocular signs and symptoms of seasonal

allergic conjunctivitis.

The usual dose of diclofenac sodium by mouth or rectally is 75 to 150 mg daily in

divided doses. In children of 1 to 12 years old, the dose by mouth for juvenile idiopathic

arthritis is one to three mg/kg daily in divided doses.


There may be pain and, occasionally, tissue damage at the site of injection when

diclofenac is given intramuscularly. Diclofenac suppositories may cause local irritation.

Transient burning and stinging may occur with diclofenac ophthalmic solution; more

serious corneal adverse effects have also occurred. The main effects on the blood reports

are haematological abnormalities including haemolytic anaemia (López, et al., 1995)

agranulocytosis (Colomina & Garcia, 1989) neutropenia (Kim & Kovacs, 1995) and

thrombocytopenia (George & Rahi, 1995) happening in patients given diclofenac.

Localized slow bleeding and inhibition of platelet aggregation (Price & Obeid, 1989)

bruising and prolonged bleeding time (Khazan, et al., 1990) have also been reported.

Rectal management of diclofenac suppositories may origin local reactions such as

burning and itching. Nephrotic syndrome (Beun, et al., 1987; Yinnon, et al., 1987;

Tattersall, et al., 1992) described in patients taking diclofenac. Rise of clinical hepatitis

and serum amino transferase activity (Ryley, et al., 1989) including fatal fulminant

hepatitis (Purcell, et al., 1991) have occurred in patients getting diclofenac.

12

1.4.4 Precautions

Use of intravenous diclofenac is contra-indicated in patients with moderate or severe

renal impairment, hypovolaemia, or dehydration. Intravenous diclofenac should not be

used in patients with a history of haemorrhagic diathesis, cerebrovascular bleeding

(including suspected), or asthma nor in patients undergoing surgery with a high risk of

haemorrhage.

1.5. Brufen

Fig. 1.4. Structural formula of Ibuprofen (IBP) Chemical name: [2-{4-(2-methylpropyl)phenyl} propanoic acid] Molecular formula: C13H18O2 =206.3

1.5.1. Pharmacopoeias According to British, European and United States Pharmacopoeias ibuprofen is a

colourless crystals or white crystalline powder having a minute characteristic odor. It is

practically insoluble in water, freely soluble in acetone, in dichloromethane, chloroform

and in methyl alcohol. It dissolves in dilute solutions of alkali hydroxides and carbonates.

Store in airtight containers.

1.5.2. Uses and Administration Ibuprofen, a propionic acid derivative, is an important anti-inflammatory, analgesic and

antipyretic medicine with considerably less gastrointestinal adverse effect than other

NSAIDs (Velasco, et al., 2010; Donald, et al., 2010; Whittle, et al., 2003) employed in

13

the administration of mild to moderate pain in situation such as headache,

dysmenorrhoea, postoperative pain, including migraine, musculoskeletal and dental pain;

joint disorders such as osteoarthritis, rheumatoid arthritis and ankylosing spondylitis;

peri-articular disorders such as tenosynovitis and bursitis; soft-tissue disorders such as

strains and sprains. NSAIDs is also used to lower fever (Clara, et al., 2009; Santini, et al.,

2006).

Patients with rheumatoid arthritis generally require higher doses of ibuprofen than those

with osteoarthritis. The recommended dose for fever reduction in adults is 200 to 400 mg

every 4 to 6 hours to a highest of 1.2 g every day (Nanda et al., 2010). In children the

usual dose by mouth for the treatment of pain or fever is 20 to 30 mg/kg daily in divided

doses. Ibuprofen is also applied topically as a 5% cream, foam, gel, or spray solution; a

10% gel is also available.


Symptoms of vomiting, tinnitus and nausea have been detailed after ibuprofen an

excessive dose. Moreover severe toxicity is unusual, but gastric emptying chased by

supportive measures is suggested if the amount ingested within the previous hour exceeds

four hundred mg/kg.

Effect on the blood including agranulocytosis, aplastic anaemia (Gryfe & Rubenzahl,

1976) pure white-cell aplasia (Mamus, et al., 1986) and thrombocytopenia ( Jain, 1994 )

have been reported in patients taking ibuprofen. Fatal haemolytic anaemia occurred in a

man taking ibuprofen and oxazepam ( Guidry et al., 1979 ). A review of NSAID-related

CNS adverse effects summarized twenty three literature reports of NSAID-associated

14

aseptic meningitis (Hoppmann, et al., 1991), while seventeen reports involved ibuprofen

patients with a diagnosis of systemic lupus erythematosus. Typically the reaction is seen

in patients who have just restarted NSAID therapy after a gap in their treatment. The

authors believed this to be the first reported case of ibuprofen-induced aseptic meningitis

in a patient with rheumatoid arthritis (Horn & Jarrett, 1997). Hyponatraemia has been

described in patients receiving ibuprofen (García, et al., 2003). Ibuprofen may be

associated with a lower risk of upper gastrointestinal effects than some other NSAIDs,

but nonetheless it can cause vomiting, nausea, dyspepsia, gastrointestinal bleeding,

perforation and peptic ulcers. Colitis and its exacerbation have also occurred (Ravi, et al.,

1986, Clements, et al., 1990). Adverse renal effects with ibuprofen include an increase in

serum creatinine concentration (Whelton, et al., 1990), Acute renal failure (Fernando, et

al., 1994) and nephrotic syndrome (Justiniani, 1986) cystitis, haematuria, and interstitial

nephritis may occur as well.

1.5.4. Overdosage and Treatment Usually after ingestion of large dose of ibuprofen serious toxicity may occur, including

seizures, hypotension, apnoea, coma, and renal failure. Treatment of Ibuprofen

overdosage is entirely supportive. Gastric lavage and activated charcoal may be of benefit

within 1 hour of ingestion. Multiple doses of activated charcoal may be useful in

enhancing elimination of Ibuprofen with long half-lives.

15

Chapter-02

LITERATURE REVIEW 2.1. Analytical Techniques for Assessment of NSAIDs Various methods are has been developed for NSAIDs determination in different types of

samples (Sirajuddin, 2007). These include, titrimetry , thin layer chromatography (TLC),

colorimetry, Gas Chromatography/ Mass Spectrometry (GC/MS), high performance

liquid chromatography (HPLC), Voltammetry, Flow injection analysis (FIA),

Spectrofluorimetry, UV/visible Spectrophotometry, Chemiluminescence, NMR, FTIR,

Raman and Solid-state Linear Dichroic Infrared (IR-LD) Spectroscopy.

Ayora et al., 2000, reported a direct sensor based determination for paracetamol analysis

using easy flow-through ultraviolet-visible optosensing device based on the solid support

packed in the flow cell after that continuously observing their absorbance on the solid

phase at 264 nm. The proposed method was profitably applied to the PC in

pharmaceuticals formulations.

Fujiwara et al., 2002, developed a systematic approach for the aqueous crystallization of

paracetamol. The solubility curve and the solution concentration of paracetamol in water

were determined using Fourier transform infrared (FTIR) spectroscopy coupled with

attenuated total reflection (ATR) mode and chemometrics.

Sun et al., 2003, reported a sensitive method on HPLC coupled with UV detector for the

simultaneously find out of non-steroidal anti-inflammatory drugs (NSAIDs) possessing

an arylpropionic acid moiety in plasma and pharmaceutical formulations.

16

Šatínský et al., 2004, been described the molecularly imprinted polymer as a carbon fiber

microelectrode coating for determining paracetamol using cyclic voltammetry (CV).

A voltammetric method for the determination of ascorbic acid with Carbon Paste

Electrodes (CPEs), was applied with good results to several dosage forms (tablets, vials)

and even to an effervescent dosage form containing a mixture of ascorbic and

acetylsalicylic acid (Sandulescu et al., 2000).

2.2. Electrochemical Techniques Use of electrochemical methods, especially the voltammetric and amperometric in

analysis paid attention as sensitive, cost-effective and accurate way of analysis in last

decades.

Voltammetric methods have been developed due to high sensitivity these techniques

offer, principally when a variety of redox mediators (Rover et al., 2000; Lobo et al.,

1997, Fung & Luk, 1989) are employed, decreasing the interference levels in the

determinations of compounds present in complex matrices such as pharmaceuticals and

body fluids (Rover et al., 2000). Square wave voltammetry (SWV) is regarded as very

responsive and direct analytical techniques, which has used for the fast and sensitive

determination of a wide range of organic molecules, with low nonfaradaic current and

high sensitivity. In most instances this method presents further advantages such as no

need for sample pretreatment and less sensitivity to matrix effects than other analytical

techniques. (Codognoto et al., 2002; Souza et al., 2003 ; Pedrosa et al., 2003;

Osteryoung., 1985).

17

Nowadays a number of polymer films are used in the electroanalytical field due to easy

generated on the electrode surface than monolayers. To improve the selectivity and

sensitivity for the determination of PC various polymeric based films modified electrodes

has been developed (Chenghang Wang, et al., 2006; Sandik & Wallace, 1993; Piro et al.,

2000).

The electroanalytical features and performances of CPEs for fabrication of chemo and

biosensors through the modification of carbon paste and their analytical applications are

well documented (Goyal, & Singh, 2006; Vire et al., 1994; Kalcher, 1990; Kalcher et al.,

1995; Gorton, 1995; Campanella et al., 1992; Kalcher et al., 1997).

It is well known from the molecular structure of paracetamol, that it is electrochemically

active, and its electrochemical properties on a choice of electrodes, such as glassy carbon

electrodes (GCE) (Wang et al., 2006; Bolado et al., 2009; Santos et al., 2008;

Wangfuengkanagul et al., 2002; Gimenes et al., 2010), screen print electrode (Fanjul-

Bolado et al., 2009), gold electrodes (Santos et al., 2008, Pedrosa et al., 2006), and

diamond electrode have been investigated (Wangfuengkanagul et al., 2002).

The cyclic voltammetric studies concerning the electrochemical oxidation of

acetaminophen were also explained (Sandulescu, 2000; Miner et al., 1981; Benschoten et

al., 1983).

Zen et al., 1997, used the Nafion/ruthenium oxide pyrochlore chemically modified

electrode using SWV for the simultaneous determination of acetaminophen and caffeine

in drug formulations. The detection limits were 2.2 and 1.2 μM for caffeine and

18

acetaminophen, respectively. The analytical application of the proposed method was

applied in several commercially available drugs, without any preliminary treatment. Fang

Fang et al., 1997, proposed an oxidation process of acetaminophen at a platinum

electrode by parallel incident spectroelectrochemistry. The results exposed that the

acetaminophen redox reaction was a diffusion-controlled one-electron quasi reversible

process, this process confirm by cyclic voltammetric experiments. Capsules were

analyzed without pretreatment using linear range of acetaminophen with a R2 =0.9985.

The voltammetric and spectrophotometric methods which were used to determine

ascorbic acid and acetaminophen in different quantity forms (tablets, effervescent and

vials). The electroanalytical study of acetaminophen, ascorbic and some mixtures of these

compounds in different ratios has been made by using a (CPE-graphite:solid paraffin 2:1)

as working electrode (Sandulescu et al., 2000). Wangfuengkanagul et al., 2002, reported

the electrochemical behavior of acetaminophen in phosphate (pH 8) buffer solution at a

boron-doped diamond thin film electrode using hydrodynamic voltammetry. Torriero, et

al., 2004, reported a cyclic and DPV method for the electrochemical oxidation of SA on a

glassy carbon electrode (GCE). The method was linear over the salicylic acid

concentration at the range of 1–60 µgml-1.

Goyal, et al., 2005, a reproducible method for determined the paracetamol using a

nanogold modified indium tin oxide electrode were developed. Under conditions of

differential pulse voltammetry, 2.0x10-7–1.5x10-3 M was range of linear calibration curve

with a correlation coefficient of 0.997 was obtained.

19

An additional approach by Goyal, et al., 2006, for voltammetric analysis of PC was

carried out at C60-modified glassy carbon electrode, which demonstrates steady response

with better sensitivity and selectivity. A linear calibration curve was obtained in the range

of 0.05–1.5 mM paracetamol concentration with sensitivity of the method as 13.04

µAmM−1 having correlation coefficient of 0.985.

Chenghang et al., 2006, fabricated a novel L-cysteine film modified electrode effectively

applied for the analysis of acetaminophen by means of an electrochemical oxidation

procedure in tablets and human urine.

Jia et al., 2007, reported the electrochemical determination of acetaminophen on the

AMP SAMs/Au in Britton–Robinson (BR) solution of buffer by SWV. The modified

electrode showed a significant enhancement in the oxidation current response for

acetaminophen in comparison to a bare gold electrode. Linear calibration curve were

obtained in the range of 2.0 ×10 –6 – 4.0 × 10 –3 M.

Kachoosangi et al., 2008 reported a sensitive and selective electroanalytical method for

the analysis of paracetamol utilizizng adsorptive stripping voltammetric technique at a

multi walled carbon nanotube modified basal plane pyrolytic graphite electrode

(Kachoosangi, et al., 2008).

Saraswathyamma et al., 2008, reported gold electrode modified with dipyrromethene-Cu

(II) derivatives possessing two dodecane alkyl chains. The presence of dipyrromethene-

Cu(II) redox centers on the electrode surface was proved by CV and Osteryoung square-

wave voltammetry for determination of PC in plasma. Skeika et al., 2008, investigated

20

the simultaneous electrochemical determination of paracetamol and dypirone by

differential pulse voltammetry technique (DPV) using an unmodified carbon paste

electrode. The multivariate calibration methodology based on partial least square

regression (PLSR) was employed for the voltammetric peaks of paracetamol and

dypirone, due to overlapping.

Parviz et al., 2009 proposed the highly stable, convenient method for the electrochemical

oxidation of naproxen and PC using dysprosium nanowire modified carbon paste

electrode With SWV. A low detection limit for PC and naproxen, were observed by this

method.

Elen et al., 2009, reported a direct determination of acetylsalicylic acid (ASA) in

pharmaceutical formulations using SWV and a boron-doped diamond electrode (BDD).

The obtained relative standard deviation was smaller than 1.4% with a detection limit of

2.0 µmol L-1. Nada & Mehar, 2009 reported a promising voltammetric sensors based on

the modification of platinum (Pt) and poly (3 methylthiophene) (PMT) electrodes with

palladinum (Pd) nanoparticles were achieved for the determination of paracetamol.

Shahrokhian & Elham, 2010, have simultaneous determined acetaminophen and ascorbic

acid in the presence of isoniazid by CV and DPV using CPE modified with thionine

immobilized on multi-walled carbon nanotube (MWCNT).

Irena Baranowska & Marta, 2009, proposed a sensitive and fast method for the

quantification of PC and its glucuronide (PG) and sulfate (PS) metabolites. The

electrochemical properties of the compounds were examined by CV on GCE.

21

Bruna et al., 2009, developed simple and highly selective SWV or DP voltammetric

method for the single or simultaneous determination of caffeine and paracetamol in

aqueous media (acetate buffer, pH 4.5) on a BDDE. The limits of detection for the

simultaneous determination of caffeine and paracetamol were 3.5×10−8 mol L−1 and

4.9×10−7 mol L−1, respectively.

It was also planned to develop a selective methods for PC analysis in pharmaceutical

determinations, and the methods were based on reaction between ethylacetoacetate and

PC by sulfuric acid (dehydrating agent) producing a coumarinic compound, which was

spectrofluorimetrically calculated (Schirmer et al., 1991; Walily et al., 1999; Gikas et al.,

2003).

Xu et al., 2009, FI analysis for the determination of acetaminophen investigated by an

amperometric detector with gold nanoparticle modified carbon paste electrode. The

obtained results were found to be comparable with HPLC.

A vast deal of interest has been point out on the Substances immobilized onto the

electrode surface and chemically modified electrodes under the effect of external electric

fields able to mediating fast electron transfer (Majdi, 2007; Pournaghi-Azar & Sabzi,

2004, Golabi & Irannejad, 2005).

2.3. Spectroscopic Methods Spectrofluorimetry for the UV–visible region can be employed to achieve the

measurements in solid matrix (Moreira et al., 2004), leading to favorable characteristics

of sensitivity, straightforwardness, selectivity, rapidity and ruggedness etc.

22

Nondestructive analyzes were carried out that follow the current tendency towards clean

chemistry. Moreover, the opportunity of optical-fiber accessories for in situ and/or on-

line analysis (Bosch, 2006; Utzinger and Richards-Kortum, 2003) becomes possible.

Now a day, this approach has scarcely been exploited in relation to solid samples of

pharmaceutical importance. Regarding the paracetamol analysis, a literature review tells

that it generally involves the derivative reactions (Pulgarin & Bermejo, 1996; Murillo &

Garcia, 1996) because paracetamol was not basically fluorescent in aqueous solutions. In

this view beginning experiments confirmed that paracetamol was fluorescent in the solid

phase (Bosch, 2006).

Various spectrofluorimetric methods reported for the quantification of single PC or as a

mixture with other drugs in pharmaceutical preparations, such as indirect determination

using Ce (IV) as an oxidant agent (de los, 2005; Amann et al., 1980), reaction with

fluorescamine (Nakamura & Tamura., 1980), 1-nitroso-2-naphthol (Shah & Balaraman,

1999), oxidation with 2,2-dihydroxy- 5,5-diacetyldiaminebiphenyl (Vilchez et al., 1995),

potassium hexacyanoferate (III) (Pulgarín & Bermejo, 1996). Conversely, several of

these methods show low selectivity and interference with other medicines and excipients

can be predicted.

Continuous flow systems are valuable devices for the automation, miniaturization and

preliminary operations, particularly as regards simplification of analytical processes.

These systems afford the development of various chemical reactions and the

implementation of reliable separation techniques with a view to increasing sensitivity and

selectivity (Criado et al., 2000; Valcarcel et al., 1998;). The on-line microwave assisted

23

hydrolysis followed by chemical reaction was used in the analysis of PC (Bouhsain et al.,

1996).

Usually it not possible to determined the PC by both batch and FIA modesin direct US

spectrophotomtery in the presence of compounds that were often found along with it, due

to the interference caused by them. To solve this problem occurred from the non-specific

absorption in this spectral region, derivative reactions have often been used in order to

give colored compounds (Ayora Canada, 2000; Murfin, & Wragg, 1972; Hassan et al.,

1981).

An additional optical determinative technique such as spectrofluorimetry more sensitive,

also needs the use of derivative reactions (Ayora, 2000; Vilchez et al., 1995), because

paracetamol was not an intrinsic fluorophor and, in any case, the determination will be

quicker and cheaper than that obtained by spectrofluorimetry if direct UV

spectrophotometry is used.

Pereira et al., 1998, proposed a method based on the on-line microwave-assisted alkaline

hydrolysis of acetylsalicylic acid to salicylic acid that reacts with Fe (III) to form a

complex that absorbs at 525 nm. The precision for ten successive measurements of 200

μg/ml acetylsalicylic acid presented a relative standard deviation of 0.40%. The detection

limit was 4.0 μg/ml and recoveries of 99.1–101.0% were obtained for acetylsalicylic acid.

Another approach described by Criado, et al., 2000, for the analysis of PC and its major

metabolites using fully automated screening system in human urine samples. The

detection limit reached, 0.1 mg ml-1.The proposed method is based on direct acid

24

microwave assisted hydrolysis of the drug to p-aminophenol after that the reaction with

o-cresol in alkaline medium.

The benefit of derivative spectroscopy has reported by Bermejo et al., 1991, for the

simultaneous determination and identification of PC and plasma salicylate using second

order derivative spectra after a normal extraction process (Jelena, et al., 2003). Damiani

et al., 1995, also reported the applications of the first derivative spectrophotometric

technique for the analysis of PC in blood serum.

Ragno et al., 2004, have reported that the UV analysis of a multicomponent mixture

contains tripelenamine, paracetamol, salicylamide and caffeine using partial least-squares

regression (PLS) and principal component regression (PCR). The PCR and PLS models

were compared and their predictive performance was inferred by a successful application

to the assays of synthetic mixtures and pharmaceutical formulations (Ragno, 2004).

Knochen et al., 2003 setted a extremely precise and sensitive FI method depends on the

nitration of paracetamol with sodium nitrite. The method was useful to the determination

of PC in oral solutions and tablets.

In view of reality that a luminol based CL system is applicable due to permanganate is an

oxidant and in a basic medium the oxidation of paracetamol can be done

(Easwaramoorthy, 2001; Seitz & Crit., 1981; Martinez & Gomez-Benito, 1990; Vilchez

et al., 1995).

A novel spectrophotometric method has developed by Afshari, et al., 2001, for rapid

quantification of acetaminophen in serum. Free unconjugated acetaminophen is separated

25

from other endogenous interferents by extracting the drug into ethyl acetate and

hydrolysis to p-aminophenol by treatment with acid and heat. The proposed method is

suitable for screening of drug overdose in an emergency situation with a linearity range

from 25 to 600 µgml−1.

2.4. Chromatographic Methods There were various methods has been reported for the quantification of paracetamol in

biological fluids as well in pharmaceuticals mainly, liquid chromatography coupled with

mass spectrometry (Lohmann & Karst., 2006; Lou et al., 2010), high performance liquid

chromatography (HPLC) (Šatínský et al., 2004; Goicoechea et al., 1995; Bari et al.,

1998), gas chromatography (Santos et al., 2007), and Gas Chromatography/ Mass

Spectrometry (El Haj et al., 1999).

However, all these process involve derivatisation or e extraction procedures. Urinary

screening of paracetamol was generally carried out by using acids (Bosch, 2006; Ray et

al., 1987; Davey & Naidoo, 1993) or enzymatic (Hammond et al., 1984; Edwardson et

al., 1989; Dasgupta & Kinnaman, 1993). The presented methods for the determination of

PC in biological fluids (urine, blood, plasma) largely use chromatographic technique

(HPLC and GC) (Wong et al., 1976; Lo and Bye, 1979; Ameer et al., 1981; Al-Obaidy et

al., 1995; Lau and Critchley, 1994; Goicoechea et al., 1995), and other chromatographic

methods. Plasma proteins are precipitated by a sulfotungstic acid reagent, and the

supernatant liquid was mixed with pyridine containing the internal standard, and

determined by gas chromatography. No interference was found, while the method was

suitable for 1x10-4 M levels of paracetamol in plasma (Pegon & Vallon, 1981).

26

Evans et al., 1991, determined salicylic acid in serum samples using liquid

chromatography, with amperometric detection. The serum extracts was found to be 0.06

mol dm–3 acetate buffer in 8% methanol (pH 5.0). The average recovery from serum was

found to be 60% with a relative standard deviation of 5.8%.

A easy and fast HPLC method by phenacetin as internal standard for simultaneous

determination of chlorphenamine maleate, caffeine and acetaminophen in the product

compound PC and chlorphenamine maleate granules (Sun et al., 2006). The accuracy,

precision and linearity of the method were satisfactory for the analyzed drugs.

The application of the ratio spectra derivative spectrophotometry and HPLC has been by

(Erk et al., 2001) for the direct analysis of PC and methocarbamol jointly in

pharmaceutical tablets. Erdal et al., 2001, developed the HPLC based first derivative ratio

amplitudes method for determination of paracetamol, caffeine and propyphenazon in

ternary mixtures and tablets, the amounts of caffeine and paracetamol in the ternary

mixture were determined using propyphenazon as a divisor.

A chromatographic method (HPLC) for the analysis of 4-aminophenol and major

adulteration of paracetamol has reported by Wyszecka-Kaszuba et al., 2003. The method

was sensitive up to 4 ng ml-1 and 1 ng ml-1 in capsules and tablets respectively. The

proposed method was effectively useful for the determination of commercially available

drugs.

27

2.5. Other Techniques In addition to above techniques there are several of analytical methods existing for the

analysis of PC in various types of samples. These include colorimetric (Knochen et al.,

2003), capillary electrophoresis (Chu et al., 2008, Heitmeier et al., 1999), micellar liquid

chromatography (Love et al., 1985), and many others.

Due to the hydrolysis of PC to 4-Aminophenol, it produced a colored compound with the

suitable reactions, they are time-consuming, however these kind of produres are not

enough convenient for PC determination in Pharmaceutical preparation. Flow-injection

(FI ) with chemiluminescense method was therefore sought to serve this purpose.

Chemiluminescence methods are regarded as sensitive and selective having various

advantages for pharmaceutical formulations (Townshend, 1990; Robards & Worsfold,

1992; Hindson & Barnett, 2001). The analysis of PC in biological fluids by using FI with

chemiluminescence based on the oxidation of paracetamol with cerium (IV) were also

reported (Koukli et al., 1989; Easwaramoorthy, 2001).

2.6. Quantification of Paracetamol in Biological Samples and Pharmaceuticals

Several analytical methodologies have been proposed for the determination of

paracetamol in pharmaceutical formulations and biological samples (Bosch et al., 2006,

Fanjul-Bolado et al., 2009). Clinical (blood and urine) determination of PC formulations

using urinary excretion data has been well documented (Goyal, 2006; Welch & Conney,

28

1965; Wong et al., 1976; Mattok et al., 1971; Sotiropoulus et al., 1981; Vila-Jato et al.,

1986; DomÃnguez et al., 2000; Su and Cheng, 2010).

The combination of mefenamic acid and paracetamol was described for the simultaneous

determination by (Dinç et al., 2002).

Criado et al., 2000, reported the method for the analysis of PC which was further diluted

and continually hydrolyzed in an alkaline medium by spectroscopic method. The average

R.S.D. of 2.4% shows the good validity of the method.

For the first time, a multiparameter responding flow-through system with solid phase

detection (a multiparameter optosensor) was described for the Simultaneous

determination of a mixture of three active principles (caffeine, paracetamol and

propyphenazone) using univariate calibration by UV spectrophotometric coupled with a

multiparameter optosensor detector. The application of the detector successfully

determined the analyte in commercial pharmaceuticals (DomÃnguez et al., 2000).

Jelena et al., 2003, reported a method to facilitate the direct and simple determination of

total paracetamol in urine by UV at the wavelength range 220–400nm.

The selective and simple spectrofluorimetrical method has been designed by de los et al.,

2005, for paracetamol determination in tablets.

Altair et al., 2005 demonstrated a rapid and simple method for direct analysis of

paracetamol in the solid state pharmaceutical formulations by fluorescence. Results were

29

compared with those obtained by the BP recommended method at the 95% confidence

level.

The FI analysis of PC was setted, based on its inhibitory effect on a luminol-

permanganate chemiluminescence system (Easwaramoorthy, 2001). The paracetamol

contents were analyzed in locally available pharmaceuticals. The recovery obtained was

in the range of 98.2–104.4%. The viability of the method was confirmed on actual

samples (Easwaramoorthy et al., 2001).

Ruengsitagoon et al., 2006 developed a simple chemiluminometric method for the

determination of paracetamol in pharmaceutical formulations using FI based on the CL

produced by the reduction of tris (2,2-bipyridyl) ruthenium(III) (Ruengsitagoon, 2006).

Pedrosa et al., 2006, reported FIA method for the analysis of PC in pharmaceutical

Formulation with a modified self-assembled monolayer (SAM) gold electrode with a of

3-mercaptopropionic acid. Fifteen days lifetime of the modified electrode was found.

Obtained results of amperometric proposed method will found comparable with

spectrophotomtery.

FIA with amperometric detection has been employed by Felix et al., 2007, for

quantification of paracetamol using a carbon film resistor electrode. This sensor exhibited

sharp and reproducible current peaks for acetaminophen without chemical modification

of its surface.

Cervini et al., 2008, evaluated a bare graphite-polyurethane composite as an

amperometric FI detector for the quantification of paracetamol in pharmaceuticals. The

30

results showed that graphite-polyurethane composite can be used as an amperometric

detector for flow analysis in the proposed determination.

Santos et al., 2008, reported a simple and fast approach for simultaneous determination in

pharmaceutical formulations of ascorbic acid and PC using FI method with multiple pulse

amperometric detection. In this method chemometric technique were also employed for

the better achievement of the results.

A novel type of modified glass carbon electrodes with the nickel magnetic nanoparticles

was made-up to determine the electrochemical properties of N-acetyl-p-aminophenol.

Differential pulse voltammetry (DPV) was used for the determination of ACOP in

effervescent dosage samples (Wang et al., 2007).

Safavi et al., 2008, investigated an easy electrochemical method for the simultaneous

determination of p-aminophenol and paracetamol in drugs. The peak potentials (oxidation

and reduction) in cyclic voltammetry for paracetamol on carbon ionic liquid electrode

were significantly improved in contrast to the usual carbon paste electrode.

Zhao et al., 2006 reported the application of the indirect determination of paracetamol by

a capillary electrophoresis–chemiluminescence (CE–CL) detection based on its inhibitory

effect on a luminol-potassium hexacyanoferrate (III).

Santhosh et al., 2009, determined the concentration of paracetamol in human blood

plasma and commercial drugs by modification of Au electrode with tetraoctylammonium

bromide stabilized gold nanoparticles attached to 1,6-hexanedithiol. (ATR)-FT-IR were

use to confirmed the modified Au surface.

31

Recently Lou Hong-gang et al., 2010, developed a highly sensitive and simple LC–

MS/MS method after one-step precipitation for the simultaneous determination of

pseudoephedrine, paracetamol, chlorpheniramine and dextrophan,, in human plasma

using diphenhydramine as internal standard. The method was successfully validated for

the determination of pharmaceutical formulation.

2.7. Quantification of Aspirin in Pharmaceutical and Biological Samples Aspirin was begin famous in the late 1890s and has been deal with range of inflammatory

conditions (Supalkova et al., 2006). Many folk remedies used since pre-historic times

have depended upon salicylates for their effect. 100 years ago aspirin was prepared from

acetic and salicylic acids. It was the first drug to be synthesized and its formulation is

regarded as the base of the current pharmaceutical industry.

Commonly, acetylsalicylic acid (ASA) is indirectly determined after its conversion to

salicylic acid (SA) and acetic acid by alkaline hydrolysis according to a British

Pharmacopoeia method, (British Pharmacopoeia, 1980). Another reference method for the

determination of salicylic acid was the spectrophotometric method based on the Trinder

reaction, (Trinder, 1954).

The SA was the most important metabolite of the ASA, which obtained by the hydrolysis.

A large number of analytical approaches such as potentiometry (Kubota et al., 1999),

spectrophotometry (Sena et al., 2000; Merckle & Kovar 1998; Loh et al., 2005; Vidal et

al., 2002; Ruiz- medina et al., 2001), amperometry (Rover et al., 2000; Rover et al.,

1998; Pasekova et al., 2001), chromatography (Nogowska et al., 1999), UV detection

32

(Glombitza & Schmidt, 1994; Matias et al., 2004) and fluorescence (Martos et al., 2001),

spectrofluorimetry (Criado et al., 2000; Arancibia et al., 2002; Moreira et al., 2004),

methods have been also expressed for the analysis of ASA and SA in pharmaceutical

formulation. Conversely it seems that they are not commonly used, maybe due the

tedious and difficult sample preparations, usually liquid chromatographic methods were

used (Pirola et al., 1998; Franeta et al., 2002; Šatínský et al., 2004; Stolker et al., 2004).

Some time atomic absorption spectroscopic methods were also used but such methods are

expensive and need extraction procedures. However ion selective electrode showing high

specificity, good detection limit and comparably cheapest (Pasekova et al., 2001). Some

other electrode has been also used for the determination aspirin such as nickel oxide-

modified nickel electrode in alkaline solution (Kowal et al., 1997; Jafarian et al., 2003;

Yousef Elahi et al., 2006; Majdi et al., 2007), and glassy carbon electrode (Houshmand et

al., 2008). A number of flow injection spectrophotometric methods were also use on-line

hydrolysis (Koupparis & Anagnostopoulou, 1988; Quintino et al., 2002; Catarino et al.,

2003; Quintino et al., 2004, Garrido et al., 2000; Rover et al., 1998).

Many developed methods has shows benefit regarding this containing some limitation

also for the particular demand of analysis. In contrast with methods, electrochemical

sensors are selective option for electroactive species because of high sensitivity and

economical (Prasek et al., 2006, Babula et al., 2006).

Supalkova et al., 2006, developed the method for indirect determination of acetylsalicylic

acid in pharmaceutical drug. Electrochemical sensor for acetylsalicylic detection was also

33

suggested. SWV using both CPE and graphite pencil as working electrode was

successfully applied for determination of ASA.

Majdi et al., 2007 and Houshmand et al., 2008, studied the electrocatalytic oxidation of

aspirin on a nickel oxide-modified nickel electrode and nanoparticles of cobalt hydroxide

electro-deposited on the surface of a glassy carbon electrode respectively in alkaline

solution by chronoamperometry, cyclic voltammetry and electrochemical impedance

spectroscopy techniques as well as steady-state polarization measurements. The

investigative facility helped for the determination of aspirin by using of modified

electrodes. The method was proven to be valid for analyzing these drugs in urine samples

(Houshmand et al., 2008).

López-Cueto et al., 2002, proposed that when ASA was added to a bromine solution,

slow decompose of the bromine concentration occurs, while hydrolysis of aspirin yields

SA slowly, and bromine reacts quickly with salicylic acid. This behavior can be utilized

to develop kinetic methods for resolution of mixtures of SA and PC.

Matias et al., 2004, described the quantitative reflectance spot test procedure for the

analysis of acetylsalicylic acid in pharmaceutical formulations. In this method the

reaction of SA were carried out by the hydrolysis of acetylsalicylic acid with Fe (III)

forming a deep blue-violet complex. Drugs having acetylsalicylic acid can be easily

determined by the suggested method without any separation.

Šatínský et al., 2004 reported the simultaneous determination of paracetamol, caffeine,

acetylsalicylic acid, and internal standard benzoic acid on a novel reversed-phase

34

sequential injection chromatography technique with UV detection. The analysis time was

about 6 min. The method was found to be applicable for the routine analysis of the active

compounds paracetamol, caffeine, and acetylsalicylic acid in pharmaceutical tablets.

A simple and rapid analytical procedure was proposed by Sena & Poppi 2004, for

simultaneous determination of caffeine paracetamol and acetylsalicylic acid by UV

spectrophotometric method using multivariate calibration and measurements at 210–300

nm.

The on-line microwave assisted alkaline hydrolysis of acetylsalicylic acid has been

developed by Pereira et al., 1998, where the hydrolysis product (salicylic acid) reacts

with iron (III) to form a complex that absorbs at 525 nm. The method was applied to the

determination of the drug in tablets.

Tsikas et al., 1998, described gas chromatographic–tandem mass spectrometric method

for the accurate analysis of ASA in human plasma after a only low-dose oral

administration of 2 guaimesal or aspirin, an acetylsalicylic acid releasing pro drug.

Hansen et al, 1998, studied the separation three metabolites such as salicyluric, and

gentisic acid of acetylsalicylic acid in a non-aqueous capillary electrophoresis system

with reversed electro osmotic flow.

Amperometric biosensor for the salicylate determination in blood serum has been

described by Rover et al., 2000.

35

The quantification and separation of the anti-nerve agent pyridostgmine bromide, the

analgesic drugs ASA and acetaminophen, and the stimulant caffeine in rat plasma and

urine has been reported by Abu-Qare et al., 2001. The resulting chromatograms showed

no interfering peaks from endogenous plasma or urine components.

2.8. Quantification of Diclofenac Sodium in Pharmaceutical and

Biological Samples Different analytical methods have been employed for the quantification of diclofenic

sodium (DS), such as spectrophotometry (Matin et al., 2005), fluorimetry (Aranciba et

al., 2000), FT-Raman spectroscopy (Mazurek & Szostak, 2006; 2008), potentiometry

(Pimenta et al., 2002), chromatography (Lala et al. 2002), voltammetry (Yang et al.,

2008) and polarography (Xu et al. 2004). Most of these methods face certain problems

such as the use of additional reagents, complex formation, long time and hazardous

matrices. A method has been devised by Carreira et al., 1995, for the analysis of diclofenac sodium

in bulk using Eu3+ ions as the Fluorescent probe. The method was developed around the

hypersensitive property of the transitions of the fluorescent probe ion at 616 nm.

Fernandez de Coardova et al., 1998, have reported spectrophotometric method which was

a sensitive, easy and very selective for the quantification of diclofenac sodium in

authentic pharmaceutical drugs.

Gonzalez et al., 1999, developed a method for the simultaneous determination of

diclofenac, betamethasone, and cyanocobalamin (Vitamin B12), by HPLC. Linearity,

interday precision and accuracy for each active ingredient were analyzed.

36

Klimes et al., 2001, have reported the HPLC methodology to select a suitable acceptor

medium for permeation experiments and also determined the release of diclofenac and its

in vitro permeation through the human skin.

Lala et al., 2002, has been developed the method using high performance thin layer

chromatographic for the determination of diclofenac sodium from serum. Standard

diclofenac sodium was spotted on silica gel precoated plates using the mobile phase

toluene:acetone:glacial acetic acid.

Tubino & Rafael, 2006, reported a method for quantification diclofenac in

pharmaceutical drugs by diffuse reflectance. The finding results were compared with the

HPLC procedure recommended by the USP.

Rodriguez et al., 2007, investigated a tubular bismuth film electrode, installed as part of a

multisyringe FI system and utilized it as an amperometric detector for analysis of

diclofenac sodium in drugs samples.

Payán et al., 2009, described an extraction method using a polypropylene membrane

supporting dihexyl ether for the analysis of some analgesic formulations in human urine

samples by HPLC using a monolithic silica column such as ibuprofen, salicylic acid and

diclofenac.

Blanco-López et al., 2003, have developed a method for the determination of diclofenic

sodium by voltammetric sensor based on the molecular recognition of the analyte by

37

molecularly imprinted methacrylate ethyleneglycol dimethacrylate co-polymers. The best

results were obtained in 0.025 M citrate solution (pH 6) containing 10% of acetonitrile.

Fernández-Llano et al., 2007, reported the molecularly imprinted polymer for diclofenac,

prepared thermal polymerization over silica beads using 2-(dimethylamino) ethyl-

methacrylate as functional monomer. A selective solid-phase extraction of the drug from

urine followed by its quantification by ADPV.

Goyal et al., 2010, proposed the voltammetric determination of diclofenac using SWV at

edge plane pyrolytic graphite (EPPG) sensor from real urine samples. Jin & Zhang, 2000

have developed the for the detection of diclofenic sodium in urine sample by using

capillary zone electrophoresis using carbon fiber microelectrode, at a constant potential.

Xu et al., 2004 have investigated the polarographic response characteristics of diclofenac

sodium in 0.25M HAc-NaAc (pH 5.0) supporting electrolyte in the absence and the

presence of dissolved oxygen.

Mazurek & Szostak, 2006, have studied the FT-Raman quantification of minophylline

and diclofenac sodium. The efficiency of many spectra treatment protcols including

multivariate partial least squares and classical univariate intensity ratio and principal

component regression (PCR) methods was compared.

Mazurek & Szostak, 2008, have reported the method for quantitative determination of

diclofenac sodium in capsules and tablets by FT-Raman. PLS, PCR counter-propagation

artificial neural networks (CP-ANN) strategies were also emlyed in this method for its

fastness and convenient to the other pharmacopoeial methods. Wang et al., 2009, have

38

investigated a method for quantitative analysis of diclofenac sodium powder on the basis

of NIR spectroscopy.

Hajjizadeh et al., 2007, have developed a sensitive and simple amperometric procedure

for the analysis of mefenamic acid, diclofenac and indomethacin in bulk form and for the

direct assay of tablets, using the NHMN electrode. The electrocatalytic oxidation of these

drugs was examined on a nickel hydroxide-modified nickel electrode in alkaline solution.

Another sensitive, simple, and time-saving amperometric approach by Heli et al., 2009,

have investigated based on the electro-oxidation of several NSAIDs such as mefenamic

acid, diclofenac and indomethacin on nanoparticles of Ni–curcumin-complex-modified

glassy carbon electrode in alkaline solution. Atomic force microscopy and scanning

electron microscopy were used for surface studies.

Yilmaz, 2010, developed a procedure based on GC-MS for the determination of

diclofenac in human plasma. This assay was successfully applied in Turkey to healthy

volunteers after an oral administration of 50 mg diclofenac.

García et al., 1998, have reported a rapid FI spectrophotometric method for the analysis

of diclofenac sodium in pharmaceuticals and urine samples.

Pimenta et al., 2002, have reported the two autonomous methods for the analysis of

diclofenac simultaneously applied in an automated analytical system, based on the

concept of sequential injection determination, providing real-time assessment of results

quality. ISE based on cyclodextrin were used for the potentiometric detection.

39

Pérez-Ruiz et al., 1997 reported the spectrophotometric determination the trace quantity

of diclofenic were determined by liquid–liquid extraction. Furthermore New, rapid and

accurate spectrophotometric method for the analysis of diclofenac sodium in drugs

sample which is based the reaction of nitric acid (Conc. at 63% w/v) proposed by Matin

et al., 2005.

2.9. Quantification of Ibuprofen in Pharmaceutical and Biological

Samples A rapid quantitative analysis of ibuprofen in plasma of human blood was carried by

Jonkman et al., 1985, using a SFE on “Baker” C-18 disposable extraction column. The

proposed method was a simple and sensitive.

Stefan and Heitmeier & Blaschke, 1999, have investigated reliable screening method that

allows determination of the drugs its metabolites in human urine after oral administration

(Heitmeier, 1999).

Damiani et al., 2001, have described the determination of ibuprofen in pharmaceutical

creams, tablets and syrups without any interference of the excipients by

spectrofluorimetric method. It involved emission at 288 nm and excitation at 263 nm. The

linear range was 2–73 mgL−1.

Palabiyik et al., 2004, described the spectrophotometric methods for the simultaneous

determination of pseudoephedrine hydrochloride and ibuprofen in their combination. The

range 100–1300 µg ml−1 for pseudoephedrine hydrochlorides and 300–1300 µg ml−1 was

found for ibuprofen. Hamoudová et al., 2006, have illustrated that the capillary zone

electrophoresis with by using spectrophotometric for the determination of flurbiprofen

40

and ibuprofen. A fused silica capillary with UV detection was used for separation which

carried out at 232 nm. The method was successfully validated for the analysis of 10

commercially available tablets, syrup, gel and cream. Hassan, 2008, presented three

methods for simultaneous analysis of PC and ibuprofen without any earlier separation. 1st

derivative UV with zero-crossing measurement was the first method. Second depends on

1st derivative of the ratio-spectra by measurements. 3rd method was depends on

multivariate spectrophotometric calibration for the simultaneously analysis of

components.

Costi et al., 2008, have proposed the practically and quantitative solvent-free solid-phase

extraction (SPE) of ibuprofen and naproxen from sewage samples. Khoshayand et al.,

2008, have reported the simultaneous determination of caffeine paracetamol and

ibuprofen in pharmaceuticals by chemometric approaches using UV spectrophotometry.

Rapid, simple and accurate economical spectrophotometric method for simultaneous

determination of PC and ibuprofen in combined soft gelatin capsule dosage form has

been developed using two wavelengths at 224 and 248 nm simultaneously (Riddhi et al.,

2010).

Sochor et al., 1995, described the HPLC method for the analysis of ibuprofen in plasma

and isolated erythrocytes. C-18 column were for the Practical and methanol-water

(220:100, v/v) were used as mobile phase.

41

A HP-TLC method for quantitative determination of ibuprofen in plasma was explained

(Save et al., 1997). The limit of detection 50 ng was found for ibuprofen from human

plasma.

Kang et al., 1998, studied the quantitative aspects of HPLC with a column-switching

system and CE for the determination of ibuprofen in plasma. Farrar et al., 2002,

developed a fast and easy method of analysis the ibuprofen in small volumes of human

plasma (50 µl) by HPLC. The calibration curve was linear and limit of quantitation of

1.56 µg/ml.

De Oliveira et al., 2005, described an easy, sensitive and fast off-line solid-phase

microextraction process coupled with HPLC for the stereoselective analysis of the major

metabolites of ibuprofen in human urine samples.

Agatonovic-Kustrin et al., 2000, studied the enantiomeric purity of ibuprofen in a simple

manner by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy with

artificial neural networks (ANNs) methodology. Russeau et al., 2009, have used the

ATR-FTIR and target factor analysis to explore the pharmaceutical gel containing

ibuprofen in human skin. Results revealed that the data were effectively correlated

between reference spectra of the components and factors from the data.

Hergert & Escandar, 2003, have studied the complexation of ibuprofen in ß-cyclodextrin

using spectrofluorimetry at both acid and alkaline pH successfully.

Valderrama & Poppi, 2010, reported the spectrofluorimetric second order standard

addition method for the determination of ibuprofen in human plasma and urine. The

methodology was based on chiral recognition of ibuprofen by formation of complex with

42

a β-cyclodextrin, in the presence of 1- butanol. The obtained results were in the molar

fraction range from 50 to 80% of ibuprofen, providing absolute errors lowers than 4.0%

for plasma and urine. Glówka, & Karazniewicz, 2005, developed a method for the

quantitative determination of ibuprofen in biological matrices, human serum and urine

using direct and stereospecific capillary zone electrophoresis (CZE). Bauza et al., 2001,

used in situ chiral derivatisation to get diastereomeric amides of ibuprofen for their

subsequent extraction with supercritical carbon dioxide. Urine sample containing

ibuprofen were spiked to reveal the suitability of this method

Sádecká et al., 2001, quantified ibuprofen and naproxen in tablets by capillary

isotachophoresis. Linearity was from 40.0 to 200.0 mg L−1 of ibuprofen, with a

coefficient of determination of 0.999.

Tapan et al., 2010, have developed simple reproducible simultaneous equation method,

requiring no prior separation, for the assessment of PC and ibuprofen in combined dosage

form. Ibuprofen has absorbance maxima at 220 nm and paracetamol at 248.40 nm using

ethanol as solvent. The method obeys Beer’s Law in concentration ranges employed for

the estimation.

Recently Issa et al., 2010, reported a simple, rapid and accurate method for the

simultaneous spectrophotometric determination of ibuprofen and paracetamol in two

components mixture and Cetofen tablets. Calibration graphs were linear in the range 2–32

µg ml−1 (LOD 0.53 µg ml−1) and 2–24 µg ml−1 (LOD 0.57 µg ml−1) ibuprofen and

paracetamol respectively.

43

Chapter-03

EXPERIMENTAL

3.1. Material and Methods for Paracetamol using UV-Visible Spectrophotometry 3.1.1. Reagents and Chemicals Acetonitrile, chloroform, 2-propanol, hydrochloric acid, ammonia, sodium hydroxide,

paracetamol (PC), acetyl salicylic acid, ibuprofen, caffeine, diclofenic sodium, with

>99% purity were obtained from Merck (Germany). Ascorbic acid, sodium sulfide,

ammonium iron (III) sulfate, sulfuric acid and ethanol were from Fluka Chemicals.

Methanol was obtained from a local sugar industry and triply distilled before use.

Doubly distilled water was used throughout for final washings and preparations of all

aqueous solutions. Aqueous PC stock solution was prepared by warming the solution

for 10–20 min at a boiling water bath until complete dissolution of the analyte. The

solution was then cooled to room temperature and made up with doubly distilled

water.

3.1.2. Instruments and Apparatus Lambda 2 UV/visible spectrometer of Perkin-Elmer company was used for the effect

of all parameters and analysis of samples by recording the respective absorbance

value at definite wavelength. FTIR spectra of the respective reagents and compounds

were recorded by means of a Nicolet Avatar Model 330 FTIR of Thermo Electron

Corporation. Temperature adjustment during experiments was carried out with

controlled temperature Water Bath, Model, GMBH D-7633, Julabo HC5, Germany in

which a netted tray was fitted for test tube holding in vertical position.

44

3.1.3. Procedure for Determining PC PC was determined in dilute standard solution after taking it in the specific 1 cm

quartz cell against a blank (doubly distilled water) at a λmax value of 243 nm by

recording the respective signal or absorbance value using a controlled unit. A

calibration range was set for PC by recording the spectra of various standard

solutions. The locally available tablets containing PC were also analyzed by the same

procedure after dissolution of five replicate portions (each tablet containing 0.01

g/100 mL water) randomly selected from weighed three powdered tablets of each

manufacturer company in the similar manner as accurate for preparing standard PC

stock solution. During temperature related experiments, the temperature of PC and

blank solutions were first regularized with controlled temperature water bath and then

transferred to quartz cells very smartly but carefully for quick UV analysis. After

several experiments it was found that there was a variation of ±1 ◦C temperature. The

dilute samples analyzed were evaluated for their PC contents after fitting their average

of replicate spectra in calibration plot and multiplying the result with dilution factor.

3.1.4. Analysis of PC in Urine Samples Collection of urine samples from four volunteers was conducted in the same way as

described elsewhere [Jelena et al., 2003]. Each individual was instructed not to use

any medication before two weeks of urine collection. The urine samples were then

collected in thoroughly washed and clean plastic bottles in the morning time from full

bladder without any dose of PC. After this, each individual was instructed to eat the

same diet he has used yesterday. Each of them was administered with a PC tablet of a

specific brand containing 500 mg PC after 12 h of previous urine collection. The next

day the urine samples were collected after 12 h in the same way as true for samples

without PC. After same proper dilutions, the samples were processed for UV

45

spectrophotometric determination of PC with the procedure described earlier by

taking the diluted sample without PC as blank and PC containing as the actual sample.

The difference gave the PC contents in the urine sample which was converted to

actual value after adjusting for dilution factor.

3.2. Material and Methods for Diclofenac Sodium using UV-Visible Spectrophotometry

3.2.1 Apparatus The apparatus were used as same describe in section 3.1.2.

3.2.2. Washing of Glassware All glassware was washed by soaking in 3 M HNO3 overnight followed by washing

with detergent water. It was then thoroughly washed with tap water and finally rinsed

at least 3 times with doubly distilled water. The glassware was then dried in an oven

at 110 0C.

3.2.3. Reagents and Solutions All the reagents used in this study were of analytical grade ultra pure quality from

Merck, Fluka and BDH, etc. Diclofenac sodium (DS) and other pharmaceutical

standards were provided by Birds Chemotec Karachi, Pakistan. The identity of

pharmaceutical standards was checked by Fourier Transform Infrared spectra and

comparing these with the relevant data found in literature. Stock standard solution

(w/v) of diclofenac sodium (1 mgmL-1) was prepared in 100 mL calibrated volumetric

flask and diluted to the mark with doubly distilled water. Dilute working standards

were prepared from time to time as per requirement. Solutions of other reagents were

also prepared in doubly distilled water in the desired concentration.

46

3.2.4. Procedure for Determining DS in Tablets Four brands of tablets containing DS from different manufacturers were purchased

from local market and analyzed by using the current method. Ten tablets from each

brand were finely powdered and mixed. An amount equal to the average weight of

one tablet was collected randomly, transferred to a 100 mL volumetric flask,

dissolved and made up with doubly distilled water. Dilute solutions were made from

each sample and analyzed by UV-Vis Spectrometer. The concentration of DS per

tablet was calculated with the help of equation for linear calibration curve of DS and

multiplying with relevant dilution factor.

3.2.5. Procedure for DS in Serum and Urine Samples The blood and urine samples were collected before the intake of DS (blank) at day 1

and after taking DS at day 2. The individuals were instructed not to use any analgesics

including DS one week before examination and use the same common diet at day 1

and day 2. The DS was administered just after the collection of blank urine samples.

The blood samples were collected after 2 hour of dosage while urine samples were

collected after 12 hours of administration of 50 mg of tablet. Serum was obtained as

supernatant from blank and DS containing clotted blood samples by centrifugation

method. In order to freed the urine and serum sample from water insoluble impurities,

1 mL of blank as well as DS containing sample were passed through a column DSC-

18 (used in solid phase extraction), which was pre-washed with 2 mL of methanol.

The recovered urine or serum sample was diluted to 10 mL with doubly distilled

water and analyzed for DSF contents by UV-Visible spectrometer taking water as

actual blank (reference) to record the spectra of blank and DS containing serum and

urine samples. The difference of absorbance between blank and actual sample gave

the concentration of DS after fitting the value in linear equation and multiplying the

47

result with dilution factor. This treatment was carried out due to the difficulty in

finding out the absorbance in case of taking actual serum or urine sample as blank

against DS containing serum or blood sample. Standard solution of DS was spiked to

each serum or urine sample in order to confirm the peak of DS.

3.3. Material and Methods for the Determination of Paracetamol using

Differential Pulse Voltammetry 3.3.1. Reagents and Solutions The stock solution of Paracetamol (N-acetyl-4-aminophenol, purity 98%, Aldrich,

Germany) (c = 1×10-3 mol.L-1) was prepared by means of dissolving 0.0076 g of the

substance in 50 mL of de-ionized (DI) water. Further dilutions were prepared by

required volume of the stock solution with DI water. All solutions were stored in the

dark, at laboratory temperature and in glass vessels. Other chemicals such as boric

acid, glacial acetic acid, phosphoric acid, sodium hydroxide, all % purity were

purchased from Lachema Brno, Czech republic and potassium chloride was

purchased from Lach-Ner, Czech republic. MilliQ plus system (Millipore, USA) was

used for deionized water. The conductive carbon ink solution was arranged by

combination 0.01 g of polystyrene (expanded – packaging box, Merck, Germany),

0.09 g of carbon powder (crystalline graphite 2 µm, CR 2 Maziva Tyn, Czech

Republic) in 0.5 mL dichlorethane (purity 99.5%, Merck, Germany). The mixture was

thoroughly homogenized by agitation.

3.3.2. Apparatus Eco-Tribo Polarograph coupled with POLAR PRO software version 5.1 (Polaro-

Sensors, Prague) was employed for all voltammetric measurements. The instrumental

software joined under the operational system Microsoft Windows XP (Microsoft

Corp.). All measurements were carried out in a three-electrode system. Silver/silver

48

chloride reference electrode ETP CZ R00308 (1 mol.L-1 KCl, Monokrystaly Turnov,

Czech republic), platinum wire as an auxiliary electrode ETP CZ P01406, and CFE

(based on AgSAE, no. 2-05-19 from Eco-trend Plus, Czech Republic, disk diameter

0.5 mm, covered by carbon ink film) as a working electrode were used.

The optimized parameters such as scan rate 20 mV.s-1, the pulse amplitude 50 mV,

sampling time of 20 ms beginning at 80 ms after the onset of the pulse were used.

Better reproducibility of voltammetric measurements at CFE was assured by a

suitable electrochemical regeneration [Fischer et al., 2007] of CFE. Optimal results

were obtained for pH 4 with electrochemical regeneration by the application of

periodical switching every 0.1 s between potentials –400 mV (Ereg1) and 1300 mV

(Ereg2) in the given measured solution for 30 s (150 cycles).

3.3.3. Procedures The voltammetric measurements were carried out by taking appropriate amount (1 µL

– 1000 µL). Paracetamol solution in water was added into a 10 mL volumetric flask,

which was filled up to the mark with corresponding Britton-Robinson buffer and then

transferred into an electrochemical cell. Oxygen was removed from measured

solutions by purging with nitrogen for 5 minutes.

Current of peak was measured from the straight-line connecting minima on both sides

of the peak. Current of background electrolyte was subtracted from current of peak.

The calibration curves were measured in triplicate and evaluated by the least squares

linear regression method. Limit of determination (LOD) was calculated using a

10S/slope ratio, where S is the standard deviation of the mean value for 12 analyte

determinations at the concentration corresponding to the lowest peak on the

49

appropriate calibration straight line according to IUPAC recommendations (Inczedy et

al., 1998). All the measurements were carried out at laboratory temperature.

Determination of Paracetamol in pharmaceutical samples was carried out by standard

addition method. Given tablet was dissolved in 100 mL of deonized water and 100 µL

of this solution was diluted by Britton-Robinson buffer (pH 4.0) up to 10 mL and used

for voltammetric measurement, and standard additions of Paracetamol stock solution.

For testing of the quantitative investigation of paracetamol in samples of human urine

the method of calibration curve was used. Calibration curves and series of

determination for analyzed drug in model solutions of human urine were determined.

3.4. Material and Methods for the Analysis of Ibuprofen using FT-IR Spectroscopy 3.4.1. Reagents and Samples The stock solution of Ibuprofen purity 99%, (Merck, Germany) (c = 1000 ppm) was

prepared by means of dissolving 0.1 g of the substance in 100 mL of chloroform

99.98 % purity (Fisher scientific UK limited). Further dilutions were prepared by

required volume of the stock solution with chloroform. All solutions were stored in

the dark, at laboratory temperature and in glass vessels with tight cover. The different

pharmaceutical tablet samples containing ibuprofen as an active component were

purchased from pharmacy unit of Hyderabad, Pakistan.

3.4.2. FT-IR Spectral Measurements For the obtaining of infrared spectra of standard, tablet and urine samples, FTIR

spectrometer (Thermo Nicolet 5700) was employed fitted with detachable liquid cell

(KBr) and DTGS detector was attached. OMNIC software version 7.3 was used to

control the instrument. The mid-IR spectral range from 4000 cm-1 to 400 cm−1 was

50

selected, total 16 scans with the resolution of 4 cm−1. A new background spectrum of

chloroform was taken before recording the spectra of all sample and standard.

3.4.3. FT-IR Calibration Ten standard of Ibuprofen ranging from 10 to 100 ppm in chloroform were prepared

for biological fluids and pharmaceutical samples and the samples were quantitatively

determined by using Turbo Quant (TQ) analyst software.

The already recorded spectra by Omnic software of ibuprofen standards were opened

in TQ analyst program for the selection of particular region of carboxylic peak (1807-

1461 cm-1). Some peak parameters such as peak height, peak width for all Ibuprofen

standards were selected and these were calculated by TQ software which makes the

use of FI-IR as powerful analytical technique for both qualitative and quantitative

analysis also obtained excellent calibration curve between actual and predicted value.

3.4.4. Sample Preparation Procedure This method requires only grinding of tablet samples followed by dissolution in

chloroform for FTIR measurement. After weighing the tablet samples were grinded to

fine powder in mortar to minimize the particle size. Quantitative analyses of solutions

of ibuprofen in chloroform were performed in a cell with KBr windows and variable

optic pathway (Wilks). The KBr windows were scanned from 4000 cm-1 to 400 cm-1

on Thermo Nicolet 5700-FTIR spectrometer.

3.4.5. Collection and Preparation of Urine Samples Urine samples were collected from healthy volunteers. 2 ml of urine sample passed

through a column DSC-18 (used in solid phase extraction), which was pre-washed

with 2 ml of methanol. Then sample was washed with 5 ml of 5% methanol followed

51

by 1 ml chloroform. After washing the urine sample was spiked with known

concentration of ibuprofen.

3.5. Material and Methods for Investigation of Aspirin using Voltammetry 3.5.1. Reagents The stock solution of acetylsalicylic acid and salicylic acid (Aldrich Chem. Co.)

(c = 1×10-1 mol.L-1, c = 1×10-3 mol.L-1) were prepared respectively in de-ionized (DI)

water. Further dilutions were prepared by required volume of the stock solution with

DI water. All solutions were stored in the dark, at laboratory temperature and in glass

vessels. Other chemicals boric acid, glacial acetic acid, phosphoric acid, sodium

hydroxide, all p.a. purity were supplied by Lachema Brno, Czech republic and

potassium chloride was supplied by Lachner Tovarni Neratovice Czech republic.

MilliQ plus system was used for deionized water. The conductive carbon ink solution

was arranged by combination 0.01 g of polystyrene, 0.09 g of carbon powder

(crystalline graphite 2 µm, CR 2 Maziva Týn, Czech Republic) in 0.5 mL

dichloreathane (purity 99.5% Merck). The mixture was thoroughly homogenized by

agitation.

3.5.2. Apparatus Eco-Tribo Polarograph coupled with POLAR PRO software version 5.1 (Polaro-

Sensors, Prague) was employed for all voltammetric measurements. The software

connected with computer system Microsoft Windows XP (Microsoft Corp.) and P-

LAB a.s. Stuart Block heater model no (SBH 130 DC) was used for the hydrolysis of

Acetysalicylic acid.

52

3.5.3. Voltammetric Procedure All measurements were carried out in a three-electrode system. Silver/silver chloride

reference electrode ETP CZ R00308 (1 mol.L-1 KCl, Monokrystaly Turnov, Czech

republic), platinum wire as an auxiliary electrode ETP CZ P01406, and CFE (based

on AgSAE, disk diameter 0.5 mm, covered by carbon ink film) No. 2-05-19 as a

working electrode were used. The optimized parameters including the pulse amplitude

50 mV, pulse width 80 ms and scan rate 20 mV.s-1 were used.

Better reproducibility of voltammetric measurements at CFE was assured with a

proper electrochemical activation of CFE at 2200 for 120 seconds. The Optimum

results were achieved by the application of electrochemical regeneration potential

between same peak potential at 750 mV.

3.5.4. Indirect Determination of ASA After hydrolysis of ASA give DPV signal on the surface of CFE, according to

increasing pH and temperature of Britton-Robinson buffer. 1 mL ASA (900 µL of

Britton-Robinson buffer pH 12 and 100 µL of of ASA); (total concentration of ASA

was 0.2-100 μmol-1 L) were hydrolyzed at 90 °C for 60 min and analyzed by DPV on

the surface of CFE in the presence of 9ml Britton-Robinson buffer (pH= 2) up to total

volume of 10 mL.

3.5.5. Indirect Determination of ASA in Pharmaceutical Drugs and Urine

Samples by DPV at CFE The above-mentioned method for indirect determination of ASA has been applied for

analysis of pharmaceutical drugs. The tablets were homogenized and given mass were

dissolved in water. We took 40 µl of this solution, added 60 µl water and added, into

it Britton-Robinson buffer (900 µl, pH 12) for 60 min at 90 °C. After that, the

53

obtained extract (1 ml) was added to the 9 ml of supporting electrolyte (Britton-

Robinson, pH 2) and analyzed by DPV at CFE.

For checking of quantitative investigation of ASA in urine samples of human the

method of calibration curve was used.

54

Chapter-04

RESULTS AND DISCUSSION

4.1. Simpler Spectrophotometric Assay of PC in Tablets and urine Samples

Analysts do always try to process the analyte in a way which is suitable for its

optimum analytical signal response. In case of PC determination by UV/visible

spectrometry the standard or sample is hydrolyzed with alkali (Chunli and Baoxin,

1995, Andres, et al., 2003) to convert it to p-aminophenol which is then reacted with a

suitable ligand to get a signal at a specific wavelength. However, such treatment

makes the process not only time consuming but costly as well. In view of the aim of

the current study, the hydrolysis step was overcome by taking directly the photoactive

properties of the PC in aqueous solution. Moreover, the process was made further

economical and safe by avoiding the use of chemicals. Various optimization studies

conducted during this work are presented below.

4.1.1 Effect of Water Addition to PC (FTIR studies)

Fig. 4.1.1. describes the behavior of PC after adding doubly distilled water. It is seen

from the spectra that in case of original PC spectrum (A) the –OH and –NH stretching

is dominant along with absorption by groups like amides. Increase in water addition

(B) to (C) results in domination of –OH stretching over –NH stretching due to

addition of more and more –OH ions from water to PC along with diminishing of

spectra between 1700 to 800 cm−1 region. The spectra obtained at very low

concentration of PC in aqueous solution are almost of the same shape as true for pure

water. However, the FTIR spectra can be calibrated for higher concentration ranges as

quoted previously by Mitsuko et al., 2002 who proposed calibration model for PC

55

concentration at various temperature ranges based on the increment of spectral signals

with increase in concentration of PC in water in the range of 1800–1100 cm−1. Similar

FTIR studies for PC in water at room temperature have also been conducted in

another citation (Maiella, et al., 1998).

Fig. 4.1.1 FTIR spectra of (A), pure PC (B), aqueous PC paste and (C) aqueous solution of 5 µgml-1 PC.

In contrast to FTIR studies of water treated PC, the solid-state IR-LD spectral analysis

of monoclinic and orthorhombic PC, polymorphs of aspirin and arginine-containing

peptides have been described by (Ivanova 2005, Koleva, 2006 and Kolev, 2006),

respectively. Another reference is the application of FTIR and Raman spectroscopic

methods for identification and quantification of orthorhombic and monoclinic PC in

powder mixes (Al-Zoubi, et al., 2002).

4.1.2. Optimization of Time for Measurement and Stability of Analytical Signal

Fig. 4.1.2. describes the effect of time on the absorbance value for a 5 µg ml-1 aqueous

solution of PC during one hour duration at room temperature of 30 ± 10C against a

reagent (PC) blank.

56

0

0.1

0.2

0.3

0.4

0 10 20 30 40 50 60

Time (minutes)

Ab

sorb

ance

Fig. 4.1.2. Dependence of absorbance of PC on time

The effect of time on absorbance of 5 µgml−1 aqueous solution of PC during 1 h

duration at room temperature of 30±1 ◦C against a reagent (PC) blank was studied. It

was observed that absorbance remained constant throughout the whole period and

thus independent of time. Further observation showed that absorption of the

mentioned PC solution was constant for 72 h while it showed a 2.94% decrease after

96 h. It means that one can analyze PC in standard or sample at his own choice. So the

analysis is very much easy to determine quickly with little energy consumption. Such

constant absorbance vs time has also been demonstrated elsewhere (Jalil and Tsan-

Zon, 2001). As another comparison to current study, a stability time of 10 min has

been reported (Chunli and Baoxin, 1995), who used Fe+3 ions in the presence of S−2

ions for formation of methylene blue-like dye after reaction with p-aminophenol (the

hydrolysis product of PC).

4.1.3. Effect of Temperature The effect of temperature on absorbance of 5 µgml-1 PC at 243 nm was checked out in

the range of 5–50 ◦C with 5 ◦C intervals between two readings. It was seen that the

temperature at lower (5–15 ◦C) and higher (40–50 ◦C) range showed some

enhancement of absorbance which corresponds to a 2.94% increase as compared to

medium (20–35 ◦C) range.

57

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40 50

Temperature (0C)

Ab

sorb

ance

Fig. 4.1.3. Effect of temperature on absorbance of PC

The higher absorbance value at lower temperature may be due to the possibility of

adsorption of PC molecules which could be condensed on the inner wall of quartz

cell. The higher absorbance at higher temperature may be due to the possibility of

evaporation of some water molecules which could make the PC solution concentrated

and hence adding to absorbance value. However, room temperature (30±1 ◦C) was

taken as optimum temperature to avoid the possibility of false analytical signal due to

adsorption at lower or evaporation of water at higher temperature. A temperature

range of 20–60 ◦C has also been described for evaluation of PC by spectrophotometry

(Andres, et al., 2000). They have also chosen room temperature for PC determination

as other temperature values had no effect on its determination.

4.1.4. Effect of Polar Solvents The effect of addition of 0.01 ml of different solvents on the absorbance of a standard

solution of 5 µgml−1 PC at other optimized conditions is outlined in Table 4.1.1. It is

seen that the addition of aliphatic alcohols in minute quantity has no effect at all.

58

Table 4.1.1. Effect of different polar solvents on absorbance of PC.

Solvent Absorbance Effect (%)

Methanol 0.34 0.00

Ethanol 0.34 0.00

2-propanol 0.34 0.00

N-dimethyl formamide 0.32 -5.88

Acetonitrile 0.32 -5.88

Chloroform 0.35 +2.94

It may be due to increased miscibility and structural similarity of these alcohols with

water. In case of dimethyl formamide and acetonitrile, the negative effect may be due

to greater structural differences of these solvents with water. In case of chloroform,

the little bit higher absorbance value is because of increased concentration of PC in

water due to immiscibility of former in the later. However, the effect of solvent on PC

determination is rarely studied in literature but some authors (Marcelo and Ronei,

2004) have used a 20:80 (v/v) ethanol/water as better solvent mixture for

determination of PC, caffeine and acetyl salicylic acid.

4.1.5. Interference by various Analgesic Drugs The interference of various analgesic drugs to PC in various proportions was checked

to investigate the possibility of its determination in the presence of other drugs

because some companies use the combination of two or more analgesics for more

efficient response. Table 4.1.2 describes the interference caused by various analgesics

with various ratios present in a solution containing 5 µgml−1PC. It is evident from the

table that in a 1:1 ratio of drug to PC, there is little or no interference while in a 5:1

ratio, caffeine, diclofenic sodium and ascorbic acid show >±5.0% interference during

PC determination. In a 10:1 ratio the caffeine and ascorbic acid are showing minimum

59

interference as compared to their previous combinations. However, the last

combination is rarely taken in analgesic formulations. So we can say that except

caffeine and ascorbic acid, all other drugs are considered as producing no or little

effect in analysis of PC up to certain possible limits. Similar interference studies have

also been carried out by other workers (Chunli and Baoxin, 1995, Wirat and

Saisunee., 2006). According to later reference, ascorbic acid has been included among

the major interferers while caffeine has been declared as non-interfering one.

Moreover, in the same citation (Wirat and Saisunee., 2006) acetyl salicylic acid has

also been included in the major interferers but in that particular case the conditions

and solution parameters were quite different than ours (Table 4.1.2).

Table 4.1.2. % interference by various analgesics in different ratios on 5 µg ml-1 PC

Ratio to PC

% interference by drug

Caffeine Diclofenic

sodium

Acetyl

salicylic

acid

Ibuprofen Ascorbic

acid

1:1 +2.94 0.00 0.00 -2.94 -2.94

5:1 +8.82 +5.88 0.00 0.00 +11.76

10:1 0.00 +17.64 -14.71 0.00 -2.94

60

Table. 4.1.3. Effect of strong and weak acids and bases on PC determination

Acid / base (1M) Volume of acid

/ base (ml)

% effect pH of PC

solution

λmax

(mid point)

(nm)

Hydrochloric acid 0.1 -2.94 2.13 243

0.5 -2.94 1.72 243

1 -2.94 1.35 243

Acetic acid 0.1 -2.94 3.65 243

0.5 0.00 3.29 243

1 +2.94 3.16 243

NaOH 0.1 +2.94 11.43 257

0.5 0.00 12.28 257

1 +5.88 12.50 257

Ammonia 0.1 0.00 10.40 254

0.5 0.00 10.70 255

1 0.00 10.85 256

4.1.6. Effect of Acidic and Basic Solutions The effect of adding various volumes of 1Mof various strong and weak acids and

bases on the absorbance and wavelength shift of 5 µgml−1 PC solution was

investigated and the results are given in the following table (Table 4.1.3). It is

observed from the data that the addition of various quantity of 1MNH3 has no effect at

all on the absorbance of PC. HCl and CH3COOH have positive or negative

interference within the acceptable limit of ±5.0%. The only positive effect of +5.88 is

shown after the addition of 1ml of 1M NaOH. It is seen from Fig. 4.1.4 that

dependence of absorbance on concentration of PC is not much affected by variation in

pH in this case and we can say that it is independent of pH. This quality helps in the

analysis of PC in various types of acidic or basic solutions.

61

Fig. 4.1.4. Shift of wavelength with pH: (A) 3.3 and (B) 12. 3 for 5 µgml-1 PC solution.

4.1.7. Calibration Range The dependence of various concentrations of PC standard solution on absorbance in a

measurable working range was evaluated at optimized parameters.

Fig. 4.1.5. Calibration range of absorbance vs. concentration for PC from 0.3 to 20 µgml-1.

62

Fig. 4.1.5 shows linear calibration range for standard PC solutions from 0.3 to 20

µgml−1. The lower detection limit for this was 0.1 µgml-1 with linear regression

coefficient of 0.9999. The relative standard deviation for a 5 µgml−1 PC (n = 11) was

found out to be 1.6%. The calibration range obtained by this method is better than that

given by Erk et al., 2001, who reported a range of 2–30 µgml−1 for PC determination

by ratio spectra derivative spectrophotometry. The lower detection limit of 0.1 µgml−1

in our case is also closer to that of the later (0.097 µgml−1). The detection limit of

current method is better than that reported by Wirat et al., 2006. Who described lower

detection limit of 0.2µgml-1 for PC determination. The linearity of the current

calibration plot is also better than that reported by other workers who described a

linear regression value of 0.9974 for determining monoclinic form of PC (Ivanova,

2005) using IR-LD spectroscopy. The r value of 0.9999 for the current method also

proves the better linearity of our calibration plot in comparison to that described by

Al-Zoubi et al 2002 who obtained the r value of 0.9965 and 0.9954 for eight

calibration points each in case of monoclinic PC by FTIR and FT-Raman

spectroscopy, respectively. Moreover, the lower standard deviation and lower

detection limit values of the current method prove the advantage of its better

repeatability and greater sensitivity over all these solid state methods mentioned

above.

4.1.8. Analysis of Tablets Table 4.1.4. shows the results of various locally manufactured tablets containing PC

with mentioned and determined concentration of the analyte. Each true result was

obtained after multiplying the actual result with dilution factor 50. The results

obtained by the currently developed method are very close to those reported earlier

(Chunli and Baoxin, 1995), which prove its validity for determination of PC by this

63

method. Moreover, the standard deviation values by the current method are better than

later.

Table 4.1.4. Determination of PC in tables of various companies by proposed method

Chemical

formulation

Mentioned

concentration

(mg unit-1 )

Found concentrationa

(mg unit-1 )

By proposed method Reported method

[Chunli and Baoxin, 1995]

Paracetamol 125 121 ± 2.2 121 ± 2.0

Disprol 500 494 ± 2.2 491 ± 3.4

Rascodal 500 497 ± 2.7 499 ± 4.8

Calpol 500 485 ± 3.5 481 ± 3.8

Panadol 500 466 ± 4.2 468 ± 4.4

a, average value; ±, standard deviation (n=5)

Table 4.1.5. Comparative analyses of PC in urine samples.

Paracetamola

Urine sample By current method By reported method

[Jelena, et al., 2003]

(µg ml-1) % age (µg ml-1) %age

1 464 ± 13 92.8 459 ±18 91.8

2 475 ± 14 95.0 472 ± 12 94.4

3 434 ± 07 86.6 435 ± 16 87.0

4 427 ± 05 84.5 430 ± 09 86.0

N=11, other abbreviations, as for table 4.

4.1.9. Analysis of Urine Samples PC determination in four urine samples of four volunteers was conducted by

recording absorbance of 1000 times diluted samples of urine without PC and with PC

64

against double distilled water as blank in each case. The results obtained were

interpreted after adjustment for dilution factor. The data are presented in Table 4.1.5.

For determining PC in urine by comparative method (Jelena, et al., 2003), the dilution

factor of 100 was used for urine sample and the data obtained after proper adjustment.

It is seen from the table that the results obtained with the current method are very

close to those obtained with the reported method. The results show that ≤95%

paracetamol is excreted in urine after 12 h. Similar studies with 95% average

excretion of PC in urine after 12 h have been reported earlier (Jelena, et al., 2003),

which strengthens the confirmation of our results. UV spectra of diluted urine samples

with and without PC are presented in Fig. 4.1.6. An average difference of absorbance

<0.05 which corresponds to a <0.5 µgml-1 PC (<500 µgml-1 PC after adjustment for

dilution factor) has been observed in this and other cases. As already evident from

Fig. 4.1.4. that there is no prominent effect of pH on the absorbance of PC, so a λmax

of 234 is true in case of urine rather than 243 in case of standard solutions of PC.

Other samples also showed variation of wavelength with pH.

Fig. 4.1.6. A 1000 times diluted sample of urine (a) with PC and (b) without PC.

65

4.2. Simpler and Faster Spectrophotometric Determination of Diclofenac Sodium (DS) in Tablets, Serum and Urine Samples

The absorption spectra of DS in aqueous medium have been described by several

workers (Matin et al. 2005; Ferreyra and Ortiz 2002). However up to the best of our

knowledge, no attempt has been made so far to utilize the aqueous medium as one of

the most reliable matrices for determining DS with excellent working linear range,

sufficiently low detection limits and its application to many types of samples. So,

various parameters were studied for their effect upon the UV-Visible

spectrophotometric determination of DS.

4.2.1. Influence of Time The effect of time on the absorbance behavior of 5 µg ml-1 aqueous solution of DS at

276 nm was studied in the range of 1–90 minutes at room temperature of 25 ± 10C

using double distilled water as a blank solution (Fig. 4.2.1). It was observed that the

absorbance remained constant throughout the whole period and thus independent of

time.

0.1

0.12

0.14

0.16

0.18

0 10 20 30 40 50 60 70 80 90

Time (min)

A

Fig. 4.2.1. Time effect on absorbance of Diclofenac Sodium

66

Time effect has been described by other workers (Matin et al. 2005; Sirajuddin et al.

2007) as well. The constant absorbance with time confirms the stability of the analyte

and faster analysis of DS in aqueous solution.

4.2.2. Effect of Temperature The effect of temperature on absorbance of 5µg ml-1 DS at 276 nm was observed in

the range of 5–60 0C (Fig. 4.2.2). It was seen that lower temperature range of 5–25 0C

showed maximum absorbance, with a little bit decreased value for the range of 30–45

0C was observed followed by a further decrease thereafter.

0.12

0.13

0.14

0.15

0.16

5 15 25 35 45 55

Temperature 0C

A

Fig. 4.2.2. Temperature effect on UV absorbance of 5 µg ml-1 DS solution.

The possible reason of higher absorbance value at lower temperature may be due to

the adsorption of DS and/ water molecules on the wall of quartz cell and thus

absorbing a little bit higher amount of UV light (Sirajuddin et al. 2007). The lower

absorbance at higher temperature may be due to the instability of DS molecules.

67

However the room temperature (25 ± 1 0C) was taken as optimum temperature in

order to make the process simple by avoiding additional steps. Temperature studies

have also been reported earlier (Matin et al. 2005) regarding the assay of DS.

4.2.3. Interference by Various Analgesic Drugs The interference of various analgesic drugs at the absorbance on 5µg ml-1 DS solution

in various proportions was checked to investigate the possibility of its determination

in the presence of other drugs (Table 4.2.1).

Table 4.2.1. % interference by various analgesics in different ratios on 5 µg ml-1 DS

Ratio

to DFS

(%) Interference

Caffeine Paracetamol Aspirin Ibuprofen Ascorbic

acid

1:1

5:1

10:1

+5.47

+49.72

0.00

+39.72

+13.69

-12.32

-18.49

-11.64

+13.01

+ 1.36

-6.16

-10.27

-7.53

-58.90

-62.32

In a 1:1 ratio caffeine and Ibuprofen show little interference while PC and aspirin are

the major positive and negative interferers respectively. The positive interference by

PC may be due to its somewhat structural similarity with or interaction with DS. The

negative value shown by aspirin may be due interaction of central N atom of DS with

the –COO group of aspirin thus hindering its presence to some extent as actual

diclofenac. Other higher values of interference for a 5:1 ratio showing +49.72 %

interference in case of caffeine may be due to indistinguishable wavelength (275 nm)

(Ferreyra and Ortiz 2002) to that of DS (276 nm) which adds up to increase the total

absorbance of DS. No interference at 10:1 ratio of caffeine to DS may be due to rate

68

of interaction of DS with UV light in a first order manner where the higher

concentration of second analyte is usually constant. The highest negative interference

value for 5:1 and 10:1 ratio of ascorbic acid:DS may be due to the interaction of –

COO groups of DS with attached -OH groups of ascorbic acid through H-bonding

which then hinders the actual absorbing activity of DS. As the lower ratio (1:1, 1:2 or

2:1) is usually taken in most of combined drugs, hence we can say that at usual

combination level the mentioned drugs are showing interferences in acceptable

values. However, the presence of PC or ascorbic acid will interfere with the true

analytical value of DS.

This has been observed that the amount of caffeine is quite high in the biological

fluids such as urine, serum or whole blood because various natural foods contain

sufficient amount of caffeine which is ultimately transferred in these fluids. So, no

interference by the highest combination of caffeine and DS in 10:1 ratio is a good

indicator for application of this method in the presence of high amount of caffeine

especially in biological samples.

4.2.4. Effect of Acidic and Alkaline Conditions Spectrophotometric study of 5 5µg ml-1 DS solution was carried out in various

concentrations of strong and weak acids and bases. UV spectra of a 5 µg ml-1 DS

solution are given (Fig. 4.2.3) to describe the shift in wavelength and absorbance of

DS at extreme conditions of acidity and alkalinity.

69

200 225 250 275 300 325 3500.0

0.1

0.2

0.3

0.4

0.5

A

nm

Fig. 4.2.3. UV spectra of DS at acidic pH, 3.56 (lower) and basic pH, 11.68 (higher).

Moreover, a detailed sketch of such effects upon the addition of different strong and

weak acids and bases was also observed (Table 4.2.2). According to the data, more

negative interference is true at lower pH values of DS solution. However as the pH is

increased, the negative interference is decreased and reaches to acceptable limit. The

value of λmax shifted from 273 nm to 276 nm by entering from acidic into basic

conditions.

70

Table 4.2.2. Effect of strong and weak acids and bases on DFS determination

Acid / base (1M) Volume of acid

/ base (ml) % effect

pH of DS

solution

λmax

(mid point)

(nm)

Hydrochloric

acid

0.1 -10.67 2.44 273

0.5 -14.44 2.07 273

1.0 -18.31 1.73 273

Acetic acid

0.1 -5.97 3.78 273

0.5 -8.32 3.56 273

1.0 -12.06 3.24 273

Ammonia

0.1 0.00 9.85 276

0.5 -2.23 10.30 276

1.0 -2.98 10.65 276

NaOH

0.1 -1.0 10.93 276

0.5 -1.86 11.68 276

1.0 -2.23 12.34 276

Lower absorbance value of DS is linked with 2 factors. First is the solubility which

depends upon pH and it is reported that solubility of DS is decreased in acidic

solution. Secondly, DS is subjected to intramolecular cyclization at acidic pH and

hence inactivated (Palomo et al. 1999). In basic conditions a reverse of cyclization

restores the actual molecule along with maximum efficiency. However, a different

situation is presented elsewhere (Matin et al. 2005) where acidic medium (HNO3)

results in the formation of yellowish nitrated derivative of DS which absorbs heavily.

According to our opinion in a protons rich (highly acidic) medium all the Na+ ions are

not easy to freed the diclofenac ions available for maximum absorption because the

available protons (H+ ions) try to repel the formation of free Na+ ions due to similar

charges. So when the number of H+ ions decreases, the number of free diclofenac ions

is increased accordingly that results in increased absorption. In case of basic solution,

71

the Na+ ions are accepted by the negative OH- ions and hence sufficient diclofenac

ions are available for increased absorption. However the very little negative effect in

basic solution is attributed to base hydrolysis of very limited number of diclofenac

ions into smaller metabolites.

4.2.5. Calibration Plot UV spectra were recorded at 276 nm for standard solutions of DS with different

concentrations in the range of 0.1–30 µg ml-1 (Fig. 4.2.4.).

200 225 250 275 300 325 3500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

A

nm

Fig. 4.2.4. Calibration plot of absorbance verses concentration for DS solutions from below to above as 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 and 30 µg ml-1. Linear equation obtained from calibration plot is represented as; Y= 0.0269X+0.0041 with regression coefficient of 0.9998 and detection limit of 0.01 µg ml-1 at 276 nm.

A relative standard deviation of 0.33 % was observed for a solution of 5 µg ml-1 DS

(n=11) which describes an excellent reproducibility and repeatability of the method.

4.2.6. Comparison with Other Reported Spectroscopic Methods A comparison of linear calibration range and detection limits of the current method

with those of some other spectroscopic methods was also described (Table 4.2.3).

72

Table 4.2.3. Comparison of current method with other spectroscopic methods for determination of DS

Method Reference Linear range LOD

Spectrofluorimetry Damiani et al.,

1999

0.2 – 5.0 µg ml-1 0.2 µg ml-1

FI spectroscopy Garcia et al.,

1998

0.2 – 8 µg ml-1 0.023 µg ml-1

UV-Vis. Spectrometry Matin et al. 2005 1-30 µg ml-1 0.46 µg ml-1

UV-Vis. Spectrometry Botello J.C. and

Perez-Caballero.,

1995

0.8 –6.4 µg ml-1 0.37 µg ml-1

UV-Vis. Spectrometry Mitic et al., 2007 1.59-38.18 µg ml-1 1.29 µg ml-1

UV-Vis. Spectrometry Current method 0.1–30 µg ml-1 0.01 µg ml-1

It is quite clear that despite using various complexing agents, still the working ranges

and detection limits of the reported methods (Matin et al. 2005; Damian et al. 1999;

Garcia et al. 1998; Botello et al. 1995; Mitic et al. 2007) could not compete with that

of the currently developed method. The addition of other reagents for complex

formation makes the other methods time consuming, complicated and expensive. Due

to the lack of the mentioned problems, better sensitivity, broader linear range and

environmental friendly nature, the newly investigated method has a clear edge over

described methods. Moreover, the method is also better than some other reported

methods described in section 1, which possess nearly similar problems as true in case

of reported spectroscopic methods.

4.2.7. Analysis of Tablets A representative UV spectrum of DS in a randomly selected sample of tablets

(Diclofen) diluted to 5 µg ml-1 DS according to the mentioned concentration is

described (Fig. 4.2.5). The clarity of the signal proves no interference from the matrix.

73

2 0 0 2 2 5 2 5 0 2 7 5 3 0 0 3 2 5 3 5 00 .0

0 .1

0 .2

0 .3

0 .4

0 .5

A

n m

Fig. 4.2.5. Representative UV-spectrum of expected 5 µg ml-1 DS in (Diclofen) tablet.

The average results of various locally manufactured tablets containing DS with

mentioned and determined concentration for 5 replicate runs were recorded (Table

4.2.4). Each actual result was obtained after multiplying the determined concentration

with dilution factor of 100.

Table 4.2.4. Determination of DS in tablets of various companies by proposed

method Chemical

formulation

Mentioned

concentration

(mg unit-1 )

Actual Concentration a Recovery

(%)

mg unit-1

Fenac

Dichloran

Voltral

Ardifenac

Diclofenac

50

50

50

50

50

51.153 ± 0.005

50.646 ± 0.005

49.388 ± 0.008

50.923 ± 0.004

49.837 ± 0.003

102.31± 0.011

101.21 ± 0.011

98.68 ± 0.016

101.85 ± 0.008

99.87 ± 0.006

a, average value; ±, standard deviation (n=5)

74

The closeness of mentioned and calculated concentrations of DS in the collected

tablets samples proves the validity of the method and lack of interference from the

excipients.

4.2.8. Analysis of Urine and Serum Samples 10 fold diluted urine or serum sample was processed according to the procedure

mentioned in experimental section. Representative spectra of each of urine and serum

sample are demonstrated respectively (Fig. 4.2.6 and Fig. 4.2.7). For each spectral

observation, the diluted blank and DS containing urine or serum sample of the same

individual was processed.

200 225 250 275 300 325 350 375 4000.0

0.4

0.8

1.2

1.6

2.0

A

nm

Fig. 4.2.6. UV spectra showing blank (black), DS containing (blue) and DS spiked (red) urine.

75

200 225 250 275 300 325 350

0.00

0.05

0.10

0.15

0.20

0.25

0.30

A

nm

Fig. 4.2.7. UV spectra showing blank (black), DS containing (blue) and DS spiked (red) serum.

In case of blank urine sample a hump is seen which reflects the presence of sufficient

amount of caffeine because it has nearly similar λmax (275 nm) Ferreyra and Ortiz,

2002, as DS (276 nm). In all samples, the absorbance of blank was considered as zero

for DS. A clear signal represented by blue line indicates the presence and absorption

spectrum of DS in the urine sample of the individual. The value is however blue

shifted due to the acidic pH and presence of some other ingredients in case of urine

samples. In case of serum sample the lower spectrum shows the presence of caffeine

at lower concentration indicating that very little of it is retained by the serum while

most is removed in urine. The results also show that DS containing serum sample

from the individual administered with 50 mg tablet has a higher absorbance value as

compared to his blank serum sample showing the presence of DS because caffeine is

constant for both samples. The spiking of each diluted urine and serum sample was

76

performed with 40 µl of DS solution in order to confirm the peak signal and hence the

presence of DS in the serum sample.

The results of DS in 4 urine and 4 serum samples by the currently developed method

were observed (Table 4.2.5). Each sample of urine and serum with specific number

(e.g. urine 1 and serum 1) was collected from same individual at different times as

described in experimental section.

Table 4.2.5. Concentration of determined DS and its relation with ingested DS by oral administration

Sample type DS found (µg ml-1) a Actual DS (µg ml-1) a DS % of ingested

tablet

Urine 1 3.49 ± 0.010 34.9 ± 0.10 69.8

Urine 2 2.93 ± 0.0120 29.3 ± 0.12 58.6

Urine 3 2.34 ± 0.0160 23.4 ± 0.16 46.8

Urine 4 2.56 ± 0.020 25.6 ± 0.20 51.2

Serum 1 1.14 ± 0.004 11.4 ± 0.04 11.8

Serum 2 1.09 ± 0.004 10.9 ± 0.04 21.8

Serum 3 1.22 ± 0.005 12.2 ± 0.05 24.4

Serum 4 1.15 ± 0.006 11.5 ± 0.06 23.0 a, average of three replicates

DS in each sample was determined with the help of linear equation and multiplied by

dilution factor 10 in order to get the actual concentration of the ingested DS

transferred to respective urine or serum sample. The data show that the urine and

serum contents of DS have the range of 46.8–62.0 % and 10.09–12.2 % respectively

for a 50 mg ingested tablet. The remaining DS may be present in plasma and/ or

converted to inactive metabolites. As there is no satisfactory data available for DS

contents in these fluids hence we rely upon the currently investigated data. Further

studies in this regard could throw sufficient light upon actual kinetics of this drug and

its fate in the body fluids.

77

4.3. Differential Pulse Voltammetric Determination of PC in Tablet

and Urine Samples at Carbon Film Electrode 4.3.1. Influence of pH on PC at DC and DP Voltammetry. Firstly, the electrochemical responses of PC on CFE in Britton-Robinson buffer in pH

range from 2-12 were investigated by DC and DP voltammetry. As can be seen in

Figure 4.3.1.

200 500 800

0

1

2

2 7 12

200

400

600A E1/2

, mV

6

23

45

I, µA

E, mV

7

8

91011

12

pH

200 500 8000

2

4

2 7 12

200

400

600B

E, mV

6

23

4

5

1

I, µA

E, mV

78

910

1112

pH

Fig. 4.3.1. DC voltammograms (A) and DP voltammograms (B) of Paracetamol (c = 100 μmol L-1) at CFE in Britton-Robinson buffer pH 2 to 12 (numbers above curves correspond to given pH) without electrode regeneration. Inset is corresponding dependence of peak potential on the pH.

The influence of different pH from 2-12 in B-R buffer are summarized in (table. 4.3.1)

which shows different peak currents at different peak potentials of paracetamol at DC

and DP voltammetry.

78

Table 4.3.1. The influence of pH on DCV and DPV of Paracetamol, c = 100 μmol L-1

pH

DC Voltammetry DP Voltammetry Ip, nA Ep, mV Ip, nA Ep, mV

2 817 597 1633 584 3 905 553 1633 541 4 1360 508 2524 464 5 1170 463 2285 410 6 736 419 771 367 7 784 374 7123 317 8 748 329 598 297 9 1141 285 598 238 10 896 240 700 212 11 822 195 889 178 12 344 151 305 159

PC gives one well defined anodic wave / peak which is similar to that observed for an

unmodified glassy carbon electrode Miner et. al, 1981, Van Benschoten et al. 1983,

investigated the electrochemical oxidation of PC using cyclic voltammetry. The first

reaction step is an electrochemical oxidation involving two electrons and two protons

to generate N-acetyl-p-quinoneimine. All subsequent reaction steps are non-

electrochemical, but pH-dependent processes (slope around –50 mV per pH unit or –

43 mV per pH unit for DCV or DPV, respectively). For oxidations at pH values

higher than 6, the final product is a benzoquinone Skeika, et. al. 2008. The best

developed wave and also peak was obtained in BR buffer at pH 4 in aqueous medium.

This medium was further used for measuring of calibration dependences. DPV was

used for further measurements due to its higher sensitivity and easy evaluation.

4.3.2. Optimization of Parameters and Calibration Curve Repeated measurements revealed passivation of the electrode, probably by products of

the electrode reaction, resulting in decreasing peaks moving toward more negative

potentials. Effect of passivation of the electrode surface was reduced by

electrochemical regeneration of electrode surface with settings of regeneration

79

potentials to values Ereg1 = –400 mV and Ereg2 = 1300 mV (values were optimized by

series of trials) as shown in Fig.4.3.2.

200 500 8000

1500

3000

0 25 500

1500

3000 I

p, nA

I, nA

E, mV

N

Fig. 4.3.2. Repetitive measurements of 100 μmol L-1 Paracetamol using DPV at CFE in Britton-Robinson buffer pH 4.0 with regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV Inset is corresponding dependence of peak current on the number of scans.

Linear calibration curves were obtained using optimal regeneration potentials in the

concentration ranges from 0.02 to100 µmol L-1. These parameters are summarized in

Table 4.3.2.

Table 4.3.2. Parameters of the calibration straight lines for the determination of Paracetamol in Britton-Robinson buffer pH 4.0 using DPV at CFE with regeneration potential Ereg1 = –400 mV, and Ereg2 = 1300 mV.

Concentration

range

[μmol L-1]

Slope

[nA.μmol L-1]

Intercept

[nA]

R

LOD

[μmol L-1]

20 – 100

2 – 10

0.2 – 1

0.02 – 0.1

29.7

48.3

48.7

58.5

49.5

15.8

1.15

2.28

0.9976

0.9981

0.9987

0.9982

–

–

–

0.034

80

The voltammograms connected with the concentration ranges from 20-100 μmol L-1,

2-10 μmol L-1, 0.2-1.0 μmol L-1 and 0.02-0.1 μmol L-1 with correlation coefficients

(R) of 0.9976, 0.9981, 0.9987 and 0.9982 respectively. This shows that the developed

method is very sensitive for salicylic acid determination as depicted in Fig. 4.3.3, Fig.

4.3.4, Fig. 4.3.5. and Fig. 4.3.6. respectively, for illustration.

200 500 8000

1500

3000

0 50 1000

1500

3000

Ip, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM

Fig. 4.3.3. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 μmol L-1. Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

81

200 500 800

200

400

600

0 5 100

250

500

Ip, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM

Fig. 4.3.4. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 μmol L-1. Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

200 500 800100

150

200

0.0 0.5 1.00

25

50

Ip, nA

6

2

3

45

1

I, nA

E, mV

c, µM

Fig. 4.3.5. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 0,2 (3) 0,4, (4) 0,6, (5) 0,8, (6) 1 μmol L-1. Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

82

200 500 800100

150

200

0.00 0.05 0.100

5

10

Ip, nA

6

234

5

1

I, nA

E, mV

c, µM

Fig. 4.3.6. Differential pulse voltammograms of Paracetamol at CFE in Britton-Robinson buffer pH 4.0, c (PC): (1) 0, (2) 0,02 (3) 0,04, (4) 0,06, (5) 0,08, (6) 0,1 μmol L-1. Regeneration potential Ereg1 = –400 mV and Ereg2 = 1300 mV. Background current = 1.94 nA. Inset is corresponding calibration dependence.

Adsorptive stripping voltammetry was tested for increasing the sensitivity of this

method but no accumulation effect was observed in range accumulation potential

from –300 mV to +300 mV and accumulation time up to 500 s.

4.3.3. Analysis of Pharmaceutical Drugs Commercial pharmaceutical samples (tablets) containing PC were analyzed in order

to evaluate the validity of this proposed method. Recovery experiments were carried

out to evaluate matrix effects after standard-solution additions yielded a good average

recovery (Table 4.3.3).

83

Table 4.3.3. The amount of Paracetamol determined by DPV in tablets of

commercial drugs with declare contents 500 mg of Paracetamol.

Name of tablet Company name Contents of Paracetamol

[mg]

Recovery

[%]

Paralen

Panadol

Paracetamol

Coldrex

Zentiva. Cz

Glaxosmithkline

Glaxosmithkline

Glaxosmithkline

494

496

503

493

98.8 ± 3

99.1 ± 2

100.7 ± 2

98.5 ± 4

Indicating that there were no important matrix interferences for the samples analyzed

by the proposed DPV method, and had the corresponding results to reference

spectrometric determination electrochemically active ascorbic acid (vitamin C) is

often combined as a medicine with PC based drugs. As documented in Fig. 4.3.7. no

significant interference effect was observed in the developed DPV method up to

tenfold concentration of Ascorbic acid in comparison to concentration of PC.

200 500 8000

100

200

0 50 1000

100

200

Ip, nA

50

102030 40

0

I, nA

E, mV

6070

8090100110

Ascorbic Acidc, µM

Fig. 4.3.7. Differential pulse voltammograms of Paracetamol 10 μmol L-1 with Ascorbic acid from 10 to 100 μmol L-1 (concentrations are written above curves in plot) at CFE in Britton-Robinson buffer pH 4.0, regeneration potential Ereg1 = –400 mV, and Ereg2 = 1300 mV. Inset is corresponding dependence of peak current of PC on concentration of Ascorbic acid.

84

4.3.4. Analysis of Urine Samples For testing the possibility of determination of PC in human urine samples the method

of calibration curve was used. Calibration curves and ranges of determination for

analyzed drug in model solutions of human urine gave linear response in the

concentration ranges of 2 – 10 (Fig. 4.3.8.A) and 20 – 100 mol.l-1 (Fig. 4.3.8.B).

200 500 800

80

160

240

0 5 100

75

150

Ip, nA

10

2

4

6

8

1

I, nA

E, mV

A

c, µM

200 500 8000

800

1600

0 50 1000

700

1400

Ip, nA100

20

40

60

80

0

I, nA

E, mV

B

c, µM

Fig. 4.3.8. Differential pulse voltammograms of 0.1 ml urine with model sample of Paracetamol (2 – 10 µmol L-1 (A), and 20 – 100 µmol L-1 (B), concentration is written next to curves in plots) sample at CFE in Britton-Robinson buffer pH 4.0, regeneration potentials Ereg1 = –400 mV, and Ereg2 = 1300 mV. Inset is corresponding calibration dependence.

The calibration curves show linear response over the whole range of concentration

used in the assay procedure. The parameters associated are summarized in Table

4.3.4. Further parameters of the calibration straight line are seen in Table 4.3.4.

85

Table 4.3.4. Parameters of the calibration straight lines for the determination of model samples of Paracetamol in urine, media of Britton-Robinson buffer pH 4.0 using DPV at CFE with regeneration potentials Ereg1 = –400 mV and Ereg2 = 1300 mV.

Concetration range

[μmol-1.l]

Slope

[nA.μmol-1.l]

Intercept

[nA]

R LOD

[μ mol-1.l]

20 – 100

2 – 10

12.91

14.17

31.66

–7.61

0.9975

0.9994

–

0.48

86

4.4. Quantification of Ibuprofen in Pharmaceuticals and Biological Samples by Transmission FTIR Spectroscopy.

The main object of the current study was to develop a new rapid method by FTIR

spectroscopy which is equally sensitive for standards, pharmaceutical and biological

samples in liquid form containing ibuprofen under similar conditions that can be

frequently used for the routine quality control in pharmaceutical industries and for

quantification in diagnostic laboratories as no method already exists. The existing

methods using high cost instruments which although provide good sensitivity but at

the same time they suffer from the draw backs of time consumption as well as

intensive labor involved. The current method uses the approach to quantify ibuprofen

in pharmaceuticals as well as biological sample such as urine by FTIR Spectroscopy

under optimized parameters. PLS multivariate calibration model was applied here in

order to correlate the measurements collected between different variables on the same

observations in multi-component mixtures as PLS works on the principle of

computing large amount of information which is common between the different

variables with maximal covariance.

4.4.1. Analysis of Pharmaceutical Samples For the quantification of ibuprofen in pharmaceutical samples, calibration was

prepared in the range of 10-100 ppm on FTIR under optimized parameters with very

good linearity R2 =0.998 as shown in (Fig. 4.4.1) by excellent response of the FTIR

with the increasing concentration.

87

Fig. 4.4.1. Calibration plot in the range of 10-100 ppm for biological samples

The calibration results clearly depict the significant sensitivity and selectivity for the

analyte under examination hence proving the method to be useful. The results

expressed in Table 4.4.1 were highly reproducible with negligible variation as taken

for five replicate measurements.

Table 4.4.1. Results for the ibuprofen concentration found in the tablet samples

Sample Ibuprofen labeled

mg/Tablet

Ibuprofen found

mg/Tablet

Sample 01 400 412 ± 1.21

Sample 02 400 397 ± 0.71

Sample 03 400 408 ± 1.73

Sample 04 400 403 ± 1.14

Sample 05 400 389 ± 0.85

Sample 06 400 403 ± 1.52

88

There was very good conformity between obtained results and the values labeled on

the formulations, which determines the suitability of the proposed procedure for the

determination of ibuprofen in pharmaceutical sample and are according to the limits

of pharmacopoeia. To check the validity of the newly developed method the known

amounts of ibuprofen was added to the commercial formulation as in Fig. 4.4.2.

Fig. 4.4.2. Pharmaceutical Tablet sample and spikes of 30 ppm, 50 ppm and 70 ppm Ibuprofen standard.

The recovery was measured by comparing the concentration of pharmaceutical

sample before spiking with that obtained from the spiked samples. After the addition

we observed that the amount of standard added was almost fully recovered with the %

recovery of (98.53, 99.66, 102.02) and were within acceptable limits according to

AOAC guidelines for single laboratory validation of chemical methods for dietary

supplements and botanicals [AOAC, 2002] as given in Table 4.4.2. The coefficient of

variation (CV) values was also small showing that the results were acceptable.

Spike 1

Spike 2

Spike 3

Tablet sample

89

Table 4.4.2. Recovery result of ibuprofen from tablet samples after spiking with known concentrations of standard

(A)

ppm

(B)

ppm

By FT-IR

(C) Recoverya CVb

(%) (%)

Acceptable recovery (%)

[AOAC, 2002]

1 30 20 49.56. ± 1.25 98.53 1.6

90-108 2 50 20 69.83 ± 0.8 99.66 1.3

3 70 20 91.42 ± 1.4 102.02 1.5

(A) Exogenous addition

(B) Before addition

(C) After addition

aRecovery (%)=(C–B)/A x100.

bCoefficient of variation was obtained from the mean of five replicate tests.

4.4.2. Analysis of Urine Samples For the analysis of ibuprofen in urine samples the same method worked effectively as

the results were precise and reproducible listed in Table 4.4.3. The same calibration

range was used as it also covered the lower range of the analyte specie in urine

sample.

90

Table 4.4.3. Results for the ibuprofen concentration found in urine samples

The spectra of extracted urine as blank was collected on FTIR, then a selected urine

sample were spiked with 10 and 20 ppm respectively (these spectra are shown in Fig.

4.4.3.) to check the response and accuracy of the method.

Fig. 4.4.3. Sample urine and 2 spikes of 10 and 20 ppm ibuprofen standard.

Sample

Ibuprofen

concentration found

(ppm)

Standard Deviation

Sample 01 11.32 0.81

Sample 02 11.64 0.34

Sample 03 10.75 0.73

Sample 04 11.03 0.24

Sample 05 11.94 0.85

91

The results of blank urine sample and spiking are given in Table 4.4.3 which show

very good recovery of the standard added to the sample as recovery is ranging from

98.6 to 100.15 % with very small variation which is clear indication of the reliability

of the method for biological fluid.

Table 4.4.4. Recovery results of ibuprofen in urine samples after spiking with known

concentration of standard

(A) ppm

(B)

ppm

By FT-IR

(C) Recoverya CVb

(%) (%)

1 10 11 20.86 ± 1.3 98.6 2.1

2 20 11 31.03 ± 0.8 100.15 1.4

(A) Exogenous addition

(B) Before addition

(C) After addition

aRecovery (%)=(C–B)/A x100.

bCoefficient of variation was obtained from the mean of five replicate tests.

All the measurements were taken in five replicates to ensure reproducibility and this

can be explained by very low values of standard deviation. Fig. 4.4.5. shows group

spectra of the ibuprofen standards acquired for the calibration to analyze ibuprofen in

urine and pharmaceutical sample analyzed using this method.

92

Fig. 4.4.5. Group Spectra of ibuprofen Standards

From the spectra we can understand that there is very negligible interference from the

other matrix present in the sample while analyzing the analyte of our interest. The

residual mean standard error of calibration (RMSEC) values of 1.06 was achieved

after comparison of actual and computed concentration for all the standards which

explain the accuracy of the method with very good precision.

4.4.3. Limits of Detection and Quantitation The analysis at the lowest concentration which produced substantial signal was

repeated eleven times and calculated by the following formula:

LOD = 3 × SD × C / M

Where SD is the standard deviation; C is the concentration of analyte and M is the

mean peak area. While LOQ was determined by the same way with following

equation: LOQ = 10 × SD × C / M

The lowest concentration of Ibuprofen to be detected through this method was found

to be 0.77 ppm and quantification limit was found to be 2.54 ppm for IBP

determination.

93

200 700 12000

1500

3000

2 4 6 8 10 12

600

900

E1/2

, mV

6

2

3

45

I, nA

E, mV

7

8

910

1112

pH

200 700 12000

1500

3000

2 4 6 8 10 120

600

1200

E, mV

6

2

3

4

5

I, nA

E, mV

8

11

7

10

912

pH

4.5. Differential Pulse Voltammetric Determination of Salicylic acid and Acetylsalicylic acid in Tablet and Urine Samples at Carbon Film Electrode

4.5.1. Influence of pH on Salicylic Acid in DC and DP Voltammetry Fig. 4.5.1A describes the influence of pH from 2-12 of BR buffer on Ip salicylic acid

using DC voltammetry. The results showed that at the maximum ions mobility at pH

2 of BR buffer, the peak height is increased for salicylic acid while others are

decreased due to over extent of ions of salicylic acid from pH 3-10 and others pH of

BR buffer did not show peaks of salicylic acid at pH 11-12 due to over the surface of

CFE electrode. So pH 2 of BR buffer was selected for further study. Fig. 4.5.1B

shows the effect of pH of BR buffer from 2-12 using DP voltammetry to compare the

sensitivity of salicylic acid for both techniques.

A B

Fig. 4.5.1. A and B. DC & DP voltammograms of Salicylic Acid (c = 100 μmol L-1)

at CFE in Britton-Robinson buffer pH 2 to 12 (numbers in above curves correspond to given pH). Inset is corresponding dependence of peak potential on the pH.

We concluded that the response of salicylic acid with enhanced peak current was at

pH 2 with smooth background current. Due to sensitivity of DP voltammograms, we

selected DP voltammetry for further studies. The influence of different pH from 2-12

94

in B-R buffer are summarized in (table. 4.5.1) which shows different peak currents at

different peak potentials of Salicylic acid at DC and DP voltammetry.

Table 4.5.1. The influence of pH on DCV and DPV of Salicylic Acid (c = 100 μmol L-1)

pH

DC Voltammetry DP Voltammetry

Ip, nA Ep, mV Ip, nA Ep, mV 2 2097 1074 2793 1065 3 934 1036 1185 1028 4 1078 993 1944 990 5 820 963. 1060 944 6 713 909. 866 899 7 731 888 746 871 8 1423 875 1302 864 9 838 881 736 817

10 895 825 736 817 11 721 862 372 864 12 168 580 83 521

For the sake of comparison between DC and DP voltammetric determination of

salicylic acid at optimal conditions was made (Supalkova, et al., 2006). All the results

were obtained at pH 2 of BR buffer. The results indicate that better sensitivity can be

obtained at DP voltammetry in comparison to DC voltammetry.

4.5.2. Reproducibility of Salicylic Acid The peak current for stability 100 µmol L-1 of salicylic acid was studied. Figure 4.5.2

displays voltammogram from series of 45 successive measurements with different

activation potentials at -2000 mV, 1500 mV and 2200 mV. In these three activated

potentials the best stability peak current was seen at activation potential 2200 mV. It

means that activation potential 2200 mV reveals excellent reproducibility of the

salicylic acid at pH 2 of BR buffer at CFE.

95

0 4 80

1500

3000

I, n

A

N

1 2

3

Fig. 4.5.2. Repetitive measurements of 100 μmol L-1 Salicylic Acid using DPV at

CFE in Britton- Robinson Buffer pH 2.0 with activation potential (1) –2000,(2) 1500 and (3) 2200mV

4.5.3. Linear Calibration Curves of Salicylic Acid Under optimized experimental conditions, calibration plots were performed for

salicylic acid using activation potential 2200 mV at CFE. Both high and low

concentration ranges in Britton-Robinson buffer pH 2 were obtained. The peak

current increased linearly with the increasing concentrations of salicylic acid in the

concentration ranges of 20-100 μmol L-1, 2-10 μmol L-1 and 0.2-1.0 μmol L-1 with

correlation coefficients ( R) of 0.9993, 0.9994 and 0.9996 respectively . This shows

that the developed method is very sensitive for salicylic acid determination as

depicted in Fig. 4.5.3, Fig. 4.5.4 and Fig. 4.5.5. respectively.

96

750 1000 1250

800

1600

2400

0 50 1000

750

1500

Ip, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM

Fig. 4.5.3. Differential pulse voltammograms of Salicylic Acid at CFE in Britton-

Robinson buffer pH 2.0, c(SA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 8,0 (6) 100 μmol L-1. Activation of potential = 2200 mV for 120 sec. Inset is corresponding calibration dependence.

750 1000 1250

250

500

750

0 5 100

100

200

Ip, nA

6

2

3

45

1

I, nA

E, mV

c, µM


Robinson buffer pH 2.0, c(SA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 μmol L-1. Activation of potential=2200mV for 120 sec Inset is corresponding calibration dependence.

97

800 1000 120040

60

80

0.0 0.5 1.00

10

20

Ip, nA

6

2345

1

I, nA

E, mV

c, µM


Robinson buffer pH 2.0, c (SA): (1) 0, (2) 0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 μmol L-1. Activation of potential = 2200 mV for 120 sec. Inset is corresponding calibration dependence.

The observed splitting of the peak can be connected with different mechanism of the

oxidation of salicylic acid. The limit of detection was obtained 0.21 µM of salicylic

acid. Relatively big intercept complicated the use of the calibration curve but it does

not make it impossible (see table 4.5.2.).

Table 4.5.2. Parameters of the calibration straight lines for the determination of

Salicylic Acid in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

Concentration range

[μ mol L-1] Slope

[nA μmol L-1]

Intercept [nA]

R

L O D [μ mol L-1]

20 – 100 2 – 10 0.2-1

13.43 16.73 18.52

35.77 9.58 0.84

0.9993 0.9994 0.9996

– –

0.21

4.5.4. Hydrolysis of Acetylsalicylic Acid In figure 4.5.6. DP voltammogram shows the hydrolysis of acetylsalicylic acid into

salicylic acid at CFE of pH 2 in B-R buffer. The peak current shows the same peak

98

current of salicylic acid and acetylsalicylic acid (Aspirin) at 2 minutes with activation

potential 2200 mV for 120 sec.

750 1000 1250

300

600

900

23

4

1

I, nA

E, mV

Fig. 4.5.6. Differential pulse voltammograms of 10 μmol L-1 hydrolyzed Acetylsalicylic

Acid at CFE in Britton-Robinson buffer pH 2.0, (1) Electrolyte, (2) 10 μmol L-1 without hydrolysis (3) 10 μmol L-1 Salicylic Acid (4) 10 μmol L-1 after hydrolysis Acetylsalicylic acid. Activation potential=2200mV for 120 sec

4.5.5. Linear Calibration Curves of Hydrolyzed Acetylsalicylic Acid at CFE The calibration dependences of hydrolyzed acetylsalicylic acid for DPV at CFE were

measured in concentration ranges, 20-100 μmol L-1, 2-10 μmol L-1 and 0.2-1.0 μmol

L-1 depicted in Fig 4.5.7, Fig 4.5.8. and Fig 4.5.9. in Britton-Robinson buffer pH 2

with pulse amplitude 50 mV, pulse width 80 ms and scan rate 20 mV s-1 with

correlation corresponding coefficients (R) of 0.9996, 0.9991 and 0.9996 respectively.

99

750 1000 12500

1000

2000

0 50 1000

750

1500

Ip, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM

Fig. 4.5.7. DP voltammograms of hydrolyzed Acetylsalicylic Acid at CFE in Britton-

Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 μmol L-1. Activation potential = 2200 mV for 120 sec. Inset is corresponding calibration dependence.

750 1000 1250

100

200

300

0 5 100

100

200

Ip, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM


Robinson buffer pH 2.0, c (ASA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 μmol L-1. Activation potential = 2200 mV for 120 sec. Inset is corresponding calibration dependence.

100

750 1000 125040

80

120

0.0 0.5 1.00

10

20

Ip, nA 6

2345

1

I, nA

E, mV

c, µM


Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 μmol L-1. Activation potential = 2200 mV for 120 sec. Inset is corresponding calibration dependence.

The limit of detection obtained was 0.15µM for acetylsalicylic acid. This shows that

the developed method is very sensitive for hydrolyzed acetylsalicylic acid. The

optimized parameters obtained for linear calibration curves are summarized in table

4.5.3.


Concentration range

[μmol L-1] Slope

[nA μmol L-1]

Intercept [nA]

R

L O D [μmol L-1]

20 – 100 2 – 10 0.2-1

13.93 18.20 20.17

17.2 3.11

0.43

0.9996 0.9991 0.9996

– –

0.15

4.5.6. Determination of Salicylic Acid in Different Drugs The developed method was tested for salicylic acid determination in 4 µM Duofilm

and Saloxyl drugs. The different concentrations of salicylic acid were spiked upto

101

10µM for the determination of salicylic acid at CFE. Good linear calibration curves

were found as given in figure 4.5.10.

750 1000 1250

80

160

240

2

3

4

5

1

I, nA

E, mV

Fig. 4.5.10. Differential pulse voltammograms of 4 μmol L-1 Duofilm sample with

spikes of salicylic acid standard at CFE in Britton-Robinson buffer pH2.0, c (SA): (1) electrolyte, (2) expected 4 μmol L-1 salicylic acid in sample of Duofilm (3) 6 μmol L-1 spike, (4) 8 μmol L-1 spike and (5) 10 μmol L-1 spike. Activation potential 2200 mV.

The results confirmed the applicability of the method. The parameters of calibration

curves in same medium are thus obtained and summarized in table 4.5.4.


S. no Name of Tablet

Company Name

Tablets (%)

Recovery ± stdev from 5 measur. (%)

1

2

Duofilm

Saloxyl

Stiefel

Herbacos-bofarma

16.7

10

103 ± 3

105 ± 1

4.5.7. Determination of Acetylsalicylic Acid from Different Drugs The developed method was tested for 4 µM Aspirin, Acypyrin, Acypyrin + C,

Disprin, Acefein and Acefein + C drugs. The different concentration of hydrolyzed

102

acetylsalicylic acid were spiked upto 10 µM for the determination of hydrolyzed

acetylsalicylic at CFE. Good linear calibration curves were found as given in figure

4.5.11.

750 1000 1250

100

200

300

2

3

4

5

1

I, nA

E, mV

Fig. 4.5.11. Differential pulse voltammograms of 4 μmol L-1 Aspirin sample with

spikes of hydrolyzed acetylsalicylic acid standard at CFE in Britton-Robinson buffer pH 2.0, c(SA): (1) electrolyte, (2) expected 4 μmol L-1 salicylic acid in sample of Aspirin (3) 6 μmol L-1 spike, (4) 8 μmol L-1 spike and (5) 10 μmol L-1 spike. Activation potential 2200 mV.

The results confirmed the applicability of the method. The parameters of calibration

curves in same medium are summarized in table 4.5.5.

Table 4.5.5. The amount of hydrolyzed Acetylsalicylic Acid determined by DPV in

Tablets

S. no Name of Tablet

Company Name

Tablets (mg)

Recovery ± stdev from 5 measur. (%)

1 2 3 4 5

Aspirin

Acylpyrin

Acylpyrin + C

Disprin

Acifein

Bayer

Herbacos-bofarma

Herbacos-bofarma

Reckitt Benckiser

Herbacos-bofarma

500

500

320

300

250

106 ± 2

106 ± 2

95 ± 1

101 ± 3

100 ± 2

103

4.5.8. Determination of Salicylic Acid in Urine Samples at CFE The proposed method was also applied to test urine samples for the determination of

salicylic acid at CFE. The linear calibration curves were obtained after the spiked

0.1ml urine samples in ranges from 2 µM to 100 µM concentration of salicylic acid

(see fig. 4.5.12 and fig 4.5.13 respectively).

750 1000 1250

250

500

750

0 50 100

200

400

600

Ip, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM

Fig. 4.5.12. Differential pulse voltammograms of 0.1 ml urine sample at CFE in

Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 μmol L-1. Activation potential 2200 mV. Inset is corresponding calibration dependence.

750 1000 125060

120

180

0 5 100

40

80

Ip, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM


Britton-Robinson buffer pH 2.0, c(SA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 μmol L-1. Activation potential 2200 mV. Inset is corresponding calibration dependence.

104

These results show the applicability of the method using DP voltammetry at CFE. The

coefficient of regression (R) and limit of detection are obtained from different

calibration curves are summarized in below table 4.5.6.

Table 4.5.6. Parameters of the calibration straight lines for the determination of

Salicylic Acid in 0.1 ml urine samples in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

Concentration range

[μ mol-1 L] Slope

[nA μmol L-1]

Intercept [nA]

R

L O D [μ mol-1 L]

20 – 100 2 – 10

5.41 8.12

39.21 –9.03

0.9886 0.9976

–

0.64

4.5.9. Determination of Acetylsalicylic Acid in Urine at CFE Fig. 4.5.14 shows the determination of hydrolyzed acetylsalicylic acid in urine

samples at CFE at pH value of 2 in BR buffer. The concentration of spiked

acetylsalicylic acid in 0.1 ml urine samples was from 20-100µM. The lower

concentration results of acetylsalicylic acid from 2-10µM is given in fig 4.5.15.

105

750 1000 1250

150

300

450

0 50 1000

150

300 I

p, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM


Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 20 (3) 40, (4) 60, (5) 80, (6) 100 μmol L-1. Activation potential 2200 mV.

Inset is corresponding calibration dependence.

750 1000 1250

30

60

90

0 5 100

20

40 I

p, nA

6

2

3

4

5

1

I, nA

E, mV

c, µM


Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 2 (3) 4, (4) 6, (5) 8, (6) 10 μmol L-1. Activation potential 2200 mV. Inset is corresponding calibration dependence.

These results show the applicability of the method using DP voltammetry at CFE. The

coefficient of regression (R) and limit of detection are obtained from different

calibration curves and summarized in table 4.5.7.

106

Table 4.5.7. Parameters of the calibration straight lines for the determination of hydrolyzed Acetylsalicylic Acid in 0.1 ml urine sample in a Britton-Robinson buffer pH 2.0 using DPV at CFE with activation potential 2200 mV

Concentration range

[μmol L-1] Slope

[nA μmol L-1]

Intercept [nA]

R

L O D [μmol L-1]

20 – 100 2 – 10

3.06 4.51

17.99 – 3.92

0.9997 0.9998

–

0.71

4.5.10. Interference Study No interference was observed in hydrolyzed acetylsalicylic acid (see fig 4.5.16.) The

concentration from 5 µM to 100 µM ascorbic acid added in 5 µM hydrolyzed

acetylsalicylic acid at CFE was observed. The DP voltammogram of ascorbic acid

show peak potential around 700 mV, while that of acetylsalicylic acid at 1100 to 1200

mV. It means ascorbic acid does not interfere in the presence of hydrolyzed

acetylsalicylic acid.

200 700 1200150

300

450

0 50 1000

50

100

Ip, nA

I, nA

E, mV

c, µM

Fig. 4.5.16. Differential pulse voltammograms of hydrolyzed Acetylsalicylic acid 5 μmol L-1 with ascorbic acid 5-100 μmol L-1 at CFE in Britton-Robinson buffer pH 2.0. Activation potential 2200 mV.

107

4.6. Conclusions Various analytical methods were developed for the determination of pharmaceuticals

such as paracetamol, diclofenac sodium, aspirin and brufen based on simplicity,

sensitivity, selectivity, rapidity, low cost as well as environmental safety. For this purpose

voltammetric and spectroscopic techniques were employed. The first proposed method

based on uv-visible spectrophotometric technique for paracetamol determination

possesses many advantages over other analytical methods due to its rapidity, lower cost,

environmental safety and better sensitivity. The method can be successfully employed for

paracetamol quantification in all types of pharmaceutical preparations and liquid samples,

such as urine, serum, gastric juice, etc.

In another approach method was developed for determining diclofenac sodium which is

more superior to reported spectrophotometric methods due to its better sensitivity,

simplicity, stability, economy, environmental safety, broader linear working and lower

detection limits. Application of the developed method for quantification of diclofenac

sodium in tablets with good recovery and lower standard deviation proved its suitability

for analysis of diclofenac sodium in other pharmaceutical preparations. Its successful use

in the determination of diclofenac sodium in urine and serum samples of patients makes

this method as biomarker for identification and hence diagnosis of some diseases

recognized by elevated or decreased level of diclofenac sodium. It may also be concluded

that voltammetric determination of paracetamol using CFE is a suitable for the

micromolar and submicromolar concentrations. The proposed methodology was

successfully applied to the determination of paracetamol in four types of commercial

108

drugs and for model human urine samples and satisfactory results were obtained.

Preparation of the sample was easy and the method is less time-consuming and

economical. Therefore, this quick and simple analytical procedure is suitable for practical

applications. This method may be considered in lot of cases as a suitable alternative to

existing more time-consuming and expensive chromatographic methods.

The main goals achieved through this method include analytical simplicity, remarkable

efficiency, better selectivity and lower cost for the quantification of Ibuprofen. Finally

FTIR spectroscopic method has been developed using KBr windows for the

determination of ibuprofen. A PLS model was successfully developed with the help of

TQ analyst software. The developed method was easy to handle, cheapest and selective

for the quantification of ibuprofen in pharmaceutical drugs and biological fluids i.e. urine

samples.

A suitable voltammetric method was developed using AgA-CFE for the determination of

micromolar concentrations of Acetylsalicylic acid (aspirin) and salicylic acid. The limit

of determination of Aspirin using DPV at AgA-CFE is lower due to efficient

electrochemical activation; the AgA-CFE gives better reproducibility with excellent

signal stability given by less passivation of electrode surface. Furthermore, fairly good

reproducibility of surface renovation has been observed.

109

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List of Publications

1. Simpler Spectrophotometric Assay of Paracetamol in Tablets and Urine Samples. Sirajuddin, Abdul Rauf Khaskheli, Afzal Shah, Muhammad Iqbal Bhanger, Abdul Niaz and Sarfaraz Mahesar; Spectrochimica Acta Part A, 2007, 68, 747-751.

2. Simpler and faster spectrophotometric determination of diclofenac sodium in tablets, serum

and urine samples. Abdul Rauf Khaskheli, Sirajuddin, Kamran Abro, S.T.H Sherazi, H.I Afridi, S.A. Mahesar, Munawar Saeed; Pakistan Journal of Environmental and Analytical Chemistry, 2009, 10, 53-58.

3. Highly sensitive spectrometric method for determination of hydroquinone in skin lightening creams: application in cosmetics. S. Uddin, A. Rauf, T.G. Kazi, H. I. Afridi and G. Lutfullah; International Journal of Cosmetic Science, 2010, 1- 8.

4. Differntial pulse voltammetric determination of paracetamol in tablet and urine samples at a

carbon film electrode. Abdul Rauf Khaskheli, Jan Fischer, Jiří Barek,Vlastimil Vyskočil, Sirajuddin and Muhammad Iqbal Bhanger. (Submitted in Electroanalysis).

5. Evaluation of ibuprofen in tablet formulation and urine samples by transmission FTIR

spectroscopy in conjunction with partial least squares (PLS). A. R. Khaskheli, S. T. H. Sherazi, Sirajuddin, S. A. Mahesar, M. Ali, Nazar Kalwar and Aftab Kandhro. (Submitted in Talanta).

6. Differntial pulse voltammetric determination of salicylic acid and acetyl salicylic acid in tablet and urine samples at carbon film electrode. Abdul Rauf Khaskheli, Jan Fischer, Jiří Barek,Vlastimil Vyskočil, Sirajuddin, Muhammad Iqbal Bhanger and Munawar Saeed. (Submitted in Electroanalysis).

Other Publications

1. Ultra-trace level determination of hydroquinone in waste photographic solutions by UV–Vis

spectrophotometry. Sirajuddin , M. Iqbal Bhanger, Abdul Niaz, Afzal Shah and Abdul Rauf; Talanta, 2007.

2. Abdul Niaz, Sirajuddin, Afzal Shah, S. A. Mahesar, Abdul Rauf. Adsorptive stripping

voltammetric determination of hydroquinone using an electrochemically pretreated glassy carbon electrode. Pakistan Journal of Environmental and Analytical Chemistry, 2008, 9, 110-117.

3. GC-MS Quantification of Fatty Acid Profile including trans FA in the Locally Manufactured

Margarines of Pakistan. Aftab Kandhro, S. T. H Sherazi, S. A. Mahesar, M. I. Bhanger, M. Younis Talpur and Abdul Rauf Khaskheli; Food Chemistry, 2008, 109, 1, 207-211.

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4. Simultaneous assessment of zinc, lead, cadmium and copper in poultry feeds by differential

pulse anodic stripping voltammetry. S. A. Mahesar, S. T. H Sherazi, A. Niaz, M. I. Bhanger, Sirajuudin and Abdul Rauf Khaskheli; Food and Chemical Toxicology, 2010, 48, 2357-2360.

5. Fast voltammetric assay of water soluble phthalates in bottled and coolers water. Munawar Saeed, Sirajuddin, Abdul Niaz, Afzal Shah, H.I Afridi and Abdul Rauf; Analytical Methods, 2010, 2, 844-850.

6. Assessment of Azithromycin in pharmaceutical formulation by FTIR transmission spectroscopy. M. Ali, S.T.H Sherazi, S.A. Mahesar and Abdul Rauf. (Submitted in Journal of Brazillian chemical Society).

7. Appraisal of Erythromycin in pharmaceutical formulation by FTIR transmission spectroscopy. M. Ali, S.T.H Sherazi, S.A. Mahesar and Abdul Rauf. (Submitted in Pakistan Journal of Pharmaceutical Sciences).

Documents

prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1260/2/1163S.pdf · ii Certificate This is to certify that Mr. ABDUL RAUF KHASKHELI has carried out his research work on the