Ph. D Thesis - Higher Education Commission

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

DEDICATED 

To My Affectionate Supervisors, Beloved Parents and Friends 

who’s Prayers, Encouragement and Cooperation Enabled Me for 

this Achievement 

i

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 

ii

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 Cosupervisor 

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, 

iii

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 

iv

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. 

v

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 (R 2 ) 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 

vi

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. 

vii

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 

1.1. 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 






1.4. Diclofen 10 





1.5. Brufen 12 





1 – 14 

viii 

viii

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 


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 

45 

3.2. Material and Methods for Diclofenac Sodium Using UV- 

Visible Spectrophotometry 

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 



3.3.3. Procedures 48 

49 

3.4. Material and Methods for the Analysis of Ibuprofen 

Using FT-IR Spectroscopy 

3.4.1. Reagents and Samples 49 

3.4.2. FT-IR Spectral Measurements 49 

ix

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 

Voltammetry 

51 

3.5.1. Reagents 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 

54 

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

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 



4.2.4. Effect of Acidic and Alkaline Conditions 68 

4.2.5. Calibration Plot 71 

4.2.6. Comparison with Other Reported Spectroscopic 

Methods 

71 


4.2.8. Analysis of Urine and Serum Samples 74 

4.3. Differential Pulse Voltammetric Determination of PC in 

Tablet and Urine Samples at Carbon Film Electrode 

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

77 

x

4.3.2. Optimization of Parameters and Calibration Curve 78 

4.3.3. Analysis of Pharmaceutical Drugs 82 


4.4. Quantification of Ibuprofen in Pharmaceuticals and 

Biological Samples by FTIR Transmission Spectroscopy. 

4.4.1. Analysis of Pharmaceutical Samples 

86 


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 

93 

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

Voltammetry 

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 

86 

xi

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 


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 


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 ) 

Table. 4.3.2. Parameters of the calibration straight lines for the 

78 

determination of Paracetamol in Britton-Robinson buffer 

pH 4.0 using DPV at CFE with regeneration potential 

Ereg1 = –400 mV, and Ereg2 = 1300 mV. 

Table. 4.3.3. The amount of Paracetamol determined by DPV in tablets 

79 

of commercial drugs with declare contents 500 mg of 

Paracetamol. 


determination of model samples of Paracetamol in urine, 

83 

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 


91 

Table. 4.5.1. The influence of pH on DCV and DPV of Salicylic Acid (c 

= 100 µmol L -1 ) 

94 

xii

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 


determination of hydrolyzed Acetylsalicylic Acid in a 

Britton-Robinson buffer pH 2.0 using DPV at CFE with 

activation potential 2200 mV 

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

tablets 

Table 4.5.5. The amount of hydrolyzed Acetylsalicylic Acid 

determined by DPV in tablets 


determination of salicylic acid in 0.1 ml urine samples in a 




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 

97 

100 

101 

102 

104 

106 

xiii

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). 

Figure. 4.2.4. Calibration plot of absorbance verses concentration for DS 

69 

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 


and DS spiked (red) serum. 

Figure. 4.3.1. DC voltammograms (A) and DP voltammograms (B) of 

Paracetamol (c = 100 μmol 

75 

-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. 

Figure. 4.3.2. Repetitive measurements of 100 µmol L 

77 

-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. 

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 

79 

-1 . Regeneration 

potential Ereg1 = –400 mV and Ereg2 = 1300 mV. 

Background current = 1.94 nA. Inset is corresponding 

calibration dependence. 

80 

xiv



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. 


in Britton-Robinson buffer pH 4.0, c(Paracetamol): 0 (1), 

81 

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. 


in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 0,02 

81 

(3) 0,04, (4) 0,06, (5) 0,08, (6) 0,1 µmol L -1 . Regeneration 




Figure. 4.3.3. Differential pulse voltammograms of Paracetamol 10 µmol 

82 

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 

83 

corresponding dependence of peak current of 

Paracetamol on concentration of Ascorbic acid. 

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 

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 

93 

95 

xv

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 


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 



0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 µmol L -1 . Activation of 



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 

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 




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 



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 

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 

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 

96 

96 

97 

98 

99 

99 

100 

101 

102 

xvi

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) 


potential 2200 mV. Inset is corresponding calibration 

dependence 



2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L -1 . Activation potential 

2200 mV. Inset is corresponding calibration dependence 


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 


dependence 





dependence 

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 

103 

103 

105 

105 

106 

xvii

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 

xviii

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 

xix

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 






1.3. Aspirin 6 






1.4. Diclofen 10 





1.5. Brufen 12 




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 



27 

2.7. Quantification of Aspirin in Pharmaceutical and Biological 

Samples 

31 



35 

2.9. Quantification of Ibuprofen in Pharmaceutical and 


39 

Chapter - Three EXPERIMENTAL 43 - 53 

3.1. Material and Methods for Paracetamol Using UV-Visible 


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 


3.2.2. Washing of Glassware 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 



3.3.3. Procedures 48 

49 

3.4. Material and Methods for the Analysis of Ibuprofen 

Using FT-IR Spectroscopy 

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 

Voltammetry 

51 

3.5.1. Reagents 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 


4.1.4. Effect of Polar Solvents 57 


4.1.6. Effect of Acidic and Basic Solutions 60 

4.1.7. Calibration Range 61 




Diclofenac Sodium (DS) in Tablets, Serum and Urine 

Samples 

65 

4.2.1. Influence of Time 65 



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 


4.2.8. Analysis of Urine and Serum Samples 74 

4.3. Differential Pulse Voltammetric Determination of PC in 77

4.3.1. 

Tablet and Urine Samples at Carbon Film Electrode 

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 


4.4. Quantification of Ibuprofen in Pharmaceuticals and 

Biological Samples by FTIR Transmission Spectroscopy. 

86 

4.4.1. Analysis of Pharmaceutical Samples 86 


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 


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 


63 

Table. 4.1.5. Comparative analyses of PC in urine samples. 63 


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 


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 

determination of Paracetamol in Britton-Robinson buffer 

pH 4.0 using DPV at CFE with regeneration potential 

Ereg1 = –400 mV, and Ereg2 = 1300 mV. 

Table. 4.3.3. The amount of Paracetamol determined by DPV in tablets 

79 

of commercial drugs with declare contents 500 mg of 

Paracetamol. 


determination of model samples of Paracetamol in urine, 

83 

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 


90

Table 4.4.4. Recovery results of ibuprofen in urine samples after 


Table. 4.5.1. The influence of pH on DCV and DPV of Salicylic Acid (c 

= 100 µmol L -1 ) 


determination of Salicylic Acid in a Britton-Robinson 

buffer pH 2.0 using DPV at CFE with activation potential 

2200 mV 


determination of hydrolyzed Acetylsalicylic Acid in a 



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

tablets 

Table 4.5.5. The amount of hydrolyzed Acetylsalicylic Acid 

determined by DPV in tablets 


determination of salicylic acid in 0.1 ml urine samples in a 




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 

91 

94 

97 

100 

101 

102 

104 

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). 

Figure. 4.2.4. Calibration plot of absorbance verses concentration for DS 

69 

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 


and DS spiked (red) urine. 

74 


and DS spiked (red) serum. 

Figure. 4.3.1. DC voltammograms (A) and DP voltammograms (B) of 

Paracetamol (c = 100 μmol 

75 

-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. 

Figure. 4.3.2. Repetitive measurements of 100 µmol L 

77 

-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



40, (4) 60, (5) 80, (6) 100 µmol L -1 . Regeneration 






80 

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. 


in Britton-Robinson buffer pH 4.0, c(Paracetamol): 0 (1), 

81 

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. 


in Britton-Robinson buffer pH 4.0, c(PC): (1) 0, (2) 0,02 

81 

(3) 0,04, (4) 0,06, (5) 0,08, (6) 0,1 µmol L -1 . Regeneration 




Figure. 4.3.3. Differential pulse voltammograms of Paracetamol 10 µmol 

82 

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 

83 

corresponding dependence of peak current of 

Paracetamol on concentration of Ascorbic acid. 

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 

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 



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 


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 





0.2 (3) 0.4, (4) 0.6, (5) 0.8, (6) 1 µmol L -1 . Activation of 



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 


CFE in Britton-Robinson buffer pH 2.0, c(ASA): (1) 0, (2) 





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 



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 

93 

95 

96 

96 

97 

98 

99 

99 

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 

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 





dependence 



2 (3) 4, (4) 6, (5) 8, (6) 10 µmol L -1 . Activation potential 

2200 mV. Inset is corresponding calibration dependence 





dependence 





dependence 

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 

101 

102 

103 

103 

105 

105 

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

INTRODUCTION 

1.1. Analgesics / NSAIDs -An overview 

Chapter-01 

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). 

1

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. 

2

1.2. Paracetamol 

1.2.1. Pharmacopoeias: 

Fig. 1.1. Structural formula of Paracetamol (PC) 

Chemical name: 4´-Hydroxyacetanilide; N-(4-Hydroxyphenyl)acetamide 

Molecular formula: C8H9NO2 =151.2 

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). 

3

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). 

4

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). 

5

1.3. Aspirin 

1.3.1. Pharmacopoeias 

Fig. 1.2. Structural formula of Aspirin (ASA) 

Chemical name: O-Acetylsalicylic acid; 2-Acetoxybenzoic acid 

Molecular formula: C9H8O4 =180.2 

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). 

6

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). 


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. 

7

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). 

8

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. 

9

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 

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 

10

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. 

11

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 

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. 


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 

12

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 

13

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. 

14

LITERATURE REVIEW 

2.1. Analytical Techniques for Assessment of NSAIDs 

Chapter-02 

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. 

15

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). 

16

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 

17

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 R 2 =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. 

18

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 

19

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. 

20

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. 

21

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 

22

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 

23

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 

24

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). 

25

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. 

26

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). 



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, 

27

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 

28

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 

29

esults 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. 

30

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 

31

(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 

32

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 

33

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. 

34

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. 



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 Eu 3+ 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. 

35

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 

36

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 

37

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. 

38

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 

39

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. 1 st 

derivative UV with zero-crossing measurement was the first method. Second depends on 

1 st derivative of the ratio-spectra by measurements. 3 rd 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. 

40

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 

41

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. 

42

EXPERIMENTAL 

3.1. Material and Methods for Paracetamol using UV-Visible 


3.1.1. Reagents and Chemicals 

Chapter-03 

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. 

43

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 

44

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 


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 0 C. 

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. 

45

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 

46

esult 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 

47

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 

48

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 

49

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 

50

y 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. 

51

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 

52

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. 

53

RESULTS AND DISCUSSION 

Chapter-04 

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 

54

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 ± 1 0 C against a 

reagent (PC) blank. 

55

Absorbance 

0.4 

0.3 

0.2 

0.1 

0 

0 10 20 30 40 50 60 

Time (minutes) 

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. 

56

Absorbance 

0.5 

0.4 

0.3 

0.2 

0.1 

0 10 20 30 40 50 

Temperature ( 0 C) 

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. 

57

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 −1 PC. 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 

58

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 

Caffeine Diclofenic 

sodium 

% interference by drug 

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 

59

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 

4.1.6. Effect of Acidic and Basic Solutions 

0.5 0.00 10.70 255 

1 0.00 10.85 256 

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. 

60

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 . 

61

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 

62

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 concentration a 

(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. 

Paracetamol a 

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 

63

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 


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 ± 1 0 C 

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. 

A 

0.18 

0.16 

0.14 

0.12 

0.1 

0 10 20 30 40 50 60 70 80 90 

Time (min) 

Fig. 4.2.1. Time effect on absorbance of Diclofenac Sodium 

65

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 0 C (Fig. 4.2.2). It was seen that lower temperature range of 5–25 0 C 

showed maximum absorbance, with a little bit decreased value for the range of 30–45 

0 C was observed followed by a further decrease thereafter. 

A 

0.16 

0.15 

0.14 

0.13 

0.12 

5 15 25 35 45 55 

Temperature 0 C 

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. 

66

However the room temperature (25 ± 1 0 C) 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 

1:1 

5:1 

10:1 

(%) Interference 

Caffeine Paracetamol Aspirin Ibuprofen Ascorbic 

acid 

+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 

67

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. 

68

A 

0.5 

0.4 

0.3 

0.2 

0.1 

0.0 

200 225 250 275 300 325 350 

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. 

nm 

69

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

Acid / base (1M) 

Hydrochloric 

acid 

Acetic acid 

Ammonia 

NaOH 

Volume of acid 

/ base (ml) 

% effect 

pH of DS 

solution 

λmax 

(mid point) 

(nm) 

0.1 -10.67 2.44 273 

0.5 -14.44 2.07 273 

1.0 -18.31 1.73 273 

0.1 -5.97 3.78 273 

0.5 -8.32 3.56 273 

1.0 -12.06 3.24 273 

0.1 0.00 9.85 276 

0.5 -2.23 10.30 276 

1.0 -2.98 10.65 276 

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, 

70

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.). 

A 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0.0 

200 225 250 275 300 325 350 

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). 

nm 

71

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 

FI spectroscopy Garcia et al., 

1998 

0.2 – 5.0 µg ml -1 0.2 µg ml -1 

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. 

72

A 

0.5 

0.4 

0.3 

0.2 

0.1 

0.0 

200 225 250 275 300 325 350 

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 

Fenac 

Dichloran 

Voltral 

Ardifenac 

Diclofenac 

Mentioned 

concentration 

(mg unit -1 ) 

50 

50 

50 

50 

50 

a , average value; ±, standard deviation (n=5) 

nm 

Actual Concentration a Recovery 

(%) 

mg unit -1 

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 

73

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. 

A 

2.0 

1.6 

1.2 

0.8 

0.4 

0.0 

200 225 250 275 300 325 350 375 400 

nm 

Fig. 4.2.6. UV spectra showing blank (black), DS containing (blue) and DS spiked 

(red) urine. 

74

A 

0.30 

0.25 

0.20 

0.15 

0.10 

0.05 

0.00 

200 225 250 275 300 325 350 

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 

nm 

75

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. 

76

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. 

2 

I, µA 

1 

0 

A 600 

E , mV 1/2 

400 

10 9 

11 

8 

12 

6 

7 

4 

5 

200 

2 7 12 

pH 

3 2 

200 500 

E, mV 

800 

4 

I, µA 

2 

0 

B 

9 

10 

11 

12 

7 

8 

6 

5 

1 

4 

600 

E, mV 

400 

200 

2 

3 

2 7 pH 12 

200 500 800 

E, mV 

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. 

77

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 

78

potentials to values Ereg1 = –400 mV and Ereg2 = 1300 mV (values were optimized by 

series of trials) as shown in Fig.4.3.2. 

3000 

I, nA 

1500 

3000 

I p , nA 

1500 

0 

0 25 N 50 

0 

200 500 800 

E, mV 

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 ] 

20 – 100 

2 – 10 

0.2 – 1 

0.02 – 0.1 

Slope 

[nA.μmol L -1 ] 

29.7 

48.3 

48.7 

58.5 

Intercept 

[nA] 

49.5 

15.8 

1.15 

2.28 

R 

0.9976 

0.9981 

0.9987 

0.9982 

LOD 

[μmol L -1 ] 

– 

– 

– 

0.034 

79

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. 

3000 

I, nA 

1500 

6 

5 

4 

3 

2 

3000 

I , nA p 

1500 

0 

0 50 c, µM 100 

1 

0 

200 500 E, mV 800 

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. 

80

600 

I, nA 

400 

200 

6 

5 

4 

3 

2 

1 

I , nA p 

250 

0 

0 5 c, µM 10 

200 500 800 

E, mV 

500 


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. 


dependence. 

200 

I, nA 

150 

6 

5 

4 

3 

2 

50 

I p , nA 

0 

0.0 0.5 

c, µM 

1.0 

100 

200 

1 

500 

E, mV 

800 

25 


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. 


dependence. 

81

200 

I, nA 

150 

6 

I , nA p 

5 

5 

4 

3 2 

0 

0.00 0.05 

c, µM 

0.10 

100 

200 500 800 

E, mV 

10 

1 


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). 

82

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 

Paralen 

Panadol 

Paracetamol 

Coldrex 

Zentiva. Cz 

Glaxosmithkline 



Contents of Paracetamol 

[mg] 

494 

496 

503 

493 

Recovery 

[%] 

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 

I, nA 

100 

0 

110 

90 

70 

50 

30 

20 

10 

0 

100 

80 

60 

40 

200 

I , nA p 

100 

0 

0 50 100 

Ascorbic Acid 

c, µM 

200 500 E, mV 800 

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. 

83


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). 

240 

I, nA 

160 

80 

A 

10 

8 

6 

4 

2 

1 

150 

I p , nA 

0 

0 5 10 

c, µM 

200 500 

E, mV 

800 

75 

1600 

I, nA 

800 

B 

20 

100 

80 

60 

40 

1400 

I p , nA 

700 

0 

0 50 100 

c, µM 

0 

200 

0 

500 

E, mV 

800 

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. 

84


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] 

20 – 100 

2 – 10 

Slope 

[nA.μmol -1 .l] 

12.91 

14.17 

Intercept 

[nA] 

31.66 

–7.61 

R LOD 

[μ mol -1 .l] 

0.9975 

0.9994 

– 

0.48 

85

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 R 2 =0.998 as shown in (Fig. 4.4.1) by excellent response of the FTIR 

with the increasing concentration. 

86

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 

87

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. 

Spike 3 

Spike 2 

Spike 1 

Tablet sample 

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. 

88

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) Recovery a CV b 

(%) (%) 

1 30 20 49.56. ± 1.25 98.53 1.6 

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 

a Recovery (%)=(C–B)/A x100. 

Acceptable recovery (%) 

[AOAC, 2002] 

90-108 

b Coefficient of variation was obtained from the mean of five replicate tests. 


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. 

89

Table 4.4.3. Results for the ibuprofen concentration found in urine samples 

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 

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. 

90

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) Recovery a CV b 

(%) (%) 

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 

a Recovery (%)=(C–B)/A x100. 

b Coefficient 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. 

91

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. 

92

3000 

I, nA 

1500 

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. 

900 

E , mV 1/2 

600 

2 4 6 8 10 12 

pH 

12 

11 

8 

4 

10 

9 

5 

6 

0 

200 700 E, mV 1200 

3 

2 

7 

3000 

I, nA 

1500 

0 

1200 

E, mV 

600 

0 

2 4 6 8 10 12 

pH 

A B 

12 

200 700 

E, mV 

1200 

4 

8 

5 

10 11 

6 

9 7 

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 

2 

93 

3

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. 

94

I, nA 

3000 

1500 

0 

0 4 8 

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. 

N 

1 

2 

3 

95

2400 

I, nA 

1600 

800 

1500 

I p , nA 

750 

0 

0 50 c, µM 100 

750 1000 E, mV 1250 

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 

I, nA 

500 

250 

200 

I p , nA 

100 

0 

0 5 c, µM 10 

750 1000 E, mV 1250 


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 


6 

5 

4 

3 

2 

1 

6 

5 

4 

3 

2 

1 

96

80 

I, nA 

60 

40 

20 

I , nA p 

10 

0 

0.0 0.5 

c, µM 

1.0 

6 

5 

4 

3 

2 

800 1000 E, mV 1200 


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 


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.). 


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 ] 

20 – 100 

2 – 10 

0.2-1 

Slope 

[nA μmol L -1 ] 

13.43 

16.73 

18.52 

4.5.4. Hydrolysis of Acetylsalicylic Acid 

Intercept 

[nA] 

35.77 

9.58 

0.84 

1 

R 

0.9993 

0.9994 

0.9996 

L O D 

[μ mol L -1 ] 

– 

– 

0.21 

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 

97

current of salicylic acid and acetylsalicylic acid (Aspirin) at 2 minutes with activation 

potential 2200 mV for 120 sec. 

900 

I, nA 

600 

4 

3 

2 

300 

750 1000 E, mV 1250 

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. 

1 

98

2000 

I, nA 

1000 

1500 

I p , nA 

750 

0 

0 50 c, µM 100 

1 

0 

750 1000 E, mV 1250 

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 


300 

I, nA 

200 

100 

200 

I p , nA 

100 

0 

0 5 c, µM 10 

750 1000 E, mV 1250 


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 


6 

5 

4 

3 

2 

6 

5 

4 

3 

2 

1 

99

120 

I, nA 

80 

20 

I , nA p 

10 

0 

0.0 0.5 1.0 

c, µM 

40 

750 1000 E, mV 1250 


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 


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. 


hydrolyzed Acetylsalicylic Acid in a Britton-Robinson buffer pH 2.0 

using DPV at CFE with activation potential 2200 mV 


[μmol L -1 ] 

20 – 100 

2 – 10 

0.2-1 

Slope 


13.93 

18.20 

20.17 

Intercept 

[nA] 

17.2 

3.11 

0.43 

6 

4 

3 

2 

5 

1 

R 

0.9996 

0.9991 

0.9996 

4.5.6. Determination of Salicylic Acid in Different Drugs 

L O D 

[μmol L -1 ] 

– 

– 

0.15 

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 

100

10µM for the determination of salicylic acid at CFE. Good linear calibration curves 

were found as given in figure 4.5.10. 

240 

I, nA 

160 

80 

750 1000 

E, mV 

1250 

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. 

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

S. no Name of Tablet 

1 

2 

Duofilm 

Saloxyl 

5 

4 

3 

2 

Company Name 

Stiefel 

1 

Herbacos-bofarma 

Tablets 

(%) 

16.7 

4.5.7. Determination of Acetylsalicylic Acid from Different Drugs 

10 

Recovery ± stdev 

from 5 measur. (%) 

103 ± 3 

105 ± 1 

The developed method was tested for 4 µM Aspirin, Acypyrin, Acypyrin + C, 

Disprin, Acefein and Acefein + C drugs. The different concentration of hydrolyzed 

101

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. 

300 

I, nA 

200 

100 

750 1000 

E, mV 

1250 

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 

1 

2 

3 

4 

5 

Aspirin 

Acylpyrin 

Acylpyrin + C 

Disprin 

Acifein 

Company Name 

Bayer 



Reckitt Benckiser 


5 

4 

3 

2 

1 

Tablets 

(mg) 

500 

500 

320 

300 

250 

Recovery ± stdev 

from 5 measur. (%) 

106 ± 2 

106 ± 2 

95 ± 1 

101 ± 3 

100 ± 2 

102

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 

I, nA 

500 

250 

600 

I , nA p 

400 

200 

0 50c, µM 100 

750 1000 E, mV 1250 

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 


180 

I, nA 

120 

80 

I p , nA 

40 

0 

0 5 c, µM 10 

60 

750 1000 

E, mV 

1250 


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 


6 

5 

4 

3 

2 

1 

6 

5 

4 

3 

2 

1 

103

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. 


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 


[μ mol -1 L] 

20 – 100 

2 – 10 

Slope 


5.41 

8.12 

Intercept 

[nA] 

39.21 

–9.03 

R 

0.9886 

0.9976 

4.5.9. Determination of Acetylsalicylic Acid in Urine at CFE 

L O D 

[μ mol -1 L] 

– 

0.64 

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. 

104

450 

I, nA 

300 

150 

300 

I p , nA 

150 

0 

0 50c, µM 100 

750 1000 

E, mV 

1250 


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. 

90 

I, nA 

60 

30 

40 

I , nA p 

20 

0 

0 5 c, µM 10 

750 1000 

E, mV 

1250 


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 


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. 

6 

5 

4 

3 

2 

1 

6 

5 

4 

3 

2 

1 

105


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 


[μmol L -1 ] 

20 – 100 

2 – 10 

4.5.10. Interference Study 

Slope 


3.06 

4.51 

Intercept 

[nA] 

17.99 

– 3.92 

R 

0.9997 

0.9998 

L O D 

[μmol L -1 ] 

– 

0.71 

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. 

450 

I, nA 

300 

150 

I p , nA 

100 

50 

0 

0 50c, µM 100 

200 700 

E, mV 

1200 

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. 

106

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 

107

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. 

108

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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. 

133

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). 

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Ph. D Thesis - Higher Education Commission

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