Eid Eid Elraghyi Mohamed

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do not fluoresce due to rapid relaxation by internal conversion and intersystem .... also accounts for the observation that the absorption spectra of the lanthanide ions are only ..... saving, compared to all of the previously reported methods. 2.3.2.4. ... quantitative determination in dissolution tests of benazepril–HCl (BNZ) and.

Genetic Engineering & Biotechnology Research Institute (GEBRI)

MODERN METHODS FOR MICRODETERMINATION OF SOME ORGANIC COMPOUNDS USED IN PHARMACEUTICAL INDUSTRIES

PRESENTED By

Eid Eid Elraghyi Mohamed B. Sci. (Chemistry Department), South Valley University, (2005)

A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science In

Genetic Engineering and Biotechnology TO Industrial Biotechnology Department Genetic Engineering & Biotechnology Research Institute (GEBRI) Menoufia University

2012

1

APPROVAL SHEET

MODERN METHODS FOR MICRODETERMINATION OF SOME ORGANIC COMPOUNDS USED IN PHARMACEUTICAL INDUSTRIES

PRESENTED By

Eid Eid Elraghyi Mohamed B.Sc. (Chemistry Department), South Valley Univ. (2005).

Examiners’ Committee:

Approved

Prof. Dr. Abdelfatah Bastawy Farag Saad............................. Prof. of Analytical Chemistry, Faculty of Science, Helwan University

Prof. Dr. Mona Abdelaziz Ahmed Hussein ……………. Prof. of Analytical Chemistry, Women's College, (Arts, Science and Education) Ain Shams University.

Prof. Dr. Abdelhameed Mahmoud Othman …............. Prof. of Analytical Chemistry and Head of Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia, University.

(Pharmaceutical Industrial) Industrial Biotechnology Department Date of Examination: 25 / 6 /2012

2

MODERN METHODS FOR MICRODETERMINATION OF SOME ORGANIC COMPOUNDS USED IN PHARMACEUTICAL INDUSTRIES

PRESENTED By

Eid Eid Elraghyi Mohamed B.Sc. (Chemistry Department), South Valley Univ. (2005).

Under the supervision of Prof. Dr. Abdelhameed Mahmoud Othman …... Prof. of Analytical Chemistry and Head of Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia, University. Prof. Dr. Nashwa Mokhtar Hassan Rizk………. Prof. of Analytical Chemistry, Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia, University. Dr. Hoda Mahrous Abass Ibrahim…………… Assistant Prof. of Dairy Microbiology Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia University.

2012

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Supervision committee: Prof. Dr. Abdelhameed Mahmoud Othman….. Prof. of Analytical Chemistry and Head of Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia, University. Prof. Dr. Nashwa Mokhtar Hassan Rizk………. Prof. of Analytical Chemistry, Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia, University. Dr. Hoda Mahrous Abass Ibrahim……………. Assistant Prof. of Dairy Microbiology Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia University.

2012

4

MODERN METHODS FOR MICRODETERMINATION OF SOME ORGANIC COMPOUNDS USED IN PHARMACEUTICAL INDUSTRIES

PRESENTED By

Eid Eid Elraghyi Mohamed B.Sc. (Chemistry Department), South Valley Univ. (2005).

A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science In

Genetic Engineering and Biotechnology TO

Industrial Biotechnology Department Genetic Engineering & Biotechnology Research Institute (GEBRI) Menoufia University

Under the supervision of Prof. Dr. AbdelHameed Mahmoud Othman Prof. Dr. Nashwa Mokhtar Hassan Rizk Dr. Hoda Mahrous Abass Ibrahim 2012

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ABSTRAC

1

ABSTRACT The studies presented in this thesis include novel analytical methods based on spectrofluorimetric techniques for determination of some organic compounds used in pharmaceutical preparations. The thesis is divided to three chapters. The first chapter includes general introduction of spectrofluorimetric activity of lanthanide elements and the parameters affects on these phenomena. Also, the chapter involved the review of literature for previous methods of metoclopramide and hydrochlorothiazide drugs determination. The second chapter deals with the materials and apparatus used during this work including the types, grades, sources and preparations of these materials. Also, include the methods for measurements of different factors as absorbance, excitation and emission, effect of pH, effect of reagents and the validity tests of the metoclopramide and hydrochlorothiazide proposed methods as selectivity, recovery, precision and stability. The third chapter includes the results and discussion of the proposed methods for determination of metoclopramide hydrochloride (MCP) and hydrochlorothiazide (HCT). 1- Metoclopramide hydrochloride (MCP): A new, simple and accurate spectrofluorimetric method for the determination of metoclopramide hydrochloride (MCP) was developed. The method depend on measuring the luminescence intensity of the Tb3+ ion doped in PMMA matrix at 545 nm in methanol and pH 6.9. The effect of different parameters, e.g., pH, temperature, Tb3+ ion concentration, foreign ions that control the fluorescence was critically investigated. The calibration curve of the emission intensity at 545 nm shows linear response of metoclopramide over a concentration range of 5×10−5–5.0×10−8 mol L−1 with detection limit of 8.7×10−10 mol L−1. The method was used successfully for the determination of metoclopramide in pharmaceutical preparations and human serum. 2- Hydrochlorothiazide (HCT): This part reports on a facile technique combined with a simple, sensitive and selective spectrofluorimetric method for the determination of hydrochlorothiazide (HCT). At the optimized experimental conditions, the enhancement of the characteristic emission band at 617 nm of Eu3+ ion doped PMMA is directly proportional to the concentration of hydrochlorothiazide with a dynamic range of 5 ×10-8 – 1.0 ×10-5 mol L−1 and detection

a

limit of 8.0 ×10−9 mol L−1. Application of the suggested method was successfully applied to the determination of hydrochlorothiazide in pharmaceutical preparations and human serum samples, with high percentage of recovery, good accuracy and precision.

b

b

PUBLISHED PAPER

c

Published paper from this thesis 1- Spectrofluorimetric

assessment

of

metoclopramide

hydrochloride using terbium doped in PMMA matrix optical sensor. M. S. Attia & A. M. Othman & E. Elraghi and Hassan Y. Aboul-Enein Journal of Fluorescence, 21(2011), 739–745.

2- Excited state interaction between hydrochlorothiazide and europium ion in PMMA polymer and its application as optical sensor for hydrochlorothiazide in tablet and serum samples.

M. S. Attia & A. M. Othman & A. O. Youssef and E. ElRaghi Journal of Luminescence , 132(2012), 2049–2053.

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ACKNOWLEDGEMENT

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ACKNOWLEDGEMENT First of all prayerful thanks to our merciful GOD for helping me to finish this work. I wish to express my sincere appreciation to Prof. Dr. Abdel-hameed Mahmoud Othman Prof. of Analytical Chemistry and Head of Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia, University, for suggesting the point of this work, his support and excellent supervision during this work. I wish to express my greatest, deepest gratitude, with full thanks to Prof. Dr. Nashwa Mokhtar Rizk Prof. of Analytical Chemistry, Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia, University for unlimited encouragement and excellent supervision. My deepest gratitude and appreciation to Prof. Dr. Hoda Mahrous Abass Ibrahim Assistant Prof. of Food and Dairy Biotechnology, Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia University. for her guidance supervision and continuous assistance throughout this work. I wish to express my greatest, deepest gratitude, with full thanks to Prof. Dr. Ashraf Farag El-Baz Hawas Prof. of Microbiology, Head of Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menoufia University for introducing the facilities and the materials for carrying out this work, unlimited encouragement. Sincere thanks are also due to Prof. Dr. mohamed said attia Assistant Professor of analytical Chemistry, Faculty of Science, Ain Shams University, for his help and supporting during the work in this thesis. I wish to express my deep and sincere thanks for Chemist Eman Farouk Safwat and Pharmacist Laila Mohamed Elemiry for helping me. Finally, I would like to thank every person helped me to finish this work.

f

g

List of Contents

Page

Contents ‫أ‬

List of Contents……………………………………………………...

i

List of Figures………………………………………………….........

vi

List of Tables………………………………………………………...

Ix

List of Abbreviations………………………………………………..

X

1. INTRODUCTION………………………………………………..

1

2. REVIEW OF LITERATURE………………………………....

3

2.1. Spectrofluoremetric technique …………………………….

3

2.1.1. Theory of fluorescence…………………………………...

3

2.1.2. The fluorescence process…………………………………

4

2.1.3. Fluorescence spectra……………………………………...

7

2.1.4. Fluorimetric methods……………………………..............

8

2.1.4.1. Physicochemical modifications……………………..

8

2.1.4.2. Chemical modifications……………………………...

9

2.1.5. Environmental effects on luminescence spectra………….

10

2.1.5.1. Solvent effects……………………………….............

10

2.1.5.2. The influence of pH………………………………….

12

2.1.5.3. The influence of concentrations. .……… ………...

14

2.1.5.4. Fluorescence and structure…………………………..

17

2.1.5.4.1. Chemical structure effects on luminescence spectra

19

2.1.5.4.2. Chemical structure and luminescence intensit….

19

2.1.5.4.3. Chemical structure and position of the luminescence maximum…………………………………………

20

2.1.6. Some applications of fluorimetry………………………..

20

2.2. Definition of the f elements: Elementary concepts…..........

23

2.2.1. Historical perspectives …………………………………...

23

2.2.1.1. Discovery of the lanthanide………………………...

23

‫ب‬

2.2.1.2. Occurrence, extraction, and synthesis of the lanthanide elements…………………………………………….

24

2.2.2. Photophysical properties of trivalent lanthanide ions…..

25

2.2.2.1. Electronic energy levels…………………................

25

2.2.2.2. Absorption………………………………………….

26

2.2.2.3. Luminescence……………………………………....

27

2.2.3. Luminescent lanthanide complexes…………………….

30

2.2.3.1. Coordination chemistry of lanthanide ions…………...

30

2.2.3.2. Energy transfer…………………………..................

32

2.2.3.3. Applications of energy transfer ………………………

35

2.3. Metoclopramide hydrochloride……………………………...

39

2.3.1. Introduction……………………………………………...

39

2.3.2. Determination of metoclopramide hydrochloride................

39

2.3.2.1. Spectrophotometric methods………………………...

39

2.3.2.2. Chromatographic methods. ………………………….

40

2.3.2.3. Voltammetric methods………………………………..

42

2.3.2.4. Other methods for the quantitative determination of metoclopramide (MCP). . …………………………. .. .. 2.4. Hydrochlorothiazide. ………………………………………...

42 44

2.4.1. Introduction……………………………………………..

44

2.4.2. Determination of hydrochlorothiazide………….............

44

2.4.2.1. Spectrophotometric methods………………………….

44

2.4.2.2. Chromatographic methods………………………….. 2.4.2.3. Other methods for the quantitative determination of hydrochlorothiazide (HCT)…………. .…………..... ....

46

2.4.2.4. Poly(methyl methacrylate) (PMMA)…… …………

49

3. MATERIALS AND METHODS………………………………

51

‫ج‬

48

3.1. Materials and methods for metoclopramide (MCP) determination…………………………………………………

51

3.1.1. Chemicals and reagents………………………………...

51

3.1.2. Apparatus………………………………………………….

52

3.1.3. General procedure………………………………………

53

3.1.3.1. Preparation of Tb3+ ion doped in PMMA matrix ……

53

3.1.3.2.Preparation of metoclopramide hydrochloride solutions..

53

3.1.3.3. Measurement procedures of the luminescence spectrum of the optical Sensor Tb3+ doped in PMMA matrix….

53

3.1.4. Validation for MCP proposed method……………………

54

3.1.4.1. Selectivity …………………………………………...

54

3.1.4.2. Linearity……………………………………………..

54

3.1.4.3. Precision……………………………………………..

55

3.1.4.4. Recovery …………………….....................................

55

3.1.4.5. Stability ………………………………………………..

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3.1.5. Determination of (MCP) in pharmaceutical preparations.

55

3.1.6. Determination of (MCP) in serum solution……………….

56

3.2. Materials and methods for hydrochlorothiazide (HCT) 57 determination……………… 3.2.1. Chemicals and reagents…………………………………

57

3.2.2. Apparatus………………………………………………….

58

3.2.3. General procedure……………………………………….

58

3.2.3.1. Preparation of Eu3+ ion doped in PMMA matrix …...

58

3.2.3.2. Preparation of (HCT) solutions………………………..

59

3.2.3.3. Measurement procedures of the luminescence spectrum of the optical sensor Eu3+ ion doped in PMMA matrix…...

59

3.2.4. Validation for hydrochlorothiazide proposed method……..

59

‫د‬

3.2.4.1. Selectivity……………………………………………..

59

3.2.4.2. Linearity…………………………………………….

60

3.2.4.3. Precision…………………………………………….

60

3.2.4.4. Recovery………………………………………………

60

3.2.4.5. Stability………………………………………………..

61

3.2.5.Determination of (HCT) in pharmaceutical

preparation.

3.2.6. Determination of (HCT) in serum solution…………………. 4. RESULTS AND DISCUSSION…………………………………. 4.1.

Spectrofluorimetric assessment of metoclopramide hydrochloride using terbium doped in PMMA matrix optical sensor ……………………………………. …….

61 61 63

63

4.1.1. Spectral characteristics for metoclopramide determination.

63

4.1.1.1. Absorption spectra ……………………………………

63

4.1.1.2. Emission and excitation spectra…………………….

63

4.1.2. Effect of experimental conditions for metoclopramide determination……………………………………………….. 4.1.2.1.Effect of the amount of metoclopramide hydrochloride.

64 64

4.1.2.2. Effect of the amount of Tb3+…………………………

67

4.1.2.3. Effect of pH on Tb-MCP compound ………………..

67

4.1.3. Analytical application for MCP determination………......

69

4.1.3.1. Linear range and limit of detection……………...

69

4.1.3.2. Determination of MCP in pharmaceutical preparations and in serum solution……... ………….

70

4.1.4. Stability……………………………………………………

71

4.15. Conclusion………………………………….......................

72

‫ه‬

4.2. Excited state interaction between hydrochlorothiazide and europium ion in PMMA polymer and its application as optical sensor for hydrochlorothiazide in tablet and serum samples………………………………………………………... 4.2.1. Spectral characteristics for hydrochlorothiazide (HCT) determination………………………………………………

77 77

4.2.1.1. Absorption spectra………………...............................

77

4.2.1.2. Emission and excitation spectra…….. ………………

77

4.2.2.

Effect of different experimental conditions of hydrochlorothiazide (HCT) determination…………….

78

4.2.2.1. Effect of the amount of (HCT)………………………

78

4.2.2.2. Effect of Eu3+ concentration…………………….

78

4.2.2.3. Effect of pH…………………………………………

79

4.2.3. Analytical application for HCT determination ………….. ..

83

4.2.3.1. Linear range and limit of detection …………………. 4.2.3.2. Determination of HCT in pharmaceutical preparations and in serum .…………… ………..

83

4.2.4. Stability…………………………………………………….

85

4.2.5. Conclusion …………………………………………..........

85

5- SUMMARY AND CONCLUSION……………………………

89

6. LITERATURE CITED………………………………………...

91

7- ARABIC SUMMARY……………………………………………

‫و‬

83

List of Figures

‫ز‬

List of Figures List of Figures

Page

Figure (2.1.) Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence……………………………………………. fluorescence and Figure (2.2.) Energy diagram comparing phosphorescence…………………………………………

4

Figure (2.3.)

Fluorescing aromatic compounds……………………….

7

Figure (2.4.)

Excitation of a fluorophore at three different wavelengths (EX 1, EX 2, EX 3) does not change the emission profile but does produce variations in fluorescence emission intensity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum…………………… Jabloński diagram for luminescence with solvent relaxation…………………………………………………

Figure (2.5.)

Figure (2.6.) pH dependences of the relative fluorescence intensities (If/If 0) of a base (B) and its conjugate acid (BH +) resulting from B becoming a stronger base in the lowest excited singlet state (pkbh+ =11.5) but having insufficient time, prior to the fluorescence of B, for complete protonation. Acid–base equilibrium is therefore not truly attained during the lifetime of the lowest excited singlet state……………………………………… Figure (2.7.) Intensity of fluorescence and radiation damping. ……….

6

9

10

13 15

Figure (2.8.)

Intensity of fluorescence and concentration……………..

17

Figure (2.9.)

The modern periodic table……………………………….

23

‫ح‬

The energy levels diagram of some trivalent lanthanide ions Nd3+, Er3+, Yb3+, Eu3+, Tb3+, Sm3+, Gd3+, and Pr3+…

29

Ionic radius of trivalent lanthanide ions with coordination number eight……………………………………………..

30

Jablonski diagram for sensitized emission of lanthanide ions by a sensitizer……………………………………….

34

Figure (2.13.)

Homogenous flouroimmunoassays. …………………….

38

Figure (3.1.)

Structure of Metoclopramide hydrochloride (MCP)… ….

51

Figure (3.2.)

Structure of Hydrochlorothiazide (HCT)………………...

57

Figure (4.1.)

The absorption spectra of 5x10-4 M of (Metoclopramide hydrochloride) in different molar concentration of Tb3+ doped in PMMA matrix curves (2, 3 and 4). .................

65

Figure (2.10.) Figure (2.11.) Figure (2.12.)

Figure (4.2.)

Figure (4.3.)

Figure (4.4.)

Figure (4.5.)

The fluorescence excitation of (1x10 −3 mol L−1of Tb3+ with 5x10-4 mol L−1 MCP), curve (1), and emission spectrum of 5x10-4 mol L−1 MCP only, curve (2) and the excitation and the emission spectrum of 1x10 −3 mol L−1 of Tb3+ only curve (3), where curve (4) is the emission spectrum of (1x10−3 mol L−1of Tb3+ and 5x10−4 mol L−1 MCP complex in PMMA matrix at λex /λem=360/545 nm……………………………………………………….. Luminescence spectra of 1 x 10-3 mol l-1 of Tb3+ doped in PMMA in the presence of different molar concentration of (5 × 10−5 mol L−1 to 5 × 10−9 mol L-1 MCP ) in methanol at λex = 360 nm…………………… −5

−1

68

−9

Calibration curve of MCP (5x10 mol L to 5 x10 mol L-1) at λex 360 / λem 545 nm in methanol using Tb3+ doped in PMMA matrix optical sensor………………… Absorption spectra of 1x10-5 mol/L of HCT with different concentrations of Eu3+ in PMMA. ……………………..

‫ط‬

66

73

80

Figure (4.6.)

Figure (4.7.)

Figure (4.8.)

The fluorescence excitation spectrum of: "1" Eu3++HCT and emission spectra of: "2" HCT, "3" HCT+1x10 -5 mol/L of Eu3+, and "4" HCT+2x10-5 M of Eu3+ in PMMA matrix at λex /λem = 340/617 nm……………… Luminescence spectra of 2x10-5 mol L-1 of Eu3+ doped in PMMA in the presence of different molar concentration of HCT in methanol at λex = 340 nm……………………

81

82

Calibration curve of the different molar concentration of HCT in methanol and the luminescence intensity of Eu3+ 86 doped in PMMA matrix at λex = 340 nm………………….

‫ي‬

List of Tables

‫ك‬

List of Tables List of Tables

Page

Table (2.1.)

Examples of fluorescent indicators ………………...

22

Table (2.2.)

The typical emission bands of the lanthanide ions Sm3+, Eu3+, Tb3+, Nd3+, Er3+, and Yb3+ in solution…. Solvents and reagents used…………………………

31

Determination of MCP in serum and pharmaceutical preparations using Tb- MCP optical sensor………...

74

Freeze–thaw stability of MCP in pharmaceutical tablets and human serum (n=3)…………………….

75

Table (3.1.) Table (4.1.) Table (4.2.) Table (4.3.)

Table (4.4.)

Table (4.5.)

Comparison of spectrofluorimetric technique with some existing methods for the determination of MCP… Determination of HCT in serum and pharmaceutical preparations using optical sensor Eu3+ ion doped in PMMA………………………….. Freeze–thaw stability of HCT in pharmaceutical tablets and human serum (n=3)……………………

‫ل‬

52

76

87 88

List of Abbreviations

‫م‬

List of Abbreviations ACE:

Angiotensin-Converting Enzyme inhibitors.

ARBs:

Angiotensin II Receptor Blockers.

B.P.:

British Pharmacopoeia.

C:

Concentration.

CPE:

Carbon Paste Electrode.

CV%:

Coefficients of Variation.

DAP:

Dapsone.

DDT:

Dichlorodiphenyltrichloroethane.

DdMCP:

Dideethylated-MCP.

DOP:

Dioctylphthalate.

ECL:

Electrogenerated Chemiluminescence

ET:

Efficiency of Transfer.

F:

Fluorescence.

FI:

Flow-Injection.

FRET:

Fluorescence Resonance Energy Transfer.

FSCCV:

Fast Stripping Continuous Cyclic Voltammetry.

GC—MS:

Gas Chromatographic—Mass Spectrometric.

GCE:

Glassy Carbon Electrode.

HCT:

Hydrochlorothiazide.

HFT:

Hydrofluorothiazide.

‫ن‬

HPLC:

High Performance Liquid Chromatography.

h

EM:

Emission Photon Energy.

h

EX:

Excitation Photon Energy.

ICH:

International Conference on Harmonisation.

If :

Emission light intensity.

I0 :

Intensity of The Excitation Radiation.

IS:

Internal standard.

ISC:

Intersystem Crossing.

It :

Transmitted light intensity.

K:

Global Constant.

LC:

Liquid Chromatography.

LCMEDs:

Electroluminescent Light Conversion Molecular Devices.

LC–MS/MS:

Liquid Chromatographic–Electrospray Tandem Mass Spectrometry.

LF:

Total Ligand-Field.

Ln(III):

Trivalent Lanthanide Ions.

LOD:

Limit Of Detection.

LOQ:

Limit Of Quantitation.

LSD:

Lysergic Acid Diethylamide.

MCP:

Metoclopramide.

mdMCP:

Monodeethylated-MCP.

MRI:

Magnetic Resonance Imaging.

MRM:

Multiple Reaction Monitor.

MRS:

Magnetic Resonance Spectroscopy.

MS/MS:

Tandem Mass Spectrometric.

‫س‬

Ionization

NIR:

Near-Infrared Light.

P:

Phosphorescence.

PABA:

p-Aminobenzoic Acid.

PAH:

Polynuclear Aromatic Hydrocarbons.

PGC:

Porous Graphitized Carbon.

PMA:

Phosphomolybdic Acid.

PMMA:

Poly(methyl methacrylate).

PVC:

Polyvinyl Chloride.

QC:

Quality Control.

R.S.D.:

Relative Standard Deviation.

RuDS:

(Ru(bpy)32+)-doped silica.

S.D:

Standard Deviation.

SE:

Sensitized Lanthanide Ion Emission.

SET:

Singlet Energy Transfer.

SIM:

Selected Ion Monitoring.

SPE:

Solid-Phase Extraction.

STPB:

Sodium Tetraphenylborate.

SWAS:

Selective Square Wave Anodic Stripping.

UV:

Ultraviolet.

VIS:

Visible Light

фƒ :

Fluorescence Quantum Yield.

‫ع‬

‫ف‬

INTRODUCTION

0

1. INTRODUCTION Some organic or inorganic compounds, liquids or solids (molecular or ionic crystals), whether pure or in solution, emit light when they are excited by photons from the visible or the near ultra-violet regions. Among the analytical applications of this phenomenon, known as photoluminescence, is fluorimetry, a selective and highly sensitive method for which a wide range of measurements are accessible (Francis R., et al., 2007). These applications are derived from the relationships between analyte concentrations and luminescence intensities and are therefore similar in concept to most other physicochemical methods of analysis. Other features of luminescence spectral bands, such as position in the electromagnetic spectrum (wavelength or frequency), band form, emission lifetime, and excitation spectrum, are related to molecular structure and environment and therefore also have analytical value (Leno O., and Anthony S., 2002; and Francis R., et al., 2007). Spectrofluoremetry is becoming more frequently used to analyze an extensive and growing number of pharmaceuticals and /or metabolites (Baeyens W. R., et al., 1991; and Braun R. D., 1982). The extreme sensitivity of this technique allows the identification and quantification of pharmaceuticals in both biological fluids and pharmaceutical preparations. Fluorimetric methods can be classified according to the nature of the fluorescence measured. The latter may be native fluorescence from the pharmaceutical itself or fluorescence obtained chemically or by structure modification (Baeyens, W. R. et al., 1991). The emission of a complexed trivalent rare earth ion arises from radiative transitions among the energy levels in the 4fn electronic

1

configuration of these ions. In the absence of any interaction between these n electrons, the levels would be degenerate (Koppe M., 2002). Metoclopramide (MCP), 4 – amino – 5 – chloro – 2 – methoxy – N (2diethylamino-ethyl) benzamide, is a dopamine-receptor antagonist active on gastrointestinal motility. It is used as an anti-emetic in the treatment of some forms of nausea and vomiting and to increase gastrointestinal motility. It is also used at much higher doses for the prevention of cancer chemotherapy-induced emesis (Tas C., et al., 2006). Hydrochlorothiazide (HCT), 6 – chloro – 3. 4 – dohydro – 2 H 1. 2. 4, benzothiadiazine-7-sulfonamide-1.1-dioxide, is a diuretic and antihypertensive agent that reduces plasma volume by increasing the excretion of sodium, chloride and water and, to a lesser extend, that of potassium ion as well (Vonaparti A., et al., 2006; and Patel R. B., et al., 1984). Poly(methyl methacrylate)

(PMMA) is a clear,

colorless polymer

extensively used for optical applications (Kim, G. H., 2005; and Jiu, H., et al., 2006). The optical properties of PMMA such as colorimetric changes, luminescence effects and light refraction changes are widely used in fiber optic sensors and optical waveguides or claddings. However, PMMA alone is an inactive material and to fabricate a fiber optic sensor, a composite polymer with an opto-active sensing element entrapped within the polymer matrix has to be prepared. (Chen C. H., and Lee W. C., 1999; and Ferriol M., et al., 2003). PMMA is frequently used in chemical sensors (ion, gas, humidity and enzyme sensors) (Chatzandroulis S., et al., 2004). The aim of the present study is to develop a simple, fast and sensitive analytical approach for the quantification of MCP and HCT using a Tb3+ and Eu3+ doped PMMA film as an optical sensor, respectively. The proposed methods are successfully validated for the routine analysis of MCP and HCT in pharmaceutical formulations and human serum samples.

2

3

REVIEW OF LITERATURE

2

2. REVIEW OF LITERATURE 2.1. Spectrofluoremetric technique: Some organic or inorganic compounds, liquids or solids (molecular or ionic crystals), whether pure or in solution, emit light when they are excited by photons from the visible or the near ultra-violet regions. Among the analytical applications of this phenomenon, known as photoluminescence, is fluorimetry, a selective and highly sensitive method for which a wide range of measurements are accessible (Francis R., et al., 2007). These applications are derived from the relationships between analyte concentrations and luminescence intensities and are therefore similar in concept to most other physicochemical methods of analysis. Other features of luminescence spectral bands, such as position in the electromagnetic spectrum (wavelength or frequency), band form, emission lifetime, and excitation spectrum, are related to molecular structure and environment and therefore also have analytical value (Leno O., and Anthony S., 2002; and Francis R., et al., 2007). Spectrofluoremetry is becoming more frequently used to analyze an extensive and growing number of pharmaceuticals and /or metabolites (Baeyens W. R., et al., 1991; and Braun R. D., 1982). The extreme sensitivity of this technique allows the identification and quantification of pharmaceuticals

in

both

biological

fluids

and

pharmaceutical

preparations.

2.1.1. Theory of fluorescence: Fluorescence is caused by the absorption of radiant energy and the reemission of some of this energy in the form of visible light. The light

3

emitted is almost always of higher wavelength than that absorbed. In true measurable time of the order 10-12 – 10-9 second. If the light is emitted with a time delay (> 10-8 second) the phenomenon is known as phosphorescence (Bassett J. et al., 1979). This time delay ranges from a fraction of a second to several weeks, so that the difference between two phenomena may be regarded as one term photoluminescence; the latter is therefore the general term applied to the process of absorption and reemission of light energy.

2.1.2. The fluorescence process: Fluorescence is the result of a three-stage process that occurs in certain molecules (generally polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. The process responsible for the fluorescence of fluorescent probes and other fluorophores is illustrated by the simple electronic-state diagram (Jablonski diagram) shown in Figure (2.1.) (Baeyens W. R. et al., 1991). Relaxation

Figure (2.1.): Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence.

4

The labeled stages 1, 2 and 3 are explained in the adjoining text. Stage 1 : Excitation: A photon of energy h

EX

is supplied by an external source such as an

incandescent lamp or a laser and absorbed by the fluorophore, creating an excited electronic singlet state (S1'). This process distinguishes fluorescence from chemiluminescence, in which the excited state is populated by a chemical reaction. Stage 2 : Excited-State lifetime: The excited state exists for a finite time (typically 1–10 nanoseconds). During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. These processes have two important consequences. First, the energy of S1' is partially dissipated, yielding a relaxed singlet excited state (S1) from which fluorescence emission originates. Second, not all the molecules initially excited by absorption (Stage 1) return to the ground state (S0) by fluorescence emission. Other processes such as collisional quenching, fluorescence resonance energy transfer (FRET) and intersystem crossing (see below) may also depopulate S1. The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a measure of the relative extent to which these processes occur. Stage 3 : Fluorescence emission: A photon of energy h

EM

is emitted, returning the fluorophore to its

ground state S0. Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon h represented by (h

EX

–h

EX. EM)

The difference in energy or wavelength is called the Stokes shift. The Stokes shift is

fundamental to the sensitivity of fluorescence techniques because it allows

5

emission photons to be detected against a low background, isolated from excitation photons (Baeyens W. R., et al., 1991). The short arrows correspond to mechanisms of internal conversion without emission of photons. The fluorescence results from transfers between states of the same multiplicity (same spin state) while the phosphorescence results from transfers between states of different multiplicity. The state T1 produces a delay in the return to the fundamental state, which can last several hours. The ‘Stokes shift’ corresponds to the energy dissipated in the form of heat (vibrational relaxation) during the lifetime of the excited state, prior to photon emission. Figure (2.2.) The real situation is more complex than this simplified Jablonski diagram suggests (Francis R., et al., 2007).

Figure

(2.2.):

Energy

diagram

comparing

fluorescence

and

phosphorescence. Sensitivity in fluorimetry is often 103 more than spectrophotometry. However, the correct use of these techniques requires a sound knowledge of the phenomenon in order to avoid a number of errors. Fluorescence, which is often encountered with rigid cyclic molecules possessing π bonds, is enhanced by the presence of electron-donating groups while being reduced by electron-withdrawing groups (Figure

6

(2.3.)). Equally, there is a dependency upon the pH of the solvent. In contrast, non-rigid molecules readily lose their entire absorbed energy through degradation and vibrational relaxation.

Figure (2.3.): Fluorescing aromatic compounds. The names are followed by their fluorescence quantum yield фƒ, for which the values are obtained by comparison with compounds of known fluorescence (Francis R., et al., 2007).

2.1.3. Fluorescence spectra: The entire fluorescence process is cyclical. Unless the fluorophore is irreversibly destroyed in the excited state, the same fluorophore can be repeatedly excited and detected. The fact that a single fluorophore can generate many thousands of detectable photons is fundamental to the high sensitivity of fluorescence detection techniques. For polyatomic molecules in solution, the discrete electronic transitions represented by h

EX

and h

EM

in Figure (2.1.) are replaced by rather broad energy spectra called the fluorescence excitation spectrum and fluorescence emission spectrum, respectively. Under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength, due to the partial dissipation of excitation energy during the excited-state lifetime, as illustrated in Figure (2.1.). The emission intensity is proportional to the

7

amplitude of the fluorescence excitation spectrum at the excitation wavelength (Figure (2.4.)) (Baeyens W. R., et al., 1991).

2.1.4. Fluorimetric methods: Fluorimetric methods can be classified according to the nature of the fluorescence measured. The latter may be native fluorescence from the pharmaceutical itself or fluorescence obtained chemically or by structure modification. Many compounds of interest do not present native fluorescence, but they may be derivatized, bound, labeled-in a word, modified to give rise to fluorescent products. The modifications induced to produce fluorescence in the molecules or in non-fluorescence systems may be chemical or physiochemical. All these methods have the aim of producing fluorescence or else of intensifying quantum yield. These processes are classified as follows.

2.1.4.1. Physicochemical modifications: Physicochemical modifications usually tend to improve the quantum yield of already fluorescent molecules. A great variety of phenomena and circumstances may occur: Modifications and changes in the molecule environment caused by changes in the polarity of the solvent. While some solvents favor the intensity of the fluorescence emission, others may quench it. Addition of chemical substance that might protect the fluorophores from their molecular environment.

8

Figure (2.4): Excitation of a fluorophore at three different wavelengths (EX 1, EX 2, EX 3) does not change the emission profile but does produce variations in fluorescence emission intensity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum. Electron or energy transfer processes among fluorescent and nonfluorescent molecules. These methods will be called sensibilizattion (Baeyens W. R., et al., 1991). 2.1.4.2. Chemical modifications: A fluorescent derivative is obtained from a non-fluorescent one, or the emission intensity of fluorescent compounds or systems may be increased. This treatment will be called derivatization. Chemical treatments including several reactions: redox, substitution, formation of binary and tertiary complexes, etc. These chemical modifications, called derivatization reactions and are also frequently used to produce stable derivatives from unstable compounds, enabling their determination by avoiding their decomposition during sample manipulations (Baeyens W. R., et al., 1991).

9

2.1.5. Environmental effects on luminescence spectra: 2.1.5.1. Solvent effects: Emission from fluorophores generally occur at wavelengths which are longer than those at which absorption occurs. This loss of energy is due to a variety of dynamic processes which occur following light absorption see Figure (2.5.). The fluorophore is typically excited to the frist singlet state (S1), usually to an excited vibrational level within S1. The excess vibrational energy is rapidly lost to the solvent. If the fluorophore is excited to the second singlet state (S2), it rapidly decays to the S1 state in 10-12 s due to internal conversion. Solvent effect shift the emission to still lower energy owing to stabilization of the excited state by the polar solvent molecules. Following excitation, the solvent dipoles can reorient or relax around µ E, which lowers the energy of the excited state. As the solvent polarity is increase, this effect becomes larger, resulting in emission at lower energyies or longer wavelengths (Lakowicz Joseph R., 2006).

Figure (2.5.): Jabloński diagram for luminescence with solvent relaxation. Examination of Figure (2.5.) reveals why absorption spectra are less sensitive to solvent polarity than emission spectra. Absorption of light occurs in about 10-15s, a time which is too short for motion of the

10

fluorophore or solvent. Absorption spectra are not affected by the decrease in the excited-state energy which occurs after absorption has occurred. The solvents in which fluorescence spectra are observed play a major role in determining the spectral positions and intensities with which fluorescence bands occur. In some cases, the solvent may determine whether or not fluorescence is observed at all. The effects of the solvent on the fluorescence spectra are determined by the nature and degree of the interactions of the solvent molecules with the ground and lowest electronically excited singlet state of the fluorescing solute molecules. The influence of the solvent on the appearance of phosphorescence is much smaller than that on fluorescence. Solvent interactions with solute molecules are electrostatic in nature and may be classified as dipolar or hydrogen bonding. The position of the fluorescence band maximum in one solvent, relative to that in another, depends on the relative separations between the ground and excited states in either solvent and therefore the relative strengths of ground- and excitedstate solvent stabilization. Hydrogen bonding in the lowest excited singlet state occasionally results in the loss of fluorescence intensity in molecules whose lowest excited singlet states are of the intramolecular charge transfer type. This is observed as a decrease in fluorescence quantum yield upon going from hydrocarbon to hydrogen-bonding solvents. Solvents containing atoms of high atomic number (e.g., alkyl iodides) also have a pronounced effect on the intensity of fluorescence of solute molecules. However, this effect is not directly related to the polarity or hydrogen-bonding properties of the ‘‘heavy-atom solvent’’ )Leno O., and Anthony S., 2002).

11

2.1.5.2. The influence of pH: The influences of pH on luminescence spectra are derived from the dissociation of acidic functional groups or the protonation of basic functional groups, associated with the aromatic portions of fluorescing molecules. The spectral shifts observed when basic groups are protonated or when acidic groups are dissociated are greater in magnitude, but qualitatively similar in direction, to the shifts resulting from interaction of lone or nonbonded electron pairs with hydrogen-bond donor solvents or from interaction of acidic hydrogen atoms with hydrogen bond acceptor solvents, respectively. Thus, the protonation of electron-withdrawing groups such as carboxyl, carbonyl, and pyridinic nitrogen results in shifts of the luminescence spectra to longer wavelengths, while the protonation of electron-donating groups such as the amino group produces spectral shifts to shorter wavelengths (Leno O. and Anthony S., 2002). If excited-state proton transfer is much slower from fluorescence, the fluorescence intensity-versus-pH curve will be exactly like the absorbanceversus-pH curve (i.e., no excited-state proton transfer occurs). If excited-state proton transfer is much faster than fluorescence, the fluorescence

intensity-versus-pH curve will

reflect

the acid–base

equilibrium in the lowest excited singlet state and the dissociation constant taken from the fluorescence intensity-versus-pH curve will be that of the excited-state reaction. Equilibrium in the excited state is a rare phenomenon and will not be dealt with further here. If the rate of proton transfer in the excited state is comparable to the rates of deactivation of acid and conjugate base by fluorescence, the variations of the fluorescence intensities of acid and conjugate base with pH will be governed by the kinetics of the excited state proton-transfer

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reactions. In general, the pH region over which the emissions of both conjugate acid and conjugate base are observed will be much wider (Figure (2.6.)) than, say, pKa

-2

< pH < pKa

+2

because in some cases it will be

possible to excite the conjugate acid and in others the conjugate base exclusively, and yet in both cases see emission from the conjugate acid and base. This obviously represents a potential interference in analytical fluorimetry. It is worth noting that buffer ions act as proton donors and acceptors with excited, potentially fluorescent molecules (Leno O., and Anthony S., 2002).

Figure (2.6.): pH dependences of the relative fluorescence intensities (If/If 0) of a base (B) and its conjugate acid (BH+) resulting from B becoming a stronger base in the lowest excited

singlet

state

(pKBH+

=11.5)

but

having

insufficient time, prior to the fluorescence of B, for complete protonation. Acid–base equilibrium is therefore not truly attained during the lifetime of the lowest excited singlet state (Leno O., and Anthony S., 2002).

13

In solutions containing high concentrations of buffer ions, the latter may induce excited-state proton transfer in molecules which would not ordinarily enter into this process in water. Consequently, the intelligent application of fluorimetry in buffered aqueous solutions requires the use of very dilute buffers and, therefore, a compromise between optimal buffer capacity and fluorimetric sensitivity (Leno O., and Anthony S., 2002). 2.1.5.3. The influence of concentrations: At each point of the solution the intensity of fluorescence is different because a part of the excitation radiation is absorbed before reaching the point being considered and because a part of the emitting radiation light finds itself trapped before it can exit the cell. Globally the fluorescence received by the detector corresponds to the sum of the fluorescence emerging from each of the individual small volumes constituting the space delimited by the entrance and exit slits (Figure (2.7)). This is why the calculation of the absolute fluorescence intensity (emission If ) for the sample is difficult. The phenomenon of radiation damping, called internal quenching is due to the partial overlap of the absorption and emission spectra (colour quenching), and is increased by transfers of energy from excited species to other molecules or ions through collisions or complexes formation (chemical quenching). That is why the presence of oxygen can cause an underestimation of fluorescence. For solutions, the quantum yield of fluorescence фf (between 0 and I inclusive), which is independent of the intensity emitted by the light source, is defined by the ratio of the number of photons emitted to the number of photons absorbed, this latter being equivalent to the ratio of fluorescence intensity If over that absorbed Ia (expression 2.1).

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(2.1)

Accepting that Ia = I0 − It (It representing the transmitted light intensity), the following reasoning allows to relate If to the concentration C, of the compound:

(2.2)

Figure (2.7.): Intensity of fluorescence and radiation damping. Modelling of expression (2.7) reveals the effect of the concentration upon fluorescence intensity. A maximum of fluorescence is observed beyond which, with a continuing increase in concentration, it diminishes. After the maximum the more concentrated the solution then the weaker is the fluorescence – a kind of roll-over or self-quenching. The illustrations correspond to three recordings, at the same scale of biacetyl tetrachloromethane. The curve records the light collected from a small volume situated at the centre of the solution as indicated on Figure (2.7). Knowing that the absorbance A is equal to log I 0/I, expression (2.2) becomes:

15

(2.3) with (2.4) If the solution is diluted, the term A is close to 0 and the term 10 −A is therefore close to 1−23 A. The expression (2.4) can then be simplified, becoming (Fancis R., et al., 2007; and Fifield F. W., and Kealey D., 2000): (2.5) with I0 the intensity of the excitation radiation, C the molar concentration of the compound, £ its molar absorption coefficient, l the cell thickness and фf the fluorescence quantum yield. This last expression reveals that the intensity of fluorescence depends upon the concentration (C), the experimental conditions (l, I0) and of the compound (£,фf ). If all the parameters due to the instrument and most due to the compound are factored into a global constant K, then the following equation can be used for weak concentrations (A N > S (Parra F. D., et el., 2002). Furthermore, it is generally agreed that Ln 3+-ligand coordination occurs predominantly via ionic bonding interactions, leading to a strong preference for negatively charged donor groups that are also “hard” bases, and neutral donors that possess large ground-state dipole moments such as amide carbonyl oxygn. Water molecules and hydroxide ions are particularly strong ligands for Ln 3+, so that in aqueous solution only ligands containing donor groups having negatively charged oxygens (such as carboxylate, sulfonate, phosphonate, phosphinate) can bind strongly (Parra F. D., et el., 2002). 2.2.3.2. Energy transfer: The terms ‘sensitized emission’ and ‘antenna effect’ are being used. These terms refer to the same phenomenon, i.e. a chromophoric group in a complex is first excited followed by energy transfer to the lanthanide ion, there by exciting this ion. Sensitizers are being used to excite the lanthanide ions via an energy transfer from the sensitizer (or antenna) to the lanthanide ion. This energy transfer is schematically depicted in a Jablonski diagram in Figure (2.12.). 32

3. Materials and Methods Upon absorption of light the sensitizer is excited to the singlet-excited state. This state can transfer its energy to the lanthanide ion (singlet energy transfer, SET), can convert to the triplet state (intersystem crossing, ISC), or can decay to the ground state (by luminescence or non-radiatively). From the triplet state energy is transferred to the lanthanide ion or the triplet state decays to the ground state. In general, energy is transferred via the triplet state because the intersystem crossing is enhanced by the nearby, paramagnetic lanthanide ions, and because energy transfer via the singlet state is not fast enough to compete with the luminescence or the intersystem crossing. Energy transfer can proceed via dipolar or multipolar interactions between sensitizer and acceptor (lanthanide ion), a froster mechanism of transfer, (Förster T., 1959). or via an exchange mechanism, the Dexter mechanism of transfer (Dexter D. L., 1953). The main difference between these two types of energy transfer is that in the Dexter mechanism orbital overlap is needed, whereas in the forester ِmechanism the transfer is through space and is strongly dependent on the spectral overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor. The distance dependence of the Dexter mechanism is exponentially, in the forster mechanism this is to the power -6. In the Dexter mechanism, the spectral overlap is independent of the oscillator strength of the transitions (emission and absorption spectra are normalized such that the areas of the spectra are unity (Hebbink G. A., 2002). Energy transfer by the Dexter mechanism is only efficient at very small distances (