EFFECT OF SOLVENT, IONIC STRENGTH AND
Transcript of EFFECT OF SOLVENT, IONIC STRENGTH AND
ii
EFFECT OF SOLVENT IONIC STRENGTH AND
METAL IONS ON THE PHOTOLYSIS OF RIBOFLAVIN
AND ITS NANOPARTICLES
Thesis
Presented by
Zubair Anwar Pharm D M Phil (BMU) R Ph
for the degree of
Doctor of Philosophy
in
Baqai Medical University
Department of Pharmaceutical Chemistry
Faculty of Pharmaceutical Sciences
Baqai Medical University Karachi
Pakistan June 2017
iii
AUTHORS DECLARATION
I Zubair Anwar hereby state that my PhD thesis titled ldquoEffect of Solvent Ionic
Strength and Metal Ions on the Photolysis of Riboflavin and its Nanoparticlesrdquo is my own
work and has not been submitted previously by me for taking any degree from Baqai
Medical University or anywhere else in the countryworld
At any time if my statement is found to be incorrect even after my Graduation the
university has the right to withdraw my PhD degree
Name of Student Zubair Anwar
Date
iv
v
PLAGIARISM UNDERTAKING
I solemnly declare that the research work presented in the thesis titled ldquoEffect of
Solvent Ionic Strength and Metal Ions on the Photolysis of Riboflavin and its Nanoparticlesrdquo
is solely my research work with no significant contribution from any other person Small
contributionhelp wherever taken has been duly acknowledged and that complete thesis
has been written by me
I understand the zero tolerance policy of the HEC and Baqai Medical University
towards plagiarism Therefore I as an Author of the above titled thesis declare that no
portion of my thesis has been plagiarized and any material used as reference is properly
referred cited
I undertake that if I am found guilty of any formal plagiarism in the above titled
thesis even after the award of PhD degree the University reserves the rights to withdraw
revoke my PhD degree and that HEC and the University has the right to publish my
name on the HEC University website on which names of students are placed who
submitted plagiarized thesis
Student Author Signature
Name Zubair Anwar
vi
CERTIFICATE OF APPROVAL
This is to certify that the research work presented in this thesis entitled ldquoEffect of
Solvent Ionic Strength and Metal Ions on the Photolysis of Riboflavin and its Nanoparticlesrdquo was conducted by Mr Zubair Anwar under the supervision of Prof Dr Iqbal Ahmad
No part of this thesis has been submitted anywhere else for any other degree This
thesis is submitted to the Department of Pharmaceutical Chemistry Baqai Institute of
Pharmaceutical Sciences Baqai Medical University in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in the field of Pharmaceutical
Chemistry Department of Pharmaceutical Chemistry Baqai Institute of
Pharmaceutical Sciences Baqai Medical University Karachi
Student Name Zubair Anwar Signature ___________
Examination Committee
a) External Examiner 1 Name Signature ___________
(Designation amp Office Address)
_____________________________
_____________________________
_____________________________
b) External Examiner 2 Name Signature ___________
(Designation amp Office Address)
_____________________________
_____________________________
_____________________________
c) Internal Examiner Name Signature ___________
(Designation amp Office Address)
_____________________________
_____________________________
_____________________________
Supervisor Name _______________________ Signature ___________
Name of DeanHOD _____________________ Signature ___________
vii
ABSTRACT
The present investigation is based on the study of the evaluation of the following
factors on the photolysis of riboflavin (RF) in aqueousorganic solvents
1 Solvent Effect on the Photolysis of RF
The kinetics of photolysis of RF in water (pH 70) and in organic solvents
(acetonitrile methanol ethanol 1-propanol 1-butanol ethyl acetate) has been studied
using a multicomponent spectrometric method for the assay of RF and its major
photoproducts formylmethylflavin and lumichrome The apparent first-order rate
constants (kobs) for the reactions range from 319 (ethyl acetate) to 461times10minus3
minminus1
(water) The values of kobs have been found to be a linear function of solvent dielectric
constant implying the participation of a dipolar intermediate along the reaction pathway
The degradation of this intermediate is enhanced by the polarity of the medium This
indicates a greater stabilization of the excited-triplet state of RF with an increase in
solvent polarity to facilitate its photoreduction The rate constants for the reaction show a
linear relation with the solvent acceptor number showing the magnitude of solutendashsolvent
interaction in different solvents It would depend on the electronndashdonating capacity of the
RF molecule in organic solvents The values of kobs are inversely proportional to the
viscosity of the medium as a result of diffusion-controlled processes
2 Ionic Strength Effects on the Photodegradation Reactions of RF
It involves the study of the effect of ionic strength on the photodegradation
reactions (photoreduction and photoaddition) of RF in phosphate buffer (pH 70) using
the specific multicomponent spectrometric method mentioned above The rates of
photodegradation reactions of RF have been found to be dependent upon the ionic
viii
strength of the solutions at different buffer concentrations The values of kobs for the
photodegradation of RF at ionic strengths of 01ndash05 M (05 M phosphate) lie in the range
of 735ndash3032 times 10minus3
minminus1
Under these conditions the rate constants for the formation
of the major products of RF lumichrome (LC) by photoreduction pathway and
cyclodehydroriboflavin (CDRF) by photoaddition pathway are in the range of 380ndash
1603 and 170ndash607 times 10minus3
minminus1
respectively A linear relationship has been observed
between log kobs and radicμ1+radicμ A similar plot of log kko against radicμ yields a straight line
with a value of ~+1 for ZAZB indicating the involvement of a charged species in the rate
determining step NaCl promotes the photodegradation reactions of RF probably by an
excited state interaction The implications of ionic strength on RF photodegradation by
different pathways and flavinndashprotein interactions have been discussed
3 Metal Ion Mediated Photolysis of RF
The effect of metal ion complexation on the photolysis of RF using various metal
ions (Ag+ Ni
2+ Co
2+ Fe
2+ Ca
2+ Cd
2+ Cu
2+ Mn
2+ Pb
2+ Mg
2+ Zn
2+ Fe
3+) has been
studied Ultraviolet and visible spectral and fluorimetric evidence has been obtained to
confirm the formation of metal-RF complexes The kinetics of photolysis of RF in metal-
RF complexes at pH 70 has been evaluated and the values of kobs for the photolysis of RF
and the formation of LC and LF (0001 M phosphate buffer) and LC LF and CDRF
(02ndash04 M phosphate buffer) have been determined These values indicate that the rate of
photolysis of RF is promoted by divalent and trivalent metal ions The second-order rate
constants (kprime) for the interaction of metal ions with RF are in the order Zn
2+ gt Mg
2+gt
Pb2+
gt Mn2+
gt Cu2+
gt Cd2+
gt Fe2+
gt Ca2+
gt Fe3+
gt Co2+
gt Ni2+
gt Ag+ In phosphate buffer
(02-04 M) an increase in metal ion concentration leads to a decrease in the formation of
ix
LC compared to that of CDRF by different pathways The values of kobs for the photolysis
of RF have been found to increase with a decrease in fluorescence intensity of RF The
photoproducts of RF formed by pathways have been identified and the mode of
photolysis of RF in metal-RF complexes has been discussed
4 Preparation Characterization and Formation Kinetics of RF-Ag NPs
Riboflavin conjugated silver nanoparticles (RFndashAg NPs) have been prepared by
photoreduction of Ag+ ions and characterized by UVndashvisible spectrometry
spectrofluorimetry dynamic light scattering (DLS) atomic force microscopy (AFM) and
FTIR spectrometry These NPs exhibit a surface plasmon resonance (SPR) band at 422
nm due to the interaction of RF and Ag+ ions The fluorescence of RF is quenched by Ag
NPs and the total loss of fluorescence is due to complete conversion of RF to RFndashAg NPs
conjugates FTIR studies indicate the appearance of an intense absorption peak at
2920 cmndash1
due to the interaction of RF and Ag DLS has shown the hydrodynamic radii
(Hd) of RFndashAg NPs in the range of 579ndash722 nm with polydispersity index of 275ndash290
AFM indicates that the NPs are spherical in nature and polydispersed with a diameter
ranging from 57 to 73 nm The effect of pH ionic strength and reducing agents on the
particle size of NPs has been studied At acidic pH (20ndash62) aggregation of RFndashAg NPs
occurs due to an increase in the ionic strength of the medium The rates of formation of
RFndashAg NPs on UV and visible light irradiation have been determined in the pH range of
80ndash105 and at different concentration of Ag+ ions The photochemical formation of RFndash
Ag NPs follows a biphasic firstndashorder reaction probably due to the formation of Ag NPs
in the first phase (fast) and the adsorption of RF on Ag NPs in the second phase (slow)
x
ACKNOWLEDGEMENTS
ldquoO My Lord Increase Me in My Knowledgerdquo
ldquoO Allah I Ask You for Knowledge that is of Benefitrdquo
(Quran 20114)
I am highly thankful to ALLAH ALL MIGHTY who gave me courage in all
difficulties and provided me strength to overcome the problems during this work
All and every kind of respect to the prophet Hazrat Muhammad (صلى الله عليه وسلم) for
complete and endless guidance and knowledge
Words are limited and are inoperative to express my gratitude to my dignified
supervisor Prof Dr Iqbal Ahmad TI Department of Pharmaceutical Chemistry for his
supervision keen interest and above all giving his valuable time throughout the course of
this work His personality and individuality has been a source of permanent motivation
throughout my study period and research work He not only groomed me with his
valuable suggestions and moral support but also guided me at every step during my
research work My deepest regards are due for his time and efforts
I am highly thankful to Professor Dr Syed Fazal Hussain CEO and Professor
Dr Shaukat Khalid Dean Faculty of Pharmaceutical Sciences for providing me an
opportunity to be a part of their organization and to complete my degree in this
institution
I am very thankful to Professor Dr Moinudin (Late) for providing me the
materials for this study
xi
I am very thankful to Associate Professor Dr Sofia Ahmad Chairperson
Department of Pharmaceutics Associate Professor Dr Muhammad Ali Sheraz
Chairman Department of Pharmacy Practice for their encouragement innovative ideas
and support during this work
I am highly thankful to Professor Dr Syed Abid Ali and Professor Dr Raza
Shah International Center for Chemical and Biological Sciences HEJ Research Institute
of Chemistry for their guidance and help in my research work
I acknowledge with sincere thanks to Associate Professor Dr Kiran Qadeer
Chairperson Department of Pharmaceutical Chemistry Associate Professor Dr Raheela
Bano and Associate Professor Dr Adeel Arsalan Department of Pharmaceutics for their
kind support in my Ph D studies
I am thankful to Ms Tania Mirza Ms Saima Zahid Ms Sadia Kazi Ms Sadia
Ahmed Zuberi Ms Nafeesa Mustan Ms Marium Fatima Khan Ms Qurat-e-Noor
Baig and Mr Muhammad Ahsan Ejaz for their moral support
I am very grateful to Mrs Professor Dr Iqbal Ahmad for her affection during my
visits which gives me motivation to do hard work and to be consistent
I feel prodigious contentment to pay my sincere and exclusive benediction to
Ms Adeela Khurshid and Aqeela Khurshid for their moral and ethical support
I am highly thankful to Mr Syed Haider Abbas Naqvi Mr Shahzaib
Ms Samina Sheikh Ms Perveen Nawaz Ms Syeda Mahwish Kazmi Ms Laiba
xii
Saleem Sultan Ms Laraib Saleem Sultan Ms Kinza Khan Ms Zuni and Ms Nazia
Ishaque for their love care and support
I am thankful to Mr Sajjad Ali Mr Anees Hassan Mr Wajahat Mr Mohsin
Ali and Mr Azharuddin for providing their technical services during my research work
In the last but not the least I would like to thank and express my gratitude to My
Father (Muhammad Anwar) Late Mother (Gul) Beloved Brother (Zeeshan
Anwar) Sisters (Shahbana Anwar and Rizwana Anwar) Sister-in-Law (Bushra
Ejaz) my Nephews (Musa Alam Essa Alam and Hassan Alam) and my Nieces
(Inshrah Hamna Anushay Aymen) for their moral support kindness and
encouragement throughout my life
Z A
xiii
To my beloved parents
and my niece
Anushay Zeeshan
xiv
CONTENTS
Chapter Page
ABSTRACT vi
ACKNOWLEDGEMENTS ix
I INTRODUCTION
11 INTRODUCTION 2
12 BIOCHEMICAL IMPORTANCE 2
13 CHEMICAL STRUCTURE OF RIBOFLAVIN 5
14 PHYSICAL PROPERTIES OF RIBOFLAVIN 7
15 CLINICAL USES 8
16 ABSORPTION FATE AND EXCRETION 9
17 THERAPEUTIC USES 10
18 PHARMACOKINETICS 10
19 LITERATURE ON RIBOFLAVIN 11
II ANALYTICAL METHODS USED FOR THE
DETERMINATION OF RIBOFLAVIN
21 SPECTROPHOTOMETRIC METHOD 13
211 UV-visible Spectrometry 13
212 Spectrofluorimetric Method 17
213 Infrared Spectrometry 23
214 Mass Spectrometry 23
22 CHROMATOGRAPHIC METHODS 25
221 High-Performance Liquid Chromatography (HPLC) 25
222 Liquid Chromatography 30
223 Ion Chromatography 31
23 ELECTROCHEMICAL METHODS 32
24 PHOTOCHEMICAL METHODS 34
25 ENZYMATIC ASSAY 35
26 FLOW INJECTION ANALYSIS (FIA) METHOD 36
xv
III PHOTOCHEMISTRY OF RIBOFLAVIN
31 INTRODUCTION 38
32 ANAEROBIC PHOTOREACTIONS 39
33 AEROBIC PHOTOREACTIONS 42
34 TYPES OF PHOTOCHEMICAL REACTIONS 43
341 Photoreduction 43
3411 Intramolecular photoreduction 43
3412 Intermolecular photoreduction 46
342 Photodealkylation 50
343 Photoaddition Reactions 51
344 Photooxidation 52
345 Photosenstization Reactions 52
346 Photostabilisation Reactions 57
347 Factors Affecting Photochemical Reactions of Riboflavin 59
3471 Radiation source 59
3472 pH effect 60
3473 Buffer effect 61
3474 Effect of complexing agents 63
3475 Effect of quenchers 66
3476 Effect of solvent 67
3477 Effect of ionic strength 68
3488 Effect of formulation 68
IV INTRODUCTION TO NANOPARTICLES AND
APPLICATIONS TO RIBOFLAVIN
41 INTODUCTION 71
42 RIBOFLAVIN AND NANOTECHNOLOGY 73
421 Photosenstizer 73
422 Stabilizer 74
423 Photoluminescent 74
424 Biosensor 76
xvi
425 Target Drug Delivery 79
426 Photochemical Interaction 80
427 Colorimetric Sensor 82
OBJECT OF PRESENT INVESTIGATION 83
PROPOSED PLAN OF WORK 84
V MATERIALS AND METHODS
51 MATERIALS 86
52 REAGENTS 88
53 METHODS 89
531 Thin-Layer Chromatography (TLC) 89
532 pH Measurements 90
533 Fourier Transform Infrared (FTIR) Spectrometry 90
534 Ultraviolet and Visible Spectrometry 92
535 Fluorescence Spectrometry 92
536 Dynamic Light Scattering (DLS) 93
537 Atomic Force Microscopy (AFM) 93
538 Photolysis of Riboflavin Solutions 94
5381 Choice of reaction vessel 94
5382 Choice of radiation source 94
539 Methods of Photolysis of Riboflavin 96
5391 Photolysis in aqueous and organic solvents 96
5392 Photolysis at various ionic strengths 96
5393 Photolysis in the presence of metal ions 96
5310 Assay of Riboflavin and Photoproducts 97
5311 Calculation of Molar Concentrations in the Spectrometric Assay of RF
and Photoproducts
97
53111 Two-component spectrometric assay (additive absorbances) 100
53112 Three-component spectrometric assay (additive absorbances) 101
xvii
VI SOLVENT EFFECT ON THE PHOTOLYSIS OF RIBOFLAVIN
61 INTRODUCTION 106
62 RESULT AND DISCUSSION 108
621 Photoproducts of RF 108
622 Spectral Characteristics 108
623 Assay of RF and Photoproducts 111
624 Kinetics of Photolysis 116
625 Effect of Solvent 128
626 Effect of Dielectric Constant 131
627 Effect of Viscosity 132
628 Mode of Photolysis 132
VII IONIC STRENGTH EFFECTS ON THE
PHOTODEGRADATION REACTIONS OF RIBOFLAVIN IN
AQUEOUS SOLUTION
71 INTRODUCTION 135
72 RESULTS AND DISCUSSION 138
721 Assay of RF and Photoproducts 138
722 Spectral Characteristics of Photolysed Solutions 152
723 Kinetics of RF Photolysis 152
724 Fluorescence Studies 156
725 Ionic strength Effects 160
VIII EFFECT OF METAL IONS ON THE PHOTODEGRADATION
REACTIONS OF RIBOFLAVIN IN AQUEOUS SOLUTION
81 INTRODUCTION 165
82 RESULTS AND DISCUSSION 170
821 Photoproducts of Metal-RF Complexes 170
822 Spectral Characteristics of Metal-RF-Complexes 171
823 Spectrometric Assay of RF and Photoproducts in Photolyzed
Solutions
174
xviii
824 Fluorescence Characteristics of Metal-Flavin Complexes 181
825 Kinetic of Photolysis of Metal-Flavin Complexes 181
826 Mode of Interaction of Metal Ions with RF 213
IX PHOTOCHEMICAL PREPARATION CHARACTERIZATION
AND FORMATION KINETICS OF RIBOFLAVIN
CONJUGATED SILVER NANOPARTICLES
91 INTRODUCTION 217
92 RESULTS AND DISCUSSION 220
921 Characterization of RF-Conjugated Ag NPs 220
9211 Optical studies 220
9212 Spectral characteristics of RF-Ag NPs 220
9213 Fluorescence characteristics of RF 222
9214 FTIR studies 224
9215 Dynamic light scattering (DLS) 228
9216 Atomic force microscopy (AFM) 230
922 Factors Affecting the Particle Size of RF-Ag NPs 230
9221 pH 232
9222 Ionic strength 232
923 Kinetics of Formation of RF-Ag NPs Conjugates 235
924 Mode of Photochemical Interaction of RF and Ag+ Ions 241
CONCLUSIONS 248
REFERENCES 252
AUTHORrsquoS BIODATA 321
xix
No LIST OF FIGURES Page
11 Chemical structures of riboflavin (1) and its analogues (flavin
mononucleotide (2) and flavin adenine dinucleotide (3))
3
12 Conversion of RF to FMN and FAD 6
31 Scheme for the photodegradation pathways of RF 40
32 Formation of αndashketone from flavin 45
33 Photoreduction of flavins to form 15ndashdihydrogen flavin and alkyl
adducts in the presence of unsaturated hydrocarbons
47
51 FTIR spectrum of riboflavin 91
52 Spectral emission of HPLN lamp 95
61 Absorption spectra of RF photolyzed in methanol at 0 30 60 90
and 120 min
110
62 Kinetic plots for the photolysis of RF in water
RF () FMF () LC () LF(diams)
117
63 Kinetic plots for the photolysis of RF in acetonitrile
RF () FMF () LC ()
117
64 Kinetic plots for the photolysis of RF in methanol
RF () FMF () LC () LF(diams)
118
65 Kinetic plots for the photolysis of RF in ethanol
RF () FMF () LC ()
118
66 Kinetic plots for the photolysis of RF in 1ndashpropanol
RF () FMF () LC ()
119
67 Kinetic plots for the photolysis of RF in 1ndashbutanol
RF () FMF () LC ()
119
68 Kinetic plots for the photolysis of RF in ethyl acetate
RF () FMF () LC ()
120
69 Apparent firstndashorder plot for the photolysis of RF
(5 times 10ndash5
M) in water (pH 70)
121
610 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
acetonitrile
121
611 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
methanol
122
612 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
ethanol
122
613 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
1ndashpropanol
123
614 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
1ndashbutanol
123
615 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
ethyl acetate
124
616 Plot of kobs for the photolysis of RF versus dielectric constant (x)
ethyl acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol ()
methanol (+) acetonitrile () water
126
617 Plot of lnkobs for the photolysis of RF versus acceptor number (x)
ethyl acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol ()
methanol (+) acetonitrile () water
127
xx
618 Plot of kobs for the photolysis of RF versus inverse of viscosity(x)
ethyl acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol ()
methanol (+) acetonitrile () water
129
619 Plot of dielectric constant versus inverse of viscosity 130
71 Absorption spectra of the photolysed solutions of RF (5 times 10ndash5
M) at pH 70 (a) at zero and (b) at 05 M ionic strength
153
72 Plots of fluorescence of RF solutions (pH 70) versus ionic
strength at different buffer concentrations (diams) 0001 M () 0025
M () 005 M (times) 01 M () 02 M (∆) 03 M () 04 M ()
05 M
158
73 A plot of log kobs versus radicμ1 + radicμ at 05 M phosphate buffer 161
74 A plot log k1k0 () and k3k0 () versus radicμ at 05 M phosphate
buffer
161
81 The photoreduction and photoaddition pathways of riboflavin
(RF)
166
82 Formation of the metalndashRF complex 168
83 Absorption spectra of RF (5 times 10ndash5
M) (pH 70) (____
) in the
presence of metal ions (1times 10ndash3
M) (ndashndashndash) (a) Fe2+
ions (b) Zn2+
ions and (c) Cu2+
ions
172
84 The percent decrease in fluorescence intensity of RF solutions
(pH 70 0001 M phosphate buffer) in the presence of metal ions
() Ni2+
ions (∆) Co2+
ions (loz) Ca2+
ions (+) Fe2+
ions () Cd2+
ions (ndash) Cu2+
ions (diams) Mn2+
ions () Pb2+
ions () Mg2+
ions
() Zn2+
ions and () Fe3+
ions
182
85 Excitation spectrum of RF (5 times 10ndash5
M) (pH 70) (a)
Fluorescence spectra of RF pure RF (b1) RF + Fe2+
ions (1 times 10ndash
3 M) (b2) RF + Fe
2+ ions (2 times 10
ndash3 M) (b3)
183
86 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Ag+ ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
185
87 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Fe2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
185
88 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Cu2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
186
89 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Zn2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
186
810 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Mg2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 () 50
187
xxi
811 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Pb2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
187
812 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Ni2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
188
813 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Ca2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
188
814 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Mn2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
189
815 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Cd2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
189
816 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Co2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
190
817 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Fe2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
190
818 Firstndashorder plots for the photolysis of RF (0001 M phosphate
buffer pH 70) in the presence Fe3+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
191
819 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Ag+ ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
191
820 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Fe2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
192
821 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Cu2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
192
822 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Zn2+
ions at different
193
xxii
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
823 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Mg2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
193
824 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Pb2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
194
825 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Ni2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
194
826 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Ca2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
195
827 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Mn2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
195
828 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Cd2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
196
829 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Co2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
196
830 Firstndashorder plots for the photolysis of RF (02 M phosphate
buffer pH 70) in the presence Fe3+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
197
831 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Ag+ ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
197
832 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Fe2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
198
833 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Cu2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
198
xxiii
834 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Zn2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
199
835 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Mg2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
199
836 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Pb2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
200
837 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Ni2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
200
838 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Ca2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
201
839 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Mn2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
201
840 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Cd2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
202
841 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Co2+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
202
842 Firstndashorder plots for the photolysis of RF (04 M phosphate
buffer pH 70) in the presence Fe3+
ions at different
concentrations (M times 104) (diams) 10 () 20 () 30 () 40 ()
50
203
843 A plot of kobs for the photolysis of RF versus fluorosecne loss
in the presence of different metal ions () Ni2+
ions (∆) Co2+
ions (loz) Ca2+
ions (+) Fe2+
ions () Cd2+
ions (ndash) Cu2+
ions (diams)
Mn2+
ions () Pb2+
ions () Mg2+
ions () Zn2+
ions () Fe3+
ions
211
844 Scheme for the photolysis of RF in metalndashRF complex 215
91 Photodegradation pathway of RF 219
92 Colour change for the formation of RFndashAg NPs from yellow
green to brown
221
93 Absorption spectra of RF and RFndashAg NPs 223
xxiv
94 Excitation spectrum of RF (green colour) and Fluorescence
spectra of RFndashAg NPs at different time 0 min (blue) 60 min
(black) 120 min (pink) 180 min (orange) 240 min (dark blue)
300 min (purple)
225
95 A plot of fluorescence loss versus time (h) for the formation of
RFndashAg NPs
226
96 FTIR spectrum of RF (a) and RFndashAg NPs (b) 227
97 Dynamic light scattering measurements of RFndashAg NPs 229
98 AFM micrograph (25 times 25 microm) of RFndashAg NPs 231
99 Absorption spectra of RFndashAg NPs at different pH values 20
(black) 40 (red) 60 (blue) 80 (green) 100 (pink) 120 (light
green)
233
910 Absorption spectra of RFndashAg NPs at different ionic strengths
(mM) 01 (black) 10 (red) 50 (blue) 100 (light green) 500
(purple) 100 (green) 250 (dark blue) 500 (maroon) 1000
(pink)
234
911 A plot of log absorbance versus time for the formation of RF-Ag
NPs
237
912 A scheme for the formation of Ag NPs (first phase) and the
adsorption of RF on the surface of Ag NPs (second phase)
238
913 Plots of k1 () (left hand side) and k2 () (right hand side) versus
pH for the formation of RF-Ag NPs in UV light
242
914 Plots of k1 () (left hand side) and k2 () (right hand side) versus
pH for the formation of RF-Ag NPs in visible light
243
915 Plots k1 () (left hand side) and k2 ()(right hand side) versus
Ag+ ion concentrations (mM) for the formation of RF-Ag NPs in
UV light
244
916 Plots k1 () (left hand side) and k2 ()(right hand side) versus
Ag+ ion concentrations (mM) for the formation of RF-Ag NPs in
visible light
245
xxv
No LIST OF TABLES Page
41 Definition of Nanoparticles (NPs) and Nanomaterials
(NMs) according to different Organizations
72
52 Molar Absorptivities (Mminus1
cmminus1
) of RF and
Photoproducts
99
61 Rf values and Fluorescence of RF and Photoproducts 109
62 Concentrations of RF and Photoproducts in Water
(pH 70)
112
63 Concentrations of RF and Photoproducts in Acetonitrile 112
64 Concentrations of RF and Photoproducts in Methanol 113
65 Concentrations of RF and Photoproducts in Ethanol 113
66 Concentrations of RF and Photoproducts in 1ndashPropanol 114
67 Concentrations of RF and Photoproducts in 1ndashButanol 114
68 Concentrations of RF and Photoproducts in Ethyl acetate 115
69 Apparent FirstndashOrder Rate Constants for the Photolysis
of Riboflavin (kobs) in Organic Solvents and Water
125
71 Concentrations of RF and Photoproducts in 01 M
Phosphate Buffer (pH 70) at 01 M Ionic Strength
139
72 Concentrations of RF and Photoproducts in 01 M
Phosphate Buffer (pH 70) at 02 M Ionic Strength
139
73 Concentrations of RF and Photoproducts in 01 M
Phosphate Buffer (pH 70) at 03 M Ionic Strength
140
74 Concentrations of RF and Photoproducts in 01 M
Phosphate Buffer (pH 70) at 04 M Ionic Strength
140
75 Concentrations of RF and Photoproducts in 01 M
Phosphate Buffer (pH 70) at 05 M Ionic Strength
141
76 Concentrations of RF and Photoproducts in 02 M
Phosphate Buffer (pH 70) at 01 M Ionic Strength
141
77 Concentrations of RF and Photoproducts in 02 M
Phosphate buffer
(pH 70) at 02 M ionic strength
142
78 Concentrations of RF and Photoproducts in 02 M
Phosphate Buffer (pH 70) at 03 M Ionic Strength
142
79 Concentrations of RF and Photoproducts in 02 M
Phosphate Buffer (pH 70) at 04 M Ionic Strength
143
710 Concentrations of RF and Photoproducts in 02 M
Phosphate Buffer (pH 70) at 05 M Ionic Strength
143
711 Concentrations of RF and Photoproducts in 03 M
Phosphate Buffer (pH 70) at 01 M Ionic Strength
144
712 Concentrations of RF and Photoproducts in 03 M
Phosphate Buffer (pH 70) at 02 M Ionic Strength
144
713 Concentrations of RF and Photoproducts in 03 M
Phosphate Buffer (pH 70) at 03 M Ionic Strength
145
714 Concentrations of RF and Photoproducts in 03 M
Phosphate Buffer (pH 70) at 04 M Ionic Strength
145
715 Concentrations of RF and Photoproducts in 03 M 146
xxvi
Phosphate Buffer (pH 70) at 05 M Ionic Strength
716 Concentrations of RF and Photoproducts in 04 M
Phosphate Buffer (pH 70) at 01 M Ionic Strength
146
717 Concentrations of RF and Photoproducts in 04 M
Phosphate Buffer (pH 70) at 02 M Ionic Strength
147
718 Concentrations of RF and Photoproducts in 04 M
Phosphate Buffer (pH 70) at 03 M Ionic Strength
147
719 Concentrations of RF and Photoproducts in 04 M
Phosphate Buffer (pH 70) at 04 M Ionic Strength
148
720 Concentrations of RF and Photoproducts in 04 M
Phosphate Buffer (pH 70) at 05 M Ionic Strength
148
721 Concentrations of RF and Photoproducts in 05 M
Phosphate Buffer (pH 70) at 01 M Ionic Strength
149
722 Concentrations of RF and Photoproducts in 05 M
Phosphate Buffer (pH 70) at 02 M Ionic Strength
149
723 Concentrations of RF and Photoproducts in 05 M
Phosphate Buffer (pH 70) at 03 M Ionic Strength
150
724 Concentrations of RF and Photoproducts in 05 M
Phosphate Buffer (pH 70) at 04 M Ionic Strength
150
725 Concentrations of RF and Photoproducts in 05 M
Phosphate Buffer (pH 70) at 05 M Ionic Strength
151
726 Apparent FirstndashOrder Rate Constants (kobs) for the
Photodegradation of Riboflavin in the presence of
Phosphate Buffer (pH 70) at different Ionic Strength
(01ndash05M) for the formation of Lumichrome (k1)
Lumiflavin (k2) and Cyclodehdroriboflavin (k3)
157
81 Concentration of RF (M times 105) and LC (M times 10
5) (0001
M Phosphate Buffer) in the presence of 10ndash50 times 10ndash4
M Metal Ions Concentration
175
82 Concentration of RF (M times 105) and CDRF (M times 10
5) (02
M Phosphate Buffer) in the presence of 10ndash50 times 10ndash4
M Metal Ions Concentration
177
83 Concentration of RF (M times 105) and CDRF (M times 10
5) (04
M Phosphate Buffer) in the presence of 10ndash50 times 10ndash4
M Metal Ions Concentration
179
84 Apparent Firstndashorder Rate Constants (kobs) for the
Photolysis of RF in the Presence of Various Metal Ions at
pH 70 (0001 M Phosphate Buffer) for the formation of
LC (k1) LF (k2) and the SecondndashOrder Rate Constants
for the Interaction of RF and Metal Ions (kʹ)
205
85 Apparent Firstndashorder Rate Constants (kobs) for the
Photolysis of RF in the Presence of Various Metal Ions at
pH 70 (02 M Phosphate Buffer) for the Formation of
LC (k1) LF (k2) CDRF (k3) and the SecondndashOrder Rate
Constants for the Interaction of RF and Metal Ions (kʹ )
207
86 Apparent Firstndashorder Rate Constants (kobs) for the 209
xxvii
Photolysis of RF in the Presence of Various Metal Ions at
pH 70 (04 M Phosphate Buffer) for the Formation of
LC (k1) LF (k2) CDRF (k3) and the SecondndashOrder Rate
Constants for the Interaction of RF and Metal Ions (kʹ)
91 First-order Constants for the Photoinduced Electron
Transfer Reaction of RF and Ag+ ions (k1) and
Adsorption of RF at Ag NPs Surface (k2) in UV and
visible light at 001 mM of Ag+ Ion Concentration
239
92 First-order Constants for the Photoinduced Electron
Transfer Reaction of RF and Ag+ ions (k1) and
Adsorption of RF at Ag NPs (k2) in UV and visible light
at Different Ag+ ion Concentrations
240
1
CHAPTER I
INTRODUCTION TO RIBOFLVAIN
2
11 INTRODUCTION
Riboflavin (RF) (1) (Fig 11) belongs to the family of vitamin B complex and is
also called as vitamin B2 It belongs to the chemical class of yellow coloured flavins
(isoalloxazines) RF was named due to its color which is derived from the Latin word
ldquoFlavinsrdquo meaning ldquoyellowrdquo It was discovered by the isolation of a heatndashstable fraction
from yeast that contained a yellow growth factor This factor after purification was
named riboflavin (Emmett and Luros 1920) Warburg and Christian (1931) isolated RF
from yeast as a coenzyme complex and named it as an antioxidant ferment The
physiological role of the yellow growth factor was later shown by Warburg and Christian
(1932) who described It as ldquoold yellow enzymerdquo composed of an apoenzyme and a
yellow factor as coenzyme The coenzyme was found to have an isoalloxazine ring (Stern
and Holiday 1934) and a phosphate containing sidendashchain ie riboflavinndash5rsquondashphosphate
(Theorell 1934) that was found to be essential for the human metabolism growth and
health RF was first synthesized by Kuhn et al (1935) and Karrer et al (1935) It is
synthesized by most of the green plants bacteria fungi and the richest sources of the
vitamin are meat legumes dairy products and eggs (Ortega et al 2004)
12 BIOCHEMICAL ROLE
RF plays a critical role in the body energy production in the form of flavin
mononucleotide (FMN) (2) or flavin adenine dinucleotide (FAD) (3) (Fig 11) When RF
is converted into FAD and FMN forms as coenzymes it is attached to protein enzymes
and allows oxygenndashbased energy production to occur Proteins with FAD or FMN
attached to them are often referred to as flavoproteins (Rivlin 2007 Moffat 2013)
3
OCH3N
NNH
N
CH3
CH2
OHH
OHH
OHH
CH2OH
O
(1)
OCH3N
NNH
N
CH3
O
CH2
OH
H
OH OH
HH
CH2O P
OH
OH
O
(2)
N
CH
C
N
C
C
N
CH
N
OCH3N
NNH
N
CH3
O
CH2
OH
H
OH OH
HH
CH2O P
O
OH
O P
O
OH
CH2O
CH
OHH
OHHO
CH
NH2
(3)
Fig 11 Chemical structures of riboflavin (1) and its analogues (flavin mononucleotide (2)
and flavin adenine dinucleotide (3))
4
These flavoproteins are found throughout the body and particularly in that
location where oxygenndashbased energy production is constantly needed (Merrill et al
1981)
RF plays an important role in maintaining the supplies of other B vitamins One
of the pathways used in the body to produce vitamin B3 (niacin) is by conversion of the
amino acid tryptophan This conversion is accomplished with the help of an enzyme
kynureninendashmonondashoxygenase and RF in its FAD form RF is the precursor of the two
flavocoenzymes (FMN and FAD) required by the two flavoproteins of the mitochondrial
electron transport chain (McCormick 1989)
Glutathione reductase is a FAD ndashdependent enzyme that precipitates in the redox
cycle of glutathione The glutathione redox cycle plays a major role in protecting
organisms from reactive oxygen species Glutathione reductase requires FAD to
regenerate two molecules of reduced glutathione (an antioxidant) from oxidized
glutathione (Beutler 1969)
Xanthine oxidase is another FAD dependent enzyme that catalyzes the oxidation
of hypoxanthine and xanthine to uric acid Uric acid is one the most effective waterndash
soluble antioxidant in the blood RF deficiency can result in decreased xanthine oxidase
activity reducing blood uric acid levels (Bohles 1997) Recent studies on migraine
patients show some evidence that indicates impaired mitochondrial oxygen metabolism in
the brain that may play a role in the pathology of migraine headaches
5
13 CHEMICAL STRUCTURE OF RIBOFLAVIN
Chemically RF is 78-Dimethyl-10-[(2S3S4R)-2345-
tetrahydroxypentyl]benzo[g]pteridine-24-dione (British Pharmacopoeia 2016) The
planar isoalloxazine ring not only provides the basic structure for RF but also for the
naturally occurring phosphorylated coenzymes that are derived from RF These
coenzymes include FMN FAD and flavin coenzymes linked covalently to specific tissue
proteins generally at the 8ndashα methyl position of the isoalloxazine ring RF exists in the
cationic and anionic forms with the pKas of 19 and 102 (Moffat et al 2013)
respectively and due to strong conjugated system it has a high molar absorptivity as well
as high fluorescence characteristics due to the presence of a strong conjugated system
(Rivlin 2007) RF in the presence of flavokinase and FMN phosphatase is converted into
FMN which is further converted into FAD by the action of FAD pyrophosphorylase and
pyrophosphatase (Powers 2003) (Fig 12) Initially flavokinase which is biosynthetic
enzyme initiates the phosphorylation of RF from ATP for the formation of FMN This
FMN in small portion is used as a coenzyme and the major portion of FMN is further
combined with a second ATP molecule for the formation of FAD The formation of FAD
is catalysed by FAD synthetase and these flavins are further covalently attached to the
different tissues after the formation of FAD (Powers 2003)
6
OCH3N
NNH
N
CH3
CH2
OHH
OHH
OHH
CH2OH
O
OCH3N
NNH
N
CH3
O
CH2
OH
H
OH OH
HH
CH2O P
OH
OH
O
N
CH
C
N
C
C
N
CH
N
OCH3N
NNH
N
CH3
O
CH2
OH
H
OH OH
HH
CH2O P
O
OH
O P
O
OH
CH2O
CH
OHH
OHHO
CH
NH2
FlavokinaseFMN Phosphatase
FAD Pyrophosphorylase Pyrophosphatase
(2)
(1)
(3)
Thyroid Harmone
Fig 12 Conversion of RF to FMN and FAD
7
14 PHYSICOCHEMICAL PROPERTIES OF RIBOFLAVIN
The physicochemical properties of RF that affect its stability or the physiological
functions are as follows (Moffat et al 2013 Sweetman 2009 British Pharmacopoeia
2016)
Empirical formula
C17H20N4O6
Molar mass 3764
Crystalline form fine needles
Melting point 278 to 282 oC
[α]D
ndash112 to ndash122o
pH of saturated solution ~6
pKa 19 102 (20o)
Redox potential
(riboflavindihydroriboflavin) pH 70 ndash0208 V
Solubility mg 100 ml
Water 33ndash606
Absolute ethanol 045
Acetone chloroform ether benzene insoluble
Absorption maxima (pH 70) 223 267 373 and 444 nm
Fluorescence emission (pH 70) 520 nm
Principle infrared peaks (KBr disk) 1544 1575 1641 1715 1235
1070 cmndash1
25
8
15 CLINICAL USES
RF is used in both clinical and in therapeutic conditions It is also used in the
phototherapy of a condition termed as neonatal jaundice RF in high doses with betandash
blockers is used in the treatment of migraine (Sandor et al 2000 Schoenen et al 1998)
It has been used in the management of the muscle pain RF along with the UV light is
effective against the pathogens that cause disease while present in the blood (Goodrich et
al 2006 Kumar et al 2004) RF is also used in the treatment of the corneal disorder
named keratoconus (Spoerl et al 2004a 2004b)
RF as a precursor of FMN and FAD shows a powerful antioxidant activity It
provides protection against peroxidase of lipids in glutathione redox cycle (Dutta 1993)
The breakdown of lipid peroxidase is mediated by glutathione peroxidase and it requires
reduced form of glutathione (GSH) which results in the regeneration of the oxidized form
of glutathione (GSSG) by glutathione reductase a FAD containing enzyme If
glutathione reductase activity is compromised then the GSH concentration is decreased
which serves as a substrate for glutathione reductase and glutathione Sndashtransferase This
results in decrease in the degradation of lipid peroxides and xenobiotic substances
(Rivilin and Dutta 1995) It has also been found that in RF deficiency glucosendash6ndash
phosphate dehydrogenase activity is also stopped (Taniguvhi and Harm 1983 Dutta et
al 1995) Miyazawa et al (1983 1984) stated that in RF deficiency the oxidant defense
system is compromised and if the RF supplement is taken then the oxidant response
system is progressively improved Deficiency of RF is also related to the lipid
peroxidation and on the use of its supplement the process is restricted (Taniguchi and
Harm 1983 Dutta et al 1995)
9
Deficiency of RF in animals and humans is found to be protective against malaria
(Kaikai and Thurnham 1983 Das et al 1988) Glactoflavin and 10ndash(4ʹndashchlorophenyl)ndash
3ndashmethlflavin are isoalloxazine derivatives that are inhibitors of glutathione reductase
and possess antimalarial activity (Becker et al 1990 SchonlebenndashJanas et al 1996)
RF is also involved in the regulation and metabolism of homocysteine (HC) HC
is mainly involved in cardiovascular peripheral vascular and cerebrovascular diseases
(Graham et al 1997) The conversion of Nndash5ndashmethyltetrahydrofolate to methionine
which is a condashsubstrate for HC and FAD are required by methyltetrahydrofolate
reductase for the conversion of Nndash5 10ndashmethylenetetrahydrofolate to Nndash5ndash
methylatetrahydrofolate For this conversion RF is required for the effective utilization of
dietary folic acid In the patients who are homozygous for genetic mutation RF controls
the HC metabolism (Rozen 2002 Yamada et al 2001) In USA it was reported that as
the dietary intake of RF increases the concentration of serum HC decreases (Ganji and
Kafai 2004)
16 ABSORPTION FATE AND EXCRETION
RF is readily absorbed from upper gastrointestinal tract by a specific transport
mechanism in which phosphorylation of the vitamin to FMN takes place (Jusko and
Levy 1975) RF is distributed to all tissues but its concentration is uniformly low and
little amount is stored in the body If RF is taken according to its daily requirement then it
is only excreted up to 9 in urine but if it is taken more than the daily requirement then
it is excreted in urine in the unchanged form If RF is present in the feces it is due to the
synthesis of the vitamin by intestinal microorganism (Tillotson and Karcz 1977) In the
10
case of boric acid poisoning RF forms a complex with boric acid and this promotes
urinary excretion that may induce riboflavin deficiency (Roe et al 1972)
17 THERAPEUTIC USES
RF at its nutritional doses is helpful in the treatment of cataracts in combination
with other B vitamins (Niacin B3) (Sperduto et al 1993) It is also used in the treatment
of sicklendashcell anemia (Ajayi et al 1993) and also in the treatment of HIV infection (Tang
et al 1996)
RF is used in the treatment of its deficiency a condition called as ariboflavinosis
It is also used in other nutritional disorders Recent randomized controlled trial of highndash
dose RF (400 mgday) in patients suffering migraine headaches showed significant
reductions in attack frequency and illness days (Schoenen et al 1998)
18 PHARMACOKINETICS
RF is mainly found in nature in the form of FMN and FAD It is used for the food
fortification RF and FMN are the principal nutritional supplement forms of riboflavin
with riboflavin being the major form Coenzyme forms of RF (FMN FAD) that are not
covalently bound to proteins are released from proteins in the acid environment of the
stomach (Zempleni et al 1996)
FMN and FAD are converted to RF in the small intestine via the action of
pyrophosphatase and phosphatase It is mainly absorbed in the proximal small intestine
by the saturable system The presence of the bile salts appears to facilitate absorption of
RF (Nath 2000)
11
19 LITERATURE ON RIBOFLAVIN
Books (Chemistry Biochemical Function and Clinical Uses)
Chapters in Books
Dyke (1965) Penzer and Radda (1971) Dollery (1999) Chapman et al (2002) Rivlin
and Pinto (2001) Baxter (2003) Delgado and Remers (2004) Rivlin (2007)
Reviews
Penzer and Radda (1967) Hemmerich (1976) Walsh (1980) Heelis (1982 1991)
Powers (2003) Ahmad and Vaid (2006)
Chemical and Photostability
Macek (1960) Garrett (1967) Hashmi (1973) DeRitter (1982) Allwood and Kearney
(1998)
Chromatography and Assay
Bolliger and Konig (1969) HoffmanndashLa Roche (1970) Hashmi (1973) Shah (1985)
Song et al (2000) Eitenmiller et al (2008)
Physiochemical Data
British Pharmacopeia (2016) United States Pharmacopeia (2009) Moffat et al (2013)
Sweetman (2009) OrsquoNeil (2013)
CHAPTER II
ANALYTICAL TECHNQIUES USED FOR THE
DETERMINATION OF RIBOFLAVIN AND
RELATED COMPOUNDS
13
Several analytical methods have been used for the determination of riboflavin
(RF) and related compounds in pure solutions pharmaceutical preparations and
biological samples These methods are described in the following sections
21 SPECTROMETRIC METHODS
211 UVndashVisible Spectrometry
The method reported for the determination of RF in British Pharmacopoeia (BP)
(2016) involves the measurement of the absorbance of aqueous solutions at 444 nm and
calculating the concentration using the value of A (1 1cm) as 328 However since RF
is sensitive to light the major problem associated with the determination of RF in
photodegraded solutions is the presence of its photoproducts that interfere at the
absorption wavelength Ghasemi and Abbasi (2005) have determined RF in vitamin B
preparations containing folic acid thiamin and pyridoxine using a multicompartment
spectrometric method This method is based on the measurement of absorbance in the pH
range of 20 to 120 at 25 oC using parallel factor analysis (PARAFA) The calibration
curves were found to be linear in the concentration range of 4ndash22 1ndash20 6ndash26 and 4ndash20
mg Lndash1
for pyridoxine riboflavin thiamin and folic acid respectively This method
shows recovery of 906ndash107 for each vitamin The kinetics of photodegradation of
RF as a function of pH has been studied using a multicomponent spectrometric method
for the determination of RF and its photoproducts formylmethylfalvin (FMF)
lumichrome (LC) and lumiflavin (LF) formed by intramolecular photoreduction reaction
(Ahmad and Rapson 1990 Ahmed et al 2004a) The photolysis of FMF a major
14
intermediate in the photodegradation of RF has also been studied by the application of
this method (Ahmed et al 1980 2006ab 2008 2013) These methods have also been
used for the study of thermal degradation (Ahmad et al 1973) and photodegradation of
RF by photoaddition reactions (Ahmad et al 2004b 2005 2006 2010) Some other
applications of these methods include the study of the buffer effect (Ahmad et al 2014
Sheraz et al 2008) solvent effect (Ahmad et al 2015) ionic strength (Ahmad et al
2016) and metal ion effect (Ahmad et al 2017) on the photodegradation of RF
A multindashcomputed flow method for the determination of RF and B vitamins in
pharmaceutical products has been reported by Rocha et al (2003) At 997 confidence
interval the calibration curve was found to be linear for RF The average recovery
obtained for the commercial and pharmaceutical products lies between 956 and 100
Mohamed et al (2011) developed a derivative and multivariate spectrometric
method for the determination of pharmaceutical preparations containing a mixture of RF
and other B vitamins in the wavelength range of 200ndash500 nm using a
01 M HCl solution The results showed a linear response in the range of
25 to 90 microg mLndash1
with a recovery range of 961 to 1012 and 970 to 1019 for the
derivative and multivariate methods respectively A method involving spectrometric
determination based on total absorbance measurement of a complex mixture containing
folic acid (FA) RF pyridoxine (PY) and thiamine (TH) has been developed by partial
least regression The calibration matrix constructed for FA RF PY and TH determined
their concentration in the ranges between 102ndash143 microg mLndash1
102ndash102 microg mLndash1
15
101ndash162 microg mLndash1
and 600ndash200 microg mLndash1
respectively The estimated detection limits
of 008 microg mLndash1
009 microg mLndash1
045 microg mLndash1
and 017 microg mLndash1
have been found for FA
RF PY and TH respectively (Aberasturi et al 2002)
A comparison between FTndashNIRS and UVndashvis spectrometry for the evaluation of
mixing kinetics for the assay of a low quantity of RF in tablets has been made NIRS is a
nonndashdestructive technique which is used for the analysis of pharmaceutical dosage forms
In this study binary mixtures of microcrystalline cellulose and RF were used to prepare
tablets by direct compression The partial least square regression fit method was used to
build the prediction model The assay of RF was carried out by NIR transmission and the
results were compared with those of the UVndashvis spectrometry method and found that
NIR spectroscopy is faster nonndashdestructive and shows less variability in results (Bodson
et al 2006)
A study has been carried out for the simultaneous spectrometric determination of
FA TH RF and PY in artificial mixtures using multivariate calibration method The
calibration curves were found to be linear in the concentration range of 04ndash150 07ndash30
02ndash11 and 08ndash30 microg mlndash1
for FA TH RF and PY respectively The optimization of
calibration matrices by PLSndashI method was carried out by absorption spectra of quaternary
mixtures The recovery for these vitamins was found to be 95ndash105 (Ghasemi and
Vosough 2002)
The simultaneous multicomponent spectrometric determination of FA TH RF
and PY using doublendashdivisorndashratio spectra derivative zero crossing method has been
16
carried out for the assay of these vitamins in synthetic mixtures This method was based
on the derivative signals of the ratio spectra employing double divisor The spectral
measurements were carried out in the range of 225ndash475 nm The calibration curves were
found to be linear in the concentration range of 1ndash26 microg mlndash1
4ndash50 microg mlndash1
1ndash28 microg mlndash1
and 6ndash42 microg mlndash1
for FA TH RF and PY respectively in phosphate buffer (pH 580)
(Ghasemi et al 2004)
The simultaneous determination of waterndashsoluble vitamins (TH PY RF and CA)
in binary ternary and quaternary mixtures has been carried out by two spectrometric
methods (derivative and multivariate methods) The derivative method was divided into
first derivative and first derivative of ratio spectra method and multivariate method into
classical least squares and principal component regression method These methods were
based on the spectrometric measurements of the vitamins in 01 M HCl in the wavelength
range of 200 to 500 nm The methods showed good linearity in the concentration range of
25ndash90 microg Lndash1
with a regression in the range of 09991ndash09999 The mean recovery
( recovery) for derivative and multivariate methods ranged from 9611 (plusmn12)ndash
1012 (plusmn10) and 970 (plusmn05)ndash1019 (plusmn13) respectively (Mohamed et al
2011)
The principle of surface Plasmon resonance with onndashchip measurements has been
developed for the quantification of RF in milkndashbased products It has been carried out by
the determination of excess RF binding protein (RBP) that was free after complexation
with RF molecules In this method the modification was done at N(3) position to
17
introduce an ester group for the binding of amino groups at the surface of the chip RF
content in the milk based products was measured in comparison with the calibration
curve obtained from the standard RF with optimized RBP LOD and LOQ were found to
be 234 microg Lndash1
and 70 microg Lndash1
respectively for the 160 microLndash1
injections (Caelen et al
2004)
A catalytic photokinetic method has been developed for the microdetermination
of RF and riboflavin 5primendashphosphate This method is based on the rate of photoreduction of
these compounds by EDTA The rate of photoreduction was monitored by spectrometry
by the formation of ferroin The ferroin was produced by the reduction of Fe (III) via a
1ndash5 dihydro form of RF in the presence of 110ndashphenanthroline This method shows
linearity in the concentration range of 3 times 10ndash8
to 96 times 10ndash7
M (PerezndashRuiz et al 1987)
212 Spectrofluorimetry
Spectrofluorimetry is the method used for the assay of RF and its preparations
United States Pharmacopeia (USP) (2016) The method involves the measurement of
fluorescence of RF solution at 530 nm The concentration of RF solution is calculated by
comparing it with the USP reference standard taking 440 nm as the excitation
wavelength
A spectrofluorimetric method has been developed for the determination of RF in
tablets The emission and excitation wavelength used were 535 and 435 respectively
This method was found to be linear for RF in the concentration range of
18
01ndash06 microg mlndash1
with regression of 09978 The mean recovery was found to in the range
of 93ndash102 with a coefficient of variation of 232 (Junqing 1997)
One of the methods for the assay of RF in total parenteral nutrition (TPN) for
neonates involves the measurement of its fluorescence in the range of 400ndash700 nm using
360 nm as the excitation wavelength (Ribeiro et al 2011) RF flavin mononucleiotide
(FMN) and flavinadenine dinucleotide (FAD) have been quantified in human plasma at
530 nm using capillary electrophoresis and laser induced fluorimetry The 4 and 9
withinndashday and betweenndashday coefficient of variance values have been reported for RF
with a linear calibration falling in the concentration range of 03 and 1000 mol Lndash1
(Hustad et al 1999)
Synchronous fluorescence spectrometry has been used for the determination of
TH RF and PY in commercial preparations (Garcia et al 2001) RF and PY have been
determined using acetate buffer (pH 6) by a sensitive fluorimetric method The
concentration found lies in the range of 10ndash500 microg mLndash1
with a standard deviation
between 046 to 1002 and the recovered amount in the range 976 to 1012
(Mohamed et al 2011) RF determination in commercial preparations such as skimmed
milk 2 partially skimmed homogenized milk 2 partially skimmed chocolate and
nonndashfat dry milk has been made using fluorimetry with the help of extracted samples
Depending on the product assayed the RSD lies between 171 to 316 with a recovery
range between 90 to 110 (Rashid and Potts 2006) The analysis of RF in anchories
has also been carried out by synchronous spectrofluorimetry by the measurement of
19
fluorescence spectra in 300ndash600 nm region The excitation and emission slit widths were
set to 5 mm and the difference in wavelengths was 65 nm Fluorescence measurements
were carried out by peak area base of 430 to 509 nm and recovery was found to be higher
than 908 (LoperndashLayton et al 1998) A synchronous spectrofluorimetric method has
been developed for the simultaneous determination of vitamin B2 and B6 in beverages
The limits of detection have been found to be 002ndash006 mg Lndash1
and 012ndash036 mg Lndash1
for
B2 and B6 respectively (TorresndashSequeiros et al 2001)
A spectrofluorimetric study has been conducted for the evaluation of interaction
between RF and isolated protein from egg white at different pH values It has been found
that in phosphate buffer (01 M pH 70) the complex formation between RF and protein
(11) occurs with an association constant (Ka) of 77 times 107 M
ndash1 The complex was
dissociated in the presence of sodiumndashdodecyl sulphate (0033 ) with a rate constant of
4 times 10ndash2
secndash1
at 29 oC The binding affinity of RF to protein has been found to decrease
in the pH range of 70ndash40 and below pH 40 the binding affinity does not exist The
fluorimetric studies showed that carboxyl group 1ndash2 tryptophan residues and 2ndash3
disulphide bridges are necessary for binding The quantum yield (Φ) and energy transfer
from tyrosine to tryptophan have been calculated by excitation of the complex at 280 and
295 nm (Murthy et al 1976)
An investigation has been carried out on the molecular interaction between
quinine sulfate (QS) and RF by fluorimetry and UVndashvis spectrometry It has been found
that in the presence of QS the RF fluorescence is quenched At different temperatures
20
(294 301 307 314 oK) the thermodynamic parameters enthalpy change (∆H) and Gibbs
energy change (∆G) were determined via a Vanrsquot Hoff equation By calculating all these
thermodynamic parameters it was found that hydrogen bond helps in the stabilization of
the complex The critical energy transfer distance (Ro) was calculated as 4047 oA and
this showed that efficient resonance energy transfer takes place between QS (donor) and
RF (acceptor) Cyclic voltammetry (CV) of QS and RF complex showed that electron
transfer occurs in the excited singlet state (Patil et al 2011)
A fluorimetric method has been developed for the simultaneous determination of
TH PY and RF in pharmaceutical multivitamin formulations In this method TH
determination is based on the measurement of thiochrome formed by oxidation using Nndash
bromosuccinimide (NndashBS) in isopropanol whereas pyridoxine and RF measurements
were made in phosphate buffer (pH 70) For TH PY and RF sensitivity ranges were
found to be 15ndash35 05ndash25 and 04ndash20 microg mlndash1
respectively (Barary et al 1986)
A fluorimetric method for the determination of RF in hemoglobinndashcatalyzed
enzymatic reaction has been developed In this method two reactions occur
photochemical reaction of RF and hemoglobin catalyzed enzymatic reaction This
method has been found to be linear in the concentration range of 50 times 10ndash9
to 10 times 10ndash7
mol Lndash1
and the detection limit is 305 times 10ndash9
mol Lndash1
For 11 determination of 70 times 10ndash2
mol Lndash1
the RSD of measurements is 23 (XiaondashYan et al 2002)
A multivariate method for the rapid determination of caffeine caramel (class III
and IV) and RF in energy drinks using synchronous fluorimetry has been developed The
21
synchronous spectra are measured in the wavelength range of 200ndash500 nm Partial least
squares (PLS) models are created by the determination of the analyte with HPLC with a
fluorescence detector This method has been found to be linear in the concentration range
of 02ndash42 025ndash525 04ndash100 and 0007ndash0054 mg Lndash1
for caffeine caramel and RF
respectively (Ziak et al 2014) In nutritional beverages the simultaneous determination
of FA and RF have been carried out by synchronous fluorescence measurments In this
method FA has been detected by treating it with H2O2 plus Cu (II) (oxidation system) to
form pterinendash6ndashcarboxylic acid that is fluorescent The method shows good linearity in
the concentration range of 100ndash250 microg Lndash1
and 1ndash250 microg Lndash1
and the detection limits of
20 and 0014 microg Lndash1
for FA and RF respectively (Wang et al 2011)
A synchronous spectrofluorimetric method has been developed for the
simultaneous determination of RF and PY Synchronous scanning is carried out at ∆λ of
58 nm The measurements were carried out in phosphate buffer (pH 70) Two peaks have
been found at 526 and 389 nm in the synchronous fluorescence spectra for RF and PY
respectively The method shows linearity in the concentration range of 0ndash10 microg mlndash1
and
0ndash15 microg mlndash1
and recovery of 935ndash1057 for RF and PY respectively
(Li et al 1992)
The determination of RF in blood in newborn babies and their mothers has been
carried out by a spectrofluorimetric microndashmethod It is based on the hydrolysis of blood
in tridichloroacetic acid medium separation of RF and FMN on florisil column and
measurements by spectrofluorimetry by standard additional method after elution with
22
collidine buffer This method shows a sensitivity of 001 microg mlndash1
in the blood sample of
05ndash10 ml with an average concentration of 171 plusmn 24 microg100 ml and 142 microg100 ml of
RF in new born baby and women respectively (Knobloch et al 1978)
A synchronous fluorimetric method has been used for the simultaneous
determination of B1 B2 and B6 It is difficult to analyse them individually as their spectra
overlap and to overcome this problem parallel factor analysis (PARAFA) is used to
enhance the resolution of the overlapped spectra of the mixture The excitation
wavelength was in the range of 200ndash500 nm and ∆λ was in the range of 20ndash120 nm In
this study PARAFA has been established and applied to the synthetic and commercial
samples of the vitamins (Ni and Cai 2005) Synchronous fluorescence spectrometry in
organized media has been used for the determination of TH RF and PY in
pharmaceuticals in the presence of bisndash2ndashethoxyndashsulfosuccinate sodium salt (AOT)
micelles It has been found that RSD for repeatability is less than 14 and the LOD
has been found to be 12 microg Lndash1
10 microg Lndash1
and 9 microg Lndash1
for TH PY and RF respectively
(Garcia et al 2001)
Artificial neural network and LavenvergndashMarquardt backndashpropagation tanning
have also been used for the simultaneous determination of B1 B2 and B6 In this method
fluorescence were measured out at 15 wavelengths which were considered as
characteristic of artificial neural network The mean recoveries were found to be 9986
9980 and 9949 for B1 B2 and B6 respectively with RSDs of 17 16 and 17
respectively for these vitamins (Wu and He 2003)
23
213 InfrandashRed Spectrometry
A study has been carried out for the determination of femtosecond time resolved
infrared spectroscopy in vibrational response of RF in dimethyl sulfoxide (DMSO) for
photoexcitation at 387 nm In this study the vibrational cooling of the excited electronic
state was evaluated and its characterization was carried out by a time constant of 40 plusmn
01 ps The characteristic pattern of excited state vibrational frequencies of RF is useful
for its determination and identification in the spectral region of 1000 to 1740 cmndash1
The
calculation for vibrational spectra of ground and excited singlet state was carried out by
HartreendashFock (HF) and configuration interaction signals (CIS) methods It has been
found that upon photooxidation of RF the double bond position C(4a) and N(5)
disappeared (Wolf et al 2008)
214 Mass Spectrometry
Depending on the molecular fragmentation laser desorption mass spectrometry
(LDMS) has been developed for the analysis of RF TH HCl retinoic acid (RA) ascorbic
acid (AA) and PY HCl vitamins in commercial preparations (McMahon 1985) A
triplendashquad mass spectrometric method (LCUVMSndashMRM) has also been designed for
the determination of RF and other B vitamins in multivitamin and multimineral
supplements using a photodiode array detector (PAD) The method is simple as it does
not involve sample cleaning (Chen and Wolf 2007) Another method employed for the
determination of RF and other B vitamins is by comparing peaks of labeled vitamins with
those of unlabelled vitamins using LCndashisotopes dilution mass spectrometry (LCIDMS)
24
(Chen et al 2007) Electrondashspray ionization mass spectrometry (ESIMS) has been
employed for the determination of RF PY CF nicotinamide (NA) and taurine (TU) in
energy drinks Linear calibration curves have been observed in the range 08 to 15
with a recovery of 81 to 106 (Aranda and Morlock 2006) The analysis of waterndash
soluble vitamins in an infant formula has been performed using ultrandashperformance liquid
chromatographyndashtanden mass spectrometry (UPLCndashMSMS) The vitamins are extracted
using BEH Shield RP 18 column and the recovery range for RF has been found to be
818 to 106 using methanol and ammonium acetate (aqueous) as mobile phase
(Zhang et al 2009)
Planar chromatographicndashmultiple detection with confirmation by electrospray
ionization mass spectrometric method has been carried out for the simultaneous
determination of vitamin B2 B6 B3 caffeine and taurine in energy drinks For the
analysis of caffeine 10 samples of energy drinks and six samples of beverages were
prepared after degassing on ultrasonic bath for 20 min Chromatographic separation and
multindashwavelength scanning is carried out at 261 and 275 nm for B3 and caffeine
fluorescence measurements at 366400 and 313340 nm for RF and pyridoxine
respectively and 325 nm for taurine after post column chromatographic derivatization by
ninhydrin The overall recoveries for these vitamins and other substances have been
found to be in the range of 81ndash105 The intermediate precision for B2 B6 B3 caffeine
and taurine is in the range of 36ndash74 28ndash63 25ndash44 21ndash29 and 05ndash40
respectively Mass confirmation for each substance is carried out by MS in positive
25
electrospray ionization (ESI) positive scan mode except for taurine in negative mode
(Aranda and Morlock 2006)
A simple and precise method has been designed using HPLCndashMS for the assay of
RF in crude products The analysis has been carried out using methanol and water as
mobile phase and all the components have been separated and identified efficiently using
a C18 column (Guo et al 2006)
22 CHROMATOGRAPHIC METHODS
221 High Performance Liquid Chromatography (HPLC)
A simultaneous method for the determination of various B vitamins including RF
involves reverse phase liquid chromatography using the ionndashpair technique The
separation of the vitamin (RF at 280 nm) has been carried out at pH 36 using methanol
and water (1585 vv) with triethylamine (005) as a mobile phase The average
recovery for RF has been found to be 982 to 10202 with RSD of 102ndash55 (Li
2002) HPLC has been employed to study the chemical stability of total parenteral
nutrition (TPN) containing several vitamins using diode array detector RF PY AA and
other B vitamins are separated using Bondapak (C18 column) and methanolwater (2773
vv) as mobile phase with 14 sodium 1ndashhexanesulfonate for ionndashpair formation
(Ribeiro et al 2011) The RPndashHPLCndashdiode arrayfluorescence detector using ODS
column has been employed for the assay of multivitamins preparations containing RF and
26
other B vitamins The gradient elution system is used for the determination of RF (Chen
et al 2009)
Another reverse phase HPLC method reported for the determination of water
soluble vitamins in nutraceuticals has been reported This method quantitatively
determines the amount of RF PY cyanocobalamin (CA) and FA using gradient elution
The quantities of RF PY CA and FA determined by UV detection have been found to be
013 mgg 0235 mgg 00794 mgg and 00966 mgg respectively Recoveries for the
method have been found to be in the range of 986 to 1005 with RSD values of less
than 1 (Perveen et al 2009)
Stability studies of certain pharmaceutical preparations containing vitamins have
been carried out using a reverse phase HPLC method The detection has been made at
280 nm using gradient elution with a mobile phase of 0015 M sodium salt of 1ndashhexane
sulphonic acid and methanol Vitamins B2 B6 B3 and B1 show 151 199 63 and 427
min retention time respectively with coefficient correlation values of 0999 (Thomas et
al 2008)
Yantih et al (2011) reported a validated HPLC method for the quantitative
determination of vitamins in syrups containing multivitamins RF TH HCl NA and PY
HCl are separated using a C18 column with 10 microm particle size The separation of the
effluent is achieved within 20 min monitored at 280 nm using a mixture of methanolndash
acetic acid (1) and sodium salt of 1ndashhexane sulphonic acid in the ratio of 2080 vv as
mobile phase
27
The stability of total parenteral nutrition containing multivitamins has been
studied using a HPLC method NA is determined using UV detector where as PY and RF
5primendashphosphate via fluorescence detection without pretreatment of the sample FA and TH
are quantified using UV detector after prendashcolumn enrichment Detection of vitamin C
(AA) is done by determining the concentration of AA as well as dehydroascorbic acid
(DHA) DHA is determined by fluorescence detection after it was converted to a
quinoxaline (Van der Horst et al 1989)
The determination of total RF phosphates by immobilized sweet potato and
phosphatase (prendashcolumn reactor) has been carried out by a chromatographic method
Hydrolysed RF is eluted using methanol as a mobile phase and the measurements are
carried at 280 nm This method shows good linearity in the concentration range of 05ndash
500 nmol mlndash1
for total RF phosphates The LOD has been found to be 25 pmol mlndash1
with
an average transformation of RF phosphates to RF to be 97 The intrandash and interndashday
precisions ( RSD) have been found to be 12 and 26 respectively (Yamato et al
2000)
The simultaneous determination of waterndashsoluble vitamins (TH RF NA PY
CA FA) in multivitamin pharmaceutical formulations and biological fluids (urine blood
serum) has been carried out by HPLC A Phenomenex Luno C18 column with gradient
elution (CH3COONH4CH3OH (991 vv) H2OCH3OH (5050 vv)) and flow rate of
05 ml minndash1
has been used The detection is carried out by PDA detector at a wavelength
of 280 nm LOD for these vitamins has been found to be 16ndash34 ng with a linearity range
28
of 25 ng microLndash1
In this method theobromine (2 ng dlndash1
) is used as internal standard (IS)
The mean recoveries () have been found to be in the range of 846ndash103
(Chatzimichalakis et al 2004)
A study has been carried out for the determination of RF by HPLC in RF depleted
urine samples as calibration and control matrix In this method 1 mg mlndash1
of RF in RF
depleted urine is used to validate the HPLC method with fluorescence detection This
method shows good linearity in the concentration range of 10ndash5000 ng mlndash1
The
coefficients of variations for intrandash and interndashday precision have been found to be 39 and
9 respectively (Chen et al 2005)
An HPLC method has been developed for the simultaneous determination of
vitamin B1 B2 B6 and sorbic acid in Alvityl syrup The samples are diluted with water
and separated by C18 column with a mobile phase of 1ndashsodiumhexane sulfonate (8 mmol)
solution containing triethylamine (025 ml) acetic acid (92 ml) and methanol The
detection for these compounds is carried out at 280 nm This method shows good
linearity in the concentration range of 002ndash04 ng mlndash1
002ndash04 ng mlndash1
0007ndash01
002ndash04 ng mlndash1
and 003ndash06 002ndash04 ng mlndash1
for vitmain B1 B2 B6 and sorbic acid
respectively (Yang et al 2010)
The determination of B1 and B2 has been carried out in four vitamin glucose
calcium particles for children by HPLC In this method a ORBAXndashEclipse XDBndashC18
column with a mobile phase of 1ndashheptane solution (0005 mol Lndash1
) containing acetic acid
(05 ) and triethylamin (005) has been used The detection is carried out at 260 nm
29
and the flow rate was 1 ml minndash1
This method shows good linearity in the concentration
range of 713ndash2296 microg mlndash1
and 812ndash323 microg mlndash1
for B1 and B2 respectively The
recoveries for B1 and B2 have been found to be 1011 and 1014 respectively with a
RSD of 06 (Yuan et al 2008)
A reversed phase ionndashpair HPLC method has been developed out for the
determination of TH RF PY and NA in the chewable tablets of vitamins The water
microndashBondapak C18 column is used with a mobile phase of sodium hexane sulfonate buffer
(0005 M) and methanol The detection is carried out at 280 nm and the method shows
good linearity in the concentration range of 06ndash288 microg mlndash1
96ndash288 microg mlndash1
15ndash45
microg mlndash1
and 100ndash300 microg mlndash1
for TH RF PY and NA respectively Mean recoveries
have been found to be 1008 1003 998 and 992 for TH RF PY and NA
respectively with RSDs of 14 12 05 and 09 respectively (Xinhe et al
1999)
The determination of vitamin Bndashcomplex (TH RF NA nicotinc acid (NC) PY
cyanocobalamin (CA) and FA) has been carried out by HPLC in pharmaceutical
preparations (multivitamin formulations) and biological fluids (blood serum and urine)
after sold phase extraction (SPE) In this method a Phenomenex luna C18 column is used
and gradient elution is carried out at a ratio of 991 of CH3COONH4CH3OH (005 M)
and H2OCH3OH (5050 vv) with a flow rate of 08 ml minndash1
with detection using a
photodiode array (PDA) detector at 280 nm The method showed good linearity upto
30
25 ng microL with a detection limits in the range of 16ndash34 ng for each vitamin
(Chatzimichalakis et al 2004)
A HPLC method has been developed and used for the determination of RF and
aromatic amino acids in the form of shrimp hydrolysates This method is based on the
acid hydrolysis (01 M HCl) of RF followed by an enzymatic digestion and protein
precipitation by trichloroacetic acid A Chrom SEPSS C18 column (5 microm) column with a
mobile phase of ammonium acetate (5 mM) and methanol (7228 vv) at a flow rate of
10 ml minndash1
has been used The method shows good linearity reproducibility accuracy
and LOD in the studied range (BuenondashSolano et al 2009)
RF has been determined in milk and nonndashdiary imitation milk during refrigeration
by HPLC with UV detection The content of RF has been found to be in the range of
116ndash131 microg mlndash1
and 133ndash144 microg mlndash1
for cows milk and nonndashdiary imitation milks
respectively These open containers when stored in a refrigerator (8 oC) in the dark the
loss of RF content ranged from 160ndash234 and 125ndash165 in cows milk and nonndash
diary imitation milk respectively (Munoz et al 1994)
222 Liquid Chromatography (LC)
A ionndashpair RP liquid chromatographic (IPndashRPndashLC) method has been developed
for the determination of RF in cooked sausages In this method the sausage samples have
been subjected to acid and enzymatic hydrolysis The samples are directly injected
without any purification and concentration treatment into the column In this method
31
heptansulfonic acid (5 mM pH 27) and acetronitrile (7525 vv) are used as a mobile
phase The intrandash and interndashday precisions have been found to be 13 and 26
respectively with LOD of 0015 mg100 g This method shows a mean recovery of gt 95
(Valls et al 1999)
The selective detection of RF has been made by liquid chromatography with a
series of dualndashelectrode electrochemical detectors In this method two electrodes
(upstream downstream) are held at ndash04 V and +01 V versus SCE This method shows
good linearity in the concentration range of 4 ngndash26 microg with a LOD of 4 ng There is no
interference in absorbance and electrochemical detection of RF in the presence of 13
different vitamins (Hou and Wang 1990)
223 Ion Chromatography (IC)
Ion chromatography (IC) with photochemical fluorimetry (PCF) has been used for
the determination of RF in health protection products The chromatographic separation is
carried out at a Low Pac AsHndashHC column using NaOH (40 mmol Lndash1
) as the mobile
phase The column effluents are subjected to UVndashirradiation (245 nm) to transform RF
into a strongly fluorescent component and detection is carried out by spectrofluorimetry
This method shows good linearity in the concentration range of 10ndash100 mg Lndash1
with LOD
of 05 ng Lndash1
The means recovery for RF was found to be 10146 plusmn 25 (Cao et al
2013)
32
23 ELECTROCHEMICAL METHODS
Cyclic voltammetry and differential pulse voltammetric (DPV) methods with
glass electrode have been employed to investigate the electrochemical behavior of RF
The sensitivity of RF peaks and the detection accuracy is enhanced using glass electrode
made up of poly (3ndashmethylthiophene) Diffusivity (Do) and the electron transfer number
lsquonrsquo using cyclic measurements have been found to be 0000026 cm2s and 2 respectively
DPV has been used for the quantitative determination of RF with a detection limit of
50 times 10ndash8
mol Lndash1
A linear peak current in the range of 1 times 10ndash7
to 2 times 10ndash4
mol Lndash1
along with a RSD of 15 has been determined (Zhang et al 2010)
A simultaneous electrochemical method has been developed for the determination
of waterndashsoluble vitamins by the use of a pretreated glassy carbon electrode (PGCE)
PGCE has been prepared by potential cycling (ndash08 to +10 V) and voltammetry is carried
out following anodic oxidation (18 V) Increase in electrochemical responses and wellndash
defined peaks (Epa = ndash0073 V Epc = 0044 V) of certain waterndashsoluble vitamins have
been achieved using PGCE (Gu et al 2001) In pharmaceutical dosage forms a
voltammetric method has been described for the determination of RF and LndashAA Using
GCE both the compounds have been investigated for their electrochemical behavior at
pH 68 (KH2PO4Na2HPO4) The concentration range for the determination of RF is
15 times 10ndash6
ndash3 times 10ndash5
M giving an anodic peak at ndash047 where as for LndashAA acid it is
15 times 10ndash4
ndash3 times 10ndash3
M with a peak at +035 V (Mielech 2003)
33
Square wave adsorptive stripping voltammetry (SWASV) is another method that
has been used for the assay of RF A mercury film electrode (MFE) is used in this
method Subsequent reductive stripping step is carried out at pH 12 after RF has been
adsorbed at 00 V (AgAgCl) A 8 precision has been found with a recovery over 90
and the limit of detection to be 05 nmolL (Economou and Fielden 2005)
The electrochemical determination of RF on glass carbon cyclic voltammetry
electrode has been studied by using cyclic voltammetry This electrode is activated by 80
mol Lndash1
HNO3 solution with an electrode potential in the range of +06 ~ +20 V The
adsorption scanning has been studied in the range of 08 ~ 70 V by changing the RF
concentration from 60 times 10ndash8
to 70 times 10ndash6
mol Lndash1
at 90 mVsec RF shows
characteristics reversible adsorption at the carbon electrode and the calibration curve is
linear in the concentration range of 60 times 10ndash8
ndash70 times 10ndash6
mol Lndash1
with a LOD of
10 times 10ndash8
mol Lndash1
(Yang et al 2001)
The voltammetric determination of RF and Lndashascorbic acid (LndashAA) has
simultaneously been carried out in multivitamin pharmaceutical preparations The
electrochemical behavior of RF and LndashAA has been studied in the presence of phosphate
buffer (pH 60) using a glassy carbon electrode RF and LndashAA gave anodic peaks at
ndash 047 and + 035 V versus SCE respectively The oxidation peaks are directly related to
the concentrations of RF and LndashAA This method has been found to be useful for the
determination of RF and LndashAA in the concentration ranges of 15 times 10ndash4
ndash30 times 10ndash5
M
and 15 times 10ndash4
ndash30 times 10ndash3
M respectively (Mielech 2003)
34
24 PHOTOCHEMICAL METHODS
RF and RF 5rsquondashphosphate have been assayed by photochemical method using
injection flow technique Photondashreduction of both the compounds has been carried out
using ethylenediaminetetraacetic acid A linear curve has been obtained at low
concentration using chemiluminescent hydrogen peroxidendashluminol reaction RF a result
of photochemical process has been observed to form 1 5ndashdihydro derivative obtained by
the peroxidation of hydrogen peroxide A linear calibration curve has been obtained in
the concentration range of 1 times 10ndash7
to 3 times 10ndash6
mol Lndash1
(PerezndashRuiz et al 1994)
RF in photodegraded samples and aged vitamin preparations has been determined
by a stabilityndashindicating photochemical method This method is based on the conversion
of RF into lumichrome (LC) in alkaline solution under a control set of conditions (ie
light intensity pH temperature distance and time of exposure) In these conditions the
twondashthird of the RF is converted in to LC and the concentrations of RF in degraded
solutionssamples is determined by the RFLC ratio In this method the photolysed
solution of RF are adjusted to pH 20 and extracted with chloroform The determination
of LC and lumiflavin (LF) is carried out by a twondashcomponent spectrometric method at
356 and 445 nm respectively This method shows a percent recovery of 99 to 101 with
a precision of around 2 (Ahmad et al 2015)
35
25 ENZYMATIC ASSAYS
The homogenousndashtype enzymendashRF complex based determination of RF and its
binder protein has been performed using synthetic enzymendashbiotin and avidinndashRF
conjugates Amount dependant addition of RF binding protein (RBP) in the determination
of RF results in reversal of observed inhibition and enzymendashbiotin conjugate activity In
the mixture free RF addition results in rendashinhibition of the activity which has been found
concentration dependant Glucose 6ndashphosphate dehydrogenase adenosine deaminase and
alkaline phosphate are the three enzymes determined in this process Significant
inhibition of the catalytic activity of the enzyme has been observed (gt 90 ) when
enzymendashbiotin conjugates were determined using avidinndashRF conjugate binding and the
process has been reversed when RBP was added (Kim et al 1995)
A RF assay based on homogenous type enzyme linked determination has been
developed This method is based on the ability of binding of either analyte vitamin
molecule or glucose 6ndashphosphate dehydrogenasendash3ndashcarboxymethylflavin conjugate on
limited RBP sites which have previously been immobilized using sepharose particles
The catalytic activity of the conjugate is increased significantly Detactability has been
observed using optimal conditions An effect of pH and different organic solvents with
different proportions on the reaction has been studied The ratio of protein binding sites
to the conjugates has been found as the main factor on which the calibration curve
sensitivity and the detection limit for the assay depends The proposed method based on
36
the RBP sites agrees well with the selectivity and results of the method
(Cha and Meyerhoff 1987)
26 FLOW INJECTION ANALYSIS (FIA) METHOD
The flow injection analysis with chemiluminescence (CL) detection has been
carried out for the determination of RF In this method reduction of RF is carried out with
chromium VI which results in the formation of chromium III The chromium III reacts
with luminal and H2O2 in alkaline solution to produce CL The CL intensity is related to
the concentration of RF which has been found to be linear in the concentration range of
10 times 10ndash10
to 10 times 10ndash5
mol Lndash1
with a detection limit of 30 times 10ndash11
mol Lndash1
This
method shows a mean recovery of 1013 with a RSD of 18 (Xie et al 2005)
The various analytical methods used for the assay of RF in pharmaceutical
preparations food materials and biological fluids have been described in the above
sections The specificity and sensitivity of these methods would depend on the nature of
the samples vitamin content interference accuracy requirement and other factors The
fluorimetric methods are inherently more sensitive than the spectrometric and
chromatographic methods for the assay of RF in different systems However
spectrometric and chromatographic methods are widely used for the assay of RF in
pharmaceutical preparations
CHAPTER III
PHOTOCHEMISTRY OF RIBOFLAVIN
38
31 INTRODUCTION
Riboflavin (RF) (1) is a photosensitive compound and therefore its stability in
the pharmaceutical preparations may alter when exposed to light (ie UV light visible
light sunlight) Various studies have been carried out on the photostability of RF in
pharmaceutical preparations (Macek 1960 Deritter 1982 Ahmad and Vaid 2006) and
parenteral nutrition (Allwood 1984 Allwood and Kearny 1998 Buxton et al 1983
Chen et al 1983 Ribeiro et al 2011 Smith and Metzler 1963 Martens 1989
Yamaoka et al 1995 Min and Boff 2002 Casini et al 1981 Asker and Habib 1990
Loukas et al 1995 1996)
RF undergoes a number of photochemical reactions in aqueous solution which
include intramolecular and intermolecular photoreduction photodealkylation (Ahmad
and Vaid 2006 Ahmad et al 2004ab 2013 2014 2015 Heelis 1982 1991 Sheraz et
al 2014b Song 1971) intramolecular and intermolecular photoaddition (Ahmad et al
2004b 2005 2006a Sheraz et al 2014ab) photooxidation (Jung et al 1995)
photosensitization (Huang et al 2004 2006) and photostabilization reactions (Ahmad et
al 2008 2011 2016a Habib and Asker 1991 Sheraz et al 2014b) When RF is
exposed to light it degraded into a number of photoproducts which include
formylmethylflavin (FMF) (4) lumichrome (LC) (5) lumiflavin (LF) (6)
carboxymethylflavin (CMF) (7) cyclodehydroriboflavin (CDRF) (8) 23ndashbutanedione
(9) and isoalloxazine ring cleavage products (Ahmad and Vaid 2006 Ahmad et al
1980 2004ab 2005 2006ab 2008 2009 2010ab 2011 2013 2014 2015ab 2016ab
Cairns and Metzler 1971 Smith and Metzler 1963 McBride and Metzler 1967 Heelis
et al 1980 1991 Schuman Jorns et al 1975 Sheraz et al 2014ab Song et al 1965
39
Treadwell et al 1968) In the presence of divalent anions (HPO42ndash
SO42ndash
) RF undergoes
photoaddition reactions to form CDRF and in the absence of divalent anions it follow
normal photolysis pathway to form FMF LC and LF A scheme for the photodegradation
pathways is given in Fig 31
Two main types of photoreactions including anaerobic and aerobic photoreactions
are discussed below
32 ANAEROBIC PHOTOREACTIONS
RF at neutral pH when exposed to light results in the fading of yellow colour by
the formation of leucodeuteroflavin The leucodeuteroflavin leads to the formation of
deutroflavin by dehydrogenation caused by oxygen The deuteroflavin in alkaline
solution is converted into LF (Kuhn and WagnerndashJauregg 1934) In the first step of
photodegradation reaction the 2ndashhydroxy group of RF sidendashchain is oxidized to a keto
group to form 78ndashdimethylndash10ndashformylmethyl isoalloxazine (FMF) (4) (Smith and
Metzler 1963) which leads to the formation of LC (5) in acidic and LC (5) and LF (6) in
alkaline solutions (Song et al 1965)
RF photolysis depends on the presence of an electron donor (photoreduction) or in
the absence of an electron donor (photobleaching) The irradiation of an aqueous solution
of RF in the presence of disodium ethylenediamine (EDTA) leads to the loss of colour
but when this solution is exposed to oxygen the colour is regained (Oster et al 1962)
40
N N
NNH
O
O
OH
OH
OH
OH
CH3
CH3
H
H
H
HH
excited singlet state excited triplet state
N
NNH
N
O
OCH3
CH3
O
OH H
OH H
CH2OH
(8) (5)
(4)
(7) (6)
intr
amol
ecula
r phot
oadditi
on
intramolecular photodealkylation
intramolecular photoreduction
[O] neutral and alkaline pH
acid neutral and alkaline pH
N N
NNH
O
O
OH
OH
OH
OH
CH3
CH3
H
H
H
HH
N N
NNH
O
O
OH
OH
OH
OH
CH3
CH3
H
H
H
HH
N
NNH
NH
O
OCH3
CH3
N
NNH
NH
O
OCH3
CH3
CH2
CHO
N
NNH
NH
O
OCH3
CH3
CH2
COOH
N
NNH
NH
O
OCH3
CH3
CH3
(1)
CH3
C
C
CH3
O
O
(9)
Fig 31 Scheme for the photodegradation pathways of RF
41
This photoreduction of RF in the presence of an external donor results in the
intermolecular reduction of the isoalloxazine ring (Enns and Burgess 1965) whereas
photobleaching is due to the intramolecular reduction of isoalloxazine nucleus by the
ribose sidendashchain (Holmstrom and Oster 1961) This leads to the formation of a 2ndashketo
compound (deutroflavin) that was predicted by Karrer et al (1935)
Under anaerobic and aerobic conditions a variety of alcoholic type sidendashchains on
N(10) position of the isoalloxazine nucleus is photobleached At neutral pH the anaerobic
photolysis of these flavins leads to the formation of alloxazine and a cyclic intermediate
which is oxygen sensitive The ratio of these two degradation products depends on the
length of the sidendashchain Under anaerobic photolysis conditions the primary secondary
and tertiary alcoholic groups attached on the side chain lead to the formation of
aldehydes ketones and regenerated alcohols respectively (Moore and Bayler 1969)
RF and other flavins containing N(10)ndashsubstituted isoalloxazine rings when
irradiated in alcohol and alcoholndashwater mixtures result in the formation of FMF and LC
(Moore and Ireton 1977) Another photoproduct (78ndashdimethylndash10(1ndashdeoxyndashDndasherythrondash
2primendashpentolosyl) isoalloxazine) of RF is formed by its photolysis in the pH range of 4ndash10
and its formation is similar to that of FMF (Cairns and Metzler 1971) At neutral pH
another photoproduct (4primendashketoflavin) of RF is formed like LC and this product is not
easily quenched by the addition of potassium iodide This product is formed by the
abstraction of 2prime and 4primendashα hydrogens in the excited ring (Cairns and Metzler 1971)
Heelis et al (1980) proposed that the triplet state [3RF] of RF is involved in the formation
of FMF below neutral pH whereas an increase in the rate of photolysis of RF at higher
42
pH is due to the anion radical This anion radical increased the rate of photodegradation
as compared to that at neutral pH (neutral radical)
33 AEROBIC PHOTOREACTIONS
RF on exposure to light in the presence of oxygen forms LC and LF (Kuhn and
WagnerndashJauregg 1934 Holmstrom and Oster 1961 Strauss and Nickerson 1961) and
also results in the breakdown of ribityl side chain (Oster 1951 Shimizu 1955
Fukumachi and Sakurai 1955) This aerobic photolysis of RF and other flavins at acid
pH is said to be a case of general acidndashbase catalysis The degradation rate of aerobic
photolysis is dependent on the buffer components (Halwer 1951)
In aerobic photolysis of RF FMF (deuteroflavin) is an intermediate which on
further photolysis leads to the formation of LF (Svobodova et al 1953) During the
aerobic photolysis of RF at alkaline pH another photoproduct carboxymethylflavin
(CMF) is also formed This photoproduct is formed by the photooxidation of 2ndashcarbonyl
of the sidendashchain of FMF by peroxides (H2O2) (Fukumachi and Sakurai 1955) During
the aerobic photolysis of RF the acidity of the aqueous solution increases due to the
formation of formic acid by the oxidation of the sidendashchain Anaerobic photolysis at pH
72 gives the same product distribution on 28 of photobleaching as that at 50 of
bleaching in aerobic photolysis This shows greater photobleaching of RF on aerobic
photolysis as compared to that of the anaerobic photolysis (Treadwell et al 1968)
In the presence of macormolecules (ie polyvinyl pyrrolidine (PVP) polysorbate
80 sodium dodecyl sulfate (SDS)) the rate of aerobic photobleaching is increased This
increase in the rate of photobleaching is due to the reversible binding of excited RF [RF]
43
to macromolecules which leads to the formation of the triplet state [3RF] This catalytic
effect of polymer is due to the protection of [3RF] by polymer from quenching by oxygen
(Kostenbauder et al 1965) Under aerobic photolysis RF at pH greater than 60 in the
presence of divalent phosphate (HPO42ndash
) anion or sulfate (SO42ndash
) anion leads to the
intramolecular photoaddition reaction which results in the formation of
cyclodehydroriboflavin (CDRF) (Schuman Jorns et al 1975)
34 TYPES OF PHOTOCHEMICAL REACTIONS
Flavins undergo a variety of photochemical reactions which occurs separately as
well as simultaneously These reactions depend on the nature of flavin and the reaction
conditions Flavins undergo both intermolecular and intramolecular reactions
(Hemmerich 1976 Heelis 1982) Different types of photochemical reactions are
discussed in the following sections
341 Photoreduction
RF undergoes intramolecular as well as intermolecular photoreduction as
discussed below
3411 Intramolecular photoreduction
RF undergoes anaerobic photoreduction in the absence of external electron donor
by the process of intramolecular disproportination This disproportination results in the
oxidation of ribityl sidendashchain and leads to the reduction of isoalloxazine ring
(Holmstrom and Oster 1961 Moore et al 1963 Radda and Calvin 1964) This
reduction in the isoalloxazine ring results in the degradation of the RF which leads to the
44
formation of FMF LC and LC (Smith and Metzler 1963) This photoreduction or
photodehydration leads to the dehydrogenation of ribityl sidendashchain with the formation of
ketonic or aldehydic functional group in the ribityl sidendashchain (Cairns and Metzler
1971) The intramolecular photoreduction of flavinRF is dependent on the pH and on the
cationic triplet [3RFH
+] and neutral triplet [
3RF] species which react differently (Cairns
and Metzler 1971)
A study has been carried out on the kinetic isotope effect on flavin (10) which
results in the replacement of αndashhydrogen in the ribityl sidendashchain (11) However no
hydroxyl hydrogen replacement has been observed (Moore and Bayler 1969 Moore and
Ireton 1977) In this reaction the αndashhydrogen removal from αndashCH results in the
formation of an intermediate biradical (12) which then disproportionate to form an
αndashketone (13) (Fig 32)
Intramolecular photoreduction of flavinRF involves singlet excited state [1RF]
and the triplet excited state [3RF] (Cairns and Metzler 1971) In an intramolecular
hydrogenndashtransfer reaction the ribityl side chain should be condashplanar with isoalloxazine
ring system (Song and Kurtin 1969) The intramolecular photoreduction rate is
dependent on the solvent polarity and this could be due to the conformational changes in
the ribityl side chain in different solvents (Moore and Ireton 1977 Ahmad et al 2015)
45
N
NNH
N
CH2
C HOH
R
O
O
(10)
N
NH
NH
N
CH2
COH
R
O
O
(11)
N
NNH
N
CH2
COH
R
O
OH
(12)
N
NH
NH
NH
CH2
O
O
CO
R
(13)
hv
Fig 32 Formation of αndashketone from flavin
46
3412 Intermolecular photoreduction
Flavins (10) in the presence of amino acids αndashhydroxyndashcarboxylic acids thiols
aldehydes unsaturated hydrocarbon (Knappe and Hemmerich 1972 1976) and αndash
substituted acetic acids (Ahmad and Tollin 1981a) results in the photoredcution that
leads to the formation of 15ndashdihydrogen flavin (H2Flred) (14) or its alkyl adducts
(RndashFlredH) ((15)ndash(17)) (Fig 33)
This H2Flred is reoxidized in the presence of oxygen (O2) to form hydrogen
peroxide (H2O2) and oxidized flavin (Eq 31) (Massey et al 1973)
H2Flred + O2 H2O2 + Flox
(31)
Intermolecular photoreduction of flavins has two different mechanisms In the
first step the photoreduction occurs by initial one electron involvement by transferring
from the substrate to the flavin and leads to the formation of flavosemiquinone radical
(33)
Fl hv 1Fl
(32)
FlH + R1Fl + RH
(33)
Fl- + RH+1Flo + RH
(34)
47
N
NNH
N
O
O
CH2
COH H
R
+ RH
N
NH
NH
NH
O
O
R
(10)
(14)
N
NNH
NH
O
O
R
R
H
N
NH
NH
NH
O
O
R
R
N
NNH
NH
O
O
R
R
(15)
(16)
(17)
Fig 33 Photoreduction of flavins to form 15ndashdihydrogen flavin and alkyl adducts in the
presence of unsaturated hydrocarbons
48
Photoreduction of flavins in presence of carboxylate anions or substrates is
expressed by the following equation
Fl hv 1Fl
(35)
Fl- + RCOO1Fl + RCOO-
(36)
R + CO2RCOO-
(37)
In this mechanism when flavin is exposed to light it is converted into the excited
singlet state (Eq 35) The excited singlet state when reacts with the carboxylate substrate
(Eq 36) leads to the formation of radicals (ie Flndash and RCOO
) The carboxylic radical
forms an alkyl radical and carbon dioxide (CO2) (Eq (37))
Photodegradation products are formed when two semiquinone radicals
disproportionate to form one reduced and the other oxidized flavin (Eq (38)) or by
radical addition
HFl + HFl
H2Flred + Flox
(38)
ProdcutsR
(39)
RFlredHHFl + R
(310)
Fritz et al (1987) presented a mechanism for the photoreduction of flavins in the
presence of external donor (EDTA) at pH 70 When the flavin is exposed to light it is
excited from the ground state to the excited singlet state (Eq (311))
1FloFl hv
(311)
49
This excited singlet state [1Fl] then through internal conversion is deactivated to
the ground state with release of heat energy (Eq (312))
1Fl oFlic
(312)
The flavin singlet excited state is converted into flavin excited triplet state through
intersystem crossing (Eq (313))
1Fl 3Flisc
(313)
Triplet excited state [3Fl] may be deactivated with release of heat energy by
coming back to ground state (Eq (314))
3Fl oFl+ heat
(314)
In the presence of a quencher the excited triplet state is quenched which leads to
the conversion of triplet state to the ground state with release of energy (Eq (315))
3Fl + oxygen quencher oFl + heat
(315)
When [3Fl] reacts with EDTA the flavin is reduced and EDTA is oxidized
(Eq (316))
3Fl + EDTAoFlred + EDTAox
(316)
The reoxidation of [oFlred] form occurs in the presence of oxygen which leads to
the formation of ground state flavin [oFl] and peroxide (Eq (317))
oFlred + O2
oFl + H2O2
(317)
50
342 Photodealkylation
Photodealkylation of flavins occurs via an intramolecular mechanism which is
due to the involvement of excited singlet and triplet states (Gladys and Knappe 1974)
Flavin photodealkylation occurs due to the simultaneously breakage of N(10)ndashC(1ʹ) and
C(2ʹ) bond via a direct proton transfer in cisndashperiplanar confirmation that leads to the
formation of LC (Hemmerich 1976) When flavins are photolysed in acetonitrile it
results in the formation of LC (5) and the corresponding alkene or cycloalkene (Gladys
and Knappe 1974)
9
6
8
7
N10
N5
2
N-
3
N1
4
CH3
CH3
O
O
CH2
C
R1
OH R2
N
NN
-
NH
CH3
CH3
O
O
pH 70hv
(1) (5)
Photodealkylation occurs by two mechanisms The first step involves homolytic
fission of the N(10)ndashC(1ʹ) bond in the biradical intermediate (Moore and Ireton 1977)
However the second step results by a synchronous process that does not involve radical
intermediates (Song 1971) The photodealkylation of RF takes place by the excited
singlet state which leads to the formation of LC (5) and its formation is not retarded by
the addition of triplet state quenchers (Cairns and Metzler 1971) It has been found that
intramolecular photodecarboxylation and dealkylation of flavins is mediated by excited
singlet and triplet state reactions (Gladys and Knappe 1974 Knappe 1975)
Carboxymethyl flavin (CMF) (flavinndash10ndashacetic acid) (7) is formed by the excited triplet
51
state which results in the formation of a biflavin intermediate This biflavin intermediate
when exposed to light forms LC (5) and other products (Knappe 1975)
343 Photoaddition Reactions
The solvent (R=H or alkyl) when introduced at position Cndash6 or Cndash9 positions of
the benzenoid subnucleus leads to the formation of hydroxy or alkoxyndashdindashhydroflavins
(Eq 318) as an intermediate (Schollnhammer and Hemmerich 1974) When ammonia or
cyanide is introduced in the system containing the flavin the reaction occurs by the attack
of a nucleophile (CNndash NH3
ndash) on the excited triplet state (Traber et al 1981a) These
reactions involve intermolecular photoaddition to RF
1Fl + CH3OH CH3O-Fl redH
(318)
The intramolecular photoaddition reactions are similar to that of the
photodehydration of flavin (Schollnhmmer and Hemmerich 1974) These reactions lead
to the formation of CDRF via autoxidation of an intermediate (dihydroriboflavin)
(Schuman Jorns et al 1975) This reaction occurs due to the presence of a nucleophilic
group in the ribityl sidendashchain It has been proposed that in this reaction the addition of a
proton takes place at N(1) and simultaneous deprotonation at C(9) position This leads to
the formation of a stable compound 15ndashdihydrondash9ndashalkoxylndashflavin which is then
converted into the CDRF by the process of autoxidation (Fig 31)
Quenching studies have been carried out to evaluate the involvement if [1Fl] and
[3Fl] states in the reactions of flavins It has been found that excited singlet state of flavin
is involved in photoaddition reaction while excited triplet state is involved in the normal
photolysis (photoreduction) reaction The excited singlet state reaction is dominant when
52
the triplet state is quenched ie oxygen quenching The photoaddition reaction occurs in
the presence of divalent anions (HPO42ndash
SO42ndash
) above pH 60 This photoaddition
reaction occurs by the formation of a flavinndashdivalent complex that results in the
C(4)O(2ʹα) interaction to form the cyclic product CDRF (8)
344 Photooxidation
Flavins in the presence of oxygen initiate the oxidation of a number of
compounds such as amino acids (Penzer 1970) indoleacetic acid (AmatndashGuerri et al
1990) cyanocobalamin (Hussain 1987) retinol (Futterman and Rollins 1973) bilirubin
(Sanvordeker and Kostenbauder 1974) lipids (Chan 1977) DNA and nucleotides
(Speck et al 1975) and phenothiazines (Uekama et al 1979)
Photooxidation of flavins occurs by electron abstraction from the substrate by
radical mechanism These substrate radicals and flavosemiquinone radicals react and
inhibit the radical back reaction (Vaish and Tollin 1971) Flash photolysis studies have
been carried out to determine the rate of photooxidation of flavin semiquinone radicals It
has been found that the neutral semiquinone radical is unreactive to oxygen as compared
to that of the anionic form of the flavin radical
345 Photosenstization Reactions
RF when exposed to light forms singlet oxygen species from triplet oxygen by
excited triplet state of RF [3RF] and triplet oxygen annihilation mechanism This plays an
important role in the photosensitized reactions (Choe et al 2005 Jung et al 2007)
53
RFhv 1RF
(319)
3RF1RF isc
(320)
3RF RF + 3O2
O2
(321)
Aerobic RFndashsensitized photodegradation of the endocrine disruptor
44rsquondashisopropylidenebisphenol (BPA) and of similar compounds like 26ndashdibromophenol
and 26ndashdimethyl phenol has been studied in water and waterndashmethanol mixtures by
continuous photolysis using visible light the uptake of oxygen being detected by
polarography stationary and time resolved fluorescence spectroscopy time resolved near
IR phosphorescence detection and laser flash photolysis techniques Bisphenols (BPs)
quench the excited singlet and triplet states of RF and have rate constants near to the
diffusion limit BPs and dissolved molecular oxygen are added in similar concentration
and they competitively quench the excited triplet state of RF As a result of this reaction
singlet molecular oxygen (O2 (1∆g)) and superoxide radical anions (O2
ndash) are produced by
electron and energy transfer The photooxidation products of BPA resulting from
oxidation dimerization and fragmentation have been identified These reactions indicate
that BPs in natural water are photodegraded under environmental conditions in the
presence of an adequate photosenstizer (Barbieri et al 2008)
RF is sensitive to light but it is relatively stable during thermal and nonndashthermal
food processing RF can accept and donate a pair of hydrogen atoms Under the influence
of light RF acts as a photosensitzer or prooxidant for food components During the
54
photosensitization of RF there is production of reactive oxygen such as singlet oxygen
hydroxyl radical superoxide anion and hydrogen peroxide Reactive oxygen and radicals
produced in this process potentiate the decomposition of proteins lipids carbohydrates
and vitamins RF acts as an excellent photosenstizer for singlet oxygen formation (Choe
and Min 2006)
RF is present in the eye as a normal component and which when exposed to light
triggers photosensitizing activity When this photosensitized RF is influenced by short
wavelength light below 400 nm it damages vitamin C that is present in the lens for the
inhibition of the photosensitization process (Rochette et al 2000)
It has been observed that RF photosensitized singlet oxygen oxidation of vitamin
D is not observed in samples without RF stored in a dark room and also in those samples
containing RF that are stored in dark Vitamin D containing RF is oxidized under the
influence of light Singlet oxygen quenched rate of αndashtocopherol is 250 times 108 M
ndash1s
ndash1 and
for ascorbic acid it is 223times107 M
ndash1s
ndash1 (King and Min 1998)
RF when exposed to light forms LC and LF and this formation is also influenced
by the pH RF when exposed to neutral or acidic pH form LC and when it is exposed to
basic pH it forms LF This conversion of RF to LF and LC is due to the type 1
mechanism of RF photosensitized reaction and singlet oxygen is also involved in the
conversion of RF to LF and LC The rates of reaction of RF LF and LC with singlet
oxygen are 966 times 108 850 times 10
8 and 821 times 10
8 M
ndash1s
ndash1 respectively (Huang et al
2006)
55
A study has been carried out on the RF sensitized decomposition of ascorbic acid
(AA) under the influence of light and it has been found that light and RF increases
photodecomposition of AA The photosensitizing activity of RF methylene blue and
protoporphyrin IX is 21511 at Indash2 ppm at different pH values (75 60 and 45) and the
rate constants for the reactions of AA are 663times108 577times10
8 and 527times10
8 M
ndash1s
ndash1 It has
been found that RF and methylene blue sensitize photooxidation of AA cyestine shows
strong antioxidant activity that is concentration dependent Alanine and phenylalanine
(01 ) show antioxidant effect on the RF sensitized photooxidation of AA and
prooxidant effect on the methylene blue sensitized photooxidation Tyrosine at 01
concentration shows prooxidant effect on both RF and methylene blue sensitized
photooxidation of AA but tryptophan (01 ) shows antioxidant or prooxidant effect on
the photooxidation of AA depending on the storage time (Jung et al 1995)
The photodegradation of tryptophan in oxygen saturated aqueous solution
resulting in the generation of reactive oxygen species 1O2 OH H2O2 and O2
ndash is
sensitized by RF Photodegradation experiments have been runs with 14
CndashRF and 14
Cndash
tryptophan The photoproducts have been separated by Sephadex Gndash15 and C18ndashHPLC
and detected as aggregate forms of RF indolic products associated to flavins indolic
products of molecular weight higher than tryptophan formyl kynurenine and other
tryptophan photoproducts (Silva et al 1994)
RF and amino acids such as phenylalanine tryptophan leucine isoleucine and
valine are present in milk RF as a photosensitzer results in the destruction of essential
amino acid by the process of oxidation It has been found that in aqueous samples that
contain increased concentration of trolox (TX) and AA show an increased head space
56
oxygen depletion and this is due to the oxidation of trolox AA and amino acid in the
presence of RF HPLC has shown that trolox and ascorbic acid decrease the
photodegradation of phenylalanine tryptophan and tyrosine and this is due to the
presence of trolox and AA acting as singlet oxygen quenchers of tryptophan and tyrosine
(Reddy 2008)
The effect of pH and ionic micelles on the rate of formation of products on the
irradiation of RF in the presence of tryptophan has been studied by absorption and
fluorescence spectroscopy In anaerobic conditions the formation of RFndashtryptophan
complex is inhibited in acid solution by the addition of anionic (sodium dodecylsulphate)
and cationic (cetyltrimethylammonium bromide) micelles In the presence of RF the
oxidation of tryptophan is faster in alkaline solutions than in acid solutions (Silva et al
1991)
A study has been carried out in the presence of flavins as sensitizers on the
photooxidation of substituted phenols under aerobic condition to determine the fate of
synthetic chemicals in environment RF is easily decomposed to form LC by the
influence of several minutes illumination with simulated sunlight It has been found that
LC is extremely stable toward sunlight and it is the major flavin component in natural
water The order of photolysis rate is pndashmethoxyphenol gt pndashchlorophenol gt phenol gt
nitrophenol in the LC sensitized photodecomposition of substituted phenols It has been
found that the total organic carbon (TOC) is decreased from the reaction solutions of all
the phenols except pndashnitrophenol (Tatsumi et al 1992)
57
In the presence of RF 4ndashhydroxyquinolone (4ndashOHQ) and 8ndashhydroxyquinolone
(8ndashOHQ) are photooxygenated under the influence of visible light in watermethanol
(91 vv) mixture RF in this reaction acts as a dye sensitizer Both of the quinolones are
transparent under the influence of visible light but 8ndashOHQ has five time faster
degradation than that of 4ndashOHQ The kinetic data shows that 4ndashOHQ degrades by the
mechanism of superoxide radical anion where as 8ndashOHQ degrades by the mechanism of
singlet molecular oxygen along with superoxide radical anion RF as a sensitizer is
photodegraded under the influence of visible light and is regenerated in the presence of
either of these two quinolones by an electron transfer process that produces superoxide
radical anion (O2-) (Criado et al 2003)
The aerobic irradiation of methanolic solutions either of phenol type compounds
pndashphenylphenol (PP) pndashnitrophenol (NP) and phenol (Ph) or other phenolic derivatives
pndashchlorophenol (CIP) and pndashmethoxyphenol (MeOP) in the presence of RF as sensitizer
results in the photodegradation of ArOH and the sensitizer A complex mechanism is
involved in the photodegradation of ArOH in which superoxide radical anion (O2ndash
) and
singlet molecular oxygen (O2 (1∆g)) is involved This mechanism is highly dependent on
the concentration of ArOH (Haggi et al 2004)
346 Photostabilisation Reactions
The effect of certain stabilizers on the aerobic photobleaching of RF has been
examined under the influence of fluorescent light The greatest photostabilizing effect is
seen by disodium ethylenediamine (EDTA) which is followed by thiourea
methylparaben DLndashmethionine and sodium thiosulfate The photostabilizing effect of
58
these compounds increases with an increase in their concentration The photodegradation
of RF solutions is influenced by pH and buffer species and EDTA (Asker and Habib
1990)
The quantum efficiency (Φ) of RF under aerobic conditions has been determined
by a microirriadiation method It has been found that the initial quantum yield of RF is
independent of light intensity wavelength of light and concentration The quantum
efficiency of RF is decreased in the presence of phenols and there is linear relation
between Hammettrsquos Sigma values and rates of photodegradation As compared to
phenols benzyl alcohol and benzoic acid are ineffective as photochemical stabilizers
The photodegradation of RF is enhanced by cinnamyl alcohol which acts as an electron
donor (Shin et al 1970)
A study has been carried out on the photostablization of RF in liposomes in
aqueous solution under various irradiation conditions liposomal composition
concentration pH and ionic strength It has been found that the photostability of RF is
increased in the presence of neutral and positively charged liposomes and by increasing
the concentration of dimyristoylndashphosphatidylcholine (DMPC) in the composition of
liposome The photostability of RF in the presence of 5ndash8 mM DMPC increases up to 23
fold as compared to a control buffer solution It has been found that the pH of the
medium effects the photostability of RF and the ionic strength of solution does not affect
The photodegradation of RF follows firstndashorder kinetics in the presence and absence of
liposomes (Habib and Asker 1991)
59
A study has been carried out on the formulation of liposomal preparations of RF
with a change in the concentration of phosphatidylcholine (PC) showing an increase in
their entrapment efficiency from 26 to 42 Physical characterization of these liposomes
has been carried out by dynamic light scattering (DLS) and atomic force microscopy
(AFM) RF encapsulated in liposomes when subjected to visible light follows firstndashorder
kinetics for its degradation RF and its photoproduct (LC) in liposomes were assayed by a
twondashcomponent spectrometric method at 356 and 445 nm and to compensate for the
interference of liposomal components an irrelevant absorption correction method was
used It has been found that with an increase in PC concentration from 1215ndash1485 mM
the rate of RF photodegradation is decreased This decrease in the rate is due to the
interaction of RF with PC and its reductive stabilization (Ahmad et al 2015b)
347 Factors Affecting Photochemical Reactions of RF
There are a number of factors which affect the photochemical reactions of RF
These factors are discussed below
3471 Radiation source
In the photolysis reactions of drugs the radiation source plays an important role
RF in the milk when exposed to sunlight degraded around 30 in 30 mins (Wishner
1964) In the powder forms RF is much stable as compared to that of the solution form in
which when exposed to light it is degraded into different photoproducts (FMF LC LF
CMF etc) (Ball 2006 Cairns and Metzler 1971 Smith and Metzler 1963 Ahmad and
Vaid 2006 Treadwell et al 1968 Ahmad et al 2004ab 2005 2006ab 2008 2009
2010 2011 2013ab Sheraz et al 2014a McDowell 2000) Different studies have been
60
carried out on the photolysis of RF using low and high intensity radiation sources
(Ahmad et al 2004a 2006 Ahmad and Rapson 1990 Becker et al 2005 Dias et al
2012 Mattivi et al 2000 Sato et al 1982) A comparison has been made on the effect
of UV and visible radiation on the rate of photolysis of RF (Ahmad et al 2004 2006)
The photoproducts formed in both cases are similar however the rate of reaction is
higher in the case of UV radiation as compared to the visible light This increase in rate is
due to the intensity of UV radiation (219plusmn012 times 1018
qsndash1
) as compared to that of visible
light (114 plusmn01 times 1017
qsndash1
(125 W) (Ahmad et al 2004a)
A study has been carried out on RF tablets exposed to a xenon lamp emitting in
the range of 300ndash800 nm It has been found that the greater colour change in samples
(yellow to green) was at 250 Wm2 after initial exposure to xenon lamp This change in
colour (yellow to green) is due to the visible light gt 400 nm and only LC was found as
the degradation product (SuendashChu et al 2009)
3472 pH effect
The pH of an aqueous solution influences the photodegradation reactions of RF
and its photoproducts The major photoproducts FMF and LC are formed in both the
acidic and alkaline pH while LF is formed in the pH range of 70 to 120 The formation
of all these products is due to the oxidation of the ribityl sidendashchain CMF βndashketoacid
and a diketo compound are minor photoproducts CMF is formed at pH 10ndash120 while
βndashketoacid and the diketo compound are formed at pH 100ndash120 The βndashketoacid and the
diketo compound are formed by the cleavage of the isoalloxazine ring by the alkaline
hydrolysis of RF (Song et al 1965 Treadwell et al 1968 Ahmad et al 2004a 2013
61
Ahmad and Rapson 1990) LC and LF are formed by the excited triplet state via an
intermediate photoproduct FMF (Ahmad and Rapson 1980 Ahmad et al 2004ab 2005
2006ab 2008 2009 2010 2011 2013ab) LC is stable at lower pH as compared to that
of higher pH which is due to its protonation at lower pH However LF is further
degraded at pH 140ndash146 to form 78ndashdimethylisoalloxazine anionic
methylisoalloxazine and quinoxaline derivatives (12ndashdihydrondash2ndashketondash167ndashtrimethylndash
1Hndashquinoxalinendash2ndashone) by cleavage of the isoalloxaine ring (Penzkofer et al 2011)
Another photoproduct (23ndashbutanedione) of RF which has buttery smell is formed in 01
M phosphate buffer at different pH (450 650 850) after light exposure This product is
formed via a ribityl sidendashchain cleavage through the effect of anion singlet oxygen (Jung
et al 2007)
A detailed study has been carried out on the photolysis of RF in the pH range of
10ndash120 It has been found that under UV and visible light the maximum stability is
achieved at pH 50ndash60 which is due to the lower redox potential of RF at this pH The
rate of photolysis at pH 100 is 80 fold higher as compared to that of 50 which is due to
the higher redox potential and higher reactivity of the flavin triplet state at this pH Above
pH 100 the rate of photolysis decreases due to the anion formation of RF (Ahmad et al
2004a)
3473 Buffer effect
The photolysis of RF has been found to be influenced by the kind and
concentration of the buffer used Several studied have been carried out on the catalytic
effect of buffers ie phosphate acetate and carbonate (Schuman Jorns et al 1975
62
Ahmad et al 2004ab 2005 2006 2010 2013) However borate (Ahmad et al 2008)
and citrate (Ahmad et al 2011) have a photostabilizing effect on RF In borate buffer RF
forms a complex with borate ion to inhibit its photolysis The divalent citrate ions
decrease the fluorescence of RF due to quenching of the excited singlet state and thus
decrease the rate of photolysis The trivalent citrate ions show a greater stabilizing effect
due to the quenching of the excited triplet state (Ahmad et al 2008 2011) Acetate
(pH 38ndash56) and carbonate (pH 92ndash108) buffers exert a catalytic effect on the
photolysis of RF The acetatendash and carbonatendashcatalyzed reactions represent bell shaped
and steep curve type kndashpH profiles respectively The rate of photolysis of RF has been
found to be catalyzed by HCO3ndash and CO3
2ndash ions in the alkaline solution and there is a
major role of CO32ndash
ions in the catalysis of RF (Ahmad et al 2014a)
The intramolecular photoreduction and photoaddition reactions of RF in the
presence of phosphate buffer have been studied in detail The analysis of RF and its
photoproducts of both reactions (CDRF FMF LC LF) is carried out by a
multicomponent spectrometric method It has been found that H2PO4ndash and HPO4
2ndash species
of phosphate buffer play a major role in the degradation of RF The H2PO4ndash species are
involved in the photoreduction reaction to form LC and LF while HPO42ndash
(02 M ge)
catalyze the photoaddition reaction to from CDRF (Ahmad et al 2005) The effect of
pH buffer and solvent viscosity on the aerobic and anaerobic photolysis of FMF has been
studied It has been found that the rate of photolysis under aerobic conditions is higher at
pH 40 and above pH 100 The rate of photolysis at alkaline pH is higher due to
sensitivity of flavin triplet state to alkaline environment The rate of photolysis of FMF is
linearly increased with the inverse of solvent viscosity (Ahmad et al 2013)
63
3474 Effect of complexing agents
In the presence of divalent species (ie HPO42ndash
SO42
tartarte succinate
malonate) RF is rapidly degraded via an intramolecular photoaddition pathway through
the formation of a RFndashdivalent ion complex (Schuman Jorns et al 1975 Ahmad et al
2004b 2005 2006 2010) The rate of photodegradation is lower in the case of organic
species (Ahmad et al 2010) In the presence of sulfate anions the rate of photolysis is
much higher as compared to that of phosphate anions This is probably due to the
formation of a strong divalent anion complex higher electronegative character and higher
amount of anionic species in the case of sulfate (Schuman Jorns et al 1975 Ahmad et
al 2010) These reactions can be expressed (Ahmad et al 2005 Ahmad and Vaid 2006)
as follows
RF [1RF] LC
hv H2PO4-
(322)
[3RF][1RF] RFH2
isc
phosphateleucodeutroflavin
(323)
RFH2
O2 FMF + side-chain products
(324)
FMFhv LC + side-chain products
(325)
FMFHOH LC + LF + side-chain products
(326)
In the presence of HPO42ndash
RF undergoes photoaddition reaction involving the
formation of a RFndashHPO42ndash
complex which on the absorption of light forms an excited
64
singlet state [1RF] [
1RF] is then converted into a dihydroflavin intermediate which upon
autoxidation gives CDRF
RFHPO4
2-
RF-HPO42- hv [1RF]
complex
(327)
dihydroflavin autoxidation[1RF]intermediate
CDRF
(328)
A study has been carried out on the effect of caffeine complexation on the
photolysis of RF in the pH range of 20ndash105 The rate of photolysis decreases with an
increase in the caffeine concentration which shows that caffeine exerts inhibitory effect
on the photolysis of RF It has been found from the kndashpH profile that initially the rate of
photolysis increase upto 100 and at pH 20 and 105 the lower photolysis rates are due to
the ionization of RF The interaction of RF with caffeine gives a bell shape curve in the
pH range of 30ndash60 and then a sigmoid curve in the pH range of 70ndash100 This shows
that a decrease in the rate of photolysis of RF in the presence of caffeine is due to
monomeric interaction and complex formation between RF and caffeine (Ahmad et al
2009)
A photodegradation study of RF (50 times 10ndash5
M) in phosphate buffer (02ndash10 M)
in the presence and absence of caffeine (250 times 10ndash4
M) has been carried out at pH 60ndash
80 In the presence of phosphate buffer RF undergoes photoreduction and photoaddition
reactions simultaneously that result in the formation of LC and CDRF respectively as
the major photoproducts It has been found that an increase in phosphate concentration
leads to greater formation of CDRF The formation of CDRF in the presence of caffeine
65
is enhanced by the photoaddition reaction due to suppression of the photoreduction
pathway of RF (Sheraz et al 2014a)
Fluorimetric studies have been carried out on RFndashcyclodextrin (CD) complex
formation using a nonndashlinear least square model Differential scanning calorimetry (DSC)
and 1H NMR spectrometry have been used for the confirmation of a RFndashβndashCD complex
in the solid state and in aqueous solution respectively (Loukas and Vraka 1997)
Spectroscopic and solubility methods have been used to study inclusion complex
formation of hydroxypropylated αndash βndash and γndashCD with RF and alloxazine Alloxazine
which is an analog of RF has been used to evaluate the role of ribityl and methyl
substituent in complexation It has been found that the cavity of hydroxypropylndashβndashCD is
appropriate for the formation of stable RF complexes Because of van der Waals forces
and hydrogen bonding these complexes were stabilized 1H NMR and computer modeling
was used to confirm the insertion of RF in the CDndashcomplex (Terekhova et al 2011a)
A thermodynamic study has been carried out on the inclusion complex formation
of αndash βndash and γndashCD with RF and alloxazine The influence of reagents structure on the
complex formation has been related to thermodynamic parameter (K ∆cG0 ∆cH
0 ∆cS
0)
It has been found that αndashCD shows less bonding affinity to RF and alloxazine as
compared to βndashCD This binding is associated with negative enthalpy and entropy
changes that involve van der Waals forces and hydrogen bonding Ribityl sidendashchain
prevents the penetration of RF in the macrocyclic cavity (Terekhova et al 2011b) Nonndash
inclusion complexes between RF and CD have been prepared to investigate the molecular
interaction between βndashCD (HPβndashCD) and their anticancer activity UVndashvis and NMR
spectrometry fluorimetry and DSC have been used for the physiochemical
66
characterization of these formulations The interaction between RF and CD has been
evaluated by molecular dynamics simulation cytotoxicity of RFndashCD against prostate
cancer by inndashvitro cell culture tests It has been found that there are no physicochemical
changes in RF on complexation with βndashCD and HPβndashCD At low concentration βndashCD
and HPβndashCD interaction is due to hydrogen bonding between flavinoid and external ring
of CDs RFndashCDs complexes have increased RF solubility and antitumor activity (de
Jesus et al 2012)
3475 Effect of quenchers
In pharmaceutical preparations of RF the external quenchers are added for the
improvement of quantum yield of photochemical reactions without the fluorescence
quenching of RF (Holmstrom et al 1961) A variety of external quenchers have been
used to deactivate the RF excited states These includes βndashcarotene and lycopene
(Cardoso et al 2007) glutathione and Dndashmannitol (Baldursdottir et al 2003) phenol
(Song and Metzler 1967) polyphenols (ie catechin epigallocatechin rutin) (Bucker et
al 2005) potassium iodide (Baldursdottir et al 2003) purine derivatives (ie uric acid
xanthine hypoxanthine) (Cardoso et al 2005) vitamin B6 (Natera et al 2012)
tocopherols (Cardoso et al 2007) xanthone derivatives (Hiraku et al 2007) 14ndash
diazabicylol [222] octane 25ndashdimethylfuran (Bradley et al 2006) ascorbic acid and
sodium azide In RF solution ascorbic acid quenches both the singlet oxygen and the
excited triplet states of RF whereas sodium azide only quenches singlet oxygen (Huang
et al 2004)
67
3476 Effect of solvent
Solvent polarity affects the rate of photolysis of RF due to conformational
changes in ribityl sidendashchain of RF in organic solvents (Moore and Ireton 1977) RF is
more stable in less polar solvents (Koziol 1966a) while in alcohol and alcoholndashwater
mixtures exposed to light it is degraded to FMF and LC (Moore and Ireton 1977) LC
has been found to be the major photoproduct of RF in organic solvents (ie acetic acid
acetone dioxane ethanol pyridine) (Koziol 1966ab Koziol and Knobloch 1965) The
rate of photodegradation of RF in greater in organic solvent as compared to aqueous
solution (Koziol 1966a Koziol and Knobloch 1965) This may be due to the effect of
physical properties of the solvents (ie viscosity polarity etc) (Ahmad et al 2006
2013a Ahmad and Fasiullah 1990 1991 Moore and Ireton 1977)
The photodegradation of RF is also influenced by the quality of water (ie D2O
distilled water) The rate of photodegradation is higher in D2O (66) as compared to that
of the distilled water (40) (Huang et al 2004) UVndashvisible spectrometric methods have
been used to study the effect of aqueous and organic solvent on the photolysis of FMF
(Ahmad et al 1990 1991 2006 2013a) It has been found that the photolysis of FMF
does not follow firstndashorder kinetics in organic solvents and water The rate of photolysis
of FMF is dependent on the dielectric constant and increases with an increase in the
dielectric constant of the solvent (Ahmad et al 2013a)
A study has recently been made on the photolysis of RF in water (pH 70) and in
organic solvents (ie acetonitrile methanol ethanol 1ndashpropanol 1ndashbutanol ethyl
acetate) using a multicomponent spectrometric method The rate of photolysis of RF is a
68
linear function of solvent dielectric constant due to the participation of a dipolar
intermediate in the reaction pathway (Ahmad and Tollin 1981a) The rate of photolysis
also shows that with an increase in electron acceptor (EA) number the rate of photolysis
is increased This shows the degree of solutendashsolvent interaction in the reaction (Ahmad
et al 2015a)
3477 Effect of ionic strength
The effect of ionic strength (01ndash05 M) on the photodegradation reactions
(photoreduction and photoaddition) of RF in phosphate buffer (pH 70) has been studied
The results show that with an increase in the ionic strength the rate of photolysis of RF is
also increased The effect of phosphate buffer concentrations (01ndash05 M) on the
phororeduction and photoaddition pathways of RF has also been evaluated An increase
in buffer concentration leads to an increase in the photodegradation of RF by both
pathways In the presence of NaCl the excited singlet state of RF forms an exciplex with
NaCl which leads to the formation of photoproducts at a faster rate (Ahmad et al 2016a)
3488 Effect of formulation
There are various formulation characteristics such as source (ie synthetic
biosynthetic natural) irradiation (ie occasional continuous) tablet processing (ie
direct compression wet granulation) that affect the photochemical reactions The change
in colour in synthetic powder samples on irradiation was found gradual while in
biosynthetic samples the change was instant at a radiation of greater than 450 kJm2
(SuendashChu et al 2009) In solid dosage forms RF colour change is due to the phenomena
69
of photochromism This change in colour is only on the surface and does not affect RF
quantitatively (SuendashChu et al 2008 2009)
The photostability of RF could be improved by encapsulating it in liposomes The
stability of RF in liposomal preparations depends on the composition of liposomes pH of
the preparation and concentration of ingredients (Habib and Asker 1991 Chauhan and
Awasthi 1995 SenndashVarma et al 1995 Arien and Dopuy 1997 Loukas 1997 Ionita
and Ion 2003 Bhowmik and Sil 2004 Ahmad et al 2015b) Dimyristoylndash
phosphatidylcholine (DPC) concentration affects the photostability of RF An increase in
DPC concentration leads to an increase in the photostability of RF (Habib and Asker
1991 Loukas 2001)
CHAPTER IV
INTRODUCTION TO NANOPARTICLES AND
APPLICATIONS TO RIBOFLAVIN
71
41 INTODUCTION
The word nano is derived from a Greek word dwarf and nanometer is
onendashbillionth of a meter (10ndash9
m) The word nanotechnology (NT) was first used by Norio
Taniguchi in Japan in 1974 (Royal Society 2004) Eric Drexler (1986 1992) who is
known to be a God Father of NT defined NT as a molecular nanotechnologyprocess
which deals with the transfer of molecules and atoms to the nanoscale products NT is a
vast term and it deals with more than one disciplines based on the scientific and
technological principles for the design preparation and characterization of nanomaterials
(NMs) (Farokhzad and Langer 2009 Ferrari 2005 Fox 2000 Jiang et al 2007
BrannonndashPeppas and Blanchette 2004 Sinha et al 2006 Uchegbu 2006) It is also
defined as the activity which is aimed to understand the natural laws on the level of
nanoscale (Balzani 2005) NT is referred as science technology and engineering for the
preparation of NMs on the scale of 1ndash100 nm (Alexis et al 2010) In NT NMs are
defined as any small material or object which itself behaves as a simple single unit for
transportation and exhibiting its properties These NMs cover the range of 100ndash2500 nm
Ultrafine particles are in the size range of 1ndash100 nm and their physical and chemical
properties depend on the nature of material through which they are prepared NPs are the
engineered structures with a diameter of less than 100 nm and are prepared by the
physical and chemical process with many definite properties (Gwinn and Vallyathan
2006) Different organizations have defined NPs which is given in Table 41
72
Table 41 Definition of Nanoparticles (NPs) and Nanomaterials (NMs) according to
different Organizations (Horikoshi and Serpone 2013)
Organization NPs NMs
International Organization
for Standardization (ISO)
1ndash100 nm ndash
American Society of
Testing and Materials
(ASTM)
Ultrafine particle whose
length in 2 or 3 places is
1ndash100 nm
ndash
National Institute of
Occupational Safety and
Health (NIOSH)
Particle diameter in the range
of 1ndash100 nm or fiber
spanning range in 1ndash100 nm
ndash
Scientific Committee on
Consumer Products (SCCP)
At least one dimension in
nanoscale
Internal structure or one
side in nanoscale range
British Standards Institution
(BSI)
All the dimensions are in the
nanoscale range
Internal structure or one
side in nanoscale range
Bundesanstalt fuumlr
Arbeitsschutz und
Arbeitsmedizin (BAuA)
All the dimensions are in the
nanoscale range
Material consisting of a
nanostructure or a
nanosubstance
73
There are certain limitations which have been applied to NT as the utilization of
materials with structural orientation between the atom and at the molecular scale but at
least the dimensions must be in the nanoscale range (Rao and Cheetham 2001 Rao et al
2002 Jortner and Rao 2002) NPs are gaining importance in modern science and
technology due to the ability of a scientist to manipulate their properties according to
onersquos requirements
42 RIBOFLAVIN AND NANOTECHNOLOGY
Riboflavin (RF) has been used as a photosensitizer stabilizer of nanoparticles
(NPs) biosensor and for other purposes in nanotechnology These aspects are described
in the following sections
421 Photosenstizer
A study has been made for the photosensitization of colloidal ZnO NPs with RF
and the determination was carried out by absorption fluorescence and time resolved
fluorescence spectrometry RF is strongly adsorbed on the ZnO NPs surface and the
association constants have been obtained by fluorescence quenching The Rehem Weller
equation has been used for the calculation of free energy change (∆Get) for the electron
transfer reaction (Vaishnavi and Renganathan 2012)
RF acts as a photosensitizer in the photooxidation of impurities present in water
courses lakes and seas It is known that RF interacts with aromatics sorbed on silica
sediments or on suspended silica particles In a study the characterization and
modification of silica NPs has been carried out by the condensation of silanol groups of
74
the particles with Endashcinnamic alcohol This reaction has been confirmed by FTIR solid
state 13
C and 29
Si cross polarization magic angle spinning (CPMAS) NMR and also by
the reduction of specific surface area measured by BET thermal analysis and
fluorescence spectrometry It has been found that RF fluorescence is quenched in the
presence of Endashcinnamic alcohol in aqueous media or in suspensions The quenching may
be due to the formation of 11 complexes between ground state of RF and free or
adsorbed cinnamic alcohol This complex formation has been confirmed by density
functional theory (DFT) calculations in aqueous medium and also by RF fluorescence
quenching on the addition of cinnamic alcohol (Arce et al 2014)
422 Stabilizer
Gold (Au) NPs are stabilized by using RF against trisndashbufferndashinduced
aggregation In the presence of Hg2+
ions RF could be released from AundashNPs surface
resulting in the formation of a RFndashHg2+
complex and leading to the aggregation of Aundash
NPs in trisndashbuffer This aggregation depends upon the concentration of Hg2+
ions This
method helps in the detection of Hg2+
ions in the concentration range of 002ndash08 microM
with the detection limit of 14 nM It indicates that Hg2+
ions shows good selectivity over
other metal ions (Cu2+
Co2+
Cd2+
Pb2+
Mg2+
Zn2+
Ag+
Ce3+
Al3+
K+) (Xu et al
2012)
423 Photoluminescence
A study has been carried out on the interaction of luminescent water soluble ZnS
NPs with flavin RF quenched ~60 of the photoluminescence of ZnS NPs but FMN
and FAD showed different quenching pattern of photoluminescence under these
75
conditions It has been found that there is no effect on luminescence intensity of ZnS NPs
when flavin are bonded with proteins such as glucose oxidase (scavenging of
photogenertaed electron of ZnS NPs by the flavin molecules may be attributed) to the
decrease in luminescence intensity The quenching of ZnS NPs with flavin shows a linear
SternndashVolmer plot and SternndashVolmer constants are decreased in the order of Ksndashv(RF) gt
Ksndashv(FAD) gt Ksndashv(FMN) This study gives a beneficial protocol for the fluorimetric
determination of RF content in biological systems (Chatterjee et al 2012)
The grapheme oxide (GO)ndashRF hybrids have been decorated by AgndashNPs with
different compositions Scanning electron microscopy of GOndashRndashAg shows a helical
fibrillar morphology that is different from the bar and wrinkled sheet of R and GO
respectively The FTndashIR spectra show that GO gives a supra molecular complex with R
and AgndashNPs that are stabilized by R and GO The UVndashvis spectra of these complexes
show a larger shift of surface Plasmon band from 390 to 570 nm The spectra of cellular
dichorism show a sudden change in the GOndashRndashAg system as compared to the GOndashR
system for a weight ratio of GO to R of 13 This suggests that AgndashNPs are enveloped in
GOndashR hybrid and R moieties The photoluminescence intensity of R is increased in the
GOndashR hybrids as compared to that of GOndashRndashAg ones The dcndashconductivity is increased
for GOndashR hybrids by the magnitude of addition of AgndashNPs Characteristics curves for
GOndashRndashAg (GOR) show negative differential resistance due to charge trapping on the
silver of NPs followed by stabilization by R (Routh et al 2012)
76
424 Biosensor
A study has been carried out for the fabrication and testing of RF as a biosensor
It is based on the use of Cr doped SnO2 NPs The CrndashSnO2 NPs are prepared by the
microwave irradiation method using different chromium concentrations (0ndash5 ww) In
this study the magnetic studies have also been carried out which show that only 3 wv
Crndashdoped nanondashSnO2 particles have ferromagnetic properties at room temperature It has
also been found that CrndashSnO2 NPs modified electrode response to RF is linear in the
concentration range of 02 times 10ndash6
to 10times 10ndash4
M with a limit of detection of 107 nM This
fabricated sensor shows good antindashinterference ability against electroactive species and
metal ions Hence it has proved to be beneficial for the determination of RF in
pharmaceutical samples (Lavanya et al 2013) The in vitro detection of RF has been
carried out by a RF binding aptamer (RBA) in combination with gold NPs (AuNPs)
These RBAndashAuNPs conjugates respond colorimetrically in the presence of RF This
method has been used as a model study to check the modification of aptamer sequence
effect on the RBAndashAuNPs stability and their response to the specific target The length of
the aptamer affects RBAndashAuNPs stability as observed by dynamic light scattering and
UVndashaggregation kinetic studies (Chavez et al 2008)
A simple and sensitive electrode has been prepared which is based on nickel
oxide NPsRFndashmodified glass carbon (NiONPsRFG) for the determination of hydrogen
peroxide This electrode is immersed in the RF solution for 5 to 300 seconds and the
projected molecules are immobilized on the surface of the electrode as a thin film This
electrode shows well defined redox couples in the pH range of 2 to 10 having surface
confined properties The results obtained from this electrode show that RF is adsorbed on
77
the surface of NiO NPs The surface coverage and hetergenous electron transfer rate
constants (ks) of RF immobilized on NiOndashGC electrode are 483 times 10ndash11
molcm2 and
54s respectively This sensor has a powerful electrocatalytic activity for H2O2 reduction
The sensitivity catalytic rate constant (kcat) and limit of detection of this electrode for the
reduction of H2O2 are 24 nA microM 73 (plusmn02) times 10ndash3
Mndash1
sndash1
and 87 nM respectively and
found to be linear in the concentration range up to 30 mM (Roushani et al 2013)
The composite film of Au fine particles and RF are used for the circular dichorism
(CD) studies in the visible region It has been found that the chiral molecules bound on
the surface of Au particles are not essential for Plasmonndashinduced CD and composite
films that contain a dye and glucose in place of Au particles and RF induced signal of CD
at wavelengths of their absorption maixma The polarity of CD is altered by using
different enantiomer of glucose (Kosaka et al 2012)
A simple novel sensitive and selective aptasensor has been developed for the
detection of cocaine an addictive drug by using an electrochemical transduction method
This sensor has been constructed by the covalent immobilization of Ag NPs (aptasensor
functionalized) on a nanocomposite (MWCNTsILChit) for the sensing interface that
improves the performance characteristics and conductivity of the aptasensor and
increases the loaded amount of the aptamer DNA sequence RF for the first time has been
used as a redox probe for the development of an aptasensor to detect cocaine In this
study it has been found that Ag NP leads to speed up the electron transfer kinetics that is
related to the reduction of RF The differential pulse voltammteric (DPV) signal of RF is
decreased with the increased concentration of cocaine in the range of 2 nMndash2 5 microM with
a limit of detection of 150 pM (Roushani and Shahdostndashfard 2015)
78
Membranes of nafionndashRF have been constructed and characterized by scanning
electron microscopy transmission electron microscopy UVndashvisible spectroscopy and
cyclic voltametry The average diameters of prepared NPs are 60 nm and these
membranes exhibit quasindashreversible electrochemical behavior with a potential of ndash562 plusmn1
mV by using a gold electrode By studying electrochemical parameters of this system it
has been found that the system has good and stable electron transfer properties In this
study horsereddish peroxide (HRP) has been immobilized on the RFndashnafion membrane
and electrochemical behavior of HRP has been found to be quasindashreversible with a
potential of 80 plusmn5 mV This film shows good catalytic activity via the reduction of H2O2
(RezaeindashZarchi et al 2008)
The NPs of ferric oxide (Fe3O4) and binary mixture of Fe3O4 via an ionic liquid
1ndashhexylndash3ndashmethylimidazolium bromide (ILndashFe3O4) have been prepared and used for the
adsorption of ascorbic acid (AA) folic acid (FA) and RF The morphology and size of
NPs have been studied by transmission electron microscopy Xndashray diffraction
thermogravimetric analysis and FTIR spectroscopy The immersion technique is used for
the determination of pH of the point of zero charge (pHpze) for both NPs This
determination is based on experimental curves and results obtained are under the
operational condition (40 mg of NPs contact time 10 mins initial concentration of
vitamins 20 mgL) The thermogravimetric analysis shows that Freundlich model lies on
the equilibrium data as compared to that of DubininndashRadushkevich model The
adsorption capacities of RF FA and AA are 48 225 and 69 mgg respectively of
adsorbent These capacities are dependent upon the pH of the solution chemical structure
of the adsorbent and temperature The pseudondashfirst order and pseudondashsecond order
79
kinetic models have been predicted by the comparative analysis of rate parameters
correlation coefficient and equilibrium adsorption capacity It has also been found that
the adsorption of FA and AA is endothermic and could be desorbed from ILndashFe3O4 NPs
at pH 30 by using NaCl for the recyclization of NPs (Kamran et al 2014)
The free radical polymerization of Nndashisopropylacrylamide is used for the
preparation of hybrid hydrogels of RF and poly(Nndashisopropylndashacrylamide) (PNIPAAM)
N Nˊndashmethylene bisacrylamide is used as a cross linker for RF in the concentration
range of 1ndash3 mM It has been found that the invariance of storage (Gˊ) and loss (Gˊˊ)
moduli at a wide range of angular frequency and Gˊ gt Gˊˊ for RFndashPNIPAAM systems
behave like a gel in a hybrid state The Gˊ and Gˊˊ are decreased with an increase in RF
concentration but this decrease is four times higher in case of Gˊ than that of Gˊˊ As
compared to PNIPAAM gels RFndashPNIPAAM gels have higher critical strain value that
increase with an increases in RF concentration This indicated that RF acts as a
supramolecular crossndashlinker and the intensity of RndashPNIPAAM gels increases with an
increase in RF concentration This variation with temperature and different pH shows a
higher intensity with temperature The maximum intensity is at ~ 30 oC which is due to
coilndash tondashglobule transition of PNIPAAM gels and could be used for temperature
detection as a probe (Chakraborty et al 2014)
425 Target Drug Delivery
In the malignant cells of human breast and prostate cancers the RF receptors are
overexpressed and these cells contain potential surface markers that are important for
targeted delivery of drugs and for the imaging of molecules In a study the fabrication
80
and characterization of core shell NCs having gold NPs (Au NPs) and coating of RF
receptor poly (amido amine) dendrimer has been carried The aim of this study was to
design NCs as a cancer targeted imaging material which is based on its surface Plasmon
resonance of Au NPs Atomic force microscopy (AFM) is utilized as a technique for
probing the binding interaction between NCs and RF binding protein (RFBP) in solution
The AFM technique also enables the precise measurement of the height of Au NPs before
and after chemisorptions of RF conjugated dendrimer as 135 and 205 nm respectively
This binding of RFndashBP to the Au NPs dendrimer results in the increase of height (267
nm) which then decreases 228 nm after coincubted with RF as a competitive ligand for
supporting interaction of Au NPs dendrimer and its target protein (Witte et al 2014)
The RF behavior adsorbed on Ag NPs and its interaction with serum albumins
(BSA HSA) has been studied The plasmonic features of the formed complexes by
RFBSAHAS and Ag NPs with an average diameter of 100 (plusmn 20 nm) have been
studied by UVndashvis absorption spectrometry The stability structure and dynamics of
serum albumins have been studied by using steadyndashstate and time resolved fluorescence
spectrometry The effectiveness of energy transfer reaction mechanisms between Ag NPs
and RF has been predicted and the mechanism of the reaction has also been proposed It
is illustrated by the participation of Ag NPs by the redox process of RF and RFndashserum
albumin interaction in Ag NPs complexes (Voicescu et al 2013)
426 Photochemical Interaction
The interaction and formation of a complex between RF and Ag NPs has been
studied by fluorescence spectrometry UVndashvis spectrometry and TEM AgNO3 and
81
trisodium citrate (TSC) have been used for the preparation of Ag NPs by the process of
chemical reduction By this method NPs of the size of 20 nm have been obtained with a
surface Plasmon resonance band at 426 nm The absorption maxima of RF (264 374 444
nm) shift significantly in the presence of Ag NPs due to the chemical interaction of Ag
NPs and RF The fluorescence of RF solutions is quenched by the addition of Ag NPs
and that may be due to the rapid adsorption of RF on AgNPs (Mokashi et al 2014)
The evaluation of the optical behavior of RF in aqueous solution in the presence
of Ag NPs has been made This Ag NPs were prepared by the oxidation and reduction
method and found that absorption intensity of RF was found to be enhanced It has been
found that when Ag NPs are added to an aqueous solution of RF the 372 and 444 nm
peaks are red and blue shifted respectively The fluorescence studies show that as the Ag
NPs concentration is increased the fluorescence intensity of RF solution is quenched
(Zhang et al 2011)
The NPs of copper have been prepared by the photoirradiation of doped solndashgel
silica by mixing Cu2+
ions ethylenediamine tetraacetic acid (EDTA) and RF into the solndash
gel solution of tetramethoxysilane (TMS) The absorption maxima of RF and Cu2+
ndash
EDTA is found to be at 442 nm and Cu2+
ndashEDTA at 740 nm respectively When the
photoirradaition is carried out the solndashgel silica develop reddish brown colour with an
absorption band around 580 nm because of Plasmon band CundashNPs Copper NPs are also
formed by solndashgel silica doped with lumichrome (LC) and lumiflavin (LF) The
photostability of the flavin dyes have been found to be in the order of LC gt LF gt RF in
solndashgel silica with Cu2+
ions The fluorescence intensities of LC LF and RF are reduced
82
by the photoirradiation of the solndashgel silica doped with Cu2+
ions without flavin dyes
(Noguchi et al 2011)
A study has been carried out on RFndashconjugation with ZnO NPs and their potential
application in jaundice The conjugation between RF and ZnO NPs has been confirmed
by UVndashvis spectrometry and photolumisence (PL) intensity In the RFndashconjugated NPs
the crystallinity and functional groups have been confirmed by Xndashray diffraction (XRD)
analysis and FTIR spectroscopy respectively Fieldndashemission scanning electron
microscopy (FESEM) and highndashresolution transmission electron microscopy (HRTEM)
have been used for the determination of the diameter of conjugated RFndashZnO NPs The
NPs shows significant ameliorative efficiency against the stress of jaundice at cellular
and molecular level in mice (Bala et al 2016)
427 Colorimetric Sensor
A study has been carried out to prepare Ag NPs using βndashcyclodextrin (βndashCD)ndash
grafted citrate as a stabilizer and reducer These NPs have been characterized by UVndashvis
spectrometry Xndashray diffraction and transmission electron microscopy (TEM) It has been
found that in the presence of RF the aggregation of Ag NPs occurs to a greater extent as
evident by the colour change (yellow to red) The formation of inclusion complexes
between RF and βndashCDndashgrafted citrate have been confirmed by 1H NMR spectroscopy
The interaction between βndashCD and RF is due to hydrogen bonding Ag NPs have been
used to develop a colorimetric sensor for the detection of RF This colorimetric
sensorprobe shows good response (selectivity and sensitivity) with 167 nM detection
limit for RF (Ma et al 2016)
83
OBJECT OF PRESENT INVESTIGATION
Vitamins are essential micronutrients required for the normal human growth
development and maintenance They are part of the enzyme systems and are involved in
the transformation of energy and for the regulation of metabolism A lack of the vitamins
results in clinical manifestations known as deficiency diseases In view of their
pharmaceutical importance it is necessary to ensure their stability in vitamin
formulations Riboflavin (RF) a component of vitamin B-complex is a photosensitive
compound and may degrade in vitamin formulations to give inactive products Several
studies have been carried out to investigate the photodegradation of RF and the effect of
factors enhancing or inhibiting these reactions These factors include pH solvent light
intensity buffers ionic strength metal ions etc Extensive work has been carried out on
the effects of pH light intensity and buffers on the photodegradation of RF However
some aspects still need to be investigated to understand the photochemical behavior of
RF under different conditions The object of present investigation is to conduct studies on
aspects such as the effect of solvent characteristics (ie dielectric constant and viscosity)
ionic strength and metal ions on the photodegradation of RF So far no quantitative and
kinetic studies have been carried out on these aspects and this work would facilitate the
formulation chemist in the development of better and more stable vitamin formulations
for the benefit of the users Moreover this work would provide a better insight into the
mechanism of RF photodegradation in aqueous and organic media In addition to this an
attempt would also be made to prepare RF nanoparticles and to study their spectrometric
fluorimetric and kinetic behavior under different experimental conditions
84
PROPOSED PLAN OF WORK
A brief outline of the proposed plan of work on various aspects of the photolysis
of riboflavin (RF) is presented as follows
1 Selection of appropriate radiation vessel and the radiation source for the
photolysis of RF in aqueous and organic solvents
2 Photolysis of RF in aqueous and organic solvents and identification of the
photoproducts in different media
3 Assay of RF and photoproducts by a suitable stability-indicating assay method
such as multicomponent spectrometric method or a HPLC method
4 Photolysis of RF in aqueous solution at different ionic strength of buffer species
at specific pH values
5 Photolysis of RF in aqueous solution at specified pH values in the presence of
different metal ions (eg Fe3+
Fe2+
Cu2+
Zn2+
Cr2+
Ag+ etc)
6 Evaluation of the kinetics of photolysis reactions as mentioned under No 24 5
7 Development of correlations between rate constants and dielectric
constantviscosityionic strengthmetal ion concentration
8 Determination of rate constants for the interaction of RF and metal ions at specific
pH values and proposed mechanism of interaction
9 Study of the photochemical formation and characterization of RF conjugated
silver (Ag) nanoparticles (NPs)
10 Evaluation of the effect of pH irradiation wavelengths (UV and visible light) and
concentration of Ag+ ions on the formation kinetics of RFndashAg NPs
CHAPTER V
MATERIALS AND METHODS
86
51 MATERIALS
Riboflavin 78-Dimethyl-10-[(2S3S4R)-2345-tetrahydroxypentyl]benzo[g]pteridine-
24-dione Merck
C17H20N4O6 Mr 3764
It was found to be chromatographically pure Rf 037 (1ndashbutanolndashacetic acidndash
water 415 vv organic phase silica gel G) [lit (Treadwell et al 1968) Rf 036] and
was stored in the dark in a refrigerator
Lumiflavin (7810ndashTrimethylisoalloxazine) Sigma
C13H12N4O2 Mr 2563
Lumiflavin was stored in a light resistant container in the dessicator below 0 degC
Lumichrome (78ndashDimethylalloxazine) Sigma
C12H10N4O2 Mr 2423
It was stored in the dark in a refrigerator
Formylmethylflavin (7 8ndashDimethylndash10ndashformylmethylisoalloxazine)
C14H12N4O3 Mr 2843
Formylmethylflavin was synthesized according to the method of Fall and Petering
(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute
methanol dried in vacuum and stored in the dark in a refrigerator
87
Carboxymethylflavin (78ndashdimethylflavinndash10ndashacetic acid)
C14H12N4O4 Mr 3003
It was prepared by the method of Fukumachi and Sakurai (1954) by aerobic
photolysis of riboflavin in alkaline solution in the presence of 30 H2O2 The material
was purified by column chromatography with Whatman CC31 cellulose powder using 1ndash
butanolndash1ndashpropanolndashacetic acidndashwater (5030218 vv) as the solvent system (Ahmad et
al 1980)
It was stored in the dark in a refrigerator
Cyclodehydroriboflavin
C17H18O6N4 Mr 3744
Cyclodehydroriboflavin was prepared by the method of Schuman Jorns et al
(1975) via aerobic photolysis of riboflavin in phosphate buffer (20 M) and recrystallized
by acetic acid (20 M)
It was stored in the dark in a refrigerator
Method of Preparation of Nanoparticles
RFndashconjugated Ag NPs were prepared by the photoreduction method A 001mM
AgNO3 solution was prepared in 50 ml in a screw capped transparent glass bottle to
which 50 ml of 0002 mM of RF solution was added To this solution 3 to 5 drops of
NaOH (18 mM) were added (pH 80ndash105) and it was placed in a thermostat bath
maintained at 25 plusmn 1oC the solution was irradiated with a Philips HPLN 125 W high
88
pressure mercury vapor fluorescent lamp (emission at 405 and 435 nm the later band
overlapping the visible absorption maximum of RF at 444 nm (British Pharmacopoeia
2016)) horizontally fixed at a distance of 25 cm from the center of the bottle The
solution was also irradiated with a Philips TUV 30 W UV tube vertically fixed at a
distance of 25 cm from the center of the bottle Samples were withdrawn at various
intervals for absorbance measurements The solutions were irradiated till there was no
change in absorbance at the maximum (422 nm)
Metal Salts
The various metal salts used in this study were obtained from Merck and are as
follows
AgNO3 (999) FeSO47H2O (999) MgSO4H2O (995) CaSO42H2O
(999) Fe2(SO4)3H2O (970) CuSO45H2O (999) NiCl26H2O (980)
ZnSO47H2O (990) PbSO4 (980) CdSO4H2O (999) MnSO4H2O (999)
CoSO47H2O (999)
52 REAGENTS
All reagents and solvents (1ndashbutanol 997 acetonitrile 998 ethanol 998
ethyl acetate 995 methanol 999) were of analytical grade obtained from
BDHMerck The following buffer systems were used KCl + HCl pH 20 CH3COONandash
CH3COOH pH 45 and KH2PO4ndashNa2HPO4 pH 70 The ionic strength was kept constant
in each case unless otherwise stated
89
Water
Freshly boiled glassndashdistilled water was used throughout the work
53 METHODS
In photochemical studies care was taken to protect the solutions from light during
the experimental work The photolysis chromatography and assay procedures of
riboflavin were carried out in a dark chamber provided with a safe light All the solutions
of riboflavin were freshly prepared for each experiment to avoid any photochemical
change
531 ThinndashLayer Chromatography (TLC)
The details of TLC systems including the adsorbents and solvents used for the
separation and identification of riboflavin and its photoproducts are as follows
Adsorbent a) Silica gel GF 254 precoated plates (Merck)
b) Whatman Mirogranular CC41 cellulose
(Merck)
Layer thickness 250ndashmicrom
Solvent systems Z1 1ndashbutanolndashacetic acidndashwater (415 vv
organic phase) silica gel G (Treadwell et al
1968)
Z2 1ndashbutanolndashacetic acidndashwater (415 vv
organicphase) cellulose powder (Ahmad et
al 1980)
90
Z3 1ndashbutanolndash1ndashpropanolndashacetic acidndashwater
(5030218 vv) cellulose powder
(Ahmad et al1980)
Z4 Chloroform-Methanol (92 vv) cellulose
powder (Schuman Jorns et al 1975)
Temperature 25ndash27 degC
Location of spots UV light 254 and 365 nm (UVtech lamp UK)
532 pH Measurements
The pH measurements of the solutions were carried out with an Elmetron LCD
display pH meter (modelndashCP501 sensitivity plusmn 001 pH units Poland) using a
combination electrode The calibration of the electrodes was automatic in the pH range
10ndash140 (25 degC) using the following buffer solutions
Phthalate pH 4008 phosphate pH 6865 disodium tetraborate pH 9180
533 Fourier Transform Infrared (FTIR) Spectrometry
The purity and identity of riboflavin used in this study was confirmed by FTIR
spectrometry using a Nicolet iS5 FTIR spectrometer (Thermo Fisher Scientific USA)
The IR spectrum was collected in the range of 4000ndash600 cmndash1
at a resolution of 4 cmndash1
using OMNIC software (version 90) and is shown in Fig 51
FTIR analysis of RF-conjugated silver nanoparticles was carried out by using a
Nicolet iS5 FTIR spectrometer (Thermofischer Scientific USA) in the range of 4000
cm-1
to 400 cm-1
The sample was centrifuged at 15000 rpm (60 min) and the supernatant
91
Fig 51 FTIR spectrum of riboflavin
Wavelength (cmndash1
)
Ab
sorb
an
ce
92
was discarded while the residue was dried for analysis The dried sample was used for the
measurement of the spectrum in transmission mode as a function of wavenumber (cm-1
)
OMNIC 90 software was used to process data
534 Ultraviolet and Visible Spectrometry
The absorbance measurements and spectral determinations on pure and
photolysed solution of riboflavin were carried out on a Thermoscientific UVndashVis
spectrophotometer (Evolution 201 USA) using matched silica cell of 10 mm path length
The cells containing the solutions were always employed in the same orientation using
appropriate control solutions in the reference beam The baseline was automatically
corrected by the builtndashin baseline memory at the initializing period Autondashzero
adjustment was made by onendashtouch operation The wavelength calibration was carried
out automatically by the instrument The absorbance scale was periodically checked
using the following calibration standards
Absorbance scale 0050 g l of K2Cr2O7 in 005 M H2SO4
Absorbance at 257 nm = 0725 350 nm = 0539 plusmn 0005 (Rand
1969)
Riboflavin solution pH 40 (acetate buffer)
A (1 1 cm) at 444 nm = 328
(British Pharmacopoeia 2016)
535 Fluorescence Spectroscopy
Fluorescence measurements were carried out by using Spectromax 5 flourimeter
(Molecular Devices USA) and Jasco Spectrofluorimeter (FPndash8500 Japan) with a Xenon
arc lamp
93
The measurements were carried out by using a 10 mm quartz cell and the
excitation and emission wavelengths were adjusted to 374 and 520 nm respectively
(United State Pharmacopoeia 2016) The fluorescence intensity was recorded in relative
fluorescence units using a pure 005 mM RF solution (pH 70) as a standard
536 Dynamic Light Scattering (DLS)
DLS measurements were carried out by Laser Spectroscatter-201 system (RiNA
GmbH Berlin Germany) having a He-Ne laser source providing 690 nm light source with
an output power range of 10-50 mW The measurements were performed by an
autopiloted run of 50 measurements in 20s at room temperature (25 oC) The RF
conjugated Ag NPs as such or filtered through a 022 microm filter (Millipore USA) were
placed in a SUPRASIL reg cell (15 mm light path) for measurements (Hameed et al
2014) at a fix scattering angle of 90o and the scattered light was collected
Autocorrelation functions were performed using a program CONTIN to measure the
hydrodynamic radius (RH) distribution The Einstein-Stokes equation was used to relate
RH to the diffusion coefficient The PMgr v301p17 software was used for the analysis of
data
537 Atomic Force Microscopy (AFM)
The sample was prepared by pouring 10 microl of the desired solution on freshly
cleaned mica for 2-3 min which was then rinsed with Milli-Q water and dried with
nitrogen (Shah et al 2014) Agilent 5500 AFMSFM microscope was used to obtain
images immediately operating the instrument in tapping mode using soft silicon probes
(NCL nominal length = 225 microm mean width-38 microm and nominal resonance frequency =
94
190 KHz nominal force constant = 48 Nm) The images of the RF-conjugated silver
nanoparticle solutions were measured at random spot surface sampling
538 Photolysis of Riboflavin solutions
5381 Choice of reaction vessel
In the photochemical work a reaction vessel is to be chosen on the basis of the
absorption characteristics of the reactants and the transmission characteristics of the
reaction vessel The aqueous solutions of riboflavin absorbs at 223 267 373 and 444 nm
in the UV and visible region (British Pharmacopeia 2016) therefore a pyrex vessel can
be used for absorption above 300 nm region Pyrex vessels have previously been used for
the photolysis of riboflavin (Ahmad et al 2004a 2004b 2005 2006 2008 2009 2010)
5382 Choice of radiation source
Riboflavin exhibits a strong peak at 444 nm in the visible region This necessities
a radiation source with strong emission in this region Philips HPLN highndashpressure
mercury vapour fluorescent lamp strongly emits at 405 and 436 nm The 436 nm
wavelength is close to the major absorption maximum of riboflavin (444 nm) This
radiation source has previously been used by Ahmad et al (2004a 2004b 2005 2006
2008 2009 2010) for the photolysis of riboflavin The spectral power distribution of the
fluorescent lamp is shown in Fig 52
95
Fig 52 Spectral emission of HPLN lamp
96
539 Methods of Photolysis of Riboflavin
5391 Photolysis in aqueous and organic solvents
A 3ndash5 times 10minus5
M solution of RF (100 ml) was prepared in water (pH 70 0001 M
phosphate buffer) or in organic solvents in volumetric flasks (Pyrex) and immersed in a
water bath maintained at 25plusmn1degC The solution was exposed to a Philips HPLN 125 W
highndashpressure mercury lamp (emission bands at 405 and 435 nm the later band overlaps
the 444 nm band of RF (British Pharmacopoeia 2016)) fixed at a distance of 25 cm from
the center of the flask for a period of 2ndash3 h depending upon the nature of the solvent
used Samples of photolyzed solution were withdrawn at various time intervals for
thinndashlayer chromatographic separation and spectrometric assay of RF and photoproducts
5392 Photolysis at various ionic strength
A 10minus4
M aqueous solution of RF (100 ml) at pH 70 (01ndash05 M phosphate
buffer) with varying ionic strengths (01ndash05 M at each buffer concentration) was
prepared in a Pyrex flask and placed in a water bath maintained at 25 plusmn 1 degC and
proceeded further as stated above
5393 Photolysis in the presence of metal ions
A 5 times 10ndash5
M aqueous solutions of RF at pH 70 (0001ndash04 M phosphate buffer)
containing different metal ions at various concentrations (10ndash50 times 10ndash4
M) were
prepared in 100 mL Pyrex flasks and proceeded further as stated in section 5391
97
5310 Assay of RF and Photoproducts
RF and its major photoproducts in degraded solutions (aqueous and organic
solvents and in the presence of metal ions) detected by TLC were assayed using a
specific multicomponent spectrophotometeric method previously developed by Ahmad
and Rapson (1990) and Ahmad et al (2004b) The methods are based on the prendash
adjustment of photolysed solutions to pH 20 (02M HClndashKCl buffer) chloroform
extraction (3 times 10 ml) to remove the photoproducts lumichrome (LC) and lumiflavin (LF)
and their determination after chloroform evaporation and dissolution of the residue at pH
45 (02 M acetate buffer) by a twondashcomponent assay at 445 nm and 356 nm The
aqueous phase was assayed for RF and formylmethylflavin (FMF) by a twondashcomponent
assay at 445 nm and 385 nm and for RF FMF and cyclodehydroflavin (CDRF) at 445
410 and 385 nm Using this method it is possible to determine the concentrations of RF
and its major photoproducts (FMF CDRF LC LF) in photolysed solutions
The analytical scheme for the assay of RF and its photoproducts (Ahmad and
Rapson 1990 Ahmad et al 2004a) is given in Scheme 51 The molar absorptivites of
RF and photoproducts used in this study are reported in Table 52
5311 Calculation of Molar Concentrations in the Spectrometric Assay of RF and
Photoproducts
The assay of RF FMF CDRF LC and LF was carried out by onendashcomponent
twondashcomponent or threendashcomponent spectrometeric methods using specific wavelengths
and molar absorptivities given in Table 52 The methods of calculation of molar
concentrations are described as follows
98
Scheme 51 Assay of riboflavin and photoproducts
The assay of RF and photoproducts in photodegraded solutions (pH 2ndash11)
containing nonndashdegraded RF and several products has been carried out by prendashadjusted
of the solution to pH 20 and extracted with chloroform The variations in the
composition of the photoproducts in different reactions are monitored by TLC
RF and Photoproducts
Aqueous phase Chloroform extract
RF FMF minor components LC (acid photolysis)
Twondashcomponent assay (RF FMF) at 445 and
385 nm
Single component assay at 356 nm
LF LC ( neutral and alkaline
photolysis)
Twondashcomponent assay at 445 and 356
nm
Threendashcomponent assay (RF FMF CDRF)
at 445 385 and 410 nm
LF LC ( neutral and alkaline
photolysis)
Twondashcomponent assay at 445 nm and
356 nm
Assumed not to interfere in the assay
99
Table 52 Molar Absorptivities (Mminus1
cmminus1
) of RF and Photoproducts
(Ahmad and Rapson 1990 Ahmad et al 2004b)a
Compound pH 356 nm 385 nm 410 nm 445 nm
Riboflavin 20 97 804 125
Formymethylflavin 20 164 114 47
Cyclodehydroriboflavin 20 86 118 391
Lumichrome 45 108 013
Lumiflavin 45 74 104
a The values of molar absorptivities of RF and photoproducts were confirmed by using
pure reference compounds
100
Onendashcomponent assay
When a compound follows Beer Law its absorbences at a particular wavelength
are additive and therefore on the choice of a suitable wavelength (eg absorption
maximum) it is possible to calculate the concentration of the compound by applying the
following equation
A1 = 1a1 1C (51)
where
A1 is the absorbance at wavelength λ
1a1 is the absorptivity at waelenght λ
1C is the concentration of component 1
Using the same absorption cell in the measurement
A1 = 1ε1 1C (52)
where
1ε1 is the molar absorptivityndashcell path product used in the calculations
53111 Twondashcomponent spectrometric assay (additive absorbances)
In a twondashcomponent assay absorbance measurements on the solutions are made
at two selected wavelengths and the concentrations are determined by solving two
simultaneous equations
A1 = 1ε1 1C + 2ε1 2C (53a)
A2 = 1ε2 1C + 2ε2 2C (53b)
where
A1 is the absorbance at wavelength λ1
101
A2 is the absorbance at wavelength λ2
1ε1 is absorptivityndashcell path product for component 1 at wavelength λ1
1ε2 is absorptivityndashcell path product for component 1 at wavelength λ2
2ε1 is absorptivityndashcell path product for component 2 at wavelength λ1
2ε2 is absorptivityndashcell path product for component 2 at wavelength λ2
1C is concentration of component 1
2C is concentration of component 2
Equations (53a) and (53b) are solved for 1C and 2C as follows
1C = (2ε2 middot A1 ndash 2ε1 middot A2)(1ε1 middot 2ε2 ndash 2 ε1 middot 1ε2) (54a)
2C = (1ε1 middot A2 ndash 1ε2 middot A1)(1ε1 middot 2ε2 ndash 2 ε1 middot 1ε2) (54b)
53112 Threendashcomponent spectrometric assay (additive absorbances)
A threendashcomponent assay involves the measurement of absorbances of solutions
at three selected wavelengths and the concentrations of individual components are
determined by solving three simultaneous equations using matrix methods The
measurements of A1 A2 A3 at λ1 λ2 λ3 are carried out for the determination of 1C 2C
and 3C
A1 = 1ε1 1C + 2ε1 2C + 3ε1 3C (55a)
A2 = 1ε2 1C + 2ε2 2C + 3ε2 3C (55b)
A3 = 1ε3 1C + 2ε3 2C + 3ε3 3C (55c)
102
Wavelength Absorbance Absorbance Sum
λ1 A1 1 ε 1 1C + 2 ε 1 2C + 3 ε 1 3C
λ2 A2 1 ε 2 1C + 2 ε 2 2C + 3 ε 2 3C
λ3 A3 1 ε 3 1C + 2 ε 3 2C + 3 ε 3 3C
(55d)
The matrix equation is as follows
A1 1ε1 2ε1 3ε1 1C
A2 = 1ε2 2ε2 3ε2 = 2C
A3 1ε3 2ε3 3ε3 3C
(AM) (ASM) (CM)
where
AM = Absorbance matrix
ASM = Absorbance sum matrix
CM = Concentration matrix
The solution of eq 55d for each concentration involves the replacement of the
particular column in the absorbance sum matrix in its determinant form and by dividing
the resultant by absorbance sum matrix (ASM) again in its determinant form
103
A1 2ε1 3ε1 1ε 1 2ε 1 3ε1
1C = A2 2ε2 3ε2 1ε2 2ε2 3ε2
A3 2ε3 3ε3 1ε3 2ε3 3ε3
1 ε 1 A1 3 ε 1
1ε 1 2ε 1 3ε1
2C = 1 ε 2 A2 3 ε 2 1ε2 2ε2 3ε2
1 ε 3 A3 3 ε 3 1ε3 2ε3 3ε3
1ε1 2ε1 A1
1ε 1 2 ε 1 3 ε 1
3C = 1ε2 2ε2 A2 1 ε 2 2 ε 2 3 ε 2
1ε3 2ε3 A3 1 ε 3 2 ε 3 3 ε 3
104
The above matrices are expanded to determine the concentration of the three components
using Laplacersquos method
1C =
A1 2ε2 3ε2
2ε3 3ε3
ndash 2 ε 1
A2 3ε 2
A3 3ε3
+ 3 ε 1
A2 2ε2
A3 2ε3
ASM expanded
A1(2ε 23ε3ndash3ε22ε3)ndash2ε1(A23ε3ndash3ε2A3)+3ε1(A22ε3ndash2ε2A3)
ASM expanded
1ε1(A23ε3ndash3ε2A3)ndashA1(1ε23ε3ndash3ε21ε3)+3ε1(1ε2A3ndashA21ε3)
ASM expanded
1ε1(2ε2A3ndashA22ε3)ndash2ε1(1ε2A3ndashA21ε3)+A1(1ε22ε3ndash2 ε 21ε3)
ASM expanded
1C =
2C =
3C =
CHAPTER VI
SOLVENT EFFECT ON THE PHOTOLYSIS OF
RIBOFLAVIN
106
61 INTRODUCTION
The influence of solvents on the rates of degradation of drugs is an important
consideration for the formulation chemist The effects of dielectric constant and viscosity
of the medium may be significant on the stability of pharmaceutical formulations
Theoretical basis of the effects of solvent on the rates and mechanism of chemical
reactions has been extensively dealt by many workers (Amis and Hinton 1973 Buncel et
al 2003 Connors et al 1986 Heitele 1993 Laidler 1987 Reichardt et al 1988
Sinko 2006 Yoshioka and Stella 2000) The effect of dielectric constant on the
degradation kinetics and stabilization of chloramphenicol (Marcus and Taraszka 1959)
barbiturates (Ikeda 1960) methanamine (Tada 1960) ampicillin (Hou and Poole 1969)
prostaglandin E2 (Roseman et al 1973) chlorambucil (Owen and Stewart 1979) 2ndash
tetrahydropyranyl benzoate (Hussain and Truelove 1979) indomethacin (Ghanem et al
1979) aspirin (Baker and Niazi 1983) phenoxybenzamine (Adams and Kostenbauder
1985) azathioprine (Singh and Gupta 1988) polypeptides (Brennan and Clarke 1993)
neostigmine (Yoshioka and Stella 2000) triprolidine (Mao et al 2000)
10ndashmethylisoalloxazine (Ahmad and Tollin 1981) formylmethylflavin (Ahmad et al
2006) levofloxacin (Ahmad et al 2013a) moxifloxacin (Ahmad et al 2014) and
norfloxacin (Ahmad et al 2015) has been reported The viscosity of the medium may
also affect the stability of a drug A linear relation has also been found between the rate
constant and the inverse of solvent viscosity for the photodegradation of 10ndash
methylisoalloxazine (Ahmad and Tollin 1981) formylmethylflavin (Ahmad et al
2013b) levofloxacin (Ahmad et al 2013a) moxifloxacin (Ahmad et al 2014) and
norfloxacin (Ahmad et al 2015) in organic solvents
107
Some kinetic studies of the photolysis of riboflavin (RF) in carboxylic acids
(Koziol 1966 Szezesma and Koziol 1977) alcoholic solvents (InsinskandashRak et al
2012 Moore and Ireton 1977 Schmidt 1982 Song and Metzler 1967) and pyridine
(Kurtin et al 1967) have been conducted However the method used for the
determination of RF is based on the measurement of absorbance at 445 nm without any
consideration of the interference caused by photoproducts formed during degradation
Thus the kinetic data obtained may not be accurate and specific methods may be required
for assay of RF in degraded solutions (Ahmad and Rapson 1990 Ahmad and Vaid
2006) Studies on the photolysis of formylmethylflavin (FMF) a major intermediate in
the photolysis sequence of the RF in organic solvents have been conducted (Ahmad et
al 2006a Ahmad et al 2013b) Solvent effects on flavin electron transfer reactions have
been found to be significant (Ahmad and Tollin 1981 Sheraz et al 2014a) The present
work involves a detailed study of the kinetics of photolysis of RF in a wide range of
organic solvents using a specific multicomponent spectrometric method for the assay of
RF and photoproducts (Ahmad and Rapson 1981 Ahmad and Vaid 2006 Sheraz et al
2014b) and to develop correlations between the kinetic data and solvent parameters such
as dielectric constant and viscosity These considerations are important in the formulation
of drugs with different polar character using condashsolvents and those whose oxidation is
viscosity dependent to achieve stabilization
The details of the experimental work involved in the study are given in section
53 (Chapter 5)
108
62 RESULT AND DISCUSSION
621 Photoproducts of RF
TLC of the photolysed solutions of RF in organic solvents on cellulose plates
using the solvent systems (Z1) and (Z3) showed the presence of FMF and LC as the main
photoproducts of this reaction CMF was also detected as a minor oxidation product of
FMF in these solvents (Ahmad et al 2006a 2013b) These products have been identified
by comparison of their fluorescence emission and Rf values with those of the authentic
compounds The formation of FMF and LC as the main photoproducts of RF in organic
solvents have previously been reported (Ahmad et al 2006a 2013b Koziol 1966) The
formation of LC in organic solvents may take place through FMF as an intermediate in
the photolysis of RF as observed in the case of aqueous solutions (Ahmad et al 2004
2006a 2013b Ahmad and Rapson 1990) The fluorescence intensity of the
photoproducts on TLC plates is an indication of the extent of their formation in a
particular solvent during the irradiation period In aqueous solutions (pH 70) LF is also
formed in addition to FMF and LC as previously reported (Ahmad et al 2004 Song and
Metzler 1967) The Rf values of RF and photoproducts are reported in Table 61
622 Spectral Characteristics
RF exhibits absorption maxima in organic solvents in the region of 440ndash450 nm
344ndash358 nm and 270ndash271 nm (Koziol 1966) A typical set of absorption spectra for the
photolysis of RF in methanol is shown in Fig 61
109
Table 61 Rf values and Fluorescence of RF and Photoproducts
Solvent System Fluorescence
Aa B
b C
c D
d
Riboflavin 034 048 027 yellow green
Formylmethylflavin 057 070 069 yellow green
Lumichrome 063 067 064 Sky blue
Lumiflavin 035 052 040 yellow green
Carboxymethylflavin 019 037 020 yellow green
Cyclodehydroriboflavine
045 Non-
fluorescent a1ndashButanolndashethanolndashwater (702010 vvv Silica gel G) (Ahmad et al 1980)
b1ndashButanolndashacetic acidndashwater (401050 vvv organic phasecellulose powder CC41)
(Ahmad et al 1980)
c1ndashButanolndash1ndashpropanolndashacetic acidndashwater (5030218 vvv cellulose powder CC41)
(Ahmad et al 1980)
d Chloroform-Methanol (92 vv cellulose powder CC41) (Schuman Jorns et al 1975)
e See section 721 for TLC identification of CDRF
110
Fig 61 Absorption spectra of RF photolyzed in methanol at 0 30 60 90
and 120 min
250 300 400 500 600
Wavelength (nm)
Ab
sorb
an
ce
00
10
15
111
There is a gradual loss of absorbance around 445 nm with a shift of the 358 nm
peak to 350 nm with time due to the formation of LC (λmax in methanol 339 nm)
(Sikorski et al 2003) the major of RF in organic solvents LC is formed through the
mediation of FMF an intermediate in the photolysis of RF (Song and Metzler 1967)
FMF has an absorption spectrum similar to that of RF due to the presence of a similar
chromophoric system and therefore it could not be distinguished from the absorption
spectrum of RF in organic solvents
623 Assay of RF and Photoproducts
The photolyzed solutions of RF have been assayed at pH 20 (02 M KClndashHCl
buffer) by extraction of LC with chloroform and its determination at pH 45 (02 M
acetate buffer) at 356 nm The aqueous phase was used to determine RF and FMF by a
twondashcomponent assay at 385 and 445 nm corresponding to the absorption maxima of
these compounds The molar concentrations of RF and its photoproducts FMF LC and
LF determined in the photolysis reactions in aqueous solution (pH 70) by the method of
Ahmad and Rapson (1990) are reported in Table 62 In the case of organic solvents the
photolysed solutions were evaporated under nitrogen at 40 oC the residue dissolved in
pH 20 buffer and the solution extracted with chloroform as stated above The RF and
FMF were determined at 384 and 445 nm and LC separately at 356 nm The results of the
assay of these compounds in organic solvents are reported in Table 63-68 The assay
method shows uniformly increasing values of FMF and LC in the photolysis reactions
with an almost constant molar balance with time indicating a good reproducibility of the
method
112
Table 62 Concentrations of RF and Photoproducts in Water (pH 70)
Time
(min)
RF
(Mtimes 105)
FMF
(Mtimes 105)
LC
(Mtimes 105)
LF
(Mtimes 105)
Total
(Mtimes 105)
0 300 000 000 000 300
30 263 028 008 004 305
60 229 060 012 007 308
90 197 078 023 009 309
120 173 086 030 012 311
Table 63 Concentrations of RF and Photoproducts in Acetonitrile
Time
(min)
RF
(Mtimes 105)
FMF
(Mtimes 105)
LC
(Mtimes 105)
Total
(Mtimes 105)
0 300 000 000 300
30 269 023 012 304
60 239 040 021 308
90 213 058 031 304
120 194 066 045 311
113
Table 64 Concentrations of RF and Photoproducts in Methanol
Time
(min)
RF
(Mtimes 105)
FMF
(Mtimes 105)
LC
(Mtimes 105)
Total
(Mtimes 105)
0 300 00 00 300
30 255 036 015 306
60 215 058 029 308
90 201 071 032 306
120 191 079 037 312
Table 65 Concentrations of RF and Photoproducts in Ethanol
Time
(min)
RF
(Mtimes 105)
FMF
(Mtimes 105)
LC
(Mtimes 105)
Total
(Mtimes 105)
0 300 000 000 300
30 273 017 014 306
60 245 032 024 310
90 223 042 036 308
120 199 049 052 306
114
Table 66 Concentrations of RF and Photoproducts in 1ndashPropanol
Time
(min)
RF
(Mtimes 105)
FMF
(Mtimes 105)
LC
(Mtimes 105)
Total
(Mtimes 105)
0 300 000 000 300
30 268 020 015 305
60 245 031 028 307
90 223 040 039 304
120 202 049 050 302
Table 67 Concentrations of RF and Photoproducts in 1ndashButanol
Time
(min)
RF
(Mtimes 105)
FMF
(Mtimes 105)
LC
(Mtimes 105)
Total
(Mtimes 105)
0 300 000 000 300
30 269 022 012 303
60 245 037 022 304
90 222 052 031 307
120 204 060 039 309
115
Table 68 Concentrations of RF and Photoproducts in Ethyl acetate
Time
(min)
RF
(Mtimes 105)
FMF
(Mtimes 105)
LC
(Mtimes 105)
Total
(Mtimes 105)
0 300 000 000 300
30 275 017 011 308
60 251 031 023 309
90 227 037 039 306
120 208 046 050 304
116
Since the concentration of FMF (an intermediate product in the photolysis reactions) and
determined in aqueous and organic solvents is less than 1 times 10ndash5
M due to its loss to LC
and LF CMF a minor oxidation product of FMF in organic solvents (Ahmad et al
2006) accounting to less than 1 (Ahmad et al 2013) does not interfere with the assay
method
624 Kinetics of Photolysis
The photolysis of RF in aqueous solution (Ahmad et al 2004 2014a Song and
Metzler 1967) and in organic solvents (Kurtin et al 1967 Song and Metzler 1967)
follows firstndashorder kinetics The kinetic plots for the photolysis of RF in water and
organic solvents (Fig 62ndash68) show that LC is the final product in these reactions as
observed by previous workers (Ahmad et al 2004a InsinskandashRak et al 2012 Moore
and Ireton 1977) The firstndashorder plots for the photolysis of RF in water and organic
solvents are shown in Fig 69ndash615 and the rate constants (kobs) determined from the
slopes of these plots range from 319 (ethyl acetate) to 461times10minus3
minminus1
(water)
(correlation coefficients 0997ndash0999) (Table 69) The values of kobs increase with an
increase in the dielectric constant indicating the influence of solvent on the rate of
reaction The value for the photolysis of RF in aqueous solution (pH 70 0005 M
phosphate buffer) is also included for comparison A plot of kobs for the photolysis of RF
as a function of solvent dielectric constant is presented in Fig 616 It shows that the rate
constants are linearly dependent upon the solvent dielectric constant Similarly a linear
relation has been found between the values of kobs and the solvent acceptor number
indicating the degree of solutendashsolvent interaction (Fig 617)
117
Fig 62 Kinetic plots for the photolysis of RF in water
RF () FMF () LC () LF(diams)
Fig 63 Kinetic plots for the photolysis of RF in acetonitrile
RF () FMF () LC ()
000
100
200
300
0 30 60 90 120
Co
nce
ntr
ati
on
times1
05
M
Time (min)
000
100
200
300
0 30 60 90 120
Co
nce
ntr
ati
on
times10
5M
Time (min)
118
Fig 64 Kinetic plots for the photolysis of RF in methanol
RF () FMF () LC () LF(diams)
Fig 65 Kinetic plots for the photolysis of RF in ethanol
RF () FMF () LC ()
00
10
20
30
0 30 60 90 120
Co
nce
ntr
ati
on
times1
05
M
Time (min)
000
100
200
300
0 30 60 90 120
Co
nce
ntr
ati
on
times10
5M
Time (min)
119
Fig 66 Kinetic plots for the photolysis of RF in 1ndashpropanol
RF () FMF () LC ()
Fig 67 Kinetic plots for the photolysis of RF in 1ndashbutanol
RF () FMF () LC ()
000
100
200
300
0 30 60 90 120
Co
nce
ntr
ati
on
times1
05
M
Time (min)
000
100
200
300
0 30 60 90 120
Co
nce
ntr
ati
on
times10
5M
Time (min)
120
Fig 68 Kinetic plots for the photolysis of RF in ethyl acetate
RF () FMF () LC ()
00
10
20
30
0 30 60 90 120
Con
cen
trati
on
times10
5M
Time (min)
121
Fig 69 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in water
(pH 70)
Fig 610 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
acetonitrile
-480
-475
-470
-465
-460
-455
-450
0 20 40 60 80 100 120 140
Time (min)
log
co
nce
ntr
ati
on
(M
times10
5)
-475
-470
-465
-460
-455
-450
0 20 40 60 80 100 120 140
Time (min)
log c
on
cen
tra
tio
n (
M times
10
5)
122
Fig 611 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
methanol
Fig 612 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
ethanol
-475
-470
-465
-460
-455
-450
0 20 40 60 80 100 120 140
Time (min)
log
co
nce
ntr
ati
on
(M
times10
5)
-475
-470
-465
-460
-455
-450
0 20 40 60 80 100 120 140
log c
on
cen
tra
tio
n (
M times
10
5)
Time (min)
123
Fig 613 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
1ndashpropanol
Fig 614 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
1ndashbutanol
-475
-470
-465
-460
-455
-450
0 20 40 60 80 100 120 140
log
co
nce
ntr
ati
on
(M
times10
5)
Time (min)
-475
-470
-465
-460
-455
-450
0 20 40 60 80 100 120 140
Time (min)
log c
on
cen
tra
tio
n (
M times
10
5)
124
Fig 615 Apparent firstndashorder plot for the photolysis of RF (5 times 10ndash5
M) in
ethyl acetate
-470
-465
-460
-455
-450
0 20 40 60 80 100 120 140
log
co
nce
ntr
ati
on
(M
times10
5)
Time (min)
125
Table 69 Apparent FirstndashOrder Rate Constants for the Photolysis of Riboflavin
(kobs) in Organic Solvents and Water
Solvents Dielectric
constant (isin)
(25 oC)
Acceptor
Number
Inverse
viscosity
(mPasndash1
)
(25 oC)
kobs times 103 min
ndash1
plusmnSDa
Ethyl acetate 602 171 2268 319plusmn014
1ndashButanol 178 368 0387 328plusmn013
1ndashPropanol 201 373 0514 334plusmn016
Ethanol 243 371 0931 345plusmn015
Methanol 326 413 1828 364plusmn017
Acetonitrile 385 189 2898 381plusmn016
Water 785 548 1123 461plusmn025
aSD standard deviation
126
Fig 616 Plot of kobs for the photolysis of RF versus dielectric constant (x) ethyl
acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol () methanol (+) acetonitrile
() water
00 100 200 300 400 500 600 700 800
Dielectric constant
00
10
20
30
40
50
60 k
ob
s times
10
3 (
min
-1)
127
Fig 617 Plot of lnkobs for the photolysis of RF versus acceptor number (x) ethyl
acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol () methanol (+) acetonitrile
() water
00 100 200 300 400 500 600 -70
-65
-60
-55
-50
-45
Solvent acceptor number
lnk
ob
s times
10
3 (
min
-1)
128
In order to observe the effect of viscosity on the rate of photolysis a plot of kobs versus
inverse of solvent viscosity was constructed (Fig 618) It showed a linear relation
between the two values indicating the influence of solvent viscosity on the rate of
reaction These results are supported by the fact that a plot of dielectric constant versus
inverse of viscosity of organic solvents is linear (Fig 619) However the values of kobs
for RF in ethyl acetate and water do not fit in the plot probably due to different behaviors
of RF in acetate (compared to alcohols) and water (eg degree of hydrogen bonding)
625 Effect of Solvent
It is known that the solvents could influence the degradation of drugs depending
on the solvent characteristics and solutendashsolvent interactions Solvents may alter the rate
and mechanism of chemical reactions (Abraham 1985 Amis and Hinton 1973 Laidler
1987 Parker 1969 Reichardt 1982 Sheraz et al 2014) and thus play a significant role
in the stabilization of pharmaceutical products (Connors et al 1986) Pharmaceutical
formulations of ionizable compounds such as RF may be stabilized by an alteration in the
solvent characteristics A suppression of the ionization of a drug susceptible to
degradation in water may be achieved by the addition of a cosolvent (eg alcohol
propylene glycol glycerin) This would result in the destabilization of the polar excited
state and therefore a decrease in the rate of reaction as observed in the case of many
drugs (Wypych 2001) The use of organic solvents as cosolvent can have a
photostabilizing effect on the product as a result of a change in the polarity and viscosity
of the medium (Tonnesen 2001)
129
Fig 618 Plot of kobs for the photolysis of RF versus inverse of viscosity
(x) ethyl acetate (diams) 1ndashbutanol () 1ndashpropanol () ethanol () methanol
(+) acetonitrile () water
100
10 15 20 25 05 30 00
20
40
60
80
00
Viscosity (mPa s)-1
ko
bs
times 1
03 (
min
-1)
130
Fig 619 Plot of dielectric constant versus inverse of viscosity
000
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
00 05 10 15 20 25 30 35
Die
lect
ric
con
sta
nt
Viscosity (mPas)-1
131
These considerations are important in the formulation of drugs with different polar
characters and those whose oxidation is viscosity dependent These aspects with respect
to the photolysis of RF as a model compound used in the clinical treatment of neonatal
jaundice (Tan 1996) keratoconus (Caporossi et al 2010) and HIV infection (Montessori
et al 2004) would now be considered and correlations would be developed between the
solvent characteristics and the rate of reaction
626 Effect of Dielectric Constant
The rate of degradation reactions between ions and dipoles in solution depends on
the bulk properties of the solvent such as the dielectric constant Any change in the
dielectric constant of a solvent can lead to variation in the energy of activation (ΔG) and
hence in the rate constants (Yoshioka and Stella 2000) This can be applied to the
degradation of RF since its rate of photolysis is a linear function of dielectric constant
This can be explained on the basis of the participation of a polar intermediate in the
reaction pathway to facilitate the reaction (Ahmad et al 2006a Ahmad and Tollin
1981) The rate of RF photolysis is affected by solvent polarity probably due to changes
in the conformation of the ribityl side chain in different solvents (Moore and Ireton
1977) Quenching of flavin excited triplet state [3FL] by oxygen during the reaction has
been suggested (Ahmad et al 2006a InsinskandashRak et al 2012) and this may affect the
rate of RF photolysis However under the present reaction conditions (ie solvents in
equilibrium with the air) the firstndashorder plots are linear for RF solutions photolyzed up to
30 and the values of kobs are relative to these conditions The electronndashdonating
capacity of a molecule (eg fluoroquinolone RF) is affected by the nature of the solvent
(Ahmad et al 2015 Peng et al 2014) and hence its rate of degradation The acceptor
132
number is a measure of the ability of solvents to share electron pairs from suitable donors
(Schmidt and Sapunov 1982 Wypych 2001) and this could affect the rate of photolysis
The results obtained and the degradation behavior of RF in organic solvents suggest that
the stability of such polar drugs can be improved by alteration of dielectric constant of
the medium
627 Effect of Viscosity
The viscosity of the medium can also influence the rate of degradation
particularly of an oxidizable drug The photolysis of RF involves oxidation of the ribityl
side chain (Moore and Ireton 1977) and thus may be affected by the solvent viscosity
The values of kobs for RF in ethyl acetate and water do not follow the relation (Fig 5)
probably due to its different structural orientation (Moore and Ireton 1977) and degree of
hydrogen bonding (Sikorski et al 2003) compared to those of the organic solvents The
behavior of RF in organic solvents indicates that the viscosity of the medium suppresses
the rate of photolysis probably as a result of solute diffusionndashcontrolled processes
(Ahmad and Tollin 1981 Turro et al 2010) It has been observed that the flavin triplet
state [3RF] quenching depends on solvent viscosity (Ahmad and Tollin 1981) and that
would affect the rate of reaction Similar effects of viscosity have been observed on the
photooxidative degradation of formylmethylflavin (Ahmad et al 2013b) and
fluoroquinolones (Ahmad et al 2013a 2014b 2015)
628 Mode of Photolysis
The photochemistry of RF has widely been studied by several workers and the
various modes of its photodegradation reactions (ie intramolecular and intermolecular
133
photoreduction photodealkylation and photoaddition) have been discussed (Ahmad et
al 2006a 2013b Ahmad and Vaid 2006 Choe et al 2005 Heelis 1982 1991 Sheraz
et al 2014a) The pathway of RF degradation in organic solvents appears to be similar to
that of the aqueous solution involving intramolecular photoreduction followed by sidendash
chain cleavage (Ahmad and Vaid 2006) However the rate of the reaction is solvent
dependent due to the participation of a dipolar intermediate (Ahmad and Tollin 1981)
whose degradation is promoted by polar environment and suppressed by nonpolar media
It has been observed by laser flash photolysis that the reduction of [3FL] in organic
solvents proceeds through the mediation of the dipolar intermediate according to the
following reaction (Ahmad and Tollin 1981)
3FL + AH (F
σndash hellip H hellip A
σndash+) FLH (61)
The flavin semiquinone radical [FLH] undergoes further reactions to give an
oxidized and a reduced flavin (Eq (62)) The reduced flavin is then oxidized by air to
form degraded products (Eq (63))
2FLHbull FL + FLH2 (62)
FLH2 degraded FL + side chain products (63)
The extent of the photolysis reaction to form radicals is controlled by the degree
of solutendashsolvent interaction The polar character of the reaction intermediate would
determine the rate of reaction and the rate would be higher in solvents of greater polarity
Thus the solvent characteristics play an important role in determining the rate of RF
degradation An appropriate combination of waterndashalcohol mixture would be a suitable
medium for the stabilization of RF and drugs of similar character
O2
CHAPTER VII
IONIC STRENGTH EFFECTS ON THE
PHOTODEGRADATION REACTIONS OF
RIBOFLAVIN IN AQUEOUS SOLUTION
135
71 INTRODUCTION
The ionic strength of a solution can have a significant effect on the rate of a
chemical reaction and is known as the primary kinetic salt effect The relationship
between the rate constant and the ionic strength for an aqueous solution at 25 oC may be
expressed by the BronstedndashBjerrum equation (Bronsted 1922 Bjerrum 1924)
log k = log ko + 102 Z
AZ
B radicmicro (71)
where ZA and Z
B are the charges carried by the reacting species in solution micro the
ionic strength k the rate constant of degradation and ko the rate constant at infinite
dilution A plot of log kko against radicmicro should give a straight line of slope 102 Z
AZ
B
Eq (71) is valid for ionic solutions up to micro = 001 At higher concentrations (micro le 01) the
BronstedndashBjerrum equation can be expressed as
log k = log ko + 102 Z
AZ
B radicmicro (1 + β radicmicro) (72)
In Eq (72) the value of β depends on the ionic diameter of the reacting species
and is often approximated to unity
If the rate constants for a chemical reaction are determined in the presence of a
series of different concentrations of the same electrolyte then a plot of log k against
under root of ionic strength is linear even in the case of solutions of high ionic strength
(Florence and Attwood 2006) The influence of ionic strength on the kinetics of drug
136
degradation and chemical reactions has been discussed by several workers (Florence and
Attwood 2006 Lachman et al 1986 Carstensen 2000 Guillory and Post 2002 Sinko
2006 Yoshioka and Stella 2000 Laidler 1987 Koppenol 1980) Ionic strength has
been found to effect the aggregation kinetics of TiO2 (French et al 2009) and the
stability of Ag nanoparticles (Badawy et al 2010) The primary salt effects on the rates
and mechanism of chemical reactions have been discussed (Frost and Pearson 1964
Corsaro 1977)
In drug degradation and stability studies the reactions are normally carried out at
a constant ionic strength to minimize its effect on the rate of reaction (Sankara et al
1999 Stankovicova et al 1999 Yeh 2000 Chadha et al 2003 Jumaa et al 2004
Ahmad et al 2004a) However a large number of studies have been conducted to
evaluate the influence of ionic strength on the kinetics of chemical (Pramar and Gupta
1991 Hoitink et al 2000 Zang and Pawelchak 2000 Matos et al 2001 Miranda et al
2002 Alibrandi et al 2003 Sato et al 2003 Aloisi et al 2004 Lallemand et al 2005
Rexroad et al 2006) and photodegradation of drug substances (Khattak et al 2012) The
ionic strength effects have important implications in photoinduced electron transfer
reactions and the binding ability of proteins to flavin species (Fukuzumi and Tanaka
1988) Laser flash photolysis studies of the kinetics of electron transfer between flavin
semiquinone and fully reduced flavins and horse rate cytochrome c have shown that the
presence of a charged phosphate group in the Nndash10 ribityl side chain leads to small ionic
strength effects on the rate constant whereas a charged group attached to the
dimethylbenzene ring produces a large ionic strength effect (Ahmad and Cusanovich
1981) Attempts have been made to describe the dependence of bimolecular rate
137
constants on ionic strength for small molecules and protein interactions (Ahmad and
Cusanovich 1981 Ahmad et al 1982 Hazzard et al 1987 1988 Watkins et al 1994
Zhong and Zewail 2001) A temperature dependent study of the effect of ionic strength
on the photolysis of riboflavin (RF) has been conducted RF undergoes biphasic
photolysis with a lowndashintensity light source In higher ionic strength phosphate buffer
(031 M) an initial faster phase is followed by a slower second phase and vice versa in
lower ionic strength buffer (005 M) (Sato et al 1984) In the presence of higher
concentration (gt 01 M) of divalent phosphate anions (HPO42ndash
) and pH values above 60
the normal course of RF photolysis (photoreduction) involving 10ndashdealkylation to form
formylmethyflavin (FMF) lumiflavin (LF) and lumichrome (LC) (Ahmad et al 2004b)
is shifted to photoaddition to yield cyclodehydroriboflavin (CDRF) (Schuman Jorms et
al 1975 Ahmad et al 2005) The present study involves the evaluation of ionic strength
effects on the photodegradation of RF with a change in the mode of reaction at higher
buffer concentrations These effects may significantly influence the rates and mechanism
of RF degradation reactions flavinndashprotein interactions and the kinetics of electron
transfer reactions The study of ionic strength effects is also necessary since the single
and multivitamin parenteral and total parenteral nutrition (TPN) preparations containing
RF are isotonic and the amount of NaCl present (09 wv) may influence the stability
of RF on exposure to light The effects of ionic strength on a change in the mode of
photodegradation of RF need to be investigated Some related work on the effect of
factors such as pH (Ahmad et al 2004b) buffer (Ahmad et al 2013 2015ab) and light
intensitywavelengths (Ahmad et al 2006) on the photodegradation of RF has been
reported
138
The details of the experimental work involved in the study are given in section
53 (Chapter 5)
72 RESULTS AND DISCUSSION
721 Assay of RF and Photoproducts
An important consideration in kinetic studies is the use of a specific assay
procedure to determine the desired compounds in the presence of degradation products
The multicomponent spectrometric method used in this study is capable of simultaneous
determination of RF and its photoproducts with reasonable accuracy (Ahmad et al
2004a) It has the advantage of determining these compounds without mutual
interference Under the present reaction conditions (ie simultaneous photolysis and
photoaddition reactions) the photodegraded solutions of RF contain a mixture of RF
FMF LF LC and CDRF as photoproducts as detected by TLC (Section 531) on
comparison with the Rf values and fluorescence of difference compound and reported
previously (Ahmad et al 1990 2004ab) Therefore a specific rapid and accurate
method is required for the assay of such a complex mixture The method used for this
purpose (Ahmad et al 2004b) fulfils these requirements and has previously been applied
to the assay of these compounds during the kinetic studies of photodegradation of RF
(Ahmad et al 2004a 2009 2010 2013 2015) Such an analysis cannot be carried out
rapidly by HPLC methods The assay of RF and photoproducts in various reactions
carried out at pH 70 with an ionic strength of 01ndash05 (01ndash05 M phosphate buffer) is
reported in Table 71ndash725
139
Table 71 Concentrations of RF and Photoproducts in 01 M Phosphate Buffer
(pH 70) at 01 M Ionic Strength
Time
(min)
RF
(M times105)
FMF
(M times105)
LC
(M times105)
LF
(M times105)
TOTAL
(M times105)
0 500 00 00 00 500
30 451 019 021 010 501
60 398 039 045 019 506
90 373 053 059 022 507
120 340 064 071 027 508
Table 72 Concentrations of RF and Photoproducts in 01 M Phosphate Buffer
(pH 70) at 02 M Ionic Strength
Time
(min)
RF
(M times105)
FMF
(M times105)
LC
(M times105)
LF
(M times105)
TOTAL
(M times105)
0 500 00 00 00 500
30 446 020 022 016 504
60 386 044 049 021 508
90 332 069 073 029 509
120 309 076 081 035 501
140
Table 73 Concentrations of RF and Photoproducts in 01 M Phosphate Buffer
(pH 70) at 03 M Ionic Strength
Time
(min)
RF
(M times105)
FMF
(M times105)
LC
(M times105)
LF
(M times105)
TOTAL
(M times105)
0 500 00 00 00 500
30 435 020 031 016 502
60 381 039 052 029 505
90 331 065 071 035 508
120 288 078 089 046 501
Table 74 Concentrations of RF and Photoproducts in 01 M Phosphate Buffer
(pH 70) at 04 M Ionic Strength
Time
(min)
RF
(M times105)
FMF
(M times105)
LC
(M times105)
LF
(M times105)
TOTAL
(M times105)
0 500 00 00 00 500
30 417 031 035 020 503
60 361 054 058 031 504
90 308 069 082 043 507
120 269 081 099 052 508
141
Table 75 Concentrations of RF and Photoproducts in 01 M Phosphate Buffer
(pH 70) at 05 M Ionic Strength
Time
(min)
RF
(M times105)
FMF
(M times105)
LC
(M times105)
LF
(M times105)
TOTAL
(M times105)
0 500 00 00 00 500
30 404 032 044 022 502
60 336 056 075 036 505
90 290 068 097 047 507
120 245 079 118 059 501
Table 76 Concentrations of RF and Photoproducts in 02 M Phosphate Buffer
(pH 70) at 01 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 435 015 016 039 008 513
60 378 026 028 048 020 508
90 329 035 046 071 030 511
120 280 048 060 092 042 522
142
Table 77 Concentrations of RF and Photoproducts in 02 M Phosphate buffer
(pH 70) at 02 M ionic strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 416 024 036 057 006 539
60 353 040 059 075 016 543
90 293 079 081 134 028 615
120 251 089 091 175 034 640
Table 78 Concentrations of RF and Photoproducts in 02 M Phosphate Buffer
(pH 70) at 03 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 386 023 032 059 006 500
60 307 040 056 083 014 511
90 239 059 069 119 021 516
120 194 064 081 131 033 503
143
Table 79 Concentrations of RF and Photoproducts in 02 M Phosphate Buffer
(pH 70) at 04 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 369 030 036 062 009 506
60 280 045 060 093 023 501
90 217 060 073 122 033 509
120 153 071 089 145 048 506
Table 710 Concentrations of RF and Photoproducts in 02 M Phosphate Buffer
(pH 70) at 05 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 338 036 046 074 009 503
60 238 055 081 112 014 510
90 164 064 116 131 027 502
120 119 073 126 149 037 504
144
Table 711 Concentrations of RF and Photoproducts in 03 M Phosphate Buffer
(pH 70) at 01 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 398 016 031 045 010 510
60 327 031 055 066 022 508
90 267 042 065 085 041 503
120 224 050 076 101 049 506
Table 712 Concentrations of RF and Photoproducts in 03 M Phosphate Buffer
(pH 70) at 02 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 367 027 037 056 013 504
60 286 047 051 096 020 511
90 221 059 069 120 031 513
120 178 057 082 139 044 509
145
Table 713 Concentrations of RF and Photoproducts in 03 M Phosphate Buffer
(pH 70) at 03 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 354 024 049 059 014 504
60 236 049 069 108 038 508
90 168 068 076 139 049 503
120 108 078 096 158 060 509
Table 714 Concentrations of RF and Photoproducts in 03 M Phosphate Buffer
(pH 70) at 04 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 295 040 051 100 015 506
60 160 056 108 143 033 505
90 097 069 121 168 045 502
120 076 075 132 177 051 506
146
Table 715 Concentrations of RF and Photoproducts in 03 M Phosphate Buffer
(pH 70) at 05 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 282 046 060 106 006 511
60 145 076 088 154 037 505
90 079 091 104 175 051 509
120 052 100 110 200 057 507
Table 716 Concentrations of RF and Photoproducts in 04 M Phosphate Buffer
(pH 70) at 01 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 397 029 026 035 017 504
60 309 036 049 076 037 507
90 239 048 061 105 051 504
120 180 067 075 126 062 508
147
Table 717 Concentrations of RF and Photoproducts in 04 M Phosphate Buffer
(pH 70) at 02 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 361 029 042 047 023 508
60 256 048 056 095 047 512
90 183 061 077 118 063 502
120 127 073 095 145 071 514
Table 718 Concentrations of RF and Photoproducts in 04 M Phosphate Buffer
(pH 70) at 03 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 314 032 050 075 035 506
60 195 055 090 113 050 513
90 130 070 108 133 062 508
120 075 085 130 145 071 506
148
Table 719 Concentrations of RF and Photoproducts in 04 M Phosphate Buffer
(pH 70) at 04 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 292 042 052 079 039 504
60 148 069 083 135 066 511
90 078 093 103 155 076 509
120 042 103 114 163 084 506
Table 720 Concentrations of RF and Photoproducts in 04 M Phosphate Buffer
(pH 70) at 05 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 217 049 070 113 055 504
60 113 060 096 157 074 509
90 057 073 106 178 086 511
120 024 082 117 187 093 506
149
Table 721 Concentrations of RF and Photoproducts in 05 M Phosphate Buffer
(pH 70) at 01 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 425 013 028 027 009 502
60 338 032 041 065 024 509
90 251 045 074 091 043 514
120 157 066 085 135 059 512
Table 722 Concentrations of RF and Photoproducts in 05 M Phosphate Buffer
(pH 70) at 02 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 313 041 046 085 019 506
60 214 056 068 115 047 509
90 140 072 085 150 057 506
120 099 081 096 164 067 507
150
Table 723 Concentrations of RF and Photoproducts in 05 M Phosphate Buffer
(pH 70) at 03 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 298 037 062 075 030 506
60 179 061 079 125 056 511
90 099 076 097 155 075 502
120 049 088 108 169 087 508
Table 724 Concentrations of RF and Photoproducts in 05 M Phosphate Buffer
(pH 70) at 04 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 249 049 068 099 036 501
60 099 071 118 145 067 509
90 049 082 128 167 077 506
120 023 088 137 178 086 512
151
Table 725 Concentrations of RF and Photoproducts in 05 M Phosphate Buffer
(pH 70) at 05 M Ionic Strength
Time
(min)
RF
(M times 105)
CDRF
(M times 105)
FMF
(M times 105)
LC
(M times 105)
LF
(M times 105)
TOTAL
(M times 105)
0 500 00 00 00 00 500
30 210 062 086 126 026 508
60 078 088 112 179 049 506
90 034 094 120 190 069 509
120 013 099 132 201 080 511
152
The assay results show that a good molar balance is achieved during the reactions
indicating the accuracy and precision of the method in the determination of RF and
photoproducts
722 Spectral Characteristics of Photolysed Solutions
The absorption spectra of RF determined during a photolysis reactions at pH 70
with an ionic strength of 01 and 05 show a decrease in absorbance at the maximum at
445 (Ahmad and Rapson 1990 Ahmad et al 2004a) indicating the gradual loss of RF
and an increase in absorbance around 356 nm (Ahmad et al 2004a) indicating the
formation of LC in the reaction (Fig 71) There is no change in the shape of absorption
spectra with a change in the ionic strength of the solutions However the variations in
ionic strength affect the magnitude of spectral changes for instance an increase in ionic
strength shows a greater decrease in absorbance at 445 nm and a greater increase in
absorbance at 356 nm This supports the view that an increase in ionic strength leads to
an increase in the rate of photolysis reactions
723 Kinetics of RF Photolysis
A large number of studies have been conducted on the photolysis of RF under
different conditions (Ahmad et al 2004ab 2005 Schuman Jorms et al 1975 Sato et
al 1984) It has been established that the photolysis of RF in aqueous solution follows
firstndashorder kinetics (Ahmad et al 2004b 2005 2015ab Song et al 1965) In this study
the effect of ionic strength on the phorodegradation of RF under different conditions has
been studied
153
Fig 71 Absorption spectra of the photolysed solutions of RF (5 times 10ndash5
M) at pH 70
(a) at zero and (b) at 05 M ionic strength
154
Considering the photolysis of RF as parallel firstndashorder reactions leading to the
formation of LC (k1) and LF (k2) as final products by phororeduction and CDRF (k3) as
final product by photoaddition pathways the values of the rate constants k1 and k2 can be
calculated as previously reported (Ahmad et al 2004a 2010) These reactions can be
expressed as follows
RF
LC
LF
CDRF
k1
k2
k3
The mathematical treatment of the analytical data to determine k1 k2 k3 for these
reactions is given by Frost and Pearson (1964) Using the concentration values of RF
LC LF and CDRF and RF0 for the initial concentration
ndashdRFdt = k1 RF + k2 RF + k3 RF = (k1+ k2+ k3) RF = kobs RF (73)
kobs= k1+ k2+ k3 (74)
and
ln (RF0RF) = kobst (75)
or
RF = RF0 endashkt
(76)
Similarly
dLCdt = k1 RF0 endashkt
(77)
155
and
LC = + constant (78)
or
LC = LC0 + (1 ndash endashkt
) (79)
LF = LF0 + (1 ndash endashkt
) (710)
CDRF = CDRF0 + (1 ndash endashkt
) (711)
If LC0 = LF0= CDRF0 = 0 the equation simplifies and is readily seen that
LFLC = k2 k1 CDRFLC = k3 k1 (712)
LC LF CDRF = k1 k2 k3 (713)
The products are in constant ratio to each other independent of time and initial
concentration of the reactant The method has been applied to the determination of rate
constants for all the three primary processes in the pure liquidndashphase pyrolysis of
αndashpinene (Fuguitt and Hawkins 1947)
The values of k1 k2 k3 determined as a function of the ionic strength at different
phosphate buffer concentrations along with k1k3 ratios are reported in Table 726 The
values of k1 show a greater increase compared to those of k3 with an increase in ionic
strength at a constant buffer concentration It has been observed that a change in k1k3
ratios in favor of k1 occurs with a change in ionic strength This indicates that the ionic
strength has a greater effect on k1 (photoreduction pathway) leading to the formation of
k2 RF0 kobs
k3 RF0 kobs
ndash RF0 endashkt
kobs
k1 RF0
kobs
156
LC The mechanism of promotion of the rate of photoaddition reactions (k3) of RF by Clndash
is not clear
The values of apparent firstndashorder rate constants (kobs) (Table 726) for the overall
photodegradation of RF in reactions carried out at a phosphate buffer concentration of 01
M (photoreduction pathway) (Ahmad et al 2004b) indicate the effect of ionic strength
on this particular reaction However the photodegradation reactions carried out at
phosphate buffer concentrations above 01 M involve both photoreduction and
photoaddition pathways the latter due to the buffer effect (Ahmad et al 2005 Schuman
Jorns et al 1975) Under these conditions the values of kobs for RF would not distinguish
the ionic strength effects on the rates of the two distinct reactions where as the individual
rate constants (k1 k2 for photoreduction pathway and k3 for photoaddition pathway)
would indicate the effect of ionic strength on these reactions The values of rate constants
are relative and have been observed under controlled conditions of light intensity and
other factors
724 Fluorescence Studies
RF exhibits an intense yellow green fluorescence at 520 nm in aqueous solution
(United States Pharmacopoeia 2016) that vanishes in strongly acidic and alkaline
solutions due to ionization of the molecule (Weber 1950) In order to observe the effect
of NaCl on the fluorescence intensity of RF fluorescence measurements were made on
5times10minus5
M RF solutions (pH 70) at different ionic strengths at constant buffer
concentrations (Fig 72) These results indicate that at a 0001 M buffer concentration
there is a 334 to 422 loss of florescence at 01 to 05 M ionic strength
157
Table 726 Apparent FirstndashOrder Rate Constants (kobs) for the Photodegradation
of Riboflavin in the presence of Phosphate Buffer (pH 70) at different Ionic
Strength (01ndash05M) for the formation of Lumichrome (k1) Lumiflavin (k2) and
Cyclodehdroriboflavin (k3)
Buffer
Concentration
(M)
Ionic
Strength
(M)
kobs times 103
(minndash1
)
k0 times 103
(minndash1
)
k1 times 103
(minndash1
)
k2 times 103
(minndash1
)
k3 times 103
(minndash1
)
k1 k3
01 01 201 079 145 055 ndashndash ndashndash
02 301 210 090 ndashndash ndashndash
03 396 261 134 ndashndash ndashndash
04 490 321 168 ndashndash ndashndash
05 625 416 208 ndashndash ndashndash
02 01 276 085 139 063 072 193
02 485 284 070 144 197
03 715 407 102 198 205
04 978 535 177 255 209
05 1190 684 201 321 213
03 01 445 120 224 109 111 201
02 825 425 151 185 229
03 1185 632 240 265 238
04 1505 835 253 345 242
05 1860 1042 296 521 248
04 01 525 135 259 127 121 214
02 1150 501 282 226 221
03 1571 756 370 325 232
04 2030 1115 487 466 239
05 2491 1279 561 522 245
05 01 735 141 380 166 170 222
02 1250 660 285 277 238
03 1891 991 478 402 246
04 2421 1220 615 482 253
05 3032 1603 638 607 264
158
Fig 72 Plots of fluorescence of RF solutions (pH 70) versus ionic
strength at different buffer concentrations (diams) 0001 M () 0025 M
() 005 M (times) 01 M () 02 M (∆) 03 M () 04 M () 05 M
40
50
60
70
80
90
100
0 01 02 03 04 05 06
F
luore
sen
ce I
nte
nsi
ty
Ionic Strength (M)
159
With an increase in buffer concentration (01ndash05 M) there is a gradual increase in the
loss of florescence reaching a value of 271 to 332 at 01 to 05 M ionic strength
respectively in 05 M buffer concentration Since phosphate buffer also quenches the
florescence of RF (Ahmad et al 2005) a combined effect of buffer and NaCl is being
observed at each buffer concentration with an increase in ionic strength This is in
agreement with a previous observation that NaCl (01 M) quenches the fluorescence of
RF solutions (Ellinger and Holden 1944) Since the kinetic results show an increase in
rate with an increase in ionic strength at each buffer concentration the loss of florescence
cannot be attributed exclusively to the excited singlet state quenching and some
interaction between RF and NaCl may be stipulated This could be analogous to the
excited singlet state quenching of RF by complexation with HPO42minus
ions leading to the
formation of CDRF by the photoaddition pathway (Schuman Jorms et al 1975) On the
basis of the kinetic results it can be suggested that a similar mechanism may operate
between RF and NaCl as explained below In the present case RF on the absorption of
light is promoted to the excited singlet state [1RF] (Eq (714)) [
1RF] could react with Cl
minus
ions to form an excited state complex (exciplex) as suggested for the exited state
reactions of organic compounds (Turro et al 2010) (Eq (715)) and observed in the case
of [1RFndashHPO4
2minus] complex leading to the formation of CDRF (Ahmad et al 2004b) In
both cases RF complexation with Clminus ions observed in the present study or with HPO4
2minus
ions (Ahmad et al 2005) results in the quenching of fluorescence involving the [1RF]
state as well as an acceleration of the photodegradation process The role of Clminus
ions
appears to be analogous to that of the HPO42 minus
ions in promoting the rate of degradation
of RF This would lead to the formation of the photoproducts of RF (eg LC) (Eq (716))
160
RF [1RF] (714)
[1RF] + NaCl [
1RFhelliphellipCl
ndash] + Na
+ (715)
[1RF helliphellipCl
ndash] Photoproducts (716)
Clminus appears to form a nonndashfluorescent complex with the ground state RF molecule
by static quenching as suggested in the case of quinine (Gutow 2005) Thus the role of
Clminus ions in the photodegradation of RF is to promote the degradation of RF by different
pathways
725 Ionic Strength Effects
In order to correlate the rate constants for the photodegradation of RF by
photoreduction (LC LF) and photoaddition (CDRF) pathways with ionic strength the log
values of rate constants (kobs) were plotted against radicμ1 + radicμ (Eq (72)) which yielded
straight lines indicating a linear relationship Extrapolation to zero ionic strength yielded
the value for k0 the rate constant for the photodegradation of RF at zero ionic strength
(Fig 73) Further plots of log k1k0 and k3k0 against radicμ (Eq (71)) gave straight lines
with a positive slope of 102 ZAZB (Fig 74) shown for a typical photodegradation
reaction of RF at 05 M buffer concentration (ionic strength 01ndash05 M) The rate
constant k2 for the formation of LF by photoreduction pathway is a minor reaction and
has been neglected The number of unit charges ZAZB can be calculated from the slope
of the plots
ZAZ
B = 105 102 = 103 = ~ + 1 (for k1)
ZAZ
B = 161 102 = 157 = ~ + 160 (for k3)
exciplex
hv
161
Fig 73 A plot of log kobs versus radicμ1 + radicμ at 05 M phosphate buffer
Fig 74 A plot log k1k0 () and k3k0 () versus radicμ at 05 M phosphate buffer
-325
-305
-285
-265
-245
-225
-205
-185
-165
-145
000 010 020 030 040
radicμ1 + radicμ
log
kob
s(m
in-1
)
000
020
040
060
080
100
000 010 020 030 040 050 060 070 080
log
k1k
0 k
3k
0
radicμ
162
The values of ZAZB (+1) for photoreduction suggest that a charged species is
involved in the rate determining step of the reaction (k1) It has been earlier suggested by
flash photolysis experiments that the flavin triplet reduction takes place via a dipolar
intermediate (Ahmad and Tollin 1981) as follows
[3F + F F ỏndashndashndashndashndashndashndash F ỏ+
] (717)
The degree to which this intermediate proceeds to form the products would be
affected by the interaction with NaCl at a particular ionic strength The higher the ionic
strength the greater is the interaction leading to the degradation and hence an increase in
the rate of the reaction A positive slope of the reaction indicates an increase in the rate of
reaction between similarly charged species as a result of an increase in the ionic strength
of the solution The degradation of RF by the photoaddition pathway also involves the
participation of a charged species in the form of a [1RFndashHPO4
2minus] complex Although Eq
(71) is essentially true for dilute solutions an effect due to ionic strength is in fact
observed at higher concentrations (Florence and Attwood 2006) as found in the present
case Since the value of ZAZB for the photoaddition reaction (k3) is 080 This value is not
an integer suggesting a complex mode of reaction between RF buffer species and Clminus
ions It has been suggested (Schuman Jorms et al 1975) that the photoaddition pathway
is not affected by the ionic strength These authors studied the analytical photochemistry
of RF by absorbance changes at the λmax at 445 nm Their analytical data may not be
reliable due to the fact that all the photoproducts of RF absorb at this wavelength and an
accurate assay of RF is not possible Thus any kinetic data obtained may not represent the
true rate constants for the reactions involved
FH F
+H ndashH+
163
The present study involves a specific analytical method to determine RF
accurately in the presence of various photoproducts and therefore the rate constants
derived from such analytical data would be reliable as reported in several previous
studies (Ahmad et al 2004a 2009 2010 2013 2015)
The effect of ionic strength has also been observed in studies carried out on the
photolysis of RF and related reactions under conditions different from those of the
present work These include the biphasic photolysis of RF in the ionic strength range of
003ndash046 M using phosphate buffer (pH 74) (Sato et al 1984) the photolysis of RF in
the presence of magnesium perchlorate at pH 70 (Schuman Jorns et al 1975) and the
alkaline hydrolysis of 67ndashdimethylndash9ndashformylmethylisoalloxazine (an intermediate in the
photolysis of RF) under various conditions of ionic strength and pH (Song et al 1965)
Ionic strength effects play a significant role in studies involving flavinndashprotein
interactions A charged phosphate group attached to the dimethylbenzene ring of flavins
has been found to produce a large ionic strength effect on the rate of interaction (Ahmad
et al 1981) The kinetics of electron transfer reactions and the binding ability of flavins
to proteins are dependent upon the ionic strength due to electrostatic interactions (Ahmad
et al 1981 1982 Hazzard et al 1987 Meyer et al 1984 Hurley et al 1999) and may
be significantly influenced at large values of ionic strength
CHAPTER VIII
METAL ION MEDIATED PHOTOLYSIS
REACTIONS OF RIBOFLAVIN
165
81 INTRODUCTION
Riboflavin (RF) (1) (Fig 81) is a photosensitive compound
(British Pharmacopoeia 2016) which undergoes degradation in aqueous solution on
exposure to light (Ahmad et al 2004a Astanov et al 2014 Sheraz et al 2014) The
degradation takes place by different mechanisms depending upon the reaction conditions
(pH buffer kind and concentration light intensity and wavelengths aerobic or anaerobic
condition) (Heelis 1982 1991 Ahmad and Vaid 2006) The photolysis of RF in aqueous
solution leads to the formation of a number of compounds including formylmethylflavin
(FMF) (2) lumichrome (LC) (3) lumiflavin (LF) (4) carboxymethylflavin (CMF) (5)
and cyclohdehydroriboflavin (CDRF) (6) by photoreduction and photoaddition pathways
given in Chapter 3 (Smith and Metzler 1963 Treadwell et al 1968 Cairns and Metzler
1971 Ahmad and Rapson 1990 Ahmad et al 2004ab 2008 2010) (Fig 31) The
kinetics of photolysis reactions of RF has been evaluated (Ahmad et al 2004a Cairns
and Metzler 1963 Ahmad et al 2004b 2008 2010 2014 2016) using specific
spectrometric methods (Ahmad and Rapson 1990 Ahmad et al 1980 2004ab 2014)
Flavins are known to interact with metal ions to form complexes For example
10ndashmethylisoalloxazine forms a complex with Cu+ ions (Hemmerich et al 1965 Yu and
Fritchie Jr 1975) RF with monovalent ions (Ag+) (Weber 1950 Wade and Fritchie Jr
1973) divalent ions (Fe Cu Cd Mg Mn Co Ni Zn Ru) (Isaka 1955 Isaka and Ishida
1953 Sakai 1956 Garland Jr and Fritchie Jr 1974 Mortland and Lawless 1983
Kaim et al 1999 Hussain et al 2006 Jabbar et al 2014) and trivalent ions (Cr3+
Fe3+
)
(Rutter 1958 Varnes et al 1971) flavin mononucleotide (FMN) with divalent ions (Mg
Ca Sr Ba Mn Co Cu Zn Cd) (Sigel et al 1995) and trivalent ions (Fe3+
) (Mortland
166
N N
NNH
C
C
C
O
O
C
OH
OH
OH
CH2OH
CH3
CH3
H
H
H
HH
N N
NNH
CH2
CHO
O
OCH3
CH3
N NH
NNH
O
OCH3
CH3
N N
NNH
CH3
O
OCH3
CH3
photo
additi
on
N N
NNH
O
OCH3
CH3
CH2
CHO
C
C
CH2OH
OH
OHH
H
HPO 4
2-
photoreduction(1)
(8)
(4) (5)
(6)
N N
NNH
CH2
O
OCH3
CH3
COOH
(7)
H+ OH-
H + O
H -
OH-O2
Fig 81 The photoreduction and photoaddition pathways of riboflavin (RF)
167
and Lawless 1984) flavin dinucleotide with Hg2+
and Cd2+
ions (Picaud and Desbois
2006) and flavin analogues (3ndashmethylndash10ndashphenylisoalloxazine and 3ndashmethylndash10ndash
phenylndash5ndashdeazaisoalloxazine) with Mg2+
and Zn2+
ions (Fukuzumi et al 1985
Fukuzumi and Kojima 2008) Structural characteristics (Wade and Fritchie Jr 1973
Isaka and Ishida 1953 Kaim et al 1999 Clarke et al 1979 1980) and redox reactivity
(Kaim et al 1999 Fukuzumi and Kojima 2008 Fukuzumi and Okhubo 2010) of the
metalndashflavin complexes have been studied in detail
It has been shown (Kaim et al 1999 Fukuzumi and Kojima 2008 Clarke et al
1978) that metal centres can bind to flavin in the N(5)ndash C(4a)ndashC(4)ndashO(4) site to form a
planar fivendashmembered chelate ring (Fig 82) Electrochemical and spectroscopic data on
the structural features of these complexes have been reported (Kaim et al 1999
Fukuzumi and Kojima 2008 Clarke et al 1978) The metalndashflavin interactions have
important implications in the electron transfer reactivity of flavins in biological systems
(Kaim et al 1999)
The aerobic photolysis of RF is promoted by Fe2+
Fe3+
Cu2+
Sn2+
Co2+
Mn2+
Cr2+
Al3+
in the decreasing order of reactivity The anaerobic photolysis of RF is
promoted by Fe3+
ions and inhibited by Fe2+
and Cu2+
ions (Sakai 1956) RF catalyzes
the photooxidation of Fe2+
(oxygen dependent) and photoreduction of Fe3+
(inhibited by
oxygen) Both ions have been found to quench the fluorescence of RF (Rutter 1958)
Metalndashflavin complexes presumably involve extensive charge transfer from metal d
orbitals to flavin π orbitals (Varnes et al 1971)
168
N10
N1
N5
NH3
4
2
9
6
8
7
O
O
OH
OH
OH
OH
CH3
CH3
H
H
H
HH
(1) (81)
M2+
N N
NNH
O
O
OH
OH
OH
OH
CH3
CH3
H
H
H
HH
M2+
Rearrangment
(82)
N N
N+ NH
O+
O
OH
OH
OH
OH
CH3
CH3
H
H
H
HH
M2+
4a5 44a
10a
Fig 82 Formation of the metalndashRF complex
169
The fluorescence of RF is quenched by Ag+ ions various divalent ions and Fe
3+
ions due to the formation of nonndashfluorescent metalndashRF complexes (Weber 1950 Isaka
1955 Isaka and Ishida 1953 Varnes et al 1971) The quenching of excited singlet states
of organic molecules by metal ions has been observed (Kemlo and Shepherd 1971) [41]
Fe2+
ions promote photolysis of RF strongly followed by the effect of Fe3+
Cu2+
Al3+
Sn2+
Co2+
Mn2+
Cr3+
and Zn2+
ions Ag+ ion inhibits the photolysis of RF (Sakai 1956)
Trace quantities of metallic impurities in pharmaceuticals may catalyze the
degradation of drug substances (British Pharmacopoeia 2016) particularly in the
presence of light These processes occur by onendashelectron oxidative reactions and result in
an increase in the rate of formation of radicals that lead to the degradation products
Oxidative reactions are often initiated by metal ions such as Fe3+
Cu2+
Co3+
Ni2+
Mn2+
These metal ions act as initiators since they are capable of acting as radicals in their
oxidation states for example Cu 2+
ion has 27 electrons and it requires one electron to
complete the electron pair The metal ion can react with a drug to form radicals
M2+
+ RH M(nndash1)+
+ H+ + R
˙ (81)
The radical can then participate in the propagation cycle or can react with a
hydroperoxide to catalyze the degradation
Mn+
+ RʹOOH M(nndash1)+
+ H+ + RʹO2
˙ (82)
RʹOOH could be a hydroperoxide of the drug (eg RF) itself or of some other
component present in the system (Connors et al 1982) Thus the metal ion can directly
react with oxygen to form an oxygen radical which can then initiate an autoxidation
reaction The metal ion can also form a complex with oxygen to produce a peroxy radical
170
or it can react with a drug (eg RF) to form a radical to initiate a photochemical chain
reaction
The object of this work is to conduct a study of the photolysis of RF in metalndashRF
complexes using various metal ions to identify the photoproducts to determine the
absorption and fluorescence characteristics and to evaluate the influence of metal ions on
the kinetics of photolysis reaction at different buffer concentrations It may have
important implications in the understanding of the reactivity of flavoenzymes since these
complexes are known to modify the redox reactivity of enzymes in the biological system
The experimental details involved in these studies are presented in 53
(Chapter 5)
82 RESULTS AND DISCUSSION
821 Photoproducts of MetalndashRF Complexes
The TLC studies of the photolyzed solutions of various metalndashRF complexes
indicated the formation of FMF an intermediate product LC LF and CMF (solvent
systems (Z1) and (Z2)) (Section 531) at low buffer concentration and FMF LC LF
CMF and CDRF (solvent system (Z3)) as the sidendashchain products of RF at pH 70 on
comparison of the Rf values and fluorescence emission (RF FMF LF CMF yellow
green LC skyblue) and CDRF (red colour) with those of the authentic compounds The
fluorescence intensity of the spots of these photoproducts varied with the concentration of
metal ions An increase in metal ion concentration leads to an increase in the
concentrations of the photoproducts as a result of enhancement in the rate of photolysis
All these photoproducts have previously been observed in the photolysis of RF
171
(Ahmad et al 2004a 2008 Smith and Metzler 1963 Treadwell et al 1968 Cairns and
Metzler 1971 Ahmad and Rapson 1990 Isaka 1955) Divalent ion impregnated silica
gel G TLC plates have been used for the separation of RF and other B vitamins on the
basis of complexation (Bushan and Parshad 1994)
822 Spectral Characteristics of MetalndashRFndashComplexes
The spectral characteristics of free RF and metalndashRF complexes have been
studied The UV and visible absorption spectra of some typical complexes (Fe2+
Zn2+
and Cu2+
) are shown in Fig 83 Aqueous solutions of RF (pH 70) exhibit absorption
maxima at 223 267 374 and 444 nm (British Pharmacopoeia 2016) On the addition of
Fe2+
ions to RF solution a big spectral change is observed in the UV and visible region
with disappearance of the 445 maximum and increase in absorption in the 200ndash400 nm
region The greater effect of Fe2+
ions (1 times 10ndash3
M) at a high concentration (20 fold)
compared to that of RF (5 times 10ndash5
M) on the spectral changes of RF is probably due to the
11 RFndashFe2+
complex formation as well as the chemical reduction of RF resulting in the
loss of the 445 nm band RF is easily chemically reduced by electron donors such as
sodium dithionite (Na2S2O4) (Burn and OrsquoBrien 1959) with a loss in absorption at 445
nm due to the disappearance of the N(5)ndashC(4a)ndashC(10a)ndashN(1) conjugated system (Fig
82) as a results of the formation of RFH2 molecule
RF + 2Fe2+ +2HRFH2 + 2Fe3+
(83)
172
Fig 83 Absorption spectra of RF (5 times 10ndash5
M) (pH 70) (____
) in the presence of
metal ions (1times 10ndash3
M) (ndashndashndash) (a) Fe2+
ions (b) Zn2+
ions and (c) Cu2+
ions
173
On the contrary the changes in the absorption spectra of RF are not very
prominent in the presence of Zn2+
and Cu2+
ions (Fig 83) These spectral changes could
result from disturbance in the conjugated system of the pteridine ring in RF as mentioned
above A slight increase in the absorption of RF in the presence of Cu2+
ions appears to
be due to an increase in the intensity of colour as a result of RFndashCu2+
complex formation
Similar minor changes in the absorption spectra of RF have been observed in the
presence of other divalent ions studied Such spectral changes have previously been
observed in the spectra of metalndashRF complexes (Isaka and Ishida 1953 Fukuzumi et al
1985) These changes in the absorption spectra of RF are not very prominent in the
presence of Zn2+
and Cu2+
ions These spectral changes could result from disturbance in
the conjugated system of the pteridine ring in RF Such changes have previously been
observed in the absorption spectra of metalndashRF complexes (Isaka and Ishida 1953
Fukuzumi et al 1985)
It is well known that various metal ions bind to flavins in the N(5)ndashC(4a)ndashC(4)ndash
O(4) chelate site to form planar 5ndashmembered redoxndashactive αndashiminoketo chelate rings
(81) (Fig 82) (Kaim et al 1999 Fukuzumi and Kojima 2008 Kemlo 1977) [28 37
40] Electrochemical and spectroscopic data on the structural features of these metalndash
flavin complexes have been reported (Kaim et al 1999 Fukuzumi and Kojima 2008
Kemlo 1977) Since O(4) and N(5) atoms of the αndashiminoketo function in the chelate ring
of RF are connected in a asymmetric πndashconjugated system the redoxndashactive metal
chelate undergoes rearrangement of the C(4)ndashC(4a) bond to a symmetrical (C(4a)ndashC(4))
form (82) (Fig 82) as suggested for αndashdiimines (Juris et al 1988 Constable 1989
Greulich et al 1996) and αndashdiketones (Burns and McAuliffe 1979) This would result in
174
the disappearance of the πndashconjugated system affecting the UVndashabsorption maxima (444
nm) of the complex The gradual loss of these maxima with an increase in metal ion
concentration (Fig 83) is indicated by a shift in the equilibria to form the symmetrical
metalndashRF complex (82) through the intermediate form (81) (Fig 82)
823 Spectrometric Assay of RF and Photoproducts in Photolyzed Solutions
The assay of RF and photoproducts (FMF LC LF CDRF) in the photolyzed
solutions of metalndashRF complexes (pH 70) has been carried out by a multicomponent
spectrometric method extensively used for the assay of RF and photoproducts in the
photolysis reactions of RF (Ahmad et al 1980 2004a 2008 2014 2016 Ahmad and
Rapson 1990) The pH of the photolyzed solutions is adjusted to pH 20 to form the
protonated species of RF and FMF (Suelter and Metzler 1960) and the solutions are
extracted with chloroform to remove LC and LF followed by their twondashcomponent assay
at 356 and 445 nm The aqueous phase is used to assay RF and FMF (at low buffer
concentration 0001 M) (Table 81) or RF FMF and CDRF (at high buffer
concentrations 02ndash04 M) (Table 82ndash83) by a twondashcomponent assay at 385 and 445
nm or a threendashcomponent assay at 385 410 and 445 nm respectively CMF is a minor
oxidation product of FMF (Ahmad et al 2004a) (Fig 81) and is not accounted in the
assay The metal ions at the concentrations used do not interfere in the assay The assay
method gives good molar balance of RF and photoproducts with a RSD of plusmn5 as
observed in earlier studies (Ahmad and Rapson 1980 Ahmad et al 2014 2016)
175
Table 81 Concentration of RF (M times 105) and LC (M times 10
5) (0001 M Phosphate
Buffer) in the presence of 10ndash50 times 10ndash4
M Metal Ions Concentration
10a
20a 30
a 40
a 50
a
Metal
Ion
Time
(min)
RF
LC
RF
LC
RF
LC
RF
LC
RF
LC
Ag+
0 500 000 500 000 500 000 500 000 500 000
60 485 008 489 006 486 007 489 006 490 005
120 470 014 477 012 472 015 478 014 479 012
180 447 026 454 023 458 020 466 019 468 018
Fe2+
0 500 000 500 000 500 000 500 000 500 000
60 472 015 463 017 457 019 450 019 446 022
120 442 032 431 037 421 035 413 038 398 046
180 416 045 398 049 384 052 371 059 355 066
Cu2+
0 500 000 500 000 500 000 500 000 500 000
60 474 014 461 018 453 022 442 026 432 030
120 450 027 424 032 413 036 393 047 373 055
180 418 039 389 044 365 055 346 065 324 076
Zn2+
0 500 000 500 000 500 000 500 000 500 000
60 472 014 462 017 442 025 429 030 421 033
120 444 024 423 033 395 044 375 052 354 061
180 414 039 385 052 352 067 322 075 295 086
Mg2+
0 500 000 500 000 500 000 500 000 500 000
60 474 015 465 018 464 019 459 021 441 028
120 450 024 436 029 430 031 415 036 389 048
180 427 036 407 045 393 051 358 062 339 068
Pb2+
0 500 000 500 000 500 000 500 000 500 000
60 474 015 459 019 450 022 440 026 427 032
120 450 026 422 038 403 044 386 051 363 065
180 417 041 381 056 355 066 338 071 309 081
Ni2+
0 500 000 500 000 500 000 500 000 500 000
60 489 006 475 013 472 014 472 014 467 016
120 465 016 449 024 446 026 443 027 437 029
180 437 032 427 036 419 039 414 041 408 045
Ca2+
0 500 000 500 000 500 000 500 000 500 000
60 475 014 467 017 463 018 457 021 451 023
120 449 024 434 030 429 033 411 040 406 042
180 426 035 407 040 390 047 374 054 363 060
176
Mn2+
0 500 000 500 000 500 000 500 000 500 000
60 472 015 463 019 453 022 442 025 433 030
120 443 029 428 035 412 042 390 051 373 057
180 416 039 394 047 371 057 342 068 322 076
Cd2+
0 500 000 500 000 500 000 500 000 500 000
60 474 014 473 016 463 019 457 021 441 028
120 447 027 444 028 428 034 416 039 390 051
180 429 036 411 042 391 051 375 057 346 068
Co2+
0 500 000 500 000 500 000 500 000 500 000
60 478 011 476 013 472 015 466 017 464 019
120 454 022 450 024 442 027 436 029 430 032
180 433 030 423 034 414 039 405 043 399 048
Fe3+
0 500 000 500 000 500 000 500 000 500 000
60 476 013 473 019 464 020 463 020 457 022
120 451 022 444 026 430 033 431 039 416 042
180 426 036 412 044 398 055 393 060 380 066 a Metal ion concentration 10ndash50 times 10
ndash4 M
Table 81 continued
177
Table 82 Concentration of RF (M times 105) and CDRF (M times 10
5) (02 M Phosphate
Buffer) in the presence of 10ndash50 times 10ndash4
M Metal Ions Concentration
10a
20a 30
a 40
a 50
a
Metal
Ion
Time
(min)
RF
CDRF
RF
CDRF
RF
CDRF
RF
CDRF
RF
CDRF
Ag+
0 500 000 500 000 500 000 500 000 500 000
60 448 014 457 014 457 014 465 014 467 013
120 405 015 416 015 416 015 424 015 436 014
180 363 017 374 016 381 015 395 014 408 013
Fe2+
0 500 000 500 000 500 000 500 000 500 000
60 425 015 416 015 416 016 398 016 389 016
120 369 017 346 018 338 019 323 019 309 020
180 322 019 298 020 279 021 257 023 245 025
Cu2+
0 500 000 500 000 500 000 500 000 500 000
60 436 015 426 015 407 016 407 016 387 016
120 371 017 363 017 338 019 323 020 300 020
180 319 019 302 020 279 023 259 024 234 026
Zn2+
0 500 000 500 000 500 000 500 000 500 000
60 416 015 398 016 380 017 371 017 352 018
120 346 018 323 019 295 021 275 022 255 024
180 291 021 257 023 229 027 203 029 177 034
Mg2+
0 500 000 500 000 500 000 500 000 500 000
60 436 015 426 015 421 015 416 015 406 016
120 380 017 363 017 354 018 338 018 320 020
180 331 019 310 020 298 021 279 022 262 024
Pb2+
0 500 000 500 000 500 000 500 000 500 000
60 436 015 426 015 416 015 416 015 396 016
120 380 017 363 017 346 018 338 018 323 019
180 328 019 308 020 295 022 274 023 256 025
Ni2+
0 500 000 500 000 500 000 500 000 500 000
60 454 014 426 015 426 015 426 015 416 015
120 406 015 371 017 363 017 354 018 338 018
180 367 019 316 022 311 023 295 025 281 026
Ca2+
0 500 000 500 000 500 000 500 000 500 000
60 436 015 426 015 416 015 407 016 396 016
120 381 017 363 017 346 018 331 019 323 020
180 334 019 311 020 293 022 274 027 251 029
178
Mn2+
0 500 000 500 000 500 000 500 000 500 000
60 436 015 416 015 407 016 398 016 381 017
120 371 017 346 018 331 019 316 020 293 022
180 319 019 291 022 266 023 244 025 228 028
Cd2+
0 500 000 500 000 500 000 500 000 500 000
60 426 015 426 015 407 016 398 016 361 018
120 371 017 354 018 338 019 323 019 262 024
180 320 021 299 025 279 028 259 031 189 037
Co2+
0 500 000 500 000 500 000 500 000 500 000
60 436 015 426 015 416 016 416 016 406 016
120 381 017 363 017 354 018 346 018 330 019
180 328 021 314 024 299 025 286 029 273 033
Fe3+
0 500 000 500 000 500 000 500 000 500 000
60 426 015 416 015 421 015 416 015 404 016
120 371 017 354 017 354 018 346 018 330 020
180 325 019 305 019 289 022 275 022 262 025 a Metal ion concentration 10ndash50 times 10
ndash4 M
Table 82 continued
179
Table 83 Concentration of RF (M times 105) and CDRF (M times 10
5) (04 M Phosphate
Buffer) in the presence of 10ndash50 times 10ndash4
M Metal Ions Concentration
10a
20a 30
a 40
a 50
a
Metal
Ion
Time
(min)
RF
CDRF
RF
CDRF
RF
CDRF
RF
CDRF
RF
CDRF
Ag+
0 500 000 500 000 500 000 500 000 500 000
60 416 011 421 010 428 009 436 009 447 007
120 347 027 354 026 369 021 389 018 402 015
180 292 034 303 030 319 028 343 026 359 021
Fe2+
0 500 000 500 000 500 000 500 000 500 000
60 403 018 372 026 375 026 358 030 347 033
120 325 024 282 037 276 038 251 045 244 046
180 252 032 216 042 194 044 171 053 165 055
Cu2+
0 500 000 500 000 500 000 500 000 500 000
60 393 019 381 020 375 021 358 024 347 026
120 307 028 289 031 276 033 254 038 244 041
180 237 037 215 039 200 041 181 044 170 048
Zn2+
0 500 000 500 000 500 000 500 000 500 000
60 375 016 347 022 334 024 319 027 295 022
120 272 030 246 033 219 035 195 036 182 035
180 200 040 167 045 143 048 122 051 103 061
Mg2+
0 500 000 500 000 500 000 500 000 500 000
60 393 011 381 015 384 015 375 016 364 018
120 319 025 298 029 289 031 276 033 263 036
180 251 033 233 036 221 038 209 041 197 043
Pb2+
0 500 000 500 000 500 000 500 000 500 000
60 393 011 381 014 384 014 375 017 364 021
120 317 022 298 025 289 027 276 031 263 035
180 251 029 229 032 223 034 207 037 194 039
Ni2+
0 500 000 500 000 500 000 500 000 500 000
60 393 014 384 016 384 016 375 018 364 020
120 303 022 298 023 289 025 276 027 263 029
180 241 031 229 033 221 035 203 037 191 039
Ca2+
0 500 000 500 000 500 000 500 000 500 000
60 393 013 384 015 384 015 367 017 345 018
120 315 019 298 021 289 023 272 026 237 029
180 255 025 225 029 215 033 198 035 169 039
180
Mn2+
0 500 000 500 000 500 000 500 000 500 000
60 393 011 375 018 367 019 347 021 337 025
120 302 022 282 026 26 030 242 035 231 038
180 237 033 207 036 188 038 169 041 155 044
Cd2+
0 500 000 500 000 500 000 500 000 500 000
60 393 014 375 019 367 020 347 022 337 024
120 302 019 282 025 266 029 242 032 231 034
180 234 027 213 031 171 041 171 043 159 047
Co2+
0 500 000 500 000 500 000 500 000 500 000
60 393 013 384 014 367 019 363 019 350 022
120 309 019 295 021 272 026 263 027 251 031
180 242 026 226 029 207 032 195 034 183 036
Fe3+
0 500 000 500 000 500 000 500 000 500 000
60 393 016 381 019 375 021 358 022 347 024
120 315 027 289 033 276 034 254 038 244 040
180 242 036 218 041 202 043 185 047 174 051 a Metal ion concentration 10ndash50 times 10
ndash4 M
Table 83 continued
181
824 Fluorescence Characteristics of MetalndashFlavin Complexes
The complexation of metal ions with RF results in the quenching of RF
fluorescence This is due to the fact that metalndashRF complexation involves charge transfer
from metal d orbitals to RF π orbital in the excited state (Varnes et al 1971)
The quenching of RF fluorescence by different metal ions at pH 70 is shown in
Fig 84 and the loss of intensity in the fluorescence spectrum of RF (530 nm) in the
presence of increasing concentrations of divalent ions such as Fe2+
ions is shown in
Fig 85 The increase in fluorescence loss of RF at 5 times 10ndash4
M metal ion concentration is
in the order
Ni2+
lt Co
2+lt Fe
3+ lt Ca
2+ +lt Fe
2+ lt Cd
2+ lt Cu
2+lt Mn
2+lt Pb
2+ lt Mg
2+lt Zn
2+lt Ag
+
Thus Ni2+
ions on interaction with RF produces the lowest loss in the
fluorescence intensity (37) and Ag+
ions produce the highest loss in fluorescence
intensity (224) of RF There is a gradual loss of RF fluorescence with an increase in
the metal ion concentration for all the metal ions studied This appears to be due to a
greater degree of metalndashRF complexation
825 Kinetic of Photolysis of MetalndashFlavin Complexes
The photochemistry of RF has been studied in detail (see Introduction) and its
modes of photolysis are well known (Heelis 1982 1991 Ahmad and Vaid 2006 Ahmad
et al 2008) (Fig 81) Metal ions are known to modify the redox reactivity of flavins
(Fukuzumi and Kojima 2008)
182
Fig 84 The percent decrease in fluorescence intensity of RF solutions (pH 70
0001 M phosphate buffer) in the presence of metal ions () Ni2+
ions (∆) Co2+
ions
(loz) Ca2+
ions (+) Fe2+
ions () Cd2+
ions (ndash) Cu2+
ions (diams) Mn2+
ions () Pb2+
ions
() Mg2+
ions () Zn2+
ions and () Fe3+
ions
900
920
940
960
980
1000
00 10 20 30 40 50 60
Metal ion concentration (M times 104)
F
luo
rese
nce
In
ten
sity
183
Fig 85 Excitation spectrum of RF (5 times 10ndash5
M) (pH 70) (a) Fluorescence spectra
of RF pure RF (b1) RF + Fe2+
ions (1 times 10ndash3
M) (b2)
RF + Fe2+
ions (2 times 10ndash3
M) (b3)
184
However no work on the kinetics of photolysis of metalndashRF complexes has been
conducted to study the behaviour of these complexes on UV or visible irradiation and to
identify the photoproducts formed RF is known to undergo photolysis in aqueous
solution by an apparent firstndashorder kinetics (Ahmad et al 1980 2004a 2008 2010
2014 2016 Sheraz et al 2014)
In the present study the photolysis of 5 times 10ndash5
M RF solutions (pH 70) at low
(0001 M) and high (02ndash04 M) phosphate buffer concentrations has been carried out in
the presence of various metal ions to evaluate the kinetics of these reactions The various
rate constants for the photolysis of RF (kobs) and for the formation of LC (k1) and LF (k2)
(photoreduction pathway) and CDRF (k3) (photoaddition pathway) (Heelis 1982 1991
Ahmad and Vaid 2006) by parallel firstndashorder reactions have been determined by the
method described by Ahmad et al (2016) A typical set of firstndashorder plots for the loss of
RF concentration on photolysis as a function of the increasing concentration of metal
ions at low (0001 M) and high buffer concentrations (02ndash04 M) are shown in Fig 86ndash
818 and 819ndash842 respectively The greater loss of RF in the presence of increasing
concentrations of Fe2+
ions may be due to a change in the equilibria of RF and the metalndash
RF complexes and their greater susceptibility of photolysis
RF + Fe2+ RF-Fe2+
(84)
Significant enhancement of the electronndashtransfer reactivity of the singlet excited
state of flavins has been observed by complexation with metal ions (Fukuzumi et al
1985 Fukuzumi and Kojima 2008 Clarke et al 1979)
185
Fig 86 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Ag+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 87 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Fe2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-436
-434
-432
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-450
-448
-446
-444
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
Time (min)
log co
nce
ntr
ati
on
186
Fig 88 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Cu2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 89 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Zn2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-450
-448
-446
-444
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
187
Fig 810 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Mg2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 811 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Pb2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-444
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
188
Fig 812 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Ni2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 813 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Ca2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-440
-439
-438
-437
-436
-435
-434
-433
-432
-431
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-446
-444
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
189
Fig 814 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Mn2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 815 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Cd2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-448
-446
-444
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
190
Fig 816 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Co2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 817 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Fe2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
191
Fig 818 Firstndashorder plots for the photolysis of RF (0001 M phosphate buffer pH
70) in the presence Fe3+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 819 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Ag+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-444
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
Time (min)
log c
on
cen
trati
on
-446
-444
-442
-440
-438
-436
-434
-432
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
192
Fig 820 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Fe2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 821 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Cu2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
193
Fig 822 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Zn2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 823 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Mg2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-480
-475
-470
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
194
Fig 824 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Pb2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 825 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Ni2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
195
Fig 826 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Ca2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 827 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Mn2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-470
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
196
Fig 828 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Cd2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 829 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Co2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
197
Fig 830 Firstndashorder plots for the photolysis of RF (02 M phosphate buffer pH 70)
in the presence Fe3+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 831 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Ag+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log c
on
cen
trati
on
Time (min)
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
198
Fig 832 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Fe2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 833 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Cu2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-490
-480
-470
-460
-450
-440
-430
0 50 100 150 200
Time (min)
log
con
cen
trati
on
-480
-475
-470
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
Time (min)
log
con
cen
trati
on
199
Fig 834 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Zn2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 835 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Mg2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-510
-500
-490
-480
-470
-460
-450
-440
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
-475
-470
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
200
Fig 836 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Pb2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 837 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Ni2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-475
-470
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
-475
-470
-465
-460
-455
-450
-445
-440
-435
-430
-425
0 50 100 150 200
log
con
cen
trati
on
Time (min)
201
Fig 838 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Ca2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 839 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Mn2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-490
-480
-470
-460
-450
-440
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
-490
-480
-470
-460
-450
-440
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
202
Fig 840 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Cd2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
Fig 841 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Co2+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-490
-480
-470
-460
-450
-440
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
-480
-475
-470
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
203
Fig 842 Firstndashorder plots for the photolysis of RF (04 M phosphate buffer pH 70)
in the presence Fe3+
ions at different concentrations (M times 10ndash4
) (diams) 10
() 20 () 30 () 40 () 50
-480
-475
-470
-465
-460
-455
-450
-445
-440
-435
-430
0 50 100 150 200
log
con
cen
trati
on
Time (min)
204
This would suggest an increase in the photoreduction of RF (Heelis 1982 1991
Ahmad and Vaid 2006) and hence an increase in the rate of photolysis The apparent
firstndashorder rate constants (kobs) for the photolysis of RF in metalndashRF complexes along
with the rate constants for the formation of LC (k1) LF (k2) and CDRF (k3) at different
buffer concentrations are reported in Table 84ndash86 The values of kobs k1 k2 and k3 show
that the photolysis of RF and the formation of LC LF and CDRF are enhanced with an
increase in the metal ion concentration indicating that the metal ions promote the
photolysis reactions of RF as observed by earlier workers (Isaka 1955 Isaka and Ishida
1953 Sakai 1956 Rutter 1958 Varnes et al 1971) In order to develop a correlation
between the rate of photolysis and the fluorescence quenching of RF a plot of kobs versus
fluorescence loss of RF has been prepared as shown in Fig 843 It indicates an increase
in kobs of RF photolysis with an increase in the fluorescence loss of RF in the presence a
metal ion Thus the higher the fluorescence loss the higher the values of kobs due to the
greater complexation of RF and metal ions The photolysis of RF at low buffer
concentration (eg 0001 M) follows photoreduction pathway in aqueous solution
(Ahmad et al 2004a 2008 2014 Sheraz et al 2014 Ahmad and Vaid 2006) and at
high phosphate buffer concentration (eg 02ndash04 M) the photoaddition pathway (Heelis
1982 1991 Ahmad and Vaid 2006 Ahmad et al 2010 2016) (Fig 81) Therefore a
difference in the rate of photolysis of RF with a change in buffer concentration in the
presence of various metal ions could be expected
205
Table 84 Apparent Firstndashorder Rate Constants (kobs) for the Photolysis of RF in the
Presence of Various Metal Ions at pH 70 (0001 M Phosphate Buffer) for the formation
of LC (k1) LF (k2) and the SecondndashOrder Rate Constants for the Interaction of RF and
Metal Ions (kʹ)
Metal Ion Metal ion
concentration
(Mtimes104)
kobs times 103
(minndash1
)
kʹ times 10
3
(Mndash1
minndash1
)
k1 times 103
(minndash1
)
k2 times 103
(minndash1
)
00 063 016 006
Ag+ 10 059 050 041 017
20 054 038 015
30 049 035 014
40 044 033 010
50 038 029 008
Fe2+
10 089 256 070 018
20 115 080 034
30 142 101 040
40 169 129 039
50 191 143 047
Cu2+
10 099 360 078 020
20 136 084 051
30 172 107 064
40 206 138 067
50 243 164 078
Zn2+
10 105 462 073 031
20 155 113 041
30 199 138 060
40 245 164 080
50 294 190 094
Mg2+
10 101 416 071 029
20 142 099 042
30 184 131 052
40 225 160 064
50 271 182 088
Pb2+
10 106 410 079 026
20 145 105 039
30 185 128 056
40 224 152 071
206
50 268 180 087
Ni2+
10 075 104 058 016
20 085 062 022
30 095 068 026
40 105 075 029
50 115 083 031
Ca2+
10 089 232 063 025
20 112 075 036
30 136 092 043
40 158 106 051
50 179 120 058
Mn2+
10 102 360 072 029
20 132 089 042
30 167 110 056
40 210 140 070
50 243 162 081
Cd2+
10 091 284 069 021
20 118 086 031
30 148 104 043
40 176 122 053
50 205 139 065
Co2+
10 078 128 054 023
20 091 063 027
30 104 071 032
40 116 080 035
50 127 087 039
Fe3+
10 082 180 060 021
20 099 075 023
30 118 091 026
40 135 151 029
50 153 174 035
Table 84 continued
207
Table 85 Apparent Firstndashorder Rate Constants (kobs) for the Photolysis of RF in the
Presence of Various Metal Ions at pH 70 (02 M Phosphate Buffer) for the Formation of
LC (k1) LF (k2) CDRF (k3) and the SecondndashOrder Rate Constants for the Interaction of
RF and Metal Ions (kʹ )
Metal
Ion
Metal ion
concentration
(Mtimes104)
kobs times 103
(minndash1
)
kʹ times 10
3
(Mndash1
minndash1
)
k1 times 103
(minndash1
)
k2 times 103
(minndash1
)
k3 times 103
(minndash1
)
k1 k3
00 204 111 038 054 205
Ag+ 10 182 184 125 027 028 446
20 164 112 025 026 430
30 144 094 023 025 376
40 127 084 019 023 365
50 112 072 016 022 327
Fe2+
10 243 384 195 020 027 722
20 285 231 022 031 724
30 325 256 032 035 726
40 363 291 033 040 728
50 396 315 036 043 730
Cu2+
10 249 410 201 021 027 724
20 285 229 025 031 726
30 325 256 034 035 728
40 365 290 036 039 730
50 409 329 033 045 732
Zn2+
10 285 742 226 027 031 729
20 358 283 036 038 733
30 435 343 043 048 736
40 505 402 048 054 738
50 575 446 059 060 741
Mg2+
10 235 246 180 024 029 620
20 265 201 030 032 628
30 295 223 036 034 655
40 325 245 039 036 671
50 358 286 035 041 697
Pb2+
10 235 334 180 024 029 620
20 269 207 029 033 625
30 302 228 035 036 629
40 335 243 044 038 633
208
50 371 284 045 044 637
Ni2+
10 227 232 149 035 042 354
20 260 179 032 049 360
30 283 195 035 053 360
40 304 210 038 056 365
50 332 230 041 061 369
Ca2+
10 235 358 178 025 030 593
20 270 207 029 034 605
30 305 231 035 037 624
40 334 253 041 040 631
50 373 284 045 044 636
Mn2+
10 251 462 196 025 031 625
20 301 233 031 036 647
30 345 268 036 039 687
40 385 303 041 043 699
50 427 333 048 046 711
Cd2+
10 254 410 179 032 043 411
20 285 201 039 043 467
30 323 231 044 048 475
40 362 259 049 054 479
50 404 289 056 059 483
Co2+
10 236 256 168 029 039 425
20 255 184 032 038 484
30 280 204 034 040 510
40 300 220 038 042 519
50 319 232 043 044 523
Fe3+
10 237 308 189 021 026 726
20 271 218 024 029 730
30 302 238 030 032 734
40 332 265 030 036 736
50 358 284 036 038 738
Table 85 continued
209
Table 86 Apparent Firstndashorder Rate Constants (kobs) for the Photolysis of RF in the
Presence of Various Metal Ions at pH 70 (04 M Phosphate Buffer) for the Formation
of LC (k1) LF (k2) CDRF (k3) and the SecondndashOrder Rate Constants for the
Interaction of RF and Metal Ions (kʹ)
Metal
Ion
Metal ion
concentration
(Mtimes104)
kobs times 103
(minndash1
)
kʹ times 10
3
(Mndash1
minndash
1)
k1 times 103
(minndash1
)
k2 times 103
(minndash1
)
k3 times 103
(minndash1
)
k1 k3
00 351 222 049 075 296
Ag+ 10 315 332 184 061 069 263
20 280 171 042 066 259
30 247 152 035 059 257
40 214 129 033 051 252
50 190 114 030 046 247
Fe2+
10 402 528 262 059 079 331
20 462 290 070 101 287
30 515 310 094 109 284
40 570 335 098 136 246
50 615 363 104 147 246
Cu2+
10 407 496 259 069 079 325
20 460 295 072 092 320
30 509 328 077 103 317
40 560 357 089 113 315
50 599 373 099 126 296
Zn2+
10 475 1048 302 075 096 314
20 580 359 106 115 310
30 681 414 128 137 302
40 784 475 151 158 299
50 875 505 173 196 257
Mg2+
10 390 348 257 058 073 352
20 425 275 066 082 335
30 458 296 071 090 328
40 490 315 075 099 318
50 525 335 082 107 313
Pb2+
10 386 348 273 050 061 447
20 427 301 057 068 442
30 458 321 060 075 428
210
40 490 336 068 084 400
50 525 355 077 091 390
Ni2+
10 387 508 254 058 073 347
20 424 273 069 081 337
30 494 317 080 096 330
40 545 347 089 107 324
50 605 380 104 119 319
Ca2+
10 389 600 271 057 060 451
20 426 287 070 067 428
30 494 327 080 085 384
40 545 359 089 095 377
50 651 432 103 116 370
Mn2+
10 415 600 282 057 075 376
20 475 318 071 085 374
30 535 363 074 098 370
40 605 405 090 110 366
50 651 423 109 117 361
Cd2+
10 413 570 287 060 065 441
20 470 320 072 077 415
30 530 337 091 101 333
40 590 370 102 116 318
50 636 392 110 132 296
Co2+
10 395 414 273 059 061 447
20 438 296 069 071 416
30 479 321 076 081 396
40 524 350 084 089 393
50 558 369 093 095 388
Fe3+
10 405 468 260 055 083 313
20 455 290 072 093 310
30 505 322 077 104 309
40 548 346 086 115 300
50 585 363 093 128 283
Table 86 continued
211
Fig 843 A plot of kobs for the photolysis of RF versus fluorosecne loss in the
presence of different metal ions () Ni2+
ions (∆) Co2+
ions (loz) Ca2+
ions (+) Fe2+
ions () Cd2+
ions (ndash) Cu2+
ions (diams) Mn2+
ions () Pb2+
ions () Mg2+
ions ()
Zn2+
ions () Fe3+
ions
000
050
100
150
200
250
300
350
00 30 60 90
Fluorescence loss
ko
bs times
10
3
212
For example the values of kobs for the photolysis of RF in the presence of Fe2+
ions (10ndash50 times 10ndash4
M) at 0001M buffer concentration (089ndash191 times 10ndash3
minndash1
) (Table
84) are lower than those obtained at 02 M buffer concentration (243ndash396 times 10ndash3
minndash1
)
(Table 85) and 04 M buffer concentration (402ndash615 times 10ndash3
minndash1
) (Table 86) The
bimolecular rate constants (kprime) for the interaction of Fe
2+ ions and RF in these reactions
are 256 384 and 528 times 10ndash3
Mndash1
minndash1
respectively These results indicate that the
metal ions not only accelerate the photolysis of RF but also influence the reaction
pathways by altering the ratio of the products formed by the photoreduction (LC) and
photoaddition (CDRF) pathways (Heelis 1982 1991 Ahmad and Vaid 2006) in the
presence of high buffer concentration This is evident from the values of the ratios of
k1k3 in the presence of Fe2+
ions at 02 M buffer concentration (72ndash73) and at 04 M
buffer concentration (33ndash25) It also shows that at the highest buffer concentration
(04 M) the formation of CDRF is increased with an increase in metal ion concentration
These observations suggest that the formation of the 5ndashmembered chelate ring (Fig 82)
in the metalndashRF complex may be affected by an increase in metal ion concentration at
high buffer concentration to influence the formation of the two photoproducts The
increase in metal ion concentration may alter the photoreduction pathway leading to the
formation of LC by k1 in favour of the photoaddition pathway leading to the formation of
CDRF by k3 and hence a change in k1k3 ratios with a change in buffer concentration A
similar pattern of product formation ratios (k1k3) has been observed in the presence of
other divalent ions (Cu2+
Zn2+
Pb2+
Ni2+
Mg2+
Ca2+
Cd2+
Co2+
) and monovalent
(Ag+) and trivalent (Fe
3+) metal ions at high buffer concentrations (Table 85 and 86)
213
Thus all the metal ions studied behave in a similar manner to affect the product
formation by different pathways in the photolysis of RF at higher buffer concentration
The secondndashorder rate constants (kprime) for the interaction of metal ions with RF are in the
order Zn2+
gt Mg2+
gt Pb2+
gt Mn2+
gt Cu2+
gt Cd2+
gt Fe2+
gt Ca2+
gt Fe3+
gt Co2+
gt Ni2+
gt Ag+
This indicates that Zn2+
has the highest rate of interaction and Ag+ has the lowest rate of
interaction with RF The metal ion effect on the reaction is probably due to the
facilitation of the photoaddition pathway which originates from the excited singlet state
interaction of RF and HPO42ndash
ions (Schuman Jorns et al 1975) This would inhibit the
photoreduction pathway occurring through the excited triplet state of RF (Heelis 1991
Ahmad and Vaid 2006 Cairns and Metzler 1971)
826 Mode of Interaction of Metal Ions with RF
The present study shows that the divalent and trivalent metal ions promote the
photolysis reactions of RF in aqueous solution Earlier studies suggested that RF
catalyzes the photooxidation of Fe2+
ions and photoreduction of Fe3+
ions (Rutter 1958)
It was later suggested that metalndashflavin complexes involve extensive charge transfer from
metal d orbitals to flavin π orbitals and the excited states of flavins should interact much
more strongly than the ground state with metal ions (Varnes et al 1971) The mechanism
of photolysis reactions of RF in the absence of metal ions has been discussed in detail
(Heelis 1982 1951 Ahmad and Vaid 2006) The mode of interaction or complexation
of different metal ions with RF to enhance its degradation appears to be different It has
been shown that the monovalent metal ions (eg Ag+) form a 11 red complex with RF in
which the Ag+ atom binds to the flavin (isoalloxazine) ring (Weber 1950 Baarda and
Metzler 1961 Bamberg and Hemmerich 1961) The divalent ions (eg Fe2+
) bind to RF
214
in the N(5)ndashC(4a)ndashC(4)ndashO(4) site to form a planar fivendashmembered chelate ring (Kaim et
al 1999 Fukuzumi et al 1985 Fukuzumi and Kojima 2008) (Fig 82) Similarly the
trivalent ions (eg Fe3+
) also form a planar fivendashmembered chelate ring similar to that of
the divalent ions with RF (Fukuzumi et al 1985 Fukuzumi and Kojima 2008
Fukuzumi and Okhubo 2010) Thus all the divalent and trivalent metal ions enhance the
photolysis of RF through metalndashRF complexation
In view of the results obtained in this study indicating the role of metal ions as
promoters of photolysis of RF a scheme for the sequence of reactions involved may be
presented (Fig 844)
RF reacts with a metal ion eg Fe2+
ion to form a [RFhellipFe2+
] complex (Eq
(85)) This complex on absorption of a photon of light is promoted to the excited singlet
state [1RFhellipFe
2+] (Eq (86)) In this state charge transfer takes place resulting in the
formation of a loosely bound semireduced semiquinone radical [RFH] and an oxidized
[Fe3+
] ion (Eq (87)) followed by their separation to give free [RFH] radicals and Fe3+
ions (Eq (88)) 2[RFH] radicals react to give a reduced RF molecule [RFprimeH2] with an
altered side chain (Eq (89)) The [RFprimeH2] molecules are oxidized by air to form FMF
and sidendashchain products (Eq (810)) FMF then undergoes hydrolysis to give LC LF and
sidendashchain products as the final photoproducts of RF (Eq (811)) The [1RFhellipFe
2+] state
in the presence of HPO42ndash
ions leads to the formation of a CDRF molecule and a Fe3+
ion
(Eq (812))
215
RF + Fe2+ [RFFe2+]
metal-RF complex
[RFFe2+] [1RFFe2+]
excited singlet state complex
[1RFFe2+] [RFHFe3+]
[RFHFe3+] RFH
+ Fe3+
2RFH RFH2
RFH2 FMF + side-chain products
FMF LC + LF + side-chain products
[1RFFe2+] CDRF + Fe3+ HPO
42-
H+ OH_
O2
(85)
(86)
(87)
(88)
(89)
(810)
(811)
(812)
Fig 844 Scheme for the photolysis of RF in metalndashRF complex
The reaction scheme described for the photochemical interaction of Fe2+
ions and
RF (Eq (81)ndash(812)) may be considered analogous to that presented for the
photostabilization of RF by phosphatidylcholine (PC) in liposomes It involves the
formation of a photoinduced charge transfer complex between RF and PC (Ahmad et al
2015 Bhowmik and Sil 2004) and norfloxacin and PC (Ahmad et al 2016) as a basis of
the stabilization of these drugs in liposomes
CHAPTER IX
PHOTOCHEMICAL PREPARATION
CHARACTERIZATION AND FORMATION
KINETICS OF RIBOFLAVIN CONJUGATED
SILVER NANOPARTICLES
217
91 INTRODUCTION
Nanoparticles (NPs) are a rapidly growing field in nanotechnology due to their
size (nm) and unique characteristics which make them an ideal candidate for application
in physical chemical and biological systems (Nairn et al 2006 Noguchi et al 2011
Routh et al 2012 Arce et al 2014 Bala et al 2016 Foresti et al 2017) NPs exhibit a
particle size of less than 100 nm and possess versatile properties as compared to the bulk
material of a compound They need high pressure energy or temperature for their
formation They also require some toxic material for their stabilization which may lead to
adverse effects when subjected to biomedical and pharmaceutical applications (Goodsell
2004 Abbasi et al 2016 Rajavel et al 2017)
Different methods have been used for the preparation of silver (Ag) NPs ie
sequential injection method (Passos et al 2015) chemical reduction (Wei et al 2015)
photochemical reduction (Chen et al 2007 Frattini et al 2005) irradiationndashassisted
chemical reaction (Sotiriou et al 2010) electrochemical reduction (Abbasi et al 2016)
biosynthesis (Ramanathan et al 2013) lithography (Ahmed et al 2016) and physical
methods (Dang et al 2014 Tien et al 2008) The mechanism of formation of Ag NPs
(Hussain et al 2011) RF conjugated ZnO NPs (Bala et al 2016) and Cu NPs (Noguchi
et al 2011) has been described Ag NPs are of great importance due to their unique
features and different applications in the fields of drug delivery (Benyettou et al 2015)
food technology (Costa et al 2011 De Moura et al 2012) agriculture (Kim et al
2012) environmental technology (Benn and Westerhoff 2008) catalysis (Huang et al
2012) water purification (Das et al 2012) and textile industry (Ilic et al 2009
Montazer et al 2012)
218
Riboflavin (RF) (1) is a photosensitive vitamin (British Pharmacopoeia 2016)
and acts as an important precursor for the synthesis of flavin mononucleotide (FMN) and
flavin adenine dinucleotide (FAD) (Foraker et al 2003) It is widely used for the
treatment of neonatal jaundice (Ebbesen et al 2015) HIV induced infections (Leeansyah
et al 2015 Fernandez et al 2015) and keratoconus (Henriquez et al 2011 Farjadina
and Naderan 2015) In photodynamic therapy RF is used as a potential drug to kill tumor
tissues (Ionita et al 2003) and colorectal adenomas (Figueiredo et al 2008) RF along
with magnesium citrate and condashenzyme Q10 is effectively used for the prevention of
migraine (Gaul et al 2015) When exposed to light RF is rapidly degraded to form
different photoproducts (ie formylmethylflavin (FMF) (4) lumichromre (LC)
(5) lumiflavin (LF) (6) and carboxymethylflavin (CMF) (7)) (Smith and Metzler 1963
Cairns and Metzler 1971 Ahmad et al 2004 2014 2016) (Fig 91) Due to the
photosensitive nature of RF different attempts have been made for its stabilization using
liposomal preparations (Habib and Asker 1991 Loukas et al 1995ab Senndashverma et al
1995 Bhowmik and Sil 2004 Ahmad et al 2015) complexation with chemical agents
(Evstigneev et al 2005 Ahmad et al 2009 Sheraz et al 2014a) and cyclodextrins (CD)
(Loukas et al 1995ab Terekhova et al 2011ab) stabilizers (Asker and Habib 1990)
and borate (Ahmad et al 2008) and citrate buffers (Ahmad et al 2011)
RF is known to form complexes with Ag+ ions and other metal ions (Weber
1950 Wade and Fritiche 1973 Ahmad et al 2017) Different studies have been carried
out on the interaction of RF with Ag NPs (Voicescu et al 2013 Routh et al 2012
Mokashi et al 2014) photoactivation of RF by Ag NPs (Khaydukov et al 2016)
detection of RF by Ag NPs (Ma et al 2016) effect of Ag NPs on the photophysics of RF
219
N N
NNH
O
O
OH
OH
OH
OH
CH3
CH3
H
H
H
HH
N N
NNH
O
OCH3
CH3
CH2
CHO
N NH
NNH
O
OCH3
CH3
N N
NNH
O
OCH3
CH3
CH2
COOH
N N
NNH
O
OCH3
CH3
CH3
(1)(4)
(5)(7) (6)
[O] neutral and alkaline pHacid neutral
and alkaline pH
Fig 91 Photodegradation pathway of RF
220
(Rivas Aiello et al 2016) preparation of RF conjugated Zn NPs (Bala et al 2016) and
Cu NPs (Noguchi et al 2003 2011) and adsorption of RF on the surface of silver (Liu et
al 2012 Akhond et al 2016) However there is a dearth of information on the effect of
some factors on the formation of RFndashAg NPs in these studies The object of present
investigation is to sprepare RFndashconjugated silver nanoparticles (Ag NPs) by
photoreduction their characterization by physical methods and the evaluation of the
effect of pH ionic strength concentration of Ag+ ions and irradiation source (visible
light UV light) on the formation kinetics of RFndashAg NPs
The experimental details involved in these studies are presented in 53
(Chapter 5)
92 RESULTS AND DISCUSSION
921 Characterization of RFndashConjugated Ag NPs
9211 Optical studies
A colour change of the RFndashAg NPs solution (yellow green to brown) was
observed which indicated the formation of RFndashconjugated Ag NPs (Fig 92) This
change in colour was due to the reduction of Ag+ ions into Ag NPs (AbdelndashHafez et al
2016 Krupa et al 2016 Mosae Selvakumar et al 2016 Alzahrani et al 2017)
9212 Spectral characteristics of RFndashAg NPs
RF exhibits absorption maxima at 223 267 374 and 444 nm in aqueous solution
(British Pharmacopoeia 2016) Ag NPs absorb in the visible region with the appearance
of a surface Plasmon resonance (SPR) band depending on the size and shape of Ag NPs
221
Fig 92 Colour change for the formation of RFndashAg NPs from yellow green
to brown
222
(Haes and Van Duyne 2002 Lee et al 2008 Amendola et al 2010 Hou and Cronin
2013 Mogensen and Kneipp 2014) The absorption maxima of SPR band of Ag NPs
have been reported in the wavelength range of 408ndash422 nm (Chairam and Somsook
2008 Tai et al 2008 Chairam et al 2009)
In the present study the effect of photochemical interaction between RF and Ag+
ions and the formation of Ag NPs on changes in their spectral characteristics has been
investigated The absorption spectrum of RF and the changes occurring on the addition of
AgNO3 formation of Ag NPs and interaction of RF with Ag NPs during a period of 6 h
are shown in Fig 93 There is a significant change in the 374 and 444 nm bands of RF
which undergo bathochromic (red) and hypsochromic (blue) shift respectively to form
the SPR band of Ag NPs with a maximum at 422 nm Similar spectral shifts of RF
maxima to form a SPR band of Ag NPs (426 nm) have been observed by Zhang et al
(2011) and Mokashi et al (2014) These spectral changes have been attributed to the
interaction of RF and Ag NPs through the hydroxyl group or methyl groups (Mokashi et
al 2014) The spectra also show a gradual increase in the absorption at 267 nm
maximum of RF during the interaction with Ag NPs An increase in RF absorption in
250ndash300 nm region with an increase in Ag NPs concentration is probably due to greater
interaction between the two species (Mohashi et al 2014)
9213 Fluorescence characteristics of RF
RF is a highly fluorescent compound and emits fluorescence in the 520ndash530 nm
region (Weber 1950 Varnes et al 1972 Heelis et al 1981 Sikorska et al 2005
Ahmad and Vaid 2006 Arce et al 2014 Ahmad et al 2017)
223
Fig 93 Absorption spectra of RF and RFndashAg NPs
224
Its fluorescence is quenched by acid and alkali (Weber 1950) complexation with organic
compounds (Penzer and Radda 1967) and metal ions including Ag+ ions (Weber 1950
Wade and Fritchie 1973 Ahmad et al 2107) The fluorescence of aqueous solutions of
RF is also quenching by Ag NPs (Zhang et al 2011 Mokashi et al 2014 Rivas Aiello
et al 2016) Cu NPs (Noguchi et al 2011) and cinnamic alcohol chemisorbed on silica
NPs (Arce et al 2014)
The fluorescence quenching of RF by Ag NPs observed in this study is shown in
Fig 94 and a plot of fluorescence loss versus irradiation time is shown in Fig 95 The
loss of fluorescence intensity of RF at 525 nm is due to the interaction of RF and Ag NPs
and the total loss of fluorescence indicates complete conversion of RF to form the RFndashAg
NPs conjugates It has been suggested that the fluorescence quenching of RF by Ag NPs
is due to the fluorescence energy transfer (FRET) from RF (donor) to Ag NPs (acceptor)
on the adsorption of RF (Mokashi et al 2014) A photoinduced electron transfer from
excited RF to metal ions such as Cu2+
ions resulting in loss of fluorescence and copper
deposition has been reported (Morishita and Suzuki 1995 Noguchi et al 2003 2011)
Such photoinduced electron transfer reactions have been observed in the formation of Ag
colloids (Mennig et al 1992 Lei et al 2017) and Cu NPs (Giuffrida et al 2004)
9214 FTIR studies
FTIR studies have been carried out to confirm the structure of RF and to ascertain
the nature of interaction between RF and Ag NPs The FTIR spectra of RF and RFndashAg
NPs conjugates are shown in Fig 96 RF (Fig 96a) exhibits strong absorption peaks at