Wajiha Gul Pharmaceutical Chemistry 2019 Uok Karachi Prr.Pdf

INTRAMOLECULAR PHOTOADDITION REACTIONS OF RIBOFLAVIN USING DIFFERENT DIVALENT IONS

For the partial fulfillment of Ph.D degree

WAJIHA GUL B.Pharm., M.Phil., R.Ph.

Department of Pharmaceutical Chemistry Faculty of Pharmacy and Pharmaceutical Sciences University of Karachi Karachi 75270 Pakistan 2019

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My Beloved Father

Late Mohammad Younus Fareed

ABSTRACT

The present study is based on an investigation of the effect of non-reducing and reducing anions (0.2-1.0 M) on the photolysisof riboflavin (RF) at pH 6.0-8.0. These anions convert the normal degradation (photoreduction) pathway in favor of photoaddition pathway of RF, depending on the catatlytic activity of RF-dianion complex that initiates phototaddition to form cyclodehydroriboflavin (CDRF). This is in contrast to photoreduction to give formylmethylflavin (FMF), lumichrome (LC) and lumiflavin

(LF). The degree of change in reaction pathways of RF depends on the strength of RF- dianion complex as observed by the loss of fluorescence resulting from complex formation. All the products have been found to be formed by simultaneous first-order reaction. The values of first-order rate constants for the overall photolysis of RF and second-order rate constants for photochemical interaction of RF and the dianions

(carbonate, phosphate, oxalate , phthalate) range from 1.81 to 8.12 min-1 and 0.76-5.48 x10-3 m1, respectively. The maximum rate of reaction has been found to occur at pH 7.0 in the order: carbonate > phosphate > oxalate > phthalate.

The photolysis of RF in presence of sulfur containing reducing dianions is different from those of the non-reducing compounds as described above. The reducing dianions cause photoreduction, phtotaddition as well as chemical reduction of RF solutions during irradiation to give LC, CDRF and dihydroriboflavin (DHRF) , respectively, as the final products. The first-order rate constants for the overall photolysis of RF, and the formation of LC, CDRF and DHRF at pH 7 are 1.52 to 3.07 and 0.68 to

1.61, 0.36 to 0.92, and 0.21 to 0.68 x10-3 min-1, respectively. The second-order rate constants for photochemical interaction of RF and dianions and the chemical reduction of

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RF in the absence of light are 1.42 to 2.01 and 2.31 to 3.25 x 10-3 M-1min-1, respectively.

Thiosulfate dianions show highest chemical reduction of RF having a lower redox potential compared to that of sulfite dianions.

RF photoaddition is maximum at pH 7.0 and is catalyzed in the order of dianions: metabisulfite> sulfite > thiosulfate and chemical reduction is enhanced in the reverse order. The photolysis quantum yield in presence of the dianions is reported. The degradation products of RF have been identified by chromatographic and UV-vis spectrometric methods. RF and photoproducts in degraded solutions have been determined by a multicomponent spectrometric method to avoid any interference in the assay of these compounds at the analytical wavelengths. The UV-vis spectral and fluorescence characteristics of RF solutions containing various dianions are reported. The mode of photolysis reactions of RF under these conditions is discussed.

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ACKNOWLEDGEMENTS

First of all, I am grateful to Almighty Allah for protection and ability to conduct this work. My whole-hearted respect to the prophet Hazrat Mohammad (S.A.W) for complete and endless guidance and knowledge.

I am very much thankful to my supervisor Prof. Dr. FaiyazVaid, Department of

Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, for his guidance and support during my Ph.D studies.

I would like to express my sincere gratitude to my co-supervisor Prof. Dr. Iqbal

Ahmad (T.I), Department of Pharmaceutical Chemistry, Baqai Medical University, for the continuous support of my research work and also for his patience, motivation and guidance which helped me in completion of my work. I could not have imagined a better co-supervisor than him for my Ph. D. study.

I am thankful to Prof. Dr. Nosheen Mushtaq, Chairperson, Department of

Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, for her kind support in my work.

I am very much grateful to Prof. Dr. Shukat Khalid, Dean Faculty of

Pharmaceutical Sciences, Baqai Medical University, for allowing me to work in her

Faculty. I am also thankful to Dr. Kiran Qadeer, Chairperson of the Department of

Pharmaceutical Chemistry for her support. My work wouldn’t have been completed without the support of Prof. Dr. Sofia Ahmed and Prof. Dr. Sheraz Ali, for which I will always be thankful.

I would also wish to recognize the valuable help provided during my research by

Dr. Zubair Anwar who helped me selflessly and was always available for guiding me.

My thanks and appreciation is also due to Dr. Ahsan Ejaz Khan and Dr. Adeela

Khursheed for their immense help. I am also grateful to Dr. Mona Mehboob Khan,

Chairperson, Pharmaceutical Chemistry Department, Dow College of Pharmacy, for her kind support.

I would also like to recognize the help of Mr. Rashid and Mr. Amir, laboratory staff of Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of

Karachi and also Mr. Sajjad, Mr. Aneesand Mr. Wajahat of Research Laboratories of

Baqai Institute of Pharmaceutical Sciences.

A special gratitude goes to Mrs. Prof. Dr. Iqbal Ahmad for her love and hospitality during my visits.

I am grateful to my mother (Fareeda Fareed), sister (Sadia Fareed), husband

(Nasheed Irshad Hussain) for their encouragement and prays throughout my work, my late guardian (Nadira Rehmatullah) and maternal uncle (Abdul Hameed) for their prayers and help, they did throughout my life. Last but not the least I am very thankful to my children (Maliha and Samiullah) as they deserved the time I spend on my research work.

W.G.

List of Tables

Table Title Page 1. Physicochemical properties of riboflavin 4 2. Analytical methods used for the detection of RF 13

2.1. Rf values of RF and photoproducts 69 3. Product composition at 30% photodegradation of RF solution (pH 7.0) in 71 the presence and absence of 1.0 M divalent anions 4. Concentration of RF and photoproducts in the presence of carbonate 76 dianions (0.2 - 1.0 M) at pH 6.0 5. Concentration of RF and photoproducts in the presence of carbonate 77 dianions (0.2 - 1.0 M) at pH 6.5 6. Concentration of RF and photoproducts in the presence of carbonate 78 dianions (0.2 - 1.0 M) at pH 7.0 7. Concentration of RF and photoproducts in the presence of carbonate 79 dianions (0.2 - 1.0 M) at pH 7.5 8. Concentration of RF and photoproducts in the presence of carbonate 80 dianions (0.2 - 1.0 M) at pH 8.0 9. Concentration of RF and photoproducts in the presence of phosphate 81 dianions (0.2 - 1.0 M) at pH 6.0 10. Concentration of RF and photoproducts in the presence of phosphate 82 dianions (0.2 - 1.0 M) at pH 6.5 11. Concentration of RF and photoproducts in the presence of phosphate 83 dianions (0.2 - 1.0 M) at pH 7.0 12. Concentration of RF and photoproducts in the presence of phosphate 84 dianions (0.2 - 1.0 M) at pH 7.5 13. Concentration of RF and photoproducts in the presence of phosphate 85 dianions (0.2 - 1.0 M) at pH 8.0 14. Concentration of RF and photoproducts in the presence of oxalate dianions 86 (0.2 - 1.0 M) at pH 6.0 15. Concentration of RF and photoproducts in the presence of oxalate dianions 87 (0.2 - 1.0 M) at pH 6.5

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16. Concentration of RF and photoproducts in the presence of oxalate dianions 88 (0.2 - 1.0 M) at pH 7.0 17. Concentration of RF and photoproducts in the presence of oxalate dianions 89 (0.2 - 1.0 M) at pH 7.5 18 Concentration of RF and photoproducts in the presence of oxalate dianions 90 (0.2 - 1.0 M) at pH 8.0 19. Concentration of RF and photoproducts in the presence of phthalate 91 dianions (0.2 - 1.0 M) at pH 6.0 20. Concentration of RF and photoproducts in the presence of phthalate 92 dianions (0.2 - 1.0 M) at pH 6.5 21. Concentration of RF and photoproducts in the presence of phthalate 93 dianions (0.2 - 1.0 M) at pH 7.0 22. Concentration of RF and photoproducts in the presence of phthalate 94 dianions (0.2 - 1.0 M) at pH 7.5 23. Concentration of RF and photoproducts in the presence of phthalate 95 dianions (0.2 - 1.0 M) at pH 8.0

24. Apparent first-order rate constants for photodegradation of RF (kobs), 119

formation of CDRF (k1) and LC (k2) in the presence of divalent anions and second-order rate constants (kʹ) for photochemical interaction of RF and divalent anions 25. Product distribution at 30% photodegradation of 5 × 10-5 M riboflavin 132 solution (pH 7.0) in the presence of 1.0 M dianions 26. Concentrations of RF and photoproducts in the presence of thiosulfate 134 dianions (0.2-1.0 M) at pH 6.0 27. Concentrations of RF and photoproducts in the presence of thiosulfate 135 dianions (0.2-1.0 M) at pH 6.5 28. Concentrations of RF and photoproducts in the presence of thiosulfate 136 dianions (0.2-1.0 M) at pH 7.0 29. Concentrations of RF and photoproducts in the presence of thiosulfate 137 dianions (0.2-1.0 M) at pH 7.5 30. Concentrations of RF and photoproducts in the presence of thiosulfate 138 dianions (0.2-1.0 M) at pH 8.0

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31. Concentrations of RF and photoproducts in the presence of sulfite dianions 139 (0.2-1.0 M) at pH 6.0 32. Concentrations of RF and photoproducts in the presence of sulfite dianions 140 (0.2-1.0 M) at pH 6.5 33. Concentrations of RF and photoproducts in the presence of sulfite dianions 141 (0.2-1.0 M) at pH 7.0 34. Concentrations of RF and photoproducts in the presence of sulfite dianions 142 (0.2-1.0 M) at pH 7.5 35. Concentrations of RF and photoproducts in the presence of sulfite dianions 143 (0.2-1.0 M) at pH 8.0 36. Concentrations of RF and photoproducts in the presence of metabisulfite 144 dianions (0.2-1.0 M) at pH 6.0 37. Concentrations of RF and photoproducts in the presence of metabisulfite 145 dianions (0.2-1.0 M) at pH 6.5 38 Concentrations of RF and photoproducts in the presence of metabisulfite 146 dianions (0.2-1.0 M) at pH 7.0 39. Concentrations of RF and photoproducts in the presence of metabisulfite 147 dianions (0.2-1.0 M) at pH 7.5 40. Concentrations of RF and photoproducts in the presence of metabisulfite 148 dianions (0.2-1.0 M) at pH 8.0

41. First-order rate constants for photodegradation of riboflavin (kobs), formation 172

of cyclodehydroxyribiflavin (k1), lumichrome (k2), and dihydroxyriboflavin

(k3) in the presence of dianion and quantum yields (Φ) of the reaction 42. Second-order rate constants for the photochemical (kʹ) and chemical dark 177 interaction(k″) of riboflavin and dianions at pH 7.0

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List of Figures

Figure Title Page 1. Chemical structures of RF, FMN and FAD 3 2. Structures of RF and its photoproducts 27 3. Scheme for the photodegradation pathways of riboflavin 28 4. Hydrolysis of FMF to LC and LF 34 5. Formation of 1,5-dihydrogen flavin and alkyl adducts from flavin 35 through photodegradation 6. Photodegradation reaction of riboflavin and formation of 68 photoproducts 7. UV and visible absorption spectra of RF solutions (pH 7.0) on 72 photodegradation in the presence of 1.0 M divalent anions: carbonate (a), oxalate (b) and phthalate (c). Times indicated are in minutes. 8. % age fluorescence of RF in the presence of divalent anions (0.2-1.0 74 M): (●) phthalate; (■) oxalate; (♦) phosphate; (▲) carbonate 9. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 6.0) in 96 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 10. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 6.5) in 97 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 11. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 7.0) in 98 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 12. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 7.5) in 99 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and formation of photoproducts: (▲) CDRF; (■) LC;

(♦) FMF; (×) LF. 13. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 8.0) in 100 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 14. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 6.0) in 101 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 15. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 6.5) in 102 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 16. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 7.0) in 103 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 17. Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 7.5) in 104 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 18 Kinetic plots of photogegradation of (5.0×10-5) RF solution (pH 8.0) in 105 the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and formation of photoproducts: (▲) CDRF; (■) LC; (♦) FMF; (×) LF. 19. Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 6.0) 106 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 20. Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 6.5) 107 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0

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M oxalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 21. Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 7.0) 108 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 22. Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 7.5) 109 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 23. Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 8.0) 110 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 24 Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 6.0) 111 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 25 Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 6.5) 112 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 26 Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 7.0) 113 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 27. Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 7.5) 114 in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 28. Kinetic plots of photodegradation of (5.0×10–5 M) RF solution (pH 8.0) 115

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in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the formation of photoproducts: (▲) CDRF; (■)LC; (♦) FMF; (×) LF. 29. Second-order plots for the photochemical interaction of RF and 118 divalent anions: (●) phthalate; (■) oxalate; (♦) phosphate; (▲) carbonate.

30. Plots of kobs for the photodegradation of RF in the presence of divalent 123 anions (1.0 M) versus pH: (●) phthalate; (■) oxalate; (♦) phosphate; (▲) carbonate.

31. Plots of loss of RF (kobs) and formation of CDRF (k1) and LC (k2) as a 125 function of % fluorescence loss of RF in the presence of divalent anions (1.0 M): (●) phthalate; (■) oxalate; (♦) phosphate; (▲) carbonate. 32. UV and visible spectra of RF solutions (Ph 7.0) on photodegradation in 149 2- 2- 2- the presence of 1 M dianions: S2O3 (a); SO3 (b); S2O5 (c). Times indicated are in minutes 33. UV and visible absorption spectra of RF solutions (Ph 7) on chemical 150 2- 2- 2- reduction in the presence of 1 M dianions: S2O3 (a); SO3 (b); S2O5 (c). Times indicated are in minutes 34. Percent fluorescence of RF in the prescence of 0.2-1.0 M dianions: (●) 152 2- 2- 2- S2O3 ; (▲) SO3 ; (♦) S2O5 . 35. Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.0) in the 154 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 36. Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.5) in the 155 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 37. Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.0) in the 156 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M

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thiosulfate anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 38. Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.5) in the 157 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 39. Photodegradation of 5 × 10-5 M RF (●) solution (pH 8.0) in the 158 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 40. Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.0) in the 159 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 41. Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.5) in the 160 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 42. Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.0) in the 161 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 43. Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.5) in the 162 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 44. Photodegradation of 5 × 10-5 M RF (●) solution (pH 8.0) in the 163 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 45. Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.0) in the 164

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presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 46. Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.5) in the 165 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 47. Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.0) in the 166 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 48. Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.5) in the 167 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 49. Photodegradation of 5 × 10-5 M RF (●) solution (pH 8.0) in the 168 presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfite anions and formation of (▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF. 50. Second-order plots for: (a) photochemical interaction; (b) chemical 176 2- 2 reduction of RF in the presence dianions: (●) S2O3 ; (▲) SO3 ; (♦) 2- S2O5 .

51. Plots of kobs for RF versus pH in the presence of 1 M dianions (●) 179 2- 2 2- S2O3 ;(▲) SO3 ;(♦) S2O5 : (a) photodegradation; (b) chemical reduction.

52. Plots of photodegradation of RF (kobs) and formation of CDRF (k1), LC 182

(k2) and DHRF (k3) versus percent fluorescence loss in the presence of 2- 2 2- 1 M dianions: (●) S2O3 ; (▲) SO3 ; (♦) S2O5 . 53. Scheme for photodegradation reactions of RF in presence of sulfur 185 containing dianions. .

LIST OF ABBREVIATIONS

AM Absorbance matrix ASM Absorbance sum matrix CDRF Cyclodehydroriboflavin CM Concentration matrix CMF Carboxymethylflavin DHRF Dihydroriboflavin FAD Flavin adenine dinucleotide FMN Flavin mononucleotide HPLC High-performance liquid chromatography ic Intersystem crossing LC Lumichrome LF Lumiflavin nm Nanometers PLS Partial least square PVP Polyvinylpyrrolidone RF Riboflavin

Rf Retention factor RSD Relative standard deviation SDS Sodium dodecyl sulfate TLC Thin-layer chromatography

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CONTENTS

Chapter Page

ABSTRACT vii

ACKNOWLEDGEMENT x

1 INTRODUCTION TO RIBOFLAVIN

1.1 HISTORIC BACKGROUND AND IMPORTANCE OF 2 RIBOFLAVIN 1.2 SOURCES 5

1.3 BIOCHEMICAL FUNCTIONS 5

1.4 PHARMACOKINETICS 6

1.5 RIBOFLAVIN DEFICIENCIES 7

1.6 RIBOFLAVIN INTERACTIONS 8

1.7 PHARMACEUTICAL PREPARATIONS AND DOSAGE FORMS 9

1.8 SOURCES OF LITERATURE 9

2 ANALYTICAL METHODS FOR THE DETERMINATION 11 OF RIBOFLAVIN AND RELATED COMPOUNDS 2.1 SPECTROSCOPIC METHODS 12

2.1.1 Ultraviolet and Visible Spectrometry 12

2.1.2 Spectrofluorimetry 15

2.1.4 Mass Spectrometry 17

2.2 CHROMATOGRAPHIC METHODS 19

2.2.1 Thin-layer Chromatography 19

2.2.2 High-performance Liquid Chromatography 20

2.3 ELCTROCHEMICAL METHODS 22

2.3.1 Potentiometry 22

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2.3.2 Polarography 23

3 PHOTOCHEMISTRY OF RIBOFLAVIN AND RELATED COMPOUNDS 3.1 TYPES OF PHOTOREACTIONS OF RIBOFLAVIN 29

3.1.1 Aerobic Photoreactions 30

3.1.2 Anaerobic Photoreactions 30

3.2 PHOTOCHEMICAL REACTIONS OF RIBOFLAVIN 32

3.2.1 Photoreduction Reactions 32

3.2.1.1 IntramolecularPhotoreduction 32

3.2.1.2 Intermolecular Photoreduction 33

3.2.1.3 IntramolecularPhotodealkylation 37

3.2.2 IntramolecularPhotoaddition Reactions 38

3.2.3 Photosensitization Reaction 38

3.2.3.1 PhotooxidationReactions 39

3.2.3.2 Photodecarboxylation Reactions 40

3.2.3.3 Photoisomerization 40

3.2.3.4 Photomoomerization 40

3.2.3.5 Photoinactivation 41

3.2.3.6 Photomodification and Photoinduction 41

3.3 FACTORS AFFECTING THE PHOTOCHEMICAL 41

REACTIONS OF RIBOFLAVIN

3.3.1 Intensity, Wavelength and Light Source 41

3.3.2 pH 43

3.3.3 Buffers 44

3.3.4 Quenchers 45

xxiii

3.3.5 Solvents 45

3.3.6 Ionic Strength 46

3.3.7 Metal Ions 46

3.3.8 Formulations 46

OBJECT OF PRESENT INVESTIGATION 48

PLAN OF WORK 50

4 MATERIALS AND METHODS

4.1 MATERIALS 51

4.1.1 Chemicals 51

4.1.2 Reagents and Solvents 52

4.1.3 Buffers 52

4.1.4 Radiation Chamber 52

4.2 METHODS 53

4.2.1 Thin-layer Chromatography 53

4.2.2 Spectral Measurements 54

4.2.3 Fluorescent Measurements 54

4.2.4 pH Measurements 54

4.2.5 Reaction Vessel 54

4.2.6 Radiation Source 55

4.2.7 Determination of Quantum yield (Φ) 55

4.2.8 Photolysis 57

4.2.9 Assay Method 57

4.2.10 Assay Parameters 58

4.2.11 Methods of Calculation of Concentration in the Assay 59

xxiv

4.2.11.1 One Component Assay 59

4.2.11.2 Two Component Assay 60

4.2.11.3 Three Component Assay 61

5 EFFECT OF DIVALENT ANIONS ON THE PHOTODEGRADATION OF RIBOFLAVIN IN AQUEOUS SOLUTION 5.1 INTODUCTION 66

5.2 RESULT AND DISCUSSION 67

5.2.1 Identification of Riboflavin Products 67

5.2.2 Composition of Photoproducts of Riboflavin 70

5.2.3 Spectral characteristics of Photodegraded Solutions of Riboflavin 70

5.2.4 Riboflavin Fluorescence in the Presence of Divalent Anions 73

5.2.5 Assay of Riboflavin and Photoproducts 75

5.2.6 Photodegradation Kinetics 75

5.2.7 pH Effect 117

5.2.8 Riboflavin-Divalent Anion Complex Formation 124

5.2.9 Photolysis Pathways of Riboflavin 126

5.2.10 Mode of PhotoadditionReaction 126

6 SIMULTANEOUS PHOTOADDITION, PHOTOREDUCTION AND CHEMICAL REDUCTION OF RIBOFLAVIN BY SULFUR CONTAINING DIANIONS: A KINETIC STUDY 6.1 INTRODUCTION 129

6.2 RESULTS AND DISCUSSION 130

6.2.1 Identification of Photoproducts of Riboflavin 130

6.2.2 Distribution of Photoproducts 131

6.2.3 Assay of Riboflavin and Photoproducts 131

xxv

6.2.4 Ultra violet and Visible Spectra of Photodegraded Solutions 133

6.2.5 Fluorescence Characteristics of Riboflavin 151

6.2.6 Kinetics of Photodegradation 151

6.2.7 pH Effect 178

6.2.8 Redox Potential Effect 180

6.2.9 Interaction of Riboflavin with Dianions 180

6.2.10 Photolysis of Quantum Yield 181

6.2.11 Mode of Riboflavin Photodegradation in the Presence of Sulfur 183 Containing Dianions CONCLUSION 186

REFERENCES 188

xxvi

CHAPTER 1

INTRODUCTION

1.1 HISTORIC BACKGROUND AND IMPORTANCE OF RIBOFLAVIN

The presence of a yellow-green fluorescent compound in milk was first observed by an English chemist Alexander Blyth in 1872 and he named it as lactochrome because of its color and flavin origin (Norhrop-Clewes and Thurnham, 2012). However, the physiological role of this yellow component was discovered by Warburg and Christian who found it to be a part of the yeast ‘Zwischenferment’ or ‘old yellow enzyme’

(Warburg and Walter, 1932). Previously it was also referred to as ovoflavin, uroflavin

(depending on its source) and vitamin G (as was known as the dietary factor required for growth). The vitamin was given its name as ‘riboflavin’ because of the sugar alcohol present in its structure (ribitol) and its yellow colour (flavus means yellow in Latin)

(Monteiro and Perrone, 2013). The synthesis of riboflavin (RF) was carried out by Kuhn et al. (1935) and Karrer et al. (1935) while its coenzymes, flavin-5-mononucleotide

(FMN) and flavin adenosine dinucleotide (FAD), were identified in 1937 and 1938 by

Theorell and Warburg and Christian respectively (Rivlin and Pinto, 2001) (Fig. 1). The

RF molecule is comprised of an isoalloxazine ring and a ribose side- chain. The structure- activity relationship of many of the biologically active flavins has been studied extensively (Merrill et al., 1981).

RF is available either in free form or as its coenzymes flavin mononucleotide

(FMN) and flavin dinucleotide and (FAD) (Fig. 1), which occur as a prosthetic group of flavoproteins. It is involved in energy production and acts as a catalyst for redox reactions in many metabolic pathways (Rivlin and Pinto, 2001). The physicochemical properties of RF are summarized in Table 1.

HO H HO H HO H H H

H3C N N O

NH H3C N O Riboflavin (RF)

H H H OH

H2C CH 2O P O OH OH OH OH N N O H3C

NH N H3C O

Flavin mononucleotide (FMN)

H H H O O

H2C CH 2O P O P CH 2O OH OH OH OH OH N N O CH H3C H OH NH NH 2 N O H OH H3C N C C N O HC C CH CH N N Flavin adenine dinucleotide (FAD)

Fig. 1: Chemical structures of RF, FMN and FAD

Table 1: Physicochemical properties of RFa

IUPAC name 7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5-

tetrahydropentyl]benzo[g]pterine-2,4-dione

Molecular formula C17H20N4O6

Molecular weight 376.3636 g/mol

Crystalline form fine needles

Melting point 280 0C (decomposes) pH, saturated solution ~ 6

o pKas 1.9, 10.2 (20 )

Redox potential

(riboflavin/ dihydroriboflavin), pH 7.0 -0.208 V

Solubility, g/100 ml

Water 0.033-0.606

Ethanol 0.0045

Acetone, chloroform, ether, Insoluble

Benzene

Ultraviolet and visible absorption maxima 223, 267, 373 and 444 nm

(pH 7.0)

Fluorescence emission (pH 7.0) 520 nm

Principle infrared peaks (KBr disk) 1544, 1575, 1641, 1715, 1235, 1070 cm-1 a (O’ Neil, 2013; Rivlin and Pinto, 2001; Watson, 2005; Sinko, 2006;British Pharmacopoeia, 2016)

1.2 SOURCES

Milk is an excellent source of this vitamin and four cups of milk can fulfill the daily requirement (Lopez et al., 1980). Vegetables and animal proteins, even when cooked, are good sources of RF. It is also found in the culture media of a number of fungi and bacteria (flavinogenic microorganisms) (Schlee and Strauba, 1984). Legumes, cereals, pasta, rice, eggs, fish and organ meats are also good sources of vitamin B2 (Moss,

1989), fruits, however, are relatively poor sources (Yoneda, 1984). RF from animal source shows better absorption and higher bioavailability as compared to vegetable source.

1.3 BIOCHEMICAL FUNCTIONS

RF can be used in clinical and therapeutic scenarios. It is a must for many cellular processes (Powers, 2003; Sarubin and Thomson, 2007; Escott-Stump, 2008). Various studies have been performed in the last two decades regarding the role of RF in metabolism and in several diseases. The coenzymes of RF i.e. FMN and FAD are also used by the flavoproteins for many metabolic reactions. Nutritional state, hormones, drugs and other stimuli regulate the formation of these coenzymes (Rivlin, 2007). In higher doses RF can prevent migrane (Schoenen et al., 1998; Silberstein et al., 2000;

Thompson and Saluja, 2017), anemia, visual impairment and infant neurological abnormalities (Leshner, 1981). It is also effective against cataract (Cumming et al.,

2000). Coenzymes of RF participate in oxidation-reduction reactions involved in metabolic pathways and in energy production via the respiratory chain. The flavins act as an oxidizing agent and play their role in the oxidation of aldehydes and aliphatic side

5 chains and in the transfer of hydrogen atoms from pyridine nucleotides to chromosomes c. RF with pyridoxine is involved in the formation of niacin from tryptophan (Ottaway,

2012).

RF is an important antioxidant and it plays a major protective action against lipid peroxidase involved in the glutathione redox cycle (Dutta, 1993). Thus RF nutrition may be critical in regulating the inactivation rate of lipid peroxidase (Rivlin and Dutta, 1995)

1.4 PHARMACOKINETICS

Primary absorption of riboflavin occurs in the proximal small intestine and because of this chewable RF tablets provide better absorption as compared to enteric- coated or sustained release preparations. Food and bile salts increase the rate of absorption of RF (Jusko et al., 1971; Mayersohn et al., 1969). It binds with albumin

(Zhao et al., 2006); however a large portion of vitamin binds with other proteins (Rivlin and Pinto, 2001). Small quantities are stored in liver, kidney, spleen and cardiac muscles but like most of the water soluble vitamins, there are no major body stores. Limited gastrointestinal absorption, rapid urinary excretion and limited capacity of tissue binding sites are the major reasons due to which the large doses of RF are wasted. Zempleni and his co-workers (1996) administered 20, 40 and 60 mg of oral doses and one intravenous bolus injection of RF to healthy humans and it was found that the maximum amount of

RF that can be absorbed from a single dose was 27 mg per adult. The renal excretion of

RF involves saturable tubular reabsorption as well as tubular secretion (Jusko and Levy,

1970). RF, lumiflavins and acetyl and propionyl esters of RF, because of their hydrophillic or moderately hydrophobic character, are cleared rapidly from the serum and

6 can be recovered from liver and kidneys in comparable quantities. However, the longer- chain butiryl and palmotoyl esters of RF show prolonged retention in serum and accumulate largely in the liver and spleen as compared to that in kidneys. This occurs due to their highly hydrophobic character causing slow release from serum lipoproteins

(Edwards et al. 1999). RF toxicity is not a problem because of limited intestinal absorption (Said and Mohammed, 2006). Large doses (0.1-40mg / kg) of RF and its coenzyme FMN do not produce any changes in the blood pressure, heart rate and respiration in man or animals (Unna and Greslin, 1942; Tsutsumi, 1957). However, FAD has been found to produce hypotensive action in rabbits through its AMP portion (Rivlin,

2007).

1.5 RIBOFLAVIN DEFICIENCIES

RF deficiency is common in developing countries and across demographic groups

(Nichoalds, 1981). Clinical features of its deficiency include weakness, fatigue , sores at the corners of the mouth, cracks, eye disorders, inflammation of tongue, dermatitis, cheilosis and skin lesions. Diseases like cancer, diabetes mellitus and cardiac disease are known to precipitate RF deficiency (Coles et al, 1976; Moshe et al, 1976 and Rivlin and

Pinto, 2001). No adverse effects related to higher consumption of RF have been reported

(Institute of Medicine, 1998).

Other than dietary inadequacy, there are also other causes of RF deficiency.

Adrenal and thyroid hormone insufficiency, interference of certain drugs and diseases with the vitamin utilization are some factors which may lead to RF deficiency (Ciminoet al., 1987). Many drugs like anti-cancers, e.g. adriamycin; antimalarials like quinacrine

(Dutta et al., 1985); psychotropic agents such as chlorpromazine and antidepressants including amitriptyline and imiparamine (Pinto et al, 1981) inhibit the conversion of RF into its active coenzyme derivatives. Alcohol inhibits both digestion and absorption of RF

(Pinto et al., 1987). The metabolism of some vitamins like folate and vitamin B6 may occur due to the deficiency of RF (Powers, 2003).

1.6 RIBOFLAVIN INTERACTIONS

Anticholinergic drugs, probenecid, oral contraceptives and alcohol inhibit the absorption of RF. RF absorption is also decreased by the diets high in psyllium fiber, however, wheat bran has shown no such effect (Roe et al., 1988). Tricyclic antidepressants, aluminum hydroxide, magnesium hydroxide and thiazide diuretics, like hydrochlorothiazide increase the excretion of RF (Pinto and Rivlin, 1987). Tricyclic antidepressant also inhibits the synthesis of FMN and FAD (Stargrove et. al., 2008).

All the transition metals which play important role in biological oxidation like iron, zinc, copper, chromium and molybdenum form complexes with RF, hence effecting its bioavailability and also of the metal as well (Clarke et al., 1980; McCormick, 1990).

In the case of zinc-RF complex the bioavalibility of zinc is markedly increased (Agte et. al.,1992). Boron also forms a complex with RF reducing absorption and increasing urinary excretion and its continous use leads to RF deficiency. In addition to metals, many drugs (cloxacillin, resorcinol, theophylline, timolol and pindolol) (Meisel et al.,

1980; Criado and Garcia, 2004; Roy et al., 2006), amino acids (histidine, tryptophan and tyrosine) (Spector, 1980) and dietary constituents (caffeine and nicotinamide) [Ahmad et al., 2009; Sevrioukova, 2009] also form complexes with RF and FMN by excitation

8 energy transfer mechanisms involving singlet oxygen state, intramolecular charge transfer state and intramolecular proton transfer.

RF metabolism is altered and its activity is interfered by the anticonvulsants especially those that induce cytochrome P450. Most of the antimalarial drugs act as RF antagonists and therefore the vitamin is contraindicated in both prevention and treatment.

The simultaneous intake of RF and tetracycline reduces the absorption and bioavailability of both the agents (Stargrove et. al., 2008).

1.7 PHARMACEUTICAL PREPARATIONSAND DOSAGE FORMS

Different pharmaceutical preparations of riboflavin are available in the form of oral and parenteral preparations alone and in combination with other vitamins and minerals. Riboflavin alone is available as Bervine, Ribon, Vita-B2, Beflavine, B2-

ASmedic, FAD Ophthalmic solution, Hibon, Alinamin, Bioflavin.

In combination is is available in the following preparations: Antioxidant forte tablets, Extralife eye care, Liv-Detoxt, BeneuranVit B-Komplext, Sulfatofer, Kwim.

Alinamin-F, Hepa-Merz, Matase, Emazian B12, Emoantitossina, Facovit, Fosforilas,

Neuroftal, Neurovitan, Pangavt, Pediatrico, Godex, Biovision, Aftasone B C, B-100

Complex, Quiet life,

1.8 SOURCES OF LITERATURE

A number of reviews have been published on different aspects of RF, some of these are:

Biochemical Functions, Chemistry, Deficiency Diseases, Interactions and Physiology

Rosenberg (1945), Williams et al. (1950), Robinson (1951), Hertz (1954),

Wagner-Jauregg (1954), Wagner and Folkers (1964), Dyke (1965), Ehrenberg and

Hemmerich(1968), Friedrich(1988), Bitsch (1997), Delgado and Remers(1998),

Dollery (1999), Rivlin and Pinto (2001), Chapman et al. (2002), Powers (2003),

McCormick (2004), Mestdagh et al. (2005), Stargrove et al. (2008).

Stability

Hertz (1954), Macek (1960), Mader (1961), Garrett (1967), Dale and Booth (1971),

Carstensen (1972), Hashmi (1973), Linter (1973), DeRitter (1982), Allwood (1984),

Friedric (1988), Allwood and Kearney (1998), Tonnesen (2004), Ball (2006), Ahmad and

Vaid (2006), Anwar and Ahmad (2017), Sweetman (2007), Huynh-Ba (2009),

British Pharmacopoeia (2016).

Methods of Identification and Assay

Mader (1961), Freed (1966), Bolliger and Konig (1969), Udenfriend (1969),

Roche (1970), Pearson (1972), Hashmi (1973), Dryhurst (1977), Shah (1985)

Eitenmiller and Friedrich (1988), Song (2000), Moffat et al. (2004), Landen (2008).

Physicochemical and Biological Data

Dawson et al. (1986), Edmondson and Gisla (1999), Massey (2000), Miura (2001),

O’Neil (2013), Rivlin and Pinto (2001), Moffat et al. (2004), Rivlin (2007),

Sweetman (2007), United States Pharmacopeia (2007), British Pharmacopeia (2016).

CHAPTER 2

ANALYTICAL METHODS FOR THE

DETERMINATION OF RIBOFLAVIN AND

RELATED COMPOUNDS

A number of analytical methods based on different techniques have been used for the determination of RF (Table 2). These methods are described in the following sections.

2.1 SPECTROMETRIC METHODS

2.1.1 Ultraviolet and visible spectrometry

Ultraviolet and visible Spectrometry has been extensively used for the identification and determination of RF and its co–enzymes, FMN and FAD. RF on reacting with cupric chloride–triphenylphosphine in alkaline medium forms an orange coloured complex and this has been made the basis for the spectrometric analysis of RF at 460 nm in pharmaceutical dosage forms (Hashmi et al., 1969). RF and FMN were determined by this method in the range of 1×10–7 – 5×10–6 M/L–3. In this method the sensitizing effect of RF and FMN on the photo–oxidation of dianisidine was studied. The

%RSD of the method was 0.68% and the method can be applied successfully to foods and pharmaceutical preparations (Perez–Ruiz et al., 1994). A rapid, sensitive, accurate and reproducible method developed for the determination of RF both in pure solutions and pharmaceutical preparations shows the linearity within 0.1–1.5 mg/mL. The cupric– spermine complex formed by the reaction of cupric chloride and amine group in spermine, is further reacted with RF producing a coloured compound and is measured at the maxima of 520 nm (Sherwani et al., 2014). RF has shown solubility in citric acid and has been assayed in aqueous media containing citric acid at 440 nm. The method has proved to be accurate and sensitive (Bartzatt and Follis, 2014).

Aberasturi et al. (2002) and Ghasemi and Vosough (2001) proposed methods for the simultaneous determination of RF, folic acid, pyridoxine, pyridoxal and thiamine by

Table 2. Analytical techniques used for the determination of RF in

pharmaceutical preparations, biological samples and degraded solutions

Techniques Method Application

Spectrophotometry Single-component UV and Assay of RF in raw materials and Visible spectrophotometry pharmaceutical preparations

Multi-component UV and Assay of RF and photoproducts Visible spectrophotometry

Infrared spectrophotometry Characterization of RF and related compounds

Single-component Assay of RF in raw materials and spectrofluorimetry pharmaceutical preparations

Multi-component Assay of RF and photoproducts spectrofluorimetry NMR spectrometry NMR (1H, 13 C) Characterization of RF and related compounds

Mass spectrometry Mass spectrometry Characterization of RF and (MC/LC-MS) related compounds

Chromatography Thin-layer chromatography Characterization of RF and related compounds

High-performance Characterization of RF and liquid chromatography related compounds

Electrochemical Potentiometry Biosensors, assay of RF techniques Polarography Assay of RF in vitamins mixtures

13 partial least–square (PLS) regression. The concentrations used were 1.02–10.2 and 0.2–

11 µg/ mL, respectively. The estimated limit of detection of the former method was found to be 0.09 µg/ mL. The latter one gave satisfactory results with the mean recovery values within 95–100%. Another spectrometric method has been reported for the simultaneous estimation of RF, thiamine, nicotinamide and pyridoxine in pharmaceutical preparations in the concentration range of 2–26 mg/L. The recoveries were higher than

95% (Lopez–de–Alba, 2006). A spectrophotometric method along with least–squares support vector machines has been used for the estimation of RF in pharmaceutical preparations and biological samples, in the presences of thiamine and pyridoxal with the detection limit and linear range of 0.5 and 1.0 – 10.0 µg/mL (Niazi et al., 2007). RF has also been assayed by both UV and visible lights at 440 nm using borate buffer at pH 7.52

(Zimmer and Huyck, 1955) and the concentration range of 3 × 10–4 to 0.0463 g/L has shown good accuracy and sensitivity (Bartzatt and Wol, 2014).

Rocha et al. (2003) proposed a multicommuted flow system for spectrophotometric determination of RF along with ascorbic acid, thiamine and pyridoxine in tablets. Linear response was found (R2 = 0.999) for the concentration range of 0.50–8.0 mg/L. the recoveries were within 95.6–100%. A group of researchers presented a simple, accurate, cost–effective, precise and rapid method for the estimation of raw RF at 445 nm. The Beer’s law was obeyed in the range of 5–30 ppm with the correlation coefficient of 0.999. The inter– and intra–day precision was 0.66–1.04% and

1.05–1.39% respectively (Shah et al., 2012).

Multicomponent spectrometric methods have been used for the assay of RF and its thermal isoalloxazine ring cleavage degradation products (Ahmad et al, 1973) and the

14 photochemical side–chain leavage degradation products, FMF, LC, LF, CDRF (Ahmad and Rapson, 1990; Ahmad et al.2004b). These methods have successfully been applied to the study of the kinetics of photodegradation reactions of RF (Ahmad et al., 2004a, 2005,

2006, 2008, 2009, 2010, 2011, 2012, 2014, 2015, 2016, 2017). Prolonged diffused daylight affects the concentration of RF less as compared to that of prolonged artificial sunlight in acidic solutions due to the degradation of RF. The ratio of the absorbance was compared to detect the changes at 265 and 270 nm (Zimmer and Huyck, 1955).

It has been found that the presence of riboflavin can cause severe aggregation of the nanoparticles in the form of color change from yellow to red. Nano particles were prepared by using β–cyclodextrin–grafted citrate in the form of reducer and stabilizer in order to prepare silver nanoparticles. UV–visible spectroscopy, powder x–ray diffraction spectroscopy and transmission electron microscopy were used to characterized the

AgNPs. The formation of the nanoparticles between riboflavin and β–cyclodextrin– grafted citrate was confirmed by 1H NMR. Hydrogen bond was considered the main driving force of the interaction between the riboflavin and external rim of β– cyclodextrin. The method has been found to be highly selective and sensitivie for riboflavin with a detection limit of 167 nM (Ma et al., 2016).

2.1.2 Spectrofluorimetry

Spectrofluorimetric methods have been extensively been used for the assay of RF due to high sensitivity of this technique.

A spectrofluorimetric method showing good reproducibility has been proposed for the determination of RF in oral pharmaceutical dosage forms where the linear

15 response was observed over the concentration range of 0–25 µg mL–1 (Ramirez–Munoz,

1974). RF and thiamine in infant formulas were determined by automated fluorimetric and AOAC manual fluorimetric methods. The mean results were between the ranges of

104–113% and 90–112% and the coefficient of variation for RF and thiamine ranged from 1.05–3.90% and 1.48–3.86%, respectively. The automated method gave better results than the manual method (Dunbar and Stevenson, 1979). Diaz et al. (1993) after

separating a mixture of RF, thiamine and niacin by TLC (Rf value of RF, 0.86) and scanned the developed plate by a bifurcated fiber–optic, transmitting emission radiation to the plate and collecting the emission signal by the fluorimeter. RF showed native fluorescence with the calibration curve in the range of 48–320 ng. Aqueous two–phase system for the extraction of analytes in the presence of salts can be developed using small molecular water–soluble organic solvents. Based on this, the trace amounts of RF in pharmaceutical formulations were developed using the acetone/ethanol/actonitrile–salt– water system. The technique was found to be simple with high extraction efficiency, low toxicity and little interference (Long, 2007). For the determination of RF in the presence of vitamin B1 and B6, the parallel factor analysis (PARAFAC) was applied for the resolution of the overlapped spectra. The excitation wavelength range was within 200–

500 nm and the delta wavelength range was between 20–120 nm (Ni and Cai, 2005). A simple and specific method has been developed for the simultaneous determination of vitamin B2 and B6 in multivitamin formulation by synchronous fluorimetric technique.

The fluorescence relative intensity and the detection limit were found to be 0–1 µg/ml and 0.5ng/ml, respectively (Li et al., 1992). Wang et al. (2011) developed a rapid synchronous specrtrofluorimetric method for the simultaneous determination of RF and

16 folic acid in nutritional drinks showing satisfactory results. The detection limit for RF was 0.014 µg/L and the calibration curves were linear within the range of 1–250 µg/L.

Synchronous fluorescence technique was used by (Ziak et al., 2014) for the determination of RF along with caffeine and caramel in cola–type and energy drinks. The fluorescence spectra were recorded between 200 to 500 nm at the constant wavelength difference of 90 nm. High coefficients of determination (> 0.99) were obtained in the 7–5 mg/L range and the results were compared to those of the standard HPLC method. RF along with pyridoxine and cyanocobalamine in the acetate buffer (pH 6) has been determined in binary, ternary and quaternary mixtures. The linear calibration curves were obtained from

0.1-5 mg/mL and the correlation coefficients ranged from 0.999 – 0.9999. There was no interference observed from excipients and the RSD and recoveries were determined as

0.46–1.02% and 97.6 ± 0.7–101.2 ± 0.8 %. The method indicated good precision

(Mohammed et al., 2011). Studies have also been conducted for the determination of RF in foods [Mann, 1946; Rettenmaier and Vuilleumier, 1983; Miquel Becker et al., 2003;

Zandomeneghi et al., 2003; Zandomeneghi et al., 2007], biological fluids (Molotkov,

1965; Clarke, 1969; Clarke, 1977; Canales et al., 2005) and in pharmaceutical formulations (Barary et al., 1986).

2.1.3 Mass spectrometry

A number of researchers have worked on the determination of RF alone (Odanaka et al., 2017; Guo et al., 2006) and in the presence of other water–soluble vitamins

(McMahon, 1985; Chen et al., 2009; Sim et al., 2016; Salavati et al., 2016). Mass spectrometry in combination with liquid chromatography (Wang and Lei, 2016; Ren et al., 2015; Halvin et al., 2013; Zand et al., 2012; Hampel et al., 2012; Bishop et al., 2011;

Goldschmidt and Wolf, 2010) has been used for the assay of riboflavin in dietary products and pharmaceutical products. RF along with all the other B vitamins has been determined in fish. For excellent peak shape and sensitivity acetonitrile, 80%, v/v, was used. the regression coefficient values were greater than 0.99 and the linear range of the vitamins ranged from 0.025 to 0.50 ng/g, and. LOQ values for different vitamins was within the range of 0.4 to 50 × 10-3 µg. The recovery values at 50 and 100 ng/g ranged from 87.5 to 97.5%. The method has been found to be precise and accurate (Chaterjee et al., 2017). A method has been reported for the analysis of multivitamins and multimineral product which do not need any treatment prior to analysis. In the reaction liquid chromatography is used along with mass spectrometry in the multiple reaction modes

(Chen and Wolf, 2007). Another simple method is reported which require low sample volume and short run time and can measure all known plasma forms of riboflavin and pyridoxine (Midttun et al., 2005) and in case of inflammation can be useful in large scale epidemiological studies (Midttun et al., 2009).

A simple, rapid and effective method has been developed for the analysis of RF along with other water–soluble vitamins in infant formula. The average intermediate precision is 3.4 ± 2.4% (n= 160). Its advantage over previous methods is that it offers facile and rugged sample preparation with improved chromatographic conditions. The method has high sample throughput capacity and is precise and accurate (Cellar et al.,

2016).

Mass spectrometry has also been utilized for the determination of RF with caffeine and other water–soluble vitamins using a single quadruple MS in positive electrospray ionization scan mode. The method proved to be simple and reliable

(Arandaand Morlock, 2006). Multivitamins in dietary products and pharmaceutical preparations can be analysedtendem mass spectrometry with capillary electrophoresis combined with electrospray ionization has proved to be sensitive, selective, simple, low cost and flexible (Marakova et al. 2014).

2.2 CHROMATOGRAPHIC METHODS

2.2.1 Thin–layer chromatography (TLC)

RF has been determined successfully in the presence of other vitamins in foods and pharmaceuticals by TLC (Bhushan and Arora, 2002; Cimpoiu and Hosu, 2006;

Cimpoiu et al., 2007; Kartsova and Koroleva, 2007). RF after separating from vitamin B1,

B6 and B12 (Joneidi et al., 1975) and vitamin A, B1, C and D3 was assayed on TLC using acetone: methanol: benzene (1:2:8, v/v) as the mobile phase (Kouimtzis and

Papadoyannis, 1979). The spots were examined by direct densitometric light emission measurement. No interference was observed from the excipients. Photodensitometric detection has also been applied to the determination of RF by HPTLC in the presence of other water–soluble vitamins. With the mobile phase consisting of a mixture of n– butanol: pyridine: water mixture in the ratio of 50:35:15 v/v, the procedure proved to be accurate and specific with satisfactory relative standard deviation and good recovery

(Postaire et al., 1991). Bhushan and Prashad (1994) developed a technique for the estimation of RF along with other B vitamins using TLC plates impregnated with transition metal ions like Mn+2 Fe+2, Co+2, Cd+2, Ni+2, Zn+2, Mg+2 (0.1–0.4% of each ion) with three different solvent systems having water, ammonia, n–propanol and n–butanol in different proportions.

RF, along with other water–soluble vitamins, in biological samples of

Helisomatrivolvis snails (Pennsylvania strains) has been determined and quantified by videodensitometry. The mobile phase used was benzene: methanol: acetone: acetic acid in the ratio of 70:20:5:5 v/v and RF was detected in the visible light as yellow coloured zone (Ponder et al. 2004).

2.2.2 High–Performance Liquid Chromatography (HPLC)

HPLC is the most widely used method for the analysis of not only RF but also other drugs. It has been used for the analysis of RF in foods (Wehling and Wetzel, 1984;

Ollilainen et al., 1990; Rizzolo and Polesello, 1992; Gliszczynsky and Koziolowa, 1999),

B vitamin mixtures (Bhushan and Arora, 2002) and degradation solutions (Ahmad et al.,

1990, 2004a, 2004b, 2005, 2006, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016,

2017).

A stability–indicating method proposed for the determination of RF and other B vitamins in pharmaceutical liquid dosage forms at 280 nm has been reported. The reverse–phase C 18 column, flow rate of 1.5 ml/min and mobile phase comprised of

0.015 M 1–hexane sulphonic acid sodium salt, pH 3.0 ± 0.05 and methanol was used in gradient elution in the concentration range of 9.61–17.84 µg/ml. The method was also validated for precision, robustness and recovery (Thomas et al., 200). RF in human plasma has been determined by RP–HPLC and detected fluorimetrically (Basic et al.,

2007). The method proved to be linear, precise and accurate for the determination of RF

(excitation 450 nm; emission 520 nm). (Petteys and Frank, 2011; Soni et al, 2012) developed a simple, accurate and precise method for the determination of RF at 290 nm

20 in multivitamin tablets and biological fluids. The C–18 column was used and the mobile phase consisted of methanol: water (22:78 v/v), with a flow rate of 1ml/min.

RF in pharmaceutical products along with other multivitamins has been determined using the HPLC technique with diode array detector (Vidovic et al., 2008). C

18 column was used with phosphate buffer–acetonitrile (85:15, v/v) as the mobile phase and the flow rate was kept at 0.5 ml/min. The detection wavelength was 282 nm. The method proved to be simple, stability–indicating, reliable and convenient and showed good sensitivity, linearity, specificity and precision (Jin et al., 2012).

Certain beverages, due to the presence of RF, FMN and FAD, on exposure to light develop onion/garlic like odour because of the formation of sulphur compounds.

This property of RF has been utilized for its determination by a precise, accurate and sensitive HPLC method. The column used was Hypersil C18 ODS with a flow rate of 0.6 ml/min (Andres–Lacueva, 1998). RF, FAD and FMN have also been simultaneously determined in food material, successfully by florescence detection (Vinas et al., 2004) on polymer–based columns using acetonitrile in citrate–phosphate buffer at pH 5.50

(Russell, 1992).

The determination of low levels of RF alone with thiamine from single extracts following oxidation and sample per concentration steps are reported. The method was found to be rapid, accurate, sensitive and economical and can be used for the routine analysis of RF (Fellman et al., 1982). Albala–Hurtado et al. (1997) have proposed a rapid, simple and reliable method for the simultaneous determination of RF with thiamine, nicotinamide, pyridoxine, pyridoxal, pyridoxamine, cyanocobalamin and folic

21 acid in infant’s milk (powdered and liquid). Satisfactory separation of vitamins has been achieved with the use of a revered–phase C18 column, a mobile phase consisting of methanol: water (15:85 v/v), 5mM octane sulfonic acid with 0.5% triethylamine (pH 3.6) and a flow–rate of 1.0 ml/min. The method showed good linearity, precision, recovery and sensitivity. RF after extraction from casein using the enzyme takadiastase for the conversion of all forms of RF to the free form has been determined by RP–HPLC with fluorescence detection. The recovery was satisfactory, limit of detection was less than 0.1 mg/kg and RSD was 3% (Hewavitharana, 1996). Three different methods were proposed for the determination of FMN, FAD, FMF and other derivatives with (method C) and without 7α– hydroxyriboflavin (method A) and to confirm their presence (method B) in the sample of raw egg yolk/ white, egg powder, pasteurized and fermented milk products and liver. Alphabond C18 column was used for method A and B, while Symmetry C18 for method C, which proved to be more effective than the former ones (Gliszczynska–

Swiglo and Koziolowa, 2000). RF in the presence of thiamine in edible marine seaweeds has been determined. The vitamins were sampled from Himanthaliaelongata,

Laminariaochroleuca, Undariapinnatifida, Palmaria sp., and Porphyra sp. and also from the canned food of H. elongata and Saccorhizapolyschides. With the flow rate of 1.35 ml/min and a mobile phase of the mixture of 0.005 M ammonium acetate (pH 6.7)– methanol (72:28, v/v), 90.08% of RF was recovered and the %RSD was 2.21%

(Sanchez–Machado, 2004).

2.3 ELECTROCHEMICAL METHODS

2.3.1 Potentiometry

Potentiometric biosensor using binding protein has also been proposed for the selective determination of RF giving rapid and reproducible response (Yao and Rechnitz,

1987). Another group of researchers prepared a sensitive vitamin B2 electrochemical sensor on a glassy carbon electrode. The method exhibited good sensitivity, reproducibility and selectivity towards RF in the concentration range of 10–120 × 10-3nM and the average recovery rate of 98.41% (Xu, 2012). A rapid, simple and selective chronopotentiometric method developed using a glassy carbon electrode for RF in pharmaceutical dosage forms showed no interference from other vitamins and excipients.

RF provided reduction signals at –0.12 V vs Ag/AgCl (3.5 mol/L KCl) electrode in 0.025 mol/L HCl in the concentration range of 0.2–70 mg/L. the limit of detection and limit of quantitation were 0.076 and 0.23 mg/ml respectively (Brezo et al., 2015).Incorporating manganese (III) tetraphenylporphyrine into a carbon paste matrix, a chemically modified electrode has been constructed which served as a sensitive electrochemical sensor for the assay of RF in pharmaceutical preparations and food samples. The electrode proved to be a fast and easy means for the determination of RF in the concentration range of

1.0 × 10–8 – 1.0 × 10–5 M with the detection limit of 8.0 × 10–9 M (Khaloo, 2016).

2.3.2. Polarography

RF in biological fluids (Hoffman, 1948; Portillor and Varela, 1949), microbial cultures (Sebek, 1947) and in combination with other vitamins and drugs (Brdicka and

Knobloch, 1941; Maquinay and Brouhon, 1957; Asahi, 1958; Arizanet al., 1961;

Tikhomirova et al., 1965; Kruze, 1969; Jozan et al., 1980) has been determined polarographically. Methods have been developed where the results of the neat solutions of RF were compared with those of partially purified and solubilized RF and also

23 multivitamin mixtures (Seagers, 1953). RF has also been determined oscillopolarographically (Dusinsky and Faith, 1967) and by means of AC polaroraphy in minute concentrations such as 2 × 10–7 M (Breyer and Biegler, 1960).

A new, accurate, sensitive and simple method proposed for the determination of

RF in multivitamin tablets is based on the single–sweep polarographic reduction wave of the photolysis products of RF. When irradiated under an iodine–tungsten lamp in the presence of pH 9.3 NH3–NH4Cl buffer. A single adsorptive wave at –0.63 V is produced which is linearly propotional to RF concentration within the range of 0.01–17.5 µg/mL.

The correlation coefficient and the limit of detection have been found to be 0.996 and

0.005 µg/mL respectively (Zhu and Luo, 2010).

RF in the presence of thiamine and ascorbic acid, in pharmaceuticals and fruit juices, has been determined using renewable silver amalgam annular band electrode, which was refreshed before each measurement. The procedure is based on the adsorptive accumulation of RF at the electrode in a phosphate buffer. The method provided linear results in the concentration range of 0.05–3 mg/L and the detection limit was found to be

0.009 mg/L (Bas et al., 2011).

CHAPTER 3

PHOTOCHEMISTRY OF RIBOFLAVIN AND RELATED COMPOUNDS

RF (1) is a highly photosensitive compound and the photochemistry and its analogues have been studied in detail during the last several decades. Many reviews have been published regarding the photodegradation, mechanism and pathways (Oster et al.,

1962; Holmstrom, 1964a; Penzer, 1970; Penzer and Radda, 1967; 1971; Song, 1971;

Hemmerich, 1976; Walsh, 1980; Muller, 1981; Heelis, 1982, 1991; Tollin, 1995;

Massey, 2000; Miura, 2001; Ahmad and Vaid, 2006; Ahmad et al., 2009; Encinas and

Previtali, 2006; Silva and Edwards, 2006; Silva and Quina, 2006; Garcia et al., 2006;

Anwar et al., 2017), photostability of RF (Macek, 1960; DeRitter, 1982; 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). Photostabilization of RF in pharmaceutical preparations (Casini et at. 1981; Asker and Habib, 1990; Habib and Asker, 1991; Loukas et al., 1995, Loukas et al., 1996; DeRitters, 1982), parenteral nutrition admixtures (Buxton et al., 1983; Chen et al., 1983; Allwood, 1984; Smith et al.,

1988; Zhan and Yin, 1992; Yamaoka et al., 1995; Allwood et al., 1998; Martens, 1989) and food products (Min and Boff, 2002) is also of great interest for drug development, quality assurance and formulation studies and also to determine the shelf-life of the vitamin. These aspects are discussed in Photostability Guidance, FDA Drug Stability

Guidance (Geigel, 2004), International Conference on Harmonization (ICH, 2005) and other literatures (Tonnesen, 2004; Tonnesen and Karlesen, 1997).

RF is destroyed in the presence of both visible light and UV radiation and the rate increases with an increase in temperature and pH (British Pharmacopoeia, 2016).

However, under specific conditions, RF remains unchanged and stable for prolonged

25 period in the dark (Huang et al., 2004; Huang et al., 2006). In the solution form, on exposure to light it is degraded rapidly into a number of photoproducts under both aerobic and anaerobic conditions (Ahmad et al., 2009; Ahmad et al., 2011; Ahmad and

Rapson, 1990; McDowell, 2000; Sheraz et al., 2014; Anwar et al., 2017). In neutral and acidic conditions the main photodegradation product is lumichrome (LC) (5), while LC and lumiflavin (LF) (6) are formed under alkaline conditions and both of these products have been found to be inactive. Cyclodehydroriboflavin (CDRF) (8) is the photodegradation product formed in the presences of divalent anions like sulphate and phosphate (Schuman Jorns et al, 1975; Ahmad et al, 2004b, 2005, 2006, 2010; Anwar et al., 2017). Formylmethylflavin (FMF) (4) is an intermediate product in the photodegradation of RF which undergoes hydrolysis. LC and LF in aqueous solution

(Ahmad et al, 1980, 2006a, 2013b; Vaid, 2006) and carboxymethylflavin (CMF) (7) is its oxidative product of RF and FMF (Ahmad et al, 2006b; Ahmad et al, 2013, Treadwell et al, 1968). FMF is more sensitive to light than RF (Ahmad et al, 2013b; Mcbride and

Metzler, 1967). The structures of RF and its photoproducts are shown in Fig. 2. The wavelength range of 350-520 nm has been found to be destructive for the aqueous solutions of RF while 415-455 nm range has been found to be most destructive (Sattar and Alexander, 1977). A scheme for the photodegradation of RF is shown in Fig. 3. The photoproducts of RF have not been found to be biologically active.

HO CHO

CH 2 HO H H3C N N O HO H

HO H NH H3C N H H 9 10 1 O H3C N 10a N O 8 2 7 3NH H C N 4a 4 3 6 5 O (RF) (FMF) COOH H H3C N N O CH 2

H3C N N O NH H3C N NH O N H3C O (CMF) (LC)

CH3 CH 2OH

H3C N N O H OH H OH NH H3C N O CH 2 O H3C N N O

NH N H3C O (LF) (CDRF)

Fig. 2: Structures of RF and its photoproducts

* HO HO HO

HO H HO H HO H HO H HO H HO H HO H HO H HO H CH H H H H H H 3 H C N N O H C N N O 3 3 H3C N N O C O

NH NH NH C O H C N H C N 3 3 H3C N O O O H3C (1) excited singlet state excited triplet state (9)

intramolecular photoreduction

intramolecular photodealkylation CHO intramolecular photoaddition

CH2 CH OH H 2 H3C N N O HO H

HO H NH N H3C O (4) O H H C 3 N N O H3C N N O acid, neutral and alkaline pH NH NH N H C H C N 3 3 [O] neutral and alkaline pH O O (8) (5)

COOH

CH 2 CH3 H H H C 3 N N O H3C N N O

NH NH H C N N 3 H3C O O (7) (6)

Fig. 3. Scheme for the photodegradation pathways of riboflavin

One of the problems encountered in the study of the photodegradation of RF is the determination of RF and its photoproducts in degraded solutions. This is due to the fact their spectral properties are similar. Although RF can be determined at 445 nm but its degradation products may cause interference by absorbing at this wavelength during photolysis and therefore such studies (Smith and Metzler, 1963; Halwer, 1951;

Holmstrom, 1964; Radda and Calvin, 1964; Song et al., 1965; McBride and Moore, 1967;

Sato et al., 1982, 1983, 1984; Montana et al., 2003; Diaz et al., 2004) may give inaccurate kinetic data.

Specific multicomponent spectrophotometric methods have been developed for the analysis of RF and its major photoproducts like FMF, LC and LF or FMF and its hydrolytic products LC and LF (Ahmad and Rapson, 1990, Ahmad et al., 2004, 2008) for the study of their hydrolysis (Ahmad et al., 1980) and photolysis reactions (Heelis et al.,

1980). Further multicomponent spectrophotometer methods have been developed to study simultaneous photoaddition (to form cyclodehydroriboflavin, CDRF) and photoreduction reactions (to form FMF, LC and LF) of RF (Ahmad et al., 2004b, 2005, 2006, 2010,

2017). These methods are described in Section 2.1.1 Chapter 2 and have extremely been used for the study of the kinetics of photodegradation reaction of RF and FMF.

3.1 TYPES OF PHOTOREACTIONS OF RIBOFLAVIN

Aerobic and anaerobic photoreactions are the two main types of photoreactions which are discussed below:

3.1.1 Aerobic Photoreactions

When exposed to light, RF forms LC and LF in the presence of oxygen (Kuhn and

Wagner-Jauregg, 1934; Holmstrom and Oeter, 1961; Strauss and Nickerson, 1961) and

the ribityl side chain is broken (Oster, 1951; Shimizu, 1955; Fukumachi and Sakurai,

1955). Such aerobic photolysis of RF and other flavins depends on the buffer components

used and the acid-base catalysis involved (Halwer, 1951).

FMF is an intermediate product of RF that leads to the formation of LF in aerobic

photolysis (Svobodova et al., 1953). Under alkaline pH CMF (7) is also formed by the

photooxidation of 2-carbonyl of the side-chain of FMF by peroxides (Fukumachi and

Sakurai, 1955). Due to the formation of formic acid by the oxidation of the side-chain,

the acidity of the aqueous solution increases. The product distribution at pH 7.2 of

anerobic photolysis is similar to 50% bleaching in the aerobic photolysis (Treadwell et

al., 1968). Macromolecules like polyvinyl pyrrolidine (PVP) and sodium dodecyl

sulphate (SDS) cause an increase in the rate of aerobic photobleaching and this increase

is due to the reversible binding of excited RF (RF*) to macromolecules forming the

triplet states (3RF) by the polymer through quenching by oxygen (Kostenbauder et al.,

1965). At pH greater than 6, under aerobic photolysis, RF in the presence of divalent

anions like phosphate and sulphate, gives CDRF by photocycloaddition (Schuman Jorns

et al., 1975).

3.1.2 Anaerobic Photoreactions

RF photolysis may occur through photoreduction or photobleaching depending upon the presence or absence of an electron donor, respectively. Aqueous solution of RF

30 in the presence of EDTA undergoes discoloration of the solution; however, the color is regained on exposing the solution to oxygen (Oster et al., 1962). The photoreduction occurs due to intramolecular reduction of isoalloxazine ring while photobleaching occurs due to the intramolecular reduction of isoalloxazine nucleus by ribose side chain

(Holmstorm and Oster, 1961) causing the formation of deuteroflavin (Karrer et al., 1935).

At pH 7, when RF is exposed to light, the yellow colour of the solution is faded resulting in the formation of a photoproduct, leucodeuteroflavin, which is converted into deuteroflavin by oxygen dehydrogenation. From deutroflavin the RF side chain of 2- hydroxy group is oxidized forming a keta group giving FMF (7,8-dimethyl-10- formylmethyl isoalloxazine) (Smith and Metzler, 1963), and FMF forms LC under acidic conditions and LC and LF under basic conditions (Song et al., 1965). LC is also formed from deuteroflavin in alkaline solution (Kuhn and Wagner-Jauregg, 1934).

FMF and LC are also formed when N (10) – substituted isoalloxazine ring containing compounds (including RF and other related compounds) are irradiated in the presence of alcohol or a mixture of alcohols and water (Moore and Ireton, 1977). RF forms different photoproducts at different pH, like the formation of a compound (4’- ketoflavin) similar to LC at neutral pH while within the pH range of 4-10 a new product is formed (7,8-dimethyl-10(1-deoxy-D-erythro-2’-pentolosyl)isoalloxazine) which is similar to that of FMF (Cairns and Metler, 1971). According to Heelis et al. (1980) the triplet state

(3RF) is responsible for the formation of FMF at acidic pH values.

3.2 PHOTOCHEMICAL REACTIONS OF RIBOFLAVIN

Hemmerich (1976) and Heelis (1982, 1991) have worked on the photochemistry of

RF and concluded that the main photochemical reactions undergone by RF are intramolecular and intermolecular photoreduction, intramolecular photoaddition and intramolecular photodealkylation. All these reactions, however, are controlled by the pH, divalent ion concentration (SchumannJorns et al., 1975; Ahmad et al., 2004a), ionic strength (Sato, 1984; Ahmad et al., 2016), buffers (Ahmad et al. 2004b, 2005; Jorns et al.,

1975), wavelength and light intensities (Sato, 1982; Ahmad et al., 2006a), polarity of solvent (Song, 1971; Ahmad et al., 2006, 2015) and the excited state participating in the reaction (Schuman Jorns et al., 1975; Cairns and Metzler, 1971). These reactions may occur individually or simultaneously to form different products.

3.2.1 Photoreduction Reactions

3.2.1.1 Intramolecular photoreduction

In aqueous solutions RF undergoes photolysis through intramolecular photoreduction (Heelis, 1982, 1991; Ahmad and Vaid. 2006). However, the photochemical reactions of RF are pH dependent (Cairns and Metzler, 1971; Ahmad et al.,

2004a). In intramolecular photoreduction, the dehydrogenation of the N (10)-ribityl side chain occurs along with the reduction of the isoalloxazinering system, forming FMF

(Moore et al., 1963; Smith and Metzler, 1963; Cairns and Metzler, 1971) which later hydrolysed to LC and LF (Song et al., 1965; Ahmad and Vaid, 2008; Ahmad et al, 1980) and oxidized to CMF (Treadwell et al., 1968; Ahmad et al., 2006, 2013, 2019) (Figure 4).

3.2.1.2 Intermolecular photoreduction

H 1.5-dihydrogen flavin (H2FIred) (14) or its alkyl products (R-FIred ) (15-17) are formed as a result of photoreduction of RF (Fig. 5). The alkyl compounds may also form

α-hydroxy-carboxylic acids in the presence of thiols, aldehydes, amino acids (Knappe and

Hemmerich, 1972, 1976) and α-substituted acetic acid (Ahmad and Tollin, 1981a). In the presence of oxygen, I,5-dihydrogen flavin is reoxidized to form hydrogen peroxide along with oxidized flavin (Massey et al., 1973) (Eq. 1).

( 1) H Fl + O H O + Fl Eq. 2 red 2 2 2 ox

Such type of intermolecular photoreduction may occurs by two different

procedures. In one case an electron is transferred to the flavin (10) from the substrate,

resulting in flavosemiquinone formation (Eq. 2-4).

(Eq. 2) Fl hv 1Fl

(Eq. 3) 1Fl + RH FlH. + R.

- + (Eq. 4) 1Fl + RH Fl + RH o

In the other case, the presence of carboylate anions, flavins get photoreduced. On

exposure to light flavin is converted into its excited state (singlet) (Eq. 5)

(Eq. 5)

This excited state on reacting with a carboxylate substrate, forms radicals.

1Fl + RCOO- Fl- + RCOO. (Eq. 6)

The carboxylate gives rise to carbondioxide and alkyl radical

CHO

CH 2 CH3 H H C N N O H C N N O 3 H3C N N O 3

NH NH NH H3C N H3C N H3C N O O O

Fig. 4a. Hydrolysis of FMF to LC and LF

R R H N N O

H R NH N HO C H (15) O CH2 R H N N O N N O + RH NH NH N N (10) O R O (16) R H N N O

NH N H R R O H N N O (17)

NH N H O (14)

Fig. 5: Formation of 1,5-dihydrogen flavin and alkyl adducts from flavin through

photoreduction.

- . (Eq. 7) RCOO R + CO2

Disproportionation of two semiquinone radicals (either reduced and oxidized, or on addition of a radical) forms the photodegradation products (3h-3j).

. . (Eq. 8) HFl + HFl H Fl + Fl 2 red ox

. (Eq. 9) R Prodcuts

. . (Eq. 10) HFl + R RFl H red

Excitation of flavin to singlet state may also occur at pH 7.0 in the presence of

EDTA as an external donor (k) (Fritz et al., 1987). Afterwards through a internal conversion it gives off energy on returning back to the ground state (l).through intersystem crossing, the excited singlet state is converted into excited triplet state (m), which finally returns back to the ground state with the release of energy (n). The excited triplet state of flavin is quenched resulting in the triplet state conversion to the ground state, the heat is also evolved (o).

(Eq. 11) oFl hv 1Fl

(Eq. l2) 1Fl ic oFl

(Eq. 13) 1Fl isc 3Fl

3 (Eq. 14) Fl oFl+ heat

3 (Eq. 15) Fl + oxygen quencher oFl + heat

This triplet excited state on reacting with EDTA oxidizes EDTA and is itself reduced.

o (Eq. 16) 3Fl + EDTA Fl + EDTA red ox

o (Eq. 17) oFl + O Fl + H O red 2 2 2

1 R 2 HO C R

CH2 9 H H3C 8 N N 2 O 10 1 H3C N N O hv - 7 N pH 7.0 - 3 N H3C N 4 6 5 H3C N O O (1) (5) (Eq. 18)

3.2.1.3 Intramolecular Photodealkylation

Another intramolecular reaction involves photodealkylation, which occurs by the loss of N (10) side-chain resulting in the formation of LC. Such reaction can also occur when the same N (10) is substituted with an alkanoic, methyl, cycloalkyl group (Knappe,

1975, 1976) an alkene/cycloalkene (Gladys and Knappe, 1974). When it take place by the excited singlet state, it leads to the formation of LC but not through excited triplet state. the first step of photodealkylation involves hemolytic fussion of N(10)-C(1’) bond into a biradical intermediate (12) (Moore and Ireton, 1977) while the triplet doesn’t involve any radical intermediate (Song, 1971).

3.2.2 Intramolecular Photoaddition

The intramolecular photoaddition reactions are just like the photodehydration of flavin (Schollnhmmer and Hemmerich, 1974). In these reactions CDRF is formed through autooxidation of 1,5-dihydro-9-alkoxyl-flavin (an intermediate product)

(Schuman Jorns et al., 1975) because of nucleophilic group in the ribityl side chain (11).

In this case deprotonation occurs at C (9) while a proton is added at N (1) resulting in the formation of a stable compound which then forms CDRF. In normal photolysis

(photoreduction) excited triplet state is involved, however, in photoaddition reactions the excited singlet state is involved. These reactions occur in the presence of divalent anions at a concentration more than 0.1M. RF forms a complex with the divalent anions during photoaddition reaction at pH above 6.0 to form CDRF.

RF + divalent anion RF-divalent anion complex (Eq. 18a)

The kinetics of these reactions has been studied by Ahmad et al., (2004b, 2005,

2006, 2010, 2017).

3.2.3 Photosensitization reactions

When exposed to light triplet oxygen (molecular oxygen) is transformed to singlet oxygen species and this singlet form plays an important role in photosensitized reactions

(Choe et al., 2005; Jung et al. 2007).

1 (Eq. 19) 3O O 2 2

hv 1 (Eq. 20) RF RF

(Eq. 21) 1RF isc 3RF

O (Eq. 22) 3RF 2 RF + 3O 2

In the presence of light, RF acts as a photosensitizer for food products and for singlet oxygen formation (Choe and Min, 2006). It can both accept and donate pair of hydrogen and remains stable during both thermal and non-thermal food processing.

Singlet oxygen, hydroxyl radical, hydrogen peroxide and superoxide anions are some of the products resulting from photosensitization of RF. Photosensitization of RF can also take place in the eye as it present there too. Vitamin D in the presence of RF is oxidized in the presence of light (King and Min, 1998). Reactive oxygen species like H2O2, OH,

1 2- O2 and O are formed as the result of photodegradation of tryptophan in oxygen saturated aqueous solution when sensitized by RF (Silva et al., 1994). There are number of compounds that undergoes the photosensitized degradation through flavins, some of such compounds includes; amines (Encinas et al., 2002), cyanocobalamine (Ahmad and

Hussain, 1992; Ansari et al., 2004, Ahmad et al., 2012), atrazine (Cui et al., 2002; Glover et al., 2003), lactoglobulin (Jung et al., 2000), norflurazon (Massad et al., 2004), pyrimidine (Haggi et al., 2002), sulfa drugs (Diaz et al., 2004), retinoids (Fu et al., 2003),

DNA (Korycka-Dahl and Richardson, 1980) and RNA (Burgstaller et al., 1997).

3.2.3.1 Photooxidation reactions

In these reactions, abstraction of an electron from the substrate takes place, by radical mechanism (Vanish and Tollin, 1971). The anionic form of the flavin radical is more reactive to oxygen as compared to the semiquinone radical. The RF exhibits

39 photosensitizing properties both in vivo and in vitro and acts as an endogenous singlet oxygen sensitizer (Malgorzata and Marek, 2014). It also has an important role in photosensitized reactions involving many substrates, acting as electron donors. RF- sensitized photooxidation of amino acids (Silva et al., 1991, 1993, 1994; Silva, 1996) and proteins (Silva and Godoy, 1994; Silva and Edwards, 1996; Lu and Liu, 2002; Viteri et al, 2003; Cardoso et al., 2004) is among the most studied reactions. Other reactions include, RF-sensitized photooxidation of lipids (Silva et al., 1998), bilirubin (Knobloch et al., 1991), folic acid (Akhtar et al., 2000), ascorbic acid (Heelis et al., 1981; Jung et al.,

1995; de La Rochette et al., 2000, 2003; Vaid et al., 2005), glucose (Silva et al, 1999), glucose-6-phosphate (Silva et al., 2005), doxorubicin (Ramu and Mehta, 2000), homatropine and scopolamine (Criado et al., 2002).

3.2.3.2 Photodecarboxylation

RF also sensitizes the photodecarboxylation of α–substituted acetic acids by radical formation (Ahmad and Tollin, 1981; Novak et al., 1980).

3.2.3.3 Photoisomerization

In the presence of biliribin RF undergoes photoisomerization and the degradation products of the tetrapyrrole skeleton undergo photooxidation through biliverdin as an intermediate product (Chapman and Reid, 1999).

3.2.3.4 Photomonomerization

1,3-Dimethylthymine, the dimer of cis, syn-cyclobutane has been used for the flavin sentitized photomonomerization using riboflavin tetraacetate (RFTA) (Miyake et

40 al., 1993). In another study, carboxymethyllumiflavin-sensitized splitting of pyrimidine dimer has been carried out through a photochemically induced dynamic nuclear polarization (Hartman et al., 1992).

3.2.3.5 Photoinactivation

Blood components, dihydroorolate, lysozyme, stress kinase some compounds which are photoinactivated by RF. The spectrum of RF-photosensitized inactivation of lambda phage has been reported (Martin et al., 2005).

3.2.3.6 Photomodification and photoinduction

RF-sensitized photomodification of kininogen that was isolated from the plasma of a sheep has been reported (Baba et al., 2000). When lens proteins are photosensitized, it leads to the modification of protein of higher molecular weights. RF can also undergo photoinduction reaction with lens proteins (Ugarte et al., 1992) and also forms photoinduced products with indole-3-acetic acid (Edwards et al., 1999).

3.3 FACTORS AFFECTING THE PHOTOCHEMICAL REACTIONS OF RF

Some factors involved in the photochemical reactions of RF are discussed below.

3.3.1 Intensity, Wavelengths and Radiation Source

Radiation source is considered as one of the important factor in the photochemical reactions of RF. Studies have been conducted by using both high and low intensity radiation at different wavelengths to perform the photolysis of RF (Ahmad, 1968; Sato et al., 1982; Fasihullah, 1988; Ahmad and Rapson, 1990; Mattivi et al., 2000; Ahmad et al.,

2004, 2006; Becker et al., 2005; Dias et al., 2012). As compared to the dried form, the

41 solution of RF is easily degraded into different photoproducts like CDRF, FMF, LC, LF,

DHRF etc. (Ahmad et al., 2004a,b, 2005, 2006a,b, 2008, 2009, 2010, 2011, 2013a,b;

Ball, 2006; Cairns and Metzler, 1971; McDowell, 2000; Sheraz et al., 2014a; Smith and

Metler, 1963). The intensity of light is also involved in the degradation of RF.The magnitude of the formation of photoproducts was higher in aqueous solutions of RF exposed to UV light as compared to visible light and this difference may be due to the higher intensity of UV radiations as compared to the visible source (Ahmad et al., 2004a,

2006). It has been found that the RF tablets undergo greater colour change with LC as the major degradation product, after being exposed to xenon lamp, which may be due to the visible light below 400 nm (Sue-Chu et al., 2009).

The photodegradation of RF in aqueous solution under UV and visible radiation gives similar photoproducts, however, both the rate of photodegradation and magnitude of formation of photoproducts were found to be slower on visible irradiation as compared to UV irradiation (Ahmad et al., 2004, 2006). It has also been reported by some researchers that the wavelengths within the range of 350-520 nm are capable of damaging the RF solution (Sattar et al., 1977), especially in the range of 415-455 nm (Ball, 2006;

Sattar et al., 1977). As their methods were not selective enough therefore neither the accurate determination of the vitamin was possible not the correct rate of degradation was able to be confirmed. However, more selective methods to determine the accurate rate of degradation under different light conditions can give proper results (Ahmad et al., 2004,

2006; Ahmad and Rapson, 1990).

3.3.2 pH

RF has been found to be stable with the pH range of 5.0-6.0 because of the lower redox potential of RF while at pH 10.0 the photolysis was found to be around 80 times higher than at pH 5.0 as at this pH not only the redox potential is higher but also flavin triplet reactivity is increased. Due to the anion formation of RF, the photolysis rate decreases above pH 10.0 (Ahmad et al., 2004a). The photostability of RF is greatly affected by the pH of the solution. RF is photodegraded to LC in neutral and acidic conditions, however in alkaline pH LC is formed along with LF. Both of these photoproducts are formed through the triplet excited state where FMF acts as an intermediate in the photolysis of RF (Rivlin, 2007; Ball, 2006; Ahmad and Vaid, 2006;

Ahmad et al., 1980, 2004, 2005, 2006, 2006, 2008, 2009, 2010, 2011, 2013; Ahmad and

Rapson, 1990; Sheraz et al., 2014; Huang et al., 2006). Different pH is required for the formation of specific photoproducts like FMF and LC can formed at both acidic and alkaline pH but LF requires basic environment for its formation (ribityl side chain is involved in their formation). CMF needs the pH range of 1.0-12.0, LF at pH 7-12, while the other minor photoproducts like β-ketoacid and diketo compound are formed within the pH range of 10.0-12.0 (these two compounds requires cleavage of the isoalloxazine ring by alkaline hydrolysis) (Ahmad and Rapson, 1990; Ahmad et al., 2004a, 2013, 2019;

Song et al., 1965; Treadwell et al., 1968). At pH 14.0-14.6, LF can be further degraded to

1,2-dihydro-2-keto-1,6,7-trimethyl-1H-quinoxaline-2-one, 7,8-dimethylisoalloxazine and anionic methylisoalloxazine (Penzkofer et al., 2011)

3.3.3 Buffers

The type and concentration of buffers play an important role in the photolysis of

RF. The catalytic effect of different buffers like phosphate, acetate and carbonate have been studied while borate and citrate have a photostabilizing effect (with bell shaped k- pH profiles) (Ahmad 2004a,b, 2005, 2006, 2008, 2010, 2013, 2014a; Asker and Habib,

1990; Halwer, 1951; Schuman Jorns et al, 1975; Sheraz et al., 2014; Holmstrom, 1964; ).

Due to the decreased rate of photolysis and quenching of the excited singlet state, the citrate ions decrease the fluorescence of RF while on the quenching of the excited triplet

- 2- state the buffer shows photostabilizing effect. In alkaline solutions HCO3 and CO3 catalyze the photolysis of RF (Ahmad et al., 2014a). The rate of photolysis has been found to be higher at alkaline pH (because the flavin triplet state is sensitive to alkaline pH) while under aerobic conditions it is above pH 10.0 (Ahmad et al., 2013). An increase in the concentration of the buffer will cause an increase in the rate of photodegradation

(Ahmad et al., 2004, 2005, 2006, 2010; Sheraz et al., 2014; Holmstrom, 1964; Halwer,

1951; Sato et al., 1984). It has also been observed that the formation of CDRF will increase while that of LC will decrease on increase in the divalent ions concentration

(Ahmad et al., 1990, 2005, 2006, 2010; Sheraz et al., 2014). The increase in the buffer concentration does not affect much on the formation of FMF and LF because of the involvement of the excited triplet state. But this may also be true for LC as some of it is also formed from RF directly (Ahmad et al., 2005, 2006, 2010; Sheraz et al., 2014;

Schuman Jorns, 1975).

3.3.4 Quenchers

In order to deactivate the excited states of RF, a number of external quenchers are utilized like 1,4-diazabicyclo [2,2,2] octane, 2,5-dimethylfuran (Bradley et al., 2006), β- carotene, lycopene and tocopherols (Cardoso et al., 2007), glutathione, D-mannitol and potassium iodide (Baldursdottir et al., 2003), phenols (Song and Metzler, 1967), polyphenols (Bucker et al., 2005), purine derivatives (Cardoso et al., 2005), vitamin B6

(Natera et al., 2012), xanthine derivatives (Hiraku et al., 2007) , octane and 2,5- dimethylfuran (Bradley et al., 2006), ascorbic acid and sodium azide. Ascorbic acid has the property that it can quench both the singlet and the excited state of the RF. Because of the dual activity of ascorbic acid it has proved itself to be a better quencher. In the presence and absence of ascorbic acid, the destruction of RF destruction was calculated as 94% and 16%, respectively (Haung et al., 2004). An increase of sodium azide from 0-

0.5 mM in RF showed 86% reduction in the formation of 2,3-butanedione (Jung et al.,

2007).

3.3.5 Solvents

The polarity of the solvent causes changes in the conformation of the ribityl side chain and hence affects the photolysis of RF (Moore and Ireton, 1977). The quality of water affects the photodegradation of RF like the photodegradation is lower (40%) in distilled water as compared to D2O (66%) (Huang et al, 2004). In the presences of alcohol-water mixtures RF degrades to form FMF and LC (Moore and Ireton, 1977), however it is more stable in less polar solvents (Koziol, 1966a).In organic solvents the photodegradation of RF is found to be more as compared to aqueous solutions (Koziol,

1966a; Koziol and Knobloch, 1965) and this occurs due to the physicochemical properties of the solvent like viscosity, polarity, dielectric constant etc (Ahmad et al.,

2006, 2013a; Ahmad and Fasihullah, 1990, 1991; Moore and Ireton, 1977).

3.3.6 Ionic strength

It has been reported that the rate of photolysis of RF increases with an increase in the ionic strength. Researchers has confirmed that an increase in the ionic concentration of phosphate buffer causes an increase in the photodegradation of RF. RF forms exited singlet state in the presence of NaCl that results in a faster rate of degradation (Ahmad et al., 2016a).

3.3.7 Metal ions

RF forms complexes with different monovalent ions (Cu+, Ag+) (Weber, 1950;

Wade and Fritchie Jr., 1973), divalent ions (Cd2+, Co2+, Cu2+, Fe2+,Mg2+, Mn2+, Ni2+,

Zn2+) and trivalent ions (Fe3+, Cr3+) (Mortland et al., 1984). After complexation with metal ions, the photolysis of RF is enhanced (Ahmad et al., 2017). Monovalent anions can affect the photolysis of RF to lesser extent as compared to the divalent and trivalents.

3.3.8.Formulation

There are certain characteristics of formulation which may affect the photochemical reactions of RF. Encapsulating in liposomes may be useful for improving the stability of RF depending upon the concentration, pH and composition of the liposome (Ahmad et al., 2015b; Arien and Dopuy, 1997; Bhowmilk and Sil, 2004;

Chauhan and Awasthi, 1995; Habib and Asker, 1991; Ionita and Ion, 2003; Loukas,

1997;Sen-Varma et al., 1995). Sometimes the colour of the solid dosage forms containing

RF may undergoes change of colour because of photochromism. However, this colour change only affects the outer surface of the formulation, the RF inside remains unaffected

(Sue-Chu et al., 2008, 2009). There are certain chemicals which may increase the phtostability of RF like dimyristoylphosphatidylcholine (DPC) (Habib and Asker, 1991;

Loukas, 2001).

OBJECT OF PRESENT INVESTIGATION

Riboflavin (vitamin B2) (RF) is a photosensitive compound and is degraded by side-chain cleavage to several products including formylmethylflavin (FMF), lumichrome (LC), lumiflvain (LF) and carboxymethylflavin (CMF) in aqueous and organic solvents through the normal photoreduction pathway. Several studies on the mode of RF degradation have been conducted in the past. With the discovery of the effect of divalent anions on the photolysis of RF, it was found that RF also undergoes degradation by another pathway involving the photoaddition of RF by the cyclization of the ribose side-chain to the isoalloxazine nucleus. Thus the divalent anions (pH > 6, > 0.2

M) alter the photoreduction pathway to the photoaddition pathway depending on the nature and concentration of the dianions used and the catalytic activity of the RF-dianion complex involved in the reaction. This pathway leads to the formation of a new compound, cyclodehydroriboflavin (CDRF), instead of FMF, LC, LF and CMF, produced by the photoreduction pathway.

The present investigation aims at studying the effect of certain non-reducing (e.g. carbonate, oxalate, phthalate) and reducing divalent anions (e.g. thiosulfate, sulfite, metabisulfite) on the product distribution and kinetics of photolysis reactions of RF that give rise to different products by the two pathways. It would also involve the determination of the extent of deviation in the normal photoreduction pathway towards the photoaddition pathway on the basis of the kinetic data. The study would also include a comparison of the light and dark reaction in presence of reducing anions. It is also

48 intended to determine the quantum yields of the photolysis reactions of RF under the experimental conditions used.

PLAN OF WORK

A brief outline of the plan of work involved in this study is as follows.

1. Identification of the photoproducts of RF formed in the presence of divalent anions.

2. Photolysis of RF in the presence of non-reducing and reducing divalent anions using

visible radiation.

3. Determination of the rates of RF photolysis by simultaneous photoreduction and

photoaddition pathways.

4. Determination of the rate constants for the photochemical interaction of RF and

divalent anions.

5. Study of the effect of RF fluorescence quenching by the anions on the kinetics of

photoaddition reaction.

6. Study of the effect of pH on the kinetics of photoaddition reaction and determination

of conditions to achieve maximum yield of CDRF.

7. Study of the effect of reducing anions on simultaneous photoreduction, photoaddition

and chemical reduction reactions of RF.

8. Study of the kinetics of chemical reduction (in dark) of RF and its contribution in the

overall photolysis of RF.

9. Determination of the photochemical quantum yields of RF in presence of anions.

10. Presentation of schemes for the photodegradation pathways of RF in the presence of

non-reducing and reducing dianions.

CHAPTER 4

MATERIALS AND METHODS

4.1 MATERIALS

4.1.1 Chemicals

Riboflavin (7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl] benzo [g] pteridine-2,4-dione)

Mr 376.4 C17H20N4O6 Merck

It was stored in a desiccator in dark and was found to be chromatographically pure.

Lumiflavin (7,8,10-trimethylisoalloxazine)

Mr 256.3 C13H12N4O2 Sigma

It was stored in a light resistant dessicator below 0 oC.

Lumichrome (7,8-dimethylalloxazine)

Mr 242.3 C13H10N4O2 Sigma

It was stored in the dark in a dessicator.

Formylmethylflavin (7,8-dimethyl-10-formylmethylisoalloxazine)

Mr 284.3 C14H12N4O3

It was prepared by periodic acid oxidation of RF according to the method of Fall and

Petering (1956). It was recrystallized from absolute methanol, dried in vacuum and stored in the dark in a refrigerator.

Carboxymethylflavin (7,8-dimethylflavin-10-acetic acid)

C14H12N4O4 Mr 300.3

It was synthesized by aerobic photolysis of RF in alkaline solution in the presence of 30% H2O2, by the method of Fukumachi and Sakurai (1954). It was purified by column chromatography with Whatmann CC31 cellulose powder and acetic acid-water-1 butanol-1 propanol (2:18:50:30 v/v) as solvent system (Ahmad et al., 1980).

4.1.2 Reagents and Solvents

All the reagents and solvents used were of analytical grade and were obtained from Merck/BDH. Freshly boiled glass-distilled water was used throughout the work.

4.1.3 Buffers

HCl/KCl buffer (0.2 M), pH 2

Acetate buffer (0.2 M), pH 4.5

Phosphate buffer, pH 7

The ionic strength of all the buffers was kept constant in each case.

4.1.4 Radiation chamber

The photodegradation reactions were performed in a special 2 × 11/2 × 13/4 foot wooden radiation chamber along with a wooden cover.The chamber had a 5× 4 inches oval shaped hole for the fixation of the radiation source outside the box in order to

prevent the heating effect. The chamber also had several small holes for the passage of air.

4.2 METHODS

All the solutions used were freshly prepared to avoid any bacterial action or chemical effects and care was taken to protect RF solutions from light during handling.

4.2.1 Thin-layer Chromatography

The details of the adsorbents and solvent systems used for the separation and identification of RF and its photoproducts are given below:

Adsorbent : Silica gel GF 254 (Merck) precoated plates

Layer thickness : 250 - µm

Solvent system : WI; 1-butanol-acetic acid-water (4:1:5, v/v, organic phase) / silica gel G (Moore and Baylor, 1969)

W2; chloroform-methanol (9:2, v/v) / silica gel G (Schuman

Jorns et al., 1975)

Temperature : 25-27 oC

Location of spots : UV light, 254 and 365 nm (Uvitec lamp, UK)

4.2.2 Spectral Measurements

The spectral measurements of pure and photodegraded solutions of RF wereperformed on a Thermoscientific UV–vis recording spectrometer (Evolution 201,

USA) using quartz cells of 1 cm path length.

4.2.3 Fluorescence Measurement

The fluorescence measurements of RF solutions were carried out at room temperature (25 ± 1 oC) using Jascospectrofluorimeter (FP–8300, Japan). The solutions were excited at 374 nm and the fluorescence emission measured at 530 nm. The fluorescence intensitywas measured in relative fluorescence units using a pure 0.001 mM

RF solution (pH 7.0) asstandard.

4.2.4 pH Measurements

All pH measurements were carried out with an Elmetron LCD display pH meter

(model – CP501, sensitivity ± 0.01 pH units, Poland) using appropriate electrodes. The electrodes were calibrated using the following buffer solutions.

4.2.5 Reaction Vessel

The choice of a reaction vessel for photochemical studies depends on the nature of the reaction, emission spectrum of the radiation source, absorption properties of the drug and transmittance of the reaction vessel. In the study of the photolysis of RF, Pyrex vessels and Quartz cells have been used (Fasihullah, 1988; Ahmad et al. 2004a; Ahmad et al., 2004b; Ahmad et al., 2005; Ahmad et al., 2006a; Ahmad et al.,, 2006b, Ahmad et al.,

2008). The Pyrex vessels are transparent to visible light and these vessels have been used

in the present work since RF absorbs in the visible region (λmax 444 nm) (British

Pharmacopoeia, 2016).

4.2.6 Radiation Source

The absorption characteristics of RF suggest the use of radiation source emitting in the visible region. Therefore, a Philips HPLN high pressure mercury vapour florescent lamp was found suitable for the present study. This radiation source has previously been used by Ahmad et al. (2004a, 2004b, 2005, 2006a, 2006b, 2008) for photolysis studies of

RF. Mercury lamps and arcs emitting in both UV and visible region have been used for photochemical work (Applied Photophysics, 1981; Hanovia, 1977; Schenck, 1955) but these are of high intensities. Different low and high intensity UV and visible radiation sources have also been used (Ahmad and Rapson, 1990; Byrom and Turnbull, 1967;

Heeliset al., 1980; Holmstorm and Oster, 1961; Sato et al., 1982; Schuman Jorns, 1975;

Treadwell et al., 1968).

4.2.7 Determination of Quantum Yield (Φ)

A Philips HPLN 125 W mercury lamp has been used in this study. The intensity of the lamp has been determined by ferrioxalateactinometry (Hatchard and Parker, 1956) as 1.15± 0.11 ×1017quata s-1. This has been used to determine the quantum yield of RF photolysis. This has been carried out by measuring the area under the emission bands ofthe lamp to obtain the amount of energy available over the wavelengths absorbed by

RF. From these values a factor R, is determined to obtain a relation between the number of quanta, Qr, emitted over the wavelength absorbed by RF and the total number of

quanta, Q, available from the lamp. Using the Qr value, the quantum yield of RF is determined.

Thus A = K × E (Eq. 23)

Where

A = Area (square cm) under a band

E = Energy emitted over the wavelength of the band

K = A constant for variation in relationship of bands

Therefore

Al + Al 1 2 = R Al + Al + Al + Al 1 2 3 4 (Eq. 24) where

λ1, λ2, λ3and λ4 are 405, 435, 545 and 578 nm, respectively. λ1and λ2 (405 and 435 nm) only are absorbed by RF, and

Qr = Q × R (Eq. 25)

The rate of photolysis = ks-1 × initial concentration of RF = Ms-1

Therefore,

Rate of photolysis × number of molecules per mole Φ = R × Q (Eq. 25)

4.2.8 Photolysis

A 5 × 10-4 M aqueous solution (100 ml) of RF containing 0.2-1.0 M 0f divalent anions (phosphate, carbonate, oxalate, phthalate, sulfite, thiosulphate and metabisulfite) was prepared in a 100 ml volumetric flask. The solution was immersed in a water bath with the temperature maintained at 25 ± 1 oC. It was irradiated with a Phillips HPLN 125

W, high-pressure mercury vapour fluorescent lamp fixed at a distance of 25 cm from the center of the flask in a radiation chamber. Samples for thin-layer chromatography and spectrometric assay were withdrawn at appropriate intervals. Control solutions were placed in the dark covered with aluminum foils during the irradiation period.

4.2.9 Assay Method

A 5 ml aliquot of the photolysed solution of RF was placed in a 25 ml beaker and the pH of the solution was adjusted to 2.0 by using 1 M HCl solution. The solution was transferred into a 10 ml volumetric flask and the volume was made up with 0.2 M HCl-

KCl buffer (pH 2.0). Afterwards the solution was extracted with 3 x 10 ml chloroform.

The chloroform layer was washed with water and evaporated to dryness at room temperature. To the residue a few drops of 0.5 N acetic acid solution was added and then diluted to 10 ml using 0.2 M acetate buffer (pH 4.5). The absorbance of the solution was measured at 356 and 445 nm and the concentrations for lumichrome (LC) and lumiflavin

(LF) were determined by a two-component assay. The aqueous phase ions used for the assay of RF (riboflavin), FMF (formylmethylflavin) and CDRF (cyclodehydroxy riboflavin) by a three-component assay at 385, 410 and 445 nm according to the method of Ahmad et al., (2004b). For the three-component assay of riboflavin (RF)

formylmethylflavin (FMF) and cyclodehydroriboflavin (CDRF) the aqueous layer was centrifuged to break any emulsion and absorbance measurements were made at 385, 410 and 445 nm.

4.2.10 Assay Parameters

The analytical scheme used in the present study for the assay of RF and its photoproducts is based on the methods reported by Ahmad and Rapson (1990) and

Ahmad et al. (2004b). Different parameters related to the assay are briefly described as follows:

The success of a multicomponent spectrophotometric assay depends on the selection of appropriate analytical wavelengths. The analytical work has greater accuracy on selecting the wavelengths that show maximum sensitivity on absorbance measurements and negligible interference from instrumental factors. Methods have been proposed regarding the factors affecting the choice of analytical wavelengths (Ahmad and Hussain, 1992; Ahmad et al., 1992; Faber et al., 2003; Frans and Harris, 1985;

Garrido et al., 1995; Jaffe and Orchin, 1962; Knowles and Burgess, 1984; Saski et al.,

1986; Steams, 1969). In the present study the wavelengths chosen for the assay of RF and its photoproducts correspond to their absorption maxima that provide maximum specificity and sensitivity as reported in many previous studies (Ahmad et al. 1990,

2004b; Fasihullah, 1988).

The absorption spectra of RF and its photoproducts are highly pH sensitive and change due to ionization of the molecule (pka 1.7, 10.2, Moffat et al., 2004) affecting thereproducibility and accuracy of the method. A pH of 2.0 has been used to assay these

compounds since RF and FMF at this pH exist in the protonated form before the extraction of LC and LF by chloroform. RF is not extractable in chloroform and FMF is extractable in chloroform in aqueous solution at pH above 2.0. This is also necessary to distinguish RF (λmax 445 nm) and FMF (λmax 385 nm) along with CDRF (λmax 410 nm) during the multicomponent assay. The assay of LC and LF at pH 4.5 can be carried out since these give clear distinction in their absorption maxima i.e. LC (356 nm) and LF

(445 nm).

4.2.11 Methods of Calculation of Concentration in the Assay

The calculation of concentrations in both single and multicomponent assay of RF and its photoproducts, were carried out as follows.

4.2.11.1 One-Component Assay

The solution of a compound following Beer’s law will have its absorbance additive at all wavelengths and hence the concentration can be calculated by choosing a suitable wavelength with the help of the following equation.

A1 =1a1 1C (Eq. 27)

Where,

A1 = absorbance at wavelength λ1

1a1= absorptivity at wavelength λ1

1C = concentration of component 1

If the same cell is used throughout then:

A1 = 1K1 1C (Eq. 28)

Where.1K1, is absorptivity-cell path product of, C at λ,

4.2.11.2 Two-Component Assay (Additive Absorbencies)

Here the absorbance measurements are taken at two suitably selected wavelength and by solving the simultaneous equations the concentrations are evaluated. It can be done directly or by using matrix methods.

A1 = 1K1 1C + 2K12C (Eq. 29)

A2 = 1K2 1C + 2K22C (Eq. 30) where,

A1= absorbance at wavelength at λ1

A2= absorbance at wavelength at λ2

1K1 = absorptivity-cell path product for component 1 at wavelength λ1

1K2 = absorptivity-cell path product for component 1 at wavelength λ2

2K1 = absorptivity-cell path product for component 2 at wavelength λ1

2K2 = absorptivity-cell path product for component 2 at wavelength λ2

1C = concentration of component 1

2C = concentration of component 2

The solution of equation (c) and (d) for 1C and 2C is:

1C = (2K2 . A1 - 2K1. A2) / (1K1 . 2K2 - 2K1 . 1K2) (Eq. 31)

2C = (1K1 . A2 - 1K2 . A1) / (1K1. 2K2 - 2K1 .1K2) (Eq. 32)

4.2.11.3 Three component assay (additive absorbences)

In this assay, the solution of three simultaneous equations is required. This may be conveniently carried out by matrix method using a computer program. Thus for measurements A1, A2, A3 at λ1, λ2, λ3 on a mixture of a components 1, 2, 3 at concentrations 1C, 2C, 3C:

Wavelength Absorbance Absorbance Sum

λ1 A1 1K11C + 2K12C + 3K13C

λ2 A2 1K21C + 2K22C + 3K23C

λ3 A3 1K31C + 2K32C + 3K33C

(Eq. 33)

The matrix equation is as follows:

A1 1K1 2K1 3K1 1C

A2 1K2 2K2 3K2 2C

A3 1K3 2K3 3K3 3C

(AM) (ASM) (CM) (Eq. 34)

Where,

(AM) = absorbance matrix

(ASM) = absorbance sum matrix

(CM) = concentration matrix

The solution of (h) for each concentration is effected by replacing the appropriate column in the absorbance sum matrix in its determinant form and dividing the resultant by the absorbance sum matrix (ASM) again in its determinant form.

A12K13K1 1K12K13K1

1C = A22K23K2 1K22K23K2

A32K33K3 1K32K33K3 (Eq. 35)

1K1 A13K1

2C = 1K2 A23K2 (ASM)

1K3 A33K3 (Eq. 36)

1K12K1 A1

3C = 1K22K2 A2 (ASM)

1K32K3 A3 (Eq. 37)

The matrix are then expanded by any convenient method, e.g. for 1C using the top row and Lap lace method;

2K2 3K2 A2 3K2 A2 2K2 A1 -2K1 +3K1 2K3 3K3 A3 3K3 A3 2K3 1C = ASM expanded

(Eq. 38)

A1 (2K2.3K3- 3K2. 2K3) - 2K1 (A2 . 3K3 - 3K2 . A3) + 3K1 (A2. 2K3- 2K2 . A3) 1C = ASM expanded

(Eq. 39)

The matrices for 2C and 3C are expanded in the similar manner.

CHAPTER 5

EFFECT OF DIVALENT ANIONS ON THE

PHOTODEGRADATION OF RIBOFLAVIN IN

AQUEOUS SOLUTION

5.1. INTRODUCTION

Riboflavin (RF) (1) is sensitive to light (British Pharmacopoeia, 2016; Sweetman,

2007; O’Neil, 2013) and is degraded to several products in aqueous, organic and pharmaceutical systems, including formylmethylflavin (FMF) (2) (a major intermediate product), lumichrome (LC) (3), lumiflavin (LF) (4), carboxymethylflavin (CMF) (5) (a minor intermediate product) and cyclodehydroriboflvain (CDRF) (6) (Smith and

Metzler, 1963; Treadwell et al., 1968; Cairns and Metzler, 1971; Ahmad and Rapson,

1990; Ahmad et al., 2004, 2006, 2013; Hemmerich, 1976; Heelis, 1991; Ahmad and

Vaid, 2006) (Fig. 2). There are several modes of degradation of RF that involve intramolecularphotoreduction, photoaddition and photodealkylation (Hemmerich, 1976;

Heelis, 1991; Ahmad and Vaid, 2006; Sheraz et al., 2014; Jorns et al., 1975; Muller,

1981; Song, 1971; Holmstrom, 1964; Anwar and Ahmad, 2017). These aspects have been dealt in detail in Chapter 3. The kinetics of photodegradationreactions of RF has largely been evaluated by multicomponent spectrometric methods (Ahamd and Rapson, 1990;

- Sheraz et al., 2014). The photodegradation of RF is catalyzed by monovalent (HCOOP ,P

- - 2- 2- CHR3RCOP ,P HR2RPOR4RP )P (Halwer, 1951; Ahmad et al.,2004, 2014), divalent (HPOR4RP ,P SOR4RP ,P

2- COR3RP ,P malonate, succinate, tartrate) (Schuman Jorns et al., 1975; Ahmad et al., 2004,

3- 3- 2005, 2006, 2010, 2016; Sheraz et al., 2014) and trivalent anions (POR4RP ,P BOR3RP ,P citrate)

(Holmstrom, 1964; Amad et al., 2004, 2005, 2006, 2008, 2011; Sheraz et al., 2014;

Holmstrom and Oster, 1961). These anions catalyze the decomposition of drugs including

RF (Florence and Attwood, 2016; Carstensen and Rhodes, 2000; Connors et al., 1986;

Sinko, 2006) and the type and concentration of buffers in pharmaceutical systems to minimize decomposition. The present study involves the investigation of the catalytic

66 effect of certain divalent anions as the photolysis of RF and any change in the mode of the reaction. Structures of RF and photoproducts are shown in Fig. 6.

The experimental work involving the materials and methods used in this study have been described in Chapter 4.

5.2 RESULTS AND DISCUSSION

5.2.1 Identification of RF Products

2- - 2- The photodegradation of RF in the presence of CO R3RP ,P (COO)R2RP ,P HPOR4RP ,P phthalate anions at pH 6.0–8.0 gives FMF, LC, LF and CMF by photoreduction (Treadwell et al.,

1968; Cairns and Metzler, 1971; Ahmad and Rapson, 1990; Ahmad et al., 2004, 2006) and CDRF by photoaddition pathways by parallel reactions (Schuman Jorns et al., 1975;

Ahmad et al., 2004, 2005, 2006, 2010, 2014; Sheraz et al., 2014). These compounds have been identified by observing their fluorescence (RF, FMF, LF, CMF, yellow green; LC,

sky blue) or color (CDRF, red) and RRfR values on comparing with reference compounds.

LC and LF are hydrolytic products and CMF is an oxidation product of FMF (Cairns and

Metzler, 1971; Ahmad and Rapson, 1990; Ahmad et al., 2004, 2006, 2013; Fukumachi and Sakurai, 1954). CDRF is formed by photoaddition of RF in the presence of divalent anions (Schuman Jorns et al., 1975; Ahmad et al., 2004, 2005, 2006, 2010; Sheraz et al.,

2014). The RRfR valves of RF and photoproducts are reported in Table 2. The identification of the photoproducts is necessary prior to the assay of these compounds in the degraded solutions.

CH 2OH H OH H OH

O CH 2

H3C N N O

NH H3C N HO O CDRF

HO H HO H Photoaddition CHO COOH HO H CH CH H H 2 2 H C N N O H C N N O H C N N O 3 3 3 Photoreduction Oxidation NH NH NH H C N H3C N 3 H3C N FMF O O O CMF RF

H+, OH- OH-

H CH3 H3C N N O H3C N N O

NH NH H3C N H3C N O O LC LF

Fig. 6: Photodegradation reactions of riboflavin and formation of photoproducts.

Table 2.1: RRf RR Rvalues of RF and photoproducts

Compounds Solvent systems

WR1 WR2

Riboflavin (RF) 0.36 0.45

Formylmethylflavin (FMF) 0.61 0.55

Lumiflavin (LF) 0.42 0.71

Lumicrome (LC) 0.69 0.82

Cyclodehydroxyriboflavin (CDRF) 0.26 0.12

Solvent systems

WR1R; 1-butanol-acetic acid-water (4:1:5, v/v, organic phase) / silica gel G (Moore and

Baylor, 1969)

WR4R; chloroform-methanol (9:2, v/v) / silica gel G (Schuman Jorns et al., 1975).

5.2.2. Composition of Photoproducts of RF

The composition of photoproducts at 30% degradation of RF in presence of different divalent anions (1.0 M) at pH 7.0 is given in Table 4. The formation of CDRF increases in the order of divalent anions:phthalate˂ phosphate˂oxalate˂ carbonate, and that of LC decreases in the same order. Theseresults indicate that the formation of LC by pathway of RF is altered by these anions in favor of the photoaddition to give CDRF. It is evident from the increasing values of CDRF/LC in the above order (Table 3). The values of FMF decrease with an increase in the amount of CDRF showing an increase in the catalytic effect of these anions in favor of photoaddition reaction in the above order.

5.2.3 Spectral Characteristics of Photodegraded Solutions of RF

The spectral variations on the photodegradation of RF in the presence of 1M divalentanions have been recorded. The spectral changes vary with the nature of the divalent anion. A set of absorption spectra of photodegraded RF solution (pH 7.0) in the

2- presence of COR3RP P anions shows distinct changes in the 300-500 nm regions (Fig. 7). The

445nm peak of RF decreases with a gradual shift towards 410 nm, the λRmaxR of CDRF, indicating its formation on photoaddion. The formation of CDRFdepends on the nature and reactivity of the divalent anion used as discussed later in section 5.2.8. The 374 nm peak of RF is decreased with a shift and increase in absorbance in the 350–360 nm regions showing the formation of LC by photoreduction. Thus, the spectral changes on

2- photodegradation of RF in presence of COR3RP P anions show the formation of CDRF and

LC.

Table 3: Product composition at 30% photodegradation of RF solution (pH 7.0) in

the presence and absence of 1.0 M divalent anions

Divalent Time CDRF FMF LC LF CDRF/LC % -5 -5) -5 -5 anion min (1×10P )P (1×10P (1×10P )P (1×10P )P Fluorescence a LossP Un- 155.0 - 5.60 22.09 2.31 - - buffered

Carbonate 49.3 13.52 5.21 8.92 2.37 1.51 78.6

Phosphate 59.8 10.59 5.30 11.71 2.40 0.90 48.1

Oxalate 70.6 8.71 5.56 12.95 2.78 0.67 41.5

Phthalate 118.5 4.35 5.60 17.10 2.95 0.25 20.8

a P PureP RF solution containing 1.0 M divalent anions

(a)

LC CDRF RF

0 40 80 120

(b)

LC RF CDRF 0 90 180

(c)

LC RF 0 60 120 240

Fig. 7.UV and visible absorption spectra of RF solutions (pH 7.0) on photodegradation in the presence of 1.0 M divalent anions: carbonate (a), oxalate (b) and phthalate (c). Times

indicated are in minutes

The spectral changes observed on the photodegradation of RF in presence of other

2- 2- divalent anions (i.e. (COO)R2RP P and phthalate (Fig. 7) are lower than that of the COR3RP P anions. This may be due to a difference in complex formation with RF and the catalytic activity of the anions to cause photoaddition of RF to give CDRF. The spectral changes in presence of these anions depend on the contribution of the photoproducts of RF, particularly the CDRF/LC ratio in degraded solutions. A previous study has shown

2- similar spectral variations on photodegradation of RF in the presence of HPOR4RP P and

2- SOR4RP P anions (Ahmad et al., 2010).

5.2.4 RF Fluorescence in the Presence of Divalent Anions

RF exhibits yellow green fluorescence in 520–530 nm region (Heelis, 1982;

Ahmad et al., 2005; 2016, 2017; Weber, 1950; Penzer and Radda, 1967). It is affected by the presence of divalent anions (Schuman Jorns et al.,1975; Ahmad et al., 2004, 2005,

2006, 2010, 2014; Sherazet al., 2014) and metal cations (Ahmad et al., 2017; Sakai,

2- 1956; Varnes et al., 1971). In the case of HPOR4RP anions,P the loss of fluorescence occurs

2– due to the formation of a RF–HPOR4RP complexP (Schuman Jorns et al.,1975). The % fluorescence of RF in the presence of divalent anions (0.2–1.0 M) is shown in Fig 8. The loss of RF fluorescence in presence of these anions increases in the order: phthalate˂oxalate˂ phosphate ˂ carbonate, indicates an increase in the degree of complexation of these anions with RF to form CDRF.

100.0

80.0

60.0

40.0 % Fluoresnece% 20.0

0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Concentration (M) of divalent anion

Fig. 8: % Fluorescence of RF in the presence of divalent anions (0.2-1.0 M): (●) phthalate;

(■) oxalate; (♦) phosphate; (▲) carbonate.

5.2.5 Assay of RF and Photoproducts

The photodegraded solutions of RF in presence of divalent anions contain a complex mixture, therefore, a previously developed multicomponent spectrometric method (Ahmad et al., 2004) has been used for the assay of the products of both photoreduction (FMF, LC, LF) and photoaddition reactions (CDRF) as well undegraded

RF (Ahmad and Rapson, 1990; Sheraz et al., 2014; Ahmad et al., 2004) (Chapter 4). The assay values of RF and photoproducts on the degradation of RF in the presence of different anions are presented in Tables 4-23. The little increase intotal molar balance in some cases is probably due to contribution of some minorunknown photoproducts absorbing at the wavelengths used for analysis. This method has previously beenused in the study of the kinetics of photodegradation reactions of RF (Sheraz et al., 2014; Ahmad et al., 2004, 2005, 2006, 2010, 2017). The assay method has been validated and has a precision of ±5%.

5.2.6 Photodegradation Kinetics

The photodegradation kinetics of RF solutions containing 0.2-1.0 M divalent anions is evaluated to determine their effect on the rates of photolysis. The plots for the loss of RF by different pathways (Ahmad et al., 2004, 2005, 2006, 2010; Jorns et al.,

1975)and the formation of FMF, LC, LF and CDRF, respectively, at pH 6.0-8.0 are shown in Fig.9-28. The two end products, LC and CDRF are formed on photodegradation and photoaddition of RF,respectively.

CDRF (kR1R) and LC (kR2R) are formed by parallel first–order reactions (Ahmad et al.,

2004, 2005, 2010, 2016, 2017) as follows.

Table 4: Concentration of RF and photoproducts in the presence of carbonate anions (0.2 - 1.0 M) at pH 6.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion (M × 10P )P (M ×10P )P (M × 10P )P (M ×10P )P (M ×10P )P (M × 10P )P conc. 0 0.2 5.00 - - - - 5.00 30 4.31 0.33 0.1 0.21 0 4.95 60 3.66 0.63 0.13 0.45 0.02 4.89 90 3.11 0.85 0.15 0.64 0.04 4.79 120 2.77 1.05 0.18 0.79 0.06 4.85 150 2.56 1.19 0.22 0.90 0.08 4.95 180 2.43 1.26 0.28 0.96 0.10 5.03 0 0.4 5.00 - - - - 5.00 30 4.20 0.36 0.11 0.22 0.02 4.91 60 3.50 0.65 0.13 0.42 0.04 4.74 90 2.97 0.88 0.17 0.64 0.06 4.72 120 2.65 1.08 0.22 0.80 0.08 4.83 150 2.44 1.25 0.27 0.92 0.10 4.98 180 2.34 1.30 0.31 0.97 0.12 5.04 0 0.6 5.00 - - - - 5.00 30 4.16 0.41 0.13 0.23 0.05 4.98 60 3.47 0.70 0.18 0.48 0.06 4.89 90 2.95 0.98 0.22 0.68 0.08 4.91 120 2.59 1.17 0.26 0.81 0.11 4.94 150 2.37 1.31 0.30 0.92 0.13 5.03 180 2.23 1.35 0.34 0.98 0.15 5.11 0 0.8 5.00 - - - - 5.00 30 4.13 0.42 0.16 0.25 0.06 5.02 60 3.40 0.78 0.19 0.50 0.08 4.95 90 2.85 1.05 0.23 0.68 0.10 4.91 120 2.48 1.24 0.29 0.83 0.13 4.97 150 2.24 1.35 0.33 0.92 0.15 4.99 180 2.12 1.41 0.38 1.00 0.17 5.08 0 1.0 5.00 - - - - 5.00 30 4.09 0.50 0.14 0.26 0.06 5.05 60 3.40 0.87 0.22 0.55 0.08 5.12 90 2.83 1.15 0.28 0.74 0.10 5.1 120 2.46 1.32 0.33 0.89 0.12 5.12 150 2.22 1.41 0.39 0.97 0.16 5.15 180 2.00 1.47 0.42 1.03 0.19 5.05

Table 5: Concentration of RF and photoproducts in the presence of carbonate anions (0.2 - 1.0 M) at pH 6.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.20 0.38 0.12 0.19 0.02 4.91 60 3.45 0.72 0.17 0.41 0.05 4.80 90 2.87 0.97 0.21 0.60 0.08 4.73 120 2.49 1.20 0.26 0.78 0.12 4.85 150 2.22 1.34 0.30 0.94 0.14 4.94 180 2.07 1.42 0.35 1.07 0.17 5.08 0 0.4 5.00 - - - - 5.00 30 4.17 0.40 0.13 0.20 0.04 4.94 60 3.45 0.75 0.20 0.43 0.07 4.90 90 2.83 1.05 0.24 0.64 0.11 4.87 120 2.38 1.27 0.30 0.82 0.14 4.91 150 2.11 1.40 0.34 0.96 0.16 4.97 180 1.94 1.49 0.38 1.09 0.19 5.09 0 0.6 5.00 - - - - 5.00 30 4.11 0.42 0.14 0.22 0.07 4.96 60 3.30 0.79 0.23 0.47 0.10 4.89 90 2.71 1.13 0.28 0.67 0.12 4.91 120 2.27 1.35 0.31 0.84 0.14 4.91 150 1.98 1.49 0.36 0.99 0.17 4.99 180 1.81 1.54 0.41 1.10 0.21 5.04 0 0.8 5.00 - - - - 5.00 30 4.07 0.47 0.14 0.23 0.07 4.98 60 3.25 0.82 0.24 0.48 0.10 4.89 90 2.61 1.11 0.31 0.70 0.16 4.89 120 2.22 1.34 0.36 0.90 0.19 5.01 150 1.92 1.48 0.40 1.02 0.22 5.04 180 1.68 1.59 0.44 1.11 0.25 5.08 0 1.0 5.00 - - - - 5.00 30 3.99 0.50 0.17 0.27 0.09 5.02 60 3.14 0.91 0.27 0.50 0.12 4.94 90 2.45 1.25 0.36 0.70 0.17 4.93 120 1.93 1.44 0.40 0.89 0.22 4.88 150 1.63 1.57 0.43 1.03 0.25 4.91 180 1.51 1.65 0.48 1.13 0.28 5.05

Table 6: Concentration of RF and photoproducts in the presence of carbonate anions (0.2 - 1.0 M) at pH 7.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.11 0.41 0.10 0.27 0.01 4.90 60 3.31 0.77 0.27 0.55 0.03 4.93 90 2.62 1.07 0.40 0.77 0.07 4.93 120 2.16 1.36 0.49 0.95 0.10 5.06 150 1.80 1.59 0.56 1.15 0.14 5.24 180 1.61 1.78 0.61 1.32 0.19 5.11 0 0.4 5.00 - - - - 5.00 30 4.03 0.43 0.13 0.31 0.03 4.93 60 3.14 0.84 0.29 0.62 0.05 4.94 90 2.37 1.19 0.40 0.84 0.09 4.89 120 1.79 1.47 0.51 1.03 0.13 4.93 150 1.48 1.68 0.60 1.20 0.18 5.14 180 1.29 1.89 0.65 1.34 0.22 5.04 0 0.6 5.00 - - - - 5.00 30 3.94 0.48 0.15 0.33 0.04 4.94 60 2.99 0.96 0.31 0.64 0.06 4.96 90 2.20 1.36 0.47 0.90 0.09 5.02 120 1.53 1.62 0.56 1.11 0.14 4.96 150 1.17 1.83 0.63 1.23 0.21 5.07 180 0.99 1.98 0.70 1.36 0.26 5.11 0 0.8 5.00 - - - - 5.00 30 3.82 0.51 0.16 0.36 0.06 4.91 60 2.86 1.02 0.35 0.68 0.11 5.02 90 2.00 1.42 0.48 0.96 0.15 5.01 120 1.31 1.70 0.59 1.14 0.20 4.94 150 0.85 1.93 0.69 1.28 0.26 5.01 180 0.59 2.08 0.76 1.40 0.31 5.06 0 1.0 5.00 - - - - 5.00 30 3.75 0.54 0.19 0.40 0.08 4.96 60 2.76 1.03 0.38 0.74 0.13 5.04 90 1.87 1.45 0.52 1.04 0.17 5.05 120 1.18 1.78 0.65 1.21 0.22 5.04 150 0.60 2.03 0.74 1.35 0.28 5.00 180 0.22 2.20 0.83 1.45 0.34 5.08

Table 7: Concentration of RF and photoproducts in the presence of carbonate anions (0.2 - 1.0 M) at pH 7.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.20 0.32 0.13 0.18 0.03 4.86 60 3.47 0.56 0.25 0.40 0.05 4.73 90 2.85 0.82 0.35 0.63 0.08 4.73 120 2.40 1.04 0.45 0.80 0.11 4.8 150 2.11 1.24 0.51 0.92 0.15 4.93 180 1.96 1.38 0.56 1.00 0.18 5.00 0 0.4 5.00 - - - - 5.00 30 4.18 0.38 0.14 0.18 0.05 4.93 60 3.44 0.72 0.27 0.46 0.07 4.96 90 2.83 0.99 0.39 0.67 0.10 4.98 120 2.37 1.18 0.48 0.83 0.13 4.99 150 2.03 1.35 0.56 0.95 0.17 5.06 180 1.79 1.45 0.60 1.02 0.20 5.02 0 0.6 5.00 - - - - 5.00 30 4.10 0.40 0.15 0.19 0.06 4.9 60 3.31 0.78 0.31 0.41 0.10 4.91 90 2.64 1.06 0.44 0.59 0.14 4.87 120 2.13 1.27 0.52 0.77 0.17 4.86 150 1.82 1.40 0.61 0.92 0.2 4.95 180 1.63 1.52 0.65 1.05 0.23 5.08 0 0.8 5.00 - - - - 5.00 30 4.04 0.42 0.16 0.22 0.08 4.92 60 3.15 0.77 0.34 0.50 0.12 4.88 90 2.39 1.05 0.47 0.68 0.17 4.76 120 1.92 1.29 0.60 0.84 0.21 4.86 150 1.58 1.47 0.66 0.99 0.25 4.95 180 1.42 1.60 0.71 1.08 0.26 5.02 0 1.0 5.00 - - - - 5.00 30 3.93 0.48 0.17 0.23 0.08 4.89 60 3.04 0.91 0.36 0.47 0.12 4.90 90 2.23 1.24 0.51 0.70 0.17 4.85 120 1.65 1.44 0.64 0.87 0.21 4.81 150 1.36 1.58 0.72 1.03 0.25 4.94 180 1.14 1.69 0.78 1.12 0.29 5.02

Table 8: Concentration of RF and photoproducts in the presence of carbonate anions (0.2 - 1.0 M) at pH 8.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.34 0.27 0.18 0.19 0.03 5.01 60 3.74 0.52 0.28 0.40 0.06 5.00 90 3.23 0.75 0.37 0.54 0.09 4.98 120 2.79 0.92 0.46 0.69 0.13 4.99 150 2.49 1.04 0.53 0.81 0.17 5.04 180 2.21 1.14 0.60 0.91 0.20 5.17 0 0.4 5.00 - - - - 5.00 30 4.36 0.29 0.16 0.19 0.04 5.04 60 3.73 0.58 0.30 0.40 0.07 5.08 90 3.20 0.82 0.40 0.56 0.09 5.07 120 2.72 0.99 0.48 0.67 0.12 4.98 150 2.40 1.10 0.54 0.80 0.17 5.01 180 2.17 1.18 0.62 0.92 0.22 5.11 0 0.6 5.00 - - - - 5.00 30 4.32 0.31 0.17 0.20 0.03 5.03 60 3.66 0.59 0.33 0.37 0.06 5.01 90 3.13 0.84 0.45 0.55 0.10 5.07 120 2.65 1.02 0.54 0.67 0.18 5.06 150 2.26 1.16 0.62 0.83 0.22 5.09 180 1.99 1.24 0.65 0.94 0.25 5.10 0 0.8 5.00 - - - - 5.00 30 4.20 0.33 0.17 0.23 0.1 5.03 60 3.55 0.63 0.34 0.43 0.12 5.07 90 2.98 0.89 0.47 0.62 0.18 5.14 120 2.48 1.09 0.57 0.78 0.2 5.12 150 2.09 1.22 0.63 0.9 0.25 5.09 180 1.85 1.3 0.68 0.97 0.27 5.07 0 1.0 5.00 - - - - 5.00 30 4.10 0.35 0.20 0.25 0.08 4.98 60 3.34 0.64 0.33 0.45 0.12 4.88 90 2.70 0.88 0.44 0.61 0.17 4.80 120 2.23 1.10 0.55 0.79 0.20 4.87 150 1.94 1.28 0.63 0.92 0.24 5.01 180 1.73 1.38 0.70 1.00 0.28 4.96

Table 9: Concentration of RF and photoproducts in the presence of phosphate anions (0.2 - 1.0 M) at pH 6.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.60 0.14 0.08 0.23 0.05 5.10 60 4.20 0.26 0.11 0.39 0.06 5.02 90 3.90 0.37 0.12 0.53 0.07 4.99 120 3.67 0.45 0.16 0.69 0.09 5.06 150 3.49 0.54 0.17 0.81 0.10 5.11 180 3.33 0.62 0.19 1.01 0.12 5.27 0 0.4 5.00 - - - - 5.00 30 4.58 0.15 0.09 0.24 0.06 5.12 60 4.18 0.3 0.10 0.47 0.08 5.13 90 3.85 0.42 0.13 0.65 0.10 5.15 120 3.57 0.50 0.15 0.78 0.11 5.11 150 3.37 0.58 0.18 0.91 0.12 5.16 180 3.20 0.65 0.20 1.03 0.13 5.21 0 0.6 5.00 - - - - 5.00 30 4.47 0.16 0.10 0.25 0.08 5.06 60 4.03 0.32 0.11 0.48 0.09 5.03 90 3.64 0.45 0.14 0.68 0.11 5.02 120 3.38 0.53 0.16 0.83 0.13 5.03 150 3.20 0.63 0.19 0.94 0.14 5.10 180 3.05 0.70 0.21 1.06 0.15 5.17 0 0.8 5.00 - - - - 5.00 30 4.44 0.19 0.11 0.27 0.09 5.1 60 3.96 0.36 0.12 0.51 0.11 5.06 90 3.57 0.50 0.15 0.70 0.13 5.05 120 3.28 0.59 0.17 0.85 0.15 5.04 150 3.04 0.69 0.20 0.95 0.16 5.04 180 2.83 0.75 0.22 1.08 0.17 5.05 0 1.0 5.00 - - - - 5.00 30 4.38 0.20 0.12 0.28 0.12 5.1 60 3.81 0.39 0.13 0.55 0.14 5.02 90 3.33 0.55 0.16 0.80 0.15 4.99 120 3.01 0.66 0.18 0.96 0.16 4.97 150 2.77 0.72 0.20 1.07 0.17 4.93 180 2.68 0.81 0.23 1.12 0.18 5.02

Table 10: Concentration of RF and photoproducts in the presence of phosphate anions (0.2 - 1.0 M) at pH 6.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.60 0.14 0.13 0.20 - 5.07 60 4.23 0.25 0.17 0.36 0.02 5.03 90 3.90 0.35 0.28 0.46 0.04 5.03 120 3.67 0.41 0.32 0.50 0.06 4.96 150 3.51 0.45 0.36 0.59 0.08 4.99 180 3.40 0.48 0.39 0.68 0.10 5.05 0 0.4 5.00 - - - - 5.00 30 4.58 0.16 0.13 0.22 0.03 5.12 60 4.20 0.27 0.18 0.40 0.05 5.10 90 3.87 0.37 0.29 0.49 0.06 5.08 120 3.63 0.42 0.34 0.53 0.08 5.00 150 3.44 0.46 0.37 0.60 0.10 4.97 180 3.35 0.51 0.41 0.69 0.12 5.08 0 0.6 5 - - - - 5 30 4.55 0.15 0.14 0.23 0.05 5.12 60 4.16 0.26 0.20 0.40 0.07 5.09 90 3.82 0.36 0.31 0.52 0.09 5.1 120 3.53 0.42 0.36 0.58 0.11 5.00 150 3.33 0.50 0.40 0.65 0.12 5.00 180 3.20 0.56 0.45 0.74 0.13 5.08 0 0.8 5.00 - - - - 5.00 30 4.47 0.18 0.16 0.25 0.08 5.14 60 4.02 0.33 0.21 0.45 0.09 5.10 90 3.64 0.46 0.33 0.59 0.10 5.12 120 3.36 0.51 0.40 0.66 0.12 5.05 150 3.17 0.55 0.45 0.72 0.14 5.03 180 3.02 0.62 0.49 0.79 0.15 5.07 0 1.0 5.00 - - - - 5 30 4.4 0.19 0.17 0.25 0.08 5.09 60 3.91 0.35 0.22 0.48 0.09 5.05 90 3.50 0.48 0.34 0.65 0.10 5.07 120 3.18 0.56 0.42 0.76 0.12 5.04 150 2.94 0.62 0.48 0.82 0.14 5.00 180 2.75 0.69 0.54 0.86 0.16 5.00

Table 11: Concentration of RF and photoproducts in the presence of phosphate anions (0.2 - 1.0 M) at pH 7.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anions (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P conc. M/L 0 0.2 5.00 - - - - 5.00 30 4.42 0.12 0.13 0.18 - 4.85 60 3.90 0.36 0.18 0.45 0.02 4.91 90 3.48 0.53 0.25 0.63 0.06 4.95 120 3.21 0.64 0.32 0.73 0.09 4.99 150 2.99 0.70 0.38 0.84 0.12 5.03 180 2.86 0.77 0.45 0.92 0.15 5.15 0 0.4 5.00 - - - - 5.00 30 4.32 0.14 0.10 0.20 0.03 4.79 60 3.70 0.39 0.20 0.48 0.07 4.84 90 3.29 0.58 0.32 0.68 0.09 4.96 120 3.01 0.69 0.36 0.74 0.10 4.90 150 2.81 0.77 0.41 0.85 0.12 4.96 180 2.68 0.82 0.48 0.96 0.17 5.11 0 0.6 5.00 - - - - 5.00 30 4.36 0.16 0.10 0.26 0.06 4.94 60 3.76 0.40 0.19 0.54 0.10 4.99 90 3.26 0.63 0.31 0.74 0.13 5.07 120 2.90 0.75 0.39 0.84 0.15 5.03 150 2.64 0.83 0.46 0.94 0.18 5.05 180 2.48 0.90 0.50 1.03 0.20 5.11 0 0.8 5.00 - - - - 5.00 30 4.33 0.18 0.13 0.29 0.07 5.00 60 3.68 0.42 0.22 0.56 0.10 4.98 90 3.13 0.65 0.34 0.77 0.14 5.03 120 2.68 0.81 0.42 0.94 0.17 5.02 150 2.36 0.91 0.48 1.02 0.20 4.97 180 2.19 0.99 0.52 1.11 0.22 5.03 0 1.0 5.00 - - - - 5.00 30 4.26 0.19 0.15 0.32 0.09 5.01 60 3.58 0.41 0.21 0.58 0.13 4.91 90 3.01 0.66 0.35 0.82 0.17 5.01 120 2.53 0.85 0.41 1.01 0.20 5.00 150 2.19 0.98 0.49 1.13 0.22 5.01 180 1.92 1.09 0.54 1.20 0.24 4.99

Table 12: Concentration of RF and photoproducts in the presence of phosphate anions (0.2 - 1.0 M) at pH 7.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.66 0.17 0.13 0.22 - 5.18 60 4.37 0.28 0.15 0.37 - 5.17 90 4.15 0.33 0.17 0.46 0.01 5.12 120 3.93 0.40 0.19 0.51 0.02 5.05 150 3.69 0.49 0.23 0.59 0.04 5.04 180 3.52 0.55 0.28 0.67 0.06 5.08 0 0.4 5.00 - - - - 5 30 4.52 0.18 0.14 0.24 - 5.08 60 4.17 0.33 0.15 0.38 0.01 5.04 90 3.90 0.42 0.19 0.48 0.03 5.02 120 3.69 0.47 0.23 0.50 0.04 4.93 150 3.53 0.53 0.28 0.63 0.06 5.03 180 3.38 0.58 0.32 0.70 0.08 5.06 0 0.6 5 - - - - 5 30 4.49 0.19 0.16 0.26 - 5.1 60 4.07 0.36 0.21 0.41 0.02 5.07 90 3.77 0.47 0.25 0.48 0.04 5.01 120 3.55 0.54 0.29 0.56 0.06 5.00 150 3.36 0.58 0.33 0.63 0.08 4.98 180 3.24 0.62 0.37 0.73 0.10 5.06 0 0.8 5.00 - - - - 5.00 30 4.41 0.20 0.18 0.28 0.02 5.09 60 3.92 0.40 0.25 0.48 0.04 5.09 90 3.60 0.52 0.35 0.59 0.06 5.12 120 3.35 0.57 0.38 0.66 0.07 5.03 150 3.20 0.62 0.41 0.70 0.09 5.02 180 3.09 0.67 0.44 0.77 0.11 5.08 0 1.0 5.00 - - - - 5.00 30 4.36 0.21 0.20 0.30 0.04 5.11 60 3.88 0.40 0.27 0.52 0.06 5.13 90 3.53 0.56 0.37 0.66 0.08 5.20 120 3.27 0.63 0.41 0.71 0.10 5.12 150 3.06 0.69 0.44 0.78 0.11 5.08 180 2.89 0.73 0.48 0.82 0.13 5.05

Table 13: Concentration of RF and photoproducts in the presence of phosphate anions (0.2 - 1.0 M) at pH 8.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.64 0.14 0.10 0.22 0.02 5.12 60 4.3 0.30 0.11 0.42 0.04 5.17 90 4.00 0.42 0.14 0.56 0.06 5.18 120 3.74 0.48 0.15 0.63 0.08 5.08 150 3.50 0.54 0.17 0.70 0.10 5.01 180 3.28 0.63 0.19 0.79 0.12 5.01 0 0.4 5.00 - - - - 5.00 30 4.54 0.15 0.11 0.23 0.03 5.06 60 4.18 0.28 0.12 0.41 0.05 5.04 90 3.85 0.41 0.14 0.59 0.07 5.06 120 3.60 0.52 0.16 0.68 0.09 5.05 150 3.34 0.58 0.18 0.77 0.11 4.98 180 3.18 0.67 0.20 0.84 0.13 5.02 0 0.6 5.00 - - - - 5.00 30 4.42 0.16 0.13 0.25 0.06 5.02 60 3.93 0.33 0.15 0.49 0.08 4.98 90 3.56 0.51 0.18 0.67 0.11 5.03 120 3.30 0.62 0.19 0.80 0.12 5.03 150 3.12 0.68 0.21 0.87 0.13 5.01 180 2.99 0.74 0.22 0.93 0.14 5.02 0 0.8 5.00 - - - - 5.00 30 4.36 0.17 0.14 0.28 0.09 5.04 60 3.83 0.36 0.16 0.54 0.11 5.00 90 3.42 0.54 0.20 0.74 0.12 5.02 120 3.13 0.68 0.22 0.88 0.14 5.05 150 2.93 0.77 0.23 0.96 0.15 5.04 180 2.8 0.81 0.24 1.02 0.16 5.03 0 1.0 5.00 - - - - 5.00 30 4.30 0.18 0.15 0.31 0.11 5.05 60 3.72 0.38 0.17 0.58 0.13 4.98 90 3.24 0.60 0.19 0.81 0.14 4.98 120 2.93 0.74 0.21 0.97 0.15 5.00 150 2.73 0.82 0.23 1.05 0.16 4.99 180 2.62 0.88 0.25 1.11 0.17 5.03

Table 14: Concentration of RF and photoproducts in the presence of oxalate anions (0.2 - 1.0 M) at pH 6.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.58 0.07 0.08 0.28 - 5.01 60 4.27 0.10 0.10 0.48 - 4.95 90 3.98 0.20 0.14 0.66 - 4.98 120 3.73 0.26 0.20 0.81 0.01 5.01 150 3.50 0.32 0.28 0.97 0.03 5.10 180 3.31 0.33 0.33 1.03 0.05 5.05 0 0.4 5.00 - - - - 5.00 30 4.59 0.13 0.10 0.26 - 5.08 60 4.20 0.11 0.12 0.49 - 4.92 90 3.90 0.21 0.16 0.66 0.01 4.94 120 3.66 0.27 0.21 0.83 0.03 5.00 150 3.45 0.32 0.29 0.92 0.05 5.03 180 3.29 0.34 0.34 0.97 0.07 5.01 0 0.6 5.00 - - - - 5.00 30 4.52 0.12 0.14 0.25 - 5.03 60 4.15 0.20 0.17 0.49 - 5.01 90 3.84 0.24 0.21 0.68 0.02 4.99 120 3.6 0.31 0.25 0.82 0.05 5.03 150 3.42 0.36 0.30 0.91 0.07 5.06 180 3.27 0.36 0.35 0.94 0.09 5.01 0 0.8 5.00 - - - - 5.00 30 4.47 0.12 0.16 0.24 - 4.99 60 4.07 0.25 0.22 0.48 0.01 5.03 90 3.79 0.31 0.26 0.65 0.03 5.04 120 3.56 0.37 0.30 0.80 0.05 5.08 150 3.40 0.41 0.34 0.88 0.08 5.11 180 3.24 0.39 0.37 0.90 0.10 5.00 0 1.0 5.00 - - - - 5.00 30 4.46 0.15 0.20 0.23 0.02 5.06 60 4.05 0.28 0.24 0.45 0.04 5.06 90 3.73 0.34 0.29 0.62 0.06 5.04 120 3.50 0.40 0.33 0.73 0.08 5.04 150 3.34 0.46 0.37 0.82 0.10 5.09 180 3.19 0.40 0.40 0.88 0.12 4.99

Table 15: Concentration of RF and photoproducts in the presence of oxalate anions (0.2 - 1.0 M) at pH 6.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.63 0.07 0.12 0.20 - 5.02 60 4.33 0.10 0.17 0.36 - 4.96 90 4.09 0.13 0.20 0.49 - 4.91 120 3.87 0.18 0.28 0.59 - 4.92 150 3.69 0.24 0.34 0.70 0.01 4.98 180 3.60 0.29 0.40 0.78 0.03 5.1 0 0.4 5.00 - - - - 5 30 4.54 0.09 0.14 0.22 - 4.99 60 4.19 0.12 0.19 0.40 - 4.90 90 3.97 0.16 0.24 0.53 - 4.90 120 3.76 0.20 0.30 0.66 0.01 4.93 150 3.61 0.25 0.37 0.77 0.02 5.02 180 3.47 0.33 0.41 0.84 0.04 5.09 0 0.6 5.00 - - - - 5.00 30 4.44 0.12 0.18 0.23 - 4.97 60 4.07 0.17 0.22 0.43 - 4.89 90 3.79 0.21 0.27 0.59 - 4.86 120 3.55 0.26 0.33 0.70 0.02 4.86 150 3.42 0.30 0.39 0.79 0.04 4.94 180 3.34 0.35 0.43 0.83 0.06 5.01 0 0.8 5.00 - - - - 5.00 30 4.46 0.14 0.20 0.26 0 5.06 60 4.04 0.23 0.28 0.48 0 5.03 90 3.69 0.29 0.32 0.65 0.01 4.96 120 3.47 0.32 0.37 0.76 0.03 4.95 150 3.28 0.38 0.41 0.83 0.05 4.95 180 3.12 0.40 0.46 0.88 0.08 4.94 0 1.0 5.00 - - - - 5.00 30 4.40 0.15 0.21 0.28 0 5.04 60 3.95 0.24 0.33 0.52 0.01 5.05 90 3.58 0.34 0.37 0.69 0.02 5.00 120 3.33 0.40 0.42 0.85 0.05 5.05 150 3.09 0.44 0.48 0.94 0.07 5.02 180 2.88 0.48 0.50 1.00 0.10 4.96

Table 16: Concentration of RF and photoproducts in the presence of oxalate anions (0.2 - 1.0 M) at pH 7.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.56 0.13 0.09 0.27 - 5.05 60 4.18 0.21 0.11 0.55 0.06 5.11 90 3.80 0.35 0.14 0.82 0.10 5.21 120 3.44 0.47 0.18 1.05 0.13 5.27 150 3.10 0.58 0.22 1.24 0.16 5.30 180 2.73 0.67 0.25 1.42 0.18 5.25 0 0.4 5.00 - - - - 5.00 30 4.50 0.15 0.12 0.26 0.07 5.10 60 4.03 0.22 0.17 0.52 0.09 5.03 90 3.64 0.36 0.22 0.75 0.12 5.09 120 3.32 0.51 0.25 1.02 0.14 5.24 150 2.98 0.62 0.26 1.25 0.17 5.28 180 2.66 0.70 0.28 1.40 0.20 5.24 0 0.6 5.00 - - - - 5.00 30 4.46 0.17 0.13 0.25 0.09 5.10 60 3.95 0.24 0.16 0.53 0.12 5.00 90 3.5 0.38 0.18 0.79 0.15 5.00 120 3.18 0.53 0.22 1.01 0.17 5.11 150 2.86 0.63 0.26 1.21 0.19 5.15 180 2.58 0.74 0.30 1.37 0.22 5.21 0 0.8 5.00 - - - - 5.00 30 4.38 0.19 0.16 0.24 0.11 5.08 60 3.83 0.30 0.19 0.53 0.15 5.00 90 3.35 0.48 0.23 0.79 0.17 5.02 120 2.98 0.63 0.27 1.01 0.20 5.09 150 2.65 0.70 0.31 1.18 0.22 5.06 180 2.45 0.79 0.33 1.33 0.24 5.14 0 1.0 5.00 - - - - 5.00 30 4.30 0.20 0.18 0.30 0.12 5.1 60 3.69 0.33 0.28 0.55 0.15 5.00 90 3.19 0.52 0.34 0.80 0.18 5.03 120 2.76 0.66 0.40 0.98 0.22 5.02 150 2.44 0.77 0.47 1.12 0.24 5.04 180 2.17 0.85 0.53 1.26 0.26 5.07

Table 17: Concentration of RF and photoproducts in the presence of oxalate anions (0.2 - 1.0 M) at pH 7.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.73 0.12 0.13 0.23 - 5.21 60 4.45 0.17 0.19 0.39 - 5.20 90 4.19 0.21 0.24 0.53 - 5.17 120 3.94 0.28 0.30 0.65 - 5.17 150 3.72 0.34 0.36 0.73 - 5.15 180 3.55 0.39 0.40 0.82 0.01 5.17 0 0.4 5.00 - - - - 5.00 30 4.67 0.11 0.15 0.22 - 5.15 60 4.38 0.17 0.21 0.39 - 5.15 90 4.11 0.23 0.27 0.52 - 5.13 120 3.85 0.30 0.32 0.62 - 5.09 150 3.65 0.36 0.38 0.73 0.01 5.13 180 3.47 0.41 0.42 0.83 0.03 5.16 0 0.6 5.00 - - - - 5.00 30 4.60 0.12 0.15 0.25 - 5.12 60 4.26 0.19 0.22 0.44 - 5.11 90 3.98 0.25 0.27 0.60 - 5.10 120 3.72 0.31 0.34 0.68 0.01 5.06 150 3.52 0.37 0.40 0.75 0.03 5.07 180 3.35 0.43 0.44 0.84 0.05 5.11 0 0.8 5.00 - - - - 5.00 30 4.50 0.15 0.18 0.30 - 5.13 60 4.06 0.26 0.25 0.50 0.01 5.08 90 3.76 0.30 0.31 0.63 0.02 5.02 120 3.53 0.35 0.37 0.71 0.03 4.99 150 3.35 0.40 0.43 0.78 0.05 5.01 180 3.19 0.46 0.47 0.85 0.07 5.04 0 1.0 5.00 - - - - 5.00 30 4.48 0.16 0.23 0.28 - 5.15 60 4.03 0.28 0.30 0.52 0.01 5.14 90 3.69 0.35 0.36 0.67 0.03 5.1 120 3.41 0.40 0.41 0.77 0.05 5.04 150 3.19 0.45 0.46 0.81 0.07 4.98 180 3.07 0.49 0.50 0.85 0.09 5.00

Table 18: Concentration of RF and photoproducts in the presence of oxalate anions (0.2 - 1.0 M) at pH 8.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.66 0.12 0.09 0.23 - 5.10 60 4.38 0.16 0.11 0.43 - 5.08 90 4.12 0.21 0.14 0.56 - 5.03 120 3.92 0.26 0.18 0.70 - 5.06 150 3.73 0.29 0.22 0.81 - 5.05 180 3.60 0.34 0.27 0.89 - 5.10 0 0.4 5.00 - - - - 5.00 30 4.66 0.15 0.11 0.25 - 5.17 60 4.35 0.18 0.15 0.46 - 5.14 90 4.10 0.22 0.18 0.62 - 5.12 120 3.85 0.27 0.21 0.76 - 5.09 150 3.66 0.31 0.24 0.87 - 5.08 180 3.49 0.38 0.29 0.95 - 5.11 0 0.6 5.00 - - - - 5.00 30 4.62 0.17 0.12 0.25 - 5.16 60 4.27 0.22 0.15 0.45 - 5.09 90 4.00 0.27 0.19 0.61 - 5.07 120 3.73 0.32 0.23 0.74 - 5.02 150 3.53 0.37 0.28 0.85 - 5.03 180 3.37 0.40 0.32 0.93 0.02 5.04 0 0.8 5.00 - - - - 5.00 30 4.59 0.19 0.18 0.28 - 5.24 60 4.20 0.25 0.20 0.50 - 5.15 90 3.90 0.32 0.23 0.66 - 5.11 120 3.64 0.37 0.28 0.80 - 5.09 150 3.42 0.41 0.32 0.87 0.02 5.04 180 3.25 0.45 0.36 0.97 0.04 5.07 0 1.0 5.00 - - - - 5.00 30 4.52 0.19 0.20 0.28 - 5.19 60 4.12 0.27 0.24 0.54 - 5.17 90 3.81 0.34 0.29 0.73 - 5.17 120 3.53 0.39 0.32 0.86 0.02 5.12 150 3.30 0.45 0.36 0.97 0.04 5.12 180 3.09 0.50 0.40 1.02 0.06 5.07

Table 19: Concentration of RF and photoproducts in the presence of phthalate anions (0.2 - 1.0 M) at pH 6.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.72 0.05 0.03 0.31 - 5.11 60 4.48 0.06 0.05 0.54 - 5.13 90 4.28 0.07 0.06 0.72 - 5.13 120 4.13 0.08 0.07 0.81 - 5.09 150 4.01 0.09 0.08 0.86 - 5.04 180 3.95 0.10 0.09 0.90 - 5.04 0 0.4 5.00 - - - - 5.00 30 4.75 0.07 0.05 0.33 - 5.20 60 4.49 0.08 0.06 0.59 - 5.22 90 4.29 0.09 0.07 0.76 - 5.21 120 4.15 0.10 0.08 0.87 - 5.20 150 4.03 0.11 0.09 0.92 - 5.15 180 3.92 0.12 0.10 0.95 - 5.09 0 0.6 5.00 - - - - 5.00 30 4.68 0.07 0.06 0.30 - 5.11 60 4.43 0.09 0.07 0.54 - 5.13 90 4.22 0.10 0.08 0.74 - 5.14 120 4.04 0.11 0.09 0.90 - 5.14 150 3.93 0.12 0.10 0.97 - 5.12 180 3.85 0.13 0.11 1.00 - 5.09 0 0.8 5.00 - - - - 5.00 30 4.66 0.10 0.07 0.31 - 5.14 60 4.38 0.11 0.08 0.55 - 5.12 90 4.13 0.12 0.09 0.76 - 5.10 120 3.94 0.13 0.10 0.90 - 5.07 150 3.80 0.15 0.11 0.99 - 5.05 180 3.74 0.17 0.12 1.06 0.01 5.10 0 1.0 5.00 - - - - 5.00 30 4.60 0.11 0.08 0.34 - 5.13 60 4.31 0.13 0.09 0.61 - 5.14 90 4.07 0.15 0.10 0.81 - 5.13 120 3.86 0.17 0.11 0.96 - 5.10 150 3.72 0.19 0.12 1.06 0.01 5.10 180 3.60 0.20 0.13 1.11 0.03 5.07

Table 20: Concentration of RF and photoproducts in the presence of phthalate anions (0.2 - 1.0 M) at pH 6.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.74 0.02 0.06 0.23 - 5.05 60 4.50 0.04 0.07 0.43 - 5.04 90 4.30 0.06 0.08 0.61 - 5.05 120 4.15 0.07 0.09 0.77 - 5.08 150 4.00 0.08 0.10 0.88 - 5.06 180 3.88 0.10 0.11 0.99 - 5.08 0 0.4 5.00 - - - - 5.00 30 4.72 0.05 0.07 0.24 - 5.08 60 4.52 0.06 0.08 0.42 - 5.08 90 4.34 0.07 0.09 0.56 - 5.06 120 4.22 0.08 0.10 0.70 - 5.10 150 4.07 0.09 0.11 0.84 - 5.11 180 3.94 0.11 0.12 0.95 - 5.12 0 0.6 5.00 - - - - 5.00 30 4.68 0.07 0.09 0.27 - 5.11 60 4.43 0.08 0.10 0.48 - 5.09 90 4.24 0.09 0.11 0.62 - 5.06 120 4.09 0.10 0.12 0.73 - 5.04 150 3.98 0.11 0.13 0.83 - 5.05 180 3.90 0.13 0.14 0.92 0.01 5.10 0 0.8 5.00 - - - - 5.00 30 4.70 0.09 0.1 0.28 - 5.17 60 4.46 0.1 0.11 0.49 - 5.16 90 4.25 0.11 0.12 0.61 - 5.09 120 4.11 0.12 0.13 0.72 - 5.08 150 3.99 0.13 0.14 0.8 0.01 5.07 180 3.89 0.15 0.15 0.88 0.03 5.10 0 1.0 5.00 - - - - 5.00 30 4.65 0.12 0.11 0.32 - 5.20 60 4.38 0.13 0.12 0.51 - 5.14 90 4.20 0.14 0.13 0.64 - 5.11 120 4.02 0.15 0.14 0.72 0.01 5.04 150 3.90 0.16 0.15 0.80 0.03 5.04 180 3.79 0.17 0.16 0.85 0.05 5.02

Table 21: Concentration of RF and photoproducts in the presence of phthalate anions (0.2 - 1.0 M) at pH 7.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P conc. M/L 0 0.2 5.00 - - - - 5.00 30 4.66 0.07 0.07 0.27 - 5.07 60 4.37 0.08 0.09 0.52 - 5.06 90 4.15 0.09 0.11 0.73 0.01 5.09 120 3.92 0.10 0.13 0.91 0.03 5.09 150 3.72 0.11 0.14 1.07 0.04 5.08 180 3.58 0.12 0.15 1.20 0.06 5.11 0 0.4 5.00 - - - - 5.00 30 4.62 0.07 0.08 0.24 - 5.01 60 4.30 0.08 0.10 0.45 - 4.93 90 4.07 0.09 0.12 0.63 0.02 4.93 120 3.86 0.10 0.14 0.83 0.04 4.97 150 3.71 0.12 0.16 0.96 0.06 5.01 180 3.54 0.15 0.17 1.07 0.08 5.01 0 0.6 5.00 - - - - 5.00 30 4.62 0.08 0.09 0.24 - 5.03 60 4.38 0.10 0.12 0.42 0.01 5.03 90 4.14 0.11 0.14 0.56 0.03 4.98 120 3.98 0.12 0.16 0.68 0.05 4.99 150 3.81 0.14 0.18 0.80 0.08 5.01 180 3.70 0.16 0.19 0.88 0.10 5.03 0 0.8 5.00 - - - - 5.00 30 4.61 0.11 0.10 0.26 0.01 5.09 60 4.31 0.12 0.13 0.45 0.03 5.04 90 4.13 0.14 0.15 0.56 0.05 5.03 120 3.95 0.16 0.16 0.68 0.07 5.02 150 3.82 0.17 0.17 0.78 0.09 5.03 180 3.72 0.18 0.21 0.87 0.11 5.09 0 1.0 5.00 - - - - 5.00 30 4.5 0.15 0.13 0.33 0.04 5.15 60 4.19 0.16 0.16 0.55 0.05 5.11 90 4.00 0.17 0.18 0.66 0.07 5.08 120 3.82 0.18 0.20 0.71 0.09 5.00 150 3.70 0.19 0.22 0.74 0.11 4.96 180 3.65 0.20 0.24 0.78 0.13 5.00

Table 22: Concentration of RF and photoproducts in the presence of phthalate anions (0.2 - 1.0 M) at pH 7.5 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion conc. (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P M/L 0 0.2 5.00 - - - - 5.00 30 4.76 0.04 0.05 0.22 - 5.07 60 4.56 0.05 0.06 0.42 0.01 5.10 90 4.37 0.06 0.07 0.61 0.03 5.14 120 4.22 0.07 0.08 0.77 0.05 5.19 150 4.08 0.08 0.09 0.89 0.07 5.21 180 3.94 0.11 0.10 1.00 0.08 5.23 0 0.4 5.00 - - - - 5.00 30 4.75 0.06 0.06 0.20 0.01 5.08 60 4.53 0.07 0.07 0.37 0.03 5.07 90 4.35 0.08 0.08 0.54 0.05 5.10 120 4.18 0.09 0.09 0.68 0.07 5.11 150 4.03 0.10 0.10 0.80 0.09 5.12 180 3.92 0.13 0.11 0.92 0.10 5.18 0 0.6 5.00 - - - - 5.00 30 4.74 0.07 0.08 0.21 0.07 5.17 60 4.49 0.08 0.09 0.37 0.08 5.11 90 4.28 0.09 0.10 0.49 0.09 5.05 120 4.15 0.11 0.11 0.62 0.10 5.09 150 4.00 0.13 0.12 0.72 0.11 5.08 180 3.9 0.14 0.13 0.82 0.12 5.11 0 0.8 5.00 - - - - 5.00 30 4.7 0.09 0.09 0.22 0.09 5.19 60 4.45 0.10 0.10 0.37 0.10 5.12 90 4.25 0.12 0.11 0.5 0.11 5.09 120 4.08 0.13 0.12 0.62 0.12 5.07 150 3.94 0.15 0.13 0.71 0.13 5.06 180 3.83 0.16 0.14 0.8 0.14 5.07 0 1.0 5.00 - - - - 5.00 30 4.60 0.10 0.10 0.26 0.10 5.16 60 4.31 0.12 0.11 0.48 0.11 5.13 90 4.12 0.13 0.12 0.60 0.12 5.09 120 3.96 0.14 0.13 0.69 0.13 5.05 150 3.84 0.15 0.14 0.73 0.14 5.00 180 3.78 0.17 0.15 0.77 0.15 5.02

Table 23: Concentration of RF and photoproducts in the presence of phthalate anions (0.2 - 1.0 M) at pH 8.0 Time Divalent RF CDRF FMF LC LF Total 5 5 5 5 5 5 (min) anion (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P (M × 10P )P conc. M/L 0 0.2 5.00 - - - - 5.00 30 4.72 0.06 0.04 0.24 0.03 5.09 60 4.45 0.07 0.05 0.44 0.05 5.06 90 4.22 0.08 0.06 0.64 0.06 5.06 120 4.01 0.09 0.07 0.81 0.08 5.06 150 3.85 0.10 0.08 0.96 0.09 5.08 180 3.70 0.11 0.10 1.10 0.10 5.11 0 0.4 5.00 - - - - 5.00 30 4.72 0.06 0.05 0.20 0.06 5.09 60 4.50 0.07 0.07 0.35 0.07 5.06 90 4.30 0.08 0.08 0.52 0.08 5.06 120 4.14 0.10 0.09 0.67 0.09 5.09 150 4.00 0.11 0.10 0.79 0.11 5.11 180 3.89 0.12 0.11 0.92 0.12 5.16 0 0.6 5.00 - - - - 5.00 30 4.70 0.07 0.06 0.22 0.07 5.12 60 4.46 0.08 0.08 0.39 0.08 5.09 90 4.24 0.09 0.09 0.55 0.10 5.07 120 4.07 0.11 0.10 0.65 0.11 5.04 150 3.96 0.12 0.11 0.77 0.12 5.08 180 3.83 0.13 0.12 0.86 0.13 5.07 0 0.8 5.00 - - - - 5.00 30 4.70 0.07 0.07 0.23 0.08 5.15 60 4.47 0.08 0.09 0.41 0.09 5.14 90 4.27 0.09 0.10 0.53 0.11 5.10 120 4.10 0.11 0.11 0.62 0.12 5.06 150 3.99 0.13 0.12 0.70 0.14 5.08 180 3.90 0.14 0.13 0.77 0.15 5.09 0 1.0 5.00 - - - - 5.00 30 4.68 0.09 0.09 0.22 0.1 5.18 60 4.40 0.10 0.10 0.38 0.11 5.09 90 4.19 0.11 0.11 0.52 0.13 5.06 120 4.05 0.12 0.12 0.61 0.14 5.04 150 3.95 0.13 0.13 0.68 0.15 5.04 180 3.88 0.15 0.14 0.75 0.16 5.08

5.00 5.00

M M

5 (b)

5 4.00 4.00

10 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00

M (c) 6.00 5

4.00 M (d) 5

10 5.00

10 ×

3.00 × 4.00 2.00 3.00 1.00 2.00 1.00

Concentration 0.00

Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00

M (e)

5 4.00 10 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 180 210 Time (min)

–5 Fig. 9: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

5.00 5.00 M

M (a) (b) 5

5 4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 5.00

M M 5 5 (c) (d)

4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 180 0 30 60 90 120 150 180 210 Time (min) Time (min)

5.00 M

5 4.00 (e) 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 180 210 Time (min)

–5 Fig. 10: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

5.00 5.00

M M 5

5 4.00 (a) 4.00 (b)

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M M 5

5 4.00 (c) 4.00 (d)

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 M

5 4.00 (e) 10

× 3.00 2.00 1.00

0.00 Concentration 0 60 120 180 Time (min)

–5 Fig. 11: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

5.00 5.00 M

(a) M (b) 5

4.00 5 4.00

10 × 3.00 × 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00 M

M (c) (d) 5

5 4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 60 120 180 240 0 60 120 180 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00 0.00 Concentration 0 60 120 180 Time (min)

–5 Fig. 12: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

5.00 5.00

M M 5

5 4.00 (a) 4.00 (b)

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M M 5

5 4.00 (c) 4.00 (d)

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 60 120 180 Time (min)

–5 Fig. 13: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 8.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M carbonate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

100

5.00

5.00 M

M M (b) 5 5 (a)

10 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00

Concentration Concentration 0.00 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 5.00

M M 5 (d) 5 (c)

4.00 10 4.00

× × 3.00 3.00 2.00 2.00 1.00

1.00 Concentration

Concentration 0.00 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 M

5 (e)

4.00 10 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 180 Time (min)

–5 Fig. 14: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and

the formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

101

5.00 5.00

M M

(a) 5 (b) 5

4.00 10 4.00

10 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration

Concentration 0.00 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 5.00

M M

4.00 10 4.00

10 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 M 5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 180 Time (minutes)

–5 Fig. 15: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and

the formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

102

5.00 5.00

M 5

(a) 5 10

4.00 10 4.00 (b) × 3.00 × 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 30 60 90 120 150 180 -10 20 50 80 110 140 170 200 Time (min) Time (min)

5.00 5.00

M 5 5 (c) (d)

4.00 10 4.00

10 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00

(e) M

5 4.00 10

× 3.00 2.00 1.00 0.00 Concentration 0 30 60 90 120 150 180 Time (min)

–5 Fig. 16: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and

the formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

103

5.00 5.00

M 5

(a) 5 (b) 10

4.00 10 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 Time (min) 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min)

5.00 5.00

M M

5 (c) (d) 5

10 4.00 4.00

10 × 3.00 × 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 M

5 (e)

4.00 10 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 180 Time (min)

–5 Fig. 17: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and

the formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

104

5.00 5.00

M 5

5 (a) (b) 10

10 4.00 4.00 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 5.00

M 5

5 (c) (d)

4.00 10

10 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 180 Time (min)

–5 Fig. 18: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 8.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phosphate anions and

the formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

105

5.00 6.00 M (a) 5 5.00 (b) 4.00 10 × 4.00 3.00 3.00 2.00 2.00

1.00 1.00 Cocentration 0.00 0.00 0 60 120 180 0 30 60 90 120 150 180 Time (mins)

5.00 5.00

M M

4.00 10 4.00

10 × × 3.00 3.00 2.00 2.00 1.00 1.00

Concentration 0.00

Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 19: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

106

5.00 5.00

M 5

5 (a) (b)

4.00 4.00

10 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M M

5 (c) (d) 5 4.00

10 4.00

10 × 3.00 × 3.00 2.00 2.00 1.00 1.00

Concentration 0.00 0.00 Concentration 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00

M (e) 5

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 180 Time (min)

–5 Fig. 20: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

107

5.00 5.00

M 5 5 (a) (b)

4.00 4.00

10 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M M

5 (c) (d)

4.00 5 4.00

10 × 3.00 × 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 M

5 (e)

4.00 10 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 21: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

108

5.00 5.00

M 5

5 (a) (b) 10

10 4.00 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M M

5 (c) (d) 5

10 4.00 4.00

10 × 3.00 × 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 22: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

109

5.00 M 5.00

M 5

5 (a) (b) 10

10 4.00 4.00 × × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M M

5 (c) (d) 5

10 4.00 4.00

10 × 3.00 × 3.00 2.00 2.00

1.00 1.00 Concentration

0.00 Concentration 0.00 0 60 120 180 0 60 120 180 Time (minutes) Time (min)

M M 5.00

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 23: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 8.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M oxalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

110

5.00 5.00

M M M

5 (a) (b) 5

10 4.00

× × 3.00 3.00 2.00 2.00 1.00

1.00 Concentration

0.00 Concentration 0 60 120 180 0.00 Time (min) 0 60 120 180 Time (min)

5.00 5.00

M 5

M (c) (d) 5

4.00 10 4.00

× × 3.00 3.00 2.00 2.00 1.00

1.00 Concentration

Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 24: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

111

5.00 5.00

M 5

5 (a) (b) 10

10 4.00 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M 5

4.00 10 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

M 5.00 5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (minutes)

–5 Fig. 25: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 6.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

112

5.00 5.00

M 5

(a) 5 (b)

10 4.00

4.00 10

× × 3.00 3.00 2.00 2.00 1.00

1.00 Concentration

Concentration 0.00 0.00 0 60 120 180 0 30 60 90 120 150 180 Time (min) Time (min)

5.00 5.00

M 5

10 4.00

10 4.00 × 3.00 × 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00

M (e) 5

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 26: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

113

M 5.00 5.00

M 5

(a) 5 (b) 10

4.00 10 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M 5

4.00 10 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 27: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 7.5) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

114

5.00 M 5.00

M 5

5 (a) (b) 10

10 4.00 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 5.00

M 5

5 (c) (d) 10

10 4.00 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 60 120 180 0 60 120 180 Time (min) Time (min)

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 60 120 180 Time (min)

–5 Fig. 28: Kinetic plots of photodegradation of (5.0×10P P M) RF solution (pH 8.0) in the presence of (a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M phthalate anions and the

formation of photoproducts: (■) CDRF; (♦) FMF; (▲) LC; (×) LF.

115

CDRF k1 RF k 2 LC

The rate equation for the formation of these photoproducts is:

-d[RF] = (k + k ) [RF] = k [RF] dt 1 2 obs

Values of kR1R and kR2Rhave been determined from the ratio, R, of concentrations of

CDRF and LC formed at equilibrium as described by Florence and Attwood (2016).

k = k R 1 obs (R + 1)

k k = obs 2 (R + 1)

2- The values of these rate constants at pH 6.0–8.0 in presence of 0.2–1.0 M COR3RP ,P

2- 2- (COO)P ,P HPOR4RP andphthalateP (Table 24). Theyield of CDRF is maximumat pH 7.0. The

ratios of kR1R/kR2 Rshow a gradual increase in the formation of CDRF with an increase in

anion concentration. The highest increase in the values of kR1R/kR2 Rhave been found to be in the presence of carbonate anions. This could be due to a stronger interaction between RF to facilitate the formation of RF–divalent anion complex compared with other divalent anions.

The second–order rate constants for the interaction of anions with RF have been

developed from the plots of kRobs Rversus respective concentrations of different anions

116

(Fig. 29). The values of these rate constants (kʹ) were determined from the slopes of the straight lines are reported in Table 24.

The values of kʹ at pH 7.0 show the rates of interaction in the order: phthalate

ratio of kR1R/kR2Rin presences of carbonate anions (Table 24).

5.2.7 pH Effect

The effect of pH is an important factor in the drug stability and can be determined by the rate–pH profiles of drugs to find the pH of maximum degradation (Florence and

Attwood, 2016; Carstensen and Rhodes, 2000; Connors et al., 1986; Yoshioka and Stella,

2000; Khar et al., 2016).

The kRobsR–pH profiles for the photolysis of RF in the presence of the anions used

(1.0 M) are shown in Fig. 30.This show that maximum rate of reaction is at pH 7.0. This is in accordance with the findings of Schuman Jorns (1975) that the divalent anion– catalyzed photoaddition of RF takes place at pH ˃ 6 under neutral conditions. In the

present study the kRobsRvalues determined at pH 6 and 8 are lower than those of pH 7.0

(Table 24) as reported by Ahmad et al. (Ahmad et al., 2010).

117

9.0

8.0 1

- 7.0

6.0 , min ,

3 5.0

10 4.0

× 3.0 obs

k 2.0 1.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Concentration (M) of divalent anion

Fig. 29: Second–order plots for the photochemical interaction of RF and divalent anions:

(●) phthalate; (■) oxalate; (♦) phosphate; (▲) carbonate.

118

Table 24:Apparent first-order rate constants for photodegradation of RF(kRobsR), formation

of CDRF (kR1R) and LC (kR2R) in the presence of divalent anions and second-order rate constants (kʹ) for photochemical interaction of RF and divalent anions

3 1 3 3 3 - pH Divalent Concentration kRobsR× 10P P kP P × 10P P kR2R× 10P P k’ × 10P (MP P kR1R/kR2 -1 -1 -1 1 anion (M) (minP )P (minP )P (minP )P Pmin-1) ±SD ±SD ±SD 6.0 Carbonate 0.2 3.16±0.12 1.84±0.07 1.40±0.05 2.00±0.08 1.31 0.4 3.78±0.15 2.18±0.08 1.62±0.06 1.34 0.6 4.31±0.17 2.52±0.10 1.83±0.07 1.37 0.8 4.76±0.19 2.80±0.11 2.00±0.08 1.40 1.0 5.45±0.40 3.20±0.12 2.25±0.19 1.42 6.5 0.2 5.22±0.20 2.42±0.09 1.83±0.07 2.96±0.11 1.32 0.4 5.37±0.21 2.91±0.11 2.13±0.08 1.36 0.6 5.96±0.23 3.39±0.13 2.43±0.09 1.39 0.8 6.64±0.26 3.70±0.14 2.58±0.10 1.43 1.0 6.64±0.32 3.94±0.30 2.70±0.20 1.46 7.0 0.2 3.2±6±0.16 1.87±0.08 1.39±0.06 5.48±0.24 1.34 0.4 4.41±0.19 2.58±0.12 1.83±0.09 1.41 0.6 5.44±0.23 3.22±0.14 2.22±0.10 1.45 0.8 6.51±0.31 3.88±0.16 2.63±0.11 1.48 1.0 8.12±0.36 4.64±0.19 3.48±0.17 1.51 7.5 0.2 4.60±0.18 2.58±0.10 1.88±0.07 3..33±0.13 1.37 0.4 5.25±0.21 2.92±0.11 2.07±0.08 1.41 0.6 5.90±0.23 3.38±0.13 2.34±0.09 1.44 0.8 6.60±0.26 3.79±0.15 2.57±0.10 1.47 1.0 7.45±0.33 4.47±0.19 2.98±0.14 1.50 8.0 0.2 5.13±0.20 2.77±0.11 2.21±0.08 1.53±0.06 1.25 0.4 5.44±0.07 3.13±0.12 2.44±0.09 1.28 0.6 5.62±0.22 3.55±0.14 2.70±0.10 1.31

119

0.8 5.84 ±0.23 4.00±0.16 2.98±0.11 1.34 1.0 6.58±0.31 4.38±0.20 3.19±0.12 1.37 6.0 Phosphate 0.2 2.41±0.09 0.99±0.03 1.62±0.06 1.64±0.06 0.61 0.4 2.89±0.11 1.14±0.04 1.80±0.07 0.63 0.6 3.22±0.12 1.31±0.05 1.98±0.07 0.66 0.8 3.70±0.14 1.49±0.05 2.15±0.08 0.69 1.0 4.05±0.16 1.74±0.06 2.41±0.09 0.72 6.5 0.2 2.89±0.11 1.26±0.05 1.80±0.07 1.89±0.07 0.70 0.4 3.28±0.13 1.45±0.05 1.98±0.07 0.73 0.6 3.81±0.15 1.61±0.06 2.14±0.08 0.75 0.8 4.33±0.17 1.83±0.07 2.34±0.09 0.78 1.0 4.69±0.22 2.01±0.09 2.52±0.12 0.80 7.0 0.2 2.94±0.14 1.33±0.07 1.61±0.14 4.21±0.17 0.83 0.4 3.85±0.19 1.77±0.08 2.08±0.09 0.85 0.6 4.61±0.02 2.15±0.10 2.47±0.12 0.87 0.8 5.43±0.27 2.56±0.10 2.87±0.13 0.89 1.0 5.96±0.28 2.81±0.14 3.15±0.13 090 7.5 0.2 2.97±0.11 1.28±0.05 1.58±0.06 2.76±0.11 0.81 0.4 3.62±0.14 1.59±0.06 1.93±0.07 0.82 0.6 4.17±0.16 1.87±0.07 2.20±0.08 0.85 0.8 4.86±0.19 2.23±0.08 2.56±0.10 0.87 1.0 5.57±0.25 2.62±0.12 2.95±0.13 0.89 8.0 0.2 2.54±0.10 1.02±0.04 1.47±0.05 2.21±0.09 0.69 0.4 3.10±0.12 1.28±0.05 1.77±0.07 0.72 0.6 3.68±0.14 1.52±0.06 2.05±0.08 0.74 0.8 4.40±0.17 1.91±0.07 2.48±0.09 0.77 1.0 4.80±0.21 2.12±0.09 2.68±0.12 0.79 6.0 Oxalate 0.2 1.84±0.07 0.49±0.01 1.53±0.06 1.07±0.04 0.32 0.4 2.02±0.08 0.58±0.02 1.65±0.06 0.35 0.6 2.30±0.09 0.69±0.02 1.81±0.07 0.38 0.8 2.62±0.10 0.81±0.03 1.88±0.07 0.43

120

1.0 2.91±0.14 0.91±0.04 2.00±0.09 0.45 6.5 0.2 2.01±0.08 0.58±0.02 1.56±0.06 1.22±0.05 0.37 0.4 2.29±0.09 0.70±0.02 1.79±0.07 0.39 0.6 2.61±0.10 0.81±0.03 1.92±0.07 0.42 0.8 2.91±0.11 0.90±0.03 1.98±0.07 0.45 1.0 3.11±0.14 1.01±0.04 2.10±0.09 0.48 7.0 0.2 2.81±0.13 0.90±0.04 1.91±0.08 2.77±0.11 0.47 0.4 3.35±0.16 1.12±0.05 2.23±0.10 0.50 0.6 3.90±0.16 1.37±0.06 2.53±0.11 0.54 0.8 4.42±0.18 1.65±0.07 2.77±0.12 0.59 1.0 5.05±0.21 2.16±0.08 3.0±0.12 0.67 7.5 0.2 2.24±0.08 0.59±0.02 1.25±0.05 1.94±0.08 0.47 0.4 2.73±0.10 0.76±0.03 1.55±0.06 0.49 0.6 3.27±0.13 0.88±0.03 1.72±0.07 0.51 0.8 3.69±0.14 0.98±0.03 1.81±0.07 0.54 1.0 4.11±0.17 1.50±0.06 2.61±0.11 0.57 8.0 0.2 2.20±0.08 0.69±0.02 1.81±0.07 1.38±0.05 0.38 0.4 2.61±0.10 0.75±0.03 1.87±0.07 0.40 0.6 3.12±0.12 0.87±0.03 2.02±0.08 0.43 0.8 3.29±0.13 1.09±0.04 2.36±0.09 0.46 1.0 3.62±0.15 1.20±0.05 2.42±0.10 0.49 6.0 Phthalate 0.2 1.86±0.07 0.14±0.02 1.27±0.04 0.47±0.02 0.11 0.4 2.00±0.08 0.17±0.03 1.40.±0.05 0.12 0.6 2.31±0.09 0.21±0.04 1.61±0.06 0.13 0.8 2.50±0.10 0.28±0.01 1.75±0.07 0.16 1.0 2.60±0.11 0.40±0.02 2.21±0.09 0.18 6.5 0.2 1.48±0.05 0.11±0.01 1.22±0.04 0.56±0.02 0.09 0.4 1.72±0.06 0.16±0.02 1.14±0.04 0.11 0.6 2.22±0.08 0.20±0.04 1.42±0.05 0.14 0.8 2.44±0.09 0.26±0.01 1.52±0.06 0.17 1.0 2.80±0.11 0.47±0.02 2.33±0.09 0.20

121

7.0 0.2 2.40±0.11 0.22±0.01 2.18±0.09 0.76±0.03 0.10 0.4 2.55±0.10 0.32±0.02 2.23±0.10 0.14 0.6 2.73±0.11 0.44±0.02 2.45±0.11 0.18 0.8 2.86±0.11 0.49±0.02 2.376±0.10 0.21 1.0 3.01±0.12 0.61±0.02 2.40±0.11 0.25 7.5 0.2 1.87±0.07 0.21±0.01 1.90±0.07 0.51±0.02 0.11 0.4 2.13±0.08 0.29±0.01 2.07±0.08 0.14 0.6 2.38±0.09 0.36±0.01 2.11±0.08 0.17 0.8 2.67±0.10 0.46±0.01 2.30±0.09 0.20 1.0 2.94±0.12 0.53±0.02 2.40±0.10 0.22 8.0 0.2 1.76±0.07 0.18±0.01 1.80±0.07 0.32±0.01 0.10 0.4 2.01±0.08 0.25±0.01 1.92±0.07 0.13 0.6 2.32±0.09 0.33±0.01 2.20±0.08 0.15 0.8 2.45±0.09 0.37±0.01 2.05±0.08 0.18 1.0 2.69±0.11 0.45±0.02 2.24±0.09 0.20

*n = 3

122

Fig. 30: Plots of kRobsR for the photodegradation of RF in the presence of divalent anions (1.0

M) versus pH: (●) phthalate; (■) oxalate; (♦) phosphate; (▲) carbonate.

123

5.2.8 RF–divalent Anion Complex Formation

RF forms complexes with divalent anions (Ahmad and Vaid, 2006; Sheraz et al.,

2014; Jorns et al., 1975; Halwer, 1951; Ahmad et al., 2005, 2006, 2010, 2014) and divalent cations (Ahmad et al., 2017; Sakai, 1956; Rutters, 1958; Constable, 1989; Kaim et al., 1999; Hussain et al., 2006; Jabbar et al., 2014; Fukuzumi et al., 2010). The degree of complexation depends on the characteristics and extent of interaction of the anion.

RF–anion interaction results in the quenching of the fluorescence of RF (Ahmad et al., 2004, 2005, 2006, 2010; Sheraz et al., 2014). This is probably an indication of the degree of RF–anion complexation. This would affect the kinetics of the reaction to form

CDRF.

To find a correlation between the rates of photolysis of RF, the formation of

CDRF and LC and the decrease in the fluorescence of RF the values of kRobsR, kR1 Rand kR2

Rhave been plotted against % fluorescence decrease of RF (Figure31). These plots show an

increase in loss of fluorescence with an increase in kRobs Rand kR1R in the order: phthalate ˂ oxalate ˂ phosphate ˂ carbonate. Thus, higher the fluorescence loss of RF in the presence of an anion, greater the formation of CDRF due to increase catalytic activity of the anion.

Conversely, an increase in fluorescence loss results in a decrease in kR 2 Rvalues. This indicates that formation of LC decreases to greater complexation and of RF–divalent anions to form CDRF. Thus, greater the RF–anion complexation, greater is the formation of CDRF as found in the presence of carbonate anions.

The lowest rate of CDRF formation in presence of phthalate anions is supported by the observations of Schuman Jorns (1975) that the reaction undergoes to a low extent

124

1 - 9.0

8.0

, min , 3

10 7.0 × 6.0 5.0 4.0 3.0

2.0

order order rate constants -

1.0 First 0.0 0 20 40 60 80 100

% Fluoresence loss

Fig. 31. Plots of loss of RF (kRobsR) and formation of CDRF (kR1R) and LC (kR2R) as a function of

% fluorescence loss of RF in the presence of divalent anions (1.0 M): (●) phthalate; (■)

oxalate; (♦) phosphate; (▲) carbonate.

125 in presence of phthalate anions. This has been found in the present study on the photolysis of RF and yields of CDRF and LC as a function of fluorescence loss of RF

(Ahmad et al., 2005).

5.2.9 Photolysis Pathways of RF

RF undergoes photolysis through intramolecular photoreduction, photoaddition and photodealkylation reaction on irradiation under different conditions (Song, 1971;

Heelis, 1982, 1991; Ahmad and Vaid, 2006; Anwar and Ahmad, 2017).

In the present study RF photolysis has been found to occur by simultaneous intramolecular photoreduction on removal of ribose side chain in aqueous and organic solvents (Smith and Metlzer, 1963; Cairns and Metlzer, 1971; Ahmad and Rapson, 1990;

Ahmad et al. 2004a; Sheraz et al.,2014), as well as by intramolecular photoaddition in presence of divalent anions by cyclization of ribose side chain at position C(2’)-OH to position C (9) of RF. The divalent anions catalyze photoaddition reaction through a RF- divalent anion complex (Shuruman Jorns et al., 1975; Ahmad et al, 2004b, 2005, 2006,

2010, 2017; Sheraz et al.. 2014). The change in photoreduction pathway in favor of the photoaddition pathway depends on the catalytic activity of the anion involved. The

2- present study shows that COR3RP P anions exert the greatest catalytic activity to induce

2- 2- photoaddition of RF to form CDRF. This is followed by HPOR4RP ,P (COO)R2RP P and phthalate anions.

5.2.10. Mode of Photoaddition Reaction

The intramolecular photoaddition reaction of RF was first reported by Schuman

Jorns (1975) under the conditions of pH values > 6.0 in the presence of divalent anions (>

126

0.1 M). The reaction strictly required the presence of a nucleophile in N(10)-side chain.

They proposed a mechanism for the photoaddition of C(2ʹ)-OH at position C(9) of RF

2- and suggested that the reaction takes place through a RF-AP P (divalent anion) complex.

This complex enables the creation of sterically favorable condition for C(9)/O(2ʹ) interaction resulting in the formation of CDRF as expressed by the following reactions.

2- HPO4 2- hv 1 RF [RF-HPO4 ] [RFox] complex excited singlet state

1 cyclization autoxidation [RFox] [dihydroflavin] CDRF intermediate

The main function of divalent anions in photoaddition of RF is to catalyze the reaction to yield CDRF through the formation of RF-divalent anion complex.

127

CHAPTER 6

SIMULTANEOUS PHOTOADDITION, PHOTOREDUCTION AND CHEMICAL REDUCTION OF RIBOFLAVIN BY SULFUR CONTAINING DIANIONS

6.1 INTRODUCTION

Riboflavin (RF) (1) is a light sensitive compound (British Pharmacopoeia, 2016;

United States Pharmacopoeia, 2016) and its photochemistry has long been studied by several workers (see Chapter 3) (Holmstrom, 1964; Hemmerich et al., 1970; Penzer,

1971; Song, 1971, Hemmerich, 1976; Muller, 1981; Heelis, 1982, 1991; Ahmad and

Vaid, 2006; Sheraz et al., 2014; Anwar and Ahmad, 2017). Photolyzed by several mechanisms including intramolecularphotoreduction, photoaddition and photodealkylation to form formylmethylflavin (FMF) (2), carboxymethylflavin (CMF)

(3), lumichrome (LC) (4), lumiflavin (LF) (5) and cyclodehrdoriboflavin (CDRF) (6)

(Figure. 2) (Heelis, 1982, 1991; Ahmad and Vaid, 2006; Sheraz et al., 2014a; Anwar and

Ahmad, 2017, Schuman Jorns et al., 1975). The previous studies on RF photochemistry have mainly been concerned with its intramolecular photoreduction and side-chain cleavage to give FMF, CMF, LC and LF (Smith and Metzler, 1963; Moore et al., 1963;

Treadwell et al., 1968; Cairns and Metzler, 1971; Ahmad and Rapson, 1990; Ahmad et al., 2004a).

On the discovery of the mode of intramolecular photoaddition of RF catalyzed by dianions anions (Schuman Jorns, 1974, 1975), several studies were performed to study the kinetics of the reaction and the change in photoreduction pathway in favor of the photoaddition using phosphate, sulphate, tartarate, succinate and malonate anions

(Ahmad et al., 2004, 2005, 2006, 2010; Sheraz et al., 2014). It was found that the photolysis of RF in solution containing dianions involves photoaddition pathway to form

CDRF as well as the photoreduction pathway to give FMF, CMF, LC and LF (Schuman

Jorns et al., 1975, Ahmad et al., 2004a,b, 2005, 2006, 2010). The rates of these

129 simultaneous reactions depend on the catalytic activity of the dianions used (Ahmad et al., 2004, 2005, 2006, 2010). The chemical reduction of RF by dithionite anion (Burns and Obriens, 1959) and thiosulfate anion (Asker andHabib, 1990) gives the colorless derivative, dihydrorioflavin (DHRF), which is resistant to photolysis. The sulfur containg dianions have considerable pharmaceutical importance since these are used as antioxidants in pharmaceutical preparations. The present investigation is based on a study of the effect of reducing dianions on the photodegradation pathways of RF and the contribution of chemical reduction in the overall photodegradtion of RF.

6.2 RESULTS AND DISCUSSION

6.2.1Identifcation of Photoproducts of RF

The photolysis of RF solutions containing sulfite, thiosulfate and metabisulfite dianions form FMF, LC and CMF by photoreduction and CDRF by photoadditon pathways as in the absence of these dianions (Cairns and Metzler, 1971; Ahmad and

Rapson, 1990; Ahmad et al., 2004a, 2004b, 2005, 2006, 2010; Schuman Jorns et al.,

1975).The identification of these products is performed on comparing fluorescence emission (RF, FMF, LC, CMF, yellowish green, LC, sky blue) and color (CDRF, dark red) and Rf values with reference compounds. FMF and CMF both are intermediate products in the photolysisof RF (Smith and Metzler, 1963; Cairns and Metzler, 1971;

Ahmad and Rapson, 1990; Ahmad et al., 2004a, b) that is degraded to LC and LF in aqueous solution on hydrolysis (Song et al., 1965; Ahmad et al., 1980, 2013, 2018;

Mirzaet al., 2018). The Rf values of these photoproducts are given in Table 3 (Chapter 5).

130

6.2.2 Distribution of Photoproducts

The sulfur containing dianions (1.0 M) used in this study on RF photolysis affect product composition (Table 25). The ratios of CDRF and LC change on chemical reduction of RF to give DHRF during photolysis. The formation of CDRF has been found to increase in the order: thiosulfate ˂ sulfite ˂metabisulfiteand the formation of LC and

DHRF decreases in the same order. The increase in the formation DHRF results in a decrease in the formation of CDRF and an increase in the formation of LC. LC is formed by intramolecular photoreduction of RF through a reduced radical species (DHRF with an altered ribityl side chain), whereas the chemical reduction of RF gives a stable DHRF species (Burn and Oberin, 1959; Askar and Habib, 1990; Moore 1963; Heelis 1991;

Ahmad and Vaid, 2006).

6.2.3 Assay of RF and Photoproducts

The spectrophotometric method employed for the assay of RF and photoproducts

(Chapter 4) is based on chloroform extraction of LC and LF from photolyzed solution at pH 2 and their determination (pH 4.5) by a two-component method at 356 and 445 nm.

The wavelength corresponding to the absorption maxima of LC and LF. RF, CDRF and

FMF are determined at 445, 410 and 385 nm (absorption maxima of these compounds, respectively) by a three component method (Ahmad et al., 2004b). The determination of

DHRF is carried out by measuring the decrease in the absorbance at 445 nm. Its concentration in photolyzed solution has been determined by the difference in the initial concentration of RF and the total molar concentration of degraded RF and photoproducts

(CDRF + FMF + LC + LF) during the reaction.The assay values of RF and photoproducts

131

Table 25. Product distribution at 30% photodegradation of 5 × 10-5 M riboflavin solution

(pH 7.0) in the presence of 1.0 M dianions

Dianion Time CDRF FMF LC LF DHRF CDRF/DHRF CDRF/LC % Fluorescence (min) % a % a %a %a %a loss

Un-buffered 155.0 - 5.6 22.1 2.3 - - - -

Metabisufite 129.5 7.2 4.2 11.6 1.9 5.1 1.41 0.62 34.5

Sulfite 108.2 6.1 3.7 12.5 2.0 5.7 1.07 0.48 50.2

Thiosulfate 92.0 5.2 3.4 12.8 2.1 6.5 0.80 0.40 66.0

a Percentage of the amount of photodegraded RF.

132 during the photolysis of RF in the presence of dianions (0.2-1.0 M) are given in Table 26-

40. An almost constant molar balance has been achieved during the course of these reactions as an indication of the accuracy of the method.

6.2.4 Ultraviolet and Visible Spectra of Photodegraded Solutions

The absorption spectra of RF solutions (pH 7) are affected by the dianions in light and in dark. The magnitude of spectral changes during photolysis of RF depends on the strength of interaction of RF and dianions. The changes observed on the photolysis

(Figure 32) are more prominent in solutions containing thiosulfate dianions than those containing sulfite or metabisulfite dianions. A gradual decrease in absorbance of 445 and

374 nm is observed with greatest decrease in solutions containing thiosulfate dianions followed by that of sulfite and metabisulfite dianions. An increase in A374nm/A445nm ration in the same order indicates the formation of LC that absorbs in the region at 356 nm. A decrease at 315 nm is also observed in solutions containing sulfite and metabisulfitedianins as reported earlier on the photolysis of RF in solutions containing

Fe2+ dications.

The spectra of RF show a decrease in absorbance at 445 nm in dark (Figure 33) in the order: thiosulfate > sulfite > metabisulfite. These changes occur on chemical reduction of the conjugated system N(5)=C(4a)-C(10a)=N(1) in the quinoxaline ring of the isoalloxazine nucleus of RF to give DHRF which has no absorbance in visible region.

The results show that thiosulfate produces greatest reduction of RF in light and in dark.

133

Table 26.Concentrations of RF and photoproducts in the presence of thiosulfate anions

(0.2-1.0 M) at pH 6.0

Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.63 0.06 0.08 0.21 0.01 0.01 5.00 60 4.40 0.10 0.13 0.33 0.03 0.03 5.02 90 4.18 0.12 0.18 0.45 0.05 0.04 5.02 120 3.99 0.16 0.23 0.53 0.06 0.05 5.02 150 3.86 0.18 0.25 0.59 0.08 0.06 5.02 0 0.4 5.00 - - - - - 5.00 30 4.60 0.07 0.08 0.25 0.02 0.01 5.03 60 4.31 0.11 0.16 0.40 0.03 0.03 5.04 90 4.02 0.15 0.20 0.53 0.06 0.04 5.00 120 3.83 0.21 0.24 0.59 0.08 0.05 5.00 150 3.72 0.21 0.27 0.63 0.09 0.08 5.00 0 0.6 5.00 - - - - - 5.00 30 4.64 0.10 0.09 0.13 0.04 0.01 5.01 60 4.37 0.13 0.16 0.25 0.05 0.02 4.98 90 4.10 0.18 0.21 0.36 0.08 0.04 4.97 120 3.80 0.23 0.26 0.47 0.10 0.06 4.92 150 3.50 0.24 0.30 0.67 0.11 0.11 4.93 0 0.8 5.00 - - - - - 5.00 30 4.51 0.08 0.08 0.30 0.05 0.03 5.05 60 4.17 0.17 0.16 0.49 0.06 0.05 5.10 90 3.81 0.20 0.20 0.62 0.08 0.07 4.98 120 3.53 0.23 0.26 0.67 0.10 0.10 4.89 150 3.32 0.27 0.32 0.72 0.12 0.12 4.87 0 1.0 5.00 - - - - - 5.00 30 4.50 0.09 0.09 0.22 0.05 0.04 4.99 60 4.08 0.17 0.17 0.40 0.07 0.06 4.95 90 3.70 0.20 0.23 0.56 0.09 0.09 4.87 120 3.39 0.26 0.27 0.70 0.11 0.11 4.84 150 3.14 0.31 0.35 0.77 0.13 0.14 4.84

134

Table 27.Concentrations of RF and photoproducts in the presence of thiosulfate anions

(0.2-1.0 M) at pH 6.5

Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.76 0.02 0.04 0.16 0.00 0.01 4.99 60 4.58 0.05 0.06 0.29 0.02 0.02 4.99 90 4.42 0.06 0.08 0.35 0.04 0.04 4.99 120 4.30 0.07 0.12 0.40 0.06 0.05 5.00 150 4.21 0.09 0.14 0.43 0.07 0.06 5.00 0 0.4 5.00 - - - - - 5.00 30 4.73 0.04 0.04 0.17 0.01 0.01 4.99 60 4.52 0.06 0.07 0.31 0.03 0.02 4.99 90 4.32 0.08 0.10 0.37 0.05 0.04 4.92 120 4.19 0.09 0.13 0.42 0.07 0.07 4.9 150 4.07 0.11 0.17 0.46 0.10 0.09 4.91 0 0.6 5.00 - - - - - 5.00 30 4.67 0.05 0.07 0.19 0.03 0.03 5.04 60 4.42 0.07 0.11 0.33 0.05 0.05 5.03 90 4.23 0.10 0.13 0.40 0.08 0.06 5.00 120 4.08 0.12 0.17 0.45 0.10 0.08 5.00 150 3.93 0.14 0.20 0.49 0.12 0.11 4.99 0 0.8 5.00 - - - - - 5.00 30 4.62 0.06 0.08 0.21 0.07 0.02 5.04 60 4.30 0.10 0.12 0.35 0.08 0.06 4.95 90 4.07 0.13 0.15 0.42 0.09 0.09 4.86 120 3.91 0.15 0.18 0.49 0.11 0.12 4.84 150 3.80 0.17 0.22 0.53 0.13 0.15 4.85 0 1.0 5.00 - - - - - 5.00 30 4.53 0.07 0.09 0.23 0.07 0.03 5.02 60 4.22 0.11 0.13 0.39 0.09 0.07 5.01 90 3.95 0.14 0.18 0.49 0.11 0.11 4.98 120 3.75 0.18 0.22 0.54 0.13 0.14 4.96 150 3.60 0.22 0.25 0.59 0.15 0.17 4.98

135

Table 28.Concentrations of RF and photoproducts in the presence of thiosulfate anions (0.2-1.0 M) at pH 7.0 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.63 0.06 0.08 0.21 0.01 0.02 5.01 60 4.40 0.10 0.13 0.33 0.03 0.05 5.04 90 4.18 0.12 0.18 0.45 0.05 0.10 5.08 120 3.99 0.16 0.23 0.53 0.06 0.12 5.09 150 3.86 0.18 0.25 0.59 0.08 0.16 5.12 0 0.4 5.00 - - - - - 5.00 30 4.60 0.07 0.08 0.25 0.02 0.03 5.05 60 4.31 0.11 0.16 0.40 0.03 0.09 5.10 90 4.02 0.15 0.20 0.53 0.06 0.12 5.08 120 3.83 0.21 0.24 0.59 0.08 0.15 5.10 150 3.72 0.21 0.27 0.63 0.09 0.19 5.11 0 0.6 5.00 - - - - - 5.00 30 4.64 0.10 0.09 0.13 0.04 0.04 5.03 60 4.37 0.13 0.16 0.25 0.05 0.11 5.07 90 4.10 0.18 0.21 0.36 0.08 0.17 5.10 120 3.80 0.23 0.26 0.47 0.10 0.21 5.07 150 3.50 0.24 0.30 0.67 0.11 0.23 5.05 0 0.8 5.00 - - - - - 5.00 30 4.51 0.08 0.08 0.30 0.05 0.06 5.08 60 4.17 0.17 0.16 0.49 0.06 0.11 5.05 90 3.81 0.20 0.20 0.62 0.08 0.16 5.07 120 3.53 0.23 0.26 0.67 0.10 0.20 4.99 150 3.32 0.27 0.32 0.72 0.12 0.25 5.00 0 1.0 5.00 - - - - - 5.00 30 4.50 0.09 0.09 0.22 0.05 0.09 5.04 60 4.08 0.17 0.17 0.40 0.07 0.17 5.06 90 3.70 0.20 0.23 0.56 0.09 0.23 5.01 120 3.39 0.26 0.27 0.70 0.11 0.27 5.05 150 3.14 0.31 0.35 0.77 0.13 0.30 5.00

136

Table 29.Concentrations of RF and photoproducts in the presence of thiosulfate anions (0.2-1.0 M) at pH 7.5

Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.66 0.08 0.07 0.17 0.01 0.02 5.01 60 4.37 0.09 0.08 0.35 0.03 0.03 4.95 90 4.15 0.13 0.10 0.47 0.06 0.05 4.96 120 3.96 0.16 0.14 0.55 0.09 0.08 4.98 150 3.84 0.19 0.15 0.60 0.11 0.11 5.00 0 0.4 5.00 - - - - - 5.00 30 4.60 0.08 0.08 0.20 0.02 0.02 5.00 60 4.30 0.10 0.09 0.38 0.04 0.04 4.95 90 4.06 0.14 0.11 0.52 0.07 0.08 4.98 120 3.87 0.17 0.15 0.60 0.10 0.12 5.01 150 3.74 0.20 0.18 0.64 0.12 0.15 5.03 0 0.6 5.00 - - - - - 5.00 30 4.52 0.11 0.10 0.21 0.02 0.05 5.01 60 4.17 0.15 0.12 0.40 0.05 0.07 4.96 90 3.89 0.18 0.15 0.54 0.07 0.11 4.94 120 3.72 0.20 0.17 0.62 0.11 0.14 4.96 150 3.56 0.23 0.21 0.68 0.13 0.18 4.99 0 0.8 5.00 - - - - - 5.00 30 4.48 0.12 0.11 0.23 0.04 0.07 5.05 60 4.07 0.16 0.15 0.42 0.06 0.10 4.96 90 3.75 0.20 0.18 0.60 0.10 0.14 4.97 120 3.52 0.24 0.21 0.68 0.12 0.18 4.95 150 3.38 0.27 0.24 0.72 0.15 0.22 4.98 0 1.0 5.00 - - - - - 5.00 30 4.43 0.13 0.12 0.26 0.05 0.10 5.10 60 4.00 0.18 0.16 0.46 0.07 0.13 5.00 90 3.67 0.24 0.21 0.61 0.11 0.17 5.01 120 3.40 0.27 0.24 0.71 0.14 0.21 4.97 150 3.25 0.30 0.27 0.76 0.17 0.26 5.01

137

Table 30.Concentrations of RF and photoproducts in the presence of thiosulfate anions (0.2-1.0 M) at pH 8.0 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.71 0.06 0.05 0.17 0.00 0.01 5.01 60 4.46 0.10 0.08 0.31 0.01 0.04 5.00 90 4.30 0.13 0.10 0.38 0.03 0.06 5.00 120 4.15 0.16 0.13 0.44 0.05 0.09 5.02 150 4.01 0.19 0.15 0.49 0.08 0.11 5.03 0 0.4 5.00 - - - - - 5.00 30 4.65 0.08 0.06 0.18 0.02 0.02 5.01 60 4.37 0.13 0.09 0.32 0.04 0.04 4.99 90 4.15 0.16 0.12 0.42 0.07 0.07 4.99 120 3.96 0.18 0.15 0.49 0.09 0.10 4.97 150 3.82 0.22 0.18 0.53 0.11 0.14 5.00 0 0.6 5.00 - - - - - 5.00 30 4.58 0.09 0.09 0.19 0.03 0.03 5.01 60 4.26 0.16 0.11 0.33 0.05 0.05 4.96 90 3.99 0.19 0.13 0.45 0.08 0.08 4.92 120 3.76 0.24 0.18 0.52 0.11 0.13 4.94 150 3.63 0.27 0.21 0.57 0.12 0.17 4.97 0 0.8 5.00 - - - - - 5.00 30 4.55 0.10 0.10 0.20 0.04 0.04 5.03 60 4.18 0.17 0.14 0.36 0.07 0.07 4.99 90 3.86 0.22 0.17 0.49 0.1 0.13 4.97 120 3.62 0.26 0.19 0.56 0.13 0.17 4.93 150 3.50 0.30 0.23 0.61 0.15 0.21 5.00 0 1.0 5.00 - - - - - 5.00 30 4.49 0.11 0.11 0.21 0.07 0.10 5.09 60 4.10 0.18 0.16 0.41 0.10 0.14 5.09 90 3.74 0.24 0.21 0.52 0.12 0.17 5.00 120 3.50 0.27 0.24 0.61 0.15 0.21 4.98 150 3.35 0.34 0.26 0.65 0.17 0.24 5.01

138

Table 31.Concentrations of RF and photoproducts in the presence of sulfite anions (0.2-1.0 M) at pH 6.0 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.81 0.05 0.03 0.1 0.00 0.01 5.00 60 4.68 0.06 0.08 0.15 0.00 0.02 4.99 90 4.60 0.09 0.10 0.16 0.00 0.04 4.99 120 4.52 0.11 0.11 0.19 0.01 0.05 4.99 150 4.45 0.13 0.12 0.22 0.02 0.06 4.98 0 0.4 5.00 - - - - - 5.00 30 4.75 0.05 0.03 0.11 0.00 0.01 4.95 60 4.54 0.08 0.08 0.17 0.00 0.02 4.89 90 4.37 0.10 0.10 0.19 0.01 0.03 4.80 120 4.28 0.12 0.11 0.22 0.02 0.06 4.81 150 4.33 0.14 0.12 0.24 0.04 0.09 4.96 0 0.6 5.00 - - - - - 5.00 30 4.75 0.06 0.04 0.12 0.00 0.01 4.98 60 4.56 0.09 0.09 0.18 0.01 0.03 4.96 90 4.42 0.11 0.11 0.21 0.03 0.05 4.93 120 4.32 0.14 0.12 0.23 0.04 0.08 4.93 150 4.23 0.16 0.14 0.26 0.06 0.11 4.96 0 0.8 5.00 - - - - - 5.00 30 4.72 0.07 0.05 0.13 0.02 0.02 5.01 60 4.52 0.10 0.10 0.20 0.03 0.05 5.00 90 4.34 0.14 0.12 0.23 0.04 0.07 4.94 120 4.24 0.16 0.14 0.25 0.06 0.10 4.95 150 4.16 0.18 0.15 0.28 0.07 0.13 4.97 0 1.0 5.00 - - - - - 5.00 30 4.68 0.08 0.06 0.14 0.03 0.03 5.02 60 4.44 0.11 0.10 0.21 0.04 0.06 4.96 90 4.29 0.14 0.13 0.24 0.05 0.09 4.94 120 4.18 0.17 0.15 0.27 0.07 0.12 4.96 150 4.09 0.20 0.16 0.30 0.08 0.15 4.98

139

Table 32.Concentrations of RF and photoproducts in the presence of sulfite anions (0.2-1.0 M) at pH 6.5

Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.77 0.07 0.03 0.12 0.00 0.01 5.00 60 4.60 0.12 0.08 0.18 0.00 0.02 5.00 90 4.48 0.15 0.10 0.24 0.00 0.03 5.00 120 4.39 0.18 0.12 0.27 0.00 0.04 5.00 150 4.30 0.20 0.14 0.30 0.01 0.07 5.02 0 0.4 5.00 - - - - - 5.00 30 4.72 0.09 0.04 0.14 0.00 0.01 5.00 60 4.52 0.14 0.07 0.20 0.00 0.02 4.95 90 4.35 0.16 0.11 0.25 0.00 0.04 4.91 120 4.20 0.19 0.13 0.29 0.01 0.06 4.88 150 4.12 0.23 0.16 0.34 0.03 0.08 4.96 0 0.6 5.00 - - - - - 5.00 30 4.64 0.12 0.05 0.20 0.00 0.02 5.03 60 4.40 0.16 0.09 0.24 0.01 0.04 4.94 90 4.21 0.19 0.12 0.30 0.02 0.06 4.90 120 4.06 0.24 0.15 0.34 0.03 0.08 4.90 150 3.87 0.27 0.18 0.39 0.05 0.11 4.87 0 0.8 5.00 - - - - - 5.00 30 4.59 0.12 0.05 0.22 0.00 0.03 5.01 60 4.30 0.16 0.09 0.27 0.01 0.06 4.89 90 4.07 0.19 0.12 0.33 0.02 0.09 4.82 120 3.91 0.24 0.15 0.37 0.05 0.12 4.84 150 3.77 0.30 0.2 0.41 0.07 0.15 4.90 0 1.0 5.00 - - - - - 5.00 30 4.57 0.14 0.09 0.24 0.01 0.04 5.09 60 4.24 0.20 0.12 0.32 0.03 0.07 4.98 90 3.98 0.25 0.15 0.36 0.04 0.11 4.89 120 3.78 0.30 0.19 0.40 0.06 0.14 4.87 150 3.6 0.34 0.23 0.45 0.09 0.17 4.88

140

Table 33.Concentrations of RF and photoproducts in the presence of sulfite anions (0.2-1.0 M) at pH 7.0 Time Dianion RF CDRF FMF LC LF DHRF Total (min) s conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.68 0.06 0.06 0.14 0.00 0.02 4.96 60 4.44 0.11 0.08 0.25 0.01 0.04 4.93 90 4.27 0.15 0.10 0.33 0.02 0.07 4.94 120 4.10 0.18 0.12 0.42 0.04 0.09 4.95 150 3.95 0.21 0.14 0.50 0.05 0.12 4.97 0 0.4 5.00 - - - - - 5.00 30 4.70 0.07 0.07 0.17 0.01 0.03 5.05 60 4.43 0.14 0.09 0.32 0.03 0.05 5.06 90 4.19 0.17 0.11 0.41 0.04 0.07 4.99 120 3.98 0.21 0.14 0.48 0.06 0.11 4.98 150 3.80 0.24 0.16 0.53 0.07 0.15 4.95 0 0.6 5.00 - - - - - 5.00 30 4.64 0.09 0.08 0.15 0.03 0.06 5.05 60 4.32 0.16 0.10 0.25 0.05 0.08 4.96 90 4.06 0.21 0.12 0.38 0.06 0.11 4.94 120 3.82 0.25 0.15 0.49 0.08 0.15 4.94 150 3.60 0.27 0.18 0.60 0.09 0.18 4.92 0 0.8 5.00 - - - - - 5.00 30 4.55 0.10 0.09 0.16 0.04 0.07 5.01 60 4.20 0.18 0.12 0.29 0.06 0.10 4.95 90 3.90 0.27 0.16 0.40 0.09 0.14 4.96 120 3.67 0.31 0.18 0.53 0.10 0.17 4.96 150 3.47 0.29 0.22 0.63 0.12 0.20 4.93 0 1.0 5.00 - - - - - 5.00 30 4.50 0.11 0.09 0.25 0.04 0.10 5.10 60 4.10 0.22 0.14 0.42 0.07 0.13 5.08 90 3.78 0.25 0.18 0.52 0.09 0.16 4.98 120 3.52 0.29 0.21 0.60 0.11 0.20 4.93 150 3.35 0.33 0.24 0.67 0.13 0.23 4.95

141

Table 34.Concentrations of RF and photoproducts in the presence of sulfite anions (0.2-1.0 M) at pH 7.5 Time Dianion RF CDRF FMF LC LF DHRF Total (min) s conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.78 0.05 0.04 0.12 0.00 0.01 5.00 60 4.64 0.09 0.06 0.19 0.00 0.02 5.00 90 4.45 0.13 0.09 0.29 0.01 0.03 5.00 120 4.33 0.17 0.11 0.33 0.02 0.05 5.01 150 4.23 0.19 0.13 0.37 0.03 0.07 5.02 0 0.4 5.00 - - - - - 5.00 30 4.72 0.08 0.05 0.13 0.00 0.02 5.00 60 4.52 0.13 0.07 0.24 0.01 0.03 5.00 90 4.34 0.17 0.10 0.32 0.02 0.04 4.99 120 4.13 0.20 0.13 0.37 0.04 0.07 4.94 150 4.02 0.22 0.14 0.43 0.05 0.10 4.96 0 0.6 5.00 - - - - - 5.00 30 4.68 0.10 0.07 0.12 0.01 0.03 5.01 60 4.39 0.16 0.10 0.25 0.02 0.05 4.97 90 4.16 0.20 0.12 0.34 0.03 0.08 4.93 120 3.98 0.23 0.14 0.42 0.05 0.12 4.94 150 3.88 0.25 0.16 0.46 0.06 0.14 4.95 0 0.8 5.00 - - - - - 5.00 30 4.70 0.09 0.06 0.13 0.01 0.05 5.04 60 4.37 0.16 0.10 0.22 0.02 0.07 4.94 90 4.11 0.23 0.13 0.33 0.04 0.11 4.95 120 3.90 0.26 0.16 0.42 0.06 0.14 4.94 150 3.73 0.28 0.17 0.5 0.08 0.17 4.93 0 1.0 5.00 - - - - - 5.00 30 4.56 0.13 0.08 0.21 0.02 0.08 5.08 60 4.22 0.17 0.11 0.34 0.04 0.11 4.99 90 3.93 0.23 0.14 0.43 0.06 0.15 4.94 120 3.74 0.27 0.17 0.49 0.08 0.18 4.93 150 3.60 0.32 0.19 0.53 0.10 0.21 4.95

142

Table 35.Concentrations of RF and photoproducts in the presence of sulfite anions (0.2-1.0 M) at pH 8.0 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.70 0.09 0.04 0.14 0.00 0.01 4.98 60 4.47 0.18 0.08 0.21 0.02 0.03 4.99 90 4.31 0.22 0.09 0.26 0.05 0.04 4.97 120 4.20 0.26 0.10 0.30 0.07 0.06 4.99 150 4.07 0.30 0.12 0.34 0.09 0.09 5.01 0 0.4 5.00 - - - - - 5.00 30 4.71 0.05 0.04 0.15 0.00 0.03 4.98 60 4.44 0.10 0.09 0.29 0.02 0.05 4.99 90 4.28 0.12 0.10 0.38 0.04 0.07 4.99 120 4.09 0.15 0.13 0.49 0.05 0.10 5.01 150 3.96 0.17 0.15 0.55 0.07 0.12 5.02 0 0.6 5.00 - - - - - 5.00 30 4.62 0.07 0.06 0.16 0.02 0.03 4.96 60 4.33 0.10 0.10 0.32 0.04 0.05 4.94 90 4.13 0.14 0.12 0.43 0.06 0.08 4.96 120 3.95 0.17 0.14 0.53 0.07 0.11 4.97 150 3.80 0.20 0.17 0.59 0.09 0.14 4.99 0 0.8 5.00 - - - - - 5.00 30 4.59 0.10 0.07 0.16 0.03 0.06 5.01 60 4.23 0.13 0.11 0.32 0.05 0.08 4.92 90 3.98 0.17 0.14 0.46 0.07 0.11 4.93 120 3.80 0.20 0.17 0.56 0.08 0.15 4.96 150 3.65 0.23 0.19 0.63 0.10 0.17 4.97 0 1.0 5.00 - - - - - 5.00 30 4.52 0.11 0.08 0.2 0.05 0.09 5.05 60 4.12 0.15 0.13 0.36 0.07 0.11 4.94 90 3.84 0.19 0.18 0.50 0.09 0.14 4.94 120 3.66 0.22 0.20 0.58 0.10 0.17 4.93 150 3.47 0.27 0.22 0.67 0.12 0.20 4.95

143

Table 36.Concentrations of RF and photoproducts in the presence of metabisulfite anions (0.2-1.0 M) at pH 6.0 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.81 0.05 0.03 0.10 0.00 0.01 5.00 60 4.68 0.06 0.08 0.15 0.00 0.02 4.99 90 4.55 0.09 0.10 0.16 0.00 0.03 4.93 120 4.49 0.11 0.11 0.19 0.01 0.04 4.95 150 4.41 0.13 0.13 0.22 0.02 0.05 4.96 0 0.4 5.00 - - - - - 5.00 30 4.78 0.05 0.03 0.11 0.00 0.01 4.98 60 4.58 0.08 0.08 0.17 0.00 0.03 4.94 90 4.41 0.10 0.1 0.19 0.01 0.04 4.85 120 4.32 0.12 0.13 0.22 0.02 0.05 4.81 150 4.3 0.14 0.15 0.24 0.04 0.06 4.93 0 0.6 5.00 - - - - - 5.00 30 4.78 0.06 0.04 0.12 0.00 0.02 5.02 60 4.58 0.09 0.09 0.18 0.01 0.05 5.00 90 4.45 0.11 0.11 0.21 0.03 0.06 4.97 120 4.36 0.14 0.12 0.23 0.04 0.07 4.96 150 4.22 0.16 0.16 0.26 0.06 0.08 4.94 0 0.8 5.00 - - - - - 5.00 30 4.72 0.07 0.05 0.13 0.02 0.01 5.00 60 4.52 0.10 0.10 0.20 0.03 0.03 4.98 90 4.34 0.14 0.12 0.23 0.04 0.05 4.92 120 4.24 0.16 0.14 0.25 0.06 0.07 4.92 150 4.16 0.18 0.15 0.28 0.07 0.10 4.94 0 1.0 5.00 - - - - - 5.00 30 4.68 0.08 0.06 0.14 0.03 0.02 5.01 60 4.44 0.11 0.10 0.21 0.04 0.04 4.94 90 4.29 0.14 0.13 0.24 0.05 0.07 4.92 120 4.18 0.17 0.15 0.27 0.07 0.10 4.94 150 4.09 0.20 0.16 0.30 0.08 0.12 4.95

144

Table 37.Concentrations of RF and photoproducts in the presence of metabisulfite anions (0.2-1.0 M) at pH 6.5 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.84 0.04 0.03 0.06 0.00 0.01 4.98 60 4.73 0.07 0.06 0.08 0.00 0.02 4.96 90 4.60 0.10 0.08 0.12 0.00 0.04 4.94 120 4.52 0.12 0.10 0.15 0.00 0.06 4.95 150 4.42 0.14 0.13 0.18 0.01 0.07 4.95 0 0.4 5.00 - - - - - 5.00 30 4.79 0.07 0.05 0.08 0.00 0.01 5.00 60 4.65 0.09 0.07 0.11 0.00 0.03 4.95 90 4.53 0.12 0.10 0.14 0.00 0.04 4.93 120 4.43 0.14 0.13 0.17 0.01 0.07 4.95 150 4.33 0.16 0.15 0.20 0.02 0.09 4.95 0 0.6 5.00 - - - - - 5.00 30 4.72 0.08 0.07 0.11 0.00 0.02 5.00 60 4.55 0.10 0.10 0.13 0.00 0.04 4.92 90 4.40 0.13 0.12 0.17 0.01 0.06 4.89 120 4.28 0.16 0.15 0.21 0.02 0.09 4.91 150 4.17 0.19 0.18 0.24 0.03 0.11 4.92 0 0.8 5.00 - - - - - 5.00 30 4.67 0.10 0.08 0.14 0.00 0.02 5.01 60 4.45 0.12 0.11 0.18 0.00 0.06 4.92 90 4.29 0.15 0.15 0.21 0.01 0.08 4.89 120 4.14 0.19 0.18 0.24 0.03 0.11 4.89 150 4.04 0.22 0.21 0.27 0.05 0.13 4.92 0 1.0 5.00 - - - - - 5.00 30 4.62 0.10 0.09 0.17 0.01 0.03 5.02 60 4.35 0.14 0.13 0.21 0.02 0.06 4.91 90 4.14 0.18 0.17 0.25 0.04 0.09 4.87 120 3.97 0.22 0.22 0.28 0.05 0.12 4.86 150 3.84 0.25 0.23 0.33 0.08 0.15 4.88

145

Table 38.Concentrations of RF and photoproducts in the presence of metabisulfite anions (0.2-1.0 M) at pH 7.0 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.72 0.07 0.06 0.12 0.00 0.01 4.98 60 4.52 0.14 0.07 0.22 0.01 0.02 4.98 90 4.32 0.18 0.08 0.31 0.02 0.04 4.95 120 4.16 0.21 0.10 0.39 0.04 0.07 4.97 150 4.03 0.25 0.12 0.44 0.05 0.09 4.98 0 0.4 5.00 - - - - - 5.00 30 4.70 0.08 0.07 0.14 0.01 0.01 5.01 60 4.43 0.15 0.09 0.26 0.03 0.03 4.99 90 4.19 0.20 0.11 0.39 0.04 0.05 4.98 120 4.00 0.25 0.12 0.45 0.06 0.08 4.96 150 3.86 0.28 0.13 0.49 0.07 0.11 4.94 0 0.6 5.00 - - - - - 5.00 30 4.64 0.09 0.08 0.15 0.03 0.02 5.01 60 4.38 0.16 0.10 0.24 0.05 0.06 4.99 90 4.12 0.21 0.12 0.33 0.06 0.08 4.92 120 3.90 0.26 0.14 0.44 0.08 0.11 4.93 150 3.74 0.30 0.15 0.52 0.09 0.15 4.95 0 0.8 5.00 - - - - - 5.00 30 4.60 0.10 0.09 0.16 0.04 0.03 5.02 60 4.27 0.18 0.12 0.29 0.06 0.06 4.98 90 3.96 0.27 0.16 0.40 0.09 0.09 4.97 120 3.74 0.31 0.18 0.50 0.10 0.13 4.96 150 3.59 0.34 0.17 0.57 0.11 0.17 4.95 0 1.0 5.00 - - - - - 5.00 30 4.52 0.11 0.10 0.22 0.04 0.09 5.08 60 4.14 0.21 0.13 0.31 0.07 0.11 4.97 90 3.84 0.28 0.15 0.42 0.09 0.15 4.93 120 3.61 0.33 0.17 0.55 0.11 0.17 4.94 150 3.47 0.37 0.18 0.61 0.12 0.21 4.96

146

Table 39.Concentrations of RF and photoproducts in the presence of metabisulfite anions (0.2-1.0 M) at pH 7.5 Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.72 0.04 0.04 0.11 0.00 0.01 4.92 60 4.48 0.10 0.14 0.20 0.00 0.02 4.94 90 4.35 0.12 0.17 0.26 0.00 0.04 4.94 120 4.23 0.14 0.20 0.30 0.01 0.06 4.94 150 4.17 0.17 0.22 0.34 0.02 0.09 5.01 0 0.4 5.00 - - - - - 5.00 30 4.70 0.08 0.07 0.14 0.00 0.01 5.00 60 4.46 0.12 0.14 0.23 0.00 0.03 4.98 90 4.31 0.17 0.17 0.28 0.00 0.05 4.98 120 4.17 0.20 0.21 0.33 0.01 0.08 5.00 150 4.06 0.21 0.24 0.38 0.03 0.11 5.03 0 0.6 5.00 - - - - - 5.00 30 4.63 0.1 0.12 0.14 0.00 0.02 5.01 60 4.38 0.15 0.16 0.22 0.00 0.04 4.95 90 4.19 0.19 0.21 0.33 0.01 0.07 5.00 120 4.05 0.21 0.25 0.38 0.03 0.11 5.03 150 3.92 0.23 0.27 0.42 0.05 0.14 5.03 0 0.8 5.00 - - - - - 5.00 30 4.54 0.09 0.15 0.21 0.00 0.04 5.03 60 4.24 0.17 0.20 0.32 0.02 0.07 5.02 90 4.06 0.21 0.22 0.37 0.03 0.10 4.99 120 3.89 0.26 0.24 0.40 0.04 0.14 4.97 150 3.79 0.27 0.26 0.46 0.06 0.17 5.01 0 1.0 5.00 - - - - - 5.00 30 4.56 0.11 0.12 0.20 0.00 0.06 5.05 60 4.19 0.17 0.17 0.34 0.02 0.10 4.99 90 3.93 0.21 0.23 0.40 0.03 0.14 4.94 120 3.72 0.26 0.27 0.46 0.04 0.17 4.92 150 3.64 0.30 0.29 0.47 0.08 0.20 4.98

147

Table 40.Concentrations of RF and photoproducts in the presence of metabisulfite anions (0.2-1.0 M) at pH 8.0

Time Dianions RF CDRF FMF LC LF DHRF Total (min) conc. (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) (M×105) M/L 0 0.2 5.00 - - - - - 5.00 30 4.78 0.04 0.03 0.12 0.00 0.01 4.98 60 4.60 0.09 0.06 0.21 0.00 0.02 4.98 90 4.45 0.10 0.07 0.28 0.00 0.03 4.93 120 4.32 0.12 0.08 0.36 0.01 0.05 4.94 150 4.20 0.14 0.10 0.42 0.02 0.07 4.95 0 0.4 5.00 - - - - - 5.00 30 4.72 0.07 0.04 0.13 0.00 0.01 4.97 60 4.52 0.12 0.06 0.24 0.00 0.03 4.97 90 4.33 0.13 0.08 0.32 0.01 0.05 4.92 120 4.21 0.15 0.10 0.38 0.02 0.06 4.92 150 4.08 0.17 0.11 0.46 0.03 0.08 4.93 0 0.6 5.00 - - - - - 5.00 30 4.68 0.09 0.04 0.16 0.00 0.01 4.98 60 4.4 0.11 0.06 0.31 0.01 0.04 4.93 90 4.22 0.14 0.08 0.39 0.03 0.06 4.92 120 4.08 0.17 0.10 0.44 0.04 0.09 4.92 150 3.93 0.20 0.12 0.51 0.05 0.12 4.93 0 0.8 5.00 - - - - - 5.00 30 4.73 0.11 0.04 0.15 0.01 0.02 5.06 60 4.45 0.14 0.05 0.28 0.02 0.03 4.97 90 4.18 0.19 0.09 0.41 0.03 0.07 4.97 120 3.97 0.21 0.11 0.50 0.04 0.11 4.94 150 3.83 0.23 0.13 0.55 0.06 0.15 4.95 0 1.0 5.00 - - - - - 5.00 30 4.60 0.13 0.05 0.20 0.01 0.04 5.03 60 4.28 0.17 0.08 0.34 0.03 0.08 4.98 90 4.02 0.20 0.10 0.44 0.05 0.12 4.93 120 3.85 0.23 0.12 0.51 0.06 0.15 4.92 150 3.72 0.25 0.14 0.59 0.08 0.18 4.96

148

0 120

Fig. 32. UV and visible absorption spectra of RF solutions (pH 7) on photodegradation in

2- 2- 2- the presence of 1M dianions: S2O3 (a); SO3 (b); S2O5 (c). Times indicated are in

minutes.

149

0 120

Fig. 33 UV and visible absorption spectra of RF solutions (pH 7) on chemical reduction in

2- 2- 2- the presence of 1M dianions: S2O3 (a); SO3 (b); S2O5 (c). Times indicated are in

minutes.

150

6.2.5 Fluorescence characteristics of RF

RF is a strong yellow green fluorescent compound emitting in aqueous solution at

520-530 nm. The fluorescence is destroyed by acid and alkali on the ionization (Weber,

1950;Duggen, 1957; Ahmad et al., 2016, 2017, 2018). Dianions(Schuman Jorns et al.,

1975; Ahmad et al., 2004b, 2005, 2006, 2010; Vaid et al., 2019) and dications(Varnes,

1971; Rutter, 1958; Ahmad et al., 2017) influence the fluorescence of RF by the formation of complex with the molecule (Schuman Jorns et al.,1975; Ahmad et al., 2004;

Vaid et al., 2019). The plots of decrease in % fluorescence of RF in presence of 0.2-1.0

M of thiosulfate, sulfite and metabisulfite anions are shown in Figure34.The decrease in

RF fluorescence occurs in the order: metabisulfite˂ sulfite ˂ thiosulfate. This result in the formation of complexes of RF and dianions as observed for phosphate, carbonate, phthalate, oxalate and other dianions (Schuman Jorns et al., 1975; Ahmad et al., 2004b,

2010; Vaid et al., 2019). The additional factor in fluorescence decrease of RF is the chemical reduction of RF to form DHRF (Burn and Obrin, 1959).

6.2.6 Kinetics of Photodegradation

Several studies were made to evaluate the effect of dianions on the photodegradation of RF and the magnitude of change in photoreduction pathway to give

LC in favor of photoaddition pathway to form CDRF (Schuman Jorns et al., 1975;

Ahmad et al., 2004b, 2005, 2006, 2010; Vaid et al., 2019; Sheraz et al., 2014a). This study involves the evaluation of the effect of sulfur containing dianions on the photoaddition of RF. Since the dianions also chemically reduce RF to DHRF during the

151

100 90 80 70 60 50 40 30 %Fluorescence %Fluorescence 20 10 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Concentration (M)

Fig. 34: Percent fluorescence of RF in the presence of 0.2-1.0 M dianion: (●)

2- 2 2- S2O3 ; (▲) SO3 ; (♦) S2O5 .

152 photolysis they influence the overall rate of degradation of RF. The kinetic plots (Fig. 35-

49) for the photolysis show that the RF degradation is highest in the presence of

2- thiosulfate followed by that of sulfite and metabisulfite. Thus S2O3 dianions are most effective in the chemical reduction of RF to form CDRF.The photodegradation of RF

(kobs) leads to the formation of CDRF (k1) by photoaddition, LC (k2) by photoadditionand

DHRF (k3) by chemical reduction as final products by parallel first-order reactions as observed previously in formation of CDRF and LC (Ahmad et al., 2004b, 2005, 2006,

2010; Vaid et al., 2019).

k 1 CDRF

hv, k k RF obs 2 LC

k 3 DHRF

The rate equation for these reactions can be expressed as:

-d[RF] = (k + k + k ) [RF] = k RF dt 1 2 3 obs

153

M 5.00 5.00

M 5

10 4.00 4.00 10 × (a) (b) 3.00 × 3.00 2.00 2.00 1.00 1.00

Concentration 0.00 0.00 Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00

M 5

4.00 5 4.00

10 × 3.00 (c) × 3.00 (d) 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 M

5 4.00 10

× 3.00 (e) 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 35. Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.0) in the presence of

(a)0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

154

5.00 M 5.00

5 10

10 4.00 4.00 × × (a) (b) 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 M 5.00

5 10

10 4.00 4.00 × × (c) (d) 3.00 3.00 2.00 2.00

1.00 1.00 Concentration

Concentration 0.00 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

M 5.00 5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 Time (min)

Fig. 36.Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.5) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

155

M 5.00 5.00

5 5 10

10 4.00 4.00

× × 3.00 (a) 3.00 (b) 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00

M M 5

5 (c) (d) 10

10 4.00 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 Time (min)

Fig. 37.Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.0) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

156

5.00 M 5.00

5 5

10 (a) (b)

4.00 10 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

M 5.00 5.00 5

5 (c) (d) 10

10 4.00 4.00

× × 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

M 5.00

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 Time (min)

Fig. 38.Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.5) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

157

5.00 M 5.00

5 10

10 4.00 4.00

× × 3.00 (a) 3.00 (b) 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

M 5.00 5.00

M 5

10 4.00 4.00

10 × 3.00 × 3.00 2.00 2.00

1.00 1.00 Concentration

0.00 Concentration 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 M

5 (e)

10 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 Time (min)

Fig. 39.Photodegradation of 5 × 10-5 M RF (●) solution (pH 8.0) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M thiosulfate anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

158

M 5.00 5.00

5 10

4.00 10 4.00 × (a) × (b) 3.00 3.00 2.00 2.00

1.00 1.00 Concentration 0.00 Concentration 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00

M 5

5 4.00 4.00 10

10 (c) ×

× (d) 3.00 3.00 2.00 2.00

1.00 1.00 Concentration Concentration 0.00 0.00 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 M 5 4.00 10 (e) × 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 40.Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.0) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfiteanions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

159

5.00 5.00

M M 5

5 4.00 4.00

10 10 × × (a) (b) 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00 M

M (d) 5

5 4.00 4.00

10 10 × × 3.00 (c) 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 41.Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.5) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfiteanions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

160

5.00 5.00 M

M (a) (b) 5

5 4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00 M

M (c) 5 5 (d)

4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 42.Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.0) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

161

5.00 5.00

M M 5

5 4.00 4.00 10

10 (a) × × 3.00 3.00 (b) 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (minutes) Time (minutes)

5.00 5.00

M M 5

5 4.00 4.00

10 10 × × 3.00 (c) 3.00 (d) 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 43.Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.5) in the presence of

(a) 0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

162

5.00 5.00

M M 5

5 4.00 4.00

10 10 × × 3.00 (a) 3.00 (b) 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00

M M 5

5 4.00 4.00 (d)

10 10 × × 3.00 (c) 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 (e)

105M 4.00 × 3.00 2.00 1.00

Concentration 0.00 0 30 60 90 120 150 Time (min)

Fig. 44.Photodegradation of 5 × 10-5 M RF (●) solution (pH 8.0) in the presence of (a)

0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M sulfite anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

163

5.00 5.00

M M 5

5 4.00 4.00 10

10 (a) × × (b) 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00

M M 5

5 4.00 4.00 10

10 (c) × × (d) 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 M 5 4.00 10 (e) × 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 45.Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.0) in the presence of (a)

0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfite anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

164

5.00 5.00

M M 5

5 4.00 4.00 10

10 (a) (b) × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 M 5.00

M 5

4.00 10 4.00 10 (c) × (d) × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 Concentration 0.00 Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 46.Photodegradation of 5 × 10-5 M RF (●) solution (pH 6.5) in the presence of (a)

0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfite anions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

165

5.00 5.00

M M 5

5 4.00 4.00

10 10 × × 3.00 (a) 3.00 (b) 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00 M

M (c) (d) 5

5 4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 47. Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.0) in the presence of (a)

0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfiteanions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

166

5.00 5.00

M M 5

5 4.00 4.00

10 10 × × 3.00 (a) 3.00 (b) 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00 M

M (c) (d) 5

5 4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 48.Photodegradation of 5 × 10-5 M RF (●) solution (pH 7.5) in the presence of (a)

0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfiteanions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

167

5.00 5.00

M M 5

5 4.00 4.00

10 10 × × 3.00 (a) 3.00 (b) 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00 5.00 M

M (c) (d) 5

5 4.00 4.00

10 10 × × 3.00 3.00 2.00 2.00 1.00 1.00

0.00 0.00

Concentration Concentration 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

5.00

M (e)

5 4.00 10

× 3.00 2.00 1.00

0.00 Concentration 0 30 60 90 120 150 Time (min)

Fig. 49.Photodegradation of 5 × 10-5 M RF (●) solution (pH 8.0) in the presence of (a)

0.2 M, (b) 0.4 M, (c) 0.6 M, (d) 0.8 M and (e) 1.0 M metabisulfiteanions and formation of

(▲)CDRF; (♦) FMF; (■) LC; (*) LF; (×) DHRF.

168

The solutions of k1, k2 and k3 is carried out according to the method of Frost and Pearson

(1964) as described previously for the evaluation of the effect of ionic strength on the photodegradation of RF (Ahmad et al., 2016). If A, D, E and F are the respective concentrations of RF, CDRF, LC and DHRF on the degradation of RF and Ao is the starting concentration of RF, the overall rate (kobs) of reaction is:

-dA = k1A + k2A + k3A =

-dt

(k1 + k2 + k3) A = kobsA

or k1 + k2 + k3 = kobs (Eq. 40)

If the degradation of A takes place by a first-order reaction,

-kt -dA = k1A = k1Aoe (Eq. 41) -dt

And

-kt D = k1Ao e + constant (Eq. 42) kobs

-kt D = Do + (k1Ao/k) (1-e )

-kt E = Eo + (k2Ao/k) (1-e )

-kt F = Fo + (k3Ao/k) (1-e )

169

Do = Eo= Fo = Fo, the equation may be written as: (Eq. 43)

E/D = k2/k1 (Eq. 44)

F/D = k3/k1 (Eq. 45)

D : E : F = k1 : k2 : k3 (Eq. 46)

The first-order rate constants for disappearance of RF (kobs) and formation of

CDRF, LC and DHRF (k1, k2andk3, respectively) at pH 6-8 in presence of (0.2-1.0 M) are given in Table 41. As reported in previous studies (Ahmad et al., 2010; Vaid et al., 2019).

CDRF formation is maximum at pH 7. Thek1/k2and k1/k3 valuesshow an increase in the formation of CDRF with an increase in the concentration of these dianions. The k3/kobs values indicate that the chemical reduction of RF at pH 7 occurs to the extent of 12.5 to

22.3%.

The second-order rate constant (kʹ) for photochemical interaction of RF-dianions obtained from the slopes of plots of kobs versus molar concentration of dianions (Fig. 50) are reported in Table 42. The values of k’ (pH 7) are in the order: thiosulfate> sulfite-

>metabisulfite. The thiosulfate dianionsindicate highest interaction with RF but donotform the highest amount of CDRF, whereas metabisulfitedianions show smallest interaction with RF but give rise to the highest amount of CDRF. The catalytic activity of sulfite dianionsto form CDRF is less than thiosulfate and more than matabisulfite.

As thiosulfate dianions cause greatest chemical reduction of RF to give DHRF, the photoaddition to give CDRF and photoreduction to give LC are affected. An evident from the values of k1/k2 and k1/k3 (Table 42). In presence of metabisulfitedianions, the

170 chemical reduction of RF is lowest, and the formation of CDRF is highest. The relative values of k1, k2 and k3 in the degradation of RF (kobs) are a function of the reactivity of dianions to facilitate photoaddition, photoreduction and chemical reduction of RF on photolysis.

171

Table 41.First-order rate constants for photodegradation of riboflavin (kobs), formation of

cyclodehydroxyribiflavin (k1), lumichrome (k2), and dihydroxyriboflavin (k3) in the

presence of dianion and quantum yields (Φ) of the reaction

3 3 3 3 pH Dianion Conc. kobs×10 k1 × 10 k2× 10 k3 × 10 k1/k2 k1/k3 k3/kobs Φ

(M) (min-1)±sd (min-1)±sd (min-1)±sd (min-)±sd

6.0 Thiosulfate 0.2 0.76±0.03 0.10±0.01 0.58±0.02 0.08±0.01 0.17 1.25 0.11 0.011

0.4 0.92±0.04 0.12±0.01 0.68±0.03 0.10±0.01 0.18 1.20 0.14 0.014

0.6 1.07±0.04 0.17±0.01 0.73±0.03 0.15±0.01 0.23 1.13 0.17 0.016

0.8 1.22±0.05 0.22±0.01 0.81±0.03 0.21±0.01 0.27 1.04 0.19 0.018

1.0 1.57±0.06 0.35±0.01 0.95±0.03 0.33±0.11 0.34 1.03 0.20 0.023

6.5 0.2 1.07±0.04 0.16±0.01 0.79±0.03 0.11±0.01 0.20 1.45 0.10 0.016

0.4 1.38±0.05 0.23±0.01 0.96±0.04 0.18±0.01 0.24 1.27 0.13 0.021

0.6 1.53±0.06 0.28±0.01 0.99±0.04 0.24±0.01 0.28 1.16 0.15 0.023

0.8 1.84±0.07 0.36±0.01 1.14±0.05 0.32±0.01 0.31 1.12 0.17 0.028

1.0 2.13±0.08 0.45±0.02 1.25±0.05 0.42±0.02 0.35 1.07 0.20 0.032

7.0 0.2 1.67±0.06 0.36±0.01 1.08±0.04 0.21±0.02 0.34 1.61 0.14 0.025

0.4 1.82±0.07 0.42±0.02 1.13±0.04 0.28±0.01 0.38 1.53 0.15 0.028

0.6 2.30±0.09 0.55±0.02 1.35±0.05 0.40±0.01 0.41 1.37 0.17 0.035

0.8 2.60±0.10 0.65±0.02 1.45±0.05 0.48±0.02 0.45 1.36 0.18 0.039

1.0 3.05±0.12 0.76±0.02 1.61±0.06 0.68±0.02 0.47 1.11 0.22 0.046

7.5 0.2 1.68±0.06 0.35±0.01 1.00±0.04 0.23±0.01 0.35 1.52 0.13 0.025

0.4 1.84±0.07 0.40±0.01 1.07±0.04 0.28±0.01 0.37 1.42 0.15 0.028

0.6 2.14±0.08 0.46±0.02 1.22±0.05 0.36±0.01 0.38 1.27 0.16 0.032

0.8 2.61±0.10 0.57±0.02 1.37±0.05 0.44±0.02 0.39 1.18 0.18 0.039

172

1.0 2.70±0.11 0.59±0.02 1.46±0.06 0.61±0.02 0.40 0.97 0.22 0.042

8.0 0.2 1.43±0.05 0.27±0.01 1.03±0.04 0.13±0.01 0.28 2.07 0.09 0.022

0.4 1.58±0.06 0.31±0.01 1.07±0.04 0.21±0.01 0.29 1.47 0.13 0.024

0.6 2.06±0.07 0.38±0.01 1.22±0.05 0.40±0.02 0.31 0.95 0.19 0.031

0.8 2.38±0.09 0.44±0.02 1.40±0.05 0.47±0.02 0.32 0.93 0.20 0.036

1.0 2.54±0.10 0.51±0.02 1.47±0.06 0.56±0.02 0.35 0.91 0.22 0.037

6.0 Sulfite 0.2 0.81±0.01 0.19±0.01 0.41±0.01 0.11±0.01 0.46 1.72 0.13 0.012

0.4 0.94±0.03 0.23±0.01 0.45±0.01 0.20±0.01 0.51 1.15 0.21 0.014

0.6 1.07±0.04 0.27±0.01 0.51±0.01 0.25±0.01 0.53 1.08 0.23 0.016

0.8 1.22±0.05 0.31±0.01 0.56±0.02 0.30±0.01 0.55 1.03 0.24 0.018

1.0 1.28±0.06 0.34±0.01 0.60±0.02 0.34±0.01 0.57 1.00 0.25 0.021

6.5 0.2 0.86±0.01 0.21±0.01 0.57±0.02 0.09±0.01 0.36 2.33 0.10 0.013

0.4 1.14±0.04 0.33±0.01 0.80±0.03 0.15±0.01 0.41 2.20 0.13 0.017

0.6 1.58±0.06 0.39±0.01 0.89±0.03 0.23±0.01 0.43 1.69 0.14 0.024

0.8 1.85±0.07 0.44±0.01 0.91±0.03 0.31±0.01 0.48 1.42 0.17 0.028

1.0 1.90±0.08 0.55±0.02 0.94±0.03 0.40±0.02 0.59 1.37 0.21 0.029

7.0 0.2 1.53±0.06 0.41±0.01 0.86±0.03 0.26±0.01 0.47 1.57 0.17 0.023

0.4 1.80±0.07 0.52±0.01 0.94±0.03 0.34±0.02 0.55 1.53 0.19 0.028

0.6 2.15±0.08 0.65±0.02 1.06±0.04 0.44±0.02 0.61 1.47 0.21 0.032

0.8 2.30±0.09 0.70±0.02 1.11±0.04 0.49±0.02 0.63 1.40 0.16 0.035

1.0 2.45±0.10 0.75±0.02 1.14±0.05 0.54±0.02 0.66 1.38 0.23 0.037

7.5 0.2 1.43±0.05 0.41±0.01 0.77±0.03 0.37±0.01 0.38 1.10 0.15 0.022

0.4 1.60±0.06 0.48±0.01 0.83±0.03 0.44±0.02 0.48 1.09 0.17 0.024

0.6 1.75±0.07 0.51±0.01 0.89±0.03 0.48±0.02 0.51 1.06 0.21 0.026

0.8 1.89±0.07 0.55±0.01 1.00±0.04 0.54±0.02 0.53 1.01 0.24 0.028

173

1.0 2.32±0.09 0.60±0.02 1.12±0.04 0.60±0.02 0.54 1.00 0.26 0.033

8.0 0.2 1.50±0.06 0.17±0.01 0.81±0.03 0.16±0.01 0.20 1.06 0.10 0.023

0.4 1.66±0.06 0.22±0.01 0.96±0.03 0.24±0.01 0.22 0.92 0.14 0.025

0.6 1.83±0.07 0.38±0.01 1.11±0.04 0.42±0.02 0.34 0.90 0.22 0.027

0.8 2.00±0.07 0.41±0.01 1.15±0.04 0.51±0.02 0.35 0.80 0.25 0.029

1.0 2.20±0.08 0.44±0.02 1.18±0.05 0.59±0.02 0.37 0.73 0.26 0.030

6.0 Metabisulfite 0.2 0.60±0.02 0.15±0.01 0.31±0.01 0.08±0.01 0.48 1.87 0.14 0.009

0.4 0.91±0.03 0.23±0.01 0.45±0.01 0.14±0.01 0.51 1.64 0.15 0.014

0.6 1.05±0.04 0.28±0.01 0.49±0.01 0.19±0.01 0.57 1.47 0.18 0.016

0.8 1.14±0.04 0.32±0.01 0.53±0.02 0.22±0.01 0.60 1.45 0.19 0.017

1.0 1.20±0.05 0.37±0.01 0.55±0.03 0.28±0.01 0.67 1.32 0.23 0.018

6.5 0.2 0.76±0.03 0.18±0.01 0.34±0.01 0.11±0.01 0.50 1.63 0.14 0.011

0.4 0.91±0.03 0.23±0.01 0.40±0.01 0.15±0.01 0.52 1.53 0.16 0.013

0.6 1.06±0.04 0.29±0.01 0.46±0.01 0.19±0.01 0.56 1.52 0.18 0.015

0.8 1.35±0.05 0.38±0.01 0.56±0.03 0.26±0.01 0.61 1.46 0.19 0.017

1.0 1.65±0.07 0.50±0.02 0.70±.03 0.35±0.02 0.71 1.42 0.21 0.025

7.0 0.2 1.52±0.06 0.59±0.02 0.68±0.03 0.25±0.01 0.86 2.36 0.16 0.023

0.4 1.68±0.06 0.66±0.02 0.73±0.03 0.29±0.01 0.90 2.27 0.17 0.025

0.6 1.97±0.08 0.78±0.03 0.83±0.03 0.36±0.01 0.94 2.16 0.18 0.030

0.8 2.14±0.08 0.84±0.03 0.87±0.04 0.43±0.01 0.97 1.95 0.20 0.032

1.0 2.30±0.09 0.92±0.03 0.90±0.05 0.48±0.01 1.02 1.89 0.21 0.035

7.5 0.2 1.53±0.06 0.36±0.01 0.68±0.03 0.20±0.01 0.52 1.80 0.13 0.023

0.4 1.68±0.06 0.47±0.02 0.76±0.03 0.31±0.01 0.61 1.51 0.18 0.025

0.6 1.84±0.08 0.54±0.02 0.80±0.04 0.36±0.01 0.67 1.49 0.19 0.027

0.8 1.92±0.08 0.61±0.02 0.86±0.04 0.42±0.01 0.70 1.45 0.22 0.029

174

1.0 2.02±0.08 0.66±0.03 0.90±0.03 0.46±0.02 0.73 1.43 0.23 0.030

8.0 0.2 1.10±0.04 0.25±0.01 0.68±0.03 0.19±0.01 0.36 1.31 0.17 0.017

0.4 1.41±0.05 0.31±0.01 0.78±0.04 0.27±0.01 0.38 1.14 0.19 0.019

0.6 1.56±0.06 0.34±0.01 0.87±0.04 0.33±0.01 0.39 1.06 0.21 0.021

0.8 1.70±0.06 0.42±0.01 1.00±0.05 0.41±0.02 0.42 1.02 0.24 0.023

1.0 1.90±0.08 0.44±0.02 1.02±0.05 0.44±0.02 0.43 1.00 0.23 0.028

175

a a

Fig. 50. Second-order plots for: (a) photochemical interaction; (b) chemical

2- 2 2- reduction of RF in the presence dianions: (●) S2O3 ; (▲) SO3 ; (♦) S2O5 .

176

Table 42. Second-order rate constants for the photochemical (kʹ) and chemical dark

interaction(k″) of riboflavin and dianions at pH 7.0

Dianion Concentration kʹ × 103 k″ × 103 kʹ/ k″

(M) (M-1min-1) ± SD (M-1min-1) ± SD

Thiosulfate 0.2-1.0 2.01 ± 0.08 3.25 ± 0.12 0.62

Sulfite 0.2-1.0 1.65 ± 0.06 2.72 ± 0.11 0.61

Metabisulfite 0.2-1.0 1.42 ± 0.05 2.31 ± 0.09 0.61

177

The second-rate constants (k”) for chemical reduction of RF determined from the plots of kobs versus molar concentration of the dianions (Fig. 51)Table 43 arehigher than those of the chemical reduction in light. The k1ʹ/k2ʹ and k3/k2ʹvalues in the presence of the dianions are almost constant that indicate their similar role in the chemical reduction of

RF in light and in the dark.

6.2.7 pH Effect

The k-pH profiles are an indication of the pH range of maximum decomposition

(Carstensen and Rhodes, 2000; Yosheka and Stella, 2000, Florence and Attwood, 2016).

A pH > 6 is reported for the formation of CDRF in presence of dianions. To evaluate the effect of pH on the rate of reaction, kobs, k1,k2 and k3 values were plotted versus pH in the range of 6.0-8.0 (Fig. 7). The k-pH profile show the highest rate of RF degradation at pH

7.0 as observed previously (Ahmad et al., 2010; Vaid et al., 2019). The dianion-catalyzed photoaddition of RF is reported to take place under neutral conditions (Schuman Jorns, et al., 1975).

The rate-pH profile for LC (k2) by photoreduction shows a gradual increase in rate with pH as reported in earlier studies (Crains and Metzler, 1971; Ahmad and Rapson,

1990; Ahmad et al., 2004, 2010, 1980). The formation of DHRF (k3) is slightly increased with pH.

178

2- Fig. 51. Plots of kobs for RF versus pH in the presence of 1 M dianions (●) S2O3 ;(▲)

2 2- SO3 ;(♦) S2O5 : (a) photoreduction; (b) chemical reduction.

179

6.2.8 Redox Potentials Effects

The reducing effect of sulfurdianionsmay be explained on considering their redox potentials (pH 7.0). The oxidation of these dianions in aqueous solutions can be expressed as follows:

2 S O 2- S O 2- + 2e (E0 +0.08 V) (Eq. 47) 2 3 4 6

SO 2- + 2 OH SO 2- + H O + 2e (E0 -0.93 V) (Eq. 48) 3 4 2

S O 2-+ H O 2 HSO - (Eq. 49) 2 5 2 3

HSO - + 3 H O HSO - + 2 H O+ + 2e (Eq. 50) 3 2 4 3

The more easily a compound loses electrons to give the oxidized form, the better a reducing agent it is (Sinko, 2006). Thus, thiosulfate dianion(oxidation potential of

+0.08 V) is more easily oxidized and is a greater reducing agent than sulfite dianion(oxidation potential of -0.93V). Metabisulfitedianionsin aqueous solution are

- converted to HSO3 anions which have a lower reducing power.

0 Kinetic data (k3) on reduction of RF (E -0.201 V, pH 7) to DHRF in photolyzed solutions are in accordance with the redox potentials of the dianions: thiosulfate>sulfite

>metabisulfite. Thus, thiosulfate dianionare most effective in the reduction of RF.

6.2.9 Interaction of RF with Dianions

Several studies have been carried out on the interaction of RF with dianions to form a complex and catalyze photoadditionreaction (SchrumanJorns, 1975; Ahmad et al.,

180

2004, 2005, 2006, 2010; Vaid et al., 2019, Sheraz et al., 2014a), however, the effect of reducing dianions on this reaction has not been studied. The present study has shown that these dianions catalyze photoaddion as well as chemical reduction of RF during photolysis. The kinetic data indicate a higher activity of thiosulfate dianions in chemical reduction and lower activity in photoadditon of RF compared to that of sulfite (Table 42).

In both of these reactions RF fluorescence is quenched due to the interaction of RF with dianion. The plots of (kobs, k1, k2 and k3) against decrease in % fluorescence of RF show a decrease in % fluorescence in the presence of dianions are shown in Fig. 52. The values of kobs, k2 and k3 have been found to increase with an increase in fluorescence loss indicating the RF-dianion interaction in the order: thiosulfate> sulfate>metabisulfite. This is in agreement with a recent study (2019) on the degradation of RF in the presence of carbonate, oxalate and phthalate dianions. However, the values of k1 for CDRF formation decrease in the above order as a result of the chemical reduction of RF.

6.2.10 Photolysis Quantum Yield

The quantum yields (Φ) of RF photolysis in presence of dianions were determined and the values (pH 7) range from 0.023-0.046 (Table 42). These values show that the Φ of RF photolysis increase in presence of dianions in the order: thiosulfate > sulfite > metabisulfite. The Φ values are higher in the presence of thiosulfate compared to these of sulfite and metabisulfite dianions.

181

4.0 1 - 3.5

, min , k

3 3.0 obs

10 × 2.5

2.0

1.5 k2

1.0 order order rate constants

- k3 0.5

k1 First 0.0 0 20 40 60 80 100 % Fluorescence loss

Fig. 52. Plots of photodegradation of RF (kobs) and formation of CDRF (k1), LC (k2) and

2- DHRF (k3) versus percent fluorescence loss in the presence of 1 M dianions: (●) S2O3 ; (▲)

2 2- SO3 ; (♦) S2O5 .

182

6.2.11 Mode of RF Photodegradation Presence of Sulfur Containing Dianions

The photodegradation of RF involves following major reactions.

Intramolecular photoaddition

RF hv

Intramolecular photoreduction

In presence of dianions (e.g. phosphate, carbonate, oxalate), the prominent reaction of RF occurs by photoaddition pathway to form CDRF along with photoreduction pathway to give FMF, CMF, LC and LF (Crains and Metzler, 1971,

ShrumanJorns, 1975, Ahmad and Rapson, 1990; Ahmad et al., 2004a, 2004b, 2005, 2006,

2010, 2017; Vaid et al., 2019). In the present study the effect of reducing dianions

(thiosulfate, sulfite, metabisulfite) on photoaddition pathway visa-a-vis the photoreduction and chemical reduction pathways of RF is studied as presented in the ions. The only reaction in the presence of these dianions in the dark chemical reduction of

RF to give DHRF. It also occurs to some extent during photodegradation. A combined scheme for photochemical reactions of RF in the light of previous studies

(SchrumanJorna, 1975; Heelis et al., 1990; Ahmad and Vaid. 2006; Ahmad et al., 2010;

Vaid et al., 2019) and the present work is presented in Fig. 53. It shows pathways for the mode of photoaddition, photoreduction and chemical reduction of RF to form CDRF, LC and DHRF, respectively, in presence of the dianions.

A common factor in these reactions is the involvement of a dihydro intermediate in photoaddtion and photoreduction pathways of RF while direct formation of DHRF in chemical reduction. The photoadditon pathway is initiated by a RF-dianion complex to

183 give CDRF (Schuman Jorns et al., 1975) and the photoreduction pathway by intramolecular hydrogen abstraction by the isoalloxazine nucleus (N-1) from ribityl side chain to give LC and other products (Moore et al., 1963). Thus the photodegradation of

RF in presence of sulfur containing dianions involves photoaddition, photoredcution and chemical reduction reactions to form different products.

184

R R O R – HO C H HO C H O S O C H

CH2 CH2 O CH H 2 H C N N O H3C N N O 3 H3C N N O 2- SO 2- SO3 3 NH Chemical reduction NH Photodegradation NH H C N H3C N 3 H3C N H O CH2 O 2- 1,5-dihydroriboflavin (DHRF) Riboflavin (RF) RF-SO3 - Complex

hv hv

R 1 C O O R * – H3C N N O O S O C H

O CH2 NH H3C N H3C N N O H O Cylic intermediate NH H3C N O

excited singlet state

cyclization

CHO H CH 2 R H3C N N O H C N N O H+, OH- 3 NH O H C N NH H 3 H C N N O H3C N 3 O Lumichrome (LC) O Formylmethylflavin (FMF) NH H3C N H - O OH 1,5-dihydro-9-alkoxyl-flavin intermediate CH3

H3C N N O Oxidation

NH Oxidation H3C N O Lumiflavin (LF) COOH R CH2 O H3C N N O

H3C N N O NH H C N 3 NH O H3C N O Carboxymethylflavin (CMF) Cyclodehydroflavin (CDRF)

Fig. 53. Scheme for photodegradation reactions of RF in presence of sulfur containing

dianions.

185

CONCLUSUSIONS

The photochemical reactions of RF mainly involve intramolecular photoreduction and intramolecular photoaddition reactions to form lumichrome and cyclodehydroriboflavin, respectively as the major compounds. The photredcution leads to the ribityl side-chain cleavage and the photoaddition involves side chain cyclization. The photoaddition reaction is initiated by divalent anions through the formation of a RF-anion complex. Additionally it has been found that in presence of reducing anions, formation of dihydroriboflavin also takes place in the presence and absence of light. The anions tend to deviate photoreduction of RF towards photoaddition depending on the degree of their catalytic activity. The photoaddition reaction occurs through the excited singlet state and the photoreduction by the excited triplet state of RF.

All the photodegradation reactions of RF occur by first-order kinetics whereas the photochemical interaction as chemical reduction of RF take place by second-order kinetics. The order of these reactions in the presence of non-reducing anions is: carbonate

> phosphate > oxalate > phthalate and in the presence of reducing anions is thiosulfate > sulfite > metabisulfite.

The order of photochemical interaction of RF and non-reducing anions determined by second-order rate constant is: carbonate > phosphate > oxalate > phthalate.

The order of photochemical reduction and chemical interaction of RF in the presence of reducing anions is: thiosulfate > sulfite > metabisulfite.

All the reactions have a maximum rate at pH 7.0. The rate-pH profiles of the photochemical reactions show that the rate of reaction is maximum around pH 7. The rate

186 constants of these reactions depend on the loss of RF fluorescence as an indication of the degree of complexation between RF and the anions as a result of fluorescence quenching by the complex. The chemical reduction of RF is a function of the redox potential of the reducing anions and is greater is the case of thiosulfate with a lower redox potential than that of sulfite with a higher redox potential. The quantum yields for photolysis of RF under these conditions have been determined. The photoaddtion of RF involves the presence of a nucleophile in the side chain which facilitates photoaddition of C(2’)OH of side chain at C(9) of isoallozaxine ring of RF. The cyclization occurs through the oxidation of a dihydroflavin intermediate to yield cyclodehydroriboflavin in the presence of reducimg anions. The photoaddition and photoreduction reactions of RF involve a dihydro intermediate radical spieces to form cyclodihydroflavin and lumichrome, respectively, whereas the chemical reduction of RF leads to the formation of a stable dihydroriboflavin compound that does not absorb in the visible region at 445 nm due to loss of the N(5)=C(4a)-C(10a)=N(1) conjugated system in the isoalloxazine ring. The mode of these reactions has been discussed.

187

REFERENCES

188 Agte VV, Chiplonkar SA, Gokhale MK. Interaction of riboflavin with zinc bioavailability. Ann N Y Acad Sci. 1992;669:314-316.

Ahmad I, Ahmed S, Sheraz MA, Aminuddin M, Vaid FHM. Effect of caffeine on the photolysis of riboflavin in aqueous solution: a kinetic study. Chem Pharm Bull.

2009;57:1363-1370.

Ahmad I, Ahmed S, Sheraz MA, Vaid FHM, Ansari IA. Effect of divalent anions on photodegradation kinetics and pathways of riboflavin in aqueous solution. Int J

Pharmaceutics. 2010;390:174-182.

Ahmad I, Ahmed S, Sheraz MA, Vaid FHM. Effect of borate buffer on the photolysis of riboflavin in aqueous solution. J Photochem Photobiol B:Biol. 2008;93:82-87.

Ahmad I, Ahmed S,Anwar Z, Sheraz MA, Sikorski M. Photostability and photostabilization of drugs and drug products, Int J Photoenergy. 2016;2016:ID 8135608.

Ahmad I, Anwar Z, Iqbal K, Ali SA, Hassan KA, Sheraz MA, Ahmed S. Ionic strength effects on the photodegradation reactions of riboflavin in aqueous solution. J Photochem

Photobiol B: Biol. 2016;157:113–119.

189

Ahmad I, Arsalan A, Ali SA, Sheraz MA, Ahmed S, Anwar Z, Munir I, Shah MR.

Formulation and stabilization of riboflavin in liposomal preparations. J Photochem

Photobiol B:Biol. 2015B;153: 358-366.

Ahmad I, Fasihullah Q, Noor A, Ansari IA, Ali QNM. Photolysis of riboflavin in aqueous solution: A kinetic study, Int J Pharm. 2004;280:199–208.

Ahmad I, Fasihullah Q, Vaid FHM. Effect of phosphate buffer on photodegradation reactions of riboflavin in aqueous solution, J Photochem Photobiol B Biol. 2005;78;229–

234.

Ahmad I, Fasihullah Q, Vaid FHM.A study of simultaneous photolysis and photoaddition reactions of riboflavin in aqueous solution. J Photochem Photobiol B: Biol. 2004b;75:13-

20.

Ahmad I, Fasiullah Q, Vaid FHM. Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution. Journal of Photochemistry and Photobiology B: Biology. 2006;82:21-27.

Ahmad I, Fasiullah Q, Vaid FHM. Photolysis of formylmethylflavin in aqueous and organic solvents.Photochemical and Photobiological Sciences. 2006b;5:680-685.

Ahmad I, Hussain W, Fareedi AA.Photolysis of cyanocobalamine in aqueous solution.J

Pharm Biomed Anal. 1992;10:9-15.

190

Ahmad I, Hussain W. Multicomponent spectrophotometric assay of cyanocobalamine, hydroxocobalamine and riboflavin. Pak J Pharm Sci. 1992;5:121 127.

Ahmad I, Mirza T, Anwar Z, Ejaz MA, Sheraz MA, Ahmed S. Multicomponent spectrofluorimetric method for the assay of formylmethylflavin and its hydrolytic products: kinetic applications, Spectrochim Acta A: Mol Biomol Spectrosc.

2018;205:540-550.

Ahmad I, Mirza T, Iqbal K, Ahmed S, Sheraz MA, Vaid FHM. Effect of pH, buffer, and viscosity on the photolysis of formylmethylflavin: a kinetic study. Aust J Chem.

2013;66:579-585.

Ahmad I, Mirza T, Iqbal K, Ahmed S, Sheraz MA, Vaid FHM. Effect of pH, buffer and viscosity on the photolysis of formylmethylflavin: A kinetic study. Aust. J Chem.

2013a;66:579-585.

Ahmad I, Qadeer K, Iqbal K, Ahmed S, Sheraz MA, Ali SA, Mirza T, Hafeez A.

Correction for irrelevant absorption in multicomponent specctrophotometric assay of riboflavin, formylmethylflavin and degradation products: kinetic application. AAPS

PharmSciTech. 2013b;14:1101-1107.

Ahmad I, Rapson HDC, Heelis PF, Phillips GO. Alkaline hydrolysis of 7,8-10

(formylmethyl)isoalloxazine. A kinetic study. J Org Chem. 1980;45:731-733.

191

Ahmad I, Rapson HDC. Multicomponent spectrophotometric assay of riboflavin and phototproducts.J Pharm Biomed Anal. 1990;8:217-223.

Ahmad I, Sheraz MA, Ahmed S, Kazi SH, Mirza T, Aminuddin M. Stabilizing effect of citrate buffer on the photolysis of riboflavin in aqueous solution. Results Pharma Sci.

2011;1:11-15.

Ahmad I, Tollin G. Flavin triplet quenching and semiquinone formation by aliphatic α- substituted acetic acids: intermediates in flavin sensitized photodecarboxylation.

Photochem Photobiol. 1981;34:441-445.

Ahmad I, Vaid FHM, Ahmed S, Sheraz MA, Hasan S. Advances in biochemical functions and photochemistry of flavins and flavoproteins. Int J Chem Anal Sci,

2010;1:18-21.

Ahmad I, Vaid FHM. Photochemistry of flavins in aqueous and organic solvents. In:

Silva E, Edwards AM, eds. Flavins Photochemistry and Photobiology. Cambridge:

Royal Society of Chemistry, 2006, pp. 13–40.

Ahmad I, Vaid FHM. Thermal degradation of 7,8-dimethyl-10- formylmethyliaoalloxazine in acid solution: A kinetic study. J Chem Soc Pak.

2008;30:688-691.

192

Ahmad I, Ahmed S, Sheraz MA, Aminuddin M, Vaid FH. Effect of caffeine complexation on the photolysis of riboflavin in aqueous solution: a kinetic study.Chem

Pharm Bull (Tokyo). 2009;57:1363-1370.

Ahmad I, Ahmed S, Sheraz MA, Anwar Z, Qadeer K, Noor A Evstigneev MP. Effect of nicotinamide on the photolysis of riboflavin in aqueous solution.Sci Pharm. 2016;84:289-

303.

Ahmad I, Ahmed S, Sheraz MA, Vaid FH, Ansari IA. Effect of divalent anions on photodegradation kinetics and pathways of riboflavin in aqueous solution.Int J Pharm.

2010;390:174-82.

Ahmad I, Ahmed S, Sheraz MA, Vaid FH. Effect of borate buffer on the photolysis of riboflavin in aqueous solution.J Photochem Photobiol B. 2008;93:82-87.

Ahmad I, Anwar Z, Ahmed S, Sheraz MA, Bano R, Hafeez A (2015).Solvent Effect on the Photolysis of Riboflavin.AAPS PharmSciTech. 2015;16:1122-8.

Ahmad I, Anwar Z, Ahmed S, Sheraz MA, Khattak SUR. Metal ion mediated photolysis reactions of riboflavin: A kinetic study. J Photochem Photobiol B:Biol. 2017;173:231-

239.

193

Ahmad I, Anwar Z, Ali SA, Hasan KA, Sheraz MA, Ahmed S. Ionic strength effects on the photodegradation reactions of riboflavin in aqueous solution.J Photochem Photobiol

B. 2016;157:113-119.

Ahmad I, Anwar Z, Iqbal K, Ali SA, Mirza T, Khurshid Ad, Khurshid Aq, Arsalan A.

Effect of acetate and carbonate buffers on the photolysis of riboflavin in aqueous solution: A kinetic study. AAPS PharmSciTech.2014;15:550-9.

Ahmad I, Fasihullah Q, Noor A, Ansari IA, Ali QN. Photolysis of riboflavin in aqueous solution: A kinetic study.Int J Pharm. 2004;280:199-208.

Ahmad I, Fasihullah Q, Vaid FHM. Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution.J Photochem Photobiol

B:Biol. 2006;82:21-27.

Ahmad I, Fasihullah Q, Vaid FHM. Effect of phosphate buffer on photodegradation reactions of riboflavin in aqueous solution.J Photochem Photobiol B:Biol. 2005;78:229-

34.

Ahmad I, Fasihullah Q, Vaid FHM. A study of simultaneous photolysis and photoaddition reactions of riboflavin in aqueous solution. J Photochem Photobiol B:Biol.

2004;75:13-20.

194

Ahmad I, Fasihullah Q, Vaid FMH. Photolysis of formylmethylflavin in aqueous and organic solvents.Photochem Photobiol Sci. 2006;5:680-685.

Ahmad I, Sheraz MA, Ahmed S, Bano R, Vaid FHM. (2012). Photochemical interaction of ascorbic acid with riboflavin, nicotinamide and alpha-tocopherol in cream formulations.Int J Cosmet Sci. 2012;34:123-31.

Ahmad I, Sheraz MA, Ahmed S, Kazi SH, Mirza T, Aminuddin M. Stabilizing effect of citrate buffer on the photolysis of riboflavinin aqueous solution.Results Pharma Sci.

2011;1:11-15.

Ahmad I,Anwar Z, Ahmed S, Sheraz MA, Khattak SUR. Metal ion mediated photolysis reactions of riboflavin: a kinetic study. J Photochem Photobiol B: Biol. 2017;173:231-

239.

Ahmad I.A study of the degradation of riboflavin and related compounds.Ph. D. Thesis,

University of London, London, UK, 1968.

Akhtar MJ, Ataullah M, Ahmad I. Effect of riboflavin on the photolysis 0f folic acid in aqueous solution. J Pharm Biomed Anal. 2000;23:1039-1044.

195

Albala-Hurtado S, Veciana-Nogue’s MT, Izquierdo-Pulido M, Marine-Font A.

Determination of water-soluble vitamins in infant milk by high-performance liquid chromatography. J Chromatogr A. 1997:778:247-253.

Allwood MC, Kearney MCJ. Compatibility and stability of additives in parenteral nutrition admixtures.Nutrition. 1998;14:697-706.

Allwood MC. Compatibility and stability of TPN mixtures in big bags. J Clin Pharm

Ther. 1984;9:181-198.

Andres-Lacueva C, Mattivi F, Tonon D. Determination of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide in wine and other beverages by high- performance liquid chromatography with fluorescence detection. J Chromatogr A.

1998;823:355-363.

Ansari AI, Vaid FHM, Ahmad I. Spectral study of photolysis of aqueous cyanocobalamine in the presence of vitamin B and C. Pak J Pharm Sci. 2004;17:83-93.

Anwar Z, Ahmad I. Photochemistry of riboflavin and related compounds, In: Berhardt

LV ed., Advances in Medicine and Biology, Nova Science Publilshers, USA, 2017,

Chap. 4.

196

Aranda M, Morlock G. (2006). Simultaneous determination of riboflavin, pyridoxine, nicotinamide, caffeine and taurine in erengy drinks by planar chromatography-multiple detection with confirmation by electrospray ionization mass spectrometry. J Chromatogr

A. 2006;1131:253-260.

Arien A, Dupuy B. Encapsulation of calcitonin in liposome’s depends on the vesicle preparation method. J Microencapsul. 1997;14:753-760.

Arizan S, Popa M, Sterescu M. Polarographic determination of cobalt, vitamin B2 and vitamin C mixtures in tablets and ampoules. Electroanal Chem. 1961;2:177.

Asahi Y. Polarographic determination of ascorbic acid, riboflavin, pteroylglutamic acid,

2-methyl-1,4-naphthohydroquinone diacetate, thiamine, niacinamide and thiamine prophyl disulfide in multivitamin preparations. J Vitaminol (Koyto). 1958;4:118-125.

Asker AF, Habib MJ.Effect of certain stabilizers on photobleaching of riboflavin solutions. Drug Dev Ind Pharm. 1990;16:149-156.

Baba SP, Patel DK, Bano B. Modification of sheep plasma kininogen by free radicals.

Free Radical Res. 2004;38:393-403.

Ball G. F. M. Vitamins in Food Analysis, Bioavailability and Stability, CRC Press, Boca

Raton, FL, 2006, Chaps.21.

197

Barary M, Abdel-Hamid M, Hassan E, Elsayed M. Simultaneous spectrofluorimetric determination of thiamine, pyridoxine and riboflavin in pharmaceutical multivitamin preparations. Pharmazie. 1986;41:483-485.

Bartzatt R, Follis ML. Detection and assay of riboflavin (vitamin B2) utilizing uv/vis spectrophotometer and citric acid buffer. J Sci Res Reports. 2014;3:799-809.

Bartzatt R, Wol T. Detection and assay of vitamin B2 (riboflavin) in alkaline borate buffer with uv/visible spectrophotometry. Int Scholarly Res Notices.

2014;2014.ID453085.

Bas B, Jakubowska M, Gorski L. Application of renewable silver amalgam annular band electrode to voltametric determination of vitamins C, B1 and B2. Talanta. 2010;84:1032-

1037.

Basic Z, Kilibarda V, Miletic I. Vitamin B2 content determination in liver past by using acid and acid-enzyme hydrolysis. Vojnosanitetski Pregled. 2007;64:744-748.

Becker EM, Cardoso DR, Skibsted LH. Deactivation of riboflavin triplet-excited state by phenolic antioxidants: mechanism behind protective effects in photooxidation of milk- based beverages. Eur Food Res Technol. 2005;221:382-386.

198

Bhowmilk BB, Sil A. Photoinduced interaction of of riboflavin dye with different

reducing agents in aqueous and liposome media. Chem Phys Lipids.2004; 127: 189-197.

Bhushan R, Arora M. Separation of vitamin B complex and folic acid by HPLC, normal phase and reverse phase-TLC. Nat Acad Sci Lett.2002;25:159-167.

Bhushan R, Prashad V. Separation of vitamin B complex and folic acid using TLC plates impregnated with some transition metal ions. Biomed Chromatogr. 1994;8:196-198.

Bishop AM, Fernandez C, Whitehead RD Jr., Morales-A P, Barr DB, Bitsch R. Vitamin

B2 (riboflavin). In: Biesatsku HK., eds., Vitamine, Thieme, Stuttgart, 1997, pp. 75-84.

Bolliger HR, Konig A. In: Stahl E., Ed., Thin-layer Chromatography, Springer- Verlag,

Berlin, 1969, pp. 239-242. 1969.

Brdicka R, Knobloch E. About the polarographic behavior of lactoflavins.Z Electrochem.

1941;47:721-728.

Breyer B, Biegler T. Estimation of riboflavin by AC polarography. J Electroanal Chem.

1960;1:453-457.

Brezo T, Stojanovic Z, Suturovic Z, Kravic S, Kos J, Durovic A. Simple, rapid and selective chronopotentiometric method for the determination of riboflavin in

199 pharmaceutical preparations using a glassy carbon electrode. Acta Chim Slov.

2015;62:923-931.

British Pharmacopoeia, Her Majesty’s Stationary Office, London, UK, 2016; Electronic version.

Brown JA, Marsh MM. Paper chromatographic separation and determination of some water-soluble vitamins. Anal Chem. 1952;24:1252-1256.

Burgstaller P, Hermann T, Huber C, Westhof E, Famulok M. Isoalloxazine derivatives promote photocleavage of natural RNAs at G U base embedded within helicles. Nucleic

Acids Res. 1997;25:4018-4027.

Burn GP, O’Brien JRP, The kinetics of the reduction of riboflavin by dithionite, Biochim

Biophys Acta 1959;31:328-335.

Buxton PC, Conduit SM, Hathaway J. Stability of parentrovit in infusion fluids.Br. J.

Intravenous Ther. 1983;4:5-12.

Byron P, Turnbull JH. Excited states of flavin coenzymes.II.Anaerobic oxidation of amino acids by excited riboflavin derivatives. Photochem Photobiol. 1967;6:125-131.

Cairns WL, Metzler DE. Photochemical degradation of flavins, VI. A new photoproduct and its use in studying photolytic mechanism, J Am Chem Soc. 1967;93:2772–2777.

200

Canales J, Cabezas A, Pinto RM, Cameselle JC. Fluorimetric HPLC detection of endogenous riboflavin 4’, 5’-cyclic phosphate in rat liver at nanomolar concentration.Anal Biochem. 2005;341:214-219.

Capo-Chichi CD, Gueant JL, Feillet F, Namour F, Vidailhet M. Analysis of riboflavin and riboflavin cofactor levels in plasma by high-performance liquid chromatography. J

Chromatogr B Biomed Sci Appl. 2000;739:219-224.

Cardosa DR, Olsen K, Skibsted LH. Mehanism of deactivation of triplet-excited riboflavin by ascorbate, carotenoids and tocopherols in homogenous and heterogenous aqueous food model systems. J Agric Food Chem. 2007;55:6285-6291.

Cardoso DR, Franco DW, Olsen K, Andersen M, Skibsted LF. Reactivity of bovine whey proteins, peptides and amino acids towards triplet riboflavin as studied by laser flash photolysis. J Agric Food Chem. 2004;52:6602-6606.

Cardoso DR, Homem-de-Mello P, Olsen K, de Silva AB, Franco DW, Skibsted LH.

Deactivation of triplet-excited riboflavin by purine derivatives: Important role of uric acid in light-induced oxidation of milk sensitized by riboflavin. JAgric Food

Chem.2005;53:3679-3684.

Carstensen JT. Theory of Pharmaceutical Systems, Vol. 1, Academic Press, New York,

1972, pp. 209-210.

201

Carstensen, J.T. Kinetics pH Profiles, In: Carstensen, JT., Rhodes, CT., eds., Drug

Stability Principles and Practices, 3rd ed. Marcel Dekker, New York, , 2000, Chap. 3.

Casini G, DeLaurentis N, Maggi N, Ottolino S. Protective effect of hydrobenzoic acid esters on the photodegradation of riboflavin. Farm Ed Prat. 1981;36;553-558.

Cellar NA, McClure SC, Salvati LM, Reddy TM. A new sample preparation and separation combination for precise, accurate, rapid and simultaneous determination of vitamins B1, B2, B3, B5, B6, B9 in infant formula and related nutritionals by LC-MS/MS.

Anal Chim Acta. 2016;934:180-185.

Chapman S., Perham R., Scrutton N., Eds. Flavins and Flavoproteins, Proc. 14th Int.

Symp., Cambridge, UK, July 14-18, 2002, Rudolph Weber Agency for Scientific

Publications, Berlin, 2002.

Chatterjee NS, Kumar KA, Ajeeshkumar KK, Kumari KR, Vishnu KV, Anandan

R, Mathew S, Ravishankar CN. Screening Natural Content of Water-Soluble B vitamins in Fish: Enzymatic Extraction, HILIC Separation, and tandem MassSpectrometric Determination.J AOAC Int. 2017;100:579-585.

Chauhan SM, Awasthi A. Incorporation of synthetic tetramethylene diamine 8,8-bis

(riboflavin tetraacetate) in DMPC vesicles. Indian JBiochem Biophys. 1995;32:286.

202

Chen MF, Boyce HW, Triplett L. Stability of B vitamins in mixed parenteral nutrition solution.JPEN. 1983;7:462-464.

Chen P, Atkinson R, Wolf WR. Single-laoratory validation of a high-performance liquid chromatographic-diode array detector-fluorescence detector/mass spectrometric method for simultaneous determination of water-soluble vitamins in multivitamin dietary tablets.J

AOAC. 2009;92:680-687.

Chen P, Wolf WR. LC/UV/MS-MRM for the simultaneous determination of water- soluble vitamins in multi-vitamin dietary supplements. Anal Bioanal Chem.

2007;387:2441-2448.

Choe E, Huang R, Min DB. Chemical reactions and stability of riboflavin in foods. J

Food Sci. 2005;70:R28-R36.

Choe E, Min DB. Chemistry and reactions of reactive oxygen species in foods.Critical

Rev Food Sci Nutr. 2006;46:1-22.

Cimino JA, Jhangiani S, Schwartz E, Cooperman JM. Riboflavin metabolism in the hypothyroid human adult. Proc Soc Exp Biol Med. 1987;184:151-153.

203

Cimpoiu C, Casoni D, Hosu A, Miclaus V, Hodisan T, Damian G. Separation and identification of eight hydrophilic vitamins using a new TLC method and raman spectroscopy. JLCRT. 2007;28:2551-2559.

Cimpoiu C, Cosu H. Thin-layer chromatography for the analysis of vitamins and their derivatives. J Liquid Chromatography Rel Technolo. 2006;30:701-728.

Ciulu M, Solinas S, Floris I, Panzanelli A, Pilo MI, Piu PC, Spano N, Sanna G. RP-

HPLC determination of water-soluble vitamins in honey. Talanta. 2011;83:924-929.

Clarke HC. A photodecomposition fluorimetric method for the determination of riboflavin in whole blood.Int Z Vitaminforsch. 1969;39:182-191.

Clarke HC. A photodecomposition fluorimetric method for the determination of riboflavin in the various constituents of blood. Int J Vitam Nutr Res. 1977;47:356-360.

Clarke MJ, Dowling MG, Garafalo AR, Brennan TF.Structural and electronic effects resulting from metal-flavin ligation. J Biol Chem. 1980;255;3472-3481.

Cole HS, Lopez R, Cooperman JM.Riboflavin deficiency in children with diabetes mellitus.Acta Diabetologia Latina. 1976;13:25- 29.

204

Connors KA., Amidon GL., Stella VJ. Chemical Stability and Pharmaceuticals, a handbook for pharmacists, 2nd ed., Wiley, New York, 1986, Chap. 1-3.

Constable EC. Homoleptic complexes of 2,2′–bipyridine, Adv Inorg Chem. 1989;34:1–

63.

Criado S, Garcia NA. Vitamin B2-sensitized photooxidation of the ophthalmic drugs timolol and pindolol: kinetics and mechanism. Redox Rep. 2004;9:291-297.

Criado S, Guardianelli C, Tuninetti J, Molina P, Garcia NA. Scavenging of photogenerated oxidative species by antimuscarinic drugs: atropine and derivatives.

Redox Rep. 2002;7:385-394.

Cui C, Hwang HM, Zeng K, Glover H, Yu H, Liu Y. Riboflavin-photosensitized degradation of atrazine in a freshwater environment. Chemospere. 2002;47:991-999.

Cumming RG, Mitchell P, Smith W. Diet and cataract: the Blue Mountains Eye Study. .

2000;107:450-56.

Dale J. K. and Booth R. E. Compatibility of drugs, In: Martin E., Ed, Dispensing of

Medication, 7th ed., Mack Publishing Co., Easton, Pennysylvania, 1971, pp. 339-340.

205

Dawson RMC, Elliot DC, Elliot WH, Jones KM. Data for Biochem Res, 3rd ed.,

Clarendon Press, Oxford, 1986, pp. 137-138.

de Jesus MB, Fraceto LF, Martini MF, Pickolz M, Ferrira CV, de Paula E. Non-inclusion complexes between riboflavin and cyclodextrins. J Pharm Pharmacol. 2012;64:832-842.

De La Rochette A, Biriouez-Aragon I, Silva E, Morliere P. Advanced glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid: consequences for the lens. Biochim Biophys Acta. 2003;1621:235-241.

De La Rochette A, Silva E, Biriouez-Aragon I, Mancini M, Edwards AM. Morliere P.

Riboflavin photodegradation and photosensitizing effects are highly dependent on oxygen and ascorbate concentration. Photochem Photobiol. 2000;72:815-820.

Delgado JN, Remers WA. eds. (1998). Wilson and Gisvold’s Textbook of Organic,

Medicinal and Pharmaceutical Chemistry, 10th ed. Lippincott-Raven, Philadelphia, 1998, pp. 899-901, 915.

DeRitter E. Vitamins in pharmaceutical formulations. J Pharm Sci., 1982; 71:1073-1096.

Dias DA, Smith TA, Ghiggino KP, Scollary GR. (2012). The role of light, temperature and wine bottle color on pigment enhancement in white wine. Food Chem.

2012;135:2934-2941.

206

Diaz AN, Paniagua AG, Sancher CF. Thin-layeer chromatography and fiber-optic fluorimetric quantitation of thiamine, riboflavin and niacin. J Chromatogr A.

1993;655:39-43.

Diaz M, Luiz M, Bertolotti SG, Miskoshi S, Garcia NA. Scavenging of photogenerated singlet molecular oxygen and superoxide radical anions by sulpha drugs – kinetics and mechanism. Can J Chem. 2004;82:1752-1759.

Dollery C. ed. Therapeutic Drugs, Churchill Livingstone, London, 1999, pp. R24-R25.

Dryhurst G. Isoalloxazine, Flavins and Flavin Mononucleotides, Electrochemistry of

Biological Molecules, Academic Press, New York, 1977, Chap. 7.

Duggan DE, Bowman RL, Brodie BB, Udenfriend S. A spectrophotofluorimetric study of compounds of biological interest. Arch Biochem Biophys. 1957;68:1-14.

Dunbar WE, Stevenson KE. Automated fluorimetric determination of thiamine and riboflavin in infant formulas. J AOAC, 1979;62:642-647.

Dusinsky G, Faith L. An identification system for drugs by means of oscillopolarography.Pharmaize. 1967;22:475-482.

207

Dutta P, Pinto JT, Rivlin RS.Antimalarial effects of riboflavin deficiency.Lancet.

1985;2:1040-1043.

Dutta P. Disturbances in glutathione metabolism and resistance to malaria: current understanding and new concepts. J Soc Pharm Chem. 1993;2: 11-48.

Dyke SF. The Chemistry of Vitamins, Interscience, London, 1965, Chap. 3.

Edmondson DE, Ghisla S. Flavoenzyme structure and function: approaches using flavin analogues. Methods Mol Biol. 1999;131:157-179.

Edwards AM, Beeno C, Saldano A, Silva E, Kassab K, Polo L, Jori G. Photochemical and pharmacokinetic properties of selected flavins. J Photochem Photobiol B: Biol.

1999;48:36-41.

Ehrenberg A. and Hemmerich P. In: Singer TP Ed., Biological Oxidations, Interscience,

New York, 1968, pp. 239-262.

Eitenmiller RR, Ye L, Landen WO. Jr. Vitamin Analysis for the Health and Food

Sciences, 2nd ed., CRC Press, Boca Raton, FL, 2008, Chap. 7.

208

Encinas MV, Bertolotti SG, Previtali CM.The interaction of ground and excited states of lumichrome with aliphatic and aromatic amines in methanol.Helv Chim Acta.

2002;85:1427-1438.

Encinas MV, Previtali CM. Excited state interaction of flavin with amines: application to the initiation of vinyl polymerization. In: Silva E, Edwards AM, eds., Flavins The

Photochemistry and Photobiology, The Royal Society of Chemistry, Cambridge, 2006,

Chap. 3.

Faber NM, Ferre J, Boque R, Kalivas SH. Quantifying selectivity in spectrophotometric multicomponent analysis. Trend Anal. Chem. 2003;22:352-361.

Fall HH, Petering HG. Metabolic inhibitors. I. 6,7-dimethyl-9- formylmethylisoalloxazine, 6,7-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and derivatives. J Am Chem Soc. 1956;78:377-381.

Fasihullah Q. Photolysis of Flavins in Aqueous and Organic Solvents, Ph.D. Thesis,

University of Karachi, Karachi, 1988.

Fellman JK, Artz WE, Tassinari PD, Cole LC, Augustin J. Simultaneous determination of thiamine and riboflavin in selected foods by high-performance liquid chromatography. J

Food Sci. 1982;47:2048-2050.

209

Florence AT,Allwood D. Drug Stability.Physicochemical Principles of Pharmacyin

Manufacture, Formulation and Clinical Use, 6th ed., Pharmaceutical Press, London,

2016, Chap. 3, 4.

Frans SD, Harris JM. Selection of analytical wavelengths for multicomponent spectrophotometric determination. Anal Chem. 1985;57;2680-2684.

Freed M., ed., Methods of Vitamin Analysis, 3rd ed., Interscience, New York, 1966,

Chap. 7.

Friedrich W. Vitamin B2: Riboflavin and its bioactive variants, In: Vitamins.chap. 7,

Walter de Gruyter Inc., Berlin, New York., 1988, pp. 403-471.

Fu PP, Cheng SH, Coop L, Xia Q, Culp SJ, Tollesen WH, Wamer WG, Howard PC.

Photoreaction, phototoxicity and photocarcinogenicity of retenoids. J Environ Sci Health

Part C: Environ. 2003;21:165-197.

Fukamachi C, Sakurai Y. The photolytic formation of 6,7-dimethylflavin-9-acetic acid from riboflavin. JVitaminol.1955;1:217-220.

Fukumachi C, Sakurai Y. Vitamin B2 photolysis. V. the photolytic formation of 6,7- dimethylflavin-9-acetic acid ester from riboflavin. Vitamins (Kyoto). 1954;7:939-943.

210

Fukuzumi S, Okhubo K. Metal–ion–coupled and decoupled electron transfer, Coord

Chem Rev. 2010;254:372–385.

Garcia J, Silva E. Flavin-sensitized photooxidation of amino acids present in a parenteral nutrition infusate protection: by ascorbic acid. Nutr Biochem. 1997;8:341-345.

Garcia NA, Criado SN, Massad WA. Riboflavin as a visible-light-sensitizer in the aerobic photodegradation of ophthalmic and sympathomimetic drugs. In: Silva E.,

Edwards A. M., eds. Flavin Photochemistry and Photobiology, The Royal Society of

Chemistry, Cambridge, chap. 4, 2006.

Garrett ER.Kinetica and mechanism in stability of drugs, In: Bean, HS, Beckett AH,

Carless JE, eds., Advances in Pharmaceutical Sciences, Vol. 2, Academic Press, London,

1967, pp. 57-58.

Garrido Frenich A, Jouaw-Rimbarrd D, Massart DL, Kuttatharmmakul S, Martinez-

Galera M, Martinez-Vidal JL.Wavelenght selection method for multicomponent spectrophotometric determination using partial least squares.Analyst. 1995;120:2787-

2792.

Geigel MA.The stability testing program. In: Compliance Handbook for Pharmaceuticals,

Medical Devices and Biologics. Medina C. ed Marcel Dedder, New York, 2004, p.195.

211

Ghasemi J, Vosough M. Simultaneous spectrophotometric determination of folic acid, thiamine, riboflavin and pyridoxal using partial least-square regression. Spectroscopy

Lett. 2001;35,153-169.

Giri KV, Krishnawant PR. Circular paper chromatography part X. separation, identification and quantitative estimation of riboflavin and flavin compounds. J Ind Inst

Sci. 1956;38(4): p. 232.

Gladys M, Knappe WR. Photochemie des (iso) alloxazins, III.Intramolekulare photodealkylierung von 10-alkylalloxazinen, eine modellreaktion fur den riboflavin- photoabbau. Chem Ber. 1974;107:3658-3673.

Gliszczynska-Swiglo A, Kozilowa A. Chromatographic determination of riboflavin and its derivatives in food. J Chromatogr A. 2000;881:285-297.

Gliszczynsky A, Koziolowa A. Chromatographic identification of a new flavin derivative in plain yogurt. J Agric Food Chem. 1999;47:3197-3201.

Glover H, Hwang HM, Zeng K. Effect of riboflavin photoproducts on microbial activity during photsensitization of atrazine transformation. Environ Toxicol. 2003;18:361-367.

212

Goldschmidt RJ, Wolf WR. Simultaneous determination of water-soluble vitamins in

SRM 1849 infant/adult nutritional formula powder by liquid chromatography-isotope dilution mass spectrometry. Anal Bioanal Chem. 2010;397:471-481.

Guillory JK, Poust RI.Chemical Kinetics and Drug Stability. In. Banker GS, Rhodes CT eds., Modern Pharmaceuticals, 4th ed., Marcel Dekker, New York, 2002, pp. 158-159.

Guo J, Lu Y, Dong H. HPLC-MS analysis of riboflavin crude product of semisynthesis. J

Chromatogr Sci. 2006;44:552-556.

Habib MJ, Asker AF.Potostabilization of riboflavin by incorporation into liposomes. J

Parent Sci Technol. 1991;45:124-127.

Haggi E, Bertolotti S, Miskoski S, Amat-Guerri F, Garacia NA. Environmental photodegradation of pyrimidine fungicides – kinetics of the visible-light-promoted interactions between riboflavin and 2-amino-4-hydroxy-6-methylpyrimidine. Can J

Chem. 2002;80:62-67.

Hais IM, Pecakova L. Paper partition chromatography of riboflavin decomposition products. Nature. 1949;163:768.

213

Halvin K, Paalme T, Nisamedtinov I. Comparision of different extraction methods for simultaneous determination of B complex vitamins in nutritional yeast using LC/MS-

TOF and stable isotope dilution assay. Anal Bioanal Chem. 2013;405:1213-1222.

Halwer M. The photochemistry of riboflavin and related compounds, J Am Chem Soc.

1951;73:4870–4874.

Hampel D, York ER, Allen LH.Ultra-performance liquid chromatography tendem mass- spectrometry (UPLC-MS/MS) for the rapid, simultaneous analysis of thiamine, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk. J

Chromatogr B Analyt Technol Biomed Life Sci. 2012;903:7-13.

Hartman RF, Rose SD, Pouwel PJ, Kaptein R. Flavin-sensitized photochemically induced dynamic nuclear polarization detection of pyrimidine dimer radicals. Photochem

Photobiol. 1992;56:305-310.

Hashmi M. H. Assay of Vitamins in Pharmaceutical Preparations, Wiley, New York,

1973, pp. 99-102.

Hashmi MH, Ajmal IA, Qureshi T, Rashid A. Spectrophotometric determination of riboflavin from pharmaceutical preparations. Microchimica Acta. 1969;57:30-35.

214

Hatchard CG, Parker CA. A new sensitive chemical actinometer II.ferrioxalate as a standard actinOmeter. Proc R Soc (Lond.) A 235. 1956:518-536.

Heelis PF, Parsons BJ, Phillips GO, McKeller JF. The flavin sensitized photooxidation of ascorbic acid. A continuous and flash photolysis study. Photochem Photobiol. 1981;33:7-

13.

Heelis PF, Phillips GO, Ahmad I, Rapson HDC. The photodegradation of formylmethylflavin. A steady state and laser flash photolysis study. Photochem

Photobiol. 1980;1:125-130.

Heelis PF. The photochemistry of flavins. In: Chemistry and Biochemistry of

Flavoenzymes. Muller F. ed., vol. 1,CRC Press, Boca Rotan, FL, 1991, pp. 171-193.

Heelis PF. The photophysical and photochemical properties of flavins (isoalloxazines),

Chem Soc Rev. 1982;11:15–39.

Heelis PF. The photophysical and photochemical properties of flavins (isoalloxazines).

Chem Soc Rev. 1982;11:15-39.

Hemmerich P. The present status of flavin and flavocoenzyme chemistry. Fortsch Chem

Org Naturst. 1976;33:451-527.

215

Hertz JJ. A Stability and Solubility Study of Riboflavin and Some Derivatives.University of Florida. 1954.

Hewavitharana AK. Method for the extraction of riboflavin for high-performance liquid chromatography and application to casein.Analyst. 1996;121:1671-1676.

Hoffman MA. Physico-chemical methods for the determination of riboflavin in animal tissues and fluid; a review.Int Z Vitaminforsch Beih. 1948;20:238-252.

Holmstorm B. Spectral studies of the bleaching of riboflavin phosphate. Arkiv Kemi.

1964;22:281-301.

Holmstrom B, Oster G. Riboflavin as an electron donor in photochemical reaction. J Am

Chem Soc. 1961;83:1867–1871.

Holmstrom B. Mechanism of the photoreduction of riboflavin, Arkiv Kemi.

1964;22:329–346.

Huang R, Choe E, Min DB. Kinetics for singlet oxygen formation by riboflavin photosensitization and the reaction between riboflavin and singlet oxygen. J Food Sci.

2004;69:C726-C732.

216

Huang R, Choe E, Min DB. Kinetics for singlet oxygen formation by riboflavin photosensitization and the reaction between riboflavin and singlet oxygen. J Food Sci.

2004;69:C726-C732.

Huang R, Kim HJ, Min DB. Photosensitizing effect of riboflavin, lumiflavin and lumichrome on the generation of volatiles in soy milk. J Agric Food Chem.

2006;54:2359-2364.

Huang SI, Caldwell MJ, Simpson KL.Urinary riboflavin determination by C18 reverse- phase open-column chromatography.Methods Enzymol. 1997;280:343-351.

Huennekens FM, Sanadi DR, Diamant E, Schepartz AI. The occurrence of a new flavin dinucleotide (FAD-X). J Am Chem Soc. 1953;75:3611-3612.

Hussain E, Fatima RA, Ali IAF, Naseem I. Photoilluminated riboflavin/riboflavin–Cu (II) inactivates trypsin Cu (II) tilts the balance. Ind J Biochem Biophys. 2006;43:312–318.

Huynh-Ba K. ed. Handbook of Stability Testing in Pharmaceutical Development,

Regulations, Methadologies and Best Practices, Springer, New York, 2009.

Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary

Reference Intakes.Dietary refernece.intakes for thiamine, riboflavin, niacin, vitamin B6,

217 folate, vitamin B12, pantothenic acid, biotin and choline’. National Academies Press (US)

1998.

Ionita MA, Ion RM, Carstocea B. Photochemical and photodynamic properties of vitamin

B2-riboflavin and liposomes. Optalmologia. 2002;58:29-34.

Jabbar MA, Salahuddin S, Mannan RJ, Mahmood AJ. Electrochemical evidence for the enhancement of hetergenous electron transfer rates of riboflavin in the presence of copper. Dhaka Univ J Sci. 2014; 62:147–152.

Jaffe HH, Orchin M. Theory and applications of ultraviolet spectroscopy. Wiley, New

York,1962, Chap. 20.

Jin P, Xia L, Li Z, Che N, Zou D, Hu X. Rapid determination of thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, folic acid and ascorbic acid in vitamins with minerals tablets by high-performance liquid chromatography with diuode array detector.

J Pharm Biomed Anal. 2012;70:151-157.

Joneidi M, Koleva M, Budevsky O. Quantitative determination of drugs by means of densitometry of thin-layer chromatography. Part 5: determination of the vitamins B1, B2,

B6 and B12 in the mixture and in pharmaceutical preparations. Die Pharmazie.

1975;30:453-455.

218

Jozan M, Szasz G, Szemeredy K. Electro-analytical study of polyvitamin preparations. I. polarographic determination of ascorbic acid, folic acid, nicotinamide and riboflavin content of polyvitamin tablets. Acta Pharm Hung. 1980;50:153-160.

Jung MY, Kim SM, Kim SY. Riboflavin-sensitized photooxidation of ascorbic acid: kinetics and amino acid effects. Food Chem. 1995;53:397-403.

Jung MY, Lee KH, Kim SY.Riboflavin-sensitized photochemical changes in beta lactoglobulin in an aqueous buffer solution as affected by ascorbic acid. J Agric Food

Chem. 2000;48:3847-3850.

Jung MY, Oh YS, Kim DK, Kim HJ, Min DB. Photoinduced generation of 2,3- butanedione from riboflavin. J Agric Food Chem. 2007;55:170-174.

Jusko WJ, Levy G, Yaffe SJ, Allen JE. Riboflavin absorption in children with biliary obstruction.Am J Dis Child. 1971;121:48-52.

Jusko WJ, Levy G. Pharmacokinetic evidences for saturable renal tubular reabsorption of riboflavin. J Pharm Sci. 1970;59:765-772.

219

Kaim W, Schwederski B, Heilmann, O, Hornung FM. Coordination compounds of pteridine, alloxazine and flavin ligands: structures and properties, Coord Chem

Rev.1999;182:323–342.

Karrer P, Kobner T, Salomon H, Zehender F. About the light degradation of flavins.Helveta Chim Act. 1935;18:266-272.

Kartsova LA, Korleva OA. Simultaneous determination of water- and fat-soluble vitamins by high-performance thin-layer chromatography using an aqueous micellar mobile phase. J Anal Chem. 2007;62:255-259.

Khaloo SS, Moziffari S, Ali Mohammadi P, Kargar H, Ordookhanian J. Sensitive and selective determination of riboflavin in food and pharmaceutical samples using manganese (III) tetraphenylporphyrine modified carbon paste electrode. Int J Food

Properties. 2016;19:2272-2283.

Khar RK, Vyas SP, Ahmad FJ, Jain GK. Lachman/Lieberman’s The Theory and Practice of Industrial Pharmacy, 4th ed., CBS Publishers & Distributors, New Dehli, ; 2016. Chap.

28.

Kilgour GL, Felton SP, Huennekens FM. Paper chromatography of flavins and flavin nucleotides. J Am Chem Soc. 1975;79:2254-2256.

220

King JM, Min DB. Riboflavin photosensitized singlet oxygen oxidation of vitamin D. J

Food Sci. 1998;63:31-34.

Knappe WR. Electron input at the N-1 position of the flavin system. In: Singer TP.; ed.,

Flavins and Flavoproteins, Proceedingds of the Fifth International Symposium on Flavins and Flavoproteins, San Francisco, March 31-April 3, 1975, Elsevier, Amsterdam, 1975, pp. 788-792.

Knappe WR. Photochemistry of (iso)alloxazines. IV. Dealkylation and decarboxylation of short-chained isoalloxazine-10-alkanoic acids. Ber Dtsch Chem Ges.1975;108:2422-

2432.

Knobloch E, Mandys F, Hodr R, Hujer R, Mader R.Study of the mechanism of the photoisomerization and photooxidation of bilirubin using a model for the phototherapy of hyperbilirubinemia. J Chromatogr. 1991;566:89-99.

Knowles A, Burgess C., eds. Practical Absorption Spectroscopy. Chapman and Hall,

London,1984, pp. 167-168.

Korycka-Dahl M, Richardson T. Photodegradation of DNA with fluorescent light in the presence of riboflavin and photoprotection by flavin-triplet state quenchers.Biochim

Biophys Acta. 1980;610:229-234.

221

Kostenbauder HB, DeLuca PP, Kowarski CK. Photobinding and photoreactivity of riboflavin in the presence of macromolecules. J Pharm Sci. 1965;54:1243-1251.

Kouimtzis TA, Papadoyannis IN. Determination of vitamins A, B1, B2, D3 and C by densitometry of thin-layer chromatography. Microchimica Acta. 1979;71:145-149.

Koziol J, Knobloch E. The solvent effect on the fluorescence and the light absorption of riboflavin and lumiflavin.Biochim Biophys Acta. 1965;102:289-300.

Koziol J. Studies on flavin in organic solvents- II.photodecomposition of riboflavin in the presence of the oxygen. Photochem Phototbiol. 1966a;5:55-62.

Kruze IE. Polarographic determination of ascorbic acid, folic acid and riboflavin.Farmatsiia. 1969;18:59-63.

Kuhn R, Reinemund K, Kaltschmitt R, Strobele R, Trischmann H. Synthetic 6.7- dimethyl-9-d-ribo-flavin. Naturwissenchaften. 1935;23:260-260.

Kuhn R, Wagner-Jauregg T. Uber das reduction-oxidation-verhalten und eine farbreaktion des lacto-flavins (vitamin B2). Eur J Inorg Chem. 1934;67:361-363.

Laidler KJ. Chemical Kinetics. Harper & Row, New York, 3rd ed., 1987, pp. 279-280,

384-399.

222

Leshner RT.Riboflavin deficiency – a reversible neurodegenerative disease.Annuals of

Neurology. 1981;10:294-295.

Li YQ, Huang XZ, Xu JG, Chen GZ. Synchronous spectrofluorimetry for simultaneous determination of riboflavin and pyridoxine and its application in multivitamin preparations.Yao Xue Xue Bao. 1992;27:52-56.

Lingane JJ, Davis OL.Polarographic determination of riboflavin (vitamin B2) and other vitamin B factors. J Biol Chem. 1941;137:567-574.

Linter CJ.Drug Product Stability, In: Cooper MS., Ed., Quality Control in Pharmaceutical

Industry, Vol. 2, Academic Press, New York, 1973, pp. 142.Lippincott Wiliams &

Wilkins, Philadelphia, Pa, USA, 6thed., 2008,

Long WQ.Determination of trace vitamin B2 by fluorimetry after aqueous two-phase extraction based on acetone and ethanol.Guang Pu Xue Yu Guang Pu Fen Xi.

2007;27:2098-2101.

Lopez R, June VS, Jack MC. Riboflavin deficiency in an adolescent population in New

York City. AJCN; 1980;33: 1283-1286.

Lopez-de-Alba P, Lopez-Martinez L, Cerda V, Amador-Hernandez J. Simultaneous determination and classification of riboflavin, thiamine, nicotinamide and pyridoxine in

223 pharmaceutical formulations by uv-visible spectrophotometry and multivariate analysis.J

Brazilian Chem Soc. 2006;17.4:715-722.

Loukas Y. 2 (k-p) Fractional factorial design via fold over: application to optimization of novel multicomponent wesicular systems. Analyts. 1997;122:1023-1028.

Loukas YL, Jayasekera P, Gregoriadis G. Novel liposome-based multicomponent systems for the protection of photolabile agents. Int J Pharm. 1995;11:85-94.

Loukas YL, Varaka V, Gregoriadis G. Use of nonlinear last-square model for the kinetic determination of the stability constant of cyclodextrin inclusion complexes. Int J Pharm.

1996;144:225-231.

Loukas YL. A Plackett-Burnam screening design directs the efficient formulation of multicomponentDRV liposomes. JPharm Biomed Anal. 2001;26:255-263.

Lu CY, Liu YY. Electron transfer oxidation of tryptophan and tyrosine by triplet states and oxidized radicals of flavin sensitizers: a laser flash photolysis study. Biochim

Biophys Acta. 2002;1571:71-76.

Ma Q, Song J, Zhang S, Wang M, Guo Y, Dong C. Colorimetric detection of riboflavin by silver nano particles capped with β-cyclodextrin-grafted citrate. Colloids and surfaces

B: Biointerfaces. 2016;148:66-72.

224

Macek TJ. Stability problems with some vitamins in pharmaceuticals. Am J Pharm.

1969;132:433-455.

Mader WJ. Vitamins, In: Higuchi T., Brochmann-Hanssen, E., Eds., Pharmaceutical

Analysis, Interscience, New York, 1961, Chap. 11.

Malgorzata I, Marek S. Riboflavin interactions with oxygen – a survey from the photochemical perspective. Chem Eur J. 2014;20:15280-15291.

Mann TB. A fluorimetric method for the estimation of riboflavin in egg white and egg yolk.Analyst. 1946;71:166-171.

Maquinay A, Brouhon N. Determination of vitamin B2 (riboflavin, lactoflavin) by polarography in pharmaceutical preparations.J Pharm Belg. 1957;12:350-359.

Marakova K, Piestansky J, Hauranek E, Mikus P. Simultaneous analysis of vitamins B in pharmaceuticals and dietary supplements by capillary electrophoresis hyphenated with triple quadrupole mass spectrometry. Pharmazie. 2014;69:663-668.

Martens HJ.Stability of water soluble vitamins in various infusion bags.Krankenhauspharmazie. 1989;10:359-361.

225

Martin CB., Wilfong E, Ruane P, Goodrich R, Platz M.An action spectrum of the riboflavin-photosensitized inactivation of lambda phage. Photochem Photobiol.

2005;81:474-480.

Massad W, Criado S, Bertolotti S, Pajares A, Gianotti J, Escalada JP, Amat-Guerri F,

Garcia NA. Photodegradation of the herbicide norflurazon sensitized by riboflavin.A kinetic and mechanistic study.Chemosphere. 2004;57:455-461.

Massey V, Palmer G, Ballon D. Oxidases and Related Redox System. In: King TE,

Mason HS, Morrison M, eds., University Park Press, Baltimore, 1973.

Massey V.The chemical and biological versatility of riboflavin. Biochem Soc Trans.

2000;28:283-296.

Mattivi F, Monetti A, Vrhovsek U, Tonon D, Andres-Lacueva C. High-performance liquid chromatographic determination of the riboflavin concentration in white wines for predicting their resistance to light. J Chromatogr A. 2000;888:121-127.

Mayersohn M, Feldman S, Gibaldi M. Bile salt enhancement of riboflavin and flavin mononucleotide absorption in man. J Nutr. 1969;98:288-296.

McBride MM, Metzler DE. Photochemical degradation of flavins – III.Hhdroxyethyl and formylmethyl analogs of riboflavin.PhotochemPhotobiol. 1967;6:113-123.

226

McBride MM, Moore MW.The photochemistry of riboflavin. II. Polarographic studies on the hydroxyethyl and formylmethyl analogs of riboflavin. Photochem Photobiol.

1967;103-113.

McCormick DB. On becoming a nutritional biochemist. Ann Rev Nutr. 2004;24:1-11.

McCormick DB. Riboflavin.In. Present Knowledge in Nutrition, 6th ed., ed., Brown M.

L., 146, International Life Sciences Institute , Washington DC, 1990, p.146.

McDowell LR. Riboflavin. In. Vitamins in animals ans human nutrition; Iowa State

University Press: Iowa, USA; 2000, pp. 311-346.

McMahon JM. The analysis of six common vitamins by laser desorption mass spectroscopy. Anal Biochem. 1985;147:535-545.

Meisel P, Amon I, Huller H, Jahrig K. Effect of theophylline on the riboflavin-sensitized photodegradation of bilirubin in vitro. Biol Neonate. 1980;38:30-33.

Merrill Jr, AH, Edmondson DE, McCormick DB.Formation and mode of action of flavoproteins.Annual Review of Nutr. 1981;1:281-317.

227

Mestdagh F, De Meulenaer B, De Clippelleer J, Devlieghere F, Huyghebaert A.

Protective influence of several packaging materials on light oxidation of milk. J Dairy

Sci. 2005;88:499-510.

Methods in Molecular Biology, vol. 131; Flavoprotein protocols, Chapman SK, Reid GA

Stephen K (eds.) Humana Press, Totowa, NJ, 1999.

Midttun O, Hustad S, Solheim E, Schneede J, Ueland PM. Multianalyte quantification of vitamin B6 and B2 species in the nanomolar range in human plasma by liquid chromatography-tandem mass spectrometry. Clin Chem 2005;51:1206-1216.

Midttun O, Hustad S, Ueland PM. Quantitative profiling of biomarkers related to B- vitamin status, tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom.

2009;23:1371-1379.

Min DB, Boff JM.Chemistry and oxygen of singlet oxygen in foods.CRFSFS. 2002;1:58-

72.

Miquel Becker E, Christensen J, Frederiksen CS, Hauquaad VK. Front-face spectroscopy and chemometrics in analysis of yogurt: rapid analysis of riboflavin. J Dairy Sci.

2003;86:2508-2515.

228

Mirza T, Anwar Z, Ejaz MA, Ahmed S, Sheraz MA, Ahmad I, Multicomponent spectrofluorimetric method for the assay of carboxymethylflavin and its hydrolytic products: kinetic applications, Luminescence. 2018;33:1314-1325.

Miura R. Versatility and specificity in flavoenzymes: control mechanisms of flavin reactivity. Chem Rec. 2001;1:183-194.

Miyake K, Masaki Y, Miyamoto I, Yanagida S, OhnoT, Yoshimura A, Pac C. Flavin- sensitized monomerization of dimethylthymine cyclobutane dimer in the presence of magnesium perchlorate. Photochem Photobiol. 1993;58:631-636.

Moffat AC, Osselton MD, Widdop B. Clarke’s Analysis of Drugs and Poisons, 3rd ed.,

Pharmaceutical Press, London, 2004, pp. 736-737, 1533.

Mohamed AM, Mohamed HA, Abdel-Latif NM, Mohamed

MR.Spectrofluorimetric determination of some water-soluble vitamins.J AOAC.

2011;94:1758-1769.

Monteiro MC, Perrone D. ‘Chemistry and biochemistry of riboflavin and related compounds’. In. Preedy VR., ed., B Vitamins and Folate, Chemistry, Analysis, Function and Effects. RSC Publishing. The Royal Society of Chemistry,London, 2012, pp. 93-107.

Moore WM, Spence JT, Raymond FA, Colson SD. Photochemistry of riboflavin. I. the hydrogen transfer process in the anaerobic photobleaching of flavins. J. Am. Chem. Soc.

1963; 85: 3367-3372.

229

Moore WW, Ireton RC.The photochemistry of riboflavin - V.The photodegradation of isoalloxazines in alcoholic solvents. Photochem Photobiol. 1977;25:347-356.

Moore WW, Ireton RC.The photochemistry of riboflavin-V.The photodegradation of isoalloxazines in alcoholic solvents. Photochem Photobiol. 1977;25:347-356.

Mortland MM, Lawless JG, Hartmann H, Frankel R. Smectite interactions with flavomononucleotide. Clays &Clay Materials.1984;32:229-282.

Moshe S, Lopez R, Cooperman JM. Riboflavin deficiency in infantsand children with heart disease. Am Heart J. 1976;92:139-143.

Moss AJ, Levy AS, Kim I, Park YK. Use of Vitamin and Mineral Supplements in the

United States: Current Users, Types of Products, and Nutrients. Hyattsville, MD:

National Center for Health Statistics; 1989. Advance Data, Vital and Health Statistics of the National Center for Health Statistics, No. 174.

Mototkov V. Quantitative determination of riboflavin in the urine by the fluorimetric method.Lab Delo. 1965;9:559-561.

Muller F. Spectroscopy and photochemistry of flavins and flavoproteins, Photochem

Photobiol. 1981;34:753–759.

230

Natera J, Massad W, Gracia NA. The role of vitamin B6 as an antioxidant in the presence of vitamin B6-photogenerated reactive oxygen species.A kinetic and mechanistic study.

Photochem Photobiol Sci. 2012;11:938-945.

Ni YN, Cia YJ. Simultaneous synchronous spectrofluorimetric determination of vitamin

B1, B2 and B6 by PARAFAC.Guang Pu Xue Yu Guang Pu Fen Xi. 2005;25:1641-1644.

Niazi A, Zolgharnein J, Afiuni-Zadeh S. Spectrophotometric determination of ternary mixtures of thiamine, riboflavin and pyridoxal in pharmaceuticals and human plasma by least-squares support vector machines. Anal Sci. 2007;23:13111316.

Nichoalds GE. Riboflavin Symposium in Laboratory Medicine. In: Labbae RF, editor. Symposium on Laboratory Assessment of Nutritional Status. Vol. 1. Philadelphia:

WB Saunders; 1981. pp. 685–698. Clinics in Laboratory Medicine Series.

Northrop-Clewes CA, Thurnham DI.The discovery and characterization of riboflavin.Annals of Nutrition and Metabolism. 2012;61:224-230.

Novak M, Miller A, Bruce TC, Tollin G. The mechanism of flavin 4a substitution which accompanies photolytic decarboxylation of α-substituted acetic acids.Carbanion vs. radical intermediates. J Am Chem Soc. 1980; 102: 1465-1467.

231

O’Neil M. J., The Merck Index, 13th ed., Merck & Co., Inc., Rahway, N.J. USA, 2013,

Electronic version.

Odanak K, Iwatsuki M, Satho T, Watanabe M. Identification and characterization of a brilliant yellow pigment produced by Bordella pertussis. Microbiol Immunol.

2017;61:490-496.

Ollilainen V, Mattila P, Varo P, Koivistoinen P, Huttunen J. The HPLC determination of total riboflavin in foods.J Micronutrient Analysis. 1990;8:199-207.

Oster G, Bellin JS, Holmstorm B. Photochemistry of riboflavin.Experientia.

1962;18:249-253.

Ottaway PB.Stability of vitamins in food.In. The Technology of Vitamins in Food:

Springer Science and Business Media. 2012.

Pearson W. N. In: Gyorgy P., Pearson W. N., eds., Vitamins, Vol. 5, Academic Press,

New York, 1972, pp. 1-96.

Penzer GR, Radda GK. Photochemistry of flavins.Methods Enzymol. 1971;18B:479-506.

Penzer GR, Radda GK. The chemistry and biological functions of isoalloxazines

(flavins). Q Rev. 1967;21:43-65.

232

Penzer GR. The chemistry of flavins and flavoproteins: aerobic photochemistry. Biochem

J. 1970;116:733-743.

Penzkofer A,Tyagi A, Kiermaier J. Room temperature hydrolysis of lumiflavin in alkaline aqueous solution. J Photochem Photobiol A: Chem. 2011;27:369-375.

Perez-Ruiz T, Martinez-Lozano C, Tomas V, Val O. Photochemical spectrophotometric determination of riboflavin and riboflavin 5’-phosphate by manual and flow injection methods. Analyst. 1994;119:1199-1203.

Petteys BJ, Frank EL. Rapid determination of vitamin B2 (riboflavin) in plasma by

HPLC. Clinica Chim Acta. 2011;412;38-43.

Pinto JT, Huang JP, Rivlin RS.Mechanisms underlying the differential effects of ethanol upon the bioavailability of riboflavin and flavin adenine dinucleotide. J Clin Invest.

1987;79:1343-1348.

Pinto JT, Huang YP, Rivlin RS.Inhibition of riboflavin metabolism in rat tissues by chlorpromazine, imipramine and imitryptyline. J Clin Invest.1981;67:1500-1506.

Pinto JT, Rivlin RS.Drugs that promote renal excration of riboflavin.Drug Nutr

Interactions. 1987;5:143-151.

233

Ponder EL, Fried B, Sherma J. Thin-layer chromatographic analysis of hydrophilic vitamins in standards and from Helisoma trivolvis snails. Acta Chromatographica.

2004;14:70-81.

Portillor R, Varela G. Polarographic determination of riboflavin in liver extracts. An Real

Acad Farm. 1949;15:787-792.

Postaire E, Cisse M, Hoang LMD, Pradeau D. Simultaneous determination of water- soluble vitamins by over-pressure layer chromatography and photodensitometric detection. J Pharm Sci. 1991:80:368-370.

Powers HJ. Riboflavin (vitamin B2) and health.Am J Clin Nutr. 2003;77:1352-1360.

Radda CK, Calvin M. Chemical and photochemical reduction of flavin nucleotides and analogues. Biochemistry. 1964;3:384-393.

Ramirez-Munoz J. Determination of riboflavin in pharmaceutical products by automatic discrete-sample analysis. Anal Chim Acta. 1974;72:407-413.

Ramu A, Mehta MM. The riboflavin-mediated photooxidation of doxorubicin. Cancer

Chemoth Pharm. 2000;46:449-458.

234

Ren XN, Yin SA, Yang Z, Yang XG., Shao B, Ren YP, Zhang J. Application of UPLC-

MS/MS method for analyzing B-vitamins in human milk. Biomed. Enviornmental Sci.

2015;28:738-750.

Rettenmaier R, Vuilleumier JP. A simple method for the determination of riboflavin in human milk. Int. J. Nutr. Res. 1983;53:32-35.

Riberio DO, PintoDC, Lima LMTR, Volpato NM, Cabral LM, Sousa VP. Chemical stability study of vitamins thiamine, riboflavin, pyridoxine and ascorbic acid in parenteral nutrition for neonatal use. Ntur J. 2011;10-47.

Rivlin RS, Dutta P. Vitamin B2 (riboflavin), relevance to malaria and antioxidant activity.

Nutr Today. 1995;30:62-67.

Rivlin RS, Pinto JT, ‘Riboflavin’. In. Rucker RB, Suttie JW, McCormick D B, Machlin

LJ. eds. Handbook of Vitamins 3rd ed., Marcel Dekker Inc., New York, Basel, CRC

Press, 2001.

Rivlin RS.Riboflavin (vitamin B2). In: Zempleni J, Rucker RB, McCormick DB, Suttie

JW. eds., Handbook of Vitamins, 4th ed., Taylor & Francis, CRC Press Boca Rotan, FL,

2007, Chap. 7.

235

Rizzolo A, Polesello S. Chromatographic determination of vitamins in foods. J

Chromatogr A. 1992;624:103-152.

Robinson F. A. The Vitamin B Complex, Wiley, New York, 1951, Chap. 3.

Rocha FRP, Filho OF, Reis BF. A multicommuted flow system for sequential spectrophotometric determination of hydrosoluble vitamins in pharmaceutical preparations.Talanta. 2003;59:191-200.

Roche. Analytical Procedures for the Determination of Vitamins in Multivitamin

Preparations, -La Roche, Basle, 1970, pp. 69-70.

Rochette AD, Silva E, Birlouez-Aragon I, Mancini M, Edwards AM, Morliere P.

Riboflavin photodegradation and phototsensitizing effects are highly dependent on oxygen and ascorbate concentrations. Photochem Photobiol. 2000;72:815-820.

Roe EA, Kalkwarf H, Stevens J. Effect of fiber supplements on the apparent apsorption of pharmacological doses of riboflavin. J Am Diet Assoc. 1988;88:211-213.

Rosenberg HR. Chemistry and Physiology of the Vitamins, Interscience, New York,

1954, pp. 153-194.

236

Roy DK, Saha A, Mukherjee AK. Spectroscopic and thermodynamic study of charge transfer complex formation between cloxacillin and riboflavin in aqueous ethanol media of varying composition. Spectrochim Acta Part A: Mol Biomol Spectroscop.

2006;63:649-699.

Russell LF, Vanderslice JT. Non-degradative extraction and simultaneous quantitation of riboflavin, flvin mononucleotide and flavin adenine dinucleotide in foods by HPLC. Food

Chem. 1992;43:151-162.

Rutter WJ.The interaction of riboflavin. FMN and FAD with various metal ions: The riboflavin catalyzed photochemical reduction of Fe III and photooxidation of Fe II, Acta

Chem Scand. 1958;12:428–446.

Said HM, Mohammed ZM. Intestinal absorption of water-soluble vitamins: an update.

Curr Opin Gastroenterol. 2006;22:140-146.

Sakai K.On the influences of several metal ions upon photolysis of riboflavin, Nagoya J

Med Sci. (Japan). 1956;10:222–231.

Salavati LM, McClure SC, Reddy TM; Cellar N. A. (2016). Simultaneous determination of total vitamins B1, B2, B3 and B6 in infant formula and related nutritionals by enzymatic digestion and LC-MS/MS: single-laboratory validation, first action 2015.14. J

AOAC Int. 2016;99:776-785.

237

Sanchez-Machado DI, Lopez-Cervantes J, Lopez-Hernandez J, Paseiro-Losada P.

Simultaneous determination of thiamine and riboflavin in edible marine seaweeds by high-performance liquid chromatography. J Chromatogr Sci. 2004;42:117-120.

Sarubin FA, Thomson C. The Health Professional’s Guide to Popular Dietary

Supplements, American Dietetic Association, Chicago, Ill, USA, 2007.

Saski K, Kawata S, Minemi S. Optimal wavelength selection for quantitative analysis.

Appl Spectrosc. 1986;40:185-190.

Sato Y, Chaki H, Suzuki Y. Biphasic photolysis of riboflavin. Chem Pharm Bull.

1984;32:1232-1235.

Sato Y, Chaki H, Suzuki Y. Biphasic photolysis of riboflavin. Chem Pharm Bull.

1984;32:1232-1235.

Sato Y, Yamasato S, Suzuki Y. Biphasic photolysis of riboflavin. II. Effect of gelatin on the photolysis. Chem Pharm Bull (Japan). 1983;31:4167-4171.

Sato Y, Yokoo M, Takahashi S, Takahashi T. Biphasic photolysis of riboflavin with a low-intensity light source. Chem Pharm Bull (Japan). 1982;30:1803-1810.

238

Sattar A, Alexander JC. Light-induced degradation of vitamins I. Kinetic studies on riboflavin decomposition in solution. Can Institute of Food Sci and Technol J.

1977;10:61-64.

Schlee D, Strauba G. Physiology and biochemistry of riboflavin formulation. Die

Pharmazie. 1984;39:805.

Schoenen J, Jacquy J, Lenaerts M. Effectiveness of high-dose riboflavin in migraine prophylaxis. Arandomized controlled trial. Neurology. 1998;50:466-70.

Schollnhamer G, Hemmerich P. Nucleophilic addition at the photoexcited flavin cation:

Synthesis and properties of 6- and 9-hydroxy-flavohydroxy-flavocoenzyme chromophores. Eur J Biochem. 1974;44:561-577.

Schuman Jorns, Schollnhammer G, Hemmerich P. Intramolecular addition of riboflavin side chain. Eur J Biochem. 1975;57:35-48.

Seagers W. J. The polarographic determination of riboflavin. J. Pharm. Sci. 1953;42:317-

318.

Sebek O. Production of riboflavin by the butylogenic becteria.Chem Listy. 1947;41:238-

240.

239

Sen-Varma C, Ghosh S, Bhowmilk BB. Photo-effect in phospholipid liposome containing riboflavin.Chem Phys Lipids. 1995;76:49-54.

Sevrioukova I. F. Redox-linked conformational dynamics in apoptosis-inducing factor. J

Mol Biol. 2009;390:924-938.

Shah H, Patel S, Patel B, Solanki N, Jivani NP, Kumar DB. Development and validation of uv-visible spectrometric method for estimation of water soluble vitamin riboflavin.Int

J Pharm Sci Res. 2012;3:3462-3466.

Shah JJ. Riboflavin, In: Augustin J, Klein BP, Becker D, Venugopal PB, Eds., Method of

Vitamin Assay, Wiley, New York, 1985, Chap. 14.

Sheraz MA, Kazi SH, Ahmed S, Anwar Z, Ahmad I. Photo, thermal and chemical degradation of riboflavin. Beilstein J Org Chem. 2014;10:1999–2012.

Sheraz MA, Kazi SH, Ahmed S, Mirza T, Ahmad I, Evstigneev MP. Effect of phosphate buffer on the complexation and photochemical interaction of riboflavin and caffeine in aqueous solution: a kinetic study. J Photochem Photobiol A: Chem. 2014;273:17-22.

Sheraz MA, Kazi SH, Ahmed S, Qadeer K, Khan MF, Ahmad I. Multicomponent spectrometric analysis of riboflavin and photoproducts and their kinetic applications.

Cent Eur J Chem. 2014;12:635–642.

240

Sherwani SK, Ashraf M, Javed S, Abbas Q, Khokhar MY, Nangyal H, Kausar R.

Spectrophotometric determination of riboflavin with spermine-copper chloride complexes in pharmaceutical preparations. American-Eurasian J Agri Enviorn Sci.

2014;14:1397-1401.

Silberstein SD, Goadsby PJ, Lipton RB. Management of migraine: al algorithmic approach. Neurology. 2000; 55:s46-s52.

Silva E, Almarza C, Berndt D, Larrondo L. Photoreactions of riboflavin with spermine and their role in tryptophan photoconsumption induced by riboflavin. J Photochem

Photobiol B. Biol. 1993;197-201.

Silva E, Edwaards AM. Flavin Photochemistry and Photobiology.The Royal Society of

Chemistry, Cambridge, 2006.

Silva E, Edwards AM, Pacheco D. Visible light-induced photooxidation of glucose sensitized by riboflavin.J Nutr Biochem. 1999;10:181-185.

Silva E, Edwards AM, Pacheco D. Visible light-induced photooxidation of glucose sensitized by riboflavin. J Nutr Biochem; 1999;10:181-185.

241

Silva E, Edwards AM. Generation of radical species by irradiation of tryptophan and tyrosine solutions sensitized by riboflavin.Biological implications. Cienc Cult (Sao

Paulo). 1996;48:47-50.

Silva E, Godoy J. Riboflavin-sensitized photooxidation of tyrosine. Int J Vit Nutr Res.

1994;64:253-256.

Silva E, Gonzalez T, Edwards AM, Zuloaga F. Visible light induced lipoperoxidation of a parenteral nutrition fat emulsion sensitized by flavins. J Nutr Biochem. 1998;9:149-154.

Silva E, Herrera L, Edwards AM, De la Fuente JR, Lissi E. Enhancement of riboflavin- mediated photooxidation of glucose 6-phosphate dehydrogenase bu urocanic acid.

Photochem Photobiol. 2005;81:206-211.

Silva E, Quina FH. Photoinduced processes in the eye lens: do flavin really play a role.

In: Silva E. and Edwards A. M. eds., Flavin Photochemistry and Photobiology. The

Royal Society of Chemistry, Cambridge,2006,Chap. 7.

242

Silva E, Ruckert V, Lissi E, Abuin E. Effect of pH and ionic micelles on the riboflavinsensitized photoprocesses of tryptophan in aqueous solution. J Photochem Photobiol B:

Biol. 1991;11:57-68.

Silva E, Salim-Hanna M, Edwards AM, Becker MI, De Ioannes AE. A light-induced tryptophan-riboflavin binding: biological implications. In. Series: Nutritional and toxicological consequences of food processing. Friedman M. ed., Springer Boston,

MA.1991, pp.33-48.

Silva E, Ugarte P, Andrade A, Edwards AM. Riboflavin-sensitized photoprocess of tryptophan. J Photochem Photobiol B Biol. 1994;23:43-48.

Silva E. Sensitized photooxidation of amino acids in proteins: some important biological implications. Biol Res. 1996;29:57-67.

Sim HJ, Kim B, Lee J. A systemic approach for the determination for B-group vitamins in multivitamin dietary supplements by high-performance liquid chromatography with diode-array detection and mass spectrometry.J AOAC. 2016;99:1223-1232.

Sinko PJ. Chemical kinetics and stability.Martin’s Physical Pharmacy and

Pharmaceutical Sciences, 5th ed., Lippincott Williams & Wilkins, Baltimore, 2006, Chap.

15.

243

Skooj DA, West DM, Holler FJ, Crouch SR. Fundamentals of Analytical Chemistry,

Thomson Asia Pte Ltd., Singapore, A14.

Smith EC, Metzler DE. The photochemical degradation of riboflavin, J Am Chem Soc.

1963;85:3285–3288.

Smith JL, Canham JE, Wells PA. Effect of phototherapy light, sodium bisulfite and pH on vitamin stability in total parenteral nutrition admixtures.JPEN. 1988;12:394-402.

Song P. S. Chemistry of flavins in their excited states, In: H, Kamin ed., Flavins and

Flavoproteins, University Park Press, Baltimore, USA, 1971, Chap. 2.

Song PS, Smith EC, Metzler DE. Photochemical degradation of flavins. IV. The mechanism of alkaline hydrolysis of 6,7-dimethyl-9-formylmethylisoalloxazine. J Am

Chem Soc. 1965;87:4181-4184.

Song WO, Beecher GR, Eitenmiller RR, eds. Modern Analytical Methods in Fat- and

Water-soluble Vitamins, Wiley, New York, 2000, Chap. 7.

Soni H, Singhai AK, Mishra K, Sharma S. Simultaneous determination of vitamins B1, B2 and B6 in multivitamin tablet and biological fluid by RP-HPLC. IJSPR. 2012;40:2163-

2167.

244

Spector R. Riboflavin transport in the central nervous system. characterization and effect of drugs. J Clin Invest. 1980;66:821-831.

Stargrove MB, Jonathan T, Dwight LM. Herb,nutrient and drug interactions: clinical implications and therapeutic strategies. Elsevier Health Science, 2008.

Stargrove MB, Treasure J, McKee Dwight. Herb, Nutrient and Drug Interactions:

Clinical Implications and Therapeutic Strategies. Elsevier Health Sciences, St. Louis,

Missouri, 2008, Chap. Vitamin B2 (Riboflavin).

Steams EI. The practice of absorption spectrophotometry. Wiley-Interscience, New York,

1969, pp. 131-135.

Strauss G, Nickerson WJ. Photochemical cleavage of water by riboflavin. II. role of activators. J Am Chem Soc. 1961;83:3187-3191.

Sue-Chu M, Kristensen S, Tonnesen HH. Influence of lag-time between light exposure and color evaluation of riboflavin in the soliod state. Die Pharmazie. 2008;63:545:546.

Sue-Chu M, Kristensen S, Tonnesen HH. Photoinduced color changes in two different qualities of riboflavin in the solid state and in various tablet formulation photoreactivity of biologically active compounds. XX. Die Pharmazie. 2009;64:428:435.

245

Suelter CH, Metzler DE. The oxidation of a reduced pyridine nucleotide analoque by flavin.Biochim Biophys Acta. 1960;44:23-33.

Svobodova S, Hais J, Kostir J. The influence of pH and light on riboflavin solution.Chemicke Listy. 1953;47:205-212.

Sweetman SC. Martindale: The Complete Drug Reference, 35th ed., Pharmaceutical

Press, London, UK, 2007, Electronic version.

Terekhova IV, Kozbial M, Kumeev RS, Alper GA. Inclusion complex formation between modified cyclodextrins and riboflavin and alloxazine in aqueous solution. J Sol Chem.

2011a;40:1435-1446.

Terekhova IV, Tikhova MN, Volkova TV, Kumeev RS, Perlovich GL. Inclusion complex formation of α- and β-cyclodextrins with riboflavin and alloxazine in aqueous solution: thermodynamic study. J Incl Phenom Macrocyccl Chem. 2011b;69:167-172.

Thomas S, Kumar R, Sharma A, Issarani R, Nagori BP. Stability-indicating HPLC method for determination of vitamins B1, B2, B3 and B6 in pharmaceutical liquid dosage form. IJCT. 2008;15:598-603.

Thompson DF, Saluja HS. The burden of neurological disease in the United States: a

summary report and call to action. Annals of Neurology, 2017;81:479484.

246

Tikhomirova GP, Belenkaia SL, Madievskaia RG, Kurochkina OA. Polarographic determination of vitamin B1, B2, nicotinic acud and nicotinamide in the presences of high vitamin C content. Vopr Pitan. 1965;24:32-37.

Tollin G. Use of flavin photochemistryto probe intraprotein and intraprotein electron transfer mechanisms.J Bionerg Biomembr. 1995;27: 303-309.

Tonnesen HH, Karlsen J. A comment on photostability testing according to ICH guidelines: calibration of light sources. PharmEuropa.1997; 9:735-736.

Tonnesen HH. ed. The Photostability of Drugs and Drug Formulations, Culinary and

Hospitality Industry Publications Services, Weimer, TX, 2004.

Tonnesen HH. Formulation and stability testing of photolabile drugs. Int J Pharm.

2001;225:1-14.

Treadwell GE, Cairns WL, Metzler DE. Photochemical degradation of flavins: V.

Chromatographic studies of the products of phototlysis of riboflavin. J. Chromatogr A.

1968;35:376-388.

Tsutsumi M. The pharmacological action of flavin adenine dinucleotide.J Vitaminol.

1957;3:123-128.

247

Udenfriend S. Fluorescence Assay in Biology and Medicine, Vol. 2, Academic Press,

New York, 1969, pp. 297-304.

Ugarde R, Edwards AM., Diez MS, Valenzuela A, Silva E. Riboflavinsensitized anaerobic modification of rat lens proteins.A correlation with age-related changes. J

Photochem Photobiol B Biol. 1992;13:161-168.

United States Pharmacopeia 30 / National Formulary 25.United States Pharmacopeial

Convention, Inc., Rockville, MD, 2007, Electronic version.

United States Pharmacopeia 30/National Formulary 25, United States Pharmacopeial

Convention, Inc., Rockville, MD, 2016 Electronic version.

Unna K, Greslin JG.Studies on the toxicity and pharmacology of riboflavin.J Pharmacol

Exp Thera. 1942;76:75-80.

Vaid FHM, Gul W, Faiyaz A, Anwar Z, Ejaz MA, Zahid S, Ahmad I.Divalent anion catalyzed photodegradation of riboflavin: A kinetic study, J Photochem Photobiol. A:

Chem. 2019;371:59-66.

Vaid FHM, Shaikh RH, Ansari IA, Ahmad I. Spectral study of the photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the presence and absence of riboflavin.J Chem Soc Pak. 2005; 27:227-232.

248

Vanish SP, Tollin G. Flash photolysis of flavins. V. oxidation and disproportion of flavin radicals.J Bioenerg Biomembr. 1971;2:61-72.

Vanish SP, Tollin G. Flash photolysis of flavins. V. oxidation and disproportionation of flavin radicals.J Bioenerg Biomembr. 1971;2:61-72.

Varnes AW, Dodson RB, Wehry EL. Interactions of transient–metal ions with photoexcited states of flavins.Fluorescence quenching studies, J Am Chem Soc. 1971;

94:946–950.

Vidovic S, Stojanovic B, Veljkovic J, Prazic-Arsic L, Roglic G, Manojlovic D.

Simultaneous determination of some water-soluble vitamins and preservatives in multivitamin syrup by validated stability-indicating high-performance liquid chromatography method. J Chromatogr A. 2008;1202: 155-162.

Vinas P, Balsalobre N, Lopez-Erroz C, Hernandez-Cordoba M. Liquid chromatographic analysis of riboflavin vitamers in foods using florescence detection. J Agric Food Chem.

2004;52:1789-1794.

Viteri G, Edwards AM, De la Fuente J, Silva E. Study of the interaction between triplet riboflavin and the α-, βH- and βL-crystallins of eye lens. Photochem Photobiol.

2003;77:535-540.

249

Wade TD, Fritchie Jr. CJ. The crystal structure of a riboflavin-metal complex: The riboflavin silver perchlorate hemihydrates.JBiol Chem. 1973;248:2337-2343.

Wagner AF, Folkers K.Vitamins and Coenzymes, Interscience, London, 1964, Chap. 4.

Wagner-Jauregg TH. In: Sebrell WH, Harris RS. eds., The Vitamins, Academic Press,

New York,1954, pp. 99-402.

Walsh C. Flavin coenzymes: at the crossroad of biological redox chemistry. Acc Chem

Res. 1980; 13: 148-155.

Wang J. M, Lei B. Proteomic analysis of retinal protein in rabbits following intravitreal

PBS injection: analysis with tendem mass tag labeling coupled with LC-MS/MS. Nan

Fang Yi Ke Da Xue Xue Bao. 2016;36:595-603.

Wang Y, Zhu PH, Tian T, Tang J, Wang L, Hu XY. Synchronous fluorescence as a rapid method for the simultaneous determination of folic acid and riboflavin in nutritional beverages.J Agric Food Chem. 2011; 59: 12629-12634.

Warburg O, Walter C. On a new oxidation enzyme and its absorption spectrum.Biochem.

1932; Z.254:438-458.

250

Watson DG. Pharmaceutical Analysis, 2nd ed., Elsevier Churchill Livingstone, 2005,p.

153.

Weber G. Fluorescence of riboflavin and flavin adeninedinucleotide, Biochem J.

1950;47:114–121.

Wehling RL, Wetzel DL. Simultaneous determination of pyridoxine, riboflavin and thiamine infortified cereal products by high-performance liquid chromatograpgy. J Agric

Food Chem. 1984;32:1326-1331.

Wilder LC, Baker SE. Quantification of riboflavin in human urine using high- performance liquid chromatography-tandem mass spectrometry. J Chromatogr B:Analyt

Technol Biomed Life Sci. 2011;879:1823-1826.

Williams RJ, Eakon RE, Beerstecher E Jr, Shine W. The Biochemistry of B Vitamins,

Reinhold, New York, 1950, Chap. 9D.

Xu P, Qiao C,Yang S, Liu L, Wang M, Zhang J. Fast determination of vitamin B2 based on molecularly imprinted electrochemical sensor. Engineering. 2012;4:129-134.

Yagi K. and Yamada S. Determination of riboflavib-5’-monosulphate. Methods in

Enzymology. 1971;465-467.

251

Yamaoka K, Nakajima Y, Terashima T. Stability of vitamins in TPN mixture in a double- chambered bag. Japan J Hosp Pharm. 1995;21:357-364.

Yao T, Rechnitz GA. Potentiometric biosensor for riboflavin based on the use of aporiboflavin- protein. Anal Chem. 1987;59:2115-2118.

Yoneda F. Riboflavin (B2). Kirk-Othmer Encyclopedia of Chemical Technology, 1984.

Yoshioka S, Stella VJ. Stability of drugs and dosage forms. New York: Kluwer

Academic/Plenum Publishers; 2000. pp. 102–104.

Yoshioka S, Stella VJ. Stability of Drugs and Dosage Forms.Kluwer Academic / Plenum

Publishers.New York, 2000, Chap. 2.

Zand N, Chowdhy BZ, Pullen FS, Snowden MJ, Tetteh J. Simultaneous dtermination of riboflavin and pyridoxine by UHPLC/LC-MS in UK commercial meal food products.

Food Chem. 2012;135:2743-2749.

Zandomeneghi M, Carbonaro L, Calucci L, Pinzino C, Galleschi L, Ghiringhelli S. Direct fluorimetric determination of fluorescent substances in powders: the case of riboflavin in cereal flours. J Agric Food Chem. 2003;51:2888-2895.

Zandomeneghi M, Carbonaro L, Zandomeneghi G. Biochemical fluorimetric method for the determination of riboflavin in milk. J Agric Food Chem.2007;55:5990-5994.

252

Zempleni J, Galloway JR, McCormick DB.Pharmacokinetics of orally and intravenously administered riboflavin in healthy humans.Am J Clin Nutr. 1996;63:54-66.

Zhan XC, Yin GK. Obtaining natural light photostability of drugs from lamp light exposure experiments.Acta Pharm Sin. 1992;27:544-548.

Zhao H, Ge M, Zang Z, Wang W, Wu G. Spectroscopic studies on the interaction between riboflavin and albumins. Spectrochim Acta A Mol Biomol Spectrosc.

2006;65:811-817.

Zhu L, Luo D. Determination of riboflavin using the polarographic reduction wave of its photochemical reaction products.Anal Lett. 2010;41: 3138-3146.

Ziak L, Majek P, Hrobonova K, Cacho F, Sadecka J. Simultaneous determination of caffeine, caramel and riboflavin in cola-type and energy drinks by synchronous fluorescence technique coupled with partial least square. Food Chem. 2014;159:282-286.

Zimmer AJ, Huyck CL. Determination of riboflavin by light absorption and polarographic methods. J Pharm Sci. 1955;44:344-348.

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BIODATA

Wajiha Gul

B.Pharm. 2000, Faculty of Pharmacy, University of Karachi

M. Phil. 2012, Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi

Current position: Working as an Asst. Professor at Dept. Pharmaceutical Chemistry, Dow College of Pharmacy, Dow University of Health Sciences, Karachi.

PUBLICATIONS

 Faiyaz H. M.Vaid, Wajiha Gul, AmbreenFaiyaz, Zubair Anwar, Mohammad AhsanEjaz, SaimaZahid, Iqbal Ahmad (2018). Divalent anion catalyzed photodegradation of riboflavin: a kinetic study, Journal of Photochemistry and Photobiology A: Chemistry, 371:59-66.  WajihaGul, Faiyaz Hussain Madni Vaid, AmbreenFaiyaz, Zubair Anwar, AdeelaKhurshid, Iqbal Ahmad (2019). Simultaneous Photoaddition, Photoreduction and Chemical Reduction of Riboflavin by Sulfur Containing Dianions: A Kinetic Study, Journal of Photochemistry and Photobiology A: Chemistry, https://doi.org/10.1016/j.jphotochem.2019.02.035  Wajiha Gul, Saima Zahid, Shaheen Perveen, Iqbal Ahmed (2014). ‘Methods of analysis of thiamine: A review’, JPPS, 2(1); 39-47.  Wajiha Gul, Zubair Anwar, Kiran Qadeer, Shaheen Perveen, Iqbal Ahmed (2014).

‘Methods of analysis of Riboflavin (vitamin B2): a review’, JPPS, 2(2); 10-21.  Wajiha Gul, Zubair Anwar, Kiran Qadeer, Shaheen Perveen, Iqbal Ahmad (2015). ‘Methods of analysis of vitamin K: A review’, JPPS, 2(3);14-22.  Wajiha Gul, Zubair Anwar, Adeela Khurshid, Aqeela Khurshid, Iqbal Ahmad (2016). Ascorbic acid: methods of analysis, JPPS, 3(2); 1-18.  Iqbal Ahmad, Faiyaz Vaid, Saima Zahid, Wajiha Gul (2018). Photolysis of methylcobalamine in aqueous solution: a kinetic study, Journal of Photochemistry and Photobiology A: Chemistry, 362:40-48.

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 Mohammad Sheraz, Iqbal Ahmad, HaiderBukhari, Zubair Anwar, Sofia Ahmed, Mohammad Furqan, Nafeesa Mustaan, Wajiha Gul (2018). Stability-Indicating Photochemical Method for the Assay of Thiamine by Spectrophotometry, (submitted).  Shaheen Perveen, Rabia Ismail Yousuf, Gauhar S, Wajiha Gul, Sheikh AF (2018). Simultaneous determination of amoxicillin and ranitidine in human plasma by RP- HPLC method, International Journal of Biology, Pharmacy and Allied Sciences (IJBPAS), 7(1); 2018.

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