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Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution
Journal of Photochemistry and Photobiology B: Biology 82 (2006) 21–27 www.elsevier.com/locate/jphotobiol
Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution Iqbal Ahmad *, Q. Fasihullah, Faiyaz H.M. Vaid Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan Received 17 January 2005; received in revised form 3 August 2005; accepted 18 August 2005 Available online 11 October 2005
Abstract A study of the effect of light intensity and wavelengths on photodegradation reactions of riboflavin (RF) solutions in the presence of phosphate buffer using three UV and visible radiation sources has been made. The rates and magnitude of the two major photodegradation reactions of riboflavin in phosphate buffer (i.e., photoaddition and photoreduction) depend on light intensity as well as the wavelengths of irradiation. Photoaddition is facilitated by UV radiation and yields cyclodehydroriboflavin (CDRF) whereas photoreduction results from normal photolysis yielding lumichrome (LC) and lumiflavin (LF). The ratios of the photoproducts of the two reactions at 2.0 M phosphate concentration, CDRF/RF (0.09–0.22) and CDRF/LC (0.54–1.75), vary with the radiation source and are higher with UV radiation than those of the visible radiation. On the contrary, the ratios of LF/LC (0.15–0.25) increase on changing the radiation source from UV to visible. The rate is much faster with UV radiation causing 25% degradation of a 105 M riboflavin solution in 7.5 min compared to that of visible radiations in 150–330 min. 2005 Elsevier B.V. All rights reserved. Keywords: Riboflavin; Photolysis; Photoaddition; Buffer effect; Light intensity and wavelength effect
1. Introduction A large number of drug substances absorb radiations in the ultraviolet and/or visible region and are thus sensitive to light. They undergo photodegradation in liquid media or in the solid state on exposure to light [1–7]. The nature and magnitude of photochemical reactions depend upon the intensities and wavelengths of light [8–14] and control of these factors is critical in photostability studies of drugs and drug formulations [15–18]. Various ultraviolet and visible radiation sources [19–29] have been used to study the photodegradation of riboflavin and analogues. Riboflavin (RF) undergoes simultaneous photoaddition and photoreduction reactions in phosphate buffer giving rise to cyclodehydroriboflavin (CDRF), and formylmethylflavin * Corresponding author. Present address: Dubai Pharmacy College, P.O. Box 19099, Dubai, United Arab Emirates. Tel.: +971 4 264 6968; fax: +971 4 264 6740. E-mail address: [email protected] (I. Ahmad).
1011-1344/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.08.004
(FMF), lumichrome (LC) and lumiflavin (LF), respectively [20,25]. The influence of UV and visible radiation sources on the variations in degradation product distribution and reaction rates of some vitamins [30–35], other drugs [36– 38], herbicides [39,40], and polymers [41] has been studied. The present work involves a quantitative study of the photodegradation reactions of riboflavin in the presence of phosphate buffer using three different radiation sources to evaluate the effect of light intensity and wavelengths on degradation product distribution and kinetics of the reactions. Some related work on these reactions has recently been reported [25,26]. 2. Materials and methods The materials and methods of photolysis using a 125 W medium pressure mercury vapour lamp (MP lamp) (Applied Photophysics Ltd., UK) [emission at 254, 313, 366 and 436 nm (Fig. 1) corresponding to the wavelengths of RF absorption (Fig. 2)], thin-layer chromatography of
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I. Ahmad et al. / Journal of Photochemistry and Photobiology B: Biology 82 (2006) 21–27
Fig. 1. Spectral emission of 125 W medium pressure mercury vapour lamp.
Fig. 2. Absorption spectrum of 5 · 105 M riboflavin at pH 7.0 (phosphate buffer).
photolysed solutions, and spectrophotometric assay of RF and photoproducts have previously been described [25]. The following method of photolysis was also used in this study. 2.1. Photolysis with visible lamps An aqueous solution of riboflavin (104 M, 200 ml) containing 0.05–2.00 M Na2HPO4 (adjusted to pH 7.0) was placed in a 250 ml volumetric flask (Pyrex) and irradiated with a Philips HPLN 125 W high pressure mercury vapour fluorescent lamp (HP lamp) [emission at 405 and 436 nm (Fig. 3) corresponding to the wavelengths of RF absorption], or a Philips 150 W tungsten lamp (TN lamp) (contin-
uum over the range 350–2000 nm) (Fig. 4) fixed at a distance of 30 cm from the centre of the flask. The temperature of the solution was maintained at 25 ± 1 C during irradiation. The solution was continuously stirred by bubbling a stream of air into the flask. Samples were withdrawn at appropriate intervals for chromatography and assay. 2.2. Light intensity measurements The intensities of the MP, HP and TN lamps were determined by potassium ferrioxalate actinometry [42] under the conditions described for the MP lamp [25] and for the HP and TN lamps (Section 2.1).
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Fig. 3. Spectral emission of 125 W high pressure mercury vapour fluorescent lamp.
Fig. 4. Spectral emission of Philips 150 W tungsten lamp.
2.3. Determination of spectral energy The energy, E, emitted by the radiation sources at various wavelengths was calculated using the equation E ðk J mol1 Þ ¼
N A hc ; knm
where NA is AvogadroÕs number = 6.0226 · 1023 mol1, h is PlanckÕs constant = 6.6256 · 1034 J s, c is velocity of light = 2.9979 · 108 m s1, k is wavelength in nm = 109 m. 3. Results and discussion 3.1. Photoproducts of riboflavin with visible radiation sources The major photoproducts of RF in aqueous solution (pH 7.0) containing 0.05–2.00 M Na2HPO4, on exposure to visible radiation sources, were identified by thin-layer chromatography as CDRF, FMF, LC, and LF. These
products have been detected earlier on UV irradiation of the RF solutions [25]. However, the magnitude of the formation of these products depends upon the extent of photolysis in a fixed period of time and is higher with the UV lamp (MP) compared to that of the visible lamps (HP and TN). 3.2. Product distribution The product distribution during the photodegradation of RF solutions (2.0 M phosphate) at 25% photolysis (7.5–330 min) and at 60 min irradiation (5.5–67% loss) using the UV and visible radiation sources (MP, HP, TN lamps) is given in Table 1. The wide exposure range is required to produce an equal amount of photolysis (25%) of RF solutions using the MP (7.5 min), HP (150 min) and TN (330 min) lamps. The plots of the values of different product ratios (CDRF/RF, CDRF/LC, LF/LC) versus phosphate concentration are shown in Fig. 5 and discussed individually in the following sections.
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Table 1 Product distribution at 25% photolysis and 60 min irradiation of riboflavin solutions (pH 7.0) in presence of 2.0 M phosphate Lamp
MP HP TN MP HP TN
Photolysis
RF
CDRF 5
FMF 5
LC
LF
min
(M · 10 )
(M · 10 )
(M · 10 )
(M · 10 )
(M · 10 )
(M · 105)
25 25 25 67 13 5.5
7.5 150 330 60 60 60
7.5 7.5 7.5 3.27 8.70 9.45
1.65 0.95 0.66 3.05 0.45 0.22
– 0.28 0.53 1.18 0.22 0.07
0.94 1.20 1.22 2.37 0.75 0.38
0.14 0.23 0.31 0.30 0.12 0.10
10.23 10.16 10.22 10.17 10.24 10.22
3.2.1. CDRF/RF ratios The CDRF/RF ratios gradually increase with an increase in phosphate concentration indicating the degradation of RF in favour of photoaddition reaction (Fig. 5). These ratios for the three radiation sources appear in the order MP lamp > HP lamp > TN lamp suggesting that for a fixed amount of RF degradation (i.e., 25%) the ratios are greater for the MP lamp (UV emission)
5
Total
%
Fig. 5. Product ratios of riboflavin and photoproducts as a function of phosphate concentration. CDRF/RF (~), CDRF/LC (s), LF/LC (h).
5
5
compared to those of the HP and TN lamps (visible emission). The UV radiation exerts much greater effect on RF degradation than that of the visible radiation since 25% degradation (2.0 M phosphate) is achieved in 7.5, 150 and 330 min by MP, HP and TN lamps, respectively. The high intensity UV emission of MP lamp at 254 and 366 nm closely corresponds to the 266 and 373 nm absorption maxima of RF [43] and has higher spectral energy (determined as 407 kJ mol1) than that of the low intensity visible emission of HP lamp at 436 nm (determined as 148 kJ mol1) corresponding to the 445 nm absorption of RF [43]. Therefore, the absorption of UV radiation from MP lamp (254 and 366 nm) by RF causes 25% photolysis in 7.5 min compared to that of the visible light of the HP lamp (435 nm) in 150 min and that of the TN lamp in 330 min. A further explanation to the values obtained for CDRF/ RF ratios may be offered on the basis of the fact that CDRF formation by intramolecular photoaddition is mediated by the excited singlet state ð1 Flox Þ whereas FMF, and hence the LC and LF formation by normal photolysis or intramolecular photoreduction occurs via the excited triplet state ð3 Flox Þ [20]. Since on UV irradiation from MP lamp at 254 and 366 nm a greater number of RF molecules are excited to the 1 Flox state compared to those on visible irradiation at 405 and 436 nm, there is a greater probability of the conversion of this state to CDRF in preference to non-radiative processes including intersystem crossing [44] to form the 3 Flox state which yields FMF and subsequently, by hydrolysis, LC and LF [45]. In normal photolysis the formation of LC directly from RF may also take place via the 1 Flox state [19,26]. The degradation of RF on irradiation with TN lamp (continuum in the visible region) is much slower than that of the MP lamp (1/44th) and HP lamp (1/20th) and, therefore, CDRF/RF values are lowest in this case. This may be due to the fact that in TN lamp (Fig. 4) no distinct wavelengths are available for absorption by RF (maximum 445 nm) and the overall energy in the desired region (400–500 nm) is relatively low. 3.2.2. CDRF/LC ratios The CDRF/LC values for the photodegradation reactions using the three radiation sources, considered as a measure of the ratios of the rates of the two reactions
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Table 2 Second-order rate constants for phosphate ions-catalysed photodegradation of riboflavin (pH 7.0) using different radiation sources Radiation source
Total no. of quanta per seconda
No. of quanta absorbed by riboflavin solutionb
k (M1 s1)
Correlation coefficient
125 W medium pressure mercury vapour lamp (MP lamp) 125 W high pressure mercury vapour fluorescent lamp (HP lamp) 150 W Tungsten lamp (TN lamp)c
2.19 ± 0.12 · 1018 1.14 ± 0.10 · 1017 1.06 ± 0.11 · 1016
1.7 · 1018 6.2 · 1016 1.1 · 1016
2.12 · 104 1.37 · 105 6.80 · 106
0.999 0.999 0.999
a
Mean ± SD, n = 3–5. Values obtained by multiplying the total no. of quanta with the % spectral energy corresponding to the wavelengths absorbed by riboflavin in each case. c It gives a continuum in the visible region (Fig. 4) and, therefore, the % spectral energy corresponding to the wavelengths absorbed by riboflavin could not be calculated. b
(photoaddition and photoreduction), are also in the same order as those of the CDRF/RF ratios. The values of CDRF/LC ratios are about 4–8 fold higher than those of the CDRF/RF ratios and increase with an increase in phosphate concentration. In the case of MP lamp, the CDRF/LC ratios are a linear function of phosphate concentration indicating the formation of CDRF and LC by parallel first-order reactions as previously reported [25]. However, the CDRF/LC ratios for the HP and TN lamps are not a linear function of phosphate concentration. This is probably due to relatively low concentrations of CDRF, compared to those of LC, in the CDRF/LC ratios and a clear indication of the fact that irradiation wavelengths have a prominent role in the product distribution of RF photodegradation. Schuman Jorns et al. [20] used a high intensity 250 W medium pressure mercury lamp with UV emission and reported a CDRF/LC ratio of 3/1 for the photoreactions of RF in 0.5 M phosphate solution at pH 7.0 which would be much higher in a 2.0 M phosphate solution. The UV radiation would facilitate RF excitation to the 1 Flox state and its subsequent conversion to CDRF at the expense of the 3 Flox state leading to LC and hence the higher CDRF/LC values. It may be concluded from the present study and the observations of Schuman Jorns et al. [20] on the photoreactions of RF in phosphate buffer that the irradiation wavelengths significantly influence its mode of degradation and hence the product distribution. 3.2.3. LF/LC ratios The values of LF/LC ratios for the individual lamps are almost constant and do not appear to be affected by an increase in phosphate concentration supporting the view that in phosphate buffer LF and LC are formed through FMF via the 3 Flox state [20] and are independent of phosphate concentration. The order of LF/LC ratios
3.3. Rate constants for photodegradation of RF The second-order rate constants for the photodegradation of RF at pH 7.0 in the presence of phosphate ions, obtained by using the UV and visible radiation sources, are reported in Table 2. The total number of quanta s1 emitted by each radiation source and the number of quanta absorbed by RF solution are also reported in Table 2. The values of rate constants for the three radiation sources are of the same order as those of the number of quanta s1 emitted by these sources, respectively. The values of the second-order rate constants indicate that the rate of reaction with MP lamp is about 15–30 fold higher than that of the HP and TN lamps. A better explanation of the kinetic data obtained using the three radiation sources may be provided by considering the values of the overlap integrals between the absorption spectra of RF and the emission spectra of the lamps. The overlap integral is a constant for a particular combination of photon source and absorbing substance and determines the rate of a photochemical reaction. The rate is expressed as the number, N, of molecules transformed per second and is a function of the photons absorbed per second · the quantum yield, u, of the reaction (i.e., Nu) [46]. Since the line emission spectra of the MP and HP lamps (Figs. 1 and 3) only indicate the relative energy distribution of the individual wavelengths involved, and the emission spectrum of the TN lamp (Fig. 4) is a continuum, there are no uniform emission characteristics of these sources to be considered for comparison. It is, therefore, not possible to calculate the overlap integrals between the riboflavin absorption spectra and the lamp emission spectra to enable the interpretation of the kinetic data on the basis of overlap integrals.
TN lamp > HP lamp > MP lamp
4. Conclusion
is inverse of the ratios of CDRF/LC obtained for the three lamps indicating that in phosphate buffer the major pathway of RF degradation, on irradiation with MP lamp, is intramolecular photoaddition which may require more energy than that of the intramolecular photoreduction. The later reaction can easily occur in the presence of visible light and is the major cause of RF instability in aqueous solution.
The present study shows the effect of light intensity and wavelengths in the UV and visible region on the mode of photodegradation of RF in phosphate buffer as well as the degradation product distribution of the two reactions involved (i.e., photoaddition and photoreduction). The pattern of the ratios of major products of these reactions, i.e., CDRF/RF, CDRF/LC and LF/LC indicates significant variations on a change from UV to visible radiation.
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This is probably due to the nature and intensity of the wavelengths absorbed, formation of the specific excited states (i.e., singlet and triplet states) and their involvement in initiating a particular reaction [26]. The magnitude of second-order rate constants for the phosphate ions-catalysed photodegradation of RF (0.68–21.20 · 105 M1 s1) using different radiation sources also reflects the influence of light intensity (i.e., number of quanta s1) and wavelengths (UV or visible) on the rates of the reaction. Such information may be utilised for the photochemical preparation or yield optimisation of a specific product and to evaluate the photostability of pharmaceutical products during formulation development [18,47]. References [1] J.V. Greenhill, M.A. McLelland, Photochemistry of drugs in vitro and in vivo, in: G.P. Ellis, J.B. West (Eds.), Progress in Medicinal Chemistry, Elsevier, Amsterdam, 1990, pp. 51–121. [2] H.H. Tonnesen, Photodecomposition of drugs, in: J. Swarbrick, J.C. Boylan (Eds.), Encyclopedia of Pharmaceutical Technology, second ed., Dekker, New York, 2002, pp. 2197–2203. [3] H.H. Tonnesen (Ed.), The Photostability of Drugs and Drug Formulations, second ed., Culinary and Hospitality Industry Publications Services, Weimer, Texas, 2004. [4] A.C. Boreen, W.A. Arnold, K. McNeill, Photodegradation of pharmaceuticals in the aquatic environment: a review, Aquat. Sci. 65 (2003) 320–341. [5] G. Cosa, Photodegradation and photosensitization in pharmaceutical products: assessing drug phototoxicity, Pure Appl. Chem. 76 (2004) 263–275. [6] B.D. Glass, C. Novak, M.E. Brown, The thermal and photostability of solid pharmaceuticals: a review, J. Therm. Anal. Cal. 77 (2004) 1013–1036. [7] S. Singh, M. Bakshi, Guidance on conduct of stress tests to determine inherent stability of drugs, Pharm. Technol. On-Line (India) 24 (2000) 1–14. [8] E.R. Garrett, Kinetics and mechanisms in stability of drugs, in: H.S. Bean, A.H. Beckett, J.E. Carless (Eds.), Advances in Pharmaceutical Sciences, vol. 2, Academic Press, London, 1967, p. 2. [9] C.J. Linter, Pharmaceutical product stability, in: M.S. Cooper (Ed.), Quality Control in the Pharmaceutical Industry, vol. 2, Academic Press, New York, 1973 (Chapter 4). [10] L. Lachman, P. Deluca, M.J. Akers, Kinetic principles and stability testing, in: L. Lachman, H.A. Lieberman, J.L. Kanig (Eds.), The Theory and Practice of Industrial Pharmacy, third ed., Lea & Febiger, Philadelphia, 1986 (Chapter 26). [11] K.J. Laidler, Chemical Kinetics, third ed., Harper & Row, New York, 1987 (Chapter 9). [12] J.K. Guillory, R.I. Poust, Chemical kinetics and drug stability, in: G.S. Banker, C.T. Rhodes (Eds.), Modern Pharmaceutics, fourth ed., Marcel Dekker, New York, 2002 (Chapter 6). [13] A. Martin, Physical Pharmacy, fourth ed., Waverly International, Baltimore, MD, 1993 (Chapter 12). [14] J. Boxhammer, Technical requirements and equipment for photostability testing, in: H.H. Tonnesen (Ed.), The Photostability of Drugs and Drug Formulations, Taylor & Francis, London, 1996 (Chapter 3). [15] N.H. Anderson, D. Johnston, M.A. McLelland, P. Munden, Photostability testing of drug substances and drug products in UK pharmaceutical laboratories, J. Pharm. Biomed. Anal. 9 (1991) 443–449. [16] H.H. Tonnesen, D.E. Moore, Photochemical degradation of components in drug formulations, Pharm. Technol. 5 (1993) 27–33.
[17] H.H. Tonnesen, J. Karlsen, Photochemical degradation of components in drug formulations. A discussion of experimental conditions, PharmEuropa 7 (1995) 137–141. [18] H.H. Tonnesen, J. Karlsen, A comment on photostability testing according to ICH guidelines: calibration of light sources, PharmEuropa 9 (1997) 735–736. [19] W.L. Cairns, D.E. Metzler, Photochemical degradation of flavins. VI. A new photoproduct and its use in studying the photolytic mechanism, J. Am. Chem. Soc. 93 (1971) 2772–2777. [20] M. Schuman Jorns, G. Schollnhammer, P. Hemmerich, Intramolecular addition of the riboflavin side chain. Anion-catalysed neutral photochemistry, Eur. J. Biochem. 57 (1975) 35–48. [21] Y. Sato, M. Yoko, S. Takahashi, T. Takahashi, Biphasic photolysis of riboflavin with a low-intensity light source, Chem. Pharm. Bull. (Japan) 30 (1982) 1803–1810. [22] P.F. Heelis, G.O. Phillips, I. Ahmad, H.D.C. Rapson, The photodegradation of formylmethylflavin – a steady state and laser flash photolysis study, Photobiochem. Photobiophys. 1 (1980) 125–130. [23] I. Ahmad, H.D.C. Rapson, Multicomponent spectrophotometric assay of riboflavin and photoproducts, J. Pharm. Biomed. Anal. 8 (1990) 217–223. [24] I. Ahmad, Q. Fasihullah, A. Noor, I.A. Ansari, Q.N.M. Ali, Photolysis of riboflavin in aqueous solution: a kinetic study, Int. J. Pharm. 280 (2004) 199–208. [25] I. Ahmad, Q. Fasihullah, F.H.M. Vaid, A study of simultaneous photolysis and photoaddition reactions of riboflavin in aqueous solution, J. Photochem. Photobiol. B: Biol. 75 (2004) 13–20. [26] I. Ahmad, Q. Fasihullah, F.H.M. Vaid, Effect of phosphate buffer on photodegradation reactions of riboflavin in aqueous solution, J. Photochem. Photobiol. B: Biol. 78 (2005) 229–234. [27] I. Ahmad, W. Hussain, Multicomponent spectrophotometric assay of cyanocobalamin, hydroxocobalamin and riboflavin, Pak. J. Pharm. Sci. 5 (2) (1992) 121–127. [28] M.J. Akhtar, M.A. Khan, I. Ahmad, Photodegradation of folic acid in presence of riboflavin in aqueous solution, J. Pharm. Biomed. Anal. 23 (2000) 1039–1044. [29] E. Haggi, S. Bertolotti, S. Miskoski, F. Amat-Guerri, N.A. Garcia, Environmental photodegradation of pyrimidine fungicides-kinetics of the visible-light-promoted interactions between riboflavin and 2amino-4-hydroxy-6-methylpyrimidine, Can. J. Chem. 80 (2002) 62– 67. [30] L.J. DeMerre, C. Wilson, Photolysis of vitamin B12, J. Am. Pharm. Assoc. Sci. Ed. 45 (1956) 129–134. [31] R.M. Wilson, T.F. Walsh, S.K. Gee, Laser photochemistry: the wavelength dependent oxidative photodegradation of vitamin K analogs, Tetrahedron Lett. 21 (1980) 3459–3463. [32] M.C. Allwood, J.H. Plane, The wavelength dependent degradation of vitamin A exposed to ultraviolet radiation, Int. J. Pharm. 31 (1986) 1– 7. [33] D. Brousmiche, P. Wan, Photogeneration of an o-quinone methide from pyridoxine (vitamin B6) in aqueous solution, Chem. Commun. (1998) 491–492. [34] A.H. Thomas, G. Suarez, F.M. Cabrerizo, F.S. Garcia Einschlag, R. Martino, C. Baiocchi, E. Pramauro, A.L. Capparelli, Photochemical behaviour of folic acid in alkaline aqueous solutions and evolution of its photoproducts, Helv. Chim. Acta 85 (2002) 2300–2315. [35] N. Failloux, I. Bonnett, E. Perrier, M.-H. Baron, Effect of light, oxygen and concentration on vitamin A1, J. Raman Spectrosc. 35 (2004) 140–147. [36] S.L. Ali, Nifedipine, in: K. Florey (Ed.), Analytical Profiles of Drug Substances 18 (1989) 221–288. [37] R. Teraoka, Y. Konishi, Y. Matsuda, Photochemical and oxidative degradation of the solid-state tretinoin tocoferil, Chem. Pharm. Bull. 49 (2001) 368–372. [38] J. Kochansky, M. Nasi, Laboratory studies on the photostability of fumagillin, the active ingredient of Fumadil B, Apidologie 35 (2004) 301–310.
I. Ahmad et al. / Journal of Photochemistry and Photobiology B: Biology 82 (2006) 21–27 [39] A. Amine-Khodja, A. Boulkamh, P. Boule, Photochemical behaviour of phenylurea herbicides, Photochem. Photobiol. Sci. 3 (2004) 145–156. [40] I.K. Konstantinou, A.K. Zarkadis, T.A. Albanis, Organic compounds in the environment. Photodegradation of selected herbicides in various natural waters and soils under environmental conditions, J. Environ. Qual. 30 (2001) 121–130. [41] A. Rivalon, J.-L. Gardette, Photooxidation of aromatic polymers, Angew. Makromol. Chem. 261-262 (1999) 173–188. [42] C.G. Hatchard, C.A. Parker, A new sensitive chemical actinometer II. Potassium ferrioxalate as a standard chemical actinometer, Proc. Roy. Soc. (Lond.) A 235 (1956) 518–536. [43] R.M.C. Dawson, D.C. Elliott, W.H. Elliott, K.M. Jones, Data for Biochemical Research, third ed., Clarenden Press, Oxford, 1986, p. 137.
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[44] D.E. Moore, Photophysical and photochemical aspects of drug stability, in: H.H. Tonnesen (Ed.), Photostability of Drugs and Drug Formulations, Tayler & Francis, London, 1996, pp. 16– 17. [45] I. Ahmad, H.D.C. Rapson, P.F. Heelis, G.O. Phillips, Alkaline hydrolysis of 7,8-dimethyl-10-(formylmethyl) isoalloxazine. A kinetic study, J. Org. Chem. 45 (1980) 731–733. [46] D.E. Moore, Standardisation of photodegradation studies and kinetic treatment of photochemical reactions, in: H.H. Tonnesen (Ed.), Photostability of Drugs and Drug Formulations, Tayler & Francis, London, 1996, pp. 74–75. [47] M.A. Geigel, The stability testing program, in: C. Medina (Ed.), Compliance Handbook for Pharmaceuticals, Medical Devices, and Biologics, Marcel Dekker, New York, 2004, p. 95.
Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution Iqbal Ahmad *, Q. Fasihullah, Faiyaz H.M. Vaid Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan Received 17 January 2005; received in revised form 3 August 2005; accepted 18 August 2005 Available online 11 October 2005
Abstract A study of the effect of light intensity and wavelengths on photodegradation reactions of riboflavin (RF) solutions in the presence of phosphate buffer using three UV and visible radiation sources has been made. The rates and magnitude of the two major photodegradation reactions of riboflavin in phosphate buffer (i.e., photoaddition and photoreduction) depend on light intensity as well as the wavelengths of irradiation. Photoaddition is facilitated by UV radiation and yields cyclodehydroriboflavin (CDRF) whereas photoreduction results from normal photolysis yielding lumichrome (LC) and lumiflavin (LF). The ratios of the photoproducts of the two reactions at 2.0 M phosphate concentration, CDRF/RF (0.09–0.22) and CDRF/LC (0.54–1.75), vary with the radiation source and are higher with UV radiation than those of the visible radiation. On the contrary, the ratios of LF/LC (0.15–0.25) increase on changing the radiation source from UV to visible. The rate is much faster with UV radiation causing 25% degradation of a 105 M riboflavin solution in 7.5 min compared to that of visible radiations in 150–330 min. 2005 Elsevier B.V. All rights reserved. Keywords: Riboflavin; Photolysis; Photoaddition; Buffer effect; Light intensity and wavelength effect
1. Introduction A large number of drug substances absorb radiations in the ultraviolet and/or visible region and are thus sensitive to light. They undergo photodegradation in liquid media or in the solid state on exposure to light [1–7]. The nature and magnitude of photochemical reactions depend upon the intensities and wavelengths of light [8–14] and control of these factors is critical in photostability studies of drugs and drug formulations [15–18]. Various ultraviolet and visible radiation sources [19–29] have been used to study the photodegradation of riboflavin and analogues. Riboflavin (RF) undergoes simultaneous photoaddition and photoreduction reactions in phosphate buffer giving rise to cyclodehydroriboflavin (CDRF), and formylmethylflavin * Corresponding author. Present address: Dubai Pharmacy College, P.O. Box 19099, Dubai, United Arab Emirates. Tel.: +971 4 264 6968; fax: +971 4 264 6740. E-mail address: [email protected] (I. Ahmad).
1011-1344/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.08.004
(FMF), lumichrome (LC) and lumiflavin (LF), respectively [20,25]. The influence of UV and visible radiation sources on the variations in degradation product distribution and reaction rates of some vitamins [30–35], other drugs [36– 38], herbicides [39,40], and polymers [41] has been studied. The present work involves a quantitative study of the photodegradation reactions of riboflavin in the presence of phosphate buffer using three different radiation sources to evaluate the effect of light intensity and wavelengths on degradation product distribution and kinetics of the reactions. Some related work on these reactions has recently been reported [25,26]. 2. Materials and methods The materials and methods of photolysis using a 125 W medium pressure mercury vapour lamp (MP lamp) (Applied Photophysics Ltd., UK) [emission at 254, 313, 366 and 436 nm (Fig. 1) corresponding to the wavelengths of RF absorption (Fig. 2)], thin-layer chromatography of
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I. Ahmad et al. / Journal of Photochemistry and Photobiology B: Biology 82 (2006) 21–27
Fig. 1. Spectral emission of 125 W medium pressure mercury vapour lamp.
Fig. 2. Absorption spectrum of 5 · 105 M riboflavin at pH 7.0 (phosphate buffer).
photolysed solutions, and spectrophotometric assay of RF and photoproducts have previously been described [25]. The following method of photolysis was also used in this study. 2.1. Photolysis with visible lamps An aqueous solution of riboflavin (104 M, 200 ml) containing 0.05–2.00 M Na2HPO4 (adjusted to pH 7.0) was placed in a 250 ml volumetric flask (Pyrex) and irradiated with a Philips HPLN 125 W high pressure mercury vapour fluorescent lamp (HP lamp) [emission at 405 and 436 nm (Fig. 3) corresponding to the wavelengths of RF absorption], or a Philips 150 W tungsten lamp (TN lamp) (contin-
uum over the range 350–2000 nm) (Fig. 4) fixed at a distance of 30 cm from the centre of the flask. The temperature of the solution was maintained at 25 ± 1 C during irradiation. The solution was continuously stirred by bubbling a stream of air into the flask. Samples were withdrawn at appropriate intervals for chromatography and assay. 2.2. Light intensity measurements The intensities of the MP, HP and TN lamps were determined by potassium ferrioxalate actinometry [42] under the conditions described for the MP lamp [25] and for the HP and TN lamps (Section 2.1).
I. Ahmad et al. / Journal of Photochemistry and Photobiology B: Biology 82 (2006) 21–27
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Fig. 3. Spectral emission of 125 W high pressure mercury vapour fluorescent lamp.
Fig. 4. Spectral emission of Philips 150 W tungsten lamp.
2.3. Determination of spectral energy The energy, E, emitted by the radiation sources at various wavelengths was calculated using the equation E ðk J mol1 Þ ¼
N A hc ; knm
where NA is AvogadroÕs number = 6.0226 · 1023 mol1, h is PlanckÕs constant = 6.6256 · 1034 J s, c is velocity of light = 2.9979 · 108 m s1, k is wavelength in nm = 109 m. 3. Results and discussion 3.1. Photoproducts of riboflavin with visible radiation sources The major photoproducts of RF in aqueous solution (pH 7.0) containing 0.05–2.00 M Na2HPO4, on exposure to visible radiation sources, were identified by thin-layer chromatography as CDRF, FMF, LC, and LF. These
products have been detected earlier on UV irradiation of the RF solutions [25]. However, the magnitude of the formation of these products depends upon the extent of photolysis in a fixed period of time and is higher with the UV lamp (MP) compared to that of the visible lamps (HP and TN). 3.2. Product distribution The product distribution during the photodegradation of RF solutions (2.0 M phosphate) at 25% photolysis (7.5–330 min) and at 60 min irradiation (5.5–67% loss) using the UV and visible radiation sources (MP, HP, TN lamps) is given in Table 1. The wide exposure range is required to produce an equal amount of photolysis (25%) of RF solutions using the MP (7.5 min), HP (150 min) and TN (330 min) lamps. The plots of the values of different product ratios (CDRF/RF, CDRF/LC, LF/LC) versus phosphate concentration are shown in Fig. 5 and discussed individually in the following sections.
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I. Ahmad et al. / Journal of Photochemistry and Photobiology B: Biology 82 (2006) 21–27
Table 1 Product distribution at 25% photolysis and 60 min irradiation of riboflavin solutions (pH 7.0) in presence of 2.0 M phosphate Lamp
MP HP TN MP HP TN
Photolysis
RF
CDRF 5
FMF 5
LC
LF
min
(M · 10 )
(M · 10 )
(M · 10 )
(M · 10 )
(M · 10 )
(M · 105)
25 25 25 67 13 5.5
7.5 150 330 60 60 60
7.5 7.5 7.5 3.27 8.70 9.45
1.65 0.95 0.66 3.05 0.45 0.22
– 0.28 0.53 1.18 0.22 0.07
0.94 1.20 1.22 2.37 0.75 0.38
0.14 0.23 0.31 0.30 0.12 0.10
10.23 10.16 10.22 10.17 10.24 10.22
3.2.1. CDRF/RF ratios The CDRF/RF ratios gradually increase with an increase in phosphate concentration indicating the degradation of RF in favour of photoaddition reaction (Fig. 5). These ratios for the three radiation sources appear in the order MP lamp > HP lamp > TN lamp suggesting that for a fixed amount of RF degradation (i.e., 25%) the ratios are greater for the MP lamp (UV emission)
5
Total
%
Fig. 5. Product ratios of riboflavin and photoproducts as a function of phosphate concentration. CDRF/RF (~), CDRF/LC (s), LF/LC (h).
5
5
compared to those of the HP and TN lamps (visible emission). The UV radiation exerts much greater effect on RF degradation than that of the visible radiation since 25% degradation (2.0 M phosphate) is achieved in 7.5, 150 and 330 min by MP, HP and TN lamps, respectively. The high intensity UV emission of MP lamp at 254 and 366 nm closely corresponds to the 266 and 373 nm absorption maxima of RF [43] and has higher spectral energy (determined as 407 kJ mol1) than that of the low intensity visible emission of HP lamp at 436 nm (determined as 148 kJ mol1) corresponding to the 445 nm absorption of RF [43]. Therefore, the absorption of UV radiation from MP lamp (254 and 366 nm) by RF causes 25% photolysis in 7.5 min compared to that of the visible light of the HP lamp (435 nm) in 150 min and that of the TN lamp in 330 min. A further explanation to the values obtained for CDRF/ RF ratios may be offered on the basis of the fact that CDRF formation by intramolecular photoaddition is mediated by the excited singlet state ð1 Flox Þ whereas FMF, and hence the LC and LF formation by normal photolysis or intramolecular photoreduction occurs via the excited triplet state ð3 Flox Þ [20]. Since on UV irradiation from MP lamp at 254 and 366 nm a greater number of RF molecules are excited to the 1 Flox state compared to those on visible irradiation at 405 and 436 nm, there is a greater probability of the conversion of this state to CDRF in preference to non-radiative processes including intersystem crossing [44] to form the 3 Flox state which yields FMF and subsequently, by hydrolysis, LC and LF [45]. In normal photolysis the formation of LC directly from RF may also take place via the 1 Flox state [19,26]. The degradation of RF on irradiation with TN lamp (continuum in the visible region) is much slower than that of the MP lamp (1/44th) and HP lamp (1/20th) and, therefore, CDRF/RF values are lowest in this case. This may be due to the fact that in TN lamp (Fig. 4) no distinct wavelengths are available for absorption by RF (maximum 445 nm) and the overall energy in the desired region (400–500 nm) is relatively low. 3.2.2. CDRF/LC ratios The CDRF/LC values for the photodegradation reactions using the three radiation sources, considered as a measure of the ratios of the rates of the two reactions
I. Ahmad et al. / Journal of Photochemistry and Photobiology B: Biology 82 (2006) 21–27
25
Table 2 Second-order rate constants for phosphate ions-catalysed photodegradation of riboflavin (pH 7.0) using different radiation sources Radiation source
Total no. of quanta per seconda
No. of quanta absorbed by riboflavin solutionb
k (M1 s1)
Correlation coefficient
125 W medium pressure mercury vapour lamp (MP lamp) 125 W high pressure mercury vapour fluorescent lamp (HP lamp) 150 W Tungsten lamp (TN lamp)c
2.19 ± 0.12 · 1018 1.14 ± 0.10 · 1017 1.06 ± 0.11 · 1016
1.7 · 1018 6.2 · 1016 1.1 · 1016
2.12 · 104 1.37 · 105 6.80 · 106
0.999 0.999 0.999
a
Mean ± SD, n = 3–5. Values obtained by multiplying the total no. of quanta with the % spectral energy corresponding to the wavelengths absorbed by riboflavin in each case. c It gives a continuum in the visible region (Fig. 4) and, therefore, the % spectral energy corresponding to the wavelengths absorbed by riboflavin could not be calculated. b
(photoaddition and photoreduction), are also in the same order as those of the CDRF/RF ratios. The values of CDRF/LC ratios are about 4–8 fold higher than those of the CDRF/RF ratios and increase with an increase in phosphate concentration. In the case of MP lamp, the CDRF/LC ratios are a linear function of phosphate concentration indicating the formation of CDRF and LC by parallel first-order reactions as previously reported [25]. However, the CDRF/LC ratios for the HP and TN lamps are not a linear function of phosphate concentration. This is probably due to relatively low concentrations of CDRF, compared to those of LC, in the CDRF/LC ratios and a clear indication of the fact that irradiation wavelengths have a prominent role in the product distribution of RF photodegradation. Schuman Jorns et al. [20] used a high intensity 250 W medium pressure mercury lamp with UV emission and reported a CDRF/LC ratio of 3/1 for the photoreactions of RF in 0.5 M phosphate solution at pH 7.0 which would be much higher in a 2.0 M phosphate solution. The UV radiation would facilitate RF excitation to the 1 Flox state and its subsequent conversion to CDRF at the expense of the 3 Flox state leading to LC and hence the higher CDRF/LC values. It may be concluded from the present study and the observations of Schuman Jorns et al. [20] on the photoreactions of RF in phosphate buffer that the irradiation wavelengths significantly influence its mode of degradation and hence the product distribution. 3.2.3. LF/LC ratios The values of LF/LC ratios for the individual lamps are almost constant and do not appear to be affected by an increase in phosphate concentration supporting the view that in phosphate buffer LF and LC are formed through FMF via the 3 Flox state [20] and are independent of phosphate concentration. The order of LF/LC ratios
3.3. Rate constants for photodegradation of RF The second-order rate constants for the photodegradation of RF at pH 7.0 in the presence of phosphate ions, obtained by using the UV and visible radiation sources, are reported in Table 2. The total number of quanta s1 emitted by each radiation source and the number of quanta absorbed by RF solution are also reported in Table 2. The values of rate constants for the three radiation sources are of the same order as those of the number of quanta s1 emitted by these sources, respectively. The values of the second-order rate constants indicate that the rate of reaction with MP lamp is about 15–30 fold higher than that of the HP and TN lamps. A better explanation of the kinetic data obtained using the three radiation sources may be provided by considering the values of the overlap integrals between the absorption spectra of RF and the emission spectra of the lamps. The overlap integral is a constant for a particular combination of photon source and absorbing substance and determines the rate of a photochemical reaction. The rate is expressed as the number, N, of molecules transformed per second and is a function of the photons absorbed per second · the quantum yield, u, of the reaction (i.e., Nu) [46]. Since the line emission spectra of the MP and HP lamps (Figs. 1 and 3) only indicate the relative energy distribution of the individual wavelengths involved, and the emission spectrum of the TN lamp (Fig. 4) is a continuum, there are no uniform emission characteristics of these sources to be considered for comparison. It is, therefore, not possible to calculate the overlap integrals between the riboflavin absorption spectra and the lamp emission spectra to enable the interpretation of the kinetic data on the basis of overlap integrals.
TN lamp > HP lamp > MP lamp
4. Conclusion
is inverse of the ratios of CDRF/LC obtained for the three lamps indicating that in phosphate buffer the major pathway of RF degradation, on irradiation with MP lamp, is intramolecular photoaddition which may require more energy than that of the intramolecular photoreduction. The later reaction can easily occur in the presence of visible light and is the major cause of RF instability in aqueous solution.
The present study shows the effect of light intensity and wavelengths in the UV and visible region on the mode of photodegradation of RF in phosphate buffer as well as the degradation product distribution of the two reactions involved (i.e., photoaddition and photoreduction). The pattern of the ratios of major products of these reactions, i.e., CDRF/RF, CDRF/LC and LF/LC indicates significant variations on a change from UV to visible radiation.
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