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Radiolysis of aqueous solutions of thiamine
Radiat. Phys. Chem. Vol. 52, Nos I-6, pp. 401-404, 1998
© 1998ElsevierScienceLtd. All rights reserved Printed in Great Britain
Pergmnon PII: S0969-806X(98)00058-9
RADIOLYSIS
OF AQUEOUS
SOLUTIONS
0969-806X/98 SI9.00 + 0.00
OF TIIlAMINE.
C. CHIJATE, G. ALBARRAN, and A. NEGRON-MENI)OZA. lnstituto de Cicncias Nuclcares, t INAM. A.P. 70-543. Coyoac,fn. 04510 Mdxico. I).F.
ABSTRACT
The results of lhe radiolysis of aqueous solutions of flfiaminc (vitamin 1~I ) arc prescnted Thc yiclds for decomposition of thiamine and Ihe production of radiolvlic products ~vc,c determined. The G values decrease as the dose increases. Some radiolxtic producl~ ~verc identified. Decomposition of thialnillC was slightly depcndenl on the presence of oxygen and on lhc pli oflhe sohllion. At pll 4.4. wilh a ctmccnlralion of 2.5 x I(}-4 tool I, -I oflhiamine in an oxygen frcc aqueous solttlion, the (;0 vahlc fi~f dccomposili~m is 5.0.
KEYWORDS Radiolysis, Thiamine, Vitamin B~, aqueous solution.
INTRODUCTION Research oil the radiolysis of vitamins is of considerable interest since these compounds arc important nutritional constituents. In spite of considerable effort in studying the radiolytic behavior of vitamins, in particular thiamine (vitamin B~), there is relatively little data and that obtained by different authors is often fragmentary and difficult to compare (Tobback 1977; Thayer el al. 1991). It is known that thiamine is the most radiation-sensitive o f the water-soluble vitamins, and it is even morc sensitive to heat than to irradiation (Kilcast 1994). To belier understand the effect o f gamma radiation on diluted aqueous solulions of thiamine tmder different conditions of plt, atmosphere and concentration, in this work we sludied lhe radiolysis of aqueous solutions o f thiamine, dcterlnining thiamine decomposition and the yields of somc radiolysis products. EXPERIMENTAL Materials.
Thialnine hydrochloride was supplied by Productos Roche, S.A. de C.V. Watcr was Ireatcd according to the standard procedures used in Radiation Cllemistry (Draganic and Draganic 1073). 401
402
C. Chijate et
al.
Irradiation. All irradiations were made with a 6~Co ganama radiation source (Gammacell 200) at an adsorbed dose rate of 1.9 Gy/min, which had previously been established by standard ferrous sulfate (Fricke) dosimetry. The irradiations were carried out in a glass vessel (3 ml). The sample vessel was sealed, either in the presence of dissolved air or after being subjected to vacuum. The ptl of the aqueous solutions was varied using thiamine hydrochloride and perchloric acid.
Analysis. Analysis of thiamine. The analysis of lhiamine was made by UV-spectropholonlet|y and by I IPI,C. "lhe LIV readings were made within 10 rain of the end of the irradiation. The absorbances at the maximum of thiamine (L = 233 nm) were measured and a standard calibration curvc for lhiamine at 233 nm, belween 10 ~ and 10.6 reel L" was made. A Varian LC Star System liquid chromatograph was used Io study thiamine disappearance and to separate some of the radiolysis producls. Thc chromatographic column was a 150 m m x 4.6 mm stainless steel column packed with Ilypersil ODS, 3rtm. The mobile phase was a aqueous mixture containing 25 % methanol, 1 % acetic acid and 5 x 10 ~ reel I," I-hexanesulfonic acid, sodium salt. 10 ~ttl aliquot o f the irradiated solution was injected into tile liquid chromatographic system. The standard curve for thiamine was constructed in the concentration interval fiom 2.5 x 10 `4 to 2.5 x I 0 -6rnol L". With a flow rate of 0.5 mL rain-', thiamine had a retention time of 19.6 rain (at 233 nln). Other standard compounds were detected using the following conditions: thiazole, 8.6 rain (~ - 250 ran), pyrimidine, 10.2 rain (L = 226 nm ), thiochrome 13.8 rain (~, = 233 am). Ammonia determination. Ammonia was quantified by an NIt~ specific electrode (Orion). A standard curve was made, and the concentration of ammonia in the samples was determinaled by comparison.
R E S U L T S AND D I S C U S S I O N
Radiolytic thiamine decomposition The data in the literature is very contradictory with respect to the decomposition yield of thiamine: The results go from very low decomposition to high values. These results werc determined by IJV spectrophotometry. We determined the decomposition by this method and we found a G=0.6 at pl I - 4 . 4 .
Figure I. l tPLC chromatogram of 2.5 x I0" reel L-' thiandne irradiated lo 500 (iy at pll 4.4.
10th International Meeting on Radiation Processing
403
This low value is due to the fact that the radiolylic products also absorb al this wavelength. The decomposilion of 2.5 x 10.4 tool L l thiamine irradiated at natural pll, oxygen-free, was quaqtified by I IPLC, without interference o f tile olher radiolytic products, because tile thiamine was separaled with good ,esolution, as is shown in Figure I .The decomposition of thiamine in aqueous solutions was a fimction of radiation dose and presents the peculiar odor of lhiazole, which increases when lhe dose increases. Figure 2 show lhe loss of thiamine as a fimction of dose. From these dala, lhe radiation chcmislry yield of decomposition of thiamine is G o= 5.0.
100 8O 6O ,,.-I
~
4o 2O O
v
0
1000
i
;
2000
3000
Dose (Gy) Figure 2. Percentage loss oflhiamine in an oxygen-flee aqueous solution as a fimclion of the irradiation dose. Initial concenlration 2.5 x 10 4 tool 1.,"
The radiolytic decomposition of thiamine was slightly lower in the presence of oxygen than in the oxygen-free solutions, suggesting that oxygen can be an effective scavenger for the radicals produced by lhe primary radiolysis o f water.
Effect of concentration To study the effect of concentration, several solutions at different concentrations were irradiated at 600 Gy, oxygen-free, and at natural pli (4.4). The percentage decomposition of thiamine was determinated by tlPLC and is shown in Table 1. It can be observed that the decomposition oflhiamine decreases when the concentration increases. These results agree with Kishore et al. (1980).
Table 1. Decomposition of thiamine irradiated to 600 Gy, as a functioq of the concentration
Thiamine concentration
Percentage toss oflhe thiamine
10 .2 tool L-'
8
10 .3 mol [,"
45
10 .4 tool L 4
67
404
C. Chijate et al.
Effect o f p t l "['he absorption spectrum of thiamine hydrochloride is a flmction of the hydrogen-ion concentration. At neutral or basic pH the thiamine is destroyed. The radiation-promoted decomposition of aqueous thiamine solutions (10 -' tool LI), as a function of pll, was deterrnined. The data indicate that the percentage loss of thiamine increases when the concentration of hydrogen-ion decreases, lhis loss being more than 30 % between ptl 3 to 5 at 370 Gy. Probably the decomposition at plI 5 increases as a function of increased e,, in the irradiated aqueous solutions. Forrester and Davison (1'990) indicated that e,, reacts with both rings.
Ammonia production The radiolysis of the thiamine in aqueous solution produces ammonia as a radiolytic product, which is formed as a function of the irradiation dose. The probable mechanism of production is by e,o attack on the amine group of the pyrimidine ring. The experimental radiation chemical yield for ammonia is G " - 0.2. The thermal decomposition of thiamine produced thiochrolne and 5-hydroxyelhyl lhiazole (Ill'T). Thcse products have been also reported as radiolytic products from thiamine, but in our study we do not find evidence of ring breaks due to the irradiation. The retention times of thiocrome and l lET were different from those of the peaks seen in I IPLC chromatogram (Figure I). The structure of this radiolytic producl should have the two rings and probably corresponds to an Oi I-adduct REMARKS The decomposition of thiamine is a function o f p t l and dose. It increases with increasing pll fl-om pll 3 to 5, and is slightly lower in a free-oxygen atmosphere, as the oxygen acts as scavenger of free radicals. The Gn~,h,,,,i,~) = 5 was obtained in oxygen fiee aqueous solutions at natural pll irradiated at 1.9 Gy nlJn I. This value was calculated from ttPLC data without interference of other radiolytic products. The spectrophotometric determination gave a G°(. . . . . ~,~= 0.2. This indicates a small amount of decomposition of the amine group of pyrimidine moiety. The principal radiolytic product, not identified, is probably formed by a addition reaction of the flee radicals from water on the double bond in the thiazole ring. The exact position of addition is tinder study. ACKNOWLEDGEMENTS
The authors are deeply indebted to Miss Carmen Peza L. for technical support and and to Mr S. ltam for making the glassware. We wish to thank Dr. Carol Collins for helpful comments on the manuscript. REFERENCES
Draganic, 1. and Draganic, Z., 1973. "Tile Radiation Chemistry of Water". Academic Press, New York, pp 203-206. Forrester A.R., Davison I.G., 1990. Identification of radicals produced by gamma-irradiation of vitamins. Radiation. l'lo,sics and Chemisto,. 36: 403-407. Kilcast, D. 1994. Effect of Irradiation on Vitamins. Food Chememisto,. 49:157-164. Kishore, K., Moorthy, P.N. and Rao, K.N. 1980. Radiation induced reactions in aqueous solutions of thialnine (vitamin B~), Radiaion Play,its and Chemistpy, 15: 669-676. Thayer, D.W., Fox, J.B., and Lakritz, L. 1991. Effects of ionizing radiation on vilamins, in "Food irradiation".. Thorne, S., Ed., Elsevier. I,ondon. pp. 285-325. Tobback. P.P., 1997. Radiation Chemistry of Vitamins, in: "Radiation Chemistry of Major Food Components", Elias, P.S. and Cohen. A.J., Eds., Elsevier. Amsterdam, pp 187-220.
© 1998ElsevierScienceLtd. All rights reserved Printed in Great Britain
Pergmnon PII: S0969-806X(98)00058-9
RADIOLYSIS
OF AQUEOUS
SOLUTIONS
0969-806X/98 SI9.00 + 0.00
OF TIIlAMINE.
C. CHIJATE, G. ALBARRAN, and A. NEGRON-MENI)OZA. lnstituto de Cicncias Nuclcares, t INAM. A.P. 70-543. Coyoac,fn. 04510 Mdxico. I).F.
ABSTRACT
The results of lhe radiolysis of aqueous solutions of flfiaminc (vitamin 1~I ) arc prescnted Thc yiclds for decomposition of thiamine and Ihe production of radiolvlic products ~vc,c determined. The G values decrease as the dose increases. Some radiolxtic producl~ ~verc identified. Decomposition of thialnillC was slightly depcndenl on the presence of oxygen and on lhc pli oflhe sohllion. At pll 4.4. wilh a ctmccnlralion of 2.5 x I(}-4 tool I, -I oflhiamine in an oxygen frcc aqueous solttlion, the (;0 vahlc fi~f dccomposili~m is 5.0.
KEYWORDS Radiolysis, Thiamine, Vitamin B~, aqueous solution.
INTRODUCTION Research oil the radiolysis of vitamins is of considerable interest since these compounds arc important nutritional constituents. In spite of considerable effort in studying the radiolytic behavior of vitamins, in particular thiamine (vitamin B~), there is relatively little data and that obtained by different authors is often fragmentary and difficult to compare (Tobback 1977; Thayer el al. 1991). It is known that thiamine is the most radiation-sensitive o f the water-soluble vitamins, and it is even morc sensitive to heat than to irradiation (Kilcast 1994). To belier understand the effect o f gamma radiation on diluted aqueous solulions of thiamine tmder different conditions of plt, atmosphere and concentration, in this work we sludied lhe radiolysis of aqueous solutions o f thiamine, dcterlnining thiamine decomposition and the yields of somc radiolysis products. EXPERIMENTAL Materials.
Thialnine hydrochloride was supplied by Productos Roche, S.A. de C.V. Watcr was Ireatcd according to the standard procedures used in Radiation Cllemistry (Draganic and Draganic 1073). 401
402
C. Chijate et
al.
Irradiation. All irradiations were made with a 6~Co ganama radiation source (Gammacell 200) at an adsorbed dose rate of 1.9 Gy/min, which had previously been established by standard ferrous sulfate (Fricke) dosimetry. The irradiations were carried out in a glass vessel (3 ml). The sample vessel was sealed, either in the presence of dissolved air or after being subjected to vacuum. The ptl of the aqueous solutions was varied using thiamine hydrochloride and perchloric acid.
Analysis. Analysis of thiamine. The analysis of lhiamine was made by UV-spectropholonlet|y and by I IPI,C. "lhe LIV readings were made within 10 rain of the end of the irradiation. The absorbances at the maximum of thiamine (L = 233 nm) were measured and a standard calibration curvc for lhiamine at 233 nm, belween 10 ~ and 10.6 reel L" was made. A Varian LC Star System liquid chromatograph was used Io study thiamine disappearance and to separate some of the radiolysis producls. Thc chromatographic column was a 150 m m x 4.6 mm stainless steel column packed with Ilypersil ODS, 3rtm. The mobile phase was a aqueous mixture containing 25 % methanol, 1 % acetic acid and 5 x 10 ~ reel I," I-hexanesulfonic acid, sodium salt. 10 ~ttl aliquot o f the irradiated solution was injected into tile liquid chromatographic system. The standard curve for thiamine was constructed in the concentration interval fiom 2.5 x 10 `4 to 2.5 x I 0 -6rnol L". With a flow rate of 0.5 mL rain-', thiamine had a retention time of 19.6 rain (at 233 nln). Other standard compounds were detected using the following conditions: thiazole, 8.6 rain (~ - 250 ran), pyrimidine, 10.2 rain (L = 226 nm ), thiochrome 13.8 rain (~, = 233 am). Ammonia determination. Ammonia was quantified by an NIt~ specific electrode (Orion). A standard curve was made, and the concentration of ammonia in the samples was determinaled by comparison.
R E S U L T S AND D I S C U S S I O N
Radiolytic thiamine decomposition The data in the literature is very contradictory with respect to the decomposition yield of thiamine: The results go from very low decomposition to high values. These results werc determined by IJV spectrophotometry. We determined the decomposition by this method and we found a G=0.6 at pl I - 4 . 4 .
Figure I. l tPLC chromatogram of 2.5 x I0" reel L-' thiandne irradiated lo 500 (iy at pll 4.4.
10th International Meeting on Radiation Processing
403
This low value is due to the fact that the radiolylic products also absorb al this wavelength. The decomposilion of 2.5 x 10.4 tool L l thiamine irradiated at natural pll, oxygen-free, was quaqtified by I IPLC, without interference o f tile olher radiolytic products, because tile thiamine was separaled with good ,esolution, as is shown in Figure I .The decomposition of thiamine in aqueous solutions was a fimction of radiation dose and presents the peculiar odor of lhiazole, which increases when lhe dose increases. Figure 2 show lhe loss of thiamine as a fimction of dose. From these dala, lhe radiation chcmislry yield of decomposition of thiamine is G o= 5.0.
100 8O 6O ,,.-I
~
4o 2O O
v
0
1000
i
;
2000
3000
Dose (Gy) Figure 2. Percentage loss oflhiamine in an oxygen-flee aqueous solution as a fimclion of the irradiation dose. Initial concenlration 2.5 x 10 4 tool 1.,"
The radiolytic decomposition of thiamine was slightly lower in the presence of oxygen than in the oxygen-free solutions, suggesting that oxygen can be an effective scavenger for the radicals produced by lhe primary radiolysis o f water.
Effect of concentration To study the effect of concentration, several solutions at different concentrations were irradiated at 600 Gy, oxygen-free, and at natural pli (4.4). The percentage decomposition of thiamine was determinated by tlPLC and is shown in Table 1. It can be observed that the decomposition oflhiamine decreases when the concentration increases. These results agree with Kishore et al. (1980).
Table 1. Decomposition of thiamine irradiated to 600 Gy, as a functioq of the concentration
Thiamine concentration
Percentage toss oflhe thiamine
10 .2 tool L-'
8
10 .3 mol [,"
45
10 .4 tool L 4
67
404
C. Chijate et al.
Effect o f p t l "['he absorption spectrum of thiamine hydrochloride is a flmction of the hydrogen-ion concentration. At neutral or basic pH the thiamine is destroyed. The radiation-promoted decomposition of aqueous thiamine solutions (10 -' tool LI), as a function of pll, was deterrnined. The data indicate that the percentage loss of thiamine increases when the concentration of hydrogen-ion decreases, lhis loss being more than 30 % between ptl 3 to 5 at 370 Gy. Probably the decomposition at plI 5 increases as a function of increased e,, in the irradiated aqueous solutions. Forrester and Davison (1'990) indicated that e,, reacts with both rings.
Ammonia production The radiolysis of the thiamine in aqueous solution produces ammonia as a radiolytic product, which is formed as a function of the irradiation dose. The probable mechanism of production is by e,o attack on the amine group of the pyrimidine ring. The experimental radiation chemical yield for ammonia is G " - 0.2. The thermal decomposition of thiamine produced thiochrolne and 5-hydroxyelhyl lhiazole (Ill'T). Thcse products have been also reported as radiolytic products from thiamine, but in our study we do not find evidence of ring breaks due to the irradiation. The retention times of thiocrome and l lET were different from those of the peaks seen in I IPLC chromatogram (Figure I). The structure of this radiolytic producl should have the two rings and probably corresponds to an Oi I-adduct REMARKS The decomposition of thiamine is a function o f p t l and dose. It increases with increasing pll fl-om pll 3 to 5, and is slightly lower in a free-oxygen atmosphere, as the oxygen acts as scavenger of free radicals. The Gn~,h,,,,i,~) = 5 was obtained in oxygen fiee aqueous solutions at natural pll irradiated at 1.9 Gy nlJn I. This value was calculated from ttPLC data without interference of other radiolytic products. The spectrophotometric determination gave a G°(. . . . . ~,~= 0.2. This indicates a small amount of decomposition of the amine group of pyrimidine moiety. The principal radiolytic product, not identified, is probably formed by a addition reaction of the flee radicals from water on the double bond in the thiazole ring. The exact position of addition is tinder study. ACKNOWLEDGEMENTS
The authors are deeply indebted to Miss Carmen Peza L. for technical support and and to Mr S. ltam for making the glassware. We wish to thank Dr. Carol Collins for helpful comments on the manuscript. REFERENCES
Draganic, 1. and Draganic, Z., 1973. "Tile Radiation Chemistry of Water". Academic Press, New York, pp 203-206. Forrester A.R., Davison I.G., 1990. Identification of radicals produced by gamma-irradiation of vitamins. Radiation. l'lo,sics and Chemisto,. 36: 403-407. Kilcast, D. 1994. Effect of Irradiation on Vitamins. Food Chememisto,. 49:157-164. Kishore, K., Moorthy, P.N. and Rao, K.N. 1980. Radiation induced reactions in aqueous solutions of thialnine (vitamin B~), Radiaion Play,its and Chemistpy, 15: 669-676. Thayer, D.W., Fox, J.B., and Lakritz, L. 1991. Effects of ionizing radiation on vilamins, in "Food irradiation".. Thorne, S., Ed., Elsevier. I,ondon. pp. 285-325. Tobback. P.P., 1997. Radiation Chemistry of Vitamins, in: "Radiation Chemistry of Major Food Components", Elias, P.S. and Cohen. A.J., Eds., Elsevier. Amsterdam, pp 187-220.