STABILIZATION
OF C Y A N O C O B A L A M I N
STERILIZATION
OF ITS S O L U T I O N S
DURING RADIATION
IN F R E E Z I N G
E. ~ . B a r e l k o , G. S. B a b a k i n a , I. V. B e r e z o v s k a y a , V. S. D e g i l o v a , L . A. P i r u z y a n , N. V. P o m o r t s e v a , I. P . S o l y a n i n a , V. V. S u k h a n o v , V. L. T a l ' r o z e , E. A. T y r i n a , a n d M. I. T s i b u l ' s k a y a
CONDITIONS UDC 615.356"577.164.16 ].014.45
One of the unsolved problems in the radiation sterilization of pharmaceuticals is the protection of preparations in aqueous solutions from decomposition during irradiation. Usually, this is a problem encountered in conjtmction with injection preparations, and noticeable amounts of sodium chloride are sometimes dissolved along with the drug in the water. The high degree of radiolysis of the dissolved substance is related to the fact that, along with the direct influence of radiation, a much stronger indirect influence is also exerted. Active intermediate products (H atoms, free hydroxyls, solvated electrons) formed from the radiolysis of water attack the dissolved substance, causing chemical conversions. Two possible means of overcoming this problem have been examined in the literature: by decreasing the dosage of sterilizing radiation, and by chemical and physical methods of protecting the drug from decomposition at dosages sufficiently high for sterilization. The proposals for reducing the dosage are based on two main considerations: 1) to provide for, if not already existing, conditions during the actual pharmaceutical production whereby the initial contamination of the solution by potentially pathogenic microorganisms would not be very high and, therefore, the fraction of radiation-resistant organisms would als0 be insignificant, so that the widely used sterilization dosage of 2.5 Mrd could be reduced to 1 Mrd [1 ]; 2) to conduct radiation sterilization at somewhat elevated temperatures (thermal radiation method) where the dosage required for achieving the same degree of inactivation (contamination reduction) is correspondingly decreased [2]. Both approaches to dosage reduction can be useful, for example, in the radiosterilization of concentrated or dry preparations. However, the degree of drug decomposition in highly dilute aqueous solutions can be reduced only insignificantly using these approaches. With respect to the thermal radiation method, there is in general no assurance that the decrease in the degree of drug radiolysis achieved by reducing the dosage would not be offset to a significant degree by accelerated radiolytic conversion of the drug under the influence of temperature [3]. More effective methods for preventing the radiolysis of a dissolved drug involve the chemical and physical protection methods, which furthermore influence the bactericidal action of irradiation to a much l e s s e r degree. Thus far, the major portion of research in this area has been in connection with chemical methods of protection whereby other substances are added to the dilute pharmaceutical solution. These substances are aeceptors of the active intermediate fragments of water hydrolysis and, as a result of this acceptor action, give less active free radicals which are no longer capable of chemically attacking the drug molecule [2, 4-6]. However, in view of the fact that the activation energy for the reaction of the active radiolysis products from water with organic drugs is usually exceedingly low, radioprotecters for the drugs have to be used in concentrations comparable to or significantly exceeding that of the drug itself. As a result of this, very serious interference arises in the already formulated pharmaceutical composition since, in many cases, additives which are suitable from a radio protection viewpoint are unacceptable from a pharmacological viewpoint. Scientific-Research Institute for the Biological Testing of Chemical Compounds, Moscow Region. Institute of Chemical Physics, Academy of Sciences of the USSR. All-Union Scientific-Research Institute for Vitamins, Moscow. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 11, No. 7, pp. 94-100, July, 1977. Original article submitted February 22, 1977.
968
0091-150X/77/1107- 0968507.50 9 1978 Plenum Publishing Corporation
The m o s t effective p r o t e c t i o n method is one of the p o s s i b l e physical m e t h o d s , n a m e l y , that of i r r a d i a t i n g the d i s s o l v e d s u b s t a n c e at a l o w e r e d t e m p e r a t u r e in a frozen state [7, 8]. L i t e r a t u r e data with r e s p e c t to the r a d i o l y s i s of p h a r m a c e u t i c a l s indicate that such an approach should p r o t e c t the drug as a consequence of h i n d e r e d diffusion in the d i s s o l v e d - i n - i c e m o l e c u l e by the r a d i o l y s i s p r o d u c t s of frozen w a t e r . To this significant f a c t o r can be added yet another two which ought be c r e a t e d s p e c i a l l y o r found in the a l r e a d y existing s y s t e m s . One of these would be h i n d r a n c e of any type to the dissociation of the drug in a solid m a t r i x . The o t h e r is based on the f a c t that s o m e t i m e s during f r e e z i n g , b e c a u s e of a d e c r e a s e in the solubility coefficient, a a p a r t i a l o r n e a r l y c o m p l e t e p h a s e s e p a r a t i o n of the w a t e r and the drug can o c c u r , thereby again eliminating the i n d i r e c t influence of radiation. Thus, the m o s t p r o m i s i n g method of p r e s e r v i n g a drug during radiation s t e r i l i z a t i o n is that of i r r a d i a t i n g the p h a r m a c e u t i c a l solutions in a frozen state. In fact, the effect of excluding the i n d i r e c t influence of r a d i o l y s i s in the p h y s i c a l s e n s e should significantly i n c r e a s e (by s e v e r a l o r d e r s of magnitude) the radiation stability of the drug in dilute solutions while only insignificantly changing the r a d i ation r e s i s t a n c e of the m i c r o o r g a n i s m s during f r e e z i n g [9]. This would n e c e s s i t a t e only a l i m i t e d i n c r e a s e in the r e q u i r e d s t e r i l i z a t i o n d o s a g e , the d a m a g i n g effects of which would be offset by the mentioned increase in effective r a d i a t i o n stability of the p h a r m a c e u t i c a l . The l i t e r a t u r e contains l i m i t e d data on the radiation stability of drugs in solutions at lowered t e m p e r a t u r e s . R e f e r e n c e is m a d e in [7] to the f a c t that the r a d i o l y s i s of s e v e r a l p h a r m a c e u t i c a l solutions at v e r y low t e m p e r a t u r e s did not lead to noticeable decomposition. H o w e v e r , it is also mentioned, without explanations, that p r o p e r t i e s of these drugs a r e d e t e r i o r a t e d by the f r e e z i n g itself. R e c e n t l y , d a t a h a v e been published on the influence of f r e e z i n g on the r a d i o l y s i s of 10 ~ M solutions of five v i t a m i n s of the B group - thiamine, pyridoxine, folic acid, nicotinamide, and riboflavin [8]. The r a d i o l y s i s of thiamine solutions was studied in a b r o a d dosage r a n g e at 18, -14, and -18~ in the a b s e n c e and in the p r e s e n c e of 0.1 M glucose solution as a OH r a d i c a l a c c e p t o r [8, 10]. The o t h e r four v i t a m i n s w e r e studied at a single d o s a g e , 2 Mrd. In all five c a s e s significant radiation stabilization was attained on freezing; at the mentioned dosage the d e g r e e of radiation decompos ition of the studied compounds d e c r e a s e d f r o m 90-100% to 3-4% (for pyridoxine to 147c ). F o r thiamine at -14~ the d e g r e e of decomposition was 20%. The l a t t e r r e s u l t indicates that ice f o r m a t i o n itself can be insufficient for the r e q u i r e d d e g r e e of protection. In fact, the p r o p e r t i e s of ice as a m a t r i x for the stabilization of active p a r t i c l e s a r e different at different t e m p e r a t u r e s . T h e s e p r o p e r t i e s have been studied widely by p h y s i c a l methods ( r a d i o t h e r m o l u m i n e s c e n c e , ESR) with r e s p e c t to the freezing of s o l r a t e d e l e c t r o n s , H a t o m s , and OH and H O 2 r a d i c a l s [11-13]o It a p p e a r s that in p u r e ice, the deactivation t e m p e r a t u r e f o r a solvated e l e c t r o n is -169~ f o r OH r a d i c a l s -160~ and f o r HO2 r a d i c a l s -113~176 E s s e n t i a l l y , the t e m p e r a t u r e f o r deactivating the r a d i c a l s depends on the c o m p o s i t i o n of the solution. T h u s , it is p r o b a b l y i m p o s s i b l e to p r e d i c t in g e n e r a l t e r m s t e m p e r a t u r e s below which ice is a sufficiently effective p r o t e c t i v e m a t r i x f o r drugs so that a s p e c i a l investigation should be conducted for each individual substance. With view to the p r a c t i c a l application of the r e s u l t s obtained, it is expedient in such investigations to study the r a d i o l y s i s of solutions s i m i l a r in c o m p o s i t i o n to the p h a r m a c e u t i c a l p r e p a r a t i o n i n a s m u c h as the p r e s e n c e of n o t i c e a b l e amounts of sodium chloride in the solution can significantly influence the s t r u c t u r a l and o t h e r p r o p e r t i e s of the ice being f o r m e d . In addition to c h e m i c a l analysis data (by s p e c t r o s c o p y and o t h e r m e t h o d s ) , a study should be conducted also of the biological activity, toxicity, and the s t o r a g e p r o p e r t y of the i r r a d i a t e d p h a r m a c e u t i c a l . T e s t i n g of the biological activity is n e c e s s a r y not only in connection with a opinion e x p r e s s e d e a r l i e r [7] c o n c e r n i n g the v u l n e r a b i l i t y of p h a r m a c e u t i c a l s to d a m a g e during f r e e z i n g (this, a p p a r ently, does not apply to v i t a m i n s and m a n y o t h e r drugs), but also b e c a u s e biological activity is the m o s t d i r e c t method of indicating the d e g r e e of p r e s e r v a t i o n of the substance. The n e c e s s i t y of testing a p r e p a r e d p h a r m a c e u t i c a l which has been i r r a d i a t e d in a f r o z e n state f o r toxicity and s t o r a g e p r o p e r t y a r i s e s f r o m g e n e r a l c o n s i d e r a t i o n s , GOST r e q u i r e m e n t s , as well as f r o m the f a c t that i r r a d i a t i o n of a s u b s t a n c e in frozen s t a t e to p r e v e n t decomposition of the drug does not p r e v e n t the f o r m a t i o n of s m a l l amounts of new s u b s t a n c e s , f o r e x a m p l e , hydrogen peroxide. Vitamin B12, c y a n o c o b a l a m i n , was s e l e c t e d for study in the p r e s e n t investigation since the r a d i o l y s i s of its nonfrozen aqueous solutions has been the s u b j e c t of a l a r g e n u m b e r of investigations [6, 14, 15], in which the m e c h a n i s m of the i n d i r e c t influence of radiation is d i s c u s s e d . The role of a solvated e l e c t r o n in this p r o c e s s (formation of v i t a m i n B12-r ) has been d e t e r m i n e d and it has been d e m o n s t r a t e d that this action can be r e v e r s e d during the p o s t i r r a d i a t i o n effect of oxygen. It is shown also that the p a r t i c l e s m a i n l y r e s p o n s i b l e f o r d e c o m p o s i t i o n of the v i t a m i n s a r e the OH r a d i c a l s , which can be r e m o v e d during r a d i o l y s i s by m e a n s of a c c e p t o r s to d e c r e a s e the l o s s on v i t a m i n . The a c c e p t o r s p r o p o s e d have concentrations exceeding by three to 969
four t i m e s the vitamin content in the p h a r m a c e u t i c a l p r e p a r a t i o n , t h e r e f o r e , they cannot be used in p h a r m a c e u t ical s y s t e m s and, apparently, a r e dangerous for use in an injection m a t e r i a l . In our investigation irradiation was conducted in an apparatus having ~~ as the s o u r c e of g a m m a r a d i ation and capacity 200,000 g-eq Ra. The dosage rate in most experiments was 5 Mrd/h; a rate of 1 M r d / h was used f o r obtaining r e s u l t s at low dosages. The dosage range was 0.02-5 Mrd. The irradiation of c y a n o cobalamin was conducted at 18, 0, -22, - 5 0 , -78, and -196~ in sealed glass ampuls containing the p r e p a r a t i o n , as a rule, in concentration 200 # g / m l . The d e g r e e of cyanocobalamin decomposition was d e t e r m i n e d using absorption s p e c t r o s c o p y , t h i n - l a y e r c h r o m a t o g r a p h y , and biological activity. T h e s e methods s a t i s f a c t o r i l y c o m p l e m e n t one another with r e s p e c t to the p r o b l e m being studied. The instrumental a c c u r a c y of absorption s p e c t r o s c o p y is the highest (several percent), but consideration m u s t be given to the f o r m a t i o n during radioiysis of products which absorb at the v e r y same wavelengths as cyanocobalamin.* This i n c r e a s e s the e r r o r to approximately • at low d e g r e e s of cyanocobalamin d e c o m position. T h i n - l a y e r c h r o m a t o g r a p h y using c o l o r intensity of spots provides for a less accurate c o m p a r i s o n of the s y s t e m s being analyzed f o r cyanocobalamin content with calibrated solutions. The a c c u r a c y of c y a n o cobalamin d e t e r m i n a t i o n at low d e g r e e s of decomposition is +15%, but the complete disappearance of the vitamin in the i r r a d i a t e d solutions can be fixed with an a c c u r a c y of •176 which allows for g r e a t e r reliability in considering the contribution of radiolysis p r o d u c t absorption to the s p e c t r u m in the s p e c t r o s c o p i c method d i s c u s s e d above [16]. The d e t e r m i n a t i o n of biological activity by diffusion of a component of the solution in agar using E s c h e r i chia coli 113-3 as the test o r g a n i s m gave an a c c u r a c y of around +15~ at low d e g r e e s of decomposition, but was the m o s t d i r e c t method for establishing the p r e s e r v a t i o n of the basic effective p r o p e r t i e s of the vitamin
[i7]. As an example, d i r e c t e x p e r i m e n t a l r e s u l t s are given in Fig. 1. of a s e r i e s of tests at +18~ obtained by the s p e c t r o p h o t o m e t r i c method and c h a r a c t e r i z e d by a d e c r e a s e in absorption by the solution with i n c r e a s ing dosage of g a m m a radiation (curve 1). Curve 2 shows the r e s u l t of c o r r e c t i n g these s a m e experimental r e s u l t s for the light absorption by the r a d i o l y s i s products and c o r r e s p o n d s to the true d e c r e a s e in absorption and, consequently, in cyanocobalamin concentration. E x p e r i m e n t a l data f r o m biological activity and t h i n - l a y e r c h r o m a t o g r a p h y for an analogous s e r i e s of tests a r e likewise given. The e r r o r in m e a s u r i n g the dosage did not exceed • As can be seen, the r e s u l t s obtained by all t h r e e methods agree s a t i s f a c t o r i l y . Data for a s e r i e s of tests at lowered t e m p e r a t u r e s are given in Fig. 2. All data show that at -78~ and --196~ the r a t e of radiolysis drops sharply in c o m p a r i s o n with radiolysis in a liquid state (18 and 0~ The d e c r e a s e in radiolysis r a t e is on the o r d e r of ~ 102. At t e m p e r a t u r e s -22 and -50~ even though the solution is already f r o z e n , the r a d i o l y s i s yield is noticeably higher than at t e m p e r a t u r e s -78 and -196~ but is lower by m o r e than 1-1.5 o r d e r s of magnitude than in the liquid state. It will be noted also that the radiolysis r a t e *The absorption (at 2~ = 361 nm) by radiolysis products in a solution which has r e c e i v e d in liquid state a g a m m a radiation dosage g r e a t e r than 0.3-0.5 Mrd and for all p r a c t i c a l purposes no longer containing cyanocobalamin c o m p r i s e d 25% of the absorption D o of the initial solution and changed little with f u r t h e r i n c r e a s e in dosage. If it is a s s u m e d that absorption g e n e r a l l y is a l i n e a r superposition of the absorption by cyanocobalamin and the mentioned radiolysis products and that absorption by these products does not change with dosage, then the concentration n c of the cyanocobalamin can be calculated f r o m the quantity D in the e x p r e s s i o n n e = (D 0"25D0)/0"75 Se l (1), where Se is the m o l a r extinction coefficient f o r eobalamin, D O is the absorption of the initial solution, and l is cuvette thickness. In the initial portion of the curve f o r the d e c r e a s e in D with dosage, this e x p r e s s i o n gives values deviating little f r o m n c calculated simply f r o m n c -- D / ~ c l . F o r example, at D = 0.9D 0, the difference is 4%; at D = 0.7D 0 it is 15%. T h e r e f o r e , if only by some type of approximation consideration is given to the principle of l i n e a r superposition of the spectra of cyanocobalamin and the d e composition products in the t e r m given above, use of Eq, (1) f o r calculating n e and the c o r r e s p o n d i n g yield of eyanoeobalamin f r o m the initial portion of the c u r v e f o r the dependence of D on dosage does not in itseff cause e r r o r s of m o r e than s e v e r a l p e r c e n t .
970
mg/lite~ 20o
m~/liter 2 0 0
~
~.~
160
I00 lOO3
!
SO
0
6-0-
o,25
o,~ Fig. I
I
aTg
,
11'2
t
goMrd
o
I
l
~ 2
~
3
i 4
1
s Mrd
Fig. 2
Fig. 1~ Dependence of cyanocobalamin conch, on dosage of gamma radiation during radiolysis at 18~ 1 O) Uncorrected spectrophotom et r i c data at h = 361 nm; 2 O) spectrophotometric data c o r r e c t e d for absorption by radiolysis products at k = 361 nm; z~) biological activity data; @) thin-layer chromatography data. Fig. 2. T e m p e r a t u r e dependence of cyanocobalamin radiochemical decomposition. 1) 18~ 2) 0~ 3) -22~ 4) -50~ 5)-78~ 6) -196~ O) spectrophotometrie data; ,~) biological activity data.
at -50~ is somewhat lower than the rate at -22~ Qualitatively, the results of the conducted measurements on protecting vitamin 1312from radiolysis by freezing agree with results obtained e a r l i e r [8] for a s e r i e s of other vitamins of the B group. The magnitude of the obtained stabilization coefficient is unusually high and, within experimental limits, practically does not increase on transition from the temperature of solid carbon dioxide to liquid nitrogen temperature. This latter phenomenon can be c o r r e l a t e d with data on the c h a r a c t e r istic freezing temp er a t ur es of active particles in ice obtained in [11-13]. In all these works significant freezing of the mobility of active intermediate products of water radiolysis is observed at t em perat ures below -78~ Such data can be used in the future to ascertain ff there are not mechanisms other than inhibition of the reaction of active radiolysis products with organic molecules frozen in ice that figure in the radiostabilization of large organic molecules such as vitamins. The high radiostability of pharmaceuticals in the described conditions is essential, as is mentioned above, for maintaining a sufficiently high degree of bactericidal action of the gamma radiation. We conducted a s e r i e s of p r e l i m i n a r y experiments in which, along with the measurements described above, the degree of contamination of the sample by microorganisms before and after irradiation in conditions typical for production was determined. Solutions taken for the investigation were not heat sterilized. The initial contamination by all types of microorganisms studied consisted of several thousand p e r sample. If the prel i m i nary results fo r the d e c r e a s e in contamination with increasing dosage are described by a simple exponential equation, they c o r r es p o n d to D10 = 0.15-0.2 Mrd. Thus, to attain a safety coefficient in the o r d e r of 105-10 ~, an irradiation dosage of 1.5-2.0 Mrd is required. At this point it will only be mentioned that for several hundreds of investigated samples with known initial contamination, no contamination is found already after dosages of g r e a t e r than 0.5 Mrd during i r r a d i ation at room temp e r at ur e or in a frozen state. Investigation of the critical toxicity of the irradiated preparations was conducted on male mice of 18-24g weight by intraperitoneally injecting an irradiated dose into six animals. The observation time was 14 days. The preparations investigated were irradiated with one of the highest dosages, 2.5 Mrd, a t 1 8 and -196~ (vitamin 1312concentration in the samples was 200 and 1000 ~g/mliterL Both in control tests using nonirradiated preparations and in tests with the irradiated preparations, doses of 2, 5, and 25 mg/kg weight were introduced. Signs of intoxication and death were not observed in the animals even in tests with preparations irradiated at 18~ i.e., even at nearly complete degradation of the vitamin. This means that for preparations irradiated in frozen state where the substance is hardly degraded at all
971
TABLE 1. Control Data for I r r a d i a t e d Vitamin/312 P r e p a r a t i o n s Daet/D2'*
Dosage, Mrd o 0,02 0,05 0,10 0,20 0,25 0,30 0,50 0,75 1,0 1,5 2,0 2,5 3,0 4,0 5,0
18o
1,7--I ,88 1,35 1,23
oo
-22 ~
D~,~/Ds~ -78 ~
1,7--1:88
,,7--1,88
1,7--1 ,g8
1,34
1,75
1,80 1,80 1,75
0,94 0,71
0,93 0,76
1,73
0,58 0,61
(I,63
0,52
0,5,
0,47
0,46
, ,50 1,50 1,35 1,26
0,47
0,43
1,72 1,54
1,13 0,96 0,74
1,74 1,70
,,72 1,70
1,70 1,70
1,69
1,66
-196 ~
1,7--1,88 1,7o
1,70 1,70
1,70
1,75
18 ~
3,0--3,4 3,1 2,5 2,7 2,l
2-;
1,7
,53
1-)
1,70
2,1
1,69 1,71
1,70 1,70
2,5
0"
3,0--3,4 3,2 3,1
2-3
-92 ~
3,0--3,4
3,0--3,4
3-5
3,4 3.4 3,3
3,2 3,1 3,0
35
3,2 3,4
3,0 2,9 2,0
-78"
2,7
2,4
-196 ~
3,0--3,4 3,0 3,0
3,3 3,2 3,3 3,2 3,3 3,3 3,2 2,8 3,3
3,0 2,9 3,2
35
3,1 3,4 3,2 3,5 3,1
and the stability i n c r e a s e s by some 10S-fold, we have a c o r r e s p o n d i n g "safety m a r g i n " in the i r r a d i a t e d p h a r maceutical preparation. P o s s i b l e r a d i o l y s i s b y - p r o d u c t s which accumulate during i r r a d i a t i o n in a f r o z e n state (for example, s m a l l amounts of hydrogen peroxide) at a s t e r i l i z i n g dosage and at n e a r l y complete p r e s e r v a t i o n of the p h a r m a c e u t i c a l and the biological activity a r e h a r m l e s s . Inasmuch as the f o r m a t i o n of hydrogen peroxide in i r r a d i a t e d ice should o c c u r analogously f o r highly dilute solutions of various organic substances, this c o n clusion can be applied in future investigations to any o t h e r solution of a p h a r m a c e u t i c a l p r e p a r a t i o n being s t e r i l i z e d by the f r e e z i n g method. In conclusion, it should be noted that technological control of the r a d i a t i o n - t r e a t e d p r e p a r a t i o n s of vitamin B12 in manufacturing conditions r e m a i n s as b e f o r e in a c c o r d a n c e with the p h a r m a c o p o e i a . All p r e p arations i r r a d i a t e d at - 7 8 and -196~ with the highest s t e r i l i z i n g dosages m e t t h e GFKh (State Standard for P h a r m a c e u t i c a l C h e m i s t r y ) r e q u i r e m e n t s based on the optical density D ratios of absorption m a x i m a at wavelengths 278 + 1, 361 ~: 1, and 548:~ 2 nm: Ds61/D54 S = 3.0-3.4 and Dsel/D2? S = 1.7-1.88 (see Table 1). F r o m the table it can be s e e n that during i r r a d i a t i o n with even s m s l l dosages at 18 and 0~ ation of these ratios f r o m p h a r m a c o p o e i a data o c c u r s .
a strong d e v i -
The authors p r o p o s e that the approaches developed in the p r e s e n t e x p e r i m e n t a l investigation can be used in many c a s e s to s t e r i l i z e p h a r m a c e u t i c a l s which cannot be s t e r i l i z e d by heat due to t h e r m a l instability. LITERATURE .
2. 3. 4. 5.
6. 7. 8. 9.
10. II, 12, 13. 14. 15. 16. 17.
972
CITED
V. V. Bochkarev, E. P. Parlor, V. V. Sedov, and V. G. Khrushchov, Khim. Farm. Zh., 7, 31 (1972). M. A. Tumanyan and D. A. Kaushanskii, Radiation Sterilization [in Russian], Moscow (1-974). E. V. Barelko, L. I. Kartasheva, and M. A. Proskurnin, Dokl. Akad. Nauk SSSR, 121, 677 (1958). J. Galatzeanu and F. Antoni, Int. J. Appl. Radiol., 17, 369 (1966). R. Blackburn, D. L. Cox, and G. D. Phillips, J. Chem. Soc. Faraday Trans. I, 68___,1687 (1972). R. Blackburn, A. J. Erkol, G. O. Phillips, etal.,J. Chem. Soc. Faraday Trans. I, 70, 1693 (1974). S. A. Grachev, B. A. Chakchir, and L. D. Ryabykh, Khim. Farm. Zh., w5, 50 (1973). K. Kishore, P. N. Moorthy, and K. Rao, Radiat. Effects, 29, No. 3,163 (1976). F. J. Ley, in: Manual on Radiation Sterilization of Medical and Biological Materials (Teeh. Rep. Series No. 149), Vienna (1973), p, 37. K. Kishore, P. N. Moorthy, and K. Rao, Radiation Effects, 27, No. 3,167 (1976). V. M. Sharpatyi, M. G. Nadzhimiddinova,etal., Khim. Vys.-~nerg., 32, 469 (1969). I. E. Makarev, B. G. Ershov, and A. K. Pikaev, Khim. Vys. Energ., 5, 187 (1971). ]3. G. E r s h o v and A. K. Pikaev, Dokl. Akad. Nauk SSSR, 209, 889 (1973). R. Blackburn, M. Kyaw, G. O. Phillips, et al., J. Chem. Soc. F a r a d a y T r a n s . I, 71, 2277 (1975). R. Blackburn, ]3. Iddon, J. S. Moore, et aL, in: Radiosterflization of Medical P r o d u c t s . P r o c e e d i n g s of a Symposium, Bombay, Int. Atomic E n e r g y Agency, Vienna (1975), p. 351. R. S t r o h e c k e r and H. M. Henning, Vitamin Assay. T e s t e d Methods, Weinheim (1965). S. M. Chaikovskaya and E. N. Druzhinina, Mikrobiologiya, 2 6 , 6 0 9 (1957).
