RADIATION STERILIZATION OF TRISAMINE SOLUTIONS FOR INJECTION S. A. Safarov, L. M. Kotlyarov, E. P. Pavlov, and V. V. Sedov

UDC 615.243.4:615.456].014.453

Results are given in the present work on microbiological and physicochemical investigations carried out with the aim of determining the possibility of sterilizing solutions of trisamine for injection with radiation. Trisamlne solution for injection is a 3.66% aqueous solution of 2-amino-2-hydroxymethyl1,3-propanediol (AHMP) manufactured by industry in hermetically sealed polyethylene vessels. EXPERIMENTAL Samples of trisamine prepared in the "Moskhlmfarmpreparaty" Industrial Association were subjected to irradiation in hermetically sealed packages made of low pressure polyethylene (type 10803-020, GOST 16337-77). The irradiation source was "~ and the irradiation dose rate was varied from 0.20 to 2.00 Gy/sec. With the aim of studying the effectiveness of sterilization, spores of Bae. 8ubtiZ~8 were put into the trisamine solution at approximately 107 cells per ml. The culture of spores was prepared according to the Pharmacopoeia procedure. Strains of Bae. 8ubtiZ~8 isolated in manufacture were used in the experiments. These microorganisms display high radiation stability. The D,o characteristic for them (doses of radiation causing a tenfold reduction in the number of populations of microorganisms) was 2-2.5 kGy. Suspensions of spores in trisamine solution were irradiated at various doses, and directly at the end of irradiation samples of liquid were inoculated onto Hottinger's agar (75 mg % amino nitrogen, pH 7.0-7.2). Cultures were incubated at a temperature of 32"C. Results were assessed first after 48 h and finally after 14 days. Data were processed statistically on the basis of the Pouisson equation. The A H ~ content was determined by the method specified in the Pharmacopoeia document for trisamlne (VFS 42-717-78). The AHMP was also isolated from irradiated and unirradiated solutions after lyophilization of trisamine samples and was subjected to analysis. The content and melting point of AHMP samples isolated from lyophilized irradiated and unirradiated solutions was determined as was also the pH of the latter. RESULTS The results of microbiological investigations are shown in Table i from which it is seen that irradiation of trisamine solutions at a dose of i0 kGy provided an inactivation factor (i.e., ratio between number of initial and irradiated populations) of approximately l0 s for radioresistant spores of the hay bacillus but irradiation at a dose of 15 kGy inactivated upwards of 107 radioresistant spores. TABLE i. Effectiveness of Sterilization of Trisamine Solutions for Injection with Gamma Irradiation Radiation doses,kGy

croorga:-

nism

Bae. subtilis 8211 Bac. subtilis 8113

o

I

I

f

,o

n u m b e r of v i a b l e ceils per ml*

(2,3=1=0,3). 107

(8,O=hO,4).lO s

(1,81-+-0,8).10'

(7,3-+0,2).101

(1,2=t=0,2) 9l0 T

(1.9+0,8).10 s

(5,7=h0,16).103

(2,1-4-0,9).101

*Here and in Table 2 the confidence range is stated for a probability level of 95%. Institute of Biophysics, Ministry of Public Health of the USSR, Moscow. Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 19, No. i, pp. 95-97, January, 1985. Original article submitted July 20, 1983.

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0091-150X/85/1901- 0068509.50

9 1985 Plenum Publishing Corporation

TABLE 2. Values of pH and Content of 2-AHMP in Solutions of Trisamine for Injection Unirradiated and Irradiated with a Dose of 15 kGy Sample tested Uhirradiated trisamine solution Irradiated trisamine solution

Drug content, [Solution mg/ral* ~[pH"

36,704-0,92

10,1

37,704-1,10

10,0

*Arithmetic mean of i0 determinations. It is known that the size of the sterilizing dose of radiation depends both on the radiation resistance of the microorganisms contaminating the drug and on the amount of viable microorganisms present in the objects before sterilization [I]. Under modern conditions two methods are used for selecting sterilizing doses of ionizing radiation. One of them, morerecently, is based on calculation of the inactivation factor of microorganismson irradiation. In this case the objects being tested are seeded with radioresistant microorganisms, irradiated at various doses, and then the number of microorganisms that have been inactivated is determined. The second method is based on calculation of the radiosensitivity of the microflora seeding the objects according to the D,o characteristic. To calculate the sterilizing dose the found values of D,o were processed in a computer with a program also taking into account the initial microbial contamination of production and the safety coefficient of sterilization. The first method of selecting sterilizing doses was used in the present study. A high degree of inactivation was achieved in our investigations for highly radioresistant microorganisms of irradiation of trlsamine solutions at a dose of I0 kGy. This means that in manufacture under good hygienic conditions where the initial microbial contamination of the preparation does not exceed i0 per ml it is fully permissible to use a dose of radiation for sterilization equal to i0 kGy since highly radioresistant microorganisms (usually spore-forming gram-positive bacteria) are found in the industrial microflora of pharmaceutical contamination (within the limits i00-i000 cells per ml) it is necessary to increase the radiation dose to 15 kGy, In accordance with the rules operative in this country regulating radiation sterilization of drugs, preparations with a higher level of microbial contamination are not subject to radiation sterilization. Results are reflected in Table 2 of the determination of AHMP in irradiated and unirradiated solution of trisamine and of the pH of these solutions. A dose of 15 kGy did not influence the content of main substance or the solution pH. No differences were detected in the melting point of AHMP isolated from the irradiated and unirradiated solutions which were 170171 and 169-171~ respectively. An increase in dose rate from 0.20 to 2.00 Gy/sec did not lead to a change in the statistical data (in Table 2 statistical data applying to a dose rate of 1 Gy/sec are given). With the aim of establishing the nature of the products of radiolysis an enrichment of these products was carried out in trisamine solutions irradiated with a dose of 15 kGy. In other experiments to accumulate these products in trisamine solutions the latter were irradiated with high doses of gamma irradiation which exceeded the sterilizing doses by tenfold or more. In these solutions among the radlolysis products detected by known methods of analysis were ammonia [3, 4, 6], formic acid [7, 8], and dimerized compounds of the initial AHMP molecule withpolyalcoholic chemical functional groups (molecular weight was 242 • 15 by mass spectroscopy). The l~tter were isolated as "start" products (Rf = 0.0) after chromatography on FN-14 paper (system acetone-ethyl alcohol, ].:5) of the irradiated solutions (dose 30-50 kGy). The radiochemical extent of decomposition of A H ~ determined at doses of radiation of 30 kGy and more was 7.9 • 0.9 molecules per i00 eV. The relative error of the method of analysis of AHMP was more than 10% and the sensitivity of the methods of analysis noted above for the products of irradiated solutions did not permit confident assessment of the amount of products, by the method of extrapolation, resulting at a radiation dose of 15 kGy. The yield

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of radical products (determined as described in [5]) presumably consisting of C(CH=OH)~ and NH2C=(CH2OH)2, was assessed at a value approximately one order of magnitude lesso It might be supposed that this is linked with the radiochemical decomposition of AHMP as a result of ion--molecule reactions which are more probable in a medium of p H ~ i 0 (see Table 2) under conditions of electrolytic dissociation. The formation of the stable products named above indicates fission of C--N snd C--C bonds. However, the sequence of the conversion processes has not been finally established by us although it was found that the presence in an irradiated soltuion of acceptors (N=O, sulfanilamides) of a hydrated electron (e-aq) depresses the rate of formation of NH~ and acceptors of OH radicals (alcohols, glucose, etc.) prevent the formation of dimerized AHMP molecules. The most effective protective substances were the sulfites of sodium, potassium, and ammonia. The presence of 1% sodium or potassium sulfite in the irradiated solution proved to be sufficient to prevent the formation of dimerized products and showed an appreciable protecting influence on the preparation up to an irradiation dose of 25-30 kGy. The quantitative characteristics obtained in this way did not differ within the limits of experimental error from those given in Table 2 and at an irradiation dose of 25 kGy were 36.6 • 1.09 mg/ml AHMP and pH 10.3 (n = i0). Glucose showed a similar protective influence at a concentration of 5-10% (range investigated by us at an irradiation dose of 25 kGy). The latter circumstance has special importance since, according to instructions for the use of trisamine in hypoglycemia, glucose is administered intraveneously simultaneously with trisamine. In the absence of sodium sulfite or glucose, solutions of trisamine irradiated with a dose of 25 kGy in a polymeric container became yellowish with time (on storage for 5-6 months), and an absorption band appeared with a maximum at 455 nm with an insignificant absorption density ( D I c m = 0.i). Consequently for the sterilization of solutions of trisamine for injection by the radiation method a dose of gamma irradiation equal to i0 and 15 kGy is recommended in cases of initial microbial contamination of the preparation of up to I0 and i000 microorganisms per ml respectively. The introduction of additives such as sodium or potassium sulfite, glucose or alcohols reduces appreciably the extent of radiochemical conversions of the preparation. The authors are grateful to E. A. Tyrina for active participation in this work. LITERATURE CITED i.

V.V.

Bochkarev, E. P. Pavlov, V. G. Khrushchev, et al., Khim.-farm. Zh., No. 2, 145-

147 (1978)o 2. 3. 4. 5. 6. 7. 8.

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V . V . Bochkarev, V. I. Vashkov, E. P. Pavlov, et al. Zh. Mikrobiol., No. 8, 56-60 (1973). State Pharmacopoeia of the USSR, 10th edn. [in Russian], Moscow (1968), p. 868. International Pharmacopoeia, 2nd edn. [in Russian], Moscow (1969), p. 50. S . A . Safarov, in: Disinfection and Sterilization. Prospects for Progress [in Russian], Volgograd (1983), pp. 116-118. W . L . Doyle and I. H. Omoto, Anal. Chem., 22, 603 (1950). E . P . Kennedy and H. A. Barker, Anal. Chem., 23, 1033 (1951). K . R . Tsai and F. Ying, Anal. Chem., 21, 818 (1949).