Sterility and Radiostability of Amoxicillin and Cefaclor Antibiotics Sterilized by Gamma Irradiation

Aim: The present investigations aimed at studying the effect of sterilization by gamma irradiation on amoxicillin and cefaclor antibiotics. They have been irradiated in solid dry state and the probable changes in physicochemical and microbiological properties were studied Place and Duration of Study: The study was carried out from 2011 to 2013 in the Drug Radiation Research Department, Egyptian Atomic Energy Authority. Methodology: Amoxicillin and cefaclor compounds in solid states were exposed to γ Original Research Article British Journal of Pharmaceutical Research, 4(17): 2046-2067, 2014 2047 irradiation in air atmosphere at room temperature, with a dose of 25kGy and afterwards they had been subjected to microbiological and analytical tests checking their sterility and antibacterial activitiy it was tested against different pathogenic bacterial species by measuring MIC using Microdilution technique and microplate reader. Then their chemical stability were evaluated by different techniques. EPR, FTIR, UV analysis, mass spectroscopy, and melting point. Results and conclusion: The results showed that the majority of initial unirradiated compounds had a slight degree contamination with Bacillus, Micrococcus genera, and fungi. By applying γ irradiation at 25kGy it showed sterilization of the tested antibiotics and keeping their antibacterial activity. The EPR analysis results showed formation of free radicals. The other analytical tests (FTIR), (UV) analysis ,mass spectroscopy, and melting point results proved that the antibiotics analyzed are radioresistant and can be sterilized by irradiation with a dose of 25kGy, without any detrimental effect on their properities and antibacterial activity.

Sterilization is intended to kill or remove all vegetative and sporing microbes from the environment or material [4]. In the case of medical substances, choice of the sterilization method depends on the type, properties, and production method of the substance in question. Irradiation of drugs, or other medical products, by a suitable dose of ionizing radiation, conducted in an appropriate environment, ensures sterile conditions [5][6][7][8]. Basic terms and sterilization protocols can be found in ISO11137-1 [9]. Radiation sterilization is especially useful in the case of thermolabile products, because irradiation causes only a small rise in the temperature of sterilized substances [8] In the case of gamma radiation, a substance to be sterilized does not directly interact with the reagents and, as a result, lacks any traces of chemical pollution [2] Moreover, packaged products may also be irradiated as gamma radiation possesses excellent penetrative properties; this constitutes one of its economic advantages [1][2]. Medical products subjected to gamma radiation sterilization do not become radioactive [2]. It was found that the sterility assurance level (SAL) of 10 -6 is normally achieved at 25kGy according to pharmacopoiea, which is a dose generally applicable to products manufactured under good manufacturing practice [10,11].

Materials
In the present study Chemotherapeutic agents as raw materials of antibiotics were supplied from companies agents in Egypt ,i.e. amoxicillin samples were mainly supplied by Glaxo Smith kline, and cefaclor from Ranbexy.

Irradiation
The process of irradiation was carried out at NCRRT. It was performed by using Cobalt 60 source (Gamma cell 4000-A-India) at the dose rate 2.78kGy/h at activity was 2914 curie (ci) All samples were stored at room temperature in air, in the dark. Then 0.1g of each sample was placed in colourless jars and closed with a plastic stopper, then every tested antibiotic was subjected to a radiation dose equal to 25kGy according to [10].

The microbial load isolation
The isolated strains were recovered using Nutrient agar (oxoid) and Tryptone glucose yeast extract agar (oxoid), and Sabraud's dextrose agar then identified by, API20Strep for streptococci/enterococci and API50CHB for Bacillus species and other endospore-forming genera (bioMérieux, Marcy 'Etoile, France) except one strain was identified according to the method described by [12]) biolog TM ID assay biochemical features were analyzed using 96 well BIO LOG TM ID technology (Biolog Inc Haywar, CA, USA) the GN 3 (Gram positive bacteria). The data were analyzed with Microlog 3, 5 software (Biolog inc.)

Microdilution technique (mIcrobiological assay and MIC)
The microbial inoculum of the test organisms under Study (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633), and Pseudomonas aeruginosa ATCC 27853, were prepared according to the method described by [13]).Isolates were removed from storage, streaked onto a Trypticase soy agar plate (Becton Dickinson Microbiology Systems, Cockeysville, Md.), and incubated for 18 to 24h at 37ºC. A working bacterial suspension was prepared by suspending 3-5 isolated colonies in 3 ml of Mueller-Hinton media. The turbidity of this suspension was carefully adjusted photometrically to equal that of a 0.5 McFarland standard. For the test, the final inoculum was further diluted in Mueller-Hinton media to achieve a final concentration of 0.5×10 5 CFU/ml. Microdilution plate was prepared according to the method described by [14]) as the following: Sterile 96-well U bottomed, microtitre plates (Tarsons, India) were used. The stock suspensions of the drugs were prepared in Mueller-Hinton medium and 0.2ml of the highest concentration of each drug was added to the respective wells of the first row of the plates except the first and the last well.

The plates were read by two methods
a-Visually by comparison with the drug free controls. b-With micro plate reader at a wavelength of 450nm

Sterility Test
Sterility tests were carried out according to USP 34 [15] by filtration method.

Statistic Analysis
The microbiological stability tests were carried out in triplet. For comparison and statistical analysis, observations were obtained for each combination of isolate and antibiotic agent. then was analyzed by statistics program SPSS-version 15 (independent sample .T. test).

UV Spectrophotometry
UV spectrophotometric determinations were carried out on aqueous solutions of the unirradiated and irradiated samples of antibiotic using JASCO UV 560 spectrophotometer (JASCO International Co. LTD ,Japan).

IR Spectroscopy
The dry powder of both unirradiated and irradiated samples of antibiotics were mixed and compressed with KBR then analysed by (FT/IR-6300 FT-IR Spectrometer).

Mass Spectroscopy
The dry powder of antibiotics was dissolved in 95%methanol. Then analayzed by mass spectrophotometer by direct inlet unit (DI-50) of Shimamadzu GC/MS-QP5050A.

Electron Paramagnetic Resonance (EPR)
It has matured into a powerful, versatile, non-destructive, and non intrusive analytical method using Bruker EMX spectrometer (X-band) product of Bruker, Germany.

Melting Point
The samples were analyzed in solid state by using stuart melting point apparatus (Stuart Analogue Melting Point Model SMP11).

The Microbial Load Isolation
The results obtained from isolation and identification of isolated strains from antibiotics were shown in (Table 1).

Evaluation of the Stability of Biological Activity of Antibiotic Amoxicilin & Cefaclor
Evaluation the stability of biological activity of the both antibiotics amoxicillin and cefaclor was tested against the previously mentioned four standard bacterial organisms (2.3.2); the effect of the antibiotic was measured by micro plate reader. It was found that the antibacterial activity of un-irradiated amoxicillin and cefaclor are the same as the irradiated samples at 25 kGy. The results of evaluation of biological activity of antibiotic amoxicillin and cefaclor against the bacterial test organisms are summarized in (Figs. [1][2][3][4][5][6][7][8]. In order as follows, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, and Pseudomonas aeruginosa ATCC 27853, respectively when unirradiated sample (blue) and irradiated sample (red). Pseudomonas showed the same resistance to both unirradiated and irradiated antibiotics samples it showed growth for all used concentrations. Pseudomonas auregienosa. recorded a great resistance to many antimicrobials [16], and specially resistance to amoxicillin and cefaclor [17] MIC values are shown in (Table 2).

UV and Discoloration
Irradiation discolored the amoxicillin powders from off-white to yellow, as shown in (Table 3) and (Figs. 11,12) but kept the same λ max absorption peak. Irradiation did not change the color of cefaclor as shown in (Table 4) and (Figs. 13,14) and kept also the same λ max absorption peak.

IR Spectroscopy
The infrared spectrum of unirradiated amoxicillin and cefaclor showed the same strong absorption peaks of irradiated at dose 25kGy as shown in (Tables 5-6) and (Figs. [15][16][17][18] for amoxicillin and cefaclor respectively.

Mass Spectroscopy
The mass spectra of the unirradiated and irradiated samples of both antibiotics showed almost the same fragmentation pattern. Summarized in (Tables 7 and 8) and (Figs. [19][20][21][22] for amoxicillin and cefaclor respectively.

Electron Paramagnetic Resonance (EPR)
Unirradiated antibiotics samples showed no signal for amoxicillin but for cefaclor there was a small singlet. The irradiated samples showed a slight increase in peak intensity indicating formation of free radical after irradiation. (Figs. 23 and 24) for amoxicillin and (Figs. [25][26] for cefaclor.

Melting Point Result
The results showed there is no difference in the melting point of the antibiotics before and after irradiation (Table 9).

DISCUSSION
The results of the of microbiological purity tests of unirradiated antibiotics revealed that there was a different range of contamination with microorganisms. The microorganisms isolated were Gram positive Bacillus sphearicus and Bacillus pumilus for amoxicillin and Grampositive Microccocus luteus and Bacillus subtilis for cefaclor and a slight contamination with fungi for both drugs. These results are in accordance with [19] who found that some penicillins and their salts, gentamycin and neomycin had been contaiminated to a slight degree by bacteria from genera Bacillus and Micrococcus and fungi.
After gamma irradiation of the antibiotics under study at 25kGy their sterility was tested and it was found that there was no observed microbial growth.This result is inaccordance with [20] who recorded that some penicillins (piperacillin, cabenicillin and benzyl penicillin ) did not reveal any microbial growth after gamma irradiation at 25kGy.
Biological & antibacterial activity of the antibiotics that evaluated by microdilution technique suscepility tests ,was not affected significantly (P>.05). These results were in accordance with [21] who reported that the biological activity of penicillin and ampicillin was not affected significally after irradiation at doses 10 and 25kGy.
UV absorption of unirradiated sample of amoxicillin was the same as irradiated sample at the λ max 280, except darkening from off-white to yellow color was observed in the irradiated sample, same results were obtained by [22] that recorded that gamma radiation has effected on the color of the ampicillin and crystalline penicillins, at 25kGy caused discoloration. While for cefaclor no change in color was observed and absorption was at λ max 580 nm. These results are in agreement with [23] who found that no changes of the UV spectra were noticed up to 50kGy for cephradine.
The melting point of unirradiated and irradiated amoxicillin was the same the mass spectra of unirradiated and irradiated amoxicillin samples showed nearly the same fragmentation pattern. Also similar results were recorded by [24] that there were no differences of melting point of both unirradiated and irradiated cephradine.
The EPR results for unirradiated and irradiated amoxicillin and cefaclor showed that there is a slight increase in peak intensity after treating with gamma indicating formation of free radicals, [25] mentioned that in the commercial market of drugs, radicals should be detected up to two years after irradiation. Also our results are ingreement with [22,26] who observed that decrease in free radicals concentration for irradiated samples of ampicillin is as a function of storage time can be explained by the interaction of the free radicals with oxygen molecules O 2 .
It was found that the FT-IR of unirradiated and irradiated samples of amoxicillin and cefaclor have the same characteristic absorption bands indicating that there is no change in the structure of both antibiotics, also [23] observed that infrared spectra of both unirradiated and irradiated cephradine were identical.
The mass spectra of unirradiated and irradiated amoxicillin and cefaclor samples showed nearly the same fragmentation pattern, the same result was obtained by [27] who found that the fragmentation pattern of MS-MS spectrum of cefatoxime and those of radiolytic compounds were very similar suggesting that they were structural analogues to the main drug.
These collective results disagree with those obtained by [28] who reported that the sterilized penicillin-derived antibiotics: piperacillin, ampicillin, and crystalline penicillin antibiotics drugs following gamma sterilization showed very different properties therefore may reduce the therapeutic properties of the pharmaceuticals. Free radicals formed in sterilized drugs may have an effect not only on the pharmacological activity of the drug [29], but also on its pharmacokinetic properties [30], which is often neglected in the scientific literature.

CONCLUSION
This study showed that amoxicillin and cefaclor antibiotics are radioresistant from a chemical point of view and for the antimicrobial activity our tests also showed no change in the antibiotics activity i.e no qualitative or quantitative differences were observed. Therefore they could be suitable candidates for radiosterilization studies by gamma rays in solid state. Briefly, this research considered the feasibility of radiation sterilization for cefaclor and amoxicillin antibiotics in dry state.

CONSENT
Not applicable.

ETHICAL APPROVAL
Not applicable.