Molecular structures of investigated drug/drug raw materials: a) Sulfa drugs, b)SA, c) SFZ, d) STZ, e) SS, f) SMH, g) BHA, h) ALB. (Reproduced from [34], [36], [38], [37], [39] with permissions from Elsevier and Taylor & Francis).
Abstract
In this study, the feasibility of radiation sterilization of drugs/drug raw materials is investigated by using Electron Spin Resonance (ESR) spectroscopy. Experimental data and their theoretical correspondings are presented for Sulfanilamide (SA), Sulfafurazole (SFZ), Sulfatiazole (STZ), Sulfacetamide Sodium (SS), Sulfamethazine (SMH), Butylated Hydroxyanisole (BHA), and Albendazole (ALB). Unirradiated samples exhibited no ESR signal whereas the irradiated samples showed ESR spectra consisting of different number of resonance lines indicating that radiolytic intermediates were produced upon irradiation. Increase in the absorbed dose did not create any pattern change in the ESR spectra of these samples. The results of ESR microwave power studies indicated that saturation is observed to be faster for the studies held below room temperatures. Low radiation yield (G=0.1-0.5) calculated by ESR data for the gamma-irradiated samples showed that these materials can not be used as sensitive dosimetric materials. No significant differences were observed between FT-IR spectra of the unirradiated and irradiated samples and this result is considered to be in agreement with the relatively small G value derived from ESR studies. The decay rates of the ESR peak heights of the samples irradiated at different doses and stored at normal and stability conditions were found to be independent of the irradiation doses. The contributing radical species were determined to decay with different decay characteristics and the decay rates but decaying faster at stability conditions. The discrimination of the samples irradiated at even a low absorbed dose from unirradiated samples was possible for a long storage time after irradiation. Cooling the sample temperature down to room temperature did not create any pattern change in the ESR spectra of irradiated samples except slight reversible increases in the peak heights and at high temperatures irreversible decreases in the peaks heights were observed. Annealing studies indicated that the decay rates of the radical species at high temperatures were higher than the decay rates at low temperatures and the decay activation energies for the radical species were calculated by using Arrhenius plots. Spectrum simulation calculations were also performed and it was concluded that, the molecular ionic fragments and ionic radicals were the main responsible units from the resonance lines of ESR spectra of the gamma-irradiated sulfanomides such as SA, SFZ, STZ, SS, and SMH. Besides these two radical species, some other radical types were also likely produced after irradiation in STZ, SS, and SMH. Besides these two radical species, some other radical types were also likely produced after irradiation in STZ, SS and SMH. As for BHA and ALB, again two other type radical species were believed to produce upon irradiation. Basing on the derived experimental and theoretical data it was concluded that SA, SFZ, STZ, SS, SMH, BHA, and ALB could be safely sterilized by gamma radiation up to permitted drug sterilization radiation doses without causing high amount of molecular damages upon irradiation, and ESR spectroscopy could be used as a potential technique in monitoring the radiosterilization of the drugs, drug raw materials, and drug delivery systems containg present samples as active ingredient.
Keywords
- ESR
- Radiation Sterilization
- Sulfanilamide
- Sulfafurazole
- Sulfatiazole
- Sulfacetamide Sodium
- Sulfamethazine
- Butylated Hydroxyanisole
- Albendazole
1. Introduction
EN 552 and ISO 11137 publications recognize standard for implementing radiation technology on sterilization [19, 20]. For the gamma sterilization method, the reference absorbed dose for terminal sterilization is accepted to be
Nevertheless, besides its advantages, radiosterilization also has some drawbacks. Radiation cannot only eliminates microorganisms included in pharmaceuticals but also can cause a molecular decrease in the amount of active drug by destroying it and, therefore, creating reactive molecular fragments which may result in a toxicological hazard [2, 5, 18, 21-24]. Although the radiolytic products induced upon irradiation are generally in very small quantities [14], the characterization of the radio-induced radicals is very important and necessary, both to determine the feasibility of the radiation treatment and to control it. Therefore, to prove the safety of radiosterilization, the determination of physical and chemical features of the radiolytic products and mechanism of radiolysis should be determined [5, 6, 23, 24]. Thus, it is desirable to establish an effective experimental method to discriminate between irradiated and unirradiated drugs as the regulations of irradiated drugs vary from country to country. Besides, radiation effects on drug molecules cannot be generalized; thus, response to ionizing radiation of each molecule has to be individually studied.
ESR is a technique that is based on the absorption of electromagnetic radiation in the microwave frequency region by a paramagnetic sample when it is placed in an external magnetic field [32]. ESR resonance can occur basing on the equation (1) where h is the Planck’s constant, υ is microwave frequency, g-value is a constant that is dependent on the nature of the radical type (g = 2.0023 for a free electron), β is Bohr magneton and H is the applied magnetic field.
The results of the ESR studies performed in our laboratory relevant to the structural and thermal properties of the radicals produced in gamma-irradiated
2. Materials and methods
The investigated drugs/drug raw materials (Sulfanilamide, Sulfafurazole, Sulfatiazole, Sulfacetamide Sodium, Sulfamethazine, Butylated Hydroxyanisole, Albendazole) were provided from local drug providers and stored at room temperature in a well-closed container protected from light. No further purification was performed and they were used as they were received. Stabilization studies for the samples stored in stability conditions (75% relative humidity; 40°C) were also investigated for some group of drugs.
All irradiations were performed at room temperature (293 K) in dark using a 60Co gamma cell supplying a dose rate of ∼2.5 kGy/hr as an ionizing radiation source at the Sarayköy Establishment of Turkish Atomic Energy Agency in Ankara, Turkey. The dose rate at the sample sites was measured by a Fricke dosimeter and ESR investigations were performed on samples irradiated at different doses (5 kGy-50 kGy).
ESR measurements were carried out using both
IR spectra of unirradiated and gamma-irradiated samples were also recorded using Nicolet 520 FT-IR spectrometer and a comparison between the principal IR bands of interested drugs/drug raw materials was performed to monitor the radiolytic products silent to ESR spectroscopy.
Digitized signal intensity data derived from room temperature ESR spectrum of each sample irradiated at different doses were used as input data for spectrum simulation calculations. The simulation calculations based on models of different tentative radical species anticipating from the results of microwave saturation, variable temperature, decay at normal and stability conditions, dose response, and annealing studies were performed to determine the spectroscopic features of the contributing free radicals.
3. Experimental results and discussion
Experimental results are presented under two different subsections. The sulfanilamides which include
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3.1. General features of the ESR spectra
Although unirradiated samples exhibited no ESR signal, irradiated samples showed ESR spectrum consisting of different number of resonance lines depending on the sample investigated [16, 34-39]. The presence of ESR signals in irradiated but not in unirradiated samples is the indication that radiolytic intermediates were produced because of the irradiation mechanism. Increase in absorbed dose did not create any pattern change in the room temperature spectra of the samples. Thus, it was concluded that irradiation dose was not an important parameter in the formation of the shape of the ESR spectra of the investigated samples in the adopted radiation dose range. The ESR spectra of the investigated samples are given in Figure 1 with their assigned peak numbers for different gamma-irradiation doses.
Irradiated
3.2. Microwave power studies
Variations of the signal heights, which were measured with respect to base line and normalized to the receiver gain, masses of the samples, and the intensities of the standart, of these resonance peaks with applied microwave power in the range of 0.5-80 mW, were examined for all the investigated samples. The results of microwave power studies indicated that heights of the assigned peaks increase rather linearly at low microwave powers and saturate homogeneously or inhomogeneously broadened resonance lines at room temperature. Saturation is observed to be faster for the microwave saturation studies which were held below room temperatures. Theoretical functions best fitting to microwave saturation data were calculated assuming
3.3. Dose-response curves and dosimetric features of the samples
Gamma radiation produces damages in the molecular structures of the irradiated samples where the amount of damage will depend on the absorbed dose level of the sample. From the experimental results it was concluded that the discrimination of the irradiated samples at a dose as low as 0.5 kGy, from unirradiated samples was possible even long after irradiation due to the relatively high stabilities of the produced radical species, even if gamma radiation yield of the samples is low. A higher concentration of radicals, generated at the same absorbed dose of radiation, indicates a higher sensitivity of the substance toward the type of radiation used. For the samples, variations of the heigths of the resonance peaks assigned as numbers with absorbed gamma radiation doses were generally found to follow a
In the dosimetric studies, G value (
3.4. Long-term stability of the radiation-induced radicals
Room temperature stabilities of the radicals induced in the irradiated drugs/drug raw materials upon irradiation are as important as the radiosensitivity of these materials. ESR spectra of the samples which were open to air, were recorded in regular time intervals over a long period of time (approximately, 3 months) without changing the position of the sample in the microwave cavity throughout the experiment on normal conditions (
The decay of the peak heights of the samples irradiated at different doses and stored in well-closed container at normal and stability conditions was found to be independent of the irradiation dose. Contributing radicals were determined to decay much faster at stability conditions. The peak heights or spectrum area were observed to experience fast decreases during the beginning of the storage period; after the first days of storage, the decay rate of the induced radicals in the samples upon irradiation was decreased. Model based on the assumptions that different numbers of radicals for each sample with different decay kinetics were produced and that they undergo
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2 | A B |
48.11 (±19.65) 201.64 (±20.49) |
0.00053 (±0.00002) 0.14487 (±0.03659) |
0.98 | A B C D |
1483 (±107) 8400 (±420) 1178 (±95) 9800 (±450) |
0.98 |
3 | A B |
70.84 (±6.27) 155.24 (±13.02) |
0.00053 (±0.00002) 0.14487 (±0.03659) |
0.94 | |||
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A B C D |
3 9901 (±1) (±285) 2 7856 (±1) (±200) 46 11764 (±5) (±325) 167 9677 (±13) (±210) |
0.98 0.99 |
A | 0.4219 0.3996 0.6062 | 0.008 | ||
B | 0.5181 0.6004 0.3938 ±0.8484 ±0.8731 ±0.8193 |
0.224 |
3.5. Variable temperature study
The results of variable temperature studies found from the variations of the peak heights with temperature in the range of 290-110 K and 295-400 K are given in Figure 4. The sample was first cooled down starting from room temperature with an decrement of 20 K. Then, the temperature was increased up to high temperatures with the same increment. Spectra were recorded ∼5 min after setting the temperature. For the irradiated samples, cooling the sample down to room temperature did not create any pattern change in the spectra except slight reversible increases in the peak heights likely due to classical paramagnetic behavior of the contributing species, as obeying Curie’s Law. But irreversible decreases in the intensities at high temperatures were observed for the samples.
3.6. Radical decays in annealed samples
Basing on the drastic decreases observed in the peak heights of the samples above room temperature, annealing studies were also performed to determine the kinetic features of the radical species which were responsible from experimental ESR spectra of gamma-irradiated samples and calculating the activation energies relavant to the radical decay processes. Investigation of the contributing radical species by using ESR signal intensities in annealed samples is very important from the kinetic point of view. The fact that radical decay rates depend on the nature of the matrix containing radicals and annealing is a constant process with local diffusion of radicals and molecules in some softening of defects or irregularities [43]. At room temperature, the decay is very slow and many radical-molecule reactions observed in the liquid state are not observed in the solid state. Irreversible decreases in the intensities at high temperatures would be expected to originate from the decay of the radical species.
Thus, irradiated samples were annealed at different temperatures above room temperature; that is, below their melting temperature range for predetermined times. The decay rates of the radicals at high temperature are found to be higher than the decay rates at low temperatures. Signal intensity decay results obtained for the samples irradiated at different doses and annealed at different temperatures for different times were used to get the decay curves of the resonance lines (Figure 5). Experimental peak height decay data obtained for the samples annealed at different temperatures were used to calculate the decay constants of the contributing species at the annealing temperatures, assuming that radical species induced upon irradiation follow a
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310 | A B C D |
9 (±1) 52 (±7) 10660 (±325) 2451 (±95) |
0.96 | 358 | A B E F |
9 (±1) 7 (±1) 69 (±4) 31 (±3) |
0.98 | ||
348 | A B C D |
59 (±8) 380 (±30) 27000 (±430) 11000 (±360) |
0.97 | 393 | A B E F |
1436 (±115) 1234 (±80) 2755 (±180) 1257 (±105) |
0.99 | ||
365 | A B C D |
344 (±45) 1230 (±75) 46000 (±550) 17000 (±280) |
0.98 | 413 | A B E F |
4536 (±185) 3547 (±155) 7215 (±220) 5390 (±205) |
0.99 | ||
393 | A B C D |
1575 (±105) 5500 (±120) 68000 (±310) 35200 (±180) |
0.99 | ||||||
413 | A B C D |
1675 (±120) 6462 (±110) 102000(±220) 56207 (±205) |
0.99 | ||||||
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365 | A B H |
0.9 3.6 80.0 |
0.99 | 370 | L M |
118.65 3.17 |
0.98 | ||
375 | A B H |
1.1 7.9 180.3 |
0.99 | 380 | L M |
127.71 6.99 |
0.99 | ||
385 | A B H |
3.7 67.8 221.2 |
0.99 | 390 | L M |
172.71 7.43 |
0.97 | ||
390 | A B H |
4.5 118.1 389.2 |
0.99 | 400 | L M |
397.14 12.16 |
0.95 | ||
395 | A B H |
10.7 212.6 440.1 |
0.99 |
3.7. Radical type
Excited molecules are produced both directly and through radical-cations neutralization reactions [44]. They may decompose to radicals by rupture of chemical bonds. However, all species produced after irradiation are expected to undergo immediate germination termination reactions [43] due to cage effect. Consequently the amounts of the species responsible from the ESR spectra would be different depending on the capacity of these species participating to the germination reaction. Excited molecules and, as a result, radicals are localized along the track in region of high local concentration.
3.7.1. Proposed radical species for sulfanomides: SA, SFZ, STZ, SS, SMH
It is believed that the
3.7.2. Proposed Radical Species for BHA and ALB
3.8. Spectrum simulation calculations and proposed tentative radical species
Simulation calculations were performed to support the idea put forward with the species responsible from the observed experimental resonance peaks of ESR spectra of gamma-irradiated samples and to determine correct spectroscopic parameters of the contributing species. For the simulation calculations, the room temperature experimental signal intensity data obtained from the ESR spectra of the irradiated sample were used as input to perform simulation calculations. A model of different numbers of radical species depending on the samples was adopted throughout the calculations. Spectral parameter values determined by this technique for contributing radical species and theoretical ESR spectra calculated using the corresponding experimental counterpart are presented together in Table 4. The agreement between experimental and theoretical spectra is fairly good, which indicates that the modelings based on the expected species of different characteristic features explains well the experimental ESR spectra of gamma-irradiated samples.
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Radical Species | Relative Weight | Line width (G) | g factor | ||||
A | 7.32 | 2.43 | gII=2.0089 g(=2.0035 |
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B | 226.47 | 1.59 | gav=2.0052 | ||||
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Radical Species | g factor | Line Width (G) |
Relative Weight |
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A | 2.0042 | 5.10 | 0.52 | ||||
B | 2.0090 2.0047 |
1.78 | 0.48 | ||||
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Radical Species |
g factor | Line Width (G) |
AN AH (G) (G) |
Relative Weight |
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A | 2.0051 | 1.5 | - - | 0.06 | |||
B | 2.0106 2.0030 |
1.4 | - - | 0.21 | |||
C | 2.0059 | 0.8 | 13.7 3.6 | 0.01 | |||
D | 2.0039 | 8.0 | - 3.2 | 0.73 | |||
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Radical Species | g factor | Line Width (G) | Hyperfine Splitting A (G) |
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A | 2.0052 | 1.21 |
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B | 2.0092 2.0031 |
2.15 |
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E | 2.0033 | 4.25 | 19.7 | ||||
F | 2.0040 | 3.50 | 8.6 | ||||
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Radical Species |
g factor | Line Width (G) | Hyperfine Splitting AN(mT) AH(mT) |
Relative Weight | ||
A | 2.0072 | 0.21 | - - |
0.47 | |||
B | 2.0059 2.0075 |
0.35 | - - |
0.51 | |||
H | 2.0066 | 0.16 | 2.03 0.21 |
0.02 | |||
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Radical Species |
g factor | Line Width (G) | Hyperfine Splitting AN(mT) AH(mT) |
Relative Weight | ||
J | 2.0057 | 0.50 | - - |
0.834 | |||
K | 2.0047 2.0067 |
0.14 | 0.1584 0.9009 |
0.166 | |||
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Radical Species |
g factor | Line Width (G) |
Relative Weight |
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L | 2.0161 | 3.8 | 0.10 | ||||
M | 2.0088 | 6.16 | 0.90 |
3.9. FT-IR studies
IR spectroscopy is also used as a complementary technique for ESR spectrocopy in the determination of the radical species induced upon irradiation for the investigated samples. For this purpose, FT-IR spectra of
4. Conclusion
Experimental results derived for studied drugs/drug raw materials showed that they were not sensitive (small G value) to high energy radiations. Therefore, they could be sterilized by gamma radiation up to a radiation dose of 50 kGy without causing very much molecular damages upon irradiation. Also, they do not present the features of a good dosimetric material. That is, they cannot be used as an effective dosimetric material in the drug sterilization radiation dose limits. Nevertheless, the detection and discrimination of an unirradiated sample from irradiated samples turned out to be possible even at low radiation doses by ESR spectroscopy. Radical species created upon irradiation were found to decay much faster at stability conditions (40°C and 75% humidity) than at normal conditions as in the case of the samples in liquid forms. This point was considered presenting the possibility of diminishing even getting rid of radiolytical intermediates produced in irradiated samples. Therefore, it was concluded that gamma radiation produces relatively low amounts of radiolytic intermediates in the studies drugs/drug raw materials and that ESR spectroscopy could be used as a potential technique in monitoring the radiosterilization of drugs, drug raw materials, and drug delivery systems containing present samples as active ingredients.
Acknowledgments
I am grateful to
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