Application oF EPR spectroscopy to examination oF gamma

Application oF EPR spectroscopy to examination oF gamma

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Abstract
Streszczenie
EPR spectra of erythromycin after gamma-irradiation by
using a 60Co source at a dose of 25 kGy were analysed. Irradiation was done in THERATRON 780E. Spectroscopic
measurements were performed by the use of an X-band
(9.3 GHz) EPR spectrometer with magnetic modulation of
100 kHz. Concentration of paramagnetic centers after irradiation in the studied drug was 5x1019 spin/g and its value
decreases with increasing time of storage. Influence of microwave power in the range of 0.7-70 mW on amplitudes
and linewidths of the erythromycin’s lines was evaluated.
The EPR lines saturated at lower microwave powers when
storage time increased. Increase of spin-lattice relaxation
time in gamma-irradiated erythromycin points out that its
molecular structure changes during storage.
Analizie poddano widma EPR gamma napromieniowanej erytromycyny. Próbki zostały gamma napromieniowane dawką 25 kGy przy użyciu aparatu THERATRON
780E zawierającego izotop kobaltu 60Co. Pomiary spektroskopowe zostały przeprowadzone przy użyciu spektrometru EPR na pasmo X (9.3 GHz) z modulacją pola
magnetycznego wynoszącą 100 kHz. Koncentracja centrów paramagnetycznych w badanym leku wynosiła
5 x 1019 spin/g a wartość ta spadała wraz z czasem przechowywania próbki. Analizowano wpływ mocy mikrofalowej
w zakresie 0.7-70 mW na amplitudę i szerokość linii EPR
erytromycyny. Wraz z czasem przechowywania próbki linie
EPR nasycają się dla niższych mocy mikrofalowych. Przyspieszenie czasu relaksacji spin-sieć wskazuje na zmiany
w strukturze chemicznej leku podczas jego przechowywania.
Key words: paramagnetic centers, EPR, microwave saturation, gamma-irradiation, erythromycin
Słowa kluczowe: centra paramagnetyczne, EPR, nasycenie mikrofalowe, promieniowanie gamma, erytromycyna
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In the last years drug sterilization by gamma-irradiation
is very active investigate, because there are a lot of advantages of this method: high penetrating power (drugs can be
sterilize in their final containers), low chemical reactivity,
isothermal character of the gamma-rays, and radiosensitivity of microorganism [1]. The knowledge about formation
of paramagnetic centers in drugs during radiosterilization is
still not enough. There is known EPR studies of gammairradiated drugs [2-11], but a lot of antibiotics were not examined so far. Paramagnetic centers in radiosterilized antibiotics may be responsible for toxic effects in organism.
In this work we examined radiosterilized antibiotic,
which is often used in dermatology [12-14]. The aim of this
work was to study concentrations of paramagnetic centers
and spin-lattice interactions in gamma-irradiated erythromycin. Evolution of paramagnetic centers during storage of
this antibiotic after irradiation was described. We compared
microwave saturation of erythromycin’s EPR spectra for
different times after samples irradiation.
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Radiosterilized erythromycin was studied. Chemical
structure of its molecule is presented in Figure 1 [14]. Erythromycin is a macrolide antibiotic produced from a strain of
actinomycetes Saccaropolyspora erythraea. It interferes
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netic modulation of 100 kHz. Microwave frequency was
measured with MCM 101 recorder.
We used ultramarine as the reference for paramagnetic
centers concentration. Additionally we measured for ultramarine and for sample EPR signals of the second reference-a
ruby crystal.
Concentration of paramagnetic centers in the sample was
calculated as follow:
Fig. 1. Chemical structure of
erythromycin [14].
with protein synthesis in sensitive bacterial cells [1214]. Erythromycin binds to the 23S rRNA molecule in
the 50S of the bacterial ribosome, blocking the exit of the
growing peptide chain thus inhibiting the translocation
of peptides. Erthromycin is active against aerobic and
anaerobic gram-positive cocci, Mycoplasma pneumoniae, Legionella and Bordetells pertussis. Erythromycin
is also used for skin infections. Erthromycin is available
in enteric - coated tablets, slow release capsules, oral
suspensions, ophthalmic solutions, ointments, gels and
injections [12]. Paramagnetic centers may be important
in radiosterilized erythromycin existing in gels or injections.
In this work erythromycin tablets were irradiated with
a 60Co source. On the basis of Polish and European Norms
[15] of radiosterilization erythromycin was given a dose of
25 kGy.
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EPR spectra of erythromycin in solid state at room temperature were obtained as first-derivative curves. The measurements we performed by the use of an X-band (9.3 GHz)
electron paramagnetic resonance spectrometer with mag-
N = nu [WuAru/Pu][Pp/(WpArpmp],
where: nu - amount of paramagnetic centers in reference
(ultramarine); Pu, Pp - areas under the absorption curve for
ultramarine and sample, respectively; Wu, Wp - receiver gain
for ultramarine, and sample, respectively; Aru, Arp - receiver
gains for ultramarine, and sample, respectively; mp - mass of
the sample. Double integration was done for calculation of
area under the absorption curves.
Changes of integral intensity of erythromycin’s EPR
line with increasing time of storage after irradiation were
analyzed. Concentration of paramagnetic centers is proportional to integral intensity.
Influence of microwave power from 0.7 to 70 mW on
amplitudes and linewidths of EPR spectra was examined.
Correlations between parameters of EPR lines and microwave power let us to determine type of paramagnetic centers
distribution in the sample. Decrease of EPR amplitudes and
broadening of EPR lines are characteristic for their homogenous distribution in sample [16]. For inhomogeneous distribution amplitudes do not change with microwave power
after reaching the maximum and linewidths remain constant
independently of microwave power [16]. We compared microwave saturation of erythromycin’s EPR lines at 1, 9, and 37
days after gamma-irradiation. Changes in microwave saturation
of EPR lines reflect changes of chemical structure of sample.
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Fig. 2. EPR spectra of gamma-irradiated erythromycin for times after
irradiation: 3, 5, 10, and 15 days. The data are presented for attenuation of 15 dB.
EPR spectra were not obtained for the original erythromycin, while strong paramagnetism characterized the
studied gamma-irradiated antibiotic. For gamma-irradiated
erythromycin we observed complex EPR spectra. Exemplary EPR spectra of erythromycin for 3, 5, 10, and 15 days after irradiation are compared in Figure 2. Paramagnetic centers concentration after 3 hours from irradiation was 5x1019
spin/g. The amount of paramagnetic centers in erythromycin
depends on storage time. Change of integral intensities of
erythromycin’s EPR line with increasing time after sample
irradiation is presented in Figure 3. The integral intensity
strongly decreases during several days after irradiation.
From 10 days after radiosterilization only slow decrease of
erythromycin’s EPR intensity we detected. The examined
gamma-sterilized antibiotic sample remains paramagnetic
even after 80 days of storage.
Lineshape of the analysed EPR spectra strongly depends
on microwave power. Influence of microwave power on
erythromycin’s EPR spectra 3 hours after drug irradiation
is shown in Figure 4. Complex character of the spectra is
not so visible at higher microwave powers (attenuation: 0.5
dB).
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Fig. 3. Changes of integral intensity of erythromycin’s EPR line with
increasing time of storage after gamma-irradiation.
Influence of microwave power on amplitudes and linewidths of
EPR spectra of erythromycin after 1, 9, and 37
days of gamma-irradiation is presented in Figures 5 and 6, respectively. Amplitudes rise with
increasing of microwave
power and decrease for
higher microwave power
value (Fig. 5). A weak
broadening of EPR lines
with increasing of microwave power was observed
(Fig. 6). Correlations in
Figures 5 and 6 are typical for homogenous distribution of paramagnetic
centers in the sample, so
gamma-irradiation forms
paramagnetic centers in
whole tablet.
While unirradiated
erythromycin presents
no EPR signal, for gamsamples
Fig. 4. Influence of microwave power ma-irradiated
on EPR spectra of gamma-irradiated was observed complex
erythromycin. The spectra were me- EPR spectrum (Fig. 2).
asured 3 hours after irradiation with
Lineshape of the EPR
attenuations 15, 10, 5 and 0.5 dB.
spectra changes with microwave power (Fig. 4)
and with storage time (Fig. 2). Complex shape of resonance
absorption curve indicates the presence of more than one
type of free radical species induced by gamma radiation in
erythromycin. For the studied drug we obtained apparent
g value of 2.0033. Unpaired electrons in free radicals in
irradiated erythromycin are probably localized on oxygen
atoms. It seems that glicoside bounds are especially sensitive for gamma irradiation. Probably rupturing of glicoside
bounds forms oxygen free radicals in gamma-irradiated
erythromycin.
Free radicals in gamma-irradiated erythromycin are not
stable (Fig. 3). The number of free radicals decay was observed. Decrease of integral intensity of erythromycin’s EPR
line/free radicals concentration is caused by interactions with
oxygen molecules in the sample environment. This effect is
the most intensive during the first stage after sterilization. It
can be concluded that radiosterilized erythromycin should be
used in therapy after several days from gamma-irradiation.
Radiosterilized antibiotic should contain the lowest amount
of free radicals. Free radicals of sterilized antibiotic may be
responsible for toxic effects in organism stimulated by their
reactions with different biological structures.
Continuous microwave saturation of gamma-irradiated
erythromycin’s EPR lines indicates that slow spin-lattice relaxation processes occur in this sample (Fig. 5). EPR lines
of the gamma-irradiated erythromycin saturate at low microwave powers independly on time after irradiation (Fig.
5). EPR lines of the antibiotic saturate at lower microwave
power for longer storage times.
Microwave saturation of EPR lines we examined to check
the hypothesis about changes of erythromycin’s chemical
structure during storage. Time evolution of molecular struc-
Fig. 5. Influence of microwave power on amplitudes of EPR lines of
erythromycin for 1, 9, and 37 days after gamma-irradiation. Mo – total
microwave power (70 mW) produced by klystron, M – microwave
power used during the measurement.
Fig. 6. Influence of microwave power on linewidths of EPR lines of
erythromycin for 1, 9, and 37 days after gamma-irradiation. Mo – total
microwave power (70 mW) produced by klystron, M – microwave
power used during the measurement.
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ture and paramagnetic centers system in sterilized drug may be
responsible for negative interactions in organism. As result the
efficiency of erythromycin may be considerably lower.
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Application of EPR spectroscopy to examination of
gamma-irradiated erythromycin indicates that: 1) Paramagnetic centers homogenously distributed in tablet are formed
during gamma-irradiation of erythromycin. 2) Paramagnetic
centers in irradiated erythromycin are not stable. 3) Slow
spin-lattice relaxation processes exist in irradiated erythromycin. 4) Spin-lattice relaxation time increases with increasing of storage time of the drug what indicates changes
of its chemical structure.
References
1. Basly JP, Basly I, Bernard M. Influence of radiation
treatment on doubutamine. Int J Pharm 1998; 170: 265269.
2. Basly JP, Longy I, Bernard M. ESR identification of radiosterilized pharmaceuticals: latamoxef and ceftriaxone. Int J Pharm 1997: 158, 241-245.
3. Basly JP, Basly I, Bernard M. ESR spectroscopy applied
to the study of pharmaceuticals radiosterylization: cefoperazone. J Pharm Biomed Anal 1998; 17: 871-875.
4. Gibella M i wsp. Electron spin resonance of some irradiated pharmaceuticals. Rad Phys Chem 2000; 58: 69 -76.
5. Varshney L, Dodke PB. Radiation effect studies on anticancer drugs, cyclophosphamide and doxorubicin for radiation
sterilization. Rad Phys Chem 2004; 71: 1103-1111.
6. Fauconnet AL, Basly JP, Berdard M. Gamma radiation
induced effects on isoproterenol. Int J Pharm 1996; 144:
123-125.
7. Basly JP, Basly I, Bernard M. Electron spin resonance
identification of irradiated ascorbic acid: Dosimetry and
influence of powder fineness. Anal Chim Acta 1998;
372: 373-378.
8. Onori S i wsp. ESR identification of irradiated antibiotics:
cephalosporins. Appl Rad Isotop 1996; 47: 1569-1572.
9. Basly JP, Doroux J, Bernard M. Radiosterylization dosimetry by ESR spectroscopy: application to terbutaline.
Int J Pharm 1996; 142: 247-249.
10. Yurus S, Ozbey T, Korkmaz M. ESR investigation of
gamma irradiated sulbactam sodium. J Pharm Biomed
Anal 2004; 35: 971-978.
11. Katušin – Ražem B i wsp. Radiation sterilization of ketoprofen. Rad Phys Chem 2005; 73: 111-116.
12. Kostowski W, Herman ZS. Red. Farmakologia. Podstawy farmakologii. Wydawnictwo Lekarskie PZWL. Warszawa 2003.
13. Janiec W, Krupińska J. Red. Farmakodynamika. Podręcznik dla studentów farmacji. Wydawnictwo Lekarskie PZWL. Warszawa 2002.
14. Zejc A, Gorczyca M. Red. Chemia leków. Wydawnictwo
Lekarskie PZWL. Warszawa 2002.
15. Polish Norm PN-EN 552, Warszawa 1999.
16. Wertz JE, Bolton JR. Electron Spin Resonance. Elementary Theory and Practical Applications. Chapman and
Hall. New York, London 1986.
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