Analytica Chimica Acta 535 (2005) 331–337

ESR identification of�-irradiated redoxon and determination of ESRparameters of radicals produced in irradiated ascorbic acid

Mustafa Polat∗, Mustafa KorkmazPhysics Engineering Department, Hacettepe University, Beytepe, 06800 Ankara, Turkey

Received 22 September 2004; received in revised form 6 December 2004; accepted 6 December 2004Available online 19 January 2005

Abstract

In the present work, electron spin resonance (ESR) identification of irradiated redoxon and its potential use as a normal and/or accidentaldosimetric material were investigated in details. The ground redoxon samples exhibited a single weak resonance line of peak-to-peak width�Hpp = 0.9 mT appearing atg= 2.0057.�-Radiation produced many resonance lines beside four different intense lines (denoted asI1, I2, I3andI4) of different spectral features in the studied dose range of 2.5–25 kGy. Experimental dose–response curves associated withI1, I2, I3 andI tures it wasc s.S le irradiateda e formationo e discussed.©

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4 resonance lines were found to follow an exponential function. From stability and kinetic studies at room and above room temperaoncluded that radicals contributing toI3 andI4 resonance lines were more stable than the radical species contributing toI1 andI2 resonance lineimulation calculations based on the room temperature ESR intensity data of an ascorbic acid (active ingredient of redoxon) sampt 10 kGy was performed to determine the structure and spectral parameters of the radiation-induced radical species involved in thf experimental ESR spectrum of ascorbic acid. The contributions of these species to the ESR spectrum of irradiated redoxon wer2004 Elsevier B.V. All rights reserved.

eywords:Dosimetry; ESR; Temperature; Kinetics; Ascorbic acid

. Introduction

Radiation sterilization technology and its applications inhe manufacture of pharmaceuticals and cosmetics are beingore actively investigated now than at any other time[1–4].he advantages of sterilization by irradiation include highenetrating power, low measurable residues, small tempera-

ure rise and the fact that there are fewer variables to control.hus, sterilization can be carried out on the finally pack-ged product and is applicable to heat-sensitive drugs. The

ncreased use of radiation processing for other industrial pur-oses (such as the sterilization of medical devices) has led to

he development of more efficient and economical irradiationquipment and processes. With the advances made in asepticrocessing, we now have products and materials, which areuch cleaner from a microbiological point of view and thus

∗ Corresponding author. Tel.: +90 312 297 72 13;ax: +90 312 299 20 37.

E-mail address:polat@hacettepe.edu.tr (M. Polat).

are likely to require much lower radiation doses to ach10−6 sterility assurance level (SAL). This change provian opportunity to terminally sterilize, or at least enhanceSAL, of a much larger number and range of drugs[5,6].

On account of the destructive nature of ionizing radiaand the difficulty in predicting the radiolytic effects, the stuof radio-induced radicals and chemical products in drunecessary, both to determine the feasibility of the radiatreatment and to control it. Moreover, the regulationsfrom country to country: radiosterilization is permittedsome countries and not in others[7–9], which leads to thnecessity for a method to differentiate between irradiatednon-irradiated samples. Electron spin resonance (ESR)of the leading methods for identification of irradiated fostuffs[10–12]and recently has proven to be an accuratereliable technique for dosimetry irradiation of pharmacecals [13–18]. ESR yields both qualitative information (iwhether or not a sample has been irradiated) and quantiresults (i.e. the dose it received). The aim of the presentwas: first, to investigate the ESR spectral features of the

003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2004.12.010

332 M. Polat, M. Korkmaz / Analytica Chimica Acta 535 (2005) 331–337

ical species produced in�-irradiated redoxon and ascorbicacid, second, to explore potential use of redoxon tablet as anormal and/or accidental dosimetric material through detailESR studies carried out at room and at high temperatures on�-irradiated redoxon.

2. Materials and methods

Redoxon tablets were provided from Roche Pharmaceu-tical Company (Istanbul). It is a Vitamin C supplementand its active ingredient is pure Vitamin C. It has yellowcolor, lemon odor and taste. Citric acid, cornstarch, flavor, ri-boflavin, sodium bicarbonate, sodium chloride, sodium sac-charin, sucrose and tartaric acid are present in redoxon asnon-medicinal ingredients. Redoxon tablets of 25 mm diam-eter and 7 mm thickness have been used in the present work.They can be easily found in all drug stores and in all housesall over the world. Thus, redoxon can be a potential candidatein the detection of accidental irradiation dose if it exhibits thefeatures of a good dosimetric material.

Redoxon tablets were ground mechanically prior to irradi-ation and sieved through a no. 60 screen (0.25 apertures/mm)and then transferred to polycarbonate vials for irradiationtreatment. Ground redoxon in the powder form was usuallyk eat-m outo al ra-d sibled ormala ed atr ad ykN ncyi eter.A , 15,2 ponsec iatedt turala andt ls atf Thei 0 K)b

ea-s uartzE X-b ippedw ra-t avep iverg ona ime,8 avityw kerE as

not changed during the long term signal intensity decay ex-periment to avoid any error ing factor and intensity measure-ments arising from changes in the cavity-filling factor. Theresults are given as the replicate of three different samples.

3. Experimental results and discussion

3.1. Unirradiated (control) and irradiated redoxonsamples

Unirradiated ground redoxon exhibited an ESR spectrumconsisting of a single resonance line of very low intensityhaving a peak-to-peak width of�Hpp = 0.9 mT and ag valueof 2.0057 (Fig. 1a). This signal was considered to origi-nate from mechanical damage of the molecules during thegrinding. However, irradiated samples were found to havean ESR spectrum consisting of many resonance lines likelydue to the creation of radical species arising from active andnon-medicinal ingredients present in redoxon. A typical ESRspectrum of the ground redoxon irradiated at 10 kGy is givenin Fig. 1b. This spectrum is fairly different from that obtainedfor pure ascorbic acid[19,20]. It is certainly due to the fact thatredoxon tablets contain many non-medicinal ingredients suchas citric acid, cornstarch, flavor, riboflavin, sodium bicarbon-a rtarica radi-c con-t SRs purea o theE

a umo n the

F unir-r Gy)a

ept at room temperature (290 K) before irradiation trent. All irradiation and ESR experiments were carriedn samples open to air in order to stay under commerciiation sterilization conditions and to determine the pososimetric use of the studied redoxon samples as a nnd/or accidental dosimeter. Irradiations were performoom (290 K) temperature using a60Co-� source supplyingose rate of 2.0 kGy/h in the sample position at the Saraoyuclear Research Center of Turkish Atomic Energy Age

n Ankara. The dose rate was measured by Fricke dosimset of 21 samples irradiated to doses of 2.5, 5.0, 7.5, 10

0 and 25 kGy was employed to construct the dose–resurve. However, samples prepared from redoxon irrado a dose of 15 kGy were used both to investigate strucnd spectral features of the contributing radical species

o study the kinetic features of radiation-induced radicaour different temperatures (313, 333, 353 and 373 K).rradiated samples were kept at room temperature (29efore measurement.

After being irradiated in polycarbonate vials, ESR murements were carried out on samples transferred into qSR tubes of 4 mm inner diameter using a Bruker EMXand ESR spectrometer operating at 9.5 GHz and equith a high sensitive cylindrical cavity (conditions of ope

ion: central field, 348.6 mT; scan range, 20 mT; microwower, 0.5 mW; microwave frequency, 9.769 GHz; receain, 2× 104; modulation frequency, 100 kHz; modulatimplitude, 0.2 mT; time constant, 327.68 ms; sweep t3.886 s). Sample temperature inside the microwave cas monitored with a digital temperature control unit (BruR 4111-VT). The position of the sample in the cavity w

te, sodium chloride, sodium saccharin, sucrose and tacid beside ascorbic acid as active ingredient. Namely,al species originating from non-medicinal ingredientsribute in large extent to the formation of experimental Epectra of irradiated redoxon. ESR spectra of irradiatedscorbic acid were also recorded to see its contribution tSR spectra of irradiated redoxon (Fig. 1c).Four intense central resonance lines denoted asI1, I2, I3

ndI4 (Fig. 1b) were found to dominate the ESR spectrf irradiated redoxon and the ESR studies carried out i

ig. 1. ESR spectra of ground redoxon and pure ascorbic acid: (a)adiated (control); (b) irradiated (10 kGy) redoxon; (c) irradiated (10 kscorbic acid.

M. Polat, M. Korkmaz / Analytica Chimica Acta 535 (2005) 331–337 333

Fig. 2. Variation of ESR line intensities with the square root of appliedmicrowave power for a sample irradiated at 15 kGy: (�) I1; (�) I2; (�) I3;(�) I4.

present work were based on the behaviors of these lines. Theshape of the spectrum was independent from applied dose inthe range of 2.5–25 kGy. Microwave saturation characteris-tics of the dominating resonance lines were investigated firstin the microwave power range of 0.1–10 mW using a sampleirradiated at a dose of 15 kGy. The results are given inFig. 2as variation of the line intensities with the square root ofapplied microwave power. As is seen,I3 line exhibits fairlydifferent saturation characteristics compared to those ofI1,I2 and I4 lines. Although, latters saturate at a microwavepower of nearly 2 mW,I3 line is not saturated even at thepower of 10 mW. Basing on this result, a microwave powerof 0.5 mW was adopted throughout the present study toavoid the effect of signal saturation.

3.2. Variable temperature studies

The variations of the ESR line intensities of a ground re-doxon sample irradiated at a dose of 15 kGy with temperaturewere also investigated in the temperature range of 130–390 K.Lines I1, I2, I3 andI4 were found to exhibit similar temper-ature behaviors in the studied temperature range. Variationwith temperature of the intensity of lineI3 is given inFig. 3as an example for these behaviors. Cooling the sample fromroom temperature (290 K) down to 130 K produced an in-c teda rvef ed toi plep g thes ngesi thet atedc es ino .T 90 Kp e rad-i

Fig. 3. Variation of the intensity of lineI3 with temperature for a sampleirradiated at dose of 15 kGy: (�) cooling; (©) heating.

3.3. Room temperature long term variations in the lineintensities

Stabilities of the radical species is an important aspectin radiation dose measurement by ESR spectroscopy. Thus, astudy relative to the variations of the signal intensities at roomtemperature in long term was performed. A sample of groundredoxon irradiated at 15 kGy was used to determine long termroom temperature variations in theI1, I2, I3 andI4 intensities.Thus, a sample was stored at room temperature (290 K) andits ESR spectra were recorded in regular time interval over aperiod of 92 days.I1 andI2 intensities were found to decreaserelatively fast disappearing completely in the first 50 and10 days, respectively, following the cessation of irradiation.However, linesI3 andI4 were very stable and their intensitiesstayed almost constant in the experimental error limits at theend of 92 days storage period. The results concerning longterm room temperature variations in the intensities of studiedresonance lines are given inFig. 4. Decreases of 2.5 and5.0% in the intensities of linesI3 andI4, respectively, werecalculated at the end of 92 days storage period. Although,

F ture(

rease in the intensity of lineI3. When the sample was heagain from 130 K to room temperature, the intensity cu

ollowed the same path, in a reversible way, and reachts initial value before cooling. Further heating of the samroduced an irreversible decrease in the intensity. Coolinample to room temperature from 390 K created no chan the signal intensity and it remains almost constant inemperature range of 390–290 K. Although, heating crehanges in intensity, it did not produce significant changther spectral parameters such asg factors and line widthshese results indicate that heating of the sample up to 3roduces an important decrease in radiation-induced fre

cal populations in redoxon.

ig. 4. Variations in long term of the line intensities at room tempera

290 K) for a sample irradiated at 15 kGy: (�) I1; (�) I2; (�) I3; (�) I4.

334 M. Polat, M. Korkmaz / Analytica Chimica Acta 535 (2005) 331–337

Fig. 5. Experimental decay data forI1, I2, I3 and I4 line intensities at theannealing temperature of 353 K for a sample irradiated at a dose of 15 kGy:(�) I1; (�) I2; (�) I3; (�) I4.

fairly small decreases in the intensities ofI3 and I4 linesoccurred at the end of storage period (92 days), thus, to getactual dose received by sample, correction must be performedon the dose values calculated from dose–response curves.

3.4. Variations of the line intensities in annealedsamples

Studying the behaviors ofI1, I2, I3 and I4 line intensi-ties above room temperature would be interesting from ki-netic point of view of the contributing radical species. Irre-versible decreases in ESR signal intensities at high temper-atures would be expected to originate from the decay of themagnetic units responsible for these signals. The decay rate ofthese units should depend on the sample temperature. To testhis idea and to get more insight into the decay processes ofthe studied resonance lines (I1, I2, I3 andI4), some irradiatedredoxon samples were annealed at four different tempera-tures (313, 333, 353 and 373 K) for predetermined times (3,6, 10, 20, 40 and 60 min). Although samples were annealedat high temperatures, all spectra were recorded at room tem-perature after cooling the samples to room temperature. Theresults obtained forI1, I2, I3 andI4 line intensity at annealingtemperature 353 K are given inFig. 5 for a sample irradi-ated at a dose of 15 kGy. The line intensities were found toe tem-p saves or thea . Thee singfi thano hts.T con-t thei e di-v ,

decay relatively fast,I3 andI4 intensities stay almost constantover the storage period (Fig. 4) at room temperature. But athigh annealing temperatures,I1, I3 and I4 experience simi-lar relatively slow decay behaviors, butI2 continue to decayfaster. This definitely means that radicals of short live takepart in the formation ofI1 and I2 intensities but the weightof the short live radicals involved in the formation ofI1 islow compared to that ofI2. Short live radicals contributing toI1 decay very quickly at high temperatures so that long liveradicals governs the decay ofI1 intensity as in the case ofI3andI4 intensities (Fig. 5).

3.5. Simulation of ESR spectrum of�-irradiatedascorbic acid

As is emphasized in the preceding sections, redoxon con-tains ascorbic acid beside many other non-medicinal ingredi-ents. Thus, radical species originating from active ingredientnamely ascorbic acid and from other ingredient are expectedto be present in�-irradiated redoxon. The presence of manyradical species of different origins and structures makes dif-ficult the analysis of the ESR spectrum of irradiated redoxon.Nevertheless, ascorbic acid, which is the only active ingredi-ent of redoxon is expected to have large contribution to theESR spectrum of this pharmaceutics. Thus, ESR spectrumo lsor -ta ngo cen-t es ofd xroad

F d at ad ); (b)r

xhibit similar decay characteristics at other annealingeratures; however, they are not presented in the work topace. As is expected, the higher the temperature and/nnealing time the faster the decays of the line intensitiesfforts made to describe the line intensity decay data urst, second and third order decay expressions of morene radical species to each intensity with different weighe problem, in this respect, is to guess the number of

ributing radical species and their contribution weights tontensities. However, the decays of line intensities can bided into different groups, whileI1 andI2 intensities, both

t

f ascorbic acid,�-irradiated at a dose of 10 kGy, was aecorded at room temperature (Figs. 1c and 6a). This specrum is similar to those given by Rexroad and Gordy[19],nd Basly et al.[20] for pure solid ascorbic acid consistif a doublet with weaker components positioned at the

er and both side of the doublet lines. Two radical speciifferent characteristic features were suggested by Re

ig. 6. Room temperature ESR spectrum of ascorbic acid irradiateose of 10 kGy: (a) experimental (solid line), theoretical (dashed lineadical A; (c) radical B.

M. Polat, M. Korkmaz / Analytica Chimica Acta 535 (2005) 331–337 335

Table 1ESR spectral parameters calculated from simulation calculations for proposed radical species

Radicalspecies

Relative weight Hyperfine splitting (mT) g factor �Hpp line-width (mT)

Ax Ay Az gx gy gz

A 0.355 (0.005) H1 0.535 (0.005) 3.318 (0.010) 2.551 (0.009) 2.0013 (0.0003) 2.0029 (0.0003) 2.0048 (0.0003) 0.183 (0.002)

B 0.645 (0.005) H1 1.397 (0.007) 0.646 (0.006) 1.266 (0.007) 2.0026 (0.0003) 2.0016 (0.0003) 2.0115 (0.0003) 0.140 (0.002)H2 0.560 (0.005) 0.779 (0.006) 0.452 (0.004)H3 0.238 (0.003) 0.372 (0.003) –

Values in brackets are the error estimated for relevant parameters.

and Gordy[19] to contribute to the formation of ascorbic acidESR spectrum but, no attempt was made to calculate spectralparameters of these radical species up to present days. Simu-lation calculations based on a model predicting the presenceof the radical species suggested by these authors were per-formed in the present work to determine spectral parametersof these species. Intensity of doublet is much higher thanthe intensities of all other lines. This means that concentra-tions of the radical species giving rise to doublet structureis much higher than the concentration of the other radicalspecies present. The results of several trials of simulationcalculations indicated that with a model based on the follow-ing A and B radical species[19] having anisotropicg andhyperfine splitting tensors, it was possible to describe bestroom temperature spectra of�-irradiated ascorbic acid. Inspecies A, H1 proton interacts with unpaired electron local-ized on carbon atom bound directly to OH proton and givesthe observed high intensity doublet, but H2 does not inter-act or interacts very weakly so that it produces no hyperfinesplitting. However, in species B: H1, H2 and H3 (a protonfrom R group) protons are involved in the production of hy-perfine splitting. Hyperfine interactions due to H1, H2 andH3 protons are orientation dependent and they are character-ized with three principalgparameters and hyperfine splittingconstants. The results of spectrum simulation calculationsa eterv c-t entedi

3

2.5,5 t the

dose–response curves relevant to the dominant resonancelines (I1, I2, I3 and I4). It is important to emphasize oncemore that in the studied dose range (2.5–25 kGy) the po-sitions and the relative distances between studied resonancelines did not change and nor did other lines appear. The math-ematical functions given inTable 2were tried to describe thevariations of the intensities of linesI1, I2, I3 andI4 with ab-sorbed radiation dose without forcing these functions to passthrough origin. In these functions,YandD stand for the ESRline intensity and absorbed dose in kGy, respectively, anda,b, c are constants to determine. As can be seen fromTable 2,power and exponentially varying functions describe best ex-perimental intensity data obtained for interested resonancelines. However, the intercepts, which reflect the line intensi-ties at zero applied dose, are the smallest for exponential func-tion although, it has similar correlation coefficient to thoseobtained for the power function. Theoretical dose–responsecurves calculated using parameter values given inTable 2for exponential function are also represented as dashed line,with their experimental counterpart inFig. 7. As is seen fromthis figure, neither of the dose–response curves relevant toI1, I2, I3 andI4 resonance lines is linear in the studied doserange (2.5–25 kGy), but they experience high increases.I3 isthe most sensitive line among others to the�-radiation. Lineintensity values of 467.2 (I1), 405.1 (I2), 756.4 (I3) and 617.8( pec-t were

e . ThefaI

nd theoretical spectrum calculated using derived paramalues are given inTable 1andFig. 6a, respectively. Sperum of A and B species were also calculated and represn Fig. 6b and c.

.6. Dose–response curve

Samples of ground redoxon irradiated to doses of.0, 7.5, 10, 15, 20 and 25 kGy were used to construc

I4) derived for studied resonance lines from the ESR srum of a sample irradiated at a measured dose of 15 kGy

mployed to get theoretical dose delivered to the sampleollowing dose values of 15.5± 2.5, 14.9± 2.3, 15.8± 2.5nd 15.8± 2.4 kGy were found for the resonance linesI1, I2,

3 andI4, respectively.

336 M. Polat, M. Korkmaz / Analytica Chimica Acta 535 (2005) 331–337

Table 2Mathematical functions used for fitting dose–response data obtained forI1, I2, I3 andI4 line intensities

Function Line Calculated parameters r2

a b c

Y=a+b×D I1 24.6 (2.1) 58.8 (29.9) – 0.9651I2 22.4 (1.9) 58.2 (26.6) – 0.9669I3 39.3 (3.8) 95.1 (55.0) – 0.9543I4 33.2 (3.3) 60.7 (46.9) – 0.9537

Y=a+b×D+c×D2 I1 −35.0 (12.8) 44.8 (2.3) −0.74 (0.08) 0.9966I2 −9.4 (31.8) 36.9 (5.7) −0.53 (0.20) 0.9753I3 −70.2 (38.1) 74.8 (6.9) −1.30 (0.24) 0.9886I4 −69.9 (45.6) 61.3 (8.3) −1.02 (0.29) 0.9771

Y=a× [1 − e−D/b] I1 1009.5 (56.3) 25.0 (1.6) – 0.9928I2 952.6 (57.2) 25.9 (2.2) – 0.9903I3 1574.5 (42.3) 24.1 (1.5) – 0.9901I4 1460.2 (52.7) 28.8 (1.7) – 0.9846

Y= 1 +b×Dc I1 −248.2 (151.0) 207.6 (113.6) 0.45 (0.12) 0.9938I2 −128.3 (148.9) 122.8 (99.7) 0.55 (0.19) 0.9848I3 −472.1 (404.1) 390.3 (315.8) 0.42 (0.17) 0.9872I4 −289.0 (321.6) 232.6 (230.6) 0.49 (0.22) 0.9787

Values in brackets are the error estimated for relevant parameters.

Fig. 7. Variation of ESR line intensities with absorbed radiation dose: (Sym-bols) experimental, (dashed) calculated, (�) I1; (�) I2; (�) I3; (�) I4.

4. Conclusion

Unirradiated but mechanically ground redoxon exhibits asingle ESR line of very low intensity havingg= 2.0057 and�Hpp = 0.9 mT likely due to the creation of radical speciesafter mechanical grinding. However,�-irradiated redoxonpresents an ESR spectrum consisting of a doublet and manyweak lines with different spectroscopic features. Microwavesaturation studies carried out forI1, I2, I3 andI4 resonancelines (Fig. 2) suggested the presence of more than one radi-cal species in irradiated redoxon. However, variations of theseline intensities with temperature over a temperature range of130–390 K were similar indicating similar thermal behaviorsfor contributing radical species. Fast decays of the line in-

tensitiesI1 and I2 and stable features ofI3 and I4 at roomtemperature (Fig. 4) were considered to be originating fromthe presence of different radical species of different spec-troscopic features contributing to these intensities. Althoughshort-half life species contribute, in large extent, to the lineintensitiesI1 and I2, species of long-half life contribute tothe line intensitiesI3 andI4. I2 line intensity continue to de-cay faster in samples annealed at high temperatures than theline intensitiesI1, I3 and I4, which were observed to decaywith nearly similar decay rate. The appeared change in thedecay rate of the lineI2 is likely due to the fact that at hightemperatures short life radical species contributing toI2 in-tensity decay completely just at the beginning of annealingtreatment, then long life species dominates the decay charac-teristics of this line intensity as in the case of line intensityI3andI4.

The results of simulation calculations performed usingESR signal intensity values of�-irradiated ascorbic acidwhich is the active ingredient of redoxon, showed that tworadical species produced after irradiation of ascorbic acidare the principal species dominating the ESR spectrum of�-irradiated redoxon and that with a model of two radicalspecies of different ESR parameters it is possible to describewell experimental spectrum.

Variations of studied line intensities with absorbed dosear isn ever,rBa sentsw etricm oxona

re different. The most sensitive line intensity of�-irradiatededoxon to radiation isI3. Dose–response curve of this lineot linear in the studied dose range (2.5–25 kGy). Howadiation sensitivity of redoxon is relatively high (G= 0.13).asing on this and high stability of line intensityI3 and I4t room temperature, it is concluded that redoxon preanted characteristics of a normal and accidental dosimaterial and that it can be used to detect irradiated rednd as a dosimetric materials.

M. Polat, M. Korkmaz / Analytica Chimica Acta 535 (2005) 331–337 337

Acknowledgement

This work was supported by Turkish State Planning Or-ganization for which we are deeply indebted (Project No.: 97K 121380).

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