ARTICLE IN PRESS

0969-806X/$ - s

doi:10.1016/j.ra

$This work w�CorrespondE-mail addr

Radiation Physics and Chemistry 73 (2005) 111–116

www.elsevier.com/locate/radphyschem

Radiation sterilization of ketoprofen$

Branka Katugin-Ramema,�, Katia Hamitoucheb, Nadica Maltar-StrmeWkic,Karmen Kosd, Irina Pucica, Smiljana Britvic-Budicina, Dugan Ramema

aRuXer Bogkovic Institute, P.O.B 180, Zagreb 10000, CroatiabCentre de Recherche et Developpement Saidal, El Harrach, Algeria

cFaculty of Veterinary Medicine, University of Zagreb, Zagreb 10000, CroatiadBelupo Pharmaceutical Works, Koprivnica 48000, Croatia

Received 23 February 2004; accepted 14 July 2004

Abstract

Radiation sterilization of ketoprofen (KP) dry powder was investigated by selected physico-chemical methods. High-

performance liquid chromatography, ultraviolet spectrophotometry, infrared spectrophotometry, differential scanning

calorimetry, X-ray diffraction and electron spin resonance spectroscopy did not show any significant degradation at

sterilization dose 25 kGy. To determine the nature, extent and direction of radiation-induced changes, KP was

irradiated to extremely high doses, much higher than necessary to achieve sterility. The irradiated KP did not show any

difference of XRD patterns up to 200 kGy; with DSC and IR some changes were detected only above 1000 and

2000 kGy, respectively; HPLC has shown about 5% destruction at 2000 kGy. Acetyl benzophenon (AcBph) was

generated by irradiation with G(AcBph)=(1.670.1)� 10–8mol J–1. Ames test has shown no mutagenicity of KP

irradiated with 3000 kGy or of the oily mixure of radiolytic products isolated from it. Solid KP has proven to be very

stable on irradiation, and irradiation has been found to be a suitable method for its sterilization.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Ketoprofen; Radiation sterilization; Radiation degradation; DSC; ESR; HPLC; IR; UV; XRD; Ames test

1. Introduction

Profens are potent nonsteroidal anti-inflammatory

drugs. Ketoprofen (KP) [2-(3-benzoylphenyl) propionic

acid] is widely used and is one of the most popular

profens. Sterility of profens is required for the intrave-

nous applications, and intramuscular and subcutaneous

injections. An attractive alternative to traditional

methods of sterilization, recognized by all major

pharmacopeias, has been afforded by irradiation. This

ee front matter r 2004 Elsevier Ltd. All rights reserv

dphyschem.2004.07.004

as presented at IMRP-2003, Chicago, USA.

ing author. Fax: +385-1-468-0098.

ess: brazem@irb.hr (B. Katugin-Ramem).

work deals with the feasibility of radiation sterilization

of ketoprofen in dry powder form.

2. Materials and methods

Ketoprofen (KP) was obtained from Belupo Pharma-

ceutical Works (Koprivnica, Croatia). Ketoprofen

CRS (chemical reference substance), ketoprofen impu-

rities A CRS (3-acetylbenzophenon) (AcBph) and C

CRS (2-(3-carboxyphenyl)-propionic acid), (CRSs avail-

able from European Directorate for the Quality of

Medicines, Council of Europe, Strasbourg, France),

were also obtained from Belupo. Benzophenon (Bph)

ed.

ARTICLE IN PRESSB. Katugin-Razem et al. / Radiation Physics and Chemistry 73 (2005) 111–116112

was supplied by Merck (Darmstadt, Germany).

Acetonitrile (HPLC grade), dichloromethane, glacial

acetic acid, sodium hydrogen carbonate and anhydrous

sodium sulphate (all reagent grade) were from Kemika

(Zagreb, Croatia). Potassium dihydrogen orthopho-

sphate was obtained from Analar (Poole, England).

Water was triply distilled.

Irradiation of 300mg samples was performed by 60Co

gamma rays at a dose rate of 10Gy s�1 in the presence

of air.

Isolation of lipophilic radiolytic products of KP for

Ames mutagenicity test was accomplished by removal of

the parent compound and acidic impurities by extraction

of dichloromethane solution of irradiated KP (dose

3000 kGy) with concentrated aqueous solution of

sodium hydrogen carbonate. After washing of the

remaining dichloromethane layer with water and drying

with anhydrous sodium sulphate, the evaporation of

solvent afforded an oily residue of lipophilic radiolytic

products.

Purity control and quantification of radiolytic pro-

ducts in irradiated KP were performed by high-

performance liquid chromatography (HPLC) following

two approaches. The first one, prescribed by the

(European Pharmacopeia (EuPh), 2001), was based on

the comparison of chromatograms of reference solutions

of CRS chemicals with chromatograms of the solutions

of investigated substances. A ProStar HPLC system by

Varian (Walnut Creek, CA, USA) was used. The system

was fitted with an LC-18, 5m, 150mm� 4.6mm column

(Supelco, Bellefonte, PA, USA). The pump flow rate was

1.2mlmin�1, detection wavelength 233 nm, and mobile

phase consisted of acetonitrile, phosphate buffer (0.5M

aqueous solution of potassium dihydrogen phosphate

adjusted to pH 3.5 with orthophosphoric acid) and

water (43:2:55, v/v)–mobile phase A. The second

approach was based on the use of external and internal

standards. Radiolytic decomposition of KP was

quantified using KP CRS as an external standard,

while Bph was used as an internal standard in the

quantification of radiolytic products. Besides the same

chromatographic system and the same column applied

under the same conditions as described above, an

additional chromatographic system, WellChrom HPLC

by Knauer Scientific Instruments (Berlin, Germany)

with a diode array detector was applied. A somewhat

faster analysis was made using a mobile phase acetoni-

trile, 0.005M aqueous acetic acid (55:45, v/v)–mobile

phase B.

Ultraviolet (UV) spectrophotometry was used for

purity control and determination of response factors of

KP and its radiolytic products at the detection

wavelength (233 nm) in mobile phases A and B. The

measurements were made at 20 1C using a Cary 2200

spectrophotometer by Varian (Mulgrave, Victoria,

Australia).

Infrared (IR) spectrophotometry was performed

by a RXI FT-IR spectrophotometer by Perkin-Elmer

(Norwalk, CT, USA) in anhydrous sodium bromide

discs.

Differential scanning calorimetry (DSC) was carried

out using a DSC 7 by Perkin-Elmer (Norwalk, CT,

USA). The thermograms were recorded under nitrogen

from 20 to 120 1C, at the heating rate of 10 1Cmin�1.

X-ray diffraction (XRD) patterns were taken by a

Model 1738 Automatic Diffractometer by Philips

(Almelo, The Netherlands) using CuKa radiation

(l ¼ 1:5418 (A) and a Philips PW 1877 AAPD v 3.6 g

program.

Electron spin resonance (ESR) spectroscopy was done

by a model E109 X-band spectrometer by Varian (Palo

Alto, CA, USA), operating at 9.5GHz with 100 kHz

modulation, equipped with a variable temperature unit

ER4111VT by Bruker Instruments (Karlsruhe, Ger-

many). The number of spins in irradiated KP was

determined by comparison of the second integral of

irradiated sample and a Bruker strong pitch reference

standard in a double microwave cavity.

Ames mutagenicity test of unirradiated KP, irradiated

KP (3000 kGy) and isolated radiolytic products was

conducted by the standard plate incorporation test

(Maron and Ames, 1983). Two test strains of Salmonella

typhimurium TA98 and TA100 (kindly provided by B.N.

Ames, University of California, Berkeley, CA, USA),

were used to detect frame-shift and base-pair mutations,

respectively. Dimethyl sulfoxide samples of 2, 20, 100

and 200 ml (equivalent to 100, 1000, 5000 and 10 000mlof KP and its radiolytic products, respectively) were

plated onto Vogel–Bonner basal agar plates with 2ml of

soft agar to which 0.5mM L-histidine and 0.5mM

biotin solutions had been previously added. Overnight

cultures of TA98 and TA100 (0.1ml), both without or

with metabolic activation (0.5ml of -S9 mixture or

0.5ml of +S9 mixture, respectively), were added to the

plates.

The S9 mixture contained 50ml of hepatic S9 preparedfrom male Wistar rats pretreated with an intraperitoneal

injection containing Aroclor 1254 (500mg/kg) dissolved

in corn oil. Immediately before mutagenicity testing, the

S9 fraction was passed sequentially through Millipore

membrane filters (0.45 and 0.22 mm filter units) to

remove any contaminating microorganisms. Each sam-

ple was plated in triplicate, and its revertants were

scored manually after 72 h incubation at 37 1C. As a

positive control (diagnostic mutagens) for this assay,

2-aminofluorene at a concentration of 10 mg/plate wasused to monitor the sensitivity of the bacterial strains

and the activity of the rat liver S9. Daunomycin at a

concentration of 10mg/plate without S9 and 645mg/plateof methyl methanesulfonate were used as an additional

control of the TA98 and TA100 strains, respectively.

The mutagenicity was expressed as the number of

ARTICLE IN PRESSB. Katugin-Razem et al. / Radiation Physics and Chemistry 73 (2005) 111–116 113

revertants per plate and per microgram of KP and its

radiolytic products, respectively.

Although there is no evidence supporting this specific

requirement, it is an arbitrary criterion for a test

substance to be considered positive in a bioassay, that

it must cause a twofold or a threefold increase over the

background of the mean number of revertants per plate

of at least one tester strain with a minimum of two

concentrations of the test substance.

Fig. 1. HPLC of KP obtained with Knauer HPLC system and

mobile phase acetonitrile: phosphate buffer: water (43:2:55, v/v)

(mobile phase A). The respective doses (from the bottom up)

are: 0, 25, 200 and 1600kGy.

0

20

40

60

80

100

0 1000 3000 4000 6000 7000

mm

ol A

cBph

/ kg

KP

2000 5000

Dose / kGy

Fig. 2. Radiolytic formation of AcBph with dose. Filled circles:

mobile phase A (acetonitrile, phosphate buffer, water (43:2:55,

v/v)). Open circles: mobile phase B (acetonitrile, 0.005M

aqueous acetic acid (55:45, v/v)).

3. Results and discussion

Unirradiated KP was a white powder which turned

pink on irradiation, probably as a result of trapping

radiolysis electrons by the Bph moiety or the conversion

of Bph to ketyl radical, or both. The intensity of the

colouration was related to dose. The white appearance

was recovered after several days of storage in a

dessiccator, in the dark, and at room temperature. No

attempt was made in this work to quantify these

changes.

Radiation-induced chemical changes in irradiated KP

were investigated by HPLC. The amounts of KP

impurity A (permitted level 0.2%), KP impurity C

(permitted level 0.2%), and the sum of other impurities

(permitted level 0.4%) were followed in unirradiated KP

and samples irradiated with 25, 50, 100 and 200 kGy by

the method prescribed by the European Pharmacopeia.

While the impurity C stayed at the same level found in

unirradiated KP, impurity A reached 0.1% at 200 kGy.

However, the sum of other impurities at 100 kGy rose to

0.7%, which is above the permitted level, and reached

1.4% at 200 kGy.

To determine the nature, extent and direction of

radiation-induced changes, KP was irradiated to ex-

tremely high doses, much higher than necessary to

achieve sterility; up to 4000 kGy was applied to produce

sizeable radiolytic changes, to be able to quantify them

and express them as radiation chemical yields.

The comparison of chromatograms obtained by

HPLC of unirradiated and irradiated KP (Fig. 1) shows

hardly any change at 25 kGy, but the presence of four

major radiolytic products eluting at 5, 11, 30 and 37min

respectively, was evident at higher doses.

Ultraviolet spectra of these products taken by a diode

array HPLC detector show that all probably retain a

Bph chromophore. The radiolytic product eluting at

11min was not unique to radiolysis, as its trace was

present in unirradiated sample, where it may have arisen

during production and handling. It has been known as

KP impurity A (AcBph). The build-up of AcBph with

dose is shown in Fig. 2, as quantified against the Bph

internal standard using two mobile phases, A and B.

The slope of this straight line gives radiation chemical

yield, G(AcBph)=(1.670.1) 10–8mol J–1. Taking this

value the level of KP impurity A at 200 kGy could be

calculated more accurately as 0.08%, which compares

well with 0.1% as estimated according to the EU Ph

procedure. It is remarkable that the linearity of

radiolytic products formation holds throughout the

dose range and that there is no trend to sublinearity.

On the other hand, it is not surprising that a direct

action of radiation on essentially non-interacting

molecules in the solid phase follows a linear pattern

as long as the probability of hitting the same molecule

for the second time remains low, and that is assured

by a low-radiation chemical yield of the change.

Assuming similar radiation chemical yields of the

other three major radiolysis products, radiation

chemical yield of decomposition could be estimated as

G(KP)=6.4� 10�8mol J�1. This would give about 3.3%

decomposition at 2000 kGy.

Infrared spectra of irradiated KP were inspected

for changes in comparison with unirradiated samples.

Two of the strongest, sharp and symmetrical peaks at

1697 and 1655 cm�1 have been attributed to carboxylic

and keto carbonyl stretching vibrations, respectively

(Fig. 3).

ARTICLE IN PRESS

4000 kGy

2000 kGy

200 kGy

100 kGy

25 kGy

0 kGy

Rel

ativ

e in

tens

ity

2000 1800 1600

Absorbance/Wavenumber

1400 1200 1000 800 600 400

Fig. 3. Selected parts of IR spectra of KP; the respective doses

are marked on the spectra.

0

1

2

3

4

5

60 70

P /m

W

25 kGy

1000 kGy

2000 kGy

0 kGy

11010080T /˚C

90

Fig. 4. DSC thermograms of KP; the respective doses are

marked on the thermograms.

03 23 43 63

2θ

0 kGy

Rel

ativ

e in

tens

ity

100000

80000

60000

40000

20000

33 5313

25 kGy

200 kGy

Fig. 5. XRD patterns of KP; the respective doses are marked

on the roentgenograms.

B. Katugin-Razem et al. / Radiation Physics and Chemistry 73 (2005) 111–116114

In unirradiated KP, the former (carboxylic) band was

slightly more intensive than the latter (carbonyl), while

the reverse was true at and above 2000 kGy, indicating

the progress of radiation-induced decarboxylation. The

position of the carboxylic band at 1697 cm�1, common

to many carboxylic acids, was assigned to the inter-

molecular hydrogen bonds present in the dimeric

skeleton formed by carboxylic groups. Its shifting

towards a higher frequency has been related to the

dissociation of hydrogen bonding after disruption of a

crystal lattice by mechanical treatment (for example by

grinding) (Nakai et al., 1978). This shift was not

observed in irradiated KP, from which we conclude

that either no breaking of intermolecular hydrogen

bonds occurs in the crystal lattice, or that it was not

visible at the level of about 3% molecules.

The melting endotherms of unirradiated KP and KP

irradiated with 25 kGy practically coincided (Fig. 4).

The onsets of the melting endotherms, their peak

temperatures and heats of fusion of irradiated KP did

not change until about 200 kGy, when a slight decrease

of the three quantities was observed. Their shift to lower

values continued with the dose, the heat of fusion

reaching 87.5% of its initial value at 1000 kGy, but still a

single fusion process was observed. At 2000 kGy the

DSC trace showed a double peak of fusion. A possible

explanation would be the loss of crystallinity, but that

was not supported by XRD data, at least up to 200 kGy.

It is interesting that DSC changes were reversible and

that the recovery towards the values in unirradiated

samples was observed after some time. This effect was

not quantified in this work, but it would suggest that

radiation-induced changes of the chemical identity of

individual molecules produced a temporary change of

the physical properties of the crystal lattice, notwith-

standing the absence of evidence by IR and XRD.

X-ray diffraction patterns of unirradiated and

irradiated KP were virtually the same up to 200 kGy

(Fig. 5), which is not surprising considering relatively

poor sensitivity of XRD patterns and very low

concentration of radiolytic products at this dose—only

about 0.3%.

As evidenced by UV spectroscopy, all major radiolytic

products in the radiolysis of solid KP contained a Bph

group, and should have been similar in size to the parent

molecule. Therefore, they could be accomodated within

the original crystal lattice when individual KP molecules

were transformed into radiolysis products in situ.

However, the process of accomodation of the larger

Bph fragment and exit of the smaller, presumably

volatile fragment of a radiolysis product, may take

some time. For example, radiation-induced decarbox-

ylation of KP results in AcBph and carbon dioxide. Bph

rings in KP and in AcBph are not co-planar but twisted,

having almost the same angles of torsion (Briard and

Rossi, 1990; Matak et al., 1994). The accomodation of

Bph should present no problem; detrapping of CO2could take some time leading to the observed recovery of

DSC properties after irradiation.

ESR spectroscopy is specific to the radiation treat-

ment. The insert to Fig. 6 shows ESR spectra of KP

irradiated with 25 kGy, obtained with 1G modulation

ARTICLE IN PRESSB. Katugin-Razem et al. / Radiation Physics and Chemistry 73 (2005) 111–116 115

amplitude at 100 kHz with 0.4mW (lower trace) and

1mW microwave power (upper trace).

Room temperature microwave saturation features of

this signal were studied in the range 0–150mW

microwave power and a complex behaviour was

observed, indicating the presence of more than one

radical species. The identification of these species was

not attempted in the present work, but likely candidates

would be: KP� and/or Bph ketyl radical, dCO�2 radical

ion, and possibly peroxyl radicals.

1 100 1000 10000

Num

ber

of s

pins

/g

342 349

1 mW

Dose /kGy

10

Magnetic field/mT

335

EPR

inte

nsity

0.4 µW

1E+19

1E+18

1E+17

1E+16

Fig. 6. Radiolytic accumulation of KP free radicals with dose

at room temperature. Insert: ESR spectra of KP irradiated with

25 kGy; lower trace: 0.4mW; upper trace: 1mW.

Table 1

Number of revertants by unirradiated and irradiated KP and by KP ra

Salmonella typhimurium TA98 and TA100 his� strains without (S9) a

Chemicals (mg /plate) No. of TA98 revertants/plate

Without S9a With

Unirradiated KP

100 3872 5971000 4072 5275000 2371 427

Irradiated KP (3000kGy)

100 5171 5471000 4773 5375000 2774 317

Radiolytic products only (3000 kGy)

100 4472 5671000 4372 4675000 3671 15710,000 1072 77

Solvent control

(DMSO) 4476 417

Positive controls 1150720 21787(1,2,3) (1) (2)

S9—metabolic activation with microsomal fraction from rat liver hom

1. Daunomycin hydrocloride (10mg/plate), 2. 2-aminofluorene (2-AF)aMean7SD of three plates.

The build-up of free radicals with the dose is also

shown in Fig. 6. The concentration increases linearly up

to 500 kGy, and in this range the radiation chemical

yield was G(free radicals)=2.3� 10�9 radicals J�1. The

decay of free radicals followed a complex kinetics, the

decay being faster at lower dose (not shown), which

indicates that recombination was not responsible

for the decay. From the above radiation chemical

yield, the saturation dose of about 1000 kGy and KP

density 1.28 g cm�3 (Liversidge, 1981), the radius of

the sphere of radical migration could be estimated as

about 5 nm. This gives a very large recombination

volume encompassing 860molecular packing units, so

that it is inconceivable that a radical could visit them in

solid. The upper value of the recombination radius

critical for stable trapping in solid is considered to be

about 1 nm per radical (Krushev et al., 1994). These

considerations then also support the view that recombi-

nation is not involved in the decay of free radicals in

irradiated KP.

All tested substances gave a negative response to both

strains, TA98 and TA100 in the Ames mutagenicity test

in comparison with known mutagens (positive controls)

as shown in Table 1. The number of revertants per plate

produced by the test substances was the same as the

number of spontaneous revertants on the negative

control plates. Both unirradiated, as well as irradiated

diolytic products in the Salmonella plate incorporation test using

nd with (+S9) metabolic activation, respectively

No.of TA100 revertants/plate

S9a Without S9a With S9a

1 160720 13574

2 14578 14276

5 134710 13574

5 13574 13078

4 134710 13878

8 13674 142710

1 172720 15374

2 12278 11776

5 152710 11374

4 13072 11474

8 150714 152712

23 2000730 1560748

(3) (2)

ogenate.

(10mg/plate), 3. Methyl methanesulphonate (625 mg/plate).

ARTICLE IN PRESSB. Katugin-Razem et al. / Radiation Physics and Chemistry 73 (2005) 111–116116

KP were not mutagenic to either of the two strains,

and that was also true for the isolated radiolytic

products of KP irradiated with 3000 kGy. There was

no indication of dose dependent responses to any

substance.

These data corroborate the published data on the

absence of mutagenicity of unirradiated KP (Philipose

et al., 1997). The new information is represented

here by the data on irradiated KP and its radiolytic

products; these substances were not investigated

before, and pertinent data do not significantly deviate

from those of unirradiated KP. This strongly supports

the conclusion on the absence of mutagenicity of

irradiated KP.

4. Concluding remarks

The feasibility of radiation sterilization of solid KP

was demonstrated almost up to 100 kGy, which

is well beyond any real needs. In fact, it is likely

that the sterilization dose much lower than the

standard 25 kGy, a routine value until recently, will be

required if the bioburden, its radiation sensitivity

and sterility assurance level were considered. The

high resistance to irradiation of KP in the dry powder

form, established in this work, enables the use of the

pre-sterilized drug in the preparation of sterile

solutions by aseptic processing and filling, which

is the second best option to terminal sterilization. The

study of radiation chemical mechanism of radiolytic

decomposition of neat KP may lead to better under-

standing of and practical solutions for the radiation

sterilization of liquid formulations of KP in final

packages.

Acknowledgement

The fellowship for training in Radiation Steriliza-

tion of Pharmaceuticals (ALG 02017 P) by the Inter-

national Atomic Energy Agency to K.H. is gratefully

acknowledged.

References

Briard, P., Rossi, J.C., 1990. Ketoprofene. Acta Crystallogr. C

46, 1036–1038.

European Pharmacopeia, 2001. Fourth ed. Directorate for

the Quality of Medicines, Council of Europe, Strasbourg,

pp. 1432–1433.

Krushev, V.V., Koizumi, H., Ichikawa, T., Yoshida, H.,

Shibata, H., Tagawa, S., Yoshida, Y., 1994. Relation

between track structure and LET effect on free radical

formation for ion beam-irradiated alanine dosimeter.

Radiat. Phys. Chem. 44, 521–526.

Liversidge, G.G., 1981. Ketoprofen. In: Florey, K. (Ed.),

Analytical Profiles of Drug Substances, vol. 10. Academic

Press, New York, pp. 443–471.

Maron, D.M., Ames, B.N., 1983. Revised methods for the

Salmonella mutagenicity test. Mutation Res. 113, 173–215.

Matak, D., Vinkovic, M., Dumic, M., 1994. 1-(3-Benzoylphe-

nyl)ethanone(I) and 3-benzoyl-a-methylbenzeneacetamidemethylene chloride solvate (2/1) (II). Acta Crystallogr. C

50, 1339–1342.

Nakai, Y., Nakajima, S.I., Yamamoto, K., Terada, K., Konno,

T., 1978. Effects of grinding on physical and chemical

properties of crystalline medicinals with microcrystalline

cellulose. III. Infrared spectra of medicinals in ground

mixtures. Chem. Pharm. Bull. 26, 3419–3425.

Philipose, B., Singh, R., Khan, K.A., Giri, A.K., 1997.

Comparative mutagenic and genotoxic effects of three

propionic acid derivatives ibuprofen, ketoprofen and

naproxen. Mutation Res. 393, 123–131.