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Effects of ionizing radiations on a pharmaceutical compound, chloramphenicol
Radiat.
Pergamon
Phys.Chem.Vol. 43, No. 5, pp. 471-480, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0969-806X/94
$6.00 + 0.00
EFFECTS OF IONIZING RADIATIONS ON A PHARMACEUTICAL COMPOUND, CHLORAMPHENICOL L.
VAR~HNEY
and K. M. PATEL
ISOMED, I&IPO, Board of Radiation and Isotope Technology, B.A.R.C., Bombay 400 085, India (Received I7 September 1992; accepted 21 April 1993) Abstract-Chloramphenicol, a broad spectrum antibiotic, has been irradiated using Cobalt-60 y radiation and electron beam at graded radiation doses upto 100 kGy. Several degradation products and free radicals are formed on irradiation. Purity, degradation products, free radicals, discolouration, crystallinity, solubility and entropy of radiation processing have been investigated. Aqueous solutions undergo extensive radiolysis even at low doses. Physico-chemical, microbiological and toxicological tests do not show significant degradation at sterilization dose. High performance liquid chromatography (HPLC), differential scanning calorimetry (DSC), UV-spectrophotometry, diffuse reflectance spectroscopy (DRS) and electron spin resonance spectroscopy (ESR) techniques were employed for the investigations.
INTRODUCITON
Radiation sterilization of medical products is one of the ideal applications of high energy radiation. It has been accepted as an industrial process of sterilization world over (Gopal, 1978; Jacob, 1985; Varshney and Iya, 1989; Wogl, 1985). Sterilization of several polymeric and metallic medical devices is carried out using
high energy y radiation from Cobalt-60, Cesium-137 radioisotopes and electron beam from electron accelerator. There is a possibility of using this technique for sterilizing solid pharmaceutical compounds which are other wise sensitive to the traditional methods of sterilization employing heat and ethylene oxide (EtO). Besides, use of Et0 for sterilization is under criticism because of its chemical and residual toxicity (Jacob, 1985; Varshney and Iya, 1989; Wogl, 1985). Microfiltration is yet another technique routinely employed for sterilization of Et0 and heat sensitive solids and solutions. In the process, filtration, filling and drying (lyophilization, for obtaining dry powder) steps are carried out under aseptic conditions which are difficult to maintain. In radiation sterilization, the process could be carried out in final shipment cartons. Consequently, a much higher sterility assurance level of one in one million (probability of finding one item non sterile in 1000,000 items) is normally achieved at 25 kGy for products manufactured under good manufacturing practice. for especially compounds, Pharmaceutical parenteral application, need to be sterilized. The process of sterilization should not affect the physicotoxicological and chemical, microbiological properties specified in pharmacopoeia. Majority of solid pharmaceuticals undergo l-3% radiation degradation at sterilization dose of 25 kGy (Wogl, 1985). Our earlier studies have shown that chloroamphenicol (CPL), a broad spectrum antibiotic, when
irradiated in powder form using y rays, undergoes less than 1% degradation at 25 kGy (Varshney and Iya, 1989). In the present study, effects of y radiations from Cobalt-60 on chemical purity, nature of degradation products, free radicals, discolouration, crystallinity, solubility and entropy of radiation processing (Srp, entropy of irradiated minus entropy of non irradiated CPL), microbiological and toxicological properties have been investigated. These results have been compared with the electron beam irradiated samples. Based on the results of the above mentioned studies, feasibility of radiation sterilization of CPL in dry powder form is being reported and certain points pertaining to the evaluation of irradiated solid pharmaceuticals have been discussed. Effects of y radiation on aqueous solution containing varying concentration of CPL are also reported. EXPERIMENTAL
Sample
CPL sample obtained from a local manufacturer was recrystallized from hot water. The recrystallized sample was of 99.99 mole % DSC purity value. Irradiation y-Irradiation. Solid CPL samples were irradiated in glass vials and aqueous solutions containing varying concentration of CPL were irradiated in 25 ml flask in a y chamber (GC-900) to various graded dose. The absorbed dose was measured using ceric-cerous sulphate dosimeter. The dose rate was 3.0 kGy/h. Electron beam irradiation. An electron accelerator, model ILU6-M3 type from USSR was employed for electron beam irradiation. Solid samples were taken in polyethylene sachets (3 x 2 x 0.25 cm). Radiochromic film dosimeters were employed for absorbed dose measurement. The absorbance of the 471
412
L. VARSHNEY and
films at 510 nm were initially calibrated using Cobalt60 y irradiation. Some of the beam parameters used were: Energy: 1.3 MeV; Pulse time: 500 ps; Beam cross section: 900 x 70 mm; Dose rate: 3200 kGy/h at sample position. Purity and degradation products evaluation HPLC. Normal phase (NP) and reverse phase (RP) chromatograhic techniques were employed for purity estimation and separation of degradation products (Varshney and Iya, 1989; Varshney, 1990). The following HPLC system configuration was employed. Pump: ConstametricIII, LDC, U.S.A.; flow rate: 1.5 ml/min; injection port: Rheodyne model 7125, LDC, U.S.A.; injection loop: 50~1; detector: Spectromonitor II, LDC, U.S.A., 273 nm, 0.16 AUFS; data logger: PDP 11/23 computer system U.S.A. Column (NP): Partisil PXS DEC, 4.6 mm x 25 cm, analytical column; mobile phase (NP): chloroform, methanol, formic acid, 90 : 2 : 0.2 (v/v). Column (RP): ODS C-18, 25 cm, 4 mm o.d., NOVAPAK(R) analytical column. Mobile phase (RP): 0.1 N sodium acetate and acetonitrile (85: 15, v/v). DSC-30. DSC-30 of Mettler’s TA-3000 thermal analysis system was employed for purity evaluation of non irradiated and irradiated CPL. Samples weighing about 2-5 mg were encapsulated in aluminium crucibles. The samples were subjected to a dynamic temperature programme between 130-175°C at a heating rate of Z”C/min. The purity values were obtained using vant Hoffs melting point depression method. Peroxides Peroxides were detected using solution of ferrous ions in dilute sulphuric acid and xylenol orange (Gupta et al., 1985). Absorption spectra of saturated solutions of CPL in ferrous-xylenol orange solution were recorded in 37&700 nm range. Discolouration DRS technique was employed to study absorption characteristics of the non irradiated and irradiated powder samples in visible region (Wendtlandt and Hecht, 1966). Hitachi-200, UV-vis spectrophotometer with an integrating sphere accessory was employed for recording DRS spectrum between 400-700 nm. The recorder output of the instrument was connected to PDP-11/23 (DEC, U.S.A.) computer system via-RS-232C interface using a locally available digital multimeter (HIL-2655). Computer software programmes developed at our laboratory were employed for various mathematical evaluations and plotting spectra. Free radicals Varian V4502 ESR spectrometer was employed for detecting free radicals. Samples weighing about 100 mg were taken in a quartz tube for scanning. The
K. M.
PATEL
samples were scanned using X-Band frequency in the range of 3385 + 500 g. Diphenyl picryl hydrazyl (DPPH) was employed as standard for determining number of spins in irradiated CPL. Crystallinity Crystallinity is directly proportional to enthalpy of fusion. DSC technique was employed to measure enthalpy of fusion. Pure Indium was used as calibration standard (mp. 156.6”C and enthalpy of fusion = 28.45 J/g). Solubility and entropy of radiation processing Saturated solutions of non irradiated and irradiated CPL in distilled water were made and kept in water bath at room temperature. After 5 h, the solutions were filtered, diluted and CPL concentration was determined using RP-chromatography as described earlier. Enthalpy values and melting point as determined by DSC were used to evaluate Srp (Grant and York, 1986). Microbiological test (inhibition zone diameters) Paper discs (4 mm dia, 1 mm thickness) impregnated with chloroamphenicol(30 pg) were irradiated. Standard bacterial strain was cultured in a test tube containing 10ml of Soyabean Casein Digest (SCD) broth. 1 ml of well mixed, 24 h SCD Broth culture was added to 3 ml of molten (45°C) top agar. The contents of the tube were mixed and spread over SCD agar medium plates (Patel, 1986). Non-irradiated and irradiated discs were kept in the same plate and incubated at 37°C for 24 h. The inhibition zone diameters were measured in millimeter under a magnifying glass and ratio of zones of non-irradiated and irradiated discs were calculated. Data from four such plates were recorded. E. coli, S. typhimurium, B. Subtilis bacterial strains were employed for these investigations. Toxicological tests Prescribed procedures were followed to evaluate CPL for undue toxicity in mice and histamine like substances in cat (Indian Pharmacopoeia, 1985). Mutagenesity Ame’s tests were conducted on concentrated fractions of degradation products separated using semipreparative HPLC column (Patel, 1986). RESULTS AND DISCUSSION
The structure
of CPL is as below: CH20H I C-NH’CO’CHCb
HOH-
C-H
3 0
EjO*
Effects of ionixing radiations on chloramphenicol
473
1
PNB
&CPL
FCPL-BASE
1
0
I
I
I
I
I
I
2
4
6
8
IO
12
ELUTION
TIME
(mln
I
I
14
16
2 3
1
Fig. 1. HPLC (reverse phase) chromatograms of chloramphenicol. (a) Non-irradiated, 20 pg; (b) irradiated, 30 kGy, 20 pg; (c) irradiated, 100 kGy, 20 pg; (d) irradiated, 11 kGy, 0.05% aqueous solution, 10~~. Scale shifted for clarity.
Tbe results of present study shows that several degradation products are formed on irradiating CPL
products (Tables 1 and 2 and Fig. 2). Lower elution times of the degradation products in comparison to
using y radiation and electron beam (Figs l-3). It is observed that both the irradiation techniques result in similar quantitative degradation and degradation
CPL on the silica gel column (NP) indicate the degradation products to be less polar and therefore, of lower molecular weight fragments of CPL. Such
0
I
I
I
I
I
2
4
6
B
IO
ELUTION
TIME
(min)
Fig. 2. HPLC (normal phase) chromatograms of chloramphenicol. (a) Non-irradiated; (h) irradiated, 50 kGy; (c) irradiated, 53 kGy (electron beam); (d) exposed to sun light 100 h. Scale shifted for clarity.
I2
L. VARSHNEYand K. M. PATEL
474
E
I
0,016
AU CPL
PNBA
(cl;.
0
16
48
32
64
96
80
ELUTION
TIME
112
126
14
(min)
Fig. 3. HPLC (normal phase, semipreparative column) chromatograms of chloramphenicol used for separation of degradation products. (a) Non-irradiated, 600 pg; (b) irradiated 100 kGy, 550 pg; (c) irradiated 250 kGy, 500 pg.
are also observed in non-irradiated powder CPL. Irradiation enhances their concentration (Varshney, 1990; Varshney and Iya, 1989a; Varshney et al., 1989). Impurities in non irradiated CPL could arise due to combined effect of diffused light, heat and moisture during storage. Considering molecular structure of CPL, probable fragments of CPL
molecule like benzene, nitrobenzene, p-nitrobenzyl alcohol and CPLbase [l-p (nitrophenyl)-2-amino-l,3 propanediol] could not be detected in the chromatograms. The unidentified degradation products have retention time closer to the mentioned fragments and therefore, indicating similarity in their chemical structure. The UV spectrum of degradation
products
Table
I. Purity,
G value,
enthalpy
of fusion and chloramphenicol
melting
point
of %o
y irradiated
Purity Dose (kGv)
HPLC
DSC
0.0 15.0 30.0 50.0 100.0
99.99(?0.3) 99.8(kO.2) 99.3 (kO.4) 98.8 (kO.3) 96.5 (kO.2)
99.99(?0.1) 99.81 (kO.1) 98.90(+0.1) 97.42 (50.1) 96.00(~0.4)
Note: numbers
Table
2. Purity
in parentheses
values,
C-
indicate
enthalpy
3.78 6.87 7.11 10.42
standard
Hr fJ/l?)
m.p. 1°C)
117.5(+1.0) 111.8(~1.0) 106.9 (2 1.0) 102.2 (& 1.0) 89.1 (k3.0)
150.8(+0.1) l50.7(+0.1) 150.4(&0.1) 149.7(*0.1) 147.9 (kO.3)
G CPL. HPLC)
deviation
for n = 5.
of fusion and melting point chloramohenicol samoles
of electron
beam
irradiated
Purity Dose (kGy)
HPLC (%)
DSC (mole X)
0.0 12.0 24.0 36.0 53.3
99.99(kO.3) 99.69(+0 .2) 99.42 (kO.3) 99.14(*0.3) 98.54 (kO.2)
99.99(kO.l) 99.97(*0.1) 99.92 (f0.2) 99.34(kO.2) 97.70 (fO.3)
Note: numbers
in parentheses indicate
(-
standard
CPL:“PLC) 7.46 7.09 7.05 8.13 deviation
Enthalav of fusion (J/g. DSC)
m.p. (“C, DSC)
ll5.85(~l.O) llO.34(~1.0) 107.29(+1.0) 105.81 (kl.0) 102.83(?1.0)
l50.6(~O.l) 150.6(fO.l) 150.3(~0.1) 150.2(fO.l) 149.4(*0.2)
for n = 4.
415
Effects of ionizing radiations on chloramphenicol
0.156
0,024
0
.ooo 370
400
450
500
550 WAVELENGTH
600 (nm
650
700
)
Fig. 4. Spectra of chloramphenicol in visible region for peroxide detection. (A) Solution without CPL; (B) solution with non-irradiated CPL; (C) solution with irradiated CPL, 100 kGy. (Inset A,) ESR signal for free radicals in irradiated CPL, 30 kGy. (Inset Ar) ESR signal of irradiated CPL, 30 kGy, recrystallized from methanol.
products
separated
from
semipreparative
column
(NP, Fig. 3) had UV absorption maximum varying from 261 to 272 nm indicating presence of nitrobenzene ring in the degradation products. No significant change in nitrite ion concentration could be detected in aqueous solution of irradiated CPL using diazotization method (Varshney, 1990; Howel, 1975). Decrease in specific rotation value of 5% (w/v) solution of irradiated (100 kGy) CPL in ethyl alcohol by 7% indicates the fission of carbon-carbon bond between the two optically active carbon atoms (Varshney, 1990). Free radicals were detected in irradiated samples which linearly increased with increasing radiation dose. The radicals could be detected even in a two years old irradiated sample. Peroxides and hydroperoxides were detected in irradiated CPL samples (Fig. 4). Presence of free radicals in irradiated samples, detection of peroxides and restricted mobility of active species in solid state, indicate that some of the peaks in the chromatograms could be due to peroxides and hydroperoxides of fragments of CPL molecule. The degradation products which could be identified contain p-nitrobenzaldehyde (PNB), p-nitrobenzoic acid (PNBA), p-nitrosobenzoic acid and HCl (Fig. 1). Calculated G values from the concentration of degradation products for PNB, PNBA and Cl- are 0.85, 0.73 and 2.40, respectively, which remain almost constant up to 100 kGy. It may be noted from HPLC profiles that PNB and PNBA are exclusively formed by ionizing radiation in solid CPL. Greater constancy in G (- CPL) is observed in elec-
tron beam irradiation than in y irradiation (Tables 1 and 2). CPL has orthorhombic crystal structure. The molecules in the unit cell have curled staggered configuration. The energy could be absorbed by any part of the molecules but channeled only to specific bonds as indicated by linear increase in degradation products with increasing radiation dose. Considering the results of present study it appears that the absorbed radiation energy is mainly concentrated on the two optically active carbon atom and C-Cl bond. To account for the several degradation products with varying concentration, it could be suggested that the absorbed energy degrades the molecules in probablistic way. The probability of breaking of bonds connected to the optically active carbon atoms being the function of associated strain energy. Most of the radiation degradation products are also formed when exposed to sun light. PNB and PNBA are exclusively formed by ionizing radiation in solid CPL. From these observations it could be suggested the PNB and PNBA are formed from ionized species. Free radicals formed on irradiation remain in crystalline matrix even for a few years as observed in irradiated CPL. These free radicals (in the absence of autoxidation reactions) normally react with oxygen during storage or with dissolved oxygen in solvents like water forming trace level of peroxides or hydroperoxides which lead to formation of products like aldehydes and acids. The concentration of such products formed on storage in irradiated solid samples is not expected to exceed the molar concen-
476
L. VARSHNEYan d K. M. Table 3. Purity of non-irradiated and irradiated aqueous solutions of chloranmhenicol Concentration Dose Wy) 0.0
2.8 5.6 8.4 Il.2
0.2%
0.1%
0.05%
100
loo
loo
91.4 71.9 65.3
82.9 69.1 56.2 44.8
69.3 53.5 35.4 22.3
tration of free radicals which is about lo-‘mol in CPL at 30 kGy. Aqueous solutions expectedly undergo extensive percentage radiolytic degradation (Table 3). Chromatogram reveals formation of several degradation products (Fig. 1). A few new products are also observed in addition to the products observed in irradiated solid CPL. Peaks corresponding to retention time of p-nitroso benzoic acid and CPL base were quite prominent in irradiated aqueous solution. Discolouration Irradiation using either technique discolours CPL samples from off white to yellow. The intensity of the colour increases with increasing radiation dose (Fig. 5) as shown by increased absorption in blue region. Samples irradiated in air using y rays were less discoloured than: (1) deoxygenated, y irradiated (Fig. 7); (2) electron beam irradiated samples. Drastic reduction in colour intensity is observed in deoxygenated sample (Fig. 6) having greater number of trapped free radicals and electrons which react and disappear during dissolution in methanol. In a separ-
PATEL
ate experiment, the ESR spectrum of the CPL sample irradiated to 30 kGy contained 18 x 10” spins/g which disappeared after dissolution (Fig. 4). These free radicals react with dissolved oxygen in the solvent forming peroxides/hydroperoxide as detected in the present study. 0.5% (w/v) solution of irradiated CPL in acetone showed linearly increasing discolouration with increasing dose in comparison to non irradiated sample (Varshney, 1990). Therefore, discolouration in CPL on irradiation could be attributed to: (i) formation of trace level degradation products as shown by increased discolouration of solution of irradiated CPL and (ii) trapped free radicals in the crystalline matrix of CPL as shown by ESR and DRS. DRS difference spectrum of y irradiated CPL samples in: (1) air and vacuum; (2) air and electron beam gave similar absorption spectrum with peak maximum at 492 nm (Fig. 7). Both radiation degradation products and free radicals should absorb in visible region to impart colour to the irradiated samples. The colour centers due to the free radicals which exist in vacuum, could be destroyed by oxidation and recombination reactions during y irradiation in air and therefore, the intensity of the colour is reduced. Increased discolouration and similar DRS of samples y irradiated in vacuum and electron beam indicate that anaerobic conditions exist during electron beam radiation. This point should be considered while using electron beam for sterilization as microorganisms are known to be more radiation resistant in the absence of oxygen (Varshney et al., 1990).
-0.15 370
400
430
500 550 WAVELENGTH
600
650
(rim)
Fig. 5. Diffuse reflectance spectra of chloroamphenicol. (a) Non-irradiated; (b) irradiated, 15 kGy; (c) irradiated, 30 kGy; (d) irradiated, 50 kGy; (e) irradiated, 100 kGy.
700
Effects of ionizing radiations on chloramphenicol
370
400
450
500
550
WAVELENGTH
600
650
700
(nm 1
Fig. 6. DRS difference spectra of chloramphenicol.
(a) Irradiated, 45 kGy, air, recrystallized from methanol; (b) irradiated, 45 kGy, deoxygenated sample, recrystallized from methanol.
Crystallinity
Enthalpy of fusion of CPL samples irradiated using either technique linearly decreases with increasing radiation dose. This indicates the extent of reduction in crystallinity. The reduction in the enthalpy is attributed to the strain forces caused by the radiation degradation products in the crystalline matrix of CPL. X-ray powder diffraction pattern of irradiated samples also showed broadening of peaks (Fig. 8)
with reduced signal intensity indicating reduction in crystallinity (Varshney, 1990; Varshney et al., 1988). The changes in crystallinity have been reported to affect dissolution profiles and hence bioavailability (Grant and York, 1986). Solubility
The solubility of a compound is dependent on thermodynamic functions. Dissolution of irradiated
0.37
: ~0.24 2 a 5: 0.11 :
370
400
450
500 550 WAVELENGTH
600
650
(nm 1
Fig. 7. DRS difference spectra of chloramphenicol. (a) Irradiated, 45 kGy, deoxygenated sample; (b) irradiated, 45 kGy, air; (c) difference spectra (a-b); (d) difference spectra (electron beam irradiated, 45 kGy - b).
700
478
L. VARSHNEY and K. M. PATEL
35
I
I
I
I
1
I
37
39
41
43
45
47
29
49
(DEGREES)
Fig. 8. X-ray powder diffraction pattern of chloramphenicol.
CPL in water could be represented as: [CPL]solid-irradiation +[CPL+Al+Bl-tCl+..]solid l&O V
(a) non-irradiated;
(b) irradiated, 30 kGy.
the degradation products than the quantitative decrease in purity of CPL. This observation implies that small concentration of radiation degradation products can bring significant physical changes like solubility and colour. Entropy of radiation processing
CPL(aq.) + Al(aq.) + Bl(aq.) + Cl(aq.) Where Al, Bl, Cl are radiation degradation products in CPL matrix. The free energy change during dissolution (AG) is related to enthalpy (AH) and entropy (AS) changes given by: AG=AH-TAS AG = - RT LnCs Where Cs is the molar solubility of CPL in water at a temperature T. Negative contribution from AH (reduction in enthalpy of fusion) and possible positive contribution by AS (entropy of mixing of degradation products in solvent) could facilitate dissolution and solubility (AG negative). It is observed that the solubility initially increases and then becomes almost constant (Table 4). The constancy in solubility could be attributed to combined effect of radiation degradation and compensation by increased solubility of irradiated CPL. The initial increase in solubility at 15 kGy could be due to more disruptive influence of
On irradiating solid CPL crystalline matrix, degradation products are formed at random positions in the crystal lattice. These products could have a disruptive influence and thus the lattice is activated affecting enthalpy and entropy values. The change in entropy of solid CPL due to radiation processing can be defined as Srp, which can be determined by calorimetry (Grant and York, 1986) Srp is defined by the following equation: Srp = - S (CPL)irrad. + xa S (CPL) +xbSb+xcSc+xdSd+...+Smix Table 4. Solubilityand entropy of radiation processing data of non-irradiated 7 irradiated CPL DO%
WY) 0 I5 30 50 100
(HPLC) @g/ml) at 25°C 2.54f 0.10
Solubility
2.72 + 0.1 I 2.71 kO.14 2.74+O.l2 2.75 f 0.14
Srp WC) (J mol-’ Km’) 0 4.3 8.0 II.5 21.2
Effects of ionizing radiations on chloramphenicol
479
Table 5. Ratio of inhibiton zone diameter on SCD agar plate Dose (kGy): Ratio
Strain coli S. typhimurium B. subtilis E.
0
I5
30
M
100
I .of 0.0
i.o*o.o
0.995 f 0.010 0.966 f 0.030
0.956 f 0.024 0.920 f 0.032
0.908 * 0.019 0.887 f 0.042
1.0*0.0
1.0+0.0
0.989 f 0.025
0.999k 0.010 0.992f 0.020 0.962f 0.030 0.903k 0.020
Note: the value zero means no distinction using mm wale could be made between zone diameters under magnifying glass
Where xa is mole fraction of CPL in irradiation CPL and xb, xc, xd are mole fraction of the degradation products and Smix is entropy of mixing. Using DSC, Srp is determined as:
CPL solid - srp
[CPL + A + B + C + . . .]solid
irradiation
E
rnl1
Melt
c
rln2
Melt
= -H,, /T,,,, (irradiated) + Hn/Tm2 (non irradiated)
where Hr,, HO and T,,,, , T,,,, are enthalpy of fusion and melting point of non-irradiated and irradiated samples respectively. The calculated Srp values for gamma irradiated CPL samples are given in Table 4. Linear increase and similar magnitude in Srp values are observed in y and electron beam irradiated CPL samples. Increase in Srp could be linked with increase in solubility of irradiated CPL in water. Microbiological
sensitivity
tests
The inhibition zone diameters do not show any significant change up to 30 kGy when the three bacterial strain were tested. This observation is in accordance with the insignificant change in chemical purity of irradiated CPL determined using HPLC and DSC techniques. Significant reduction in antibacterial property is observed at 100 kGy (Table 5). Toxicological
tests
toxicity and histamine like undue substances were observed in non-irradiated as well as CPL samples irradiated up to 30 kGy. No mutagenic behaviour was shown by the degradation products. Most of the products formed on irradiation of CPL are also present in non irradiated product. Irradiation merely increases their concentration to an insignificant level at lower dose such as 15 kGy. Several radiation degradation products are formed in irradiated CPL and there are practical limitations in identifying all the degradation products. Some or all products could be toxic individually at certain concentration. In the present study p-nitrobenzaldehyde formed on irradiation is a known toxic compound having LDSO-ORAL(Rat) = 4700 mg/kg. CPL itself No
is toxic and has TDLO-ORAL value of 1700 mg/kg in women of average weight 50 kg (Irving, 1984). Toxicity examination of such irradiated drug containing several degradation products, totalling less than 1% could be far less than the toxicity of drug itself. Although, the changes in toxicity characteristics are not expected to change at sterilization dose, the drug must be screened for undue toxicity and histamine like substances. CONCLUSIONS
(1) Chloroamphenicol in solid dry powder form can be radiation sterilized using cobalt-60 or electron beam at lower radiation dose of 15 kGy at which insignificant radiation effects have been observed. Aqueous solutions of CPL undergo extensive degradation on irradiation and hence can not be sterilized. (2) Although, high chemical purity of irradiated CPL is retained at sterilization dose of 15 kGy, changes could be observed in physical properties like discolouration, crystallinity and solubility. (3) Irradiated CPL should not be considered as a new drug, if its physico-chemical integrity is established using sensitive analytical techniques by experts in the field. The low concentrations of radiation degradation products at the sterilization dose of 15 kGy are not expected to cause any untoward reactions. (4) In addition to pharmacopeal tests, irradiated CPL should be evaluated using sensitive analytical techniques such as HPLC and DSC for purity and degradation products. Acknowledgements-We are grateful to Shri K. Krishnamurthy for his support and encouragement. Thanks are due to Dr V. K. Iya and Shri S. Sabharwal for discussion and valuable suggestions and Shri R. S. Deshpande for help in carrying
out electron
beam irradiation. REFERENCES
British Pharmacopoeia (1988) p. 117. HMSO, London. Gopal N. G. S. (1978) Radiation sterilization of pharmaceuticals and polymers. Radiat. Phys. Chem. 12, 35. Gupta B. L., Bhat R. M., Narayan G. R. and Nelekani S. R. (1985) A snectrophotometric read out method for free radical dosim&y. Radial. Phys. Chem. 26, 647. Howe1 F. N. (1975) Standard Methods of Chemical Analvsis. VIth edn, Kreiger, New York, p. 745. Irving S. N. (1984) Dangerous Properfies of Industrial Materials. VI edn. Van Nostrand Reinhold, New York. Jacob G. P. (1985) A review: radiation sterilization of pharmaceuticals. Radiat. Phys. Chem. 26, 133.
480
L.
VARSHNEY and
Pate1 K. M. (1986) Effects of repeated exposure of gamma rays on the survivors of different strains of staphylococcus aureus. Ph.D. thesis Bombay University, India and references therein. The Indian Pharmacopoeia (1985) III edn. Controller of Publications, Delhi, India. pp. A-42-A-44. Varshney L. (1990) Effects of ionizing radiation on chloramphenicol and feasibility studies on its radiation sterilization. Ph.D. thesis University of Bombay, India. Varshney L. and Iya V. K. (1989) Effects of Cobalt-60 gamma radiation on chloramphenicol. Ind. J. Pharm. Sci. 51, 25.
Varshney L., Sharma G. and Iya V. K. (1988) Cobalt-60 gamma irradiation of chloramphenicol uis-a-ois its crystallinity. Indian Pharmaceutical Cong. Calcutta, West Bengal, India.
K. M.
PATEL
Varshney L., Sharma G. and Iya V. K. (1989) Natural and Radiolytic degradation products of chloroamphenicol. In Radio Chemistry and Radiation Chemistry Symp., Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamilnadu, India. Varshney L., Iya V. K., Sharma G. and Pate1 K. M. (1990) A comparitive study of irradiation of chloramphenicol using electron beam and cobalt-60 gamma rays. In Naf. Semin. on Industrial Radiation Processing and Technology,
IIT Powai, Bombay, India. Wendtlandt W. W. and Hecht H. G. (1986) Rejlectance Spectroscopy, Chapt. 3 and 4. Interscience, New York. Wogl W. (1985) Radiation sterilization of pharmaceuticals, chemical changes and consequences. Radiat. Phys. Chem. 25, 425.
Pergamon
Phys.Chem.Vol. 43, No. 5, pp. 471-480, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0969-806X/94
$6.00 + 0.00
EFFECTS OF IONIZING RADIATIONS ON A PHARMACEUTICAL COMPOUND, CHLORAMPHENICOL L.
VAR~HNEY
and K. M. PATEL
ISOMED, I&IPO, Board of Radiation and Isotope Technology, B.A.R.C., Bombay 400 085, India (Received I7 September 1992; accepted 21 April 1993) Abstract-Chloramphenicol, a broad spectrum antibiotic, has been irradiated using Cobalt-60 y radiation and electron beam at graded radiation doses upto 100 kGy. Several degradation products and free radicals are formed on irradiation. Purity, degradation products, free radicals, discolouration, crystallinity, solubility and entropy of radiation processing have been investigated. Aqueous solutions undergo extensive radiolysis even at low doses. Physico-chemical, microbiological and toxicological tests do not show significant degradation at sterilization dose. High performance liquid chromatography (HPLC), differential scanning calorimetry (DSC), UV-spectrophotometry, diffuse reflectance spectroscopy (DRS) and electron spin resonance spectroscopy (ESR) techniques were employed for the investigations.
INTRODUCITON
Radiation sterilization of medical products is one of the ideal applications of high energy radiation. It has been accepted as an industrial process of sterilization world over (Gopal, 1978; Jacob, 1985; Varshney and Iya, 1989; Wogl, 1985). Sterilization of several polymeric and metallic medical devices is carried out using
high energy y radiation from Cobalt-60, Cesium-137 radioisotopes and electron beam from electron accelerator. There is a possibility of using this technique for sterilizing solid pharmaceutical compounds which are other wise sensitive to the traditional methods of sterilization employing heat and ethylene oxide (EtO). Besides, use of Et0 for sterilization is under criticism because of its chemical and residual toxicity (Jacob, 1985; Varshney and Iya, 1989; Wogl, 1985). Microfiltration is yet another technique routinely employed for sterilization of Et0 and heat sensitive solids and solutions. In the process, filtration, filling and drying (lyophilization, for obtaining dry powder) steps are carried out under aseptic conditions which are difficult to maintain. In radiation sterilization, the process could be carried out in final shipment cartons. Consequently, a much higher sterility assurance level of one in one million (probability of finding one item non sterile in 1000,000 items) is normally achieved at 25 kGy for products manufactured under good manufacturing practice. for especially compounds, Pharmaceutical parenteral application, need to be sterilized. The process of sterilization should not affect the physicotoxicological and chemical, microbiological properties specified in pharmacopoeia. Majority of solid pharmaceuticals undergo l-3% radiation degradation at sterilization dose of 25 kGy (Wogl, 1985). Our earlier studies have shown that chloroamphenicol (CPL), a broad spectrum antibiotic, when
irradiated in powder form using y rays, undergoes less than 1% degradation at 25 kGy (Varshney and Iya, 1989). In the present study, effects of y radiations from Cobalt-60 on chemical purity, nature of degradation products, free radicals, discolouration, crystallinity, solubility and entropy of radiation processing (Srp, entropy of irradiated minus entropy of non irradiated CPL), microbiological and toxicological properties have been investigated. These results have been compared with the electron beam irradiated samples. Based on the results of the above mentioned studies, feasibility of radiation sterilization of CPL in dry powder form is being reported and certain points pertaining to the evaluation of irradiated solid pharmaceuticals have been discussed. Effects of y radiation on aqueous solution containing varying concentration of CPL are also reported. EXPERIMENTAL
Sample
CPL sample obtained from a local manufacturer was recrystallized from hot water. The recrystallized sample was of 99.99 mole % DSC purity value. Irradiation y-Irradiation. Solid CPL samples were irradiated in glass vials and aqueous solutions containing varying concentration of CPL were irradiated in 25 ml flask in a y chamber (GC-900) to various graded dose. The absorbed dose was measured using ceric-cerous sulphate dosimeter. The dose rate was 3.0 kGy/h. Electron beam irradiation. An electron accelerator, model ILU6-M3 type from USSR was employed for electron beam irradiation. Solid samples were taken in polyethylene sachets (3 x 2 x 0.25 cm). Radiochromic film dosimeters were employed for absorbed dose measurement. The absorbance of the 471
412
L. VARSHNEY and
films at 510 nm were initially calibrated using Cobalt60 y irradiation. Some of the beam parameters used were: Energy: 1.3 MeV; Pulse time: 500 ps; Beam cross section: 900 x 70 mm; Dose rate: 3200 kGy/h at sample position. Purity and degradation products evaluation HPLC. Normal phase (NP) and reverse phase (RP) chromatograhic techniques were employed for purity estimation and separation of degradation products (Varshney and Iya, 1989; Varshney, 1990). The following HPLC system configuration was employed. Pump: ConstametricIII, LDC, U.S.A.; flow rate: 1.5 ml/min; injection port: Rheodyne model 7125, LDC, U.S.A.; injection loop: 50~1; detector: Spectromonitor II, LDC, U.S.A., 273 nm, 0.16 AUFS; data logger: PDP 11/23 computer system U.S.A. Column (NP): Partisil PXS DEC, 4.6 mm x 25 cm, analytical column; mobile phase (NP): chloroform, methanol, formic acid, 90 : 2 : 0.2 (v/v). Column (RP): ODS C-18, 25 cm, 4 mm o.d., NOVAPAK(R) analytical column. Mobile phase (RP): 0.1 N sodium acetate and acetonitrile (85: 15, v/v). DSC-30. DSC-30 of Mettler’s TA-3000 thermal analysis system was employed for purity evaluation of non irradiated and irradiated CPL. Samples weighing about 2-5 mg were encapsulated in aluminium crucibles. The samples were subjected to a dynamic temperature programme between 130-175°C at a heating rate of Z”C/min. The purity values were obtained using vant Hoffs melting point depression method. Peroxides Peroxides were detected using solution of ferrous ions in dilute sulphuric acid and xylenol orange (Gupta et al., 1985). Absorption spectra of saturated solutions of CPL in ferrous-xylenol orange solution were recorded in 37&700 nm range. Discolouration DRS technique was employed to study absorption characteristics of the non irradiated and irradiated powder samples in visible region (Wendtlandt and Hecht, 1966). Hitachi-200, UV-vis spectrophotometer with an integrating sphere accessory was employed for recording DRS spectrum between 400-700 nm. The recorder output of the instrument was connected to PDP-11/23 (DEC, U.S.A.) computer system via-RS-232C interface using a locally available digital multimeter (HIL-2655). Computer software programmes developed at our laboratory were employed for various mathematical evaluations and plotting spectra. Free radicals Varian V4502 ESR spectrometer was employed for detecting free radicals. Samples weighing about 100 mg were taken in a quartz tube for scanning. The
K. M.
PATEL
samples were scanned using X-Band frequency in the range of 3385 + 500 g. Diphenyl picryl hydrazyl (DPPH) was employed as standard for determining number of spins in irradiated CPL. Crystallinity Crystallinity is directly proportional to enthalpy of fusion. DSC technique was employed to measure enthalpy of fusion. Pure Indium was used as calibration standard (mp. 156.6”C and enthalpy of fusion = 28.45 J/g). Solubility and entropy of radiation processing Saturated solutions of non irradiated and irradiated CPL in distilled water were made and kept in water bath at room temperature. After 5 h, the solutions were filtered, diluted and CPL concentration was determined using RP-chromatography as described earlier. Enthalpy values and melting point as determined by DSC were used to evaluate Srp (Grant and York, 1986). Microbiological test (inhibition zone diameters) Paper discs (4 mm dia, 1 mm thickness) impregnated with chloroamphenicol(30 pg) were irradiated. Standard bacterial strain was cultured in a test tube containing 10ml of Soyabean Casein Digest (SCD) broth. 1 ml of well mixed, 24 h SCD Broth culture was added to 3 ml of molten (45°C) top agar. The contents of the tube were mixed and spread over SCD agar medium plates (Patel, 1986). Non-irradiated and irradiated discs were kept in the same plate and incubated at 37°C for 24 h. The inhibition zone diameters were measured in millimeter under a magnifying glass and ratio of zones of non-irradiated and irradiated discs were calculated. Data from four such plates were recorded. E. coli, S. typhimurium, B. Subtilis bacterial strains were employed for these investigations. Toxicological tests Prescribed procedures were followed to evaluate CPL for undue toxicity in mice and histamine like substances in cat (Indian Pharmacopoeia, 1985). Mutagenesity Ame’s tests were conducted on concentrated fractions of degradation products separated using semipreparative HPLC column (Patel, 1986). RESULTS AND DISCUSSION
The structure
of CPL is as below: CH20H I C-NH’CO’CHCb
HOH-
C-H
3 0
EjO*
Effects of ionixing radiations on chloramphenicol
473
1
PNB
&CPL
FCPL-BASE
1
0
I
I
I
I
I
I
2
4
6
8
IO
12
ELUTION
TIME
(mln
I
I
14
16
2 3
1
Fig. 1. HPLC (reverse phase) chromatograms of chloramphenicol. (a) Non-irradiated, 20 pg; (b) irradiated, 30 kGy, 20 pg; (c) irradiated, 100 kGy, 20 pg; (d) irradiated, 11 kGy, 0.05% aqueous solution, 10~~. Scale shifted for clarity.
Tbe results of present study shows that several degradation products are formed on irradiating CPL
products (Tables 1 and 2 and Fig. 2). Lower elution times of the degradation products in comparison to
using y radiation and electron beam (Figs l-3). It is observed that both the irradiation techniques result in similar quantitative degradation and degradation
CPL on the silica gel column (NP) indicate the degradation products to be less polar and therefore, of lower molecular weight fragments of CPL. Such
0
I
I
I
I
I
2
4
6
B
IO
ELUTION
TIME
(min)
Fig. 2. HPLC (normal phase) chromatograms of chloramphenicol. (a) Non-irradiated; (h) irradiated, 50 kGy; (c) irradiated, 53 kGy (electron beam); (d) exposed to sun light 100 h. Scale shifted for clarity.
I2
L. VARSHNEYand K. M. PATEL
474
E
I
0,016
AU CPL
PNBA
(cl;.
0
16
48
32
64
96
80
ELUTION
TIME
112
126
14
(min)
Fig. 3. HPLC (normal phase, semipreparative column) chromatograms of chloramphenicol used for separation of degradation products. (a) Non-irradiated, 600 pg; (b) irradiated 100 kGy, 550 pg; (c) irradiated 250 kGy, 500 pg.
are also observed in non-irradiated powder CPL. Irradiation enhances their concentration (Varshney, 1990; Varshney and Iya, 1989a; Varshney et al., 1989). Impurities in non irradiated CPL could arise due to combined effect of diffused light, heat and moisture during storage. Considering molecular structure of CPL, probable fragments of CPL
molecule like benzene, nitrobenzene, p-nitrobenzyl alcohol and CPLbase [l-p (nitrophenyl)-2-amino-l,3 propanediol] could not be detected in the chromatograms. The unidentified degradation products have retention time closer to the mentioned fragments and therefore, indicating similarity in their chemical structure. The UV spectrum of degradation
products
Table
I. Purity,
G value,
enthalpy
of fusion and chloramphenicol
melting
point
of %o
y irradiated
Purity Dose (kGv)
HPLC
DSC
0.0 15.0 30.0 50.0 100.0
99.99(?0.3) 99.8(kO.2) 99.3 (kO.4) 98.8 (kO.3) 96.5 (kO.2)
99.99(?0.1) 99.81 (kO.1) 98.90(+0.1) 97.42 (50.1) 96.00(~0.4)
Note: numbers
Table
2. Purity
in parentheses
values,
C-
indicate
enthalpy
3.78 6.87 7.11 10.42
standard
Hr fJ/l?)
m.p. 1°C)
117.5(+1.0) 111.8(~1.0) 106.9 (2 1.0) 102.2 (& 1.0) 89.1 (k3.0)
150.8(+0.1) l50.7(+0.1) 150.4(&0.1) 149.7(*0.1) 147.9 (kO.3)
G CPL. HPLC)
deviation
for n = 5.
of fusion and melting point chloramohenicol samoles
of electron
beam
irradiated
Purity Dose (kGy)
HPLC (%)
DSC (mole X)
0.0 12.0 24.0 36.0 53.3
99.99(kO.3) 99.69(+0 .2) 99.42 (kO.3) 99.14(*0.3) 98.54 (kO.2)
99.99(kO.l) 99.97(*0.1) 99.92 (f0.2) 99.34(kO.2) 97.70 (fO.3)
Note: numbers
in parentheses indicate
(-
standard
CPL:“PLC) 7.46 7.09 7.05 8.13 deviation
Enthalav of fusion (J/g. DSC)
m.p. (“C, DSC)
ll5.85(~l.O) llO.34(~1.0) 107.29(+1.0) 105.81 (kl.0) 102.83(?1.0)
l50.6(~O.l) 150.6(fO.l) 150.3(~0.1) 150.2(fO.l) 149.4(*0.2)
for n = 4.
415
Effects of ionizing radiations on chloramphenicol
0.156
0,024
0
.ooo 370
400
450
500
550 WAVELENGTH
600 (nm
650
700
)
Fig. 4. Spectra of chloramphenicol in visible region for peroxide detection. (A) Solution without CPL; (B) solution with non-irradiated CPL; (C) solution with irradiated CPL, 100 kGy. (Inset A,) ESR signal for free radicals in irradiated CPL, 30 kGy. (Inset Ar) ESR signal of irradiated CPL, 30 kGy, recrystallized from methanol.
products
separated
from
semipreparative
column
(NP, Fig. 3) had UV absorption maximum varying from 261 to 272 nm indicating presence of nitrobenzene ring in the degradation products. No significant change in nitrite ion concentration could be detected in aqueous solution of irradiated CPL using diazotization method (Varshney, 1990; Howel, 1975). Decrease in specific rotation value of 5% (w/v) solution of irradiated (100 kGy) CPL in ethyl alcohol by 7% indicates the fission of carbon-carbon bond between the two optically active carbon atoms (Varshney, 1990). Free radicals were detected in irradiated samples which linearly increased with increasing radiation dose. The radicals could be detected even in a two years old irradiated sample. Peroxides and hydroperoxides were detected in irradiated CPL samples (Fig. 4). Presence of free radicals in irradiated samples, detection of peroxides and restricted mobility of active species in solid state, indicate that some of the peaks in the chromatograms could be due to peroxides and hydroperoxides of fragments of CPL molecule. The degradation products which could be identified contain p-nitrobenzaldehyde (PNB), p-nitrobenzoic acid (PNBA), p-nitrosobenzoic acid and HCl (Fig. 1). Calculated G values from the concentration of degradation products for PNB, PNBA and Cl- are 0.85, 0.73 and 2.40, respectively, which remain almost constant up to 100 kGy. It may be noted from HPLC profiles that PNB and PNBA are exclusively formed by ionizing radiation in solid CPL. Greater constancy in G (- CPL) is observed in elec-
tron beam irradiation than in y irradiation (Tables 1 and 2). CPL has orthorhombic crystal structure. The molecules in the unit cell have curled staggered configuration. The energy could be absorbed by any part of the molecules but channeled only to specific bonds as indicated by linear increase in degradation products with increasing radiation dose. Considering the results of present study it appears that the absorbed radiation energy is mainly concentrated on the two optically active carbon atom and C-Cl bond. To account for the several degradation products with varying concentration, it could be suggested that the absorbed energy degrades the molecules in probablistic way. The probability of breaking of bonds connected to the optically active carbon atoms being the function of associated strain energy. Most of the radiation degradation products are also formed when exposed to sun light. PNB and PNBA are exclusively formed by ionizing radiation in solid CPL. From these observations it could be suggested the PNB and PNBA are formed from ionized species. Free radicals formed on irradiation remain in crystalline matrix even for a few years as observed in irradiated CPL. These free radicals (in the absence of autoxidation reactions) normally react with oxygen during storage or with dissolved oxygen in solvents like water forming trace level of peroxides or hydroperoxides which lead to formation of products like aldehydes and acids. The concentration of such products formed on storage in irradiated solid samples is not expected to exceed the molar concen-
476
L. VARSHNEYan d K. M. Table 3. Purity of non-irradiated and irradiated aqueous solutions of chloranmhenicol Concentration Dose Wy) 0.0
2.8 5.6 8.4 Il.2
0.2%
0.1%
0.05%
100
loo
loo
91.4 71.9 65.3
82.9 69.1 56.2 44.8
69.3 53.5 35.4 22.3
tration of free radicals which is about lo-‘mol in CPL at 30 kGy. Aqueous solutions expectedly undergo extensive percentage radiolytic degradation (Table 3). Chromatogram reveals formation of several degradation products (Fig. 1). A few new products are also observed in addition to the products observed in irradiated solid CPL. Peaks corresponding to retention time of p-nitroso benzoic acid and CPL base were quite prominent in irradiated aqueous solution. Discolouration Irradiation using either technique discolours CPL samples from off white to yellow. The intensity of the colour increases with increasing radiation dose (Fig. 5) as shown by increased absorption in blue region. Samples irradiated in air using y rays were less discoloured than: (1) deoxygenated, y irradiated (Fig. 7); (2) electron beam irradiated samples. Drastic reduction in colour intensity is observed in deoxygenated sample (Fig. 6) having greater number of trapped free radicals and electrons which react and disappear during dissolution in methanol. In a separ-
PATEL
ate experiment, the ESR spectrum of the CPL sample irradiated to 30 kGy contained 18 x 10” spins/g which disappeared after dissolution (Fig. 4). These free radicals react with dissolved oxygen in the solvent forming peroxides/hydroperoxide as detected in the present study. 0.5% (w/v) solution of irradiated CPL in acetone showed linearly increasing discolouration with increasing dose in comparison to non irradiated sample (Varshney, 1990). Therefore, discolouration in CPL on irradiation could be attributed to: (i) formation of trace level degradation products as shown by increased discolouration of solution of irradiated CPL and (ii) trapped free radicals in the crystalline matrix of CPL as shown by ESR and DRS. DRS difference spectrum of y irradiated CPL samples in: (1) air and vacuum; (2) air and electron beam gave similar absorption spectrum with peak maximum at 492 nm (Fig. 7). Both radiation degradation products and free radicals should absorb in visible region to impart colour to the irradiated samples. The colour centers due to the free radicals which exist in vacuum, could be destroyed by oxidation and recombination reactions during y irradiation in air and therefore, the intensity of the colour is reduced. Increased discolouration and similar DRS of samples y irradiated in vacuum and electron beam indicate that anaerobic conditions exist during electron beam radiation. This point should be considered while using electron beam for sterilization as microorganisms are known to be more radiation resistant in the absence of oxygen (Varshney et al., 1990).
-0.15 370
400
430
500 550 WAVELENGTH
600
650
(rim)
Fig. 5. Diffuse reflectance spectra of chloroamphenicol. (a) Non-irradiated; (b) irradiated, 15 kGy; (c) irradiated, 30 kGy; (d) irradiated, 50 kGy; (e) irradiated, 100 kGy.
700
Effects of ionizing radiations on chloramphenicol
370
400
450
500
550
WAVELENGTH
600
650
700
(nm 1
Fig. 6. DRS difference spectra of chloramphenicol.
(a) Irradiated, 45 kGy, air, recrystallized from methanol; (b) irradiated, 45 kGy, deoxygenated sample, recrystallized from methanol.
Crystallinity
Enthalpy of fusion of CPL samples irradiated using either technique linearly decreases with increasing radiation dose. This indicates the extent of reduction in crystallinity. The reduction in the enthalpy is attributed to the strain forces caused by the radiation degradation products in the crystalline matrix of CPL. X-ray powder diffraction pattern of irradiated samples also showed broadening of peaks (Fig. 8)
with reduced signal intensity indicating reduction in crystallinity (Varshney, 1990; Varshney et al., 1988). The changes in crystallinity have been reported to affect dissolution profiles and hence bioavailability (Grant and York, 1986). Solubility
The solubility of a compound is dependent on thermodynamic functions. Dissolution of irradiated
0.37
: ~0.24 2 a 5: 0.11 :
370
400
450
500 550 WAVELENGTH
600
650
(nm 1
Fig. 7. DRS difference spectra of chloramphenicol. (a) Irradiated, 45 kGy, deoxygenated sample; (b) irradiated, 45 kGy, air; (c) difference spectra (a-b); (d) difference spectra (electron beam irradiated, 45 kGy - b).
700
478
L. VARSHNEY and K. M. PATEL
35
I
I
I
I
1
I
37
39
41
43
45
47
29
49
(DEGREES)
Fig. 8. X-ray powder diffraction pattern of chloramphenicol.
CPL in water could be represented as: [CPL]solid-irradiation +[CPL+Al+Bl-tCl+..]solid l&O V
(a) non-irradiated;
(b) irradiated, 30 kGy.
the degradation products than the quantitative decrease in purity of CPL. This observation implies that small concentration of radiation degradation products can bring significant physical changes like solubility and colour. Entropy of radiation processing
CPL(aq.) + Al(aq.) + Bl(aq.) + Cl(aq.) Where Al, Bl, Cl are radiation degradation products in CPL matrix. The free energy change during dissolution (AG) is related to enthalpy (AH) and entropy (AS) changes given by: AG=AH-TAS AG = - RT LnCs Where Cs is the molar solubility of CPL in water at a temperature T. Negative contribution from AH (reduction in enthalpy of fusion) and possible positive contribution by AS (entropy of mixing of degradation products in solvent) could facilitate dissolution and solubility (AG negative). It is observed that the solubility initially increases and then becomes almost constant (Table 4). The constancy in solubility could be attributed to combined effect of radiation degradation and compensation by increased solubility of irradiated CPL. The initial increase in solubility at 15 kGy could be due to more disruptive influence of
On irradiating solid CPL crystalline matrix, degradation products are formed at random positions in the crystal lattice. These products could have a disruptive influence and thus the lattice is activated affecting enthalpy and entropy values. The change in entropy of solid CPL due to radiation processing can be defined as Srp, which can be determined by calorimetry (Grant and York, 1986) Srp is defined by the following equation: Srp = - S (CPL)irrad. + xa S (CPL) +xbSb+xcSc+xdSd+...+Smix Table 4. Solubilityand entropy of radiation processing data of non-irradiated 7 irradiated CPL DO%
WY) 0 I5 30 50 100
(HPLC) @g/ml) at 25°C 2.54f 0.10
Solubility
2.72 + 0.1 I 2.71 kO.14 2.74+O.l2 2.75 f 0.14
Srp WC) (J mol-’ Km’) 0 4.3 8.0 II.5 21.2
Effects of ionizing radiations on chloramphenicol
479
Table 5. Ratio of inhibiton zone diameter on SCD agar plate Dose (kGy): Ratio
Strain coli S. typhimurium B. subtilis E.
0
I5
30
M
100
I .of 0.0
i.o*o.o
0.995 f 0.010 0.966 f 0.030
0.956 f 0.024 0.920 f 0.032
0.908 * 0.019 0.887 f 0.042
1.0*0.0
1.0+0.0
0.989 f 0.025
0.999k 0.010 0.992f 0.020 0.962f 0.030 0.903k 0.020
Note: the value zero means no distinction using mm wale could be made between zone diameters under magnifying glass
Where xa is mole fraction of CPL in irradiation CPL and xb, xc, xd are mole fraction of the degradation products and Smix is entropy of mixing. Using DSC, Srp is determined as:
CPL solid - srp
[CPL + A + B + C + . . .]solid
irradiation
E
rnl1
Melt
c
rln2
Melt
= -H,, /T,,,, (irradiated) + Hn/Tm2 (non irradiated)
where Hr,, HO and T,,,, , T,,,, are enthalpy of fusion and melting point of non-irradiated and irradiated samples respectively. The calculated Srp values for gamma irradiated CPL samples are given in Table 4. Linear increase and similar magnitude in Srp values are observed in y and electron beam irradiated CPL samples. Increase in Srp could be linked with increase in solubility of irradiated CPL in water. Microbiological
sensitivity
tests
The inhibition zone diameters do not show any significant change up to 30 kGy when the three bacterial strain were tested. This observation is in accordance with the insignificant change in chemical purity of irradiated CPL determined using HPLC and DSC techniques. Significant reduction in antibacterial property is observed at 100 kGy (Table 5). Toxicological
tests
toxicity and histamine like undue substances were observed in non-irradiated as well as CPL samples irradiated up to 30 kGy. No mutagenic behaviour was shown by the degradation products. Most of the products formed on irradiation of CPL are also present in non irradiated product. Irradiation merely increases their concentration to an insignificant level at lower dose such as 15 kGy. Several radiation degradation products are formed in irradiated CPL and there are practical limitations in identifying all the degradation products. Some or all products could be toxic individually at certain concentration. In the present study p-nitrobenzaldehyde formed on irradiation is a known toxic compound having LDSO-ORAL(Rat) = 4700 mg/kg. CPL itself No
is toxic and has TDLO-ORAL value of 1700 mg/kg in women of average weight 50 kg (Irving, 1984). Toxicity examination of such irradiated drug containing several degradation products, totalling less than 1% could be far less than the toxicity of drug itself. Although, the changes in toxicity characteristics are not expected to change at sterilization dose, the drug must be screened for undue toxicity and histamine like substances. CONCLUSIONS
(1) Chloroamphenicol in solid dry powder form can be radiation sterilized using cobalt-60 or electron beam at lower radiation dose of 15 kGy at which insignificant radiation effects have been observed. Aqueous solutions of CPL undergo extensive degradation on irradiation and hence can not be sterilized. (2) Although, high chemical purity of irradiated CPL is retained at sterilization dose of 15 kGy, changes could be observed in physical properties like discolouration, crystallinity and solubility. (3) Irradiated CPL should not be considered as a new drug, if its physico-chemical integrity is established using sensitive analytical techniques by experts in the field. The low concentrations of radiation degradation products at the sterilization dose of 15 kGy are not expected to cause any untoward reactions. (4) In addition to pharmacopeal tests, irradiated CPL should be evaluated using sensitive analytical techniques such as HPLC and DSC for purity and degradation products. Acknowledgements-We are grateful to Shri K. Krishnamurthy for his support and encouragement. Thanks are due to Dr V. K. Iya and Shri S. Sabharwal for discussion and valuable suggestions and Shri R. S. Deshpande for help in carrying
out electron
beam irradiation. REFERENCES
British Pharmacopoeia (1988) p. 117. HMSO, London. Gopal N. G. S. (1978) Radiation sterilization of pharmaceuticals and polymers. Radiat. Phys. Chem. 12, 35. Gupta B. L., Bhat R. M., Narayan G. R. and Nelekani S. R. (1985) A snectrophotometric read out method for free radical dosim&y. Radial. Phys. Chem. 26, 647. Howe1 F. N. (1975) Standard Methods of Chemical Analvsis. VIth edn, Kreiger, New York, p. 745. Irving S. N. (1984) Dangerous Properfies of Industrial Materials. VI edn. Van Nostrand Reinhold, New York. Jacob G. P. (1985) A review: radiation sterilization of pharmaceuticals. Radiat. Phys. Chem. 26, 133.
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L.
VARSHNEY and
Pate1 K. M. (1986) Effects of repeated exposure of gamma rays on the survivors of different strains of staphylococcus aureus. Ph.D. thesis Bombay University, India and references therein. The Indian Pharmacopoeia (1985) III edn. Controller of Publications, Delhi, India. pp. A-42-A-44. Varshney L. (1990) Effects of ionizing radiation on chloramphenicol and feasibility studies on its radiation sterilization. Ph.D. thesis University of Bombay, India. Varshney L. and Iya V. K. (1989) Effects of Cobalt-60 gamma radiation on chloramphenicol. Ind. J. Pharm. Sci. 51, 25.
Varshney L., Sharma G. and Iya V. K. (1988) Cobalt-60 gamma irradiation of chloramphenicol uis-a-ois its crystallinity. Indian Pharmaceutical Cong. Calcutta, West Bengal, India.
K. M.
PATEL
Varshney L., Sharma G. and Iya V. K. (1989) Natural and Radiolytic degradation products of chloroamphenicol. In Radio Chemistry and Radiation Chemistry Symp., Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamilnadu, India. Varshney L., Iya V. K., Sharma G. and Pate1 K. M. (1990) A comparitive study of irradiation of chloramphenicol using electron beam and cobalt-60 gamma rays. In Naf. Semin. on Industrial Radiation Processing and Technology,
IIT Powai, Bombay, India. Wendtlandt W. W. and Hecht H. G. (1986) Rejlectance Spectroscopy, Chapt. 3 and 4. Interscience, New York. Wogl W. (1985) Radiation sterilization of pharmaceuticals, chemical changes and consequences. Radiat. Phys. Chem. 25, 425.