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Nigerian Journal of Pharmaceutical Sciences Vol. 8, No. 1, March, 2009, ISSN: 0189-823X All Rights Reserved

LIGHT INDUCED DEGRADATION OF AQUEOUS SOLUTION OF

CHLORAMPHENICOL

1Bakare-Odunola, M. T. 2Bello-Mustapha, K. B. and 2Enemali, I. S.

1Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University, Zaria, Nigeria 2Department of Medicinal Plant and Traditional Medicine,

National Institute for Pharmaceutical Research and Development, Abuja-Nigeria

*Author for Correspondence: mojitaibat@yahoo.com, 08035896043

ABSTRACT Some pharmaceutical preparations exhibit chemical and physical instabilities leading to decomposition and deterioration of the chemical compounds, hence, loss of therapeutic benefits of the drug. Aqueous solution of chlorampenicol reference solution and eye drops were exposed to sunlight, ultraviolet (U.V.) radiation at 365nm wavelength and red light for varying lengths of time. The kinetics of decomposition was studied using thin layer chromatography (TLC) techniques and U.V. spectrophotometric methods of analysis. The half - lives (t½) of decomposition were 20.47h, 22.02h and 1052. 0 h; the rate constant (K) values were 3.39 X 10-2 h,-1 3.15 X10-2 h-1 and 0.07X10-2 h-1 in sunlight, U.V. light and in red light respectively. The average half - lives of decomposition were 21.34 ± 2.70, 19.66 ± 1.16, 861.36 ± 87.95h; the rate constant (K) values were 3.29 ± 0.42 X 10-2h-1,3.52 ± 0.21 X10-2h-1 and 0.08 ± 0.01 X 10-2h-1 for the chloramphenicol preparations in sunlight, U.V. light and in red light respectively. The time taken for the half reactant to decompose (t½) was higher compared with in sunlight or U.V. light. The rate constant of decomposition in red light was also lowered in red light than in sunlight or in U.V. light. The chloramphenicol preparations were more stable in red light than in sunlight or U.V. light. The study showed the importance of proper storage condition of pharmaceuticals. Key words: Stability, Chloramphenicol, Sunlight, Ultra Violet and Red light. INTRODUCTION The large number of possible reactions leading to drug degradation that do occur can be classified as either hydrolysis or oxidation. In part, it is a consequence of the nature of the nearly ubiquitous occurrence of water and oxygen (Cori, 1980). Because many pharmacopoeial substances contain water in absorbed form, the British pharmacopoeia (B.P.), for example gives limits for the water content of some substance (Olaniyi and Ogunbamila, 1991). Such substances must keep within limits to avoid hydrolysis. The environmental factors that are known to

influence the extent and rate of deterioration are heat, moisture, oxygen, light and pH changes. Oxygen in the presence of light causes oxidation of many pharmaceutical compounds, especially light – sensitive formulation (Olaniyi, 2000). Information about the stability of drug components and drug formulations is also needed to predict the shelf –life of the final products (Esche et al., 2003). Chemical stability studies may be conducted under normal storage or experimental conditions or under exaggerated conditions (accelerated stability testing) and the results

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extrapolated to normal condition (Cater, 1984). The procedure usually involves the monitoring of the rate of decomposition of the parent compound or the rate of formation of the degradation product (Kenneth and Gorden, 1986). Chloramphenicol is a broad spectrum antibiotic with wide range of clinical applications for systemic and topical uses (Lawrence and Bennett, 1987). Commercial chloramphenical preparations are packed in different type of containers, ranging from plain glass, transparent plastic, coloured plastic to amber coloured bottles. The chemistry of the photo degradation products of chloramphenicol aqueous solution (pH 5.4) at room temperature (21 – 30oc) and in the presence of sunlight, suggests that under the influence of light and water the drug undergoes oxidation, reduction, and condensation reactions. The photolysis degradation product were isolated and identified (James and Leach, 1970., Shih, 1971). Since the degradation of chloramephenicol is initiated by light the rate of decomposition of chloramphenicol may therefore be dependendent on the source of light and the light intensity. A knowledge of decomposition of chloramphenicol is essential in order to access the storage conditions of chloramphenicol preparations. There is also no gain saying in the fact that instability posses serious problems for drug products distributed and used in tropical climates because of extremes of temperature and humidity conditions (Olaniyi, 1997). Such instability may decrease the therapeutic activity of the preparation or cause the appearance of a toxic substance formed as a degradation product. This work reports the light influenced decomposition of chloramphenicol preparations formulated and marketed in Nigeria.

METERIALS AND METHODS Materials Chloramphenicol eye drugs of varying brands, Beltacol®, Optachlor®, Franol Elcee®, and MCA® were bought from pharmacy shops in Zaria. Chloramphenicol authentic sample was obtained from the Department of pharmaceutical chemistry, Ahmadu Bello University. A.B.U. Zaria. Ethanol 96% (BDH Chemical Limited), iso propanol, methanol (Daykson chemical industries, Lagos, Nigeria), chloroform (May and Baker Limited, Dugenham, England) silica gel H 254 (F.Woelm ICN Pharm. Gonbit and Co. W. Germany) were used for the analysis. A fixed wavelength 254nm and 365nm u.v. lamp; (Eagle scientific Nottingham England) and Red coloured bulb (Philips) purchased from an electrical shop in Zaria, were used for the degradation process. A double beam spectrophotometer SP8-100 (PYE-UNICAM, Cambridge, England) was used to take spectrophotometric readings. Methods Pretreatment of chloramphenicol solutions: A 5 ml volume of the different test samples (commercial chloramphenicol eye drops coded A, B, C, D, E) and aqueous solution of the authentic sample, were measured into set of different conical flasks. Each of the conical flask was wrapped with foil paper until it reaches the place of exposure. The pH of each of the sample was taken to be sure there had not been any change in pH before exposure. The samples were then exposed to different sources of light (sunlight, ultraviolet light (365nm) and red light at room temperature (29-31oc). Sample (100ml) were withdrawn at 1, 2, 3, 6, 12, 24 and 168 h for analysis. Qualitative analysis of Chloramphenicol and degraded Products: Analytical thin layer chromatographic (TLC) plates were spotted with the chloramphenicol solutions of reference sample in water and of each sample of the eye drops before and after exposure to

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sunlight, red bulb and UV lamp (365nm) for varying length of time, the stationary phase was silica gel HF 254. Two different mobile phases: (a) chloroform: Iso propanol (80:20) and (b) Chloroform: carbontetrachloride (2:1) were used in developing the plates (Shih, 1971). After developing, the plates were air dried at room temperature for about 10 minutes; separated spots were identified by viewing under ultraviolet light at 254nm. Rf values for chloramphenicol and degraded products were calculated from the ratio of the distance moved by sample to the distance of the solvent front. Quantitative Estimation of Residual Chloramphenicol: Residual chloramphenicol was estimated using modified method of (Shih, 1971). Preparative TLC chromatographic plates were spotted with 100ul capacity capillary pipette. The plates were developed for 30 mins using the chloroform: Isopropanol (80:20) mobile phase. The plates were air dried and the chloramphenicol spots identified under UV light, and scraped off into a test tube. 10ml Ethanol (96%) was added to the test tube shaken and allowed to stay in the dark overnight. The ethanolic solution was centrifuged at 2000g for 5 min. The supernatant was decanted, and the residual chloramphenicol estimated using a double beam ultraviolet spectrophotometer at wavelength 278nm. Concentration of chloramphenicol was generated from the calibration curve and the percentage drug remaining (residual chloramphenicol) was calculated from the formular: = Concentration obtained X Expected concentration 1

100

The rate of decomposition K was calculated from the slope of the plot of residual concentration of chloramphenicol against time

on a semi log graph sheet. Half- life of the composition was calculated from the relationship: First order rate of decomposition. t ½ = K

0.693

RESULTS AND DISCUSSION The colourless aqueous solution of the reference and eye drops changed to an orange yellow colour solution with precipitation on exposure to sunlight and U.V. light in the presence of air. The colour change was light cream with red light. TLC analysis of the exposed chloramphenicol solution showed two degraded products with Rf values of 0.19 and 0.12 with mobile phase (a) chloroform: iso propanol (80:20), and Rf values 0.17 and 0.61 with mobile phase (b) chloroform: carbon tetrachloride (2:1). This is in agreement with the findings of Shih (1971) who also reported two degraded products on exposure of chloramphenicol solution to light. There was no evidence of degradation in the reference sample solution.

Tables 1, 2 and 3 show the effect of sunlight, U.V. light (365nm) and red light respectively, on chloramphenicol aqueous solution of the reference and samples. The percentage drug contents of the samples just before exposure were 100% reference sample (Rs); A 65%, B 31%, C 80%, D 95% and E 60%. The percentage drug content of chloramphenicol in all the samples decreased with time of exposure. Thus providing evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of factors for example light. Table 4 shows the rate constant K and half lives (t½) of degradation of the reference and sample solution. The values in sunlight for reference are rate constant 3.39 x 10-2h-1, t½ 20.47h and mean values for samples are rate constant, 3.29 ± 0.42 x 10-2 h-1, t½ 21.34 ± 2.70 h. In UV light (365nm), for reference rate constant, 3.15 ± 10-2 h-1, t½ 22h and mean values for samples are, rate constant 3.52 ± 0.21 x 10-2 h-1, t½ 19.66 ± 1.16 h and in

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red light , reference solution rate constant , 0.07 x 10-2 h-1, t½ 1052. 0 h. The mean values of the sample are; rate constant 0.08 ± 0.01 x 10-2 h-1 and t½ 861.36 ± 87.95h. The K and the t½ values of samples in direct sunlight did not differ significantly from those obtained with ultraviolet light. The K and t½ values obtained

in this study may infer that chloramphenicol is much stable in red light than in sunlight or UV light (365nm).

This work showed the stability of chloramphenicol in coloured or amber coloured – bottles and also in air tight containers.

Table 1: Effect of direct sunlight (Temp. 32-35oC) on the percentage drug content of chloramphenicol aqueous solution of reference sample and eye drops

Time (h) 0 1.0 2.0 3.0 6.0 12.0 24.0 168.0

Ref 100 45 23 22.3 16.6 12.1 7.5 0.5

A 65 43 40 34 26.8 20.8 14.8 3.0

B 31 28.6 23 19 18.0 4.0 4.0 1.2

C 80 70 43 42 28.0 12.0 5.0 2.4

D 95 84 44.6 42 31.0 22.0 12.0 0

E 60 44.4 40.0 33.2 20.0 5.8 2.2 0

Table 2: Effect of U.V. light (365nm) on the percentage drug content of chlorampenicol

aqueous solution of reference sample and eye drops

Time (h) 0 1 2 3 6 12 24 168

Ref 100 90 84 84 75 47 31 2.2

A 65 62 40 36 25 24 7.0 4.0

B 31 30 27 20 17 14 6.2 0

C 80 70 46 43.40 29.40 16 6.20 0

D 95 90 92 80 60 38 19 1.2

E 60 40 40 36.40 24 20.0 5.80 0

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Table 3: Effect of red light on the percentage drug content of chlorampenicol aqueous solution of reference sample and eye drops

Time (h) 0 1 2 3 6 12 24 168

Ref 100 100 100 100 91 90.0 89.4 87.6

A 65 65 65 64.2 60.4 58.1 57.3 56.5

B 31 31 31 31 29.2 28.2 27.5 27.0

C 80 80 80 78.0 74.4 73.9 73.0 72.40

D 95 95 95 91.8 88.0 87.0 87.0 86.60

E 60 60 60 58.1 54.7 53.9 52.0 51.70

Table 4: Rate constant (K) of decomposition and half live (t½) of chlorampenicol aqueous solution of reference sample and eye drops under sunlight, UV light (365nm) and red light

Sunlight UV light (365nm) Red light

Sample Kx10-2h-1 t½(h) x10-2h-1 t½(h) Kx10-2h-1 t½(h)

Ref 3.39 K20.47 3.15 22.00 0.07 1052. 0

A 3.52 19.70N 3.76 18.44 0.07 931.45

B 2.73 25.23 3.57 19.42 0.08 841.02

C 3.39 20.43 3.75 18.57 .07 991.42

D 2.92 23.74 3.2 21.53 .09 783.05

E 3.88 17.86 3.4 20.36 .09 759.87 Mean ±S.E.M 3.29± 0.42 21.34 ±2.70 3.52± 0.21 19.66±1.16 0.08±.01 861.36±87.95

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