African Journal of Pure and Applied Chemistry Vol. 3 (8), pp. 135-139, August, 2009 Available online at http://www.academicjournals.org/AJPAC ISSN 1996 - 0840 © 2009 Academic Journals
Full Length Research Paper
Effect of heating on the chemical stability of two brands of metronidazole suspension Enoche Florence Oga1* and Nelson Agaba Ochekpe2 1
Department of Medicinal Chemistry and Quality Control, National Institute for Pharmaceutical Research and Development, PMB 21 Garki, Abuja, Nigeria. 2 Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of Jos, Plateau State, Nigeria. Accepted 8 July, 2009
Metronidazole suspension is available in most developed countries as a prescription only medicine and in the form of an extemporaneous preparation of 90 days shelf-life. In Nigeria, it is available on the shelf with shelf lives of 2 - 4 years. It is available from at least 20 local manufacturers. This work has determined the shelf life of two brands of metronidazole suspension using accelerated stability testing method. A relatively simple analytical method has been utilized for stability studies of metronidazole suspension because of known research constraints in the laboratories of most developing countries. The analytical procedure employed for quantization of metronidazole in the suspension involved; liquid-liquid extraction followed by the use of ultraviolet/visible Spectrophotometry. This method has been successfully used for the determination of the shelf-life of two branded metronidazole suspensions. The technique offers the advantage of simplicity, rapidity and sensitivity without the need for very expensive equipment. The shelf lives calculated for Brand 1 at the temperatures of 25 and 30°C were 4.96 and 3.86 years, respectively; while that for Brand 2 was 7.7 and 5.87 years, respectively. The labeled shelf life for Brand 1 was 2 years while that for Brand 2 was 3 years. Key words: Metronidazole suspension, accelerated stability testing, shelf-life, spectrophotometric assay method. INTRODUCTION In the Nigerian Essential Drugs List (Federal Ministry of Health Abuja, 2003), the Metronidazole preparations identified for local use are: tablet, intravenous infusion, and suspension. The British Pharmacopoeia (British Pharmacopoeia, 1998) and United States Pharmacopoeia (United States Pharmacopoeia XXI) contains monographs on the following preparations: suppositories, tablets, capsules, injections, topical gel, vaginal gel, and topical cream. Metronidazole is a nitro imidazole anti infective agent used mainly against susceptible anaerobic bacteria and protozoa organisms. Its mechanism of action involves the disruption of DNA and nucleic acid synthesis in the bacteria (The Access Project - Metronidazole, 2009; McEvoy, 1996). The aromaticity of metronidazole (whose structure is
*Corresponding author. E-mail:
[email protected]. Tel.: 234-807 350 1659.
shown below) is due to the presence of pie bonds with free moving electrons making it possible for the compound to absorb radiation in the electromagnetic spectrum. The aliphatic ethanolic group is attached to position one which can undergo normal aliphatic oxidation and reduction reactions. The nitro group on position five can be reduced by reducing agents like zinc in concentrated hydrochloric acid to an amino function (Saffaj et al., 2004). This forms the basis of the differential ultraviolet spectrophotometric assay of Metronidazole. Differential ultraviolet spectroscopy is a technique that is based on the effects of pH changes on the absorption spectra. The difference in absorbance of the drug in acid or alkaline media can be readily related to the concentration of the drug. Here the wavelength of maximum absorption, max of one component can be shifted by the addition of acid or base to permit measurement of the other while the absorbance due to each component is determined. The ability of Metronidazole to undergo reduction reaction in which the reduced form does not absorb at the same max of the pure Metronidazole has been made used
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C H O
2
N
2
C H
2
O H C H
N
3
N Chemical structure of metronidazole.
used of in this work. Any absorbance observed in the reduced metronidazole solution at that wavelength represents that of a background or interfering substance or impurity that may contribute to the absorbance of pure Metronidazole. Therefore the true absorbance due to Metronidazole has to be determined by the difference between the reduced and unreduced Metronidazole (Deepika et al., 2008). To ensure the quality, efficacy and safety of pharmaceutical products, stability studies form an essential part of the pharmaceutical pre- and post-formulation studies (Loyd et al., 2003; Watterman et al., 2007; Yunqi and Reza, 2005). Instabilities in modern formulations are often detectable only after considerable storage under normal conditions. To assess the stability of the formulation, it is usual to expose it to high stress conditions to enhance its deterioration and therefore reduce considerably the time required for testing (Olaniyi, 2000; Tson et al., 2003; Djira et al., 2008). Accelerated stability studies have proved an effective alternative to the time consuming and uneconomical practice of storing the product at room temperature for the time the products would be likely to remain in stock and use. A comprehensive stability plan is an essential and pertinent extension of the quality assurance programme. The effect of temperature on the rate of decomposition as described by the Arrhenius equation (Alfonso et al., 1985) states that:
k=Ae
–Ea/RT
Where k = specific rate constant, T= absolute temperature, Ea = Energy of activation, R =Gas Constant (8.314J/K.Mol), A = Frequency factor. The constants A and Ea may be evaluated by determining k at several temperatures and plotting log k against 1/T. The slope of the graph is –Ea/2.303R and the intercept on the y-axis is log A from which the values of Ea and A may be calculated. Stability of a pharmaceutical product may be defined as the capability of the particular formulation in a specific container/closure system to remain within its physical, chemical, microbiological, therapeutic and toxicological specifications. It is the time from the manufacture and packaging of the formulation until its chemical and biological activity is not less than a predetermined level of labeled potency while its physical characteristics remain unchanged (Wolfgang, 1987; Repeto, 2000; Sprandel et al., 2005).
The physical stability of a suspension is normally assessed by the measurement of its rate of sedimentation, the final volume or height of the sediment and the ease of re-dispersion of the product. An assessment of these three parameters at elevated temperatures would give speedier indication of a rank of order of instability but it is essential to correlate these results with those taken from suspensions stored at ambient temperatures (Sethi, 1997; Jiben, 2002). There have been several reports on Metronidazole stability (Irwin et al., 1987; Mathew et al., 1994; Allen et al., 1996; Alexander, 1997; Trissel, 2000; Bempong et al., 2005; Glass et al., 2006; Vu Nicole et al., 2008). Irwin et al. (1987) evaluated the stability and bioavailability of a Metronidazole 15 mg/ml oral suspension compounded from tablets. Mathew et al. (1994) evaluated the stability of the benzoate ester of Metronidazole prepared as oral solutions. Allen et al. (1996) evaluated the stability of three Metronidazole 50 mg/ml oral suspensions extemporaneously compounded from powder. Trissel (2000) reported Metronidazole and Metronidazole hydrochloride to be stable in air but may darken upon exposure to light and thus recommended that such products should be packaged in light resistant containers. Refrigeration of metronoidazole may result in crystal formation, which redissolves upon warming to room temperature. Reconstituted Metronidazole hydrochloride is stable for 96 h when stored below 30°C and on exposure to normal room light. Vu Nicole (2008) assessed the stability of metronidazole benzoate suspension using SyrSpend suspension system, validating the assay method by High Performance Liquid Chromatography and stability indicating characteristics through a forced degradation study. MATERIALS AND METHODS Equipment Some of the apparatus and equipment used included; hot air oven and water bath (Gallen Kamp, England), Zeal’s laboratory thermometer (0 - 360°C), UV/Visible spectrophotometer (Jenway 6305, England) , hot plate, filter paper (Whatmans No. 1), Analytical balance ( Metler Toledo, Switzerland), and refrigerator. Chemicals and samples Sodium hydroxide and dichloromethane (May and Baker, England), Brands of Metronidazole suspension, [Brand 1 (MTZ1) and Brand 2 (MTZ2)], Metronidazole (Chemical Reference sample) was obtained from the Department of Pharmaceutical Chemistry laboratory, University of Jos, Jos. Methodology A 20% sodium hydroxide solution was prepared. Metronidazole reference standard solution was also prepared and stored in the refrigerator. Using a metronidazole solution of 50 mg/ml, a scan from 240 to380 nm was carried out to determine the max. A calibration as a
Oga and Ochekpe
Figures 1. MTZ1:Log k against 1/T.
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Figures 2. MTZ2:Log k against 1/T.
validation procedure for the method was carried out using the drug solutions of 40, 20, 10, 5, 2.5 mg/ml obtained by serial dilution. The absorbances of these solutions were obtained at the wavelength for maximum absorbance of 315 nm. The mean of five replicate absorbance readings for each sample was calculated and used to obtain the corresponding concentration using the linear regression equation of the best fit line that was obtained from the calibration. Liquid-liquid extraction was carried out in triplicates using dichloromethane as the extraction solvent. The dichloromethane layer for each extraction was combined and evaporated to dryness on a hot water bath. The residue was dissolved in distilled water and made up to 100 ml. From this solution, 2 ml was added to 3 ml 20%NaOH solution, kept at room temperature for about 8 min, made up to 10 ml with distilled water and the absorbance taken at 315 nm. The method of accelerated stability testing employed was based on that demonstrated in Rhodes et al. (1996). Irregular energy source and lack of climatic chamber led us to the use of aluminum pots filled with dry fine sand to maintain a constant temperature. A thermostat hot plate was the heating device and a generator as the source of energy.
RESULTS AND DISCUSSION The need for stability tests cannot be over emphasized. Stability testing is the basis for among other things, the selection and establishment of suitable containers/ closure systems, the shelf life, the use life, storage and the directions for storage for the drug product. Some effects of instability in metronidazole suspension include loss of content uniformity (sedimentation impactation), production of potentially toxic degradation products, and loss of pharmaceutical elegance (e.g. colour changes/development of unpleasant odours). Accelerated stability tests serve to identify the weak points in a formulation, to select the test criteria and to check the suitability of the analytical techniques. The result of these investigations provides estimates of shelf life and predicts stability. During this work, the wavelength of maximum absorbance obtained was 315 nm. The linear regression equation for Metronidazole Chemical Reference Standard was
Figure 3. MTZ1 Conc. against time.
y = 0.0034x + 0.0079 with a correlation coefficient of 0.9929. This gave a good plot specifying agreement to Beer-Lamberts law. For MTZ 1, on plotting the graph of log k (logarithm of the rate of decomposition) against 1/T (reciprocal of absolute temperature in Kelvin) and concentration against time [Figures (1) and (3)], the integral form of the Arrhenius equation log K= log A – Ea/ (2.303RT), and the linear regression equation of the best fit was: Y = -1819.7x + 6.4991 6
Where A= 3.16 x 10 , gradient or slope = Ea/2.303R. From which Ea = 34.8 KJ/Mol. When T = 25°C, using the first order rate equation, t = 4.96 years and when T = 30°C, t = 3.86 years. For MTZ 2, on plotting the graph of log k (logarithm of the rate of decomposition) against 1/T(reciprocal of
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tion is simple, inexpensive and economical, hence could be used for routine shelf life determination in less endowed laboratories. Therefore, based on this method of accelerated stability studies using elevated temperature, there is no overt advantage of extemporaneously prepared Metronidazole suspension over the pre-formulated suspension obtained over the counter. REFERENCES
Figures 4. MTZ2: Conc. against time.
Absolute temperature in Kelvin) and concentration against time [Figures (2) and (4)], Y = -1942.7x + 6.7215, 6 where A = 5.27 x 10 , gradient or slope = Ea/2.303R. From which Ea = 37.2 KJ/Mol. When T = 25°C, using the first order rate reaction, t= 7.7 years and when T = 30°C, t = 5.87 years. The Ea for most drug decomposition reactions are known to range from 50 - 100 KJ/Mol. On conducting the work, the Ea for the hydrolytic decomposition of MTZ 1 was 34.8 KJ/Mol. That for MTZ2 was 37.2 KJ/Mol. They both did not fall within the range. However Ea values are not really useful parameters in reaction kinetic studies. For MTZ 1, storage at 25°C gave a shelf life of 4.96 years while storage at 30°C gave 3.86 years. Both temperature values were used in the calculations since Nigeria falls within the tropics and the temperature fluctuates generally within 23 - 30°C though in some states, higher ambient temperatures of up to 35 - 45°C may be recorded. On the other hand, for MTZ2, storage at 25°C gave a shelf life of 7.7 years while at 30°C, it was 5.87 years. The shelf life obtained for both brands of Metronidazole suspension were above that stated on the labels of 2 years for MTZ 1 and 3 years for MTZ 2. Thus the stated shelf lives would appear appropriate bearing other factors such as humidity and light that were not examined in this study. In this method applied, considering the resource limited setting in our laboratories, an innovative technique has been used whereby the suspension was subjected to elevated temperature by heating in an enclosed chamber (sand pot) with the sand and the desired temperature being regulated appropriately. This proposed method offers the advantage of speed and sensitivity. Unlike, other techniques, the instrumenta-
Alexander KS (1997). The Formulation, Development and Stability of Metronidazole Suspension. Int. J. Pharm. Compounding.1: 200-205. Alfonso R, Gennaru Ed. (1985) Remington’s Pharmaceutical Sciences. 17th ed. Easton, Pennsylvania: Mack Publishing Company. p. 1478. Allen LV, Erickson MA III (1996). Stability of Ketoconazole, Metazolone, Metronidazole, Procainamide HCl and Spirinolactone in Extemporaneously Compounded Oral Liquids. Am. J. Health-Syst Pharm. 53: 2073-2078. Bempong DK, Manning RG, Mirza T, Bhattacharya L (2005). A Stabilityindicating HPLC assay for Metronidazole benzoate. J. Pharm. Biomed. Anal. 38(4): 776-780. British Pharmacopoeia (1998). Her Majesty’s Stationery Office. London. (1): 893. Deepika M, Jain A, Maheshwari RK, Patidar V (2008). Simultaneous spectrophotometric estimation of metronidazole and norfloxacin in combined tablet formulations using hydrotrophy. Asian J. Pharm. 1(4): 357-361. Djira GD, Horthorn LA, Tsong Y (2008).Equivalence Tests for Shelf life and average Drug Content in Stability Studies. J. Biopharm. Stat. 18(5): 985-95. Federal Republic of Nigeria (2003). Essential Drugs List.4th Revision. Federal Ministry of Health, Abuja in collaboration with World Health Organization. Glass BD, Haywood A (2006). Stability Considerations in Liquid Dosage Forms Extemporaneously prepared from Commercially Available Tablets. J. Pharm. Pharm. Sci. 9(3): 398-406. Irwin DB, Dupuis LL, Prober CG, Tesoro A (1987). The Acceptability, Stability and Relative Bioavailability of an Extemporaneous Metronidazole Suspension. Can. J. Hosp. Pharm. 40(2): 42-46. Jiben J (2002). Pharmaceutical Impurities, a mini review. Roy J Pharmaceutical Impurities. AAPS Pharm. Sci. TECH. 3 Suppl 2: 6. Lawrence AT (2000). Trissels Stability of Compounded formulations. 2nd ed. Am. Pharm. Assoc. pp. 250-253. Loyd VA, Martin AE (2003). Stability of extemporaneously prepared Paediatric formulations using Ora-Plus with Ora-Sweet SF. Secundum Artem. 5 Suppl 4: 1-5. Mathew M, Das Dupta V, Bethea C (1994). Stability of Metronidazole Benzoate in Suspensions. J. Clin. Pharm. Ther. 19(1): 31-34. McEvoy GK Ed. (1996). Metronidazole. AHFS-96 Drug Information. Bethesda (MD): Am. Soc. Health Sys. Pharm. p. 1197. Olaniyi AA (2000). Principles of Drug Quality Assurance and Pharmaceutical Analysis. Mosuro Publishers p. 104-109. Repeto P (2000). Some Considerations about Stability Study Design. J. Biopharm. Stat. 10(1): 73 -82. Rhodes CT, Banker GS, Eds (1996). Modern Pharmaceutics. 3rd Ed. New York: Marcel Dekker p. 329. Saffaj T, Charrouf M, Abourriche A, Abboud Y, Bennamara A, Berrada M (2004). Spectrophotometric Determination of Metronidazole and Secnidazole in Pharmaceutical Preparations. II Farmaco 59(10): 843-846. Sethi PD (1997). Quantitative Analysis of Drugs in Pharmaceutical Formulations. 3rd Edition. CBS Publishers. Sprandel KA, Styrczula DE, Deyo K, Sibley C, Rodvold KA (2005). Stability and Compatibility of Levofloxacin and Metronidazole during Simulated and Actual Y-Site Administration. Am. J. Health-System Pharm. 62(1): 88-92. The Access Project- Metronidazole. http:/www.aidsinfonyc.org/network
Oga and Ochekpe
/access/drugs/metr.html. [Accessed 20th January, 2009]. Tson Y, Chen WJ, Lin TY, Chen CW (2003). Shelf life Determination based on Equivalence Assessment. J. Biopharm. Stat. 13(3): 431-4. United States Pharmacopoeia XXI. United States Pharmacopoeial Convention Inc. p. 691. Vu Nicole T, Aloumanis V, Ben MJ, Kupiec TC, Patterson EK, Radke J, Erisckson MA, Schneider G (2008). Stability of Metronidazole Benzoate in SyrSpend SF One-step Suspension System. Int. J Pharm. Compounding. Nov/Dec. 555-564. Watterman K, Carella A, Gumkowski M, Lukulay P, MacDonald B, Roy M, Shamblin S (2007). Improved Protocol and Data Analysis for Accelerated Shelf life Estimation of Solid Dosage Forms. Pharm. Res. 24(4): 780-790.
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Wolfgang G (1987). Stability testing of Drug Products. Stuttgart: Wissenschaftliche Verlagsgesellschaft mbH; p. 146-150. Yunqi Wu, Reza F (2005). Stability of Metronidazole, Tetracycline Hydrochloride and Famotidine alone and in Combination. Int. J. Pharm. 290(1): 1- 13.
