Indian Journal of Pharmaceutical and Biological Research (IJPBR)

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1 Indian J. Pharm. Biol. Res. 2016; 4(3):5-14 Research Article Stability analysis and quantitative evaluation of metronidazole suspension Patil P. D *, Desai S. R, Disouza J. I CODEN (USA): IJPB07 ISSN: Indian Journal of Pharmaceutical and Biological Research (IJPBR) Tatyasaheb Kore College of Pharmacy, Warananagar, Maharashtra, India Journal homepage: ARTICLE INFO: Article history: Received: 10 July 2016 Received in revised form: 2 August 2016 Accepted: 10 August 2016 Available online: 30September 2016 Keywords: Suspension, Sedimentation, Drug content ABSTRACT Suspension is a heterogeneous mixture including solid particles that are satisfactorily bulky for sedimentation. Generally they must be larger than one micrometer. It is when particles are left floating around freely in a solvent. The inner phase (solid) is discrete all through the external phase (fluid) during mechanical stir, with utilizing of certain excipients or suspending agents. Metronidazole or 2-(2 methyl-5 nitro-1 H imidazol-1 yl) ethanol belongs to the nitro imidazole group of antibiotics whose antimicrobial property through derive from the formation of toxic free radicals by intracellular reduction. The objective of present study was to prepare suspension of Metronidazole by using different suspending agents and stability testing was to provide evidence on how the quality of suspensions varies with the time under of influence of environmental factors such as temperature, light, oxygen, moisture, other ingredient or excipients in the dosage form, particle size of drug, microbial contamination etc. and to establish a recommended storage condition. The FTIR and DSC techniques are use for characterization of active ingredient MBZ. This suspension was evaluated for appearance, ph, sedimentation volume and dissolution study for in vitro drug release. Result of evaluation indicates 1% w/v xanthan gum give optimal characteristic suspension. Suspension containing 1% w/v was exposed for the stability studies as per ICH guidelines. The suspensions were evaluated for their physicochemical parameter, particle size, drug release, drug content and microbiological assessment. The suspension shows good stability at 25 º C ± 2 º C /60%± 5% RH, 30 º C ± 2 º C /65%± 5% RH and good quantitative analysis result throughout the period of study. Suspension stored at 40 º C ± 2 º C /75%± 5% RH shows noncompliance with the analysis parameter after 4 week of storage. This indicate high temperature storage was shows degradation of the suspension which results in crystal growth in formulation and affect the physicochemical parameter as Appearance, ph, viscosity, specific gravity, drug dissolution profile and drug content. Introduction Convenience of administration and patient compliance are gaining significant importance in the design of dosage forms. Recently, more stress is laid down on the development of organoleptically elegant and patient friendly drug delivery system for pediatric and geriatric patients [1-2]. Metronidazole or 2-(2 methyl-5 nitro-1 H imidazol-1 yl) ethanol British pharmacopoeia (B.P.2003) belongs to the nitro imidazole group of antibiotics whose antimicrobial property through derive from the formation of toxic free radicals by intracellular reduction. Metronidazole activity is partially inhibited by the presence of oxygen, thus it is usually used in anaerobic infections, although it is also effective in the treatment of trichomonasis. Additionally Metronidazole is the agent of choice in patients with antibiotic associated colitis due to Clostridium difficile. [3-4]Both internal and parental routes have been used to administer metronidazole. Metronidazole benzoate often used in oral liquid dosage forms due to its nonbitter taste. All products under go changes, depending on the way that they are stored after they are manufactured, and Metronidazole suspensions are no exception; the World Health organization has issued Guidelines for Assessment of suspensions which suggest that the suspensions should also be assessed for their stability or shelf-life. [5-6] The objective of stability testing is to provide evidence on how the quality of suspensions varies with the time under of influence of environmental factors such as temperature, light, oxygen, moisture, other ingredient or excipients in the dosage form, particle size of drug, microbial contamination, trace metal * Corresponding Author: Patil PD, Tatyasaheb Kore college of pharmacy, Warananagar, Maharashtra, India. E- Mail:srdesai.tkpc@gmail.com 5

2 contamination, leaching from container etc. and to establish a recommended storage condition, retest period and shelf-life. Therefore evaluation of the parameters is based upon chemical, physical, microbiological, therapeutic and toxicological studies can serve as an important tool in stability studies. [7] Stability studies ensuring the maintenance of product quality, safety and efficacy throughout the shelf life are considered as pre-requisite for the acceptance and approval of any pharmaceutical product. These studies are required to be conducted in a planned way following the guidelines issued by ICH regulatory agency. Material and method Materials Metronidazole Benzoate, Sodium Methyl Paraben, Sodium Propyl Paraben, Propylene glycol, Xanthan Gum, Sorbitol, Colour sunset yellow, Pineapple flavour Method Preformulation Study Oraganoleptic Properties The Sample of metronidazole was evalutated visually for its physical state, color and odor Identification Tests The Metronidazole Benzote was studied for its identity, purity and physico chemical nature by using FTIR, DSC and UV Spectra. Meting point and other physico chemical tests were performed Formulation development of Suspension [8] Selection of Medium for Suspension The component in the suspension that is dispersion medium was selected on the basis of solubility of drug, less GI irritation, more stable in the room temperature, and palatable to patients. Screening of Suspending agent The various suspending agents were used in the development of suspension. The suspending agent HPMC ( 0.5-1% w/v), Tragacanth (0.5-1%w/v), Guar gum (0.1-1% w/v), Na CMC (0.5-1% w/v), Bentonite ( 0.5-1% w/v) and methyl cellulose (0.5-1% w/v), xanthan gum (0.5-1% w/v) were tried. The formulations were observed for sedimentation, redispersibility and cake formation. Additional Suspension Excipients The chief constituents of a well-formulated suspension, apart from the API and suspending agent(s), include excipients that wet the API, influence flocculation, control viscosity, adjust ph and the external medium, usually water. In addition, flavoring, sweetening and colouring agents are employed as well as suitable preservatives in order to minimize microbiological contamination. The quality of medicines depends not only on the API and production processes, but also on the performance of the excipients. Evaluation and selection of Batch [9-12] Appearance The appearance of the suspension sample was visually examined for color, homogeneity and for the appearance of sediment. The physical stability of a suspension is normally assessed by the measurement of its rate of sedimentation, ph The ph of the, was measured using a ph meter. Each suspension was well shaken for 20 seconds prior to ph measurement. It was checked in first week and after 6 months Determination of viscosity The viscosity of the suspension determined at room temperature. It was checked in first week and after 6 months using Brookfield rheometer. Assay of suspension Take 10 ml of suspension in 100 ml volumetric flask and make up the volume up to 100 ml with 0.1N HCl. Now withdraw 2 ml of solution from flask and add into 200 ml volumetric flask. Make up the volume with 0.1N HCl filter it measure the absorbance at λmax 278 nm using UVspectrophotometer. Particle size analysis Particle size analysis is carried out by using Malvern instrument.distilled Water is used as dispersant. Sedimentation volume Sedimentation volume (F) is ratio of the final or ultimate volume of sediment (Vu) to the original volume of sediment (Vo) before settling. Re-despersbility The suspension is filling up in the container. Settle for specific time and after that container is keep in hand, the container is rotated in 180º. The shakes required to redispersed is noted. As +++ indicate excellent, ++ indicate moderate, + indicate poor. In vitro drug release Dissolution studies of sample were performed using USP XXIII type 2 apparatus. Suspension equivalent to 200 mg of drug added to activated dialysis bag. The bag was tied from one side add 10 ml of suspension, bag was tightetened from other side. The bag was put into dissolution medium (900 ml of 0.1 N HCl at a temperature of 37 º C ) which was stirred with rotating paddle at 50 rpm. At suitable time intervals 10 ml samples were withdrawn, filtered,diluted and analyzed at 278 nm using UV. Determination of drug content The amount of metronidazole present in the formulation was determined by UV-Visible spectrophotometer. 0.5 ml of suspension taken and diluted to the 10 ml of 0.1N HCl and 1 ml was taken diluted to 10 ml and same procedure is follow for the blank. The absorbance was taken at λmax 278 nm by UV-Visible spectrophotometer. The % drug content was calculated. Microbiological analysis Microbiological studies were carried out by using cup and plate assay method. The plates were incubated at 25 o C for five days. The fungal count was estimated using a colony counter and then multiplied by the dilution factor to obtain the total count. Accelerated stability studies Research Article 6

3 Metrinidazole suspensions were packed in 60 ml glass bottle. The packed bottle kept maintained at following condition. Samples were collected at time at 0 week, 2 week, 4 week, 8 week and 12 week and after 6 months. Further studies was carried out to analyzed parameters which possibly change during storage like ph, viscosity, drug content, sedimentation, microbiological parameter. Result and discussion Preformulation study The identification test of Metronidazole Benzoate was tested by the respective methods. From the results shown in table 1, it is clear that the Metronidazole Benzoate is in pure form. Table 1: Identification test Sr. Test Methods Description No. 1. Physical state Visual observation Anhydrous solid 2. Colour Visual observation Creamy white 3. Odour Visual observation Odorless 4. Melting point Capillary method 98 0 C 5. IR spectra IR spectroscopy The IR absorption spectrum was concordant with the reference spectrum. 6. DSC Differential scanning colorimetry Melting point endotherm was observed is U. V. Spectra U.V. visible spectrophotometer U.V spectra shows λmax of 278nm in 0.1 N HCl Fourier Transform Infrared Spectrophotometer (FTIR) Fig 1: FTIR spectrum of Metronidazole benzoate Presence of aromatic ring (1) cm -1, cm -1, cm -1 and cm -1 which attributed to c-c vibrational modes conjugation with system or NO 2 causes splitting in cm -1 without considerable band shifting as seen by extra band at The ester is recognized by the high intensity absorption band of carbonyl stretching at (2) cm -1 and control band of the C-O, Single bond vibration at (3) cm -1. The benzoate ester is characterized by (4) the c-o group bands at cm -1, cm -1, cm- 1.The aromatic C-N stretching vibration characterized by band (5) cm -1, and aliphatic CN vibrations shows absorption bands at (6) cm -1 and band at cm -1 and cm -1 shows indicate nitro group(7). Research Article 7

4 Absorbance Patil P.D et al. / Indian J. Pharm. Biol. Res., 2016; 4(3):5-14 Differential scanning colorimetry (DSC) Thermal analysis by Differential scanning colorimetry showed a characteristic sharp endothermic peak at º C indicating the melting point of the drug. This fact confirmed that the drug is in pure form. Fig 2: DSC spectrum of Metronidazole benzoate UV Spectroscopy Determination of λmax Metronidazole Benzoate λmax was determined in 0.1 N HCl, Methanol and results are shown in table 2. Table 2: Determination of λmax Sr. No. Solvent λmax (nm) N HCl Methanol Standard calibration curve of drug The graph of absorbance v/s concentration for pure Metronidazole Benzoate was found to be linear in concentration 2-20 μg/ml at 278nm y = x R² = Concentration(μg/ml) Fig 3: Calibration curve for Metronidazole benzoate Formulation development of Suspension Medium for suspension For formulation of suspension purified water is used as medium. Propylene glycol is used as co solvent. Screening of Suspending Agent The various suspending agents were used in the development of suspension as shown in table 3. Research Article 8

5 Drug release % drug release% Patil P.D et al. / Indian J. Pharm. Biol. Res., 2016; 4(3):5-14 (Redispersibilty: +++ Excellent, ++ moderate, + Poor) Table 3: Screening of Suspending Agent Suspending Quantity Sedimentation Redispersibility Comments agent % w/v ratio HPMC Cake formed Cake formed Tragacanth No cake No cake Methyl cellulose No cake No cake Bentonite No cake No cake Na CMC No cake No cake Xanthum gum No cake No cake Evaluation and selection of Batch ph The ph of the suspension having concentration of xanthan gum 0.55, 0.65, 0.75, 0.85 and 1 %w/v as follow Table 4: ph of suspension (Xanthan gum) Xanthan gum (%w/v) ph time(min) 0.55%m/v xanthan gum 0.65%m/v xantahn gum 0.75 %m/v xanthan gum Fig 4: Dissolution curves for suspension with different concentration of xanthan gum Time (min) Fig 5: Dissolution curve of Metronidazole suspension in 0.1 N HCl Research Article 9

6 Suspension containing 1% w/v xanthan gum shows proper dissolution, good redispersability, and drug release when compared with standard suspension. Thus it was selected for further studies.70-75% drug was released in 60 min. The formulation batch containing 1% w/v xanthan gum was selected for further evaluation and stability study. Evaluation data for the suspension containing 1% xanthan gum is as shown in table 5 in first week of formulation and after 6 months of formulation Table 5: Evaluation of suspension in 1st week and after 6 months Time In first week After 6 month Sr. No. Test Specification Specification 1. Appearance Pale yellow color Pale yellow color 3. Average fill volume 60 ml 60 ml 4. Taste Sweet Sweet 5. ph Viscosity (mPa s) (mPa s) 7. Mass per liter cm cm Assay 101% 100% Appearance The bulk visual appearance of the metronidazole benzoate suspension remained the same in first week and after 6 months stored at room temperature. ph ph of the specification range for a xanthan gum suspension was found 5.94 in first week and 5.89 after 6 months.the ph value of the suspension are in specification range. Viscosity Viscosity of the optimized suspension was found to be mpa. s using Brookfield rheometer. Detail result is shown in table 6. Table 6: Viscosity of suspension in 1 st week Research Article 10

7 Particle size analysis Particle size analysis was as in ranged d(0.1) 0.082μm, d(0.5) 0.225μm, d(0.9) μm. The result is shown in Fig. 6. Fig 6: Particle size analysis Specific gravity The mass per liter was found to be gm/cm 3 in 1 st week and 1.17gm/cm 3 after 6 months. Assay of suspension The assay value of metronidazole benzoate suspension was found within range that complies with the desired specification. Table 7: Assay of suspension Sr. No. Theoretical conc.(mg) Conc. Of API(mg) % recovery Redispersibility: Formulation exhibits good redispersibility on mild shaking. Table 8: Redispersability in 1 st week to after 6 months Time Redispersibility Time Redispersibility First week +++ After 4 month +++ After 1 month +++ After 5 month +++ After 2 month +++ In 6 month +++ After 3 month ++ After 6 month ++ (+++ excellent, ++ moderate, + poor) Research Article 11

8 Drug release % Patil P.D et al. / Indian J. Pharm. Biol. Res., 2016; 4(3):5-14 Sedimentation volume The satisfactory choice usually accounts to Sedimentation volume (F) is 1 or 0.9. Sedimentation volume near to one means the particles tend to flocculate easily. The result of sedimentation volume was shown in table 9. Table 9: Sedimentation volume Time Sedimentation volume Week Week Week After 6 month 0.89 Dissolution studies In vitro drug release studies were carried out in 1 st, 2 nd, 4 th, 12th and 12 weeks. From fig. 7 it was observed that there is uniform release pattern at various time interval and Drug release was % in 90 min first week 2 week 4 week 8 week 12 week After 6 month Time(min) Fig 7: % Drug release Microbiological analysis Cup plate method showed zone of inhibition of drug, formulation and marketed preparation as indicated in fig. 8. From result it was observed that developed suspension showed zone of inhibition similar to drug and marketed preparation. Research Article 12

9 Fig 8: Zone of inhibition Stability study Stability result showed that developed suspension was stable after 6 months. Test parameters results were found to be within the acceptance limit. Table 10: Evaluation of the suspension at 25 C ± 2 C / RH 60 % ± 5 % RH Time Appearance Taste ph Viscosity Specific Redispersibility (mpa) gravity 1 week Pale yellow colour sweet week Pale yellow colour Sweet week Pale yellow colour sweet week Pale yellow colour Sweet week Pale yellow colour Sweet After 6month Pale yellow colour sweet Conclusion Metronidazole benzoate suspension was successfully developed. Different suspending agent was tried evaluated for ph, redispersibilty, dissolution profile, stability and sendimentation ratio. 1% w/v Xantan gum was found good suspending agent as it shows required ph, excellent Redispersibility, stability and drug % drug release in min time period. Other excipients used were methyl paraben and propyl paraben which prove to be good preservative throughout study. By the expolartion of the whole study it was proved that the suspension of metronidazole benzoate is stable at 25 ºC ± 2ºC / RH 75 % ± 5 % RH and 30 ºC ± 2ºC / RH 65 % ± 5 % RH but there is effect of temperature on stability of suspension. References 1. Chemistry: Matter and Its Changes, 4th Ed. by Brady, Senese, ISBN Ausutosh, M. Formulation, development and evaluation of patient friendly dosage form of Metfromin soft gel: Asian journal of pharmaceutics, 2008;2: Verma, P. Nambodiary, V. A stability indicating HPLC method for the determination of metronidazole using ecofriendly solvent as mobile phase components: Int J of Pharmacy & Pharmaceutical Sciences,2013; Nayak. S. Goupale, DC. Comparative stability study of metronidazole in aqueous and non-aqueous vehicle: Journal of Applied Pharamacy. 2011;3, Ancha, MJ. Kumar, KL. Formulation and evaluation of pediatric azithromycin suspension :Indian journal of pharm bio science, 2010;1(2), Chinthala, CH. S.K.., Reddy Kota, K.S., Hadassah, M., Metilda, E. H., Sridevi, S. Formulation and evaluation of gastro retentive dosage form of gabapentin using effervescent technology: International Journal of Pharmaceutical and Biomedical Research, 2012;3(4), Abue, SK, Jamiu OM, Abue- saeed MB. Quality control of some pediatric preparations in Nigerian Market, FSJ Pharm Rs, 2012;1(3), Latchman, L., Lieberman, H.A., Kanig, J.L., The Theory and Practice of Industrial Pharmacy, fourth ed. Varghese publishing house, Bombay. 9. Bora, D., Borude, P., Bhise, K., Formulation and Evaluation of Self microemulsifying drug delivery systems of low solubility drug for enhanced solubility and dissolution. International Journal of Pharmaceutical Innovation, Kulkarni, A. P., Shahnawaz, M., Zaheer, Z., Dehghan, M. H. G. Development and Validation of a Dissolution Research Article 26

10 Method for Pioglitazone Tablets. Dissolution Technologies.2012; van Zwet AA, de Boer WA, Schneeberger PM, Weel J, Jansz AR, Thijs JC. Prevalence of primary Helicobacter pylori resistance to metronidazole and clarithromycin in The Netherlands. Eur J Clin Microbiol Infect Dis. 1996;15: Akiram E. I, Ibrahim K.E,studies on the photochemical decomposition of Metronidazole, International journal of pharmaceutics 1991; Cite this article as: Patil P. D *, Desai S. R, Disouza J. I. Stability analysis and quantitative evaluation of metronidazole suspension Indian J. Pharm. Biol. Res.2016; 4(3):5-14. All 2016 are reserved by Indian Journal of Pharmaceutical and Biological Research This Journal is licensed under a Creative Commons Attribution-Non Commercial -Share Alike 3.0 Unported License. This article can be downloaded to ANDROID OS based mobile. Research Article 14