Al-Neelain University Graduate College Development and Validation of UV-Spectrophotometric Method for the Analysis of Meronidazole (MTZ) Tablets A Thesis Submitted In Partial Fulfillment of the Requirements for M.Sc. in Chemistry By Aida Adam Ameer Haroun Supervisor Dr. Omer Abdalla Ahmed Hamdi Jan. 2019 Sudan 2 الاستهلال ٹ ٹ ئې ئې ئې ئى ئى ئى ی ی ی ی ئج چ ئي بج بح بخ بم بى بي تج تح تخ ئىئح ئم چثم ثى ثي جح جم ثجتم تى تي صدق الله العظيم 3 Dedication To my Parents, Brothers, sisters and Uncle Mobark M. Noor 4 Acknowledgements I would like to express my sincere appreciation to supervisor Dr. Omer abdalla Ahmed Hamdi for the continuous supports, motivation and immense knowledge. My appreciation extended to Dr. Abdalla Al-Abied for his guidance and help through all the time of the research and writing up of this thesis. I would like to thank the staff members of Wafra-Pharma Laboratories for giving me an access to the laboratory and research facilities. I also want to thank of the all staff of Chemistry Department for technical support. 5 ABSTRACT A simple, reliable, precise and isocratic, rapid and accurate UV- spectrophotometric method was developed and validated for estimation of metronidazole in the pharmaceutical dosage, using hydrochloric acid as diluent which does not show any interference with UV- spectrophotometric measurements where the method use the advantage of useless amount of metronidazole and less time in the analysis. The maximum wave length of absorbance λmax was found 277nm. The proposal method was developed and validated according to ICH guidelines. The values of linearity, accuracy, precision and other statistical and analysis parameters were found to be in good accordance with the prescribed values. Beer-Lambert law’s was obeyed in the working concentration range 2- 25mg/ml with the coefficient of determination (R 2 ) being 0.999. The intra-RSD (n= 1.04) was < 2.0% for five reading of samples. The developed method was successfully applied for determination of metronidazole and thus enabling the utility of this new method for routine analysis of metronidazole in pharmaceutical dosage forms. 6 UV 722 57599000 n = 1.0475 7 Table of contents Content Page No. I Dedication II Acknowledgements III Abstract IV V List of contents VI List of Tables IX List of Figures X List of Abbreviations XI Chapter one: Introduction and Literature Review 1.1. Introduction 1 1.1.1 Ultraviolet – spectroscopy or Ultraviolet – visible spectroscopy (UV or UV/Vis) 3 1.1.2 High Performance Liquid Chromatography (HPLC) 4 1.1.3 Uses of metronidazole 6 1.1.4 Drug Interactions 6 1.1.5 Side effects 7 1.2 Objective of research 7 1.3 Literature review 8 1.3.1 Specificity 8 8 1.3.2 Accuracy 8 1.3.3 Precision 8 1.3.3.1 Repeatability 9 1.3.3.2 Intermediate precisions 9 1.3.3.3 Re-product-ability 9 1.3.4 Limit of Quantitation 9 1.3.5 Limit of Detection 9 1.3.6 Linearity 9 1.3.7 Robustness 10 1.3.8 Range 10 1.3.9 Stability 10 Chapter two: Materials and Methods 2.1 Materials 13 2.1.1 Instruments 13 2.1.2 Standard used 13 2.1.3 Physical tests 13 2.1.4 Chemical used 14 2.1.5 Dissolution 14 2.2 Method 14 2.2.1 Sample preparation for UV analysis 14 2.2.2 Standards preparation for UV analysis 14 2.2.3 Sample and standard preparation for HPLC analysis 15 9 2.2.4 Mobile phase preparation for HPLC analysis 15 Chapter three: Results and Discussion 3.1 Validation parameters 16 3.1.1 accuracy 16 3.1.2 Precision 16 3.1.2.1 Repeatability 16 3.1.2.2 Intermediate Precision 17 3.1.3 Linearity 18 3.1.3.1 Analyst A 18 3.1.3.2 Analyst B 21 3.1.3.3 Analyst C 24 3.1.4 Range 27 3.1.5 Specificity 27 3.1.6 Robustness 27 3.1.7 Solution stability 28 Chapter four: Conclusion and Recommendations 4.1 Conclusion 29 4.2 Recommendations 29 4.3 References 30 10 List of Tables Table Page No. Table 3.1: Repeatability and actual assay 17 Table 3.2: Intermediate precision 17 Table 3.3: Concentration vs. Absorbance for linearity and study and actual concentration and recovery obtained by analyst A 18 Table 3.4: Parameter for Linearity obtained by analyst A 19 Table 3.5: Theoretical assay against actual assay obtained by analyst A 20 Table 3.6: Concentration vs. Absorbance for linearity study and actual concentration and recovery obtained by analyst B 21 Table 3.7: Parameter Linearity results obtained by analyst B 22 Table 3.8: Theoretical assay against actual assay obtained by analyst B 23 Table 3.9: Concentration vs. Absorbance for linearity study and actual concentration and recovery obtained by analyst C 24 Table 3.10: Linearity results obtained by analyst C 25 Table 3.11: Theoretical assay against actual assay obtained by analyst C 26 Table 3.12: Specificity 27 Table 3.13: Parameter results for The Robustness 27 Table 3.14: Parameter results for The solution stability at room temperature 28 11 List of Figures Figure Figure. No Figure 1.1: Chemical structure of Metronidazole 2 Figure 1.2: UV-visible instrument 4 Figure 1.3: HPLC instrument 5 Figure 3.1: Linearity curve for the Metronidazole obtained by analyst A 19 Figure 3.2: Theoretical assay against actual assay obtained by analyst A 20 Figure 3.3: Linearity Curve for the Metronidazole obtained by analyst B 22 Figure 3.4: Theoretical assay against actual assay obtained analyst B 23 Figure 3.5: Linearity curve for the Metronidazole obtained by analyst C 25 Figure 3.6: theoretical assay against actual assay obtained by analyst C 26 12 List of Abbreviations UV Ultra violet radiation Vis Visible radiation Mg Micro gram Nm Nano gram MTZ Metronidazole λmax Maximum absorbance SD Standard Deviation RSD Relative Standard Deviation ICH International conference on Harmonization HPLC High Performance Chromatography SE Standard Error LOD Limit of Detection LOQ Limit of Quantitation PDA Photo Diode Array 13 Chapter One Introduction and Literature Review 14 Chapter One Introduction and Literature Review 1.1 Introduction The development of the pharmaceutical drugs brought a revolution in human health. These products serve their intended of only if they are free form impurities and are administered in an appropriate amount to make drugs serve their purpose. Various chemical and instrumental methods were developed at regular intervals which are involved used for the estimation of drugs composition. These pharmaceutical products may develop impurities at various stages of their development, transportation and storage which makes the risky to be administered thus they must be detected and quantized for this purpose where analytical instrumentation and methods play an important role. Drugs are either in form of raw material or in the form of formulation they can be assayed by dissolving the drugs in a suitable solvent then measuring the absorbance at specific wavelength (1) . Spectrophotometric techniques are mainly based on measurement of interaction of electromagnetic radiation with the quantized matter at specific energy levels. It is the branch of science dealing with the study of interaction between electromagnetic radiation and matter. It is a most powerful tool available for the study of atomic and molecular structures and is used in the analysis of a wide range of samples. Optical spectroscopy includes the region on electromagnetic spectrum between 200-400 nm. The regions of electromagnetic spectrum one shown blew (2) . 15 Figure 1.1: Chemical structure of metronidazole [MTZ] Metronidazole fig (1.1) as the chemical name 2-(2-methyle-5-mitro-1H- imidazole-1-yle) ethanol is cytostatic drug for the treatment of rosacea, a common chronic syndrome characterized by persistent facial erythema, flushing, edema, pustules and papules. It is available in gel formulation for treatment of bacterial vaginitis as well as in topical gel and cream for the treatment of inflammatory lesions and any themarosacea. The molecular formula of metronidazole is C6H9N3O3 and its molecular weight 171.2 - 443-48-1. The content of metronidazole (95%-105%) dried substance (MTZ) as the white or yellowish, crystalline powder. (MTZ) is slightly soluble in water, in acetone, in alcohol and acid chloride (methylene chloride). It is also used to treat bacterial upper and lower respiratory tract infections and structure infections and sexually transmitted diseases. (MTZ) represents a significant improvement in the treatment of selected community acquired infections (3) . Beer-Lambert law when of beam of light is assessed through a transparent cell containing a solution of an absorbing substance, reduction of the intensity of light may occur Beer-Lambert law is expressed mathematically as: Where A = absorbance or optical density A = a b c 16 a = absorptivity or extinction coefficient b = path length of radiation through sample (cm) c = Concentration of solute in solution Both (b) and (a) are constant so A is directly proportional to the concentration when (c) is in gm/100ml, then the constant is called A (1%1 cm) (4) 1.1.1 Ultraviolet- spectroscopy or Ultraviolet – visible spectroscopy (UV- or UV/Vis) : Region Wave length For (vacuum) 10- 200 nm Ultraviolet 200 – 400 nm Visible 400 – 750 nm Near infra-red 0.75 – 2.2 µm Mid infra-red 2.5 – 50 µm Far infra-red 50 – 1000 µm UV-spectroscopy photometry is one of the most employed techniques in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible radiation by a substance in solution. Instrument which measure the ratio or function of ratio of the intensity of two beams of light in the UV-visible region are called ultraviolet-visible spectrophotometers. In qualitative analysis, organic compound can be identified by use of spectrophotometric, if any recorded data is available, and quantitative A = A ** 1%/1 cm b c 17 spectrophotometric analysis is used to ascertain the quantity of molecular species absorbing the radiation. Spectrophotometric technique is simple, rapid moderately specific and applicable to small quantities of compound (5) . Figure 1.1: UV-Spectrometer In UV absorption spectroscopy although many organic compounds absorb quite strongly, only limited number of inorganic ions do, and it is the normal procedure of inorganic absorption spectrophotometry to add a reagent species to the solution of the inorganic Ion that reacts with it and in the process, bring about a marked change in the spectral characteristic of the reagent. In such reaction colour are produced and having selected the λmax by a spectrophotometer, colorimeters are used for quantifying the amount of absorbing species. UV-spectrophotometers measure the visible regions of ultraviolet light and can provide valuable information about the levels of active ingredients present in pharmaceutical compound, and detect any impurities by measuring the absorption of UV-radiation of light; spectrophotometric analysis can quantify these levels at a highly accurate rate (6) . 1.1.2: High performance liquid chromatography (HPLC): High-performance liquid chromatography (HPLC) formerly referred to as high- pressure liquid chromatography is an analytical chemistry technique used to separate, identify and quantify each component in a mixture. It relies on pumps to 18 pass pressurized liquid solvent containing the sample interacts through a column filled with an adsorbing material. Each component in the sample interacts slightly differently with the adsorbent material, at different flow rates for the different component and leading to the separation of component as they elute out the column. Figure 1.2: HPLC Instrument HPLC has been used manufacturing (during the production process of pharmaceutical and biological products (separating the component of a complex biological sample, or similar synthetic chemical form each other), medical (e.g. Vitamins levels in blood serum). Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbents leading to the separation of the sample component. The active component of the column, the adsorbent, is typically a granular material made of solid particles (e.g. silica polymers, etc.) 2.50 nm in size. The components of the sample mixture are separated from each other due to their different degree of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water – acetonitrile and for methanol) and is referred to as a “mobile phase” it’s composition and 19 temperature play a major role in the separation process by influencing the interaction taking place between sample components and adsorbent. These interactions are physical in nature, such hydrophobic (dispersive) dipole- dipole and Ionic, rephrase. The schematic components of an HPLC instrument typically include a degasser, sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of mobile phase through the column. The detector generates a signal proportional to the amount of sample components emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in HPLC instrument can mix multiple solvents together in rations changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/VIS photodiode array (PDA) or based on mass spectrometry most HPLC instruments also have a column over that allows for adjusting the temperature at which the separation is performed (7) . 1.1.3: Uses of metronidazole: This medication is orally administered directed by doctors to prevent stomach upset, this medication is taken food a full glass of water or milk. This dosage is based on patients medical condition and response to treatment. For the effect, antibiotic are also taken at specified times to help you remember, take this medication out the sometime every day. This medication is taken until the full prescribed dose is finished, even if symptoms disappear after a few days. 20 1.1.4 Drug interaction Interactions may change how the your medication work or increase the risk of side effects, this document does not contain all possible drug interaction keep a list of all the products you use (including precision/ non-prescription drugs and herbal products) and share it with doctors and pharmacist. Some products that may insert with this drug include Alcohol- containing products (such as cough and cold syrups, after share), products containing propylene glycol, copinarir, ritonavir solution, lithium. Although most antibiotics are unlikely is affect hormonal birth control such as pills, a few antibiotics (such as rifampin, Rifabutin) decreasing their effectiveness. This could result in pregnancy. If hormonal birth control, ask doctors, pharmacist for more details. Medication may interfere with certain lab tests, possibly, causing false test results. Make sure lab personnel and doctors know you use this drug (8) . 1.1.5 Side effects Dizziness, Headache, Stomach, Nausea, vomiting, loss of appetite diarrhoea, constipation, or metallic taste in your mouth may occur. If any of these effects last of get worse, tell your doctor or pharmacist promptly. This medication may cause your urine to turn darker in color. Remember that doctors have prescribed this medication because he or she has judged that the benefit to you is greater than the risk of side effects, many people using this medication do not have serious sides effects. Tell doctors right away if you have any serious side effects, including signs of a new infection (such as sore threat that does not go away, fever) easy bruising/ bleeding, stomach/ abdominal pain, painful urination. Get medical help must be right any if you have very serious side effects, including: unsteadiness, seizures, mental/ mood changes (such as confusion), 21 trouble speaking, number ness/tingling of arms/legs eye pain, sudden vision changes, headache that is severe or does not go away stiff/painful neck (9) . 1.2 Objective of research: The project objectives and aims could be summarized as:  To develop a UV spectrophotometric method for determination of metronidazole in tablet.  To confirm the validity of the method by statistical analysis. 1.3 Literature review: Validation methodology 1.3.1 Specificity Specificity is the ability to access, unequivocally, the analyte in the presence of components which may express to be present. Typically, these might include impurities, degradants matrix etc. Specificity of an individual analytical procedure may be compensated by other supporting analytical procedure. This definition has the following implications: Identification to ensure the identity of analyte Purity tests: to ensure that all the analytical procedures performed allow an accurate statement of content of impurities of an analyte i.e. related substances tests, beany metal, residual solvent content. Assay (content or potency) To provide an exact result this allows an accurate statement on content or potency of the analyte in a sample. 1.3.2 Accuracy: The accuracy of an analytical procedure express the closeness or agreement between the value which is accepted either as a conventional true or an accepted references value and the value found. This is some Imus termed trueness. 22 1.3.3 Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the some homogenous sample under the prescribed conditions. Precision may be considered at levels: repeatability, intermediate precision and reproducibility (10) . Precision should be investing using homogenous, authentic sample. However, if it is not possible to be obtain a homogenous sample it may be investigated using artificially prepared samples or sample solution. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. 1.3.3.1: Repeatability: Repeatability expresses the precision under the same operating conditions over a start interval of true. Repeatability is also termed intra-assay precision. 1.3.3.2: Intermediate precision Intermediate precision express within laboratories variations: different analysis, different equipment. 1.3.3.3 Reproducibility Reproducibility expresses the precision between laboratories (collective studies, usually applied to standardization of mythology). 1.3.4 Limit of Quantitation: The limit of quantitation of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is parameter of quantitative assay for low levels of compound in sample matrices, and is used particularly for the determination of impurities and/ or degradation products. 23 1.3.5 Limit of Detection (LOD) The limit of detection is individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessary quantitated as an exact value. 1.3.6 Linearity: Linearity of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including this concentration) for which it has been demonstrated that the analytical procedure has a suitable level of precision accuracy and linearity. 1.3.7 Robustness: Robustness of an analytical procedure is the measure of it is capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. 1.3.8 Ranges: The range of analytical procedure is the interval between the upper and lower concentration (amounts) of analytical in the sample (including this concentration) for which it has been demonstrated that analytical procedure has a suitable level of precision, accuracy and linearity (11) . 1.3.9 Stability Chemical degradation and physical properties of drug substance may change their pharmacological effects resulting in altered therapeutically efficacy as well as toxicological consequence. Because pharmaceutical at used therapeutically is based on their efficacy and safety, they should be stable and maintain their quality during the time of usage or until the expiration data. The quality should be maintained under the various condition that pharmaceutical encounter, during production storage in warehouses, transportation, and storage in hospital and community pharmacies, as we has in the home. Therefore, understanding the 24 factors that alter the stability of pharmaceutical and identifying ways to guarantee their stability are critical. Since the early 1950s may studies on the stability of pharmaceutical degradation pathway, rates of reaction, and the means of stabilizing drugs have been well documented in primary continuously adding to increase knowledge base. New assay methodologies are being developed and new ways of treating stability data are also evolving. This is especially the case with the newer complex drugs and dosage forms. Stability indicating assay method may be defined as validated, quantities analytical methods that detect changes chemical composition, Physical and microbiological properties and drug substance and drug products, and that are specific so that content of active ingredients, degradation products, and other component of interest can be accurately measured without interference. Stability is required for any new or a wanted to ensure it is capable of giving reproducing and reliable results, when used by different operators employing the same equipment in the some different laboratories. The type of stability program required depends on the particular method and it proposed application. Typical analytical parameters used in assay validation include: precision, accuracy, linearity, range, ruggedness, robustness, and limit of detection, limit of quantitation, selectivity and specificity. Stability testing forms an important part of the process of drug product development the purpose of stability testing to provide evidence on how the quality of drug substance or drug product varies with time under the influence of a variety of environment factors such as temperature, humidity, and light and enable recommendation of storage conditions, retest period, and shelf life to be established. 25 The two main aspects of drug products that play an important role in shelf life determination are assay of active drug and degradation products generated, during the determined using stability indicating method, as recommendation by the international conference on harmonization (ICH). To establish the stability indicating nature of the method, forced degradation of drug substance and drug product was performed under stress conditions (thermal, humidity, acid/base hydrolysis and oxidative). Information on the stability of drug substance is an integral part of the systematic approach to stability evaluation. Stress testing or force degradation studies are a critical component of the development process. The ICH guideline indicates that stress is designed to help determine the intrinsic stability of the Molecule by stability degradation path ways in order to identify the likely degradation products and validate the stability indicating power of the analytical procedures used. Stress testing also is becoming increasingly important in testing new molecules. Method development by stress and stability information gained from those methods can hare significant effect. 26 Chapter Two Materials and Methods 27 Chapter Two Material and Methods 2.1 Materials - Metronidazole tablets (250 mg) Wafrapharma Ltd. Sudan. - Metronidazole standard (purity 95%) wafrapharma Ltd. Sudan. - Distilled water. - Hydrochloric acid 0.1 HCl. - 1.36 potassium di-hydrogen phosphate wafrapharma Ltd, Sudan. - Acetonitrile (70 ml) wafrapharma Ltd, Sudan. - Methanol (30 ml) wafrapharma Ltd, Sudan. 2.1.1 Instrumentation: - UV-Vis spectrophotometer Model No. s.1130, s-11530 VAC220, 60Hz, Serial No 31100-30-010300bp. - Dissolution test apparatus - HPLC UV-visible (254-265 nm), column ops length 150 nm size: f = 0.25 m, Ф = 4.6 nm, stationary phase: octadecylsillyl silica gel for chromatography for (5 µm). Mobile phase isocratic flow ratio 1 ml/min, injection 10 ml, retention time = about 1 min). - Analytical Balance. 2.1.2. Standard used - Metronidazole working standard 2.1.3. Physical tests - Potency = 99.7% - Water content = 0.07% - Friability = 0.34% - Hardness = 3.48 nm - Thickness = 3.49 nm 28 - Diameter = 10.3 - Disintegration = 0.48 nm - Assay = 0.280 gm 2.1.4. Chemical used: Dissolution media 0.1N HCl 2.1.5 Solution - Media 900 ml of 0.1 N HCl - Temperature = 37 i - Speed 100 RPM 2.2. Method: 2.2.1 Sample preparation for UV analysis: Twenty tablets of wafrazole weighted and grand. 0.284 mg of wafrazole were transferred to a 100 volumetric flask, dissolved in hydrochloric acid (0.1 N) and the solution was of stirred for 20 min. The above solution was further diluted using (0.01) HCl to a 100 ml volumetric flask by 10 ml to 100 volumetric flasks and diluted to the mark with (0.01) HCl. 2.2.2 Standard preparation for UV analysis 0.200 mg of metronidazole STD were weighted, dissolved in HCL and transferred to volumetric flask (100 ml) completed to volume with dissolution media, and transfer 10 ml to 100 ml volumetric flaks complete to volume with dissolution media. Serial 5, 10, 15, 20, 25 mg/ ml were prepared from standard stock solution of metronidazole for linearity test. 29 2.2.3 Sample and standard preparation for HPLC analysis: 0.284 g of (MTZ) tablets was transferred to 100 ml volumetric flask. The content was dissolved in mobile phase and diluted up to the mark with mobile phase. The above solution was further diluted using mobile phase by taking 10 ml to 100 ml volumetric flask and diluted up to the mark with mobile phase. The standard solution of (MTZ) tablets was prepared by dissolving 0.284 g as the sample preparation above. 2.2.4 Mobile phase preparation for HPLC analysis: 1.3 g of potassium di-hydrogen phosphate was weighed then dissolved in 100 volumetric flasks using distilled water then 70 ml of acetonitrile and 30 ml of methanol were added the solution was completed up to mark using distilled water. 30 Chapter Three Results and Discussion 31 Chapter Three Results and Discussion 3.1 Validation parameters 3.1.1 Accuracy Express the closeness of agreement between the values which is accepted either as a conventional true value or an accepted reference value and the value found. Actual assay for accuracy in concentrations 5, 10, 15, 20 – 25 mg/ml were found to be accurate the mean of actual assay is 99.2% RSD is 1.04 (less than 2%) as shown Table 3.1. The method was found to be accurate according to recovery results from linearity study as presented in Tables 3.3, 3.6 and 3.9 (99.6, 99.8%, 99.8%) respectively. 3.1.2 Precision Precision of analytical method is ascertained by carrying out the analysis as per the procedure and as per normal weight taken for analysis rep eat the analysis six times. Calculate the % assay, % mean assay, % deviation and % RSD. The developed method was found to be precise as reported by the %RSD values (less than 2%) for repeatability and intermediate precision studies as shown in Table 3.1 and 3.2. 3.1.2.1. Repeatability Expresses the precision under the same operating conditions from six samples: Recovery = actual assay/ Theoretical assay * 100 32 Table 3.1: Repeatability and actual assay: Standard absorbance Sample absorbance Actual assay% 0.5524 0.5524 99.1 0.5409 0.5498 100.7 0.5487 0.5392 98.2 0.5493 0.5495 100.4 0.5665 0.5592 98.7 0.5484 0.5406 98.6 Mean 99.3 STD 1.04 RSD 1.05 3.1.2.2 Intermediate precision: Intermediate precision was conducted within laboratory variation (different analyst on different days), using a concentration 25 mg/ml with three replicates. Repeat the analysis three times for the entire analyst A, B and C. Table 3.2 Intermediate precision No Analyst A Analyst B Analyst C 1 98.4 100.5 102 2 98.9 97.2 98.8 3 96.5 100 99.3 Mean 98 99 100 SD 1.3 1.8 1.7 RSD 1.3 1.8 1.7 33 3.1.3 Linearity The ability (within a given range) to obtain test results that directly proportional to concentration (amount) of analyte in the sample. 3.1.3.1 Analyst A Six points calibration curve were obtained in concentration ranges from 5- 25 mg/ml for metronidazole. The response of the drug was found to be linear in the investigation concentration ranges and the linear regression A = 0.0219 * 0.091 with correlation coefficient 0.998 and 1.88% less than 2% the results are shown in table 3.3 figure 3.1 * linearity = fit% = 100% = 99.8. And the relationship between the actual concentration and theoretical concentrations found to be linear as shown in figure 3.2. Table 3.3 concentration vs. absorbance for linearity study and actual concentration and recovery obtained by analyst A: Standard concentration mg/ml Absorbance Actual concentration mg/ml Recovery% 5 0.1163 4.9 97.9 10 0.2253 9.9 98.8 15 0.2253 15.2 101.5 20 0.3425 20.3 101.2 25 0.4522 24.3 97.3 Sample 25 g/ml 0.5636 25.3 101.3 Mean 99.6 SE 0.004 SD 1.9 RSD 1.9 LOD 1.3 LOQ 4.2 34 Figure 3.1 parameters for metronidazole obtained by analyst A Table 3.4 parameters for linearity Parameters Value Accuracy 99.6 ± 1.8 Slop 0.0219 Intercept 0.0091 Linearity range (2- 25) mg/ml Correlation coefficient 0.998 RSD 1.8 0 0 1/5 2/7 2/5 1/2 3/5 5/7 5 10 20 30 40 50 Absorbance Concentration mg/ml Y = 0.0219x + 0.0091 Absorbance Concentration 35 Table 3.5 actual assay against Theoretical assay obtained by analyst A Theoretical concentration mg/ml Actual concentration mg/ml 5 4.89 10 9.87 15 15.22 20 20.28 25 24.32 Sample 25 g/ml 25.31 Figure 3.2 actual assays against theoretical assay obtained by analyst A 0 5 10 15 20 25 30 5 10 15 20 25 30 Actual concentration Thereotical concentration R2 = 0.998 Series 1 Series 2 36 3.1.3.2 Analyst B Six points calibration curve were obtain in concentration range 5-25 mg/ml for metronidazole. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y = 0.224 + 0.007 with correlation coefficient 0.9982 and RSD 1.71% less than 2% the results are shown in table 4.6, figure 3.3. * Linearity = Fit% = 100% = 99.82. And the relationship between the theoretical concentration and actual concentration found to be linear as shown in figure 3.4: Table 3.6: concentration vs. Absorbance for linearity study and actual concentration and recovery by analysis B Standard concentration mg/ml Absorbance Actual concentration Mg/ml Recovery 5 0.1116 5 99 10 0.222 9.9 98.8 15 0.3438 15.3 102 20 0.4467 19.9 99.6 25 0.5488 24.5 97.9 Samples 25 g/ml 0.5708 25.5 101.8 Mean 99.9 SE 0.01 SD 1.72 RSD 1.72 LOD 3 LOQ 10.1 37 Figure 3.3: linearity curve for metronidazole obtained by analyst B Table 3.7: parameters for linearity test obtained by analyst B Parameter Value Accuracy 98.8 ± 1.71 Slope 0.0224 Intercept 0.0007 Linearity range (2-25) mg/ml Correlation coefficient 0.9982 RSD 1.71 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 5 10 15 20 25 30 Absorbace Concentrationxis Title Y = 0.224x + 0.0007 R = 0.9882 Absorbance Concentration 38 Table 3.8 actual assay against Theoretical assay obtained by analyst B: Theoretical concentration mg/ml Actual concentration mg/ml 5 4.95 10 9.87 15 15.31 20 19.91 25 24.46 Sample 25 g/ml 25.45 Figure 3.4: Theoretical assay against actual assay by analyst B 0 5 10 15 20 25 30 10 20 30 40 50 60 Theoretical Concentarion Actual Concentration R2 = 0.999 Theoretical Actual 39 3.1.3.3. Analyst C: Six points calibration curve were obtain in concentration ranges from 5-25 mg/ml for metronidazole. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y = 0.222x + 0.0015 with correlation coefficient 99.92 and RSD 1.21% less than 2% the results are shown in table 3.9, figure 3.5. * Linearity = Fit% = 100% = 99.9. And the relationship between the actual concentration and theoretical concentration found to be linear as shown in figure 3.6: Table 3.9: Concentration vs. Absorbance for linearity study and actual concentration and recovery by analysis B Standard concentration mg/ml Absorbance Actual concentration Mg/ml Recovery 5 0.1113 4.9 98.9 10 0.2183 9.8 97.7 15 0.3281 14.7 98.1 20 0.4462 20 100.2 25 0.5453 24.5 98 Samples g/ml 0.5593 25 100.5 Mean 98.9 SE 0.01 SD 1.2 RSD 1.2 LOD 2.02 LOQ 6.1 40 Figure 3.5: linearity curve for metronidazole obtained by analyst C Table 3.10 Parameters for linearity test obtained by analyst C Parameter Value Accuracy 98.8 ± 1.71 Slope 0.0224 Intercept 0.0007 Linearity range (2-25) mg/ml Correlation coefficient 0.9982 RSD 1.71 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 5 10 15 20 25 30 Absorbance Concentraion mg/ml R2 = 0.9992 Absorbance Concentration 41 Table 3.11 Theoretical assay against actual assay obtained by analyst C Theoretical concentration mg/ml Actual concentration mg/ml 5 4.94 10 9.76 15 14.71 20 20.03 25 24.49 Sample 25 g/ml 25.12 Figure 3.6: Theoretical assay against actual assay by analyst C 0 5 10 15 20 25 30 10 20 30 40 50 60 Theoretical concentration Actual concentration R2 = 0.999 Theoretical Actual 42 3.1.4. Range: The interval between the upper and lower concentration amount of analyte (including these concentrations) for which is has between demonstrated that the analytical procedure has a suitable of precision, accuracy and linearity (5-25) mg/ml. 3.1.5 Specificity The specificity to asses unequivocally the analyte in the presence of components that may expect to be present its specific method because there is no interference from the recipients. Table 3.12: Specificity Blank Standard Sample Absorbance 0.0 0.626 0.620 3.1.6 Robustness The robustness of an analytical procedure is measure of its capacity to remain unaffected by small deliberate variations in method parameters and provides an indication of it is reliability during normal usage. This experiment involves, changing the method parameters, reliability, one at time, and establishing the data for any minor changes occurring inadvertently during the routine analysis. Table 3.13: Parameter Result of Robustness for the different ware length: Parameter result at the wave length 279 nm Parameter result at the wave length 275 nm Parameter result at the wave length 277 nm Average result of Robustness control 101.1% Average result of Robustness control 101.4% Average result of Robustness control 101.1% Mean assay value 100% Mean assay value 100% Mean assay value 100% Difference 1.1% Difference 1.4% Difference 1.1% 43 3.1.7: Solution stability The solution stability of an analytical procedure is measure of its capacity to remain unaffected by variation of time, variation of solution stability parameters provide an indication of its reliability during normal usage. Table 3.14 Parameter results for “solution stability” at room temperature Parameter Experiment Result % Acceptance criteria Solution stability Comparison of sample solution analyzed initially and after determined interval 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 24 hours Initial result: 100% 1 Hour result: 100% RSD at all-time interval should not exceed 2.0% Initial result: 100% 2 hours result: 100% Initial result: 100% 3 hours result: 100.6% Initial result: 100% 4 hours result: 100.5% Initial result: 100% 5 hours result: 100.5% Initial result: 100% 6 hours result: 100.6% Initial result: 100% 24 hours result: 100% % RSD for all time interval: 1.2% 44 Chapter Four Conclusion and Recommendations 45 Chapter Four Conclusions and Recommendations 4.1 Conclusions The official method of analysis of Metronidazole tablets in pharmaceutical industry is HPLC method. It is expensive and takes more time for analysis in addition to that it consumes large amount of solvents comparing with UV-method. The UV analytical method was validated as per ICH guideline for determination of assay content in metronidazole tablets (250 mg). It based on the measurement of inter action of electromagnetic radiation with the quantized matter at 277 nm. The validation method result of Metronidazole in this study showed good linearity, accuracy selectivity, range, intermediate precision, robustness, ruggedness and repeatability comparable with previous studies of metronidazole (MTZ). The proposed method represents a promising approach in area of pharmaceutical monitoring with low cost high speed, simplicity and sensitivity therefore can be recommended for the drug quality control. Moreover, the method is economical, simple and rapid, hence can be employed for routine analysis in quantity control laboratory for estimation of metronidazole form marked formulation and raw material. 4.2 Recommendation: It’s recommended to pursue this research by pharmaceutical research on the method used for the analysis of metronidazole (MTZ). 46 References 1- Masoom, S.Zeid, A. and Nafisur R. (2013) Analytical techniques in pharmaceutical analysis. Arabian journal of chemistry, 4: 1878, 5352. 2- Siladity a, B, Subhjjt, G, Faha’d, A, Saayak, S. and Sritoma, B. (2012). UV- visible spectrophotometric method development and validation of assay paracetamol tablet formulation, Analytical and bio-analytical techniques; 3: 2155, 9872 3- Metronidazole WH. The extra pharmacopoeia 27ed London: The pharmaceutical press; 1977.p.1570. 4- Willard H. H, Merriltl, L., dean J. A. and Settle, F. A (1988) Instrumental method analysis- (7-Thedn) wad worth publishing company, California, 544-545. 5- Davidson, A.G. (2002). Ultra-violet Absorption Spectrophotometry. (4 th edn)., CBS publishers and distributors, New Delhi, 275-278. 6- Alhalabi Z, AL-Khayat MA Haidar S. (2012): Separation and assay antiprotozoal imidazole derivatives (Metronidazole, Tinidazole and secnidazole) by RP-HPLC. International journal of pharmaceutical sciences Review and Research 13: 12,18. 7- Metronidazole oral: http://www.webmd.com/drugs/2drug-6424/metronidazole- oral/details. 8- Barwick, V (2006). Introduction to Method Validation,. IGC limited 2.6. Patel K, Green. Hopkins I, Luis Tunkel AR 2008. Cerebellar ataxia following prolonged use of Metronidazole: case report and literature are view. Int. Infect Dis – 12: e 111-114.http://dx.doi.org/10.10/j.ijid.2008.03.006. 9- Agudelo M. Vesgao (2012) Therapeutic equivalence requires pharmaceutical, pharmacokinetic, and pharmacodynamics identities: true-bioequivalence of generic product of intravenous Metronidazole Antimicrobial Agents chemothor 56:2659, 2665. http://www.webmd.com/drugs/2drug-6424/metronidazole-oral/details http://www.webmd.com/drugs/2drug-6424/metronidazole-oral/details 47 10- AL-sabea N (2008) Assay of Metronidazole from different manufacturing source in large markets. AJPS 1: 16, 25. 11- Bernstein L. H, Frank MS, Brandt L, Baley S,. 1980. Healing of perineal croh’s disease with metronidazole. Gastroenterology 79:599. 12- Bradley WG.Karlsson1J. Rassol Co.1977. Metronidazole neuropathy. BMjii:610-611. http://dx.doi.org/10.1136/bmj:2.6087.610. 13- British pharmacopoeia 2012 Volume III formulated preparations: specific Monographs Metronidazole Tablets. 14- Budhiraja RD (2009) Elementary pharmacology & Toxicology, popular par kashan Mumbai Pl: 323433. 15- Chystal E J, Koch RL Mcl Afferty MA. Goldman P (1980). Relationship between Metronidazole in Metabolism and Bacterial Activity Antimicrobial Agents chemother 25: 573, 665. 16- Cosar C and Julan L. Activity of (Hydroxy- 2’ethyl1-1methyl-2-nitro-5- imidazole (882) RP in experimental tri-chomonas-vaginalis infections: Annales del’ institute Pasteur. 1959; 96(2):238-241. 17- Foye. W.O.(1975). Principles of Medicine Chemistry, Philadelphia, USA 726. 18- Harrison’s Principles of internal Medicine, International edition-cd-ram of Great Britain. 19- Ingham HR. Eaton S.venables CW. Adams pc.1978.Bacteriodes Fragilis resistant to Metronidazole after long-term therapy. Lancet: 214. 20- KalioV.Vibhui, Saggar K.2010.Case report MRI of the brain in metronidazole toxicity. Indian J. Radial Imaging 20:195-195-197: http://dx.doi.org.10.4/0971.3026.69355. 21- Kim G, Na DG, Kim EY, Kim, H son KR, change KH. 2007. MR imaging of metronidazole- include encephalopathy: lesion distribution and diffusion weighted http://dx.doi.org/10.1136/bmj:2.6087.610 http://dx.doi.org.10.4/0971.3026.69355 48 imaging findings. Am). Neuroradiology, 28:1652-1658. http//dx.oloi.org/10:3174/ajnr.Aot55. 22- Kishore Kumarhotha (2013): Forced degradation studies practical approach – overview of regulatory guidance and literature for the drug products and drug substances. 23- Lloyd DR, Kinzer KF and T seng HS: Micro-porous membrane formation via thermally include phase separation. 1. solid- liquid phase separation, Journal of Membranes sciences. 1990, 52(3): 239, 261. 24- SeokJI, 41H, SongYM, leeWY.2003. Metronidazole induced encephalopathy and inferior alivary hypertrophy: lesion analysis with diffusion weighted imaging and apparent diffusion coefficient maps.Arch.Neurol.60:1796-1800. http://dx.doi.org/10.1001/archneur.60.121496. 25- Siladity a, B, Subhjjt, G, Faha’d, A, Saayak, S. and Sritoma, B. (2012). UV- visible spectrophotometric method development and validation of assay paracetamol tablet formulation, Analytical and bio-analytical techniques; 3: 2155, 9872. 26- Thulasamma P and Venkateswarlu P (2009), Spectrophotometric method for the determination of Metronidazole in pharmaceutical pure and dosage forms. Rasayan journal: 865-868. http://dx.doi.org/10.1001/archneur.60.121496