ABOUT AUTHORS:
Shahin*,Vemavarapu Satish kumar1
*Shadan Women’s College of Pharmacy. khairtabad, Hyderabad. A.P
1IPQC team member at GRANULES INDIA LIMITED, M.Pharmacy (pharmaceutics) Deevena College of Pharmacy.
*shahins333@gmail.com
INTRODUCTION
A drug may be defined as a substance meant for diagnosis, cure, mitigation, prevention or treatment of diseases in human beings or animals or for alternating any structure or function of the body of human being or animals. Pharmaceutical chemistry is a science that makes use of general laws of chemistry to study drugs i.e. their preparation, chemical nature, composition, structure, influence on an organism and studies the physical and chemical properties of drugs, the methods of quality control and the conditions of their storage etc. The family of drugs may be broadly classified as.
1. Pharmacodynamic agents.
2. Chemotherapeutic agents.
It is necessary to find the content of each drug either in pure or single, combined dosage forms for purity testing. It is also essential to know the concentration of the drug and it’s metabolites in biological fluids after taking the dosage form for treatment.
The scope of developing and validating analytical methods is to ensure a suitable method for a particular analyte more specific, accurate and precise. The main objective for that is to improve the conditions and parameters, which should be followed in the development and validation.
A survey of literature reveals that even though good analytical methods have been available for the drugs like Metronidazole, as a part of B.Pharm project we have selected the same drug for which method development and validation has been carried out and the results obtained were all within the limits. The method which we followed has good accuracy, sensitivity and it was best suited for the drug i.e.; Metronidazole.
For the method development and validation of Metronidazole we have selected UV-Visible double beam spectrophotometer and the best suited solvent for the determination of Metronidazole was found to be 0.1 N HCl.
REFERENCE ID: PHARMATUTOR-ART-1921
1.2 Introduction to Ultraviolet (UV) Spectroscopy
Ultraviolet spectroscopy is one of several forms of spectroscopy that we will study this semester. Accordingly, it is important that you understand the capabilities and limitations of each of these forms of spectroscopy. The word spectroscopy implies that we will use the electromagnetic spectrum to gain information about organic molecules. The modifier ultraviolet means that the information will come from a specific region of the electromagnetic spectrum called the ultraviolet region. The electromagnetic spectrum includes all radiation that travels at the speed of light c (3 x 1010 cm/sec). The electromagnetic spectrum includes radio waves, which have long wavelengths, x-rays, which have short wavelengths, and visible light, which has wavelengths between those of radio waves and x-rays. All of these waves travel at the speed of light. We normally describe these waves in terms of their energy. Of the three kinds mentioned, x-rays are most energetic, visible light next, and radio waves least energetic. Thus, the shorter the wavelength, the greater the energy of an electromagnetic wave.
Electromagnetic radiation (EMR) has a dual nature; it has the characteristics of both waves and particles. These particles are so tiny that they are indistinguishable from a wave. Both forms of EMR are important. From the wave nature of the waves we get the wavelength or distance between two crests. The wavelength is related to the frequency, how many wavelengths pass a given point in a given time, by the velocity of the wave c. From the particulate nature of EMR, we get the energy E of a given wave, which is proportional to its frequency. Plank’s constant h turns the proportionality into an equation. The mathematical relationships among these variables are shown below.
Visible light includes the rainbow colors red, orange, yellow, green, blue, indigo, and violet. A handy acronym for these colors is ROY G BIV, said like the name of a person called Roy. Note red is at the low-energy end of the visible spectrum and violet is at the high-energy end. These facts allow us to quickly understand the terms infrared and ultraviolet. The prefix infra means below, and the prefix ultra means above. Thus, infrared radiation is outside the visible range and lies just below red on the energy scale. That is, infrared radiation is less energetic than visible light. Ultraviolet radiation is outside the visible range and is just above violet on the energy scale. Thus, infrared literally means “below” red (in terms of energy), and ultraviolet means “above” violet (in terms of energy). We know UV is more energetic than visible light or IR because UV light gives us sunburns.
We learned in general chemistry that visible yellow light is observed when sodium ions are heated in a Bunsen burner. The heat excites some ground-state electrons to higher energy levels, then when the electrons “fall” back to the ground state, they “emit” energy that corresponds to the energy difference between the energy states (orbitals) where the electrons are found. When this energy difference falls within the energy range of visible light, we can see it as a color. In the case of sodium, we see yellow light. Note that it takes the same amount of energy to make the electrons jump from the lower to higher states as the amount of energy the electrons emit when they fall from higher to lower states. We generally add more energy than is absolutely necessary for the transition to ensure that the transition occurs. When we add energy to a system, we give it a positive sign (endoenergetic). When a system gives off energy, we give it a negative sign (exoenergetic).
Just as heat causes some of sodium’s electrons to move to higher energy states, ultraviolet radiation causes electrons in certain organic compounds to move from their ground state locations to orbitals of higher energy. The energy of the ultraviolet light acts just like the energy of the heat. In this case, the molecules are said to “absorb” ultraviolet radiation. A measurement of this phenomenon is called an absorption spectrum as opposed to an emission spectrum. When electrons move from lower to higher energy levels, we call the movement an electronic transition. Thus, the basic interaction between UV light and organic compounds is that UV light causes electronic transitions in certain organic structures.
The organic compound is dissolved in a solvent that does not absorb UV light. Such a solvent is said to be transparent to UV light. The sample (compound in its solvent) is placed in a cuvette. A cuvette is a sample holder that has very precise dimensions. The cuvette is placed in an ultraviolet spectrophotometer. The instrument produces ultraviolet light over a range of wavelengths between 200 and 400 nanometers (nm), and the UV light is directed through the solution of the organic compound.
We will then make use of certain relationships that govern how much UV light can be absorbed by a sample. Namely, that the amount of light absorbed (A) is proportional to how many molecules or the concentration (c) of molecules that are absorbing light, and how far the UV light must pass through this concentration or the path length l.
A= εcl
This last proportionality says that the absorbance A is directly proportional to the concentration of the sample and to the path length (width of the cuvette). This is why the dimensions of the cuvette must be precise. The proportionality is useful for one given concentration or sample. A far more useful form of the relationships above is the Beer-Lambert equation, which makes the proportionality into an equation by addition of the proportionality constant.
1.3 METHOD DEVELOPMENT
SPECTROPHOTOMETRIC METHODS:
Spectrophotometry is generally preferred especially by small-scale industries as the cost of the equipment is less and the maintenance problems are minimal. The method of analysis is based on measuring the absorption of a monochromatic light by colorless compounds in the near ultraviolet path of spectrum (200-380nm). The photometric methods of analysis are based on the Bouger-Lambert-Beer’s law, which establishes the absorbance of a solution is directly proportional to the concentration of the analyte. The fundamental principle of operation of spectrophotometer covering UV region consists in that light of definite interval of wavelength passes through a cell with solvent and falls on to the photoelectric cell that transforms the radiant energy into electrical energy measured by a galvanometer.
The important applications are-
¨ Identification of many types of organic, inorganic molecules and ions.
¨ Quantitative determination of many biological, organic and inorganic species.
¨ Quantitative determination of mixtures of analytes
¨ Monitoring and identification of chromatographic of effluents.
¨ Determination of equilibrium constants.
¨ Determination of stoichiometry and chemical reactions.
¨ Monitoring of environmental and industrial process.
¨ Monitoring of reaction rates.
¨ Typical analysis times range from 2 to 30 min for sample
1.4 SPECTROPHOTOMETRIC ESTIMATION
Spectrophotometry is generally preferred especially by small-scale industries as the cost of the equipment is less and the maintenance problems are minimal. The method of analysis is based on measuring the absorption of a monochromatic light by colorless compounds in the near ultraviolet path of spectrum (200-380nm). The photometric methods of analysis are based on the Bouger-Lambert-Beer’s law, which establishes the absorbance of a solution is directly proportional to the concentration of the analyte. The fundamental principle of operation of spectrophotometer covering UV region consists in that light of definite interval of wavelength passes through a cell with solvent and falls on to the photoelectric cell that transforms the radiant energy into electrical energy measured by a galvanometer.
The important applications are18
¨ Identification of many types of organic, inorganic molecules and ions.
¨ Quantitative determination of many biological, organic and inorganic species.
¨ Quantitative determination of mixtures of analytes.
¨ Monitoring and identification of chromatographic of effluents.
¨ Determination of equilibrium constants.
¨ Determination of stoichiometry and chemical reactions.
¨ Monitoring of environmental and industrial process.
¨ Monitoring of reaction rates.
¨ Typical analysis times range from 2 to 30 min for sample.
PRINCIPLE OF SPECTROPHOTOMETRIC ESTIMATION OF MEDICINAL SUBSTANCES
The assay of an absorbing substance may be quickly carried out by preparing a solution in a transparent solvent and measuring its absorbance at a suitable wavelength. The concentration of the absorbing substance calculated from the measured absorbance using one of three principal procedures.
Use of a standard absorptivity value:
This procedure is adopted by official compendia, e.g. British Pharmacopoeia, for substances such as methyl testosterone that has reasonably broad absorption variation of instrumental parameters e.g. slit width, scan speed.
Use of a calibration graph:
In this procedure the absorbances of a number (typically 4-6) of standard solutions of the reference substance at concentrations encompassing the sample concentrations are measured and a calibration graph is constructed. The concentration of the analyte in the sample solution is read from the graph as the concentration corresponding to the absorbance of the solution.
Single or double point standardization:
The single-point procedure involves the measurement of the absorbance of a sample solution and of a standard solution of the reference substance. The standard and sample solutions are prepared in a similar manner. Ideally, the concentration of the standard solution should be close to that of the sample solution. A 'two-point bracketing' standardization is therefore required to determine the concentration of the sample solutions. The concentration of one of the standard solutions is greater than that of the sample while the other standard solution has a lower concentration than the sample.
1.5 METHOD VALIDATION
Method validation can be defined as (ICH) “Establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics”.
Method validation is an integral part of the method development; it is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, quality, purity, and potency of the drug substances and drug products. Simply, method validation is the process of proving that an analytical method is acceptable for its intended purpose.
Method Validation, however, is generally a one-time process performed after the method has been developed to demonstrate that the method is scientifically sound and that it serves the intended analytical purpose.
All the variables of the method should be considered, including sampling procedure, sample preparation, chromatographic separation, and detection and data evaluation. For chromatographic methods used in analytical applications there is more consistency in validation practice with key analytical parameters including
(a) Recovery (b) Response function (c) Sensitivity (d) Precision (e) Accuracy (f) Limit of detection (g) Limit of quantitation (h) Ruggedness (i) Robustness (j) stability (k) system suitability.
(a) Recovery
The absolute recovery of analytical method is measured as the response of a processed spiked matrix standard expressed as a percentage of the response of pure standard, which has not been subjected to sample pre-treatment and indicates whether the method provides a response for the entire amount of analyte that is present in the sample. It is best established by comparing the responses of extracted samples at low, medium and high concentrations in replicates of at least 6 with those non-extracted standards, which represent 100 % recovery.
If an internal standard is used, its recovery should be determined independently at the concentration levels used in the method.
(b) Precision
The purpose of carrying out a determination is to obtain a valid estimate of a ‘true’ value. When one considers the criteria according to which an analytical procedure is selected, precision and accuracy are usually the first time to come to mind. Precision and accuracy together determine the error of an individual determination. They are among the most important criteria for judging analytical procedures by their results.
Precision refers to the reproducibility of measurement within a set, that is, to the scatter of dispersion of a set about its central value. The term ‘set’ is defined as referring to a number (n) of independent replicate measurements of some property. One of the most common statistical terms employed is the standard deviation of a population of observation. Standard deviation is the square root of the sum of squares of deviations of individual results for the mean, divided by one less than the number of results in the set. The standard deviation S, is given by
Standard deviation has the same units as the property being measured.
The square of standard deviation is called variance (S2). Relative standard deviation is the standard deviation expressed as a fraction of the mean, i.e., S/x. It is some times multiplied by 100 and expressed as a percent relative standard deviation. It becomes a more reliable expression of precision.
% Relative standard deviation = S x 100 / x
(c) Accuracy
Accuracy normally refers to the difference between the mean x****, of the set of results and the true or correct value for the quantity measured. According to IUPAC accuracy relates to the difference between results (or mean) and the true value. For analytical methods, there are two possible ways of determining the accuracy, absolute method and comparative method.
CHAPTER II
2.1 REVIEW OF LITERATURE
DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF METRONIDAZOLE IN TABLET FORMULATION
The present research work discusses the development of a UV spectrophotometric method for Metronidazole. Simple, accurate and cost efficient spectrophotometric method has been developed for the estimation of Metronidazole (MND) in Tablet dosage form. The optimum conditions for the analysis of the drug were established. The maximum wavelength (λ max) was found to be 277nm. The percentage recovery of Metronidazole was in the 98.2±0.129. Beers law was obeyed in the concentration range of 1-10μg/ml. Calibration curves shows a linear relationship between the absorbance and concentration. The line equation y=0.0283x+0.0407 with r2 of 0.9902 was obtained. Validation was performed as ICH guidelines for Linearity, accuracy, precision, LOD and LOQ. The sample solution was stable up to 36 hours. The proposed method may be suitable for the analysis of MND in tablet formulation for quality control purposes.
SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF METRONIDAZOLE IN PHARMACEUTICAL PURE AND DOSAGE FORMS
Two simple, precies, rapid, sensitive and accurate Spectrophotometric methods have been developed for the estimation of metronidazole either in pure form or in tablet dosage forms. The proposed methods are based on the reduction of metronidazole was carried out with Zinc powder and 5N HCl at room temperature in methonal.The resulting amine was used to two methods. Method A is based on oxidation Coupling with 1,10- Phenanthrolin to form Orange red colored chromogen exhibiting absorption maxima at 510 nm with apparent molar absorptivity of 2.32x103(Lm-1cm-1) and obeyed beer’s law in the concentration range 5-55 μg/ml.Method B is based on diazotization and coupling reaction with NaNo2 and 4-chloro3-nitro Aniline to form Yellow colored chromogene exhibiting absorbance maximum at 480 nm with apparent molar absorptivity of 2.71x103(Lm-1cm-1) and obeyed beer’s law in the concentration range of 5-60 μg/ml.The assay of results was found to be in good agreement with label claim. The proposed methods were Simple, Sensitive, Precise, Accurate, quick and useful for routine quality control.
SIMULTANEOUS ESTIMATION OF METRONIDAZOLE AND AMOXICILLIN IN SYNTHETIC MIXTURE BY ULTRAVIOLET SPECTROSCOPY
Two simple, accurate, precise, reproducible, requiring no prior separation and economical procedures for simultaneous estimation of metronidazole and amoxicillin in combined dosage form have been developed. First method employs formation and solving of simultaneous equation using 319 nm and 273.8 nm as two analytical wavelengths for both drugs in phosphate buffer pH 7.4. The second method is Q value analysis based on measurement of absorptivity at 319 nm and 289 nm (as a iso-absorptive point). Metronidazole and amoxicillin at their respective λmax 319 nm and 272 nm and at isoabsorptive point 289 nm shows linearity in a concentration range of 10-50 mcg/mL. The results of analysis have been validated statistically, the standard deviation lies in the range of 0.012 - 0.071 for amoxicillin and 0.028 - 0.153 for amoxicillin in case of simultaneous equation method and 0.020 - 0.098 for amoxicillin and 0.032 - 0.231 for amoxicillin in case of Q - analysis method. Recovery studies range from 98.45 - 99.0% for amoxicillin and 101.10 – 102.06% for metronidazole in case of simultaneous equation method and 99.60 – 101.0% for amoxicillin and 100.20-100.90% for metronidazole in case of Q - analysis method confirming the accuracy of the proposed method.
DEVELOPMENT AND VALIDATION OF RP-HPLC AND UV METHODS OF ANALYSIS FOR FLUCONAZOLE IN PHARMACEUTICAL SOLID DOSAGE FORMS
A RP-HPLC and an UV spectrophotometric assay method were developed and validated for quantitative determination of fluconazole in pharmaceutical solid dosage forms like capsules, uncoated tablets, and dispersible tablets. The chromatography was carried out on a C-18 (150 mm x 4.6 mm, 5 μm) column with water and acetonitrile (65:35 v/v) as mobile phase at 260 nm detector wave length. The UV method was performed at 260 nm using 0.1M HCl as solvent. The linearity was established in the range of 1 to 100 μg/ml and 50 to 400 μg/ml for HPLC and UV methods respectively. The HPLC method was accurate and precise for all the dosage forms studied with a recovery of 98 to 102%. The UV method correlated well with HPLC for the analysis of fluconazole only in capsule dosage form.
CHAPTER III
3.1 DRUG PROFILE OF METRONIDAZOLE
Structure of Metronidazole:
|
Category: Anti-amoebic IUPAC Name: 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol |
|
|
Trade names: |
Flagyl |
|
Formula : |
C6H9N3O3 |
|
Melting point: |
159–163 °C (318–325 °F) |
Mechanism of Action:
Metronidazole, taken up by diffusion, is selectively absorbed by anaerobic bacteria and sensitive protozoa. Once taken up by anaerobes, it is non-enzymatically reduced by reacting with reduced ferredoxin, which is generated by pyruvate oxido-reductase. Many of the reduced nitroso intermediates will form sulfinamides and thioether linkages with cystein bearing enzymes deactivating these critical enzymes. As many as 150 separate enzymes are affected.
In addition or alternatively, the metronidazole metabolites are taken up into bacterial DNA, and form unstable molecules. This function only occurs when metronidazole is partially reduced, and because this reduction usually happens only in anaerobic cells, it has relatively little
Pharmacokinetics:
Absorption:
Oral Metronidazole is well absorbed; topical application is less complete and more prolonged. Following administration, Tmax is 1 to 2 h, and Cmax is 25 mg/mL. Oral bioavailability is not affected by food, but peak serum levels will be delayed to 2 h. Following vaginal administration, Cmax and Tmax are 281 ng/mL and 9.5 h. respectively.
Distribution:
Metronidazole appears in CSF, saliva and breast milk in concentrations similar to those found in plasma. Less than 20% is protein bound.
Metabolism:
Metabolites are 2-hydroxymethyl and acidic metabolite.
Elimination:
Routes of elimination are via urine (60% to 80%) and feces (6% to 15%). Renal clearance is approximately 10 mL/min per 1.73 m2. The half-life is 8 h in healthy adults and the hydroxyl metabolite half-life is 15 h.
Therapeutic uses of Metronidazole:
Metronidazole is indicated for the treatment of:
- Bacterial vaginosis, commonly associated with overgrowth of Gardnerellaspecies and coinfective anaerobes (Mobiluncus, Bacteroides), in symptomatic patients.
- Pelvic inflammatory diseasein conjunction with other antibiotics such as ofloxacin, levofloxacin, or ceftriaxone.
- Pseudomembranous colitis due to Clostridium difficile.
- Dental infection of bacterial origin, such as periapical abscess, periodontal abscess, acute pericoronitis of impacted or partially erupted teeth; often used in conjunction with Amoxicillin.
- Amoebiasis: Infections caused by Entamoeba histolytica.
- Giardiasis: infection of the small intestine caused by the ingestion of infective cysts of protozoan Giardia lamblia.
- Trichomoniasis: infection caused by Trichomonas vaginalis, which is a common cause of vaginitis and is the most frequently presenting new infection of the common sexually transmitted diseases.
Adverse effects:
Common adverse drug reactions(≥1% of patients) associated with systemic metronidazole therapy include: nausea, diarrhea, and/or metallic taste in the mouth. Intravenous administration is commonly associated with thrombophlebitis. Infrequent adverse effects include: hypersensitivity reactions (rash, itch, flushing, fever), headache, dizziness, vomiting, glossitis, stomatitis, dark urine, and/or paraesthesia.
High doses and/or long-term systemic treatment with metronidazole is associated with the development of leukopenia, neutropenia, increased risk of peripheral neuropathy and/or CNS toxicity.
CHAPTER – IV
4.1 AIM OF WORK
The aim of present work is to develop a new method which becomes necessary to find the content of drug in pure form or single dosage forms for purity testing.
The scope of developing and validating analytical methods is to ensure a suitable method for a particular analyte more specific, accurate and precise. The main objective for that is to improve the conditions and parameters, which should be followed in the development and validation.
The existing physicochemical methods are inadequate to meet the requirements, hence it is proposed to improve the existing methods and to develop new methods for the assay of Metronidazole in pharmaceutical dosage forms adapting available analytical technique like UV-Spectrophotometry.
OBJECTIVE OF THE STUDY
The main objective of the proposed method is to develop simple and accurate methods for the determination of Metronidazole by UV-Spectrophotometric method.
4.2 PLAN OF WORK
The plan of the proposed work includes the following steps:
- To undertake solubility studies and analytical studies of Metronidazole to develop initial UV conditions.
- Setting up of initial UV conditions for the assay of Metronidazole in pure and pharmaceutical dosage forms.
- Optimization of initial spectrophotometric conditions.
- Analytical method validation of the developed UV method and evaluation of analytical method validation report generated for the developed method
4.3 EXPERIMENTAL DETAILS
LIST OF INSTRUMENTS USED
|
S. No. |
Instruments/Equipments |
|
1 |
UV-Visible double beam Spectrophotometer (ELICO) |
|
2 |
Electronic Balance |
|
3 |
Ultra Sonicator |
LIST OF CHEMICALS, REAGENTS AND STANDARDS
|
S. No. |
Chemicals / Reagents / Standards |
|
1 |
Metronidazole Sample |
|
2 |
Metronidazole Standard |
|
3 |
Hydrochloric acid |
|
4 |
Deionised water |
4.4 METHOD DEVELOPMENT OF METRONIDAZOLE
During the method development stage, the initial sets of conditions that have evolved from the first stages of development are improved or maximized to quantify the specific analyte of interest. From the literature survey it was found that the best suitable solvent for the analysis of Metronidazole was found to be 0.1 N HCl, in which the drug has shown maximum absorbance at 277 nm. Now in the present method of analysis of Metronidazole for its method development the drug was analyzed by using different solvents such as 0.1 N HCl, 0.1 N NaOH and Sodium hydrogen phosphate buffer as a part of trial and error method. By using the above reagents it was found that the drug has shown maximum solubility in 0.1 N HCl and also it has shown maximum absorbance i.e; 277 nm. The results obtained by using 0.1 N HCl were found to be within the limits and hence the best suited solvent for the analysis of Metronidazole was found to be 0.1 N HCl.
Method for Metronidazole:
Preparation of 0.1 N HCl solution (Blank solution):
Measure carefully 8.5 ml of concentrated Hydrochloric acid and dissolve it in sufficient amount of deionised water in a 1000 ml volumetric flask and make up to volume with deionised water.
Preparation of standard solution of Metronidazole:
The stock solution of Metronidazole (1mg/ml) is prepared by dissolving 100 mg of pure drug in 100 ml of volumetric flask with 20 ml of 0.1 N HCl.The solution is sonicated for 10 minutes and then made up to volume with 0.1 N HCl. Working standard solutions of Metronidazole was prepared by suitable dilution of the stock solution with blank solution.
Preparation of sample solution of Metronidazole:
Twenty tablets were weighed, finely powdered and an accurately weighed sample of powdered tablets equivalent to 100 mg of Metronidazole was dissolved with 0.1 N HClin a 100 ml volumetric flask using ultra sonicator. This solution was filtered through 0.45µm filter paper. The solution obtained was diluted with the 0.1 N HCl so as to obtain a concentration in the range of linearity previously determined.
Procedure for calibration curve:
The contents of the blank solution were filtered before use through 0.45µm filter paper, and prepare a series of solutions for standard drug (Metronidazole) ranging from 1 µg/ml to 7 µg/ml. Then the prepared series of dilutions were scanned for its maximum absorbance and the maximum absorbance of Metronidazole both for sample and the standard was found to be 277 nm. The calibration curve of Metronidazole is plotted by taking concentration (µg/ml) on X-axix and absorbance on Y-axix. From the calibration curve correlation coefficient value for Metronidazole was found to be 0.999. The linearity range and linearity graphs were shown in table.
Analysis of formulation:
The amount of drug present in pharmaceutical formulation was calculated through absorbance values of formulation to that of standard drug by using the standard calibration curve (concentration in mg/ml was taken on X-axis and absorbance on Y-axis).
4.5 METHOD VALIDATION OF METRONIDAZOLE BY UV
Method validation can be defined as “Establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics”.
Method validation is an integral part of the method development; it is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, quality, purity, and potency of the drug substances and drug products. Simply, method validation is the process of proving that an analytical method is acceptable for its intended purpose.
Method Validation, however, is generally a one-time process performed after the method has been developed to demonstrate that the method is scientifically sound and that it serves the intended analytical purpose.
In the present method, the developed method for Metronidazole is validated for parameters which includes linearity, precision and accuracy. From the results obtained for validation of Metronidazole it was found that the results were all within the limits.
The developed method was validated for:
(1) Linearity
(2) Precision
(3) Accuracy
(1) Linearity:
To carry out linearity for Metronidazole, sample solutions were prepared over a concentration range of 1 µg/ml to 6 µg/ml. The absorbance for prepared sample was recorded in duplicate. The observed absorbances were calculated for its correlation coefficient.
(2) Precision:
To carry out precision for Metronidazole, sample solution of same concentration is prepared six times and the absorbance are recorded in duplicate. The observed absorbances were calculated for its mean, %RSD and standard deviation.
(3) Accuracy:
To carry out accuracy for Metronidazole, sample solutions are prepared in triplicate at three levels over a range of 80% to 120%. The observed absorbances were calculated for its % RSD.
CHAPTER – V
5.1 RESULTS AND DISCUSSIONS
METHOD DEVELOPMENT OF METRONIDAZOLE
Linearity curve of Metronidazole:
|
Concentration (µg/ml) |
Absorbance |
|
1 |
0.0385 |
|
2 |
0.067 |
|
3 |
0.095 |
|
4 |
0.125 |
|
5 |
0.153 |
|
6 |
0.1825 |
Acceptance criteria: The Correlation coefficient (R2) value should not be less than 0.9990
Discussion: From the linearity curve for Metronidazole, the R2 value for the present method was found to be 0.9990 and hence the developed method is validated for its linearity.
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METHOD VALIDATION OF METRONIDAZOLE
Precision:
|
Concentration |
Sample No |
Absorbance – I |
Absorbance – II |
Average |
|
10 µg/ml |
1 |
0.410 |
0.408 |
0.409 |
|
2 |
0.403 |
0.407 |
0.405 |
|
|
3 |
0.410 |
0.408 |
0.409 |
|
|
4 |
0.406 |
0.408 |
0.407 |
|
|
5 |
0.425 |
0.417 |
0.421 |
|
|
6 |
0.407 |
0.404 |
0.4055 |
Average: 0.4094
% RSD: 1.45%
S.D: 0.00592
Acceptance criteria:% RSD should not be more than 2.0%
Discussion: From the precision table the % RSD was found to be 1.45% which is less than 2.0% and hence the developed method for Metronidazole is said to be validated for its precision.
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