Analytical Method Development and Validation for Quantitative Estimation of Metronidazole in Pharmaceutical Dosage Forms by RP- HPLC

The chemical definition of metronidazole is 2 (2-methyl-5-nitro-1H-imidazol-1-yl) ethanol [1]. It is an artificial derivative of nitroimidazole mostly applied to deal with the infection caused by anaerobic bacteria and protozoa such as Trichomonas vaginalis, Entamoeba histolytica, and Giardia lamblia [2]. Action of the drug involves blocking the production of nucleic acid by destroying the DNA of microbial cells. This activity can only take place when Metronidazole is partially reduced, which only normally happens in anaerobic cells.

Quantitative estimation of Active Pharmaceutical Ingredients (APIs) is an important process in Quality Control (QC) and Quality Assurance (QA) in the pharmaceutical industry. Although different techniques like UV-Visible spectrophotometry, titrimetric, as well as electrochemical methods are available, they cannot be sufficient to identify the API as distinct to impurities, degradation products, or excipients [3]. RP-HPLC can separate and quantify Metronidazole accurately and precisely hence it is the choice of assay when it comes to meeting regulatory compliance.

PHYSICOCHEMICAL PROFILE

Knowledge of the physicochemical characteristics of the analyte is the key to the development of the methods.

Chemical Structure: Metronidazole has both a nitro group and an imidazole ring.

Molecular Weight: 171.15 g/mol

Solubility: It is insoluble in water and alcohol and freely soluble in acidic solutions. The solubility profile determines what diluents are used in HPLC, and commonly it uses mixtures of water, methanol or acetonitrile [4].

pKa: pKa is around 2.5 meaning that it is slightly basic. This parameter plays a very important role in buffer selection to retain the drug in a non-ionized state (or controlled ionization) throughout the chromatography so that the drug would not tail [5].

Strategy Of Analytical Method Development In Rp-Hplc.

The process of developing the method is a progressive screening and optimizing of the chromatographic conditions in an attempt to obtain a robust separation.

Choice Of Detection Wavelength

The detection wavelength is very important in the sensitivity of HPLC method. Metronidazole is associated with typical UV absorption. An absorption peak at a wavelength of less than 200 -400nm is generally detected after the scan of the standard solution (typically 10-20 mL) in the wavelength range between 200 and 400 nm. usually reveals an absorption maximum at around 277 nm, although certain techniques have reported the detection at 254 nm or 318 nm in different solvents [6]. When analyzing Metronidazole with other drugs (e.g., Ofloxacin or Norfloxacin) it is often desirable to select the isosbestic point in order to have a consistent sensitivity.

Selection Of Stationary Phase (Column)

The stationary phase is selected with the objective of reducing turbulent shear stress effects on the columns and the stationary fluid phases. Most of the reported techniques use columns of Octadecyl silane (C18) based on their harshness and ability to separate polar and non-polar substances.

1. Common Dimensions: 250 mm, 4.6 mm, 5 um particle size.
2. Alternative Columns: C8 columns are sometimes employed in place of C18 to achieve higher elution rates but C18 is the most common in that case (Rs > 2).
3. Brands: Columns like Hypersil BDS, Phenomenex Luna and Waters XBridge are among the commonly mentioned brands in successful validations [7].

Mobile Phase Optimization

The mobile phase composition defines the retention time (Rt), the peak shape and resolution.

1. Buffer Selection: Phosphate buffers (Potassium dihydrogen phosphate, K H 2 PO 4) are most prevalent. Orthophosphoric acid is usually used to adjust the pH between 3 and 4.5 to prevent the silanol groups on the column to ionize and enhance the symmetry of peaks [8].
2. Organic Modifiers: Acetonitrile (ACN) and Methanol (MeOH) are conventional. CNC is usually favored by below back pressure and steeper peaks.
3. Ratios: The usual isocratic ratios are Buffer:ACN (80:20 v/v) or Buffer:Methanol (60:40 v/v). The use of gradient elution can be applied in the event that there are impurities that have significantly differing polarities [1].

Flow Rate And Temperature

Flow rate: the normal flow rates include 0.8-1.2 mL/min. The starting point of a rate of 1.0 mL/min balances between the run time and column pressure.

Temperature: Ambient temperature (25 o C) is usually used, but maintaining the column oven at 30 o C or 35 o C may enhance reproducibility and decrease the viscosity of the mobile phase.

Method Validation (Ichq2(R1)Guideline)

The method should then be tested after being optimized to prove that it is applicable in its target application. The parameters described by the International Council of Harmonisation (ICH) are as follows [9]:

System Suitability

System suitability tests (SST) are done to verify the instrument and method are working well before the samples are analyzed.

Criteria:

1. Theoretical Plates > 2000 (Shows efficiency of columns).
2. Tailing Factor < 2.0 (Peak symmetry).
3. Resolution > 2.0 (When analysing mixtures).
4. Relative Standard deviation of retention time and peak area less than 2.0 percent replicate injections.

Specificity

Specificity is the characteristic of measuring the analyte without any doubt in the presence of other components such as impurities, degradants and excipients. This is confirmed by injecting a blank (mobile phase) and a placebo (excipients only contain no drug). Interference is not expected at the retention time of Metronidazole (usually 3-6 mins) [10].

Linearity And Range

The presence of linearity proves that the test results are proportional to the concentration of analyte.

1. Procedure: A standard series of solutions (e.g., 5, 10, 15, 20, 25 mug/mL) is injected.
2. Acceptance: The correlation coefficient (R 2) is supposed to be 0.999.

Accuracy (Recovery Study)

The accuracy is determined with the help of spiking known quantities of standard drug to the placebo matrix at three levels (e.g., 50, 100, 150 or 80, 100, 120).

Acceptance: The average refer to 98.0% -102.0% [11] of recovery.

Precision

1. Repeatability (Intra-day): Repeat injection on the same sample on the same day of identical homogenous sample.
2. Intermediate Precision (Inter-day): Results from the analysis on other days, other analysts or other equipment.
3. Acceptance: The percentage Relative Standard Deviation (%RSD) is expected to be less than 2.0%.

Limit Of Detection (Lod) And Limit If Quantification (Loq)

The parameters characterize the sensitivity.

1. LOD: Lowest amount detected
2. LOQ: Minimum amount that is measured with precision (10).

Where is the standard deviation of response and is the slop of calibration curve. Metronidazole is reported to have an LOQ range of 0.05 -0.5 mug/mL [12].

Robustness

Robustness is an assessment of the ability of the method to stay unchanged when small, intentional changes occur in the method parameters such as:

1. Flow rate (pm 0.1 mL/min).
2. Wavelength (pm 2 nm).
3. Mobile phase pH (pm 0.2 units).
4. Mobile phase composition (pm 2% organic).

Force Degradation Studies (Stability Indicating Method)

To assert that a method is Stability Indicating, the substance is exposed to conditions of stress to make sure the degradation products do not affect the primary peak.

1. Acid Degradation: 0.1 N HCl at 60 o C during 1-2 hours. Metronidazole is stable moderately here.
2. Base Degradation: 0.1 N NaOH. Metronidazole is very vulnerable to degradation by alkaline hydrolysis and is usually affected by 20-30 per cent degradation [13].
3. Oxidative Degradation: Degradation peaks of 3% are seen frequently.
4. Thermal/Photolytic: Dry heat (60 o C) and UV light.
5. Result: A validated procedure should exhibit Peak pure, such that the purity angle is less than the purity limit (PDA detector), and that no co-elution of degradants with the Metronidazole peak occurs.

Review Of Reported Method Of Literature

To draw a comparison between several reported RP-HPLC methods of Metronidazole estimation, a summary of the methods is presented below so as to point out the trends in chromatographic conditions.

Table 1: Overview of different RP-HPLC conditions that were reported in the literature.