Metronidazole, a commonly used antibiotic, targets anaerobic bacterial infections, serving as a standard therapeutic agent across various infection types. Due to its wide application in medical and pharmaceutical fields, accurate quantification methods for metronidazole are crucial to ensure proper dosing, quality control, and pharmacokinetic studies. High-Performance Liquid Chromatography (HPLC) is a robust, sensitive, and precise analytical method ideal for both polar and non-polar compounds. In metronidazole quantification, using oxytetracycline as an internal standard enhances the accuracy of measurements.
Experimental Methodology
A. Chromatographic Conditions and Parameters
Wavelength Selection
Identifying the maximum absorbance wavelength of metronidazole is crucial for its effective detection and quantification. For this purpose, metronidazole’s peak absorbance wavelength is selected, ensuring the highest sensitivity in HPLC analysis.
Choice of Mobile Phase
A mobile phase consisting of methanol and an aqueous solution of oxalic acid (0.002 mol/L) has proven effective for metronidazole quantification. This solvent system optimizes retention time and peak resolution, providing reliable separations and enhanced peak sharpness.
B. Preparation of Internal Standard Solution
For quantitative analysis, oxytetracycline, chosen as the internal standard, is prepared at a precise concentration of 100 mol/L. The similarity in chemical properties between oxytetracycline and metronidazole, combined with its lack of interaction with the target compound under specified chromatographic conditions, makes it a suitable standard.
C. Preparation of Standard and Sample Solutions
Metronidazole Standard Solution: An accurately weighed quantity of metronidazole is dissolved to yield a 100 mol/L solution, serving as the reference concentration for quantification.
Sample Solution: Tablets containing metronidazole are finely ground, and an exact amount is weighed and dissolved to produce the test solution. This solution is prepared in a manner identical to the standard, ensuring consistency in analytical outcomes.
D. Retention Time Measurement
Retention times for both metronidazole and oxytetracycline are determined under identical HPLC conditions. Recording retention times for each analyte is crucial for calculating the concentration of metronidazole in samples based on the ratio of the analyte peak area to the internal standard peak area.
E. Determination of Relative Correction Factor
The relative correction factor (RCF) is calculated by combining standard solutions of metronidazole and oxytetracycline. This factor adjusts for any minor variations in analyte detection, further improving the p recision of the quantification process.
F. Quantification of Metronidazole in Samples
Sample and internal standard solutions are mixed and analyzed under consistent chromatographic conditions, with peak areas recorded for both metronidazole and oxytetracycline. The concentration of metronidazole in each sample is determined by comparing the peak area ratio of the sample to the known concentration of the internal standard.
