Pharmaceutica Analytica Acta

Ultra-Performance Liquid Chromatography: A Superior Method for Metformin Detection in Rat Plasma

Silvia Tejeda^* Department of Pharmacy, University of Pavia, Pavia, Italy
^*Correspondence: Silvia Tejeda, Department of Pharmacy, University of Pavia, Pavia, Italy, Email:

Received: 26-Feb-2024, Manuscript No. PAA-24-25872; Editor assigned: 28-Feb-2024, Pre QC No. PAA-24-25872 (PQ); Reviewed: 13-Mar-2024, QC No. PAA-24-25872; Revised: 20-Mar-2024, Manuscript No. PAA-24-25872 (R); Published: 27-Mar-2024, DOI: 10.35248/2153-2435.24.15.775

Description

Metformin, a widely used oral hypoglycaemic agent, plays a crucial role in the management of type 2 diabetes mellitus. Its primary mechanism involves the inhibition of hepatic glucose production and the improvement of insulin sensitivity. To understand the pharmacokinetics and pharmacodynamics of metformin, precise and reliable quantification in biological matrices like rat plasma is essential. Ultra-Performance Liquid Chromatography (UPLC) has emerged as a powerful analytical technique for this purpose, offering significant advantages over traditional High-Performance Liquid Chromatography (HPLC) in terms of speed, resolution, and sensitivity.

Introduction to UPLC

UPLC represents a significant advancement in liquid chromatography, utilizing smaller particle sizes (sub-2 microns) for the stationary phase compared to traditional HPLC (typically 3-5 microns). This reduction in particle size increases the surface area, enhancing the separation efficiency and allowing for faster analysis with better resolution. UPLC operates at higher pressures, up to 15,000 psi, compared to the 6,000 psi limit of HPLC, facilitating quicker elution times without compromising performance.

Sample preparation

Accurate determination of metformin in rat plasma necessitates meticulous sample preparation. Common techniques include protein precipitation, liquid-liquid extraction, and solid-phase extraction. For UPLC analysis, the preferred method often involves protein precipitation due to its simplicity and effectiveness in removing proteins that might interfere with the analysis.

Protein precipitation method

Blood samples are collected from rats and centrifuged to obtain plasma. A known volume of plasma (typically 100 μL) is mixed with an organic solvent like acetonitrile in a 1:3 ratio to precipitate proteins. The mixture is centrifuged at high speed (e.g., 10,000 rpm) for 10 minutes to separate the precipitated proteins. The clear supernatant is carefully collected and subjected to UPLC analysis.

UPLC method development

Developing a robust UPLC method for metformin involves optimizing several parameters, including the choice of column, mobile phase composition, flow rate, and detection wavelength.

A C18 column (e.g., 2.1 × 50 mm, 1.7 μm) is commonly used due to its excellent retention and separation properties for metformin. A combination of aqueous and organic solvents, such as water with 0.1% formic acid (solvent A) and acetonitrile (solvent B), is typically employed. A gradient elution is often used, starting with a high proportion of aqueous solvent and gradually increasing the organic solvent to achieve better separation. A flow rate of 0.3-0.5 mL/min is suitable for UPLC to ensure adequate resolution and short run times. An injection volume of 2-5 μL is generally sufficient to achieve good sensitivity without overloading the column. Metformin is commonly detected using UV or PDA detectors at a wavelength of around 233 nm, where it exhibits maximum absorbance.

Calibration and quantification

A series of metformin standard solutions in the concentration range of 10-1000 ng/mL are prepared and analysed. The peak area of metformin is plotted against its concentration to construct a calibration curve. Linear regression analysis is performed to obtain the equation of the calibration curve, which is used to quantify metformin in plasma samples.

Validation

The UPLC method is validated in terms of specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Specificity is assessed by analyzing blank plasma samples to ensure no interfering peaks at the retention time of metformin. Linearity is evaluated by the correlation coefficient (r^2) of the calibration curve, which should be greater than 0.99. Accuracy and precision are determined by analyzing quality control samples at different concentration levels. LOD and LOQ are calculated based on the signal-to-noise ratio, with typical values for metformin being in the low ng/mL range.

Application to pharmacokinetic studies

The developed UPLC method is applied to pharmacokinetic studies to measure metformin concentrations in rat plasma over time. Rats are administered metformin, and plasma samples are collected at various time points. The concentration-time data are analysed to determine pharmacokinetic parameters such as the maximum concentration (C_max), time to reach maximum concentration (T_max), half-life (t_-1/2), and Area Under the Concentration-time curve (AUC).

Conclusion

The determination of metformin in rat plasma using UPLC methods offers a highly efficient, sensitive, and accurate approach for pharmacokinetic studies. UPLC's advantages, including faster analysis, better resolution, and higher sensitivity, make it an ideal choice for the quantification of metformin in biological samples. This method provides valuable insights into the pharmacokinetics of metformin, aiding in the optimization of its therapeutic use in managing type 2 diabetes mellitus.