Method Development of Determination of Alliin in Gallic Using Ultra Performance Liquid Chromatographic Technique

Method Development of Determination of Alliin in Gallic by UPLC

Abstract

        Alliin is a major bioactive compound found in garlic (Allium sativum L.). It exhibits pharmacological activities including antibacterial, anti-inflammatory, and antihypertensive effects. Therefore, the quantitative determination of alliin in garlic can be used to evaluate its quality. The objective of this study was to develop an analytical method for the quantitation of alliin in garlic using Ultra Performance Liquid Chromatography (UPLC). A 150 mg sample of garlic was extracted with 25 mL of 50% methanol by vortex mixing for 1 minute. The supernatant was filtered through a PTFE syringe filter, and a 1 μL aliquot of the filtrate was injected into the chromatographic system. The separation was performed on an Acquity^TM BEH^® Amide column (2.1 × 100 mm, 1.7 μm). The mobile phase consisted of acetonitrile and water, beginning at a ratio of 85:15 for 0.65 minutes, then adjusted to 75:25 at 1.42 minutes, and subsequently to 86:14 at 1.52 minutes, and maintained at 86:14 until 2.30 minutes. The flow rate was 0.55 mL/min, the column temperature was maintained at 40°C and detection was carried out with a Photodiode Array (PDA) detector at 220 nm. It was found that the retention time of alliin was 2.02 minutes. As a result, the method validation demonstrated that the calibration curve for alliin was linear over the concentration range of 0.049–0.582 mg/mL with a coefficient of determination (R²) of 0.999791. Accuracy ranged from 98.64% to 106.02%, while precision (RSD) was between 0.51% and 1.19%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.006 mg/mL and 0.033 mg/mL, respectively. Analysis of eight garlic samples revealed an average alliin content of 9.87±2.75 mg/g. This study demonstrates that the developed UPLC method is suitable for the quantification of alliin in garlic and can be applied for quality control as well as for the selection of high-quality garlic for pharmaceutical or health product manufacturing.