Abstract
<title>Abstract</title> <p>Fatty acids (FAs) are key components of plant metabolism with significant implications for nutrition, bio fortification, and industrial applications. Accurate profiling of FA composition is therefore essential, particularly for high-throughput screening of plant tissues. However, conventional lipid extraction and analysis methods are often labor-intensive and require large amounts of biomass, limiting their applicability for small tissue samples. Here, we describe a rapid, sensitive, and reproducible protocol for fatty acid profiling from minimal quantities of plant leaf tissue. The workflow involves efficient total lipid extraction using a chloroform–methanol system, followed by trans-methylation to generate fatty acid methyl esters (FAMEs), and subsequent identification and quantification using gas chromatography–flame ionization detection (GC-FID). The method is optimized for micro-scale samples (50–100 mg fresh weight) and enables reliable detection of major fatty acid species, particularly C18 fatty acids, without the need for internal standards. This protocol provides a streamlined approach for fatty acid analysis, offering improved sensitivity, reduced sample requirement, and compatibility with routine laboratory workflows. It is well-suited for applications in plant phenotyping, metabolic studies, and crop improvement programs requiring rapid and efficient lipid profiling.</p>