## 1 Introduction  
Carotenoids, a class of naturally occurring pigments, play a critical role in plant biology and human health [1; 2; 3]. These compounds contribute to the vibrant red, orange, and yellow hues of fruits and vegetables and are essential for photosynthesis and plant photoprotection [1; 2]. In humans, carotenoids such as \(\beta\)-carotene and lycopene are known for their antioxidant properties and their role as precursors to vitamin A, contributing to immune function, vision, and cellular health [2]. Given their significant role in human health and nutrition, precise quantification of carotenoids in plant-based foods is essential [1; 2; 3]. This analysis is pivotal for improving food quality, optimizing bioavailability, and informing nutritional guidelines. Understanding carotenoid composition in plant foods is particularly important due to their health benefits and role in dietary interventions for chronic diseases, including cardiovascular conditions and certain cancers [4]. Although advancements in analytical methods have improved carotenoid quantification, their structural diversity, susceptibility to degradation, and interactions with other compounds within food matrices present persistent challenges. For instance, carotenoids exhibit various molecular structures, including differences in chain length, functional groups, and conjugated double bonds, all of which influence their physical, chemical, and biological properties. Figure 1 illustrates the molecular structures of major carotenoids, highlighting their structural variability and the functional groups that contribute to their unique properties. Understanding these structures is essential for addressing analytical challenges and optimizing quantification methods. The accurate analysis of carotenoids also aids in understanding their broader health implications. These compounds have modulated oxidative stress and inflammation, reducing the risk of chronic diseases such as macular degeneration, cardiovascular diseases, and certain types of cancer [5]. Furthermore, as precursors to vitamin A, carotenoids are crucial in preventing deficiency-related conditions like xerophthalmia. Recent studies, such as those utilizing spectroscopic methods, have linked carotenoid biomarkers to dietary intake, highlighting their potential to enhance immune responses and promote skin health [6]. Chromatographic methods have become the cornerstone of carotenoid analysis due to their precision and reliability in quantifying individual compounds [7]. Techniques such as high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) offer robust approaches for achieving rapid separation and quantification, even in complex plant matrices [8]. Emerging methods like supercritical fluid chromatography (SFC) offer eco-friendly alternatives with high separation efficiency,  
Figure 1: Structural representation of major carotenoids and related compounds. especially for nonpolar carotenoids, while advancements in sample preparation, including enzymatic treatments and green solvents, are enhancing recovery rates and reducing analytical errors, driving the field toward more sustainable practices. [9; 10]. Despite these advancements, challenges like carotenoid degradation during analysis, matrix interferences, and the lack of standardized reference materials persist. Overcoming these hurdles requires interdisciplinary approaches integrating chemistry, biochemistry, and food science [11]. Additionally, regulatory frameworks must evolve to accommodate novel analytical techniques and ensure global standardization. Despite significant advancements in carotenoid analysis, there remains a need for a comprehensive evaluation of how different methodologies compare in terms of accuracy, efficiency, and applicability. Traditional techniques, such as HPLC, have provided reliable quantification for decades, but newer technologies, including UHPLC and SFC, are demonstrating enhanced precision and sustainability.