ASHS 2025 Conference

Carotenoids are natural pigments that give fruits and vegetables their yellow, orange, and red colors, and their consumption is associated with numerous health benefits. Most carotenoids occur in isomeric forms (primarily cis and trans); this poses challenges for efficient separation and identification during chromatographic analysis. Tomato (Solanum lycopersicum), an economically important crop, has high nutritional and health benefits, attributed mainly to carotenoids. Lycopene, a major carotenoid in tomatoes, predominantly exists in the trans isoform; however, the trans isoform is less bioavailable than the cis isoform. To accurately measure these isoforms in tomatoes and thus provide information for breeding tomatoes with more cis lycopene, we aimed to develop an Ultra-High Performance Liquid Chromatography (UHPLC) method based on mobile phase (MP) modifiers to measure a wide range of carotenoids (cis and trans) across different tomato varieties (heirloom and cherry) with different colors (green, yellow, orange, purple, and red). The chromatographic parameters, such as temperature (15-30 °C), flow rate (0.3-1.5 ml/min), column lengths (50, 150, and 250 mm), and gradients, and MP modifiers (water, acetonitrile, tetrahydrofuran, triethylamine, ammonium acetate, and ethyl acetate) were optimized. Among the tested parameters, methanol and tert-butyl methyl ether as MP, including ethyl acetate (10%) as a modifier, and C30 column (150 x 4.6 mm) at 20 °C gave the best resolution, allowing the separation of up to 20 peaks indicating individual compounds and isoforms in 35 min. The optimized UHPLC method was also validated for complete profiling, recovery, precision, limit of detection, and quantification. The complexity of the profile based on the number of peaks followed the following order: Orange Heirloom (15) > Orange Cherry (15), Red Cherry (15) > Red Heirloom (13) > Green Heirloom (13) > Purple Cherry (13) > Yellow Cherry (2). Among all the tested tomatoes, trans-lycopene, 5-cis-lycopene, and β-carotene were the predominant carotenoids, followed by 9- and 13-cis-lycopene, and for the first time, mutatoxanthin was detected in Orange Heirloom tomatoes. This method provided efficient peak separation resolution, although not all of them were identified as potential carotenoids; however, their identification could be confirmed by mass spectrometry analysis in future studies. The efficiency, separation, and quantification in this method make it robust for carotenoid analysis in any tomato variety regardless of color. USDA-NIFA-2024-51181-43464 supported this work through the Vegetable and Fruit Improvement Center and partially funded by the Texas A