Carotenoids are a diverse group of secondary metabolites, with more than 700 structures identified in numerous biological pathways.1 Because past studies have identified associations between carotenoid intake and beneficial health effects, researchers such as Van Meulebroek and colleagues are interested in metabolomics profiling to better understand these health benefits and possibly increase the content of beneficial carotenoids found in foods.
This research group has recently published a screening method to detect carotenoids in tomato fruit.2 Their experiments relied on a full-scan high-resolution Orbitrap mass spectrometry and targeted lutein, zeaxanthin, α-carotene, β-carotene, and lycopene.
For their experiments, the researchers used a D-optimal design to optimize eight variables affecting the extraction efficiency. They used stock solutions of all-trans-lycopene, all trans-β-carotene, all-trans α-carotene, trans-lutein and all-trans-zeaxanthin for the optimization. Additionally, they also used β-apo-8’-carotene as an internal standard.
The researchers based their final experimental design around the most efficient scenario optimality criterion, also called the G-efficiency. The selected design included 28 experiments (25 design runs and 3 center points), with each of the experiments consisting of a specific combination of settings for the eight variables. The results were also carefully calculated and statistically evaluated based on P<0.05.
Once the researchers were confident in their optimizations, they were ready to put real samples to the test. The researchers grew tomato plants (S. lycopersicum L. cv Moneymaker) in a 60 m2 greenhouse compartment within the Institute for Agricultural and Fisheries Research. They harvested both red-ripe and green-colored tomatoes. Tomatoes from both developmental stages were cut, lyophilized, ground up and sieved to form a homogeneous powder.
They added MgCO3 to 25 mg of homogenized tomato fruit material to neutralize trace levels of organic acids. Next, they used liquid–liquid extraction with methanol/tert-butyl methyl ether (1:1, v/v) to extract the carotenoids. They also added β-apo-8’-carotene to the extraction solvent.
Once the carotenoids were extracted, the researchers analyzed the extracted samples using high resolution mass spectrometry on an Exactive Orbitrap mass spectrometer (Thermo Scientific). They determined a mass deviation of <2 ppm and the mass resolving power up to 100,000 full width at half maximum proved to be both sensitive and accurate for these samples.
The researchers found that all of the targeted carotenoids were present at detectable concentration levels in the red-ripe samples. The green-colored samples, representing the earliest in development and some of the lowest amount of carotenoids, were used for validation studies. The researchers also followed the Commission Directive directives 2002/657/EC as a guideline for validating the extraction and detection procedures.
In addition to the identification of the target carotenoids, the researchers also performed an efficient metabolomics screening of the full-scan data for carotenoid compounds using the software program ToxID 2.1.2 (Thermo Scientific). They constructed a ToxID database based on the molecular formula of 355 relevant carotenoids. They demonstrated the profiling capabilities of this method by surveying one red and one green tomato and were able to detect 59 and 97 peaks, respectively. This screening method for carotenoids by Van Meulebroek and colleagues may bring rise to future studies of unknown compounds involved in carotenoid metabolism.
1. Oliver, J. (2000) ‘Chromatographic determination of carotenoids in foods.’, Journal of Chromatography, June8, 888 1(1-2) (pp. 543-555)
2. Van Meulebroek, L. (2014) ‘High-resolution Orbitrap mass spectrometry for the analysis of carotenoids in tomato fruit: validation and comparative evaluation towards UV-VIS and tandem mass spectrometry.’, Analytical and Bioanalytical Chemistry, Apr;406(11) (pp. 2613-26) doi: 10.1007/s00216-014-7654-6. Epub 2014 Feb 20.