Abstract
High-performance liquid chromatography (HPLC) has become the method of choice for carotenoid analysis. Although a number of normal-phase columns have been reported, reverse-phase columns are the most widely used stationary phases for the analysis of these molecules. C18 and C30 stationary phases have provided good resolution for the separation of geometrical isomers and carotenoids with similar polarity. More recently ultra high-performance liquid chromatography (UHPLC) has been used. UHPLC has a number of distinct advantages over conventional HPLC. These include: faster analyses (due to shorter retention times), narrower peaks (giving increased signal-to-noise ratio) and higher sensitivity. High strength silica (HSS) T3 and C18 and ethylene bridged hybrid (BEH) C18 stationary phases, with sub-2 μm particles have been used successfully for UHPLC analysis and separation of carotenoids. A number of spectroscopic and mass spectrometric techniques have also been used for carotenoid qualitative and quantitative analysis. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS), atmospheric-pressure solids-analysis probe (ASAP) and Raman spectroscopy are used to profile rapidly and qualitative carotenoids present in different crude extracts. Such detection methods can be used directly for the analysis of samples without the need for sample preparation or chromatographic separation. Consequently, they allow for a fast screen for the detection of multiple analytes. Quantitative carotenoid analysis can be carried out using absorbance or mass detectors. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) is efficient for carotenoid identification through the use of transitions for the detection of analytes through precursor and daughter ions. This approach is suitable for the identification of carotenoids with the same molecular mass but different fragmentation patterns. Here we review critically the latest improvements for carotenoid resolution and detection and we discuss a number of analytical techniques for qualitative and quantitative analysis of carotenoids.
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