The complexity of many food samples places a great demand in terms of both separation capabilities and specificity of detection. In this article, a novel system for fully automated comprehensive two-dimensional liquid chromatography (LC×LC) is discussed. The on-line coupling of the two separation dimensions was achieved using two six-port, two-position switching valves. High orthogonality was achieved by using a micro-bore cyano column for the first dimension separation, interfaced to a secondary C18 column packed with fused-core particles. The hyphenation to a triple quadrupole mass spectrometer generates a powerful analytical system, capable of extremely high resolving power, as well as targeted and untargeted analysis. The so-called multiple reaction monitoring (MRM) mode in fact enhanced selectivity, reducing sample consumption and the need for tedious clean-up procedures, specifically for beta-carotene quantification in a red pepper extract.
The high complexity of many food samples places a great demand in terms of both separation capabilities and specificity of detection. As far as separation is concerned, the implementation of multidimensional liquid chromatography (LC) techniques has provided enhanced resolving power for highly complex samples, especially in the "comprehensive" mode (LC×LC), in which the whole effluent from the first chromatographic dimension (1D) is transferred to a second chromatographic dimension (2D). As far as operation mode is concerned, "continuous on-line" techniques bring in additional advantages, including no need for flow interruption, no increase in overall analysis time, and full automation of two-dimensional LC; for example, involving off-line transfer between the two dimensions, or the "stop-flow" techniques (1–5).
The coupling of LC×LC separation to mass spectrometry (MS) detection generates the most powerful analytical tool for nonvolatile analytes. Such a combination offers a number of analytical advantages, and may help to overcome some limitations of the two techniques, when considered individually. With respect to conventional LC–MS, the combination of two LC separations enhances physical separation of the components, reducing undesirable matrix effects arising from co-elutions. Maximizing the resolution is in fact beneficial for subsequent MS detection, in terms of sensitivity and dynamic range, since it alleviates ion suppression effects resulting from insufficient separation, which may cause high abundant species to obscure the detection of less abundant ones. Unlike the UV detector, MS systems can also be used with non-absorbing analytes, and can be operated in the full-scan mode (TIC) or, more specifically, in tandem MS (MS–MS) experiments or in the selected ion monitoring (SIM) mode. Constant neutral loss or precursor ion scanning techniques help to distinguish the ions of interest from unspecific matrix components by monitoring only those m/z values that originate from a characteristic fragmentation pattern. The so-called multiple reaction monitoring (MRM) mode enhances selectivity and lowers detection limits, therefore reducing sample consumption; in addition, the MRM approach can also decrease analysis times by reducing the need for clean-up procedures, which are often mandatory, prior to the analysis of complex samples, such as many foodstuffs (6).Among these complex samples, carotenoids represent a challenging analysis task for a number of reasons. These include high variability in the chemical structures, isomerization, poor stability, and the lack of commercially available standards for reliable identification and quantification in real samples. Commonly found in plants, algae, fungi, and bacteria, carotenoids consist of a C40-tetraterpenoid structure with a symmetrical skeleton (7), and are usually divided into two groups: Hydrocarbon carotenoids, generally known as carotenes (such as β-carotene, lycopene), and oxygenated carotenoids, known as xanthophylls (for example, lutein, β-cryptoxanthin) (8). These compounds can be found in nature in their free form, or in a more stable fatty acid esterified form. The study of esterified carotenoids in natural sources is rather limited, especially because of the high degree of complexity; a saponification step is instead often used prior to LC analysis. However, such a strategy does present some drawbacks as during the saponification procedure, strong conditions are used and, as a consequence, carotenoid loss, as well as isomerization, can occur (9).
As far as separation is concerned, reversed-phase LC with both C18 and C30 stationary phases has been extensively used to achieve the separation of molecules differing in hydrophobicity within a given structural class (10). On the other hand, normal-phase LC on silica-based columns is largely used for carotenoid class separation, according to different polarity (with retention increasing from hydrocarbons to xanthophylls). A major limitation of this approach is diminished resolving power when separating carotenoid classes lying at the two extremes of polarity scale (hydrocarbons and xanthophylls ) from the rest of the matrix.
Furthermore, the true potential of coupling different C18 columns (12) and two-dimensional LC approaches (13,14) has been investigated to increase the separation power and thus resolution and efficiency for the analysis of carotenoids in extremely complex natural samples.
Identification of carotenoids in foodstuffs is generally accomplished by the complementary information provided by LC retention times, photodiode array (PDA), and MS data. Although commonly used for carotenoid identification, PDA detection nevertheless fails in the case of analytes that exhibit similar — or even identical — spectra. On the other hand, MS was excellent for the analysis of these substances, allowing structure elucidation on the basis of both molecular mass and fragmentation pattern. Several ionization methods have been reported for MS analysis of carotenoids including: Electron impact (EI), fast atom bombardment (FAB), matrix-assisted laser desorption–ionization (MALDI), electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and, more recently, atmospheric-pressure photoionization (APPI) and atmospheric pressure solids analysis probe (ASAP) (15). Sometimes, LC–MS–MS or MSn can be advantageously applied to carotenoid analysis through the use of specific transitions and daughter ions for the identification of analytes via precursor ion selection, eventually allowing carotenoids of equal molecular masses but different fragmentation patterns to be discriminated between (16).
This article describes a novel LC×LC–PDA–MS–MS system capable of extremely high-resolving power, as well as targeted and untargeted analysis, simultaneously. The system was successfully used for the characterization of native carotenoids in red chili pepper, in addition to quantification of beta-carotene at sub-ppm level.