Elsevier

Journal of Chromatography A

Volume 1313, 25 October 2013, Pages 212-227
Journal of Chromatography A

Reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry as a powerful tool for metabolic profiling of vegetables: Lactuca sativa as an example of its application

https://doi.org/10.1016/j.chroma.2013.07.020Get rights and content

Highlights

  • RP-UHPLC-ESI-QTOF-MS has proved to be a powerful analytical tool for metabolites profiling.

  • A total of 171 compounds belonging to several structural classes were tentatively characterized in L. sativa.

  • Lettuce was qualitatively rich in phenolic compounds, especially the cultivar “baby”.

  • Novel sulfate and amino acid derivatives were tentatively proposed for the first time in lettuce.

Abstract

Lettuce (Lactuca sativa), a leafy vegetal widely consumed worldwide, fresh cut or minimally processed, constitutes a major dietary source of natural antioxidants and bioactive compounds. In this study, reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-QTOF-MS) was applied for the comprehensive profiling of polar and semi-polar metabolites from three lettuce cultivars (baby, romaine, and iceberg). The UHPLC systems allowed the use of a small-particle-size C18 column (1.8 μm), with very fine resolution for the separation of up to seven isomers, and the QTOF mass analyzer enabled sensitive detection with high mass resolution and accuracy in full scan. Thus, a total of 171 compounds were tentatively identified by matching their accurate mass signals and suggested molecular formula with those previously reported in family Asteraceae. Afterwards, their structures were also corroborated by the MS/MS data provided by the QTOF analyzer. Well-known amino acids, organic acids, sesquiterpene lactones, phenolic acids and flavonoids were characterized, e.g. lactucin, lactucopicrin, caftaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, isochlorogenic acid A, luteolin, and quercetin glycosides. For this plant species, this is the first available report of several isomeric forms of the latter polyphenols and other types of components such as nucleosides, peptides, and tryptophan-derived alkaloids. Remarkably, 10 novel structures formed by the conjugation of known amino acids and sesquiterpene lactones were also proposed. Thus, the methodology applied is a useful option to develop an exhaustive metabolic profiling of plants that helps to explain their potential biological activities and folk uses.

Introduction

Diets containing high proportions of fruit and vegetables have been advocated as one of the best practices to reduce the incidence of chronic disease in the modern world. These beneficial effects have been partially attributed to the increased consumption of phenolic compounds and other bioactive phytochemicals [1]. Although a great number of studies have focused on discovering and characterizing these metabolites, a large percentage still remain unknown and need to be identified before their relation to health can be fully understood [2]. For this, different pretreatment procedures and analysis techniques have been developed and recently reviewed [3]. Among the analytical methods, liquid chromatography (LC) coupled with mass spectrometry (MS) has become a useful tool in the metabolic profiling of plants extracts. For improved speed, resolution, and sensitivity, HPLC methods can be successfully transferred to UHPLC, which operate with smaller particle sizes (<2 μm) of the stationary phase and at higher pressures (up to 600 bar) [4]. In addition, MS is a powerful structural-characterization technique, for which good prior separation is crucial because of the complexity of plant extracts and the presence of large numbers of isomers [5], which could be undifferentiated by MS.

The hybrid mass analyzer quadrupole-time-of-flight (QTOF) provides excellent mass accuracy over a wide dynamic range and measurements of the true isotope pattern that elucidates the molecular formula of unknown metabolites with a high degree of reliability, and taking into account the seven golden heuristic and chemical rules for selecting elemental compositions [6]. This instrument also performs tandem MS, which is useful as a structural confirmation tool when standard compounds are not available. Furthermore, QTOF provides high selectivity by the extracted-ion chromatogram (EIC) mode when there are overlapping peaks, where spectrophotometric detection could be limited. Thus, LC coupled to highly sensitive and high-resolution MS, such as QTOF, enables the separation and detection of minor compounds that could co-elute and be underestimated vs. major ions, not being identified or even detected by older methodologies. LC coupled to QTOF-MS has been applied in targeted analysis but is suitable also for the extensive profiling of hundreds of natural plant metabolites [7], xenobiotics and their degradation products [8], [9]. Among the ionization sources, electrospray ionization (ESI), particularly in the negative mode, is a good choice for studying small molecules [4], [5]. In our previous studies, it has been demonstrated that RP-RRLC-ESI-QTOF-MS is well suited to the untargeted characterization of edible vegetables, and enabled a wide overview of the composition (amino acids, nucleosides, organic acids, and phenolics) of zucchini [10]. Following a similar strategy, 73 phenolic compounds have been characterized in cucumber [11]. Lettuce (Lactuca sativa), a leafy vegetable of the family Asteraceae (Compositae), and one of the most widely used food crops for the so-called “fourth-range” vegetables (cleaned, possibly chopped, mixed, ready to be seasoned and eaten fresh) [12]. Thus, minimally processed, it retains many naturally occurring components. Besides, lettuce is one of the major vegetable crops in the world [FAOSTAT (2010), [13] and a good source of nutrients, as well as phytochemicals [14], [15]. Among these, dietary phenolics are of the particular interest due to their potential as antioxidants, not only as natural food preservatives, but also for their role on human heath, as commented above. In this respect, antioxidant activity of lettuce has been reported both in vitro [1] and in vivo [16]. Furthermore, lettuce contains other important phytochemicals such as sesquiterpene lactones, a characteristic and diverse class from Asteraceae. This type of terpenoids together with phenolic compounds have different bioactivities and contribute to sensory properties of plants, but also, in some cases, presents allergenic potency [17]. Several techniques have been used to identify lettuce phytochemicals, e.g. LC coupled to UV and diode array detection (DAD), MS and MS/MS [18], [19], [20], [21], [22], [23], and GC–MS [24]. However, only one NMR-based approach has been applied for more comprehensive characterization of lettuce [25]. Sensitivity is perhaps the most important requirement for metabolomic profiling, and NMR is several orders of magnitude less sensitive than MS [26]. Thus, the main objective of this study is to evaluate the polar and semi-polar fraction, including primary and secondary metabolites, of three highly consumed lettuce cultivars (baby, romaine, and iceberg) by RP-UHPLC-ESI-QTOF-MS and -MS/MS. In addition, this methodology was used as starting point for structure elucidation of new molecules, based on the suggested molecular formula, structural information provided by MS/MS fragmentation, and the literature.

Section snippets

Chemicals

Acetic acid, methanol, and acetonitrile were purchased from Fluka (Sigma–Aldrich, Steinheim, Germany) and Fisher chemical (Acros Organics, Geel, Belgium), respectively. Water was purified by a Milli-Q system from Millipore (Bedford, MA, USA). All chemicals were of HPLC–MS grade, except methanol (analytical grade), and used as received.

Plant material and extraction of lettuce metabolites

Fresh lettuce (cv. baby, romaine, and iceberg), purchased from a local market, were from crops cultivated in Almeria (Spain) in the year 2011 with no prior

RP-UHPLC-ESI-QTOF-MS as a powerful tool for metabolic profiling

Preliminarily, positive and negative ionization modes were evaluated. Finally, negative ionization mode was chosen for profiling based on larger numbers of compounds found and the stronger response in that mode. Thus, the base peak chromatograms (BPC) of the extracts from baby, iceberg and romaine lettuces and the compounds characterized in negative ion mode are shown in Fig. 1. It revealed a complex mixture of polar and semi-polar metabolites, including some regions of the chromatograms that

Conclusion

This study demonstrates the power of the RP-UHPLC-ESI-QTOF-MS for “non-targeted” metabolic profiling of vegetables in one run without tedious sample pretreatments. It allowed the tentative characterization of a total of 171 compounds belonging to various structural classes: amino acids and peptides (42), organic acids (11), nucleosides (3), alkaloids (2), phenolic compounds (92), sesquiterpene lactones (17) and one iridioid. Our results indicate that genotype, at least in part, was an important

Acknowledgments

The authors are grateful to the Spanish Ministry of Economy and Competitiveness (MINECO) for the project AGL2011-29857-C03-02 and to Andalusian Regional Government for the excellence projects P09-CTS-4564, P10-FQM-6563, P11-CTS-7625 and contract No. 2661. The author I. Abu-Reidah thanks to the Ministry of Foreign Affairs and the Spanish Agency for International Cooperation for the postgraduate study funding MAEC-AECID Scholarship 2009, and M. M. Contreras thanks to the MINECO for the Juan de la

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