Elsevier

Journal of Chromatography A

Volume 1316, 5 November 2013, Pages 78-91
Journal of Chromatography A

A new ultrahigh performance liquid chromatography with diode array detection coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry analytical strategy for fast analysis and improved characterization of phenolic compounds in apple products

https://doi.org/10.1016/j.chroma.2013.09.075Get rights and content

Highlights

  • Development of a rapid and selective UPLC-DAD–ESI-Q-ToF analytical method.

  • A breakthrough in the analysis of phenolic compounds.

  • MSE technology applied for the first time in flavonoids.

  • 52 phenolics identified in apple product extracts in 18 min.

  • 2 phloretin and 3 isorhamnetin new derivates detected in apple.

Abstract

A new, rapid, selective and sensitive ultrahigh performance liquid chromatography with diode array detection coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (UHPLC-DAD–ESI-Q-ToF-MS) strategy using automatic and simultaneous acquisition of exact mass at high and low collision energy, MSE, has been developed to obtain polyphenolic profile of apples, apple pomace and apple juice from Asturian cider apples in a single run injection of 22 min. MSE spectral data acquisition overcomes chromatographic co-elution problems, performing simultaneous collection of precursor ions as well as other ions produced as a result of their fragmentation, which allows resolving complex spectra from mixtures of precursor ions in an unsupervised way and eases their interpretation. Using this technique, 52 phenolic compounds of five different classes were readily characterized in these apple extracts in both positive and negative ionization modes. The spectral data for phenolic compounds obtained using this acquisition mode are comparable to those obtained by conventional LC–MS/MS as exemplified in this work. Among the 52 phenolic compounds identified in this work, 2 dihydrochalcones and 3 flavonols have been tentatively identified for the first time in apple products. Moreover, 2 flavanols, 4 dihydrochalcones, 9 hydroxycinnamic acids and 4 flavonols had not been previously reported in apple by ToF analysis to our knowledge.

Introduction

Apples contain a variety of phenolic compounds that can be classified into several sub-classes with procyanidins being the most abundant class followed by hydroxycinnamic acids, dihydrochalcones, flavonols, anthocyanins, and flavan-3-ols [1], [2], [3], [4], [5]. In cider apple cultivars, this kind of compounds is responsible for the color and the balance of bitterness to astringency, which defines the “overall mouthfeel” of ciders [6]. Furthermore, they are implicated in the alcoholic and malolactic fermentations as metabolites, providing cider aroma, and as inhibitors of the microbiological growth, controlling fermentation rates and cider spoilage [7]. However, since apple peel has a higher content of polyphenols than apple pulp, cider and apple juices only keep 3–10% of the antioxidant activity of the whole fruit [4], [5], [8]. In view of the fact that most of the phenolic compounds remain in the apple pomace, a great interest is focused on this by-product as a potential source of bioactive phenolics [4], [9], [10].

Apart from their beneficial properties in food, which have conferred on them a relevant role as nutraceuticals (e.g. Applephenon®, Apple Synergic®) [11], polyphenols are chemotaxonomic markers due to their specifity and ubiquity, and they have proven to be chemical markers for food authentication demanded by food producers, consumers and regulatory bodies [12], [13], [14].

For the investigation of structure–activity relationships and food quality control, it is important to have access to rapid and reliable methods for the analysis and identification of these natural phenolic compounds in all their many forms. Among the methods used for the determination of polyphenols, the most powerful tool are based on high performance liquid chromatography (HPLC) coupled with diode array detection and mass spectrometry (HPLC–PDA–MSn) is a powerful tool for structure elucidation [15]. Apple pomace extracts were analyzed using this technique, with a total of 30 different phenolic compounds identified, some of which tentatively [1]. Compared with conventional high performance liquid chromatography (HPLC), ultrahigh performance liquid chromatography (UHPLC) achieves rapid analysis and can obtain better peak separation than is usually possible with standard LC methods [16]. This technique has been already applied to the quantification of 39 polyphenols in apples [17]. With the emergence of UHPLC-coupled ToF or Q-ToF instruments, it is possible to develop a highly attractive analytical method with very high resolution and accurate mass measurements of the precursor and fragment ions [18].

Normally, obtaining this information requires several experiments to be performed. Firstly, low collision energy (CE) MS experiments to obtain molecular ions and secondly, higher collision energy MS/MS experiments to obtain the fragment ion data, which is often essential for structure elucidation. Clearly, such MS/MS information can be acquired in a variety of different ways, the simplest method being the selection of a precursor ion (using Q1 in a Q-ToF instrument) and then fragmenting in the collision cell and monitoring fragment ions using a scanning mode in ToF. In this approach, it is necessary to have prior knowledge of the ions of interest, usually obtained via a previous full-scan MS run. Therefore, these approaches are less efficient than it would be desired for the rapid analysis of complex multicomponent samples such as plant extracts.

Technological advances have provided superior hybrid mass spectrometers such as the Q-ToF over the more conventional tandem quadrupole or linear ion trap in terms of enhanced mass accuracy or spectral resolution and an outstanding sensitivity in full scan mode. A clear example is the ability of the Q-ToF to conduct many precursor and neutral loss acquisitions over a single experimental run using an instrument acquisition mode called MSE [19]. During a MSE acquisition, two alternative scan functions are used for data acquisition. Namely, the first scan function acquires a wide mass range at low collision energy and collects information about the unfragmented ions in the ionization source. The second scan function acquires data over the same mass range but using higher collision energy (fixed or ramped). This scan function allows the selective collection of fragmented ion data based on all ions acquired in the first scan, which is equivalent to a simultaneous batch of selective tandem mass spectrometric (MS/MS) product ions scans from all precursor ions coming from ion source.

In this paper, a new, rapid, selective and sensitive UHPLC-DAD–ESI-Q-ToF-MSE methodology to separate and identify simultaneously the complete phenolic of apples and apple products (apple pomace and apple juice) in a single run injection has been developed. The method applicability was demonstrated for analysis of apple and apple juice of three different cultivars and an apple pomace. Structural assignment for 52 phenolic compounds is reported. So far, this acquisition MS method, MSE, has been used for the structural elucidation of phenolic compounds in complex plant extracts in this work for the first time, providing excellent MS efficiencies.

Section snippets

Apples, apple pomace and apple juice

Cider apples of Meana and Florina varieties were harvested at the stage of maturity during 2012 season in Villaviciosa (Asturias, Spain). Besides, other 3 samples from a progeny of ‘Meana’ × ‘Florina’ from a genetic improvement program were selected. Apples were cut, cores were removed and a formic acid solution (3%) was sprayed on them before they were frozen with liquid nitrogen. They were kept at −20 °C until their freeze-drying (FreeZone Plus, Labconco, Kansas City, MO, USA). Apple pomace

Results and discussion

The UHPLC conditions (mobile phase, flow rate, injection volume, column temperature,…) were checked taking into account the chromatographic behavior of the analytes present in the samples under study, analysis time, sensitivity, peak shape, etc. Different gradients and solvents (methanol and acetonitrile, and aqueous formic and acetic acid of different pH values) were tested, reaching the conclusion that the best mobile phase consists of methanol–acetic acid (99.9:0.1, v/v) as an organic

Conclusions

A new, rapid, selective and sensitive UHPLC-DAD–ESI-Q-ToF-MS strategy using MSE scan mode was developed to obtain phenolic profiles in complex apple product extracts.

The MSE spectra together with complementary information obtained from retention times and UV–vis spectra allowed identifying 52 phenolic compounds in apple extracts. Although some of the phenolic compounds have been previously described in literature, others have been tentatively identified for the first time using a Q-ToF

Acknowledgments

This research was supported by Gobierno Vasco/Eusko Jaularitza (project number IT413-10), Ministerio de Ciencia e Innovación (project number CTQ2009-08390) and Plan Nacional de I+D+I 2008–2011 (project number RTA2012-00118-C03-03). Maria Ramirez-Ambrosi, Sergio Garmon-Lobato and Maria Viloria-Bernal thank Gobierno Vasco/Eusko Jaurlaritza and Universidad del País Vasco/Euskal Herriko Unibertsitatea for their Ph.D. grants. Technical and human support provided by SGIker (UPV/EHU, MICINN, GV/EJ,

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