Fast analysis of polyphenols and alkaloids in cocoa-based products by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap-MS/MS)

Highlights

• Main polyphenols and alkaloids in 80 cocoa-based samples were determined

• UHPLC-Orbitrap HRMS for putative identification of cocoa varieties was developed

• Method performance fulfilled the validation criteria established in legislation

• The developed method could help to identify suspicious adulterations in cocoa

• Promising results here obtained require an extended study to confirm preliminary data

Abstract

The cocoa varieties Criollo, Forastero and Trinitario, which have different organoleptic properties, quite often are mixed together in cocoa-based products. The objective of this work was to develop a rapid method for the simultaneous determination of polyphenols (n = 35) and alkaloids (n = 2) in cocoa-based products by using the high throughput advantages provided by the Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (HRMS). The proposed procedure was optimized and validated in terms of selectivity and specificity (mass accuracy <5 ppm), sensitivity (instrumental limit of detection from 15 to 30 fg), linearity (r² > 0.990), accuracy (recovery range from 89.1 to 112.4%) and precision (relative standard deviation <10%). The method was applied to 80 cocoa-based samples. Alkaloids represented on average the 57.7% of assayed compounds. Flavanols and procyanidins were the most abundant polyphenols being quantified in a range between 261 and 668 mg/kg and from 127 to 405 mg/g, respectively. Mass equivalents but structural isomers bioflavonoids were characterized by using the HRMS-Orbitrap methodology here developed.

Introduction

In recent decades, nutrition research has focused on the investigation of bioactive dietary compounds, widely found in many plant-based foods and beverages, in order to elucidate their beneficial properties to human health. Cocoa (Theobroma cacao L.) and chocolate products appear to be one of the most promising foods due to their high polyphenol and alkaloid contents (Capriotti, Cavaliere, Foglia, Piovesana, & Ventura, 2015; La Barbera, Capriotti, Cavaliere, Montone, et al., 2017). The cocoa polyphenols have an important antioxidative effect as previously reported in literature (Andujar, Recio, Giner, & Rios, 2012). Cocoa bioactive compounds have also been related to blood pressure lowering, cancer prevention, and inflammatory processes modulation in the human body, and even to significant activity in skin health maintenance (Camps-Bossacoma et al., 2017; Ludovici et al., 2017; Marsh et al., 2017; Żyżelewicz et al., 2016). In addition, help in maintain endothelium-dependent vasodilation contributing to healthy blood flow have also been associated to these phenolic compounds as claimed effect accepted by EFSA (EFSA, 2012). Based on that, some studies have reported that eating a moderate amount of chocolate, mainly dark chocolate, could induce beneficial health effects (Kerimi & Williamson, 2015). Cocoa-based foodstuffs are a widely consumed all around the world. The latest available consumption data reported by FAO showed an annual per capita consumption of 2.1 kg in Europe, being the population from Southern Europe the largest consumers with a consumption per capita of 2.27 kg/person (FAOSTAT, 2013).

Traditionally, the trade has considered two main genetic groups, Criollo and Forastero, based on morphological traits and geographical origins. A third group, Trinitario, has been recognized and consists of Criollo and Forastero hybrids. Although a new nomenclature based on genetic clusters was proposed by Motamayor et al. (2008), the term Forastero is still used by the trade to refer to mainstream cocoas as reported the Federation of Cocoa Commerce and the European Cocoa Association (CAOBISCO/ECA/FCC, 2015). The identification of compounds responsible of bitterness and astringency in different cocoa varieties has been widely studied to assess both consumer taste preferences and to explorer the consumer healthy perception about the different types of chocolate (Afoakwa, Paterson, Fowler, & Ryan, 2008; Gámbaro & Ellis, 2012; Hofmann, 2008; Li et al., 2012; Loureiro et al., 2017).

The cocoa industry needs a sustainable and consistent supply of cocoa beans with the quality attributes to meet the diverse requirements. Among the current quality parameters for cocoa in quality control, the cocoa must: (i) be properly fermented and dried; (ii) be free from any foreign odours; (iii) comply with limits in contents of slaty, flat, clusters, broken, mouldy, insect-damage, foreign matter and germinated beans; (iv) conform to the required moisture level; and (v) comply with bean count requirements (BISCO/ECA/FCC, 2015). Nonetheless, the concentrations of some bioactive compounds, such as polyphenols, can be reduced markedly during post-harvest cocoa beans. To minimize that fact, factors affecting quality of cocoa beans from post-harvest to the factory gate are taken into account throughout standardized processing treatments by industries to produce products that are wholesome and comply with European legislation and other international food safety standards.

The analysis of bioactive compounds in food samples is complex because of the wide range of compounds which differ in polarity and size. In the literature, many solvent types are used to extract antioxidant compounds from various plant materials. The most widely used solvents for extracting phenolic substances are methanol, acetone, and their water mixtures, acidified or not. In a study conducted by Michiels, Kevers, Pincemail, Defraigne & Dommes (2012) acetone-based mixtures showed greater extraction efficiency than the methanol-based mixtures for phenolic extraction yields from fruits and vegetables, being in agreement with the results recently reported by Złotek, Mikulska, Nagajek & Swieca (2016) who studied the effect of different solvents to extract the phenolic compounds from basil leaves. Nonetheless, Tomsone, Kruma & Galoburda (2012) reported that the best solvents for phenolic extraction from horseradish roots were ethanol and ethanol/water solutions. Similarly, Sowndhararajan and Kang, (2013) concluded that the maximum polyphenolic extraction was obtained in the methanol extract of Bauhinia vahlii followed by acetone, hot water and chloroform extracts. Therefore, it was highlighted by Złotek, Mikulska, Nagajek, and Świeca (2016) that selection of the most efficient solvent for phenolic compounds extraction must depend on the used food matrices, as also discussed in other studies (Luthria, 2008; Michiels, Kevers, Pincemail, Defraigne, & Dommes, 2012).

Regarding the analysis of phenolic and alkaloid compounds in cocoa samples, several extraction procedures followed by high performance liquid chromatography (HPLC) with mass spectrometry (MS) (Kelm, Johnson, Robbins, Hammerstone & Schmitz, 2006) and tandem MS (MS/MS) (Ortega et al., 2010; Sánchez-Rabaneda et al., 2003) have been proposed in scientific literature, but all of them with considerable chromatography run time. In the last decade, there have been improvements in the LC technique with the development of ultra-high performance LC (UHPLC) using sub-2 μm particle packed columns. This leads to a shorter analysis time, higher peak efficiency and higher resolution. Another advantage is that UHPLC methods can be considered more cost effective because they typically consume around 80% less organic solvents than conventional HPLC methods. The reversed-phase UHPLC–MS/MS technique has been applied by Cooper et al. (2007) to quantify the major polyphenols in chocolate. Today, UHPLC coupled to MS/MS is one of the most widely employed techniques in food analysis and the number of works focusing on the determination of polyphenols is growing up as recently reviewed Lucci, Saurina & Nunez (2017). High resolution MS (HRMS), like Orbitrap mass spectrometer, has been successfully employed to the analysis of phenolic compounds in several matrices such as fruit products and vegetables including coffee and coffee-based products (Capriotti et al., 2014; Budryn et al., 2014; Passo Tsamo et al., 2015; Lucci, Saurina, & Nunez, 2017; Zenezini et al., 2017; La Barbera, Capriotti, Cavaliere, Piovesana, et al., 2017). Among the bioactive agents of the cocoa beans the above-mentioned technique was used for the determination of biogenic amines (Oracz & Nebesny, 2014). Orbitrap-based mass spectrometers offer full scan acquisition mode, without limitations in the number of monitored compounds, and retrospective analysis can be performed with exact mass measurement.

Therefore, the aim of this study was to develop a fast methodology to simultaneously determine the main polyphenols (n = 35) and alkaloids (n = 2) in cocoa-based products by using the high throughput advantages provided by ultra-high performance liquid chromatography and the Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer.