Original research article
A rapid and simple method for the analysis of bioactive compounds in olive oil refining by-products by liquid chromatography with ultraviolet and mass spectrometry detection

https://doi.org/10.1016/j.jfca.2018.02.013Get rights and content

Highlights

Abstract

In this study, a simple and rapid reversed phase high performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed and validated for the simultaneous analysis of erythrodiol, tocopherols, phytosterols and squalene in olive oil and olive pomace oil chemical and physical refining by-products. A sample pretreatment without saponification, solely based on n-hexane extraction, allowed direct analysis of the target compounds from the by-product samples. α-tocopherol, β-sitosterol and squalene were quantified by external calibration. The highest concentrations of these compounds were found in olive oil chemical refining by products (i.e. 0.19%, 4.1% and 19.1% m/m, respectively). Afterwards, the method was adapted to high performance liquid chromatography with electrospray ionisation mass spectrometry detection (HPLC–MS). Positive ESI (+ESI) allowed better sensitivity than negative ESI (−ESI), and detection of all the target compounds except squalene. In addition to α-tocopherol and β-sitosterol, the presence of erythrodiol in the complex by-product samples could be confirmed due to the excellent selectivity of MS detection.

Introduction

The nutritional and health benefits of consuming olive oil are well known worldwide (Aparicio-Soto et al., 2016; Buckland and Gonzalez, 2015; Parkinson and Cicerale, 2016). Olive oil production is concentrated in the Mediterranean area, and Spain, Italy, Tunisia, Morocco and Greece are the most important producers and exporters (International Olive Council, 2016). The EU legislation (regulation (EEC) 2568/91 (European Union, 1991) amended by regulations (EC) 1989/2003 (European Union, 2003), (EU) 299/1213 (European Union, 2013a) and (EU) 1348/2013 (European Union, 2013b)) classifies olive oil in eight categories (i.e. extra virgin olive oil, virgin olive oil, lampante olive oil, olive oil composed of refined and virgin olive oils, crude olive pomace oil, refined olive pomace oil and olive pomace oil) depending on several chemical and organoleptic characteristics determined by different methods of analysis (European Union, 2013a, European Union, 2013b, European Union, 2003, European Union, 1991). In accordance to these categories, as with other vegetable oils, non-edible lampante and olive pomace oils must be refined to achieve the minimum requirements to be marketed for human consumption (Antonopoulos et al., 2006; Bondioli, 2006). The refining process can be chemical refining, where chemicals such as NaOH or KOH are used to reduce the acidity caused by free fatty acids, or physical refining, where this acidity is eliminated by distillation (Antonopoulos et al., 2006). During the refining process, several by-products are generated, which may contain substantial amounts of compounds with interesting biological properties and a great economic value (Bondioli, 2006; Fernández-Bolaños et al., 2006). Recycling, reuse and value enhancement of these refining by-products and other oil processing waste is regarded by the oil industry as an opportunity to decrease waste disposal, while improving the sustainability of the production process and the economic revenues.

Several investigations have been focused on purification or bioconversion of different compounds found in olive oil refining by-products (Fernández-Bolaños et al., 2006). The terpene hydrocarbon squalene (Fig. 1), which is also found in human sebum and shark liver oil, is the most interesting component in olive oil refining by-products and can be found in high concentration (Aguado Ramos et al., 2009; Gunawan et al., 2008; Gutiérrez et al., 2014; Margnat et al., 2011). Squalene is a precursor of cholesterol biosynthesis in vivo, which regulates the normal values of dehydroepiandrosterone hormone (DHEA). DHEA improves neurological performance, memory, concentration and reduces fat accumulation in the abdominal area. Squalene has also important applications in cosmetic and medical industry because of the antioxidant, antiviral, antifungal, antibacterial and antitumor properties. Olive oil refining by-products also contain lower concentrations of erythrodiol, tocopherols and phytosterols (plant sterols) (Fig. 1), which have also important applications. Erythrodiol is a pentacyclic triterpene alcohol with anti-inflammatory, immunomodulatory and antioxidant properties (Fernández-Bolaños et al., 2006). Tocopherols such as α-tocopherol, are well known for their antioxidant properties (Czuppon et al., 2006; Eloy, 2004; Margnat et al., 2011; Raja Rajan and Gopala Krishna, 2014). Finally, phytosterols have a similar structure to cholesterol, and their consumption reduces cholesterol blood levels (Czuppon et al., 2006; Eloy, 2004; Margnat et al., 2011; Raja Rajan and Gopala Krishna, 2014).

Several chromatographic methods have been developed to analyse erythrodiol, tocopherols, phytosterols and squalene in vegetable oil refining by-products, as a way to assess the by-product economic value, and hence the feasibility of purification (Naz et al., 2014; Ruiz-Méndez and Dobarganes, 2007; Verleyen et al., 2001; Zarrouk et al., 2009). Because of their significant differences in polarity and solubility, it is not easy to find a unique method for their simultaneous analysis. Most methods described in the literature to analyse vegetable oil refining by-products use gas chromatography (GC) and a laborious sample preparation procedure including alkaline hydrolysis (saponification), followed by extraction from the unsaponifiable material and chemical derivatisation (Bondioli, 2006). As an alternative, Verleyen et al. described a GC method for the analysis of the complex deodoriser distillates from vegetable oil refining without sample saponification (Verleyen et al., 2001). Similarly, Ruiz-Mendez et al., described a method based on adsorption chromatography to separate into a polar and a nonpolar fraction the deodoriser distillates before size exclusion chromatography and GC (Ruiz-Méndez and Dobarganes, 2007). On the other hand, the use of mass spectrometry (MS) detection is an excellent option to avoid chemical derivatisation in GC. Naz et al. described a simple and rapid GC–MS method without derivatisation for the simultaneous determination of different compounds, including squalene, tocopherols and phytosterols, in the non saponifiable fraction of a deodorizer distillate from canola and palm oils (Naz et al., 2014, Naz et al., 2012). As an alternative to GC, the use of high performance liquid chromatography (HPLC) for the analysis of different components in olive oils, including squalene, sterols and tocopherols, have been described with ultraviolet (UV) (Cunha et al., 2006; Manai-Djebali et al., 2012; Rocco and Fanali, 2009; Salvo et al., 2017) and MS detection (Bartosińska et al., 2016; Gu et al., 2016a; Jiang et al., 2015; Lanina et al., 2007; Lerma-García et al., 2009; Rocco and Fanali, 2009; Zarrouk et al., 2009), with atmospheric pressure chemical ionisation (APCI) or electrospray ionisation (ESI). However, application of HPLC in oil refining by-product analysis is very rare (Yuan et al., 2015), despite the benefits of avoiding chemical derivatisation prior to the separation and the versatility of the separations. Recently, Yuan et al. reported an HPLC-UV method for the rapid analysis of squalene, tocopherols and phytosterols, in soybean, cottonseed, tea seed and rice bran oil deodorizer distillates after sample saponification (Yuan et al., 2015). To the best of our knowledge, there are no publications on the analysis of olive oil refining by-products by HPLC.

In this paper, an HPLC-UV method was developed and validated for the simple, rapid and simultaneous analysis of erythrodiol, tocopherols, phytosterols and squalene in olive oil and olive pomace oil chemical and physical refining by-products without saponification. Afterwards, the method was adapted to HPLC–MS to confirm unequivocally the presence of the analysed compounds.

Section snippets

Chemicals and reagents

Squalene (>98% purity), (+)-α-tocopherol (>98% purity), β-sitosterol (>95% purity) and erythrodiol (>97% purity) were purchased from Cymit Química (Barcelona, Spain). (+)-γ-tocopherol and (+)-δ-tocopherol (>95% purity) were acquired from Supelco (Bellefonte, Pennsylvania, United States). Stigmasterol (>95% purity) was provided by Tecknokroma (Sant Cugat del Vallès, Spain). Methanol (>99.9% purity, UHPLC Supergradient and LC–MS grade) and n-hexane (>95% purity, HPLC-grade) were purchased from

HPLC-UV analysis of standards

The chromatographic method was optimised by analysing a standard mixture of 100 mg L−1 erythrodiol; 20 mg L−1 δ-tocopherol, γ-tocopherol and α-tocopherol; 100 mg L−1 stigmasterol and β-sitosterol; and 10 mg L−1 squalene (Fig. 1), which were expected to be at high concentration in the by-products samples (Naz et al., 2014; Ruiz-Méndez and Dobarganes, 2007; Verleyen et al., 2001). Using a Zorbax SB-C18 column (2.1 ID x 150 mm LT, 5 μm particle size, Agilent Technologies), a very simple isocratic

Conclusions

A simple HPLC-UV method was successfully developed and validated for the simultaneous analysis in less than 15 min of erythrodiol, squalene, tocopherols and phytosterols in olive oil and olive pomace oil chemical and physical refining by-products without saponification. The method was sensitive and reproducible, and α-tocopherol, β-sitosterol and squalene were quantified in the different by-products, obtaining a higher concentration in samples from olive oil chemical refining. Accuracy of the

Acknowledgements

The authors thank Coreysa S.A. for financial aid and for kindly supplying the olive oil by-product samples. This study was in part supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTQ2014-56777-R) and the Cathedra UB Rector Francisco Buscarons Ubeda (Forensic Chemistry and Chemical Engineering).

The authors have declared no conflict of interest.

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