Elsevier

Talanta

Volume 74, Issue 1, 15 November 2007, Pages 91-103
Talanta

Screening of volatile composition from Portuguese multifloral honeys using headspace solid-phase microextraction-gas chromatography–quadrupole mass spectrometry

https://doi.org/10.1016/j.talanta.2007.05.037Get rights and content

Abstract

The volatile composition from four types of multifloral Portuguese (produced in Madeira Island) honeys was investigated by a suitable analytical procedure based on dynamic headspace solid-phase microextraction (HS-SPME) followed by thermal desorption gas chromatography–quadrupole mass spectrometry detection (GC–qMS). The performance of five commercially available SPME fibres: 100 μm polydimethylsiloxane, PDMS; 85 μm polyacrylate, PA; 50/30 μm divinylbenzene/carboxen on polydimethylsiloxane, DVB/CAR/PDMS (StableFlex); 75 μm carboxen/polydimethylsiloxane, CAR/PDMS, and 65 μm carbowax/divinylbenzene, CW/DVB; were evaluated and compared. The highest amounts of extract, in terms of the maximum signal obtained for the total volatile composition, were obtained with a DVB/CAR/PDMS coating fibre at 60 °C during an extraction time of 40 min with a constant stirring at 750 rpm, after saturating the sample with NaCl (30%). Using this methodology more than one hundred volatile compounds, belonging to different biosynthetic pathways were identified, including monoterpenols, C13-norisoprenoids, sesquiterpenes, higher alcohols, ethyl esters and fatty acids. The main components of the HS-SPME samples of honey were in average ethanol, hotrienol, benzeneacetaldehyde, furfural, trans-linalool oxide and 1,3-dihydroxy-2-propanone.

Introduction

Honey is a natural product produced by Apis mellifera bees from the nectar of plants and has for long been an excellent nutritional option for many generations due to its health benefits (one of the traditional sources for treatment of flue and common cold in the region) [1], has been reported to be effective in gastrointestinal disorders, in healing of wounds and burns, as an anti-microbial agent [2]. The healing effect of honey is due to the enzyme glucose oxidase, this enzyme is virtually inactive in full-density honey but becomes active in diluted honey producing hydrogen peroxide and gluconic acid from glucose. In addition, many natural antibacterial compounds have been identified from different types of honey [2]. Honey, as a source of antioxidants has been proven to be effective against deterioative oxidation reaction in food [3]. The antibacterial activity of honey is attributed both to physical factors, acidity and osmolarity and chemical factors, hydrogen peroxide, volatiles, beeswax, nectar, pollen and propolis [2], [3].

Honey includes over 400 different chemical compounds, more than 95% mainly formed by sugars and water, whereas proteins, vitamins (mainly vitamin B6, thiamin, niacin, riboflavin, and pantothenic acid), essential minerals (including calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), pigments, flavours, free amino acids and volatile compounds constitute minor components [4]. The sugars present in honey are mainly fructose (about 38.5%) and glucose (about 31.0%). The remaining carbohydrates include maltose, sucrose, and other complex carbohydrates.

The chemical composition of honey is highly dependent to the nectar source and the botanical origin of the nectar foraged by bees [1]. Aroma compounds are present in honey at very low concentrations as complex mixtures of volatile components of different chemical families belonging, in general, to monoterpenes, C13-norisoprenoids, sesquiterpenoids, benzene derivatives [5] and in lowest content, higher alcohols, esters, fatty acids, ketones, terpenes and aldehydes. Some of these substances are present in honey collected by bees, and have been described as characteristics of the floral source (could be related to plant characteristics), and other compounds, like some alcohols, branched aldehydes, and furfural derivatives, may be related to the microbial purity of processing and storage conditions of honey [6]. The quantitative analysis of volatile compounds present in such samples is extremely demanding due: (1) to complex chemical composition of the volatile fraction and (2) the fact of individual volatile compounds can be present in a wide range of concentration. Honey volatile fractions have been used as quality markers for the authenticity of the floral origin [7], [8]. It also prevents overpayment and helps to identify frauds [7].

Traditional analytical methods employing organic solvents such as liquid–liquid extraction [9], simultaneous distillation–extraction (SDE) [10], supercritical fluid extraction (SFE) [11], solid-phase extraction (SPE) [12] and ultrasound extraction [13] were commonly used. These are hazardous since requires large amounts of toxic and expensive solvents, are labour-intensive and time-consuming and requires the pre-concentration of the extract. Each procedure of the sample preparation is subject to inconveniences, but offers specific advantages under determined circumstances. Nowadays, alternative to these classical methods that may overcome their disadvantages, more easier and selective, are used such as solid-phase microextraction (SPME), developed by Pawliszyn and coworker [14], [15] in the early 1990s and more recently stir bar sorptive extraction (SBSE) developed in the late 1990s by Baltussen et al. [16]. This technique uses a TwisterTM, a glass stir bar onto which is bonded a sorptive phase, often polydimethylsiloxane (PDMS), in quantities far in excess of those found on SPME fibres [17]. SPME is an equilibrium technique that requires a previous optimisation of the extraction parameters that can affect extraction efficiencies, in order to obtain high recoveries of volatiles. SPME sampling can be performed in three basic modes: direct extraction (the analytes were transported directly from matrix to the extracting phase), headspace extraction (the analytes are extracted from the gas phase equilibrated with the sample) and extraction with membrane protection (the fibre is separated from the sample with a selective membrane). The selection of the sampling mode is dependent of the nature of the compounds to be analysed and the sample type. Bearing in mind that one of the goals of this study was to screen volatile compounds from honeys, the headspace sampling mode was the most appropriate. The headspace SPME process protects the fibre from adverse effects caused by non-volatile compounds present in the sample matrix namely sugars, and allows modifications, as for example: pH, with no effect in the fibre. Moreover, the equilibration times for volatile compounds are shorter for headspace SPME extraction than for direct extraction under the same conditions.

Since the first SPME fibres becomes commercially available, it has been more and more used and the fields of application have been continuously growing, including a wide range of food analysis, namely the volatile composition of wines [18], [19], [20], [21], [22], beers [23], whiskeys [24], [25], [26], several kinds of fruits [27], [28], [29], [30], [31] and honeys [32], [33], [34], [35], [36], with nowadays about 3000 research papers published. The technique gained growing acceptance and increasing use in routine laboratories and industrial applications. This method shows clear advantages compared with traditional techniques, eliminates the use of (toxic) organic solvents, allows the quantification of a large number of molecules, no or little manipulation/preparation of samples, substantially shortness the time of analysis and moreover are simple and faster techniques, and covers a wide range of sampling techniques, including field, in situ and air sampling. Generally accepted disadvantages are relatively lot-to-lot variations, sensitivity against organic solvents and the limited range of stationary phases which are commercially available.

In this study, headspace SPME combined with GC–qMS, was developed and applied to evaluating the volatile composition profile of different multifloral honey samples (H1–H4). A preliminary screening of fibre of various polarities was carried out in order to select the best coating for the matrix. Comparison between the performance of the five sorbent materials is given. To confirm the applicability of the SPME, comparative study on the characteristic GC–qMS volatile honey profiles were performed. The possibility of differentiation from the investigated honeys was evaluated.

Section snippets

Chemicals and materials

All reagents used were analytical quality and all solvents were HPLC grade. Sodium chloride (99.5%) used to obtain the adequate ionic strength (decrease the solubility of the aroma molecules which then partition more readily into the headspace improving the adsorption of analytes in SPME analysis), was supplied by Merck (Darmstadt, Germany). Water was purified through a Milli-Q purification system (Millipore). The C8–C20 n-alkanes series, and the chemical standard used as internal standard,

Results and discussion

Sixteen honey samples from different multifloral origins were analysed with the objective to identify and compare their volatile compounds profiles. Differences in the total ion current (TIC) chromatographic profiles were observed when comparing the studied honeys. TIC chromatograms from H1–H4 samples were compared in terms of total areas of the volatile compounds and number of compounds. The identified compounds were organized in different groups according to their chemical structure. This was

Conclusions

Headspace solid-phase microextraction sampling followed by GC–qMS analysis provides an appropriate and selective way to characterize the volatile compounds in honey. Is a simple procedure of extraction with a great capacity of concentration and combines extraction to a rapid, sensitive and solvent-free method suitable for determination of volatile and semivolatile compounds. The chromatographic profiles obtained after extraction with PDMS, PA, CAR/PDMS, CW/DVB and DVB/CAR/PDMS coatings suggest

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