Original Research Article
Application of QuEChERS-EMR-Lipid-DLLME method for the determination of polycyclic aromatic hydrocarbons in smoked food of animal origin

https://doi.org/10.1016/j.jfca.2020.103420Get rights and content

Highlights

  • QuEChERS + EMR-Lipid + DLLME is the best procedure for PAH determination in smoked food.

  • The method is fast, effective, provides clean extracts and good validation results.

  • This procedure is approved to be used in the heat-treated food of animal origin with high fat content.

Abstract

The aim of this study was to develop an effective sample preparation procedure for the determination of polycyclic aromatic hydrocarbons (PAHs) in smoked fatty products of animal origin (fish, cheese and sausage). Two different approaches were tested: classical QuEChERS and procedure with the use of Enhanced Matrix Removal (EMR)-Lipid material. Two techniques of extract preconcentration: under nitrogen stream and with the use of dispersive liquid-liquid microextraction (DLLME), were also taken into consideration. All samples were analysed using gas chromatography-mass spectrometry. The results showed the optimised sample preparation procedure was composed of three steps: 1) QuEChERS extraction, 2) clean-up by EMR-Lipid material and 3) extract preconcentration by DLLME. The obtained recovery rates within the range of 50–120% were received for all compounds with relative standard deviation (RSD) values lower than 16.7%. The proposed method is fast and effective and can be successfully applied for PAHs determination in difficult matrices such as heat-treated food of animal origin with high fat content. The research also discovered the significance of the quality of the laboratory disposables. Contaminants present in plastic consumables can be transferred to the sample extract contributing to its contamination and can also lead to failure of analytical equipment.

Introduction

Smoking is one of the oldest food preserving technologies. It has been used by mankind for over 10,000 years. It is believed that man would hang his catch over the fire as a protection against canines and subsequently the preserving effect of smoke was probably discovered (Šimko, 2002, 2009). The first evidence of smoking as a technological process dates back 90,000 years to Poland where the oldest smoking house was discovered by archaeologists in a Stone Age colony located in Zwierzyniec, near Krakow (Möhler, 1978). Ever since, smoking started to be widely used not only for special organoleptic profiles of smoked products, but also for the inactivating effect of smoke (and heat) on enzymes and microorganisms (Essumang et al., 2010; Šimko, 2002). Smoking is usually used for preservation of fish and its products as well as meat and meat products. Apart from that, other foods can also be subjected to smoke treatment e.g. cheeses or even fruits (Suchanová et al., 2008; Fasano et al., 2016; Surma et al., 2018). According to Stołyhwo and Sikorski (2005) in Europe about 15 % of the total quantity of fish for human consumption is offered on the market in the form of either cold- or hot-smoked products. Currently, we suppose, the technology is mainly used to enrich the foods with its specific taste, odour, and appearance, as there is a high demand for it on the market (Šimko, 2005; Hui et al., 2001; Essumang et al., 2013). It is assumed that the technology is today applied in many forms to treat 40–60 % of meat products (Sikorski, 2004) and 15 % of fish (Stołyhwo and Sikorski, 2005).

The preservation effect is generally attributed to antioxidant and antimicrobial properties of phenolic compounds contained in smoke. The rate of deposition of different components depends on temperature, humidity, flow rate, and density of the smoke, water solubility and volatility of particular compounds, as well as shelf life, and wholesomeness of the product (Borgstrom, 2012; Stołyhwo and Sikorski, 2005). Generation of wood and charcoal smoke during curing is a typical example of incomplete combustion, and it is known that polycyclic aromatic hydrocarbons (PAHs) are generated and released, and, in consequence, due to the contact of food with smoke and high temperature of this process, PAHs are transferred to smoked food. PAHs are a large group of hydrophobic organic compounds, containing two or more aromatic rings. The compounds containing five or more aromatic rings are known as ‘heavy’ PAHs, whereas those containing less than five rings are named ‘light’ PAHs. Both kinds of PAHs are non-polar compounds, showing high lipophilic nature, although heavy PAHs are more stable and toxic than the other group (Raters and Matissek, 2014). PAHs originate mainly from environmental sources (natural and anthropogenic) and food processing (e.g. heating, drying, smoking, grilling, roasting and frying) (Singh et al., 2016). PAHs show clear evidence of mutagenicity/genotoxicity in somatic cells in experimental animals in vivo and are classified by International Agency for Research on Cancer (IARC) in both groups 2A and 2B and benzo(a)pyrene in group 1 (carcinogenic to humans) (IARC, International Agency for Research on Cancer, 2014). In European Union, as PAHs indicator in food sum of four of them (∑4 PAHs) including benzo(a)anthracene, chrysene, benzo(b)fluoranthene, and benzo(a)pyrene has been designated (European Commission, 2011a). So far, maximum levels (MLs) were established only for several groups of smoked food, including smoked fish and smoked fishery products as well as smoked meat and smoked meat products. However, for other smoked products such as traditional smoked cheeses from east and central Europe, no MLs have been set until this date.

One of the main challenges in the determination of PAHs in smoked food of animal origin is their high fat content (e.g. lipids, triglycerides and fatty acids) and the extraction of PAHs from these complex matrices is usually laborious and often not effective enough. Fat residues in analysed extracts can contribute to the deterioration of chromatographic system (especially GC) but also can suppress signal of analytes. Therefore, there is a constant need of search for effective techniques for fat removal from smoked fatty samples.

The most common approach for the determination of PAHs in fatty foods involves saponification of lipids by methanolic or ethanolic KOH or NaOH solution followed by the isolation of the PAHs by liquid-liquid extraction (LLE) with cyclohexane, hexane, dichloromethane or its mixtures. Obtained extracts are then cleaned up using gel-permeation chromatography (GPC), solid phase extraction (SPE) or adsorption chromatography with the use of silica or Florisil sorbents. For the detection and quantification of PAHs, gas chromatography with mass spectrometry (GC-MS) and high-performance liquid chromatography with fluorometric detection (HPLC-FLD) are usually used (Silva et al., 2017; Slámová et al., 2017; Urban and Lesueur, 2017; Zachara et al., 2017).

The QuEChERS (quick, easy, cheap, effective, rugged, safe) method is another concept that can be applied for the PAHs determination in fatty food samples. It is characterized by short extraction and purification times, as well as low solvent consumption. In clean-up step mainly PSA (primary secondary amine), C18 (octadecyl), and Z-Sep (zirconium dioxide-based) sorbents are used for the fat removal, but also an implementation of freezing out has been reported in the literature (Rejczak and Tuzimski, 2015; Sadowska-Rociek et al., 2016; Kim et al., 2019). However, in the case of food with higher fat content, even these modifications might be insufficient, to achieve adequate sample cleanup and in consequence matrix co-extractives can affect analyte signals and even destroy the elements of analytical equipment (Lucas and Zhao, 2015). Additionally, these sorbents can exhibit nonselective interactions with analytes providing the loss of analysed compounds (Lucas and Zhao, 2015; Rejczak and Tuzimski, 2015).

Recently, a new material “enhanced matrix removal” (EMR-Lipid) has been proposed for the fat removal from fat-rich food products. The structure of EMR-Lipid is a proprietary secret, and it does not function as a conventional sorbent, but it dissolves to saturation in sample extract solution, and its mechanism is said to involve both size exclusion and hydrophobic interactions. Long-chain hydrocarbons associated with lipids fit within the EMR-Lipid structure, where they are trapped. The EMR-Lipid complex is either precipitated out of solution or remains in the aqueous phase during the final salting-out step (Lucas and Zhao, 2015; Han et al., 2016). The manufacturer claims that EMR-Lipid selectively removes lipids from QuEChERS extracts without loss of analytes (Huang et al., 2019).

Depending on the final determination method, the low levels of PAHs sometimes require application of an extract preconcentration step, such as e.g. evaporation in a stream of nitrogen and dissolution of the residues in a small volume of solvent that will then be injected to chromatographic system. However, in case of lighter PAHs, the stream of gas can lead to the loss of analytes. An alternative to this operation is the direct transfer of analytes from the extract into a small volume of another non-miscible solvent. This approach is used in dispersive liquid-liquid microextraction (DLLME) method that is based on the system of three solvents: aqueous sample, dispersive solvent and extraction solvent. The mixture of an extraction solvent (e.g. chloroform) and a dispersive solvent (water-organic miscible solvent, e.g. acetonitrile) is rapidly injected into an aqueous sample, forming a cloudy solution. After centrifugation, the analytes are preconcentrated into the phase of extraction solvent (Viñas et al., 2014; Kamankesh et al., 2015). Until now, DLLME has demonstrated promising results in extract preconcentration without any loss of analytes, also in the case of the determination of PAHs in food samples (Sadowska-Rociek et al., 2015; Petrarca and Godoy, 2018).

Therefore, the aim of this study was to develop an effective sample preparation procedure for the determination of PAHs in smoked fatty products of animal origin. Two different approaches were employed: 1) classical QuEChERS with PSA and C18 sorbents 2) procedure with the use of EMR-Lipid according to the manufacturer. We have also compared two different methods of extract preconcentration: under nitrogen stream and with the use of DLLME method. All samples were analysed using gas chromatography-mass spectrometry. Finally, some findings resulting from the use of plastic laboratory consumables have been also discussed.

Section snippets

Chemicals and reagents

Polycyclic aromatic hydrocarbons suitable for EPA Method 610, anthracene-d10 (Internal Standard 1; IS1), chrysene-d12 (Internal Standard 2; IS2), hexachlorobenzene (Syringe Standard; SS) were obtained from Sigma-Aldrich, Saint Louis, Missouri, USA. Magnesium sulphate anhydrous p.a. and sodium chloride p.a. were purchased from Krakchemia SA, Krakow, Poland. Acetonitrile, chloroform and hexane, were purchased from Merck KGaA, Darmstadt, Germany. PSA, C18, SPE Bulk Sorbents and EMR-Lipid material

Comparison of different methods of sample preparation

Fig. 2 shows the comparison of PAHs recoveries obtained for the tested variants (“QuEChERS” – 1, “EMR-Lipid” – 2, in the combination with evaporation to dryness using the nitrogen stream). Generally, the recovery values within the acceptable range (50–120%, according to EU recommendation) were obtained only for QuEChERS method, but with the exception for NaP and MeNaP1, for which the recovery was below 50%. For two other compounds, Ant and B[a]a, the results of the recovery were exceptionally

Conclusions

The experiment conducted in this study revealed that the combination of QuEChERS extraction method with clean-up step by EMR-Lipid and DLLME technique as an extract preconcentration resulted in successfully purified samples providing in the same time acceptable recoveries of PAHs in smoked fatty products. Although the proposed method is composed of several steps, it is fast and effective and can be successfully applied for PAHs determination in difficult matrices such as heat-treated food of

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

CRediT authorship contribution statement

Tereza Slámová: Conceptualization, Investigation, Writing - original draft, Writing - review & editing. Anna Sadowska-Rociek: Methodology, Formal analysis, Validation, Investigation, Writing - original draft, Writing - review & editing. Adéla Fraňková: Conceptualization, Formal analysis, Investigation. Magdalena Surma: Formal analysis, Validation, Writing - review & editing. Jan Banout: Conceptualization, Supervision.

Declaration of Competing Interest

All authors declare that they have no conflict of interest.

Acknowledgment

This research was performed with the financial support from Ministry of Science and Higher Education of Republic of Poland within the statutory R & D activities (DS-3700/KTGiK/2018), and by Internal Grant Agency of Faculty of Tropical AgriSciences: grant number 20195006.

References (32)

  • G. Borgstrom

    Fish as Food V3: Processing

    (2012)
  • D.K. Essumang et al.

    Distribution, levels, and risk assessment of polycyclic aromatic hydrocarbons in the soot of some kitchens in the Cape Coast Metropolis of Ghana

    Toxicol. Environ. Chem.

    (2010)
  • European Commission

    Commission Regulation (EU) No 835/2011 of 19 August 2011 Amending Regulation (EC) No 1881/2006 As Regards Maximum Levels for Polycyclic Aromatic Hydrocarbons in Foodstuffs

    (2011)
  • European Commission

    Commission Regulation (EU) No 836/2011 of 19 August 2011 Amending Regulation (EC) No 333/2007 Laying Down the Methods of Sampling and Analysis for the Official Control of the Levels of Lead, Cadmium, Mercury, Inorganic Tin, 3-MCPD and Benzo(a)pyrene in Foodstuffs

    (2011)
  • E. Fasano et al.

    Detection of polycyclic aromatic hydrocarbons in smoked buffalo mozzarella cheese produced in Campania Region, Italy

    J. Sci. Food Agric.

    (2016)
  • Y. Huang et al.

    Simultaneous determination of acrylamide and 5-Hydroxymethylfurfural in heat-processed foods employing enhanced matrix removal - lipid as a new dispersive solid-phase extraction sorbent followed by liquid chromatography-tandem mass spectrometry

    J. Agric. Food Chem.

    (2019)
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