Method development for the determination of PFOA and PFOS in honey based on the dispersive Solid Phase Extraction (d-SPE) with micro-UHPLC–MS/MS system
Introduction
Perfluorinated compounds (PFCs) comprise a class of environmentally persistent chemicals that have a wide range of industrial applications, such as fire-fighting foams, pesticides and consumer applications including surface coatings for carpets, furniture and paper products used as food packaging [1], [2], [3]. The two most frequently studied PFCs as xenobiotics and also as contaminants in the environment are perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), generally considered reference substances [4]. It is fully justified, because all of the perfluorooctanesulfonyl fluoride (POSF)-based compounds biologically break down or become metabolized to PFOS [3]. PFOA, in addition to its production and use as a surfactant, is also formed as a degradation product of several fluoropolymers and fluorotelomer alcohol. Analyses of PFOA and PFOS in wildlife samples from all over the world have identified them as global pollutants and have shown their bioaccumulation in higher trophic levels in the food chains [5], [6], [7], [8]. There is currently no legislation for perfluorinated organic substances such as PFOA and PFOS in food or feed within the EU [9]. The limited scope of research and preliminary findings of EFSA indicate the need to identify PFCs in raw material as well as in food of plant origin, which would assess the current scale of their occurrence and in consequence a risk for human health [10]. In those circumstances, it is clear that pollution by PFCs, is a global issue, given the transboundary movement of these compounds. EFSA's Scientific Panel on Contaminants in the Food Chain therefore recommended that further data on perfluoroalkylated substance levels in food and humans would be desirable, particularly with respect to monitoring trends in exposure to these substances [9]. In order to estimate the relevance of their presence in food, in March 2010 Commission Recommendation 2010/161/EU invited the Member States to monitor the presence of PFOS and PFOA, compounds similar to them but with different chain length and their precursors [11]. For this purpose, according the abovementioned document, it is required to use a method of analysis that has been proven to generate reliable results. Ideally, the recovery rates should be in the range 70–120%, with limits of quantification (LOQ) of 1 μg kg− 1.
Honey is a natural product produced by Apis mellifera bees from the nectar of plants. Its composition is mainly dependant on the floral origin of the nectar [12], as well as regional and climatic conditions [13]. The quality of honey is mainly determined by its sensorial, chemical, physical and microbiological characteristics. International honey standards are specified in the European Honey Directive and in the Codex Alimentarius Standard for Honey, both of which are presently under revision [14]. In those documents, the present knowledge on the different quality criteria is reviewed. The standard drafts, mentioned above include standards and methods for the determination of the following quality factors: moisture, ash, acidity, hydroxymethylfurfural, apparent reducing sugars, apparent sucrose, diastase activity and water-insoluble matter. These analyses help the food analyst to determine the quality of the honeys analyzed.
Bee products are produced in an environment polluted by different sources of contamination since these contaminants can reach the raw materials of bee products (nectar, pollen, plant exudates) by air, water, plants and soil and then be transported into the bee hive by the bees [15]. Honey is a food product with world-wide consumption especially among children and in terms of food safety concern it must be free of chemical contaminants particularly from POPs. The specific composition of any batch of honey, as well as the contaminants present in it, are dependent on the crops surrounding the beehive [16], [17]. PFCs can contaminate soil, air, and water thereby/thus the flowers from which bees collect nectar for honey production. Consequently, honey may serve as indicators of environmental pollution since honeybees are greatly affected by contaminants and transport them to the colony as contaminated nectar which ends as a contaminated honey. Therefore, a sensitive method of PFOA and PFOS determination in honey samples is extremely required.
Their analysis is a challenging task not only because the low concentration levels expected for these compounds in food samples but also on account of the complexity of matrices, so efficient sample preparation procedures and very sensitive determination technique are needed. Methods employed for sample preparation consisting usually one of three most common approaches which are as follows: pressurized liquid extraction [18], alkaline digestion [19], [20], [21] and ion-pair extraction [22]. In the first two approaches, the next step was the sample cleanup using Solid Phase Extraction (SPE) usually with WAX (week anion exchanges) [23], [24] or HLB (hydrophilic–lipophilic balanced sorbent) [20] cartridges. Because each of these methods showed some limitations, there is a need to implement an innovative solution that would enable not only high throughput sample handling, but also accurate determination of an entire set of analytes of concern at an ultra-trace level. In the last few years, a fast and inexpensive extraction QuEChERS method has been developed. Simplicity, minimum steps, and effectiveness for cleaning up complex samples have shown its usefulness in the analysis of residues in foods. QuEChERS methodology involves two steps. Extraction based on partitioning via salting out extraction involving the equilibrium between an aqueous and an organic layer is the first step. Dispersive Solid Phase Extraction (d-SPE) that involves further clean up using combinations of anhydrous salt and various sorbents to remove interfering substances is the second step. This sample treatment has been extensively used for sample preparation for pesticides residues in vegetables [25], but it has been extended to other residues and matrixes [26], [27], [28], [29].
Currently, due to its high sensitivity and selectivity, liquid chromatography hyphenated with tandem mass spectrometry (LC–MS/MS) operated in the multiple reaction monitoring mode (MRM) is the preferred technique for a quantitation of PFCs trace levels. Moreover, micro-UHPLC technique provides higher peak capacity, greater resolution, increased sensitivity and a higher speed of analysis compared to conventional LC system [30], mainly in combination with MS/MS.
According to our knowledge, the QuEChERS methodology has not been used for the PFCs analysis in honey samples. Therefore, the aim of this study was to develop a method for the determination of two most frequently studied PFCs (PFOA and PFOS) in honey samples based on the dispersive Solid Phase Extraction (d-SPE) with micro-UHPLC–MS/MS system. An application of different types of sorbents such as silica based (PSA, SAX, NH2, FL and C18) and polymer based (ENV), and acetonitrile as an extraction solvent was addressed. Finally, the analysis of real honey samples was performed.
Section snippets
Chemicals and reagents
Reagents in MS grade including acetonitrile (MeCN), methanol (MeOH), formic acid (FA) were purchased from Sigma Chemical Co., St. Louis, MO. Water was purified with a Milli-Q system, Millipore, Bedford, USA. Acetonitrile (for extraction) HPLC grade was purchased from Merck KGaA, Germany. Magnesium sulfate anhydrous p.a. and sodium chloride p.a. were purchased from POCh SA, Poland. PSA (primary and secondary amine), SAX (strong ion exchange), NH2 (aminopropyl), C18 (octadecylsilane), ENV
Chromatographic determination
The PFCs involved in this study were identified by the retention time and MRM ion pairs. Calibration curves were constructed by plotting the ratio of the peak area against concentration of the analyte. The retention times and MRM ion pairs for analyzed PFCs are summarized in Table 1.
Comparison different variants of QuEChERS methods — recovery study
The recovery values (fortification level of 0.005 mg kg− 1) for all tested sorbents ranged from 40 to 84% for PFOA and from 47 to 87% for PFOS. The values are presented in Fig. 4. The obtained results showed that the
Conclusion
The developed analytical method, using dispersive Solid Phase Extraction (d-SPE) and micro-UHPLC–MS/MS detection can be successfully applied for the determination of PFOA and PFOS in honey samples. The use of the polymer based sorbent — ENV for the efficient honey sample preparation based on QuEChERS method is recommended. The developed method has a potential for assessing the current scale of PFCs occurrence and in consequence a risk for human health since level of PFOS and PFOA in honey may
Compliance with ethics requirements
Magdalena Surma declares that she has not received any research grants and any honoraries from any commercial companies. Ewa Cieślik declares that she has not received any research grants and any honoraries from any commercial companies. Henryk Zieliński declares that he has not received any research grants and any honoraries from any commercial companies. Wiesław Wiczkowski declares that he has not received any research grants and any honoraries from any commercial companies.
This article does
Acknowledgments
This research was performed with the financial support of EU project REFRESH (FP7-REGPOT-2010-1-264105) — Unlocking the potential of the Institute of Animal Reproduction and Food Research for strengthening integration with the European Research Area and region development, which are hereby gratefully acknowledged.
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2020, Microchemical JournalCitation Excerpt :Therefore, the monitoring of PFOS is urgently required. Currently, the major analytical methods for the detection of PFOS are chromatography technologies, including liquid chromatography-mass spectrometry (LC-MS) [16–18], liquid chromatography-tandem mass spectrometry (LC-MS-MS) [19–21], and gas chromatography-mass spectrometry (GC–MS) [22,23], etc. Despite of good sensitivity and accuracy, the chromatography-based methods require expensive equipment, complicated operation and long time.