Fabric phase sorptive extraction combined with high-performance-liquid chromatography-photodiode array analysis for the determination of seven parabens in human breast tissues: Application to cancerous and non-cancerous samples

https://doi.org/10.1016/j.chroma.2020.461530Get rights and content

Highlights

  • A novel, ecological and simple fabric phase sorptive extraction is applied in bioanalysis of parabens in human breast tissue.

  • An improved and modernized sample preparation protocol based on FPSE is proposed.

  • A well-designed optimization procedure for the FPSE workflow is described.

  • Successful application in cancerous and non-cancerous tissue samples is achieved.

Abstract

An improved pretreatment approach of human breast tissue is demonstrated for subsequent analysis of seven parabens including methyl paraben (MPB), ethyl paraben (EPB), propyl paraben (PPB), butyl paraben (BPB), isopropyl paraben (iPPB), isobutyl paraben (iBPB), and benzyl paraben (BzPB). Specifically, a well-designed homogenization procedure, conjugated with an optimized fabric phase sorptive extraction (FPSE) protocol, resulted in a carefully outlined sample preparation process as part of a green, simple, sensitive, economical and fast HPLC-PDA analytical method in agreement with Green Analytical Chemistry (GAC) demands. Among all tested FPSE membranes, the highest extraction efficiency was achieved by employing sol-gel poly(tetrahydrofuran) (sol-gel PTHF) coating on 100% cotton cellulose fabric that represents a medium polarity microextraction device, which combined the advanced material characteristics of sol-gel sorbent and the rich surface chemistry of an inherent porous cellulose fabric substrate. The chromatographic separation was accomplished with a Spherisorb C18 column and an isocratic mobile phase consisted of ammonium acetate and acetonitrile at a flow rate of 1.4 mL/min. The total analysis time was 13.6 min. The analytical adequacy of the composite sample preparation and chromatographic separation method was strongly evidenced by its successful application in the bioanalysis of real cancerous and non-cancerous tissue samples originated from different sub regions of human breast including axila, the upper left and the right quadrant. In all samples, at least one paraben was detected, while 35% of the samples were tested positive for all seven target parabens. Moreover, concentration levels of parabens in cancerous tissues were unambiguously higher than in healthy tissues. The obtained results underlined bioaccumulation potential of parabens in human breast tissue as a consequence of constant low-dose exposure of humans, despite the statutory concentration limits. The developed methodology has demonstrated to be suitable and efficient for future epidemiological and toxicological studies.

Introduction

Parabens (PBs) is a general denomination describing a well-recognized category of homologue chemical compounds, which are alkyl esters of p-hydroxybenzoic acid (PHBA). These molecules arise from the esterification reaction at C-4 position of the PHBA with common alcohols including methyl paraben (MPB), ethyl paraben (EPB), propyl paraben (PPB), butyl paraben (BPB), isopropyl paraben (iPPB), isobutyl paraben (iBPB) and benzyl paraben (BzPB). These substances exhibit efficient antimicrobial, antibacterial and antifungal activity, which has positioned them as very popular preservative agents since the 1920s with no impact on odor, taste, color or pH on final products. Nowadays, PBs are ubiquitous in personal care products, pharmaceuticals, cosmetics, food stuff, drinks, packaging materials and daily use products. Although their use is considered harmless when it falls within certain limits, constant long-term low-dose human exposure has raised health concerns in recent years. Furthermore, their universal utilization inevitably brought about a significant accumulation in environmental constituents like air, water, and soil [1,2].

Chronic and daily absorption of PBs through all possible routes and sources, as for example from topical application of cosmetics like creams, deodorants or antiperspirant, lead to their permanent existence at low concentration levels and thus to a bioaccumulation in different human biological matrices, such as urine, serum, breast milk, placental tissue, umbilical cord blood and amniotic fluid [3,4]. This has been connected with various unwanted malfunctions, as they deemed to be endocrine disrupting compounds (EDCs) that induce deregulation of the hormone system [5]. Their estrogenic activity may be a causative factor of the growth of human breast cancer, as PBs have genuinely been measured in breast tumors [3,4]. In recent years, concerns have been raised about the role of parabens in other various disease processes connected with the human reproductive system or about their ability to promote mitochondrial dysfunction [[1], [2], [3], [4]]. In the human body, PBs can be hydrolyzed to PHBA from enzymes called carboxylesterases that are present in organs affiliated to the metabolic system such as the liver, but also in skin and subcutaneous tissue [3,4]. As such, there is a justified presumption that PBs may accumulate as intact esters in human breast tissue from dermal application. Indeed, PBs have been quantified not only in tissue samples from different locations of the breast, but also breast tumors [6]. As a consequence, utilization of PBs in commodities is limited by international and European organizations. For instance, the European Union (EU) and the United States Food and Drug Administration (US FDA) have defined that theamount of Pbs should not exceed the mass proportions of 0.4% and 0.1% for one PB type or 0.8% and 0.3% for a PB combination in cosmetics and drugs, respectively [1].

The physicochemical and biological characteristics of PBs are significantly affected by the elongation and branching of the alkyl chain (see Table S1 in the Supporting Information) [3,7,22]. The increase in the length of the PB alkyl chain increases their antimicrobial potency, lipophilicity, cellular penetrability as well as estrogenic activity [[1], [2], [3]].

Due to constant human exposure to PBs, it is of great importance for epidemiological and toxicological studies to analyze PBs in human breast tissue. Nevertheless, existing scientific literature reports only a few methods for this target tissue type, while there is a much larger number of published studies on the determination of PBs in other biological matrices [3]. Published data on PB detection in human breast cancerous and non-cancerous tissue are scarce and some recently published researches employed thin-layer chromatography (TLC), high-pressure liquid chromatography followed by tandem mass spectrometry (HPLC-MS/MS) and gas chromatography coupled with mass spectrometry (GC-MS) [4,6,8,9]. The reasons for such shortage of bibliographic data may be found in laborious and complex analytical work needed for tissue analysis, genuine challenges related to handling and pretreating solid tissue samples compared to liquid biological samples, as well as difficulties to apply bioanalytical method validation processes, as described in formal guidelines (US FDA, European Bioanalysis Forum-). Several review articles have provided useful information and suggestions as to how to manipulate and analyze tissue samples in general [10,11]. Sample preparation and extraction methodology have a very profound role in any type of tissue bioanalysis. When the sample pretreatment path is elaborate and efficient, it ensures proper homogenization, exhaustive recovery of analytes from the tissue, sampleclean up, avoidance of interferences and moderation of matrix effects [10,11]. In the case of PB analysis, the first part of the sample pretreatment process is the homogenization of the solid tissue material and the extraction of PBs in the liquid phase. Homogenization may range from physical or mechanical to biochemical depending on the sample requirements and the type of targeted analytes. Breast tissue is usually treated by mechanical homogenization. During the second part of the pretreatment process, the sample clean up and the selective PB extraction from the tissue homogenate liquid solution is performed by means of extraction techniques such as solid phase extraction (SPE) combined with GC-MS [8] and liquid-liquid extraction (LLE) followed by HPLC-MS/MS [4,6]. However, there are some drawbacks that are inherent to these techniques. The LLE demands augmented amounts of organic solvents, is not time-saving, has relatively poor extraction capacity and limited selectivity. The SPE is also time-consuming, includes a large number of steps and is prone to increased jeopardy of analyte loss despite that it performs with higher selectivity and sensitivity. Recently, Xiong Shen et al proposed a simple and ecological sample preparation method for the isolation of PBs from human breast tumors and peripheral adipose tissue that was based on ultrasound-assisted dispersive liquid-liquid microextraction followed by analysis in GC-MS [9] in accordance with the philosophy and principals of green analytical chemistry [12,13]. Even a superior approach, namely FPSE, has been introduced by Kabir and Furton in 2014 [14]. The FPSE method fulfills the strong need for a contemporary, innovative, miniaturized and sorbent-based microextraction technique that overcomes the shortcomings of the classical techniques including also the advantages of eco-friendliness, cost-effectiveness and simplicity [14]. Its entire rationale and physicochemical basis has been extensively optimized and well-testified elsewhere [[15], [16], [17], [18]]. This technique exploits the noteworthy benefits of the FPSE extraction device, emerging from the combination of diverse natural or synthetic thin fabric substrates made by cellulose or polyester, chemically coated by sol–gel acquired hybrid organic-inorganic sorbents, which essentially represent all the used sorbent materials in all the extraction techniques. The FPSE membranes possess substantially high chemical, thermal and solvent stability, augmented primary contact surface area, inherent porosity, open geometry and thus huge retention capacity and extraction efficiency, motivating to a fast and direct analyte extraction by both the exhaustive and equilibrium extraction mode. Although it is a new technique, it has already been applied with distinct advantages over classical sample preparation techniques, both exhaustive and equilibrium driven, for many analytes in a variety of samples such as environmental, pharmaceutical, food samples, toxicological and biological ones, combined with various analytical techniques like HPLC-PDA, HPLC-MS/MS, GC-MS [[16], [17], [18], [19], [20], [21]].

This study aimed to introduce a novel, green and easy-to-apply FPSE-based pretreatment method for the selective extraction of PBs from the breast tissue samples by applying efficient sample cleaning and removal of detrimental and obnoxious matrix burdens. In addition, we also aimed to establish a useful method for the tissue homogenization and deliverance of PBs from the solid tissue material to the liquid homogenate solution. Applied sol-gel PTHF coated FPSE membrane demonstrated the most advantageous behavior among nineteen tested membranes. This medium polarity FPSE membrane, which is expedient when the polarity of targeting analytes ranges from medium to high, has already been applied to other biosamples [[16], [17], [18], [19], [20], [21]]. Subsequently, a new and quick HPLC-DAD method was developed and validated. The entire methodology was successfully implemented to real cancerous and non-cancerous tissue samples derived from different sub regions of women's breast obtained from diverse individuals.

Section snippets

Instrumentation and materials

The chromatographic analysis was conducted with a quaternary low-pressure gradient HPLC–PAD system purchased from Shimadzu (Kyoto, Japan). The distribution of the mobile phase to the analytical column was carried out by means of an FCV-10ALVP mixing system and an LC-10ADVP pump equipped with a Shimadzu SCL-10ALVP System Controller. Sample injection was fulfilled with a Rheodyne 7725i injection valve (Rheodyne, Cotati California, USA) geared with a 20 μL loop. The detection, data acquisition and

Selection of sol-gel sorbent coated FPSE membrane

The holistic modus operandi and tactic of the production process of FPSE membrane consists of a series of sequential steps including (a) the pretreatment of cellulose and polyester fabric in order to render them suitable for sol-gel coating process, (b) the design and preparation of sol solution by making use of organic polymer, organically modified inorganic precursor(s), an appropriate solvent system and an aqueous acid solution as a catalyst, (c) the process and mechanism of the sol-gel

Conclusions

The presented method is characterized by clear advantages. A well-designed homogenization strategy using the least amounts of organic solvents and based on mechanical means, which are omnipresent in every laboratory, permitted the deliverance of parabens from the solid tissue, in combination with ordinary practices like centrifugation.

Subsequently, FPSE-based procedure was adopted for further sample preparation. FPSE exploits the outstanding idiosyncrasy of the microextraction devices resulting

CRediT authorship contribution statement

Vasileios Alampanos: Methodology, Validation, Data curation, Investigation, Writing - original draft. Abuzar Kabir: Conceptualization, Investigation, Writing - review & editing. Kenneth G. Furton: Supervision. Željka Roje: Investigation, Methodology. Ivana Vinković Vrček: Conceptualization, Writing - review & editing, Data curation. Victoria Samanidou: Conceptualization, Supervision, Data curation, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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