Development, validation, and application of an ultra-performance liquid chromatography–sector field inductively coupled plasma mass spectrometry method for simultaneous determination of six organotin compounds in human serum
Introduction
Organotin compounds (OTCs) are a class of organometallic species comprised of a tin (Sn) atom bound covalently to one or more alkyl or aryl groups [1]. The number and structure of the organic substituents bound to Sn can significantly alter its physicochemical characteristics, and as a result, Sn has the greatest variety of organometallic derivatives that are currently in use by industry among any other element [2]. Since the 1940s, the plastics industry has employed mono and dialkyl OTCs as heat and light stabilizers for the production of polyvinyl chloride and other materials [3]. More recently, dialkyl OTCs have been employed in thin film, transparent conductive coatings for liquid crystal display panels [4]. Many OTCs are biocides, with maximum toxicological activity observed for trisubstituted compounds. Tributyltin (TBT) and triphenyltin (TPT) have historically been used in marine antifouling paints to minimize organism growth on ship hulls and as insecticides, miticides, or fungicides for wood preservation or agricultural crop protection [5], [6], [7].
The industrial utility of OTCs has led to significant amounts of the chemicals being found in household products and the environment, resulting in widespread potential for human exposure. Several cases of OTC poisoning or exposure were recently reported in workers involved in leather, plastic, or other manufacturing activities [8], [9], and OTCs originating from floor wax, diapers, baking paper, clothing, and other consumer products were reported in house dusts [10]. The extent of exposure in the general population is less characterized. The predominant environmental sources of TPT and TBT have been agricultural runoff and the degradation of marine antifouling paints [11], [1]; both TPT and TBT are toxic and endocrine disruptors in aquatic organisms, even at sub ng L−1 levels [13]. Numerous reports of gastropod imposex, resulting in permanent female masculinization and species decline [13], [14.], [15], coupled with OTC bioaccumulation spanning several marine food chain trophic levels [5], [12], [16], [17], [18], [19] have prompted regulatory agencies across the world to restrict use of tin-containing antifouling marine paints to larger ships [20], [21]. Even though the use of these paints has been limited, OTCs have persisted in the environment by binding with sediments. When exposed to favorable environmental conditions, aquatic sediments can release sequestered OTCs back into the water column for biological uptake [18].
The differential toxicity, industrial utility, environmental prevalence, and potential for human exposure have fueled the demand for analytical methods capable of determining the concentrations of OTCs in a variety of matrices. These methods must be capable of differentiating target organotin species at very low concentrations in an environmental or biological matrix of interest. Fluorescence spectrometry and bioluminescent assays have recently been reported for detection of organotin compounds [22], [23], but the most common analytical approach remains coupling of chromatography with a specific detector. Gas chromatography (GC) coupled to mass spectrometry (MS) [12], tandem mass spectrometry (MS/MS) [13], high resolution MS [24], atomic absorption spectrometry (AAS) [3], flame photometric detection (FPD) [25], pulsed flame photometric detection (PFPD) [20], atomic emission detection (AED) [26], and inductively coupled plasma mass spectrometry (ICP-MS) detection [5] have all been used to quantify organotin species in a variety of matrices. Regardless of the instrumental technique used for detection, OTCs must be extracted and converted to fully alkylated, volatile species to allow analysis by GC. Extraction of ionic OTC species into non-polar solvents has been achieved with complexing agents like tropolone or dithiocarbamate, followed by sample cleanup and concentration. A second analytical approach involves the use of ethylating agents including sodium tetraethylhydroborate and Grignard reagents to convert OTCs to volatile forms that are amenable to analysis by gas chromatography (GC) [20], [27]. More recently, headspace solid-phase microextraction (HS-SPME) with in situ derivitization has been employed to simultaneously volatilize, extract, and concentrate OTCs prior to GC separation [28.], [29], [30], [31].
Although GC has been successfully employed for OTC determinations, challenges associated with the method have prompted a search for alternative methods. Extraction, clean-up, and concentration procedures for OTC measurements by GC can be tedious and time-consuming [32], [33]. In addition, GC methods require derivatization of extracted OTCs to more volatile forms, and the extent of derivatization can be dependent on the sample matrix and Sn species present [34], [35]. Consolidated extraction and derivatization HS-SPME techniques are promising, but are prone to inconsistent matrix effects for OTC measurements [26]. In contrast, liquid chromatography (LC) does not require derivatization prior to analysis and could be capable of separating common OTC environmental contaminants when used with complexing agents. Further, coupling of LC with ICP-MS could provide sensitive detection of OTCs and relatively low limits of detection compared with other detection methods. The objective of this investigation was to develop and validate a rapid, sensitive, and selective ultra-performance liquid chromatography (UPLC) method coupled with sector field inductively coupled plasma mass spectrometry (SF-ICP-MS) detection for the determination of a suite of OTCs, including monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), monophenyltin (MPT), diphenyltin (DPT), and triphenyltin (TPT) (structures presented in Supplementary information, Table S1) in human serum collected from women participating in the Snart-Foraeldre/MiljØ (Soon-Parents/Environment) Study, a cohort of Danish women who have recently discontinued birth control in order to become pregnant.
Section snippets
Reagents
The human serum matrix used throughout this investigation to prepare matrix standards and quality control (QC) samples was pooled from six adult female donors and was received and stored frozen (nominal −20 °C) from BioChemed Services (Winchester, VA, USA). Several ampules of a custom organotin standard containing nominal 2000 µg mL−1 concentrations of MBT, DBT, TBT, MPT, DPT, and TPT chlorides in methylene chloride were obtained from Restek (Bellefonte, PA, USA). Semiconductor grade methanol,
Results and discussion
The analytical method utilized UPLC coupled with SF-ICP-MS. The small (sub-2 µm) particle size of the UPLC column stationary phase and the sensitivity of the SF-ICP-MS enabled separation and sensitive determination of the analyte suite in approximately 3 min per sample (Fig. 1). To the best of our knowledge, the chromatographic run time and detection limits for the suite of analytes are superior to those other literature methods for OTC determinations in biological fluids. The analytical method
Conclusion
A UPLC–SF-ICP-MS method was successfully developed and validated for the determination of a suite of six OTCs (MPT, MBT, TPT, DPT, DBT, and TBT) in human serum. The employed sample preparation and chromatographic procedures allowed for separation of the analytes in less than three minutes without derivatization, resulting in considerably less sample preparation time than that required for many equivalent HPLC or GC separations. This shortened analysis time may offer a significant advantage in
Acknowledgements
This research was supported by the National Institute of Health/National Institute of Environmental Health Sciences (NIH/NIEHS; HHSN27320110003C). The authors would like to acknowledge Mr. Bradley J. Collins, Dr. Michelle Hooth, and Dr. Esra Mutlu for their review of this manuscript prior to formal submission.
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