Analytical capabilities of high performance liquid chromatography – Atmospheric pressure photoionization – Orbitrap mass spectrometry (HPLC-APPI-Orbitrap-MS) for the trace determination of novel and emerging flame retardants in fish

Highlights

• Simultaneous determination of twenty seven BFRs is presented.

• HPLC-APPI-Orbitrap-MS is an efficient tool for detection of BFRs.

• Recoveries between 80 and 119% and repeatability between 1.2 and 15.5% were obtained.

• Method met the sensitivity criteria stated in Recommendation 2014/118/EU.

Abstract

A new analytical method was established and validated for the analysis of 27 brominated flame retardants (BFRs), including so called “emerging” and “novel” BFRs (EBFRs and NBFRs) in fish samples. High performance liquid chromatography (HPLC) coupled to Orbitrap mass spectrometry (Orbitrap-MS) employing atmospheric pressure photoionization (APPI) interface operated in negative mode was used for the identification/quantitation of contaminants. HPLC-Orbitrap-MS analysis provided a fast separation of selected analytes within 14 min, thus demonstrating a high throughput processing of samples. The developed methodology was tested by intralaboratory validation in terms of recovery, repeatability, linear calibration ranges, instrumental and method limits of quantitation (i-LOQ and m-LOQ), and where possible, trueness was verified by analysis of certified reference materials (CRMs). Recoveries of analytes were between 80 and 119%, while the repeatability in terms of relative standard deviations (RSDs) was in the range from 1.2 to 15.5%. The measured values for both analyzed CRMs agreed with the provided consensus values, revealing the recovery of reference concentrations in 72–119% range. The elaborated method met the sensitivity criterion according to Commission Recommendation 2014/118/EU on monitoring of BFRs in food products for majority of the compounds. The concentrations of polybrominated diphenyl ethers (PBDEs) in real samples determined by HPLC-APPI-Orbitrap-MS method and validated gas chromatography–high-resolution mass spectrometry (GC–HRMS) method were found to be in a good agreement.

Introduction

During the past decades, at least seventy five BFRs have been commercially produced and their use has increased dramatically since 1970s [1]. Due to the persistency and bioaccumulation properties, one of the largest group of BFRs, PBDEs, has been banned [2], [3], [4] and several EBFRs and NBFRs have been suggested as replacements for the banned formulations [5]. By EBFRs are considered chemicals which are documented regarding production and use as BFRs that have been shown to occur/distribute in the environment and/or wildlife, humans or other biological matrices, while NBFRs are compounds which are documented as potential BFRs that have been shown to be present in materials or products [5]. There is a lack of comprehensive information on the current use and production volume, as well as experimental data both on the physical/chemical characteristics or stability/reactivity/persistence for most of the EBFRs. According to the scientific opinion of European Food Safety Authority (EFSA), some of these compounds present potential human health concerns due to genotoxic and carcinogenic properties [6]. The similarity of EBFRs and NBFRs to well known persistent organic pollutants (POPs) may result in migration through food chains, with possible long-term health and environmental impacts. Taking into account the growing attention to the BFR problems in the environment, EU recommendation on the monitoring of traces of BFRs in food products was recently proposed in order to promote the data collection on these compounds [7]. Risk assessment for the exposure of environmental objects to these compounds is a complicated procedure, and the development of accurate and comprehensive methods of analysis is one of the essential steps to implement these strategies. Currently, the possibility of chemical identification and availability of quantitative data on these potential contaminants in food or environmental samples is restricted due to the absence of specific analytical methods for the majority of EBFRs.

The tackling of complex matrices and the required trace level detection limits are of great importance in the analysis of EBFRs. A number of effective analytical methods for some of BFRs have been developed using GC–MS. However, GC–MS methods could be applied for the determination of only a limited number of these contaminants [8], and trace level analysis of the majority of BFRs (especially highly brominated ones) is quite challenging due to the thermal lability of these compounds, which restricts the field of effective application of GC methods. To overcome the thermal lability issue for highly brominated BFRs (e.g., hexabromocyclododecane (HBCD), octabromotrimethylphenyllindane (OBIND), decabromodiphenyl ether (BDE 209), and others), highly sensitive methods based on LC coupled to tandem mass spectrometry (MS/MS) have been developed and extensively used for the analysis of biota and environmental objects [9], [10], [11]. However, despite the indisputable advantages of the MS/MS approach, such as high sensitivity and a wide linearity range, selectivity issues may be encountered using this type of MS; false-positive and false-negative findings during determination of veterinary drugs were documented, showing obvious cases of inadequate selectivity of the MS/MS technique for complex matrices [12], [13]. A possible solution to the insufficient selectivity issue is the use of HRMS techniques, among which Orbitrap-MS has become more affordable in recent years [14]. Orbitrap-MS is an attractive tool for trace analysis of different compounds in complex matrices, ensuring a selectivity exceeding that provided by commonly used conventional low resolution MS/MS techniques [12]. The possibility to operate in a full scan mode and the retrospective evaluation of instrumental raw data makes Orbitrap-HRMS a much more flexible tool than MS/MS, with a future perspective that this advanced approach may dominate in multicomponent analysis.

Until now, only isolated attempts have been made to analyze BFRs by Orbitrap-MS, and these recent developments confirmed the efficiency of this mainstream MS technique in the analysis of such BFRs as HBCD [15], [16] and derivatives of tetrabromobisphenol A (TBBPA) [17]. The powerful advantages of Orbitrap-MS, such as the high mass resolution (up to 500,000 Full Width Half Maximum (FWHM)) and mass accuracy (up to 1 ppm) result in high selectivity for target analytes, and provide new perspectives in the development of confirmatory analytical methods for the determination of ultra-trace levels BFRs. To our knowledge, all these methods were used for the analysis of BFR representatives, which could be analyzed under the conditions of electrospray ionization (ESI) technique. However, it has been documented that this type of ionization was mostly useless in the analysis of the majority of non-polar BFRs of aromatic nature [10], and alternative techniques, such as atmospheric pressure chemical ionization (APCI) and APPI operated in negative mode were therefore proposed instead of ESI, as very soft ionization methods that were highly effective in the analysis of several BFRs using LC-MS/MS [9], [10], [11]. Special attention was paid to the APPI technique, providing efficient and selective ionization of hydrophobic aromatic molecules, that is of great importance in the analysis of such BFR representatives as PBDEs and related substances [10], [11], [18]. Taking into account the great potential of APPI-Orbitrap-MS technique in terms of selectivity and sensitivity, this approach may lead to the development of highly reliable and efficient methods.

The principal aim of this study was to propose a new analytical method for the simultaneous determination of various BFRs, among which several EBFRs are presented. In the present study, negative APPI in combination with detection by Orbitrap-MS operating in full scan mode was applied for the first time in analysis of a broad range of BFRs, revealing new perspectives in the analysis of these contaminants.