Validation of a confirmatory method for lipophilic marine toxins in shellfish using UHPLC-HR-Orbitrap MS

Abstract

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion ((13)C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software.

Quantification and profiling of lipophilic marine toxins in microalgae by UHPLC coupled to high-resolution orbitrap mass spectrometry

“…In recent years, these groups have been routinely monitored in shellfish using LC-MS/MS,based methods as proposed by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). However, the use of full-scan high-resolution MS has recently been proposed as an alternative analytical platform for the quantitative measurement of marine lipophilic toxins in shellfish [23,24] and excellent validation data have been obtained with this methodology [25]. Indeed the quantitative performance of HR-Orbitrap MS has proved to be even better than MS/MS and excellent validation data have been obtained with this methodology [25].…”

“…Automatic gain control (AGC) target was set at balanced range (1×10 6 ions). Ionization source working parameters were optimized according to Orellana et al (2014) [25]. An m/z scan range of 100-1200m/z was chosen, based on the m/z values of the lipophilic target compounds that are included in current literature.…”

“…This software also provides an option for internal lock mass correction of Orbitrap data. As a precondition and precautionary measure, internal lock mass was used for each compound in order to eliminate potential systematic mass drift during analysis [25].…”

“…However, the use of full-scan high-resolution MS has recently been proposed as an alternative analytical platform for the quantitative measurement of marine lipophilic toxins in shellfish [23,24] and excellent validation data have been obtained with this methodology [25]. Indeed the quantitative performance of HR-Orbitrap MS has proved to be even better than MS/MS and excellent validation data have been obtained with this methodology [25]. Furthermore, HROrbitrap MS allows to retrospectively screen for many analogues and metabolites, including untargeted compounds, using its full-scan capabilities and metabolomics software [26].…”

“…The method of detection has been developed in a previous report [25] and applied to guarantee a fast and accurate analysis. The developed method was validated for representative compounds of the five main groups of marine lipophilic biotoxins in algal matrix.…”