Results of a Saxitoxin Proficiency Test Including Characterization of Reference Material and Stability Studies

Abstract

A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories’ capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses.

Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

“…The detection limits of dcSTX and NEO were at about the same level as the detection limit for STX (~1 ng/mL, ~20 ng/g mussel), and these analogues could be quantified in the mussel samples with the quantitation limit of approximately 3 ng/mL and 60 ng/g mussel. The results for STX, dcSTX, and NEO in the PT were acceptable and in good accordance with the assigned values obtained with pre-column oxidation HPLC-FLD ( Table 7 ) [ 28 ].…”

“…STX identification in mussel samples was based on the OPCW retention time criteria [ 26 ] and tandem mass spectrometry (MS 2 ) fragmentation pattern of the analyte when compared to certified reference standard according to the EU criteria [ 27 ]. The developed method was applied and tested in the STX proficiency test (PT) of the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk) under the 7 th European Union Framework Programme for Research (FP7) [ 28 ]. Materials and methods are detailed described in the experimental Section 3 .…”

Biological toxins of potential bioterrorism risk: Current status of detection and identification technology

“…Oxidation of PSP toxins into fluorescent iminopurine derivatives followed by LC separation and FLD detection [66] Post-column oxidation HPLC-FLD Official PSP toxin method for shellfish: LC separation and post-column oxidation of PSP toxins into fluorescent iminopurine derivatives followed by FLD detection [67] Spectrometric assay HILIC LC-MS/MS: Positive ESI with SRM detection of PSP toxins in shellfish [70] Positive or negative ESI with MRM transitions of PSP toxins in shellfish [77,78] Positive ESI and product ion scan of STX in algal and mussel matrices [72] * To the best of our knowledge, the thoroughly characterized [30]. The materials of highest relevance for humans (BoNT/A, B and E) were spiked in different concentrations (0.5-1000 ng/mL) into buffer, serum, milk, and meat extract.…”

“…The emphasis of these exercises has been on seafood safety and the total PSP content in the samples provided. The aim of the STX PT organized in the context of the EQuATox project was the verification of STX from the CWC point of view in real samples, including mussel samples, algal cell samples, and diluted algal extracts [72]. According to the OPCW, unambiguous identification of an analyte should be based on at least two independent analytical methods.…”

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method Coupled with Dispersive Solid-Phase Extraction for Simultaneous Quantification of Eight Paralytic Shellfish Poisoning Toxins in Shellfish

“…Another concern about analysis is that generally PSP toxins do not have natural ultraviolet or fluorescence absorption and thus need derivation pretreatments before detection by liquid chromatography. Post-column derivatization [ 13 , 14 , 15 , 16 ] or pre-column derivatization [ 17 , 18 , 19 , 20 ] with fluorescence detection (LC-FLD) shows high sensitivity but requires a complex equipment with daily maintenance. In recent years, the development methods of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for determination of cyanobacterial toxins and STXs were carried out in previous studies [ 21 , 22 , 23 , 24 , 25 ] with a hydrophilic interaction liquid chromatography (HILIC)-based column for separation.…”