Revisiting the HPLC-FLD Method to Quantify Paralytic Shellfish Toxins: C3,4 Quantification and the First Steps towards Validation

Abstract

Paralytic shellfish toxins (PSTs) are a large group of biotoxins that cause paralytic shellfish poisoning. Their appearance in natural waters and their ingestion by aquatic species have a huge socio-economic impact, whereby their monitoring is of the upmost relevance to minimize the consequences. For earlier detection and faster response/action by stakeholders, validation of adjusted analytical methods, particularly for lower concentration levels, is important. This work proposes a derived High-Performance Liquid Chromatography method, with fluorescence detection (HPLC-FLD). The main differences from the official method are the size of the HPLC column and the gradient elution conditions. It covers the current eleven certified reference materials (CRM) available on the market, including the most recent-C3,4. This first calibration report for C3,4 suggests limits of detection (LOD) and limits of quantification (LOQ) of 6 nM and 19 nM (~5 µg STX.2HCl eqv./kg and 17 µg STX.2HCl eqv./kg), respectively. For the remaining CRM, LODs ranged between 3 and 28 nM (~0.9 and 127 µg STX.2HCl eqv./kg), while LOQs varied between 11 and 94 nM (~3 and 409 µg STX.2HCl eqv./kg, considering toxicity equivalency factors (TEFs) reported by EFSA).

Keywords: C3,4; monitoring; quantification; saxitoxins; validation.

Figure 1

Chromatograms of a mix solution containing 11 CRMs, previously oxidized with periodate (black) or peroxide (pink). Identification of peaks with peroxide oxidation: 1—dcGTX2,3 (1st peak); 2—dcGTX2,3 (2nd peak); 3—C1,2; 4—dcSTX (1st peak); 5—dcSTX (2nd peak); 6—GTX2,3; 7—GTX5; 8—STX. Identification of peaks with periodate oxidation: 1—dcGTX2,3 (1st peak); 2—GTX1,4 + C3,4 + dcGTX2,3 (2nd peak); 3—C1,2; 4—dcNEO (1st peak); 5—NEO + GTX6 + dcSTX (2nd peak) + dcNEO (2nd peak); 6—GTX2,3 + products d and h (Figure S2) of GTX1,4 and C3,4, respectively; 7—GTX5; 8—STX + products f and j (Figure S2) of NEO and GTX6, respectively. (c, g, e and i correspond to the secondary products identified in Figures S1 and S2, in SI).