Quantitative Determination of Gymnodimine-A by High Performance Liquid Chromatography in Contaminated Clams from Tunisia Coastline

Abstract

Quantitative determination by high performance liquid chromatography (HPLC) was performed for gymnodimine-A (GYM-A), a phycotoxin responsible for the contamination of Tunisian clams. This study demonstrates a rapid and reproducible HPLC-ultraviolet (UV) method for extraction, detection and quantification of GYM-A in toxic clams. The extraction of GYM-A from the digestive gland of clams in acetone, subsequent clean-up with diethyl ether and extraction with dichloromethane is the more valid protocol. Chromatography analyses were performed using a gradient of acetonitrile-water (10:90 to 90:10), containing trifluoroacetic acid (0.1%) for 20 min at 1 mL/min rate with a C18 column. Recovery rates exceeded 96%, and limits of detection and quantification were 5 ng/mL and 8 ng/g digestive gland, respectively. Repeatability and reproducibility were tested for various samples containing different levels of GYM-A. A significant correlation was observed between toxicity level of samples and the determined amount of GYM-A. Also, the persistence of GYM-A in contaminated clams from Boughrara lagoon was demonstrated. The kinetics discharge study of GYM-A in controlled medium, during 1 month, showed that the process of depuration was biphasic with an exponential discharge of 75% of the total amount of sequestered GYM-A during the first 12 days followed by a slow discharge (>10%) for the subsequent days up to the seventeenth day. This is the first time that a quantitative study of GYM-A in clams from Tunisian coasts is performed through the development of a new method for detection and quantify of this phycotoxin. We found HPLC-UV a reliable and suitable alternative to the mouse bioassay.

Prevalence and persistence of gymnodimines in clams from the Gulf of Gabes (Tunisia) studied by mouse bioassay and LC–MS/MS

“…These results confirmed the previous reports of presence of GYMs in the Gulf of Gabes (Biré et al, 2002;Marrouchi et al, 2010), but also showed the great prevalence of this toxin group in the area. According to our results, synergistic effects with other lipophilic toxins proposed by Dragunow et al (2005) are unlikely.…”

“…Apparently, GYM-B is 10-fold less toxic than GYM-A (Kharrat et al, 2008), and a potential synergistic effect with other toxins has been proposed since GYMs were able to sensitize Neuro2a cells to other marine toxins such as okadaic acid (Dragunow et al, 2005). Nowadays, GYM-A has shown a worldwide distribution including New Zealand coastlines (Stirling, 2001b), Tunisia (Biré et al, 2002;Marrouchi et al, 2010), Australia (Takahashi et al, 2007), Europe & North America coast (Kharrat et al, 2008), and more recently South Africa (Krock et al, 2009) and China (Liu et al, 2011). They may persist in oysters for years (MacKenzie et al, 2002), although an enhanced detoxification capability has been recently demonstrated in clams (Medhioub et al, 2010).…”

“…As the information about the persistence of GYMs is still limited apart from oysters in New Zealand, the magnitude of the potential arrival of GYMs in the market is difficult to assess. In this work, we studied the causative origin of toxicity in clams from the Gulf of Gabes, Tunisia, where the presence of GYMs has been previously reported (Biré et al, 2002;Marrouchi et al, 2010). Tunisia has 1300 km of coastline and considerable resources for shellfish production.…”

Cyclic imines, as emerging marine toxins: Chemical properties, distribution, toxicological aspects and detection methods

“…A HPLC-UV method was developed for the routine analysis of GYMs in shellfish at a wavelength measuring up to 210nm and the LOD of GYM A was reported to be 2.4 μg/kg of digestive gland of clams (Marrouchi et al 2009). However, cyclic imines lack chromophores and, therefore, optical detection methods, such as ultraviolet (UV), do not provide sufficient selectivity because the UV absorption of cyclic imines is low (EFSA 2010).…”

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems

“…there is evidence that CIP toxins and their producers also are found in europe, the USA, and North Africa (Aasen et al, 2005a;Bire et al, 2002;Cembella and Krock, 2008;Ciminiello et al, 2006;John et al, 2003;MacKinnon et al, 2006;Marrouchi et al, 2010;Rundberget et al, 2011;touzet et al, 2008;Villar Gonzalez et al, 2006), and their presence, along with PSP to CTX also primarily in predatory fish consumed by humans (snapper, barracuda, amber jack and moray eel), and more investigations should be conducted on the role on Mtx in human intoxication.…”