domoic acid ( da ) ( figure 1 ) is a neurotoxin produced by several species of phytoplankton , predominantly of the diatom genus pseudo - nitzschia . this toxin enters food webs through feeding interactions and can accumulate in higher trophic levels ( costa et al . , 2005 ; doucette et al . , human consumption of da - contaminated shellfish causes amnesic shellfish poisoning ( asp ) . a regulatory action level of 20 g da / g shellfish tissue is employed world wide for the harvesting and consumption of shellfish resources to protect human health ( reg . high performance liquid chromatography ( hplc ) with photodiode array detection ( dad ) is the most popular method used for the determination of da in shellfish tissue , developed by quilliam and wright ( 1989 ) now modified as the eu - harmonised - sop - asp - hplc - uv version 1 ( aeasan , 2008 ) , using a reversed phase column , under isocratic elution , and an optical absorbance detection at 242 nm . lc coupled to mass spectrometry ( ms ) detection has also been used for the determination of da ( wang et al . , 2007 ) but to today this method was complex and costly . we here report a method for the determination of the neurotoxin domoic acid ( da ) in shellfish using lc - tof / ms , which is really sensitive and provides a higher resolution allowing for the detection of also all the isomers of domoic acid . studies in fish zebrafish ( danio rerio ) ( tiedekena et al . , 2005 ; hong et al . , 2015 ) , treated with low but constant levels of domoic acid , showed damage to the brain system as well as to the cardiovascular system . domoic acid reference material was purchased from sigma aldrich ( saint louis , mo , usa ) . the official method was performed on an agilent 1200 instruments equipped with quaternary pump and an auto sampler . phenomenex synergi 4 polar - rp 80a ( 2504.60 mm ) thermostated at 40c was used for chromatographic separation . the isocratic mobile phase , consisting of a mixture of 1% aqueous trifluoroacetic acid and acetonitrile in the ratio 90:10 ( v / v ) , was used . sample solutions ( 8 , 4 , 2 , 0.4 ppm ) were prepared in acn / w ( 1:9 ) and 20 l was injected . the hplc - uv analysis worked in optical absorbance detection at 242 nm and major peaks of da was eluted approximately at 12. the high - resolution method was performed using an agilent 1100 instruments equipped with binary pump and an auto sampler . phenomenex luna 3  pfp ( 1502.00 mm ) was used for chromatographic separation , this column has the unique chemistry of a pentafluorophenyl which increases selectivity and reproducibility of compounds . the isocratic mobile phase consisted of a mixture of 0.1% aqueous acetic acid and acetonitrile in the ratio 90:10 ( v / v ) was used . sample solutions ( 0.4 , 0.2 , 0.1 , 0.05 , 0.025 , 0.0125 , 0.00625 ppm , r=0.985 ) were prepared in acn / w ( 1:9 ) and 5 l was injected . the esi / tof analysis worked in positive mode , and mass range was set at m / z 100 - 650 u at a resolving power of 10,000 . the conditions of esi source were as follows : drying gas ( n2 ) flow rate , 8 ml / min ; drying gas temperature , 300c ; nebuliser , 45 psig ; capillary voltage , 4000 v ; fragmentor 225 v ; skimmer voltage , 60 v. acquisition and analysis of data were controlled by agilent lc - ms tof software ( agilent , santa clara , ca , usa ) . tuning mix ( g1969 - 85001 ) was used for lock mass calibration in our assay . under these conditions , major peaks of da appeared as the protonated ion at m / z 312 , being accompanied by minor peaks consisting of sodium - binding ions at m / z 334 and the ion fragment m / z 266 represents loss of hcooh from the protonated da ion ( figure 2 ) . the fragmentation pattern observed was found to be highly diagnostic for these molecules which , combined with the retention time , provide a high specificity for the molecule identification . the quantitation limit for domoic acid by this method is , at least , 0.005 mg / kg . the method has been tested for domoic acid determination by quasimeme proficiency test providing always satisfactory results . 182 mussel samples were analysed since january 1 till july 31 , 2015 by the official method in hplc - uv ( quilliam and wright , 1989 ) and among these , 78 were selected randomly for esi - tof analysis . the procedure for handling shellfish tissue bivalve mollusc were cleaned free of shell before initiating the extract protocol on the remaining soft components . da was extracted from the mussels tissue by homogenisation with methanol - water ( 1:1 , v / v ) . 4 g of this homogenate tissue were placed in a 50 ml plastic conical tube and combined with 16 ml of extraction solvent ( 50% aqueous methanol ) and homogenised again for 2 min to facilitate extraction of da by the solvent . the mixture was then either centrifugated at 3000 rpm for 10 minutes and the supernatant , representing the sample extract , was injected in the hplc - uv or , for the esi - tof analysis , filtered again through a 0.22 m pore diameter membrane ( millipore ) and centrifugated at 12,000 rpm for 10 minutes . the results of 182 samples of mussels have always been below the limits of sensitivity of the official method ( with the exception of one sample quantified by 3.1 mg / kg ; figure 3 ) . in the mussel sample quantified by 3.1 mg / kg the official method is capable to identify only the domoic acid itself , whereas the esi - tof method allowed us to identify the presence of the toxin and its isomers ( figure 4 ) . the identity of the domoic acid isomers is only supposed because of the lack of their reference material for the certain identification . among the 78 mussel samples analysed by esi - tof , 51 ( 65% ) showed the presence of domoic acid whereas in 27 ( 35% ) the amount of biotoxin was not quantifiable ( < 0.00125 mg / kg ) . current analytical techniques used to detect environmental contaminants are generally designed to target only a fraction of these compounds and have not yet been adapted to the detection and the identification of unknown substances . it is however crucial to study the presence of contaminants at lower levels compared to the official limits . new analytical techniques are thus necessary to screen for the presence of contaminants and their degradation products in environmental samples . high resolution - mass spectrometry is a promising technique that has the potential to address the shortcomings of present analytical techniques . the analysis performed by the official method , from january 2015 to late july 2015 , gave always accordant results : the amount found showed lower levels than the regulatory action level ( 20 mg / kg ) . the analysis performed by esi - tof , more sensitive , has instead revealed a frequent presence of domoic acid . the presence of this toxin , albeit minimal , it still indicates the presence of toxic algae blooms such pseudonitzschia whose sudden must always be kept under control . although the monitoring programs under way seems to be effective in preventing acute poisoning by algal biotoxins , repeated exposure to low levels of toxins may cause long - term consequences ( which have yet to be identified ) .
algal biotoxins , chemical compounds produced by some microscopic algae , constitute the phytoplankton . 
 the mussels , feeding on phytoplankton , can accumulate these compounds to become themselves toxic . 
 there have been several cases of food poisoning by consumption of contaminated shellfish . 
 such food poisoning have pushed our health care system to provide monitoring of shellfish in the framework of the monitoring plans carried out by aassll . in this paper 
 we report the results obtained monitoring the presence of amnesic shellfish poisoning biotoxins , like domoic acid and its isomers , produced by pseudonitzschia algae . 
 the analyses were carried out by using both the high - performance liquid chromatography - ultraviolet official method and an experimental method performed with a time - of - flight mass spectrometer ( esi - tof ) . 
 the 100% of samples analysed by the official method have always been below the limits of sensitivity ( except one sample ) , and the 65% of samples analysed by esi - tof showed the presence of domoic acid .