A Novel Diagnostic Method for Detection and Quantitation of Paralytic Shellfish Poisoning in Humans

Abstract

Paralytic shellfish toxins(PSTs) are potent neurotoxins which bind to the voltage gated sodium channels and prevent the conduction of action potentials leading to respiratory paralysis and death. Gonyautoxins(GTXs) 1,2,3,4 are the most potent PSTs and testing them in human matrices is the best approach to quantify the exposures. Here, we describe a solid phase extraction method for extracting GTXs from human plasma using HILIC HPLC-MS/MS analysis. Pooled plasmas were spiked with GTXs and extracted using strong cationic exchange cartridges. These cartridges were conditioned with methanol and acetate buffer and eluted with 5% ammonium hydroxide in methanol. Calibrants were prepared at the following concentration ranges: GTX1 8.13-517.66 ng/mL, GTX2 6.98-473.25 ng/mL, GTX3 2.96-200.68 ng/mL and GTX4 2.56-162.91 ng/mL. Eight plasma specimens were spiked with toxin at a concentration of 173.85ng/ml. All the samples were extracted and injected onto HPLC-MS/MS for analysis. The developed method was validated according to FDA guidance for bioanalytical method validation. The method showed good percent accuracies for all the toxins: GTX1 95-104%, GTX2 92-114%, GTX3 92-117%, GTX4 92-107%. Precision ranged from 3.5 to 10.9% for GTX1, 3.03 to 11.25% for GTX2, 3.01 to 12.72 for GTX3 and 2.08 to 10.49 for GTX4. Recovery of GTXs from specimen plasmas were between 83.27% to 110.55%. We have successfully developed a fast and simple diagnostic method accessible to common clinical laboratories.