Optimized extraction and analytical protocols for guanitoxin: an organophosphate cyanobacterial neurotoxin of environmental concern

Abstract

This study evaluated cell growth, chlorophyll-a production, and guanitoxin (GNT) synthesis by the cyanobacterium Sphaerospermopsis torques-reginae (strain ITEP-24) in different culture media over 52 days. The maximum total GNT production was observed in Z8 medium, peaking on the 45th day, indicating potential for biotechnological studies. BG-11 with 5% nitrogen offered a good balance between yield and stability, while ASM-1 with 5% nitrogen showed higher GNT production efficiency per cell, suggesting suitability for metabolic studies. An optimized extraction protocol using 70% methanol with 0.3% acetic acid and ultrasonic bath lysis proved most effective, ensuring high intracellular recovery and molecular integrity. For semi-purification, silica gel SPE cartridges (500 mg) achieved 61% recovery, surpassing the 48% obtained with C18. Both methods resulted in considerable losses, primarily due to the toxin's inherent chemical lability. Based on these tests, the optimized methodology utilizing a silica-filled glass column facilitated the semi-isolation of 1.2 mg of GNT from 1.375 g of dry biomass. Freeze-drying of enriched fractions proved reliable when temperature fluctuations were avoided, and detection was achieved with high sensitivity and selectivity by LC-HR-QTOF-MS (ZIC-HILIC column). These advances support scale-up feasibility, reference standards, toxicological assays, environmental monitoring, and public health protection.