Optimizing LC-MS-MS determination of microcystin toxins in natural water and drinking water supplies

Abstract

The performance of a solid phase extraction (SPE) LC-MS-MS procedure for determining microcystins (MC) in water was investigated. Six MC, MCRR, MCYR, MCLR, MCLA, MCLF and MCLW, were determined using positive ion electrospray (+ESI) with separation on a C8 column eluted with a methanol–water gradient containing 15 mM ammonium formate and 0.006% acetic acid. Each MC, as well as the internal standard and surrogate, were detected by two transitions. Isocratic and gradient separations were examined to increase the resolution of MCRR and MCLF as all known naturally occurring MC elute between these two variants. Analysis of fortified laboratory reagent water as well as bloom samples from northern California provided performance information. Cell lysis, accomplished by freeze-thawing samples, was required to determine total sample toxins as 93 to 99% of the toxins were intracellular. Up to 98% of dissolved MC were lost by sorption in filter cartridges, but satisfactory recoveries were obtained with polypropylene filters after the filtrate was combined with a methanol rinse. C18 SPE required elution with trifluoroacetic acid (TFA) for acceptable recoveries of the arginine-containing MC. Freezing was required to preserve MC which degraded rapidly by microbial metabolism at 4 °C after an approximate 10 day lag period. With the optimized method the experimental Method Detection Limits (MDL) for both quantitation and confirmation transitions for the arginine-containing MC ranged from 0.03 to 0.04 μg L⁻¹. Laboratory reporting limits (or LOQ) of 0.1 and 0.2 μg L⁻¹ were confirmed by analyzing surface water samples where accuracy averaged 65 to 70%. Measurement of total MC in bloom samples was reproducible with relative standard deviations of 8.3 to 12%. The study demonstrates the importance of sample preservation, sample workup and quality control procedures in reliable MC determination using modern LC-MS-MS instruments.