Analysis of microcystins LR, YR, and RR in biological fluids by 2D-LC technology
Renner, Beatriz Jael
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Algae “super blooms” are a commonly encountered environmental issue in fresh and brackish water that occurs due to the buildup of cyanobacteria. Many of the commonly encountered cyanobacteria such as Mycrocystis aeruginosa (M. aeruginosa) produce potent cyanotoxins (microcystins) that pose serious health threats and even death to local wild life and humans. Microcystin contaminated fresh-water that empties into the ocean has been shown to lethally affect marine life in the area of contamination. Human consumption of tainted sea life and other routes of mycrocystin exposure can lead to serious liver damage and even death. Thus, a method was developed for forensic postmortem analysis of microcystins RR, LR and YR by Two-Dimensional (2D) Liquid Chromatography (LC) - tandem Mass Spectrometry (MS/MS). A final 2D LC-MS/MS method was selected from 6x6 automated method development experiments. Each microcystin were subjected to a total of 36 methods, which were completed over an 18hr period. The extraction process was performed using a reverse-phase sorbent (Oasis HLB, Waters Corporation, Milford, MA) with a 3cc solid phase extraction (SPE) barrel using sequential elution. From acetonitrile (ACN) and methanol (MeOH) stock solutions, 10 μL of the internal standard (IS) Nodularin was added to the final extract. The concept of sequential micro extraction was designed to capture the retention behaviour of the target analyte in response to various extraction parameters (sorbent strength, elution polarity, and solubility). Therefore, optimized conditions were selected to excise the region of interest during extraction. The elution solvents chosen for the microcystins were acetonitrile, methanol and acetone with 10 % sequential increments. Since microcystins exhibit a zwitterionic structure, three sets of elution solutions were created to evaluate their elution profile (pH 3, pH 7, pH 10). When the elution profile for low pH and high pH are compared, microcystin RR was eluted over the 40% to 70% methanol fractions under low pH conditions with a slight shift towards higher organic % (50%-70% fractions) under high pH. This elution behaviour suggests that the basic moieties of the structure demonstrate a stronger retention for the stationary phase. Microcystin LR and YR however, eluted at a higher organic solvent percentage under low pH conditions and at a lower organic solvent percentage under high pH conditions, indicates that the acidic moieties of the structures have stronger retention. The urine sample gave recovery values for all three microcystins in the 80-90% range, as to be expected with type of complexity associated with biological samples. The sequential extraction protocol produced an extraction method that delivered a clean extract after a 30 min workflow using a single and optimized 2D LC-MS/MS method. The total analytical run time was set at 10 minutes.