Analysis of Microcystins LR, YR, and RR in Urine by 2D-LC/MS/MS

New Organic Monitoring Techniques
Poster Presentation

Presented by J. Lewis
Prepared by B. Renner1, K. Organtini1, K. Rosnack1, C. Mallet1, S. Botch-Jones2
1 - Waters Corporation, 34 Maple St, Milford, MA, 01757, United States
2 - Boston University, 72 E. Concord St., L815B, Boston, MA, 02118, United States

Contact Information: [email protected]; 508-478-2000


Human consumption of tainted sea life can lead to microcystin exposure and even death. Several analytical techniques that have been developed for the analysis of microcystins include the mouse bioassay, phosphatase inhibition assay, enzyme-linked immunosorbent assay (ELISA), and reversed-phase high performance liquid chromatography (RP-HPLC), just to name a few. Little has been done with multi-dimensional chromatography; thus, a method was developed for analysis in urine of microcystins RR, LR, and YR by 2-dimensional (2D) liquid chromatography mass spectrometry (LC-MS/MS). For this application, finding the optimum extraction and chromatographic conditions for this multi-residue analysis posed a significant challenge. The chromatographic conditions were tested on several trapping chemistries and one separation chemistry. The loading (low pH, high pH, and neutral pH) and eluting mobile phase (methanol or acetonitrile at low and high pH) were also optimized using an automated 6 × 6 process. The extraction process was performed on pre-conditioned reversed-phase sorbent for the captive extraction for the screening extraction. The 6 × 6 chromatography method optimization was completed over ~18 hours. The option of direct large volume injections of up to 100% organic extracts made it possible to optimize an extraction protocol with a total work time of <30 minutes. This added benefit of 2D-LC-MS/MS allows direct injections of aqueous and organic extracts at various percentages to effectively eliminate time-consuming evaporation and reconstitution steps. The captive extraction protocol produced a 90% recovery of microcystins YR, LR, and RR in urine.