# US 2018/0321237 A1 · Protocol for Preconcentration and Quantification of Microcystins Using LC-MS > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2018/0321237 A1 - Document ID: US20180321237A1 - Status: Published application - Kind: A1 - Application number: 16/033,496 - Filed: 2018-07-12 - Published: 2018-11-08 - Earliest claimed: 2016-10-21 - Assignees: The University of Toledo - Applicants: The University of Toledo - Inventors: Dragan Isailovic; Dilrukshika Palagama; David Baliu-Rodriguez; Bruce S. Levison; David J. Kennedy; Steven T. Haller; Kenneth Hensley - Web page: https://patentgrep.com/patent/US20180321237A1 ## CPC - G01N 33/56911: {Bacteria} - G01N 1/405: {by adsorption or absorption} - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 2001/4027: {evaporation leaving a concentrated sample} - G01N 2333/195: from bacteria - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Methods for detecting and quantifying one or more microcystin compounds in a sample are described. The methods may include a preconcentration step, and generally utilize an LC-MS or LC-MS/MS analysis with an Orbitrap Fusion mass spectrometer or a QqQ mass spectrometer. The methods provide excellent recoveries and limits of quantification of microcystins. ## Claims ### Claim 1 A method for detecting and quantifying a microcystin compound in a sample, the method comprising: purifying a sample containing a microcystin compound in plasma, serum, or urine through a solid-phase extraction to obtain a purified sample; conducting a quantitative analysis on the purified sample to quantify the amount of microcystin in the preconcentrated sample, wherein the quantitative analysis comprises a liquid chromatography step and a mass spectrometry step; and using a calibration curve to determine the concentration of the microcystin compound in the sample. ### Claim 2 (depends on 1) The method of claim 1, wherein the quantitative analysis comprises HPLC-orbitrap-MS. ### Claim 3 (depends on 1) The method of claim 1, wherein the quantitative analysis comprises HPLC-QqQ-MS/MS. ### Claim 4 (depends on 1) The method of claim 1, wherein the sample contains a microcystin compound in plasma or serum, and the method further comprises adding ZnSO4 to the sample. ### Claim 5 (depends on 4) The method of claim 4, wherein the ZnSO4 is added at a concentration of about 100 mM. ### Claim 6 (depends on 4) The method of claim 4, further comprising acidifying the sample with formic acid. ### Claim 7 (depends on 6) The method of claim 6, further comprising subjecting the sample to SPE and analyzing the sample by HPLC-orbitrap-MS. ### Claim 8 (depends on 1) The method of claim 1, wherein the sample contains a microcystin compound in plasma or serum, and the purifying comprises eluting the sample with methanol containing formic acid. ### Claim 9 (depends on 1) The method of claim 1, wherein the sample contains a microcystin compound in urine, and the purifying comprises eluting the sample with acetonitrile and formic acid. ### Claim 10 (depends on 1) The method of claim 1, wherein the sample contains multiple microcystin compounds, and the liquid chromatography step separates the multiple microcystin compounds. ### Claim 11 (depends on 10) The method of claim 10, wherein the liquid chromatography step comprises gradient high-performance liquid chromatography. ### Claim 12 (depends on 1) The method of claim 1, wherein the method is capable of quantifying the microcystin compound at a concentration in the sample as low as about 130 ppt when the sample comprises urine, or as low as about 250 ppt when the sample comprises plasma or serum. ### Claim 13 (depends on 1) The method of claim 1, wherein the microcystin compound is selected from the group consisting of MC-LR, MC-LA, MC-LF, MC-LW, MC-YR, and MC-RR. ### Claim 14 A method of preconcentrating mycrocystin in a sample, the method comprising: conditioning a solid-phase extraction (SPE) cartridge with methanol containing formic acid; loading a sample containing a microcystin at a first concentration onto the solid-phase extraction cartridge; eluting the sample with methanol containing formic acid, and collecting the eluted sample; evaporating solvent from the eluted sample to obtain an evaporated sample, and redissolving sample in the solvent to obtain a preconcentrated sample, wherein the preconcentrated sample contains microcystin at a second concentration, the second concentration being greater than the first concentration. ### Claim 15 (depends on 14) The method of claim 14, further comprising equilibrating the SPE cartridge with formic acid. ### Claim 16 (depends on 14) The method of claim 14, further comprising a washing step. ### Claim 17 (depends on 14) The method of claim 14, further comprising subjecting the preconcentrated sample to LC-MS analysis. ### Claim 18 (depends on 14) The method of claim 14, wherein the SPE cartridge is a C18 cartridge. ### Claim 19 (depends on 14) The method of claim 14, wherein the sample comprises a plurality of microcystin species. ### Claim 20 (depends on 19) The method of claim 19, wherein the plurality of microcystin species includes two or more of MC-LR, MC-LA, MC-LF, MC-LW, MC-YR, and MC-RR.