LC/MS Analysis of 3-Methoxytyramine and Metanephrines in Horse Urine on Ascentis(R) Express Biphenyl after On-line SPE using Supel(TM)Genie RP-Amide Cartridge

LC/MS Analysis of 3-Methoxytyramine and Metanephrines in Horse Urine on Ascentis® Express Biphenyl after On-line SPE using Supel™Genie RP-Amide Cartridge


SPE tube/cartridge Supel Genie On-line cartridge:  RP-Amide 2 cm x 4.0 mm I.D. (55516-U)
column Ascentis® Express Biphenyl 10 cm x 2.1 mm I.D., 2.7 μm particles (64065-U)
column temp. 25 °C
mobile phase [A] 0.1% formic acid in water; [B] 0.1% formic acid in methanol
gradient 20 to 100% B in 10 min; held at 100% B for 1 min
flow rate 0.5 mL/min
sample 200 ng/mL in horse urine (Sample loading and washing: 0.3 mL/min for 2 min, then the valve switches to in-line with HPLC column before sample loading the trap is equilibrated with the loading solvent for 2.5 min. Sample loading solvent: 5% methanol)
injection 5 μL
detector MS, ESI(+), MRM, m/z 184>166, 198>180, 168>151


Analysis Note 3-Methoxytyramine, metanephrine and normetanephrine in horse urine are directly injected and then analyzed by online SPE LC/MS. After injection, the analytes are retained and cleaned up on an RP-Amide online SPE cartridge, then eluted and baseline separated on an Ascentis Express biphenyl column. The results show ion-pairing agent in the loading mobile phase is critical for the retention of these polar analytes on the RP-Amide cartridges. The recovery of the three analytes from horse urine is 43% - 74%, with reproducibility (RSD) 5.5%-9.1%. The method is linear over the range of 10 – 500 ng/, linearity (r2) being 0.996-0.998..
Featured Industry Forensics and Toxicology
Legal Information Ascentis is a registered trademark of Sigma-Aldrich Co. LLC, Supel is a trademark of Sigma-Aldrich Co. LLC
suitability application for LC-MS


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