Analysis of neuropeptide Y and its metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger.
A novel restricted access cation exchanger with sulphonic acid groups at the internal surface was proven to be highly suitable in the sample clean up of peptides on-line coupled to HPLC-electrospray ionization (ESI)-MS. Neuropeptide Y (NPY) and several of its fragments in plasma were subjected to the sample clean-up procedure. The peptides were eluted by a step gradient from the restricted access column, applying 10 mM phosphate buffer pH 3.5 from 5 to 20% (v/v) of acetonitrile with 1 M NaCl and transferred to a Micra ODS II column (33x4.6 mm). The separation of the peptides and their fragments was performed by a linear gradient from 20 to 60% (v/v) acetonitrile in water with 0.1% formic acid and 0.01% trifluoroacetic acid in 4 min at a flow-rate of 0.75 ml/min. An integrated and completely automated system composed of sample clean up-HPLC-ESI-MS was used to analyze real life samples. The sample volumes ranged between 20 and 100 microl. Peaks due to the fragments NPY 1-36, 3-36 and 13-36 in porcine plasma were identified by ESI-MS. The limit of detection was in the 5 nmol/ml range. The total analysis required 21 min and allowed the direct injection of plasma.
Racaityte
K
K
Kaunas University of Technology, Department of Organic Technology, Lithuania. kristinara@yahoo.com
Lutz ESM
Unger
K K
KK
Lubda
D
D
Boos
K S
KS
D000595:Amino Acid Sequence; D000818:Animals; D002411:Cation Exchange Resins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000032:analysis* / Q000378:metabolism; D013451:Sulfonic Acids / Q000737:chemistry
D002411:Cation Exchange Resins; D009478:Neuropeptide Y; D013451:Sulfonic Acids