On-line coupling of size exclusion chromatography and capillary electrophoresis via solid-phase extraction and a Tee-split interface.
An on-line size exclusion chromatography (SEC)-solid-phase extraction (SPE)-capillary electrophoresis (CE) system using a Tee-split interface has been developed for the analysis of peptides in biological fluids. The SEC column fractionates the sample by molecular size and the low-molecular-weight fraction, which contains the peptides, is directed to a C(18) SPE microcolumn, where the peptides are trapped and concentrated. The SPE column is desorbed with 425 nL acetonitrile and the effluent is sent to the Tee-split interface, which hydrodynamically splits (1:40) the flow and, thus, allows appropriate injection of analytes into the CE system. The performance of the system is investigated by the analysis of enkephalins in cerebrospinal fluid (CSF). It is demonstrated that the SEC step efficiently removes potentially interfering proteins, permitting reproducible SPE and CE. The total system provides efficient separations of the enkephalins with plate numbers up to 100,000. Concentration limits of detection (S/N = 3) for the peptides are about 100 ng/mL for injection of 20 microL spiked CSF samples. Plots of enkephalin peak areas versus concentration showed good linearity over the 0.25-10 microg/mL range (R2 > or = 0.985). Repeatability of migration time and peak area was within 2% and 10% R.S.D., respectively.
Tempels
F W Alexander
FW
Department of Biomedical Analysis, Faculty of Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, NL-3584 CA Utrecht, The Netherlands. f.w.a.temples@pharm.uu.nl
Wiese
Gerard
G
Underberg
Willy J M
WJ
Somsen
Govert W
GW
de Jong
Gerhardus J
GJ
D002850:Chromatography, Gel / Q000379:methods*; D019075:Electrophoresis, Capillary; D006801:Humans; D009862:Online Systems; D010455:Peptides / Q000134:cerebrospinal fluid* / Q000737:chemistry; D013997:Time Factors
D010455:Peptides