title: On-line coupling of size-exclusion chromatography and capillary zone electrophoresis via a reversed-phase C18 trapping column for the determination of peptides in biological samples.
abstract: Since biologically active peptides usually exhibit their effects in low concentrations, the development of sensitive analytical methods has become a challenge. In this paper, a multidimensional system is presented, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on a 4 mm x 3 mm ID reversed-phase C18 (RP18) column with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE). The system has been tested with mixtures of six enkephalins and albumin, mimicking biological matrices such as plasma and cerebrospinal fluid. After separation of albumin from the enkephalins in the SEC dimension a heart-cut of 200 micro L, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution with a 20 micro L plug of 80% acetonitrile, electrokinetically injected into the CZE system, where stacking and separation is achieved. While validation shows generally good linearity and reproducibility, the quantitation limit with UV detection is acceptable (2.5 micro g/ mL with an injection volume of 50 micro L).
authors.affiliation: Department of Biomedical Analysis, Faculty of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Thom
authors.initials: T
authors.lastname: Stroink
authors.forename: Pim
authors.initials: P
authors.lastname: Schravendijk
authors.forename: Gerard
authors.initials: G
authors.lastname: Wiese
authors.forename: Jan
authors.initials: J
authors.lastname: Teeuwsen
authors.forename: Henk
authors.initials: H
authors.lastname: Lingeman
authors.forename: Joop C M
authors.initials: JC
authors.lastname: Waterval
authors.forename: Auke
authors.initials: A
authors.lastname: Bult
authors.forename: Gerhardus J
authors.initials: GJ
authors.lastname: de Jong
authors.forename: Willy J M
authors.initials: WJ
authors.lastname: Underberg
mesh_terms: D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004745:Enkephalins / Q000032:analysis* / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008970:Molecular Weight; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry*
chemical_list: D004745:Enkephalins; D012709:Serum Albumin