Capillary Electrophoresis of Insulin-like Growth Factors: Enhanced Ultraviolet Detection Using Dynamically Coated Capillaries and On-Line Solid-Phase Extraction

Abstract

Insulin-like growth factor-I and -II (IGF-I and IGF-II) are difficult to separate and measure as a result of their homology, both structurally and immunologically. A number of binding proteins (BPs) which interact with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly. Current methodology for measuring IGF is immuno-based and involves dissociation from the IGFs and removal of the binding proteins through sample acidification and removal by solid-phase adsorption. However, the net result is an assay that is time-consuming and, at best, semiquantitative. In an attempt to improve the reproducibility and accuracy of IGF-I and -II measurement, electrophoretic systems employing dynamically coated and bare silica capillaries were evaluated. Separations in bare silica capillaries in the presence or absence of the cationic additive, decamethonium bromide were ineffective for resolving IGF-I and IGF-II. However, when the capillary was coated dynamically with polybrene, IGF-I and -II could be resolved in a BSA sample matrix using a low pH buffer. Despite the fact that the IGFs could be resolved in the presence of an IGF-I analog used as an internal standard, polybrene recoating was required after as few as 12 runs and poor coating-to-coating reproducibility was observed. Use of polydiallyldimethylammonium chloride (PDMAC) as a dynamic cationic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -II. It was found that PDMAC need not be included in the separation buffer to obtain reproducible analyses regarding IGF separation. Subsequently, functionality remained intact for as many as 35–40 consecutive analyses before recoating was required. Without the need for PDMAC in the buffer, on-line solid-phase extraction–capillary electrophoresis could be accomplished for detection of IGF-I and -II at concentrations as low 195 ng/ml.