CAPILLARY ELECTROPHORESIS OF INSULIN-LIKE GROWTH-FACTORS - ENHANCED ULTRAVIOLET DETECTION USING DYNAMICALLY COATED CAPILLARIES AND ONLINE SOLID-PHASE EXTRACTION

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 structural ly and immunologically, A number of binding proteins (BPs) which inter act with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly, Current methodology for measuri ng IGF is immune-based and involves dissociation from the IGFs and rem oval 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 impro ve the reproducibility and accuracy of IGF-I and -II measurement, elec trophoretic systems employing dynamically coated and bare silica capil laries were evaluated, Separations in bare silica capillaries in the p resence or absence of the cationic additive, decamethonium bromide wer e ineffective for resolving IGF-I and IGF-II. However, when the capill ary was coated dynamically with polybrene, IGF-I and -II could be reso lved in a BSA sample matrix using a low pH buffer, Despite the fact th at 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 catio nic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -I I, It was found that PDMAC need not be included in the separation buff er to obtain reproducible analyses regarding IGF separation, Subsequen tly, functionality remained intact for as many as 35-40 consecutive an alyses before recoating was required, Without the need for PDMAC in th e buffer, on-line solid-phase extraction-capillary electrophoresis cou ld be accomplished for detection of IGF-I and -II at concentrations as low 195 ng/ml. (C) 1998 Academic Press.