Monitoring the purity of a synthetic peptide by capillary electrophoresis: utilization of an on-line preconcentration method for improved separation and detection sensitivity.

: To date, there has been a considerable amount of interest and success in the pharmaceutical industry in the discovery of drug targets and diagnostics utilizing peptides. The success of peptide pharmaceuticals has, however, been accompanied by some failures, both prior to entry and in the clinic. Progress has been made in various areas to improve the effectiveness of the final drug product. One major advance has been in the area of peptide synthesis and control of the purity of the peptide of interest. Recent advances in analytical instrumentation, including advances in capillary electrophoresis, have had a great impact on the ability to separate and detect low quantities of impurities and degradation products during the synthesis of a peptide drug. In this work, affinity capillary electrophoresis (ACE) was developed for the identification and characterization of a chemically synthesized peptide fragment of a snake toxin called fasciculin. The affinity capillary electrophoresis technology utilized in this study employed two powerful techniques coupled on-line for the direct and rapid determination of analytes in simple and complex matrices. The first technique, aimed for the nonselective extraction and concentration of one or more analytes of interest, utilizes a solid-phase analyte concentrator device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated target analyte(s), after elution from an analyte concentrator device. The on-line preconcentration step has proved valuable in terms of improving separation conditions as well as enhancing detection sensitivity values for the peptide fragment with a sensitivity increase ranging from 100- to 10,000-fold. Different types of analyte concentrator devices and a few binding-desorption conditions were tested. Bare and internal-wall-coated fused-silica capillaries were used. Comparative performance with HPLC in terms of selectivity and sensitivity is also discussed.