Online large volume sample staking preconcentration and separation of enantiomeric GHRH analogs by capillary electrophoresis

Abstract

AbstractA capillary electrophoresis method is proposed to analyze the four most well‐known growth hormone–releasing hormone (GHRH) analogs that are misused by athletes. Dimethyl‐β‐cyclodextrin used as a chiral selector allowed, for the first time, the separation of those basic peptide analogs, including enantiopeptides (sermorelin and CJC‐1293) that differ by the chirality of only one amino acid. To increase the method sensitivity, electrokinetic preconcentration methods have been investigated. The large volume sample stacking with polarity switching (PS‐LVSS) method with an injected sample volume corresponding to 80% of the capillary one was found superior to the sweeping in terms of signal enhancement factor (SEF). Acid and organic solvent addition to the sample (0.1 mM phosphoric acid with 30% methanol) led to a twofold signal improvement, when compared to water as a matrix. We increased capillary dimensions to provide a signal enhancement through the injection of a larger sample volume. Finally, using a combination of the optimized PS‐LVSS preconcentration with the chiral capillary zone electrophoresis (CZE), the GHRH analogs were separated and limits of detection between 75 and 200 ng/mL were reached. This method was successfully applied to urine after a desalting step. An optimized C18 SPE was used for that purpose in order to provide low sample conductivity (<130 µS/cm) and preserve the efficiency of LVSS preconcentration. SEF of 640 was obtained with desalted urine spiked with sermorelin by comparison to the CZE (without preconcentration) method.