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

Study · human · Electrophoresis · 2023 · DOI 10.1002/elps.202200278 · PMID 36787346

Plain-language summary

Paraphrased from the published abstract below — not a verdict on whether anything works.

This method-development study describes a capillary electrophoresis technique to separate four GHRH analogs, including enantiopeptides sermorelin and CJC-1293, using dimethyl-β-cyclodextrin as a chiral selector. A large volume sample stacking with polarity switching (PS-LVSS) method, with injected sample volume corresponding to 80% of capillary volume, showed a greater signal enhancement factor (SEF) than sweeping. Adding acid and organic solvent to the sample (0.1 mM phosphoric acid with 30% methanol) produced a twofold signal improvement versus water, and larger capillary dimensions further improved signal via larger injection volumes. Combining optimized PS-LVSS with chiral capillary zone electrophoresis (CZE), the GHRH analogs were separated with limits of detection between 75 and 200 ng/mL; applied to desalted human urine spiked with sermorelin, an SEF of 640 was obtained versus CZE alone.

Abstract

A 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.

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