Online capillary liquid–liquid electroextraction of peptides as fast pre‐concentration prior to LC‐MS

Abstract

In this research paper, we show that capillary electroextraction (cEE) is capable of fast online peptide concentration and that it can be coupled online to LC-MS to result in a fast and sensitive method. Electroextraction takes place when an electrical field is applied in a two-phase liquid-liquid system. Sample molecules in the organic phase migrate very fast into the aqueous phase and are concentrated in a small zone. In this work, cEE of peptides is developed and coupled online to LC-MS via a switching valve. Comparison of 10 min of cEE-LC-MS with a normal LC-MS injection showed more than 100-fold increased peak heights. Of five model peptides, good calibration curves in the range of 0.05-5 μmol/L were obtained. The linearity was good (R(2) values between 0.984 and 0.996) and RSD between 5% at the highest to 25% at the lowest concentration (n=3). The LOD of bradykinin, angiotensin I-converting enzyme inhibitor and angiotensin I was in the low nmol/L range. Analysis of a tryptic digest of eight model proteins resulted in more than 170 peptides, without bias for pI or hydrophilicity. Urine analysis is demonstrated, resulting in an LOD around 0.04 μmol/L urine for tryptic cytochrome C peptides spiked to urine and an increase of 42% in the number of chromatographic peaks compared with the conventional LC-MS. In summary, cEE-LC-MS is a fast electrophoresis-driven sample preconcentration technique that is quantitative, able to extract a wide peptide range and applicable to bioanalysis.