Quantitative analysis of pharmaceutically active peptides using on-capillary analyte preconcentration transient isotachophoresis

Abstract

An on-capillary adsorptive phase in combination with capillary electrophoresis (CE), frequently referred to as preconcentration CE, for quantitative analysis of low peptide concentrations was developed. The capillary containing the on-line analyte preconcentrator can be constructed within 5 min from commercially available extraction disks. These disks contain poly(styrenedivinylbenzene) adsorbent particles incorporated in a matrix of inert Teflon, creating a mechanically stable sorbent. Therefore, no frits are needed in the capillary to hold the stationary phase in place. Several parameters, such as the required minimal elution volume, required elution strength, sample application speed or ionic strength, and the capacity were investigated and special interest was given to the quantitative properties of the method. Instead of nL injections, volumes up to a least 25 microL are possible, yielding improvements in detection limits of 3-4 orders of magnitude. The observed limit of detection for both model peptides was 20 pg, corresponding to a 20 microL injection of a 1 ng/mL solution of both model peptides. Using low-wavelength UV detection, reproducibility and linearity in the low nanogram range were satisfactory. No influence of matrix salt concentrations was observed, extending the use of CE to all kinds of samples.

Quantitation in capillary electrophoresis-mass spectrometry

“…The SPE-CE-MS approach has especially been applied in the context of peptide analysis [33,[64][65][66]. The usefulness of online SPE-CE-MS for quantitative measurements has been shown by Waterval et al [67]. Different materials enable the selective enrichment of analytes from complex matrices [68].…”

Recent advances in capillary electrophoresis of peptides

“…The effect of sample composition on the initial stacking conditions was shown by computer simulation of CE separation of oligoglycines samples with different content of residual acetic acid [40]. Combination of on-capillary adsorptive phase with polymeric adsorbent placed between preconcentration and separation capillary and transient ITP as preconcentration technique for substance eluted from the sorbent has been applied for quantitative analysis of pharmaceutically active peptides [41]. Transient ITP contributed to the enhanced sensitivity in the CE-MS analysis of tryptic peptides of cytochrome c [42].…”

“…The relationship between injection volume of a dilute sample and its concentration at peak maximum in an ITP zone entering the ZE phase of ITP-CZE separation and the correlation between sensitivity of counterflow ITP-CZE system and injection volume of diluted solution of angiotensins was investigated by Chen et al [34]. Transient ITP has been employed to increase the sensitivity of CE analysis of pharmacologically active peptides [41] and tryptic peptides of cytochrome c. CITP is especially suitable for determination of low-molecularmass ionic admixtures in peptide preparations, as it was reported in the previous review [1]. Transient ITP has been employed to increase the sensitivity of CE analysis of pharmacologically active peptides [41] and tryptic peptides of cytochrome c. CITP is especially suitable for determination of low-molecularmass ionic admixtures in peptide preparations, as it was reported in the previous review [1].…”

Recent progress in capillary isotachophoresis

“…The same preconcentration principle was used to enhance the sensitivity of peptide analysis by CZE-MS with a sheath liquid electrospray interface [112]. Transient cationic ITP preconcentration was combined with the use of an on-capillary adsorptive phase to analyze pharmaceutically active peptides (BGE = TE: citric acid; LE: NH 4 OH; elution solvent: 80% v/v acetonitrile/ water) [113]. Interfacing of ITP with Fourier transform ion cyclotron resonance-MS was tested on analyses of proteins and oligonucleotides and has shown to be effective for separating minor components of protein mixtures for on-line mass spectral analysis (LE: 7 mM HCl, 13 mM balanine; TE: 10 mM caproic acid) [114].…”