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.

Robust and cost-effective capillary electrophoresis-mass spectrometry interfaces suitable for combination with on-line analyte preconcentration

“…In a previous paper, using UV detection, the analyte preconcentration system was validated for two model peptides on factors like capacity, linearity, reproducibility, and robustness [21]. The on-line stationary phase is able to trap the analytes from several injected capillary volumes of sample based on nonspecific hydrophobic interactions between the analytes and the adsorptive polymer.…”

“…An on-line preconcentration system [21], comprising a small piece of absorbing polymer at the beginning of the capillary traps the analytes in a nonselective way when several capillary volumes of sample are injected. After desorption, electrophoretic separation of the concentrated analytes takes place in the capillary.…”

On-line sample preconcentration in capillary electrophoresis, focused on the determination of proteins and peptides

“…These chromatographic mechanisms can be of low specificity or of high specificity. Basically, three methods are available using a low-specific chromatographic mechanism: (i) solid adsorptive phase chromatography, including solid-phase extraction (SPE) and membrane preconcentration (mPC) [2,[16][17][18][19][20][21][22][23][24], (ii) liquid distribution phase chromatography, including electroextraction (EE) [2] and supported liquid membranes (SLM) [25,26], and (iii) hollow fibers [27,28]. Low-specific chromatography is probably the most widely used application of chromatographic retention.…”

“…In a study, using three different types of reversed-phase preconcentration, a limit of detection of 20 pg, using UV detection, could be obtained, corresponding to a 20 mL injection of a 1 ng/mL solution of a model peptide [17]. mPC and spPC are valuable methods of CZE preconcentration, especially for analysis of biological samples.…”

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration

“…However, CE has intrinsic poor concentration sensitivity, due to a low loading capacity. Several solutions have been brought up to increase the sample loadability without loss of resolution, such as field-amplified sample stacking [2][3][4][5][6], transient isotachophoresis [7][8][9][10][11][12][13][14][15][16][17][18][19], and on-capillary preconcentration on a stationary phase [20][21][22][23][24][25][26][27][28][29][30][31]. The on-capillary preconcentration has the benefit that sample loading is not limited to the capillary volume and that on-line purification of the applied sample can be accomplished by employing washing steps.…”