Several strategies have been developed to decrease the concentration limits of detection (LODs) in capillary electrophoresis (CE). Nowadays, chromatographic-based preconcentration using a microcartridge integrated in the separation capillary for in-line solid-phase extraction capillary electrophoresis (SPE-CE) is one of the best alternatives for high throughput and reproducible sample clean-up and analyte preconcentration. This review covers different designs (geometrical configurations, with frits or fritless, capillary types, compatibility with commercial instrumentation, etc.) and materials (sorbents, supports, affinity ligands, etc.) applied for almost 30 years to prepare in-line SPE-CE microcartridges (i.e. analyte concentrators), with emphasis on the conventional unidirectional configuration in capillary format. Advantages, disadvantages and future perspectives are analyzed in detail to provide the reader a wide overview about the great potential of this technique to enhance sensitivity and address trace analysis.

Protein precipitation and centrifugal filtration are well-established methods for concentrating and purifying peptides with a low relative molecular mass (Mr) from human blood plasma before proteomic and peptidomic studies using high-performance separation techniques, but there is little information on peptide recoveries. Here, we evaluate acetonitrile precipitation followed by a range of centrifugal filtration conditions for the analysis of low Mr peptides in human blood plasma before CE–MS and SPE coupled online to CE–MS. Three opioid peptides were used as model compounds, that is, dynorphin A 1–7, endomorphin 1, and methionine enkephalin and 3, 10, and 30 K Mr cut-off cellulose acetate filters (Amicon® Ultra-0.5) and 10 K Mr cut-off polyethersulfone filters (Vivaspin® 500) were studied. Unexpectedly, recoveries and repeatability were only optimum after passivating the 10 K Mr cut-off cellulose acetate filters with PEG to avoid peptide adsorption on the inner walls of the plastic sample reservoir.

In this paper, an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using magnetic beads (MBs) is described for the analysis of serum transthyretin (TTR), which is a protein related to different types of amyloidosis. First, purification of TTR from serum was investigated by off-line immunoprecipitation and CE-MS. The suitability of three Protein A (ProA) MBs (Protein A Ultrarapid Agarose(TM) (UAPA), Dynabeads(®) Protein A (DyPA) and SiMAG-Protein A (SiPA) and AffiAmino Ultrarapid Agarose(TM) (UAAF) MBs to prepare an IA sorbent with a polyclonal antibody (Ab) against TTR, was studied. In all cases, results were repeatable and it was possible the identification and the quantitation of the relative abundance of the six most abundant TTR proteoforms. Although recoveries were the best with UAPA MBs, UAAF MBs were preferred for on-line immunopurification because Ab was not eluted from the MBs. Under the optimized conditions with standards in IA-SPE-CE-MS, microcartridge lifetime (>20 analyses/day) and repeatability (2.9 and 4.3% RSD for migration times and peak areas) were good, the method was linear between 5 and 25 μg/mL and LOD was around 1 μg/mL (25 times lower than by CE-MS, ≈25 μg/mL). A simple off-line sample pretreatment based on precipitation of the most abundant proteins with 5% (v/v) of phenol was necessary to clean-up serum samples. The potential of the on-line method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was demonstrated analysing serum samples from healthy controls and FAP-I patients.

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Transthyretin (TTR) is known to misfold and aggregate, causing different types of amyloidosis. Familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, is associated with a TTR variant that presents a single amino acid substitution of valine for methionine at position 30 (Met 30). To screen for TTR-related amyloidosis rapidly and reliably, we have developed a novel procedure based on the analysis of monomers from the homotetrameric protein (∼56 kDa). First, we established a CZE-ESI-TOF-MS method to detect wild-type (normal) TTR with or without several PTMs, as well as an extra minor isoform in TTR standard solutions. Later, a sample pretreatment based on immunoprecipitation (IP) and centrifugal filtration was optimized to analyze serum samples from healthy controls and FAP-I patients (including an asymptomatic patient, a symptomatic patient, a liver-transplanted patient with the specific mutation, and a patient originally without the mutation who received a liver transplant from an FAP-I patient (iatrogenic FAP-I)). The mutant TTR (Met 30) variant with a relative molecular mass 32.07 higher than the wild-type TTR was found in the asymptomatic, the symptomatic and the iatrogenic FAP-I patients, who interestingly also presented the same concentration ratio between both variants of TTR (abnormal and normal). In contrast, as in the healthy controls, the abnormal TTR variant was not detected in the liver-transplanted patient with the specific mutation, which confirms the effectiveness of the treatment. The proposed procedure could be regarded as a suitable screening system for individuals with suspected TTR amyloidosis, and to gain insight into TTR structure, to understand the mechanism underlying the disease.