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.

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.

In this paper, an on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and characterization of endogenous microRNA (miRNA) and their post-transcriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-5p) and hsa-let-7g-5p (let-7g-5p). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions with standards, the microcartridge lifetime (>25 analyses) and repeatability (2.8% RSD for the migration times; 4.4 and 6.4% RSD for the miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25 and 100 nmol·L, and the limit of detection (LOD) was around 10 nmol·L (50 times lower than by CE-MS). In order to analyze human serum samples, an off-line sample pretreatment based on phenol/chloroform/isoamyl alcohol (PCA) extraction was necessary prior to SPE-CE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated by an analysis of serum samples from healthy controls and patients. MicroRNAs, specifically miR-21-5p and a 23 nucleotide long 5'-phosphorylated miRNA with 3'-uridylation (iso-miR-16-5p), were only detected in the CLL patients.

In this study, we present the use of microreactors packed with immobilized trypsin particles for the rapid and efficient bottom-up analysis of proteins by on-line immobilized enzyme microreactor capillary electrophoresis mass spectrometry (IMER-CE-MS). The results obtained digesting β-lactoglogulin (β-LG) off-line with free trypsin in solution and with immobilized trypsin particles were taken as a reference for the optimization of the on-line protein digestion. Under the optimized conditions, online digestion, separation and characterization of the protein digests were possible in less than 30 min. The limit of detection for complete sequence coverage was around 10 µg mL-1 (~500 µM) of β-LG, the repeatability was comparable to the off-line digestion methods and the microreactor could be reused until thirty times. The good performance of IMER-CE-MS was also demonstrated for several other proteins as α-casein (α-CSN), β-casein (β-CSN), and κ-casein (κ-CSN), as well as for a complex protein mixture (an Escherichia coli whole cell lysate).

In this paper, an on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry method is described for the purification, preconcentration, separation, and characterization of α-synuclein (α-syn) in blood at the intact protein level. A single-stranded DNA aptamer is used to bind with high affinity and selectivity α-syn, which is a major component of Lewy bodies, the typical aggregated protein deposits found in Parkinson's disease (PD). Under the conditions optimized with recombinant α-syn, repeatability (2.1 and 5.4% percent relative standard deviation for migration times and peak areas, respectively) and microcartridge lifetime (around 20 analyses/microcartridge) were good, the method was linear between 0.5 and 10 µg•mL -1 and limit of detection was 0.2 µg•mL -1 (100 times lower than by CE-MS, 20 µg•mL -1 ). The method was subsequently applied to the analysis of endogenous α-syn from red blood cells lysate of healthy controls and PD patients.Capillary electrophoresis-mass spectrometry (CE-MS) is regarded nowadays as a powerful technique for the highly efficient separation and characterization of biomolecules, including peptides, protein isoforms and post-translational modifications (PTMs) or protein complexes [1][2][3][4] . However, as in many other microscale separation techniques, the small sample volume injected for optimum separation (typically 1-2% of the capillary volume) compromises the concentration sensitivity for most analytes and is very often a limitation that hinders a more widespread application [5][6][7][8][9] . To improve the limits of detection (LODs), the use of more selective and sensitive mass spectrometers is in many cases not satisfactory enough. Therefore, for the on-line preconcentration of the target analytes after the injection of a large volume of sample, CE-MS has been often combined with different electrophoretic 5,6 and chromatographic 6-9 techniques.Within the chromatographic preconcentration techniques, on-line solid-phase extraction capillary electrophoresis (SPE-CE) is widely recognized as an excellent option to preconcentrate and clean up the target analytes, minimizing sample handling and increasing analysis throughput. In the most widely used SPE-CE configuration, a microcartridge with an appropriate sorbent to selectively retain the target analyte is integrated in-line near the entrance of the separation capillary and no valves are necessary for the operation. After loading a large volume of sample (∼50-100 μL), the capillary is rinsed to eliminate non-retained molecules and filled with background electrolyte (BGE). Then, the analyte is eluted and preconcentrated in a small volume of an appropriate solution (~25-50 nL) before the separation and the detection 8,9 .Nowadays, the availability of a wide variety of commercial or lab-made sorbents has broadened the applicability of SPE-CE. SPE-CE has been explored using conventional chromatographic sorbents (e.g. C18 or HLB) 7-9 , but there is an urgent need of high selective affinity sorbents to anal...