This review highlights recent applications of on-line sample preconcentration techniques in capillary electrophoresis (CE) from 2019 to 2021. Various preconcentration techniques based on the analyte velocity change in two or three discontinuous solutions system including field-amplified sample stacking, transient isotachophoresis, pH-mediated stacking, sweeping, and their modified and combined techniques have been employed to enrich and separate biological, environmental, food samples, and so on, in CE. More than 60 published research articles collected from Scopus databases described the dynamic on-line sample preconcentration techniques. This review provides comprehensive tables listing the applications of the on-line sample preconcentration techniques.

A simultaneous determination method for seven β₂-agonists (clenbuterol, ractopamine, zilpaterol, cimaterol, isoxsuprine, salbutamol and terbutaline) was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) to evaluate their residual status in livestock products commercially available in Japan. The livestock sample was homogenized with ethyl acetate under alkaline conditions and degreased with n-hexane. The target β₂-agonists were then purified by dispersive solid-phase extraction with C18 particles. For the LC separation, a semi-micro scale column packed with an octadecylsilica (ODS) stationary phase was employed, and gradient elution was performed for 10 min using aqueous acetonitrile containing ammonium acetate as the mobile phase solvent, where the target compounds were eluted within 5-8 min. The developed method was validated using pig muscle, a processed pork food, cattle muscle and a processed beef food, and the obtained trueness values were 60.1-103.5%. The obtained relative standard deviation (RSD) values were 1.26-8.94% (repeatability) and 1.93-10.55% (intermediate precision). In some of the imported livestock products, small but clear peaks of ractopamine were detected by the developed LC-MS/MS method, and the presence of ractopamine was further confirmed by an LC-MS/MS/MS analysis with higher selectivity.

Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has the great ability to accurately and precisely quantify various biomolecules, but there is a concern about its analysis time, especially during the analysis of a high number of samples. Sample-multiplexing in the same injection is a promising strategy for reducing the total analysis time. This strategy can be accomplished by derivatization of multiple samples with multiple isotopologous reagents. In this study, a sample-triplex LC/ESI-MS/MS assay was developed for quantifying the urinary hexanoylglycine (HG), a diagnostic marker of medium-chain acyl-coenzyme A dehydrogenase deficiency, in three different samples within a single run. For this purpose, the 1-[(4-diethylaminophenyl)carbonyl]piperazine (DEAPPZ) isotopologues (²H₀-, ²H₃- and ²H₆-forms) were synthesized. When compared to the non-derivatization method, which analyzed one sample in each run, the analysis time after the sample pretreatment was reduced to 55% (390 min → 210 min) for 30 samples in the sample-triplex method, which also had an acceptable precision (intra- and inter-assay precisions; ≤ 5.9% and ≤ 9.1%, respectively) and accuracy (91.7–97.1%). Thus, the sample-triplex strategy using the DEAPPZ isotopologues could successfully reduce the analysis time in the urinary HG quantification.

For the simultaneous determination of lactate (LA), 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB) and malate (MA) enantiomers, a new separation technique in a two-dimensional high-performance liquid chromatography (2D-HPLC) system following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole has been developed. The 2D-HPLC system was composed of the reversed-phase (1D) and enantioselective (2D) separations. In the first dimension, the target hydroxy acids were separated as their D plus L mixtures on a Singularity RP18 column (1.0 mm i.d. x 250 mm), and the enantiomers were separated in the second dimension using a polysaccharide-based chiral stationary phase, Chiralpak IG (2.0 mm i.d. x 250 mm). By using the Chiralpak IG column, the LA, 2HB, 3HB and MA enantiomers were separated with resolution values of 3.47, 3.63, 6.81 and 3.72, respectively. The developed 2D-HPLC system was applied to human plasma, and trace levels of D-LA and L-3HB in addition to their major antipodes were determined. The acquired %D of LA (the percentage of D-LA over total LA) and %L of 3HB (the percentage of L-3HB over total 3HB) were 1.0 and 2.7, respectively. For 2HB, only the L-form was detected, while the MA enantiomers were not found in the human plasma.

Super absorbent polymer (SAP) particles, i.e., sodium polyacrylate, were introduced for purge and trap (PT) extraction of methanol in water samples. The purged methanol was extracted by a needle-type extraction device and determined using a gas chromatograph equipped with a flame ionization detector. A functional porous polytetrafluoroethylene membrane was fixed on a solid-phase extraction glass cartridge to retain the aqueous sample while allowing the purge gas to pass through. A sponge was formed by adding 2 g of SAP particles into 10 mL of water sample. Then, the purge gas was collected from the tip of the cartridge and the purged methanol was extracted on the adsorbent of the extraction needle. The limit of detection of the method was 2.5 ng/mL. The method was applied to the determination of methanol in fruit juice samples. The proposed method showed higher sensitivity than conventional PT methods. Furthermore, bubbles are not produce during PT extraction, showing that the developed method is especially suitable for samples that form bubbles in typical PT extraction processes.

Nonaqueous capillary zone electrophoresis (NACZE) utilizing cationic reagents as an electrophoresis promoter has the potential for separating water-insoluble synthetic polymers and that with tetrahydrofuran based electrophoretic media containing cationic reagents has been reported. In this study, NACZE using hexafluoroisopropyl alcohol (HFIP)-based media was developed for separating polyesters and polyamides, which were insoluble in tetrahydrofuran. Nonaqueous media of HFIP containing 400 mM cetyltrimethylammonium chloride (CTAC) and 50 mM trihexyl(tetradecyl)phosphonium chloride (P66614Cl) were used, and it was found that hydrophobic P66614 cation enhanced the electrophoresis of both polyesters and polyamides compared with cetyltrimethylammonium cation. The media containing CTAC resulted in partially separated poly(ethylene terephthalate)/poly(hexamethylene terephthalate) and poly(ethylene terephthalate)/poly(ethylene naphthalate), but P66614Cl failed to separate them. By contrast, nylon 6I and nylon 6/6I were separated with the P66614Cl media, whereas they were co-eluted in the media with CTAC.

The success of the phosphoproteome analysis depends on the separation efficiency of phosphorylated peptides in ion-pair reversed-phase liquid chromatography coupled with tandem mass spectrometry. Here, we report that the phosphorylated peptides were less susceptible to the column temperature than the unphosphorylated peptides when trifluoroacetic acid (TFA) was used as an ion-pair reagent. This trend was partially reversed when TFA was replaced by acetic acid, and the retention order of phosphorylated and unphosphorylated peptide pairs was also partially reversed. Our results indicate that the retention behavior of phosphorylated peptides is relatively predictable in the TFA system, which has long been used in ion-pair liquid chromatographic analysis of peptides, but in the acetic acid system, which is compatible with mass spectrometry, the retention behavior is more diverse, and further data accumulation is needed to quantitatively understand the retention behavior.

A hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS)-based highly sensitive method utilizing limited sample volumes has been developed to analyze 5-aminolevulinic acid (5-ALA) in cell samples. Under the constructed HILIC conditions, 5-ALA is completely separated from biomolecules with similar molecular weights (leucine, isoleucine, hydroxyproline, and creatine), allowing for selective quantification through MS/MS. To analyze 5-ALA using limited amounts of 5-aminolevulinic acid synthase (ALAS) reaction solution, we established a pretreatment method to handle smaller sample volumes. A calibration curve of 5-ALA standard solutions in the range of 7.5–375 nM shows good quantitation with R² = 0.999 and satisfactory precision in the range of 0.7–5.3%. The mentioned concentration range corresponds to 1–50 nM in 5-ALA synthase ALAS reaction solution and is below the quantification limit (3.9 nM) of the existing ALAS activity assays. Thus, the proposed method successfully quantifies the 5-ALA concentrations in cell lysates after the ALAS reaction.

Sample pretreatment is an important step in chromatographic analysis of peptides and proteins. In this study, we investigated the purification of peptides and proteins by using monolithic silica disk-packed spin columns modified with octadecylsilyl moieties. First, digested bovine serum albumin (BSA) peptide samples were purified using the spin columns to examine the purification of peptides. The spin columns removed polar compounds from the samples and reduced the salt concentration. When adrenomedullin, a vasodilating peptide, was spiked into a control serum sample, the addition of trifluoroacetic acid was necessary to remove the pigment derived from the serum while maintaining high recovery. Next, protein purification was investigated and its recovery was evaluated using samples containing ribonuclease A, insulin, lysozyme, BSA, and myoglobin. When ethanol was used as the organic solvent, almost 100% recovery was achieved for all proteins, except BSA. Furthermore, we investigated the quantity of protein loaded onto the spin column. Insulin was more retained on the spin column than BSA. Insulin, a small molecule, can easily penetrate the pores of the stationary phase and thus is easily retained in the stationary phase of the spin column. These results indicate that this method is suitable for the purification of peptides and proteins using spin columns.