A high-throughput, ultrafast, and online three-phase electro-extraction method for analysis of trace level pharmaceuticals.

Sample preparation is often reported as the main bottleneck of analytical processes. To meet the requirements of both high-throughput and high sensitivity, improved sample-preparation methods capable of fast analyte preconcentration are urgently needed. To this end, a new three-phase electroextraction (EE) method is presented that allows for ultrafast electroextraction hyphenated to flow-injection analysis mass spectrometry (FIA-MS). Four model compounds, i.e., propranolol, amitriptyline, bupivacaine, and oxeladin, were used to optimize and evaluate the method. Within only 30 s extraction time, enrichment factors (EF) of 105-569 and extraction recoveries (ER) of 10.2%-55.7% were achieved for these analytes, with limits of detection (LODs) ranging from 0.36 to 3.21 ng mL-1, good linear response function (R2 > 0.99), low relative standard deviation (0.6%-17.8%) and acceptable accuracy (73-112%). Finally, the optimized three-phase EE method was successfully applied to human urine and plasma samples. Our three-phase electroextraction method is simple to construct and offers ultrafast, online extraction of trace amounts of analytes from biological samples, and therefore has great potential for high-throughput analysis.

A novel method for serum lipoprotein profiling using high performance capillary isotachophoresis.

A new capillary isotachophoresis (cITP) method for lipoprotein profiling with superior lipoprotein coverage compared to previous methods has been developed, resolving twice as many lipoprotein species (18 peaks/fractions) in serum or plasma in less than 9.5 min. For this, a novel mixture of 24 spacers, including amino acids, dipeptides and sulfonic acids, was developed and fine-tuned, using predictive software (PeakMaster) and testing of spiked serum samples. Lipoprotein peaks were identified by serum-spiking with reference lipoproteins. Compatibility with common lipophilic stains for selective lipoprotein detection with either UV/Vis or laser-induced fluorescence was demonstrated. A special new capillary with a neutral coating (combining water-compatible OV1701-OH deactivation and methylation) was used for the first time for electrodriven separations, allowing very stable separations in a pH 8.8-9.4 gradient system, being functional for more than 100 injections. Excellent reproducibility was achieved, with coefficients of variation lower than 2.6% for absolute migration times. Comparison was performed with human plasma samples analyzed by NMR, leading to similar results with cITP after multivariate statistics, regarding group-clustering and lipoprotein species correlation. The new cITP method was applied to the analysis of serum samples from a LDL receptor knock-out mice model fed either a normal diet or a western-type diet. Differences in the lipoprotein levels and in the sublipoprotein types were detected, showing a shift to more atherogenic particles due to the high cholesterol diet. In summary, this novel method will allow more detailed and informative profiling of lipoprotein particle subtypes for cardiovascular disease research.

Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations.

A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10(-9) m(2) V(-1) s(-1)) when compared with unmodified fused silica (5.9 ± 0.1 10(-8) m(2) V(-1) s(-1)). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1-1.8% coefficient-of-variation (CV) within a day) and 2-3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use.

Electroextraction and electromembrane extraction: Advances in hyphenation to analytical techniques.

Electroextraction (EE) and electromembrane extraction (EME) are sample preparation techniques that both require an electric field that is applied over a liquid-liquid system, which enables the migration of charged analytes. Furthermore, both techniques are often used to pre-concentrate analytes prior to analysis. In this review an overview is provided of the body of literature spanning April 2012-November 2015 concerning EE and EME, focused on hyphenation to analytical techniques. First, the theoretical aspects of concentration enhancement in EE and EME are discussed to explain extraction recovery and enrichment factor. Next, overviews are provided of the techniques based on their hyphenation to LC, GC, CE, and direct detection. These overviews cover the compounds and matrices, experimental aspects (i.e. donor volume, acceptor volume, extraction time, extraction voltage, and separation time) and the analytical aspects (i.e. limit of detection, enrichment factor, and extraction recovery). Techniques that were either hyphenated online to analytical techniques or show high potential with respect to online hyphenation are highlighted. Finally, the potential future directions of EE and EME are discussed.

Lab-on-a-Chip hyphenation with mass spectrometry: strategies for bioanalytical applications.

The Lab-on-a-Chip concept aims at miniaturizing laboratory processes to enable automation and/or parallelization via microfluidic chips that are capable of handling minute sample volumes. Mass spectrometry is nowadays the detection method of choice, because of its selectivity, sensitivity and wide application range. We review the most interesting examples over the last two-and-a-half years where the two techniques were used for bioanalytical applications. Furthermore, we discuss the merits and limitations of such hyphenated systems. We inventorize the reported applications and approaches. We see an ongoing trend towards chip-based liquid chromatography-mass spectrometry usage and small volume analysis applications, particularly in the field of proteomics where bottom-up approaches profit from chip-based technologies and hyphenation with complex cell cultures.

Continuous-flow microelectroextraction for enrichment of low abundant compounds.

We present a continuous-flow microelectroextraction flow cell that allows for electric field enhanced extraction of analytes from a large volume (1 mL) of continuously flowing donor phase into a micro volume of stagnant acceptor phase (13.4 µL). We demonstrate for the first time that the interface between the stagnant acceptor phase and fast-flowing donor phase can be stabilized by a phaseguide. Chip performance was assessed by visual experiments using crystal violet. Then, extraction of a mixture of acylcarnitines was assessed by off-line coupling to reversed phase liquid chromatography coupled to time-of-flight mass spectrometry, resulting in concentration factors of 80.0 ± 9.2 times for hexanoylcarnitine, 73.8 ± 9.1 for octanoylcarnitine, and 34.1 ± 4.7 times for lauroylcarnitine, corresponding to recoveries of 107.8 ± 12.3%, 98.9 ± 12.3%, and 45.7 ± 6.3%, respectively, in a sample of 500 µL delivered at a flow of 50 µL min(-1) under an extraction voltage of 300 V. Finally, the method was applied to the analysis of acylcarnitines spiked to urine, resulting in detection limits as low as 0.3-2 nM. Several putative endogenous acylcarnitines were found. The current flowing-to-stagnant phase microelectroextraction setup allows for the extraction of milliliter range volumes and is, as a consequence, very suited for analysis of low-abundant metabolites.

Simple capillary electrophoresis-mass spectrometry method for complex glycan analysis using a flow-through microvial interface.

A flow-through microvial is used to interface capillary electrophoresis and mass spectrometry (CE-MS) to develop a method for simultaneous profiling both neutral and sialylated glycans without derivatization or labeling. The CE separation was performed at near-zero electroosmotic flow in a capillary with neutral, hydrophilic coating, using 50 mM ammonium acetate in 20% methanol (pH 3.1) as the background electrolyte. The method was optimized with reversed CE polarity and negative ion ESI-MS. Enzymatically released N-glycans from human immunoglobulin G (IgG) were used as the test sample. The approach was also used to study the more complex N-glycans from recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary (CHO) cells. Glycoscreening of rHuEPO was performed using a triple quadrupole MS and an ultrahigh resolution TOF-MS. The high sensitivity and high mass accuracy of the TOF-MS revealed the presence of more than 70 glycans. Three mono- and di-sialylated tetra-antennary N-glycans and one mono-sialylated tri-antennary N-glycan of rHuEPO are reported for the first time. Further glycan heterogeneity was identified of the highly sialylated N-glycans of rHuEPO by extensive acetylation, Neu5Ac/Neu5Gc variation and the presence of N-acetyl-lactosamine repeats. For comparative purposes, porous graphitic carbon-based LC-MS/MS was also used to glycoprofile rHuEPO. This work demonstrates the potential of CE-MS to provide a comprehensive glycosylation profile with detailed features of the secondary glycan modifications. The CE-MS based method eliminates the need to label the N-glycans, as well as the requirement to desialylate before analysis, and could complement other established techniques for glycan characterization of therapeutic glycoproteins.

Capillary electrophoresis-mass spectrometry using a flow-through microvial interface for cationic metabolome analysis.

The application of CE-MS in the field of metabolomics is underrepresented, even though it is in principle highly suited for the analysis of small charged compounds, as many metabolites are. Moreover, a robust coupling, using the sheath liquid (SL)-assisted interface was already presented more than a decade ago. A lack of concentration sensitivity is often mentioned as a reason for the underrepresentation of CE-MS in metabolomics. This is caused by postcolumn dilution of the sample with SL, which is typically delivered at a flow rate of 1-10 µL/min. In this study, we investigated the performance of the flow-through microvial (MV) assisted CE-MS interface for cationic metabolomics. With this interface, only a little liquid is added postcolumn, that is, typically 100-500 nL/min. For the evaluation, we used a metabolite mix comprising 45 important cationic metabolites and compared the sensitivity and LOD of both devices. The performance of the CE-MS system was significantly improved by using the MV-assisted interface; the sensitivity was increased more than three times and the LOD decreased more than five times. Then, we analyzed single zebrafish embryos to demonstrate the method on a volume-limited biological sample. In comparison with SL-assisted CE-MS, twice as many molecular features were found, of which several could be identified. These results demonstrate the good potential of the MV interface for enhancing the coverage of the metabolome.

The potential of electrophoretic sample pretreatment techniques and new instrumentation for bioanalysis, with a focus on peptidomics and metabolomics.

This Review highlights the potential of new electromigration-based sample pretreatment techniques for bioanalysis. Sample pretreatment is a challenging part of the analytical workflow, especially in the fields of peptidomics and metabolomics, where the analytes are very diverse, both in physicochemical properties and in endogenous concentration. Electromigration-based techniques have several strengths, such as fast selective analyte concentration and that complementary information on the content of a sample can be obtained when compared with more conventional (chromatography-based) techniques. In the past decade, various new electromigration-based sample pretreatment techniques have been developed, and importantly, new instrumental setups. In this Review, we provide an introduction on electromigration and its strengths. Then, selected examples of electromigration-based sample pretreatment techniques and instrumentation are discussed, namely free-flow electrophoresis, isoelectric focusing, isotachophoresis, electrodialysis, electromembrane extraction and electroextraction. Finally, the promising perspectives of electromigration-based sample pretreatment techniques are outlined.

Potential of polyE-323 coated capillaries for capillary electrophoresis of lipids.

In this note the feasibility of a polyamine-based capillary coating, polyE-323, for capillary electrophoresis (CE) of lipids is explored. PolyE-323 has previously been demonstrated to be suitable to suppress analyte-wall interaction of proteins in CE. However, the full applicability range of polyE-323 has not been exploited yet and it might be useful in the analysis of hydrophobic analytes, such as lipids. In this study, the stability of polyE-323 when using highly organic background electrolytes (BGEs), which are needed to solubilize the lipid analytes, was studied. For this, we used three different lipid samples: sphingomyelin, cardiolipin and a lipid extract from a cell culture. The highly organic BGEs that were used in this study consisted of 94.5% of organic solvents and 5.5% of an aqueous buffer. First, the influence of pure acetonitrile, methanol, propylene carbonate, isopropanol and chloroform on the polyE-323 coating was investigated. Then BGEs were developed and tested, using sphingomyelin and cardiolipin as test analytes in CE-UV experiments. After establishing the best BGEs (in terms of analysis time and repeatability) by CE-UV, sphingomyelin was used as a test analyte to demonstrate that method was also suitable for CE with mass-spectrometry detection (CE-MS). The LOD of sphingomyelin was estimated to be 100 nM and its migration time repeatability was 1.3%. The CE-MS analysis was further applied on a lipid extract obtained from human glioblastoma cells, which resulted in the separation and detection of a multitude of putative lipids. The results of our feasibility study indicate that CE systems based on polyE-323 coated capillaries and highly organic BGEs are promising for fast electromigration-based analysis of lipids.

Feasibility of electroextraction as versatile sample preconcentration for fast and sensitive analysis of urine metabolites, demonstrated on acylcarnitines.

In this work, we demonstrate the applicability of electroextraction (EE) to urine metabolites. To investigate which urine metabolite classes are susceptible to EE, off-line EE experiments were carried out with a prototype device, in which urine metabolites were electroextracted from ethyl acetate into water. The obtained extracts were examined with direct infusion MS and the results demonstrated that several compound classes could be extracted, amongst which amino acids and acylcarnitines. Acylcarnitines were selected for evaluation of the performance of EE. For this, the EE setup was adapted to capillary EE (cEE) to be able to analyze large urine sample series, and it was coupled online to LC-MS. cEE-LC-MS of acylcarnitines was optimized and characterized. The recovery, linearity, repeatability, and detection limit of the cEE-LC-MS method was good to excellent. To demonstrate the versatility of EE for sample preparation in analytical procedures, extracts were injected into a CZE-MS system, resulting in detection of the acylcarnitines along with more than 100 presumed metabolite peaks. The results presented here indicate that EE can be used as a fast sample preconcentration technique of low abundant urine metabolites, in combination with both LC and CZE.

On-line large-volume electroextraction coupled to liquid chromatography-mass spectrometry to improve detection of peptides.

Electroextraction (EE) takes place in a two-phase liquid-liquid system, consisting of an aqueous and an organic phase, where an applied electric field causes ions to be extracted from one phase into the other, to be concentrated close after the liquid-liquid interface. The extraction takes place in a wide-bore capillary that is connected to a 2-way 10-port switching valve, which serves to couple capillary EE (cEE) with LC-MS. In this set-up, volumes as high as 100 µL can be extracted, which is a ten times larger volume than has been reported, earlier. After a feasibility study using the cationic purple dye crystal violet, the method was coupled to LC-MS and large volume cEE of several model peptides was optimized. The cEE-LC-MS method had good repeatability, good linearity and LODs between 0.5 and 10nM. The whole procedure was automated and could be used routinely. Finally, the method was applied to plasma analysis and calibration curves of the relevant plasma peptides angiotensin 1 and 2 as well as the fragment angiotensin 2 (3-8) showed good linearity and repeatability; LOD values were 10-50 nM. Analysis of unspiked plasma resulted in 60 putative endogenous peptides, underlining the great potential of EE as on-line sample concentrating technique. On-line large volume cEE-LC-MS allows for enrichment, separation and detection of plasma peptides from large sample volumes, minimizes sample handling and can be an important step in full automation of analytical procedures.

Online capillary liquid-liquid electroextraction of peptides as fast pre-concentration prior to LC-MS.

In this research paper, we show that capillary electroextraction (cEE) is capable of fast online peptide concentration and that it can be coupled online to LC-MS to result in a fast and sensitive method. Electroextraction takes place when an electrical field is applied in a two-phase liquid-liquid system. Sample molecules in the organic phase migrate very fast into the aqueous phase and are concentrated in a small zone. In this work, cEE of peptides is developed and coupled online to LC-MS via a switching valve. Comparison of 10 min of cEE-LC-MS with a normal LC-MS injection showed more than 100-fold increased peak heights. Of five model peptides, good calibration curves in the range of 0.05-5 µmol/L were obtained. The linearity was good (R(2) values between 0.984 and 0.996) and RSD between 5% at the highest to 25% at the lowest concentration (n=3). The LOD of bradykinin, angiotensin I-converting enzyme inhibitor and angiotensin I was in the low nmol/L range. Analysis of a tryptic digest of eight model proteins resulted in more than 170 peptides, without bias for pI or hydrophilicity. Urine analysis is demonstrated, resulting in an LOD around 0.04 µmol/L urine for tryptic cytochrome C peptides spiked to urine and an increase of 42% in the number of chromatographic peaks compared with the conventional LC-MS. In summary, cEE-LC-MS is a fast electrophoresis-driven sample preconcentration technique that is quantitative, able to extract a wide peptide range and applicable to bioanalysis.

Open tubular capillary electrochromatography: technique for oxidation and interaction studies on human low-density lipoproteins.

A novel, open tubular capillary electrochromatographic method was developed for the in vitro oxidation of low-density lipoprotein (LDL) particles. Low-density lipoprotein particles with molar mass of approximately 2.5 MDa yielded a stable stationary phase at temperatures 25 and 37 degrees C and at pH values from 3.2 to 7.4. The quality of the coatings was not influenced by variations in the LDL concentration in the coating solutions (within the range of 2-0.015 mg/mL) with the coating procedure used in the study. Radiolabeled LDL stationary phases and scanning electron microscopy, employed to shed light on the location and coating density of LDL particles on the inner surface of the capillary wall, confirmed the presence of an LDL monolayer and almost 100% coating efficiency (99 +/- 8%). In addition, the radioactivity measurements allowed estimation of the amount of LDL present in a single capillary coating. Capillaries coated with human LDL particles were submitted to different oxidative conditions by changing the concentration of the oxidant (CuSO4), oxidation time, pH value, and temperature. The oxidation procedure was followed with electroosmotic flow mobility, which served as an indicator of the increase in total negative charges of LDL coatings, and by asymmetrical field flow fractionation, which measured the changes in size of the lipoprotein particles. The results indicated that oxidation of LDL was progressing with increasing time, temperature, and concentration of the oxidant as expected. The oxidation process was faster around neutral pH values (pH 6.5-7.4) and inhibited at acidic pH values (pH 5.5 and lower).