Capillary Reversed-phase Liquid Chromatography Research Articles

Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms.

Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteomics include the analysis of large proteoforms and the sensitivity required to examine proteoforms from minimal amounts of sample. To address these challenges, we have developed a new method termed "small-scale serial Size Exclusion Chromatography" (s3SEC), which incorporates a small-scale protein extraction (1 mg of tissue) and serial SEC without postfractionation sample handling, coupled with online high sensitivity capillary reversed-phase liquid chromatography tandem MS (RPLC-MS/MS) for analysis of large proteoforms. The s3SEC-RPLC-MS/MS method significantly enhanced the sensitivity and reduced the proteome complexity across the fractions, enabling the detection of high MW proteoforms previously undetected in one-dimensional (1D)-RPLC analysis. Importantly, we observed a drastic improvement in the signal intensity of high MW proteoforms in early fractions when using the s3SEC-RPLC method. Moreover, we demonstrate that this s3SEC-RPLC-MS/MS method also allows the analysis of lower MW proteoforms in subsequent fractions without significant alteration in proteoform abundance and equivalent or improved fragmentation efficiency to that of the 1D-RPLC approach. Although this study focuses on the use of cardiac tissue, the s3SEC-RPLC-MS/MS method could be broadly applicable to other systems with limited sample inputs.

One-step preparation of cyclen-containing hydrophilic polymeric monolithic materials via epoxy-amine ring-opening reaction and their application in enrichment of N-glycopeptides

Considering the special structure of 1,4,7,10-tetraazacyclododecane (cyclen) which is easy to form complexes with ions, it is beneficial to achieve particular selectivity. Cyclen was selected as a precursor to react with triglycidyl isocyanurate (TGIC), and a novel kind of hydrophilic polymeric monolithic material was facilely prepared via epoxy-amine ring-opening reaction in the presence of a binary porogenic system of acetonitrile (ACN) and polyethylene glycol. The resulting poly (TGIC-co-cyclen) monolithic column was used to separate both nonpolar alkylbenzenes using mobile phase of ACN/H2O (35/65, v/v) and polar phenolic compounds and anilines under the mobile phase of ACN/H2O (60/40, v/v) in reversed-phase capillary liquid chromatography (cLC). It should be pointed that the monolith was further used for separation of a mixture of toluene, DMF, acrylamide and thiourea under the mobile phase of ACN/H2O (95/5, v/v) by hydrophilic interaction chromatography (HILIC). These results indicated that the poly (TGIC-co-cyclen) column exhibited mixed-mode retention mechanism. As a result, the prepared monolithic material was employed for enrichment of glycosylated peptides from the tryptic digest of human immunoglobulin G (IgG) and serum protein tryptic digests. A total of 531 N-glycopeptides and 329 N-glycosylation sites, mapped to 166 glycoproteins, were identified from 2 μL human serum digest. The results indicated the prepared monolith had ability for enriching N-glycopeptides from complex biological samples.

Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples.

Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-μm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 μL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n-Dodecyl β-d-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ∼49,000-94,000 unique peptides, corresponding to ∼6,700-8,300 protein groups using only 100-1000 ng of HeLa tryptic digest (equivalent to ∼500-5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ∼20,000 phosphopeptides from 100 μg of MCF-7 cell lysate.

Simultaneous Determination of Paracetamol and Chlorzoxazone in Their Combined Pharmaceutical Formulations by Reversed-phase Capillary Liquid Chromatography Using a Polymethacrylate Monolithic Column.

Recently, analytical separation techniques have the potential toward green approaches to reduce the environmental impact. This study focuses on the development of an analytical method for the determination of paracetamol and chlorzoxazone in their pharmaceutical combination. The separation was achieved using a home-made capillary column (0.10 mm i.d. × 200 mm length) filled with porous cross-linked hexyl polymethacrylate as monolithic stationary phase. The method proved to be simple, fast, sensitive, efficient, cost-effective and green approach due to the combination of the amazing properties of a monolithic material and a miniaturized liquid chromatography, which would be considered as a step toward reducing the analytical costs and the environmental impact of chromatographic applications. Both components were detected using a 3-nL nano-UV cell fixed at 270 nm wavelength. The optimized mobile phase was composed of 1% aqueous formic acid solution and acetonitrile at 40:60 ratio, 1.0 μL/min flow rate, 4.0 nL injection volume and 50°C column temperature. Under the optimized conditions, paracetamol and chlorzoxazone have been separated in about 6.5 min with chromatographic resolution of 2.37. The prepared column and the analysis method was fully validated and compared with other reported works. All findings allow to conclude that the prepared column and proposed method are applicable for quality control and routine analysis of the two drugs.

Poly(2-carboxyethyl acrylate-co-ethylene glycol dimethacrylate) precursor monolith. Part I. Carbodiimide assisted post-polymerization modification with octadecyl ligands for use in reversed phase capillary liquid chromatography

In this proof of concept study, a new precursor monolithic column was prepared by the in situ co-polymerization of 2-carboxyethyl acrylate (CEA) and ethylene glycol dimethacrylate (EDMA) in a 300 μm i.d. fused silica capillary yielding a poly(CEA-co-EDMA) monolithic column. This carboxy monolith precursor with its surface carboxyl groups was subsequently subjected to post-polymerization modification with octadecylamine (ODA) via a carbodiimide conjugation reaction. The carbodiimide with its zero-length carboxyl-to-amine coupling ability works by activating the surface carboxyl groups of the monolith for direct reaction with the primary amines of ODA via amide bond formation. This reaction, which is applied for the first time in monolithic column fabrication, yielded the octadecyl-poly(CEA-co-EDMA) monolithic capillary column for use in reversed phase capillary LC. This octadecyl column was evaluated for its RPC retention property with alkylbenzenes (ABs) and alkyl phenyl ketones (APKs). The methylene group selectivity (αCH2) in the particular homologs was about the same for both ABs and APKs (αCH2 = 1.6) using the same mobile phase composition. This may indicate that the retention mechanism is the same for both homologous series. The retention behaviors of polyaromatic hydrocarbons and chlorophenol compounds were also examined on the octadecyl capillary column.

Protonation of polyaniline-coated silica stationary phase affects the retention behavior of neutral hydrophobic solutes in reversed-phase capillary liquid chromatography.

Because of its high conductivity when acid doped, polyaniline is known as a synthetic metal and is used in a wide range of applications, such as supercapacitors, biosensors, electrochromic devices, or solar and fuel cells. Emeraldine is the partly oxidized, stable form of polyaniline, consisting of alternating diaminobenzenoid and iminoquinoid segments. When acidified, the nitrogen atoms of emeraldine become protonated. Due to electrostatic repulsion between positive charges, the polarity and morphology of emeraldine chains presumably change; however, the protonation effects on emeraldine have not yet been clarified. Thus, we investigated these changes by reversed-phase capillary liquid chromatography using a linear solvation energy relationship approach to assess differences in dominant retention interactions under a significantly varied mobile phase pH. We observed that hydrophobicity dominates the intermolecular interactions under both acidic and alkaline eluent conditions, albeit to different extents. Therefore, by tuning the mobile phase pH, we can even modulate the retention of neutral hydrophobic solutes, such as aromatic hydrocarbons, because the pH-dependent charge and structure of polymer chains of the emeraldine-coated silica stationary phase show a mixed-mode separation mechanism.

Preparation and characterization of vinylimidazole-based polymer monolithic stationary phases for reversed-phase and hydrophilic interaction capillary liquid chromatography

In this study, we applied 1-vinylimidazole (VIM) as the functional monomer to prepare a series of VIM-based monolithic stationary phases for both reversed-phase and hydrophilic interaction capillary liquid chromatography (LC) using various dimethacrylates (EDMA: ethylene dimethacrylate; HDDMA: 1,6-hexanediol dimethacrylate; DDDMA: 1,10-decanediol dimethacrylate) as cross-linkers. With a simple thermally initiated free-radical cross-linking polymerization process, VIM-based monolithic stationary phases have been successfully prepared. The porosity, permeability, and column efficiency of synthesized VIM-based monolithic stationary phases were characterized. With similar total porosity (85–90%), the VIM-HDDMA monoliths showed the lowest permeability among the three sets of VIM-based stationary phases. Various sets of non-polar (alkyl benzenes and polycyclic aromatic hydrocarbons) and polar analytes (phenol derivatives and amphenicol antibiotics) were applied as model compounds to further investigate the retention behavior of the VIM-based monolithic stationary phases for reversed-phase capillary LC analysis using selected VIM-based monolithic columns. While a mixture of organic acids was employed to perform HILIC analysis using the selected VIM-based monolithic columns. The separation selectivity and retention behavior of the VIM-based monolithic stationary phases were compared to those obtained using three previously prepared alkyl methacrylate-based monolithic columns. Strong retention and good resolution of polar analytes (such as phenol derivatives, amphenicol antibiotics, and organic acids) were observed using the selected VIM-based monolithic columns. The strong retention and good resolution might be attributed to the additional hydrogen-bonding between the hydrogen-donating analytes and the hydrogen-accepting imidazolium functionality on the VIM-based stationary phase. The applicability for both reversed-phase and HILIC capillary LC analysis has also been demonstrated using the selected VIM-based monolithic columns.

Synthesis of polymeric monoliths via thiol-maleimide polymerization reaction for highly efficient chromatographic separation

One-step thiol-maleimide polymerization reaction was firstly adopted for direct preparation of polymeric monoliths via alkaline-catalyzed reaction of 4,4′-bis(maleimidophenyl)methane (BMI) and trimethylolpropane tris(3-mercaptopropionate) (3SH)/pentaerythriol tetra(3-mercaptopropionate) (4SH) in the presence of a small amount of triethylamine (TEA). The polymerization could be performed within 3h, which was faster than thermal-initiated free radical polymerization. Two kinds of monoliths, poly(BMI-co-3SH) (marked as I) and poly(BMI-co-4SH) (marked as II), were characterized with scanning electron microscopy (SEM), attenuated total reflection Fourier-transformed infrared spectroscopy (ATR-FTIR), thermal gravimetric analysis (TGA) and mercury intrusion porosimetry (MIP). Satisfactory chromatographic separation ability and column efficiency were gained for analysis of small molecular compounds such as alkylbenzenes, polynuclear aromatic hydrocarbons (EPA 610) and phenols in reversed-phase capillary liquid chromatography (cLC). High column efficiency (180,500N/m) for butylbenzene was acquired on poly(BMI-co-3SH) column I-2, which was higher than those on most reported polymeric monoliths. A retention-independent efficient performance of small molecules was obtained by plotting of plate height (H) of alkylbenzenes versus the linear velocity (u). A term values in van Deemter equation of I-2 (1.72-0.24μm) and poly(BMI-co-4SH) column II-2 (5.28-4.14μm) were smaller than those of traditional organic/hybrid monoliths. Finally, as a practical application, 53 and 2184 unique peptides from the tryptic digests of bovine serum albumin (BSA) and HeLa cell proteins were positively identified with poly(BMI-co-3SH) monolith in cLC–MS.

Residual brewing yeast as a source of polyphenols: Extraction, identification and quantification by chromatographic and chemometric tools

A method combining aqueous extraction, reversed-phase high-performance capillary liquid chromatography with photodiode array detection (cLC-DAD) and chemometric tools, was developed to determine phenolic compounds in residual brewing yeast. The effect of temperature, nature of extraction solvent and method for separation of extract solution were studied to optimize the extraction conditions on the basis of total phenolic content (TPC), total flavonoids content (TFC) and antioxidant capacity. Polyphenols were determined by cLC-DAD. Flavonols as rutin and kaempferol, flavonoids as naringin, phenolic acids as gallic acid and antioxidants as trans-ferulic and p-coumaric acids were found and quantified in the brewing residue. Data were subjected to evaluation using multifactor ANOVA and principal component analysis (PCA), both showing that lyophilization pretreatment affects the content of individual polyphenols and that residual brewing yeast contains higher polyphenol amounts than the liquid beer waste. The obtained results suggest that residual brewing yeast could be a source of polyphenols.

New Tools for Characterizing Metallic Nanoparticles: AgNPs, A Case Study.

Currently, transmission electron microscopy (TEM) is the main technique for estimating the sizes of spherical nanoparticles (NPs) and through them, their concentrations. This paper demonstrates for the first time that C18 reversed-phase capillary liquid chromatography (Cap-LC) coupled to diode array detection (DAD) has the potential to estimate mean concentrations of silver nanoparticles (AgNPs) and thereby determine their average size. Direct injection of the sample without previous extraction or separation steps is carried out. Only a unique standard with a known AgNP size is needed for the calibration. In a first approach, the new method has been tested over silver nanoparticles, produced using different methods of synthesis, and their water dilutions. Good results were achieved: relative errors ranged up to 5% compared with TEM. Also stability and functionality-related NP properties, as well as nonspherical AgNPs, can be studied using this method. Moreover, by coupling online in-tube solid-phase microextraction (IT-SPME) to Cap-LC-DAD, the effect of the dilution can be studied as particles distribute by polarity in two groups, a distribution that responds to average particle size of not only AgNPs, but also gold nanoparticles (AuNPs). In such a distribution, the average particle size is correlated with the peak area ratio. Additionally, besides higher sensitivity and concentration-dependent signals, IT-SPME-Cap-LC responds to changes in the particle's hydrodynamic diameter allowing, for instance, the detection of cationic surfactants. Size-exclusion and hydrophobic effects are the mechanisms involved to explain this behavior.

Separation Behavior of Short Oligonucleotides by Ion-Pair Reversed-Phase Capillary Liquid Chromatography Using a Silica-Based Monolithic Column Applied to Simple Detection of SNPs

The separation behaviors of short oligonucleotides using capillary liquid chromatography have been investigated comparatively on an octadecylsilane (ODS) packed column (0.3 mm inner diameter, particle size 3 μm) and a monolithic ODS column (0.5 mm inner diameter, through pore size 2 μm). The present paper clearly demonstrates that the resolution obtained using the monolithic ODS packed column is much higher than that using a semi-μ-high-performance liquid chromatography (HPLC) column on a semi-μ-HPLC system. Furthermore, it was surprising that complete baseline resolution of oligonucleotides, typically shorter than 30-mer, with single nucleotide units was achieved using the monolithic column, although this was not possible using the ODS packed column. Good separation of short oligonucleotides was achieved at 40–60 °C with a linear 40–60 % gradient of methanol content. Separation conditions on the monolithic column were optimized regarding the type of counterion, methanol content, temperature, and flow rate. Using the present method, detection of single-nucleotide polymorphisms was achieved without using a mass-spectrometry (MS) detector.

Sensitive determination of parabens in human urine and serum using methacrylate monoliths and reversed-phase capillary liquid chromatography–mass spectrometry

A method for the determination of parabens in human urine and serum by capillary liquid chromatography (cLC) with UV–Vis and mass spectrometry (MS) detection using methacrylate ester-based monolithic columns has been developed. The influence of composition of polymerization mixture was studied. The optimum monolith was obtained with butyl methacrylate monomer at 60/40% (wt/wt) butyl methacrylate/ethylene dimethacrylate ratio and 50wt% porogens (composed of 36wt% of 1,4-butanediol, 54wt% 1-propanol and 10wt% water). Baseline resolution of analytes was achieved through a mobile phase of acetonitrile/water in gradient elution mode. Additionally, dispersive liquid–liquid microextraction (DLLME) was combined with both cLC–UV–Vis and cLC–MS to achieve the determination of parabens in human urine and serum samples with very low limits of detection. Satisfactory intra- and inter-day repeatabilities were obtained in UV–Vis and MS detection, although the latter provided lower detection limits (up to 300-fold) than the UV–Vis detection. Recoveries for the target analytes from spiked biological samples ranged from 95.2% to 106.7%. The proposed methodology for the ultra-low determination of parabens in human urine and serum samples is simple and fast, the consumption of reagents is very low, and very small samples can be analyzed.

Effect of different glycation agents on Cu(II) binding to human serum albumin, studied by liquid chromatography, nitrogen microwave-plasma atomic-emission spectrometry, inductively-coupled-plasma mass spectrometry, and high-resolution molecular-mass spectrometry

The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)-DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)-DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation.

High efficiency polyethylene glycol diacrylate monoliths for reversed-phase capillary liquid chromatography of small molecules

Highly cross-linked monolithic networks (i.e., polyethylene glycol diacrylate, PEGDA) synthesized from monomers containing varying ethylene oxide chain lengths were fabricated inside fused silica capillary columns for use in liquid chromatography (LC) of small molecules. Tergitol was used as a surfactant porogen in combination with other typical organic liquid porogens. Column performance was correlated with quantitative descriptors of the physical/chemical properties of the monomers and porogens using a statistical model. Solubility and viscosity values of the components were identified as important predictors of monolith morphology and efficiency. The chromatographic retention mechanism was determined to be principally reversed-phase (RP) with additional hydrogen bonding between the polar groups of the analytes and the ethylene oxide groups embedded in the monolith structure. The fabricated monolithic columns were evaluated under RPLC conditions using phenols, hydroxy benzoic acids, and alkyl parabens as test compounds. Isocratic elution of hydroxy benzoic acids at a linear velocity of 0.04cm/s using a PEGDA-700 monolith gave chromatographic peaks with little tailing (i.e., tailing factor<1.28). The chromatographic efficiency measured for a non-retained compound (uracil) using this column was 186,000plates/m when corrected for injector dead volume. High resolution gradient separations of selected pharmaceutical compounds and phenylurea herbicides were achieved in less than 18min. Optimized monoliths synthesized from all four crosslinking monomers exhibited high permeability and demonstrated little swelling or shrinking in different polarity solvents. Column preparation was highly reproducible, with relative standard deviation (RSD) values less than 2.1%, based on retention times of the phenol standards (3 different columns).

Incorporation of metal-organic framework HKUST-1 into porous polymer monolithic capillary columns to enhance the chromatographic separation of small molecules

Metal-organic framework (MOF) HKUST-1 nanoparticles have been incorporated into poly(glycidyl methacrylate-co-ethylene dimethacrylate) (HKUST-1-poly(GMA-co-EDMA)) monoliths to afford stationary phases with enhanced chromatographic performance of small molecules in the reversed phase capillary liquid chromatography. The effect of HKUST-1 nanoparticles in the polymerization mixture on the performance of the monolithic column was explored in detail. While the bare poly(GMA-co-EDMA) monolith exhibited poor resolution (Rs<1.0) and low efficiency (800–16,300plates/m), addition of a small amount of HKUST-1 nanoparticles to the polymerization mixture provide high increased resolution (Rs≥1.3) and high efficiency ranged from 16,300 to 44,300plates/m. Chromatographic performance of HKUST-1-poly(GMA-co-EDMA) monolith was demonstrated by separation of various analytes including polycyclic aromatic hydrocarbons, ethylbenzene and styrene, phenols and aromatic acids using a binary polar mobile phase (CH3CN/H2O). The HKUST-1-poly(GMA-co-EDMA) monolith displayed enhanced hydrophobic and π–π interaction characteristics in the reversed phase separation of test analytes compared to the bare poly(GMA-co-EDMA) monolith. The experiment results showed that HKUST-1-poly(GMA-co-EDMA) monoliths are an alternative to enhance the chromatographic separation of small molecules.

One-step strategy for the synthesis of a derivatized cyclodextrin-based monolithic column.

Derivatized β-cyclodextrin (β-CD) functionalized monolithic columns were prepared by a "one-step" strategy using click chemistry. First, the intended derivatized β-CD monomers were synthesized by a click reaction between propargyl methacrylate and mono-6-azido-β-CD and then sulfonation or methylation was carried out. Finally, monolithic columns were prepared through a one-step in situ copolymerization of the derivatized β-CD monomer and ethylene glycol dimethacrylate. The sulfated β-CD-based monolith was successfully applied to the hydrophilic interaction liquid chromatography separation of nucleosides and small peptides, while the methylated β-CD-functionalized monolith was useful for the separation of nonpolar compounds and drug enantiomers in capillary reversed-phase liquid chromatography. The structures of the monomers were characterized by Fourier transform infrared spectroscopy and mass spectrometry. The physicochemical properties and column performance of monoliths were evaluated by scanning electron microscopy and micro high performance liquid chromatography. This strategy has considerable prospects for the preparation of other derivatized CD-functionalized methacrylate monoliths.

Highly crosslinked polymeric monoliths with various C6 functional groups for reversed-phase capillary liquid chromatography of small molecules

Three crosslinking monomers, i.e., 1,6-hexanediol dimethacrylate (HDDMA), cyclohexanediol dimethacrylate (CHDDMA) and 1,4-phenylene diacrylate (PHDA), were used to synthesize highly cross-linked monolithic capillary columns for reversed-phase liquid chromatography (RPLC) of small molecules. Selection of porogen type and concentration was investigated in detail. Isocratic elution of alkylbenzenes at a flow rate of 300nL/min was performed using HDDMA and CHDDMA monolithic columns. Gradient elution of alkylbenzenes using all three monolithic columns showed good separations. Optimized monoliths synthesized from all three crosslinking monomers possessed high permeabilities. Poly(HDDMA) monoliths demonstrated column efficiencies up to 86,000 plates/m. Column preparation of poly(HDDMA) monolithic columns was highly reproducible; the relative standard deviation (RSD) values (n=3) for run-to-run and column-to-column were less than 0.26% and 0.70%, respectively, based on retention times of alkylbenzenes.

Ion-paired extraction of cephalosporins in acetone prior to their analysis by capillary liquid chromatography in environmental water and meat samples

Ion-pair extraction of cephalosporins from aqueous solution into acetone by the addition of ammonium sulfate to a 1:2 (v/v) acetone–water solvent was carried out followed by their determination using reversed-phase capillary liquid chromatography. The analytes included are cephoperazone, cefquinome, cephalexin, cephapirin, cephaloniun, cephamandole, cephazolin and cephadroxile. In order to form the ion-pair, hexadecyltrimethylammonium bromide (CTAB) was selected as cationic ion-pairing agent at a concentration of 0.9mM using 10mM phosphate buffer at pH 8 as the optimum condition for the aqueous solution. The applied methodology, named salting-out assisted liquid/liquid extraction (SALLE) involves the use of 1.25g of ammonium sulfate as salting-out agent.The separation of cephalosporins using a Luna C18 (150mm×0.3mm, 5µm, 100Å) column was achieved under the following conditions: a gradient program combining solvent A (0.1% formic acid in water, pH 4) and solvent B (acetonitrile–methanol (50:50, v/v)), at a flow rate of 20µlmin−1, column temperature 35°C and injection volume 7µl with UV detection at 250nm. The limits of quantification for the studied compounds were between 4.3 and 22.7μg/L for water samples and 4.1 and 73.3μg/kg in the case of beef samples, lower than the maximum residue limits permitted by the EU for this kind of food. The developed methodology has demonstrated its suitability for the analysis of these widely applied antibiotics in environmental water and meat samples, including beef and pork muscle, with high sensitivity, precision and satisfactory recoveries.

Facile preparation of a stable and functionalizable hybrid monolith via ring-opening polymerization for capillary liquid chromatography

An organic–inorganic hybrid monolith was prepared by a single-step ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (POSS) with poly(ethylenimine) (PEI). The obtained hybrid monoliths possessed high ordered 3D skeletal microstructure with dual retention mechanism that exhibits reversed-phase (RP) mechanism under polar mobile phase and hydrophilic-interaction liquid chromatography (HILIC) retention mechanism under less polar mobile phase. The high column efficiencies of 110,000N/m can be achieved for separation of alkylbenzenes in capillary reversed-phase liquid chromatography (cLC). Due to the robust property of hybrid monolith and the rich primary and secondary amino groups on its surface, the resulting hybrid monolith was easily modified with γ-gluconolactone and physically coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC), respectively. The former was successfully applied for HILIC separation of neutral, basic and acidic polar compounds as well as small peptides, and the latter for enantioseparation of racemates in cLC. The high column efficiencies were achieved in all of those separations. These results demonstrated that the hybrid monolith (POSS–PEI) possessed high stability and good surface tailorbility, potentially being applied for other research fields

Identification and confirmation of haptoglobin as a potential serum biomarker in hypertrophic cardiomyopathy using proteomic approaches

Introduction. Aiming at identifying biomarkers for hypertrophic cardiomyopathy (HCM), the serum proteome was explored through a two-dimensional gel-based proteomic approach (2D-DIGE) coupled with mass spectrometry and database interrogation.Methods. Serum samples from 20 male HCM patients and their sex- and age-matched controls were cleaned from interfering components. Patients and controls were pooled in five matched groups with the same age, and proteins extracts from each pool were labelled with cyanine dyes. Then, gel images were analysed using a fluorescence scanner and proteins were identified. Tryptic peptides were analysed by capillary reversed-phase liquid chromatography coupled online with tandem mass spectrometry (MS/MS).Results. Four different proteins were observed to be differentially expressed between HCM patients and their matched controls. Of them, decreases in haptoglobin levels were confirmed to be associated with HCM in an independent set of 181 consecutive HCM patients from our monographic clinic and 114 controls with similar age and sex using a nephelometer-based technique. Moreover, a significant negative correlation was observed between haptoglobin and subaortic gradient, thus highlighting the role of haptoglobin in HCM.Conclusion. All these observations point out the utility of the 2D-DIGE proteomic strategy for the identification of serum proteins indicative of the presence of cardiac injury.